August 2003

Volume 205

Number

BIOLOGICAL
BULLETIN
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THE BIOLOGICAL BULLETIN

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SEEKERS
THE SOCIETY
OF CELLS.
Simon

says.

"We

At Dr. Simon Watkins'

lab,

they look at

of traders and communicators, of connections

and

relationships.

never jump to conclusions.

to us." His mantra?

"We

We

are the observers,"

the conclusions

let

Which

"Imaging is everything."
the brightest of tomorrow's seekers and solvers find
and the Watkins Lab.

ROCKET SCIENCE

me ca

jump
why the best and
their way to Pittsburgh

is

o.

OLYMPUS*
(From

Ana

to K)

Bursick

Stuart

Research Specialist

Shand Research

Simon

Specialist

C. Watkins, Ph.D.

Director

Glenn Popworth - Research Associate

Romesh Draviam Graduate Student
Center

for Biologic

Imaging,

University of Pittsburgh
Pittsburgh,

PA

Medical School,

cells

way anthropologists look at human culture:

as communities of good guys and bad guys,
the

AUG

2 5 2003

"

'

various species; the crab

Cover

is

Chionocetes

sp.

The

top right shows octopus eggs (length,

on
being sampled by the suction sampler

at the

occupied; but
in restricted areas and under unusual conditions,
such as cold seeps, vents, and seamounts, dense

The deep sea

is,

in general, sparsely

and persist for generations.

also
aggregate temporarily to
Sparse populations

communities do

exist

and such
mating, breeding, and brooding,
are well known in varireproductive aggregations
but not in the deep sea, where only
ous habitats
three such aggregations have previously been doc-

facilitate

umented.

The Biological Bulletin (p. 1 ), Jefand

colleagues at the Monterey Bay
frey C. Drazen
Institute (MBARI, California)
Research
Aquarium
In this issue of

describe, for the first time in the

deep

sea, a multi-

or reproductive

species reproductive aggregation

w ith an unusually high population dennot S p 0t
This aggregation is featured on
biomass.
and
sity

the cover;

it

is

located in

1500-1600 meters of

water on the Gorda Escarpment, a submarine plateau off Cape Mendocino in northern California.
The site was discovered in the course of 1 5 exploratory dives by

MBARI's

remotely operated vehicle

the
(top left image on the cover);
the
identifiable
are
cameras
main
two
by
vehicle's
white protective collars around their glass domes.
The map on the cover locates the hot spot (red
and the ROV
circle). Cape Mendocino (red dot),

(ROV) Tiburon

dives (the line of small, irregular black areas extending westward).

an ocReproductive aggregations of two species

blob
the
a
sculpin
topus (Graneledone sp.), and fish,
co-occurred at this site.
(Psychrolutes phrictus)

image shows three octopuses (body

width, -16 cm) in a characteristic brooding posi-

The bottom

of the eggs hatched during samone

hatchling appears in the sampler tube,
pling;
and another is swimming away.' In the lower right
which is
image watching from behind a rock,
is a
anemones
and
stars
sea
in
covered
brisingid

ROV. Many

the

blob sculpin (length,

~60 cm)

with a nest of large,

Another fish is just visible in

pinkish eggs behind it.
the image. Most blob sculof
corner
left
the upper
to their egg masses (e.g.,
pins were seen attending
observations of padirect
first
the
4).
3A,
p.
Fig.
rental care by an oviparous deep-sea fish.

The

of this reproductive hot spot

particular location

could be due to environmental heterogeneity; that

at the crest of the
is, the animals were concentrated
local topography and near cold seeps. In these situations, they might benefit from enhanced current
flow and local productivity, critical resources for

the deep sea. where oxygen

reproductive success in
tension is very low and food is in short supply.

Thus, for some deep-sea species, the fortuitous occurrence of critical environmental features may be
essential for reproduction.

The four images

are frames selected

from videos

taken during dives in 2001 and 2002. The videos

were produced collaboratively by the crew of the
the ROV Tiburon
support ship R/V Western Flyer,

and the scientists. Photo credit is to MBARI.

contributed to the cover and
Jeffrey C. Drazen
was designed by Beth Liles
legend. The final cover
pilots,

(Marine Biological Laboratory,

Woods

Hole, Mas-

sachusetts).

left

to the
eggs are underneath them, attached
of
anemones
are
several
attached
rock outcrop. Also

tion; their

image
40 mm)

The octopus hatchlings

cago Field Museum), an

are being described by Janet Voight (Chi-

MBARI

collaborator.

THE

BIOLOGICAL BULLETIN
AUGUST
Editor

MICHAEL

Associate Editors

Louis E. BURNETT
R.

J.

GREENBERG

2003

The Whitney Laboratory, University of Florida

Grice Marine Laboratory, College of Charleston
California Institute of Technology

ANDREW CAMERON

CHARLES D. DERBY

Georgia State University

MICHAEL LABARBERA

University of Chicago

Section Editor

SHINYA INOUE, Imaging and Microscopv

Marine Biological Laboratory

Online Editors

JAMES A. BLAKE, Keys

ENSR

to

Marine

Woods Hole Region

WILLIAM D. COHEN, Marine Models
Electronic Record and Compendia

Marine

&

Coastal Center,

Woods Hole

Invertebrates of the

Editorial

Board

New York

PETER B. ARMSTRONG

University of California, Davis

JOAN CERDA

Center of Aquaculture-IRTA, Spain

Bodega Marine Lab., University of California, Davis

ERNEST S. CHANG
THOMAS H. DIETZ
RICHARD B. EMLET

DAVID EPEL
KENNETH M. HALANYCH
GREGORY HINKLE

NANCY KNOWLTON
MAKOTO KOBAYASHI
ESTHER M. LEISE
DONAL T. MANAHAN
MARGARET MCFALL-NGAI
MARK W. MILLER
TATSUO MOTOKAWA
YOSHITAKA NAGAHAMA
SHERRY D. PAINTER

HERBERT WAITE

Louisiana State University

Oregon Institute of Marine Biology, Univ. of Oregon
Hopkins Marine Station, Stanford University

Auburn University, Alabama

Millennium Pharmaceuticals, Cambridge, Massachusetts
Scripps Inst. Oceanography & Smithsonian Tropical Res.
Hiroshima University of Economics, Japan
University of North Carolina Greensboro
University of Southern California

Kewalo Marine Laboratory, University of Hawaii

Institute of

Tokyo

Neurobiology, University of Puerto Rico

Institute of

Technology, Japan

National Institute for Basic Biology, Japan

Marine Biomed. Inst., Univ. of Texas Medical Branch

RICHARD K. ZIMMER

University of California, Santa Barbara

University of California, Los Angeles

J.

Editorial Office

Hunter College, City University of

PAMELA CLAPP HINKLE

Managing Editor

VICTORIA R. GIBSON

Staff Editor

CAROL SCHACHINGER

Editorial Associate

WENDY CHILD

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CONTENTS
VOLUME

205, No.

RESEARCH NOTE

AUGUST 2003

Eyster, L. S., and L. M. van Camp

Extracellular lipid droplets in Idiosepiiu nutoides, the

Drazen, Jeffrey C., Shaiia K. Goffredi, Brian Schlining,

S. Stakes

47

Southern pygmy squid

and Debra

Christensen,

of egg-brooding deep-sea fish and

cephalopods on the Gorda Escarpment: a reproduc-

Ana Beardsley, James M. Colacino, and

Celia Bonaventura

Aggregations
tive

1:

Functional and biochemical properties of the hemoglobins of the burrowing brittle star Hemipholis elon-

hot spot

54

Say (Echinodermata, Ophiuroidea)

frtiiii

EVOLUTION
Zigler, Kirk S.,

SYMBIOSIS AND PARASITOLOGY

and H. A. Lessios

250 million years

of

bindin evolution
Davy, Simon K,, and John R. Turner
Early development and acquisition of zooxanthellae
in the temperate symbiotic sea anemone Anthopleura

NEUROBIOLOGY AND BEHAVIOR

ballii

Painter, Sherry- D., Bret Clough, Sara Black,

T. Nagle

Behavioral

characterization

and Gregg

of attractin,

borne peptide pheromone in the genus

Bergman, Daniel A., and Paul A. Moore

DEVELOPMENT AND REPRODUCTION

water-

Aplysifi

Neumann,

Field observations of intraspecific agonistic behavior

of two crayfish species,

conectes

i>i>ilis,

Orconectes

in different habitats

66

(Cocks)

and Or..............

On

nisticus

26

PHYSIOLOGY AND BIOMECHANICS

Dietrich,

and Heike Kappes

the growth of bivalve

gills initiated

from

a lobule-

producing budding zone

Beninger, Peter G., Gael Le Pennec, and Marcel Le
Pennec
Demonstration of nutrient pathway from the digessystem to oocytes in the gonad intestinal loop of
the scallop Pecten maximus L

73

tive

Etnier, Shelley A.

Twisting and bending of biological beams: distribution of biological beams in a stiffness mechanospace .....................................

36

Annual Report of the Marine Biological Laboratory

...

83

Rl

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Bi,>l. Bull. 205: 1-7. (August 2003)

2003 Marine Biological Laboratory

Reference:

Aggregations of Egg-Brooding Deep-Sea Fish and

Cephalopods on the Gorda Escarpment:
a Reproductive Hot Spot
JEFFREY

C.

DRAZEN*. SHANA

K.

DEBRA

GOFFREDI, BRIAN SCHLINING.

S.

Monterey Ba\ Aquarium Research

Moss Landing,

AND

STAKES
7700 Sandholdt Road.

Institute,

California 95039-9644

Localized areas of intense biological activity, or hot

spots, in the deep sea are infrequent but important features

an otherwise sparsely occupied habitat (1). Hydrothermal vents, methane cold seeps, and the tops of seamounts

critical resource for reproductive success in

may be a

some

deep-sea species.
Fifteen

in

ROV

dives were conducted on the

Gorda Escarp-

are well documented areas where dense communities per-

ment and Mendocino Ridge during three visits in August

2000, August 2001. and July 2002 (Fig. 1). The Gorda

sist

Escarpment

for

generations

Reproductive

(2-5).

aggregations

California.

ing or egg brooding could be thought of as transient hot

spots. It is likely that they occur in populations with low

its

maximize mate location and increase reproducsuccess (6). However, only a few deep-sea reproductive

densities to
tive

aggregations have ever been documented (7-9). demonstrating the paucity of present-day information regarding

reproductive behavior of deep-sea animals. In this paper we

describe a unique mitltispecies reproductive aggregation

Gorda Escarpment, California. We document

highest fish and octopus densities ever reported

located on the

some of the
the

in

deep

sea,

with most individuals of both species

We

describe the nesting behavior of the blob

sculpin, Psychrolutes phrictus, and the egg-brooding behav-

brooding eggs.

is

submarine plateau offshore of northern

The Mendocino Ridge extends westward from

northern edge at 40.35 N. The Escarpment's northern

where conspecifics concentrate for the purposes of spawn-

side

is

characterized by steep topography, frequent rocky

outcrops and talus fields, sediment slumps, and drainage

channels ( 10). The depth of investigation ranged from 1300
to 3000 m.

Reproductive aggregations of both blob sculpin and octopus were present at Site 1 (Fig. 1 ). The biomass of P.
phrictus alone at this site was equivalent to the average total
biomass of fishes on the continental slope. Likewise, the
density of Graneledone sp. was considerably greater than
previously published estimates (Fig. 2). Eighty-four individuals off. phrictus and 64 nests (Fig.

They were present

at

two

sites,

3A) were observed.

with the highest density

observed during annual

dives of a remotely operated vehicle (ROV) on the Gorda
Escarpment. The animals are concentrated at the crest of

occurring

and near cold seeps where they may

enhanced
current flow and local productivity.
benefit from
These findings provide new information on the reproductive
behaviors of deep-sea animals. More importantly, they
highlight how physical and bathymetric heterogeneity in the

more gently sloping top (Fig. 4). P.

phrictus and associated nests were absent in July 2002. Two

ior of an octopus,

Graneledone

sp.

the local topography

environment can result

in

reproductive hot spots, which

(Fig.

and

1).

at a

at Site

The

fish

both August 2000 and August 2001

were found over the steepest topography

in

topographic break between the steep northern side

of the ridge and the

hundred and thirty-two individuals of Graneledone sp. (Fig.

3B) were observed across all locations, with the highest
densities observed at Site

during all three visits (Fig. 1 ).

The octopus co-occurred with the blob sculpin, with 51% of
the octopus observed within 5
of sculpin adults or nests
1

Received 14 February 2003; accepted 12

*

To

whom

jdrazen@mban.org

correspondence

should

in

May
be

2003.
addressed.

E-mail:

2001. Smaller aggregations of brooding blob sculpin and

octopus were observed

Site

at Site 2.

(depth 1547-1603 m; dives T208, T349, T448) was

J.

C.

DRAZEN ET AL

12600'W

12500'W

o
O

Density (# ha

oo
O
O
')

0-10
11-20

21-30

31 +

Figure

1.

are in meters.

25

75

50

100

Km

Balhymetric map of the Mendocino Ridge and Gorda Escarpment, showing all dive sites. Depths
One hundred and fourteen hours of video from ROV bottom time was recorded, annotated, and

analyzed. Annotations of all occurrences of discernible animals and geologic features were stored in a searchable
database with corresponding environmental (CTDO), observational (time, position), and system (camera zoom)
data. Bathymetry is derived from a hull-mounted EM300 sonar system with 20-m pixel resolution. Ultrashort
baseline Transponders (Sonardyne. Houston.

TX) mounted on

the

ROV

and the ship determine position.

Tracklines are derived in a real-time ArcView-based (Environmental Systems Research Institute) navigation

system. Closed circles, open circles, and hatched circles are densities (# ha~'l of blob sculpin (yellow) and
octopus (red) from dives in 2000, 2001. and 2002 respectively. For each dive the densities reflect the number
of animals observed over the surveyed area of seafloor. Areas for density estimates were calculated using the
navigation to determine track length and assuming an average observational width of 4 m. Overlap of the dive
track

was accounted

for in the calculations.

characterized by small rocky cliffs and bouldered slopes

ambient (0.1-0.2

which the gravel and

boulders were interspersed with sediment. Site 2 (depth
1534-1583 m; dive T351; Fig. 1) was on a shallowly
sloping mud and sand bottom interspersed by boulders,

age from the substrate (10).

Blob sculpin attended nests of large (4.0

and small rock outcrops. Diffuse cold seeps at the

base of several bouldered slopes at both sites were evident
by the presence of small patches of vestimentiferan tube

on or touching the eggs. Some nests and fish were observed

by themselves primarily in the roughest terrain where it was
difficult to see behind nearby rocks and ledges. Eggs were

worms and vesicomyid clams

free

that shoaled to a sloping talus field in

talus,

(10).

Sites

and 2 were

characterized by an average bottom water temperature of

2.4 C (range = 2.3 - 2.7 C) and very low oxygen concentration
~
'

).

(mean =

1.07

The temperature

ml 1"'; range

at Site

was

0.73-1.46 ml

slightly elevated

above

C) due

to local subsurface fluid seep-

0.6

mm;

n =

50) pinkish eggs (Fig. 3A). The majority of the nests had
fish in close attendance (within 3 m). often sitting directly

of sediment, suggesting that the adults cleaned or

sites. Brooding fish were almost always

fanned their nest

found very close

to

each other, and nests were often on

neighboring boulders separated by only 1-2 m. Generally

the parent fish did not

move when

the

ROV

approached;

DEEP-SEA REPRODUCTIVE HOT SPOT

A)

17-

-- 10

'

"

C.

J.

DRAZEN ET

AL.

Figure 3. Egg-brooding fish and octopus. (Al Three blob sculpin. Psychrolutes phrictus. attending nests.
fish on the left has a nest just outside of the field of view. Size-calibrated images were used to determine

The
fish

egg

size

and fecundity.

When

the

camera had zoomed such

that the plane of focus

horizontal dimension of the field of view (field widthl could be determined (30).

From

was narrow, then

the

the resulting calibrated

images. Optimas image analysis software (ver. 6) (Optimas Corporation. Bothell. WAl was used to measure fish
egg diameters. Occasionally when field width could be used to calibrate the size of objects in the video, the
Optimas software was used to calculate the area of fish egg masses. The eggs appeared to be laid in a thin layer
across the rocks, and in a few cases they were piled on top of each other near the center of the mass.

Consequently, egg numbers were estimated by assuming that a single layer of eggs was placed across the nest
area as closely together as possible. (B) Eight egg-brooding individuals of Graneledone sp. on a rock outcrop.
(C)

specimen of Graneledone

describe have been found

wise, on

(J.

more than 200 dives

sp.

showing eggs protected under arms and mantle.

Drazen. unpubl. data). Likearea at

in the

Monterey Bay
and often in areas of rocky
substrate (i.e., canyon walls and slopes), no brooding octopuses were observed (although octopuses are common) and
depths greater than 1000

only 13 blob sculpin

and the distribution of nesting blob sculpin was much

broader than that of the seeps (Fig.

were seen, none with eggs.

The presence of cold seeps can dramatically influence

local productivity of surrounding deep-sea

Cold seeps are related

communities by
were

transfer of organic nutrients (2). Diffuse cold seeps

observed

at both sites of sculpin and octopus aggregations,

suggesting that enhanced local productivity from cold seeps

on the Gorda Escarpment may also influence the aggregations. This is unconfirmed, however, because only six octopus were seen in the immediate vicinity of seep organisms

the

4).

upward flow of warm,

methane-rich pore fluids from depth;

this

flow has also

generated slight increases in temperature (0.1-0.2

ambient)
the

to

at

Site

(10).

above

Increases in temperature could

shorten egg development times, which would be an advanw of

tage to species that invest parental care. Assuming a

2,

be required for a 10%

incubation time. Similar conclusions were

an increase of 1.5

reduction

drawn

in

C would

for benthic octopus brooding near cold seeps at the

Baby Bare

site

off of Washington

State

(8).

However,

temperature elevations of this magnitude around cold seeps

are very unlikely. Furthermore, animal occurrences did not

DEEP-SEA REPRODUCTIVE HOT SPOT

iveT342
,-^~

T448

Figure

4.

Three-dimensional sunshaded

octopus, and cold seeps

The compass

is

also a scale

map

of dive tracks and locations of

all

sightings of blob sculpin,

Mapping information was generated as for Figure 1.

bar with each arm equivalent to 500 m. Note that, due to the typical perspective of

at Site

1.

Contours are

in meters.

a three-dimensional rendering, the apparent distances for each axis are not equal.

correlate with the highest temperature anomalies. Therefore,

we conclude

that cold seeps

physically, but they

may

do not

benefit these animals

provide a food source that could

adequate oxygen for embryogenesis. A reduction in the

need to ventilate or fan the eggs could be an energetic
benefit to the adults. In addition, benthic

egg brooding and

play a role in the location of the animal aggregations.

In addition, elevated currents may influence site selection

hatching implies a demersal larval/juvenile phase (23). Bottom currents in the deep sea are generally low, so these

by brooding aggregations. All blob sculpin and most octopus were observed near the ridge crest where exposure to

organisms may take advantage of intensified currents at this

site to enhance the dispersal of larvae or juveniles within the

elevated currents

is

likely (1,3, 20).

As on seamount

crests,

abundant suspension feeders such as brisingid sea stars,

tunicates, gorgonians, and venus flytrap anemones were
found

at

the crest of the

Gorda Escarpment, providing

demersal habitat.

At one time the deep sea was thought to be a sparsely

). Today, dense
populated and homogenous environment
localized communities such as the chemosynthetic communities of hydrothermal vents and methane cold seeps (2) and
(

evidence of accelerated current speeds. Some shallow-living sculpins have a strong preference for nesting sites that

the suspension-feeding

are exposed to the current: this exposure aids in gas ex-

well known.

change and waste removal and accelerates embryogenesis

(21. 22). At Site 1, where oxygen concentrations are very

another unique type of biological hot spot in the deep sea.

The site is connected to the continental margin but topo-

low, enhanced water

movement may be

required to deliver

Our study

communities of seamounts
site

(3) are

on the Gorda Escarpment

is

graphically exhibits characteristics of a seamount environ-

J.

C.

DRAZEN ET

We

merit. In addition, small cold seeps are present.

hypothesize that the local topography interacting with the physical

5.

two very

different

6.

This information has several important implications. The

reproductive hot spot on the Gorda Escarpment (and future

7.

determined to be similar) might qualify as an area to be

protected from fishing. The protection of habitats associated

8.

with vulnerable

9.

development of deep-sea fisheries to depths of

2000 m, and currently fishers regularly operate at depths of

10.

m off of the west coast of the United States (25).

deep-sea ecosystem as well as providing sites where scientists can predictably observe reproductive
biology in deepsea animals, a prospect that is exciting for the study of these

1.

12.

provided helpful advice and software for measuring egg and

nest sizes from video framegrabs. We are indebted to Dave

Vrijenhoek was the principal investigator on dives T349 and

T351. Thanks to the pilots of the ROV Tiburon and the crew

Western Flyer. Thanks to Jim Barry, Brad Seibel.

Cailliet for discussion and com-

Bruce Robison, and Greg

Waldo Wakefield,

P. A. Tyler. 1991.

Stakes, D.

S.,

M. Trehu, S. K. Goffredi, T. H. Naehr, and R. A.

Mass wasting, methane venting, and biological comMendocino transform fault. Geology 30: 407-410.

A.

Smith, C., and R.

J.

The

Wootton. 1995.

costs of parental care in

Silverberg, N., H.

M. Edenborn, G.
fish,

Ouellet,

and

P.

Beland. 1987.

Melanostigma atlanticum (Zo-

Stein, D. L. 1980.

Aspects of reproduction of liparid fishes from the

Cambridge.
Tunnicliffe, V., A. G. McArthur, and D.

16.

Haedrich, R. L. 1997.

1998.

17

Reproduction

Distribution and population ecology. Pp.

J.

Randall and A.

P.

Farrell. eds.

H. L. Sanders, R. R. Messier, G. T. Rowe, and T.

Pattern

and zonation: a study of the bathyal

the research submersible Alvin.

Deep-Sea Res. 22:

D. S. M., and B. Hansen. 1982. Abyssal aggregations of

Kolga hya/inu Danielssen and Koren (Echinodermata: Holothurioidea)
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Billett,

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Tyler, P. A., C.

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the deep-sea holothurian

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Mar.

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447-462.

20. Fock, H., F. Uiblein, F. Roster,

and H. von Westernhagen. 2002.

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fish

assemblage

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at

Meteor Seamount (subtropical

NE

Atlan-

21.

sampled by different trawls. Mar. Biol. 141: 185-199.

DeMartini, E. E. 1978. Spatial aspects of reproduction in buffalo
sculpin. Enophrys bison. Environ. Biol. Fishes 3: 331-336.

22.

DeMartini, E.

tic),

E., and B. G. Patten. 1979.

Egg guarding and
reproductive biology of the red Irish lord. Hemi/epidotiis hemi/epidotus (Tilesius). Syesis 12:

Hochberg,

F. G..

41-55.

M. Nixon, and

R. B. Toll. 1992.

Order Octopoda

Leach. 1818. Pp. 213-280 in "Larval" ami Juvenile Cephalopods: A

Manual for Their Identification. M. J. Sweeney. C. F. E. Roper. K. M.
Mangold. M. R. Clarke, and S. v. Boletzky. eds. Smithsonian Contri-

Genin, A., P. K. Dayton, P. F. Lonsdale, and F. N. Spiess. 1986.

Corals on seamount peaks provide evidence of current acceleration

butions to Zoology 513. Smithsonian Institution Press, Washington,

DC
24

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megafauna using
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Adv. Mar. Biol. 34: 353-442.

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Van Dover, C. L., C. R. German, K. G. Speer, L.

Bertelsen,

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and D. M. Cohen. 1964.

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Deep-Sea Biology: a Natural

Press.

4.

J. R., and A. J. Grehan. 2000.

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octopuses in the North Pacific Ocean. Biol. Bull. 198: 94-100.
Young, C. M., P. A. Tyler, J. L. Cameron, and S. G. Rumrill. 1992.
Seasonal breeding aggregations in low-density populations of the
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Voight,

among deep-sea fishes. Deep-Sea Res. 11: 569-596.

Merrett, N., and R. L. Haedrich. 1997. Deep-Sea Demersal Fish
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Literature Cited

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New

14.

Undersea Research Program awarded to Robert

Duncan (Oregon State University). J. C. Drazen was supported by a postdoctoral fellowship from MBARI.

and

Spawning dynamics of orange roughy,

continental slope and abyssal plain off Oregon, with notes on growth.

the National

J. D.,

B.H.

195-202.
13.

Eric Hochberg, and an anony-

mous reviewer provided valuable insight and revisions.

Dives T348, T350, and T352 were funded by a grant from

Gage,

Atolls.

Boehlert, eds. Geophysical

arcidae) spawning within bottom sediments. Environ. Biol. Fishes 20:

Clague, Robert Young, and Jenny Paduan for their assistance with dive T448. Janet Voight also provided assistance
on that dive and helped to confirm the octopus identity. Bob

1.

G.W.

Copeia 687-699.

Special thanks to Linda Kuhnz. Kyra Schlining, Susan

Von Thun, and Kris Walz for video annotation. Dan Davis

ments.

Seamount biota and

1987.

Seamounts. Islands, and

Direct evidence of a mesopelagic

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understanding of habitat heterogeneity within the broader

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Domeier, M. L., and P. L. Colin. 1997. Tropical reef fish spawning
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Keating. P. Fryer, R. Batiza, and

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Wilson, R. R.,

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Reference: Bio/. Bull. 205: 8-15. (August 2003)

2003 Marine Biological Laboratory

250 Million Years of Bindin Evolution

KIRK
1

S.

Smithsonian Tropical Research

Duke

Bindin plays a central role

Abstract.

ment and fusion

ZIGLER

in

1 -

Institute,

University;

LESSIOS

Department of Biology,

Durham, North Carolina

sperm-egg attach-

DNA sequence of bindin in two orders of the

determined the

H. A.

Balboa, Panama: and

urchins (echinoids). Previous studies

in sea

AND

level, often within species,

sometimes within genera, but

rarely across an entire class. There are good reasons for this
focus: such studies are likely to uncover mutational changes

mate recognition and

class Echinoidea, representing \Q9c of all echinoid species.

that are important in

We

However, comparisons across broad taxonomic

report sequences of mature bindin from five additional

genera, representing four new orders, including the distantly
related sand dollars, heart urchins, and pencil urchins.
six orders in

which bindin

is

now known

include

70%

The

of

all

was present in the comextant sea urchins more than 250 million

echinoids, and indicate that bindin

mon

ancestor of

all

in speciation.

levels can

They can
which features of these molecules are conserved (and
thus essential for basic functions) and which features are

offer insights into the evolution of such molecules.

reveal
are

For the parts that do vary, such comparisons

can determine common features of evolution. Most of all,
free to vary.

years ago.

Over this span of evolutionary time there has

remarkable conservation in the core region of
bindin, particularly in a stretch of 29 amino acids that has

the comparisons can address the question of the universality

been

of a particular molecule by asking how far back in evolution

one needs to search to find the point at which a completely

motif of basic

different molecule has taken over the essential functions

not changed
amino acids

at

all;

(2) conservation of a

at the

between preprobindin and

site

in gamete binding and fusion.

Echinoids (sea urchins, heart urchins, and sand dollars),
with their readily obtainable gametes, have long been model

cleavage
mature bindin; (3) more than a twofold change in length of
mature bindin; and (4) emergence of high variation in the

involved

sequences outside the core, including the insertion of gly-

organisms for fertilization studies. Because fertilization is

external, the molecules involved in gamete recognition and

cine-rich repeats in the bindins of

some

orders, but not

others.

fusion are associated exclusively with the gametes. Bio-

chemical studies

Introduction
Various studies have shown

that

(and

molecules involved

in

interactions)

in

particularly
gamete
reproduction
evolve rapidly, often under the influence of positive selection

(reviewed

in

Swanson and Vacquier, 2002). Among

these proteins there are examples of both high (Metz and

Palumbi,

1996) and low (Metz

intraspecific

variation.

In

et

1998b) levels of

al.,

some cases

a single

molecule

displays domains that are highly conserved and other domains that are highly variable (Vacquier et ai, 1995). Variation in such proteins

is

usually studied

at

a low taxonomic

in sea

urchins identified the

first

"gamete

recognition protein," bindin (Vacquier and Moy, 1977).

Bindin is the major insoluble component of the sperm

acrosomal vesicle and has been implicated in three molecular interactions (Hofmann and Glabe, 1994). First, after the
acrosomal reaction, bindin self-associates, coating the acrosomal process. Second, it functions in sperm-egg attachment by binding to carbohydrates in the vitelline layer on

egg surface. Third, it is involved in the fusion of sperm

and egg membranes (Ulrich et ai, 1998. 1999).
Bindin is translated as a larger precursor, from which the
the

N-terminal preprobindin portion is subsequently cleaved to

produce mature bindin (Gao et al., 1986). The mature bindin

molecule contains an amino acid core of about 55 residues

Received 25 February 2003; accepted
*

To whom correspondence should

June 2003.

be addressed. Current address: Fri-

day Harbor Laboratories. University of Washington. 620 University Road,

Friday Harbor.
98250. E-mail: zilerk@u. washinaton.edu

WA

that is highly

conserved among

date (Vacquier

et al..

1995).

all

An

bindins characterized to

18

amino acid section of

shown to fuse lipid

this conserved core (B18) has been

EVOLUTION OF BINDIN
vesicles in vitro, suggesting that this region functions in

differences in molecular size,

til..
1998. 1999).
sperm-egg membrane fusion
Thus far, bindin is known only from echinoids; no homol-

number of

(Ulrich ft

ogous molecules have been

identified in

(Metz and Palmnbi. 1996). Strongylocentrotus (Gao el

1986; Minor etui., 1991; Biermann. 1998; Debenham et
2000). and Heliocidaris (Zigler et

al..

2003), there are

nl..
nl..

many

sequence rearrangements among individuals and species,

and indications of positive selection in regions on either side
of the core. In Arhacia (Glabe and Clark. 1991; Metz

et al..

1998a) and Tripneustes (Zigler and Lessios. 2003), there are

fewer sequence rearrangements and no evidence for positive

(Minor

et al..

1991

so the

),

mode

of evolution of the

molecule remains unknown.

The

five

genera

in

which bindin was previously

two echinoid

se-

orders, the Echinoida and

quenced belong

to

the Arbacioida.

These two orders contain only 10% of

extant echinoid species (Kier.

wood and
in the

that bindin is present outside the

Echinoida and Arbacioida comes from

who

Moy

and Vacquier

of Strongyreported that an antibody

from
one species
locentrotus purpuratus reacted with sperm
of the order Phymosomatoida and two species of the order
1979).

to bindin

Clypeasteroida. As Vacquier 1998) has pointed out. molein contrast to those central
cules that mediate fertilization
(

often differ between taxa. For

to other basic life processes

example,

in the

molluscan class Bivalvia. completely difgamete recognition of oysters

ferent proteins are involved in

(Brandriff >//., 1978) and of mussels (Takagi et

It

is.

dence

therefore, not safe to

pencil urchins (order Cidaroida) were represented in

The

our study by Eucidaris tribuloides, collected on the Atlantic

coast of Panama; the order Diadematoida by Diadema an-

from the Atlantic coast of Panama. The sand

were represented by Encope
stokesii from the Pacific coast of Panama; the heart urchins
(order Spatangoida) by Moira clotho collected at the Perlas
ti/lcinnn. also

dollars (order Clypeasteroida)

Islands in the

Bay of Panama. Heliocidaris erythrogramma

was collected near Sydney, Australia.

(order Echinoida)

al.,

1994).

assume without empirical

evi-

that bindin is present in all orders of echinoids. or that

it has the same

general structure as in the taxa in which it
has already been characterized.
As a first step in determining which orders of echinoids
possess bindin and, if they do. how its structure varies, we

cloned and sequenced mature bindin from

five

DNA

isolation

We

genera of sea

which belong to orders in which bindin was

We combined our data with those of
unknown.
previously
urchins, four of

and sequencing

injected various individuals of each species with 0.5

M KC1

all

1977; Smith. 1984: Little-

Smith. 1995). The molecular structure of bindin

The only evidence

Samples

testes

other 13 orders of the class Echinoida has not been

studied.

Materials and Methods

one sequence has been pub-

selection. In Lvtechinus, only

lished

repeats.

any other organism

(Vacquier. 1998).
To date, the nucleotide sequence of bindin has been
determined in six genera of sea urchins. In Echinometra

amino acid sequence, and

until we encountered one that produced sperm. The

of this ripe male were removed and used either

directly for

mRNA extraction, or after preservation in either

RNALater (Ambion
for

mRNA

Inc.) or in liquid nitrogen.

The methods

isolation, reverse transcription reactions, initial

polymerase chain reactions. 3' and 5' rapid amplification of

cDNA ends (RACE) reactions, and DNA sequencing were
as described in Zigler and Lessios (2003). with the follow1
A fragment of the core region of
ing modifications.
(

bindin was amplified from the reverse transcriptase reaction product or from genomic DNA. using primers

MB1 130+ (5'-TGCTSGGTGCSACSAAGATTGA-3')

either core200-

core 157-

and
or

(5'-TCYTCYTCYTCYTGCATIGC-3')

(5'-CIGGRTCICCHATRTTIGC-3'). These prim-

ers correspond to

VLGATKID. ANIGDP, and

(Vacquier et al., 1995). (2) When

amino acids

AMQEEEE.

respectively
mature bindin sequences were not obtained
during the first round of 5' RACE, new primers were
designed at the 5' end of the obtained sequence; then a

complete

5'

second round of

RACE

amplification

was conducted.

(3)

5' preprobindin primer was designed based on a comparison

of preprobindin sequences of Moira clotho (this study) to
preprobindin sequences of Arbacia (Glabe and Clark. 1991).

Strong\locentrotus (Gao et

Lvtechinus (Minor et

al.,

al..

1986;

Minor

et al..

1991), and

1991). This primer. prolSO (5'-

AAGMGIKCIAGYSCIMGIAAGGG-3').

which corresponds

previous studies of bindin in genera belonging to the orders

to the

Echinoida and Arbacioida. The

preprobindin, was used in combination with exact primers

from the bindin core to amplify mature bindin sequences 5'
of the core from Eucidaris tribuloides testis cDNA. (4)
Bindin sequences obtained from RACE were subsequently

final

data set includes bindin

from 10 genera of sea urchins, pencil urchins, sand dollars,

and heart urchins, and the results indicate that the molecule

was present

in the

common

ancestor of

all

extant echinoids

from each other over 250 million years ago.

The core sequence has remained remarkably unchanged
that diverged

in great

KR(A/S)S(A/P)RKG of

confirmed by amplification, cloning, and sequencing of

mature bindin sequences from testis cDNA.

over this period of time, whereas the areas flanking the core

have undergone substantial modification, resulting

conserved amino

acids

an

the

full

Sequencing of both DNA strands was performed on

ABI 377 automated sequencer, and sequences were

10

K. S.

ZIGLER

AND

Sequencher 4.1 (Gene Codes Corp.). Sehave

been
quences
deposited in GenBank (Accession numbers AY126482-AY126485. AF530406). Published mature
edited

using

bindin sequences from a single exemplar from each of

the five genera in which bindin had been previously sequenced were taken from GenBank. These representatives

were Strongylocentrotus purpuratus (Accession number:

M14487, Gao et aL 1986), Lytechimts variegatus (M59489,
Minor et ul., 1991), Arbacia punctitlata (X54155, Glabe and
Clark,

1991), Echinometra oblonga (U39503,

Metz and

Palumbi, 1996), and Tripneustes ventricosus (AF520222,

Zigler and Lessios, 2003). Three amino acids of the core
region of the bindin of Lytechinus variegatus [numbers 367
(N), 368 (L), and 385 (Y) in the alignment of Vacquier et
(1995)] were changed to A, V, and D, respectively,
based on our own sequence data of Lytechinus bindin from
al..

all 25 sequences had

and Lessios. unpub.).
In Echinometra oblonga, sequences for the extreme 3' end
of preprobindin are not in GenBank. They were inferred
from the primer sequences used by Metz and Palumbi

H. A.

LESSIOS

for the mature bindins both for the core region (10 se-

quences, 55 amino acids per sequence) and for mature

bindin sequences outside the core 10 sequences of varying
length for a total of 1909 amino acids). The program
(

CODONS

(Lloyd and Sharp, 1992) was used to calculate

number of codons (ENC), a measure of codon

the effective

usage bias (Wright, 1990), for each sequence. ENC values

can range from 20 to 61, with 61 indicating that all synonymous codons are used in equal frequency (no codon bias),
and 20 indicating that only a single codon is used for each

amino acid (maximum codon

amino acids

statistical analysis

separated repeats, simple tandem repeats, and periodic repeats in each mature bindin sequence (Brendel et al.. 1992).

25 individuals representing 5 species;

these

The

bias).

of protein sequences (SAPS, http://www.isrec.isb-sib.ch/

software/SAPS_form.html) program was used to identify

Results and Discussion

at the 3 sites (Zigler

(1996) to amplify mature bindin sequences.

Figure 1 shows the phylogenetic relationships among the

echinoid orders from which bindin was sequenced, as they

have been reconstructed from molecular, morphological,

and fossil evidence (Littlewood and Smith. 1995; Smith et

As the figure indicates, bindin is present not only

Echinoida and the Arbacioida (from which it was
previously known), but also in the sand dollars (Clypeas-

al.,

1995).

in the

Sequence analysis

We

aligned the mature bindin amino acid sequences with

ClustalXver. 1.81 (Thompson et

alignment by eye

in

Se-Al

al.

1997), and adjusted the

(ver. 2.0a5,

Rambaut, 1996).

We

characterized the amino acid changes observed in the core

region of bindin as either radical or conservative with respect to charge and polarity (Taylor, 1986;
1990).

The

PROTPARAM

tool of the

Hughes

EXPASY

et al.,

proteomics

server of the Swiss Institute for Bioinformatics (http://www.

expasy.org) was used to calculate Kyte and Doolittle ( 1982)

11 amino acids) for

hydrophobicity plots (window size
each mature bindin sequence. The PROTSCALE tool of the

same server was used

to calculate

amino acid composition

Millions of years ago

teroida) and the heart urchins (Spatangoida), as well as the

phylogenetically much more distant Diadematoida and CiAlong with the sequence of Heliocidaris, reported

daroida.

in this paper, and the previously known sequences from

Arbacia, Strongylocentrotus, Tripneustes, Lytechinus, and
Echinometra. the data set covers orders that contain more

70% of all extant echinoid species (Kier, 1977). The

Cidaroida, the only extant order of the subclass Perischoechinoidea. is the lineage most divergent from all other
than

It was separated from the Euechinoidea approximately 250 mya. Bindin's presence in both extant subclasses of the Echinoidea indicates that it was present in

echinoids.

Species

Order

Source

Eucidaris tributoides

Cidaroida

this

Diadema antillarum

Diadematoida

this

study

Clypeasteroida

this

study
study

Encope

stokesii

study

Moira clotho

Spatangoida

this

Arbacia punctulata

Arbacioida

Glabe and Clark. 1991

Strongylocentrotus purpuratus

Echinoida

Gaoetal.. 1986

Tripneustes ventricosus

Echinoida

Zigler and Lessios, 2003

Lytechinus variegatus

Echinoida

Minorca/., 1991

Heliocidaris erythrogramma

Echinoida

this

Echinometra oblonga

Echinoida

Metz and Palumbi. 1996

study

Figure 1. Phylogenetic relationships, divergence times, and systematic position of genera in which bindin
has been sequenced. Echinoid phylogeny and divergence times are from Smith 1988) and Smith el al. 1995).
Source of bindin sequence data is also indicated.
(

EVOLUTION OF BINDIN

11

their

common ancestor and that it has been evolving along

each of the branches of the sea urchin phylogenetic tree for

gent regions. Over the past 250 my, the 55 residues of the
core (ami no acids 155-209) have been remarkably con-

more than 250 my. Whether bindin is present in other

echinoderms remains uncertain. Moy and Vacquier (1979)
found that their antibody to Strongylocentrotus purpiirutiis

served. This region does not contain any insertions or deletions in any echinoid lineage. Of the 55 amino acids, 45

bindin did not react with sperm from three species of sea

stretch of

and "zoo blots" using

probe genomic

DNAs

are conserved across all of the 10 exemplars, including a

stars.

Moira clolho
Arbacia punctulala
SlronKvlocenlroms purpuratus
Tripneustes venlncosus
Lvlechinus \-ariegalus
Helioctdaris en-lhrogramma

Echtnomelra oblonea

RC F
K Q R R
RV
RG
RG FJP
RK
RK

exhibit a singleton

(i.e..

core region

a change found

in only one of the sequences). Four of these changes are

conservative with respect to charge and polarity (amino

sites in the

amino acid change

GIT --- YT RGGGHC YV A T RPGE l[(fT

OC
PT GN V GRA GAOOGG|GTFAAYPPAQSGRPNYY|GPR A NN POPA YAG
Y PMMM
MP
PN Q A P A A PS P Y^N R
A AVMD
QGL GMPGD V|GG QEV
POMGLPVQGYOGNQ|L
AQGA
P V
MN Q G
p PM
GGMOGGYG Y P Q A GGAQY
V NTM
--C YPC |AMSPQW
GQPA ....
GN R
1NOQM
PGQ- P
GNM
N
rtN Q P M C
PGQP
fN POM GGJG
GNMM
PGPG -AMI
PVPGOAPMGOPAddG
YJG ............. N YPQ AMN P PMGGG
-

row (amino acids 164-192). The

conserved section. Seven amino acid

quences are a mosaic of highly conserved and highly diver-

Diadema antillantm
Encope stokesu

in a

18 amino acids implicated in membrane

fusion (Ulrich et ill., 1998. 1999) is part of this perfectly

negative (Minor et at., 1991). No attempt has been made to

determine bindin's presence in the ophiuroids or crinoids.
Figure 2 indicates that the aligned mature bindin se-

Eucidaris trihulaides

29 residues

B18 sequence of

purpiininis bindin sequences to

of a sea cucumber and a sea star were
S.

12

K. S.

ZIGLER

AND

acids at positions 155, 157, 164. and 208), and three are

Each of positions 196,

amino acids across the 10
have been at least two changes

radical (positions 193, 194, and 200).

199, and 203 contain three

genera, indicating that there

at each of these sites. At least one of the changes at each site
must have been a radical change. Thus, radical changes are

in only six amino acid positions of the core region,

of them concentrated in a small portion of the core close

observed
all

to the

203).

terminus (amino acids 193, 194, 196, 199, 200, and

rest of the core (amino acids 155 through 192 and

The

204 through 209) contains only four conservative singleton

amino acid substitutions.

A second conserved region is the cleavage site at the

border between preprobindin and mature bindin (Fig. 2). In
Strongylocentrotus piirpuratus, the cleavage site is marked
by a motif of four basic amino acids (RKKR) (Gao et al.,
1986). Multibasic motifs are also present in the other nine

genera (Fig.

2).

Such multibasic motifs

typically

mark

the

of proproteins from the mature molecule

during the secretory process through the action of proprotein convertases (Steiner, 1998; Seidah and Chretien, 1999).
cleavage

sites

The conservation of
forces the idea that

this multibasic

motif

in

bindin rein-

functions as a signal for the cleavage

of preprobindin from mature bindin in all echinoids.
In contrast to the core and to the cleavage site, the rest of
the molecule

is

it

so variable between orders that

we have

confidence that the alignment of these regions depicted

Figure 2 is correct. There is a great amount of variation

little

in

in the length

of mature bindin both on the 5' and on the

side of the molecule (Table

3'

This study identifies both the

and
the
shortest
bindins
described to date. Bindin in
longest
Diadema antillarum (418 amino acids) is more than twice
1

).

Encope stokesii (193 amino acids).

Bindin length 5' of the core ranges from 78 to 148 amino
acids, while bindin length 3' of the core ranges from 56 to
215 amino acids. There seems to be no discernible evoluas long as bindin in

tionary trend in bindin length. Closely related orders do not

Table

Number of amino

acids in three regions of I he mature bindin

in

10

genera

Core
Eucidaris

Total

H. A.

LESSIOS

EVOLUTION OF BINDIN

13

such as toxins of cone snails (Duda

idly evolving proteins
and Palumhi. 1999) and pheromones of the marine ciliate
Euplotefi (Luporini et

ten

among

al.,

1995)

cysteine residues are of-

most conserved amino

the

serving as
guides for aligning sequences. Thus, the lack of cysteine
residues in bindin may have important structural conse-

When all sequences

most common amino acid

are pooled, glycine

quences.
the

nearly a quarter of

all

is

by

glycine-rich repeats (Echinoida and Spatangoidu) are separated from those that do not. glycine remains the most
common amino acid in both categories, constituting 29.6%

non-core residues

The

E.s.

M.c.
<Y\?Vv

jyvW^

former and 16.4% of

in the

in the latter.

D.a.

far

outside the core, constituting

residues. If the orders that possess

of the non-core amino acids

E.i.

acids,

six

most

common

resi-

dues outside the core (G, A, P, Q, N. and E) compose 63.9%

of all non-core residues. Leucine is the most common amino

A.p.

S.p.

acid in the core, present in 10 completely conserved amino

acid positions, including 6 of the 18 amino acids in the B 18
region. There

much higher

proportion of charged residues in the core (31.8%) than in the rest of the molecule
(

is

15.6%). Each of the five charged amino acids (E, D. R. H,

and K) is more common in the core.

Another common feature of all bindins

is

with an average of 56.4. Low levels of codon usage bias

have also been observed in sex-related genes in Drosopliila
(Civetta and Singh. 1998) and in the Chlamvdomonas mat-

Given the

Mid and Fusl

large divergence in

(Ferris et

it

is

not sur-

prising that hydrophobicity plots (Fig. 3) from these bindins

are diverse. The conserved amino acid sequence of the core

and its flanking regions causes all plots to be similar through

the middle of the molecule. Plots of the closely related
Tripneustes ventricosus, Lytechinus variegatus. Heliocidaris erythro gramma, and Echinometra oblonga bindins are
similar throughout their lengths.

The

rest

along its extended length. A second is the highly hydrophobic region 3' of the core of Arbacia bindin. noted by Glabe

and Clark (1991).

The only other gamete recognition protein that has been

studied in marine invertebrates separated for as long as 250
my is the gastropod sperm protein lysin. Lysin opens a hole
envelope of free-spawning snails and thus

enables sperm to penetrate to the plasma membrane of the

egg. It has been studied in the abalones (Hciliotis) (Lee and

Vacquier, 1992; Lee et

viewed

al.,

1995:

Yang

H.e.

~^\/

E.o.

uy

Figure 3.
Hydrophobicity plots of mature bindin in 10 genera. The
genera are presented in the same order as in Figure 1 and abbreviated as
follows: E.t.: Eucidaris tribuloides, D.a.: Diadema aniilliinini, E.s.: En-

cope

stokesii, M.c.:

Moira

locentrotus pnrpn ranis,

clotho. A.p.:

T.v.:

et al.,

2000;

re-

longu.

box encloses the core, and a bracket indicates

region in Arbacia.

and

1,

The

scale bars

S.p.:

L.r.:

Stmngv-

Lytechinus

the

above and below each zero

hydrophobic
line

mark +1

respectively.

additional bindin sequences reported here reinforce the conclusions of Hellberg and Vacquier ( 1999) from comparisons
between the modes of evolution of these two proteins.

Although they are both involved in gamete recognition and

both lack cysteine residues, they evolve in different fashions. There is no equivalent of a bindin core region in lysin:

amino acid

substitutions are spread throughout the mole-

cule, with only three

amino acids conserved between

its

function by conserving secondary structure through conamino acid substitutions (Hellberg and Vacquier.

servative

same time

from 126

from the euechinoids. The

all

Haliotis species and the two teguline genera. Instead of

conserving a section of the molecule, lysin has maintained

ture bindin length varies

the cidaroids separated

Arbacia punciiilata.

Tripneustes ventricosus.

variegatus, H.e.: Heliocidaris etythrogramma, and E.o.: Echinomelra ob-

Kresge et al., 2001 and in two genera of turban

snails, Tegula and Norrisin (Hellberg and Vacquier. 1999).
Abalones and turban snails diverged 250 mya. roughly the
in

of the hydropho-

bicity plots are not clearly similar. One particularly distinct

region is the long hydrophilic stretches in Diadema bindin

in the vitelline

"/V

v.

2002).

al.,

amino acid sequence and

length (and the uncertainties in alignments),

L.

their lack of

codon usage bias. ENC values among the 10 genera range

from 61 (for Eucidaris and Diadenui) to 48.1 (for Arbacia),

ing-type locus genes

T.v.

1999). Length variation

is

lysin length (at least in the

to

another obvious difference.

from 193

to

418 amino

two groups studied

138 amino acids.

Ma-

acids, but

to date) only

14

K. S.

ZIGLER

AND

H. A.

LESSIOS

sequence variation and selection

Conclusions

The comparisons of bindin from 10 genera of echinoids

reveal the results of long-term evolution under two opposing selective forces acting on gamete recognition molecules.
sections of the molecule involved in the basic functions

The

of gamete fusion and post-translational cleaving of the

preprobindin have been remarkably conserved over 250 my
of evolution, presumably through purifying selection. The
sections involved in species recognition have been evolving

rapidly in seemingly unpredictable directions, presumably

under diversifying selection; such changes are likely to be
specific to each species.

A
in

number of

need of functional explanations.

Among

the conserved

core region is the only one

that can be easily explained. We do not yet know whether
there is a particular reason for the low codon usage bias of
all

F.,

in the

change

bindins, for the absence of tryptophan or cysteine resi-

dues, or for the absence of major hydrophobic regions in all

bindins except that of Arbacia. The differences between the

orders are equally puzzling. Is there a functional reason for

the length variation of the regions outside the core? Why do
the Echinoida and the Spatangoida have glycine-rich repeats
in the regions flanking the core, while other orders do not?

Comparisons alone cannot provide answers to these questions; but they can identify features of the molecule that are
worthy of functional study.

and

R. Palumbi. 1999.

S.

J.

Mol. Evol. 51: 481-490.

Molecular genetics of ecological

and rapid evolution of toxin genes of the

Natl. Acad. Sci. USA 96: 6820-

diversification: duplication

venomous gastropod Conns. Proc.

6823.

Armbrust, and U. W. Goodenough. 2002. Genetic

mating-type locus of Chlamydomonas reinhardtii.

Ferris, P. J., E. V.
structure of the

Genetics 160: 181-200.

Gao,

B., L. E. Klein, R. J. Britten,

and E. H. Davidson. 1986.

Se-

mRNA

quence of

coding for bindin, a species-specific sperm protein

required for fertilization. Proc. Natl. Acad. Sci. USA 83: 8634-8638.
Glabe, C. G., and D. Clark. 1991. The sequence of the Arbacia punclulata bindin

Hellberg,

cDNA

and implications for the structural basis of speciesfertilization. Dev. Biol. 143: 282-288.

sperm adhesion and

specific

features identified by these comparisons are

features, the lack of

Duda, T.

hindin locus of the red sea

in the

urchin, Strongylocentrotus franciscanus.

M.

E.,

and V. D. Vacquier. 1999.

ization selectivity

and

lysin

cDNA

Rapid evolution of

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fertil-

in teguline gastropods.

Mol. Biol. Evol. 16: 839-848.

and C. G. Glabe. 1994. Bindin, a multifunctional sperm

new species. Semin. Dev. Biol. 5: 233-242.
Hughes, A. L., T. Ota, and M. Nei. 1990. Positive Darwinian selection

Hofmann.

A.,

ligand and the evolution of

profile diversity in the antigen-binding cleft of class I

major-histocompatibility-complex molecules. Mol. Biol. Evol. 7: 515524.

promotes charge

Kier, P.

M.

The poor

1977.

fossil

record of the regular echinoid. Paleo-

biology 3: 168-174.

Kresge, N., V. D. Vacquier, and C. D. Stout. 2001. Abalone lysin: the

dissolving and evolving sperm protein. Bioessays 23: 95-103.

A simple method for displaying the

J., and R. F. Doolittle. 1982.
hydrophobic character of a protein. J. Mol. Biol. 157: 105-132.
Lee, Y.-H., and V. D. Vacquier. 1992. The divergence of speciesKyte,

specific abalone

sperm lysins is promoted by positive Darwinian

97-104.

se-

lection. Biol. Bull. 182:

Positive selection is a
Lee, Y.-H., T. Ota, and V. D. Vacquier. 1995.
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Acknowledgments

Evol. 12: 231-238.

We

are grateful to A.

and

L.

Calderon for providing

support in the laboratory, to M. McCartney for primer

design and advice on the RACE technique, to T. Duda for

Moira clotho, and to E. Popodi for providing

testis RNA from Heliocidaris ery thro gramma. Comments
from C. Cunningham, D. McClay, R. Sponer. W. Swanson,
V. Vacquier, and two anonymous reviewers improved the
manuscript. This work was supported by National Science

collecting

Foundation and Smithsonian predoctoral fellowships to

KSZ, by the Duke University Department of Zoology, and

by the Smithsonian Molecular Evolution Program.

A combined morphological
J., and A. B. Smith. 1995.
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program
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Positive selection and sequence

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Reference: Biol Bull 205: 16-25. (August 2003)

2003 Marine Biological Laboratory

Behavioral Characterization of Attractin, a WaterBorne Peptide Pheromone in the Genus Aplysia

SHERRY

D.

PAINTER*. BRET CLOUGH, SARA BLACK,

The Marine Biomedical

Institute

and

the

AND GREGG

NAGLE

T.

Department of Anatomy and Neitrosciences,

University of Texas Medical Branch, Galveston. Texas 77555-1069

reduce the latency to mating. The effects are larger

is considered: in addition to

Pheromones play a significant role in coordinating reproductive activity in many animals, including
opisthobranch molluscs of the genus Aplysia. Although
solitary during most of the year, these simultaneous her-

attractin

maphrodites gather into breeding aggregations during the

reproductive season. The aggregations contain both mating

tractin stimulating both

Abstract.

when hermaphroditic mating

reducing latency, attractin doubles the number of pairs

mating as hermaphrodites. The effect may result from atanimals to mate as males and would

be consistent with behaviors previously seen in the T-maze.

Attractin may thus be contributing to the formation of

and egg-laying animals and are associated with masses of

egg cordons. The egg cordons are a source of pheromones

copulatory chains and rings seen in aggregations in the field.

These results may be interpreted in two ways: ( 1 ) attrac-

reduce their latency to

mating, and induce egg laying. One of these water-borne
egg cordon pheromones ("attractin") has been characterized
that attract other Aplysia to the area,

and shown
first

be attractive in T-maze assays. Attractin

to

water-borne peptide pheromone characterized

is

ment and maintenance of the aggregation; or (2) the induced

desire to mate may make attractin attractive when it is

the

presented in conjunction with an animal. In either case, the

results open the door for cellular and molecular studies of

in inver-

tebrates.
In the current studies, behavioral assays

mechanism of

to

action.

examine whether
can induce mating. Although the two activities

better characterize the attraction,

attractin

were used

has multiple activities that contribute to the establish-

tin

could be related

(i.e.,

to

Introduction

attraction occurring because animals

were looking for a partner),

assays showed

and

that attractin

was not

this

works

tested.

Chemical communication

T-maze

why

may

multiple species

2002; Saudan

attractin are equally attractive, verifying that /V-glycosyla-

worms (Ram

Mating

is

studies

most ancient form of

protozoans (Luporini et al.. 1995), yeast (Kodama et ai,

2003), insects (Monsma and Wolfner, 1988; Roelofs et al.,

be associated with one aggregation. Native and recombinant

tion at residue 8

the

communication and is used by most, if not all, animals

examined. The organisms include, for example, ciliated

odors: the peptide is attractive only when

is part of the stimulus. The animal does not need to be a
conspecific, perhaps explaining

is

bouquet of
Aplysia brasiliana

as part of a

not required for attraction.

that both native and recombinant

(Kikuyama

showed

al.,

et al.. 2002),

molluscs (Painter et

al.,

1998),

el al.. 1999), fish (Li et al.,

et al..

2002), amphibians
1995; Rollmann et al., 1999; Wabnitz et

1999), rodents (Stowers et ai, 2002; Novotny, 2003)

and humans (Savic

Received
*

et al.. 2001.). The number of pherocharacterized in each species depends, at least in

part, on the chemical nature of the pheromones and on
whether the pheromones are water-borne.

mones

October 2002: accepted 16 April 2003.

To whom correspondence

should be addressed. Marine Biomedical

2.138 Medical Research Building. University of Texas Medical

Branch, 301 University Blvd., Galveston, Texas 77555-1069. E-mail:
Institute.

Opisthobranch molluscs of the genus Aplysia are simultaneous hermaphrodites that do not normally fertilize their
own eggs. Field studies (Kupfermann and Carew, 1974;

sdpainter@houston.rr.com
Abbreviations: ASW, artificial seawater; Att.
nitrile;

HFBA,

heptafluorobutyric acid;

Educational Products;

RP-HPLC,

attractin; CH,CN, acetoM-REP. Marine Research and

Audesirk, 1979; Susswein et

reversed-phase high performance liquid

that they are solitary

chromatography.
16

al..

animals that

1983, 1984) have

move

shown

into breeding ag-

PHEROMONAL ATTRACTANT

APLYSIA

17

76 aa

iQNCDIGNITSQCQMQHKNCEDANGCDTIIEECKTSMVERCQNQEFESAAGSTTLGPQ
QNCD I GN I TSOCQMQH!lNc3DANGCDTI

I E

EC

KT S MVE RC Q NQE FE S A

A) Schematic diagram of

the precursor to the uttractin pherotnone from the albumen gland of

Cleavage of the signal sequence (arrow) generates the 58-residue pheromone attractin. The
disulfide-bonding pattern of cyxleine residue-. (S) is I-IV. II-V. and 11I-1V, where the Roman numeral indicates

Figure

1.

californica.

.A/>/v.v/'(i

the order of occurrence in the primary sequence (Schein el

pheromones

2001). Unlike attractin. the precursors for

al..

group of scents often contain sequences of more than one scent. (B) The amino
from the two species of Aplysia used in the current studies, A. californicu and A.

that act as part of a

acid sequences of attractin

briisiliiinu (Painter el al..

1998, 2000).

Amino

acid residues that are identical to those in A. californica attractin

are indicated h\ the black background.

gregations during the reproductive season. The aggregations

typically contain both mating and egg-laying animals and

potential mating behaviors (1-10

are associated with masses of recently deposited egg cordons, often deposited one on top of another. Most of the

water) was in the range of concentrations normally observed

with pheromones, demonstrating that attractin has phero-

egg-laying animals mate simultaneously as females even

though mating does not cause reflex ovulation (Blankenship

monal

ft al., 1983),

tions of the

suggesting that egg laying precedes mating

the aggregation and that egg laying

may

release

in

pheromones

and maintain the aggregation.

Similar observations have been made in the laboratory
when animals were not individually caged (Audesirk. 1979;

that establish

Blankenship

et al.. 1983;

behavioral studies have

Susswein

shown

et al..

1983, 1984). and

was

that

tin

attractive

to

and induced the

conspecifics
in

pmol

artificial sea-

activity.

There

is

no geographical overlap between the distribu-

two

species, suggesting that attractin or an

attractin-related peptide

brasiliana.

A peptide

is

pheromonal

was subsequently

attractant in A.

isolated

from the A.

brasiliana albumen gland and sequenced. It is 58 amino

acids in length and differs from A. californica attractin at

only 3 amino acids (Fig.

Painter et al.

1;

2000).

It

is

animals with

deposited on the egg cordon and elutes into the seawater

cordons are more attractive than sexually mature but nonlaying conspecifics (Aspey and Blankenship, 1976; Jahan-

following deposition. It could thus serve a pheromonal

function in A. brasiliana, but its pheromonal activities have
yet to be tested.

that egg-laying

Parwar. 1976; Audesirk. 1977; Painter et

assays show

some of the

al..

1989).

T-maze

the egg cordon and are waterborne: (1) recent egg-layers

In the present study, behavioral assays were used to better

characterize the attraction and to examine whether mating is

without egg cordons are no more attractive than non-laying

conspecifics; (2) recently deposited egg cordons are attrac-

works as

that at least

attractants derive

from

with or without a non-laying conspecific, but sham egg

cordons are not; and (3) both recently deposited egg cordons
and their eluates increase the attractiveness of non-laying
tive,

conspecifics
(Painter et

when placed

al..

in

the

surrounding

seawater

1991: Painter, 1992).

One of
isolated

the water-borne pheromonal attractants has been

from eluates of the egg cordon and characterized.

Named

attractin,

cysteines
(Fig.

was

1;

that

it

is

a 58-residue peptide that has

six

form three intramolecular disulfide bonds

Painter et

al..

1998; Schein et

al..

2001). Attractin

The

current T-maze assays showed that attractin

of
a bouquet of water-borne odors: the peptide
part
attractive only when individuals of A. brasiliana or A.

induced.

is

californica are part of the stimulus.

need

The animal does not

be a conspecific, perhaps explaining why multiple

species of Aplysia may be associated with one aggregation
for example, A. vaccaria with A. californica aggreto

gations

(Kupfermann and Carew. 1974; Pennings, 1991);

A.

californica with A. vaccaria aggregations (S. LePage, Marine Research and Educational Products (M-REP), pers.
comm.); and A. depilans with A. fasciata aggregations

(Achituv and Susswein, 1985). Recombinant attractin was

two reasons:

from a Pacific Coast species (A. californica)

and bioassayed in a species from the Gulf of Mexico (A.

also tested for

was done because individuals of .4. calitend

to
crawl
out of T-maze chambers before they
fornica
are exposed to the stimulus. A. californica attractin was

glycosylated and recombinant attractin is not); and (2) to see

whether the two are equally attractive, so that recombinant

isolated

brasiliana). This

attractive to A. brasiliana

and produced behaviors

that

were

suggestive of mating (Painter et al.. 1998), but these behaviors were not further analyzed. The amount of attrac-

ation at

uttractin

Asn

is

( 1

to see

whether W-glycosyl-

necessary for attraction (native attractin

could be used in

3D

nuclear magnetic resonance

solution structure studies (Garimella et

series

al.,

2003) and for

mechanism of action at the receptor level.

of mating assays was performed because behav-

future studies of

is

S.

18
iors in earlier

T-maze assays suggested

D.

PAINTER ET AL
Beach. CalAlacrity Marine Biological Services (Redondo
California).
They were
ifornia) and M-REP (Escondido,

that both the stim-

ulus and test animals wanted to mate (Painter et at.. 1998).

The assays showed that attractin reduces the latency to
with a pheromone. Atmating at concentrations consistent

maintained as described above, except that the water tem2C. This species was used as a stimperature was 14
of T-maze assays and as the source of
set
one
ulus animal in

hermaphroditic mating

tractin also reduces the latency to

mating as hermaphrodites.
attractin
from
result
stimulating both aniThis effect may
with behavconsistent
be
would
and
males
as
mate
mals to

and

doubles the

number of

iors seen in earlier

These

where

pairs

T-maze assays

(Painter et

al.,

1998).

results suggest that attractin, acting in an aggregation

there are more animals, could be at least partially

chains and rings that have

responsible for the copulatory
been observed. Recombinant attractin also induces mating,

both one-way and as a hermaphrodite, showing that Nfor the induction of either type
glycosylation is not required
of mating. The attraction and mating data demonstrate that
contribute to the establishment and mainteattractin

may

nance of breeding aggregations, and to successful reproduction.

Purification of native

and recombinant

attractin

Procedures. Attractin from the albumen gland of A. cali-

fomica was

purified by analytical

CIS reversed-phase high

chromatography (RP-HPLC) as previTo prepare recombiously described (Painter et al.. 1998).

nant attractin. the A. califomica albumen-gland attractin
cDNA (Fan et al., 1997) was subcloned into the baculovirus
vector pFastBac 1, and recombinant virus was

performance liquid

expression

Baculovirus Expression
generated using the Bac-to-Bac
was
expressed in Sf9 insect
System (Invitrogen). Attractin
medium
cells grown at 27-28 C in Sf-900 II serum-free

and the pellet was

Expressing Sf9 cells were centrifuged,
0.1%
heptafluorobutyric
resuspended in 20 ml of ice-cold

and Results

(HFBA) and sonicated. The resulting lysates were

Vac cartridges (5 g; Waters Corp.)
purified on CIS Sep-Pak
10 ml of 100% acetonitrile
with
that were pretreated

acid

Animals
in

Specimens of Aplysia brasiliana (Rang), ranging

collected from South Padre
weight from 100 to 500 g, were
between June
Island, Texas, and were used in experiments
and September. A. brasiliana was used as the experimental
in the

T-maze and mating experiments because

more reproductively

active than A. califomica (see

fig.

it is

2 in

Painter et al., 1998), does not crawl out of T-mazes, makes

fewer false choices, and can be collected in large numbers

from the south Texas coast during the reproductive season.

Previous T-maze assays (Painter et al., 1998) showed that
an individual of A. brasiliana is attracted to a non-laying
behaviors suggestive of mating
and
displays

conspecific
when 10 pmol of attractin

seawater. even though

placed in the adjacent artificial

attractin is a product of the A. caliis

fornica albumen gland.

The animals were housed

cages in
seawaartificial
one of five aquaria containing recirculating
Pet
Supply,
ter (ASW; Instant Ocean Marine Salt, Longhorn
in individual plastic

Houston, Texas). Water was maintained

ture (20

2C);

the salinity ranged

at

room tempera-

from 30

to

32

ppt.

aquarium
14:10 light:dark cycle was maintained
with the light period starting at 0600. Animals were
in

the

facility,

fed dried laver in the late afternoon (1600-1800) after

experiments were completed.

Egg-laying activity was checked twice every day (08000900, 1600-1800), egg-laying activity was recorded, and
used in assays were
egg cordons were removed. All animals
to lay eggs sponsexually mature, as defined by the ability
of
atrial
to
gland extracts
injection
taneously or in response

(made

for purification of native attractin.

(Invitrogen).

Materials, Methods,

animal

albumen glands

as described in Painter et

al.,

1991).

obtained from
Specimens of A. califomica (Cooper) were

(CH,CN) containing 0.1% HFBA and rinsed with

0.1% HFBA. The peptides were loaded, eluted with

20 ml of
15 ml

0.1% HFBA, and lyophilized.

The lyophilizate was resuspended in 2.5 ml of 0.1% HFBA
and applied to a Vydac analytical CIS RP-HPLC column
of

50% CH 3 CN

containing

X 250 mm).
The column was eluted with a two-step linear gradient of
0.1% HFBA in water and 100% CH 3 CN containing 0.1%
HFBA. The first step was 0%-10% CH 3 CN in 5 min,

(4.6

followed by a shallower gradient from 10% to 34% CH 3 CN

and
in 85 min. The column eluate was monitored at 215 nm,
1-min

ml) fractions were collected. The attractin-contain-

and repurified by
ing fractions were combined, lyophilized,
were
same
The
RP-HPLC.
C18
gradient conditions

Vydac

used as described above, except

acid

was

that

0.1%

trifluoroacetic

the counterion.

The RP-HPLC peak fractions containing A.

attractin, identified by comparison to
recombinant
fomica
attractin, were characterized by
native
of
time
the elution
and
acid
microsequence analyses; the
amino
compositional
cali-

Results.

58-residue peptide sequence was identical to A. califomica

to matrixalbumen-gland attractin except that, according
assisted laser desorption/ionization

native peptide

is

/V-glycosylated

nant peptide

is not.

Pheromonal

attraction

at

mass spectrometry, the

8
Asn and the recombi-

is
Procedures. The T-maze, and its associated cages,
was
of
6
each
Before
2.
assay,
illustrated in Figure
1

ASW

APLYSIA

PHEROMONAL ATTRACTANT

cm

101

the third criterion in

was used

animal (Painter

test

et al..

A. califoniica

was always

998).

the attractiveness of a non-laying

compared

hnuilitimi with

The

first

specimen of

pmol of either native or recombinant

ASW

to a non-laying conspecific
with nothing added. We were asking several questions. Are
smaller amounts of native attractin attractive? Is recombiattractin in the adjacent

cm
cm

30.5

when

of assays,

set

Several series of experiments were performed.

cm

10.2

one

as the stimulus animal. A. brasi liana

used as the

12.7

19

Would

nant attractin as attractive?

recombinant

be feasible to use

it

attractin in future behavioral, molecular, or

3D

The second series examined whether a

non-laying conspecific was needed for pmol attractin to be
structural studies?

cm

10.2
Figure

2.

We

attractive.

Schematic diagram of the T-maze with removable stimulus

in place. T-maze depth: 10.2 cm.

cages (dashed outlines)

as part of a

were asking: does

attractin function alone or

"bouquet of scents," as other pheromones do in

third series examined whether the non-

many systems? The

laying animal must be a conspecific.

put into the maze; the
ments. To minimize the

ASW

was stationary during experiamount of stress experienced by the

animals during transfer to the maze, the

temperature and salinity to that
the animals were taken. The

in the

ASW

ASW was similar in

aquarium from which

in the maze had

placed

not previously contacted A. californica or A. brusiliana,

because there are animal-derived factors that make a nonlaying conspecific attractive (Painter er

al.,

1991

).

non-laying conspecific was placed in one of the stim-

ulus cages and a potential attractant added to the adjacent

ASW; this is the stimulus animal. After 5 min, a non-laying

known as the test animal, was placed in the base of

maze and watched for up to 20 min. In most cases, the
test animal moved directly to the top of the maze and
exhibited one of two patterns of behavior.
It stopped,
moved its head from side to side, then either moved into one
arm or returned to the base of the maze and remained there.
animal,
the

the presence of multiple species at

We

were asking: could

one breeding aggregation

be due. partially or completely, to attractin? If animalderived factors are necessary, do they differ among species?
Results.

The

results of the

experiments comparing the

attractiveness of native and recombinant attractin are

shown

Figure 3. In the negative control (non-laying conspecific

with nothing placed in the adjacent ASW). two animals
in

arm and remained, two (20%)

arm and remained, and six (60%) did
neither. Of the four animals making a choice, only two went
to the stimulus animal, one of which was in the right arm
and the other of which was in the left arm of the maze.
(20%) traveled

to the right

traveled to the

left

These bioassays verify that there is no directional bias in the

maze and establish chance levels of attraction at two animals.

The response

pattern

changed when
pmol of either
was placed in the seawater
I

native or recombinant attractin

It swam around in the maze, often visiting both cages

before deciding where to stop. A response was considered to
be positive if the test animal traveled to the stimulus within

adjacent to the stimulus animal (Fig. 3): 9 of 10 animals

(90%) were attracted to recombinant attractin, and 8 of 10

20 min, and then maintained contact with the stimulus cage

for 5 min. It was negative if the test animal traveled to the

cases, fewer animals

(2)

cage

The

opposite arm and maintained contact for 5 min.

response was considered to be no choice if the test
in the

animal did neither. Ten assays were performed for every

potential attractant. and the attractant was alternated be-

tween arms

consecutive assays. Statistical significance

was assessed by chi-square analysis. In each case, test
in

one arm and

Animals for each assay were selected on the basis of three

animals must have been sexually mature
but not have laid eggs or been used in a bioassay during the
criteria. First, the

h.

Second, the test animal

must not have been

exposed previously to the fraction being tested. Third, stimulus and test animals must have been housed in the same
aquarium (Painter

et al..

998).

An

exception was

made

to

went

to native attractin; in

to the opposite

both

arm and fewer

failed to make a choice. The response patterns for each

differed significantly from that for a non-laying conspecific

alone [recombinant:
2

(2)

icantly

The

in

animals were choosing between a stimulus

no stimulus in the other.

preceding 24

animals (80%) were attracted

animal

10.44. 0.05

from each other

results
is

(2)

< P <

13.75:

P <

0.005; native:

0.1]. but did not differ signif-

L\ (2)

2.1. 0.25

< P<

0.5].

of the experiment examining whether an

needed for attraction are shown

in

Figure

3.

When

pmol recombinant attractin was placed in the seawater

without a stimulus animal. 3 of 10 animals (30%) were
1

attracted to

recombinant

and

attractin.

two animals (20%) went

animals (50%) did neither (Fig.

The
to
3).
response pattern
pmol recombinant attractin
alone differed significantly from that to 1 pmol recombinant
to the opposite arm.

five

attractin with an

animal

:
],v

(2)

6.00: 0.025

<P<

0.05].

but did not differ from that to a non-laying conspecific alone

2
= 0.277: 0.95
P
0.975].
[X (2)

<

<

20

S.

D.

PAINTER ET

AL.

Positive

Negative

No Choice

Nonlaycr

No Animal

Konlayer

Nonlayer

Native Att

RccombAn

Recomb

A brasiliana
Recomb AR

Alt

A. californica

RecombAn

Added

Material

Figure 3. Both native and recombinant attractin are attractive; attractin acts in conjunction with other odors;
and the animal-derived factor is not species-specific. The number of Aplysia brasiliana individuals attracted to
a non-laying conspecific (Nonlayer)

was increased by placing

Att) or recombinant attractin (Nonlayer

pmol of

either native attractin (Nonlayer Native

Att) in the adjacent seawater. In each assay, animals chose

one arm and no stimulus in the other. Fewer A. brasiliana individuals were attracted to
when the stimulus did not contain a non-laying conspecific (No Animal Recomb Att; 1
same number of A. brasiliana individuals were attracted to the specimen of A. californica with

between a stimulus
recombinant

Recomb

in

attractin

pmol). About the

recombinant

attractin (A. californica

recombinant

attractin (A. brasiliana

Recomb Att; pmol) as were

Recomb Att; pmol).
1

attracted to the

Animals do not release attractin unless they are laying

eggs; therefore, the combined odor of a non-laying animal

that for a

and

7.50; 0.005

produces a qualitatively different stimulus

from attractin alone. The data confirm that attractin funcattractin

tions as part of a bouquet of scents

and led us

to ask.

it

(2)

non-laying A. brasiliana alone [A. brasiliana,

2
10.44; 0.005 < P < 0.01; A. californica, * (2) =

<P<

0.01].

Does

pheromone have to come from a concome from a different species of Aplysia,

the animal-derived
specific or can

specimen of A. brasiliana with

perhaps accounting for the presence of multiple species at

an aggregation? This would also be consistent with reports

Pheromonal induction of mating

Procedures.

were used

As

in the

activity

T-maze bioassays,

three criteria

in the

animals for each experiment. First, the

animals must have been sexually mature but not have laid

experiments examining whether the stimulus animal needs to be a conspecific in order for attractin to

eggs or been used in a bioassay during the previous 24 h.

Second, the animals must not have been exposed previously
to the fraction being tested or have been paired with the

of multiple species showing up

at

one aggregation

field.

The

results of

be attractive are shown

recombinant

attractin

in

Figure

was placed

A. brasiliana. 8 of 10/4. brasiliana

the non-laying conspecific.

3.

in the

When

When

(80%) were
1

pmol of

seawater adjacent to
attracted to

pmol of recombinant

was placed in the seawater adjacent to A. califor1 of 10 A. brasiliana (70%) were attracted to the

attractin

nica,

non-laying A. californica. The response patterns for the two

=
species did not differ significantly from each other [^(2)
0.265; 0.75

<P<

0.9], but

each differed significantly from

to select

must have been

same aquarium (Painter et ai, 1998).
Each assay was performed in 3 of aerated ASW in a 4-1
plastic beaker. The ASW had approximately the same osmolarity and temperature as the ASW in the aquarium from
which the animals were removed, and had not previously

same animal

housed

twice. Third, both animals

in the

contacted A. brasiliana. Animal-conditioned

ASW

not only

increases the attractiveness of a non-laying cospecific, but

also reduces the latency to mating (Painter et al., 1991).

APLYSIA

PHEROMONAL ATTRACTANT

21

Native Attractin

Native Attractin

100

80
tn

60
40
-

20

I
80

40

120

Time

200

160

ASW

240

(min)

Recombinant Attractin

10 pmol

pmol

Material

Added

Recombinant Attractin
150

100

o>

120

a- a- a-

80

ft- ft- ft-

a-

O)

pmol

90
a)

60

-o- ASW

60

40
0)

20

30

I
40

80

120

Time

160

200

ASW

240

Material

(min)

pmol

Added

Figure 4. Both native and recombinant attractin reduce the latency to mating in Aplysia brasiliana. (A) The
percentage of animals mating at early time periods was increased when native attractin was placed in the adjacent
seawater. (B) The latency to mating was reduced by placing either 1 pmol or 10 pmol native attractin in the
seawater. (C) The percentage of animals mating at early time periods was increased when recombinant attractin

was placed

in the adjacent seawater.

(D) The latency to mating was reduced by placing

pmol recombinant

attractin in the seawater.

Animals were rinsed

in fresh

non-conditioned

ASW

before

being introduced into the experimental beaker.

Two individuals of A. brasiliana and a test sample were

added

and behaviors were assessed at 10-min

270 min. Three categories of behavior were
mating as a female or male (one-way mating),
a hermaphrodite, and (3) laying eggs. Since an

to a beaker,

intervals for
identified:
(2)

mating as

egg cordon

is

a source of multiple contact and water-borne

pheromones that modify reproductive behaviors, egg-laying

activity was noted and the bioassay stopped; the bioassay
for that

sample was repeated with other animals. Egg laying

occurred rarely with any stimulus.

Test samples included
with nothing added (negative control),
with 1 or 10 pmol native attractin added,

ASW

ASW

and

ASW

statistical

points

with

pmol recombinant

significance

of the

attractin added.

differences

was determined by chi-square

The

between time

analysis; the statistical

significance of differences in

mean

was determined

latency

by one-way analysis of variance. The same number of

assays was performed for each treatment.
Results (native attractin).
attractin

was placed

in

When

ASW

pmol of native
two
containing
non-laying
1

or 10

specimens of A. brasiliana, the percentage of animals mating at each time point 10-min intervals) was recorded. The
(

percentage of animals mating was significantly increased

for 10 pmol attractin at 10, 20, 30, and 40 min, and there

was a nonsignificant trend in this direction for

attractin (Fig. 4 A). The mean latency to mating was
icantly reduced for 10

P <

0.05; n

10),

this direction for

pmol

attractin

(%

( 1 )

>

pmol
signif-

3.84 for each;

and there was a nonsignificant trend

in

Although the latency to

mating was reduced, the overall percentage of animal pairs
mating during the 270-min period was not affected (negative controls: 90% mated; native attractin: 100% mated).
1

pmol

(Fig. 4B).

22

S.

D.

PAINTER ET

AL.

was not

perhaps reflecting the long duration of the assay or animal

dites

housing in individual cages. In these experiments, nearly all

animal pairs eventually mated during the 270-min time

point, the percentage of animal pairs that

period, regardless of whether attractin

was

present. Never-

theless, the results suggest that attractin facilitates, but

does

rodites at

some

70% mated

Results (recombinant attractin).

When

pmolofrecom-

as hermaphrodites; recombinant attrac-

as hermaphrodites).

not tested, which

not induce, mating

any particular time

mated as hermaph-

point during the assay did increase (negative

40% mated

controls:
tin:

significantly increased at

may account

dose of 10 pmol was

for the lack of statistical

significance.

ASW

binant attractin was placed in

containing two nonlaying specimens of A. brasiliana. the percentage of animals
at

mating

and 240 min was significantly

170,

10, 20,

in-

creased compared to negative controls [^(l) > 3.84 for

each; P < 0.05; n = 10; Fig. 4C]. The mean latency to

mating was significantly reduced for 1 pmol recombinant

(P < 0.05; one-way analysis of variance; Fig. 4D).

attractin

Although the latency

to

mating was reduced, the

binant attractin:

We

native attractin from extracts of Aplysia

albumen
californica
gland (Painter et al., 1998) and recombinant attractin from insect cells to better characterize the
purified

biological activity of the peptide and to see whether recombinant attractin could be used in future molecular studies.

total per-

centage of animal pairs that mated during the entire 270-min

period was similar (negative controls: 90% mated; recom-

100%

tin facilitates, rather

Discussion

mated), suggesting again that attracthan induces, mating.

Pheromonal

attraction

T-maze, the attractiveness of a stimulus animal was

In the

increased

recombinant

attractin

verifying that

Pheromonal induction of hermaphroditic mating

sistent with

is

a re-analysis of the data collected in

on whether attractin can induce

pheromonal

the mating assays, focusing

each other, demonstrating

or facilitate hermaphroditic mating. As noted above, hermaphroditic mating was recorded during the mating assays.

studies.

Results (native attractin).

When

native

attractin

was

ASW

placed in the
surrounding two non-laying specimens
of A. brasiliana, the percentage of animal pairs mating as
hermaphrodites was significantly increased for 10 pmol

every time point between 20 and 170 min and for

2
190, 200, 230, and 250 min (^ (1) > 3.84 for each; P <

attractin at

0.05; n

10); the

same was

240, and 250 min (\

2
( 1

>

true for

pmol

3.84 for each;

attractin at 230,

P<

0.05;

n=

10)

(Fig. 5A). The mean latency to hermaphroditic mating was

significantly reduced for 10 pmol attractin (P < 0.05; oneway analysis of variance), and there was a nonsignificant

trend in this direction for

pared

mated

pmol

attractin (Fig. 5B).

Com-

percentage of animal pairs that

to control assays, the

as hermaphrodites during the

270-min period was

about doubled (negative controls: 40%:

pmol: 70%; 10
This
that
attractin
rather than
80%).
induces,
pmol:
suggests
1

facilitates,

hermaphroditic mating, perhaps by stimulating

both animals to mate as males. This induction could be
responsible for copulatory rings and chains in the field,
which may result because there are usually more than two

animals

in

an aggregation.

Results (recombinant attracting The percentage of animals mating as hermaphrodites at any given time point and
the latency to hermaphroditic mating were not significantly

increased upon addition of

pmol of recombinant

attractin.

although there were trends in this direction (Fig. 5 C, D).

Although the percentage of animals mating as hermaphro-

activity,

and confirming

N-

that

not required for attraction. The response

patterns for the two peptides do not differ significantly from
is

glycosylation

Procedure. This

when
pmol of either native or
was placed in the adjacent seawater,
both peptides are attractive in amounts con-

significantly

Recombinant

that either

attractin

was

could be used in future

therefore used in subse-

quent T-maze bioassays. Since it was not W-glycosylated,

recombinant attractin was also used to determine the solution structure of the

pheromone by 3D nuclear magnetic

resonance (Garimella

et al.,

Fewer individuals of
combinant

attractin

2003).

A. brasiliana

when

were attracted

to re-

the stimulus did not contain a

non-laying conspecific. demonstrating that attractin acts in

concert with other unidentified odors to stimulate attraction.

These
cordon

results,
is

combined with

earlier observations (the

attractive without a stimulus animal. Painter et

egg
al.,

1991: attractin elutes from the egg cordon, Painter et al.,

1 998 ).
suggest that the composition of the bouquet of scents

can vary.

To

identify other attractive chemical factors in the

egg-cordon bouquet of scents,

we have begun

isolating

other peptides/proteins that elute from the cordon for bioassay.

To

begin

looking for animal-derived attractants,

we

be a conspecific. It does not. A. californica with attractin and A. brasiliana with attractin each attracted a similar number of A.
tested

whether the stimulus animal needs

to

brasiliana. This pairing may seem inappropriate since the

two species do not overlap in their geographic distributions
(A. californica, Pacific Coast; A. brasiliana.
ico), but

Gulf of Mex-

help explain why multiple Aplysia species

are sometimes associated with one aggregation. For examit

may

ple, A. californica

and A. vaccaria have been observed

in

same breeding aggregations off the coast of California

(Kupfermann and Carew, 1974; S. LePage, M-REP, pers.
the

23

PHEROMONAL ATTRACTANT

APLYSIA

Native Attractin

Native Attractin

90
,

80

0>

70

S2

250

10 pmol

pmol

200

60

jg

-&--

50

40

.C
0.

-o- ASW

30

CO

20

o
10

40

80

120
Time

200

160

240

ASW

(min)

10pmol

pmol

Added

Material

Recombinant Attractin

Recombinant Attractin
.,

300

200

.c
Q.
CO

100

X
o

B
40

80

120
Time

Figure

5.

160

ASW

240

200

Material

(min)

Both native and recombinant

attractin

pmol

Added

induce hermaphroditic mating in Aplysia brasiliana. (A)

The percentage of animals mating as hermaphrodites was increased when native attractin was placed in the
was reduced by placing either pmol or 10 pmol
adjacent seawater. (B) The latency to hermaphroditic mating
was
native attractin in the seawater. (C and D) The percentage of animal pairs mating as hermaphrodites
to
mean
The
seawater.
the
in
was
latency
attractin
recombinant
when
1
adjacent
increased
placed
pmol
1

hermaphroditic mating was also reduced.

comm.), and have been seen mating with each other in the
A. fasciata
aggregation (S. LePage, M-REP, pers. comm.).
and A. depilans have also been seen associated with the
same aggregation (Achituv and Susswein, 1985), but mating
has not been observed because their reproductive cycles are
not

entirely

found

synchronized.

that A. californica

Audesirk

was not

(1977)

previously

attracted to conspecifics in

showed
assays, and Audesirk and Audesirk (1977)
that there was no seasonal effect on the sensitivity to con-

Y-maze

of the A.
specifics. Furthermore, experimental perfusion
californica rhinophore nerve with seawater that had bathed
A. californica, A. vaccaria, or Pleurobranchia californica
evoked about the same increase in afferent activity, suggest-

of Aplysia species in the field are not

species-specific chemical cues (Chase,

ing that aggregations

determined
1979).

by

Pheromonal induction of mating

Mating assays were performed because behaviors seen in
T-maze assays suggested that exposure to attractin
could stimulate behaviors suggestive of mating as a male
earlier

(Painter er

/.,

1998).

The

current studies

showed

that

when

seawater adjacent to a pair of A.

brasiliana, the latency to mating is reduced relative to
controls. However, the overall percentage of animal pairs
attractin is

added

to the

mating during the prolonged assay period was not

signifi-

but
cantly different, suggesting that attractin facilitates,

does

not induce, mating.

Attractin also significantly reduces the latency to hermating when added to the seawater surround-

maphroditic
of animal pairs
ing a pair of A. brasiliana. The percentage
about
mating as hermaphrodites during the assay period was
doubled, suggesting that attractin induces hermaphroditic

24

S.

PAINTER ET

D.

AL.

mating. This effect may result from attractin stimulating

both animals to mate as males, as suggested by T-maze

strong selection pressure against novel blends and response

preferences (Roelofs et al., 2002). Although airborne sex

behaviors. Overall, these data suggest that attractin contributes to the establishment and maintenance of breeding ag-

pheromones capable of inducing spatial orientation of conspecifics "downwind" are well established in insects (Carde

gregations.

and Minks, 1996),

identified sex

effectiveness; in fish, the

Attractin does not stimulate species-specific attraction

have a small range of

to

known

sex pheromones are go-

nadal steroids, prostaglandins, or bile acids (Li et

Mate

whose

this is not the case in vertebrates,

pheromones tend

2002).

al.,

genus Aplysia, and perhaps

appear to be a structurally diverse family of

peptides, each of which is sequence-specific for a given
species. Attractin has recently been characterized from A.

gastropods, appears to involve long-distance signaling via

waterborne pheromone blends. Attractin by itself is not

brasiliana, A. fasciata, A. vaccaria, A. depiluns, and Bur-

attractive to Aplysia species, but

The

attracting

satella leachii

21%

and found

be 95%, 91%, 43%, 41%, and

to

identical to A. California! attractin, respectively (Paint-

er et al, 2000,

and unpubl. data). Nevertheless,

attractin

attractive to all aquatic gastropods tested to date:

californica attractin
al.,

is

( 1 )

siliana (unpubl. data);

and

attractive to the freshwater

mones. Once aggregations of multiple Aplysia species form,

pulmonate Lymnaea stagnalis

Amsterdam, pers. comm.).

invertebrates that

is

first

al..

peptide

2003).

species-specific

To our knowl-

pheromone family

in

not species-specific. In contrast, water-

borne peptide pheromonal attractants

in

amphibians are

et al.. 2002).

(Kikuyama

There may be advantages

to attracting multiple species to

same breeding aggregation. If members of a second

species lay eggs on those of a different species, the mixed
egg mass becomes larger, which might in some way protect
the

the eggs of both species. Another possibility

is

that

egg

laying by one Aplysia species attracts a second species that

then lays eggs and releases attractin. which may eventually
attract

members of

the

first

species.

Because

reception and mechanoreception (Chase, 1979).

is

Although the primary structures of attractin-related peptides

are divergent, their 3D structures may be similar to A.
edge, the attractins are the

ing that the cordons themselves contain a blend of phero-

appropriate intraspecific mating may be achieved through

the use of specific proximal cues involving contact chemo-

(A. ter Maat, Free University,

californica attractin (Garimella et

egg cordons alone are

"downstream," indicat-

A.

attractive to A. bra-

is

(3) A. californica attractin

sufficient to attract Aplysia species

in other

is

attractive to A. brasiliana (Painter et

1998); (2) A. vaccaria attractin

attraction in the

attractin is

Acknowledgments

We

M. Miller
comments and acknowledge the Uni-

thank Drs. A. Susswein, A. ter Maat. and

for their interesting

versity of

Texas Medical Branch

try Laboratory,

which

is

(UTMB)

Protein Chemis-

supported by the

UTMB

Educa-

Cancer Center, for compositional and microsequence

analyses. This work was supported by NSF Grant IBNtional

9985778 (S.D.P.). and John Sealy Memorial Endowment

Fund for Biomedical Research Development Grant 2579-

02R

(G.T.N.).

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Reference: Biol. Bull. 205: 26-35. (August 2003)

2003 Marine Biological Laboratory

Field Observations of Intraspecific Agonistic Behavior

of Two Crayfish Species, Orconectes rusticus and

Orconectes
DANIEL

viriliSj

A.

in Different Habitats

BERGMAN AND PAUL

A.

MOORE*

Laboratory for Sensory Ecology, Department of Biological Sciences itiul the J. P. Scott Center for
Neuroscience, Mind, and Behavior, Bowling Green State University, Bowling Green, Ohio 43403;
and University of Michigan Biological Station. 9008 Biological Road. Pellxtun, Michigan 49769

Introduction

Agonistic behavior is a fundamental aspect of

ecological theories on resource acquisition and sexual se-

Abstract.

lection.

Crustaceans are exemplary models for agonistic

behavior within the laboratory, but agonistic behavior

Many

is often neglected. Laboratory studies do not

achieve the same ecological realism as field studies. In an
attempt to connect laboratory results to field data and in-

natural habitats

vestigate

how

habitat structure affects agonistic

shelter acquisition (Capelli and Hamilton,

nl..

interac-

Michigan

lakes.
in

that

less

likely

to

fight

end with

tailflip.

show

Asymmetries

May

size, sex,

in

fighting

ability

intrinsic features or extrinsic

may be produced by

circumstances that favor

dom-

1984; Pavey and Fielder. 1996). chelae size (Garvey and

al.. 1995). and sex (Stein. 1976;

the

Stein. 1993: Rutherford et

Peeke

et al.,

1995. 1998). Extrinsic asymmetries such as

prior residence (Peeke et

al..

1995, 1998), differing fight

strategies (Guiasu and Dunham, 1997). and previous history

in agonistic encounters (Rubenstein and Hazlett, 1974;

Daws

2003.

he addressed. E-mail:

intrinsic

1976). Intrinsic asymmetries are accurate predictors of

Extrinsic and intrinsic factors affect intraspecific aggression in many ways, and both should always be recognized as having the potential to alter agonistic behavior.

To whom correspondence should

and

inance during interactions between pairs of decapod crustaceans; they include physical body size (Bovbjerg, 1953,
1970; Rubenstein and Hazlett, 1974; Berrill and Arsenault,

ies.

extrinsic

one contestant (Parker, 1974; Maynard Smith and Parker,

dynamics associated with laboratory stud-

Received 16 December 2002: accepted 19

clarifying the

have been shown to affect aggression are

some

compared with

but do

in

resource availability, prior residence, and shelter presence.

laboratory fights, those in nature are shorter, less intense,

fundamental

1999, 2001),

reproductive state, hunger state, and social experience,

while extrinsic factors are status and individual recognition,

than macrophytes. In addition, observations of aggressive

behavior within a natural setting can add validity to labo-

and

al..

factors that affect agonistic interactions. Intrinsic factors

presence of detritus patches had higher average intensities

and ended with more tailflips away from an opponent,
suggesting that detritus was a more valuable food resource

fights in nature are

1956; Zulandt Schneider et

been invaluable

longer and more intense, suggesting that shelters have a

higher perceived value than food resources. Fights in the

When

1984; Peeke et

1999). chemical communication

Stocker and Huber. 2001). Laboratory experiments have

two food resources macroand one sheltered habitat. The overall

habitats:

phytes and detritus

observations reinforce the concept that resources influence
agonistic bouts. Fights in the presence of shelters were

ratory studies.

al..

mating (Hill and Lodge, 1999), food preferences (Capelli

and Munjal. 1982). and starvation (Hazlett et al., 1975;

agonistic interactions were

Intraspecific

three

1995; Figler et

(Bovbjerg,

behavior of two crayfish species was

observed by scuba diving and snorkeling in two northern
tions, the nocturnal

analyzed

made

observations of crayfish behavior have been

under controlled laboratory conditions. These studies generally focus on intraspecific aggressive behavior in terms of

in

et al..

2002; Bergman

outcome of agonistic

pmoore@

et al.,

interactions.

2003) contribute

to the

Seasonal variations in

food availability can also increase activity levels that lead to

bgnet. bjjsu.edu

26

FIELD STUDY OF CRAYFISH AGONISM

27

increased social contact and consequently to increased aggressive interactions (Hazlett et al.. 1975). Laboratory ex-

but less emphasis has been placed on agonistic behavior in

a natural setting. For this reason, we examined agonistic

periments are an invaluable aid to understanding behavioral

behavior under natural nocturnal conditions in two northern

mechanisms, but they have limitations

in their applicability

ecosystems (Bovbjerg, 1953. 1956; Peeke et al.,

1995). One severe constraint on laboratory studies of aggression is the restriction of space, which reduces an anito natural

mal's ability to escape from an opponent.

Dominance

hierarchies, territorial defense,

tion, substrate preferences,

and escalation of

mate selec-

fight

behavior

Michigan

lakes.

The study was conducted

in three different

habitats within the lakes to provide a global

view of

in-

traspecific agonistic behavior in nature that could be correlated to laboratory results on aggression. The results of this

study also allowed us to examine differences in agonistic

may be correlated to differing extrinsic factors

behavior that

in the laboratory

observed under laboratory conditions may not be representative of behaviors in a natural setting (Karnofsky et al.,

and nature.
Materials and Methods

1989). These changes in agonistic behavior observed within

largely be caused by an inability to
escape an agonistic conflict (Hediger, 1950). To circumvent
the laboratory

may

this artifact, studies

have been conducted

or streams that are less restrictive

in artificial

ponds
than the aquaria used in

standard laboratory experiments. By increasing the complexity of the experimental environment, studies in these
semi-natural settings attempt to obtain a more natural repertoire of behavior. They provide useful information about
agonistic interactions, foraging, mating, orientation, shelter

and molting (Abrahamsson, 1966; Ranta and

Lindstrom, 1992; Tomba et al., 2001 ). However, even studies in semi-natural environments cannot illustrate the "true"
acquisition,

behavioral ecology of the crayfish. Because of this shortfield studies are invaluable to the understanding of
crayfish behavior. They minimize laboratory bias and allow

coming,

for an integration of behaviors observed in laboratories with

those in a natural setting.

Crustaceans, particularly crayfish, have been used as a

to study aggression (Dingle, 1983; Hyatt,
1983) because of the ritualized nature of their agonistic

Dunham. 1987), the presence of formidable chelipeds (Garvey and Stein, 1993; Schroeder and
Huber, 2001), and the use of sensory information during
such encounters (Zulandt Schneider et al, 1999, 2001;
bouts (Bruski and

Bergman

et al.,
is

2003).

The ultimate goal of any aggressive

to obtain an elevated social status that gives

an

individual an advantage in obtaining a resource, such as

food, mates, and shelters (Wilson,

1975; Atema,

1986).

Conversely, a subordinate individual may lose access to

resources through unsuccessful bouts, but may obtain a net
benefit by avoiding costs such as increased energy expenditure, injury

from a conspecific, or increased predation risk

Edsman and Jonsson, 1996). If a subordinate

(Wilson, 1975;

does not gain a benefit, then the lower status will have a
negative effect on fitness. Consequently, a subordinate will
less food and shelter and fewer mating opportunities.
Extrinsic environmental factors can have a profound ef-

have

on aggressive

site

The study was sited in two remnant glacial lakes in the

northern part of the lower peninsula of Michigan: Douglas
Lake

(lat.

4528' N,

4533' N. long. 8457' W) and Burt Lake (lat.

long. 8440' W). The Burt Lake substrate is

predominantly sand and small gravel. Water depth ranges

from 0.4 (shallow) to 2.0
(deep). A mixture of sand and

gravel containing intermittent patches of detritus dominates

the shallow-water substrates.

The deep water contains a

sand substrate with a population of macrophytes (dominated

by Potamogeton sp. and Vallisneria sp.) and their associated

epiphytes. Water temperatures range from 14 to 23 C.
Observation points were accessed by snorkeling. The Douglas Lake substrate is sand that contains a small band of iron
substrata forming natural holes that crayfish use as shelters
in depth and
(burrows). This site ranges from 7.5 to 18.0

devoid of macrophytes. The water temperature ranges

from 10 to 15 C. Observation points were accessed through
is

model system

encounter

Stud\

thus a connection between

scuba diving.
Studv animals

Both the

Bun Lake and Douglas Lake

sites

contained two

species of crayfish, Orconectes rusticus and Orconectes

virilis. Crayfish species were determined by the color of the

periopods (chelae and legs), which are bright blue in O.

virilis and brownish-green in O. rusticus. The determination
of species allowed for an analysis of conspecific fights. In
Douglas Lake, only O. rusticus conspecific fights were
in the shelter habitat. In the Burt Lake population
conspecific interactions for O. virilis were observed only on
the macrophyte beds and not on the detritus patches, even

observed

although both species were present in the two regions. The

observers took care to avoid physically disturbing any of the
animals; they remained as motionless as possible by using
intermittent kick strokes to drift over the observation areas

(Karnofsky

et al.. 1989).

None of

the animals

were handled

their effects in nature

before or captured after behavioral observations. Consequently, male and female crayfish could not be distin-

need further validation. Agonistic behavior has been studied

extensively in the laboratory and in semi-natural conditions.

guished when aggressive interactions were analyzed, but the

relative size difference between crayfish was determined on

fect

activities;

extrinsic factors in the laboratory

and

D. A.

28
a

video

PVM-

(Sony Trinitron

screen
1

3 1 5Q) by calcu

color
'-''it

'

BERGMAN AND

monitor;

P.

A.

MOORE
Table

model

size difference of

Crayfish ellmgram codes

the opponents.
Intensity

Level

Behavioral

Description

oh'-'

away from opponent or fast retreat

back
away from opponent
Slowly
lanore opponent with no response or threat display

and August between

.inu 0100 (nocturnal activity period) from
the hours of 2
of
1996 to 2002 (no observations were made in the summer
calm
nights
1999) All observations were made on clear,
Observations

v.

Tailflip

.ade during July

when

the water surf

was below

8.0 cm. Interactions

and/or
Approach with threat display using meral spread
antennal whip

were

Initial

recorded on a video camera (Sony Hi-8 Handycam; model

# CCD-TR700) that was illuminated with white lights

on the underwater housing noticeably disturbed an

animal, the interaction was removed from the analysis.
animals, and any behavCrayfish are primarily nocturnal
sudden
the
exposure to white
ioral alterations caused by
this
from
could not be determined
study. For this
light

reason, any animal that tailflipped away or used a meral

in the absence of an interaction was removed from

spread
the data analysis; however, this does not take into account
in response to the
any unnoticeable changes in behavior
their behavior
alter
to
do
artificial light. Crayfish
appear
and Dunham,
altered
are
(Bruski
when light intensities
1987); however, since uniform white lighting
all

was used

in

observations, there should be no differential effects on

the behavior.

Two
was

to

first technique
sampling techniques were used. The
an agonistic
had
it
until
a
follow
single crayfish

interaction with a conspecific.

The second method was

Active claw use by grabbing opponent with open claws

Unrestrained fighting by grasping and pulling opponent's

were within two body lengths of one another. When

with either of these
agonistic interactions were observed
was
encounter
the
videotaped from
sampling techniques,
initiation to termination of the fight and the interactions

that

were

later

claws or appendages

closed chelae (intensity 3). When the chelae are opened and
used to grab an opponent, a new intensity level is reached
of
4). The most intense interactions have periods
(intensity

unrestrained fighting in which an individual appears to

at chelae, legs, or
attempt to injure an opponent by grasping

antennae (intensity

VHS
analyzed by playing the tape on a Panasonic
AG-7530-P) onto the Sony Trinitron

recorder (model #

monitor.

conflict

is

concluded when one

-1). usually signified by a

the
from
opponent (intensity -2), and usually
tailflip away
followed by a submissive posture (Bruski and Dunham,
assume a
1987). A subordinate will retreat consistently and

claws are

abdomen, and
posture in which the cephalothorax.
near the substrate. Typically, crayfish did not respond to
each other when separated by greater than two body lengths
of these changes in
(intensity 0). The temporal dynamics
behavior were recorded to include the total duration of the
encounter and the time it took to reach the different intensity

intensity

levels

maximum
maximum intensity

Duration, time to different intensities,

levels.

level

reached, and average

were analyzed using a one-way

MANOVA

and a

hoc
honestly significant difference (hsd) post

Tukey
The retreating animals

test.

away) and maximum intenan encounter were recorded and anasity achieved during
for proportions continlyzed using a multiple comparisons
::
that allows for testing
3 633
gency table (90.05.-.4
tests
analogous to the Tukey or Student-Newman-Keuls
(tailflip

<7oo5*4

and a

All videotaped fight trials were analyzed using an ethogram modified from Bui :.i and Dunham (1987) (Table 1).
An agonistic encounter in a laboratory setting with no

when an

individual

approaches a potential opponent (intensity 1). The encounter may then progress to a series of agonistic threat displays
a meral spread (intensity 2). If neither individual
using

bout gradually increases in fight intensity, startcontact and progressing to pushing with
with
chelae
ing

retreats, the

>

a
(Zar. 1999). Significant results are represented by giving
test
3 314 from the mult 'P le comparisons
value

Analvsis of fight heluivinr

resources available typically begins

5).

individual retreats (intensity

to

scan detritus patches (Burt Luke), macrophyte beds (Burt

Lake), and the shelter areas (Douglas Lake) for two crayfish

claw use by boxing, pushing, or touching with

closed claws

mounted on an underwater housing (Stingray video housthat contained the camera. Animals
ing; model # SR-700)
were filmed from a minimum distance of 0.4 m. Slow
when
swimming motions were made to follow animals, and
the lights

a threat display

Approach without

P
--

|3)

comparisons
size

>
< 0.05. An
-

value

additional

was included

for the

power analysis (Power

ANOVA

and multiple

for proportions contingency table tests.

differences of agonistic opponents were obtained

in

The
1

of the fights. Size differences are presented as a percentage

of the larger animal in the pairing. Thus, a value of 20%
means that the smaller animal is 20% smaller than the larger
size difference in
regression analysis between
was
analyzed using an expopercentage and fight duration
the
nential regression using
least-squares method.

animal.

FIELD STUDY OF CRAYFISH AGONISM

Results

29

A)

l^B Shelter

Qualitative description of fight dynamics

In

general, as

the laboratory, crayfish quickly apanother and immediately began to interact

in

proached one
(Bruski and Dunham, 1987; Bergman

et at. 2003). In

instances, fights, unlike those in the laboratory,

tively short and did not always show a stepwise
in intensities

were

et

at.

N=

away

2-

Bergman

09
08
07
06
05
04

of the stereo-

0.3

in

a different

direction. Fights rarely progressed to the

high intensities
seen in the laboratory (Stocker and Huber, 2001;
et at,

2003), but did seem to include

many

typical agonistic behaviors

(Huber and Kravitz, 1995). Surthe

number
of
prisingly,
fights ending in tailflips was low
(45%) compared to fights in a laboratory (>90% for laboratory fights; Moore, pers. obs.). In addition, multiple interactions between the same opponents within a short time

were

virtually

recognition

nonexistent, which

(Daws

may

be due to social

et at, 2002).

specific fights for O. rusticus for the three habitat types.

Conspecific fights for O. virilis were observed only in the
habitat,

and

all statistical tests

were done on

these animals. Within the macrophyte bed habitat, no

significant differences were found for any of the
following
statistical tests.

habitat fights

For

this reason, the data for the

were pooled

to provide a

macrophyte

more global

descrip-

tion of the parameters of average agonistic encounters in

nature. In the subsequent statistical tests, encounters

were

separated on the basis of the habitat in which the encounter

occurred. The mean duration of all encounters was 5.3
s (mean
SE); (n = 246; Fig. 1A), and 0.45 (111 of
246 encounters) of the conflicts ended with the behavior
"tailflips away from an opponent" (Fig. 2). Intensity 2 was

0.4

reached

0.49 of the encounters (121 of 246; Fig. 3 A).

was reached in 0.39 (95 of 246; Fig. 3A), and
4
was
reached in 0.12 (29 of 246; Fig. 3A). The
intensity
maximum
average
intensity of all encounters was 2.6
in

intensity 3

0.04 on the crayfish ethogram scale (Table 1; Fig. 3B). The

rate of escalation is a measure of time to different levels of

0.0
4-6

1-3

Figure

I.

(A) The

significant

significant difference

(SEM)

22-24

19-21

25-28

29-31

(s)

tight duration

of

all fights

(hatched),

difference

were not categorized

between

P <

test;

into a habitat type

the

habitat

0.05). (Note:

types (one-way
Nine interactions

and are only included

in the

"All"

category). (B) Frequency histogram

the proportion of fight dura-

tions in the shelter,

habitats in 3-s bins.

showing
macrophyte, and detritus

using a one-way
1A). The

(Fig.

0.7

s;

patch (2.9

MANOVA with a Tukey post hoc analysis

fight duration in the shelter habitat (11.1

85) significantly differed from both the detritus

0.3 s; n = 33) and macrophyte bed interactions

(1.80.1s;n =

= 1.00) (P < 0.05). There was

1 18; Power
no significant difference in fight duration between the conflicts occurring on detritus
patches and on macrophyte beds
(P > 0.05). Fight durations for encounters in the shelter
habitat ranged between 1 and 3 1 s, whereas the duration of
encounters on macrophyte beds and detritus patches did not
exceed 6 s (Fig. IB).

Tailflip-away

contingency table for multiple comparisons of propor-

tions demonstrated that agonistic encounters

tailflip significantly
==

shelter (61/85

more often when

0.72; q

detritus patch habitats (20/33

19.01;

0.61;

the fight

Power

=
q

ended

was

==

10.36;

in

in the

0.84) and

Power

Fight intensity

showed a

mean

Tukey-hsd post hoc

Fight duration
overall fight duration in the three habitats for the

16-18

on detritus patches (white), and fights

among macrophytes beds (Crosshatch). Values above bars (N =) indicate
numbers used for the statistical calculations. The letters above the bars

0.14) than

The

13-15

fights near shelters (black), fights

0.1 s for escalation to intensity

intensity and averaged 1.5
2 (246 of 246 encounters; Fig. 4), 3.9
0.2 s to intensity 3
0.9 s to intensity
(124 of 246 encounters; Fig. 4), and 9.5
4 (28 of 246 encounters).

collective pool of crayfish

I
10-12

7-9

Fight Duration

ANOVA,

Two hundred and forty-six encounters were included in

the data analysis. Statistical tests were performed on con-

Habitat

0.2

denote

Quantitative description of all fights

macrophyte

118

rela-

2003). Crayfish retreated

quickly from opponents by moving

Macrophyte

6-

progression
(Stocker and Huber, 2001; Zulandt Schneider

2001; Bergman

at,

Detritus

most

B)

et

i':':?'!l

when in macrophyte bed habitats (30/1 18

0.25;
Power = 0.98) (P < 0.05; Fig. 2). No significant difference
was found between conflicts in the shelter and detritus
habitats (q

3.29;

P >

0.05).

significantly greater proportion of agonistic encounters

on macrophyte beds (0.85; Power

1.00) reached a

max-

30

BERGMAN AND

D. A.

MOORE

A.

P.

All

All

Shelter

Shelter

Detritus

Detritus
:

>:

:
:

:
:

Macrophyte

Macrophyte

Intensity 2

4-]

I
1

Intensity 4

Intensity 3

Maximum
B)

Intensity

3 -

2 ~

00

0-

<

Habitat

Figure
that

3.

(A) Frequency histogram showing the proportion of fights

achieved each

maximum

intensity level for all fights (hatched), fights

near shelters (black), fights on detritus patches (white), and fights among
macrophytes beds (Crosshatch). The letters above the bars denote a significant difference

between the habitat

multiple comparisons of proportions;

mum

fight intensity level

intensities

P <

(contingency table for

0.05). (B)

The average maxi-

achieved per habitat type. The

bars denote a significant difference between the average

sity

per habitat (P

<

letters

above the

maximum

inten-

0.05).

Habitat
Figure 2. Frequency histogram showing the proportion of lights that
ended in a tailliip for all fights (hatched), fights near shelters (black), fights
on detritus patches (white), and tights among macrophytes beds (crosshatch). The letters above the bars denote a significant difference between
the habitat types (contingency table for multiple
tions;

P <

imum

comparisons of propor-

mum

had

detritus patches

mum

<

0.05; Fig. 3B). Interactions on

a significantly higher

average maxi-

0.09) than encounters on macro-

intensity (2.67

phyte beds (average

(P < 0.05; Fi2. 3B).

0.05).

0.05) than encounters in either of the

intensity (3.33

other two habitats (P

maximum

intensity of 2.16

0.03)

intensity level of 2 (meral spread display) than either

encounters

in the shelter (0.0:

detritus habitat (0.36; q

14.86

44.31;

Power =

Power =
0.21

1.00) or

(P

<

0.05;

Fig. 3A).
significantly greater proportion of encounters on
detritus patches reached the maximum intensity of 2 than

did encounters in the shelter habitat (q

Fig. 3A).

A maximum

was reached
when in the

16.57;

P <

in a significantly greater proportion

Macrophyte

of fights

Power = 0.62) and

Power = 0.11) than in

13.99;
q
= 0.62) (P < 0.05; Fig. 3A).
beds
Power
(0.15;
macrophyte
There was no significant difference between fights in the
detritus

Detritus

22.01:

detritus habitats (0.61;

Shelter

0.05;

intensity of 3 (pushing with chelae)

shelter (0.67; q

and macrophyte habitats (q =

1.89;

P >

2-

0.05). In

maximum

intensity 4 (open chelae use by grabbing) was reached by a greater proportion of conflicts in the

addition,

shelter habitat (0.33;

Pov\u

detritus patches (0.03: q

rophyte beds (0.0; q

no

0.24) than by interactions on

Power = 0.15) or mac-

1.23:

Intensity 2

Intensin

<

0.05; Fig. 3 A). There was

significant difference between the detritus and macro-

20.0) (P

2.83; P > 0.05). No fights in any habitat

phyte fights (q
achieved intensity 5 (unrestrained fighting). Encounters in
the shelter habitat had a significantly higher average maxi-

Figure

4.

The mean

(SEM)

time to intensity levels of

all

fights

(hatched), fights near shelters (black), fights on detritus patches (white),

and fights among macrophytes beds (Crosshatch). The letters above the bars

denote a significant difference for the time to reach intensity levels for each
habitat (one-way
Tukey hsd post hoc test; P < 0.05).

ANOVA

FIELD STUDY OF CRAYFISH AGONISM

Rate of escalation

The average time

the shelter habitat (4.3

0.2

(3.0

Power =

was significantly longer in

than on the detritus patches

to intensity 3

0.5

s)

or macrophyte beds (3.1

s)

1.00; Fig. 4). Intensity 2

difference

s)

(P

<

0.05;

primarily achieved in the shelter habitat; however, no statistical test could be performed because of the lack of
fights
in the

macrophyte

(n

0) and detritus (n

1) habitats.

when

method demonstrated that the duration of ag= 117) was longer when the size
differential between opponents was smaller (P < 0.05; Fig.
5). Encounters were longer when opponents were sizematched within 10%, whereas fights with a size difference
least-squares

did not exceed 4

sp.) they

intrinsic factors

(detritus
itat

were more

al.,

phytes. Moreover, crayfish have been observed foraging on

both species of macrophyte (Potamogeton

ria sp.)

and on

sp.

and Vallisne-

detritus, suggesting that all three are viable

food resources (Lodge and Lorman, 1987; Hill et al., 1993;

et al., 2002). Among these food resources, detritus

on or near food-resource habitats

in the shelter

in distinct patches,

their associated epiphytes

whereas the macrophytes

far more abundant and

were

consistently distributed in Burt Lake. Moreover, shelters

of agonistic behavior

and macrophytes). Interactions

and higher

rates

Physiologically, it appears as if detritus is more

nutritious and thus a more valuable resource than macro-

and

Crayfish agonistic interactions were longer (Fig. 1A),

more intense (Fig. 3A, 3B, 4), and more likely to end with
a tailflip (Fig. 2) when the interaction took place near a
shelter (burrow) than

have slower growth

1993).

was located

and

availability of a shelter or a

food resource, seem to influence aggressive fighting behavior in crayfish. With reference to food resources, when

Cronin

s.

Discussion
Extrinsic

they occurred on detritus patches as opposed

macrophyte beds.
Extrinsic factors, such as the

to

levels of mortality than crayfish fed detritus (Hill et

onistic interactions (n

10%

alluring

defense from

predators (Hill and Lodge, 1999). Conflicts were more

intense (Fig. 3A, B) and ended more often with a tailflip

Lymnaea

significant exponential regression analysis using the

greater than

may be
in

crayfish are fed a strictly macrophyte diet (Anmicola sp. and

Effect of size differential on fight duration

macrophytes or detritus patches. Shelters

because of their use to attract mates or

(Fig. 2)

significant

whereas intensity 4 was

the habitats,

among

0.2

showed no

31

hab-

likely to reach higher intensities, but they

also took longer to reach those intensities (Fig. 4). These

results indicate that shelters were more valuable than either

and detritus patches are limited resources, hence more easily

defended. Conversely, macrophyte beds are usually an easily

accessible and abundant food source (Capelli, 1982), and

defense becomes difficult and unnecessary when they are

widely available. Given the increased nutritional value and
limited distribution of detrital food sources,

on

intraspecific encounters

detritus patches

we

predict that

would be more

intense and longer than fights in a macrophyte habitat.

Indeed, in our sample, intraspecific fights lasted longer,

reached a higher average maximum intensity, and ended

more often with a tailflip. These results may be caused by
the relative scarcity and temporal unpredictability of detritus patches within Burt Lake. Patches are often destroyed or

25-

Duration

~, fc *-

.2

(267948-0218825i7edifTcrcnccl
656 + exp

moved
2

20-

=0.85586

A
O

Detritus

and when

Macrophyte

become more

15-t

may

wave

action. Detrital patch

limit this potential nutritional resource,

finds a rare patch, the interactions

a crayfish

intense

macrophyte beds and

Shelter

overnight by physical

heterogeneity

in

defense of

it.

In

contrast,

the

had a more

their associated epiphytes

homogeneous distribution and greater temporal stability

than detrital patches. As a result, macrophytes interactions
were the least intense of the habitat types.

10-

Our
5-

results for the crayfish interactions in the

macrophyte

and detritus habitats are consistent with the idea

that detritus

is

more valuable than macrophytes because of its increased

al., 1993). However, no definitive

nutritional value (Hill et

0-

conclusion about the relative merits of detritus and macro5

10

15

20

25

30

35

% Size Difference of Combatants

5.

The percentage

size differences of agonistic

opponents analyzed with an exponential regression using the least-squares method.

Size-matched fights lasted longer than fights between unevenly sized

Figure

opponents (P

<

0.05).

phyte diets can be drawn from our study due to the unknown
and varying composition of the detritus. Nevertheless, both

macrophyte and

detritus food resources appear to be less

valuable than shelters. Shelters have been

effect

on agonistic outcomes

more

likely to retain a shelter

shown

to

have an

previous owner is
and initiate more interactions

in that the

32

D. A.

BERGMAN AND

Edsman and Jonsson, 1996). Capelli and

Hamilton 1984) have shown that fond and prior residencies

(Peeks

eta!.. 1995:
(

affect agonistic behavior in a

ment. They reported

the

d laboratory environ-

ip

activity decreases with

thai

both shelters and food.

increased availabilir

In

ncrease in shelter availability

addition, they shov

:un an increase in food availabilreduces aggression mou

more macrophytes than
ity. Thus, high food availability,

and low shelter availability would lead

detrital patches,

more

intense conflict over shelters, followed

by

to

detritus

MOORE

A.

P.

social or individual recognition of conspecifics (Bruski

Dunham, 1987; Karavanich and Atema, 1998a,

and

b). Intrinsic

such as size and recognition, and extrinsic factors,

factors,

such as environmental surroundings, are important in determining intraspecific agonistic outcomes. However, the extent of the role
to

each

and extrinsic factor plays

intrinsic

is

yet

be conclusively determined.

Cursory review of laboratory- studies

in relation to field

observations

patches, and then macrophyte beds.

Conspecific conflicts can usually be thought of as a

"limited war." in which serious injury is avoided (Maynard

Smith and

However, conspecific

Price, 1973).

conflicts be-

tween crayfish involve potentially lethal chelae that allow

for an "unlimited war" with the possibility for more intense
and

lethal

fights.

common

High-intensity fights are

in

laboratory environment, largely because the opponents have

been closely matched for size of carapace and chelae (Huber

and Kravitz, 1995; Karavanich and Ateina, 1998a.

b).

In

nature, an advantage in size directly confers an advantage in

resource holding power (RHP) to the larger individual.

Parker (1974) noted that as RHP disparity (size difference)
increases, conflicts

combatants

may

become

less intense

and shorter. Both

increase their overall fitness by minimizing

and reducing energy expenditure from

and
intense
fights. The winners of such interactions
long
the chance for injury

gain access to

more valuable resources such

as mates, food,

or shelters, while the losers reduce their risk of predation,

minimize energy costs, and emigrate

Our

results are typical for

Smith and Parker, 1976)

in

to find a

new

resource.

asymmetric contests (Maynard

which a larger individual holds

more valuable resources (shelters), and conflicts are longer

when the opponents are size-matched. Moreover, when resource availability is asymmetrical, conflicts will generally
be shorter when the least valuable resource
macrophytes
in this

study

is

in dispute.

The

have some significance tied to

were in this habitat, and these

shelter habitat appears to

because the longest fights
fights were the most closely
it

size-matched (Fig. 5). The longest fights in all three habitats

occurred when the opponents were within 10% of each

Intraspecific aggressive behavior between decapod crustaceans can be influenced by a myriad of extrinsic factors.
For example, an extrinsic factor such as small aquarium size
will

sometimes

elicit

a "critical reaction" (Hediger. 1950).

when antagonists are crowded

together in an aquarium with no possibility of escape. The
inability to escape a competitor can cause changes in fight
reaction occurs

critical

duration, retreat behavior, and intensity levels reached in

ct al., 2000). The presence of a defendable
resource can also cause an escalation in fight

(Peeke

fights

extrinsic

intensities in small aquaria.

will be

more

intense than

When

when

shelters are present, fights

they are absent (Peeke ct

al.,

1995). Intrinsic factors such as size. sex. and social expe-

rience can also affect aggressive activities. Size-matched

large crayfish escalate

more slowly

to high intensities

and

have longer fight durations than size-matched small crayfish

(Schroeder and Huber, 2001). Generally, male crayfish are
more aggressive than females (Bruski and Dunham. 1987),

and social experiences

in

the

form of winner and loser

effects influence the likelihood of success in subsequent

(Daws

fights

extrinsic

and

et ai,

so that

in the laboratory

same

In general, fights

than

maximum

in

were shorter

maximum

2003). These

fight

studies

s;

Fig. 1)

3B)

and

in the

(Table

and 2.8) and Bergman

(Table

0.4

(5.3

intensities (2.6; Fig.

2). The average

was
lower than in
intensity in the field
and
Huber (2001
seen by both Schroeder

laboratory

laboratory fights
(2.7

ct al.,

change the dynamics of fights

they do not necessarily show the

characteristics as fights in a natural setting.

had lower average

field

2002; Bergman

intrinsic factors

2). In addition, the

et al..

(2003) (4.2 and 3.5)

time to different intensity levels

Dunham. 1987; Schroeder and

has been used as a measure of the rate of escalation in

Huber, 2001; Stocker and Huber. 2001; Bergman ct al.,

2003). However, the shelter habitat appears to be more

violence during fights and was considerably shorter for all

intensities in the field than in the laboratory fights of Stocker

closely matched than

and Huber (2001 and Bergman et al. (2003) (Table 2).

Within a laboratory environment, all aspects of a con-

other in size (Bruski and

iln

food resource habitats; conse-

quently, the valuable resource (shelter)

individuals,

may

which causes smaller individuals

attract larger

to

move

to the

periphery or into other habitats (detritus and macrophyte).

Moreover, a hierarchy has likely been established in the

whereas the macrophyte and detritus

habitats do not provide the same temporal stability and do
stable shelter habitat,

not function to decrease predation.

archical status

is

The recognition of

hier-

probably reinforced by visual or chemical

frontation can be controlled to lengthen conflicts or increase

fight intensities.

Sex, species, size of opponents, size of

aquarium, reproductive state, status/individual recognition,

social experience, and hierarchy establishment can all be
controlled in the laboratory.

An example

of a controlled

size-matched opponents (Bruski and Dunham,

1987; Rutherford et al., 1995; Stocker and Huber, 2001:

variable

is

FIELD STUDY OF CRAYFISH AGONISM

Table
Ciir.\ory

review of crustacean agonistic experiments

Reference

in the

laboratory

33

34

D. A.

BERGMAN AND

MOORE

A.

P.

Homarus americanus. Can.

tion in the lobster.

Summary

J.

Fish. Ac/uat. Sci. 43:

2283-2290.

These

observations sugge
have
a signihY
surroundings

ihe

field

nistic bouts in crayfish

in resoiiR

asymmetries

environmental

on intraspecific ago>ted by Parker (1974),

i

<ower can be an important

Conflicts in the presence of

'
1

factor in fight prop-

were longei

Bergman, D. A., C. P. Kozlowski, J. C. Mclntyre, R. Huber, A. G.

Daws, and P. A. Moore. 2003. Temporal dynamics and communication of winner-effects in the crayfish. Orconectes nisticus. Behaviour
(In Press).

M., and M. Arsenault. 1984. The breeding behaviour of the

northern temperate orconectid crayfish. Orconectes nisticus. Anim.

Berrill,

suggesting that shel-

Belmv. 32: 333-339.

have a higher nn . ;s value than detritus or macrophyte

food resources. A shelter's protective value may outweigh

Bovbjerg, R. V. 1953.

shelters

;e intense,

ters

the value of the food sources

when

the threat of predation

is

especially high. Detrital food sources are likely more valuable than macrophyte food sources because of their patchy
distribution

and the nutritional inadequacy of macrophytes

It is
quite evident from this study's results

(Hill etui., 1993).

an intricate influence on the

that extrinsic resources are

examined both

in the

that aggressive

behavior must be

laboratory and in the field to better

understand the factors that influence crayfish aggression.

Each experimental environment has unique benefits and
problems. Observations in nature contribute to an understanding of habitat usage, movement patterns, shelter occu-

and food

availability.

Laboratory experiments are

mechanisms and the

invaluable in elucidating the behavioral

environmental components that affect aggression. By controlling different aspects of agonistic interactions, such as
food preferences, and shelter accessibility, a researcher can test facets of agonistic behavior that are not
size, sex,

However, such invesdo not answer the question of whether the behavior

easily controlled in a natural setting.

tigations

an artifact of laboratory confinement or a behavior that is

displayed in nature. Consequently, one must be hesitant
is

when using

laboratory results to explain agonistic behaviors

in the wild. Laboratory and field observations show considerable differences in fight dynamics.

two

is

needed

to

develop a

combination of the

realistic picture

of aggressive

behavior.

factors affecting aggressive behavior in

crayfish. Physiol. Zool. 29: 127-136.

Bovbjerg, R. V. 1970. Ecological isolation and competitive exclusion in

two crayfish (Orconectes virilis and Orconectes immunis). Ecology 51:
225-236.
Bruski, C. A., and D. \V.

Dunham.

The importance of

1987.

communication of the crayfish Orconectes

agonistic

vision in

rusticus.

Behav-

M. 1982. Displacement of northern Wisconsin crayfish by

Orconectes rusticus (Girard). Limnol. Oceanogr. 27: 741-745.
Effects of food and shelter
Capelli, G. M., and P. A. Hamilton. 1984.
Capelli, G.

on aggressive activity in the crayfish Orconectes rusticus (Girard).

Cmstac. Bio/. 4: 252-260.

and

Capelli, G. M.,

in relation to

of the genus Orconectes.

J.

J.

Aggressive interactions and

B. L. Munjal. 1982.

resource competition

species displacement

Crustac. Biol. 2:

among

cray-

486-492.

Cronin, G.. D. M. Lodge, M. E. Hay, M. Miller, A. M. Hill, T. Hovath,

R. C. Bolser, N. Lindquist, and M. Wahl. 2002.
Crayfish feeding
preferences for freshwater macrophytes: the influence of plant structure

and chemistry.

Daws, A.

J.

Crustac. Biol. 22: 708-718.

G., J. L. Grills, K.

Konzen, and

Moore. 2002.

P. A.

Previous

experiences alter the outcome of aggressive interactions between males

in the crayfish.

Procambarus

clarkii.

Mar. Freslm: Behav. Physiol. 35:

139-148.
Dingle, H. 1983.
1

Strategies of agonistic behavior in Crustacea. Pp.

Rebach and D. W. Dunham,

Edsman,

85-

Studies in Adaptation: The Behavior of Higher Crustacea, S.

in

L.,

eds.

and A. Jonsson. 1996.

John Wiley and Sons. New York.

The effect of size, antennal injury,

ownership, and ownership duration on fighting success in male signal

crayfish. Pacifastactts /eniusculits (Dana). Nord. J. Freshw. Res. 72:

80-87.
Figler,

M.

H., H.

ciiltun' 178:

Garvey,

The authors thank the Laboratory for Sensory Ecology

for comments on the manuscript and the
University of
Michigan Biological Station for the use of their facilities.
Thanks also to Robert Schneider, Rebecca Zulandt
Schneider, and

Mary Wolf

for help in collecting the data.

was supplied by the National

Science Foundation (DAB 9874608, IBN-95 14492. and
IBN-0131320).
Support for this research

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2003 Marine Biological Laboratory

Twisting and Bending of Biological Beams:

Distribution of Biological Beams in
a Stiffness Mechanospace
SHELLEY

A.

Department of Biologv, Duke University,

Most

Abstract.
tively easily

beams bend and twist relahuman-made structures. This pain 57 diverse biological beams in

compared

per investigates flexibility

common

an effort to identify

between

stiffness.

The

North Carolina 27708-0338

drostatic pressures.

tory

swimming (McHenry

by mapping both ideal and biological

mechanospace defined by flexural and tor-

been implicated

beams

(Gal, 1993). In

sional stiffness.

random, but

is

The

distribution of biological

beams

is

that are stiff in

are stiff in torsion, while those that

of a

fish

backbone

influ-

in

1995; Long and Nipper.

mammalian backbones has

et al.,

locomotor differences between species

many

sessile organisms, flexibility allows

structures to passively adjust their posture relative to the

not

generally limited to particular regions of the

mechanospace. Biological beams

by muscular contractions or hy-

flexibility

1996), while the flexibility of

pat-

terns are investigated

into a

The

ences the mechanical behavior of the body during undula-

patterns in the relationship

and torsional

flexural stiffness

Di/iiuiin,

internal forces generated

biological
to

ETNIER*

forces experienced (Wainwright et

al..

1976; Vogel, 1984).

Leaf petioles (Vogel, 1989; Niklas, 1991) and herbaceous

plants (Ennos, 1993; Etnier and Vogel, 2000) reduce flow-

bending

bend easily also twist

properties of

induced drag by bending or twisting in response to wind,

and similar drag-reducing mechanisms have been found in
hydroid colonies (Harvell and LaBarbera, 1985) and anem-

closely resemble theoretical expectations

ones (Koehl, 1977a). Other flexible organisms take advan-

beams. Both distributions are potentially being

driven by the interdependence of the material and structural
properties determining stiffness. The mechanospace can be

tage of external forces to passively orient their filter-feeding

structures in response to ever-changing flow (Wainwright

easily. Unoccupied regions of the mechanospace represent

rare combinations of mechanical properties, without prov-

ing that they are impossible.

biological

beams

The mechanical

for ideal

and Dillon, 1969; Koehl, 1977b; Harvell and LaBarbera,

1985; Best, 1988). Thus, the ability to deform in response to

used as a broadly comparative tool to highlight systems that

fall outside the general pattern observed in this study. These
outlying
gists

beams may be of

loads

particular interest to both biolo-

and engineers due to either material or structural

property of mosl

Denny, 1988), yet the

deform

in

response to a load,
1984;

:>1

beam

Received 14 February 2003; accepted 21 May 2003.

*Present address: University of North Carolina at Wilmington. 601

NC

terrestrial

(a structure that

environment, apparently

nr) represents the resistance of a

is

long relative to

resistance of a

beam

to

its

width) to

nr) represents the

stiffness and
Flexural
twisting.

bending, and torsional stiffness (GJ

consequences of flexibility

vary widely. In motile organisms, flexibility permits the

relative movements of structural elements in response to

College Road, Wilmington.

both motile and sessile organisms living

ural stiffness (El in

Mnlogical beams (Vogel,

bii.l

in

an aquatic or a

importance of flexibility in the design of biological organisms (Lauder, 1982; Vogel, 1998).
flexFlexibility is measured in terms of stiffness, where

Introduction
Flexibility, or the ability to

observed

independent of phylogenetic affiliations. Such convergence

may be viewed as a red flag indicating the tremendous

innovations.

is

is

in either

in

torsional stiffness are composite variables that are influ-

enced both by material and structural properties (Waina

wright et al.. 1976). Every beam is characterized by
combination of flexural stiffness and torsional stiffness, and

S.

the relationship

28403. E-mail: etniers@uncwil.edu

36

between these two variables determines

TWISTING AND BENDING BIOLOGICAL BEAMS

how

the

beam responds

to a

given load. The ratio of flexural

commonly termed the twist-

stiffness to torsional stiffness,

to-bend

ratio,

has been used as a dimensionless (and, thus,

size invariant) index describing the relationship

between
two variables (Niklas. 1992; Vogel, 1992, 1995; Etand Vogel, 2000). The twist-to-bend ratio indicates the

these
nier

beam

relative resistance of a

More

bending versus twisting.

to

intuitively, a higher twist-to-bend ratio indicates a

structure that twists

more

readily than

bends, without

it

reference to the magnitude of either variable.

flexibility is a common property of a phylogenetidiverse
cally
group of organisms, are there any common
or
trends
in the relationship between flexural stiffpatterns
ness and torsional stiffness in biological beams? This paper

While

37

Material properties. The material properties influencing

flexural stiffness

and torsional

stiffness are

and the shear modulus (G in N m~ ),

respectively. Young's modulus and the shear modulus are
related to one another by Poisson's ratio (v), which is the
)

dimensionless ratio of the induced

while rubber has a ratio closer to 0.50 (Denny, 1988) and

materials such as cornstalks have moderate ratios around
0.23 (Prince and Bradway. 1969). Commonly occurring
metals have Poisson's ratios between 0.25 and 0.30 (Niklas,

space, similar to Raup's (1966) classic morphospace,

Young's modulus (Roark. 1943)

For isotropic materials, the shear modulus

broadly comparative tool used to visualize the relationships

between mechanical variables in biological beams. The

concept is based on the premise that the mechanical properties of flexural and torsional stiffness are common to all
biological beams. Three variations of this

be used to compare the patterns of

large diversity of biological structures.
will

be used

structural properties will

in

mechanospace

flexibility

seen in a

First, material and

combination

to predict,

beams. Second, experimentally measured values of

and torsional stiffness for biological struc-

flexural stiffness

tures will be

examined within

the context of the theoretical

distribution. Finally, the relative contribution of overall size

to the

mechanical properties of biological beams will be

The results suggest that the distribution of biobeams within the mechanospace is not random, due

explored.
logical

to the interdependence of material

and structural properties

stiffness.

determining

G =
2(1

related to

(I)
v)

Thus, the Young's modulus for a typical isotropic material

will range from 2 to 3 times its shear modulus as Poisson's
ratio varies

from

to 0.50

(Wainwright

et ai,

1976: Niklas,

Structural properties.
flexural stiffness

The

structural variables influencing

and torsional

m4

stiffness are the

second mo-

and the polar moment of area (J in

m 4 ), respectively. These variables reflect the geometry of a
cross section of a beam and are influenced by size, shape,

ment of area

(/ in

and orientation (Roark, 1943). The relationship between 7

and J depends upon the cross-sectional shape of the structure in question (Roark. 1943).

Formulas for the calculation

of / and J for most simple shapes can be found in any basic

engineering handbook (e.g., Gere and Timoshenko, 1984). /

and J are both proportional to radius to the fourth power,

hence radius

is
a very strong determinant of stiffness
(Roark, 1943). For example, for a beam with a circular
cross-sectional area

Materials and Methods

Distribution of ideal

beams
/

The

is

by:

1992).

on the basis of principles of engineering beam theory, the

theoretical relationships between bending and twisting in
ideal

lateral

causing

can vary from

to 0.50 for naturally occurring isotropic
materials. Mollusc shell has a Poisson's ratio of about 0.10,

1992).

strain,

contraction of the specimen, to the applied strain, causing

the specimen to elongate (Vincent, 1990). Poisson's ratios

investigates such patterns with a mechanospace defined by

values of flexural and torsional stiffness. The mechanois

Young's mod-

N m

ulus (E in

beams was determined using

from engineering beam theory. Importantly, this

and

77T

(2)

distribution of ideal

principles

material

II

is

0.50. Small changes in the cross-sectional

beam can

limited to structures built of a single, isotropic

shape of a

(i.e.,

the material properties are not directional ly

thus, the value I/J for noncircular cross sections

beam
beam
engineering
shape of the

early elastic,

and

theory stipulates that the material is linbeam is straight and does not vary

shape, depending on its orientation with respect to the

applied load (Table 1 ).

that the

shape along

its

length, nor does the

10%

of

total length

solutions are required for

dergo larger deflections

Morgan, 1989).

can be

much

question (Roark, 1943). Additionally,

in

deflections greater than

More complex

greatly influence the values of / and J;

higher (Table 1). Note that there may be several

values of II J for a beam with an asymmetric cross-sectional

variable), with precise specifications for the cross-sectional

in size or

the value of

is

distribution

(e.g.,

beam undergo
(Roark. 1943).

beams

that un-

Morgan and Cannell, 1987;

Relationship benveen flexural

El and

GJ

and

torsional stiffness.

are dependent variables, with Poisson's ratio

linking the material properties (E and G), and geometry

linking the structural properties (/ and /). Theoretically, the
relationship

between

flexural stiffness

and torsional

stiff-

38

S.

A.

ETNIER

Table
Theoretical

re/tin.

^ tv/; flexural

and

torsional stiffness

Ratio of El/GJ

onal shape

TWISTING AND BENDING BIOLOGICAL BEAMS

39

o antennae
Elliptical

beam

n crinoid arms

v = 0.50
o horsetails

Z
A stems
<U

petioles
C/3

2
3
X

Daffodil

Circular

= 0.25

Sedge

+ root

beam

A vines

OJD

-3

gorgonians
tree branches

-6

-3

log Torsional Stiffness

Figure

1.

(Nm

The mechanospace defined by values of flexural stiffness and torsional stiffness. The lines
beams based upon an assumed cross-sectional shape and Poisson's ratio. The

represent the distribution of ideal

symbols represent

the nine groups (Table 2), with each individual point representing an average value for a

The upper line represents an elliptical beam (4:1 major to minor axis) with /// calculated
about the short axis and a Poisson's ratio of v = 0.50. The lower line represents that same beam with I/J
species (appendix).

calculated about the long axis and a Poisson's ratio of v

a Poisson's ratio of v

0.

The dashed

line represents a circular

assumptions were made for the gorgonian corals, with

m (Jeyasuria and Lewis,

author's research to obtain average flexural stiffness and

ilar

torsional stiffness

measures for 25 species of plants and

animals (see appendix). These data were obtained following

an estimated diameter of 0.002

protocols similar to those discussed above. In other cases

affect the overall

(n

32), published data consisted only of

and G.

measures of

In these cases, the researchers experimentally deter-

mined El and GJ, but then factored out / and J based on the
cross-sectional size and shape of the structures. But note
experimental data were
mechanical behavior of the beam

that the

load), rather than direct

beam with

0.25.

still

based on the overall

(i.e.,

deformation due to a

measures of material properties. In

cases where published data consisted only of measures of

and G, both size and shape were estimated to determine

1987).

The assumption of

a relatively small diameter will

and

tor-

sional stiffness, but will not affect the ratio of the two.

The

magnitudes of flexural

stiffness

assumption of a circular cross-sectional shape will result in

lower estimated values of I/J than would be seen in noncir-

beams (Table
The species (total

cular

).

57) were divided into nine groups

based upon broad morphological similarities (Table 2).
Structures varied greatly in size, with diameters ranging

from 0.001

(red

/;

maple

petioles) to 0.05

branches). Average values for log

GJ

(small tree

versus log El for

all

values for / and J (appendix). Flexural stiffness and torsional stiffness were then calculated, based on these esti-

57) were plotted in the mechanospace (Fig. 1 ).

species (n
Note that each point represents an average value for a

mates. The estimates of beam diameter were deliberately

conservative, while cross-sectional shape was always assumed to be circular. For example, for 14 of the 16 vines

and material properties. For asymmetric beams, such as the

daffodils, crinoid arms, and crustacean antennae, flexural

and 5 of the
the

tree

branches included in the mechanospace.

to have a circular cross section with

beam was assumed

a diameter of 0.02 m.

species were 0.02-0.05

The reported diameters

for these

(Putz and Holbrook. 1991). Sim-

species,

and thus does not

reflect individual variation in size

stiffness is reported as the

average of the different orienta-

tions. Coefficients of variation for these

between 357r and 196% for

to

110%

organisms varied
and from 36%

flexural stiffness,

for torsional stiffness (Etnier and Vogel, 2000;

40

S.

A.

ETNIER

Table 2
Basic group characteristics for beams

Results

ui\il,-;t'd

this

study

The

K characteristics

Groups

jicssive interactions.

Not loaded

gravitationally. Aquatic animal.

Cnnoid arms

Multi-jointed beam.
filter

feeding.

Arms used

Not loaded

for passive

gravitationally.

Aquatic animal.
Multi-jointed beam. Stem must maintain

Horsetails

in

are based

upon the assumpand

theory
represent values for

Figure

beam

beams. The distribution of biological beams closely

matched that of the ideal beams, with 93% (53 of 57) of the

supporting. Terrestrial plant.

supporting. Terrestrial plant.

Continuous beam. Petiole must support leaf

against gravity and withstand wind, so selfsupporting. Terrestrial plant.

Tree roots

Continuous beam. Root

Loaded
Vines

not self-supporting.

in tension. Terrestrial plant.

Continuous beam. Vine

Loaded

is

is

not self-supporting.

in tension. Terrestrial plant.

Continuous beam. Support individual polyps

against water flow. Not loaded

Gorgonian corals

gravitationally. Aquatic animal.

Continuous beam. Tree must maintain upright

Tree branches

position for photosynthesis, so self-

supporting. Terrestrial plant.

biological

beams investigated were divided

on broad differences

2001),

in

into nine groups,

based

morphology and function.

reflecting

the

individual

variation

within the bounded region (Fig.

noted

tional

shapes.

Flexural

stiffness

each species was calculated

and compared to the predicted values as an additional descriptor of flexibility in biological beams (Fig. 2). Standard
ratio for

deviations for the twist-to-bend ratios,

when

and torsional

in the biological

hard to bend were also hard to

stiffness

and torsional

stiffness

).

The

stiffness

beams. Thus, beams

twist.

were

Overall, flexural

each varied over 9 orders of

stiffness

and torsional

stiffness

available, are

reported in the appendix.

of group members were of

similar magnitude.

Due

to the finite nature of the data set, the

unoccupied

regions of the mechanospace suggest that other combinations of mechanical values are rare without proving that
they are impossible. Biological beams may exist within
these unoccupied regions, given sufficient variation in material

or structural design.

The four samples

falling outside

of the predicted boundaries for ideal circular and elliptical

beams included two herbaceous stems (daffodils Narcissus

pseudonarcissus and sedges Carex acutiformis), a tree

branch (Dendropanex arboreus), and a tropical vine (Marcgravia rectiflora). These systems were all characterized by
high flexural stiffness relative to torsional stiffness that is,
they twisted more easily than they bent (Fig. 2).
Twist-to-bend ratios varied dramatically among different
species (Fig. 2), with both the

above.

The twist-to-bend

magnitude. Species within the defined groups occupied similar regions of the mechanospace, implying that the flexural

position for photosynthesis, so self-

Leaf petioles

falling

boundaries reflect possible extremes for biological beams,

based on assumed material values and chosen cross-sec-

that bent easily also twisted easily, while those that

Continuous beam. Stem must maintain upright

Herbaceous stems

points

changed concurrently

upright position for photosynthesis, so self-

Etnier.

shown

ideal
Mil beam. Antennae used for sensory
ution and in some cases, for

Crustacean antennae

The

lines

tions of engineering

minimum

twist-to-bend ratio for

falls

maximum

(52.9) and the

(1.4) occurring in tropical vines.

all

The average

groups combined was

between the predicted values for

ideal

7.2,

beams

which

that are

circular or elliptical in cross-sectional shape.

When

the data

were normalized for

size (Fig. 3), the

observed distribution changed slightly. In particular, the

relative position of the antennae, horsetail rushes, and gorSize-normalized mechanospace
Size greatly influences the stiffness of a biological
beam, both in twisting and in bending. The structural
variables,

and

J.

are both proportional to radius to the

fourth power; thus, small increases in size will greatly

increase beam stiffness, regardless of cross-sectional
shape or material composition. Values of El and GJ were

normalized for size by dividing by radius to the fourth

power to determine if size alone was driving the observed
patterns. These values were then mapped into a sizenormalized mechanospace (Fig. 3). This normalization
accounts for size alone, rather than equating to calculations of

or

for a given

beam.

gonian corals shifted up and to the right relative to the other

beams. This result suggests that these structures are all
relatively stiff for their size.

changed

While the position of groups

were stiffer in bending were

slightly, structures that

also stiffer in torsion, regardless of their overall size.

Discussion

The close resemblance in the distribution of ideal and

biological beams suggests that both are limited by the
interdependence between the material properties,

and between the geometric properties,

and

and G,

Yet, keep in
theory, which are violated
/

J.

the assumptions of beam

almost universally by biological beams. The theoretical
relationships apply only to beams consisting of a single.

mind

TWISTING AND BENDING BIOLOGICAL BEAMS

Circular

4J

5/1

0.0

i
"o

Elliptical

beam

= 0.50

>

5?

4*

beam

41

o
DC

2.
Twist-to-bend ratios for the nine groups. The horizontal lines indicate the predicted values for
beams with circular (El/GJ = 1.00. v = 0) and elliptical (EI/GJ = 12.75. v = 0.50) cross sections. The
beams are arranged by size within each group, with the smallest diameter beam to the left. Standard deviations
for the twist-to-bend ratios, when available, are reported in the appendix.

Figure

ideal

isotropic material (Roark,

more

1943). Biological materials are

typically anisotropic, and values for Poisson's ratio

can vary greatly from the theoretical expectations (Vincent,

1990). More importantly, biological beams are almost al-

ways composite

structures built of multiple materials that

mechanical properties. Thus, it is not

El and GJ would be highly interdependent in

differ greatly in their

obvious that

biological beams, particularly in the beams that deviate

most from the theoretical assumptions, such as the jointed
crinoid arms or crustacean antennae.

The four samples

falling outside of the predicted

aries are all characterized

by high flexural

bound-

stiffness relative

that is, they will twist more easily

than bend (Fig. 2). The two herbaceous stems have flowers
or seed heads that extend perpendicular to the long axis of
to torsional stiffness

the stem, potentially causing the stem to bend or twist when

the wind blows. Rather than resisting this load with a high

torsional stiffness, daffodils (Narcissus pseudonarcissus)

and sedges (Carex acntifonnis) have a low torsional stiffness, which allows them to twist in the wind. Daffodils
reduce flow-induced drag with

this action (Etnier

and Vo-

and a similar function has been suggested for

sedges (Ennos, 1993). The functional relevance of the high
ratios of flexural stiffness to torsional stiffness in the tree
gel, 2000),

trunk and vine has not been explored.

Conservative estimates of size and shape were

many

made

for

of the tree branches, vines, and gorgonian corals

(appendix).

Beam

size affects

El and

GJ

equally; thus,

errors in the size estimates will not affect the position of

data points relative to the predicted boundaries (Fig. 4).

Rather, an increase or decrease in diameter will cause these
points to shift parallel to the boundaries. In contrast, shape
estimates will differentially affect El and GJ, causing data
points to shift relative to the predicted boundaries (Fig. 4).

Although branches and vines are fairly circular in cross

assumption may not be valid for the gorgonian
corals, whose cross-sectional shape can be circular, elliptisection, this

or even triangular (Jeyasuria and Lewis, 1987). The

assumption of a circular cross section potentially underestimates the range of values seen in the gorgonian corals. For
cal,

example,

assumed

if

to

the cross-sectional shape of the gorgonians

be a

4:

is

ellipse rather than circular, then all of

42

S.

1Z.UU

A.

ETNIER

TWISTING AND BENDING BIOLOGICAL BEAMS

43

small circular
r

= 0.002

beam

O large circular beam

r

= 0.004

X elliptical beam
rmax=

0.004 m,

rmin = 0.001

-3

-2

-1

log Torsional Stiffness

(Nm

Figure 4. Changes in the assumptions of size and shape of beams will affect their distribution in the
and were based on an
mechanospace. The small filled circles are the gorgonian values graphed in Figure
assumed radius of 0.002 m. The lines represent the predicted values for ideal beams discussed previously.
1

in radius will affect the magnitude of / and J equally; thus, an increase or decrease in radius will cause
these points to shift parallel to the predictions. For example, increasing the radius from r = 0.002
to r = 0.004

Changes

shifts the distribution

J. If the cross-sectional

and

/ is

up and

shape

is

assumed

to

be a

4:

ellipse (r max

calculated about the short axis, the data points

cross-sectional area of the elliptical

do not

reflect

an increase

shape estimates differentially affect / and

0.004 m, r mn = 0.001 m) rather than a circle,

to the right (large circles). In contrast,

move

outside of the upper boundary (stars). Note that the

beams is equivalent to that of the

amount of material, but rather

in the total

small, circular beams; thus, these changes

a

change

in its distribution.

extend the entire length of the beam, but this explanation

is inadequate for the jointed beams, which have no materi-

determined by inherent principles governing the relationships between E and /, as well as G and J. Conversely,
empty areas within the distribution may not be an indication

extending uninterruptedly along their entire length.

similarity in values for flexural stiffness and torsional

of physical impossibility, but of evolutionary history. There

may be an absence of environmental patterns of change

both jointed and continuous systems sug-

causing natural selection for particular combinations of mechanical properties (Raup and Stanley, 1971 ). Alternatively,

chanical

als

The

stiffness

properties

for

gests that these

may

be attributed to materials that

beams may represent

meet the functional need for

tures.

Two

alternative designs to

flexibility in biological struc-

of the jointed systems, horsetails and antennae,

are relatively stiff for their size, suggesting that the presence

of joints does not necessarily equate with increased

flexi-

once an evolutionary pathway has been

canalization

initiated,

phyloge-

future

options for change

within
the mechano(Lauder, 1982). Finally, empty spaces
reflect
rather
than
space may
temporal,
physical, limitations

netic

may

limit

distributed uniformly throughout the

(Raup, 1966). The empty spaces will evenbe

tually
occupied, given enough time. Although the identification of boundaries within this mechanospace may not

1).

reveal

bility.

Neither the ideal beams nor the biological beams are

mechanospace (Fig.
Unoccupied regions of the mechanospace correspond to
beams that bend but do not twist and beams that twist but do
not bend. The distribution within the mechanospace may be

to those areas

their

factors that

ultimate source, the boundaries do identify

may

Patton, 1989).

influence the observed pattern (Lessa and

44

S.

A.

Despite the structural diversity of the samples used in

study, they are merely a subset of the biological

this

possibilities.

n is the

notable limitati

wrapped beams

in the

absence of

>

wrapped with
The fibers
fibers parallel and

pically
al.,

1978).

orthogonally with the

perpendicular to the long axis of the structure, or they
may be in a helical array with fibers running in right- and
left-handed helices around the

long axis. Orthogonal

resistance to twisting, while being rel-

atively stiff in bending

(Wainwright

et al.,

1978). Thus,

beams would potentially fall into the upper lefthand corner of the mechanospace. In contrast, helical
fiber arrays allow pressurized beams to bend smoothly

these

without

kinking,

tions

(Wainwright

these

beams

while

Etnier, S. A. 1999.

resisting

torsional

deforma-

et al.,

1978), potentially positioning

in the lower right of the mechanospace.

The mechanospace presented here

is

a useful approach

Importantly, the mechanospace does not imply that flexifunctional

relevance

in

each system.

should be used as a broadly comparative tool to

highlight systems in which flexibility may be biologically
important. Biological beams that do not follow the basic
Rather,

it

seen in the mechanospace may be of particular

interest to both biologists and engineers, either due to ma-

pattern

terial

or structural innovation.

and S. Vogel. 2000. Reonentation of daffodil (Narcissus:

Amaryllidaceae) flowers in wind: drag reduction and torsional flexibility. Am. J. Bot. 87: 29-32.

am

manuscript, as well as informative

and invaluable discus-

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manuscript was greatly improved by the comments of the

reviewers.

;>s

cantilever

control of local velocities in hydroid colonies. Biol. Bull. 168: 312

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297.

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Steve Wainwright, and Dr. Bill Hoese on early drafts of this

II.

Gallenmuller,

nautical

grateful for the

spinal biomechanics.

experiments and mechanisms of bending resistance.

281-297.

Morgan,

Mammalian

Gal, J. 1993.

fish

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dis-

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has critical

Flexural and torsional stiffness in biological beams:

Etnier, S. A. 2001.

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of mechanical properties. Thus, their inclusion in the
mechanospace may greatly expand the observed distribu-

bility

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Jeyasuria,

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mei .ham- pace. Internally pressur-

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M.

Stanley. 1971.

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Roark. R.
Hill.

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1943.

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Vincent,

J.

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Structural Biomateriuls. Princeton University Press,

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Vogel, S. 1984.
37-44.
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flexibility in sessile

organisms. Am. Zooi 24:

Drag and reconfiguration of broad leaves

40: 941-948.

Twist-to-bend ratios of

Vogel, S. 1995.

in

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E. R.

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Wainwright,

A.

1988.

Weibel, C. R. Taylor, and L. Bolis, eds.

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Axis and Circumference:

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The Cylindrical

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Wainwright,

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R. Dillon. 1969.

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Shark skin:

46

S.

A ETNIER

Appendix
Species (n

57) used

in the

mechanpsp^v

Species for which assumptions were

parenthesis,

when

available

;ilong with their source, diameter, flexural stiffness

iboul their size

and shape are marked with an

(7), torsional stiffness (G/), and the ratio EI/GJ.

asterisk.

The standard deviations

for

EI/GJ are given

in

Reference: Biol. Bull. 205: 47-53. (August 2003)

2003 Marine Biological Laboratory

Extracellular Lipid Droplets in Idiosepius notoides, the

Southern Pygmy Squid

L.

S.

EYSTER* AND

L.

VAN CAMP

M.

School of Biological Sciences, Flinders Universitv, Adelaide, South Australia 5001, Australia

Cephalopod metabolism

Abstract.

Despite evidence of limited lipid metabolism in cephalopods, lipid storage has been reported for the digestive gland,
the only cephalopod organ consisting of more than a few

typically involves car-

bohydrates and proteins, so that the lipid content of the

mantle and all internal organs except the digestive gland is
very low. Despite clear evidence of nonlipoid metabolic
trends in cephalopods, we observed extracellular spheres, or
droplets, in the

cecum and

lected juvenile, male,

digestive gland of newly coland female individuals of Idiosepius

notoides. the southern

pygmy

Idiosepius notoides Berry 1921 is a small sepioid found

beds in southern Australian waters, from Cock-

burn Sound

cecum about

in

ture of these droplets.

3 h

also speculate on the origin,

et ai,

metabolize

or

store

lipids

1975; Storey and Storey, 1983;

O'Dor

capacity

to

was collected by seining over sea39

C,
ppt salinity near the mouth of the Port
South
River, Adelaide,
Australia, on 29 March 2002 (Day
Idiosepius notoides

grass beds

0). All

and Webber. 1986; Moltschaniwskyj and Semmens. 2000).

In 1975, cephalopod metabolism was described as poorly

Ulva

squid were recognized by their small size (about 10

sp.

using dorsal duoadhesive glands

(Norman and

Reid, 2000). Because idiosepiids are morphologically unusual, they historically have been placed with the teuthids

1975). That mystery arises because lipids, efficient energy

storage molecules due to their high per-gram energy content, are typically not abundant in cephalopods (Hochachka
al.,

rear of the body, and by their attachment to seagrasses and

and squid storage substrates were "a well

known mystery in marine biochemistry" (Hochachka et ai,

1975; Castro et ai, 1992: Clarke et

( 1

mm dorsal mantle length), by a pair of rounded fins near the

understood,

et ai,

We

Materials and Methods

Although cephalopods require considerable energy for

rapid movement, growth, and reproduction, they apparently

(Hochachka

Morton Bay. Queens-

function, and fate of the droplets.

Introduction

limited

to

digestive gland of field-collected specimens of /. notoides

subjected to starvation, and explore the possible lipoid na-

after feeding.

have

Western Australia

land (Shepard and Thomas, 1989). We report the existence

and retention of yellow spheres within the cecal lumen and

spheres are lipid and that they derive

from the squid's normal field diet. When newly collected
squid were starved in the laboratory, the droplets disapin the

Boucaud-Camou and Boucher-Rodoni,

1984: Westermann and Schipp. 1998).

et al.,

in seagrass

We conclude that these

7-8 d and then reappeared

The cecum is not reported to be a typical lipid

although cecal cells have been ascribed a va-

1966;

O'Dor

1983;

dense than water, hydrophobic, and sudanophilic, stainSudan III. Sudan IV, and Sudan Black B.

in

site,

(Bidder.

squid. Prior to staining, the

ing positively with

peared

lipid.

storage

riety of functions, including fat absorption in several species

droplets were various shades of yellow and were often large

enough to detect at 7X magnification. The droplets were
less

percent

but are currently classified as sepiids (Berry. 1932; Hylle-

berg and Nateewathana, 1991); despite their cuttlefish

1994).

ances, they are

commonly

called

pygmy "squid" and

alli-

will

be

referred to as squid in this paper.

Received 24 September 2002; accepted 12 June 2003.
*
To whom correspondence should be addressed. Current address: Sci-

Squid

ence Department, Milton Academy, Milton, Massachusetts, 02186. E-mail:

Linda_Eyster@ Milton.edu
Ahhreviation:

ML =

that died

on collection

(/;

2)

were examined on

Live squid were kept unfed in an aquarium conDay

nected to the recirculating seawater system
16 C, 40 ppt
0.

mantle length.

salinity.

47

12 h light:12 h dark cycle) until natural death or

48

L.

S.

EYSTER AND

on Days 4-15. To investigate whether "starved"

sacrifice

squid ate small

organisms

vidually

seawater from the

Before

per second)

total.

measuring 19.3

cm

mg one

re.

sacrifice,

present in the

were videotaped

trmv

recirculating seawaten.
(2.5

>ds

(f.;;

tig

endi

liter

in

indi-

a tank

of unfiltered

at

random) was

about 10X magnification in a small, flat dish for

an attempt to locate droplets prior to anesthesia or
dissection. The dark-pigmented mantle of active, stressed

observed
15

min

at

in

animals often hid droplets. However, during the squids'

we could see through the
mantle tissue and determine droplet location.

occasional flashes of transparency,

At

squid were transferred (in seawater) to a

sacrifice,

freezer for terminal anesthesia,

measured (dorsal mantle

= ML), and then decapitated (Boyle. 1991). (Alcold water may be analgesic rather than anesthetic

length

though

[Boyle, 1991], in this paper the treatment is referred to as

cold anesthesia.) The mantle was cut open to expose but not

We

recorded gender, stage of sexual maturation (based on Lipinski's maturation scale as

damage

internal organs.

interpreted by Moltschaniwskyj,

1995), droplet presence,

and sometimes droplet diameter. Droplets to be tested for

lipid content were obtained by puncturing the cecum. Because

is

it

unknown

if

handling of squid

into smaller droplets (and

we minimized

testing),

we wanted

Sudan

may

break drops

larger drops for stain

handling of squid prior to anesthesia

and during dissection.

Droplets were tested for

lipid content using three stains:

Sudan IV, and Sudan Black B (one stain per

Because these stains have very low water solubilities
III,

squid).

(ranging from <0.1

Sudan IV;

mg/ml for Sudan III to 0.7 mg/ml for

Green, 1990) compared to solubility in ethanol.
were prepared

staining solutions

as saturated solutions in

70% ethanol. Staining solutions were prepared only a few

days before use to avoid the deterioration that can occur in
Sudan/alcohol solutions (Gurr, 1962).

We

tried three

approaches

M.

VAN CAMP

other squid, its movements were less vigorous in the small

observation dish, and it seemed to have a transparent mantle

more

frequently.

feeding, but

it

after feeding.

No

was used on

anesthesia

to cecal droplet staining

third days
Before feeding, the squid had no cecal drops
at

end

the anterior

of the digestive gland, one on the left and one on the right
side. After it caught the live shrimp provided (Hippolyte sp.,

mm

-19
long), the squid was left undisturbed until it
discarded the intact but almost empty exoskeleton 45 min
later. No effort was made to locate oil droplets during
feeding because preliminary work showed that disturbed
squid tended to abandon their prey. We examined the squid
for droplets immediately after its meal, at hourly intervals

for the next 7 h,

were detected

at

and then up

to twice daily until no drops

a magnification of about 30X.
mea-

We

sured the cecal droplets

if

the squid

remained stationary and

was transparent during observations.

To

address the possibility of droplet expulsion,

four immature squid (4.4-6.9

mm ML)

in

we

kept

individual glass

bowls (colorless) until their oil droplets were undetectable.

We used small bowls 100 ml of filtered seawater, 16 C) to
(

we would have

decrease the surface area

to search for oil.

These squid, from a separate collection made in late April

2002 near Noarlunga (south of Adelaide), all had oil drops

when

collected and when placed in the glass bowls. Prior to

use in this experiment they were maintained in an aquarium
connected to the recirculating seawater system and fed
field-collected

mysid shrimp

for

week. After placement

in

individual dishes, these squid were kept without food and

were examined with a dissecting microscope once per day

to determine whether droplets were present in the digestive
system. Prior to the daily cleaning and refilling of the bowls,
the water surface was examined with a dissecting micro-

scope for floating droplets and then was patted with white
paper toweling, which was also examined for droplets.

(one

method per squid). For ceca cut open unsubinerged, stain

was pipetted directly onto the body. For ceca punctured

the day of

had to be used on the second and

but did have two small, equal-sized drops

system).

squid (chosen

L.

Results

submerged, filtered stain was added to the seawater (i.e.,

drops were stained floating on the water surface). Additional

Of the 16 squid collected in March, 11 were males, 3

were juveniles, and 2 were females. Dorsal mantle length
ranged from 6.5 to 16.5 mm. Females tended to be larger

droplets collected from the water surface by patting with

than

strips

of

filter

paper were flooded with stain for about 10

min, followed by ethanol rinses to de-stain the paper. Con-

were conducted using food-grade vegetable oil.

Positive stain for lipid includes yellow-orange for Sudan 111.
orange-red for Sudan IV, ancJ blue-black for Sudan Black B
trol

tests

(Conn. 1961; Gurr, 1962, 1065).

To determine

if

changes

in extracellular
lipid

volume or

seemed

less

was chosen because it

stressed by handling: compared to most of our
7,

Table

mm

mm

mm

juvenile squid.

Droplets were easiest to locate in dissected, fresh squid or

in healthy, stationary squid in transparent phase.

mantle tissue

location could be detected after feeding, we fed one squid

and then repeatedly examined it for droplets. This squid (see

9.7-mm-ML male on Day

males, as previously recorded (Norman and Reid,

1.1
(mean
2000); mantle length was 15.4
SD) in
in
0.9
in males, and 6.8
0.3
females, 8.4

in

Opaque

preserved and moribund squid hid the

droplets. For active squid,

it

was

difficult or

impossible to

obtain accurate droplet counts or measurements.

In the first
nile,

week

after collection,

we examined one juveOf these 12 unfed

nine male, and two female squid.

squid.

had droplets (Table

).

Droplets were not detected

EXTRACELLULAR
Table
'r.

reproductive mutiiritv singe,

stanvd Idiosepius notoides

utjti ilorstil

individuals

collection for extracellular droplets in the

Days
(post-collection)

tminlle length of

examined Days 0-15 post-

i/i^omr

rruc!

LIPID IN

PYGMY SQUID

49

50

L. S.

EYSTER AND

comparisons for cecal droplets beoaise the variable bluerig. la vs. Ib) probably
green hues of the cecal flui
er
altered our color pen.drops of yellow food
;

(almond, olive,

oils

.-getable) all appeared colunuer the same lighting condicollected onto white filter paper

si'

tions.

Unstained

from two Port Riv_j

<

<

orless floating in

,.;iud

were conspicuous

at

10X due

to

on vellow color, while droplets from three

of the four Noarlunga squid looked colorless in the cecum.
their brilliant

!>

Number and

size of droplets varied

among

squid. For

mm

in
example, one squid had a huge cecal drop (-0.9
diameter; Fig. la), and another had dozens of tiny droplets

(Fig.

Ib).

However, on Days 0-7 after collection, most

2-14 droplets with a typical diameter of

starved squid had

0.05-0.2

mm

per droplet.

Evidence supporting the

summarized in Table 2 and

lipid nature of the droplets is

Figure Ic and d. The yellow

were
hydrophobic. typically forming small spheres
droplets
in the cecum. When we cut a submerged cecum, droplets
in

rapidly escaped and floated to the water surface.

the air-water interface, larger drops

On

hitting

"popped" from a sphere

into a thin disk on the water surface. One large drop pipetted
onto a piece of thin brown-paper bag and pressed onto the
1
in
paper using Parafilm left a translucent window
( 1

diameter)

in

the

for

paper
whereas other random fluids

mm

48-h observation period,

from the same squid did not.
a

Floating droplets collected onto filter paper changed from

yellow (before staining) to orange with Sudan III, orangered with Sudan IV, and blue-black with Sudan Black B.
Floating droplets exposed to

Sudan IV changed from yellow

(Fig. Ic) to

orange (Fig. Id).

There was no evidence of feeding

All squid

were accounted

for, so

in any "starved" squid.

no cannibalism occurred.

Two

of the videotaped squid explored the aquarium walls

with their arms for about 10% of the filmed time, and they

sometimes made motions as

if catching something, but

frame-by-frame viewing of those times gave no conclusive
evidence of feeding.

In the squid fed the large shrimp, no cecal droplets were

seen until 3 h after feeding ended (Table 3). Cecal droplet
volume increased on the day of feeding, then decreased,

becoming negligible by

later.

When

cecal drop total

Table 2

Summary
l,

of evidri*:.

Idiosepiux

is

-Hire

of cecal droplets

in

southern

pygmy

L.

M.

VAN CAMP

EXTRACELLULAR

LIPID IN
Table 3

after feeding: the

PYGMY SQUID

51

52

L.

receives and processes fine particles, has

to isolate contents, and

from

food

cecum of

/.

own

sphincter

the primary site of absorption

is

1966: Boucaud-Camou and

O'Dor and Webber, 1986). Perhaps

(Bidder,

fluids

Boucher-Rodoni, 1983;
the

its

EYSTER AND

S.

noioiilcs can absorb dietary lipids, as re-

ported for Octopus. Loligo, Sepia,

and Nautilus (Bidder,

Boucaud-Camou and Boucher-Rodoni, 1983; O'Dor

et al.. 19S4: Westermann and Schipp, 1998).
In this study, we saw extracellular lipid in two organs: the
cecum and the digestive gland. In other cephalopods, the
1966:

only organ with significant lipid (reported as cellular deposits) is the digestive gland; therefore, it has been considered
the only storage site for lipid molecules.

These

lipid

mole-

cules might be oxidized during reproductive maturation or

starvation,

as

other marine organisms (Voogt, 1983;

1971. cited in Blanchier and Boucaud-

in

Boucaud-Camou,
Camou, 1984; Kreuzer, 1984,
Clarke

et al.,

cited in Castro et

al..

1992;

1994). Besides storing lipids, the digestive

may be absorptive in some but not all species (Bidder.

Boucaud-Camou and Boucher-Rodoni, 1983). In fact,

gland
1966;

lipid seen inside digestive gland cells of two squid species

appears to be packaged for expulsion, not storage (Semmens, 1998); because expulsion would be energetically

wasteful and would

moved

lipid is

make the squid denser, perhaps some

cecum rather than expelled from the

to the

organism. Although very few cephalopods produce and

store lipids for buoyancy (Clarke. 1988; O'Dor, 2002).

perhaps the cecal lipid

in

notoides aides

/.

in the

support of

this small but

It

lipid

negatively buoyant cephalopod.

clear from our study that retention of extracellular

is

occurs in

/.

notoides;

it is

less clear

for 7 to 8 days qualifies as "storage."

used

in the literature

whether retention

The term storage

is

without reference to length of reten-

without evidence of retention versus replacement of

molecules, and with the implication of future use. Labeling

tion,

studies

may be

useful in determining if "storage" of extracellular lipid in the cecum and digestive gland of /. notoides
is

due to slow

utilization and/or

slow elimination, either or

new molecules from the

both coupled with the addition of

next meal.

/.

notnides, lumenal oil

a period of

days (7-8

our field-fed squid; 3-4 days in our mysid-fed

not
hours or minutes. This slow disappearance of
squid),
in

lipid suggests

slow absorption, slow expulsion, or both

starved squid.

We

drops

in the field.

rapid vertical

cunnoi
It

is

nil'.-

also

movements

in

out rapid expulsion of large

unknown
in

the

M.

VAN CAMP

vide a quick increase in negative buoyancy during a dive,

and new drops could apparently be formed at the next meal.

Our squid could

not deep dive in our expulsion study

because the water was only a couple of centimeters deep;
the water level in our holding tank was about 10 cm deep,

and the tank contained no predators that might induce

swimming up and down in the water column.
Extracellular lipid droplets have not been previously reported in Idiosepius species. Perhaps the drops are specific
to

/.

notoides, which

is

not a well-researched species. Per-

haps droplet presence and color vary with

lipid content of

the field diet; less fatty prey might not lead to droplets,

some prey might

and

lead to paler droplets that are harder to see.

Perhaps droplets were overlooked in previous studies of /.

notoides because tiny droplets in live squid can resemble the
yellow chromatophores in size and color, or because the
droplets were obscured or extracted by preservatives

alcohol can turn the

We

cecum opaque

(e.g.,

white).

considered using chemical anesthesia on

/.

notoides

and measure droplets in live squid, but this

can move material from organ to organ in the digestive tract
to help us locate

of cephalopods (Bidder, 1966). Decapitation and dissection

can also cause movement of material between organs (Bid-

Although movement or breakage of drops due to

handling cannot be ruled out in our study, we minimized
post-collection handling and confirmed for some squid that
droplets were in the cecum before chilling.
der, 1966).

We

provide preliminary evidence that cecal oil droplets

from food a few hours after consumption. Although droplets were admittedly less obvious in the digesoriginate

tive gland than in the

cecum

at

3-7 h

cecum, the amount of lipid in the

exceeded the amount

after feeding far

detected in the digestive gland before feeding. Thus, the

"new" cecal lipid probably derived from the latest meal.

Cephalopod digestion times (defined as time from food

capture to return of stomach and cecum to "hunger condition") include 15-20 h in Octopus and Sepia and 4-12 h in
Loligo (Bidder. 1966; Lipiriski. 1990). Although cephalopods "digest quickly, convert efficiently, and grow but do
not store energy during their 'live fast, die young' lives"

During our laboratory study with

droplets disappeared slowly, over
days

L.

if this

field.

In

species

makes

the laboratory,

(O'Dor and Webber, 1986), we cannot say with certainty

/. notoides was
empty after 7 d of

that the digestive gland of

starvation.

However, because digestive gland

lipid in

Octo-

pus dropped from 0.3% to 0.06% of body weight with a 6-d

starvation (O'Dor et al.. 1984), 7-d starvation in /. notoides
may deplete most non-membrane lipid from its digestive
gland.

The

large

volume of "new" cecal

coupled with a week

lipid after feeding,

of prior starvation, leads us to conclude

these squid spent most of iheir time sitting on

aquarium
walls or on the undersurface of plastic plants, and move-

duced

ments were mostly horizontal. Anesthetized squid sank to

the bottom, indicating that even with lipid droplets in the

pearance of lipid drops in the squid fed after a 7-d starvation

and the continued absence of drops in all squid starved more

cecum, these squid are negatively buoyant. Rapid expulsion

than 7 d support this conclusion.

This study describes extracellular lipid droplets

of large lipid drops by these small cephalopods might pro-

that the

post-meal cecal lipid seen in /. notoides was profew hours from the recent meal. Both the reap-

in a

in

/.

EXTRACELLULAR

LIPID IN

notoides but leaves unanswered whether these tiny cephalopods expel the material over time, whether they are metabolically capable of obtaining energy

from the

and

lipid.

whether the drops confer a buoyant advantage. The

fact that

lipid droplets did not disappear until the eighth or ninth day
of starvation in field-fed animals suggests that these squid
may use the droplets as an energy source. However, slow

expulsion of the lipid as a dietary waste cannot yet be ruled

out.

PYGMY SQUID

Green,

53

The Sigma-A/drich Handbook of Stains. Dyes, and

Chem. Co.. Milwaukee, WI. 776 pp.

F. J. 1990.

Indicators. Aldrich

Staining Animal Tissues: Practical

1962.

(iurr, E.

and

Theoretical.

London. 631 pp.

Gurr. E. 1965. The Rational Use of Dyes in Biology and General
Siainiii.x Methods. Leonard Hill Ltd.. London. 422 pp.

Leonard

Hill Ltd..

Hochachka,

P. VV., T.

W. Moon,

W.

T. Mustafa, and T.

Metabolic sources of power for mantle muscle of a

squid. Comp. Biochem. Physiol. 52B: 151-158.

Storey. 1975.

fast

swimming

J., and A. Nateewathana. 1991.

Redescription of IJiosepius
p\Xiaeus Steenstrup. 1881 (Cephalopoda: Idiosepiidae), with mention

Hylleberg,

of additional morphological characters. Phuket Mar. Biol. Cent. Res.

Bull. 55:

Acknowledgments

We

Havenhand

are grateful to Jon N.

for use of his

laboratory facilities, to J. Robertson and A. R. Dyer for the

use of their boats, to O. A. Pechenik for typing and data

Cat Darrow for technical help in preparing the

color
digital
plate. Research was supported by a sabbatical
leave and research grant from Milton Academy to L. S.
entry, and

33-42.
Cephalopods: handling, processing and products.

Kreuzer, R. 1984.

to

Eyster.

FAO

Fish. Tech. Pap. 254: 1-108. (Cited in Castro et al.

M.

l.ipinski.

R. 1990.

Photololigo

sp.:

What

M.

Feeding and digestion in cephalopods. Pp. 97-124

in Physiology ofMolhisca. Vol II. K. M. Wilbur and C. M. Y'onge. eds.
Academic Press, New York.

Bidder, A.

1966.

Boucaud-Camou.

Blanchier, B., and E.

1984.

Lipids in the digesiiM.-

gland and the gonad of immature and mature Sepia ofjicianalis (Mollusca:

Cephalopoda). Mar.

Boucaud-Camou,
officianalis.

Camou. 1984.)
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Westermann,

Reference: Biol. Bull. 205: 54-65. (August 2003)

2003 Marine Biological Laboratory

Functional and Biochemical Properties of the

Hemoglobins of the Burrowing Brittle Star Hemipholis
elongata Say (Echinodermata, Ophiuroidea)
ANA BEARDSLEY CHRISTENSEN '*, JAMES M. COLACINO
CELIA BONAVENTURA 3
1

2
,

AND

^Biology Department. Lainar University, PO Box 10037. Beaumont. Texas 77710; 'Department of
Biology, Clemson University. 132 Long Hall, Clemson, South Carolina 29634; and ^Marine Biomedical

Laboratory,

Duke Marine Laboratory. Duke

University,

135 Duke Marine Lab Road.

Beaufort, North Carolina 28516

Abstract:

The burrowing

brittle star

1975; Hajduk, 1992: unpubl. data). The presence of RBCs

imparts a bright red color to the tube feet, which

Hemipholis elongata

WVS

coelomocytes
possesses
hemoglobin-containing
(RBCs) in its water vascular system. The RBCs, which

in the

between the arms

oxygen transport. The hemoglobin of adult animals
has a moderate affinity for oxygen (P 5U = 1 1.4 mm Hg at
pH 8, 20 C, measured in cellulo) and exhibits cooperativity
(Hill coefficient > 1.7). The hemoglobin of juveniles has a
= 2.3 minHg at pH 8.0. 20 C) and also
higher affinity (/%,

guishes H. elongata from other burrowing ophiuroids (fam-

(Say)

and body, are thought to play a

circulate
role in

exhibits cooperativity.

The oxygen-binding

are relatively insensitive to

hemoglobin
and hydrogen

and the

properties of the

same

WVS

into the water

are circulated throughout the

body

transports

column

oxygen from arms extended

body parts (Beardsley and

to buried

H. elongata

is

often found in the lower intertidal zone in

protected, low-energy areas of the southeast coast of the

United States (Hendler et a!.. 1995), and the sediments it
inhabits are often soft, poorly oxygenated and

hydrogen

may

contain

(Windom and

sulfide

The

1982; unpubl. data).

Kendall, 1979; Camargo,

distribution of H. elongata is

sporadic, but densities in a given area

hemoglobins.

may

be as high as

2000/nr (Valentine. 1991a). The juveniles of H. elongata

often settle out onto the arms of the adults (adult disc

Introduction

diameter 5 to 12

the

burrowing brittle star Hemipholis

elongata Say (Echinodermata, Ophiuroidea) is contained in
anucleate coelomocytes (red blood cells, RBCs) present in

(WVS) (Hajduk and

Received 4 September 2002; accepted 27

*
To
whom correspondence should

RBCs

locations.

Colacino, 1998).

amino acid sequence (about 40 amino acids) of

hemoglobin reveals little homology with holothurian

the water vascular system

distin-

series of

globin in the

pH, temperature,

partial

The hemoglobin of

WVS

fluid of the

in the

and readily

synchronous contractions of the tube feet

(Beardsley and Colacino, 1998). H. elongata does not ventilate its burrow, and it has been hypothesized that hemo-

by a

ing experiments performed on purified fractions indicate

that the native hemoglobin is in the form of homopolymers.

adult

Amphiuridae) occurring

ily

sulfide. Adult hemoglobin is a heterogeneous

mixture composed of three major fractions. The combined
results of electrospray mass spectrometry and oxygen-bind-

WVS,

are external projections of the

May
be

crawl

burrow

Cosgrove,

2003.
addressed.

down

mm)

(Mortensen, 1920; Valentine, 199 la,

adult, and grow in the

which they can establish
their own burrows. Recently settled juveniles (disc diameter > 0.48 mm) also possess RBCs. Although juveniles
smaller than 0.43 mm apparently do not have RBCs (unb),

the

until they

arms of the

reach a size

publ. data), the stage at

E-mail:

at

which they

start

producing

RBCs

is

unknown.

christenubls hal.lamar.edu
1

List

of abbreviations: RBCs. red blood

cells;

WVS.

Only three of the approximately 2000 species of

water vascular

stars

system.

54

possess hemoglobin;

Ophiactis

virens

brittle

(Foettinger,

HEMOGLOBINS OF A BURROWING BRITTLE STAR

1880: Cuenot,

1891), Hemipholis elimgata (Hajduk and

Cosgrove, 1975; Heatwole. 1981; Beardsley et ai. 1993),

and Ophiactis simplex (Christensen. 1998). Of these three,

55

remnants and debris. The hemolysate was diluted with

appropriate buffer to an absorbance of 0.4 to 0.5 OD units
cell

at

540

nm

to

minimize photometric error (van Assendelft.

only H. elongata burrows; the other species inhabit the

fouling communities of rock jetties and pilings. As for the

1970).

functional and biochemical properties of ophiuroid hemoglobins, very little has been reported in the literature

spectrum of the solution was recorded

(Hajduk and Cosgrove, 1975; Beardsley et al.. 1993: Christensen. 1998; Weber and Vinogradov, 2001). Hajduk and

Cosgrove (1975) reported

that the

hemoglobin of H. elon-

= 9 mmHg) and is
gata has a high affinity for oxygen (P
of
five
components separable by acrylamide gel
composed

also reported

They

electrophoresis.

two components with

Da separable by

molecular weights of 19,000 and 23,000

SDS

gel electrophoresis.

rians, the other

The hemoglobins of

the holothu-

group of extant echinoderms possessing the

For determination of

total

hemoglobin, the absorption

in a

Beckman DU-65

The

hemoglobin concentration, as
spectrophotometer.
heme, was calculated using the Beer-Lambert law and the
extinction coefficient for human hemoglobin at 542 nm (van

was multiplied by the

sample volume to give total hemoglobin (as heme) in
millimoles, and then divided by animal wet weight to obtain
total hemoglobin per gram of wet weight.
Hemolysates used in the oxygen-binding equilibria and
Assendelft, 1970). This concentration
total

the sulfide sensitivity experiments were prepared as above,

but with RBCs taken only from excised arms with the aim

tigated (Terwilliger and Terwilliger, 1988 [review]; Suzuki.

1989; Mauri et al.. 1991; McDonald et ai. 1992: Baker and

of avoiding potential interfering effects due to enzymes

released from the gut and gonads. Such material may have
increased the formation of methemoglobin in the whole

Terwilliger. 1993; Kitto et ai. 1998).

animal hemolysates (see Discussion for methemoglobin

iron-containing pigment, have been

more thoroughly

inves-

the present study was to characterize the

biochemical, and structural properties of the

The goal of
functional,

hemoglobins of Hemipholis elongata in more detail. Comparisons were made with the findings of Hajduk and Cosgrove (1975), and the functional and structural properties
observed in this study were compared to those of the holothurian hemoglobins.

and care of animals

Animals were collected with a shovel and sieve during

tide from Johnson Creek. Hunting Island. South Carolina. Animals were transported to the laboratory and kept at

low

room temperature (24 C)

in

an aquarium containing sedi-

ments from the collection

site

and aerated natural seawater.

All experiments using

ducted within 6 weeks of collection.

noted,

all

RBCs were

whole animals or

con-

Unless otherwise

experiments used hemoglobin or

Intracellular

RBCs

heme concentration

collected

from an animal

in isotonic filtered

resuspended
at 20 C with 50

as described

above were

seawater buffered to

pH

8.0

mM TRIS. A few drops of a suspension of

polystyrene microbeads (Poly sciences. Inc.) of 5.85 /u.m

0.13 jam diameter were added to the cell suspension. A
small drop of this mixture was placed on a glass microscope

Materials and Methods

Collection

ef-

fects).

RBCs from

adult

slide, and a glass coverslip was placed on top of the drop.

Excess water was wicked away with a tissue until the
coverslip was resting on the microbeads. and the cells were

flattened between the coverslip and the slide. This technique

serves to set the pathlength for the subsequent absorbance
measurements (Colacino and Kraus. 1984). The slide was

then placed on the stage of the microspectrophotometer

(Mangum et al.. 1989). The light transmission spectrum

through an area of the slide containing only buffer was

collected as a reference. Cells were picked at random on the

and the transmission spectrum was recorded through

The absorbance at 540 nm. 560 nm, and 577 nm was
computed from light transmission data. The intracellular
hemoglobin concentration was calculated as heme concen-

animals.

slide,

each.

Preparation of hemolysates and determination of total

hemoglobin per animal

tration

RBCs were

extracted by cutting the animal into small

fragments in a small quantity of buffered seawater (50
TRIS. pH 8.0 at 20 C) and then rinsing the fragments until

mM

no red color was

The body fragments were removed

mixture was placed on ice. The cells

visible.

and the cell/buffer

were washed three times

the supernatant

thawed
in

in fresh buffer. After a final

wash,

was removed and the cell pellet frozen and

The thawed pellet was resuspended

using the Beer-Lambert

coefficients for

Law and

human hemoglobin

at

the

the

extinction

chosen wave-

lengths (van Assendelft, 1970). Concentrations calculated at

were averaged for each cell. A total of

were measured (12 cells on each of four slides

the three wavelengths

48

cells

prepared from the same cell suspension).

Separation of hemoglobins

to lyse the cells.

about

ml of 50

mM TRIS,

water and centrifuged

at

pH 8 at 20 C in distilled
14.000 X g for 5 min to remove

The presence of multiple hemoglobins was determined

using

Pharmacia

fast

protein

liquid

chromatography

56

A. B.

CHRISTENSEN ET

AL.

(FPLC) system. Crude hemolysates were collected as previously described and resuspended in 50 mM TRIS, pH 8.0,
at 20 C. Hemolysates IP n 7-10 individuals were pooled

at

or hemoglobin collected from

due to the small qu;iv
d samples were equilibrated with 50
each animal. The p(
TRIS. pH N.<
y
dialysis. The hemolysates were then
loaded onto a D!AE Q Sepharose Hi-Load 2610 column,

tics

60-ml column solume. The resin had been equilibrated with

four volumes of 50 mM, pH 8.0, TRIS buffer. A linear salt

and 580 nm) using a two-wavelength modification of a

standard analysis (Rossi-Fanelli and Antonini. 1958). The

mM

gradient (50

was used

mM TRIS

for elution.

to

The

M NaCl

0.075

Fractions were collected at a rate

absorbance was read

at

mM

+ 50
TRIS)
volume was 300 ml.
of 6 min per fraction, and
(for protein) and 415 nm

total gradient

280

nm

(hemoglobin peak).

The

showing peak absorbance were electrophoresed on a Pharmacia Phast system using a native gel.
fractions

Samples were run on a 10% acrylamide gel with

Coomassie

electrophoresis buffer and stained with

pH

protein.

in these

To examine

total

of 60 cells taken from 47 animals

experiments.

the equilibrium oxygen-binding characteris-

of the hemoglobin in cellulo, cells were exposed to

to > 150
gases at nine oxygen tensions, ranging from

mmHg

(room

air).

Fractional saturation values were

puted from transmitted

light intensities

com-

(540 nm, 560 nm,

pH on oxygen affinity was determined from oxygen equilibrium experiments conducted at pH 7.0, 8.0, and
9.0. Temperature effects on oxygen binding were determined from oxygen affinity experiments conducted at 10
effect of

C, pH 8.0. and 20 C, pH 8.0. The heat of oxygenation

(AH) was calculated from the van't Hoff equation.

8.3

blue.

Oxygen-binding equilibria

(upper fraction) was used to

sharpen the banding pattern. Samples contained 5-25 /ag of
low-porosity

100 ml/min.

were used

in vitro

stacking gel

Crude hemolysate and human hemoglobin

were

For measurements on crude hemolysates, about 50 /nl of

was placed in the working chamber of the

the hemolysate

electrophoresed as references.

gas slide along with a 0.5-cm piece of monofilament nylon

mesh (105-ju.m mesh opening) (Small Parts, Inc.). A freshly

Determination of molecular weight

prepared hemolysate solution was used in each experiment

because repeated freezing and thawing increased methemo-

The molecular weights of

the purified hemoglobin fracwere determined by electrospray ionization mass spectrometry. Samples were prepared by the method described

tions

by Stevens et al. (1994). Measurements were made on a

Fissons-VG BIO-Q triple quadrupole mass spectrometer
equipped with a pneumatically assisted electrospray ionization source operating at atmospheric pressure (supplied by

VG

Biotech. Altrincham. UK).

Hemoglobin spectra and oxygen-binding

equilibria in

A small portion (< 5 mm) of the distal end of an arm was

excised from an unanesthetized animal and rinsed in buffer
remove any adhering mud. The arm

tip was placed in a

containing a small amount of
0.45-ju.m filtered isotonic seawater buffered with 50
TRIS of the desired experimental pH (7. 8, or 9). The tube

large

depression

measurements. Oxygen-binding equiwere measured as previously described for in cellulo

measurements at 20 C. Measurements on purified FPLC
for light transmission
libria

fractions
at

pH

were made using standard tonometric techniques

20 C.

7 at

Oxygen-binding equilibria of juvenile hemoglobin

cellulo

to

globin formation (see Discussion for methemoglobin effects). The nylon mesh served to ensure a stable pathlength

slide

mM

Oxygen-binding equilibria for hemoglobins of juvenile

H. elongata were measured on both isolated cells and intact
animals. For the isolated-cell measurements, cells were collected

from individuals of disc diameter

cells.

was loaded onto

tained

(Mangum

et al.,

1989).

The

slide

was main-

constant temperature (20

0.5 C) with water
from a refrigerated water bath (Forma Scientific).

at a

pumped
The internal gas tension of the slide was controlled by a
gas-mixing flowmeter (Cameron Instrument Co.). The gases
were humidified and brought

to

experimental temperature
before flowing through the sample chamber of the gas slide

mm

(//

5)

by

mM

gas slide (Colacino and Kraus, 1984). The gas slide was
placed on the microscope stage of a diode array microspec-

trophotometer

excising a small portion of an arm in a small quantity of

TRIS. pH 8.0, at 20 C. The cells were
buffer, 50

were manually stimulated to contract, forcing the RBCs

out of the cut end of the radial canal. About 50 p.\ of the
buffer/RBC mixture was transferred to a specially designed
feet

<

slide, and the oxygen binding was

measured by the method previously described for the adult

loaded onto the gas

For the whole-animal measurements, an

intact juvenile

The juvenile was anesthetized

it
in
a
small
quantity of buffer (< 200 /nl)
by placing
7% MgCU. The individual was
a
few
of
containing
drops
then placed centrally in the gas slide chamber in a small
the gas slide.

drop of buffer/MgCK. Hemoglobin spectra were taken

through a tube foot with RBCs in it. Oxygen-binding experiments were conducted as before. Four individuals were

measured

in this

manner.

HEMOGLOBINS OF A BURROWING BRITTLE STAR

Hemoglobin

The
libria

Aniino acid seanence of hemoglobin fractions

sensitivity to sulfide

effects of hydrogen sulfide on oxygen-binding equiwere examined using hemolysates prepared as de-

scribed earlier (hemoglobin 50

mA/ TRIS

in distilled

water,

20 C, adjusted to give an absorbance reading of

0.4-0.5 at 540 nm in an 0.5-cm cuvette). Four milliliters of
8.0 at

pH

were placed into each of two glass tonometers

this solution

equipped with 0.5-cm pathlength cuvettes. The initial oxygenated spectra were recorded from 650 nm to 400 nm on a

DU

65 spectrophotometer. The samples were

deoxygenated with 99.999% N 2 and a deoxygenated specNa 2 S solution was
trum was taken. Then 100 ju,l of 10

Beckman

57

mM

amino acid sequences of each purified hemoglobin

were determined by the automated Edman method
using a Porton Instruments PI 2090 integrated micro-sequencing system. The amino acids were identified by visual
inspection of hardcopy plots, using retention times from a
Partial

fraction

standard of

PTH amino

acids.

Proteins were not digested with proteases prior to se-

quencing. Each analysis used 150-200 pmol of protein.

and 2 were processed for 40 cycles,
Samples of fractions
1

which time it became difficult to distinguish the amino

acid peaks from the background noise. Fraction 3 was run

at

one tonometer with a syringe; 100 p.] of

deoxygenated buffer was added to the control tonometer.

for 21 cycles.

The spectrum was again recorded. Oxygen was gradually

introduced in a stepwise fashion by the injection of room air
samples after an equal volume had been removed from the

protein sequences using the

injected

into

The

partial

was compared to other

MacVector sequence analysis

sequence for fraction

software (Oxford Molecular Group

PLC) and

the Entrez

database (National Center for Biotechnology Information).

tonometers, and the spectra were measured after a 10-min

equilibration. This experiment was repeated three times

Results

with freshly prepared hemolysates pooled from three to five

individuals.

Total hemoglobin per individual

Stopped-flow measurements of ligand kinetics

The average wet weight of the adult animals used for this
0.16 g (mean
measurement was 0.39 g
SD, n = 9).

Kinetics of the hemoglobin-oxygen reaction were estimated using the stopped-flow technique on the crude cell

mM

HEPES. pH 7, at 20 C;
hemolysates suspended in 50
the crude hemolysates represent pooled samples. Measurements were made for the O 2 "off reaction (dissociation)
and the

CO

CO

"on" reaction (association). The

was also examined using flash photolysis. The presence

of modulator effects was determined by performing the
in the

figure translates

0.35% of

into

the total

body mass

ac-

counted for by the hemoelobin.

"on" reac-

tion

above reactions

4.4 X
These individuals contained 8.5 X 10~ mmol
5
as
heme.
10~ mmol of hemoglobin, measured
Using a
mass
of
16,000 Da, this
hemoglobin subunit molecular

Intracellnlar hemoglobin concentration

and estimation

of

hematocrit

presence of ATP.

RBC

Gibson-Durrum
Durrum model
13000 light source and monochromator and Durrum model
110 stopped-flow instrument with pneumatic drive. The

hemoglobin concentration, as heme, was

SD, n = 48 cells). Using a mean
cell diameter of 9 /LUTI (Hajduk and Cosgrove, 1975; Heatwole, 1981; unpubl. data) and an intracellular hemoglobin

was determined by reacting oxyA;,,,,

from
crude
hemolysates, with sodium dithiohemoglobin,
nite (Na 2 S 2 O 4 (about 0.5%). The time course of the reaction
was monitored by measuring transmitted light

concentration of 19.5

intensities.

equal to 4.3

Experiments

were

performed

on

stopped-flow apparatus that consists of a

dissociation constant.

Light intensity data were collected by a

acquisition,

storage,

and

retrieval

DASAR

system with a

data

DW-2

4052 computer. Initial data analyses

were carried out with the ASYST program (Macmillan
Software Co.) prior to curve-fitting analysis by a nonlinear

The

5.0

19.5

X 10~ 7
If
it

mM (mean

mm

an animal has a

total

has approximately

volume of

mM,

the

volume of a

and contains 7.4 X 10"

of 8.5

10

X 10~ 5

cells.

single cell

is

3.8

12

mmol hemoglobin.
mmol of hemoglobin,

The

total cell

volume

is

mm3 and for an animal with a total WVS

18.1 mm (Beardsley and Colacino. 1994). the

fraction of the

WVS

taken up by cells

is

0.24.

interface to a Tektronix

program (Johnson et at, 1981

The carbon monoxide (CO) association

least-squares

).

reaction

was

monitored by the stopped-flow technique and flash photol-

Separation of the hemoglobins

The hemoglobin is a heterogeneous mixture. Separation

of oxygenated crude cell hemolysates by DEAE Q Sepharose FPLC resulted in three fractions that absorb at 280 and

nm

Native gel electrophoresis of the crude

five bands, two of the major bands

Flash photolysis was performed with dual fast extinguishing (approximately 30 /us) flash tubes and a Xenon

415

Corp. model B micropulser. The subsequent association of

CO and the hemoglobin was then monitored as before.

corresponding to FPLC fractions and 2, a third major band

of uncertain identity, and two minor bands (Fig. 2). Lack of

ysis.

(Fig.

1).

hemolysates yielded

A. B.

CHRISTENSEN ET

AL.

The P 50 values measured on

FPLC

the

fractions are lower

than those measured on the crude hemolysates and

lulo. P 50 at pH 7.0 at 20 C for fraction 1 is 6. 1

in cel-

mmHg

mmHg.

due

to the formation of

methemoglobin

The

Hill

numbers

Values for
Cliromatogram of crude hemolysates from Ht'mipholis elun-

1.

Figure

FPLC

gata, separated by

achieved using a

volume.

DEAE Q

ion exchange chromatography. Separation

Sepharose Hi-Load 2610 column. 60-ml column

Tris to 0.75
was established from 50

mM Tris;

absorbance

at

280

gradient volume

nm

mM

linear salt gradient

NaCl + 50

was

was 300

The

ml.

profile represents

and 415 nm.

P5{) measured

fractions

and 2

).

at the three

are significantly

pHs

from one another (Student's

different

(see Discussion).

FPLC

for the purified

are greater (about 1.8) (Table

and

These low values may be

that for fraction 2 is 2.5

test,

P =

0.05)

increases slightly as pH decreases. This is opposite to the usual pH effect. The slope of
the Bohr plot is 0.072. The temperature dependence of the

(Table

oxygen

affinity is small

mmHg

at

).

Oxygen

20

C).

affinity

(P S()

mmHg at

8.9

10

The heat of oxygenation (AH)

v.v.

is

1.4

-4.1

kcal/mol.
in lane 5

banding

(FPLC

fraction 3)

attributed to

is

low

concentration applied to the gel.

As
it

the sample placed on the

unknown

is

if all

Oxygen-binding equilibria of juvenile hemoglobin

column was

FPLC

of the

a pooled sample,
fractions are found in a

single individual or in separate individuals.

However,

the

was performed on two occasions using animals

from different collections, and both experiments yielded the
same results.
separation

Molecular weight of hemoglobin subunits

The technique used to determine molecular weight, electrospray mass spectrometry, breaks polymeric hemoglobins
into monomeric subunits and causes the heme to dissociate
and 2
from the protein. The signals for FPLC fractions
indicated that each was composed of a single protein and a
heme peak. The molecular weights of the subunits were as

The hemoglobin of juveniles

mm)
4.0

(disc diameter 0.48 to 0.8

has a higher affinity (P ?(l = 2.3

in tube feet of intact animals,

mmHg

mmHg

pH

in cellulo

and

8.0 at 20

C)

oxygen than the adult hemoglobin (P 50 =11.4 mmHg,

disc diameter 5 to 12 mm) (see Table 1). This was true for
for

RBCs and intact animal

apparent P 50 for measurements

both isolated

measurements. The

greater

using intact animals

may be explained by oxygen consumption of the animal.

The Po-, within the tissues is lower than the external Po 2 due
oxygen consumption by the tissues. The difference in Po 2
can lead to an overestimation of the P 50 Even with the
to

follows: fraction
tion 3

616.

16,143.

The

16,080, fraction 2

The hemes

16,1 19.

and frac-

had a molecular weight of

all

differences in the protein weight indicate differ-

amino acid composition for the three subunits.

The mass spectrum generated by the third FPLC fraction
indicated the possibility of more than one subunit. However,

ences

the

in

low concentration of

mass spectrometry

data,

this

sample resulted

making

Oxygen-binding equilibria

in high-noise
conclusion uncertain.

this

and

in cellulo

in vitro

The hemoglobin of Hemipholis elongata has a moderate

= 11.4 mmHg at pH 8.0, 20 C)
affinity for oxygen (P 5I
The P 50 at pH 8.0 is similar to those reported for
(Table
holothurian hemoglobins (Table 1). The Hill numbers (n)
,

12345

).

were greater than

for both in cellulo

and

in vitro

measure-

ments, indicating cooperativity and functional hemoglobin

composed of at least two subunits (a dimer) (Table 1 ). Then
n values are greater for the

2.81) than

difference in

//;

cellulo

measurements

0; =

crude hemolysates (n =
1.91). The
Hill coefficients may be due to concentration

for

the

differences (19.5

mM

/;;

cellulo

v.v.

0.1

Discussion for concentration effects).

mM

in vitro) (see

Native gel eletrophoresis of crude hemolysates and purified

Figure
FPLC fractions of Hemiplmlis elimgata hemoglobin. Samples were run on
a W7c acrylamide gel with pH 8.3 electrophoresis buffer and stained with
2.

blue. A low-porosity stacking gel (upper fraction) was used to

sharpen the banding pattern. Lack of banding in lane 5 is attributed to low
concentration applied to the gel, since mass spectrometry results showed

Coomassie

fraction 3 to contain hemoglobin. Lane

Crude hemolysate; lane 3: FPLC fraction

5:

FPLC

fraction 3.

Human hemoglobin A:
lane 4: FPLC fraction

1;

lane 2:
2: lane

HEMOGLOBINS OF A BURROWING BRITTLE STAR

Table

Oxygen P S( values and

,

Hill

Species

numbers ofechinoderm hemoglobins (mean

SE)

59

60

CHR1STENSEN ET AL

A. B.

mM

holothurian RBCs (12.5

for Sclerodactyla (Thyone)
briareus [Colacino, 1973] and 15.8
for Cucumaria
miniata [from data in Man well. 1959, and Terwilliger and
Read, 1972]). These values are similar to those of human

06

>
00
.2

04

02

00

mM

mM

RBCs

(20
heme) (calculated from Guyton, 1991) and
heme) Vanphoronid (Phoronis architecta) RBCs 14
dergon and Colacino, 1989). If the hemoglobin of//, elon-

mM

gata exists as dimers, this would mean that the intracellular

hemoglobin concentration is 9.8 mM.

-06
-08

-1

The computed hematocrit value of H. elongata

comparable
-1

(0.24)

is

to the tube foot hematocrits of the holothurian

briareus (0.24, by direct measurement) (Colacino, 1973).

These values are greater than the reported hematocrits of the

S.

-14

02

0.0

04

08

06

10

Cucumaria pseudocuand
Paracaudina chilen1984)

perivisceral fluids of the holothurians

rata (0.032) (Roberts et

LogPO, (mmHg)

al.,

(0.015) (Baker and Terwilliger,

sis

those for

(>

humans

1993), but less than

(0.4) (Guyton, 1991)

and other mammals

0.4) (Schmidt-Nielsen, 1990).

Structural properties of the hemoglobin

The hemoglobins of H. elongata are a heterogeneous

mixture of three components, as evidenced by FPLC. Because the samples were pooled from several individuals, it

Figure 3. Hill plots for the oxygen-binding equilibria of Hemipholis

elongate hemoglobin in the absence () and presence of Na 2 S (O).
Hemoglobin is crude hemolysate suspended in 50 mAf Tris. pH 8.0. at 20

is

not

known whether hemoglobin

heterogeneity

is

a normal

characteristic of H. elongata blood or an artifact of

fractions

and 2

by eight amino

differ

acids.

The

sequence for fraction 3 was identical to that of fraction 2.

Neither hemoglobin fraction appears to be blocked at the

three fractions are present within an individual. Hajduk

and Cosgrove (1975) reported only two components separable by gel filtration, one apparently composed of monoall

N-terminus of the globin, unlike the hemoglobins of many

holothurians (Terwilliger and Terwilliger, 1988). Proteins
blocked

at the

N-terminus are resistant

to the

Edman

mers and the other of dimers. The difference

reac-

sequencing (Kitto et ai, 1976). No

digestion with proteases) of the H. elon-

tion used in protein

modification

(e.g.,

fractions

The

intracellular

mM)

is

heme

ile

ser

ala

gly

and hematocrit

F2.

val

ile

ser

ala

F3:

val

ile

ser

lys

thr

leu

ile

asp glu

lys

asn

leu

ala

asp

glu

lys

asn

phe gin

ile

gly

phe gin

val

gly

1 and 2. Because this

sample was a crude hemolysate,
two minor bands may have been due to proteins other

tions
the

trp

ala

pro

val

tyr

ala

gly

asp

trp

phe thr

val

tyr

ser

gly

asp

ser

trp

phe

thr

val

tyr

ser

asn

phe

ala

tyr

pro

ala

asn

phe

ala

tyr

arg

asp

ser

ile

arg

ser

leu

ile

arg

val

asn val phe

thr

val

asp val

thr

glu

Fractions, continued

Fl:
F2:

arg
arg

Figure

Amino

4.

Partial

phe

amino acid sequences

for the three

FPLC

fractions of

Hemipholis elongata hemoglobin.

acids in bold type represent differences in primary sequence;? represents

definitively identified. Fl

fraction

1.

number of

Both the present study and Hajduk and Cosgrove (1975)

obtained five bands by acrylamide gel electrophoresis. Two
of the major bands reported here correspond to FPLC frac-

concentration of Hemipholis elonto the values reported for

val

in
in

DEAE Q Sepharose FPLC (ion exchange) separates

molecules on the basis of charge.

comparable

Fl:

attributable to difference

shape;

Discussion

gata (19.5

may be

separation
techniques. Gel filtration separates on the basis of size and

gata protein was necessary to obtain amino acid sequences.

Intracellular hemoglobin concentrations

sample

mixing. However, the identical results (e.g., same ratio of

fractions) obtained on two separate occasions suggest that

partial

F2 = fraction

2.

F3

fraction 3.

amino acids

that

could not be

HEMOGLOBINS OF A BURROWING BRITTLE STAR

than hemoglobin, found within or associated with the

Due

RBCs.

to the presence of herne at high relative concentration,

structural studies

61

on H. elongata hemoglobin are needed

investigate the nature of the interactions of

its

to

subunits.

two of the major bands from the crude hemolysate were

visible on the gel prior to staining with Coomassie blue. The

Comparison of the amino acid sequence of fraction 1

from H. elongata with sequences reported for the globins of

two minor bands were not

the holothurians Caiidina arenicola (Mauri ct

minor bands do

visible. If the

low concentration, as evidenced

the
faintness
of
the
bands
on the gel. could explain why
by
these fractions were not isolated by FPLC.
represent hemoglobins, the

The molecular masses of

the various fractions (approxi-

McDonald

et al.,

1991;

al..

1992) and Paracaudina chilensis (Suzuki,

1989) reveals little homology. The lack of homology between the ophiuroid and holothurian globins has contributed
to the inability to identify the brittle star globin

gene by

mately 16.000 Da) are comparable to human )3 chain

(15.860 Da) (Dickerson and Geis. 1980) and holothurian

using holothurian primers (Kitto, pers. comm.). Furthermore, no successful primers for the hemoglobin gene have

hemoglobin monomers (17.000-18.000 Da) (Terwilliger

and Terwilliger. 1988). These values are smaller than those

been generated based on the 39 amino acid sequence of the

ophiuroid (Kitto, pers. comm.).

by Hajduk and Cosgrove (1975) (19.000 and

23.000 Da). Differences between the previously reported

Oxygen-binding characteristics of the hemoglobin

reported

weights and those of the present study may be attributed to

differences in techniques. Mangum (1992) remarked that

many of

the early studies on holothurian

hemoglobin reported similarly large molecular weights that were later

found to be smaller (-17,000 Da). She attributed these
differences to refinement of molecular techniques.

The cooperative binding of oxygen, both in cellulo and in

vitro (Hill number > 1), indicates that the functional heexists as a polymer.

moglobin

of the holothurian

Many

hemoglobins are known to exist as dimers (Terwilliger and

Terwilliger. 1988) and possibly tetramers (Baker and Terwilliger. 1993).

The

purified fractions,

and

2,

The hemoglobin of H. elongata has

for oxygen, both
this

are able to

typically observed at

than

low concentrations, somewhat

less

are consistent with the formation of cooperative

2.

homodimers. Larger assemblies may form under some circumstances, as evidenced by Hill numbers greater than 3
that

were observed under some conditions

The normally

existing

(e.g., in cellulo).

homopolymers of

several inverte-

brate hemoglobins exhibit cooperativity (Scapharca inaei/iiivul\-i\

[Chiancone

et

ai,

1981; Royer el ai,

1985];

holothurians [Terwilliger and Read. 1972; Bonaventura et

ill..
1976]). although homodimers and homotetramers of

human (and
the
to

other vertebrate) hemoglobins do not. In fact,

mechanism

for cooperativity in invertebrates

is

thought

be different from that utilized by vertebrates (Riggs,

is thought to be due to interactions

1998). Cooperativity

between the E and F helices of the hemoglobin subunits,

first described in the arcid clam Scapharca inaequivalvis
(Royer

et ai,

1985, 1990). This

same association has been

served

inaequivalvis as the residues involved at the crucial

contact points are different for the two species. Further

It is

not certain

fractions exist in the

each

treatment.

This

suggests

at

same
same

mixture

that

is

The

ratio of this

unknown.

The oxygen affinity results of the present study differ

from those reported by Hajduk and Cosgrove 1975)
= 9 mmHg). On the basis of the Hill numbers from the
90

greatly

the

FPLC

hemoglobins represented by the purified

fractions are present within a single cell.

present study, a hemoglobin with a P

should have a P
of about 3 mmHg.

w of 9 mmHg (pH 7.2)

vitro analysis in the present study

tween 8 and 9

oxygen

mmHg

affinities

may

at

pH

7.0.

The

in cellulo

demonstrated a

20

and

P 50

in

be-

C. This difference in

be attributable to the formation of

methemoglobin. The oxygen

affinity

of mammalian hemo-

globin increases with increasing percentages of

methemo-

globin in the sample (Darling and Roughton, 1942)

Several of the holothurian hemoglobins are prone to
oxidization and denaturation at

Read,

1972; Bonaventura

Parkhurst, 1979).

When

et

pH

al.,

7.0 (Terwilliger and

1976;

Steinmeier and

the crude hemolysates of H. elon-

gata were repeatedly frozen and thawed, the P ?0 decreased.

Tests for the presence of methemoglobin showed an in-

amount present in the sample. Freshly prepared

H. elongata hemolysates were typically 5%-l% methemocrease in the

globin as determined by the ferrocyanide method (van As-

one experiment, the methemoglobin fracinitially and rose to 45% by the end. The P so

sendelft, 1970). In

the

S.

within

different

However, Kitto m//. 1998) believe that the

mechanism
in C. arenicola differs from that in
cooperativity
(

FPLC

viduals were examined microspectrophotometrically and litvariation was seen within the measured P 50 values ob-

tion

1995).

of the

tle

described for the innkeeper worm, Urechis caupo (Kolatkar

ct /.il.. 1994) and a sea cucumber. Caiidina arenicola (Mitchell etui..

all

a moderate affinity

separate cell populations, or even within the

animal. However, a large number of cells from many indi-

of H. elon-

oxygen binding, suggesting that both

form cooperative homodimers. The Hill numbers

cellulo and in vitro.

cell, in

gata hemoglobin have only one globin chain type but exhibit cooperativity in

time whether

//;

was 13%

was 3.6 mmHg. This corresponds to

value estimated from the data of Hajduk and Cos-

calculated for this run

5( j

grove (1975).
The formation of methemoglobin may explain the low
P50 of the purified fractions. Although it is not unusual for

62

A. B.

different

hemoglobins within an individual

to

CHRISTENSEN ET

have different

P50

of the mixture usually

binding affinities, the apparent
oarate fractions, not above them.
lies between those of the

AL.

very small and may not have any adaptive

under
significance
physiological conditions.
There is not a large temperature dependence, as evi-

however,

is

L \periments on the purified fracmethemogiob.!; rose from 10% to 16% of the total in
fraction 1 and from 32% to 42% in fraction 2. While the

denced by the small change in affinity with a change in

= -4.1 kcal/mol, is
temperature. The heat of oxygenation. AH

large proportion of methemoglobin, particularly in fraction

2, makes comparisons of P 50 difficult, the importance of this

mol

During oxygen-bim

tions,

experiment is the demonstration of the cooperative binding

of homopolymers.

Another explanation for the difference in oxygen affinities may be the presence of intracellular modulators that

would have been removed during the purification of the

fractions. However, many invertebrate hemoglobins, including the holothurian hemoglobins, are insensitive to or1988;
ganic phosphates (Terwilliger and Terwilliger.
Scholnick and Mangum. 1991; Baker and Terwilliger,
1993).

The

rate constant for the

oxygen "off reaction of H.

elongata crude hemolysates exhibited a small, but significant, increase in the presence of ATP.

The differences
cellule)

and

apparent cooperativity between the in

vitro measurements may be due to concen-

in

in

tration effects. Dilute preparations

may be

less

aggregated

than concentrated ones, with the consequence that the Hill

coefficient increases due to subunit interactions as the concentration of hemoglobin increases. This
the fact that the

hemoglobins of

may account for

worms were

capitellid

et at.,

1992).

Other than those reported here, no data on ligand-binding

kinetics are available for the brittle star hemoglobins.

The

smaller of the two dissociation rate constants for H. elon-

(40

s~',
gata hemoglobin is similar to those of human
Antonini and Brunori. 1971) and holothurian hemoglobin

briareus, 4.8 s~' [Colacino, 1973]; Thyonella

(S.

3.6 to 8 s~
the

'

[Steinmeier and Parkhurst, 1979]).

two dissociation

largest reported.

elongata
briareus.
4

is

gemmata,
The larger of

rate constants (134.6 s~') is

CO

The

among

the

association rate constant of H.

between those of holothurian hemoglobin

~10 3

'

's

[Colacino, 1973];

T.

gemmata, 10

(S.
3

to

1979]) and human

5
X
A/~'s~'
and
10
Brunori, 1971]).
(30
[Antonini
hemoglobins
Most hemoglobins exhibit a decrease in oxygen affinity
as the pH decreases (Weber, 1980). It is generally accepted

10

M~'s~' [Steinmeier and

8 kcal/mol (Antonini and Brunori, 1971; Colacino,

to

The possession of

1973).

Parkhurst,

most holothurian hemoglobins are insensitive to pH

(Terwilliger and Terwilliger, 1988). A weak pH dependence
that

ism, because

it

would make

relatively insensitive to the

it

that

large temperature changes

can occur

in the intertidal

environment (Cochran and Burnett, 1996; unpubl.

The high

data).

affinity of the

hemoglobin of juvenile brittle

stars is clearly different from that of the adults. This functional difference could be adaptive in their normal habitat.

The planktonic
arms of the
into the

the

The juveniles then crawl down

burrow of the

burrow

mm)

larvae often settle out onto the extended

adults.

adult,

(pers. obs).

do not extend

the

arm

where they move around within

Small juveniles (disc diameter < 1

arms into the water column as the

their

must obtain oxygen from the

burrow environment or from the adult. The measured Po 2 of
adults do; therefore, they

the

burrow water

FOXY

very low (undetectable with

is

system [Ocean Optics, Inc.]; unpubl. data), but some oxygen must escape into the environment from the adult, as
evidenced by the

fact that the

sediments lining the burrow

The possession of high-affinity hemoglobin by

the juveniles would aid them in the acquisition of oxygen
from this low-oxygen environment. The switch from highaffinity

hemoglobin

in

the adult has

RBCs

is

affinity

in

many

hemoglobin occurs is not known. Also unwhether the hemoglobin present in the juvenile
one of the hemoglobins found in the adults or an

is

Due

entirely different one.

to the small size of the juveniles

and the small numbers collected, insufficient hemoglobin

has been isolated to make comparisons with adult hemoglobin by gel electrophoresis.

The hemoglobins of H. elongata have a higher affinity for

oxygen than those of another ophiuroid species. Ophiactis
simplex (Christensen. 1998) (see Table 1). The differences
in the

ences

oxygen-binding properties could be related to differH. elongata burrows in anoxic mud,

in their lifestyles.

ensis (Baker and Terwilliger, 1993) and

been documented

low-affinity

known

roids.

Molpadia arenicola
The hemoglobin of H. elongata,
however, shows a slight increase in its oxygen affinity, both
in cellulo and in vitro, with a decrease in pH (Table
). The
magnitude of the Bohr shift of H. elongata hemoglobin.

lower

animals (Barcroft, 1935; Riggs, 1951). The time in development in H. elongata when the switch from high-affinity to

does not ventilate

et ai, 1976).

in the fetus/juvenile to

hemoglobin

(normal direction) on the oxygen affinity of hemoglobin has

only been reported for two holothurians, Paracauding chil-

(Bonaventura

may

perature dependence

hemoglobin with a small tem-

be adaptive to an intertidal organ-

are oxidized.

reported to have greater cooperativity in cellulo than in vitro

(Mangum

6 kcal/

smaller than that reported for other hemoglobins,

known

its

burrow, and lacks genital bursae,

to serve as sites of gas

exchange in ophiuand
commonly occurs in
epibenthic
with
rock
communities
associated
jetties and wharf
fouling
environments
The
levels
in
these
may not be
pilings.
oxygen
structures

O. simplex

is

mud. The function of hemoglobin

not known, but is under investigation.

as limiting as in the

simplex

is

in O.

