Close-range attraction in Lygocoris pabulinus (L.)

Article abstract of DOI:10.1023/A:1010311928256

Males of the green capsid bug, Lygocoris pabulinus, exhibit a specific courtship behavior, i.e., a vibration of the abdomen. When both live and dead females were offered to males, this vibration behavior was elicited in most of the males tested. When females were dissected into separate body parts, heads, wings, and legs elicited equal responses, while thorax plus abdomen elicited a much lower response. When separate body parts were extracted, the leg extracts elicited significantly stronger responses than any other extract. This suggests that female L. pabulinus legs are either the source of a close-range sex pheromone or that pheromone is accumulated on the legs due to grooming behavior. The leg extracts contained several hydrocarbons such as n-alkenes, n-alkanes, and some methylalkanes. Female extracts contained more (Z)-9-pentacosene and male extracts contained more (Z)-9-heptacosene. Substrates on which females had walked elicited similar responses as female legs, indicating that the pheromone is deposited on the substrate. This enlarges the functional range of low-volatility compounds, which are thought to function only when sexes are in close vicinity or in contact.

Full text of DOI:10.1023/A:1010311928256

Journal of Chemical Ecology, Vol. 27, No. 6, 2001
CLOSE-RANGE ATTRACTION IN Lygocoris pabulinus (L.)
FALKO P. DRIJFHOUT1, and ASTRID T. GROOT2
1Laboratory of Organic Chemistry, Phytochemical Section
Wageningen University
Dreijenplein 8, 6703 HB Wageningen, The Netherlands
2Plant Research International, Wageningen UR
P.O. Box 16, 6700 AA Wageningen, The Netherlands
(Received May 1, 2000; accepted February 10, 2001)
Abstract—Males of the green capsid bug, Lygocoris pabulinus, exhibit a spe-
cific courtship behavior, i.e., a vibration of the abdomen. When both live and
dead females were offered to males, this vibration behavior was elicited in most
of the males tested. When females were dissected into separate body parts, heads,
wings, and legs elicited equal responses, while thorax plus abdomen elicited a
much lower response. When separate body parts were extracted, the leg extracts
elicited significantly stronger responses than any other extract. This suggests that
female L. pabulinus legs are either the source of a close-range sex pheromone or
that pheromone is accumulated on the legs due to grooming behavior. The leg
extracts contained several hydrocarbons such as n-alkenes, n-alkanes, and some
methylalkanes. Female extracts contained more (Z)-9-pentacosene and male
extracts contained more (Z)-9-heptacosene. Substrates on which females had
walked elicited similar responses as female legs, indicating that the pheromone
is deposited on the substrate. This enlarges the functional range of low-volatility
compounds, which are thought to function only when sexes are in close vicinity
or in contact.
Key Words—Heteroptera, Miridae, contact sex pheromone, male vibration,
(Z)-9-pentacosene, (Z)-9-heptacosene, cuticular hydrocarbons, close-range
pheromone, SPME.
INTRODUCTION
Sex pheromones are commonly used by insects to locate mates at long range
and to stimulate mating at close range (Carlson et al., 1971; Carde´ et al., 1975;
Muhammed et al., 1975). Long-range sex pheromones were first described and
chemically identified in moths (Butenandt et al., 1959) and are now widely used for
To whom correspondence should be addressed. e-mail: Falko.Drijfhout@bio.oc.wau.nl
1133
C
0098-0331/01/0600-1133$19.50/0 2001 Plenum Publishing Corporation

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DRIJFHOUT AND GROOT
monitoring of lepidopterous pests (e.g., Minks and Van Deventer, 1992; Carde´ and
Minks, 1997). In recent years identification of nonlepidopteran sex pheromones
has received growing attention (Hardie and Minks, 1999). In mirids (Heteroptera:
Miridae), whose virgin females attract males, long-range sex pheromones have
been identified for three species so far (Smith et al., 1991; Millar et al., 1997;
Millar and Rice, 1998; McBrien and Millar, 1999).
Close-range sex pheromones initiate courtship behavior. Such pheromones
are usually less volatile than long-range pheromones (Blomquist et al., 1993).
Despite their low volatility, close-range pheromones may play an important role
in the decision of an insect to land at a certain spot (Carlson et al., 1971). Without
the addition of such pheromones, arriving males may not enter a trap (Carde´
et al., 1975; Kennedy, 1977). In mirids close-range sex pheromones have not yet
been reported. Major focus has been on attractive and alarm compounds from
the metathoracic and accessory scent glands (e.g., Carayon, 1971; Staddon, 1979;
Aldrich, 1988). Compounds identified from these glands have carbon chain lengths
of 2–15 and are most commonly acids, aldehydes, ketones, alcohols, and esters
(Staddon, 1979; Aldrich, 1988). Close-range pheromones may have carbon chain
lengths of 20–30 or even more (Blomquist et al., 1993). The source of long-
range pheromones in mirids has been suggested to be the metathoracic scent gland
(Aldrich, 1988), or at least the thoracic region (Millar et al., 1997), although
Graham (1988) identified the ovipositor region as source of attraction. Since the
chemicalnatureofclose-rangepheromonesmaydiffercompletelyfromlong-range
pheromones, their sources probably differ as well.
To identify close-range sex pheromones, a specific arousal or courtship be-
havior of one of the sexes should be distinguished. Males of the green capsid bug
[Lygocoris pabulinus (L.), Heteroptera: Miridae] exhibit a characteristic sex-
specificcourtshipbehavior, arepeatedvibrationoftheabdomen;onlymalesvibrate
in the presence of females and only when they are sexually mature (Groot et al.,
1998). We used this vibration behavior of male L. pabulinus to determine the
source of attraction in females at close range. Additionally, we attempted to iden-
tify compounds involved in this close-range attraction.
METHODS AND MATERIALS
Insects. Lygocoris pabulinus was reared on potted potato plants, (cultivar
Bintje) in wooden cages in a greenhouse at 22 2 C, 65 5 % relative humidity,
and 18L : 6D photoregime, following the procedure of Blommers et al. (1997). Ev-
ery two to three days newly emerged adults were collected from the rearing cages,
after which the sexes were isolated in separate rearing cages. In this way, virgin
males and females of known age were continuously available for the experiments
(Groot et al., 1998).

