8/6/2019 Blatt et al. 1998

1/19

Journal of Chemical Ecology, Vol. 24, No. 6,1998

ALARM PHEROMONE SYSTEM OF THE WESTERNCONIFER SEED BUG, Leptoglossus occidentalis

S. E. BLATT,1,* J. H. BORDE N,1 H. D. PIERCE, JR., 2 R. GRIES,1and G. GRIES1

1Centre for Pest Management, Department of Biological Sciences2Department of ChemistrySimon Fraser University

Burnaby, British Columbia, V5A 1S6Canada

(Received March 31, 1997; accepted February 5,1998)

AbstractThe alarm pheromonesfor adult and nymphal western conifer seedbugs, Leptoglossus occidentalis, were collected from the headspace volatilesof agitated bugs and from extracted adult thoraxes and nymphal abdomens.Adult bugs secreted a blend from the metathoracic glands that consisted ofhexyl acetate, hexanal, hexanol, heptyl acetate, and octyl acetate (ratio of152:103:8:1.5:1). Nym phal alarm pheromone produced by the dorsalabdominal glands consisted of ()-2-hexe nal. Agitated adults emitted~ 24%of the pheromone contained withinthe glands, while nym phs released ~ 33%of their constitutive supply.Th e complete blend from both adults and nymphs,tested in a laboratory headspace bioassay, elicited a dispersal (or alarm)response in >70% of individuals tested. Nymphs in the field exposed to

synthetic adult or nymphal pheromones, or a mixture of both, responded with>50% dispersing. When single components weretested on adults rearedunder summer conditions in a forced-air one-way bioassay, hexanal and hexylacetate, the major components of the secretion, were responsible for elicitingthe alarm response. Adultscollected in the fall from the field were unrespon-sive to the tested blend, suggesting that adults seeking aggregation sites inthe fall become refractory to alarm pheromone stimuli that would cause aggre-gations to disperse. The weak dispersal responses elicited in both adults andnymphs by either nymphal or adult pheromones are consistent with a trade-off in the advantage gained by avoiding predation and the disadvantage ofleaving a food source. Because of these weak responses, use of alarm pher-omones as pest management tools against L. occidentalis is unlikely.

Key WordsLeptoglossus occidentalis, western conifer seed bug, Hemip-tera: Coreidae, alarm pheromone, metathoracic glands, hexylacetate, hex-anol, hexanal, octyl acetate and heptyl acetate.

*To whom correspondence shouldbe addressed at: 41 McMichael Street, Kingston, Ontario, K7MIM8, Canada.

1013

0098-0331/98/0600-1013/$15.00/0 1998 Plenum Publishing Corporation

8/6/2019 Blatt et al. 1998

2/19

1014 BLATT ET AL.

INTRODUCTION

Defensive secretions in some Homoptera and Heteroptera have been well doc-umented. The secretions from aphids (Wohlers, 1981; Dawson et al., 1987);the coreids Hotea gambiae (Westwood) (Hamilton et al., 1985) and Leptoglos-sus zonatus (Dallas) (Leal et al., 1993); the alydids Megalotomus quinquespi-nosus (Say), Alydus eurinus (Say), and Alydus pilosulus Herrich-Schaeffer(Oetting and Yonke, 1978); the pentatomids Eurydema rugosa Motschulsky(Ishiwatari, 1974) and Eurydema pulchra Motschulsky (Ishiwatari, 1976);Nezara varidula (L.) (Lockwood and Story, 1987); and Erthesino ful lo Thung-berg (Kou et al., 1989) have been characterized as eliciting alarm behaviors(e.g., agitated walking, dropping from the host, flight attempt) among conspe-cifics.

These species share the following common characteristics: (1) they are

easily disturbed and readily emit their offensive odor, (2) they form aggrega-tions, and (3) most possess hexanal as a component of their defensive secretion.Only one Leptoglossus sp. has had its alarm pheromone system characterized(Leal et al., 1993). Given the variety of habitats and plant species used bymembers of this genus and the allopatric ranges of many species, we hypothe-sized that the alarm pheromone system would be fairly similar for related Lep-toglossus species. The propensity toward gregariousness may be a prerequisiteto the evolution of alarm pheromones (Nault and Phelan, 1984). Individuals inaggregations can benefit by perceiving volatile secretions used by other membersof a group to repel predators and can disperse to avoid being targets for pre-dation.

The western conifer seed bug, Leptoglossus occidentalis Heidemann (Hem-iptera: Coreidae), is a common and potentially severe pest of conifer seedorchards in western North America (Hedlin et al., 1980; Schowalter and Sexton,1990; Connelly and Schowalter, 1991). Throughout the summer, groups ofadults and nymphs are easily disturbed and emit a defensive secretion. Ashypothesized for other Hemiptera, this scent apparently elicits dispersal or alarmbehavior in adults and nymphs. As defined by Starr (1990), alarm is the "com-munication of a shared danger. To show that it exists we need only find acorrelation between the responses of an individual which perceives an intrusionand one which does not."