63

HEMOGLOBINS OF A BURROWING BRITTLE STAR

sillfide

Effects of

Vertebrate and

many

invertebrate hemoglobins bind with

form sulfhemoglobin.

sulfide to

Acknowledgments

on oxygen binding

In

human hemoglobin A,

the binding of sulfide takes place at the

iron,

and

Carrico

effectively irreversible (Berzofsky et

is

et

heme. but not

til..

1978). This also decreases

oxygen

cil.,

Barry Kitto and Pei

Thomas

and discussions on

1971;

for their work in trying to identify the brittle star hemoglobin gene; Robert Cashon for his analysis of the hemoglobin

affinity so

much
hemoglobin functionless under normal conditions (Cameo et ai. 1978). Many invertebrate
sulfide

this

thank the following people for their contributions to

research: Ed Ruppert for his aid in animal collections

to the

as to render the

hemoglobins show reversible binding with

We

(Arp and

Childress. 1983: Childress et ai, 1984; Doeller et ai, 1988;

mass spectromeof
the protein sework; Ellen Beedle for operation

kinetics;
try

brittle stars;

Bob Stevens

for the electrospray

their help in
quencer; Gerald Godette and Shirley Tesh for
and
oxygen binding of
purification of the hemoglobins

many cases the binding of sulfide is

it harbors endosymbionts that
because
organism
source
sulfide
as
an
(Felbeck, 1983; Hand
energy
require
and Somero. 1983; Powell and Somero. 1986). No such

on H.
purified fractions; Steve Stancyk for his discussions
in
DAPI
for
his
staining and
help
elongata: John Wourms

endosymbionts have been observed

C; William C. Bridges for his help in the statistical analfor their

ysis; and Jim Schindler and Stuart Mellinchamp

Somero

et ai, 1989). In

vital to the

in H.

elongata (unpubl.

data.)

No

abnormal spectral changes were observed during the

oxygen equilibrium experiments on H. elongata hemoglo-

fluorescence microscopy to look for the presence of nuclei

in the RBCs; Will Johnson for his measurements made at 10

and use of the Coulter Counter

instruction

RBC

present.

However, due

to the nature of the

Literature Cited

hemo-

any enzymes present would be greatly dithus decreasing their efficiency. The lack of abnormal

lysate solution,
luted,

to
spectra points toward a hemoglobin that is insensitive
sulfide. The hemoglobins of the phoronid Phoronis arclii-

tecta (Vandergon and Colacino, 1991) and the polychaete

Abarenicola affinis (Wells and Pankhurst. 1980) also appear
to be insensitive to sulfide. If sulfide does bind to the

Antonini, K., and

alter

oxygen

it

does so

in a

way

that

does not

affinity or the visible absorption spectra.

that live in sulfide-rich

For

environments but do not

organisms
rely on sulfide-requiring symbionts.
adaptive to

it
would be highly
is not diswhose
function
possess hemoglobin

rupted by exposure to sulfide.

M. Brunori.

Hemoglobin and Myoglobin

1971.

in

Their Reaction.', with Ligands: Frontiers of Biology, Vol. 21. North-

Holkind Publishing, Amsterdam. 436 pp.

Sulfide binding by the blood of the
J., and J. J. Childress. 1983.
hydrothermal vent tube worm Riftiu pachyptila. Science 219: 295-297.

Arp, A.

Baker, S. M., and N. B. Terwilliger. 1993. Hemoglobin structure and

function in the rat-tailed sea cucumber. Paracaudina chilensis. Biol.
115-122.

Bull. 185:

Barcroft,

hemoglobin of H. elongata.

determine

size.

bin exposed to sulfide. This indicates that either the hemoglobin is insensitive to sulfide or there are detoxifying

enzymes

to

1935.

J.,

Foetal

respiration.

Proc.

R.

Lomi

Soc.

B.

118:

242-263.
Beardsley, A. M., and J.
and oxygen transport

M. Colacino.

Red blood

1994.

cell circulation

Hemipholis elongata (Ophiuroidea. Echinodermata). Am. Zool. 34: 35A.

Beardsley, A. M., and J. M. Colacino. 1998. System-wide oxygen
in

the burrowing ophiuroid.

transport by the water vascular system of

Hemipholis elongata (Say). Pp. 323-328

in

Echinoderms: San Fran-

Proceedings of the 9th International Echinoderm Conference, R.

Mooi, and M. Telford. eds. Balkema, Rotterdam.
cisco.

Summary

Beardsley, A. M.,

appears that the hemoglobins of Hemipholis elongata

are well suited to the habitat and lifestyle of the animal. H.
It

elongata does not ventilate its burrow, and its aerobic mefluid
tabolism must be supported by circulation of the
and
column
and RBCs between arms exposed to the water

WVS

buried body pans.

at

20

The moderate

C) and cooperativity

/%<>

(Hill

1.4

mmHg, pH

number ^

8.0,

1.7) of the

extract oxygen from the

hemoglobin would allow it to

overlying water column and deliver it to buried body parts
over a wide range of external and internal Po 2 values. The
relative insensitivity of the

perature and

hemoglobins

to

changes

in

tem-

when con-

preserve hemoglobin function

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Reference: Biol. Bull. 205: 66-72. (August 2003)

2003 Marine Biological Laboratory

Early Development and Acquisition of Zooxanthellae

in the Temperate Symbiotic Sea Anemone
Anthopleura ballii (Cocks)
SIMON
'

DAVY

K.

'*

AND JOHN

R.

TURNER 2

of Marine Studies, University' of Plvinouth, Drake Circus, Plvmouth PL4 8AA, UK; and
School of Ocean Sciences, University of Wales - Bangor, Marine Science Laboratories,
Menai Bridge, Anglesey LL59 5EY, UK

Institute
~

The ova of Anthopleura

Abstract.

become

ballii

Hosts

infected

with zooxanthellae (endosymbiotic dinoflagellates) of maternal origin just prior to spawning. After fertilization, the

by invagination

to

form

and

later,

ciliated planulae.

Because

on one side of the ovum

the zooxanthellae are localized

within the blastomeres

at

of the adult

1986; Glynn et ai, 1991), the vast majority

of coral species spawn gametes that lack zooxanthellae

(Babcock

endoderm and hence to the gastrodermal cells

anemone. We propose that maternal inheritance

of zooxanthellae plays an important part

these temperate sea anemones, which live

ses transmission

These include the

which

inherits zooxanthellae

abundant

not immediately obvious in temperate waters,

in nutri-

et

have

et al.,

2000, 2001), soft corals (Benayahu et

1992;

al.,

jellyfish

1988,

(Montgomery

(Manuel, 1988; Turner. 1988; Davy et al., 1996, 1997). The

zooxanthellae harbored by A. ballii belong to the genus
Symbiodinium (Davy et al., 1997), though they have yet to

1996; Muller-Parker and Davy, 2001).

7 August 2002; accepted

May 2003.
Author for correspondence and current address: School of Biological
1

PO Box

cells

The sea anemone Anthopleura ballii (Cocks) is locally

abundant along the southwestern coasts of Europe, where it
is found from intertidal regions to depths of about 25 m

associations between invertebrates and zooxanthellae are

Sciences, Victoria University of Wellington,

New Zealand.

endodermal

been reported for tropical scleractinian corals (Hirose

and Kremer, 1995), but not for temperate corals or sea

anemones.

and seasonal abundance of planktonic food (Davy

1996, 1997; Muller-Parker and Davy, 2001). Indeed,

Davy

symbionts (reviewed by Shick,

Benayahu and Schleyer, 1998), and

which show

temperate latitudes (Turner, 1988;

algal

their eventual restriction to the host's

fluctuations in irradiance, high levels of

Astrangia danae. which

1991; Muller-Parker and Davy, 2001). Moreover, the cellular events leading to the acquisition of zooxanthellae and

nutrients,

Capnella gaboensis,
from the parent colony (Far-

whose ova contain

conservation and recycling of essential nutrients (Muscatine. 1990; Davies, 1992). These nutritional advantages are

Received

soft coral

rant, 1986); the scleractinian coral

ent-poor tropical seas, where the zooxanthellae supply photosynthetically fixed carbon to their hosts and facilitate the

at

symbiofew

identified in only a

spawns zooxanthella-free gametes (Szmant-Froelich et al.,

980); and a small number of sea anemones, the majority of

biotic dinoflagellates (zooxanthellae) are

uncommon

modes have been

cases.

Associations between marine invertebrates and endosym-

a/..

1986).

regions where

Introduction

et al.,

et al..

In contrast to tropical symbioses, for temperate

potential sources of zooxanthellae are scarce.

marked seasonal

et at.,

success of

in the
in

is

Babcock

one end of the embryo

invagination leads to the zooxanthellae being restricted to

the planular

acquire zooxanthellae either by maternal in-

probably the rarer mechanism in the tropics. For

example, while some reef corals inherit their symbionts
(Lewis, 1974; Kojis and Quinn, 1981; Richmond, 1981;
itance

zygotes undergo radial, holoblastic cleavage, and then gastrulate

may

heritance or from the surrounding seawater. Maternal inher-

be subjected to molecular characterization. In this study, we

cellular events from gametogenesis through to

documented

600, Wellington,

planula development in A. ballii. paying particular attention

66

EARLY DEVELOPMENT
to the transmission

and distribution of zooxanthellae within

the host's tissues.

Specimens of the zooxanthellate sea anemone Anthoand 25 in

pleura bnllii were collected, from between

Lough Hyne Marine Nature Reserve,

Eire.

then maintained for up to one year in 30-1

10-15 C. Irradiance of 96

recirculating seawater tanks at

2

/nmol photons m~ s~' was provided on a 12-h

dark regime, and the anemones were fed twice

light:

12-h

weekly with

Anemia

that

are

67
of actiniarians; Larkman,
The cytoplasm was hetero-

characteristic

geneous, dense and granular, and no nuclei were visible

under low power in unstained preparations. When the ova
were optically sectioned by interference contrast micros-

Experimental organisms

Anemones were

rovilli

KALLII

1980), about 23 jum in length.

Materials and Methods

depth, in the

ANTHOPLEURA

IN

copy, the zooxanthellae could be observed in the cytoplasm,

membrane. Moreover, the zooxanthellae

just inside the cell

were concentrated in one hemisphere of the ovum (Fig. IB).

Out of a total of 380 ova examined, only one aposymbiotic
ovum was observed.

Spermatozoa were examined under high power X 1000)

preparation. The head was rectangular, 3.5 /j.m in
length and 2.3 ;am across, with dense cytoplasm and a large,
(

in a live

dark nucleus.

sp. nauplii.

50

No

basal

body was

visible.

The

tail

was about

;u.m long.

Microscopical examination
Fertilization

Spawning of A. bnllii, which is dioecious, was induced

during summer. This was done by exposing anemones to air
for between 3 and 5 h. The expelled gametes were collected
by pipette and maintained in 100-ml sterile flasks containing
seawater at 15 C. Fertilization occurred within
hours and, every second day. the embryos were pipetted into
artificial

new

which also contained artificial seawater. This

ensured
that the only possible source of zooxanprocedure
flasks,

thellae

was

the adult

ful

later

once

daily, for microscopical observation.

search for zooxanthellae

was made, using

care-

interference

contrast microscopy, by optical sectioning at each

developmental stage.
Leitz Dialux 20 microscope with Vario-

orthomat photographic system was employed, and a photographic record of early development was produced. In
addition, cellular events occurring during gametogenesis

were documented, again using interference contrast microscopy. This was made possible by anesthetizing anemones in
equal parts artificial seawater and 7.5% MgCU 6H 2 O for
12 to 24 h, and then teasing gametes out of the
gonads.

usually

occurred

within

3.5

of

spawning, and unfertilized ova disintegrated after about 7 h.

liberating their zooxanthellae. Spermatozoa were still active
at this

stage and

became

inactive after

20-32

h.

Cleavage

About

h after

spawning.

2-,

4-.

8-.

and 16-cell

16-cell stages

shown

in

Fig. 1C-1E; 2-cell stage not shown).

radial,

complete

embryo

(i.e..

Cleavage was equal,

holoblastic). and rapid, dividing the

into a ball of cells (blastomeres).

Due

to

the

of the zooxanthellae, symbionts were

distributed unevenly in the blastomeres, being concentrated in those cells at only one end of the
embryo. The
initial localization

zooxanthellae remained just inside the cell membrane of

each blastomere. Blastulas of 32 cells were observed
after about 5.5 h, and blastulas of 64 cells or more were

apparent after 6

h.

The blastomeres became ever smaller

repeated cleavage, and after 8 h. a coeloblastula

consisting of many cells and one cell layer was formed

Results

Gametogenesis and gametes

to

(Figs. IF, 2B).

Dissection occasionally revealed germ cells in the gonadal tissue on the mesentery. The mesenterial tissue was
densely packed with zooxanthellae, and the tissues around
zooxanthellae, but the oocytes
themselves were never observed to contain zooxanthellae

many

(Fig. 1A).

examined by interference phase microscopy immediately upon release from the adult anemone,
were spherical, yellow-brown, and 300 /xm in diameter
(Figs. IB, 2A). The surface of each ovum was covered in
Unfertilized ova,

fine translucent cytospines (stiffened

3.5

blastulas were observed (4- to

due

the oocytes contained

Fertilization

spines.

anemone.

Gametes, fertilization, and subsequent early development

were examined by taking samples, first at hourly intervals

and

The gametes were shed into open water, where fertilizaEach released ovum was surrounded by numerous sperm, which were aggregated between the cytotion occurred.

bundles of long mac-

The blastomeres were now 20-30 jim

in

diameter, even in size, and rarely contained more than

one zooxanthella each (Fig. 1G). The cytospines were
resorbed and replaced by cilia, which soon began to
exhibit the characteristic metachronal rhythm that rendered the coeloblastula motile.

Gastrulation

Few

gastrulae (Figs. 1H-I. 2C-E) were seen, suggesting

that this

developmental stage

is

very short. Twenty hours

spawning, the motile coeloblastulas began to show a

slight depression at the pole about which the algae were

after

68

S.

K.

DAVY AND

J.

R.

TURNER

-^*

H
Jtef

Figure

Larval development and zooxanthella acquisition in the temperate sea

hullii. (A) Developing oocyte. The oocyte contains no zooxanthel-

1.

anemone Anthoplcnni
lae,

which

are.

however, scattered throughout the surrounding

tissues. (B)

The zooxanthellae

are concentrated within the left hemisphere of the

has cytospines on

its

at

the

ovum

surface. (Cl 4-cell hlastula. (D) S-cell blastula. (E) 16-cell blastula.

(F) Coelohlastula. Zooxanthellae continue to be localized

Blastomeres

Released ovum.

ovum, and

coeloblastula stage.

Many

on one side of the embryo. (Gl

blastomeres contain only a single zooxanthella

with the remaining blastomeres not being infected at this stage. (H) Mid-gastrula.
Note the blastopore on the left-hand side. (I) Late gastrula. The blastopore and blastocoel

cell,

by the clear central region. Zooxanthellae are largely restricted to the

endoderm, while the ectoderm is largely zooxanthella-free. (J) Cell layers of gastrula,
showing distribution of zooxanthellae. Most zooxanthellae are in the endoderm, but two
are indicated

can be seen

in

the ectoderm.

One

of these zooxanthellae (see arrow) appears to be

degenerating. (K) Early planula. (L) Late planula. Zooxanthellae are distributed along the
mesenteries. (M) Aposymbiotic mid-planula. Scale bar in A applies to all images except
G and J and represents about 100 jum; scale bars for G and J represent about 50 /.ini.

EARLY DEVELOPMENT

IN

ANTHOPLEURA

69

BAl.l.ll

tentacle

cytospine

rudiment

cytoplasm

mouth
symbiotic alga

blastocoel

actinopharynx

coelenteron

mesentery

cilia

archenteron
blastopore

ectoderm

endoderm

SOOum
i

Localization of zooxanthellae during early development of Anthopleura ballii. (A) Ovum.

Figure
showing localization of zooxanthellae. (B) Coeloblastula. (C) Early gastrula, showing invagination. (D) Midgastrula. showing localization of zooxanthellae in the endoderm. (E) Late gastrula-early planula. (F) Mid2.

planula.

showing zooxanthellae distributed along the mesenteries. (G) Late planula, prior

concentrated. Gastrulation by invagination (and perhaps

epiboly) followed (Figs. 1H. 2C). with the blastomeres

aggregated, at first, around the blastopore. Gastrulation led

to the formation of an embryo with two cell layers encom-

passing a central cavity

the archenteron (Figs.

During gastrulation, almost

ing zooxanthellae

moved

the blastopore region.

all

II,

thellae seen in the ectoderm,

peared to disintegrate (Fig.

and many of these

cells ap-

1J).

Planulation

2D).

of the blastomeres contain-

into the

to settlement.

endoderm from around

Only very occasionally were zooxan-

After 27

h,

most embryos had become

late gastrulae

or

early planulae (Figs. IK, 2E). By this stage, the developing

larvae had shown no growth, remaining about 300 ^im in

70

S.

K.

DAVY AND

diameter. However, after 2 days, most planulae began to

J.

R.

TURNER

elongate along their vertical axis, tapering slightly towards

the posterior end. The zeoxanthellae were clearly visible,

infected oocytes were never observed. In contrast, spawned

ova almost always harbored zooxanthellae, indicating that
infection must occur at, or just prior to, release. We could

-ining the length of the endoderm.

arva was completely ciliated, and an

not ascertain whether infection occurs in the gonadal tissue

or after the ova have been released into the coelenteron. But,

aggregated in

striatinn-

The surface of

ea<. ^

apical tuft of longer cilia

larvae began

!;

was

After 3 days, the

visible.

exhibit signs of differentiation (Figs.

1L,

2F), with (he development of nematocysts, and a ciliated

actinopharynx, which replaced the blastopore. Between 3
and 5 days, the planulae began to grow to about 400-600
/urn in length

were not

fed.

and 300 /j,m in diameter, even though they

The number of zooxanthellae also increased

(not quantified),

quently.

As

and dividing zooxanthellae were seen

the mesenteries developed,

it

became

fre-

clear that

as the

anemones were kept

in artificial

(and so zooxanthella-

and as spawning occurred in

the zooxanthellae were of ma-

free) seawater in sterile flasks,

air,

we can be

ternal origin,

certain that

and

that infection occurs prior to release into

the surrounding seawater and hence prior to fertilization.

While the mode of zooxanthella acquisition has been

in relatively few species of cnidarians, early

determined

indications are that infection prior to fertilization

uncommon. For example,

is

quite

the vast majority of scleractinian

most zooxanthellae were located along these structures.

Interestingly, only one aposymbiotic planula was observed

corals investigated

throughout the course of this work (Fig.

(Lough Hyne). Tentacle rudiments were seen very

son and Wallace, 1990), though some species of Pocillopora and Montipora do release zooxanthellate ova (Babcock et al., 1986; Harrison and Wallace, 1990; Glynn el al.,

occasionally in some planulae (Fig. 2G). Although care was

taken to isolate the surviving planulae, they could not be
kept alive for more than 7 days and so settlement was not

1991; Himseetai, 2001). Of note, the eggs of the hard coral

Montipora digitata become infected just 24 h prior to spawning (Harrison and Wallace, 1990), suggesting that the delayed

1M), which

is

consistent with the absence of aposymbiotic A. ballii at the

field site

observed.

(Szmant-Froelich

Gametogenesis, spawning, and early development

in

An-

thopleura ballii follows the pattern exemplified by many

anemone species (Siebert, 1973; Chia, 1976; Jennison,
).

All these species are dioecious, shedding their

gametes into open water where

fertilization occurs.

The

undergoes
cleavage and
forms a hollow coeloblastula. Gastrulation follows by invagination to form a ciliated, pelagic planula larva. This
zygote then

radial,

holoblastic

notably different from that shown

the
meroblastic
ova of the anemones
by
larger, yolky,
Tealia crassicornis (Chia and Spaulding, 1972) and Cri-

of development

is

brinopsis femaldi (Siebert and Spaulding, 1976), in which

cleavage is incomplete, unequal, and relatively slow. The

were very similar

zooxanthellate
or zootemperate

sequence and timing of events

to those described for the

some other

anemones Anthopleura elegantissima and Anthopleura xanthogrammica (Siebert, 1973). However, unanemones, A.

ballii

spawned ova

that contained

zooxanthellae. In A. ballii, concentration of the zooxanthel-

one hemisphere of the ovum, and invagination (and

perhaps epiboly) during the gastrula stage, led to the localization of symbionts within the host's endoderm; this same
lae in

process occurs in the temperate

(Turner, 1988).

anemone Anemonia

endodermal

mitted maternally, infection does not occur until the later

stages of embryogenesis or larval development (Benayahu et
al.,

1988; Benayahu and Schleyer. 1998).

The mechanism of entry

may

into the

ovum

is

unknown, but

well be similar to that described for the soft coral

Litophyton arboreum (Benayahu et al.. 1992). In L arboreum, zooxanthellae pass through gaps in the mesogloeal
covering of the oocytes, accumulate in the perioocytic zone,

and ultimately bulge through the oolema and enter the

mature oocyte. A similar "phagocytosis" of algal symbionts
has been reported for the oocytes of several scleractinian
corals (Hirose et

Hydra

al..

2001), as well as for the freshwater

viridissima (Campbell, 1990).

Spawning, early development, and the localization

of zooxanthellae

The sperm of

tissue surrounding the devel-

oping oocytes was heavily laden with zooxanthellae, though

A.

ballii

are

similar to those of other

Anthopleura spp. (Siebert, 1973). Moreover, as in other

symbiotic Anthozoa, the heads are too small (3.5 X 2.3 /urn)
to act as vectors for paternal transmission

of zooxanthellae;

zooxanthellae in A. ballii are about 10 /urn in diameter

(Turner, 1988;

viridis

Gametogenesis and zoo.\anthella acquisition

In A. ballii. the

hosts.

in

in A. ballii

chlorellate

like these

eggs

brooding species like the soft corals Xenia

itmbellata and Anthelia glauca. where zooxanthellae are trans-

Discussion

1979, 1981

in their

1980; Babcock el al, 1986; Harri-

infection seen in A. ballii eggs also occurs in

Furthermore,

mode

do not harbor zooxanthellae

et ai.

During

Davy

et ai.

1996).

the early stages of development, and throughout

cleavage, the zooxanthellae remain localized at one end of

the embryo. By the time a coeloblastula forms, most zooxanthellae are located in individual blastomeres, at one end

of the coeloblastula. That this positioning is of paramount

importance for the ultimate localization of the zooxanthellae

EARLY DEVELOPMENT
becomes evident during

when zooxanthellae
become
the mechanism is so

gastrulation,

are situated within invaginating blastomeres and so

localized within the endoderm. Indeed,

IN

ANTHOPLEURA
nous sources

71

BALl.ll

to

infect

Fautin, 1993; Kinzie et

potential
al.,

hosts (Buddemeier and

2001).

In contrast, while transmission

mechanisms have been

successful that "stray" zooxanthellae, which end up in the

investigated in relatively

ectoderm, are rare (Fig.

vertebrate, initial observations (including those presented

The

initial

1J).

localization

of zooxanthellae seen here

is

similar to that seen in the corals Pocillopora vernicosa and

P. eydou.\i (Hirose et ai, 2000).

However,

coral species (Szmant-Froelich et

lation in P.

some other

as in

1980, 1985). gastru-

al.,

vernicosa and P. eydouxi occurs via delamina-

tion rather than imagination. This

means

that, in

marked

contrast to events observed in A. ballii. blastomeres containing zooxanthellae

move

into the blastocoel of develop-

ing embryos, eventually filling the space and forming a

stereogastrula (Hirose et

al..

2000).

The

precise

mechanism

by which the zooxanthellae move into the blastocoel

is

few species of zooxanthellate

in-

here) suggest that maternal transmission of zooxanthellae is

more likely to occur in temperate regions than in the tropics

(reviewed by Muller-Parker and Davy, 2001). A predominance of maternal transmission mechanisms at high latitudes would not be surprising, as it could be related to a
scarcity of

exogenous sources of zooxanthellae and,

fore, selection against hosts that acquire their

there-

symbionts

from exogenous supplies (Muller-Parker and Davy, 2001).

Indeed, a scarcity of sources of zooxanthellae could explain
the temperate coral Astrangia danae, which does not

why

acquire

its

zooxanthellae maternally,

sometimes found

is

unknown.

devoid of these symbionts (Szmant-Froelich et al., 1980). In

addition, maternal transmission, combined with the ability

Planulation

of temperate algal-invertebrate symbioses to tolerate a wide

range of environmental variables (Kevin and Hudson, 1979;

As

the position of zooxanthellae in the

stated above,

embryo, and the subsequent localization of zooxanthellae in

the endodermis by invagination, means that "stray" zoo-

xanthellae in the epidermal cells of planulae are very rare.

similar paucity of stray zooxanthellae was also reported for
the reef corals Pocillopora vernicosa and P. eydouxi (Hi-

2000). However, the planulae of some scleractinian corals (Szmant-Froelich et ai, 1985; Schwarz et al.,

rose et

al.,

1999), soft corals (Farrant,

1986; Benayahu et

al.,

Squire, 2000; Howe and Marshall, 2001), could explain the

persistence of zooxanthellate organisms at high latitudes

(Davy

et al..

1997; Muller-Parker and Davy, 2001).

More

analyses of zooxanthellar transmission mechanisms at different latitudes, and of the ecological advantages conveyed

by symbioses

in nutrient-rich

temperate waters, will help

resolve this matter.

1988,

1992; Benayahu and Schleyer, 1998), and jellyfish (Montgomery and Kremer. 1995) may contain zooxanthellae in

Acknowledgments
This work was funded by a

NERC

award

to

JRT and was

JRT thanks

epidermal cells more frequently. In these cases, the

zooxanthellae infect either the planulae or, as in the jellyfish

conducted,

Linuche unguiculata, both the embryos and planulae (Montgomery and Kremer, 1995), as opposed to the gametes. The

thanks the Institute of Marine Studies, University of Ply-

their

zooxanthellae

may

then be transferred to the endodermal

tissue via cell migration

degrade in the host or be expelled as a result of being

harbored by an inappropriate cell type. Degrading zooxan-

have been observed

corals Stylophora pistillata.

in

planulae of the scleractinian

Sehatopora caliendrum, and

Pocillopora verrucosa, though always in the endodermis,

rather than the epidermis (Titlyanov et al., 1998).

Mode

of transmission as

a function of latitude

Professor Sir David Smith

mouth

in tropical

seas and

regularly release zooxanthellae into the surrounding seawa-

(Hoegh-Guldberg

FRS FRSE

for support.

SKD

for financial assistance.

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Reference: Bio/. Bull. 205: 73-82. (August 2003)

2003 Marine Biological Laboratory

On

the

Growth

of Bivalve Gills Initiated

From

Lobule-Producing Budding Zone

DIETRICH
Zoological

NEUMANN* AND HEIKE KAPPES

Institute, University

of Cologne. D-50923 Koln, Germany

The growth of bivalve gills proceeds at the

end
of the gill from a meristem-like budding zone,
posterior
that is, an undifferentiated terminal organ, which continuAbstract.

series

ously proliferates new gill elements in growing bivalves. In

representatives of protobranch, filibranch, and eulamelli-

branch

gills

allel disks.

sition zone.

The lobules elongate,

and transform into

first

it

fenestrated, or transformed

mass and mantle

in

cavity,

and two additional

body

functions

evolved: feeding on inhaled particles, facilitated by mucous

secretion and followed by food-string transport along food
grooves; and in some taxa, brood care within the interla-

by tissue

junctions.
distally located main growth zone for each lobe
is suggested. With regard to the delayed onset of the dif-

demibranch

that creates a flow of

valves, the size of the gills increased in relation to

fila-

ferentiation of the outer

pump

system that carries the oxygenated hemolymph to the heart.

During the evolution of filibranch and eulamellibranch bi-

differentiate into lobes,

ments represent the differentiated outer margins of each

lobe, of which the central tissue (interlamellar septum)

becomes incised or

a peripheral

oxygenated water over

and through the demibranchs, and an internal circulatory

ciliary

forming a tran-

and eulamellibranchs). The

filament structure also appears on the sur-

Ridewood. 1903).
These gills all share two functional elements:

leaflet-like structures (protobranchs) or

into filaments (filibranchs

The

gills; however,
demibranchs are much more complex organs, because
the filaments are connected by various tissue junctions (see

produces either transverse folds that split after a transition

zone into parallel pairs of lobules (which themselves later

and outer demibranchs), or

the

their

Palaeoheterodonta, and Heterodonta), the first growth steps

demonstrate a uniform basic pattern. The budding zone

produces the lobules directly, without

gills,

face of the demibranch in eulamellibranch

(13 species from Protobranchia, Pteriomorphia,

differentiate into the inner

of ciliated leaf-like discs. In filibranch

demibranchs are considerably longer and consist of exrather than parthe filaments
tended parallel structures

mellar spaces (for reviews, see Purchon. 1968; Bayne et

a/..

1976; Morton, 1996).

juvenile unionids. an

additional temporary growth zone for filaments is suggested

to exist at the anterior end of the outer demibranch.

Bivalve gills develop new elements from their posterior

end as they grow (Wasserloos, 1911; Ansell, 1962; Korniushin. 1997). In the past, however, studies on gill growth

Introduction

processes focused only on the early organogenesis of the

review of the
gill during the postlarval development (for
older literature, see Raven, 1966). In

unique organs that show a continuous

terminal growth by adding new elements in correlation with
the lifelong increase in shell size. The gills consist of two
Bivalve

plate-like

gills are

demibranchs

that are

branch (Jackson,

extended anterior-posteri-

on each side of the visceral mass (the only exception:

Lucinidae with only one demibranch; Ridewood, 1903). In
the case of the phylogenetically primitive protobranch gill,

al..

2001).

addressed.

E-mail:

et ai,

The row of filaments extends

1911;

1998; Chaparro et
in anterior-poste-

first

display knob-like thickenings at their distal

ends and then transform to widened, roughly V-shaped

filaments; no bending or reflexion was involved in this

Received 29 August 2002; accepted 30 April 2003.

should be

1901; Wasserloos,

of postlarval stages of the

eulamellibranch Veneridae revealed that the unidirectional

demibranchs are comparatively small and consist of a

To whom correspondence

subclasses except

rior sequence. Detailed studies

filaments

1890; Drew.

Ansell, 1962; Waller. 1981; Gros

orly

the

all

Protobranchia, these early stages start with a short row of

unidirectional slender filaments of only the inner demi-

dietrich.

process (Ansell, 1962;

neumann@uni-koeln.de
73

Moueza

et al..

1999). At the end of

74

D.

development, the new filaments of the inner

from the posterior end of the gill base in

this postlarval

demibranch
the

NEUMANN AND

H.

KAPPES

Histology of subtidult Unio

gills

arise

form of V-shaped

elc;

nts (Ansell,

1962).

.opmental results on postlarvae

^tudy is focused on the continual
growth of the i\'<" .ntiated bivalve gill from its posterior
.re new filaments are added. Bivalves of
growth zone,
In contrast to these

and early juveniles

For histological analysis of the budding zone, two specimens of U. pictorum (shell lengths 4.85 mm and 20.1 mm)
were fixed in Bouin-Allen's fluid (2 h, 37 C). After rinsing
in

70%

ethanol followed by standard dehydration, the

sues were

embedded

tis-

via Rotihistol (15 h) and Rotihistol-

>

were examined. For juvenile unionids,

we further describe the beginning of the outer demibranch,
which lags behind the early formation of the inner demidifferent subclasses

The results offer new insights into the increase in

filament number, the differentiation of the filaments, and

Rotiplast 1:1

point 58

with

1.5

61

at

C). Serial 10-/u,m

stain

Domagk's

C) in Rotiplast (paraffin, melting

microtome sections were stained

(Romeis, 1968).

branch.

bivalve

gill

growth

Dissection of juvenile gills

in general.

Early juvenile eulamellibranchs possess only slender filWe estimated the shell size

aments of the inner demibranch.

at which the outer demibranchs first developed. Specimens

of U. pictorum and U. tumidus (shell length between 3.5 and

Materials and Methods

16.95

mm)

Material
in

To examine

gill

development

in

bivalves possessing pro-

gills, we studied three Nucula species (subclass

Protobranchia) preserved in ethanol (Museum Senckenberg.
Frankfurt a.M, FRG): Nucula nucleus Linnaeus (Helgoland

tobranch

ex Wolf/2, Coll.-No.

SMF 320968/2). N. sulcata Bronn

SMF 320966/3). and N. tennis
101 Ku/6. Coll.-No. SMF 320967/6).

(Me5/51 Ku/3. Coll.-No.

(Montagu) (Gauss-St.

As examples of

filibranch gills,

we examined

species
belonging to the subclass Pteriomorphia: Mytilus ednlis
Linnaeus (sampled at Gromitz on the shore of the Baltic
Sea, Schleswig-Holstein, Germany), M. galloprovincialis

Lamarck (Ria de Vigo, Galicia,

(Kakinada Bay, Andhra Pradesh,

Spain), and
India; soft

Anadara
body

sp.

were studied. Corbiciilu was collected from the

Rhine River near Cologne (Rh.-km 683); the other species

from waters of the flood plain of the Lower Rhine (Haffensche Landwehr near Rees, Rh.-km 840).

We

also in-

from Gromitz and Venempis

spected Mya arenaria (L.)

decussatu (L. from Ria de Vigo.
)

embedded

slides (after dehydration

in Rotihistokitt (Roth,

Karlsruhe-

length could be measured to the nearest 0.01 mm. Linear

regression lines were calculated using SPSS 7.5 and Stat-

graphics 4.0.

Terms used for

gill structures

Various anatomical terms have been used for the description of bivalve

others).

fluminea (O.F. Miiller), and Pisidium casertanum

ethanol were dissected for this

image analyzing system attached to a CCD-camera linked to

a Leitz microscope. With a pointer on the monitor, the

preserved in Bouin's fluid).

Eulamellibranch gills from the subclasses Palaeoheter-

(Poli)]

in

were placed on

FRG). The longest filament of each demibranch was then

measured from its dorsal base to its ventral tip with an

Yonge,

biculci

gills

ethanol) and

directly

odonta [Unio pictorum (Linnaeus), U. tumidus (Philipsson)]

and Heterodonta [Dreissena polymorpha (Pallas), Cor-

preserved

The

purpose.

gills

(Mitsukuri,

1881; Ridewood,

1903;

1956; Beninger et al.. 1988; and

avoid terminological confusion, we summarize

1947; Kilias,

To

most of these terms and mark (by single quotation marks

and italics) those that we will use in this study. In most
aspects we follow Ridewood (1903). However, with regard
to the posterior growth zone of the gill, we will introduce

new

terms.

When

juvenile filibranch and eulamellibranch bivalves

have passed the early period of gill differentiation, they

possess two 'demibranchs' (also gill plates) in an anteriorposterior extension on each side of the foot. These two
demibranchs, that is, the 'inner and 'outer' ones (Fig. Ib,

and od), are attached by a 'gill base' (also gill

root) on the dorsal side of the 'mantle cavity' (also

right side: id
axis, gill

between the visceral mass and the mantle.

Each dorsoventrally lengthened demibranch is formed into
two 'lamella' -like structures (also membrane plates, leaves)

Scanning electron microscopy

pallial cavity)

Fresh gills (posterior sections) of U. pictorum. C. fluminea. D. polymorpha, and P. casertanum were fixed in 2%

consisting of vertically ciliated 'filaments' in parallel (also

in

0.133 mol phosphate buffer (pH 7.2) for 2

bars, ciliated discs) (Fig.

la).

The terms 'descending

glutaraldehyde
h. This material, as well as prefixed gills from Anadara sp.,
N. sulcata. and./V. tennis, were then dehydrated in ethanol.

gill

After two rinses in pure acetone for 2 h each, the gills were
stored overnight in pure acetone, then dried with COS,

base) and 'ascending limb' (the other part of the filament

dorsally unattached or fused with foot or mantle) will be

mounted, and sputte r ed

(ca.

140-nm gold

layer).

limb' of the filament (also descending portion of the

ment,

i.e..

that part of the filament

circumvented as

far as possible

connected to the

fila-

gill

because of developmental

ON THE GROWTH OF BIVALVE GILLS

75

slits) through which the inhaled water passes. The filaments

of each demibranch are strengthened by skeletal rods and

are joined at their ventrodistal margins, thus forming the

ciliated 'food groove' (also marginal groove). As the central
structures of the lamellae increase in complexity,

of

eral types

come

two gen-

behomorhabdic and heterorhabdic

different species. Homorhabdic gills con-

gills

evident in

only 'ordinary filaments' whereas heterorhabdic gills

contain both 'ordinary' and 'principal' filaments (Ridetain

wood, 1903).
Gills of protobranch bivalves are smaller, restricted to the

posterior part of the mantle cavity, and characterized by a

simple anatomy. However, the gross design is the same as

of the filibranch and eulamellibranch types, that is, two

demibranchs on each side. Each consists of a series of
that

extended

leaflets (also discs), ciliated

and attached

each

to

other by ciliated knobs.

Results

Budding zones of protobranch and filibranch

On

gills

the basis of our material, the posterior growth zone of

two

these

branch

gill

gill

of

types can be demonstrated best in the

Anadara (Pteriomorphia)

fili-

(Fig. 2a). In this

species, the posterior part of the gill base ends in a small,

rounded projection of undifferentiated

cells,

from which the

separation of new filaments starts (Fig. 2b). We name this

meristem-like cell complex the 'budding zone'
As was observed in all dissections, the budding zone of

Anadara

is

not attached to the mantle but projects into the

mantle cavity. The budding zone of the specimen presented

(Fig. 2b) is already
1.

Figure

(a)

Schematic view of

shell

shape and the relative position

of the two adductor muscles (aa: anterior adductor, pa: posterior adductor)

and the

gill in

increasing

Unio tumidus.

number of

The budding zone

filaments

(fi)

is

located

at

(bz) of the successively

the posterior end.

The

filaments lengthen during shell growth, (b) Schematic view of a transverse

section

showing

the age-dependent difference in gill organization

juvenile unionids of shell length 3.5

mm

(left half)

and

8.1

mm

between

(right half),

id:

inner demibranch, showing early differentiation and later differentiation

on

the

left

attached to
junction,

and right sides, respectively (in most parts of the gill, not yet
the foot), od: outer demibranch, gb: gill base, ilj: interlamellar

ils:

interlamellar space, m: mantle,

ft:

foot.

medial

line.

This

is

marked on

that separates the inner

ventral side

is

'lobules' of the inner

the outer demibranchs in a characteristic

were able

to

and

relationship.

confirm the Ill-ratio of demibranch

How-

lobules in the filibranch mussel Mytilus (not shown).

ever, the undifferentiated

lowed by a

fine

the appearance of transverse

which form undifferentiated

We

by a

and outer demibranchs. The second

step of early differentiation

folds

its

the onset of the deep longitudinal groove

budding zone of Mytilus

'transition zone' characterized

is fol-

by a number of

transverse folds that have not yet split medially into the
lobules of the two demibranchs (as in Unio, compare Fig.

The length of the transition zone differed

imens. In M. galloprovincialis from Vigo (/;

and functional connotations. 'Interlamellar junctions' may

stabilize the elongated filibranch filaments, and adjacent

4a).

filaments are held together by 'ciliated knobs' (also ciliated

discs), which are arranged dorsoventrally, and more or less

specimens (0.5-0.7 cm) and larger ones (1.0-3.3 cm) revealed 4-6 and 8-12 transverse folds, respectively (excep-

two types
of tissue bridges: 'interlamellar junctions' (also septa) between the descending and ascending limbs of the filaments,

tion:

equidistantly (Fig. 2). Eulamellibranchs possess

among

spec-

12), small

one 3.7-cm specimen with only four folds). In M.

= 7) from Gromitz, the length of the transition
zone had no relation to shell length (2 folds in specimens of
edulis (n

and

cm

shell length. 5 folds for sizes of 1.8

and

and 'interfilamentar junctions' between adjacent filaments.

1.7

The

variety of tissue junctions increases the complexity of

the branchial architecture, with 'interlamellar spaces' or

cm. 7 for a 1.2-cm specimen, 9 and 10 folds for sizes of 3.2

cm and 2.0 cm, respectively). It is possible that such vari-

'gaps' (also suprabranchial chambers, vertical water tubes,

ations are correlated with different rates of gill increase.

Similar to Anadara. in Nucula tennis (Protobranchia) the

interlamellar cavity) and 'interfilamentar pores' (also ostia,

2.1

2.1

76

D.

NEUMANN AND

H.

KAPPES

budding zone of the gill is represented by the posterior

apex of the gill base and has no contact to the mantle (Fig.

tiny

2c).

The same was observed

in dissected

specimens of the

other two Nucula species. In Nuculu, both the transverse

folds and the separation of inner and outer demibranchs

occur simultaneously. Thus, the lobules appear from the

very beginning.

Lobule differentiation

in

protohranchs and filibranchs

In the protobranch Nucula. the lobules of the demibranchs extend laterally and dorsoventrally and form the

margin of each lobe becomes

leaf-like lobes. Part of the

thickened and ciliated, whereas the expanded inner portion

of the lobe
its 'lamina' (also interlamellar
septum) re-

mains unchanged. These are then the differentiated leaflets.

The lobules in the filibranchs Anadara and Mytilus
mainly increase dorsoventrally and form elongated lobes.
Their margins differentiate into descending and ascending
limbs of the filament, as already described for Arcidae and
Mytilidae (Ridewood, 1903). Simultaneously, the lamina of
each lobe becomes transformed. In Anadara, a gap (interlamellar space) occurs within the lamina and separates the
filament's margins (Fig. 3a).
as

little

as

50%

or as

much

The

as

length of the gap may be

of the filament's length.

90%

filament now resemble descending

and ascending limbs. Adjacent filaments are attached to
each other by nearly equidistant ciliated knobs that are
arranged in vertical rows, one on either side of the two

The outer margins of the

ciliated margins. Lateral

views of the lamellae reveal

that

knobs on adjacent filaments are aligned, and

the number of equidistant lines of knobs increases as

the equidistant
that

the filaments elongate.

tion,

new

ment's

It

was obvious

that,

during elonga-

knobs appeared stepwise near the

end. Thus, a main growth zone of each

lines of

distal

ment must be localized

in

this

distal

area

(Fig.

filafila-

3a).

However, it can also be seen that a new line of knobs

becomes inserted in a few areas along the length of the
lamellae after the distance between two rows has increased
(not shown). This fact suggests that, in

Anadara

gills,

incremental elongation of the filaments also occurs

the dorsoventral length of the lamina.

all

some
along

In Mytilus, the lobules occur after the medial splitting of

the transition zone into inner and outer demibranchs (see

above). Each lobule elongates and forms a lobe. Then, its

lamina becomes transformed. A few interlamellar junctions

Tr J^
i?4

JhW'i

'^r^*/i

-r-R

^v
.'^,'

IkJ
*

>--

'
.

14 i
>;

Figure
branchs

2.

Scanning electron micrographs of the differentiating demiAnailara sp. {Asp; full-grown specimen) and the

in the filibranch

protobranch Nuculu r?iutis (Nt; 6.5 mm), (a) Overview of the posterior end
of the left gill of an adult Anadara specimen showing the budding zone
(bz). (b)

Budding zone of Anadara

and outer dennhranch

(id. od). (c)

differentiating into lobules of the inner

Budding zone and separating inner and

outer demibranchs of Nucula', the lobules are partly covered by

cilia.

mucus and

ON THE GROWTH OF BIVALVE GILLS

77

similar to that of protobranchs and filibranchs: the folds split

into inner and outer demibranchs. Apart from these com-

mon

differentiation events, species-specific differences ex-

in

ist

budding zone and

the relative size of the

in

the

extension of a segmented transition zone before the two

parallel rows of lobules begin to emerge (Fig. 4). Cilia
already occur on the early lobules.

Unio

(Fig. 4a):

the

Only

growing

left gill

and the adjacent

mantle epithelium are visible; the right gill is obscured. The

budding zone is located on the posterior process of the gill
base,

which

curved inwards. At

is

extend from

it;

least six transverse folds

this is the transition zone.

These folds then

become separated by the prospective dorsal food groove

into two parallel rows of further enlarged lobules. The
lobules become ciliated as they grow ventrally. As can be
seen in the outer demibranch (Fig. 4a), the outer edges of
the lobules (later forming the outer lamellae) are attached to

from the very beginning. The correspond-

the mantle tissue

ing processes occur on the other side of the foot, on the

growing right gill.

Dreissena (Fig. 4b): In

view from the ventral

this

side,

only the left gill is horizontally positioned, so that the details

of the transitions between the budding zone and the trans-

The budding zone

verse folds can be seen.

Unio and
Figure

Gill filaments of

3.

two

filibranch bivalves demonstrating the

lengthened lobules with the equidistantly arranged ciliated knobs (kn),

which increase
zone (gz)

is

in

number from

indicated, (al

the distal end.

Anadara

The proposed

sp. (shell size

ils:

growth

about 5 cm); (b) \f\tilnx

edulis (shell size of 2.2 cm), fg: food groove, gb: gill base,

junction,

distal

ilj:

tions

to

is

smaller than in

followed by a short transition zone.

is

the

No

excep-

number between
relationship
and outer demibranchs was found. As was
in

1:1

lobule

developing inner
demonstrated during dissections, the budding zone

is

not

attached to the mantle.

interlamellar

Corbicula (Fig. 4c): The separation of the transverse

same principles as described above. The

interlamellar space.

folds follows the

remain and stabilize the small distance between the outer

margins (Fig. 3b); the first ones appeared at about the 30th
filament (counted anteriorly from the budding zone). Again,

budding zones of the left and right gills lie close together
and seem to be attached to the mantle. The dorsal margins
of the outer demibranch lobes are also attached to the
mantle from the beginning on. However, two peculiarities

the margins strongly resemble filamentary structures. All

can be noticed

adjacent filaments are attached to each other by equidistant

ciliated knobs (Fig. 3b). In dissections of the gills of adult

fold starts at the inner

Mytilus,

we observed

tant lines,

the

whole

that the ciliated

which are arranged

gill.

knobs form equidis-

lines increased with the size

of the demibranchs and the length of their filaments. In

contrast to Anadara, Mvtilus shows new lines of knobs only
near the ventral end of the filaments.

knobs were detected

in lateral

No

inserted lines of

views of the lamina. Thus,

unlike growth in Anadara, the main growth zone responsible for elongation must be restricted to this area.

does not

gills

The four

representatives of the Palaeoheterodonta (Unio)

and Heterodonta (Dreissena, Corbicula, and Pisidiinn) stud-

is

found

gill.

Secondly, a

Unio and Dreissena,

in

exist.

The convergent budding zones were

The separation of lobules and their
appear to be somewhat advanced in the

Pisidiinn (Fig. 4d):

as short as in Corbicula.

early differentiation

most recent section of the inner demibranch, without

dis-

relationship of the older inner and outer

lobules (compare idl and odl in Fig. 4d).

turbing the

1:1

Dissection of two other lamellibranchs revealed a differentiation of

new

lobules

more or

less identical to that

shown

Figure
Venerupis decussata specimens, neither
the budding zone nor the transition zone was fused to the
mantle; and the transition zone extended over only two or
4. In adult

in

Budding -ones of eulamellibranch

demibranch of each

transition zone, such as

parallel to the gill base along

The number of

in this species. Firstly, the first transverse

three transverse folds.

ied possess eulamellibranch gills. Again, an undifferentiated

rated, the

budding zone lies at the posterior end of each species' gills

(Fig. 4). In each example, the transverse folds form in a way

still

Mya

When

the

demibranchs were sepa-

most posterior lobules of both demibranchs were

solid, lacking

arenaria (shell length 3.8

mm)

small specimen of
showed a similar pat-

any transformation.

78

NEUMANN AND

D.

8-

Figure

4.

H.

KAPPES

-,

Scanning electron micrographs of the budding zones and the start of both lobule and demibranch
Unio piclorum (Up; shell length 1.7 cm); (b) Dreissena

differentiation in four eulamellibranch bivalves, (a)

polymorplui (Dp; 1.8 cm); (c) Corhicula fluminea (Cf; 0.9 cm); and (d) Pisidium casertanum (Pc; 2.1 mm). The
budding zones of the left and right gills (bzl, bzr) may lie next to each other, as in Dp. Cf. and PC. Each is
differentiating into the lobules of the separating inner and outer
transition

zone

(trz)

of about six transverse folds

is

demihranchs

shown, m: mantle.

(idl, odl.

and

idr, odr). In

Unio, a

ON THE GROWTH OF BIVALVE GILLS

The

tern.

transition

zone consisted of three folds

at

this

the row; Fig. la). In

Figure 6 the height of the

stage.

in

Lobule differentiation

in

were examined

euliimellihranchs

lobules

into

the

to follow the differentiation

filaments of eulamellibranch

rately

of the

gills.

In the

budding zone. The

The

linear regressions of

was observed

we never found such

shown

demibranch formation. At

first, these rods seem to be conbetween the inner and outer derni-

extend into the filaments.

differentiation can be seen

Figure 5c. Tissue bridges representing interfilament
junctions occur between
adjacent lobes (ifj in Fig. 5c). As
in

the three

main

increase in

bears the ciliary

machinery and the vertical
vessels of the gill.

Outer demibranch formation

The

in

hemolymph

juvenile unionids

dorsal mantle cavity of a

Unit) pictoruni revealed

3.5-mm-long specimen of
no indication of filaments of the

outer demibranch (Fig. Ib). However, six short filaments

of
the outer

demibranch were identified in frontal sections of a

U. pictonun specimen of 4.9-mm shell
length. Apart from
the anterior one, these were
differentiated in that
already

they were fused

at

their outer

13

Because the development of the outer demibranch

lags
that of the inner, the two demibranchs
differ in
filament number, in gill-base
length, and in demibranch
behind

height (as expressed by the length of the longest filament in

species

-arrangement

filaments in

formation

in juveniles and adults of

and uniform pattern. The
the number of leaflets in
protobranchs, and of
filibranchs and eulamellibranchs, starts from an
gill

shows

undifferentiated cell

common

complex

that

we termed

the 'budding

zone'. This growth zone

generates a series of transverse,
paired lobules that constitute, in a Ill-relationship, both the
inner and outer demibranch. The lobules
into extended

grow

and elongated lobes that become transformed into leaflets

in
protobranch gills and into filaments in filibranch and eulamellibranch

gills.

The budding zone should be seen as a

entiated complex of
dividing cells that is

specific, undiffer-

active in growing
bivalves. This terminal zone can be characterized as
meris-

tem-like because

it
produces new gill elements during the
of these animals, similar to the formation of new
leaves from a shoot apical meristem in
higher plants, or the
development of new polyps from a terminal cell
in

whole

life

complex

the

elongating

(Berking

margins with the mantle

epithelium.

Figure 5a.

types (protobranchs, filibranchs, and

gill

remains unchanged and constitutes an interlamellar

junction
(ilj in Fig. 5c). When the demibranchs of eulamellibranchs
are observed in situ, the lamellae
appear filamentous because the outer ciliated
margins of the lobes have differentiated into the
descending and ascending limbs of the so-

However, this filamentary appearance is due

no more than the outermost 50
/xm of the tissue, which

in

The terminal growth zone of bivalve

gills was described
based on dissections,
scanning electron micrographs, and
histological sections. Despite the anatomical differences of
eulamellibranchs),

to

the

Discussion

documented further, interlamellar spaces

appear in the laminae of most lobes (ils in
Fig. 5c). In some lobes, the lamina

called filament.

mm),

larger
a large difference in fila-

filaments already

skeletal rods
(grayish opaque, no nuclei) are
in the late transition zone
during the beginning of

first

later they

which an

mm). Along the whole gill axis, the skeletal rods of

parallel
filaments of inner and outer demibranchs touched each
other
at the gill base,
resulting in the strict 1
of

5a-c).

The other processes of lobule

in

(shell length 4.9

ment number at the anterior

margins of inner and outer
demibranchs; usually the difference was 10-12 filaments
(n= 10 specimens with shell lengths between 6.9 and 14.6

development. As they cannot be followed in only one

we present sections of different levels (Fia

branchs (Fig. 5a);

fila-

shell length

case of the smallest Unio

specimen

specimens,

frontal section,

fined to the gill base

maximum

anteriormost filament of the outer demibranch was located

next to the 27th filament of the inner demibranch. In

continuously
lengthening
lobes into the
complexly structured filaments of the eulamellibranchs. Three processes can be
distinguished during

formed

sepa-

O.I).

outer demibranch

longitudinal splitting of the folds into two rows of lobules

(representing the inner and outer demibranch) is
clearly
visible in Figure 5b. The
development of these lobules into
extended lobes (still unfenestrated) is followed
by those
differentiations that
transform the

The

>

(P

were significantly different

with respect to the
for
the inner and outer demiintercepts
branchs (P < 0.001). The
slopes differed only marginally
(P = 0.051).

gill, that is, the budding zone, reveals

the character of an undifferentiated tissue
with a high density of nuclei (Fig. 5a). New transverse folds are added to
the already differentiated
from the

this

mm

ment length versus

The

posterior end of the

gill

demibranch

is

juveniles

plotted against shell length, up to 17

(years 1-3, based on the number of growth rings). The data
for both Unio
species were pooled because no speciesspecific deviation was found when they were tested

Histological sections of a suhadult specimen of Unii>

(Fig. 5)

79

et al..

stems or stolons of thecate

hydrozoans
2002).

One may assume

that

projection of the postlarval

this
gill

terminal growth zone

axis

is

composed of peripheral

ectodermic and internal mesodermic cells. The

budding
zone either first produces transversal folds (in cases of
delayed splitting into inner and outer demibranch lobules, as
in

Mytilus. Unio, Dreissena, Venerupis.

A/v)

or

it

directly

80

D.

forms lobules

(in

NEUMANN AND

H.

KAPPES

cases of simultaneous splitting of the

demibranchs: Nucula, Anailuni. Crbicula, Pisidium). The

segregation may resemble the first steps of somitic segmen-

demibranch
inner

embryology of segmented animals (Wol1998). accompanied by a change in cell adhesion

tation in the early

pert et

<//..

between

distinct blocl-

->

of ectodermic

outer

cells.

10

Figure 6.
demibranchs
U.

Length of the longest filaments of the inner and outer

and open triangles, respectively) in Unio pictorum and

The regression
13), (r

shell

lengths (pooled data of both

= 0.22(0.01) A + 0.17(O.I5), (;
demibranchs: and y^ = 0. 19(0.01)

lines are

0.97) in the case of the inner

0.93(0.

(mm)

(solid

tumidus specimens of different

species).

20

15

shell length

y jd

.v

0.97) in the case of the outer demibranchs.

The conformity of initial lobule formation in all bivalves

monophyly of this class. In Nucula, the

tested supports the

protobranch gill closely resembles the ctenidia of prosobranch gastropods, because both consist of a series of leaflets

along a

ture

is

gill

bivalves.

axis (Yonge, 1947). This simple gill struc-

from

the more complex gills in the rest of the

Based on morphological and molecular data sets,

distinct

the Protobranchia are therefore considered to be a sister

group

which

to the other bivalves,

are

grouped as Auto-

branchia (Hoeh et ai, 1998; Giribet and Wheeler, 2002).

In the Autobranchia, the gills are adapted to additional

functions, such as feeding and breeding. The decisive evolutionary step was the strong elongation of the lobe. The

lobe's transformation into filibranch or eulamellibranch

aments can be understood as a

fil-

series of

developmental steps
correlated with increasing efficiencies of the gill's various
functions. Fossil records (Cope, 1996) as well as morphological

and molecular data (Hoeh

Wheeler, 2002) indicate

m
.'

"^
:"

Figure

5.

(bz),

(a)

1998; Giribet and

Frontal (horizontal) sections (slightly slanted) through the

posterior end of the

mm),

et ai,

that the filibranch gill represents

left gill

Section near the

and with a

transition

ation ria lobules, (b)

of a subadult Unio pictorum (shell-length 20

base with the undifferentiated budding zone

gill

zone

The same

(trz)

and the beginning of lobe differenti-

section (about 30 /j.m ventral of section a),

the increasing gap between inner and outer demibranch (id, od).
Section of the outer demibranch, ventral to the left part of section b,

showing
(c)

revealing further details of the differentiation of lobules into early filaments

with interfilamentar junctions

rnellar junctions

(ilj).

(ifj),

interlamellar spaces

(ils),

and

interla-

'af: ascending limb of filament, 'df: descending

limb of filament, m: mantle

tissue,

r:

rod structure inside the

connecting the inner and outer demibranchs.

gill

base

ON THE GROWTH OF BIVALVE GILLS

the plesiomorphic type, and that the eulamellibranch

gill

81

inidus. Firstly, a certain

size

body

characters evolved polyphyletically.

Because lobules and lobes are the primary structural
elements, it is interesting to follow their successive trans-

outer demibranch development

formation into the so-called filaments. The present study

confirms that neither bending nor folding of filamentary

mens of

structures occurs in juvenile and adult bivalves.

V-shape of the filaments

results

The

from the continuous

final

trans-

formation of a lobular anlage via lobes into filaments by a

dominating ventral growth zone near the tip of the filaments.

zone was ob-

Evidence of high mitotic

activities in this

served

(Crenomytilus, Mytilus) by

in adult filibranchs

thymidine

Movchan,

based on the pattern of equidistant

Movchan (1975) de-

is

of ciliated knobs. Leibson and

tected
in

and
(Leibson
labeling
autoradiographical
1975). This result perfectly correlates with our

conclusion, which
lines

two additional areas of DNA-synthesizing activities

Both were situated close to the dorsal food

Mvtilus.

we

population
branch started

must be reached before

initialized. In the

is

lengths of about

at shell

this size

showed one growth

4-4.5 mm. Speciring, indicating the

cessation of growth during the first winter. In contrast,

Korniushin ( 1997) found the first filaments in 2.4-mm Unio

specimens. Whether such a difference in the start of outer

demibranch formation is due to environmental or genetic
factors remains unclear. Secondly, at the anterior

the

other eulamellibranch species (Komiushin, 1997). A reduction of the foremost filaments of the inner demibranch

seems

to

be unlikely, because

differentiated at the

4.9-mm

all

filaments were completely

stage;

i.e.,

they were fused to

the foot and

We

that

its

investigate whether

anterior end

further studies are

needed

to

two areas represent additional growth zones of the

filaments or a higher renewal rate of epithelium cells. In any
event, during the transformation of lobes into filaments and
these

the succeeding elongation, no bending or reflection occurs;

Yonge 1947. p. 501 "this mode of origin is

The convenient terms 'descending limb' and

as declared by

impossible."

):

'ascending limh' are referring neither to the direction of

end of the

number of filaments in inner and outer demibranchs differs. The difference is size-dependent and decreases in larger juveniles. Such a decrease in the difference
of anterior filament number was also observed in several
gill axis,

grooves, one at the gill base, the other one at the dorsal apex
of the filament's ascending limb. As these authors already
stated,

Unio

studied, differentiation of the outer demi-

were functionally integrated.

the outer demibranch also extends

hypothesize

range

at

its

where

during a short developmental peria limited number of lobules differentiate from the gill

od

axis in parallel to the filaments of the inner demibranch.

In summary, the generation of simply structured lobules
from the posterior budding zone and their differentiation
into protobranch leaflets, filibranch filaments (interlamellar

junctions and ciliated knobs between adjacent filaments), or

more intricate structures (with complex interfilamentar

growth of the filaments, nor to the direction of hemolymph

flows, because both arterial and venous lacunae (separated

junctions, as in pseudolamellibranchs and eulamellibranchs)

by an intrafilamentar septum; Ridewood. 1903) are located

studies,

inside the limbs (Yonge. 1947; Kilias, 1956; our observa-

the various gill types that occurred during the phylogeny of

tions

on Anadara).

In the early

which may also

bivalves.

development of the unidirectional slender

filaments in postlarvae. developmental processes similar to

those described above seem to occur. One could term them
'pro-filaments' because they strongly differ from the filaments in adults. Based on scanning electron microscopy
figures of juveniles of the pseudolamellibranch Ostreu chit-

ensis (Chaparro et

a!.,

2001)

it

may be

inferred that

growth

occurs without bending; the gill rudiments (e.g., pp. 201203: Fig. Ic, Fig. 2a) perfectly correspond to compact
transverse structures,

i.e..

lobules.

During postlarval devel-

opment of the eulamellibranch Veneridae,

the unidirectional

filaments of the inner demibranch display a thickened distal

into V-shaped filaments without any

end and transform

bending (Ansell. 1962; Moueza etui. 1999). This thickened

end may be recognized as a kind of lobule. Corresponding
conclusions may be derived from the thickened ends of the
filaments presented in figures of Pecten (Beninger et
1994). freshly

an interesting model for further developmental

offer insight into the evolution of

may be

<//.,

metamorphosed Unio (Herbers. 1913) and

juvenile Sphaerium (Wasserloos, 1911.

figs.

K. L).

The onset of the outer demibranch formation and its delay

in the Autobranchia reveals two further interesting developmental aspects, as shown in Unio pictarum and U. tu-

Acknowledgments
Our

sincere thanks are due to Dr. Ronald Janssen (For-

schungsinstitut und
a.

M.)

Naturmuseum Senckenberg, Frankfurt

Nucula specimens from the museum

Gerard Van de Velde (Dept. of Ecology,

for lending

collection; to Prof.

University of Nijmengen) for acquiring the Anadara specimens from India; to Hans-Peter Bollhagen, Helmut Wratil
(University of Cologne), and Dr. Markus Weitere (Dept. of
assistance; to
Biology, Free University of Berlin) for
Frederic Bartlett for linguistic comments; and to Prof. M. J.

SEM

Greenberg and anonymous referees for valuable suggestions

for

improving the

text.

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Reference: Biol. Bull. 205: 83-92. (August 2003)

2003 Marine Biological Laboratory

Demonstration of Nutrient Pathway From the

Digestive System to Oocytes in the Gonad Intestinal

PETER
1

G.

maximus L.

of the Scallop Pecten

Loop

BENINGER'

*,

2
GAEL LE PENNEC 2 AND MARCEL LE PENNEC
,

Laboratoire de Biologie Marine, Faculte des Sciences, Universite de Nantes, 44322 Nantes Cedex,
2
Institut Universitaire Europeen de la Mer, Universite de Bretagne Occidentale, Site

France; and

Technopole Brest-Iroise. 29280 Plouzane, France

Abstract.

The mechanism of

digestive system

to the

gonad

nutrient transfer

acini

Introduction

from the

and developing oocytes

In the Bivalvia, the digestive and reproductive systems
and often intertwined, either within the

loop system of the

was injected
L.
Ferritin
maximus
Pecten
queen scallop
of
intestine
specimens from the
directly into the purged

was investigated

in the gonad-intestinal

are closely situated

begun to be elucidated in this class (Pipe, 1987a, b; Dorange

and Le Pennec, 1989; Eckelbarger and Davis, 1996a, b).
However, gametogenesis must rely on the transfer of nutri-

territin

stored in variously sized inclusions, as well as in the

the 12
general cytoplasm. In all sections examined for

ents,

in
experimental individuals, hemocytes were always found
from
the
association with connective tissue fibers extending

min

individuals. Ferritin-bearing cells

were rarely found

sociation with male acini or gametes, nor with

is

bivalve species (Goddard and Martin, 1966; Vassallo, 1973;

Ansell, 1974; Comely, 1974; Gabbott, 1975, 1983; Adachi,

in as-

mature

1979; Zaba,

als, transfer is

the

first

from the
to

1991a,

among

1981; Lubet el ai,

1987; Le Pennec el al,

Although successful gamete production

little

is

known about

relies

on

the underlying

pathways and mechanisms. The elaboration of oocyte reserves has been the subject of considerable research in many

individu-

not synchronized on short time scales. This

b).

such transfers, very

suggesting that nutrient transfer to oocytes

either not a continuous process, or that

tissues

gonad

all

female gametes, but often with developing female gametes.

Not all individuals showed the same temporal dynamics of
ferritin transport,

which are acquired almost exclusively by other

or organs and transferred to the gonad.

Transfer of nutrients from storage or digestive sites to the
has been inferred or demonstrated in a number of

After 30-

incubation, ferritin appeared inside the acini of

distinctly

tural characteristics of

was

acini.

more

Le Pennec, 1991; Morse and Zardus, 1997). The ultrastrucgametogenesis have only recently

various cell types. Ferritin was rapidly absorbed by the

intestinal epithelium, and then appeared in hemocytes in the

base of the intestinal epithelium to gonad

(the majority of bivalves), or

Pectinidae (Galtseparated from the visceral mass, as in the

soff, 1964; Morales- Alamo and Mann, 1989; Beninger and

wild. Subsequently, a histochemical reaction and transmission electron microscopy were used to localize ferritin in

surrounding connective tissue. In the hemocytes,

mass

visceral

is

invertebrates, but

is

largely lacking in bivalves (see Eckel-

demonstration of a pathway of nutrient transfer

barger and Davis, 1996a, for review). Regardless of whether

and more generally the digestive system,

bivalve gametes ultimately elaborate vitelline reserves using

autosynthetic (Suzuki el al., 1992) or heterosynthetic path-

intestine,

developing oocytes

in the Bivalvia.

ways
both,

(as suggested
it

largely

is

by Eckelbarger and Davis, 1996a), or

must be made available,

clear that nutrients

from the

diet, for the synthesis

of the gametes and

their reserves.

Received 21 November 2000; accepted 4 June 2003.

* To whom
correspondence should be addressed.

E-mail:

A summary

Peter.

nutrients to the

Beninger@Isomer.univ-nantes.fr

83

of

known

gonad

or inferred pathways that transfer

and gametes has been outlined

acini

84

BEN1NGER ET AL

G.

P.

queen scallop. Pecten imuiniitx, based on anatomical, ultrastructural, and histoch -iiical observations (Le Pentransfer of nutrients from
nec el ul.. 199 la). In p:;
was proposed. Althe intestine to the A
ig gametes
extracellular and
that
both
nown
h.
though it has long
for the

:;

ike place in the intestine

intracellular di

(Zacks.

1955:

Reid,

1966; Payne et

of bivalves

1972: Mathers,

/.,

1973; Teo and Sabapathy. 1990). the persistence of the

"conventional wisdom" that the intestine merely serves as a

struction of histochemical sections (Kiernan, 1990). Control

for the eventual presence of naturally occuring ferritin can
be accomplished through the use of control subjects.
Pectinids are ideal candidates for such experiments, be-

cause the gonad-intestinal complex is well-separated from

the other organs. Pecten maximus was chosen in part be-

cause

it

is

a simultaneous hermaphrodite, thus allowing

within
investigation of both male and female components
same individual under identical experimental condi-

the

The gonad

intestinal

loop of pectinids also presents

most of the

conduit for undigested matter prompted Purchon (1971) to

call for a reexamination of the role of this organ. In the
the otherwise
family Pectinidae, the intestine loops within
Pennec et til.
indeed
Le
and
distinct
gonad.
anatomically

tions.

(199 la) provided data suggesting that nutrients are trans-

ally lacks circulating respiratory

ferred from

also

developing gametes. They

heproposed a transfer mechanism and pathway involving
substudies
ultrastructural
detailed
and
mocytes. Enzymatic
this structure to

sequently showed

that the scallop intestinal loop is

of digestion and assimilation (Le Pennec

et

til.,

capable

1991b). This

research provided a framework for the demonstration of

transfer

pathways using

direct

physiological

techniques

such as labeling. In this study, therefore, we have used

ferritin as a marker to examine the proposed transfer paththe gonad intestinal loop to gonadal tissue in the
maximus.
Pecten
scallop
Ferritin is an iron-containing transfer protein, consisting

way from

of a core of up to 4300 iron cations

in the

form of

the advantage of being easily visible throughout

reproductive cycle. No respiratory function has yet been

ascribed to bivalve hemocytes, and bivalve plasma gener-

gum.

pigments (Booth and Man-

1978). obviating possible artifacts.

Materials and Methods

Twelve specimens of Pecten maximus

(size range

9-10

cm

shell length, antero-posterior axis)

were collected from

the

Bay of Brest

The valves of each

(Finistere, France).

dorsal
scallop were kept open with a wedge in the posterior
intestinal
of
the
the
and
descending
proximal part
region,
the male
loop was located by directing a cold light source at
Into this
1 ).
portion of the translucent gonad (see Fig.
a
4
1
ml
of
each
in
of
the
intestine
mg ml
scallop.
portion

solution of cadmium-free ferritin (Sigma horse spleen

Type

ferric

oxyhydroxide and ferric phosphate, and a protein shell of

approximately 450,000 Da (Miksys and Saleuddin. 1986).
In the specific tissues of living organisms which contain
molecules are often grouped into variously sized
with a near-crystalline appearance (Bottke and
Sinha. 1979; Miksys and Saleuddin. 1986). A specific stain
for iron can therefore be used to distinguish it from other

ferritin, the

clusters,

proteins

(Bockman and Winbom. 1966; Heneine

1969; Block et ah.

1981: Bottke

et

til..

Rodoni and Boucaud-Camou, 1987; Paar

til.,

1992). Ferritin

is

et

et

til..

1982; Bouchertil..

1992; Ito et

also visible in uncontrasted transmis-

sion electron microscopy

(TEM)

sections as small, vari-

ously sized electron-dense clusters (Bottke and Sinha. 1979;

Miksys and Saleuddin. 1986). Ferritin has been used both to

demonstrate intestinal absorption mechanisms (Bockman

and Winborn, 1966; Boucher-Rodoni and Boucaud-Camou.
1987) and to study mechanisms of uptake into the ferritinrich yolk of snail oocytes (Bottke et

we

til..

PO

1982). In this study

use ferritin as a substrate model with which to follow the

transfer of nutrient

molecules from the intestine to the

gonadal tissue of Pecten maximus. Although hemoglobin is

present in the hemocytes of some bivalve families (see
reviews by Reid. 1966; Bonaventura and Bonaventura.
1983), none has yet been reported in the pectinids. and in

Schematic diagram to show planes of

median (M). and posterior (PO) gonad levels.
such that the region
Histological sections were performed on (hese planes,
of the median level surrounding the descending branch of the intestinal
whereas the region of the
loop (DB) contained predominantly male acini,
Figure

1.

AYr/cii

;<I.V/HI.V.

section in anterior (A),

any event, this substance cannot confound histochemical

detection of injected ferritin since the iron of hemoglobin

median level surrounding the ascending branch of intestinal loop (AB)

contained predominantly female acini. AM, adductor muscle; DC. digestive gland: FT. fool: I., lips; R. rectum: S. stomach; 8. male, and 9. female

cannot be demonstrated histochemically without

parts of gonad.

total de-

PECTEN NUTRIENT PATHWAY TO OOCYTES

85

0.4

pm

Figure 2. Transmission electron micrographs of uncontrasted oocytes and hemocytes in posterior (female)
2.2 Details of early-developing
region of gonad. from individuals which had not been injected with ferritin. 2.1.
Detail of rounded hemocyte. showing
oocytes at two magnifications (20.000 and 40.000 x. respectively). 2.3
non-ferritin-containing inclusions

(I);

2.4 Detail of pseudopod-bearing hemocyte. Note absence of ferritin

clusters in all micrographs. N. nucleus.

nm

diameter molecules) was slowly injected.

Complete distribution of ferritin throughout the length of
the intestinal lumen was monitored visually by the appear1,

10-12

ance of the colored solution

in the

each, corresponding to 10. 30. 120. and 300 min of exposure

to the ferritin solution. Exposure was carried out in 15C

which was aerated with a pump and

air-

stone. Following the designated period of exposure, the

were immediately
gonad (about 1-2 mm

scallops

dissected. Transverse slices of

were removed

Histology
Slices

nol-xylene series, and

levels along the antero-posterior axis of the gonad as shown

in Figure 1. and immediately processed for histology and

level

To

control for the eventual presence of

clusters of endogenic ferritin molecules or other electrondense particles, a control group of three individuals was

electron microscopy.

embedded

in

paraffin.

Transverse

sections corresponding to each exposure time and

level

thick)

of gonad were fixed for histology in aqueous

h), dehydrated in an ascending etha-

Bouin's solution (24

at three

the

and processed for

ascending branch. The

scallops were divided into four groups of three individuals

filtered seawater.

injected with 0.8 /urn filtered seawater,

histology as detailed below.

slides

were cut

at 5

gonad

p.m. and each exposure series for a given

were positioned on a single microscope slide. The

were then immersed in 35% ammonium sulfite for 2 h

and rinsed with Milli-Q-filtered ultrapure water (all glassware at this stage was also prerinsed with ultrapure water).
Sections were stained using the Turnbull blue protocol.

86

P.

Exposure time
(min)
Distribution star?

G.

BENINGER ET

AL.

87

PECTEN NUTRIENT PATHWAY TO OOCYTES

BL
BL

IE

CT

CT

HF

.^p
'

-%

'

^11

CF

VO

HF
\

CF
HF

HF
HF

DO

DO
'

RMO

HF

RVO

P.

Figure

5.

G.

BENINGER ET AL

Pecten maximus. Uncontrasted transmission electron micrographs of cells in the intestine-gonad

ferritin injection in the intestinal lumen. 5.1 Detail of cytoplasm of absorptive cell from

complex following

median (male + female)

intestirul epithelium, in

level of

gonad showing

ferriting clusters (F) in inclusions

(FCI). 5.2 Enlargement of indicated region in Fig. 4.1. Note presence of ferritin both within the inclusions and
distributed

I'reel;.

in

surrounding the acini

inclusions. 5.4

>). 5.3 Detail of a large, rounded hemocyte in the connective tissue

cytoplasm
median (male + female) level of gonad. Note presence of ferritin (F) in variously sized

the
in

Enlargement of region indicated

in Fig. 4.3..

showing

ferritin (F) in inclusions. 5.5,

5.6 Portion

PECTF.N NUTRIENT

they

(detached from the acinal wall). Fol-

may be mature

lowing spawning, some oocytes of both types may remain

the acinus; these are termed residual oocytes.
this

adopt

No
or

terminology

We

in

will thus

in the present paper.

was detected using either the Turnbull method

observation (Fig. 2) in any of the control individmay thus conclude that the ferritin observed in our

ferritin

TEM

uals;

PATHWAY TO OOCYTES

we

histological and electron microscopical sections was

in-

jected.

The

histological observations of the entire set of individ-

uals and
ritin in
tial

gonad

the

levels revealed that the distribution of fer-

sampled
(1)

steps:

appearance

in

tissues could be divided into 5 sequeninto

uptake

intestinal

epithelium.

hemocytes among the connective

(2)

tissue surat the

rounding the intestine, (3) appearance in

hemocytes

exterior faces of acini, (4) appearance in

hemocytes within

the acini,

and

(5)

hemocytes/follicle cells
not possible to ascertain whether

appearance

appressed to oocytes.

It is

in

these cells were hemocytes or follicle cells in the histological sections (only
profiles can distinguish these cell

TEM

types).

The

TEM

profiles of these cells described

which are rich

not correspond to follicle cells,

below do
in

rough

endoplasmic reticulum (Dorange and Le Pennec. 1989).

absent in the two pectinid hemocyte types (Beninger and Le
Pennec, 1991) and

in the cells

observed appressed to the

TEM

sections were uncontrasted,

oocytes; however, as the
it is not possible to distinguish these cells with certainty.

Although ferritin-containing hemocytes were very rarely

observed associated with male spermatogonia. they were
never observed appressed to developing male gametes. The
distribution of ferritin

among

the sections

is

summarized

in

which presents each step in the sequence, for each

and each intestinal branch (ascending and

Figure

3,

gonad

level,

descending), for each exposure time.

Light microscopy of the histological sections showed that

was distributed in cells of the intestinal epithelium at

ferritin

three antero-posterior levels of the gonad-intestinal complex, from the apex to the basal region of the columnar
all

appeared very rapidly in the

and was observed in all preparations, even

intestinal cells (Fig. 4). Ferritin

intestinal cells,

after 10
ferritin

min of exposure.

TEM

micrographs revealed that

in

appeared predominantly

variously sized inclu-

appeared

in these

(Fig. 5.3-5.6). In all sections

examined

for the 12 experi-

mental individuals, ferritin-containing hemocytes were always observed in close association with connective tissue
fibers

extending from the basal lamina of the intestinal

epithelium and the acini (Fig. 4.2, 4.3).

Further transport of ferritin to the gonad acini and oocytes
appeared to be somewhat independent of exposure time
(Fig. 3).

Specimens

observed

showed

in

which further

ferritin transport

ferritin-containing

only a 10-min exposure (Fig. 4.2). Ferritin

both at the outside faces of gonad acini (including

those which were remote from the intestine) and inside the
cells

gonad acini (Fig. 4.3-4.8). These hemocytes/follicle cells

were typically found appressed to developing oocytes (Fig.
4.3-4.8). Although no positive Turnbull blue reaction was
in female gametes, transmission electron micrographs revealed dispersed ferritin clusters within the cytoplasm of oocytes to which ferritin-containing hemocytes/

observed

follicle cells

The

were appressed

(Fig. 5.7, 5.8).

localization and distribution of ferritin within the

various cell types involved in the transfer sequence presented notable differences, which also explains the absence

of a visibly positive Turnbull reaction

in

some

cell types.

and the transport hemocytes possessed variously sized inclusions with considerable concentrations of ferritin, and these cells presented visibly positive

Both the

intestinal cells

Turnbull reactions.

Upon
scallops

histological examination, 3 of the 12 experimental

were observed

to

be mature and ready to spawn,

while the remaining 9 had already spawned and had begun

producing a new cohort of female gametes. Although fer-

appeared in the intestinal epithelium and surrounding

connective tissue of the mature scallops, ferritin-containing
ritin

hemocytes were

virtually never observed, either in the acini,

or appressed to the oocytes of these individuals. Moreover,

despite the presence of mature residual oocytes in the acini

of the individuals which had spawned previously, ferritincontaining hemocytes/follicle cells were never observed

appressed to them. Ferritin-containing hemocytes/follicle

however, observed appressed to late-developing

cells were,

residual oocytes in these individuals (Fig. 4.8).

Discussion

The uptake of
Pecten

imi.\innis.

ferritin

observed

in

in

the

intestinal

membrane; MT. mitochondria.

pseudopods of hemocyte/follicle
cytoplasm of both

cells

>).

of association between early developing oocyte (EO) and

(HP), in posterior (female) level of gonad. Note ferritin freely distributed

5.7, 5.8 Detail

cell

N. nucleus.

epithelium of

the present study,

of a large, rounded hemocyte within an acinus in posterior (female) level of gonad. showing ferritin-containing
inclusions (FCI). as well as ferritin molecules and clusters distributed freely in the cytoplasm ( >). CM. cell

in

was

hemocytes/follicle

hemocytes were also detected beneath

the intestinal basal lamina within the surrounding connective tissue after

in variously sized inclusions,

hemocytes

as well as being generally distributed within the cytoplasm

sions of the intestinal cells, although isolated granules could

also be found in the cytoplasm (Fig. 5. 1. 5.2). Large, round,
ferritin-containing

89

demon-

90

P.

strates that proteinaceous substrates are

scallop intestinal epithelii'

quently appear in heir

and ubiquity of this u

at

is

the

enzymatic equipment of the

Pennec

intestinal epitHe'

the scallop intestine

absorbed by the

some of which subsecell bases. The rapidity

least

viserved in the present study, as

id

well as the cyi<

BENINGER ET

G.

el ai, 1991b),

suggest that

well-adapted for both a digestive

a transfer fiMCiinn. This result

is

and

consistent with the view of

the intestine as a digestive organ in bivalves (Zacks, 1955;

Reid.

al..
1972; Mathers, 1973; Teo and
The development of the gonad around the

1966; Payne ft

Sabapathy, 1990).

intestine optimizes the potential for the transfer of nutrients

to

developing gametes.
The results of the present study allow us to identify the
various cell categories and pathways that mediate the en-

tero-gonadal transfer system in bivalves: intestine epithelial

cells; large hemocytes which concentrate ferritin in cyto-

plasmic inclusions, in addition to that present freely in the

cytoplasm; and connective fibers which are often associated
with the hemocytes. Previous studies have shown that hemocytes may move across the intestinal epithelium: those
containing material of little or no nutritional value move
toward the lumen, while those containing nutritionally valuable material move from the lumen to the tissues surrounding the intestinal epithelium (see Cheng, 1996, for review of
bivalve hemocyte types and functions). The present study

shows

move

that

the scallop intestinal cells

nutrients

from the lumen

may, themselves,

to the basal lamina;

hemo-

cytes subsequently act as transport vectors to the surround-

This line of investigation does not seem to

have been pursued previously, despite long-held anatomical
ing

gonad

tissue.

knowledge of the scallop intestine-gonad

The pathway of
form largely

relationship.

intestine-oocyte transfer seems to con-

to that postulated

by Le Pennec

detailed histological observations (1991a).

et ai,

based on

These authors

proposed that nutrients assimilated by the intestinal epithelium are transferred to hemocytes at the base of the basal
lamina, as observed in the present study. They further
proposed

that the efficiency of this transport relied

upon

connective tissue fibers linking the basal lamina to the acini,

such that incorporated nutrients could be directed specifically to acini. In the present study, ferritin-containing he-

mocytes were always observed in association with connective tissue fibers between the base of the intestinal
epithelium and the bases of the acini.

The asymmetry in ferritin distribution between the male

and female parts of the simultaneous hermaphroditic gonad
is consistent with the difference in the composition and

AL.

spermatozoa with few energy reserves (see Beninger and Le

Pennec, 1991, for the sizes of spermatozoa and oocytes in
pectinids. and Beninger and Le Pennec, 1997, for the sizes
of spermatozoa and oocytes in bivalves generally); and
ferritin-containing hemocytes were never observed appressed to developing male gametes. This asymmetry suggests that the entero-gonadal

The

specific to female
is

described

fact that ferritin-containing hemocytes/follicle cells

the ferritin-containing cells might be able to distinguish

between these two

states, and can supply nutrients to the

oocytes most in need, i.e., developing oocytes. It should be
noted that the follicle cells detach from the mature oocytes

of Pecten maximus (Dorange and Le Pennec, 1989). Although both residual mature and residual developing oocytes

were

observed

in

individuals

pressed to
residual

that

had

cells

mature oocytes. This finding suggests

spawning

recently

were only observed apthe residual developing oocytes, and never to the

spawned, ferritin-containing

status

(i.e.,

that

the

prespawning or postspawning) does

not influence nutrient transport; rather, the oocyte developmental stage appears to be the determining feature of such

even when these gametes are destined for atresia

and metabolic recycling (Pipe, 1987b; Dorange and Le
Pennec, 1989; Le Pennec et a!., 199 la). The data of Figure
3 show that ferritin transfer to developing oocytes does not
transport,

occur

at a

uniform

rate for all individuals; indeed, after

300

min, some individuals had no ferritin-containing cells appressed to developing oocytes. While this could be due to
the stress induced by the experimental procedure, it could
also indicate that nutrient transfer from the intestine is not a

continuous physiological activity, or that among individuals, transfer is not synchronous on such short time scales.

We

unaware of any studies that present the dynamics of

oogenesis on such short time scales, but this is an interesting
are

physiological question.

While

the particular scallop gonad-intestine anatomical

is not common in bivalves, the digestive system

and gonad are generally closely associated and intertwined,
with loose connective tissue containing abundant fibers

relationship

between these epithelia (Galtsoff, 1964; Morales-Alamo

and Mann, 1989; Morse and Zardus, 1997). These similarities to the pectinid system suggest that transfer from digesdeveloping oocytes via a pathway similar to
above may be a general feature of bivalve

tive epithelia to

that described

physiology.

Acknowledgments

which

elaborate substantial vitelline reserves, they were rarely

observed within male gonad acini, which produce small

is

were always found in association with developing oocytes,

and never in association with mature oocytes, suggests that

consequent energetic demand of male and female gametes.

While ferritin-containing hemocytes/follicle cells were
readily observed appressed to developing oocytes,

pathway

gametes. Another such possible distinction

below.

The authors wish

to

thank Dr.

Ita

Widowati, Diponegoro

University, Semarang, Indonesia, for having carried out

PECTEN NUTRIENT PATHWAY TO OOCYTES

which
previous unpublished experiments on this subject,
inWe
are
work.
of
the
the
present
methodology
guided
debted to the staff of The Biological Bulletin for their very
rigorous editorial work on this paper, which allowed us to

improve earlier versions of the manuscript. As

a study of no direct economic or medical impact on humans,
the
it is not
possible to acknowledge funding from any of
French fundina aaencies.
substantially

91

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Zacks.

MBL

annual report 2002

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Rl

TABLE OF CONTENTS

R2

REPORT OF THE DIRECTORS CEO

R7

RESEARCH

R44 EDUCATION
R63 MBL/WHOI LIBRARY

R66 FINANCIAL INFORMATION

Dogfish thrombocytes,

Kyeng-Gea Lee

R69 GIFTS
R85

GOVERNANCE & ADMINISTRATION

R2

CEO

report of the director and

am

pleased to share with you

highlights from the Marine
I

Biological Laboratory for the

year 2002.

I've

Director and

now

served as

CEO of the MBL

for nearly three years.

being done here, the impact that our basic

research continues to have on human health

able

commitment our

staff

have to

their

and the remark-

scientists, faculty,

and

work and to the Marine

Biological Laboratory

first

strategic plan

organization.

who

I'm

most

grateful to

everyone

has contributed to the experience.

continue to be impressed with

the breadth of the science

as well as the environment,

Developing the Laboratory's

has been an exciting and illuminating process

involving participation at all levels of the

Establishing the Program

Infectious Diseases

One

Global

of the highlights of the year

ing our

was

establish

newest resident research program

in

Global Infectious Diseases. Funded by a

$5

itself.

in

million grant from the Ellison Medical

Foundation, the Program

who

links scientists

study disease-causing organisms with experts

(Developing

a Strategic Plan

in

molecular biology, phylogenetics, and

environmental microbiology and creates a

Much

my time in 2002 was devoted to

directing, with MBL President John Dowling
of

one-of-a-kind international center for research

and

training dedicated to studying pathogens

and the complex relationships they have with

and the aid of the consulting firm McKinsey &

Company, a planning effort aimed at devel-

their hosts.

oping a strategic roadmap for the Laboratory

over the next five to ten years. This process,

We

and the recommendations emerging to date,

are outlined in detail on our web site
(www.mbl.edu/inside/what/planning/
index.html). Although we continue to

tune the plan, the

initial

fine-

phases of the

a result of this effort,

we

initiatives.

These include developing a graduate

program with Brown University, hiring

Academic and
a

new

Birmingham where he was

a Professor in the

of Biochemistry

and Molecular

a Chief

program

in

in

the AIDS

Center and the Comprehensive Cancer Center.

Steve brings with him a number of students,
postdocs, and technicians.

welcomed two new

Scientific Officer, establishing

resident research

and molecular biologist Stephen L.

Hajduk to direct the Program. Steve comes to
the MBL from the University of Alabama at

Genetics and Senior Scientist

are already

undertaking a number of key

recruit leading parasi-

tologist

Department

process are largely complete.

As

were fortunate to

In

addition,

we

Assistant Scientists

Robert Sabatini and Andrew McArthur

Program

in

to the

early 2003.

Cellular

Dynamics and Imaging, expanding the Board

Steve's research

of Trustees, preparing for major renovations

the area of molecular and biochemical basis of

of the

Whitman

Building for

visiting research,

summer and

and working with a

site

planner to help us further reconfigure

and

renovate space to meet the emerging needs

of the strategic plan.

pathogenesis.

program

Many

is

broadly based

of his studies focus on

African trypanosomes, which cause

sleeping sickness, a

in

fatal

human

disease that has

reemerged as a major health problem

sub-Saharan Africa.

in

R3
The MBL has

The importance of research in infectious disease

cannot be overstated. No single reason can

a rich history in studying the basic

explain our inability to eradicate or minimize

science of parasitism and infectious disease.

Twenty years ago, the laboratory launched the

the global impact of infectious agents on

field of

human

health. In total,

25%

of

all

deaths

molecular parasitology with the estab-

lishment of

its Biology of Parasitism course,

which continuously reinvents itself as it trains

worldwide are caused by bacterial, viral, fungal,

and parasitic pathogens. Every year, one to
three million people die from malaria, a disease

new

in this

caused by the organism Plasmodium. Tuberculosis infects one person every second, and over

includes a strong training

the coming decade, at least 30 million

scientists

institutions.

Infectious Disease

within

Program is part
of the MBL's Josephine Bay Paul Center for
Comparative Molecular Biology and Evolution.

genome.

gens, Giardia

is

a principal

cause of diarrheal

in children and adults and

therapeutic
treatments for the parasite are almost as

devastating as the disease

of the Giardia

The

itself.

genome may

availabil-

Zettler

and

Erik Zettler

capability to integrate visiting

and give them access to

will

the field of infectious diseases globally

have on
is

very

exciting.

Other Resident Research Highlights

The MBL's

resident research program

other areas during 2002 as well.

Frederick Goetz
staff

in

January,

have

effects.

We

grew in
welcomed

when he joined

the

of the Marine Resources Center (MRC) as

Director of the Program

lead to the

identification of novel treatments that

minimal side

Heliozoan, Linda Amaral

lie

Excluding bacterial patho-

disease

ity

its

impact that the training component

ties

have contributed many important insights

about the evolution of parasitic protists using

G/ard/a

home

instruments and technology that would

otherwise be unavailable to them. The broad

to infectious disease. Bay Paul Center scientists

modern genomic approaches. Recently they

embarked upon the sequence analysis of the

will

The Program's unique strengths

scientists into the lab

Directed by Mitchell Sogin, the Bay Paul Center

has an active research program with strong

that

from the world to conduct research

they would be unable to do at their

from the disease.

The Global

component

allow tropical health and infectious disease

die

will

field.

ever-expanding
The Global Infectious Diseases Program
investigators

in Scientific

Aquacul-

Through exploring factors such as an

organism's nutritional and water quality

ture.

requirements, physiological characteristics,

The technology necessary to sequence entire

genomes is found at very few institutions.

reproductive biology, diseases, and genetic

background, the MBL's

Scientific

Aquaculture

Program
on the unique expertise of the Bay
Paul Center to provide more traditional

Program is developing novel research techniques and addressing problems faced by both
scientific and commercial aquaculture interests.

parasitologists the opportunity to

spearheading the Program's efforts to

use DNA technology as a tool to understand

The MBL's Global

will

Infectious Diseases

build

research and better apply

genomics to

research areas. This blending of

and parasitology

expand

their

their

genomics

create a uniquely producenvironment and provide the

exciting new discoveries on the
will

Rick

is

the growth, reproduction, and disease

tance

in

fish

resis-

and

commercially important
He is also focusing on enhancing

tive research

shellfish.

catalyst for

the MRC's culture and husbandry of biomedical

important pathogens.

models such as squid, clams,

and zebrafish.

toadfish,

Continued...

Clam egg, Robert Palazzo

R4

Woods Ho/e
Pigment

aerial,

cells

Doug Weisman

on the surface of the cunner spinal cord, Steven

Gabriele Gerlach also joined the

in

2002 as an Associate

Zottoli

staff of

Scientist.

MRC

the

specialist

in

behavioral ecology and population genetics,

Gabby

is

using the zebrafish

MRC

the

facilities at

The MBL's summer and

further strengthened in

a remarkable

and farsighted

2002 with

gift of $2.3 million

from long-time summer investigators Laura and

Arthur Colwin. Their

to explore the genetic basis of behavior.

visiting research

program was

gift

established the Laura

and Arthur Colwin Endowed Summer Research

Scientists at

The Ecosystems Center received

Fellowship Fund, which,

strong support from the National Science

provide

Foundation through the competitive peer-review

grant process again this year. Of special note is

in

the $2.7 million, multi-year award

in

support of

the Long-Term Ecological Research project at

Plum

Island

Sound, a research

Here

of Boston.

site

located north

scientists are studying

effects of land-use change on watersheds. The

Center also received $850,000 from the Andrew

W. Mellon Foundation

to support research on

nitrogen transformation

in terrestrial

landscapes.

when mature,

at the

MBL for a minimum of two months during

MBL is committed to ensuring

the summer. The

that the very best scientists have the opportu-

conduct research here each summer.

On

the recommendation of the Science

Awards
Visiting

MBL

for outstanding presentations at the

annual General Scientific Meetings

Research

in

During the meeting, which was held

During a typical

life in

organisms from squid to surf clams to zebrafish.

They ask how nerve cells communicate, how cells
regulate their complex processes, and

how

they

They explore how organisms reproduce and develop, how they fight disease, how
sense organs gather information, and how brains
proliferate.

cess

it.

The

investigators

who

gather each

approaches and
research. In 2002 we wel-

iner bring a diversity of

questions to their

comed 129

principal investigators

and 237 other

researchers from 124 institutions representing 12

countries.

2002.

in

the

Lillie

56 presentations
were made. After peer-review of all papers and

Auditorium August 12 to

Woods Hole summer, MBL

researchers look for basic principles of

We

can help do this by providing financial support

in the form of fellowships like these.

Council, the Laboratory reinstated the

[Summer and

will

support for approximately 10

independent investigators conducting research

the fields of cell and developmental biology

nity to

the

full

talks, four

14,

awards and two honorable mentions

were presented

in

the categories of Senior

Investigator (Peter Armstrong), Junior Investigator (Michael

Smotherman), Graduate Student

(Beate Mittman), and Undergraduate Student

(Jane La Du).

R5

Education

MBL/WHOI

The MBL/WHOI

The 2002 Education Program provided 499

graduate and advanced-level students from
288

tion

study a range of biological topics with

of the leading scientists in the world

nity to

some

and

serving as course faculty

lecturers.

and

and 203

staff

Library

The

lecturers to the courses

serials

in

day or
In

The

MBL

offered two

"Advances

in

new courses

2002.

was co-directed by

scientists'

early 2002, the

was delivering approximately 52%

and 100% of

its

databases

of

its

electronically,

night.

addition, the Library

new

actively involved

is

in

the

electronic research tools.

development
Thanks to a grant of $500,000 from the Andrew
W. Mellon Foundation, Library and IT staff are

Mitchell

developing the Universal Biological Indexer and

Organizer (uBio), an exciting new database and

Sogin, director of the MBL's Bay Paul Center,

and Claire M. Fraser of The

the Internet.

of

Genome Technology and

Bioinformatics,"

via

In

extending the Library's reach beyond its walls to

wherever its patrons may be at any time of the

2002. They represented 175 institutions and

33 countries.

in

being delivered directly to

is

desktops

members

Laboratory welcomed 554 faculty

grow in nonMore and more informa-

Library continues to

traditional directions.

and 31 countries an opportu-

institutions

Library

Institute of

Genomic Research. Twenty-four students from

Internet tool that provides an innovative

the U.S. and Europe participated

of accessing, sorting,

in

the inaugural course, held October 6 to

November 1, 2002. The course integrates

and

information contained

in

collating

means

taxonomic

databases distributed

throughout the Internet.

bioinformatics with the latest laboratory

techniques for
analysis,

genome

sequencing,

genome

was
portion of the Library's archival collection
summer of 2002 in the

also featured during the

and high-throughput gene

"Wall Charts as Art" exhibit

expression.

The

in

the Meigs Room.

showcased
reproductions
16 charts produced by Rudolf Leukart

exhibit

life-size

The second course, "Neuroinformatics,"

of the

was directed by Partha Mitra of Lucent

Technologies and Emery Brown of Massachu-

these charts were used as teaching aids

between

877 and

892.

Noted

for their detail,

General Hospital. Twenty-six students

held
participated in that course, which was

one

August 17 to September

of the original chromolithographs.

universities

setts

1.

of the few remaining complete collections

grateful to
In

MBL

also offers a

"Semester

Ann Weissmann

We

are

for curating this

outstanding exhibit.

addition to our programs for graduate

students, the

in

around the world. The Archives has

in

Environmental Science" program for undergraduates each fall. In 2002, 19 undergradu-

Outreach
|

ates from 14 colleges and small universities

around the country participated in the program, which is hosted by The Ecosystems
Center.
in its

The goal

6 th year,

is

of the

SES program, now

to help prepare the next

generation of leaders

in

environmental

science and policymaking.

During the

fall

of 2002, the

Avari Studios to reorganize

MBL's

web

site.

The new

MBL worked

with

and redesign the

was launched in

site

January 2003 to very positive reviews. Early data

show

that the site is being visited by nearly

50,000 unique users a month. We are continuing
to build and enhance the site, which is becoming

an increasingly important tool for institutional

advancement, marketing, communications, and

outreach. If you haven't seen the new site,
I

invite

you to

visit

it

at

<www.mbl.edu>.

Continued.

R6

[Construction

We

and

were fortunate

Facilities

in

Thomas

2002 to be able to

S.

Crane were elected to serve as

Continue to fund depreciation and upgrade

our facilities. Lab and office space in both the

and

Marine Resources Center and the Crane Wing

also elected to serve a final

year as Chair of

of the

Lillie

President of the Corporation, Treasurer,

building were substantially

Sheldon Segal was

Clerk, respectively.

the Board.

renovated for new Marine Resources and

On November

Global Infectious Disease program staff. The

Grass Fellows also enjoyed 1400 square feet
of newly renovated laboratory
space

Whitman

Building during the

2002. This

fully

and also

Fellows' research

foster the cooperative

and

to Al Zeien, the former

Chairman and

Board. Shelly has served as a

needs

Board

collegial

nature of the program. Educational programs

for

20

years, 10 as

Shelly has been,

force at the

MBL.

Loeb teaching

community,

building.

CEO of

its

member

Chair.

He

of the

now

will

also purchased a five-acre farm

and

will

On

continue to be, a

behalf of the

thank Shelly for

all

vital

MBL
over

his efforts

know we can count on

guidance and wisdom in the future.
these

MBL

as

gavel

serve as an Honorary Trustee.

program for undergraduates

from Williams College, were moved to the

The

his virtual

this renovation, including

displaced by

SPINES and

handed

the Gillette Company, who assumed the duties

of Chair at the February 2003 meeting of the

of

modernized space was

meet the

designed to

the

in

summer

down

2002, Shelly stepped

8,

Chair of the Board and

in

many

years.

his

Newbury, MA. The property will enable the

expansion of the MBL's Plum Island Research

Conclusion

Program, which focuses on understanding

Eel

Pond through the

front

doors of Ebert Hall

how

coastal ecosystems are affected by

changing land cover, climate, and sea

Upon

level.

the departure of Ecosystems Center

staff to

the C.V. Starr Building, the Homestead

Building

was gutted and renovated

for

use by

the administrative departments of Financial

Human

Services,

The Biological

now home
ment

Resources, Education, and

Bulletin.

The Candle House,

to the Director's Office,

Develop-

Office, Associates Office,

nications

and Public

received a modest

and

Commu-

are living

in

challenging times, and the

Marine Biological Laboratory is not immune to

the impact that the downturn in the national
having on businesses, foundations,

economy

is

and

and

state

local governments. Like everywe've

had
to tighten our belts and
one,

carefully consider the

impact new

may have on our budget. So

been able

to

in

positive

achieving the goals we've

2002.

far,

initiatives

the

MBL

has

weather the economic storm and

continue to work

Affairs Office, also

facelift in

We

ways towards

begun

to establish

through the strategic planning process.

This

[Trustees

is

an exciting time for the Marine Biological

Laboratory.

The Board of Trustees elected

members whose terms began

five
in

new

2002.

Margaret C. Bowles of Woods Hole, MA;

Martha W. Cox of Hobe Sound, FL; Walter

Massey of
Boston,

E.

look forward to working with you

further build

B.

M.D., of

Conrad, and

of this

Biological Laboratory continues to have a

E.

all
serving four-year terms
of the Class of 2006. In addition,

Dowling, Mary

upon the strengths

to ensure that the Marine

disproportionate impact on the biological,

biomedical, and environmental sciences long

into the future.

NY, are

members

John

we

vital institution

GA; Marcia C. Morris of

MA; and Gerald Weissmann,

New York,
as

Atlanta,

as

William

T.

Speck

R7

research
Throughout

its

history,

the

MBL

their research, not distracted

university

life.

that enables

Today 47
round

in

has been a place where the world's top biologists can focus on
by departmental affairs, committee work, or other aspects of

The MBL provides both the resource support and the

many

do

scientists to

principal investigators

areas such as

cellular,

intellectual

and and

their staff

conduct research

at the

Laboratory year-

developmental, and reproductive biology; molecular biology and

and sensory physiology; ecology and ecosystems

and marine biotechnology and aquaculture.

evolution; neurobiology
infectious diseases;

environment

their best work.

studies; global

Zebrafish cardiovascular

system, Jonathan

Muyskens

The population of
guished

scientists

investigators

grows dramatically each summer when hundreds of

distin-

from around the world gather here to do research. During a typical MBL
for basic principles of life in organisms from squid to surf clams to

summer, researchers look

They ask how nerve cells communicate, how cells regulate their complex processes,
and how they proliferate. They explore how organisms reproduce and develop, how they fight
disease, how sense organs gather information, and how brains process it. The investigators who
zebrafish.

gather each

number

summer

bring a diversity of approaches and questions. Along with the large

of faculty associated with the

exciting biological laboratory

Top photo, Elizabeth Armstrong

in

summer

the world.

courses, they

make

the

MBL the

largest

and most

R8

Resident Research

THE ECOSYSTT

CENTER

The Ecosystems Center, founded in 1975, is a collegia! association

scientists led by co-directors John Hobbie and Jerry Melillo. Its
mission

is

how ecosystems

to understand

are structured and

how

of

they

function, to predict their response to changing environments, to apply

the best scientific knowledge to the preservation and management of
natural resources,

and to educate

scientists

In

and

citizens of the future.

2002, the Center continued

the Semester

in

Environmental

Science. This program brings

undergraduates from a consortium of nearly 60 small

arts colleges

and

MBL campus

the

universities to
for

an inten-

sciences.

The complex nature

of

modern
CO-DIRECTORS

ecosystems research requires a

John

multi-disciplinary collaborative

Jerry M. Melillo

approach to address a variety of

questions. Accordingly, Center
scientists collaborate

than 60 projects.
The Ecosystems Centers

V.

Starr Building

our

We

field studies in

on more
conduct

many

Arctic to Brazil, from the

New

England to the estuaries of the eastern U.S.

One

question addressed

in

2002 was the

temperate forests of

effect that a

warmer climate

have on the high amounts of organic matter accumulated in forest

soils over the centuries. If microbes decompose most of the organic

will

matter, the forests

would switch from

a global sink of

carbon dioxide

gas to a source causing an acceleration of global warming. Jerry

Melillo and Paul Steudler of the Center have collaborated with University

the

of

New

soil

Hampshire

scientists in a

was heated

of 6 x 6

plots

decade-long experiment in which

above the temperature of similar

They found that soil warming did accelerate soil organic

matter decay and carbon dioxide fluxes to the atmosphere but that the
able to
response was small and short-lived because microbes were

control plots.

decompose

only a small proportion of the total organic matter.

Hobbie

E.

SENIOR SCIENTISTS
John E Hobbie
Charles
Jerry

locations, from the North

American and European

Eel grass. Rick Crawford

environmen-

sive introduction to
tal

liberal

S.

M.

Hopkinson

Melillo

Knute

J.

Nadelhoffer

Bruce

J.

Peterson

Edward B Rastetter
Gaius R Shaver

ASSOCIATE SCIENTISTS
Linda A- Deegan
Anne E. Giblin
ASSISTANT SCIENTISTS
Christopher Neill

Joseph

J.

Vallino

SENIOR RESEARCH
SPECIALIST
Paul

Steudler

R9

Robert Holmes at the Yem'sey

River, Russia

International Study Shows River Discharge

in Arctic Ocean is Increasing

Researchers from The Ecosystems Center, along with an interna-

Another question investigated microbes in nature.

These are responsible for many of the transformations
the carbon and nitrogen cycles that control important

ecological processes such as carbon storage and nutrient

between the
recycling. This study looked at the link
structure of the microbial populations in an Arctic lake

and

seasonal changes. This research, carried out by Byron

Crump and John Hobbie

of the Center, could only

be done with the

tance

in

assis-

molecular tech-

niques from Mitch Sogin of

the Bay Paul Center. New
techniques allowed the
identification of

microbes

and species changes,

An Ecosystems
collects

scientist

which

is

the

first

samples from the

Kuparuk River in Alaska

ecological function.

The

bacterial species

was

group

Northern Europe.

Ecosystems Center researchers Bruce Peterson, Robert (Max)

Holmes, and James McClelland led the team of scientists from
the United States, Russia, and Germany. They analy2ed discharge
data from the six largest Eurasian rivers that drain into the Arctic

Ocean. These

40%

rivers, all

located

in

Russia,

its

kind for

a resident population of
year.

of species

more than

1999. They contend that this measured increase in runoff is an

observed confirmation of what climatologists have been saying
that freshwater inputs to the Arctic Ocean and North
for years
Atlantic will increase with global warming.

discharge continues into the future, Arctic

of

for

Peterson and his colleagues found that combined annual

discharge from the Russian rivers increased by 7% from 1936 to

of linking species to
first

account

of total riverine freshwater inputs to the Arctic Ocean.

positive relationship

throughout the

distinctly different

step

the global climate, perhaps leading to the cooling of

towards the long-term goal

study, the

lakes, revealed that there

team of hydrologists and oceanographers, have documented that the flow of freshwater from Arctic rivers into the
Arctic Ocean has increased significantly over recent decades.
If the trend continues, some scientists predict this could impact
tional

in

"If

the observed

between global temperature and

increase to levels that impact Atlantic

river

Ocean

river

discharge

may

and

circulation

climate within the 21st century," says Peterson.

However, a

appeared when large

lake during the

and perhaps
This
runoff.
meltwater
matter,
organic
spring
the bacteria, came from the plant litter and soil in the

Continued.

amounts of organic matter entered the

surrounding watershed.

This project was funded by the Arctic System Science Program of the
National Science Foundation.

RIO
Arctic River Discharge, continued

v.vater flow to

significant increase

the Arctic

Ocear

}wn or shut

:3ntic

formation

off the

Water. The

drivir

underwater

"

Deep

-ehind the great

-sit"

current

known

Nation. Thermohaline

as thermoh.-

risible for moving great

mal energy around the

circuL

amounts

Staff
|

globe, influencing the planet's climate.

One of the potential effects could be

RESEARCH STAFF

Martin

Toby Ahrens, Research Assistant

Erica

Michele

Kristin S. Tholke,

Benstead, Postdoctoral Scientist

Neil D. Bettez. Research Assistant
Biesinger, Research Assistant
Mary S. Booth, Postdoctoral Scientist

Zy

F.

Elizabeth H. Burrows, Research Assistant

Alvarus

Data analyzed

December

magazine,

is

study, published in
2002, issue of Science

in this

13,

Bahr. Research Assistant

P.

Laura C. Broughton, Postdoctoral Scientist

Donald W. Burnette, Research Assistant

cooling of Northern Europe.

the

P.

Jonathan

important because

it

Chan. Postdoctoral Scientist

Colman, Research Assistant

S. K.

Benjamin

P.

Christopher

P.

Crockett, Research Assistant

Byron Crump, Postdoctoral Scientist

Jeffrey A. Evans, Research Assistant
Felzer,

Research Associate

represents net precipitation (precipitation

Benjamin

minus evapotranspiration) over

Solange Filoso, Postdoctoral Scientist

Robert H Garritt, Research Assistant

in

contrast to point

precipitation

a vast area,

measurements of

and evapotranspiration which

are difficult to extrapolate to a large area.

"These data are a unique measure of an

environmental trend both in terms of how
long the time series is and in that it
integrates over a vast area rather than just

Marcus O. Gay, Research Assistant

Joshua H. Goldstein, Research Assistant
Adrian C- Green, Research Assistant
Heather Haas, Postdoctoral Scientist
Diana C Garcia-Montiel, Staff Scientist
Darrell A. Herbert, Staff Scientist

Robert M. Holmes, Staff Scientist

Shaomin Hu, Research Assistant

locations," says co-author Stefan

Hughes, Staff Scientist

Samuel Kelsey, Research Assistant
David W. Kicklighter, Research Associate
Bonnie L- Kwiatkowski, Research Assistant

Rahmstorf of the Potsdam

James A Laundre, Research

measuring

a precipitation trend at a

few

Institute for

Climate Impact Research.

which

links

the work of hydrologists

and oceanographers,
fields of

will

stimulate the two

science to better communicate

their scientific findings with each other.

The

and oceanic components of the

Arctic

hydrologic cycle and on the biogeochemical tracers that allow scientists to follow
the circulation of riverine freshwater

throughout the northern oceans. This

research is needed to better understand
the current functioning of the linked land-

ocean-atmosphere hydrologic system and

improve confidence

in

predictions of the

future behavior of the vvstem.

researcher from .';:

osystems
Center collects water from a stream in
the Ipswich River watershed in the Plum
Island

Ecosystem Long Term Ecological

Research

site in

northeastern

Massachusetts. Investigators study

different land uses and their impacts on
nutrient loading into the estuaries of
Plum Island Sound.

R.

Tobias, Postdoctoral Scientist

Jane Tucker. Research Assistant

Zhenwen Wan, Postdoctoral Scientist

Ian J. Washboume, Research Assistant
Michael R Williams, Postdoctoral Scientist
Yuriko Yano, Postdoctoral Scientist
Qianlai Zhuang, Postdoctoral Scientist

ADJUNCT

SCIENTISTS

Robert W. Howarth, Cornell University

Paul A. Colinvaux. Smithsonian Tropical
Research

Institute (retired)

VISITING SCIENTISTS

James Galloway,

ADMINISTRATIVE STAFF
Kenneth H. Foreman, Associate Director,
Semester in Environmental Science Program
Dorothy

Berthel, Administrative Assistant

Anthony J Cave, Research Administrator

Suzanne J Donovan, Executive Assistant
Frances Johnson-Horman, Administrative
Assistant

Guillermo Nunez. Research Administrator

Deborah G Scanlon, Executive Assistant

CONSULTANTS

James

Staff Scientist

University of Virginia

Robert Naiman, University of Washington

Ann

Roxanne Marino,

Research Assistant

Thomas, Research Assistant

Mary Ann

Seifert,

Administrative Assistant

P. Bowles, Research
Designs
Margaret C. Bowles

Francis

McClelland, Postdoctoral Scientist

Patricia Micks,

group will focus their future work on the

links between the atmospheric, continental,

Assistant

Suzanne
Craig

Postdoctoral Scientist

Stieve, Research Assistant

Corey R. Lawrence, Research Assistant

John M. Logan, Research Assistant
L.
Lezberg, Research Assistant
Heidi Lux, Research Assistant

Project scientists are hopeful that this

study,

Jeffrey E.

Sommerkom,

L.

Research Assistant

Sarah Morrisseau. Research Assistant

Marshall L Otter. Research Assistant

Suzanne Randazzo, Research Assistant

Heather M. Rueth, Postdoctoral Scientist
Diane M Sanzone, Postdoctoral Scientist
Carol

Schwamb, Laboratory

Assistant

Karie A. Slavik, Research Assistant

Boardwalks protect vegetation from disturbance at research sites in the Arctic heath and
mountain birch ecosystem around the Abisko
Naturvetenskapliga Station in northern
Plots shown here are part of a pilot

Sweden.

soil-warming project begun

Abisko, Rose Crabtree

dunng

993 at

Rll

The Ecosystems Center's

Long-Term Ecological
Research

site at

Toolik

Knute

Lake, Alaska,

Nadelhoffer

Publications

Barren,

S.,

C. F.

Weber,

R.

Marino,

A.

E.

Currle,

W. Howarth.
2002. Effects of varying salinity on phytoplankton growth in a low-salinity coastal pond under
two nutrient conditions. Bfol. Bull. 203: 260-261
Davidson, G. Tomasky, and

N., and R. W. Howarth. 2002.

Modern Biogeochemistry. Kluwer, Dordrecht.

S.,

and

K.

J.

The

Nadelhoffer. 2002.

Harvard Forest. Ecosystems 5:446-460.

.

Currie,

Bashkln, V.

W.

Imprint of land use history: patterns of carbon

and nitrogen in downed woody debris at the

R.

W.

S.,

K.

Nadelhoffer, and

J.

B.

Colman.

2002. Long-term movement of 1 5N tracers Into

woody debris under chronically elevated N

S.,

D.

W.

Kicklighter,

J.

M.

Mellllo, C.

Using a Biogeochemistry Model. Report No. 90,

MIT Joint Program on the Science and Policy of
Global Change, Cambridge, MA.

fine

Garcia-Montiel, D. C. 2002. Legacy of

inputs. Plant Soil 238: 31 3-323.

S61 pp.

Felzer, B.

Wang, Q. Zhuang, and R. G. Prlnn. 2002. Ozone

Effects on Net Primary Production and Carbon
Sequestration In the Conterminous United States

human

97activity in the present neotropical forests. Pp.

Bettez, N., P. Rublee,

W. J. O'Brien, and M.

C.

2002. Changes in abundance, composition and cont rols within the plankton of a
Miller.

Freshw.

fertilized arctic lake.

Bio/. 47:

303-311.

Dargaville, R.

J.,

M. Heimann, A. D. McGuire,

C. Prentice, D.

W.

Clein, G. Esser,

J.

J.

M.

Melillo, B.

Kicklighter, F. Joos,

Foley,

Moore

Reichenau, A. Schloss,
E.

Boyer,

W.,

and

R.

W. Howarth, eds. 2002.

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Cycle. Kluwer, Dordrecht. 561 pp.

Williams,

and

J.

III,

R. A.

Kaplan,
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J.

Tian,

T.

U. Wittenberg. 2002. Evaluation

C0 2 measurements:

W., C.

E.

L Goodale,

N. A. Jaworski,

and

W. Howarth. 2002. Anthropogenic nitrogen

sources and relationships to riverine nitrogen
R.

export
istry

in

the northeastern

from

results

Bret-Harte, M.

S.,

G.

R.

Shaver,

and

F. S.

growth

in arctic

shrubs: implications for

16: 1092.

DOI:10.1029/

2001 GM001 426.

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M.

B.,

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A. Colinvaux. 2002. Orbital forcing signal in

sediments of two Amazonian

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Clein,

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and

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Zhang, D. W.

Wofsy, P. G.
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242:15-32.

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Garcia-Montiel, D.

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Res. Lett. 29: 14.1 -14.3.

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light manipulation. Arct. Antarct. Alp.

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of nutrient enrichment on the support of nekton

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and G.H.

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community response to environmental change.

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2002. Primary and secondary stem

III.

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57/58: 137-169.

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CO

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of terrestrial carbon cycle models with

atmospheric

Forests, M.R. Guariguata

Meier,

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Plant Biol.

4:

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the feasibility of using the nitrogen and oxygen

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Arctic National Wildlife Refuge,

Laura Broughton

R13
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F P. Bowles,

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to the climate system. Science 298: 2173-2176

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warming and carbon-cycle feedbacks

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responses of tropical forest

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J.

Laundre, A.

E. Giblin,

R Shaver. 2002 Fine root production and nutrient use

wet and moist arctic tundras as influenced by chronic
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S. Sheldon, L. Deegan, and R. Garritt. 2002.

and freshwater inputs from sewage effluent
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Pond, Falmouth, Massachusetts. Bio/. Bull. 203: 261-263

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in

R14

JOSEPHINE BAY

MOLECULAR

CENTER FOR

OGY AND EVOLUTION

The underlying theme

Bay Paul Center

is

of the Josephine

to explore the

evolution and interaction of

genomes

of diverse organisms that play signifi-

cant roles

DIRECTOR
Mitchell Sogin

SENIOR SCIENTISTS
Stephen Hajduk
Monica Riley

ASSISTANT SCIENTISTS

human

environmental biology and

in

health. This

dynamic research

program integrates the powerful tools

of

genome

science, molecular

phylogenetics, and molecular ecology

advance our understanding of how

living organisms are related to each
to

Giardia lamblia. Barb Davids (UCSD)

Michael Cuirtmings
Robert Sabatmi

other, to provide the tools to quantify

Jennifer

assess biodiversity, and to identify genes

Wernegreen

underlying mechanisms

and

and

of biomedical

importance.

Three interlocking programs define the scope

of research

in

the Program

in

Global Infectious Diseases, the

in

Molecular Evolution, and the

Program

in

Molecular Microbial Diversity. This

past year has marked significant growth

We

Bay Paul Center.

in

the

Mat Meselson's

attracted

molecular evolution program to the MBL.

Meselson is an esteemed member of the

Academy

ration with David

of Science

and

Linda Amaral Zettler

evolution group

explores

in

genome

A generous

in

collabo-

Mark Welch and Jessica

Mark Welch, he has established

Ocean-dwelling ancantnanan,

"River of Fire"

the Bay Paul Center. They are

Program

National

Extremes Molecular Technology

Uncovers Astonishing Diversity in Spain's
Life at the

a molecular

the Bay Paul Center that

evolution

award by the

in

asexual

rotifers.

Living conditions are

Medical

for bacteria, algae,

the countryside of southwestern Spain.

since

3000

B.C., the Rio Tinto contains

and

Mined
heavy metal

concentrations that are several orders of magnitude

higher than those of typical fresh water. New
findings from the Rio Tinto, published

in

the

May

9,

2002, issue of the journal Nature, present the first

molecular description of eukaryotes in a highly
acidic, high

metal environment and reveal the

The results
show that adaptation to extreme conditions is much
more widespread than originally expected and
provide a new understanding of the range of
River's incredible eukaryotic diversity.

organisms capable of
Ellison

tough

other microscopic organisms in the Rio Tinto, the

highly acidic, vividly crimson river that flows through

living at life's

extremes and

perhaps on other planets.

Foundation allowed us to move forward with a

dramatically

expanded program

in

Global

Infectious Diseases. This grant provided

support for a major renovation that accommodates 24 scientists and visitors to the Bay Paul
Center. Dr. Steve Hajduk

new

initiative

is

the director of this

and has brought

six

graduate

and post doctoral fellows to the MBL.

His

research emphasizes the post transcriptional

processing of RNA in African trypanosomes,

the cause of

human sleeping

sickness.

RNA

Eukaryota describes those organisms whose genetic

material is contained within a membrane-bound
nucleus. This includes plants, animals, and humans.
Previous studies of the Rio Tinto relied on morphol-

ogy to describe the

scientists to only a
ties

between

its

river's diversity

and alerted

few of the evolutionary

similari-

eukaryotic organisms. By examin-

ing the DNA of organisms extracted from the Rio

Tmto's sediment and biofilm, the slimy substance
that coats the surface of the River's water
scientists

and

rocks,

have uncovered new eukaryotic lineages

R15

editing results

in

nucleotides into

reading frames.

the post-transcriptional insertion or deletion of

mRNA,
It

at specific sites, creating functional

open

has been suggested that the enzymes involved

in

RNA

editing might be potential targets for drug development. As part

of this major expansion we have recruited two other new investigators
to the

the

GID program.

MBL

since 2000.

Dr.

Andrew McArthur has been

Andrew

directs

a Staff Scientist at

NIH-funded programs to explore

gene expression during different stages of the life cycle of the parasite
Giardia lamblia and in Trypanosoma brucei. Robert Sabatini studies
the role of unusual base modifications

"X-base." The enzymes involved

in

in

trypanosomes referred to as
may be

these genetic alterations

unique to Trypanosomes and therefore may serve as valuable targets

for

Finally,

we

offered a

new course

titled

Advances

in

Genome

Sciences

and Bioinformatics, which is co-directed by Mitchell Sogin of the

and Claire Fraser from The Institute of Genome Research (TIGR).

J. L.

de Lope,

J.

M. Sanchez

(pH levels between 1 .7 and 2.2) in the presence of iron concentrations

that can exceed 20 milligrams/ml; descriptions of eukaryotic diversity in
warm, anoxic sediments that are proximal to hydrothermal vents; the
the primitive eukaryote, Giardia lamblia; and
transfer of genes from several different bacteria into

discovery of introns

evidence of

genome

lateral

in

of Giardia lamblia.

Rio Tinto, continued

MBL

Important research publications this past year include descriptions of

eukaryotic microbial populations that thrive in very acidic environments

the

Rio Tinto,

drug design.

escaped detection by traditional methods.

The work, led by Linda Amaral Zettler and Mitchell
that

Sogin of the Josephine Bay Paul Center for

Comparative Molecular Biology and Evolution, has
also revealed

completely new
eukaryotes as well
as others which

have never been

seen before

in

such

a highly-acidic

environment.

In

Camponotus

nearcticus, collected in

Fa/mouth, MA. Adam Lazarus

mapping out the

Linda Amaral Zettler. Erik Zettler

evolutionary family
tree (or phylogeny)
for the Rio Tinto,

Amaral

Zettler, Sogin,

and

their

colleagues have

detected a close relationship between the River's

acid-loving eukaryotes

and other species that

The short evolutionary

prefer neutral environments.

distance between the two

tells

the scientists that

addition to being widespread,

occurring rapidly when measured on an evolution-

adaptations are,

in

ary time scale.

The project was funded by the National Science

Foundation's Life in Extreme Environments (or LexEn)
Program and NASA's Astrobiology Institute.

R16

Staff

Publications

RESEARCH STAFF

Cross,
c

Kieft, R. Sabatini,

Dirks-Mulder,

David Beaudom, Research A:

Chaves, and P Borst 2002 J-binding protein

increases the level and retention of the unusual

Steven

base

Linda Amaral Zettler, Staff

Patrick

Resean.

J. Biller,

Amy Crump,

Resear
:

..

Daniel Golde

Gosv

:.,

>

Scientist

ih,

II

Research Assistant

Ulandt Kim, Research Assistant

Abby Laatsch, Research Assistant

Moi Microbiol

1575-1584

P., Y Satou, R. K. Campbell, J. Chapman,

B Degnan, eta). 2002. The draft genome of
Ciona intestina/is insights into chordate and

Podar,

Group

vertebrate origins. Science 298 2157-2167.

vent environment App/ Environ. Microbio/

Lazarus, Research Assistant

Edgcomb, V P., D T Kysela, A. Teske, A.

de Vera GUmez, and M. L. Sogin 2002 Benthic
eukaryotic diversity in the Guaymas Basin
hydrothermal vent environment Proc Nat/.
Acad. Sci. USA 99: 7658-7662.

II

Mark Welch, Postdoctoral Scientist

David Mark Welch, Staff Scientist

Mullmeaux, H -R Huang, P S

Perlman, and

L Sogin

introns in a

II

2002

Bacterial

deep-sea hydrothermal

6392-6398

68:

Erica Lasek-Nesselquist, Research Assistant

Bruce Luders, Research Assistant

DNA

II

Adam

trypanosome

Wernegreen 2002.

J. J

strong effect of AT mutational bias on

ammo acid usage in Buchnera is mitigated at
high-expression genes. Mol. Biol. Evol 19

46: 37-47

Dehal,

...itdoctoral Scientist
,

J in

C and

Palacios,

ate Student

-earch Assistant

Josh Herb
Jennifer

Distant

Degnan,

Ashita Dhillon

Sulip

Sabatini, R

N Meeuwenoord,

H van Boom,

Borst 2002 Recognition of base J

duplex DNA by J-bmding protein J Biol

and

P.

in

Chem

277 958-966.

Jessica

II

Andrew McArthur,

Assistant Scientist

Hilary Morrison, Staff Scientist

Maria Murray, Research Assistant
Daniel Myers, Lab Assistant
II

Laila

Nahum, Postdoctoral

Olsson, Senior Research Assistant

Sarah Pacochal, Research Assistant

Carmen Palacios, Postdoctoral Scientist
II

Gretta Serres, Staff Scientist

Lynn Sherrer, Graduate Student

Jillian Ward, Research Assistant

Sabatini, R

Garcla-Verela,

M.

Cummings, G
Gardner, and J. P
P.

Gomez, E

Perez-

Ponce de Leon, S L
Laclette
2002 Phylogenetic analysis based on 18S
nbosomal RNA gene sequences supports the

N Meeuwenoord,

VISITING SCIENTISTS

Camargo, University of Sao Paolo,

Brazil

J.

H van Boom,

B., L

A.

Amaral

Zettler, B

Keenan, R Amils, and

Zettler, F.

M L Sogin 2002 Heavy-metal, acid-loving

"
Nature
eukaryotes from Spain's "River of Fire
417 137

existence of class Polyacanthocephala

(Acanthocephala) Mol Phy/ogenet. Evol

Simpson,

23: 288-292.

D D

G. B

Leipe, V. P.

Patterson,

Maristela

and P. Borst. 2002 Site-specific interactions of

JBP with base and sugar moieties in duplex
J-DNA. J. Bio/. Chem. 277 28,150-28,156
Simpson, A. G-

Scientist

Lorraine Olendzenski, Postdoctoral Scientist

Bertil

V. P., A. G. B. Simpson, L. A. Amaral

T A. Nerad, D J Patterson, M E
Holder, and M. L Sogin 2002 Pelobionts are
degenerate protists: insights from molecules and
morphology Mol Bio/. Evol. 19 978-982

Edgcomb,

Zettler,

and

Roger,

Edgcomb,

L.

Sogin 2002. Evolutionary

history of "early diverging" eukaryotes. the

Laan, M., H. Richmond, C. He, and R K

Campbell 2002 Zebrafish as a model for

excavate taxon Carpediemonas is a close relative

of Giardia. Mol Biol. Evol. 19: 1782-1791

Robert Campbell, Ares Pharmaceutical

vertebrate reproduction characterization of

the first functional zebrafish (Danio reno)

Lynne McAnelly, University of Texas, Austin

gonadotropin receptor Gen. Comp. Endocnnol

Sun, C.-H., D. Palm, A. G. McArthur,

125: 349-364.

and F

ADJUNCT SCIENTISTS
Matthew Meselson, Harvard

Silberman,

S Jermnn,

2002

Gillin

S.

G. Svard,

novel Myb-related

in
transcnptional activation of
encystation genes in Giardta lamblia Mo/.
Microbio/- 46: 97 1-984

protein involved
University

Roger Milkman, Professor Emeritus, University

of Iowa

Langford, T
Weiland, S

Sogin, and F

D. Silberman,

E -L

McCaffery, M L
2002 Giardia lamblia
and characterization of Rab
Svard,

J.M

Gillin

Klasson, B Canback,

Harold Zakon, University of Texas, Austin

identification

HIGH SCHOOL INTERN

and GDI proteins in a genome survey of the

ER to Golgi endomembrane system. Exp

Alexandria Papa

Parasite/ 101: 13-24

million years of

Tamas,
S

K,

Naslund,

Wernegreen, J P. Sandstrom,
N A Moran, and S G Andersson 2002 50
Eriksson, J

genomic

stasis in

endosymbtotic

bacteria Science 296- 2376-2379

VISITING
Alissa

UNDERGRADUATE

Cohen

Morrison,

H G

G Zamora,

K.

Campbell,

Sogin. 2002 Inferring protein function

from genomic sequence Giardia lamblia

and M.

L.

HHMI SUMMER UNDERGRADUATES

expresses a phosphatidyhnositol kinase-related

Sarah Biber

kinase similar to yeast and

Marsha Wheeler

Comp. Siochem

Physio/.

mammalian TOR.
B Siochem Mol Biol.

Chad Brock
Andrew Magis

Amy

McCurley
John Sander

ADMINISTRATIVE STAFF

Nixon, J E

J,A Wang,

J.

Field,

A G

McArthur, M. L Sogin, B
Samuelson. 2002 Evidence for

of hydrothermal sediments in the Guaymas

Basin: evidence for anaerobic methanotrophic

H G Morrison,
Loftus,

and

lateral transfer of

Wernegreen, J J 2002. Genome evolution in

bacterial endosymbionts of insects Nat Rev.

genes encoding ferredoxins, nitroreductases,

NADH oxidase, and alcohol dehydrogenase 3

Genet

from anaerobic prokaryotes to Giardia lamblia

Wernegreen, J J A. B Lazarus, and P. H

Degnan 2002 Small genome of Candiclatus

and Entamoeba
181-190

histolytica

Eukaryotic Cell

Nixon, J E J

A Wang, H G

Morrison, A. G.

McArthur, M. L Sogin, B Loftus, and J

Samuelson 2002 A spliceosomal mtron
Giardia intestina/is. Proc. Nat/. Acad.

99 3701-3705

3:

850-861

b/ochmannia, the bacterial endosymbiont of

Pauline Lim
Tara Nihill

V Edgcomb, A de

Vera Gomez, D. Kysela, S P. Sylva, M. L Sogin,

Jannasch 2002 Microbial diversity
and H

communities App/. Environ. Microbiol.

68: 1994-2007

133:477-491

NATIONAL SCIENCE FOUNDATION

RESEARCH EXPERIENCE FOR
UNDERGRADUATE STUDENTS

Teske, A., K -U Hmrichs,

Sci.

Camponotus, implies irreversible specialization

to an mtracellular lifestyle Microb/o/ogy 148:
2551-2556.

in

USA

R17

ARCHITECTURAL DYNAMICS

The

CELLS

IN LIVING

Architectural

Dynamics

in

PROGRAM

Living Cells Program, established

MBL

by Dr. Shinya Inoue in 1992, continues the pioneering

research and educational activities in biophysical inquiries directly
at the

that Dr. Inoue started at Princeton University

in living cells

1949. The Program focuses on architectural dynamics

cells

the timely and coordinated assembly and disassembly of

macromolecular structures essential

M/crotubu/es radiating
all directions from a

in

centrosome, Rudolf
Oldenbourg, with Robert
E.

Palazzo

and

in

in living

for the

proper functioning

and temporal organization

physiological and genetic control.

differentiation of cells, the spatial

and

of these structures,

The Program
of powerful
living cells

also

is

their

devoted to the development and application

new imaging

tools that permit such studies directly

and functional

have special expertise

in

cell-free extracts.

in

DISTINGUISHED SCIENTISTS
Shinya Inoue

SENIOR SCIENTIST
Rudolf Oldenbourg

Program members

the use of polarized light for analyzing

the local arrangement of molecular bonds and fine structure

STAFF SCIENTIST

II

Michael Shribak

in

biological specimens. Unique instrumentation developed by

Program members include the universal light microscope,

centrifuge polarizing microscope, the liquid-crystal based Pol-

Scope, and related technology. Biological phenomena currently

under investigation include mitosis/meiosis and related motility,

amoeboid movement, microtubule-centrosome

optical properties of green fluorescent protein.

Dynamics

in

Living Cells

MBL's resident

cell

and

Architectural

an active component of the

research group and promotes interdisciplinary

research and training

investigators,

Program

is

interaction,

The

among

its

resident core researchers, visiting

and collaborating manufacturers.

Meiosis

II

the crane

spermatocyte of
recorded with the

in
fly,

new

Pol-Scope, James R.
Lafountain (U. at Buffalo) and

Rudolf Oldenbourg

R18

Polarized fluorescence of single crystal of GfP rotated

between

parallel polarizers, Shinya

Inoue

|Pub//cat/ons

Inoue,

2002. Polarization microscopy.

S.

Current Protocols

In

Cell Biology, Suppl. 13, J.

Lippincott-Schwartz, ed. John Wiley & Sons,
in

pp. 4.9.1-4.9.27.
Inoue,

and

S.,

O. Shimomura, M Goda, M. Shribak,

2002. Fluorescence polarization

P.T. Tran.

of green fluorescent protein (GFP) Proc. Nat/.

Acad. So. USA 99(7): 4272-4277.
Inoue, S., and T. Inoue. 2002. Direct-view highspeed confocal scanner theCSU-10. In
Cell Biological Applications of Confocal

Microscopy, 2nd Edition, B. Matsumoto, ed.

Press, pp. 87-123.

Academic

Oldenbourg, R 2002. Retardance measurement

method. US Patent, Number 6,501,548. USA,
Assignee: Cambridge Research
tion Inc.

&

Instrumenta-

(Woburn, MA).

Shribak, M.,

S.

Inoue,

and R. Oldenbourg 2002

caused by differential

Polarization aberrations

transmission and phase

theory,

shift in

measurement and

high

NA

rectification.

lenses

Opt.

Eng. 41(5):943-954.
I

Staff
I., and R. Oldenbourg. 2002
Scanning aperture polarized light microscope:

Shribak, M.

RESEARCH STAFF
Diane Baraby, Research Assistant
Grant Harris, Software Engineer

observation of small calcite crystals using

oblique illumination. In Three-Dimensional and
Multidimensional Microscopy: Image Acquisition

Robert Knudson, Instrument Development Engineer and Research Associate

Szent-Gyorgyi, Research Assistant

and Processing IX, J.-A. Conchello, C. J.

Cogswell and T. Wilson, eds. San Jose,

Gwen

Proceedings of SPIE 4621: 104-109.

VISITING INVESTIGATORS
Michael Bennett, Albert Einstein College of Medicine
Michael Braun, Woods Hole Oceanographic Institution

Shribak. M., and R. Oldenbourg. 2002. Sensitive

measurements of two-dimensional birefringence

Robert Campbell, Serono

distributions using near-circularly polarized

beam. In Polarization Analysis, Measurement

Cohen, Yale University School of Medicine

Makoto Goda, Japan Biological Informatics Consortium, Tokyo, Japan
Peter Hepler, University of Massachusetts at Amherst
Joseph Hoffman, Yale University School of Medicine
David Keefe. Rhode Island Women & Infants Hospital
Larry

James R LaFountain, University

Un Uu, Rhode Island Women &

Woods

Hole,

ADMINISTRATIVE STAFF
Jane MacNeil

Inc

H. Goldstein, D. B.

Meng,
L

R. J.

Hackett, R.

Keefe. 2002 Rigorous

thermal control during intracytoplasmic sperm

injection stabilizes the meiotic spindle and

improves

fertilization

Fertil. Steril.

at

Chapel

Hill

Wobum, MA

MA

Japan

Universal Imaging Corporation,

H., L

Oldenbourg, and D.

INDUSTRIAL COLLABORATORS
Cambridge Research and Instrumentation,

Electric,

Wang,

Edward D Salmon, University of North Carolina

Orion Vanderlinde, Florida State University

Duggin, eds Seattle,

Proceedings of SP/E 4819: 56-67

at Buffalo

Mark Welch, Bay Paul Center, MBL

Timothy Mitchison, Harvard University
Robert Palazzo, Rensselaer Polytechnic Institute
Prem Ponka, McGill University, Montreal

Yokogawa

Infants Hospital

Jessica

Nikon, Japan
Technical Video,

and Remote Sensing V,

Egan and

Chenault,

Downingtown, PA

and pregnancy

77: 1274-1277.

rates.

R19

BOSTON UNIVERSITY MARINE PROGRAM

In

2002, the Boston University Marine Program realized

expanded

research focus

We appointed
I

two new

in

vision for an

assistant professors, Paul Barber (Berkely,

PhD;

Harvard, postdoc) and Gil Rosenthal (University of Texas PhD; UC San

Diego, postdoc). In addition, joint planning and advertising with the MBL's

staff

Marine Resources Center resulted

DIRECTOR
Jelle

its

behavioral ecology and population genetics.

in

additional strength

in this

area with

the appointment of MBL associate scientist Gabi Gerlach (University of

Konstanz). Their scientific perspectives complement the sensory and

Atema, Professor of Biology

FACULTY

behavioral ecology strengths

Paul Barber, Assistant Professor of Biology

John Finnerty, Assistant Professor of Biology

Stjepko Golubic, Professor of Biology

Les Kaufman, Associate Professor of Biology
Associate Professor of Biology
Gil Rosenthal, Assistant Professor of Biology

in

the labs of senior faculty

Phil

Lobel and

director, Roger Hanlon. Newly appointed MRC

senior scientist Rick Goetz further enhances the scientific goals of this

Jelle

Atema and

MRC

research focus. Gerlach and Goetz are both

Phillip Lobel,

BUMP

adjunct professors.

Ivan Valiela, Professor of Biology

The

ADJUNCT FACULTY
Roger Hanlon, MBL
Gabriele Gerlach, MBL
Anne Giblin, MBL
Frederick Goetz, MBL
Norman Wainwright, MBL

continuous stream of graduate students, postdocs, and international

visiting scientists. Its Research Experience for Undergraduates Program

coastal ecology

program headed by

Ivan Valiela flourished with a

continues to bring 10 outstanding undergraduates to

Woods

Anne

research that ends

Hole doing

Giblin of the MBL's

up being published regularly.

Ecosystems Center has an adjunct appointment at BUMP.

SENIOR STAFF COORDINATORS

Sharifa

Gulamhussein

With the new

Jennifer Ripley

ADMINISTRATIVE STAFF
Sheri Hall,

Stefano

Program Manager

Mazzilli,

Research Assistant, Valiela Lab

Michelle McCafferty, Program Coordinator

faculty,

the Program immediately took

in a

graduate class of

17 highly competitive students for both PhD and Masters degrees,

continuing its mission to provide exceptional educational opportunities to
students in Marine Biology. Several of our current 35 students work
directly with scientists at the

Woods

Hole Oceanographic

Institution,

and

the National Marine Fisheries Service.

Continued

The undergraduate program

also continued

its

mission successfully

by providing eight challenging research-based courses to 16 students

primarily from Boston University. Here too, student research has led
to several publications.
Cuttlefish

embryos.

Lisa Kerr

Lobel

R20

Publications

Armstrong, Peter

R.

Margaret

8.,

T.

Armstrong,

and Norman

Pardy, Alice Child,

Wainwright- 2002. Immunohistochemical

demonstration of a lipopolysaccharide in the
eukan/ote. the green alga,

cell wall of a

Ch/ore/la. Biol. Bull. 203: 203-204.

Rohrkasse, Sarah M., and Jelle Atema 2002.

whelks.
Tracking behavior of Busyconinae
Bio). Bull. 203: 235-236
Rosenthal, G.

Wagner,

preference for

P. H.,

M.

Moosa, and

K.

S. R.

of larval dispersal:
spatial scale

from Krakatau. Proc

examples
B 269:

Soc. Lond.

R.

S.,

J. Y.

invisible

1591-1597.

RESEARCH TECHNICIANS

Janelle

Morano

David Portnoy

VISITING FACULTY
Bill Simmons, Sandia
National Laboratory
Nathalie Ward, Lecturer

Amee Mehta
Karen Morschauser
Catherine O'Keefe
Mollie

Oremland

Aaron Rice
Deborah Rutecki
Andrea Shriver
Elizabeth Soule

Todd Stueckle

Summers
Melissa Sweeny

Erin

PhD STUDENTS
Brendan Anrrett
Jennifer

Bowen

Ruth Carmichael
Marci Cole

UNDERGRADUATE
STUDENTS
Alaina Avery
Colleen Butler

Eric Crandall

Sean Dixon

Heidi Fisher

Yael

J.

Vision

D.

Sara Grady
Kevin Kroeger

Alfred

Carolyn Miller

Laura Kloepper

Huang

Vanessa Miller-Sims

Heather Marlow

Elizabeth Neeley
Jason Philibotte

William

Mindy Richlen
Gregory Skomal

James Saenz

Molly Steinbach
Mirta Teichberg

Gabrielle

Tomasky

Joanna York
Erik Zettler

MASTERS STUDENTS
Abby

Atkinson

Andrea Bogomotni
Lisa

Bonacci

Chelsea Bouchard-Harnish
Jessica

Buckingham

Melissa Penn

Elisse Ruiz

Josiah Sewell

Jami Whitney
Kristen Wright

Moosa. 2002. Sharp genetic

K.

S. R.

breaks among populations of a benthic

marine crustacean indicate limited oceanic
larval transport: patterns,

consequence. Mol.

Ecol.

cause, and
1 1

659-674

Young
Chandra Ziegler

Marino, Eric Davidson, Gabrielle Tomasky,

and Robert Howarth. 2002. Effects of varying
in a lowsalinity on phytoplankton growth

pond under two

nutrient

Valiela. 2002.
A., and
of a
Response of growth and density
demissa to
population of Geukensia

land-derived nitrogen loading, in Waquoit

Massachusetts Estuar. Coast. Shelf So.
Bay,

Sarah Rohrkasse

Dianne Suggs

James Estrada

Jeremy Testa

Christopher Florio

Carolyn

Sophia Fox

Bradley Williams

Good
Dawn Grebner

SUMMER VOLUNTEER

Weber

Sarah

Jillian

Barber

SUMMER STUDENT
RESEARCHERS
Rachel Allen
Jillian

Barber

Margaret Johnson

Valiela.

and

mortality of

bay scallops.

response to
changes in food resources.

irradians, in

H. Carmichael, Sara
Suggs, Dianne N.. Ruth
Effects of
Grady, and Ivan Valiela- 2002.

on pairing

in

P.

horseshoe crabs

203: 225-227.

Jeremy M., Matthew A. Charette,

Edward R. Sholkovitz, Matt C Allen, Adarn
2002 Dissolved
Rago, and Craig W. Herbold.

Testa,

iron cycling in the subterranean estuary of a

I.,

and

J.

L Bowen.

2002,

and estuaries:
Nitrogen sources to watersheds
Role of land cover mosaics and losses within
watersheds. Environ. Pollut. 118: 239-248.

125-138

and K. D Kroeger.
I., J. L. Bowen,
2002. Assessment of models for estimation of
land-denved nitrogen loads to shallow

Valiela,

Gaines, Emily F., Ruth H. Carmichael,

Sara P. Grady, and Ivan Valiela 2002.
Stable isotopic evidence for changing
nutritional sources of juvenile horseshoe

crabs

Biol. Bull.

203: 228-230

estuaries. Appl,

Valiela,

I.,

Grasso,

F.

W., and

J.

Atema. 2002.

turbulent flows. Environ. Fluid Mech.

Weber, Carolyn
Tomasky, and

Cape Cod

Biol. Bull.

203:

247-248.
Millman, Melissa, Mirta Teichberg,
Paulina Martinetto,

and Ivan

Valiela

2002

to landResponse of shrimp populations

derived nitrogen in Waquoit Bay, MassachuBiol. Bull.

203: 263-264.

Philibotte, Jason. 2002 Pelagic larval

duration of the Caribbean wrasse,
Biol. Bull.

F.,

Stacy Barron, Roxanne

Gabrielle

W. Howarth,

Eric A.

Davidson. 2002

Nutrient limitation of phytoplankton growth

in

Falmouth,
Vineyard Sound and Oyster Pond,
Massachusetts. Biol. Bull. 203: 261-263
E. T., R. H Carmichael, and I. Valiela
2002. The effect of nitrogen loading on the

Weiss,

growth rates of quahogs (Mercenaria

mercenaria) and soft-shell clams (Mya arenaria)
through changes in food supply. Aquacu/ture
211: 1-4.
S., Jeffrey E. Hughes, and
Hunter-Thomson. 2002. Influence of
on fish predation
algal coverage

Williams, Bradley
Kristin

Thalassoma bifasciatum
203: 245-246

Cole 2002. Comparative

marshes and mangroves

L.

meadows from landprotect seagrass

derived nitrogen loads Ecosystems 5: 92-102.

Marino, Robert

2:95-114.

setts

salt

17: 935-953.

may

Integration of flow and chemical sensing

for guidance of autonomous marine robots
in

Geochem.

and M.

evidence that

habitats of

Jane La Du
Melissa Millman

I.

eutrophic-driven
J. Exp. Mar. Biol. Ecol. 279: 21-40.

Valiela,

Emily Gaines

Hunter-Thompson

and

2002. Growth, condition, reproductive

I.

Evgenidou,

Stacy Barron
Kristin

flicker.

coastal bay: Waquoit Bay, Massachusetts

Biol. Bull. 203: 255-256.

conditions. Biol. Bull. 203: 260-261

Hunter-Thomson, Kristin, Jeffrey Hughes,

and Bradley Williams, 2002. Estuarinefish community
open-water comparison of
structure in eelgrass (Zostera marina L.)

SUMMER REU INTERNS

H. S. Fisher,

Weber, Roxanne

Barron, Stacy, Carolyn

55.

MacLeod,

Shriver, A. C., R- H. Carmichael,

Biol. Bull

salinity coastal

A.

68.

2:

individual size

Julia

Pieter deHart

David Martel

and M.

Rowan

Michael Cermak

Andrea Hsu
Alison Leschen
Mark Lever
Vanessa Malley

Palumbi, M. V. Erdmann.

Barber, P.

I.

luminance and chromatic

potential,

Erlitz

Deirdre Grant

E.

from
Liang. 2002. Adaptation

Argopecten

Sarah Boyce
Devin Drown

W.

Shady,

Palumbi. 2002. Rapid recovery of genetic

the temporal and
diversity on coral reefs and

continued

Ryan, and

detection, retention and orientation to reefs.

Mar. Ecol. Prog. Ser. 241: 151-160

Barber,

Staff,

J.

Secondary loss of
swords in the pygmy

swordtail Xiphophorus nigrensis (Pisces:

Poeciliidae). Anim. Behav. 63: 37-45.

and

BUMP

M.

M. K Kingsford, and G Gerlach

2002. Larval reef fish could use odour for

Atema. J

Ruth Carmichael,

2002

Jr

epiphytic
rates in simulated eelgrass habitats. Biol
Bull.

203: 248-249

R21

MARINE RESOURCES CENTER PROGRAMS

DIRECTOR/SENIOR

The Marine Resources Center (MRC) is a national center for the development and use of aquatic organisms in basic biological research, biomedical research, aquaculture, and fisheries science. The research programs

SCIENTIST

focus on biological processes integrated at the level of the whole

Staff

Roger Hanlon

organism.

SENIOR SCIENTIST
Frederick Goetz

We made three primary faculty

ASSOCIATE SCIENTISTS

research ranks during 2002: Frederick Goetz,

Gabriele Gerlach

Senior Scientist; Gabriele Gerlach, Associate

Alan Kuzirian

Scientist;

ASSOCIATE SCIENTIST/
VETERINARIAN
Roxanna Smolowitz

and Scott

Lindell,

hires in the

Research

addition, two Assistant

Specialist.
Professors affiliated with the MRC were hired
In

by the Boston
Gil

University Marine Program:

Rosenthal and Paul Barber. Collectively,

these hires help establish a

year-round researchers

Marine Resources Center photos, Elizabeth Armstrong

in

critical

the

mass

MRC.

of

R22

in Sensory Biology, Behavioral Ecology

Population Genetics

Program

&

Our

studies of the physiological, sensory and genetic mechanisms of

behavior bridge neuroscience, behavior, and ecology. Such an ap-

proach allows us

(1)

to study evolutionary processes of natural

sexual selection that shape the lives of animals

investigate the genetic

and

and humans, and

consequences of behavioral interactions

(2)
in

to

an

ecological context, including the population level.

In

DNA fingerprinting, we discovered that there

2002, using

genetic stocks of squids

in

are five

the northwest Atlantic, and that each stock

tends to return to certain spawning grounds each summer. This finding

will not only revise the federal fishery management plan, but also
highlights the sophistication of the squids' sensory

and behavioral

abilities.

Social signals such as

fish

as well as

we found

in

pheromones regulate reproductive behavior

in

other vertebrates including humans. Using zebrafish,

male and female pheromones released during complex

behavioral interactions greatly influenced female reproduction. This
research

MRC Staff,

William Grossman, Marine Specimen Collector/Diving

Safety Officer
Janice Hanley, Water Quality and Animal Health Technician
William Klimm, Licensed Boat Captain - R/V Gemma

Scott Lindell, Manager, Aquatic Resource Services and

Aquaculture Research Specialist

Beth Linnon, Special Projects Coordinator
William Mebane, Aquaculture and Engineering Division

Superintendent
Gabrielle Santore, Executive Assistant

Andrew Sexton, Marine Organism Shipper

Daniel Sullivan, Boat Captain
Eugene Tassinari, Senior Biological Collector

Sean Whelan, Diver/Marine Specimen Collector

SEASONAL EMPLOYEES AND VOLUNTEERS

Brianne
at

Carroll, Intern,

Como,

Lawrence School
Massachusetts

Intern, University of

Dartmouth

Jay Dimond, Diver/Collector

George Gannon, Intern, Massachusetts Bay Community

College

Andrew

Sterling, Diver/Coilector

Christian Sterling, Diver/Collector

Monica Weedo.i,

will lay

background

continued

James Carroll, Life Support Technical Assistant

Edward Enos, Aquatic Resources Division Superintendent

Amanda

that

Intern, Pratt Institute

Photos by Elizabeth Armstrong

of

the foundation for future analysis of the genetic

fertility

and reproduction

in

vertebrates.

Learning and

memory experiments centered upon defining the

memory formation. Using the marine mollusc
Hermissenda, we were able to correlate memory acquisition and

different stages of

retention with the animals' responses to an accessory sensory stimulus.

By quantifying the immunocytochemistry, we demonstrated that levels

of calexcitin increase and persist during the formation stages of longterm memory. Calexcitin
calcium release

in

is

memory

key element for regulating internal

acquisition.

R23

in Scientific

Program

Aquaculture

program focuses on biotechnology research, applied research on

biomedical and commercial organisms, and policy development in
both of those areas. The biotechnology research is aimed at basic

AMERICORPS VOLUNTEERS (SENIOR MEMBERS)

mechanisms

Birgit

This

that control growth, behavior, reproduction,

and disease

commercially important finfish and shellfish. This includes studies on

novel regulators of growth and reproduction in fish and shellfish,
in

pathogen-regulated genes

in fish,

Pat Kosky

Joan Lemieux
Haskell

Maude

Nelson

Joseph Sheeny
Judith Sheehy
Joyce Wynne

and the development of molecularLaboratory of Roger Han/on

based diagnostic techniques.

STAFF
In

2002, with collaborators

in

any

fish

species

tiated trout

in

primary

macrophages.

Spain,

we

established

for the

first

time

technique to obtain differenthen demonstrated that only these

cell culture

We

macrophages can respond to pathogenic antigens by

upregulating early response genes such as tumor necrosis factors and
interleukins. It will now be possible to use global genomic techniques
differentiated

to obtain

of the

all

exposure and

this

macrophage genes
is

that are regulated

Roger Hanlon, Senior Scientist

Jean Boal, Adjunct Scientist
Kendra Buresch, Research Assistant
Martha Delaney, Research Assistant
Chris Florio, Graduate Student, Boston University
Nicole Gilles, Research Assistant

Mary Beth

by antigen
VISITING INVESTIGATORS

being pursued.

Chuan-Chin Chiao, Postdoc, Howard Hughes

received funding from the Southeastern Massachusetts Aquacul-

and developed new technology to help commercial

growers overwinter quahog clam seed, which will circumvent

ture Center
shellfish

the

60-80%

"winter-kill"

common

in

field-planted seed.

The

Policy Center for Marine Biosciences and Technology, directed by

former MBL Director Harlyn Halvorson, has now been aligned jointly

with the University of Massachusetts (Boston)

and the Marine

Resources Center. The Policy Center defines major problems in the

marine aquaculture and biotechnology, and conducts

fields of

international

Adjunct Scientist

Shashar, Adjunct Scientist

Mollie Tubbs. Research Assistant

Medical

We

Saffo,

Nadav

workshops to address these important

societal issues.

Institute

Melissa Grable, Graduate Student, Boston University

Marine Program
Nuutti Kangas. Postdoc,

Academy

of Finland

Miranda Karson, Graduate Student, Michigan

State University
Allen Mensinger, University of Minnesota at Duluth

Marie-Jose Naud. Graduate Student, Flinders University

MMBR Student, University of
California, Santa Barbara

Andrew Simpson,

INTERNS
Angela Abbott, Massachusetts Maritime Academy
Melissa Cox, Purdue University
Robert Nobuhara, Colorado State University

Reshma

Patel,

Emory

Camille Riviere,

University

EN. S.A.I. A.

Eric Stone, University of Massachusetts,

Dartmouth

Kate Sweeney, Colby College

Photos by Elizabeth Armstrong

Continued.

R24
|

Publications

Laboratory of Alan Kuzirian

STAFF

Atema.

Alan Ku:inan, Associate Scientist

Hemant Chikarmane, Investigator

fish

Herman

M.

J.,

Larval reef
Kingsford. and G. Gerlach. 2002
for detection, retention and orientation

could use odour

to reefs. Mar. Ecol. Prog. Ser. 241: 151-160.

Epstein, Investigator

G.

Basil, J. A.,

8.

Lazenby,

L.

Nakanuku, and R T. Hanlon.

to male conspecific

VISITING INVESTIGATORS

2002 Female Nautilus are attracted

Frank Child

odor. Bull. Mar. Sd. 70: 217-225

John

Clay,

National Institutes of Health

New York

Robert Gould,

State Institute for Basic Research

Borley, K. A., H.

T Epstein, and

M. Kuzirian. 2002 Effects

A.

of a sensory block on calexcitin levels in the photoreceptors

of Hermissenda crassicornis. Biol. Bull. 203: 197-198

INTERNS
Kimberly Borley, Ohio University
Alex Hangsterfer, Roger Williams University
Justin Walker, Massachusetts Maritime Academy

John R and Alan M. Kuzirian 2002. Trafficking of

axonal K' channels potential role of Hsc70. J. Neurosci. Res.
67: 745-752.

Clay,

Laboratory of Roxanna Smolowitz

N. Hanney, J. Lusk-Yablick,
Delaney, M., C Follet, N. Ryan,
and G. Gerlach. 2002 Social interaction and distribution of

STAFF

female zebrafish (Danio

Roxanna Smolowitz, Veterinarian

203: 240-241.

rerio) in a

large aquarium

Biol. Bull.

Kevin Uhlinger, Research Assistant

Gerlach, G., and S Bartmann. 2002 Reproductive skew,
costs and benefits of cooperative breeding in female wood

INTERNS
Krystal Baird,

Amy

AmeriCorps Member

Summer Veterinary
AmeriCorps Member

Hancock,

Jen Hsieh,

mice (Apodemus

Behav. Ecol. 13 408-418

M M N. Shashar, N. L. Gilles, C.-C. Chiao, and R

Hanlon. 2002 Cuttlefish body patterns as a behavioral

Grable,

Andrea Hsu, Graduate Student, Boston University

Kyle Hunt,

sylvaticus).

Intern

T.

Mashpee High School

assay to determine polarization perception.

Biol. Bull.

203:

232-234

Laboratory of Frederick Coetz

C and

Hall, K.

Hanlon 2002

R. T.

of the
Principal features

STAFF

of the giant
mating system of a large spawning aggregation
Australian cuttlefish Sepia apama (Mollusca: Cephalopoda)

Frederick Goetz, Senior Scientist

Mar. Biol. 140: 533-545.

Scott Lindell, Manager, Aquatic Resource Services

Marine models,

JoAnna DeNobi/e,
Irina

Chaikhoutdmov,

and

Lydia Louis

and Aquaculture Research Specialist

Hsieh,

M. Chikarmane,

H.

J. L..

Linda McCauley, Research Assistant

Steven Roberts, Postdoctoral Researcher

W. Mebane, and

Raquel Sussman

Bull 203: 266-267.

Andrew Sweetman, Graduate Student,

of

ozone

Iliev,

disinfection

in

R.

Kuzirian

Smolowitz,

K. R. Uhlinger,

2002 Microbial analysis of

a recirculating seawater system

Biol.

University

Bergen

Dimitar

A.

Graduate Student, University of Notre

Dame

Kusakabe, M., T, Todo, H. J. McQuillan, F. W. Goetz, and G.

of steYoung 2002. Characterization and expression
MLN64 cDNAs in
roidogenic acute regulatory protein and
trout. Endocrmology 143: 2062-2070.

Laboratory of Gabhele Gerlach

Roberts,

STAFF
Gabriele Gerlach, Associate Scientist
Jenny Lusk-Yablick, Research Assistant

S.

B 2002 Charactenzation of growth hormone

in several teleost species

yellow perch and myostatin
Abst. Int. B Sci. Eng 63: 1710

in

Oiss.

VISITING INVESTIGATORS
Thomas Breithaupt, Konstanz University

M B. 2002 Themes from variation: probing the

commonalities of symbiotic associations. Integr Comp.
42: 291-294

INTERNS

Shashar,

Martha Delaney, University of Massachusetts at Amherst

Chris Follett, Acton Boxborough Regional High School
Nick Hanney, Bishop Stang High School

and
cephalopods: neuroanatomical
behavioral features that illustrate aspects of form and
function. Mar. Freshwat. Behav. Physio/. 35 57-68

Saffo,

Nick Ryan, Thayer

N C A
,

Polarization vision

Milbury,

Biol.

and R T Hanlon 2002

in

Academy

Smolowitz, R J. Hanley, and H. Richmond. 2002

tumors in zebrafish
year retrospective study of abdominal
maintained in an aquatic laboratory animal facility Biol. Bull.
three-

203: 265-266.

M.

A. Stokes, R Smolowitz, R C. Karney, and E.

I.. N
Burreson. 2002. Haplosporidium costale (seaside organism),
a parasite of the eastern oyster, is present in Long Island
Sunila,

Sound

J.

Shellfish Res. 21

113-118

R25

Rat cardiac muscle

PROGRAM

cell.

Peter

molecular bases of cellular physiology. The several laboratories making

up the PMP focus on cellular plasticity and the properties of molecular

A variety of experimental approaches are used

ranging from molecular and biochemical methodologies, through

transport mechanisms.

niques.

advanced

An example

laboratories within

drion

in

health

optical

and electrochemical imaging tech-

of a research area spanning the

PMP

is

the role

and disease. How,

contribute to insulin secretion,

independent
of metabolism and the mitochon-

for

example, does the mitochondrion

heme

synthesis, or channel modulation?

Does the aging process, targeting metabolic disorders, contribute to

reproductive and neural malfunction, degeneration, and apoptosis?
In

addition to our interests

carry

on

in

basic biology the laboratories of the

a strong tradition within the

PMP

Marine Biological Laboratory

resident programs for instrumentation development.

Research Center

(NCRR)

has pioneered the use of electrochemical

in the

sensors to define cellular activity through monitoring conditions

extended boundary

(Staff

BioCurrents Research Center (NIH:

Transport of Bioactive Molecules; Development of
Electrochemical

and Optical Sensors

DIRECTOR/SENIOR SCIENTIST
Peter

J.

S Smith

RESEARCH ASSISTANTS
Katharine Hammar
Laurel

Moore

Richard Sanger

TECHNICIAN
Robert Lewis

Laboratory of Peter J.5. Smith: Molecular Physiology

of Transport and Sensor Development

SENIOR SCIENTIST
Peter

J. S.

Smith

The BioCurrents

a national bioengineering resource of the National

Institutes of Health

Smith

MOLECULAR PHYSIOLOGY

IN

The Program in Molecular Physiology (PMP) brings together a group of

resident and visiting scientists who share common interests in the

biophysics, to

J. 5.

STAFF SCIENTIST
Mark Messerli

POSTDOCTORAL RESEARCHERS
Abdoullah Diarra

layer.

VISITING SCIENTIST

notable characteristic of the

rative

PMP

is

the extensive year-round collabo-

outreach to regional universities and hospitals.

Members

ute to three Boston based NIH Program Project Grants

trafficking, diabetes,

rapidly advance

in

in

contrib-

protein

Radwan Khawaled

ADJUNCT

SCIENTIST
George Holz

and anemia. Collaboration also allows the group to

areas of topical interest

as with an

ongoing

study the molecular physiology of the multi-drug resistant

transporters, players of critical interest to cancer research and our
initiative to

understanding of infectious diseases. Annually, the member laboratories

host more than 40 national and international visitors, taking advantage
of the unique combination of scientific

and technical expertise concen-

trated at the Marine Biological Laboratory. Access

is

provided to

Laboratory of Stefan

Biophysics

ASSISTANT SCIENTIST
Stefan

McDonough

Laboratory of Orian
of Mitochondria

Shirihai:

Molecular Physiology

ASSISTANT SCIENTIST

experimental platforms, cutting edge imaging techniques, and a diverse

array of marine models suitable for studying both basic and biomedical

Orian Shirihai

problems. Strong collaborative and joint research projects are also

underway with other members of the resident MBL community

Sarah Haigh

notably the Architectural Dynamics Program and the Bay Paul Center.

McDonough: Channel

POSTDOCTORAL RESEARCHERS
Shana Katzman

Continued...

R26

Staff,

Publications

continued

Erica

Cooper, R

RESEARCH ASSISTANTS

and S Jung. 2002 Single

Electrochemistry: 9 (Biochemistry),

Corson

cell

In

electrochemistry.

&

S Wilson, ed Wiley

Encyclopedia of

Sons,

New York

Solomon Graf
R. T., L.

Kennedy,

Gil Palchik

M.

Kauri. G.

inp-cells Diabetes 51 (suppl.

Keefe, Adjunct

Maria A. Blasco,

Liu, L..

DIRECTOR
L.

Scientist,

Brown

functional telomeres in
University

Biol.

James R Trimarchi, and David L. Keefe. 2002. An essential role for

mouse germ cells during fertilization and early development Dev.

249: 74-84.

ADJUNCT SCIENTISTS
Lin Liu,

James

Brown University
Brown University

Smith, and D.

R.

mitosis after oocyte activation. Mo/. Reprod. Dev. 62 277-288

first

P. J. S.

Trimarchi,

L.

DNA integrity at

Keefe. 2002. Checkpoint for

Liu L, J

the

Trimarchc,

Liu, L, Maria A Blasco, and David L Keefe 2002 Requirement of functional telomeres for
metaphase chromosome alignments and integrity of meiotic spindles EMBO Reports 3:
230-234

POSTDOCTORAL RESEARCHER
Eva Czerwiec, Brown University

Laboratory of Ayse Dosemeci: Synaptic

Liu L, J

Plasticity

R.

Trimarchi,

and D

Keefe 2002 Haploidy but not parthenogenetic activation

in mouse embryos. Biol. Reprod. 66: 204-210

leads to increased incidence of apoptosis

ADJUNCT SCIENTIST
Ayse Dosemeci

oscillations

Liu, L., and David L. Keefe. 2002 Aging-associated aberration in meiosis of oocytes from
Senescence-Accelerated Mouse (SAM). Hum. Reprod. 17: 2678-2685.

/e Medicine:
Laboratory for Reprouu
Molecular Physiology of Reproduction

David

M. Dahlgren, and S -K Jung 2002 Metabolic

S152-S161

1):

Liu,

I.

R Trimarchi.

P. J. S.

Smith, and D. L Keefe 2002. Mitochondnal dysfunction

genomic instability Aging Cell 1 40-46.

leads to telomere attrition and

McDonough,

S.

I.,

L.

M. Mintz, and B. P Bean. 2002. Interactions among

and P-type calcium channels J. Gen. Physio/.

M. Boland.

toxins that inhibit N-type

I.

119 313-328
Robinson.

K.

and

A. Messerli

2002.

Pulsating ion fluxes and growth at the pollen

tube tip. (Online]. Science's STKE 2002 (162):

PE51.

Trimarchi,

J.

L.

Liu, P, J. S.

Smith,

and

D.

L.

Keefe 2002 Apoptosis recruits two-pore

domain potassium channels used for
homeostatic volume regulation.
Cell Physio/. 282:

Am

J.

Physio/.

C588-C594.

Twig. G R. P. Malchow, K Hammar. P J S.

Perlman 2002 A novel
Smith, H Levy, and
,

turtle retinal

preparation for simultaneously

measuring light-induced electrical

changes in metabolite levels Biol.
198-200

Representative immunofluorescence images of spindles (green!, actin filaments

oocytes from young and old mice. Lin Liu

(red),

and chromosomes

activity
Bull.

(blue) of

and

203

R27

A non-adhenng dam
(Mya arenaria)
leukemia cell, stained
reddish via the

LABORATORY OF AQUATIC BIOMEDICINE

monoclonal antibody
"1E10", atop a mat
of spreading, normal

clam hemocytes,
Carol Reinisch

This laboratory

is

dedicated to using

marine invertebrates as biomedical

models to study
the molecular

model,

we

issues of health at

level.

In

IBM

%. \

an embryo

are examining

how

chemicals influence neural

industrial

and function.

Specifically, polychlorinated
plasticity,
biphenyls (PCBs) or chemicals found in the wells of Brick, NJ, a site of
autism in children, are added to developing clam embryos and

development,

(Staff

SENIOR SCIENTIST
Carol L Reinisch

ADJUNCT SCIENTIST
Raymond Stephens. Boston

neuronal development assessed. We evaluate how chemicals pinpoint

molecular targets such as the p53 gene family. We are dissecting how

p53 gene expression and function are altered by chemical exposure.

Currently we are focusing on the p73 gene, which is critically important

in

regulating neuronal development.

University

POSTDOCTORAL

The second

RESEARCHERS
Rachel Cox

clams or mussels

line of research

examines the induction of leukemia

at industrially

sites.

polluted

We

Publication
in

have developed

technology to grow the tumor cells for genetic analyses.

(Supported by the Alternatives Research and Development.)

in

Jill

Kreiling

STUDENT

collaboration with the Division of Fisheries and

Daniel Kabat

we
VISITING SCIENTISTS
Sylvie St Jean. Division of

Fisheries and Oceans,

Moncton, Canada
Greg McCallum, Atlantic

Veterinary College,
Chartottetown, Prince

Edward

Island,

Canada

the blue mussel. Mussels are placed

Canadian harbors. Five to

and assessed

six

months

in

this laboratory.

Myt/7us edulis,

Thus

far

dirty sites in

family

is

developm en tally

expressed and responds to

the polychlorinated
biphenyls(PCBs) Environ.

Health Perspect. 110: 3

77-385.

the animals are retrieved

for cancer using a leukemia-specific

body generated by

in

both clean and

later,

In

Oceans (DFO) Canada,

are examining the rate of induction of leukemia

Jessen-Eller, eta/., 2002.

A new invertebrate
member of the p53 gene

vitro

monoclonal

anti-

we have determined that

exposure of mussels to PAHs, PCBs, heavy metals, and other industrial

compounds increases both the rate and severity of leukemia. This
research

is

funded by DFO, Canada.

72-hour-old Spisula

solidissima embryo,

stained with an

antibody to clam
neurofilament/
intermediate filament
protein (NF/IF).
Kreiling

Photo by Elizabeth Armstrong

Jilt

R28

LABORATORY OF BARBARA FURIE AND BRUCE FURIE

P-Carboxyglutamic acid

Brown, M.,

amino

a calcium-binding

acid that

is

found

[Staff

in

Hambe,

B.

is

the conopeptides of the predatory marine cone snail, Conus. This

laboratory has been investigating the biosynthesis of this amino acid

|Pub/ications

B.

Conus and the

structural role of

p-carboxyglutamic acid

in

ADJUNCT SCIENTISTS
in

Barbara C. Furie. Harvard

Medical School

the

Furie, B. C. Furie, J.

and L

Stenflo,

Stenberg. 2002. Detection

of vitamin K-dependent
proteins

in

venoms

with a

conopeptides. This satellite laboratory relates closely to the main

laboratory, the Center for Hemostasis and Thrombosis Research, on the
Harvard Medical School campus

in

Boston,

whose main focus

monoclonal antibody

synthesis and function of p-carboxyglutamic acid

specific for p-

proteins and the

carboxyglutamic acid,
lexicon 40: 447-453,
G.

Czerwiec,

E.,

K Taylor,

Stenflo,

and B

Furie,

S.

Furie.

Structural similarity

Begley,
B. C.

2002.

and

functional differences

between invertebrate and

vertebrate carboxylases:

expression and characterization of recombinant

vitamin

K-dependent

(J-

glutamyl carboxylase from

Conus

textile.

in

ur. J.

Biochem. 269:6162-6172.

blood clotting

VISITING SCIENTIST

Johan

Stenflo, University of

known

to synthesize the vitamin K-dependent

amino

acid, p--

carboxyglutamic acid (Gla), but the work of this laboratory and others
has shown that this synthetic pathway has been preserved in most animal phyla. The cone snail
produces neurotoxic conopeptides, some rich in Gla, which it injects into its prey to immobilize
To examine the biosynthetic pathway for Gla, we have studied the Conus carboxylase which
it.
converts glutamic acid to p-carboxyglutamic acid

in

the presence of vitamin K.

We

examined

the diversity of animal species that maintain vitamin K-dependent carboxylation to generate
Gla.

We

toadfish

have cloned

(Opsanus

full

tau),

length carboxylase from the beluga whale (De/ph/napterus /eucas), the

and the cone

rat,

crab (L/mu/us po/yphemus).

snail

and mouse

In

text//e)

to

sequences.

In

(Conus

cDNA

gallus), hagfish

addition, the Drosophi/a

compare these
addition,

most regions are nearly

sequence

similarity.

as a carboxylase

-dependent

structures to the

we have

(Myxine g/ut/nosa),

genome

gene. The predicted amino acid sequence of the carboxylase

Volker Steger

II

Eva Czerwiec

the marine cone snail had been the sole invertebrate

the carboxylase gene from chicken (Ga//us

snails,

Alan Rigby, Harvard

Medical School

the

role of vitamin K.

known bovine, human,

Cone

is

STAFF SCIENTIST
Until recently,

J.

Bruce Furie, Harvard

Medical School

partially

cloned

and horseshoe

contains the p-carboxylase

cDNA from Conus

textile

shows

mammalian sequence, and that there is about 40%

has been expressed, and the recombinant enzyme identified

identical to the

This protein

and epoxidase. These

results

demonstrate the broad distribution of the vitamin

carboxylase gene, including a highly conserved motif that

is

likely critical for

The vitamin K-dependent biosynthesis of Gla is a highly conserved function in

enzyme
the animal kingdom and we are now searching for a novel Gla containing protein that is critical
function.

for survival of animal species.

Lund

R29

LABORATORY OF NORMAN WAINWRIGHT

The mission
lar
in

of this laboratory

is

to understand the molecu-

defense mechanisms exhibited by marine invertebrates

response to invasion by bacteria, fungi, and viruses.
immune systems demonstrate unique and

Their primitive

powerful strategies for survival in diverse marine environments. The key model has been the horseshoe crab
Limulus polyphemus. Limulus hemocytes exhibit a very
sensitive LPS-triggered protease

cascade that

results in

blood coagulation. Several proteins found in the hemocyte

and hemolymph display microbial binding properties that
contribute to antimicrobial defense.

Commensal

may also augment

mechanisms of macroscopic marine species.

symbiotic microorganisms
crobial

or

the antimi-

Secondary metabolites are being isolated from diverse

marine microbial strains in an attempt to understand their
role. Microbial participation in

hydrogen

sulfide

is

oxidation of the toxic gas

also being studied.

Photo by Volker Sieger

I

Staff

DIRECTOR/SENIOR SCIENTIST
Norman Wainwright

Publication
|

RESEARCH ASSISTANTS
Alice Child

Kendra Williams
VISITING SCIENTIST

Armstrong, Peter
Alice Child,

B.,

Margaret

Armstrong,

R.

Pardy,

tochemical demonstration of a lipopolysaccharide in the

cell wall of a eukaryote. the green alga. CMore/la. Bio/. Bull.
203: 203-204.

Porter Anderson

Limulus, Volker Steger

T.

and Norman Wainwright. 2002- Immunohis-

Limulus

trilobites,

Beate Mittmann

R30

DSS section. Jack

Mertidal mats, John Spear

Farmer

CENTER FOR ADVANCED STUDIES

In

1995, NASA's

life

sciences programs and the

Crystalline

IN

MBL

THE SPACE

increase awareness of NASA's

life

NASA's interactions with talented

in

2002,

CASSLS had

several meetings

its

Advanced

CASSLS

strives to

sciences interests and to expand

biologists. In support of these goals

busiest year ever with the presentation of

and workshops.

meetings ranged in content

from information technology to evolutionary biology, and served more
than 125 participants. Additionally, in another workshop, 17 East Coast
Scientific

The

ADMINISTRATIVE
ASSISTANT

sciences.

Three Scientific Conferences were held at the

in

the National

Biological

Erik

Jonsson Center

for

Academy of Sciences:

Bulletin:

April 22-24, 2002.

"Limits to Self-Organization in Biological

"

Systems

Includes 12 peer-reviewed
articles

presented

in

Ph.D., of the

ranging from

"Combating Uncertainty with Fusion,"

collaboration with meeting Chair Misha Pavel,

Oregon Graduate

Institute

computational to
behavioral studies of

self-

organizing phenomena.
Bio/. Bull. 202. 243-320.

June 2002.

DIRECTOR

Heather K

Meeting proceedings published

Staff

Diana E Jennings

teachers spent four days learning about astrobiology and space

life

SCIENCES

established a

with the formation of the Center for

cooperative agreement
Studies in the Space Life Sciences (CASSLS at MBL).

LIFE

gypsum, John Spear

May

1-3,

2002. "Outcomes of

genome-genome

interactions,"

presented in collaboration with meeting Chair Mitchell Sogin,

Ph.D., of the Marine Biological Laboratory

September 22-24, 2002. "Understanding Mechanisms

Evolution," presented

in

of

collaboration with meeting Chair Eric

Davidson, Ph.D., of the California Institute of Technology

Teacher Enhancement Workshop held at the MBL:

November

22-24, 2002: "Life and Living in Space," co-directed

Diana
by
Jennings and Lorraine Olendzenski

Farrell

R3I

SUMMER AND

VISITING

RESEARCHERS

Many

visiting

MBL

investigators use marine organisms as

models

for

studying basic biological processes. Research using squids, sea

horseshoe crabs, dogfish, clams, toadfish, and sea slugs, for

example, has increased our fundamental understanding of a broad
urchins,

range of diseases and medical conditions including cancer, diabetes,

epilepsy, hypertension, multiple sclerosis, arthritis,

and neurological

disorders.

During 2002, the

MBL welcomed

129

and 237

Principal Investigators

other researchers from 124 institutions, representing 12 countries.

Members

of the

summer community come from

Harvard and Howard,

from the University of Alabama and the Universitat de Barcelona, from

the
Sea urchin

cell, Philip

Presley

Food and Drug Administration and the National

Institutes of

Health, from Canada, Argentina, England, and Switzerland,

many

MBL summer

researchers find an infrastructure and an informal,

interactive scientific

community

research almost immediately

always

seem

veteran

summer

upon

them

their arrival.

academic duties

to launch into

Advice and equipment

at their

home

summer
some

institutions,

do more hands-on research in

do
they
during the rest of the year at

scientists report they

three months at the

home

that allows

available from other researchers or from the

courses. Free from

their

among

other institutions, universities, agencies and countries.

MBL than

institutions.

Spider crab embryo

Gundrun Aspoek

R32

Spisub

:.

Jissima nuclei,

Anne Goldman

Elizabeth Armstrong

2002 Summer Investigators

Douglas, John K.
University of Arizona

Armstrong, Clay

Burgess, David

University of Pennsylvania

Boston College

Armstrong, Peter B

Camargo, Maristela
University of Sao Paulo,

University of California, Davis

Augustine, George J
Duke University Medical Center

Eckberg, William
Brazil

Howard

University

Canessa, Cecilia

Edds-Walton, Peggy

Vale University

Parmly Hearing

Institute of

Loyola University
Baker, Robert

New

York University Medical Center

Chang, Fred
Columbia University

Ellenberg, Jan

European Molecular Biology

Barlow,

Jr.,

Robert

State University of

Chappell, Richard L

B.

New

York

Upstate Medical University

Laboratory,

Germany

Hunter College, City University of

New

York

Clay,

John

fay, Richard

Loyola University of Chicago

Barry,

Susan

Mount Ho/yoke College

National Institutes of Health

Field, Christine

Harvard University Medical School

Cohen, Lawrence

Luis

Beauge,

B.

de Investigacion Medica
"Mercedes y Martin Ferreyra,"

Yale University School of Medicine

Argentina

Cohen, William D.
Hunter College, City University of

Institute

Bennett, Michael

Douglas

National Institutes of Health

New York

V. L.

Fields,

Albert Einstein College of Medicine

Fishman, Harvey M.
University of Texas Medical Branch,
Galveston

Crawford, Karen
Bodznick, David

St.

Mary's College of Maryland

Wesleyan University

Gadsby, David
The Rockefeller University

De
Botto, Florencia

Universidad Nacional de
Plata,

Mar del

Polavieja, Gonzalo
University of Cambridge, United

Galione, Antony

Kingdom

Oxford

University,

United Kingdom

Argentina

De Weer,
Boyer, Barbara

Gandhi, Sunil

Paul

Union College

University of Pennsylvania School

of Medicine

Brady, Scott

Denk, Winfried

The Salt

Institute

Garber, Sarah
T.

The Chicago Medical School

The University of Texas Southwestern Medical Center, Dallas

Max-Planck-lnstitute for Medical

Brown, Joel

Desai,

Albert Einstein College of Medicine

Yale University School of Medicine

Research,

Germany

Gerhart, John
University of California, Berkeley

Rooma
Giuditta, Antonio

Universifa di Napoli "Federico

Browne. Carole

Dickinson,

Wake

Children's Hospital

Forest University School of

Bonny

Italy

Goldman, Robert D.

Medicine
DiPolo, Reinaldo

Burbach, Peter
Rudolf Magnus Institute for

/nvestigaciones Cientificas,

Neurosciences, The Netherlands

Venezula

Institute

Venezolano

Northwestern University Medical

School
Gould, Robert

New York

Burger, Max
Novartis International

Switzerland

"
II,

Research

Dodge, Frederick

AG,

State University of

New York

Upstate Medical University

State Institute for Basic

R33

Groden, Joanna
University of Cincinnati

Gruenbaum, Yosef

Koonce, Micnael
Wadsworth Center

Pant,

Hansh

National Institutes of Health

Sugimon, Mutsuyuki

New

York University Medical Center

R34

Mitochondria/ dysfunction
and oxidative stress lead to
attrition and
chromosomal end-to-end

te/omere

fusions (indicated by

arrows)

in

mouse embryos,

Lin Liu

MBL

Research Fellows

Twenty-two

Peter Armstrong, Ph.D.

Cecilia

scientists received

University of California, Davis

awards to conduct

immune defense proteins and

defense processes of arthropods that show evolutionary

New Haven, Connecticut

"Cloning and characterization of ASIC channels in marine
"
Dr Canessa was funded by The Erik B Fries
vertebrates

research at the

MBL

in

2002.

M. Canessa, M.D.

Yale University,

His research focused on

conservation. Dr. Armstrong was funded by The Laura and

Arthur Colwin Endowed Summer Research Fellowship Fund.

Fellowship, the M.G F. Fuortes Memorial

Fellowship Fund, The Stephen W. Kuffler Fellowship Fund,
an MBL Research Fellowship, and the Ann E Kammer

Endowed

Memorial Fellowship Fund

Florencia Botto, Ph.D
Universidad Nacional de Mar del

Plata,

Mar del

Plata,

Argentina

burrowing species (e.g., crabs) on the

dynamics of organic matter in estuarine environments."
Dr. Botto was funded by the MBL Associates, The Catherine

"The

role of intertidal

Filene

Shouse Foundation, and the Lucy

B.

Lemann

Fellowship Fund.

J.

Fred Chang, M.D., Ph.D.

Columbia University College of Physicians and Surgeons,

New

York,

New

York

"Placement of the

"

cell division

Dr. Chang
was funded by The Universal Imaging Corporation

plane

Fellowship Fund.

Peter H. Burbach, Ph.D.

Karen Crawford, Ph.D.

Rudolf Magnus Institute for Neurosciences University

Medical Center, Utrecht, The Netherlands
"The stellate ganglion of the squid as a model for

neurodevelopment gene cascades." Dr, Burbach was

funded by The Stephen W. Kuffler Fellowship Fund and the
Baxter Postdoctoral Fellowship Fund.

Mary's College of Maryland, St. Mary's City, Maryland

"Molecular analysis of B-catenin expression, axes formation

St.

and

early

embryogenesis

insights into evolution."

in

the squid, Loligo pealei,

Dr Crawford was funded by

the Evelyn and Melvin Spiegel Fellowship Fund, the

MBL Associates, and the James A and Faith Miller

Fellowship Fund

David Burgess, Ph.D.

Boston College, Chestnut

Hill,

Massachusetts

"Cytokinesis in embryonic cells." Dr. Burgess was funded by

the Josiah Macy, Jr Foundation, the Robert Day Allen

Fellowship Fund, and the William Townsend Porter

Foundation.

Camargo, D.V.M., Ph.D.

Sao Paulo, Sao Paulo, Brazil
"An evolutive study of Th1/Th2 differentiation." Dr.
Camargo was funded by The Catherine Filene Shouse
Foundation, The Frederik B Bang Fellowship Fund, and an
Maristela
University

MBL

Research Fellowship

Bonny Dickinson, Ph.D.

Harvard Medical School and Children's Hospital, Boston
"Calmodulm and the unconventional myosins play key roles
in

FcRn

trafficking

by mediating interaction with the actin

cytoskeleton" Dr Dickinson was funded by The Laura and

Arthur Colwin Endowed Summer Research Fellowship, The
Frederik B Bang Fellowship Fund, the MBL Associates, and

an

MBL

Research Fellowship.

R35
John

K.

Oscar

Iribarne, Ph.D.
Universidad Nacional de

Douglass, Ph.D.

University of Arizona, Tucson

"An electrophysiological and anatomical study of central

visual pathways in Limulus po/yphemus." Dr. Douglass was
funded by the H. Keffer Hartline Fellowship Fund, the Plum
Foundation, John E. Dowling Fellowship Fund, and the
Herbert W. Rand Fellowship.

"The

role of the

Mar del

SW Atlantic

Plata,

intertidal

Chasmagnathus granu/ata in the dynamics of nutrients."

Iribame was funded by the Lucy B. Lemann

Dr.

Fellowship Fund.

Jan Ellenberg, Ph.D.

Diane Lipscombe, Ph.D.

Brown University, Providence, Rhode

European Molecular Biology Laboratory,

"The

Heidelberg,

identification of novel

among

Germany

"Mechanism of nuclear envelope breakdown (NEBD) in

echinoderm oocytes and embryos." Dr. Ellenberg was the
2002 Nikon Fellow, funded by Nikon Instruments, Inc.

Argentina
burrowing crab

Island

conus toxins to discriminate

voltage-gated calcium channels and their splice

Lipscombe was funded by The Catherine

variants." Dr.

Filene

Shouse Foundation and the

MBL Associates

Ido Perlman, Ph.D.

Sarah Garber, Ph.D.

Technion-lsrael Institute of

Chicago Medical School, North Chicago, Illinois

"Correlation of ion flux and regulation of cell volume."
Dr. Garber was funded by The Erik 8. Fries Endowed

Technology, Haifa, Israel

"Nitric oxide synthesis in the
vertebrate retina and

its

physiological and cellular

"
Dr. Perlman was
functions

Fellowship.

funded by The Gruss Upper

Yosef Gruenbaum, Ph.D.

Institute of Life Sciences at The Hebrew

Foundation.
University of

Jerusalem, Jerusalem, Israel

"Molecular and functional dissection of the nuclear lamina
in

the surf clam." Dr.

Gruenbaum was funded by The Gruss

Upper Foundation, The Frank

R. Lillie

Fund, The Erik

B.

Endowed

Fellowship, the Robert Day Allen

Fellowship Fund, and the H. Burr Steinbach Memorial
Fries

Fellowship Fund-

Prem Ponka, M.D., Ph.D.

McGi/l University, Montreal,

Canada
"Iron Trafficking in Erythroid Cells:
"

A Collaborative

Program
Ponka was funded by the Frank

Dr.

R. Lillie

Fund.

University of California, Riverside

Nancy

Ratner, Ph.D.

Her research focused on how molecular motors are

University of Cincinnati College of

Leah Haimo, Ph.D.

regulated to control organelle transport.

Dr.

Haimo was

funded by The Laura and Arthur Colwin Endowed Summer

Research Fellowship Fund.

Medicine, Cincinnati, Ohio

The title of her research project
was "Cyclin-dependent kinases

in

axonal transport." Dr. Ratner

was funded by the Frank R. Lillie
fast

Jorg Hardege, Ph.D.

Fund and The Herbert

Hull University, Hull, United Kingdom

"Do sex pheromone differences in Nereidid polychaetes
lead to reproductive isolation?" Dr. Hardege was funded

Fellowship Fund.

by the Lucy B. Lemann Fellowship Fund, The Charles R.

Crane Fellowship Fund and The John O. Crane Fellowship

J.

Fund.

Brunswick,

Mary-Ellen Harper, Ph.D.

"The Sea Squirt's Secret: How We Discovered Our

Chordate Ancestry." Dr. Whittaker was funded by the
Frank A. Brown, Jr. Readership Fund

University of Ottawa, Ontario, Canada

"Use and construction of self-referencing microelectro-

chemical probes for studies into the role of Uncoupling

Protein-3 (UCP3) in myocellular energy metabolism."
Dr. Harper was funded by The Laura and Arthur Colwin

Endowed Summer Research

Fellowship and the H. Burr

Steinbach Memorial Fellowship Fund.

Rand

Kevin Begos

Richard Whittaker, Ph.D.

University of

New

Brunswick

in

Fredericton,

New

Canada

R36

Grass Fellows
Nine

scientists participated in the

2002 Grass Fellowship Program

in

Neuroscience at the Marine Biological

Laboratory. The program is sponsored by The Grass Foundation and offers independent research opportunities to young neuroscientists. The 2002 program was directed by Dr. Susan R. Barry of Mount Holyoke
College. Dr. Melissa Ann Vollrath of Baylor College of Medicine was the program's Associate Director.

Rooma

Desai, Ph.D., Yale School of Medicine,

"Isolation of K' Currents Underlying the 'Chopper

Response' of the Principal Cells of Lateral Superior

Gonzalo Garcia de

Polavieja, Ph.D.,

UCLA

School

of Medicine, "Behavioral Algorithm and Circuitry

for Visual Motion Detection in the Leech"

Olive (LSO)"
Sunil Gandhi,

The Salk

Institute,

"Evanescent

Microscopy of Single Vesicle Recycling

in

Wave

Goldfish

Dima Rinberg, Ph.D., Bell Laboratories Lucent

Technologies, "Optical Recording of Multineuron
Activity Using Ballistic Delivery of Voltage Sensitive

Retinal Bipolar Terminals"

Dyes"

Matthias Gruhn, Ph.D., Cornell University,

"Correlation of Extracellular Nerve Recordings

Adrian Rodriguez-Contreras, Ph.D., University of

and Behavioral

and Morphology of Inhibitory Neurons in the

Midbrain Auditory Pathway of Chicken"

Activity in Live Crayfish Using

Implantable Electrodes and High-Speed

California, Davis, "Intrinsic Properties, Distribution

Video Technology"
Michael

Beate Mittmann,

Institut fur Biologic,

"The

Development of the Nervous System in the

Horseshoe Crab Limulus po/yphemus (Chemicerata,
Ziphosura) and its Implication for Arthropod
Relationships"

S.

Ph.D.,

Damian G. Wheeler, McGill

"Multiprotein

Nucleus"

Limulus centra/ nervous system, Beate Mittmann

Smotherman,

ing Control of Chromatophore

Cephalopod Brain"

Complex

UCLA, "DescendMotoneurons in the

University,

Signaling from Synapse to

R37

Other Research Personnel

Abe. Teruo, Niigata

University,

Ge, Lan, University of California, Riverside

Gifford, Raeann, University of Kansas

Japan

Christina, Williams College

Adams,

Akingbade, Kathenne, University of Oxford,

Gilland, Edwin,

Alber, Merryl, University of Georgia

Alimi,

Mariam, Wake Forest University

Health Sciences Center

Brown University
Guo, YiFan, Williams College
Greer, Jonathan,

Nikiya, Williams College

Ayliffe, Harold, University of Utah

Asomoah,

Gyoeva, Fatima,

Denmark

Hull University,

Brown

Berbenan, Graciela,

"Mercedes

University

Institute

de Investigacion Medica

y Martin Ferreyra," Argentina

Bertetto, Lisa,

Wesleyan

Jill,

Research, Russia

Hanney, Nicholas, Marine Biological Laboratory

Harrington, John, University of California, Davis

Harwood, Claire, University of Pennsylvania

Hatoum, Nagi, New York University Medical School
Helbig, Anmka, Institut fur Biologische Informationswerarbeltung,

Germany

University

Earlham College
Binion, Samantha, Emory University

Hellemons, Anita, Rudolf Magnus

The Netherlands

Biber, Sarah,

Bodily,

Institute of Protein

Hangsterfer, Alexandra, Marine Biological Laboratory

United Kingdom

Beach, Rebecca, Hollins University

Bearer, Elaine,

University Medical School

University School of Medicine

Grant, Philip, National Institutes of Health

Gratton, Michael Anne, University of Pennsylvania

Arnolds, David. Williams College

Artigas, Pablo. Rockefeller University

Bartels-Hardege. H

York University School of Medicine

Goldman, Anne, Northwestern

Gomez, Maria del Pilar, Boston

Alliegro, Mark, Louisiana State University

Banta, Gary, Roskilde University,

New

Gioio, Anthony, National Institutes of Health

Goda, Makoto, Japan Biological Information Research Center, Japan

United Kingdom

Neuroscience,

Institute for

Helm, Jessica, Yale University School of Medicine

Hepler, Peter, University of Massachusetts
Hernandez, Carlos, New York University School of Medicine

Stanford University

Bordenstem, Seth, Marine Biological Laboratory

Ohio University
Bernstein, Gil, Technion, Israel
Borley, Kimberly.

Hernandez, Ruben, University of Texas

Braun, Alexander, Hunter College

Brewton, Luke, University of Texas Medical Branch

Hess, Sam, National Institutes of Health

Brown, Jeremiah, Dartmouth College

Bucior, Iwona, Friedrich Miescher Institut, Switzerland

Hoffman, Mathew, Boston College

Holtz, Scott, Northwestern University Medical School
Homsi, Sara,

Wake

Forest University

Horseman, Nelson, University of Cincinnati

Cameron, Lisa, Univesity of North Carolina, Chapel

Cameron, Luiz, University of Rio de Janeiro, Brazil
Carroll, Amanda, Marine Biology Laboratory

Hill

Irina,

Dimitar, University of Notre

Dame

Jackson, Ticaria, Howard University

Johnson, Michael, University of Connecticut

Cefaliello, Carolina, University of Naples, Italy

Chaikhoutdin,

Iliev,

Hunter College

Chang, Donald, Kong Kong University, Kong Kong

Chen, Xiaobing, National Institutes of Health

Jurkovicova, Dana, National Institutes of Health

Chiao, Chuan-Chin, Massachusetts General Hospital

Chludzinski, John, National Institutes of Health

Katar,

Churchill, Grant, University of Oxford, United

Clark, Michael, Medical College of

Collis,

Kingdon

Mazkhan, Wayne State University

Khavandgar, Simin, Albert Einstein College
King, Curtis, University of Utah
Knowles, James, Colgate University
Koester, Helmut, Baylor College of Medicine

Georgia

Leon, University of Rhode Island

Conrad, Mara, Hunter College

Konnerth, Arthur, University of Munich, Germany

Koop-Jabobsen, Ketil, Roskilde University, Denmark

Coric, Tatjana, Yale University School of Medicine

Erica, Mt Holyoke College
Couch, Ernest, Texas Christian University
Cox, Melissa, Marine Biological Laboratory

Corson,

Lee, Kyeng-Gea, Hunter College

Hanna, Technion, Israel

YuLong, Duke University Medical Center
Lober, Robert, Medical Collegte of Georgia

Levy,
Li,

De

Stefano, Rosanna, University of Naples, Italy

Delaney, Martha, Marine Biological Laboratory

Lockard, Jon, National Institutes of Health

DeNobile, JoAnna, Hunter College

Dmeen, Shauna, Williams College
Djunsic, Maja, Yale University School of Medicine

Louis, Lydia, Rutgers,

Lowe,

Maddox,
Ehsanian, Reza,

NASA Ames

Research Center

Evans. Louise. Harvard Medical School

Eyman, Maria, Universita

di

Napoli "Fedenco

II,"

Italy

Fernandez-Busquets, Xavier, Universitat de Barcelona, Spam

Ferrara, Eugenia, Unversita di Napoli "Federico II," Italy
Salem, Williams College
Marine Biological Laboratory
Franzmi-Armstrong, Clara, University of Pennsylvania
Fevrier,

Follett, Christopher.

School of Medicine
Frick,

Andreas, Baylor College of Medicine

The State University of

New Jersey

Chris, University of California, Berkeley

Paul, University of

North Carolina

Marangoni, Maria Natalia, University of Buenos Aires, Argentina

Martinez, Gabnela, University of New Hampshire
Masgrau, Roser, Oxford University, United Kingdom

Maude,
Mbanu,

Haskell, Marine Biological Laboratory

Chijioke,

Wayne

State University

McAnelly, Lynne. University of Texas, Austin

McCurley. Amy, Richmond University

Molina, Anthony, University of Illinois at Chicago
Momose-Sato, Yoko, Tokyo Medical and Dental University, Japan

Montanez, Marlena, Mt Holyoke College

Montgomery, John, University of Auckland, New Zealand
Moran, Kimberly, New York University School of Medicine

Garnham, Give,

Moreira, Jorge, University of Sao Paulo, Brazil

Morfini, Gerardo, University of Texas Southwestern Medical Center

Gaszewska, Anna, Medical College of Georgia

Morgan, Anthony, University of Oxford, United Kingdom

Morse, Thomas, Yale University

Gainer, Harold, National Institutes of Health

University of Oxford, United Kingdom
Garza, John, University of Texas at San Antonio

Continued.

R38
Domestic

Najera, Julia, University of Texas

Nedoluzhko, Aleksey, Wadsworth Cent

Ng, Michelle, Boston College
Nobuhara, Robert, Colorado S*

Represented

Albert Einstein College of Medicine

Arizona State University

ersity

Nonaka, Mio, Kyoto Univere^

Normand,

Institutions

,-

Hampshire

Danielle, Unive':

Connecticut Health Center

Nuccitelli, Richard. Unive'

Barnard College
Baylor College of Medicine

scleston

O'Neal, Jessie;,
Obata, Shuictv
Olsen, Gary,

Ur.ivei

Beth

City University,

Illinois,

Japan

Urbana

Israel

Deaconess Medical Center

Boston College
Boston University School of Medicine

Ortiz, Christopi:e., University of California, Irvine

Brown

Palmer, Luc/, University of Minnesota

Pascal, Akil, Williams College

California Institute of

Passianoto, Caio, Marine Biological Laboratory

California, University of, Davis

Reshma, Marine Biological Laboratory

Peacock-Villa, Elizabeth, Dartmouth College

California, University of, Irvine

Brown

Cory,

Technology

California, University of, Berkeley

Patel,

Pelletier,

University

California, University of, Los

Angeles

California. University of. Riverside

University

Petersen, Jennifer, National Institutes of Health

California, University of,

Pollema, Sarah, University of Minnesota, Duluth

Prasad, Kondury, University of Texas Health Science Center

Cincinnati, University of
Colorado School of Medicine, University of

San Francisco

Columbia University
Quigley, James, Scripps Research Institute

Connecticut, University of
Cornell University

Rabbitt, Richard, University of Utah

Courant

Institute

Radojicic, Mihailo, Yale University

Redenti, Stephen, Hunter College

Remick, Kathenne. University of Texas Medical Branch

Rhodes, Paul,

New York University

Medical School

Dartmouth College

Duke
Duke

University

University Medical Center

Richmond, Hazel, University of Minnesota

Ridings, Corey, Occidental College
Rieder, Leila,

Albany High School

Rinkwitz, Silke,

Ripps, Jeff,

New York

Towson

Rummel, John,

University Medical School

University

NASA

Sanchez, Carlos, University of Texas

Sato, Katsushige, Tokyo Medical and Dental University, Japan
Satpute, Prasanna,

Brown

University

Schnackenberg, Bradley, University of Carolina, Chapel Hill

Scotto Lavina, Zeno, National Institutes of Health
Short, Michelle, Marine Biological Laboratory
Shumaker, Dale, Northwestern University Medical School
Barbara
Simpson, Andrew, University of California, Santa
Smillie, Darren, University of Edinburgh, United Kingdom
Smith, Kalmia, Cornell University

Steeds, Craig. Kansas University

Eric, Marine Biological Laboratory
Sweeney, Catherine, Marine Biological Laboratory

Stone,

Takahashi, Hajime,

Olympus

Optical

Co

Ltd.,

Japan

Tanner, Geoffrey, Wesleyan University

Terasaki. Mark, University of Connecticut Health Center

Thompson, Reid, Dartmouth College

Tokumaru, Hiroshi, Duke University Medical Center
Tokumaru, Keiko, Duke University Medical Center
Twig, Gilad, Technion, Israel
Tzur,

Yonatan, Hebrew University,

Israel

Emory

University

Vautrin, Jean, University of Montpellier, France

Vetrano, Anna, Rutgers University

Villalba-Galea, Carlos,

Duke

University Medical Center

Federal Department of Agriculture

Flower Garden Banks National Marine Sanctuary

Vucinic, Dejan, Yale University

Food and Drug Administration

Wachowiak, Matt, Yale University School of Medicine

Weedon, Monica, Marine Biological Laboratory

Gladstone

Wetherington, Jonathan. Medical College of Georgia

Hartford, University of

Weyand,

Ingo, Institut fur Biologische Informationswerarbeitung,

Germany
Wheeler. N'sreha, Earlham College
Williams, Keuuirsh. Howard University
Wollert. Torsten. Universitat Rostock,

Germany

Yamasaki, Michiko. jnivc-rsity of Oxford, United Kingdom

Young, lain, University of Pennsylvania
Zakevicius, Jane, University of Illinois College of Medicine
Zakon. Harold, University of Texas, Austin

Institute of Neurological

Harvard University
Harvard University Medical School
Hawaii, University of

Howard

University

Hunter College
Illinois,

University of

Disease

R39

Kansas. University of

Foreign Institutions Represented

Louisiana State University

Loyola University of Chicago

Auckland, University

Maryland, University of
Massachusetts, University of

New

of,

Zealand

Barcelona, Universitat de, Spain

Buenos

Medical College of Georgia

Miami School of Medicine, University of

Aires, University of,

Cambridge, University

of.

Argentina

United Kingdom

Michigan State University

Millersville University

Minnesota, University of

Edinburgh, University of, United Kingdom

European Molecular Biology Laboratory, Germany

NASA

Friedrich Miescher Institute, Switzerland

National Institutes of Health

National Institutes of Mental Health

New York
New York
New York

Hebrew

State Institute for Basic Research

University

University of Jerusalem, Israel

Hong Kong

University,

Hong Kong

Hospital for Sick Children,

University School of Medicine

North Carolina State University

North Carolina, University of

Northwestern University Medical School

Hull University,

Institut fur

Biologische Informationswerarbeitung,

Germany

Institute of Protein Research, Russia

Institute

Ohio University

Canada

United Kingdom

de

Investigacion Medica

"Mercedes

y Martin Ferreyra,

Argentina
Institute

Venezolano Investigaciones

Cientificas,

Venezuela

Penn State University

Pennsylvania, University of

Japan Biological Information Research Center, Japan

Pomona College
Providence College
Puerto Rico, University of

Kyoto

University,

Japan

Max-Planck-lnstitute for Medical Research,

Rutgers, the State University of

New

Jersey

McGill University,

Germany

Canada

Montpellier, University of, France

Scripps Research Institute

Munich, University

Germany

of,

South Carolina, University of

Mary's College of Maryland
Stanford University
St

State University of

New

York Upstate Medical University

Napoli "Federico II", Universita di, Italy

New Brunswick, University of, Canada

Nugata

University,

Japan

Syracuse University

Novartis International

Texas Health Science Center, University of

Texas Southwestern Medical Center, University of

Olympus

Texas,

University of,

Texas, University

The Rockefeller

of,

Austin

Optical

Co

AG, Switzerland
Ltd

of,

Canada

Oxford, University

of,

United Kingdom

San Antonio

University

Rio

de Janeiro,

University of, Brazil

Roskilde University,

Denmark

Union College

Rostock, Universitat,

Utah, University of

Rudolf

Virginia, University of

Sao Paulo,

Wadsworth Center

Tokyo Medical and Dental

Wake

Japan

Ottawa, University

Magnus

Germany

Institute for

Neuroscience, The Netherlands

University of, Brazil

Technion-lsrael Institute of Technology, Israel
University,

Japan

Forest University

Washington University School of Medicine

Wayne State University School of Medicine

Universidad Nacional de Mar del Plata, Argentina

University of College London, United Kingdom

Wesleyan University

Utrecht, University of,

The Netherlands

Williams College

Women

and

Infants Hospital

Yale University
Yale University School of Medicine

Yokohama

City University,

Japan

R40

2002
FRIDAY EVENING LECTURES

June 21
Barry Bloom, Harvard School of Public Health
"Economic and Political Implications of

Global Infectious Diseases"

June 28
R.

Alan

B.

Ezekowitz, Massachusetts
Hospital for Children

"Fighting Infections from Flies to

Gen
Man"

JulyS
Lang Lecture
Michael J. Ryan, University of Texas, AL
"Sexual Selection and The Brain"
July 12

Naughton, Advanced Tissue Sciences

"Stem Cells and Tissue Engineering:

Gail K.

General

Scientific

Meetings Awards

From Science

On

recommenda-

the

tion of the Science

MBL
the MBL

Council, the
reinstated

Award

for outstand-

ing presentations at
the Laboratory's

annual General
Scientific

Meetings.

The award

in

each

category consists
a crystal clock

is

and

Senior Investigator:
Peter Armstrong, Margaret Armstrong, R. L. Pardy,
Alice Child, and Norman Wainwright, "Histochemical

demonstration of lipopolysaccharide in the

green alga Chlorella"

cell

wall of a eukaryote, the

motoneurons

the Meetings, which

standing population dynamics of the toxic

were held August 72

dinoflagellate A/exandrium fundyense"

Auditorium. After
all

talks,

four

awards and two

Student Honorable Mentions:

Jeremy M. Testa, Matt Charette, Edward Sholkovitz,
Matt Allen, Adam Rago, and Craig Herbold,
"Dissolved iron cycling in the subterranean estuary
of a coastal bay: Waquoit Bay, Massachusetts"
D. E. Arnolds,

p; -sented.

"Seeing Motion: Linking Neurophysiology

to Perceptual Psychology"

Michael Brown, University of Texas

Southwestern Medical Center at Dallas
"Genetic Defenses Against Heart Attacks"

August 2
Steven Hyman, Harvard University
"Reflections on Behavior in
the Postgenomic Era"

Dan

Barry,

NASA

"Sensations of Space Flight"

August 16
Tim Hunt, Cancer Research UK,
Clare Hall Laboratories

honorable mentions

were

University
I.Thursday, July 18
"Making Decisions: The Brain's Link Between

August 9

Lillie

peer-review of

Newsome, Stanford

July 26

were given during

papers and

in

Graduate Student:
Beate Mittmann, "Early neurogenesis in the
horseshoe crab Limu/us po/yphemus and its

Undergraduate Student:
Jane La Du, Deana Erdner, Sonya Dyhrman, and
Don Anderson, "Molecular approaches to under-

presentations

to 14 in the

William

implication for arthropod relationships"

a $300 cash prize.

Fifty-six

July 18, 19
Forbes Lectures

Perception and Action"

II.
Friday, July 19

Junior Investigator:
Michael Smotherman, "Acetylcholine mediates
excitatory input to chromatophore
the squid, Loligo pealei"

Fiction to Medical Fact"

5. J. Zottoli,

C. E.

Adams,

S.

M.

Quo, and A. J. Pascal,

"Physiological effects of tricaine on the
Dineen,

S. Fevrier, Y.

supramedullary/dorsal neurons of the cunner,

Tautogo/abrus adspersus"

Nerve branches and synaptic endings

mouse, Jeff Lichtman

in a

musc/e of a transgenic

"What

How

is

the Cell Cycle and

Controlled?"

is it

R41

Pub/ications

induced

C 2002. Coiled body heterogeneity

Alliegro,
+ bumetanide. Exp. Cell Res.
by G1 arrest with amilonde
279 111-117

Cox,

B. L.,

Belz,

inclusions

2002 Nuclear injection

Alhegro, M C and M A Alliegro
of anti-pigpen antibodies inhibits endothelial cell division
,

Bio/.

Chem. 277: 19,037-19,041

Margaret T Armstrong, R L Pardy,
and Norman Wainwright 2002 Immunohis-

Armstrong, Peter B
Alice Child,

tochemical demonstration of a lipopolysacchande in the

Biol Bull
cell wall of a eukaryote, the green alga, Ch/orella

203 203-204

R.,

Popa,

Bazylinski,

D A

Lanoil

Douglas,

and Nealson, K.H 2002

and
Organization and elemental analysis of P-, S-,
S

Engler,

in

Fe-rich

a population of freshwater magnetococci.

Geomicrobio/.

19.

J.

387-406

Crawford, Karen 2002 Culture method for in vitro

fertilization to hatching of the squid, Loligo pealeit
Bio/. Bull- 203 216-217,
2002.
DeGiorgis, J A T S Reese, and E L Bearer
with axoplasmic organelles
Association of myosin
Mol. Biol Cell 13
implications for axonal transport
,

II

1046-1057

D E

Arnolds,

W.. S

Zottoli,

E Adams,

S.

M. Dineen,

S Fevrier, Y Guo, and A J Pascal 2002. Physiological

effects of tncaine on the supramedullary/dorsal neurons
Biol Bull 203:
of the cunner,

Tautogolabrus adspersus

188-189
of
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the cytoskeleton in the life cycle of viruses and mtracellular
bacteria tracks, motors, and polymerization machines.
Curr. Drug Targets Infect. Disord 2 247-264
,

Eddleman, C S

SEM comparison

G D

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and H

Fishman 2002

of severed ends of giant axons isolated

from squid (Loligo pea/en) and crayfish (Procambarus

B.ol. Bull. 203: 219-220
darkii)
Edds-Walton, P

and R

R.

Fay 2002 Directional

auditory processing by the oyster toadfish,

Bioacoustics 1 2 202-204

Opsanus

tau.

Edds-Walton, P L L A Mangiamele, and L C Rome

2002 Variations of pulse repetition rate in boatwhistle
sounds from oyster toadfish (Opsanus tau) Bioacoustics
13. 153-173
,

and Robert Barlow. 2002 Orcadian

horseshoe crabs.
rhythms in locomotor activity of juvenile
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Borst, Douglas,

S M Highstem, J P Carey, and J P Xu 2002

Functional recovery of anterior semicircular canal afferents

Boyle R

following hair cell regeneration

in

birds

JARO

tics

12

fragment of myosin-V displaces vesicleassociated motor and blocks vesicle transport in squid
nerve cell extracts Biol Bull 2 210-211
tail

and

Edds-Walton 2002 Preliminary

evidence for interpulse

interval selectivity of cells in the

torus semicircularis of the oyster toadfish (Opsanus

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escape behavior of prey?

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Cermak, Michael J 2002 Caranx /atus (Carangidae)

chooses dock pilings to attack silverside schools a tactic to
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in fishes.

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adhesion-related proteins as specific markers of sponge

cell types involved in allogeneic recognition Dev. Comp

Immunol. 26: 313-323

203. 241-243.

H Ripps 2002
L., E Schuette, R. Anton, and
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and Mary

Hoffman 2002.

Cl

and

Chappell, R

Garber, Sarah S

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anion
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Clay,

John R

K+ channels

and Alan Kuzinan 2002 Trafficking of axonal

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the squid giant axon neurotransmitter
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and Alvin Shner 2002 Temperature

mtracellular

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Koenig 2002. Axonal and presynaptic protein synthesis
new insights into the biology of the neuron. Trends

Giuditta,

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and R S Blumberg 2002 Functional reconstitution of

human FcRn in Madm-Darby canine kidney cells requires
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Chem
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Golan.

A., Y.

Yudkovsky, and A. Hershko 2002 The cyclm-

ubiquitin ligase activity of the cyclosome/APC is jointly

activated by protein kinases Cdkl-cyclin B and Plk.
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Ludlam, John P., David H. Shull, and Robert Buchsbaum

2002. Effects of haying on salt-marsh surface invertebrates
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and Robert Baker 2002 Central pathways mediating
oculomotor reflexes in an elasmobranch, Scyltorhinus
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203:250-251.

Ma. W.-L., and R R Fay 2002 Neural representations of

the axis of acoustic particle motion in the auditory midbrain
of the goldfish, Carassius auratus J. Comp. Physio/ 188:
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canicu/a. Biol. Bull. 203: 236-238.

P., A. Desai, K. Oegema, T. J. Mitchison, and E
D. Salmon. 2002. Poleward microtubule flux is a major

Maddox,
B B Slepchenko, M M. Rolls, T. C. Walther, P. A
Stem, L M. Mehlmann, J Ellenberg, and M. Terasaki 2002
Chromosomal association of Ran during meiotic and mitotic
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L Kristensen, L Sottnjp- Jensen 2002. Localization of
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L.

in

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their bait region

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Islas-Flores,

I.,

S.

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L.

Bearer, J C. Raya,

and M.-A Villanueva

B.

Li.

M. Eyman, 2 Scotto Lavina, A. E. Gioio, K.

R van der Schors, W. P. M. Geraerts A Giuditta, B
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J.

van Minnen. 2002. Protein synthesis

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fratj,

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fish

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cells

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horseshoe crab Limulus po/yphemus and its implication
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Montgomery, John, Guy Carton, and David Bodznick 2002.

Error-driven motor learning in fish. Biol. Bull. 203: 238-239.
Morgan, J R., G J Augustine, and E M Lafer 2002
Synaptic vesicle endocytosis: the races, places, and
molecular faces Neuromolecular Med 2: 101-114.
Homsi, Hilary

Gould 2002 mRNAs located

in

Mornson, and Robert

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M. Gomez, and

E.

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adaptation of molluscan microvillar

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Rakowski, R F

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microtubules

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follicle

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Jones, and

A R W. Cole, and C
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kmase C

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Anderson 2002. Molecular approaches to understanding
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mouse

neural telemetry from free-swimming

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Ridings,

Borst, K. Smith, F

Dodge, and R B Barlow.

2002. Visual behavior of juvenile Ltmulus in their natural

habitat and in captivity Bio/. Bull. 203 224-225.
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mammalian

and R

Cole. 2002. Cold shock and the

cycle Cell Cycle

cell

J. S., S. Grego, E D Salmon, and T J Mitchison.

2002 EB1-microtubule interactions in Xenopus egg
extracts: role of EB1 in microtubule stabilization and
mechanisms of targeting to microtubules Mo/ Biol. Cell

Tirnauer

13:

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and

B.,

separate event
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P J

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Weeg, M

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R. R.
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Hammar,

Wachowiak, M., L B Cohen, and M Zochowski. 2002

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junctions and the 'bystander'

K.

I.

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Malchow,

Perlman 2002 A novel turtle retinal preparaLevy, and

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D. R Burgess 2002 Transitions
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Robert

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and Marc Bartman 2002

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Jurkovicova, V.

Pickel,

E Gioio, and

Kaplan 2002 Identification of a novel membraneassociated protein expressed in neurons of the squid and
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203

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Freshwater ferromanganese stromatolites from Lake
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Thompson R

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Julie C.

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Mitchison 2002 EB1 targets to kinetochores with attached,

polymerizing microtubules Mo/ Biol. Cell 13 4308-4316

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R44

education
The 2002 Education Program provided 499 students from 288

institutions

and 30 countries an

opportunity to study a range of biological topics with some of the best and brightest scientists
in the world serving as course faculty and lecturers. The Laboratory welcomed 554 faculty
members and staff and 203 lecturers to the courses in 2002. They represented 175 institutions

summer, we were especially

Tim
Hunt, who gave the Arthur K.
pleased to host two Nobel Laureates, Michael Brown and
Parpart and the Irvin Isenberg Lectures, respectively, in the Physiology course.

and 31 countries.

In

Among

the

many outstanding

addition to the MBL's six major

summer

lecturers last

courses,

new

throughout the year, including two exciting

we

offered 14 special topics courses

courses:

Advances

in

Genome Technology and

Bioinformatics, directed by Claire M. Fraser, TIGR, and Mitchell Sogin, MBL; and

Neuroinformatics, directed by Partha Mitra of Lucent Technologies, Emery Brown of Massachusetts

General Hospital, and David Kleinfeld of the University of California, San Diego.

At the end of the 2002 season,

we

bid farewell to Chris Tschudi and Elisabetta Ullu, directors of

the Biology of Parasitism course. Jay Bangs of the University of Wisconsin, Madison,

will

take

Bialek and Rob de Ruyter Van

the helm of that course in 2003. We also said goodbye
Steveninck of the Computational Neuroscience course. Bard Ermentrout of the University of
to

Pittsburgh and John White of Boston University

2003.

In

addition, Sandra Masur, of

Mount

Sinai

as co-director of the Vision Research course

in

will

Bill

assume the directorship of the course

in

School of Medicine, joined David Papermaster

2002.

The MBL's educational program once again received

stamp of approval from the National

Institutes of Health's competitive peer review process with renewed funding for the Embryology, Neural Systems & Behavior, and Neurobiology courses, and new funding for the inaugural
Neuroinformatics course.

