0022-1910/84$3.00+ 0.

00
Copyright 0 1984Pergamon Press Ltd

J. Insect Physiol. Vol.

30, No. 4, pp. 331-340, 1984

Printed in Great Britain. All rights reserved

THE EFFECT OF JUVENILE HORMONE ON

PROTHORACIC
GLAND FUNCTION DURING THE
LARVAL-PUPAL
DEVELOPMENT
OF MANDUCA
SEXTA :
AN IN SITU AND IN VITRO ANALYSIS
M. C. GRUETZMACHER*, L. I. GILBERT?~, N. A. GRANGER~. W. GOODMANQ:and
W. E. BOLLENBACHER~
*Committee on Virology, The University of Chicago, Chicago, Illinois 60637. TDepartment of Biology,
The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27514, fDepartment of
Anatomy, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27514 and
Department of Entomology. University of Wisconsin, Madison, Wisconsin 53706. U.S.A.
(Receioed 21 September 1983)
Abstract-The application of juvenile hormone I or ZR 512 to neck-ligated. day-5 fifth instar (V,) larvae
reduced the time to pupation in a dose-dependent manner when compared to neck-ligated controls treated
with methyl epoxy stearate. Haemolymph
ecdysteroid
titres determined
by radioimmunoassay
(RIA)
reflected the ability of juvenile hormone I and ZR 512 to stimulate larval-pupal
development,
i.e. the
ecdysteroid
tilres were similar to those of normally developing
larvae although
the ecdysteroid
peak
elicited by ZR 512 lagged that in the normal titre by 1 day, while that elicited by juvenile hormone I lagged
the ecdysteroid
peak in normal larvae by 2 days. Neck-ligated
V, larvae that were untreated ultimately
pupated
and the haemolymph
ecdysteroid
peak eliciting pupation
in these animals was 7 pg/ml
haemolymph,
almost double that of normal animals and ZR 512- and juvenile hormone I-treated, ligated
larvae. The data indicated that juvenile hormone I does stimulate the prothoracic
glands but to determine
whether this stimulation
was direct or indirect, an in vitro approach was taken. Prothoracic glands from

V,, V, and V, larvae were incubated in vitro under conditions in which they could be stimulated by
prothoracicotropic hormone, and were exposed to concentration
of free juvenile hormones I, Il. III or
ZR 512 ranging
from 10m5 M to IO-M.
In no case were the prothoracic
glands stimulated
in a
dose-dependent
manner that would be indicative of hormone activation.
Similar results were obtained
when juvenile hormone bound to binding protein was incubated with the prothoracic
glands. Studies with
the acids of the three juvenile hormone
homologues
revealed them to be ineffective in activating
prothoracic
glands, although juvenile hormone III acid does appear to inhibit the synthesis of ecdysone
by day-0 pupal prothoracic
glands. The significance of the latter effect is unknown. It is concluded from
these data tha? juvenile hormone can, indeed, activate late larval prothoracic
glands in situ, but does so
indirectly.

Key Word Index: Juvenile hormone. Manduca sexta, prothoracicotropic

INTRODUCTION
Although the prothoracicotropic
hormone (PTTH) is
accepted as the primary effector of prothoracic
gland
activity, studies as early as 1959 suggested a direct
interaction
between the corpora
allata and prothoracic glands. Ichikawa
and Nishiitsutsuji-Uwo
(1959) were able to elicit adult development
in brainless Philosamia Cynthia ricini pupae by implanting
corpora allata and concluded
that PTTH released
from the implanted
corpora
allata elicited prothoracic
gland activation
and subsequent
development. Williams (1959) conducted similar studies on
brainless, diapausing Hyalophora cecropia pupae and
concluded that juvenile hormone from the corpora
allata stimulated the prothoracic glands. At the same
time, Gilbert and Schneiderman
(1959) succeeded in
eliciting development
in brainless saturniid
pupae
with crude juvenile hormone
extracts
and postulated
qcorrespondence
to: Professor L. I. Gilbert, Department
of
Biology, Wilson Hall 046A, The University
of North
Carolina
at Chaoel Hill. Chaoel Hill, North Carolina

27514, U.S.A.

titre

that the stimulatory effect of these extracts was due

to the presence of juvenile hormone.
These early
studies were subsequently interpreted to indicate that
juvenile hormone stimulates the prothoracic
glands
directly, although the possibility remains that PTTH
was released from the implanted corpora allata since
in some species this gland is the neurohaemal
organ
(see Agui et al., 1980). Further, if juvenile hormone
was the active factor, it could have elicited its effect
indirectly by stimulating the synthesis and/or release
of a prothoracicotropic
factor from other tissues, or
one or more juvenile hormone metabolites could have
been responsible for the observed effects (e.g. Bhaskaran et al., 1980).
In recent years, several in vitro studies utilizing a
variety of Lepidoptera
have supported the idea that
juvenile hormone affects prothoracic
gland activity
(e.g. Hiruma et al., 1978; Cymborowski
and Stolarz,
1979; Hiruma, 1980; Safranek et al., 1980; Hiruma
and Agui, 1982). There are indications that juvenile
hormone
can either inhibit or activate the prothoracic glands depending
on developmental
state
(e.g. Cymborowski
and Stolarz, 1979; Hiruma and
331

,.P 30,4--E

effects, ecdysteroid

M. C. GRUETZMACHER e6 al.

332

Agui, 1982); that is, juvenile hormone inhibits the

prothoracic glands of young iast-instar larvae but
stimulates the glands late in the last instar.
Although more than 2 decades have passed since
the first studies suggested that juvenile hormone acts
on the prothoracic glands to stimulate the synthesis
of ecdysone, there is still no evidence that the effect
is direct. The recent availability of techniques to
assess prothoracic gland activity in vitro has now
provided the means to study the putative effects of
juvenile hormone upon prothoracic gland activity.
The present study addresses the question of juvenile
hormone control of prothoracic gland activity using
the in vitro techniques utilized so successfully in
studies of prothoracic gland activation by PTTH
(Bollenbacher et al., 1979; see Gilbert et al., 1981).
MATERIALS