Our objectives were to capture, isolate, identify, and bioassay the alarmpheromones of adults and nymphs of L. occidentalis.

METHODS AND MATERIALS

Insects, Adult males, females, and nymphs of L. occidentalis for chemicalanalysis and most bioassay experiments were obtained from a laboratory colony

8/6/2019 Blatt et al. 1998

3/19

ALARM PHEROMONEOF L. octidentalis 1015

maintained at a 15L:9D photoperiod regime, 32C and 20C peak photophase

and scotophase temperatures, respectively, and ~ 70% relative humidity. Thecolony was established in 1992 and revitalized using field-collected bugs eachsummer. Adults were segregated by sex for all but one phase of the study, butnymphs were not. One set of bioassay experiments was done with bugs collectedin the field from Skimikin Seed Orchards, BC Forest Service, Tappen, BritishColumbia and Kalamalka Seed Orchards, Vemon, British Columbia, in mid-September 1994.

Headspace Bioassay for Adult an d Nymphal Volatiles. Glass jars (130 ml)were washed and air dried prior to use and between replicates. Two sets eachof five males or females were placed in separate jars covered with Parafilm.One set of insects, designated nonagitated, were not disturbed. The jar contain-ing the second set of insects, designated agitated, was roughly handled andshaken until the putative alarm pheromone could be detected by the human nose.

Fo r bioassays (Figure 1), a single nonagitated test insect was isolated in aParafilm-covered jar and allowed to settle. Headspace air (10 cc), equal toapprox. 0.4 bug equivalents, was drawn from the jar containing the nonagitatedinsects into a disposable syringe and injected into the jar containing the testinsect. The test insect was then observed for any change in behavior, and then

FIG. 1. Apparatus used in headspace bioassays testing for response by adult and nymphalL. occidentalis to headspace samples taken from agitated bugs.

8/6/2019 Blatt et al. 1998

4/19

1016 BLATT ET AL.

was treated with either 10 cc of headspace air drawn from the jar containing the

nonagitated insects (control) or the agitated insects (treatment). Again the testinsects' behavior wa s observed. Any insect responding to air from nonagitatedinsects was discounted. An alarm response was recorded if the test insect exhib-ited agitated behavior, such as a sudden, rapid increase in movement or attemptedflight. No change in behavior was deemed a negative response.

Fifteen individuals (replicates) of females, males, and nymphs were tested.Crossover experiments were then conducted with the same bioassay and 15replicates obtained for all combinations of adults exposed to volatiles fromnymphs and for nymphs exposed to volatiles from male or female adults. Allresults were analyzed by x 2 analysis and Fisher's exact test (Zar, 1984).

In both laboratory and field bioassay, one bug equivalent was equal to theamount of volatiles given off by one agitated bug as determined by volatilecapture.

Analysis of Volatiles. Volatiles from live adults or nymphs were collectedon Porapak Q (Waters Associates Inc., Milford, Massachusetts). Twenty-fivegroups of 10 agitated males or females and five groups of 20 nymphs wereplaced in a glass chamber (6.5 cm high, radius 4.7 cm). Bugs were then agitatedwith a glass rod until the putative alarm pheromone was detected by the humannose. Then air was drawn by a water aspirator through a charcoal scrubber andover the insects for 10 min at 1.65 liters/'min, allowing for ~36 exchanges ofair in the aeration chamber. Volatiles were collected in a glass trap (6 mm ODx 30 mm) filled with Porapak Q (50-80 mesh). Trapped volatiles were extractedby eluting the Porapak with 2-3 ml of double-distilled pentane, and the volumewas concentrated under a nitrogen stream to 1 ml. A !-/*! sample of the extractwas analyzed with a Hewlett Packard 5830A gas chromatograph (GC) with anSP1000 nonbonded polar column (30 m X 0.32 mm ID) (Supelco, Oakville,Ontario) and flame ionization detector and with a Hewlett Packard 5890 gaschromatograph with a fused silica coated DB-5 nonpolar column (30 m x 0.25mm ID) (J&W Scientific, Folsom, California).

Volatiles also were analyzed by coupled gas chromatographic-electro-antennographicdetection (GC-EAD) (Arn et al., 1975, Gries et al., 1993), witha Hewlett Packard 5890A gas chromatograph and a custom built amplifier witha passive low-pass filter and a cutoff frequency of 10 kHz. An antenna wasgently pulled out of the insect's head; the exposed nerve endings were suspendedin a saline solution that contained the indifferent electrode and the distal end ofthe antenna was pierced with a recording electrode. Antennally active com-pounds were analyzed by coupled GC-mass spectroscopy (MS) with a HewlettPackard 5985B GC equipped with a fused silica (30 m X 0.25 mm ID) DB-5column in full-scan and selective ion monitoring (SIM) mode. Compounds wereidentified by comparison with published spectra (Jennings and Shibamoto, 1980)and identification was verified by GC with authentic standards. Quantities of