R45

SUMMER COURSES
Biology of Parasitism:

Modern

Approaches
June 13- August

10,

Martmdale. Mark, University of Hawaii

Niswander, Lee, Sloan-Kettermg Institute

2002

Nipam, University of Chicago

DIRECTORS

Patel,

Tschudi, Christian, Yale University Medical School

Saunders, John, Retired

Ullu, Elisabetta, Yale University

Sherwood, David, California

Medical School

Institute of

Technology

FACULTY

Zeller,

Robert, San Diego State University

Bangs. James, University of Wisconsin-Madison

LECTURERS

Grencis, Richard, University of Manchester

Hajduk, Stephen. University of Alabama-

Davidson,

Matthews. Keith, University of Manchester

McFadden, Geoff,

University of

Greenwald.

Melbourne

Parsons. Marilyn. Seattle Biomedical Research

Rathod, Pradip, University of Washington

Reiner. Steven, University of Pennsylvania
Tarleton. Rick. University of

Cross, George, Rockefeller University

Deitsch. Kirk, Weill Medical College of

Texas

A&M

Bridegam,

Patrick,

McKmnon,

Nicole. University of Victoria. 8-C

University

Andrea, Inst de
Parana-IBMP

Biol

Molecular do

Institute
Eric. Case Western Reserve University
Meg, UT Southwestern Medical Center
Eleanor, London School of Hygiene &

Pearlman,
Phillips.

Tropical Medicine

Rudenko, Gloria. University of Oxford

Sacks. David. NIAID,

NIH

Wray, Gregory, Duke University

Cockburn,

Ian, University of

Yelon, Deborah,

Edinburgh

White. Michael, Montana State University

Wilson, lain. National Institute for Medical

Washington

Klotz, Christian, Humboldt-University-Berlin

KOOIJ, Taco,

Lee,

Wynn, Thomas. National

Hongiie, Yale University

Meissner, Markus. Imperial College of Science,
Li,

Technology & Medicine, UK

Dominique, Imperial College of Science

& Technology

MERYL ROSE LECTURER

Gerhart, John, University of California. Berkeley

TEACHING ASSISTANTS
Baker. Clare. University of

Cambridge

Cheeks, Rebecca, University of North Carolina-

Chapel

Hill

Medicine, Heidelberg
Nkmin, Wuyika, University of Yaounde
Slavin, lleana, Universidad Nacional de Cordoba

Gamse, Joshua, Carnegie Institution of

Washington
Gerberdmg, Matthias, University of Chicago
Gross, Jeffrey. Duke University

Stubbs. Janine, Royal Melbourne Hospital

Khokha. Mustafa, University of California,

Mueller, Ann-Kristin. University School of

Berkeley/MCB
Kuhlman, Julie, University of Oregon
Lartillot. Nicolas, Centre Genetique Moleculaire

Embryology: Concepts and

Techniques in Modern
Developmental Biology

Institutes of Health

Dobbelaere. Dirk, University of Bern

Roditi, Isabel, University of Bern

University School

Leiden University Medical Centre

SooHee. Johns Hopkins School of Medicine

June 16 -July 28, 2002

Research

New York

of Medicine

Stanley.

Netherlands

Princeton University

Nathalie, Institut Pasteur

Scherf, Artur, Institut Pasteur

Sam, Washington University

Striepen. Bons, University of Georgia
Waters, Andrew. Leiden University. The

Eric,

Chamond,

Karnataki, Anuradha, University of

Panigrahi, Aswini, Seattle Biomedical Research

University

Research

Wieschaus.

Englund, Paul, Johns Hopkins Medical School

Hunter, Christopher, University of Pennsylvania
Muller. Miklos. The Rockefeller University
Nutman, Thomas. National Institutes of Health

Columbia

Tramor. Paul, Stowers Institute for Medical

Garside, Paul, University of

Glasgow

Sanes, Joshua, Washington University

Shankland, Martin. University of Texas at Austin
Struhl. Gary.

Fenn, Katelyn, University of Edinburgh

Green, Heather New York University

Cornell University

University of California.

Robertson, Elizabeth, Harvard University

COURSE ASSISTANTS

Avila,

Edinburgh

Columbia University
New York Umversity/HHMI

San Diego

Georgia

LECTURERS
Allen, Judith, University of

Iva,

McGmnis. William,

Kevin Begos

STUDENTS

Soldati,

Technology

Joyner, Alexandra,

Institute

Riley.

Eric. California Institute of

Keller, Ray, University of Virginia

Birmingham

Liu.

Karen. University of California, Berkeley

Maduro, Morris, University of

California,

Santa Barbara

Nederbragt, Alexander, Unversity of Hawaii/PBRC

Solomon, Keely, Emory University
Tobey. Allison, Memorial Sloan Kettenng

Cancer Center

DIRECTORS
Richard Harland, University of California. Berkeley
Joel Rothman, University of California,

Santa Barbara

Wallmgford, John, University of California,

Berkeley
Walsh, Emily. Whitehead

Institute for

Biomedical

Research

TEACHING ASSISTANTS

FACULTY

Jiang, Lei, University of

Fraser,

Washington

Scon, California

Institute of

Technology

Karthikeyan, Ganesan, University of Washington

Mollard, Vanessa, University of Melbourne

Levine, Michael. University of California. Berkeley

David, University of Pennsylvania

Cummings, Kara. University of Georgia

Tabm.

Artis,

Jensen, Bryan, Seattle Biomedical Research

Institute

Mair,

Gunnar, Queen's University Belfast

Rokhsar, Dan. University of California. Berkeley

Clifford,

Harvard University

Seth, University of Wisconsin-Madison

Bronner-Fraser. Marianne. California Institute
Blair,

House Ear

Institute

Martin, Diana, University of Georgia

Ettensohn, Charles, Carnegie Mellon University

Pennock, Joanne, University of Manchester

of
Halpern, Marnie, Carnegie Institution

Ralph, Stuart, University of Melbourne

Triggs, Veronica, University of Wisconsin-Madison

Henry, Jonathan, University of

van Deursen, Fredenck, The University

Krumlauf, Robb, Stowers Institute for Medical

of Manchester

Balligan, Sarah, University of Missouri-Columbia

McCluskey. Kathryn, Marine Biological

Laboratory
Tai, Phillip,

Washington

Research

Biomedical Research
Wilson. Valene. Centre for Genome Research
Wolfe. Adam. University of Illinois. Urbana

COURSE ASSISTANTS

of Technology
Collazo. Andres.

Weatherbee. Scott. Memorial Sloan Kettenng

Cancer Center
Wiellette, Elizabeth, Whitehead Institute for

University of California, Berkeley

Illinois

Continued.

R46

STUDENTS
Berry, Katy, University of Sheffield

Brown, Ann, Medgar Evers College

Emory University School

Caracino, Diana,
of Medicine

Copf, Tijana, University of Crete

Crotwell, Patricia, University of South Dakota

Dash, Satya, University of East Anglia

Delalande, Jean-Marie, University College

London
Drago, Grazia, Universita Degli Studi di Palermo
Extavour, Cassandra, University of Cambridge
Guest, Jennifer, National Institute for Medical
Research
Kee, Yun, California Institute of Technology
Kerney, Ryan, Harvard University
Koziel, Lydia, Max-Planctc-lnst. for

Molecular

Genetics
Carolina, University of Texas Health

Livi,

Science Center, San Antonio

Malartre, Marianne, University of Portsmouth

Maslakova, Svetlana, Smithsonian Institution

Matus, David, University of Hawaii
Mitchell, Tracy, University of

Kevin Begos

Wisconsin-Madison

Muyskens, Jonathan, University of Oregon

TEACHING ASSISTANTS

Neural Systems

Princeton University
Orsborn, Apnl, University of Missouri-Columbia

Behrens, Sebastian, MPI for Marine Microbiology

Martiny, Adam, Danmark Tekniske Universitet

June 16

Primus, Alexander, University of Texas, Austin

Roche, Daniel, University of California, Berkeley

Schaefer,

Nouri,

Ali,

Su, Yi-Hsien, Scripps

Inst.

of

Oceanography,

MBRD
Van

Mueller, Jochen, Stanford University

Amy, University of Iowa

&

Behavior

2002

DIRECTORS
Carr, Catherine, University of

Maryland

Hawkins, Andrew, University of Iowa

FACULTY

LAB ASSISTANT

Calabrese, Ronald. Emory University

Waterbury, Matthew, Marine Biological Laboratory

Chitwood, Raymond, Baylor College of Medicine

Baines, Richard, University of Warwick

Graeme,

Davis,

Microbial Diversity

STUDENTS

June 16- August

Boucher, Yan, Dalhousie University

Case, Rebecca, University of New South Wales
Clement, Barbara, Doane College

2,

2002

DIRECTORS
Harwood, Caroline, University of Iowa
Spormann, Alfred, Stanford University

FACULTY

Denef, Vincent, Michigan State University

Dethlefsen, Les, Michigan State University
Dick, Gregory, Scripps Institute of

Oceanography

Buckley, Daniel, University of Connecticut

Erbs, Marianne, Swiss Fed. Inst. for

Gentile, Margaret, Stanford University

LECTURERS

Ginder-Vogel, Matthew, Stanford University

Graco, Michelle, University of Pierre et Marie

Boetius, Antje,

MPI

Marine Mikrobiologie

fur

Environmental Science

& Technology

Curie

Breznak, John, Michigan State University

Harrison, Faith, University of Iowa

Delong, Edward, Monterey Bay Aquarium

Koren, Omry, Tel Aviv University

Lostroh, Phoebe, University of Iowa College
of Medicine

Elhai, Jeff, Virginia

Commonwealth

University
State University

Giovannoni, Stephen, Oregon

Gottschalk, Gerhard, Institut fur Mikrobiologie

Genet

Julia,

Pennsylvania State University

Pinel, Nicolas, University of

Handelsman, Jo, University of Wisconsin

Larimer, Frank, Oak Ridge National Laboratory
Stephen, Harvard Medical School

Loveley, Derek, University of Massachusetts

Meeks, John, University of California
Wii:..

Maresca,

'

'-.vr'sity of Illinois

Rocap, Gabnelle, University of Washington

:>f
Strous. Marc, b
rsil
Nymegen
,

Thauer, Rudolf, MHI fur Terrestr. Mikro

Wackett, Lawrence, University of Minnesota

Wolfe. Ralph, University of Illinois

University of California,

San Francisco

Gibson. Jane. Cornell University (Emerita)

Marsh, Terence, Michigan State University

Metcalf.

10,

COURSE COORDINATOR
Melante, Boston University School of

Stry,

Lory.

August

Levine, Richard, University of Arizona

Medicine

Washington
Rajagopal, Soumitra, University of Nebraska
Remold, Susanna, Michigan State University
Sharp, Katherine, Scripps Institute of

Oceanography
Spain, Jim, United States Air Force
Walker, Jeffrey. University of Colorado

French, Kathleen, University of California, San Diego

Glanzman, David, University of California,
Los Angeles
Golowasch, Jorge, Rutgers University
Kristan, William, University of California, San Diego

Mooney, Richard, Duke University

Nadim, Farzan, Rutgers University
Philpot, Ben, Brown University
Pnjsky, Glen, University of Lethbridge

Reyes, Alex, New York University

Ribera, Angeles, University of Colorado Health

Sciences Center
Roberts, William, University of Oregon
Simon, Jonathan, University of Maryland
Stein,

Wolfgang, Universitaet Ulm

Szczupak, Lidia, Universidad de Buenos Aires

Weeks. Jams, University of Oregon

Wenning-Erxleben. Angela, Emory University

Wessel, Ralf, Washington University
Wilson, Richard. University of Calgary
Wood, Debra, Case Western Reserve University

Wood, Emma,

University of Edinburgh
Zhang, Bing, University of Texas at Austin

LECTURERS
Augustine, George, Duke Medical Center
Bate. Michael, University of Cambridge

Feldman, Daniel, University of California, San Diego

Thomas. University of Colorado Health

Finger,

Sciences Center

R47

TEACHING ASSISTANTS

Neurobiology
June 16- August

Allana, Tariq. Boston University

2002

17.

Chang, Paul, New York University

Godinho, Leanne, Washington University School
of Medicine
Guan, Zhuo, Massachusetts Institute of

DIRECTORS
Faber. Donald, Albert Einstein College of

Medicine
Lichtman,

Jeff,

Washington University School

Technology
School
Misgeld, Thomas, Washington University

of Medicine

of Medicine

SECTION DIRECTOR

Stewart, Bryan, University of Toronto

Oregon Health & Science

Trussell, Larry.

University

White, Stephanie, University of California, Los

Angeles

TEACHING ASSISTANTS
Beenhakker, Mark, University of Pennsylvania
Bradford, Yvonne, University of Oregon
Chen, Shanping, House Ear Institute

Coleman, Melissa, Duke University

Ezzeddine, Youssef, University of California, Los

Angeles
Fairies, Michael, University of Pennsylvania

Heiser, Ryan, University of

Colorado Health

Sciences

MacLeod, Katnna,

University of Maryland,

College Park

Adam.

Arizona

University of California,

Duke

University

Siegel, Jennifer, Bowling

Green State

University

Scares, Daphne, University of Maryland

Svoboda. Kurt, University of Colorado Health

Sciences Center
Villareal,

COURSE ASSISTANTS

Buchanan, JoAnn, Stanford University

Hall.

Conchello. Jose-Angel, Washington University

Medical School

Satterlee, Danielle, Texas

Coyle, Joseph, Harvard University

Denk, Winfried, MPI fur Medical Research
Gan, Wenbiao, New York University

STUDENTS

Hart,

David. Marine Biological Laboratory

A& M

University

Boassa, Daniela, University of Anzona College

Anne, Massachusetts General Hospital

Heuser, John, Washington University

Jacob, Michele, Tufts University
Einstein College
Kaprielian. Zaven. Albert

of Medicine
Campbell, Susan, University of Alabama,

Birmingham
Ewald, Rebecca, Cold Spring Harbor Laboratory
Hobbs, Steven, University of Colorado

of Medicine
Khodakhah, Kamran, Albert Einstein College
of Medicine

Hu, Hailan, University of California, Berkeley

of
Ihring. Alexandra, Max-Planck-Institute

Lambert, Nevin, Medical College of Georgia

Koirala, Samir, University of

Levinthal, David, University of Pittsburgh

Kozhevnikov, Alexay. Bell Labs/Lucent

Jen-Wei, Boston University

Massachusetts Institute

of Technology
Logothetis. Nikos, MPI for Biological Cybernetics
McMahon, Lori, University of Alabama

Pimenta, Aurea, University of Pittsburgh School

Thomas, Albert

Southern California

Institute of

Technology
Petersen, Rasmus, International School

for

Advanced Study (SISSA)

Health Systems
Ryan, Amy, University of Virginia
Zhou. Zhaolan

of Medicine

Preuss,

Neurobiology

Technologies
Montana, Enrico, Massachusetts

Littleton, J. Troy,

Los Angeles
Roy. Arani,

FACULTY

Lin,

Miller, Julie. University of

Roberts,

DeFranco, Donald. University of Pittsburgh

School of Medicine

Rosenberg, Madelaine, Tufts University

Szabo, Theresa. Albert Einstein College
of Medicine

(Joe),

Harvard Medical School

Einstein College of

Medicine

Greg, University of California.

Los Angeles
Zee. M Jade. University of

Oregon

Donald, Johns Hopkins Universtiy

Reese, Tom, NIH
Price,

Physiology: The Biochemical

and Molecular Basis of Cell

Schweizer, Felix, University of California.

Los Angeles

COURSE ASSISTANTS
Cardon, Aaron, Texas

A&M

University

Rodrigues, Elizabeth, University of Oregon

Signaling

Smith, Stephen, Stanford University

Wesley, University of Texas

Thompson.
Wong, Rachel, Washington

June 16- July 27, 2002

University

DIRECTORS

Shaw, Abigail, Stanford University

Garbers, David,

LECTURERS

UT Southwestern

Medical

Center/HHMI

STUDENTS

Aimers. Wolfhard, Vollum Institute

Chang, Andrew, Oregon State University

De Labra. Carmen, University College London

Auerbach, Anthony, SUNY at Buffalo

Bear, Mark, HHMI/Brown University

Reed, Randall, Johns Hopkins University/HHMI

Brodin, Lennart. Karolinska Institute!

Ed, University of Wisconsin-Madison

FACULTY

Harlow, Mark, Stanford University

Ehrlich, Barbara, Yale University

Dellen. Babette,

Washington

University, St. Louis

Doiron, Brent, University of Ottawa

Dulcis, Davide, University of Arizona. Tucson

Chapman,

of Pennsylvania
Higley, Michael. University

Harris,

Hughes, Cynthia, Indiana University

Julian, Glennis, University of Arizona

Hoh. Jan, Johns Hopkins School of Medicine

Massachusetts Institute of

Khalil,

Mona. Columbia

University

Miranda, Jason, University of Texas at Austin

Pfeiffer,

Keram, Philipps-Umversitat Marburg

Ken Rutgers

University

Hopkins, Nancy,

Technology

Lipscombe, Diane, Brown University

Rutherford. Mark, University of Oregon

Sebe, Joy. University of Washington

Magee,

Nadja, Georgia State University

Steinberg, Rebecca, University of Texas

McMahan,

Spitzer,

Sternson, Scott, Rockefeller University

Witney. Alice. University of Birmingham

Medical School

Wohlgemuth, Sandra, Humboldt-Universitat

zu Berlin

Zomik,

Erik,

Columbia University

Elly,

in

the goldfish brain,

Schultz, Nikolaus, University of Texas

Southwestern Medical Center

,

Stanford University

Massachusetts

Institute of

Nicolelis, Miguel,

LECTURERS
Bennett, Anton, Yale Medical School

Technology

Duke

Ogden, David, National

University
Institute for

Medical

Research
Ryan, Tim, Weill Medical Cornell
Sigworth, Fred, Yale University

Cold Spring Harbor Laboratory

Tsien, Roger. University of California. San Diego
Harbor Laboratory
Tully. Tim, Cold Spring

Svoboda.

Vestibular spinal neurons

Steven Zottoli

Health

Science Center

Nedivi,

Kao, Ling-Rong, University of Texas

Southwestern Medical Center

Southwestern Medical Center

Jeffrey, Louisiana State University

Del J

Franco, Peter, University of Minnesota

Furlow, David. University of California, Davis

Lim, Wayland. University of California, Davis

Megraw, Timothy, University of Texas

Huguenard, John, Stanford University

Kernan, Maurice, SUNY at Stony Brook

Renart, Alfonso, Brandeis University

Duncan, Tod, Imperial Cancer Research Fund

Karel.

Yellen, Gary, Harvard Medical School

Clapham, David, Harvard Medical School

Comerford, Sarah, University of Texas
Southwestern Medical Center
Devreotes. Peter. Johns Hopkins University
School of Medicine
Dietnch, William, Harvard Medical School
Gardner, Kevin. University of Texas Southwestern
Medical Center

Continued

R48

2002 SPECIAL LECTURE SERIES

Meryl S. Rose Lecture (June 17)
John Gerhart, University of California, Berkeley
Embryos, and Evolution. Toward a Cellular and Developmental Understanding of
Phenotypic Variation and Evolutionary Adaptability"

"Cells.

Hammer,

R<

South

--cdical

"rsity of

Hepler

of Texas

Center

/rv;n

"Protein Synthesis

.J^vid, University of Texas

Mang*:
Southwestern Medical Center
Nambu, John, University of Massachusetts
3

Stock, Ann, University of Medicine

New Jersey-RW Johnson

&

/senberg Lecture (June 21)

Timothy Hunt, Nobel Laureate, International Cancer Research Fund, Clare

Massachusetts

and the Control

Hall

Lab

of the Cell Cycle"

Gertrude Forfcosh Waxier Lecture

(July

7)

Robert A. Weinberg, Massachusetts Institute of Technology

"Rules for Making Human Tumor Cells"

Dentistry,

Medical School/HHMI

Tilney, Lewis, University of Pennsylvania

Teru Ha/ashi Lecture (July 23)

Melanie H. Cobb, University of Texas Southwestern Medical Center
"Information Flow in MAP Kinase Cascades"

Welsh, Michael, University of Iowa

IRVIN

ISEN8ERG LECTURER

Hunt, Timothy, International Cancer Research

Fund, Clare Hall Laboratories

Arthur K. Parpart Lecture (July 26)

Michael Brown, Nobel Laureate, University of Texas Southwestern Medical Cente

"The SREBP Pathway: How the Membrane Tells the Nucleus What it Needs"

GERTRUDE FORKOSH WAXLER LECTURER

Weinberg, Robert, Whitehead

ARTHUR

K.

Institute

Ruth Sager Lecture in Genetr'cs (August 30)

Mark Fishman, Massachusetts Hospital and Harvard Medical School
"Genetic Modules: Fashioning Organs in Zebrafish"

PARPART LECTURER

Brown, Michael, University of Texas

Southwestern Medical Center

SPECIAL TOPICS COURSES

TERU HAYASHI LECTURER

Cobb, Melanie,

University of Texas

Southwestern Medical Center

Advances
TEACHING ASSISTANTS

&

Anyatonwu, Georgia, Yale University

October 6

in

Genome Technology

Bioinformatics
-

November

2,

Peterson, Scott,

2002

DIRECTORS
Rossi, Kristen. University of

Fraser, Claire,

Texas Southwestern

Grellhesl, Dana, University of Texas Southwestern

Medical Center

Swaney, Sara-Love, University of Texas

Southwestern Medical Center

The

Institute for

Genomic Research

Aguilar, Arturo, Institute Politecnico National,

Mexico

Medical College

Chong,

Curtis,

Johns Hopkins School of Medicine

Davis, Kevin, University of Pittsburgh

Dojcinovic, Danijel, Arizona State University

FACULTY
Bateman, Alex, Sanger Institue
Blake, Judith, Jackson Laboratory
Churchill, Gary, Jackson Laboratory
Cummings, Michael, Marine Biological Laboratory
deJong, Pieter, Children's Hospital Oakland

Feldblyum, Tamara, The Institute for Genomic

Research
Felsenstein, Joe, University of

Washington

Florens, Laurence. Scripps Research Institute

Gill, Steven, The Institute for Genomic Research

Gray, Michael, Dalhousie Institute

Heidelberg, John, The Institute for

Research

Whitehead

Genomic

Institute for

Biomedical

of Kentucky College

Kydd, Alison, University of Calgary

LaPointe, Nichole, Northwestern University

McVaugh, Cheryl, University of Pennsylvania

Oh, Ji-Eun, University of Illinois at Chicago
Pace, Margaret, University of Texas
Pignatelli, Vincenzo, University of Pisa

Pineda, Gabn'el, University of Texas

Southwestern Medical Center

Ramos, Arnolt, Children's Hospital, Boston

Rossi, Chiara, University of Pisa

White,

Jennifer,

Owen, The

Marine Biological Laboratory

Genomic Research

Institute for

Kent, Jim, University of California, Santa Cnjz

Kim, Ulandt, Marine Biological Laboratory

Lee, Norman, The Institute for Genomic Research

Mann, Barbara,

The

Genomic Research
Genomic Research
The Institute for Genomic

Institute for

Radune, Diana, The

Institute for

Vamathevan, Jessica,
Research

Davidsen, Tanja, The Institute for Genomic Research

Gill, John, The Institute for Genomic Research
Bhagabati, Nirmal, The Institute for Genomic Research

Saeed, Alexander, The Institute for Genomic Research

White, Joseph, The Institute for Genomic Research
Thiagarajan, Mathangi, The Institute for

Genomic

Research

Research

of Medicine

Venter, Craig,

Tallon, Luke,

Lawrence Berkeley

Ge, Lan, University of California, Riverside

Goentoro, Lea, Princeton University
Kaynar, Murat, Beth Israel Deaconess
Kelly, Melissa, University

The Institute for Genomic Research

The Institute for Genomic Research

Tettelin, Herve,

TEACHING ASSISTANTS

Eisen, Michael.

Jaffe, David,

Illinois

Roos, David, University of Pennsylvania

Salzberg, Steven, The Institute for Genomic Research
Smith, Hamilton, Celera Genomics

Wernegreen,

DeLong, Edward, Monterey Bay Aquarium

Eisen, Jonathan, The Institute for Genomic

Gadea, Bedrick, Harvard Medical School

Medical Center

Research

Reysenbach, Anna-Louise, Portland State University

Ringwald, Martin, Jackson Laboratory

Research

Amarie, Dragos, University of Notre Dame

Chen, Yen-Chin, National Cheng Kung University

Genomic Research

Reich, Claudia, University of

Research Institute

STUDENTS

Institute for

Sogin, Mitchell, Marine Biological Laboratory

Medical Center

COURSE ASSISTANTS

The

Pop, Mihai, The Institute for Genomic Research

Quackenbush. John, The Institute for Genomic

Rengifo, Juliana, Yale University

COURSE COORDINATOR

Palmer, Jeffrey, Indiana University

Pearson, William, University of Virginia

University of Virginia Health

Wang, Hong-Ying, The Institute for Genomic Research

Gaspard, Renee, The Institute for Genomic Research
Frank, Bryan, The Institute for Genomic Research
Hasseman, Jeremy, The Institute for Genomic Research

System
McArthur, Andrew, Marine Biological Laboratory
Mesirov, Jill, Whitehead Institute for Biomedical

Research
Morrison, Hilary, Marine Biological Laboratory
Myers, Eugene, Celera Genomics

Nelson, Karen, The Institute for

Genomic

Bebout, Brad,

NASA Ames

Research Center

Brodhagen, Marion, Oregon State University

Bundy, Becky, University of Georgia
Chen, Lishan, University of Washington
da Silva, Alexandre, Centers for Disease Control

and Prevention

Research

Sha, Edward, Indiana University Medical Center

Thamatrakoln, Kimberlee, University of California,

Nierman, William, The

Research

San Diego
Vega, Rebecca, Stanford University

Nusbaum, Chad, Whitehead

Institute for

Genomic

Fenwick, Brad. Kansas State University

Francis, Susan, University of

University of Arizona

Olsen, Gary, University of

Doak, Thomas, University of Utah

Duvefelt, Kristina, Karolinska Institute!

Institute for

Biomedical Research

Ochman, Howard,

STUDENTS

Illinois

Washington
George, College of William & Mary
Golden, Daniel, University of Alabama, Birmingham
Gilchrist,

R49

Woods Hole Oceanographic

Handley, Heather.

Hildebrandt, John. Medical University of South

Carolina

Kanzok, Stefan, Yale University School of Medicine

Kiesling, Traci, University of

Ranson,

Hilary.

Fleming, Shawna, Brown University

Fu, Lianwu, University of Alabama, Birminghan
Galko, Michael, Stanford University School

August 24, 2002

DIRECTORS
Guarente, Lenny, Massachusetts

Institute of

Technology

Glover, Greta.

Oregon Health and Sciences

Wallace. Douglas, University of California, Irvine

University

Gruenbaum.

New York

University

Lore, Boehringer Ingelheim

Pharmaceuticals

Habdas,

Yale University

Tyler,

Molecular Biology of Aging

August

Green, Heather,

Tobias, Carl Zeiss

Jena-Pineda, Fernando, Johns Hopkins

Bloomberg School of Public Health
Radniecki,

John, Yale University School

of Medicine

Miami

Kimbell. Jennifer, University of Hawaii

Kinnersley, Margie, University of Montana

Neumann,

Fitzpatrick,

of Medicine

Institution

Piotr,

Emory

University

FACULTY
Culotta, Valeria, Johns Hopkins University
Kirkwood, Tom, University of Manchester

Lambeth, Dave, Emory University

Liverpool School of Tropical

Medicine
Sawyer, Sara, Cornell University

Thomas,

Bolaji, Tufts University

Williams, David,

Illinois

State University

and Quantitative Light

Microscopy

Analytical

May 9- May

2002

17,

DIRECTORS
Sluder, Greenfield, University of Massachusetts

Medical Center
Wolf, David, BioHybrid Technologies

FACULTY
Amos,

William,

MRC

Lab of Molecular Biology

Bulseco, Dylan, University of Massachusetts

Medical School
Cardullo. Richard. University of California
Gelles, Jeff, Brandeis University
Hinchcliffe, Edward, University of Notre

Dame

Inoue, Shinya, Marine Biological Laboratory

Moomaw,

Hamamatsu Photonic Systems

Butch,

Reichelt, Stefanie,

MRC

Lab of Molecular

Biology

Salmon, Edward, University of North Carolina,

Chapel
Silver,

Hill

Randi, Weill Medical College Cornell

Drosophila, April Orsborn

University

Spring, Kenneth, National Institutes of Health

Swedlow, Jason, University of Dundee
Waters Shuler, Jennifer, Harvard Medical School
Sears, Kathryn, Sensor Technologies

Hidalgo, Carlos, Institute Venezolano de

Investig- Cientificas
Iszard, Melissa,

Raytheon Polar Services

Massachusetts
Jungnickel, Melissa, University of

LECTURERS
Straight,

Medical School

Aaron, Harvard Medical School

Wachowiak, Mel, Smithsonian

Institution

Liu,

Songtao. Fox Chase Cancer Center

Luna, Elizabeth, University of Massachusetts

Medical School

TEACHING ASSISTANT

McKeown,

Ehrhardt. Anka, University of Massachusetts

Paladino, Simona. University of Naples

Medical School

Caroline, University of Utah

Panetti, Tracee,

Temple

University School of

Medicine

COURSE ASSISTANT

Poskanzer, Kira, University of California. San

Nordberg, Joshua, University of Massachusetts

Medical School

STUDENTS
Chatterjee, Samit, Weill Medical College of
Cornell University

Cooke, Emma-Louise, AstraZeneca R&D,

Charnwood. UK
Counterman, Anne, Pennsylvania State
University

Espinosa Tanguma, Ricardo, University of

San

Lui Potosi

Fischer, Robert,

Francisco

Powers, Maureen, Emory University

Raphael, Marc, The Naval Research Laboratory
Rusan. Nasser, University of Massachusetts,

Amherst
Smyth. Jeremy, University of Massachusetts,

Amherst
Tarn,

Jenny, Tufts University

Wagener, Michael. Carl Zeiss, Inc.

Welch, Jeffrey, Duke University Medical Center
Yamamoto, Akihiro. RIKEN, Japan
Zandbank-Zuker, Keren, Hebrew Universtiy

The Scnpps Research

Institute

Zhang.

LingLi, University of Pennsylvania

Weindruch, Richard, University of Wisconsin

Institute
Bishop, Nicholas, Massachusetts
of Technology

Helfand, Stephen, University of Connecticut

Health Center

Donald, Johns Hopkins University

Ruvkun, Gary, Massachusetts General Hospital
Wright, Woodring, University of Texas
Price,

Southwestern Medical Center

LECTURERS
Pelicci. Pier

Giuseppe, European

Institute of

Oncology
Austad, Steven, University of Idaho
Bohr, Vilhelm, National Institute on Aging, NIH
Campisi, Judith, Lawrence Berkeley National

Laboratory
Davenport, John,

AAAS

Baylor College of Medicine

Goldberg, Alfred, Harvard Medical School
Hanawalt, Philip, Stanford University

Donehower,

Larry,

Harley, Calvin,

Geron Corporation

Hekimi, Siegfried, McGill University

Jones, Dean. Emory University

Kim, Stuart, Stanford University
Martin, George. University of

Washington
Continued...

R50
Partridge, Linda, University College London
Richardson, Arlan, University of Texas Health

Thomas,

New

England Medical Center

Hunt, Joan, University of Kansas

Roberts, Sheila. Bridgewater State College

Launnda, University of Connecticut

Health Center

COURSE ASSISTANT

Tatar,

TEACHING
Coskun,

Kaeberlein.

?'n California

;ry University

jchusetts Institute of

'

Technology

Emory University
Kokoszka, Jason, Emory University
Visithawan. Mo, Massachusetts Institute
Kerstani-,. Keilh,

of Technology
Liszt,

Mayo. Kelly. Northwestern University

Moore, Karen, University of Florida
Overstrom,

ASSIST,

Elif Pi

Eric, Tufts University

of Veterinary

Harrison, Emily, Bridgewater State College

STUDENTS

School

Medicine

Akcali, Kamil, Bilkent University, Turkey

Schatten, Gerald, University of Pittsburgh

Shupnik, Margaret, University of Virginia

Technology
Subramaniam, Vaidya, Emory University

Emory

Health Center
Weigel, Nancy, Baylor College of Medicine

Gonsalves, Joanna, University of California,

San Francisco
Hallikas, Outi, University of Helsinki

Lecturers

Gosden, Roger, Eastern

Virginia Medical School

Hassold, Terry, Case Western Reserve University

Miele, Lucio, University of Illinois at Chicago

University

Nilson, John,

Case Western Reserve

University

COURSE ASSISTANT

Ober, Carole, University of Chicago

Wylie, Michael, University of Michigan

Richards, JoAnne. Baylor College of Medicine

Sarras Jr, Michael, University of Kansas

Ka, Hakhyun, University of Kansas Medical Center

Medicine
Kayisli, Umit, Yale University School of

Kreeger, Pamela, Northwestern University

Perez. Christian, University of Pennsylvania

Prosen, Tracy, University of Pittsburgh

Sachdeva, Geetanjali,
in

Medical Center

STUDENTS
Almeida, Claudia, Weill Medical College

University

Terasaki, Mark, University of Connecticut

Guillette, Louis, University of Florida

COURSE COORDINATOR

Bachman, Katherine, Case Western Reserve

Busso, Dolores, Buenos Aires University
Gill, Ryan, University of Kansas Medical Center

Medical Center

Gregory, Massachusetts Institute of

Burke, Rhonda,

COURSE COORDINATOR

Jaffe,

Science Center at San Antonio

Marc, Brown Unive'j'ty
Tower, John, Universe

Ducibella,

Torrens. Javier,

New

Jersey Medical School,

Wang,

NASA Ames

Eileen,

Research Center
Northwestern Medical School

Seminara, Stephanie, Massachusetts General

of Cornell University
Bishop, Glenda, Case Western Reserve University
Bokov, Alex. University of Texas Health Science
Center, San Antonio
Chen, Lishan, University of Washington

Hospital
Shenker, Andrew, Northwestern University

Fundamental Issues

Medical School

Development Center
Georgetown University

Simerly, Calvin, Pittsburgh

Suarez-Quian, Carlos,
Medical School

Harvey, Sarah, Medical College of Virginia

Hong, Eun-Jm (Erica), Yale University

Tasca, Richard,

Wells, Dagan, St Barnabas Medical Center

DIRECTORS

Johnston, Janet, Queen's University Belfast

Lledias, Fernando, Institute de Biotecnologia,

Welt, Cornne, Massachusetts General Hospital

Yin, Hang, McGill University

Masur, Sandra,

Xiangdong, University of North Carolina,

Rea, Shane, University of Colorado

Rutten, Bart. University of Maastricht

Shirasawa, Takuji. Tokyo Metropolitan Institute

of Gerontology

Tong, Liqi, University of California, Irvine

Zou, Ymg, University of Texas Southwestern
Medical Center

Vision

August

1 1

August 24, 2002

NIH

UNAM
Chapel Hill
Moynihan, Kathryn, Washington University
Ogle, William, Stanford University
Powers. Ralph, The University of Washington
Proctor, Carole, University of Newcastle

in

Research

Dunaief, Joshua, University of Pennsylvania

Fuller, Kattiryn, University of Minnesota

Lu,

Research

UMDNJ
Tou, Janet.

Schultz. Richard, University of Pennsylvania

Institute for

Reproduction

Mount

Sinai

School of Medicine

Papermaster, David, University of Connecticut

Health Center

Teaching Assistants
Agoulnik,

Irina,

Aldrich, Carrie,

Baylor College of Medicine

Roche Molecular Systems

Berard, Mark, University of Michigan

Britton,

Chad, Case Western Reserve University

Brudney, Allison, University of Illinois at Chicago

Carroll, David, Florida Institute of Technology

Combelles, Catherine, Tufts University

Curtin, Denis, University of Virginia
Galet, Colette, University of Iowa

Hadsell. Louise, Baylor College of Medicine

Huntress, Victoria, Tufts University School of

Veterinary Medicine

Jackson, Jodi, Case Western Reserve University

Kalinowski, Rebecca, University of Connecticut
Health Center

Frontiers

in

Reproduction:
Molecular and Cellular Concepts

and Applications
May 9 - June
1

29,

Kenny, Hilary, Northwestern University

Kim, Julie, University of Illinois at Chicago
Klemhenz, Andrew, Case Western Reserve
University

Payne, Christopher,

2002

Magee-Womens

Research

Limulus eye, Robert Barlow

Institute

Margaret, University of Kansas

Medical Center
Runft, Linda, University of California, Santa
Petroff,

DIRECTORS
Fazleabas, Asgerally, University of

Illinois at

Chicago
Hunt, Patricia, Case Western Reserve University

Woodruff, Teresa, Northwestern University

Barbara
Santiago. Jose, Northwestern University
Stein. Paula. University of Pennsylvania
Susiarjo. Martha,

FACULTY
Albertini, David, Tufts University

School of

Wang, Min-Kang,

Medicine
Ascoli, Mario.

The University of Iowa

Behnnger, Richard, University of Texas

James C, University of Calgary

Croy, B Anne, University of Guelph
DeMayo. Francesco, Baylor College of Medicine

Cross,

Case Western Reserve

University

Suszko, Magdalena, Northwestern University

Tufts University Veterinary

School

Wang,

Jie,

FACULTY
Beebe, David, Washington University
Bok, Dean, Univensty of California, Los Angeles
Born, Richard, Harvard Medical School
Colley, Nansi. University of Wisconsin Madison

Gordon, Marion, Rutgers University

Horwitz, Joseph, Jules Stein Eye Institute,
Masland, Richard, Harvard/MGH
Sugrue. Stephen, University of Florida

Baylor College of Medicine

UCLA

R51

Medical Informatics

LECTURERS

SUNY

Upstate Medical University

Barres, Ben. Stanford Medical School
Berson, Elliot., Harvard Medical School

May 26

Besharse, Joseph. Medical College of Wisconsin

Birk, David, Jefferson Medical College
Bok, Dean, University of California, Los Angeles

Cimmo, James, Columbia

Barlow, Robert,

Colley, Nansi, University of

Wisconsin-Madison

Dowling, John, Harvard University

Green, Carla, University of Virginia
Wisconsin Medical
Griep, Ann, University of

School

June

2,

Medical Informatics

2002

of Medicine

FACULTY

FACULTY
Friedman, Charles, University of Pittsburgh
of Medicine
Kingsland, Lawrence, National Library
Medicine
Lindberg, Donald, National Library of

Friedman, Charles, University of Pittsburgh

Hammond,

William,

Duke

Miller, Perry,

University

Kingsland, Lawrence, National Library of Medicine

Issac, Children's Hospital
Lindberg, Donald, National Library of Medicine
Miller, Perry, Yale University

Starren, Justin,

San

LaVail, Jennifer, University of California,

Columbia University

Nesbitt,

Thomas, University of

California, Davis

Ozbolt, Judy, Vanderbilt University

Shortliffe,

Edward, Columbia University

Fransico

LECTURERS
Medicine

Canese,

Kathi, National Library of

Cimmo,

Christopher, Albert Eistein College

LECTURERS
McCray, Alexa, National Library of Medicine
Ash, Joan, Oregon Health and Sciences University

of Medicine

McCray, Alexa, National Library of Medicine

Moses, Marsha, Harvard Medical School

STUDENTS

Niederkorn, Jerry, University of Texas

Southwestern Medical Center

STUDENTS

Joram, NIH/NEI
Raviola, Elio, Harvard Medical School
Stepp, Mary Ann, GWU Medical Center

Anderson, Karen, University of North Dakota

Bergman, Dale, Alberta Research Council
Binstock, Mark, Ohio Permanente Medical Group

Paul,

Eye Health Services, Weymouth,

MA
Wiggs, Janey, Massachusetts Eye & Ear Infirmary

Mount

Sinai

School of Medicine

STUDENTS
Bensinger, Steven, University of Pennsylvania
Cusato, Karen, Albert Einstein College of

Medicine

Dominy, Nathaniel, University of Chicago

Ehrhch, Jason, Stanford University School of
Medicine
Ensslen, Sonya,

Case Western Reserve

Katz, Elizabeth, University of

Hanna, Technion

University

Technology

Brock, Tina, University of North Carolina,

Brown University

Francisco

Prevention

Erdley, William, University of Buffalo

Hayes, Barrie, University of North Carolina,

Chapel

Hill

Henry, Nancy, Pennsylvania State University

Kovach, Christine, Kaiser Permanente Northwest

Prasad, Dipti, University of California, San Diego

Ramsey, David, University of Illinois, Chicago

Chad, Pennsylvania State University

College of Medicine

Reiter,

A&M

University Health

Southern California

University of Michigan
Yang, Ellen, Mount Sinai School of Medicine

Wu, David,

Zamora. David, Oregon Health & Science

University

Zandy, Anna, Washington University

in St.

Jennifer,

James Madison

University

Pond, Fred, Dartmouth College

Prendergast, Neville, Washington University

Louis

Samaritan Family

Practice Center
Virginia

Commonwealth

University

Robertson, Nan, Kaiser Permanente-Northwest

Ruiz, Jorge, University of Miami School of

Morehouse School

Elizabeth,

of

Medicine

Los Angeles

New

York University School

Dnebeek, Mary, Duke University

Hughes, Christopher, Monongahela Valley
Hospital

James, Veronica, Georgetown University

Medical Center Library
Just, Melissa, Childrens Hospital Los

Angeles

Long, Susan, Virginia Mason Medical Center

Mays, Brynn, Georgetown University Medical

Center
Meisel, Jim, Massachusetts General Hospital
Mittal, Richa, University of

Toronto

Nagle, Ellen, University of Minnesota

Perley, Cathy,

Empona

State University

Reavie, Keir, University of California, San

Francisco

Reismger, Curtis, North Shore Long Island

Jewish Health System
Schardt, Connie,

Duke

University

Colorado Health

Sciences Center

Schmidt, Heidi, University of California, San

Francisco
Sennett, Cary, American College of Cardiology
Silverman, Howard, Banner Health System

Tnmarchi, Michaeleen, The Scripps Research

Institute

Medicine
Health Care System
Smith, Scott, University of North Carolina
Sunil,

Bowen,

Contini, Janice, University of California,

Schilling, Lisa, University of

Good

Rangappa, Shantaram,

Smha,

Bird, Geoffrey, Children's Hospital of Philadelphia

Pimental, Sara, Kaiser Permanente

Marazzo, Donald, Family Health Council

Markland, Mary, University of North Dakota

Raglow, Gregory,

Science Center
Shiyi, University of

Children's Hospital of

School of Medicine

Shepard, Laura, University of Oklahoma Health

Science Center

Texas

The

Philadelphia

McCabe,

Steinle, Jena,

Ltd

Dee, Cheryl, University of South Florida

Dhara, Rosaline, Centers for Disease Control

&

Medical School
Beaudoin, Denise, Utah Department of Health
Beyea, Suzanne, AORN

Cuddy, Colleen,
of Medicine

Chuma, Chomba, Avenue Healthcare

Cowen, Janet, Maine Medical Center

Luberti, Anthony,

Eye & Ear Infirmary

Ortega, Nathalie, University of California, San

Wei, Echo

Bowles, Kathy, University of Pennsylvania

Bragdon, Lynn, Veterans Affairs Medical Center

Lambert-Lannmg, Anita, Toronto West HospitalUniversity Health Network

Maryland

Israel Instituterof

Lishko, Polina, Massachusetts

Sarah,

New Mexico

Chapel Hill
Cheng, Grace, Hospital Authority

COURSE COORDINATOR
Zekaria, Dania,

Aarstad, Robert, Louisiana State University

Alverson, Dale, University of

Piatigorsky,

McCabe,

Yale University

Randolph, Vanderbilt University Medical

Center
Nahin, Annette, National Library of Medicine
Miller,

Stead, William, Vanderbilt University

Hunter, Chyren, National Eye Institute/NIH

Lang, Richard, Children's Hospital, Cincinnati
Liberman, Ellen, National Eye Institute/NIH

Levy,

II

2002

6,

Ackerman, Michael, National Library of Medicine

Bakken, Suzanne, Columbia University

Fransico

Wasson,

October

Cimino, James, Columbia University

University

Kohane,

Hernandez, Rosano, Washington University

School of Medicine
Horton, Jonathan, University of California, San

DIRECTOR

DIRECTOR

Medical Center

Hauswirth, William, University of Florida College

September 29

VA Maryland

Suzewits, Jeffrey, Southern

School of Medicine

Illinois

University

Wax, Diane,

University of

New Mexico

White, Mary, Kaiser Permanente

Williams, Annette, Vanderbilt University

Medical Center

Zumga, Miguel, Texas

A&M

University Health

Sciences Center

Continued..

R52

Methods

in
Computational
Neuroscience

August 4

September

University

Nasir, Sazzad, University of California,

San

Pinto, Reynaldo, Institute

de

Fisica

da Univ of

Milena, Rutgers University

Shahrezaei, Vahid, Simon Fraser University
Stephens, Greg, Los Alamos National Laboratory
Raffi,

Bialek, William, F

de Ruyter van

^rsity

.ob, Princeton

University

Werner-Reiss,

FACULTV

Zhou,
3,

Aslin, Rich;

Ermeni

New

Kopell, Nancy, Boston University

Lewen, Geoffrey, NEC Research Institute, Inc
Remagel, Pamela, Harvard Medical School
Saffran, Jenny, University of Wisconsin-Madison

Schneidman, Elad, Princeton University

Shadmehr, Reza, Johns Hopkins University
Solla, Sara, Northwestern University
Sompolinsky, Haim, Hebrew University
Tank, David, Princeton University

The Hebrew

University

White, John, Boston University

Institute

y Areas, Blaise, Princeton University

Doupe, Alhson, UCSF

Fettiplace, Robert, University of Wisconsin-

Madison

Kennedy, Mary, Cal Tech

Logothetis, Nikos, Max-Planck-lnstitute
Menzel, Randolf, Freie Universitaet Berlin
,

University of

Washington
Wisconsin-Madison

Saffran, Jenny, University of

Seung, Sebastian, Massachusetts

Institute of

Technology
Srinivasan, MV, Australian National University
Zucker, Steven, Yale University

Beck, James,

University

Calderone, Richard, Georgetown University

Medical Center
Casadevall, Arturo, Albert Einstein College
of Medicine

Heitman, Joseph, Duke

Magee, Paul, University

University
of Minnesota

Rhodes, Judith, University of Cincinnati

Sanglard, Dominique, University Hospital

New

York University School

of Medicine

Laboratory

Dobbins, Heather, University of Maryland

at Baltimore

Genetics of Zebrafish
August 18 -August 31, 2002

DIRECTORS
Cecilia,

Fred Hutchmson Cancer

Talbot, William, Stanford University

FACULTY
Chien, Chi-Bm, University of Utah
Collazo, Andres, House Ear Institute

Dowlmg, John, Harvard

University

Fetcho, Joseph, SUNY at Stony Brook

Granato, Michael, University of Pennsylvania

Hanlon, Roger, Marine Biological Laboratory

Kimmel, Charles, University of Oregon

Lin, Shuo, University of California, Los Angeles
Link, Brian, Medical College of Wisconsin
Linnon, Beth, Marine Biological Laboratory
Mullins, Mary, University of Pennsylvania

Neuhauss. Stephan, ETH Zurich

Raible, David, University of

LECTURERS
Alspaugh, Andrew, Duke University Medical
Center
University of Aberdeen
Gale, Cheryl, University of Minnesota

Brown,

Washington

LECTURERS
Astrofsky, Keith, Praecis Pharmaceutical

Alistair,

Goldman, William, Washington

University

Kronstad, Jim, University of British Columbia

Kumamoto,

Carol, Tufts University

White, Ted, Seattle Biomedical Res.

Fadool, James, Florida State University

Hopkins, Nancy, Massachusetts Institute of

Technology

TEACHING ASSISTANTS
Inst

Cooke, Julie, Fred Hutchmson Cancer Research

Center

Downes, Gerald,

Wendy, Harbor-UCLA Medical Center

University of Pennsylvania

Durchanek, Charlme, University of Oregon

Hutson, Lara, University of Utah

Schumacher, Jennifer, University of Pennsylvania

Hannah, Glen Ridge High School

Ungos, Josette, University of Washington

COURSE COORDINATORS

Andes, David, University of Wisconsin

Berbes, Carlos, Virginia

Commonwealth

Pennsylvania
Gaertner, Tara, University of Texas Health
Sciences Center
Herrera-Vakj--z. Marco, University of Arizona

Huys, Quen-

~.-,mbndge University

Cheng-C-

Physiology Institute of

3.

Loebel, Alex. The \A

Ky, Califor

Robyn, Cornei

.-mann Institute of Science

.nstitute of

University
Frank, Charlotte, Yale University

Heung, Lena, Medical University of South

Carolina
Jabra-Rizk,

Man/Ann, University of Maryland,

Baltimore

MacCallum, Donna, University of Aberdeen

Farries, Michael, University of

Technology

\ersity

Mirny, Leonid, Massachusetts Institute of

Technology

for

Lawrence, Christian, Harvard University

Lesko, Suzanna, Case Western Reserve
University

Chiappe, Eugenia, The Rockefeller University

Connell, Michael, Harvard University

University h.

Inst.

Biotechnology
Wozniak, Karen, Louisiana State University
Health Sciences Center

Wilson, Carole, University College London

STUDENTS
University

Chakraborty, Santanu, Cold Spring Harbor

Miller,

Columbia

FACULTY

Mitchell,

STUDENTS

Whee

Mitchell, Aaron,

COURSE ASSISTANT

George Mason

University Medical Center

Flemish Interuniversity

Research Center/HHMI

Edwards, John, Harbor-UCLA Medical Center

Rafkin,

Jensen, Kate, Princeton University

Barreto, Ernest,

Dijck, Patrick,

Moens,

DIRECTORS

COURSE COORDINATOR

Susanne, Princeton University

COURSE ASSISTANT

Ma,

August 30, 2002

Yeaman, Michael, UCLA-Harbor Medical Center

TEACHING ASSISTANT

Lien,

August 12

Lausanne

Hopfield, John, Princeton University

Johnston, Dan, Baylor College of Medicine

Rieke, Fred

Van

Neural Development and

Molecular Mycology: Current

Approaches to Fungal
Pathogenesis

Cole, Gary, Medical College of Ohio

Filler, Scott, Harbor-UCLA Medical Center

LECTURERS
Alon, Uri, Weizmann

Duke

Vallim, Marcelo,

York

Gelpenn, Alan, Monell Chemical Senses Center

Jensen, Roderick, Wesleyan University
Koberle, Roland, University of Sao Paulo

Still,

Dartmouth College

ut, Bard, University of Pittsburgh

Tishby, Naftali,

Sturtevant, Joy, Louisiana State University Health

Boston University

Yi,

Brandets University

University of Rochester,

Faimali, Adrienne, Princeton University

Ayuera

Uri,

Wright, Geraldme, Ohio State University

Abbott.

of Medicine

Sciences Center

Sao Paulo

DIRECTORS

Reese, Amy, Washington University School

Stembach, William, Duke University

Francisco

2002

1,

Montgomery, Kimberly, Northwestern

Mayorga, Maria, Microbia Inc

Morais, Flavia, Umversidade Federal de Sao
Nielsen, Kirsten,

Duke

University Medical

HHMI

Noble, Suzanne, University of California, San

Francisco

Ramon, Ana, Georgetown

Center

Wilson, Steven, University College London

STUDENTS
Ahlgren, Sara, California Institute of Technology
Campbell, Douglas, University of Cambridge

Dambly-Chaudiere, Christine, Universite

Montpellier2
Durr, Katnn, University of Freiburg

Paulo

Center,

Perkins, Brian, Harvard University

University Medical

Gerlach, Gabnele, Marine Biological Laboratory

Hammonds-Odie, Latanya, Spelman College

Leach, Steven, Johns Hopkins University
Malaga-Tnllo, Edward, University of Konstanz

Masino, Marie, SUNY, Stony Brook

Morns, Jacqueline, Cleveland Clinic Foundation
Panzer, Jessica, University of Pennsylvania

R53

Prober, David, University of

Washington

Renier, Corinne, Stanford University

Dana Farber Cancer

Stewart. Rodney,

Bruce. Ashley. University of Chicago

Davenne. Marc, Cold Spring Harbor Laboratory
Dorman, Jennie, University of Washington
Ferris. Matthew. Los Alamos National Laboratory

Institute

Weiyi, University of Pennsylvania

Westerlund, Johanna, University of Helsinki
Young, Rodrigo. Universidad de Chile

Wang,

Gray. Annette,

Brown

University

Hooper. John, The Scripps Research Institute

Jenik, Pablo, Carnegie Institution of Washington
Kateneva, Anna, Oklahoma Medial Research

Neuroinformatics
August 17

September

Foundation

1,

2002

Case Western Reserve

Kolb, Robert,

Wake

McCauley, Anita,

DIRECTORS

McDonough,

Brown, Emery, Massachusetts General Hospital

Klemfeld, David, University of California, San

Morgan,

Diego

Herb Luther

Faculty

Fee, Michael, Bell Labs. Lucent Technologies

lyengar, Satish, University of Pittsburgh
Institute of

Technology

Barry. National Institute of

Mental

Health

Jonathan, Weill Medical College of

Cornell University

Jones, Matthew, Massachusetts Institute of

Technology
Knutsen, Per,

Weizmann

Institute of

Science

Tecumseh, Harvard University

Gardener, Dan, Cornell Medical School
Hu, Xiaoping, University of Minnesota
Fitch,

Troy,

Montana

State University

Moravcikova, Gabriela, University of Pittsburgh

Nicolaou, Nicoletta. University of Reading
Reddy, Leila, California Institute of

Technology

Smith, Spencer, University of California, Los

Wayne, Ohio State

Miller,

Arun, Johns Hopkins University

Yokoo, Takeshi, Mount Sinai School of Medicine
Sripati,

Sch.ff,

Cornell University

Vicano. David. Rutgers University

October 9

October

8,

University

Research
Bauer, Markus, University of

Nijmegen

Bodelon, Clara. Boston University

Boloori. Alireza, Harvard University
Buffalo, Elizabeth, National Institutes of Health,

NIMH
Cimenser, Aylin,

Bell

Laboratories/Lucent

FACULTY
Allen, Jennifer. University of

Janeiro

DePasquale. Joseph.

New York

State

Hoffman, Alex, National

Technology
Planck Institute for Brain

Jones. Lauren, University of Maryland at

Baltimore

on Drug Abuse

Kentucky Medical

Center
Palmer, Michael, University of Colorado Health

Sciences Center

Medical College
Murray, John, University of Pennsylvania School
of Medicine

Pomerleau, Francois, University of Kentucky

Medical Center

North, Allison, The Rockefeller University

Porterfield.

Pierini,

Lynda, Cornell University Medical

Piston, David, Vanderbilt University

Institutes of Health
Spring, Kenneth, National

Swedlow, Jason, The University of Dundee

TEACHING ASSISTANTS

Medical Center

TEACHING ASSISTANTS
NIH/NIDA

Knight, Tim, University of Kentucky

Parnsh, J Michael. University of Kentucky

Medical Center

College
Oberski, Danial, University of Buffalo
Platani, Melpomeni, The University of

Marshall, University of

Missouri-Rolla

Surgener, Stewart, University of Kentucky

College

Dundee

Sigurdson. Wade, SUNY, Buffalo

Snyder, Kenneth, University at Buffalo

Hudson, Andrew, Weill Medical College of

Cornell University

of Kentucky

Kentucky

Institute

Huettl, Peter. University of

Hao, Mmgming, Cornell University Medical

Fanselow, Enka. Brown University

Froud, Karen, Massachusetts Institute of

Kentucky

Medical Center
Daws, Lynette, University of Texas Health
Sciences Center at San Antonio

FACULTY

Caulder, Tara,

Diogo, Antonia, Federal University of Rio de

Max

Center

Davis, Heather. University of Kentucky

Albany

Technologies

Grun, Sonja,
Research

Gerhardt. Greg, University of Kentucky Medical

Medical Center

DIRECTOR

Maxfield, Frederick. Cornell University

Brown

DIRECTOR

Currier, Theresa, University of

STUDENTS
Bntt,

2002

Burmeister. Jason, Center For Sensor Technology

2002

Aldworth, Zane, Montana State University

Baron, Jerome. Max-Planck-Institute for Brain

13,

Medical Center

Department of Health
Hard. Robert. SUNY. Buffalo
Keller, H Ernst, Carl Zeiss. Inc

Anderson,

Measurements
May 9- May

Apparsundaram, Subu, University

Optical Microscopy

Izzard, Colin, University at

Schmidt, Marc, University of Pennsylvania

Strothers, Steven, University of Minnesota
Tchernikovski, Ofer, City College of New York

Rapid Electrochemical

Medical Center

University

John, Montana State University

Nicholas. Weill Medical College of

of Medicine

Walters, Katherine, University of Iowa

Angeles

Jacobs, Gwen, Montana State University

Loader, Catherine. Bell Labs. Lucent Technologies
Margoliash, Daniel, University of Chicago

Veklich, Yuri, University of Pennsylvania School

Technology

McKeehan,

Technology

Dale, Anders, Harvard University

Vaishnava, Shipra, University of Georgia

Litvak, Vladimir. Technion-lsrael Institute of

Sanjana, Neville, Massachusetts Institute of

LECTURERS

King,

University

Seth. Abhinav, University of Texas Southwestern

Medical Center at Dallas

Kronhaus, Dma, University of Edinburgh

Pesaran, Bijan, California Institute of Techology

Purpura, Ketih, Cornell Medical School

Victor,

Brown

Popoola, Joyce. King's College London

Prigozhma, Natalie. The Scripps Research Institute

Bnllinger, David, University of California, Berkeley

Bokil, Hemant. California Institute of Technology

Richmond,

Stefan, Marine Biological Laboratory

Jeffrey,

Murthy, Vmit, Rice University

Pfeifer, Andrea, National Institutes of Health

Mitra, Partha, Bell Laboratories

Mehta, Mayank. Massachusetts

University

Forest University

STUDENTS
Ahir, Alpa, University College London
Bauer, Christoph, Universiy of Geneva

Boes, Marianne, Harvard Medical School

Boxem, Mike, Dana Farber Cancer Institute

Robinson, Scott, University of Kentucky

Medical Center

COURSE COORDINATOR
Lindsay. Robin, University of Kentucky Medical

Center

Coniim

R54
TEACHING ASSISTANT

STUDENTS
Almeida, Catanna, Umversidade Avem Portugal
Baccei, Christopher, Merck Research Labs
Caldwell, Ray, University of

Yale

Concur, John,

lealth

Sciences

Dieguez, Jr
Antonio

Ur

^rsity of Illinois at

Urbana-

Champaign
Douglas, Christopher, University of Michigan
French, Kristen, Medical University of South

Dario, University of Texas at San

The Ohio State

Noll, Elizabeth,

Bngham & Women's &

A&M
of

Miami

Technology
Salas-Ramirez, Kalins, Michigan State University
Sanchez, Javier, Baylor College of Medicine

Torres-Reveron, Annelyn, Ponce School of

Medicine

Oldenziel, Weite, University Centre for Pharmacy

Overh, Oyvind, University of South Dakota

The Ohio State

Sarter, Martin,

Wrubel, Kathryn, University of Texas

Workshop on Molecular Evolution

Stephens, Jr, Robert, Ohio State University

I

vanHorne, Craig, Brigham & Women's Hospital

Amy

University of Pittsburgh

Lauren, Medical University of South

July 28

August

DIRECTOR
Cummings, Michael, Marine

Biological

Laboratory

Beerli, Peter, University of Washington

Edwards, Scott, University of Washington

in

Institute for

Genomic

June 15 -July 13,2002

Lewis, Paul, University of Connecticut

DIRECTORS
Martinez, Joseph, University of Texas at San

Antonio
Townsel, James, Meharry Medical College

Meyer, Axel, University of Konstanz

Rand, David, Brown University
Swofford, David, Florida State University
Florida State University

Thompson, Steven,

Yang, Ziheng, University College London

Yoder, Anne, Yale University

FACULTY

Antonio
Zottoli, Steven,

Williams College

Berger-Sweeney, Joanne, Wellesley College

Castaneda, Edward, Arizona State University
Etgen, Anne, Albert Einstein College of Medicine

Tom, Harvard Medical School

Gonzalez-Lima, Erika, University of Texas

at Austin

Gonzalez-Lima, Francisco, University of Texas

LECTURERS
Fraser, Claire,

The

Institute for

Genomic Research

Pearson, William, University of Virginia

Sanderson, Michael, University of California, Davis
Voytas. Daniel, Iowa State University

Yokoyama, Shozo, Syracuse University

TEACHING ASSISTANTS
University of Cape Town
State University
Rokas, Antonis, University of Wisconsin-Madison

C K

Bowie, Rauri

Mead,

Louise,

San Diego

Gonzalez, Francisco, Universidad Autonoma del

Estado de Morelos

Gopal, Shuba, The Rockefeller University

Gormley, Joseph, Massachusetts Biomedical
Initiative
Hall, Paula, University of

New Mexico

Hallam, Steven, Monterey Bay Aquarium

Research Institute

Hudelot, Cendnne, University of Manchester

Jennings, Bryan, University of Texas at Austin
Curie
Jolly, Marc, Universite Pierre et Mane

New

Winka, Katarina,

York University

WHO

Influenza Center

Lange, Martin, Staatliche Lehr-und

Forschungsanstalt fur Landwirtschaft

Langeland, James, Kalamazoo College

Lee, Dan, The Institute for Genomic Research
Leonard, Jennifer, University of California,
Los Angeles
Lim, Grace,

Scnpps

Institution of

Oceanography

Linse, Katrin, British Antarctic Survey

Woods Hole Oceanographic

Merson, Rebeka,
Institution

Opazo, Juan, P Universidad Catolica de Chile

Pearman, Peter, University of Zurich
Pie, Marcio, Boston University
Presa, Pablo, University of Vigo

Raes, Jeroen, University of

Gent

Rodriguez, Carmina, Universidad Complutense

Madrid

Rohma, University of Kansas

Sakwa, James, University of Pretoria

Sievert, Stefan, Woods Hole Oceanographic
Institution

Sipe, Tavis,

Wake

Forest University

Smith, Catherine, Centers for Disease Control

&

Prevention

Alamos National Laboratory

Stahls-Makela, Gunilla, University of Helsinki
Storz, Jay, University of Arizona
Smith, Una, Los

Umea

University

Stuart, Gary, Indiana State University

Terry,

Rebecca, University of Leeds

Thomas,

Bolaji, Tufts University

Stephen, Smith College

Wang, Zhenshan, University of Washington
Tilley,

Hildebrand, John, University of Arizona

James

American Psychological

Association
Nickerson, Kim, American Psychological
Association

STUDENTS
Banks, Michael,

Oregon

State University

Behrmann, Jasminca, University of Constance

Benavides, Edgar, Brigham Young University
Borchardt, Mark, Marshfield Medical Research

Foundation

LECTURERS
Burgess, David, Boston College
Kaplan, Barry, NIH/IVi\
D.;

Langford, George,

Mensmger,

Urbana-

Oregon

at Austin

Jones,

Illinois at

Erica, University of California,

Goetze,

Rubicz,

Hernandez, Ruben, University of Texas, San

Antonio
LeBaron, Richard, University of Texas, San

University

Martmello, Rick, Yale Universty School of Medicine

Research
Felsenstein, Joseph, University of Washington
Kuhner, Mary, University of Washington

Survival (SPINES)

Fox,

The

Eisen, Jonathan,

Neuroscience, Ethics, and

Autonomous

Champaign

Komadina, Naomi,

FACULTY

Summer Program

Cornell University

Gmzel, Matthew, University of

Kiontke, Karin,

2002

9,

Carolina

Zapata, Agustin, National Institutes of Health

Erik,

Research Center

Southern California

Holder, Mark, University of Connecticut

University

Schad, Christina, Chicago Medical School

Sokoloski, Joshua, University of Pittsburgh

Willis,

San Antonio

Mayra, University of California, Berkeley

Prather, Richard, Massachusetts Institute of

Hospitals

Wagner,

at

University

Padilla,

University
Children's

Peter, University of

Flores-Ramirez, Sergio,
of Baja California Sur

Mendes, Shannon, University

Martin, Joshua,

Countway,

York University

NASA-Ames

Conley, Catharine,

Gutierrez, Tannia, University of Georgia

Herzog, Chris, Ohio State University

Fiona, Tulane University

Medicine

of Natural

Fehling, Johanna, Dunstaffnage Marine Laboratory

Martinez, Veronica, Texas

Korean, Wayne, University of South Dakota

Li, Guichu, East Carolina University School of

New

Caufield, Page,

Dopman,

Galvan, Adnana, Emory University

Inglis,

Museum

Gallegos, Diana, San Jose State University

Hyde, Rhonda, Harvard University

Jones, Floretta, University of Texas

Carolina

Jutta, Field

History

Cruz, Nelson, Brandeis University

Diaz, Manuel, University of Puerto Rico

Gordon, University of MissouriColumbia

Burleigh,

Buschbom,

Black, Carlita, University of Virginia

Cao, Bo-Jin, University of

Center
Idil,

Burk, Robert, Albert Einstein College of Medicine

James, University of Texas, San Antonio

STUDENTS

Carolina,

Hill

Chapel

Cavus,

Orfila.

'_,:h

Allen, Univi

Palazzo, Robert, University,

.(

'

College
Minnesota

Kansas

Zakon, Harold, University oi

as, Austin
Kravitz, Edward A Harvard Medical School
,

Boudreau, Ellen, Dalhousie University

Boyce, Sarah Lyn, Natural History Museum of
Los Angeles County

Brown, Joseph, Queen's University

Budd, Aidan, European Molecular Biology
Laboratory

Bumbaugh,

Alyssa, Michigan State University

Whitford, Tracy, East Stroudsburg University

Wojciechowski, Martin, Arizona State University

Wu,

Martin,

Zmser,

Erik,

The

Institute for

Massachusetts

Genomic Research

Institute of

Technology

Zwickl, Derrick, University of Texas, Austin

R55

OTHER EDUCATIONAL
PROGRAMS

Marine Models in Biological

Research Undergraduate Program
DIRECTORS

Wake Forest University

Wake Forest University School

Browne. Carole,
Tytell,

Michael.

of Medicine

FACULTY
Augustine. George. Duke University

Eckberg. William. Howard University

Fune. Barbara. Harvard School of Medicine
Furie, Bruce.

Harvard School of Medicine

Gould, Robert New York State

Basic Research

Institute for

Hanlon, Roger. Marine Biological Laboratory

Jonas. Elizabeth, Yale University
Laufer, Hans, University of

Connecticut

University of Illinois, Chicago

Mensinger, Al, University of Minnesota-Duluth

Malchow,

R, Paul,

Palazzo, Robert, Rensselaer Polytechnic Institute

Larry, University of

Rome,

Robert,

Silver,

Wayne

Pennsylvania

State University

Wainwright, Norman, Marine Biological

Laboratory

NASA

Planetary Biology
|

Science Journalism Program

Internship Program
STUDENTS
Alimi,

FELLOWS

Manam, Wake

Bodily,

Jill.

Forest University

Stanford University

Borely, Kimberly,

Homsi, Sara,

Ohto

Wake

University

Forest University

Howard University
Montanez, Marlena, Mount Holyoke College

Jackson, Ticana,

Najera. Julia, Univ of Texas, El Paso

Normand, Danielle, University of New

Hampshire
O'Neal, Jessica, College of Charleston

Simpson, Andrew, University of California,

Santa Barbara
Steeds. Craig. University of Kansas

DIRECTORS
Dolan, Michael F, University of Massachusetts

Bellmghim, Ruth Helena. Science Reporter.

Berrcby, David, Freelance

Brazil

Biskup, Agnes, Freelance

Amherst
Margulis, Lynn, University of Massachusetts

Jennifer,

Bogo,

Audubon Magazine

Carter, Kandice,

Amherst

AAAS

Science Update

Dempsey, Dale, Freelance

INTERNS

Griffin,

Allen, Michelle,

The

University of

New

South

Chichon Garcia, Francisco

Autonoma de Madrid
Fike, David, University of

Universidad

Guarin. Alejandro, The Pennsylvania State

University

Ruben Peco,

Manier. Jeremy. Chicago Tribune

Omfade.

Cambridge

Universitatsklinikum

Richmond Times-Dispatch

The Pa'm Beach Post

King, Robert.

Wales, Australia

Navio,

Katherme, Freelance

Hosteller.

Perry.

Diran, Nigerian Television Authority

Rebecca. Los Angeles Times

Reker, Mary Lou. Library of Congress

Valentine, Vikki, National Public Radio Online

Wisby. Gary, Chicago Sun-Times

Hamburg-Eppendorf
Rosenfeld, Ane. University of Haifa

BIOMEDICAL FACULTY

Vaisanen, Katarina, University of Aberdeen

Wier. Andrew. University of Wisconsin-

Beach, Dale,

Bloom,

UNC

Kerry.

Chapel

Dale Beach,

Hill

UNC

Chapel

Hill

Palazzo. Robert. University of Kansas

Milwaukee

Pearson. Chad, UNC Chapel Hill

Schnackenberg. Brad, UNC Chapel

SPONSORS
Cabrol, Nathalie

A NASA Ames
.

Hill

Research

ENVIRONMENT FACULTY

Center

NASA Kennedy

Space Center
Hagan. William, College of St. Rose
Hinkle, C Ross. NASA Kennedy Space Center

Garland, Jay.

Foreman, Kenneth, Marine Biological Laboratory

Neill, Chnstoper, Marine Biological Laboratory
Tholke.

Kris.

Marine Biological Laboratory

Margulis. Lynn, University of Massachusetts

CO-DIRECTORS

Amherst

Summons,

Roger, Massachusetts Institute of

Technology
Trent, Jonathan.

Wofsy. Steven

NASA Ames
,

Research Center

Harvard University

Goldman. Robert D

Northwestern University

Rensberger. Boyce. Director, Knight Science

Journalism Fellowships, Massachusetts
Institute of

Technology

ADMINISTRATIVE DIRECTOR
Hinkle,

Pamela Clapp, Marine Biological

Laboratory

R56
Semester

Environmental

in

Teachers' Workshop: Living

in

the Microbial World

August 10-16, 2002

Science
September 2 - December

'

16,

20C

DIRECTORS

DIRECTOR

Dorntie, Barbara,

Hobbie, John E

School, Cambridge, Massachusetts

Olendzenski, Lorraine, University of Connecticut,

ASSOCIATE

Latin

Storrs

DIR':

Foreman, Kenne

Cambridge Rindge and

Gunnard, Jessie, University of Massachusetts

Deegan. Linda A.
Foreman, Kenneth

Offerdahl, Enka, University of Arizona

COURSE ASSISTANT:

Hobbie. John E
Hopkmson, Charles
Liles,

Jr

Institute of

Technology

Melillo, Jerry

PRESENTERS
Bermudes, David, Vion Pharmaceuticals,

Neill,

Christopher
Peterson, Bruce J.
Rastetter,

Edward B

Dyer, Betsey,

Edgcomb,

Steudler, Paul

New

Wheaton College

Virginia,

Woods

Bahr, Michele

Amherst
Rogers, Dan,

Bowen, Jennifer

Cambridge,

Hole Oceanographic

Community

Hmgham

Public Schools,

Massachusetts

Ledyard Middle School, Gales

Connecticut

Natoli, Therese,
Ferry,

Woods

School,

College, Massachusetts
Lincoln, Peter,

Guerrero, Ricardo, University of Barcelona, Spain

Margulis, Lynn, University of Massachusetts,

RESEARCH AND TEACHING ASSISTANTS

Hermon

Lee, Marge, Harrington School,

Lichtenstein, Leslie, Massasoit

Hole Oceanographic

Institution

Vallmo, Joseph J

Howie, Charles, Old Rochester Regional High

School, Massachusetts

Massachusetts

Haven, Connecticut

Shaver, Gaius R

Placentia, California

Knox, Carol, Northfield Mount

Massachusetts

George

Cape Cod Regional Technical

High School, Massachusetts
Golet, Gerie, Salem School, Connecticut
Freitas, Caroline.

Kingdom

Waksman, George, Massachusetts

S.,

Middle Years Alternative School

Humanities, Philadelphia, Pennsylvania

Hammond, Christine, Clarendon House

Grammar School, Ramsgate, Kent. United

H.

Anne E

for the

Goodding, Debbie, Kraemer Middle School,

FACULTY
FACULTY

Giblin,

Eliot, Judith,

Potrafka, Renee, Father Gabriel Richard Catholic

High School. Ann Arbor, Michigan

Institute

Creswell, Joel

Kwiatkowski, Bonnie
Micks, Patricia

Tholke. Kris

Ziemann,

Tori

Administrat've Assistant

Johnson-Horman, Frances
Students

Adams, Jacqueline
Burce, Allison

Ripon College

Harvey

E.,

Mudd

College

Copeland, Maureen T, Allegheny College

Dean, Mary D,, Ripon College
Engelhart, Gabnella J Lafayette College
,

Fila,

Laurie

A Mount Holyoke
,

Franklin, Jennifer

College

Wheaton College

Freeman, Christopher J Connecticut College

Haverford College
Havassy, Joshua
Kang, MoonKoo Simon, Clark University
,

Kennedy, Jenny L Clarkson University

Leahy, Sarah E Wheaton College
,

Leamy, Claire A Wellesley College

Lmdell, Joshua S Dickinson College
,

Roberts, Rachael

A Skidmore
,

Shea, Alexandra E

College
Earlham College

Stern, Stephanie B., Wellesley College

Webster, William
Wright, Julie

K., Trinity

University

Wellesley College

Teacher Participants
Barren, Melanie,

Cambridge

Cape Cod Regional

Donna Bedard

Technical

High School, Massachusetts

Connor, Lynn, Old Rochester Regional High
School, Massachusetts
Crook, Jolene, East Lyme High School,
Connecticut
Diehl, Penny,

Middle School, Groton,

Connecticut
Roark, Eileen, Nathan Hale-Ray High School,

Massachusetts
Berrick. Steve,

Hempfield High School,

Landisville, Pennsylvania

Microbes,

Pullan, Mary, Fitch

Public Schools,

Moodus, Connecticut
Ruston, Steve, King Etherbert School,
Birchmgton, Kent, United Kingdom

Tanigawa, Joy.

El

Rancho High School, Pico

Rivera, California

R57

SCHOLARSHIP AWARDS

BURROUGHS WELLCOME FUND-MOLECULAR

MYCOLOGY COURSE
Berbes, Carlos, Virginia

Commonwealth

University

Brown, Constance, Howard University

Heung, Lena, Medical University of South
Carolina

MacCallum, Donna, University

Aberdeen
de

of

Morais, Flavia, Universidade Federal

Sao Paulo
Noble, Suzanne. University of California.
San Francisco

Ramon, Ana. Georgetown

University Medical

Center
Reese. Amy. Washington University School
of

Medicine

Stembach, William, Duke University

Sturtevant, Joy, Louisiana State University

Health Science Center

Vallim, Marcelo,

Van

Duke

Dijck, Patrick.

University Medical Center

Flemish Interuniversity

Institute for Biotechnology

Wozniak, Karen. Louisiana State University
Heath Science Center

GARY N CALKINS MEMORIAL

SCHOLARSHIP FUND
Berry, Katy, University of Sheffield

E/izabeth Armstrong

Copf, Ti|ana. University of Crete

THE BRUCE AND BETTY ALBERTS

SCHOLARSHIP IN PHYSIOLOGY
Agutlar, Arturo,

Av

ENDOWED

CONRAD
|

Dash, Satya, University of East Anglia

Gonsalves, Joanna, University of California,

Institute PolitEcnico National

Kelly, Melissa, University of

LALOR BURDICK SCHOLARSHIP

|C

Sachdeva, Geetanjali,

Institute for

Research

in

Reproduction, India

San Francisco

Kentucky College

EDWIN GRANT CONKLIN MEMORIAL FUND

Hallikas. Outi, University of Helsinki

of Medicine

Prosen, Tracy, University of Pittsburgh

Caracino, Diana, Emory University School

BURROUGHS WELLCOME FUND

BIOLOGY OF PARASITISM COURSE

Copf, Ti|ana, University of Crete

AMERICAN SOCIETY FOR CELL BIOLOGY

of Medicine
-

Brown, Ann, Medgar Evers College

Campbell, Susan, University of Alabama,

Chamond,

Nathalie, Institut Pasteur

Davis, Kevin, University of Pittsburgh

Cockburn,

Ian, University

Gadea. Bedrick, Harvard Medical School

Karnataki. Anuradha, University of

Gonsalves, Joanna, University of California,

San Francisco

Klotz. Christian, Humbo'dt-University-Berlin

Birmingham

Green, Heather,

New York

Kooij,

Lee,

University

Harrison, Faith, University of Iowa

Livi, Carolina, University of Texas Health Sciences

Li,

Sciences Center, Dallas

New

Jersey Medical School,

Van

Stry,

Taco Leiden, University Medical Centre

Melanie, Boston University School

Vega, Rebecca, Stanford University

Hongjie, Yale University

WILLIAM
I

BIOLOGY CLUB OF THE COLLEGE OF

THE CITY OF NEW YORK
Zornik, Erik

Columbia University

JJOHN AND ELISABETH BUCK SCHOLARSHIP

Thamatrakoln, Kimberlee, University of California.
San Diego

AND

IRENE C DILLER MEMORIAL

SCHOLARSHIP FUND
Davis, Kevin, University of Pittsburgh

BURROUGHS WELLCOME FUND FRONTIERS

IN REPRODUCTION COURSE
-

Vega, Rebecca, Stanford University

Akcali, Kamil. Bilkent University

University

Ryan. University of Kansas Medical Center

Gonsalves. Joanna. University of California. San
Gill.

Francisco
I

& Technology

Pmel, Nicolas, University of Washington

of Medicine

Dalhousie University

Environmental Science

SooHee, Johns Hopkins School of Medicine

Bachman, Katherme, Case Western Reserve

UMDNJ

Yart,

Denef, Vincent. Michigan State University

Erbs, Marianne, Swiss Fed Institute for

Medicine, Heidelberg

Miranda, Jason, University of Texas at Austin

Pineda, Gabriel. University of Texas Health

BERNARD DAVIS FUND

Boucher,

Washington

Mueller. Ann-Kristin, University School of

Center, San Antonio

TorrEns, Javier,

of Edinburgh

THE ELLISON MEDICAL FOUNDATION

BIOLOGY OF PARASITISM COURSE
Chamond, Nathalie, Institut Pasteur

Ian, University of Edinburgh

Karnataki, Anuradha, University of Washington

Cockburn,

Humboldt-University-Bcrlm
Taco Leiden, University Medical Centre
Mueller, Ann-Kristin, University School of
Klotz, Christian,

Hallikas. Outi, University of Helsinki

Hakhyun, University of Kansas Medical

Center
School of Medicine
Kayisli, Umit, Yale University
Ka,

KOOIJ,

Medicine. Heidelberg

Kreeger, Pamela, Northwestern University

Perez, Christian, University of Pennsylvania

THE ELLISON MEDICAL FOUNDATION

Prosen, Tracy, University of Pittsburgh

Almeida, Claudia, Weill Medical College of

TorrEns, Javier,

New Jersey

Medical School,

UMDNJ
Wang,

Eileen,

MOLECULAR BIOLOGY OF AGING COURSE

Cornell University

Bishop, Glenda, Case Western Reserve

Northwestern Medical School

University

continued

R58
Bokov, Alex, University of Texas Health Science
Center, San Antonio

Dunaief, Joshua, University o<

Fuller, Kathryn, University of

Harvey, Sarah, Medi<

Hong, Eun-Jin (Eric
to

de Biotecnologia,

UNAM
;versity of

XiangcV

North Carolina,

Chapel H
Moynihdn Kathryn, Washington University

Rossi, Chiara, University of Pisa

Advanced Study

(SISSA)

Keram, Philipps-Universitat Marburg

Washington
Rajagopal, Soumitra, University of Nebraska

Koren, Omry, Tel Aviv University

Litvak, Vladimir, Technion-lsrael Institute of

Technology
Loebel, Alex, The

Witney, Alice, University of Birmingham

Medical School

Amane, Dragos,

Berry, Katy, University of Sheffield

Hallikas, Outi, University of Helsinki

Ihnng, Alexandra, Max-Planck-lnstitute of

Chakraborty, Santanu, Cold Spring Harbor

Chen, Yen-Chin, National Cheng Kung

University Medical College

Laboratory

Cambridge

(WILLIAM RANDOLPH HEARST FOUNDATION

Chong, Curtis, Johns Hopkins School of
Medicine
Kentucky College

of Medicine

LaPomte, Nichole, Northwestern University

Kozhevnikov, Alexay, Bell Laboratories

Nasir, Sazzad, Unversity of California, San

Cheryl, University of Pennsylvania

Pace, Margaret, University of Texas

Pinto, Reynaaldo, Institute

de

Flsica

da

University of Sao Paulo

Reddy. Leila, California Institute of Techology

Shahrezaei, Yahid, Simon Fraser University

Snpati, Arun, Johns Hopkins University
Wang, Weiyi, University of Pennsylvania

Young, Rodngo, University of Chile

Zhou, Yi, Boston University

McVaugh,

Pisa

Sha, Edward, Indiana University Medical Center

INSTITUTE

BIOLOGY OF PARASITISM COURSE

Meissner, Markus, Imperial College of Science,

Technology & Medicine, UK

Slavin, lleana,

Dame

MBL PIONEERS SCHOLARSHIP FUND

Herrera-Valdez, Marco, University of Arizona

Hu, Hailan, University of California, Berkeley
Koirala, Samir, University of Southern California

Francisco

Cheryl, University of Pennsylvania

HOWARD HUGHES MEDICAL

University of Notre

GROSCH SCHOLARSHIP FUND

Vmcenzo, University of

Amane, Dragos,

de Janeiro

Dethlefsen, Les, Michigan State University

Ge, Lan, University of California, Riverside

Pignatelli,

MBL ASSOCIATES ENDOWED

SCHOLARSHIP FUND

Diogo, Antonia, Federal University of Rio

London
Extavour, Cassandra, University of

Neurobiology
Zhou, Zhaolan (Joe), Harvard Medical School

Chiappe, Eugenia, The Rockefeller University

Copf, Tijana, University of Crete

Delalande, Jean-Mane, University College

Kelly, Melissa, University of

Dame

(SO MAST MEMORIAL FUND

Akcali, Kamil, Bilkent University

INTERNATIONAL BRAIN RESEARCH

ORGANIZATION

ICASWELL GRAVE SCHOLARSHIP FUND

McVaugh,

University of Notre

Dojcmovic, Danijel, Arizona State University

Pace, Margaret, University of Texas

Medical Center

S-

Science

IICRO-UNESCO

Tong, Liqi, University of California, Irvine

Zou, Ying, University of Texas Southwestern

DANIEL

Institute of

JACQUES LOEB FOUNDERS'

SCHOLARSHIP FUND

Werner-Reiss, Un, Dartmouth College

Shirasawa, Takuji, Tokyo Metropolitan Institute

Weizmann

Oceanography

Wright, Geraldme, Ohio State University

of Gerontology

Zandbank

Keren,

Pinel, Nicolas, University of

Renart, Alfonso, Brandeis University

Rea, Shane, University of Colorado

Rutten, Bart, University of Maastricht

THE GRUSS LIPPER FOUNDATION

SCHOLARSHIP

Pfeiffer,

Sharp, Kathenne, Scnpps Institute of

Ogle, William, Stanford University

Powers, Ralph, The University of Washington
Proctor, Carole, University of Newcastle

Chicago

Ramos, Arnolt, Children's Hospital

Neurobiology

Petersen, Rasmus, International School for

<!ty

versity Belfast

Illinois at

Pace, Margaret, University of Texas

Malartre, Marianne, University of Portsmouth

Nkinm, Wuyika, University of Yaounde

Virginia

Oh, Ji-Eun, University of

Ihnng, Alexandra, Max-Planck-lnstitute of

esota

Lu,

Kydd, Alison, University of Calgary

Research

Chen, Lishan, University of Washington

Johnston, Janet,
Lledias, Ferpjr

Guest, Jennifer, National Institute for Medical

ARTHUR KLORFEIN SCHOLARSHIP AND

FELLOWSHIP FUND

Universidad Nacional de Cordoba

Berry, Katy, University of Sheffield

Caracino, Diana, Emory University School of

Medicine
Copf, Tijana, University of Crete
Dash, Satya, University of East Anglia
Delalande, Jean-Mane University College, London
Extavour, Cassandra, University of Cambridge
Koziel, Lydia, Max-Planck-lnstitute for Molecular

Genetics
Livi,

Carolina, University of Texas Health Science

Center, San Antonio

CHARLES BAKER METZ AND WILLIAM METZ

SCHOLARSHIP FUND IN REPRODUCTIVE
BIOLOGY
Akcali, Kamil, Bilkent University

Drago, Grazia, Universita Degli Studi di Palermo

Extavour, Cassandra, University of Cambridge

Hallikas, Outi, University of Helsinki

Kee, Yun, California Institute of Technology

Koziel, Lydia, Max-Planck-lnstitute for Molecular

Prosen, Tracy, University of Pittsburgh

Kreeger, Pamela, Northwestern University

MORRELL ENDOWED MEMORIAL

Genetics
Malartre, Marianne, University of Portsmouth

Stubbs, Janine, Royal Melbourne Hospital

Brown, Ann, Medgar Evers College

Maslakova, Svetlana, Smithsonian Institution

Su, Yi-Hsien, Scripps Institute of

Oceanography,

SCHOLARSHIP
IFRANK
Petersen, Rasmus, International School for

Advanced Study

(SISSA)

MBRD
IHOWARD HUGHES MEDICAL

(MOUNTAIN MEMORIAL FUND SCHOLARSHIP

INSTITUTE

Boassa, Daniela, University of Arizona College

Aguilar, Arturo,
in

St Louis

Denef, Vincent, Michigan State University

Doiron, Brent, University of Ottawa
Dulcis, Davide, University of Arizona, Tucson

Graco, Michelle, University of Pierre et

Mane Curie

LILLIE

FELLOWSHIP AND

(SCHOLARSHIP FUND

of Medicine

Dash, Satya, University of East Anglia

Dellen, Babette, Washington University

IFRANK R

Av

Institute Politecnico

National
University of Notre Dame
Do|Cinovic, Danijel, Arizona State University
Ge, Lan, University of California, Riverside

Amane, Dragos,

Goentoro, Lea, Princeton University

Kaynar, Murat, Beth Israel Deaconess Medical

Center

Chong,

Curtis,

Johns Hopkins School of Medicine

Kentucky College

Kelly, Melissa, University of

of Medicine

LaPomte, Nichole, Northwestern University

Oh, Ji-Eun, University of Illinois at Chicago
Pignatelli,

Vincenzo, University of Pisa

Ramos, Arnolt, Children's Hospital

R59

IALBERTO

MONROY FOUNDATION

Drago, Grazia. Universita Degli Studi

Palermo

PFIZER INC.

di

ENDOWED SCHOLARSHIP
Av

Aguilar, Arturo,

Marine diatom,
Ka/ma White

Institute PolitEcnico

National

Chong. Curtis, Johns Hopkins School of

Medicine
Pignatelli,

Vmcenzo, University

of Pisa

PLANETARY BIOLOGY INTERNSHIP

SCHOLARSHIPS

Lien,

Cheng-Chang, Physiology

Julia,

Walker. Jeffrey, University of Colorado

Masmo, Mark.
I

TOWNSEND PORTER FELLOWSHIP

WILLIAM

Institute of

at

State University of

Berlin

New York

IMARJORIE

Stony Brook

Prober, David, University of

IAND SCHOLARSHIP FUND

Georgia State University

Spitzer. Nadja.

Wohlgemuth, Sandra. Humboldt-Universit zu

University Freiburg
Ma, Whee Ky, California Institute of Technology
Malaga-Trillo, Edward, University of Konstanz

Pennsylvania State University

Remold, Susanna, Michigan State University

Maresca,

STETTEN SCHOLARSHIP FUND

Witney. Alice, University of Birmingham

Medical School

Washington

Milena, Rutgers University

Ramos, Arnolt, Children's Hospital
Raffi,

Brown, Ann, Medgar Evers College

Campbell, Susan, University of Alabama,

Birmingham
Davis, Kevin, University of Pittsburgh

HORACE

STUNKARD SCHOLARSHIP FUND

Rossi. Chiara, University of Pisa

Akcali, Kamil, Bilkent University

Sha, Edward, Indiana University Medical Center

Gill,

Ryan, University of Kansas Medical Center

Gadea, Bedrick. Harvard Medical School

FLORENCE C ROSE AND S MERYL ROSE

ENDOWED SCHOLARSHIP FUND

Gonsalves, Joanna, University of California,

San Francisco

Green, Heather,

New York

Caracino. Diana, Emory University School

of Medicine

University

Harrison, Faith, University of Iowa

Livi, Carolina, University of Texas Health

Science Center, San Antonio

Arizona State University

Hu, Hailan, University of California. Berkeley

Wohlgemuth. Sandra, Humboldt-Universit zu

RUTH SAGER MEMORIAL SCHOLARSHIP

Guest, Jennifer, National Institute for Medical

Miranda, Jason, University of Texas at Austin

Pineda, Gabriel, University of Texas

Southwestern Medical Center

ISOCIETY FOR GENERAL PHYSIOLOGY

Brown, Ann. Medgar Evers College
Dojcmovic. Danijel. Fumio Mekata Scholar,

Berlin

Research
Mitchell, Tracy, University of

at Dallas

ISURDNA FOUNDATION SCHOLARSHIP

De Labra. Carmen, University College London

Wisconsin-Madison

HOWARD A SCHNEIDERMAN ENDOWED

Dellen, Babette,

(SCHOLARSHIP

POST-COURSE RESEARCH AWARDS

Curtis,

Ewald, Rebecca, Cold Spring Harbor Lab

Cambridge (Embryology)
Johns Hopkins School of Medicine (Physiology)

De

Khali!.

ENDOWED

Mona, Columbia

Zhou. Zhaolan

New

Jersey Medical School,

UMDNJ
Van

Stry,

Melanie, Boston University School

W. RAND FELLOWSHIP
[HERBERT
(SCHOLARSHIP FUND

AND

Ahlgren, Sara, California Institute of Technology

Amarie, Dragos, University of Notre Dame

IRVING WEINSTEIN

Harvard Medical School

ENDOWED SCHOLARSHIP

Su, Yi-Hsien, Scripps Institute of

University

MOSHE SHILO MEMORIAL

SCHOLARSHIP FUND

Oceanography,

MBRD
I

Koren, Omry, Tel Aviv University

of Medicine

Vega, Rebecca, Stanford University

(Joe).

Berry, Katy, University of Sheffield

Kerney, Ryan, Harvard University

Torrens. Javier,

Louis

Ryan, Amy, University of Virginia Health Systems

Labra,

MILTON L. SHIFMAN
SCHOLARSHIP

St

of Technology

Carmen, University College London

Doiron, Brent, University of Ottawa

Davis, Kevin, University of Pittsburgh (Physiology)

Ge, Lan, University of California, Riverside (Physiology)

LaPointe, Nichole, Northwestern University (Physiology)

in

Hobbs. Steven, University of Colorado

Montana, Enrico, Massachusetts Institute

Boassa, Damela, University of Arizona College

of Medicine

Extavour, Cassandra, University of

Chong,

Washington University

Doiron, Brent, University of Ottawa

WILLIAM

MORTON WHEELER

FAMILY

FOUNDERS' SCHOLARSHIP
Ihnng, Alexandra, Max-Planck-lnstitute of

Neurobiology

(CATHERINE FILENE SHOUSE SCHOLARSHIP

Avila, Andrea, Institute de Biologia Molecular
do Paran -IBMP

Boassa, Daniela, University of Arizona College

WALTER L WILSON ENDOWED

SCHOLARSHIP FUND
Dojcmovic, Danijel, Arizona State University
LaPointe, Nichole, Northwestern University

of Medicine

Brown, Ann, Medgar Evers College

Campbell. Douglas. University of Cambridge

Chen. Yen-Chin, National Cheng Kung
University Medical College

Caracino. Diana, Emory University School

of Medicine

WORLD ACADEMY OF ARTS AND SCIENCES

EMILY MUDD SCHOLARSHIP

Dojcmovic, Danijel, Arizona State University

Ewald, Rebecca, Cold Spring Harbor Lab

Fenn, Katelyn, University of Edinburgh

Akcali, Kamil, Bilkent University

Durr, Katrin, University of Freiburg

Gaertner, Tara, University of Texas Health

Science Center

Goentoro. Lea. Princeton University

Hammonds-Odie,

Kaynar, Murat, Beth Israel

Medical Center

Spelman College
Deaconess

Latanya,

Bachman, Katherme. Case Western Reserve

Graco, Michelle, University of Pierre et

Mane Curie

Hallikas, Outi. University of Helsinki

Kreeger, Pamela, Northwestern University

April, University of Missouri-Columbia

Wang,

University

Perez, Christian, University of Pennsylvania

Orsborn,

Eileen,

Northwestern Medical Sch>"

Sharp. Katherme, Scripps Institute of

Oceanography

WORLD HEALTH ORGANIZATION

Busso, Dolores, Buenos Aires Un:v.

R60

INSTITUTIONS REPRESENTED

Albert Einstein Colleqp o

Medicine

(students)

Harvard University

American College

Hebrew

c'

Carjtology

AORN

New
New
New

Harvard Medical School

Alberta Research Cc-.ncil

Universtiy

AstraZeneca

Howard

R&D Charnwood, UK

University

Northwestern University

State University
Imperial College of Science, Technology

Ohio Permanente Medical Group

Ohio State University

Illinois

Banner Health System

Baylor College of Medicine

Indiana State University

Indiana University

Laboratories/Lucent Technologies
Beth Israel Deaconess Medical Center

Indiana University Medical Center

Institute

Bilkent University

Institut

Boehringer Ingelheim Pharmaceuticals

Boston University

Institute

Boston University School of Medicine

Institute

Brandeis University

Institute

Oklahoma Medical Research Foundation

Oregon Health & Science University

& Medicine

Brigham

& Women's

University

British Antarctic

Survey

Cambridge

Venezolano de Investig Cientificas

de

Institute for

State University

P Universidad Catolica

de

Chile

Pennsylvania State University College

Pasteur
Fisica

da Univ of Sao Paulo

Research

in

Reproduction
de Biologia Molecular do Parana-IBMP

of Medicine

Pennsylvania State University

Philipps-Universitat

Marburg

Physiology Institute of University Freiburg

Ponce School of Medicine
Princeton University

James Madison

Brown University
Buenos Aires University
California Institute of

Oregon

de Biotecnologia, UNAM
International School for Advanced Study (SISSA)

Hospital

Bngham Young

York University School of Medicine

Humboldt-Universitat zu Berlin

Autonomous University of Baja California Sur

Av Institute Politecmco National #2508
Avenue Healthcare Ltd.

Bell

UMDNJ

North Shore Long Island Jewish Health System

Northwestern Medical School

Hospital Authority

Arizona State University

Jersey Medical School,

York University

University

Johns Hopkins Bloomberg School of Public

Queen's University Belfast

Health

Technology

Raytheon Polar Services

Johns Hopkins School of Medicine

Johns Hopkins University

Rice University

RIKEN, Japan

University

Carl Zeiss

Permanente
Kalamazoo College

Rockefeller University

Kansas State University

Karolmska Institute!

Rutgers University

Kaiser

Carnegie Institution of Washington

Case Western Reserve University
Centers for Disease Control

&

Prevention

Royal Melbourne Hospital

San Jose State University

Chicago Medical School

King's College

Children's Hospital Boston

Children's Hospital of Philadelphia
Children's Hospital Los Angeles

Leiden University Medical Centre

Liverpool School of Tropical Medicine

Cleveland Clinic Foundation

Los

Cold Spring Harbor Lab

& Mary
Columbia University

Louisiana State University Health Sciences Center

Louisiana State University Medical School

College of William

London

Alamos National Laboratory

Scnpps Institute of Oceanography

Simon Fraser University
Smith College
Smithsonian Institution

Southern

Illinois

University School of Medicine

Spelman College
Staatliche Lehr-und Forschungsanstaltfur

Landwirtschaft

Maine Medical Center

Dalhousie University
Dana Farber Cancer Institute

Marine Biological Laboratory

Marshfield Medical Research Foundation

Stanford University
Stanford University School of Medicine

Dartmouth College

Massachusetts Biomedical

State University of

Doane College
Duke University
Duke University Medical Center

Massachusetts Eye & Ear Infirmary

Massachusetts General Hospital

Initiative

Technion

Max-Planck-lnstitute for Molecular Genetics

Tel Aviv University

East Carolina University School of Medicine

East Stroudsburg University

Max-Planck-lnstitute of Neurobiology
Medgar Evers College

Temple

Emory
Emory

Medical College of Virginia

Medical University of South Carolina

University

York at Stony Brook

& Technology

Massachusetts Institute of Technology

Max-Planck-Institute for Brain Research

Dunstaffnage Marine Laboratory

New

Swiss Federal Institute for Environmental Science

Israel Institute of

University School of Medicine

ASM
Texas A&M
Texas

Technology

University
University Health Science Center

European Molecular Biology Laboratory

Michigan State University

The Institute for Genomic Research

The Naval Research Laboratory
The Scnpps Research Institute

Microbia Inc

Tokyo Metropolitan

Family Health Council

Federal University of Rio

Monongahela Valley Hospital

Montana State University

Toronto West Hospital-Univ Health Network

University School of Medicine

Empona State University

Field

Museum

de Janeiro

of Natural History

Flemish Interumvo^ity Institute for

Monterey Bay Aquarium Research

Morehouse School of Medicine

Mount

Biotechnology
Fox Chase Cancer

George Mason University

Georgetown University Me

MerckResearch Labs

Sinai

Center

Georgia State University

Good Samaritan Family Practic-= Center

Tufts University

School of Medicine

National Institute for Medical Research

National Institutes of Health
Natural History

Museum

Gerontology

Institute

NASA-Ames Research Center

National Cheng Kung University Medical College
iical

Institute of

of Los Angeles County

UMass Medical School

United States Air Force
University of Massachusetts, Amherst
Universidad Autonoma del Estado de Morelos

Universidad Complutense Madrid

Universidad de Chile
Universidad Nacional de Cordoba

Universidade Aveiro

R61

COUNTRIES REPRESENTED
Argentina
Australia

Belgium
Brazil

Cameroon
Canada
Ch.le

China

Cyanobactena, Rolf Schauder

Finland

France

Universidade Federal de Sao Paulo

Umversita Degli Studi

di

Palermo

Germany
University of

Nijmegen

Greece

University of North Carolina

India

Universite Montpellier 2
Umversite Pierre et Mane Curie-Pans 6

University of North

Dakota

University of Notre

Dame

University Centre for

University of

Oklahoma Health Sciences Center

University

University of

Oregon

University of

Ottawa

Pharmacy
College London
of Aberdeen

University
University of

Alabama, Birmingham

University of Arizona
University of Arizona College of Medicine
University of Birmingham Medical School
University of Buffalo
University of Calgary
University of California, Los Angeles
University of California, Berkeley

Israel
Italy

Japan
Kenya
Mexico

University of Pennsylvania

Netherlands

University of Pennsylvania School of Medicine

University of Pierre et Mane Curie

Portugal

Scotland

University of Pisa

South Africa

University of Pittsburgh
University of Portsmouth

Spam
Sweden

University of Pretoria

Switzerland

University of Puerto Rico

Taiwan

University of California, Irvine

University of California, Riverside

University of Reading
University of San Lui Potosf

Turkey
United

University of California, San Diego

University of California, San Francisco

University of Sheffield

United States of America

University of

Cambridge

University of South Florida

Chicago
Colorado
Colorado Health Sciences Center

University of Southern California

University of Texas at Austin

University of
University of
University of

University of Connecticut

University of South

Dakota

Venezuela

University of Texas Health Science Center,

San Antonio

University of Constance
University of Crete

University of Texas Southwestern Medical

University of East Anglia

University of Toronto

University of Edinburgh

University of Utah

University of Freiburg
University of Gent

University of Virginia
University of

University of Hawaii

University of Wisconsin

Illinois at
Illinois at

of Science

Washington

American Psychological Association

University of Wisconsin-Madison

Chicago
Urbana-Champaign

(faculty)

Albert Einstein College of Medicine

American Association for the Advancement

Vigo

University of Georgia

University of
University of

INSTITUTIONS REPRESENTED

Center at Dallas

University of

University of Helsinki

Kingdom

University of

Arizona State University

Yaounde
Baylor College of Medicine

University of Zurich

Bell Labs,

Lucent Technologies

University of Iowa

University School of Medicine, Heidelberg

University of Iowa College of Medicine

University of

University of Kansas

Utah Department of Health

Boston College
Boston University

University of Kentucky College of Medicine

VA Maryland

Bowlrng Green State University

University of Konstanz

Vanderbilt University Medical Center

BioHybnd Technologies

Geneva

University of Kansas Medical Center

University of

Leeds

University of Maastricht
University of

Manchester

Health Care System

Veterans Affairs Medical Center

Virginia
Virginia

Commonwealth University
Mason Medical Center

University of
University of

Miami
Miami School of Medicine

Wake

Carnegie

Forest University

Washington University

in St

Louis

University of Michigan

Weil! Medical College of Cornell University

Weizmann Institute of Science

University of Minnesota

WHO Influenza Center

University of Missouri-Columbia

Woods

University of

New
New

Mexico
South Wales
University of Newcastle
University of
University of

Hole Oceanographic

Institution

Montana

University of Naples
University of Nebraska

Bndgewater State College

Brown University
California Institute of Technology

University of Maryland

University of Massachusetts Medtcal School

Brandeis University

Yale University
Yale University School of Medicine

Institution of

Washington

Carnegie Mellon University

Case Western Reserve University

CCNY
Center For Sensor Technology
for Genome Research

Centre

Centre Genetique Moleculaire

Cold Spring Harbor Laboratory
Columbia University
Cornell Medical School
Cornell University

Contmued

R62
Dartmouth College

New

Danmark Tekniske Universitet

Duke University Medical Center
Duke University

Northwestern University
Northwestern University Medical School

York University School of Medicine

Ohio State
Eastern Virginia Medical

Emory University
ETH Zurich
European

Institute o\

Florida Institute

J|

Fred Hutchir,

Praecis Pharmaceuticals

University of Guelph
University of Hawaii

Princeton University

University of Idaho

ancer Research Center

University Medical School

Dundee

University of Georgia
University of Glasgow

University

Queen's University Belfast

Freie Universitaet Berlin

Georgetown
Geron

University

ogy

Florida State Ui

University of

University of Edinburgh
University of Florida

Oregon Health & Science

Oregon State University

University of Connecticut
University of Connecticut Health Center

University of

Illinois

University of

Illinois,

University of

Illinois,

Chicago
Urbana

Roche Molecular Systems

University of Iowa

Rockefeller University

University of Kansas

Rutgers University

University of Kentucky
University of Kentucky Medical Center

San Diego State University

Seattle Biomededical Research

Hamamatsu Photonic Systems

Harbor-UCLA Medical Center

University of Konstanz
Institute

University of Lethbridge
University of

Manchester

Harvard Medical School

Sensor Technologies
Smithsonian Institution

Harvard University

St

Hebrew University
HHMI/Brown University
HHMI/Fred Hutchmson Cancer Research Center
HHMI/UMDNJ-RW Johnson Medical School

Stanford Medical School

University of Massachusetts
University of Massachusetts Medical School

Stanford University

University of

Barnabas Medical Center

Stowers

Institute for

HHMI/UT Southwestern Medical Center

SUNY
SU NY

Stony Brook

HHMI/Johns Hopkins

Syracuse University

University

University of Maryland

Medical Research

at Buffalo
at

University of

Missoun-Columbia

University of Missouri-Rolla

HHMI/NYU

University of North Carolina,

House Ear

Institute

ICRF Clare

Hall Laboratories

Texas

Imperial Cancer Research Fund

Imperial College of Science Technology
Institut

Melbourne

University of Michigan
University of Minnesota

Pasteur

A&M

University of Notre

University

The Hebrew University

The Institute for Genomic Research
The University of Iowa
The University of Manchester

University of Utah

Medicine

Tufts University School of Veterinary

Medicine

Umea University
University of Pittsburgh
University of Texas Southwestern Medical

University of

Washington

University of Wisconsin

UT Health Science

University of Virginia

London

University of Victoria, B
University of Virginia
University of Warwick

University of Wisconsin

Center

Karolinska Institutet

Kent State University

King's College,

University of Texas

University of Texas at Austin

University of Toronto

Tufts University School of

Johns Hopkins School of Medicine

Johns Hopkins Universtiy
Jules Stem Eye Institute, UCLA

Hill

University of Oregon
University of Oxford

Tufts University

Iowa State University

Chapel

Dame

Madison

Center, San Antonio

University of Texas at Austin

Vollum

University of California, Davis

Lawrence Berkeley National Laboratory

London School of Hygiene and Trop

Med

University of California, San

Institute

Diego

University of California, Santa Barbara

Louisiana State University Health Science Center

University of California, Berkeley

Washington University
Washington University School of Medicine

Leiden University, The Netherlands

University of California, Los

Weill Medical College Cornell University

Magee-Womens Research

Institute

Angeles

University of California, San Francisco

Weizmann

University of North Caroline,

Wellesley College

Chapel

Hill

Institute

Marine Biological Laboratory

Massachusetts General Hospital

University of

University of Kentucky Medical Center

Wesleyan University
Whitehead Institute for Biomedical Research

Massachusetts

University of Maryland, College Park

Williams College

Institute of

Technology

Illinois,

Chicago

McGill University

University of Rochester (NY)

Medical College of Georgia

Medical College of Ohio

University of Pittsburgh

Medical College of Wisconsin

Meharry Medical College

University of British

Michigan State University

Monell Chemical Senses Center

University of Kansas Medical Center

Montana State University

Mount Sinai School of Medicine

University of Pennsylvania

MPI

for

University of Southern California

MPl
MPI

for Biological

for

University of

Medical Research

Cybernetics

Manne Microbiology

Colorado Health Sciences Center

Lab of Molecular Biology

Memorial Sloan Kenenng Cancer Center
National Institute on Aqing, NIH
National Institute for M.-J.ca! Research

Yale University

Columbia
University of Colorado Health Sciences Center
University of

University of

Sao Paulo

Ulm

University College

London

University Hospital

Lausanne

University of

Argentina
Australia

University of Pittsburgh School of Medicine

Universitaet

COUNTRIES REPRESENTED

Oregon

Universidad de Buenos Aires

MRC

Yale Medical School

Aberdeen

Lebanon
Mexico

Austria

New Zealand

Brazil

Russia

Canada

South Africa

China

Sweden

Columbia

Switzerland

Denmark

Taiwan

University of Alabama
University of Arizona

France

The Netherlands

Germany

National Institute on Druq

National Institutes of Health

University of Bern
University of Calgary

India

Turkey
United Kingdom
United States of Americ

National Library of Medicine

University of

National Institute of Me";,

'^alth

NEC Research Institute, Inc

New England Medical Center
New York University

University

Cambridge
of Cape Town

University of

Greece
Indonesia
Ireland
Israel

Chicago

University of Cincinnati

Italy

Jamaica

R63

mbl/whoi

library

REPORT OF THE LIBRARIAN

computer-age doorways to information systems

Libraries are the

worldwide. Today the

MBL/WHOI

Library uses an integrated set of

resources for online cataloging, resource sharing and reference

services, with local

large universities

are

now

and regional systems

and museums

all

essentially borderless. With increases

shrinking budgets, libraries

more and more

Woods

linking

Hole with

over the world. Libraries

in

like

ours

journal pricing and

must collaborate with other

institutions

to deliver information that their users require.

which enables the one-time transformation

Digitization technology

of information from print to electronic format

will

only increase

collaborations and encourage the sharing of information over time.

Thanks to
at risk of

this

being

lost forever

on

books, and images once

articles,

technology, important

dusty shelf can

be stored and

delivered electronically to users around the world. The

The

MBL/WHOI

Library maintains

world's largest print

and

one of

electronic collec-

tions of biomedical, oceanographic,

marine biological

literature.

operated with the

graphic
located

jointly

Woods Hole Oceano-

Institution.
in

It is

and

the MBL's

The main
Lillie

library

Building, with

in

Woods

since

996. Over the years, hundred of thousands of people from

th
around the world have rediscovered these 19 century teaching

charts,

which are

still

very useful today.

is

branches on the campuses of the Woods

Hole Oceanographic and the National

Marine Fisheries Service

the

MBL/WHOI

of the
Library has shown this to be true with the successful digitization
web
site
on
the
have
been
available
which
Leuckart charts,
Library's

Hole.

The MBL/WHOI
tions.

We

important

Library continues to

grow

in

non-traditional direc-

are developing innovative digitization services and

new

research tools, including X-ID, an online taxonomic key,

funded by the Jewett Foundation, and uBio, a network taxonomic

name server funded by the Andrew W. Mellon Foundation. Through
these two pilot projects

we have

established global alliances with

GBIF (Global Biodiversity Information

Taxonomic Information

museums.

Currently,

Herbarium of
of

Service)

we

local fauna

MBL Associates

is

Facility)

and

and bolstered our

ITIS (Information

relationships with

are creating a digital archive for the

MBL

and marine algae. A very enthusiastic group

scanning the specimens for the project, which

funded by SeaGrant.
Continued

is

R64
In

2002,

we

aggressively

moved more Library content and

web for direct delivery to the

supporting services to the

end of 2002, the MBL/WHOI Library was

-ly 52% of our serials and 100% of
delivering appro-r
patron. By the

our database?
the Library'

patrons

week.

;rh

"

We

outed, electronic form extending

'

beyond our walls

to

be twenty-four hours

wherever our

a day, seven

days a

continued to improve existing services with the

of our web site (www.mblwhoilibrary.org/).

enhancement

We

also continued to invest

in

the Library's future through

serial

and database usage

statistics

and surveyed the

community about how current subscriptions and

exchange programs were meeting their needs. Discusscientific

sions resulted

substantive changes to the 2003

in

serial

back of long-standing exthe MBL and WHOL These changes

collection, including a cut

change programs
reflect input
nity

at

from librarians and members of the

on a number of

commu-

factors: survey responses; the

community's request that we subscribe to the Web of

Science database; rising serials prices; expiring ejournal

major expenditures, projects, technological platforms,

contracts being replaced with higher-cost alternatives;

support systems and replacement of current infrastructure

and computer technology that support existing library

space constraints
in

the

in

Lillie

the MBL-serials budget

Building;

and a

4%

decrease

line.

services.

The

Library hosted

1 1

9 Readers taking sabbaticals,

working on writing projects, or conducting long-term

research projects. The wireless network installed in 2002

brought Internet access to the stacks

and reading rooms.

we have declared the Grass Reading Room to

"technology free" zone to maintain a quiet, contem-

Ironically,

be

plative

space

in

a Library that has resolutely

embraced

Monographs

We continued
named

to acquire titles for the

book and

special/

budget and specific

funds including the Atwood, Aron, and Mullin gift funds.
The WHOI ship libraries were updated with the addition
of

new

collections using our general

reference as well as recreational reading materials.

The Clark Reading Room was completely dismantled

electronic information delivery.

reorganize
als

were

staff

to

workspace. Non-duplicate Clark materi-

filed with

the

mam book

which was completely

stacks were relabeled.
Lillie),

collection (3rd floor

shifted.

In

addition,

all

Special Collections

Dedicated

staff

and volunteers continued to make great

The MBL Archives pro-

strides in Special Collections.

cessed the reprint collection of Viktor Hamburger, John

Burris' Director's

papers,

Director's papers,

James

Ebert's scientific

and the Arthur

glass plates, slides,

Humes

and photographs.

and

collection of

We

also restored

and rebound 130 items from Rare Books/Special Collections with the support of the Florence Gould Foundation.

The

WHOI

Data Library

process and catalog

Serials

&

Archives (DLA) continued to

their extensive collection of technical

reports,

We made

smooth

transition to a

new

serials

vendor,

EBSCO, and escaped the turmoil our former vendor

created when it declared bankruptcy and stranded major
academic libraries with loss of access to journals. The
Library realigned serial holdings

support current

and services

scientific research interests.

to better

We

analyzed

maps,

scientist's

papers, ship cruise reports and

They spent a considerable amount of time

migrating Alvm and other data from old media to newer
logs, etc.

formats. Both

awareness

ment

MBL

for the

plans.

and

need

WHOI

Archivists are raising

of institutional records

manage-

R65

LIBRARY READERS

Courses
Library staff

gave 37 general

library

sessions and taught 25 courses

in

2002. Trainers were brought

in

from

several specific databases to

do

in-

depth

training sessions.

tion with our Spring

and

Informatics courses,

new

In

conjunc-

Fall

Allen. Garland.

Lisman, John. Brandeis University

Laderman, Aimlee. Yale University

Washington University
Anderson, Everett, Harvard University

Linck, Richard, Umersity of

web pages were designed,

and course packs were made available over the web.
student

Our
|

staff actively

professional

participated

development

in

activities

and contributed to the profession as

\a whole. This serves to

MBL/WHOI

promote
and it

Library projects

exposes our staff to new ideas and

methods. DLA staff hosted a meeting
of the

Marine Technology Society

concerning

The

Baccetti.

Bacoo,

NRC

Minnesota

New

York University
Loewenstem, Werner. Journal of Membrane

of Siena. Italy

Rudolfo,

Benjamin. Thomas Harvard Medical School

Bernhard. Jeffery, University of Massachusetts
Medical Center

Biology
Lorand. Laszlo, Northwestern University

Borgese, Thomas, Leaman College, CUNY

Bower. James, University of Texas, San Antonio
Boyer, John, Union College

Luckenbill, Louise,

Medical

interactive

York University

Laczko, Jozsef.

Llinas.

orientations or department-oriented

New

Abbott, Jayne, Marine Research. Inc

Ahmad|ian, Vernon, Clark University

Medical School

Ohio

University

Menmi, Anna, CNR-SISSA

Candelas, Graciela, University of Puerto Rico

Cariello, Lucio, Stanzione Zoologica "A Dohrn"

Milkman, Roger, University of Iowa

Miller, Andra, NIH
Mitchell, Ralph, Harvard University

Chang, Donald, Hong Kong University

Morrell, Leyla,

Rush Presbyterian

Lukes

St

Child, Frank, Trinity College

Clarkson. Kenneth, Bell Labs

Cohen, Seymour, American Cancer Society

Cook. Erik, Howard Hughes Medical Center

Nagel. Ronald. Albert Einstein College of Medicine

Narahashi, Toshio. Northwestern University

Naugle. John.

NASA

Cooperstein, Sherwin, University of

Plummer Cobb, Jewel,

Connecticut

Copeland. Eugene.

Woods

Hole,

MA

California State University

Prendergast, Robert, Johns Hopkins University

Corwin, Jeffery, University of Virginia

Couch, Ernest, Texas Christian University

D'Alessio, Giuseppe, Universita di Napoli

Fedenco

II

Davis, Jonathan, Lexigen Pharmaceuticals

Donavan, Erin, Auburn University

Dube, Francois. St. Luc, Canada
Duncan. Thomas, Nichols College

scientific instruments.

Library Director

and Associate

Director also served on various task

forces involved

in

developing a

strategic plan for the

MBL.

Epstein,

Herman, Brandeis University

Fmkelstein, Alan. Albert Einstein College of

Medicine
Fraenkel, Dan, Harvard Medical School
Frenkel, Krystyna, New York University
School of Medicine

The MBL/WHOI

Library continues to

serve as an electronic bridge between

Woods

Hole and the world providing

access to collections at universities,

and

in

public, corporate, private,

museum, and

laboratory libraries

around the globe.

Galatzer-Levy, Robert, University of Chicago

Rabmowiu. Michael, Harvard

Gancia-Blanco, Mariano. Duke University

Medical School

Rafferty,

German, James, Cornell University

Grossman, Albert, New York University Medical

Rome, Larry, University of Pennsylvania

Ruderman, Joan, Harvard Medical School

University

MA

Reynolds, George, Princeton University

School

The Population Council

Sheng, Morgan, Massachusetts Institute

Gruner. John, Cephalon, Inc

Segal, Sheldon,

Halvorson, Harlyn, University of Massachusetts,

of Technology
Shephard, Frank,

Boston

Catherine N. Norton

Nancy, Falmouth.

Harrington, John, SUNY New Paltz

Herskovits. Theodore, Fordham University

Woods

Hole Data Base

Shimomura, Osamu, Falmouth,

MA

Smith. Tim, Northeast Fisheries Science Center

Solomon, Dennis, Yarmouth

Port.

MA

Abraham, College of Physicians

& Surgeons
Spotte, Stephen, Mote Marine Lab

Inoue, Sadayuki. McGill University

Spector,

Jaye, Robert, Boston

Jacobson, Allan, University of Massachusetts

Medical Center

Steinberg. Martin. Boston University School

Josephson, Robert, University of California

Stuart,

of Medicine

Ann. University of North Carolina

Sullivan, Gerald, Savio

Prep-Boston

Kaltenbach, Jane, Mount Holyoke College

Karlin, Arthur,
Kelly,

Robert.

Tnnkhaus, John, Yale University

Columbia University

Woods

Keynan, Alexander.
& Humanities

Hole,

Israel

MA

Academy

of Sciences

MA

King. Kenneth, Falmouth,

Knox, Carol, Northfield Mount

Tweedell. Kenyon. University of Notre Dame

Tycosmski, Mark. University of Pennsylvania

Walton, Alan, Cavendish Laboratory

Herman

Kornberg. Hans. Boston University

Krane. Stephen, Harvard Medical School

Warren. Leonard, University of Pennsylvania

Weissmann, Gerald. New York University
Medical Center
Whittaker. J Richard. University of

Woods Hole

Research Center

New

Brunswick

R66

financials

REPORT OF THE TREASURER

The

financial results for

ment

in

a year

where

2002

all

reflected the difficult operating environ-

non-profits were adversely affected by two

major trends: the continued decline

in

the investment markets and a

softening of philanthropic support.

The Unrestricted Operating results showed a loss of $1.76 million,

which was a vast improvement over the $2.9 million loss reported in
2001. This was due to a more rapid increase (14.4%)

in

Unrestricted

Operating Support when compared to Operating Expense growth

(9.6%). On the revenue side, Government Grants increased by 8.2%,
Lab Rental & Net Tuition increased by 12%, Fees for Conferences and

rebounded 21 .7% from depressed levels in 2001 as a result

cancelled events due to September 1 1"\ and Investment and Other
Services

of

Revenues increased by 30%.

On

the expense side. Research activities represented two-thirds of the

increase, growing by approximately $2 million (9.7%) as the Laboratory

added more than two dozen

scientists mainly to

gear up for expanded

Aquaculture and Global Infectious
Disease. As a result, grant applications hit an all-time high and grant
dollars awarded increased almost 30% over 2001
Looking at the
research programs

in

Scientific

underlying components, double-digit increases were experienced

in

Salaries (13.6%), Fringe Benefits (16.2%), Supplies (19.4%), Utilities

(12.1%),

and Depreciation (16.8%).

Philanthropic support

fell

dramatically from the unusually strong levels

the Laboratory had experienced in the three previous years when the
MBL received gifts exceeding $10 million each year. Total Contributions including those to Plant

were $4.6

million, the lowest level since

1995. The long-term investment portfolio, however, performed quite

fund
competitively in a year when the average common stock mutual

declined by 28%. The 3.9% decline in market values was in the top
how a universe of 1 22 foundations and
quartile when compared to

endowments performed

in

2002. The

MBL had

investment losses, which totaled $2 million

in

realized

and unrealized

2002.

in
Taking into account the challenging near-term market environment,
Assets
in
Total
Net
decline
its
first
2002 the Laboratory experienced

since 1994.

MBL's Net Assets declined $6.2 million.

R67

The MBL's Balance Sheet Assets

declining by $7.1 million.
trated

in

The

reflected this impact,

entire

decline was concen-

permanently

restricted gifts.

summary,

it

was

to the receipt of
Property, Plant

planning effort

also held steady as $2.4 million

in

new
& Equipment

improvements more

than offset the accrued depreciation. Liabilities declined

by approximately $1 million, principally due to the relin-

quishment of a Unitrust to the benefit of the Laboratory.

we have

some financial performance ratios, our Return

on Average Net Assets was a negative 6.7%, which is in
line with most non-profits during this period. The MBL's
Considering

&

Temporarily Restricted Net

already started implementing

steps that will position the Laboratory for a strong

rebound

in

our Government Grants and to ultimately

gear-up for a new capital campaign that should improve

our philanthropic support. Our education, summer/
visiting scientist,

Leverage Ratio (Unrestricted

a challenging year financially mainly

to the effect the third consecutive year of investment

market declines had on MBL's philanthropic support and

investment portfolio. Coming out of our strategic

Short Term Investments and Pledge Receivables.

The Endowment held up due

In

due

and conference

Coverage

permanently
at

ratio of

restricted

year-end are well

in

.72X for 2002 and our non-

Cash & Investments

remained

research programs should help us continue to improve

our operating results

near future.

in

the

Assets/ Debt) remains sound at 4.48X. Also, both our Debt

Service

activities

strong and when combined with the expanded resident

Mary

B.

Conrad

of $25.6 million

excess of the financial covenants of

the Letter of Credit supporting the MBL's Long Term Debt.

Lobster eyes, Diane Heck. David Ramsey, Lydia Louis, and Jeff Laskin

R68

FINANCIAL STATEMENTS

Operating History and Balance Sheet as of December 31 2002 and 2001

,

BALANCE SHEET

(In

Thousands)

2002

2001

$4.357

$8.993

8.794

11,265

ASSETS:

Cash and Short Term Investments

Pledges and Other Receivables
Assets Held by Bond Trustee

Other Assets

269
631

711

42.290

42.181

Property Plant and Equipment (Net)

31.729

31,519

TOTAL ASSETS:

87.801

94.938

2,797

3,133

Endowment and

Similar Investments

LIABILITIES.

Accounts Payable
Annuities and Unitrusts Payable

535

1,383

2.557

2,318

Long Term Debt

10,200

10,200

Total Liabilities:

16.089

17,034

Deferred Revenue and Other

Liabilities

NET ASSETSThe

financial

statements of the

Marine Biological Laboratory,

the fiscal year ending
31. 2002.

for

December

Unrestricted

20,381

22.261

Temporary Restricted

25.278

29.941

Permanently Restricted

26,053

25,702

Net Assets:

71.712

77.904

$87.801

$94,938

were audited by

Pr/cewaterhouseCoopers. LLP

Total

Complete financial statements are

upon request from:

TOTAL

LIA8ILITES

AND NET ASSETS:

available

Mr Homer Lane

OPERATING HISTORY

(In

Thousands)

Chief Financial Officer

Marine Biological Laboratory

OPERATING SUPPORT:

M8L Street
Woods Hole. MA

Government Grants

$15.849

$14,648

Private Contracts

1.495

1,675

02543-1015

Lab Rental and Net Tuition

2,188

1,954

5.333

4.383

Contributions

4.522

10.886

Investment and Other Revenues

3,321

2.555

32,708

36,101

Fees

Total

for

Conferences and Services

Operating Support

EXPENSES:
Research

22,371

20.399

Instruction

5.998

5.637

Conferences and Services

1.460

1.133

Other Programs

5.027

4.643

34.856

31.812

(2,148)

4.239

(149)

2,244

Total Expenses:

CHANGE

IN NET ASSETS BEFORE

NON. OPERATING ACTIVITY.

Non-Operating

Activities.

Contributions to Plant and Other Expenses, Net

Total Investment

Income and Earnings

Less Investment Earnings Used for Operations

Reinvested

(Utilized)

TOTAL CHANGE

IN

Investment Earning

NET ASSETS:

(1,994)

(2.930)

(1,901)

(1.475)

(3.895)

(4,405)

$(6.192)

$2.128

R69

gifts

REPORT OF THE DEVELOPMENT COMMITTEE

As the

MBL engaged

in

institution-wide strategic planning during this past year as described by

2002 was a pivotal year for Development efforts. Moving forward

successful
our
after
Discovery Campaign with new research and educational prospectacularly
attention to broadening our outreach and education activities
in
our
we
turned
place,
grams now
Dr.

earlier in this report,

Speck

through a series of events and communications.

P/anlcromc sarcodme The Host

DNA

is

heated

within the dark.

centra/ nuc'ear capsule. The

symbionts are the sma// bright

specks radfatmg outward along the

host spines, David Caron

summer, we held an enjoyable and informative Day of Science attended by 92 guests from
nearby communities that included presentations by MBL scientists and lab tours. The Council of
Last

meeting drew 80 friends from across the country to learn about Modem Molecular
Approaches to Global Infectious Diseases and to tour the totally refurbished suite of laboratories

Visitors

lectures and
newly established research program in this area. As in past years, our summer
the
MBL.
We
word
about
the
in
and
Martha's
Nantucket
childrens' programs
Vineyard spread
North
in
alumni
Carolina,
first-ever
branched out in 2002 and held our
Chapel Hill,
reception

for the

MBL

Corporation Members, and friends of the Lab.

And, turning to the Internet, Development information is now featured on MBL's web site so we
can effectively extend our message to a world-wide community.

attended by 65 course alumni and

In

2002,

MBL

raised $4,944,803

Mellon Foundation
project designed to

in

faculty,

from the
private support. This included $1 ,350,000

Andrew W.

renewed support for terrestrial forest research and support for a new pilot
index and organize information about organisms that is distributed on the

in

MBL

Council of Visitors member, Robert Shifman, provided an additional gift of

$564,502 to the Milton Shifman Endowed Scholarship The G. Linger Vetlesen Foundation
Internet.

Bay Paul Center and unrestricted

and the Burroughs Wellcome
support. The Ellison Medical Foundation, the Grass Foundation,
Fund all provided renewed support for MBL's advanced courses in biology and biomedicme.
continued

its

generous and long-standing support

for the

The 2002 Annual Fund had another strong year with $553,620 raised from 898 donors both new
records in annual fund giving at the MBL. The amount raised and numbers of donors are both up
whose gifts of
by 9% over last year. As in past years, the Whitman Society, comprised of donors
$1,000 or more accounted
serving as Annual
Associates.

On
I

We

for

much

Fund Chair and

are

of this success.

would

Mr. Michael Fenlon for

enormously grateful

to thank Dr. Peter

Armstrong for
drive
for the
Annual
Fund
leading the

for this unrestricted

like

support for the Laboratory.

behalf of the Development Committee, the Board of Trustees, and the entire MBL community,
like to express my appreciation to the donors whose names appear on the following

would

all most grateful for their generous

pages, and to those who requested anonymity. We are
educational programs.
and
research
support for the Marine Biological Laboratory's

Christopher M. Weld, Chair, Development Committee

R70

HIGHLIGHT!

During 2002,

Ecosystems Center

the following

members map out

foundations and

research plan,
Elizabeth Armstrong

individuals

provided major
support for the
Laboratory.

Burroughs Wellcome Fund awarded $170,000

support of the Molecular

Mr. Robert Shifman

made

an additional

for continuing

$564,502

to support the Milton

Mycology course.

Endowed

Scholarship.

member
The

Ellison

Mr.

L.

gift

of

Shifman

Shifman was

of the Council of Visitors.

Medical Foundation awarded

for continuing support of the

Molecular Biology of Aging Colloquium for

$282,670

G. Linger Vetlesen Foundation provided

$150,000

for the

Josephine Bay Paul Center

the period 2002 through 2004.

in

The Grass Foundation awarded $226,500 in

renewed funding for the Neurobiology and

support of the
program to develop marine models for
biomedical research; and $100,000 to

Neural Systems and Behavior courses.

underwrite veterinary services

Comparative Molecular Biology and

Evolution; $100,000

in

Resources Center.

Andrew W. Mellon Foundation awarded two

grants totaling $1,350,000: $850,000 to

support research on nitrogen transformation

landscapes conducted by The

Ecosystems Center and $500,000 to launch
the Universal Biological Indexer and Orgain terrestrial

nizer (uBio), a

database and internet tool to

provide up-to-date biological information.

Neura/ system cone mossaic, Inigo Nova'<s F/amanque

Staff
a

in

the Marine

R7I

SPECIAL GIFTS
Other

Gifts

American Society for Biochemistry & Molecular Biology

Mrs KimballC Atwood, III
Aquatic Eco-systems, Inc
Ms Susan M Barnes

Josephine Bay Paul

Bio-Rad
Dr Barry R Bloom

& C Michael

Paul Foundation

Mr and Mrs Thomas C Bolton

Mrs Carolyn S Carlson
Dr Eloise E Clark

Cohen

Drs William and Marion

Dr Seymour S Cohen
Elsevier Science Ireland Ltd
Drs Lawrence

Cohen and Barbara

Ehrlich

Federation of American Societies for Experimental Biology

Fluke Corporation
Friendship Fund

Dr and Mrs Harold Gainer

Anne Goldman
Woodland Hastings
Haubnch

Drs Robert and

Dr and Mrs

Dr Robert R

and companies provided

funds for special projects and programs at the Marine Biological
Laboratory. We gratefully acknowledge their support.
variety of foundations, individuals,

Mr and Mrs Irwm Herskowitz

Mrs Carmela J Huettner
Mr and Mrs Richard A Huettner
Dr and Mrs Shmya Inoue
Mr David Isenberg and Ms Paula Blumenthal
Invitrogen

Dr Wallace

Major

Anonymous

and Mrs Benjamin Kaminer

Dr Andrew Kropmski and Dr Peggy Pntchard
Dr Kiyoshi Kusano
Dr.

(2)

American Society
American Society

Ip

Mr Daniel Isenberg

Gifts

for Cell Biology

for

Reproductive Medicine

Dr Hans Laufer

Mr Richard Mawe
Merck Research Laboratories

Applied Biosystems

Ms

Cox Foundation, Inc

Estate of Emily Ann Cramer

Natalie Miller

Environmental Data Research Institute

Dr Betty C Moore
Ms Mary Musacchia and Dr James Faber

ExxonMobil Foundation

Mr Telephone,

Georgia-Pacific Corporation
Mr William Golden and Dr Jean Taylor

Howard Hughes Medical

Institute

England Biolabs, Inc

Ms Catherine N Norton
Novartis

Dr and Mrs Kurt J Isselbacher

Mr and Mrs George
Logan

Dr and Mrs

The Mantia Family

PhotoArk

Pharmacia

Trust

Inc

New

Philip

Person

& Upjohn Company

Madelene

Massachusetts Environmental Trust

Estate of

The Honorable and Mrs G William Miller

New York Times Company Foundation

Princeton Day School

Nikon Instruments

Qiagen, Inc
Dr and Mrs Harris Ripps
Dr Raja Rosenbluth

Inc

The Pfizer Foundation

Mrs Robert
Pierce
William Townsend Porter Foundation
Dr and Mrs John
Rowe
The Schooner Foundation

Pierce

Promega Corporation

Mr and Mrs

William Rugh

Sholley Foundation, Inc

The Catherine Filene Shouse Foundation

Schenng-Plough Research Institute

Smauer Associates Inc Publishers
Dr Sol Sepsenwol
Dr Abraham Spector and Ms Marguerite Filson

Alfred P Sloan Foundation

Drs

Society for Neuroscience

The Seth Sprague Education

Mrs Eleanor Stembach

&

Charitable Foundation

Drs William Speck and Evelyn Upper

Melvn and Evelyn Spiegel

Syngenta
Dr and Mrs Andrew

Szent-Gyorgyi

The Sprague Foundation

Drs Albert Stunkard and Margaret Maurin
Mr and Mrs Gerard I Swope

Ms

Universal Imaging Corporation

Dr and Mrs. Gerald Weissmann

Mr Sidney

Mrs Clare M Wilber

World Health Organization
World Precision Instruments
The Worthmgton Family Foundation,

Wemberg, Jr
The Irving Wemstein Foundation, Inc.
Mr and Mrs Christopher M Weld
J

Natalie Trousof

United States Geological Survey

Dr Dale A Webster

Inc

R72

THE WHITMAN SOCIETY

Mem.'

"

(e

'

j in

the

Whitman Society

$1,000 or more

open each year

New York, New

Anonymous

to individual

donors who

or Alumni Funds.

Mr William T. Golden
American Museum of Natural

Director's Circle
|

Dr. Porter

is

MBL Annual

to the

Gmny and

Pete Nicholas

Boston, Massachusetts

History,

York

Arthur B. Pardee and

W. Anderson,

Mr.

Jr.

and Mrs. Amos

Hosteller

B.

Ann

B.

Goodman

Cambridge, Massachusetts

Jr.

Boston, Massachusetts

Key Largo, Florida

Manus

Mr.

and Mrs William C Cox,

Hobe Sound, Florida

Mr.

Dr.

Jr

Laurie

J.

Northport,

Landeau

New

A. Robinson

Key Biscayne,

Florida

York

Drs Mitchell Sogin and Laurel Miller

Dr.

and Mrs. Kurt

Newton

J.

Fa/mouth, Massachusetts

Robert A. Prendergast
Fa/mouth, Massachusetts

Isselbacher

Center, Massachusetts

Mr.
Mr.

Mr.

and Mrs. George W. Logan

New

Salem, Virginia

The Honorable
Washington,

and Mrs. Richard

York,

Harriet

William Miller

DC

New

Reiss

New

C.

Arbor, Michigan

Drs.

Ann

Stegeman

York

and Sheldon Segal

Hartsc/ale,

and Mrs. John

Ann

Chapel

E.

Hill,

Stuart

and John W. Moore

North Carolina

York

Mrs. Robert W. Pierce

Boca Grande, Florida

Patron

Member

Mr. Vin

Ryan and Ms. Carla Meyer

Boston, Massachusetts
Dr.

and Mrs. John W. Rowe

New

York,

New

Woods

York

Speck and Evelyn Upper

West Fa/mouth, Massachusetts

Mr.

Mr. Sidney

J.

Mr.

New

New

Wemberg,

New

Frank and Mardi Bowles

Drs. William

York,

Edward and Marion Adelberg

Anonymous

Jr.

York

Hofe, Massachusetts

and Mrs. Malcolm

and Mrs. Murray H Bring

East Hampton,

New

York

Drs Claire Fraser and

New

Potomac, Maryland

Mr.

New

York

and Mrs. Christopher

Weld

Essex, Massachusetts
Mr.

and Mrs Alfred

Zeien

Dr Louise Adler
Fa/mouth, Massachusetts
Dr Garland E Allen
Saint Louis, Missouri

Dr and Mrs Gerald Weissmann

York,

Brachman

K.

Dallas, Texas

Haven, Connecticut

J.

Craig Venter

Mr and Mrs Douglas

Bloomfield

Hills,

F Allison

Michigan

Drs Clay and Clara Armstrong

Philadelphia, Pennsylvania

Frances and Howard Jacobson

Westborough, Massachusetts

Boston, Massachusetts

and Mrs. Stephen M. Krane

Waban, Massachusetts

Benefactor

Catherine C. Lastavica, M.D.

Manchester, Massachusetts

Diane and Norman Bernstein

Dr.

Dr.

Peter and Margaret Armstrong

Davis, California

Dr Samuel C Armstrong
Everett,

Washington

Jean and Bob Ashton

New

York,

New

York

Mr and Mrs David Bakalar

Washington,
Mr,

York,

New

Woods

Laster

Chestnut

Hill,

Massachusetts

Hole, Massachusetts

Mr and Mrs Charles A Baker

and Mrs. Jonathan Conrad

New
Mr.

DC

and Mrs. Leonard

York

and Mrs. Diarmaid Douglas-Hamilton

Beverly, Massachusetts

Dr.

Anna Logan Lawson

Princeton,

New Jersey

Daleville, Virginia

Drs Robert and Harriet Baker

Birgit Rose and Werner K Loewenstem
Fa/mouth, Massachusetts

White

Plains,

New

York

David Baltimore and Alice S Huang

Pasadena, California

R73
Mrs Fredenk B Bang
Woods Hole, Massachusetts

Mr and Mrs Frank

East Fa/mouth, Massachusetts

Woodside,

Hope and Mel Barkan

Dr and Mrs Richard

Judith

Boston, Massachusetts

New York, New

Dr and Mrs Robert B Barlow

Frank and Julie Child

The Jane and

Woods

Vero Beach, Florida

Jamesville,

New

York

C.

Carotenuto

Chappell

Mr and Mrs David

Douglass

California

and John Dowhng

Boston, Massachusetts

York

Hole. Massachusetts

Allan

Dragone Foundation

Fred and Christine Bay

Dr Eloise E Clark

Mr and Mrs George Edmonds

Landgrove, Vermont

Bowling Green, Ohio

Osterville,

Bruce Anthony Beal

Woods Hole, Massachusetts

Mr.

Drs Eugene and Millicent

Boston, Massachusetts

Mr and Mrs James

Mr and Mrs Hans A Ege

Mahwah, New Jersey

and Mrs Hays Clark

Hobe Sound,

Florida

Massachusetts

Palm Beach, Florida

Herman N. Eisen and Natalie Aronson

Waban, Massachusetts

Drs Michael and R Suzanne Bennett

Arnold and Connie Clark

Mrs Ariana Fairbanks

New

Woods

Honolulu, Hawaii

Rochelle,

New

Bell

York

Hole, Massachusetts

Mrs. Beth Berne

Drs Alexander

Woods

Seattle,

Hole, Massachusetts

Clark

Clowes and Susan E Detweiler

Drs

Dr.

Verdi Farmanfarmaian

Princeton,

Washington

New Jersey

Jewelle and Nathaniel Bickford

Mrs George H A. Clowes

Ms

New

Dover, Massachusetts

Newton, Massachusetts

Yoric,

New

Mr and Mrs

Yorlc

Eric F Billings

Dr Jewel

PlummerCobb

Linda Sallop

Mrs James

and Mr Michael Fenlon

Ferguson Jr

Potomac, Maryland

Los Angeles, California

Dr and Mrs Elkan R Blout

Drs. Arthur

Cambridge, Massachusetts

Key Biscayne, Florida

Northbroolc,

Mr

Dr and Mrs D Eugene Copeland

Woods Hole, Massachusetts

Drs Bruce and Barbara

Allen Bragdon

South Yarmouth, Massachusetts

Dr and Mrs Goodwin

New

Yorlc,

New

Breinm

York

Dr and Mrs. John B Buck

Sykesvtlle,

Maryland

Mr and Mrs N
Princeton,

Dr Max

Harrison Buck

New Jersey
Burger

Molly

L,

and Laura H Colwin

Furie

Wellesley, Massachusetts

C Gadsby

New

Dr William B Cosgrove
Pittsboro, North Carolina

Ms

Dr Joseph T Coyle
Belmont, Massachusetts

Rebeckah DuBois Glazebrook

Thomas and Geraldine Crane

Dr and Mrs Murray Glusman

Woods Hole, Massachusetts

Dr and Mrs Mario Burgos

Mendoza, Argentina

Dr and Mrs Stephen

Bethesda, Maryland

and Nonnie Burnes

Hole Fund

E Foreman, Jr

Illinois

Dr and Mrs David

Cornell

Weston, Massachusetts

Butler's

Mr and Mrs Harold

Falmouth, Massachusetts

Basel, Switzerland

Rick

Chevy Chase, Maryland

Mrs

Sally

York,

New

Yorlc

SalheGiffen

Falmouth, Massachusetts

Osprey, Florida

Crocker

Suzanne and Maynard Goldman

Boston, Massachusetts

Drs Robert and

Cross

Anne Goldman

Illinois

Falmouth, Massachusetts

Chicago,

Dr and Mrs Anthony

Drs Timothy and Mary Helen Goldsmith

Northford, Connecticut

Boston, Massachusetts

Mr and Mrs Malcolm Campbell

Upper Montcla/r, New Jersey
Vineyard Haven, Massachusetts

Mr and Mrs Bruce G Daniels

Lincoln, Massachusetts

Dr Graciela C Candelas
San Juan, Puerto Rico

Cutaia

Ballwin, Missouri

Dr

Eric

H Davidson

Pasadena, California

Dr and Mrs Moise H Goldstein,

Woods Hole, Massachusetts
Susan and
Teahclcet,

Jr

Tom Goux
Massachusetts

Continued

R74

Hinsch

Dick and Eleanor Grace

Dr Gertrude

The Brain Center

Thonotosassa, Florida

Boca Grande, Florida

John and Ohvann Hobbie

Mr and Mrs Rudy Landry

Fa/mouth, Massachusetts

Pocasset, Massachusetts

Drs Joseph F Hoffman and Elena Gtkowitz

New Haven, Connect/cut

Homer W and Mary Cronan Lane

Mew

Lambrecht

Seabury, Massachusetts

Grant

Philip

Washington,

Dr Michael

DC
and Mr

<

:a

H Greenberg

St Augustine, Florida

Dr Mary

New

Mr and Mrs Robert

York,

Dr. Francis C G Hoskin and

Elizabeth M Farnham

Greer

New

North Fa/mouth, Massachusetts

Mrs Nancy Norman Lassalle

New

New

York,

York

Canton, Massachusetts

York

Dr Hans Laufer
Mr.

and Mrs William H Greer,

Jr

Mrs.

Carmela

Woods

Chevy Chase, Maryland

Huettner

Storrs,

Mr
Dr and Mrs Thomas C Gregg
Fa/mouth, Massachusetts

Mr and Mrs

and Mona Gross

Jamaica Plain, Massachusetts

Dr.

Dr and Mrs Lawrence Grossman

Dr and Mrs Shmya Inoue

Fa/mouth, Massachusetts

Kansas

Connecticut

Hole, Massachusetts

Charles Hunter

Joel

Leavitt

Boston, Massachusetts

Missouri

City,

J K Lilly,
West Fa/mouth, Massachusetts

Mrs
Paul R

and Mrs Hugh E Huxley

III

Concord, Massachusetts

Baltimore, Maryland

Dr and Mrs Anthony Liuzzi

Boston, Massachusetts
Laszlo

Dr.

and Mrs Harlyn

Woods

Halvorson

Hole, Massachusetts

Mary D Janney

and Joyce Lorand

Glencoe,

Illinois

DC

Washington,

Dr and Mrs Robert E

Diane and Robert Jaye

Newton, Massachusetts

Ms Penelope Hare
West Fa/mouth, Massachusetts

Mamer

Way/and, Massachusetts
Walter and Shirley Massey

Dr.

Dr William R Jeffery

and Mrs. Robert Haselkorn

Silver Spring,

Illinois

Chicago,

Ms

Woody and Hanna

Hastings
Cambridge, Massachusetts

Barbara

Atlanta, Georgia

Maryland

Mr and Mrs Robert Mastroianni

Jones

Fa/mouth, Massachusetts

Fa/mouth, Massachusetts
Luigi Mastroianni,

Freda and Benjamin Kaminer

Woods Hole, Massachusetts

Dr Robert R Haubnch

Ohio

Granville,

Synnova Hayes
Princeton, West

Anastasia and

Dr Diane E Heck
Rumson,
Dr.

New Jersey

and Mrs Thomas Hedges,

Jr

New Jersey

Moorestown,

Dons and Howard

Tom

Sir

Hilhs

and Dr

Paul

Colmvaux

Mr Timothy T

Hilton

Cambridge, Massachusetts
Gregory

and Pamela Clapp Hmkle

Plymouth, Massachusetts

Hole, Massachusetts

Jr

Dr and Mrs Jerry M Melillo

Falmouth, Massachusetts

Hans and Lady Kornberg

Fa/mouth, Massachusetts

Mr

Ms

Dr and Mrs Roger D Milkman

Fa/mouth, Massachusetts

Ellyn

Chatham,

V Korzun

New

Dr William

Jersey

Kuhns

Toronto, Ontario

Ph

Andrew Mattox and Ms Sandee Parmelee

Woods

Wi//iamstown, Massachusetts

Mr and Mrs David Hibbitt

Bristol, Rhode Island

Hole, Massachusetts

Mr.

Mr and Mrs Michael Meehan

Israel

Raleigh, North Carolina

Woods

Bockman

Dr and Mrs Alexander Keynan

Cambridge, Massachusetts

Dr Llewellya

MD

Jerusalem,

Mr and Mrs A Sidney Knowles,

Hiatt

and

Dr and Mrs Robert T McCluskey

Brooklme, Massachusetts

Kelly

New Jersey

River Vale,

D.

Haverfbrd, Pennsylvania

Drs Darcy B. Kelley and Richard S

Sag Harbor, New York

Virginia

Elaine Pierson-Mastroianni,

Richard P Mellon

LaugMmtown, Pennsy/vanja

Mr Gernsh

New

York,

H. Milliken

New

York

R75
Ralph and Muriel Mitchell
Cambridge, Massachusetts

Tom and

Patty Pollard
Branrbrd, Connecticut

Drs Dorothy M Skinner and John S Cook

Falmoufh, Massachusetts

Dr and Mrs Merle Mizell

Drs Frank and

Drs

New

Washington,

Orleans, Louisiana

Dr Leyla deToledo-Morrell
Chicago,

Press

Mr and Mrs John S

Charlotte and Irving

Morris

Price

Rabb

Boston, Massachusetts

Cambridge, Massachusetts

Professor and Mrs Toshio Narahashi

Lionel

Chicago,

Mr and Mrs Frank

and Mildred Rebhun

Nickerson

Fa/mouth. Massachusetts

Mr and Mrs

Peter

New

Hampshire

Mrs Connnc Steel

Little

Neck,

New

York

Dr and Mrs Malcolm S Steinberg

Princeton,

New

Jersey

Mr and Mrs James

Stewart

Nanrucket. Massachusetts

Char/o tteswl/e, Virginia

///<nois

Melvm and Evelyn Spiegel

Hanover,

Bryn Mawr, Pennsylvania

Illinois

Ms Marcia

Billie

DC

Renaghan

Carol and Joseph T Stewart, Jr

New

North Fa/mouth, Massachusetts

Skillman.

Mr and Mrs Richard Rhoads

Mr Richard H Stowc

Boston, Massachusetts

New York, New

Dr Monica Riley
Fa/mouth, Massachusetts

Drs Alfred and Dorothy Suacher

Dr and Mrs Harris Ripps

Gerard and Mary Swope

Chicago,

Dr and Mrs Jack Rosenbluth

New

New

Rochelle.

Allan

and Clare Rosenfield

New

York

DC

John and Malory Swope

Concord, New Hampshire

Andrew G and

Ursula Szent-Gyorgyi

Wa'tham, Massachusetts

York

Mr and Mrs Edward

York

York

Washrngton,

Illinois

Hartsda/e,

New

Ros'yn,

Jersey

Rowland

Mr and Mrs Samuel Thome

Harm/ton, Massachusetts

Manchester, Massachusetts

Drs Joan and Gerald Ruderman

Mr and Mrs Tom Tierney

Wellesley, Massachusetts

Wellesley, Massachusetts

Mr Andrew Sabm

Mr D Thomas

Andrew Hawkins
East

Ronald P and Karen E O'Hanley

Ipswich, Massachusetts

Hampton, New

Dr and Mrs Edward

Chapel

Hill.

Trigg

York

Westwood, Massachusetts

D Salmon

Elaine

North Caro//na

New

and Walter

York.

New

Troll

York

David and Anastasia Palmer

Waquoif. Massachusetts

Dr.

and Mrs Hidemi Sato

Dr and Mrs George

Chicago,

D Pappas
Dr and Mrs John

Illinois

Mr and Mrs Edward

Tsoi

Arlington, Massachusetts

Toba M/e, Japan

Saunders, Jr

Drs Walter Vincent and Dore Butler

Waquo/f, Massachusetts

Woods

Mr Morton T Saunders

Dr and Mrs Byron H

Ho'c. Massachusetts

Drs Thoru and Judith Pederson

Worcester, Massachusetts

G/adwyne, Pennsylvania

New

York,

New

Waksman

York

Mr and Mrs Joseph Pellegnno

Boston, Massachusetts

Mrs Nancy Pendleton

Fa/mouth, Massachusetts

Dr Cecily Cannan Selby

Leonard and Eve Warren

New

Bato Cynwyd, Pennsylvania

York,

New

York

Dr and Mrs Douglas R Shanklm

Memphis, Tennessee

Mr and Mrs Henry Wheeler

Boston. Massachusetts

Bertha and Philip Person

Flushing.

New

York

Marilyn and David

Woods
Mr and Mrs Frederick H
Amherst,

New

Boston, Massachusetts

Ms

Rosalind

New

York,

C Wh.tehead

New

York

Pierce

Hampshire

Mr and Mrs Robert

Sheprow

Hole, Massachusetts

Mrs Jacqueline N Simpkins

Barnstab'e, Massachusetts

Mrs Annette

Williamson

Fort Worth, Texas

Pierce. Jr

Dr Matthew Wmkler
Austin, Texas

R76

ANNUAL FUND

2002

Gifts to the

Annual Fund are unrestricted and used immediately where they

MBL

the current activities of the

Corporate and Foundation Donors

best benefit

The Century Club

The Sponsors Club

$250 $499

$1 00- $249

Amengroup Foundation

Dr and Mrs Daniel

Mr Thomas H

Bay Foundation

Drs James and Helene Anderson

George Botelho, Inc

The Commonwealth Fund
Cox Foundation, Inc

Mr and Mrs Michael Angelini

Dr and Mrs George P. Baker, Jr
Dr Andrew Bass and Ms Margaret Marchaterre

ExxonMobil Foundation, Inc

Gilbane Building Company

Dr Martha B Baylor
Dr Miriam F Bennett
Mr and Mrs Thomas C Bolton

Aetna

will

research community.

Inc

The Gillette Company

Doreen Grace Fund

Alkon

A Abt
Dr Nina Stromgren Allen
Mr and Mrs David S Ament
Mr and Mrs Duncan P Aspmwall
Mr and Mrs Richard Asthalter
Mrs Kimball C Atwood,
Mr Nathaniel Atwood and Ms Susan Parkes
Mr and Mrs Donald R Aukamp
Dr. and Mrs David S Babm
Mr and Mrs John M Baitsell
Drs Barbara-Anne Battelle and James Alligood
Dr and Mrs Thomas L Benjamin
Mr and Mrs Maks Birnbach
Dr, and Mrs. Thomas P Bleck
III

Harken Foundation

Drs Harry Conner and Carol Scott-Conner

Mr John Cromie and Dr Gloria Gallo-Cromie

Johnson & Johnson

The Henry J Kaiser Family Foundation
The Gruss Lipper Foundation
McDonald's Corporation

Dr and Mrs. Paul J De Weer

Drs Lmda Deegan and Christopher
Prof and Mrs Frederick A Dodge
Mr and Mrs David Fausch

Aal

Dr and Mrs Donald

Neill

National Grid

Dr Rachel

Oracle Corporation
The David and Lucile Packard Foundation

Dr and Mrs Harold Gainer

Dr and Mrs David

Josephine Bay Paul and C Michael Paul

Foundation

Mrs Mary L Goldman

Dr Stephen L Hajduk
Dr and Mrs John P Harrington

George Botelho, Inc

Drs Barbara and John Boyer
Mr and Mrs Peter Boyer

Ralph F Peo Foundation, Inc

Saint-Gobain Corporation Foundation

Mr William A Raskins
Mr and Mrs Gary G Hayward

Mrs Eleanor

The Schooner Foundation

Esther Simon Charitable Trust

Mrs Elizabeth Heald

Dr Yukio Hiramoto
Mr and Mrs Lon Hocker

Fink

Dr and Mrs Daniel Johnston

The 7888 Club

$500 - $999

Dr and Mrs Morns John Karnovsky

Mr and Mrs Gilbert King
Dr and Mrs Edward

Drs Ted Begenisich and Sherrill Spires

Mr and Mrs Robert
Bigelow
Drs Colleen Cavanaugh and Philip Gschwend

Mr and Mrs Gorham L Cross

Drs Robert and Ellen DeGroof
Dr Philip Dunham and Ms Gudrun Bjarnarson
Mrs Ruth E Fye
Dr Joseph Gall and Ms Diane Dwyer
Mr and Mrs Charles M Ganson, Jr
Mr and Mrs William Gilbane, Jr

Mr and Mrs John G McMillian

Mr Andrew E Norman
Mr and Mrs Jonathan O'Herron
Dr Michael Rabinowitz and Ms Diane Hoxmeier
Dr Edward B. Rastetter
Dr and Mrs Frederick R, Rickles
Mr Michael C Ruettgers
Prof, and Mrs Howard K Schachman
Mr. and Mrs Hans L Schlesmger
Dr and Mrs William Trager
Mr and Mrs John J Valois
Dr Dyann an M- Peter Wirth

Drs Richard and

Kuzman

Hermme Makman

D Bronson

Mr and Mrs

Mrs Barbara Gates Burv/ell

Mr and Mrs William O Burwell

Mr
Mr
Mr
Mr
Mr

and
and
and
and

Mrs Bruce E Buxton

Mrs John

N.ckerson

Dr and Mrs Santo

Mr and Mrs William

Nicosia
J

Pechilis

Dr Khela Ransier
Rev Michael Robertson and
Dr Emmy Robertson

Mr and Mrs Phillip S Robertson

Mr Kenneth Rosenthal
Dr Cynthia V Stauffacher
Dr Dusan Stefoski
Dr Andreas C Stemmer
Drs Maurice and Raquel Sussman
Mr and Mrs Stephen E Taylor
Drs William and Mary Telfer
Mr and Mrs Richard Verney
Mrs. Lynn

H Wilke

Leslie J

Wilson

Robert

J.

Callahan, Jr

Carney

Father Joseph

Cassidy.

Ph

Caudill

Estill L

Dr Mary Clutter
Dr Carolyn Cohen
Dr and Mrs Maynard

Cohen

Mr and Mrs Alexander Colby

Mr and Mrs Franz Colloredo-Mansfeld
Dr.

LeBaron C.

Colt, Jr

Mr and Mrs Nathaniel S Coolidge

Dr and Mrs Sherwm J Cooperstem
Dr and Mrs John O Corliss
Dr Helen

Mr
Mr
Dr
Mr

Costello

and Mrs William G Coughlin

Charles Crane and Ms Wendy Breuer
Karen Crawford

and Mrs Richard D Cutler

Dr and Mrs Giuseppe D'Alessio
Dr and Mrs Nigel Daw
Dr Martha Bridge Denckla
Dr Wolf-Dietrich Dettbarn

A Dickson

Dr Charles Yanofsky

Dr William

Mr John F

Zietlow, Jr

Mr and Mrs David

Dr Michael

Zigmond
Mr and Mrs Hans Zimmer

Mrs David J Campbell

Mrs D Bret Carlson

Ms Martha Chen
Mrs Vera S Clark

Drs William and Madeline Burbanck

Ms Jane A McLaughlm

Dr Peter

A Buckingham
Co Inc

Darryl

Bufftree Building

Dr and Mrs

Dr DeForest Mellon, Jr
Drs Timothy Mitchison and Christine Field

Bodznick

Sarah Dixwell Brown

Dr and Mrs Julian B Marsh

Mr and Mrs

Hakhyun Ka

Kravitz

Attorney and Mrs William K Mackey

Ms Lynn Snyder Mackler

Mr and

Fertilized sea urchin egg,

Dr and Mrs Alan

Ms

Ms Dorothy

Donovan

Drummey

R77

Annual Fund Volunteers

Chairman
Dr Peter B Armstrong
Associates Chairman

Mr Michael Fenlon
Volunteers

Dr Robert B Barlow.
Dr

Mr Homer
Dr.

Jr

Verdi Farmanfarmaian

Lane. Jr

Hans Laufer

Dr Anthony Liuzzi
Dr Luigi Mastroianni. Jr
Dr Merle Mizell
Dr Thoru Pederson

Dr Robert

Prendergast

Dr Cecily C Selby
Dr Byron H Waksman

Annual Fund, continued

Dr and Mrs Thomas K Duncan

Mr and Mrs Hoyt Ecker

Dr Frank Egloff
Drs Paul Englund and Christine Schneyer
Dr and Mrs Herman T Epstein
Mr Gordon C Estabrooks
Dr and Mrs Thomas Evans
Dr Alan Ezekowitz and Ms Debbie Lunder
Dr

Patricia

Failla

Mr and Mrs Daniel D Federman

Mr and Mrs Howard G Freeman
Larry Jay Friedman
Dr and Mrs John J Funkhouser

Dr

Dr and Mrs Robert

Galatzer-Levy

Dr and Mrs Frank Gallagher

Miss Eleanor Garfield

Ms Margaret A

Geist

Mr and Mrs Vincent Geoffrey

Dr and Mrs Martin Gibbs

Mrs Janet

F.

Gillette

Drs Alfred and Joan Goldberg

Dr. and Mrs Herbert Graham

Dr and Mrs Philip Green

Mr Noah Greenberg
Dr Roger L Greif
Dr and Mrs Newton H Gresser
Dr June Harngan and Mr Arnold Lum
Dr Glenn
Harnngton

Dr and Mrs Neils Haugaard

Mr and Mrs Edmund Hazzard
Dr and Mrs Peter K Hepler
Dr and Mrs Theodore T Herskovits
Mr and Mrs William Hobart
Dr Edward G Horn
Dr and Mrs James C Houk
Prof Ruth Hubbard
Bruce Hunter
Dr and Mrs
Dr and Mrs Kurt J Isselbacher

Elizabeth Armstrong

Dr David Klein

Mr and Mrs

Mr Mark Koide

Mr.

Dr Kiyoshi Kusano
Drs Eugene and Mitsuko Laforet

Dr

Ms. Judith E Laster

Mr William Lawrence and Mrs Barbara Buchanan

Dr and Mrs John J Lee
Dr Marian E LeFevre
Dr David P Lenzi

Dr and Mrs John B Pearce

Dr and Mrs Aaron B Lerner

Mr and Mrs David Pearson

Dr and Mrs Jack Levin

Dr. and Mrs Richard B Levine

Dr and Mrs Courtland

Alice

C Leech

Lillie
Mr and Mrs David
Dr and Mrs Daniel M Lilly
Dr. and Mrs Richard W. Linck

Dr

Raymond

Dr Stephen

Lipicky

Dr.

Lipson
and Mrs. John E Lisman

Dr.

Robert B Loftfield

Dr and Mrs. Irving M London

Dr Louise M Luckenbill

Dr Richard Lum
Mr Gerald Lynch
Drs Richard and Sylvia Manalis
Mr and Mrs Ronald Marcks
Dr Andrew C Mannucci
Mr and Mrs Lowell V Martin
Dr and Mrs William M McDermott

Mr
Dr

Mr
Dr

Paul

McGonigle
and Mrs David McGrath
and Mrs James McSherry
Martin Mendelson
Melanie and Mr Klein Merriman

Dr

Dr Launnda A Jaffe
Mr and Mrs Ernest G Jaworski
Mrs Sally S Joslm
Drs Jane Kaltenbach and Robert Townsend

Dr Nancy S Milburn
Dr and Mrs Rtcardo Miledi
Drs David and Virginia Miller

Sally

Karush

Mr and Mrs H Ernst Keller

Mr Louis M Kerr
Mr and Mrs Arthur King

Mr
Dr

Mr
Ms Marina H Park
Dr and Mrs Rodenc B

Ms

Dr Marietta Radovic Issidorides

Mrs

Dr

Brian Nickerson

and Mrs Michael O'Rand

and Mrs Rudolf Oldenbourg
Janice and Mr Albert Olszowka
James G Ottos
and Mrs Ray D Owen
Malcolm A Park

and
Mr and
Dr. and
Dr. and

Mrs Robert
Morey. Jr
Mrs Samuel Murphy
Mrs X J Musacchia
Mrs John E Naugle
Messrs William and David Newton

Mr.

Dr Bruce

Park

Perkins

Peterson

Drs David and Jeanette Pleasure

Dr Jeanne S Pomdexter
Dr and Mrs Harvey B Pollard
Drs Mary Porter and Tom Hays
Dr Dale Purves and Ms Shannon Ravenel

Mr and Mrs George Putnam,

Ms Kathenne

III

E Putnam

Dr and Mrs James P Quigley

Dr and Mrs Robert F Rakowski
Dr Carol Reinisch and Mr Robert Suitor
Dr Judith Rhodes
Drs Paul Rmgel and Michele Lorand

Mr and Mrs John Ripple

Dr Hope Rmer
Mr Dana Rodin
Dr and Mrs Lawrence

Dr Joel

C Rome

Rosenbaum

Drs William Ross and

Nechama

Lasser-Ross

Dr Uldis Roze
Dr William Devme Russell-Hunter

Mrs Anne
Mrs Lilian

Sawyer

Scherp

Dr Herbert Schuel

Dr and Mrs Robert Seidler

Ms

Barbara Park Shapiro

J Sheridan

Mr and Mrs John

Drs Roy Silverstem and Jacquelyn

Joseph-Silverstein

Dr Maxine

F Singer

Continued

R78
Mr and Mrs

Alain Singer

Mrs Cynthia C Smith

Dr and Mrs. Paul F Smith
Drs Roxanna and Ronald

Mr David Space

L
;

>icwitz
'

ar

in/is

Dr and Mrs Alan

and Mrs
Mr and Mrs
Dr Elijah Ste~
Dr.

phenson

'.

"

Drs.

Alber

rd

Bihler

and Margaret Maurin

and Mrs James E

Mrs Barbara B Glade
Dr Joel S Gordon
Mr.

Gifford

Dr Martin Gorovsky
Mrs Enka A Green
Dr Nancy Carole Greep

Mr and Mrs Joseph Guttenplan

Mr and Mrs Peter A Hall
Mr and Mrs Robert Hall

Drs Clifford and Drusilla Harding

Dr, and Mrs Richard Bennet
Harvey

i.^utsuyuki

Mr and Mrs.

ewart

Ms Jasmin

Mr and Mrs Alvin Fossner

Dr Krystyna Frenkel
Drs Patricia Garrett and Oliver Woshmsky
Dr Stephen E Gellis

E.

Kent

Sugimori

Swift, Jr

Dr Margaret
Taft
Drs. Bruce Telzer and Leah T Haimo
Mr. and Mrs
Nicholas Thorndike

John F Towle, DOS

Mr and Mrs William Traver
Dr and Mrs David M. Travis
Dr and Mrs Steven N Treistman
Miss Ruth Tucker

and Mrs. Kenyon S Tweedell

Mr and Mrs Volker Ulbrich
Dr and Mrs. Ivan Valiela
Drs. Claude and Dorothy Villee
Mr and Mrs Samuel Vincent
Dr Talbot H Waterman
Dr Annemarie Weber
Dr and Mrs George Weiffenbach
Dr Gary Wessel
Mrs RoseT Wheeler
Dr and Mrs Martin Keister White
Dr and Mrs Roland L Wigley
Drs Jonathan and Beatrice Wittenberg
Drs Joshua Zimmerberg and Teresa Jones
Dr.

Other
|

Felix Inigo

Charles

and Mrs Alan K Karplus

Mark D Kirk
Dr Peter Kivy and Ms Joan Pearlman
Mr and Mrs Paul
Knaplund
Mrs Phyllis Kuffler
Mr and Mrs Ted Kulesza
Mr and Mrs Ray La Ranger
Mr and Mrs Steven V Launa
Mr.

Prof

Le Bovit

Briana

Dr Irene Loewenfeld

and Mrs Frank

Lord

Mr Fred

Lux

Mrs

Priscilla

Longo

M, Makay

Dr Robert P Malchow
Dr Dawn Morin Manck

Mr and Mrs Joseph S Maranchie

Dr and Mrs Joe L Martinez, Jr

Mr and Mrs Arnold H Burrough

Mr and Mrs E Brewster Buxton

and Mrs Frank J Mather,

Mathews
Mrs Dorothea J Mautner
Mrs Polly Miles
Dr and Mrs Daniel G Miller
Cdr and Mrs Lloyd C Morris U5N (RET)
Mr and Mrs Dana S. Morse
Mr Thomas A Mulholland
Mr,

and Mrs George Cadwalader

Mr Arthur D Calfee
Mr.

Dr Peter Clark and Ms Ellen Barol

Mr and Mrs James M deary
Mrs Elizabeth Ann Cohen

Mr and Mrs Peter Connolly

Mr and Mrs J Sterling Crandall
Mrs Marcia Donovan
Dr

Comer

Dr

III

Rita

(Ret)

Pantazis

Dr and Mrs Charles Parmenter

Dr Leonard M Passano
Ms Joan Pearlman and Dr Peter Kivy
Ms Joyce S Pendery
Dr and Mrs Murray E Pendleton
Dr and Mrs Ronald J Pfohl
Mr and Mrs Harold Pilskaln
Mr and Mrs Kenneth Poehls
Dr and Mrs Richard A Polin
Dr.

and Mrs Daniel A. Pollen

Mr and Mrs Abraham Pressman

Mr and Mrs Gerald B Reynolds
I

Ms Kathleen Lake Shaw

Mr Richard
Dr Jeffrey

Shrmer

Silberman

M Specht
Rev and Mrs William A
Mrs Eleanor Steinbach
Mrs. Louise

A Johnson

Dr and Mrs James E Johnson

Dr Harry S Kahn
Dr Edna S Kaneshiro

Ms Lena T

USN

Dr and Mrs Gaius R Shaver

Hill

and Mrs Robert B Hill

Mr and Mrs Gerald J Holtz
Dr Linda A Hufnagel-Zackroff
Dr.

and Mrs John H. Lochhead

Mr and Mrs Edward Loessel

Mrs Jennie P Brown

Mr and Mrs Thomas A Brown
Dr Roberto Bruzzone

Mrs Ruth Alice Fitz

Mr John H. Ford

Drs Richard and Susan

Dr.

Mulloy

Mr and Mrs Peter J Romano

Mr Dorothy C Ryder
Mr and Mrs Herbert Shanker

Mrs Jane M Heald

Dr Simone Helluy

Mr and Mrs
Mr and Mrs

Paul J

Ms Ins Nelson
Ms Kathryn Paine
Mr and Mrs Nicholas
Mr David Parker, Jr

Dr Robert V Rice
Dr Morris Rockstem

Dr Teru Hayashi
Dr David S. Hays

Dr.

Ms Jane Berger and Mr Roger Gittmes

Ms Pauline F Blanchard
Ms Casey Bliss

Mrs Helen M Erickson

Dr Aian Scott Fanning

E Hathaway

Dr and Mrs Stephen B Leighton

Mr and Mrs Edwin M Libbm

Dr and Mrs Richard H Backus

Mr and Mrs Joe Barnngton

Patricia L Dudley
Dr Quan-Yang Duh and Ms Ann
Mrs Frances E Eastman
Dr Hugh Young Elder

Ms Elizabeth
Mr John Hay

Ms Corinne

Mr and Mrs David C Ahearn

Mr and Mrs Scott M Allard
Mr and Mrs John J Aziz

Mr and Mrs Anthony

Dr Audrey E V Haschemeyer

RADM

Dr Raymond

Spurrier,

III

Stross

K Taylor
Dr Leana and Mr Joby Topper
Prof and Mrs Michael Tytell
Mrs Alice H van Buren
Mrs. Belle

Mr James Ware and Ms Sharon McCarthy

Mr Michael S Weinstein
Dr and Mrs Charles R Wyttenbach
Mrs Marilyn J Young
Mr and Mrs Kenneth H Zimble
Mrs Margery P Zinn
Dr and Mrs Steven J Zottoli

R79

ALUMNI RELATIONS
ADVISORY BOARD

ALUMNI FUND 2002

Dr.

Robert B Barlow, Jr. (Physiology. 1963)

SUNY Upstate Medical University

Dr.

Thomas

L.

Benjamin (Physiology. 1959/

Neurophysiology. 1966). Harvard Medical School

Oihs to the Alumni Fund are used

MBL

to

meet

the basic

needs of

Dr.

Richard

Born (Neural Systems & Behavior, 1987)

Harvard Medical School

T.

courses, including tuition support for students.

C. Cannon (Neurobiology, 1988)
of Texas Southwestern Medical Center

ephen
sity

Anonymous
Dr Joan Abbott
Prof and Mrs Laurence F Abbott
Dr William Agnew
Drs Dianne and

Thomas

Dr. Eloise E. Clark

(Physiology,

Ms Joan Edstrom
Ms Marcia Edwards
Dr.

Manlynn

Etzler

Dr and Mrs Arnold

Dr. Jeffrey T.

Eversole

Donald Faber
and Mrs Richard R Fay

Prof.

Jr

Dr.

Drs Robert and Lynne Angerer

Dr Carolina V Arancibia

Dr.

Marta Feldmesser

Dr

Cyril

Drs Carol Arnosti and Andreas Teste

Dr Thomas R Flanagan

Dr Michael 5 Ascher

Dr. Karl

Ms

Dr and Mrs Robert Barker. Jr

Dr Edward Joseph Behrman

Dr William H Bergstrom

V Fmnegan,

Dr.

Jr

Dr.

Marvin

Dr

Dr. Elizabeth

Ari Berkowitz

Dr Anne E Fry

D Brown

Dr Ka Hou Chu
Dr Carl Cyrus Clark

Dr.

Dr.

Washington

Dr and Mrs R John Collier

Dr and Mrs Marc D Coltrera
Dr Clark E. Corliss
Dr Jeffrey T Corwin

Mr John Cromie and Dr

Mamie

Daniel P. Kiehart (Physiology, 1975)

Dufce University Medical Center

George M, Langford

(Physiology, 1972),

Dartmouth College
Dr William M. McDermott (Invertebrate Zoology.

E Halpern

Dr Lisa M Halvorson
Dr Cadet Hammond Hand,
Dr. Susan M Harding

Dr.

Robert

Dr.

Norman B Hecht

1950), U.S.
Jr

Dr.

Navy

(retired)

Melanie Pratt Merriman (Embryology, 1975/

Physiology, 1976), Touchstone Consulting

Harvey
Dr.

Joseph Heitman
Drs. Joseph and Barbara Hichar
Dr Raymond
Holton
Dr and Mrs Seymour Holtzman

Thomas

Dr.

William T Clusin

Mr Timothy E Holy
Dr Xudong Huang

D. Pollard (Physiology, Director 19891

Dr Joshua

993), Yale University

R.

Sanes (Neurobiology, 1971)

Washington University Medical Center

Dr.

Wise Young (Neurobiology, 1972)

Drs Deborah Hursh and Mark Mortin

Rutgers University

Dr Jerard Hurwitz

Gloria Gallo-Cromie

Curry

Dr. Stephen C Dahl

Dr and Mrs Harry
Dickerson
Dr William J Dickinson

Joan Eiger Gottlieb

Dr Esther M Goudsmit
Dr and Mrs Eugene Grebner

Dr

Joiner Cartwnght. Jr, Ph

Dr Clarissa M. Cheney
Dr Jonathan Chernoff
Dr Chi-Bin Chien
Dr Carson C Chow

Dr Bruce

Glazier

Calie

T.

Institution of

Alexander Keynan (Physiology, 1961)

Israel Academy of Sciences and Humanities

Dr Lewis J. Greene
Dr Warren M Grill
Dr Jerome Gross

Harley P Brown
Dr Richard R Burgess

Dr Alice

James A

Halpern (Neurobiology. 1986)

Dr.

Dr.

Dr.

Dr.

Prof

E.

Carnegie

Dr Joel S Gordon

Dr Emil Borysko
Dr Peter Brodfuehrer

Mr and Mrs Patrick

Dr David Campbell
Dr James R Campi

Marnie

Theresa Gaasterland

Dr and Mrs Donald

966)

Leah T. Haimo (Physiology, 1974),

University of California, Riverside

Dr Paul E. Gallant
Dr. Helen
Gjessing

Elayne Bornslaeger-Bednar and

Dr.

Fritzler

Dr.

Dr.

Mr Michael Bednar

Fowler (Physiology,

Millennium Pharmaceutica/s

Dr Emmanuel C Besa

Dr.

Behavior,

Dr,

Dr Mark Boothby
Dr Richard T Born

&

1984), State University of New York

Elizabeth Fowler

Gerald Bergtrom

Blumenthal

Fetcho (Neural Systems

R.

Joseph

Foreman
and Dr James Parmentier
Dr Kenneth Freedman
Drs Hugo and Anita Freudenthal
Dr.

T. Coyle (Neurobiology, 2001)

Harvard Medical School

Joseph

Flessa

Christine

Dr.

Corwin (Neurobiology, 1975)

University of Virginia, School of Medicine

Allen

Dr C Ronald Anderson
Dr William DeWitt Andrus,

Mr Edward

1956)

Bow/ing Green State University

Diner

Jonathan S and Dr Thelma Dixon

Mr and Mrs Richard Drucker
Dr Catherine Asleson Dundon
Dr Kathleen Dunlap
Mr.

Dr and Mrs David Durica

Dr Richard Intres
Dr Allen Isaacson
Dr and Mrs Stephen K

Ex-Officio

Itaya

Dr Peter

B. Armstrong (Invertebrate Zoology, 1961)

Chairman, MBL Annua!/Alumni Fund

Jon
Jacklet
Dr Nancy A Johnson
Dr.

Dr

Leslie

and Mr James

University of California, Davis

Jolly

Dr.

John

E.

Dowling (Neurobiology, 1970)

MBL Corporation
Harvard University

President.

Continued

R80

Students get
dockside instruction

from Marine

Resources

Center

staff,

Elizabeth Armstrong

and Mrs James

Kalat
M Kane
Dr Alvm M. Kaye
Or Gordon
Kaye
Drs Thomas and Laura Keller
Dr and Mrs R Emmett Kenney
Dr.

Or Mananna

Dr Dana T Nojima
Mrs Phyllis Norris

Dr Joel P Stafstrom

Drs Victor and Ruth Nussenzweig

Dr Richard

Kessel

Dr Lynda A Kiefer
Dr Elizabeth L Kimberly

Dr Donald

Dr Leonard

King

B- Kirschner

Dr and Mrs Paul

Knopf

Dr Robert E Knowlton

A Koenigsberger
Dr WieslawJ Kozek
Dr Ajit Kumar
Dr Carol

Dr Michael

Dr
Dr
Dr
Dr
Dr

Holly

Landzberg

LeMosy

Ethan Lerner

Drs Brian Link and Vivian Lee

Ms Anne M

Linton

Drs Joan and James Lisak

Mr and Mrs Edward K Lobenhofer

Dr and Mrs Robert J Loeffler
Dr Ann M Lohof
Dr Jane Lubchenco
Dr Stephen E Malawista
Dr David E Mann, Jr
Dr and Mrs. Phillip 6 Maples
Dr Junko Munakata Marr
Dr Michael Massanan
Dr. Jon M McCormick
Dr Duane

P.

McPherson

Dr Anne Messer
Dr Laurie Miller
Dr Maria

Dr Catherine L Olsen
Dr Ingnth Deyrup Olsen
Prof. John M. Olson
Dr and Mrs Brett

Ms

C Sweet

Dr.

Hyla

Swartz

Ben G Szaro

Ms Penny A Tavormma
Mrs Vera A Taylor

A Oxberry

Mrs Mary G. Pacifici

Drs Kimberly Paul and Charles Thomas

Mr Matthew Person and Ms

Dr Norman J Pieniazek

Dr Frank

Jill

Dr Saul Teichberg
Dr Wesley J Thompson
Dr Barbara Holland Toomey

Enckson
Dr Jeanine

Ursitti

Dr Louis Pierro

Dr Carl B PNcher
Dr and Mrs Anthony Pires
Dr Sabrina and Mr Bradford Powell

Dr Raghunath Virkar

Ms

Melissa

Vollrath

Dr Susan Volman

Lavoie

Matthew K Lee
William J Lehman
Ellen K.

Dr Michael D. Oberdorfer

Dr Gary R Strichartz
Dr David T Sullivan

Morasso

Dr Yasuhiro Morita

Mr Stephen h Munroe
Dr David P Nagle, Jr
Drs Angus Nairn and Marina Picciotto
Dr Lee Niswander

Dr Esther

Dr Jennifer

Racoosm
L

Raymond

Dr Jean and Mr Ronald Regal

Dr and Mrs Robert Alan Resnik
Dr

Kristin Lester Revill

Dr.

Randall

Reyer

Ms

Marian

Rice

Drs Duncan and Grace Saunders Rollason

Ms

Carol

Dr Deborah Williams Ward

Ward and Zail Berry

Mr and Mrs Herman Ward
Dr and Mrs Samuel Ward
Drs Gary E

MS

Ann Ryder

Ms Kay E Wellik
Dr Harold Bancroft White
Dr Clayton Wiley
Dr Ulrike Wille
Dr Kathenne Wilson
Dr Andrea G Witlin
Dr Anne

Dr.

David R Samols

Dr Noriyuki Satoh
Dr Richard L Saunders
Dr Charles H Sawyer
Dr RolfSchauder
Dr.

G Wadsworth
Wang

Dr Susanna H Weerth

Dr Austen F Riggs
Dr Regma M Robbins
Brian K Romias, MD,
Dr James T Russell

Dr William
Dr Brant G

Paul R. Schloerb

M Wood

Drs Lawrence Wysocki and Judith Spiegel

Dr Ayako Yamaguchi
Dr Naoyuki Yamamoto

Ms
Dr.

Judith L Yanowitz

Karen P York

Dr James H Schwartz
Dr Carla Shatz

Dr Lin Yue

Dr and Mrs Alan Sher

Dr and Mrs

Dr David R Sherwood

Dr Richard E Zigmond

Dr Joyce M Simpson
Dr Max Snodderly

Dr Sandra C Souza
Mr Nelson Spruston

Cheng Zhu

R81

FELLOWSHIPS

AND SCHOLARSHIPS

Endowed and expendable funds for scholarships and fellowships are an integral part of the MBL's successful research
and education programs. In 2002, twenty-two scientists from the U.S. and abroad were awarded fellowship grants that
allowed them to work in our uniquely collaborative research environment. The Laboratory also awarded scholarships to
97 highly qualified students, enabling them to participate in our total-immersion courses.

We

gratefully

scholarships

acknowledge the donors

in

listed below,

who provided $245,270

for research fellowships

and $726,558

2002.

American Society of Cell Biology

Summer Research Awards
American Society of

Robert Day Allen Fellowship Fund

Drs. Joseph and Jean Sanger

MBL

Associates

Endowed

Technic, Inc

Scholarship Fund

MBL Associates
Mrs.

Just Endowed Research Fellowship Fund

The Cole Memorial Family Fund
William Townsend Porter Foundation

E. E-

Nawne Meigs-Brown

Bang Fellowship Fund

Dr and Mrs Jack Levin

Frederik B.

Georgia-Pacific Corporation

Endowed Library Readership

Dr and Mrs Laszlo Lorand

Fred Karush

Dr and Mrs Arthur

I

Charles

R.

John O. Crane Fellowship Fund

Mrs Eleanor Steinbach

Dr

Drs Joseph and Jean Sanger

Bernard Davis Fellowship Fund

Daniel

Ms

S.

F Hartline

Dr and Mrs Thomas R Hedges,

Ms Rebecca Kucera

Jean and Katsuma Dan Fellowship Fund

Mr William A Haskms

Mrs Elizabeth

and Edith

T.

Grosch Scholarship Fund

Weidner

R. Lillie

Fellowship and Scholarship

Ann Cramer

Estate of Emily

Jr

Kuffler Fellowship Fund

Dr and Mrs Edward A Kravitz

Frank
|

Davis

Laura Grosch and Mr Herb Jackson

Earl

Mountain Memorial Fund

Dr and Mrs Dean C Allard.
Ms Brenda J Bodian

Jr

Mr and Mrs Amos L Roberts

Mr and Mrs Thomas H Roberts

Mouse

Fund

Peter Hartline

Mr and Mrs Frederick

Friendship Fund

Silverstein

Keffer Hartline Fellowship

Dr.

Crane Fellowship Fund

Friendship Fund

Fund

Dr and Mrs Benjamin Kaminer

Dr and Mrs Lewis P Rowland

Cell Biology

Aline D. Gross Scholarship

Dr and Mrs Paul R Gross

oocyte, Umit Ali Kayisli

Continued

for

R82

James A and

Faith Miller Fellowship

Fund

The Catherine Filene Shouse SES Scholarship Fund

The Catherine Filene Shouse Foundation

Drs David and Virginia Miller

Frank Morrell Endowed Memorial Scholarship

Dr Leyla de Toledo Morrell

(The Catherine Filene Shouse Scholarship Fund

The Catherine Filene Shouse Foundation

Emily Hartshorne Mudd Scholarship Fund

World Academy of Art and Science

Neural Systems and Behavior Scholarship Fund

Dr and Mrs Alan Gelpenn
Dr Warren M Gnll
Drs William Knstan and Kathleen French
Dr and Mrs Richard B Levine
Dr Mark
Miller
Drs. Jams C Weeks and William M Roberts
Drs Harold Zakon and M Lynne McAnetly
Ms M Jade Zee

The Catherine
The Catherine

Filene Shouse Fellowship

Shouse Foundation

The Gruss Upper Fund

The Gruss Upper Foundation

The

and Phoebe Speck Fund

Bill

Dr William T Speck

The Evelyn and Melvm Spiegel Fellowship Fund

Dr and Mrs Jack Levin
The Sprague Foundation
Drs Joseph and Jean Sanger

Nikon Fellowship
Nikon Instruments

Inc

The Plum Foundation John

Fellowship Fund

E.

'

Horace

C Rose and

Stunkard Scholarship Fund

Drs Albert Stunkard and Margaret

William Townsend Porter Scholarship Fund

for Minority Students
William Townsend Porter Foundation

Florence

Fund

Stembach Fellowship
Mrs Eleanor Stembach

H. B.

Dowlmg

The Plum Foundation

S.

Meryl Rose

Ms Kaaren Janssen

Maunn

Eva Szent-Gyorgyi Scholarship Fund

Dr and Mrs Benjamin Kammer
Dr and Mrs Laszlo Lorand
Drs Joseph and Jean Sanger
Dr Andrew and Ms Ursula Szent-Gyorgyi

Endowed

Scholarship Fund
Dr Gwynn Akin Bowers
Dr and Mrs D Eugene Copeland
Mrs Edna B Hill

Dr and Mrs Benjamin Kammer

K Kellogg Foundation

Universal Imaging Fellowship

Universal Imaging Corporation

Walter

L.

Dr Paul

Wilson

Endowed

Fund

Scholarship

N Chervm

Dr Jean R Wilson

Dr Hans Laufer
Dr and Mrs Anthony Liuzzi
Dr and Mrs Roger D Milkman

Mrs Eleanor M, Nace

Drs Keen A and Nancy S. Rafferty
Mrs Florence C Rose
Dr and Mrs John
Saunders. Jr
Dr and Mrs J P Trrnkaus

Mouse cell tnvas'cn pores

and membrane, mouse
b'astosist,

Umit

Ali Kayislt

Young Scholars/Fellows Program

Drs James and Helene Anderson
Drs Harry Conner and Carol Scott-Conner

Or and Mrs James

German

Dr and Mrs Anthony LIUZZI

Drs Luigi and Elaine Mastroianm
Drs David and Miriam Mauzerall
Drs Matthew and Jeanne Meselson

Milton L Shifman Endowed Scholarship

Milton L Shifman Scholarship Trust

Fund

Filene

Drs Dorothy Skinner and John Cook

Dr and Mrs Edward A Spiegel

Dr Annemarie

Weber

Drs Jonathan and Beatrice Wittenberg

Mr and Mrs Kenneth H Zimble

R83

MEMORIAL AND TRIBUTE GIFTS

The following donors have chosen

Laboratory as a special

way

to

to

support the Marine Biological

a relative or friend.

remember or honor

In
|

Memory

of

John Helfnch

Colleen Cavanaugh and

Drs Bruce and Teresa Corliss
Drs.

Philip

Gschwend

Harken Foundation

Ms Ken Holland
Dr Max Holmes
Drs Knute Nadelhoffer and Barbara Billings

Ms Andrea Ricca
Dr and Mrs Gaius R Shaver
Mr

Jeffrey Shelkey

Ms Jane
Dr.

and Ms Joanne Willey

Tucker

and Mrs Lawrence

Wangh

Dr Xiangming Xiao
In
|

Memory

of Mr. Robert Huettner

Mrs Carmela J Huertner

Mr and Mrs Richard A Huettner

Ms
In
|

Memory

Dr
In
|

Catherine

N Norton

of Dr. Arthur G.

Patricia L

Humes

Dudley

Honor of Macy Lawrence

Dr and Mrs Gerald Weissmann

In
|

of Dr. Frank Morrell

Memory

Dr Thomas

Honor of Drs. Clay and Clara Armstrong

Ms Lynn Snyder Mackler

|ln

Bequests
Estate of Emily

Madelene E

Pierce

Memory of Dr. Kimball C. Atwood,

Mrs Eleanor Steinbach

In

Bleck

Honor of Cooper Neely

Mr and Mrs Joe Barnngton

In
|

Ann Cramer
|

Estate of

P.

III

In
|

of Dr. Lionel

Memory

Drs Robert and

In
|

of Dr. Frank A. Brown,

Memory

Mrs Jennie

P.

In

Rebhun

Jr.

In

Brown

of Elizabeth Russell (Tibby)

Memory

Dr and Mrs Ray

|

I.

Anne Goldman

D Owen

of Dr. Francis D. Carlson

Memory

Mrs Carolyn

S.

In

Carlson

Honor

of

Marjone Salmon

Drs David Forkosh and Linda Hirshman

In
|

Honor of

Mr. Richard D. Cutler

Gilbane Building

In

Company

Honor of

Ms

Memory of Eugene Floyd DuBois

Mrs James R Glazebrook

Mr. Arthur D. Traub

Natalie Miller

|ln

In
|

Memory

of Dr.

George E Wheeler

Dr Khela Ransier
|

Mrs Rose T Wheeler

Memory of Dr. and Mrs. James D. Ebert

Dr and Mrs Charles R Wyttenbach

In

In
|

In

Memory

of Morris

Dr and Mrs Harris Ripps

|

In
|

Memory

of Elizabeth K. Hartline

Dr and Mrs Thomas R Hedges,

Memory

Mrs Clare

Goldman

Jr

In

Honor

of Dr Charles G. Wilber

Wilber

of Alfred

and Joyce Zeien

Mr Michael C Ruettgers

R84

OTHER GIFTS AND PROGRAMS

The quality and success of the MBL educational program is maintained
and computers by:
through the loan of research equipment, reagents,

Equipment Lenders

Accelrys Inc

Adams &

Matching
Inc

Agilent Technologies.

Amersham Pharmacia
A.M P.I

Biotech Inc

GlaxoSmithKlme

Discoveries/ Robbins

Solutions

Grass Instrument Company/

Astro-Med, Inc GrassTelefactor

PCS Assembly
Peak Performance Technologies. Inc
Perkm Elmer Applied Biosystems

Perkm Elmer Life Sciences

Photon Technology International
Photonic Instruments

Computing Inc
Promega Corporation

Platform

Scientific

Hamamatsu Photonic Systems

Inc

Harvard Apparatus, Inc

Quantitative Imaging Corporation

Aquatic Habitats

BP Foundation, Inc
The Commonwealth Fund
ExxonMobil Foundation, Inc
Johnson & Johnson

The Henry

Genomic

Division

Applied Precision, Inc

Aquatic Eco-Systems.

Aetna

General Valve Corporation

Genetronics, Inc / BTX Instrument

Inc.

Apogent

Gifts

Associates

List

Elizabeth Armstrong

Arcturus Engineering, Inc

Atto Bioscience

Improvision Inc

AutoQua,nt

Instrutech Corporation
Inc
Intelligent Imaging Innovations,

Research Precision Instruments,

Roche Applied Science

Axon

Invitrogen

Roper

Jackson ImmunoResearch

San Diego Instruments

Imaging, Inc
Instruments, Inc

Scientific

Kaiser Family

Foundation

K Kellogg Foundation
McDonald's Corporation

Barnstead/Thermolyne

Beckman Coulter

Instruments. Inc
IS

National Grid

Becton Dickinson
Biocare Medical

Oracle Corporation
The David & Lucile Packard

BD

Foundation
Saint-Gobain Corporation
Foundation

Biosciences

Biophotonics international

Bioptechs
Bio-Rad Confocat

Santa Cruz Biotechnology

Laboratories, Inc

Scanalytics

Scion Corporation
SD Instruments

Kepco Power Co
Kinetic Systems, Inc

Kipp

&

Zonen/Division of SCI-TEC

Sigma
Stnauer Associates, Inc

Instalments, Inc

Soma

Scientific Instruments

Bio-Rad Laboratories

Lab Line

SONY

Bnnkmann

Laser Science

Stoelting

Brownlee Precision Co

Leica Confocal

Sutler Instrument

Burleigh Instruments, Inc

Leica Microsystems Inc

Ludl Electronic Products, Ltd

Synaptosoft, Inc

Cairn Research LTD

Ludlum Measurements,

Technical Manufacturing

Instruments

Cambridge Electronic Design

Cambridge Research
Instrumentation, Inc

CBS

Scientific Company, Inc

Chroma Technology Corporation

Inc

COMPAC

Computer Corporation

Inc

Imaging Systems

Crest Technologies

CyBio, Inc

The Company
Inc

Microway

MJ Research

ThermolEC
ThermoNeslab
ThermoSavant
ThermoShandon

Molecular Dynamics
Molecular Probes

ThermoSpectronics

MWG

Universal Imaging Corporation

Biotech

Nanodrop Technologies

Vashaw

Nanshige USA,

Vibratome

David Kopf Instruments

Del Imaging Systems, LLC

National Instruments

Diagnostic Instruments, Inc

Nikon. Inc

Inc

Inc

Olympus Confocal
Olympus Corporation
Optical, Inc

Eastman Kodak

Omega

Edinburgh Biocomputing Systems

Ophthalmic Instrument Co
Optiquip

Eppendorf

Scientific Inc.

Optronics

FHC
Fine Science Tools
Fisher Scientific/Zylux Corp.

Fluke Corporation

Scientific

Vincent Associates

Nikon Confocal

Star, Inc

DVC Co

of Biologists, Ltd

ThermoElectron

Dagan Corporation
Dako Corporation

DNA

Company

Corporation

MatTek Corporation
McCrone Microscopes
Micro Video Instruments,
Miltenyi Biotec, Inc

Coherent

Compix

Inc

Medical Systems

VWR
Warner Instrument Corporation
World Precision Instalments
Carl Zeiss, Inc
Carl Zeiss Confocal
Carl Zeiss Imaging

Co

R85

&

governance

administration

BOARD OF TRUSTEES
Chairman of the Board of Trustees
Sheldon

Segal,

The Population Council

William T Speck, Director and Chief Executive Officer

Robert E Palazzo, Chair of the Science Council

Vice Chair of the Board of Trustees

George

Ex Officio Trustees

Logan. Pine Street Partners

Mary B Conrad, Fiduciary

Trust International

President of the Corporation

John E Dowlmg, Harvard University
Director

and Chief Executive

Executive Committee
of the Board of Trustees

Officer

William T Speck, Marine Biological

Laboratory*

Margaret C Bowles
Mary B Conrad'

Treasurer of the Corporation

Mary B Conrad. Fiduciary Trust International"

George

Logan

Marcia Morris

Clerk of the Corporation,

Thomas

S Crane. Mintz. Levin.

Ronald P O'Hanley

Cohn.

Ferris,

Glovsky

Robert E Palazzo'
Sheldon J Segal

&

Popeo, PC*

William T Speck-

Chair of the Science Council

Chnstopher

Weld

Robert E Palazzo, Rensselaer Polytechnic Institute'

Class of 2003

Darcy Brisbane Kelley. Columbia University

Laurie J Landeau, Marmetics, Inc
Burton J Lee, III. Vero Beach. FL
Ronald P O'Hanley, Mellon Institutional Asset

William T Golden. New York. NY

Robert E Mainer, Wayland, MA

Mgt

Jean Pierce, Boca Grande. FL

Vincent J Ryan. Schooner Capital LLC

\Classof2004
M Howard Jacobson,

George

William

Langford, Dartmouth College

G William Miller & Co Inc
.

Frank Press, The Washington

Advisory Group
Christopher M Weld, Sullivan and Worcester. Boston
Torsten N Wiesel. The Rockefeller
University

Claire

George

Anderson. Key Largo. FL

Fraser,

The

Institute for

Logan. Salem,

Genomic Research

VA

Robert Prendergast, Falmouth, MA

Rowe, Aetna U S Healthcare

John

Donald Eugene Copeland, Woods Hole,

Gerald Weissmann.

New

York University School of Medicine

Haven.

CT

MA

Sears Crowell, Jr. Indiana University.

Bloomington, IN
Teru Hayashi. Woods Hole. MA

C Ladd

MA

John

Prosser, University of

Illinois.

Urbana,

Saunders. Waquoit.
David Sheprow. Boston, MA

D Thomas Tngg

Wellesley,

Woods

MA

MA

Hole.

MA

iDirectors Emeriti
Paul Gross. Falmouth,

Harlyn

IL

Russell-Hunter. Syracuse University. Syracuse.

Walter S Vincent.

Class of 2006
Margaret C Bowles, Woods Hole, MA
Martha
Cox, Hobe Sound, FL
Walter E Massey, Morehouse College
Marcia C Morris, Boston, MA

New

Seymour S Cohen, Woods Hole, MA

Arthur L Colwm. Key Biscayne. FL
Laura Hunter Colwm, Key
Biscayne. FL

Ruth Hubbard, Cambridge.

Class of 2005
Porter

Trustees Emeriti
Edward A Adelberg, Yale University.
John B Buck, Sykesville, MD

Bankers Trust

Miller,

Honorary Trustees

Halvorson.

'Ex officio

As of April I 2002

MA

Woods

Hole.

MA

NY

R86

STANDING
COMMITTEES

STRATEGIC PLANNING COMMITTEES

Development Committee
Christopher Weld, Chair
Porter

Anderson

Peter Armstrong

Robert Barlow
Mardi Bowles

Martha Cox

John Dowling
Claire Fraser

M Howard Jacobson
Burton Lee

William Miller

Jean Pierce
Robert Prendergast

John

Rowe

Torsten Wiesel

&

Facilities

Capital

Equipment Committee
Mardi Bowles, Chair
Porter

Anderson

George Langford
Walter Massey
Andrew McArthur
Frank Press

Christopher

Weld

Nominating Committee
John W. Rowe, Chair
Martha Cox
'John E Dowling, President of
the Corporation
Claire Fraser

'Robert Palazzo, Science Council

Chair

Jean Pierce
Vincent J Ryan
William T Speck, Director

& CEO

Gerry Weissmann

Finance Committee
Ronald P O'Hanley, Chair
Mary B Conrad

Thomas

Crane

Maynard Goldman
M Howard Jacobson
Darcy Kelley
Laurie

Landeau

George

Logan

Robert Prendergast

Investment Committee
Mary B Conrad. Chair
Thomas S Crane

George

Logan

William Miller

Marcia Morris

Ronald O'Hanley
Vincent J Ryan

Steering

Committee

R87

CORPORATION
I

Life

Members

A Adelberg, New Haven, CT

Dr Bjorn Afzelius. Stockholm University
Dr Ernest Amatniek, (address unknown)
Dr Edward

Dr John

Arnold, Falmouth,

Dr Herbert Levitan, National Science Foundation

Dr John H Lochhead, London, England
Dr Birgit Rose Loewenstein, Falmouth, MA
Dr Frank A Loewus, Washington State University
Dr Robert B Loftfield, University of New Mexico
Dr Laszlo Lorand, Northwestern University

MA

Mrs Betsy G Bang, Woods Hole, MA

Dr Alan
Bernheimer, New York University
Medical Center

Dr Lloyd M Bertholf, Bloomington,

Dr Herman F Bosch, Falmouth, MA

Medical School

IL

Dr Robert E Mainer, The Boston Company, Inc

Dr Julian B Marsh, Chestnut Hill, MA

Institutes of Health

Dr F J- Brmley, Jr National
Dr John B Buck, Sykesville,
,

MD
GA
GA

Dr Madeline P Burbanck, Atlanta,

Dr William

Burbanck, Atlanta,

Mr Lowell V

Martin,

Woods

MA
MA

Hole,

Dr

Rita

Dr,

Jams Metuzals, Ontario, Canada

John A Moore, University of California

Dr.

Mathews, Southfield,

(deceased 2002)

Dr Alfred B Chaet, Maitland, FL

Dr Arnold

Mr James

M
M

Clark,

Woods

Dr.

MA

Hole,

Dr Marjone McCann Collier, Effie, LA

Dr Arthur L Colwin, Key Biscayne, FL
Dr Laura Hunter Colwin, Key Biscayne, FL
Dr Sherwin

Moore, Duke University Medical

Dr Aron

A Moscona, New

Dr X

Musacchia, Bella Vista,

Dr

Maimon

Nasatir, Ojai,

Dr Leonard
Dr.

NY
AR

York,

CA

Passano, University of Wisconsin

Potts, University of Lancaster

William T

A Price, Falmouth, MA
Dr Margaret McDonald Prytz (address unknown)
Dr Carl

Cooperstein, University of

John

Center

Clark, Palm Beach, FL

Dr Maynard M Cohen, Rush Medical College
Dr Seymour S Cohen, Woods Hole, MA
Dr Jack R Collier, Effie, LA

Connecticut

Dr

D Eugene

Dr John

Copeland,

Corliss, Bala

Woods

Hole,

Dr Helen M. Costello, Chapel Hill,

Dr Helen Crouse, Hayesville, NC

Dr Nigel
Dr Robert

Daw, Branford,

Dr Charles E Renn (address unknown)

Dr George T Reynolds, Princeton University
Dr Robert V Rice, Falmouth, MA
Dr Morris Rockstem, Coral Gables, FL

NC

Dr Raphael R Ronkin, Washington, DC

Dr John D Roslansky, Woods Hole, MA

CT
University School

DeHaan, Emory

MA

Cynwyd, PA

of Medicine

Dr Patncia L Dudley,

Seattle,

(deceased 2003)

Dr

WA

F Roslansky, Associates of

Priscilla

Cape Cod,

Inc.

Dr Jay S Roth,

Dr Charles Edwards, Longboat Key, FL

Dr Gerald F Elliott, The Open University

Dr. Allan

Patricia M Failla, Johns Island, SC

Dr Donald T Frazier, University of Kentucky
Medical Center

Dr

Dr Arthur

L Gabriel,

Dr Howard

Woods

Hole,

Dr. Francis

Hamilton, University of Virginia

C G

Hoskin, Canton,

F.

Sjodin, Baltimore,

Smith,

Woods

Hole,

MD

MA

Speer, Portsmouth, Rl

Dr Melvm Spiegel, Dartmouth College

Dr Graver C Stephens, University of California

Dr. Clifford

Dr Teru Hayashi, Woods Hole, MA

Dr Frederick L Hisaw, McMinnville,

Dr Nicholas Sperelakis, University of Cincinnati

Dr Evelyn Spiegel, Dartmouth College

MA

V Harding, Jr, Falmouth, MA

Dr Audrey E V Haschemeyer, Woods Hole,

Paul

Mr John

York State

Psychiatric Institute

Dr Herbert Graham,

MA

Colby College
Silverstein, Johns Hopkins

Scott,

Dr Raymond

Brooklyn College

New

MA

University

Dr.

Dr Murray Glusman,

Hole,

Dr Hidemi Sato, Nagoya University

Dr R Walter Schlesmger, North Falmouth,

Research Unit

Dr Mordecai

Woods

MA

OR

MA

Mrs Jane Lazarow Stetten, Chevy Chase, MD

Dr Bernard L Strehler, Laguna Niguel, CA
Dr Maurice Sussman, Falmouth, MA
Dr Raquel B Sussman, Marine Biological
Laboratory
Gwen P Szent-Gyorgyi,

Mrs

Woods

Hole,

MA

Prof Ruth Hubbard, Harvard University

Bruce Hunter, Peterborough, NH

Dr Charles Hurwitz, Stratton VA Medical Center
Dr

Dr George Katz, Sarasota, FL

Dr John M Kingsbury, Cornell University
Dr Kiyoshi Kusano, National Institutes of Health

Mr

Nicholas Thorndike, Wellington

Management Company
Dr William Trager, The Rockefeller University
Dr J P Tnnkaus, Yale University (deceased 2003)

Dr Claude A Villee, Jr Harvard Medical School

Dr Walter S Vincent, Woods Hole, MA
,

Mr

Ezra Laderman, Yale University

Dr Paul H LaMarche, Eastern Maine Medical

Center
Dr

Max A

Lauffer,

Medical Center

Penn State University

Dr Talbot H Waterman, Yale University

Dr Roland L Wigley, Woods Hole, MA
Dr Lon

Wilkens, University of Missouri

NYU Medical Center

Dr Paul Witkovsky,

RSS

Members

Dr Donald A Abt, Univer;ity of Pennsylvania

School of Veterinary Medicine

A Adams.

Dr James

Tallai

assee, FL

Dr William J A. .-?i'iian. Jr. Falmouth, MA

Dr Daniel L Alkon, Rockefeller Neuroscience
Institute

Garland E Allen, Washington University

Dr Nina Stromgren Allen, North Carolina

Dr Robert

Alhegro, Louisiana State University

Medical Center

Dr Everett Anderson, Harvard Medical School

Dr John M Anderson, Ithaca, NY

Dr Porter
Anderson, Jr, Key Largo, FL
Dr Christine Armett-Kibel, University of
Massachusetts, Boston

Prof Clay

Technology
Dr Stephen C Brown, SUNY at Albany
Mr William L Brown, Weston, MA
Dr.

State University

of

Carole L Browne,
School of Medicine

Dr.

Dr Mark

Dr Barbara C Boyer, Union College

Dr. Bruce P Brandhorst, Simon Fraser University
Dr Marianne Bronner-Fraser, California Institute

MA

Dr Peter B Armstrong, University of California

Mr Robert
Ashton, Bay Foundation
Dr Jelle Atema, Boston University Marine

Bucklin, University of

Dr Robert G Baker,
Medical Center

New York

University

Dr David Baltimore, California Institute

of Technology
Robert B Barlow, Jr,

SUNY Upstate
Medical University
Dr Daniel T Barry, South Hadley, MA
Dr. Susan R Barry, Mount Holyoke College
Dr.

Dr Andrew H

Bass, Cornell University

Dr. Barbara-Anne Battelle, University of Florida

Dr.

Frederick Bay, Bay Foundation

Elaine L Bearer, Brown University

Dr.

John M.

Beatty, University of

Minnesota

Alberto Beauge, Institute de

Investigacion Medica, Argentina

Dr. Luis

Dr.

Ted Begenisich, University of Rochester

David A Begg, University of Alberta, Canada

Dr.

Eugene

Dr.

Bell,

TEI Biosciences Inc

Thomas

L Benjamin, Harvard Medical School

Michael V L Bennett, Albert Einstein College
of Medicine

Dr.
Dr.

Dr Miriam F Bennett, Colby College

Dr.

R Suzanne Bennett, Albert Einstein College

of Medicine
Dr.

New

Mr Norman

Berlin, York,

Bernstein,

ME

Columbia Realty Venture

Health Science Center

Dr Francisco Bezanilla,
Dr. John D Biggers, Harvard Medical School

Dr.

Dr David

Dr. Francis

P.

Laboratory

Sherman Oaks, CA
Borgese, Lehman College, CUNY

Borst, Jr, Illinois State University

Bowles. Marine Biological

Marine Biological

Cutler,

Laboratory

Laboratory
Dr Paul J De Weer, University of Pennsylvania
Dr Linda A Deegan, Marine Biological

Dr Ronald
Dr.

Calabrese, Emory University

Andrew Cameron,

California Institute

of Technology

Mr Richard H Campbell, Bang-Campbell

C Candelas,

University of

Puerto Rico

Dr Lucio Canello, Stazione Zoologica "A

Dohrn," Italy
Dr Catherine Emily Carr, University of Maryland
Prof James F Case, University of California
Father Joseph D Cassidy, O P, Ph D
,

Providence College
Dr Colleen M Cavanaugh, Harvard University

Dr Edward

Chambers, University of Miami

Dr Donald

of

Eric

H Davidson,

C Chang, Hong Kong

Laboratory

Dr Robert C DeGroof, Doylestown, PA

Dr Martha Bridge Denckla, Johns
Hopkins University
Dr Henry A DePhillips, Jr

Trinity College
Dr Douglas
DeSimone, University of Virginia
Dr Wolf-Dietrich Dettbarn, Nashville, TN

Science and Technology

L Chappell, Hunter College
Dr Frank M Child, Woods Hole, MA

Dr Rex Leslie Chisholm, Northwestern University

Dr Elena Citkowitz, Hospital of St Raphael
Dr David E Clapham, Children's Hospital

Dr.

Vincent E Dionne, Boston University Marine

Program
Dr John E Dowling, Harvard University
Dr Arthur Brooks DuBois, John B, Pierce
Foundation Laboratory
Dr Thomas K Duncan, Nichols College
Dr Philip B Dunham, Syracuse University

Dr William R Eckberg, Howard University

Dr Kenneth T Edds, Bayer Diagnostics
Dr Howard A Eder, Albert Einstein College
of Medicine

Ms Joan Edstrom, Falmouth,

Dr Barbara

E. Ehrlich,

State University
Clark, Hobe Sound, FL
Walhs H Clark, Jr., Madison, ME
Dr John R Clay, National Institutes of Health

of Technology
Dr Hugh Young
Scotland

Dr Alexander

Dr. Paul T.

Mr Hays
Prof

Clowes, University of

Washington

Dr Carolyn Cohen, Brandeis University

Dr Lawrence B Cohen, Yale University School

D Cohen,

Hunter College
Dr Annette
Coleman, Brown University
Dr Paul Colmvaux, Marine Biological Laboratory

Harvard Medical School

Dr Jeffrey T Corwm, University of

School of Medicine

Virginia,

Dr Ernest F Couch, Texas Christian University

Dr Rachel Llanelly Cox, Marine Biological
Laboratory
S Crane, Esq
Ferns, Glovsky

Mintz, Levin, Cohn,

& Popeo,

Glasgow,

Englund, Johns Hopkins

Medical School

Dr David Epel, Stanford University

Herman T. Epstein, Woods Hole,

Mr Ray L Epstein, Taunton, MA
Prof

of Medicine

Thomas

Elder, University of

Dr.

State University

Collier,

MA

Yale University

Dr Arthur Z Eisen, Washington University

Dr Herman N Eisen, Massachusetts Institute

Dr Eloise E Clark, Bowling Green

Dr R John

University of

Dr Richard

California Institute

Technology

Dr Paul V Dunlap, University of Michigan

School of Medicine

Dr James P Collins, Arizona State University

Dr D. Wesley Corson, Jr, Storm Eye Institute

Boolootian,

Laboratory

Mr Richard D

Dr Sherry Burszta|n, Dartmouth Medical School

Dr Kerry S Bloom, University of North Carolina

Dr. David A Bodznick, Wesleyan University
Dr Edward G Boettiger, Rochester, VT

A
Thomas A

Crowther, Shriners Hospitals

for Children

Dr Daniel B Davison, Bristol-Myers Squibb PRI

Dr EliezarA Dawidowicz, Marine Biological

Dr William

Dr Richard

Medical School

Mr Robert

Dr John E Burns, Beloit College

Harold L Burstyn, Syracuse University

Stephen H Bishop, Ames, IA

Dr Dieter Blennemann, Riverside, CT
Dr George S Bloom, University of Texas
Southwestern Medical Center
Dr.

Dr Gertrud Cremer-Bartels, Muenster, Germany

Dr Terry J Crow, University of Texas

Dr

Dr Jewel Plummer Cobb, California

Suzanne T

College

Hampshire
Dr Max M Burger, Novartis International AG
Dr David R Burgess, Boston College
Dr Mario H Burgos, IHEM Medical School

Dr Graciela
Dr Baccio Baccetti, University of Sienna, Italy

St Mary's

of Maryland

Dr Michael P Cummings, Marine Biological

Browne, Wake Forest University

Associates

Program

Mr

Forest University

Dr.

Armstrong, University of

Pennsylvania School of Medicine

Mrs Ellen Prosser Armstrong, Woods Hole,

Dr Anne

Wake

Dr Karen Crawford,

MA

Donald Faber, Albert Einstein College

of Medicine

Dr David H Farb, Boston University School

of Medicine

Dr A Verdi Farmanfarmaian, Rutgers University

Dr Barry William Festoff, VA Medical Center
Dr Rachel D Fink, Mount Holyoke College
Dr Alan Finkelstem, Albert Einstein College
of Medicine

Dr Gerald

Fischbach, Columbia University

Dr Harvey M. Fishman, University of Texas

Medical Branch

Mr Dennis Flanagan, New

York,

NY

R89
Dr Richard Allen Fluck, Franklin and
Marshall College

Mr A Robert Gunning, Falmouth, MA

Dr G. Francis Gwilham, Reed College

Dr Robert D. Hunter, Oakland University

Dr Hugh E Huxley, Brandeis University

Prof Leah T Haimo, University of California

Dr Stephen L. Hajduk, Marine Biological

Dr Joseph

Dr Kenneth H Foreman, Marine Biological

Laboratory

Dr Thomas

Fox, Harvard Medical School

Dr Clara Franzim-Armstrong, University of

Dr.

Kathleen

French, University of California.

San Diego
Dr Robert J French, University of Calgary
Dr Chandler M Fulton, Brandeis University
Dr Barbara C Fune, Beth Israel Deaconess
Israel

Deaconess

Howaida Gabr, Suez Canal University

Dr David C Gadsby, The Rockefeller University
Dr Harold Gainer, National Institutes of Health
Dr Robert M Galatzer-Levy, Chicago, IL
Dr Joseph G Gall, Carnegie Institution
Dr Michael A Gallo, UMDNJ-Robert Wood
Dr.

Johnson Medical School

Dr Alan Gelperm, Monell Chemical
Senses Center
Dr James L German, III, Weill Medical College
of Cornell University
Dr Martin Gibbs, Brandeis University
Dr Anne E Giblin, Marine Biological Laboratory
Dr A. Jane Gibson, Enta, NH

Dr Prosser Gifford, Library of Congress

Prof Giovanni Giudice, Universita di

II,"

di

Napoli

Italy

Dr Paul Glynn, Brunswick, ME

Mr William T Golden, Chairman Emeritus,
American Museum
of Natural History
Dr.

Robert

Genomics

Tatsuji Haneji,

The

D Goldman, Northwestern

University

Medical School

Dr Paul K Goldsmith, National

Institutes

of Health

Dr Timothy H Goldsmith, Yale University

Dr Moise H Goldstein, Jr, The Johns Hopkins

Dr Robert Michael Gould, NYS

Laboratory
Dr Ferenc Harosi,

Dr Marietta Radovic Issidondes, Theodor

Theohan Cozzika Foundation

New

University

College of the
of South Florida

Washington
Peter Greenberg, University of Iowa
J Greenberg, University of Florida

Dr Raymond L Hayes, Jr, Howard University

Dr Diane E Heck, Rutgers University
Dr Jonathan Joseph Henry, University of Illinois
Dr Peter K Hepler, University of Massachusetts

Dr Diana

Dr Walter R Herndon, University of Tennessee

Prof

Avram Hershko, Technion-lsrael

Institute of

Israel

Technology,

Dr Theodore T Herskovits, Fordham University

Dr Howard H Hiatt, Bngham and Women's
Hospital

Dr Stephen

Highstem, Washington

University School of Medicine

Dr John

Dr Richard

Hildebrand, University of Arizona

Hill, Michigan State University

Dr Robert B. Hill, University of Rhode Island

Dr Susan D Hill, Michigan State University
Dr Llewellya

Hillis,

Marine Biological

Dr Edward H

Hinchcliffe, University of

Prof Lawrence Grossman,

The Johns

Hopkins University
Yosef Gruenbaum, The Hebrew University

of Jerusalem
Gruner, Cephalon, Inc

Jaffe,

E. J

Marine Biological Laboratory

Jeffery, University of

Maryland

Jennings, Marine Biological

Laboratory
Dr Daniel Johnston, Baylor College of Medicine
Dr Teresa L Z Jones, National Institutes
of Health

Dr Robert K Josephson, University of California

Dr Leonard K Kaczmarek, Yale University School
of Medicine
Dr Gabor Kaley, New York Medical College
Dr Jane C Kaltenbach, Mount Holyoke College

Dr Benjamin Kaminer, Boston University

Medical School
Dr Edna S Kaneshiro, University of Cincinnati
Dr Ehud Kaplan, Mount
Medicine

Sinai

School of

Dr Stephen J Karakashian, Milwaukie,

Dr Arthur Karlin, Columbia University

OR

Dr Morns John Karnovsky, Harvard Medical

School

Dr Gregory J Hinkle, Millenium

Pharmaceuticals

Dr

Dr Gertrude W. Hinsch, University of

South Florida
Dr Jan Hinsch, Leica, Inc

M Kerr, Marine Biological Laboratory

Dr Alexander Keynan, Israel Academy of

Dr John E Hobbie, Marine Biological

Dr Shahid

Laboratory
Dr Alan J Hodge, San Diego,

CA

Biological Laboratory

Houk, Northwestern University

Medical School
Dr Ronald R Hoy, Cornell University

Technology
Dr Linda A Hufnagel-Zackroff, University of

York University

Connecticut

Mr H

Mary J Greer, New York, NY

Dr Donald R Griffin, Harvard University

New

Jaffe, University of

Massachusetts Medical School

Dr Michael Hmes, Yale University School
of Medicine

Dr Alice S Huang, California

SUNY Albany

Health Center

Dr William R

Dr Michael
Dr.