AND METHODS

Animals
Munduca sexta larvae were reared on an artificial
diet under a long-day photoperiod (16L:8D) at 25C
(Vince and Gilbert, 1977) and approximately 70%
relative humidity. Fifth-instar larvae were separated
into gates and only gate-2 animals were used (Truman, 1972; Granger et al., 1979).
Juvenile ~zormone b~nd~ngprotein
Juvenile hormone binding protein was partially
purified by affinity chromatography (Goodman and
Goodman, 1981). Haemolymph was collected from
late fourth-instar larvae and concentrated by ultrafiltration (Amicon UMIO). The binding protein in
the concentrated haemolymph was labelled by adding [3Hljuvenile hormone I (New England Nuclear
Corp., 10 Ci/mmol) and the labelled material was
separated by gel filtration on an S-200 (Pharmacia)
column (2.5 x 135cm) (Goodman et al., 1978). The
[HIjuvenile hormone binding protein fractions were
pooled and dialyzed overnight against a borateglycerol buffer (0.1 M sodium borate, 20% glycerol,
pH 7.8). Juvenile hormone was removed from the
dialyzed binding protein by addition of an equal
amount of heptane-toluene
(9: 1, v/v), and then
gently shaking the mixture at 25C for 3 h. The
delipidated protein (aqueous fraction) was removed
and centrifuged at 12,OOOg for 30min (4*C) to
remove denatured protein. The protein solution was
then incubated
for 30min with phenylmethylsulphonylfluoride
( 10m3M), mixed and passed
through the affinity column which would be the
column bed. The resin mixture was incubated for 2 h
at 4C with occasional stirring, brought to room
temperature and the unbound protein eluted with
glycerol borate buffer. The column was washed with
sufficient borate buffer to reduce the U.V. (280nm)
absorbance of the effluent to zero. Another 50 ml of
buffer was then passed through the column before
eluting the juvenile hormone-associated protein. The
latter was accomplished by incubation of the column
at room temperature with a hormone-saturated
buffer (2 mg juvenile hormone I, 0.25 m1 of ethanol
and 49.75ml of borate buffer) for 2 h. During the
incubation period the column bed was occasionally
stirred to ensure complete distribution of the hormone. The hormone-containing
buffer was then col-

lected and the column washed with an additional

50 ml of hormone-free buffer. The combined eluates
were pooled and concentrated.
The partially purified binding protein was stored at
-20C until use and the amount of protein recovered
was quantified by U.V. s~ctrophotometry
at 230 nm.
For in vitro incubations with binding protein, juvenile
hormone-saturated
binding protein was added directly to the incubation medium in concentrations of
juvenile hormone ranging from 1O--6to lo-M.
Juvenile hormone and juvenile hormone acids
Juvenile hormones I, II and III were purchased
from Calbiochem. The hormone analogue ZR 512
was a gift from Zoecon Corp. and methyl epoxy
stearate was purchased from Allied Science Laboratories.
Juvenile hormone acid(s) was prepared by incubating 10 mg of the appropriate hormone homologue
with 5 ml methanol- 1 N NaOH (1: 1, v/v) in the dark
in a shaking water bath at 40C for 4 h. The reaction
mixture was then carefully titrated to pH 5.0 with
I-ICI and extracted 5 times with chloroform-toluene
(9: 1, v/v). The aqueous methanol phase containing
the juvenile hormone acid was then dried under NZ,
taken up in chloroform and the acid purified by high
performance liquid chromatography (HPLC). The
purity of the generated juvenile hormone acid was
established by NMR and i.r. spectroscopy. The juvenile hormone acids were stored dry at -20C until
use and were quantifi~
spectrophotometrically
in
spectra-grade
methanol (A,,,,
213-217 nm; extinction coefficient, 14,770).
Uptake of ffifjuvenile

hormone I

To determine the degree to which topically applied

juvenile hormone homologues were taken up by
treated larvae, 100 pg of [-HIjuvenile hormone I (sp.
act. 0.015 pCi/mmol) was applied to neck-ligated
day-5 fifth instar (V,) larvae. To assess uptake and
metabolism of the hormone, haemolymph samples
were taken periodically over 48 h from different
animals at each point to minimize the potentially
adverse effects of repeated bleeding from a single
animal. Samples were immediateIy extracted for juvenile hormone I with hexane-methanol
(1:2, v/v>
(Granger et al., 1979). The efficiency of extraction of
[HIjuvenile hormone by this partition was 8004. To
separate juvenile hormone I from its metabolites, an
aliquot of the hexane epiphase containing
the
[3H]juvenile hormone I was mixed with unlabelled
juvenile hormone I to serve as a carrier, and the
sample was subjected to thin layer chromatography
(TLC) in hexane-ethyl acetate (3 : 1, v/v). The developed plates were divided into 1 cm fractions, the
fractions scraped individually into scintillation vials
with Aquasol (NEN) and the samples radioassayed
by liquid scintillation counting. The efficiency of
recovery from the TLC plate was 80%. The aqueous
hypophase from the organic solvent partition contained polar metabolites of juvenile hormone, e.g.
juvenile hormone acid, and the amount of this material present in the haemoiymph was determined by
counting an aliquot of the hypophase added to
Aquasol. The total amount of [HIjuvenile hormone
I metabolites in the haemolymph was determined by

Effect of juvenile hormone on prothoracic gland

adding the radioactivity in the aqueous hypophase
and the radioactivity on the TLC plate that did not
co-chromatograph with juvenile hormone I.