GREEN CAPSID BUG ATTRACTION
1135
Bioassays. One to two hours before each test, virgin males 6–9 days old were
collected from the separate rearing cages and isolated in small glass tubes. Glass
Petri dishes 5 cm diam. were cleaned with acetone, and the bottoms were covered
with white filter paper disks of the same diameter. The stimuli to be tested were
placed in the Petri dishes, after which one male per dish was introduced. Stimuli
consisted of one bug equivalent per Petri dish, and originated from bugs that were
virgin and 6–9 days old. All males were observed for 15 min. If a male in a dish
started to vibrate within this period, that dish was set aside and counted as a positive
response. The number of Petri dishes with positive responses was calculated as a
fraction of the total number of Petri dishes in which the stimulus had been applied.
Different stimuli were tested at the same time, and stimuli were tested on several
different days. All experiments were carried out at 19–23 C between 10:00 and
14:00 hr C.E.T.
In a second experiment, a solid-phase microextraction (SPME) needle
(100 m polydimethylsiloxane coating from Supelco, Bellefonte, Pennsylvania)
was positioned on the bottom of the Petri dish through a hole in the side. A metal
strip was placed in a V-shape around the needle to reduce the amount of space the
bug had, thus increasing the chance of bug–needle contact. One or two bugs were
then placed in the Petri dish for 2–3 hr.
Stimuli Tested. First, a series of live females, dead females, live males, and
dead males were tested. Bugs were anesthetized with CO2, after which the heads
were clipped off. In a following series freshly anesthetized females were dissected
into heads, wings, legs, and thorax plus abdomen. Thorax and abdomen were
not subdivided, since clipping would mean cutting through several organs and
glands that run from thorax to abdomen, which may then release a variety of
chemicals. Third, extracts were made of the different body parts of females, i.e.,
heads, wings, legs, and thorax plus abdomen. After anesthetizing fresh females
with CO2, the body parts were dissected and placed in 1.8- or 4-ml vials. After
dissecting all available females, 15–50 l of either dichloromethane, pentane,
pentane–ether (2 : 1), or water per female was added, the amount of which was set
as one female equivalent of the particular extract. The extracts were stored in a
freezer ( 20 2 C) until used. All extracts were used 1–14 days after the initial
dissections. In a fourth series, one female equivalent of a synthetic mixture (2.5
g
in total) was tested. The synthetic mixture consisted of 1-hexanol (40 ng), hexyl
butyrate (500 ng), (E)-2-hexenyl butyrate (25 ng), (Z)-9-tricosene (150 ng), (Z)-7-
pentacosene (200 ng), (Z)-9-pentacosene (1000 ng), (Z)-9-heptacosene (200 ng),
tricosane (80 ng), and pentacosane (80 ng) (Table 1 below).
Chemical Analysis. Extracts were analyzed with a dual-column GC (HP
6890) equipped with an apolar DB-1 column (J&W Scientific, Folsom, California;
60 m 0.25 mm; 0.25- m) and a polar Stabilwax column (Restek, Bellefonte,
Pennsylvania; 60 m 0.25 mm; 0.25- m) and two flame ionization detectors.
Oven program: 30 C (2 min hold) to 238 C (25 min hold) at 4 C/min. Hydrogen

1136
DRIJFHOUT AND GROOT
TABLE 1. AVERAGE COMPOSITION (%) OF EXTRACTS OF LEGS FROM FEMALE AND
MALE L. pabulinus
Composition(%, mean SD)
No.a
Compound
Hexan-1-olb
Females
Males
1
2
3
4
5
1.7 1.8
19.2 13.7
0.8 0.5
6.3 1.9
3.1 1.7
0.2
0.6 0.4
22.3 23.4
0.8 0.9
0.4 0.7
0.1 0.1
0.2
Hexyl butyrateb
(E)-2-Hexenyl butyrateb
Tricosaneb
(Z)-9-Tricoseneb
(Z)-9-Tetracosene
Tetracosane
0.2
0.2
8
9
10
11
2-Methyltetracosane
Pentacosaneb
(Z)-9-Pentacoseneb
(Z)-7-Pentacoseneb
9-Hexacosene
3.3 1.5
8.6 2.8
40.8 9.2
8.7 1.9
0.2
1.8 0.2
10.6 4.9
3.4 2.3
20.5 4.8
0.2
2-Methylhexacosane
(Z)-9-Heptacoseneb
(Z)-7-Heptacosene
Heptacosane
0.2
7.6 4.6
0.2
0.2
39.6 13.2
0.2
14
0.2
0.2
(Z)-9-Nonacosene
0.2
0.2
aNumbers according to Figures 3 and 4.
bCompounds used in the synthetic mixture.
was used as the carrier gas (constant flow of 2.4 ml/min, linear velocity: 48 cm/sec).
GC-MS analyses were carried out on Varian 3400 GC connected to a Finnigan
MAT95 mass spectrometer. The BP5 column (SGE; 25 m 0.25 mm; 0.25- m)
was programmed from 50 C to 270 C (4 min. hold) at 5 C/min. The mass spec-
trometer was operated in EI mode (at 70 eV) and scanning was done from mass
24 to 500 at 0.7 sec/dec. 1H NMR (200 MHz) spectra were recorded on a Bruker
AC200 spectrometer. FTIR spectra were recorded on a Perkin-Elmer 1725X spec-
trometer. Identification of compounds in extracts was carried out with GC-MS and
by comparison of retention times of reference compounds with GC. The position
of the double bond in the alkenes was determined by derivatization with DMDS
according to Carlson et al. (1989).
Chemicals. Hexanol, tricosane, pentacosane, 1-bromotetradecane, 1-bromo-
hexadecane, 1-bromooctadecane, triphenylphosphine, nonanal, heptanal, n-butyl-
lithium in hexane, DMDS, and urea were all purchased from Acros Organics (Geel,
Belgium) and hexyl butyrate from Roth (Karlsruhe, Germany). (E)-2-Hexenyl
butyrate was synthesized according to Drijfhout et al. (2000). (Z)-9-Tricosene,
(Z)-9-pentacosene, (Z)-7-pentacosene, and (Z)-9-heptacosene were all synthe-
sized as described below. All the chemicals used were >98% pure. All solvents
used were distilled twice before use.