Dr Laurmda
Dr Lionel

Dr James

Dr John

Izzard,

Dr Robert Haselkorn, University of Chicago

J Woodland Hastings, Harvard
University

Dr Judith P Grassle, Rutgers University

Dr Katherine G. Graubard, University of

Dr.

Dr Colin S

Dr

Mr Dick Grace, Doreen Grace Fund

Dr Werner M Graf, College of France, France
Dr Philip Grant, National Institutes of Health

Dr Albert Grossman,
Medical Center

Jersey

Dr KurtJ Isselbacher, Massachusetts General

Hospital Cancer Center

Dr Joseph F Hoffman, Yale University School

of Medicine
Dr George G Holz, IV, New York University
Medical Center
Dr Charles S Hopkinson, Jr, Marine

Institute of

Basic Research

E.

New

Tokushima, Japan
Dr Roger T Hanlon, Marine Biological

(deceased 2002)

University

Dr

Ingoglia,

Medical School

Dr Saduyki Inoue, McGill University, Canada

Dr Shinya Inoue, Marine Biological Laboratory

University of

Laboratory

Italy

Dr Antonio Giuditta, Universita

"Fedenco

Functional Insect

Case Western Reserve

Dr Edwin J Furshpan, Harvard Medical School

Dr Robert P Futrelle, Northeastern University

Palermo,

Hall,

June F Harngan, Ph D Honolulu, HI

Dr John P Harrington, SUNY - New Paltz
Dr Stephen C. Harrison, Harvard University

Medical Center
Dr Bruce Fune, Beth
Medical Center

Institute

Dr

of Technology

Dr Nicholas

Laboratory

Dr Linda

Pennsylvania
Dr Scott Fraser, California Institute

llan,

University

Rhode

Institute of

Island

Dr William D Hummon, Ohio University

Dr Susie H Humphreys, Food and Drug
Administration

Tom Humphreys, University of Hawaii

Dr Tim Hunt, ICRF Clare Hall Labs, England
Dr.

Ernst Keller, Carl Zeiss,

Darcy B.
Dr Robert E
Dr.

Norman

Mr John
Mr Louis

Kelley,
Kelly,

Columbia

Woods

Inc.

University

Hole,

MA

University of Michigan
P Kendall, Faneuil Hall Associates

Kemp,

Sciences/Humanities,

M M

Israel

SUNY Upstate
Medical University
Dr Kamran Khodakhah, Albert Einstein College
of Medicine
Khan,

Dr Daniel P Kiehart, Duke University

Medical Center
Dr

Irving

Klotz,

Northwestern University

Mr Robert A Knudson, Marine

Biological

Laboratory

Dr Samuel S Koide, The Rockefeller University

Sir Hans Kornberg, Boston University
Dr Edward M Kosower, Tel-Aviv University, Israel
Dr Stephen M Krane, Massachusetts
General Hospital
Dr Robert Krauss, Denton, MD
Dr Edward

Kravitz,

Dr William B Knstan,
San Diego
Dr Andrew

Canada
Dr Damien P

Harvard Medical School

Jr.,

University of California,

Kropmski, Queen's University,

Kuffler, Institute

of Neurobiology

William J Kuhns, The Hospital for Sick

Children, Canada

Dr.

R90
Dr Joseph G. Kunkel, University of Massachusetts
Dr Alan M, Kuzinan, Marine Biological Laboratory

Dr Frances
Ms. Jane

Dr Aimlee
Dr Laurie

D laderman.
Landeau,

J.

Dr Dennis

M D

Li

Vale University
,tov.

'

'!,

La-"-'

-y

Hospital

Story

La",

is,

National Institutes of Health

Dr David L,v
>.vnr, University of Miami
Dr. George M. Langford, Dartmouth College
Dr Jeffrey Laskin, University of Medicine and

New

Dentistry of
Dr,

Nechama

Jersey

Lasser-Ross,

New

York

Murray, Dartmouth

McLaughlin, Marine Biological

Laboratory
Dr Robert F
Dr.

McMahon, University of Texas

Thomas Meedel, Rhode Island College

Prof Ian

of Cleveland
Dr.

V McCann

Medical School

Meinertzhagen, Dalhousie

Canada

University,

Dr Dennis E Meiss, Immunodiagnostic

Laboratories

Dr Jerry M Melillo, Marine Biological Laboratory

Dr DeForest Mellon, Jr, University of Virginia
Mr. Richard P Mellon, Laughlmtown, PA
Dr Michael E Mendelsohn,
Medical Center

Dr Alan

Dr Melanie

Scheie Eye Institute

Dr Hans Laufer, University of Connecticut

Dr Paul B Lazarow, Mount Sinai

School

of Medicine

Mr Maurice

Lazarus, Federated

Department

Stores

Dr Edward R Leadbetter, University of

Connecticut

Dr Joshua Lederberg, The Rockefeller University

Dr John J Lee, City College of CUNY

Mr Donald B

Lehy, North Falmouth,

MA

Stephen B Leighton, Beecher Instruments

Dr Aaron B. Lerner, Yale University School

Dr.

Dr Jack Levin, University of California School

of Medicine
Dr Michael S Levine, University of California
Dr Richard B. Levine, University of Arizona

Consulting
Dr Matthew Meselson, Harvard University
Dr Ricardo Miledi, University of California, Irvine

Dr Roger D Milkman, University of Iowa

Dr Andrew L Miller, Hong Kong University
of Science and Technology

of Basel, Switzerland

Dr Ralph Mitchell, Harvard University

Dr Timothy Mitchison, Harvard University
Medical School

Pharmacy, Japan
Dr David M Miyamoto, Drew University
Dr. Merle Mizell, Tulane University

Dr Jorge E Moreira, National

Dr.

James G Morin,

Dr.

Leyla

School of Medicine

St

Linck, University of

Minnesota

School of Medicine
J

Lipicky,

Dr.

Anthony

Lisman, Brandeis University

Liuzzi,

Dr Rodolfo R

Boston,

Llinas,

New

MA
York Unversity

Medical Center

Dr

S Lobel, Boston University Marine

Phillip

Program, Marine Biological Laboratory

Dr Werner R Loewenstein, Falmouth, MA
Dr Irving M London, Harvard-MIT

Longo, University of Iowa

Dr Louise M Luckenbill, Falmouth, MA
Dr Frank

J.

Jr,

Boston University

Dr Jane Ann Maienschein, Arizona State

University

Dr Craig C Malbon, State University of New York

Dr Robert P Malchow, University of Illinois,

Chicago
Richard S Manalis, Indiana-Purdue University

Dr Lynn Margulis, University of Massachusetts

Dr Andrew C Marmucci, Mercerville, NJ

Dr Joe L Martinez, Jr University of Texas

Dr Luigi Mastroianni, Jr, Hospital of University
,

of Pennsylvania

Dr David Mauzerail, Rockefeller University

Dr M Lynne McAnelly, University of Texas

Dr Andrew G McArthur, Marine Biological

Laboratory

Robert E Palazzo, Rensselaer Polytechnic

Institute

Dr John D Palmer,

University of Massachusetts
Pant, National Institutes of Health

Dr George D Pappas, University of Illinois

Dr Arthur B Pardee, Dana-Farber Cancer Institute
Dr Rosevelt

Dr James

Parmentier, International Health

Nebraska

Pardy, University of

Organization

Dr Thoru Pederson, University of Massachusetts

Medical Center

Dr Courtland D Perkins, Alexandria, VA

Dr Philip Person, Flushing, NY
Dr Bruce J Peterson, Marine Biological Laboratory
Dr Ronald Pethig, University College of North
Wales, UK
Dr Ronald J Pfohl, Miami University
Dr Sidney K Pierce, Jr, University of South Florida

Dr David E Pleasure, Children's Hospital

Dr Jeanne S. Poindexter, Barnard College
Dr Harvey B Pollard, U S U H S

Dr Thomas D
Dr Beverly H

Pollard, Yale University

Columbia, MD
Dr Mary E Porter, University of Minnesota
Dr. David D. Potter, Harvard Medical School
Dr Maureen K Powers, San Pablo, CA

Dr Robert
Dr David

J.

Porter,

Prendergast, Johns Hopkins University

Northern Arizona University

Prior,

Dr Robert D. Prusch, Gonzaga University

Dr Dale Purves, Duke University Medical Center

Cornell University
Worrell, Rush-Presbyterian-

Dr.

James P Quigley, The Scripps Research

Mr

Rabb, Cambridge, MA
Irving
Harvey Rabin, Rockville, MD
Michael B Rabinowitz, Boston, MA

Institute

Lukes

Dr Stephen S Morse, Columbia University

Dr Andrew
Murray, Harvard University

Dr.
Dr.

Dr Samuel M Nabrit, Atlanta, GA

Dr Knute J Nadelhoffer, National Science
Foundation
Dr. Ronald L Nagel, Albert Einstein College
of Medicine

Dr Nancy S Rafferty, Marine Biological Laboratory

Dr Robert F Rakowski, Ohio University
Dr Fidel Ramon, Universidad Nacional Autonoma
de Mexico, Mexico
Dr Edward B Rastetter, Marine Biological

Dr Yasuko Nakajima, University of

College of Medicine

Dr Lionel

Illinois,

Prof Toshio Narahashi, Northwestern University

Nasi, Boston University School

Rebhun, University of

Virginia

(deceased 2002}

Dr.

Thomas

Dr Carol

of Medicine

Christopher

Laboratory

Dr John R Reddan, Oakland University

Medical School

Dr Enrico
Dr.

Dr Edward F MacNichol,
School of Medicine

Dr.

de Toledo

Institutes of Health

Food and Drug

Administration

John

MA

Dr Gradimir Misevic, University Hospital

Prof Irwin B. Levitan, University of Pennsylvania

Dr.

Concord,

J. Miller,

Dr Francoise Levmthal, Columbia University

Dr Raymond

England

Dr Hiroyoshi Miyakawa, Tokyo College of

of Medicine

Dr Richard

New

Dr Allen F Mensmger, University of Minnesota

Pratt Mernman, Touchstone

Mr Thomas

Dr,

Dr. Harish

Medical College
Dr Leonard Laster, University of Massachusetts
Medical School
Laties,

Dr Ada L. Olins, Foundation for Blood Research

Dr Donald E. Olins, Foundation for Blood Research
Dr James L Oschman, Dover, NH

Neill,

Reese, National Institutes of Health

Reinisch, Marine Biological Laboratory

Marine Biological

Dr Frederick R

Laboratory

Margaret C Nelson, Ph.D., Cornell University

Peter A. Nickerson, SUNY, Buffalo
Dr Santo V Nicosia, University of South Florida
Ms Catherine N Norton, Marine Biological
Dr.

George Washington

Dr Conly L Rieder, Wadsworth Center

Dr. Monica Riley, Marine Biological Laboratory
Dr Harris Ripps, University of Ilinois at Chicago
Dr

Laboratory

Rickles,

University

Murdoch

Ritchie, Yale University

School

of Medicine

Dr Michael P Nusbaurn, University of

Pennsylvania School of Medicine

Dr Lawrence C Rome, University of

Mr Jonathan O'Herron, Lazard Freres & Company

Dr Ana Lia Obaid, University of Pennsylvania

Dr Jack Rosenbluth,
School of Medicine

New

Dr

Simon Fraser

Pennsylvania

School of Medicine
Dr Shinpei Ohki, SUNY at Buffalo
Dr Rudolf Oldenbourg, Marine Biological
L

University

Dr Allan Rosenfield, Columbia University School

of Public Health

Dr Herbert S Rosenkranz, Florida Atlantic

Laboratory

Dr James

Raja Rosenbluth,

York University

Olds,

George Mason

University

University

R9I
N

Dr William

Mr Rudi

Ross,

New

York Medical College

Rottenfusser, Carl Zeiss Inc

Dr Lewis P Rowland, Neurological Institute

Dr Joan V Ruderman, Harvard Medical School
Dr John D Rummel, NASA Headquarters
Dr Norman

B.

Rushforth,

Case Western

Reserve University
Dr William Devine Russell-Hunter, Easton,

Dr Abraham Spector, Columbia University

Dr Johanna E Speksnijder, Odikj, The
Netherlands

Dr David

of Medicine

Dr John H Steele,

Woods

Hole Oceanographic

North Carolina

Dr Abigail Salyers, University of

Illinois

Prof Brian

Salzberg, University of
Pennsylvania School of Medicine

Dr Jean

Sanger, University of Pennsylvania

School of Medicine

Dr Joseph W. Sanger, University of Pennsylvania

Medical Center
Dr. John
Saunders, Jr. Waquoit, MA
Prof- Howard K Schachman, University of

Gerald P Schatten, University of Pittsburgh

Dr Arlene C Schmeer, Mercenene Cancer
Research Institute
Herbert Schuel,

Dr

Felix

SUNY

at Buffalo

Nicola Schweitzer, Brooklme,

MA

E Schweizer, University of California,

Los Angeles
Dr Sheldon J Segal, The Population Council
Dr Stephen Lament Senft, Woods Hole, MA

Dr Douglas R Shanklin, University of Tennessee

Dr Nadav Shashar, The Interuniversity Institute
of

E. Shashoua, Harvard Medical School

Dr Gaius R Shaver, Marine Biological

Laboratory

John

R.

Shaver, Michigan State University

(deceased 2003)

Dr Michael P Sheetz, Columbia University

Dr David Sheprow, Boston University
Dr Irwin
Sherman, University of California
Dr Osamu Shimomura, Falmouth, MA

Mr Alan

Shipley, Forestdale,

MA

Dr Robert B Silver, Wayne State University

Dr Kathleen K Siwicki, Swarthmore College

Dr Dorothy M Skinner, Falmouth, MA

Dr Roger D Sloboda, Dartmouth College
Dr Greenfield Sluder, University of
Massachusetts Medical Center

Dr Peter

J S

Laboratory

Paul

Darrell R. Stokes,

Biological

Ph

University of

Dr

Elijah

Emory University
Stommel, Dartmouth Hitchcock

Medical Center
Dr.

Alfred Stracher,

SUNY

Health Science Center

at Brooklyn

Strumwasser, East Falmouth,

Felix

Dr Ann E

Stuart, University of

MA

North Carolina at

Hill

Chapel

New

York University

Dr William C Summers, Western Washington

University

Dr Kathy
Prof

Suprenant, University of Kansas

Mary Anne

Dr Andrew
Dr Marvin

Sydlik,

Hope College

Szent-Gyorgyi, Brandeis University

L Tanzer, University of

Connecticut,

School of Dental Medicine

Dr

Tasaki, National Institutes of Health

Ichiji

Taylor, University of

Dr William H
Dr Bruce

Telfer, University

Telzer,

Chicago

of Pennsylvania

Pomona College

Prof Mark Terasaki, University of Connecticut

Health Center

Dr James

Dr David

Townsel, Meharry Medical College

Travis, Woods Hole, MA

Dr Steven N Treistman, University of

Massachusetts Medical Center
Dr Walter Troll, NYU Medical Center
Dr Robert

F.

Troxler,

Boston University School

Dr Kenyon S Tweedell, University of Notre Dame

Dr Mark L Tykocinski, Case Western Reserve
University

Prof Michael Tytell, Wake Forest University

School of Medicine

Ueno, Kyoto

Valiela,

University,

Japan

Boston University Marine Program

Dr Richard Vallee, University College of

Mr.

John

&

Surgeons

Valois,

Sorenson, Cidade Universitana-

Wangh, Brandeis

University

Warner, Laguna Beach,

Dr Leonard Warren, Wistar Institute

CA

John B Waterbury, Woods Hole

Oceanographic Institution
Dr Stephen G Waxman, Yale Medical School
Dr Annemane Weber, University of Pennsylvania
School of Medicine
Dr Janis C Weeks, University of Oregon
Weidner, Louisiana State University
Dr Alice Sara Weiss, Silver Spring, MD
Dr. Earl

Dr.

Dieter

Weiss, University of Rostock,

Dr Leon P Weiss, University of Pennsylvania

School of Veterinary Medicine
Dr Mansa

Woods

Hole,

MA

and Mary
Dr Kensal E Van Holde, Oregon State University

Weiss, Paoli Memorial Hospital

New York University

Dr Gerald Weissmann,
Medical Center
Dr Jennifer

Wernegreen, Marine Biological

Laboratory
Dr Monte Westerfield, University of Oregon
Dr J Richard Whittaker, University of New

Brunswick
Dr Torsten N Wiesel, The Rockefeller University
Dr Darcy B. Wilson, Torrey Pines Institute

Dr T Hastings Wilson, Harvard Medical School

Dr Beatrice Wittenberg, Albert Einstein College
of Medicine
Dr Jonathan B Wittenberg, Albert Einstein
College of Medicine

Dr William F Wonderlin, West Virginia University

Dr Mary Kate Worden, University of Virginia
Dr Basil V Worgul, Columbia University
Dr Chau Hsiung Wu, Northwestern University
Medical School
Dr Charles R Wyttenbach, University of Kansas
Harold H. Zakon, University of Texas
Dr Seymour Zigman, Falmouth, MA
Dr Michael J Zigmond, University of Pittsburgh
Dr.

Dr Joshua

of Medicine

Dr Ivan

Germany

Dr Cindy Lee Van Dover, The College of William

UFRJ, Brazil
Dr William T Speck, Marine Biological
Laboratory

Steudler, Marine Biological Laboratory

Physicians

Sogm, Marine

Laboratory

Dr Martha

Mr
Dr

Dr. Hiroshi

Dr Stephen J Smith, Stanford University School

of Medicine
Dr Roxanna S Smolowitz, Marine Biological
L

MD

Stenflo,

Sweden

Smith, Marine Biological

Laboratory

Dr Mitchell

Johan

Dr Edwin

Eilat, Israel

Dr Victor

Dr.

Switzerland

Lund,

Dr Lawrence

Dr.

Dr Mutsuyuki Sugimon,
Medical Center

Dr Lawrence Schwartz, University of

Massachusetts
Dr

Puerto Rico

Dr Malcolm S Steinberg, Princeton University

Dr Andreas C Stemmer, Institut fuer Robotik,

Dr

California
Dr.

Dr.

Dr Antoinette Stemacker, University of

Prof

Laboratory

Dr Byron H Waksman, New York University

Medical Center
Dr Betty Wall, Woods Hole, MA
Dr Robert

Institution

Dr Mary Beth Saffo, Harvard University

Dr Guy Salama, University of Pittsburgh
Dr Edward D Salmon, University of

Wadsworth, University of

Patricia

Massachusetts

Dr Norman R Wamwnght, Marine Biological

Spray, Albert Einstein College

Dr Kenneth R Spring, National Institutes

of Health

MD

Dr

J.

Zimmerberg, National

Zottoli, Williams

Institutes

of Health

Dr Steven

College

R92

ASSOCIATES

Patron

Dr.

Mr and Mrs. MaicoIm Campbell

The Honorable and Mrs. John S Langford
Dr and Mrs Edward F MacNichol, Jr
Mrs William O.

(Miles.

II

Mr and Mrs. David Bakalar

Mr and Mrs Norman Bernstein
Mr Bnan Bragmton-Smith
Bufftree Building

Co

Inc.

Mrs Martha Saunders Ferguson

Dr David Forkosh and Dr Linda Hirshman

Dr and Mrs. Leonard Laster

Dr and Mrs John
Rowe

Supporting Associate
Mrs Margaret Clowes
Mrs Sally Cross

Ms Anne
Mr

Clark

Mr and Mrs James M. Clean/

Dr Robert R Haubnch

Mrs Elizabeth Heald

Drs Harry Conner and Carol Scott-Conner

Mr and Mrs Donald B Cook

Mr and Mrs Edward S Heard

Dr and Mrs Howard H. Hiatt
Mr and Mrs David Hibbitt
Dr Llewellya Hillis and Dr Paul Colmvaux
Dr and Mrs John E Hobbie
Dr Peter A Hoenig

Mr and Mrs Theron

[Sustaining

Dr and Mrs Laurence P Cloud

Mr and Mrs

Associate

Dr and Mrs Richard Bennet Harvey

Dr and Mrs J Woodland Hastings

and Mrs Frank M, Child

Dr and Mrs Arnold

S Curtis, Jr

Joel Davis

Mr and Mrs Joseph L Dixon

Mr and Mrs F, Gerald Douglass
E. Dowlmg
Mr and Mrs
J Doyle
Dr and Mrs Arthur Brooks DuBois
Mr and Mrs Robert Elias
Mr and Mrs Jerome Fanger

Dr and Mrs Michael

R DuBois

H Eaton
Dr and Mrs Harold S Ginsberg
Mrs Rebeckah DuBois Glazebrook
Drs Alfred and Joan Goldberg

Ms Penelope Hare
Mr and Mrs. Gary G Hayward
Mr and Mrs William K Mackey, Esq
Dr. and Mrs William M. McDermott
Mr and Mrs Robert Parkinson
Mr. and Mrs. William J. Pechilis
Dr and Mrs Alan D Perlmutter
Mr and Mrs Walter J Salmon
Mrs Anne
Sawyer
Dr John Tochko and

Drs Francis

Mrs Carmela

Huettner

Ms Susan A Huettner
Dr and Mrs Shmya Inoue
Dr and Mrs Kurt J Isselbacher

Fishbein

Mr and Mrs Howard G Freeman

Dr and Mrs Robert A Frosch
Dr and Mrs John J. Funkhouser
Dr and Mrs Mordecai L Gabriel
Dr and Mrs David Garber

Mrs Mary D Janney

Dr and Mrs James E. Johnson
Mrs Sally S, Joslin

Miss Eleanor Garfield

Dr and Mrs George Katz

Mr and Mrs Arthur King

Dr and Mrs James L German,

Dr and Mrs Prosser Gifford

Clifton

Mr and Mrs Gerald J Holtz

C Hoskm and Elizabeth Farnhan

Dr and Mrs John

Dr and Mrs Benjamin Kammer

Dr and Mrs Morns John Karnovsky

HI

Mr and Mrs Paul

Knaplund
Mr and Mrs. A. Sidney Knowles,
Mr and Mrs Walter E Knox

Dr and Mrs Murray Glusman

Dr. and Mrs Moise H Goldstein

Mrs Ann

Goodman and

Mr and Mrs
Mr and Mrs
Dr and Mrs
Dr and Mrs

Ms

Dr.

Hans and Lady Kornberg

and Mrs S. Andrew Kulin
Mr Ezra Laderman
Dr and Mrs George M Langford
Dr Hans Laufer
Dr and Mrs John J Lee
Mr Russ Lemcke
Mr. and Mrs James E Lloyd
Sir

Arthur Pardee

Charles Goodwin,

Dr.

III

Frederic Greenman
Thomas C Gregg
Newton H Gresser

Kathryn Hackett

Dr and Mrs Harlyn O Halvorson

Mr and Mrs Benjamin Handelman

Mrs. Christina Myles-Tochko

[Family

Dr and Mrs David E Adelberg

Dr and Mrs Dean C Allard, Jr
Dr Peggy Alsup
Mr and Mrs Douglas

Amon

C Armstrong

Mr and Mrs Duncan P. Aspinwall

Mr and Mrs Donald R, Aukamp
Mr and Mrs John M Baitsell
Dr and Mrs Robert B Barlow, Jr
Mr and Mrs John
Barnes
Dr and Mrs Harriet P Bemheimer

Mr and Mrs Robert

Dr and Mrs Edward

Mr and Mrs

Bigelow

Boettiger
Kendall 8 Bohr

Dr and Mrs Thomas

October

MBi Associates

A Borgese

Dr and Mrs Francis P Bowles

Mr and Mrs Peter Boyer

Mr and Mrs Thomas A. Brown
Dr and Mrs John B Buck
Mr and Mrs William O Burwell
Mr and M's Bruce E. Buxton
Dr and Mr, Richard L Chappell

5,

2001

Sing in the Shower"

Keith Lockhart, Conductor, The Boston Pops Orchestra

"Why

Drs James and Helene Anderson

Dr and Mrs Samuel

THE 2001-2002 FALMOUTH FORUM SERIES

sponsored by the

Membership

October 19, 2001

"Memoirs of a Geisha: The Making of a Novel"
Arthur Golden, Author, Memoirs of a Geisha

December

7,

2001

"An Evening with Norm Abram"

Norm Abram, Master Carpenter and host of The New Yankee Workshop
January 18, 2002
"The Great Powers and the East Mediterranean World"
Erik Goldstein,

Jr

Chairman, Department of International Relations;

Professor of International Relations, Boston University

February 1 2002
"Around the Other Round Stone Barn: The History, Restoration,
and Relevance of the Hancock Shaker Community"
Mary Rentz, Past-President, Board of Trustees, Hancock Shaker Village
,

Friday,

March

1,

2002

"Osteoporosis: The Research Frontier Hopes for a Cure"

Bjorn R. Olsen, Hersey Professor of Cell Biology;
Harvard-Forsyth Professor of Oral Biology; Chairman,
-

Harvard-Forsyth Department of Oral Biology, Harvard Medical School

R93

ASSOCIATES EXECUTIVE BOARD

Ruth

Ann

taster, President

Sallle Giffen, Vice-President

Kitty Brown, Treasurer
Ruth Shephard. Secretary

Mr.
Dr.

Helen Barnes
Peter Boyer
Bruce Button

Mr

Julie Child

Alice Knowles

Hans Kornberg
Rebecca Lash
Susan Loucks
Jack Pearce

Reynolds

members
Speck, Director & CEO, MBL
Dowling, President of the
Corporation,

J.

Segal,

MBL

Chairman of the Board

of Trustees,

MBL

Associates Administrator

Susan Joslin

Pothier,

Mr. Everett E

Jr.

Mr and Mrs John D Ross

Dr and Mrs John

Saunders,
Mr and Mrs Savely Schuster
Mr and Mrs Harold H Sears

Jr

Mr and Mrs
Jr.

MacNeil

Mr and Mrs Joseph Martyna

Drs Luigi and Elaine Mastroiann:
Dr and Mrs Robert T McCluskey

Mr and Mrs Derek J McDonald

Mr and Mrs James McSherry
Mrs Nawrie Meigs-Brown
and Mrs Jerry M Melillo
Dr Martin Mendelson
Dr.

Mr and Mrs
and Mrs
Dr and Mrs
Dr. and Mrs
Mr and Mrs
Mr James V
Mr. and Mrs.
Dr. and Mrs
Mr.

Richard Meyers

Charles

Mitchell

Merle Mizell
Charles H

Bagley

Ms.

Megan

E Barrasso

Ms

Patricia

M. Barry

Mr Mike

Barry

Montgomery

Stephen A Moore
Moynihan
Lewis Nassikas

John E Naugle
Dr. Pamela Nelson and Mr Christopher Olmsted
Mr. and Mrs Frank L Nickerson
Dr. and Mrs Clifford T O'Connell
Mr and Mrs. James J O'Connor
Mr and Mrs Daniel O'Grady
Mr and Ms David R Palmer
Dr and Mrs Clement E Papazian

Dr Millicent Bell
Mr C John Berg

Ms Olive C Beverly
Mr George Billings
Mrs Ellen F Binda
Ms. Avis Blomberg
Dr. Robert H Broyles

Burke
Joseph
Mrs Barbara Gates Burwell
Mr.

G. Butch
Dr Graciela

Dr and Mrs
Dr and Mrs
Mr and Mrs
Dr and Mrs
Dr. and Mrs
Mr and Mrs
Mr and Mrs
Mr and Mrs

Sheldon

Segal

Robert Seidler
Daniel Shearer

David Sheprow
Irwm Sherman
Bertram R

Silver

Jonathan O Simonds
John A Simounan

Dr and Mrs Thomas R Stetson

Dr and Mrs Laszlo Lorand

Phyllis

Mr Dean N Arden

Mrs June Atwood

Andrew H Plevin
George H Plough

Dr and Mrs Guy L. Steele, Sr

Dr and Mrs Alan B Steinbach
Dr and Mrs William K Stephenson

Associates, continued

Mrs

Mr Richard A Ahern
Dr Nina Stromgren Allen
Mrs Fredenca Z Alpert

Drs Frederick and Marguerite Smith

Drs William Speck and Evelyn Lipper

Mr Richard C Levering
Mr and Mrs Francis C Lowell,

Membership

Mrs Barbara Atwood

Harold Pilskaln

Rev Michael Robertson and Dr. Emmy Robertson

Drs. Priscilla and John Roslansky

Ex-officio

Sheldon

Individual

Marjorie Salmon
Volker Ulbrich

E.

Peters
E Joel Peterson

Aubrey

Mrs. Ellen Prosser Armstrong

Mr Garfield
Arthur

Allan Ray Putnam

Mrs Lionel Rebhun
Dr and Mrs George T Reynolds
Dr and Mrs Harns Ripps
Ms Jean Roberts

Alan Perlmutter

T.

Mr and Mrs
Mr. and Mrs
Mr and Mrs
Mr. and Mrs
Dr and Mrs
Mr and Mrs

Thomas Gregg
James Johnson

John

Frederick S Peters

Mrs. Grace

Martha Ferguson

William

Jr

Mrs Nancy Pendleton

Mr and Mrs John 8 Pen
Dr and Mrs Courtland D. Perkins
Dr and Mrs Philip Person

Tammy Amon

Virginia

and Mrs Richard M Paulson,

and Mrs John B Pearce

E Kent Swift, Jr

Mr and Mrs Gerard

Mr and Mrs Emil D
Mr D. Thomas Trigg

Swope

Tietje, Jr

and Mrs Walter Troll

and Mrs Michael Tytell
Mr. and Mrs Volker Ulbrich
Mr and Mrs. John J Valois
Drs. Claude and Dorothy Villee
Dr John Waterbury and Ms Vicky Cullen
Mr and Mrs John T. Weeks
Dr and Mrs Gerald Weissmann
Dr. and Mrs. Paul S Wheeler
Ms Mabel Whelpley and Mr George Rollins
Dr.

Prof

Mrs. Geoffrey Whitney

Mr. and Mrs. Lynn H Wilke

Mr and

Mrs. Leslie J Wilson

Dr and Mrs T Hastings Wilson

Mr and Mrs. Leonard M. Wilson

Mr and Mrs Richard Yoder

Mrs Manlyn G Zacks
Mr and Mrs Bruce Zimmerli

C Candelas

Mr Frank C Carotenuto
Dr Robert H Carner
Dr Chia-Yen Chen
Dr

Ms

Chisholm

Sallie

Paula Ciara

Mrs Octavia C Clement

Dr Jewel Plummer Cobb
Mrs Margaret H Coburn
Dr Seymour S Cohen
Ms Genevieve Coleman

Ms Anne S Concannon
Ms Margaret S Cooper
Dr

D Eugene Copeland

Mrs. Molly N. Cornell

Dr Vincent Cowling
Mrs Marilyn E Crandall

Ms Cathleen Creedon
Ms Helen M. Crossley
Ms Dorothy Crossley
Mrs Villa B Crowell
Mrs Dons M Curran
Mrs Elizabeth M Davis
Ms Maureen Davis
Ms Carol Reimann DeYoung
Mrs Virginia A Dierker

Mr David

Donovan

Ms Suzanne Droban
Ms Maureen J. Ougan
Ms. Deborah

Dunn

Mrs Frances E Eastman

Dr Frank Egloff
Ms. Elizabeth Egloff
Mrs Eleanor B Faithorn

Ms Helen C Farnngton
Mrs Ruth Alice Fitz

Ms

Sylvia

Mr John

Flanagan
Folmo, Jr

R94

GIFT SHOP VOLUNTEERS

Marion Adeiburg
Beth Berne
Avis

Blomberg

Gloria Borgese
Julie Child

Janet Daniels
Carol

De Young

Frances Eastman
Pat Ferguson

Barbara Grossman

Jean Halvorson

Hanna Hastings

Associates, continued

Marcella Katz

Donna Kornberg

Mr

Barbara

Mrs Ruth E Fye

Little

Florence Mixer

Paul

J.

Freyheit

Bertha Person

Mr Joseph C Gallagher
Mrs Lois E Galvin

Julie Ranlcin

Miss Lan

Arlene Rogers

Mrs Matilda L Gellis

Mrs Dons D Gerace

Atholie

Rosen

Cynthia Smith
Alice

Todd

Ms

Ge

Sallie Giffen

Mrs. Janet F Gillette

Ms Mary W McKoan
Ms Jane A McLaughlm
Ms Louise McManus
Ms Cornelia Hanna McMurtne
Ms Paula A Mealy
Dr Carmen Merryman
Ms Vivagean V. Merz
Mrs Marianne F Milkman

Mr Michael P Goldring

Ms

Clare Wilber

Ms

Betty Wilson

Mrs Winifred

Paul J Mulloy
Mrs Carol Murray

Grace Witzell
Bunnie Zigman

Gould

Muriel

Ms Janet

Green

Gregg

Mrs Jeanne B Griffith

Mrs Barbara Grossman
Mrs Valerie A Hall

Ms Mary

Elizabeth

Hamstrom

Mrs Jane M Heald

Mr Mark Hollander
Mrs Betsy Honey

TOUR GUIDES
Gloria

Borgese

Frank Child

Nancy Fraser
Sallie Giffen

Ronald Glantz
Charles

Mahoney

Haskell

Maude

Andrew McArthur
Vivagean Merz
Carl

Ollmer

William Philips
Julie Rankin

Howard Redpath
Sheila Silverberg

Mary Ulbrich
John Valois

Mr Roger W Hubbell
Ms Alexandra Izzo
Dr Diana E Jennings
Mrs Megan H. Jones

Ms Carolyn L Parmenter
Ms Joan Pearlman
Dr Daniel A Pollen

Mrs

Dr Peter

Kivy

Ms Kathryn M Kleekamp
Ms Margaret D Lakis
Ms Meryl Langbort
Ms Rebecca Lash
Mr William Lawrence
Dr Marian E LeFevre

Mr Edwin M Libbin
Mr Lennart Lindberg
Mrs. Barbara C Little
Mrs. Sarah J

Loessel

Julia S

Rankin

Dr Magaret M Rappaport
Mrs Carol V Rasic

Mr

Nancy R Malkiel
Mandel and

Brett

Ms

Laura

Wembaum

Mrs Diane B Maranchie

Mrs Marjorie Marshall
Mrs Mary Hartwell Mavor

Mr
Mr

Bill

McGoey

Pauf

McGonigle

Dr Susan

Mcllwain

III

Ms

Natalie Trousof

Mr Louis C Turner

Ms
Ms
Ms
Ms
Ms
Mr
Mr

Eleanor S Uhlmger

Ciona Ulbrich
Sylvia Vatuk

Dawn Vaughn
Susan Veeder
Lee

Arthur

Vincent

Voorhis

Ms

Dr Monica Riley
Mrs- Lola E Robertson

Dr Steven R Rodermel
Mrs Arlene Rogers

Mrs Atholie K Rosett

Dr Virginia F Ross
Dr John D Rummel
Mrs Rosalind Russell

Mrs.

Mr Edward Stimpson,
Mrs Elizabeth Stommel
Ms Maren Studlien
Mr Albert H Swam
Mr Dorman J Swartz
Mr James K Taylor
Mrs Alice Todd
Mr Arthur D Traub

Dr Mary Elizabeth Rice

Ms Sandy L Richardson

Mr Jeremy Loyd
Dr Zella Luria
Lyon
Mrs Margaret M MacLeish
Mrs Annemarie E Mahler

Mrs. Jane Lazarow Stetten

Mrs Ann Wadsworth

Mrs Eve Warren

Mrs

Dr Malcolm S Steinberg
Ms Eleanor Sterling

Mr Fred J Ravens, Jr
Ms Mary W Rianhard
Ms Andrea Ricca

Ms Susan Loucks

Sallie

(Ret)

Dr Renee Bennett O'Sullivan

Dr Jack S Parker
Mr David Parker. Jr

Ms

Keoughan

Mrs Eleanor Stembach

USN

Nolan

Mr Barry Pratt
Ms Elizabeth T Price
Ms Dianne Purves

Patricia E

Dr Robert E Steele

Ms Peggy Schiffer Noland

Ms Catherine N Norton

Jones
Mrs Joan T Kanwisher
Mrs Sally Karush

Ms Barbara

Ms

Neuron. Peter Koulen

C Smith

Specht
Dr Evelyn Spiegel
Mrs Helene E Spurrier

Mrs Eleanor M Nace

Mrs Anne Nelson
Dr Eliot H Nierman

Mr Edmund

Dr Jeffrey D Silberman
Mrs Phyllis J Silver

Cynthia Moor

RADM

Ms Dorothy Shiebler
Ms Enid K Sichel

Mrs. Cynthia
Mrs Louise

Mrs Florence E Mixer

Mr Ken Moffitt
Mr Lawrence A Monte

Natalie Trousof

Mrs Ruth Shephard

Mr Frank Shephard

Wendy

Rose

Mr. Michael S
Lillian

Wemstein

Wendonf

Dr Gary Wessel
Mr George R Wezntak

Mr Gerry White
Mrs Barbara Whitehead
Mrs Clare

Wilber

Mrs Helen Wilson

Mrs Grace Witzell

Ms Nancy Woitkoski
Mr Dale Wolfgram

Mr Raymond A Sanborn

Mrs Dorothy M York

Dr Linda Amaral Zettler
Mrs Bunnie Rose Zigman

Dr Thomas Sbarra

Mrs Margery P Zinn

Dr Albert Samuel

Ms Mary M Scanlan
Mr Claude Schoepf
Mr Samuel C Schon
Mrs

Elsie

Scott

Dr Cecily Cannan Selby

Mrs Deborah G Senft

Ms Dorothy

Sgarzi

Mrs Charlotte Shemin

R95

COUNCIL OF VISITORS
The purpose of the Council of Visitors is to increase awareness of the Marine Biological Laboratory and to inform
the broad range of activities in research and educational programs. COV members serve as

members about

ambassadores of the Laboratory, and thus

Mr Robert

Dr Goodwin

Ashton

York.

New

Mr Donald
Continental

MBL.

Breinin

New York University Medical

New York, New Yorlt

Bay Foundation

New

raise visibility of fhe

York

Mr Murray H

Bainton

New

Can Co

York,

Dr Sylvia

Center

Bring
York

New

Earle

Deep Ocean Engineering

Oakland. California

Mr & Mrs Hoyt Ecker

Vero Beach, Florida

Boca Raton, Florida

Mr John Callahan

Mr David

Carpenter, Sheperd & Warden

New Lone/on, New Hampshire

Bakalar

Chestnut

Hi/I,

Massachusetts

Dr George
Massachusetts General Hospital
Boston, Massachusetts
P Baker

Mrs. Elizabeth

Campanella
West Fafmouth, Massachusetts

Mr Anthony B Evnin
Venrock Associates

New

York,

New

York

Mr Michael Fenlon
Nathan Sallop Insurance Agency.

Inc

Boston, Massachusetts

Dr R John Collier
Dr Sumner

A Barenberg

Bernard Technologies
Chicago,

Judah Folkman,

Boston, Massachusetts

Children's Hospital

Illinois

Boston, Massachusetts

Dr Stephen D Crocker
Bethesda, Mary/and

Mr. Mel Barkan

The Barkan Companies

Mr Michael

Mr

Cognition Corporation
Bedford, Massachusetts

Frederick Bay
Josephine Say Paul

& C Michael

New

Cronin

New

York,

New

York

Dr Anthony

Anheuser-Busch,

Mr George Berkowitz
Legal Sea Foods
Massachusetts

Inc

Dr,

Lorenzo DiCarlo

Mrs. Sally

Ann

Stegeman DiCarlo

New

Mr Maynard Goldman
Maynard Goldman & Associates

York

Ms

Charlotte

Hall

Edgartown, Massachusetts

Ms Penelope

Hare

West Falmouth, Massachusetts

Arbor. Michigan

Jewelle and Nathaniel Bickford

York,

Mr and Mrs Huib Geerlings

Cutaia

St Louis, Missouri

New

Eleanor Ford House

Boston, Massachusetts
International, Inc

Mason, Ohio

Al/ston.

&

Grosse Pomte Farms. Michigan

Boston, Massachusetts

Mrs. Lynn
Piasecki Cunningham
Film and Videomaker, Piasecki Productions

York

Mr Robert P Beech
Component Software

Paul

Foundation, Inc
York,

Mrs Hadley Mack French

Edsel

Boston, Massachusetts

New

MD

Harvard Medical School

Dr Charles Di Cecca

Mrs Elizabeth Heald

West Falmouth, Massachusetts

Medford, Massachusetts
Dr Elkan R Blout

Dr Thomas R Hedges, Jr

Harvard Medical School

Mr Diarmaid H Douglas-Hamilton

Boston. Massachusetts

Hamilton Thorne Research

Neurological Institute, Pennsylvania Hospital

Philadelphia. Pennsylvania

Beverly, Massachusetts

Mr Malcolm K Brachman
Northwest Oil

Company

Dallas. Texas

Drs Linda Hirshman and David Forkosh

Mr Benjamin F du Pont
Du Pont Company
Deepwater,

New

Brandeis University & FMH Foundation

Waltham, Massachusetts

Jersey

Continued
Mitch Sogm, Elizabeth Armstrong
Nosema locustae spores, Linda Amaral Zertler

R96

COUNCILOR VISITORS MEETING

JUNE

27, 28,

Mr Robert

2002

St

Modern Molecular Approaches

John

R.

S Shifman

Simon's Island, Georgia

Mr and Mrs Gregory Skau

Grosse Pomte Farms, Michigan

to Global Infectious Diseases

David, M-D., Moderator

Harvard School of Public Health

Mr John C Stegeman
Campus Rentals
Ann Arbor, Michigan

A 21st Century Challenge:

Immunology

Using Genomics, Proteomics, and Molecular

Develop Vaccines for Malaria and Lung Cancer

to

Stephen

L.

Hoffman, M.D.

Mr Joseph T

Senior Vice President, Biologies

Celera Genomics

AIDS

in Africa:

Max

Skillman,

Mr

New Epidemics, New Viruses

Stewart, Jr

New Jersey

Richard H Stowe

Capital Counsel LLC

New York, New York

Essex, D.V.M., Ph.D.

Chairman, Department of Immunology and Infectious Diseases

Harvard School of Public Health

Mr Gerard
The Conundrum of Tuberculosis and HIV/AIDS
Jerrold J. Eilner, M.D.
Director, Center for Emerging and Re-Emerging Pathogens

Swope

DC

Washington,

Mr John

New Jersey School of Medicine

Concord,

Swope

New

Hampshire

Mr and Mrs Stephen

E Taylor

Mi/ton Massachusetts

Mrs Barbara

Hostetter

Mr Samuel Thome
Thorne Trading Campany, LLC

Mr Michael T Martin

Manchester, Massachusetts

Barr Foundation

SportsMark, Inc

Boston, Massachusetts

New

Mr Charles Hunter

Mrs Christy Swift Maxwell

Grosse Pomte Farms, Michigan

Kessinger Hunter & Co

Kansas City, Missouri

New

York,

York

Mrs Karen Tierney

Wellesley, Massachusetts
Mrs Donna Vanden Bosch-Flynn

Mr Ambrose Monell
Mr Thomas

&

Meredith

Hynes, Jr
Grew, Inc
J,

Spring Lake,

New Jersey

Linger Vetlesen Foundation

Palm Beach, Florida

Mr Benjamin S Warren III

Grosse Pomte Farms, Michigan

Boston, Massachusetts

Dr Mark Novitch

Mrs Mary D Janney

Nancy B Wemstein. R.N

The Hospice, Inc
Glen Ridge, New Jersey

DC

Washington,

Washington, D.C.

Mr David R Palmer
Dr Morris

Karnovsky
Harvard Medical School

David Ross Palmer

&

Associates

Stephen S Wemstein, Esq

Mornstown, New Jersey

Waquoit, Massachusetts

Boston, Massachusetts

Ms

Ellyn

Mr and Mrs Joseph

Korzun

P.

Pellegnno

John C West.

Boston, Massachusetts

Goldman, Sachs & Co

New

York,

New

York

Mr Robert Pierce, Jr
Aluminum Co

Mr

Pierce

Mr and Mrs

Robert Lambrecht

MD

Danville, Pennsylvania

Franklin,

Frederick J Weyerhaeuser

Beverly, Massachusetts

Massachusetts

Boca Grande, Florida

Mr Rudy Landry

Mrs Robin Wheeler

West Falmouth, Massachusetts

Mr Manus A Robinson
Fundamental Investors Ltd

Pocasset. Massachusetts

Key Biscayne,

Catherine C Lastavica, M D
Tufts University School of Medicine

Mr Edward Rowland

New

Boston, Massachusetts

Tucker Anthony. Inc.

Boston, Massachusetts

Mrs Annette Williamson

Dr Anna Logan Lawson

Mr Andrew E Sabin

Daleville, Virginia

Sabin Metal Corporation

East Hampton, New York

Florida

Ms

Rosalind
York.

C Whitehead

New

York

Forth Worth. Texas

Mr

Joel A. Leavitt

Boston, Massachusetts

Ms

Linda Sallop

Nathan Sallop Insurance Agency,

Mrs. Margaret

Lilly

Inc.

Boston, Massachusetts

West Falmouth, Massachusetts

Mr Gregory A Sandomirsky
Cohen Ferns Glovsky & Popeo, PC

Mr. Richard
Lipkin

Mintz Levin

Shipan Capital

Boston, Massachusetts

New

York.

New

York

Dr Cecily

New

York.

Selby

New

York

R97

ADMINISTRATIVE SUPPORT STAFF

[Biological

Services Office

Bulletin

Greenberg, Michael

[Financial
Lane. Jr. Homer

W.

Chief

Financial Officer

Editor-in -Chief

Pamela Clapp,
Managing Editor

McLean, David. Controller

Hinkle.

Child.

Mullen, Richard J

Manager,
Research Administration

Wendy

Adams, Taryn
Aguiar, Deborah

Gibson, Victoria R

Schachmger, Carol H

Bliss,

Casey

Brady, Caroline
I

Director's Office

Crosby, Kenneth

Speck. William T.. Director

and Chief Executive Officer

Griffin,

Donovan. Marcia H

Newman,

Bonnie Jean

Lancaster, Cindy

Melissa

Nunes, Kenda
Solchenberger, Carolyn

Equal Employment Opportunity

MacNeil. Jane L

Stellrecht. Lynette

Stock

Veterinarian Services

Smolowitz, Roxanna.

Room

Schorer. Timothy

Campus

Supervisor

:
Galatzer-Levy. David

Veterinarian

Olive, Jr, Charles

Allen, Taylor

Photo by Volker Steger

Bonacci, Lisa'

Dalpe. Heather

Purchasing

Ehlen.

Hall Jr, Lionel E., Supervisor

Jill-

Hancock,

Amy

Hunt, Lisa

[Housing and Conferences

Beckwith, Mary M Director
Fuglister. Charles K
Grasso, Deborah

|Educational Programs
Dawidowicz. Eliezar A Director
Hamel, Carol C

Holzworth, Kelly
Central Microscopy Facility

Knjger, Sally J
Livingstone. Suzanne

and

Oldham. Pamela

General Use Rooms

Kerr, Louis

Widdiss, Brittany'

Stukey. Jetley

Pento, Diana

Supervisor

7
Histen, Gavin

Stackhouse. Barbara

Inzina, Jessica-

Wagner, Carol

Luther, Herbert

Ogomo.

Christopher On'
:
Parmenter, Marjone

Housekeeping

Peterson, Martha B

Barnes, Susan

?
Scanlon, John

:
Barron, Laura

Bailey. Jeffrey Jr

Photo by Elizabeth Armstrong

Bernos. Jessica

Chen, Zhi
(External Affairs

Xm

Doherty. Bryant-'
I

Carotenuto, Frank

Johnston, David-"

Director

Hannigan, Catherine

Butcher, Valerie

Faxon,

Wendy

George, Mary
Johnson, A Knstme
Patch-Wing, Dolores
Quigley. Barbara
Shaw, Kathleen L

Santiago. Crystal

Susan

Sgarzi.

Dorothy

Waterbury. Matthew-"

J. Erik
|

Jonsson Center,

Carlisle, Ann-'

Langill, Christine'

Doherty. Joanne

Samantha
Mock-Munoz De Luna, DanaMansfield,

Rullo.

Gina

Ehchalt,

Manager

Safety Manager
of Research Administration

Cathy Norton. Director of the MBL/WHOI Library

William T Speck, Director and Chief Executive Officer

Snow, Linda

Hlista. Laurel'

Lane. Chief Financial Officer

Richard Mullen.

Carmen

Houser,

Beth R

Homer

Andrew Mattox, Environmental Health and

P, Director

Damery, Angela"

J.

Communications Office
Hinkle. Pamela Clapp Director

Liles,

Roger Hanlon, Director of the Marine Resources Center

Pamela Clapp Hinkle. Director of Communications

|Human Resources
Goux, Susan

Hemmerdinger, Catherine
Hartmann. Kelley'

Lenny Dawidowicz, Director of Education

Susan Goux, Director of Human Resources

Shum, Mei Wah

Associates Program
Joslin,

Mary Beckwith, Director of Housing and Conferences

Frank Carotenuto, Director of External Affairs
Richard Cutler, Director of Facilities and Services

MacDonald. Cynthia C
McNamara, Noreen M

Senior Staff

NAS

Donald
Joan :

Shurtleff,

Including persons who joined or

or temporary

Summer

left

the

staff

during 2001

R98
Support

staff,

continued

Josephine Bay Paul Cents

Comparative Molecular
Biology and Evolution

for

Information Technology Division

Inzina, Barbara, Network Manager
Callahan, Michael2

Cohen, Alex
Dematos, Christopher
Fournier, Pamela

Lim, Pauline
Tara

Nih.ll,

Marine Resources Center

Leary, Patrick

Hanlon, Roger T, Director

Maddux, Betty L S -

Lowell, Gregory

Aquatic Resources Department

Enos,

Jr.,

Edward

Mountford, Rebecca
Renna, Denis

Space, David

B.

P,

NASA

2
Stackhouse, Aaron

2
Rattacasa, Frank

Apparatus
Atwood, Paul

Shepherd, Denise
Sullivan,

for

Advanced
Life

2
Sterling, Christian

Diana, Administrator

Heather

Farrell,

Ware, Lynn
Security

& Grounds

Blunt,

Mebane, William N

Systems

Health,

and Safety Manager

Elder, Kristopher

Robert F

Rogers, William

Nunez, Guillermo, Center

Research Administrator

Berthel,

Research Space Administration

Kaufmann, Sandra J

Hanley, Janice S
Kuzirian, Alan

Linnon, Beth

Satellite/Periwinkle Children's

Programs
Robinson, Paulina H

IMBL/WHOI LIBRARY
Norton, Catherine

Director

Uhlmger, Eleanor, Assistant

MBL

Library

Deveer, Joseph

Langill,

Matthew

Reuter, Laura

Stafford,

Stout,

Noonan,

Nancy

Orfila,

Amy

Digital Processing

Jason

Goehl, George
Gonsalves, Jr, Walter W.

Henderson, Jon R
House, James
Howell, Robert

Caitlin 2

Anna 2

Sturbaum, Sonja 2

Center

Michael

Ficher,

Patrick 2

Swetish, Margaret

Bailey, Jeffrey B

Elias,

Cecelia 2

Plourde,

Walton, Jennifer

Atwood, Paul R
Auclair, Donald

2
Cormier, Garrett

Susan 2

O'Connor,

Jacqueline

Abbott, Thomas E., Supervisor

Anderson, Lewis B

Horace 2
Cadose, James W.
Callahan, John J

Sarah 2

Halter,

Langill,
2

Swiniarski, Kathryn

Richard

Laurmo, Frank
2
Mancevice, Connne

Clark, Sarah

Thamm,

Jennifer

McAdams

Hadway, Nancy
2
Mannix, Jessica

Urciuoli,

Karen 2

McHugh, Michael

Vachon,

Julia

Reuter, Laura

Westburg, Joanne

and Maintenance

Bryant,

2
Leveque, Rachel
Noonan, Brendan 2

Nelson, Heidi

Riley,

Guiffrida, Beth

Plant Operations

Barnes, John S

Meredith 2

Butler,

2
Camire, Aaron

2
Karalekas, Nina

Monahan, A Jean
Person,

Dorothy

J.

Donovan, Suzanne

James

Director,

The Ecosystems Center

Operator
Carroll,

Cave, Anthony, Center Research

Administrator

McHugh, Clare
2
Mendoza, Guy
Santoro, John

J.

Scanlan, Melanie

Support System

Lochhead, William

Environmental,

David

Matthew 2
Duane, James 2
Malchow, Robert 2

Cutler,

Illgen,

(Safety Services
Mattox, Andrew H

Projects

M Manager

Rozum, John

Tassman, Eugene

MRC Life

and

Hathaway, Peter
Kelley, Kevin

Clayton, Daniel
Collins, Paul J

Sullivan, Daniel A.

Whelan, Sean P

Hugh

Fish Jr,

L.

Brereton, Richard S

Brendon

Toner, Michael

Transportation Services

Sciences
Blazis,

Donald

Fleet, Barry

the Space

in

D
A

Hayes, Joseph H., Supervisor

Bailey, Jeffrey B

Center

Studies

Andrew 2

Sterling,

Barry

Pratt, Barry

2
Wagner, Paul

Stephen

Pratt,

Boucher, Richard

Mendoza, Duke

Facilities

Mills,

Haskins, William

W
Sexton, Andrew W

and

Director

Enos, Joyce B

Barnes, Franklin

Superintendent

Dimond, James L 2
Grossman, William
Klimm III, Henry

Baptiste, Michael

Remsen, David

Santore, Gabnelle

Services, Projects
Cutler, Richard

Settlemire,

Jones, Patricia L
|

III,

Herbert

McQuillan, Jeffrey

O
2

Seifert,

Mary Ann

ARINE IXESOURCES
WOODS

MARINE BIOLOGICAL LABORATORY

HOLE.

CENTER

MA 02543

(508)289-7700

WWW.MBL.EDU/SERVICES/MRC/INDEX.HTML

Animal and Tissue Supply for

Education

& Research

150 aquatic species available for

shipment via
online catalog: <http://vwwv.mbl.edu/animals/

index.htmb; phone: (508)289-7375; or

e-mail:

specimens@mbl.edu

zebrafish colony containing limited mutant strains

custom dissection and furnishing of specific organ

and tissue samples

zebrafish facilities

MRC Services

Available

basic water Quality analysis

veterinary services (clinical, histopathologic,

microbial services, health certificates, etc.)

aquatic systems design (mechanical, biological,

engineering, etc.)
educational tours and collecting trips aboard
the R/V Gemma

Using the

MRC for Your Research

capability for

advanced animal husbandry (temperature,

availability of_year-round,

developmental

adaptability of tank system design for

live

life

light control, etc.)

stages

marine animal experimentation

New

The evolution

ApoTome. Nothing
revolution

in

less

than a minor

fluorescence microscopy.

collected from the

maximum

in

full

contrast

sample with

& optimal image

Even with the thick

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of optical sections

Carl Zeiss Microlmaging, Inc.

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For

more information,

call

800.233.2343.

Thornwood, NY 10594

micro@zeiss.com

fluorescence microscopy

zeiss.com/micro

ZEISS
We make

it

visible.

October

Volume 205

200:?

Number

BIOLOGICAL
BULLETIN

Published by the Marine Biological Laboratory

www.biolbuW.org

THE BIOLOGICAL BULLETIN

ONLINE
The Marine
to

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is

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SEEKERS
THE SOCIETY
OF CELLS.
Simon

says.

"We

At Dr. Simon Watkins'

look

at cells

way anthropologists look at human culture:

as communities of good guys and bad guys,
of traders and communicators, of connections

and

relationships.

never jump to conclusions.

to us." His mantra?

lab, they

the

"Imaging

is

everything."

the brightest of tomorrow's seekers

and

"We

We

are the observers,"

let

Which

the conclusions
is

why

solvers find their

jump

the best and

way

to Pittsburgh

and the Watkins Lab.

ROCKET SCIENCE

OLYMPUS
{From

Ana

L to R)

Bursick

Stuart

Research Specialist

Shond Research

Simon

Specialist

C. Watkins, Ph.D.

Director

Glenn Papworth - Research Associate

Romesh Droviam Graduate Student
-

Center

lor Biologic

Imaging,

University of Pittsburgh
Pittsburgh,

PA

Medical School,

800455-8236
.CO ^/microscope

Cover

image on the cover, microtubules of the peripheral

outward along the F-actin-containing
cortex, away from a microtubule-poor central region. At about 20 min post-activation, with the aster
aster curve

Mature eggs of the surf clam Spisula solidissima are

arrested in prophase of the first meiotic division.
When the eggs are activated by sperm or KC1, the
first meiotic division is completed, the second meiotic division follows, and then (with sperm) embryonic development ensues. As in all meioses, the
cleavages are very eccentric, and each produces a

small polar body. Because very large numbers of

Spisula eggs can be activated simultaneously, and
thus form their polar bodies in near synchrony,
these eggs are an excellent model with which to
study, not only the usual embryonic cell division,
but also polar body formation
an example of ex-

chromosomes are now arranged in

anaphase (upper-right image), and a "bulls-eye"
F-actin ring
the cytokinetic ring
appears on the
diminishing, the

cortex (side view, upper right; computer-generated

face view, lower left). Finally, at about 26 min
post-activation, the peripheral nucleus and its remaining centrosomal material enter the F-actin-

poor center of the ring to produce the

first

polar

body (lower- right image).

In this issue of

These stages include critical activities particularly, docking of the spindle with the cell cortex,
and signaling to generate the cytokinetic contractile

193),

ring

ies in Spisula eggs.

immediately precede the formation of polar bodThe movements and locations

of these structures were revealed by confocal

approximate those of the F-actin ring at anaphase.

This correspondence suggests that generation of the

fluorescence microscopy after appropriate staining (F-actin, red-orange; microtubules. green;

contractile ring

tremely asymmetrical cytokinesis.

The Biological Bulletin (pp. 192Rafal Pielak, Valeriya Gaysinskaya, and

William D. Cohen report on the organization of
F-actin and microtubules in meiotic stages that

chromosomes, blue-violet). Four images from the

set upon a background of diagrammatic
report
surf clams
appear on the cover (see scale bars in
Fig.

At aboi
phasc

193).

p.
t

13

,s|

min

form,

astral

is

triggered by signals from the

microtubule-cortex contact.

Rafal Pielak and Valeriya Gaysinskaya were sum-

mer 2003 research interns in Hunter CollegeHoward Hughes Medical Institute (HHM1) Undergraduate Biological Science Education Program

after activation

of the

first

(23C), the meta-

meiotic division

is

already

and eccentrically positioned; it then

moves toward the cell surface. In the upper left
fully

that occur in all sexually reproducing animals

by mechanisms yet unknown. But note that, at
metaphase, the pattern and dimensions of the contact between the astral rays and the egg cortex

at

Marine Biological Laboratory. The surf clam

pattern on the cover was designed by William D.
Cohen. The cover was designed by Beth Liles (Mathe

rine Biological Laboratory).

THE

BIOLOGICAL BULLETIN
OCTOBER
Editor

MICHAEL

Associate Editors

Louis E. BURNETT
R.

J.

2003

The Whitney Laboratory, University of Florida

GREENBERG

Grice Marine Laboratory, College of Charleston

California Institute of Technology

ANDREW CAMERON

CHARLES D. DERBY

Georgia State University

MICHAEL LABARBERA

University of Chicago

Section Editor

SHINYA INOUE, Imaging and Microscopy

Marine Biological Laboratory

Online Editors

JAMES A. BLAKE, Keys

ENSR

to

Marine

Woods Hole Region

WILLIAM D. COHEN, Marine Models
Electronic Record and Compendia

Marine

&

Coastal Center.

Woods Hole

Invertebrates of the

Editorial

Board

New York

PETER B. ARMSTRONG

University of California. Davis

JOAN CERDA

Center of Aquaculture-IRTA, Spain

Bodega Marine Lab., University of California, Davis

ERNEST S. CHANG
THOMAS H. DIETZ
RICHARD B. EMLET

DAVID EPEL

Louisiana State University

Oregon Institute of Marine Biology. Univ. of Oregon
Hopkins Marine Station, Stanford University

KENNETH M. HALANYCH
GREGORY HINKLE
NANCY KNOWLTON

Auburn University. Alabama

MAKOTO KOBAYASHI

Hiroshima University of Economics. Japan

University of North Carolina Greensboro

ESTHER M. LEISE

DONAL T. MANAHAN
MARGARET MCFALL-NGAI
MARK W. MILLER
TATSUO MOTOKAWA
YOSHITAKA NAGAHAMA
SHERRY D. PAINTER
J. HERBERT WAITE
RICHARD K. ZIMMER
Editorial Office

Hunter College. City University of

Millennium Pharmaceuticals. Cambridge. Massachusetts

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University of Southern California

Kewalo Marine Laboratory, University of Hawaii

Institute

Tokyo

of Neurobiology. University of Puerto Rico

Institute of

Technology. Japan

National Institute for Basic Biology, Japan

Marine Biomed. Inst.. Univ. of Texas Medical Branch
University of California. Santa Barbara
University of California. Los Angeles

PAMELA CLAPP HINKLE

Managing Editor

VICTORIA R. GIBSON

Staff Editor

CAROL SCHACHINGER

Editorial Associate

WENDY CHILD

Subscription

&

Advertising Administrator

Published by

MARINE BIOLOGICAL LABORATORY

WOODS HOLE, MASSACHUSETTS

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CONTENTS
VOLUME

205, No.

OCTOBER 2003

2:

RESEARCH NOTE
and Heidi M. Dierssen
Cascading trophic impacts of reduced biomass

Seibel,

Brad

PHYSIOLOGY AND BIOMECHANICS

Hamdoun, Amro

A.,

Ross Sea, Antarctica: |usl the

Lee, Raymond W.

tip

in tin

of the iceberg?

M., Daniel P. Cheney, and Gary N.

Cherr

93

Phenotvpic

of

plasticity

HSP70 and HSP70 gene

ex-

pression in the Pacific oyster (Crassostrea gigas): implications for thermal limits and induction of thermal

Thermal tolerances of deep-sea hydrothermal vent

animals from the Northeast Pacific.

98

tolerance

160

SHORT REPORTS FROM THE

2003

GENERAL

SCIENTIFIC MEETINGS OF THE MARINE

NEUROBIOLOGY AND BEHAVIOR

BIOLOGICAL LABORATORY
Robison, Bruce H., Kim R. Reisenbichler, James C.
Hunt, and Steven H. D. Haddock

arm

Light production by the

cephalopod Vampyroteuthu

tips

The Editor
The MBL Awards

of the deep-sea

for

I>l \l

ECOLOGY OF PARASITES
Ann Zimmer, and

Patterns and processes of larval

estuarine parasite system

175

1(11.'

iii/midli*

Fingerut, Jonathan T., Cheryl

ard K. Zimmer

y 103

nl'MI

MU

BlOl.OC.Y

and Winfried Denk

Long duration three-dimensional imaging of calcium

Gilland. Edwin, Robert Baker,

waves in zebrafish using mnltiphoton fluorescence

Rich-

microscopy
Opher, and Alon Sabban
Squid sperm to clam eggs: imaging wet samples in a
scanning electron microscope
Wadeson, P. H., and K. Crawford
Formation of the blastoderm and yolk syncytial layer

7(>

Gileadi,

emergence

in

an
1

DEVELOPMENT AND REPRODUCTION

in early squid development

Crawford, K.
Lithium chloride inhibits development along the animal vegetal axis and anterior midline of the squid

Gibson, Glenys D.
Larval development and metamorphosis in I'l/'iixibranchaea maculata, with a review of development in

the Notaspidea (Opisthobranchia)

PJ1

179

181

embryo
Hill,

177

Susan D., and Barbara C. Boyer

HXK-1 N-CAM immunoreactivitv

iary patterns
Capitella sp.

correlates with

cil-

during development of the polychaete

182

ECOLOGY AND EVOLUTION

('.ill

and Irving L. Weissman

Effects of allogeneic contact on life-history traits of
the colonial ascidian Botryllm schlossen in Monterey

Chadwick-Furman. Nanette

E.,

Bav

Heck, D.
133

and D. K. A. Barnes
Effect of disturbance on assemblages: an example

BeU,

J. J.,

using Porifera

E.,

and

J.

D. Laskin

Rvanodine-sensitive calcium llux regulates motility of

Arbaciri punctulata
Gallant, P. E.

Axotomv
144

in the

sperm ......................

185

inhibits the slow axonal transport of tubulin

squid giant axon .......................

187

Delacruz, John, Jeremiah R. Brown, and George M.

Molina, Anthony J. A., Katherine Hammar, Richard

Sanger, Peter J. S. Smith, and Robert P. Malchow

Langford
Interactions

br-t'

squid kinesin
DeSelm, Carl f

George M. Lai
Ral>GDI
in.

ombinant

conventional

Intracellular release of caged calcium in skate hori-

188

zontal cells using fine optical fibers

Palmer, L. M., B. A. Giuffrida, and A. F. Mensinger
Neural recordings from the lateral line in free-swim-

190

Child, F. M., H. T. Epstein, A.

>id
its

i!

port

myosin-V
vaiiah R. Brown, Renne Lu, and
'c

invosin \'-dependent vesicle trans-

axon

^q.i,a giant

Pielak, R, M., V. A. Gaysinskaya,

ming

and W. D. Cohen

Memorv
192

Shribak, Michael, and Rudolf

Wollert, Torsten,

Ana

S.

DePina, Carl

J.

DeSelm, and

Mann, K.

and H. Ripps
An experimental approach to the study of gap-junc-

Apoptosis in Microrinna

immune

J.

Kuhns, M. M.

and Margaret T. Armstrong

clot:

binding of agents of the innate

Limuhis blood
1201

Isakova, Victoria, and Peter B. Armstrong

Imprisonment in a death-row cell: the fates of mi-

crobes entrapped in the Limit/in blood clot

Harrington, John M., and Peter B. Armstrong

203

Mate choice

rerio)

224

in zebrafish

(Danio

rerio)

analyzed with

225

MOLECULAR BIOLOGY, PATHOLOGY, AND MICROBIOLOGY

liposome-permeating activity from the surface of

the carapace of the American horeshoe crab, Limuhis
205

W. Goetz

Expressed sequence tag analysis of genes expressed

in the bay scallop, Argopecten irradians
Hsu, A. C., and R. M. Smolowitz

Scanning
epizootic
canus

jjolyphemus

(Danio

Mann, G. G. Rosenthal, and G.

Roberts, S. B., and F.

system to the fibrils of the

clot

juvenile zebrafish

video-stimulus techniques

199

prolifrra allografts

in

Gerlach
197

Tepsuporn, S., J. C. Kaltenbach, W.

Burger, and X. Fernandez-Busquets
B.,

222

Atema, and G. Gerlach

based on olfactory cues

Turnell, E. R., K. D.

J. Zakevicius,

death

D., E. R. Turnell, J.

Kin recognition
195

Armstrong, Peter
The decorated

220

Atema

to chemical stimuli in Hoinants americanus

cytes

cell

Savage, Anna, and Jelle

Neurochemical modulation of behavioral response

is

tion-mediated

E. Motta,

calexcitin in Hennissenda

194

required for myosin-II-mediated vesicle

transport during M-phase in extracts of clam ooCusato, K.,

218

M. Child, H. T. Epstein, M.
Oldenburg, and D. L. Alkon

Training alone, not the tripeptide RGD, modulates

George M. Langford
Rho-kinase

L.

reconsolidation in Hermssenda

Kuzirian, A. M., F.
C. E.

Oldenbourg

Three-dimensional birefringence distribution in reconstituted asters of Spisula oocytes revealed by

scanned aperture polarized light microscopy

21(i

M. Kuzirian, and D.

Alkon

Cytoskeletal events preceding polar bodv formation

in activated Spisula eggs

toadfish, Opsriiiu\ inu

215

electron
lobster

microscopy

shell

disease

in

227

of
investigation
Homarus ameri-

228

Orchard, Elizabeth, Eric Webb, and Sonya Dyhrman

Characterization of phosphorus-regulated genes in

230

Trichodesmium spp
Galac, Madeline,

Deana Erdner, Donald M. Anderson,

and Sonya Dyhnnan

Molecular quantification

NEUROBIOLOGY AMI BEHAVIOR

I'IIM-

Sangster, C. R.,

Bogorff, Daniel
chow, and Peter

J.,

Mark

J. S.

Smith

233

onis.

a self-referenc-

207

ing glutamate-selective micro-biosensor

Chappell, R. L., J. Zakevicius, and H. Ripps

209

oocytes

Chambers, R. Eseh,

Zinc chelation enhances the sensitivity of the

b-wave in dark-adapted skate retina

Hemant M. Chikarmane, Roxanna

in

Opsanus tauby

polvmerase chain reaction

Weidner, Earl, and Ann Findley

235

Catalase in microsporidian spores before and during

236

discharge

Transient use of tricaine to remove the telencephalon has no residual effects on physiological recordings of supramedullary/dorsal neurons of the cunner, Tautogplabrui adspersus
Redenti, S., and R. L. Chappell

Baird, Krystal D.,

Smolowitz, and Kevin R. Uhliiiger

Detection of Edwardsiella infections

Zinc modulation of hemichannel currents in Xenopits

Zottoli, S. J., O. T. Burton, J. A.
L. M. Gutierrez, and M. M. Kron

231

Description of Vibrio alginolyticus infection in mltured Sepia officinalis, Sepin tipamn, and Si'pi/i phara-

A. Messerli, Robert P. Mai-

Development and characterization of

ot toxic Alexundriiim fundf-

Maine
and R. M. Smolowitz

in the Gull of

ECOLOGY

A.\<D

POPULATION BIOLOGY

211

ERG
213

Agnew, A. M., D. H. Shull, and R. Buchsbaum

Growth of a salt marsh invertebrate on several species
of marsh grass detritus

238

Cavatorta, Jason R.,

Morgan Johnston, Charles Hopkin-

Morgan,

son, and Vinton Valentine

Patterns of sedimentation in a

I.

salt

marsh-dominated

Thonis, T., A. E. Giblin, K. H. Foreman

242

I.

Valiela

and relationand trophic position of the Atlanhorseshoe crab, I./i/iii/u\ /mly/ilti'iiius, within Cape

Cod

254

estuaries

Walker, C., E. Davidson, W. Kingerlee, and K. Savage

Incubation conditions of forest soil yielding maximum dissolved organic nitrogen concentrations and

245

minimal residual
Holden, M. T., C.

256

nitrate
Lippitt, R. G. Pontius, Jr.,

and C.

Williams

Importance of metabolism in the development of salt

marsh ponds
I.

Carmichael, and

tic

244

Johnston, M.
V. Valentine

Agniar, A. B.,

252

H.

ships between size

Abraham, D. M., M. A. Charette, M. C. Allen, A. Rago,

and K. D. Kroeger
Radiocheniical estimates of submarine groundwater
discharge to Waquoit Bay, Massachusetts
E., J. R. Cavatorta, C. S. Hopkinson, and

lactuca in estu-

Waquoit

Stable isotopic assessment of site loyalty

J.

Nitrogen flux and speciation through the subterranean estuary of Waquoit Bay, Massachusetts

and nutrient supply on

Bay, Massachusetts
O'Connell, C. W., S. P. Grady, A. S. Leschen, R.

M., K. D. Kroeger, A. Rago, M. C. Allen, and

M. A. Charette
Talbot,

M. Teichberg, and

growth of the green macroalga Ulva

aries of

Multiple approaches to tracing nitrogen loss in the

\\Vst Falmouth wastewater plume

A. B. Aguiar, S. Fox,

Relative influence of grazing

239

estuary

J. A.,

Valiela

J.

Building a database of historic land cover to detect

248

257

landscape change

A. Morgan, M. Teichberg, S. Fox, and

Valiela

ORAL PRESENTATIONS

Transplantation and isotopic evidence of the relative

effects of ambient and internal nutrient supply on
the growth of L'lva lactuca

250

Published bv

title

onlv.

259

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Reference: Bid/. Bull. 205: 93-97. (October 2003

2003 Marine Biological Laboratory

Cascading Trophic Impacts of Reduced Biomass in the

Ross Sea, Antarctica: Just the Tip of the Iceberg?
BRAD

A.

SEIBEL*

AND

Montere\ Ba\ Aquarium Research

in the

austral

due

to the

food link between

in sea-ice retreat

presence of an immense iceberg, B15

(1) (Fig.

Reduced phytoplankton stocks

clearly affected

pteropod Limacina helicina (Phipps. 1774} (Gastropoda: Mollusca), an abundant primary consumer in the

tween

and C.

antarctica are important

polar ecosystems and

"
ecosvstem "health in

L.

components of

Southern

50<7r to

CT

in

is

known

terminants of metabolism
(3).

and

is

to be

in all

ence,

among

among

L. helicina

the primary de-

organisms, including ptero-

especially important in the highly seasonal

Antarctic environment (9. 10).

Flagg Road. Biological Sciences Center. Biological Sciences Department.

University of Rhode Island. Kingston. RI 02881. E-mail: seibel@uri.edu
t Current address: Department of Marine Sciences. University of Con-

1080 Shennecosset Road, Groton.

a (Chi) concentrations, and high

was observed

Nutritional state

Received 20 November 2002; accepted 21 July 2003.

* To whom
correspondence should be addressed. Current address: 100

Point.

reduction in phytoplankton biomass, es-

by a series of metabolic measurements made on

and C. antarctica between 1999 and 2002.

is

pods

Averv

75%

the region, causing cascading effects through higher trophic

levels in the following year. This assertion is supported here

linked strongly to the seasonal oscillations in the extent of

the sea ice (6. 7) and survival of higher trophic levels is

necticut at

with the

). We believe that the

only 60% of the previous year
reduced phytoplankton stocks in 2000-2001 had pronounced impacts on the condition of primary consumers in

consumers may be further

Ocean, phytoplankton production

in concert,

and C. antarctica provides a unique

study the ecological and trophic conse-

still

impacted.
the

They grow

December 2000-2001 relative to previous years (Table 1; Fig. 2: 8). A limited bloom
did form by February, but annual primary production was

In this last austral summer. 2002-2003, sea-ice

was much higher and phytoplankton stocks were
dramaticall\ lower than any reported previously, effects
possiblv associated with El Nirio conditions, and we hypoth-

In

of

helicina

sea-ice cover

extent

their

timated as chlorophyll

may be good indicators of overall

McMurdo Sound and perhaps in the

and

web

Sea.

Ross Sea.

esize that pteropods

helicina

opportunity
quences of a depression in primary productivity in the Ross

1902). an abundant pteropod mollusc (Gastropoda) that

(5). Metabolic rates ofC.

helicina

life histories.

to

feeds exclusively on L. helicina

2001-2002. Both

ment requires precise timing to ensure that predator and

prey coexist. The coevolved predator-prey relationship be-

was absent from McMurdo Sound. Manyimportant predators, including whales and fishes, relyheavily on L. helicina for food (3, 4). However, most obviouslv impacted by its absence was Clione antarctica (Smith,

in

C. antarctica.

preferred prey size increasing with predator size (3). Such

specificity within the context of a highly seasonal environ-

record. L. helicina

50%

and

water column by extending a

that traps

have parallel

region (2, 3), as indicated by depressed metabolic rates in

2000-2001. The following season, for the first time on

were depressed by

L. helicina

in the

phytoplankton and. to a lesser extent, small

zooplankton (3). L. helicina is the exclusive food source of
C. antarctica throughout the life cycle, and the two species

mucus

the

antarctica

and feeds

lives

}.

Our obsen'ations in McMurdo Sound suggest temporally

and trophically cascading impacts of that depression in
productivitv.

95039

California

dependent on reproductive cycles that are synchronous with

phytoplankton blooms. This is especially true of the direct

summer of 2000-2001,

a possible consequence of a disruption

Moss Landing,

Institute,

significant reduction in phytoplankton biontass in the

Ross Sea was reported

HEIDI M. DIERSSENt

Food

availability will influ-

other things, the rates of protein synthesis,

oxygen consumption, growth, and reproduction (9-1

1).

We

collected L. helicina and C. antarctica at four sampling

stations along

06340.

93

Ross Island

(Fig.

and measured the oxygen

94

B. A.

SEIBEL

AND

H.

M. DIERSSEN

C. which

1.86

McMurdo
helicina in

is the year-round ambient temperature

Sound. The oxygen consumption rates of
2001 were reduced by more than 30% relative

those measured

1999 (Table

in

to

This reduction was

1).

food deprivation due to reduced

we cannot rule out a possi-

result of

presumably a

in
L.

phytoplankton stocks, although

ble additional influence of changes in food quality

species composition

may

The following season, phytoplankton stocks were

2001).

elevated; but for the

50%

are only

We

L.

station sampled.

any

was heavily
Sound. The
species in 2002

predator, C. antarcticu

the absence of

oxygen consumption
Fig. 3).

at

monophagous

impacted by

time on record (see below),

first

was not found

helicina

As

(i.e.,

also have changed from 1999 to

its

prey

in

McMurdo

measured for

rates

of those measured

in

this

previous years (Table

1;

also conducted laboratory experiments in 2001

which specimens of C. cmiarctica were deprived of food

weeks. Over the first 14 days, metabolic rates declined
gradually to about 50% of control (wild-caught and laborain

for 3

The 2002 rates correspond closely

of individuals deprived of food in 2001, strongly

tory-fed animals) levels.

to those

supporting the suggestion that the depressed rates resulted

from the extended absence of L. helicina in the region.

True-color imagery of McMurdo Sound and the iceberg

Ross Sea. Antarctica, on 26 December 2001. Imagery is from
the Moderate Resolution Imaging Spectroradiometer (MODIS) (33) at

Figure

B ISA

250-m

1.

resolution. Sites on

collected are

and

C.

in the

marked

1.

Ross Island where pteropod specimens were

McMurdo

Station; 2.

Cape Royds;

3,

Cape

Bird:

many

polar zooplankton (14.

15).

(5% wet mass) during

lipid stores

the

released the following spring (16). With a depressed metabolic rate of 0.99 /Limol (0.022 ml )O 2 g~' h~' (Table 1 ), an
oxy-calorific conversion of 4.7 kcal 1~'

consumption rates of both species in January of 1999. 2001.

and 2002. using end-point analysis as described previously

The measurement temperature

in all

analyses

content of 9.4 kcal g~

was

nearly 6 months on

Table
Impacts of reduced hiotnass

tin

trophic dynamit

chlorophyl a ling

m"

')

in the

'

lipid, a

lipid

O2

and an energy

100-mg animal could survive

alone,

but

at

the expense of

I997-199X

Mean

like

productive spring and summer months, presumably for survival through the winter and production of eggs that are

4. ice edge.

(12, 13).

antarctica.

accumulates large

1998-1999

1999-2000

2001-2002

2000-2001

2002-2003

Western Ross Sea

(See Fig. 2 for details)

December

2.1

3.9

3.4

1.0

5.4

0.56

January

1.6

1.5

3.1

2.2

3.4

0.56

December

0.72

0.50

0.31

0.66

0.30

0.88

January

0.52

0.29

0.16

0.57

0.20

0.78

Fraction of the Western Ross Sea covered with sea ice

[See Fig. 2 for details)

Oxygen consumption
data pooled from

rate (jumol
all

O2

g~'h~'), mean

collection sites (Fig.

SE();

:
1

Limacina

n.d.

5.51

0.4(12)

n.d.

3.78

0.20(22)*

Clione

n.d.

1.93

0.21 (10)

n.d.

2.04

0.12 (31)

0.96

0.10(7)*

Clmne
1

Sea

n.d.,

starved

ice

cover determined as the fraction of Western Ross Sea not covered by open water, as shown in Fig. 2.
that oxygen consumption rates were significantly different from those in 1998-1999 (P

no data; "indicates

<

absent

0.99

0.01).

0.05 (30)*

present

present

TROPHIC IMPACTS OF REDUCED B1OMASS

A) Dec. 1997

THE ROSS SEA

IN

B) Dec. 1998

C) Dec. 1999
1

0.5

-0.5

-1

Figure
at

9-km

2.

(mg

70 e

180 E

<7n

"

1.5

log chlorophyll a

95

3
)

Ross Sea chlorophyll a (Chi) concentrations, representing the monthly mean of sea-ice-free pixels
from satellite ocean color imagery obtained from Sea-viewing Wide Field-of-view

resolution, derived

Sensor (SeaWiFS; Level 3 Standard Mapped Image, Reprocessing #4) (33) for December 1997 (Al-2002 (F).
Gray areas designate land and white areas indicate the presence of sea ice. The dashed magenta line represents
Ihe average extent of sea ice determined

The sea
of the

reproduction.
tion

ice extent

B15A

and Chi data reported

iceberg

is

shown

from passive microwave satellite data (SSM/I NASA Team Algorithm).

in Table 1 were determined from the area within this line. The location

as a solid

magenta shape.

positive correlation between egg produc-

and availability of food

(i.e..

Limacina) has been dem-

onstrated in the laboratory for C. limacina (3).

L. helicina is typically abundant throughout the Southern

ocean-atmosphere feedback loops. The state of pteropod

populations is almost certainly indicative of overall ecosys-

tem "health"
the

McMurdo

in

Sound, and perhaps throughout

Ross Sea.

Ocean, sometimes displacing krill as the dominant zooplankton (17). In McMurdo Sound. L. helicina may constitute more than 20% of the zooplankton biomass and reach

at all

concentrations exceeding 300 individuals per cubic meter

along the ice edge ( 18, 19). L. helicina is also an important

reported in McMurdo Sound in every systematic zooplankton sampling study to date (2, 5, 18. 19, 25, 26). The

prey item for a number of other species in the Antarctic,

including whales and myctophid and notothenioid fishes (4,

Antarctic Biology Training Course sponsored by the U.S.

National Science Foundation also confirmed an abundance

20), themselves important components in the diet of penguins and mammals (21, 22). Although Clione limacina, the
northern hemisphere congener of C. antarctica, has also

of

been reported in the diet of fishes and whales (3), C.

antarctica may have limited importance for higher trophic
levels in McMurdo Sound because it produces a novel

helicina in

"anti-feedant"

compound

(19).

However, both

L.

helicina

Large aggregations of both pteropod species were found

in January of 1999 and
four sampling stations (Fig.
1

2001. Equally large aggregations of both species have been

L.

helicina in

McMurdo Sound

every year of

its

operation

(1994-1996. 1999-2001; D. Karentz. University of San

Francisco. California, pers. comm.). Thus, the absence of L.

2001-2002 appears

to

be unprecedented

Murdo Sound, although we cannot

that L. helicina

Sea

in

Mc-

rule out the possibility

was recruited from other

parts of the

Ross

later in the year.

and C. antarctica are functionally important components of

the ecosystem with the potential to influence phytoplankton
stocks (18), carbon flux (23). and dimethyl sulfide (DMS)

life

levels (24) that, in turn, influence global climate through

abundant

The absence of
sulted

from food

cycle of
in

.5

late

L.

helicina in

2001-2002 may have

re-

limitation. In the Arctic. L. helicina has a

to 2 years,

summer

and veliger larvae are most

to early fall

(27).

Assuming

96

B.

SEIBEL

A.

AND

M. DIERSSEN

H.

conditions and thus contributed to the low biomass observed

2000-2001. Among the most compelling is that the

immense iceberg B15 prevented the retreat of pack ice out

in

of the Ross Sea, causing a reduction in open water and a

shortened growing season that delayed and stunted the

O
o
o

phytoplankton bloom (1). However, substantial interannual

variability exists in both sea-ice extent and phytoplankton
production. For example, both 1997 and 2002 had high ice

cover (Fig.

2;

Table

even though the iceberg was no

longer preventing the retreat of pack ice in those years.

f
o

Phytoplankton biomass was reduced somewhat in 1997, but

was dramatically reduced in 2002 (mean chlorophyll con-

centration of 0.56

mg

3
).

Interestingly, both 1997

2002 experienced El Nino events

that are

known

and

to influ-

ence Antarctic waters (30).

-H

Continued monitoring is required to assess the causes of

variability in Ross Sea phytoplankton stocks, the role of sea

-H-

0.01

Mass
Figure

3.

Oxygen consumption

rates of Clione antarctica plotted as a

function of wet body mass. All rates were measured

at

.86

C, the

year-round ambient temperature in McMurdo Sound. The rates from ani028

mals captured in 2002 (open circles, y = 0.43.v~
) were significantly

lower than those measured

(grey

deprived of food

measured

in

2001 (black

(ANCOVA; P <

circles)

in

0.01).

circles,

= 0.93*"-*)

Consumption

laboratory in 2001

the

the Southern

ice in

(g)

are

rates

(31, 32),

and thus primary productivity, with consequences

or 1999

of animals

Acknowledgments

similar to those

in

We

thank

B. Robison,

helicina in the region.

J.
J.

Smith, and S.

life

history for L. helicina in the

Ross Sea, veligers

may not have metamorphosed and grown to adult sizes

by summer 2001-2002. Relatively short delays in food
availability are known to lead to failed metamorphosis of
larval zooplankton (28). Unfortunately, we have no data
outside of McMurdo Sound in 2002. An alternative hypothesis is that L. helicina was simply excluded from McMurdo
there

Sound by changes

in the local

currents due to an

immense

which ran aground along

2000-2001
(Fig. 1 ). The iceberg and associated ice cover in 2001-2002
may have prevented the typical flow of water from the Ross
Sea gyre around Cape Bird and southward into McMurdo
iceberg, B15. a large fragment of

the eastern edge of

Sound

(29).

absence of

and

Ross Island

this

comments on

the

improved this manuscript. We thank Raytheon Polar

Services and the National Science Foundation
Office of
Polar Programs for facilitating and funding this work. This

work was

additionally

Aquarium Research

supported by the Monterey Bay

and the NSF-sponsored Ant-

Institute

Biology Training Course and its instructors, including

A. Marsh. D. Karentz. G. Somero, G. Hoffman, C. Mar-

arctic

shall, L.

Goff, and D. Manahan.

Literature Cited
1

more localized

3.

the position of the

2000-2001 and 2001-2002, but

it

is

more

Stanford,
4.

recent observations. Substantial populations of both C. cintarctica and L. helicina were found in McMurdo Sound in

2002-2003 (Luke Hunt. Hopkins Marine Station, pers.

comm.) even, though the iceberg continues to block the
mouth of McMurdo Sound.
A number of factors may have influenced the sea-ice

Time and depth comparisons of sub-ice zooplankton in McMurdo Sound. Antarctica. Polar Biol. 9: 431-435.
Lalli, C. M., and R. W. Gilmer. 1989.
Pelagic Snails: The Biology
Foster, B. A. 1989.

of Holoplanktonic Gastropod Molliixks, Stanford University Press.

iceberg between

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Reference: Biol. Bull. 205: 98-101. (October 2003)

2003 Marine Biological Laboratory

Thermal Tolerances of Deep-Sea Hydrothermal Vent

Animals From the Northeast Pacific
RAYMOND

W. LEE

School of Biological Sciences, Washington State University. Pullman, Washington 99164

Dense biological communities on

suljide structures at

deep-sea hvdrothermal vents survive in one of Earth's most

extreme environments. The thermotolerance of vent animals
siilfide chimneys in the Northeast Pacific was
determined b\ maintaining them in pressurized chambers
under controlled temperature and chemical conditions. Ob-

dwelling on

sen>ations

indicated

that

lethal

temperature

correlates

strongly with distributions observed in nature. One species

studied, the alvinellid siilfide worm Paralvinella sulfincola,

exhibited a thermal limit of 50-56

C. Since observations

of

survival under controlled conditions are the only unambig-

uous means of demonstrating that an animal can tolerate a

given environmental condition, the documented thermal

of these conclusions

For animals inhabiting less remote

(2).

environments, corroborative evidence has often

come from

laboratory investigations of live animals. Such an approach

has not been extensively used in studies of vent animals due

requirement that experiments be conducted at high

pressure. However, this kind of evidence is necessary to
to the

determine actual physiological limits. Documented survival

under controlled conditions provides unambiguous evidence
for thermotolerance. In the present study, this type of direct

approach was taken

thermal tolerance of

to investigate the

several species of vent animals.

Some of the sulride-chimney assemblages

Fuca

at

the Juan de

and Explorer ridges in the Northeast Pacific are ideal

for metazoan life at hydrothermal vents should be

considered to be above 45 C, but less than 60 C.

for investigation of environmental tolerance since the

Although the biology of hydrothermal vents has been

actively investigated over the past 20 years, delineating

are small

linkages between the physical environment and the biota

moderate, making

Gradients and temporal changes are pronounced. Therefore, to know what conditions a vent organ-

experiments, and most organisms are motile, allowing behavioral investigations. In the present study, thermal limits

ism routinely encounters, measurements would ideally be

conducted with spatial resolution at the sub-centimeter

were investigated

limit

has been

difficult.

with temporal resolution over the course of days to

weeks, and without modifying fluid flow by the presence of
the sensor or submersible. Consequently, investigators are
level,

generally cautious in inferring physiological tolerance from

environmental measurements. A recent study presents en-

vironmental data suggesting that Alvinel/a pompejana. a

vent-chimney alvinellid worm, lives under sustained temperatures of

60

C, which could

thermotolerant metazoan

).

make

it

the Earth's

However, steep thermal

most
gra-

dients and the difficulty of sampling fragile alvinellid tubes

from a submersible have raised questions about the

validity

inant invertebrates, unlike those

and can

relatively

inexpensive pressure

vessels. Pressures at these sites (depths

it

1500-1800 m)

are

easier to maintain in situ pressure in

for four

abundant species of chimney

and

invertebrates: the paralvinellids Paralvinella sulfincola

P.

pahnifonms, the limpet Lepetodrilus fucensis, and the

snail

Depressigyra globulus.
distributions of these organisms on vent chimneys
were described by Sarrazin et al. (3) and exhibit a "zona-

The

tion" pattern of distinct invertebrate assemblages

through V) that differ

tics (3).

Assemblage

in
I.

(named

temperature and flow characteris-

closest to hot vent fluids, consists

almost entirely of P. sulfincola, which suggests that this

species may be the most thermotolerant metazoan at Northeast Pacific vents.

blage

II. is

assemblage

The second warmest assemblage, assem-

dominated by

The gastropods
Received 3 March 2003; accepted 24 June 2003.

in

fit

dom-

from other vent systems,

II.

P. sulfincola

and P. palmiformis.

fucensis and D. globulus are also found in

but are more common, and dominant, where
L.

Illl

the influence of venting

is

RMAL TOLERANCES OF VENT ANIMALS

weaker (assemblages

III-V).

It is

not clear what factors govern the distribution of organisms

on .sulride structures. It has been postulated that tolerance to

1.

3.

99

d
*

active
active/not active after

2.

reduced

4.

not active

activity

abiotic factors such as high temperature or

hydrogen sultide
be
may
important regulators, but analysis of available environmental data and faunal distributions indicates that less

P. sulfincola

than 3()9r of the variance in species distribution can be

P.

palmiformis

accounted for by abiotic factors that have been measured so

far (4). If thermal tolerance is a factor governing distribu-

D.

globulus

OO

thermotolerance should be highest in P. sulfincola. followed by P. palmifimnis, then L. fucensis and D.

L.

fucensis

O OOO D

AA

tions, then

globulus. Specimens of P. sulfincola were collected from

assemblage I, P. palmiformis from assemblage

and gastropods from assemblage III. Differences
tolerance

among

collections were not tested for.

that further study

could reveal acclimation

II

and

in

thermal

It is

III,

10

Figure

Observations of mixed assemblages of these species in

pressurized chambers subjected to temperature increases are
summarized in Figure 1. Each data point shown represents

outcome of a single experiment in one pressure chamThe following endpoints were measured:
activity at
(

60

Effects of experimental temperature on activity of

1.

of activity following return to low temperatures; (3) activity

at experimental temperature, but no activity at higher temperatures and no activity after return to low temperature in
all individuals; or (4) no activity in all individuals, with no
return of activity at lower temperatures. In

ROPOS

The

chimney

submersible was used to collect animals from

on the Explorer Ridge at a depth of 1800 ms. Immediately

upon arrival at the surface, animals were placed in 30-ml pressure chambers and returned to an in situ pressure of 2600 psi. Depressurization during
sultide structures

recovery was unavoidable since pressurized recovery systems are only

the developmental stages. Within minutes after animals were placed

in
in

experimental temperature by one or more individuals, with

continued activity following return to low temperatures; (2)
obvious reduction or cessation of activity, with restoration

category 2

50

degrees Celsius

likely

invertebrates.

ber.

40

30

to microhabitat

conditions.

the

20

some

cases,

was not

readily observable; i.e., D. global us

to
exhibit
appeared
high activity or none at all. In some
trials, the threshold temperature at which activity ceases was

not monitored. In these cases, animals exhibited no activity

at a given temperature, but the threshold may have been

pressure chambers, activity was generally observed. Animals not repressurized and kept at
aim also exhibited activity, but appeared to be less
I

active than pressurized animals. For experiments, high-pressure liquid

chromatography pumps were used to continuously perfuse (0.3 ml/min) the

chambers with filtered seawater equilibrated with 20% oxygen at pH 8.
Sulride

was metered

liter"

Chamber temperature was

'.

culating waterbath. Temperature

2-3

h.

then ramped

in

experiment

and 40

two or three experimental temperaexperiments and all P. siilfincola

until

thermotolerant, with reduction or cessation of activity be-

same

rate as

identical unpresexperimental cham-

text.

4 are

to

Experiments consisted of a temperature increase

to

10-15

C.

C. D. globulus and P. palmiformis exhib-

ited reduction or cessation of activity in the ranges of

individuals exposed to

in nature. The temperature above which

activity
ceased (with no recovery at lower temperatures) was interpreted to be the thermal limit. L fucensis was the least

at the

one pressure chamber. Behavior categories

followed by return of temperature

was

served

for

through 1.25-cm diameter viewports, either by using a video camera or by

Each data point represents the outcome of a single

and 2 of 8 experiments were from

one temperature increase could have resulted in lower thermal limits for activity in those experiments.
Thermal tolerance was correlated with distributions ob-

to experimental temperatures.

direct observation.

mis, 9 of 23, 8 of 17,

experiments consisted of a single elevated temperature exposure). It is possible that exposure of animals to more than

C/h

against a NIST-traceable digital thermometer). Activity

tween 30 and 35

ture increases; all other

a rate of 10

recir-

programmable
10-15

initially at

Yellow Springs Instruments temperature probe (calibrated

was monitored

bers, using a

some

temperatures) were conducted on the same sets of

individuals (for D. globulus. L. fucensis. and P. pabnifor-

was maintained

surized control chamber, perfused

described in the

cases, multiple experiments (at sequentially higher

at

of 100-200 micromoles

controlled using a

Chamber temperature was determined by monitoring an

lower. These instances were designated as category 4. Experiments consisted of a temperature increase (10 C per
hour) followed by return of temperature to 10-15 C. In

maximum

to give concentrations

in

respectively.

The

sultide

worm

35-40

P. sulfincola

clearly highly thermotolerant. Activity did not cease

temperatures of 50-56 C were achieved (Fig. 2; 7/20,

7/28 expts.). P. sulfincola survived sustained exposure to 45
C on the order of h or longer (Fig. 2; 8/2 expt.). This
1

indicates that temperatures of

exposure

to

activity at

50

or above, rather than

lower temperatures, accounted for cessation of

50-56 C

(Fig. 2; 7/20, 7/28 expts.).

video from experiments can be found

at

Time-lapse

(http://www.wsu.

edu/~rlee/sulfideworm/psulf.html).

These findings are consistent with, and may account

for,

the distributions observed in nature, and indicate that these

organisms inhabit microenvironments close to

their thermal

100
70

THERMAL TOLERANCES OF VENT ANIMALS

Coast

and

Polar

Regions

Undersea

National

Research

7.

Dahlhotf, E., and G. N. Somero. 1991.

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S. C.. T.

J. Stein. 1998.

Worms

bask

in

extreme

Choaldonnr.

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Jnllivet. K. Zal.

"Pompeii worms."
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Sarrazin.

J.,

C. R. Fisher.

J. J.

and A. Toulmond.

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Childress, D. Desbruveres, D.

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and

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1490-1508.

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D. Jollivet, P.

Desbruyeres, and

M. Sarradin,

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1992.

Dahlhoff, E., J. O'Brien, G. N. Somero, and R. D. Vetter. 1991.

Temperature effects on mitochondria from hydrothermal vent invertebrates: evidence for adaptation to elevated and variable habitat tem-

Gaill, F.,

Idrissi Slitine, J.

Engel. 1991.

Sarrazin.

Chevaldonne, P., D. Desbruyeres, and J. J. Childress.

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Hemoglobin from

Extracellular hemoglobins of hydrothermal vent an-

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A.. F. El

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Reference: Bio/. Bull. 205: 102-109. (Month 2003)

2003 Marine Biological Laboratr: v

Light Production by the Arm Tips of the Deep-Sea

Cephalopod Vampyroteuthis infernalis
BRUCE

ROBISON*, KIM

H.

REISENBICHLER, JAMES

R.

AND STEVEN
Monterey Bay Aquarium Research

The

Abstract.

this infernalis

600

off

archaic, deep-sea cephalopod

occurs

in

Monterey Bay. California. Living specimens,

undescribed means of bioluminescent

from the arm

particles is released

are

apparently linked to

the

tip-lights is readily

educed by contact

stimuli, while fluid

the light they produce

is

dim

or absent altogether,

observed

impression

and

oxygen content
to

is

low,

6C

in its natural habitat,

Vampyroteuthis has

is

somewhat misleading. Manipulation in situ

body is very soft, with

in the laboratory reveals that its

is

1996: Seibel et

al.,

1997. 1998. 1999).

Vampyroteuthis has been reported to feed upon copepods,

prawns, and cnidarians (Young. 1977: Nixon. 1987). but
dietary evidence is very scarce. In the laboratory, members

Introduction
is the lone
Vampyroteuthis infernalis Chun, 1903 (Fig.
occupant of the cephalopod order Vampyromorpha. Its
unique morphological characteristics, combining features of
1

species

watery tissues and little dense musculature. It has a very low

metabolic rate and lives at extremely low oxygen concentrations, yet it is capable of relatively high swimming
speeds, relying on its fins rather than jet propulsion (Hunt,

incorporated into behavioral patterns.

of this species will take euphausiids and pieces of fish when

the food is placed in contact with the oral surface of the

although this is hardly natural feeding. In turn.

\'amp\n>teuthix beaks have been reported from the stom-

arms,

both the octopodiformes and decapodiformes, suggest that it

represents an evolutionary position intermediate between

achs of large, deep-diving fishes, pinnipeds, and whales, and

from benthopelagic fishes (e.g., Pearcy and Ambler, 1974;

two groups (Young, 1977: Healy, 1989); and it may be

relic from an ancestral cephalopod line (Pickford, 1946).

the
a

sunlight

When

production. This paper presents observations on the structure and operation of the arm-tip light organs, the character

how

status of the

the appearance of a robust and substantial animal, but this

much

of the luminous cloud, and

the enigmatic

1998).

centered around 0.4 ml/1.

higher triggering threshold. Coelenare the chemical precursors of light

luciferase
terazine and

expulsion has a

(Young

et al,

have found Vampyroteuthis throughout the year at depths

between 600 and 900 m and at oxygen concentrations

form a glowing

tips to

modes of light production

anti-predation strategies. Use of

to

California 95039

(Pickford,
and temperatures range from
1946). In waters over the Monterey Submarine Canyon, we

luminous

cloud around the animal. Both

about 2

expression for the species. In the first, light is produced by

a new type of organ located at the tips of all eight arms. In
the second, a viscous fluid containing microscopic

Vampyroteuthis inhabits temperate

and tropical waters of the Pacific. Atlantic, and Indian
Oceans, typically between 600 and 1200 m. At these depths,

dark, oxygen-poor waters below

collected gently with a remotely operated vehicle (ROV)

and quickly transported to a laboratory ashore, have re-

vealed two hitherto

Moss Landing,

/?</..

which only adds

Vampyroteu-

HUNT

HADDOCK

H. D.

7700 SamihoUlt

Institute,

C.

Antonelis

This phylogenetic issue has not yet been clearly resolved.

et al.,

1987; Fiscus et

al.,

1989; Clarke et

al..

1996; Clarke and Young. 1998; Drazen et al.. 2001 All but
the whales are visually cued predators with large eyes that
).

Received 10 February 2003; accepted 15 July 2003.

* To whom
correspondence should he addressed.
mbari.org
Current address: University of
04005.
Biddeford,
1

New

England.

function effectively in dim. monochromatic light.

E-mail:

Hills

robr@

Cephalopods. particularly deep-water squids, employ a

diverse suite of light-producing organs that can occur on the

Beach Rd..

ME

mantle,
102

fins,

arms, tentacles, head, eyes, viscera, or else-

\:\MPYROTEVTHIS BIOLUMINESCENCE

103

1.
Vampyroteuthis infenhili.\. frame grab from high-definition video footage shot at a depth of 717 m
= 10.2 cm. The fin-base photophore is located behind the dark patch
Monterey Bay. California. Mantle length
of skin, posterior to (to the right of) the fin. The composite organ is the small, elongate white patch dorsal to and

Figure

in

on a

line just

behind the eye. Minute epidermal organs are scattered over the surface of the mantle and the arms,
The new arm-tip light organs described here are located on the oral surface of the filamentous

but not on the web.

portion of each arm, beyond the margin of the web.

where, depending on the species (Herring, 1977). The light

is used for attracting prey, deterring predators,

light produced by organs at the tips of all eight arms, and

luminous fluid released by the arm tips.

and presumably for intraspecific communication. Luminous

secretions are found in a number of deep-living inverte-

Materials and Methods

they produce

brates but are rare

among cephalopods and

fishes (Herring,

1977. 1988).

Three types of light-emitting organs have been described

in

Vampyroteuthis infenuilis:

phores

at

large, paired,

complex photo-

the bases of the fins; small, simple, epidermal

organs scattered over the surface of the animal; and comtwo clusters of small, pale nodules located
posite organs
dorsally on a line just behind the eyes (Pickford,

1949).

Light production has been observed only from the fin-base

photophores; emission spectra of these organs were measured at 460 nm by Herring (1983) and 461-466 nm by

Widder

et al.

(1983). Herring et

al.

(1994) examined

all

In

situ

behavioral observations and quantitative video

surveys of meso- and bathypelagic cephalopods have been a

component of MBARI's midwater research program since

1991 (Hunt, 1996). The program

remotely operated vehicles, or

span

a 10-year time

roteuthis in

carefully observed 57 individuals of Vampysitu and have collected 18 to establish in labo-

Specimens in this study included adult males

and females with mantle lengths ranging from 7.9 to 12.1

ratory aquaria.

cm. All were gently collected with the ROV Ventana (Robison, 1993) at a time-series station 1600 m deep over the
axis of the Monterey Submarine Canyon. Field observations
and collections occurred under

dence, concluded that the composite organs are probably

extraocular photoreceptors. while the epidermal organs are

ROVs

They also found that the reflective surfaces in the light organs were collagen instead of the
iridisomal platelets found in other modern cephalopods. To
date, no one has observed light from the epidermal organs,

tainers

likely light producers.

based on the use of

we have

three organ types and, based on detailed histological evi-

most

is

ROVs. Over

was

full

illumination from the

four 500-W, broad-spectrum lights.

recovered, the animals

were placed

in

Once

the

ROV

darkened con-

and were quickly transferred to our laboratory

ashore. In the shoreside facility they were maintained in the
dark, at 4 to 6 C, in circular, 260-1 kreisel tanks (Hamner,

nor has there been any behavioral evidence of light sensitivity by the composite organs. We have discovered two

1990) for as long as 2 months.

Most of the specimens appeared to be temporarily
blinded by the vehicle's lights during capture. After several

new forms of bioluminescent expression

hours

in

Vampyroteuthis:

in the dark, they

responded

to point sources of white

104

B.

H.

ROBISON ET AL

by moving away, and by contracting the iris-like

sphincter muscle (Pickford. 1949) that surrounds the front
of the eye. Laboratory observations were made both under
light

red light and in

classified as Ge:

.ark, often with an

il

intensitier

image

,1+ according to the U.S.

Army Night
Vision Laboratory's criteria. Light production was recorded
with a variety of low-light video cameras.
For chemical assays of arm-tip light organs, we removed
the distal portions of arms from several specimens and used

them

either fresh or after they

nitrogen. Light output

measured with
in a

had been frozen

from each assay

Hamamatsu HC-124

listed

in

liquid

below was

photomultiplier tube,

custom-built integrating sphere, for at least 20 s.

To test for the presence of coelen-

Coelenterazine assay:
terazine,

we homogenized

individual

arm

tips in

500

/tl

of

by volume). One milliliterof

Tris
purified Oplophorus luciferase in a solution of 20
and 100
NaCl was injected into 200 jul of the sample
methanol (approximately

10:1

mM

mM

Mantle tissue with epidermal light organs and web

(which lacks the epidermal light organs) were also

solution.
tissue

assayed for the presence of this luciferin.

Luciferase assay: Sample arm tips to be tested for

addition caused no light output, indicating that a calciumactivated photoprotein was not involved. The test solution

and the

to

light

20

/A!

of coelenterazine in 0.5

/Ag/jul

described in Hastings et al. (1978), with the flavin reduced

by bubbling with H 2 gas in the presence of platinized
carbon. Cultured Vibrio han'eyi were used as positive con-

We

for this assay.

trols

MeOH,

production was measured. For negative con-

in

4C

Fluorescence microscopy: Autofluorescence images of

arm-tip light organs and ejecta were obtained with a Zeiss

Axioplan microscope using 10X and

under DAPI illumination.

from the web was homogenized, and the exwere added to methanol.

fixed using

osmium

tetroxide and

embedded

the

same region were

stained with uranyl acetate and lead

In the laboratory

we observed

that the tips of all eight

arms often glowed when an animal was handled

The

outward.

(Fig. 2).

bright blue lights usually appeared as a tight chain of

6 small discs, tapering in size distally along the oral

surface of each arm tip. Occasionally there was a different

to

pattern, in

which

two parallel lines

With a mild contact stimulus, the
outward, with the arm tips glowing.

the light appeared as

flared

With stronger prodding, the arms were

curled, writhing up

Frame grab from a low-light video recording, showing the glowing arm tips of Vampyroteulhis
The animal is oriented such that Us head and beak are directed toward the camera, with the arms and
to flare

Epon. Thick

citrate.

arms and web

Bacterial luciferase assay: Assays for luminous bacteria

web beginning

in

1-2 /Am) sections through the light-producing region of the

arm tip were stained with toluidine blue. Thin sections from
(

tracts

2.

Neofluar objec-

Electron microscopy: Material was fixed in 2% glutaraldehyde with 0.1

cacodylate buffer. Samples were post-

separated by a dark gap.

Figure

40x

tives,

trols, tissue

infernulis.

also tested for the presence of

samples of arm-tip exudate that were

for 2 weeks.
streaked on seawater agar plates kept at

luminous bacteria

Results

mM

was added

from the organs, and

the surrounding water followed the reduced flavin assay

lucif-

erase activity were extracted in an aqueous solution of 100

Tris pH 8.1 and 50
EDTA. Calcium chloride

mM

in the arm-tip light organs, the ejecta

VAMPYROTEUTHIS B1OLUMINESCENCE
over the head

105

the apex of the mantle, exposing the

and placing the glowing arm tips in a
the top. When an animal rolled the arms and

suckers and
cluster at

to

cirri

down

mantle back

tucked the arm

normal position, it frequently

the web, where they were

to their

tips

within

shielded from view. This behavior, which was observed

both

the

in

field

and

in

the

laboratory,

similar to a

is

nonluminous pattern seen in octopuses attacked by moray

(Hanlon and Messenger. 1996). The eight arm-tip light
organs of Vaiiiiiyniteiithis always glowed and dimmed sieels

multaneously. They flashed

glowed

to 3

times per second, or

one minute. The

steadily, but rarely for longer than

pulsing could include complete extinction of the light, or

just dimming, before returning to the previous level of
intensity. There is a dark, densely pigmented layer of skin

on the aboral surface and on the sides of each arm

the oral surface

The

is

tip,

but

generally unpigmented.

structure of the oral surface of the

arm

tips

continues

the basic pattern found along the entire length of the arm
a
series of central plates alternating with paired lateral plates

proximal and medial portions of the arm, the

support cirri, while the central plates are the

(Fig. 3). In the

lateral plates

bases for suckers (Pickford. 1949, plate VI. fig. 20). Plates,
cirri, and suckers get smaller toward the distal end; near the
tip. the plates bear mere rudiments. Proximally. the plates
are pale and opaque, but as they approach the distal tip they
become translucent. Within this window at the tip of the arm

are subdermal clusters of particles that impart an iridescent

When

green and yellow sheen to the plates.

the

arm

is

viewed from the

side, the central plates appear bulbous and

extend outward beyond the lateral plates (Pickford, 1949,

Figure 3.
showing the

Arm

tip

of a living specimen of Vampyroteuthis infemalis,

Light expressed from the central plates

alone may be the source of the pattern that appears as a
chain of discs, while light coming from just the lateral plates

cp

would show as

to patches of iridescent particles that are not present after

plate VI, fig.

the

arm

19).

The light-producing area of

parallel lines.

can be occluded by the edges of the dark skin

tip

distal

light-producing region, and patches of small, green

particles in the lateral plates that are associated with the

central plate; Ip

lateral plate; scale

bar

mm;

luminous

ejecta.

the arrows point

an extensive

luminous cloud has been created.

along both sides, which close together along the midline of

the oral surface. This means of controlling light output is
similar to that described for the arm-tip photophores of

stimulated to glow, the dense clusters of particles in the arm

tips were gone. The fluid matrix that bears the luminous

Tuningiu danae by Herring et

particles

al.

1992).

Given a strong contact stimulus to the arms or body, the

arm tips exuded a viscous fluid containing small glowing
particles.

As

arms swept up over the head and mantle,

luminous
To all observers, the light from the cloud was

the

the particles dispersed, enveloping the animal in a

cloud (Fig.

4).

much dimmer
were unable

than that of the

to

measure

cles released varied

its

and arm tips, but we

The number of parti-

fin lights

intensity.

from a few dozen

to several hundred,

usually related to the strength of the stimulus.

Cloud lumi-

nescence persisted for 2 to 3 min. and individual particles

glowed for as long as 9 min (Hunt. 1996). Once the particles

had gone dark,

stirring the water did not re-initiate lumi-

nescence. After several such displays, production of the

luminous fluid ceased, and while the arm tips could still be

viscous and somewhat sticky. Arm tips that

brushed across the inner surface of a kreisel during a bioluminescent display usually left behind a lingering streak of
light.

is

The

release of luminous particles often preceded an

escape response by the animal.

The chemical assays provided clear evidence of the presence of coelenterazine (luciferin) and luciferase in the armtip light

organs of Vampyroteuthis (Fig.

that these

compounds

5),

which indicates

are the basis for light production.

No

calcium-activated photoprotein activity was detected in any

assay. Small amounts of coelenterazine were found in the

mantle epidermal

tissue.

sion by Herring et

al.

These

results support the conclu-

(1994) that the epidermal organs

web
produce light. Assays
tissue were negative. The assav for bacterial luciferase in
for luciferin and luciferase in the

106

B.

H.

ROBISON ET

AL.

4.
Frame grab from a low-light video recording showing the release of glowing particles from the
of Vampyroteuthis infernalis. The head of the specimen is directed toward the camera. Particles in the
cloud are swirled by movements of the arms and web. and by water jetting through the siphon. "Tails" on the

Figure

arm

tips

glowing

particles are caused

was negative,

by electronic lag

in the

were the culturing efforts to

demonstrate the presence of luminous bacteria in the arm
tips and their exudate.
the tip lights

as

Microscopic examination of the iridescent clusters in the

tips of animals that had not yet secreted luminous

arm

material revealed extensive patches of rounded yellow par-

glowed blue-green under fluorescent illumination

No pores that might release the fluid were evident

ticles that

(Fig. 6).

on the arm
sites.

although the rudimentary suckers are likely

The particles matched, in size and configuration, partips,

ticles culled

from the arm-tip exudate and from the water

in

which a luminous cloud had been produced. Sections of the

arm tips showed a low-density central core with prominent
nuclei on the oral side and sparse muscle tissue on the
aboral.

We saw no evidence of an

iridosomal reflective layer

camera's image

intensifier.

together, they could operate independently of the other two

light sources. Light emission from the fin lights was regulated

by chromatophores and by

ilar to

those that shield the eyes.

glowed without

sim-

iris-like skin closures

The arm-tip

the fin lights also being on,

lights

and

all

seldom

10 could

concert. The luminous ejecta was never observed

pulse in
without the tip lights glowing as well.
On one occasion, male and female specimens were collected on the

same day and were then placed

in separate

darkened laboratory
kreisels less than a meter
was
disturbed
and began to flash
ashore. When the female
apart, in the

her arm-tip lights, the undisturbed male quickly and vigorously responded with tip-light flashes. This reaction was

repeated twice (Hunt. 1996).

ferential

light

We

saw no evidence of

dif-

production by females and males. In the

nor of layered collagen fibers like those found by Herring et

Cranchiidae and Lycoteuthidae, arm-tip photophores de-

1994) in the fin-base photophores.

A comparison of our specimens with others collected by
trawling in Monterey Bay and elsewhere in the North Pa-

We

al.

velop as secondary sexual characters (Herring et al., 1992).

detected no sexual dimorphism in the light organs of
Vampyroteuthis. Luminous suckers on the deep-sea octopus

may be used

for intraspecific

com-

revealed that, in almost every case, the arm-tip light

organs had broken off the trawl-caught specimens. This
observation is similar to that made on Octopoteuthis (Her-

Suini-ntciitlii.'i

1992) and may explain why the arm-tip light

of
organs
Vampyroteuthis were not discovered until we
could collect the animals in perfect condition. On two of our

lights of Vampyroteuthis. Although animals in kreisels reacted to point sources of artificial light by shading their eyes
with their arms and web, or by moving away from the light,

ROV-caught specimens, we found

the

cific

ring et

al.,

a short, apparently reeach

with
what
arm,
generated
appeared to be a small light
source at its tip.

Over

a gradient of stimuli, the fin lights

readily illuminated,

and although

this pair

were the most

always worked

syrtensis

munication (Johnsen

et al..

1999a.

the light organs in this species

is

b). but the structure

not

response of Vampyroteuthis to
included luminescence.

at all like

artificial

of

the arm-tip

light

never

Supplemental images (in situ video, laboratory low-light

stills, and electron micrographs) are available

video, digital

online

at

http://www.mbari.org/midwater/vamp.

VAMPYROTEUTHIS BIOLUMINESCENCE

107

behavior, such as chromatophore displays and unpredictable

protean behavior, is often coupled with locomotion in

cephalopod

escape

strategies

of

1996). In the darkness

its

substitute luminescence for

(Hanlon

habitat,

and

Messenger,

Vampyroteuthis

chromatophore displays

may
in

an

otherwise familiar cephalopod behavior pattern of deception, diversion,

and

flight.

Arm-tip light organs, which can be bitten or broken off

and then regenerated, may serve as sacrificial diversions for

<u

predators (Herring, 1977). Tip lights are found in several

deep-living squids such as Chiroteuthis and Octopoteuthis,

at

where they may also serve

as lures for prey, thus function-

escae of anglerfish and the barbels of stomiid

fishes (Herring, 1977; Young, 1983). Our observations of
ing like the

-A-NwJWV-^
1

Time

20

apparently regenerated light organs at the ends of shortened

arms in Vampyroteuthis may be evidence of their potential

(s)

as sacrificial structures.

The

displays indicate that there

characteristics of the arm-tip

direct neural control of their

is

luminescence.

The production of luminous clouds is common among

other deep-living pelagic invertebrates but rare in cephalo-

3
g.

pods. Anecdotal evidence for the production of luminous

clouds by squids was summarized by Young et al. 1979),
(

who

might be the luminous substrate. The only well-documented case is the sepiolid Heteroteuthis, which ejects a cloud of luminous particles when
suggested that renal

fluid

disturbed, presumably as a distraction to predators

(Herring, 1977). The ejecta is produced by glands within the
it

16

12

Time

(s)

is

mantle that contain dense populations of light-producing

bacteria, which are combined with ink and mucus during
through the siphon (Herring. 1988, 2002). The
emit light and have complex internal
themselves
glands
which
reflectors,
suggests that they have multiple uses (Herrelease

Figure

5.

Luciferin and luciferase assays for arm-tip light organs of

Vampyroteuthis infernalis. (A) Light produced by methanolic extracts upon

the addition of

Oplophorus luciferase indicates

the presence of the luciferin

coelenterazine. (B) Addition of coelenterazine to aqueous extracts

shows

high luciferase activity. Negative controls for both assays (omitted here for
clarity) did not deviate

measurably from the pre-injection baseline. The

higher noise associated with the coelenterazine assay is due to the higher
gain setting required to detect the presence of that molecule.

ring,

1988). Structurally and operationally, the release of

fluids appears to be a completely different process

luminous

Vampyroteuthis than it is in Heteroteuthis.

Visual trickery is common within the depth range and
light regime that Vampyroteuthis occupies (Robison, 1995,
in

1999; Herring, 2002), and our observations suggest some

ways that its luminescence may be employed.

additional

Discussion

Because the luminous

The

effect of arm-tip luminescent displays

on the dark-

adapted human eye is striking; coupled with the bright blue

light emitted by the two fin-base photophores, these displays produce a complex and dynamic visual

field.

The

fundamental question they raise is, how does Vampyroteuthis use the light? Because these responses can be elicited

by mechanical stimuli, we assume that production of light

from the arm-tip organs and the cloud of luminous particles

sticky,

it

initiate a

fluid released

would adhere

by Vampyroteuthis is
and might

to a potential predator

"burglar alarm" consequence by painting it with

that cannot be turned off or readily re-

bioluminescence

moved, thus making the attacker vulnerable to secondary

predators. A similar behavior has been described for the
bathypelagic
1992).

holothurian

Glowing

particles

Enypniastes
in

eximia

(Robison.

the ejecta might be used to

startling

prey such as copepods. which would then

become trapped by the viscous matrix. This function has

or distracting a potential predator, thus allowing for escape

been proposed for the twinkling bioluminescent suckers and

(Young, 1983). The visual predators that we know about are

all better swimmers than Vampyroteuthis, so its escape
strategy must rely on more than speed. Deceptive, deimatic

mucous glands of the

are elements of an anti-predation strategy based

on

attract smaller

(Johnsen

et

til.,

cirrate

199%).

and abundance of

It

octopus Stauroteuthis syrtensis

is

tempting to correlate the size

in the cloud with the epi-

light sources

ROBISON ET

AL.

Epitluore.sence micrograph of the oral surface of an

arm

108

B. H.

Figure

6.

patches of clustered particles.

their rudiments. Scale bar =

dermal organs of Vampyroteuthis. but

tip

of Vampyroteuthis

!ntfrihili.\,

showing

The dark
1

mm.

areas along the midline are in the central plates, which bear suckers or
Individual particles ranged from III to 15 /nm in greatest dimension.

we have never

seen

the latter luminesce.

ication of the

ROV

Ventana's pilots and the crew of the

R/V

of Vampyro-

Point Lobo.s. Coelenterazine, synthesized by S. Inoue, and

Oplophorus luciferase, purified by Y. Haneda, were kindly

unique among the known cephalopod bioluminescent systems. Likewise, the arm-tip light organs are structurally distinct from all others. Predator avoidance seems

provided by O. Shimomura. M. Haygood and J. W. Hastings

gave us advice on the bacterial luciferase assays. We thank
J. F. Case for a helpful review of the
paper. Supported by

The luminous
teuthis

the

secretion from the

arm

tips

is

most

likely function of the

luminous behavior we have

seen, but clearly, much is yet to be learned

these animals in their natural habitat.

the

David and Lucile Packard Foundation.

from observing
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Brain, behaviour and evolution of cephalopods.

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Young, R. E. 1983.
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C. F. E. Roper. K. Mangold, G. Leisman, and F. G.

Luminescence from non-bioluminescent tissues in

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Reference: Biol. Bui/. 205:

10-120. (October 2003)

2003 Marine Biological Laboraton

Patterns and Processes of Larval Emergence in an

Estuarine Parasite System
JONATHAN

T.

FINGERUT,

CHERYL ANN ZIMMER.

'*

Department of Biology,

Abstract.

Trematode parasites

in intertidal estuaries

tween hosts. Given the short

life

spans

emergence timing should be optimized

ex-

K.

ZIMMER

'

(<24
to

h) of cercariae,

Introduction

field

Parasite larvae typically disperse prior to finding and

As with propagules of free-living organisms, such as crabs (Forward ct ai. 1986; Morgan, 1996),
infecting a host.

sponges (Amano. 1988), and plants (Horn ct al., 2001;

Sehauber ct al., 2002), external cues may direct emergence
of parasite larvae under favorable conditions. Because dis-

species-specific temperature ranges, exclu-

sively during daylight hours, and only

when

snails

were

submerged. The three factors operate over different temposcales: temperature

and water depth

of California,

gence of trematode cercariae, mitigating the vagaries of an

intertidal environment.

enhance the prob-

of successful transmission. In the present study,

emerged over

Institute, University

exogenous and endogenous factors control emer-

gether.

measurements and laboratory experiments identified processes that regulate the emergence of cercariae from their
first intermediate snail hosts in an intertidal marsh. Larvae

riod),

AND RICHARD

Neurosciences Program and Brain Research

Los Angeles. California 90095-1606

perience constantly varying conditions, with the presence or

absence of water potentially limiting larval transport be-

ral

ability

persal stages of digenetic trematodes have life spans of 24 h

or less (McCarthy, 1999; Toledo et al.. 1999), timing emer-

monthly, light diurnally (24-h petidally (12-h period). Each stimulus

correspond with host availability would be espeadvantageous (Combes et ai, 1994; Pechenik and
Fried, 1995). Moreover, the widespread distribution of
trematodes in fresh water (Pages and Theron, 1990; Gerard,

gence

creates a necessary condition for the next, forming a hierarchy of environmental cues. Emergence as the tide floods

to

cially

would favor transport within the estuary, and light may

trigger direct (downward or upward) swimming toward host
habitats. Abbreviated dispersal would retain asexually re-

2001) and saltwater (Martin, 1972; Bartoli and Combes,

1986; Jonsson and Andre. 1992; Curtis, 1997) environments

marsh, and local mixing

would diversify the gene pool of larvae encysting on subsequent hosts. In contrast to the timing of cercarial release,

produced cercariae within the

allows for cross-habitat comparisons of emergence characteristics.

Trematode emergence has been studied

emergence duration was under endogenous control. Duration of emergence decreased from sunrise to sunset, perhaps

largely in fresh-

water systems, with much of this research addressing medical and agricultural concerns (Bergquist, 2002; McKerrow

response to the diminishing lighted interval as the day

progresses. Circadian rhythms that control cercarial emer-

and

gence of freshwater species (including schistosomes) are

often set by the activity patterns of subsequent hosts. In this

somes, larval emergence from intermediate host snails varies on a circadian cycle and is synchronized with definitive
host availability (Pages and Theron, 1990; N'Goran et al.,

in

estuary, however, the synchronizing agent

is

the tides. To-

Salter. 2002).

In

common

parasites, such as schisto-

1997). Circadian rhythms are usually entrained by photoperiod or thermoperiod (Theron, 1984; Mouchet ct al.. 1992;
Received 12 February 2003; accepted 16 June 2003.
* Current
address: Patrick Center for Environmental Research, Academy
of Natural Sciences. 1900 Benjamin Franklin Parkway, Philadelphia. PA

Combes

19103-1 19?

gence

t Formerl) Cheryl

Correspond

i,"j

ct al..

from aquatic
scales

Ann Butman.

author. E-mail: z@biology.ucla.edu

1991).

no

1994). Freshwater parasite larvae

to terrestrial vertebrate hosts

to coincide with waterfront activities

of hours

(Theron,

1989;

moving

time their emer-

of the hosts, on

Raymond and

Probert,

PATTERNS AND PROCESSES OF LARVAL EMERGENCE

In southern California intertidal marshes, there

a guild

is

of more than 18 digenetic trematode species (Martin. 1972).

Sexual reproduction occurs in definitive shorebird hosts,

which defecate parasite embryos into the marsh. Free-living

miracidia hatch and infect the California horn snail, Cerithideu California.! (Haldeman), causing castration

and other

sublethal effects (Sousa, 1983: Sousa and Gleason, 1989).

Asexual reproduction ensues, producing tens to thousands

of cercariae per snail per day. The cercariae are produced in
the area previously filled by the snail gonad.

and the larvae

111

paired with a day collection on the preceding or proceeding

tide to compare emergence within the same group of snails.

The field site (tidal channel) was located in Carpinteria

Marsh Reserve (CSMR), east of Santa Barbara. California (3424'16" N, 11931'3()" W). Detailed physical
Salt

characteristics are given in Fingerut et

al.

(2003). Biological

measurements took place along the centerline of a channel

400 m long, 5 m wide, and 0.6 m deep (at high tide). A
centrally located 3-m-wide mudflat was carpeted (460
16/nv) with C. culifomica, the

first

intermediate host for the

then crawl within the snail hindgut to emerge from tissues in

the rectum. Once released into the environment, cercariae

trematodes studied here. The snail population extended hundreds of meters in the along-channel direction. The hydro-

encyst on second intermediate hosts, such as benthic snails

(including C. culifomica). crabs, and fishes. Ingestion of

dynamic regime

these intermediate hosts by birds completes the parasite life

cycle. Swimming cercariae are short-lived, so they must

move toward

or remain near host habitat for effective trans-

mission. In marshes, tidally varying water depth and currents

may

The purpose of

the present study was to identify factors

controlling cercarial emergence in an intertidal estuary, as

tested.
in

1 )

to

(e.g..

in

and similar southern California

this

Mission Bay and Newport Bay) was domi-

nated by slow flows

(<

5 cm/s), with shear velocities

Two

freshwater systems.

Timing of cercarial release

hypotheses were

varies over the day, as

freshwater trematodes. Light (24-h period) may impose a

in both systems, but tidal (12-h

similar diel periodicity

1%

0.8 cm/s) occurred only about

of the time. Intertidal

1.3 m above MLLW

(mean lower low water) and were inundated twice a day by
the semidiurnal tide. Water depth exceeded this height for

an average of 4 h during each

variations in salinity were

28-34

12-h tidal cycle. Daily

psu, consistent across

seasons (Fingerut et al., 2003). Salinity and temperature

were relatively constant throughout the water column, indi-

period) effects (presence or absence of water, currents)

would be unique to the estuary. (2) Exogenous factors

cating well-mixed conditions in the shallow channel.

control cercarial

larval collector

whereas

in

emergence

many

in intertidal estuarine habitats,

freshwater species, larval release

is

con-

endogenously. Circadian rhythms in freshwater cercariae are often associated with innate activity patterns of
trolled

intermediate or definitive hosts

(Combes

et al., 1994).

Such

not be important in an estuarine system, where submergence is limited by the tidal

predictable host signals

may

cycle.

Our research was conducted

in

measured emerging parasite larvae

two

stages. Field studies

as a function of external

A specially designed
measure (non-intrusively) cer-

Collections of emerged cercariae.

was used

to

emergence from a specified group of snail hosts. The

main body of the collector was a flat, clear acrylic chamber
(50 cm wide by 50 cm deep by 2.5 cm tall) with 34-jii,m-

carial

mesh side panels and a solid bottom. Penetrating the top of

the chamber was a 4 by 4 array of 8-cm-diameter holes
(covering 32% of the surface area), each fitted with an
inverted funnel (i.e., small end pointing away from the

chamber)
single

intake. Contents of all funnels

12.5-mm (ID) tube

(Masterflex IP variable speed).

tory experiments identified the role of the specific factors in

tered water from the

We

herein
controlling the onset and duration of emergence.
define "emergence" as the shedding of cercariae from in-

termediate host snails. This study does not distinguish between the effects of larval behavior and rates of asexual

reproduction on cercarial emergence patterns.

over a 34-|iun
times during each 10-min sampling interval.
During each sampling event, 200 snails from the largest

(=2.5-cm-long) size class in the marsh were placed in the

collector. Once on the mudflat, the chamber was gradually

with oxygenated water

from

snails

collected emerging cercariae of

all

species

(Cerhhidea culifornica) while simultaneously

measuring environmental variables. Data were collected on

38 days from 1999 to 2002. Measurement intervals were
selected to span

Moreover,

all

hours of the day and months of the year.

six night collections

(>

h after

fil-

chamber at a rate of about 5 liters/min

mesh. The chamber volume was flushed 8

at

the

same time

as the

free-ranging snail population. There was no movement of

cercariae into or out of the chamber. Captives were provided

Field observations

we

pump

The pump continuously

inundated by the incoming tide

Materials and Methods

were united into a

that fed into a peristaltic

variables (temperature, salinity, tidal height). Then, labora-

In the field,

(//*)

of 0.02 cm/s or less occurring more than 80% of the time

>
(Fingerut et al., 2003). Rarer, storm-driven currents (H*
mudflats in channels were located

limit transport.

compared

marshes

sundown) were

cariae collected on the

at

ambient temperature. Thus, cercould be ascribed solely to the

filter

enclosed host population. Size ranges of marsh cercariae are

50-100 /im (width) and 200-1000 p.m (length) (Martin,
1950; Adams and Martin, 1963), so the mesh retained all
species.

The system self-primed when

the water level reached the

112

FINGERUT ET AL

J.T.

bottom of the funnels

min

the filter

was

ethyl alcohol.

cm

(5

after the tide first rose

>

Every 10

i^oved (and replaced), dipped

in

90%

a sealed petri dish for later

in

placed

water depth), usually 5-10 min

the level of the mudflat.

Lugol's solution) and counting. Samples

staining (0.5'

were also examined

numerically dominant spethe
and
cies throughout
year
day. Each collection lasted 4 h,
to identify

matching the average period of

snail inundation.

was assembled from

lative frequency distribution

cumu-

the total

number of emerged cercariae during each 10-min collecThe time for 95% of all cercariae to emerge was

tion.

compared between collections.

Environmental measurements. Properties of the physical
environment during tidal inundation (4-h interval during
flood or ebb) were measured on the 38 periods of quantified
cercarial

emergence. The mudflat was exposed to

about 8 h between each

inundation.

tidal

air for

Maximal water

depth was 60 cm during each tide. Using an ORION model

140 probe, seawater temperature and salinity were recorded
1

cm above the bed every 10 min. An underwater quantum

(LICOR model 190SA) was flush-mounted at the

sensor

mudflat surface before tidal inundation to register light

intensity at 1 Hz over the duration of the flood and ebb tides.

were randomly assigned

placed

cm

in trays (21

to

one of two treatments, and were

cm wide by 3 cm deep),

long by

Both groups were then situated in an

environmental chamber at 21C (average summer water
at

20

snails per tray.

temperature) with constant light (40 jumol/nr/s). For one

group, trays were kept dry for 4 h; for the other group, trays
were filled with water from CSMR. At the end of 4 h the dry

were briefly filled to suspend any emerged cercariae.

and the water from both groups was filtered through a
34-/j,m mesh. The number of emerged cercariae from each
trays

was determined using the same processing methfield samples. The following day, the treatments were reversed for the two snail groups. This 2-day
experiment (one 4-h test period each day) was replicated
three times, with a new batch of snails each time.
treatment

ods as for the

Effects of light intensity'. Snails

intensity

(2000

intensity

(40

/imol/nr/s

were exposed

to a

midday

or to simulated dawn/dusk

jumol/nr/s), created by reducing midday

filter. Snails (100 per group)

intensity with a neutral density

in water-filled trays (see Effects of host inundation)

were randomly assigned to one of the light conditions.
Every 15 min, trays were overturned onto a 1-mm-mesh

placed

panel. Snails remained in the trays, but water and cercariae

passed through. Trays were immediately refilled. Water and

larvae were filtered over 34-/xm mesh and processed as in

Laboratory- experiments

the field study.

Field measurements (see Results] indicated that the fol-

To

stronger sunlight,

control for the

21C

warming

trays were held

snail

in

effect of the

a water bath

summer months).

control cercarial emergence: temperature, host inundation (water depth), light, and time of day.

maintained

Because we used only infected

consecutive days, and was replicated three times, each with

lowing factors

may

sample of the host population)

snails (rather than a

in laboratory

random

experiments,

all

laboratory

carial

emergence

performed

(27

time series were conducted

each using the same 53 snails (C. califorThe animals were placed individually in chambers

cm 3

filled

were held

with seawater. In the

first

7-day

Effects of time of day.

temperature

in the laboratory,

nica).

different snails.

water-filled trays (see Effects of host inundation) at constant

during the spring (March-April).

Effects of temperature.

series, snails

determined as
trials,

later. (All

daylight time). Snails

times are reported as Pacific

at a constant 18
C

were kept dry

between runs (average spring air temperature for daylight

hours). The second 7-day series followed the same protocol,

1C

except that exposure temperature was decreased

per
day, from 19 to 3 C. At the end of a 4-h incubation period,
contents of individual chambers were processed separately
1

to

determine species (Martin, 1972) and number of emerged

h, start-

the

light

snails,

experiments. Three replicate

at all four start times.

were run

the second series of experiments was to

an endogenous rhythm, stimulated by light,
might control emergence duration. This research is neces-

but

if

not

sufficient

to

establish

circadian

rhythm

(Aschoff. 1960; Pittendrigh, 1993; Dunlap. 1999). The experiment held lightidark ratio constant while varying the
onset of daylight. The prediction was that emergence duration would track with an internal clock set by incipient

dawn, independent of the absolute time of day.

Two temperature- and light-controlled chambers were
at 21

and 40 /j,mol/nr/s. The control chamber was

set

set

on

a light:dark (14:10) cycle, with the natural (0600) sunrise.

cercariae.

Effects of host inundation.

The goal of

sary

in

each with new

were collected 4 h

light (40 /j,mol/m /s) for

time each day (0900, 1200. 1500. and

1800). During each 4-h interval, emergence duration was

determine

higher

(21C) and

ing at a different

C) water temperature on consecutive days, over the range 13-19 C. Water was changed
to the new temperature at 1000 h each day, and cercariae
at a

Trials

both conditions on

were

as a function of water temperature

Two

group exposed

to

Trials to evaluate cer-

experiments were conducted during

summer months (June-September).

(average for

Two series of experiments were

one
time
of day (TOD) on emergence
performed,
testing
duration and the other testing for the existence of an endogenous rhythm. In the first series, 100 snails were placed in

numbers of cercariae emerging were 10-100 times higher

than those reported for field observations. With one exception,

at

lasted 2 d, with each

Two

groups of 100 snails each

The experimental chamber used

the

same

light:dark ratio

PATTERNS AND PROCESSES OF LARVAL EMERGENCE

with sunrise shifted either 8 h forward to

hul

backward
placed
light

to 2200. In the first experiment,

1400 or

100 snails were

the control and another 100 in the forward-shifted

in

chamber

for

week before

the

trial

acclimation period, emerging cercariae

began. After this

both chambers

in

were monitored over 4 h, beginning at 800, as in the light

experiments. In the second experiment, new snails were
acclimated to the control and backward-shifted light regime
1

for

1000.

week, and snails were monitored for 4 h beginning at

In both experiments, the control and light-shifted

snails were tested on sequential days. Moreover, as a check

on the repeatability of the results, the same set of snails was
monitored over an additional 4 d. Both experimental series

were conducted once

in

2000 and once

in

2001.

Results
Field observations

The magnitude of cercarial emergence correlated significantly with water temperature, but not with salinity or total
irradiance (Table

nicu)

first

and Fig.

appeared

in

Snails (Cerithicleti califor-

1).

channels

tidal

temperatures rarely exceeded 13

did not begin until March, however,

to

15

and above

1).

(Fig.

in

February,

(Fig. 2 A).

when

Emergence

when seawater warmed

Despite continued warming

during April and May, emergence was moderate until June.

correspondence between temperature and

evident for the spring cercariae. which vacated

relatively close

emergence

is

their hosts at about the same temperature threshold 15 C)

during each tidal cycle (Fig. 2B). As cool ocean water
flooded the marsh, the shallow water mass was warmed first
(

from contact with the mudflat and then by the sun. Cercarial
emergence following snail inundation was typically delayed
up to 2 h, until water temperature exceeded 15 C.
During the warm summer months, June to September,
when water temperature was above 18 C (average of
21 C), the number of emerged cercariae increased substantially

(Fig.

1).

Larvae

left

snails

as

inundated (Fig. 2C). Despite similarity

soon as they were

in temperature (1-2

difference) between paired day/night collections,

no cercariae were collected

cooled by about 3

in

October, but emergence remained

Table

Stepwise multiple regression analysis of environmental cues

of variation

in the

few or

night (Table 2). Seawater

at

number of emerged

tc/< LIIUK

tt.\

source*

13

114

J.T.

FINGERUT ET

AL.

B
March-May

aary-Febmary

18

100
-10/99

4/16/99

Temperature
Cercanae
16

50-

25

0-

GO
03

0)

.S

14

16

PATTHRNS AND PROCESSES OF LARVAL EMERGENCE

Table 3
2.0
viiriiitiim in fmcr.ifr/iir iliiriitinn

'
i

Source

Time
Time

47.96

1/19

<0.0()()l

11(13

1/19

0.86

<1.0

<O.C)1

1/19

(1.45

<I.O

.7S

1/19

0.19

S.I

oUl.ii
ol

vc.n iinoiuhl

Temperature

nl variation

ill

-\alue

Salinil\

explained

Laboratory experiments
Hn-,1 inundation.

Throughout all laboratory studies, cercariae emerged onl\ if snails were totally submerged. The
average number of cercariae to emerge in paired treatments
with submerged (3861
599 SEM) and dry (0
SEM)

showed unequivocally that cercariae would not leave

it
test: / =
was underwater (Student's
=
<
df
P
channel
8.497.
0.0001 The muddy
5,
containing
the highest snail densities was at a tidal height of about
snails

the host unless

).

250

200-

100

e
00

50-

W
10

12

14

16

18

20

10

12

14

16

18

20

B
'

'

'

"J

y
-^

Zi

^
"3

O
Ef-

-= ua

H
Time of Day
Figure

3.

Effect of time of day

(TOD) on

(h)

cercarial

emergence duration

95% of all cercariae to emerge

during a given event (4 h) as a function of TOD. (B) The total number of
cercariae emerged per snail during a given event as a function of TOD.
in

the tield. (A)

The amount of time

for

115

J.T.

116
4000

FINGERUT ET AL

I'.\ITI-:RNS

225

Q
C
u
E?
<u

UJ

AND PROCESSES OF LARVAL EMERGENCE

17

18

left

J.T.

submerged

FINGERUT ET

snails only during daylight hours. In inter-

AL.

Endogenously regulated emergence

differs

between the

tidal estuaries, tidal currents are a predictable signal that

estuarine intertidal trematodes studied here and

determines both submersion time of the hosts and aquatic

transport of the larvae. Thus, emergence of cercariae

water species. This result was not predicted by our second

hypothesis (see Introduction), and may be explained by the

tracked u

ith

according

to the

major synchronizing agents in these systems. In freshwater,

emergence timing is often under endogenous control and is

which changes daily

The daylight requirement may

the daytime flood tide,

lunar cycle.

indicate that light

is

a critical cue. In laboratory Hume

downward swimming of H. rhi^c-

studies, photo-triggered

CSMR

was

many

fresh-

linked to presence of the subsequent host. For

freshwater schistosomes, definitive vertebrate hosts fre-

directly

effective in slow flows typical of

(Fingerut
2003). This activity quickly brings larvae to the bed
for contact with benthic hosts. Moreover, we observed that

quent the waterfront on a predictable innate cycle. Circadian

rhythms of cercarial emergence are tuned to the biological
clocks and activity patterns of these vertebrates (Combes.

swam upward in response to light

Such behavior could increase con-

et al.. 1994). For example, maximal emerof

some
schistosome cercariae corresponds with the
gence
of
their
bovine hosts. Cercariae emerge and swim
proximity

tltiiui

el

ill..

/:.

culifomienxis cercariae

(authors' unpubl. data).

tact

with host fish in the water column. Likewise, most

freshwater cercariae are shed on the bottom, and light or

gravity directs swimming up or down, depending on the
location of the next host

(Combes

el

<//..

1994). Within the

CSMR

estuary, cercarial emergence during flood tides

would transport larvae 100-300 m/h (Fingerut, 2003). Yet.
emerged H. rhigedana were dispersed only about 1-2 in

before encystment (Fingerut, 2003). Directed swimming by

larvae thus may have greatly shortened their transport distances.

Abbreviated dispersal would retain asexually reproduced

where local mixing could diversify

larvae within the marsh,

the gene pool of cercariae encysting

on subsequent

hosts.

All except 2 of 18 species within the southern California

estuarine trematode guild have benthic second intermediate

hosts (snails and crabs) that are interspersed within

first

intermediate host populations. In fact, some trematodes

(e.g., H. rhigedana, P. acanthus) use the same first and

second host species. Turbulent mixing could commingle

larval genotypes throughout the marsh, enhancing genetic
diversity of the multiple cercariae that encyst each second

1991;

Combes

to the surface in the

while they drink

morning, thus infecting the animals

waterfront (Mouahid et ill., 1991;

and
Probert.
1991). Likewise, afternoon emerRaymond
at the

gence peaks occur in trematode species infecting human

hosts that wash, play, or drink at midday (Theron, 1984;
Pages and Theron. 1990). Emergence in non-schistosome
species, such as Proterometra edne\i (Uglem) also occurs

on a circadian cycle timed to the presence of fish that are

their second intermediate hosts (Lewis el <//.. 1989). There
is

no emergence rhythm in cercariae of Fasciolti liepaticu

however, because second intermediate host

(Linnaeus),

plants are always available (Bouix-Busson et

<//.,

1985).

making synchronization unnecessary.

CSMR

In the

intertidal estuary, duration rather than tim-

emergence was apparently under endogenous control,

and the tide was the synchronizing agent. There is a finite,
ing of

tidally

determined period of sufficient immersion for larval

Emergence duration decreased

transport in lighted hours.

throughout the day

in

response to dwindling daylight.

An

endogenous rhythm associated with the light:dark cycle

host.

Ensuing sexual reproduction between dissimilar ge-

may be

netic

parasites within

the course of a suitable flood tide. Apportioning larvae over

the definitive

shorebird host

may

enhance

responsible for the changing emergence rate over

fitness of the trematode population (e.g., Scheltema, 1971; Jablonski, 1986; Pechenik, 1999, for marine

the entire lighted

invertebrates).

throughout the day

As

digenetic trematodes, larval release in tree-living

estuarine invertebrates is contingent on tidal flows. For
in

example,

estuarine

mud-dwelling isopods

(e.g.,

Parag-

luitliia

formica [Hess]) release larvae only when high

reach

their

burrows,

Reilly, 2002).

facilitating

Shore crabs living

transport

(Tinsley

in high-intertidal

limit larval release to nighttime spring tides

tides

and

refuges

(Morgan and

Hovel and Morgan, 1997). Darkness reduces

predation on spawners, whereas large-amplitude tides flush
away from diurnal predators. Similarly, in tidally

Christy, 1995;

hours.

submerged

envelope.

persal

interval

Contraction

may

of

maximizes the

emergence

dis-

duration

optimize use of remaining daylight

As

emerge

the day progresses, larger pulses of larvae must

over a shorter interval to take advantage of the

light. Such diel adjustments to the emergence

period are unnecessary in freshwater systems, where water
depth is relatively stable. Thus driven by the submergence

vestigial

emergence

strategies of

estuarine cercariae involve both exogenous and

endogenous

constraints of their natal habitat, the

factors that optimize transport to tidally accessible hosts.

Acknowledgments

influenced riverine habitats, adult terrestrial crabs, such as

Sesannn huematochcir (de Haan), release larvae on

a night-

time semilunar cycle that minimizes predation on adults

while providing optimal conditions for survival and dispersal of larvae (Saigusa, 1982).

This study was supported by awards from the National

Science Foundation (OCE 97-22972 and OCE 02-47834)

and the

UCLA

Council on Research.

and E. Maldonado

We

thank M. Grattan

for their tireless help in the field. A.

PATTERNS AND PROCESSES OF LARVAL EMERGENCE

Kuris contributed valuable insights on trematode parasites
that substantially improved earlier drafts of

119

Levy, O., L. Mizrahi, N. E. Chadwick-Furman, and Y. Achituv. 2001.

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Reference: Biol. Bull. 205: 121-132. (October 2003)

2003 Marine Biological Laboratory

Larval Development and Metamorphosis in

Pleurobranchaea maculata, With a Review of
in the

Development

Notaspidea (Opisthobranchia)

GLENYS

D.

Department of Biology. Actulin University.

Abstract.

Pleurobranchaea maculata

taspidean that is

common

in

New

is

is

known

14

C). Larval devel-

In

Introduction

The Notaspidea is a small, specialized order of opisthobranchs that are considered to be phylogenetically intermediate between the highly derived Nudibranchia and the more
basal

show

from veligers of other opisthobranch

ative embryological studies of the Notaspidea are therefore

species of pleurobranchids. mantle and shell growth

taxa.

Canada B4P 2R6

Scotia.

Cephalaspidea (Schmekel, 1985; Willan. 1987;

Mikkelsen, 1998) or monophyletic with the Nudibranchia to
form the Nudipleura (Wa'gele and Willan, 2000). Compar-

in detail for

striking differences

Nova

a carnivorous no-

only two other notaspidean

species. P. japonica and Beithellina citrina. In all three

opment

Wolfi'ille,

Zealand. This species

produces small eggs (diameter 100 p.m) and planktotrophic

veligers that hatch in 8 d and are planktonic for 3 weeks
before settling on biofilmed surfaces

GIBSON

young veligers of pleurobranchids, the shell

new

added by

is

significant for phylogenetic analyses but also for under-

cells other

standing morphological evolution in the Opisthobranchia, a

than those of the mantle fold, and an operculum does not

form. Thus some "adult" traits (e.g.. notum differentiation,

clade rich in homoplastic similarities (Gosliner, 1991, 1994;

mechanism of

taspidean development.

overgrown

by the mantle,

pressed early

shell

is

growth, lack of operculum) are exlarval development. This suggests that

shell
in

characteristic
of
adult
apomorphies
pleurobranchids
evolved through heterochrony. with expression in larvae of

Mikkelsen, 1998). Unfortunately,

opment

in

My

goal

little is

is

known about

Pleurobranchaea maculata (Quoy and Gaimard,

1832) and to provide a preliminary analysis of development

in

Notaspidea.

Adult notaspideans are carnivores and opportunistic scav-

of adults of other clades. The protoconch is

dissolved post-settlement and not cast off as occurs in other

engers.

opisthobranch orders, indicating that shell loss

ctenidium on the right side, rolled rhinophores, and a

traits typical

is

apomormaculata veligers are atypical of opisthobranchs in

having a field of highly folded cells on the lower velar
surface, a mouth that is posterior to the metatroch. and a
phic. P.

richly glandular, possibly

chemodefensive mantle. These

data indicate that notaspidean larvae are highly derived in

terms of the novel

traits

and the timing of morphogenic

events. Phylogenetic analysis must consider embryological

origins before assuming homology. as morphological similarities (e.g..

tinct

shell loss)

may have developed

mechanisms.

through dis-

no-

to describe larval devel-

They

are

characterized

by

single,

external
flat-

tened shell (Willan, 1983: Schmekel, 1985; Willan. 1987).

The order

traditionally

includes

the

Umbraculomorpha

(families Tylodinidae and Umbraculidae) with large limpetlike external shells

and a small mantle, and the Pleuro-

branchomorpha (Pleurobranchidae) with a prominent mantle and shells that are internal and reduced or lost in adults
(Thompson. 1976; Willan. 1983. 1987). Mantle secretions
provide chemical defense in many species, both shelled and
through the release of acid (Thompson and Slinn.
1959; Thompson. 1988), secondary metabolites (Ciavatta et

shell-less,

til..

1993, 1995: Spmella et ai. 1997). or dietary alkaloids

(Ebel et ai.

1999).

On

the basis of adult anatomy,

some

Received 27 August 2002: accepted 23 June 2003

investigators suggest that the Notaspidea are polyphyletic

E-mail: glenys.gibson@acadiau.ca

(Schmekel,

1985).

while

others

argue

that

they

are

122

G. D.

paraphyletic

and

uire

re-

(Wagele and Will

Phylogenetic
relatedness

which

;.

lysis

laxa.

ai

of the

inclusion

Nudibranchia

00).

promotes an understanding of the

and of morphological evolution, for

developmental data are essential. Alno reason to assume that larval and adult

.utive

coi:

though tl re is
traits have coevolved, inclusion of larval
analyses by increasing the data
revealing homologies in

set,

traits

but

traits

strengthens

more importantly, by

with similar embryological

While we have an abundance of data on nudi-

origins.

known

some of

GIBSON
Live embryos, larvae, and juveniles were observed,
sketched, and photographed with a Nikon AFX photomicroscope. Measurements were taken to the nearest 5 /urn.
Pediveligers and early juveniles were fixed for

speciose orders. For the Notaspidea in particular, detailed

descriptions of larval development are limited to two species of Pleurobranchidae: Berthellina citrina Rueppell and

Draw

poorly

for

2.5%

imens were observed with a Phillips XL 30S SEM at 5 kV.

were composed in Corel Photo Paint 9.0 and Corel

Plates

is

in

mens were dehydrated in ethanol, critical-point-dried with a

Polaron B3000 Series II critical-point drier, and coated with
gold-paladium with a Polaron SC7640 sputter coaler. Spec-

the less

branchs, development

SEM

glutaraldehyde followed by post-fixation in 1% osmium

tetraoxide, both in filtered seawater. After fixation, speci-

and

9.0, using

digital

both scanned negatives (light micrographs)

images (electron micrographs).

Leuckart, 1828 (Gohar and Abul-Ela, 1957: Usuki, 1969),

Results

and Pleitrobranchaea japonica Thiele, 1925 (Tsubokawa

and Okutani. 1991). Partial information on larvae is avail-

The

cylindrical egg masses

were 171.60

mm

59.61

in

range 88-240 mm) and about 8-12

able for less than a half-dozen additional species. Even these

limited data suggest that larval pleurobranchids are diverse

mm in width, as produced by two adults

and can be planktotrophic, lecithotrophic, or direct developing (Gohar and Abul-Ela, 1957; Usuki, 1969; Thompson,

maculata spawned frequently, and these two

length.
individuals produced 15 egg masses in about 5 weeks of

Tsubokawa and Okutani. 1991; Wagele, 1996; God-

laboratory culture. Usually adults deposited the loosely

coiled egg strings on the side walls of the tank near the

1976;

We

dard, 20()lb).

lack descriptions of development for

all

(X

length

SD; n

5;

mm in

10 and 145

P.

The eggs were

species of the Tylodinidae and Umbraculidae.

air-water interface.

My
development of Pleiirobranchaea maculata, and to compare development of nota-

and deposited in egg capsules housed

within long strings that formed a double spiral at the pe10.92 by
riphery of the jelly mass. Capsules were 259.50

objectives are to describe

spideans with other opisthobranchs. P. maculata is an opportunistic carnivore found in New Zealand, southeastern
Australia. China, Sri Lanka, and Japan (Willan. 1983;

Mar-

cus and Gosliner, 1984). Notes by Willan (1983) indicate

that

P maculata

produces long, cylindrical egg masses that

The present study extends

in

45).

10.54

/urn

diameter (n

250.00

in

size

(;i

white, 100.91

20).

and contained an

= 30
eggs per capsule (range 2-6; n
each of 7 egg masses). Small globules of yolk
with lipid droplets and several times larger than polar

average of 3.97

.02

capsules in
(filled

release planktotrophic veligers.

bodies) detached from developing young in about

record to include embryology, larval structure, and

metamorphosis using bright field and scanning electron

the

microscopy (SEM).

yolk

this

2.18 ju,m

egg capsules observed

(n

= 244

capsules, from a

18%

of

total

of

8 egg masses). Early veligers ingested this extra-embryonic

hatching. Fecundity was estimated to be

110,602 embryos per egg mass (/; = 5). Egg
masses were as figured by Willan 1983).

before

318.417

Materials and Methods

Spawning generally occurred over a period of 2-3 h in

the early and mid-morning. A chronology of early embry-

Specimens of Pleurobranchaea maculata were collected

from intertidal sandflats at the Tahuna Torea Reserve, Ta-

maki Estuary. Auckland.

Adults were maintained
temperature
stiitclibinyi

New
at

Zealand, in August 2001.

ambient conditions (seawater

C) and were fed cockles (Austrcivcmts

14

Wood, 1828)

or oysters (Ostrea lutaria Hutton,

1873) daily. Egg masses were cultured in flowing seawater

(14 C). and small sections were removed for observation.

Larvae were cultured

17-19
saliiui

It-

17-19 C) v

surface.

in
n!i

galbana Parke, 1949,

Green, 1975 three times weekly. Set-

''itheri

tlement occun
(

idoresco. 1905. Isocluysis

and Pin

jar

of

-jam-filtered seawater at
C. Larvae were fed a 1:1:1 mixture of Dunalit'lla
in 1-1 jars

pediveligers that were cultured

a

in a

1-1

1-week growth of biofilm on the glass

ological events

is

chophores, with a

summarized
flat

in

Table

1.

Elongate

tro-

velar field and small pedal rudiment,

developed by 4 d. The shell was visible by 4.5 d and had

grown to cover the visceropallial mass by 5 d. Also at 5 d.
the statocysts. each with a single statolith, were visible, and

embryos were capable of ingesting extra-embryonic yolk.

By 5.5 d, the dark red pigmented mantle organ was visible
and the internal organs, although

still

yolky. were better

The elongate

foot lacked an operculum. and velar

cilia were capable of metachronic beating and reversals. By
defined.

pigmented mantle
The
larval
retractor muscles,
were
defined.
organ,
sharply
visible at 6 d. were functional by 7 d although the embryos
ft

d.

the internal organs, including the

were capable of incomplete retraction only, leaving the

velar lobes and foot partially exposed.

DEVELOPMENT OF PLEUROBRANCHAEA
Table
of the

SiimiiHirv

Time

14

1" polar
2'"'

8.5 h

9.45 h

into the shell, possibly

nd

cleavage
rd

cleavage

effective shell aperture.

About 3 weeks after hatching, the first pediveligers seton the biofilmed surface of the culture container. The
23 p.m in length (;/ = 20), was almost comshell, 480

Blastula formation

48-72 h

tled

Gastrulation

4 d

Trochophores: pedal rudiment and velar

field present.

embryo elongate
4.5 d

pletely covered

Cap-like shell visible

Early veligers: larval shell complete, pigmented mantle

5 d

organ, statocysts, right and

left

digestive gland

present, toot visible but lacks an

o d

operculum

Pigmented mantle organ well developed, retractor

muscle present but not functional, digestive glands

becoming more

distinct

Hatching: subvelar ridge prominent, kidney rudiment

present, pigmented mantle organ, mantle edge

branchial aperture on the right side of the "neck" and along

the upper right side of the foot (Fig. 2F). The densely

rounded, shell surface granulated. Eyes and

mouth was located

ciliated

operculum lacking
period.

Timing of early events

is

relative to the onset of oviposition.

Veligers hatched

lateral

at

8 d, with a shell length of 135

5.5

and began feeding on phytoplankton immediately (Table 1, Fig. 1A). The digestive glands were still
yolky and the ciliated stomach had hyaline rods in the
20),

posterior wall, as described in

nudibranchs (Thompson,

1959). Just above the pigmented mantle organ

was

a small,

ventral and posterior to the

subvelar ridge (Fig. 2F). The foot was well developed and
covered with fine cilia ventrally and with tufts of cilia on the

and upper surfaces (Fig. 2F). The pedal

described for veligers

jum (n

2A-F).

was thin as it extended over

the shell, while older mantle tissue became thickened as the
mantle glands developed (Fig. 2C). The mantle glands in-

openings of the mantle glands (Fig. 2E). A lateral ciliary

tract was present externally from the opening of the pre-

still yolky but yolk depleted from

stomach, mantle fold well developed

Spawning occurred over a 2-3-h

thick, glandular mantle (Fig.

the mantle

vaginated from the epidermis to the shell margin, forming

digestive glands

8 d

by the

The growth zone of

elongate, simple tubular glands (Fig. 2D, E). Small tufts of

cilia were scattered over the mantle surface, between the

Retractor muscle functional, velar lobes large.

7 d

because of the size of the velar lobes

and also because the growth of the mantle reduced the

Morula

22 h

2448

concurrent with overgrowth by the mantle, during larval

development. The veliger was unable to completely retract

hod\

polar hod\

P' cleavage

).15h

the rhi-

envelope the larval shell by growing up and over the dorsal

shell aperture (Fig. IB). The larval shell continued to grow,

Spawning

4.5 h

larger, slightly

Buds of

nophores were present on the anterior velar field (Fig. ID).

Also in the first week of larval life, the mantle began to
Event

Oh

was much

bilobed. and lacking any visible contents.

id. h)

2.5 h

IB. D), and the kidney rudiment

major events in emhrynnic development of

Pleurnhranchaea maculata

123

In late-stage larvae

week

tuft,

as

was absent.
velum showed

after hatching,

and pediveligers, the

additional structures on both the upper and lower surfaces.

The rhinophores extended from the upper velar surface as

two curved ridges of tissue that were covered with fine cilia
(Fig. 2 A, F). As the rhinophores developed, they grew
remained open on the

anteriorly, then laterally; but they

transparent organ, presumably the rudiment of the definitive

lateral surface, thus

kidney (Gohar and Abul-Ela. 1957). The kidney rudiment

in close association with the pigmented mantle

that is typical of adults (Willan,

giving rise to the scroll-like morphology

1983).

The

oral veil

was

on the upper surface of

remained

also visible as a broad, ciliated ridge

organ and anus throughout larval development. Larvae

lacked eyes at hatching, and the finely ciliated foot lacked
an operculum throughout development. The Type
shell

but not connected to them (Fig. 2F).

The lower

was covered by

diameter) rounded cells

the velum, located immediately ventral to the rhinophores

large

(-15 /im

in

velar surface

finely

with a highly folded, microvillar surface (Fig. 2F. G). These

granulated on the surface; ridges, as occur in P. japonica

(Tsubokawa and Okutani. 1991), were not observed.

post-velar cells were tightly packed together and covered

the lower surface of each velar lobe from the subvelar ridge

(types by

Thompson. 1961) lacked pigment and was

One week
15 /Jim {n

after hatching, the larval shell was about 190

20) in length, and the velar lobes had increased

substantially in size (Fig. IB. C, D).

New

sensory structures

had formed, including a pair of black eyes and the pedal

a prominent cluster of elongate cilia on the tip of the

tuft,

foot.

The stomach was

greatly enlarged relative to shell size,

and the hyaline rods were more numerous in the posterior

stomach wall. The larval heart was also present and beating.

The pigmented mantle organ, now

black,

was

larger (Fig.

to the

body

Some

wall.

internal organs

were

difficult to

observe

in

live

pediveligers because of the thick mantle. The eyes and

statocysts were well developed, and the buccal mass was

prominent

in

the anterior digestive tract (Fig.

2 A).

The

pigmented mantle organ was darker, and the enlarged, transparent kidney was easily observed though the thick mantle
tissue (Fig. 2B. D).

Acquisition of a juvenile morphology occurred gradually

124

G. D.

GIBSON

svr

rdg
a f

Idg

100 |um

rh

vl

Idg

pmo

1.
Early veligers of Pleurobranchaea maculata. (A) Veliger on the first day of hatching, drawn from
(B) Veliger 7 d after hatching, drawn from life. (C. D) Bright field micrographs of veligers 7 d after
hatching. Scale bar is the same for all four illustrations, a. anus; e. eye; f, foot; hr. hyaline rods; i. intestine; k,

Figure

life.

kidney rudiment; Idg,

tuft; rdg. right

left

digestive gland;

Ih, larval heart;

m, mantle; pmo. pigmented mantle organ; pt. pedal

s. stomach: st.

digestive gland; rh. rhinophore bud; rm. retractor muscle; rvl. right velar lobe;

statocvst; svr. suhvelar

nde;

vl,

velar lobes.

and involved development of the mantle throughout most of

larval life. The final stage of metamorphosis primarily in-

became smooth and

volved loss of the velum. As the velar and subvelar

the lower velar surface, followed

Figure

2.

Pediveligers of Pleurobranchaea imicitlatu. 3 weeks after hatching. (A. B)

micrograph of

field

cilia

were shed, the highly folded surface of post-velar cells

the cells were gradually resorbed into
by resorption of the velar

Drawing and

bright

Scanning electron micrograph of the overgrowth of the larval shell by

bright field and scanning electron micrographs. (F) Scanning electron

live pediveligers. (C)

the mantle. (D. E) Mantle glands in

micrographs of a pediveliger. (G) Scanning electron micrograph of the post-velar cells on the lower velar
surface; the subvelar ridge is shown in the upper right. (H) Scanning electron micrograph of the partially
i

Mirhed velar lobes during metamorphosis, bm. buccal mass; cso, cephalic sensory organ;

tract;

f.

gland; mo, mouth; ov. oral veil;

svr.

Mihxd.u ridge:

ctr. lateral ciliary

kidney rudiment; Ih. larval heart; m. mantle; mg. mantle

pmo, pigmented mantle organ; pvc, post-velar cells; rh. rhinophore; sh, shell;

foot: glz, glandular zone; grz.

vr. velar ridge.

growth zone;

k.

DEVELOPMENT OF PLEUROBRANCHAEA

125

126

G. D.

Loss of the ve!

es for several

a prominent

two velar

ii

riorly fiv;

uie

days (Fig. 3A-D).

scvealed the cephalic sensory organ,

.ated organ located dorsally between the

.;

(Fig. 2H).

The rhinophores extended

remnant of the velar

field (Fig.

(Fig.

ante-

3A, C), and

the oral veil projected as a broad ridge to cover the

and anterior foot

mouth

3A-C). The prebranchial aperture,

open on the right side, led to the lateral ciliary tract. The gill
had not yet formed (Fig. 3D). The opaque mantle, both
glandular and also with a scattering of red pigment, made it
difficult

to

Discussion

Early juveniles retained lateral

lobes into the head (Fi

remnants of the

GIBSON

determine when the shell was dissolved,

al-

Morphogenesis of Pleurobranchaea maculata

Development of Pleurobranchaea maculata is similar to

(Tsubokawa and Okutani, 1991 in terms
of egg mass characteristics and overall larval morphology.
that of P. ja/xinica

However,

the present study revealed several additional traits

that warrant discussion, including the

mantle glands, post-

velar cells, sensory organs, and the position of the mouth.

The mantle of P. maculata becomes richly glandular as it

grows back over the larval shell. These simple, tubular

easily

glands project through the entire thickness of the mantle,

appear fairly early in ontogeny, and persist through meta-

observed through the ventral body wall (Fig. 3B). The

pigmented mantle organ appeared to be lost during late
metamorphosis. The kidney remained next to the pre-

morphosis. Thompson and Slinn (1959) and Thompson

(1988) reported the secretion of sulfuric acid from the
mantle of adult Pleurobranchidae from both simple co-

branchial aperture.

lumnar

though the buccal

mass and digestive glands were

cells

and flask-shaped glands. Adults of Pleuro-

3.
Early juveniles of Pleurnhraiichai'ii nnuiilahi. (A) Newly settled juvenile, dorsal aspect, drawn
(B) Bright field micrograph of a newly settled juvenile, ventral aspect. (C) Bright field micrograph of
load. (D) Scanning electron micrograph of a newly settled juvenile, a. anus; bm, buccal mass; ctr. lateral

Figure
oni

01 al

life.

tiact; dg. digestive gland; e. eye; es, esophagus; f. foot; k. kidney; m. mantle; mp. mantle pigment; ov,
>nl: pha, prebranchial aperture; pvc, post-velar cells; rh, rhmophore; s, stomach; v, remnant of velar lobe.

DI-V1

OI'MI-NT

01-

/'//

KOHK

\\CII\I

127

hranchaea and Pleurobranchus also synthesize a variety of

tract

defensive

from the prebranchial aperture, which houses the anus and

1997).

compounds (Ciavatta et ai, 1995; Spinella ct <//..

The abundance and differentiation of mantle glands

serve to generate water currents directed

may

nephroproct: this function is likely taken over by the gill

once the gill has formed. Gill formation was not

inacidata pediveligers suggest that they function in

late-stage larvae and likely confer chemical protection to

cilia

planktonic pediveligers and newly settled juveniles. The

capacity of larvae to synthesize their own chemical defense

Okutani (1991) describe the

in P.

is unusual. Chemical defense occurs in eggs or egg masses

of some opisthobranchs; however, in these other taxa. the
defensive compounds are maternally derived, as occurs in

the

Anaspidea (Pennings. 1994). Ascoglossa (Paul and Van

1988). Nudibranchia (Pawlik et at., 1988). and

Alstyne.

away

observed

in the

present study, although

gill

in

P.

Tsubokawa and

japonica

in early

juveniles.

Pattern and process

Egg mass

in

structure.

notaspidean development

Egg masses have been described for

As is typical of

several species of Notaspidea (Table 2).

edge, have not been reported elsewhere. These cells develop

opisthobranchs. eggs are located in capsules within an elongate string that runs in a double spiral around the periphery
of a jelly mass, although in some notaspideans the string is

in larvae, persist through early metamorphosis, and are

resorbed with the rest of the velum. The post-velar cells

poorly defined (Milieu, unpubl. obs. cited in Strathmann.

1987) or highly modified (Bandel. 1976). In some species,

greatly increase the surface area of the lower velar surface

through their abundance and highly folded apical surface.

capsules contain a single egg; in others, as many as 37 eggs

per capsule are common (Table 2). Egg size is variable

seems

species and. although the sample size is small,

appears correlated with developmental mode (Table 2), as
expected for opisthobranchs (Hadfield and Miller. 1987).

tylodinid Notaspidea (Ebel et ul.. 1999).

The post-velar cells are also unusual and. to

While the function of these

cells

is

my

unknown,

it

knowl-

reasonable to suggest that they are secretory because of their

size,

number, and morphology. If secretory, they may prohead and velum by neutralizing secretions of the

tect the

mantle glands.

The rhinophores of P. macitlata originate from the center

of the upper velar surface; appear fairly early in larval
development; and grow anteriorly as curved, ciliated ridges
of tissue. In contrast, the rhinophores of P. japonica arise
from a pair of depressions at the lateral edges of the oral veil
(Tsubokawa and Okutani. 1991 ). The intravelar location of
the rhinophores in P.

niucnlata

is

similar to that of the

nudibranch Rostanga piilchra MacFarland, 1905 (Chia and

Koss, 1982. 1991 ). although the definitive morphology difrhinophores of nudibranchs are solid and lack
groove. A cephalic organ, as observed in P. inacu-

among

However, in some Notaspidea, egg size can vary considerably within one species; in the lecithotrophic Berthellina
example, variation in egg size is reported to span
140 urn, ranging from 270 to 410 ^.m over 15 egg masses

citrina, for

(Usuki, 1969).

Lan'al morpholog\. Morphogenesis in notaspidean veligcharacteristic of benthic

is modified from the pattern

ers

opisthobranchs

that is

observed

morphogenesis appears similar

a lateral

branchs.

was not reported in P. japonica (Tsubokawa and

Okutani. 1991) but would be difficult to observe without

SEM. However,
laspids

a cephalic organ

is

present in

some cepha-

and Ruthensteiner, 2001) and nudi-

(Schaeffer

branchs (Chia and Koss. 1984).

Also of

cilia,

interest is the position of the

mouth posterior

to

or prototroch) and the subvelar ridge (i.e.. the post-oral

or metatroch). Whether this is the original embryolog-

ical location

of the mouth

(e.g..

protostomal) or represents a

secondary mouth opening is not known. Comparative work

is needed to clarify the embryological origins and potential
migration of the definitive mouth from the protostome.
The ciliary tract on the right lateral foot has not been
reported elsewhere in notaspideans, but is reported for nudibranchs (e.g.. Bonar and Hadfield. 1974; Goddard, 1996).

The

ciliary

tract

in

P.

to that of other opistho-

In most notaspideans (Table 2). larval shells are Type

whorled; typical of most classes of opisthobranchs) or less
commonly. Type 2 (inflated and cuplike; occurs in some
1

nudibranchs). However, once the protoconch has formed,

larval shell in pleurobranchids is atypical of

growth of the

the subvelar ridge in pediveligers. Veligers typically have a

mouth positioned between the velar cilia (i.e.. the pre-oral
cilia,

Cephalaspidea, As-

notum formation, and lack of an operculum. Otherwise,

fers in that the

lata,

in the

coglossa. Anaspidea. and Nudibranchia (e.g.. Thompson,

1976). Major differences include shell growth and loss,

maculata.

occupies the position of the adult

not present in adults,

gill

(Willan. 1983).

The

other opisthobranchs in mechanism and timing. In other

opisthobranchs. the larval shell grows via secretions by the

mantle fold, a ridge of tissue next to the shell aperture

(Tardy. 1991 ). In pleurobranchids. the mantle fold is lost in
early veligers at the

same time

as the mantle extends past

the aperture to ultimately cover the entire larval shell. This

occurs after hatching for planktotrophic species of Pleuro-

hranchaca (Tsubokawa and Okutani. 1991;

within the egg

this study) or

capsule for lecithotrophic species of Ber-

(Usuki. 1969) and direct-developing Bathyberthella

(Wa'gele, 1996). Thus larval shell growth is concurrent with
tliellina

mantle overgrowth and occurs despite the migration of the

mantle edge away from the aperture; presumably, the region
of mantle adjacent to the aperture retains shell-secretion

128

G. D.

GIBSON

DEVELOPMENT OF PLEUROBR.\NCHAE.\

Ontogenetic Stage

Trait

Larva

Embryo

Shell

129

Growth:
(growth

from mantle

Adult

Metamorphosis

fold)

Cephalaspidca
Notaspidea
-

Umbraculidae
Tylodinidae
Pleurobranchidae

(shell +/- in adults)

Nudibranchia

Protoconch:

(larval shell retained)

Cephalaspidea

Notaspidea
- Umbraculidae
-

Tylodinidae
Pleurobranchidae

Nudibranchia

Growth

of

(dissolved/ internalized)

(shell cast off)

Notum:

Cephalaspidea

Notaspidea
- Umbraculidae
-

Tylodinidae
Pleurobranchidae

Nudibranchia

Operculum:
Cephalaspidea
Notaspidea

(operculum +/-

Umbraculidae

Tylodinidae
Pleurobranchidae

sj(does not develop

in adults)

?)

Nudibranchia

4.
Heterochrony and pleurobranchid development. Data summarize the time of onset and offset of
growth, protoconch (formation/loss), notum growth, and operculum (formation/loss). Timing is generalized
to ontogenetic stage (embryo, larva, and adult) for Cephalaspidea (light gray bars). Notaspidea (white bars), and

Figure

shell

Nudibranchia (dark gray bars). Different patterns within a bar indicate differences in the developmental process
underlying a particular trait; for example, shell growth occurs at the mantle fold in early stages and the mantle
gland

in

later stages,

(Nudibranchia).

Indicates that the

of onset

is

and

Denotes
trait is

shell

(some pleurobranchids) or being cast off

pleurobranchids, relative to other indicated groups. +/
the adults of only some species in each family or order. ? Indicates that time

loss occurs through dissolution

change

present

in

in

time of onset

in

not known. References are provided in the text and in Table

activity in larvae. Migration of the

mantle edge away from

2.

growth of epipodial

larvae

(e.g., Anaspidea) or parapodial (e.g.,

Cephalaspidea) lobes in juveniles of other shelled opisthobranchs (Tardy. 1991).

occurs by means of the early onset of an ontogenetic process

found in juveniles and adults of species that retain their

larval shell is also atypical of opisthobranchs (Fig. 4). In

the aperture suggests that shell

modified

in

several

ways.

growth

in

shell

First,

pleurobranchids

growth

in

is

In shell-less notaspideans, the

mechanism of

loss of the

(Fig. 4). Second, morphogenesis of the shell and

mantle are decoupled from other morphological changes
that occur during metamorphosis. Third, shell-less nota-

other opisthobranch orders, the retractor muscles are severed and the shell cast off at metamorphosis (e.g., Asco-

spideans have retained the plesiomorphic mechanism of

adult shell overgrowth found in shelled notaspideans. This

less

shells

mechanism of

shell

overgrowth

is

also distinct

from the

glossa, Nudibranchia.

Gymnosomata).

Pleurobranchidae

lose

the

In contrast, the shell-

larval

shell
through
overgrowth and dissolution by the mantle during or shortly
after metamorphosis (Tsubokawa and Okutani, 1991). This

130

G. D.

distinctive

mechanism

that in the Notaspk!<

the opisthobranci
phic.

It

lacking in non-planktotrophic notaspideans, including Ber-

-as has been suggested throughout

thellina citrinn (Usuki, 1969), the probably lecithotrophic

Berthella plumula (Montagu, 1803) (Thompson, 1976) and
the direct-developing Bathyberthella antarctica Willan and

liner,

1994)

shell loss

suggests that similarities

in shell los-

,!

in

of shell loss supports the hypothesis

i

add i'

is

apomoradults

among

notum morphology have evolved through

Notaspidea and Nudibranchia.

homoplasy
Mantle growth

in

GIBSON

notaspideans

is

also atypical. In

most

opisthobranchs the mantle remains small until after metamorphosis. In contrast, the mantle in Notaspidea begins to

Bertsch. 1987 (Wa'gele, 1996). Although the pigmented

mantle organ appears similar in the Notaspidea and Cephalaspidea, ultrastructural and detailed embryological studies
are needed to determine if they are homologous. All three

studied species of Pleurobranchaea (P. japonica. P. cali-

Tsubokawa and Okutani, 1991;

take on adult characteristics at an early larval stage. This

fornica, and P. maculata:

early development includes formation of the notum (Gohar

and Abul-Ela. 1957; Thompson, 1976; Tardy, 1991; Tsu-

Goddard, 200 Ib; present study) also have a large, transparent organ positioned adjacent and dorsal to the pigmented
mantle organ, which is here considered to be the rudiment of

bokawa and Okutani. 1991;

Wa'gele,

1996) and also the

and glands (present study). Early

onset of notum differentiation may provide the larva and
differentiation of cilia

settled juvenile with an effective

newly
(e.g.,
(e.g.,

means of defense

potential acid cells),

mantle ciliary

ciated

increased sensory perception

and a larger size, with the asso-

tufts),

advantages of predator deterrence and increased

buoyancy.
Larvae of pleurobranchid notaspideans lack an operculum. The single recorded exception is Willan's 1983) note

Settlement
thellina

lata all settle

Ela,

maculata; however, an operculum

the present study of this species. Lack

are opportunistic carnivores.

in

in

benthic lifestyle) occurs over a relatively short time span,

metamorphosis (the acquisition of an adult morphology)

sinicum (Gmelin, 1791)

described as having an operculum (Ostergaard, 1950). Larvae of all other opisthobranch

orders have an operculum. with individual exceptions such
is

as the nudibranch Aegires alhopunctatus

in

2()()la).

Settlement and metamorphosis are also decoupled events

pleurobranchids. Whereas settlement (acquisition of a

in P.

of an operculum may be characteristic only of pleurobranchids, however, as larvae of the tylodinid Umbracitliun

(Goddard,

on biofilmed culture dishes (Gohar and Abul1969; Tsubokawa and Okutani, 1991:

1957; Usuki,

present study). Preferences for specific substrates were not

tested, but a nonspecific cue is probable as all three species

of an operculum
was not observed

Gohar and Abul-Ela, 1957).

and metamorphosis. Pediveligers of Bercitrina, Pleurobranchaea japonica, and P. macu-

the kidney (following

The operculum

most opisthobranchs. except

MacFarland, 1905

is lost at

for a

metamorphosis
few species of Cepha-

laspidea (Thompson, 1976). This suggests that loss of an

operculum in pleurobranchid larvae is an apomorphic trait

begins early in larval life with an accelerated onset of some

processes of differentiation typical of adults (e.g.. shell and

notum growth), while

differentiation of other organ systems

velum, foot) appears similar to that of other opisthobranch orders. For example, the larval shell is produced by
a mantle region other than the mantle fold; thus we see an
(e.g.,

early onset of a shell-growth

mechanism common

in

juve-

and adults of shelled species (Fig. 4). Also, in the

mantle of early larvae, "adult" structures such as glands and

niles

a con-

external cilia undergo rapid growth and differentiation pro-

of other orders (Fig. 4).

As with other opisthobranchs, the mantle cavity of nota-

spidean veligers contains several cell clusters that have been

cesses that also are associated with juvenile development in

other orders. Absence of an operculum in pleurobranchid
larvae is also a trait typical of adult opisthobranchs. Collec-

referred to as "larval kidneys" and "anal cells"

tively, these observations suggest that in the Pleurobranchi-

that represents

dition

common

Abul-Ela.

an earlier

embryonic) onset of

(i.e.,

to the adults

1957; Usuki,

1969;

(Gohar and

Tsubokawa and Okutani,

The function and ultrastructure of these organs are

unknown, and the terms are used inconsistently across gastropod taxa. Thus, it is more appropriate to use the descrip1991).

"pigmented mantle organ" to refer to the darkly pigmented structure associated with the anus. A pigmented

dae, aspects of the specialized

morphology of the

evolved through heterochronic changes

adult have

in specific

morpho-

genetic events associated with metamorphosis.

tor

mantle organ

is found in the planktotrophic veligers of

several notaspideans, including P. californica MacFarland,

1966 (Goddard. 2001a), P. japonica (Tsubokawa and Oku1991), P. maculata (present study), Berthella califor-

Phylogenetic implications

Development in the Notaspidea is relatively poorly

known, and the data we have primarily includes the Pleurobranchidae, while details are lacking for the Umbraculi-

1900) (Goddard, 1984), B. xtnmgi (MacFarland,

dae and Tylodinidae. The descriptions of development that

are available for the Pleurobranchidae support the current

1966) (Goddard, pers. comm.), Berthellina engeli Gardiner,

1936 (Goddard, pers. comm.) and Umbraculum xinicnni

hypothesis that the Notaspidea are phylogenetically closely

linked with the Nudibranchia (Schmekel. 1985; Gosliner.

tani,

nicci (Dall,

(Ostergaard. 1950).

similar organ

phic species of Cephalaspidea.

is

found

in

planktotro-

pigmented mantle organ

is

1994; Wa'gele and Willan, 2000) yet share some, possibly

plesiomorphic. traits with the Cephalaspidea. Potentially

DEVELOPMENT OF PLEUROBRANCHAEA
include the pigmented mantle
to planktotrophic species of the Cephu-

plesiomorphic larval

(common

organ

traits

laspidea and Notaspidea), the cephalic sensory organ (found

in

Cephalaspidea and Nudibranchia), and a type

shell

(common

larval

to all orders).

tion of the rhinophores support

homology

in

the Pleuro-

branchoidea and Nudibranchia. despite differences

(rolled

phology

in the

Apomorphies
larval

versus

Chia. F.-S.. and R. Koss. 1984.

solid

structure,

in

mor-

respectively).

Pleurobranchidae include lack of the

operculum, the mechanism of shell loss or internaland the pattern of notum formation. These apomor-

Fine structure of the cephalic sensory

nudibranch Rnstiinga pulchra (Mollusca.

organ
Opisthobranchia. Nudihranchia). Zoomorphology 104: 131-139.
Chia, F.-S.. and R. Koss. 1991.
Sensory structures and behaviour in
the larva of the

in

opisthobranch veliger larvae. Bull.

16:

Synapomorphies with the Nudibranchia include the shape

of the egg mass and the presence of rhinophores that arise
from the upper velar field. This agrees with Wa'gele and
Willan (2000). who suggest that similarities in innerva-

131

lust.

polypropionates from the skin of the mediterranean

mollusc Pleurobrunchiis membranaceus. Tetrahedron Lett. 34: 6791-

6794.
Ciavatta, M., G. Villani, E. Trivellone,

of dietary alkaloids

Biochem.

and ultrastructural work.

Goddard,

.1.

Goddard,

769-777.

The opisthobranchs of Cape Arago, Oregon, with

1984.

at the

New

Lecithotrophic development

Leigh Marine LaboI thank the

Zealand.

Goddard.

University of Auck(School of Engineering),

at the

(School of Engineering) for their kind assistance and providing access to their lab facilities. J. Goddard generously

provided unpublished observations and insight into opisthobranchs. which have considerably added to the manuscript.
This manuscript benefited from the comments of an anonymous reviewer and of M. Gibson and I. Paterson. J. Ha-

venhand and

L. Page provided insight on veliger structure

and physiology. This research was supported by the Natural
Science and Engineering Research Council of Canada

(NSERC).

J.

The

2001a.

J.

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Reference: Biol. Hull. 205: 133-143. (October 2003)

2003 Marine Biological Laboratory

Effects of Allogeneic Contact on Life-History Traits of

the Colonial Ascidian Botryllus schlosseri
in

NANETTE
^

E.

Monterey Bay

CHADWICK-FURMAN
Eilat, Israel;

Stanford University.

The formation of chimeric colonies following

allogeneic

contact

between

benthic

strongly affect colony fitness. Here

invertebrates

WEISSMAN 2

we show

that, in a field

allogeneic contact with conspecifics.

We

Stoner

et al.,

1999).

shown that the eggs of one partner

brooded
be
retained
and
may
by the other partner over
several reproductive cycles (Sabbadin and Zaniolo, 1979).

Botryllus schlosseri have

experi-

In laboratory studies, fusion

several fitness parameters (Rinkevich and

terms of

Weissman, 1987,

1992a, b); indeed an inevitable result of such fusion

terms of most life-history traits measured.

We propose that one of the benefits of precise allorecognition is that, in fused colonies, it limits the unit of selection
in

composed of

between allogeneic colonies of

B. schlosseri leads to costs rather than benefits in

also with colonies that rejected alloge-

to chimeric individuals

at a

terms of germ and somatic cell

Studies of fused allogeneic colonies of the protochordate

neic neighbors, colonies that fused with neighbors incurred

reduced fitness

in

parasitism (Stoner and Weissman, 1996; Rinkevich, 1996;

following life-history traits under natural field conditions:

growth, age and size at first reproduction, and egg production (fecundity). When compared with isolated colonies,

some cohorts

may come

different genotypes also

cost to individuals,

mentally assessed the effects of allogeneic contact on the

in

L.

However, fusion of

may

population of the colonial ascidian Botryllus schlosseri in

Monterey Bay, California, more than 20% of all colonies

and

AND IRVING

Ramat Can, Israel, and Interuniversity Institute for

and 'Department of Pathology, Stanford Medical School,
Stanford, California 94305-5323

Abstract.

in

Life Science Department, Bar-llan University;

Marine Science, P.O. Box 469,

occur

death and resorption of

all

is

the

zooids (colonial units) of one

colony, and the survival of the zooids of the other colony for

up

closely related kin.

to

many weeks

(Rinkevich and Weissman,

after fusion

1992a, b). In nature,

however, resorption

is

not the inevita-

ble conclusion of fusion prior to the onset of reproductive

Introduction

competence (Chadwick-Furman and Weissman,

Further, the resorbed partner also

Tissue fusion and chimera formation between allogeneic

individuals occurs in sessile invertebrates such as sponges

and Loya, 1990),

Rinkevich. 1994; Hidaka
(Ilan

corals
et al..

til.,

and Weissman, 1996; Stoner

1997),

Previously

Thus, the genetic

may be more

nature

in the laboratory.

reported on seasonal variation

in life his-

Monterey Bay, California (Chadwick-Furman and Wiessman, 1995b). Here we determine natural frequencies of
allogeneic contact in the same field population, and assess
the resulting impacts on life-history traits in this colonial

3 July

whom

we

in

tory traits of B. schlosseri colonies in a field population in

and increased survival following partial predation

(Buss, 1982; Grosberg and Quinn, 1986; Sabbadin. 1994).

tion,

*To

et al., 1999).

composition of chimeric colonies

complex than previously observed

between kin may confer benefits on colonial invertebrates

due to increased body size, early onset of sexual reproduc-

2002; accepted 8 July 2003.

correspondence should be addressed. E-mail: furmants

germ

of the resorbing partner in chimeras under both

laboratory and field conditions (Pancer et al., 1995; Stoner

and protochordate ascidians (Chadwick-Furman and Weissman. 1995a: Rinkevich. 1996). The formation of chimeras

Received

1995a).

parasitize the

cell line

(Chadwick-Furman and
1997; Frank et

may

ascidian.

We

also describe the

morphology and

chimeric colonies under natural

mail.biu.ac.il

133

field conditions.

stability

of

134

N. E.

CHADWICK-FURMAN AND

WEISSMAN

L.

I.

Materials and Methods

Under

field conditions, individuals

of the cosmopolitan

ascidian Botiyllits schlosseri pallas form compact, discshaped colonies (Fig. la), which occur in protected shallow

marine environments, such as bays, harbors, and marinas, in

temperate areas of both the northern and southern hemispheres (Chadwick-Furman and Weissman, 1995b, and references therein). Our experimental studies were conducted
in the Monterey Municipal Marina, Monterey Country, Cal-

(3637.4'N;12154'W), where colonies of B. schlosdominant component of the fouling community on

hard submerged surfaces (Chadwick-Furman and Weissifornia

seri are a

man, 1995b).

We

observed colonies of B. schlosseri on submerged

columns and docks

the surface to

between

contacts

in the

Monterey Marina

m and determined the

these

colonies

depths from
frequencies of natural

and

at

other

encrusting

macroorganisms. This survey was conducted during November 1990, in the season of low abundance of fouling
organisms in the marina (Boyd et til.. 1986; Carwile, 1989);
thus our estimates represent minimal contact rates. Three

hundred and nine colonies of B. schlosseri were observed

the

marina for determination of

To

test the effects

traits in B. schlosseri,

in

their contact status.

of allogeneic contact on life-history

set up three treatments using each

we

of four cohorts of newly settled offspring from field-collected colonies. The four cohorts settled on 19 May 1990, 3
July 1990,

15

October 1990, and 25 January 1991 (after

In each cohort,

Chadwick-Furman and Weissman. 1995b).

newly settled, one-system colonies of 1-7 zooids each

one circular system of zooids. Fig. la), were either
isolated

on

(2)

plates,

placed

in

(1)

incompatible pairs that

rejected each other, or (3) placed in compatible pairs that

We

fused.

distinguished between fusible and incompatible

by placing the small, one-system colonies into contact

and observing the outcome. We used only colony pairs that
pairs

established contact during the one-system stage of develop-

Bulbous ampullae, or sacs of the circulatory system, are visible around the
perimeter of the colony. This colony settled in May 1990. began sexually
producing eggs in August, and produced a total of 683 eggs in four clutches
died in September. (B) Fused chimeric colony at 142 days old.
genotype, according to developmental characters (see text), conof 151 zooids; the right genotype, which is slightly darker, consists of

before

The
sisis

it

left

215 zooids. The

members of

this

line

of fusion of their tunics

chimera

is

visible at center.

October 1990. came

settled in

Both

into contact

and

January 1991, and died simultaneously in March 1991 at 149 days

old, without having produced any eggs. (C) Pair of rejecting colonies at 68
fused

Figure

1.

grown under

i-.onies

of the ascidian Btitryllux xchloxxcri that were

days

in

old.

Both colonies

settled in

Monterey Bay.
colony at 49 days old,

rejected during June 1990.

consisting of 70 clonal units, teimed zooids, that are arranged into six

colony, which consists of

three Upe.s oi allogeneic contact conditions in

California. Scale

.bar-.

circular groups (systems) of

in a clear

mm.

(A)

An

isolated

10-14 zooids each. The zooids

are

embedded

gelatinous tunic and connected by a closed circulatory system.

Along

May

1990, and

came

into contact

and

their interacting borders at center. 25

pairs of blood-system ampullae are in allogeneic tissue contact.

The

right

process of
colony, which consists of 89 zooids, died one

129 decaying zooids.

is

in

senescing and dying. The left

week later at 75 days old. Neither colony produced eggs.

the

ALLOCONTACT AND ASCIDIAN FITNESS

merit. In each cohort, offspring from 10 field-collected
colonies were assigned randomly to each of the three treatments (n = 8-36 newly settled colonies per treatment). A
total of 274 colonies from all cohorts were monitored.

Each experimental colony or

on

LI

glass plate measuring 5.0

attach firmly for

week

these

traits.

135

Log-transformed values of

had

approximately equal
groups within each cohort, so

all life-history traits

between

variances

treatment

ANOVA tests were applied to

the data.

pair of colonies was placed

7.5

cm

and allowed to

in the laboratory (after

Results

Rinkevich

and Weissman, 1992a; Chadwick-Furrnan and Weissman.

Frequencies of natural contacts

1995b). Colonies that did not firmly attach to the plates, or

observed high frequencies of natural contact between

colonies of Botiyllus schlosseri and other encrusting macro= 309)
organisms. About one-third of all colonies (28.2%, n

that

appeared damaged, were removed from the study

point. All well-attached colonies

the marina field

schlosseri

B.

site, in

grow

at this

were then transferred

to

an area where abundant colonies of

naturally on fouling surfaces.

About

every 7 days, depending on the time of year, all the zooids

in each colony passed through an asexual growth cycle
(hereafter termed "cycle"). During each cycle, the zooids

produced buds, then shrank and were replaced by their buds;

new asexual generation of zooids was formed in each

thus a

colony.

To examine

cycle-related life-history traits, every

collected all the experimental colonies, ob-

4-7 days we

served them under a dissecting microscope in the laboratory, and returned them to the field within a few hours (after

Chadwick-Furman and Weissman. 1995a. b).

We examined the following life-history parameters
each colony:

growth

rate

for

of somatic tissues, as measured

We

contacted

encrusting bryozoans. Many colonies of B.

schlosseri contacted the other colonial ascidians Botrylloides

violaceous

or

(23.6%)

macdonaldi

Diplosoma

(4.8%), or individuals of solitary ascidians (5.5%). In addition, 21.4% of Botrylliis schlosseri colonies occurred in
allogeneic contact with conspecifics. Only one colony was
observed to contact macroalgae (0.3%), and some colonies

were isolated from contact with other

sessile

macroorgan-

isms (16.2%).

Morphology and growth

Colony morphology was similar

in all

imental treatments. All colonies were

cohorts and exper-

flat

and disc-shaped

by the number of clonal units (zooids. Fig. 1) produced per

cycle; (2) age and size at sexual maturity, defined as the

when small, with closely spaced groups of zooids

As they grew, some of the colonies developed

beginning of egg production; and (3) sexual reproductive

output, as measured by the number of eggs produced by
each zooid during each cycle, the number of cycles in which

outlines, but the zooid systems

eggs were produced (# clutches), and the total number of

eggs produced by each colony throughout its lifespan (fecundity) (after Sabbadin and Zaniolo, 1979; Sabbadin and

systems straddled the area of initial fusion (Fig. Ib). The

zooids of all genotypes in fused colonies appeared to grow

Astorri.

We

1988; Chadwick-Furman and Weissman. 1995b).

assigned zooids

in

chimeric colonies to genotype on the

basis of morphological and developmental characters, such

as their relative positions in the chimera, the number of buds

some cases, color patterns (after Chadwick-Furman and Weissman, 1995a; Yund et til., 1997).
Since colonies were observed every 4-7 days, we counted
directly the number of buds produced by each zooid at each
cycle, and thus accurately assigned each new budded zooid
produced, and

to original

in

colony genotypes

All statistical analyses

in

chimeras.

were performed using STATA,

version 7.0 (Statacorp. 2001). Effects of allogeneic contact

treatment on life-history traits were examined only within

each cohort, since between-cohort comparison of

life-his-

were made previously (Chadwick-Furman and

Weissman, 1995b). For life-history traits that were examined on a per-cycle basis (i.e., number of zooids produced
tory traits

per cycle and

above),

colony,

number of eggs per zooid per

cycle, see

we measured the value for each cycle within a

but we present only the mean of these values for

each colony. Thus,

at

one-way model was used

in

analyzing

(Fig. la).

irregular

remained compact and close

together (Fig. Ib. c). In fused chimeric colonies, the area of

fusion became barely visible over time, and some zooid

constantly and to coexist in chimeras during their entire

lifespan (Fig. Ib). We did not observe any shrinkage or

somatic resorption of one genotype by another in chimeras.

Until the time of chimeric colony senescence and death,
robust blastozooids from

all

partners appeared to coexist

within a single fused colony (Fig. Ib).

Colonies that contacted noncompatible partners underwent rejection reactions that persisted along an extensive

border of contacting tissues (Fig.

Ic).

As colonies grew,

the

contact area expanded along this border, and the number of

points of rejection increased. Up to 15 points of rejection

were observed during each sampling period throughout the

lifespan of rejecting colonies. All rejecting colonies maintained a long, continuous border throughout their lifespans,
until one of them senesced and died (Fig. Ic). Pairs of

grew actively, and

away from each other.

rejecting colonies were compact,

retreated nor grew

Colonies grew
culture plate, then

ued

to spread

colonies

1).

reached the edges of the glass

grew around

the plate edges,

over the back sides of the

filled all

plate (Fig.

until they

neither

plate.

and contin-

None of

the

of the space available on both sides of the

136

N. E.

ponentially, regardless of treat-

Juvenile colonies givw

ment

(Fig.

January and October, exponential

.:g time of 3-5 cycles (= 32 to 64
cohorts, colonies in the isolated treat-

Durir

2).

growth began

days. Fig. 2).

ment reachf

:;ie

tern persis...

even

,.;

CHADWICK-FURMAN AND

largest

maximum

in the

size (Fig. 2). This pat-

October cohort,

in

which some

isolated colonies experienced partial predation during cycle

9 that reduced their size to almost zero, after which they

recovered and became the largest colonies

(Fig. 2).

Growth

reproduction in
In

rate
all

2).

significant effect of treatment

rate (Tables

and

2, Fig. 3a). Isolated

colonies grew faster than did both rejected and fused colonies in the cohorts born during January and May (Table 2).

oooo

L.

WEISSMAN

two of the cohorts, rejected colonies also grew

did colonies that fused to

January

-Rejected

yses of life-history differences among colonies that reached

sexual maturity (Fig. 3, Tables 1 and 2).

Sexual reproduction

There was no effect of allogeneic contact treatment on the

which colonies reached

age

at

the

May

maturity

cohort,

first

reproduction, except in

where rejected colonies reached sexual

at a significantly later

age than did both isolated

and fused colonies (Tables 1 and 2, Fig. 3b). The age at

which colonies began to reproduce sexually appeared to be

May

10000

Isolated

1000

Rejected

Fused pairs

-Fused pairs

10

15

20

25

2). In the

October cohort, none of the fused colonies grew past the

juvenile stage, and so were not included in statistical anal-

Isolated

1000

faster than

become chimeras (Table

cohort

slowed upon commencement of sexual

cohorts (Fig.

most cohorts, there was a

on colony growth

in the

In

I.

30

10

10000

July

15

20

25

30

October
isolated

Isolated

1000
Rejected

Age

(cycles)

Figure
Typical growth curves of colonies of the ascidian Botrylhis schlosseri for three allogeneic contact
treatments and four cohorts in Monterey Bay. California. The shape of growth curves varied widely within each
treatment, so mean and error values cannot be shown clearly here. Thus, only the largest colony in each treatment
is shown for each cohort (for mean
growth rates, see Fig. 3a). Note that colony size is plotted on a logarithmic
2.

:ile. Arrows mark the commencement of sexual

reproduction (egg production) for the first colonies to reach
maturity in each cohort. Data on isolated colonies were published previously as Figure 1 in Chadwick-Furman

and VvVissman (I995b).

Rejected

Fused pairs

Fused pairs

ALLOCONTACT AND ASCIDIAN FITNESS

Table

One-wa\ ANOVAs of life-history

grown

in

traits hetu'cen

Monterey Bay, California

Life-history

trait

137

allogeneic contact treatments within each of four cohorts of the colonial ascidian Botryllus schlosseri

138

N. E.

CHADWICK-FURMAN AND

Table 2
Tnk.e\-Kramer

mii!i

!i

"'act

traits beftveen all

the colonial a-

m)

California

Life-history

trait

TO h wv for differences in life-history

treatments within each of 4 cohorts of

llus schlosseri

grown

in

Monterey Bay,

1.

L.

WEISSMAN

AU.OCONTACT AND ASCIDIAN FITNESS

D Isolated

H Rejected

139

Fused

(18X6X12)

C5K6IC5)

Cl

1(131(16)

(/X3)(0)

6000 i

??4000

= 2000

January

October

July

October

January

Cohort
Figure

3.

Variation in life-history

traits

among

three allogeneic contact treatments and four cohorts in the

Monterey Bay, California. Note that for fused colonies, traits are
presented for each genotype within the colony. Means plus one standard deviation are shown. Sample sizes for
all life-history traits are given in parentheses in graph A. Sample sizes are low in some groups due to mortality

colonial ascidian Botryllus .schlnsseri,

grown

in

some colonies before reaching sexual maturity (compare sample sizes with those in Fig. 5). Data on isolated
colonies were published previously as Figure 2 in Chadwick-Furman and Weissman (1995b).
of

Monterey are even higher than those presented

ceous were especially high at our site (see Results). Yet,

because our survey of contact frequencies was based on a

organisms

one-time observation, which inherently underestimates contacts throughout the life of a colony, lifetime contact rates

one of the cohorts of Botnilux schloxseri (born on

between colonies of Botrvllus xchlosseri and other

Botrylloides violaceous results in a level of fecundity inter-

sessile

at

here (see Results).

Our

limited manipulation of colonies in

15

October 1990) indicates

that

xenogeneic interaction with

140

N. E.

CHADWICK-FURMAN AND

100 T

10

I.

L.

WEISSMAN

May

100 i

January

.
-

1o

Isolated

(N=28)

Isolated

Rejected (N=14)

Fused (N=36)

Rejected (1M=8)

Fused (N= 16)

(N=35)

7
30

25

10

20

15

25

30

u
100 q

10

July

100

Isolated

(N=26)

4.

reproduction

15

20

30

25

in

each cohort. The

last

point

in

10

each

(x

Chadwick-Furman,

pers.

obs.; compare with October cohort in Fig. 3f], Thus, xenogeneic contact appears to affect colony fecundity, but not as

severely as allogeneic contact.

may

The process

fitness

of colonies following fusion or re-

from energetic or physiological costs

recognizing and reacting to non-self tissue.

result
ith

oi

30

(cycles)
Arrows

indicate the

line represents the last surviving

9 xenocontacted colonies of Botryllus schlosseri

associated

25

in

Monterey Bay,

commencement of

sexual

colony of each group. Note

plotted

that survived to maturity, N. E.

jection

20

15

on a logarithmic scale. Data on isolated colonies were published previously as Figure

Chadwick-Furman and Weissman (I995b).
is

number of eggs produced = 1383 + 769

The reduced

(N=33)

Survivorship curves for colonies of the ascidian Bittnllits schlosscri grown

mediate between those of isolated and allocontacted colo-

Fused (N=12)

Fused (N=24)

that survivorship

SD), n

Rejected (N=14)

Rejected (N=28)

California, in four cohorts and three allogeneic contact treatments.

3 in

Isolated

Age
Figure

10

nies [total

October

interaction along the borders of rejecting

colonies involves extensive tissue

damage and resource

demand on both
reviewed

colonies (Scofield and Nagashima, 1983:

Rinkevich, 1992). In addition, competition between somatic and germ cell lines within fused chimeras
in

may draw

heavily on the physiological resources of the

involved
(Buss, 1982). Colonies that are isolated
partners
also

from allogeneic contact do not face these costs.

The lack of resorption observed here in field-raised

chi-

meras of Botiyllus schlosseri is in striking contrast to previous results from laboratory studies (Rinkevich and Weiss-

man, 1987, 1992a,

b;

Pancer

et al.,

1995).

The reduced

chimeric stability of laboratory colonies has been demonstrated by growing genetically identical replicates of chime-

ALLOCONTACT AND ASCIDIAN FITNESS

somatic or germ
However, germ

D Isolated
CD

Rejected

141

cell

parasitism as a result of colony fusion.

cell

parasitism has been reported in this

species (Rinkevich and Weissman, 1987; Sabbadin and

Astorri. 1988; Pancer et al.. 1995), and recent work has

Fused

verified that

occurs

it

in

both male and female gametes

capable of fertilization (Stoner and Weissman, 1996; Stoner

et al., 1999). The process of germ cell parasitism, in which

one partner

in a

chimera uses the somatic resources of the

other to produce its own germ cells, may alter the relative
fitness of fused genotypes in chimeras (Buss, 1982; Stoner

and Weissman, 1996; Stoner et al., 1999; Weissman, 2000).

However, because the fitness of fused pairs of genotypes

was

less than half that of isolated colonies in all cohorts

genotypes comchimeric colonies was less than that of genotypes in

(Fig. 3f), the reproductive output of all the

'

(281(81(161

(351(141(361

(261(28)124)

(33H14M12)

May

July

October

January

bined

in

isolated colonies. Thus,

amount of

relative

germ

cell parasitism

fitness lost

due

may

alter the

to fusion in chimeric

Cohort
colonies, but cannot prevent an overall reduction in fitness
percent survivorship to first reproduction among
four cohorts and three allogeneic contact treatments of colonies of the

Figure

Variation

5.

in

ascidian Bottyllus schlosseri

grown

in

Monterey Bay, California. Sample

sizes for each treatment are given in parentheses.

to fusion. Even if germ cell parasitism were extensive in

the chimeras tested here, chimera formation causes reduced

due

fecundity, regardless of which genotype dominates (Fig.

30%

chimeras examined by Stoner and

same Monterey marina site, little or
no germ or somatic cell parasitism was found. Thus, the
3f). In

of the

Weissman 1996)
(

ras

under

field

versus laboratory conditions (Chadwick-

1995a). The present results show

conditions in Monterey, the partners of a
chimera appear to grow in a stable manner and do not
undergo somatic resorption. Previous field studies indicate a

Furman and Weissman,

that,

under

field

high level of environmentally dependent plasticity in fitness-related life-history traits of B. schlosseri (Chadwick-

Furman and Weissman, 1995a; Yund et

The reduced reproductive success of

al.,

1997).

field

at the

values presented here for genotype-specific measures of

fitness (Fig. 3) may represent realistic estimates for at least

some chimeras

that retain a stable genetic

composition

in

the wild.

drawback of the present study is that we could not set

as
controls, undissected pairs of isogeneic colonies, to
up,
determine whether isogeneic contact affects fitness. Thus,

reveals effects of

an evaluation of the actual costs of allogeneic contact per se

is problematic. However, set-up of this control group would

allogeneic contacts on sexual reproduction as well as on

somatic tissue production. Recruitment of larvae at

have required dissecting apart and re-uniting systems from

multi-system colonies, thus introducing further manipula-

isolated colonies (Fig. 3

Monterey

in the

and Table

springtime

may be

1 )

interacting versus

derived from a small

tion of all colonies in this experiment.

As

the colonies grew,

number of parent colonies that overwinter (Carwile, 1989;

Chad wick- Furman and Weissman, 1995b). Thus, the costs
of interactions in this experiment would have resulted in

they produced lobes of tissue that contacted along their

edges and fused along the undulating margins of the colony

reduced representation of the offspring of winter allo-contacting colonies in the summer bloom.

affected fitness,

Allogeneic interactions

do not

colonies in most cohorts (Fig.

5).

3b and 4) and lower survivorship

alter the survivorship

The longer

of

lifespans (Figs.

(Fig. 5) of colonies during

in

treatments (Fig. Ib. c). Thus, if isogeneic contact

it did so
equally in all treatments here.

all

We

that egg production in fused colonies is

reduced
(Fig. 30. possibly due to competition begreatly
tween the genetically different individuals that fused to

show here

make up

Thus, one benefit of precise allorecogspecies may be that it limits the unit of

that colony.

summer, appear to be due to a

compared
of
slowing
colony growth and development during low
temperatures in the winter in Monterey Bay (Boyd et al.,
1986; Chadwick-Furman and Weissman. 1995b). As found

nition

whole-colony senescence causes the death of

most colonies (Chadwick-Furman and Weissman. 1995a, b)
and is genetically controlled (Rinkevich et ui. 1992).
At the time of our experiments, we did not have markers

Because of the high polymorphism of the Fii/HC gene locus

winter, as

to

in past studies,

in

this

chimeras composed of closely related kin

and
Quinn. 1986; Rinkevich and Weissman.
(Grosberg
1987; Stoner and Weissman, 1996; Stoner et al.. 1999).
selection

(that
et

to

permits fusion rather than rejection to occur; Scofield

1982), fused individuals in the wild most likely

al.,

to identify the

represent kin rather than a random assortment of genotypes

(Grosberg and Quinn. 1986). In Botryllus schlosseri. the

metes

proportion of fusions occurring between siblings

in

genotypes of blood cells, bud cells, or gathe fused colonies, and so we did not test for

is

higher

142

N. E.

CHADWICK-FURMAN AND

than between nonsiblinus (Scofield et ai, 1982;

til.,

colonies of fusee

competition

sperm

thai

by Fu/HC

:i

rfti

r^ed

Magor

et

to genetic inheritance for chimeric

would be the outcome of germline

1999). Thus. Mr

mother colony and the diverse

the

her. In addition to kin fusion, regulated

matching, kin cosettlement

is

WEISSMAN

L.

I.

acknowledged for advice on statistical analysis.

was
provided by a Frederick B. Bang Scholarship
Funding
from the American Association of Immunologists, and PHS
Grant CA09302 awarded by the National Cancer Institute.
gratefully

DHHS.

encouraged by

limited dispersal of tadpole larvae from the maternal colony

and nonrandom cosettlement according to shared Fu/HC

genotype (Grosberg and Quinn. 1986). A common selected
these chimeras

trait in

(Weissman

et

is

1990). Kin selection

til.,

shared genes other than the selected

common

Fu/HC

allele-sharing at the

to these siblings.

would

Fu/HC

locus

act also

on

types that are

in these

Reproductive outcomes

could be as complex as the outcomes of selective resorption

germ

cell

parasitism that generate skewing from that

simple representation (Pancer et til.. 1995; Stoner and

Weissman, 1996; Stoner et til., 1999; Weissman, 2000).

No

matter whether allogeneic colony contact results in

if it leads to reduced fitness, as measured

fusion or rejection,

by growth and fecundity, with no increase in survivorship,

why have these organisms developed and maintained an
elaborate system of allorecognition? Perhaps, in this spe-

based allorecognition

cies, genetically

is

It

nonadaptive.
linked to other processes that are adaptive, and thus
have evolved as a by-product of processes such as disease

may be

recognition (Buss and Green. 1985; Magor ct til., 1999) or

gametic compatibility (Scofield et til.. 1982). However, the
reject nonrelated colonies, and to
fuse only with closely related kin that share alleles at the
Fii/HC locus, may be directly beneficial in that it reduces
ability to recognize

the costs of

germ

and

cell

parasitism in colonies (Stoner et

til.,

1999).
fusion, and

to

I.L.W.

C., S. K. Brown, J. A. Harp, and I. L. Weissman. 1986.

Growth and sexual maturation of laboratory-cultured Monterey Botryl-

Boyd H.

development

of reproductive competence in chimeras are not limited to

protochordates, and may be important selective factors in
other sessile organisms as diverse as fungi, sponges, and

91-109.

lus schloxseri. Biol. Bull. 170:

W.

Somatic

1982.

parasitism and the evolution of somatic

cell

Pmc.

tissue compatibility.

USA

Nut/. Acini. Sci.

79: 5337-5341.

W., and D. R. Green. 1985. Histoincompatibility in

brates: the relict hypothesis. Den Com/). Imnninol. 9: 191-201.

Buss,

I..

verte-

Settlement of larvae, colony growth and longevity

of ascidians and the effect on the species composition

Carwile, A. H. 1989.
in three species

of a marine fouling community. Ph.D. dissertation. University of Cal-

Los Angeles.

ifornia,

Chadwick-Furman.
nition

system

N.,

in a

and

A complex

B. Rinkevich. 1994.

allorecog-

reef-building coral: delayed responses, reversals and

nonlransitive hierarchies. Cera/ Reefs 13: 57-63.

Chadvvick-Furman, N.

E..

and

Weissman. 1995a.

L.

I.

Life history

chimeras of the colonial ascidian Botiylhis schlosseri.

Pmc. R. St: Loml. K 262: 157-162.
plasticity

in

Chadvvick-Furman, N.
and

E.,

and

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senescence of Botryllits

I..

Weissman. 1995b.

Life histories

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(Chordata.

.iclili>.\seri

Monterey Bay. Biol. Bull. 189: 36-41.

Frank, U., U. Oren, Y. Loya, and B. Rinkevich. 1997.
in the coral

Alloimmune

Grosberg, R. K., and

is

St\lphi>ra /nstillata

F.

.1.

The genetic control and

Quinn. 1986.

consequences of kin recognition by the larvae of a colonial marine

invertebrate. Nature 322: 456-459.

Hidaka. M., K. Yurugi, S. Sunagawa, and R. A. Kinzie III. 1997.

Contact reactions between young colonies of the coral Pocillopora
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dtiinicornis.

M., and Y. Loya. 1990.

He Tomaso,

G., A.

Magor, B.

in

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L.

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Ontogenetic variation

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279-286.

and

B. Rinkevich,

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in colonial tunicates: protection against

Allorecognition

predatory cell

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Pancer, Z., H. Gershon. and B. Rinkevich. 1995.

Coexistence and

cnidarians (reviewed in Buss. 1982; Rinkevich and Weiss-

possible parasitism of somatic and

man, 1987; Pancer etui, 1995). Our findings that allogeneic

contact, and especially chimera formation, reduce individ-

colonial urochordate Botiyllits schlosseri. Biol. Bull. 189: 106-112.

ual fitness

under natural

field

conditions

may have broad

implications for the evolution of allorecognition systems.

We

a hypothesis for links

and resulted from these studies.

We

also

thank Kathi Ishizuki and Kurlu Palmeri for assistance

Bart!

that

improved

the manuscript.

in

and Robert Lauzon for dis-

cussions, and Uri Frank and Buki Rinkevich for

between natural

comments

Karen Tarnaruder assisted

with the graphics. Prof. Maureen Lahiff of the School of

Public Health at the University of California at Berkeley is

chimeras of the

in botryllid

and evolutionary ecology.

B.,

li.

and

I..

I.

E.v/>.

Rinkevich,

B.,

63:

colonial urochordates:

din.

Weissman. 1987.

Iniiiiiiitoxcnct.

Chimeras

in

13:

61-69.

colonial inver-

and germ-cell parasitism?

17-134.

and

homogeneous

in

tissue transplantation, allogenet-

tebrates: a sMicrgistic symbiosis or somatic-

thank Buki Rinkevich for discussions and collabora-

Simona

Bi- versus multichimcrism

Rinkevich, B. 1996.

SvmbioM.-, 4:

data collection.

cell lines in

ascidians. .4m';n. Biol.

ics

tions that led to

germ

Aspects of the incompatibility nature

1: 17-28.

Rinkevich, B. 1992.

UinkiMi

Acknowledgments

in

achieved through three

distinctive stages. 4 months post-metamorphosis. Proc. R. Sot: Loud. B
264: 99-104.
maturation

Ilan,

The phenomena of cosettlement.

Grant CA42551

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Reference: Bio/. Bull. 205: 144-159. (October 2003)

2003 Marine Biological Laboratory

Effect of Disturbance on Assemblages: an

Using Porifera
J.

BELL

J.

AND

'*

Example

BARNES 2

D. K. A.

Department of Zoology ami Animal Ecology, University College Cork, Lee Mailings, Co. Cork, Ireland;
2
and British Antarctic Sun>ey, N.E.R.C., High Cross, Madingley Road, Cambridge CB3 GET, United Kingdom

Abstract.

number of

Introduction

Extensive sponge assemblages are found in a

Lough Hyne Marine Nature Reserve.

habitats at

These habitats are unusual

in

The development of any community

experiencing a range of envi-

ronmental conditions, even though

by small geographic distances (1-500 m), reducing the
sites (e.g.,
possibility of confounding effects between study

to

both rock and

of 96 species) were

cliff habitats.

ence some similar conditions

total

found in the least-developed assem-

disturbance

where

had

dif-

to large rocks,

compared
of which had similar assem-

small rocks, and panels, all

blages irrespective of environmental

ences

in

conditions.

assemblages were partially attributed

morphology (shape),

as certain

morphologies

cent species) were excluded from

mechanises

were

2-D rock

considered

also

(e.g.,

responsible

To whom correspondence

stitute

of Biological Sciences.

