Entomologia Experimentalis et Applicata 84: 127–135, 1997.

127

c 1997 Kluwer Academic Publishers. Printed in Belgium.

Semiochemistry of aposematic seed bugs

J. R. Aldrich1; , W. S. Leal1 , R. Nishida2 , A. P. Khrimian3; , C.-J. Lee3 & Y. Sakuratani4

1
National Institute of Sericultural & Entomol. Science, Laboratory of Chemical Prospecting, Tsukuba,
Ibaraki 305, Japan; 2 Pesticide Research Institute, Kyoto University, Kyoto 606, Japan; 3 USDA/ARS, Insect
Chemical Ecology Laboratory, B-007, BARC-West, Beltsville, MD 20705, USA; 4 Entomology Laboratory, Faculty
of Agriculture, Kinki University, Nara 631, Japan;  Permanent address: USDA/ARS, Insect Chemical Ecology
Laboratory;  Present address: Halocarbon Products, Inc., North Augusta, SC 29861, USA

Accepted: May 1, 1997

Key words: Hemiptera, Heteroptera, Lygaeidae, Oncopeltus, Tropidothorax, Neacoryphus, pheromone, attractant,
aposematic, Asclepiadaceae

Abstract

(E )-2,7-Octadienyl acetate and (E )-2-octenyl acetate (1:10 by volume) were identified as a pheromone attractive
to both sexes of the lygaeid bug, Tropidothorax cruciger. In a parallel investigation of Neacoryphus bicrucis
(Lygaeidae), (E; E )-2,4-hexadienyl acetate and phenethyl acetate (9:1) were identified from males, and found
attractive to both sexes of adults in the field plus a tachinid fly parasitoid of the bugs. In N. bicrucis, the pheromone
was clearly shown to come from the tubular accessory glands of the metathoracic scent gland; this evidence,
plus earlier literature reports for other species, indicate that male lygaeids are the pheromone emitters. In another
lygaeid, Oncopeltus fasciatus, 2-isobutyl-3-methoxypyrazine was identified in the cardiac glycoside-laden fluid
sequestered from milkweed hosts and expelled by these bugs when they are attacked. Alkyl methoxypyrazines are
warning odorants associated with poisonous insect secretions, and their presence in O. fasciatus indicates that the
plant-derived chemical defense of lygaeines is more elaborate than previously appreciated.

Introduction eroptera) (Feir, 1974; Aldrich, 1988). The sequestra-

There are more than 4000 species of so-called ‘seed
bugs’ in the world (Heteroptera: Lygaeidae) (Slater
& O’Donnell, 1995). Most lygaeids inconspicuously
feed on fallen seeds, but species in the derived subfam-
ily, Lygaeinae, expose themselves to birds and other
visually oriented predators by feeding on developing
seeds (Aldrich, 1988). Lygaeines counter their vulner-
ability by sequestering poinsonous cardiac glycosides
and pyrrolizidine alkaloids from host plants such as
milkweeds (Asclepiadaceae) and asters (Asteraceae)
(Scudder & Duffey, 1972; Von Euw et al., 1971;
McLain & Shure, 1985). To advertise their unpalatab-
ility, lygaeines have evolved conspicuous red and black
color patterns and are frequently gregarious (Slater &
Baranowski, 1978).
As showy and often abundant insects, lygaeines
have been studied more than most other true bugs (Het-
tion of toxins has, and continues to be, extensively stud-
ied in the large milkweed bug, Oncolpeltus fasciatus
(Dallas) (e.g., Detzel & Wink, 1995; Scudder et al.,
1986). This species has also been a research animal of
choice for investigations of insect migration (Dingle,
1985) and population ecology (e.g., Dingle, 1992), as
have other lygaeines such as Neacoryphus bicrucis Say
(Solbreck & Pehrson, 1979; McLain, 1992). Neverthe-
less, attractant pheromones have yet to be identified for
any lygaeid bug.
Here we report identification of the first attract-
ant pheromones for lygaeids: Tropidothorax cruciger
(Motschulsky), and N. bicrucis (Lygaeinae). In addi-
tion, evidence is presented indicating that the pher-
omones of these species are produced in the gland
usually thought to be solely responsible for chemical
defense in Heteroptera. We also report the identifica-
tion of a heretofore overlooked warning odor present

