Journal of Chemical Ecology, Vol. 24, No.

6, 1998

ALARM PHEROMONE SYSTEM OF THE WESTERN CONIFER SEED BUG, Leptoglossus occidentalis

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

1Centre

for Pest Management, Department of Biological Sciences 2Department of Chemistry Simon Fraser University Burnaby, British Columbia, V5A 1S6 Canada

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

AbstractThe alarm pheromones for adult and nymphal western conifer seed bugs, Leptoglossus occidentalis, were collected from the headspace volatiles of agitated bugs and from extracted adult thoraxes and nymphal abdomens. Adult bugs secreted a blend from the metathoracic glands that consisted of hexyl acetate, hexanal, hexanol, heptyl acetate, and octyl acetate (ratio of 152:103:8:1.5:1). Nymphal alarm pheromone produced by the dorsal abdominal glands consisted of ()-2-hexenal. Agitated adults emitted ~ 24% of the pheromone contained within the glands, while nymphs released ~ 33% of their constitutive supply. The 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 were tested on adults reared under summer conditions in a forced-air one-way bioassay, hexanal and hexyl acetate, the major components of the secretion, were responsible for eliciting the alarm response. Adults collected in the fall from the field were unresponsive to the tested blend, suggesting that adults seeking aggregation sites in the fall become refractory to alarm pheromone stimuli that would cause aggregations to disperse. The weak dispersal responses elicited in both adults and nymphs by either nymphal or adult pheromones are consistent with a tradeoff in the advantage gained by avoiding predation and the disadvantage of leaving a food source. Because of these weak responses, use of alarm pheromones as pest management tools against L. occidentalis is unlikely. Key WordsLeptoglossus occidentalis, western conifer seed bug, Hemiptera: Coreidae, alarm pheromone, metathoracic glands, hexyl acetate, hexanol, hexanal, octyl acetate and heptyl acetate. *To whom correspondence should be addressed at: 41 McMichael Street, Kingston, Ontario, K7M IM8, Canada.

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

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Defensive secretions in some Homoptera and Heteroptera have been well documented. The secretions from aphids (Wohlers, 1981; Dawson et al., 1987); the coreids Hotea gambiae (Westwood) (Hamilton et al., 1985) and Leptoglossus zonatus (Dallas) (Leal et al., 1993); the alydids Megalotomus quinquespinosus (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 fullo Thungberg (Kou et al., 1989) have been characterized as eliciting alarm behaviors (e.g., agitated walking, dropping from the host, flight attempt) among conspecifics. These species share the following common characteristics: (1) they are easily disturbed and readily emit their offensive odor, (2) they form aggregations, 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 by members of this genus and the allopatric ranges of many species, we hypothesized that the alarm pheromone system would be fairly similar for related Leptoglossus species. The propensity toward gregariousness may be a prerequisite to the evolution of alarm pheromones (Nault and Phelan, 1984). Individuals in aggregations can benefit by perceiving volatile secretions used by other members of a group to repel predators and can disperse to avoid being targets for predation. The western conifer seed bug, Leptoglossus occidentalis Heidemann (Hemiptera: Coreidae), is a common and potentially severe pest of conifer seed orchards in western North America (Hedlin et al., 1980; Schowalter and Sexton, 1990; Connelly and Schowalter, 1991). Throughout the summer, groups of adults and nymphs are easily disturbed and emit a defensive secretion. As hypothesized for other Hemiptera, this scent apparently elicits dispersal or alarm behavior in adults and nymphs. As defined by Starr (1990), alarm is the "communication of a shared danger. To show that it exists we need only find a correlation between the responses of an individual which perceives an intrusion and one which does not." Our objectives were to capture, isolate, identify, and bioassay the alarm pheromones of adults and nymphs of L. occidentalis.
METHODS AND MATERIALS

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

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maintained at a 15L:9D photoperiod regime, 32C and 20C peak photophase and scotophase temperatures, respectively, and ~ 70% relative humidity. The colony was established in 1992 and revitalized using field-collected bugs each summer. Adults were segregated by sex for all but one phase of the study, but nymphs were not. One set of bioassay experiments was done with bugs collected in the field from Skimikin Seed Orchards, BC Forest Service, Tappen, British Columbia and Kalamalka Seed Orchards, Vemon, British Columbia, in midSeptember 1994. Headspace Bioassay for Adult and Nymphal Volatiles. Glass jars (130 ml) were washed and air dried prior to use and between replicates. Two sets each of five males or females were placed in separate jars covered with Parafilm. One set of insects, designated nonagitated, were not disturbed. The jar containing the second set of insects, designated agitated, was roughly handled and shaken until the putative alarm pheromone could be detected by the human nose. For bioassays (Figure 1), a single nonagitated test insect was isolated in a Parafilm-covered jar and allowed to settle. Headspace air (10 cc), equal to approx. 0.4 bug equivalents, was drawn from the jar containing the nonagitated insects into a disposable syringe and injected into the jar containing the test insect. 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 nymphal L. occidentalis to headspace samples taken from agitated bugs.