May

arbores-

the

disturbance

is

smaller rocks are more likely to be

moved

causing differences

(all

in

community

than larger rocks,

species) and assem-

man. 1997).

habitats.

Sponges are an important component of hard-substratum

communities throughout most polar (e.g., Dayton, 1978).
temperate (e.g., Hiscock et al., 1983; Bell and Barnes.
2000a), and tropical regions (e.g., Alcolado, 1990). There is

2003.

Building.

less well-developed commuexperiencing slight current flow,

reduced (Maughan and Barnes,

may have

newly created habitats or space (Lilly et al., 1953; Osman.

1977; Sousa, 1979; Barnes et al.. 1996; McCook and Chap-

Other

should be addressed. Current address: In-

Edward Llwyd
SY23 3DA.

high,

often colonized by fast-growing, opportunistic, and r-straof

tegic species that are able to quickly take advantage

sponge

for

is

blage (component phyla) composition (Barnes et al., 1996).

Unstable or highly disturbed (e.g., small rocks) habitats are

Differ-

habitats.

sponge assen, vuges associated with different

Received 27 August 2001: accepted 23

to

temperature,

climax community states may be achieved only under certain environmental conditions. In the case of rocks, size may
also be important to community development because

analysis and Multi-Dimensional Scaling, which enabled differences and similarities between sponge assemblages to be
sites

rate,

2000a), even though they are separated by less than 300 m.

The same may be true for sublittoral cliff habitats where

blages inhabiting cliffs varied considerably. Assemblage

composition was visualized using Bray-Curtis similarity

from high- and low-energy

flow

nities than rocks in areas

tween high- and low-energy environments, whereas assem-

ferent assemblage compositions

(e.g.,

food concentration) but are at different stages of development. For example, rocks in areas with fast currents, where

blages (slate panels) and were common to all habitats.

Sponge assemblages on rocks and panels varied little be-

visualized. Cliffs

as

species) are well developed (ma-

of development in proximity to one another. Lough Hyne is

such an example, where adjacent communities may experi-

com-

Seven species (of a

some marine environments, such

(all

such as ice-scoured polar shores, all communities tend to be poorly developed (young). Yet there are
other localities that contain communities in various stages

assemblages varied considerably between habitat types such

mon

In

ture); in others,

energy environments that were used to represent two measures of disturbance (flow rate and habitat stability). Sponge
(25 species) of species reported were

1979).

caves, communities

and temperature). Sponge assemblages

were examined on ephemeral (rocks), stable (cliffs), and
artificial (slate panels) hard substrata from high- and low-

26%

mature or

is

Jackson,

silica concentrations

that only

(e.g.,

controlled by a suite of biological and physical

factors that may be closely related and interlinked (Buss and

young)

they are only separated

The University of

a wide body of literature that considers the influence of

Wales. Aberystwyth, Ceredigion

144

SPONGB COMMUNITY COMPOSITION

environmental parameters on the composition ot sponge
assemblages (e.g., Wilkinson and Cheshire. 1989; Alcolad,
1990; Alvarez et

Witman and Sebens,

1990;

common

flow rates). Also, the use of a

between

panel, rock, and cliff)

sites

with

different environmental conditions (flow regime) gives a

1990). There are also examples

second environmental gradient based on flow-rate-generated disturbance (rather than habitat stability). This study

ul..

of studies comparing sponge assemblages on large (1001000 km) spatial scales (Maldonado and Uriz, 1995; Hooper

2002) and on smaller scales (2-20 km) between

et al..

fast
(i.e..

1990; Schmahl.

1990; Diaz et

til.,

(e.g.,

substratum

gime

145

attempts to answer four questions: ( 1 ) How do sponge

assemblages vary with environmental conditions and habitat

How

does the composition of sponge assemblages vary with water flow rate compared with different
rock sizes; both are surrogate measures of disturbance, so do

sponges inhabiting mangrove pools (Riitzler et til.. 2000).

However, comparisons between different habitat types (e.g.,
between loose rock and cliffs) under fixed environmental

stability? (2)

conditions or on local scales (hundreds of meters) where

they have similar consequences? (3) Are there discriminating species for (local) habitats of differing stability and flow

confounding effects are reduced are less common.

Studies of sponge assemblages suggest that a number of
factors
physical factors control species distributions; these
include water flow rate (Bell and Barnes, 2000a), sedimen-

(Konnecker,

tation

depth (Alvarez
light (Sara et

et

al.,

til..

(Storr,

1976),

Witman and Sebens,

1990).

nutrient

1973).

1990;

levels

1978; Cheshire and Wilkinson, 1991

habitat availability (Konnecker, 1973; Barthel

),

For example,

ical characteristics.

flow,

where sedimentation
on

falling

own

amount of sediment

inclined, vertical, or overhanging cliff surfaces

varies considerably, resulting in different assemblages on

c).

not as well studied, biological factors are also

Although

known

to

composition of sponge assemblages (see

Paine. 1974; Wulff, 1995, 2000), and predation may also
limit the local distribution of sponge species (Dunlap and
influence

the

Pawlik, 1996; Wulff. 2000; Bell, 2001). Rich sponge as-

semblages on

sublittoral cliffs at

Lough Hyne have been

the

focus of recent studies, but abundant and diverse sponge

populations also occur on the undersides of loose rocks
1953: van Soest and Weinberg, 1980; van Soest
1981). At many sites within Lough Hyne, these two

(Lilly et
et al.,

til..

m, providing the
investigate differences between sponge as-

habitats can be separated

opportunity to

semblages occurring
els

of

stability,

by

less than

in habitats

experiencing different lev-

without the confounding effects created by

larger spatial scales.

The high

overall diversity and richness found on hard

Lough Hyne, coupled with the large range

substrata within

of local habitat stability, results in assemblages at various

stages of development existing in proximity. Such environ-

mental characteristics provide opportunities to examine the

contribution of certain taxa (in this case sponges) to the
overall

community. This study investigates assemblages

inhabiting three

substratum types

most ephemeral), rocks, and

artificial

cliffs (the

most

stability?

Materials and Methods

Stutl\ site

predefined physof slight water

each surface type (see Bell and Barnes, 2000b.

extremes of flow rate and habitat

and Tendal.

at a site

high, the

is

in

assemblage composition

similarities or differences in

and

1993). Habitats classified within these physical parameters

often contain many
(e.g., fast water flow at a depth of 20 m)

smaller or cryptic habitats with their

Are there consistent

rates? (4)

panels (the

stable)

which

represent a qualitative series in terms of habitat stability

(i.e.. amount of disturbance). The degree of development of

sponge assemblages at different levels of habitat stability

can be investigated within a particular environmental re-

Lough Hyne Marine Nature Reserve

(Fig.

is

a small

(0.5 km ) temperate sea lough on the southwest coast of

Ireland (5129'N, 918'W). It is characterized by a large

number of

habitats within a small area (Kitching, 1987).

Habitats range from current-swept cliff faces to soft-sediment basins where water currents are slight. The lough is

connected to the adjacent Atlantic coast by a shallow and

narrow channel (The Rapids). This constriction results in an
unusual tidal regime whereby water flows into Lough Hyne
for about

4 h and out

The squeeze causes

for 8 h.

fast current

(>250 cm

s~') in the eastern parts of the lough

during inflow, but only slight surface currents in the vicinity
of the rapids during outflow (Bassindale et al.. 1957). As
velocities

water moves from east to west across the lough, there is a

quick reduction in current flow rate with a corresponding
increase in sedimentation.

The small

size of

Lough Hyne means

that

communities

scales (hundreds of

separated on small spatial

meters). Nevertheless, these communities are discrete be-

are only

cause

cliff

and rock habitats

in different parts

of the lough

are separated by soft sediments. Sponge assemblages are

thus isolated and predictable, rather than occurring along a

continuous gradient. Different substratum types (i.e., rocks,

cliffs, and panels) within a specific environment were separated by less than several meters and therefore not discrete.
Panels were sited at Labhra Cliff and Whirlpool Cliff, and

rock habitats were sampled at West Cliff and Whirlpool

Cliff (Fig. 1). West Cliff and Labhra Cliff have similar
sedimentation and rates of water flow, and the sponge
assemblages are also similar. Both sites are characterized by
slight current flow rates

(<5 cm

s~") and heavy sediment

accumulation, which increases with depth. Hard substratum

at both of these sites. Rocks were
extends more than 30

not sufficiently abundant at Labhra Cliff to sample. Whirlunidirecpool Cliff experiences a very fast (>200 cm s~'),
tional flow regime, resulting in high disturbance.

This

site

146

.1

BELL AND

D. K. A.

BARNES

Three panels (forming one panel array) were

attached by cable ties to welded steel bars. The panel array
was positioned with the blue surfaces facing down (to
translucent.

simulate the undersides of a boulder). Bolts

each

were 20
ployed

at

the corner of

frame allowed the panels to be adjusted so they

above the substratum. Panel arrays were de-

steel

mm

at

m. 6 m, and 12

depths of

The

at

Labhra Cliff and

Whirlpool
panels were deployed at the start
of October 1997 and were replaced bimonthly until March
Cliff.

first

2001. Before panels were replaced, they were cleaned using

a razor blade. Each panel was examined under a binocular

microscope, and the number of sponge recruits was recorded. Spicule preparations of sponge recruits were made
to confirm identification. Additional panel arrays were
placed at the same depths, but were not replaced bimonthly.

Each panel was photographed

1,

2, 6,

and 12 months

after

the date of deployment. Photographs were then projected

onto a grid composed of 400 random dots, and the percent-

age cover of sponges on each panel was estimated. The

mean percent cover was calculated for panels submerged at

each time

interval.

Twenty-five rocks were randomly selected within three

size classes

large (501-1000 cm
'

such

examined

cm 2 medium 151-500 cm 2
and very large (1001-2000 cm

small (10-150

that equal

s~'),

),

),

numbers of boulders of each

size

were

does not represent the true boulder size

distribution). The surface areas of both upper and lower

1.

Figure
in

Sites

where sponge rock and

cliff

assemblages were sampled

extends to about 18 m, and current flow rates decrease with

cm

100

were taken from the

s~'

at

18

literature (Bell

(Fig. 2).

Some

data

and Barnes, 2000a) to

enable greater comparison between sponge assemblages

from different habitats within Lough Hyne. These data,

which were included

in

the analysis, concerned

vertical

assemblages inhabiting
sublittoral cliffs at Labhra

sponge
and inclined surfaces on

Cliff, West Cliff, Whirlpool Cliff.

and Bullock Island (an adjacent Atlantic coastal site with a
turbulent flow regime). The tidal range within Lough Hyne
is

about

1.5

this

rock surfaces were measured using a transparent cloth

marked in square centimeters. The number of sponges

(number of patches) for each species on each rock was

recorded. Each species was photographed, and samples
were taken for spicule analysis to confirm identification.

Lough Hyne Marine Nature Reserve.

depth, falling to

(i.e..

Sponge samples were dissolved

in

or bleach as required, and were

concentrated nitric acid,

examined through a com-

pound microscope at high power (X400). All observations

were made between October 1999 and February 2000,
which eliminated fuunistic inter-site differences caused by
seasonal growth or tissue retraction over the study period.
The morphologies of all sponges observed on rocks were
classified within the following

groups (after Boury-Esnault

and Ruizler, 1997; Bell and Barnes, 2000d): encrusting
(EN), flabellate (FL), clathrate (CL), massive (MA), arbo-

rescent

AR). repent (RE), tubular (TU), ficiform (FI),

sive globulose (MG), and papillate (PA).

m.

mas-

Sampling and observation methods

The

Data visualization and

substrata (panels) used were square machined slate panels (15 x 15 cm), prepared and assembled

Turner and Todd

They were used to investigate an ear!> pioneer stage in community succession. A blue
square (10 cm K 10 cm) was drawn in the center of each
as for

statistical

procedures

artificial

1994).

panel with a permanent blue marker pen. Panels were placed

in running water iVr 24 h and then dried; this
process was

repeated twice more prior to deployment.

ground makes

it

The blue back-

easier to identify recruits that are small or

Data were subjected to Bray-Curtis similarity analysis

using hierarchical agglomerative group-average clustering
for all habitats and depths (including data from Bell and
Barnes, 2000a). This analysis was performed using the
unweighted pair group method using arithmetic averages

with the PRIMER program (ver. 5.2.8. Plymouth

Marine Laboratory, Plymouth. England). Data were log
transformed to reduce the importance of extreme
(.v +

(UPGMA)
1

SPONGK COMMUNITY COMPOSITION

147

60
Glannafeen

40
30

20

T3

OT

10

APR 99

MAY 99

JUN 99 JUL

99

AUG 99

SEP 99

OCT

99

NOV 99 DEC 99 JAN 00 FEBOO MAR 00 APR 00 MAY 00

60
50

Goleen

40

c
a
C

30

"-^

20

3
OJ
00

10

APR 99 MAY 99 JUN 99 JUL

99

AUG 99

SEP 99 OCT 99

NOV 99 DEC 99 JAN 00

FEB 00

MAR 00 APR 00 MAY

00

60
50

Labhra Cliff

40
30

20
10

APR 99

MA Y 99

JUN

99

JUL 99

AUG 99

OCT 99 NOV 99 DEC

SEP 99

99

JAN 00 FEB 00

MAR 00 APR 00 MAY 00

60

West

Cliff

50

40
30

c 2?
3

on
20

10

APR 99

MAY 99

JUN 99 JUL

99

AUG 99 SEP 99 OCT 99 NOV

99

DEC 99 JAN 00 FEBOO MAR 00 APR 00

MAY 00

Month
Figure

2.

:
d ') at 6-m depth intervals at tour
sedimentation rates (g sediment m
24
6
m
12
18
(O) 30 m (). Standard error bars
(D)
(T)
()
Lough Hyne.

Annual variation

sublittoral cliff sites within

in

are shown. [After Bell (2001).]

values (rare species). Ordination by non-metric Multi-Di-

mensional Scaling
dissimilarity
analysis.

(MDS

matrix

SIMPER

in

PRIMER)

created from

analysis (in

was undertaken on a

Bray-Curtis similarity
used to de-

PRIMER) was

termine the contribution of each

species to the

average
Bray-Curtis dissimilarity between habitats and sites. This
method of analysis determines which species are responsi-

ble for any differences that occur.

Data were transformed as

for Bray-Curtis analysis. Because sampling areas differed

between habitat types, the area of each habitat and the

abundance of each species were scaled up or down to

standardize sampling areas between sites, depths, and habitats

(sponges m~~).

Patterns of species diversity for sponge assemblages on

148

J.

rocks were described

v-i;!i

1989) informal!'"-

n tion

dent's

tests

we-

(GLM

\)

BELL AND

Shannon and Weiner (Krebs,

H'

Paired Stu-

-neral linear

model analysis of variance

-S/?,/ /',.
d to examine differences between rock

surface typ

the

J.

was used

to

.--.lornied

Whirlpool Cliff. The same method was used to compare the

untransformed relationship between sponge density and
rock surface area. This method compares the average linear
relationship to an average linear relationship fitted to all the

were used

data. Kruskall-Wallis tests

BARNES

than between different habitat types (Table 3). However, the

proportion of species shared between sites was higher for

rock and panel habitats

(>85%)

than for cliff habitats

compare logarithmically

sponge species diversity, richness, and

rock surface area relationships between West Cliff and

(LogH

D. K. A.

to

compare

Sponge morphology

On rocks, encrusting morphologies were the most abundant form (20 of 44 species), followed by massive (15 of 44
species) and repent forms (7 of
data).

44 species)

The remaining species exhibited

(all

pooled rock

cylindrical (Scypha

ciliatum) and clathrate forms (Clathrina coriacea).

On

cliff

the differ-

ences between percentage cover on submerged panels after

1, 2, 6, and 12 months.

were encrusting, 19 were massive, and

6 exhibited repent forms (pooling cliff data). Sponge species
on panels had a wide range of morphologies (given the
surfaces, 30 species

small

Results

number of species), including

Assemblage composition

tubular, repent, encrust-

and clathrate forms. Differences were ob-

ing, massive,

served between the proportions of sponge morphologies

between certain habitats and sites (Table 4). There was a

A total of 96 species were reported during this study, 44

of which were found on rocks and 76 from cliff habitats

considerable difference between the proportion of morphol-

Twenty-five sponge species were shared between

rock and cliff habitats. All but two of the species (Hvmeni-

more abundant

(Table

).

acidon perlevis and Halichondria bowerhanki) of sponge

found on rocks occurred on the undersides. These two
species were often found growing on the fronds of algae as
well as on rock surfaces. Although sponge assemblages
varied considerably between habitat types, there was little
variation between rock sponge assemblages inhabiting the
different sites (extremes of water flow rate) (Table 2).

(Mycale
being found exclusively at Whirlpool Cliff.
The remaining 6 species were only found at West Cliff.
Nineteen of the 44 species found on rocks were exclusive to
rotalis)

rock habitats, illustrating the high degree of species exclusivity within this habitat type. The panels at Labhra Cliff

and Whirlpool Cliff showed no exclusive species, and only

7 species of sponge were reported (Table
). Therefore, no
1

was recorded

for the majority of

sponge

species reported during this study despite the nearly 4-year

duration of panel deployment. All species found on panels
in both rock and cliff habitats. Because panels can
be considered a similar habitat to rocks (but younger in
terms of community development), community age was the

occurred

most

likely determinant of the

assemblage.
cliff

Of the

habitats,

31

total

composition of the sponge

76 species of sponge reported from

species

Barnes, 2()00a) were found

(data

included from Bell and

Bullock Island, compared to

40 species at Whirlpool Cliff and 52 from both Labhra Cliff
and West Cliff (Table 3). Although many of these species
at

were shared between cliff habitats, only 10 of the total 76

species were shared between all cliff habitats. Environmental

the

parameters, therefore, proved important in determining

sponge assemblage

in

cliff habitats.

at the

3).

high-energy

Encrusting forms were

(Bullock Island

cliff sites

and Whirlpool Cliff) than at low-energy sites (Labhra Cliff

and West Cliff), where they were replaced by arborescent

and papillate forms. In rock habitats, high proportions of

encrusting and massive forms were found, with no differences between sites. Panel assemblages were also very
similar in the proportions of morphologies between sites.

Of the

44 species inhabiting rocks, only 7 (16%) differed between

West Cliff and Whirlpool Cliff, with only of these species

juvenile settlement

ogies between cliff sites (Table

Identification of different

sponge assemblages

Small sponges were difficult to identify, (particularly on

panels) and potentially represent an error in the analysis.

However,

this

problem was reduced by the transformation

of the data prior to analysis, which decreased the importance

of extreme values or apparent rare species.
Bray-Curtis similarity analysis and Multi-Dimensional
Scaling (MDS) (Fig. 4) were used to distinguish between

sponge assemblages within different habitats and

identified

five

sites.

analysis
major
groups that showed increasing similarity, as follows: rocks
(all sites and sizes), panels (all sites), Whirlpool Cliff,
Bullock Island, and Labhra Cliff/West Cliff. However, each

Bray-Curtis

major assemblage group had a very low similarity (<25%)

with the others.

MDS

distinguished two additional, smaller

assemblages, which were not obvious from the Bray-Curtis

analysis. The first of these assemblages was at 30 m at

Labhra

Cliff;

it

showed

the greatest affinity with the other

Labhra Cliff and West Cliff

cluster at

(cliff habitat).

The

second assemblage was composed of 0-m sponges at Labhra

Cliff and West Cliff (cliff habitat). These assemblages were
most similar to those at Whirlpool Cliff. The subtidal (6 and
12

m) panels had high

between

sites,

levels of correspondence

(=65%)

but differed considerably from those in the

A much

higher

intertidal

same

habitat

morphological differences created by the nature of the

proportion of species were shared within the

The

assemblage

zone (0 m)

(=30%

similarity).

To account

for
dif-

SPONGE COMMUNITY COMPOSITION

Table
Sponge species found within Lough Hyne on

sublittoral cliff

and rocks and on

the adjacent Atlantic coast

149

150

J.

J.

BELL AND

D. K. A.

BARNES

Table 2

Sponge species

tin::

wei

'I

iroiti

the

under (*) and upper

sides of rocks

found

at

rim

sites at

Lough Hyne

SPONGE COMMUNITY COMPOSITION

Table 3
The number of sponge species shared between

Site

sites

151

152

J.

J.

Spring Tide

250

BELL AND

D. K. A.

BARNES
Neap Tide

SPONGE COMMUNITY COMPOSITION

153

Stress = .17
1

2 -

WHO (V)
WH6 (V)

28-LA30(I)

29-WCO(V)
30-WC6(V)
31-WCI2(V)
32-WC18(V)
33 - WC24 (V)
34-WCOd)

3-WHI2IV)
4-WH18(V)
5 -

WHO (I)

6-WH6(I)
7-WH12(l)
S-WHIS(I)
9-BUO(V)
IO-BU6(V)
11-BU12(V)
12-BU18(V)

35-WC6(I)
36 -

-WH 12 panels

41
42 43 44 -

45
46
47
48
49

18-LA6(V)
19-LA12(V)
21

(I)

40 - LO panels

13-BUO(I)
14-BU6(I)
15-BU12(I)
16-BU18(I)
17- LAO (V)

20

WC12

37- WCI8(I)
38 -L12 panels
39 - L6 panels

-LA 18 (V)
-LA 24 (V)

22 - LA30 (V)
23 - LAO (I)
24 - LA6 (I)

WH6 panels
WHO panels

WC Sm boulder
WC Med. boulder
WC Lar boulder
WC V.Lar boulder

WH Sra. boulder
WH Med. boulder
WH Lar Boulder
- WH V Ur boulder

50 51

25-LA12(I)
26

-LAI 8 (I)

27-LA24(I)

to compare the sponge

Bray-Curtis similarity and Multi-Dimensional Scaling (MDS) analysis
and
cliff at a number of sites, depths, and surface inclinations from Lough
rocks,
inhabiting
panels,
assemblages
produced for loose rock assemblages.
Hyne. The dashed circle indicates the grouping that

Figure

4.

MDS

between West Cliff and Whirlpool Cliff (GLM

ANOVA; F ratio = 0.07, P = 0.78. denominator df = 1,
intercept

numerator df

278). This

was

also true for the transformed

between species richness and rock surface area

Cliff and Whirlpool Cliff (graph not shown)

relationships
at

West

ANOVA;

(GLM

F-ratio

1.1.

P =

0.25, denominator df

1,

154

J.

J.

BELL AND

D. K. A.

BARNES

_^

JS

E ?

SPONGE COMMUNITY COMPOSITION

140

12

5?

Labhra Cliff

10

2
s

1
"3

155

120

100

H.

Numbers

60

= 73
Cliff
R-squared
=
Whirlpool Cliff R-squared 0.54
West

12

o
Whirlpool Cliff

10

500

2500

2000

1500

1000

")

Boulder surface area cm"

{

between sponge densities and rock

high-energy (Whirlpool Cliff) and low-energy (West Cliff)
sites on the undersides of rocks at Lough Hyne. Number of sponges (West
= 60.3
35.84) + 14.8
0.85) x surface area. Number of
Cliff)
Figure

-3

a,

The

7.

surface area

linear relationship

at

sponges (Whirlpool

Cliff)

47.3

46.54)

22.6

1.87)

surface

area.
--

Number of months
The proportion of

5.

Figure

10

12

since panels deployed

artificial

settlement panels covered in

(Whirlpool Cliff)

months of deployment at high-energy

2. 6,
and low-energy (West Cliff) sites at Lough Hyne.

numerator df

sponges after

1.

and

278).

12

However, when

GLM ANOVA

at

West

increase in surface area resulted in a greater increase in

at West Cliff than at Whirlpool Cliff. Also,
rock
size
harbored a greater number of sponges at
any given
West Cliff than at Whirlpool Cliff.

sponge density

was

used to compare the linear relationship between sponge

numbers and rock surface area, a significant difference was

found between the slopes of the relationships

and Whirlpool Cliff (GLM ANOVA; F ratio = 16.16. P <

0.001, denominator df = 1, numerator df = 278). Any unit

Cliff

Discussion

Animal communities

are

known

to vary

between large

geographic areas (scale of kilometers), but the variability

between localized habitats (1 to 100 m) under similar environmental conditions

is

far less well

known. This

is

true

for sponge assemblages: broad distributions have been described by habitat at Lough Hyne (Picton, 1991; van Soest

and Weinberg, 1980; van Soest et /., 1981). and recently

more detailed studies have added environmental variables to
such distributions (Bell and Barnes, 2000b, c). As with
organisms, environmental factors are critical
to the distribution of sponge species. To date, little quantimost,

if

not

all

tative information has

been provided on localized (where

factors are minimized) habitat-associated dif-

234
c

Whirlpool Cliff

R-squared

confounding
ferences between sponge assemblages

in

temperate locali-

ties.

= 0.54

Morphological variability with habitat

environmental characteristics

stability

and

Boulder surface area (Log 10)

The studied sponge assemblages (within Lough Hyne)

Figure
richness

6.

v.s.

The

relationships

rock surface area

energy (West Cliff)

sites

on the undersides of rocks

Species diversity (antilog 10)

area.

at

between sponge species diversity and

high-energy (Whirlpool Cliff) and low-

2.09

0.15)

2.09

<

at

Lough Hyne.
X surface

0.04)

varied considerably between habitats of differing stability

conditions (i.e.,
(i.e., cliff and boulders) and environmental

high and low flow), even when habitats were separated by

hard subonly several meters or less. Sponges inhabiting

156

J.

stratum have been

logical adaptation

ing flow regime,

both assemblage
levels (Mancr.-v

shown

in

in exhibit

iio;ise to

BELL AND

considerable morpho-

a variety of factors, includ-

icntation,
t.rli

J.

and substratum type,

It

seems, tluivture, that morphology alone may account for

some of these differences in assemblage composition between sites and habitats. The two-dimensional nature of
rock (under-surface) habitats undoubtedly accounts for the
high proportion of encrusting forms because the overt threeto cliff habitats

is

localized areas of negligible current flow, even in high-

current areas.

at

and Barnes, 2000d) and individual

,nd Pronzato, 1991; Bell et ai. 2002).

other sponge species common

inhibited (e.g., arborescent forms). High-

dimensional nature of

many

energy cliff sites were dominated by encrusting and massive

forms and showed greater levels of similarity (in assemblage composition) to rock habitat assemblages than to
cliffs. However, high proportions of

Assemblage composition

is not directly important in

determining the comof
position
sponge assemblages. Sponge diversity and richness did, as with most organisms, increase with area (rock

flow rate

size),

encrusting and massive morphologies were found in both

habitats, although the species

compositions

Morphology alone cannot account for

all the differences found. Organisms
living on the undersides of rocks have a number of advantages, including
different.

reduced competition from fast-growing algae found on upper rock surfaces, since many sponges are considered slow-

growing (Ayling, 1983), and protection against potentially

harmful UV radiation. Underside rock communities also
experience reduced sediment settlement in areas of low
current flow, reduced effects of desiccation (in the inter-

and reduced predation from fish and other large

invertebrates (Dunlap and Pawlik, 1996; Wulff, 2000). A

tidal),

combination of these factors

may

account for the presence

or absence of any single species on the undersides of loose

rocks. Therefore, the distribution and habitat of each species

should be considered individually.

Morphology alone has already proved valuable

in

sepa-

sponge assemblages inhabiting sublittoral cliffs (Bell

and Barnes, 2000a). Increased numbers of delicate morpholrating

ogies, exhibited by a

number of species exclusive

to low-

energy sites, have been associated with decreasing flow rate

and as a mechanism to reduce sediment settlement (Bell,
2001). However, the rate of water flow had

little

effect

on

the sponge assemblages found on rocks and apparently was

not a regulatory factor of such assemblages (in the present
study).

One

aspect of morphology that

was not considered

within this study is the potential importance of the surface

texture of encrusting sponges in response to habitat distur-

bance or

For example, sponges with smooth surbe

suited
to areas of high current flow since they
may
reduced
experience
drag. Also, the actual flow (in terms of
stability.

faces

which

is

usually termed an area effect.

at

low-energy environments from superior spatial

competitors such as colonial ascidians and anthozoans
in

(Maughan and Barnes, 2000b;

Increased

2003).

sponges

may

adaptation, has been

credited for the shift in competitive ability (Bell, 2001a).

However, since the organisms inhabiting the undersides of

rocks experience little direct sedimentation, other factors

must be responsible for the lack of superior competitors,
such as increased sediment loading (rather than direct settlement) in the water, a condition that sponges appear to
tolerate (Lilly et al., 1953). Also important is the difference

between upper and lower rock surfaces. The domination of

sponges on lower surfaces relates to the absence of macroalgae,

which has been considered

to control both large-

and small-scale sponge distributions (Witman and Sebens,

1990). Sponge proliferation on upper surfaces may also be
prevented by interference competition from the sweeping
action of algal fronds, as suggested for other invertebrates
(Jenkins ct

til.,

1999).

The

linear increase in the

number of

sponges with increasing surface area of rock may appear

unusual. One might expect that the dominant sponge species

would occupy most of the primary substratum (Russ, 1982;

Maughan and Barnes, 20()()b). However, competitive encounters and interactions between sponges may be nonhierarchical and resemble a network (Buss and Jackson,
1979; Bell and Barnes, 2003), thereby preventing monospe-

dominance. Even if certain sponge species inhabiting

rocks are dominant over others, these species may show
seasonal growth and retraction of tissue that prevents them

cies

from monopolizing rock surfaces (Sara, 1970; Stone, 1970;

Elvin,

1976; Barthel.

1989).

The

distribution of sponges

also be limited by other biological factors, in particular

predation (e.g., Wulff, 1995); but in the observations of

may

sponge assemblages

was

be influenced by microscale environmental characteristics, which are themselves difficult to characterize. For

2001a; Bell and Barnes,

show morphological and physiological

1983), such that their

distribution

Bell,

sedimentation, to which

speed and direction) experienced by sponges may be significantly influenced by seabed characteristics (Hiscock,

morphology and species

The density of

any given rock size was greater at West Cliff

Whirlpool Cliff, most likely due to reduced compe-

sponges for
tition

and rock

and

Negligible (between-site) differences in sponge diversity

or species richness attributable to flow rate were found on
the under-surfaces of rocks. This suggests that in rock
habitats unlike cliff habitats (Bell and Barnes, 2000a), water

than

were very

variability with flow rate

rock size

those of low-energy

cliff

BARNES

D. K. A.

at

Lough Hyne, predation of sponges

rarely observed. Fish feeding

on algae sometimes broke

may

branches from arborescent species on

example, the overlying nature of loose rocks

predation was not seen. Sponge population heterogeneity is

also affected by competitive processes (Becerro et <//.,

may

lead to

cliffs,

but extensive

SPONGE COMMUNITY COMPOSITION

157

fluence the direction of growth (in encrusting species) and

lead to the patchiness observed within habitats.

all species found on panels, which simulated early

development of rock communities, were also found in cliff
and rock habitats, we suggest that habitat is not as critical in

Disturbance has been considered an important factor

structuring marine epifaunal communities, and rock size has

determining species composition at this early stage in development as it is in more mature communities. However,

such disturbance (Dayton.

977). This theory is based upon the principle

sponge assemblages (rather than species) found on panels

1994), because interactions between spatial competitors in-

been considered
1

97

Osman.

a surrogate of

more likely to be moved than larger

However, all but the smallest rocks at Lough Hyne
showed very similar sponge assemblages across sites experiencing differential flow rates. Sponge assemblages do vary
between some surrogate measures of disturbance (flow
rate), but not between actual extremes of habitat stability.
that smaller rocks are

rocks.

Since

show

greater affinities with rock assemblages than with

cliffs, which is to be expected given that the panels were

did

used as a surrogate for young rock habitats. Even though a

number of sponges were reported from the panels, it is also
true that

most of the species found

in this

study did not settle

on the panels and thus can still be considered late colonizers

of benthic communities. For a complete evaluation of the

This suggests that treatment of rock surface area as a surrogate measure of disturbance is unsatisfactory and shows

contribution of sponges to communities of different ages

(since panels represent a pioneer succession stage, while

no correlation between extremes of surrogate (i.e., small and

large rocks) and true (i.e., high- and low-energy sites)

cliff

measures of disturbance. At Lough Hyne, small rocks are

study. Small rock assemblages had a

often held in place by overlying larger rocks, as a result, the

probability of motion can be lower (rather than higher) for

with the species found to inhabit panels, with two species

(Leucosolenia complicata and Scypha ciliatum) being of

small rocks than for larger rocks, because the movement of

smaller rocks is dependent on the movement of larger rocks

particular interest.

a priori. Thus, lack of space rather than level of community

development (and age) can explain why impoverished
sponge assemblages are seen more frequently on small

and Barnes, 2000b) and of the early development of hardsubstratum communites (Osman. 1977). These were two of
only three calcareous sponges reported during the study, and

rocks. For organisms (including sponges) that inhabit disturbed habitats (such as loose rocks), succession will be

they appear to be r-strategic, or opportunistic, in the use of

newly available space. It is unclear if the calcareous nature

important in structuring the community. Occasional disturbance will prevent the development of a climax community

of these sponges provides them with some adaptive advantage over siliceous sponges.

and permit colonization by weaker competitors that might

have been excluded from a habitat that had reached a state
of climax.

Sponge assemblages on panels had little similarity with

other habitats examined and, in contrast to findings with
taxa (Maughan and Barnes. 2000a; Barnes and
Maughan, 2001), showed negligible inter-site differences.
However, the space occupation (percentage cover) of

other

sponges during the development of the (pioneer succession)

panel assemblage varied significantly over the 12-month
deployment period (continuous immersion for 12 months).
greater area occupied by sponges on the undersides of
panels at Labhra Cliff than Whirlpool Cliff is consistent
with the results of other studies of sponge assemblage

The

differences between cliff environments at these

(Bell

and Barnes. 2000a.

d).

two

sites

Sponges are more abundant on

panels, rocks, and cliffs at low-energy sites in Lough Hyne

than at high-energy sites. Again, this is most likely due to

low-energy environments from superior spatial competitors, such as colonial ascidians and
anthozoans (Maughan and Barnes, 2000b; Bell. 2001; Bell
reduced competition

in

and Barnes, 2003). All species found on panels submerged

for 2-month periods were also found on rocks and cliffs.

These early colonizers were important to both mature and

immature communities, even though sponges are widely
regarded as

late

colonizers (Dayton, 1971

Maughan, 2000).

communities may be many years

extend over

species

is

many more time

old),

monitoring must

intervals than in the present

The presence of

much

these

greater affinity

two calcareous

consistent with other studies of panels

(Maughan

Are there discriminating species for (local) habitats of

differing stability and experiencing different flow rates?

Most habitats and sites could be characterized by several

abundant sponge species. Different cliff sites were easily
distinguishable from each other and from other habitats on

many species. In rock habitats seven species

differed between the high- and low-energy sites, though
the basis of

they were too rare to be between-site discriminating species

(as

determined by

SIMPER

analysis).

Given

the similarities

assemblage composition, it is not surprising that the five

most important discriminating species between rock and
other habitats did not differ between sites. The low number
in

and ubiquitous nature of species on artificial substrata made

the identification of true inter-site discriminating species in
this habitat irrelevant.

Are there consistent

similarities

or differences

in

assemblage composition from extremes of flow rate and

habitat

stability'.'

Although certain environmental characteristics have been

found to influence sponge communities, patterns of sponge
assemblages on rocks are not consistent with those described from other hard-substratum environments (Storr,

1976; Alvarez et ai. 1990;

Witman and Sebens,

1990; Bell

158

and Barnes,

200()a).

sponges on rocks.

seemed

blage,

From our
,

results,

J-

BELL AND

not the composition of the assem-

to be manifestly affected

by

rate of

water

Despite ;iie late-colonizer "tag" applied to sponges in

marine communities (Dayton, 1971: Maughan 2000). the

lithophyllic
for only a

number of species contributing

determining sponge assemblage composition. Disturbance seemed to be more important in determining sponge
assemblage composition on cliffs than in loose rock habiin

tats.

This study has shown that habitat stability is an imporbe taken into account along with other mea-

distribution of

when considering

Bell,

and D. K. A. Barnes.

.1.

J.,

of sponges

and

Shaw

the

morphology of sublittoral sponge populations

Mar. Biol. As.mc. UK 80: 707-718.

at

The importance of competitor

morphology and ranking methodology to outcomes in interference competition: an example of sponges. Mar. Biol. (In Press).
identity,

K. A. Barnes, and

Bell, J. J., D.

J.

The importance

R. Turner. 2002.

micro and macro morphological variation

toral demosponge to current extremes. Mar.
nt

in

adaptation of a sublit-

Biol. 140:

75-81.

Washington DC.

Buss, L. W., and

for assisting in data col(Irish

We

also thank Enterprise Ireland for financial assistance.

Thanks also to the anonymous referees for significantly

manuscript.

J.

B. C. Jackson.

1979.

Competitive networks:

nontransitive competitive relationships in cryptic coral reef environ-

ments. Am. Nut. 113: 223-234.

Cheshire, A. C., and C. R. Wilkinson. 1991.

Wildlife Service) for granting permits for research at Lough

Hyne, and Chris Todd for provision of artificial substrata.

this

The influence of bathymetry

K. A. Barnes. 2000d.

Thesaurus of Sponge
Morphology. Smitlison. Contrih. Zool. 596. Smithsonian Institution

and manuscript revision, Declan O'Donnell

improving

1).

Lough Hyne MNR. J.

Bell, J. J., and D. K. A Barnes. 2003.

Press,

Claire

and

How regime on

Boury-Esnault, N., and K. Rutzler, eds. 1997.

Acknowledgments

lection

20(IOc.
The distribution and prevalence
environmental gradient within a temperate sea
surfaces. Divers. Distrih. 6: 305-323.

in relation to

lough: inclined cliff

Bell, J. J.,

the

sponge species.

The authors thank

Lough Hyne Marine Nature

The distribution and prevalence

of sponges in relation to environmental gradients within a temperate
sea lough: vertical cliff surfaces. Divers. Distrib. 6: 283-303.

tant factor to

sures of disturbance such as flow rate,

at

HyJrohiologia 440: 55-64.

island.

and D. K. A Barnes. 2000b.

be more important than current flow rate

to

marine

com-

position from extremes of environmental stability, and rock

was judged

The ecology of sponges

2001.

J.

.1.

Bell, J. J.,

to

communities on panels that had been deployed

few months. Rock habitats did not display a

consistent trend of decreasing similarity in assemblage

size

Bell,

Reserve. Co. Cork, Ireland. Ph.D. thesis. University College Cork.

Bell, J. J., and D. K. A Barnes. 2000a.
A sponge diversity centre within

flow.

present study found a

occupation by the encrusting sponge Cramhe crambe: variation in

shape as a function of size and environment. Mar. Biol. 121: 301-307.

abundance of

the

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D. K. A.

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Modelling the photosynby sponges on Davies Reef. Great Barrier Reef. Mar. Biol. 109:

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Reference: Biol. Bull. 205: 160-169. (October 2003)

2003 Marine Biological Laboratory

Phenotypic Plasticity of HSP70 and HSP70 Gene

Expression in the Pacific Oyster (Crassostrea gigas):
Implications for Thermal Limits and Induction of
Thermal Tolerance
AMRO
1

DANIEL

'*,

P.

CHENEY", AND GARY

tidal heights, routinely

We

limits of oysters are relatively plastic,

and

Marine invertebrates

iologically challenging. Littoral invertebrates routinely enstress, and wide thermal fluctua-

counter anoxia, osmotic

tions.

We

responses are particularly important in the adaptive response of sessile intertidal invertebrates such as mussels and

prior to laboratory heat shock) of HSC77 and

are positively correlated with increases in ambient

oysters.

temperature and were significantly higher in August than in

January. The elevated level of HSCs during the summer was
thermal limits for survival.

We

Many

HSP70. Total hsp70

(7"on

ciently labile to interact with their substrates, they are often

highly susceptible to protein-denaturing stresses such as

required for

reduced intracellular

mRNA

expression reflected the seasonal changes in the expression of inducible

but not cognate members of the HSP70 family of proteins.
induction of

potential cost of increased

ron

in the

summer

is

domains of proteins and cause

nonspecific protein interactions. Under these conditions,

that there

several families of proteins

the upper thermal limits for survival

of thermotolerance) after sublethal heat

stabilizing functions (Lindquist,

at

brook, 1992; Morimoto el ai.

critical protein-

1986: Gething and Sam1994). Under "non-stress"

hydrophobic protein domains exposed to aqueous cellular

environments during protein synthesis and translocation (for
review, see Hartl and Hayer-Hartl, 2002). Stress-induced
of
expression of HSPs contributes dramatically to tolerance

Present Address: Hopkins Marine Station. Stanford University. Pacific

To whom correspondence

as heat-shock proteins

conditions, molecular chaperones are required to stabilize

Grove, California 93950.

t

known

(HSP), or molecular chaperones, perform

Received 12 April 2002; accepted 24 June 2003.

Bodega Marine Laboratory Contribution Number: 2175
*

or elevated temperature (Somero,

the revealing of hydrophobic

temperatures that were sufficient to induce thermotolerance during the winter months.

shock

pH

1995). Protein-denaturing stressors such as heat can result in

was no extension of
induction

of these natural stresses can directly alter protein

stability. Because proteins must be suffi-

conformation and

C, increases in the upper

measured concomitant in-

creases in the threshold temperatures

Withstanding these conditions usually requires the

of coordinated cellular responses to stress. These

initiation

(i.e..

associated with moderate, 2-3

tolerate a remarkable array of nat-

ural

tored for changes in expression of cognate (HSC) and

inducible (HSP) heat-shock proteins during the progression
found that the "control"
from spring through winter.

(i.e.,

'

and anthropogenic stresses. Although the nearshore

environment supports great invertebrate diversity, it is phys-

that these

changes in the expression of one

family of heat-shock proteins (HSP70). Oysters were cultured in the intertidal zone, at two tidal heights, and moni-

Introduction

living at a

encounter large seasonal

demonstrate that the ther-

limits are correlated with

HSC72

CHERR 3 ^

PO Box 247. Bodega Bay, California 94923; ^Pacific Shellfish Institute.

3
#142, Olympia, Washington; and Departments of Environmental Toxicology and
Nutrition. University of California Davis. Davis. California 95616

fluctuations in temperature.

levels

N.

NE

Pacific oysters, Crassostrea gigas.

Abstract

mal

HAMDOUN

Bodega Marine Laboratory,

120 State Ave.

range of

M.

otherwise lethal conditions (Parsell and Lindquist, 1994),

should be addressed. E-mail: gncherr

known

ucdavis.edu

160

as "induced thermotolerance."

HSP GENE EXPRESSION IN OYSTER

161

Materials and Methods

Natural variation in the heat-shock response appears to be

correlated with distribution along environmental gradients

Tomanek and Somtemperatures for HSPs are

of stress (Hofmann and Somero, 1996;

ero. 1999).

Threshold induction

positively correlated with ambient environmental temperatures (Dietz and Somero, 1992; Buckley et ai, 2001 ). In the

marine
higher

mussel

animals living

in

threshold

Mytilns.
at

higher

temperatures

tidal

can be

heights (Roberts et

1997). In congeneric species of the marine snail Tegnla,

nl.,

threshold temperatures are positively correlated with the

maximal tidal height at which each species is found. In

maintained even after labora-

Tcgiilu, these differences are

tory acclimation, suggesting a role for gene regulatory factors that establish set points for HSP induction or interspecific variation in the stability

of cellular proteins (Tomanek

and Somero. 1999).

The

basic features of the heat-shock response in the

Pacific oyster. Crassostrea gigas, have been well character-

HSP70

ized (Clegg et ai, 1998).

primary family of
Three isoforms of

HSP

has been

shown

to

be the

that is responsive to thermal stress.

HSP70

family protein are resolved after

one-dimensional electrophoresis and western blotting. Two

HSC77 and HSC72.

are constitutively expressed,

their level of expression

and accumulation increases

proteins.

and

after acute thermal stress. In contrast to other bivalve spe-

Animals and study

site

A commercial oyster bed in Totten Inlet, south Puget

Sound. Washington, was selected as an experimental study
site. The juvenile "seed" (about 5-10 cm in length) oysters
for this experiment, 1999 age class,

June 1999 and planted

tidal plot at 0.3

at

two

were obtained

in late

tidal elevations: a low-inter-

mean lower low water (MLLW) and a

m MLLW. The seed were initially

high-intertidal plot at 1.2

in rectangular. 0.5-cm mesh, plastic grow-out bags

on top of metal racks at each tidal height. The oysters
were later transplanted to larger, 2-4-cm mesh, grow-out

placed
set

bags. Oysters were sampled monthly during low tide events

starting in April

External

site

2000 and continuing until January 2001.

temperature data were recorded every 15

min using Hobo temperature

sensors. Internal temperature

Hobo 4-channel

data were recorded using

external data

loggers and TMC6-HA temperature sensors. Temperature

data were recorded at each tidal height from three oysters

with internally mounted sensors and one externally mounted

sensor (TMC6-HA sensor), which was placed next to the
oysters either in a grow-out bag or on the ground. The
internal temperature sensors were placed inside the shell of
live oysters through a 6mm opening. The sensors were
secured and sealed by using marine epoxy.

cies such as Mytilus. C. gigas can express a third protein.

it is
typically only detectable after acute thermal
Several days after heat shock, the levels of HSP69
account for roughly one-third of total HSP70. In the absence

HSP69, but

Heat shock

stress.

of additional

stress.

HSP69

completely disappears within

mRNAs

that

to the constitutively expressed (cognate)

and

7-10 days of heat shock.

Two

genes encoding

correspond
inducible forms of oyster HSP70 family have been sequenced and characterized. Distinct gene products encode a
cognate protein of about 72 kDa (Gourdon et til.. 2000) and
an inducible protein of about 68-70
2003).

In

Pacific

kDa

(Boutet

oysters, a single sublethal

sufficient to cause significant

HSP69

et ul..

heat shock

expression also results

induced thermotolerance for a remarkably prolonged

period of up to 2 weeks (Clegg et ai, 1998).
in

In this study,

the

we attempted

mechanism of adaptive

to

determine the extent and

plasticity

of the heat-shock

group of sibling Pacific oysters planted at two

tidal heights ( + 0.3 m and +1.2 m) at Totten Inlet, Southern
Puget Sound. Washington State. We measured the seasonal
response

in a

in HSP70 and hsp70 gene expression in these

and
we examined these findings in the context of
oysters,
the observed, concomitant changes in control and induc-

changes

upper thermal limits for survival. Our findings suggest that phenotypic plasticity of the heat-shock response
ible

is

a significant

mal

stress.

component of adaptation to natural ther-

Immediately after collection oysters were shipped overC) coolers to Bodega Marine Lab-

night in chilled (12-15

Bodega Bay. California, for use in experiments.

After 24 h of recovery in flow-through tanks with raw
2C), animals were
Bodega Bay seawater (RSW; 13C,
oratory.

in fine mesh bags and heat-shocked at a variety of

temperatures (see Results) by being submerged in preheated, aerated filtered seawater baths for 65 min. Animals

placed

were returned

to the flow-through

seawater tanks immedi-

ately after heat shock. Five to six oysters

were heat-shocked

at each temperature for sampling of gills for northern and

western blots. After heat shock, oysters were returned to the

RSW tanks
and mRNA
the point at

gills were sampled for protein

h
has
previously been shown to be
analysis; 48
which accumulation of HSP70 protein is max-

for

48 h before

imal, and de novo synthesis of

HSP70

is

still

elevated

relative to control specimens of C. gigas (Clegg et ai,

1998). This protocol allowed us to compare the level of

mRNA

HSP70

protein and
important to note that

from

HSP70

identical gill sections.

levels

It is

remain elevated

in

oysters for at least one week following heat shock. Thermal

limits were determined by heat-shocking three groups of

seven animals

(in

fine-mesh bags)

at

each temperature.

Survival was determined after 10 d of recovery

(

2C)

in

RSW

tanks.

at

13C

A. M.

162

HAMDOUN ET

To

ensure that shipping mcl brief holding did not alter the
.sion. we compared samples of gill
profile of HSP70
oysters (prior to shipping), from
arrived at the laboratory, and from
u been held for 2 days in ambient RSW. No

from freshly o
oysters that

ist

oysters thai

HSP70

protein levels was observed between

these
not shown).
of
(data
groups
any
difference

in

AL.

synthesized from minipreps (confirmed to be positive for

the hsc72 gene product by sequencing). Briefly, a digoxi-

genein-labeled

and sample preparation

Oysters were opened and

oyster

hybridize with

The

into

two sections

for protein

and

RNA

isolation. Gill

sam-

were prepared according to Clegg et

they were rinsed in double-distilled H 2 O,

ples for western blots

(1998). Briefly,
blotted dry. weighed, and flash-frozen in liquid nitrogen.

nl.

These samples were stored at 70C and later homogenized

on ice in glass tissue grinders containing potassium glu-

mM MgSO
mM gluconic acid.

conate buffer (5

HEPES, 70

mM NaH,PO 4 40 mM
mM sorbitol; pH 7.5) at
,

150

a concentration of 100 milligrams of tissue per milliliter of

Homogenates were prepared

buffer.

in

1:1

mixing

2x sample

RNA

for electrophoresis by
buffer and boiling for 5 min.

were immediately preserved in 5 volumes of RNAlater (Ambion) solution, frozen

in liquid nitrogen, and stored at
70C. Gills were later
in
3
ml
homogenized
guanidine thiocyanate denaturing soGill sections for

lution:

900

jul

isolation

of this homogenate was mixed with 100

ju,l

of

M sodium acetate and incubated on ice for 5 min. Samples

were next mixed

1:1

with ice-cold phenol/chloroform (pH

ice for 20 min. After centrif-

Sigma) and incubated on

ugation for 25 min at 10.000

4.3:

removed, and

RNA

was

g, the

aqueous layer was

precipitated overnight in an equal

volume of isopropyl alcohol. The next day the RNA was

pelleted, washed, and resuspended in DHPC H 2 O. Purity
and concentration were checked using a spectrophotometer.
cloning and probe

Total

RNA

was

synthesis

isolated

from heat-shocked oysters and

cDNA

with Superscript Reverse Transcriptase (Gibco-BRL) according to manufacturer's instructions. A 660-bp fragment of oyster HSC72 cDNA was

used to synthesize

amplified from this template using degenerate

HSP70

the dual promoter

pCRII-TOPO

genes,

of the

all

vector (Invitrogen) and

cloning
Aliquots of plasmid minipreps (Qiagen) of
clones
were sent to Davis Sequencing (Davis, Calpositive

in this

we expected

known gene

in

the antisense

region of the
this

probe to

products of the

HSP70

was determined by
probe versus a digoxigenein-RNA

yield of resulting probe

in-

structions.

Western blotting

Western blots of oyster HSP70 family were prepared

according to Clegg et al. 1998) and 10 /j,l of each solubi(

lized
gel.

sample was resolved on a 10% SDS-polyacrylamide

To normalize between blots, multiple aliquots of a

single sample of heat-shocked oyster gill

were made. Each

time gels were run, an aliquot of this sample was loaded on

the center lane of each gel. The internal control sample was

chosen from a previous experiment and was

arbitrarily

known

to

have

all

malized relative

HSP70

of the corresponding band from

variation in standard

to the level

maximal

the internal standard (the

between gels was

level

HSP70 family protein.

family member were nor-

three isoforms of

Therefore, levels of each

less than 10-fold).

To ensure

that the

large differences in HSP70 protein levels between summer

and winter controls were not attributable to blot variation,
the

and

summer and

winter samples were run on a single blot

heat-shocked oysters were ad-

their values relative to

justed according to the level of the corresponding standard

sample run on that blot. Control levels of HSP70

mRNA

were compared from samples run on a single northern blot.

Proteins were transferred onto nitrocellulose and probed
a

monoclonal

anti-HSP70 antibody

rat

(Affinity

Bioreagents, MA3-001), and an HRP-conjugated goat secondary antibody (Sigma). Signal was detected by chemilu-

minescence using enhanced detection reagents (Supersigon a Bioirnaging Systems digital gel scanner or
by exposing blots to photographic film (Kodak, Biomax
MR). Relative levels of band were intensified using gel
nal. Pierce)

prim-

(Cochrane et til., 1994). PCR products were checked for

size and yield on 1% agarose gel prior to TA cloning using

ers

Ambion) of

probe standard (Roche) according to manufacturer's

using

cDNA

HSP70

using a dot blot of the

were dissected and divided

gills

(Roche) probe was synthesized by

Because of the high homology

strand.

known
family.

Tissue sampling

UTP

vitro transcription (Maxiscript:

plotting

macros available on Scion Image

(ver.

1.61) soft-

ware.

Northern blotting

kit.

ifornia) for sequencing. Results

BLAST

program

at

were analyzed by using the

the National Center for Biotechnology

Information to compare existing sequences. A 660-bp fragment was cloned that was identical to a portion of a previously submitted sequence of Pacific oyster
(Gourdon ct til., 2000).

An

antisense

RNA

probe for total

HSC72

HSP70

mRNA

For each northern

blot,

loaded onto each lane of

;ag of

denatured

agarose

(in

total

RNA

was

X MOPS), 6.66%

formaldehyde gels and run for 3-5 h at 50 V. RNAs were

transferred overnight onto nylon membranes (Hybond) using standard capillary methods in 10x SSC buffer. After
was cross-linked to the blots (Stratalinker),
transfer.

RNA

and blots were stored

probe was

1%

RNA

at

20C

probes were prepared

at

until

probed. DIG-labeled

a final concentration of

50

HSP GENE EXPRESSION

ng/ml

in

SDS

high

buffer

buffers in

(all

RNA

detection

were prepared according to instructions from DIG applications manual. Roche). Blots were incubated overnight at

50C

probe solution with gentle rotation. The next day,

nonspecific hybridization was removed using four 15-min
in

60C

in decreasing concentration of
stringency washes at
SSC solution (2X-0.5X). Blots were then washed twice in

Tween

maleic acid buffer containing 0.3%

for

30 min using

x blocking

20,

(Roche)

used 1:100

buffer (Roche). Blocked blots

CSPD

solution (Roche)

was

detection buffer to detect hybridized probe.

in

animals
both

at

163

Chemiluminescence was enhanced by 15-min incubation of

the blot at 37C, followed by detection using a Bioimaging

was

less than 1(Y7c annually.

est

the

at

months (June-September).

HSP70

protein ami Iisp70

mRNA

levels

The

HSC70 (HSC77 and HSC72)

levels of

Statistics

mRNA level were analyzed for

protein and

statistical significance

using one-way

ANOVA.

and multi-

pairwise comparisons were performed according to

Dunn's method: all tested data conformed to equal variance
and normality assumptions. All analyses were conducted

ple

using Sigmastat software ver. 2.03.

in control

(non-heat-shocked) Pacific oysters were associated with the

level of environmental thermal stress (Fig. 4). At both study
sites the control levels of HSC77 and HSC72 were as much

summer

as 100-fold greater in

protein levels, as

in

Animals

m) showed a cumulative mortality of

high
16.5% (62 out of 375 animals): those at the low tidal height
(0.3 m) experienced an 18.4% (69 out of 375 animals)
mortality. Most of the mortalities occurred during the wanntidal height (1.2

than in winter (P

These differences were not attributed

Systems digital gel scanner.

Differences

during the experiment. Total mortality

at either site

sites

blocking buffer.

in

OYSTER

and blocked

h with gentle rotation at room temperature in a 1:10.000 solution of anti-DIG AP Fab fragments

were incubated for

IN

mean

<

0.001).

to differences in total

control protein levels were 41.5

mg/g wet weight and 46.2 mg/g wet weight in winter and
summer, respectively (Bradford protein assay, BioRad). and

>

these differences were not significantly different (P

0.07).
the
winter
months
there
was
no
statistically signifDuring
icant difference in the control levels of

high and low

The threshold temperature

to

(P

height sites

tidal

HSC70 between

the

0.917).

for induction of

HSP69

ap-

be associated with the level of environmental

peared
thermal stress (Figs.
!). In January. HSP69 accumulation
was induced in oysters from both sites after heat shock at
1

Results

Animals and studv

37C. In contrast, limited or no induction of HSP69 and

HSC72 was observed following heat shock at 37C in

site

Temperatures recorded in living oysters generally

matched those recorded by external data temperature loggers placed alongside the oysters in the culture bags (Fig.

This suggests that despite

its

thickness, oyster shell

1).

is

relatively poor insulator against thermal stress. Oysters at

MLLW

1.2 in
generally experienced higher temperatures
and longer durations of exposure to elevated temperature
than oysters at 0.3 m. To estimate the duration of exposure

temperatures, measurements from data

were
sorted
into
5C intervals, and measurements in
loggers
each class were summed together. Each data point was
to elevated

assumed

(>20C)

to

represent about

15

min of exposure

to

the

living at high or

August oysters

was not observed

ters

encountered elevated

No

(>20C)

and reduced

(<15C)

or above the point of emersion.

significant difference in mortality was recorded in

temperatures

at

August

until after heat

shock

at

40-

mRNA

in summertime high tidal

of hsp70
relative to control
increased
dramatically
height oysters
after heat shock at 40 and 43C, but not after heat shock at

The

level

33 and 37

(Fig.

HSP69

increases in

heat-shocked

compared to 44 h at the lower tidal

The
thermal
height.
regime based on mean emersion is
summarized in Figure 3. The majority (>70%) of the time
(not shown), oysters at both tidal heights were submerged
and encountered temperatures in the 15-20C range. Oys-

in

icant seasonal plasticity.

weeks preceding the

summer sampling. During the summer 2000 period, the total
exposure to temperatures in excess of 20C was about 67 h
the high tidal height

height (Fig. 4: see

Thus, both the threshold temperature for HSP69

induction and the control levels of HSC70 exhibited signif-

peratures in the

at

tidal

43C.

recorded temperature. In nearly all cases the approximate

duration of exposure to warm temperatures was longer at
the high tidal height (Fig. 2) for the 2

low

also Fig. 6A). Full induction (i.e.. HSP69 expression at

levels approximating those of HSC77 or HSC72) of HSP69

5).

This result reflected the observed

protein after heat shock at these tem-

same animals.

site was sampled and

monthly during the summer, beginning in April and ending in September (not shown). Based
on the results of these observations, the greatest change in
threshold temperature for HSP69 induction and control

subset of oysters from each

at

37C

level of HSC77/HSC72 temperature occurred between our

June and July sampling dates and winter (January) (Fig. 6A,

B; Fig.

7).

In June, oysters at the

pletely induced

by

HSP69

July, induction of

after a

37C

high tidal height comheat shock. In contrast,

HSP69 was much more limited in

HSC70 also increased mark-

these oysters. Control levels of

edly during this period. Although there were slight increases

A. M.

164

HAMDOUN ET

AL.

Totten 0.3m

Totten 1.2m

Ovster 2

CH'Sler 1

Figure
Totten

1.

Inlet,

Ch'Ster 3

Internal temperatures of three living oysters (solid lines) at

Washington) and external temperatures from

Bag Temp

two

a temperature

tidal heights

1.2

and 0.3

at

probe placed alongside the oysters

(dotted lines). Internal temperatures closely track external temperatures.

in

mean

levels of control

hsp70

mRNA

to winter oysters, the differences

in

were not

summer

relative

significant

(P =

compared to their low tidal height counterparts during the

summer. August survival of high tidal height oysters after

44C

0.13; Fig. 7).

low

Thermal

limits

and induction of thermal tolerance

significant difference in lethal temperatures

between

oysters at the two tidal heights. However, by August there

were significant increases in the baseline thermal limits of
at both study sHes. Moreover, oysters at the high
height appeared to exhibit elevated thermal limits

oysters
tidal

was

significantly higher than that of the

height oysters (P

occurred in the absence of

Laboratory heat shock of oysters at both tidal heights

suggested both seasonal and tidal height influences on the
upper thermal limits for survival (Fig. 8). In January there

was no

heat shock
tidal

0.03).

HSP69

These differences

expression, suggesting

that control thermal limits in Pacific oysters are likely to

be

more closely associated with cognate HSC70 expression

than to inducible

HSP70

expression. In contrast, induced

thermal tolerance appeared to be associated with inducible

HSP70

at the high tidal height

during the winter were able
to completely withstand a subsequent lethal treatment of
44C. Heat shock at 37C did not induce thermotolerance

that

9).

expression (Fig.

were heat-shocked

at

Oysters

37C

HSP GENE EXPRESSION

35

IN

OYSTER

165

A. M.

166

HAMDOUN ET

AL.

37C

Control

33C

Control

37C

January

40C

August

B.
37C

Control

:HSC77
:HSC72
HSP69

June

<HSCT7
<H5C72
July

~i..

Figure 6. (,4) Western blots, captured on digital scanner, of HSP70 family expression in high tidal height
(1.2-m) oysters (3 individuals per treatment) in August and in January. HSC77 and HSC72 are dramatically
elevated in control oysters sampled in August. (B) Western blots, exposed to film, of HSP70 protein family in
oysters (5 individuals per treatment) living at high tidal height (1.2-m). In June, a single 1-h heat shock (37C)
completely induces HSP69 expression. In July. HSP69 expression is absent in most animals sampled, hut the
control levels of HSC77 and HSC 72 are elevated relative to June.

33-37C

from about

in

winter to

37-40C

in

summer.

JANUARY

In

high tidal height oysters, the temperature required for induction of thermal tolerance also increased from 37C in

40C

winter to

Tonen

Tolten

3m
2m

summer.

in

Although we did not determine the changes to the absolute limits for control and inducible thermal tolerance, the
observed changes suggest potential costs for plasticity of the
heat-shock response. It is clear that seasonal increases in
thermal limits are associated with concomitant increases

in

the temperatures required for induction of tolerance to oth-

4500

[3

4000

AUGUST

January

a August

3500

Tonen
Tonen

3m
2m

3000

2500

2000

s
1500
1000

500

IISP7l}mRNA

Figure

HSP70

l/i

Control levels of total Iisp70

7.
r-

'
i

prior to heat

the high tidal height

on the same hku

as

measured

in

.2

m).

mRNA

(n

4) and total

summer and winter from oysters from

Summer and winter protein samples were run

shock

in

mRNA

samples, and relative levels were

arbitrary units using Scion Image software.

were

Figure

8.

summer and
each

Survival of oysters after

winter. Points are the

s.e.m.).

h of

means of

submerged heat shock

in

three groups of seven oysters

HSP GENE EXPRESSION

IN

OYSTER

specific effect

JANUARY O AUGUST

167

due

Moreover, by planting

to selection.
at

each

site,

we

sib-

limited

ling juvenile
oysters
any
confounding effect of selection for thermal tolerance at
settlement. Thus, we conclude that our results and perhaps

those previously observed in Mytiliis (Roberts et

al..

1997;

2001) are probably the consequence of

Buckley
significant phenotypic plasticity in the heat-shock response
rather than of fixed genotypic differences between bivalves
et

al..

living along gradients of stress.

height

Induced thermal tolerance

9.

Figure

1.2 m).

from the high

in oysters

Oysters were either heat-shocked directly

ature or shocked for

at lethal

tidal

temper-

h at a sublethal temperature and then allowed to

recover for 2 days before lethal temperature treatment.

Numbers below

initial treatment temperature, followed by the temperature

of the subsequent treatment (LT = lethal temperature as determined tor
each group of oysters 1 week prior to the start of the experiments). LT was

bars indicate the

44C

in the

winter and 46

in the

groups of seven oysters each

summer. Bars represent means of

three

s.e.m.l.

changes

in

thermal limits

in nature.

Clearly most of
below those

the temperatures encountered in nature are far

required to measure changes in thermal limits. Thus the

consequences of changes in inducible thermal tolerance

be most significant during periods when elevated temperatures are extreme or when they occur in concert with
other stresses.

may

Over-expression of HSPs is known to have deleterious

consequences. Initiation of heat-shock responses usually
results in a concomitant reduction in the synthesis of all
other proteins (Parsell and Lindquist. 1994). During stress.
HSPs can account for a large fraction of cellular protein;
thus

it

hypothesized that their synthesis represents a

is

significant energetic cost. In Drosophila,

levels of

HSP70

artificially high
can reduce thermotolerance and slow the

development of larvae (Krebs and Feder, 1998). Thus it is

possible that the observed plasticity of the heat-shock response

most useful

in differentiating

components of the heat

re-

sponse that are genetically fixed from those that exhibit

adaptive plasticity or intra-specific variability. Oysters may
be an ideal system for these studies. Because oysters are a

commercially valuable species, recent studies (e.g., Launey

and Hedgecock, 2001) have focused on characterizing the
genetics of Pacific oysters from known lineages. The possibility of producing oysters with characterized genotypes

erwise lethal temperatures. However, induction of thermal

tolerance is a laboratory phenomenon and may not directly
relate to

Further experiments using genetically characterized famof bivalves, and other model organisms, may be the

ilies

in Pacific oysters represents a trade-off

between the

and improved culture performance under a wide range of

environmental conditions is being explored (Langdon et al..
2003).

major challenge facing oyster growers

mortality during the warmest

al., 2000). It is not yet known

is

reducing

summer months (Cheney

if

the

et

improved performance

of these characterized families of oysters

is

related, at least

in part, to genetically fixed differences in their ability to

mount functional heat-shock responses and withstand

mal

ther-

stress.

Our

results suggest that both

rates of
stress,

accumulation of

gene expression are responsive

to

HSP

and

environmental

but that changes in gene expression alone are not

sufficient to account for the differences in protein

accumu-

Remarkably. hsp70 mRNA levels were elevated 2

days after heat shock in high tidal height oysters that were
heat-shocked at 40 and 43C. It is possible that elevated
lation.

mRNA

levels

would have also been measured

at

lower

temperatures if tissues were sampled sooner than 48 h after

heat shock. Our results suggest that cognate HSP70 protein
expression in oysters

is

not directly controlled at the tran-

costs of maintaining elevated control thermal tolerance and

the cost of the resting state in which the heat shock proteins

scriptional level.

are maintained at lower levels.

regulatory components of the long-term modulation in control HSP70 level. Consistent with this hypothesis, Clegg et

In the laboratory, other bivalve species such as Mvtilus

exhibit significant plasticity in various features of the heat-

shock response (Buckley et al., 2001). However, two factors

confound the interpretation of results from experiments with
adult bivalves collected

selection

2001

),

occurs

and

it

is

at

from the

settlement

likely to

field.

First,

significant

(Launey and Hedgecock,

correspond with distribution along

gradients of stress (Michener and Kenny, 1991). Second,

large-scale mortality may occur within adult bivalve beds

during

was no
ments,

warm

seasons (Cheney

et al.,

2000). Because there

significant differential mortality during our experi-

we can downgrade

the likelihood of a strong site-

HSP70

al.

It

is

possible that changes in the rate of

protein synthesis and degradation

may

be important

HSC77 and HSC72

HSP70 synthesis has re-

(1998) demonstrated that levels of

remain elevated long

after total

turned to control levels.

Other authors (Tomanek and Somero. 2002) have proposed that level of cognate HSP70 isoforms may be an
index of protein synthesis capacity, whereas the levels of
inducible isoforms

may be more

accurate indices of adapit was the threshold for

tation to heat stress. In our study

HSP69 expression that reflected adaptation to thermal stress

associated with seasonal changes. The seasonal plasticity of
the heat-shock response

was

reflected

most dramatically

in

168

A. M.

the large seasonal change- in the control levels of

and HSC72, sugge-

hit these isoforms

ii

Jtmg the

important roles in
In

shock

a;

cr,

effects of thermal stress.

experiments using Drosophila

2001), there is evidence that the heat-

may be

se

res;

HSC77

also play

ction

labor;]i'

(Lerman

may

HAMDOUN ET

regulated by genetic factors that are

fixed during evolution in a particular environment.

How-

ever, in sessile intertidal species, several key features of the

heat-shock response (e.g., control levels of HSP and threshold temperatures of HSP induction) appear to be sensitive to

AL.

be adaptive

This study has shown that a change in the threshold

temperature for HSP70 induction, in response to chronic
stress, is associated with a concomitant change in the thresh-

We

old temperature required to induce thermotolerance.

have shown that the changes in threshold temperature for

induction of thermal tolerance match the seasonal changes

in threshold temperature for HSP70 induction and expression.

Of particular

ecological significance

acclimation to laboratory and natural seasonal conditions

an increase

1997; Buckley et
/.,
2001). In sessile
such
as
or
behavioral
mussels,
species
oysters
adaptation

temperatures at
acquisition of thermotolerance

(Roberts et ai.

plays a

more limited

mental

stress.

Thus,

role in regulating exposure to environ-

we hypothesize

that

the heat-shock

response, and particularly the levels of HSC70 in Pacific

oysters, must exhibit sufficient phenotypic plasticity to al-

low organisms

to

Most

studies of the ecological physiology of stress pro-

have two key limitations (Feder and Block, 1991;

Feder and Hofmann, 1999). First, most relevant stressors

teins

initiate

responses from a variety of cellular and systemic

stress-response systems. Second, single stresses are rarely

encountered

environment.

in the

More

typically,

organisms

encounter multiple stresses that interact with a variety of

stress-responsive systems. In the case of sessile bivalves, a
critical stressor that

has been overlooked

is

the simultaneous

tions interact with heat-shock responses in intertidal

Plasticity of the heat-shock response

mon

is

apparently a

com-

other marine invertebrates

(Tomanek and Somero, 1999;

et ai., 2002).

ioral regulation

more

of thermal environment

is

possible. Perhaps

significantly, these results could indicate that although

heat-shock proteins and the genes that encode them are

highly conserved, the composition and expression of the
regulatory components that collectively constitute the cellular thermostat may exhibit significant interspecific variability.

Thus

oysters, mussels,

and other species

that exhibit

significant adaptive plasticity of the heat-shock response

may have

evi

mechanisms.

<l

which control

o.

the most complex and robust regulatory

nresolved question is the mechanism by
K
ng levels of HSP70 are regulated

during chronic stress exposure.

mechanisms provide

It

is

not yet clear

distinct regulatory

pathways

if

that

these

might

Although

this

The authors thank Brian McDonald, Andrew Suhrbier

their technical assistance. The authors

and Sheila Walsh for

thank Drs. Jim Clegg, Fred Griffin, and Lars Tomanek for
their many helpful comments and thought-provoking discussions about this work.

and Ernie Chang for

We

greatly thank Drs. Jeff Specs

generous help during the analysis

of hsp70 mRNA. The work was supported by a grant from
the National Sea Grant College Oyster Disease Research
their

Program (ODRP) Grant No. NA96RG0488 and the

UC

Toxic Substances Research and Teaching Program.

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benefits in species and life-history stages for which behav-

Specs

oysters.

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from selected parents.

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Insect Physiol. 44: 1091 -I 101.

Evans. D. Jacobson, and M. Blouin. 2003.

IN

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snails (Genus Tegula) from different thermal habitats: implications for
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Tomanek,

L..

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Reports of Papers Presented at

THE GENERAL SCIENTIFIC MEETINGS

OF THE MARINE BIOLOGICAL LABORATORY,

Woods

Hole, Massachusetts

11 to 13

August 2003

Program Chairs:
Mary's College of Maryland

KAREN CRAWFORD, St.

ROBERT GOULD, New York State Institute for Basic Research
ROBERT PAUL MALCHOW, University of Illinois at Chicago
JOSEPH VALLINO, The Ecosystems Center, MBL

Each of these reports was reviewed by two members of a special editorial board
drawn from the research community of Woods Hole, Massachusetts.
Reviewers included scientists from
THE MARINE BIOLOGICAL LABORATORY
AND THE WOODS HOLE OCEANOGRAPHIC INSTITUTION

SHORT REPORTS FROM THE 2003 GENERAL SCIENTIFIC MEETINGS

OF THE MARINE BIOLOGICAL LABORATORY
The Editor
The MBL Awards

Wollert, Torsten,
for

Ana

S.

DePina, Carl

J.

DeSelm, and

George M. Langford

175

2003

Rho-kinase

is

required for myosin-II-mediated vesicle

in extracts of clam oo-

transport during M-phase

DEVELOPMENTAL BIOLOGY

cytes

195

Zakevicius, and H. Ripps

experimental approach to the study of gap-junc-

Cusato, K.,
(

An

Edwin, Robert Baker, and Winfried Denk

duration
three-dimensional imaging of calcium
Long
waves in zebrafish using multiphoton fluorescence

.ill.

in.

I.

microscopy
Opher, and Alon Sabban
Squid sperm to clam eggs: imaging wet samples

tion-mediated

176

177

Armstrong, Peter
The decorated

Apoptosis in Microcinna
in a

scanning electron microscope

immune

Kuhns, M. M.
199

prolifera allografts

and Margaret T. Armstrong

clot:

binding of agents of the innate

fibrils of the Limulus blood

system to the

201

Isakova, Victoria, and Peter B. Armstrong

Imprisonment in a death-row cell: the fates of mi-

179

Crawford, K.
Lithium chloride inhibits development along the animal vegetal axis and anterior midline of the squid

crobes entrapped in the Limulus blood clot

Harrington, John M., and Peter B. Armstrong

181

Susan D., and Barbara C. Boyer

HNK-1/N-CAM immunoreactivity correlates with

B.,

J.

clot

syncytial layer

embryo

197

death

cell

Tepsuporn, S., J. C. Kaltenbach, W.

Burger, and X. Fernandez-Busquets

Gileadi,

Wadeson, P. H., and K. Crawford

Formation of the blastoderm and yolk
in early squid development

J.

liposome-permeating

activity

203

from the surface of

the carapace of the American horeshoe crab, Limulus

Hill,

cil-

205

po/\phemus

iary patterns during development of the polychaete

Capitella sp.

182

NEUROBIOLOGY AND BEHAVIOR

CELL BIOLOGY

Bogorff, Daniel

J.,

chow, and Peter J.

Heck, D.

E.,

and

J.

self-referenc-

207

ing glutamate-selective micro-biosensor

Chappell, R. L., J. Zakevicius, and H. Ripps

185

Zinc modulation of hernichannel currents in Xenopus

Gallant, P. E.
inhibits the slow axonal transport of tubulin

squid giant axon

Delacruz, John, Jeremiah R. Brown, and George M.
in the

between recombinant conventional

squid kinesin and native myosin-V
DeSelm, Carl J., Jeremiah R. Brown, Renne Lu, and
George M. Langford
Rab-GDI inhibits myosin V-dependent vesicle transport in squid giant axon
Pielak, R. M., V. A. Gaysinskaya, and W. D. Cohen

Shribak, Michael, and Rudolf

asters

190

Redenti, S., and R. L. Chappell

Zinc chelation enhances the sensitivity of the
b-wave in dark-adapted skate retina

192

Molina, Anthony J. A., Katherine Hanimar, Richard

Sanger, Peter J. S. Smith, and Robert P. Malchow
Intracellular release of caged calcium in skate hori-

light

in re-

revealed

microscopy

by

194

ming
173

toadfish, Opsamis

tail

211

ERG

zontal cells using fine optical fibers

Palmer, L. M., B. A. Giuffrida, and A. F. Mensinger
Neural recordings horn the lateral line in free-swim-

Oldenbourg

of Sp/sula oocytes

scanned aperture polarized

ings of supramedullary/dorsal neurons of the cunner, Tautognlahnu adspersus

188

body formation

Three-dimensional birefringence distribution

Chambers, R. Eseh,

Transient use of tricaine to remove the telencephalon has no residual effects on physiological record-

Interactions

Cytoskeletal events preceding polar

in activated Spisula eggs

209

oocytes
Zottoli, S. J., O. T. Burton, J. A.
L. M. Gutierrez, and M. M. Kron

187

Langford

constituted

A. Messerli, Robert P. Mai-

Smith

Development and characterization of a

D. Laskin

Ryanodine-sensitive calcium flux regulates motility of

Arbaria punctulata sperm

Axotomy

Mark
S.

213

215

216

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

174
Child, F. M., H. T. Epst

'

i.

A.

M. Kuzirian, and D.

L.

Cavatorta, Jason R.,

Alkon

Memory red
Kuzirian, A.
C. E.

Morgan Johnston, Charles Hopkin-

son, and Vinton Valentine

Ok'

i'."ii

!V

.<

;.

in

ne, not the tripeptide

Train

E. Motta,

ROD, modulates
220

Savage, Anna, and Jelle Atema

Neurochemical modulation of behavioral response
to chemical stimuli in H<ntini\ innmrtums
Mann, K. D., E. R. Turnell, J. Atema, and G. Gerlach

Kin recognition in juvenile zebratish (Danio reno)

based on olfactory cues
Turnell, E. R., K. D. Mann, G. G. Rosenthal, and G.

224

225

and

F.

epizootic

electron
lobster

microscopy

shell

disease

in

I.

Molecular quantification of toxic Alexandrium fundyense in the Gulf of Maine

Sangster, C. R., and R. M. Smolowitz

Valiela

tic

size

254

estuaries

Davidson, W. Kingerlee, and K. Savage

Walker,
Incubation conditions of forest soil yielding maxiC., E.

Hemant M. Chikarmane, Roxanna

mum

by

235

and Aiui Findley

dissolved organic nitrogen concentrations

minimal residual
Holden, M. T., C.

and
256

nitrate

Lippitt, R. G. Pontius, Jr.,

and C.

Williams
Building a database of historic land cover to detect

and during
236

discharge

252

Grady, A. S. Leschen, R. H.
Valiela
S. P.

and relationand trophic position of the Atlanhorseshoe crab, Limulus polyphemus, within Cape
between

ships

Cod
233

onh

Catalase in microsporidian spores before

250

M. Teichberg, and

Stable isotopic assessment of site loyalty

231

Description of Vibrio alginolylicus infection in cultured Sepia officinalis. Sepia ripnmri, and Sepia phara-

Earl,

I.

O'ConneU, C. W.,
Carmichael, and I.

and Sonya Dyhrman

tail

Transplantation and isotopic evidence of the relative

effects of ambient and internal nutrient supply on

and nutrient supply on

the
of
green macroalga Ulva lactuca in estugrowth
aries of Waquoit Bay, Massachusetts

MadeUne, Deana Erdner, Donald M. Anderson,

Smolowitz, and Kevin R. Uhlinger

Detection of Edwarduella infections in Opsanus
polymerase chain reaction

248

Relative influence of grazing

Characterization of phosphorus-regulated genes in

Trichodesmium spp

Weidner,

246

Valiela

the growth of Ulva lactuca

Morgan, J. A., A. B. Aguiar, S. Fox,

of
investigation
Homarus ameri-

228

Baird, Krystal D.,

discharge to Waquoit Bay, Massachusetts

Johnston, M. E., J. R. Cavatorta, C. S. Hopkinson, and

227

Orchard, Elizabeth, Eric Webb, and Sonya Dyhrman

Galac,

244

Abraham, D. M., M. A. Charette, M. C. Allen, A. Rago,

and K. D. Kroeger
Radiochemical estimates of submarine groundwater

Importance of metabolism in the development of salt

marsh ponds
S. Fox, and
Aguiar, A. B., J. A. Morgan, M. Teichberg,

W. Goetz

Expressed sequence tag analysis of genes expressed

in the bay scallop, Argopecten irradians
Hsu, A. C., and R. M. Smolowitz

Scanning

Nitrogen flux and speciation through the subterranean estuary of Waquoit Bay, Massachusetts

V. Valentine

MOLECULAR BIOLOGY, PATHOLOGY, AND MICROBIOLOGY

S. B.,

242

M. A. Charette

in zebrafish (Dania rnio) analyzed with

video-stimulus techniques

Multiple approaches to tracing nitrogen loss in the

West Falmouth wastewater plume
Talbot, J. M., K. D. Kroeger, A. Rago, M. C. Allen, and

222

Gerlach

Roberts,

239

estuary

Thorns, T., A. E. Giblin, K. H. Foreman

calexcirin in Hermissenda

Mate choice

Patterns of sedimentation in a salt marsh-dominated

218

Hermissenda

M. Child, H. T. Epstein, M.
and D. L. Alkon

257

landscape change

ECOLOGY AND POPULATION BIOLOGY

Agnew, A. M., D. H. Shull, and R. Buchsbaum
Growth of a salt marsh invertebrate on several species
of marsh grass detritus

ORAL PRESENTATIONS
238

Published by

title

only

259

Reference: Bio/. Bull. 205: 175. (October 2003)

2003 Marine Biological Laboratory

The

MBL

Awards

for 2003

MBL

Awards are given for the best paper presented at the General Scientific Meetings by a senior investigator,
a junior investigator, a graduate student, and an undergraduate. The awards are based on both the oral presentation
and the short report, and additional papers are selected for Honorable Mention.
The four presentations selected

Among

we

these reports,

for

MBL

Awards

this

year reflect the broad scope of science

at the

Laboratory.

find a classic vegitalizing agent used to study

morphogenesis in squids, multiphoton

zebrafish embryos, a dermal exudate that may

fluorescence microscopy revealing complex. 3-D signaling events in

This
keep the horseshoe crab's exoskeleton clean, and serotonin prolonging the response of lobsters to food odors.
title of their presentation and the page on which their short report appears.
with
the
are
listed
below
awardees
year's

The Editor
August 2003

Senior Investigator

Junior Investigator

Axotomy

Edwin Gilland

WINNER

WINNER

Paul Gallant

with Robert Btiker

inhibits the slow axonal transport of tu-

buliti in the

squid giant axon

(p.

HONORABLE MENTION

Long duration three-dimensional imaging of

187)

cium waves

Karen Crawford

(p.

in zebrafish using

cence microscopy

Lithium chloride inhibits development along the

animal vegetal axis and anterior midline of the
squid embryo

and Winfrifd Denk

(p.

multiphoton

176)

HONORABLE MENTION
Jane Zakevicius

with

181)

cal-

fluores-

Karen Cusato

and Harris Ripps

An

experimental approach to the study of gapjunction-mediated cell death (p. 197)

Graduate Student

Undergraduate

WINNER

Anna Savage
Atema
Neurochemical modulation of behavioral response to chemical stimuli in Homarus americanus

WINNER

John M. Harrington

with Peter B. Armstrong

withjellf

activity from the surface of

American horseshoe crab,

liposome-permeating

the carapace of the

Limulus polyphemus

(p.

205)

HONORABLE MENTION
Hammar, Richard
and Robert P. Malchow

with Katherine
Smith,

(p.

Sanger, Peter J.

with

Matthew

with

S.

Charette,

Abraham
Adam Rago,

Danial

Matthew

Allen,

and Kevin Kroeger

Radiochemical estimates of submarine groundwater discharge to Waquoit Bay, Massachusetts

Intracellular release of caged calcium in skate horizontal cells using fine optical fibers (p. 215)

HONORABLE MENTION

222)

HONORABLE MENTION

Anthony Molina

Cheryl Sangster

(p.

246)

HONORABLE MENTION

Roxanna Smolowitz

with Valeriya Ga\sinskaya

Description of Vibrio alginolyticus infection in cultured Sepia officinalis, Sepia apama, and Sepia pharaoms (p. 233)

Rafal Pielak

and William Cohen

Cytoskeletal events preceding polar body

tion in activated Spisula eggs (p. 192)

175

forma-

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

176
Reference: Biol. Bull.
2003 Marine Bioloi

Lone

2115

7.

(October 2003)

Itor)

Imaging of Calcium Waves in Zebrafish Using Multiphoton

Fluorescence Microscopy
2
Edwin Gil land '*, Robert Baker', and Winfried Denk

.on Three-Dimensional

Max

Both luminescent (1,2) and fluorescent

used

to

image

continue for

from

(2) reporters

periodic large-scale intercellular

begin during zebrafish gastrulation,

a variety of locations

about

waves

12 hours. These

at least

at

School of Medicine. New York. NY

Plank Institute, Heidelberg, Germany

NYU

have been

Embryos were

injected while in

30%

Danieau's solution (1) and

calcium waves that

then transferred to

10% Danieau's

65%

diameter grooves

agarose-coated dishes, and maintained

epiboly, and

arise every 5 to 10

min

and traverse the blastoderm margin and

in

solution, placed into

5-W Mira

titanium-sapphire pulsed laser

waves that likely propagate by a

calcium-induced calcium
mechanism
feedback
involving
positive
release from intracellular stores, possibly including diffusion of

either parasagittal, frontal, or horizontal, starting just

1 ).

the category of fast calcium

calcium or IP3 through gap junctions (3). Likely targets of the

waves include calcium-sensitive proteins involved in epiboly (3)
and convergent extension

may

(4),

and others such as calreticulin

activity (5). Long-distance signaling

an

that

nuclear receptor
play a role in the temporal regulation of

mechanism

by rhythmic calcium waves

is

for

synchronizing calcium-triggered
events throughout the embryo with high temporal precision. Since
appealing

the zebrafish

600 p,m

embryo

in diameter,

is

a roughly spherical

imaging these

waves

body approximately

in a single optical plane,

approximate their three-dimenMoreover, other calcium signals that may be

28

on a thermoelectric heating stage. Imaging was performed with

a custom-built multiphoton fluorescence microscope using 890 nm
excitation light from a

mm
at

During somitogenesis they appear as a series

of pulses of elevated calcium levels centered on the tailbud region.
The waves travel at approximately 5-10 /j,m per s and thus fall into

main body axis

(Coherent) and a 20X, wide aperture, infrared objective, N.A. 0.8

(Olympus). Embryos were imaged in a series of optical planes,

below the

surface of the embryo, with successive planes separated along the

by distances from 5 to 20 /xm, for total imaging depths of up

to approximately 300 /urn. Scanning each image plane in a 256 X
256 pixel pattern required 0.4 to 0.8 s. depending on the beam
; axis

dwell time. In a typical experiment, a sequence of 15 optical slices

was repeated every 14 s over 8 h. Collected image sequences were
analyzed using the IDL (SRI) and ImageJ (NIH) software packages.

The experiments showed

that the periodic increases in

levels seen in previous studies (1, 2) pass through

all

calcium
cellular

through most or all of

shows data from an experiment in which 45

regions of the embryo and.

at later stages,

as in previous studies, can only

the yolk cell. Figure

sional trajectories.

calcium waves were imaged over 4 h. Time lapse movies of the

imaging sequences can be viewed on the Biological Bulletin's web

occurring

at the

same time, but

in different optical

planes within

embryo, cannot be documented. Ideally, free calcium levels

should be imaged for many hours throughout the entire volume of

the

shorter than the lifetimes of individual

site

www. mbl.edu/BiologicalBulletin/VIDEO/BB. video, html).

Single images (X-Y) from different parasagittal optical planes

Luminescent techniques require considerable

and
temporal integration to achieve adequate spatial resolution

in Figure
during one imaging cycle 15 planes in 14.3 s) are shown
to the
area
dorsal
for
an
values
time,
of
A
a-e.
1,
through
pixel
plot
tailbud, shows calcium levels oscillating with a mean period of

thus cannot approach this goal. Conversely, scanning laser micros-

315

the

embryo

at intervals

signaling events.

copy using

visible light has the necessary spatial

resolution, but cannot be used for prolonged

and temporal

imaging

at

high

Gilsampling rates due to phototoxic damage to the embryo (E.

lund, pers. obs.). The present study demonstrates that multiphoton
fluorescence microscopy has the potential to achieve the goal of

sampling calcium dynamics throughout the entire zebrafish embryo for long durations with sufficient spatial and temporal reso-

complex three-dimensional signaling events.

lution to reveal

Glass micropipettes with outside tip diameters of 7 to 10 /nm

were used to pierce the chorion and inject zebrafish embryos (2- to
4-cell stages) with

(calcium

Kd =

to 2 nl

3 ju,M;

MW

of a

2%

solution of Fluo-4 dextran

10 kDa; Molecular Probes) dis-

KC1. The injections were made by a vegetal

approach and placed into the dorsal half of the yolk mass. Dye was
carried into the cytoplasm by diffusion and cytoplasmic streaming.
solved in 0.2

Corresponding author: egillandfs'mbl.edu

over many hours (Fig. If). As found in previous studies 1.

was seen in the
strongest modulation of calcium levels
(

caudal half of the embryo centered around the region of the

bud, with the brightest signals in the

tail

mesoderm, endoderm. and

densities and dye

subjacent yolk syncytial layer. Since cellular

shown) vary in different tissue layers, ratio imbe required to determine whether the greater intensities
in specific locations represent genuinely higher calcium

distribution (not

aging will
of signal
levels.

The movement of calcium waves through

the

embryo can

be roughly assessed by eye in the time lapse movies, and more

the time coordinates of peaks at
precisely, either by comparing
different X-Y-Z locations (not shown), or by reslicing an X-Y time
series

along a line

in

the

X-Y

plane (Fig.

and dark bands

Ig).

The calcium

in the resliced

image,
and the slopes of the bands indicate the direction of wave movement. In the case shown, most of the oscillations appeared earliest

oscillations appear as light

at
*

2), the

dorsal and ventral locations, and latest at a region dorso-anterior

to the tail bud, indicating that the

waves were sweeping caudally

DEVELOPMENTAL BIOLOGY

Figure

1.

a ratal of 200
is

Lateral views of a 5-somite zebrafish embryo showing 5 parasagittal imaging planes (a-e). The planes arc separated by 40 /j,m and span
^m in depth. Plane (a) is nearest the surface of the embryo while plane (e) is nearest the midline. The lime interval represented by (a-e)

near 200 min

the

in

(f).

The circle

X-Y plane along which

in (d)

inverse pattern,

i.e.,

shows a sampling area whose pixel intensity profile is shown in (f). The cun-ed line in (e) indicates the path
was re-sliced, as shown in (g). The white line in (g) shows the approximate time profile of one wave.

tail

bud. Other waves

they started near the

The three-dimensional

showed

Literature Cited

the

bud and traveled

tail

structure of the calcium

waves

Gilland, E., A. Miller, E. Karplus, R. Baker, and S.

Proc. Natl. Acad. Sci.

initial experiments are far more complex than

could be inferred from sequences imaged at single optical planes.

hinted at by these

To completely

2.
3.

Webb,
Webb,

S.,
S.,

and A.
and A.

USA

Webb.

1999.

Bio/. 4:

539-

96: 157-161.

Miller. 2003.

Miller. 2003.

Zygote 11: 175-182.

Nat. Rev. Mot. Cell

waves through space and time, the

temporal phase of wave peak values must be quantified for all X-Y
positions in all optical planes through the time series. Such anal-

4.

Wallingford, J., A. Ewald, R. Harland, and S. Eraser. 2001.

Biol. 11: 65 2- 6d 1.

provide further clues to the developmental role of the

5.

Holaska,

yses

may

in

the data stack

through the embryo towards the

rostrally.

177

reconstruct the

55

panembryonic calcium signaling system.

J.,

B. Black, F. Rastinejad, and B. Paschal. 2002.

Cell. Biol. 22:

Curr.

Mol.

6286-6297.

Reference: Biol. Bull. 205: 177-179. (October 20031

2003 Marine Biological Laboratory

Squid Sperm

to

Clam

Eggs: Imaging

Wet Samples

in a

Scanning Electron Microscope

Opher Gileadi* and Alon Sabban

Quantomix Ltd., Re/wvot, Israel

We

introduce

Wet SEM,

new imaging technology

electron microscopy of wet samples.

The samples

sealed specimen capsules and are separated from the

vacuum

in the

microscope chamber by an impermeable, electron-transparent

membrane. Imaging is performed in a standard scanning electron
microscope (SEM) using a backscattered electron detector (BSE)
(Fig. 1A).

tion

The technique

is

described

in a

pending patent applica-

).

The Wet

that allows

are placed in

SEM

technique presents,

respects, a

new

micro-organisms, plants, animal and human tissues, and a

electron
variety of fluid samples. Second, the use of backscattered

detection and the elimination of charging allow the internal struc-

is

Corresponding author: opherls'quantomix.com

many

cells,

by scanning electron microscopy. This

ture of cells to be visualized

in

the complete separation of the

sample
imaging modality.
from the vacuum allows direct imaging of fully hydrated, wholemount samples in an electron microscope operating at moderate
beam energies. Such samples include primary or cultured animal
First,

in

contrast

with customary

electron detection), which

is

SEM

imaging (using secondary

restricted to the surface.

The depth

ot

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

178

Wet SEM imaging of dam egg nuclei. IA) Schematic cross-sectional view of the capsule enclosing u sample. The generally rigid capsule (a)
Figure 1.
topped by a window, covered by an electron-transparent partition membrane (h). The sample (c) is in close contact with the partition membrane. When
placed in the evacuated chamber of a SEM. the sample is maintained in a wet state at atmospheric pressure. The microscopic image is obtained when the
is

scanning electron beam (d) penetrates the partition membrane and interacts with the sample, and hackscattered electrons (BSEt
detector

(/).

(B)

Clam egg

nucleus, adhering to the capsule

electron microscope (FEl

's

partition

membrane, was stained with uranyl acetate and imaged

XL-BO ESEM-FEGl Bar = 10 ^m. ICI Clam egg

nucleus, adhering in the capsule's partition

anti-nuclear pore complex antibodies and 0.8-nm gold secondarv antibodies, followed h\ xilver enhancement.
as in (C) but with unti-lamin antibodies. Bar = 2 fun.

imaged layer is limited by the penetration of the beam electrons, and is estimated to be 2-3 /urn. Furthermore, the depth can
the

be varied by changing the acceleration voltage of the electron

beam (typically within the range of 10-30 keV), yielding threedimensional information.

The method presents some additional

beneficial features,

some

of which were not wholly anticipated. We obtain resolutions

between 10-100 nm, an order of magnitude smaller than could he
predicted from the

volume of

interaction of the electron

beam with

an aqueous sample. Contrast between materials of low atomic

number, such as carbon and oxygen, can be readily detected. Thick
samples, such as tissue biopsies, can be imaged without thin
sectioning: only the top layer of up to 3 /J,m is seen. Both the global

and high-resolution distribution of colloidal gold labels on

can be readily determined.

The Wet

SEM

is

cells

uniquely suitable for samples, including lipid-

we have explored
scientists

egg nuclei were fixed

whole

cells, the

imaging of tissue

BSE

an scanning

membrane, labeled with

(D) Clam egg nucleus, labeled

capsule.

One example

in

adhere stably to the poly-L-lysine-coated partition membrane,

and all subsequent treatments were performed in the capsule.
to

Figure

IB shows a nucleus stained with uranyl acetate; the

condensed chromosomes are clearly

visible, as are the outline of

the nucleus and diffusely stained nuclear proteins.

somes seem to be surrounded by

which is not yet clear.

The chromo-

a dark "halo," the significance of

Figure 1C shows a portion of a nucleus stained with antibodies

complex, followed by secondary antibodies that

to the nuclear pore

conjugated to 0.8-nm colloidal gold particles. The silverenhanced gold particles are visible as bright dots. Note the edge
(asterisk) between the region of the nuclear membrane that is
attached to the partition

at

in

several applications of the technology with

visitors.

niques that derive from the simplicity of sample preparation; these

advantages include the ability to process and image numerous

samples, the ability to look

ij.m.

are detected by a

is shown in Figure 1. Clam

formaldehyde, then placed in the imaging
brief centrifugation (500 X g, 5 min) caused the nuclei

and

are

EM

Bar = 2

<e)

a wet state

specimens (especially of epithelial tissues), and the imaging of

myelin sheaths in neural tissue.
During our 2-month stay at the Marine Biological Laboratory,

and hydrated gels, which are adversely affected by

standard process.-, of dehydration that use organic solvents. The
method has several practical advantages over standard
techrich structures

in

region that

is

exposed

membrane of
to the solution

the capsule (att)

U-.Y/J);

the

and the

immunolabel,

which recognizes the outer aspect of the nuclear pore, has bound

DIM
in the

only the exposed region (as depicted

of gold labels in Fig. A).

OI'MI.M

Figure

ID shows
Note

179

(Hi'i

This work was performed in collaboration with Yosef Gruenof the Hebrew University and Robert Goldman of North-

schematic distribution

baum

to lamin.

HKII

Al

a portion of a nucleus stained with antibodies

Figure 1C. the immunolabeling

that, in contrast to

XL-30 scanning

western University. The

a generous loan

electron microscope

from FEI Company. Peabody.

was

MA.

extends through the entire visible region of the nucleus. We

attribute the difference to the location of lamin inside the nucleus,
so access by labeling antibodies

is

Literature Cited

not blocked by adhesion to the

capsule's partition membrane.

These

show

results

that the

Moses,

SEM

Wet

technique can derive

at high resolution from samples that were

subjected to treatments comparable to those used to prepare for

meaningful information

O. Zik. and

Thiberge. 2002. Device and method for the

non-vacuum environment using a scanning

S.

in a

electron microscope. International patent application

published as

WO0245125.

PCT/1LO 1/01 108

[Online] Available: http://ep.espacenet.com/

[2003. August 25].

microscopy.

light

E.,

examination of samples

Reference: Biol. Bull. 205: 179-180. (October 2003)

2003 Marine Biological Laboratory

Formation of the Blastoderm and Yolk Syncytial Layer in Early Squid Development
2 3
1 3
and K. Crawford '*
P. H. Wadeson
'

'

Rochester Institute of Technology, Rochester,

2
"

lot

eyepiece, and camera.

by winching.

Nearly all of what we know of cephalopod development has

been drawn from live or fixed embryos collected from naturally
capsules

(1.

2.

3).

aspects of development

Many

including pronuclear migration and cleavage (4). cytoplasmic and

cellular

computer-controlled Axiovision software

program was used for image acquisition from an MRc5 Zeiss

X 1936). Digital
digital camera set to optimal resolution (2584

Yogi Berra

laid jelly

MD

Mary's City,
Mary's College of Maryland,
Marine Biological Laboratory, Woods Hole, MA

St.

You can observe a

NY

St.

movements

(5

1.

differentiation

and organogenesis

would

be better understood and more effectively compared to other

embryos if examined in the living state. To begin this work, we

images were collected

h.

at 5-

or 7-min intervals at 21

Throughout these periods, embryos appeared

for 2-12.5

to cleave

and

develop normally.

Ten separate time-lapse sessions were carried out and images

from two are presented. In the first session, images were collected
at 5-min intervals, from first cleavage through blastoderm forma-

development
squid embryos. Loligo pealeii, from early

images are presented in Fig. la-c. The first

image was taken 8 h post-fertilization (hpf) after the fourth cleavage (Fig. la). Each cleavage furrow is numbered in the order of its

cleavage through the formation of the external yolk syncytial layer

(YSL) and the early phase of epiboly. Adding the dimension of

occurrence, and the polar bodies (pb and arrow) are visible resting
in the first cleavage furrow. The larger blastomeres. formed by the

time to our analysis revealed new and intriguing elements in the

development of this organism.
In vitro- fertilized squid embryos were prepared (6) and oriented

emunequal third cleavage furrow characteristic of cephalopod

The
bryos ( 1 ). identify the future anterior midline of the embryo.
second image (Fig. Ib) was taken 9.6 hpf at sixth cleavage (arrowhead). This cleavage separates the central blastomeres from the

have used time-lapse video microscopy

of

in

for

v/rro-fertilized

imaging

in

depressions

made

in

to record the

0.2% agarose (Sigma (-lined

plastic petri dishes (Falcon) filled with Millipore (0.22 /j.m) filtered

seawater. Dishes of

embryos were placed on

a universal transmit-

ted light illuminator with an adjustable reflector that allowed for

bright field or oblique illumination, or a combination of the two.

To minimize

KL

1500 constant-color-temperature
filter was used to illuminate the
source
with
an
infrared
fiber-optic
specimen. A Zeiss Stemi SV 11 stereomicroscope with a 1.6x
heat transfer, a

Planapochromat lens was used for time-lapse imaging. An intermediate mount was placed between the lens and microscope body
to align the light path with the center of the

Corresponding author:

kcrawford@smcm.edu

front lens, right

tion: three of these

outer syncytial layer of the embryo, which is continuous with the

yolk cell. The final image (Fig. Ic) was taken 16.25 hpf and reveals
a well-defined blastoderm surrounded by radiating clusters of cells,
the outermost of

boundary created

which are continuous with the yolk

at

cell.

The

sixth cleavage (arrowhead) remains well pre-

served.
In the second session, images were collected at 7-min intervals,
from blastoderm formation to the onset of epiboly, 26-27.5 hpf:
three of these images are presented in Fig. Id-f. At 26 hpf, the

boundary formed
cells have moved

at sixth

cleavage has become more distinct, as

into the blastoderm;

and pairs or small clusters

of sister blastomeres formed during earlier cleavages radiate

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

180

Figure 1.
linages of developing squid embryos Delected from two time-lapse sessions, (a. b, c) Early cleavage through blastoderm formation, 4-16.5
h post-fertilization (hpf); an arrow indicates the polar bodies (ph) in each panel in this session, (a) Fourth cleavage. 8 hpf. (/>) Sixth cleavage (arrowhead),
9.6 hpf. (ct Blastoderm formation, 76.25 hpf. The boundary created at sixth cleavage {arrowhead} is well preserved at this stage of development, (d, e, f)
to the onset of epiboly, 26-27.5 hpf. (d) A distinct blastoderm with radiating pairs or clusters (*) of outer blastomeres, 26 hpf. (e)
Blastoderm, 26. 7 hpf: the inner blastomeres from each marked pair have migrated toward the developing blastoderm while their outer sister cells and the
one lone cell simultaneously collapse into the cytoplasmic cortex of the yolk cell. If) Blastoderm. 27.5 hpf: the migrating inner blastomeres have reached

Blastoderm formation

the blastoderm while their outer sister cells are

Video Note. Supplementary video

no longer

clips are available

visible.

Scale bar

= 200

jj.m.

For further

description, see

for viewing on The Biological Bulletin web

site at

text.

[http://www.mbl.edu/BiologicalBu/letin/

VIDEO/BB.video.htmll.

around the blastoderm.

An

asterisk (*) has

been placed by each of

four pairs of these radiating sister blastomeres. in addition to one

lone cell (Fig. Id).

marked

By

26.7 hpf, the inner blastomeres from each

toward the developing blastoderm,

while their outer sister cells simultaneously collapse, a cell behavior first described by J. P. Trinkaus in fish (7), into the cortex of the
pair have migrated

One hour

migrating inner blastomeres

have reached the blastoderm, while the nuclei of their outer sister

yolk

cell (Fig. le).

cells

have entered the yolk

later, the

cell,

contributing to the yolk syncytium

made possible by support from a Faculty DevelGrant

and
the Aldom-Plansoen Distinguished Endowed
opment
Professorship in Contemporary Studies to K.C. from St. Mary's
This work was

College of Maryland. K.C.

is

most grateful

to

Bill

Eckberg,

University, who graciously provided laboratory space,

collaborative guidance, and digital imaging assistance.
thank

Howard

We

Rudi Rottenfusser.

summer

MBL

Zeiss representative, for allowing his

intern (P.H.W.) to participate in this work. Finally, K.C.

wishes to thank

J.P.

Trinkaus and his

many

students for reminding

us to "watch."

(Fig. If I.

YSL is critical to patterning and development (7.

and time-lapse video analysis has played an important role in
our understanding of this model system (9). As in squid, the YSL
In teleosts. the

8),

separates the yolk from the

nutrients

embryo and

regulates the transfer of

all

and factors present within the yolk

to the developing
important layer involves the collapse of
the boundary between the developing blastoderm

embryo. Formation of
blastomeres

at

this

cytoplasm. That cephalopods form their YSL

through similar developmental mechanisms to those of teleosts
exemplities the fundamental similarities that exist between em-

and the yolk

Literature Cited
1.

Arnold.

3.

Segawa,

J.

M.

S.,

1965.

W.

Anniv.

Mem. Boston

Biol. Bull.

Soc. N.H. 1-22.

128: 24-32.

T. Yang. H.-J. Marthy,

and R.

T. Hanlon. 1988.

Vcliser 30: 230-243.

Crawford, K. 2000.
Crawford, K. 2001.

Biol. Bull.

199: 207-208.

Biol. Bull.

201: 2? 1-252.

f>.

Crawford, K. 2002.

Btol. Hull.

203: 216-217.

Trinkaus,

J. P. 1993.

Trinkaus.

J. P.

9.

Concha. M.

4.

cell

bryos faced with similar structural constraints and highlights the

importance and need for further comparative study.

Brooks. VV. K. 1880.

2.

L.,

1996.

and R.

Exp. Zoo/. 265: 258-284.

Dev. Biol. 177: 356-370.

J.

J.

Adams.

1998.

Development 125: 983-994.

DEVELOPMENTAL BIOLOGY

181

Reference: Biol. Bull 205: 181-182. (October 2003)

2003 Marine Biological Laboratory
>

Lithium Chloride Inhibits Development Along the Animal Vegetal Axis and Anterior Midline of the
Squid Embryo
K. Crawford
Marine Biological

Laboratory, Woods

When

squid embryos. Loliga pealeii, are cultured in vitro ( )

they may be individually manipulated with classic and molecular
techniques, providing insights into the conservation of develop1

mental pathways. Dorsoventral polarity in many embryos is associated with precise gene transduction cascades involving the Wnt
signaling pathway

(2).

Results from experiments with frog (3), fish

and mouse (5) embryos suggest that a component of this

cascade, /3-catenin. plays a major role in axis formation. Additional support for the role of j3-catenin in the early development of
(4).

many embryos comes from studies using

LiCl is a known vegetalizing agent for
echinoderms generally,

lithium chloride (LiCl).

sea urchins (6), and in

enhances and expands levels and regions

it

MD

Mary's College of Maryland,

St.

8 of 16

Hole,

MA

embryos cultured

in the

presence of 60

As

anterior midline structures that were inhibited.

were abnormally placed:

either

(2/16), or cyclopic (1/16).

mM

LiCl had

a result, the eyes

converged (5/16, Fig.

If),

In contrast, structures that

form on the posterior body wall such as the funnel or paired

statocysts, although reduced, were always present in these embryos. These observations suggest that various regions in the

embryo

are differentially sensitive to LiCl treatment.

Lithium chloride treatment inhibits development along the animal-vegetal axis in the squid embryo and causes convergence and
fusion of anterior cephalic structures. This result

is

consistent with

the notion that LiCl treatment induces vegetalization of the squid

of nuclear /3-catenin localization coincident with increases in

embryo and thereby enhances mesodermal and endodermal

endoderm and mesoderm

tures at the expense of ectodermal derivatives, as

embryos

is

(7, 8). In contrast, its effect

on amphibian

species- and stage-specific. For example,

when

gastru-

embryos are treated with LiCl, they develop reduced notochords and enhanced vegetal structures, but when treatments are

fused

normally

invertebrate and vertebrate

embryos

(3,

7.

6,

8,

it

does

9).

struc-

in other

That LiCl

induce convergence, fusion, and cyclopia

lating

treatment

given

for a survey of the

embryos strengthens this interpretation (see
literature
on
early
cyclopia produced by experimental means) and

at earlier

izing effects,

or later cleavage stages, dorsalizing and anterior-

respectively,

embryos cultured

in vitro

are

observed

(3,

9).

In

this

study,

were treated with lithium chloride

to

on development: this is the first step towards

the
molecular
mechanisms of patterning in squid.
understanding
Embryos were fertilized in vitro (1) and cultured at 17 C in

determine

its

effect

may

in

squid

provides insights into the possible conservation of developmental

mechanisms

in cephalopods.
This work was made possible by support from a Faculty Development Grant and the Aldom-Plansoen Distinguished Endowed

were lined with 0.2%

Professorship in Contemporary Studies to K.C. from St. Mary's

College of Maryland. K.C. is most grateful to Bill Eckberg.

agarose (Type II-A. Sigma), filled with Millipore (0.22 ;u.M) filtered seawater (MFSW), and supplemented with bovine serum

Howard University, who graciously provided laboratory space,

collaborative guidance, and digital imagine assistance.

60-mm

plastic petri dishes (Falcon) that

albumin (BSA) (0.5%). Each LiCl treatment dish also contained

60
LiCl (Sigma). Three trials of 15 embryos per

20, 40, or

mM

Literature Cited

treatment were performed. Dishes and solutions were changed

every other day. Embryos were treated with LiCl for the first 6 d

Crawford, K.

of development, by which time epiboly of the outer yolk cell was

Sokol,

complete. Development was observed until the control embryos

began to hatch from their chorions. at 19-20 d after fertilization.

Cell 67: 741-752.

The

classical stages of

J.

M. Arnold

10)

embryonic development. Embryos cultured

were used
in the

to describe

presence of LiCl

exhibited a dosage-dependent inhibition of development that was

evident by 6 d in culture (Fig. la. b. 6 d post-fertilization (dpf),
stage 18). but

it

esis (Fig. Id. e.

many

was more
f,

easily detected later, during organogen-

17 dpf. stage 27).

Development was

and funnel. Moreover, convergence

and inhibition of anterior midline structures was observed in
treated with

40 and 60

E-mail: kcrawford@snicm.edu

mM LiCl. In one

trial,

203: 216-217.

Moon, and D. A. Melton.

1991.

4 Haegel, H., L. Larue, M. Ohsugi, L. Fedorov, K. Herrenknecht,

and R. Kemler. 1995. Development 121: 3529-3537.
Mech. Dev.
5. Kelly, G. M., D. F. Erezylimaz, and R. T. Moon. 1995.
53: 261-273.
6.

Lallier, R. 1975.
istry

7.

Pp.

473-507

in

The Sea Urchin Embryo: Biochemed. Springer. New York.

and Morphogenesis. G. Czihak,

Y.. J. R. Miller, M. J. Ferdowicz, and D. R. McClay.

Development 126: 345-357.
Kitazawa, C., and S. Amemiya. 2001. Dev. Growth Differ. 43:

Logan, C.
1999.

as tentacles, eyes, mantle, fins,

embryos

Biol. Bull.

Schneider, S., H. Steinheisser, R. M. Warga, and P. Hausen. 1996.

Mech. Dev. 57: 191-198.

inhibited in

structures normally associated with ectodermal tissues, such

20(12.

L. Christian, R. T.

S., J.

8.

73-82.

for example.

and R.

9.

Kao, K.

R.,

10.

Arnold,

J.

11.

Rogers, K. T. 1963.

M.

1965.

P. Klinson. 1989.
Biol. Bull.

Dev. Biol. 132: 81-90.

128: 24-32.

Dev. Riol. 8: 129-150.

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

182

Figure 1. Lithium chloride inhibits the uninml vegetal body <an and tjonnal organogenests in si]iiid embryos, fti) Control embryo, 6 davs post-fertilization (dpf} stage
an arrow marks the developing tail bud. Note the well-defined indentation that indicates the boundary betw'een the embryo ami yolk sac (arrowheads), (b) Embryo
treated with 20
Lid, 6 dpf. Tlie arrow indicates the developing tail bud. When compared to the control, this embryo Li rounder and /ess well developed, (c ami d)
18:

mM

Embryos from control

17

(side view).

dpf. stage 27.

UCI-treated embryo, although differentiation

organs within
lightl\

it

and 20

occurred

mM LiCI (anterior view) cultures,

in the

eyes

and

tentacles, (el

respectively.

visible in their centers that

yolk sac.

a,

b and

c,

(/)

Embryo treated with 60 mM

have converged toward the anterior mid-line.

and e and fare

the

same

The mantle and body have failed

Anterior view of an embryo treated with

and tentacle primorida (*)are present but pot >rlv differentiated.

are inhibited: e\es

pigmented eves with lenses

side of the embryo, ys

lias

magnification. Scale bars

= 500

An
jum

* indicates
in

40

to

mM LiCI.

LiCI. 1 7 dpf.

develop properly

17

dpf. Tile

in the

mantle and

Large arrowheads indicate

each of the four tentacle pnmordia on the

left

each view.

Reference: Biol. Bull. 205: 182-184. (October 2003)

2003 Marine Biological Laboratory

HNK-1/N-CAM

Immunoreactivity Correlates with Ciliary Patterns During Development of the

Polychaete Capitella sp. I
2
Susan D. Hill '* and Barbara C. Bayer
1

'

Capitt'l/i:

and

is

amoni:

-.he

is

Michigan State University, East Lansing,

Union College, Schenectady, NY

an opportunistic polychaete that

is

widespread

early colonizers of disturbed areas that are high

in organic content.
settle

within a lew

MI

Larvae are lecithotrophic and are competent to

moments of emerging from the brood tube,

although they can remain viable in the water column without

feeding for two or three days if environmental cues for settling are
'

Corresponding author: hillss@msu.edu

not present.

Under experimental conditions

larvae demonstrate a

DEVELOPMENTAL BIOLOGY
notable ability to select the appropriate environment for settlement

and metamorphosis

(1. 2).

Fertilized eggs develop in a

brood tube, emerging

after eight or

nine days as segmented, motile metatrochophores. The muscular

framework that will be used by the juvenile worm after metamoralready in place (3). but

is

phosis

swimming

cilia,

are not only important in locomotion, but

role in settlement

mosensory

may

also have a che-

and metamorphosis

We

used a monoclonal antibody against HNK-l/Nexpressed on neuroepithelial cells in early embryos

as a tool to follow neural development; we have found that it

locomotion.

(6),
is

which

is

also a marker of ciliary pattern formation in capitellid larvae. In

the present study.

quence of

HNK-1

antibody

used

is

to visualize the se-

development in Capitella sp. 1.

were
cultured in filtered seawater and mud in finger
Capitellids
bowls (J. P. Grassle. pers. comm.). and brood tubes were collected.
Larvae at different developmental stages were removed from the
ciliary

brood tubes and fixed

in

in

4%

formaldehyde

in

PBS. They were then

acetone, treated with blocking serum, and incu-

permeabilized
bated in anti-HNK-1/N-CAM (Molecular Probes). This was fol-

lowed by incubation in a Texas red-labeled secondary antibody.

Whole mount preparations were made and observed with an Olympus

BX60

fluorescence microscope and imaged with an

Olympus

Magnifier digital camera (model S99860).

There is no evidence of HNK-1 reactivity during cleavage or
gastrulation. If larvae are removed from the brood tube immedi-

no locomotion or

to

labeled prototroch. there

the

is

not yet a distinct band of

telotrochal label (Fig. Ib). At this stage the larvae are able to glide
on the substrate.

After reaching

its

maximum

width, the prototrochal fluores-

cence begins to recede towards the

initial

band, which

is

now

prominently labeled. Concurrently the neurotroch becomes evident

as a midventral mass of punctate fluorescence, with the densest
reactivity just posterior to the

stomodeum. Label

becoming dense and organized except in

there is no fluorescence. This reflects the
is

(5).

This study is part of an ongoing investigation of the interaction

between nerves and muscles during development and its role in

CAM.

continues to increase. Antibody labeling reveals a distinct perianal

ring and beginning organization of the pygidial region. In contrast

powered by two

is

an anterior prototroch. and a posterior telotroch. A

cluster
of
cilia, the neurotroch. is apparent on the ventral
large
surface in scanning electron micrographs (4). These ciliary bands

bands of

183

in the

pygidium
where

the dorsal region

ciliary patterns seen in

scanning electron micrographs in which the dorsal area of the

pygidium in the hatched larva is devoid of cilia (4). The telotroch
is

now

prominently labeled, forming a band of fluorescence several

Ic). Although they have not yet emerged from the

dots wide (Fig.

brood tube, the larvae are now capable of swimming.

Next, the anterior boundary of the prototroch remains
labeled,

and some scattered fluorescence

The neurotrochal

labeling

is

lightly

persists in the episphere.

greatly diminished, while the pygidial

region and telotroch are heavily labeled (Fig. Id). By this stage, the
ciliary patterning appears to be complete.
The patterns of cilia in newly emerged metatrochophores of
Capitella sp. I have been described by Eckelbarger and Grassle
(4). These include well-developed prototrochal and telotrochal
a midventral cluster of cilia
bands, and a distinct neurotroch

beginning posterior to the prototroch and terminating a short

distance anterior to the telotroch. There is no apical tuft, although
there are scattered cilia and mucous glands in the episphere.
Discrete rays of cilia radiate from the perianal ring toward the
telotroch except in the dorsal area, which is free of cilia. Our

characteristics are visible. For a brief period before they begin to

labeling, which proceeds from anterior to posterior and corresponds to these patterns, indicates that the HNK-1 /N-CAM protein

swim, the larvae are able to glide slowly over the substrate. Most
of the changes in patterns of fluorescence occur as the larvae

down.

ately after gastrulation,

acquire

swimming

morphological

ability.

positive reaction

is first

larvae several days prior to

seen

distinct

observed

in

nonmotile, postgastrula

emergence from the brood

tube.

The

developing prototrochal region in the form of

a row of sparse fluorescent dots. This is followed by a small
amount of punctate fluorescence in the perianal region and in the
label

is

in the

episphere, the region anterior to the prototroch. which will

the prostomium (Fig. la

is

being expressed sequentially as the ciliary organization

We suggest that anti-HNK-1/N-CAM

fluorescent dots then

the prototrochal region

around the

become more numerous,

and forming a

slightly

become
Literature Cited

delineating

thickened band

4.

to be

The width of

the prototrochal

band of fluorescence next

in-

creases significantly, although the posterior edge remains less

defined than the anterior. The amount of label in the episphere

Butman, C.

A., J. P. Grassle.

and C. M. Webb. 1988.

Nature 333:

771-773.

episphere. In the pygidial region, posterior to the telotroch. scattered fluorescence is seen.

larva.

to

in creating the figure.

2.

seems

laid

and the Union College Faculty Research Fund. The authors gratefully acknowledge the generous assistance of Dr. William Eckberg

Concurrently the punctate fluorescence, which at

randomly arranged, continues to appear in the

first

is

may prove

be an important tool for studying the development of cilia.

This work was supported in part by Michigan State LIniversity

I.

The

labeling

Butman. C. A., and J. P. Grassle. 1992. J. Mar. Res. 50: 669-675

Biol. Bull. 201: 257-25S.
Hill. S. D., and B. C. Boxer. 2001.
Eckelbarger. K. J.. and J. P. Grassle. 1987. Biol. Soc. Wash. Bull.
1:

62-67.

5.

Biggers,

6.

Kruse,

W.

J.,

J.,

and H. Laufer. 1999.

Biol. Bull. 196:

R. Mailhamnier. H. \\ernecke, A. Faissner,

C. Goridis, and

M. Schathner.

1984.

187-198.
I.

Sommer,

Nature 311: 153-155.

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

184

Figure 1. Whole mounts of developing Capitella ,s/>. / larvae showing labeling of developing ciliary hands. Anterior is to the left. Early embryos
measure approximately 240 X 175 JLUH. As development proceeds they become more elongated without increasing in size, (a) The first HNK-1 expression
developing prototroch (arrow), (b) The wide prototrochal band is apparent. Note scattered fluorescence in the epispliere. In the p\gidial
is labeled (arrow), (c) Labeling of the prototroch has narrowed and the ventral neurotroch and the telotroch are visible.
Kays of fluorescence (arrow) extend from the perianal ring toward the telotroch except on the dorsal surface of the pygidinm. which remains cilia-free,
(d) Labeling is most prominent in the telotroch and pygidiufn. The prototroch continues t<> be lightlv labeled. Ep. episphere: N. neurotroch: P. prototroch:
is

seen

in the

region a distinct perianal ring

Py, pygidial region: T. telotroch.

CELL BIOLOGY

185

Reference: Biol. Hull. 205: 185-186. (October 2003)

2003 Marino Biological Laboratory
1

>

Ryanodine-Sensitive Calcium Flux Regulates Motility of Arbacia punctulata

D. E. Heck

anil

J.

Rutgers University, Piscataway,

2

The
their

UMDNJ -Robert Wood Johnson

motility of sea urchin spermatozoa and the direction of

are due to the beat of the flagellum, which is

movement

controlled by an ion flux across the plasma membrane. When

sea urchin sperm contact egg jelly, channels are activated that
rapidly

and transiently elevate intracellular calcium,

a cascade of events that lead to the sperm acrosome

(<5

initiating

s)

reaction (1-3). This process, in turn, induces a complex sequence of events that include protein phosphorylation and elevation of calcium levels thought to be dependent on intracellular

Recent reports have identified

intracellular calcium stores as critical for

calcium mobilization

ryanodine-gated
controlling

the

present studies,
lar

motility of mammalian sperm

we determined whether increases

calcium and motility

cia punctulata
are

also

(4).

that

(5).

In

the

in intracellu-

sperm of the sea urchin Arbaare mediated by egg-derived products

in the

mediated by ryanodine-gated intracellular calcium

stores.
In

initial

NJ

Medical School, Piscutawav, NJ

the addition of the nitric oxide/peroxynitrite-releasing drug 3-mor-

pholinosydnonimine (SIN-1). We found that SIN-1 (1 /u,Af) also

stimulated sperm calcium mobilization (Fig. 1, panel B). Increases
calcium were approximately 3- to 4-fold, persisted
10 min. and were also independent of extracellular

in intracellular

for at least

calcium. Motility and calcium mobilization induced by SIN-1 in

Arbacia punctulata sperm were also found to be inhibited by
ruthenium red (Fig. 1, panel D. and not shown). These data
indicate that nitric oxide, like egg water,

is effective at mobilizing
oxide appears to function in
sea urchin sperm upstream of ryanodine-sensitive calcium chan-

intracellular calcium.

we found

that

Arbacia punctulata egg-

derived products (egg water) that stimulate sperm motility also

increase sperm intracellular free calcium (Fig. 1, panel A). The
increase was about 10-fold, occurred within 2 min, and persisted for at least 10 min. These experiments and all further

experiments were repeated three times with similar results. The

increase in intracellular calcium did not require calcium in the
seawater. indicating that the ion was likely released from internal stores (data not shown). In further experiments, therefore,
we used calcium-free seawater. Sperm motility and increases in

Moreover,

nitric

nels.

The lower panel in Figure 1 shows a schematic representation

of events leading to the activation of Arbacia punctulata sperm.
In this model, the interaction of the egg-derived mediator with
activity of the

sperm receptors activates the guanylate cyclase

cGMP. The

the formation of

in

receptor, resulting
studies,

Sperm

D. Laskiir

resulting

cascade of events, including an intermediate calcium-induced

release of sequestered intracellular calcium ("gray box") that

provokes the activity of high capacity sodium channels

plasmalemma. ultimately culminates

in motility.

We

in the

hypothe-

produced in response to enzymatic binding of calcium/calmodulin made available by the initial brief
size that nitric oxide,

calcium, interacts with ryanodine-gated ion

channels to mediate the release of calcium from internal stores.
rise in intracellular

We

be stimulated 2- to

speculate that, in a manner similar to that of ryanodinegated release of calcium from stores in mammalian skeletal
muscles (7, 14), nitric oxide-mediated alterations to channel

3-fold by ryanodine (0.3-1 ^M) or caffeine (1-3 p,M). both

agonists of ryanodine-gated ion channels (for reviews see refs.

proteins are central to the prolonged and hyperactive motility of

egg mediator-activated sperm.

intracellular free calcium

were also found

and 6) (data not shown).

We

to

next examined the effects on sea

urchin sperm of ruthenium red. a potent inhibitor of ryanodine-

We found that H.M ruthecalcium mobilization and sperm

motility initiated by egg water, or by ryanodine and caffeine
(Fig. 1, panel C and not shown). Taken together, these data
sensitive channel activity (6-8).

nium red would

Literature Cited

inhibit the

1.

2.

residues (9, 10). In previous studies,

strated that nitric oxide

sea urchin sperm, and

is

we and

others have

demon-

further that this process

is

be dependent on the activity of GTP-binding proteins

(11-13). In these reports, sperm motility and nitric oxide production were inhibited by cholera toxin, and motility was recovered by

4.
5.

Corresponding author: heck@eohsi.rutgers.edu

Schackmann, R.

\V.,

and

P. B.

Chock. 1986.

Biol. Client.

261:

Proc. Natl. Acad. Sci.

USA

J.

Su, Y. H., and V. D. Vacquier. 2002.

Schackmann, R.

1986.

Methods Cell

Bio/. 27:

57-71.

VValensky, L. D., T. M. Daw son, J. P. Steiner, D. M. Sabatini, J. D.

Suarez, G. R. Klinefelter. and S. H. Snyder. 1998. Mol. Med. 4:

502-514.
6.

Ehrlich, B. E., E. Kaftan, S. Bezprozvannaya, and

Trends Phunmicol. Set. 15: 145-149.
vanny. 1994.

7.

8.

Salama, G., E. V. Menshikova, and J. J. Abramson. 2000.

oxid. Redox Signal. 2: 5-16.
Ozawa, T. 2001. Int. J. Mol. Med. 7: 21-25.

9.

Galione, A., and G. C. Churchill. 2002.

likely to

Scie?ice

99: 6743-6748.

also important in regulating motility in

we have found

D. L. Garbers, and V. D. Vacquier. 1985.

8719-8728.
3.

Recent studies indicate that ryanodine receptors are regulated by

nitric oxide-induced oxidation and reduction of critical sulfhydryl

E.,

227: 768-770.

indicate that a ryanodine-sensitive calcium flux regulates the

motility of Arbacia piinctiilata sperm.

Ward, G.

10.

Heck, D. E. 2001.

Antioxid.

Redox

I.

BezprozAnti-

Cell Calcium 32: 343-354.

Signal. 3:

249-260.

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

186
I

1.

Heck, D.

W.

E.,

Troll, anri J. D. La.skin. 1996.

Biol.

191:

Bull.

275-276.
12.

Heck, D.

E.,

i
.

Herrem,
Plwiii,

14.

Sun.
./.

J..

B.,

L.

D. Laskin. 1996.

Moncada,

Xu,

Biol. Cliem.

S.

Gross,

Pp.
J.

16-17

in

Tin-

Stamler, and A.

ME.

E. de Lamirande, and C.

Gannon.

20(13.

Ciin:

419-425.

>:

J.

S.

u Press. Portland.

Higgs. ed13.

i-i

Biology of Ni

J. P.

Eu,

J. S.

Stamler, and G. Meissner. 2(103.

278: 8184-8189.

01

1000

100

Log Fluorescence

MOTILITY

"^

1000

100

10

1.0

(free calcium)

Egg-derived Mediators

Cytosol

Ca

Figure

from

1.

Effects

of egg water and SIN-1 on calcium mobilization

the sea urchin Arhacia punctulata. Freshly isolated sea urchin

in sperm
spenn were

incubated with ihe cell permeant calcium-sensitive fluorescent indicatorfluo-4

(Molecular Prohes. Eugene. OR. 5

jLtM.

10

mill}

and

water (panels A and C) or SIN- 1 (Molecular Probes,

mill,

[hen stimulated with

/xM, panels

B and D}.

mlracellular levels of free calcium lure analysed using a Coulter

AM
(%'t;

After

EPICS

flaw cytitmeter (excitation wavelength 488 nm. emission wavelength 525 mn).

Data are presented on a 4-deende log

scale. US, iinstimulated spenn. In experi-

ments where cells were treated with ruthenium

red, the

spenn were first transiently

penneahili:ed using a Live Cell Penneahili:ation Kit (Gihco. Grand Island, NY)
to

allow uptake of the compound (panels C and D). Note that spenn treated with
to mobilise calcium in response to egg water or SIN-1 (panels

ruthenium red tailed

C and D).

(Lower panel): Schematic representation of events leading

to activation

o/"Arbacia punctulata spenn. Events originate with the binding of egg-dcriml

mediators to their receptors on the surface of the spenn and progress through
signaling to stimulate the release of calcium from internal stores. Tliis release
potentially involves protein interaction with intracelhilarly

and presumably occurs

produced

opening of high-capacity sodium channels

in the phisnialetnina

PM, plasma membrane: RYR. minodinc

plasmic reticulum: NO. nitiic oxide.
leads to motility.

nitric

oxide

within the endoplasmic reliculuin. which mediates the

and

ultimately

receptor: ER. endo-

CELL BIOLOGY

187

/-/. Hull. 2(15: IS7-IS8. (October 2003)

Reference:
2003 Marine Biological l.ahoraton,

<D

Axotomv

Axonal Transport of Tubulin

Inhibits the Slow

P. E.

National Institutes of Health, Bethesda,

MD
MA

Marine Biological Laboratoiy, Woods Hole,

Slow uxonal
In

transport

our previous work

is

essential to axonal

we demonstrated

many macromolecules,
move slowly down the

including fluoreseently labeled tuhulin.

squid giant axon (1,2).

We

growth and survival.

that

(1,2) and others (3) have proposed that

slow transport of tuhulin might be due to the intermittent

associations of axonal tubulin with a fast motor or motor-earner
the

cytoplasm becomes disorganized (5). Though most of these signs

of damage could be seen at distances closer than 1 mm from the
transection site (not shown), the rest of the

these or other transected axons (n

(compare E with

neuronal cell bodies and transported down the

ously synthesized
axons (4), separating the axon from its cell bodies by axotomy
should eventually reduce the amount of this hypothetical tubulin

swollen (n

To

and thus

inhibit tubulin transport.

test this possibility, the lateral giant

axon was transected, and

was then assayed. For each experiment a squid

/'irr/.v. obtained most months of the year from the

normal

Since protein loss

MA)

was

5 min.

anesthetized with

first

The
flat

Hole,

was

was measured

excised

in

pH

6.8),

verify that optical density

axons was transected (axotomized). The squid

to a fresh seawater tank for 2 days. Squid were

usually fed once a day for 2 days after return to the seawater tanks.

in

then

M urea,

in total

was

axonal proteins, the separated

protein stain (Molecular Probes).

Ruby

dilution series of control axons

tration

The isolated axon was

(2% SDS, 2% BME, 8

and the solubilized proteins were separated by 7.5%

PAGE. To measure changes

inserted through the mantle opening, and one of the

was then returned

in

in

calcium-free seawater.

dissolved in solubilization buffer

Tris

damage

as follows: Equal lengths (from 1-4 cm in

axotomized axon and its paired control were

proteins were stained with

were not

squid as
well as in other axons, we compared the levels of neurofilament
and other axonal proteins in transected and control axons. Total

ganglia were then located by eye through the

pair of Vannas micro-dissection scissors angled

lateral giant

first

Woods

ethanol in seawater for about

stellate

mantle opening.

on the

1%

illustrated in

and neurofilament proteolysis

in general,

protein

Resource Center, Marine Biological Laboratory.

and as

particular (6). are sensitive indicators of axonal

different squids) of an

Resource Center for Cephalopods. Galveston TX, or

May or October from the Marine

12);

10).

(Lulifitiiicnlii

National

in

F), the cytoskeletal elements appeared

the transected axon, and the mitochondria

in

tubulin transport

Loligo pealeii, obtained during

Figure

carrier,

1-4 cm axons showed

no signs of axonal damage. As illustrated in Figure

(compare
with D), no change in darkfield light scattering was detected

complex. Since most rapidly moving axonal proteins, including

this presumed rapidly moving tubulin carrier, should be continuin

Squid Giant Axon

in the

Gallant

was run with each experiment

linearly related to protein

each run (not shown). As illustrated

in

to

concen-

Figure

1G.

axotomy produced no changes in neurofilament (NF200 and

NF60), tubulin, or any other major axonal protein. Equal lengths of

same squid (squid

After 2 days the squid was sacrificed, and the distal end of the
transected lateral axon as well as the uncut control axon from the

control (Ic) and transected (It) axons from the

other side were removed, cleaned, and prepared for the slow

axons from a smaller squid (squid 2) had proportionately less

protein (2c and 2t), but there was no visible difference in protein

The

transport assays.
pressure system described previously (2)
was used to inject both fluorescently labeled tubulin (Cytoskeleton,

and a marker

air

drop into the control and transected axons.

Axons were injected in the middle of the 2-4 cm isolated axons.
Transport was visualized by recording the fluorescent image over
Inc.)

oil

time with a Zeiss microscope equipped with a Photometries Cool

Snap HQ camera driven by Openlab software (Improvision).
In the control

axons (n

6). tubulin diffused while simulta-

neously being transported anterogradely (to the right in Fig. 1A).

injection site is marked by an arrowhead. As illustrated in

The

Figure IB. this anterograde tubulin transport

after

the

was

arrested 2 days

axotomy. The tubulin diffused in both directions away from

site (arrowhead) but was not transported antero-

injection

gradely (n

determine whether the inhibition of tubulin transport after

axotomy might be due to some nonspecific damage or degeneration of the isolated axon. a

number of

of axonal health were performed.

onstrated that

when

virtually identical protein-staining profiles. Smaller paired

had

levels

axon

between the control axon (2c) and the previously transected

= 6).
squids tested (n
the loss of conventional kinesin might be

(2t) in this or other

To determine

if

responsible for the loss of slow tubulin transport (3), we measured

kinesin levels in the control and transected axons. Since conventional kinesin

and other potential motors are minor constituents of

axoplasm. Ruby protein stain would not detect these protein. To

measure the kinesin levels, axons were cut, cleaned, dissolved, and
run on

PAGE

as above.

The PAGE-separated

proteins were then

PVDF

paper by western blotting in Tris-glycine

buffer, and exposed to an anti-kinesin antibody (3) (Chemicon
1614). The amount of antibody was detected with chemilutransferred onto

MAB

minescence. As with the protein measurements, axoplasm stan-

6).

To

tests

1 )

We

and biochemical

have previously dem-

damaged or becomes leaky,

mitochondria become swollen, and

a squid

light scattering increases,

structural

axon

is

dards were run to insure that the recorded optical densities varied
linearly with kinesin concentration.

(Kinesin), there

was no

As

illustrated in Figure

between the control and the transected axon

Ic with It) or squid 2

its

(compare

its

other animals tested (n

1H

significant difference in kinesin levels

9).

in either squid

(compare 2c with

2t) or

with any

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

188

Transected

Control

Kinesin

Proteins

NF200

Kinesm

<- NF60

Ic

Figure

I.

away from

Actin

2t

Ic

It

2c

2t

ofaxotomy on axonal transport, structure and proteins. Fluorescent tuhulin injected into an axon moved ante rogradely (to the right)
(arrowhead in A Tubuiin injected into tin axon that had been separated from its cell bodies 2 d prior showed no anterograde
This tubulin just diffused in both directions around tile injection site (arrowhead in B). Darkfield light scattering was unaffected by axotomy.

Light scattering was the same

showed no

Tubulin

Effects

the injection site

transport (B).

2c

It

).

in

control (C) as

difference between control (El

was

it

in

transected axons (D). Higher resolution video enhanced differential interference microscopy also
(F) axons. Mitochondria (arrows in E and F) and smaller organelles (arrowheads) were

and paired transected

in control and transected axons. Scale bars: 100 /jjnforA and B. 50 fjjnfor C and D. and I fjjn for E and F. Solubilized axonal proteins (G)from
equal lengths of axon from the control (Ic) and the transected side (It) of a squid that had been axotomized 2 d earlier showed no change in protein-staining
intensity. Paired axons from a smaller squid (2) with shorter axons had proportionately less protein than the larger squid (1 ). but there was no significant

present

and
same nvo

difference in protein levels between the control side (2c)

the transected side (2t). Similarly,

kinesin levels (H) in the paired axons from these

squids. (Ic vs. It

The present experiment suggests

essential to axonal tubulin transport

that

some

is lost

and 2c

1.

axotomy. This inhibition is not due to the depletion of conventional kinesin. It could be due to the inactivation of conventional
kinesin or to the inactivation or depletion of
transport in the squid giant axon

some of

may

facilitate the identification

the factors essential to the control

of

and maintenance of

on

total

Galbraith,

J. A.,

T. S. Reese,

M.

immunochemically measured

A. Galbraith, P. E. Gallant, and T. S.

USA

M.

USA

92:

L. Schlief,

1,500-1 1.503.

and

P. E. Gallant. 1999.

96: 11.589-11.594.

Kinjo, and N. Hirokawa. 2000.

Terada,

4.

Gallant, P. E. 2000.

5.

Gallant, P. E.,

S.,

J.

Proc. Nail. Acad. Sci.

Proc. Null. Acad. Sci.

slow axonal transport.

J.

and

J.

Cell 103: 141-155.

Neurocytol. 29: 779-782.

A. Galbraith. 1997.

J.

Neurotrawmi

14:

XI 1-822.

The author thanks James Galbraith and Thomas Reese

helpful discussions and

Terasaki. M., A. Schmidek,

Reese. 1995.

2.

some other motor or

This model of protein

effect

Literature Cited

unidentified factor

or inhibited 2 days after

factor essential to axonal tubulin transport.

axotomy had no

vs. 2ti.

for

comments.

b.

Gallant, P. E., H. C. Pant, R.

J.

M.

Pruss, and H. Gainer. 1986.

Neurochem. 46: 1573-1581.

Reference: Biol. Bull. 205: 188-190. (October 2003)

2003 Marine Biological Laboratory

Recombinant Conventional Squid Kinesin and Native Myosin-V

John Delacruz, Jeremiah R. Brown, and George M. Langford
Dartmouth College, Hanover, NH

Interactions Between

Axoplasm from

the squid giant

axon

is

one of a small number of

which axonal transport can be studied in

vitro (1). In squid <\oplasm, one can observe both microtubulebased and actin-based vesicle transport and the seamless transition
of vesicles from microtubules to actin filaments (2). Based on
cell-free extra.t^ within

studies of vesicle transport in this cell-free preparation, a

new

model of axonal transport has emerged called "dual transport" in

which long-range vesicle transport is microtubule-based while
short-range transport

An

is

actin-based

exciting recent discovery

is

(3).

the

finding that the cargo-

binding domains of myosin-V, an actin-based motor, and kinesin.

a microtubule-based motor, interact to form a hetero-motor com-

CELL BIOLOGY
The

plex (4. 5, 6. 7).

interaction of

myosin-V with kinesin has been

established through yeast 2-hybrid assay, co-immunoprecipitation.

co-affinity isolation, and co-purification with myosin-V (4, 5, 6. 7).
The distal/globular tail domain of myosin-V binds to the rod-tail
domain of kinesin in the "hetero-motor" complex. The members of

been shown

the kinesin super family that have

to bind to

include conventional or ubiquitous kinesin (kinl) and

Evidence

6. 7).

that

myosin-V and kinesin

interact

In this report,

we performed experiments

sin-V.

Our working hypothesis

is

that

to

on the

(4. 5.

determine the func-

between kinesin and myotail-tail interactions between

For example, one motor may become inactive

actively transporting a vesicle along a filament

is

partner

shown

Several studies have

when

inhibited

the

ATPase

that the

head and

may be
when its

interact (8, 9). Auto-inhibition

one motor becomes inactive

filament. Therefore, only

ment and

a tug-of-war

one motor

is

the

116

200
116

207
129
85

45

40
32

66
40

Assays

Motility

SB: 22: 43 8"

<

S"

activity of kinesin

domains of

tail

200

Blot

Gel

from microtubules

when
(3).

Blot
207
129
85

to actin filaments.
is

Affinity Isolation

45

activity during the transition of vesicles

its

Gel

mem-

these motors provide feedback and thereby allow coordination of

motor

His-SK Purification

myosin-V

Smylp

brane surface has not yet been demonstrated.

tional significance of the interactions

189

the

molecule

mechanism by which

partner motor binds to a

actively

between motors

is

engaged

in

move-

prevented. Such feed-

back between motors could explain the seamless transition of

vesicles from microtubules to actin filaments observed in the squid
giant axon.
In this study we used a histidine-tag bound to the tail fragment
of squid conventional kinesin (His-tagged) to study the interac-

between kinesin and myosin-V.

tions

A cDNA

construct coding

domain of conventional squid kinesin (SK KhcU;

of K. Kosik) was engineered into a vector containing a His-tag.

for the rod-tail

gift

The

1.5

entire
insert

kb

SK KhcU

contained most of the rod

II

domain and

the

domain including the stop codon. The sequence of the

was confirmed by PCR. The His-kinesin vector was ex-

tail

pressed in E.

coli.

and the recombinant protein was then purified

A gel of the fraction eluted from the column

on a nickel-column.

mM

imidazole showed a prominent band at 45 kDa, the

expected molecular weight of the fragment (Fig. A, lane 1 ). This
with 40

band was
cellulose

1A. lane

Next,

identified as the His-labeled kinesin fragment

membranes

that

on

nitro-

were probed with the His-antibody

(Fig.

2).

we

applied a clarified extract of His-kinesin

tail fragment
column. Nonspecific
proteins were removed by several buffer washes; and a clarified
squid optic lobe extract was then passed over the column, followed

to a

Ni-column

to

make

a kinesin

tail

affinity

by buffer washes. The proteins that bound specifically to the tail

domain of kinesin were eluted with imidazole and analyzed by

SDS-PAGE.

1.

Figure

(At The presence of the His-tagged recombinant squid

kinesin tail construct

was

verified using

SDS-PAGE and western

blots,

and

sequencing data. Lane 1 shows an SDS-PAGE gel confirming the presence

of a protein with a molecular weight of 45 kDa, which matches the
expected weight of the kinesin construct. Lane 2 shows a western blot

probed with anti-His antibody:

the blot confirms that the

45 kDa protein

contains a His-tag. (B) The squid kinesin tail fragment is shown to interact
with native squid myosin-V obtained from homogenized optic lobes. The

homogenized extract was run through a His-kinesin affinity column. Lane

I shows an SDS-PAGE gel of the diction fraction and shows that the 45
kDa kinesin tail and the 196 kDa native myosin-V are present. The elution

was blotted and probed with myosin-V antibody ct-QLLQ (lane 2)

and confirms myosin-V. (Ct Allen Video Enhanced Contrast-Digital Interference Contrast (VEC-DIC) microscopy is used to image microtubules
and vesicle moti/itv in extruded squid axoplasm. Experiments are being
fraction

performed

to

determine whether the recombinant kinesin

tail

fragment

conclusively inhibits vesicle motility along microtubules.

Multiple protein bands were visible on the gel of the

1). The proteins were transferred to

membrane and probed with an antibody to myosin-V, a known
binding partner of kinesin. A prominent band was observed on the
blot (Fig. IB. lane 2). These data showed that the recombinant

eluted fraction (Fig. IB, lane

His-kinesin fragment interacted with myosin-V and several other

proteins in the squid brain extract. Finally,

we asked whether

His-kinesin fragment blocked microtubule-based vesicle

ment
in

in motility

the

move-

assays. Preliminary experiments were performed

which the His-kinesin

tail

fragment was added to extruded

axoplasm from the squid giant axon (Fig. 1C). Vesicle transport
was measured by counting the number of vesicles moving/micro-

tubule/min (v/mt/m: motile activity)

at

tion of motile activity occurred but the

15-niin intervals.

number of

reduc-

replicates

was

not sufficient to provide conclusive evidence.

In summary, recombinant. His-labeled, squid kinesin tail fragment binds to squid brain myosin-V. as demonstrated by affinity

The recombinant

tail fragment is thus an excelbinding partners of kinesin and

potentially for studies of kinesin-mediated vesicle transport.
This work was supported by NSF Grant IBN-0131470, and the

column

isolation.

lent tool for identifying specific

Leadership Alliance Grant and MBL-Shifman Award

to

JMD.

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

190

Literature Cited

Brown,
Bid.

Allen, R. D., D.

G
.

M. Langford, and D. G. Weiss.

1992.

LangtW

4.

Brn-.-ii.

G. M.

M. 2002.

Traffic 3:

859-865.

K. R. Simonetta, L. A. Sandberg, P. Stafford, and

Ki,,l. Bull. 201: 240-241.
angford. 2001.
R.,

Huang,

M. Peacock-Villada, and G. M. Langford.

2002.

203: 210-211

J. D., S.

T. Brady, B. VV. Richards, D. Stenolen, J. H. Resau,

N. G. Copeland, and N. A. Jenkins. 1999.

Nutiir?

356: 72
3.

tV/Y Mutil. CytoskcU-t. I: 291-302.

'

K. Ha.vden, D. T. Brown, H. Fujiwake.

and M. Simpsi.
Ku/netsm,

J. R., E.

Bull.

and

7.

Lillie, S. H.,

Freidman, D.

9.

Coy, D.

L.,

C.,

S. S.

Brown. 1998.

Nature 397: 267-270.

Cell Biol. 140: 873-883.

J.

and R. D. Vale. 1999.

Mir. Cell Biol. 1:

W. O. Hancock, M. Wagenbach, and

Mir. Cell Biol. 1:

J.

288-292.

Howard.

1999.

288-297.

Reference: Biol. Bull. 205: 190-191. (October 2003)

2003 Marine Biological Laboratory

Rab-GDI
Carl

Myosin V-Dependent Vesicle Transport in Squid Giant Axon

DeSelm, Jeremiah R. Brown, Rcnne Lu and George M. Langford
Dartmouth College, Hanover, NH

Inhibits

J.

'Boston Biomedical Research

Myosin-V. an uetin-based motor, and kinesin, a microtubuleto form a "hetero-motor" complex

Axo-

based motor, interact

1 ).

plasmic vesicles containing these hetero-motor complexes move

on microtubules in the axon and on actin filaments at the cell
cortex (2). Negative feedback between these two motors

is

thought

of vesicles from microtubules to actin

to facilitate the transition

filaments (3). Proteins that couple the hetero-motor

complex

vesicles have not been identified. Recent studies have

shown

the

Rab family of GTPases

involved

is

the

in

that

recruitment of

myosin-V to vesicles. In melanocytes, myosin Va is

melanosomes by Rab 27a (4, 5. 6). Melanophilin, an

Rab

to

to

axoplasmic vesicles

in the squid giant

is

known

axon has not been

synaptic vesicles in squid brain (8). In this study

Rab-GDI

down myosin-V by

pulled

domain of myosin-V binds

we show

affinity isolation

vesicle transport in motility assays.

We

also

to tubulin dimers,

show

that

Rab GTPase involved

S-transferase (GST)-labeled globular

(GST-GTD) and
(GST-GDI)

in

GST-labeled Rab

motility

and

we showed

affinity

presumably through

dissociation inhibitor

isolation experiments.

In

GST-GTD

binds to squid brain

vesicles and displaces the native myosin-V. The displacement of
native myosin-V blocked transport of axoplasmic vesicles on actin

previous study,

filaments
the

in

assays using the squid giant axon (9, 10). In this study,

GST-GTD

partners ot

that

GST-GDI were

and

for analysis by

myisin-V

used to pull

2-D

down

binding

gel electrophoresis

and

protein sequel"

A plasmid corrtai
AF6/Cno
pressed

ii

tail-glubu.

in E.

coli (9).

myosin-V globular

tail

.1

cDNA

insert for

GST-mouse myosin-V

from Dr. Huang) was exThe hacterially expressed 84 kDa GSTK'liiain

present in the eluted fraction from the

revealed by

SDS-PAGE

in the fraction

(Fig. 1A. lane

was

GST-GTD

column, as

There were no proteins

the absence of GST-GTD
in the

blots of this fraction with

known binding

identified in the fraction eluted

also applied to

).

column in
The presence of GST-GTD

eluted from the

(Fig. 1A, lane 2).

was

GST-GTD. GST-GTD was

eluted fraction

GST antibody

partner of myosin-V,

from the

GST-GTD column

was analyzed further by 2-D gel

A
of
3-10
was used in the first dimenelectrophoresis.
pH range
sion and an 8.5% SDS-PAGE gel for the second dimension. The
2-D gel was electroblotted to PDVF membrane (ProBlott, Applied
(Fig. 1A, lane 4). This fraction

Biosystems), and the protein spots were stained with Coomassie

Blue R-250. Approximately 35 spots were clearly visible on the

membranes

domain of myosin-V

GDP

a control, clarified brain extract

N-terminal sequences were determined using an Applied Biosystern Precise sequenator. The identification of the proteins was

in

tail

As

and eluted from a column without

that the tail

myosin-V/kinesin binding to axoplasmic vesicles, and to identify all binding partners of

the hetero-motor complex, we used a recombinant glutathione
identify the

glutathione.

was applied to the

was eluted with

extract

GST-GTD

and blocked

kinesin or another linker protein.

To

myosin-V tail. Clarified squid brain

column and, after extensive washes,

(Fig. 1A, lane 3). Kinesin. a

be associated with

to

generate an affinity column for isolation of binding partners of the

activator of

shown to be required for the binding of

Rab 27a (7). Therefore. Rab 27a and melanophilin
have been identified as the receptor complex for myosin Va on
melanosomes. The Rab GTPase responsible for myosin-V recruitment

MA

was confirmed by probing

to

determined, although Rab 3a

Watertown,

recruited to

27a, has been

myosin-V

Institute,

(gift

fragment was bound to a GST-column to

(Fig.

IB).

The major

spots were excised and the

based on sequence homology using BLAST.

Sequence information has been obtained for eight proteins excised from the
identified

are

2-D

gel.

The two most

interesting proteins thus far

a- and /3-tubulin. These data

show

tubulin

that

dimers are retained on the column, suggesting that the tail domain
of myosin-V binds directly or indirectly to microtubules. An
indirect link
sin,

between myosin-V and tubulin may be through kine-

which binds

directly to the

Several studies have

and other proteins

shown

tail

of myosin-V (Fig. 1A, lane

interactions

between myosin-V

that bind to microtubules. Therefore,

4).
tail

we con-

clude that tubulin binds to myosin-V indirectly, either through

kinesin or another microtubule-associated protein.

plasmid containing the full-length

cDNA

for

Drosophila

GST-Rab-GDI (gift from Dr. C. Cheney) was expressed in

The 75 kDa GST-tagged protein was purified on a GST
column

and used

E. coli.
affinity

pull-down experiments with

was
identified
as one of the probrain
extracts.
Myosin-V
squid
teins in the fraction eluted from the GST affinity column (Fig. 1C,
(Fig. 1C, lane 1)

in

CELL BIOLOGY

GST-GTD

Control

191

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

192

Reference: Biol. Bull 2(15: 19 -193. (October 2003)

2003 Marine Bioloci
atorv
;

skeletal

Events Preceding Polar Body Formation in Activated Spisula Eggs

R. M. Pielak, V. A. Gaysinskaya, and W. D. Cohen*
Hunter College, New York. NY

Marine Biological Laboratory, Woods Hole,

Polar body formation

is

of interest both as a fundamental pro-

MA

Zeiss Laser Scanning System

LSM

PASCAL, and

images were

cytokinesis in cell biology. Eggs of the surf clam Spisula solidis-

processed with Zeiss LSM Image Examiner software.

occurred in
Using the KCl-activation method at 23 C,

sima, released in the germinal vesicle stage, are readily induced to

about 7 min. and was followed

form polar bodies by activation with KC1 or by fertilization.

However, although Spisula eggs are utilized in current studies of

At

cess in sexual reproduction and as an extreme example of unequal

centrosomes and the

cell

cycle

(e.g.,

1),

polar

body formation

in

GVBD

approximately 13 min postactivation by the appearance of the first metaphase meiotic spindle.
first,

the

at

metaphase spindle was slightly eccentric (Fig. la).

it moved toward the cell surface and
approached the

Subsequently,

model system is described only in older literature (2, 3). We

have examined changes occurring in major cytoskeletal elements microtubules and F-actin in the stages immediately pre-

cortex while remaining in metaphase (Fig. Ib;

ceding formation of the first polar body. These stages are thought
to be critical for docking of the meiotic spindle with the cell cortex,

from

this

and for meiotic apparatus-cortex signaling

tioning and generation of the contractile

that

mediates the posi-

ring.

These processes

occur by as yet unknown mechanisms.

localize actin

and tubulin

relative to

Ripe Spisula were obtained from the Aquatic Resources Division

of the Marine Biological Laboratory (MBL) and maintained at
1

1-13 "C

until used.

was removed, and

Clams were opened by

the eggs

were

dissection, the

gonad

through cheesecloth.
Before use, the eggs were washed 3 times in 0.2-^m filtered
seawater (FSW), and resuspended to a concentration of 1:10
filtered

(^eggs/^Hsw)- The eggs were activated, either by adding excess

KC1 to the seawater or by fertilization with freshly obtained sperm,
according to standard methods for this species (4. 5). Time of KC1
or sperm addition

was recorded

as

0. After

germinal vesicle

breakdown (GVBD). the eggs were resuspended to a concentration

Veags/V FSW ). Time-course samples, taken approximately

of 1:100

every 2 min after

fixation of eggs

formaldehyde

in

GVBD.
in

PEM

were prepared by simultaneous

medium
(100

lysis

and

consisting of 0.6% Brij-58,

4%

mM

PIPES, 5

mM

EGTA,

niM

a central microtubule-poor region (Fig. Ib, b' arrow).

in diameter, in

which

tated

images

(Fig.

Id',

lecular Probes), and

supplemental

ro-

animation

it

became apparent that the

Id. d') was the region

through which the polar body nucleus and associated remaining

centrosomal material passed (22-24 min post-activation; Fig.
le). The first polar body appeared fully formed at about 26 min; it
was enclosed by an actin-containing cortex that followed an out-

wardly and inwardly bulging contour (Fig. If). Results similar to

shown in Figure 1 were obtained with KCl-activated eggs

those

from several different clams, and also with sperm-activated eggs.

These observations are consistent with, and extend, older electron
microscopic work on polar body formation in eggs of Spisula and
Tubifex (2,7) and are of value for comparison with current findings

on unequal

cell division in

elegans

The sequence of
gram,

is

Saccharomyces cerevisiae and Caeno-

(8. 9).

cytoskeletal events observed in Spisula eggs

body formation, as illustrated in the Figure 1 diasuggestive of mechanisms. The initially eccentric meta-

moves toward

eral aster contacts the surface

the cortex,

and the periph-

Curvature of the

previous

astral
(diagram
microtubules and spreading outward along the cortex at the metaphase-to-anaphase transition follows (diagram c. d), rather than

localization in a variety of cell types (6). and

shortening of straight astral microtubules on contact. Such behav-

chromosomes and

fragments

nuclei were stained with

DAPI. These procedures followed protocols developed

work on microtubule

online

bulls-eye center of the F-actin ring (Fig.

phase meiotic apparatus

(Zenon. Mo-

and

thinner,

3-D computer-generated

visible along the cortex. Subsequently,

anti-a and anti-0 tubulins (Sigma T9026, T-4026) pre-labeled with

F ab

20

www.mbl.edu/BiologicalBulletinA/IDEO/BB.video.html). The
peripheral aster was now greatly diminished in size, and no longer

prior to polar

fluor 488-iabeled anti-mouse

away

At

at

rhodamine or Alexa Fluor 568-phalloidin. Microtubules were stained with a 1:1 mass mixture of mouse monoclonal
Alexa

was considerably

the F-actin

creating a "bulls-eye" appearance in

rhtihilitis

stained with

ob-

min the spindle entered anaphase (Fig. Ic), and a ring of thickened
17-20 p,m in diameter, then appeared in the cortex (Fig.
F-actin.
7-9
Id, d'). This ring surrounded a small circular cortical area,

MgCl,. pH 6.8 using NaOH). After incubation for h. the samples

were washed in PEM and then stained. For F-actin, the eggs were
1

now

served to curve outward along the cell cortex, symmetrically

fim

microscopy was used to

meiotic chromosomal stage.

In this study, eonfocal fluorescence

18 mini. Micro-

tubules of the peripheral aster, initially straight, were

in

our measurements showed that normal Spixula egg diameters

50-55 jam) were retained after such treatment. Confocal fluo(

rescence microscopy of stained samples was performed using the

ior

is

b).

consistent with models in which spindle

movement toward

brought about by the capture of plus ends of microtubules and microtubule transport, either by cortical dynein. or by
a formin-based mechanism (10. 11
The thickened ring of cortical
the surface

is

).

F-actin
:

Corresponding author: cohen(s'genectr.hunter.cuny.edu.

the inner layer of

which

(at least) is

sent formation of the contractile ring

is

presumed

to repre-

not established until late

CELL BIOLOGY

Figure

summon'

Singes

1.

(black

in

193

KCI-activated eggs at 23 C, with triple staining for F-actin, microtubiiles, ami chromosomes (white letters) and a diagrammatic
= 16 min post-activation, (b) Metaphase spindle at cell cortex
metaphase meiotic spindle, eccentrically positioned; t

letters), (a) First

with astral microtubiiles curving outward from central microtubitle-poor region; ~!8 min post-activation, (b
Higher magnification view of stage (b);
arrow: microtubule-poor central region, (c) Early anaphase with microtubiiles spread along cortex, (d) Edge view of thickened cortical F-actin ring, only
20 min post-activation. Inset: same stage, with microtubiiles also stained, (d' ) Computer-generated, rotated image of
chromosomes and actin stained;
1

the F-actin ring

same

cell,

bar for

shown

in (d). (e)

24 min post-activation. Inset:

Te/ophase chromosome set within F-actin ring; only chromosomes and actin stained;
26 min post-activation. Magnification bars = 10 \im;
(f) First polar body, enclosed by an actin-containing cortex;

microtubiiles also stained,

nil tii>urcs

other than b'

anaphase (diagram

d).

as

shown

in

it.

This suggests the involvement of anaphase

Literature Cited

signaling following a metaphase checkpoint.

It is evident that the sequence involves a mechanism for peripheral aster disassembly (Fig. Id, inset), but such disassembly is

spreading along the cortex. In addition,

the earlier pattern of contact between peripheral aster microtubules
and the cortex, with its central microtubule-poor area (diagram b).

delayed

is

its

match

to the "bulls-eye" pattern of

central thinner area,

dimensions of the
earlier aster (~-

which appears

later pattern also

Longo, F.
495-514.

J.,

and

3.

Kuriyama,

R.,

G. G. Borisy, and Y. Masui. 1986.

is

and C. Henley. 1971. Methods for Obtaining and

nd
Handling Marine Eggs and Embryos. 2 ed. Marine Biological Lab-

(diagram

correspond

to those of the

Undergraduate Science Education Program in

52002679), and by PSC-CUNY64249 and
gratefully acknowledged.

Dev. Biol. 114:

Allen, R. D. 1953.

Biol. Bull.

Woods

Hole.

Lee, K-G., A. Braun,

and W. Cohen. 2002.

7.

Shimizu, T. 1983.

8.

Knoblich,

9.

10

105: 213-239.

Costello, D. P.,

oratory,
6.

We

(grant

33:

5.

thank Kyeng-Gea Lee, Dr. David Burgess, Dr. Robert

Palazzo, Dr. Shirley Raps, Ruben Pinkhasov. and Alex Braun, for
assistance and helpful discussion. Support by the Howard Hughes

NSF9726771.

Res.

4.

microtubules.

Biology

Ultrastmct.

J.

The

d).

eter).
together, these observations strongly suggest that the
mechanism
for contractile ring generation involves the
signaling

Institute

Anderson. 1970.

thickened F-actin,

later

16-20-jxm outer diameter, ~7-9-|xm inner diam-

Medical

E.

151-160.

Taken

astral

Palazzo, R. E., E. A. Veisberg, D. G. Weiss, S. A. Kuznetsov, and

W. Steffen. 1999. J. Cell Sci. 112: 1291-1302.

until after astral

a close

with

J.

MA.
Chaikhoutdinov,

I.

Kiol. Bull.

Eur.

A. 2001.

J.

J.

DeNobile,

M. Conrad,

203: 204-206.

Cell Biol. 30: 74-82.

Nut. Kev. Mol. Cell Biol. 2: 11-20.

Guertin, D. A., S. Trautmann, and D. McCollum. 2002.

biol. Mol. Biol. Rev. 66: 155-178.

Micro-

S., D. Dujardin, A. Moreau, J. Dompierre, and J. R. De

Mey. 1998. Cnrr. Biol. 8: 541-544.
Gundersen, G. G., and A. Bretscher. 2003. Science 300: 2040-

Busson,

2041.

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

194
Reference: Bio/. Bull. 205
2003 Marine BioloL

Three-Di

(October 2003)

i" .; -195.

itwatory

al

Birefringence Distribution in Reconstituted Asters of Spisula Oocytes Revealed by

Scanned Aperture Polarized Light Microscopy

Michael Shribak and Rudolf Oldenbourg

Marine Biological Laboratory, Woods Hole, MA 02543

Ten years ago we reported the first measurement of the distribution of birefringence in asters dispersed in lysates of Spisulu
of polarized
oocytes: the observations were made with a new type
light

microscope, the Pol-Scope

1 ).

CCD

Since then, the Pol-Scope and

objective lens

MA

commercial version, the LC-PolScope (CRI, Inc., Woburn,

(www.cri-inc.com)), have become a mature microscopical techits

laboratories around the world for studying

nique used

in

living cells

and other specimens

many

animal

cells.

specimen on

directions

all

condenser lens

from

microtubule-organizing centers found in almost all

Nevertheless, both the structure of centrosomes and

their orientation within the plane of focus.

But when microtubules

away from the plane of focus, the measured retardance

reduced, and the angle of inclination cannot be discerned.

are inclined

Now we

are introducing a

new technique

called scanned aper-

microscopy (Fig. 1). The Scanned Aperture

Pol-Scope not only measures the microtubule orientation within
the plane of focus, but it also measures the angle of inclination

ture polarized light

the focal plane. In addition, the Scanned Aperture

measures
the retardance of all microtubules equally,
Pol-Scope
irrespective of their orientation and inclination angle.

away from

Figure 2 shows images of an aster reconstituted by polymerizing

tubulin off a centrosome purified from Spisulu oocytes (3).

The

images were recorded with the LC-PolScope (panel B) and with

the Scanned Aperture Pol-Scope (panel C and D). As illustrated in
the schematic of panel A. the focal plane
relative to the center of the aster (focal

was

offset by 5

jum

depth approximately 0.3

lacks retardance in the center

juiii). While the LC-PolScope image
of the structure, the image recorded with the Scanned Aperture

Pol-Scope reveals the retardance of the dense microtubule arrays

and are oriented almost parallel to

that are located near the center

microscope axis. In addition, panels C and D also indicate the

measured inclination and orientation angle of the birefringence

linear polarizer:

LC-M
linear polarizer]

a.

interference

mechanism by which they nucleate microtubules are still

poorly understood. The Pol-Scope reveals the birefringence retar-

is

LC-B
LC-A
,

>

the

dation, also called retardance, of astral microtubules irrespective of

slide

CD

(2).

Asters consist of microtubules radiating in

centrosomes

camera

circular analyzer

filter

lamp

Figure 1. Schematic of optical left and electronic components of the

Scanned Aperture Pol-Scope. The optical set-up is based on a traditional
device and an analyzer
light microsi ope enhanced by an aperture .scanning
for circular polarized light. The scanning device includes the liquid crystal
devices LC-A, LC-B. and LC-M. LC-M is sandwiched between two linear
I

retarder plates with .S or l>

polarizers. All liquid crv.stal devices are linear
individuull\ controlled sectors. LC-M and the two linear polarizers are

used

to block

of sectors

in

or transmit light passing through 8 pie-shaped sectors. A pair

in series, function as a universal

LC-A and LC-B. arranged

All
compensator, controlling the polarization state of the passing light /-//.
components of the scanning device are bonded together and form a 7

mm-tlnck

171/11 (//

flat

with electrical connections

and an approximate

diameter of 25 mm. The computer-controlled scanning device is placed in

the front aperture plane of the condenser lens and is used to sequentially
illuminate the specimen with a polarized, converging light beam whose
central ia\ is tspically tilled to the microscope a.\i.s. Tilt angles are

controlled bv blocking or passing the light in the K pie sectors.

the light in all

fs

sectors, the

tilt

angle

is

zero,

By passing
and recorded images are

recorded at ?
equivalent to LC-PolScope images. Currently, images are
Jil/ereni till angles and are combined using specially developed algorithms
for

measuring the .^-dimensional birefringence distribution

solved specimen point {see

instrument see /5/J.

t-'ig.

2: for

in

every re-

a more detailed description of the

the

which also indicates the orientation of the

axis,

astral

microtu-

bules.

We

here are typical of our observations of

position. All results reported
at least 3 asters and repeated measurements at different focus
positions.

images after adjusting the focal plane to

include the centrosome in the center of the aster (images not
also recorded

shown).

In

this

configuration, the retardance values in images

recorded with the Scanned Aperture Pol-Scope were below 0.3 nm

near the center of the aster, increased rapidly at a radius of 3 ju.m.

and peaked

at a

radius of 7.4 /xm (peak retardance

addition, the inclination angle

was close

3 nm). In

to zero for all retardance

Our findings indicate that the centrosome essentially lacks

of astral microaligned microtubule arrays. However, the density
tubules increases sharply at the surface of the centrosome.
to interpret the radius at which the measured retardance

We

propose
has the steepest gradient as the location of the centrosome surface
(radius = 3.1 ju.m). In the future we plan to measure the density of
microtubule arrays as a function of radius from the center of asters
exposed to various physiological conditions to determine

values measured in the focal plane that included the center of the

that are

This latter result conforms to the expectation that all astral

microtubules are oriented parallel to the focal plane at this focal

their potential for microtubule nucleation.

aster.

In conclusion,

we have demonstrated

that the

Scanned Aperture

CELL BIOLOGY

195

Pol-Scope can be used to measure the orientation and inclination

angle of the birefringence axis of objects exhibiting low birefrin-

Side View
microscope axis

gence, such as asters.

retardance of

all

In

addition, the technique measures the

birefringent objects equally, regardless of their

orientation and inclination angle.

at

The measurements

resolved specimen points simultaneously,

all

(~ 0.3 /urn), and high sensitivity

This seems to be the first reported technique
resolution

focal plane

are performed

at

high spatial

(0.1

nm

retardance).

that

can measure the

3-dimensional distribution of birefringence in microscopic specimens. We expect this technique to impact many application areas
of the traditional polarized light microscope, including the imaging

of living cells, tissues, and functional model systems

and medicine.
Purified

components

for

reconstituting

asters

in

biology

were

in-vitro

kindly provided by Robert E. Palazzo of Rensselaer Polytechnic

Institute

and the Marine Biological Laboratory. The research

NIH grants GM49210 and EB002045.

is

supported by

Literature Cited
I

Figure

2.

An

ami

aster reconstituted from purified centrosomc

was imaged with

the

LC-PolScope and the Scanned Aperture Pol-Scope

and objective lens \Zeiss Neoftuar 100 X

NA

Poll. IAI

of the plum'

panels B, C. and

in

and

the definition of the

LC-PolScope image of microtubule retardance in the

focal plane. The measured retardance in the center of the image is close to
~ero because the microtubule arrays are oriented nearly perpendicular to
I

parallel to the microscope

a.\is>.

(Cl Microtubule retar-

dance measured with the Scanned Aperture Pol-Scope. Gray scale image
shows retardance of all microtubule arra\s, regardless of inclination

Biul. Bull. 185:

In Lire Cell Imaging: A Laboratory Manual. D. L.

and
R.
D.
eds. Cold Spring Harbor Laboratory Press,
Goldman,
Spector
Cold Spring Harbor. NY. (In press).

3.

Schnackenberg. B.

inclination angle. IB)

the focal plane

G. Mei, and R. E. Palazzo. 1993.

Oldenbourg, R.

Schematic side view of the aster indicating the position

tui if.

i'/

R.,

2.

using mi nil immersion condenser

/1. 3

Oldenbourg,
288.

tuhiilin

J.,

and R.

Methods Cell Bioi

E. Palazzo. 2001.

67: 149-165.
4.

Shribak, M., and R. Oldenbourg. 2003.

5.

Shribak, M.

I.,

Appl. Opt. 42:

and R. Oldenbourg. 2002.

Pp.

3009-3017

104-109

In

Three -

Dimensional and Multidimensional Microscop\: Image Acquisition and

Processing IX. Proceedings of SPIE 4621, San Jose. CA.

angle. Over/av indicates lines of equal inclination angle in steps of 20. (D)

Same microtubule relardance image

as

in

panel C: the image

is

overlaid

with lines indicating the orientation of measured birefringence axis in the

plane offocus. For

and
In

clarity,

both overlays show only a subset of orientation

measured in every resolved image point.

inclination angles, which tire

images of panels

and D. black

B. C.

is

~ero retardance.

and white

is

run retardance.

Reference: Biol. Bull. 205: 195-197. (October 2003)

2003 Marine Biological Laboratory

Rho-kinase

Required for Myosin-II-Mediated Vesicle Transport During M-Phase in Extracts of

Clam Oocytes
Torsten Wollert', Ana S. DePiiur. Carl J. DeSelnr, and George M. Langford'

Is

Rostock University, Rostock, Germany

~
Dartmouth College, Hanover, NH

In

mammalian

cells.

Rho

proteins

Rho/Rac/Cdc42 regulate

the

formation of the actin cytoskeleton in stress fibers, lamellipodia,

and filopodiu
). One of the
ways in which Rho proteins mediate
(

effects

on the actin cytoskeleton

myostn phosphatase pathway.

phosphatase (MyoP)
thereby inhibited

is

(2).

is

via the

In this

pathway, myosin

phosphorylated by

The

net result

Rho-ROK/Rho

is

ROK/Rho

kinase-

light

chain

kinase and

the activation of

is

myosin-

11-mediated activities. In addition, Rho-family proteins have also

been shown

to regulate the actin cytoskeleton during cell division

(3).

To

study the role of

transport during the

inhibit

Rho

Rho

proteins in myosin-II mediated vesicle

M-phase of

the cell cycle,

we used Y27632

kinase activity in extracts of clam oocytes.

shown previously

that actin filaments

We

to

have

assemble spontaneously

in

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

196

such extracts and organize into a three-dimensional network of

This self-organized network of actin
interconnected til
<

ytokinetic ring of dividing cells in the

filaments resent'"

following v
sliding of

it

li

M-ph:;

vesicles are

motor

.nents (4, 5), and

cell cycle.

extracts co-align to

are visible by

exhibits myosin-II-mediated, anti-parallel

form bundles (10-20

AVEC-DIC

moved along

Y27632, blocks

it

assembles during the

microscopy.

parallel filaments) that

We

the actin filaments

we show

have also shown

by a class

II

that

myosin

shifted

at

the

G2/M

from 6.8

to 7.2

by diluting 2-fold with pH-8 buffer to

progression through M-phase. Nocodazole (30 JJ.M) was

added to the extracts to block microtubule assembly; an ATP
initiate

regenerating system was added to maintain

vesicle transport in these extracts, thereby

reconstitute

45'

actin in

is

80 C. To
phase of the cell cycle were snap frozen and stored at
begin an experiment, the pH of the cytoplasmic extracts was

preparation

that a specific inhibitor of

movement on

mediated by myosin-II.
Extracts prepared from mature oocytes arrested

these extracts

Rho

(4). In this report,

kinase.

(ii)

Fortuitously, actin filaments in the

providing additional evidence that vesicle

was incubated
motor

at

activity.

to

ATP

levels;

and the

assemble actin filaments and

Rhodamine-phalloidin (0.5 ^.M) was

CELL BIOLOGY
added

to stain the net in filaments,

and the myosin-Il motor activity

was monitored by AVEC-D1C and fluorescence microscopy.

In control extracts, vesicle transport was measured at regular
time points to determine whether motile activity varied upon entry
into M-phase of the cell cycle. Vesicle transport was measured by

counting the number of vesicles moving per video field per min
(v/f/m: motile activity) at 15-min time intervals. We found that the
motile activity was high during the first 15 min of incubation at 18
C, then declined during the 15-30-min interval but rose again and
remained stably high between 45 and 60 min (Fig. 1A). Motile
activity declined again after 120

min

C.

18

at

The

actin network,

as revealed by rhodamine-phalloidin staining, did not change during the 2-h period of incubation. To determine when the extracts

were

M-phase of the cell cycle, a Xenopus sperm-nucleus

shape-change assay was performed. Xenopus sperm nuclei were
added to the extract, stained with DAPI, and observed by fluoresin the

cence microscopy
initial

regular intervals during incubation. In the

period, the Xenopus sperm nuclei remained elongated, but
at

at

197

low concentrations of the

To determine whether Rho

port, extracts

were incubated

kinase
at

18

for

ine (Fig. 1C), a general kinase inhibitor.

Y27632 was added at a concentration of 300 nM. We

Y27632 inhibited vesicle transport by 65% compared to
the control (Fig. 1C). The inhibitor had no effect on the actin
cytoskeleton. The fact that vesicle transport was strongly inhibited

most sensitive

volved

Based on the

results with

is

required for

is

directly in-

Because there was no effect on the

in vesicle transport.

downstream

actin cytoskeleton in this case, the

kinase

target of

mM staurospor-

Y27632 inhibitor, we conclude that Rho kinase

movement of vesicles on actin filaments.
In summary, these data show that Rho kinase

Rho

target of

mostly likely myosin II. This is the only myosin-mediated

pathway known to be regulated by Rho GTPases. The downstream
effector of the Rho proteins is most likely myosin light chain
is

Therefore, we can conclude that the Rho-ROK/

phosphatase
Rho kinase-myosin phosphatase pathway regulates vesicle trans1

).

M-phase

port during

clam oocytes.

in

This work was supported by

Shifman award

to

NSF Grant IBN-0131470

MBL

and

CJD.

Literature Cited
1

2.

Bishop, A. L., and A. Hall.

Kimura,
fuku, B.

3.

2(100.

Biochem.

348: 241-255.

J.

M. Ito. M. Amano, K. Chihara, Y. Fukata, M. NakaYamamori, J. Feng. T. Nakano, K. Okawa, A. Iwamatsu,

K.,

and K. Kaibuchi.

Science 273: 245-248.

1996.

Yoshizaki, H., Y. Ohba, K. Kurokawa, R. E. Itoh, T. Nakamura, N.

Mochizuki. K. Nagashima, and M. Matsuda. 2003.

45 min, and then the

inhibitor

found

kinase, the

also achieved with 2

the

required for vesicle trans-

is

was

this inhibitor. Inhibition

they began to expand and appear uniformly bright at 30 min (Fig.

IB). At 45 min. the nuclei assumed an irregular shape as the

chromosomes condensed. Chromosome condensation is diagnostic

of M-phase. The nuclei remained irregular in shape with condensed chromosomes from 45-120 min, the period when motile
activity was high (Fig. 1A). Based on this assay, extracts incubated
for 45 to 60 min were judged to be in M-phase.

inhibitor suggested that the specific

was Rho

target of the inhibitor

J.

Cell Bil.

162: 223-232.
4.

that

Wollert, T., A. S. DePina, R. F. Reid, and G.

5.

M. Langford.

Wollert, T., A. S. DePina, L. A. Sandberg, and G.

2001.

20(12.

203: 20S-210.

Biol. Bull.

Biol. Bull.

M. Langford.

201: 241-243.

Reference: Biol. Bull. 205: 197-199. (October 2003)

2003 Marine Biological Laboratory

An Experimental Approach

Cusato'

A'.

The

vertebrate retina

tissue derived

is

',

J.

'Albert Einstein College of Medicine, Bronx, NY

University of Illinois College of Medicine, Chicago, IL

a highly specialized sheet of neural

from an undifferentiated population of neural pro-

genitor cells, which,

upon completion of

their final division, mi-

grate to their laminar positions and differentiate

retina,

Study of Gap-Junction-Mediated Cell Death

2
Zakevicius and H. Ripps"*

to the

almost every class of neuron and

).

In the adult

glial cell is linked to its

neighbors by gap junctions

aqueous channels that allow the
intercellular exchange of ions, second messengers, and other small

molecules (<

kDa). Thus, this communication pathway aids

in

the synchronization of cellular activity, and plays a significant role

maintaining cellular homeostasis

in

(2).

During development,

however, many retinal cells undergo programmed cell death, or

apoptosis, and both intracellular and intercellular communication
are

known

to regulate the process (3). Indeed,

Corresponding author: harrripp@uic.edu

we

recently reported

that

gap junctions mediate

form of "bystander"

cell

death

in the

developing retina (4). Although this process is largely arrested in

the mature retina, differentiated neurons and glia undergo apoptosis in neurodegenerative diseases (5), as well as in ischemia and
trauma. In

dying

all

of these cases, the spread of cell death from one

cell to its

increase the total

otherwise unaffected neighbors (bystanders)

number of cells

Because the retina

is

that enter the apoptotic

complex

tissue,

stander cell death are technically unfeasible

some

may

pathway.

studies of by-

at this time.

We

have

developed a model system for the study of gap junction-mediated

cell

death using Xenopus oocytes which express an endogenous

gap-junctional protein (connexin38, [Cx38]). Oocytes are paired at

their vegetal poles following removal of their vitelline membranes

coupled via gap junctions. Because the

to express many different connexins, the
can
serve
Xenopus oocyte
system should enable us to identify which gap junctional channels

and become

electrically

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

198

Cell

Bystander

CcCell

CELL BIOLOGY
membrane

remain open under apoptotic conditions, to study the dynamics of

gap junctional coupling during cell death, and to determine which
molecules may pass from a dying cell to its neighbors to trigger the

potential

gap-junction-mediated cell
essential molecules (e.g.. ATP) passing from the healthy

apoptotic agent

cell to its

of caspase

3.

have shown

a progressive loss of

and

DNA

membrane

fragmentation

that injection of

into

present study we
one oocyte of a

stances, since antisense to

in future

UIC Department

Research to Prevent Blindness.

This result suggests that the intercellular channels

Award from

tor

longer than 40 min for bystander cell death to occur.

Alcon Research

also

RPB

the

1.

injections failed to induce cell death in either injected or nonin-

jected cells of electrically coupled pairs.

Dual electrode voltage-clamp using GeneClamp 500 amplifiers

CA) was

(Axon Instruments, Foster

2.

used to monitor gap junctional coupling between cell

one of the cells with cytochrome c. The two

were voltage-clamped

to

Marine Biological Labora-

Institute

Inc.; a

Senior Research InvestigaAward of Merit from the

(HR); and an
(HR).

the

same

3.

4.

responses of both cells were recorded

(7).

(-40 mV).

potential

voltage steps were applied to the noninjected

cell,

5.

and the current

As shown

6
in

Figure 1
(c.d), cells remained electrically coupled despite the gradual death
of the injected cell. In addition, we found that the loss of mem-

c,

Andrade-Rozental, A.

F..

R. Rozental,

M. G. Hopperstad,

J.

K.

Wu,

and D. C. Spray. 2000. Brain Res. Rev. 32: 308-315.

Linden, R. 2000. Brain Rcy Rev. 32: 146-158.
Cusato, K., A. Bosco, R. Rozental, C. A. Guimaraes, B. E. Reese, R.
Linden, and D. C. Spray. 2003. J. Neurosci. 23: 6413 6422
Ripps, H. 2002. E.\p. Eye Res. 74: 327-336.
Bhuyan, A. K., A. Varshney, and M. K. Mathew. 2001.
Differ. 8:

Cell Death

63-69.

Dahl, G., T. Miller, D. L. Paul, R. Voellmy, and R. Werner. 1987.

Science 236: 11.290-11,293.

brane potential resulting from cytochrome c injection reported by

Bhuyan et al. (6) in single oocytes could be seen also in cell pairs.
After injecting one cell with cytochrome

S. R. 1991.
Pp. 69-128 in Vision and Visual Dysfunction.
Vol 3: Neuroanatomy of the Visual Pathwavs and their Development. B.
Dreher and S. R. Robinson, eds. Macmillan, London.

Robinson,

F. D. Vrionis,

City,

pairs after injecting

cells

at the

Literature Cited

close apposition to the dying cell but remained intact. Vehicle

8 software

induce bystander

not mediated by contact or by

extracellular toxins, since the surviving cell continued to be in

pClamp

to

Grass Foundation Fellowship (KC); an unrestricted award to the

of Ophthalmology and Visual Sciences from

joining the cytoplasm of the coupled cells must remain intact for

controlled by

coupled partners

Woods Hole, Massachusetts, and were supported by grants

from the NIH (HR: EY-06516 and EY-01792; KC: HL-07675); a

It

its

These studies were conducted

lysed in less than 40 min. the noninjected cell did not die (Fig.

is

Cx38 prevented bystander cell death,

Of particular interest is the fact that gap

cell death.

tory,

indicates that bystander killing

and

cell

had been injected did

die, its paired neighbor did not. Similarly, in cases where the cells
were electrically coupled, but where the cytochrome c injected cell
la.b. left pair).

too great to pass through gap junctions

to identify the intercellular signals that pass

experiments

between a dying

exhibit bystander cell killing following cytochrome c injection;

which cytochrome

death by intracellular injection

junctional coupling persists during the apoptotic process, encouraging us to use the Xenopus oocyte and other expression systems

jected cells over a period of 2-3 h (Fig. la,b). Pairs of oocytes

preinjected with an antisense oligonucleotide to Cx38 did not

although the cell into

is

but not primary cell death.

pair causes death in both the injected and nonin-

Cx38-coupled

kDa)

13

cell

evident that the molecular mass of this

is

it

carrying the death signal to bystander cells. Likewise, bystander

killing is unlikely to be mediated by contact or diffusible sub-

potential, activation

(6). In the

cytochrome c

c,

Clearly, cytochrome c itself cannot be the toxic substance

(8).

dying neighbor.
Previous studies of single Xenopus oocytes have shown that
microinjection of cytochrome c induces apoptotic cell death, ac-

companied by

rapidly following injection (Fig. le).

Although we have induced

is

it

membrane

depolarization, with the injected cell losing

more

of cytochrome

important to recognize that

death may result from the depletion of

apoptotic process. Conversely,

199

Bennett,
1988.

both cells underwent

M. V.

Pp.

L.,

287-304

V. K. Verselis. R. L. White, and D. C. Spray.

in Gap Junctions. E. L. Hertzberg. and R. G.

New

Johnson, eds. Liss,

York.

Reference: Biol. Bull. 205: 199-201. (October 2003)

2003 Marine Biological Laboratory

1
S.

Tepsuporn

'*, J.

Apoptosis in Microciona prolifera Allografls

2
W. J. Kuhns M. M. Burger and

C. Kaltenbach'
1

Mount Holyoke

College. South Hadley,

X.

Fernandez-Busquets

MA

Hospital for Sick Children. Toronto, Canada

Friedrich Miescher Institute, Basel, Switzerland
University of Barcelona, Barcelona, Spain

When two

sponge tissue fragments from the same individual are

adjoined, isogeneic recognition occurs, and the fragments fuse.

Corresponding author: stepsupo@mtholyoke.edu

On

the other hand,

if

the

two pieces are from different individuals,

allogeneic recognition occurs, followed by failure of fusion and.

presumably, death of cells at the graft contact zone (1. 2). It has

been proposed that two cell types, archaeocytes and gray cells, are
involved in sponge allogeneic recognition. Archaeocytes are large

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

200
phagocytic cells with

wound

:m<

healing

are motile cell'

Gray

is

sponges

that

camera on

a Zeiss

microscope.

24-h period (Fig. 1A). On the contrary, treatment of allograft

sections with the same primary antibody showed that cell death at

1 );

pseudopodia and are commonly

il-defined

cell death) in

(programmed

TUNEL

by

digital

dem-

granules

region

onstrated

mounted, and photographed with a

egions of the sponge but are rare in the marginal

in

cells

Isograft sections treated with anti-active caspase-3 primary antibody did not stain, indicating that apoptosis did not occur in
either the line of contact or any of the cells in the grafts during a

many dense cytoplasmic

ai

'

in gi

commonly found

-ting areas of the sponge (1).

they do nut

found

:iudeoli; they are

In, L-C

nick-end

had undergone

ring winter event (4).

assay

labeling

in

first

hibernating

tissue regression as a naturally occur-

The development of an

was

detecting apoptosis

sponges was

alternate

method

for

the finding that a series of caspases (pro-

enzymes leading to cell death (5). Of

enzyme was the final and most important protease. From this have emerged antibodies for the detection of
caspase-3 (6). Biochemical methods were used by Wiens et al. (7)
teases) delineated sequential
these, caspase-3

the contact zone

their identity as archaeocytes.

the existence of archaeocytes

However,
contact area

prompted the use of

marine sponge Geodia

cydonium, where caspase-3 activity was found to be greatly in-

when compared with

result of apoptosis. Cells near the

IB). The longer the period of allogeneic contact, the higher the
number of large, elongated apoptotic cells accumulated at the
contact zone. The morphology of these cells was consistent with

to study apoptotic events in extracts of the

creased in allograft extracts

was indeed a

contact zone started to undergo apoptosis 6 h after grafting (data

not shown). The line of contact became most apparent at 24 h (Fig.

and gray

cells in the

a gray cell identity

marker

in

The
immu-

isografts.

objectives of the present research are (a) to determine by

nohistochemistry whether cell death at the allograft contact zone is

a result of apoptosis rather than necrosis (death by injury that may

from mechanical damage

result

to the cells)

cell type or types, if any, that

and

(b) to identify the

undergo apoptosis

at the

contact

zone.

To examine whether the death

we used an

result of apoptosis,

of cells

at the

contact zone

is

a
'

indirect labeling technique that

provides precise tissue localization of active caspase-3 as a

marker

were visualized by the presence of a dark brown precipitate when

color was developed with diaminobenzidine (DAB).
Individuals of Microciana prolifera were collected by the

Resources

(Woods Hole,

Center of the

MA)

Marine

-''',;";;;;.

-,^'-

;,-.',

-?' -^
'

;^M#?. Sm:%?

were reactive

for cells undergoing apoptosis. Processed cells that

rine

S>V$V!K J'^vOii>

Ma-

Laboratory
and were maintained until use in a tank with
Biological

The two sponge pieces for isografts and

were held together with number zero stainless steel pins
attached to a Styrofoam board floating in a tank of running cold
running cold seawater.
allografts

seawater.

The

grafts

were fixed

6-h intervals from

at

to

24 h

in

3.7% formaldehyde in MBL artificial seawater (MBLASW) overnight, then washed and dehydrated in a series of ethyl alcohol
concentrations from

30%

to

70%

in

MBLASW.

Spicules were

dissolved by overnight treatment of grafts with 4% hydrofluoric

acid in 70% ethanol. The grafts were then embedded in paraffin

and sectioned

The

at 7 jum.

slides of sectioned isografts

finized, hydrated.

and treated with

and allografts were deparaf-

3%

hydrogen peroxide

to re-

move endogenous

peroxidase activity. Tissue presumed to possess

caspase-3 activity was incubated using purified rabbit IgG anti-

human

active

caspase-3, which had been generated by affinity

from a caspase-3 enzyme fragment (Pharmingen

#559565); the antibody was used at a concentration of 0.25 jug/ml
purification

for 2 h

:it

room

temperature. The

phosphate-buffered saline (PBS.

slides

pH

were then washed with

7.5) (Sigma),

followed by

treatment with appropriate biotinylated secondary antibody (Vector Laboratories).

ABC
DAB

wash in PBS, the slides were

enzyme complex VectaStain Elite

After another

treated with avidin-biotinylated

Vector Laboratories) before the peroxidase substrate

(Sigma) was applied. The slides were washed, dehydrated.
Kit,

Figure 1. Apoptotic response of allografts prepared from the marine

sponge Microciona prolifera. (A) Control isograft: fixed tissue stained with
anti-active caspase-3 antibody. Arrows indicate the zone of contact. No
reaction occurs in this zone or in cells in the area. Only occasionally do a

few

cells within the tissue

show

positive staining (arrowhead).

under phase contrast. (B) Allograft fixed al 24

caspase-3 antibody.
(arrows},

and

in

A marked

cells

in

the

h.

Viewed

stained with anti-active

reaction is seen at the zone of contact

neighborhood of the contact area. (C)

Allograft fixed at 6 h double stained with anti-active caspase-3 antibody,

visualized as a

brown precipitate with DAB-H,O 2 and with anti-CD44

Brown apoptotic cells desig.

antibodv. visualized by pink fluorescence.

nated as archaeocytes surround the contact

distinct

from the pink fluorescent

head). Colocalization of caspase-3

represents

50

[Jim.

line (arrows),

CD44 positive)
and of CD44 was

(i.e..

and appear to be

gray

cells

(arrow-

not obsen'ed.

Bar

CELL BIOLOGY
addition to identification by morphology.

been demonstrated

in

gray

cells,

CD44

has previously

but not in archaeocytes

),

and

therefore anti-CD44 antibodies were used in combination with

anti-active caspase-3 antibodies in double labeling experiments.

CD44

staining

was done using

a rat anti-mouse

CD44

(Pharmin-

201

the Boston University

tute

at

/xg/ml for 12 h at 4

were

slides

above (microscope setting for

fluorescent double staining: 647 nm filter,

mercury and halogen lamp as light sources). Colocalization of

anti-CD44 was not observed (Fig. 1C).

anti-active caspase-3 and

conclude

that cell death at the allograft contact

result of apoptosis rather than tissue necrosis,

totic cells are

The

Literature Cited

treated as described

single field imaging in

We

Insti-

Ramon

y Cajal tenure-track position from the Ministerio de Ciencia y

Tecnologfa (MCyT). Spain, and acknowledges the support of grant
BIO2002-00128 from the MCyT.

C, followed by wash and biotinlabeled secondary antibody (Vector Laboratories). Fluorescence

was developed using Texas red-avidin (Vector Laboratories). The
gen)

Marine Program; Friedrich Miescher

of the Novartis Research Foundation. X. F.-B. holds a

most

ability to

and

zone

that the

is

1.

26: 313-323.
2.
3.

apop-

likely archaeocytes. not gray cells.

recognize self from non-self has been conserved

A better knowledge of this process in the

throughout evolution.

sponge model should enhance our understanding of allogeneic

recognition and its regulation in more complex species, including

humans. The knowledge gained from such basic mechanisms

should be of use in developing effective specific drugs that can
abrogate the pathological effects of allogeneic responses

5.

Biol. Bull. 191: 159-167.

Yin, C., and T. Humphreys. 1996.
Simpson, T. L. 1968. P. 23 in The Structure and Function of Sponge

Cells: New Criteria for tin- Taxonomy of Poecilosclerid Sponges. Peabody Museum of Natural History. New Haven, CT.
Kuhns, VV. J.. M. Ho, M. M. Burger, and R. Smolowitz. 1997. Biol.
Bull. 193: 239-241.

Kaufmann,
11:

S. H.,

and M. O. Hengartner. 2001.

Trends Cell Biol.

526-534.

Volm, M..

J.

Mattern, and R. Koomaegi. 1999.

Anricuncer Res. 19:

1669-1672.
7.

(8).

Acknowledgments: Mount Holyoke College HHMI Cascade

Mentoring Program: Research Experience for Undergraduates of

Fernandez-Busquets, X., W. J. Kuhns. T. L. Simpson, M. Ho, D.

Gerosa, M. Grob, and M. M. Burger. 2002. Dei. Comri. Immunol.

Wiens. M.. A. Krasko,

chim. Biophys.

Ada

S. Perovic,

and W.

E. Miiller. 2003.

Bin-

1593: 179-189.

Kuhns, W. J., M. M. Burger, M. Sarkar, X. Fernandez-Busquets,

and T. L. Simpson. 2000. Biol. Bull. 199: 192-194.

Reference: Biol. Bull. 205: 201-203. (October 2003)

2003 Marine Biological Laboratory

The Decorated

Immune System to the Fibrils of the Limulus

Blood Clot
1 2
1 2
Peter B. Armstrong '*, and Margaret T. Armstrong
1
Marine Biological Laboratory, Woods Hole, MA

Clot: Binding of Agents of the Innate

'

The
innate

fibrillar

'

University of California. Davis,

blood clot functions as an important element of the

its ability to entrap and immobilize

immune system by

bacteria that have entered the

body

viti

wounds, thereby preventing

their systemic dissemination throughout the

body of

the injured

The coagulin clot of the horseshoe crab appears to play

a similar role in protecting that animal from pathogenic attack.
host (1,2).

Bacteria entrapped in the coagulin clot are held so tightly as to

abolish even thermal (Brownian) motion, and the clot synergizes
with plasma in the killing of entrapped microbes (3). The present

study investigates the proteins of the innate

immune system of

Limidus that bind

to the fibrils of the coagulin clot, potentially

supplementing the entrapment actions of the clot in two ways: first,
by the lethality of clot-bound proteins for the entrapped microbes
and second, by the ability of these proteins that decorate the clot
fibrils

to

bind and inactivate the toxic products of entrapped

establish the clot, the blood cells contained in

collected under sterile conditions were dispersed

3% NaCI

plastic petri dish (Falcon

attachment of the cells

with

50%

or

100%

Cat # 35-1008). After 5 min

to the dish surface, the saline

sterile

in

drop of blood

(Baxter Healthcare Corp.. Deerfield. IL)

ml of
in

sterile

35-mm

clotting system.

dense coagulin clot then formed above the

monolayer of attached blood cells. After 0.5-2 h of washing with

several changes of wash buffer, this was either fixed directly in 4%
paraformaldehyde dissolved
extracted with
in

in

3%

0.5% Triton X-100

NaCI, 10

in the

mM

CaCU

or

same buffer and then

paraformaldehyde. All of the antibodies used for

showed

was
fixed

this report

two preparations. The

prepare antibodies were purified as fol-

identical staining patterns for the

various proteins used to

lows: coagulogen. the structural protein of the blood clot, as

described by Srimal ci ai
described by Armstrong el

ultracentrifugation

(4);
at.
al.

(285,000 X

Limulus a 2 -macroglobulin. as
the Limulus pentraxins, as

(5):
(6):

and hemocyanin. purified by

followed by gel filtration

g, 8 h)

ehromatography (Sephacryl S-300). Antibody production in rabbits and immunocytochemical staining utilized standard methods
(7).

Corresponding author: pbarmstrong(s'ucda\ is.edu

allow

Limulus plasma. Under these conditions,

The polyclonal antibodies were checked for specificity by

(8) and in some cases were affinity-purified on

Western blotting
*

to

was replaced

the blood cells rapidly degranulated. releasing the coagulin blood-

described by Armstrong et

microbes.

To

CA

antigen-Sepharose affinity columns

(7).

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

202
The

coagulin clot showed to advantage

'.copy an d by immunocytochemical

i'v

fibrillar structi'

both by phase
staining with

same

highly

pi

shown

i.

The

.-parations of Liimilus a-,-macroglobulin (data not

and hemocyanin (data not

nndus os-macroglobulin functions in the binding and
inilus pentraxins (Fig.

shd\'

cle

>gen antibodies (data not shown).

iiiunostained with antibodies prepared against

fibril^

),

uce of proteases, including, presumably, the proteases of

:.

pathogenic microbes

The Limn/us pentraxins show

(9).

a potent

and may operate

to assist in

the cytolytic destruction of microbial invaders (6, 10).

Hemocya-

cytolytic activity against foreign cells

nin
ally

is

the respiratory protein in solution in the blood but addition-

shows

a phenoloxidase activity that potentially functions to

microorganisms by the generation of oxygen radicals (11).

The binding of os-macroglobulin may be covalent because

+2
12) or
oligomeric structure of the hemocyanin molecule on Ca
+2
a true Ca
-dependent binding of hemocyanin to the coagulin
(

There are two potential sources for the clot-bound a r

macroglobulin: the plasma (13) and the 2 -macroglobulin released
fibrils.

from the secretory granules of the blood

bound

protein,

degranulate

because the clot

in saline (0.5

prior to fixation with boiling

it

SDS-

polyacrylamide gel sample buffer containing 2-mercaptoethanol.

Most of the known ligand-recognition properties of the Limulus
+2

Ca -dependent (6). In contrast, binding to the

+
coagulin clot is Ca "-independent, because immunostaining is not
+2
diminished by treatment of the clot with a Ca
-ehelating agent,
pentraxins are

ethylenediaminetetraacetic acid

NaCl, 10

mM

Tris,

pH

this

is

Most of

0.1

the

I.

0.5

EDTA,

M
is

have not

fibrils

10

produced by

mM CaCU)

cells

in the

that

absence

known to bind
The blood

a suite of proteins that assist in the functions of the clot.

clot of

mammals

binds fibronectin, which potentiates the immi-

gration of wound-repair fibroblasts (15); FGF-2. which promotes

proliferation of clot-associated endothelium (16, 17);
pins,

and the

ser-

plasmin activator inhibitor-2 (PAI-2) and os-antiplasmin.

which are presumed to protect the clot from proteolysis (18).

However, we are not aware of any reports of agents of the immune
system binding to the fibrin clot of mammals. Thus the observation
that

immune

effector proteins bind to the Liimilus clot suggests the

novel idea that the clot

for

invading microbes:

is
it

more than a passive entrapment device

is

potentially

a delivery

vehicle for

proteins that are lethal to the entrapped microbes and proteins that

simply a reflection of the dependence of the

inactivate toxic products of those microbes. Indeed, the clot syn-

the clot with

but

it

is

'.'

<.'

Binding <'/ ///( LiMHilus jiciniii \ms in Ithiils >>! ihc l.muilus hlooil tl{>t in spci'itticns not c\Irticicil inY// Itifon X-llKI. The fibrils oj the clot
and iiiiniiinnxttiin \\-ilh tin untihtnly against the Limulus
monoluyers of Limulus blood cells me visible />\ phu.\c conlruxl niicroscn/n f.-l.
'i
The (/nvnr.v in A and
nhlicule the MI/IIC tihril r;.M/)/c />v />/;
cuntniM inn TOM -o/'v (A) and immunofluorescence (B). The darkish bodies

prodiK
pentnnii:'
H'<'" b\

EDTA.

bound hemocyanin

Ki;-

We

not certain

removed by treatment of
whether

7.3).

(EOT A

M NaCl,

of plasma are decorated with a,-macroglobulin.

The fibrin fibers of the mammalian blood clot are

kill

was not removed by treatment

cells (14).

binding of plasma a 2 -macroglobulin. but secretorygranule-derived os-macroglobulin does contribute to the clotruled out

/>'

trasl niifn>\c<>/>\ (A.

/',.

antibodv.

ll;c

ill

,'\

Cl arc

that are clearly visible

were manipulated niciU'cally

in

ilie

ninici n/ the hlcnj cell\ atlac/icil In the culture \iiifaee. \Vlien

b\ phuse contrast micros-coin' fC)

Photoshop, so are directly comparable.

fail to slain ID),

8 and

normal rahbit serum replaces

specific

were I'lmlo^rapheJ under identical conditions unJ

CELL BIOLOGY
in the plasma in effecting the active killing of
clot-entrapped microbes (3).
This research was supported by Grant No MCB-2677 from the
National Science Foundation.

ergizes with factors

8.

203

Towbin.

USA

Sci.
9.

2.

Dunn.

I).

and

I...

Rotstein. O.

R.

[..

1992.

I).

11.

Simmons. 1982. SurK cry 92: 513-519.

Em: J. Clin. Mk-rohiol. Intcci. O/.v 11: 1064-

3.

12.

Isakova, V.. and P. B. Armstrong. 2003. Bi,>l. Bull. 205: 203-204.

Srimal, S., T. Miyata. S. Ka\vabata, and S. hvanaga. 1985. J. Bio-

13.

chem. Tokyo 98: 305-3 S.

Armstrong. P. B., R. Melchior. and

14

Pliyxiol.
6.

J. P.

Quigley, and P. B. Armstrong. 1995.

J.

Bin/.

Swarnakar,

R. Asokan,

S..

Bio, hem.

./.

P.

.1.

Quigley. and P. B. Armstrong.

347: 679-685.

Nagai, T., and S. Kawabata. 2000.

J.

Biol.

Chem. 275: 29.264-

Van Holde, K.

E.,

and K.

and

P. B.

Miller. 1982.

I.

Q. Rev. Biorihy*.

15:

J. P.

Quigley. 1996.

S.
J.

Srimal. S. Misquith, E. A. Hahn,

P. Quigley. 1996.
J. Biol. Chem. 271: 14,717-

Swarnakar.

S.

Armstrong. 1983.

J.

Biol.

Chem. 258:

Armstrong,

P. B., J. P. Quiglev,

and

F. R. Rickles. 1990.

Biol.

137-143.

16.

Biol. Chem. 250: 6614-6621.

Mosher, D. F. 1975.
Blood 96: 3772-3778.
Sahni, A., and C. W. Francis. 2(100.

17.

Sahni, A., T. Odrljin, and C. VV. Francis. 1998.

15.

42: 53-64.

Armstrong. P. B.,
R. T. Aimes, and

J. P..

Bull. 178:

In.wt

./.

Quigley,

7903-7906.

./.

J.

Biol.

Chem.

273: 7554-7559.

14.721.
7.

Proc. Nail. Acail.

1-129.

5.

Gordon. 1979.

.1.

29.267.

1068.

and

4350-4354.

Melchior. R.,

2000.

1.

76:

Chem. 270: 13.496-13.502.

10.

Literature Cited

H.. T. Staehelin,

Harlow. E.. and I). Lane, eds. 1988. Antibodies, a Laboratory ManCold Spring Harbor Laboratory. Cold Spring Harbor. NY.

ual.

18.

Ritchie, H.. L. C. Lawrie. P. \V.

N. A. Booth. 2000.

./.

Biol.

Crombie. M.

VV.

Mosesson, and

Chem. 275: 24.915-24.920.

Biol. Bull. 205: 203-204. (October 2003)

2003 Mamie Biological Laborators

Reference:

Imprisonment

Death-Row

in a

Cell:

The Fates of Microbes Entrapped

Victoria Isakova
'

'

fibrillar

blood clot

'

and Peter

Hunter College of the City University of New York,

is

University of California, Davis,

an extracellular matrix established

at

of damage to the walls of the blood-vascular system. In

humans, the clot is a polymer of the protein, fibrin. In the horsesites

shoe crab. Limulus polyphemus. the clot is a meshwork of fibrillar

The blood clot functions to
polymers of the protein, coagulin
(

seal the

wound

).

to staunch bleeding, operates as a transient extra-

wound-healing epithelial and

and serves as a barrier to entry of microbes

process

wound. This study concerns

of Limulus.
teria

We

investigated the extent of immobilization of bac-

by the coagulin blood clot and the viability of the

clot-

entrapped bacteria.

Plasma was collected from adult horseshoe crabs under

sterile,

lipopolysaccharide-free conditions by cardiac puncture.

Blood

cells

were removed immediately

after collection

and the plasma

was

sterile-filtered through a filter with a pore size of 0.22 /j.m

(Corning. Inc.. Cat # 4307691. The marine bacterium \'ihri<> a/i;inolyticus was grown in liquid culture on Marine Broth 2216

growing populations were established by culroom temperature. Blood clots were established by

(Difco). Log-phase
ture for 12 h at

Corresponding author: pbarmstrong@ucdavis.edu

result

is

form

that

polymerizes into the fibrillar

numbers of bacterial cells

the entrapment of

suspension. V. uli;iii<>l\ticus shows rapid flagellar-driven

swimming locomotion. Bacteria entrapped in the coagulin clot are
In

immotile. and are held so tightly as to lack even thermal (Brownian) motion. When bacteria were killed to eliminate swimming

more than 909r of

motility.

the

killed bacteria in

showed thermal (Brownian) motion:

meshed in the clot were absolutely

suspension

in contrast, all bacteria en-

stationary, without thermal

Entrapped bacteria survive and proliferate in clots maintained in

seawater. which is isotonic to Limulus blood. In a typical

trial.

87%

of the bacterial clusters enmeshed

in the clot

contained

immediately after capture, but 54% of the bacterial

clusters contained two or more cells by 4 h of incubation.
only one

The

immobilization of bacteria by the clot and the killing of clotentrapped bacteria by components of the plasma operating in
synergy with the clot.

artificial

Under these conditions, the blood

coagulogen and the proteases that

meshwork of fibers of the coagulin clot. The fate of the

entrapped bacteria was investigated by direct light microscopic
observation. The two parameters of greatest interest were the

Corp.. Deerfield. IL) in a 35-mm plastic

#
dish
Cat
(Falcon.
35-1008). Bacteria suspended in O.I
petri

NaCl (Baxter Healthcare

NY

in the

motion.

to release

into coagulin. the

it

drop of Limulus blood collected by

cardiac puncture under sterile conditions in 2 ml of sterile 3%
plating the cells contained in

York,

CA

cells degranulate

clot (2).

into the interior of the animal via the

MA

New

to the culture surface of the dish.

connective tissue

the characterization of this last function for the coagulin blood clot

Limulus Blood Clot

sucrose. 3% NaCl. equivalent to the bacteria contained in 250 /nl

of the original culture medium, were introduced into the culture
dish before the blood cells were added to facilitate direct presentation of bacteria to the blood cells while the latter were attaching

cellular matrix for the migration of

cells,

in the

1 ' '*

B.

Armstrong
Marine Biological Laboratory, Woods Hole,
3

The

1 2

cell

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

204

"->

t^^.f *A^

1&&N
^4"
Figure

\.

Proliferation

and killing of Vibrio

individual bacteria entrapped in the clot (A.

(C).

Proliferation
bacteria,

is

h) survive

and

proliferate to establish

clot.

same microscopic
= 2 h) when
t
when the preparation

Sequential views of the

clumps of two or more

cells (B,

field

shmving

the clot with

is exposed to
in bacteriologic culture medium. Bacteria entrapped in the clot are efficiently killed
marine broth culture medium, the clot-entrapped bacteria show as dense phase-bright bodies when viewed by phase contrast
Clot-entrapped bacteria become nonrefractile ghost cells when incubated for 4 h in plasma (D).

contained bacteria

is

incubated

plasma. After 4 h incubation

microscopy

alginolyticus entrapped in the Limulus blood

is

rapid

in

if

the

clot,

with

its

transferred to bacterial culture

cargo of entrapped
(Fig. 1A, B).

medium

involved in killing. The cytotoxic agent of plasma appears to be a

protein, because heat-treated plasma 100 C, 0.5 h) lost bacterio(

After 4 h of incubation of the clot with entrapped bacteria in

lytic

cells.

medium, 84% of bacterial clusters contained two or more

However, bacteria were killed when the preparation of clot
with entrapped bacteria was transferred to sterile Limulus plasma.

molecules are not necessary for cytotoxicity.

In summary, we have defined two important contributions of the

Killing was monitored by loss of retractility of the bacterial cells

under phase -contrast microscope examination (Plan 100/1.3 NA

coagulin blood clot to immunity

culture

The non-refractile cells ("ghost cells") are

be dead or seriously damaged. Loss of bacterial
refractility shows a lag period of 1.5 h, after which killing is rapid.
objective) (Fig. 1C, D).

presumed

to

Dialyzed plasma, which lacked all components

smaller than 10-12 kDa, was fully active, indicating that small
activity.

in Linutliis.

The

clot

immobilizes

microbes, which presumably impedes their dissemination throughout the animal after gaining access via a wound. Plasma also shows

an immobilizing action by its inhibition of rlagellar swimming

motility. Neither clot nor plasma alone kill the bacteria but the two

appeared normal and strongly retractile at

of the cells had become transparent at 2 h. We

synergize to effect the cytotoxic destruction of clot-entrapped

did not observe bacterial proliferation in the plasma-treated clot

even after 8 h of incubation; whereas, as noted above, the seawa-

This research was supported by a grant

26771 from the
National Science Foundation (PBA) and a fellowship from the
Howard Hughes Medical Institute Undergraduate Science Educa-

In a typical trial, cells

1

.5 h,

but about

80%

ter-treated clot did support bacterial growth.

25%

The LD50 was

at

dilution of plasma. Interestingly, plasma-based cytotoxicity

was dependent on entrapment

bacterial cells diluted into

These

in the

coagulin clot because free

plasma remained phase-dense and pre-

did lose the capacity for flagellargenerated motility, but showed the same capacity for proliferation
when transferred to nutrient agar as did cells treated under equiv-

sumably

alive.

microbes.

MCB

Program in Biology, grant 52002679 (VI). We thank Ms.

Alice Child, Mr. Joseph Lee, and Drs. William Cohen and Norman
Wainwright for help with the research.
tion

cells

Literature Cited

alent conditions in plain seawater.

Hemocyanin-free plasma produced by ultracentrifugation

8 h) retained the bacteriolytic activity of whole
(285,000 X

1.

plasma; and purified hemocyanin was not bacteriolylic at 40 mg/

ml, its concentraiion in plasma, indicating that hemocyanin is not

Iwanaga,

S.,

T. Miyata, F. Tokunaga, and T. Muta. 1992.

Thromb.

Res. 68: 1-32.

t-,

Armstrong,
15-24.

P. B.,

and

F. R.

Rickles. 1982.

Ev/>

Cell Res.

140:

CELL BIOLOGY

205

Reference: Biol. Hull. 205: 205-206. (October 2003)

2003 Marine Biological Lahoratorv

<D

A Liposome-Permeating

Activity

From

the Surface of the Carapace of the

American Horseshoe Crab,

Limulus polyphemus
1'

and Peter B. Armstrong '*

John M. Harrington
1
Marine Biological Laboratory; Woods Hole, MA
2

University of California, Davis,

Colonization by sessile fouling organisms (epibionts) is the

fate for solid surfaces in the marine environment. The

usual

'

CA

cein (CF), a self-quenching fluorescent dye.

The

resulting mul-

were made unilamellar through

tilamellar liposomes

five suc-

upon reaching maturity and

lives several years as an adult in an

cessive freeze-thaw cycles. Untrapped CF was removed from

the liposome suspension by passage over a Sephadex G-50

epibiont-rich milieu, yet

typically maintains a cuticle that

column. The release of

American horseshoe

largely free of

crab. Limulus polyphemus, ceases molting

it

and fauna. Indeed,

flora

is

in the

is

CF from

monitored as an indicator of

the interior of liposomes

was

have investi-

permeation. Assays
liposomes in 20
Tris, pH 7.2. Fluorescence intensity was observed with a Photon
technologies fluorometer. Excitation and emission wavelengths
were 460 nm and 550 nm respectively. The slit widths were

gated the antibiological properties of a potential anti-fouling system of Limulus, a viscous secretion of a system of dermal glands
that discharge their product onto the surface of the carapace (2).

adjusted such that the Raman scatter peak of distilled, deionized

water at 397 nm gave an intensity of approximately 350.000
counts/s when excited at 350 nm. Release of entrapped CF from

We

liposomes was seen as an increase

interest of the

macroscopic
animal to maintain

its

it

carapace free from such

organisms, as colonization of the cuticle by green and blue-green

algae can be fatal 1 ). The mechanisms by which Limulus main(

We

tains a clean carapace are not well understood.

propose

In the past

we have

lytic activity

dermal exudate (DE), contributes

integrity, of the cuticle.

that this substance,

to maintaining the cleanliness,

identified

present in

DE

and thus the

and

partially characterized a

(3, 4). It

was shown

hemo-

that the presence

of macromolecular osmolites in the hemolysis assay medium,

dextran-8 and to a lesser extent dextran-4, prevented lysis of the
red blood cells. This effect

macromolecular osmolites

attributed to the ability of these

is

to establish an

osmotic balance between

the interior and exterior of the cell so that

from

the cell occurs, protecting the cell

no

DE-induced

effect of dextrans suggests that

net water flow into

cytolysis.

The

lysis

inhibitory

results

from

hydrophilic pore formation in the lipid bilayer of the red blood cell
rather than from a detergent-like disruption of lipid packing or

from phospholipase
tions of

system

DE

in

activity.

To

investigate the potential interac-

directly with lipid bilayers,

which

we have

utilized a

inside liposomes; an increase in the fluorescence of the

marker

for

model

a self-quenching fluorescent dye has been trapped

membrane permeation. Here we

dye

is

report the ability of an

DE to permeabilize liposomes. The

agent or agents responsible for these hemolytic and liposomepermeating activities may function as deterrents against potential
acid-precipitable constituent of

colonizers of the Limulus cuticle.

The secretion of

DE

can be stimulated by housing Limulus in

stressful conditions such as a cage stocked with decaying fish
material. Dermal exudate was collected by scraping the dorsal
carapace.

NaN 3

-20 C or with 0.02%

Liposomes were prepared
phosphatidylcholine from Sigma. Lyoph-

The exudate was stored

at

to prevent bacterial growth.

with soybean type II

ilized lipid was dissolved

in

chloroform and dried under a

nitrogen stream to a thin film. The film was placed under

vacuum overnight to remove any traces of solvent. The lipids

were hydrated

in

20

mM Tris

pH

7.4. 100

mM carboxyfluores-

Corresponding author: pbarmstrong@ucdavis.edu

were performed with

unit of activity

is

sponding

mM

in

fluorescence intensity. A
an initial increase in

arbitrarily defined as

5%

fluorescent intensity of
to

lipid bilayer

a 1:1000 dilution of

100%

per minute. Intensity values corre-

were obtained with the addition of

lysis

Triton X-100.

Crude dermal exudate showed potent liposome-permeating acCaCl 2 markedly

1:100 dilution. Addition of 10

mM

tivity at a

reduced

activity, as did acidic

pH. The membrane-permeating

DE was retained by a 10-kDa cutoff filter (Amicon P-10) and was precipitated by
HC1, suggesting that the
responsible agent is a large molecule. When, however, the material
that precipitated during the acid extraction was resuspended in an
activity of crude

equivalent volume of 20

mM Tris,
was

pH

7.2, a 71-fold increase in

seen. Presumably, acid treat-

liposome-permeating
ment removes an inhibitor. The activity of the resuspended material was, like that of crude DE, inactive at low pH. The active agent
activity

present in the acid-induced precipitate also failed to pass through

a 10.000 molecular weight cutoff filter. Addition of NaCl or CaCU

attenuated the activity in a concentration-dependant fashion. This

suggests that the lytic agent might initially bind the lipid bilayer

through ionic interactions.

The secretion is most

whose ducts open onto

likely the product

of transdermal glands

the surface of the carapace (2. 5).

The

DE may

provide for a matrix that slows the diffusion

of cytolytic effector molecules into the surrounding aqueous environment. Direct cytolytic attack is a method of defense from
viscosity of

used by

many animal

documenting

the diversity

potential pathogens that

nous

literature exists

is

phyla.

volumi-

and mechanisms

of proteins and peptides capable of permeabilizing lipid bilayers

(6).