GREEN CAPSID BUG ATTRACTION
1137
Synthesis of Alkyltriphenylphosphonium Bromide. A mixture of 0.3 mmol
of the alkylbromide (1-bromotetradecane, 1-bromohexadecane, or 1-bromoocta-
decane) and 0.3 mmol of triphenylphosphine was heated to 140 C under a nitrogen
atmosphere for 5 hr. The reaction mixture formed a solid when cooled down. Dry
acetone (5 ml) and dry diethyl ether (12 ml) were added to the solid and cooled to
20 C (overnight). After filtration, the alkyltriphenylphosphonium bromide was
obtained as white crystals.
Synthesis of Alkenes. A slurry of 5 mmol of powdered alkyltriphenylphos-
phonium bromide in 10 ml of THF was prepared under nitrogen. The mixture
was cooled in an ice bath, 5 ml of DMSO was added after which 5 mmol of
n-butyllithium in hexane was injected. The butyllithium was added at such a rate
that the temperature of the mixture remained at 10–15 C. After 5 min, 5 mmol
of the aldehyde (nonanal or heptanal) was injected, and the resulting mixture was
stirred for 30 min at ambient temperature. The mixture was diluted with water
and extracted with petroleum ether 40/60. The dried (MgSO4) extract was filtered
and concentrated to give a 90% yield of the alkene. The alkene was further purified
with column chromatography on silica gel and eluted with hexane to give a mixture
of Z and E isomers in a ratio of 85 : 15.
Separation of Z and E Isomers. The two isomers of the alkenes obtained
during the synthesis were separated making use of their different complexation
with urea (Leadbetter and Plimmer, 1979). One part of alkene and five parts of
urea were dissolved in 20 parts of methanol. This was left to crystallize at room
temperature. The white crystals were separated by filtration. The methanol fraction
was evaporated to obtain the (Z)-isomer in 97% purity. The procedure was repeated
to improve the purity of the Z isomer. If the alkene could not be dissolved in
methanol, isopropanol was added.
1
(Z)-9-Tricosene. H NMR (CDCl3) 0.83–0.89 (t, 6 H, CH3), 1.2–1.6 (m,
34 H, CH2CH2 ), 2.0 (m, 4 H, CH CH2), 5.3–5.35 (m, 2 H, CH CH). MS:
m/z = 322 (M+); Kovats indices: 2320 on Stabilwax and 2271 on DB-1. IR (film):
1
CH(cis) 722 (m) cm
HC
1
(Z)-7-Pentacosene. H NMR (CDCl3) 0.83–0.89 (t, 6 H, CH3), 1.2–1.4, (m,
38 H, CH2CH2 ), 1.9–2.0 (m, 4 H, CH CH2), 5.3, (m, 2 H, CH CH). MS:
m/z = 350 (M+); Kovats indices: 2526 on Stabilwax and 2477 on DB-1; IR (film):
1
722 cm
HC CH (cis)
1
(Z)-9-Pentacosene. H NMR (CDCl3) 0.83–0.89 (t, 6 H, CH3), 1.2 (m, 38
H, CH2CH2 ), 1.9–2.2 (m, 4 H, CH CH2), 5.3–5.4 (m, 2 H, CH CH). MS:
m/z = 350 (M+); Kovats indices: 2519 on Stabilwax and 2470 on DB-1; IR (film):
1
722 cm
HC CH (cis)
1
(Z)-9-Heptacosene. H NMR (CDCl3): 0.9 (t, 6 H, CH3), 1.1–1.5 (m, 40 H,
CH2CH2 ), 2.2 (m, 4 H, CH CH2), 5.3 (m, 2 H, CH CH). MS: m/z = 378
(M+); Kovats indices: 2721 on Stabilwax and 2671 on DB-1; IR (film):
722 cm
HC CH (cis)
1

