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Sexual orientation of true bugs to their mates was first investigated systematically ... The spined soldier bug, Podisus maculiventris (Say) (Heteroptera: Penta-.
Journal of Chemical Ecology, Vol. 24, No. 6, 1998

COMPARATIVE ELECTROPHYSIOLOGICAL STUDIES OF OLFACTION IN PREDACEOUS BUGS, Podisus maculiventris and P. nigrispinus

JOSUE SANT'ANA1 and JOSEPH C. DICKENS2,* ' Universidade Federal do Rio Grande do Sul Faculdade de Agronomia, Departamento de Fitossanidade Av. Bento Gonfalves, 7712 Porto Alegre, Rio Grande do Sul Brasil 91540-000 *USDA-ARS, Vegetable Laboratory Plant Sciences Institute Beltsville Agricultural Research Center Beltsville, Maryland 20705 (Received August 8, 1997; accepted February 2, 1998) Abstract—Electroantennograms (EAGs) were recorded from both sexes of spined soldier bug (SSB), Podisus maculiventris, and Brazilian SSB (BSSB), P. nigrispinus to determine antennal olfactory responsiveness of 23 compounds in SSB and 14 compounds in BSSB, including the multicomponent male-produced aggregation pheromone and plant volatiles. EAGs of both species were similar. (E)-2-Hexenol and (E)-2-hexenal elicited the greatest EAGs, followed by heptanol, nonanal, hexanal, and the pheromonal compounds, (±)-a-terpineol and benzyl alcohol. Both sexes of SSB and BSSB were more sensitive to components of the male-produced aggregation pheromone [(±>a-terpineol, (±)-linalool, and benzyl alcohol] and nonanal than either (E)-2hexenol or (£)-2-hexenal (a component of the aggregation pheromone). BSSB were more sensitive to (±)-a-terpineol, (±)-linalool, benzyl alcohol, and nonanal than were SSB. EAGs to the plant volatile 1-hexanol and the pheromonal components (E)-2-hexenal and (±)-a-terpineol decreased significantly with removal of antennal segments, suggesting that receptors for these compounds are distributed over the distal three segments of the five-segmented antennae. Key Words—Pheromone, plant volatile, olfactory receptors, electroantennogram, predators, Podisus maculiventris, Podisus nigrispinus, insect.

*To

whom correspondence should be addressed.

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INTRODUCTION

Sexual orientation of true bugs to their mates was first investigated systematically by Mitchell and Mau (1971). These investigators showed that male southern green stink bugs, Nezara viridula (L.), release a pheromone that attracts conspecific males and females, as well as parasitoids. This pioneering study was extended by the identification of the specific chemicals responsible for the observed attraction (Aldrich et al., 1987). The spined soldier bug, Podisus maculiventris (Say) (Heteroptera: Pentatomidae) (SSB), and Brazilian spined soldier bug, P. nigrispinus (Dallas) (BSSB), are common generalist predators (O'Neil, 1988; McPherson, 1982) in areas of North America (Aldrich et al., 1984a) and South America (Freitas et al., 1990), respectively. Predaceous bugs are important because inundative releases of species such as P. maculiventris and Perillus bioculatus (F.) may be used to manage a number of pestiferous insects including the Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae) (Hough-Goldstein and Keil, 1991; Hough-Goldstein and McPherson, 1996). The dorsal abdominal glands (DAGs) associated with abdominal segments 3 and 4 of male P. maculiventris and P. nigrispinus release a blend of seven chemicals that attracts conspecifics and associated parasitoids (Aldrich et al., 1984a). These glands in both species have the same compounds, but in different proportions. The blend of chemicals is attractive not only to adults, but also to nymphs (Sant'Ana et al., 1997). Although many compounds of bugs have been identified (Gilby and Waterhouse, 1965; Games and Staddon, 1973; Ishiwatari, 1974, 1976; Aldrich, 1988, 1995; Blumenthal, 1978; Slaymaker and Tugwell, 1984), few studies have investigated peripheral reception of these compounds by antennal receptors (Chinta et al., 1994; Dickens et al., 1995). We report here results of investigations to determine selectivity and sensitivity of antennal receptors of both sexes of SSB and BSSB to components of the male-produced aggregation phermone of SSB. As both species produce the same compounds but in different proportions in their attractant glands, comparative studies between the two were done to determine similarities in olfactory reception. Since the putative pheromone components are common plant odors, antennal olfactory responsiveness of both species was determined for related plant odors and their analogs. To localize pheromone responsive neurons, electroantennogram recordings were made following ablation of terminal antennal segments. METHODS AND MATERIALS