We

propose

may employ

that the ancient arthropod

just such a

method

Limulus polyphemus

for maintaining a

carapace free

from colonizing organisms. We have, in past publications, documented the ability of DE to lyse red blood cells and presented
evidence for a mechanism of pore formation

in the lipid bilayer

of

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

206
the target cell.

Here we present further evidence for direct interith lipid bilayers in such a manner
nt in Dl
a\

Literature Cited

action of a lytic
as to

are

make them

componen

of the car,"
of

thi

.>

.
,

We

lytic activities

1.

u.Iensive system that operates at the surface

2.

.1

-able.

propose that these

We

MCB-26771 from

DE

David

for donation of striped bass heads for the stimulation of

production by caged horseshoe crabs.

21-115

Biitl.

Bull. 173: 430.

In Chelicerale

Harrison and R. R. Foelix, eds. Wiley-Liss,

New

Anhropoda,

York.

Harrington,
274-275.

J.

M., and P. B. Armstrong. 1999.

Biol.

Bull.

197:

Harrington,
189-190.

J.

M.. and P. B. Armstrong. 2000.

Bi,,l.

Bull.

199:

5.

Stagner,

van't Hof, W., E. C.

thank Mr. Louis Kerr for im-

portant assistance with use of the fluorometer and Dr.

Gadsby

the

W.

Pp.

3.

cuticle.

This research was supported by Grant No.

and G. A. Lewbart. 1987.

Fahrenbach, W. H. 1999.
F.

Hindus to maintain the cleanliness and integrity

National Science Foundation.

Liebovitz, L.,

J.

I.,

ongen. 2001.

and

R.

J.

Biol.

Redmond.

Veerman,

1975.

Mar. Fish. Rev. 37:

E. J. Helmhorst.

Chem. 382: 597-619.

1-19.

and A. V. Amer-

NEUROBIOLOGY AND BEHAVIOR

207

Reference: Bio/. Bull. 205: 207-208. (October 2003)

2003 Marine Biological Laboratory

Development and Characterization of a Self-Referencing Glutamate-Selective Micro-biosensor

2
Daniel J. Bogorff' Mark A. Messerli', Robert P. Malchow ami Peter J. S. Smith
'
Marine Biological Laboratory', Woods Hole. MA
1

Glutamate

is

University' of Illinois at Chicago, Chicago. IL

the primary excitatory neurotransmitter in the

CNS

ot

and metabotropic receptors

Glutamate, along with other amino acids, has also been identified as
an osmoticum used to regulate the water potential across the plasma
vertebrates, activating both ionotropic

membrane of animal

cells (2).

The molecular mechanisms

regulating

the release and uptake of glutamate are consequently of considerable

The

physiological interest.

release of glutamate from single cells can

be detected with outside-out patches of glutamate receptors pulled

from nerve cells (3), but these sensors lack consistency, are technically arduous to construct,

and lose

sensitivity

over time. Fluorescent

detection methods have also been used, but are also difficult to

and quantitate

4).

Our

specific

in these other techniques,

micro-biosensor

that

and uptake by single

will

aim

is

make

to avoid the difficulties inherent

by developing a noninvasive. real-time

quantitatively measure glutamate release

cells.

High-sensitivity, real-time detection of glutamate can be achieved

using electrochemical detection of H-,O 2

a byproduct of the enzy-

matic conversion of L-glutamate to a-ketoglutarate catalyzed by glutamate oxidase (5). Glutamate o.xidase itself has approximately 61-

and 325-fold

over aspartate and glutamine

(5). H^O 2 can be directly

selectivity for glutamate

when immobilized on macroelectrodes

detected with a platinum wire electrode polarized to +0.6

(6).

method can be problematic due to (a) electrical drift of

electrode response, which reduces the useful sensitivity and reli-

However,
the

this

ability of the measurement, and (b) oxidation of other compounds

released by cells, most notably ascorbate. Electrical drift can be

eliminated
this

if

the electrode

mode, measurements

is

used in a self-referencing format

are

made

glutamate influx or efflux, and then

at a

second location a

the difference in the readings at the

away;
measurement of the

flux.

Electrical drift

is

process, provided

more quickly than

the rate of electrical drift:

also be subtracted.

set distance

two locations

is

the

subtracted out by this

generated by the electrode occurs

that the signal

itself will

(7). In

alternately, near the source of

if it is

The oxidation of

too slow, the signal

interfering

compounds

can be eliminated by using enzyme-coupled detection of H 2 O 2 at a

lower electrical potential. This is accomplished by coating the elec-

polymer containing horseradish peroxidase (HRP)

and osmium. HRP catalyzes the reduction of H 2 2 to water and is
trode with a redox

itself

reduced by osmium(II), which

electrode then donates electrons to

um! II) and.

(8.

9).

Our

in the process,

goal

in

is

converted to osmium! III). The

osmium! Ill), regenerating osmi-

producing a measurable electrical current

work is to determine if such a

the present

hydrogel-based glutamate-selective electrode could be miniaturized

for use in a self-referencing mode.

(BAS, W. Lafayette. IN) and allowed to dry for 10 min. Electrodes

were then dip-coated in 50 units/ml glutamate oxidase (Sigma) and
allowed to dry for 10-30 min. Glutamate electrodes were polaror

ized to either

physiological saline.
trodes

+100 mV against a Ag/AgCl reference in

The average response of platinum-based elec-

was 0.45 pA

0.

pA/ftM of glutamate, whereas gold-

based electrodes produced a weaker signal of 0.21

pA/juAl
0.01 pA/p.M. Figure 1A
and carbon gave the weakest signal. 0.1
shows the electrical current detected from a platinum gel electrode;
it was
placed 20 jum from a source pipette containing 25 /uM
glutamate and moved alternately to a position 50 /urn away. The
initial period of oscillation was 0.1 Hz, and the electrode com-

pleted

translation to each location in 1.5

its

s.

At the arrow, the

period of oscillation of the electrode was slowed to approximately

30 s. The record clearly shows that the electrical current induced

by glutamate had reached more than 90% of its maximal response

within the 10-s time frame of oscillation. While this sensor could
be used in a static configuration to detect glutamate, the electrical
in the electrode, which can limit detection at low

drift inherent

concentrations of glutamate, can also be plainly seen as the slow

IB shows the

rise in the baseline current (Fig. 1A). Figure

ential responses obtained

self-referencing

was

electrode

mode

initially

when

the electrode

differ-

was employed

in a

reduce the impact of this drift. The

placed 20 jim from a 25 /iM glutamate
to

source pipette, and differential recordings were made by subtracting responses obtained at a point 50 /im distant; the rate of
oscillation

was

0.1

Hz. In this condition, a steady differential

650 fA could be detected. The electrode

signal of approximately

was then progressively moved

to positions

more

distant

from

the

glutamate source, and differential recordings were obtained in the

same fashion. The decline in the differential signal as a function of
distance

is

apparent. Note also the clear, steady, small signal of

approximately 50 fA that can be detected with the electrode 100

ju.m

away from

the source pipette

a signal that

is

significantly

smaller than the electrical drift and noise depicted in the raw-

recordings presented in Figure 1A.

Our work demonstrates that glutamate-selective

electrodes

based on a redox polymer hydrogel system can be miniaturized

sufficiently to permit detection of glutamate.

It

also

shows

that the

response time of these electrodes is short enough that they can be

used in a self-referencing mode, which is useful for enhancing the
signal-to-noise ratio for measuring slowly changing glutamate
The sensitivity of these electrodes is suitable for mea-

gradients.

oxygen

surements of glutamate release from living cells (5, 1 ).

This work was supported by grants from the National Center for

sensors (10). except that 8-^.m carbon fiber, 12-p.m gold wire, and
10- and 25-p.m platinum wire were used at the reactive surface.

Research Resources (P4I RR01395) and National Science Foundation (009-1240). Special thanks to Robert Lewis, Richard H.

Microelectrodes were fabricated

Electrodes

in

manner

similar to

were dip-coated with Os-gel-HRP redox polymer

Sanaer. and Kasia

Hammar

for their efforts

and assistance.

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

208

-12.8

probe movement

probe movement under

under manual control

autematie-eontrol

-13.6

20

40

60

Time

20 urn from 25

uM

80

100

120

(s)

glutamate source

-800
5

10

15

Time (min)
Figure

moved
used

is

(A) Electrical current from a glutamate-selective micro-biosensor placed 211

1.

alternately to a position
in

50

fjjn distant, first at

a rate o/O.

a self-referencing mode at increasing distances from

Literature Cited
1.

Jahr, C. E., and R. A. Lester. 1992.

6.

Curt: O/nn. Neiirohiol. 2:

7.

270-274.
2.

3.

Pasantes-Morales, H., R. Franco, L. Ochoa, and B. Orda/. 2002.

Copenhagen, D.

and C.

Nature 341: 536-

E. Jahr. 1989.

Si,

and D. R. Copenhagen. 1991.

}.

Neuroxci. Methods

Hu,

10

Y.,

K.

M.

Mitchell, F. N. Albahadily, E. K. Michaelis,

Bruin Res. 659:

17-125.

and G.

S.

Portertield, R. H. Sanger,

Y., L.

and

Microxc. Res. Tech. 46: 398-417.

Gordon, and T. Solomon. 2002.

Ctirr.

Anal.

Separations

119-125.

Land,
Biol.

Wilson. 1994.

M.

Smith, and O. S.

Yigzaw,
19:

Ayoub, G.

K. Hanimar. D.

J. S..

R. Trimarchi.

the electrode

9.

37: 7-14.
5

Smith, P.

J. S.

The electrode was

when

Kulagina. N. V.. L. Shankar, and A. C. Michael. 1999.

Chem. 71: 5093-5100.

539.
4.

pipette.

8.

59-65.

R.,

from a 25 jjM glutamale source

Twig, G., S.-K. Jung, M. A. Messerli, P.

Shirihai. 2001.
Biol. Bull. 201: 261-262.

J.

Neiirocltcin Res. 27:

IJ.IH

H:. then ai 0.01 H:. IB) Recording of differentia/ currents obtained

the source.
I

S. C., D.

M.

Porterheld, and P. J. S. Smith. 1999.

./.

Ev/>.

202: 211-218.

Takahashi, M., B. Billups,

I).

D. Altwell. 1997.

Biol.

./.

E.v/i.

Rossi,

M.

Sarantis,

200: 401-409.

M. Hamann, and

209

NEUROBIOLOGY AND BEHAVIOR

Reference: Bio/. Bull. 205: 209-21 1. (October 2003)
2003 Marine Biological Laboratory

Zinc Modulation of Hemichannel Currents

J.

R. L. Chappell,
'

Zakevicius,

Hunter College and The Graduate Center, CUNY, New York,

2
Universitv of Illinois College of Medicine, Chicago, IL

Connexins are a multigene family of structural proteins comthat link elecprising gap-junctional channels, the aqueous pores
cells in tissues throughout the body. These narrow

coupled
= 16 A) allow the intercellular exchange of ions.
passages (d
second messengers, and other small molecules having a molecular

Data were analyzed

In the course

of forming gap junctions, the conknown as "connexons"

nexins oligomerize into hexameric arrays

or "hemichannels" that assemble in the plasma

membrane

before

NY

ClampFit (Axon) and plotted with software

in

Origin (Microcal Inc., Northampton, MA).

Membrane currents recorded in a sodium-free modified Barth's
in

programs

trically

mass <1 kDa.

in Xenopus Oocytes
2
ami H. Ripps '*

Xenopus oocytes expressing Cx35 are shown

The
1A.
slowly developing outward currents characterFigure
istic of hemichannel activity are seen with depolarizing voltage
solution from

(MB)
in

> +20 mV.

steps

taining

After changing the bath solution to one conzinc, substantially greater hemichannel currents

10 n.M

When

There is now
docking with the connexons of adjacent cells ( 1 ).
abundant evidence that, at this penultimate stage of gap-junction
formation, hemichannels can be activated both chemically and

were

reflect those of fully

electrically (2. 3), that their properties often

The I-V data obtained with an oocyte expressing Cx35

formed gap junctions

and

(4),

that the modulation of

hemichannel

activity may be of physiological significance (5).

of the verLight-induced changes in the chemical environment

tebrate retina have profound effects

and

electrical synapses:

on both neurotransmitter-gated

and these effects can,

that transmit the visual

CNS. One

putative neuro-

aroused a great deal of interest in recent years

zinc, which has been found in the synaptic vesicles of glutamaneurons in brain and retina (6). In the retinas of amphibia

modulator
is

message

to the

in turn, alter the

and signalling pathways

sensitivity, receptive field organization,

that has

tergic

(7). fish (8).

and mammals

which signal second-order

(6),

cells

zinc

is

present in photoreceptors,

by regulating glutamate release

in

of zinc
response to photic stimulation. Although the co-release
with glutamate remains conjectural (9). the effects it exerts on
retinal neurons have not been explored extensively (8, 10); and no
studies have addressed the question of

its

effect

on connexins or

the channels they form.

In the present study, we used the two-electrode voltage-clamp
currents
recording technique to examine the effects of zinc on the

mediated by the connexons formed by the endogenous connexin

(Cx38| of stage V-VI Xenopus oocytes, and those formed by perch
Cx35, a connexin expressed in neurons of the vertebrate retina (11.
72 h
12). To study the behavior of Cx35, cells were tested 48 to

were injected with 46 nl of a mixture of 10 ng/cell Cx35

and 10 ng/cell of an antisense oligonucleotide to Cx38. The
+
in a Na -free medium
recordings were made with the cells bathed
after they

cRNA

Na* -dependent currents that are similar in

time course to the hemichannel currents, but of opposite polarity

to eliminate the large

(13);

were
of

zinc chloride

was added without

elicited with a series of 10-s pulses

-40

mV

to

+60

mV

substitution.

from

(see protocol. Fig.

Responses

a holding potential

IB

inset),

and were

recorded with low resistance electrodes (0.7-1.5 Mfl) connected

to a GeneClamp 500 amplifier (Axon Instruments, Foster City,

CA) and

controlled by protocols generated in

pClamp

8 (Axon).

elicited at these voltages (Fig.

Corresponding author: harrripplS'uic.edu

the bathing

me-

mM

to

for the

in Figure ID.
range of zinc concentrations tested are illustrated
is

evident that relatively low concentrations of zinc (1

^M and

It

10

/xM) produce enhanced current responses at depolarizing voltages

>40 mV, and the effect is reversed when the Zn concentration is

slOO

increased to

juM.

It

should be noted that hemichannel

currents recorded during experimental runs in which the solutions

were delivered in reverse order (i.e.. decreasing zinc concentra-

mM zinc through 1 /iM zinc to Na-free MB) gave rise

same biphasic behavior. Comparable results were obtained
with Cx38 (Fig. IE), where we show, in addition, that the blocking
from

tions

to the

effect of

of

mM zinc could be completely reversed by coapplication

mM histidine,

a zinc chelator.

Hemichannel currents

elicited

+40 mV from all

by depolarizing voltage steps from -40 mV to
to their control current in Na-free
normalized
were
tested
oocytes
MB solution and averaged according to connexin type for each
concentration of zinc applied.

The

results

from 5 Cx35 oocytes

1G) were similar. Currents

(Fig. IF) and 11 Cx38 oocytes (Fig.
and 10 pM zinc were greater than in the control
recorded in
(MB) solution, but decreased when the cells were bathed in 100
1

/LtM

and

mM

zinc.

series returned the

return to

hemichannel

MB

(Fig.

IF) following a zinc

currents to their control values.

Cells injected with the antisense to

Cx38

alone (controls) did not

exhibit significant hemichannel activity (results not shown).

The biphasic effect of zinc on membrane currents is not unique.

As shown
greatly

receptors
rents
zinc.

in

an earlier study

(8),

the addition of 10

enhanced GABA-induced currents mediated by

(GABA A R)

pM zinc
GABA A

of skate bipolar cells; in contrast, the cur-

mM

were markedly reduced when the cells were exposed to 1

Moreover, the zinc enhancement of hemichannel currents
to be insensitive to voltage; currents in
ju,A/ and 10 IJ.M

appears
zinc recorded
greater than

at
in

+40 and +60

MB. a finding

GABA.x R-mediated
suggest that zinc

high affinity

site,

mV

were approximately

1.7 times

consistent with that obtained for

currents of bipolar cells. These observations

interact with connexins at two external

may

with very different affinities for zinc. The

activated at low concentrations of zinc, gives rise

membrane binding
*

IB).

one containing 1
zinc, the hemichannel
currents were suppressed below those recorded in MB (Fig. 1C).

dium was switched

sites,

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

210

B
Na-ftce

MB

10

jjMZinc

mM Zinc

Cx35

04uA
*-60

-20
-40

mV

E
020-,

08
07

18-

Cx35

16-

06

14-

05

12-

04

10-

Ci38
-

Io

Na-Fcee

MB

10 M M Zinc
1
Zinc
1

mM
mM Histidme + mM Zinc
1

*. 008-

006-

02

0041

002-

00
0.00-0

mV

mV

10

MM

100

MM

mM

10

MM

100 (iM

mM

Zinc Concentration

Zinc Concentration

mV

mV

40
to +60
Figure 1. (Al Depolarising voltage steps from
expressing Cx35. With the cell bullied in a sodium-free modified Earth's

in

20

(MB)

mV

increments (inset)

solution, in

elicit

hemichannel currents

which most of the sodium

is

in

a Xenopus oocyte

replaced by choline (Na

reduced

> +20 mV.

<3 mM),

a slowly developing nutward current, attributable to the opening of membrane hemichannels, is evident at voltages
The "Na-free"
MB solution contained I in mM] C,,H I4NOCI (881, KCI [I], NaHCO, [2.41. N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) [15], Ca(NO,),
10.33], CaCl 2 10.41], and MgSOj [0.82]: 10 mg/l gentamvcin was added, and the solution titrated with NaOH to pH 7.6. (Bl With the addition of 10 juM
to

.-/HC"

chloride, the

mM

hemichannel currents are greatly enhanced. (C) Increasing the zinc concentration to I
suppresses the hemichannel currents to levels
in MB. (D) The effects of zinc concentration on the current-voltage relation (corrected for leakage currents) obtained for an oocyte

below those obtained

expressing Cx35. (E) The l-V relation for the endogenous cimnexin (Cx38) displays a similar response to zinc: i.e., a large enhancement of hemicurrents
in III juM zinc and suppression in I
:inc. With the addition of I
histidine, a zinc chelator, the suppressive effect of I
zinc is reversed. (F-G)

mM

mM

mM

Bar graphs show averaged Jala obtained with Cx35 and C.\38 at different concentrations of zinc. In each data set,
a voltage step from the holding potential
40 mV) to +40 mV have been normalized to the value obtained initially
= i for Cx35; with Cx3S, n = 6
= 12 for the higher concentrations).
(n
for I fjM ZH, and n
(

to an

of hemichannel currents, whereas the low

-merit

attinity SH

uires high concentrations of zinc to

produce

its

in

possibility that zinc modulation of

Na-free

MB. Error bars =

SEM

hemichannel activity contrib-

utes to the processing of visual information in the distal retina.

at the Marine Biological LaboraHole, Massachusetts, and supported by grants from

These studies were conducted

inhibitory efiV.

Thepresein
synaptic termin.

the currents recorded in response to

>.

Is

ui

and evidence
i

that zinc

is

located within the

rtehrate photoreceptors (7, 8). raise the

tory.

the

Woods

National Eye Institute (EY-06516 and EY-01792), a

PSC/

NEUROBIOLOGY AND BEHAVIOR

CUNY

hold. T. Sjoerdsma,

grant (65711-0034) to RC. an unrestricted award to the

of Ophthalmology and Visual Sciences from

Senior Research Investiga-

6.

(HR), and an Award of Merit from the

7.

Research to Prevent Blindness,

Award from

Alcon Research

RPB

the

Inc., a

(HR).

9.

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10.

and D. A. Goodenough. 1991.

1.

Musil, L.

2.

DeVries, S. H., and E. A. Schwartz. 1992.

3.

Malchow. R.

Cell Biol.

J.

P..

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Physiol. 445:

H. Qian, and H. Ripps. 1994.

Gen. Physiol.

J.

201-230

L., V.

M. Berthoud, and

E. C. Beyer. 1995.

Biophys.

Kamermans, M.,

5.

I.

Fahrenf'ort. K. Schultz,

II.

Rosenstein, F.

and R. L. Chappell. 2003.

J.,

J.

Neurosci.

Neuro-

Lett.

345:

O'Brien,

J.,

M. R.

Al-Ubaidi, and H. Ripps. 1996.

Mot. Biol. Cell

233-243.

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White, T. W., M. R. Deans, J. O'Brien, M. R. Al-Ubaidi, D. A.

Goodenough, H. Ripps, and R. Bruzzone. 1999. Em: J. Neurosci.

13.

Ripps, H.. H. Qian, and

121: 81-92.

11: 1883-1890.

J.

68: 1796-1803.

Qian. H.. L. Li, R. L. Chappell, and H. Ripps. 1997.

physiol 78: 2402-2412.
Kay, A. R. 2003. J. Xeurosd. 23: 6847-6855.

7:

104: 1039-1055.

Ebihara,

Ugarte, M., and N. N. Osborne. 2001.

Prog. Neurobiol. 64: 219-249.
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81-84.

115:

1357_1374.

4.

Science 292: 1178-

Res. 33: 2611-2616.

Institute

8.

S.,

and R. Weiler. 2001.

1180.

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211

Janssen-Bien-

J.

Zakevicius. 2002.

J.

Neurosci. Melh.

Reference: Biol. Bull. 205: 211-212. (October 2003)

2003 Marine Biological Laboratory

Remove the Telencephalon Has No Residual Effects on Physiological

of
Recordings
Supramedullary/Dorsal Neurons of the Gunner, Tautogolabrm adspersus
S. J. Zottoli, O. T. Burton, J. A. Chambers, R. Eseh, L. M. Gutierrez, and M. M. Kron

Transient Use of Tricaine to

MA

Williams College, Williamstown.

When

used as a general anesthetic, tricaine significantly alters

parameters recorded from supramedullary/dorsal

physiological

water adjusted to pH 8. When respiration ceased, the fish were

transferred to an operating chamber where tricaine (100 mg/1) in

was

recirculated through the

cells

chilled seawater adjusted to

alent recordings

mouth and over the gills. Ice packs were placed in the operating
chamber on either side of the fish. The skull was removed to
expose the telencephalic hemispheres, and these structures were

of the cunner. Tautogolabrus adspersus. Specifically, tricaine

reduces spike height, increases the current needed to elicit an
action potential, and blocks afferent input
In contrast, equiv).
(

this

from

locally anesthetized fish are not altered in

way. However, although the use of local anesthetic

is

a better

alternative to tricaine general anesthesia for physiological record-

ings

( 1

),

there are limitations. Local anesthetics have a limited

lifetime, are difficult to reapply while recording,

and can enter the

bloodstream.

As in other vertebrates, the telencephalic hemispheres of fish are

considered to be the "highest" brain centers. For example, the
telencephalon of goldfish has been implicated in spatial and avoidance learning

(e.g.. 2, 3).

The removal of

transient tricaine anesthesia

many

is

used

laboratories (e.g.. 4. 5).

conducted

to

in lieu

the central nervous system.

We

of general anesthesia in

However, no

determine whether tricaine

fects physiological parameters of

the telencephalon under

studies have been

after its

removal

neurons whose somata

lie

af-

within

have therefore studied whether

either the transient use of tricaine or telencephalon removal have

any residual effects on resting potential, spike height, and current

needed to elicit a spike.

removed. The

mg/kg)
ter

fish

were injected with tubocurarine chloride

with anesthetic was replaced with anesthetic-free, chilled sea-

was exposed. In the second

condition no anesthetic was used. Fish were injected with tubocuwater. Finally, the rostral spinal cord

rarine chloride and placed in an operating chamber. Their telen-

cephalic hemispheres and the rostral spinal cord were exposed. In

seven experiments, the telencephalon was stimulated to determine

whether any input

to the dorsal cells

initial

dorsal cell

hemispheres.

Comparison

hemispheres, or were not anesthetized.

ences

tricaine (ethyl-w-aminobenzoate;

300 mg/1. Sigma-Aldrich)

in sea-

MO

potentials

16) in amplitude could be evoked by 4.5

anesthetized with tricaine during the removal of the telencephalic

initially anesthetized in

5-20

43.4 ^m (mean
The recordings were all made within 78
SD; n = 26) of the surface of the brain.
After tricaine was used transiently to remove the telencephalic
102.1
9.4 mV (mean + SD, n =
action

nA) was

were

KCl-filled,

tions.

mV,

condition, the fish

were made from somata of supramedullary/

neurons in both anesthetic and anesthetic-free condi-

resistance)

1.2 cm (mean
operculum were recorded from cunner, 10.5
SD: n = 22) in body length. The fish were either transiently

first

could be elicited.

Single microelectrode recordings (3

recordings were started 68

the

(0.1

block neuromuscular transmission, and then the seawa-

to

Responses of supramedullary/dorsal cells to depolarizing current pulses and to electrical stimulation of the skin on the right

In

pH

2.9

nA

anesthetic-free experiments neither the spike height

n=

-74.1

current (initial

55 min after removal of

(

tricaine). In

103.3

12.5

11) nor the current needed to evoke the spike (2.4

significantly different
Test).

in resting

5.8

potentials (PSPs)

>

In addition, there

membrane

mV;

(P

1.7

0.05; Bonferroni's Multiple

were no significant

differ-

potential (transient use of tricaine

anesthetic free

= -72

7.1

mV). Post-synaptic

were readily evoked by stimulation of the skin of

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

212

hemispheres were stimulated again at roughly the same location

and the same voltages. The response to stimulation
persisted.
These results indicate that activation was through current spread to

adjacent structures such as the trigeminal nerve, which

contain processes of dorsal cells.

General anesthetics, such as tricaine, are

currents, and their effects are reversible

known

dium

(6).

is

to

The

known

to

reduce so-

transient use

of tricaine and the removal of the telencephalic hemispheres

in this

study appear to have had no residual effect on spike height, on the

current needed to elicit an action potential, or on the ability to elicit

PSPs from

the supramedullary/dorsal cells.

Although Rose (7) provides a compelling argument that it is

implausible for fish to experience pain, implausible is not conclu-

sive. Nociceptors have been identified in the trout (8, 9).

Endogenous opioid peptides (10) and opioid receptors in fish (e.g., 11,
12) may serve an anti-nociceptive function as in mammals (11). If
is no telencephalic influence on the
system being studied,
then the transient use of tricaine followed by removal of the

there

telencephalon serves as a reasonable precaution against the possibility that fish

experience pain.
This work was supported in part by Howard Hughes Medical
Institute and Essel Foundation grants to Williams College. All

MBL

experimental procedures were approved by the

IACUC.

Literature Cited
Comparison of PSPs evoked hy electrical stimulation of the
skin in supramedullary/dorsal cells. (A, B, C> A calibration
pulse of 80 in \
2 ms is present at the beginning of each
recording. Cells fired action
Figure

1.

'.

2.

potentials in response to a short intracellular depolarising pulse in one of

the ni'o superimposed traces. This pulse was
followed bv electrical stimiilution

the

of the skin of the right

opeiriili/in (to

nm superimposed traces this stimulus

are designated with arrowheads).

tricaine

was used

transiently to

provide a baseline,

was not given: stimulus

190-199.
3.

one of

4.

from an unanesthetized

operculum

in

(1; Fig.

6.

in

at

dorsal cells.

To

C. H., and D. Bodznick. 1996.

Duman,

J.

Rohregger, M., and N. Dieringer. 2002.

Frazier, D. T.,

and T. Narahashi. 1975.

only the high voltages (80-100 V) activated the

test whether this activation was the result of direct

or indirect telencephalic input to the

supramedullary/dorsal cells
and not current spread to adjacent nervous tissue, in three exper-

iments the telencephalic hemispheres were removed and then

replaced in their original positions. The disconnected telencephalic

D. 2002.

7.

Rose,

8.

Sneddon. L. V. 2003.

9.

Sneddon. L.

II.,

R. Soc. Loiul.

fi

1).

In anesthetic-free experiments, stimulation of the

telencephalic

hemispheres

Brain

Phvsiol.

Comp.

179:

J.

Neurophvs. 87: 385-

Eur.

./.

Pharmacol. 33:

313-317.

both conditions. This was not the case

animals under general anesthesia

Salas. 2002.

398.

rise to

electrical stimulation of the right operciilitm.

the right

and C.

797-807.

an action potential. (C) Recordings from

a fish under general tricaine anesthesia. No PSPs could be evoked to

p\h.

Portavella, M., J. P. Vargas, B. Torres,

Res. Bull. 57: 397-399.

artifacts

A) Recordings from a fish in which

remove the telencephalon. A PSP (arrow)

gives rise to an action potential. IB I Recordings

A PSP farrow) gives

in

J. Zottoli, C. E. Adams, S. M. Dineen, S.

Guo, and A. J. Pascal. 2002. Binl. Bull. 203: 188-189.
Overmier. J. B., and M. R. Papini. 1986. Behav. Neiirosci. 100:

Arnolds, D. E. W., S.

Fevrier, U.

10.

A/,./.

Darlison,

M.

Sliihmer. H.

Acud.
1

2.

Sci.

Rev. Fish. Sci. 10: 1-38.

Brain Res. 972: 44-52.

V. A. Braithaite,

and M.

J.

Gentle. 2003.

Proc.

270: 1115-1121.

Gonzalez-Nunez,
2003.

11

J.

V., R.

Gonzalez-Sarmiento, and R. E. Rodriguez.

Bruin Rc\. 114: 31-39.

G., F. R. Greten, R. J. Harvey, H-J.

Z \viers, K.

Kreienkamp, T.

Lederis, and D. Richter. 1997.

Proc. Natl.

94: 8214-8219.

Rodriguez, R. E., A. Barrallo. F. Garcia-Malvar,

R. Gonzalez-Sarmiento, and J. R. Traynor. 2000.
2X8: 207-210.

I.

J.

McFadyen,

Neiirosci. Lett.

213

NEUROB1OLOGY AND BEHAVIOR

Reference: Biol Bull. 205: 213-214. (October 2003)
2003 Marine Biological Laboratory

Zinc Chelation Enhances the Sensitivity of the

S.

Redenti

ami

R.

ERG
L

b-Wave

in

Dark-Adapted Skate Retina

Chappc'Il'"*

CUNY, New York, NY

CUNY, New York, NY

'The Graduate Center.

-Hunter College,

Wu

decade ago,

et at. reported

evidence of a dense band of

of the
region of the photoreceptor terminals
salamander retina 1 ). They speculated that zinc may play a neurorole in the outer retina, including possible feedback onto
ionic

zinc

the

in

modulatory

as well as feedphotoreceptors to down-regulate transmitter release,

a
cells.
order
onto
second
forward
high ionic zinc
Subsequently,

was

concentration

photoreceptors

and Osbome

identified in a similar region near the base of the

in the all-rod retina

(3)

of the skate

(2). In addition,

Ugarte
dense band of ionic

that a

have reported recently

zinc, located in the photoreceptor region of the light-adapted

is redistributed under dark-adapted conditions.

Zinc has been

number of retinal

known

rat retina,

to affect the response of receptors

on

cell types to various neurotransmitters (4). In the

skate, zinc regulates both

GABA (2) and glutamate (5) receptors of

isolated cells. Furthermore, the zinc chelator histidine (an

amino

acid endogenous in the retina, where it may play various roles in

cell metabolism and disease (6, 7, 8. 9, 10)) has been shown to

enhance the

the skate (5)

membrane

and zebrafish

currents recorded postsynaptically from horizontal cells

in the skate retinal slice

during voltage-clamp

The

the electroretinogram (ERG) of

can also increase the
Histidine
(11).

b-wave of

size of the

effects of histidine support the notion that

preparation (12).

endogenous zinc may

be playing a role in the physiological response of the retina to light.

Recent studies of the effects of histidine on the zebrafish ERG
have demonstrated an increase in sensitivity of its mixed rod-cone
retina in the presence of a zinc chelator (11). Here, we report
studies of the effect of histidine on the retina of the skate. Raja

erinacea. indicating that application of this zinc chelator enhances

the sensitivity of the b-wave of the ERG in an all-rod retina.

Skates were obtained through the Marine Resources Center of

the Marine Biological Laboratory (Woods Hole. MA). The ani-

one hour prior to an

were enucleated
the
euthanasia,
After
eyes
approved
experiment.
and dissected under dim red light. The anterior portion of the eye,
mals were allowed to dark-adapt for

including cornea and lens,

at least

was removed; and

eyecup preparation was used for

ERG

the remaining retinal

recordings. Eyecups were

reference electrode
placed into a chamber over a silver chloride
within a Faraday cage.

connected

to

The

active silver chloride electrode

superfusion solutions

in

the

eyecup

was

a glass

via

solution
superfused (-0.5 ml/min) with skate-modified Ringer's
(2) alone, or to which 200 fj.M picrotoxin (to block GABAergic

100 /i/W histidine plus

known

found that responses

to be zinc-sensitive (2)),

and then

200 p,M picrotoxin had been added.

in

picrotoxin did not increase after the

Corresponding author: rchappellCs'gc. cuny.edu

ERG responses

in histidine

could continue to increase

up to 30 min. Therefore, the preparation was kept in the dark

for 30 min in Ringer, at least 10 min in picrotoxin. and at least 30
for

min

We

had

first

10

recording an intensity-

in histidine plus picrotoxin prior to

response

series.

One-second

flashes

were used

ERG

to elicit

re-

insponses from which b-wave amplitudes were measured. The

was 260 /nW/cnr.
tensity of the unattenuated beam (Log 1=0)
Individual ERG responses recorded from one skate eyecup prepa-

ration in response to stimulation at an intensity of

shown

Log

= -3.0

are

1A. Note that in addition to increased b-wave

in

Figure
amplitude observed when picrotoxin, or histidine plus picrotoxin,
were applied, an increased a-wave as well as a more prominent OFF

component were often observed,

as described previously

by Chappell

and Rosenstein (13) using picrotoxin alone.

Similar data at all intensities were obtained from five preparations. Intensity-response data

from one such preparation

are plot-

A V max

under normal Ringer conditions for each

a Naka-Rushton
eye was determined by obtaining the best fit of
curve (14) to the intensity-response data obtained in the control
ted in Figure IB.

Ringer solution. The value of

V max

obtained was used to normalize

and plotting the results, which are

shown in Figure 1C and ID. Figure 1C is a bar graph of the
= -4,
I
averaged data from five preparations at the intensity Log

the data before averaging

it

an intensity near the half amplitude intensity (a) for the intensitycurves plotted with the
response curve obtained in Ringer. The
data in Figure D are the best fit of the data in
1

intensity-response

to
Ringer, picrotoxin, and histidine plus picrotoxin. respectively,
the Naka-Rushton relation, using the least-squares approximation
in Oriain.

Using

this

approach,

we could

obtain the value of the

the half amplitude (a) of the Nakaintensity corresponding to

as
the
well
Rushton curves, as
mjx for each of these curves. The

values of

Vm

lx

obtained increased from 0.95

in

Ringer, to 1.09 in

100 ;uM histidine plus 200 \j.M

u
shifted from a Log I of -4.3 to
of
values
while
the
picrotoxin,
-4.52 to -5.13. respectively.
The shift of a toward dimmer intensities represents an increase

200

nM

picrotoxin. and

in sensitivity

1.4 in

of 0.8 log units (roughly a factor of 6)

of histidine. Such an increase in sensitivity

is

in the

presence

consistent with the

in which they suggest

feedback model proposed by Wu et til.
2 +
that Zn
may feed back onto photoreceptor terminals to reduce
and thereby reduce vesicular transmitter release from
Ca'*
(

containing Ringer/agar. ERG responses to increasing

intensities of illumination were recorded while the preparation was

capillary

receptors in the retina

min, but that

).

entry

the photoreceptor terminals.

It

would

also be consistent with a

reduced inhibition of glutamate receptors on second-order retinal

neurons (5), or possibly even an action of zinc on hemichannels
(15).

since hemichannels have been suggested as a

means of

onto cones in the carp retina, where

feedback from
the hemichannel blocker cambenoxolone has been shown to alter
horizontal cells

feedback-mediated responses (16). Whatever mechanism

is

in-

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

214

80-

40 M V

nM

200

200

.1

1'n

Picrotoxin

nM

Histidine

+200 niM Picrotoxin

L^_

rutcmn

roti.

I'll

(JV1

60-

100 jiM Hislidine

i-

Ringer

- 200
- 100

>3.

MM

40-

20-

Ringer
0Log

-3

-5

Log

20-,
Ringer
1.2

Log

1.8-

-4

200nM
00

Histidine

+200 (iM

1.0

Ringer

Control

04-

.2

Picrotoxin

nM

Picrotoxin

Pic;

Picrotoxin

5 02

1.

Figure

The

c/iir

chelntor histidine increases sensitivity of the skate electroretinogram (ERG) b-wave response. (A) ERG responses recorded
from
= -3 (Unattenuated beam intensity.
=
in response to a I -x flash of white light at an intensity of
Log I
Log 1

a dark-adapted skate eyeciip preparation

was 260 nW/cnr). The amplitude of the b-wave ("b". upper trace) of the ERG recorded from a dark-adapted skate eyecup preparation increased when
/J.M histidine (in the presence of 200 u.M picrotoxin to block :inc-sensitive GABA receptors} was added to the superfusate. A small increase in the

0,

100

"a ". upper trace) was sometimes seen as well. IB) Intensity-response data from one e\ecup preparation in Ringer. 200 juM
picrotoxin. and 100
p.M histidine plus 200 fj.M picrotoxin. C) Nonna/i-ed data at Log I = -4 from 5 preparations. (D) Intensity-response data, averaged and plotted (mean
SEM). with curves representing the best fit to the Naka-Rushton equation fur each condition. The Log I of the half-amplitude intensity (a) of these curves
determined in Ringer. 200 fjM picrotoxin, and in 100 /xM histidine plus 200 p.M picrotoxin were
4.3,
4.5. and
5.7. respectiveh: Thus, in addition
a-wtive

<

to

50%

increase

in

V,,,,

a for

the histidine intensity-response curve irm shifted

C..S'

lot;

units to the

left,

representing a 6-fold increase in sensitivity in

the presence of histidine.

volved. the evidence that removal of extracellular zinc by chelation

alters the sensitivity of the physiological response to light in an all-rod
retina suggests that zinc

may be

8.

Yasunuira,

playing an important role as a

neuromodulator of rod afferent pathways in the vertebrate

Supported by PSC/CUNY grant 6571 1-0034.

9.

M., X. Qiao,
Res. 33: 261 l-2hl<v
S.

,1.

L. Noebels,

and \.

L.

Yang. 1993.

11

Qian, H., L. Li, R. I,. Chappell, and H. Ripps. 1997.

/>/ivs;,./. 78: 2402-2412.

3.

Ugarte, M., and N. N. Oshorne. 1999. Ev/>. Eye Res. 69: 459-461.
Pro K NeuroNol. 64: 214-249.
Ugarte, M.. and N. N. Osborne. 2(H)1.
Rosenstein, F. J., and R. L. Chappell. 2003.
,\'cnrosci. Lett. 345:

5.

Med.

4:

S.,

A. Ruiz,

S. L. Bernstein,

Touchman.
Vis. 8:

Rrdenti,

J.

Hu. D. Bok, and R. R. Rando. 2001.

489: 14-18.

(.',.

M. K. Wyatt,

R. N. Fariss, A. Behal.

Bnuffard, D. Smith, and K. Peterson.

20(t2.

205-220.

and R.

S.,

L.

Chappell. 2002.

Hiol.

Hull.

203:

200-

12
13.

Chappell, R. L., and S. Redenti. 2001. Hiol.

Chappell. R. L., and F. J. Rosenstein. 1996.

/lull.
./

201: 265-267.

Gen.

Pliysiol. 107:

535-544.
14

Naka, K. L, and W. A. H. Rushton. 1966.

J.

Pliysiol.

(Loud.) 185:

587-599.

Jia,i, Y., V. C. Vu, F. Buchholz, S. O'Connell, S. J. Rhodes, C.

Bio/.

Mi;.

202.

Neiiro-

S4.

Camltloro. V. R. Xia, A.
7

J.

6.

Mol.

Vision

Lett.

Wistow, G.,
J. VV.

4.

Mondal, M.

FEBS
10.

Wu,

Nishikawa, D.

S.

C. Flannery, and M. M. La Vail. 1998.

J.

467-971.

retina.

Literature Cited
I

CHIT. Eye Res. 16: 600-604.

Lewin, A. S., K. A. Drenser, \V. \V. Hauswirth.

ft,

Kusakari.

J.

Lusis,

and M.

(;. J.

271: 10.723-10.730.
..

S.

Nishikawa.

S.

15.

Id.

Ishiguro.

and M. Tamai. 1997.

Chappell. R.
205: 2IW-21

Rosenfeld. 1996.

L., J.

Zakevicius, and H. Ripps. 2003.

Hiol.

Hull.

Kumermans, M.,
T. Sjoerdsma.

I.

Fahrenfort, K. Schultz,

and R.

\\eiler. 2001.

II.

Janssen-Bienhold,
178-1 180.

Science 292:

NEUROBIOLOGY AND BEHAVIOR

215

Reference: Biol. Bull. 205: 215-216. (October 2003)

2003 Marine Biological Laboratory
>

Caged Calcium in Skate Horizontal Cells Using Fine Optical Fibers

2
Katherine Haminar, Richard Sanger~, Peter J. S. Smith and Robert P. Malchow

Intracellular Release of

Anthony

J.

Molina

A.

1
,

University of Illinois at Chicago, Chicago, IL

MA

Marine Biological Laboratory; Woods Hole,

direct input

were then incubated with this solution for 30 min at 14 C,

washed twice with skate Ringer's solution, and allowed to stand

the formation of the inhibitory surround portion of the classic

cleave off the

Horizontal cells are second order retinal neurons that receive

from photoreceptors and are involved in establishing a

number of key features of visual perception. These cells mediate

cells

for a

minimum

of

h.

During

this time,

endogenous esterases

AM

center-surround receptive fields of retinal neurons

). The centersurround receptive fields are important for enhancing the contrast

portion of the dye and the caged calcium

compound, thereby trapping them inside the cell. Because NPEGTA enters the cell unbound to calcium, it is important to

of visual objects and are also involved in color perception. The

molecular mechanisms by which horizontal cells send lateral in-

pre-expose the preparation to a brief rise in intracellular calcium. Application of 100 /aM glutamate for 20 s permits cal-

under

cium entry and loading of NP-EGTA compounds trapped within

the cell. The cells were then thoroughly washed with fresh

hibitory signals to photoreceptors and bipolar cells are

still

debate, but protons released from horizontal cells have been hy-

pothesized to alter the flow of visual information within the outer

retina (2). Indeed, small

changes

in extracellular

can dramat-

pH

ically alter neural signals within the retina, in part because photoreceptor calcium channels are highly sensitive to protons. When
protons bind to photoreceptor calcium channels, the voltage acti-

vation range of the channels shifts to

more depolarized

and the overall conductance of the

cell to

which

calcium

is

significantly reduces neurotransmitter release (3).

vious work has

shown

potentials

reduced,

Our

pre-

that glutamate, the neurotransmitter re-

leased by photoreceptors onto horizontal cells, modulates the flux

of hydrogen ions from skate retinal horizontal cells

(4).

Glutamate-

induced changes in H* flux depend on the presence of extracellular calcium and likely reflect the activation of plasma membrane
calcium/H* ATPases. These transporters extrude intracellular cal-

cium

in

exchange for extracellular hydrogen

concentration of protons

at the extracellular

ions, decreasing the

face of the horizontal

cells (5).

We

would

know whether

changes in calcium cause

localized alterations in proton flux from horizontal cells. In the
experiments reported here, we sought to develop methods to locally

raise

like to

intracellular

local

calcium levels

in

horizontal cells.

We

use of small optical fibers that can apply focal

explored
ultraviolet stimuli to cells loaded with the caged calcium buffer
the

NP-EGTA.

This

compound

ing site that releases

its

contains a UV-sensitive calcium bind-

bound calcium upon absorption of

ultra-

violet light (6).

Ringer's solution, and experiments were typically conducted

10-30 min after calcium loading. Imaging of intracellular cal-

cium concentration was performed with

Multi-mode

optical fibers.

ical

aperture

(NA) of

puller.

The

forms of

NP-EGTA

membrane

and the calcium-

dye Oregon Green, prepared as follows. NP-EGTA

(50 /ag) and Oregon Green (50 /ag) were dissolved in
with 2(} (7c pluronic acid and added to 8 ml of skate Ringer's
sensitive

solution,

yielding

Green-AM and

final

p.M

concentrations

NP-EGTA-AM. The

of

/aM

was

fiber

The

inserted into a borosilicate capillary tube

fiber

was then glued

were coupled

to another

an ultraviolet

laser.

Figure

1A shows

in

place with cyanoacrylate glue. Fibers

multi-mode

fiber,

which was coupled

the ultraviolet light output

to

from one such

pulled fiber. In this experiment, the dish was filled with a fluores-

cent solution, and the fluorescence of the solution, caused by

ultraviolet light stimulation,

was examined. The fluorescence was

cone-shaped, with the region of highest intensity localized at the

aperture of the optical fiber. A Narashige hydraulic manipulator
was then used to place the fiber in a dish with the tip positioned 5

/am away from the membrane of a horizontal cell. Figure IB shows

change in Oregon Green fluorescence measured from a single

ultraviolet light

ester

a high coupling

previously pulled to a 5-/am tip diameter and placed so that the tip
of the optical fiber protruded about 5 /am from the tip of the glass.

where they were readily identified due to their distinct morphology and large size, about 150 /am. Cells plated on Falcon
culture dishes were loaded with the cell

which gives them

pulled to a final tip diameter of 1-2 /am with a Sutler P-2000 laser

EGTA-loaded

AM

0.22.

prepared these fibers as follows. First, the coating

and cladding were burned off the fibers with a heated tungsten coil,
leaving a 5-cm portion bare. This portion of the fiber was then

dissociated horizontal cells were placed in primary culture,

35-mm

the

We

efficiency.

the

3001

F-MCB-T. were obtained from

Newport Corp.; they have a core diameter of 100 /am, a cladding

diameter of 100 /am, a coating diameter of 140 /am, and a numer-

Horizontal cells were isolated from skate retinas by enzymatic dissociation, as described by Malchow et al. (7). The

permeable

a Zeiss Attofluor im-

aging system.

horizontal cell upon the photolytic release of calcium from

in

cells.

from

fluorescence

When

the cell

is

stimulated for 5

NPwith

the pulled optical fiber, a significant increase

detected, indicative of a rise in intracellular

is

calcium concentration. The stimulus was then repeated two more

times, which induced calcium increases that were smaller with

DMSO

each subsequent stimulation. In control experiments, with cells

loaded only with Oregon Green, application of ultraviolet stimuli

Oregon

were about

cultured horizontal

led to increases in

85%

measured fluorescence. Note

smaller

NP-EGTA. Moreover,

(;;

2) than those

that these increases

on

cells containing

the control fluorescence disappeared

imme-

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

216

was removed. In contrast,

NP-EGTA-loaded cells persisted for
stimulus was turned off. Additionally,

diately after the ultraviolet stimulus

in

fluorescence with

changes
many seconds

after the

UV

the size of the fluorescent signal did not decay with subsequent

UV

stimuli.

Our work demonstrates

that local stimulation of

cium trapped within horizontal

cal-

caged

cells

by ultraviolet light delivered by small optic fibers can be used to increase intracellular
levels of calcium in isolated horizontal cells. For future studies,
this

approach must be modified

achieve the proper spatial

to

resolution of calcium uncaging. Currently,

various methods to decrease the

UV

we

are examining

of the pulled

light output

optical fiber by adding neutral density filters and optimal align-

ment of

We

the laser source.

hope

to use this technique,

H+

conjunction with self-referencing recordings of

60

in

from

flux

horizontal cells, to examine the spatial dependence of proton

flux

from these

cells.

This work was supported by grants from the National Center

for Research Resources (P4I RR01395), the National Science

Foundation (009-1240), and

40

Grass Foundation

Summer

Fel-

lowship.

Literature Cited
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M. G.

Baylor, D. A.,

Fuortes, and P.

M. O'Bryan.

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Physiol.

214: 265-294.

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100

150

200

2.

Kamermans, M., and H.

Spekreijse. 1999.

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2449-

2468.

seconds
Figure

1.

<A)

A pulled

capiilarv tithe with

ti

optical fiber, which has been inserted into

5-^jLin

opening mul placed

in

tli\h

containing

4.

is

inducing fluorescence of the fluorescein solution. Fluorescence was

detected through a 520-nm emission

from a

retinal horizontal cell

ultraviolet light

Oregon Green fluorescence

loaded with NP-EGTA and stimulated with

from a nearb\ optical

fiber.

Arrows indicate

5-.v

ultraviolet

light pulses delivered via the pulled fiber optic.

V. Merchant, and F.

Mahmud.

Proc. Null. Acad.

1993.

90: 10.081-10,085.

Molina, A.

J. A., P. J. S.

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B 253: 285-289.

Proc. R.

Soc. Loud.

IB)

filter.

S.,

USA

Hull.

fluorescein. Laser-generated ultraviolet light has been coupled to the fiber

that

Barnes,
Sci.

ci

M.

6.

Nerbonne.

7.

Malchow, R.
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Reference: Biol. Bull. 205: 216-218. (October 2003)

2003 Marine Biological Laboratory

Neural Recordings From the Lateral Line in Free-Swimming Toadfish, Opsanus tan
1 2
2
L. M. Palmer
B. A. Giuffrida
and A. F. Mensinger1 2
'

'

University of Minnesota, Dithith,

Fish and aquatic amphibians have evolved a unique lateral line

system that detects local water displacements. The lateral line
functions in surface feeding, rheotaxis, localization of underwater

and subsurface prey detection

The detection of biomust often be accomplished during reafferent stimulation from self-generated motion (swimming, ven-

objects,

).

logically relevant stimuli

tilation).

However,

recording techniqiu

ment

is

due
..

to

the

constraints

of

conventional

the activity of the lateral line during

difficult to quantify.

MN

Marine Biological Laboratory, Woods Hole,

move-

MA

The development of an inductive telemetry system

neural activity to be recorded from free-swimming

tem

utilizes inductive telemetry to transmit biological

from the
are free

fish to

(2) enables

fish.

The

an external recording device. Consequently,

from constraints and

environment. Using

are able to

this technique,

we

behave

sys-

information
fish

in a quasi-natural

investigated the activity of

primary afferent fibers of the anterior lateral line nerve during

self-induced motion
toadfish (28

1.4

in

the oyster toadfish,

SE cm

standard length. 675

Opsumis
46 SE

tun.

Adult

g) of either

Ni-uKomomr.Y AND BEHAVIOR

sex

were

lightly

0.001%

anesthetized in

tricaine

increased firing in response to swimming and ventilatory movements. During forward swimming, the firing rate of the anterior
lateral line increased above spontaneous rates (Fig. 1 A), and neural
activity returned to spontaneous rate within 2 s. There was no

(Sigma) and

lightly paralyzed with pancuronium bromide (Sigma) (600 jug/kg).

microwire electrode was inserted into the dorsal rumus of the

which innervates the supraorbital and

or evoked activity of

anterior lateral line ner\e.

intraorbital lateral line (3).
I

to 3 afferent fibers

Once spontaneous

was obtained,

cylindrical telemetry tag (15

mm

was attached

the electrode

diameter

X 38

mm

to a

length) and

(Fig. IB).

active) fibers to

and three spontaneously active fibers fired in correlation

with the exhalation phase of the ventilation cycle. The other nine
fibers were not modulated by ventilation; however, we were unable
fibers

the aquarium; however, data acquisition

and tag charging are only possible when the fish remains on or near
the stage. Fish were placed on the stage in an experimental tank (1.6

were also stimulated by ventilatory moveThe responses of 15 (6 silent and 9 spontaneously

the ventilatory cycle were monitored. Three silent

lateral line afferents

ments

inductive telemetry signal and actively to produce the magnetic

field necessary to recharge the capacitors on the tag. The fish is

move throughout

<

0.07) between swimming speed and firing rate,

suggesting that firing was saturated at all swimming events. This
contrasted with previous work that indicates lateral line afferent
activity is reduced during vigorous body movement (4). Anterior
correlation (r

mounted externally on the dorsal surface of the fish. The rechargeable telemetry tag was inductively coupled to a bimodal recording
stage (45 cm diameter). The stage acts passively to receive the

free to

217

to

cm

determine whether

this

was due

to the distant location of the

water depth), and allowed to recover from the

for
at least 3 h before experiments were conducted.
surgical procedure
Fish movement was monitored with a digital camera (30

neuromas! from the operculum or to efferent inhibitory activity.

Efferent stimulation has been shown to reduce the activity of

frames/s) and correlated with nerve firing offline (ADInstruments,

efferent inhibition of the lateral line in response to visual octavol-

Chart4: Cambridge Electronic Designs. Spike2). Spontaneous neural activity was recorded in each fiber and correlated with venti-

ateralis stimuli (6).

diameter. 20

lation cycles.

Nerve

firing

was

also recorded

when

the fish

afferent fibers of the lateral line (5). Other studies also illustrated

However,

in this study, all fibers

were activated

by swimming and 40% were activated by ventilatory activity. Thus

reafferent noise does not appear to be inhibited by efferent activity

moved

independently or was provoked into swimming by gently prodding

its caudal fin with a rod. All swimming events consisted of short

at the

swimming bursts that displaced the fish up to a body

forward. Swimming speeds (range: 3.6-15.1 cm/s) were

length

and Montgomery

deter-

contain an adaptive filter capability that cancels inputs consistently

associated with an animal's own movements. Fish are generally

mined by videotape analysis.

The primary afferents of the

primary afferent level. Consequently, self-generated noise is

possibly filtered from the signal in higher order neurons. Bodznick
(7) indicate that the lateral line

mobile animals; however, the

anterior lateral line in the toadfish

medullary nuclei

ability of the lateral line to function

B.
I

200

100
1)

J4

'.

Ofi

.C

80

40

12

10

Speed (cm

14

16

s)

Figure 1. Neural activit\ of the anterior lateral lint' in Op.sanus tail in response to reafferent self-generated motion. lAI The response of two fibers to
short-range swimming events. The dashed black line rcpic\i-nt\ llic spontaneous aclivitv of each fiber. The solid black line represents a linear regression
through

all

data point* (upper: y

= -5.5x +

158.4,

r =

0.07; lower: y

= -0.9\ +

5S.3,

r =

0.02). IB)

Neural trace of a

silent fiber (i.e.. fiber with

The fiber innervated a neiiroinast located on the infraorbital canal

indicate the period of maximum operculum abduction during the ventilation cycle I measured by video umihsist

no spontaneous

activity) that

was responsive

to the ventilation cycle.

line.

Arrow,

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

218
K

during self movement

preliminary

lateral line cli:r

mechanos,

wo

This

.igely

Literature Cited

activity of the anterior

..rated motion, indicating that perhaps

not inhibited in afferent activity.
supported by the Minnesota Sea Grant College
c

unknown. This study reports

janced nerve

rin.lii

1.

is

Program supported by the NOAA Office of Sea Grant. United

States Department of Commerce, under grant No. NOAA-NA16RG1040 (AFM). The U.S. Government is authorized to reproduce
and distribute reprints for government purposes, not withstanding
any copyright notation that may appear hereon. This paper is
journal reprint No. JR-494 of the Minnesota Sea Grant College
Program. This work was also supported by Sigma Xi Grants in Aid
of Research (LMP).

Montgomery,

2.

Mensinger, A.
194-195.

3.

De Rosa,

4.

Russell,

F.,

I.

Coombs, and M. Halstead.

S.

J.,

1995.

Rev. Fish.

399-416.

Bio/. Fish. 5:

and M. Deffenhaugh. 1998.

F.,

and M.
and

J.,

5.

Flock, A., and

f>.

Tricas, T. C.,

I.

L. Fine. 1988.

J. Russell.

S.

M.

1976.

Bull.

195:

Brain Belnn: Evoi 31: 3 2-3

1

B. L. Roberts. 1974.

and

Biol.

J.

/.

Camp.

7.

Physiol. 94: 7-15.

Physiol. 257: 45-62.

Highstein. 1991.

Com/'. Physiol. A. 169:

J.

25-37.
7.

Montgomery,

J. C.,

and D. Bodznick. 1994.

Neurusci.

Lett.

174:

145-148.

Reference: Biol. Bull. 205: 218-219. (October 2003)

2003 Marine Biological Laboratory

Memory
M. Child

H.

Reconsolidation in Hermissenda

M. Kirjrian

ami D. L. Alkoif
Marine Biological Laboratory, Woods Hole, MA 02543
Blanchette Rockefeller Neiiroscience Institute, Rock\'ille, MD 20850
F.

*.

Epstein', A.

T.

Remembering seems a

priori to be

composed of

three

main

processes: acquisition (or input), storage (or consolidation), and

retrieval (or recall)

the
<iL

In this study the acquisition of

memory

is

form of Pavlovian conditioning elaborated by Lederhendler

er

).

The consolidation of
studied by Epstein et

known

in

it

this

al. (3).

humans

which memory reaches

ences) no longer inhibit

conditioning in Hennissenda was

Consolidation of memories has been

least

at

experimentally

demonstrated

in

since Muller and Pilzecker (4)

1400.

recall

It is

defined as the process by

which interventions

a state in

(or interfer-

of what was presented to be remem-

bered. This does not mean, as initially inferred (5. 6). that the

memory cannot

installed

be altered;

respect to the said interferences.

it

is

just that

it

is

stable with

Examples of such interventions

include the four treatments (chemicals, sensory input) used in this

study.

Reconsolidation (a

much newer

finding)

is

defined as the ending

of the interference-insensitive state

brought about by the recall of
what was memorized. That is. after a memory has been consoliif

it

recalled

is

it

inhibitors of
affect

mRNA

becomes

it

sensitive to agents such as

synthesis and protein synthesis

that did not

just before the recall. Since the consolidated

whether the

fact

memory

is

become important to determine

of reconsolidation has more to do with temporary

regenerated after recall,

it

will

degradation or with a weakening of the retrieval process.

Consolidation and reconsolidation are being studied in many
organisms from molluscs to humans (5. 6. 7, 8). The existence of

memory

reconsolidation

may allow more

aspects ot consolidation.

We

study of reconsolidation

in

proven

Our

(3, 9,

10).

training

conditioning

procedure with Hennissenda was a Pavlovian

regime

Animals were placed

(2).

in

transparent

acrylic plastic trays (with 16 fluid-filled lanes about 0.9

(2).

dated,

vertebrate nervous system, especially in the area of learning and

memory

to !x

detailed study of

many proposed

and deep and 15

for the

cm wide

incubator for 10 min

(the conditioned stimulus, CS), paired, after a 2-s delay, with a 4-s

vigorous orbital shaking of the tray containing the animals (the

unconditioned stimulus, US). The animals respond to the shaking

by contracting lengthwise. This combination of the two stimuli is

a paired training event (TE) and is
repeated at 1-min

called

intervals.

Testing of recall was done by four presentations of the CS at

1-min intervals, and was recorded using a videocassette recorder

whose input was

camera placed below the transwas measured on the video monitor.

registered by a

parent trays. Animal length

The measure used was

the percentage by

changed between light-on and

age changes
mean.

we compute

which the animal's length

light-off times.

mean and

From

those percent-

the standard error of the

We

used four interventions after training to probe the acquisiand consolidation of memory in Hennissenda. The first intervention is the sensory input used by Epstein et ai (3). The other
three are drugs dissolved in seawater. pH 8. and administered by
tion

replacement of the seawater bathing the animal.

1.

Sensory input: the tray containing the animals

hand 180 around its long axis and.

rotated by

Hennissenda. This nudibranch has

model

long) in a closed

ot dark adaptation before training or

testing. Training consists of
exposing the animals to a bright white light (650-700 lux) for 6 s

therefore undertook a preliminary

a high-connectivity

cm

quickly rotated back to

more complex

its

is

quickly

after 5

s.

original position. This produces a

vestibular input (in addition to the

TE) and

is

termed

sensory block (SB).

*

Correspondirg author: fchildlS'nibl.edu

2.

Anisomycin

(AND

interferes with protein synthesis by in-

NEUROBIOLOGY AND BEHAVIOR

In the

219

experiment, specimens of Hermissenda were given

first

TEs and tested at 4 h; this is the time at which our previous

studies have shown recall to become insensitive to all four inhib-

nine

itors we are using, thereby showing that the stored memory is

consolidated long-term memory (CLTM). After testing to verify
arrival at CLTM, ANI was added either immediately, 10 min later,

min

or 30

later.

The data

cin immediately or 10

in

min

Figure 1A

loss of recall; adding the

recall has

We

anisomycin
been re-established.

24

40

32

48

56

TIME OF TESTING

72

64

80

96

88

adding anisomy-

after

30 min showed

that

wiped out recall if added

whereas waiting for 30 min before

IB). All three inhibitors

immediately after testing

16

that

next studied whether the other three inhibitors had similar

effects (Fig.

show

after testing for recall resulted in the

at

h,

using the inhibitors revealed that recall had been re-established, as

had been found for ANI (Fig. 1A).

Thus, these results demonstrate the existence of reconsolidation

(h)

Hermissenda. Reconsolidation was triggered by testing for

4 h and then probing with each of the four inhibitors.

in

recall after

The reconsolidation was found to be reached by about 30 min after

recall: thus it is a much more rapid process than the initial acquisition and consolidation phase, which has been shown to take

40

nearly 4

h.

The sensory block (SB) was previously shown by Epstein

L-i

(3) to

(LTM) and

el nl.

memory (STM) as well as long-term

long-term memory (CLTM). The fact that

short-term

inhibit

consolidated

works on both consolidation and reconsolidation raises again the

question of what steps it is affecting. Molecular and physiological
it

studies will be needed to get at this question.

and reconsolidation deserve study in

for learning and schooling. If the
be
ramifications
may
consolidation study by Miiller and Pilzecker (3) is correct,

Finally, both consolidation

that there

TIME OF TESTING

first

(h)

teaching additional novel material within 6 to 10

of inhibitors on the behavior tnut memory recall (us
measured hv cluinge in body length) of Hermissenda. (A) Effects of ant M>m\cin (ANI). Animals were trained with nine training events ana then
1.

Figure

Effects

tested for recall at 4 h post-training.

For three groups of animals. ANI was

added

either immediatel\ after (curve ani4), or at 10 inin (curve ani4 + 10)

or 30

inin after the testing at

relested at

8.

24. 48.

4 h

Iciin'e

ani4+30). Animals were then

weakens

These aspects need serious

study by researchers in the field of education.

Literature Cited

actinom\cin-D (act-D). anisomycin (anil the tripeptide CAM

and the sensory block (sh). Animals were treated as

1.

in A.

with the inhibitors added at the times indicated (on the

graph) after the initial testing at 4

recall at 5. 6. or 8 h post-training.

h.

2.

Lederhendler,

3.

The animals were tested again for

4.

5.

translation-inhibition

was administered

interferes with

inhibiting transcription.

It

transcription-inhibition

ROD
tween
at

at

ju,g/ml.

The

was not checked.

Actinomycin-D (Act-D)

The
4.

It

mRNA

was administered
was

synthesis by

at

O.I

jug/ml.

adhesion molecules (CAMs).

It

was administered

Opin.

S.

Gart, and D. L. Alkon. 1986.

Epstein, D. A., H. T. Epstein, F. M. Child, A.

Biol. Bull. 199: 182-183.
D. L. Alkon. 2000.
Miiller,

Neurohiol.

11:

J.

Neurosci. 6:

G.

E.,

and A. Pilzeckvr. 1900.

Z.

M.

Kuzirian, and

Psychol Erganzungsband

1-300.

Riccio, D. C., E.

2000.

Sara, S.

7.

Nader, K. 2003.

8.

Pedreira,

W. Moody, and

P.

M.

Millin. 2002.

Integr.

J.

M.

E.,

Learn.

Mem.

7:

73-84.

Trend', Neurosci. 26: 65-72.

and H. M. Maldonado. 2003.

Neuron 38: 863-

869.
9.

cell

1. 1.,

6.

bond formation be-

10 /xg/ml. The bond formation inhibition was not checked.

Curr.

Physio/. Behav. Sci. 37: 245-253.

not checked.

(arginyl-glycyl-aspartate) inhibits

Lattal. 2001.

1325-1331.

1:

hihiting translation.

M.

Abel, T., and K.

180-187.

inhibitor (rgd).

3.

memory, then new information might prevent recon-

the

recall or retention of the

were not treated with ANI. Data points below the

and positive recall of the training:

positive numbers indicate body elongation as in normal locomotion with
recall inhibition and blocked memory. (B) Combined results of the four

described

having

solidation and thus should be avoided.

line indicate foot contraction

inhibitors.

after

and for some. 72 h for

training. Controls (con)

-em

min

well
taught a primary point that is meant to be remembered could
weaken retention of that point. Similarly, if recalling something

Kuzirian. A. M., H. T. Epstein, D. Buck, F. M. Child, T. Nelson,

J. Neurocytal. 30: 993-1008.
L. Alkon. 2001.

and D.
10

Epstein, H. T., F.
Netirobiol. Learn.

M. Child, A. M. Kuzirian, and D.

Mem. 79: 127-131.

L. Alkon. 2003.

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

220

Reference: Biol. Bull. 205: ^0-222. (October 2003)

2003 Marine Biologu.il
Moratory

Hermissenda
ing Alone, Not the Tripeptide RGD, Modulates Calexcitin in
4
3
2
1
1
1 -*
F. M. Child, H. T. Epstein, M. E. Motta, C. E. Oldenburg. and D. L Alkon
Kuzirian,
ii

A.

Marine Biological Laboratory, Woods Hole. MA

'
University' of New Hampshire, Durham. NH
?
Carnegie Mellon University, Pittsburgh, PA
Blanchette Rockefeller Neuroscience Institute, Johns Hopkins University,
1

"

In the

nudibranch mollusc Hermissenda erassieomis. the inten2

sity

citin

of immunostaining for the Ca VGTP-binding

correlates positively with the degree of learning obtained
( 1
)

and the level of memory expressed

3).

protein calex-

When

after

Pavlovian conditioning (2.

Act-D) and

inhibitors of transcription (actinomycin-D;

translation (anisomycin;

ANI)

are applied after training, they affect

the animal's ability to recall the learned behavior. Epstein el

have recently described the time windows for these

a/.

effects.

(4)

They

Rockville,

MD

response. UR). The two stimuli are designated a paired training

event (TE), which was repeated at 1-min intervals for nine repetitions. Recall of training was tested by four presentations of the

CS

alone

at

1-min intervals. Behavioral recall was assessed by cal-

beculating the percentage by which the animal's length changed

tween light-on and light-off. Untrained (naive) animals show no recall

and typically lengthen as a normal response to light, as do animals
given

light

and agitation

in

an unpaired or random fashion

10).

noted that there are two phases of sensitivity to the inhibitors: an

early phase, immediately after training (0-13 nun), and a later

Experimental conditions shown by Epstein et ill. (5) to demonstrate the RGD-sensitive transition between LTM and CLTM were

phase (70-160 min for Act-D, and 70-220 min for ANI). Subse-

repeated to test for possible correlations between different times of

memory also has

quently. Epstein
two distinct biological configurations: long-term memory (LTM).
which lasts about 24 h. and consolidated long-term memory
(CLTM), which persists up to 6 days. The amount of memory
retained by conditioned animals appears to depend, in part, on how
much consolidation has been completed; and the transition from

RGD application after training and changes in calexcitin intensities. RGD was diluted to an effective working dosage of 10 jag/ml

et al. (5)

LTM

to

inhibitor,

CLTM
the

is

reported that

long-term

sensitive to a cell adhesion molecule

tripeptide

arginyl-glycyl-aspartate

(CAM)
One

(RGD).

working hypothesis predicts that increased calexcitin levels are

needed to establish LTM. and that RGD-sensitive CAMs are
subsequently involved in the transition from LTM to CLTM. What

was unknown was

levels

the possible relationship

and the effects of

RGD during

between calexcitin

this critical transition period.

Thus, as part of a continuing study to describe and define the

memory stages demonstrated in Hermissenda (6. 4. 5), we undertook an immunocytochemical study to investigate possible correlations between calexcitin levels and the observed effects of RGD
during the transition from

LTM

to

CLTM.

Hermissenda were purchased from Sea Life Supply (Sand City.

California), and the tripeptide CAM inhibitor, arginyl-glycyl-aspartate (RGD), was obtained from Calbiochem (San Diego, CA).
Animals were acclimated to laboratory conditions for a minimum
of 3 days. The training procedure used was adapted from a Pavlovian conditioning regime developed by Crow and Alkon (7),
elaborated later by Lederhendler, Gart. and Alkon (8), and modified by Kuzirian el ul. (9) and Epstein (10). Before training or

animals were dark-adapted for 10 min in a transparent

acrylic plastic tray with 16 lanes ( 15 cm long and 0.9 cm by 0.9 cm
in cross section) in an
C incubator. In paired training, animals

testing,

were exposed

to a bright,

white light (650-700 lux) for 6

conditioned stimulus, CS) coincident with, after a 2-s delay, 4

(the
s

of

(29 /iA/) with Tris-buffered (20 mA/) natural seawater (NSW-Tris)

(pH 8.0). All solutions were administered post-training, at designated times, by bath application using a training tray with a
modified injection-port cover (4). After training, the RGD-exposed

animals were tested for retention of recall

rapidly

decapitated.

immediately fixed

in

The

4%

central

at

4 and 24

h.

then

nervous systems (CNS) were

paraformaldehyde/NSW-Tris

to preserve,

as accurately as possible, the expressed calexcitin levels.

To discern the temporal effects of training alone on the immunostaining intensities of calexcitin,

CS

and

to test for possible effects

from

were similarly conditioned with 9

TEs in natural seawater. but they were not exposed to RGD or tested
for recall. They were decapitated and fixed at time points similar to
testing with the

those for the

alone, animals

RGD-exposed

animals. Naive, untrained animals were

used as overall experimental controls and were tested after 24 h.

All fixed CNSs were then processed for embedding in polyeth-

ylene glycol-400-distearate and were sectioned (6 jum). Calexcitin

was immunolabeled with a primary polyclonal antibody (25U2;
cloned from squid optic lobe and raised in rabbit: 1:1000 dil) (2,

secondsubsequently, color was developed with a biotinylated

the
with
reacted
and
avidin-bound
microperoxidase
ary antibody,
3);

chromogen 3-amino-9-ethylcarbazole (AEC). Gray-scale intensity

was measured (using NIH Image software) from digital photomicrographs of serial sections of the B-photoreceptors in the eyes of
each animal. Intensity differences between conditions were statisand Student's t tests.
tically analyzed using

ANOVA

The data indicate that the levels of calexcitin remained steady

from 40 to 200 min post-training, whether the animals were
exposed to RGD or not (Fig. ). Levels then fell to a baseline about
240 min after training. ANOVA and pairwise Student's / tests
among and between intensities measured during the first phase
1

vigorous agitation (the unconditioned stimulus, LIS). Animals respond to the agitation by contracting lengthwise (unconditioned

Corresponding uuilmr: akuziria@mbl.edu

(40-200 min post-training) indicated no

between all intensity values (/" = 0.67,

significant differences
tx

<

2.0;

P = >0.7;

NEUROBIOLOGY AND BEHAVIOR

221

Post-Training Treatment Times

Figure
of

Immune/staining intensity levels for calexcitin under riro experimental conditions. First, specimens of Hermissenda were exposed to 10 fig/ml
adhesion molecule inhibitor, at the designated post-training (nine training events) application times through to 240 min

1.

RGD. a

tripeptide. cell

(designated RGD.x (mint: donhlc-hatched columns). At 240 min. the animals were tested, the RGD removed, and the animals transferred in the training
tray to a nih with running setiwater heing siphoned through each lane until they were tested again at 24-h post-training. Second, replicate sets of animals
(solid black columns) were similarly trained but not exposed to RGD or rested, and then decapitated and fixed at time points similar to those for the

RGD-exposed animals. An overall control group (single-hatched column) consisted of naive, untrained animals held under similar conditions until they
were also decapitated and filed. All central nen'ous svstems were processed collectively for immunocytochemistry. Calexcitin initnunostaining intensity was
measured from

digital

NIH Image

photomicrographs using

sofnvare. Results were statistica/lv

compared using

ANOVA

and painvise Student 's

tests (see

text).

= 4-10

eyes measured), whether animals were treated with

RGD and tested at 4 and 24 h. or simply

trained and fixed at similar

time points. The same was true for the second phase (240 min to
24 h post-training). Again, the intensities were statistically identical,

whether animals were tested or

0.07.

ts

<

the overall

2.0:

P = >0.9;

ANOVA

between

differences between the

<0.001).
The principal

first

not. at

240 min or

at

24 h

F =

= 4-10 eyes measured). However.

all

conditions did

show

significant

and second phases (F = 7.49:

P =

results obtained

from

this

study indicate that the

working hypothesis must be redefined to indicate that, to preserve

memory, calexcitin levels must remain elevated through the

RGD

LTM

to

CLTM.

inhibition of this

Also, the previously demonstrated

LTM/CLTM

transition does not appear to

operate by affecting calexcitin levels, but rather, as predicted by

Epstein et til. (5). through the competitive inhibition and functioning of

CAMs.

Calexcitin itself

may

contribute to the

transition through the initiation of early

translation of

The

RGD

inhibitor

is

LTM/CLTM

gene transcription and

CAMs.

calexcitin. Therefore, the behavioral effects related to

sure reported by Epstein et

calexcitin immunostaining intensities observed during this

LTM

intensity occurred

by 90 min post-training, the

(3).

The

in ealexcitin

the

al.

(5)

RGD

were not mediated

expo-

directly

through changes in calexcitin levels. The similarity of intensity

levels in tested and nontested animals also suggests that calexcitin
levels

be insensitive to the immediate perturbation of recall

may

caused solely by

testing: a

phenomenon known

Memory

sociative conditioning regime can be

as reconsolidation

generated by an astesting with the

weakened by

conditioned stimulus (CS) alone and must be reconsolidated to

remain

fixed.

The data indicated

quite clearly that there

were no

differences in calexcitin intensity related to testing alone.

Previous reports

(3.

show

12)

that

calexcitin

is

involved in

LTM.

This was accomplished by exposing Hennissenda to a sensory block (an additional vestibular input) at time
points before LTM is established (at 60 min post-training) (5) and
establishing

afterward.
levels until

study were similar to levels reported previously for

initial study by Kuzirian et al. (3) showed that the rise

Hermissenda with

independent of the expressed levels of

(11) (also described as extinction).

transition of

further demonstrated that the treatment of

CAM

The sensory block suppressed this rise in calexcitin

LTM was fixed. However, the current data indicate that

calexcitin levels remain high through to the period coinciding with

the establishment of

involved in some

earliest time point

Calexcitin

sampled. However, this study indicates that the intensity rise

begins earlier, by 40 min or even sooner. The results of this study

Epstein et
phase, a

was
al.

CLTM

(4. 5),

way with

and thus calexcitin may also be

the consolidation of

LTM

to

CLTM.

also elevated during the time period designated

phenomenon known

by

being an intermediate-term memory

to occur in Hermissenda and Aplysia,

(4) as potentially

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

222

among other animals

PKC, is known
t>

>

U>

(1?

e<,

binding to ryam

lie

Since calexcitin, once activated by

release of internal calcium stores

on the ER, and

;>l"is

early gene aci

recall (15),

sition, storat

is

of which are involved in

all

would be

it

responsible for

memory

logical to

broaden

this

::,mg the effects of transcription and translation

inhibik)'.'

on the immuno-expression levels of calexcitin under

experimental conditions similar to those just described. Indeed,

such studies are underway.

Alkon and Thomas Nelson provided Hennissenda

antibodies for this study. MEM and CEO acknowl-

and calexcitin

Kuzirian, A. M., H. T. Epstein, D. Buck, F. M. Child, T. Nelson,

L. Alkon. 2001.
J. Neurocytol. 30: 993-1008.

and D.
4.

M. Child, A. M. Kuzirian, and

Mem. 79: 127-131.
A. M. Kuzirian, F. M. Child, and

Epstein, H. T., F.

5.

Epstein, H. T.,

Mem.

Neurobiol. Learn.

D. L. Alkon. 2000.

and D.

7.

Crow,

8.

Lederhendler,
sci. 6:

9.

T.,

L. Alkon. 1974.

H.

Kuzirian, A. M., F.

10.

Center, Marine Biological Laboratory.

Epstein, H. T. 1997.

11.

Sara, S.

12.

Nelson, T.
P. A.
J.

Cavallaro, C. L. Yi, D. McPhie, B. G. Schreurs,

Beushausen, G. Ascoli,

Olds, R. Neve, and D. L. Alkon. 1996.

Proc. Natl. Acad. Sci.

Kim,

J.

M.

Kuzirian, and

Science 201: 1239-1241

Gart, and D. L. Alkon. 1986.

J.

Neuro-

M.

J.

2000.

Child, H. T. Epstein, P.

Biol. Bull. 191:

Biol. Bull. 193:

Learn.

Mem.

1:

J. S.

Smith, and

2002.

Biol. Bull.

260-261.
255-257.

73-84.

Borley, K. A., H. T. Epstein, and A.

M. Kuzirian.

203: 197-198.

S.

USA
2.

J., S.

Gusev, A. Favit, O. Zhar,

S.

Child, A.

1325-1331.

C. T. Tamse. 1996.

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M.

Bull 199: 182-183.

Biol.

I. I..

D. L. Alkon. 2003.

(In press).

Epstein, D. A., H. T. Epstein, F.

edge the assistance of Beth Linnon. program coordinator, and the

student internship program sponsored by the Marine Resources

D. L. Alkon. 2003.

Neurobiol. Learn.

acqui-

study b\

Drs. Daniel

by

Crow,

13

T., J. J.

14

M.

Sutton,

93: 13808-13813.

2001.

Kuzirian, A. M., H. T. Epstein, T. J. Nelson, N. S. Rafferty, and

D. L. Alkon. 1998. Biol. Bull. 195: 198-201.

Xue-Bian, and V. Siddiqi. 1999.

J.

Neurophysiol.

82: 495-500.

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A., S. E. Masters,

M. W.

Bagnall, and T. J. Carew.

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Alkon, D.

L., T. J.

Nelson,

W. Zhao, and

S.

Cavallaro. 1998.

Trends Neurosci. 21: 529-537.

Reference: Biol. Bull. 205: 222-223. (October 2003)

2003 Marine Biological Laboratory

Neurochemical Modulation of Behavioral Response to Chemical Stimuli

Anna Savage 1 2 and Jelle Atema 2 '*

in

Homarus americanus

'

Ainherst College, Amherst, MA

Boston University Marine Program, Woods Hole,
1

Serotonin (5-HT) and octopamine have been implicated

ulating the behavioral phenotype of lobsters

aggression

in agonistic interactions

).

by

in reg-

altering levels of

High-dosage injections of

these amines consistently evoke extension (5-HT) and flexion

MA

were injected with serotonin, octopamine, dopamine (Sigma Chemior lobster saline. Each injection of amine was administered in

cals),

random order over 4 consecutive days of testing, and consisted of 0.3

mg of neuromodulator per kg of animal dissolved in ml lobster
1

were made intramuscularly

(octopamine) postures in both lobsters and crayfish. These postures

have been likened probably erroneously to aggressive and sub-

saline. Injections

missive postures, suggesting a specific role for these neuromodulators in social behavior (2). Dopamine injections into freely

(4).

After injection, each lobster was placed in an observation tank

(36

X 46 X

moving

have also induced motor

lobsters

tension of claws, legs, and

However,

nistic posture (3).

lator treatment

tail,

activity, including ex-

originally interpreted as an ago-

the relationship

between neuromodu-

and overall crustacean behavior

is not
simple, with
responses varying with the stimuli presented and the aminergic
manipulations performed (4, 5). Serotonin is known to regulate

feeding behavior

in

other animal species (6), but

lobster feeding behavior

investigated.

signals

As

animals

lobsters are

for food

reaction of H.

and responses

known

to

and social information

americanus

to

its

effects

on

have not yet been

rely heavily on chemical

to odors

(7),

we examined

the

food and social odors after the

been injected with serotonin and two other biological

amines o .union to the lobster central nervous system (CNS).
li.i

Seven adui

ntermolt male lobsters (81-93

mm

segment

into the

first

to the right of the ventral nerve cord, following

abdominal

Peeke

el al.

72 cm) with a constant flow of unfiltered seawater. Each

tank had a plastic, two-entrance shelter in front of the

window and

water circulation system with funnel interconstantly flowing

ruption (7). Food odor and body odor were injected into the funnel,
air-lift

which delivered an irregular flow of tank water into the shelter at a

mean rate of 4.8 ml/s. Lobsters were not fed for the 4-d duration of the
experiment. Tanks were kept dark during observations except for a
25-watt bulb covered in black plastic with a small hole cut through
that

allowed a narrow beam of

light to illuminate the shelter interior.

were prepared by collecting water samples from

one male and one female lobster isolated in 10 1 of aerated standing

Body odor

seawater for

stimuli

at least

h.

Food odor consisted of store-bought clam

minutes after drug injection, lobsters were presented

with 0.5 ml of each stimulus at 5-min intervals, and the resulting

juice. Fifteen

behaviors and their durations were recorded to the nearest second. The

carapace length)
order of stimulus introduction was held constant over the four days of

randomly among the animals. We selected beAtema and Cowan (7) based on frequency and quanti-

injections but varied

*

Corresponding author

itema@bu.edu

haviors from

NEUROBIOLOGY AND BEHAVIOR

223

Serotonin

Octopamine

Dopamine
Saline

50

Time

200

150

100

250

after injection (min)

Figure 1. The effect of injections with 0.3 mg/kg of serotonin, octopamine. dopamine. or saline on the duration of lobster dactyl clasping behavior in
SE shown for 7 lobsters or 6 time points post-injection. The serotonin-affected response was significantly higher,
response to clam juice. Mean duration
and the dopamine-affected response was significantly lower, than the saline control (see te.\t>.

They included

liability.

"locate source," turning toward the glass at

and probing the stimulus inflow tube; "check

entrance." walking over to. and standing still at, one of the two
entrances: and "dactyl clasping," a typical feeding response marked
the front of the shelter

by opening and closing the dactyls of the first two pairs of walking
legs. Stimulus introductions were repeated every 15 min for the first
min post-injection to test for a time
hour, and then again 20 and 2
1

delay in response. The study was conducted blind for six of the eight
animals tested, with the observer unaware of the amine injected from

day

to day.

Lobsters injected with serotonin and presented with clam juice

mean duration of dactyl clasping that was twice that

displayed a

observed

after saline injections. Statistical analysis

was

difference in duration
6.69.

P =

"

significant

The serotonin

(ANOVA

showed

post-hoc

injection,

).

effect

= 120 min
by t
was
also
response
significantly longer than
15 min (t = -2.69, P = .0082). In contrast

saline control; the effect then returned to initial levels

(Fig.

= 60 min
=
response at

The

1 ).

the initial

dopamine induced a shorter duration of clasping in

clam juice at all times examined: the mean response was

in

507c that of

less than

cant

saline.

sponse to

behaviors induced by serotonin cannot be attributed to simple extensor effects. The observed inconsistency of dactyl clasping follow ing

octopamine injection reflects the complex relationship between neuromodulator action and lobster behavior (4). Dopamine-induced depression of response

a novel result and merits further behavioral and

is

neurochemical investigation.
Financial

0097498

site

support

is

awarded

to

statistical analysis.

Literature Cited

3.

not significant

observed

to saline

The

4.

5.

6.

of dactyl clasping after injection

with serotonin and the decrease after injection with dopamine suggest
haviors,

feed

neuromodulators are priming lobsters toward certain beless (dopamine) motivated to

making them more (5-HT) or

when given

the opportunity. This serotonin-induced persistence

Brain Behav. Eml. 46: 72-83.

and E. A. Kravitz. 1995.

R..

M. Harris-Warrick, and

Barthe. J. Y., N. Mons, D. Cattaert,

1989.
Brain Res. 497: 368-373.
Peeke. H. V.

Comp.

S.,

Doernberg,

S.

S. B., S.

Mancilla-Diaz,
S. E.

G.

Blank,

M. H.

E. A. Kravitz. 1980.

M. Geffard, and

Figler,

and

E. S.

F. Clarac.

Chang. 2000.

Phyxiol. 186: 575-582.

Kravitz. 2001.

significant increase in length

that these

Huber,

Livingstone, M. S., R.
Science 208: 76-79.

J.

in

appeared unrelated to treatment condition.

stimuli,

Octopamine-induced clasping in reclam juice varied widely between animals, and the overall

(/
1.254. P
0.21). Dactyl clasping was not
response to body odor. The occurrence of "locate source"
and "check entrance" behaviors, infrequent for all three chemical

was

acknowledged from NSF-REU (OCEBoston University Marine Program). We

thank Sean Sapcariu for overseeing blind injection schedules.

Molly Steinbach for design advice, and Gabi Gerlach for her aid in

.0078).

compared

seroto-

it

clasping requires sequences of rapid flexion and extension: thus the

2.

difference in duration for octopamine injections

However,

appears to act as
a general motivator of lobster behaviors, including both agonistic
encounters and feeding responses. Additionally, prolonged dactyl

statistically signifi-

= -2.68. P =

(t

This difference was

(8).

nin's effects are not limited to aggression. Rather,

to serotonin,

response to

where 5-HT-treated

agonistic encounters,

subordinate lobsters are less willing to retreat

that this
test, t

peaked at 60 min postwhen the duration of clasping was over 5 times that of the
7e

has been demonstrated

J.

J.

I.

Cromarty. R. Heinrich, B.

Comp.

S. Beltz,

and

E. A.

Physinl. 187: 91-103.

M., R. E. Escartin-Perez, V. E. Lopez-Alonso, and

Cruz-Morales. 2002.

Eur. Neitropsychophannacoi.

12:

445-

451.
7.
S.

Atema.
Huber.
1997.

J.,

and D.

F.

Cowan.

1986.

/.

Chem.

Ecol. 12:

2065-2080.

K. Smith, A. Delago. K. Isaksson, and E. A. Kravitz.

Proc. Natl. Acad. Sci. 94: 5939-5942.
R.,

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

224
Reference: Bio/. Bull.
2003 Marine Bu !.-

-225. (October 2003)

2(!-:

.!ory

/i

Recognition in Juvenile Zebrafish (Danio rerio) Based on Olfactory Cues

1 '"
A'. D. Mann', E. R. Tnrnell
J. Ateimr, ami G. Gerlach
1

'

'

Marine Biological Laboratory; Woods Hole, MA

Boston University Marine Program, Woods Hole, MA

Genetic analyses of numerous fish species have shown that

shoals formed by larvae often consist of closely related kin ( 1 ).
Aggregating with kin may be an altruistic trait that evolved
through kin selection

(2).

would increase

Individuals

sive fitness by sharing the benefits of

shoaling
individuals (3). Laboratory experiments on

their inclu-

among

related

recognition of kin

v\.

non-kin groups of Atlantic Salmon (Salnw sular) (4) demonstrated

possible advantages: kin groups had fewer aggressive interactions,
used a greater proportion of "threat" behavior as opposed to

and subordinates especially had improved growth.

The mechanisms by which these kin groups develop and stay
separate from each other are not known. The genes of the major

fighting,

histocompatibility

complex (MHC)

are

odors released into the water via urine

be involved

in

(5).

source of individual

Such pheromones might

olfactory kin recognition.

Here,

we

tested the

Figure 1. Diagram of the choice flume, and a xraplt ofte.il result, (a)
Water inflow area: (b) collimator to hoinogcni-e turbulent flow; (c) barrier-separated channels: (d) area of flume where water columns remain

separated without the harrier

[%

SEM]

(fine

dotted center line): fe) screen to contain

outflow channel. Black bar

test subjects: (/)

in

graph indicates preference

= P < 0.05).

for kin over non-kin hy test subjects (*

hypothesis that zebrafish can recognize kin based on olfactory

cues.

Zebrafish (Danio

rt-rin)

live

in

freshwater streams and rice

the flume

(A or B,

Fig.

the fish

was swimming

on.

The number

paddies
Ganges River of East India, Bangladesh, and Burma.
Although this species is widely used as a model in genetic and
developmental research, little is known about its natural behavior.

of times each animal was recorded on the side with kin stimulus

Zebrafish

(random

in the

hundred eggs at a time, and these

without any parental care. Larvae (G.G.
develop
pers. obs.), and sometimes adults, can be observed in shoals (6. 7),

spawn up

to several

in the substrate

but their genetic relatedness

is

unknown.

We observed wild-type juvenile

zebrafish. aged 6-8 weeks; the

were kept in 2.5-1 aquaria under a day/night cycle of 14/10 h
and fed on a standard diet of brine shrimp nauplii and dry fish food.
fish

Twenty-four hours before an experiment started, 2 separate kin

groups consisting of 12 full siblings each were placed into two 9-1
aquaria with standing water.

From each

kin group, 3 fish were

was expressed
observations

as a percentage of the total

(i.e.,

kin plus non-kin).

number of recorded

score greater than 507r

distribution) indicated a preference for the kin stimulus;

and when the percentages of

all the fish were

compared using a
Wilcoxon matched-pairs signed-ranks (WSR) test, the preference
for kin was significant (WSR = 45.0, P = 0.050)
(Fig. 1). Our
is

study

the

first

to

show

that juvenile zebrafish

prefer their siblings to unrelated conspecifics

can recognize and

based on olfactory

cues.

There are two general categories of kin recognition mechanisms,

both based on learning processes as Tang-Martinez (9) emphasized. The first ("indirect") mechanism is based on
familiarity,

procedure was repeated 3 times for a total of 6 kin groups and X

test animals. Water from each of the two
aquaria was used as the

where individuals behave nepotistically to conspecifics with whom

they grow up. The second ("direct") kin recognition mechanism
allows individuals to identify even unfamiliar kin. Direct recogni-

two

tion

tested in the flume, one at a time, for a total of 6 fish. This

I

stimuli in an olfactory preference

test.

Single individuals of

group were used as test fish and were placed into a choice
flume (20 cm long X 4 cm wide, water level 2.5 cm) that main-

either

tained

two separate water columns (Fig.

was maintained at

unidirectional water flow

ml/min

3.5 mm/s). Periodic

dye

tests

1)

(8).

Uniform and

a constant rate of

showed

that the

40

plate for

he subject to acclimate. Each

periods, during

presented
bility of um

trial

which an

comparison with the phenotype of other individuals. Our

between these recognition mechanisms.

Literature Cited

consisted of four 3-min

which water from kin and non-kin aquaria was

alternate sides of the flume to correct for the

possi>Y-d side bias. Everv 10 s, we recorded which side of

Krause.

J.,

Ruxton,

J.

B
2.

Biol. Sci.

D. J. Hoare, D. Croft, J. Lawrence, A.

Godin. and R. James. 2000.

267: 2011-2017.

G.

J.

Ward, G. D.

Prot: K. Sot: Land.

The Behaviour ofTc/eosr Fishes. Croom Helm,

Pitcher, T. J. 1986.

London.
3.

Hamilton. \V. D. 1964.

4.

Brown, G.

'

Corresponding autho/: ggerlach@mbl.edu

in

two water

thought to be based on 'phenotype matching',

results cannot distinguish

columns remained well separated. Prior to each trial, formulated

fresh water was run through both channels of the flume for 5 in in
to allow

is

individual must learn cues, either from the phenotypes of close

relatives (familial imprinting) (10), or from itself, to form a tem-

225-23

E.,

and

J.

./.

Theoi: Biol. 1: 1-16.

A. Brown. 1993.

Belui\: Ecol. Sociobiol. 33:

NEUROBIOLOGY AND BEHAVIOR

Apanius, V., D. Penn,

5.

Pritchard, V.

h.

P. R. Slev, L. R. RulT.

and W.

Potts. 1997.

Atema,

8.

I..,

J.

Lawrence. R. K. Butlin, and

J.

Krause. 2001.

2002.

Mar. Ecol. Pro K

Tang-Martinez, Z. 2001. Hclmv. Process. 53: 21-40.

Sherman. P. W., H. K. Reeve, and D. W. Pfennig. 1997.

9.

10.

Anim. Belitu: 62: 1085-1088.

Ecology: An Evolutionary Approach,

and N. B. Davies. eds. Blackwell Scientific, Oxford. UK.

Delaney, M., C. Follet, N. Ryan, N. Hanney. J. Lusk-Vablick, and

G. Gerlach. 2002. Biol. Bull. 203: 240-24 1.

7.

M. Kingsford. and G. Gerlach.

J.,

Set: 241: I5I-IM).

Immitnol. 17: 174 -224.

Crit. Rev.

225

(>')-%

in Bi'liuvionil

J.

Pp.

R. Krebs

Reference: Biol. Bull. 205: 2:5-226. (October 2003)

2003 Marine Biological Laboratory

Mate Choice

(Danio rerio) Analyzed With Video-Stimulus Techniques

2
and G. Gerlach'-*
Turnell', K. D. Mann', G. G. Rosenthul

in Zebrafish

E. R.

Marine Biological Laboratory, Woods Hole, MA

Boston University- Marine Program, Woods Hole. MA
1

In

many

a
species, individuals of both sexes have developed

which to
variety of visual signals and behavioral patterns with
broadcast their quality as mating partners 1 ). The complexity of
(

these signals

makes

it

difficult to distinguish those that are

most

important in mate selection. Animated models offer a solution to

problem by allowing for the alteration of single parameters in

this

complex stimulus presented.

the

In this study,

we have

tested the

use of computer animated three-dimensional models to analyze

mate choice criteria in zebrafish; we applied this tool to examine
the roles played by

two

visual characteristics in mate selection.

Sexually mature wild-type zebrafish. aged at least 8 months,

were kept in 9-1 aquaria under a day/night cycle of 14/10 h.
Subjects were tested in a 50 cm X 32 cm tank: a vertical line

drawn on the wall of the tank divided it into two equal sections. A
and B. The tank was placed between two 17-inch Dell computer
monitors on which animated models of swimming zebrafish were
displayed. The models were created using 3D Studio Max 1.0
(Kinetix) on a Dell Optiplex GXPro computer and a Targa 1000

used

in all

[2]

vertical versus horizontal stripes (both with

female

[3] vertical versus horizontal stripes (both with

male

had equal
shape).
amounts of blue coloration. Female-shaped images differed from
male-shaped images only in that their bellies were 10% larger in
side view. Stimulus pairs [1] and [2] were presented to both male

The

vertical

and horizontal

stripe

patterns

and female subjects, while stimulus pair [3] was presented to

female subjects only. In addition, stimulus pair (1] was shown to
females who had spawned on the day of the trial. (The females

Corresponding author: ggerlach@mbl.edu

(WSR)

signed-ranks

The

results

Males did not

and

The

or

Wilcoxon matched-pairs

test.

evaluations are

statistical

shown

in

Figure

1.

between the male and female-shaped

significant preference (19.6%) for the

differentiate

images but showed

horizontal stripe pattern over the vertical stripe pattern when

the images were female-shaped. Female zebrafish preferred a

male-shaped stimulus over a female-shaped stimulus by 20.3%.

However, females that had just spawned eggs on the morning of
the trial did not show a preference for either the male or the
female shape. Females also showed a significant preference
(10.7%) for the horizontal stripe pattern over the vertical stripe

images were male-shaped, but did not differentiate between stripe patterns when the images were femaleshaped.

shape), and

trial.)

were alternated between the monitors to

B) was calculated and compared using

Before each trial, an individual fish was placed in the tank and
allowed to acclimate for 5 min. During each trial, the subject was
simultaneously shown two different animated stimuli. Each trial

stripes).

did not spawn on the day of the

subject spent in proximity to each stimulus (presence on side

pattern

monitors. Three pairs of stimuli were shown to the subjects: [1]

male versus female body shape (both with natural horizontal

trials

stimuli

balance for side effects. During each viewing period, the location
of the fish was recorded every 10 s. The percentage of time the

board for digital/analog conversion of video signals, as described

by Rosenthal (2, 3).

consisted of four 5-min viewing periods separated by 1-min intervals during which black covers were gently slid in front of the

other

two animated

when

the

The preference of females

for the

male-shaped stimulus over the

allows fefemale-shaped stimulus indicates that belly size alone

male zebrafish to distinguish between sexes. The indifference of
the females
interest

in

who hud

recently laid eggs suggests that females'

males correlates with

their reproductive

stage.

The

and females against vertical stripe pattern

significant bias of males
in the opposite-sex animation may result from selection against
mating with heterospecifics.
The failure of males to distinguish between the male- and
female-shaped images has at
the belly of the female image
realistically simulate a

least

may

two possible explanations: (a)

not have been large enough to

fecund female zebrafish: or (b) male ze-

on visual cues than on olfactory cues to select

brafish may
have
shown that male zebrafish are attracted to
studies
Prior
mates.
rely less

pheromones released by females (4-6).

This study shows that video-stimulus techniques can be used to
further study mate choice and visual preferences in zebrafish.

the

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

226

50

SEM) of males (white bars) and females (black bars) for each stimulus image, as measured by time spent in proximity to
Figure 1. Preference (%
each one. [la] Females significantly preferred the male-shaped stimulus (M) over the female-shaped stimulus (F) ( WSR = 27.0, n = 12, P = 0.034); males
showed no preference (WSR = 3.0. n = 15. P = 0.868). [Ib] Females who had spawned the day of the trial also showed no preference behveen images

M and F
(F,

WSR =

FV) (WSR =

17.5, n

37.0. n

75,

/5.

horizontal to the vertical stripe

P =

0.288). [2] Males significantly preferred the horizontal to the vertical stripe pattern when both images were female
P = 0.017); females showed no preference (WSR = 2.0. n = 15. P = 0.923). 13] Females significantly preferred the
= 38.0. n = 16. P = 0.049). * = P < 0.05.
pattern when both images were male (M. MV) (WSR

Literature Cited

Rosenthal, G. G.,

W.

E.

Wagner, and M.

J.

Ryan. 2002.

Aniin.

Bchav. 63: 37-45.

1.

Dugatkin, L. A., and G.

J.

FitzGerald. 1997.

havioral Ecology ofTe/eost Fishes. J.-G.

sity Press,
2.

J.

Pp.

266-291

Environ. Biol. Fishes 56: 307-316.

and

van den Hurk,

2381-2387.

5.

Bloom, H.

Delaney, M., C. Follet, N. Ryan, N. Hanney,

Gerlach. 2002. Bio/. Bull. 203: 240-241.

Be-

Godin, ed. Oxford Univer-

Oxford.

Rosenthal, G. G. 1999.

in

D.,

R.,

J.

G. D. Lambert. 1983.

and A. Perlmutter. 1977.

J.

Can.

./.

Zoo/. 61:

Exp. Zoo/. 199: 215-226.

J.

Lusk-Vablick, and G.

MOLECULAR BIOLOGY. PATHOLOGY, AND MICROBIOLOGY

227

Reference: Biol. Bull. 205: 227-228. (October 2003)

2003 Marine Biological Laboratory

Expressed Sequence Tag Analysis of Genes Expressed

S. B.

Roberts ami F.

in the

IV.

Bay

Scallop, Argopecten irradians

Goetz

MA

Marine Biological Laboratory', Woods Hole.

The bay scallop (Argopecten irradians) is a marine bivalve

found along the eastern United States. As in other pectinids, the
bay scallop has a single, large adductor muscle which acts to open
and close the

This muscle

shell with great force.

feature observed

when

the shell

removed, and

is

both scientists and consumers, as

it

is
is

a prominent

a favorite of

offers an excellent

model

for

understanding muscle physiology and provides a healthy, highprotein food source. Consumer demand has caused the increase in

cloning kit (Stratagene), starting with 5 /ng of

described
).
(

mRNA as previously

To obtain ESTs. the library was mass excised to pBK-CMV

phagemids and plated. From these plates. 454 randomly chosen
cDNA clones were picked and sequenced from the 5' region by
using the dideoxy chain termination method, with Big Dye Terminator (Applied Biosystems) and a vector-specific primer. The
reactions were precipitated and resuspended in Hi-Di

EDTA

Formamide

ABI Prism 3730

aquaculture of bay scallops for direct marketing or to seed local

with

waters.

automated sequencer (Applied Biosystems). Gene identification

analysis was performed using Phred (base-calling) and Cross_

The objective of
factors

the present study

was

to better

understand the

contribute to the growth and development of the

that

scallop adductor muscle.

Because

little

is

known about

these

(Applied Biosystems) and run on an

match (vector removal) software (http://www.phrap.org/); then

EST sequences were compared with those in the NCBI database

processes in scallops, we have started by isolating and identifying

genes from adductor muscle tissue. The results from this work
could help facilitate future research. One example is in aquaculture. where these genes could be used as markers for a selective

(nr) using the

breeding programs. The use of such techniques could result in

increasing the yield of adductor muscle or altering the size and

not used for sequence analysis.

density of the muscle fibers.

distinct

To

identify the factors or genes involved in scallop

structure and function, a

cDNA

muscle

was constructed from bay

scallop adductor muscle, and expressed sequence tags (ESTs) were
sequenced.

ESTs

library

are small pieces of

DNA

sequence (usually 100

800 nucleotides long) generated by sequencing randomly selected cDNA clones from a library. In this paper, the sequences of
to

bay scallop adductor muscle are reported.

To construct the cDNA library, adductor muscle tissue was

genes expressed

in the

dissected from four adult bay scallops obtained from Woods Hole.
Massachusetts. Total
was extracted with Tri-Reagent (Mo-

RNA

lecular Research Center Inc.). and

mRNA

was

mRNA

isolated using the

Isolation System (Promega). The cDNA

Poly-A-Tract
library was constructed using the A Zap Express cDNA/Gigapack

Cell Structure

33%

Blast-X and Blast-N programs (http://www.ncbi.

nlm.nih.gov/BLAST) (2).
Of the 454 cDNA clones, 20 yielded no sequence and an
additional 9 produced sequences of less than 150 bp; these were

The average length of the remainbp. Based on top Blast hits, 137
sequences were observed and 90 of these genes were

ing 425 sequences

was 792

putatively identified; 47 lacked similarity with

could not be identified.

most similar

Of the

latter

47

known genes and

distinct sequences.

38 were

to unidentified

products {e.g., hypothetical protein,

unnamed protein product) and 9 produced no hits in the database.
Only 16 of the distinct sequences had previously been sequenced

from the genus Argopecten and were either myosins. ribosomal

proteins, or mitochondria! sequence. The mitochondrial sequence
appeared 54 times (13%) in the ESTs and does not necessarily
code for a protein; the presence of a poly-A region in the sequence
leads to

Of

its

involved in
the

mRNA.

isolation along with

the genes identified based

cDNA

cell structure (Fig.

library

were

actin

Gene

1 ).

on sequence similarity, 33% are

The two genes most prevalent in

and myosin because they are the

Protein

Expression

3%
Cell Signaling

8%

Immune Related

7%

Binding Proteins

7%

Metabolism

31%
Unclassified

11%
Figure

1.

Percentages of identifiable ESTs sequenced that could he grouped together based on gene function or that could not be

classified.

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

228
major components of
actin (2 forms) occur-

was found 68
as being

425

cDNA

clones,

>.

involved

(Fig.

the

80 times (19%) and myosin (11 forms)

The remaining ESTs could be classified

'

tin

gene/protein expression (3%). immunity

in

(31%), protein binding (7%). or cell signaling

Some identified genes (11%) could not be classified

(7%). met;"

(8%

Of

tissue.

liscle

).

CF197421-CF197787]. The ESTs generated

offer a valuable re-

wide range of disciplines including muscle

and
development, immunity, genetic identifiphysiology, growth
cation, and aquaculture.
source to scientists

in a

Funding for this research was provided by USDA grant #200203633 Program in Growth and Nutrient Utilization.

into any of these groups.

all the ESTs generated from the bay scallop
and
the
cDNA library
putative identifications determined to date
are available on a Bay Scallop EST project website (http://www.

Literature Cited

The sequences of

mbl.edu/goetz/EST.html) as well as

NCBI's GenBank database

Accession numbers

in

[GenBank

(http://www.ncbi.nlm.nih.gov/)

Garczynski,
856-864.

M.

Altschul, S. F.,
1990.

J.

and

A.,

W.

Gish,

F.

W.

W.

Goetz. 1997.

Miller, E.

Binl.

W. Myers, and

Kcpmd. 57:

D.

J.

Lipman.

Mol. Bio/. 215: 403-410.

Reference: Binl. Bull. 205: 228-230. (October 2003)

2003 Marine Biological Laboratory

Scanning Electron Microscopy Investigation of Epizootic Lobster Shell Disease

2 '*
A. C. Hsu' and R. M. Smolowitz
'
Boston University Marine Program, Woods Hole, MA
~
Marine Biological Laboratory, Woods Hole, MA

The American

Homarus americamis,

lobster,

portant fishery for

much

of the

represents an imEngland coast as well as

New

several coastal provinces in Canada. Yet during the past decade.

New England has reported dramatic decreases in the catch value in
this lucrative industry

1 ).

Shell disease

is

the deterioration of the

crustacean exoskeleton by chitinoclastic organisms occurring in

both marine and freshwater environments

(2). In the past

6 years,

and severity of shell disease has markedly increased

(K. Castro, Rhode Island Sea Grant, pers. comm). Predominately
occurring in areas from Buzzard's Bay (Massachusetts) to eastern
the prevalence

Long

Island

Sound (New York),

lobster shell disease

observed
in

in

has been termed epizootic

it

(ELSD). Recently

ELSD

Cape Cod Bay (Massachusetts),

offshore waters of

New

England

studies on lobster shell

(SEM) to observe the progression of lesion

development. For this study, SEM was used to produce threedimensional views of ELSD development from geographically

electron microscopy

England coast for

site

comparisons.
22) and without

During 2002-2003, lobsters with lesions (11

==
14) were collected. The sites sampled were the

lesions (n

4). Rhode
Long Island Sound (n
Buzzards Bay (n = 7). Cape Cod Bay (n = 3),

inshore waters of eastern

Island

13).

and Maine (n = 4), and the offshore waters of

(11

).

Animals were defined

anol on

New

Hampshire

as "healthy" or "infected"

depend-

ing on the presence of noticeable lesions on the cephalothorax.

Corresponding author:

rsmol@mbl.edu

Samples were trimmed,

in early disease

and normal
analyzed.

critical-point-dried,

The

and sput-

surface of cuticle lesions

phases and the deeper interface between lesions

edge of lesions) were compared and

cuticle (leading

To compare

the presence of morphologically distinct

bacteria identified in lesions, images were analyzed using Sigma-

Scan 4.0 (Jandel

Scientific).

Gross examination of carapace pieces showed that the carapaces

of healthy animals showed no degradation, while samples from

bacterial cells. Setal cores

filled

and natural abrasions were consistently

with bacteria embedded

in the cuticle at the lesion

surface

(Fig. IB). Additionally, bacteria were abundant at the leading edge

of the lesions (Fig. 1C). Overall, healthy carapace samples had

substantially fewer bacteria on the carapace surface. These observations were consistent for

The

all

sampling

sites.

role of bacteria in the progression of lesion

was indicated by

bacteria found

the epicuticle (Fig.

embedded

ID). In other areas

on

in

development

shallow

pits

along

the surface, halo-like

holes surrounded several bacterial types that were associated with

shallow erosions. These holes were not observed

at

the

deep

leading edges of the lesions. Bore holes appeared to match the

length/width ratio of associated bacteria and, therefore, were believed to be caused by bacteria secreting chitinase. lipase. or
noted
protease (7). Diatoms, algae, and fungi/actinomycetes were
but bactypically in low abundance within the cuticle filaments,

were consistently the dominant organisms on both the carapace surface and the leading edge of lesions.
teria

'

ice.

ter-coated with gold palladium (6).

healthy carapace revealed minimal bacterial buildup (Fig. 1A). In

contrast, carapace lesions of infected lobsters were covered with

disease have used histology and molecular techniques to define the

organisms involved in lesions (3, 4) no study has used scanning

New

Carapace pieces were collected, fixed in 10% formalin in sterile

seawater (5), and dehydrated in increasing concentrations of eth-

infected animals were severely eroded. Microscopic analysis of

For the present study, carapace lesions from wild-caught specwm-riciiiiiis were examined for the etiological agent

distinct areas along the

americamis

have been

lobsters

).

ELSD. Although previous

Homams

Kittery (Maine), and

imens of H.

responsible for

in

MOLECULAR BIOLOGY. PATHOLOGY. AND MICROBIOLOGY

229

Figure 1. SEM images from infected and healthy lobsters collected from various sampling sites. (A) Healthy setae with minima/ bacterial cells at base
of core {arrow) from a non-diseased Maine lobster. (B) Infected setae with a high abundance of bacteria (arrow) from a diseased New Hampshire lobster.
iCl Bacterial buildup (arrow) in the leading edge of a lesion from an infected Buzzard's Bay lobster. (D) Enzymatic digest by coccoid, rod, and rod linked
bacteria

from an

infected Buzzard's

Bay

lobster.

Bars represent 10 pun

Five morphologically distinct bacterial types were observed on

both healthy and infected animals. The majority of cells were
either rods 1 x 0.4 /xm). coccoid rods (0.8 x 0.5 /^m), or cocci
(

(0.5

0.45

/j.m).

Segmented rod

links (each piece 1.5

and

in A, B,

0.5 /urn)

C,

and

I p-in in ().

Findings from the present study revealed the complexity of this

its progression and development and demonstrated the

disease in

necessity for further molecular strides, including bacterial speciaand infection studies, in ELSD research.

tion

and coccoid links (each piece 1.5 X 1.0 /urn) were less abundant
and found on infected animals only. Most bacterial types were
found on animals from

all

Literature Cited

geographical areas, but coccoid links

were observed only on infected Rhode Island and Cape Cod Bay
samples, indicating that they
Bacteria are ubiquitous in

ing the causative agents in a disease can be difficult. Examination

biology are needed to identify the bacteria responsible for

ELSD.

J.,

K. A. Lund) and T. B. Hoopes. 2002.

Massachusetts

able: http://www.state.ina.us/dfwele/dmf/index.html [accessed

August

2003].
2.

of the interface between healthy and necrotic tissue provided the

evidence necessary to identify bacteria as the disease-initiating

organisms. Scanning electron microscopic imagery cannot speciate

bacteria, so additional techniques such as those used in molecular

Dean, M.

Division of Marine Fisheries, Technical Report TR-13 (Online). Avail-

may be secondary invaders.

the marine environment, so identify-

Stewart,

New
3.

Pp. 32

J. E. 1980.

Lobsters, Vol.

1.

J.

S.

-324

Cobb and

in

The Biology and Management of

B. F. Phillips, eds.

Academic

Press,

York.

Chistoserdov, A., R. Smnlowitz, and A. Hsu. 2003.

Pp. fil-f)4 in

Connecticut Sea Grunt Extension, Third Long Island Sound Lobster

Health Symposium. University of Connecticut, Storrs.

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

230
4.

5.

Smolowitz, R. M.,
99-112.

'

liis,

and D. A. Abt. 1992.

Biol. Bull.

l3:

107 in Histopathologic Methods and Color

Luna, L. G. 1'Wi.

ii</ Tissue Artifacts.

Johnson

Downers

Printers,

Jr.,

and K. L. Klomparens. 1993.

Scanning and Transmission Electron Microscopy: An

Introduction. Oxford University Press, New York.

151-167

Pp.

Alias ofSin

W. Heckman

Flegler, S. L., J.

Baross,

7.

in

A. Tester, and R. Y. Morita. 1978.

J. A., P.

Board Canada 35:

Grove, IL

Fish. Res.

J.

141-1 149.

Reference: Biol. Bull. 205: 230-231. (October 2003)

2003 Marine Biological Laboratory

Characterization of Phosphorus-Regulated Genes in Trichodesmium spp.

2
1
2 '*
Elizabeth Orchard Eric Webb and Sony a Dyhrman
,

'

Woods Hole Oceanographic

Cyanobacteria of the genus Trichodesmium are abundant

and subtropical regions and significantly contribute

tropical

carbon and nitrogen fixation

in these

environments

studies suggest Ihat phosphorus (P) supply

may

).

Institution,

primer pairs were designed

Recent

influence carbon

within four species,

related species

T.

erythraeum,

Katagnymene

T.

in

phosphorus physiology
remie. T. t/iiebiiutii, and the

nated pstSl and pstS2. These genes are

of

P-regulated scavenging mechanisms in other cyanobacteria and

The plioA gene codes for a predicted

phosphatase. an enzyme that hydrolyzes inorganic phos-

heterotrophic bacteria (4, 5).

phorus from organic phosphorus, which can then be used by the

organism for growth. The activity of this enzyme has been used as
an indicator of phosphorus stress

in field

populations

(2).

PstS

is

high-affinity binding protein for inorganic phosphorus and is part

of a phosphate-induced high-affinity scavenging system.
The sequenced genome of T. erythraeum (http://genome.jgi-

psf.org/draft_microbes/trier/trier. home. html)

was used

to identify

phoA and two copies of pstS

(pstSl and pstS2> on the basis of their

to
characterized
similarity
genes in GenBank as part of the ongo-

(6).

PCR

amplification conditions were

DNA

To optimize

amplification, the annealing temperature

was

var-

gene and species over a range of at least 25 C. Other

variables included magnesium and enzyme concentration.
ied for each

from
from

to amplify the complete phoA and pstSl gene

erythraeum and T. teiuie, and the complete pxtS2 gene

were able
T.

T.

erythraeum.

thiebautii and

A',

spimlis (Table
amplified from

pBluescript

II

We amplified gene fragments from pstSl

in T.

and from pstS2 in T. tenue and K.

With the entire 3.5 kb of the phoA gene

xpirulix,

2).
T.

erythraeum,

SK +
(

plasmid

we were
in

able to clone

Escherichia coli

it

into the

DH5a,

in order

to identify the activity associated with the putative gene.

However,

and expression work is still ongoing.

products were sequenced at the Josephine Bay Paul
Center of the Marine Biological Laboratory (Woods Hole, MA)
activity

All

PCR

using the facility's protocols. With the

T. erythraeum genome as a
were designed to obtain full coverage of the
gene on both strands. Genes were aligned between species to

guide, internal primers

entire

compare their sequence divergence using Sequencher

(Gene Codes Inc. Ann Arbor, MI).

4.1 software

Preliminary sequencing data for the genes and gene fragments

phoA in T. tenue as well as pstS2 in

Table

Gene

DNA was extracted following the method

indicate that pstS], pxtS2, and

Corresponding author: sdyhrman@whoi.edu

Primer sequences and annealing temperatures for

effort (unpubl. data). Multiple

amplify internal fragments (referred

polymerase (Stratagene, La Jolla,

using conditions optimized for each gene and species (Table

We
common components

1).

described by Orcutt et al.

performed with PfuTurbo

CA)

to

complete plioA, pstSl, or pstS2 gene (referred

to as external) (Table

spiralis.

Three genes thought to be related to phosphorus physiology

were examined in this study: plioA and two copies of pstS, desig-

alkaline

to as internal) or the

1 ).

MA

Trichodesmium annotation

ing

among Trichodesmium species (2, 3), suggesting that the

may have unique scavenging mechanisms. Here
several genes involved

Hole,

to

individual species

we examine

Woods

in

and nitrogen fixation by Trichodesmium in the Atlantic (2, 3).

However, evidence of phosphorus deficiency in field populations
differs

NY

Cornell University, Ithaca,

PCR

reactions

MOLECULAR BIOLOGY. PATHOLOGY. AND MICROBIOLOGY

Table 2
Amplified genes from

Trichodesmium and Katagnyniene

spiralis

231

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

232

-69

30'W

-68 55'W

_l

-68 20'W

14

''''

'

/?",-V
43 45'N

Cell

Number
cells/I

500

or non-detectable

43 10'N-

A
'

AlcxanJrium fundyense distribution. (At The arrow indicates a

Uirtic rihosomal snbunil ILSU) gene primer?, ami a imii>
174-hp fragment of the LSU gem: as shown In tin- Illtl-hp laddci Amplification occurred nnl\ in strains of A. fundyense that are present in the Gulf of
Maine (GTCA28. CB30I, GTPPOIl. Amp/iln ation c/icl nut incur in Alexandrium \tiams I rum other pun-. f the world (SP3B5. AL8T. TN9A): nor did
amplification occur in oilier Alexandrium species \nch n\ A. ostenfeldii ami A. andersoni that could occur with A. fundyense in the Gulf of Maine (A.
1.

Figure

Spcci/iiit\

<>l

Other Jinoflagellate species also did not amplify (CCMPIV37, SA2. Karenia breve, Prorocentrum minimum, GPES22). (B> A
Manic during a cruise from 2V May to 6 June 21103.

ostenfeldii, TC02).

map

of

A. fundyense distribution in surface water of the Gull "/

and a 60

annealing temperature. Under these conditions amplifrom the Gulf of

fication occurred only with A. finnlvfiisc isolated

during the cruise and oligonucleotide probe counts [Anderson,

unpubl. data]), which we did not expect to be exactly the same

Maine

since the samples were taken from different Niskein bottles and

(Fig. 1A).

Field samples were collected from surface water along transects

in the

Gulf of Maine from 29

May

through 6 June 21)03. At each

processed differently. Of the stations tested, station 86 and offshore stations 104 and 105 had the highest concentrations of cells.

of surface water was collected, prescreened through a

64-ju.m sieve, and collected on a 15-jum filter. The samples were
each extracted with Qiagen DNEasy kit.

To confirm

Using qPCR, the number of cells in a field sample was determined. In this study, qPCR was performed using Strutagene Bril-

using the facility's protocols and were

station,

liant

SYBR

was

Green

QPCR

Master Mix. and a fluorescence thresh-

by the analytical software for the BioRad iCycler. The

PCR cycle during which this threshold was crossed for each
sample designated the C r Sample C , can be compared to the C r

old

set

at

known

cell

count to specify the number of cells

present in the sample (4).

To ensure

that different strains

27.6

R 2 = 0.4514

R2

for the

first

strain

for the second strain).

number of

cells

was

significant

PCR

inhibition of the target

neous

DNA

shown

to

be A. fundyense.

summary. qPCR appears to be a specific and sensitive approach to monitoring the abundance of A. fundyense. This method
shows
the

great promise for mapping the A. fundyense populations

Gulf of Maine.

This research was supported by an

EPA

Star

Award through

in

the

ECOHAB

Program (R-83041501-0) and a NSF-REU site grant

(OCE-0097498) to the Boston University Marine Program.

of A. fundyense have the same

copy number of the LSU gene, standard curves were built from
two Gulf of Maine strains in culture (GTCA28 and CB301). Both
strains resulted in similar standard curves (v =
-2.5281.V +
30.664,

In

of standards with a

amplification of A. fundyense from the field samples.

products generated from samples 89 and 28 were sequenced
the Marine Biological Laboratory. Woods Hole. Massachusetts,

PCR

-2.0291* +
series of a known

and v =

dilution

also analyzed with a field sample matrix.

was detected from

No

Literature Cited
1.

3.

extra-

in the field

samples.
The number of cells detected in the Gulf of Maine ranged from
to 35 cells per liter (Fig. IB). This range is similar to the range

of cell numbers calculated from other methods (live counts done

Anderson, D. M. 1997. Lininol. Occano K r. 42: 1009-1022.

Anderson, D. M., D. M. Kulis, B. A. Kealer, and E. Berdalet. 1999.
/ Phycul. 35: S70-883.
Bowers, H. A., T. Tengs, H. B. Glasgow, J. M. Burkholder, P. A.
Ruhlee, and D. W. Oldach. 2000. Appl. Environ. Microhio/. 66:
4641-4648.

4.

La Du,
Bull.

5.

J.,

D. Erdner, S.

Dyhrman, and

D. Anderson. 2002.

Biol.

203: 244-245.

M. Herzog, M. Sogin, and D. M. Anderson.

W9-10I1.

Scholin, C. A.,
Pin-col. 30:

1994.

./.

MOLECULAR BIOLOGY. PATHOLOGY. AND MICROBIOLOGY

233

Reference: Biol. Bull. 205: 233-234. (October 2003)

200? Marine Biological Laboratory

Description of Vibrio alginolyticus Infection in Cultured Sepia officinalis. Sepia apaina,

and Sepia pharaonis
2 '*
1
C. R. Songster and R. M. Smolowitz
1

Western College of Veterinary Medicine, Saskatoon, Canada

'

Marine Biological Laboratory, Woods Hole,

Cuttlefish of the genus Sepia (S. officinalis, S.

apama. and

S.

the Marine Resources Center

phanumisl have been cultured at

(MRC) of the Marine Biological Laboratory
objectives of this retrospective study were to

The

1992.

since

MA

granulomatous-like inflammation of intermediate size was commonly found in branchial heart and its appendage (0.2-0.7

mm

diameter) (Fig. 1C).

common

Severe, necrotizing hemocytic inflammation also occurred in the

causes of morbidity and mortality in the cuttlefish populations

maintained at the MRC. and to describe the histological appear-

serosa of various organs including the branchial heart and stomach,

which are anatomically suspended in large vascular sinuses. Sim-

identify

ance of those lesions. Such information can be used in developing

more effective methods of diagnosis, prevention, and treatment.

Necropsy cases were selected for inclusion in this study if

had been obtained at necropsy. Bacterial cultures
obtained
from 53 necropsies performed between March
had been
bacterial cultures

1999 and June 2003. Those culture samples were taken from the
digestive gland, kidney sac. or gonad and were plated on marine
brain heart infusion

medium

).

Retrospective examination of archived necropsy cases of Sepia

with bactespp. showed that mortality was commonly associated
cultures positive for Vibrio alginolyticus, a marine bacterium
routinely found in coastal waters, sediment, and culture systems (2.

rial

3).

Of

the 53 cases in

positive for

Of

V.

which

bacterial cultures

were taken. 33 were

alginolyticus.

the 33 animals with cultures positive for the bacterium,

archived histological sections were available

The

19 cases.

in

sections were paraffin-embedded fixed tissues, sectioned at 6 /xm

and stained with hematoxylin and eosin (4). Examination of these
sections

showed

that the

most commonly affected

tissues

were the

kidney, branchial heart appendage, branchial heart, and gill (42%).

A high incidence of infection (47%) was noted in the reproductive

organs (nidamental gland, accessory nidamental gland, and gonads). All animals with reproductive lesions were older than 9

that the bacteria

periphery of the fronds (suspended in the renal sac), while the

hemocytic response was located in the vascular/connective tissue
core (Fig. ID).
Bacteria causing the observed lesions

9 as mild.

No

reaction

was detected

moderate

to severe,

in the digestive

and

gland of any

animal. Examination of Gram-stained tissue sections (3) confirmed

the presence of gram-negative bacteria in the infected foci.

Histopathological examination of sections

showed

that,

based

on the appearance of the response as identified

in the tissues, the

sepiod inflammatory reaction

occurred

three distinct forms.

The

to V. alginolyticus

first

loma-like lesions (0.95-1.4

was

mm

in

one of

multifocal, necrotizing, granudiameter), most often seen in

reproductive tissues such as the testicular ducts and accessory

nidamental glands (Fig.
A. B). The second form consisted of
I

multifocal. necrotizing, granulomatous-like inflammation that resulted in subtle lesions (0.1-0.25

mm

bacteria, and was predominately found

of affected animals. Finally, multifocal. necrotizing.

numbers of hemocytes and

in the gills

diameter) containing small

accessed the

was not

rec-

animals and was only mild in others, so the potential

for other routes should not be ignored. In the circulation, the

ognized in

all

bacteria lodged in the branchial heart, resulting in foci of moderate

inflammation and necrosis. Most other lesions appear to have been

spread by bacterial seeding of organs that receive circulation from
the branchial heart. Thus, bacteria-laden hemolymph that passed

from the heart

directly to the gill

resulted in foci of inflammation.

and branchial heart appendage

The branchial

heart appendage

discharged into the kidney sac and

the
fronds;
bathes the kidney
epithelium of the kidney fronds

forms an

ultrafiltrate that is

body through a renal pore.

and attack the suspended
mount
the inflammatory rewhich
fronds.
Hemocytes.
kidney
connective core of the
sponse, migrate from vessels within the
modifies the

before

filtrate

it

exits the

Bacteria can proliferate in the

kidney fronds to the

These

results

in

filtrate

of infection.

site

show

that V. alginolyticus

icance in sepoids cultured

coastal waters and

at

is

the

MRC.

\'.

is

a pathogen of signifis

alginolyticus

common

therefore likely to be present in

the

This pathogen has been associated

facility's seawater supply (2).
with tank surfaces in culture systems (5). Comparisons between
bacterial populations

on wild-caught and laboratory-reared squid.

showed

Lolligiincula hrt-vis.

that

animals reared

had higher total numbers of bacteria.

due

in the

laboratory

The increase was primarily

to Vibrio spp.. including V. alginolyticus (6). In that study, the

increase in bacteria

was

not linked to the ulceration of the epider-

mis. Similarly, V. ulginolvticus has caused disease in juvenile red

abalone, Haliotis rufescens, but large numbers can be found on the
foot of otherwise healthy individuals (5).

Such findings suggest

epidermis is an unlikely pathogenesis

cuttlefish. Routes of infection probably

that direct infection of the

for V.

alginnlvticiis

in

include infection secondary to ulceration. especially

caused by
'Corresponding author: rsmol@mbl.edu

may have

circulation via epidermal ulcerations, but ulceration

months. Of the 19 included cases. 16 (84%) had some form of

epidermal ulceration. with 7 classified as

hemocytic inflammation of the kidney fronds was unique in

and associated necrosis occurred primarily at the

ilar

jetting into tank walls colonized

a/giiiol\tii'iis

if

the injury

by the bacteria.

has also been found on the carapace of copepods

is

V.

(7).

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

234

:-l

*1
"i

Figure
necrosis

Wn/o/o.ij/Vi// rctutiim n/

1.

and

heiiiocvtic renction 13).

infiltration (2).
in the

core

Sepia

.v/'/i.

M Vibrio alginolylicus.

f/Aj

Granuloma-like

fornnitioti in lite

bacteria in the center of the granuloma (1) are surrounded by a layer of dying heinocytcs

(2).

Id

Moderate inflammation

(Dl The periphery of the kidney

A normal

tissue

frond

is

branchial heart

in the

frond.',

also present

is

typified

MRC.

are necrotic and bacteria-laden

).

reproductive-associated lesions were at or near the upper limit of

2.

suggesting that senescent tissues are more

susceptible to this pathogen and are a potential point of initiation
for a systemic infection.

alginolyticus

common pathogen in Sepia spp. cultured at the MRC. A number

of potential routes of infection exist but, as in most aquatic animal
diseases, stress and husbandry likely play an important role in the
occurrence of disease. Better sepoid husbandry methods may help
reduce or prevent this disease from occurring. Additionally, the
data collected in this study indicate that,

when systemic

is

suspected

New

York.

Hormansdorfer,
Bauer.

2(100.

S.,

Int.

.1.

H. \Ventges, K. Neugebaur-Buchler, and

H\ K Environ. Health 203: 169-175.

Humanson, G. L. 1962. Pp. 3-126

W. H. Freeman, San Francisco.

Elston, R..

Ford, L. A., S. K. Alexander, K.

./.

and G.

S.

in

Lockwood. 1983.

Animal Tissue Techniques.

Fish Dis. 6:

M. Cooper, and

1-128.

R. T. Hanlon. 1986.

Im-ertehr. Puthol. 48: 13-26.

Dumontet,

S..

K. Krovacek, S. B. Svensnn, V. Pa.squale, S. B.

Balnda, and G. Figliuolo. 2000.

Dis. 23: 53-72.

in

J.

4.

bacterial

Sepia spp., the branchial heart and branchial

heart appendage should be examined histologically, and bacterial
cultures should be obtained from the renal sac.
disease

Biol. Bull. 193:

Bulows, A., H. G. Truper, M. Dworkin, \V. Hardner, and K. Schlei1992.

Pp. 2995-2996 in The Proka notes. 2nd ed.. Springer-

Verlag,

is

R. Bullis, and R. Smiilowitz. 1997.

S.,

fer.

3.

V.

Wenuganen,
269-27(1.

(8),

This retrospective study shows that systemic

and hemocytic

Literature Cited

and thus may be an important

1

span

(It,

while a front of heinocytes infiltrates the tissue from vessels

(3).

source of infection. Finally, over half of the studied cuttlefish with

their life

mm

by large numbers of bacteria and cellular necrosis

Crustaceans, including amphipods, are the primary food fed to

cuttlefish cultured at the

accessory nUlu/ni'iituI xluiiil. (B) Coagulative

which is in
surrounded by a fibrotic

(2).

Forsythe,

J.

Coin/), linnntnol. Microbiol. Infect.

W., R. H. De Rusha, and R. T. Hanlon. 1994.

Loud. 233: 175-192.

Zoo/.

MOLECULAR BIOLOGY. PATHOLOGY. AND MICROBIOLOGY

235

Reference: Bio/. Bull. 205: 235-236. (October 2003)

21103 Marine Biological Lahoiaioi\
1

Detection of Edwardsiella Infections in Opsanus tan by Polymerase Chain Reaction

1 3
Roxanna Smolowitz 1 '*, and Kevin R. Uhlinger'
Kn-stal D. Baircl' ', Hemant M. Chikarmane
'

Marine Biological Laboratory, Woods Hole, MA

Barnstable County AmeriCorps Cape Cod, Barnstable,
'

Cape Cod Community

"'

the oyster toadtish. is an important laboratory

hearing and balance research at the Marine Biofish are
logical Laboratory (MBL) (1). Wild-caught and cultured
maintained year-round in both recirculation and flow-through

Opsanns

tan.

animal used

in

tanks in the Marine Resources Center

cause of disease in toadfish held

(

A member

1. 2).

(MRC)

at the

MRC

at the
is

MBL. A

major

Edwardsiella tarda

of the Enterobacteriaceae, E. tarda

is

a natural

inhabitant of fresh and marine water and causes gastrointestinal

Proteinase

(50 p.g) was added, and tubes were incubated overwas precipitated by the addition of

DNA

50C. Genomic

night at

MA

MA

College, W. Barnstable,

0.55 ml of isopropanol: the liquid portion was removed completely. The

pellet was rinsed twice with 0.8 ml of 70%

DNA

ethanol. After the ethanol

in 0.8

ml of

TE

was evaporated,

(Tris-EDTA:

DNA was dissolved

TRC GTG TTA ATA

Forward primer Eta -363 f (5'-GTG

1

GCA-3') was designed

the

8).

to amplify E. tarda

from human sources

Published reports of fish

tarda involve cultured \\ arm-water fish, but

ATCC 15947. Forward

(biotype 1). represented by
AGG
TGT GAA-3') was
GGA
GGA
Eta2-351
(5'-TAG
primer

the disease can also affect cold-water salmon (4). Poor water

designed to amplify E. tarda strains isolated from fish (biotype 2).

An Edwardsiella genus-specific reverse primer Edwsp-780r (5'-

and extraintestinal disease

disease caused by E.

in

humans

(3).

high water temperatures, feces. and decaying organic

matter likely contribute to the onset and severity of the disease and

quality,

probably allow for occurrence and proliferation of the bacteria in

the fish's environment. These factors, combined with capture and
holding-induced stresses, may account for the high levels of disease caused by E. tarda in the toadfish in our facility.
Currently, the only diagnostic test available for identification of
E. tarda in diseased fish is bacterial culture and identification using
traditional biochemical tests.

At

the

MRC.

these tests are sent to an

the type strain

CTC TAG CTT GCC ACT

primers.

PCR

amplification

GeneAmp

was performed with an Applied Biosystems

contained IX Taq
/n.1)

9700. The reaction mixture (10

mM

commenced

with an

cycles of denaturation

ment

and extension

often attempted before verification that the lesions identinecropsy were caused by E. tarda. Using the PCR methods
described here, we can specifically and rapidly identify E. tarda,
is

resulting in timely and appropriate

treatments for infected

management procedures and

fish.

The polymerase chain

(PCR)

reaction

is

DNA-based method

can be used for the quick and sensitive detection of microorganisms in both antemorteni and necropsied tissues. PCR primers
specific for E. tarda isolates from Japanese eels have been derived
that

from an anonymous species-specific sequence (5) or from the

hemolysin gene (6). Here we describe PCR primers based on

RNA

genes for direct identification of E. tarda. Primer development will be described in detail
elsewhere.
Eilmirdxiella small subunit (ssu)

The type

strains E. tarda

ATCC

15947 and

E. icrahiri

ATCC

33202 were obtained from the American Type Culture Collection.

E. ictalnri is an important pathogen of cultured catfish and produces very similar lesions to those caused by E. tarda in toadfish.
In addition. E. tarda has been shown to cause disease in catfish (7).
Cultures were

One

grown

in

Difco marine brain heart infusion broth.

of the liquid broth culture was transferred aseptically

to 1.5-ml microfuge tubes, which were then centrifuged at 2790 X
milliliter

g for 4 min
lysis buffer

were resuspended in 0.6 ml of

EDTA. and c7c SDS).
8.0. 5

to pellet cells. Pellets

(

10

mM Tris-Cl

pH

mM

Corresponding author: rsmol@mbl.edu.

mM

dNTP mix.
MgCK.
polymerase buffer (Promega). 1.5
100 nM each of forward and reverse primer. 0.5 units Taq polymerase (Promega) and template DNA. The thermal cycle profile

outside laboratory, and the process takes about three weeks. Treat-

fied at

CTT-3') was used with the forward

min

initial
(

at

denaturation for

min

at

min

94C). annealing

72C); and a

final

extension

at

at

30

58C),

72C

for 7

.5% agaX Tris-Borate-EDTA (TBE)

/ng/ml ethidium bromide and

min. Amplification products were electrophoresed in a

rose gel (Fisher Scientific) in 0.5
buffer (8). Gels were stained with

94C;

at

min

Images were manipulated in Adobe Photoshop. Some amplifications were carried out directly from bacterial colonies, in which case the initial denaturation time in the PCR
digitally photographed.

was increased

profile

to 5

min

at

94 C.

determined by electrophoresis. showed that DNA

of E. tarda and E. ictaluri could be distinctly amplified by the

The

results, as

appropriate primers (Fig. 1). The Etal-363f-Edwsp-780r primers

of 216 bp. and
amplified E. tarda ATCC 15947. yielding a product
did not amplify E. ictalttri ATCC 33202 (Fig. 1A. lanes 1-4) or

MRC

fish isolates biochemically identified as E. tarda (not

shown). As would be expected. Eta2-351f-Edsp-780r failed to

amplify both type strains (Fig. 1A, lanes 5-8). However. Eta2351f-Edwsp-780r primers amplified the E. tarda fish isolates (Fig.
IB. lanes 1-6). demonstrating that the strains isolated at the

MRC

were clearly of fish origin, and biotype 2. The biotype 2 amplifications were performed directly from bacterial colonies, showing
that direct identifications were indeed possible.

w ill involve development of a direct assay method

tarda from both postmortem toadfish tissues and. more
E. tarda
importantly, antemortem tissues. The ability to amplify
understand,
able
to
first
to
strains from toadfish is the
being
step
Future work

for E.

isolate,

and control future outbreaks of

E. tarda.

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

236

M123

M1234M5678

Eta2-351f

Etal-363f
Figure

Lane

7.

Eta2-351f

Gel electrophoresis of PCR amplification products from Edwardsiella. (Panel A) Amplifications from purified genomic DNA. Lanes 1. 3. 5,
15947. Lanes 2, 4, 6, 8 are E. ictaluri ATCC 33202. (Panel B} Amplifications from bacterial colonies. Lanes 1-6, E. terdafish isolates.

1.

7 are E. tarda

ATCC

Vibrio

sp.

negative control. Lane

no template control. Ed\\'sp-780r was used as the reverse primer

8,

R. Smolowitz acknowledges support from NIH/NIDCD grant

5P01 DC001837-09. H. Chikarmane acknowledges support from
the

4.

Plumb.
3,

5.

Wenuganen,
Janda,

J.

R. Bullis, R. Smolowitz,

S.,

and

E. Barbieri.

Chen,

Darwish, A.,

J. D..

Ctin. Infect. Dis. 17:

I.

479-490

and Fungal
Publishing,

indicated.

Fish Diseases and Disorders Vol.

in

Infections, P. T. K.

New

Hirono. 1995.

Foncard primers are

Woo

and D.

W.

York.

Asian Fisheries Society Special Pub-

and
J.

S. V. Lai. 1998.

A. Plumb, and

J.

Zo,,l. Stud.

37: 169-176.

C. Newton. 2000.

J.

Aquat. Anim.

Health 12: 255-266.

1997.

S.

Sambrook,

J.,

ton Manual.

M., and S. L. Abbott. 1993.

Pp.

in all cases.

10: 135-146.

7.

270-271

269-270.

Biol. Bull. 193:

3.

Biol. Bull. 193:

CABI

6.

Literature Cited
Bullis. 1997.

A. 1999.

Aoki, T., and

lication.

Smolowitz, R., and R.

J.

Viral, Bacterial,

Bruno, eds.

Wilkens Foundation and the Cape Cod Community College

Educational Foundation.

45 678

742-74X

and
3

td

Russell. 20(11.

I).

Molecular Cloning: A Labora-

Cold Spring Harbor Laboratory Press.

ed.

New

York.

Reference: Biol. Bull. 205: 236-237. (October 2003)

2003 Marine Biological Laboratory

Catalase in Microsporidian Spores Before and During Discharge

1
2
Earl Weidner and Ann Findley
1

Louisiana State University, Baton Rouge, LA

University of Louisiana at Monroe, Monroe, LA

The noted

parasitologist

crosporidians as

the

among

Horace W. Stunkard characterized mi-

characteristically

most widespread of

evidence that peroxisomal enzymes occur

parasites, possess-

ing significant survival adaptations, a consequence of their long-

term host associations

within this group

is

).

One of

the

more obvious adaptations

the extrusion apparatus of the infective spore

Here,

We

The energy source

thought to reside
(3),

for the

we inuicawd

in the posterior

that

fatty acids

of this apparatus

(2). In

the posterior vacuole

peroxisomev which function primarily

CoA

firing

vacuole

is

an earlier report

had properties of

to process very

long chain

(VLCFA) with the assistance of key enzymes, acyl(ACOX) and catalase. Although peroxisomes are

oxidase

aerobic cells with mitochondria, there

blot analyses indicate that

discharged from the spores of

mem-

ganelle.

in

in the

is

amitochondriate

microsporidian Spragttea lophii. In particular, biochemical assays

and western

brane, a polar filament, and a posterior vacuole, or swelling or-

stage. This apparatus consists of an aperture, a polaroplast

found

we

S.

ACOX

and catalase are

lophii (Findley. unpubl. data).

report the localization of catalase within the spore cell.

used a cytochemical protocol described by Angermuller and

Fahimi (4). In brief, spores were prefixed in 2.5% glutaraldehyde
(to eliminate resident

incubated
zidine
fixed

in Tris

peroxiase activity) and were subsequently

buffer

(pH

10.5) in the presence of

diaminoben-

(DAB) and hydrogen peroxide. The cells were then postin 29r osmium tetroxide and processed for transmission

electron microscopy.

MOLECULAR BIOLOGY. PATHOLOGY. AND MICROBIOLOGY

237

***N
***$
'* **

%>?*$

B
Figure

Arnws

\.

Linuli:<iHi'ii

v/iou reaction

portion of unaxe has

vacuole.

Bar

i>;

product

DAB

Spraguea lophii

ctitalase activity. (A} Electron

vacuole. IB> Similar ima^c o\

in

DAB

reaction on outside. lC> Control preparation without

scales represent 0.5

DAB

reaction

is

associated with the inner surface of the tube (Fig.

the outer surface of the discharged tube. In the

B and also along

)

control preparations without

was no catalasc-induced

DAB

vacuole (Fig. 1C). In addition to the peroxisomal enzymes, spores

S. lophii extrude the VLCFA nervonic acid into the external

and the data indicate

with the catalase and

is

that

a characteristic peroxisomal
it

ACOX

is

DAB

or hydrogen peroxide

s/iuir.s

no reaction

in the

ni;lit

spore posterior

reported here also support the Lorn and Vavra model of spore

discharge

component,

discharged from the spores along

enzymes. Of particular

interest

is

(2). In this

model, the posterior vacuole swells

signifi-

cantly after the spore is activated, the extrusion apparatus everts,

and a membraneous sac forms

accommodate

and hydrogen peroxide, there

reaction product in the spore posterior

of

medium. Nervonic acid

to the posterior \ucnole.

fj.ni.

Electron micrographs revealed

reaction product in the
vacuole
of
the
before
and
posterior
spores
during its discharge
(Fig. 1A). As the invasion tube emerges from the tiring spore, the

DAB

micrograph of alkaline-DAB reaction confined

reaction extended to discharged spore tube (arrows). Discharged tube at /our;-

If the

at the

end of the discharged tube

to

the exiting spore cell, or sporoplasm.

Lom

and Vavra model

is

correct, the

membrane

of the

extruded sporoplasm may be derived from the extrusion apparatus

and may therefore be reversed. Indeed, studies with cytoplasmic
protein probes indicate that

brane orientation

is

in

discharged sporoplasms. the

mem-

reversed as proposed (DeGiorgis and Weidner.

unpubl. obs.).

the

sizeable drop in nervonic acid levels that occurs during and utter

discharge of 5. lophii spores (Findley, unpubl. data).

These data indicate that DAB-labeled catalase is
stricted to the posterior vacuole.

Literature Cited

initially re-

subsequently moves to the ex-

1.

Stunkard,

J.

truding polar tube during spore firing, appears on the outside of the
discharged tube, and finally diffuses to the extracellular medium

2.

Lom,

3.

Weidner,

discharged sporoplasms (not shown). The results

4.

Angermuller,

that bathes the

J.,

and
E.,

W., and F. E. Lux. 1965.

J.

Vavra. 1963.

Ada

Biol. Bull. 129:

Protocol.

and A. Findley. 20(12. Iliol.

S., and H. I). Fahimi. 1981.

Bull.

1:

371-385.

81-92.

203: 212

Histochem. 71: 33-44.

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

238

Reference: Binl. Bull. 205: 238-239. (October 2003)

2003 Marine Bioloj.'f_-:!l !,.iUir:uory

<;< ivvth

Marsh Invertebrate on

of a Salt

Marsh Grass

Several Species of

Detritus

and R. Bitchsbainir
Gordon College, Wenhain, MA
Massachusetts Audubon Society, Wenluuu. MA

A. M. Agnew', D. H. Sltull''*.
'

These were placed at

were

marshes are important and productive ecosystems. Marsh

grasses fuel coastal ecosystem production, and marsh invertebrates

for each grass-species/age/site treatment.

convert abundant decomposing marsh grasses into biomass available to higher trophic levels. Changes in climate, land use. nutrient

changed about once a week, and dishes were kept wet by the
addition of filtered seawater from the marsh every few days.

Salt

and introduced species potentially threaten

input,

An

however.

this

ecosystem,

accelerated rate of sea-level rise has allowed cord

). Meanwhile,
grass (Spartina alterniflora) to migrate shoreward
the high-marsh invasive reed Phragmites australis has expanded
(

seaward, reducing the extent of indigenous high-marsh grasses

such as Spartina patens and Distichlis spicata (2). Although
changes in invertebrate community structure have been observed
following Pliragiiiitex invasion

(3), less is

known about

its

species of marsh grass equally nutritious for the

invertebrates that feed on them? These questions are closely re-

Are

lated to

all

some of

Plum

the primary goals of the

Island

Ecosystem
Long Term Ecological Research (PIE-LTER) program, which is
concerned with the processing of organic matter within the salt
marsh.

To

Growth data

some of

rates

the

above questions, our study investigated

of the salt marsh amphipod Orchestia grillus. one
the

growth
of the most abundant and best-studied detritivores at our study site
(4), feeding on four species of marsh grass. Two sites were studied.

Clubhead Creek and Greenwood Creek;

riched due to

its

proximity to a

was

to

examine

sewage

the latter

outfall.

is

nutrient en-

The purpose of our

experiment were eliminated from growth data

was allowed to run for 38 days.

Spartina patens (marsh hay). Distichlis spicata (spike

and Phraginilex uustralis (common reed). S. alternifli>ra is
the dominant low-marsh species. S. patens and D. spicata are

grass).
grass),

native high-marsh species, and P. australis

is

high-marsh inva-

sive.

in

We

sampled two differently aged grasses to assess how the

of
detritus
quality
changes over time. Old samples were sorted, cut,
and frozen at -20 C. Young grass samples were dried at 70 'C
2003.

for

24

the fresh

h,

soaked

in

seawater for 63

h,

and then dried again

at

70

24 h before freezing, to simulate the formation of fresh detritus.

Organisms collected from the marshes were allowed to acclimate
for

to laboratory conditions in a large tank containing

marsh wrack

for

of the experiment cm 22 June 2003, single

organisms were weighed and placed into petri dishes containing a
single type of grass Irom each site. Ten replicates were designated
3 days. At the

start

Corresponding

iiutht-r:

dshull(5'gordon.edu

Phenolic concentrations (deter-

detritus.

extracts at

nificantly higher in the fresh detritus, as

factor

ANOVA
= 33

with grass

P < 0.000

320 nm) were

P =

0.01

rates

on the year-old

).

sig-

demonstrated by two-

and age as fixed factors

and Orchestia mortality rates were

species
).

significantly correlated with phenolic concentrations

For these reasons,

we

(R

0.56.

focused our analysis of growth

detritus.

The mean growth rates for O. grillus consuming year-old detritus varied with marsh grass species. Rates were highest for amphipods consuming D. spicata (O. grillus increased in size by 40%
50%) and decreased in the order of S. alterniflora, S. patens, and
australis (Fig.

Growth

1A).

rates

were determined from the

and
slope of organism size versus time plots by linear regression,
rates among sites and species were compared by two-factor analysis of variance, with collection site

fixed factors. Data

in

two
the

sites

were found

growth

was highly

and marsh grass species as

were log-transformed prior

rect for heteroscedasticity.

No

(/",

to analysis to cor-

differences in growth rates between

= 0.088. P = 0.77). Variation
4l
,,

rates of O. grillus

significant (F,,^,,,

significant interaction

on different marsh grass species

= 5.7. P = 0.002), with no

(F |141)| = 0.12. P = 0.95). Post hoc

comparisons indicated that the growth rates of O. grillus on P.

australis were significantly lower than those on S. alterniflora and
D. spicata (Scheffe F-test.

P <

0.01

).

Variation in O. grillus growth rate was not correlated with the

= 0.052, P = 0.59).
percent carbon content of the detritus (R~
linear regression analysis indicated that nitrogen content
year-old detritus accounted for approximately 70%> of the vari= 13.7. P = 0.01 ).
ation in growth rates (Fig. IB,

However,
in

F,,.,,,

Our
marsh

marsh grass

mined by absorbance of methanol

the

Samples of one-year-old standing dead grasses and young newgrowth grasses were collected from each site from 2 to 6 June

The experiment

remaining alive in these treatments were not growing. This pattern

may have been due to the presence of high phenolic concentrations

P.

were Spartina alterniflora (cord

sets.

Within a few weeks, nearly 80% of the organisms feeding on

were dead, and it became clear that those individuals

terms of O. grillus growth rates and nitrogen and carbon content.

plant species studied

to

fresh grass

to

The four

them

These data were normalized by dividing measurements by the initial size of each individual. Mortality data were
collected daily, but all organisms that died before the end of the

the nutritional quality of the detritus in

research

were collected weekly by removing,

for O. grillus

their petri dish.

(F, 1.241

address

grid on a laboratory bench. Grasses

patting dry, and weighing individuals, and then returning

effect

on ecosystem function. How would changes in the species composition of marsh grasses affect food supply for higher trophic
levels?

random over a numbered

results indicate that,

when compared

to the three native

plant species studied, the invasive species, P. australis.

relatively poor food source for O.

grillus.

diet

of

salt

is

marsh

ECOLOGY AND POPULATION BIOLOGY

1.7
1.6 H
fl>

15a
'-

'35

TJ

I
|

1.3 i

1.2

1.1

10

20

Time

(d)

239

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

240

given a geographic location) using ArcGIS (version 8.3.0) software.

We

determined

main

along the

'iition

a>

:c

known

v<

where

total

suspended solids (TSS)

marshes of the lower estuary. We collected

at high water on a spring tide adjacent to

creek-, v iihm

tidal

of

estuary as well as along three third-order

of water

was examined and

on the marsh platform may be generated within the creeks themselves, presumably from eroded riverbanks. Slumping banks deliver large

amounts of sediment

where

to stream channels,

become suspended and deposited on

the

it

may

marsh surface during

flood tides. Examination of 1953 and 2001 imagery supports this

conclusion, as creek bank erosion is pronounced near the mouths

We deployed 94 sediment traps

of these creeks, especially at Nelson Island Creek where the most

sedimentation was observed.

along the estuary; these consisted of pre-weighed 9-cm glass fiber

secured with rubber bands to upside-down plastic petri dish

The spatial distributions of sedimentation and TSS along the

main axis of the estuary are complex, but generally decrease with

weighed

limentation

0.7-/j,m glass tiher filters.

filtered

it

through pre-

filters

covers

Marsh

(5).

grass

was

cut

away, and a galvanized

nail

was

down

distance

the estuary. Parker River sediments

inserted through the petri dish covers to secure the structures to the

nate at the headwaters of the river and to remain in

marsh. At West Creek. Club Head Creek, and Nelson Island Creek,

sediment source

we

set

up

transects of sediment traps perpendicular to a

ditch and a

mosquito

second-, and third-order stream (Fig. 1, inset).

Each transect consisted of three sites: 4, 20, and 50
in from the
first-,

creek edge to investigate the amount of sediment reaching the

marsh interior. Eleven sites were also selected along the Parker

River (Fig. ). We deployed two replicate sediment traps at each

site. We recovered two sets of sediment traps on 2 and 23 July
2003, after they were exposed to several spring tidal cycles.
1

Several samples (n

5) with anomalously high weight increases

were moderated by rinsing with

distilled

water to reduce high

river's

internal

why

ponds are

system. This

rare along the

upper reaches (although a higher concentration of mosquito

dug to drain the marsh, also prevent pond formation).

may not have been observed along the

Parker because of the brevity of the study and the fact that summer
is typically the season with the lowest sediment transport (5).

Greater sedimentation

Generally low sedimentation may also explain why decreasing

sedimentation was not measured farther from the creek bank.

Sediment sources from other nearby creeks may also have

interior marsh accretion.
Because of limited sediment source

in the creeks,

inflated

belowground

be more important than sedimentation in

marsh accretion, even though softer organic sediments may be
plant production

Comparisons were made using a single factor analysis of vari(6). Post-ANOVA pairs were analyzed using Student's / test

explain

to origi-

ditches,

salt

concentrations.

may

seem

its

may

ance

greatly compacted. This supposition

with a Bonferroni adjusted alpha value to account for multiple

that the

is

corroborated by the fact

area since

55% organic
matter or more. Because plant productivity is higher on creek
banks (7). depressed internal areas may develop because subter-

ponded today (several

of which cover thousands of square meters) were not ponded on

ranean accretion rates are slower away from the creek bank.
The increase in size and number of internal ponds, which

map. Few ponds appear (during either year)

coincides with observed patterns of sedimentation, may be a useful

indicator that marshes are not maintaining elevation relative to a

comparisons.
Internal

marsh ponds have increased

the early 1950s.

Many

the 1953 topographic

number and

in

areas that are largely

upper 10 km of the estuary. Broad marshes adjacent to the

lower third of the estuary are densely ponded. Pond drainage and
in the

seem to have occurred only rarely.

along the main axis clearly show the influence of Parker
River runoff: solids in the water column are most concentrated in
vegetative recovery

TSS

= 0.003),
upper estuary and decrease towards the sound (P
which receives regular exchange with the ocean (Fig. 2B). TSS

the

were also low

and increased

mouths of

at the

in the

tidal

creeks adjacent to the sound

second- and third-order reaches, where

about double the amount

in the

sound

TSS

is

= 0.009). Club Head Creek, which is located

upstream (P
between them, received an intermediate amount of sediment (Fig.

We

using sea level. Because the marshes studied are typical of New
England macro-tidal marshes, similar marsh degradation is likely
occurring in other areas.
This study received support from The

did not observe a strong relationship between

9726921. and the Atlantic Coast Environmental Indicators Consortium

maximum values, however, overlap

creeks (Fig. 2A. C). The slight overlap is not
surprising since TSS measurements were taken only once. More
perhaps

yield

strongly correlated with sedimentation.

significant difference

50

m
The

We

mean

values

Garritt

more

Gleason,

M.

D. A. Elmer, N. C. Pien, and

L.,

J. S. Fisher.

1979.

Estuaries 2(4): 271-273.

2

3.

did not observe a

and

Houghton, J. T., Y. Ding, D. J. Griggs, and M. Noguer. 2001.

Climate Change 2001: The Scientific Basis. Cambridge University
Press. Cambridge. England.
Orson, R., W. Panageotou, and
Res. 1: 29-37.

Kearney, M.

?.

Reed, D.

6.

Rooth,

from the creek.

three-

Hap

S.,

R. E. Grace,

S.

and

J.

Leatherman. 1985.
Stevenson. 1988.

J.

Coastal

Geogr. Rev,

78: 205-220.

between sediment accumulation 4

creeks considered in this study are fed from water

Our data indicate that sediment accreting

with relative's low TSS.

Special thanks to

their assistance.

Literature Cited

TSS and

in the

TSS measurements would

EPA grant number R828677.

and W. McDonald Lee for

sediment accumulation: their

somewhat

Woods Hole Marine

LTER NSF #OCE-

Sciences Consortium, the Plum Island Sound

(Fig. 2 A).

Nelson Island Creek received 0.050 g of sedimentation per 9-cm

filter, significantly more than West Creek, which is located farther

2E).

Parker River marsh sediments consist of

J.

J., J.

1989.

Esruanes 12(4): 222-227.

Cornwell, and

J.

Stevenson. 2003.

Extuaries 26: 475-

483.
7.

Frev, R., and P. Basan. 1985.

Environments, R. Davis.

Jr..

Pp. 222-301 in Coaxial Setlimentaiy

ed. Springer- Verlag,

New

York.

ECOLOGY AND POPULATION BIOLOGY

14
12

i
-

241

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

242
20v

Reference: Biot. Bull.

2003 Marine Biolo

242
'

(October 2003)

uory

>

Mu!-

!43

Tracing Nitrogen Loss in the West Falmouth Wastewater Plume

to

Approaches

Thoins' A. E. Giblin'.

T.

'

Wastewater transported through groundwater

aries

is

to receiving estu-

major contributor of nitrogen (N) in densely settled

watersheds, and contributes to coastal eutrophication (1-3). Al-

though nitrate removal by microbial denitrification, adsorption,

and uptake by plants is well documented, little is known about the
nitrogen loss in the vadose zone and aquifer

(1, 4).

This project focused on the fate of nitrogen leaving the Falmouth Wastewater Treatment Plant (FWTP) in Falmouth, Massa-

The plant lies on the Snug Harbor sub-watershed of West

Falmouth Harbor (WFH). and the wastewater it discharges perco-

chusetts.

lates through 25-30 m of vadose zone before meeting the aquifer

and traveling in groundwater to WFH (3). This site provides a
good model for a study of N loss due to the well-documented

history of wastewater discharge and previous research involving

loading and removal.

The two

to assess the
principal objectives of this study were
of
N
removal
as
wastewater
travels
from
FWTP to
efficiency
WFH, and (2) to determine where N loss is occurring. To satisfy

our

first

objective,

we

quantified

loss using

methods: a mass balance calculation, and

We

technique.

conducted
the

in

two independent

a conservative tracer

then compared these results to a previous study

1999

(3).

same conservative

the path of the

The

site

of

removal was examined using

tracer technique applied to samples within

plume

in

We

the aquifer.

further investigated

whether denitrification was the primary mechanism responsible for

N loss in the aquifer by using dissolved N^ and Ar gas analysis (5).
the development of the membrane inlet mass spectrometer, it
has recently become possible to measure dissolved N 2 /Ar ratios
with sufficient precision and accuracy to estimate denitrification

With

(6).

We

measured dissolved inorganic nitrogen (DIN; nitrate

in wastewater effluent at FWTP and

ammonium), and boron (B)

+
in

monitoring wells located within and outside of the treatment plant.

Boron, a component of laundry detergent, can be used as a conservative tracer for wastewater (7).

about to enter

WFH

We

also

sampled groundwater

within the boundaries of the Snug Harbor

watershed. Samples were taken from 0.5 to 1.5

below the
sediment surface, using well-points. These shoreline samples were
analyzed for DIN. B, and dissolved N-, and Ar gas concentrations.

Samples with elevated

rejected. Nitrate,

salinity, indicating

ammonium, and B

in

1987

during the
estimated 1

to the present,

first .7

plant to

N/y

to

WFh

seawater intrusion, were

concentrations were measured

using standard colorimetric techniques.

We compiled discharge data for the

ment

FWTP

and observed

years of operation (Fig.

from

its

establish-

that loading increased

1A).

Based upon an

ear groundwater travel time from the center of the

3),

compare

we used

\\:th

the 1993 discharge value of 13,177

the loading

and

K. H.

Foreman

Colorado College, Colorado Springs, CO

Marine Biological Laboratory, Woods Hole. MA
1

now

reaching the harbor.

kg

To

ECOLOGY AND POPULATION BIOLOGY

DIN

calculate the

centration

entering

WFH, we

used the average

shoreline groundwater. 208.5 H.M (n

ill'

27.8). multiplied

a literature value for the

by

DIN con28, se =

groundwater Hux

5070 kg/y of DIN is currently discharging from the Snug Harbor sub-watershed. To isolate DIN
originating at FWTP. we subtracted 554 kg of DIN reaching the
*

(1737

10 nrVy) (3). Thus.

atmospheric deposition, and septic

66% of the DIN discharged
1993 was lost en route to the harbor.

shore derived from

wastewater

from the

(3).

fertilizer,

Thus,

FWTP

in

we

calculate that

The conservative tracer method produced

Changes in the DIN/B were used to calculate N

similar

results.

based on the

loss

following formulas:

aquifer

was

243

to use

N 2 /Ar

used to calculate the temperature of the water entering the aquifer.

assumed that when the water entered the aquifer, both gases

were

in

atmospheric equilibrium

by which

N, measured

the

= (DIN

B measured )/B

effluent *

effluent

loss

(1

(Average (DIN measured)/

Average (DIN expected)))

100

13.3 fj.M respectively)

at

FWTP.

method corrects
from

were measured from the

This method estimates that

for dilution, but

fertilizers or

it

56%

of

last

holding pond

DIN

is

lost.

does not account for

atmospheric deposition and thus may

The
input

exceeded the amount

N (n = 18, se = 3.9), indicating that only

shore averaged 46.8
8% of the DIN in the effluent leaving the FWTP was denitrified

^M

within the aquifer. Because high rates of denitrification within the

aquifer should have been detected as N,, we believe that denitri-

as a possible site of

in the initial

interface,

100

where

of the aquifer.

N 2 gas can escape,

removal.

coupled with the DIN/B data from within the

that
denitrification within this aquifer is small and
plume, suggest
most of the removal may occur in the vadose zone, as has been
results,

reported previously

Expected and measured concentrations refer to shoreline samples

(Fig. IB), and DIN and B effluent concentrations (2017 juM and

The amount

temperature.

expected with atmospheric equilibration was used to estimate

denitrification within the aquifer. Values of excess N 2 along the

These

%DIN

at that

the water

in

is not a major process

However, we cannot ignore the

each wellpoint)

(at

gas

was

We

fication

DIN expected

N2

the water

ratios to calculate the excess

samples. The Ar content of

present in the shoreline

( 1 ).

This

consistent with the idea that dis-

is

solved organic carbon, a component of the denitrification process,

is largely consumed in vadose zones thicker than 5 m, therefore

allowing for little denitrification activity in the aquifer (8).

This research was supported by a grant from the NSF Research

Experience for Undergraduates (OCE-0097498).

slightly

DIN loss.
Using the DIN shoreline load measured in 1999 (3) compared
with 1989 effluent data, we calculated an 81% DIN loss. The lower
percent removal that we found in 2003, in addition to the two-fold
increase in DIN concentration in shoreline samples over 4 years
underestimate

Literature Cited
1.

2.

pM

in 2003). suggests that the efficiency of

109 fxM in 1999, 208
N removal may be declining over time. Alternatively, we may
have underestimated groundwater travel time, thus underestimat(

ing losses (Fig.

To

ascertain

data

show

that

in the

about

DIN/B

we used DIN/B

ratios

wastewater plume (Fig. 1C). The

of the DIN is lost between the
first

monitoring well. There-

ratios decrease little with distance traveled in

ground-

water, suggesting that the primary loss due to denitrification and

retention occurs either in the vadose zone, at the interface of the
aquifer and vadose zone, or during the

initial

100

of travel

J.

Kremer, K. Lajtha, M.

second approach to examining the loss of

and

Valiela. 1999.

I.

within the

Em-iron.

Cape Cod

2: 15-26.

Controls on Magnitude and Species Composition of Groundwater-Transported Nitrogen Exports From Glacial Out-

Program. Boston.
Jordan, M. J., K.

J.

Dissertation. Boston

Marine University

Nadelhoffer, and B. Fry. 1997.

Ecol. Appl.

864-881.

5.

Vogel, J. C., A. S. Talma, and T. H. E. Heaton. 1981.

191-200.

Kana, T. M., C. Darkangelo, M. D. Hunt, J. B. Oldham, G. E.

Bennett, and J. C. Cornwell. 1994. Ami/. Cliem. 66: 4166-4170

7.

Westgate, E.
Pabich, J. W.,
55: 247-268.

K. Kroeger,
221-223.

J.,

Biul. Bull. 199:

8.

DIN

Geist, B. Seely, J.

Kroeger, K. D. 2003.

7(3):

in the

aquifer.

4.

60%

point of effluent discharge and the

after.

G. Collins,

wash Plain Watersheds. Ph.D.

the loss occurred,

from the monitoring wells

DIN/B

I.,

Brawley, and C. H. Sham. 1997. Ecol. Appl. 7(2): 358-380.

Kroeger, K. D., J. Bovven, I). Corcoran, J. Moorman, J. Michalowski,

3.

A).

where

Valiela,

I.

Valiela,

W.

and H.

F.

J.

Pabich, and

Hemond.

2001.

I.

J.

Hytlrol. 50:

Valiela. 2000.

Bio K i-oclwinistry

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

244

Reference: Biol. Bull. 205: 244-245. (October 2003)

2003 Marine Biolor
ory

md

Nitro^c
J.

Speciation Through the Subterranean Estuary of Waquoit Bay, Massachusetts

2
2
2
K. D. Kroeger, A. Rago
M. C. Allen and M. A. Charette '*

M. Talbol'

MA

Boston University, Boston,

2

Fresh groundwater discharge

to coastal waters

gen transport

Woods Hole Oceanographic

is
1

).

an important vehicle for nitroIn near-shore aquifers, mixing

of fresh groundwater with saline pore waters produces groundwater of intermediate salinity that fluxes to sea as submarine ground-

water discharge (2). Discharge of this mixed water, known as a

subterranean estuary (3), may affect the rate of delivery of nitrogen
to the water column. In this study we estimated the flux of
+
dissolved inorganic nitrogen (NO," and
to the head of
4
Waquoit Bay through both the fresh and intermediate-salinity
portions of the near-shore aquifer and investigated the behavior of

NH

during transport through the subterranean estuary.

To quantify nitrogen concentrations in the aquifer,

we

collected

each location

above and below the mean

we

tide line

on

the beach.

At

collected groundwater samples at depth intervals

MA

Hole,

considering the linear relationship with salinity, such activity is

+
must
negligible. This conservative transport suggests that the NH 4
travel with intermediate-salinity

groundwater discharging to sea.

within the subterranean estuary, low concentrations in bay surface water, and high concentrations in fresh

Absence of

NO,

NO,~

(Fig.

discharges without mixing with the subterranean estuary.

~
that is mixing with the saline
3

Assessing the amount of

concentration

To

NO

difficult since nitrate tends to travel as a

is

the freshwater

in

endmember

estimate relative groundwater flux of

we

multiplied the average

We

About 20 groundwater samples from each

polyethersulphone
profile were obtained and stored frozen until analysis. Salinity was
measured by salinometer (4); pH. temperature, dissolved oxygen,

of 6500

and redox potential were measured oil-site with a YSI 600 multiprobe. Samples were analyzed colorimetrically for dissolved am-

discharge of 3400

AMS

filter.

monium and
chat

QuickChem FIA+ La(Zellweger Analytics, QuickChem

nitrate concentration with a

nutrient

auto-analyzer

8000 Series) within 3 weeks of

1A) increased with depth beneath

an area of fresh groundwater. This follows the Ghyben-Hertzberg
model of high-density seawater intrusion beneath the lower density

groundwater

present

This

(5). Nitrate

in the relatively

traveled as a

within the intertidal

ammonium

in

aquifer (Fig.

was

the

Freshwater flux of

IB).

plume and apparently discharged to the bay

zone. DIN was present almost exclusively as

Low

intermediate-salinity portion of the

concentrations were found near the salt-

across the head of the bay (Kroeger and Charette. unpubl.).

+
Low concentrations of
in Waquoit Bay surface water and
4

NH

groundwater

(Fig.

ID) suggested

that

ammonium

is

trans-

water

may be

flux to the

NH 4 +

in saline

ground-

explained by cation exchange and ion pairing, but

Corresponding author: mcluireiteis whoi.edu

in groundwater and bay surface waters (6). Total DIN

bay was calculated as the sum of the DIN flux through each

portion of the aquifer.

On

the basis of these calculations, the freshwater portion of the aquifer

discharged 6.2 kg N/day to the head of the bay.

intermediate-salinity zone

of the

total

These
ary of

DIN

was

flux to the

as

DIN

flux through the

kg N/day. which accounts for

20%

flux through the subterranean estu-

significant relative to

The DIN

ammonium,

.5

DIN

results suggest that

regenerated

head of the bay of 7.7 kg N/day.

Waquoit Bay may be

freshwater discharge.

transported

opposed

is

DIN

composed

flux

due

to

entirely of

to the terrestrially derived (7)

by fresh groundwater. Despite mixing of fresh and

saline groundwater masses (Fig. 1A). the subterranean estuary is not
nitrate transported

of net biogeochemical transformations of ammonium.

This research was funded by a National Science Foundation

site

Experience
to

I.

Valiela,

for

NSF

Undergraduates
grant

Woods Hole Oceanographic

site

(OCE-0095384)

Institution Coastal

Grant
to

(OCE-

M.A.C., and

Ocean

Institute

We

Postdoctoral Fellowship to K.D.K.

thank the Waquoit Bay
National Estuarine Research Reserve staff for use of their faciliSpecial thanks to Dan Abraham, Rachel Hutchinson, Ann
Mulligan, and Craig Herbold for their assistance with this project.

ties.
*

groundwater

radon activity

Research

conservatively through the intermediate-salin-

High concentrations of

A rate of intermediate-salinity

m Vday was calculated as the difference between an

+ saline) groundwater discharge rate of 9900

and the fresh groundwater discharge rate. Total groundwater
discharge to the head of the bay was based on mass-balance-derived

0097498)

(Fig. ID).

calculated using a freshwater discharge rate

m Vday

ammonium moved
zone

was

estimated total (fresh

ported into the subterranean estuary by advection through marine

sediments where organic N is mineralized. Our data show that

ity

m /day, determined from hydrological measurements of head

and Hutchinson, unpubl.).

the reducing

1C).

from an along-shore

head of the bay (Kroeger and Charette, unpubl.).

gradient and hydraulic conductivity at our sampling sites (Mulligan

most abundant DIN species

oxidizing fresh groundwater (Fig.

fresh groundwater interface and approached a uniform concentration with depth. These results are consistent with collections from

in fresh

transect across the

estimates of submarine groundwater discharge using measurements of

collection.

In each profile, salinity (Fig.

fresh

to estuarine surface

DIN

concentration in each portion

of the aquifer by estimated rates of groundwater advection through
each zone.
calculated average DIN concentrations using our
waters,

collections plus previously collected samples

tn a

plume and

not clear.

is

depth of 7 to 8.5 m below the land surface.

Samples were collected using an
Retract-a-Tip piezometer
with a peristaltic pump and filtered through an in-line 0.45-/nm

of 0.15 to 0.6

NO

IE) suggest loss of

3
by denitrification
within the reducing conditions of the saline groundwater. However, it may be that much of the fresh groundwater containing high

groundwater

groundwater

groundwater samples from four locations on the shore of Waquoit

Bay along a 12-m transect that was perpendicular to shore and
extended 6

Woods

Institution,

RCOLOGY AND POPULATION BIOLOGY

245

d)

12m

Ammonium
60
PZ1

50

OPZ2

40

OPZ4

PZ3
Bay Water

f.30
20
10

ol30

20

10

Salinity

e)

Nitrate
250
PZ1

200 I

DPZ2

150 8

OPZ4

PZ3

- 2

Bay Water

100 ,
50

I
30

20

10

Salinity

C)

Ammonium
I

_6
- s
Behavior

1.

Figure

<>

beginning

of nitrate anil

above mean

in

title

ammonium

in

line for (A) salinity. IB)

near-shore aquifer. Cross-sectional contour plots based on depth profiles collected in a transect
+
NO,~ concentration, and(C) NH 4 concentration. Concentrations of(D) ammonium and (E) nitrate

\ersus \alinity fur each profile. Different symbols indicate samples collected al pic:nmeter locations

and

of

this figure. Filled

diamond \\mhol\

indicate average

ammonium

(.1.3

Valiela,

I.,

K..

Foreman, M. LaMontagne, I). Hersh, .1. Costa. C.

P. Peckol, B. DeMen-Andrcsun, C. Sham el

al.

1992.

Estuaries 15: 443-457.

D. A. Barry, F. Stagnitti. and J.-Y. Parlange. 1999.

Kcso,,r. Res. 35: 3253-32.^).
Li. L..

3.

Moore, W.

S. 1999.

Knapp. G.

P.,

M.

Mar. Chem. 65:

C. Stalcup, and R.

J.

Stanley. 199(1.

Freeze, R. A., and

A. Cherry. 1979.

6.

Edgewood Cliffs. N.I. Pp. 375-378.

Abraham, D. M., M. A. Charette, M. C.

J.

in

panels A. B.

sin-face water.

Institution.

Woods

Grouiuhmter. Prentice Hall,

Allen, A. Rago,

and K. D.

Radiochemical estimates of submarine goundwaiei

discharge to Waquoit Bay. Massachusetts. Kiol. Bull. 2(15: 246-247.
Valiela, I., G. Collins, J. Kremer. K. Lajtha. J. Geist, B. Seely. J.

Kroeger. 2003.

7.

Technical

Waqnoil Bay

5.

\\atcr

1-12?.

in

Woods Hole Oceanographic

D'Avanzo, M. Babione,
2.

and 4 from 0-12 m. depicted

Report WHOI-90-35,
Hole. MA. 25 pp.

Literature Cited
1.

2. 3,

I,

/xMj and nitrate (1.2 JU.M) concentrations

Braley, and C. H. Sham.

1997.

Ecol. Appl. 1:

358-380.

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

246

Reference: Bio/. Bull. 205: M6-247. (October 2003)

2003 Marine Biological

<D

Estimates of Submarine Groundwater Discharge to Waquoit Bay, Massachusetts

D. M. Abniltiun
M. A. Charette2 '*, M. C. Allen 2 A. Rugo', and K. D. Kmeger

Radioed--

.i,

Bowdoin College, Brunswick, ME

Woods Hole Oceanographic Institution, Woods Hole,
1

Submarine groundwater discharge (SGD) is the flux of fresh and

brackish groundwater to the ocean through a coastal aquifer
).
Groundwater often transports large amounts of anthropogenic ni(

(A)

MA

Falmouth,

MA

(B)

trogen and phosphorus to the ocean, and understanding the magnitude of groundwater flux is important to the environmental
protection and

management of coastal waters

have employed radium isotopes

enriched

rally

in

groundwater

high uranium content

coastal systems.

(2. 3).

and radon-222

(4)

Recent studies
both natu-

(5).

relative to surface water

in aquifer

sediments, to quantify

due

to a

SGD

However, these studies have been limited by

in

poor understanding of the distribution of these isotopes in coastal

groundwater. the processes controlling these distributions, and a
lack of high-resolution time-series sampling of tracer activities in

we

surface waters. Here,

tations by

and ~~"Ra

mapping

U l/2

1600

==

SGD

dependence of

222

Rn

(r,,,

3.83 days)

in

in situ

222

Rn

At each sampling depth. 22 "Ra was extracted from -151

of groundwater on a column of Mn-impregnated fiber. In the
).

laboratory, the liber

was ashed and

For

22

''Ra quantified via

gamma

ray

222

Rn. 250 ml of groundwater was collected

in a glass bottle and directly
analyzed on a Durridge RAD7
electronic radon monitor; activities were
decay-corrected to the
spectroscopy

(6).

To quantify tracer concentrations in surface

226
water, discrete bay water samples for
Ra were collected at four
locations at the head of the bay. Continuous measurements of
time of collection.

~2

Rn

at a

using the

single station

RAD7

the

at

coupled
Burnett and Dulaiova

head of Waquoit Bay were obtained

to an air-water equilibrator as

described

The instrument recorded the 222 Rn

activity of surface water every 30 min for 3 days. Tidal height,
measured as water column depth to the sea floor at the sampling
location, was monitored with a YSI 600 series CTD. and local
in

atmospheric conditions

weather tower (#
''Ra

(5).

were obtained from

BUZM3.

nearby

activities

(<3 dpm

'

observed

at salinities

of 25

or greater arc from the landward

portion of the transect. This is in
contrast in the 'ypical distribution of radium in estuarine surface
waters,

when

.livities are

elevated

at all salinities

>0

and often

site ini.v .situated

sampling

u/ong the heath at the head of the ba\

(B).

'

Siirftuc

measurements

lo the

measurement

of rtidiiiin-22f> activity, a continuous

at radan-222. iiiul a transect a/ four depth profiles oriented

heach u-ere used

to quantify

transect of 4 depth profiles).

revealed a sharp increase

*

SGD

from

related to ion

exchange

(B,

A =

--''Ra,

perpendicular
222
Rn, * - **

O=

'

verticil/

in salinity

tC. contour plot

111

cross .sectiim of xroiintlwater

the freshwater-saltwater interface

to **).

(7).

The

pattern

we observed could be

22<1

Ra source with increasing depth and

explained by an increased
distance from shore, a function of either higher sediment content of
22(1
thorium-230 (radiogenic parent of
Ra) or a decrease in the grain
size of aquifer sediments.

This

latter

scenario would result

in

greater sediment surface area, thereby increasing the availability of

radium

for desorption.

Waquoit Bay
bilities

The
that

Buzzards Bay. MA).

in

The low

site

NOAA

the groundwater increased with increasing

activity
salimt): the highest activities occurred above a salinity of 25 (Fia.
2a).

1.
Lucullan of It tii/uoir Bay. Massachusetts, and our
sampling
during the summer of 2003. Wai/noil Ba\ is located <ni the cmicm
harder of the town of Ftilitnnil/i. Massachusetts, un Cape Cod (A). Our

Figure

Waquoit Bay.

cross section of each isotope at the groundwuter-seawater interface

1

a
a
a

Waquoit Bay

analyzer to study

Using a drive-point piezometer, four depth profiles of ground226

water
Ra and 222 Rn were collected along a transect perpendicular to the shore at the head of Waquoit Bay.
creating a vertical
(Fig.

years) across a groundwater salinity

gradient, and by deploying a new

the time

-~

attempt to overcome these earlier limi-

the distribution of

Distance (m)( c )
4
8
12 **

of

is

is

However, sediment sampling

at the

head of

necessary to determine which of these two possi-

the nmst likely explanation for the observed pattern.

222
Rn with salinity was similar to
groundwater

pattern of
~2

"Ra, with ~3-fold increases in activity at salinities higher

222
Rn also displayed an increase in activity with

than 20 (Fig. 2a).

increasing depth and distance from the

is

a noble gas,

its

berm

(Fig. 2b).

Because Rn

distribution throughout the aquifer should be

uniform, and not affected by changes

in salinity.

Also, because the

residence time of fresh groundwater beneath the bay

years (8), the activities of

222

Rn

in this

is

several

zone are likely to be

in

22

peak

at

intermeJi

salinity

(15-20) due

to a desorption reaction

"Ra.
equilibrium with the sediment activity of its parent isotope
222
Therefore, the higher observed activities of
Rn in the saline
22

Corresponding author: mdiarette&whoi.ecJu

"Ra source,
portion of the aquifer must be a function of a greater
which is consistent with the observed 22<1 Ra distribution.

ECOLOGY AND POPULATION BIOLOGY

8000

1t^

247

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

248
Reference: Bio/. Bull. 205:
2003 Marine Biological

249. (October 2003)

21

;.,

oratory

Ponds
importance of Metabolism in the Development of 3Salt Marsh
2
V.
Valentine^
and
S.
C.
R.
Cavatorta
M. E. Johnston' *, J.
Hopkinson
,

'

MA

^Marine Biological Laboratory, Woods Hole,

Ponds are a

common

feature on the salt

marsh surface and are

depressed regions of the high-marsh platform

).
They are widely held to be valuable habitat for larval and
Pools of standing water
juvenile fish as well as for avifauna.
a
not
receive
that
do
regular supply of sediment
develop in areas
typically found
(

in

with flooding. This standing water often becomes hypersaline and

anoxic. thus inhibiting marsh grass production and further exac-

where the rate of marsh

erbating marsh accretion (2). In areas
of
sea-level
rise, the formation of
the
rate
less
than
accretion is
interior

marsh ponds

marsh degradation

is

expected to increase, thus contributing to

(3).

that

formed

of the

Plum

50 years in about a 1 -hectare (ha)

marsh was examined along the Rowley River

in the past

area of intertidal salt

Island

dissolved oxygen emphasizes the extreme conditions of these

in view of the
ponds as habitat. These conditions seem paradoxical

assumed value of these

The

rates

6.98 g O-,
3.6

to 6.9

in the

Plum

O,

in

Sound estuary

in

northeastern Massachusetts

investigated using two techniques: free-

ponds at '/2-h intervals for

durations of 3 to 5 days during June and July of 2003. DO was
measured using a pulsed, polarographic O : electrode, which is
corrected for temperature and conductivity (YSI, Inc.). Rates of
change in DO attributable to gross primary production and respi-

oxygen (DO) was measured

in three

were corrected for diffusion across the air-sea interface

to wind speed (5). Net

using a gas transfer velocity proportional
the balance between gross
as
was
calculated
ecosystem production

primary production and respiration over 24-h

Sediment cores 15.5 X 50 cm) were taken
(

edge and the center of a pond and

measure the respective benthic respiration

intervals.
in the field

from

the

returned to the laboratory to

cores (10

X 50 cm) was

rates.

Another

set of

used to determine the average carbon

content of the peat in the pond environment by drying and coma

busting peat samples of known depth and volume and assuming

d~

'
.

These

found

rates are similar to those

Sound estuary (J. Vallino. Marine Biological

comm.) and eutrophic estuaries in general (6).

ponds

tion indicates that

negative value for net ecosystem producis consumed than produced

more organic matter

is

the

estimates of metabolism
highly organic marsh sediment. Our

are

internally (7).

The most

likely source of this organic matter

be conservative during this time of year to the extent that

anaerobic products of sulfate reduction are stored temporarily in
likely to

anoxic sediments

water diurnal changes in dissolved oxygen, and oxygen consumption of sediment cores. In the free-water technique, dissolved

ration

average, rates of respiration exceeded gross primary production

such that net ecosystem production was negative 8 out

the

(4).

Pond metabolism was

larval organisms.

Island

Laboratory, pers.

On

and

'

habitats to juvenile

of gross primary production ranged from 2.75 to

"
:
d
in the ponds, and the rates of respiration from

of 12 days (Fig. Ib).

This study focuses on pond metabolism, measured by dissolved

oxygen changes, as a possible mechanism of marsh pond expansion, as well as an indication of habitat quality. A network of

ponds

PA

The Pennsylvania State University, University Park,

Amherst College, Amherst. MA

Decomposition

(8).

rates of root

and rhizome material decreased

with depth, indicating that peat lability decreases with depth (Fig.
Ic). Respiration rates in benthic cores were higher in the deep
center of the pond than along the shallow edge (Fig. Id).

On

the

we expected

basis of root and rhizome decomposition, however,

to

see lower respiration rates in the benthic cores taken from the
center of the pond than in those taken from the edge where the

remains of recently dead macrophytes were evident. Higher rescan be attributed to the presence
piration rates in the pond center
of Emenimorpliii. This alga grows around the periphery of the
the deeper, more central
ponds, and its remains tend to collect in
decreased following
portions of the ponds. Benthic respiration

from the
highly labile, detrital Enteromoipha layer
Center
Id
labeled
2*).
second center core (Fig.

removal of

this

The importance of excess

respiration as a

mechanism

contrib-

our
pond enlargement over time can be seen by comparing
measurements of net ecosystem production with our independent

uting to

estimates of the rate of pond formation. Average depth-integrated

of carbon to organic matter of 0.5:1. To investigate the

relative lability of organic matter from varying depths, root and

carbon content of marsh peat surrounding ponds

rhizome material (5 g wet weight) from depths corresponding to

the sediment cores was placed in BOD bottles with seawater (297
ml) and a bacterial/sediment inoculum (3 ml), and respiration was

average pond depth of 20 cm (data not shown),

2
decomposition rate of 154 g C m~ y"

measured.

of organic and
pond depth: peat decomposition and accretion
to the ponds. Because the
inorganic material in the marsh adjacent

ratio

Dissolved o\\gen levels

(Fig. la).

ponds fluctuated greatly over

from 0<7r to 200% of saturation

in all the

a 24-h period, with values ranging

Along with high temperatures and

salinity, this

range of

38.446 g

m"

3
.

Assuming

maximum pond

Corresponding author: cnejl36@psu.edu

approximately
age of 50 y and an

we

estimate a peat

However, two mechanisms

areas of

marsh surrounding

assume

that the surrounding

are responsible for the increase in

ponds are not yet inundated, we can

marsh has been accreting sediment at

the

a rate comparable to that of sea-level

*

is

locally

(9).

rise,

Accordingly, the adjacent

which

is

2.65

mm

marsh has accreted by

at

249

ECOLOGY AND POPULATION BIOLOGY

0:00

0:00

DO

Rates

hr'

m
2
Respiration

Peat

(g

Cone.

DO

Saturation Cone.

Pond

Pond 2

Pond 3

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

250
Reference: Biol. Bull. 205:
2003 Marine Biologic

251. (October 2003)

Nutrient Supply
Isotopic Evidence of the Relative Effects of Ambient and Internal
on the Growth of Ulva lactuca
3
2
1
M. Teichberg '*, S. For*, and I. Valiela*
A. B. Agniar J. A. Morgan
id

Transpb

Lafavette College, Easton, PA

Yale University, New Haven, CT

2
3

Growth of macroalgae

Boston University Marine Program. Woods Hole,

in coastal

environments

is,

to a large

extent, limited by the available supply of nitrogen (1.2). Macroalinternal pool of

gal growth rates may be influenced by their

nitrogen, and also by the supply of new nitrogen provided by

ambient water (3). Few studies have investigated the relative role

MA
from Sage Lot Pond were

rates of U. lactuca collected

significantly

lower than those achieved by fronds collected from Childs and

Quashnet rivers when transplanted into the Childs River (ANOVA,

F =

13.66,

P = 0.000017). An ad hoc Duncan's

test

showed no

differences between growth rates of algae collected from the

when

transplanted into Childs River

rates of fronds from all

of internal and external nitrogen supply on growth of macroalgae.

In this study we investigate the net growth of Ulva lactuca, a

Childs and Quashnet rivers

widespread opportunistic species of macroalga found in the estuaries of Waquoit Bay. Cape Cod. Massachusetts, in response to
internal and external nitrogen supply by using a field transplanta-

three estuaries transplanted into Sage Lot

experiment and isotopic measurements (4).

Fronds of U. lactuca were collected from Sage Lot Pond.
Quashnet River, and Childs River, subestuaries in the Waquoit

nutrient-poor water grew slowly if at all. even when transplanted

in nutrient-rich estuaries. Second. U. lactuca fronds from nutrient-

tion

Bay

estuarine system.

these estuaries differ

The land-use
enough

to

patterns

on the watershed of

lead to substantially different

nitrogen loads of 14, 350, and 601 kg N ha" y~ ', respectively (2).
Fronds collected from these three estuaries will therefore have
'

different nitrogen regimes and have entered the

with
different internal nitrogen contents. Two fronds
experiment

grown under

(ANOVA. F =
cantly different

suggest

rich

0.

(ANOVA. F =

that, first,

estuaries

P =

2.64.

).

Growth

0.71.

Pond were not

P =

fronds of U. lactuca grown

grew

in

an estuary with

and more so when transplanted

faster,

nutrient-rich estuaries. Third, fronds

lag considerably, even

signifi-

0.40). These results

when

in

from nutrient-poor estuaries

transplanted into nutrient-rich water;

evidence of some other impairment of growth.

To further examine the effects of internal and external nitrogen,
we plotted net percent growth of U. lactuca in Childs River and

this

is

Sage Lot Pond versus

initial

percent nitrogen

in the

fronds (Fig.

from the same originating estuary were weighed (blotted wet

weight) and placed inside cages constructed out of transparent
disposable containers (GladWare) with two sides covered by mesh,

which
place
not surprising because initial percent nitrogen in the fronds was
above 0.71. the minimum <7r nitrogen content in the tissue of U.

allowing for water flow.

To assess the effect of ambient nitrogen supply on growth of U.
lactuca. the algae collected from each estuary and placed in the

lactuca required for growth (3).

cages were then transplanted into either Sage Lot Pond, which has
the lowest nitrogen load (and hence the lowest nitrogen supply for
fronds (5)). or Childs River, which has the highest nitrogen load
(and highest supply of nitrogen), with 30 cages per estuary. The
m apart within the existing macroalgal
cages were randomly set
1

above the bottom in locations with salinities

canopy, and 0.2
between 25 and 30 ppt. This experimental design was deployed
from 18 to 27 June 2003 and was repeated from 17 to 24 July 2003.

We

growth response of U. lactuca by deterand

final
wet weights of each algal frond within
the
initial
mining
each cage. Growth data from both runs of the experiment were
pooled to span possible differences across the months. Fronds were

measured the

net

dried at 60

C, ground, and sent to the Stable Isotope Facility.

University of California, Davis, for analysis of carbon and nitrogen
content and stable isotope signatures.

Net growth

rate

of U. lactuca depended on both the nitrogen

pool within the fronds, obtained from the estuary from which the
fronds were collected, and the nitrogen supply provided by the
estuary to which the fronds were transplanted (Fig. 1A).

Growth

IB).

Some growth took

Corresponding author: mirta?'bu.edu

three estuaries (Fig. IB),

is

The initial nitrogen content of

fronds from the nutrient-rich estuary was 3-fold larger than that of
fronds from the nutrient-poor estuary (Fig. IB). The steeper slope
of the growth rate for fronds transplanted into Childs River (black
rates of U. lactuca
points in the figure) suggests that net growth
were more affected by external nitrogen supply than by internal

nitrogen content, as found in studies on other macroalgae (1. 6).

The relative importance of external nitrogen supply and internal
nitrogen content were corroborated by the isotopic signatures (Fig.
1C). Fronds initially differed markedly in carbon and nitrogen
isotopic signatures; signatures in fronds

transplanted into Childs River

from Childs River and

were notably different from those of

Sage Lot Pond fronds transplanted

into

Sage Lot Pond

(Fig. 1C).

Transplanted fronds soon reflected the signature of the nitrogen

the

estuaries

in

which they were incubated, and showed

influence from the estuary of origin. Although the

initial

in

less

percent

Pond fronds (Fig.

nitrogen may have slowed growth of Sage Lot
1A. IB), the nitrogen pools of the fronds from Childs River and
from Quashnet River showed
1C).

Some

fast

responses to the ambient nitro-

factor other than internal pool size

gen supply (Fig.

must be responsible for the lag in growth of Sage Lot Pond fronds.
KS
The range of fi N between Childs River and Sage Lot Pond has
been reported

in all

(7).

and

is

associated with different land-derived

nitrogen loads, bearing different S

IS

signatures, arriving

from the

ECOLOGY AND POPULATION BIOLOGY

Transplanted

CR

SLP

SLP

12

Nitrate concentration (LiM)

>,

251

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

252
Reference: Biol. Bull. 205:
2003 Marine Bioloi'i

Relative

-253.
>l

October 2003)

.iratory

of Grazing and Nutrient Supply on Growth of the Green Macroalga Viva lactuca in
Estuaries of Waquoit Bay, Massachusetts
J.

A.

Morgan',

A. B.

Aguiar

S.

Far M.

Yale University,

and

Teichberg~\

New

Haven,

I.

Valielci"

CT

-1

Nitrogen supply
roalgae

1, 2.

1.

is

Lafayette College, Easton, PA

Boston University Marine Program, Woods Hole,

a major control on growth of coastal

Top-down

effects in

which grazing

mac-

significantly

and nutrient-grazer interactions (3) have

macroalgae
also been described. In this paper we describe an experiment in
affects

(4, 5).

growth of a common macroalga, Ulva

treatments that allowed different numbers of grazers to

which we measured
lactuca, in

net

access fronds as well as incubation of fronds in estuaries with

the

MA

end of the incubation. The grazers were sorted into four groups,

amphipods. shrimp, crabs, and isopods. The

ers in the

estuaries than the

F =

total number of grazwas

cages
significantly lower in all three
number in the 4-mm cages (Fig. la; ANOVA

1-mm mesh

P = 0.0014). The number

31.0.

18-mm mesh cages was

of grazers found in the

also significantly different, although they

contained lower grazer abundances than the

-mm

and

4-mm mesh

demonstrably different nutrient supplies. These treatments were

intended to assess the relative influence of grazer and nitrogen

cages (Fig.
with IS

supply on net growth rates of a coastal producer.

To examine the effect of grazing on growth of U. lactuca.

decreasing grazer abundances. This possible effect of predators on

grazers suggests that there might be important top-down cascade

constructed acrylic plastic cages with sides of 1-mm,

4-mm,

we
or

8-mm

mesh. The different mesh openings were intended to allow

entry to different numbers of grazers, which we took as a proxy for
grazing pressure. The cage design also allowed for light penetra1

and horizontal water flow. The 18-mm mesh permitted larger

size classes and a greater number of grazers to enter the cages,
tion

1-mm mesh excluded

while the

larger size classes

and allowed

fewer grazers. The 4-mm mesh was intended to furnish an

mediate grazer treatment.

inter-

and large shrimp entered the cages

Predatory
mesh and likely fed on the smaller grazers, thus

mm

effects in this

The

fish

la).

system waiting to be studied.

difference in nitrogen load in the three estuaries provided

quantitatively different nutrient supplies, as evident in the nitrate

concentrations cited above. Bottom-up effects from these different

nitrogen supplies on net growth of U.
factors. Rates of net

larger external nitrogen loads (Fig. Ib;

of macroalgae to nitrogen supply

experience different nitrogen loads

Sage Lot Pond. 14 kg
ha y
350
ha
and Childs River. 601
River.
Quashnet
kg
y
from their watershed (6). These nitrogen loads led to
kg ha~'y~'

effects of nutrients (Fig. Ib).

in estuaries

ANOVA

F =

receiving

P <

61.8,

0.001). Percent net growth of U. lactucu in Childs River was

almost three times that in Sage Lot Pond (Fig. Ib). This response

To evaluate the effect of nitrogen supply and grazing on algal

growth, cages with the three mesh sides were placed in three
estuaries in Waquoit Bay. Massachusetts. These three estuaries
;

lactuca were dominant

growth were higher

is

similar to that reported by

others (2).

Top-down

1-mm

effects

caused by grazers were small compared

Across all estuaries, net growth

(fewer grazers) cages was higher than growth in

to the
in the

4-mm

the estuaries

(more grazers) cages, but these differences were statistically insignificant. In contrast, there was more than a 200% increase in

during July 2002. one year prior to the time of our experiments:
1.75 /nM for Sage Lot Pond. Quashnet River, and

these results suggest that the direct and indirect bottom-up effects

mean

different

0.04, 6.1, and

nitrate concentrations

measured

in

Childs River, respectively (G. Tomasky. Boston University Marine

Program, unpubl. data). To minimize effects of differences be-

tween estuaries other than our treatments, we chose

percent net growth caused by the nutrient effects.

associated with nitrogen loading were

down

effects of grazing

much

Taken

together,

larger than the top-

on the net growth of U. lactuca.

sites similar in

Increased nutrient inputs might affect the composition of the

and algal composition. In each estuary we placed

four replicates of each of the three grazing pressure treatments, for
a total of 36 cages.

grazers found in the three estuaries (Fig. Ic). Shrimp (Palaemon-

salinity, depth,

etes sp.) and

grazers

in

amphipods (Gammaridea) accounted for over 85% of

cages. Shrimp were far more abundant in high

all

Three fronds of U. lactuca, each approximately 300 mg (blotted

wet weight), were suspended inside each cage. To measure the

nitrogen load conditions (Childs River), while amphipods were

top-down versus bottom-up factors, we measured net

growth as the dependent variable. Net growth was the growth
achieved by the fronds minus the biomass consumed by grazers.

Fig. Ic).

effect of

To determine
initially

net growth, the U.

(blotted

lactuca fronds were weighed

after 10 days of field

wet weight) and again

incubation.
First,

we

assess the successed of the treatments.

To roughly

measure the grazing pressure, we sorted and counted the potential

grazers found in the cages for two replicates of each treatment at

more

plentiful in lower nitrogen load conditions (Sage Lot

Shrimp

Pond;

are predators as well as grazers (7). so they could

have fed upon amphipods. Similar

have been reported elsewhere (3.

shifts in species
8).

composition

Increased anthropogenic

supplies of nitrogen might therefore not only change the growth

rates of U. lactuca directly, but also alter the relative

abundances

of consumers and lower abundances of grazers. This effect is

possibly linked to more frequent anoxic and hypoxic conditions in
the

more nitrogen-loaded

To

estuaries such as Childs River (9).

further assess possible grazer effects,

we

plotted percent net

ECOLOGY AND POPULATION BIOLOGY

Mesh opening

(mm)

253

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

254
Reference: Biol. Bull 205:
2003 Marine Biolnu;

Stable

2'

(October 2003)

5.

,.uory

Assessment of Site Loyalty and Relationships Between Size and Trophic Position of the
Atlantic Horseshoe Crab, Limulus polyphemus, within Cape Cod Estuaries
2
2
2
C. W. O'Connell,' S. P. Grotty,- A. S. Leschen, R. H. Cannichael, and 1. Valiela

Carmichael

Bay, Cape Cod, Massachusetts, and also found that these animals
forage within relatively small areas of estuaries. In this study, we
used

measurements and

field

stable isotope analysis (2) to deter-

mine whether the trophic position and site loyalty described by

Carmichael ci a/.
in Pleasant Bay were also found in two
additional Cape estuaries. Stage Harbor and Barnstable Harbor,
(

and

determine

to

how

the trophic position of horseshoe crabs

Increased population densities and the resulting anthropogenic

wastes within watersheds increase nitrogen loads to estuaries (2).
Pleasant

and has

Bay
a

is

Cape Cod National Seashore Reserve

part of the

low nitrogen load due to the lack of developed land in its

).
Stage Harbor and Barnstable Harbor have relatively

watershed

greater population densities in their watersheds, and as reported

in Cape Cod (2), should have greater relative nitrogen
The land-derived nitrogen loading rates to the three estuaries

elsewhere
loads.

>

Pleasant

Bay

(3, 4).

we opted

their

ences

Barnstuble

Since increased urban development

results in heavier nitrogen signatures of estuarine

(2),

>

Harbor

are likely to rank in the order of Stage

Harbor

to study these estuaries

water and biota

We

2003

collected samples during July

Stage Harbor

sites in

in

at

a site in Barnstable

Cape Cod.

We

measured

prosoma (7). To obtain samples for isotope analysis, we sampled

tissue from the last two segments of the second or third walking
of horseshoe crabs,

we

( 1 ).

To

identify

some

potential foods

sampled quahogs (Mercenariti nwr-

also

tenaria). polychaetes (Nereis sp., Nephtys sp., and Glvcera sp.),

seston filtered from

1

water samples, and sediment from two

1-1

0-ml sediment cores taken 3

single sample. All samples

cm

I5

deep, which were pooled into a

were dried

at

60 C C, ground, and

sent to

I5

the 8

and sediment, judging from

8

13

axis.

The carbon

in

their position relative to values

seston and sediment

was

on the

likely initially

derived from phytoplankton and macroalgal organic particulates

(gray lines in Fig.

la)

(H).

Polychaetes seemed to belong to a

values are

Bay
horseshoe crabs consumed
(

).

values for each taxon (shown by points within dashed

seem

ovals. Fig. la)

be heavier

to

in

Stage Harbor than

in

Barn-

from the different land-use

patterns, heavier land-derived nitrogen sources

from freshwater

inputs into Stage Harbor (5, 6).

Isotopic signatures of individual crabs varied considerably. Part

of the variation was associated with different crab size; larger

I5
crabs tended to have significantly heavier 8 N signatures (F =

P = 0.004) and

9.5,

0.0005

(Fig. Ib. c).

lighter 8

I3

values (F

The increased 8 I5 N

to increased prey size

signatures

13.94.

may

be related

The slopes of

larger crabs (9).

among

==

the

regression lines in Figure Ib did not differ, but the intercepts were
significantly different

Ic,

(ANOVA: F =

between the regressions shows

offset

l;i

we added

width and 8

I5

9.26,

P =

that Stage

0.004). The

Harbor crabs had

signature than Barnstable Harbor crabs. In Figure

a gray reference area that represents the prosomal

Bay (1). the most

The Pleasant Bay values were

values for crabs from Pleasant

of the three estuaries.

pristine

appreciably lighter than those of either Stage Harbor or Barnstable

Harbor (Fig. Ib). These isotopic comparisons suggested that the

watershed-derived nitrogen loads (and contribution by wastewater)
were greater in Stage Harbor than in Barnstable Harbor and that
both these estuaries received greater anthropogenic nitrogen loads
than Pleasant Bay.

The

significant difference in the isotopic signature of horseshoe

crabs collected in the three estuaries can also be interpreted to

mean

remain foraging in an estuary long enough

values characteristic of the estuary in which they

that crabs tend to

to acquire 8

I5

were found. These

riods of time.

the horseshoe crabs suggest that their

included a mix of polychaetes and quahogs (Fig.
Quahogs. in turn, assimilated carbon from a mixture of seston

S'

15

mass spectrometry.
The 5' 3 C signatures of

may have

These

of 2%c-4%P (Fig. la) (2). 8 N values for bivalves and polychaetes

were 2%o-5%c heavier than those of paniculate matter. In all cases,

in spite

la).

la).

combination of quahogs. polychaetes. and paniculate organic matter, given the expected fractionation from food source to consumer

the Stable Isotope Facility. University of California, Davis, for

diet

(Fig.

signatures suggested that the

in

the size of horseshoe crabs as the width at the widest region of the

leg of adult horseshoe crabs

a heavier 8

with different land use

watersheds to take advantage of potential resulting differI3

in S'^N and 8 C signatures (5, 6).

Harbor and three

macroalgae and Spartina grass

consistent with carbon signatures found in Pleasant

stable Harbor, suggesting, as suspected

as adult crabs grow.

might change

MA

Boston University Marine Program, Woods Hole,

used stable isotope analysis to define the

food webs of horseshoe crabs, Limulus polyplieniitx. in Pleasant
el al.

PA

Bucknell University, Lewisbitrg,

results corroborate earlier observations

( 1 )

that

of the evident mobility of horseshoe crabs, they do tend to

remain within relatively circumscribed areas for considerable peI3

The S C values provided

additional

information about the

foraging and feeding behavior of horseshoe crabs (Fig.

became

lighter as size of crabs increased

0.0005)
shifted

food

(Fig.

Ic).

As

to values

13.93.

adult horseshoe crabs grew,

from values near the

web

(F

8'

Ic).

I3

C
-

signatures

for the Spartina-based side of the

more closely associated with phytoplankton

separate branch ut the food web. since they had relatively heavier

(dashed boxes

signatures, and most likely incorporated a

as adult crabs grew, a larger percentage of their diet consisted of

mix of carbon from

in Fig. Ic.

and

Fig. la). This transition suggests that

ECOLOGY AND POPULATION BIOLOGY

a)

Phytoplankton

14

b)

Macroalgae

255

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

256
Reference: Binl. Bull.
2003 Marine Bin!.

Incuh-

:;'

7.

(October 2003)

.atory

onditions of Forest Soil Yielding

C.

Soil

is

Maximum

dominant long-term sink for

in forest

ecosystems

With increasing atmospheric N deposition in forests caused

combustion
of fossil fuels (2. 3). concern over effects of N
by
on
loading
ecosystem sustainability is substantial. Net primary
(

1 ).

productivity

forests (4). but

immobilized

be limited by N in most temperate

of the N from deposition appears to be

thought to

is

much

in the

taken up by plants

organic matter of the

(

).

Dissolved Organic Nitrogen Concentrations

and Minimal Residual Nitrate

E.
Walker,
Davidson,* W. Kingerlee, and K. Savage
Woods Hole Research Center, Woods Hole, MA

soil rather

than being

However, the mechanisms of

N immo-

bilization as well as the bioavailability and ultimate fate of

residual nitrate. At the end of each incubation period.

60 ml of

water was added to the saturated samples and 30 ml to the

unsaturated. All samples were shaken and immediately filtered

Whatman filter paper. Filtrate from each incuwas analyzed for nitrate, ammonium, and total N using a
Lachat 8000 flow-injection autoanalyzer with in-line persulfate

through No.

bation

digestion (7.

DON

8).

was calculated by subtracting

ammonium from

and

nitrate

total

the

sum of

N.

Nitrate concentrations in soil extracts remained at

low values

organic N remain poorly understood. One hypothesized pathway of immobilization is abiotic reduction of nitrate to nitrite

throughout the experiment in incubations under saturated and

unsaturated conditions with no added nitrate (Fig.
In incuba).

and reaction of

tions with

with dissolved organic matter to produce

dissolved organic nitrogen (DON) (5). Studying the fate of
DON would be facilitated by the development of a I5 N label in
a realistic soil

nitrite

DON

product. In the present study,

we

tested

incubation conditions that would permit nitrate immobilization

during 24 h, followed by conditions that would mini-

to

DON

mize the remaining nitrate. Water content and N concentrations

were varied in 1-8-day incubations of organic horizon to study
the dynamic of nitrate reduction and DON formation.

was collected from

Soil for these incubations

the

humus

(O,)
layer of a forest stand dominated by oak at the Harvard Forest.

Massachusetts. The stand developed after agricultural abandonment at the end of the 19th century and after a devastating
hurricane

2-mm

in

1938

sieve to

(6).

remove

The

soil

samples were passed through a

large roots.

were placed in 60-ml serum

combination of two treatments
ml of either deionized water or

Subsamples of 10 g (n = 64)
which then received a

bottles,

in a factorial design. First.

3.33

nitrate solution (50 /xg

N/g dry
was added. Second. 30 ml of deionized water was

soil as

KNO 3

added

to half

added

nitrate,

concentrations increased slightly and

similarly under saturated and unsaturated conditions during the

first

day.

after

which they declined

remainder of the

for the

experiment. Incubation time, addition of

and the interaction

nitrate,

of time by nitrate were significant (P < 0.01 in an analysis of

variance. Saturation was also found to be significant (P < 0.05),
as nitrate concentrations were lower in saturated incubations com)

pared to those remaining unsaturated.

Ammonium concentrations in soil extracts increased
fashion, with day 8 values higher (close to 74 jug

in a linear

N/g dry

with

soil

or without added nitrate) in incubations under saturated conditions

than those remaining unsaturated (53 fig N/g dry soil with and 46
^.g

soil

N/g dry

without added

ration apparently

enhanced

nitrate). In

denitrification

summary, water

satu-

and either stimulated

mineralization or inhibited nitrification, thus yielding lower nitrate

and higher

DON

ammonium

than did unsaturated conditions.

concentrations in soil extracts increased over time

in

incubations with and without nitrate addition under saturated and

unsaturated conditions.

An

analysis of variance

shows incubation

of the samples to create saturated conditions,

whereas those not receiving additional water were considered

time to be the only significant (P < 0.01 factor. Also, there was
not a stoichiometric conversion of nitrate to DON, suggesting that

The bottles were sealed using rubber stoppers and

metal
tear-away
crimping seals. Each treatment combination
was replicated 16 times so that four replicates could be destruc-

DON

unsaturated.

water

and 8 days after

the start of the incubation. The "day zero" extraction was done
immediately after sample treatment (addition of nitrogen or
water) to measure background nitrate and DON and to measure
tively

initial

sampled by extraction

in

at 0.

1.

4.

recovery of added nitrate. Ambient aerobic conditions

was produced primarily from sources other than

nitrate

immobilization. However, only a small amount of nitrate

immo-

DON

DON

would be necessary to create a labeled

pool when '^N-labeled nitrate is used.
This experiment will be repeated with a '-"'N label in the added
nitrate to determine the recovery of labeled N in the extracted
bilization to

DON, ammonium,

and

in the

small remaining nitrate pool. These

preliminary results using unlabeled

demonstrate that

may be

it

day of incubation to allow for aerobic

immobilization processes. After the first day. air was flushed

possible to create a series of incubation conditions that

from the serum bottles with dinitrogen gas

ing nitrate. Hence,

prevailed during the

conditions

in

first

to create

anaerobic

an effort to increase denitrification. thus reducing

*Corresponding author: edaudsonls'wrirc.org

permit nitrate immobilization into

labeled

DON

it

may be

DON

would

while minimizing remain-

possible to obtain an extract with

that is created naturally within the soil

and

that

can

be used to study DON bioavailability in subsequent experiments.

This research was funded by the NSF Research Experience for
Undergraduates site Grant (OCE-0097498).

ECOLOGY AND POPULATION BIOLOGY

200

246
Days of Incubation

8
DON with
A

added N

Nitrate with

added N

257

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

258
1UU
0)

98

Kddvnce:
<D

205: 259. (October 2003)

Biol. Bull.

2003 Marine Biological Laboratory

Published by Title Only

Chung, Clare, and John Dowling
Anatomical analysis of

retinal

Kuhlman, Sandra, Hiro Higashiyama, and

ganglion cells

in

Role of inhibition

zebrarish

in

timing onset of ocular dominance

Lyons, M. M., K. R. Uhlinger,

bulb

J.

Ward, and

E.

R.

Smolowitz
Role of marine aggregates

Freeman, Chris, Heather Haas, Linda Deegan, and John

in the

transmission of bivalve

parasites

Logan
Comparison of invertebrate populations between
form and aquatic border along a
New England salt marsh

the plat-

Miller-Sims, Vanessa, and Jelle

salinity gradient in a

Tuning of mechanoreceptors

Henderson, Kristin, and Rebecca Cast

Comparison of Acanthamoeba distribution between en-

Ruben,

Myra Guzman,

Randy Elizando,

Nelly

that

Mechanisms of feedback

Ileana Juarez, Joe Martinez, Jr., and

FAK

and Richard

in

hippocampus

CA

inhibition in the retina: recip-

rocal synaptic signaling between bipolar and amacrine

cells

is

differentially expressed in naive

and theta-burst stimulated hippocampal

Smolowitz, R.,

slice

Nutritional

Hopp, Joshua, Bonnie Keeler, Richard Garritt, and

Linda Deegan
Distribution of benthic chlorophyll
tidal

Myra Guzman,
Jr.,

Murphy, Gabe
Garcia,

Richard LeBaron
Evidence

lobster antennule

Indication that theta-burst stimulation influences an al-

pha5 immune-related protein

vironmental samples and enrichment cultures

Hernandez,

Atema

in the

Mrizek, Michael D., Nelly I. Garcia,

Ruben Hernandez, Joe L. Martinez,
G. LeBaron

Gherardi, Francesca, and Jelle Atema

Individual recognition and memory in hermit crabs

marsh

Josh

plasticity in visual cortex

Davison, Ian, and Kerry Delaney

GAB A(B (-mediated inhibition of receptor neuron inputs
to the olfactory

Z.

Huang

larval

among

J.

Hanley, and M. Tubbs

myopathy

in cultured toadfish

Watanabe, Shigeo, Matthew Larkum, Paul Rhodes,

Nechama Lasser-Ross, and William Ross

habitats in salt

creeks

Dendritic properties of turtle cortical pyramidal neurons

259

200-4

IUMMER RESEARCH FELLOWSHIPS

FUNDING AVAILABLE FOR SUMMER RESEARCH IN NEUROSCIENCE AT

THE MARINE BIOLOGICAL LABORATORY IN WOODS HOLE
APPLICATION DEADLINE:

The Marine Biological Laboratory is pleased to announce the availability

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These programs

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JANUARY 30, 2004

FOR
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provide up to $50,000/year/award with a

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Proposals must describe collaborative research in any area of neuroscience by teams of two or more
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Hole. Collaborative teams must consist of a minimum of two Assistant/Associate Professor level
neuroscientists. Collaborative units

may be formed

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rental, plus

housing and travel costs for the

Pis.

Learning & Memory

study of learning and memory with a minimum

Dart Foundation Scholars Program

Proposals must be devoted to the

in

stay of six

weeks

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at the

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&

in,

but are not limited

to,

APPLICATION DEADLINE:

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FOR
SUMMER RESEARCH
FELLOWSHIPS

the

Applications are encouraged from

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members of underrepresented

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minorities.

Microbiology

FOR APPLICATION FORMS AND ADDITIONAL INFORMATION, PLEASE CONTACT:

Sandra Kaufmann, Fellowship Coordinator
508-289-7441; skaufman@mbl.edu
Visit our

web

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net in this issue of The Biological Bulletin (p. 295),

the nonfeeding larvae of S. insignis (and some other

Cover
Sabellids, or feather-duster

worms,

are a diverse

group of polychaete annelids common in many

benthic marine habitats. Each adult sabellid secretes

and inhabits an organic tube, from which it extends

a crown of tentacles used for both suspension feedshows
ing and respiration. The image on the cover
in
shallow
common
sabellid
three individuals of a
waters of the northeastern Pacific, Schizobranchia
insignis;

its

generic

name

reflects

its

distinctive,

dichotomously branching tentacles (which, because

of their respiratory function, are sometimes called
branchiae). In
ual

the

life,

shown here

is

crown of

about 4

cm

the largest individ-

in

instead,

development

to

metamorphosis

is

fueled by lipids, proteins, and carbohydrates stored

Phylogenetic evidence suggests that the

nonfeeding mode of development common to sabeiiids is a specific example of a frequent evoluin the eggs.

tioiK.r;

transition

among marine

invertebrates

the

loss of ihe requirement for particulate food during

the larval stage. In most known cases, this transition
in

larval

nulrition

is

soon followed by dramatic

reduction or outright loss of the larval structures

involved in feeding. But as reported by Bruno Per-

that resemble, in

are

used by the

feeding larvae of closely related annelids to capture

food particles (see Figures 3 and 4, pp. 300 and
301). In fact, larvae of S. insignis can use these

food particles
vestigial feeding structures to capture
and transport them to the mouth; but because the

mouth

is

not connected to the midgut, these partiA functional digestive sys-

cles cannot be ingested.

tem does not develop

in these larvae until

well after

metamorphosis.

Why

diameter.

Like the planktonic larvae of other sabellids, those

of Schizobranchia insignis do not ingest particulate
food;

bands
form and behavior, bands that
sabellids) possess ciliary

the larvae of

some

sabellids retain functional

loss of both the

particle capture systems despite
need for food and the ability to ingest it is an

More
interesting puzzle that remains unresolved.
observations suggest
generally, however, these
new, testable hypotheses about the developmental
loss of larval
processes underlying the evolutionary
with embryos
feeding in annelids and other animals
that

undergo

spiral cleavage.

The image on the cover was photographed by

Bruno Pernet (Friday Harbor Laboratories, UniverThe cover was designed by
sity of Washington).
Beth Liles (Marine Biological Laboratory.
Hole. Massachusetts).

Woods

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IE

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IN
IN

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but are not limited

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CONTENTS
VOI.UMK 205, No.

PHYSIOLOGY AND BIOMECHANICS

Motokawa, Tatsuo, and Akifumi Tsuchi
l)\namic mechanical properties of body-wall dermis
in various mechanical states and their implications
for the behavior of sea cucumbers
Tomanek. Lars, and Eric Sanford
Heat-shock protein 70 (HspTO) as a biochemical
stress indicator: an experimental field test in two
congeneric intertidal gastropods (Genus: Tegula).
.