1138
DRIJFHOUT AND GROOT
Statistical Analysis. If males responded to a source, differences in responses
towards the different sources were statistically analyzed by fitting a logit regres-
sion model with overdispersion to the daily observed counts of responses of a test
(McCullagh and Nelder, 1989), using the computer program Genstat 5 (release
4.1, PC/Windows NT, 1997). In the model, source was taken as the explanatory
variable and the variance was assumed to be proportional to the binomial vari-
ance. First, a chi-square test for the residual deviance was conducted to determine
overdispersion. The overall effect of treatments was determined by performing an
F test for the ratio of the mean deviance for treatment and the mean deviance of
the rest. If the overall test was significant (P < 0.05), pairwise comparisons by t
test between treatment means on the logit scale were conducted.
RESULTS
Live and dead females elicited similar responses, the fractions of males
vibrating being 0.88
0.08 and 0.74
0.09 (mean
SE), respectively
(Figure 1A). Live and dead males elicited vibration responses in few males. When
the bodies of females were dissected, the head, wings, and legs were equally attrac-
tive and as attractive as dead females, while the thorax plus abdomen of females
were significantly less attractive (Figure 1B). Responses to freshly dissected wings
and heads may be due to grooming, which spreads attractive compounds over the
body surface. For confirmation of presence of attractive compounds on the whole
body surface, small pieces of filter paper were rubbed over female bodies (after
anesthetizing and clipping off heads). When these pieces of paper were offered
in clean Petri dishes, almost half of the males tested (0.41 0.07, mean SE)
started vibrating (N = 46).
After extraction of the separate body parts of females, leg extracts elicited sig-
nificantly more vibrational response than all other extracts (Figure 1C). Extracts
from thorax plus abdomen did not elicit a response from males, which may be
due to defensive compounds in the metathoracic gland. Therefore, extracts were
also made of female thorax plus abdomen, from which the metathoracic gland
was removed by gently cutting the cuticle with two sharp tweezers, trying to de-
stroy as little tissue as possible. Few males responded to this extract (Figure 1C).
When differences in responses between freshly dissected body parts and their
corresponding extracts were statistically compared, male responses to the leg ex-
tracts were not different from responses to freshly dissected legs, while wing and
head extracts elicited lower responses (P < 0.05) than freshly dissected wings and
heads.
Organic solvent extracts stimulated significantly more males to vibrate than
water extracts (Figure 2). The graph indicates that “water extracts” caused only
minimal vibration in males. Legs from female bugs, used for these “water extracts,”

GREEN CAPSID BUG ATTRACTION
1139
FIG. 1. Male L. pabulinus responses (mean SE) to different stimuli: (A) whole insects,
(B) body parts of females, (C) extracts of female body parts. 1Total number of males tested;
2Metathoracic gland left in thorax; 3Metathoracic gland removed from thorax. Significant
differences were determined between sources within one group (A, B, C). Different letters
above the bars indicate significant differences in each group at the 5% level. See text for
statistical methods used. Not included in the statistical analyses, as no males had responded.

1140
DRIJFHOUT AND GROOT
FIG. 2. Response of male L. pabulinus to extracts of female legs prepared in different
solvents (mean SE; n = number of males tested). Different letters above bars indicate
significant difference at P 0.05 (two-tailed).
still elicited vibratory behavior in males. No significant difference in male vibration
occurred using dichloromethane, pentane or the mixture of pentane and ether
(Figure 2).
In both male and female leg extracts, the major part of the compounds con-
sisted of hydrocarbons (Figure 3A). These hydrocarbons consisted of alkenes
(75%), alkanes (20%), and some monoalkanes (5%). (Z)-9-Pentacosene (10) was
the most abundant alkene in females, while (Z)-9-heptacosene (14) was the most
abundant in males. The ratio of (Z)-9-pentacosene to (Z)-7-pentacosene (11) was
opposite in males and females. Furthermore, sometimes the female extracts con-
tained more (Z)-9-tricosene than male extracts. (Figures 3B and C). Both male and
female extracts contained three oxygen containing compounds: hexyl butyrate (2)
and (E)-2-hexenyl butyrate (3) and sometimes 1-hexanol (1). Other minor com-
pounds identified in the leg extracts of male and female L. pabulinus are listed
in Table 1. Table 2 lists some characteristics used to identify the most abundant
alkenes present in the extracts.
The vibrational bioassays suggest that female legs are the source of close-
range sex pheromone. To determine whether the source of attraction could be
defined more precisely, female legs were subdivided into: (A) forelegs, middle
legs, and hind legs; or (B) coxae plus femorae, and tibiae plus tarsi. In series A,
one pair of forelegs, middle legs, or hind legs of three females was placed in one
Petri dish, so that six legs per dish were offered. In series B, the six coxae plus
femorae of one female were placed in one Petri dish, and the six tibiae plus tarsi
in another. Table 3 shows that all parts of the legs were equally attractive (no
significant differences were found between any pair). However, when the overall

GREEN CAPSID BUG ATTRACTION
1141
FIG. 3. Gas chromatograms of L. pabulinus leg extracts: (A) female leg extract; enlargement
of the cuticular hydrocarbon part of female (B) and male (C) extracts. DB-Wax column;
FID detector. For explanation of the numbers see Table 1.
response to fore-, middle and hind legs was compared to the overall response to
coxae plus femorae and tibiae plus tarsi, responses to entire legs were significantly
stronger than to parts.
When legs contain attractive compounds, these compounds may be deposited
on the substrate on which female L. pabulinus walk. To determine possible de-
position of attractive compounds, we tested three different substrates: a piece of
potato leaf (cultivar Bintje), a piece of green bean leaf (Phaseolus vulgaris, culti-
var Miracle), and the glass of an empty Petri dish. One L. pabulinus female was
allowed to walk in each dish for 75–140 min. As a control, we tested pieces of