*141661*
GR: 201003445, Pips nr. 141661 BIO2KAP
ento1581.tex; 20/08/1997; 14:09; v.7; p.1
128
in the sequestered toxic secretion of Oncolpeltus fas-
ciatus.
Materials and methods
Tropidothorax cruciger. About 60 adults of T. cruci-
ger were collected from an overwintering aggregation
of 100 or more individuals on the trunk of a cherry tree
near Yahata-City, Kyoto Prefecture, Japan, on Janu-
ary 28, 1996. The bugs were kept in cages contain-
ing moist cotton at ambient temperature outdoors until
use. Volatiles were collected separately from groups
of ca. 25 males and females in an all glass vessel
(ca. 1 l) contained inside a growth chamber (L16:
D8; 23  C) using SuperQTM as the adsorbent (All-
tech Associates, Inc., Deerfield, IL; 80/100 mesh, ca.
2 g/trial). Incoming air was drawn by aspiration vacu-
um (ca. 1 l/min) from outside the building, prefiltered
through charcoal and SuperQTM columns, and humidi-
fied through a water bubbler before entering the insect
chamber. Entrained volatiles were extracted with high-
grade, redistilled hexane (ca. 10 ml, plus two 2–3 ml
rinses), concentrated by rotoevaporation to ca. 200–
300 l, transferred to cone-bottom vials, and con-
centrated to ca. 100 ml under an argon stream. The
metathoracic scent glands were dissected from some
CO2 -anesthetized adults submerged in tap water, and
extracts of whole metathoracic scent glands or com-
ponents of the gland were prepared in ca. 75 l of
hexane for chemical analysis.
Samples from T. cruciger were analyzed in Japan
by gas chromatography-mass spectrometry (GC-MS)
using a Hewlett-Packard 5890 II Plus GC linked to an
HP 5972 Mass Selective Detector at 70 eV, with an
HP-5MSTM column (0.25 mm film; 30-m  0.25 mm
ID), programmed from 50  C for 2 min to 250  C at
15 /min. Vapor-phase infrared (IR) spectra were recor-
ded using a light pipe interface by gas chromatography-
Fourier transform IR (GC-FTIR) using an HP 6890 GC
with an HP-5 column (0.25 m film; 30 m  0.32 mm
ID; 50  C 1 min to 180C at 5  C/min for 1 min, then
to 230  C at 10  C/min and held for 10 min) in series
with an FTSD-40A GC/C-32 BioRad instrument oper-
ated at 200  C with the transfer line at 250  C. An
HP 5890 GC coupled with an electroantennograph-
ic detector (GC-EAD) was used to locate compounds
in the gland and volatile extracts that were likely to
be pheromone components. The GC-EAD system was
patterned after that described by Struble & Arn (1984)
with modifications originally developed for recording
from the antennae of scarab beetles (Leal et al., 1996a).
Laboratory and field bioassays were performed
for the suspected pheromone of T. cruciger. In the
laboratory, the response of male and female adults to
a 1:10 solution of (E )-2,7-octadienyl acetate: (E )-
octenyl acetate (500 ng/trial in hexane on filter paper;
5 bugs/15 min trial) were tested separately in a Y-tube
olfactometer (aspiration vacuum flow 50 ml/min).
Field tests were conducted from April 23 through
June 8, 1996, at the Kyoto Prefecture site where over-
wintering T. cruciger adults were collected, and on the
campus of Kinki University, Nara Prefecture, May 2–
19, 1996. Three sticky traps (Trap-a-roach Hoy HoyTM,
Earth Chemical Company, Ltd.) each baited with 10 l
of the neat (E )-2,7-octadienyl acetate: (E )-octenyl
acetate blend on a rubber septum were deployed at
each site along with three unbaited control traps.
Neacoryphus bicrucis. Adults were collected from
flower buds of an undetermined composite species near
the campus of the Universidade Federal de Viçosa,
Minas Gerais, Brazil, during mid-September, 1995.
The metathoracic scent glands were dissected from
chilled N. bicrucis adults submerged in tap water, and
extracted in ca. 75 l of CH2 Cl2 for later chemical ana-
lysis in the U.S. The entire gland complex was extrac-
ted for females (1 female gland/sample; 3 samples).
For males, the tubular glands and secondary reser-
voir were dissected and extracted separately from the
primary reservoir of the metathoracic scent gland com-
plex (1 male gland component/sample; 3 samples).
Samples were analyzed by GC on a DB-5TM column
(0.25 m film, 30-m  0.25-mm ID) in a Varian 3500
GC with helium as carrier (50 cm/s linear velocity), a
temperature program from 50  C for 2 min to 235  C at
15 /min, with a flame ionization detector (FID). GC-
MS was performed using a Hewlett-Packard 5890 GC-
MS instrument at 70 eV, with HP-5TM column (0.11 m
film; 25-m  0.2 mm ID), programmed from 50  C for
2 min to 250  C at 15 /min.
Field bioassays were performed for the suspected
pheromone of N. bicrucis at the Beltsville Agricultural
Research Center from July 1 through 31, 1996. Three
live traps (Aldrich et al., 1984) baited with 5 l of
(E; E )-2,4-hexadienyl acetate and phenethyl acetate
(92:8 by volume, neat) on a rubber septum, plus 3
unbaited control traps, were hung ca. 1.5 m from the
ground along a chain-link fence bordering an electrical
powerline. Traps were monitored daily, and rebaited
every 2–3 days.
ento1581.tex; 20/08/1997; 14:09; v.7; p.2