W.

Hart,

Anna

Peniet,

Joyner, Joanna

276

Suzanne M. Peyer, and Raymond W.

Possible roles of sulfur-containing

amino

acids in a

chemoautotrophic bacterium-mollusc symbiosis

Schwarz, J. A., and V. M. Weis

331

Localization of a symbiosis-related protein. Sym32, in

the Anthopleura ekgantissima-Symbiodinium musculnn-i

339

associadon

ECOLOGY AND EVOLUTION

285

Price,

Rebecca M.

Columellar muscle of neogastropods: muscle attachment and the function of columellar folds
295

351

Frick, Kinsey

Response

in

nematocyst uptake bv the nuclibranch

presence of various preda-

Flabellina vemicosn to the

tors in the

H. Rees
Cloning, characterization, and developmental
transpression of a putative farnesoic acid O-methvl
ferase in the female edible crab Cancer pagimis ....

L.,

Lee

nonfeeding

annelid lanae
Ruddell, Carolyn J., Geoffrey Wainwright, Audrey Geffen, Michael R. H. \VTiite, Simon G. Webster, and Huw

SYMBIOSIS AND PARASITOLOGY

261

Bmno

Persistent ancestral feeding structures in

319

octocoral

Cerra, and Paula

Reproduciion and larval morphology of broadcasting

and viviparous species in the Cryptasterina species
complex

DECEMBER 2003
Lasker, Howard R., Michael L. Boiler, John Castanaro,
and Juan Armando Sanchez
Determinate growth and modularity in a gorgonian

DEVELOPMENT AND REPRODUCTION

Byrne, Maria, Michael
Cisternas

3:

southern Gulf of Maine

367

ex-

308

Index for Volume 205

ERRATUM
in listing the winner of the Senior
page 175 of the October issue (Volume 205. Number 2). we erred
In\ estimator category of the awards for a Short Report presented at the 2003 General Scientific Meetings of
the Marine Biological Laboratory. Karen Crawford, who was listed as receiving honorable mention, should

On

have been named as the winner of the award; Paul Gallant received honorable mention.

377

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CONTENTS
for
No.

Volume 205
1:

AUGUST 2003

RESEARCH NOTE

Eyster, L. S., and L. M. van Camp

Extracellular lipid droplets in Idiosepius notoides. the

Drazen. Jeffrey C., Shana K. Goffredi, Brian Schlining,

arid Debra S. Stakes

Southern pvgmv squid

Christensen,

of egg-brooding deep-sea fish and

cephalopods on the Gorda Escarpment: a reproduc-

James M. Colacino, and

Functional and biochemical properties of the hemoglobins of the burrowing brittle star Hemipholis elon-

hot spot

54

gata Say (Echinodermata, Ophiuroidea)

EVOLUTION
Zigler, Kirk S..

47

Beardsley,

Celia Bonaventura

Aggregations
tive

Ana

SYMBIOSIS AND PARASITOLOGY

and H. A. Lessios

250 million vears of hindin evolution

Davy, Simon K., and John R. Turner

Early development and acquisition of zooxanthellae
in the temperate symbiotic sea anemone
Anthnpleura

NEUROBIOLOGY AND BEHAVIOR

Painter, Sherry D., Bret Clough, Sara Black,
T. Nagle

Behavioral

characterization

of attractin.

borne peptide pheromone in the genus

Bergman, Daniel A., and Paul A. Moore

DEVELOPMENT AND REPRODUCTION

water-

Afilysia

16

...

Neumann,

On

Field observations of intraspecific agonistic behavior

of two crayfish species, Orconectes rusticus and <>rconertes virilis, in different habitats

66

(Cocks)

brillii

and Gregg

26

PHYSIOLOGY AND BIOMECHANICS

Etnier, Shelley A.

Dietrich,

and Heike Kappes

the growth of bivalve

gills

initiated

from

a lobule-

producing budding zone

Beninger, Peter G., Gael Le Pennec, and Marcel Le
Pennec
Demonstration of nutrient pathway from the digestive system to oocytes in the gonad intestinal loop of
the scallop Pecti-n maximus L

83

Annual Report of the Marine

Rl

73

Twisting and bending of biological beams: distribution of biological beams in a stiffness mechano-

36

space

No.

2:

Lee,

Se.t,

in

Antarctica: Just the tip of the iceberg?

Fingerut, Jonathan T., Cheryl

the
.

93

Raymond W.

Thermal tolerances of deep-sea hydrothermal vent

animals from the Northeast Pacific.

cephalopod Vampyroteuthis

tips

iii/rninlis

ard K.

Ann Zimmer, and

Rich-

Zimmer

Patterns and processes of larval

estuarine parasite svslem

emergence

in

an
Ill)

DEVELOPMENT AND REPRODUCTION

Robison, Bruce H.. Kim R. Reisenbichler, James C.

Hunt, and Steven H. D. Haddock

arm

98

NEUROBIOLOGY AND BEHAVIOR

Light production by the

ECOLOGY OF PARASITES

Seibel,

Ross

OCTOBER 2003

RESEARCH NOTE
Brad A., and Heidi M. Dierssen
Cascading trophic impacts of reduced biomass

Biological Laboratory

Gibson, Glenys D.
Larval development and metamorphosis in Plfurobranchaea maculata, with a review of development in

of the deep-sea
102

the Notaspidea (Opisthobranchia)

121

CONTENTS: VOLUME

ECOLOGY AND EVOLUTION

205

Delacniz, John, Jeremiah R. Brown, and George M.

Langford
Nanette

Effects nl

the

coir.;

E.,

ascidian Botiyllus srhlossm in

<
:

133

and D. K. A. Barnes
J.,
Effect of disturbance on assemblages: an example

Bell, J.

144

using Porifera

plasticity

of

HSP70 and HSP70 gene

Ana

S.

DePina, Carl

J.

Rho-kinase

required for myosin-11-mediated vesicle

is

195

and H. Ripps
An experimental approach to the study of gap-junc-

GENERAL

SCIENTIFIC MEETINGS OF THE MARINE

2003

175

system to the

fibrils

of the Limit/us blood

201

203

crobes entrapped in the Limulu* blood clot

Harrington, John M., and Peter B. Armstrong

waves in zebrafish using multiphoton fluorescence

microscopy .................................
nh. Opher, and Alon Sabban
Squid sperm to clam eggs: imaging wet samples in a

176

scanning electron microscope ..................

177

Wadeson, P. H., and K. Crawford

Formation of the blastoderm and yolk syncytial layer
in early squid development ....................

179

liposome-permeating

activity

from the surface of

the carapace of the American horeshoe crab, Limit/in

.ili-.

205

polyphemui

Crawford, K.
Lithium chloride inhibits development along the animal vegetal axis and anterior midline of the squid

NEUROBIOLOGY AND BEHAVIOR

Bogorff, Daniel
181

J.,

chow, and Peter J.

Mark
S.

A. Messerli, Robert P. Mai-

Smidi

Development and characterization of a

self-referenc-

207

ing glutamate-selective micro-biosensor

HNK-1/N-CAM

immunoreactivity correlates with ciliary patterns during development of the polychaete

...............................

199

Micrurinna prolifera allografts

Isakova, Victoria, and Peter B. Armstrong

Imprisonment in a death-row cell: the fates of mi-

and Winfried Denk

Long duration three-dimensional imaging of calcium

in

J.

Kuhns, M. M.

clot

Gilland, Edwin, Robert Baker,

Capitella sp.

W.

Armstrong, Peter B., and Margaret T. Armstrong

The decorated clot: binding of agents of the innate

immune

embryo ...................................
Hill, Susan D., and Barbara C. Boyer

C. Kaltenbach,

Tepsuporn,
Burger, and X. Fernandez-Busquets
Apoptosis

for

197

death

cell

S., J.

DEVELOPMENTAL BIOLOGY

<

J. Zakevicius,

tion-mediated

BIOLOGICAL LABORATORY
The Editor
The MBL Awards

194

DeSelm, and

cytes

2003

192

transport during M-phase in extracts of clam oo-

Cusato, K.,

SHORT REPORTS FROM THE

190

George M. Langford
160

George M. Langford
Rab-GDI inhibits myosin V-dependent vesicle transport in squid giant axon
Pielak, R. M., V. A. Gaysinskaya, and W. D. Cohen

Wollert, Torsten,

ex-

pression in the Pacific oyster (Crassostrea gigas): implications for thermal limits and induction of thermal
tolerance

188

Renne Lu, and

Three-dimensional birefringence distribution in reconstituted asters of Spisula oocytes revealed by

scanned aperture polarized light microscopy

M., Daniel P. Cheney, and Gary N.

Cherr
Phenotypic

conventional

Cytoskeletal events preceding polar body formation

in activated Sp/sula eggs
Shribak, Michael, and Rudolf Oldenbourg

PHYSIOLOGY AND BIOMECHANICS

Hamdoun, Amro

recombinant

squid kinesin and native myosin-V

DeSelm, Carl J., Jeremiah R. Brown,

Monterey

Bav

between

Interactions

and Irving L. Weissman

neic contact on life-history traits of

Chadwick-Fumv

182

Chappell, R. L., J. Zakevicius, and H. Ripps

Zinc modulation of hemichannel currents in Xenapus

209

oocytes

O. T. Burton, J. A. Chambers, R. Eseh,

L. M. Gutierrez, and M. M. Kron
Transient use of tricaine to remove the telencephaZottoli, S. J.,

CELI

Heck, D.
Arh/u

in

lini has no residual effects on physiological recordings of supramedullary/dorsal neurons of the cun-

;nd J. D. Laskin

Ryanod

\<

<

-,hive
r
,

calcium flux regulates motility of

^perm ......................

185

Gallant, P. E.
inhibits the slow axonal transport of tubulin
squid giant axon .......................

Zinc chelation enhances the sensitivity of the

Axotomy
in the

211

ner, Tautogolabrus adspersus

S., and R. L. Chappell

Redenti,
1S7

b-wave in dark-adapted skate retina

ERG
213

CONTENTS: VOLUME

205

Molina, Anthony J. A., Katharine Hanimar, Richard

Sanger, Peter J. S. Smith, and Robert P. Malchow

caged calcium

[ntracellular release ol

Eioini.'i

in skate hori-

zontal cells using line optical fibers

Palmer, L. M., B. A. Ginffrida, and A. F. Mensinger
Neural recordings from the lateral line in free-swim-

-15

Ian

216

ming

(oadfish,

()/><,nin<\

Child, F. M.. H. T. Epstein, A.

M. Kuzirian, and D.

\ll(>.\

BlOLoi,}

Agnew, A. M., D. H. Shull, and R. Buchsbaum

Growth of a salt marsh invertebrate on several species
of marsh grass detritus

Patterns of sedimentation in a salt marsh-dominated

L.

239

estuary

Memon

-IS

reconsolidation in Hermissenda

Kuzirian, A. M., F. M. Child, H. T. Epstein, M. E. Motta,

C. E. Oldenburg, and D. L. Alkon
Training alone, not the tripeptide RGI), modulates

Thorns, T., A. E. Giblin, K. H. Foreman

Multiple approaches to tracing nitrogen loss in the
West Falmouth wastewaler plume
Talbot, J. M., K. D. Kroeger, A. Rago, M. C. Allen, and

220

calexcitin in liiriin\\i-nda

Homarus nmmiinni*
R. Turnell, J. Atema, and G. Gerlach

Mann, K.

D., E.

Kin recognition in juvenile zebrafish (Danio reria)

based on olfactory cues
Turnell, E. R., K. D. Mann, G. G. Rosenthal, and G.

Nitrogen flux and speciation through the subterranean estuary of Waquoit Bay, Massachusetts

222

Abraham, D. M., M. A. Charette, M. C. Allen, A. Rago,

and K. D. Kroeger
Radiochemical estimates of submarine groundwater

224

discharge to Waquoit Bay, Massachusetts

Johnston, M. E.. J. R. Cavatorta, C. S. Hopkinson, and

Gerlach

244

245

V. Valentine

Mate choice

in zebrafish (l)nnin rerio)

analyzed with

225

video-stimulus techniques

Mm n

242

M. A. Charette

Savage, Anna, and Jelle Atema

Neuroclieinical modulation of behavioral response
to chemical stimuli in

238

Morgan Johnston, Charles Hopkinson, and Vinton Valentine

Cavatorta, Jason R.,

Alkon

Importance of metabolism in the development of salt

marsh ponds
Aguiar. A. B., J. A. Morgan, M. Teichberg, S. Fox, and
I.

\n BIOLOC,}, P.viiioi.iH-.Y. A\I>

Roberts, S. B., and F.

MICROBIOLOGY

epizootic
canu\

electron
lobster

microscopy

shell

disease

227

Orchard, Elizabeth, Eric Webb, and Sonya Dyhrman

Characterization of phosphorus-regulated genes in

230

algiTiolyticui
Si'ji/f/

ri/if/iiui,

infection

and

in

231

cul-

233

Hemant M. Chikarmane, Roxanna

Smolowitz, and Kevin R. Uhlinger

Detection of Edit<nrd\iflln infections

Valiela

I.

Leschen, R. H.

and

relation-

ships between size and trophic position of the Atlantic horseshoe crab, Limnlm /M>l\/>li<'ni\. within Cape

254

estuaries

Walker, C., E. Davidson, W. Kingerlee, and K. Savage

Incubation conditions of forest soil yielding maximum dissolved organic nitrogen concentrations and

256
Jr.,

and C.

Williams
Building a database of historic land cover to detect

257

landscape change
in ()/>\n>im l/iu bv

235

polvmerase chain reaction

Weidner, Earl, and Ann Findley
Catalase in microsporidian spores before

barge

Carmichael, and

S.

minimal residual nitrate

Holden, M. T., C. Lippitt, R. G. Pontius,

Si'/nn />/i/irn-

,>is

Baird, Krystal D.,

Grady, A.

S. P.

Cod

and R. M. Smolowitz

Description of Vibrio
tured Sepia offirintili\,

252

Waquoit Bay, Massachusetts

O'Connell, C. W.,

Stable isotopic assessment of site loyalty

Galac, Madeline, Deana Erdner, Donald M. Anderson,

and Sonya Dyhrman

Sangster, C. R.,

Valiela

aries of

228

Molecular quantification of toxic Alexandrium jundyense in the Gulf of Maine

I.

250

M. Teichberg, and

Relative influence of grazing and nutrient supply on

growth of the green macroalga Ulvn lactuca in estu-

of
investigation
in Homanis ameri-

Trichodesmiiim spp

Valiela

the growth of ilva lactura

Morgan, J. A., A. B. Aguiar, S. Fox,

Hsu, A. C., and R. M. Smolowitz

Scanning

248

Transplantation and isotopic evidence of the relative

effects of ambient and internal nutrient supply on

W. Goetz

Expressed sequence tag analysis of genes expressed

in the bav scallop. Argopecten irradians

disc

\\l> I'nri

<>i; \i

/'/,'/

s/

i/vo.y.s

and during
236

Published bv

title

onl\

259

CONTENTS: VOLUME
No.

PHT

3:

DECEMBER 2003

;OGY AND BIOMECHANICS

Howard R., Michael L. Boiler, John Castanaro,

and Juan Armando Sanchez
Determinate growth and modularity in a gorgonian
Lasker,

atsuo, and Akifumi Tsuchi

mechanical properties of body-wall dermis
in \.triotis mechanical states and their implications
for the behavior of sea cucumbers
Tomanek, Lars, and Eric Sanford
Heat-shock protein 70 (Hsp70) as a biochemical
stress indicator: an experimental field test in two

261

congeneric intertidal gastropods (Genus: Tegula).

276

Motokatv:
|)

205

319

octocoral

mk

SYMBIOSIS AND PARASITOLOGY

Joyner, Joanna L., Suzanne M. Peyer, and

Raymond W.

Lee
Possible roles of sulfur-containing

amino

acids in a

chemoautotrophic bacterium-mollusc symbiosis

Schwarz, J. A., and V. M. Weis

DEVELOPMENT AND REPRODUCTION

Byrne, Maria, Michael Hart,
Cistern as

Persistent ancestral feeding structures in nonfeeding

annelid larvae

331

339

association

ECOLOGY AND EVOLUTION

285

Price,

Rebecca M.

Columellar muscle of neogastropods: muscle attachment and the function of columellar folds

295

351

Frick, Kinsey

nematocyst uptake by the nudibranch

presence of various preda-

Ruddell, Carolyn J., Geoffrey Wainwright, Audrey Geffen, Michael R. H. White, Simon G. Webster, and Huw

Flabellina verrucosa to the

H. Rees

tors in the

Cloning, characterization, and developmental

pression of a putative farnesoic acid O-methyl transferase in the female edible crab Cancer pagurus ....

Localization of a symbiosis-related protein, Sym32, in

the Anthopleura elegantissimaSymbiodinium muscatinei

Anna Cerra, and Paula

Reproduction and larval morphology of broadcasting

and viviparous species in the Crypla\lmna species
complex
Fernet, Bruno

Response

in

southern Gulf of Maine

367

ex-

308

Index for Volume 205

377

Reference: Bio/. Bull. 205: 261-275. (December 2003)

2003 Marine Biological Laboratory

Dynamic Mechanical Properties of Body-Wall Dermis

in Various Mechanical States and Their Implications
for the Behavior of Sea Cucumbers
TATSUO MOTOKAWA* AND AKIFUMI TSUCHI
Dcpanment of Biological

The dermis of

Abstract.

the sea

and Biotechnology. Tokyo Institute of

Technology, Meguro, Tokyo, 152-8551 Japan

Sciences, Graduate School of Bioscience

cucumber body wall

is

control

typical catch connective tissue that rapidly changes its mechanical properties in response to various stimuli. Dynamic

(Motokawa. 1984a; Wilkie, 1996). Connective

tis-

sue with such mutability is

mutable connective tissue. The mutability has been attributed to the changes in the mechanical properties of the

called catch connective tissue or

mechanical properties were measured in stiff, standard, and

soft states of the sea cucumber Actinopyga mciuritiana.

extracellular

materials in

the

tissue

(Motokawa,

1984a;

Sinusoidal deformations were applied, either at a constant

frequency of 0.1 Hz with varying maximum strain of 2%-

Wilkie, 2002; Tipper ct ai, 2003). The dermis in the body

wall of sea cucumbers is a favored material for studies of

or at a fixed maximum strain of 1.8% with varying

frequency of 0.0005-50 Hz. The dermis showed viscoelasticity with both strain and strain-rate dependence. The der-

connective tissue catch because of

20%

standard state showed a J-shaped stress-strain

curve with a stiffness of 1 MPa and a dissipation ratio of

mis

in the

60%;

the curve of the stiff dermis

MPa) and

stiffness (3

low

was

large size.

The me-

1981; Eylers, 1982), stress-strain tests (Motokawa. 1982),

(Motokawa, 1984b; Greenberg and

stress-relaxation tests

linear with high

dissipation ratio

its

chanical properties of the dermis have been described by

various mechanical methods such as creep tests (Motokawa,

(Motokawa. 1982), and dynamic

(Shibayama et at.. 1994; Szulgit and Shadwick, 2000).
These studies revealed the viscoelastic nature of the dermis.
Eylers, 1984), tensile tests

(30%). Soft

tests

dermis showed a J-shaped curve with low stiffness (0.3

MPa) and a high dissipation ratio (80%). The strain-induced

They drew,

however,

contradictory

conclusions

about

was observed in the soft state. Stiff samples had a

higher storage modulus and a lower tangent 8 than soft ones,
implying a larger contribution of the elastic component in
the stiff state. A simple molecular model was proposed that
accounted for the mechanical behavior of the dermis. The

which component, elastic or viscous, changed during alterations in mechanical properties. Motokawa (1984b) concluded from the results of creep tests and stress-relaxation

model suggested

Shadwick (2000) concluded that it was the elastic component that changed. The former used a fixed strain and the
latter a fixed strain and strain rate. The apparently contra-

softening

changes in the mechanical properties mainly

occurred in the viscous component, whereas Szulgit and
tests that the

that stiffening stimulation increased inter-

molecular bonds, whereas softening stimulation affected

intra-molecular bonds. The adaptive significance of each

mechanical

state in the

behavior of sea cucumbers

is

dis-

dictory conclusions of these two studies are not surprising,

because viscoelastic materials exhibit different properties

cussed.

when

Echinoderms have collagenous connective tissues that

can alter their mechanical properties rapidly under nervous

To whom

et ai,

and

strain

1976). Therefore, description of the

(Wainwright
mechanical properties of viscoelastic materials

Introduction

Received

tested under different conditions of strain

rate

is

not satis-

a limited range of strain and strain rate. The

factory
present study was undertaken to describe the dynamic mechanical properties of the holothurian dermis under wide
in

The information obmechanism

catch and how sea cucumbers adapt to

ranges of both strain and strain

2003; accepted 5 September 2003.

correspondence should be addressed. E-mail: tmotokaw

May

rate.

tained establishes a basis for understanding the

of connective tissue

bio.titech.ac.jp

261

262

MOTOKAWA AND

T.

various mechanic;'
ical propertie:

environments by changing the mechan-

TSUCHI

MgCU.
Materials and Methods

52.5;

NaHCO,,

potassium was

2.5. In

EGTA

dium concentration was adjusted

from the lagoon

in

of Sesoko Marine Science Center,

our laboratory. This sea cucumber has a thick body wall

(about 1-1.5 cm). The connective tissue dermis occupies
most of the thickness. The outer side of the dermis is

covered with a thin epidermis, and the inner side is lined

with body wall muscles. Unlike some dendrochirotid species such as Cuciunaria frondosa, the dermis of this aspi-

dochirotid sea

cucumber looks uniform, showing no differA dermis sample was dissected from

entiation into layers.

the lateral interambulacrum of the

wall.

body

Both the

epidermis and muscles were removed and the middle portion of the dermis was cut out for experiments. The dimensions of the samples were measured with a caliper to a

precision of 0.05

mm. The mean

end was 2.3

oral to aboral

mm

length of the samples from

SD; n = 79),

(0.89

mm
mm2 (1.9 mnr

and the mean cross-sectional area was 5.4

SD;

;;

The samples were

79).

of normal composition
subjected to mechanical

(ASW)
tests.

rested in artificial seawater

for

17-24 h before being

The temperature of

the sea-

water was 18-25

C, which roughly corresponded to the

of
the
water from which the sea cucumbers
temperature
were collected. Mechanical tests were performed at a con-

20

stant temperature of
that

Samples
in

ASW

the concentration of

CaFASW

con-

concentration constant.

ASW. The pH

of

ACh

all

to

keep the osmotic

was 10~ 4 mol/1

concentration

the solutions

was adjusted

to 8.2.

Experimental apparatus

dermis sample was subject to forced vibrations using

The sample was stretched and

sinusoidal displacements.

compressed
corded.

cyclically,

and the resulting forces were

The experimental apparatus

(Fig.

1)

re-

included a

EMIC, Japan) driven by sinusoidal curwere generated by a function generator (SG-4101,

Iwatsu, Japan). The force developed in the dermis was
measured by a micro load cell (LTS-200GA, Kyowa, Javibrator (511-A,

rents that

pan).

The compliance of

contributed

at

most

4%

the present experiments.

the load cell

to the

was

0.3 jum/g,

measured value of

which

strain in

The deformation of the dermis was

monitored by an eddy-current displacement sensor (502-F,

EMIC, Japan). Force signals were amplified by a strain
amplifier

(DPM-602A, Kyowa,

Japan). Both force and dis-

placement signals were displayed on an oscilloscope and

simultaneously recorded by a computer through a dataacquisition unit (Lab Stack, Keisoku Giken, Japan). The
dermis sample was glued with cyanoacrylate glue to the
holders, one attached to the vibrator and the other to the load

C.

were rested

in

ASW

for

7-24 h and

tested

are described here as being in a standard state.

Soft-state samples

were prepared by removal of calcium

ions (Hayashi and

Motokawa. 1986); they were

rested in

(CaFASW) and

tested in

calcium-free

CaFASW.

ASW

as

10.1:

N' yV'-tetraacetic acid). In both cases, the so-

Specimens of the aspidochirotid sea cucumber Actinopvgu mauritiuna (Quay and Gaimard) were collected
University of the Ryukyus, Okinawa. They were shipped to
Tokyo Institute of Technology and kept in an aquarium in

was

CaCk

(ethylene glycol bis(j3-aminoethyl-

Tissu
ether)-/V, N,

KASW,

raised to 100 mmol/1, and

tained 5 mmol/1

in front

ASW

The composition of

the mechanical testing.

follows (in mmol/1): NaCl. 433.7; KC1, 10.0;

the Jermis.

>'

A.

artificial

Stiff-state

seawater

samples are those

without a resting period.

Rough

that

were tested

in

physical handling

makes the dermis stiffen reversibly (Motokawa, 1984c).

Soon after the dissection, the dermis experienced quite
rough physical handling, so these are termed the physically
stimulated samples. This state probably corresponds to the
state the sea
ically.

Two

invoke the

cucumbers are

in after

being stimulated phys-

other kinds of stimulation were

employed

to

(possibly through stimulating the stiffchemical stimulation by the

ening iM-'-hanism in vivo)
stiff state

neurotrniismitter acetylcholine (ACh) (Motokawa, 1987)

and chemi'." simulation using artificial seawater with an

elevated pot:
stimulates a a

Vm concentration (KASW).
-

mechanism

The

latter likely

that controls stiffness,

such

as nerves, through depolarization

(Motokawa, 1981).
The samples for chemical stimulation were rested for
17-24 h in ASW, and chemicals were applied 20 min before

Figure

1.

Schematic of the experimental apparatus tor dynamic

tests.

VISCOELASTICITY OF SEA CUCUMBER

263

and an experimental solution was introduced to the

trough. The sample was usually rested for 20 min in the

cell,

stress

however, the physically stimulated sample was

trough;

tested immediately.

The trough was water-jacketed

the temperature constant at

to

keep

20 C.

We performed two series of experiments. In the constantfrequency experiments, the frequency of vibration was kept
constant

Hz and

at 0.1

varied between

2%

the

maximum

and 20%.

chosen because the differences

in

strain in a cycle

was

frequency of 0.1 Hz was

the mechanical properties

of the three states were most prominent

at this

strain

frequency

(see Results). In the constant-maximum-strain experiments,

the

maximum

was kept

strain in a cycle

at

1.8% and the

frequency was varied between 0.0005 and 50 Hz. A maximum strain of .8% was chosen because the dermis behaved
1

as a linear viscoelastic material at this strain (see Results).

The samples were preconditioned by applying oscillamin prior to the test; the amplitude and frequency were the same as those in the test. A steady-state
response was reached by the preconditioning.
tions for 10

Figure 2. Schematic of a hysteresis loop of the dermis in standard state

under a sinusoidal deformation of 10% maximum strain at 0. 1 Hz. The loop
showed clockwise rotation (arrows). The stiffness was defined as the slope

Constant-frequency experiment

experiments that examined the effects of strain, a

dermis sample was subjected to successive oscillatory tests

of the dotted

line.

The darker hatched

area represents the dissipated energy,

In

with different levels of maximum strain. The hysteresis loop

of the stress-strain relationship showed almost point symmetry. The point of symmetry was brought to the origin of

the lighter hatched areas the conserved energy,

the deformation energy.

it

was

The

inset

and the

total

hatched area

shows how the J index was measured:

the percent ratio of the hatched area to the area of the triangle

by dotted

shown

lines.

the coordinates by adjusting the length of the sample. Data

were then collected, and a

erated for that

was

maximum

typical hysteresis loop

was gen-

When the data acquisition

maximum strain, the maximum

strain.

finished at a certain

was increased by about 4%

sample was preconditioned, and data

strain in an oscillatory cycle

(range 2% 7%), the

were collected with the
Strain (e)

new maximum

was defined

as the length

original length of the sample,

and

When

change divided by the

at the original

length.

was

plotted against strain, a closed hysteresis

obtained (Fig. 2). Because the dermis behaved

stress

point,

and the other side was a

inset).

vertical

The J index

to the difference between the energy fed into

specimen and the deformation energy of an elastic body

was equivalent
the

with a linear stress-strain curve; the J index

measure of how concave the

strain.

stress (cr) as the force

divided by the cross-sectional area

from the intersecting

extending from the peak point (Fig. 2

stress-strain

is

thus a

curve was. The J

when the curve was convex. The

was defined as the slope between the two peaks of
hysteresis loop. The deformation energy (,) was defined

index was defined as

stiffness

as the hatched area in Figure 2.

It

corresponds to the energy

quite similarly in tension and in compression, the values

required to deform the dermis of a unit volume for one

cycle. In each cycle of deformation, the deformation energy

in this paper are the averages of the absolute values at

equal strains in tension and in compression. The maximum

geometry of the sample; the

loop was

given

imposed in a loop was denoted as the maximum strain

(e max ), and the maximum stress observed in a loop was
strain

denoted as the

maximum

had the shape of the

quarter of the loop

"toe" region and a
introduced an index (the J

stress (cr max

letter "J"

with a

).

flat

vertical "pole" region. We

index) to quantify the degree of concavity of the stressstrain curves in the loading and unloading phases in tensile

more

is

partly conserved

and reused as

elastic recoil to restore the

rest

is

dissipated and lost,

primarily as thermal energy. The energy dissipated

corresponds to the area enclosed by the hysteresis loop.
= EJE, and
dissipation ratio D was defined as

(/

The
was

expressed as a percentage.
The limit strain was estimated as the average of the
smallest maximum strain that caused the strain-induced

stress-strain

softening and a maximum strain one step smaller (where

strain-induced softening was not observed).
Reversibility of the response was determined by succes-

same

the stiff state caused by physical stimulation was tested,

then rested for 22 h and tested again. In the chemically

strain.

We

defined the J index as the area enclosed by the

curve and the line connecting the peak point

with the intersecting point at
strain in the curve, divided
by the area of a right triangle whose hypotenuse was the
line as before,

one side was a horizontal extending

sive mechanical tests using the

same sample. The sample

in

T.

264

MOTOKAWA AND

samples, the preparations were first tested

and then trea-.'.-d with stimulation media for 20

stimulated

stiff

was gradually stretched, a tensionwhich

the tensile strain decreased, a
unloading phase
which
the dermis was gradually comin
compressing phase
a
and
phase in which the
compression-unloading
pressed,
in

in

mm

ASW

before the second mechanical

thoroughlv

in

ASW

24

for

h.

tests. Samples were washed

and the third mechanical test

CaFASW

stretching the samples

in

was recoverable when

the

CaFASW. Then

in

by
softening
beyond the limit strain

maximum

strain

was reduced

30%

oscillations of e max of about

were

given for 10 min, and the

S m ax

= 2%.

to

tested at s max =

sample was

tested again at

strain decreased.

at
hysteresis loop of a sample in the standard state
strain is shown in Figure 3a. When stretched

The

induced

The sample was

less than the limit strain.

2%

the dermis

compressive

whether

determined

also

which

in

was performed.

We

TSUCHI

A.

10% maximum
from zero

dermis could be deformed quite easily:

strain, the

thus the slope of the curve was flat at first, corresponding to

the toe region of the J. When strain exceeded about 5%, the

became progressively

slope

steeper; thus the tensile stress-

followed a typical J-shaped curve (Wain1976). The J index averaged 43% (9.1% SD,

relation

strain

wright etui..
n
17). After the tensile stress peaked, the unloading
was
phase started and stress decreased rapidly. The slope
at each strain. As the
in
the
than
that
loading
phase
steeper
----

Constant-maximum-strain experiment
In this
at

maximum

experiment, the

strain

1.8% and the frequency was varied

in a

was kept constant

dermis sample.

sample was tested first at 0. Hz. Then the test frequency

was either increased stepwise to 0.5. 1, 5. 10. and 50 Hz or
decreased to 0.05. 0.01. 0.005. 0.001. and 0.0005 Hz. The
1

data from 10 cycles

at

experiments except

at

each frequency were averaged

the lowest

in all

two frequencies, where

data for two cycles were averaged.

In

a viscoelastic material, stress

and

strain

are not in

behind stress by a phase angle

rather, strain lags

8.

phase
For a linear viscoelastic material, the complex modulus E*
"'
are defined
the storage modulus ". and the loss modulus
,

as follows (Oka. 1974).

E* = ale =

E'

IE"

strain progressively decreased, the slope declined and the

flat. The J index of the unloading

curve became almost

(13%

17). much greater

SD. n
phase averaged 71%
than that of the loading phase (significant difference by
and unloading
paired t test, P < 0.01). In the loading

the curve
phases of the compression half of the cycle,
followed almost the same course as that of the tensile half
with the sign of stress and strain reversed. Thus the dermis

tension and compression. The four

the
phases of the hysteresis loop all exhibited a J shape, and

behaved similarly

E'

E"

tan 8

|E*

sing

(Fig. 3b, c. d).

E"/E'

dissipated.

component and

Tangent 8

is

is

measure of the energy

the ratio of the energy lost to the

energy stored.

The

soft state

was induced by

20% maximum
Such
sults).

a vibration of 0.

strain applied for

shape became

less

Thus each phase ot the

rather straight, which
hysteresis loop in stiff samples was
made the J index small. The J indices of the loading phase
a small kink

The complex modulus represents the conventional "stiffness." The storage modulus is equivalent to the elastic
modulus in phase with the stress and is a measure of the
energy elastically stored in each cycle. The loss modulus is
the out-of-phase

The

prominent: the
under
2%-4%)
toe region became short (restricted to strains
and was not flat but had a steep slope so that there was only

cos 8

\E*

in

whole loop showed approximate point symmetry.

The loops of stiff samples differed from those of samples
in the standard state both in shape and in the stress developed

==

30 min

in

Hz

with

CaFASW.

a treatment caused strain-induced softening (see Re-

The softened sample was subsequently

tested

in

between toe and

pole.

stimsamples stiffened by physical stimulation. KASW

=
8.2%
4).
were
9.0%(n
ulation, and ACh stimulation
=
=
6.0
5), respectively
1% (n
(n
19%
4), and 26
in

SD). The average J indices were significantly

(average
smaller than that of the standard state (Scheffe test. P <
0.01 ). In stiff samples, the shape of the stress-strain curve

from
during the unloading phase showed no great difference
the
J
indices
The
that of the loading phase.
unloading
during
samples stiffened by physical stimulation. KASW
8.6% (n =
stimulation, and ACh stimulation were 14%
in

phase
4).

23%

12%

(//

and 33

4),

8.9

CaFASW.
Results

the

==

5),

of the
loading phase, but the statistical comparison

averages showed no significant difference. In

Constant-fret/i.

<

the

maximum strain
When stress was plotted

Hvsteresis loop at

=
quency

(/;

SD). The J index of the unloading

respectively (average
was slightly larger than that of
curve
in
a
hysteresis
phase

0.1 Hz.

70% and

fre-

against strain, a

closed hysteresis loop of clockwise rotation was generated.

The loop could be divided into four phases: a tensile phase

maximum

stress

by a loop was
state.

was much

much

smaller than

These features implied

haved

The

stiff

samples,

larger and the area enclosed

in

samples

that the stiffened

in

standard

dermis be-

like a stiff, resilient spring.

hysteresis loop of soft samples

was

rather

flat

in

CUCUMBER

VISCOELASTICITY OF SEA

265

0.5

o
o

-0.5

-2

-4

-1

-10

-10

10

-10

10

10
strain

(%)

-2

-2

-10

10

0.6

0.3

-0.3

-0.6

-10
Figure
Stiff state

10
Typical hysteresis loops at
induced by physical stimulation

3.

10% maximum
(b),

by

KASW

loading (Fig. 3e). This is because the toe region was similar,
both in length (strain) and in height (stress) to that of the
standard, but the pole was shorter: for example, the maxi-

strain
(c),

and

0.1

Hz

and by 10~

mum

frequency, (a) Standard state, (b-d)

mol/1 acetylcholme (d). (e) Soft state.

was much smaller in the soft state than in the

The J index in loading phase averaged 27%
8.9% (average
SD, n = 9) which was significantly
stress

standard

state.

266

MOTOKAWA AND

T.

smaller than that of the standard state (Scheffe

0.01).

The shape

loading phas
loading ph;<
creased fff

if

ed marked differences from that of the

.he

peak

to give a

J index as high as

).

larger.

state

This

made

86%

The sample that experienced strain-induced softening

was softer than before, even when measured at strains
smaller than the limit strain. The stiffness of soft samples
was measured first at e max == 2% in CaFASW; values
=
). The samples were
averaged 0.24 MPa( 0.1 2 SD,;/

test.

P <

the area enclosed by the hysteresis loop

These features implied

that the

became more compliant and

Influence of
e max

statistically
/

maximum

= 0.5%-3%,

strain

dermis

in the softened

on the hysteresis loop. At

skewed

series of tests

which was

showing no
signs of the J-shaped curve seen at larger strains. Both E*
and tan 5 were constant in this strain range. Thus the dermis
behaved as a linear viscoelastic material.
ellipse regardless of the state of the samples,

The maximum

2%-4%

ments of

than the

strains

was increased stepwise by incre= 2%-3%. In standard

starting from e max

5%

to

15%, and thus the

strain

SD =

standard samples, the stiffness increased

at a

from

value of about

2% to 5%; remained
MPa above 5% (Fig.
it

samples (regardless of the stimulation used to

sample) were significantly greater than those

of standard samples (P < 0.01) at a maximum strain of

both 3%' and 5% (Table
There were no significant
).

also

differences in stiffness

at first,

was 8.0%-18.2% (average = 11.2%,

stiff

stiffen the

but once e max exceeded a certain value

(the limit strain) (Tmax decreased as e max increased. The limit
increased

among

stiff

samples produced by

different stimuli.

The

3.9%,

stiffness

was low

in the soft

samples. At strains less

-0.6

-20

-10
strain

20

-20

10
strain

(%)

strain

(%)

(%)

Figure 4. Typical examples of effects of maximum strain on hysteresis loops measured at 0.1 Hz: (a)
standard state; (b) stiff state induced by physical stimulation: (c) soft state. The maximum strain was increased
stepwise

in a single

nearest integer.

softening

sample

in

each

The curves of some

state.

steps

The number above each loop

were not shown except

was observed: the maximum stress decreased

was estimated as 12.7% (see text).

limit strain

ratio.

5a). In stiff samples, the stiffness was almost independent

of e max at a value of about 3 MPa, 2.5-3.5 times greater
than that of standard samples. The average stiffness val-

showed a marked dependence on e max

(T max

Once

values.

deformation energv. and dissipation

strain. In

ues of

e max was increased above 5%,

10),

0.01

over the limit strain but also for strains under the

almost constant

In soft samples, the shape of the hysteresis loop and the

stress both

initial

2- to 3-fold as e max increased

When

test,

P <

mum

stress-strain

max increased.
when e max exceeded about 15%.

(Fig. 4c).

SD, n

summarizes the mechanical properties of the

samples in each of the three states, and Figure 5 shows
the dependence of mechanical properties on the maxi-

The maximum stress (<rmax increased as

The samples detached from the holders

maximum

significantly smaller (paired

Stiffness,

Table

shape as e max increased

to
In
15%.
stiff
the
up
samples,
shape of the loop was quite
similar for e max between 5% and 15% (Fig. 4b) with a

constant J index.

MPa (0.005

limit strain.

-= 5%-25% were
samples, the hysteresis loops of e max
of
J
curves.
The
composed
prominent
peak stress became
as
e
increased
The
J index increased as
higher
max
(Fig. 4a).

curve showed a more pronounced

averaged 0.010

strain-induced softening occurred, the decreased stiffness was apparent not only at

strain

e max increased from

then subjected to vibrations beyond the limit strain for 10

min and tested again at 2% e max The stiffness in the second

less resilient.

the hysteresis loop followed a

18%-26%.

11^ unloading phase, stress sharply de-

TSUCHI

6). The hysteresis loop above the limit strain had a

long toe region, which was expected from the larger J index
with smaller <r max The dermis ruptured when s max exceeded

the stress-strain curve during the un-

7.1% (average
SD, n -- 9). which was
different from that of the loading phase (paired
0.01

P <

test,

A.

as

in c.

maximum

is

Note

the

maximum

strain

rounded

to the

that in the soft state, strain-induced

strain increased

from 10%

to

21%. The

VISCOELASTICITY OF SEA CUCUMBER

Table
Mechanical properties

<>/

267

MH cucumber dermis

Maximum

strain

268

MOTOKAWA AND

T.

2%

increased from
dissipation ratio increased as e max
to 5%, but it reimirvd almost constant for larger e max (Fig.

The

5c).

deformation energy was dissipated in

About 601

standard

samp

.s at

= 10%

s max

(Table

1).

strain of

10%

in the stiff

from

that of the standard state

and

soft states

were

<

(P

'

in the high-frequency range, reflecting the

5 was less than 1 at higher frequencies.
The frequency dependence of stiff and soft states is given

one-tenth of
fact that tan

stimuli coincided well, suggesting that the three stimuli

The

resumed

stiffen-

the

ASW

Figure

The curve of

No

increase in stress

was observed during

the precondi-

dermis. Stiffening of the dermis by forced vibration has

been reported in other sea cucumbers but at much larger
strains

(Shibayama

et

til..

The frequency dependence of

standard state

is

in

given

Figure

*,

",

",

and tan 8

in the

The complex modulus

7.

*
took a low and rather
gave a sigmoid curve (Fig. 7a).
constant value of about 20 kPa at frequencies lower than
Hz and higher it showed a rather constant
0.005 Hz. At

value of about 3

MPa. Tan 6 was more or

the lower frequencies with a

less constant in

maximum value of about 1. and

Hz it decreased with fre-

for frequencies exceeding 0.01

quency

to 0.06 at

similar to that of

50 Hz
*,

'

(Fig. 7b).

exhibited a curve

with the value for each frequency a

a constant value of around 3

E*

as

of the standard

after

was

every frequency

at the

stiff state (Fig. 8a).

was

rather constant at a

low value (about 10 kPa) for frequencies lower than 0.5 Hz

MPa.
but sharply increased at higher frequencies to about
1

was 1/100-1/10 the value of

seen

in the

Tan 8

* of the standard state

middle frequency region (0.05-5 Hz).

decreased with frequency to reach

in the stiff states

Hz and

higher.

than 0.005 Hz), but

less. In the soft state,

The values were

low frequencies

similar to those in the standard state at

(less

higher frequencies tan d was much

tan 8 was about 1 with a little decrease

at

with increasing frequency in the range higher than 0.005

Hz. but a sharp drop between 0.0005 and 0.005 Hz. The
values of tan 5 were similar to those of the standard in the

frequency range 0.005-0.05 Hz. but at

cies
higher or lower the values were
those in either the standard or

The curve of

in

other frequenhigher than

much

stiff states.

'

in the stiff state

appeared on the top, that

c)

50 r

10
before

stimulation

before

after

KASW

stimulation

(square), and physical

Reversibility of stiffening responses induced by ACh (triangle),
Figure 6.
were made both before and after the
stimulation (cross). For chemical stimulation, measurements in
stimulation, (a) Stiffness; (b) deformation energy; (c) dissipation ratio. Identical symbols are from the same

ASW

sample.

In the stiff state,

at

two orders of magnitude

0.1

stimulation

MPa.

by as
lower frequencies.
At frequencies higher than 0.5 Hz, however, the difference
* was different
decreased by a factor of 2. In the soft state,
in shape and value from both those in the standard state and
higher than

10

0.1

b)

before

It

rather a constant value at 5

1994).

different in shape

those in the

tioning period, which implied that the imposed vibration of

1.8% strain did not act as a mechanical stimulus for the

was

standard state (Fig. 8a). The

sharply increased with frequency

in the

X 10~ Hz): the

low frequency range (5 X 10~ of increase diminished as frequency increased, reaching

much

Constant-maximmn-stniin experiments

state.

in the stiff state

in the

samples, the samples did not show any sign of decay, and
after stimulation were
the mechanical parameters in

ASW

and position from that

curve was not sigmoid.
rate

before stimulation.

8.

invoked an identical mechanical

for 1 day (Fig. 6).

standard state after resting in
Although it took 2 days from the time of preparation to the
completion of measurements in the chemically stimulated

ASW

All curves of stiff states induced by different

in

ing response was

similar to those in

showed a

for
sigmoid curve (Fig. 7d) with values similar to that of
"
was about
frequencies lower than 0.01 Hz. However.

0.05).

successive measurements on the same sample.

stiffened samples

also

(Fig. 7c).

significantly

Reversibility of responses. Whether the stiffened dermis,

which was stimulated from the standard state, resumed the
standard state after removal of the stimuli was examined by

reversible

"

smaller than that of

little

'

The

dissipation
ratio was hah ed in stiff samples but increased to as much as
80% in soft samples. The average values at a maximum

different

TSUCHI

A.

after

VISCOELASTICITY OF SEA CUCUMBER

10

10

269

10"

10=

10"

10

10 2
10'

10'"

10'

10"

10

10

frequency

10 2

1CT

10'

10'

10'

frequency

(Hz)

10'

10

10

10

10'

10

10'

10

(Hz)

b)

10

=-

10'

10"

10-

10

10
10"

10-

10'

ID'

10

frequency
P'igurc 7.

3
=-

10"

tangent 8; (c) storage modulus

"; (d) loss

different individuals; bars are

that in the standard

E' for the

stiff state

gave

The curve of E' in the soft

was similar to that of E* except at low frequencies
than 0.005 Hz), where E' showed a sharp increase with

a curve similar to that of E*.

state
(less

SD.

standard state
(a)

at

(Hz)

1.8%

Complex modulus

ferent at different frequencies.

strain.

*; (b)

when

E* occurred

For example, a

0.005

fairly large

Hz

without a change in tan

the dermis stiffened from standard state. At this

increase in

'

stiff states.

in the

modulus E".

in-between (Fig. 8c). As for *, the difference in the values

of
became smaller with increasing frequency in the
Hz, especially the difference
frequency range higher than

between standard and

10

10'

frequency

Frequency dependence of mechanical properties of samples

on the bottom, and

10-

10-

(Hz)

Circles give the average of 8 samples from

in the soft state

10

10'

at

frequency, both increases in the elastic component (" ) and

increases in the viscous component ("') contributed equally
to the increase in

*.

At 5-50 Hz, however, a decrease

"

'

without changes in
caused a decrease
dermis softened from the standard state.

in

when

in

the

frequency, reflecting the sharp decrease of tan 8 in this

frequency range.
The curve of E" in the

Discussion
stiff state

was

rather

flat

with a

value on the order of 100 kPa. The curve of E" in the soft
state

was sigmoid.

was

as

that in the standard. E" in-

Strain

and

strain-rate

dependence

The present work studied the mechanical properties of the

catch connective tissue in the holothurian dermis and their

creased with frequency, but plateaued above 0.1 Hz in

standard state, whereas in the soft state it plateaued at a
higher frequency (5 Hz). The saturated E" value of about

changes upon stimulation. Dynamic sinusoidal strain of

0.5%-25% over a frequency range covering 5 logarithmic

200 kPa was

5-50 H/i.

decades was applied and mechanical parameters were examined. This is the first description of the dynamic visco-

the

same

in all three states

Comparison of curves
that both the viscous

(frequency range

in different states (Fig. 8)

component and the

elastic

showed

component
changed with tissue states; the relative contributions of the
two components to the changes in E* appeared to be dif-

mutable connective tissues that systematically

varied both strain and strain-rate over wide ranges. The
study revealed that the viscoelastic properties of the ho-

elasticity of

lothurian dermis

is

both strain and strain-rate dependent.

270

T.

10

io

10

10

10

MOTOKAWA AND

TSUCH1

A.

10"

10'

10'"

10'

10'

10

frequency

10

10

10'

10'

10'

10'

(Hz)

10
(Hz)

frequency

10

id)

b)

10

no

|r

10''

TO'

10'

10'

10'

10'

10

frequency
8.

Figure

10

10

10

10

10'

Frequency dependence of mechanical properties of the stiff

Complex modulus E*: (b) tangent 5; (c) storage modulus E'\

state

(KASW);

from

cross, stiff state

3 individuals.

(ACh);

Hollow

state

circle, stiff state (dissection);

filled circle, soft state.

Broken

and of the

(d) loss

modulus

10

10

(Hz)

frequency

(strain l.S^c). (a)

are averages of 6 samples

10

10'

10"'

(Hz)

soft

E".

state

Values

hollow diamond,

stiff

lines are the curves of the standard

state.

We

observed several kinds of

strain

dependence. The

dermis behaved as a linear viscoelastic material

strains (less than

3%), whereas

it

showed

at

small

nonlinearity at

Two

kinds of notable nonlinear strain dependence were observed. One was the J-shaped stress-strain
larger strains.

relationship,

which

is

common

feature of soft biological

materials (Wainwright eluL, 1976). The J-shaped curve was

apparent in the standard and soft states but not in the stiff

The

index enabled us to quantitatively describe the difference in the shape of the curves.
The other nonlinearity. which was observed only in the soft
state.

introduction of the

./

state,

was strain-induced softening

when

strain

exceeded the

the stiffness decreased

limit strain of

about 10%. These

nonlinear strain dependences were thus tissue-state dependent.

e

the

dard

obtained a modulus-frequency curve by varying

nc\. The curves of
*,
", and E" in the stan-

which clearly showed that the

mech:i
iperties of the dermis were strain-rate dependent. Thi rurves and thus the dependence were quite
s

ere sigmoid,

different in different tissue states.

stiff,

The curves of

"

in

standard, and soft states were different from each

which implied that changes in elasticity accompanied the alteration in tissue states. The curves of E" in the
other,

three states

were also

changes
Thus, both

changes
states

occurred

The curves of

different values at

showed

at tissue-state

that the

changes.

and viscosity changed with the

in tissue states.

gave

clearly

elasticity

which implied

different,

in viscosity also

tan 8 for the three

most frequencies. This

that the ratio of the contribution of the

viscous component to the contribution of the elastic

component changed with tissue states. The change in

and the change

in viscosity appeared either

or
simultaneously
independently at a given frequency.
Previous studies based on experiments with a fixed, ar-

elasticity

bitrarily selected strain rate

drew contradictory conclu-

sions on which

(elastic or viscous)

components

mainly

changed at alteration in tissue states (Motokawa, 1984b;

Szulgit and Shadwick, 2000). The present study clearly

showed

that both

components change.

CUCUMBER

VISCOELASTICITY OF SEA

When

Stiff state

The present study employed three different stimuli that

possibly acted through stimulating the stiffening mechanism
active in the intact dermis. Although the stiff state was
induced by different methods, all produced almost identical
parameter-frequency curves. Therefore we concluded that
the same mechanical state was induced by these methods.
Acetylcholine

is

a ubiquitous neurotransmitter that func-

tions in the control of dermal stiffness in sea

cucumbers

the

271

maximum

exceeded a

strain

about 10%, stiffness decreased as the

explain the rather contradictory

results reported for other sea cucumbers by this strain-

induced softening phenomenon. The soft state in the present

study was induced by calcium chelation. This procedure

was found

to cause drastic softening in creep tests

elements controlling the dermal stiffness by membrane depolarization (Motokawa, 1994). The stiffening caused by
the handling at preparation likely corresponds to the dermal

detectable

the organism

We

is

mechanically
state seen in the

disturbed in nature.
thus regard the stiff
present study as representative of the stiff state occurring
naturally in the intact dermis. The stiff state was characterized, in the constant-frequency experiments,

by high stifflow dissipation ratio, and

the constant-maximum-strain experiments,

ness, high deformation energy,

low J index.
this state

In

was characterized by high moduli and low

The low values

tan 5.

5 and dissipation ratio implied that

over viscosity. Tan 8 was less than 0.1
when the frequency exceeded 0.1 Hz, which implies that the
contribution of the viscous component was quite small. This
in tan

elasticity prevailed

much

less

the difference in the extent of softening

pretable, at least in part,

ber. In

creep
induced soft

tests, the

state,

a stiff spring. This state has

been believed

to function in

posture maintenance and mechanical defense (Motokawa,

1985).

The

stiff,

spring-like properties

features for such functions.

The high

seem

to

stiffness

be adaptive
is

helpful in

likely that the state in

elasticity

over viscosity also

helps by minimizing plastic flow.

The

soft state

is

experiments by low

characterized in the constant-frequency

stiffness,

low deformation energy, high

dissipation
high J index in unloading, and straininduced softening. In the constant-maximum-strain experiments, the soft state was characterized by low moduli and
ratio,

Tan 8 was about

over a wide frequency range,

which implies that the contribution of the viscous component was as large as that of the elastic component. At the
lowest frequency, tan 8 exceeded
indicating the domihigh tan

8.

nance of the viscous component.

significant contribution

of the viscous component was also found

in the constant-

frequency experiments that showed a high dissipation ratio

in the soft state.

CaFASW mimicked the soft state of

in CaFASW showed a drastic

when

the deformation exceeded the

unique behavior, together with the quite

high energy dissipation ratio in the soft

adaptive significance

show

bers
the

body

through
is

in

autotomy and

state,

evisceration, a kind of autotomy.

to

have

Sea cucum-

They

contract

coelomic cavity,

in the

body wall; they eject their viscera

Because the dermis in the ruptured por-

that hole.

very soft to the touch, that part

The scenario

how

for

works

in the evisceration

would

first

make

seems

fission.

to increase the pressure in the

causing a rupture

is

no doubt

process

is

as follows.

a small portion of the dermis

At

in a soft

the strain-induced softening

The animal
that to

be

softened part still contributes to the integrity of the body wall because the stiffness of
the soft state at low strain is not as low as that after having
ruptured

Soft state

it

The dermis

limit strain. This

state.

moved. The dominance of

very likely in the strain-

an intact sea cucumber that was subjected to large repetitive

1988), and thus it seems very

restore the original posture after the

re-

is

deformations (Motokawa.

tion

is

dermis

probably was not in that state.

Strain-induced softening of the dermis was observed in

defense and body support, and the springy feature helps

imposed force

inter-

but in the previous dynamic tests

decrease in stiffness

like

be

was usually subjected to fairly

large strain, while
dynamic tests, the strain imposed was
much smaller than the limit strain of the present sea cucum-

The

behaved

to

in

a linear elastic solid at frequencies higher than 0.1 Hz.

in the stiff state

by

seems

the difference in the strain used.

In creep tests, the dermis

intact animals.

dermis

in

changes (Shibayama el al.. 1994). Although

and
Shadwick (2000) measured shear modulus, not
Szulgit
elastic modulus, and thus strict comparison is not possible,

like

ulus. Thus, in short, the

(Hayashi

dynamic tests it caused

and
Shad
wick, 2000) or no
softening (Szulgit

and the low J index imply that the tissue behaved rather

high values of stiffness, moduli, and deformation energy are

the features associated with the increase in the elastic mod-

re-

we could

ported previously,

(Motokawa, 1987; Birenheide el ai. 1998). The potassiumrich media very likely worked through stimulating cellular

when

other

in

Although strain-induced softening has not been

states.

of

strain in-

phenomenon was never observed

creased. This

and Motokawa. 1986), whereas

stiffening that occurs

strain

limit

maximum

soft.

exceeded the

this stage, the

limit strain.

The animal would then

increase

coelomic pressure, causing larger deformations

softened part. Once the deformation exceeds the limit
the

in the
strain,

the stiffness drastically decreases and so the dissipation

ratio increases,

which allows the dermis

same pressure

to

continue deform-

even under lower pressure) until

rupture. This is positive feedback: the more deformed the
dermis, the more easily the dermis is deformed. Such meing at the

(or

chanical properties allow the animal to eviscerate with only

a transient increase in the coelomic pressure, and thus to

confine the rupture to the small portion initially softened,

leaving the rest of the dermis intact.

272

T.

MOTOKAWA AND

Standard state

We

havior
d the

emplo

that the
state

convention used in previous studies

ASW, was taken as the standard

.sied in

(M

.ukuwa,

1984a).

We

tested the dermis after a

resting period of
day in ASW. Such a lengthy resting
was
chosen
because
the dermis, when rested for less
period
1

than 15 h,

of the

showed

Thus, recovery from

preparation took quite a long time

The long

this species.

were between those

stiffness values that

and the standard

stiff state

the effects of handling at

in

TSUCHI

A.

state.

resting period

seems not

to ad-

versely affect the dermis,

because the sample rested for

showed
clear
day
and to ACh.
responses both to
Previous studies did not employ such a
long resting period,
which may be one reason for the notoriously
large varia1

KASW

tions in the reported

mechanical properties of non-stimulated dermis in

(Motokawa, 1984c; Hayashi and Motokawa, 1986; Szulgit and Shadwick, 2000).

ASW

By touching the living sea cucumber, we can feel the

stiffening of the body wall. If such a stiffened body wall is
then vigorously squeezed, it becomes
soft
soft

1981

is

observed not only

make covalent

to

show

isolated dermis

a viscous flow
the

in

(Motokawa, 1988). The

standard state also

showed both

stiffening and softening responses. Thus it seems reasonable

to suppose that the dermis of the intact animal at rest is in

a state that corresponds to the

present standard state; in this
state, the animal is likely to
change its body shape for

movement. The standard

strain relationship

state

showed

with a prominent

flat

a J-shaped stresstoe region followed

by a steep pole region. The toe region allows animals

change

their posture easily, with

little

to

energy expenditure,

by using their body wall muscles. In contrast, to protect the

animal from damage, the steep region resists
exterlarge,

nally imposed forces. Thus the standard state with its Jshaped stress-strain curve seems to have adaptive signifi-

cance.

The standard

showed mechanical

state

mediate between the

stiff

and

soft

properties inter-

states,

and thus the

standard state appears to be just an intermediate between

two extremes. Close inspection of the stress-strain relation-

however, suggests that the mechanisms of stiffening

and of softening from the standard state are
probably difship,

ferent (see next section),

and thus we conclude that the

dermis of the sea cucumber can assume three distinct mechanical states

stiff,

standard, and soft.

cross-links with each other.

catch

The derniv of
extracellulm

been thought

-i

aerials

lo

main componem

i.Mermine those of the whole dermis.

;

(he extracellular materials are

The

macro-

molecules such as collagen and

proteoglycans (Matsumura.
1974; Kariya et ai, 1990). The dermis shows continuous
creep to

final

breakage under even a small load. This be-

seems

to

be

in

the

meshwork of polymers. In the meshwork, polymer molecules make a noncovalent bond with
adjacent
a

is

molecules

at each crossing
point of the molecular mesh. The
chain
between adjacent crossing points is here
polymer
called a segment. Two kinds of bonds are
postulated in the
meshwork. One is the intermolecular bond that forms the

crossing point of the meshwork, and the other is the segmental bond found within the molecular chain that com-

segment (Fig. 9). A part of the molecule in the

segment is presumed to take a folded structure in which the
folds were maintained by intra-molecular or
prises the

segmental

bonds. Such bonds

resistant

to

stretch.

bonds implies

that

make the segment less flexible and

The introduction of inter-molecular
more molecules

in the

dermis are

re-

cruited into the force-bearing meshwork.

When the dermis in the standard state

is stretched, each
molecular segment freely rotates around the
crossing points
of the mesh until all the segments become
parallel to the
direction of stretch. Further stretch
directly stretches the

segment and thus stretches the segmental bonds (central

column of Fig. 9). The free rotation of segments corresponds to the toe region of the J curve, and the direct
stretching of segments corresponds to the pole region. The
unloading curve shows a higher J index than that of the
loading curve. This implies that stretching segments induces

some

plastic deformation.

Such

plastic

deformation must be

temporary rather than permanent, because the next hysteresis loop exhibited
exactly the same shape. To explain this

we postulate that some segmental bonds are

stretched plastically, but they recover their
original state in
the unloading and
that follow.
behavior,

compressing phases
Tempobehavior explains the rather high dissipation
ratio seen in the standard state.
rarily plastic

Stiffening

stimulation

induces

inter-molecular

bonds,

which reduces the segment length of the mesh (upper

right
of Fig. 9). A reduced segment
length accounts for the short
It

also increases the resistance to rotation around

crossing points because a smaller mesh size increases the

resistance to displacement of water from the mesh, and thus
increases the slope of the toe region.

cucumber is composed mainly of

whose mechanical properties have

the sea

It

Let us suppose that the

force-bearing structure

dermis

toe region.

Simple polymer model and implications for mechanism of

(Motokawa,

possible to regard dermis as a blend of non-cross-linked

polymers, and thus it is tempting to interpret the present
results in terms of polymer science.

very

enough

in the standard state

but also in soft and stiff states

(unpubl. obs.), which
suggests that the main components of the dermis do not
)

An

increase in inter-

molecular bonds recruits more molecules into the meshwork, and thus increases the resistance to stretch of the
meshwork. This explains the higher stiffness of the pole
region. The newly recruited molecules are presumed not to
contain plastic segmental bonds given the small difference
between loading and unloading curves and the low
dissipation ratio in the stiff state.

VISCOELAST1CITY OF SEA CUCUMBER

273

Figure 9. Schematic of a simple polymer model for the dermis. A single mesh is drawn. Top row portrays.
from left to right, the soft state, the standard state, and the stiff state. Hollow circle, intermolecular bond; rilled
segmental bond that deforms elastically when stretched; stippled circle, segmental bond that deforms
when stretched. The folded structure of the segment is drawn only for one side of a mesh. Central
column is the mesh of the standard state under increasing strain from top to bottom when stretched horizontally.
circle,

plastically

The mesh

is

free to

deform (middle): the segmental bonds experience deformation

after the

segments become

oriented parallel to the direction of stretch (bottom).

In the soft state, the length

of the toe region remained the

same as that in the standard

From

state.

this result

we

nous mesogloea of a sea anemone (Gosline, 1971). The

stiffening mechanism and the softening mechanism seem to

own

postulate that the segment length remains the same, and thus

have their

the number of inter-molecular bonds remained unchanged at

the transition from standard to soft states (upper left of Fig.

that stiffens the

The segmental bonds are, however, postulated to decrease in number. Removal of segmental bonds makes the
segment more flexible and stretchy, which accounts for the
low stiffness of the pole region in the soft state. A fair
proportion of the remaining segmental bonds show plastic
deformation on stretch, producing a fairly large J index at

The presence of a softening molecule has also been shown

(Koob et ai, 1999; Szulgit and Shadwick, 2000). The stiffening mechanism and the softening mechanism also seem to

9).

unloading and a large dissipation ratio in the soft state. In

bonds break when the strain ex-

the soft state, segmental

ceeds some

limit.

This endows the model with strain-soft-

ening behavior. The loss of segmental bonds that had held

the folded structure of the chain results in elongation of the

segment. This explains the longer toe region in samples that

experienced strain-induced softening. The breakage of

bonds also accounts for the decrease

in the

maximum

stress.

This meshwork model simulates well the mechanical

behavior of the dermis with only a small number of assumptions. The model suggests that the molecular mechanism
involved in stiffening is different from that involved in
Inter-molecular bonds are associated with
softening.

changes between the standard state and the stiff state,

whereas segmental bonds or intra-molecular bonds are associated with changes between the standard state and the
soft state.

An

increase in E' associated with the formation of

bonds between molecules has been reported

in the collage-

2003),

have

is

dermis by binding

to collagen

(Tipper et ai,

a candidate for the inter-molecular bonding agent.

own

their

heide et

cross-bridging molecules. Tensilin, a protein

al.,

neural pathways (Motokawa. 1987; Biren-

1998). In the present model, calcium ions are

involved in segmental bonds. Calcium ions have a number

of possible sites that they affect in both polymer systems
and in biological systems. They seem to have some roles in
a

"polymer system" of the dermis because detergent-treated

is quite sensitive to calcium ions (Motokawa, 1994).

dermis

are also probably involved in the "biological system"

of the dermis, affecting neuronal activities, secretion processes, or both (Motokawa and Hayashi, 1987; Trotter and

They

Koob, 1995). Calcium translocation in the holothurian dermis has been suggested (Matsuno and Motokawa, 1992).
Thus calcium ions no doubt play one or more important
roles in the mechanism of connective tissue catch.

A meshwork
gives an

"

of noncovalently cross-linked polymers

-frequency curve with four characteristic regions

(Ferry. 1980).

They

are,

from the high-frequency end

to the
"

low-frequency end, the glass region with a high value of

the transition region with decreasing E' as frequency de,

creases, the rubbery or plateau region of constant E' and

the flow region of decreasing E' as the frequency decreases.
,

274

T.

MOTOKAWA AND

These four regions are all apparent only when measurements are per.orrned over a very wide range of frequencies,
which is often not practicable. In the present study, the

showed an

in the suit state

dermis behaves

like a liquid,

and because the softened

dermis does exhibit flow (Motokawa, 1988). The

showed an
glass state

is

intact

10"

w
10

stiff state

"

increase in
"

a constant value of

10

'

-frequency curve with

in the lowest fredecreases
as
E'
frequency
decreasing
It is reasonable to regard this region as the
quency region.
flow region because a tan 8 value of 10 implies that the

dermis

TSUCHI

A.

with increasing frequency to reach

"
in the
of about 5 MPa. Because

10

10

2 orders of magnitude higher than this (Fuka-

hori, 2000), the

high-frequency region of the

stiff state is

very probably the plateau region, not the glass region.

In polymer science, it is possible to construct a single

E' -frequency curve from experimental data of limited

10"

10-'

iry

10

10

frequency (Hz)

fre-

"Apparent master curve" of storage modulus E' Hollow

hollow diamond, the stiff state (KASW);

HI.

Figure

quency ranges by using the time-temperature superposition

principle. For linear viscoelastic materials such as amor-

circle, the stiff state (dissection};

.-'.

the stiff state (ACh); +, the standard state; filled circle, the soft state.

phous polymers, the effects of time and temperature on

mechanical properties are equivalent; thus the curve at one

The logarithm of the shift factor was 1 .7 for the stiff states; for
it
was
2. meaning that the curve shifted to the left by

temperature can be superimposed upon those at different

temperatures by shifting the curves from lower temperatures
to the left and those from higher temperatures to the right

magnitude

along the frequency axis to generate a smooth master curve

(Ferry, 1980). This method is convenient because it generates a single curve that gives an overview of the frequency

dependence of mechanical properties.

master curve

is

in

the soft state

2 orders of

frequency.

sphere Research Center, University of the Ryukyus, for the

supply of sea cucumbers. Supported by Grant-in-aid for

on priority area (A) "Molecular synchro-

scientific research

nization for design of

made by varying the temperature. It is, however,

sometimes composed from curves derived from different

new

materials system."

usually

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concentrations of polymers or in solutions of different ionic

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al.,

We

attempted to generate an
order to get a single curve that

1968).

"apparent master curve" in

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affected the mechanical properties, acting not only directly

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ling

leaving that of the standard state as a reference.

construct a smooth curve of

"

We

could

with four different phases

10),

although there

is

no physicochemical theory

at

hand

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we could

smooth curve could also be

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fre-

changes.

Motokawa, M. Ohtani,
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Reference: Bio/. Bull 205: 276-284. (December 2003)

2003 Marine Biological Laboratory

Heat-Shock Protein 70 (Hsp70) as a Biochemical

Stress Indicator: an Experimental Field Test in Two
Congeneric Intertidal Gastropods (Genus: Tegula)
LARS TOMANEK AND ERIC SANFORD 2
1

Hopkins Marine

Station, Stanford University, Pacific Grove, California

Although previous studies have demonstrated

heat-shock protein 70 (Hsp70) can be induced by environmental stress, little is known about natural variation in

Abstract.

zone,

that

ities

response over short time scales. We examined how

Hsp70 levels varied over days to weeks in two intertidal
snail species of the genus Tegula. Sampling was conducted

Hsp70

levels reflected the different thermal sensitiv-

of the physiological systems of these two species.

this

Introduction

The synthesis of heat-shock proteins (Hsps) is induced

when environmental variation perturbs an organism's phys-

both under naturally changing environmental conditions and

in different vertical

93950-3094

zones on a rocky shore. The subtidal to

into shaded and

iological system to the extent that

its

proteins denature.

brunnea was transplanted

Under such environmental conditions Hsps and other mo-

unshaded mid-intertidal cages to assess temporal variation

in Hsps under conditions of increased stress. For comparison, the low to mid-intertidal T. funebralis was transplanted

lecular chaperones stabilize denaturing proteins, refold re-

zone of

and Craig, 1988; Parsell and Lindquist, 1994; Feige et <;/..

1996; Frydman, 2001; Haiti and Hayer-Hartl, 2002). Nu-

low-intertidal T.

into mid-intertidal cages, within this species' natural

versibly denatured proteins, and facilitate the degradation of

irreversibly denatured proteins (Lindquist, 1986; Lindquist

occurrence. Snails were sampled every 3 to 4 days for one

month, and endogenous levels of two Hsp70-kDa family

members (Hsp72 and Hsp74) were

studies have investigated the relationship between

synthesis and various potential stress factors; however,
very few studies have investigated the variation of Hsp
levels under varying natural conditions (for review see

merous

Hsp

quantified using solid-

phase immunochemistry. Following periods of midday low

tides, levels of Hsps increased greatly in transplanted T.

brunnea but not

less in

T.

in T. funebralis.

brunnea transplanted

unshaded cages, suggesting

that

Feder and Hofmann, 1999). Even fewer studies have inves-

Levels of Hsps increased

to shaded cages than to

tigated short-term variation in

Hsp levels (e.g., hours, days

response to variable physical conditions in the
field (Hofmann and Somero, 1995; Nakano and Iwama,
to

prolonged emersion and

reduction in feeding time per se are factors that are only

mildly stressful. Upregulated levels of Hsps returned to base
levels within days. In
their natural zones,

2002).

levels

showed

little

in

comprehensive understanding of such a time

in Hsp levels under natural conditions is

course of variation

unmanipulated snails collected from

Hsp

weeks)

to interpret Hsp levels from field-collected organisms and therefore to evaluate stress under natural condi-

needed

change with

thermal variation, indicating that these species did not experience thermally stressful conditions during this study.
However under common conditions in the mid-intertidal

Furthermore, predictions about the ecological role of

the heat-shock response that are made from laboratory comtions.

parisons of species that occupy widely varying thermal

environments have not been tested under natural conditions.
Received 28 May, 2003; accepted 24 September 2003.
1

Our study focuses on Hsp

To whom correspondence

should he addressed. Present address: Dept.

of Animal Science. University of California, One Shields Avenue, Davis.

CA

variation in response to phys-

organisms. Physical factors, in particular temperature, play an important role in setting the
upper limits to the vertical distribution range of intertidal
ical stress in intertidal

95616. E-mail: ltomanek@ ucdavis.edu

"

Present address: Eric Sanford. Dept. of Ecology and Evolutionary

Biology, Brown University, Providence, Rl 02412.

organisms and confine them

276

to distinct

bands on the shore

HSP70 AS STRESS

with species-specific upper and lower vertical limits (see

reviews in Benson, 2(102; Tomunek and Helmuth, 2002).
is supported by numerous laboratory studies
demonstrating that physiological resistance to physical con-

This view

ditions

is

species that live

in

greater

at

higher

tidal

INDICATOR

277

how Hsp levels vary

response to the changing physical conditions
within and beyond a species' natural thermal zone.

thus attempts to comprehensively test

over time

in

Materials and Methods

heights

(Newell. 1979; Somero. 2002).

Intraspecific variation in the expression of

found

in intertidal

species in association with seasonal ac-

climatization (Dietz and Somero. 1992;

ero.

I99v Roberts

ley et

Hofmann and Som-

1997; Chappie et

ct al..

al.,

Somero. 1996a; Roberts

et al.,

1997;

1998: Buck-

(Hofmann and

laboratory acclimation

2001).

al.,

Hsps has been

Tomanek and Somero.

1999. 2000. 2002). competition for space (Rossi and Sny-

food availability and wave exposure (Dahlhoff

2001). and microhabitat (Helmuth and Hofmann.

der. 2001).
et

cil..

2001). Interspecific differences in the heat-shock response,

especially among congeneric species, often correlate positively with thermal

extremes

in the

environment (Sanders

el

Stiu/y

organisms and distribution patterns

The two Tegula congeners used

in this study differ in

biogeographic and vertical distributions. Tegula briinnea inhabits the subtidal to low-intertidal zones of the

their

Ocean from Cape Arago, Oregon (43 25 'N)

eastern Pacific

Channel

to the

OO'N) (Abbott and

Islands. California (34

1980:

Haderlie,

Riedman

1981: Watanabe.

et al.,

19X4).

Tegula funebralis is found in the low- to mid-intertidal zone

and has a wider latitudinal range, from Vancouver Island.
British

Columbia, Canada (48

25'N), to central Baja Cal-

Mexico (28 OO'N) (Abbott and Haderlie. 1980;

Riedman et al.. 1981).

ifornia.

Hofmann and Somero.

1996a: Tomanek and Somero. 1999. 2000. 2002: Nakano
and Iwama. 2002: Tomanek, 2002). Some of these studies
have shown how Hsp levels vary over hours in response to

Experimental design

thermal stress under natural (Hofmann and Somero, 1995.

Station of Stanford University in Pacific Grove. California

1996b; Nakano and Iwama, 2002) as well as laboratory

(Tomanek and Somero. 2000). To our best

(36

ul.,

1991; Dietz and Somero, 1993;

conditions

known about

the variation of

Hsp

knowledge, nothing

is

levels in response to

changing natural conditions over days

Field experiments were conducted at Hopkins Marine

54'W). To test the role of temperature in

upper vertical limit of the subtidal to low-interbninnen, we transplanted this species above its

36'N. 121

setting the
tidal

T.

natural zone of occurrence. Snails (shell diameter

whether interspecific differences in

the heat-shock response limit a species' thermal niche and

mm. marked

contribute to setting its vertical distribution range within the

intertidal zone has not been tested under natural conditions.

stainless steel wire cloth with a

to weeks. Furthermore,

This study focuses on two herbivorous gastropod species

of the genus Tegula that occupy distinct vertical zones on
the shore:

T.

bninnea

(Philippi.

1848),

subtidal to low-intertidal zone, and

1855),
et

common

in the

work suggested

that

in

the

funebralis (Adams.
low- to mid-intertidal zone (Riedman

Our previous

1981: Watanabe.

al..

common

T.

1984).
laboratory
heat stress might prevent the low-

new thermal environment. Laboratory results (Tomanek

and Somero. 1999) also suggested that the mid-intertidal T.

its

would

funebralis

quently

activate

in its natural

the

heat-shock

zone of occurrence due

response
to the

fre-

temper-

ature extremes and fluctuations that characterize the midintertidal zone. In this study

we

test

these predictions by

cm

high;

316

opening size)

that

ment) or shaded by covering them with plastic mesh (two

lav ers of a black polyethylene mesh with a 3.8-mm opening
size).

Each treatment included seven cages, with 10

intertidal

intertidal

3-mm

over the rock surface and feed on the algal species present
naturally. Cages were either unshaded (sun-exposed treat-

bninnea transplanted upward into the midzone would express increased levels of Hsp70 in

predicted that T.

x 20 cm wide and

were positioned at similar heights in the mid-intertidal zone

0.12 m above mean lower low water). These
(+0.64
had
no bottom and thus allowed snails to move
enclosures

per cage.

T.

with yellow nail polish) were placed into

stainless steel cages (20

bninnea from occupying the mid-intertidal

zone (Tomanek and Somero. 1999, 2000, 2002). Thus, we
intertidal

= 20-25

In addition,

we

transplanted

T.

funebralis from the mid-

zone into unshaded mid-intertidal cages

positioned

at the

same

two purposes. First, it allowed us

of T. bninnea transplanted

caged
tidal

T.

its

natural

upper

(;;

7)

This treatment served

tidal height.

to

stress

zone (above

snails

compare

into the

limit)

the thermal

mid-intertidal

to that

of similarly

funebralis. the natural inhabitant of the mid-inter-

zone. Secondly,

by comparing

it

allowed us to

caged mid-intertidal

test for
T.

caging artifacts

funebralis to

unma-

time course of expression of two Hsp70

following
isoforms (Hsp72 and Hsp74) over a month-long sampling

nipulated snails

period in specimens transplanted from the low-intertidal

into the mid-intertidal zone, and in control individuals col-

with an unmarked specimen (to avoid changes in snail

density during the experiment) every 3rd or 4th day over a

the

lected

from

their natural vertical zones.

This combination of ecological and molecular approaches

found naturally in that zone.

snails
were
collected from each cage and replaced
Single

May 2000; see

month-long sampling period (31 March to
of
1
and
To
evaluate
the
snails in their
2).
Figs.
response
1

278
native thermal environment,
snails

low

TOMANEK AND

L.

from

we

also collected unrestricted

aid intertidal (T. fiinehralis)

and the

shal-

one

E.

SANFORD

mmol 1~' Tris-HCl, pH 7.5 at 20 C, 150 mmol

NaCl, 0.1% (v/v) Tween. 0.02% (w/v) Thimerosol, 5%

buffer (25
'

(T. bntnnea; specimens were always

and during collection). All collections
were mack- within 45 min of low tide to minimize any

Tris-buffered saline (TBS; 25

physiological variation that might be related to endogenous

tidal rhythms. Snails were frozen immediately on dry ice

solution of a monoclonal rat antibody (IgG) against Hsp70

(clone 7.10; Affinity BioReagent, MA3-001; 1:2500 dilu-

following collection and kept at -70 C until further proTo obtain a record of Tegula body temperature,

tion of Hsp70 antibody

bovine serum albumin

subtid;;

submen.'. o

i'ore

cessing.

temperatures

in

gelatin-filled

snail

shells

were recorded

and outside (on adjacent rocks)

inside mid-intertidal cages

(w/v) nonfat dried milk) for

20

C.

mmol

150

subsequently washed with

1~' Tris-HCl, pH 7.5 at

h,

mmol

NaCl), and then incubated with a

1~'

buffer

in

in

TBS)

membranes, we incubated them

anti-rat bridging

(BA): TBS, 2.5% (w/v)

for

for

h. After washing the

30 min with a rabbit-

antibody (IgG) solution

:2000 dilution in

by a Stow A way XTI temperature data logger (Onset Computer Corp, Pocasset, MA). For further details on this

BA; Vector. AI-4000). followed again by several washing

steps. Finally, we incubated membranes with a horseradish-

method, see Tomanek and Somero (1999). Because of

equipment failure, only one complete record, from a T.

peroxidase protein A solution (1:5000 dilution in BA; BioRad) for 30 min. Membranes were washed and overlaid

fiinebralis shell attached to

with a solution of enhanced chemiluminescent (ECL) re-

open rock adjacent

mid-

to a

was obtained from the six data loggers

and 2) therefore
Temperatures shown (Figs.

represent the thermal variation of field-acclimatized T. fiinebralis from the mid-intertidal zone. Immediately follow-

(Amersham Pharmacia) according to the manufacmin. Under dark room conditions,

we exposed membranes onto pre-flashed Hyperfilm (Amersham Pharmacia) for 5. 10. 20. 30. and 50 min after ECL

ing the experiment (and discovery of the equipment failure),

additional data loggers were deployed to characterize the

treatment to obtain various exposures that were in the linear

range of detection. All samples were run at least twice.

intertidal

cage,

installed.

difference

among our

treatments. During

midday low

agent

turer's instructions for

tides,

temperatures within unshaded cages were typically 3-6 C

cooler than on the open rock outside the cage, whereas

temperatures in shaded cages were an additional 2-5

cooler than in cages without shades.

linage analysis

and

quantification of expression of heat

shock proteins
Film images were scanned on a densitometer (Sharp
JX-330) and the digitized images were analyzed with image

Tissue preparation

analysis software (ImageMaster ID. ver. 2.01, Pharmacia)

was dissected from whole snails that were

thawed under conditions that do not induce heat shock
3
C) and immediately placed in 200 /xl (T. fiinebralis, 15.0 to
25.0 mg wet weight) or 300 /j,l (T. hrunneci, 30.0 to 45.0 mg

to quantify

Gill tissue

wet weight) of homogenization buffer (32 mmol

HC1, pH 7.5 at 4 C, 2% (w/v) SDS.
mmol T
1

mmol

1~' Tris-

EDTA,

of about 74
relative to a

to

account for variation

was

repeated and homogenates were centrifuged at

15,800 X g for 15 min. The supernatant was removed and

-70 "C. Protein concentrations were determined

Micro-BCA assay (Pierce) according to the man-

Statistical

anahsis

Variation in Hsp72 and Hsp74 was compared using a

two-factor analysis of variance (ANOVA) with experimental treatments and sampling days as the main effects. We

conducted post hoc comparisons of

(Student-Newman-Keuls

ufacturer's instructions.

separately.

protocol
In general,

we followed

the procedure described in

To-

manek and Somero

(2002). Briefly, proteins were separated

electrophoretically and subsequently transferred onto nitrocellulose membranes (Nitrobind, Schleicher and Schuell) in
transfer buffer (25

20%

mmol

methanol

1~'

(v/v),

pH

Tris-base, 0.193
8.3 at

mol

To

calculate

test)

all five

treatment groups

within each sampling day

the critical

value,

we used

the

= 0.95; m =
appropriate Studentized range statistic (a
=
number of means: df
degrees of freedom of the error term
from the ANOVA) and an adjusted H value (/;,,) to account

Gel electrophoresis and immunodetection (Western)

glycine,

two Hsp70 isoforms, one

kDa (Hsp72), the other

kDa (Hsp74). We express band intensities
known amount of a bovine heat-shock cognate

70 (80 ng: StressGen, SPP-750)

amonc Western blots.

using the

intensities of the

Pefabloc (Boehringer Mannheim), 10 ^g ml"

pepstatin, and 10 /n,g ml"
leupeptin). Tissues were incubated for 5 min at 100 C and homogenized. The procedure
I"

stored at

band

with a molecular mass of about 72

for unequal

sample sizes among the means using the

lowing equation:

1"'

20 C). After mem-

branes were dried overnight they were treated with

blocking

=
a

fol-

HM-70 AS STRESS

(a = 50) and
were not
Variances
mean.
"/;," the sample size for each
>
and
therefore
P
0.05).
heterogeneous (Cochrane's test,

with "a" the

there

total

was no

number of means compared

need to transform the data.

(MatLab Software) com-

cross-correlation analysis

minimum, maximum

pared average,

daily temperatures as

well as daily temperature range with endogenous levels of

Hsp72 and Hsp74 over the entire sampling time for field-

acclimatized mid-intertidal

T.

funebralis only.

279

INDICATOR

experimentally caged (sun-exposed) or unrestricted (fieldacclimatized) to address three issues. First, we tested for

caging artifacts by comparing caged and uncaged snails

within the same zone. Second, we tested the prediction that

shock response more frequently than

congener due

heat-shock responses (Tomanek and Somero. 1999, 2000).

Third, we compared the response to thermal stress in the

"common

heights under

that

occupy

different tidal

garden" conditions (see below).

Unrestricted individuals of

Data logger records indicate that the body temperature of

mid-intertidal Tegula varied dramatically with tidal cycle

activate the heat-

the shallow subtidal

thermal environments and

to their differing

two temperate Tegula congeners

Results

would

the mid-intertidal Tegula congener

T.

funebralis were collected in

The two groups did not

crevices next to the unshaded cages.

differ until the last

week

in April,

when Hsp72

levels

were

experiment (Figs. A and

and 2) show the
D
and
three
2 A). The
(Figs.
panels B, C,
over
the month of
and
of
levels
Hsp74
Hsp72
endogenous

sun-exposed caged snails than in field-acclimahigher

tized snails (Fig. 1C). Preceding this time period, peak
temperatures from data loggers were relatively low, but they

sampling.

increased greatly with the onset of a 10-day period of early

and date over the course of

this

in

to

Variation of
its

Hsp70

in

Tegula brunnea transplanted above

midday low

but

in

tides.

higher levels than

natural limit

Specimens of T. bninneti transplanted into unshaded

cages in the mid-zone often showed dramatically elevated
levels of both

Hsp72 and Hsp74

uals collected

from the shallow subtidal zone

relative to control individ(for statistical

IB and 2B). The overall response of both

similar.
was
Moreover, these elevated levels of
very
Hsps
in
seen
transplanted individuals were correlated with
Hsps
environmental factors: 3 days on which Hsp72 and Hsp74
levels were 2 to 4 times higher in sun-exposed mid-inter-

results, see Figs.

than in control snails from the shallow subtidal

were preceded by periods of 2 or more days of midday low
tides that greatly raised body temperatures for 1-4 h (3 and
13 April and
May 01). However, on 21 April, sun-exposed
of
T.
brunnea showed 6 times higher endogenous
specimens
levels of Hsp72 and Hsp74 than control animals, but maximal daily temperatures during the preceding 4 days were
tidal snails

Hsp74

levels

showed

caged

snails

a similar pattern,

individuals

field-acclimatized

addition,

from 10

showed

to 13 April (Fig.

2C). Thus, caged specimens of T. funebralis were apparconently more thermally stressed than their unrestricted
specifics during the last

week of

the study, perhaps because

caging limited the access of snails to

shaded and moist

microhabitats.

With time and changing temperatures, Hsp72 levels varied little until they increased in caged but not in unrestricted
individuals (Fig. 1C). Although Hsp74 levels showed
were still
greater temporal variability, the resulting changes
within the range of variation observed for shallow subtidal
T.

brunnea

(Fig.

2B,

Hsp74 showed any

field samples).

Neither Hsp72 nor

correlations with any of the temperature

variables (cross-correlation analysis,

P >

0.05).

These

relatively

low compared

In addition,

T.

brunnea

that

T.

subtidal.

to other time periods.

individuals of

funebralis does not

elevate levels of Hsps more often than T. brunnea does
under the less thermally variable conditions of the shallow
results suggest that mid-intertidal

were

trans-

shaded mid-zone cages showed elevated levels

of Hsps only slightly more often than control snails from the
shallow subtidal, and to a greater degree in the case of
Hsp74 (e.g.. 10 April and 24 April) than in Hsp72. In

planted into

contrast,

sun-exposed individuals often showed greater Hsp

shaded conspecifics that were trans-

levels relative to their

planted into the mid-zone, with

being higher

in the

Hsp72

sun-exposed

T.

levels almost always

brunnea

(Fig. IB).

Interspecific

comparisons of Tegula

mid-zone

in the midTransplanting both species to unshaded cages

zone allowed us to compare their responses to

intertidal

garden conditions. Whereas

T. brunnea responded to thermal
of
transplanted specimens
stress, specimens of 7". funebralis caged in the mid-zone
thermal stress under

(their natural

Time course of Hsps in transplanted

acclimatized Tegula funebralis

in the

common

zone of occurrence) changed

little

(Figs.

ID

and 2D). Elevated levels of both Hsps indicate a response to

thermal variation during time periods of midday low tides in

and field-

brunnea (see above for details). Although base levels of

Hsp72 in sun-exposed T. brunnea were close to levels found
T.

We quantified
of

T.

the time course of

Hsp

levels in

specimens

funebralis from the mid-intertidal zone that were either

for T. funebralis.

Hsp74

levels were, regardless of the tidal

280

L.

TOMANEK AND

SANFORD

E.

200
T.

T.

150

T.

brunnea
brunnea
brunnea

sun-exposed
shaded

field

100

50

CM

200

JO
g
S>

T.

funebralis

sun-exposed

T.

funebralis

field

T.

brunnea

r.

funebralis

sun-exposed
sun-exposed

150

100

50

200
-

150

100

50

01-May-OO

24-Apr-OO

17-Apr-OO

10-Apr-OO

03-Apr-OO

Time (days)
tal

Figure 1. Environmental variation and time course of beat-shock protein expression (Hsp72) in experimenand control snails. (Al Tidal heights (m above mean lower low water) for Monterey Bay, day and night (gray)

cycles,

and temperatures recorded

(Tegula funebralis attached

in a gelatin-filled snail shell

in the mid-intertidul

zone). Snails within unshaded (sun-exposed) and shaded cages experienced lower temperatures. (B)

Time course

of endogenous levels of Hsp72 for specimens of Tegula briinneu transplanted to unshaded and shaded
mid-intertidal cages versus
restricted (caged)

field-acclimatized conspecifics (shallow subtidal zone).

and unrestricted (field-acclimatized) individuals of

for T. funebralis and

T.

brunnea individuals transplanted

into

T.

(C) Hsp72 levels for

funebralis (mid-intertidal zone) and (D)

unshaded mid-intertidal cages. Levels are

expressed relative to an internal control (a bovine heat-shock cognate 70). Values are mean
significant differences

sun-exposed

T.

(P

0.05) among treatments; n

brunnea on 21

= 5-7

SEM;

* indicates

= 4

for

April).

in T. hninnea (with one ex7

on
ception
April), supporting our previous results from
acclimation
laboratory
experiments (Tomanek and Somero,

Discussion

regime, consistently elevated

2002).

snails for all data points (except n

Although temporal variation in levels of heat-shock pro(Hsp) has been suggested to closely track sublethal

tein

Hsp70 AS STRESS

200

T brunnea
T. brunnea
T. brunnea

100

50

sun-exposed
shaded
field

200
Q.

281

150

INDICATOR

T.

funebralis

sun-exposed

7"

funebralis

field

150

<n

100

50

9>

0>

200

-,

7.

brunnea

T.

funebralis

sun-exposed
sun-exposed

150

100

50

01-May-OO

24-Apr-OO

17-Apr-OO

10-Apr-OO

03-Apr-OO

Time (days)
tal

Figure 2. Environmental variation and time course of heat-shock protein expression (Hsp74) in experimenand control snails. (A) Tide heights, day and night cycles, and mid-intertidal temperatures (see Fig. 1 legend

Time course of endogenous levels of Hsp74 for specimens of Tegiila brunnea transplanted to
unshaded (sun-exposed) and shaded mid-intertidal cages versus field-acclimatized conspecifics (shallow subtidal
zone). (C) Hsp74 levels for restricted (caged) and unrestricted (field-acclimatized) individuals of Tegula
for details). (B)

funebralis (mid-intertidal zone) and (D) for

intertidal

stress, there

cages (see Fig.

have been few

tests

of

T.

funebralis and

this

hypothesis under

natural conditions. Furthermore, the ecological importance

of interspecific variation

from
this

in the

heat-shock response deduced

laboratory- studies has not

study

we show

been tested

in the field. In

that the subtidal to low-intertidal

brunnea transplanted above

its

T.

brunnea transplanted

into

unshaded mid-

legend for details).

Tegula

natural zone of occurrence

experienced sublethal thermal stress, as reflected

by

ele-

vated levels of two isoforms of the 70-kDa family of heat-

shock proteins (Hsp72 and Hsp74). In contrast, levels of

Hsp72 and Hsp74 varied little in control specimens of
Tegula brunnea collected from the shallow subtidal zone,

and

less in T.

brunnea individuals transplanted

to

shaded

mid-intertidal cages. In addition, T. funebralis transplanted

within

its

cages,

but

natural

not

slightly elevated

zone of occurrence,
field-acclimatized

Hsp

to

mid-intertidal

individuals,

levels during a prolonged

showed
midday

282

L.

low-tide period
course of Hsr

mly. Here
ion

thermal hiv'

and

heat-sh*'

Mise

range

(2)

we

TOMANEK AND

how

discuss (1)

the time

correlates with an organism's

how

may

interspecific variation in the

limit the vertical distribution

rtidal invertebrates.

E.

SANFORD

the field after

low

tide,

in

presumably

denaturation due to thermal stress

response to protein

(Hofmann and Somero,

1996b).

This discrepancy between our field results and our laboratory-based predictions could be due to several factors:

most of our laboratory incubations were done in water,

leading to a high rate of heating. However, heat stress in the
intertidal zone is typically experienced under aerial condi-

first,

Time course of Hsp

One of

levels

the objectives of this study

was

examine the

to

between thermal events and the organism's

correlation

re-

tions,

when

heating

missed an increase

course of levels of Hsp70 shows that periods of extreme

thermal conditions upregulate endogenous levels of Hsp70

several

in transplanted T. hnmnea, but not in individuals

of both species that were collected from their natural ther-

(Figs. 1B-C and 2B-C). One

between increased Hsp levels in

mal environment
the association

slower (Tomanek and Somero, 2000).

to 4 days may have

in levels of Hsp70 isoforms in response

thermal stress on some collecting days that followed

sponse through time to better interpret the role of Hsps as

biochemical indicators of sublethal thermal stress. The time

isoforms

is

Second, our collection interval of 3

to

midday low

tides after

which individuals may show

an attenuated response. Yet some of those collecting days

were preceded by the "first" extreme low tide following

minor

several days of

tides (e.g., 10

exception to

the recovery time of elevated

transplanted

heat stress

in

T.

Hsp

Although

April).

synthesis

in

response to

Tomanek and

short (6 h;

is

fiinebralis

and 24

brunnea and peak temperatures occurred on 21 April.

However, changes in the daily temperature range (Tomanek. 2002) during the preceding days, from 16 to 17

Somero. 2000), we should have detected elevated endogenous Hsp levels over a much longer time period. Other

April, were of similar magnitude as at the onset of a midday

low-tide series (e.g.. 8 to 9 April), even if maximal temper-

short-term acute severe heat stress (elevation lasting up to 2

weeks; Clegg et ai, 1998) and to long-term chronic mild

T.

atures were relatively low.

response

Hsp

levels

were upregulated

to physiological stress for not

more than

3 to

in

It is still

unclear what thermal signals elicit an increase in

Levels in field-acclimatized mid-intertidal T.

levels.

any of the thermal variables tested. Hsp levels will respond differently to chronic
and acute thermal stress (Helmuth and Hofmann, 2001), but

fiinebralis did not con-elate with

further studies are needed to test the response of

more complex thermal

Hsps

signals.

typical of the mid-intertidal zone (30

C;

Tomanek and

Somero, 2000). These studies also indicated that T. funehralis would activate Hsp synthesis for at least several hours
(< 6 h for Hsp70) in response to temperatures of 30 C or
exposures that were reached at least several times
during this month-long field experiment (Fig. 1A). In addition, the activation temperature of Hsp synthesis is 27 C in

above

laboratory-acclimated (constant temperature) specimens of

T. fiinehrtilis that were exposed to a wide range of incubation temperatures following rapid heating in

do not

seawater (To-

1999). Although these laboratory con-

match

conditions

completely, the
activation temperature should be within a few degrees of 27
one of the highest temperatures
C, certainly below 35 C
field

specimens of T. fiinebralis experienced during our month-long sampling period. Additionthat field-acclimatized

Hsp synthesis js upregulated for several hours in response to an acute thc/mal stress in mussels collected from
ally,

2002). Alternatively,

Hsp70

is

Nakano and Iwama,

closely regulated and quickly

granules following heat shock (Morimoto,

1998), and we may have therefore missed briefly elevated

levels of

in

Hsp70.

final factor

may be

that the

moderation

of temperatures generated by the cages themselves (even

those cages without additional shading), may have kept the
temperatures of caged snails below 30

C.

Interspecific variation in the heat-shock response

predicted increased Hsp levels in transplanted T.

hnmnea on the basis of its long recovery (> 50 h) in the
laboratory from a heat-shock-inducing thermal exposure

ditions

levels in response to

to

We

manek and Somero,

Hsp

heat stress (elevation as long as 4 days;

eliminated

days.

Hsp

studies have observed elevated

and

vertical distribution limits in intertidal invertebrates

By

T.

transplanting

brunnea above

its

natural zone of

occurrence and by following the time course of changes in

endogenous levels of Hsps over a month of thermal variation,

we were

able to directly evaluate the importance of

relation
interspecific variation in the heat-shock response in
to vertical distribution limits.

Transplanted specimens of T. brunnea increased their

levels of both Hsp72 and Hsp74 in response to

endogenous

midday low-tide periods and therefore reached,

above the activation threshold

dicted, temperatures
stress response
tality in

as

pre-

for their

(Tomanek and Somero. 1999, 2000). Mor-

transplanted

T.

hnmnea

is

also indicative of severe

(8.5% mortality in sun-exposed individuals over the

entire month; no individuals of T. fiinebralis died). Endogenous levels of Hsp72 and Hsp74 changed little in T.
fiinebralis in comparison to T. brunnea, and this could be
stress

mainly due

the laboratory,

sponse

at

higher activation temperature. In

fiinebralis activates the heat-shock re-

to this species'

27

CI

T.

versus 24

in

T.

hnmnea

(following

AS STRESS INDICATOR

H.SP70

acclimation to 13

and after rapid heating

in

good predictors of the relative levels of sublethal thermal stress and therefore the relative increases in

response are

endogenous

Hsps under

levels of

common

garden condi-

although the actual activation of the stress response

depends on the heating rate and the medium (air versus
tions,

water:

An

Tomanek and Somero,

2000).

increase in endogenous levels of Hsps

in T.

reduction

in

1988) and cellular energy levels (e.g.. ATP), which may

disrupt protein homeostasis. Individuals of T. hninneu
transplanted to shaded mid-intertidal cages differed from
unmanipulated snails collected from the shallow subtidal

experiencing slightly higher body temperatures and

longer emersion times. Yet levels of Hsp72 and

in

much

Hsp74 did

not differ consistently

between shaded mid-

and shallow subtidal controls, suggestthat

emersion
times per se were not activating
ing
longer
increased Hsp levels. Other stress factors, e.g., osmotic and
intertidal transplants

desiccation stress,
levels, but these

These
intertidal
T.

may

also contribute to changes in

were not addressed

to preventing T.

hrunnea

from inhabiting the mid-intertidal zone. This is in large part

due to the lower activation temperature (7",,,,) of the stress
response, the 6

lower temperature of maximal Hsp

synthesis (T/>CII ^. and the lower temperature

synthesis of proteins (including Hsps) ceases

(7",,,,:

Tomanek and Somero,

than in T.funebrulis

at

which the

in T.

brunnea

1999). In addition, levels of

(HSF1 are lower in T.

(Tomanek and Somero. 2002).

the heat-shock transcription factor!

hnmnea

T. finiehralis is therefore better adapted to the physical

conditions of the mid-intertidal zone, but such adaptations
in the heat-shock response may be costly. For example,

Hsp70 levels due to experimentally higher gene copy

numbers of Hsp70 can impact life-history traits (e.g.. morhigher

and developmental time) that determine fecundity in

Drosophila (Krebs and Feder. 1997a. b). and yeast strains
tality

with lower levels of

1992). Furthermore.

Hspl04 grow
Hsps can

faster

(Sanchez et

interact in detrimental

al.,

ways

1992). Thus, higher costs due to adaptations in the stress

response to the mid-intertidal environment may in part

why T. fimebralis shows slower growth rates than

explain
its

gastropods

are. the transient

low-inter-

zone shows

that

Hsps

indicators of the thermal sensitivities of physio-

good

logical systems under

common

field conditions.

Our

results

also confirm our prediction that interspecific variation in the

heat-shock response of Tegula congeners is adaptive to life
in

the thermally

variable

mid-intertidal zone

(Tomanek.

Acknowledgments
The study was supported by the David and Lucile Packard Foundation and a National Science Foundation grant
thank Michelle
(IBN-01 13184 to Dr. George Somero).

We

Phillips and Shanshan Bao for their help with the immunochemical analysis: Josh Troll for his help collecting animals
in the field: and Drs. Jim Watanabe. Mark Denny, and Chris

Harley for their comments and advice on the manuscript and

analyses. Dr. George Somero generously supour
work with advice, laboratory space, and funding.
ported
This is contribution number 137 of the Partnership for
Interdisciplinary Studies of Coastal Oceans (PISCO): A
statistical

Long-Term Ecological Consortium funded by

the

David

and Lucile Packard Foundation.

low-intertidal to subtidal congeners

T.

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Reference: Biol. Bull. 205: 2N5-294. (December 2003)

2003 Marine Biological Laboratory

Reproduction and Larval Morphology of Broadcasting

and Viviparous Species in the Cryptasterina Species

Complex
MARIA BYRNE
1

'*,

histories. C.

pentagona and

intragonadal

larvae,

C. hystera
C.

respectively.

B3H

4J1,

Canada

Introduction

asterinid sea stars

Australasia comprises cryptic species with derived

in

Department of Anatomy and Histology. F13. University

2
Department of Biology. Dalhousie University, Halifax, Nova Scotia

The Cryptasterina group of

Abstract.

AND PAULA CISTERNAS

of Sydney, NSW 2006, Australia; and

MICHAEL W. HART 2 ANNA CERRA

life

Speciation in marine invertebrate taxa

have planktonic and

enced by the evolution of

pentagona has the

is

strongly influ-

life-history traits.

Evolutionary

of reproduction with a

changes that influence speciation include modifications to

planktonic lecithotrophic brachiolaria larva. C. hyxtera is

hermaphroditic with an intragonadal lecithotrophic brachio-

gamete-binding proteins, oogenesis, larval nutrition (planktotrophic, lecithotrophic). and location (planktonic, benthic)

and the juveniles emerge through the gonopore. Both

species have large lipid-rich buoyant eggs and well-devel-

of development (Strathmann, 1985; Reid, 1990; Palumbi.

1992; Vacquier et ai. 1995; Byrne et al., 1999, 2003; Duda

gonochoric. free-spawning

mode

laria.

and Palumbi, 1999: OFoighil and Taylor, 2000; Villinski et

al.. 2002). In a large number of these cases, the combination

oped brachiolariae. Early juveniles are sustained by maternal nutrients for several weeks while the digestive tract
develops. C. hystera
phosis.

Its

was reared

in vitro

of rapid and diverse evolution of larval forms and stasis in

adult stages has resulted in congeneric species with mark-

through metamor-

brachiolariae exhibited the benthic exploration

edly different larval phenotypes and habitats but similar

adult phenotypes and ecologies. This decoupling of larval

and settlement behavior typical of planktonic larvae, and

they attached to the substratum with their brachiolar complex. These behaviors are unlikely to be used in the intragonadal environment.

and adult morphological evolution suggests that critical

examination of suspected morphospecies will reveal undiscovered marine biodiversity. Molecular and developmental

The presence of a buoyant egg and

would not be expected in an

functional brachiolaria larva

intragonadal brooder and indicate the potential for

shown that many problematic taxa include a

of cryptic species (Reid. 1990; Knowlton. 1993; Degnan and Lavin. 1995: OFoighil and Smith, 1995; Arndt et
al.. 1996; Huber et al., 2000). Application of the comparastudies have

life-

suite

history reversal to a planktonic existence. Life-history traits

of species in the Cryptasterina group are compared with

those of other asterinids in the genus Patiriella with vivip-

tive

arous development. Modifications of life-history traits and

pathways associated with evolution of viviparity in the
adaptations and clade-specific features associated with this

mode of

underlying evolution

and development, and speciation in the sea (Hart et

1997; Degnan and Lavin. 1995; Huber et al.. 2000).

Asterinidae are assessed, and the presence of convergent

unusual

approach has made many of these taxa important mod-

els for the investigation of processes

The

parental care are examined.

al.,

potential for species divergence through life-history

is common in some marine invertebrate taxa but

evolution

rare in other,

speciation

is

even closely related, taxa. Genera in which

associated with evolution of development are

found among gastropods (Littorina, Conns), clams (Lasaea), soft corals (A/cyoniiinn. asteroids (Asterina. Putiri-

Received 28 March 2003: accepted 23 July 2003.

*

To

whom

correspondence

should

be

addressed.

echinoids (Heliocidaris) and ascidians (Molgulu)

(Reid. 1990; OFoighil and Smith. 1995; Raff. 1996: Hart et

ella).

E-mail:

rnbyme@anatomy.usyd.edu.au
285

M.

286

Duda

1997;

til.,

McFadden

et

;ind

taxa are prone to de2001). Why

'ion and not others is not known. In the

u!..

is a species-rich family comderived forms of reproduction.

Asterinidae

th.

that share

prisini

and brood protection (Byrne and Cerra.

1999b). Molecular phylogenetic analy-

larval 'ij.irphology,

1996: Byrne

et

til.,

ses of these sea stars revealed

in

gence

which derived

many examples

life-history

through independent pathways (Hart

Australia,

In

the

traits

et

til.,

of conver-

have evolved
1997. 2003).

Patiriella

exigua species
temperate
group includes three species: one benthic egg layer and two
viviparous lineages (Dartnall. 1969. 1971; Keough and
Dartnall.

Until the observation of live birth,

1978).

the

presence of a second cryptic group of asterinids in the genus

Cryptasterinu, which occurs throughout Australasia (Hart et
ai, 2003; Dartnall et at.. 2003). These sea stars, formerly in

Cryptasterina pentagona was collected from five locaQueensland coast (Fig. 1) at irregular inter-

tions along the

between 1996 and 2002. This included several

vals

sites in

North Queensland (10/00: 11/00; 10/02), including Airlie

Beach (2030'S; 14845'E); Rowes Bay, Townsville
(1915'S; 14650'E); and Bingil Bay, Mission Beach

(1750'S; 14606'E). C. liystera was collected from Statue

Bay (2315'S; 15045'E) in central Queensland (8/96; 2/96;
9/97; 10/99). The samples were used to assess the condition
of the gonads and preserve samples for histology.
locality

for

P.

pseudoexigua

is

Airlie

Beach

The type
(Dartnall,

1971). In October 2002. the gonads of specimens

Beach

from

Bowen

and several sites

(201'S;
Dalrymple Point, Rose Bay, and Murrays Bay
were examined and processed for histology. Iso1)
ovaries of females from these sites and from Rowes
in

14816'E)
(Fig.

lated

Bay, Townsville, were induced to spawn through the use of

pseudoexigua species complex (Dartnall.

Gates, 1995), have been reassigned to

Patirielln

Rowe and

1971:

Cryptasteriiw in a recent taxonomic review (Dartnall et

2003).

One
n.

occurs

in

life

1992).

al.,

lineage. C. pentagona, (formerly P. pseudoex-

igua) occurs in Queensland.

erina

phic

Materials and Methods

Airlie

viviparous species were considered to be morphs of P.

exigua. Detailed analysis of mtDNA sequences revealed the

the

AL.

2000;

et ai,

some

velopment!
Asteroidea.

Palumbi, 1999; Huber

BYRNE ET

second lineage, Cryptast-

known

as P. pseudoexigua)
and
its
Taiwan,
planktonic lecithotroWanlitung,
well-documented
is
(Chen and Chen.
history

(also formerly

sp.

The viviparous species described by Hayashi

1977).

pseudoexigua pacifica, has been reassigned to C.

et til.. 2003).
(Dartnall
pacifica
On the basis of the position of C. pentagona in the
Patiriella

phylogenetic

tree,

nested between broadcasting and broodtil.,

1997), and in light of the very broad

ing species (Hart et

nominal species, it appeared likely

of
C. pentagona would have intermorphs
modes
of
development. A recent molecular study
esting
revealed the relationships among four lineages of this taxon
geographic range of

this

that Australian

called P. pseudoexigua (Hart et ai, 2003). In

Queensland these lineages comprise two species with dif-

recently

til.. 2003). One of these species,

a recently described intragonadal brooder

ferent life histories (Hart et

C.

hystera,

(Dartnall et

is

al.,

2003). In this study

tions of Cryptasterina

we examined

from the original type

popula-

locality in

Queensland and elsewhere along the coast to document

details of their reproduction and development. We compared the

life

history traits of C. liystera to those of other

viviparous asterinids in the genus Patiriella and closely

related broadcasting species to assess the

changes associ-

ated with the evolution of viviparity. This

the extreme end of the broadcast-brooding

life

history

is at

modes of prop-

agation in the Asteroidea. The pathways in the evolution of

viviparity in the Asterinidae are assessed, and the potential
for convergent

mode

Captations in

of parental .are

is

species with this unusual

examined.

Figure

known

1.

Map

of Queensland showing sample locations and current

distributions of Cr\piu\tcrinti

and C. hystera.

DEVELOPMENT

IN

CRYPTASTERINA

287

hormone 1 -methyl adenine in tillered seawater

seawater [FSW]). The eggs were fertil(10
ized with sperm removed from the testes. and the larvae
were reared in FSW at 23-25 C. Settlement substrata

of spermatogenic columns along the germinal epithelium

and a lumen filled with sperm (Fig. 2B).

including glass slides aged in seawater and pieces of coralwere introduced into some culture dishes contain-

and gastrula was typical of development in lecithotrophic

sea stars (Byrne, 1995). At 23 C the early larvae (2 days)

the ovulatory

in tiltered

line algae

ing competent larvae.

The gonads of the Statue Bay sea

contained embryos. These were removed and reared

in

24

stars

in vitro

FSW (1.0 jLim) at 22 C through metamorphosis.

For histology, the gonads were fixed in Bouin's fluid for
rinsed in distilled water, dehydrated in graded eth-

h.

embedded

and

anols,

in

paraffin.

were

sections

Serial

stained with hematoxylin and eosin. For scanning electron

microscopy, larvae and juveniles were fixed for

2.5%

in

Development of

C.

pentagona through the stages of ho-

loblastic radial cleavage, early blastula, wrinkled blastula.

had a large preoral lobe and developing larval arms (brachia) (Fig. 3A). The central brachium developed as a bulge
that

emerged from the preoral lobe, flanked posteriorly on

by two lateral braehia (Figs. 2D, 3B). Advanced

either side

brachiolariae (5 days) had a prominent brachiolar complex

comprising three braehia and a central adhesive disc (Fig.

3C). Early larvae

cover of

cilia (Fig.

swam

at

3A, B,

the surface propelled

by

their

rudiment

F); but as the juvenile

fixation,

in the posterior region, they swam at the bottom

of the culture dishes, anterior end up. An extracellular
matrix with meshlike holes covered the surface of the larvae

dried,

and juveniles

glutaraldehyde in 0.45 ju,m FSW. Use of a secondary fixative was found to be unnecessary (Byrne, pers. obs.). After
the specimens were dehydrated, critical-pointand viewed with a JEOL JSM-35C scanning electron

developed

microscope.

feet

Results

Our field survey of life-history traits in six populations of

Cryptasterina provided data on the distribution of two linone broadcasting species and one intragonadal
brooder. C. pentagona from the type locality Airlie Beach
was found to be a free-spawner with a short-lived
(Fig.
eages,

planktonic larva. This species was found from Airlie Beach

to Mission Beach in north Queensland (Fig. 1 ). Mission

Beach

is

near the northern limit of the distribution of C.

Queensland (A.J. Dartnall, James Cook Unicomm.). This species was located under rocks
the intertidal zone which dry out at low tide. Reports

pentagona

in

versity, pers.

high in
of C. pentugona south of Airlie Beach will have to be

in light of the discovery of C. hystera in Statue

Bay, Central Queensland (Fig. 1 ). C. hystera is an intragonadal brooder. Molecular data indicate that this new species

Yeppoon

(Hart et

al.,

1997).

It

occurs high in

mangrove habitats. The

two species may overlap south of Airlie

the intertidal under small rocks in

distribution of the

Beach.

thorough search of the

indicated that the

sites

used

in this

two species do not co-occur

in

study
north

Queensland.

using their braehia. As they explored the substratum, they

flexed dorsally to bring the adhesive disc to the surface.

Once

they were committed to metamorphose, permanent

benthic attachment was achieved by the adhesive disc assisted

by the braehia. The larvae attached

pentagona

is

a dioecious free-spawner with a plank-

tonic lecithotrophic brachiolaria larva (Figs. 2A, B, D;

C). Ovaries of

specimens collected

range of

to a

substrata including the walls of the culture dishes, although

they appeared to favor coralline algae (Fig. 2F). Most larvae

metamorphosed regardless of whether a specific settlement

substratum was introduced into the culture dishes.

them

settled

on the walls of

their containers.

Many

of

Newly meta-

11.8, H
morphosed juveniles (620 /u,m diameter; SE
15) had two pairs of tube feet in each radius (Fig. 3D, E).
They were a dark amber color, indicating the presence of
extensive maternal nutritive reserves. The mouth did not

open for

weeks

after settlement (Fig. 3D).

Development

the settled juvenile stage took 9 days at 23

to

C, while at

ambient temperatures (30 C) in Queensland, development

was completed in 6 days (Dartnall, pers. comm.).

Intragonadal developer
C. hystera had ovotestes that were a mosaic of oogenic
and spermatogenic areas (Fig. 2C). Gravid specimens were
present in all the samples obtained from September to

Planktonic developer
C.

was most evident over

of the juveniles. Advanced larvae adhered to surfaces

checked

also occurs in

(Fig. 3F). This feature

the braehia of the larvae and on the oral surface and tube

in

3A-

October and Novem-

ber 2000 and October 2002 were gravid (Fig. 2A).

They

contained large eggs dominated by large lipid droplets.

Spawned eggs (413 /im diameter; SE = 6.4 /urn, n = 20)

were an amber-gold color. They were positively buoyant

and floated to the surface as they emerged from the gonopore. Mature testes were typical of asteroids, having a layer

November, indicating that the reproductive period lasts at

least 3 months. In December 1999, the gonads contained
juveniles and few gametes. The amount of spermatogenic
tissue in the

gonads varied among individuals

(/;

20). In

some specimens, sperm was only

detectable histologically;
in others, white testieular regions of the gonad were evident
by direct examination. Like those of C. pentagona, the eggs

were large (440 jum diameter; SE = 6.0 /urn, /i

contained abundant lipid droplets. They floated

8) and

to the sur-

2X8

M.

Figure

2.

spermatozoa:

Histology and light microscopy. (A, B) Cryptasterina pentagona ovaries and testes. o, oocyte; s.
spermatocyte columns. (C) C. /jv.v/t-. The ovotestis contains lipid-rich eggs (o). sperm (s). and

sc.

(D, E) Brachiolaria larvae of O. pi'iiuigona and C. hyxlcnt respectively,

developing embryos (arrows);

g, gastrula.

hu, hrachia; h. hydropore. (F)

Metamorphosing juvenile

showing juveniles (arrows)

200 )j.m; G = 500 ju.ni.

liy\lciv

face

BYRNE ET AL

when removed from

the

in

C. iJeniu^onu on coralline algae (ca). (G) Dissected C.

= 100 ftm; F, H =
hysteru. Scales: A-E
gonad. (H) Newly released

gonad and were gold-orange,

with a dark vegetal pole.

Developing embryos and larvae were interspersed with

in the gonud (Fit;. 2C. G). Embryos removed from

iKimetes

the

at the early blustulu stage developed indepenof the parent through the wrinkled blustulu and

gonad

dently

gastrula stages into a planktonic highly buoyant brachiolaria. The developing brachia appeared as three bulges (Fig.

DEVELOPMENT

IN

2S9

CRYPTASTERINA

Figure 3
Scanning electron microscopy, Cryptasterina pentagonu. (A. B) Brachiolaria larvae have a
uniform cover of cilia. The central brachium (arrow) develops as a protrusion of the preoral lohe. (C) Advanced
larva with a well-developed adhesive disc (ad) at the hase of the brachia (ba). (D. E) Recently

each radius and with a mouth opening. (F) Detail of

matrix on larval surface. Scales: A. B = 100 fim; C, E = 50 /im; F = 4 jitm.

juvenile with two pairs of tube feel

in

4A). and the adhesive disc developed between the arms (not
illustrated).

As

in

C. pennigonu.

advanced larvae had

well-developed preoral lobe from which the central brachium emerged as a bulge-like protrusion (Fig. 4B. C). They

swam

metamorphosed

cilia (c)

and meshlike

anterior end up with their ciliary cover (Fig. 4F).

larvae 10 days) had a well-developed brachiolar

Advanced

complex which was used

for

benthic attachment.

They

exhibited typical settlement behavior while exploring the

290

M.

Figure
(ha

I.

4.

BYRNE ET AL

Scanning electron microscopy. Cryptiuiciinii Imtera. (A. B) Early larvae with developing hiachia
larva. The central brachium (arrow develops as a posterior protrusion of the preoral lobe.

Advanced

(C)

(D) Metamorphosing larva with resorbing larval body (arrow) and developing tube feet (t). (El Recently
metamorphosed juvenile with two pairs of podia in each radius and with a mouth opening- (F) Detail of cilia on
larval surface. Scales: A. B, E = 100 /Am: C. D = 51) jum; F = 12 /im.

substratum and adhered to the surface of the culture dishes

tract,

and

either with the tips of the brachia or by flexing the

niles

were white due

attach the adhesive disc.

in the

posterior region as

4D). Development
days.

Newly

to

body
The juvenile rudiment developed
the larval body was resorbed (Fig.

vitro to the juvenile stage took

in

settled juveniles

well-developed skeleton. Newly released juveto the color of the skeleton, and they

appeared to lack residual maternal nutrients.

16

Discussion

had a dark amber pigment due

mouth opening to develop, and by this time

maternal reserves were no longer evident (Fig. 4E). Three-

range of taxa
of
developmental
by investigation
evolution and molecular phylogeny (Reid. 1990; Degnan

week-old juveniles had

and Lavin.

to the presence of maternal

weeks

nutritive reserves.

It

took 3

for the

In aquaria, juveniles

a well-developed skeleton.
(800 ju,m diameter. SE = 6.3. n

10) with two pairs of tube feet in each radius emerged from
gonopore on the aboral surface of the adults (Fig. 2H).

the

These juveniles had

mouth opening,

a functional digestive

Recent discoveries of cryptic species

have been

in a

facilitated

1995: OFoighil and Smith. 1995; Williams,

2000; Flowers and Foltz, 2001; McFadden et ai, 2001).

Observations of juvenile birth resulted

new viviparous PatirielUi

1969, 1971; Keough and

in the description

in the P. exigiui

group

of

(Dartnall.

Dartnall, 1978), and our investi-

DEVELOPMENT
of

gallon

was

in
Cryptasterina
molecular phytogeny Hart

biodiversity

cryptic

prompted by the

results of

IN

ct

<//..

1997). The Asterinidae are a taxonomically difficult group

and detection of the cryptic species investigated here would
have been difficult with traditional taxonomy because adult

CRYPTASTERINA

291

), the gonads of C. hystera were ovotestes.

species (Table
This indicates the potential for self-fertilization, as appears
to be the case for P. vivipuru (Byrne. 1996). The amount of
1

gonads of C. h\stcra is more than sufficient to

eggs produced, and so it is likely that some

in the

sperm

fertilize all the

forms appear very similar even to taxonomic experts (Dartnall. 1971; Rowe and Gates, 1995). Once specific status has

individuals release sperm. For out-crossing to occur, the

sperm would have to gain access to eggs by swimming

been determined, however, close examination of

through the gonopore.

may

species

cryptic-

reveal the presence of diagnostic morphologi-

cal traits (Dartnall. pers.

comm.). Indeed,

and

life-history

mine
crinii

genetic study

is

required to deter-

progeny in the gonads of the viviparous Cryptastand Putiriellii are full siblings or half siblings. The

if

molecular data have guided taxonomic effort in the discovery and description of several cryptic PtitiHcllii species in

diversity of the fertilization biology in these asterinids with

New

fertilization

Zealand and Australia (O'Loughlin ct <//., 2002; Dartnall. pers. comm.). Molecular data revealed that C. pcntuxonii. the broadcasting species, occurs from the type locality Airlie

Beach north

2003). C. hvstem
10

km

apart.

An

the free-spawners, partial self-

egg

layers,

and potential for

viviparous forms provides a useful model in

which to investigate the relationships between the evolution
selling

in

Hart ct ai.

of mating systems and the genetic structure of sea star

populations (Byrne. 1995. 1996).
As characteristic of echinoderms, the evolution of leci-

Townsville (Fig.

5;

extremely limited distribution

is

also char-

acteristic of the viviparous Patirielhi

thotrophy

large

(Byrne el ai. 1999b).

survey of mangrove habitats along the central Queensland
coast will be required to determine the distribution of C.
hystera.

in

the benthic

in

only from two locations, about

to

known

is

complete out-crossing

The nominal taxon

C.

pentagona occurs through

ct ai. 1998). and it is

Cryptasterina species involved acquisition of a

in

The increase in egg size from what

egg (Table
would have been an ancestral form with a small egg and
planktotrophic development is considered to have been necI

).

Asia (Marsh. 1977; VandenSpiegel

essary to sustain development without feeding (Mortensen,

found (Dartnall
ct ii/., 2003). Other widely distributed asterinids such as
Patiriellfi e.\i^iia, which occurs from South Africa to Aus-

of extensive nutritive reserves

likely that other divergent lineages will be

tralia,

pers.

also appear
comm.).

to

be a suite of cryptic species (Dartnall,

The life-history traits and phylogenetic relationships of

species in the Ciyptasteiina pentagona group and the Patiriellti e.\ii>nu

group are shown

gonochoric. free-spawning

in

mode

pentagona and Cryptasterina

n.

Table

and Figure

5.

of reproduction seen
sp.

is

ancestral

The

in C.

for the

Echinodermata, while acquisition of hermaphroditism, a

derived character, is exhibited by most echinoderms that

brood

their young (Strathmann el ai, 1984; Byrne, 1991,

1999; Hendler. 1991). Like those of the other viviparous

Table
l.ili--ln\tor\ trtiitx

Species*

of

tin'

Cryptasterina pentugona

ami

Patiriella

1921; Strathmann, 1978; Emlet

juveniles, however,

shows

ct

<//.,

in

1987).

The presence

newly metamorphosed

that a considerable portion of

maternal reserves in the eggs of the two Cryptasterina

species investigated here is allocated to the perimetamorphic postlarval period. A substantial proportion of the maternal provisions in their large

eggs

is

stored through larval

support development of the postlarval

stages, a feature seen in other lecithotrophic echinoderms
(Emlet and Hoegh-Guldberg. 1997; Hoegh-Guldberg and

development

to

Emlet, 1997: Byrne

ct ai.

1999a. 2003: Villinski el ai.

2002). In contrast, the small eggs

P. vivi/nira

(150

exigua species groups

jiim

and

P.

135-150 jam diameter) of

pan-ivipara are similar in size to those

diameter) of their planktotrophic congener P.

M.

292

BYRNE ET AL

(600

diameter) reared in vitro were

/u,m

Juveniles of C. hystera (2-3 weeks old, 800 /xm diameter)

left the parent with a functional gut. Similarly, newly meta-

C. pacifica

morphosed juveniles of C. pacifica (650 /j.m diameter)

emerge from the gonopore (900 ju,m diameter) with a functional gut (Komatsu et al., 1990). The juveniles of P.

-\
Airlie

C. hystera

supported by maternal nutrients for several weeks until the

digestive tract became functional and juveniles could feed.

n. sp.

Cryptaslerina

and

eter)

Patiriella regularis

Beach

Rose Bay

vivipara and P. parriripara continue their growth

Townsville

become the
gonads
mm diameter) known
to

northern

in

the

largest recruiting juveniles (1.0-5.0

for the Asteroidea (Byrne, 1996).

to a year in the gonads preying on their

unconventional
source of maternal nutrients.
an
siblings,
There may be selection for extended brood care in the

They spend up
Queensland

Townsville

(lecithotrophic)
C.

Dalrymple

pentagons

Pt.

viviparous

species.

Intragonadal

through the vulnerable early

Dalrymple

Airlie

Pt.

central

Queensland

tained a

few large juveniles

(viviparous)
C. hystera

(Byrne, pers. obs.).

exigua

1-2

mm

diameter), indicating

brood cannibalism occasionally occurs

that

The eggs of species

P.

in

in this

species

the Cryptasterina group have

conspicuous lipid reserves, a feature characteristic of echi-

P. vivipara

noderms with planktonic lecithotrophy (Emlet et al.. 1987;

Emlet and Hoegh-Guldberg. 1997; Hoegh-Guldberg and

P. parvivipara

Emlet. 1997; Byrne et al.. I999a, 2003; Villinski et al..

2002). Their brachiolariae are also similar in appearance,

5.
Phylogenetic tree based on branch-and-branch searching of
sequences (COI and 5 tRNA genes) from the Cryptasterina
pentagona and Patiriella exigua groups (after Hart et a/.. 1997. 2003).
Species in bold are known to be viviparous. Bootstrapping produced strong

Figure

mtDNA

(92%) for monophyly of the six northern Queensland lineages,

two southern Queensland lineages, and for all these lineages as a clade
separate from P regularis. The Yeppoon specimen was from a museum
support

for

collection and the presence of intragonadal

embryos could not be con-

firmed.

with a prominent preoral lobe and a central brachium that

develops as a bulge-like protrusion (Komatsu et al.. 1990).

Possession of a buoyant egg and a functional larva in C.
hystera and C. pacifica would not be expected in viviparous

These features and the morphological similarity

of their larvae to those of congeners with planktonic development suggest that viviparity in these sea stars evolved
asteroids.

through retention and fertilization of a large egg by a C.

pciitagi>na-Y\ke ancestor. This suggestion

regularis and represent a secondary reduction in size (Byrne

and Cerra, 1996; Hart et ai. 1997). The eggs of P. vivipara
and P. parvivipara support intragonadal lecithotrophic de-

velopment

to a very small postlarva

(200-300 ^im diame-

indicating that the maternal reserves in these eggs may

be near the limit required to support development through
metamorphosis in the absence of feeding (Byrne, 1996).
ter),

The

larvae and juveniles of C.

pentagona have a meshlike

extracellular matrix covering regions of the body, similar to

that described for P. regularis

(Byrne and Barker, 1991).

Preservation of this matrix depends on fixation method,

suggesting it may be an artifact (Byrne, pers. obs.). Alterits presence in some regions of larvae and juvesuch as the brachiolar arms and oral surface indicates

natively,
niles

progeny

stages and their

stages of marine invertebrates is usually high (Gosselin and

Qian, 1997). Interestingly, the gonads of C. hystera con-

Yeppoon

postlarval

of

release at a large size undoubtedly conveys a survival advantage for the juveniles. Mortality of the early settlement

Beach

Statue Bay

brooding

that the extracellular matrix

Newly

may have

settled juveniles of C.

regional differences.

pentagona (612

yarn

diam-

molecular phylogenetic data (Fig.

The intragonadal

5;

is

Hart ct

brachiolariae of P.

supported by
al.,

2003).

vivipara and P.

parvivipara are vestigial, unlike those of C. hystera. Their

minute larvae have a reduced brachiolar complex comprising three small nonsticky protrusions that cannot function in
attachment, and some embryos do not develop brachia at all.
in P. vivipara and P. pan'ivipara
thought to have evolved through a P. e.\igua-\\ke ancestor
that laid benthic egg masses and had highly modified

Intragonadal development

is

benthic nondispersive larvae (Fig. 5; Byrne, 1995; Hart et

997). Evolution of viviparity through retention of eggs

al..

by an ancestor with benthic egg masses is suggested to have

been a likely pathway for the acquisition of this form of
brooding in asterinids (Strathmann et al.. 1984; Byrne,
1996).

Despite their intragonadal location, the larvae of C. hys-

DEVELOPMENT
/era exhibited exploratory settlement behavior and attached
to the substratum with their brachia and adhesive disc prior
to

metamorphosis,

in a

manner

oid larvae. This behavior

is

the intragonadal environment.

larva

typical of planktonic aster-

unlikely to serve any function in

reversal to a planktonic

readily envisaged for C. hystera and C. pacifica. a

is

suggestion also

made

for Pteruxter tesselatus

These species could potentially

1992).

(McEdward,
use two modes of

parvivipani are committed to intragonadal development.

egg and functional larva apvivipara and P. /Hirvivi/nini.

reversal to reacquire a large

Intragonadal development

Among
is

is

asteroids, intragonadal

known

rare in the

Echinodermata.

development and

live birth

for only three genera. Cryptasterina, Putiriella

and the aberrant XylopUi\ medusiformis from the

deep sea (Rowe et <//.. 1987). Morphological and molecular
evidence supports the conclusion that viviparity evolved
(Table

).

independently three times

Han

5:

(Fig.

et

/..

and Cryptasterina

in Patiriella

1997. 2003).

Parallel

evolution of

derived lecithotrophic life histories is common in echinoderms and is often associated with a suite of convergent
phenotypes (Strathmann. 1985; Emlet et ai, 1987; Wray,
1996; Hart

et nl.,

1997). For asterinids.

some convergent

adaptations associated with viviparity are evident in all

brooding species, while other features follow phylogenetic
lines (Table 1. Fig. 5). With respect to adult traits, conver-

gence

is

seen

in

possession of ovotestes and the potential for

both Cryptasterina and Patiriella. Egg

self-fertilization in

type and composition, however, is similar within these

genera, but not between them. With respect to developmen-

phenotype. Cryptasterina and Patiriella differ in the

presence of functional or nonfunctional larvae. Regardless
tal

of the pathways involved in evolution of viviparity. however, the probability of making the evolutionary switch to
intragonadal development appears high in the Asterinidae.
Selection for viviparity may be associated with colonization

of marginal habitats along the upper fringe of the

intertidal.

by sea stars. Why

unusual form of parental care evolved in Cryptasterina and
Patiriella and not in other asteroid taxa is not known.
a habitat

not generally

this

utilized

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Reference: Bio/. Bull. 205: 295-307. (December 2003)

2003 Marine Biological Laboratory

Persistent Ancestral Feeding Structures in Nonfeeding

Annelid Larvae
BRUNO FERNET
Frida\ Harbor Laboratories, 620 University Road, Fridav Harbor. Washington 98250

Evolutionary loss of the requirement for feedlarvae of marine invertebrates is often followed by

form (Emlet, 1991). Many species with nonfeeding larval

development evolved independently from ancestors that had

and digesting food.

form evolves

feeding larvae (Strathmann. 1978). This suggests a strong

evolutionary association between the loss of the require-

Abstract.
ing in

loss of structures involved in capturing

Studies of echinoderms

suggest that larval

rapidly in response to loss of the requirement for feeding,

but a lack of data from other taxa makes it difficult to assess

ment

the generality of this result. I show that many members of a

large clade of annelids, the Sabellidae, retain ancestral sys-

tems for

particle capture despite loss of the

to feed. In at least

need and

one species, Schizobranchia

the

an

and transport them

to the

is lost in

lost

lost larval feeding very

egg

development without paniculate food. These derived

may feed facultatively. However, once larvae have
the requirement for food, stabilizing selection on feed-

is weakened, and mutations that affect the

form or function of feeding structures may accumulate
(Strathmann, 1975). Selection on other larval functions like

ing performance

swimming

larvae of annelids and other

spiralians following increases in

1994).

Under

become

many

most obviously,

in

may occur by

morphology, physiology,

which may occur rapidly after

(Wray and Raff,

ancestral feeding structures,

loss of the requirement for paniculate food

including embryonic de-

1991; Hart, 1996; Wray, 1996), then leads to simplification

in nonfeeding larvae (Emlet. 1991; Byrne

of external form

genetic structure (Palumbi, 1995), species duration (Jablon1986). and perhaps

Raff, 1991; Emlet.

or behavior, but details of this process are mostly unknown

(Kempf and Todd. 1989; Wray, 1996). Further reduction of

velopment (Wray and Bely, 1994), dispersal and population

ski,

also

nonfunctional, and the resulting larvae are obli-

any of many different changes

others cannot feed and instead rely on materials stored in the

egg. These alternative nutritional modes are associated with
traits,

(Wray and

may

these conditions, feeding structures eventually

gately nonfeeding. Loss of the ability to feed

Larvae of some marine invertebrates require paniculate

food to complete development to the juvenile stage, but

other

or developing rapidly and efficiently

lead to changes in larval form

size.

Introduction

differences in

Kempf and Todd,

1987;

larvae

opposed bands are inexpensive to construct

and operate, or that opposed bands have some alternative
function. These observations also suggest a hypothesis on
the ability to feed

1975: Raff,

plete

recently, that

how

is

1996: Wray, 1996; McEdward and Janies,

1997). Increased egg energy content permits larvae to com-

mouth, but captured particles

have

that

development

1989: Hart,

are invariably rejected because larvae lack a functional gut.

The persistence of particle capture systems in larvae of

sabellids suggests that they

step in the evolution of nonfeeding

1972; Strathmann,

opposed-band system of prototrochal. food-groove, and

metatrochal ciliary bands can concentrate suspended particles

first

an increase in the energy content of the egg (Jagersten,

ability

in.signis.

for feeding and the loss of larval feeding structures

(Wray, 1996).
How does this association arise? One model suggests

larval form. Feed-

el al.,

2001

).

This scenario

is

a specific

ing larvae bear structures that function in particle capture,

general model of the reduction and

ingestion. and assimilation, while nonfeeding larvae tend to

characters (Fong et

lack such structures and are relatively simple in external

Though

al.,

example of a more

loss of nonfunctional

1995).

larval feeding has

been

lost in

many

lineages of

marine invertebrates (Strathmann. 1978). subsequent patchange in larval form have been studied primarily

terns of

Received 3 April 2003; accepted 22 September 2003.

in

E-mail: pernetb@hotmail.com

295

members of only one phylum,

the

Echinodermata.

296

B.

FERNET

Examples from other phyla would be useful in assessing the

provide such an example
generality of thev results. Here
I

from a phylum only distantly related

Vjoellid annelids are
Annelida

worms

often

known

to the

echinoderms, the

sessile,

as "feather-duster

tube-dwelling
of

worms" because

development is likely plesiomorphic in

(Rouse and Fitzhugh, 1994). The inference that
the common ancestor of serpulids and sabellids had a feedlarvae, nonfeeding

the family

ing larval stage relies primarily on the observation that

feeding larvae of sabellariids and serpulids capture particles

using similar systems of three parallel ciliary bands, the

crown of tentacles they extend from their organic, unmineralized tubes for suspension feeding. The family includes about 490 species that fall into two clades, the

prototroch, food groove, and metatroch (Fig.

subfamilies Fabriciinae and Sabellinae (Rouse and Pleijel.

2001). All fabriciins whose reproduction has been studied

unpubl. data). The cilia of the prototroch, which form a

transverse band anterior to the mouth, beat from anterior to

(about 15 of 75 species) brood embryos that undergo direct

development. Sabellins are more variable in reproductive
biology, with some species brooding embryos and larvae
through the juvenile stage; others brooding embryos and

posterior.

the

larvae for most of their development, releasing larvae for a

and still others

brief planktonic phase before settlement;

releasing gametes directly into the sea

where they are

fer-

et

1972; Strathmann,

nl..

These

current as well as

bands are transported towards the

of the food groove. This is usually referred

between these two

mouth by

cilia

ciliary

"opposed band" feeding. (Note that Rouse [1999.

200()a] argues that sabellariid larvae do not feed with op-

(about 30 of 415 species), these are broadly distributed in

phylogenies of the family (Rouse and Fitzhugh, 1994). No

posed bands of

(Rouse and Fitzhugh,

swimming

body. Cilia of the metatroch. located posterior to the

mouth, beat from posterior to anterior. Particles caught

to as

to feed

cilia create a

larval

and develop into planktonic larvae. Though reproduction and development has been studied in few sabellins

known

ids in

which

alreolala and

cilia.

However, larvae of the only

larval feeding has

S.

A look at the relationships of annelid worms

in

nonfeeding development
larval feeding (Fig.

).

suggests that

sabellids represents a loss of

Sister clade to the Sabellidae

is

the

Serpulidae, which includes both species with feeding larvae

and species with nonfeeding larval development. Sister to
the clade (Serpulidae, Sabellidae]

is

the Sabellariidae. All

sabellariids whose development has been described have

feeding larvae. These character states are shown in Figure 1,
with inferences on larval nutritional mode in ancestral

forms. As no

extant sabellid

is

known

to

have feeding

Sabellidae
Sabellariidae Serpulidae Fabriciinae

BD

ffl

Sabellinae

sabellari-

been studied, Sabelluria

cementarium, do feed

in this

mann, 1987; Strathmann and Fernet, unpubl.

1994).

Strathmann

being involved in feeding. Suspended particles passing near

them are overtaken and moved towards the surface of the

tilized

sabellid larvae are

2:

1987; Strathmann and Fernet,

way

[Strath-

data].)

The

presence of similar feeding structures in sabellariids and

serpulids suggests that ancestors of the clades [Sabellariidae
[Serpuiidae, Sabellidae]] and [Serpulidae, Sabellidae] had
larvae that fed with opposed bands of cilia. Nonfeeding
development in the sabellids thus represents a loss of larval
feeding.

Rouse (2000a) also concluded

that sabellids

were

derived from ancestors that had feeding larvae.

Here I show that despite loss of the requirement and

members of

at least eight genera of

bands
of
cilia.
In larvae of at least
opposed
one species, Schizobranchia insignia, these structures can
concentrate particles from suspension and transport them to

ability to feed as larvae,

sabellids retain

the mouth,

where they are invariably rejected because the

larvae lack functional digestive systems.

The

persistence of

ancestral systems for particle capture in nonfeeding sabellid

larvae is unexpected in light of data from echinoderms,

which suggest

that

once the requirement for

larval feeding

is

feeding structures are rapidly reduced or lost. These

observations suggest that sabellids lost larval feeding very
lost,

recently, that opposed bands are inexpensive to construct

and operate, or that opposed bands have alternative functions in these larvae. These observations also suggest a

hypothesis on
in

feeding larvae
nonfeeding larvae

Relationships of sabellariid, serpulid, and sabellid annelids,

as indicated by cladistic analyses of morphological and reproductive char-

Figure

acters
is

how development

mediates the loss of feed-

larvae of annelids and related phyla after

ing ability
increases in egg size.

Materials and Methods

1.

Collection, xf>nwnin}>,

by Fitzhugh (1989) and Rouse and Fitzhugh (1994). This topology

elongation factor-

alpha (D.

DNA

sequence data from the nuclear gene

McHugh, Colgate University, pers. comm.).

also supported by analyses of

Inferences on ancestral character slates are discussed

in

the text.

and

lurval culture

studied larvae of the sabellin sabellid Schizobranchia

insignis Bush, 1905, intensively,

and made additional ob-

servations on larvae of three other sabellins,

Denwnax me-

OPPOSED BANDS

IN

297

SABELLID LARVAH

ANTERIOR

collected adult Mvxicoln aesthetica from floating docks

Anacortes. Washington, in July 2002. Adults spawned

when removed from their tubes and subjected to gradual
warming (to room temperature) and rapid cooling of the
in

prototroch

seawater they were incubated

raised as described above.
I

in.

Embryos and

larvae were

obtained two egg masses of Demonax medius from the

zone on the west side of San Juan Island in

intertidal

food
groove

February and April 2002, and incubated them

FSW

in

at

8-10 C in the laboratory. examined larvae that had been

removed from masses, as well as planktonic larvae that had
I

metatroch

emerged from masses

mouth

naturally.

Planktonic larvae were

raised as described above.

neurotroch
Lan'al morphology

Developmental stages were examined with a

POSTERIOR
Figure

showing

a larva of a serpulid annelid,

Diagrammatic venlral view of

2.

opposed-band feeding system and the path

(arrowhead) captured from suspension. Cilia ot a preoral hand,

structures used in the

of a particle

from anterior to posterior. As they beat, panicles that

of cilia, the food
pass within their reach may be moved into a perioral band
anterior and may help trap
from
to
cilia
beat
Metatrochal
posterior
groove.
the prototroch, beat

the

in

particles

food groove. Once

in

the

food groove, particles are

transported ventrally to the mouth. Rejected particles are

by

cilia

moved

posteriorly

of the neurotroch.

adults of
species except D. medius are broadcast spawners;
D. medius brood embryos and larvae in a gelatinous mass

around the opening of the adult tube, and larvae emerge

from masses for a brief (~1 day) planktonic period
(McEuen et ai, 1983). Adult S. insignis and P. occelata

were collected from floating docks on San Juan Island,

Washington, from January to April in 2002 and 2003. The
tube of each animal was cleaned of fouling organisms and
to the length

of the

worm

it

contained.

To induce

in a
spawning. 10-20 conspecific individuals were placed
About
flowed.
seawater
which
fresh
container
through
large
half of the worms so treated spawned within 48 h. usually at

collected fertilized eggs from the bottom of the

container the following morning, rinsed them several times
night.

micro-

and seawater before examination. Body dimensions were

estimated with a calibrated ocular micrometer. For scanning
electron microscopy, relaxed larvae were killed by gradual
addition of dilute formalin. They were then rinsed in Millipore-filtered seawater

fixed for 2 h in

(MPFSW, mesh

2% OsO 4

in

size 0.45

jitin)

and

1.25% sodium bicarbonate

buffer (pH 7.2). Fixed larvae were rinsed in distilled water.

then dehydrated in ascending concentrations of ethanol.

dins (Bush. 1905). Myxicola aesthetica (Claparede. 1870),

and Pseudopotamilhi occelata (Moore, 1905). All of these

trimmed

light

scope equipped with differential interference contrast (DIG)

1:1 solution of 7.5% MgCK
optics. Larvae were relaxed in a

They were critical-point-dried, mounted on stubs with carbon adhesive disks, and sputter-coated with gold-palladium
before being examined and photographed with a JEOL

JSM-35 scanning electron microscope. Larvae of Schi-ohnmchia insignis were also sectioned for study of internal
anatomy. Relaxed larvae were fixed in 2.5% glutaraldehyde
in 0.2

Millonig's phosphate buffer for 1-3 h. rinsed in

1% OsO 4 in buffer for h. After

buffer, then post-fixed in

dehydration

in ethanol, larvae

were embedded

812 (Electron Microscopy Sciences,

oxide as an

infiltration solvent.

in

EMBed-

Inc.) with

propylene

-1 /am

thick were

Sections

cut with glass knives and stained with Richardson's stain for
light microscopy (Richardson et ai, 1960).

Functional morphology of ciliary bands

in coarsely filtered

cultured them in

seawater (FSW, mesh size

-liter

beakers of

FSW

~5

/nm). and

at densities

of

~5

Beat patterns of cilia were recorded with a high-speed

video camera (Motionscope 1000-S. RedLake Imaging Inc.)

mounted on

compound microscope with DIC

optics. Lar-

in flowing
per milliliter. Beakers were partially submerged
seawater at temperatures of 8-10 C (near field tempera-

vae were restrained under a coverslip supported with plasticene modeling clay. Images were collected at 250 frames

Wa-

ter in larval cultures

per second and replayed at 10 frames per second. Sequences

were routed from the camera through a Sony DVMC-DA2

was added, but

saved as
analog-digital converter to a Macintosh iBook and

tures)

and

stirred with

paddles (M. Strathmann, 1987).

was changed every 3-6 days. No food

cultures certainly contained bacteria and

unicellular protists. Larvae of

S.

insignis

were followed

through metamorphosis. These larvae settled on small

to
pieces of adult tube that were scored with a razor blade
provide crevices. Juveniles were fed the unicellular algae
Isochrysis sp. and

Rhodomonas

sp. at

high concentrations.

iMovie

files (Apple Computer, Inc.).

Capture and transport of particles by larvae of Sclii:obranchia insignis were visualized by videotaping larvae in a
a slide
suspension of particles. Larvae were mounted on

under a coverslip supported with plasticene modeling clay.

298

B.

FERNET

The coverslip was pressed down just enough to prevent

larvae from s miming rapidly. High concentrations of

function in an opposed-band feeding system. Using the

methods of Rivest (1981) and Moran (1999), I tested the

polystyrene ti; mvlbenzene beads (3-/Mm diameter, bluedyed, Polvi ,-:v,ces. Inc.) were added and larvae were observed with a compound microscope. Beads had previously

hypothesis that larvae of Schizobranchia insignis or Demonax medius take up proteins with the cells that bear the

been incubated

(BSA)

in a

in distilled

pended

2.5%

serum albumin

h, then rinsed and resusSuch beads are readily captured and

water for 1-2

MPFSW.

in

solution of bovine

ingested by free-swimming or restrained feeding larvae of

and serpulids (Strathmann and Fernet, unpubl.

Images were collected with a video camera (Sony

sabellariids
data).

CCD SSC-S20) and recorded on VHS

tape, with a date-time

ciliary bands. Ten larvae were placed in 1 ml of

fluoresceinisothiocyanate-conjugated bovine serum albumin
(1 mg/ml). Negative
(FITC-BSA) dissolved in

opposed

controls were incubated in

known

to take

MPFSW
MPFSW only.

sit-

kana [Moran, 1999] or Crepidula aditnca [Rivest, 1981])

were included in all vials as positive controls. Larvae were
incubated

6 h

in test solutions for

generator providing a time signal. Tapes were played frame

incubation, they were rinsed in

by frame

epi fluorescence microscopy.

for analysis.

Larvae of species

up large proteins (veligers of Littorina

At

at

10

MPFSW

in the dark.

After

and observed by

least five sabellid larvae, as

well as several positive controls, were examined from each

treatment. For S. insignis, assays were conducted every 5
days, from the 5th day after fertilization until larvae were 30
days old. For D. medius, assays were carried out with larvae

Possible functions of opposed ciliary bands in sabellid

larvae
I

tested

bands

two hypotheses on functions of opposed

in larvae

ciliary

of sabellids.

opment and later examining their guts for ingested particles.

For each assay, 20 larvae were placed in 20 ml MPFSW in

Two

types of particles were added to each vial:

their individual capsules but

had not

egg masses on their own (these larvae were about

5-15 days old, and were removed from egg masses by
yet

Opposed ciliary bands are used for capturing paniculate

food during the planktonic larval stage. I verified that larvae
of Schizobranchia insignis and Demonax medius are nonfeeding by offering them particulate "food" through devel-

had hatched from

that

left

agitation).

Results

Lan'al development and form

blue-dyed polystyrene divinylbenzene beads 3 and 6 /u,m in

diameter, previously incubated in 2.5% BSA as described

Schizobranchia insignis. Except for notes on egg size and

developmental mode (Lee, 1970. 1975; McEuen et ai.
1983), development of this species has not previously been

above, and single-celled algae (Dunaliella sp.). Final concentrations of beads were 5-10 per microliter of each di-

Table

a vial.

ameter:

did not measure algal concentrations.

several feeding larvae of the echinoid

included

echinoderm Strongy-

locentrotus droebacliiensis or the serpulid annelid Serpula

in each vial as positive controls. Vials were

coluinbiana

slowly rotated on a plankton wheel in the dark

1-2 h. Larvae of sabellariids and serpulids feed

at

10

for

at high rates
under these conditions (Fernet, unpubl. data). Larvae were
preserved at the end of feeding assays by addition of buff-

ered formalin. They were later cleared and mounted in

glycerin, and examined with a compound microscope to

described.

A summary

of

its

development can be found

In the

spawning events

observed, males always spawned

before females. Fertilized eggs were spherical, opaque, negatively buoyant, gray in reflected light,

envelope brought the total diameter to 180-190

jam (Fig. 3A). First and second cleavages were both markedly unequal (without the formation of polar lobes), leading
fertilization

to a four-cell stage with

one

cell that

the others (Fig. 3B).

pigment; both types of particles were easily visible when

present in larval guts. Ten sabellid larvae, as well as several

shaped trochophore larvae (Fig. 3C).

known

to feed,

were examined

in

each assay. For

S.

and surrounded by

one
an elevated envelope (Fig. 3 A). Mean diameters
each
three
standard deviation) of 20 eggs from
of
separate
females were 154 (3.4), 156 (2.8), and 157 (2.8) ^m; the

determine whether they had ingested particles. Through

processing, beads retained blue dye, and algae retained

larvae

in

1.

Two

days after

was much

larger than

embryos had become pearThe widest part of the

band
of
cilia, the prototroch.
preoral

fertilization,

body bore a transverse,

The prototroch was composed of three

parallel

tiers

of

insignis. assays were conducted every 5 days, from the 5th

day after fertilization until larvae were 30 days old. For D.

cilia

feeding assays were carried out with swimming

larvae that had left egg masses on their own.

Cilia of all three tiers protruded through the fertilization

nii'diiis.

bands are

Opposed ciliary
of uptake of dissolved
Moran
found
that
(1999)
proteins.
encapsulated larvae of
sites

several marine gastropods use velar cells to take

up

dis-

solved proteins from the capsular fluid. In related species

with planktonic feeding larvae, these cells bear cilia that

an anterior

compound

cilia,

tier

of short

and a posterior

cilia, a middle tier of long

tier

of short

compound

cilia.

envelope. They could be identified as preoral because a

slight depression had appeared just behind them, midventrally at the site

By

of the developing mouth.

the 3rd day after fertilization,

two additional bands of

transverse cilia had appeared (Fig. 3D). Immediately posterior to the prototroch, at the level of the larval mouth, was

OPPOSED BANDS
Table

Fertilization

Prototrochal cilia protrude through egg envelope;

Pear-shaped trochophores; prototroch well

developed; ocelli, head and anal vesicles present
Metatroch and food-groove cilia present; neurotroch

? d

Notochaetae

in

chaetiger

5 d

Notochaetae

in

chaetiger 2

Notochaetae

in

in

tion of

in

peristomium
Neurotroch between peristomium and pygidium
in all individuals;

lost

lost

prototroch lost middorsally;

on prostomium
Prostomial snout present; radiole buds each divided
into 2 lobes; anus present

4 d

Radiole buds each divided into 3 lobes, ciliated;

long

cilia

snout begins?
Radiole buds each divided into 4 lobes

6-7 d

Juvenile feeding begins

all

larvae,

if

not offered a suitable settlement substrate, continue

swimming and remain competent

fertilization.

allowed

to settle until at least 30 days postFor the schedule of post-settlement development, larvae were

to settle after a 15-d planktonic period.

was similar

post-settlement development

The timing of events

in larvae that

were so large that they completely

lumen. Eight-day-old larvae had neither an
intestine nor an anus. In 20-day-old larvae, the mouth had
cells that

its

increased in depth and led to a very narrow ciliated stomo(Fig. 3H). In serial sections of five 20-day-old larvae,

daeum
I

was unable

to find a connection

between the stomodaeum

stomodaeum. This lumen disappeared posteriorly.

served no intestine or anus in these larvae.

Timing data are summarized from observations of cohorts of offspring

from six separate females. Though competent to settle 3 days after fertilization,

a shallow, ciliated midven-

and midgut. The cells lining the midgut had shrunk slightly,
and it now had a narrow central lumen at the level of the

of larval prototroch gone; ingestion of

5 d

endodermal
occluded

radiole buds present

3 d

changes
gut morphology during development.
Transverse sections of 8-day-old larvae showed that the
depression (Fig. 3G). This depression was not connected to the midgut. The midgut wall was composed of

mucus tube formed around body

Neurotroch between peristomium and pygidium

in some indmduals. cullar lobes
present on

2 d

various ages allowed descrip-

tral

Settlement;

at

in

mouth was represented only by

chaetiger 4

Post-settlement development
II

around 30 days.

Sections of larvae fixed

chaetiger 3; neurochaetae (uncim)

Neurochaetae (uncini)

feeding tentacles, or radioles. Mortality of larvae appeared

chaetigers 2 and 3

12-1? d

on the prostomium. In normally metamorphosing worms, these bulges go on to form the adult

dorsolateral bulges

to increase greatly at

present; competent to settle

4

the plankton. In particular, they developed a pair of

still in

weakly swimming
- d

clean

mostly involving Ihe appearance of chaetae in

developing segments (e.g.. Fig. 3F) are summarized in
Table
Between the 25th and 30th day of development,
many larvae began to show signs of metamorphosis while

Stage or event

in stirred cultures in

most remained planktonic until at least 30

fertilization. Changes in form in larvae from 3-30

Planktonic larval development

were maintained

days after
days of age

Time

(I

299

glass beakers,

temperatures of 8-10

SABELLID LARVAE
substrates and

Schizobranchia msignis raised at

Si'lieilnle nj ilcreltipnicnt in

IN

in

had 9-d and 30-d

ob-

Larvae of Schizobranchia insignis were competent to

from 3 to at least 30 days after fertilization. When

settle

offered pieces of adult tube in still water, 20%-50% of

larvae settled within 24 h. Larvae usually settled in crevices
in the tubes, or on the glass bowl beneath pieces of tube.

The events following settlement of 15-day-old larvae are

summarized in Table 1. Note that prototrochal cilia had
been resorbed or shed by 4 days

planktonic periods.

after settlement, before

juveniles began feeding.

a narrow

band of

short, simple cilia.

The width of

this

band was about 4-5 /xm in rive 10-day-old larvae.

band was a postoral band of simple
These ciliary bands are shown in greater detail in an

perioral

Demonax

mediits. All larvae of D. mediits that

exam-

larvae extracted from gelatinous brood masses, and

ined

Just behind this perioral

larvae that had escaped from masses for a brief planktonic

cilia.

period

of an older larva (Fig. 3E). Positionally, these

bands of cilia are identical to the perioral food groove and
illustration

postoral

metatroch

known from

with opposed bands of

groove and metatroch

cilia,

and

in the rest

annelid larvae that feed

will refer to

of

them

this paper.

as food

Both food

had prototroch, food groove, and metatroch

five

larvae that had

left

egg masses on

of two broods of D. mediits are

the metatroch. Three-day-old larvae

that

midventral

band of short

cilia,

stretched from the metatroch to the posterior end of the

their

own. The

metatrochal band was slightly broader than that of S. insigni.\. In most other
respects my observations of development

groove and metatroch were incomplete dorsally; the dorsal

gap in the food groove was substantially wider than that in

had also developed a

the neurotroch, which

cilia

similar to those seen in Schizobranchia insignis (Fig. 4A).

The width of the food groove ranged from about 6-8 jam in

McEuen

et

til.

(1983).

One

in

accord with those of

addition to their description

is

embryos and early larval stages in gelatinous egg

masses were individually encapsulated in spherical capsules

body.

only slightly larger in diameter than the developing worms

themselves. Capsules are present in addition to fertilization

Three-day-old larvae were competent to settle and metamorphose. However, if larvae were not offered settlement

envelopes like those described above for S. insignis. In the

broods I observed, larvae hatched from capsules about 5-7

300

B.

FERNET

Figure 3.
Embryonic and larval stages of Schizobranchia insignis. (A) Fertilized egg. (Bl Four-cell embryo.
(C) Two-day-old larva. (D) Three-day-old larva. (E) Detail of opposed ciliary bands of fourteen-day-old larva.
(F) Fourteen-day-old larva. (G) Transverse section of eight-day-old larva at the level of the mouth. |H)
Transverse section of twenty-day-old larva at the level of the mouth. Scale bars = 50 pim, except for (E) where
the scale bar = 15 /im. chl = first chaetiger, ch2 = second chaetiger. ch3 = third chaetiger. fe = fertilization
envelope, fg

= food

neurotroch, po
pr3

groove,

me =

metatroch.

mg =

= mouth, ne =
midgut lumen, mo
=
second
tier
of
prototroch, pr2
prototrochal cilia,

midgut wall, mgl

pore of one of the paired anal vesicles, pr

third tier of prototrochal cilia.

days after egg deposition, and they emerged from egg

masses about 7-15 days after deposition.

Myxicola aesthetica. Fertilized eggs of M. aestlietica

were spherical, opaque, negatively buoyant, rose-colored in

reflected light, and surrounded by an elevated envelope. The
mean diameter
one standard deviation) of 10 eggs from
(

OPPOSED BANDS

IN

301

SABELLID LARVAE

was 130 (4.9) /urn. First and second cleavof

were
distinctly unequal. At incubation temperatures
ages
12-13 C. larvae began swimming by about 18 h after

a single female

fertilization.

By

totrochal.

2 days after fertilization, larvae bore proand metatrochal cilia similar to

food-groove,
those seen in Schizobranchia insignis (Fig. 4B).

The width
was about 6 jum in five larvae. As in
Demonax medius, the metatrochal band of M. aesthetica
was broader than that of S. insignis.
of the food groove

Pseudopotamilla occelata. Fertilized eggs of P. occelata

were spherical, opaque, negatively buoyant, gray in reflected light, and surrounded by an elevated envelope. The
one standard deviation) of 15 eggs from
mean diameter
(

a single female

was 142

and second cleav-

(2.9) /u,m. First

after fertilization,
distinctly unequal. By 3 days
larvae bore prototroch, food-groove, and metatroch cilia
similar to those seen in Schizobranchia insignis (Fig. 4C).

ages were

The width of the food groove was about 6

jurn in five larvae.

Functional morphology of ciliary bands

Food-groove and metatrochal ciliary bands in larvae of

four species behaved like similar ciliary bands in larvae
that feed with opposed bands of cilia (e.g., Strathmann et
/.,
1972). The directions of effective strokes of cilia were
all

to posterior (prototroch), posterior to anterior

(metatroch). and laterally towards the mouth (food groove).

anterior

All three bands of cilia could arrest their beat, apparently

independently of the others.

When

metatrochal

cilia

were

along the larval body, pointed

beat
These
patterns were confirmed by
ciliary
posteriorly.
were also clearly
analyses of high-speed video footage, but
not beating, they lay

visible

flat

by inspection of living larvae

at

40QX

final

magni-

fication.
I

examined

the ability of larvae of Schizobranchia insig-

nis to concentrate particles

them

to the

from suspension and transport

mouth using opposed bands of

cilia.

video-

of 3-/u,m beads
taped four 9-day-old larvae in suspensions
for a total of 22 min. This footage included at least 30
of these video sequences indiparticle captures. Analysis
cated that beads were caught in the current generated by the
into the food groove. Resoluprototrochal cilia and moved
tion of images was not sufficient to determine if metatrochal

were actively beating during captures. Captured beads

were transported in the food groove around the body towards the larval mouth (Fig. 5). Once at the mouth, beads

cilia

were moved along the neurotroch

posterior end of the body.
Figure 4.
and

Opposed

ciliary

bunds of

Demonax

metliiis.

Myxicola aes-

(A) Ciliary bands in the right

ventral region of larva of D. mediiis. (B) Left lateral view of ciliary bands
bands
of
-day-old larva of M. aesthetica. (C) Left dorsal view of ciliary
of 3-day-old larva of P. occelata. Scale bars = 25 /u.m. fg, food groove; me.

iltciHLi,

Psi-iul,ip<>kiiiiilla

occelata.

metatroch; mo, mouth;

Possible functions of opposed ciliary bands in sabellid

larvae
I

pr, prototroch.

until they fell off the

tested the hypothesis that planktonic larvae of Schizo-

branchia insignis or Demonax medius feed on paniculate

food by offering larvae particles that are readily ingested by

302

B.

FERNET

A
0:00

D
0:51

Figure 5. Capture and transport of a particle by a 9-day-old larva of Schi-ohranchia insignis. in ventral
A. B) Capture of a particle. (C, D) Transport to the mouth in the food groove. (E) Transport posteriorly
on the neurotroch. Time (s) is marked on each frame. Scale bar = ?() /j.m; the asterisk is adjacent to the particle.
view.

(F)

Composite diagram of

particle positions in

(A-E):

me =

metatroch. pr

positions of the prototroch and metatroch are shown. Food-groove cilia

run from the gap in the metatroch to the posterior

cilia

position of the particle

shown

related feeding larvae. Paniculate food

in the guts

of larvae of

old, or in the guts

5.

was never observed

from 5 to 30 days

of larvae of D. mediits that had hatched

from egg masses. Feeding larvae of echinoid echinoderms or serpulid annelids included as positive controls
naturally

also tested the hypothesis

that

particles.

ciliated cells

of the

prototroch, food groove, or metatroch are involved in the

uptake of dissolved proteins by endocytosis. Larvae of

Schizobranchia insignis and Demonax inedins exposed to

FITC-BSA never showed any

differences in fluorescence

relative to larvae incubated only in

MPFSW.

ments, larval chaetae autofluoresced

traviolet light.

when

In

both

treat-

excited with ul-

Larvae known to be able to take up large

dissolved proteins always showed fluorescence indicating

uptake oi FITC-BSA in velar cells (Littorimi sitkana: Moran.

194"

or

cells

of the

"larval

kidney" (Crepidiilti
adunca: RivvM. 1981) when incubated in FITC-BSA. but
not

when incubated

in

MPFSW.

of the body.

prototroch. For clarity, only

between them, and

The mouth

the neurotrochal

slightly anterior to the

is

in (D).

insignis ranging

always ingested many of both types of

tip

lie

Discussion
Evolutionary loss of the requirement for larval feeding
has occurred repeatedly in many phyla, and is typically
followed by the loss of structures formerly involved in
feeding (Strathmann, 1978: Emlet, 1991; Wray. 1996). Detailed hypotheses on how this association arises, however,

have been tested mainly in the context of comparative data

on members of only one phylum, the Echinodermata (Hart.
1996: Wray, 1996). The observations reported here on larvae of annelids permit an

initial

These data are also useful

assessment of their gener-

generating hypotheses on
other topics, including the specific sequence of events that
lead to loss of feeding ability after increases in egg size and
ality.

energy content

in

animals that develop via spiral cleavage.

Ancestral feeding structures

I

in

in

nonfeeding annelid lan'ae

interpret the transverse ciliary

larvae described here as

bands of the sabellid

homologs of

the prototroch, food

OPPOSED BANDS

IN

SABELLID LARVAE

303

where the two new rows of

form the

groove, and metatroch of closely related feeding larvae (i.e..

those of serpulids and sahellariids). Almost all annelid lar-

hmnchia

vae bear a prototroch. and in all cases where detailed

observations have been made, these arc homologous by

arrested, points posteriorly. Finally, a text description and

drawings of larvae of Chime teres also suggest the presence

developmental criteria (Rouse. 1999). The lineages of the

cells that bear food-groove and metatrochal cilia are not
known in any detail, so other criteria must be used to infer

of opposed bands. Okuda (1946) states that the "prototroch"

of larvae of C. terex is composed of one band of long cilia
followed posteriorly by two adjacent bands of short cilia.

homology of

Two

these ciliary bands.

criteria that support

my interpretation are those of position and behavior. The

perioral and postoral cilia of larvae of Schizohranchhi i/ixignis,

Dcmoiutx nicdins. Myxicola aesthetica, and Pseudo-

/><>t(iinil/ii

same positions

occe/<itii are located in the

food-groove and metatrochal

as the

respectively, of feeding

cilia,

and serpulid larxae. and they show similar beat

patterns (perioral cilia beat laterally towards the mouth, and
postoral cilia beat from posterior to anterior). Together, the
sabellariid

preoral, perioral.

and postoral

bands of 5. inxignix are

ciliary

capable of capturing particles from suspension and transporting them

mouth,
opposed bands ot related
larvae.
Given
current
hypotheses on the relationfeeding
of
sabellariids. serpulids, and sabellids, phylogenetic
ships

criteria for

like the

to the

are also

homology

met

Lander. 1994).

1;

(Fig.

Under
food-groove and metatrochal
larvae
can be identified as ancestral
bands
of
sabellid
ciliary
this interpretation, the

structures retained after loss of larval feeding in the family.

The hypothesis

that the

sabellid larvae are

opposed

homologous

bands of these

ciliary

of related feeding

to those

new phylogenetic. developmental, anatomical, or physiological data, but given current

larvae might be falsified with

knowledge

it

hypothesis

is

appears likely that

that the

opposed

it

is

correct.

ciliary

An

alternative

bands of sabellids

insignis.

cilia

food groove and metatroch, and where the metatroch,

when

Unfortunately, he does not specify the spatial relationships

of these cilia to the larval mouth. His drawings show ciliary

bands similar to those observed here

in

four genera of

sabellin sabellids. Published illustrations of larvae of several other sabellins (e.g., Anipliigleiui mitluie

[Rouse and

Gambi. 1998] and Pcrkinsiana riwo [Rouse. 1996|) may

indicate the presence of only a single band of cilia behind
the prototroch, but

more observations of these larvae

are

found no descriptions of sabellin larvae that unequivocally indicate the absence of food groove and
needed.

metatroch.

Thus, members of

at least eight genera of sabellins likely

of cilia. According to recent phybands
possess opposed
and
(Rouse
Fitzhugh. 1994; Fitzhugh and Rouse.
logenies

1999). these genera are not clustered in any particular sub-

some (e.g.. Amphicorina, Myxicola)

from deep nodes in the tree, and others (e.g., Laonome,
Schiyhnincliiii) from shallow nodes. All known sabellin
clade of the Sabellinae:
arise

reproductive strategies (brooding to the juvenile stage.

brooding with release of a planktonic larva, and freespawning: Rouse and Fit/hugh. 1994) are represented in the species for

which there

conclude
spread

that

in

the

evidence of opposed ciliary bands. I

opposed ciliary bands are probably wideis

sabellins,

a clade

that

includes over 400

with those of sabellariids and serpulids.

This seems unlikely on grounds of parsimony. Further, the

opposed ciliary bands do not appear in

the direct-developing embryos of fabriciin sabellids (Rouse

opposed ciliary bands of larvae of Schizobranchia insignis

lack obvious functions, which makes it difficult to understand how they might have evolved independently.

and Fitzhugh. 1994).

evolved

in parallel

Opposed

ciliary

bands

may

be widely distributed among

My observations

the nonfeeding larvae of sabellin sabellids.

show

that

present

in

ciliary

bands are

least four

genera of

food-groove and metatrochal

members of

larvae of

at

I examined published descriptions of nonfeeding

larvae of sabellids for further evidence on the distribution of

species. In contrast,

cilia have previously been identified

of
several species of sabellins (e.g.,
by
Rouse and Fitzhugh. 1994), their presence has more usually
gone unnoticed. That metatrochal cilia may act with pro-

Though metatrochal
position in larvae

totrochal and food-groove cilia as opposed-band systems in

these larvae has not previously been described. It is possible

bands are more widely distributed

in

sabellins.

that

opposed bands of

nonfeeding annelid larvae. Indeed, the nonfeeding larvae of

some serpulids also have opposed ciliary bands, at least by

This survey revealed

nonfeeding larvae of four more

cilia in the family.

probable opposed bands

in

genera of sabellins. Scanning electron micrographs provide

evidence for the presence of food groove and metatroch in

opposed

ciliary

(Kupriyanova et <//.. 2001: Nishi

and Yamasu, 1992). Other annelid clades that should be
examined for the presence of opposed bands in nonfeeding
the criterion of position

members of Amphicorina (Rouse and Fitzhugh. 1994) and

Laonome (Hsieh, cited in Fig. 12.5D of Fernet el /., 2002).

larvae include those that contain both species

larval

text description of the "prototrochal" cilia

of Megalomma

BrancliiommcD vesicitlosum (Wilson. 1936) is also suggestive of opposed bands. Early larvae of M. vesiculosum
(as

have three

of prototrochal cilia, but slightly later in

two
more rows of cilia appear posterior to the
development
prototroch.

tiers

The most

posteriorly. This

is

posterior row.

when

arrested, points

precisely the pattern seen in Sfhi:.<>-

whose larvae

feed with opposed bands and species that have nonfeeding

Phylogenetic hypotheses delimiting

(e.g., those including the families
Opheliidae. and Polygordiidae, all of

development.

some of

these clades

Amphinomidae.
which include species
Fernet et

some of

/..

that feed with

2002) are

illustrated

opposed bands of cilia:

by Rouse (2000b). In

these clades. nonfeeding larvae probably evolved

from ancestors

that

possessed

opposed bands of

cilia.

304

B.

Knowledge of the

distribution of

opposed bands

in larvae

FERNET

of

these groups will pro ide additional insight into the evolution of larval tV-,n in annelids.

bands continue

(e.g., feeding on
within
the
maternally provided particles
capsule: Chaparro
et ul.. 2002). A variety of alternative functions of opposed

bands

ciliary

Wliv

lu:\'i

strnl feeding structures

'

nonfeeding sabellid

One

in

to

have clear functions

free-swimming sabellid larvae are possible.

For example, opposed bands might serve as the site of

uptake of dissolved organic material; might play a role in
assessing potential settlement sites; might be involved in

been retained h\

lanwe?

juvenile tube; or might be used in feeding

possible explanation for the retention of feeding

forming the

initial

need and requirement for

by recently

settled juveniles while the definitive adult feed-

structures

is

that sabellids lost the

larval feeding only very recently.

ing structures develop.

ment

and metatrochal

If nonfeeding developplesiomorphic for the sabellids, a minimal estimate

of the age of their loss of larval feeding is the time of the
is

clade's origin. Unfortunately, scarce fossil and molecular

data make it difficult to estimate the age of this clade.

Sabellid tubes are usually unminerali/.ed, so are not ex-

pected to be easily preserved. Though putative sabellid

fossils have been described from strata ranging from the
lower Miocene to the Devonian (e.g., Howell, 1962: Plicka,
1968; Termier et

Hay ward.

It is

also possible that food groove

bands play some role

in development. Several of these hypotheses (uptake of dissolved

ciliary

organic material, feeding in juveniles) are likely false, as

suggested by data reported in this paper. There is currently
little evidence available with which to assess other hypotheses.

in

Retention of functional ancestral particle capture systems

nonfeeding larvae of sabellids contrasts sharply with data

from echinoderms, where

are as speciose

larval feeding structures appear to

very rapidly after loss of the requirement for particulate food during larval development (Wray and Raff. 1991

the serpulids,

Hart. 1996;

til..

til..

1973:

1977: Schweigert et

1998). these identifications are quite tentative. Sabellids

and widely distributed as their sister taxon,

which are known from the Devonian (Glasby,

2000; Rouse and

Pleijel,

2001).

diversification

were similar

interpreted as

weak evidence

Assuming that patterns of

two clades, this can be

in the

that sabellids are also Paleo-

zoic in origin. Accurate determination of the age of the

Sabellidae clearly requires more fossil and molecular data.

A
cilia

second possibility

is

that

food groove and metatrochal

might be very inexpensive

to construct

and operate.

It

is little cost to maintaining these structures,

they may
be relatively invisible to selection for economy during development. Reduction of these ciliary bands might still

there

be

lost

Wray. 1996). To make a more general assessment of how rapidly larval form responds to changes in

mode, studies of more independent evolu-

larval nutritional

clearly required. Since evolutionary

requirement for larval feeding have been
the history of marine invertebrates (Strathmann,

events

tionary

are

of the

losses

common

in

1978). as well-supported phylogenies

become

available for

should be possible to identify many more

specific examples of the evolutionary loss of larval feeding,
to estimate the timing of these events, and to assess how

more taxa

it

they are related to the evolution of larval form.

occur, but only as a result of the slow accumulation of

mutations in genes specifying their development. Indeed,

degeneration of the opposed-band system may well be oc-

Events

curring in sabellids. as indicated by their unusually narrow

food grooves. In annelid and mollusc larvae that feed with

The observations reported here are generally consistent

with the model discussed above to account for the associa-

opposed bands, the food groove is typically 20-30 /urn wide

(Phillips and Fernet, 1996; Fernet, unpubl. data), but in the

tion

examined here, food grooves ranged in width

from 5-8 /nm. Width of the food groove may constrain the
sizes of particles transported to the mouth (R. Strathmann.

that

1987; Hansen. 1993). Thus the opposed bands of sabellid

though "functional" in the sense that they can cap-

serpulid species that have feeding larvae.

sabellid larvae

larvae,

ture and transport particles, likely cannot capture particles

of as wide a size range as those of related feeding larvae.

Finally, one might expect ancestral feeding structures to

be retained in nonfeeding larvae if they have functions other

than feeding. Prototrochal cilia clearly function in swimming in both feeding and nonfeeding annelid larvae. However, functions other than particle capture have rarely been

in the

between

evolutionary loss of larval feeding ability

loss of the requirement for

food

in the larval

argued above
sabellids are derived from an ancestor that had a feed-

stage and loss of larval feeding structures.

ing larval stage. For purposes of comparison. I assume that

this ancestor shared characteristics of extant sabellariid and

The evolution of

increased energy content in the egg is thought to be the first

step in the evolution of nonfeeding development. Compared
to the

eggs of sabellariids and serpulids with feeding larvae,

in diameter from about 45-90 /u,m (Gian-

which range

grande. 1997 [her reference to 150-;u.m eggs

spiniilosa

anova

et

is

til..

incorrect, according to

2001

),

Wilson

in

Sabellaria

1929)]; Kupriy-

the eggs of sabellids are relatively large

considered for food groove and metatrochal ciliary bands in

free-swimming larvae. Opposed ciliary bands have been

;u.m: Rouse and Fitzhugh, 1994) and energy-rich

(Fernet and Jaeckle, unpubl. data). The second step in this
sequence is loss of the ability to feed. All known sabellid

retained in the encapsulated, "non-feeding" larvae of numerous species of gastropods, but in these cases the opposed

larvae are obligately nonfeeding. However, they retain some

ancestral feeding structures (in particular, cilia of the food

(>110

OPPOSED BANDS

IN

groove and metatroch). These larvae can thus he interpreted

intermediates in the evolutionary sequence discussed
above. Such intermediates have previously been considered
as

observations suggest
this clade of annelids.

Wray. 1996). but

rare (Hart. 1996:

my

may be common in
These data may be useful in answering

that they

a question that has

that is, what specific
previously been poorly understood
events lead to the loss of larval feeding ability after an
evolutionary increase in egg provisioning? Wray (1996)

noted that loss of the ability to feed might be the result of

any of many changes, such as loss of a particular digestive
of some aspect of ciliary coordination, or
failure to complete morphogenesis of the larval mouth. He

enzyme,

loss

also noted that the rarity of nonfeeding larvae with only

SABELLID LARVAE

305

nonfeeding larvae, however, eggs are larger. Increased egg

volume is presumably due mainly to the addition of material
used to fuel development of the larvae or juveniles. During
development most of this additional material is

early

shunted to the macromeres for storage and gradual mobilization. In annelid

then,

that

embryos

macromeres

develop from large eggs,

are proportionally larger (relative to the

develop from small

eggs (Schneider ft ai, 1992). Construction of a functional
midgut may be difficult when a large volume of endodermal

micromeres) than those of embryos

must

cells

fit

remaining

in the relatively

that

small volume delimited by the

of the embryo. In

cells

this

case,

it

may be

midgut that has

impossible to assemble the endoderm
at
all.
Indeed, nonfeeding
a substantial lumen, or any lumen
into a

the specific changes in

larvae of annelids typically lack midgut lumens (Wilson.

1936; Anderson. 1973: Heimler. 1988). In addition to sim-

larvae unable to feed.

ple size constraints,

slightly derived

morphology makes

it

difficult to identify

morphology or behavior that render

Most putative intermediates (e.g., the

sea urchin Phvlliictinllius hnpcrialis: Olson

ft nl.,

1993)

division of endodermal

have undergone changes in multiple feeding-related traits

(c.t>.. number and shape of arms, structure of the larval gut)

genesis)

since divergence from a feeding ancestor, and it is thus

difficult to identify any one key change that resulted in a

Thus,

loss of feeding ability.

As

(e.g..

vide insight into how larval feeding ability

evolutionary increases in egg size. Larvae

is

may

pro-

lost after

of Schizo-

bninchia insignis. and probably many other sabellins, possess the ciliary bands needed to capture food particles from
suspension. These ciliary bands remain capable of capturing

and moving them to the mouth. The mouth, however, does not connect to the midgut, which in any case has
no (or, later in development, a very small) lumen because
the cells that

make up

its

reserves. Loss of feeding in

in the digestive system.

with
a consideration of annelid
This observation, along
development, suggests a specific hypothesis on the link
between increased maternal provisioning of the egg and the

related primarily to a

change

loss of larval feeding ability. Embryos of annelids (and

those of members of several other phyla, together known as

spiralians)

undergo

spiral cleavage, a process in

which

the

fates of some blastomeres are specified early in development. In spiralian embryos, the descendants of four cells

3A. 3B, 3C. and

tissue that

4D

becomes

cleavages from the

form the endoderm.

the larval midgut.

As

the

embryonic

a result of

unequal

third through the fifth cleavage cycles,

known as macromeres. are usually far larger

than the remaining embryonic cells, the micromeres. At
these cells,

el ul..

subsequent gut morpho-

relative to species with small

may

to

be the

initial

egg volume,

step in the loss

These

of larval

lead directly to a loss of larval digestive ability

because of spatial constraints or delays

esis.

eggs

1992).

in annelids, a quantitative increase in

typically thought

effects

may

in gut

persist through larval

morphogen-

development,

resulting in nonfeeding larval development; alternatively,

they may last only through part of larval development,

delaying the onset of larval feeding until cells of the midgut

wall shrink as energy stores are consumed.

This hypothesis

particles

wall are swollen with energy

larvae of S. insignis may thus be

cells (and

may be delayed

Schneider

feeding,

putative intermediates, larvae of sabellins

annelid species with large eggs,

in

its

is

reduction of

because of

attractive

vulnerability to test.

One approach

is

its

simplicity and

the experimental

endoderm volume by removal of one or more

cells (f.g.. Boring, 1989; Clement.

1962; Martindale. 1986). Carrying out this manipulation in
annelid embryos that normally develop into nonfeeding

presumptive endodermal

larvae with occluded midguts might yield larvae with open

midguts. It is not clear how morphogenesis of the rest of the

affected by
(e.g., mouth and stomodaeum) might be
macromere ablation, but it is possible that a simple reduction of endodermal cell volume might permit the develop-

gut

ment of a complete

larval gut. In species that retain ancestral

particle capture systems, like the sabellids described here,

this

manipulation might result

in

conversion of a nonfeeding

larva to a feeding larva.

Another approach is to use intraspecific variation in egg

and larval nutritional mode to examine the effects of
on midgut development. For example, some indisize
egg
size

gastrulation, the

viduals of the annelid Streblospio benedicti (family Spionidae) produce small eggs (56-70 /u,m diameter) that de-

ternalized,

velop into feeding larvae, while others produce large eggs

macromeres and their descendants are inwhere they form the larval midgut (Kume and

Dun. 1968: Anderson, 1973).

cies, the

larval

descendants of

midnut

3A-C and 4D form

that has a substantial

15-152 yum) that develop into nonfeeding larvae (Levin,

1984; Schulze el ai, 2000). If the hypothesis proposed
above is correct, nonfeeding development in 5. benedicti
(

Annelids that have feeding larval stages typically have

small eggs (Schroeder and Hermans. 1975). In these spelumen.

the wall of a

In annelids

with

should be a result of reduction

in size

of the midgut lumen

or delayed development of the midgut in larvae that develop

306

FERNET

B.

from large eggs. This prediction can be

tested with

com-

parative developmental
This idea might also apply to other spiralian taxa such as

molluscs

u.id entoprocts.

As

they should be subject to

their

development is similar.
similar effects of endodermal

volume on midgut morphogenesis. Both approaches to testdeletions of endodermal blastomeres

ing the hypothesis
and comparative studies within species with variable egg
size and developmental mode (e.g., the ascoglossan Alderia
modesta: Krug, 1998) may be fruitful.
This hypothesis links changes in development

(in this

egg size and allocation to endodermal lineages) to

changes in the form and function of later stages (loss of
case,

larval feeding ability via

delayed midgut morphogenesis).

Such connections have long been sought by developmental
biologists (e.g., Lillie, 1899; Freeman and Lundelius, 1992).
In addition,

vation

it

may be

useful in explaining a peculiar obser-

that correlated intraspecitic variation in

egg

size

and larval nutritional mode (a form of "poecilogony") appears to be limited in distribution to spiralians. in particular
annelids and molluscs (Chia et ai. 1996). I propose that
annelid and mollusc species with great intraspecific variation in

egg

nutritional

size

may show

mode because

correlated variation in larval

of spatial constraints or hetero-

chronic effects on midgut morphogenesis imposed by their

conserved pattern of development.

Development of Ilyanassa following removal of

Clement. A. C. 1962.

the

data.

macromere

successive cleavage stages.

at

Exp. Zool.

J.

166:

77-88.
Functional constraints on the evolution of larval

Emlet, R. B. 1991.

forms of marine invertebrates: experimental and comparative evidence.

Am. Zool. 31: 707-725.

Body form and

Emlet, R. B. 1994.

patterns of ciliation in nonfeeding

larvae of echinoderms: functional solutions to

swimming

in the

plank-

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A systematic revision of the Sabellidae-Caobangidae-Sabellongidae complex (Annelida: Polychaeta). Bull. Am. Mus.
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Fitzhugh, K. 1989.

W. Rouse. 1999. A remarkable new

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Acknowledgments
thank A.H. Whiteley for reminding

me

of Lee's studies

of Schizobranchia insignis eggs, and B. Bingham and C.

Mills for helping locate populations of Myxicola aesthetica.

R.R. Strathmann generously provided

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access to a culture-

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Reference: Bio/. Bull. 205: 30X-318. (December 2003)

2003 Marine Biological Laboratory

Cloning, Characterization, and Developmental

Expression of a Putative Farnesoic Acid 0-Methyl

Transferase in the Female Edible Crab Cancer pagurus

CAROLYN

RUDDELL GEOFFREY WAINWRIGHT AUDREY GEFFEN

MICHAEL R. H. WHITE SIMON G. WEBSTER 2 AND HUW H. REES *
1

J.

School of Biological Sciences, University of Liverpool, Liverpool L69 7ZB, United Kingdom: and
2
School of Biological Sciences, University of Wales, Bangor, LL57 2UW. United Kingdom

Abstract.

Farnesoic acid methyl transferase (FAMTase)

catalyzes methylation of farnesoic acid to yield the crustacean juvenoid, methyl farnesoate (MF). A full-length cDNA

female specimens of C. pagurus, which segregated into

"high MF" and "low MF" groups. The high MF tilers,
which occurred before or during early vitellogenesis, coin-

encoding a 275 amino acid putative FAMTase has been

isolated from the mandibular organ of the female edible

cided with, or were preceded by, elevated levels of putalive

FAMTase

mRNA

in the

crab (Cancer pagurus) by reverse transcriptase-polymerase

chain reaction in conjunction with cDNA library screening.
A high degree of sequence identity was found between this

mandibular organs.

Introduction

and other putative crustacean FAMTases. Conceptual translation and protein sequence analysis suggested that phos-

similar to insect juvenile

phorylation could occur at multiple sites in the FAMTase.

This finding is consistent with the recent observation that

ai. 1987a, b;Borst et

endogenous FAMTase

1998). Just as the juvenile

activity in

Methyl farnesoate (MF), a sesquiterpenoid structurally

hormone III. is produced and

secreted by the mandibular organs of crustaceans (Laufer et

mandibular organ extracts

1987; Wainwright etui, 1996a, b,

hormones maintain larval char-

al..

can be regulated by phosphorylation in vitro. We demonstrated that the recombinant FAMTase could be expressed

acteristics

as a LacZ-fusion protein in Esclierichiu colt and have un-

regulates larval

partial purification from inclusion bodies. In an

established assay system, the recombinant FAMTase lacked

2000). In adult insects, juvenile hormone has been implicated in the regulation of ovarian development (Davey,

dertaken

between successive molts

its

correspondence

to

be

addressed.

Ahbre\-iatitii,\.

ul..

MF,

either

1999),

by

by the effects of eyestalk ablation (Jo

injection of MF (Reddy and

direct

Ramamurthi. 1998, Rodriguez

E-mail:

tration of

et al.,

2002). or by adminis-

MF

through the diet (Laufer et al., 1998), oocyte

growth and ovarian development were stimulated. Similarly, a significant increase in mean oocyte diameter was

\MTase, S-adenosyl-L-methionine farnesoic acid O.".

1996a) have been found

occur during vitellogenesis. In experiments where a vaof crustaceans were exposed to artificially enhanced

levels of

reeshhfe'liv ....

methyl transferase:
inhibiting hormone.

et al.,

riety

September 2003.

should

al.,

MF

hemolymph (Wainwright

'

April 2002; accepted 5

MF

MF

the

et

barnacles (Smith et

MF

fluctuate during vitellogenesis and embryonic development.

Throughout the spring of 2002. an HPLC-based method was
used to measure hemolymph MF liters in more than 70

To

in

crab Libinia e/narginata and the edible crab Cancer pagurus, increased levels of
synthesis in the mandibular
in
organs (Laufer et al., 1987a) and elevated levels of

female C. pagurus tissues. Levels of FAMTase transcripts

in mandibular organs of female C. pagurus were found to

metamorphosis

gests an analogous role for

Northern blotting demonstrated widespread expression of

an approximately 1250-nucleotide FAMTase transcript in

review,

1996; Belles, 1998), and a growing body of evidence sugin crustaceans. In the spider

activity.

Received

in insects (for a

1994), a recent report confirms that

see Riddiford,

methyl farnesoate; MO-IH, mandibular organ-

reported
308

when ovary

explants from shrimp, Penaeus van-

METHYL TRANSFERASE CHARACTERIZATION

(Tsukimura and Kamemoto. 1991) and red swamp
crayfish. Procambarus clarkii (Rodriguez el al.. 2002) were
'i

incubated

with physiologically relevant concentra-

in vitro

tions of

MF. This combined evidence

role for

MF

the reproductive

in

strongly supports a

development of female

crustaceans.

two

into

number AF249871) or from

is

development

broadly separated

phase and a

distinct phases, a pre-vitellogenic

vitellogenic phase. During the pre-vitellogenic phase, prito

accumulate rough endoplasmic

retic-

mary oocytes begin

ulum. and endogenous glycoprotein content increases. At
the end of this phase, oocyte development arrests at
prophase-I of meiosis, which synchronizes the oocytes at
the same stage of development (for a review, see Charniaux-

the recombinant

Gunawardene

The

vitellogenesis fol-

characterized by a signifivitellogenic phase

cant increase in the size of the ovary and an accumulation of
lows.

is

et ai,

2002).

No

However,

to catalyze, in

brachyuran decapod

Cancer pagurus might also have enzyme

we

In this report,

activity.

describe the isolation and characteriza-

cDNA encoding a 275-residue putative

mandibular organs of female specimens of

tion of a full-length

FAMTase from

C. pagurus (GenBank accession number AY337487). The

recombinant FAMTase was heterologously expressed, and

tive

breakdown occurs, and

activity.

shrimp Metapenaeus

has been studied to date, but these findings with shrimp

suggest that a homologous putative FAMTase from the crab

simultaneously. In locusts and shrimp, the meiotic block is

released by physiological doses of ecdy steroids (Cledon.

germinal vesicle

the

vitro, the conversion of farnesoic acid to methyl farnesoate

enzyme

1989). After meiosis resumes,

FAMTase from

(AF333042) has recently been reported

ensis

Cotton and Payen, 1988). Synchronization is critical because crustaceans fertilize and spawn all of their oocytes

1985; Lanot and Cledon.

the clawed lobster Hoimirus

FAMTase

iimericanus (U25846) have

(Silva

In crustaceans, ovarian

309

activity

was

system (Wainwright

FAMTase

investigated in an established assay

1998). The distribution of puta-

et al.,

expression

is

presented for a range of C.

throughout the course of ovarian developalso present further

during embryogenesis.

tissues,

pagurus
ment and

We

details regarding the previously reported

lymph MF (Wainwright et al.,

peak of hemo-

1996a), which occurs prior to

or during the earliest stage of vitellogenesis.

yolk protein within the oocytes of the developing ovary

tissue. In C.

by sperm

copulation,

Eggs

are

pagurus. oocytes are fertilized as they are

that has

when

been stored

the female

laid.

was soft-bodied

after molting.

brooded under the abdomen attached

hatch 6 months later (Warner, 1977). The precise timing of

vitellogenic events in C. pagitnis has not been fully deter-

mined, although vitellogenesis appears to begin during the

spring, presumably in response to environmental cues, such

and temperature. The exact role of

MF

in

crustacean ovarian development is still unclear, but in C.

concentration is reported to
pagurus the hemolymph

MF

peak during the earliest stage of vitellogenesis, suggesting

its involvement in the control of this process (Wainwright el
nl..

1996a).

Previously,

we

investigated the regulation of

MF produc-

mandibular organs of C. pagurus. isolating and

mandibular organcharacterizing two 78-residue peptides
tion in the

inhibiting

hormones (MO-IHs)

that

Animals

to ovigerous

pleopod hairs until embryogenesis is complete, and the

hatched larvae are released as free-swimming zoeae. Egglaying is believed to occur during the winter, and the larvae

as photoperiod

Materials and Methods

spermathecae since

in the

down-regulate

the

MF

Adult females of Cancer pagurus (Linnaeus), the edible

were obtained weekly from local fishermen and main-

crab,

tained in a recirculating seawater system at ambient light

and temperature. These wild-caught animals constituted a

non-synchronized population; therefore, crabs at different

stages of ovarian development could be encountered at any
given time. Crabs were dissected after cold-anesthesia, and
the stage of ovarian development was determined according
to established criteria

(Wainwright, 1995; Wainwright

genic, with steadily increasing organ size, oocyte diameter,

and quantity of accumulated yolk protein (orange color).

crabs, with cream-colored undeveloped ovarian
Stage
tissue, are classed as either "pre-vitellogenic." if the

lymph
color

color

is

circulating yolk protein.

MO-IH

and maintained

adenosyl-L-methionine farnesoic acid O-methyl transferase

(FAMTase). an enzyme that catalyses the final step of

MF

biosynthesis (Wainwright

el ai,

1998).

To

date, putative

FAMTase sequences from three decapod crustaceans have

been published in on-line databases. Of these sequences,
there is no evidence that the cloned gene products from the
spiny lobster Paniilirus interruptus

(GenBank accession

hemo-

gray, or "vitellogenic," if the hemolymph

orange; the orange color indicates the presence of
is

production of
by the mandibular organs (Wainwright et
ill.. 1996b). Furthermore, we demonstrated that the action of

on mandibular organs ultimately regulates an S-

et

1996a). In brief, crabs were assigned a stage of ovarian

to 4. Stage 1 to 4 ovaries are vitellodevelopment from

al.,

Two

egg-carrying females were caught during the winter

in individual tanks at the Marine Biological
Laboratories at Port Erin, Isle of Man. Embryonic offspring

were collected from the egg-bearing pleopod hairs in the

abdominal brooding pouch using flat-ended forceps.
Hatched larvae were collected by filtration of the tank water.
Embryos, larvae, and dissected adult tissues were stored in
Trizol reagent (Life Technologies, Inc.) at
analysis.

-80 C

prior to

310

C.

Isolation of u

PCR

-1 tiii^ineni of

FAMTase

RUDDELL ET

J.

using a nested

appii

AL.

sense

(5'-GGYTCNGGRTCNGTCCAYTC)

primers (Fig.

and with the following temperature profile: 94

min. 53
min. followed by 25 cycles of 94 C for
1

).

The
C.

mai'.iiiluilar
'i/n<.\

/>i/'s

and

total

organs were dissected from three female

specimens (ovary stage

RNA

0,

hemolymph

orange),

isolated using Trizol reagent. Nested

was

degenerate PCR primers were designed based on conserved

regions within the crustacean FAMTase sequences pub-

total

RNA.

PCR was

earned out

0.5 units

cDNA

synthesis was performed using

as described

in

the

SMART cDNA

/j.g

Library

Manual (version #PR92334)

Construction
(Clontech Laboratories. Inc.). Most of this material was
User

Kit

used to synthesize a mandibular organ cDNA library (see

below). One-tenth of the first-strand synthesis reaction was
used as cDNA template for PCR. with DMTSI sense (5'-

GCNCAYGAYGCNCAYGTNGC)

and

DMTAS1

p.M

anti-

Taq

min. and a

DNA

final

for 2

extension step of 7 min.

in a 20-jnl reaction

polymerase (Roche)

volume containing
in the

manufactur-

mM MgCK) in the presence of

(MWG Biotech) and 0.25 mM each

PCR

primers
(Promega). Nested

DMTS2
sense
(5'-CNCCNGAYATHYTNWSNGARGAR) and
DMTAS2 antisense (5'-YTTNCKYTCYTCYTCRCARC)
of the first PCR reaction as a
using 5
primers (Fig.
template. The following thermal cycle was employed: 94 C

dNTP
I

for

for 2

er's reaction buffer (with 1.5

0.5

lished online.
First-strand

min. 72

C
C

for

Other

min. 54

PCR

'

).

PCR was

performed with

/u.1

for

min, 72

for

min

for 25 cycles.

conditions and reaction mixture constituents

were as described above. Agarose gel electrophoresis of the

MADEIPAL

taagttgttggtgcttctcctgtctgtacacacccccacacaccacacaccgagatcatggctgatgagattcccgccct

GTDENKEYRFRELDGKTLRFQVKTAH
DCHVAFTSAGEETDPIVEVFIGGWEGA

tggcacggacgagaacaaggagtaccgcttcagggagcttgatggcaagacccttcgattccaggtcaaaacagcgcacg
DMTS1

attgtcatgtggcattcacgtcagccggtgaagagacagaccccatagtggaggtgttcattgggggatgggagggcgct
DMTS2

241

gcctcggccatcaggttcaagaaagctgatgatctagtgaaggtggatapgccagacattcttagcgagggagagtaccg

320

ASAIRFKKADDLVKVD(T)PDIL(S)EGE(Y)R
ncoV
GSP2
GSP
GSP3

EFWIAVDHDEIRVGKGGEWEPLMQAP
IPEPFPITHYGYSTGWGAVGWWKFMND

tgaattctggattgc tgtggaccacgacgaaataagagtaggcaagggcggagagtgggagccgctcatgcaggcgccca

tcccagagcccttccctatcacccactacggctactccacaggctggggtgctgttggctggtggaagttcatgaacgac

480

agggtcctaaacactgaagactgcctcacctacaacttcgagcctgcctacggtgacaccttctccttcagcgtcgcctg

560

SNDAHLALTSGAEETTPMYEIF1GGW
ENQHSAIRLNKGDDNAKVETPDALCCE
^

RVLNTEDCLTYNFEPA(Y)GDTFSFSVAC

561

cagtaacgatgctcatttggctcttacctctggcgccgaagagaccacgccaatgtacgagatcttcattggtgggtggg

640

641

agaaccaacactccgccatccgcctcaataagggtgacgacatggccaaagtagagactccggacgcactgtgttgtgag

720

DMTAS2

721

gaggagaggaagttcttcgtgtccttcaggaacggccatatcaaggtgggctAcaaggacac tgatcccttcctgcagtg
K
F
F
v fs) F R N G H i K v G (V) K D T D P F L Q w
E
E
R
DMTAS1

TDPEPWKVTHVGYCTGWGATGKWKLD

gactgaccctgagccctggaaggtaacccatgtgggatactgtacgggatggggcgctaccggcaagtggaagc ttgata

881

tctaagtgaacaaaaggtggaggtggtgatgtgatgtttgtcagtcatgcatcacactcaccaccactcgtcacactatc

960

961

accacccctgctgccatgctatccactaccattggtacgtaataacgcttctatccttatctttgtcttcagtttataat

1040

1041

aaagc ttccaaagcctgagaagccctatgagggtggagcgttgcgcacacacc tgttgctgcattaacctttaaatgtcc

1120

1121

tcttacatggaattaaaagtgggagttatttttcgtactctttgtagcttcacgtoaataaaUcctgaaaac tag

1225

(a)

30

Sequence ol'cDNA from the mandibular organ encoding ihe putative FAMTase. The full-length
PCR and nucleotide sequence determined (see Methods The cDNA sequence shows an
825-bp open reading frame, which encodes a 275 amino acid protein. The stop codon is indicated by an asterisk.
Figure

cDNA
and

1.

was

isolated by

(AATAAA) is enclosed in a box. Circles indicate potential phosphorylation sites

mature protein Arrows indicate positions of primers, which are identified (see Methods). A single Eco RI
322 bp is underlined.

a poly.idein lation signal

in the

site at

I.

METHYL TRANSFERASE CHARACTERIZATION

nested

PCR

products revealed a band of about 450 hp.

into the pGEM T-easy TA cloning vector

which was cloned

according to the manufacturer's instructions (Promega). Sequencing of the 450-bp cloned insert showed it to be very
similar to the existing crustacean FAMTase sequences in

cDNA fragment was used as a probe to

cDNA encoding C. pagunis FAMTase.

databases. This

additional

311

5'-TACTCT-

(GSP2,

primers

gene-specific

TATTTCGTCGTGGTCC;
GAATTCACGG), together

5'-ACAGCAATCCA-

GSP3,

with anchor primers supplied

by the manufacturer. The second-round PCR product of

about 380 bp was cloned into pGEM-T Easy Vector, and the
nucleotide sequences of several clones were determined.

isolate a full-length

FAMTase

Expression of

FAMTase cDNAfroin
organ cDNA lihrar\

Isolation of full-length

mandihiilar

From

the remainder of the first-strand synthesis reaction

cDNA

li-

brary was constructed in bacteriophage ATriplEx2. The liter

h
of the primary, unamplified library was 0.97 X 10 pfu/ml

>95%

To

positive recombinanls.

isolate clones containing full-length

cDNA

Expression of protein encoded by

Ex2 plasmid was

(see above), a unidirectional, mandibular organ

with

protein

<;

cDNAs

encoding

F'. Bacteria

within pTriplTOP 10

carried out, as follows, in E. coli

containing the pTriplEX2 plasmid were grown

6m 0.5 to 0.6. at 37 C, in LB medium

to a density of

OD

supplemented with 50

LacZ

fusion protein

of 0.4

mM (Brent.

ju.g/ml

of ampicillin. Expression of

was achieved by addition of isopropyl

j3-D-thiogalactopyranoside (IPTG) to a final concentration
1994).

hybridization screening was

the
carried out using
450-bp FAMTase fragment end-la3
beled with [a- -PrdCTP using the Ready-To-Go

SDS PAGE

Labeling Kit (Amersham Pharmacia) as a probe. Plaque lifts

were carried out with BioTrace NT nitrocellulose blotting

Extracts of proteins from E. coli were prepared to yield

both soluble protein fractions and insoluble inclusion-body

FAMTase, plaque

C. pagiirus

DNA

membrane (Gelman

and

Sciences),

washes were carried out according

lines.

hybridizations

to manufacturer's guide-

feature of the ATriplEx2 vector

embedded

bacterial plasmid

DNA,

circularization of the plasmid

DNA

and

is

that

it

contains an

pTriplEx2. Excision and

DNA

from the linear bacte-

readily achieved by a process involving /;;

riophage
vivo excision using Cre recombinase-mediated site-specific
is

recombination

at

plasmid. This

is

31

in the

two loxP
carried out

sites

embedded

flanking the

at

by incubating ATriplEx2
BM25.8 (Clontech Labo-

presence of E. coli

ratories, Inc.).

The

resullanl plasmid can be used lo express

fusion protein variants of the encoded proteins under

the regulation of a LacZ promoter. Positive clones from the
and
library screening were converted into plasmid clones

LacZ

analysis of proteins

fractions, according to a published

method

both cases, E. coli cells were harvested by centrifugation

(3000 X g. 10 min, 4 C). washed in PBS buffer, and
centrifuged (3000

HEPES [pH

7.6],

0.1%

glycerol,

hibitors (1

g.

HEMGN

in

resuspended

X
0.1

10 min, 4

C).

buffer (100

mM EDTA

[v/v] Triton

mM

[pH

tor cocktail

original culture

at

10

volume) and lysozyme

jul

Briefly,

gene-specific

in-

and

g. 15
lysed by sonication. After centrifugation
and
insoluble
min, 4 C) to separate soluble (supernatant)

fractions (pellet), the pellet

g,

30 min, 4

was extracted

into

HEMGN

guanidinium-HCl, and centrifuged

C).

The supernatant was dialyzed

once against HEMGN buffer containing

guanidiniumHCl and protease inhibitors (see above), Ihen Iwice against

-RACE)

To obtain the 5'-end of the FAMTase cDNA clone,

5 '-RACE was carried oul using total RNA from mandibular
organs. A 5 '-RACE system version 2.0 (Life Technologies)
was used to amplify the 5' terminus of the message for
sequencing.

[v/v]

per 100 ml of

(0.5 mg/ml).

(5'

10%

(27,000 x

(87,000

ends

KC1, 25

8.0],

X-100) containing protease

[Sigma. #P8340] added

buffer containing 8

cDNA

was

mM

mM dithiothreitol, 0.1 mM phenylmethylsulfonyl

mM sodium metabisulfite and protease inhibi-

Sail.

of

pellet

The

fluoride, 0.1

analyzed by restriction enzyme digestion with EcoRl and

5' -Rapid amplification

(Brent, 1994). In

primer (GSP1, 5'-

ACTCTCCGCCCTTGCC) was hybridized to ihe mRNA,

reverse
and cDNA was synthesized using Superscript

HEMGN
sate

was

buffer containing protease inhibitors. The dialycentrifuged (12,000 X g. 5 min, 4 C) to yield 8

guanidinium-HCl soluble (supernatant) and insoluble

(pel-

protein extracts (see Fig. 3). Portions of all extracts were

analyzed by eleclrophoresis on a 10% polyacrylamide gel.
let)

Protein bands were visualized by staining wilh colloidal

Coomassie blue G250.

II

The RNA was Ihen degraded with RNase mix

(RNase H and RNase Tl and the cDNA was purified using
a GlassMax spin cartridge supplied with the kit. A poly(dC)
tail was added to Ihe 3 '-terminus of the purified cDNA
transcriptase.

FAMTase

assays

).

using
the

dCTP and

cDNA

terminal deoxynucleotidyl transferase. and

region corresponding to the 5'-end of the

was amplified by two successive rounds of

mRNA

PCR

using

Broken-cell

FAMTase

extracts

of

E.

coli

were

assayed

for

activity using assay conditions previously pub-

lished (Wainwright et al., 1998). Briefly, extracts (200 /u.1)

were dialyzed against a hypotonic HEPES buffer (0.037
KF, pH 7.4) before the
HEPES, 0.3
sucrose, 0.01

312

C.

J.

RUDDELL ET AL

250
volume
of 50
S-adenosyl-L-in.
and
were
at 37 C for
h
out
were
carried
Incubationju,l.
addition of 150 ju.1 of acetonitrile. The
terminated rr.
addition of 2.4

^M

[12- H] farnesoic acid and

ionine, in a final reaction

tl

t!

reaction products were analyzed by reversed-phase HPLC

with on-line radioactivity monitoring, as described previ-

ously (Wainwright et

HiPerSolv grade), and 100 ng

internal standard.

n-hexane (Merck, HiPerSolv grade), achieving phase sepa-

by centrifugation at 500 X g for 10 min

hexane layer (top) was removed and 300
ration

directly to

HPLC
in

from a variety of

using

tissues (see Results)

was

Trizol

RNA

was electrophoresed on a formaldehyde/ 1% agarose gel for 3 h at 75 V. The RNA was blotted onto Electran
nylon membrane (BDH) with 20 x SSC (SSC is 0.15
ual tissues

NaCl/0.15

M sodium

pared, as
in

UV

citrate)

and

RNA

cross-linked to the

The FAMTase probe was predescribed above, and hybridization was carried out

membrane by

radiation.

solution (Stratagene) for 1.5 h at 68

QuickHyb

temperature, for 10 min each, in 2

SDS, and

twice,

at

45

C, for

C. After

room
X SSC containing 0.1%
10 min in 0.1 X SSC

was washed

hybridization, the blot

three times, at

containing 0.1% SDS. Autoradiographs were exposed

-70

at

C.

To compare relative levels of expression of FAMTase in

samples, a mouse 18S rRNA probe (Takeuchi et al.. 2000)
was co-hybridized under the same conditions. This procedure provided an internal calibration for each sample and
allowed for differences among lanes in the loading of RNA.
In preliminary control experiments,

when Northern

blotting

carried out with individual probes alone, using identical

hybridization and wash conditions, the hybridization pat-

showed

Under

single bands of appropriate sizes for each

rRNA. 2000

probe (18S

nf,

FAMTase mRNA, 1250

18S rRNA

the conditions used, therefore, the

FAMTase

mRNA

probes did not cross-hybridize with their

So we co-hybridized the blots

subsequent experiments. Densitometric analyses were
earned out using Quantity One software (Bio-Rad) and
in

FAMTase

mRNA

normalized against those of

18S rRNA.

in

subjected

hemolymph hexane

extracts were determined by adsorption

HPLC

on a Varian

Pro Star chromatography workstation, with a modification

of the method previously described by Borst et til. (2002).
Separation of MF isomers contained in hexane extracts (300
/LI!)

from hemolymph was achieved on a Rainin MicroSorb

/nm, 250 X 4.6 mm internal

MV silica adsorption column (5

diameter) using isocratic elution at 2 ml/min for 45 min in

0.47r diethyl ether in /i-hexane (Merck, HiPerSolv grade)
that had been dried overnight after the addition of 50 g of

A (bead), 8-12 mesh (Sigma, #M1760),

of solvent. Eluted compounds were detected by UV
absorbance at 229 nm. Peak areas were calculated using Star
workstation software (Varian). All-trans-MF content of hemolecular sieve 4

per 2.5

molymph samples was

calculated by comparison of the

cis, trans-MF peak area

all-tnins-MF peak area to the

(internal standard 100 ng).

Results
Isolation, characterization,

and expression of FAMTase

from C. pagurus inantlibular organ

and characterization of FAMTase. To obtain

encoding FAMTase, nested PCR was

Isolation

length

full-

cDNA

lowed by

nt).

and

respective target transcripts.

amounts of

20 C. The

;u,l

quantification of methyl farnesoate

was

terns

at

analysis.

C. pagurus tissues

reagent (Life Technologies). For

from individNorthern blotting, about 10 /ig of total
isolated

HPLC

Levels of all-trans-MF contained

RNA

Total

as an

partitioned against 2 ml

1998).

til.,

FAMTase

Expression of

trans-MF isomer

cis,

The mixture was

isolation of full-length

cDNA

fol-

from a mandibular

cDNA

Previously
published putative
library.
sequences provided information for the design of
degenerate oligonucleotide primers for the isolation of a ca.

organ

FAMTase

450-bp

fragment

of

cDNA

that

encoded

putative

FAMTase. Subsequent sequence

analysis suggested that

this was, in fact, a putative FAMTase
fragment of
442 bp. This partial
was used to screen a mandibular

cDNA

cDNA

Preparation of hemolymph methyl farnesoate extracts for

HPLC

organ cDNA library. Initially, approximately 8 X 10 recombinant bacteriophage were grown in two 15-cm petri
dishes and screened. This

initial screen identified four posclones that were subsequently isolated and purified. All
four phage clones were converted to their corresponding

itive

Hemolymph samples were taken from adult female specimens of C. pagurus with a hypodermic syringe; the arthwas punctured,

plasmid clones as described, and the inserts were analyzed

by restriction enzyme digestion with Sail and EcoRl: the

and 2 ml of hemolymph was extracted. Methyl farnesoate

isomers were extracted into hexane using a triphasic proce-

products were then separated electrophoretically on a 1%

agarose gel. Of the clones analyzed, two distinct types were

dure (Borst and Tsukimura, 1991) incorporating modifica-

apparent: one type, on digestion, produced two products

(900 and 300 bp) originating from the cloned insert cDNA,

rodial

membrane

at

the base of a walking leg

(1996a). Briefly, heWainwright

were
added
to
tubes containing 2
(2
ml)
samples

tions according to

molymph
ml NaCl

4%

(w/v)

et al.

in

H,O,

ml acetonitrile (Merck,

far

UV

while the other type produced only a 900-bp insert. Sequencing and BLAST searching revealed the former type.

METHYL TRANSFERASE CHARACTERIZATION

Group

1.

to be putative

FAMTase

sequence clones. The

ver.

1.1

313

software (http://www.cbs.dtu.dk/services/SignalP/)

complete sequence of the putative FAMTase clone was

obtained (see Fig. ). The clone was 1216 bp in length, and

suggested that the protein does not contain a signal peptide

cleavage site. Further analysis of the protein sequence, with

open reading frame of

825 bp encoding a 275 amino acid protein with a predicted
molecular weight of 31114. The 5'-UTR was 48 bp long.

NetPhos 2.0 (http://www.cbs.dtu.dk/services/NetPhos/) and

ScanProsite (http://ca.expasy.org/tools/scanprosite/). for potential posttranslational modifications shows multiple high-

The 3'-UTR was 336 bp long and contained

scoring (score

conceptual translation indicated an

ation signal.

A ATA A A,

5 '-RACE demonstrated that the

single

EcoKl

restriction

tail.

full

and sequenced
length of the 5'-UTR.

enzyme

cutting site occurs at

clone was 9 bp shorter than the

a polyadenyl-

13 bp upstream of the poly(A)

isolated

>

0.8) sites for possible phosphorylation at

serine, threonine,

and tyrosine side chains within the mol-

ecule (Fig.

2).

Expression and activity of

FAMTase

protein

322 bp.
ClustalW alignment of the isolated putative FAMTase
from C. pagitrus, with sequences identified in other crusta-

To determine whether the protein encoded by the isolated

FAMTase cDNA clone was indeed an active FAMTase.

cean species, demonstrated a high degree of sequence identity (Fig. 2). Analysis of the protein sequence with Signal?

expression of the protein in E. coli was carried out as

described (Methods). Just prior to the addition of 1PTG to

pagurus
americanus
M. ensis
P. interruptus

MA-DEIPALGTDENKEYRFRELDGKTLRFQVKTAHDCHVAFTSAGEETDPIVEVFIGGWE
MGDDNWASYGTDENKEYRFRDISGKTLHFQVKTAHDAHVALTSGAEETDPMVEIFIGGWE
MA-DNWPAYGTDENKEYRFRIIKGKTLRFQVKAAHDAHIALTSGEEETDPMLEIFIGGWE
MGDDNWPSYGTDENKEYRFRDIGGKCLRFKVKTAHDAHVALTSGAEETDPIVEVFIGAWE
*** **
*.**.***
***********

C pagurus

GAASAIRFKKADDLVKVE(T^DII@EGX^IREFWIAVDHDEIRVGKGGEWEPLMQAPIPEPF
GAASAVRFKKGEDLVKVE@PDII@EEB^REFWIAFDHDEIRVGKGGEGEPFMQCPIPEPF
GAASAIRFKKADDLTKVT(T^DILNAE^REFWIAFDHDNVRVGKGGEWEPFMSATVPEPF
GAASAIRFKKADDLAKVE(TpDILNEE^REFWITFDNDEVRVGKAGDWEPFMMSPSQSHS
*.*..**** *. **.*
*******
*****.**** ** ********

C.

H.

americanus
M. ensis
P. interruptus
H.

pagurus
americanus
M. ensis
P. interruptus

PITHYGYSTGWGAVGWWKFMNDRVLNTEDCLTYNFEP?(Y)GDTFSFSVACSNDAHLALTSG
GITHYGYSTGWGAVGWWQFHAEKSYNTEDCLTYNFIPV(Y)3DTLEFSVSCSNriAHVALTSA
EITHYGYSTGWGATGWWQFHSEMHFQTEDCLTYNFVP\(Y)3DTFSFSVACSNDAHLALTSG

C.

AEETTPMYEIFIGGWENQHSAIRLNK
GDDMAKVETPDALCCEEERKFF\(S)FRNGH
GDDMIKVDTPDILCCEEERKFW\(S)FKNGH
AEETTPMYELLLGGWENQHSAIRLNK
PEETTPMYEVFIGGWENQHSAIRLSKEGRSSGEDMIKVDTPDIVCCEEERKFT$|)FKDGH
GDDMIKVDSPDIVCSEEERKFWI()FKNGR
PEETTPMYEVFIGGWENQHSAIRLNK
* ******
* ** **
************
********

C.

H.

pagurus
americanus
M. ensis
P. interruptus
H.

KSPTMAIPLAGVLSAGGSFIMR-DFHTEDSQAYKFEPV^DSLTFSVSCGHDAHLALTSG
********

pagurus
americanus
M. ensis
P. interruptus
C.

H.

IKVQ@KDTDPFLQWTDPEPWKVTHVGYCTGWGATGKWKLDI
IRVd^KDTDPFMEWTDPEPWKITHIGYCTGWGATGKWKFEY
IKVG@QDSDPFMEWTDPEPWKITHVGYCTGWGASGKWKFEF
IRVQYkDSDPFMEWTDPEPWKVTHVGYTTAWGAAGKWMLEI
** *

** *

Figure 2. Amino acid sequence alignment of putative FAMTases from four crustaceans. ClustalW alignment of FAMTases from Cancer pagurus (this report, GenBank accession number AY337487), Hamarus
americanus (U25846). Metapenaeus ensis (Silva Gunawardene el al., 2001. AF333042). and Panulirus inlerni/>m\ (AF249871 ). Identical amino acids at a particular position, in all sequences, are denoted by an asterisk.
Colons denote alignment of amino acids with strong similarities; periods indicate aligned residues with weaker
according to their physicochemical properties. Hyphens denote gaps introduced to maximize the
sequence alignment. Circles indicate potential phosphorylation sites in the mature protein. EMBL ClustalW
default alignment settings were used (www.ebi.ac.uk/clustalw/).
similarities,

314

C.

the cultures, a 1-ml

protein extract
(I

ples

n.

h)

RUDDELL ET AL

was taken and

v pared as described (see Methods). Samnal culture were taken at 1, 2 and 5 h

Both soluble and insoluble extracts of

..ition.

post-IPTG

sample (lime

J.

FAMTase expression levels in

hemolymph RNA, the FAMTase tran-

eluded estimation of relative

these tissues; and in
script

was undetectable by Northern

To determine

blotting.
the developmental profile of expression of

RNA

ia were prepared and analyzed by SDS-PAGE.

of
the samples taken 5 h after the addition of IPTG
Analysis

FAMTase

shows that recombinant protein expression is induced by IPTG. and that the recombinant protein is produced predominantly in the insoluble inclusion body frac-

ovarian development, and Northern blotting was carried out

as described. A representative blot (Fig. 5a) demonstrates a

the bar

clearly

in

mandibular organs,

was extracted from

the mandibular organs of female crabs at different stages of

distinct variation in the level of expression of

FAMTase

in

Molecular weight analysis demonstrated the

induction of a protein of approximately 40 kDa. This size is

the mandibular organ during ovarian development. Follow-

FAMTase-

grams, the ratio of FAMTase to 18S rRNA was determined,

and the results were displayed graphically (Fig. 5b). Expres-

tion (Fig. 3).

entirely consistent with the expected size of the

LacZ fusion protein.