A radioimmunoassay (RIA) was used to quantify

both the ecdysteroids in haemolymph (Bollenbacher
et al., 1981) and the ecdysone synthesized by the
prothoracic glands in zlitro(Bollenbacher et al., 1983).
For determining haemolymph ecdysteroid titres, the
haemolymph was extracted with methanol (1: 2, v/v)
and aliquots of the extract were assayed by a micro
RIA (Bollenbacher et al., 1983). Culture medium was
assayed for ecdysone directly using a macro RIA
(Bollenbacher er al., 1983).
In situ assay-for juvenile hormone-evoked development
To assess the eflect of juvenile hormone on the
larval-pupal development of Manduca, V, larvae that
exhibited wandering behaviour and dorsal vessel
exposure were neck-ligated and the heads removed.
The test compounds, juvenile hormone I, ZR 512and
methyl epoxy stearate, at concentrations of 5~g/~l
cyclohexane were topically applied in a single dose of
up to 100 pg per ligated larva. For this study there
were two types of controls, ligated larvae treated with
20 p I of cyclohexane and untreated larvae. A positive
effect on development was scored in terms of the
number of days that treatment accelerated development to the pupa in comparison to the 10 days it
takes larvae that were decapitated and untreated.
In vitro assay for prothoracic gland activation
The assay for juvenile hormone activation (or inhibition) of the prothoracic glands was derived from
that described pre\iously for PTTH activation of
these glands (Bollenbacher et al., 1983). Each member of a pair of prothoracic glands from a given insect
was incubated individually for 8-48 h in a 0.025ml
standing drop of Marks medium 19 AB (C&anger and
Borg, 1976). Since the right and left glands of each
pair synthesize and release ecdysone at equal rates
(Bollenbacher et al., 1979). one gland served as the
experimental and the contralateral gland served as
the control. The experimental glands were incubated
in the presence of the test substances: juvenile hormone I, II and III, the respective acids and ZR 512
at various concentrations (10-6-10-o M). To minimize the non-specific adsorption of juvenile hormone
to glass surfaces, all glassware used was coated with
10% polyethylene
glycol (MW 20,000; Calbiochem).
A time course of ecdysone synthesis by experimental
and control prothoracic glands was determined for all
in vitro incubations. This was accomplished by taking
0.01 ml aliquots of medium at designated time points
followed by the addition of a volume of fresh culture
medium equal to that removed to maintain a constant incubation volume. The aliquots were assayed
by RIA to determine the ecdysone content in the
medium, and the cumulative synthesis of hormone by
the glands was determined. Activation of the prothoracic glands was expressed as an activation ratio
(A,), which is the amount of ecdysone synthesized by
the experimental gland divided by the amount synthesized by the control gland during that same time
(Bollenbacher ef al., 1979).

0.01

0.1

io

i 0

50

100

(pq f ommoi 1
Fig. 1. Effects of topically applied ZR 512 to neck-ligated
V, larvae. Each point represents 3-9 animais k SEM. C,
control V, neck-ligated larvae. 0 dose consists of solvent
ZR512

only (20 yl cyclohexane).

RESULTS

EfSects of juvenile hormone

larval-pupal development

and

ZR 512

on

Before the apparent prothoracicotropic

effect of
juvenile hormone on the prothoracic glands could be
probed in vitro, it was necessary to verify that the
hormone and its analogue ZR 512 accelerated the
larval-pupal
development of wandering last-instar
Manduca larvae. These experiments were performed
essentially as described by Safranek et al. (1980) in
that V, wandering larvae were neck-ligated to remove
the endogenous source of both PTTH and juvenile
hormone, and the length of time necessary for metamorphosis to the pupa was then determined. In this
study both ligated animals treated with cyclohexane
and untreated controls developed to pupae approx 10
days after wandering (Fig. l), confirming previously
reported results (Safranek et al., 1980). When the
ligated animals were treated with increasing concentrations of ZR 5 12, they pupated progressively earlier
in response to the increasing doses. A linear range of
response to ZR 512 occurred with doses from 0.1 pg
to lOOpg, with lOO~g/animal eliciting a normal
pupation time of approx 5 days. These results corroborated the reported effects of juvenile hormone and
the concentration of ZR 512 also demonstrated at
which ligated larvae would pupate within a normal
period of time, thus providing the concentration to be
used in subsequent studies.
These same topical application experiments were
then conducted with juvenile hormone I. Results
identical to those for ZR 512 were obtained, in that
the response was dose-dependent and 100 pg of juvenile hormone I elicited pupation in the normal period
of time. When methyl epoxy stearate was used as a
control compound to demonstrate the specificity of
the juvenile hormone effect, no decrease in the 10
days required for the pupation of ligated, untreated
if the
animals
was observed. To determine
brain-retrocerebral
complex influenced the effect of
juvenile hormone on development, the bioassay was
repeated with non-ligated, normal larvae. The results
revealed that topical application of ZR 5 12 to normal
V, larvae decreased the time to pupation by 1 day and
that 0.01 pg of ZR 512 was as effective as 100~8.
The topical application studies clearly showed an
acceleratory effect of ZR 512 and juvenile hormone
on larval-pupal development. but whether this effect
occurred by direct activation of the prothoracic
glands could not be concluded from these results.

M. C.

334

GRUETZMACHER

0123456789
Days

after

llgatlon

Fig. 2. Effects of treatment on the haemolymph

ecdysteroid
titre of V, neck-ligated
larvae. l normally
developing
larvae; 0 neck-ligated
larvae treated with 100 pg ZR 512;
A neck-ligated larvae treated with 100 pg juvenile hormone
I; n neck-ligated
larvae treated with solvent only (20~1
cyclohexane);
0 neck-ligated
larvae treated with IOOpg
methyl epoxy stearate. W denotes wandering and P denotes
pupation.