Insects. Colonies of P. maculiventris and P. nigrispinus were started from insects captured in pheromone-baited traps at the USDA-ARS, Beltsville Agri-

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cultural Research Center, Beltsville, Maryland, and Universidade de Viscosa, Brazil, respectively. All insects were reared in a growth room at 26 ± 2°C, 60% ± 10% relative humidity, and 16:8 L-D photoperiod, at the USDA-ARS, Insect Chemical Ecology Laboratory, Beltsville, Maryland. Insects were provided with water vials, green bean pods, and pupae of Tenebrio molitor (Coleoptera: Tenebrionidae). Odorous Stimuli. Chemicals tested were chosen based on either their presence in the segment 3-4 dorsal abdominal gland (DAG) of SSB and BSSB males or their occurrence in plants of the prey's habitat (p-h) (see Table 1). Also tested were SSB dorsal abdominal gland extract (DAG) from adult males, a topical ether extract of Heliothis virescens larvae, and a synthetic pheromone blend composed of 53.6% (£)-2-hexenal + 40% (±)-a-terpineol + 4.9% benzyl alcohol + 0.9% (±)-linaloolool+ 0.5% (+)-terpinen-4-ol for SSB, and 3.58% (E)-2-hexenal + 84% (±)-a-terpineol + 3.4% benzyl alcohol + 2.81% (±)linalool ± 1.82% (+)-terpinen-4-ol + 4.4% frans-piperitol for BSSB. Purities of all compounds were determined using gas chromatography. Stimulus dilutions in nanograde hexane were delivered from glass odor cartridges (80 mm long x 5 mm ID) as 5-zl aliquots on Whatman No. 1 filter paper ( 7 x 1 8 mm). Odor cartridges were oriented towards the antenna from a distance of 1 cm. Odor molecules evaporating from the filter paper were carried over the preparation by dry, charcoal-filtered, hydrocarbon-free air. Stimulus duration was 1 sec; interstimulus time intervals of 2-3 min allowed for recovery of the sensory cells. Air around the experimental set up was continuously exhausted. Because of the wide range in volatilities of test compounds (see Kovats indices, Table 1) (Kovats, 1961), only relative comparisons can be made between the odorous stimuli except for closely related compounds, between which comparisons are valid. Electrophysiology. Electroantennogram (EAG) techniques utilized in these studies are a modification of previous techniques (Schneider, 1957) and are described in detail elsewhere (Dickens, 1984; Dickens et al., 1995). Briefly, Ag-AgCl capillary electrodes filled with Drosophila ringer (NaCl 7.5 g/liter, KC1 0.35 g/liter, and CaCl2 0.21 g/liter) were used. Intact insects were immobilized on a cork block using adhesive tape. After puncturing the antenna with a tungsten needle in the proximal and distal segments, the recording electrode was inserted into the distal antennal segment, and the indifferent electrode was placed into the proximal segment. The signal was amplified by a Grass P-16 microelectrode DC preamplifier and viewed on a storage oscilloscope. EAGs were recorded on a stripchart recorder. Experimental Protocol. Three types of experiments were performed. The first two elucidated selectivity and sensitivity of antennal receptors of P. maculiventris and P. nigrispinus for male-produced pheromone components, prey-

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TABLE 1. CHEMICALS USED IN BAG EXPERIMENTS, THEIR PURITY, SOURCE OF SUPPLY, KOVATS' RETENTION INDICES, AND BIOLOGICAL SOURCE