8/6/2019 Blatt et al. 1998

5/19

8/6/2019 Blatt et al. 1998

6/19

1018 BLATT ET AL.

volatile stimuli. A ruler was taped to the board with 0 cm positioned at the end

of the ground glass joint. An adult was placed in the tube, near the 0 cm markand its original position recorded. The component to be tested was applied to aglass ampoule, held by a cork, and inserted into the vertical port apparatus(Figure 2). Room air, humidified by passing through water, was blown at 1.5liters/min into the horizontal tube, over the volatile stimulus and the test insect,and then out through the open end of the tube. Room temperature was maintainedat ~25C.

All stimuli were evaluated at one bug equivalent in 2 ul of pentane. Stimulitested were an extract from excised adult thoraxes, each of five antenally activecompounds found in the adult thoraxes, a five-component synthetic blend (hexylacetate, hexanal, hexanol, heptyl acetate, and octyl acetate in the ratio152:103:8:1.5:1) and (')-2-hexenal, the putative nymphal alarm pheromone.Each insect had 30 sec to respond to the stimulus, after which its final position

was recorded. Ten males and 10 females from the colony were tested in thespring and 10 field-collected individuals of each sex were tested in the fall. Tencontrol insects in each category were tested for their response to untreated air.Colony-reared "summer" females and males did not differ in their response toany of the stimuli (F = 0.02, P = 0.89) and were pooled for analysis. Meandistances moved in response to experimental stimuli were compared with dis-tances moved in untreated air control tests by means of Dunnett's one-way testat a = 0.05.

Field Bioassays. Adults and nymphs resting and feeding on cone clustersof western white pine, Pinus monticola Dougl. ex. D. Don, were located atSkimikin Seed Orchards. They were counted and their position identified withplastic flagging. Cone clusters were sprayed with a small (150-ml volume)atomizer containing 4 ml pentane spray with zero (control), two, or four bugequivalents of synthetic adult or nymphal alarm pheromone (as above), or both.The four-bug equivalent treatment of nymphal pheromone was lost by spillagein the field. Six cone clusters were sprayed for each treatment. Numbers ofadults that responded by flying away from the cones and numbers of nymphsresponding by either dropping or walking away from the cones were recorded.The numbers of nymphs were pooled for each treatment (heterogeneity chi-square values ranged from 3.14 to 14.82, P values ranged from 0.17 to 0.98),and percentage responses were compared with the response to control sprays(pentane study) by a test of difference between proportions with Z scores asdescribed by Zar(1984).

Due to the low number of adults in the field bioassay (N = 6), a laboratoryexperiment for adults was designed. Potted Douglas fir, Pseudotsuga menziesii(Mirb.) Franco, seedlings (approx. 25 cm high) were placed individually in alarge mesh screened cage measuring 76 x 61 X 36 cm. Ten adults (mixed sex)were placed on the seedling, allowed to settle, and then sprayed as above with

8/6/2019 Blatt et al. 1998

7/19

ALARM PHEROMONE OF L. occidentalis 1019

two or four bug equivalents of synthetic (adult, nymphal, or a mixture) alarm

pheromone. Ten replicates of 10 bugs each were tested for each treatment.Numbers of adults that left the seedling by flying and numbers that displayedagitated behavior were recorded. Mean percentage responses to experimentalstimuli were compared with those to pentane control treatments by Dunnett'sone-way test (a 0.05) following transformation with arcsinVx to stabilize thevariances.

RESULTS

Headspace Bioassays. Males, females, and nymphs all showed a significantpositive alarm response to the headspace volatiles from agitated males, females,and nymphs (and all combinations thereof) (Table 1). Males and females did

no t differ significantly in their responses to volatiles from their own or from theopposite sex, x 2 = 0.36, P = 0.54. Volatiles from nonagitated individualselicted little or no response.

Analysis of Volatiles. GC , GC-MS, and GC-EAD analyses revealed thatthe antenally active volatiles emitted from agitated adults and those containedwithin the metathoracic gland and reservoir were hexanal, hexyl acetate, hex-anol, heptyl acetate, and octyl acetate (Figure 3). The single antenally activevolatile emitted by agitated nymphs was identified as ()-2-hexenal (Figure 4),which is also produced by other hemipteran nymphs (Lockwood and Story,1987). Male and female adults produced the same components in approximatelythe same quantity and ratio (Table 2). During the GC-EAD analysis at doses of~4 bug equivalents, some antennae ceased to respond following exposure to

the hexyl acetate or hexanal in the extract. To solve this problem, extracts werediluted 20-fold, enabling an antenna to respond for the duration of the analysis.A similar effect was observed in Formica spp. with n-undecane (Blum andBrand, 1972). This may be an adaptive mechanism that allows the emitter tohide from aggressive conspecifics. In our analysis with L. occidentalis, theconcentration of hexyl acetate and hexanal were probably so high that there wasa toxic inhibition of olfactory response.