Oncopeltus fasciatus. Adult large milkweed bugs
were collected along roadsides from open milkweed
seed pods (Ascelepias sp.) in Prince George’s County,
Maryland, during late October, 1994. Oncopeltus
adults were squeezed with forceps causing them to
expel droplets from dorso-lateral spaces containing flu-
id enriched in cardenolides (Duffey et al., 1978). This
dorso-lateral space fluid was collected in micropipettes
from 100 milkweed bugs (mixed sexes), and expelled
into 100 l of HPLC-grade water for analysis of alkyl
methoxypyrazines. An EmporeTM extraction disk (oct-
adecyl, 1-cm OD, 3M Company, St. Paul, MN) was
placed in a 1-ml glass-fritted funnel, washed with
0.5 ml aliquots of diethylether, methanol, and water
(HPLC-grade MeOH and H2 O). The milkweed bug
fluid sample was then added to the funnel, filtered
through the disk by positive pressure, and adsorbed
organic compounds were eluted from the disk with ca.
300 l of freshly distilled ether. This extract was dried
over Na2 SO4 , and concentrated to 1–2 l for injec-
tion into the GC-MS. The sample was analyzed on
a Finnigan INCOS XL instrument operated in the EI
mode at 70 eV, with a DB-1TM column (0.25 mm film,
60-m  0.25-mm ID; J&W Scientific, Folsom, CA),
helium as carrier (50 cm/s), a temperature program
from 50  C for 2 min to 230  C at 5 /min.
Chemicals. All identifications were verified by mass
spectral comparisons to, and coinjection with known
standards, except for (E )-4-oxo-2-octenal which was
identified by the published MS (Staddon et al., 1985)
and a retention time matching that for this compound
in exocrine secretions of other Heteroptera (Aldrich,
1995). (E )-4-Oxo-2-hexenal was synthesized accord-
ing to Ward & VanDorp (1969). 2,7-Octadienol (TCI
America, Portland, OR; E :Z = 88:12) was acet-
ylated with acetic anhydride in pyridine, and puri-
fied by flash chromatography on AgNO3 -SiO2 (20%
AgNO3 ) using hexane: ethyl acetate (7:2) to give a
2:98 mixture of (Z )- and (E )-2,7-octadienyl acet-
ate, respectively. (E; E )-2,4-Hexadienyl acetate and
phenethyl acetate were prepared and purified sim-
ilarly by acetylation of the corresponding alcohols.
The following standards were purchased commer-
cially: 2-isopropyl-3-methoxypyrazine, 2-sec-butyl-3-
methoxypyrazine, and 2-isobutyl-3-methoxypyrazine,
propionic acid, and (E; E )-2,4-hexadienol (Aldrich
Chemical Company, Milwaukee, WI); (E )-2-hexenal,
(E )-2-octenal, and (E )-2-octenyl acetate (Bedoukian
Research Inc., Danbury, CT). (E; E )-2,4-hexadienyl
129
propionate was prepared from the acid and alcohol by
standard procedures.
Results
Tropidothorax cruciger. The volatile samples from
T. cruciger males (Figure 1A) and females (not shown)
produced similar GC-FID traces except that the two
major compounds eluting just prior to 10 min (peaks
a and b, Figure 1A) were more abundant in the two
samples from females than in the two samples from
males. In a series of GC-EAD experiments (n = 23),
the antennae of males and females consistently respon-
ded only to the major component eluting at 10.5 min
and to a minor component eluling about 12 s earli-
er, with the greatest antennal response correspond-
ing to the minor component (Figure 1B). The mass
spectra for these two compounds matched those of
(E )-2,7-octadienyl acetate and (E )-2-octenyl acetate,
respectively, in the Hewlett-PackardTM computerized
mass spectral library. Further GC-FID/EAD and GC-
MS experiments with synthetic standards of (E )-2,7-
octadienyl acetate and (E )-2-octenyl acetate verified
that the standards were chemically identical to the
respective natural products. In addition, the IR of
the electrophysiologically active minor natural product
matched that for synthetic (E )-2,7-octadienyl acetate,
including adsorption at 3089 cm,1 which is charac-
teristic of a terminal double bond (Leal, 1996). (E )-
2,7-Octadienyl acetate and (E )-2-octenyl acetate each
elicited strong antennal responses as seen for the nat-
ural products. Finally, the mass spectra of the major
components eluting prior to 10 min (a and b, Fig-
ure 1A) matched the library and published spectra of
(E )-2-octenal and (E )-4-oxo-2-octenal, respectively
(Staddon et al., 1985).
The major GC-FID/EAD compounds found in the
volatiles collected from T. cruciger are characteristic of
the metathoracic scent gland secretion of various heter-
opterans (Aldrich, 1988); therefore, the metathoracic
scent glands were dissected and extracted for chem-
ical analyses. The metathoracic scent gland consists
of paired tubular glands, an unpaired median reser-
voir, and a pair of accessory glands embedded in the
wall of the reservoir (Johansson, 1957; Aldrich, 1988).
The metathoracic scent gland complexes were simil-
ar in males and females, but in males the reservoir
appeared to be slightly reduced, and the tubular glands
somewhat more developed. GC-MS analyses of these
extracts substantiated the visual impression of the relat-
ento1581.tex; 20/08/1997; 14:09; v.7; p.3
130