Chemical Green leaf volatiles (GLVs) (E)-2-Hexenal Hexanal I-hexanol (E)-2-Hexenol (£)-2-Hexenoic acid (E)-2-Hexenyl acetate Monoterpenes Myreene (±)-Linalool Nerol Geraniol (±)-90 >94 99 >99 100 100

A A A A A C

965 945 980 995 nad na

I l/II 2, 3, 4, 5, 6, 7 II2, 7 II 2,77 II 2, 3, 4, 5, 7 II 7

>50 >99 >98 >88 >97 >94 >60

A

1115 na 1410 1425 1340

II 3, 4, 5, 6 I l/II 3, 4, 5, 6 II2, 5 II 2, 3, 4, 5 I l/II 2

na na

I1 I1

100 >89 >99

A A C

1260 1172

I l/II 2 I l/II 4

>90 100 >96 >60

A A A

1040

II 3, 5, 7

na 1225

C

na

II7 II3,5 I 1 (nymphs)

>97

A

na

I 1 (nymphs)

A B B

A A D

Identified from insect (I), host plant (II)c

II7

na

aA.

Aldrich Chemical Co., Milwaukee, Wisconsin; B. Pfalz & Bauer, Inc., Stamford, Connecticut; C. Fluka Chemical AG, Buchs, Switzerland; PCR Research Chemical Inc., Gainesville, Florida. bKovats indices [Kovats, (1961), Hedin et al. (1976)]. c1, Aldrich et al. (1948b); 2, Hedin et al. (1971); 3, Hedin et al. (1973); 4, Hedin et al. (1975); 5, Hedin et al. (1976); 6, Loughrin et al. (1994); 7, MacLeod et al. (1982). dNot available.

habitat volatiles, and a prey odor extract. The third localized receptors for one plant odor and two pheromone components to specific antennal segments. In the first experiment, the general responsiveness of antennal receptors to individual odorants was measured by recording EAGs to volatiles emanating from 50-fjig stimulus loads of each compound. Presentation of each odorant was randomly ordered for each sample. In both SSB and BSSB, six replicates were obtained for each sex.

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In the second experiment, odorants were selected for more detailed examination based on data obtained in the first experiment. Dose-response curves were constructed from EAGs elicited by serial dilutions of each compound (0.005-500 jig). Serial dilutions were presented in order from the lowest to the highest stimulus load. Three replicates were obtained for each sex for both species. A third experiment localized pheromone and plant odor receptors in SSB and BSSB to certain segments of their five-segmented antennae. EAGs were recorded from adult males and females in which the distal segment of the antenna were removed and from adult males and females in which both the distal and penultimate segments of the antenna were removed. EAGs obtained in this experiment were compared to EAGs obtained from intact antennae. The recording electrode was always placed at the end of the last intact antennal segment. This experiment was replicated three times for both sexes of SSB and BSSB. Odorous stimuli included 50-fg stimulus loads of 1-hexanol (a plant odor) and (E)-2-hexenal and a-terpineol (pheromone compounds). Different individuals were used for each EAG experiment. 1-Hexanol (50-ig stimulus load) was used as a standard to normalize all responses, so that responses within an individual and among individuals could be compared (Payne, 1975). Stimulation with the standard preceded and followed every two stimulations. Millivolt responses were converted into a percentage of the mean of two nearest responses to the standard. Control stimulations (5 jtl of the hexane solvent) were made at the beginning and at the end of each preparation. The mean response to the control was subtracted from each EAG. Each EAG was measured as the peak of depolarization during the stimulation period. The threshold was considered to be the lowest dose at which the lower limit of the standard error of the mean response is greater than the upper limit of the standard error for the lowest dilution tested. Saturation level was taken as the highest dose at which the mean response is equal to or less than the previous dose. Statistical Analyses. EAGs were compared statistically using analysis of variance procedure and Duncan's multiple range test (Duncan, 1955). Sexual differences between points on dose-response curves were compared for significant differences in sex using the t test for two means (Ostle, 1969). RESULTS