Haemolymph

ecdysteroid

titre

If topically applied juvenile hormone were affecting

the prothoracic
glands this effect should be reflected
by characteristic differences in the haemolymph ecdysteroid titre of the various experimental populations.
These differences should more likely be temporal
than quantitative,
since pupal development
occurs
even in ligated, untreated
animals. Therefore,
as
another means of assessing the putative tropic effect
of juvenile hormone on the prothoracic
glands, the
haemolymph
ecdysteroid
titres were determined
in
treated
larvae
during
ligated
and
normal,
larval-pupal
development.
Haemolymph ecdysteroid titres of neck-ligated larvae treated with ZR 512 and juvenile hormone I were
compared to the ecdysteroid titre of normally developing V, animals and neck-ligated
controls treated
with cyclohexane (Fig. 2). For unmanipulated,
wandering larvae, the titre was essentially that reported
previously (Bollenbacher
et al., 1981): the titre was
low (0.25 pg/ml haemolymph)
on day 0 (equivalent
to wandering V, larvae), peaked on day 1 (equivalent
to V, larvae) at 3.5 pg/ml haemolymph,
and then
declined to approximately
1.2 pg/ml haemolymph on
day 3 (equivalent to V, larvae). These animals pupated 5 days after wandering.
The haemolymph
ecdysteroid
titre of ZR 512-treated
animals was
about 0.25 pg/ml haemolymph
on day 0, and increased gradually to 0.75 pg/ml haemolymph
on day
2. The titres on days 1 and 2 in these larvae were
significantly lower than the titres on comparable days
in normal, developing animals (P < 0.01). Three days
after treatment the titre in the ZR 512-treated larvae
peaked at 4.5 pg/ml haemolymph,
a level similar to
the peak in the titre of untreated
day-2 animals
(P > 0.05), even though this peak occurred
1 day
later. By day 4 the titre fell to about 0.9 pg/ml
haemolymph
and pupation occurred at the normal
time, 5 days after ligation. Thus, it appeared that ZR
512 restored a normal developmental
time frame by
evoking an increase in the ecdysteroid titre, although
the peak in the titre occurred 1 day later than in the
untreated
larvae. In juvenile
hormone
I-treated,
ligated animals, which also pupated
5 days after

et

al.

treatment, the peak ecdysteroid titre reached a level

comparable
to that in the untreated
and ZR
512-treated larvae. However, this peak lagged that in
the normal titre by 2 days and that of ZR 512-treated,
neck-ligated animals by 1 day.
The slow development
of the ligated
and
cyclohexane-treated
larvae was characterized
by a
haemolymph
ecdysteroid
titre that increased gradually from the time of ligation to approx 5 days after
ligation, 0.5 pg/ml-0.9 pg/ml, respectively. After this
gradual rise, the titre increased rapidly, peaking at the
time of pupation on day 9 at 7 pg/ml haemolymph.
This peak is approx double those determined
for
normal animals and ZR 512- and juvenile hormone
I-treated,
ligated larvae. The significance
of this
relatively enormous ecdysteroid titre is conjectural at
present.
Lastly, to confirm the analogue specificity of the
response obtained with ZR 5 12 and juvenile hormone
I treatment of ligated larvae, methyl epoxy stearate
was applied to V, neck-ligated larvae and the ecdysteroid titre was assessed. The haemolymph
ecdysteroid titre was similar to that determined
for the
cyclohexane-treated
larvae, as suggested by the time
to pupation (10 days). From days O-9 after treatment, the titre in the methyl epoxy stearate-treated
larvae was essentially the same as that of the ligated
controls,
with a peak of approx 4.5 pg/ml haemolymph on the day of pupation. This ecdysteroid
level, as well as the level on day 12, was not
significantly different than those of the cyclohexanetreated controls at the same times (P > 0.05). This
indicated that methyl epoxy stearate had no effect on
the ecdysteroid
titre either quantitatively
or temporally, which is consistent with data on its lack of
an effect on the time required for the pupation of
ligated, control larvae.
The above results revealed that ZR 512 and juvenile hormone I appear to affect prothoracic
gland
function so that a near-normal
haemolymph
ecdysteroid titre results from their application
to ligated
larvae. The only apparent
difference between the
titres of normal animals and those of ZR 512 - and
juvenile hormone I-treated ligated larvae is the temporal lag in the rise of the haemolymph
ecdysteroid
titre by one and two days in ZR 512 - and juvenile
hormone I-treated, ligated larvae, respectively. Thus,
it appears that the prothoracic
glands could have
been activated by juvenile hormone to increase their
rate of ecdysone synthesis, but to study the changes
in the ecdysteroid
titre in response to juvenile hormone is merely to examine a covert effect for which
the overt expression
is the acceleration
of development. The results do not demonstrate
that the
effect on the prothoracic
glands is direct.
[H]Juvenile

hormone I penetration

To demonstrate

of the cuticle

a possible direct effect of juvenile

hormone on the prothoracic
glands an in vitro approach was required.
This necessitated
an initial
determination
of the effective concentration
of juvenile hormone to be used in the in vitro studies; this
concentration
could be established
by determining
the in situ haemolymph
titre of juvenile hormone
resulting from the dose of topically applied hormone
(100 /*g) needed to elicit normal pharate pupal devel-

Effect of juvenile hormone on prothoracic gland

335

present in the haemolymph was apparently due to a

relatively constant
rate of catabolism
of the
[3H]juvenile hormone I together with a similar rate of
sequestration and/or excretion of metabolites. Given
the high levels of these metabolites in the haemolymph, of which a large portion appeared to be
juvenile hormone acid, the possibility existed that
these metabolites could affect the larval-pupal development of the ligated larvae; such a possibility has
been suggested previously (Bhaskaran et al., 1980;
Granger et al., 1982).
Once the effective in situ concentration of juvenile
hormone I had been determined, it was possible to
initiate the in vitro studies probing the function of
juvenile hormone in the regulation of the prothoracic
glands during the larval-pupal development of Manduca.