Bioassay of Synthetic and Extracted Volatiles. A synthetic blend containingall five components of adult pheromone elicited an alarm response by males,females, and nymphs in the head space bioassay (Table 3). Synthetic nymphalalarm pheromone elicited a similarly ubiquitous response (Table 3). Hexanaland hexyl acetate, the most abundant components in both glands and headspacevolatiles from agitated adults, and (E)-2-hexenal from nymphs elicited a sig-nificant response from colony-reared "summer" adults in the forced air bioassay(Figure 5).

Adults collected in the fall did not respond to the synthetic alarm pheromone

8/6/2019 Blatt et al. 1998

8/19

1020 BLATT ET AL.

TABLE 1 . ALARM BEHAVIOR RESPONSES OF ADULT AN DNYMPHAL L. occidentalis INHEADSPACE BIOASSAY TO HEADSPACE VOLATILES OP AGITATED AND NONAGITATED

(CONTROL) BUGS"

Test insect andsource of volatile

stimulus

FemalesAgitated femalesNonagitated females

FemalesAgitated malesNonagitated males

MalesAgitated females

Nonagitated femalesMales

Agitated malesNonagitated males

FemalesAgitated nymphsNonagitated nymphs

MalesAgitated nymphsNonagitated nymphs

NymphsAgitated femalesNonagitated females

NymphsAgitated malesNonagitated males

NymphsAgitated nymphsNonagitated nymphs

Postiveresponse (%)

1000

1000

87

0

800

87

13

930

807

877

930

X2 probabilityexperimental vs.

control

0.001

0.001

0.001

0.001

0.001

0.001

0.001

0.001

0.001

aN = 15 bugs per test.

components (F = 1.16, P = 0.27 and F = 1.10, P = 0.34 for females andmales, respectively) (Figure 5). In many cases, the insects moved towards thestimulus.

Field Experiments. Nymphs in the field showed a significant dispersalresponse to synthetic alarm pheromone sprays (Table 4). At a dose of 2 bug

equivalents, synthetic nymphal pheromone and a mix of adult and nymphalpheromones were significantly more effective than the control (pentane only) at

8/6/2019 Blatt et al. 1998

9/19

FIG. 3. Flame ionization detector (FID) and corresponding electroantennographic (BAD)trace from adult female antenna to alarm pheromone collected in vivo from adult femalesand analyzed on a DB-5 column with a program of 50C for 1 min, 2C/min to 65C,5C/min to 120C, and then 20C/min to 240C.

FIG. 4. Flame ionization detector (FID) and corresponding electroantennographic (BAD)trace from nymphal antenna to alarm pheromone collected in vivo from third and fourthinstars and analyzed on a DB-5 column with a program of 50C for 1 min, 2C/min to65C, 5C/min to 120C, and then 20C/min to 240C.

8/6/2019 Blatt et al. 1998

10/19

1022 BLATT ET AL.

TABLE 2. COMPARISON BETWEEN QUANTITIES OF ALARM PHEROMONE CONTAINED

WITHIN METATHORACIC GLANDOF ADULTS AND DORSAL ABDOMINAL GLANDSOFNYMPHSAND QUANTITIES GIVENOFF WHEN AGITATED

Pheromonesource

Adultfemales

Adultmales

Nymphs

Compound

HexanalHexanolHexyl acetateHeptyl acetateOctyl acetate

Hexanal

HexanolHexyl acetateHeptyl acetateOctyl acetate(>2-Hexenal

Q uantity(ug/bug) in gland

(mean SE)

568.7 170.4012.2 7.10

390.7 156.900.3 0.043.1 1.18

339.7 116.70

4.6 1.04337.1 96.70

0.3 0.141.5 0.483.6 0.69

Q uantity (/ig/bug)given off when

agitated (mean SE)*

17.1 3.531.3 0.93

21.7 2.690.2 0.100.2 0.02

15.5 4.46

1.4 0.8727.2 5.25

0.2 0.060.2 0.011. 2 0.35

Percent of totalcontent given off

when agitated

3.010.65.6

92.34.9

4.6

30.88.1

63.611.632.7

"Data obtained from four replicates each consisting of five adult males or females (total = 20 ofeach sex), and four replicates of 10, and one replicate of 11 nymphs (total = 51).

*Data collected from 25 replicates each of 10 males or females (total = 250 of each sex), and fivereplicates of 20 nymphs (total = 100).

causing dispersal of nymphs from the cone clusters. Synthetic adult alarm pher-

omone at 4 bug equivalents caused a dispersal response similar to that causedby the nymphal-adult mix at the same dosage. At the 2 bug equivalent dose,the adult pheromone blend was no more effective than the control, suggestingthat nymphs are more responsive to alarm pheromone from nymphs than fromadults. While the pheromone treatments caused nymphs to walk away from thecones, the effect was short-lived, and they would return to the cones within 5min. For all treatments tested, fewer than 20% of the nymphs present respondedby dropping from the cone clusters (Table 4).