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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 test insects' behavior was observed. Any insect responding to air from nonagitated insects was discounted. An alarm response was recorded if the test insect exhibited agitated behavior, such as a sudden, rapid increase in movement or attempted flight. 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 15 replicates obtained for all combinations of adults exposed to volatiles from nymphs and for nymphs exposed to volatiles from male or female adults. All results were analyzed by x2 analysis and Fisher's exact test (Zar, 1984). In both laboratory and field bioassay, one bug equivalent was equal to the amount of volatiles given off by one agitated bug as determined by volatile capture. Analysis of Volatiles. Volatiles from live adults or nymphs were collected on Porapak Q (Waters Associates Inc., Milford, Massachusetts). Twenty-five groups of 10 agitated males or females and five groups of 20 nymphs were placed in a glass chamber (6.5 cm high, radius 4.7 cm). Bugs were then agitated with a glass rod until the putative alarm pheromone was detected by the human nose. Then air was drawn by a water aspirator through a charcoal scrubber and over the insects for 10 min at 1.65 liters/'min, allowing for ~36 exchanges of air in the aeration chamber. Volatiles were collected in a glass trap (6 mm OD x 30 mm) filled with Porapak Q (50-80 mesh). Trapped volatiles were extracted by eluting the Porapak with 2-3 ml of double-distilled pentane, and the volume was concentrated under a nitrogen stream to 1 ml. A !-/*! sample of the extract was analyzed with a Hewlett Packard 5830A gas chromatograph (GC) with an SP1000 nonbonded polar column (30 m X 0.32 mm ID) (Supelco, Oakville, Ontario) and flame ionization detector and with a Hewlett Packard 5890 gas chromatograph with a fused silica coated DB-5 nonpolar column (30 m x 0.25 mm ID) (J&W Scientific, Folsom, California). Volatiles also were analyzed by coupled gas chromatographic-electroantennographic detection (GC-EAD) (Arn et al., 1975, Gries et al., 1993), with a Hewlett Packard 5890A gas chromatograph and a custom built amplifier with a passive low-pass filter and a cutoff frequency of 10 kHz. An antenna was gently pulled out of the insect's head; the exposed nerve endings were suspended in a saline solution that contained the indifferent electrode and the distal end of the antenna was pierced with a recording electrode. Antennally active compounds were analyzed by coupled GC-mass spectroscopy (MS) with a Hewlett Packard 5985B GC equipped with a fused silica (30 m X 0.25 mm ID) DB-5 column in full-scan and selective ion monitoring (SIM) mode. Compounds were identified by comparison with published spectra (Jennings and Shibamoto, 1980) and identification was verified by GC with authentic standards. Quantities of

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components present in the extracts were calculated by comparing area counts with those obtained from external standards of known concentration. The contents of the metathoracic glands and reservoirs from 20 adult males and 20 females and the contents of the dorsal abdominal glands of 41 third and fourth instars were analyzed. Source insects were collected from the colony and held at 15 C for 20 min to facilitate handling and inhibit emission of volatiles or pheromones. Insects were processed in batches of 5-10. The thoraxes were excised, immersed in pentane chilled on dry ice, and pulverized with a glass rod. The extract was transferred to a volumetric vial, concentrated to 300 ul with a forced nitrogen stream, and placed in a glass tube containing glass wool attached to a Porapak Q trap (as described above). Volatiles were blown onto the trap by nitrogen gas for approximately 2 hr. Traps were then eluted with 1 ml of double-distilled pentane, and the extract was analyzed by GC and GCMS and the amounts of components calculated as above. Bioassay of Extracted and Synthetic Volatiles. A one-way forced-air bioassay was developed to quantify the response of adult bugs to single and blended volatiles (Figure 2). A 16/26 ground glass joint was welded to a glass tube resulting in a straight tube 48 cm long that was strapped horizontally to a board to prevent it from rolling. The ground glass joint fitted to a glass tube, 7 cm long, with a vertical open port, 3 cm long, which served as a receptacle for

FIG. 2. Apparatus used in forced air bioassays during tests for response by adult L. occidentalis to synthetic and extracted alarm pheromone candidates.