Figure 1. GC-EAD of an adult female Tropidothorax cruciger to an aeration extract of adult conspecific males. A: the entire GC-EAD run; B:
the active region of the electroantennogram.

Figure 2. Reconstructed ion chromatograms of the extracts of the metathoracic scent gland reservoir and tubular accessory glands from
Tropidothorax cruciger adults. A and C: extracts of the primary scent gland reservoir for a typical male and female, respectively. B and D:
extracts of the tubular accessory glands of the corresponding male and female.

ento1581.tex; 20/08/1997; 14:09; v.7; p.4

 131
Table 1. Attraction of Tropidothorax cruciger adults to (E )-2,7-
octadienyl + (E )-2-octenyl acetates in a Y-tube laboratory bioas-
say (1:10 volume/volume; 500 ng/trial; 3–5 Bugs/15 min trial)

Location in Y-tube Mean  SEM

Female (n = 73) Male (n = 94)

Ester arm 2:18  0:14 1:49  0:11

Control arm 0:38  0:09 0:57  0:09
Control 2:44  0:13 2:94  0:13

ive sizes of the gland components, and corroborated the

chemical pattern observed by GC for airborne extracts
of male and female T. cruciger (Figure 2). The tubular
gland extracts of a typical male (Figure 2B) and female
(Figure 2D) each contained predominantly (E )-2,7- Figure 3. Tropidothorax cruciger adults caught in three sticky traps
octadienyl and (E )-2-octenyl acetates in similar pro- baited with 10 l of (E )-2,7-octadienyl and (E )-2-octenyl acetates
portions but, based on the ion abundances, the extract per period (1:10 by volume, neat) from April 23 through June 8,
1996, Yahata-City, Kyoto Prefecture, Japan. No T. cruciger indi-
from the male was more concentrated than that for the viduals were caught in unbaited control traps.
female. The extracts of the corresponding male and
female reservoirs showed that (E )-2-octenal and (E )- Table 2. Attraction of Tropidothorax cruciger adults
to (E )-2,7-octadienyl + (E )-2-octenyl acetates (1:10
4-oxo-2-octenal were relatively much more abundant
v/v) at Kinki University, Japan, May 2–19, 1996a
in the extract from the female than that from the male
and, based on the ion abundances, that the metathoracic Date Pheromone-baited Unbaited
scent gland reservoir of the female bug contained about Females Males controls
twice as much material as the reservoir of the male
May 2 27 57 0
(Figures 2A and C).
May 4 4 25 0
In the Y-tube bioassay conducted February 28, May 6 0 3 0
1996, in Tsukuba using overwintering T. cruciger May 13b 27 94 0
adults, both males and females were highly attrac- May 17 0 2 0
ted to the olfactometer arm containing 500 ng of the May 19 0 0 0
blend, with the females being more responsive than
a Three pheromone-baited sticky traps and 3 unbaited
the males (Table 1). The first field-test showed that the
control traps/site; 10 l neat pheromone/trap.
blend of (E )-2,7-octadienyl and (E )-2-octenyl acet- b Traps rebaited with pheromone.
ates is sufficient to attract T. cruciger adults into traps.
During the entire test, more males were caught in
traps than females: 147 and 82, respectively, at the alongside early-instar nymphs. Dissection of males and
Kyoto Prefecture site (2 = 18:50, P0:005 = 7:88) females collected in copula revealed that the meta-
(Figure 3); 181 and 58, respectively, at the Kini Uni- thoracic scent glands were sexually dimorphic. In
versity site (2 = 63:30) (Table 2). However, during males, the tubular glands were pinkish and swollen
the second trapping period at Kyoto Prefecture (May 3– with secretion forming a secondary reservoir in addi-
14) significantly more females were caught than males tion to the normal orange-colored primary reservoir.
(2 = 5:40, P0:02 = 5:02) (Figure 3). Observations In females, the primary reservoir of the metathoracic
at the Kinki University site began in early afternoon scent gland was like that of males, but the tubular
under sunny skies, and within 3.5 h there were 84 bugs glands were relatively small and showed no sign of
in traps indicating that bugs can be quickly attracted storing secretion.
under ideal weather conditions (Table 2). The morphological dimorphism observed for
N. bicrucis metathoracic glands is mirrored chemic-
Neacoryphus bicrucis. Adults of this lygaeine were ally (Figure 4). (E )-2-Hexenal, (E )-2-octenal, (E )-
reproductively active at the time field collections were 4-oxo-2-octenal, and (E )-2-octenyl acetate accounted
made in Brazil, with several mating pairs observed for 86% of the total volatiles detected by GC in the