24

Time

40

(h)

Fig. 3. Haemolymph titre of juvenile hormone and metabelites after topical application of [Hljuvenile hormone I to
V, neck-ligated larvae. 0 juvenile hormone I; 0 juvenile
hormone I metabolites.
100 pig juvenile hormone
I
(0.015 ~Ci/mmol) was applied in cyclohexane at zero time.

opment in Vj wandering, ligated larvae. (It is assumed here that the endogenous juvenile hormone
titre is negligible in V, larvae -J. Bergot et al., pers.
comm.-and
especially in neck-ligated larvae.) In
addition, determination of the haemolymph juvenile
hormone titre resulting from the topical application
of juvenile hormone would provide information regarding the temporal relationship between the increased juvenile hormone titre and the increased
ecdysteroid titre. This information would aid in the
design and interpretation of in vitro studies, since a
direct effect of increased juvenile hormone titre on
the prothoracic glands should be followed by an
increase in the ecdysteroid titre. Finally, an analysis
of [3H]juvenile hormone uptake would also provide
information on the rate and nature of degradation of
juvenile hormone to metabolites that could possibly
influence prothoracic gland activity.
A time course analysis of [3H]juvenlle hormone I
levels in larval haemolymph after topical application
revealed that the hormone was present at a level in
excess of lo- M within 10 min (Fig. 3). At 0.5, 1 and
2 h, the concentration
of [3H]juvenile hormone I
fluctuated around lo- M, falling by 6 h to IO-M.
At 24 h it had dropped to 7 x 10m9M, and finally
reached approx 1 x 10m9M at 48 h. Thus, these results indicated that the topical application of 100 yg
of juvenile hormone I needed to elicit deveiopment in
ligated larvae did not result in pharmacological haemolymph titres of juvenile hormone, at least after
24 h. The titre had fallen to about low9 M 1S-2 days
before the increase in the ecdysteroid titre occurred,
an observation which suggested that the effect of
juvenile hormone on the prothoracic glands may not
be direct.
This study also revealed that a substantial degree
of degradation of the exogenous juvenile hormone I
occurred. The concentration of rH] polar metabolites
was well in excess of 10e6M, and remained at this
level for the 48 h of the time course study (Fig. 3).
This relatively constant amount of metabolites

In vitro conditions for prothoracic gland incubations

with juvenile hormone

To test for a possible direct stimulatory effect of

juvenile hormone and ZR 512 on the prothoracic
glands in vitro glands from V,, V, and V, larvae were
used. The selection of these days was based on the
observation that these stages of larval-pupal development of either normal or juvenile hormone-treated,
ligated animals occurred before or during the increase
in the haemolymph ecdysteroid titre (Bollenbacher et
al., 1981), and thus, represented times at which the
prothoracic glands were presumably responding to
juvenile hormone or ZR 512 in situ.
Time courses of ecdysone synthesis by these prothoracic glands were generated to determine the
length of time the glands could be incubated in vitro
during which potential JH effects might be observed.
Previous studies on prothoracic gland activation in
vitro indicated that an incubation time of 8 h was the
maximal time within which activation could be discerned, since beyond this time rates of synthesis by
activated glands were no longer linear relative to the
rate of synthesis by unactivated glands. This effect on
synthesis rates was presumably a result of substrate
depletion of the activated glands (Bollenbacher et al.,
1979). Thus, although incubation times longer than
8 h could be used, the resulting data might be difficult
to interpret since any activation of synthesis would be
occurring under non-linear conditions for both control and experimental glands.
To verify that the basal rates of ecdysone synthesis
by day 5, 6 and 7 larval prothoracic glands were
linear during an incubation period of 8 h, time
courses of ecdysone synthesis by these glands were
determined (Fig. 4). For day-5 and -6 prothoracic
glands, the rates of ecdysone synthesis were both
linear and comparable (_ 5 ng h-l gland-) during
the incubation period, while the mean rate of ecdysone synthesis by day-7 prothoracic glands was considerably less, approx 1.4 ng h- gland-.
The
significance of the different rates is not clear, but for
the purpose of this study it was only important to
know that the glands were capable of linear synthesis
in vitro, and that they possessed sufficient substrate to
demonstrate an increased rate of ecdysone synthesis
if activated within the incubation period. Thus, these
glands were suitable for probing the putative direct
tropic effect of juvenile hormone and ZR 512 on the

336

M. C. GRUETZMACHER

10

4
Time

(h)

rates of ecdysone synthesis by prothoracic

glands in vitro. l glands from V, larvae; 0 glands from V,
larvae: a glands from V, larvae. Bars signify + SEM.
Fig. 4. Basal

prothoracic glands. It is important to note that under

these in vitro conditions,
prothoracic
glands from
days 5-7 can be activated by PTTH (Gruetzmacher,
Gilbert and Bollenbacher, unpublished observations)
in a manner similar to that reported for day-0 pupal
prothoracic
glands (Bollenbacher
et al., 1983).
Short-term efJects in vitro of juvenile
ZR 512 on prothoracic glands

hormone and

To conduct these in vitro activation studies both

critically and thoroughly, a time course protocol was
used with a broad range of concentrations
of the test
compounds.
Prothoracic
glands from V,. V, and V,
larvae were incubated
in vitro with either juvenile
hormone or ZR 512 in concentrations
ranging from
10m6 to lO_M,
and a time course of ecdysone
synthesis by experimental
(+ test compound)
and
control glands (- test compound)
was then generated at each concentration
to monitor
potential
effects on the rate of ecdysone synthesis. A dosedependent
stimulation
of ecdysone synthesis would
be indicative of direct activation of the glands by the
test compound; i.e. the latter would be functioning as
a prothoracicotropin.
Since the corpora allata of Munduca synthesize

et al.