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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 mark and its original position recorded. The component to be tested was applied to a glass ampoule, held by a cork, and inserted into the vertical port apparatus (Figure 2). Room air, humidified by passing through water, was blown at 1.5 liters/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 maintained at ~25C. All stimuli were evaluated at one bug equivalent in 2 ul of pentane. Stimuli tested were an extract from excised adult thoraxes, each of five antenally active compounds found in the adult thoraxes, a five-component synthetic blend (hexyl acetate, hexanal, hexanol, heptyl acetate, and octyl acetate in the ratio 152:103:8:1.5:1) 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 the spring and 10 field-collected individuals of each sex were tested in the fall. Ten control insects in each category were tested for their response to untreated air. Colony-reared "summer" females and males did not differ in their response to any of the stimuli (F = 0.02, P = 0.89) and were pooled for analysis. Mean distances moved in response to experimental stimuli were compared with distances moved in untreated air control tests by means of Dunnett's one-way test at a = 0.05. Field Bioassays. Adults and nymphs resting and feeding on cone clusters of western white pine, Pinus monticola Dougl. ex. D. Don, were located at Skimikin Seed Orchards. They were counted and their position identified with plastic flagging. Cone clusters were sprayed with a small (150-ml volume) atomizer containing 4 ml pentane spray with zero (control), two, or four bug equivalents of synthetic adult or nymphal alarm pheromone (as above), or both. The four-bug equivalent treatment of nymphal pheromone was lost by spillage in the field. Six cone clusters were sprayed for each treatment. Numbers of adults that responded by flying away from the cones and numbers of nymphs responding by either dropping or walking away from the cones were recorded. The numbers of nymphs were pooled for each treatment (heterogeneity chisquare 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 as described by Zar(1984). Due to the low number of adults in the field bioassay (N = 6), a laboratory experiment for adults was designed. Potted Douglas fir, Pseudotsuga menziesii (Mirb.) Franco, seedlings (approx. 25 cm high) were placed individually in a large 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

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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 displayed agitated behavior were recorded. Mean percentage responses to experimental stimuli were compared with those to pentane control treatments by Dunnett's one-way test (a 0.05) following transformation with arcsinVx to stabilize the variances.

RESULTS

Headspace Bioassays. Males, females, and nymphs all showed a significant positive alarm response to the headspace volatiles from agitated males, females, and nymphs (and all combinations thereof) (Table 1). Males and females did not differ significantly in their responses to volatiles from their own or from the opposite sex, x2 = 0.36, P = 0.54. Volatiles from nonagitated individuals elicted little or no response. Analysis of Volatiles. GC, GC-MS, and GC-EAD analyses revealed that the antenally active volatiles emitted from agitated adults and those contained within the metathoracic gland and reservoir were hexanal, hexyl acetate, hexanol, heptyl acetate, and octyl acetate (Figure 3). The single antenally active volatile 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 approximately the 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 were diluted 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 and Brand, 1972). This may be an adaptive mechanism that allows the emitter to hide from aggressive conspecifics. In our analysis with L. occidentalis, the concentration of hexyl acetate and hexanal were probably so high that there was a toxic inhibition of olfactory response. Bioassay of Synthetic and Extracted Volatiles. A synthetic blend containing all five components of adult pheromone elicited an alarm response by males, females, and nymphs in the head space bioassay (Table 3). Synthetic nymphal alarm pheromone elicited a similarly ubiquitous response (Table 3). Hexanal and hexyl acetate, the most abundant components in both glands and headspace volatiles from agitated adults, and (E)-2-hexenal from nymphs elicited a significant 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

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TABLE 1 . ALARM BEHAVIOR RESPONSES OF ADULT AND NYMPHAL L. occidentalis IN HEADSPACE BIOASSAY TO HEADSPACE VOLATILES OP AGITATED AND NONAGITATED (CONTROL) BUGS" X2 probability experimental vs. control

Test insect and source of volatile stimulus Females Agitated females Nonagitated females Females Agitated males Nonagitated males Males Agitated females Nonagitated females Males Agitated males Nonagitated males Females Agitated nymphs Nonagitated nymphs Males Agitated nymphs Nonagitated nymphs Nymphs Agitated females Nonagitated females Nymphs Agitated males Nonagitated males Nymphs Agitated nymphs Nonagitated nymphs
a

Postive response (%)

100 0 100 0 87 0 80 0 87 13 93 0 80

0.001

0.001

0.001

0.001

0.001

0.001

7
87 7 93 0

0.001

0.001

0.001

N = 15 bugs per test.

components (F = 1.16, P = 0.27 and F = 1.10, P = 0.34 for females and males, respectively) (Figure 5). In many cases, the insects moved towards the stimulus. Field Experiments. Nymphs in the field showed a significant dispersal response to synthetic alarm pheromone sprays (Table 4). At a dose of 2 bug equivalents, synthetic nymphal pheromone and a mix of adult and nymphal pheromones were significantly more effective than the control (pentane only) at

FIG. 3. Flame ionization detector (FID) and corresponding electroantennographic (BAD) trace from adult female antenna to alarm pheromone collected in vivo from adult females and 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 fourth instars and 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.

