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