juvenile hormones II and III as well as juvenile hormone I during larval-pupal

development
(Granger
et al., 1982), these homologues
were assayed in vitro
with the same protocol used for juvenile hormone I.
Since juvenile hormone
I in the haemolymph
of
Munduca is normally
associated
with its binding
protein (Goodman
and Gilbert, 1978) the in vitro
studies testing the effect of juvenile hormone I were
conducted not only with the hormone free in solution
(unbound),
but also complexed to the binding protein. Each assay consisted of 3 replicates and was
conducted at least 2 times. More than 1100 pairs of
prothoracic
glands were utilized during the course of
this study.
Time courses of ecdysone synthesis by V, prothoracic glands in response to various concentrations
of unbound juvenile hormone
I (Fig. 5) failed to
reveal prothoracic
gland stimulation,
i.e. the activation ratio was approx one for all time points. Some
variability was noted at 10m7M and lo- M juvenile
hormone I at 1 and 2 h but the increased activation
ratios were not significantly different and were not
dose-dependent.
Since the activation ratios were less
than 2 and were not significantly different between
juvenile hormone I concentrations
at a given incubation time, nor between different incubation times at
a given hormone concentration,
it was concluded that
this hormone was not exerting a tropic effect on V,
prothoracic
glands. This same protocol was used to
test the effect of juvenile hormone I on V, and V,
glands and of juvenile hormone II and III on V,, V,
and V, glands. Overall, the time courses of ecdysone
synthesis by these various prothoracic
glands with
differing concentrations
of the juvenile hormone homologues yielded results almost identical to those
noted above for juvenile hormone I on V, prothoracic
glands, i.e. a tropic effect was not observed. These
studies are summarized in Table 1 with mean activation ratios obtained
for all the concentrations
of
unbound juvenile hormone used at a given incubation
time. The range of the activation ratios obtained at
each time point is also presented
to illustrate the
general reproducibility
and small variation occurring
over the entire concentration
range.
Careful
analysis
of these dose-response
time
course data revealed that there were isolated cases,
e.g. juvenile hormone I with V, prothoracic glands at
1 h and for hormone II with V, prothoracic glands at
1 h, when the range of activation ratios obtained with
different
concentrations
of
hormone
varied
significantly.
However,
these were isolated occurrences and the response was never observed for more

Time (h)
Fig. 5. Effects of juvenile hormone I on the in vitro activity of prothoracic
glands from V, larvae. l
10m6 M; 0 IO-M;
n lo-M;
0 10m9M; A IO-M;
A control prothoracic
glands. A, denotes
activation
ratio.

337

Effect of juvenile hormone on prothoracic gland

Table 1. The effects of unbound juvenile hormones on prothoracic

gland activity in uizro

Mean A,+ SEM*

Developmental
Stage

Treatment

Juvenile
hormone I

Juvenile
hormone II

Juvenile
hormone III

ZR 512

(Range)
lh

2h

4h

8h

v,

1.27JrO.18
(0.81-1.77)

1.06 + 0.09
(0.79.-1.32)

I .02 rfr0.05
(0.93-1.21)

1.06 rt 0.07
(0.94-1.35)

V,

0.8 1t 0.02
(0.75-0.88)

1.02& 0.05
(0.90- 1.21)

(0.85-1.19)

I .02rt 0.06

V,

0.81 2 0.07
(0.69-1.10)

0.88 i 0.04
(0.79-1.02)

0.98 f 0.02
(0.93-1.02)

1.01 rt 0.01
(0.98--l .06)

0.86*o.li
(0.62- 1.36)

0.98 f: 0.08
(0.82-I .32)

1.03 t 0.08
(0.84.--1.24)

1.08 i 0.07
(0.90.. 1.33)

0.93 +0.16
(0.36- 1.46)

0.91 10.15
(0.33-1.35)

0.94 rt 0.06
(0.82-1.84)

1.2s+o.t5
(0.91--1.89)

0.95 i 0.1 I
(0.65-I .40)

V,

1.40&0.16
(0.98-2.01)

1.31 kO.12
(0.96-l .63)

VS

1.15+0.06
(0.95-1.34)

1.10 & 0.05

(0.98-1.31)

0.88 * 0.09
(0.56-1.19)

0.89 & 0.60

(0.75-1.08)

0.97 + 0.05
(0.78-1.08)

1.03 *0.14
(0.62-I .46)

1.19 & 0.15

(0.76-1.77)

1.01 rtO.08
(0.66-1.15)

VS

1.08 t 0.05
(0.96-1.21)

1.14&0.07
(1.04 1.38)

1.00 + 0.07
(0.84-I .23)

V6

1.~21_0.13
(0.56-1.42)

1.2550.21
(0.65-1.82)

1.20f0.10
(0.89-l .43)

V,

0.90 & 0.04

(0.76-0.97)

0.96 f 0.08
(0.70-1.16)

0.90 f 0.06
(0.72-1.01)

1.08+ 0.09
(0.9% 1.08)

*The mean A, (activation ratio) values represent the mean for all con~ntrations of juvenile hormones or ZR 512 assayed. concentrations
assayed were lOme, lo-, 10w8, 10s9 and IO-M. h signifies hours of incubation.

than one time point during the incubation period.

Furthermore, a dose-dependent response was never
observed.
Except with V, prothoracic glands at 2 h, no effects
on the glands were observed with ZR 512. The lack
of an effect is exemplified by the response of V,
prothoracic glands to ZR 512 (Fig. 6) where the
activation ratio varied nominally over the 8-hr incubation period. That ZR 512 did not stimulate the
glands was a notable observation since it was the
most potent material in evoking a biological response
in situ and is not subject to catabolism by esterases
(Kramer and DeKort, 1976). Therefore, it appears
that in the unbound state, neither juvenile hormone
nor ZR 512 stimulate the prothoracic glands in vitro.
Since juvenile hormone I is bound to the binding
protein in situ, it was possible that the hormone/bind-

ing protein complex could activate the prothoracic

glands since this would be a more physiological
condition than the use of unbound hormone. The
same experimental design was employed as in the
previous in vitro studies and similar results were
obtained: juvenile hormone/binding
protein did not
elicit a significant dose-dependent effect on gland
activity over time (Table 2). Therefore, the presence
of binding protein does not appear to be adequate for
demonstrating a tropic effect of juvenile hormone I
on the prothoracic glands during 8 h in vitro.
In summary, these in vitro studies reveal that
during 8 h in vitro under defined incubation conditions, juvenile hormone does not have a tropic effect
on the prothoracic glands. Therefore, its effect on the
larval-pupal development of neck-ligated V, larvae is
probably a result of the indirect activation of the

2
t

d' ,

t:
L

6
Time

I
8

(h)

of ZR 512 on the in vitro activity of prothoracic glands from V, larvae. 0 10F6M; 0

10m7M; n 1Om8
M; 0 10d9M; A 1O-oM; A control prothoracic glands. A, denotes activation ratio.