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TABLE 2. COMPARISON BETWEEN QUANTITIES OF ALARM PHEROMONE CONTAINED WITHIN METATHORACIC GLAND OF ADULTS AND DORSAL ABDOMINAL GLANDS OF NYMPHS AND QUANTITIES GIVEN OFF WHEN AGITATED
Quantity (ug/bug) in gland (mean SE) Quantity (/ig/bug) given off when agitated (mean SE)* Percent of total content given off when agitated

Pheromone source Adult females

Compound

Hexanal Hexanol Hexyl acetate Heptyl acetate Octyl acetate Hexanal Hexanol Hexyl acetate Heptyl acetate Octyl acetate (>2-Hexenal

568.7 170.40 12.2 7.10 390.7 156.90 0.3 0.04 3.1 1.18 339.7 4.6 337.1 0.3 1.5 3.6 116.70 1.04 96.70 0.14 0.48 0.69

17.1 1.3 21.7 0.2 0.2 15.5 1.4 27.2 0.2 0.2 1.2

3.53 0.93 2.69 0.10 0.02

3.0 10.6 5.6 92.3 4.9 4.6 30.8 8.1 63.6 11.6 32.7

Adult males

Nymphs

4.46 0.87 5.25 0.06 0.01 0.35

"Data obtained from four replicates each consisting of five adult males or females (total = 20 of each 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 five replicates of 20 nymphs (total = 100).

causing dispersal of nymphs from the cone clusters. Synthetic adult alarm pheromone at 4 bug equivalents caused a dispersal response similar to that caused by 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, suggesting that nymphs are more responsive to alarm pheromone from nymphs than from adults. While the pheromone treatments caused nymphs to walk away from the cones, the effect was short-lived, and they would return to the cones within 5 min. For all treatments tested, fewer than 20% of the nymphs present responded by 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, either alone or in mixture with nymphal pheromone, caused significantly higher numbers of adults to become agitated than the pentane control treatment, but only the adult blend alone caused adults to leave the seedlings in significant numbers. Nymphal alarm pheromone had no significant effect.

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TABLE 3. ALARM BEHAVIOR OF ADULT AND NYMPHAL L. occidentalis IN HEADSPACE BIOASSAY TO SYNTHETIC BLEND OF ADULT ALARM PHEROMONE CANDIDATES AND SYNTHETIC NYMPHAL ALARM PHEROMONE, ()-2-Hexenala
Test insect and source of stimulus Females Synthetic adult blend Pentane Males Synthetic adult blend Pentane Females Synthetic nymph Pentane Males Synthetic nymph Pentane Nymphs Synthetic adult blend Pentane Nymphs Synthetic nymph Pentane Percent positive response X2 Probability experimental vs. control

80 13 87 6 93 6 87 6 73 33 80 20

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 semiochemicals found in L. occidentalis are also found in three other Leptoglossus species: L. zonatus (Dallas) (Leal et al., 1993), L. oppositus (Say), and L. clypealis Heidemann (Aldrich and Yonke, 1975). Leptoglossus zonatus produces hexanoic acid in small quantities (Leal et al., 1993), and both L. oppositus and L. clypealis produce small amounts of acetic acid (McCullough, 1968, 1969; Aidrich and Yonke, 1975), n-hexyl hexanoate, and ()-2-octenyl acetate (Aldrich and Yonke, 1975). Hexaldehydes are a common component of defensive secretions in numerous other Hemiptera and Homoptera (Waterhouse and Gilby, 1964; McCullough, 1970, 1973a,b; Aldrich et al., 1978, 1979, 1984; Everton et al., 1979; Lockwood and Story, 1987). The frequent occurrence of hexanal in such secretions suggests that it is an effective, broad spectrum irritant easily

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FIG 5. Mean distance moved away from adult and nymphal alarm pheromone and its components by L. occidentalis reared in colony under "summer" conditions or collected from 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, moved from stimulus as compared with control (pentane).