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132
Figure 4. Gas chromatograms and major components of meta-
thoracic scent gland extracts of Neacoryphus bicrucis adults. The
whole gland of a mature N. bicrucis female (A), the primary reser-
voir of a mature male (B), and the secondary reservoir and tubular
glands of the corresponding male (C).
primary reservoir and tubular gland extract of a typic-
al female (Figure 4A). In males, the same four com-
pounds were identified in the primary reservoir extract
of the metathoracic scent gland although (E )-2-octenyl
acetate was relatively much less abundant (Figure 4B).
The minor component eluting just ahead of (E )-2-
octenyl acetate (4) was not identified (nor were other
minor components) but, based on its mass spectrum,
(E )-2,7-octadienyl acetate was eliminated as a pos-
sible structure for this component. The male second-
ary reservoir secretion contained mainly (E; E )-2,4-
hexadienyl acetate (89%) and phenethyl acetate (8%)
(Figure 4C). A trace (<1%) of (E )-4-oxo-2-octenal
was also detected (from the primary reservoir), plus a
trace (<0.2%) of (E; E )-2,4-hexadienyl propionate.
During July, a total of 50 N. bicrucis adults were
caught in pheromone-baited traps, including 27 males
and 23 females (Figure 5). In addition, 17 adult
females of an unidentified tachinid fly were caught
in pheromone-baited traps. No N. bicrucis adults or
tachinid flies were caught in control traps. Two para-
sitic tachinids identical to those caught in pheromone
traps emerged from field-collected N. bicrucis adults.
Figure 5. Neacoryphus bicrucis adults caught in three live traps
baited with 5 l of (E; E )-2,4-hexadienyl acetate and phenethyl
acetate (92:8 by volume, neat) every 2–3 days at the Beltsville
Agricultural Research Center, Maryland, from July 1–31, 1996. No
N. bicrucis individuals were caught in unbaited control traps.
Oncopeltus fasciatus. Under our GC-MS condi-
tions, standards of 2-isopropyl-3-methoxypyrazine,
2-sec-butyl-3-methoxypyrazine, and 2-isobutyl-3-
methoxypyrazine (1 ng/l hexane) eluted at 17 min,
19 min 33 s, and 19 min 47 s, respectively. The
reconstructed ion chromatograph of the dorso-lateral
space fluid sample showed large amounts of unidenti-
fied compounds, but there were relatively few interfer-
ing peaks in the regions where the methoxypyrazines
in question eluted. A small peak was observed in the
space fluid sample at 19 min 47 s whose electron
impact-MS (EI-MS) (Figure 6A) was nearly identic-
al, except for ions at m/z 60 and 73 from a coe-
luting acid, to that for the standard of 2-isobutyl-3-
methoxypyrazine (Figure 6B). The efficiency of our
extraction procedure for alkyl methoxypyrazines was
not determined; however, based on the ion abundances
of the EI-MS from the standard and natural product,
we estimate that milkweed bug adults release as little
as 11 pg of 2-isobutyl-3-methoxypyrazine/bug.
Discussion
Alkyl methoxypyrazines are thought to be warning
odorants associated in many insects with poisonous
secretions (Moore et al., 1990; Dettner & Liepert,
1994; Aldrich et al., 1996). Mating aggregations of
Tropidothorax cruciger were observed on Metaplex-
is japonica (Asclepiadaceae) near the Kyoto Prefec-
ture field site (Nishida, personal observation), and the
adult bugs produced large amounts of dorsal space
fluid with a definite alkyl methoxypyrazine odor sim-
ilar to the exudate from Oncopeltus fasciatus (Aldrich,
ento1581.tex; 20/08/1997; 14:09; v.7; p.6