Adults tested on seedlings showed significant alarm response (Figure 6).The synthetic adult alarm pheromone at both 2 and 4 bug equivalents, eitheralone or in mixture with nymphal pheromone, caused significantly higher num-bers of adults to become agitated than the pentane control treatment, but onlythe adult blend alone caused adults to leave the seedlings in significant numbers.Nymphal alarm pheromone had no significant effect.

8/6/2019 Blatt et al. 1998

11/19

ALARM PHEROMONBOF L. occidentalis 1023

TABLE 3. ALARM BEHAVIOR OF ADULT AND NYMPHAL L. occidentalis IN HEADSPACE

BIOASSAY TO SYNTHETIC BLEND OF ADULT ALARM PHEROMONE CANDIDATESANDSYNTHETIC NY MPHAL ALARM PHEROMONE,()-2-Hexenal a

Test insect andsource of stimulus

FemalesSynthetic adult blendPentane

MalesSynthetic adult blendPentane

FemalesSynthetic nymphPentane

MalesSynthetic nymphPentane

NymphsSynthetic adult blendPentane

NymphsSynthetic nymphPentane

Percent positiveresponse

8013

876

936

876

7333

8020

X2 Probabilityexperimental vs. control

0.005

0.001

0.001

0.001

0.05

0.005

"Approximately 0.4 bug equivalents per stimulus. N = 15 bugs per test.

DISCUSSION

Hexanal, hexyl acetate, and hexanol, the most abundant semiochemicalsfound in L. occidentalis are also found in three other Leptoglossus species: L.zonatus (Dallas) (Leal et al., 1993), L. oppositus (Say), and L. clypealisHeidemann (Aldrich and Yonke, 1975). Leptoglossus zonatus produces hex-anoic acid in small quantities (Leal et al., 1993), and both L. oppositus and L.clypealis produce small amounts of acetic acid (McCullough, 1968, 1969; Aid-rich and Yonke, 1975), n-hexyl hexanoate, and ()-2-octenyl acetate (Aldrichand Yonke, 1975). Hexaldehydes are a common component of defensive secre-tions in numerous other Hemiptera and Homoptera (Waterhouse and Gilby,1964; McCullough, 1970, 1973a,b; Aldrich et al., 1978, 1979, 1984; Evertonet al., 1979; Lockwood and Story, 1987). The frequent occurrence of hexanalin such secretions suggests that it is an effective, broad spectrum irritant easily

8/6/2019 Blatt et al. 1998

12/19

1024 BLATT ET AL.

FIG 5. Mean distance moved away from adult and nymphal alarm pheromone and itscomponents by L. occidentalis reared in colony under "summer" conditions or collectedfrom the field in the fall. All components tested at a concentration of one bug equivalent.

An asterisk indicates significant distance, Dunnett's one-way test, P < 0.05, movedfrom stimulus as compared with control (pentane).

8/6/2019 Blatt et al. 1998

13/19

ALARM PHEROMONE OF L. occidentaUs 1025

8/6/2019 Blatt et al. 1998

14/19

1026 BLATT ET AL.

FIG. 6. Percentage of adults responding to synthetic alarm pheromone sprays in a lab-oratory test on caged seedlings. Significant response, Dunnett's one-way test, P < 0.05,compared with control (pentane) indicated by an asterisk.

8/6/2019 Blatt et al. 1998

15/19

ALARM PHEROMONEOF L. occidentalis 1027

produced or acquired by insects and useful for defense. It is a common green

leaf volatile in herbaceous plants (Visser et al., 1979; and McCall et al., 1994),but its function may differ by insect species (Visser et al., 1979; Visser, 1986;Dickens et al., 1990, 1992).

Although hexanol, heptyl acetate, and octyl acetate were antenally active(Figure 3), they did not elicit alarm behavior in the bioassays, and there wasno evidence of an additive or synergistic interaction of components in the syn-thetic blend. These minor components have been argued as inconsequential forconspecific recognition (Aldrich and Yonke, 1975). However, reanalysis of thevolatiles from previously studied bugs by GC-EAD may reveal other minorcompounds of potential behavioral importance. We hypothesize that these com-pounds, less volatile than hexanal and hexyl acetate, may serve as conspecificrecognition signals.