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

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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 was no evidence of an additive or synergistic interaction of components in the synthetic blend. These minor components have been argued as inconsequential for conspecific recognition (Aldrich and Yonke, 1975). However, reanalysis of the volatiles from previously studied bugs by GC-EAD may reveal other minor compounds of potential behavioral importance. We hypothesize that these compounds, less volatile than hexanal and hexyl acetate, may serve as conspecific recognition signals. The utilization of different pheromone components by adults and nymphs of L. occidentalis may indicate that pheromones with different properties are required to accommodate their differing release mechanisms. Adult L. occidentalis expel their pheromone through small openings in the thorax as a spray (personal observation) while nymphs release alarm pheromone through openings in their abdominal tergites. Hexanal, produced by adults, has a higher molecular weight than (E)-2-hexenal and is less volatile (bp 131 C). Hexanal may disperse into the air as an aerosol or may contact a predator prior to volatilization. (E)2-Hexenal (bp 47 C), if released in a similar manner, will volatilize rapidly on exposure to air. As (')-2-hexenal is probably released onto the surface of the tergites, rather than directly into the air, the large odor plume created would probably be as effective as a hexanal spray in warding off predators, particularly from 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 may be possible for species that are preyed upon by ants; however, ants have frequently been observed on conifer boles and branches, but have not been noted as predators of L. occidentalis or any other Leptoglossus species. Metathoracic glands and the associated reservoir of male and female adults contain several hundred micrograms of pheromone, while dorsal abdominal glands of nymphs contain < 10 ug (Table 2). Because only a portion of this amount (~24% of the total for adults and ~33% for nymphs) was emitted by agitated bugs, an individual insect should be capable of repeated emissions. However, approximately 15- and 45-min refractory periods occurred before adults and nymphs, respectively, could be provoked to reemit alarm pheromone. In the nymphs, the long refractory period may indicate that ()-2-hexenal is more useful as a repellent against predation than as a conspecific alarm pheromone. Their aposematic coloring supports this hypothesis. Unlike adults, which can fly away from a host and return, nymphs dropping from cones onto lower

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branches would escape the immediate threat of predation, but would potentially lose their food supply and increase the chances of desiccation and predation by spiders and rodents while on the forest floor. Pea aphids, Acyrthosiphon pisum (Harris), from Kamloops, British Columbia, were less likely than aphids from Vancouver to drop from their host plant in response to alarm pheromone (Roitberg and Myers, 1978). This was postulated as a means to avoid desiccation in areas where ground conditions are harsh. Lockwood and Story (1987) hypothesized that alarming conspecifics evolved as a secondary function of the defensive secretion in Nezara viridula (L.). The reserve of pheromone remaining in the adults after one emission suggests that L. occidentalis could repeatedly defend itself against persistent attempts at predation, 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 an aggregation pheromone at 0.1 bug equivalents. While this behavior is dose dependent, the differential response of summer and fall adult L. occidentalis to their defensive secretion may be physiologically dependent. In the fall, both sexes of L. occidentalis seek cryptic overwintering sites, respond to an unknown male-produced aggregation pheromone (Blatt and Borden, 1996), and are commonly aggregated in large numbers (Blatt, 1994). In these sites they are not easily accessible to predators, such as birds. During this time, alarm responses followed by dispersal would be maladaptive. It is hypothesized that the lesser produced components, hexanol, heptyl acetate, and octyl acetate may be used as conspecific recognition cues. Results obtained in the laboratory bioassays indicate that, like nymphs, adults are more responsive to their own pheromone than to the pheromone produced by bugs in a different life stage. The uniformly high responses to headspace volatiles from any life stage suggest that when stimuli are strong, there is an adaptive advantage to respond to any conspecific alarm signal. The low percentage of adults leaving the seedling suggests that other indications of danger, e.g., rapid movement and shadows, would be necessary to cause adults to depart (Clegg and Barlow, 1982). Of the six adults tested in the field, three flew away and the others dispersed from the cones by agitated walking. In both the field and the laboratory, adults that left the cones or seedlings returned within 10 min. Based on these results, we conclude that the alarm pheromone would not be operationally effective in reducing L. occidentalis populations or in preventing 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 critical reviews. This research was supported by the Natural Sciences and Engineering Research Council of Canada, the Canadian Forest Service Green Plan, the Science Council of BC, the Coast Forest

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

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WOHLERS, P. 1981. Effects of the alarm pheromone (E)-B-famesene on dispersal behavior of the pea aphid Acyrthosiphon pisum. Entomol. Exp. Appl. 29:117-124. ZAR, J. H. 1984. Biostatistical Analysis. Prentice-Hall, Englewood Cliffs, New Jersey.