1142
DRIJFHOUT AND GROOT
TABLE 2. CHARACTERISTICS USED IN IDENTIFICATION OF FOUR MAJOR ALKENES
PRESENT IN LEG EXTRACTS OF L. pabulinus
Kovats indices
DBI Stabilwax
MS characteristics
Compound
M+
m/z after DMDS derivatization
5
10
11
14
2271
2470
2477
2670
2319
2518
2526
2719
322
350
350
378
173 (C1–C9), 243 (C10–C23), 416 (M+)
173 (C1–C9), 271 (C10–C25), 444 (M+)
145 (C1–C7), 299 (C8–C25), 444 (M+)
173 (C1–C9), 299 (C10–C27), 472 (M+)
potato leaf or empty dishes on which males had walked for 60–120 min, as well
as pieces of potato leaves on which no bug had walked. Table 4 shows that males
responded to substrates on which females had walked, while no males showed
vibration behavior in any of the control dishes. During the 75–140 min that fe-
males walked around in the dishes, a characteristic pheromone-laying behavior
was not observed. In addition, desorption of the SPME needle (250 C) revealed
the same hydrocarbon profile as found in the leg extracts of the concerning sex.
From Figure 4 it is clear that when females walked on or near the needle, a high
concentration of (Z)-9-pentacosene (10) was deposited on the needle. The volatile
compounds 1-hexanol (1), hexyl butyrate (2), and (E)-2-hexenyl butyrate (3) were
not always present.
When the synthetic mixture, containing compounds derived from female
leg extracts, was tested in the vibration bioassay, no males started to vibrate.
A few males showed vibratory behavior when male legs, loaded with 5 g (Z)-9-
pentacosene (instead of 1 g), were offered (Table 5). Adding (Z)-9-tricosene
to these legs did not result in more males vibrating. As no biological active
compound was found, the possibility of (Z)-9-heptacosene acting as a repel-
lent was investigated. Female legs were therefore loaded with a high dose of
TABLE 3. MALE VIBRATION RESPONSE TO DIFFERENT PARTS OF FEMALE LEGS
Source
Fraction of males responding ( SE)
N
Female legs
Forelegs
Middle legs
Hind legs
Coxae + femorae
0.70 0.05
0.76 0.14
0.71 0.15
0.88 0.10
0.45 0.14
143
17
17
17
22
aa
a
a
a
a
Ab
A
B
Tibiae + tarsi
0.41 0.14
22
a
aDifferent letters indicate signifant differences between pairs (P < 0.05).
bDifferent letters indicate significant differences between groups (P < 0.05).
See text for statistical methods used.

GREEN CAPSID BUG ATTRACTION
1143
FIG. 4. Gas chromatographic analysis of an SPME needle on which a female bug had
walked. DB-Wax column; FID detector. For explanation of the numbers see Table 1.
(Z)-9-heptacosene (5–8 g). Yet, a similar amount of males showed vibration
behavior to these legs as when untreated female legs were offered (Table 5).
DISCUSSION
The legs of L. pabulinus females most consistently elicited vibration behavior
in males, suggesting that legs are the source of close-range attraction in this species.
Legs have been recognized as the site of sex pheromone release in the aphid
Megoura viciae (Marsh, 1972), the mosquito Culiseta inornata (Lang, 1977), the
tsetse fly Glossina morsitans morsitans (Carlson et al., 1978), the housefly Musca
domestica (Schlein et al., 1980), and the parasitoid braconid Ascogaster reticulatus
(Kainoh and Oishi, 1993). In some species, specific glands in the legs have been
TABLE 4. MALE VIBRATION RESPONSE TO SUBSTRATE ON WHICH FEMALES HAD
WALKED
Fraction of males
Source
responding ( SE)
N
Potato leaf on which female walked for 75–140 min
Bean leaf on which female walked for 75–140 min
Empty Petri dish on which female walked for 75–140 min
Potato leaf on which male walked for 60–120 min
Empty Petri dish on which male walked for 60–120 min
Potato leaf
0.68 0.10
0.64 0.13
0.37 0.09
39
22
46
15
10
10
aa
a
a
b
0
0
0
b
b
aDifferent letters indicate signifant differences (P < 0.05). See text for statistical methods used.
bNot statistically analysed, as no male had responded.

1144
DRIJFHOUT AND GROOT
TABLE 5. MALE VIBRATORY RESPONSE TO FRESHLY DISSECTED LEGS WITH OR
WITHOUT ALKENE ADDED
Fractions of males Males
Source
responding ( SE)
(N)
Synthetic mixture
Male legs
0
0
32
5
aa
a
Male legs + 5 g (Z)-9-pentacosene
Male legs + 5 g (Z)-9-pentacosene + 1 g (Z)-9-tricosene
Female legs + 5–8 g (Z)-9-heptacosene
Female legs
0.19 0.11
0.05 0.06
0.76 0.13
0.70 0.06
29
10
21
143
a
a
b
b
aDifferent letters indicate significant difference at P 0.05 (two-tailed).
identified as the site of sex pheromone excretion (Marsh, 1972; Schlein et al.,
1980). In L. pabulinus, response to fore-, middle, and hind legs was similarly
strong. The lower response to parts of the legs compared to entire legs may be due
to the lower amount of leg biomass per Petri dish in the latter group. From these
experiments no specific site of possible glands in legs became apparent.
Specific glands in the legs may not synthesize contact sex pheromones.
Cuticular hydrocarbons are probably synthesized by oenocytes, large cells that
are rich in smooth endoplasmatic reticulum and mitochondria, which appear to be
restricted to epidermal tissue in the thorax and abdomen (Gu et al., 1995; Schal
et al., 1998). After synthesis, attractive hydrocarbons may be deposited at specific
target sites, as in the German cockroach Blattella germanica, where the wings
accumulate large amounts of pheromone (Gu et al., 1995). The cuticle of the legs
may thus be the specific target deposition site of the attractive compounds.
The presence of a close-range sex pheromone on the legs may also be due
to grooming. Grooming may either accumulate pheromone from other body parts
on the legs (Howard and Blomquist, 1982), or it may spread the pheromone from
leg glands over the whole body surface, as in polistine wasps (Beani and Calloni,
1991), whose territorial marking pheromones from leg glands function as sex
attractants as well. L. pabulinus males and females groom frequently (Groot et al.,
1998) and the attractive compounds are not only present on female legs, but also
on other body parts as demonstrated by the male response to female wings and
heads and to pieces of filter paper rubbed over female bodies. In short, the site
of sex pheromone production does not have to be the site of pheromone release,
specific glands are not necessarily involved, and grooming may enhance chemical
dispersion or accumulation at specific sites.
In mirids, close-range or contact pheromones have not yet been studied,
and to our knowledge this is the first study on cuticular hydrocarbons of a mirid
species. In L. pabulinus the major part of the hydrocarbons consisted of alkenes,
followed by alkanes and some methyl alkanes. All compounds were C23–C29