The utilization of different pheromone components by adults and nymphs

of L. occidentalis may indicate that pheromones with different properties arerequired to accommodate their differing release mechanisms. Adult L. occiden-talis expel their pheromone through small openings in the thorax as a spray(personal observation) while nymphs release alarm pheromone through openingsin their abdominal tergites. Hexanal, produced by adults, has a higher molecularweight than (E)-2-hexenal and is less volatile (bp 131 C). Hexanal may disperseinto the air as an aerosol or may contact a predator prior to volatilization. (E)-2-Hexenal (bp 47C), if released in a similar manner, will volatilize rapidly onexposure to air. As (')-2-hexenal is probably released onto the surface of thetergites, rather than directly into the air, the large odor plume created wouldprobably be as effective as a hexanal spray in warding off predators, particularlyfrom a group of aggregated and alarmed nymphs. Aldrich and Yonke (1975)proposed that the difference in adult and nymphal alarm pheromones of L.oppositus and L. clypealis resulted from selection pressure by ants. This maybe possible for species that are preyed upon by ants; however, ants have fre-quently been observed on conifer boles and branches, but have not been notedas predators of L. occidentalis or any other Leptoglossus species.

Metathoracic glands and the associated reservoir of male and female adultscontain several hundred micrograms of pheromone, while dorsal abdominalglands of nymphs contain < 10 ug (Table 2). Because only a portion of thisamount (~24% of the total for adults and ~33% for nymphs) was emitted byagitated bugs, an individual insect should be capable of repeated emissions.However, approximately 15- and 45-min refractory periods occurred beforeadults and nymphs, respectively, could be provoked to reemit alarm pheromone.In the nymphs, the long refractory period may indicate that ()-2-hexenal ismore useful as a repellent against predation than as a conspecific alarm phero-mone. Their aposematic coloring supports this hypothesis. Unlike adults, whichcan fly away from a host and return, nymphs dropping from cones onto lower

8/6/2019 Blatt et al. 1998

16/19

1028 BLATT ET AL.

branches would escape the immediate threat of predation, but would potentially

lose their food supply and increase the chances of desiccation and predation byspiders and rodents while on the forest floor. Pea aphids, Acyrthosiphon pisum(Harris), from Kamloops, British Columbia, were less likely than aphids fromVancouver to drop from their host plant in response to alarm pheromone (Roit-berg and Myers, 1978). This was postulated as a means to avoid desiccation inareas where ground conditions are harsh.

Lockwood and Story (1987) hypothesized that alarming conspecifics evolvedas a secondary function of the defensive secretion in Nezara viridula (L.). Thereserve of pheromone remaining in the adults after one emission suggests thatL. occidentalis could repeatedly defend itself against persistent attempts at pre-dation, with alarm and dispersal by conspecifics being a secondary adaptation,as hypothesized for N. viridula.

Lockwood and Story (1985) demonstrated that in first-instar N. viridula,

n-tridecane serves as an alarm pheromone at 1.0 bug equivalents and as anaggregation pheromone at 0.1 bug equivalents. While this behavior is dosedependent, the differential response of summer and fall adult L. occidentalis totheir defensive secretion may be physiologically dependent. In the fall, bothsexes of L. occidentalis seek cryptic overwintering sites, respond to an unknownmale-produced aggregation pheromone (Blatt and Borden, 1996), and are com-monly aggregated in large numbers (Blatt, 1994). In these sites they are noteasily accessible to predators, such as birds. During this time, alarm responsesfollowed by dispersal would be maladaptive. It is hypothesized that the lesserproduced components, hexanol, heptyl acetate, and octyl acetate may be usedas conspecific recognition cues.

Results obtained in the laboratory bioassays indicate that, like nymphs,adults are more responsive to their own pheromone than to the pheromoneproduced by bugs in a different life stage. The uniformly high responses toheadspace volatiles from any life stage suggest that when stimuli are strong,there is an adaptive advantage to respond to any conspecific alarm signal. Thelow percentage of adults leaving the seedling suggests that other indications ofdanger, e.g., rapid movement and shadows, would be necessary to cause adultsto depart (Clegg and Barlow, 1982). Of the six adults tested in the field, threeflew away and the others dispersed from the cones by agitated walking. In boththe field and the laboratory, adults that left the cones or seedlings returned within10 min. Based on these results, we conclude that the alarm pheromone wouldnot be operationally effective in reducing L. occidentalis populations or in pre-venting or reducing damage to seeds of conifers.

Acknowledgments-We thank A. Franklin for assistance, K. Cox, and L. Langois for access

to the western white pine orchard at Skimikin, and Drs. R. A. Alfaro and B. D. Roitberg for criticalreviews. This research wa s supported by the Natural Sciences and Engineering Research Councilof Canada, the Canadian Forest Service Green Plan, the Science Council of BC, the Coast Forest

8/6/2019 Blatt et al. 1998

17/19

ALARM PHEROMONEOF L. occidentalis 1029

and Lumber Sector, the Interior Lumber Manufacturers'Association, the Cariboo Lumber Manu-facturers Association, Phero Tech Inc., and seven forest industry companies.

REFERENCES

A L D R I C H ,J. R., and Y O N K E ,T. R. 1975. Natur al products of abdominal and metathoracic scentglands of coreoid bugs. Ann. Entomol. Soc.Am. 68:955-960.