Figure 6. The electron impact-mass spectrum (EI-MS) of the natural
product isolated from the dorso-lateral space fluid collected from
100 Oncopeltus fasciatus adults (A), and the EI-MS of 2-isobutyl-
3-methoxypyrazine (B).
personal observation). The presence of 2-isobutyl-3-
methoxypyrazine in the cardiac glycoside-laden fluid
expelled by O. fasciatus indicates that the plant-derived
chemical defense of lygaeines is more elaborate than
previously appreciated (Scudder et al., 1986).
It has been proposed that acquisition of poisons
from host plants may have superseded the defensive
role of the metathoracic scent gland in Lygaeinae,
allowing the gland to secondarily evolve a sexual func-
tion (Aldrich, 1988). Our discovery that the attractant
pheromone of T. cruciger comes from its metathoracic
133
scent gland is consistent with this interpretation. Nev-
ertheless, the totality of evidence suggests that produc-
tion of sexual pheromones in the metathoracic gland
of Heteroptera did not necessarily coevolve with spe-
cialization on toxic plants.
The blend of (E )-2,7-octadienyl acetate and (E )-
2-octenyl acetate that constitutes the pheromone of
T. cruciger is produced in the paired tubular glands
attached to the main reservoir of the metathoracic
scent gland complex. In the overwintering specimens
of T. cruciger available to us the tubular glands were
only slightly larger in males than females, but the
tubular glands of sexually mature Tropidothorax males
are substantially enlarged relative to those of females
(Carayon, 1948). The metathoracic glands of mat-
ing Neacoryphus bicrucis males, in which the tubular
glands were swollen with secretion forming a second-
ary reservoir, may be more typical of lygaeines because
morphologically similar metathoracic glands have
been described for two related species: Oncolpeltus
fasciatus (Johansson 1957), and Spilostethus rivularis
(Germar) (Staddon et al., 1985). In O. fasciatus, C6 and
C8 (E )-2-alkenyl and (E; E )-2,4-alkadienyl acetates
are reportedly abundant only in the tubular glands of
males (Games & Staddon, 1973; Games et al., 1974).
In S. rivularis, whole gland extracts of both sexes con-
tained (E; E )-2,4-hexadienyl acetate and 2-phenethyl
acetate, but the former component accounted for 96%
of the volatiles in the extract from a male versus only a
trace from that of a female (Staddon et al., 1985). Thus,
the tubular gland secretion of S. rivulais males, a native
of the Ethiopian region, is probably a pheromone like
that of its New World relative, N. bicrucis.
Although Carayon (1948) noted that not all
lygaeids have sexually dimorphic metathoracic glands,
he did find that in males of various species of Blissinae
and Rhyparochrominae the tubular glands are hyper-
trophied as in lygaeines. Neither blissines nor rhypa-
rochromines are noted for feeding on toxic plants or
for being aposematic (Slater & Baranowski, 1978). In
another non-aposematic seed bug, Oxycarenus hya-
linipennes (Costa) (Oxycareninae), the metathoracic
glands appear similar in males and females, but a
day or so after emergence the tubular glands of both