Fig. 6. Effects

M. C. GRUETZMACHER ef al.

338

Table 2. The effects of juvenile hormone I-juvenile

hormone
binding protein complex on prothoracic gland activity in trilro
Developmental
stage
V,

Mean A,kSEM*
(Range)
2h
4h
_.-.
. .._-0.98 i 0.09
1.07 i 0.09
(0.80-1.31)
(0.84-1.31)

8h
-----1.25 f 0.11
(0.98-1.66)

1.17+0.16
(0.72-1.74)

1A4 * 0.30
(3.11-2.80)

1.35* 0.07
(1.21.-1.61)

V,

1.16iO.04
(1.00-1.28)

1.32 k 0.09
(1.01-1.58)

1.20 i: 0.08
(0.92-1.48)

*The mean A, (activation ratio) values represent the mean for all
con~nt~dtions ofjuvenile hormone I bound to juvenile hormone
binding protein assayed. Concentrations of juvenile hormone I
bound to the binding protein assayed were IO-, IO-, IO-, IO+
and IO- M. h signifies hours of incubation.

prothoracic glands. To ensure that our inability to

demonstrate a direct effect was not due to lack of
exposure time between the juvenile hormone and
prothoracic glands, longer incubation periods were
also examined.
Long term t$ects of ZR 512 on prothoracic gland
activity in vitro

It has been suggested that juvenile hormone and

ZR 512 may affect prothoracic gland activity during
very narrowly defined developmental periods in the
fifth instar of Manduca, and that these effects might
be observed in vitro if the glands were maintained in
culture for at least 48 h in the presence of the
hormone (Sho Sakurai, personal communication). To
test this possibility, prothoracic glands were exposed
to ZR 5 12 for 48 h in vitro under sterile conditions in
two separate, preliminary studies. For V, glands the
activation ratio values at 48 h varied more than those
for the 8-hr incubations, but still fluctuated around
one. Because of these fluctuations, only one concentration of ZR 512 (10m9M) yielding an activation
ratio of 1.5 appeared to be significantly different. This
result is probably not physiologically significant for
the same reasons detailed for the 8-hr incubation
results, i.e. lack of a dose-response.
In summary, the results indicate that juvenile hormone does not affect prothoracic gland activity
directly under either the long-term or short-term
conditions utilized here.
Other efects ?~jatie~iIe hormone and its metab~l~tes on
prothoracic glands in vitro

In addition to the possible tropic effect of juvenile

hormone on the prothoracic glands, an inhibitory
effect has also been proposed (see Introduction). A
preliminary in vitro analysis of juvenile hormone I on
V, larval prothoracic gland activity indicated a lack
of effect, although topical application of juvenile hormone I to V, larvae resulted in an arrest of development. However, preliminary analysis of the effects of
juvenile hormone acids on day-0 pupal prothoracic
glands in vitro suggests that the acid of juvenile
hormone III, but not of hormones I or II, inhibits
ecdysone synthesis by the prothoracic glands in a
dose-dependent manner (lo-* M juvenile hormone
III acid to lO-M). Neither methyl epoxy stearate
nor linoleic acid were effective and no change in the
pH of the culture medium was noted after the

addition of juvenile hormone III acid. There was no

effect of any of the juvenile hormone acids on prothoracic glands from larvae (V,, V,, V,, V,). The
physiological significance of this preliminary observation on day-0 pupal prothoracic glands is not clear:
further research is necessary to ascertain if this effect
is physiological or pharmacological.
Nevertheiess,
this is the only result from all of the in vitro studies
on juvenile hormone indicating a direct effect on the
prothoracic glands in vitro.

This investigation of the potential direct tropic

effect of juvenile hormone on the prothoracic glands
of Manduca sexta has revealed by whole-animal
experimentation, i.e. by indirect means, that juvenile
hormone exerts a positive effect on the glands of
neck-ligated, V, larvae as reported previously (e.g.
Hiruma et al., 1978; Cymborowski and Stolarz, 1979;
Safranek et al., 1980). That is, topical application of
the hormone or ZR 512 results in a decrease in the
time to pupation and this is a result of an increased
haemolymph ecdysteroid titre. Further analysis of
this phenomenon assessed the putative activation of
the prothoracic glands by juvenile hormone directly
with an in vitro protocol and revealed that neither
unbound juvenile hormone I, II, III or ZR 512, nor
the hormone bound to binding protein affected prothoracic gland activity in vitro in a dose-dependent
manner at concentrations
ranging from 10d6 to
lo-M.
However, prothoracic glands of the stages
tested can be activated by PTTH extract. At least in
the case of Manduca, juvenile hormone does not
appear to activate the glands directly, but probably
acts indirectly through another factor which, in turn,
stimulates ecdysone synthesis by the prothoracic
glands.
In the past 5 years, there have been several in situ
studies demonstrating that topically applied juvenile
hormone affects prothoracic gland activity in Lepidopteran larvae. In Mamestra brassicae, the time
required for neck-ligated, wandering-stage larvae to
pupate was reduced in a dose-dependent manner by
topically applied ZR 512 (Hiruma et al., 1978). When
the prothoracic glands of these ZR 5l2-treated larvae
were transplanted into untreated recipients, the time
to pupation was also reduced. Similar studies on
unmanipulated
larvae of Spodopkra littoral& gave
essentially the same results, in that larvae treated with
a juvenile hormone analogue after dorsal vessel exposure underwent pupal development more quickly
than untreated animals (Cymborowski and Stolarz,
1979). As in the Mamestra study, it was concluded
that juvenile hormone acted directly on the prothoracic glands and had a prothoracicotropic role at
the time of the second head-critical period (pharate
pupal development). When wandering V, Manduca
larvae were neck-ligated the length of time to the
pupal moult doubled, similar to the effect observed in
Mamestra. Topical application of ZR 512 reduced
this delay which was progressively eliminated by
the analogue in a dose-dependent manner (see also
Safranek et al., 1980).
While the in situ investigations conducted thus far
on the putative prothoracicotropic action of juvenile