A L D R I C H ,J. R., B L U M ,M. S., L L O Y D ,H. A., and PALES, H. M. 1978. Pentatomid natural products:chemistry and morphology of the III-IV dorsal abdominal glandsof adults. J. Chem. Ecol.4:161-172.

A L D R I C H ,J. R., B L U M , M. S., L L O Y D ,H. A., E VA N S ,P. H., and B U R K H A R D ,D. R. 1979. Novelexocrine secretions from two species of scentless plant bugs (Hemiptera: Rho palidae).Entomol.E x p . Appl. 26:323-331.

A L D R I C H ,J. R., L U S B Y,W. R., K O C H A N S K Y,J. P., and ABRAMS, C. B. 1984. Volatile compoundsfrom the predatory insect Podisus maculiventris (Hemiptera: Pentatomidae): Male and female

metathoracic scent gland and female dorsal abdominal gland secretions. J. Chem. Ecol.10:561-568.ARM, H., S TA D L E R ,E., and R A U S C H E R ,S. 1975. The electroantennographic detectora selective

and sensitive tool in the gas chromatographic analysis of insect pheromones.Z. Naturforsch.30c:722-725.

BLATT, S. E. 1994. An unusually large aggregation of the western conifer seed bug,Leploglossusoccidentals (Hemiptera: Coreidae), in a man-made structure.J. Entomol. Soc. B.C.91:71-72.

BLATT, S. E., and B O R D E N ,J. H. 1996. Evidence for a male-produced aggregation pheromone inthe western conifer seed bug, Leptoglossus o ccidentalis Heidemann (Hemiptera: Coreidae).C a n . Entomol. 128:777-778.

B L U M , M. S., and B R A ND ,J. M. 1972. Social insect pheromones: Their chemistry andfunction.Am. Zool. 12:553-576.

CLEOO, J. M., and B A R LO W ,C. A. 1982. Escape behavior of the pea aphidAcyrthosiphon pisum(Harris) in response to alarm pheromone and vibration. Can. J. Zool. 60:2245-2252.

C O N N E L LY,A. E., and S C H O WA LT ER ,T. D. 1991. Seed losses to feeding by Leptoglossus occiden-talis (Heteroptera: Coreidae) during twoperiods of second year cone development in westernwhite pine. J. Econ. Entomol. 84:215-217.

D AW S O N,G. W., GRIFFITHS, D. C., PIC KETT, J. A., W A D H AM S ,L. J., and W O OD C O C K ,C. M.1987. Plant-derived synergists of alarm pheromone from turnip aphid,Lipaphis (Hyadaphis)erysimi (Homoptera, Aphididae).J. Chem. Ecol. 13:1663-1671.

D I C K E N S ,J. C., J A N G , E. B., LIGHT, D. M., and ALFORD, A. R. 1990. Enhancement of insectpheromone responses by green leaf volatiles. Naturwissenschaften 77:29-31.

D I C K E N S ,J. C., BILLINGS,R. F., and PAY N E , T. L. 1992. Green leaf volatiles interrupt aggregationpheromone response in bark beetles infesting southern pines. Experientia 48:523-524.

EV ERTON,I. J., K N I G H T,D. W., and S TA D D O N ,B. W. 1979. Linalool from the metathoracic scentgland of the cotton stainer Dysdercus intermediusDistant (Heteroptera: Pyrrhocoridae). Comp.Biochem. Physiol. 636:157-161.

GRIES, G. , GRIES, R. K R A N N IT Z ,S. H., LI, J., K I N G ,G. G. S., SLESSOR,K. N., B O R D E N ,J. H.,B O W E R S ,W. W., WEST, R. J., and U N D E R B I LL ,E. W. 1993. Sex pheromone of the westernhemlock looper, Lambdina fiscellaria lugubrosa (Hulst) (Lepidoptera: Geometridae).J. Chem.Ecol. 19:1009-1019.

HAMILTON,J. G. C., G O U G H , A. J. E., S TA D D O N ,B. W., and GAMES, D. E. 1985. Multichemicaldefense of plant bug Hotea gambiae (Westwood) (Heteroptera: Scutelleridae): ()-2-Hexen olfrom abdominal gland in adults.J. Chem. Ecol. 11:1399-1409.

8/6/2019 Blatt et al. 1998

18/19

1030 BLATT ET AL.

HEDLIN, A. F., YATES, H. O., III, CIBRIAN-TOVAR,D., EBEL, B. H., K OERBER,T. W., andMERKEL, E. P. 1980. Cone and seed insects of North American conifers. Canadian ForestryService, USDA Forest Service, and Secretaria de Agriculture y Recursos Hidraulicos, Mexico.

ISHIW ATARI ,T. 1974. Studies on the scent of stink bugs (Hemiptera: Pentatomidae) I. Alarm pher-omone activity. Appl. Entomol. Zool. 9:153-158.

ISHWATARI, T. 1976. Studies on the scent of stink bugs (Hemiptera: Pentatomidae) II. Aggregationpheromone act ivity. Appl. Entomol. Zool. 11:38-44.