sexes undergo a dramatic change from synthesizing
aliphatics to the synthesis of sesquiterpenes, prin-
cipally (Z; E )--farnesene (Olagbemiro & Staddon,
1983; Knight et al., 1984). Thus, it seems that the
metathoracic scent glands have evolved sexual roles
even in lygaeid species that do not sequester plant tox-
ins for defense.
ento1581.tex; 20/08/1997; 14:09; v.7; p.7
134
Attractant pheromones emanating from the meta-
thoracic scent glands have also been identified in spe-
cies of Miridae and Alydidae, two distantly related
heteropteran families. Females of many mirid plant
bugs attract males with pheromones (Aldrich, 1996).
The first such pheromone to be chemically identi-
fied was for Campyloma verbasci (Meyer) (Mirid-
ae: Phylinae); a blend of butyl butyrate and (E )-2-
butenyl butyrate (16:1, respectively) attracts conspe-
cific males (Smith et al., 1991), and this blend prob-
ably comes from the metathoracic glands (Thistlewood
et al. 1989). Males of the bean bug, Riptortus clavatus
(Thumberg) (Alydidae), release (E )-2-hexenyl (E )-2-
hexenoate, (E )-2-hexenyl (Z )-3-hexenoate, and myri-
styl isobutyrate which attracts both sexes of adults, as
well as nymphs (Leal et al., 1995). The pheromones
of Riptortus spp. are produced in the enlarged tubu-
lar glands of males (Aldrich et al., 1993; Aldrich &
Leal, unpubl.). Other alydids both with (Leal et al.,
1996b) and without (Aldrich et al., 1993) sexually
dimorphic metathoracic glands also apparently release
attractant pheromones from this gland. Again, none
of these species are aposematic or feed on poisonous
plants although, curiously, alydids are characterized
by another form of defense: ant and wasp mimicry
(Aldrich et al., 1972; Schuh & Slater, 1995).
Our discoveries that tubular gland esters constitute
the attractant pheromone of T. cruciger and N. bicru-
cis, and that alkyl methoxypyrazines appear to advert-
ise unpalatability in milkweed bugs, should provide
impetus for semiochemical research on these and oth-
er lygaeids. Indeed, the possibility that disparate het-
eropterans produce sexual pheromones in their meta-
thoracic scent glands ought to be reconsidered inas-
much as similar pheromones occur in species from
each of the main phylogenetic branches of terrestrial
Heteroptera (Schuh & Slater, 1995).
Acknowledgments
The work in Japan was supported in part by a special
coordination fund for promoting science and techno-
logy in the basic research core system by the Science
and Technology Agency (STA) of Japan granted to
WSL, and JRA held an STA Fellowship for research
in Japan. One of us (JRA) also gratefully acknow-
ledges support from the U.S. Department of Agricul-
ture, Organization for International Cooperative Devel-
opment, for research in Brazil. We thank Dr Thomas
J. Henry, Systematic Entomology Laboratory, USDA-
ARS, Smithsonian Institution, for species determina-
tion. Finally, we thank Ms Claudia Zarbin for running
laboratory bioassays, and Ikuhisa Nishida for finding
the overwintering population of T. cruciger.
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