To determine whether

FAMTase

exhibited any

the recombinant fusion protein

activity, the protein extracts

were

assayed for their ability to convert farnesoic acid into

methyl farnesoate using conditions previously described

Wainwright et al., 1998). The broken cell exexhibited no detectable FAMTase activity (results not

ing densitometric analysis of the Northern blot autoradio-

sion of

FAMTase from

mid and

late stages

(see above;

mandibular organ

tracts

ate,

shown).

FAMTase

during ovarian development and

embryogenesis
of FAMTase

Expression

To determine

in

tissues

and developmental

the tissue distribution and develop-

FAMTase, Northern

mental expression of the putative

blot-

was

carried out using the 450-bp clone as a probe. The

detected
a single band of approximately 1250 nt in a
probe
number of tissues, including muscle, eyes, mandibular orting

is

significantly

of ovarian development, the levels of

FAMTase

expression appeared to fluctubut definitive trends could not be identified at these

stages.

Expression of

stages.

the mandibular organ

higher before the onset of vitellogenesis than after vitello= 0.03). During the
genesis has begun (unpaired t test, P

gans, epidermis,

gills, heart,

The extremely low

(Fig. 4).

ovary, hepatopancreas and gut

signals detected by Northern

and sub-epidermal adipose

blotting in Y-organs

tissue pre-

Female methyl farnesoate hemolymph liters. As part of an

investigation aimed at further characterizing events that
occur during the transition from the pre-vitellogenic to
vitellogenic phases of ovarian development, we measured

hemolymph methyl farnesoate (MF) tilers of over 100

female specimens of C. pagitnis throughout the spring of
2002. In stage
crabs (n == 70). hemolymph MF tilers

the

segregaled inlo two groups: "low" (93%) and "high" (7%)

MF, wilh a cul-off liter of about 150 ng/ml between the

groups (Fig. 6). It was noted that although one high MF crab
was at an early stage of vilellogenesis (stage 0, orange

hemolymph), four of

the five high

pre-vitellogenic

stage

MF

crabs

category (slage

0,

fell into

the

gray hemo-

lymph).
Soluble fraction

Insoluble fraction

Plasmid clone

KDa

IPTG

markers

75
50

FAMTase
ment.

expression during embryonic and lan'al developinvestigations, samples of

As an extension of our

developing embryos, up to hatching, were collected and

analyzed for expression of FAMTase Iranscripts. The resulls

==
40 kDa

37
25

(Fig. 7) show lhal, in bolh groups of embryos sampled from

each of the individual brooding females, levels of FAMTase
transcript increased noliceably during developmenl, and fell

to near the basal level just before halching.

15

Figure

Discussion
Expression of recomhinant putative

3.

FAMTase. pTriplEx2

encoding full-length FAMTase were

Protein expression was induced by addition of

Here we report the isolation and characterization of a

putalive farnesoic acid methyl Iransferase cDNA from man-

(see Methods). Extracts of E. coli prepared 5 h after induction with

w .: e analyzed for expression of recombinant
protein by SDS-PAGE

dibular organs of the edible crab Cancer pagurus. Using a

combination of a nested PCR-based approach and screening

analy-:

Methods). Soluble and insoluble post-dialysis fractions are

shown.

absence (-) or presence + of either IPTG (to induce

'em expression when plasmid is present) or plasmid clone

of a mandibular organ cDNA library, a 12 16-bp cDNA was

isolated that encodes an approximately 31 -kDa protein mol-

plasmids containing
in

gri

IPTG
IPTG

recomhm.,

CD//

in the original

gel image.

cDNA

TOPIO

F'.

inserts

'//

culture conditions

is

indicated in the table above the

ecule (Fig.
hibits

1).

The

putative

FAMTase

of C. pagurus ex-

high degree of sequence similarity wilh those

MF.THYI. TKANSFFRASF.

CHARACTERIZATION

315

//

18SrRNA
FAMTase
(~1250nt)
Northern blot analysis. Approximately 10 /Mg of total RNA from a variety of Cancer \itiv.uni\
two organ equivalents of Y-organ RNA was electrophoresed. blotted onto a nylon membrane,
'2
co-hybndi/ed at 68 C with a P-labeled-FAMTase probe and a inouse IXS rRNA probe, and washed at 45 C

Figure

4.

tissues and

'

(see Methods).

The Northern

blot

shows

the tissue distribution

si/e of the C. /xtxiirux

Stage of ovarian development

a)
0*

18SrRNA

0*

<*

FAMTase
(~1250nt)

b)

and

1.2

FAMTase

transcript.

C.

316

re

O
in

J.

RUDDELL ET AL

METHYL T.RANSFERASE CHARACTERIZATION

317

be regulated by phosphorylation/dephosphorylation in vitro.

suggesting that it may be a mechanism of regulation of

These crabs were predominantly

(Wainwright and Rees. 2001). Indeed, other work has shown that treatment of mandibular

previously reported to

enzyme

activity in vivo

organs with the peptide hormone

MO-IH

gan-inhibiting hormone) leads to an

(Wainwright ci ul.. 1999). In turn, this
in the

phosphorylation state

(mandibular or-

increase

cAMP

in

may lead to changes

of FAMTase. thereby modulat-

ing the production of methyl farnesoate (MF).

To determine whether the protein characterized from the

mandibular organ possesses

FAMTase

activity, the

recom-

of development. Interestingly, a

occur

at

a pre-vitellogenic stage

hemolymph

MF

in early vitellogenic

peak was

specimens

(Wainwright et at.. 1996a) and. in agreement with this, we

also observed a "high MF" titer in an early vitellogenic crab.
These data suggest that, in contrast to what has previously
been observed, elevated hemolymph

MF

may occur

at dif-

ferent times during pre-vitellogenesis and early vitellogenesis in C. pagurus. As only 17c of pre- and early vitellogenic

specimens were found to have high MF. we believe that the

MF peak may be relatively short-lived. The transience of

binant protein

was expressed in E. coli as a LacZ fusion

protein of approximately 40 kDa. On analysis, an approximately 40-kDa protein appeared in the inclusion body subcellular fractions: however, the inclusion body fractions had

this

no detectable FAMTase activity. This observation has three

possible explanations. First, the presence of the LacZ tag

also changes in expression in relation to ovarian development and the circulating levels of MF. Northern blot anal-

and linker sequence

ysis of the expression of the putative

may

interfere with the folding of the

nascent protein and. thus, with the biological activity of the

enzyme. Second, it may have been impossible to resolubili/e the

inclusion bodies

in vitro. In this event, the activity

FAMTase may have been disrupted. Third, previous

attempts to stabilize FAMTase activity in extracts of cytosol
of the

and. thus, to facilitate traditional

during storage
of enzyme purification and characterization

methods

have resulted

almost complete loss of activity (G. Wainwright, unpubl.

obs.). This sensitivity of FAMTase activity to its environin

ment may also explain the lack of observed

activity in the

recombinantly expressed protein, under the experimental

conditions described here. Similar difficulties have been

encountered

in

FAMTase from

expressing
the shrimp

recombinant

functional

Metapenaeus

eusis,

when

pro-

cedures largely equivalent to those described here were

followed. In the M. ensis study, biological activity was
eventually detected in a partially purified bacterial extract

when a high-level expression system was employed

Gunawardene et ai. 2002). It was surmised that only
proportion of recombinant

pressed

FAMTase may

in a correctly folded, active

in

ular

investigated, experimentally, whether the

organs

ety of tissues

FAMTase among

was demonstrated by Northern

blotting,

which

revealed a single transcript prevalent in muscle, eyes, mandibular organs, epidermis, gills, heart, ovary, hepatopancreas. and gut. Hemolymph did not exhibit a signal detect-

stages

in

mandib-

of ovarian
of
sig-

MF

appears to occur at some time during prehemolymph

or early vitellogenesis, it is apparent that FAMTase transcripts are elevated within the mandibular organ before, or
during, the stages of development

lymph

MF titer occur.

pattern of peaks in

MF

lymph
product
is

is

needed

when

the peaks in

hemo-

Consequently, the observed temporal

FAMTase

mRNA expression and hemo-

supports the view that our cloned gene

indeed a FAMTase. although additional evidence
titer

to

confirm

this.

The presence of FAMTase

transcripts has also

been dem-

onstrated in the juvenile shrimp M. ensis (Silva Gunawardene et ai.. 2001 ). Similarly, we detected the expression of

showed

a vari-

FAMTase

different

the early stages of vitellogenesis (Fig. 5b). Since the peak in

peak

FAMTase

in

embryos of

C. pagurus.

The

results

embryonic development of C. pagurus.

expression is observed during late embryogenesis,

that during

in

prior to hatching (Fig. 7).

FAMTase
distribution of the putative

at

nificantly higher in pre-vitellogenic crabs than in animals in

a putative

bacte-

from crabs

FAMTase

development showed that the relative expression

FAMTase. normalized using an 18S rRNA probe, was

a tiny

ria.

The

We

(Silva

have been ex-

conformation

peak (or peaks) would reduce the likelihood of it being

observed in a wild-caught, non-synchronized population,
and has presumably hindered its characterization to date.

in

The

significance of the role of

biochemical and developmental processes

in

embryonic and juvenile crustaceans is as yet unclear, but it

seems likely that, through regulation of production of MF.
this

enzyme

will

affect

growth and developmental pro-

cesses.

able by this method. Expression of the putative FAMTase

transcript in a range of non-mandibular organ tissues in C.

Acknowledgments

pagurus, described here, resembles that in M. ensis (Silva

Gunawardene et ai. 2001, 2002). suggesting that FAMTase

We thank NERC (DEM A initiative) for financial support.

We are grateful to Mr. N. Fullerton (Port Erin Marine

quite broadly distributed in crustacean tissues. Perhaps

Laboratory, University of Liverpool) for supplying samples

of embryos and larval C. pagurus used in this work. We also

is

the substrate specificity of the

HPLC

analysis of

MF

enzyme

levels in the

is

not

strict.

hemolymph of

pre-

vitellogenic and early vitellogenic crabs revealed a small

number of

individuals possessing "high

MF"

tilers (Fig. 6).

thank Mr. A. Tweedale (University of Wales, Bangor, U.K.)

and Mr. S. Corrigan (University of Liverpool, U.K.) for
supply and maintenance of crabs, respectively.

318

C.

RUDDELL ET

J.

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/></. Hull. 205: 319-330. (December 2003)

2003 Marine Biological Laboratory

Rcfcri.-ni.-L-:

<D

Determinate Growth and Modularity

in a Gorgonian Octocoral
HOWARD

R.

ROLLER JOHN CASTANARO,

AND JUAN ARMANDO SANCHEZ 2

LASKER*. MICHAEL

L.

Department of Biological Sciences, 109 Cooke

Hall, University at Buffalo

(The State Universin of New York), Buffalo,

Abstract.

Growth

rates of

applicable to

Images of 261 colonies were made at 6-month intervals and

colony and branch growth analyzed. Branch growth rates

module,

measurements were made. Colonies developed a

where meristems, leaves, branches,

tive

coral

or the zooids of a tunicate

ules," but in

of subsequent "generations" of
branches. The rate of branch origination decreased with
sites for the origination

many

are undoubtedly

"mod-

taxa these modules are then organized

into structures that are

themselves iteratively replicated,

such as the branches of gorgonians or the inhalant-exhalent

systems of colonial tunicates. Colony growth and development, known as astogeny. can be characterized by the

each generation of branching, and branch growth rates were

lower on larger colonies, leading to determinate colony
growth. Although colonial invertebrates like P. elisabethae
grow through the addition of polyps, branches behave as
size

groups, the basic unit of replication, the

difficult to identify. This is perhaps

study of colonial invertebrates that grow through the iterareplication of polyps and zooids. The polyps of a

were greatest at the time the branch originated and branches

seldom grew beyond a length of 8 cm. A small number of
branches had greater growth rates, did not stop growing, and

modules with determinate growth. Colony form and

many

sometimes

and leaf-branch systems can each be characterized as the

module from which the individual is constructed (Harper
and White. 1974). Many of the same difficulties exist in the

plumelike morphology through a pattern of branch origination and determinate growth in which branch growth rates

were

is

best illustrated in plants

differed between colonies and between the time intervals in

the

York 14260

of a wide variety of animal and plant communities (Jackson,

1977; Hughes, 1989). Although the concept of modularity is

branches of colonies of the

gorgonian Pseudopterogorgia elisabethae were monitored

for 2 years on a reef at San Salvador Island, Bahamas.

which

New

replication of these larger units as readily as

by the demog-

raphy of polyps and zooids (Lasker and Sanchez. 2002;

Rinkevich, 2002). Although the history of colony growth

is

generated by the iterative addition of branches.

can be portrayed using either polyps or branches, it is

unclear which level of organization should be studied to

Introduction

understand the processes controlling colony size and form.

The construction of colonies from modules is closely

Taxa ranging from algae to higher plants, and from cnidarians to protochordates, grow through the iterated replication of individual modules to form large, integrated individuals or colonies (Jackson el ai. 1985). Such modular
organisms are ubiquitous and are often dominant members

associated with indeterminate growth, that

is.

continuous

growth throughout an organism's lifetime. Indeed, modular

organisms are known to live for centuries and to attain sizes
of tens of meters. The continuous production and addition of
individuals leads to colonies that are remarkably variable in

Received 30 September 2002: accepted 3 September 2003.

* To
whom correspondence should be addressed.

and form. This mode of growth is central to a colony's

ability to survive and recover from both natural and anthrosize

E-mail:

hlasker@buffalo.edu

pogenic disturbances (Done, 1987, 1988). However, modular growth does not necessarily lead to indeterminate

'

Current address: Department of Biological Sciences, University of

Rhode Island. Kingston, RI 02881.
2

growth.

Current address: Department of Systematic Biology, National Museum

of Natural History, Smithsonian Institution, Washington. DC 20013-7012.

Many

species can be characterized by a distinct

colony form and a

319

maximum

colony

size.

The

attribution of

320

H. R.

a definable colony form and

maximum

LASKER ET

size suggests that

growth of the modules is in some fashion constrained,

which suggest* determinate growth.

The

focti^ of the current

study

is to

identify the pattern of

colony and branch growth among colonies of the Caribbean

gorgonian octocoral Pseudopterogorgia elisabethae. We
show that branches on these colonies behave as modules,
with a developmental sequence that leads to side branches
of similar length throughout the colony. We show how both
branches and entire colonies of Pseudopterogorgia elisa-

bethae exhibit determinate growth.

Materials and Methods

colony of Pseudopterogorgia elisabethae starts developing when a planula larva settles and metamorphoses into

A single, vertical branch is produced as polyps are

added alternately on both sides of the tip, thereby extending
the branch (Fig. 1A). Formation of new branches occurs
a polyp.

several centimeters

below the branch

tip (Figs.

IB and 2A).

.41.

leads, eventually, to plume-like colonies that characteristi-

have

cally

The

maximum

height of less than

pattern of branching creates a hierarchy

we characterize branch
system that we term generational

(Fig. 2B).

among

the

branches, and

order using an order-

ing

ordering (Lasker and

Sanchez, 2002; Sanchez, 2002; Sanchez

which branches

are ranked

et

2003),

<;/..

in

on the basis of the number of

branching events away from the original, primary branch

(Fig. 1; Lasker and Sanchez, 2002). The original branch has
a rank of
each branch that it produces is assigned a rank
of 2; branches that grow off of the secondary branches are
assigned a rank of 3; and so on. Each branch tip is assigned
a rank, and the rank of any single branch remains constant.
1 ;

While having some

similarities to ordering systems previously adapted to colonies (i.e., Brazeau and Lasker, 1988),
generational ordering has very different properties (Sanchez
et al.,

2003).

We

also

make

a distinction

between branches

termed mother
give
branches, and those from which no additional branches
that

rise

to

additional

branches,

termed daughter branches. Since there are no

morphological differences between mother and

This pattern is apparent in Figure 2A. in which branches 32

through 35 developed on branch 1 between December 1998

originate,

and July 1999. Each new branch then grows

fashion as the original branch, and some give

daughter branches, the distinction is based solely on the

presence of side branches. The terminology is homologous

in the

same

rise to addi-

other

and tributaries" used previously (Brazeau and

Lasker. 1988; Lasker and Sanchez, 2002). but has the ad-

branches (Fig.
This pattern of side branches themselves becoming sources of new side branches is illustrated

to "sources

by the growth of branches 2 and 5 in Figure 2 A.

This mode of branching and arowth in P. elisabethae

vantage of portraying the temporal relationships

branches (Sanchez. 2002).

tional

).

among

3rd

Generation

Branches

2nd
Generation
Branches
1st

Generation

Branches

Polyq

D
Figure 1. Colony development of Pseudopterogorgia elisabeihae. (A) Initial development and subsequent
branch elongation occurs through the addition of polyps at the branch tip. (B) The primary, "first generation"
branch generates a side branch subapically (the second generation). (C) The colony continues to add a second
generation of branches as the primary branch grows. (D) Two of the second-generation branches give rise to a
third generation of branches. Polyps are no longer depicted in C and D. (After lig 5. Lasker and Sanchez. 20021.

the

OCTOCORAL COLONY GROWTH

2nd generation
mother branch

321

3rd generation
daughter branch

2nd generation
mother branch

Primary Branch
(1st generation

mother branch)

2nd generation
daughter branch

December 1998

July 1999

Dec 1997

May1998

Dec 1998

July1999

Dec 1999

Figure 2. (A) Close-up photographs of a Pseudopterogorgia elisabethae branch in December 1998 and
1999 showing branch growth and the system for numbering branches, which enabled successive
measurements. Representative branches are labeled on the basis of type (mother, daughter) and order. Note
that only the most distal (i.e.. youngest) daughter branches grew during the interval between photos, and

July

that

mother branches continued

to

extend and

to

branch regardless of position. Branches 30-35 originated,

and 10 and 15 died, during the interval between photographs. Grid lines in the photos are 10 cm apart. See
text for explanation of branch order. (B) Photographs of a P. e/isabelhae colony from San Salvador.
Bahamas, taken over a 2-year period. All of the photographs have been normalized to the same scale, and
the grid

is

10

10 cm.

322

H. R.

To

follow

we

branches,
hcilnu'

the
!".>v

,1

;il

LASKER ET

growth of individual colonies and

and tagged 261 colonies of P. elisa-

70

transects totaling

m2

of substratum

on San Salvador, Bahamas.

located
depths of 12-15
The colonies were photographed in place at roughly
at

6-month

intervals

between December 1997 and December

1999; and the growth of individual branches was determined by measuring changes in branch lengths through

At.

When

an entire colony was included in the image, height

the length of the longest branch. If the

was measured as
entire colony was

not included in the image, height (length

of the longest branch) was measured in the field with a
flexible tape measure, to the nearest 0. 1 cm. For purposes

of analysis, colony heights were categorized into six size

classes: 0.1-10.0 cm, 10.1-20.0 cm, 20.1-30.0 cm. 30.140.0 cm. 40.1-50.0 cm, >50.1 cm.

Branch generation and

mother
vs.
were
assessed from the
type (i.e..
daughter)
and
the
number
of
branches
that
images,
originated in each

Growth

time interval was recorded as well as the branches they

developed from. Colony height was determined from either

monitored,

sequences of images, as in Figure

the

2.

images or from direct measurements

in the field.

Over

and branch measurements

measured images could be obtained by positioning colonies

between a grid 10 cm X 10 cm and a clear acrylic plastic
cover, which held the branches against the grid (Fig. 2). If

was small (<20 cm height), the entire structure

was photographed; if it was large, an arbitrarily selected
branch containing 15-25 branches was followed. In December 1997, photographs were taken with a Nikonos V underwater camera and Kodachrome 200 film. Those images
a colony

later digitized

and converted

TIFF. In subsequent

to

observations, photographs were taken at a resolution of

640 X 480 bits with a Sony Mavica digital camera (either

MVC-7
created

or

MVC-83)

an underwater housing. Distortion

in

when photographs were

shot

at

and 23,478

measurements

individual

rate

5870 branches were

length

the difference

measurements

between succes-

was then determined, and those values

were extrapolated to annual rates based on the number of

months between the measurements. Measurement error for

Pseudopterogorgia elisabethae forms colonies with most

side branches oriented in a single plane. Therefore, readily

were

the 2 years, 261 colonies with

were made. Growth

sive

analvsis

rates

a slight angle

from

was corrected in Photoshop (Version 4.0,

250 X 250 pixel grid was overlaid on the image,

1.2 cm y~', which combines the effects of

of
the
6-month intervals to 1 year and the
extrapolation
additive effect of variance in each of the two measurements

growth

rates

was

of colony length. The individual growth measurements were

categorized by the time interval in which the measurement

was made and by branch

age, based on

when

the branch

originated. Branch ages were categorized into one of five

classes; <6, 6-12, 13-18, 19-24 months, or present at the

of monitoring. In some analyses, we also designated

branches that originated during the study as "new"
start

branches. This latter category distinguished branches that

were less than 2 years old from those branches present at the
start

of the study. Each of the growth rates was also clasaccording to the branch's generation order and branch

sified

type (mother or daughter).

the perpendicular

Adobe).

and the shape of the original image was adjusted with the
free transform function of the
in the

until the

photograph matched the 250-pixel

The length of each branch

sured with the program
erick,

program

MD). Although

SCION

high accuracy, variation

although the
images,

it

grid.

photograph was mea-

(Scion Corporation, Fred-

program measures distance with

the

ments by several steps

in the

10-cm grid

is

introduced into the measure-

in the

measurement process.

First,

of a branch is easily discerned in the

also necessary to define its point of origin.

tip

was

We

chose, as the point, the intersection of the branch with

the line running along the middle of its mother branch, and

had to be identified in each image. Second, the

branches are curvilinear structures and were measured as
that point

segmenteJ
created b\
line

lines.

Small differences

iation in the

segnu

in

measurements are

number and placement of those

o assess the magnitude of

Afferent observers

To reduce

the effects of grazing on the analyses, cases in

which growth was <0.0 were dropped from the data set. By
rejecting these cases of negative growth, the most severe
effects of grazing were eliminated. Grazing also may have
reduced the observed growth of some branches with posigrowth rates, but since scars from grazing heal rapidly,

tive

such branches could not be identified. Rejecting the negative values may have inflated the calculated growth rates of
branches that were otherwise not growing. Since the measurement error was 1.2 cm y '. some branches that had not

grown would, through measurement error alone, have small

negative growth rates, and some would have small positive
growth
0.0

rates.

cm y

'

Exclusion of branches with growth less than

the underestimates of the

would have eliminated

zero growth branches but not the overestimates.

measurement

measured each of 130

branches. The bctween-measurement standard deviation
was 0.3 cm.
variation, three

Negative growth rates

Statistical analyses

Growth

(ANOVA,

rates

were compared by analysis of variance

UNIANOVA and MANOVA. SPSS

functions

OCTOCORAL COLONY GROWTH

323

using the five independent variables: branch order, branch

grew at dramatically slower rates. This is evident in Figure

2A. where the rapid extension of daughter branches (e.g.,

type (mother or daughter), branch age. time interval in

branches 22. 23. and 24) near the

which the measurement was made, and colony height.

Branch length was also included as a covariate in some of

is in

version

10. 1).

those analyses, branches were classified

In

Due

and complexity of the data set.

it was
to
examine
all of the effects in a single
impossible
Our
was
to
conduct multiple tests, each
analysis.
strategy
the analyses.

to the size

including the greatest number of variables possible, and to

use Bonferroni corrections to significance testing when mul-

were conducted on the same

tiple tests

data.

Between-colony variation and tinu interval effects. The

same branches were measured multiple times, so a repeated1

ANOVA

measures

was

the

ever, because of the large

colonies, an

ANOVA

that included all five categorical vari-

ables could not be

measures design.
sures

most appropriate design. How-

number of branches nested within

computed within

We

a single

repeated-

therefore conducted a repeated-mea-

ANOVA that tested for the random effects of branches

within colonies and time interval (the 6-month interval in

which

the

measurement was made). The

effects of inter-

colony variation were again examined in an ANOVA of the

growth of branches that were less than 6 months old (i.e..

growth during the time interval

originated).

Growth

in

which the branch had

rates in this analysis

were compared

with respect to colony and time interval (Table

A). A
was used for this analysis, as data
simple two-way
from no single branch was included in more than one
I

ANOVA

marked contrast

many

of the branch were conducted (Table IB through IE). The

analysis was repeated for each of the four time intervals,
\\hich again led to a single branch being used only once in

each of the analyses.

ANOVA.

Multi-way
included

ANOVA

Finally, a multi-way

that

of the positive growth rates was conducted

all

using branch type, branch order, branch age, and colony

height as the independent variables (Table 2).
All of the
data.

ANOVA results are reported for untransformed

Growth

rates

were heteroscedastic even

of transforms (Levene's, Bartlett's or

F mM

after a variety
tests

of homo-

geneity of variances, depending on the structure of the data

results were almost always concordant with a
set).

ANOVA
ANOVA

parallel

using the rank transformed data and with

nonparametric analyses, which could only consider a single

factor at a time.

Results

General observations

tip of the primary branch

branches closer to the base,

of which exhibited no apparent growth (11,

simplified characterization of the results

is

13, 14).

that

mother

branches grew faster than daughters, new branches grew

faster than those that were more than 6 months old (Fig. 3),

and branches on small colonies had higher growth rates than

those on large colonies (Fig. 4A).
The measured rates of branch growth were highly vari-

from negative values to 17.8 cm y~'. Large

negative values were often associated with almost total
branch loss, as in the case of branch 10 in Figure 2A; this
able, ranging

colony, for instance, lost two branches between December

1998 and July 1999. The presence of some uncharacteristically short daughter branches that did not grow during the
study suggested that severely damaged branches did not
further. When cases of negative growth rates were
eliminated, the data set was reduced to 8341 individual

grow

growth

rate

measurements.

Statistical anal\ses

of branch growth

Between-colony variation and time-interval

effects.

The

repeated-measures analysis of variance was restricted to the

608 branches for which measurements were available from

observation in this analysis.

Between-colony variation and branch a^e. Next, a series

of analyses that simultaneously considered colony and age

to that of

324

H. R.

LASKER ET

AL.

and time interval on growth, significant

were detected when the data were partitioned
into subsets based on branch type. Separate analyses of the

effects of colony

fixed effects

5.0

growth of daughter branches during each of the time intervals (Table 1. B-E) identified a significant effect of branch
age

2.5

of the four time periods (C-E). There were

between colony and branch age in all

in three

significant interactions

four intervals, and significant colony effects in two of the

time intervals (Table

1.

Multi-way ANOVA. The

and E).
effects of branch type, genera-

and colony height were compared in this large

(Table
2). Branch growth rates were significantly
analysis
affected by branch type (growth rates of mother branches >
tion, age,

Colony Height (cm)

3), branch age (younger > older.

and colony height (short > tall. Fig. 4A). In addithere were significant interactions between branch type

daughter branches. Fig.

7.5

Fig. 3).
tion,

and age. branch type and colony height, and in the threeway interaction between branch type, order, and colony

5.0

height.

The

significant interactions reflect the nonlinearity

of the patterns seen

2.5

the different factors

in

Figures 3 and 4; that

is.

the effects of

were not additive.

Variation between colonies was differentiated

analyses due

_L

8
10
6
Branch Length (cm)
4

denotes mother branches. The figure presents a

daughter branches, and
random subset of the over 10.000 points.

four time intervals. (Branches were excluded from this

if they did not originate until later in the experiment, were lost during the experiment, or had a single
interval in which the measurement could not be made due to

analysis

poor photo quality.) The analysis identified significant effects of both time interval and colony, as well as an interaction of time interval by colony

The same

(all effects.

P < 0.001

).

was obtained when the analysis was restricted to the middle two time intervals, thereby allowing
the inclusion of a total of 2496 branches.

When

result

the growth rates of

in

these

computational limits of incorporating

colonies as a fourth independent variable with 260 degrees

of freedom (i.e., 261 colonies). Branches from the different

12

Figure 4. Growth rates of Pseudopterogorgia elisubethae branches

from San Salvador. Bahamas, as a function of colony height (A) and
branch length (B). Values in (A) are means
standard error. (B)
denotes

all

to the

newly originated branches were

analyzed separately, growth rates differed among colonies,

and there was also an interaction between colony and time
interval, but no significant effect of time interval alone

colonies were represented in almost all of the combinations

of branch type and age, which should have reduced the

confounding effects of not incorporating colony identity as

an independent variable. Time interval was not tested in
these analyses because it is confounded with the age of the
branches (i.e., the later intervals for a given branch also
record the growth of an older branch). Generation, which
was marginally not significant, was confounded with branch

type because first-generation branches are by definition

mother branches. When daughter and mother branches were

analyzed separately in an analysis that was otherwise identical to that in Table 2, generation was not a significant
factor (P = 0.52 and 0.40. respectively).

The

effect of branch age

on growth

rates of the

new

Figure 3 because it assumes

that new branches grew over a 6-month period. If we
assume that branches originated continuously over the

branches

is

underestimated

in

6-month interval, then the average age of a new branch

would be 3 months, and the mean growth rate would be
twice that reported in Figure 3. Furthermore, as noted in
Materials and Methods, excluding negative values from the

(Table 1A). These analyses indicate that the growth rates

between colonies, and that the magnitude of differ-

analysis has the effect of slightly inflating growth estimates

when the true value is near zero. When cases of negative

ences between colonies varied between time intervals, but

growth were included, older (>12 mo) daughter branches

had growth rates close to zero.

differed

was no simple additive difference in variance based on

time inu al ;\lone. For instance, one colony may have had
there

its

highest growth rates in one time interval, while a differ-

ent colon) had

iiv

Between-colon

lowest growth in the same time interval.

va: union and branch ai>e. Despite the

With branch length as a covariate, growth rates of daughbranches were analyzed separately for the effects of
branch age. Both increasing age and increasing branch
length (Fig. 4B) had significant negative effects on branch
ter

OCTOCORAL COLONY GROWTH

Table
Analysis of variance tables testing colony ami temporal variation

Source of variation

in

gnm-th rates of Pseudopterogorgia elisabethae at Sun Salvador, Bahamas

325

326

H. R.

Table 2
Analysis

<>/

branch ami

r.

>!

branch growth rates as a function of

..in.cteristics

uice of variation

LASKER ET

AL.

OCTOCORAL COLONY GROWTH

05

50

327

328

H. R.

LASKER ET

supply and self-interference. A number of realistic models

of form in rr^Jular organisms have been developed, in
is controlled
which grov
by the local responses of the
individu

modules

environment (Braverman,
1976; Colasanti and Hunt.

to their local

1974; Graus and Maclntyre,

1997; Kaandorp and Kiibler, 2001; Oborny et al. 2001).

However, the decrease in branch growth among P. elisa-

bethae colonies

is

best described as an age-dependent de-

crease. Thus, although the smallest branches had the greatest

growth

rates,

many

small branches also exhibited low

Furthermore, daughter branches stop growgrowth

while
ing
adjacent mother branches continue to grow, which
indicates that position alone does not control branch growth.
(Fig. 5).

size in P. elisabethae also appears to be determi-

Colony
and observations of determinate growth have been
reported among a wide range of colonial taxa. As noted,

AL.

colonies within the population. Mortality of colonies taller

than 40 cm increased from 0.0% to 40.2% when Hurricane

Floyd, a Category 4 hurricane, struck San Salvador on 13

September 1999. Although mortality events generated by

such storms reduce the number of large colonies, the distribution of colony sizes observed in the San Salvador
population would require that the mortality of large colonies
be high almost every year, not only in those occasional
years with severe hurricanes. Decreased growth rates among
the

larger colonies appears

to

be a more parsimonious

explanation of the size-frequency distribution. Because aging and size are often correlated and the ages of most of the
colonies were not known, the causative variable

is

difficult

to distinguish.

nate,

octocoral descriptions often include

maximum

colony sizes

Bayer, 1961), and decreasing growth with increasing

colony size has been reported for a number of gorgonians
(i.e..

(Grigg, 1974; Velimirov, 1975; Mitchell etui.. 1993:

et al.,

1998; Cordes et ai,

2001).

Among

Coma

scleractinian

determinate growth of independently growing

branches generates colonies with determinate form and size
(Rinkevich, 2002). Among botryllid tunicates, groups of

corals,

zooids, referred to as systems, undergo synchronous senes-

cence (Sabbadin, 1969), and whole colonies, including iso-

from a common source, undergo simultaneous senescence (Milkman. 1967; Rinkevich et al., 1992).
In addition, graptolite colonies are believed to have had
lated explants

determinate

growth

leading

to

distinct

species-specific

forms and sizes (Mitchell, 1988).

Maximum
growth. In a

size alone

manner

does not demonstrate determinate

Mothers and daughters

branches

Age. generation, and colony height all affect rates of

branch growth and origination, but the most striking differ-

growth of branches are those between mother

and daughter branches. The data indicate that the two
branch types are fundamentally different. First, mother
ences

in the

branches continue growing while adjacent branches stop

growing; for instance, compare branch 2 to branches 4 or 17
Figure 2A. The self-shading effects hypothesized for
Plexaura homomalla (Kim and Lasker. 1997) do not acin

count for the continued growth of mother branches, which

were otherwise indistinguishable from daughter branches.

Second, mother branches exhibit high growth rates as soon

as they originate, well before they have produced their first
daughter. However, the two branch types are not immutable.

When

colonies are damaged, branches that were previously

functionally equivalent to determinate

growth, modular organisms may also stop growing, not

according to a genetically determined developmental plan,
but due to size-dependent interactions between the colony

and environment, such as the balance between nutrient

daughter
branches

Kim

and Lasker (1998). Taxa

that exhibit

growth patterns

octocorals (McFadden. 1986;

Kim and

Lasker, 1997) and

begin

to

and generate

extend

new

be essential to our understanding of developmental pro-

uptake and metabolic rates. Size-dependent change in colony growth that is mediated by metabolic rate and resource

consistent with these simple models have been reported in

branches

(Castanaro and Lasker, 2003). Understanding

whether and how branches "become" mother branches will

cesses

capture has been modeled by Sebens (1982. 1987) and by

two developmental classes of

among

these colonial organisms.

Applications

Modular growth
strategy,

is an especially advantageous growth

transplants are used to remediate populations
2000), when explants are used as stock in

when

(Rinkevich,

tunicates (Holyoak, 1997).

mariculture, and for the sustained harvest of wild popula-

Ecological processes such as size-dependent mortality

also could generate a maximum colony size and the appear-

tions

ance of determinate growth. Colonies are susceptible to

being knocked over because drag forces increase with colony size and bioerosion weakens the substratum around the
holdfast (Birkeland. 1974). During most of this study,

how-

where colonies

are

natural

1998).

branches from colonies.

6 months: 0-10 cm, 0.146: 11-20 cm, 0.108; 21-30 cm,

size

31-40 cm, 0.048; >40 cm,

mortality would have

That pattern of
led to the accumulation of large
(1.000).

at regular

time intervals

products called pseudopterosins (Mayer et al,,

Material for extraction is collected by cropping

ever, mortality decreased with colony height (mortality per

0.071;

cropped

(Castanaro and Lasker. 2003). Understanding the pattern of

growth of P. elisabethae colonies is particularly important
because this species is harvested and extracted for a class of

If

growth

is

inversely related to the

of the colony, then reductions

enhance growth and productivity.

lations, this

suggests that colonies

in

Among

colony size will

harvested popuat an

can be maintained

OCTOCORAL COLONY GROWTH

optimal size, and that naturally occurring populations might
recover from disturbance at rates greater than the growth
rates

observed before the disturbance. Alternatively,

there

if

2), the

329

National Undersea Research Center

at the

Caribbean

Marine Research Center (grants 99-301 and CMRC-99NRHL-01-OIC), and the National Geographic Society

component
growth regulation (i.e..
and
then
Connell.
1987),
recovery from either anHughes

(#?968-97). Equipment for scuba diving was provided by a

thropogenic or natural disturbance may not be as great as

suggested by colony size alone. Detailed understanding of

We

is

also an age-based

colony growth patterns

species

is

to

is

essential to determining

Sherwood Scuba to the University at Buffalo.

Buchan and the staff of the Gerace
Research Centre for their assistance in the Bahamas.

grant from

also thank K.

whether a

suitable for sustained harvesting and whether

remediation following anthropogenic disturbance

is

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thank C. Gutierrez-Rodriguez, T. Swain, and the staff

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Reference: Biol. Bull. 205: 331-338. (December 2003)

<D

2003 Marine Biological Laboratory

Amino Acids

Possible Roles of Sulfur-Containing

in a

Chemoautotrophic Bacterium-Mollusc Symbiosis

JOANNA

L.

JOYNER

-*,

SUZANNE

M. PEYER,

AND RAYMOND

W. LEE

School of Biological Sciences. Washington State University, P.O. Bo.\ 644236. Pullman.
Washington 99164-4236

Abstract.

onts face the unique challenge of

supplying their

with hydrogen sulfide while avoiding

sulfur-containing free

may

toxin hydrogen sulfide (Fenchel and Riedl, 1970; Felbeck et

1983). Hydrogen sulfide diffuses
ai. 1981;

Invertebrate hosts of Chemoautotrophic symbi-

amino

its

Cavanaugh,

symbionts

toxic effects.

The

freely across respiratory surfaces

function in sulfide detoxification by serving as sulfur

storage

compounds

symbiont and

or as transport

compounds between

host. After sulfide exposure,

both taurine and

thiotaurine levels increased in the gill tissues of the

otic coastal bivalve

globin oxygen affinity (Carrico et ai. 1978). Consequently,

animals living in these environments require physiological

symbi-

Solemya velum. Inhibition of prokary-

mechanisms

metabolism with chloramphenicol, inhibition of euand inhibition of

karyotic metabolism with cycloheximide,
reduced
sulfoximine
ammonia assimilation with methionine

otic

levels of sulfur-containing

amino

cope with hydrogen sulfide toxicity.

that harbor symbiotic Chemoautotrophic

bacteria also require sulfide. for their symbionts utilize the

chemical
by hydrogen sulfide oxidation to

acids.

fix

energy generated
carbon dioxide into carbohydrates (Felbeck

et ai.

1981;

Cavanaugh, 1983). The invertebrate host

Ruby
delivers hydrogen sulfide and oxygen to its symbionts and
et al, 1981:

absence of metabolic inhibitors, estimated rates of

sulfide incorporation into taurine and thiotaurine accounted
In the

relies

for nearly half of the sulfide removed from the medium. In

contrast, amino acid levels in the nonsymbiotic, sulfide-

upon

the

symbiont-produced carbohydrates as a
1981; Southward et al.. 1981:

source of nutrition (Ran.

Felbeck, 1985). This type of symbiotic relationship exists in

over 100 species from at least five invertebrate phyla (Ca-

Geukensia demissa and Yoldia limatula

did not change after sulfide exposure. These findings sugtolerant molluscs

amino acids function

to

Invertebrates

Chloramphenicol
treatment inhibited the removal of sulfide from the medium.

gest that sulfur-containing

and therefore cannot be

excluded from tissues (Denis and Reed, 1927; Julian and

c oxidase
Arp. 1992). It also reversibly inhibits cytochrome
hemodecreases
and
Nicholls,
1975)
(Lovatt Evans, 1967;

acids taurine and thiotaurine

vanaugh, 1994).

in sulfide

Sulfide detoxification and transport strategies have been

well studied in the coastal protobranch bivalves of the genus

detoxification in symbiotic invertebrates, and that this process depends upon ammonia assimilation and symbiont

Solemya. which harbor approximately

metabolic capabilities.

10

intracellular

Chemoautotrophic symbionts per gram wet weight of gill

tissue (Cavanaugh. 1983: Felbeck. 1983). These clams gen-

Introduction
Aquatic habitats such as deep-sea hydrothermal vents,
sites
eelgrass beds, and sewage outfall

mangrove swamps,

form U- or Y-shaped burrows in shallow-water retheir

ducing sediments and pump oxygenated water through
burrows (Frey. 1968), which enables them to simulta-

tend to be characterized by high levels of the metabolic

needed for
neously acquire the dissolved oxygen and sulfide

erally

chemoautotrophy.

two
detoxify sulfide. Solemya velum and 5. reidi,
utilifrom
several
use
well-studied species,
strategies apart

To

Received 30 January- 2003; accepted 12 September 2003.

Current address: Department of Zoology, University of Florida. 223
Bartram Hall. Box 18525. Gainesville, Florida 3261 1-8525.
1

zation by their symbionts. For example.

To whom correspondence

Abbreviations:

MSX,

should be addressed: jjoyner@zoo.ufl.edu

methionine sulfoximine;

ASW.

artificial

S.

velum has two

binds oxygen, and a

types of cytoplasmic hemoglobins: one
form ferric hemoto
sulfide
second, which combines with

seawater;

FIA. flow injection analysis; POS. polarographic oxygen sensor.

331

332

J.

globin sulfide.
onts (Doeller

may mediate
it!.,

symbi-

of sulfide exposure on levels of hypotaurine (2-aminoeth-

The sulfide-binding form of

anesulfinic acid), a possible precursor to taurine and thio-

sulfide transport to the

1988).

JOYNER ET AL

L.

not present in S. reidi (Kraus et

ai, 1992); rather, sulfide oxidation occurs in the mitochon-

cytoplasmic iiomoglobin
hernatin,

dria,

is

and sulfide-oxidizing bodies (Powell and

Somero, 1985, 1986; Powell and Arp, 1989).

were also examined. Host- and symbiont-specific

metabolic inhibitors were used to distinguish the roles of the

taurine,

velum host and its symbionts in ammonia assimilation

and the maintenance of taurine, thiotaurine, and hypotaurine

S.

Synthesis of sulfur-containing free amino acids may be

an additional strategy for sultide detoxification and transport

pools. Flow-through respirometry studies

chemoautotrophic symbioses (Alberic and Boulegue,

1990; Conway and McDowell Capuzzo, 1992; Pranal et til.,
1995; Pruski et ai. 1997). In many marine organisms,

determine whether sulfide consumption

in

including solemyid clams and

some deep-sea symbiotic

is the domiamino acid (Alberic and Boulegue, 1990; Conway

species, taurine (2-aminoethanesulfonic acid)

nant free
c/

Conway and McDowell Capuzzo,

1992;

til..

1995; Lee et ai. 1997; Pruski et

et al.,

til.,

with

S.

ations in

velum

to quantify rates

amino acid

of

is

flux

and

to

related to fluctu-

pools. In parallel studies,

correlations between sulfide and free

two

were conducted

ammonia

we

amino acid

tested for
levels in

nonsymbiotic bivalve species: the estuarine mussel Geukensia demissa and the protobranch bisulfide-tolerant,

valve Yoldia limatula.

1992; Pranal

1997). Intracel-

amino acid pools function in cell volume regula(Pierce, 1982; Yancey et ai, 1982; Rice and Stephens,

Materials and Methods

lular free

tion

1988). and the primary function of taurine

may

Specimen collection and maintenance

be as a

osmolyte, a function which is conserved

throughout the animal kingdom (Allen and Garrett, 1971;
Huxtable. 1992).

Solemya velum Say, 1822, Geukensia demissa (Dillwyn,

compatible

The exceptionally high levels of taurine and the related

amino acid thiotaurine (2-aminoethanethiosulfonic acid) in
several chemoautotrophic symbioses have

prompted invesamino acids.

tigators to consider additional roles for these

For example, taurine appears

monia assimilation

in S. reidi

involved in sulfur cycling

to be an

(Lee

in 5.

end product of amand may be

et ai, 1997),

velum (Conway and Mc-

Dowell Capuzzo, 1992). Similarly, thiotaurine may function

deep-sea symbiotic bivalves (Alberic
1990; Pruski et al., 2000; Pruski. 2001).

in sulfur cycling in

and Boulegue,
Taurine and thiotaurine may serve as important sulfide
storage compounds, allowing 5. velum to maintain low
levels of intracellular sulfide.

Under conditions where

sul-

low or absent, taurine and thiotaurine may provide

fide is

mitochondria! and symbiont sulfide oxidation

Several
pathways.
species of aerobic gram-negative soil and
enteric bacteria use taurine as a sulfur, carbon, and nitrogen
sulfide

to

source (Stapley and Starkey, 1970; Smiley and Wilkinson,

1983; Seitz et al., 1993; King and Quinn, 1997; Chien etui.,
1999;

Cook

et ai,

in

the

gram-negative chemoautotrophic symbionts. Additionally,

taurine and thiotaurine production could facilitate sulfide
detoxification under anaerobic conditions, thereby preventing deleterious effects such as the reaction of sulfide with

metalloproteins. This strategy

would be

particularly bene-

burrow environment of solemyid clams,

which oxygen and sulfide levels vary.
in

the

In the present study,

and thiotaurine levels

upon exposure

Marine Resources Center of the Marine Biological Laboratory (Woods Hole. MA) and shipped on the day of
the

collection. In our laboratory the

up

to 5 days

in a

clams were maintained for

flow-through respirometry system (see

http://www.wsu.edu/^-rlee/respirometer/respi rometer.htm
and Fig.
In each experiment, the temperature of the
).
I

respirometry system was regulated to match the ambient

water temperature at which the clams were collected, which

ranged seasonally from 5 to 15 C over a nearly 2-year

period. These temperature differences did not affect the free

amino acid composition. Consumption of sulfide, oxygen,

and ammonia was measured in a subset of experiments
conducted in the late spring and at 15 C. Solemya velum
and
limatula were maintained in 0.45-ju.m filtered, 359ft
>'.

(ASW; Instant Ocean, Aquarium SysMentor, OH) supplemented with 50 ju,M ammonia.

artificial

tems,

seawater

Geukensia demissa was maintained

in filtered,

100 rpm).

ASW

30%r

ASW

ASW

solution in

stir

bars (60 to

supplemented with 50 i^M ammonia. The

the chambers was kept mixed with magnetic

flow rates ranged from 0.18 to 0.19 ml

1999; Reichenbecher and Murrell, 1999),

and similar metabolic pathways may be present

ficial

1817). and Yoldia limatula (Say. 1831) were collected by

we

tested the hypothesis that taurine

in intact gills

to sulfide,

in

of

5.

velum increase

which would occur

if

acids are involved in sulfur storage or cycling.

these

amino

The

effects

Maintaining the clams in a flow-through respirometry

system allowed for constant monitoring of experimental
conditions.

The oxygen concentrations

in the

chamber

out-

flows were determined with a polarographic O 2 sensor

(POS), whereas the concentrations of sulfide and, in some

experiments,

ammonia were measured by flow

injection

analysis (FIA). Throughout this paper, sulfide refers to

SHiS (primarily the sum of HS~ and H : S), and ammonia
+

sum of NH 3 and

NH 4

Outflows were

refers to

SNH,

pumped

directly through a flow cell containing a sulfide-

(the

).

POS (Orbisphere 2120, Geneva) followed by a series of FIA injection sample loops
insensitive gold microcathode

SULFUR AMINO ACIDS

IN

SOLEMYA VELUM

333

334

6300 amino acu;

from Lee and
(1:30,

1%

Becki

i.

alyzer following a protocol modified

(1988). The samples were diluted

ai

by vol

thu

JOYNER ET

L.

J.

H 2 O. 1%

Li-S buffer (96.8%

ith

0.7% HC1, 0.5% benzoic

jul

or standard loaded onto the sample loops. 50 ju.1

of eacii solution was analyzed. The amino acids were sep-

of sa

arated in Li-A buffer

(98% H 2 O. 1% Li citrate. 0.5% LiCl.

Beckman Coulter) on a 10-cm ion

0.5% HC1; pH 2.8;

exchange column and
tion.

reacted in-line with ninhydrin solu-

Absorbances were monitored

at

570

nm

and 440 nm.

After preliminary experiments, only taurine, hypotaurine,

and thiotaurine were quantified. The standards were 200 /xA/
taurine (Sigma) and 20

tests

(Statistica).

Results

2.2:

CA). Of the 240-250

her. Inc.. Fullerton.

amino acid data were analyzed with two-sample

LiCl,

pH

acid;

AL.

Sulfide exposure increased taurine

in S.

velum hut not

and

thiotaurine levels

two nonsymbiotic bivalves

in

Specimens of Solemya velum exposed to sulfide had

= 0.0034) and thiotaurine
significantly more taurine (P
in their gill tissue than clams not so exposed
(P < 0.000
1

These values equate to average rates of change in

taurine and thiotaurine levels of 0.89 fimol
g~' wet
h"
and
h"
0.22
over
the
24-h
jumol
weight
g
(Table

).

'

incubation period. Hypotaurine levels were not significantly

(P = 0.064). Cysteine and methionine, two other

hypotaurine (Sigma) in Li-S

buffer. The thiotaurine standard was prepared by dissolving

affected

9H 2 O (Fisher
g hypotaurine and 0.05 g Na,S
Scientific. Fair Lawn, NJ) in deionized water, heating to 100
C. acidifying the solution with
HC1, and evaporating

sulfur-containing amino acids, were below the limits of

detection in all samples. Levels of the most abundant nonalanine. glutamate, and
sulfur-containing free amino acids

/u,A/

0.0011

the

solution

(Cavallini et

1963). This standard

/.,

by mass spectrometry.
The temperature of the column

was

verified

affected the separation of

thiotaurine from taurine and the reactivity of hypotaurine

with ninhydrin. Thiotaurine could be detected only

The values of hypotaurine reported here

at

70 C.

are only

from

analyses run at 45 C, because hypotaurine was not as

reactive with ninhydrin at 70 C. The temperature did not
affect taurine levels. Reported values for taurine are aver-

ages between levels detected at 45 C and 70 C.

Results are presented as the mean
the standard error
and as average rates of synthesis per gram wet weight over
a 24-h period [(amino acid level in

exposed
posed

to

'

g~

assuming the synthesis

sulfide)/24 h]

linear over the 24-h period. Differences

ANOVA

detected using one-way

velum samples

wet weight in clams

in clams not ex-

(amino acid level

to sulfide)

for

rate

was

among means were

each amino acid

(Statistica. Statsoft, Inc., Tulsa.

in S.

OK). The

appropriate comparisons were analyzed with Fisher's LSD

procedure (Statistica). The G. demissa and Y. limatitla

Table
Effects of sulfide exposure

and

inhibitors on tauriin; liypukiurine.

were unaffected by sulfide exposure (data not

shown). In preliminary experiments with sulfide-exposed
clams, free amino acid profiles of S. velum foot (symbiontaspartate

free)

and

Treatment

-Sulfide

were similar (data not shown), as

tissues

only taurine, hypotaurine, and thiotaurine levels in the symbiont-containing gill tissues of S. velum were quantified.
Gill tissue from the nonsymbiotic bivalve species Geukensia demissa and Yoldia limatitla contained less taurine

< 0.0001, non-sulfide-exposed) than S. velum (Table

but comparable levels of hypotaurine (P = 0.068) and
thiotaurine (P -- 0.648). The concentrations of these
(P

1),

amino acids were

exposed

levels in S.

To
onts,

the

same whether or

to sulfide (all

Metabolic

u/liihiturs

velum

comparisons,

not the bivalves

P >

decreased taurine and thiotaurine

investigate the role of the chemoautotrophic symbito

chloramphenicol, a specific

and thiotaurine

levels in gills of three bivalve species

Thiotaurine

Hypotaurine

+ Sulfide

-Sulfide

+ Sultide

-Sulfide

Solemya velum

Data are mean

'

SEM

in

/imol amino acid/g wet weight of

Significant differences (/'

Significant differences (P

gill tissue. (/(),

were

0.05).

gills

clams were exposed

Taurine

Species

gill

observed previously (Conway and McDowell Capuzzo,

1992). In subsequent experiments with metabolic inhibitors,

Number

of replicates;

MSX.

methionine sulfoximine.

0.05) between clams exposed to sulfide ( + Sulfide) and not exposed (-Sulfide).
0.05) between clams treated with inhibitor and not treated.

+ Sulfide

SULFUR AMINO ACIDS

inhibitor of bacterial protein synthesis
at

cycloheximide. a specific inhibitor of eukaryotic

exposed
protein synthesis (Burnap and Trench. 1989). at a concento

tration nontoxic to the host for the duration of the treatment.

ammonia asmethionine sulfoximine (MSX; Rees.

In additional experiments, the effects of the

similation inhibitor,

1987) were examined:

MSX

which has been detected

in

SOLEMYA VELUM

I ~

inhibitor

added
-

chloramphenicol

5 5

-1

>
u 1

-2

o>

v
-3

"o

co

-4

-5

sulfide

control

added

cycloheximide

inhibits
5.

335

(Burnap and Trench.

a concentration previously determined to disrupt

symbiont metabolism but to be nontoxic to the host (R. W.
Lee, unpubl.). To examine the role of the host, clams were

1989).

IN

glutamine synthetase.
velum tissues (Lee et al..

hours

Rees (1987). Taurine. hypotaurine. and thiotaurine levels in

clams not exposed to sulfide were not affected by exposure
to any of the inhibitors (Table 1). Additionally, the wet

MSX

To

tion, the

MSX

150

100

50

ensure complete inhibition of ammonia assimilalevel was 10-fold higher than that utilized by

1999).

stopped

weights of whole clams were not altered by treatment with

sulfide or metabolic inhibitors.

When clams were

itors, the

treated with the three metabolic inhib-

usual sulfide-induced increase

in taurine levels

was

sulfide
not observed (P values for comparisons between
and + sulfide. in the presence of inhibitors: chloramphenicol,

P = 0.552;

cycloheximide.

P = 0.451: MSX. P

Hypotaurine levels were not altered by sulfide

= 0.064). Thioexposure or treatment with inhibitors (P
taurine levels increased after sulfide exposure, even in the
0.707).

presence of metabolic inhibitors (P values for comparisons

between
sulfide and + sulfide in the presence of inhibitors:

P < 0.001; cycloheximide. P < 0.001:

MSX. P < 0.0001 This sulfide-stimulated increase, how-

chloramphenicol.

).

ever,

was reduced by treatment with chloramphenicol (P =

Figure

Effect of inhibitors

2.

on Solemya velum ammonia and

sulfide

Chloramphenicol (0.9 mA/). an inhibitor of prokaryotic protein

synthesis, blocked sulfide consumption. Cycloheximide (0.02 mA/). an
inhibitor of eukaryotic protein synthesis, had no effect. (B). Methionine
fluxes. (A).

sulfoximine

(MSX.

ammonia assimilation,
ammonia excretion. Negative

0.25 mA/). an inhibitor of

blocked ammonia uptake and resulted

fluxes denote uptake from the medium.

in

0.016. sulfide-exposed control clams versus chloramphenof symbiont metabolism decreased the sulfide-stimulated
thiotaurine synthesis. The maintenance of free amino acid

icol-treated sulfide-exposed clams).

Chloramphenicol and MSX decreased

and ammonia consumption

The average

rates of sulfide

S.

velum

pools depended upon the presence of functioning symbi-

sulfide

and oxygen consumption

in

the absence of inhibitors were 2.57

wet weight

SE

h~' [mean

0.06 (5) /nmol g~'

of
(n
experiments)] and

h~'. respectively. Chloramphenicol exposure completely halted sulfide consumption

after 10 h (Fig. 2A. hours 120-130). Cycloheximide treat3.98

0.01 (5) ju,mol

ment did not

with

MSX

g~'

affect sulfide

inhibited

consumption

ammonia uptake

rates.

(Fig.

Treatment

Discussion
This study demonstrates that taurine and thiotaurine lev-

Solemva velum

gills

symbioses.

The magnitude of changes

pools observed

increase after sulfide exposure.

in the

in

taurine

present study

is

and thiotaurine

sufficient for these

amino acids

to be physiologically significant sulfide storage

compounds.

In

sulfide for

2B, hours 10-

30).

els in

consumption, and host ammonia assimilation.

Thus, sulfur-containing free amino acids also may be a link
between cycling of nitrogen and sulfur in chemoautotrophic
onts, sulfide

24

which clams were exposed to

the increases in taurine and thiotaurine

experiments
h,

in

levels corresponded to synthesis rates of 0.89 ju.mol

g~'
wet weight h"' and 0.22 jLtmol g~' h~', respectively.
Since taurine contains one S atom and thiotaurine contains

two S atoms,

corresponds to a potential sulfide incorporation rate of 1.33 ;u,mol g~' h~'. The average rate of
whole animal sulfide consumption measured under the con-

These two amino acids may function as nontoxic sulfide

storage compounds. Inhibition of symbiont and host protein
synthesis and host ammonia assimilation blocked sulfide-

trol

stimulated taurine synthesis; in contrast, only the inhibition

al.,

this

(no inhibitor) conditions was 2.57 jumol

which

similar to the sulfide

rate

h '.
g
of Solemya

is
consumption
under similar experimental conditions (Anderson et
1987). Therefore, the contribution of taurine and thio-

reidi

336

L.

J.

to

50%

cycloheximide.

and

taurine to sulfide detoxification could account for

of the

total sulfido

r.

Treatment

MSX

Jiloramphenicol.

PR

leveK

up

increases in taurine

by the control clams. The lack of detectable

taurine x\iuhesis in chloramphenicol-treated clams likely

can be attributed to the chloramphenicol-induced cessation

of sulfide consumption. Additionally, chloramphenicol may

act to prevent synthesis of mitochondria! proteins that are
not nuclear encoded. Therefore, effects of chloramphenicol

treatment might also be ascribed to impairment of mito-

ments,

S.

However,

metabolism.

chondrial

in

preliminary

velum tolerated treatment with 0.9

at least

disrupted symbiont metabolism rather than toxicity to the

Treatment with cycloheximide, which inhibits eukaryprotein synthesis (Burnap and Trench. 1989) and is

host.
otic

functionally analogous to chloramphenicol. decreased taurine synthesis in the presence of sulfide, but did not affect

consumption. These results suggest that taurine is

synthesized by the host and that the cycloheximide treatsulfide

to

in

MSX

(Lee

results mirror those reported

1997),

who found

by Lee and coworkers

between

a direct relationship

ammonia

is

not directly involved in sulfide detoxifica-

hypotaurine
nor is it an intermediate

tion,

in

5.

velum (Cavallini

in the taurine
cr

al..

1976).

synthesis pathAlternatively,

hypotaurine could have protective functions, such as by

serving as a compatible osmolyte (Yin et

al.,

2000) or by

scavenging free radicals (Huxtable, 1992).

Thiotaurine levels were greater in the gills of sulfide-exposed clams than
exposure

to

in

non-sulfide-exposed clams, regardless of

metabolic inhibitors. Treatment with chloram-

phenicol reduced, but did not prevent, sulfide-induced thiotaurine synthesis. This reduction likely resulted

from the chlor-

of
sulfide
amphenicol-induced cessation
consumption.
Inhibition of host metabolic activity with cycloheximide did
not affect thiotaurine levels in sul fide-exposed clams. Thiotaurine

may

be produced abiotically in host tissues, in which case

host enzymati.

MSX-inducecJ
levels in
sure.

bivalves maintain free

medium in the experiments presented here, suggesting that solemyid clams synthesize taurine and thiotaurine. The biosynthesis pathways of taurine and thiotaurine
incubation

in

solemyid clams are unknown, but the results from

study suggest that these pathways require

ilation, sulfide

this

ammonia assim-

consumption, and active symbiont metabo-

lism.

Ammonia

is

present at elevated levels in the burrow

environment of solemyid clams (Lee et al., 1992; Krueger,

1996). and the clams assimilate it into amino acids, which

may

then serve as precursors for sulfur-containing amino

acids (Lee and Childress. 1994).

that

glutamine synthetase

assimilation pathway, since

is

The present study indicates

enzyme in the

the primary

MSX treatment blocked ammo-

1987). The product of ammonia assimilation, glutamate, can

then be used as a precursor in the production of taurine,
which is a major product of ammonia assimilation in S. reidi

availability and taurine levels in S. reidi.

levels
were not altered by exposure to sulHypotaurine
fide or the metabolic inhibitors. These results suggest that

way

known whether symbiotic

not

blocked ammonia assimilation, probably contribMSX-treated clams.

et al.,

external

It is

nia uptake and caused

affect

uting to the decreased taurine levels in

These

velum

any sulfide consumption which may

host tissues (Powell and Somero, 1986). Exposure

ment did not

occur

in S.

amino acid pools by absorbing amino acids from the environment or by synthesizing them. We do know that S. reidi
can take up free amino acids from sediment interstitial water
(Lee et al., 1992). However, sulfur-containing amino acids
were not detected in the pore water samples from 5. reidi
burrows (Lee et al., 1992) and were not present in the

experi-

mM chloram-

9 days, suggesting that the effects due

to chloramphenicol treatment are most likely the result of
phenicol for

Taurine and thiotaurine synthesis

i\

the sulfide-induced

JOYNER ET AL

inl

Again, these

al.,

1997; thiotaurine production was not

tested for). Therefore, in

study,

S.

velum, taurine and thiotaurine

response to sulfide, as demonstrated in this

be facilitated by the ability of the bacterium-

in

production

may

mollusc association to synthesize glutamate from inorganic

nitrogen.
Just as glutamate likely contributes organic nitrogen to

the synthesis of taurine and thiotaurine, the probable source

of organic sulfur is cysteine. All of the demonstrated taurine

mammalian and

synthesis pathways in

incorporate

(Jacobsen

cysteine

and

invertebrate tissues

Smith.

Jr..

1968;

Bender, 1975; Bishop et al., 1983; Huxtable. 1992), which

apparently cannot be synthesized by molluscs (Bishop et al..
1983). Although

cysteine

sources

some
to

intertidal

maintain

molluscs

taurine

Awapara. 1960; Jacobsen and Smith.

utilize external

pools (Allen and

1968; Allen and

Jr..

1972; Bender. 1975), it is unlikely that solemyid

clams take up cysteine from their environment (Lee et al..
1992). Therefore, the most likely source of cysteine or other
Garrett,

taurine precursors is the symbiotic bacteria. Gram-negative

bacteria cannot synthesize hypotaurine, taurine, or thiotaurine (Jacobsen and Smith. Jr., 1968; Huxtable, 1992). but

clams increased following sulfide exponil

suggest that thiotaurine may be

can make cysteine (Kredich, 1996). Cysteine synthesis in E.

coli requires sulfur in the form of sulfide or thiosulfate

''.ion

re

tissues (Lee et

excretion, similar to what

an algal-cnidarian symbiosis (Rees,

in

may not be necessary. Despite the

of ammonia assimilation, thiotaurine

pathways

MSX-u

was demonstrated

ammonia

produced abiotically from precursors already present

tissue and depend less upon ammonia availability.

in the gill

(Stauffer,

1996) and assimilated

glutamate (Reitzer,

ammonia

in the

form of

1996). Translocation of the essential

SULFUR AMINO ACIDS

Environment

IN

gram

Host

337

SOLEMYA VELUM
to

R.W.L. and

the

Western Society of Malacologists

to

J.L.J.

NH

-*
3

Glutamate

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Taurine

Allen. J. A.,

Thiotanrine

Allen, J. A.,

Figure

3.

in
Proposed model of taurine and thiotaurine biosynthesis

Solfin\;i vfliiin.

The clams

einitonmcm. Ammonia

extract

ammonia and

sulfides

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with
metabolism
of
The
inhibition
3.
symbiont
Figure

its

in

to a loss of cysteine

may equate
metabolism, thus decreasing sulfide consumption and taurine and thiotaurine synthesis by the host. Ammonia limitation, either by MSX treatment or reduced exogenous am-

chloramphenicol, therefore,

monia resources, may

velum

limit

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S.

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3).

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clams

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in

this

study and

in

Thus, taurine and thiotau-

be a link between nitrogen and sulfur cycling in

sulfide
chemoautotrophic symbioses and serve as nontoxic
rine

may

storage and transport compounds.

patterns in

The absence of

nonsymbiotic sulfide-tolerant molluscs

similar

(Geuken-

demissa and YolJin limatula) suggests these functions of

be limited to symsulfur-containing free amino acids may

sia

E.,

.1.

Childress,

J.

and

J.

thank Gerhard

Munske of Washington

State Univer-

We
conducting the amino acid analyses.
J.
Michael
also thank David Julian, Stephanie Wohlgemuth.
comfor
reviewers
helpful
Greenberg, and two anonymous
sity for his aid in

of
manuscript. The Marine Resources Center

Woods Hole

collected

Marine Biological Laboratory

of the clams and provided the information on water
Technical Services
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<D

2003 Marine Biological Laboratory

Localization of a Symbiosis-Related Protein, Sym32, in

the Anthopleura elegantissima-Symbiodinium
muscatinei Association
A.

J.

SCHWARZ* AND

Department of Zoology, 3029 Cordtey

Hull.

Oregon

V.

M. WEIS

Introduction

Cnidarian-dinoflagellate symbioses are widethe

marine environment. Growing concern over
in
spread
the health of coral reef ecosystems has revealed a funda-

Abstract.

mental lack of knowledge of

how

tions are regulated at the cellular

cnidarian-algal associa-

and molecular

level.

We

are interested in identifying genes that mediate interactions

between the

partners,

as a model.

We

ganisms and animal hosts

actions ranging from parasitic to mutualistic. Most eukaryotic microbes that infect animals reside within a membrane-

bound

protein, that

is

previ-

namic process

was

located in vesicles

the symbiont

specific

to

detect

the

We

may

also produce a fasciclin

suggest that host

vesicles to the

domain

cells

a dy-

by active invasion of

for example, the parasite

Symbiodinium

(Fitt

and Trench, 1983).

microbe

fails to

the phagocytic
ganelles that direct the vacuole through
is that the microbe
process (Mellman, 1996). The result

successfully transforms what

protein.

relocated from gastrodermal

sym32 is
symbiosome membrane when symbionts

is

in the normal
actively interferes with one or more steps
and endocytic orprocess of fusion between the vacuole

target

Symbiodinium bermudense cells. Westreof

ern blots
proteins from two Symbiodinium species
that
48
and
kDa,
45
of
vealed a protein doublet
suggesting
protein in cultured

the symbionts

host

continue through the complete endoin acidification of the vacuole

cytic pathway that culminates
and fusion with lysosomes. Instead, the microbial inhabitant
the

also present in the accumulation

sufficiently

that is initiated either

of

the vacuole

Studies on eukaryotic microbial parasites show that, in

the vast majority of cases, the nascent vacuole that contains

bodies of the symbionts. Although the anti-sym32 anti-

serum was not

cytoplasm

by the microbe
or by
(Dobrowolski and Sibley, 1996)
for
cell
host
the
phagocytic uptake of the microbe by
and
et
/..
2002)
example, the parasite Leishmania (Courret

that occur along the apical edges of gastrodermal cells. In

but sym32 was
symbiotic hosts, such vesicles were absent,
membranes. Sym32 (or a
present within the symbiosome

cross-reactive protein)

the

Toxoplasma

and aposymbiotic A. elegantissima. Here, we describe the

subcellular localization of the sym32 protein. In aposymbi-

sym32 was

within

the host cell

differentially expressed in symbiotic

otic (symbiont-free) hosts,

vacuole

(Hackstadt, 2000). The formation of

a fasciclin
ously described a host gene, sym32. encoding

domain

between eukaryotic microorencompass a wide range of inter-

Intracellular associations

and we are using the temperate sea

anemone Anthopleura elegantissima

Oregon 97331

State University, Comillis.

sosome

are

into a

compartment

would have been

a phagoly-

that is hospitable for

growth,

into various life-history stages

replication, or differentiation
(Hackstadt, 2000; and for examples, see Mukkada et <//..

taken into host cells by phagocytosis.

1985; Sinai and Joiner, 1997).

In the marine environment, associations between animals
and eukaryotic microbes include mutualistic as well as

One prevalent mutualistic association

occurs between cnidarians (most commonly sea anemones

parasitic interactions.
Received 14 April 2003; accepted 27 August 2003.
* To whom
Decorrespondence should be addressed. Current address:
Fairchild Building, 299
partment of Microbiology and Immunology, D305
Drive, Stanford University. Stanford. CA 94305. E-mail:

and corals) and photosynthetic dinoflagellates (usually Symbiodinium spp.). These associations are characterized
host and
by reciprocal nutritional interactions between

Campus

jschwarz@stanford.edu

339

340

J.

symbiont (FullD'Elia, 199K

r;

19'

Muller-Parker

and

symbionts contribute
ganic compounds to host metabolism
1971: Battey and Patton. 1987; Musca:

(Lewitine,

1984;

<//..

SCHWARZ AND

,>hotosynthetic

'

glycerol

ci

ski

A.

;.

ith.

and the host contributes nitrogen (Wang and

Douglas. 1998, 1999) and carbon dioxide (Weis, 1993) for

algal photosynthesis. These associations occur in the photic

zones of both temperate and tropical benthic habitats, and

they thrive in nutrient-poor tropical marine environments

because they conserve and recycle nutrients.

In its associations with dinorlagellates, the host cnidarian

most commonly houses symbionts within gastrodermal

cells, in

a vacuole of phagosomal origin.

The

initial infec-

during which the dinoflagellate symbionts are internalized, typically occurs when dinorlagellates that enter the
tion,

host's

mouth

are taken into phagocytic gastrodermal cells

that line the gastric cavity (Colley

and Trench, 1983: Schwarz

et ai.

and Trench, 1983;

1999, 2002).

Fitt

The phago-

some, through unknown mechanisms, fails to fuse with

lysosomes (Fitt and Trench. 1983). and the dinoflagellates

remain undigested

within

the

dinoflagellates reside within a

multiple

membranes

vacuole.

Ultimately,

the

compartment delineated by

(Taylor. 1968. 1987; Wakefield et ai.

2000). Historically, the origin of the multiple membranes

surround the symbiont has been uncertain. Recently,

that

immunolocalization

studies

using

host-specific

and

monoclonal antibodies suggest that

only the outermost membrane originates from the host, and
all of the inner membranes originate from the dinoflageldinoflagellate-specific

V. M.

WEIS

one and four repeats of three highly conserved "fasciclin"

domains that are believed to function as adhesive domains
in cell-cell

ai, 1998).

and cell-extracellular matrix interactions (Hu

this family have been identified

Members of

et

in

organisms as diverse as mycobacteria (Harboe and Nagai,

1984; Harboe et ai, 1995; Terasaka et ai, 1989), Volvox

(Huber and Sumper. 1994), insects (Zinn et ai, 1988), sea

urchins (Brennan and Robinson. 1994), and humans
(Skonier

To

et ai,

1992; Takeshita et ai, 1993).

further investigate the role played by s\in32 in

symbetween cnidarian hosts and dinoflagellate symbionts, we used immunocytochemical techniques to

examine the distribution of the sym32 protein within the A.
biotic interactions

elegantissima-S. inuscatinei association. In this paper

demonstrate

that

sym32

protein

is

we

differentially distributed

symbiotic and aposymbiotic A. elegantissima both at the

cellular and subcellular level. Of particular interest, sym32
in

antiserum labels the multiple layers of membrane that surround the symbiont within the host cell. We also show that

anti-sym32 antiserum specifically labels the accumulation

body of dinoflagellates residing within host gastroderm.
Western blots of proteins from two Symbiodiniitin species
revealed a protein doublet of 45 and 48 kDa (45/48 kDa)
that cross reacts with the sym32 antiserum; but in immu-

was insufficiently specultured specimens of S.

nolocalization studies, the antiserum

cific to detect the target protein in

bermudense. Possibly, both the host and symbiont produce

fasciclin

domain

proteins that interact via the fasciclin ad-

hesive domain.

perhaps accumulating from repeated cycles of ecdysis

within the host vacuole (Wakefield and Kempt'. 2001 ).
lates,

We

have been interested

in

play roles in symbiotic interactions in cnidarian- dinoflagel-

symbioses (Weis and Levine, 1996; Weis and Reynolds. 1999; Reynolds et ai, 2000). We have used as a model
late

system the temperate symbiotic sea anemone Anthopleura

elegantissima (Brandt. 1835), which is abundant in the
intertidal region

of the eastern Pacific, from Mexico through

Alaska. This species is able to form associations with two

species of dinoflagellates, Symbiodiniiim californium and S.
inuscatinei.

The presence of one or more of

the symbionts

within a particular host depends upon microhabitat differences that are created along temperature and light gradients
that

occur along latitudinal and

Oregon

coast,

intertidal ranges.

Along

the

most hosts contain only a single symbiont,

and Trench, 2000).

Using biochemical and molecular approaches, we have

in

Animal maintenance
Symbiotic and aposymbiotic specimens of A. elegantissima were collected at low tide from the intertidal zone at

Seal Rock, Oregon. Aposymbiotic anemones were taken

from under rock overhangs or crevices where there was
little

no

or

light

to

support

the

growth of symbiotic

dinoflagellates. Symbiotic anemones were taken from the

open rock benches that are exposed to light. Anemones were

transported to the laboratory, where they were maintained in

an 11 C recirculating seawater aquarium on a 12:12 h
light:dark cycle. Anemones were fed
adult brine shrimp about once a week.

previously frozen

S.

inuscatinei (LaJeunesse

number of

host genes that are likely to function

mediating host-symbiont interactions (Reynolds et al.,

identified a

Materials and Methods

identifying host genes that

Light-level immunocytochemistry

Anemones. Tentacles from both symbiotic and aposymspecimens of A. elegantissima were clipped and im-

biotic

2000; Weis and Levine, 1996; Weis and Reynolds, 1999).

of these, s\m32. encodes a protein that belongs to a

mediately transferred to tissue freezing medium (Triangle

Biomedical Sciences) and frozen at -80 C. Tentacles were

domain pro2000). Fasciclin domain proteins

share low overall sequence identity, but all possess between

cryosectioned

One

class of cell adhesn>n proteins called fasciclin

teins

(Reynolds

et ai.

(20-jum

at

sections)

immersed

in

4%

20

on a Reichert-Jung cryostat

and placed on polylysine

paraformaldehyde

slides,

fixative in

then

phosphate

SYM32 LOCALIZATION

mM

buffered saline (PBS: 10

mM

150

NaCl)

for 1.5 h.

min each

three times, 5

75%. 50%. 25%): and

were incubated

a blocking solution of 1:200 dilution of

in

30 min at room temperature and

5 min each time, in PBS/BSA.

Slides were incubated for

h in either a 1:2000 dilution of

antiserum from rabbit (antibody development de-

2000)

PBS/BSA

scribed in Reynolds
dilution of preimmune serum from the
</ at..

were

BSA;

with 0.5%

PBS/BSA. Sections

rinsed again in

goat serum:PBS/BSA for

then rinsed three times,

sym32

PBS

time, in

methanol series (25%, 50%, 75%, 100%,

in a

dehydrated

phosphate buffer, pH 1.2. +

Sections on slides were rinsed

rinsed three times, 5

then incubated for

IgG-5-nm

h in a

in

min each
1

same

or in a

:2000

rabbit. Sections

time, in

PBS/BSA and

:200 dilution of goat anti-rabbit

were

colloidal gold conjugate (Ted Pella). Slides

rinsed as above. Gold particle labeling

was silver-enhanced

kit (Ted Pella). To stop color

were washed in ePure water. Coverslips

using a silver enhancement

slides

development,
were affixed with a glycerol mount and sealed with finger-

ANTHOPLEURA

IN

capsules in fresh

polymerize

at

52

341

LR White. LR
C for 2 days.

White was allowed

Ultra-thin, gold-silver sections were cut with a diamond

knife and placed onto Formvar-coated nickel grids. These
grids were processed for immunocytochemistry as follows:

they were immersed in blocking solution (PBS

for 15 min, incubated in a 1:1000 dilution of

1.5

h,

sym32

anti-

0.1% Tween

20, incubated in a 1:75 dilution of

goat anti-rabbit

PBS

+ 5% BSA)

1000 dilution of preimmune serum in PBS for

rinsed 3 times. 10 min each time, in PBS/BSA +

serum or a

for

lgG-15-nm

h, rinsed as

EM

grade

colloidal gold (Ted Pella) in

above, rinsed

in

ePure water for 5

min, and then allowed to dry. Grids were stained in 2%

uranyl acetate for 5 min. rinsed by dipping in water 10 times

each

in three

0.4% lead

changes of water, then immediately stained

in

acetate for 3 min, with water rinses as above, and

then air dried. Between 5 and 10 grids of each type of

anemone were viewed under 60 kV using a CM- 12 Phillips

transmission electron microscope.

Cultured Symbiodinium bermudense

nail polish.

to

cells.

Cells were

Cultured Symbiodinium bermudense cells. Immunofluorescence was used to investigate whether the symbiont-

collected as described in the light microscopy section, fixed

as described for anemone tentacles, and processed essen-

produced 45/48 kDa cross-reactive protein (identified from

Western blots, described below) could be localized to symbionts free from host cells. Cells of 5. bermudense

tially as

(CCMP830) were grown

with f/2-Si media

at

h dark

at

light to 12

in sterile filtered

seawater enriched

about 100 p.E light on a cycle of 12 h

25 C. Cells were collected by centrif-

ugation from liquid media and resuspended into 3% paraformaldehyde in PBS. After fixation for 30 min, cells were
given two 10-min washes in PBS and transferred to PBS.
Cells were incubated 30

min

in

blocking solution (PBS

described for

anemone

tentacles. Several dilutions

of anti-sym32 antiserum and preimmune serum were tested

(1:10, 1:50, 1:200. 1:500, 1:1000, 1:2000), as well as vari-

ous dilutions of either Tween-20 or Triton

1%)

in the

(0%, 0.1%.

following solutions: blocking solution, primary

antibody solution, wash solution.

Preparation of anemone and dinoflagellate proteins: oneand nvo-dimensional SDS-PAGE and Western analysis

h in

Proteins were isolated from the host as follows: a host

preimmune serum or anti-sym32 antiserum diluted with

PBS/BSA/Triton X to 1:50. 1:200. 1:2000: 5 min in PBS,
h in 1:200 Alexa Fluor 488 goat
repeated five times:

C recirculating aquaranemone was removed from an

ium and flash-frozen in liquid nitrogen. The anemone was

3% BSA):

30 min

in

PBS/BSA/0.2%

Triton X-100:

PBS/BSA; and 5 min in PBS. repeated

IgG
times. After incubation, the cells were viewed under an
in

anti-rabbit
five

Olympus BX-60

fluorescence microscope.

1 1

minced with a razor blade, and placed into a glass grinder

with a Teflon pestle driven by a hand drill in four volumes
NaCl. 10
Tris. 100
ice-cold grinding buffer (100

mM
mM
mM EDTA) with protease inhibitors (Sigma: 5 per
/id

10 ml

The homogenate was placed into a centrifuge tube

and the grinder was rinsed with two volumes of buffer (v:w
anemone tissue), which was added to the tube and mixed
well. The remaining homogenate was centrifuged at
16,000 X g for 10 min at 4 C to remove algal cells and host
cell debris and membranes from the homogenized anemone
tissue. The supernatant was removed to a new tube and
centrifuged again. Protein concentration was determined on
buffer).

EM-level immunocytochemistry

Immunocytochemistry was performed on

anemones collected at different times and on one aposymbiotic anemone. Tentacles
from aposymbiotic and symbiotic anemones were clipped

Anemones.

three occasions using symbiotic

and immersed
fixative in

10

in

PBS

1% paraformaldehyde, 1%

for 1.5 h. Tentacles

min each time,

in

PBS and

glutaraldehyde

were rinsed three times,

then dehydrated for 15 min in

each concentration of a methanol

(MeOH)

series

(15%,

30%, 50%, 85%. 95%. 100%, 100%). Tentacles were

trated with

MeOH
night,

LR

White resin on a rotating

dilutions (1:3

LR White:MeOH

100% LR White

for 3 h),

infil-

table in a series of

overnight, 1:1 over-

and then placed

in gelatin

homogenate using the Bradford assay (Pierce

Host proteins prepared according to
contamination by symbiont proare
free
from
protocol

this cleared

Coomassie
this

teins

reagent).

(Weis and Levine. 1996).

Proteins were isolated from symbionts that had been

either continuously maintained in culture with no host contact

for

many

generations or freshly

isolated

from an

342

J.

A. elegantissim,: !X>M.

Many

l^ir hosts

be isolated

SCHWARZ AND

A.

species of Symbiodinium can

and brought into culture in

^ U seawater. These
symbionts are thereany host cell contact. We obtained frozen
:nbionts from cultures of S. benmtdense, which

nutrient-si]."':

fore

pellete.i

-!'.

igmally isolated from the tropical sea anemone Aipta100 /xl

siti/>iillida. A chunk of the pelleted symbionts (about

was

oi

volume) was

grinder with
a Teflon pestle in an equal volume of grinding buffer with
in

briefly

ground

in a glass tissue

protease inhibitor cocktail. Examination under a light microscope revealed that nearly all symbionts were still intact
after this step. An equal volume of acid-rinsed glass beads

(Sigma: 425-600 /j,m) was added and the mixture alternately vortexed for 15-30 s and placed on ice for about 30 s,
for a total of

20 times. With repeated vortexing. the homog-

became

intensely orange, likely indicating the release

of the major water-soluble accessory pigment, peridininchlorophyll protein. By this means, at least 75% of symbi-

enate

onts were broken open, as determined by light microscopy.

With a syringe and a 24-gauge needle, the homogenate was

removed from the glass beads and centrifuged at 16,000 X g

for 10 min at 4 C to remove cellular debris. The supernatant was placed into a new tube and again centrifuged. This
cleared supernatant fraction was then assayed for protein
concentration, as described above, and then prepared for

one-dimensional or two-dimensional

SDS-PAGE,

as de-

V.

M. WEIS

One-dimensional

SDS-PAGE

with

Proteins from freshly isolated symbionts were prepared

by isolating symbionts from a host and then extracting
proteins using glass beads to fracture the symbiont cell wall

and release the contents of the cytoplasm.

weighing about 2 g was removed from an

phoresis using LDS buffer + DTT (Invitrogen) according to

manufacturer's instructions; 10 /u,g of protein was loaded for

MOPS

sample. Electrophoresis was performed in

buffer according to the manufacturer's instructions. Gels
were transferred to 12.5
Tris, 100
glycine, 10%

each

mM

MeOH

for

transferred onto nitrocellulose

mM

20 min, and proteins were electrophoretically

membrane

for 1.25 h at 100

BioRad chamber.
Two-dimensional SDS-PAGE was performed using proteins extracted from freshly isolated symbionts. Proteins
were extracted as described above, except that no NaCl was
in a

used

in the buffer, as salt interferes

The SDS-PAGE was

ing.

with isoelectric focus-

carried out on a Multiphor

II

system (Amersham Pharmacia), according to the manufacturer's instructions and as described in Reynolds et al.
(2000). Thirty microliters of symbiont

homogenate contain-

of protein was used for isoelectric focusing on an

IPG strip. pH 3-10. After isoelectric focusing, the

ing 60

/jig

180-mm
IPG strip was

equilibrated and placed on a

12% ExcelGel;

electrophoresis was performed according to manufacturer's

instructions. After electrophoresis, the gel was placed into
transfer buffer (50

mM

Tris.

40

mM glycine,

0.04% SDS,

host

1 1

anemone

recirculat-

methanol) for 20 min. The gel was removed from the

plastic backing, and proteins were transferred to a nitrocellulose

membrane under

tiphor

II

a discontinuous buffer system (Mulsystem from Amersham).

For Western analysis, the membranes from one- and
two-dimensional SDS-PAGE were incubated at 4 C over-

ing aquarium and flash-frozen in liquid nitrogen. The anemone was minced with a razor blade and placed into a glass

night in blocking buffer (TBS: 20

grinder with 3 ml of grinding buffer plus protease inhibitor.

following morning, the

were performed on

was ground with a Teflon

pestle to

Bis-

20%

scribed below.

All subsequent steps

10% Nu-PAGE

was performed on proteins from host

tissue and from freshly isolated, and cultured symbiont
proteins. Samples were denatured and prepared for electroTris gels (Invitrogen)

homogenize

ice.

pestle instead of a

The anemone
ground glass

the animal tissues without shearing

homogenate was cen2000 x g for 10 min to pellet the symbionts. The

pellet, about 300 /xl in volume, was partially cleaned of
anemone debris by regrinding the pellet in grinding buffer

the cell walls of the symbionts. This

trifuged at

using a glass tissue grinder with a Teflon pestle (this regrinding step was sufficient to break up the pellet, but not

break open the symbionts) and reconcentrating the symbionts by centrifugation. The partially cleaned pellet was
ground a final time before adding 500 jul of buffer with

Ph

7.5.

+ 5% powdered

milk

membrane was washed

TBS + 0.5% Tween-20 (TBST);

1:1500

dilution

rinsed 10
in

of

min each

in

1:5000 dilution

mM Tris. 500 mM NaCl,

+ 0.1% Tween-20). The

anti-sym32

antiserum:block

min

Pharmacia); and washed as before.

buffer;

Sym32

ECL

protein was
detection re-

(Amersham Pharmacia) and exposing membranes

film for

in

in a

TBS. TBST, TBS; incubated 45 min

of HRP-antirabbit IgG (Amersham

detected by chemiluminescence using

agents

for

incubated for 45 min

to

min.

Results

Microscopy of anemone tentacles

protease inhibitor cocktail and an equal volume of prerinsed

glass beads. This resuspended pellet contained a significant

Cryosectioned tentacles. Cryosectioned tentacles of aposymbiotic and symbiotic anemones were incubated with

amount of host tissue, as revealed by the presence of numerous nematocysts. We then followed the same vortexing

preimmune serum

and centrifugation protocol as described above. The cleared

supernatant fraction (intensely orange in color) was used for
one-dimensional SDS-F

AGE,

as described below.

as a negative control

for

endogenous

sym32 antiserum. Staining for sym32 in

tentacles
was distinct from that in symbiotic
aposymbiotic
tentacles, relative to preimmune controls (Fig. 1). In both
symbiotic and aposymbiotic tentacles, preimmune controls
staining or (2)

SYM32 LOCALIZATION

ep

ANTHOPLEURA

IN

343

ga

ep

ga

Figure 1. Light micrographs showing immunolocalization of sym32 protein within cryosections of tentacles
from aposymbiotic (A and B) and symbiotic (C and D) Antlwpleura elegantissima. The spherical symbionts can
be clearly seen within the gastroderm of symbiotic tentacles. Sections were incubated in either preimmune serum
(A andC)orsym32 anti-serum (B and D). and sym32 was visualized using silver enhancement of colloidal gold
labeling. Sym32 levels are high in the gastrodermis of symbiotic anemones, low in the gastrodermis of

aposymbiotic anemones, faint in the epidermis of both types of anemones, and absent in the mesoglea of both
= epidermis, ga = gastrodermis. m = mesoglea. Scale
types of anemones. Host tissue layers are marked as ep

bar

brown

0.3

mm

for

all

panels.

staining in epidermal and gastrodernial

showed

light

tissues,

and no staining

in the

mesoglea.

In

aposymbiotic

In aposymbiotic tentacles. sym32 gold-sphere

was associated exclusively with medium-density

labeling
vesicles

antiserum. staining in the

slightly darker than

located within both epidermal and gastrodermal cells

(Fig. 2). There was no evidence of any sym32 label

in the

preimmune controls. In symbiotic tentacles, staining

epidermis was also slightly darker than in the preim-

within the mesogleal layer, and the pattern of distribution

of the sym32-containing vesicles in the epidermal cells

mune

controls,

gastrodermis, where dinoflagel-

was distinct from that in the gastrodermis. The vesicles

were relatively uncommon in the epidermis (Fig. 2 A, B)
but more abundant in the gastrodermis. where they were

tentacles

incubated

in

sym32

epidermal and gastrodermal layers was

in the

lates are

and

housed,

layer of both

it

in the

was

significantly darker.

The mesogleal

aposymbiotic and symbiotic tentacles

re-

mained unstained.

concentrated along the apical end of the gastroderm, near

Electron micniscopv. To further examine the location of

sym32 within the host-symbiont association, we performed

the

interface

between the gastroderm and the gastric

cavity (Fig. 2C. D).

EM-level colloidal gold immunocytochemistry using sym32

antiserum to label the sym32 protein within thin sections of
resin-embedded tentacles from one aposymbiotic and three

In symbiotic tentacles, the pattern of distribution in the

epidermis was the same as in aposymbiotic tentacles;

symbiotic anemones (Figs. 2-4). We examined between 15

and 25 sections from each anemone. We also performed
negative controls with preimmune serum to check for non-

cles that

specific labeling of the tissues. In all cases, the preimmune

controls were almost completely free of gold-sphere labeling (data not shown).

sym32 gold-sphere

labeling

was contained within

vesi-

were relatively sparsely distributed, most com-

monly occurring near nematocysts. In contrast, the distribution

of sym32 within
the
gastrodermis was
dramatically different. The sym32-containing vesicles
that were so abundant in aposymbiotic gastroderm were
not present in symbiotic gastroderm. Instead, the

sym32

344

J.

A.

SCHWARZ AND

V. M.

WEIS

of aposymFigure 2. Transmission electron micrographs of iramunogold-labeled sections from tentacles

anemones. Gold spheres, \isible as black dots, indicate the presence of s\m?2. lA) Epidermal cells with

biotic

near
(n). (B) Enlargement of the boxed section of A. with gold spheres labeling \esicles located
of the boxed section of
nematocysts. (C) Gastrodermal cells adjacent to the gastric cavity (go. (D) Enlargement
= 2 p.m (A. C) and ^m (B. D).
C. showing gold spheres within vesicles in the gastrodermal cells. Scale bars

nematocysts

label

was associated with

the multiple

membranes

that

enclose the dinoflagellate sxmbiont within the host cell

3A, B). Gold spheres were diffusely arranged

within these membranes, not clearly associated with any
(Fig.

membrane layer. To confirm that this labeling was

specific tn :he membranous lasers, we quantified the
to areas outside the membranes. The
staining rela
contained an average ot 12.2 gold
membranous

single

l.i

1
4.65 <SD), n =while equivalent areas
),
spheres
outside the membranous layers contained an average of
= 19).
1.0
1.1, n
I

Symbiodinium witliin host cells. In addition to the

svm32 labeling \\ithin the membranes that surround the
symbionts. there was a significant amount of labeling
within the symbionts themselves (Fig. 4). Specifically,
gold spheres were located within the accumulation body,

described organelle that is believed to function

in the endocytic pathways of dinoflagellates. The density
of labeling within the accumulation bodies was highly
variable: some contained only a few gold spheres, while

a poorly

others contained hundreds (average

2

gold spheres/ jiAm

IS).

56.4

S3. 2

[SD]

SYM32 LOCALIZATION

ANTHOPLEVRA

IN

345

ol tentacles trom symbiotic

presence of dinoflagellates within host gastrodermal cells. (Bi
of membrane that
Enlargement of boxed area in A. showing gold spheres associated with the multiple layers
surround the dinoflagellates. The arrows delineate the margins of the multiple membranes surrounding the

Figure

Transmission electron micrographs of immunogold-labeled sections

3.

Aiithnpleiim

elef>uiiti.\siitui.

dinofiagellate.

chloroplast.

Preimmune

cw =

A)

Illustrates the

controls

showed

virtually

no gold-sphere labeling (not shown),

s\rnbiont cell wall, g = gastrodermal cell of the host,

==

symbiont

dinoflagellate symbiont. Scale bar

.m.

Western Nuts

with distinctly different molecular weights and isoelecpoints (pi) (Fig. 5B). A cross-reactive spot at 32 kDa.
8.2 pi. corresponds exactly with host sym32 (Reynolds el

tric

The presence of gold-sphere

lation bodies of the

symbionts

onts were producing a

label within the

accumu-

suggested that the

sym32 homolog.

We

symbi-

performed

Western analysis using anti-sym32 antiserum to look for

cross-reactive proteins in homogenates from symbionts
freshly removed from a host anemone and from cultured
symbionts that were not in contact with host cells. Antisym32 Western blots of one-dimensional gels from ho-

mogenates of freshly isolated Symbiodinium nniscatinei

revealed three bands (Fig. 5A. lane 2). The 32-kDa band,
identical in size to a band in host-only homogenate (lane
1). is probably due to contamination from host sym32.
This was expected, as protein preparations of the symbionts are invariably contaminated by host proteins (Weis
et ill., 1998). A 48-kDa band and a faint 45-kDa band

below
that

it

are

cultured

never
the

in

suggest the presence of cross-reactive proteins

produced by the symbionts. Homogenates of
that

were

algae (Symbiodinium bermudense)

contact with host tissues (lane 3) also contained

same 45/48-kDa doublet but lacked

the

32-kDa host

band. Western blots of two-dimensional gels of freshly

isolated 5. muscat inei homogenates revealed two spots

al..

probably due to host contaminawas a 48-kDa spot with a pi range

The 45/48-kDa protein doublet that is

2000), and again

is

tion. In addition there

of 4.3 to 4.5.

present in cultured S. bermuilense and in S. muscatinei

freshly harvested from a host, and is also faintly visible
in the host lane, therefore represents a protein produced

by S\mhi<>dininm both when it

host, and when it is free-living.

is

in

symbiosis with a

Microscopy of cultured Symbiodinium bermudense

We

were interested

in

cells

determining the location of the

symbiont-produced 45/48-kDa protein doublet. We therefore used the anti-sym32 antiserum (which was devel-

oped against recombinant host sym32

ize

the

For intact

We

cells,

protein), to local-

specimens of S.
used two immunolocalization methods.

protein

target

bermudense.

in

cultured

we used immunofluorescence,

in

which

fluorescent secondary antibody is detected by fluorescence microscopy. For sectioned cells, immunoelectron

microscopy allowed us

to detect

any patterns

in staining

346

J.

A.

SCHWARZ AND

V.

M. WEIS

CW
Figure

4.

-.

Transmission electron micrographs of immunogold -labeled sections

ot Jinoflagellalc

symhionts

contained within host gastrodermal cells. (A) Dinoflagellate contained within a host gastrodermal cell. (B)
Enlargement of the accumulation body, shown boxed in A, illustrating sparse gold labeling of the dinoflagellate

accumulation body. (C) Region around the accumulation body of another symbiont (section not counterstained)
showing intense labeling specific to the accumulation body. The cell wall (cw) and membrane layers (m) are
visible as concentric gray rings

around the symbiont. Sections incubated with preimmune serum showed virtually

no gold-sphere labeling (data not shown),

cell wall, c

m =

dinoflagellate chloroplast, ab

membranes surrounding

= accumulation body

the dinoflagellate,

cw =

of the dinoflagellate. Scale bar

dinoflagellate

2.5 /urn (A).

1.0 /iin (B, C).

at

the subcellular level. Despite testing

tions of antiserum and buffer solutions,

many concentrawe were unable

to detect a pattern of staining of the target protein

cultured cells of

5.

in

hcrmudcnse. With the immunofluo-

rescence method, preimmune controls looked identical to

antiseruiii incubated specimens at all dilutions of serum
that

we

sted.

could deu
partment wi!

>

;.'

The sym32

protein

is

distributed

among

different subcel-

symbiotic and aposymbiotic anemocompartments

nes. Most notably, the sym32-containing vesicles that are so

lular

in

With immunoelectron microscopy, we

specific staining to any particular com-

in the gastroderm of aposymbiotic hosts are absent

from symbiotic hosts; instead, sym32 localizes to the symbiosome membranes. This suggests that the internalization
of symbionts is accompanied by a transfer of sym32 from

even though the background

Imost no gold-sphere labeling at the

gastrodermal vesicles to the symbiosome membranes. This

would most likely occur during phagocytic uptake of the

at the

1:10 dilutions.

symbiont. by fusion of the sym32 vesicles with the phago-

was

restricted to a

some.

the cell,

staining varied
1:2000 dilutions h
In the

Discussion

preimmune

heavy labeling

controls, labeling

few, widely scattered spheres.

abundant

The presence of sym32 within

the dinoflagellates them-

SYM32 LOCALIZATION

IN

347

ANTHOPLEURA

_43 kDa
_30 kDa
2

B
8.3

4.5

pi

- 67 kDa

-43

-30

-20
Figure 5. Ami-sym32 Western blots of protein homogenates from Symbiodinium. (A) Western blot of a
one-dimensional gel. All lanes contained ]() /Mg of soluble protein. Lane I. host-only proteins, shows a 32-kDa

band and

a faint

48-kDa band. Lane

2.

Svmbiot/iniiim nniscatinei freshly harvested from a host

therefore contaminated with host tissues),

shows

three bands: a strongly staining

anemone (and

32-kDa band,

a strongly

48-kDa band, and a faint 45-kDa band. Lane 3. cultured Symhiiuliniiim hcrimuli'iisi: contains two equal
of protein homogenate (60/ng)
intensity bands at 45-kDa and 48-kDa. (B) Western blot of a two-dimensional gel
from S. nniscatinei freshly harvested from an anemone host. Two spots are present: a 32-kDa. pi 8.2, spot,
identifiable as sym32, presumably from contaminating host tissue, and a 48-kDa spot, pi range 4.3-4.5.
staining

(Taylor. 1987; Wakefield el ai,

selves complicates the picture and adds a new dimension to

our studies of sym32. The accumulation body in dinoflagel-

as a molecular "trash

lates is postulated to function as a

ably located adjacent to the nucleus, often appearing to

displace the edge of the nucleus. If the accumulation body

lysosome. although

this

organelle has not been studied in many species, and its

function has not been examined in Symbiodinium. The freeliving

dinoflagellate Prorocentrum has multiple accumula-

tion bodies with features characteristic of eukaryotic lyso-

somes.

These

bodies

fibrous material, and

contain

electron-dense

membranous

material,

material, and they pos-

sess acid phosphatase activity, react positively with the

periodic acid/Schiff reagent, and stain with acridine orange

(Zhou and
lation

Fritz.

body

994).

Symbiodinium has a single accumuand is postulated to function

that varies in size

2000).

is

We

observed

dump"

that the

accumulation body

is

lysosome or a trash dump, host sym32 may be

invari-

trans-

ported from the vacuolar membranes, across the dinoflagellate cell wall, and into a degradative pathway within the
dinoflagellate. This ability to transport molecules

host

cell,

from the

across the vacuolar membrane, into the cytosol or

organelles of an intracellular inhabitant

is

common

in par-

protozoans, and there are many mechanisms by which

this occurs (Schwab ct <//., 1994; Raibaud et al.. 2001;

asitic

Goodyer

el ai.

1997).

348

A.

J.

also possible, however, that the protein detected in

is not a host protein but a symbiont

is

It

SCHWARZ AND

the accumula'^
protein.

sym32

'

To A

whether the symbionts also produce a

we performed anti-sym32

protein,

MS on symbiont proteins separated by one- or

SDS-PAGE. Anti-sym32 antiserum la-

Wester

two-dimensional

beled a 45/48-kDa cross-reactive protein doublet in both

cultured S. bermudensc (free from any host cell contact) and
S.

nnisccitinei that

we removed from

host cells (Fig. 5).

The

presence of a 32-kDa band in the lane containing S. nuiscatinei proteins almost certainly results from the presence of

contaminating host proteins.

symbiont 45/48-kDa protein

size

Its

It

is,

highly likely that the

is

in fact, a

consistent with the fasciclin

is

sym32 homolog.
domain

proteins,

which consist of between one and four repeats of an approximately 15-kDa domain (thus symbiont p45/48 might
consist of three repeats of the

15-kDa-domain). Further-

more, fasciclin domain proteins are diversely distributed

and have been identified in bacteria (Paulsrud and Lindblad,
2002; Terasaka

1989). photosynthetic algae (Huber

et ai,

and Sumper. 1994). invertebrate animals (Bastiani et ai,

1987; Zinn et ai, 1988; Brennan and Robinson. 1994;
Bostic and Strand. 1996: Reynolds et ui, 2000). and hu-

mans (Skonier
is

p45/48
ing to

We

ft

sequence

To confirm

1992).

ai,

in fact a fasciclin

domain

protein,

that

we

symbiont

are attempt-

V. M.

WEIS

expand upon the

greatly

protein as a

(Elkins et

1990).

It is

ing domain, they

domains

may

description of the fasciclin

adhesion molecule

cell

homophilic

ai.,

domain proteins contain

tional

initial

now known
at least

that,

while

in insects

all

fasciclin

one 15-kDa fasciclin bind-

also contain a variety of other func-

that lend diversity to their functions. All,

common

however, share the

role, via the fasciclin

domain,

of mediating recognition and specificity events in cell-cell

or cell-extracellular matrix interaction. The mechanisms by

which they do so have yet

provided by

to

be elucidated, but clues

may be

as in the insect fasciclin

their structures,

protein (domains 3 and 4) and the Mycobacteria tuberculosis

MBP70

complex

protein (Carr et ai, 2003: Clout et

Although the mycobacterial MBP70 protein conof a single fasciclin domain, and the insect fasciclin I

ai, 2003).
sists

protein contains four domains, the structures of the individual

domains are

strikingly similar.

Each domain appears

to

two functional faces on opposite sides of the

each of which probably binds independently to

fold to produce
protein,

other molecules (Can' et ai, 2003). Mutations within these

functional faces in the

human

protein )3ig-h3 are

known

to

be associated with corneal dystrophy, suggesting that specificity in binding of these molecules to their targets is

mediated by small changes

in

amino acid composition along

the functional faces (Carr et ai, 2003). This has important

this gene.

attempted to immunolocalize the 45/48-kDa symbi-

ont protein in fixed specimens from cultures of

S.

bermu-

by using the anti-sym32 antiserum to detect the

45/48-kDa target protein. Two methods (immunofluores-

implications for the possible role of a fasciclin domain

protein in a symbiosis, where specificity in molecular inter-

dense,

actions between host and symbiont

cence to examine intact

establishment and regulation of the partnership.

Recent evidence suggests that fasciclin domain proteins
function in mediating symbiotic interactions in other asso-

copy

to

cells,

examine sectioned

and immunoelectron micros-

cells) failed to detect

any pattern
of staining. This suggests that the anti-sym32 antiserum,
although able to recognize epitopes in denatured symbiont
proteins on a Western blot (Fig. 4), was not specific enough
to detect epitopes in the native protein within S. hermndense
(data not shown). Occasionally, antibodies can

work

in

Western analysis but not immunolocalization. especially

when

the antibody
than that to which

is
it

being used to detect a protein other

was developed (Harlow and Lane.

likely

mediates the

ciations. In both the rhizobium-plant association

and the

cyanobacterial fungal lichen association, homologs have

been identified from the symbiont genomes, and

deletion of this gene reduces

its

in plants,

ability to fix nitrogen

(Oke

and Long, 1999; Paulsrud and Lindblad, 2002). This is the

first report to suggest that both partners in a symbiosis may

produce fasciclin domain proteins.

The sym32

story

is

complex. The sym32 protein appar-

1999). These results strengthen the conclusion that the protein that we observed within the symbiosome membranes

ently has functions in multiple biological processes within

and the symbiont accumulation body is the host-derived

protein and not the symbiont-derived protein. If true, this

(as

indicates that the symbiont has an active role in modifying

the

structure or design

However,

of the symbiosome membranes.

remain untested until antibod-

this possibility will

that can distinguish between the host and symbiont

proteins are ilcveloped.
ies

Interest

domain proteins appears

to be gaining
recent reports that describe the functions or structures of these proteins in diverse
ii;

momentum

fasciclin

judging from the

many

organisms (for example. Kim

2002; Carr et til.. 2003; Clout

et nl.,

2002; Tamura

et a!..

2003). These reports

et

til.,

the host; both in symbiotic interactions with dinoflugellates

evidenced by the presence of sym32 within the membranes surrounding the dinoflagellates), and in other nonsymbiosis-related processes (as evidenced by the presence
of sym32 in the epidermis of both aposymbiotic and symbiotic anemones). Furthermore, the presence of a crossreactive protein in both tree-living

Symbiodinium and S\m-

biodiniwn freshly isolated from a host suggests that the

symbionts possess a sym32 homolog. Still to be elucidated
are the degree to
interact

which the host and symbiont proteins

and the roles

that

each plays

in the

biology of each

partner separately and of the partners in symbiosis.

SYM32 LOCALIZATION

ANTHOPLEURA

IN

Harlow.

Acknowledgments

Thanks

to

Mike Nesson

for his assistance with the elec-

tron microscopy, Santiago Perez for cultures of S\mhio-

dinium hcnninlcnxe. and

Kim Koch

E.,

1-39

in

M. Sonnenfeld.

S.,

fasciclin: a

for her assistance with

immunocytochemistry. This research was

light-level

supported by an
(915432) and an

EPA STAR graduate fellowship to JAS

NSF award (9728405-IBN) to VMW.

and D. P. Lane. 1999. Antibody-antigen interactions. Pp.

Using Antibodies: A Laboratory Manual. Cold Spring Harbor

NY.

Laboratory Press. Cold Spring Harbor.

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dinoflagellate lysosomes.

J.

Zinn, K., L. McAllister, and C. S.

and neuronal expression of

Cell 53: 577-587.

niticiilosum

(Dinophyceae)

are

Phycol. 30: 39-44.

Goodman.

fasciclin

in

1988.

Sequence analysis

grasshopper and Drosophila.

Reference: Biol. Bull. 205: 351-366. (December 20(Ml

2003 Marine Biological Laboratory

(0

Columellar Muscle of Neogastropods: Muscle

Attachment and the Function of Columellar Folds
REBECCA

M. PRICE

Department of Geophysical Sciences, University of Chicago, 5734

S. Ellis

Ave., Chicago, Illinois

60637

Malacologists often assume that ornamentation

on snail shells is functional, and therefore adaptive. I con-

Introduction

of the widely accepted

hypothesis that columellar folds, a type of internal ornamentation, enhance the performance of the columellar mus-

Malacologists often assume that gastropod shell ornamentation is adaptive, but experiments that demonstrate the

Abstract.

ducted the

cle,

first

comprehensive

which attaches the

test

function of these presumed adaptations are rare (Morton.

1967; for notable tests of ornamentation function, see

snail to its shell. Careful dissections

live,

1979; Bertness and Cunningham, 1981;

and
1988; Marko and Palmer. 1991; West
Palmer,
Appleton
et a I., 1991; Carefoot and Donovan, 1995; Donovan el al..
1999). Columellar folds, plications on the columella. or

is

not restricted to a small, circular patch located deep

long and narrow,

whorl along the length of the

within the shell. Instead, the attachment

extending approximately a

full

is

central

I
developed a novel technique for preparing
three-dimensional reconstructions from photographs documenting the dissections. These reconstructions were then

(2) the total contact area

muscle and the columella,

(3

the length of attachment.

between the

animal to the columella (Signer and Kat, 1984; Fretter and

Graham, 1994) and has grooves that fit between each fold.

the depth of attachment, and

None

In this paper,

to

maneuver

drawal. These

its

shell

nor

facilitate

test three

hypotheses that purport to explain

between the columellar folds and

the columellar muscle.

Columellar folds are readily visible on the inner lip of

shells, and systematists have used the impressive

first

many

diversity of fold shapes to distinguish species and higher

taxa. I consider a fold to be any ridge on the inner lip of the

indicate that columellar folds neither increase an animal's

ability

a functional relationship

parameter indicate that columellar folds

do
not
probably
guide the columellar muscle as the animal
moves in and out of its shell. Values of the other parameters
values of the

are partic-

be an adaptation that is intimately related to the columellar muscle (Fig. 1). In fact, the muscle does attach the

of these parameters
differed significantly between species with and without
folds. In light of the biomechanics of muscular hydrostats,
(4)

1 ).

may

muscle and columella.

a gastropod shell (see Fig.

Neogastropoda of the caenogastropods, the columellar folds

used to measure four parameters that describe the muscle:

the surface area of the physical attachment between the
1

column of

ularly intriguing ornaments because they evolved repeatedly

in a number of clades, such as the Caenogatropoda. Opisthobranchia. and Pulmonata (Price, 2001). In the subclade

columella.

1977.

Palmer,

non-relaxed specimens reveal that the physical attachment between the columellar muscle and the columella

of

aperture that extends along the columella for a number of

whorls, usually extending all the way to the apex. Some
folds occur at the bottom edge of the aperture, as in Nas-

deeper with-

results, and the fact that folds have evolved

convergently several times, might indicate that folds are an

easily evolvable solution to many functional problems, none

sarius

vibex,

whereas others, such as those

dislocata, are located in the center of the inner

of which are currently understood.

be

wide

(Triplofiisus

vibex), subtle

giganteus)

or

in

lip.

narrow

Terebru
Folds can

(Nassarius

(Busycon contrarium) or prominent (Vasum

muricatum).
The columellar muscle conforms exactly to the shape of
the folds where it lies over them, and this conformation has

Received 22 October 2002; accepted 29 August 2003.

inspired the hypotheses that are

E-mail: rmprice@uchicago.edu

351

most commonly cited

to

352

R.

M. PRICE

explain the fimction of folds. These hypotheses must be

considered in li;:h, of the fact that the columellar muscle

muscular hydrostat (Thompson ct al, 1998),

controlling protraction from, and retraction into, the shell.
Like hydrostatic skeletons in general, the volume of a musfunctions a

shell

shapes must account for the absence of folds

some large neogastropods, such as S\rinx amaniix. whose shell can reach almost a meter in length
in

.1

(Harasewych and
2.

cular hydrostat remains constant, so a contraction in one

direction induces elongation in an opposing direction (Kier

Petit, 1989).

Maneuverability,'. Columellar folds enhance a snail's

ability to maneuver its shell (Fretter and Graham,

1962; Vermeij,

A number

1978).

of authors have

and Smith, 1985). The columellar muscle is composed of

muscle fibers that are oriented longitudinally, transversely,

assumed

and obliquely with respect to the long axis of the columellar

muscle (Thompson el al., 1998). Thus, the columellar mus-

the adductor muscle attachment in bivalves (e.g.. Lin-

cle controls

addition to

own

twisting, shortening, and elongating

protraction and retraction.
its

shape of the muscle's physical attachment to the columella is small and circular, like

sley.

lip,

were deposited

its

maneuvering
shell back and

other

large mantle retracted into

This idea predicts that ornamentation would be

more pronounced
tation

at the

aperture (Dall, 1894), but

around the aperture where the mantle

largest

is

easily detaches
3.

all

example when

when

it

pries

it

open the

at

swings the

(Thompson

shells of prey

from

cracked

shell.

Predator avoidance. Snails with columellar folds can

withdraw more deeply into

exaggerated only in some species with determinate growth

(Paul, 1991; Vermeij and Signer, 1992; Vermeij. 2001a);
furthermore, contrary to Dall's prediction, not

shell, for

ing and discarding the shell, and because the muscle

ornamen-

is

that

(Taylor et al., 1980). This hypothesis has remained

untested because dissections usually begin by remov-

would form when an overly

the shell.

1993;

assumed

forth to fend off a predator

et al., 1998), or

wrinkles

in the

Morita,

animal with greater attachment area might be better

internal ornamentation including the parietal ridge, lirae,

that

1984;

1998). Vermeij (1978)

predicted that animals with folds would consequently

have a larger surface area of muscle attachment. An

explanation does not pertain to the columellar muscle, relying instead on the nature of the mantle, the tissue that

and teeth on the outer

Signor and Kat.

et al..

the attachment occurs immediately over the folds and

Dall (1894; restated by Fretter and Graham, 1994) pubthe only nonfunctional explanation of folds. His

all

1978;

Thompson

in

lished

secretes the shell. Dall surmised that folds, and

that the

their shells (Dall,

1894),

increasing their ability to escape from predators. Dall

(1894) commented that snails with columellar folds

of these

more deeply, but presented no data in support

of this idea. Since then, others have documented that

animals have columellar folds. Indeed, the internal mor-

retract

phology of gastropods is highly stereotyped, not random,

and it is unlikely that the mantle would always wrinkle

snails that retract

more deeply

are better at escaping

uniformly (Signer and Kat. 1984). There are also no obvious differences in mantle size between species with and

predators: they are harder to reach, and they take so

without internal ornamentation

the task (Vermeij,

in

long to handle that the predator eventually abandons

1978. 1987). Although it is not

general, or columellar

immediately obvious

folds in particular (pers. obs.).

Three interrelated hypotheses have been presented

explain

how

Guidance. Columellar folds guide the columellar musanimal moves in and out of its shell (Signor

cle as the

and Kat, 1984; Ponder, 1972). The analogy here

is

that

the folds act like a railroad track guiding a train; that

is,

they prevent the animal from slipping in

One manner
is

in

its

shell.

which the folds may guide the muscle

by protruding far into the columellar muscle, remuscle movement along the folds. In this

stricting the

event, the area of contact should be greater in species

with folds than

in

those without.

I have
developed procedures for dissecting gastropods
while keeping the columellar muscle intact; employed a
novel mathematical algorithm that converts a photograph of
a snail into a three-dimensional surface from which I can

measure areas; and examined neogastropod species

been assumed, the muscle attachment to the columella is

complex and leaves no scar. None of the three hypotheses
outlined above adequately explain the functional relationship between the columellar folds and the columellar muscle.

test this prediction.

implying that the columellar muscle would

within the shell unless folds guided

Any

attempt to

extend

this

that

represent a range of columellar fold morphologies, as well

as species with smooth columellae. Contrary to what has

Signor. and Kat (1984) limited their guidance hypothesis to high-spired, narrow (turritelliform) species,

these behaviors might be

confirm Dall's claim are easy to collect.

the folds affect the function of the columellar

muscle, thereby explaining both why folds have evolved

and why so many neogastropod lineages maintain them:

why

associated with columellar folds, the data required to

to

its

slip

movement.

hypothesis to low-spired

Materials and Methods

Sample
Quantitative data on the columellar muscle were colfrom seven species with folds (from five genera and

lected

FUNCTION OF COLUMELLAR FOLDS

some authors describe as columellar

do not continue inside

tour families) and five species lacking them (from five

genera and three families), affording phylogenetic breadth

that

(Table

1). One of the species without folds. Strombus alanot a neogastropod; it is a caenogastropod (a clade
that contains the neogastropods) with shell shape similar to

therefore

tus, is

the animal

other species considered here. At least two and up to five

specimens were studied for each species, for a total of 31

Museum

in

70%

ethanol and deposited

of Natural History

at

the

(FMNH). Chicago,

Field

Illinois.

folds (e.g.. Abbott,

the shell, and they

1974), but these

do not contact the columellar muscle, even when

is

fully

protracted.

As

such, these apertural

plications cannot affect the function of the columellar muscle, so I consider Oliva sayana to be without folds.

specimens for most measurements (36 specimens for attachment depth). These data were supplemented with qualitative
observations from an additional nine species. All specimens

were stored

353

Terminology describing orientation

description of fold

morphology requires terms

orient the reader to the snail shell (Fig.

terms refer to a snail shell

in a

).

that

In this paper, all

standard orientation, with

species have a functional

Except
operculum, eliminating any possible bias introduced by a
relationship between folds and an operculum.

apex up and aperture visible (and on the right for dextral

species). Top and bottom refer to positions along the coiling
axis. The bottom of a whorl is farthest from the apex,

The sample includes species with a variety of columellar

and numbers (Table 2), but all have fairly

whereas the top of the whorl is closest to the apex. The

width of a feature is measured along a line parallel to the
top-bottom axis, following the convention that the short axis

for Oliva xtiyana.

all

fold shapes

modest

folds.

Prominent

folds,

such as those

in the Mitridae.

Volutidae, and Cancellariidae could not be included, be-

cause they were too difficult to collect or purchase alive.

Oliva sayana has plications on the inner lip of the aperture

of the columellar muscle represents width, whereas the long

axis of the columellar muscle represents length (Fig. 1A, E,

Table

Fig. 2E). Apical

and apertural refer

to directions along the

Species examined

Collection site*

Species

Yes

299449, 299459

Quantitative

299444, 299463, 299468, 299472

Quantitative

Fasciolariidae

Yes
Yes

299450, 299492. 299493

Quantitative

Fasciolariidae

Yes

299448. 299456

Quantitative

Nassariidae

Yes

299475. 299477

Quantitative

HT

Fasciolariidae

Yes

299441, 299442

Quantitative

DIR

Muricidae

Yes

299486, 299487

Quantitative

WFS
FS, WFS
WFS

Muricidae

299451, 299458

Quantitative

Melongenidae
Muricidae

299453, 299457

Quantitative

299452, 299454. 301941

Quantitative

Strombidae

301942, 301943

Quantitative

WFS. SM. ST

Muricidae

No
No
No
No
No

299481. 299482. 299483,

Quantitative

Fasciolaria hunteria (Perry. 1811)

FS.

Fasciolaria tulipa (Linnaeus, 1758)

HT

Nassarius vibex (Say. 1822)

FS,

Triplofusus gigante us

Camharus

Kiener. 1840)

cancellarius (Conrad. 1846)t

Chiciiri'us florifer distans

(Adams, 1855)

Melongena corona (Gmelin, 1791)

Sirainniiita haemaxtoma (Linnaeus, 1767)
Strombus alalus (Gmelin, 1791)
Urosalpinx perrugala (Conrad. 1846)

Data type

Melongenidae

FS
FS

Museum ID

Folds

Melongenidae

Busycon spiratum (Lamarck. 1816)

Busycon contrarium (Conrad. 1840)

Family

WFS,

SM

WFS

299484, 299485

Columbella

ritsticoides (Heilprin, 1886)

Leucozonia nassa (Gmelin, 1791)

DIR

Columbellidae

Yes

299473. 299474

Qualitative

Fasciolariidae

Yes

299506, 299520, 299521,

Qualitative

299522, 299526. 299527,

299530, 299531

Opealostoma pseudcdon (Burrow, 1815)

Pleurnpltica salmo (Wood. 1928)

IV

Fasciolariidae

Yes

299496

Qualitative

Fasciolariidae

Yes

299494

Qualitative

Terebra dislocata (Say, 1822)

Vusum muricatum (von Born. 1778)

HT
G
G

Terebridae

Yes

299488, 299489. 299490. 299491

Qualitative

Vasidae

Yes

299500

Qualitative

Cypraeidae

299495

Qualitative

Oliva sayana (Ravenel. 1834)

SM. HT

Olividae

299460, 299461, 299466. 299469

Qualitative!

Pnlysrira nobilis (Hinds. 1843)

GC

Turridae

No
No
No

299497, 299499

Qualitative

Cypruea < en-Hi (Linnaeus. 1771

* Collection sites:
B, Bique.

Panama (Pacific Ocean); DIR. Dog Island Reef. Florida (Gulf of Mexico); FS. Florida State University Marine Lab, Turkey
Bayou. Florida (Gulf of Mexico); G, Galeta. Panama (Caribbean Sea); GC, Golfo de Chiriqui, Panama (Pacific Ocean); HT. Hammock Trail, near St. Joseph
Bay. Gulf County, Florida (Gulf of Mexico); IV. Isla Venado near Playa Veracruz. Panama (Pacific Ocean); P. Purchased from Gulf Specimen Aquarium
and Marine Biological Supply, Panacea. Florida; S. Sebastian. Florida (Indian River County); SM. St. Mark's Wildlife Refuge, Florida (Gulf of Mexico);
ST. Beach

at St.

Teresa. Florida (Gulf of Mexico);

WFS. West

t Tentatively placed in Soteneistra by Vermeij (20()lb).

f Only the attachment depth was measured.

side of Florida State University

Marine Lab on oyster

bar.

354

R.

M. PRICE

attachment morphology could be quantified in the largest

specimens of Nassarius vibex, but the available speci-

Table 2
fold inorphi

i/i

\iimined

mens of Columbella

inirium

subtle fold at the

bottom of the whorl,

at

aperture, but

Busycon spiratum

more prominent in older whorls

As in B. contrariuiii. but slightly more defined

Ctintharus cancellarius

fold at the bottom of the whorl, angled

obliquely; broader than in other species

Columbella rusticoides

fold immediately at the

bottom of the whorl

2 folds at the bottom of the whorl, angled

Fasciolaria himteria

3 folds, angled obliquely and closely spaced

1

with 3 folds

well-defined fold at the bottom of the whorl,

two whorls; see

was physically attached. Since

free, the attachment was not

when probed.

disturbed

rounded, profile
in L. nasssa

Figs. 1,2)

bottom of the muscle was

the

angled obliquely; square, rather than

As

Opeatostoma

pseudodon

As

P/europloca salmo
Terebra dislocala

Measurements

in F. tulipa

2 large, broad folds spread throughout whorl;

the bottom fold

is

more prominent than

As

Triplofusus giganteus

3)
but top fold

in F. tulipa.

is

more

subtle than

the others

5 folds in "1 prominent

nniricatiim

The measurements required

the

top one

Vasum

I used a blunt but flexible 34-gauge copper wire

probe between the tissue and columella. In all cases, only
the top of the muscle (that closest to the junction between

to

As

Oliva sayana. the

in

cannot withstand even slight pressure.

To distinguish between muscle that was physically attached and muscle that was simply pressed against the
columella,

Leucozonia nassa

was documented

morphology of attachment throughout the rest of its

length was inaccessible, because the delicate columella

coincides with the edge of the columella

Fasciolaria tu/ipa

Nassarius vibex

the attachment

obliquely; the lower edge of the bottom fold

in F. himteria, but

The

largest

distinguished by a groove and angled

obliquely; difficult to observe

rusticoides were too small.

whorl of Terebra dislocata, a high-spired species,

is only a few millimeters tall, so it could not be quantified
with these methods. Although the most apertural point of

Fold morphology

Sn

prominent

weak

folds angled

weak

prominent" pattern;

more perpendicular

to coiling

axis than in any of the other species and

spread throughout the

bottom two-thirds of

the whorl

to test the

were the standardized (see below)

hypotheses (Table
of muscle

total area

physically attached to the columella, the standardized total

area of muscle in contact with the columella throughout the

attachment, the depth of attachment, and the length of the

attachment.
It

was especially

difficult to

measure the

total area

of

muscle physically attached to the columella (ATT in Figs. 1,

2). In these species, the columellar muscle attachment does
not scar the shell, so the attachment area had to be observed
directly.

of the shell towards the

or opening respectively:
the columellur muscle narrows apically, but is wide aperspiral

tip

tried

removing

on the columella, but

the attachment.

number of revolutions between

when measured from

one revolution

is

360;

attachment might begin

it

and the aperture, where

muscle

for example, the columellar

at a

depth of 300.

this

However,

junction occurs where the bottom of one whorl

meets the top of another. The depth of a feature indicates the
turally.

Dissections were performed on live, untreated animals.

Surprisingly, any treatment of animals, such as freezing,
storing in ethanol, or relaxing in

magnesium

sulfate

caused the muscle to detach from the

(Epsom

shell,

even

when tugged only

slightly. Fresh material is therefore essential to study columellar muscle attachment.

To expose the soft tissues while keeping them and the

intact,

used a Dremel rotary tool with a

abrasive wheel to cut

1.6-mm-diame'ter carborundum

away

the exterior.

The

size

of the blade limited the

could be dissected with precision to those

specimens
with whorls larger than half a centimeter. Thus, the
that

left

procedure inevitably destroyed

was so narrow

the attachment

it

muscle

that,

could be rea-

digital photographs,
sonably estimated as a thin but long box, only one pixel
wide but many degrees long. The small bias introduced by

approximation should be the same

without folds.

Dissections

columella

of columellar muscle that

how much muscle was

relative to the total width of the columellar

this

salts),

strips

were not attached to determine

in species

with and

Another challenge was to measure the surface area of

contact between the columellar muscle and the columella

(CM

in Figs. 1,

2G). The apertural end of the muscle grades

1990: Thompson et al., 1998), so

into the foot (Voltzow.

the only point that could be consistently identified at the

apertural end of the columellar muscle was the point at

which

the

muscle attachment began (ATT in Fig. 2E). I

total area of muscle in contact with the colu-

measured the

mella: beginning with the attachment, extending down the

width of the muscle to the bottom of the whorl, and extending across the columella to include the most apical part of
(CM in Figs. 1, 2G). Because the muscle always

the muscle

spanned the columella between the attachment and the

FUNCTION OF COLUMELLAR FOLDS

355

CA
180

Apert
A.

CF
D

B.
Definition of terms describing columellar

1.

Figure

illustrated with Triplofusus gigunteus.

morphology

(A)

apex along a vertical axis dulupicul in some literature,

of that same axis (abapicul). The open arrows wrap around
e.g.. Vemieij. 1978); bottom refers to the lower part
the axis from the aperture to the apex. The apertural point of the muscle attachment is that closest to the aperture

Top

refers to the part of a whorl relatively closer to the

and the a/>iV<i/-most point is that immediately after the attachment crosses the folds (also see E).
dimension from the top and to the bottom of a structure (e.g.. the muscle is shown at its widest

(also see D).

Width (W)
point in

the

is

and E; see also Fig. 2E). (B) Close-up of the inner lip of the aperture. The fold modifier is calculated
FO:FL. where FL is the minimum length between folds, and FO is the outline of the folds (see
in Materials and Methods). (C) Intact shell. (D) Same shell rotated 180 with sections of the shell

as the ratio of

Equation

as in D, but another 360 of shell exterior has been removed, and open arrows
Cut surfaces are indicated by fine, vertical hatching. Degrees indicate the depth
the number of degrees between the landmark and the aperture. The columellar muscle

Same view

exterior removed. (E)

indicate the apertural-apical axis.

of the landmark (LM):

i.e..

shaded gray and passes over the columellar folds: the attachment area is a thick black line that is slightly
exaggerated for illustrative purposes: stipples mark the area of the columella throughout the length of the
is

attachment (CA). Abbreviations: Apert, aperture: ATT. columellar muscle attachment; C, columella; CA,
columellar area; CE, edge of the columella; CF, columellar folds; CM, area of contact between columellar

muscle and columella; FO. length of fold outline: FL. minimum length of
LM. landmark line; W. width.

bottom of the whorl,

this

measurement could be made

in

folds;

J.

junction between two whorls;

Depth of attachment was much easier

to

measure, and

relaxed, contracted, and protracted animals. The position

and length of the muscle attachment and the area under the

not subject to behavioral variation. This proxy assumes that the amount of space between the columellar

attachment remain the same regardless of the animal's behavior. The exclusion of the area between the foot and the

muscle attachment of the contracted animal and its operculum varies equally among species with folds and those

initial

that

attachment was justifiable:

conformed

to the

it

never included muscle

morphology of the folds and was

therefore not relevant to the present study; there

reason to assume that

and without

it

was no

varied differently in species with

Both the attachment area and the contact area were stan-

remove

is

without.

The length of attachment was defined as the number of

degrees between the depth of attachment and the apical edge
of the attachment, which occurred in the crease between the
columella and the

folds.

dardized to the columellar area

it

(CA

in Fig.

the effect of size. Columellar area

is

1;

Table 3)

to

the area of the

muscle
apically

fibers

rest

of the shell (Fig. 2F. G).

Some

and connective tissue extended even farther

beyond

this point, but

likely to function differently

methods were too coarse

they were small and thus

rest of the muscle. My

from the

to dissect this tiny strand of tissue

columella above, below, and including the length of attachment.

accurately.

used the depth of attachment as a proxy for depth of

measured as the number of degrees behind the
The
two depths are intuitively related, because
aperture.

Because the length of attachment and the depth of attachment were both measured as the number of revolutions
behind the aperture (in degrees), they were independent of

retraction,

an

animal cannot retract deeper than

its

attachment.

size

and did not need to be standardized.

356

R.

M. PRICE

Apex

Figure
299441
area

is

area

is

(A-D) An example illustrating the image processing technique. One photograph is shown from a
documents the whole dissection. In this example, the contact area of Triplofusus giganteus (FMNH

2.

series that
is

reconstructed.

The

central

landmark

in this

view has a depth of 540

(see Fig. ID, E). (B) Contact

highlighted (in green). (C) Comparison between contact area and a reference area (white).
a symmetrical stack of circles centered on the coiling axis (see Appendix).

middle 90

sector with the image (45

on

either side of the

landmark

line),

The black

The reference
lines

mark

the

or "target area" as described in the

Appendix. (D) Reconstructed contact urea. Read the reconstruction as a topographic map: lighter points on the
image are farther from the plane of the page. (E) Generic neogastropod with shell and viscera removed to
attachment and the manner in which the columellar muscle (in gray) coils throughout its length. The
broken edge indicates that the columellar muscle grades into the musculature of the foot. Drawing based on
figure 2 in Thompson, J. T.. A. D. Lowe, and W. M. Kier. 1998. The columellar muscle of prosobranch
illustrate the

gastropods: morphological zonation and

its

functional implications. Invertebr. Biol. 117: 45-56. (F) Photo-

graphic montage and (G) graphic representation of the columellar muscle attachment in the same specimen
illustrated in A and B. F and G are constructed from a series of photographs spliced together by aligning the
attachment, and thus creating a single image that looks like an uncoiled, flattened shell. As indicated by the blue
line, the

attachment begins

at

the top of the whorl and gradually slides

down

the columella.

Towards

the apical

end of the muscle, the angle of attachment changes dramatically, and the attachment crosses the folds. In F. much
of the muscle has been removed to expose the columellar folds, but it remains largely intact in two of the views,
demonstrating that the area of contact extends from the attachment to the bottom of the whorl. Hatching indicates
shell broken to expose the columella and columellar muscle. All scale bars, 1 cm. Abbreviations: F, foot; L,
length:

OP, operculum; W, width; other abbreviations

Image processing
All

measurements were taken from a

as in Figure

1.

sions, so the surface areas in the

series of digital

photographs that documented each dissection. However,

photographs distort three-dimensional data into two dimen-

photographs under-repre-

therefore developed a procedure to process

the series of photographs for each dissection, as well as a
sent true areas.

geometric algorithm that calculates the three-dimensional

FUNCTION OF COLUMELLAR FOLDS

and 405. and the other centered on landmarks

Table 3

Summary of measurements require J

to test

357

270, 360, and 450.

hypotheses

from the two

averaged the

final

at 180,
measurements

series.

Use modifier
to adjust for

Measurement
Standardized

fold

Hypothesis
of muscle

total area

topography?

Maneuverability

Yes

Guidance

Yes

three-dimensional reconstruction technique emhere

did not take into account the presence of folds.
ployed
for
To account
folds, I employed a scale factor ("modifier")

The

physically attached to the

columella

(ATT/CA

in Fig. 1)

Standardized surface area of

defined as the length of the projected outline of the folds

divided by the minimum distance between the two end-

contact between columella and

columellar muscle throughout

attachment when animal is

(CM/CA

relaxed

in Fig.

Depth of attachment,

in

Length of attachment,

in

Fold modifier

points of the folds (Fig. IB; Eq.

):

1)

No
No

Retraction depth

degrees

Maneuverability

degrees

modifier

= Length outline

(1)

Length mmimum
there are no folds, the minimum length equals the
length of the outline, so the lower limit of the modifier is 1.
I measured the fold modifier in each of the photographs

When
area projected into each photograph.
sented in the Appendix.

The algorithm

is

pre-

Photographs were taken with a Nikon CoolPix 950 digital

camera. The image plane of each photograph was parallel to

of a series that clearly showed the folds' silhouette. This

which the optic axis was perpendicular.

Each photograph also included a scale bar, was centered on
a landmark, and showed the muscle attachment and the edge

the endpoints of the fold outline, for which I used inflection

points. To compensate for this sensitivity, I always used the

of the columella.

statistical difference

the coiling axis, to

measurement

maximum

is

highly sensitive to shell orientation and to

fold modifier; this approach favored finding a

running perpendicular to the junctions between whorls (LM

in Fig.
every quarter of a whorl for small specimens and

between groups with and without folds.

Although there was considerable variability in fold modifier
values within a species, the variance for the entire sample of
species with folds was low (fold modifier variance. 0.003;

every eighth of a whorl for larger specimens (generally,

those with shells longer than 2 cm). These landmark lines

range. 1.0 to 1.2; 114 measurements over 17 specimens).

After measuring the surface area of the columellar folds,

were used

and transforming

The outside of each

1

Each

shell

was marked with

radial lines

to locate the

image within the series.

between 4 and 12 photographs,

series consisted of

pendix.

one photograph per quarter whorl. To create

images were scaled and oriented identically.

with

at least

each

series, all

Only the middle 90 sector within each photograph was

measured (45 on either side of the landmark line), thus
eliminating overlap

the images (Fig. 2C).

among

The algorithm employed

to estimate surface area con-

verted tracings of photographs into three-dimensional surfaces (Fig. 2A-C). I used a mouse or mouse tablet (Wacom

Area,,,^

the transformed fold area

calculate the

2D), and then areas were

the

series.

number of degrees from

to

the aperture to the

apertural and apical ends of the attachment

in the

summed

The procedure used

is

also described

Ap-

(An-

modifier)

(2)

A^

is

the similarly

from the attachment

subtracted

area, multi-

it
by the fold modifier, added the product back to the
attachment area, and then repeated the adjustment for the

contact area.

in

A, T

transformed area of the folds. In other words,

within each image. Tracings were transformed according to

the procedure described in the Appendix (implemented in
ver. 6.0; Fig.

= AT -

in the

2:

attachment area or contact area), and

plied

from each image

according to the algorithm

where A T is the area transformed by the algorithm into an

estimate of the area of a three-dimensional surface (either

Graphire2) to trace the surface areas of the muscle attachment, the muscle in contact with the shell, and the columella

MATLAB

it

applied Equation

The

total surface area

of the columella was not adjusted,

is used to standardize the other

because that measurement

metrics. If fold surface area

the

difference

would be

were added

to columellar area,

between species with and without folds

erased.

Appendix.

The 20

specimens were used to determine the

precision of each measurement. I had two quarter-whorl
series for these specimens, because they were photographed
largest

every eighth of a whorl; one series, for example, depicted

the attachment centered on landmarks at 135, 225, 315,

Statistics
Statistical comparisons between species with folds and
those lacking them were performed with a Mann-Whitney
U test using StatView 5.0 for Windows.

358

M. PRICE

moved from

Results
/filar

Thes
cle att

muscle across to the

most apertural end, the attachment lay
immediately under the junction of the two whorls. Tracing
apical edge. At

muscle attaclinient

v,ections demonstrated that the columellar

,

mnent

in

neogastropods

is

far

mus-

more complex than

previously assumed (by authors such as Linsley, 1978;

Signer and Kat, 1984; Morita, 1993; Thompson et al.,
1998).

The muscle did not leave

macroscopic feature

a scar, so there

on the columella

was no

that

approximated
attachment shape. The shape of the attachment was similar
species, regardless of the presence of folds (Fig.
2E-G). In most of the species examined, the attachment
in all

began about three-quarters of a whorl (-275) back from

the aperture (Fig. 3C, raw data for the histogram is in Table
4).

The depth of attachment might be much deeper

in

high-spired shells. In Terehra dislocata. attachments began

about two whorls back from the aperture (pers. obs.). T.

maculata and T. siihiilatii, muscles were previously reported
to attach

more deeply

about 2.5 and 4.5 whorls back from

at

the apertural edge of the

and Kat. 1984). In general, the attachment was long and sometimes extended for
more than one revolution; it was restricted to the upper edge

it

its

apically along the rising junction, the attachment shifted

progressively downward and across the columella toward the bottom of the whorl, with the rate of

in position

descent gradually increasing. Shortly before reaching the

bottom of the whorl, the angle of the attachment changed
dramatically,

becoming more oblique

down

to the

to the junction and

base of the whorl; this is where

curving sharply
the attachment crossed the folds

Here the muscle was remarkably

when

they were present.

less robust than

elsewhere

along
length. Farther apically, the muscle was only a thin
extension situated in the crease between the columella and
its

the base of the whorl. Within the area

attached, the muscle width

where the muscle was

was maximal at its apertural end

and minimal towards the apex.

Functional hvpotlieses

the aperture, respectively (Signer

of the muscle (blue line

The

is

ATT

in Fig.

2E-G).

relative position of the attachment

changed as

A.

I
D-

o
CD

it

P=0.71
with folds
- without folds

mm
1.5

2.O

2.5 3.O

3.5

it
4.O

4.

Standardized Attachment Area x 10"

Contrary to the prediction drawn from the functional

hypotheses outlined in the Introduction, no significant differences were found between species with and without folds
with respect to the surface area of muscle attachment (P =

g
8

FUNCTION OF COLUMELLAR FOLDS

Tahlu 4

Measurements for

Species

nil

specimens

359

R.

360

M. PRICE

function of columellar folds, although the species included

Columellar muscle attachment

most exuberantly developed folds. These

data cast doubt on the hypothesis that folds act as struts,
cellar muscle as the animal protracts from,
euidine the

The complexity and extent of the columellar muscle

attachment has gone unrecognized because the attachment

do not exhibit

the

its shell. Furthermore, the muscle probably

need to be guided, because it is a muscular hydrostat. The muscle probably mirrors the morphology of the
folds simply because it is physically adjacent to them. I

and

retra,

does

ti

>.

n<-.

with folds maneuver

reject the hypotheses that animals
better because their attachment area is larger, and that these

animals avoid predators by retracting deeper. While I reject

these hypotheses as general explanations for columellar fold
function in neogastropods as a whole, future

work may

demonstrate that some of them adequately explain function

within groups of species with similarly shaped folds.

remarkably delicate. A slight tug in the wrong

(away from the aperture, toward the apex) tore the
muscle from the columella. and the strength of the attach-

itself is

direction

ment deteriorated rapidly

in

preserved or relaxed animals.

between
tenuous nature probably
the physical mechanism of adhesion and the shape of the
adhesive surface. Although the muscle was easily detached
reflects the interplay

Its

from the columella, the animal never naturally experiences

a force pulling on the attachment from the angle or location
used during dissection; if the shell is opened as I have
opened it, the animal is already too exposed to save itself
from predators. An adhesive joint that can withstand large
I

shear stresses will frequently

Methodology

The muscle attachment

1986).

developed two novel methods in this research: one is a

is an analytical
practical dissection technique, and the other
I

approach for measuring distances and areas from photo-

of tape on a tabletop.

other gastropods can be dissected without damaging the

columella, and future applications of this approach will
provide a more general understanding of how soft tissues

Mutvei, 1996;

With

the analytical approach,

features.
cle in

1 1

soft tissues

and

shell

quantitatively characterized the columellar musspecies of neogastropods and one caenogastropod

1, 3, 4). This algorithm will work for any organism

echinoderms, cnidarians,
arranged as a stack of circles
foraminifera, diatoms, and mushrooms, for example.

(Tables

The measurements and

their analysis rest

on three

as-

sumptions. First, to measure the area of contact between the

columellar muscle and the columella, I assumed that the

amount of muscle
in species

that the

seems

apertural to the attachment varies equally

I also assumed

with and without folds. Second,

attachment area was only one pixel wide, which

by the observation that the attachment was

justified

quite thin relative to the total width of the muscle. Third,

areas were inflated by the algorithm that transformed the

two-dimensional data, because the columella was assumed

to be perfectly symmetrical (see Appendix). All of these

assumptions were

attempt to
muscle.
Furthermore,
quantify differences in the columellar
there is no a priori reason to believe that they would affect
justified, especially in this first

species with folds differently than species lacking them.

Note that the measurements are coarse (Table 4), and that
subtle differences in contact area, attachment area, depth of

attachment, and length of attachment cannot be resolved

with these methods.

analogous

(Portelli,

to a long piece

When

Surprisingly, none of the species studied here have

cle scars,

determined the relative position of

when peeled

peeled backward, perpendicular

to the table, the tape detaches easily, but when pulled along
its length, it has great strength.

graphs of specimens. With the dissection technique, I described the columellar muscle attachment in 20 neogastropods and one caenogastropod (Table 1). The soft tissues of

are situated within the shell.

fail
is

mus-

even though scars are found in all major groups of

shelled molluscs throughout their history (e.g., Abbott,
1974; Lindberg, 1985; Pojeta, 1985; Doguzhaeva and
muscles

Isaji et al.,

do

frequently

2002). In vertebrates, however,

not

leave

attachment

scars

(McGowan, 1982; Bryant and Seymour, 1990), especially

when they insert directly into bone instead of attaching to a
tendon which inserts into bone. In

snails, the

columellar

an epithelium that in turn inserts into the

shell (Tompa and Watabe, 1976). Additional work is required to determine whether neogastropods and vertebrates

muscle

inserts into

lack muscle scars for similar reasons.

so much longer
as Linsley,
such
than previously thought (by authors
interesthas
1978; Morita, 1993; Thompson et al.. 1998)

The

fact that the

muscle attachment

ing implications for

how

is

the columellar muscle func-

(1998) calculated, on theoretical

considerations, the force required to buckle the columellar muscle and the amount of torsion the muscle could
tions.

Thompson

et al.

The muscle, which has a crescentic cross section,

buckled more easily and could not exert as much torsional force as a cylindrical muscle with a circular cross

exert.

However, these authors assumed that the muscle

was attached at only one end rather than throughout its
the
length. The true, side-long attachment should help
section.

muscle

resist

compressive forces

(J.

T.

Thompson,

St.

Joseph's University, pers. comm.), so that the mechanical

disadvantage due to buckling may not be so severe.
Similarly, the net torsion exerted must be reconsidered in
light of the

new attachment

data.

FUNCTION OF COLLIMELLAR FOLDS

and Smith. 1985, 1990; Hodgson and Trueman. 1987; Kier,

1988; Marshall ct ni. 1989), the muscle seems to function

Guidance
hypothesized that folds guide the columellar muscle

during protraction and retraction by protruding far into the

columellar muscle, compelling the muscle to move along
the folds.

reasoned further

that, if folds act as struts,

then

they should protrude far enough into the muscle to significantly increase the amount of contact between the muscle

and columella. For the species analyzed here, however,

there was no significant difference in the contact area or
length of attachment between species with folds and those
lacking them. Therefore, the folds-as-struts hypothesis, as

was

explained here,

From another

not supported.

perspective, the methods were relatively

If these methods

coarse, and the data were poorly resolved.

are applied to species with a subtle fold topology, then

significant increase in the contact area

may

be detectable.

no
In

species with folds, I compared the unadjusted contact area

to the contact area after it had been adjusted with the fold
modifier. The two metrics differed by more than 2<7r in only

one specimen, and the average difference was only 0.2%

(n

361

17). This difference

is

less than the precision of the

in the

that

way

was previously thought

inefficient (Signer

and Kat, 1984).

Signor and Kat (1984) formulated the guidance hypothesis in part because Signor (1982) correlated burrowing
behavior

in

high-spired gastropods with columellar folds;

in 46
55 of 59 burrowing species had folds, but only
columellar
He
concluded
that
did.
non-burrowing species
1

folds guide the columellar muscle in burrowing animals

(Signor and Kat, 1984). However, 40 of his 59 burrowers

1
families
were in the Terebridae, and only 4 of his
1

included non-burrowing species. Thus, his results may be

explained by phylogenetic bias and should be reanalyzed

with comparative methods based on phylogenetic contrasts

(as in Harvey and Pagel. 1991) once the appropriate esti-

mates of relationship are available. The advantage of folds

to burrowing animals is not obvious, especially if the guidance hypothesis

is

not true. Furthermore,

species with

many

do not burrow (G. J. Vermeij. University of California,

Davis, pers. comm.). The species considered here cannot be
used to explore the relationship between folds and burrow-

folds

which is only to one decimal

the
Therefore,
although
topography of folds does not
place.
increase the contact area in the specimens considered here,

ing behavior, because species were not sampled randomly

may be greater in species with more prominent folds, such as those in the Volutidae, Mitridae. and

is an exact impression of the columellar folds, it is

to assume that the muscle moves along the
reasonable
only
folds. This assumption does not require, however, that the

measurement of contact

area,

the contact area

Cancellariidae. Thus, additional

work with these species

across burrowing and non-burrowing habitats.

In conclusion, since the inner surface of the columellar

muscle

and with methods providing higher resolution may lend

support to the guidance hypothesis and its corollary that

folds dictate the muscle's motion.

folds act as struts.

their proximity,

consideration of the association between the area of

contact and the presence of folds suggests other functional

relationships. For example, if the advantage offered by

were offset by the benefit of a large contact

strut-like folds

larity in the

suspect that the simi-

shape of the muscle and folds

and

that the direction the

governed instead by attachment

is due simply
muscle moves

to
is

morphology and muscle

fiber orientation.

Maneuverability

area, species with folds should have a significantly smaller

area of contact than those without. Similarly, species with

As with

contact area, the folds added insignificantly to the

contact due to the

attachment area. The attachment was so long, and so much

of it was distant from the folds, that the folds would need to

by a shorter attachment length.

However, no such significant differences were observed.

protrude into the muscle six times more than they do (i.e.,
multiply the fold modifier by 6) to significantly increase

The guidance hypothesis assumes that, without the resistance offered by folds, the muscle would slip along the
columella during both retraction and protraction, presum-

attachment area

ably causing the animal to expend more energy. However,

the arrangement of fibers within the muscle, including some

attachment.

folds could have the

folds,

same area of contact

provided that the increase

protruding folds

was

in

as species without

offset

wound

obliquely around the robust, apertural

end of the muscle, suggests that the fibers, and not the
columellar folds, control the path of the muscle when it
fibers that are

contracts

(Thompson

et

cil.,

1998). Because the muscle

is

in species

with folds

at the

<

0.05 level.

If

animals with folds are better able to maneuver their

it

is

shells.

not because they have a greater surface area of muscle

Signor and Kat (1984) suggested that the columellar

muscle is divided by the folds into functionally discrete
units joined only by connective tissue. However, judging by
the observations presented here, the divisions they describe
are probably part of a gradation

between the robust, most

top edge
columella when the oblique fibers contract. In light of this
newer evidence about the columellar muscle in particular

apertural, part of the muscle and the weak, more apical part
(left and right sides of Fig. 2E-G). Since Signor and Kat
1984) did not mention the frequency or placement of their

and the muscular hydrostats of molluscs

histological sections, nor illustrate their results,

attached along

its

(Fig. 2),

it

will shift across the

in

general (Kier

it

is difficult

362

R.

M. PRICE

conclusions in the light of the newly

to reevaluate thei.

morphology. The muscle histology

recognized attaci <nent

of high-spin '! species with and without folds should be

with folds) lying on coral, each with the aperture

lariid,

pointed upward and the foot hanging outside the aperture.

compared
and

if so.

at least

to determine whether the muscle is subdivided,

whether those subdivisions are constrained by, or

correspond

number of

in a

terrestrial

pulmonates
Maneuverability
does appear to be enhanced by physically distinct subdivisions of the columellar muscle (Suvorov, 1993. 1999a. b, c).
In these taxa. the columellar muscle originates from the
most apical point (this is the only part of the muscle that is
attached) and is divided into left and right pedal retractors,
and left and right buccal mass retractors. These four
branches continue to subdivide closer to the aperture. The

four functional groups of the muscle are separated by a

septum of connective tissue, but apertural teeth and colu-

mellar folds may play a secondary role in keeping subsets of

branches separated. Thus, columellar ornamentation in pulmonates apparently evolved for a different reason than it did
in

have a single function?

Although the function of columellar folds remains un-

columellar folds.

to.

Do folds

identified, there are potentially fruitful paths for future re-

search.

As discussed above,

the correlation between the

presence of folds and burrowing habit in high-spired gastropods (Signer, 1982) must be studied in more detail. Also,
columellar folds might strengthen the shell, thereby protecting the animal from predators. External features of the shell,

such as thickness and the presence of spines, have been

shown

to increase resistance to predators,

difficult for a

durophagous

making

it

more

break a snail's shell

predator to

(reviewed in Vermeij, 1993; Kohn, 1999). In fact, some

species have a corrugated shell, which presumably increases
strength while minimizing the costs associated with building and moving a heavy shell (Vermeij, 1993). Perhaps, in
a similar manner, columellar folds protect against predators

neogastropods.

by increasing the strength of the inner

lip

while minimizing

the cost of thickening the entire columella. Supporting this

Predator avoidance

idea,

Hughes and Elner

was unable

to

substantiate Dall's (1894) claim that

animals with folds retract deeper into their shells. Instead,

animals lacking folds retract more deeply, because they

have a significantly deeper attachment site. A larger sample

of species is required to confirm this conclusion. Because all
of the specimens from which quantitative data were obtained were collected in spring, seasonal variability in

growth

rate

ability in

and attachment

growth

rate

may

were not considered. Vari-

site

be especially important to con-

sider in genera such as Busycon.

which exhibit highly

epi-

sodic growth.

The depth of attachment was

surprisingly constant

among

most species considered here (median depth = 295; n =

36). Interestingly, the distribution of attachment depth was
bimodal. with medians

at

280 (n

31) and 630

(n

5)

the

inner

lip,

1979) report that crabs open NHhave thin columellae by snapping

cella lapillus shells that

breaking the shell

inner lip

makes

the

shell

at

the

outer

lip

(Vermeij,

actions directly. There

the shell fails catastrophically (G.

by retracting
laboratory and

into

their

field

observations suggest that neogastropods

shell.

are not subject to particularly intense predation. For example, a number of species (Leucozonia nassa, Stramonita

haemastoma,

S.

nistica.

Pisania

tincta,

and Melongena

quickly nor re-orient themselves

their shells are overturned (pers. obs.). I have

corona) neither

retract

quickly when
found wild specimens of Latims mediamericanus (fascio-

of

damage on

J.

Vermeij, pers. comm.).

Vermeij (1978) has observed that, within at least the

families Vasidae and Mitridae. columellar folds are more

ing of latitudinal diversity trends

escape from their predators

Moreover, my qualitative

apically.

1982; Johannesson,

may be no evidence

quantitatively, although

to

more

columella, as there are with failed attempts at predation in

other parts of the shell, because when these attacks succeed,

Despite these overall similarities, the shallower attachment depth of species with folds implies that columellar

do not help gastropods

contrast, N.

1986); folds are simply ill-placed to affect either of these

sodicity in growth.

folds

In

the columella harder to break

common

although, admittedly, there

half.

However, predators of gastropods rarely break the inner lip.

Most predators either crush the shell at its apex or peel back

were few specimens in

the higher mode. One species, Stramonita haemastoma, had
specimens in both modes. This bimodality may reflect epi(Fig. 3),

in

lapillus individuals with thickened columellae are attacked

at the apex of the shell. Both N. lapillus phenotypes lack
folds, but these observations imply that strengthening the

in tropical species, suggesting that the presence of

folds might be correlated with latitude and increased predation intensity. This observation should certainly be tested

Columellar folds

gence

is

it

may

is

difficult to interpret the

(Roy

et ai.

not have a function.

mean-

1998).
Still,

conver-

considered to be some of the best evidence for

adaptation (Raup and Gould, 1974; Harvey and Pagel, 1991;

Larson and Losos, 1996), and columellar folds have evolved
at least six

times within different families of neogastropods

on how fold shape has

(Price. 2001). Direct observations

evolved over time

may

reveal patterns of evolution that are

would require a

consistent with adaptation. This approach

phylogenetic context (Harvey and Pagel, 1991; Larson and

Losos. 1996), although there are currently no well-resolved

363

FUNCTION OF COLUMELLAR FOLDS

phylogenies that include a sufficiently large number of
an
species with and without folds to give power to such
it
method,
With
the
phylogenetic comparative
analysis.

berg provided helpful suggestions that greatly improved this

paper. This material is based upon work supported by the

determine whether folds evolved by

with
demonstrably adaptive characters
"hitchhiking" along
Smith
and
Haigh, 1974).
(Maynard

NSF

would be possible

On

to

may have evolved as a common

a number of problems, especially because the

the other hand, folds

solution to

function of folds in pulmonates obviously differs from that

in neogastropods. Functional experiments on species from a

number of smaller clades

that contain closely related species

with and without folds should be conducted to identify

functions unique to those smaller clades. If columellar folds
serve different functions in different clades, then they might
be an easy-to-evolve, flexible solution to a number of prob-

and the University of Chicago Hinds and Gurley

Funds provided additional support.

Station,

Literature Cited
American

Abbott, R. T. 1974.
hold,

New

Si-ashclls.

2nd

Columellar folds probably do not guide the columellar

muscle. Rather, the motion of the muscle is likely deter-

mined by muscle fiber orientation and by the attachment to

the shell, which is along the upper edge of the muscle. The
probably responsible for

geometry of the attachment

adhesive strength, and the weak physical connection between muscle and shell is not. The methods employed here
show no association between the presence of folds and the
is

its

length of columellar muscle attachment, surface area of

attachment, or depth of attachment. The widely observed

morphology between
mellar muscle may be due simply
similarity in

the folds

and the colu-

to their physical juxta-

with
position rather than to any functional relationship,
functions.
unknown
and
some
other
folds having
presently
Because folds have evolved multiple times, the most plausible explanation for their existence

an easy-to-evolve solution to a

might be

number of

that they are

functional de-

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Chris Degni for

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Eshel helped debug the programs that

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Greg Farley. Cheryl Swanson, Jessica Cande, the Smithsonian Marine Station, and the Florida State University Ma-

Lab helped with

field

collections.

Janeen Jones, and Martin Pryzdia

at the

Jochen Gerber,
Field

Museum

of

helped deposit specimens. Additional

thanks to Michael LaBarbera, David Jablonski, Riidiger

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FUNCTION OF COLUMELLAR FOLDS

365

Appendix
Measuring True Surface Area and Location on a Shell from a Series of Photographs

Measuring surface area

photograph defines an XF-plane that contains an object

(Appendix Fig. 1A), and the pixels within the object are
"on," whereas those outside the object are "off." That object
is a projection of a surface, Z, onto the XT-plane. The
projected surface area,

SA Z

is

defined as

dx

Because a

Thomas and

digital

(AD

is

with respect to A and the

in the object corresponded to one circle, and all

were centered about the X-axis, which is also the

Each row
circles

coiling axis.

Anyone

familiar with the shape of neogastro-

columella is somewhat asymImposing symmetry on the columella inflated true

surface area, but this deviation was uniform across ranthat the

domly sampled specimens with and without

Finney, 1984).

photograph

metrical.

\dxdy

dy

of

with respect to y by assuming that the

object was a series of circles stacked on top of each other.

inflation,

(equation 6a in

partial derivative

pods will recognize

dzV

SA 7 =

found the

partial derivative of

pixelated,

approximated

SA Z by calculating the surface area of each

summing those values over all n pixels:

pixel,

then

/,

st.

dev.

= 15%.

folds

Consider one row of pixels, /. of the photograph. The

number of "on" pixels in that row equals the diameter of the
because the circle was projected straight onto that
row. Thus, the circle's radius and origin were both known,

circle,

the surface function,

z,,

for a given

row was

(A2)

where A,
1,

is

the area of the pixel. Since the area of a pixel

A2

Equation

(28%

10).

(A4)

(Appendix Fig. IB), and the

was

partial derivative

is

-x n

reduces to

(A5)

dx n
(A3)

where p was any pixel

The

in

row

partial derivative of

of the object.

in the .v-direction

represented

the distortion of the projection due to the curvature of the

circle

Y=

Coiling Axis

about the coiling axis. Therefore, a pixel located

immediately on or above the coiling axis did not have any

horizontal distortion, and the

two pixels 90 from the

coil-

ing axis were the most severely distorted.

To calculate the partial derivative of Z with respect to y.
I

expressed the edge of the image as /( y

Equation

A4

2
.v

1.

(A)

shell in a

as one-halt the

number of "on"

define the target region. 45

is

the radius of a circle,

pixels in a row.

Dashed

on either side of the coiling axis;

v is the

curved surface projects a 180

bold is/( y) = ;. (B) The projection of a circle onto the

X-axis is perpendicular to the page. 8 is the angle from the coiling axis, and

coiling axis, the

the coiling axis always corresponds to a

known, and x

is

landmark) can be calculated.

landmark

line in these

and

(the

photo-

from the coiling

angle between P, and the

the projected distance

axis to P,, both c (the surface function)

fitting a

second-degree

derivative

dz/dy was

in pixels.

Identifying a target region

when

The curve in
XZ-plane. The

is

(A6)

lines

distance from the coiling axis to the edge of the target region.

graphs. Since r

Rewriting

photograph can be thought of as

a stack of circles centered on a coiling axis. Y. r

measured

v)

evaluated from this polynomial.

The resulting area was measured

Appendix Figure

r.

=/(.v,)

was found empirically by

polynomial to the edge. The partial

A.

gives:

v,

where /(

sector onto a row, so

a series contains photographs taken every

photographs overlap.

90 about a

eliminated overlap

by using only the middle of each image (dashed lines in

Appendix Fig. 1). This approach has the added benefit of
using the region of the photograph with the best focus and
least distortion.

366

R.

One row
onto

its

of "on" pixels represented a circle projected

The coiling axis marked the midpoint of

diamei'-T.

the diameter, and the

45

from

see that
triangle

M. PRICE

its

end points of the row were 45 and

we draw the circle, we can

Ihe coiling axis. If

radius

(Appendix

is

between the coiling axis and P,. Here, the ^-coordinate was
known, but the triangle was not necessarily isosceles, so the
angle,

Fig. IB).

The

sides of this triangle equal

that defined the arc

used the same circle to locate point

aperture (Appendix Fig.

attachment. In this case,

relative to the

edges of the
determined the X-coordinate of
to calculate the

from the photograph, which

asin

(A7)

Pj was apertural to the nearest landmark,

subtracted 6

from the landmark's depth. Suppose, for example,

Measuring degrees from aperture

1),

\r
If

the point

was not known. The angle

the hypotenuse of an isosceles right

the .v-coordinate for the boundary of the target region.

t),

between the coiling axis and P, was calculated:

is

simply the distance

that P,

was depth of attachment, the curve /(v) in Appendix Figure

1
was coincident with the landmark at 270, and
equals
20. The value of attachment depth would be 290. If P,
were apical to the nearest landmark, then
added
to the landmark's depth.

would have

Reference: Biol. Bull. 205: 367-376. (December 2003)

Marine Biological Laboratory

"' _(MI3

Nematocyst Uptake by the Nudibranch

Flabellina verrucosa to the Presence of Various
Predators in the Southern Gulf of Maine

Response

in

KINSEY FRICK

NOAA

Fisheries. Northwest Fisheries Science Center, Fish

2725 Montlake Boulevard

East, Seattle,

Introduction

Aeolid nudibranchs maintain nematocysts se-

Abstract.

questered from their cnidarian prey for protection against

predators. Selection for nematocyst incorporation is a func-

Prey anti-predator responses are crucial to prey survival,

though studies of predator-prey coexistence often focus on
predator characteristics (for reviews see Murdoch and

and prey choice, but ratios vary among nudibranchs feeding on a given diet, indicating that other factors
tion of diet

may

be involved.

It is

proposed

1978: Taylor, 1984). Prey species

reduce their mortality from predation by using a variety of
tactics. Mobile animals may be protected by armored or

Oaten, 1975; Hassell.

that the presence of pred-

ators influences nematocyst incorporation. Nematocyst up-

take in the nudibranch Flabellina verrucosa collected from

the southern

Gulf of Maine was examined

cryptic morphologies, noxious chemicals, and a variety of

response to
various potential predators, including Crossaster papposus,
Tautogolabrus adspersus, and Carcinus maenas. Nudibranchs in individual flow-through containers feeding on a
diet of the hydroids Tubiilaria spp.

were subjected

in

escape behaviors (Edmunds, 1974; Pianka, 1983; Havel,

1987). In marine communities, opisthobranch nudibranchs

would seem

and Obelia geniculata

to tanks containing a predator, then their

verrucosa responded to both

papposus by

T.

refuge, nudibranchs have developed various defensive strategies, including avoidance behaviors, cryptic or aposematic

coloration, spicules. toxic secretions, and stinging nemato-

ailspersus and C.

(summarized by Harris, 1973). They are slow-moving

and often flamboyantly colored or on contrasting substrate,
but despite their being seemingly easy prey, there are few
cysts

significantly increasing microbasic mastigo-

phore incorporation. No differential uptake was seen with

C. maenas. Response was evident in the nudibranchs both
for predators present in the collection area

reports of predation on nudibranchs

and for those

with which they had no previous exposure, indicating that

F. verrucosa modulates nematocyst incorporation in re-

in

amastigophores;

MM.

HoA,

nudibranch defense

tactics.

nematocysts that they acquire, through ingestion. from their

cnidarian prey (Thompson, 1960; Edmunds, 1966; Thompson and Bennett, 1969; Greenwood and Mariscal, 1984a. b).

II October 2002: accepted 15 September 2003.

E-mail: Kinsey.Frick@noaa.gov.

microbasic euryteles:

strating the efficacy of

of cnidarian nematocysts. For armament against predators,

these nudibranchs maintain an arsenal of functional stinging

Received

erotrichous anisorhizas: HI, holotrichous isorhizas;

1992: Proksch. 1994). thus

aspect of predator-prey interactions unique to the

defensive strategies of some aeolid nudibranchs is their use

nematocyst uptake.

Abbreviations: BI. basitrichous isorhizas; DS. desmosomes;

(Thompson. 1976: Kademon-

ruso, 1987; Faulkner.

An

sponse to the presence of predators as well as to diet. A

coevolution of nudibranchs and potential predators may

govern changes

a likely prey item since they are not protected

against predation by the shell employed by prosobranch

gastropods. To compensate for the lack of a natural physical

nematocyst distribution was examined. Although most of

the changes over the experimental period were attributable
to diet, F.

Ecology Division,
Washington 981 12

HeA,

Nematocysts pass through the digestive tract to the tips of

the dorsal cerata, where they are incorporated into special-

het-

HME, heterotrichous
MA, microbasic

ized cavities called cnidosacs and are maintained in a func-

holotrichous anisorhizas:

tional state (Conklin

microbasic mastigophores: ST. stenoteles.

367

and Mariscal, 1977: Greenwood and

K.

368

The nematocysts

Mariscal. 1984a).
ejected by the

>ich.

ibi

are then available to be

presumably

as a defense

mech-

FRICK
whether population-level variation in nematocysts sequestered by F. verrucosa provides a link between nematocyst
incorporation and predation pressure.

anism.

While

it

ell

is

understood that aeolid nudibranchs store

their prey, the dynamics of

not
are
selection
explicit. There are more than
nematocyst
25 types of nematocysts in cnidarians (see Mariscal, 1974),

Materials and Methods

cnidanan nematocysts from

prey acquisition and degiven cnidarian are a function

each with different functions

fense,

and those present

in a

Study organisms
Flabellimi

in

(formerly

branchialis) (Sars, 1829)

verrucosa

Coryphelld)
is

common

(=

rufi-

aeolid nudibranch in

of the species. Therefore, specific nematocysts are present

the shallow marine subtidal throughout the Gulf of Maine.

varying combinations and proportions among nudibranch

prey species (Mariscal, 1974: Calder, 1988). Nudibranch

Its

in

nematocyst incorporation is a function of availability in the

so depending on the cnidarian prey they consume and
on which parts of the prey, nudibranchs sequester different

distribution

is

circumboreal: in the Atlantic

its

range

includes northern Europe (British Isles, Norway, and Iceland) and Greenland south to the Gulf of Maine; in the

found off British Columbia and

diet,

Pacific F. verrucosa can be

kinds of nematocysts within their cnidosacs. Additionally,

the coast of Russia (Bleakney, 1996).

generalist predator.
F. verrucosa consumes numerous athecate hydroid species,

they can preferentially select specific types from what is

available (Grosvenor. 1903; Day and Harris. 1978). The

scyphistomae, and tunicates (Kuzirian, 1979). No predators

are known to prey primarily on this nudibranch.

nematocyst complement serves as a measure of feeding

history, as nudibranchs incorporate a small number of all

to a variety of predators,

nematocyst types found

in the

prey they have been consum-

ing. However, individuals of a given nudibranch species

feeding on the same diet sequester varying proportions of

those nematocyst types (pers. obs.), indicating that factors

other than strict availability must be involved in selection.

verrucosa was exposed

and nematocyst uptake was com-

In the following experiments, F.

pared with uptake

in

which

F.

predators to

the absence of predator cues. The

verrucosa was exposed included the

wrasse Tautogolabrm adspersus (Walbaum, 1792). the sea

star Crossaster papposus (Linnaeus, 1767), and the green
crab Carcinux maenas (Linnaeus, 1758). Crossaster pappopresent in cold deep waters in the southern Gulf of
collection area, though none were observed at the

may influence nematocyst uptake, and

therefore different nematocyst types may be selected in

sus

response to specific predators. Since nudibranch nematocysts purportedly function as predator deterrents, predator
cues may affect nematocyst incorporation such that nudi-

depths where nudibranchs were collected for this study.

While not a nudibranch specialist, C. papposus feeds on

branchs maintain weapons capable of combating predators

specific to the area in which they live. Edmunds (1966)

tered in the field

Predation pressure

is

Maine

nudibranchs, including F. verrucosa,

(Mauzey

et at.,

when

they are encoun-

1968) (pers. obs.).

Among

be more effec-

other prey, the wrasse T. adspersus (cunner) is known to

feed voraciously on some nudibranch species, but shows an

penetrants for use against fish

predators
and adherents against crustaceans, for example so nema-

aversion to consuming F. verrucosa (Harris, 1986; pers.

obs. ). Cunner are seasonally abundant in the southern Gulf

suggested that certain nematocyst types

tive against

may

some

tocyst incorporation

may be based on nudibranch

predators

its

of Maine

in the

summer and

fall

when water temperatures

Despite studies of individual species populations, few studies have examined variation with respect to

are mild, but they are absent during the colder winter and

the predation pressure encountered

nas

in the vicinity.

tion.

The objective of

this

by the organism

in

ques-

study was to identify changes in

nematocyst uptake by nudibranchs in response to chemical

cues from potential predators. My work examines the incorporation of nematocysts by nudibranchs in response to
individual predator species and considers both predation
pressure and the nudibranch' s previous experience. To further elucidate the relationship between nematocysts in nudi-

branch cnidosacs and

predation

pressures,

examined

spring seasons.

The omnivorous green crab Carcinus mae-

common

both intertidally and subtidally throughout

is

Gulf of Maine, but unlike some crab species reported to

prey on certain nudibranchs (Harris, 1970; Ajeska, 1971),

the

maenas is not a known nudibranch predator.

The experience that specimens of F. verrucosa collected
in the southern Gulf of Maine have had with the predators
used in this study ranges from common exposure (C. maeC.

nas and

T.

adspersus) to no probable exposure (C. pappo-

sus) for nudibranchs of the collected generation.

Depending

may be

nematocyst uptake in the nudibranch Flabellimi verrucosa

with and without exposure to potential predators. I hypoth-

on collection

esize that (a) the presence of specific predators differentially

affects nematocyst uptake, (b) response depends on the

Shoals are less likely to have encountered T. adspersus than

those from Nubble (description below). At Nubble, fish

nudibranch's previous exposure to the predator, and (c)

response depends on the predator's ability to prey on the

forage and live on the wall where nudibranchs were collected, but at Shoals, the mooring lines do not support

nudibranch.

By

testing these hypotheses

will

determine

differences in exposure
from
nudibranchs
collected
to cunner:
mooring chains at
location, there

substantial populations of the predatory fish. For

all

collec-

369

NUDIBRANCH RESPONSE TO PREDATORS

tion sites, nudibranchs are unlikely to be

have been exposed to C. papposus prior to collection.

However, parental generations of F. verrucosa may have
been exposed to the entire suite of experimental predators in
to

other areas within the species' range, presenting the possia

bility that nematocyst uptake could be influenced by

revolutionary response based on exposure of previous

generations to the predators used in this study.

Nematocvst types found upon examination of cnidarian prey

tissues

Nematocysts apparent
in tissue*

Hydroid species

MM. MA

Obelia geniculaiti
Ttthuiiiria indivisa, T.

ST. DS. HeA.

crocea

Nematocyst types are abbreviated as follows:

MM.

HME.

BI

microbasic mas-

MA,

microbasic amastigophores; ST. stenoteles; DS, desmonemes; HeA. heterotrichous anisorhizas; HME, heterotrichous micro-

tigophores;

Specimen collection
Nudibranchs were collected from two locations within
the southern Gulf of Maine: Cape Neddick (Nubble) in
York. Maine (439'54"N. 7035'29"W). and Gosport Harbor near Appledore and Lunging Islands of the Isles of
Shoals island group located about 10 km off the coast of

New

Table

found deep enough

Hampshire (4259'21"N, 7036'54"W). The shallow

Cape Neddick and the Isles of Shoals are both

subtidal of

algal-dominated, gradual slopes containing vertical rock

surfaces and undercuts dominated by animal communities.

Nudibranch populations at these sites are of the same genetic stock due to site proximity and widespread dispersal of
planktonic veliger larvae, but their post-settlement feeding
histories may differ due to the availability of prey items at

two locations.
Between 30 and 50 specimens of Flabellina verrucosa
were collected from vertical rock wall surfaces at Nubble in
3-8 m of water, and also from blooms of the hydroid
Tubularia crocea on mooring ball lines near the Isles of
Shoals (Appledore and Lunging Islands) in October and

the

November 2001 and January 2002. Animals were maintained following collection and for the duration of all experiments at 10 C in a constant-temperature room at the

University of New Hampshire. Tanks were filled with natural seawater obtained from the Coastal Marine Laboratory
the Portsmouth Coast Guard Station at the mouth of
Portsmouth Harbor <434'20"N. 7042'37"W). Animals for
each site were kept together and used separately in each
at

basic euryteles; BI, basitrichous isorhizas.

and O. geniculata from pilings at the Portsmouth Coast

Guard Station. The hydroids used as prey were not fed
during the experiment and have no direct ecological relationships with the predators used in the study.
Experimental flow-through containers remained in the

tanks for 2 weeks, with a fresh replacement of hydroid food

and a partial water change in the tanks after 7 days. Control
tanks had five containers for each nudibranch population
randomly distributed among the tanks. After 2 weeks of

exposure to the experimental conditions, the nematocyst

content of each nudibranch was evaluated by examining
ceras squashes for three cerata per animal via light microscopy. Cerata were removed from the anterior region in the
or second ceratal cluster by using forceps combined
with the animals' propensity to autotomize these projections. For each ceras, 100 nematocysts (identification acfirst

cording to Mariscal, 1974; see reference for visual representations) were categorized on the basis of visual

view included more than 100

were counted). Counts included

characteristics (if the field of

all in

the field

nematocysts,
both fired and encapsulated (unfired) nematocysts. The
setup was repeated with each predator and for each nudibranch collection site, adjusted as described below. The

numbers of
in

Table

replicates for each experiment are

summarized

2.

experiment.

For the C. papposus experiment, ten 2.5-gallon tanks

containing a sea star measuring 2.7-6.5 cm and three 2.5-

Experimental setup

gallon tanks without a sea star were established. Each tank

contained one container for each nudibranch collection site.

Experimental tanks containing a predator were estab-

were starved for the duration of the experiment

to eat regularly once brought into the

lished 1-2 days before experiment inception. Control tanks

The sea

did not contain a predator. Following initial nematocyst

counts (procedure described below) for each nudibranch

because they refused

population, nudibranchs were placed in individual flowthrough containers with two hydroid food sources. The

hydroids used were Obelia geniculata (Linnaeus. 1758) and

Tiihiilnria spp. (crocea and/or indivisa), each present in

excess so that food was not a limiting factor. Examination of

the tissues of these cnidarian prey species revealed mutually
exclusive nematocyst complements (Table ), so they offer
a variety of nematocyst types. Tubularia spp. were collected
1

from the nudibranch collection

sites at the Isles

of Shoals,

stars

The experiment using the cunner T. adspersus

was conducted similarly, but due to limited numbers of

laboratory.

captive fish,

it

involved six 10-gallon tanks, each containing

two flow-through containers for each experimental site. The

cunner in the tanks measured between 7 and 17 cm and were
fed locally collected Mytilus edulis meat every other day.
When the green crab Carcinus maenas was used as the
predator, tank setup and size

was equivalent

to that

used for

papposus experiment, with crab carapace size rangfrom

5.5 to 7.0 cm. Crabs were fed frozen cooked
ing
the C.

370

K.

FRICK

Table

Summary of experimental

design, including

number of replicate flow-through containers for each population

inui each treatment

NTDIBRANCH RESPONSE TO PREDATORS

Nubble
80

Shoals
7060
50

40

3020

10

ST

DS

HoA

HeA

HME

MM

Bl

Nematocyst Type

80

70

6001

50

if

40

-30
20
10

DS

HoA

HeA

HME

MM

Nematocyst Type

80-

MA

HI

371

372

K.

FRICK

Table 3
Significant clim<:

individual

<

<

t<

'.r.'cv.w

and

the

incorporation between experimental and control Flabellina verrucosa when exposed to each predator for each

combined southern Gulf of Maine

NUDIBRANCH RESPONSE TO PREDATORS

373

explain the observed increase in MM, as O. geniciilata is the

source of this nematocyst type for the experiment. However,

nematocysts from the Tubularia spp. tissue (ST, DS, and

HME) were also present at high levels in these animals.
Although nudibranch feeding preference was not quantified,
the experiments were monitored or cnidarian prey
were changed, nudibranchs were observed more often on
the Tubularia hydroids. This observation and the presence

when

of tubularian nematocysts suggest that a superficial preference for consuming O. geniculata in the face of predators
cannot solely explain the increase in microbasic mastigophores.

The

70

"

ability

of a prey species to react by optimizing

374

K.

70

FRICK

NUDIBRANCH RESPONSE TO PREDATORS

the selective pressures that have brought about their forma-

Day, R. M., and L. G. Harris. 1978.

terate

tion.

nematocysts

Dixon, C. A.,

Some

Harris and Rebecca Toppin helped with formulating ideas

and collecting specimens for this work. Additional thanks
to

go

Glen Rice

for hydroid collection

and Chris Neefus for

Comments from Lauralyn Dyer and Jenn

statistical advice.

Dijkstra improved the experimental design, and Larry HarAnne Stork, and the members of the writing seminar

ris.

made

helpful suggestions on earlier drafts of this paper. This

research was supported by funds from the University of

New

Hampshire Center for Marine Biology; the National

Sea Grant College Program of the National Oceanic and
Atmospheric Administration, Department of Commerce,
under grant number N A56RGO 59 to the University of New
Hampshire/University of Maine Sea Grant College Program: and by additional funds provided by the Open Ocean
1

NOAA UNH CoopEngland Mariculture and Fisheries

Aquaculture project component of the

erative Institute for

New

NOAA

(CINEMAR),

grant

number NA16RP1718.

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INDEX
Behavior, 218. 222. 225

ABRAHAM.

M.

D. M.. M. A. CHARETTE.

C. ALLEN, A.

RAGO, AND K. D.

KROEGER, RacJiochemicul estimates of submarine groundwater

charge to Waquoit Bay. Massachusetts, 246
Actin, 195

dis-

Behavioral characterization of attractin, a water-borne peptide pheromone

in the genus Aplysiu. 16
BELL. J. J., AND D. K. A. BARNES. Effect of disturbance on assemblages: an
example using Porifera. 144

308
S-adenosyl-L-methionine:farnesoic acid 0-methyl transferase,

BENINGER, PETER G.. GAEL LE PENNEC. AND MARCEL LE PENNEC. Demonstration of nutrient pathway from the digestive system to oocytes in
the gonad intestinal loop of the scallop Pecten maximus L.. 83

Aggregation,

BERGMAN. DANIEL

ring, 192

on the Gorda
Aggregations of egg-brooding deep-sea fish and cephalopods
Escarpment: a reproductive hot spot.
Aggression. 26

AND PAUL A. MOORE. Field observations of intraspebehavior of two crayfish species, Orconectes rusticus
and Orconectes virilis. in different habitats. 26

AGNEW, A. M.. D.

H. SHULL.

AND

BUCHSBAUM. Growth

R.

marsh grass

invertebrate on several species of

AGUIAR. A. B.. see J. A. Morgan, 252

of a salt marsh

detritus,

238

B..

J.

A.

Bindin. 8
Biological beam. 36
Bioluminescence. 102

Biosensor. 207

Fox, AND

VALIELA.
relative effects of ambiTransplantation and isotopic evidence of the
ent and internal nutrient supply on the growth of Viva lactuca. 250

AGUIAR, A.

A..

cific agonistic

MORGAN. M. TEICHBERG,

S.

I.

Bivalve. 83
gills.

73

BLACK, SARA, see Sherry D.

Painter. 16

Alexandrium, 231

Blastoderm. 179

Alkaline phosphatase. 230

ALKON. D. L., see F. M. Child, 218; A. M. Kuzirian. 220
ALLEN, M. C.. see J. M. Talbot. 244; D. M. Abraham, 246

Blob sculpin.
Blood clotting, 201. 203
BOGORFF, DANIEL J.. MARK A. MESSERLI. ROBERT P. MALCHOW. AND PETER
J. S. SMITH. Development and characterization of a self-referencing
207
glutamate-selective micro-biosensor,
BOLLER, MICHAEL L., see Howard R. Lasker. 330
1

Allogeneic interaction. 133

Ammonia. 331

Ammonium. 244
Arnphipod. 252
An experimental approach to the study of gap-junction-mediated

BONAVENTURA, CELIA,
cell death,

197
see Madeline Galac. 231

Animation. 225
Antarctica, 93
Apl\\ia, 16

Apoptosis, 197, 199

allografts.

285
species in the Cryptasterina species complex,
cell death, 197

Bystander

Limuhts blood clot. 201

M. Harrington, 205
Armstrong, Peter B.. see Victoria Isakova, 203; John
Ascidian. 133
Aster, 193

C-reactive protein, 201

Calcium. 176. 215

Astogeny, 330

mobilization. 185

Anna Savage.

see

222: K. D.

Mann. 224

Calexcitin.

220

Attractin. 16

Capitella. 182

Axon. 187, 188, 190

Axonal transport. 188. 190

CARMICHAEL. R.

H.. see C. W. O'Connell, 254

Antarctica:
Cascading trophic impacts of reduced biomass in the Ross Sea.
Just the tip of the iceberg?, 93

Axoplasm. 188
Axotomy, 187
inhibits the

slow axonal transport of tubulin

in the

Caspase-3. 199
CASTANARO, JOHN, see

Howard R. Lasker, 330

Catalase in microsporidian spores before and during discharge. 236
Catch connective tissue. 261

squid giant

axon. 187

CAVATORTA. J. R.. see M. E. Johnston, 248

CAVATORTA. JASON R.. MORGAN JOHNSTON. CHARLES HOPKINSON, AND
VINTON VALENTINE. Patterns of sedimentation in a salt marsh-domi-

B
Bacteria, 228,
Bacteriolysis,

DeSelm. 190

BURTON. O. T.. see S. J. Zottoli. 21

BYRNE, MARIA, MICHAEL HART, ANNA CERRA, AND PAULA CISTERNAS.
and viviparous
Reproduction and larval morphology of broadcasting

199

ARMSTRONG. MARGARET T.. see Peter B. Armstrong. 201

ARMSTRONG. PETER B.. AND MARGARET T. ARMSTRONG, The decorated clot:
to the fibrils of the
binding of agents of the innate immune system

Axotomy

J.

Microciona prolifera
Aquifer. 244, 246
in

J..

Beardsley Christensen, 54

detect landscape change. 257

Building a database of historic land cover to
BURGER. M. M.. see S. Tepsuporn. 199

Anthopleura, 66, 339

ATEMA,

Ana

BOYER, BARBARA C.. see Susan D. Hill. 182

Brachiolaria. 285
Brittle star. 54
BROWN. JEREMIAH R.. see John Delacruz, 188; Carl
BUCHSBAUM. R.. see A. M. Agnew. 238

AnuJara, 73

ANDERSON, DONALD M.,

see

Bottom-up. 252

233
203

nated estuary, 239

Cecum. 47

BAIRD, KRYSTAL D., HEMANT M. CHIKARMANE, ROXANNA SMOLOWITZ, AND

KEVIN R. UHLINGER, Detection of Edwardsiella infections in Opsanus

Cell

adhesion, 220

hut by polymerase chain reaction, 235

BAKER. ROBERT, see Edwin Gilland. 176

BARNES. D. K. A., see

J. J.

cycle. 195
division, unequal, 192

Bell. 144

377

INDEX TO VOLUME

378

Centrosome, 193
Cephalopod. I, 47

'

DAVIDSON. E., see C. Walker. 256

DAVY. SIMON K.. AND JOHN R. TURNER. Early development and

Cercaria, 110

na Byrne. 285

CERRA, AN\

CHADWICK-T

.,

NANETTE

E.,

^ciilosseri in

A., see S.

J.

AND IRVING

Monterey Bay.
J.

Zottoli. 21

L.

WEISSMAN, Effects of

33

fibrils

L.. see S. Redenti. 213

CHAPPELL. R. L.. J. ZAKEVICIUS. AND H. RIPPS. Zinc modulation of
hemichannel currents in Xenopus oocytes. 209
Characterization of phosphorus-regulated genes in Trichodesmiwn spp., 230

A., see

J.

M. Talbot, 244; D. M. Abraham. 246

Chemoautotrophy. 331
Chemoreception. 222

clot: binding of agents of the innate

of the tinnitus blood clot, 201

tn the

Deep-sea. 102
DELACRUZ, JOHN, JEREMIAH R. BROWN. AND GEORGE M. LANGFORD, Interactions between recombinant conventional squid kinesin and native

myosin-V, 188
Demonstration of nutrient pathway from the digestive system to oocytes
the gonad intestinal loop of the scallop Pecten maxiinim L., 83

in

DENK, WINFRIED. see Edwin Gilland. 176

DEPINA. ANA S.. see Torsten Wbllert. 195
Description of Vibrio alginolyticus infection

CHILD. F. M.. H. T. EPSTEIN. A. M. KUZIRIAN, AND D. L. ALKON,

reconsolidation in Hermissenda, 218

Memory

ANA BEARDSLEY. JAMES M. COLACINO. AND CELIA BONAVEN-

TURA, Functional and biochemical properties of the hemoglobins of

the burrowing brittle star Hemipholis elongata Say (Echinodermata.
Ophiuroidea). 54
182

squid giant axon. 190

Detection of Edwanlsiella infections
reaction.

CISTERNAS, PAULA, see Maria Byrne. 285

Cleavage. 179

Detritus.

pagurus, 308

in

Opsaiius run by polymerase chain

235

Detntivore. 238

CLOUGH, BRET, see Sherry D. Painter, 16

COHEN, W. D., see R. M. Pielak. 192
COLACINO. JAMES M., see Ana Beardsley Christensen, 54

cultured Sepia officinalis.

DESELM. CARL J.. JEREMIAH R. BROWN, RENNE Lu, AND GEORGE M.

LANGFORD. Rab-GDI inhibits myosin V-dependent vesicle transport in

Determinate growth and modularity

a putative
Cloning, characterization, and developmental expression of
farnesoic acid O-methyl transferase in the female edible crab Cancer

in

Sepia apama. and Sepia pharaonis, 233

DESELM, CARL J., see Torsten Wollert. 195

Cilia.

in a

gorgonian octocoral. 330

26
Development. 181, 182. 285
nonfeeding. 295
Development and characterization of a self-referencing glutamate-selective
micro-biosensor, 207
DIERSSEN. HEIDI M.. see Brad A. Seibel, 93
Digestive gland. 47
Dinoflagellate, 231

Colonial invertebrate, 133

Disease. 233, 235

Colony growth, 330

DON. 256

Columellar

Dorsal

fold,

immune system

Denitrincation, 242

CHENEY, DANIEL P., see Amro M. Hamdoun. 160

CHERR, GARY N., see Amro M. Hamdoun. 160
CHIKARMANE, HEMANT M.. see Krystal D. Baird, 235
CHILD. F. M.. see A. M. Kuzirian. 220

CHRISTENSEN,

Decapod, 26

The decorated

CHAPPELL. R.

CHARETTE. M.

the acquiof zooxanthellae in the temperate symbiotic sea anemone Anthopleum ballii (Cocks). 66
sition

the colonial ascidian Botallogen.ic contact on life-history traits of

CHAMULKS.

205

cell,

211

C.. SHANA K. GOFFREDI, BRIAN SCHLINING, AND DEBRA S.

STAKES. Aggregations of egg-brooding deep-sea fish and cephalopods
on the Gorda Escarpment: a reproductive hot spot. 1
Dreissena. 73
DYHRMAN. SONYA. see Elizabeth Orchard. 230: Madeline Galac. 231
Dynamic mechanical properties of body-wall dermis in various mechanical
states and their implications for the behavior of sea cucumbers. 261

DRAZEN, JEFFREY

351

muscle, 351

Columellar muscle of neogastropods: muscle attachment and the function

of Columellar folds. 35 1

Community composition. 144

Competition, 133
Conditioning, associative, 220

Connective tissue
catch, 261

mutable. 261

Connexm,

197.

Early development and the acquisition of zooxanthellae in the temperate

66
symbiotic sea anemone Anthopleura ballii (Cocks).

209

Cooperativity. 54
Coral, 339

Echinoderm, 54, 261

Edwardsiella tarda. 235

Coral reef, 330

Corbicula, 73

CRAWFORD,

K.,

Lithium chloride inhibits development

along the animal

vegetal axis and anterior midline of the squid embryo. 181

CRAWFORD, K., see P. H. Wadeson. 179

Crustacea, 26, 308

Cryptasterina, 285

Cryptic species, 285

CUSATO, K.. J. ZAKEVICIUS,

AND H. RIPPS, An experimental approach to the

study of gap-junction-mediated cell death. 197

Cuttlefish,

233

Biitryllus schlosseri in

Monterey Bay, 133

Electron microscopy, 177

Embryonic development. 308

Epizootic lobster shell disease. 228
EPSTEIN, H. T.. see F. M. Child, 218; A. M. Kuzirian. 220
ERDNER, DEANA, see Madeline Galac. 231

ESEH. R., see

Estuary.

S. J. Zottoli.

211

10

ETNIER. SHELLEY A.. Twisting and bending of biological beams: distribu-

Cx35. 209
Cx38, 209
Cyclopia,

Effect of disturbance on assemblages: an example using Porifera, 144

Effects of allogeneic contact on life-history traits of the colonial ascidian

tion of biological beams in a stiffness mechanospace. 36

Eutrophication. 242, 252
1

Cytochrome

c,

197

Cytokinesis, 192

Cytoskeletal eve:;t-, preceding polar body formation in activated Spisula

eggs, 192

Cytoskeleton, 192

Expressed sequence tag, 227

Expressed sequence tag analysis of genes expressed in the bay scallop.
ArgopecJen irradians. 227
Extracellular lipid droplets in Diosepius nowides, the Southern pygmy
squid, 47
Extrinsic factors. 26

INDEX TO VOLUME
EVSTER. L.

S..

AND

L.

M. VAN CAMP,

Diosepius nowides. the Southern

Extracellular Hpid droplets

pygmy

squid.

in

252

algal. 250,

Growth of

47

379

205

a salt marsh invertebrate on several species of marsh grass

detritus, 238
Gulf of Maine, 231
GUTIERREZ, L. M., see

Fasciclin.

S.

J.

Zottoli. 21

Feeding. 93

opposed-band, 295

FERNANDEZ-BUSQUETS,

HADDOCK, STEVEN H. D., see Bruce H. Robison, 102

HAMDOUN. AMRO M., DANIEL P. CHENEY. AND GARY N. CHERR. Phenotypic

X.. see S. Tepsuporn. 199

Fertilization. 8

Field observations of intraspecific agonistic behavior of two crayfish species, Orconectes rusticus and Orconectes virilis, in different habitats.

26
growth. 73
formation. 73

distal

FINDLEY. ANN. see Earl Weidner, 236

FINGERUT. JONATHAN T.. CHERYL ANN ZIMMER, AND RICHARD K. ZIMMER.

Patterns and processes of larval emergence in an estuarine parasite
system. 110
Fish. 211
Fltihfllinii vcrntcosti.

367

Flexibility.

H.. see T. Thorns,

242

Formation of the blastoderm and yolk syncytial layer in early squid

development. 179
Fox. S.. see A. B. Aguiar. 250; J. A. Morgan. 252
FRICK. KINSEY. Response in nematocyst uptake by the nudibranch Flabellina verrucosa to the presence of various predators in the southern

Gulf of Maine, 367

Functional and biochemical properties of the hemoglobins of the burrowing brittle star Hemipholis elongata Say (Echinodermata, Ophiuroidea). 54

GALAC. MADELINE, DEANA ERDNER, DONALD M. ANDERSON, AND SONYA

DYHRMAN. Molecular quantification of toxic Alexandrium fiindyense

GALLANT.

Gulf of Maine. 231

P. E.,

Axotomy

inhibits the

slow axonal transport of tubulin

in

the squid giant axon. 187

Gamete recognition. 8

J.

D. LASKIN, Ryanodine-sensitive calcium flux regulates

GIBSON. GLENYS D., Larval development and metamorphosis in Pleitrobranchaea maculata, with a review of development in the Notaspidea
(Opisthobranchia). 121
GILEADI. OPHER, AND ALON SABBAN, Squid sperm to clam eggs: imaging
wet samples in a scanning electron microscope, 177

GILLAND. EDWIN. ROBERT BAKER. AND WINFRIED DENK. Long duration

three-dimensional imaging of calcium waves in zebrafish using multiphoton fluorescence microscopy, 176
GIS. 257
GIUFFRIDA. B. A., see L. M. Palmer. 216
Glutamate, 207
GOETZ, F. W., see S. B. Roberts. 227
GOFFREDI, SHANA K.. see Jeffrey C. Drazen.
Gonad. 83

W. O'Connell. 254

Groundwater. 242, 244

Growth. 133. 238

Heterochrony. 121
HILL.

SUSAN

D.,

AND BARBARA

C. BOYER,

HNK-1/N-CAM immunoreac-

with ciliary patterns during development of the

polychaete Capile/la sp. I, 182
tivity

correlates

Histidine. 2 3
1

Histopathology, 233

immunoreactivity correlates with ciliary patterns during

development of the polychaete Capirel/a sp. I. 182

HOLDEN, M. T.. C. LIPPITT. R. G. PONTIUS. JR.. AND C. WILLIAMS. Building
a database of historic land cover to detect landscape change. 257
Homarus americamis. 222, 228
HOPKINSON, C. S., see Jason R. Cavatorta, 239; M. E. Johnston. 248
Horizontal

cell,

215

HSP70

family, 160
Hsu, A. C., AND R.

M. SMOLOWITZ, Scanning

electron microscopy inves-

C., see

Homarus americanus, 228

Bruce H. Robison. 102

Hydrothermal vent. 98

GAYSINSKAYA, V. A., see R. M. Pielak, 192

GEFFEN, AUDREY, see Carolyn J. Ruddell, 308
Geographic information system, 257
GERLACH, G., see K. D. Mann. 224; E. R. Turnell. 225
GIBSON. A. E., see T. Thorns. 242

Grazing. 252

Hemolysis, 205
Hennissenda. 218. 220

HUNT, JAMES

junction. 197

S. P.. see C.

Hemichannel, 209
Hemoglobin. 54

tigation of epizootic lobster shell disease in

Gastropod. 93. 276, 351

Gastrulation. 176

Graneledone,

AND

HNK-1/N-CAM

Functional morphology. 351

in the

E.,

motility of Arbacia punctulata sperm, 185

36

FOREMAN. K.

see Anthony J. A. Molina. 215

HARRINGTON, JOHN M.. AND PETER B. ARMSTRONG, A liposome-permeating
activity from the surface of the carapace of the American horseshoe
crab. Limulus polyphemus, 205
HART. MICHAEL, see Maria Byrne, 285
Heat-shock protein. 160. 276
Heat-shock protein 70 (Hsp70) as a biochemical stress indicator: an experimental field test in two congeneric intertidal gastropods (Genus:
Tegula), 276

HECK, D.

Flat worm. 110

GRADY.

HSP70 and HSP70 gene expression in the Pacific oyster

{Crassostrea gigasl: implications for thermal limits and induction of
thermal tolerance, 160
plasticity of

HAMMAR, KATHERINE,

Filament

Gap

339

Iceberg, 93
Idiosepius,

47

Imaging, 177

Immunity
cutaneous, 205
innate, 201. 203.

205

invertebrate, 199

Immunolabeling, 177
Immunolocalization, 339
Importance of metabolism in the development of salt marsh ponds. 248
Imprisonment in a death-row cell: the fates of microbes entrapped in the

Limulus blood

clot.

203

soil yielding maximum dissolved organic

nitrogen concentrations and minimal residual nitrate. 256

Incubation conditions of forest

Induced protein. 160

Interactions between recombinant conventional squid kinesin and native

myosin-V, 188
276

Intertidal zone,

caged calcium in skate horizontal cells using fine

215
Invasive species, 238
ISAKOVA, VICTORIA, AND PETER B. ARMSTRONG, Imprisonment in a deathIntracellular release of

optical fibers,

INDEX TO VOLUME

380
row

cell: the talcs

microbes entrapped

in the

Limulus blood

205

clot.

203

d2-macroglobulin. 201

Macrophytes, 26

MALCHOW. ROBERT
E.. J. R. CAVATORTA, C. S. HOPKINSON. AND V. VALENTINE.
Importance of metabolism in the development of salt marsh ponds.
24S
JOHNSTON. MORGAN, see Jason R. Cavatorta. 239
JOVNER. JOANNA L., SUZANNE M. PEYER, AND RAYMOND W. LEE. Possible

JOHNSTON. M.

roles of sulfur-containing

amino acids

in a

chemoautotrophic bacte-

rium-mollusc symbiosis, 331

MANN,
MANN,

C.. see S.

J.

Tepsuporn. 199

(Danio rerio) based on olfactory cues,

in juvenile zebrafish

J.

A. Molina.

K. D.. see E. R. Turnell. 225

J. ATEMA, AND G. GERLACH, Kin recognition

juvenile zebrafish iDanio rerio) based on olfactory cues, 224
Mantle formation. 2

in

Marine Biological Laboratory

Annual Report, v. 205(1). Rl
General Scientific Meetings, 171
Map, digital land-cover, 257
Mate choice. 225
(Danio rerio} analyzed with video-stimulus

zebrafish

in

Mechanical properties, 261

Memory, 218
consolidated long-term. 218
long-term. 220
reconsolidation, 218

Kinesin. 188

KINGERLEE. W.. see C. Walker. 256

Memory

KROEGER, K. D., see J. M. Talbot, 244; D. M. Abraham. 246

KRON, M. M.. see S. J. Zottoli. 211
KUHNS, W. J.. see S. Tepsuporn, 199
KUZIRIAN, A. M.. see F. M. Child. 218

M. CHILD. H. T. EPSTEIN, M.
OLDENBURG, AND D. L. ALKON. Training alone,

KUZIRIAN. A. M..

ROD,

Bogorff. 207; Anthony

J.

techniques, 225

KAPPES, HEIKE, see Dietrich Neumann. 73

Kin recognition. 224

Kin recognition
224

see Daniel

K. D., E. R. TURNELL,

Mate choice
KALTENBACH,

P.,

215
Mandibular organ, 308

F.

modulates calexcitin

in

E.

reconsolidation in Hermissenda. 218

MENSINGER. A.
MESSERLI,
Metabolic

MOTTA. C.

E.

not the tripeptide

Hermissenda, 220

F..

MARK

see L.

M. Palmer. 216

A., see Daniel

J.

Bogorff. 207

93

rate,

Methyl farnesoate, 308

Methyl transferase, 308
Microscope, polarizing-aperture scanning, 193
Microsporidian catalase, 236
Microtubule. 193
Modularity. 330

Landscape change, 257

LANGFORD, GEORGE M..

see John Delacruz,

188; Carl

J.

DeSelm. 190;

Torsten Wollert, 195

Larval development and metamorphosis in PU'iunhrancliaca inaculata,
with a review of development in the Notaspidea (Opisthobranchia).

Gulf of

J.

A.,

KATHERINE HAMMAR. RICHARD SANGER, PETER

SMITH. AND ROBERT P. MALCHOW. Intracellular release of caged

calcium in skate horizontal cells using fine optical fibers. 215
J.

S.

Mollusc. 16

121

HOWARD R.. MICHAEL L. ROLLER. JOHN CASTANARO, AND JUAN

ARMANDO SANCHEZ. Determinate growth and modularity in a gorgo-

LASKER.

nian octocoral, 330

J.

D.. see D. E. Heck. 185

see C.

J.

252

A., see A. B. Aguiar.

250

Mortality. 133

MOTOKAWA, TATSUO, AND AKIFUMI

TSUCHI, Dynamic mechanical properof body-wall dermis in various mechanical states and their implications for the behavior of sea cucumbers, 261

ties

MOTTA. M.

W. O'Connell, 254

LESSIOS. H. A., see Kirk S. Zigler, 8

Light production by the arm tips of the deep-sea cephalopod Vampyroteuiht\ infernalis, 102

E., see

A.

M.

Kuzirian. 220

Multiphoton fluorescence. 176

Multiple approaches to tracing nitrogen loss in the West Falmouth wastewater plume. 242

Myosin

Limulus. 201. 203. 205. 254

-II.

Lipid. 47

195

-V. 188, 190

Liposome. 205
liposome-permeating activity from the surface of the carapace of the
American horseshoe crab. Limulus polyphetnus, 205

M. T. Holden, 257
Lithium chloride. 181
Lithium chloride inhibits development along the animal vegetal axis and
anterior midline of the squid embryo. 181
Lobster, 222, 228
Localization of a .symbiosis-related protein. Sym32, in the Anthopleura
elegantissimaSymbiodinium inu\{<iiii!t'i association, 339

LIPPITT. C.. see

Long duration three-dimensional imaging of calcium waves

using multipholon fluorescence microscopy. 176

Lough Hyne, 144

Lu. RENNE, see Carl

VALIELA,

Morphology. 330

S.,

I.

Relative influence of grazing and nutrient supply on growth of the

green macroalga Ulva lactuca in estuaries of Waquoit Bay, Massa-

MORGAN.

Learning, 218
LEE, RAYMOND W., see Joanna L. Joyner, 33
LEE. RAYMOND W., Thermal tolerances of deep-sea hydrothermal vent
animals from the Northeast Pacific, 98

LESCHEN, A.

MOORE. PAUL A., see Daniel A. Bergman. 26

MORGAN. J. A.. A. B. AGLIIAR. S. Fox. M. TEICHBERG, AND

chusetts,

216
LE PENNEC, GAEL, see Peter G. Beninger. 83
LE PENNEC, MARCEL, see Peter G. Beninger. 83
Lateral line,

in the

MOLINA. ANTHONY

Larva, 110. 121. 285. 295

LASKIS.

Molecular chaperone. 160

Molecular quantification of toxic Alexandriiim fiimlyense
Maine. 23
Molecule, 220

J.

DeSelm. 190

in zebrafish

N-CAM. 182
15
N label. 256
NAGLE, GREGG

T.. see

Sherry D. Painter. 16

Nematocyst. 367

NEUMANN, DIETRICH. AND HEIKE KAPPES. On

the growth of bivalve gills

from a lobule-producing budding zone, 73
Neural recordings from the lateral line in free-swimming toadfish, Opsanus
tun. 216
Neurochemical modulation of behavioral response to chemical stimuli in
initiated

Hnnhii'u\ americanus. 222

INDF.X

TO VOLUME

381

205

Predator, 367

Neuromodulatoi, 222
Neuron, supramedullary. 211
Nitrate. 244
Nitric oxide, IS?

REBECCA M.. Columellar muscle of neogastropods: muscle

ment and the function of columellar folds. 351
Productivity, 93

Nitrogen. 242. 244. 256

Prototroch. 182

Nitrogen flux and speciation through the subterranean estuary of Waquoit

Bay, Massachusetts, 244

Psychmlutcs.
Pteropoda. 93

PRICE,

attach-

Nuclear envelope. 177

Niiculu. 73

Nudihranch.

16.

218, 220, 367

Nutrient

Rab-GDI

supply. 250
transfer,

o
S. P. GRADY, A. S. LESCHEN. R. H. CARMICHAEL, AND
VALIELA, Stable isotopic assessment of site loyalty and relationships
between size and trophic position of the Atlantic horseshoe crab,
Limn/in p<>l\plicinus, within Cape Cod estuaries. 254

O'CoNNELL, C. W.,
I.

OLDENBOURO, RUDOLF, see Michael Shribak. 194

OLDENBURG. C. E.. see A. M. Kuzirian, 220
Olfaction. 224

On

the

growth of bivalve

gills initiated

from a lobule-producing budding

zone. 73

vesicle transport in squid giant

Radiochemical
Radium. 246
Radon, 246

RAGO.

A., see

tracer,

J.

M.

246

Talbot, 244: D.

M. Abraham. 246

Ratio, twist-to-bend, 36

REDENTI. S., AND R. L. CHAPPELL. Zinc chelation enhances the sensitivity

of the ERG b-wave in dark-adapted skate retina. 213
REES, Huw H.. see Carolyn J. Ruddell. 308

REISENBICHLER. KIM R.. see Bruce H. Robison. 102

Relative influence of grazing and nutrient supply on growth of the green
macroalga Ulva lucti/cti in estuaries of Waquoit Bay. Massachusetts.

252

Oocyte. 83. 192

Ophiuroid. 54

Remote

Opsamix tan. 216. 235

ORCHARD, ELIZABETH, ERIC WEBB, AND SONYA DYHRMAN, Characterization
of phosphorus-regulated genes
Orchestia grillns, 238

Oxygen binding

myosin V-dependent

Rabs. 190
Radiochemical estimates of submarine groundwater discharge to Waquoit
Bay, Massachusetts. 246

Nutrition, larval, 295

Octopus.

inhibits

axon, 190

83

in

Trichodesmium

spp.,

230

sensing. 93

Reproduction, 83. 133, 285, 308

Reproduction and larval morphology of broadcasting and viviparous species in the Cryptusterina species complex, 285
Reproductive hot spot, 1
Resource holding power, 26

equilibrium. 54

Response

Oyster. 160

in

nematocyst uptake by the nudihranch FUihcllina verrucosa to

Gulf of Maine, 367

the presence of various predators in the southern

Retina, 213, 215

RGD, 220
Rho-kinase. 195

Rho-kinase

Pain. 211

PAINTER. SHERRY D.. BRET CLOUGH. SARA BLACK, AND GREGG T. NAGLE.
Behavioral characterization of attractin. a water-borne peptide pher-

omone

genus Aplysia. 16
PALMER. L. M.. B. A. GIUFFRIDA. AND A. F. MENSINGER. Neural recordings
from the lateral line in free-swimming toadfish. Opsanus tan, 216
in the

Paralytic shellfish poisoning. 231

Parasite, 111)

285
Patterns and processes of

required for myosin-II-mediated vesicle transport during

of clam oocytes. 195

in extracts

RIPPS. H.. see K. Cusato, 197: R. L. Chappell.

ROBERTS.

S. B..

AND

F.

emergence

in

an estuarine parasite system.

Ill)

Patterns of sedimentation in a salt marsh-dominated estuary. 239

Peptide pheromone. 16
in

nonfeeding an-

Persistent ancestral feeding structures in nonfeeding annelid larvae,

PEYER, SUZANNE M.. see Joanna L. Joyner. 331

tag analysis of

genes expressed in the bay scallop, Argopecten irradians, 227

ROBISON, BRUCE H., KIM R. REISENBICHLER. JAMES C. HUNT. AND STEVEN
H. D. HADDOCK. Light production by the arm tips of the deep-sea
infernali*.

102

ROSENTHAL, G. G., see E. R. Turnell, 225

RUDDELL, CAROLYN J., GEOFFREY WAINWRIGHT, AUDREY GEFFEN, MICHAEL
R. H. WHITE. SIMON G. WEBSTER. AND Huw H. REES. Cloning,
characterization, and developmental expression of a putative farnesoic
acid O-methyl transferase in the female edible crab Cancer pagurus,

PCR. 231. 235

PERNET. BRUNO. Persistent ancestral feeding structures
nelid larvae. 295

209

W. GOETZ. Expressed sequence

cephalopod Vampyroteuthis

Patiriella.

larval

is

M-phase

295

308
Ryanodine, 185
Ryanodine-sensitive calcium flux regulates motility of Arbacia punctulata
sperm, 185

Phenotypic plasticity of HSP70 and HSP70 gene expression in the Pacific

ovster (Crassostrea gigas): implications for thermal limits and induction of thermal tolerance. 160

Pheromone. peptide. 16
Phosphorus. 230
Phytogeny, opisthobranch. 121
PIELAK. R. M.. V. A. GAYSINSKAYA,

ANDW. D. Com v C\u>skeletal events

preceding polar body formation in activated Spixulu eggs, 192
Pisidium, 73
Polar body, 192
Polychaete. 182. 295

PONTIUS, R. G.
Porifera, 144

JR.,

see

M.

T. Holden.

Possible roles of sulfur-containing

257

amino acids

bacterium-mollusc symbiosis. 331

in

chemoautotrophic

SABBAN, ALON. see Opher Gileadi. 177

Sabellid, 295
Salt marsh. 238. 239. 248
accretion. 239
metabolism, 248
pond. 239, 248
SANCHEZ. JUAN ARMANDO, see Howard R. Lasker, 330
SANFORD. ERIC, see Lars Tomanek, 276
SANGER, RICHARD, see Anthony J. A. Molina, 215
SANGSTER. C. R.. AND R. M. SMOLOWITZ, Description of Vibrio
cits

alginolytiinfection in cultured Sepia officiiui/ix, Sepia n/'niini. and Sepia

pharaonis, 233

INDEX TO VOLUME

382

SAVAGE, ANNA, AND JELLE ATEMA, Neurochemical modulation of behavioral response to chemical stimuli in Homarus americanus. 222

SAVAGE,

256

K.. see C. Walker.

Scallop, 83. 227

Scanning electron microscopy, 228
Scanning electron microscopy investigation of epizootic lobster shell dis-

ease in Honiiim.'i iiinencantts. 228

SCHLINING, BRIAN, see Jeffrey C. Drazen,

SCHWARZ, J. A.. AND V. M. WEIS, Localization of a symbiosis-related
1

Sym32,

protein,

the

in

Anthopleura elegantissimaSymbiodinium

TEICHBERG. M., see

S.. J. C. KALTENBACH, W.
FERNANDEZ-BusQUETS. Apoptosis

urchin,

199

Thiotaurine. 331

A. E. GIBSON.

T.,

AND

copy. 194
reconstruction, 351

stimulus. 225

AND HEIDI M. DIERSSEN. Cascading

trophic impacts of
reduced biomass in the Ross Sea, Antarctica: Just the tip of the
A..

FOREMAN. Multiple approaches to

West Falmouth wastewater plume. 242

K. H.

3-D

Sedimentation, 239

BRAD

Time-lapse, 179
Toadfish, 216, 235

TOMANEK, LARS. AND ERIC SANFORD. Heat-shock

iceberg?, 93
Self-recognition, 199

Sepia, 233

Top-down. 252

Sewage, 242

Training alone, not the tripeptide

senda. 220

Shell

loss,

fringence distribution in reconstituted asters of Spisula oocytes revealed by scanned aperture polarized light microscopy. 194
SHULL, D. H., see A. M. Agnew, 238
Site loyalty,

254

Transport, 187
Trematode, 10
1

Trichodesmium

Bogorff, 207; Anthony J. A. Molina, 215

SMOLOWITZ. R. M.. see A. D. Hsu. 228; C. R. Sangster, 233; Krystal D.
Baird, 235

SMITH, PETER

J. S..

see Daniel

J.

256

23(1

Trochophore, 182
Trophic
ecology, 93
position,

254

TSUCHI, AKIFUMI, see Tatsuo Motokawa, 26

Tubulin. 187
TURNELL. E. R.. see K. D. Mann. 224

motility, 185

Spisula. 192
Sponge. 144. 199

Spore discharge. 236

Squid, 47, 179

TURNELL,

MANN, G. G. ROSENTHAL, AND G. GERLACH. Mate

E. R., K. D.

choice

in zebrafish

(Danio rerio) analyzed with video-stimulus tech-

niques. 225

embryo. 181
giant axon. 187. 188. 190
Squid sperm to clam eggs: imaging wet samples in a scanning electron
microscope. 77
Stable isotope, 250, 254
Stable isotopic assessment of site loyalty and relationships between size
1

trophic

of

position

the

Atlantic

horseshoe

crab.

TURNER. JOHN

R., see

Twist-to-bend

ratio.

Simon K. Davy, 66

36

Twisting and bending of biological beams: distribution of biological beams

in a stiffness mechanospace, 36

250 million years of bindin evolution,

Limiilus

polyphemus, within Cape Cod estuaries, 254

STAKES, DEBRA S., see Jeffrey C. Drazen.

Stiffness
flexural,

Hermis-

Skate," 2 13

and

in

of the cunner, Tautogolabms adspersus, 2 1

Transplantation and isotopic evidence of the relative effects of ambient and
internal nutrient supply on the growth of Ulva lucttu'ti. 250

Tricaine, 21

Solemva, 331

Sperm

modulates calexcitin

two conge-

Transient use of tricaine to remove the telencephalon has no residual

effects on physiological recordings of supramedullary/dorsal neurons

26

SHRIBAK. MICHAEL. AND RUDOLF OLDENBOURG. Three-dimensional bire-

Soil,

RGD.

test in

Trait-mediated indirect interaction. 367

228

121

Shelters,

protein 70 (Hsp70) as a

biochemical stress indicator: an experimental field

neric intertidal gastropods (Genus: Tegula). 276

Self-referencing, 207

lesions,

KUHNS, M. M. BURGER. AND X.

Microciona prolifera allografts,

Three-dimensional birefringence distribution in reconstituted asters of

Spisula oocytes revealed by scanned aperture polarized light micros-

185

8,

J.

in

Thermal tolerances of deep-sea hydrothermal vent animals from the Northeast Pacific. 98
Thermotolerance, 98. 160. 276

tracing nitrogen loss in the

anemone. 66, 339

cucumber, 261
level rise, 239
star, 285

SEIBEL,

A. Morgan. 252; A. B. Aguiar, 259

of, 211

TEPSUPORN.

Scuba, 26

Sea
Sea
Sea
Sea
Sea

J.

Telencephalic hemisphere, removal

Telotroch, 182

THOMS.

muscatinei association, 339

205

UHLINGER, KEVIN R., see Krystal D. Baird, 235

Viva Uictiicu. 250, 252

36
36

torsional,

Unio. 73

Stress protein. 160

Submarine groundwater discharge. 244. 246

Sulfide. 54
Supramedullary neuron.
Symbiodinium, 339
Symbiosis. 66. 331, 339

VALENTINE, VINTON. see Jason R. Cavatorta, 239; M. E. Johnston. 248

VALIELA. I., see A. B. Aguiar, 250; J. A. Morgan, 252: C. W. O'Connell.
254
Vuip\rtiti'uthiii infemalis. 102

VAN CAMP,

L. M.. see L. S. Eyster.

Veliger. 121

TALBOT. J. M.. R. D. KROEGER. A. RAGO. M. C. ALLEN, AND M. A.

CHARETTE, Nitrogen flux and speciation through the subterranean
estuary of Waquoit Bay, Massachusetts, 244

Vesicle transport, 188. 190. 195

Vibrio ulginoh'ticiis, 233

Taurine, 331

Visual sensitivity, 213

Tegula, 276

Viviparity, 285

Vision, 225

47

INDIA TO VOl.l'MH

383

2115

W
WADESON.

P. H.,

AND

K.

CRAWFORD. Formation of

the blastoderm

and yolk

Yolk syneytial nuclear

collapse. 179

syneytial layer in early squid development. 79

\I\\VRIGHT, GEOFFREY, see Carolyn J. Ruddell. 308
1

WALKER.

C., E.

DAVIDSON. W. KINGERLEE. AND K. SAVAGE, Ineuhation

conditions of forest soil yielding maximum dissolved organic nitrogen

concentrations and minimal residual nitrate. 256

WEBB. ERIC, see

WEBSTER. SIMON

Elizabeth Orchard. 230

G.. see Carolyn J. Ruddell. 308

WEIDNER. EARL. AND ANN FINDLEY. Catalase in microsporidian spores
before and during discharge. 236
WEIS. V. M, see J. A. Schwarz, 339
WEISSMAN. IRVING L.. see Nanette E. Chadwick-Furman, 133
WHITE. MICHAEL R. H.. see Carolyn J. Ruddell. 308
WILLIAMS. C.. see M. T. Holden, 257
WOLLERT. TORSTEN. ANA S. DsPlNA. CARL J. DsSELM, AND GEORGE M.
LANGFORD. Rho-kinase is required for myosin-11-mediated vesicle
transport during M-phase in extracts of clam oocytes. 195

ZAKEVICIUS,

J.,

see K. Cusato. 197; R. L. Chappell,

209

Zebrafish,176. 224. 225

ZIGLER, KIRK

S..

AND H. A. LESSIOS, 250

million years of bindin evolution.

ZIMMER, CHERYL ANN, see Jonathan T. Fingerut. 10

ZIMMER, RICHARD K., see Jonathan T. Fingerut, 10
Zinc, 209, 213
Zinc chelation enhances the sensitivity of the ERG b-wave in dark-adapted
skate retina. 213
Zinc modulation of hemichannel currents in Xenopus oocytes, 209
1

Zooplankton. 93
Zooxanthella. 66. 339

ZOTTOLI,

S.

J..

O. T. BURTON,

J.

A. CHAMBERS. R. ESEH. L.

M. GUTIERREZ,

AND M. M. KRON.

Transient use of tricaine to remove the telencephalon has no residual effects on physiological recordings of supramed-

Xenopus oocytes. 209

ullary /dorsal neurons of the cunner. Taulogolabrus adspersus, 21

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