J E N N I N G S ,W., and SHIBAMOTO, T. 1980. Qualitative Analysis of Flavor and Fragrance Volatilesby Glass Capillary Gas Chromatography. Academic Press, New York.

Kou, R., TANG, D. S., and CHOW, Y. S. 1989. Alarm pheromone of pentatomid bug, ErthesinaJulio Thunberg (Hemiptera: Pentatomidae). J. Chem. Ecol. 15:2695-2702.

LEAL, W. S., PANIZZI,A. R., and NIVA, C. C. 1993. Alarm pheromone system of leaf-footed bugLeptoglossus zonatus (Heteroptera: Coreidae). J. Chem. Ecol. 20:1209-1216.

LOCKWOOD,J. A., and STORY, R. N. 1985. Bif tinctional pheromone in the first instarof the southerngreen stink bug, Nezara viridula (L.) (Hemiptera: Pentatomidae): Its characterization andinteraction with other stimuli. Ann. Entomol. Soc. Am. 78:474-479.

LOCKWOOD, J. A., and STORY, R. N. 1987. Defensive secretion of the southern green stink bug(Hemiptera: Pentatomidae) as an alarm pheromone. Ann. Entomol. Soc. Am. 80:686-691.

McCALL, P. J., TURLINGS,T. C. J., LouGHRiN, J., PROVEAUX,A. T., and TUMLINSON,J. H. 1994.Herbivore-induced volatile emissions from cotton (Gossypium hirsutum L.) seedlings. J. Chem.Ecol. 20:3039-3050.

McCuLLOUGH, T. 1968. Acid and aldehyde compounds in the scent fluid of Leptoglossus oppositus.Ann. Entomol. Soc. Am. 61:1044.

McCuLLOUGH, T. 1969. Chemical analysis of the scent fluid of Leptoglossus clypeatus. Ann.Entomol. Soc. Am. 62:673.

McCuLLOUGH, T. 1970. Acid content of scent fluid from Acanthocephala femorata, A. declivis,and A. granulosa (Hemiptera: Coreidae). Ann. Entomol. Soc. Am. 64:1199-1200.

MCCULLOUGH,T. 1973a. Chemical analysis of the defensive scent fluid released by Hypselonotuspunctiventris (Hemiptera: Coreidae). Ann. Entomol. Soc. Am. 67:749.

McCuLLOUGH, T. 1973b. Chemical analysis of the defensive secretion fluid produced by Mozenalunata (Hemiptera: Coreidae). Ann. Entomol. Soc. Am. 67:298.

NAULT, L. R., and PHELAN, P. L. 1984. Alarm pheromones and sociality in pre-social insects, pp.

237-256, In W. J. Bell and R. T. Carde (eds.). Chemical Ecology of Insects. Sinauer, Sun-derland, Massachusetts.

OETTING, R. D., and YONKE, T. R. 1978. Morphology of the scent gland apparatus of three Alydidae(Hemiptera). y. Kans. Entomol. Soc. 51:294-306.

ROITBBRG, B. D., and MYERS, J. H. 1978. Adaptation of alarm pheromone responses of the peaaphid Acyrthosiphon pisum (Harris). Can. J. Zool. 56:103-108.

S C H O W A LTE R ,T. D., and SEXTON, J. M. 1990. Effect of Leptoglossus occidentalis (Heteroptera:Coreidae) on seed development of Douglas-fir at different times during the growing season inwestern Oregon. J. Econ. Entomol. 83:1485-1486.

STARR, C. K. 1990. Holding the fort: colony defense in some primit ively social wasps, pp. 421-461,in D. L. Evans and J. O. Schmidt (eds.). Insect Defenses: Adaptive Mechanisms and Strategiesof Prey and Predators. State University of New York Press, Albany, New York.

VISSER, J. H. 1986. Host odor perception in phytophagous insects. Annu. Rev. Entomol. 31:121-144.VISSER, J. H., VAN STRATEN, S., and MAARSE, H. 1979. Isolation and identification of volatiles in

the foliage of potato, Solatium tuberosum, a host plant of the Colorado potato beetle, Lep-tinotarsa decemlineata. Entomol. Exp. Appl. 24:538-549.

WATERHOUSE, D. F., and GILBY, A. R, 1964. The adult scent glands and scent of nine bugs of thesuperfamily Coreoidea. J. Insect Physiol. 10:977-987.

8/6/2019 Blatt et al. 1998

19/19

ALARM PHEROMONEOF L. occidentalis 1031

WOHLERS, P. 1981. Effects of the alarm pheromone (E)-B-famesene on dispersal behavior of thepea aphid Acyrthosiphon pisum. Entomol. Exp. Appl. 29:117-124.

Z A R , J. H. 1984. Biostatistical Analysis. Prentice-Hall, Englewood Cliffs, Ne w Jersey.