GREEN CAPSID BUG ATTRACTION
1145
hydrocarbons. This is also the first time that a clear-cut difference was found in
L. pabulinus between male- and female-derived compounds, i.e., females produce
a high amount of (Z)-9-pentacosene, whereas males produce high amounts of
(Z)-9-heptacosene. Because of the low amount of (Z)-9-pentacosene present in
males, the ratio of (Z)-9-pentacosene and (Z)-7-pentacosene is opposite in males
and females. To our knowledge, there are only two other studies on cuticular
hydrocarbons in heteropteran species, the milkweed bug, Oncopeltus fasciatus
(Lygaeidae) (Jackson, 1983), and Triatoma infestans and T. mazzotti (Reduvidae)
(Jua´rez and Blomquist, 1993). In all these species mostly n-alkanes, branched
monoalkanes, anddimethylalkaneswerefound. Furthermorehydrocarbonsofupto
41–43 carbon atoms were found. More importantly, male and female O. fasciatus,
T. infestans, and T. mazzotti had similar profiles, whereas L. pabulinus males and
females produce a different blend of hydrocarbons.
The hydrocarbons of L. pabulinus females appear to elicit vibratory behav-
ior in males, as extracts in water (in which no hydrocarbons are present) were
not active. Furthermore, female legs used for water extraction remained active,
indicating that the compounds were still present on the legs. Fractionation of the
extracts on silica into two fractions (hydrocarbons and oxygenated compounds)
suggested that both fractions were needed to elicit vibration behavior in males.
The composition of the oxygenated compound fraction of males and females did
not show any difference. The similarity of this fraction between males and females
was supported by the finding that this fraction from males combined with the hy-
drocarbon fraction of females did cause vibratory behavior in males (unpublished
results).
In various species cuticular hydrocarbons have been identified as contact
pheromones (e.g., Muhammed et al., 1975; Carlson et al., 1978; Bolton et al.,
1980; Dillwith et al., 1981; Blomquist et al., 1993; Gu et al., 1995; Fukaya
et al., 1996; Doi et al., 1997). Alkenes seem to be involved in sexual communication
between sexes (Howard and Blomquist, 1982). More precisely, (Z)-9-pentacosene
is often the main hydrocarbon present in beetles (Baker et al., 1979), ants (Morgan
et al., 1992), and bees (Paulmier et al., 1999). In L. pabulinus, although (Z)-
9-pentacosene is the only compound obviously present in much lower amounts
in males than in females, the activity of this compound on its own could not be
proven in bioassays. Given that the synthetic mixture did not elicit vibration be-
havior in males, one cannot rule out the possibility of other compounds being part
of the contact pheromone. The most abundant male alkene, (Z)-9-heptacosene,
did not act as a repellent, because female legs sprayed with a high dose of this
alkene still elicited vibratory behavior in males (Table 5). More experiments are
needed to determine if there are other compounds present in the extracts that act
as a vibration elicitor.
The attractive compounds on female legs may be deposited on the substrate.
Male L. pabulinus showed strong responses to substrates on which females had

1146
DRIJFHOUT AND GROOT
walked. The fraction of males responding to potato leaves was even similar to
responses to female legs. When females were allowed to walk on a SPME needle, a
similar chemical profile was found as in attractive female leg extracts. These results
support the hypothesis that these hydrocarbons are deposited on the substrate. As
a characteristic pheromone-laying behavior was not observed, we suspect that
deposition on the substrate occurs passively or that pheromone is adsorbed to
the substrate. Adsorption or deposition of attractive compounds on the substrate
increases the probability of sex encounters, as it elicits intensive search by males
in these areas (Colwell et al., 1978; Fauvergue et al., 1995). Depending on their
volatility, these pheromones are active at some distance, as in M. viciae (Pickett
et al., 1992), or they elicit response at close range or upon contact, as in the other
species mentioned. Adsorption of pheromone to a substrate also increases the
surface area from which pheromone evaporates, thereby increasing both the rate
of volatilization and the possible communication distance (Colwell et al., 1978).
Males may follow a gradient of intensity, created by the decay of the compounds
over time, to orient their movements towards females (Fauvergue et al., 1995). In
this way, the functional range of low-volatility cuticular hydrocarbons would be
greatly enlarged.
Identification of the sex pheromones of mirid species is a challenging task.
This study indicates that the sex-specific cuticular hydrocarbons, probably in addi-
tion to esters, may play a role in the sexual communication of these bugs. Evidence
is accumulating that such hydrocarbons not only function as contact pheromones,
but are also involved in attraction at a (short) distance. Uebel et al. (1978) demon-
strated that field catches of male Fannia canicularis and F. pusio increased slightly
when alkenes were loaded on the lures. Connor et al. (1980) reported that the
pheromone of the arctiid moth Utetheisa ornatrix (Z,Z,Z-3,6,9-heneicosatriene
plus a small quantity of an unidentified C21 tetraene), perhaps serves as a close-
range orientation cue for locating the female. Recently, Schiestl et al. (1999, 2000)
reported that flowers of Ophrys orchids also mimic the odor profile of bees by
using this class of alkenes in order to attract these bees. Successful pheromone
trapping of mirids might also have to take these close-range cues into account.
Acknowledgments—We thank Anneke Heijboer for rearing L. pabulinus, Dr. M. A. Posthumus
for carrying out all the GS-MS experiments, and Pieter Vereijken for conducting the statistical analy-
ses. Marcel Dicke, Teris van Beek, and Hans Visser are greatly acknowledged for critical comments
on earlier versions of the manuscript. This study was funded by the Dutch Technology Foundation
(WBI44.3383).
REFERENCES
ALDRICH, J. R. 1988. Chemical ecology of the Heteroptera. Annu. Rev. Entomol. 33:211–238.
BAKER, J. E., NELSON, D. R., and FATLAND, C. L. 1979. Developmental changes in cuticular lipids of
the black carpet beetle, Attagenus megatoma. Insect Biochem. 9:335–339.