E&et of juvenile hormone on prothoracic gland

hormone have unqu~~sti#nably demonstrated such an
effect, the indirect nature ofthe approaches employed
prevented a determination of the level at which the
effect was exerted. However, our attempt to establish
a direct causal relationship between this hormone and
increased prothoracic gland activity using an in vitro
approach was unsucc~essful. Stimulation of the glands
was not observed in the presence of concentrations of
juvenile hormone sp.anning the normal physiological
range of the hormone, nor with concentrations well
above and below this range. These same results were
obtained irrespective of the homologue used and the
manner in which it was presented to the glands (i.e.
rt: binding protein). AIthough it is possible that this
failure to stimulate the prothoracic glands was a
function of unfavourabie in vitro incubation conditions, this seems unlikely since these same conditions
support PTTH activation of the prothoracic glands.
Thus, it appears that juvenile hormone does not
activate the prothoracic glands directly but does so
indirectly. Results suggesting such a possibility have
been reported previously (Sehnal et al., 1981). Further analysis of this phenomenon in ~ff~d~cu using
the in u&o approach has revealed the existence of an
as yet uniden~~ed tropic factor whose synthesis
and/or release is controlled by juvenile hormone
(~ollenbacher
ef a&, ~~etzmacher
et al., unpubi~sh~ obs~~ations~~ It is apparently this factor
that accelerates development. The chemical properties of the factor are ~igni~~ntly different from those
of PTTH, and its nature and function iprsitu will be
the subject of future reports from our laboratories.
A physiological role for juvenile hormone as a
protharacicotropin
driving the larval-pupal
development of ~u~~~~ff is temporally consistent with the
haemolymph titres of juvenile hormone I and II
@ergot et al., personal communication), and with the
in vitro biosynthetic activity of the corpora allata
(Cranger et ul., f982j during this period. Why FTTH
and juvenile hormone are both necessary to regulate
the prothoracic glands is not clear, but one explanation may be the need to sustain an elevated level
of gland activity over a long period of time. This
possibility is supported by the extended increase in
the ecdysteroid titre during larval-pupal
development in Manduca, an increase which begins on day
5.5 and peaks on day 7.5 (BolIenbach~~ et al., 1981).
Conceivably, juvenile hormone could first exert its
indirect effect after commitment (first head-critical
period for PTTH release), elevating the basal activity
of the glands. The increase in corpora allata activity
found on day 5 supports this possibility @ranger ~1
ai., 1982). PTTH would then exert a direct and far
more dramatic effect on ecdysone synthesis at the
second head-critical period of the instar, which would
affect the sharp rise in the ecdysteroid titre on day
7-8. The experimental evidence tits this hypothesis,
since ligated, wandering larvae which lack a source of
PTTH are nevertheless capable of undergoing metamorphosis to the pupa in a normal period of time if
treated with exogenous juvenile hormone, thus indicating the possibility of multiple levels of control of
the prothoracic glands.
Under experimental conditions, either juvenile hormone or PTTH may act alone, the latter directly on
the prothoracic glands and the former indirectly, but

339

to achieve a normal developmental sequence in a

~p~ation
of insects, both must act. This, of course,
does not explain
why larvae whose entire
brain-retrocerebral
complex have been removed ultimately develop (Judy, 1972). In the neck-ligated
Munduca larvae in the present study not treated with
juvenile hormone, the haemolymph ecdysteroid titre
increased gradually until it was N 175% greater than
that of normal larvae when pupation occurred
-4 days after ligation. This corroborates the studies
conducted with cialleria (Sehnal et ai., 1981f and
C+JQ&S (Dean and Steel, 1982). Therefore, the
prothoracic glands which are never completely quiescent in the last-larval instar of ~~~u~a (Bollenbather et al., 1981) may also become active spontaneously.
In contrast to the tropic effect of juvenile hormone
on figated, wandering Vs larvae is the inhibitory effect
of the hormone on the development of last-instar
larvae treated before the first head-critical period
(pre-commitment). This phenomenon has been demonstrated with several species of Lepidoptera (Cymborowski and Stolarz, 1979; Safranek et al., 1980;
Hiruma and Agui, 1982) and has been corroborated
by this study. As with the tropic effect of juvenile
hormone, the inhibition of prothoracic gland activity
by juvenile hormone appears to occur only at specific
developmental stages. Although our attempts to
demonstrate
this effect in vitro with V, (precommitments larval prothoracic glands were unsuccessful, a metabolite of juvenile hormone XII,
juvenile hormone III acid, did inhibit day-0 pupal
prothoracic gland activity in vitro in a doscdependent manner. The physiological significance of
the inhibition of this stage of development is not
known, but is currently being investigated and may
be a fail-safe mechanism for preventing precocious
pupal-adult development.
As can be seen from the above, control of prothoracic gland activity during a single instar is more
complex than thought previously. New hypo~eses
in~rporatin~ these data derived from in &ro studies
must be formulate and tested before we can hope to
elucidate the mechanisms of prothoracic gland regulation. An understanding of these regulating processes is requisite for an understanding of moulting
and metamorphosis.
Acknowledgements-This work was supported by grants
from NIH (AM-30118) to L.G., (M-18791) to W.B. and
from NSF (PCM-8116931) to L.G.
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