GREEN CAPSID BUG ATTRACTION
1147
BEANI, L., and CALLONI, C. 1991. Leg tegumental glands and male rubbing behavior at leks in Polistes
dominilus (Hymenoptera: Vespidae). J. Insect Behav. 4:449–461.
BLOMMERS, L. H. M., VAAL, F. W. N. M., andHELSEN, H. H. M. 1997. Lifehistory, seasonaladaptations,
and monitoring of common green capsid Lygocoris pabulinus (L.) (Hemiptera: Miridae). J. Appl.
Entomol. 121:389–398.
BLOMQUIST, G. J., TILLMAN-WALL, J. A., GUO, L., QUILICI, D. R., GU, P., and SCHAL, C. 1993.
Hydrocarbon and hydrocarbon derived sex pheromones in insects: Biochemistry and endocrine
regulation, pp. 317–351, in D. W. Stanley-Samuelson and D. R. Nelson (eds.). Insect Lipids:
Chemistry, Biochemistry & Biology. University of Nebraska Press, Lincoln, Nebraska.
BOLTON, H. T., BUTLER, J. F., and CARLSON, D. A. 1980. A mating stimulant pheromone of the horn fly,
Haematobia irritans (L.): demonstration of biological activity in separated cuticular components.
J. Chem. Ecol. 6:951–964.
¨
BUTENANDT, A., BECKMANN, R., STAMM, D., and HECKER, E. 1959. Uber den Sexuallockstof des
Seidenspinner, Bombyx mori. Reindarstellung und Kostitution. Z. Naturforsch. 14B:283–284.
CARAYON, J. 1971. Notes et documents sur l’appareil odorant me´tathoraciques des He´mipte`res. Ann.
Soc. Entomol. Fr. 7:737–770.
´
CARDE, R. T., and MINKS, A. K. 1997. Insect Pheromone Research: New Directions. Chapman and
Hall, New York, 684 pp.
´
CARDE, R. T., BAKER, T. C., and ROELOFS, W. L. 1975. Behavioral role of individual components of
a multichemical attractant system in the Oriental fruit moth. Nature 253:348–349.
CARLSON, D. A., MAYER, M. S., SILHACEK, D. L., JAMES, J. D., BEROZA, M., and BIERL, B. A.
1971. Sex attraction pheromone of the house fly: Isolation, identification and synthesis. Science
174:76–77.
CARLSON, D. A., LANGLEY, P. A., and HUYTON, P. 1978. Sex pheromone of the tsetse fly: Isolation,
identification, and synthesis of contact aphrodisiacs. Science 201:750–753.
CARLSON, D. A., ROAN, C., YOST, R. A., and HECTOR, J. 1989. Dimethyl disulfide derivatives of
long chain alkenes, alkadienes and alkatrienes for gas chromatography/mass spectrometry. Anal.
Chem. 61:1564–1571.
COLWELL, A. E., SHOREY, H. H., BAUMER, P., and VAN VORHIS KEY, S. E. 1978. Sex pheromone
scent marking by females of Pectinophora gossypiella (Lepidoptera: Gelechiidae). J. Chem. Ecol.
4:717–721.
CONNOR, W. E., EISNER, T., VANDER MEER, R. K., GUERRO, A., GHIRINGELLI, D., and MEINWALD, J.
1980. Sex attractant of an arctiid moth (Utetheisa orntrix): A pulse chemical signal. Behav. Ecol.
Sociobiol. 7:35–63.
DILLWITH, J. W., BLOMQUIST, G. J., and NELSON, D. R. 1981. Biosynthesis of the hydrocarbon com-
ponents of the sex pheromone of the housefly, Musca domestica L. Insect Biochem. 11:247–253.
DOI, M., NEMOTO, T., NAKANISHI, H., KUWAHARA, Y., and OGUMA, Y. 1997. Behavioral response of
males to major sex pheromone component, (Z,Z)-5,25-hentriacontadiene, of Drosophila ananas-
sae females. J. Chem. Ecol. 23:2067–2078.
DRIJFHOUT, F. P., VAN BEEK, T. A., VISSER, J. H., and DE GROOT, Æ. 2000. On-line thermal desorption-
gas chromatography of intact living insects for pheromone analysis. J. Chem. Ecol. 26:1383–1392.
FAUVERGUE, X., HOPPER, K. R., and ANTOLIN, M. F. 1995. Mate finding via a trail sex pheromone by
a parasitoid wasp. Proc. Natl. Acad. Sci. U.S.A. 92:900–904.
FUKAYA, M., YASUDA, T., WAKAMURA, S., and HONDA, H. 1996. Reproductive biology of the yellow-
spotted longicorn beetle, Psacotheca hilaris (Pascoe) (Coleoptera: Cerambycidae). III. Identifi-
cation of contact sex pheromone on female body surface. J. Chem. Ecol. 22:259–270.
GRAHAM, H. M. 1988. Sexual attraction of Lygus hesperus Knight. Southwest. Entomol. 13:31–37.
GROOT, A. T., VAN DER WAL, A. J., SCHUURMAN, A., VISSER, J. H., BLOMMERS, L. H. M., and
VAN BEEK, T. A. 1998. Copulation behavior of Lygocoris pabulinus under laboratory conditions.
Entomol. Exp. Appl. 88:219–228.

1148
DRIJFHOUT AND GROOT
´
GU, X., QUILICI, D., JUAREZ, P., BLOMQUIST, G. J., and SCHAL, C. 1995. Biosynthesis of hydrocarbons
and contact sex pheromone and their transport by lipophorin in females of the German cockroach
(Blattella germanica). J. Insect Physiol. 41:257–267.
HARDIE, J., and MINKS, A. K. (eds.). 1999. Pheromones of Non-Lepidopteran Insects Associated with
Agricultural Plants. CABI Publishing, Oxon, UK, 466 pp.
HOWARD, R. W., and BLOMQUIST, G. J. 1982. Chemical ecology and biochemistry of insect hydrocar-
bons. Annu. Rev. Entomol. 27:149–172.
JACKSON, L. J. 1983. Cuticular hydrocarbons of the milkweed bug Oncopeltus fasciatus by age and
sex. Insect Biochem. 13:19–25.
´
JUAREZ, P., and BLOMQUIST, G. J. 1993. Cuticular hydroacorbons of Triatoma infestans and T. mazzotti.
Comp. Biochem. Physiol. 106B:667–674.
KAINOH, Y., and OISHI, Y. 1993. Source of sex pheromone of the egg-larval parasitoid Ascogaster
reticulatus Watanabe (Hymenoptera: Braconidae). J. Chem. Ecol. 19:963–969.
KENNEDY, J. S. 1977. Behaviorally discriminating assays of attractants and repellents, pp. 215–229,
in H. H. Shorey and J. J. McKelvey, Jr. (eds.). Chemical Control of Insect Behavior, Theory and
Application. John Wiley & Sons, New York.
LANG, J. T. 1977. Contact sex pheromone in the mosquito Culiseta inornata (Diptera: Culicidae). J.
Med. Entomol. 14:448–454.
LEADBETTER, G., and PLIMMER, J. R. 1979. An improved preparation of some insect sex attractants:
Synthesis and separation of geometrical isomers by formation of urea complexes. J. Chem. Ecol.
5:101–108.
MARSH, D. 1972. Sex pheromone in the aphid Megoura viciae. Nature 238:31–32.
MCBRIEN, H. L., and MILLAR, J. C. 1999. Phytophagous bugs, pp. 277–304, in J. Hardie and A.K.
Minks (eds.). Pheromones of Non-Lepidopterous Insects Associated with Agricultural Plants.
CABI Publishing, Oxon, UK.
MCCULLAGH, P., and NELDER, J. A. 1989. Generalized Linear Models, 2nd ed. Chapman and Hall,
London.
MILLAR, J. G., and RICE, R. E. 1998. Sex pheromone of the plant bug Phytocoris californicus Knight
(Heteroptera: Hemiptera: Miridae). J. Econ. Entomol. 91:132–137.
MILLAR, J. G., RICE, R. E., and WANG, Q. 1997. Sex pheromone of the mirid bug Phytocoris relativus
Knight. J. Chem. Ecol. 23:1743–1754.
MINKS, A. K., and VAN DEVENTER, P. 1992. Practical applications: The European scene, pp. 9–18, in
R. L. Ridgway, M. Inscoe, and H. Arn (eds.). Insect Pheromones and Other Behavior-Modifying
Chemicals. BCPC Monograph No 51. Farnham, British Crop Protection Council.
¨
MORGAN, E. D., HOLLDOBLER, B., VAISAR, T., and JACKSON, B. D. 1992. Contents of poison apparatus
and their relation to trail-following in the ant Deceton atmigerum. J. Chem. Ecol. 18:2161–
2168.
MUHAMMED, S., BUTLER, J. F., and CARLSON, D. A. 1975. Stable fly sex attractant and mating
pheromones found in female body hydrocarbons. J. Chem. Ecol. 1:399–412.
`
`
´
PAULMIER, I., BAGNERES, A., AFONSO, C. M. M., DUSTICIER, G., RIVIERE, G., and CLEMENT, J.
1999. Alkenes as a sexual pheromone in the leaf-cutter bee Megachile rotundata. J. Chem. Ecol.
25:471–490.
PICKETT, J. A., WADHAMS, L. J., and WOODCOCK, C. M. 1992. The chemical ecology of aphids. Annu.
Rev. Entomol. 37:67–90.
SCHAL, C., SEVALA, V. L., YOUNG, H. P., and BACHMANN, J. A. S. 1998. Sites of synthesis and transport
pathways of insect hydrocarbons: Cuticle and ovary as target tissues. Am. Zool. 38:382–393.
¨
SCHIESTL, F. P., AYASSE, M., PAULUS, H. F., LOFSTEDT, C., HANSSON, B. S., IBARRA, F., and FRANCKE,
W. 1999. Orchid pollination by sexual swindle. Nature 399:421–422.
¨
SCHIESTL, F. P., AYASSE, M., PAULUS, H. F., LOFSTEDT, C., HANSSON, B. S., IBARRA, F., and FRANCKE,
W. 2000. Sex pheromone mimicry in the early spider orchid (Ophrys sphegodes): Patterns of

GREEN CAPSID BUG ATTRACTION
1149
hydrocarbons as the key mechanism for pollination by sexual deception. J. Comp. Physiol. A
186:567–574.
SCHLEIN, Y., GALUN, R., and BEN-ELIAHU, M. N. 1980. The legs of Musca domestica and Glossina
morsitans females as the site of sex pheromone release. Experientia 38:1174–1176.
SMITH, R. F., PIERCE, H. D., and BORDEN, J. H. 1991. Sex pheromone of the mullein bug, Campylomma
verbasci (Meyer) (Heteroptera: Miridae). J. Chem. Ecol. 17:1437–1447.
STADDON, B. W. 1979. The scent glands of Heteroptera. Adv. Insect Physiol. 14:351–418.
UEBEL, E. C., SCHWARS, M., SONNET, P. E., MILLER, R. W., and MENZER, R. E. 1978. Evaluation of
the mating stimulant pheromone of Fannia canicularis, F. pusio, and F. femoralis as attractants.
Fla. Entomol. 61:139–143.