Responses to Standard. Mean electroantennograms (EAGs) to the standard, 1-hexanol (50-jug stimulus load) in Podisus maculiventris males (0.5 mV ± 0.014; N = 22), were significantly greater than those obtained for females (0.45 mV ± 0.015; N = 22). P. nigrispinus females (0.55 mV ± 0.015; N = 14)

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were more responsive to 1-hexanol than males (0.46 mV ± 0.012; N = 14) (P < 0.005). Antennal Chemoreceptive Selectivity. EAGs were significantly different (P < 0.05) for the individual compounds tested in the SSB and BSSB antennae. Differences between males and females also occurred in the magnitude of EAGs for individual odorants (Tables 2 and 3). In Podisus maculiventris (SSB), the green leaf volatiles (GLVs), (E)-2hexenol and (E)-2-hexenal, were, in general, the most effective odorants tested. Among monoterpenes, a-terpineol elicited the greatest EAGs in SSB adults (Table 2). Significant differences in EAGs between sexes in this class of compounds were observed to (±)-a-terpineol, geraniol, (+)-terpinen-4-ol, myrcene, and trans-piperitol. Females were more responsive to these monoterpenes than males, except males exhibited a higher response to myrcene. Among the benzenoids tested, benzyl alcohol was the most effective. Both benzyl alcohol and benzaldehyde elicited significantly higher responses in SSB females than males. Overall responses to 6-, 7-, and 9-carbon aldehydes were higher than those elicited by 8- and 14-carbon aldehydes. Females were generally more responsive to 6-, 7-, 8-, and 9-carbon chain compounds than were males. However, the differences proved to be significant only for heptanal and octanal. EAGs of both sexes increased to a maximum at 7-carbon chain length. The synthetic pheromone blend of SSB elicited greater EAGs in both sexes than did the DAG extract, although no significant difference was found between sexes. Heliothis virescens larval extract was barely detected by the antennal receptors of SSB males or females. In Podisus nigrispinus (BSSB), GLVs were the most effective compounds tested (Table 3). The greatest responses were elicited by (E)-2-hexenal and (E)2-hexenol. However, no differences in EAGs between sexes were recorded for these two compounds. Responses to oxygenated monoterpenes [(±)-linalool, nerol, (+)-terpinen-4-ol, and rrans-piperitol)] were not significantly different between sexes. However, EAGs to (+)-a-terpineol were higher in males than in females. Responses to benzaldehyde were similar in both sexes, but males were more selective to benzyl alcohol than females. Males exhibited significantly higher responses to hexanal and heptanal than females. EAGs to hexanal, heptanal, and nonanal obtained from females were not significantly different. Mean response to the BSSB pheromone blend did not differ significantly between sexes. n-Tridecane, a compound present in the gland of nymphs, elicited small EAGs from antennal receptors of adults. Interspecific Comparison of Antennal Selectivity. In general, no statistically significant differences in EAGs of the same sex were observed between species to most of the compounds tested with one exception: significantly greater EAGs were elicited in response to the monoterpenol, a-terpineol, and heptanal in female SSB compared with female BSSB.

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TABLE 3. MEAN EAGs OF Six MALE AND Six FEMALE Podisus nigrispinus TO 50-sg STIMULUS LOAD OF SELECTED VOLATILES, PHEROMONE COMPOUNDS, AND RELATED CHEMICALS Females

Males Chemical (E)-2-Hexenal (E)-2-Hexenol Heptanalc Hexanal (±)-a-Terpineol P. nigrispinus (synthetic) Nonanal Benzyl alcohol (+)-Terpinen-4-ol (±)-Linalool Benzaldehyde Nerol n-tridecane frans-Piperitol

Mean EAGsb 90.06 85.58 56.56 51.55 49.01

± ± ± ± ±

20.34 14.38 12.78 10.85 4.89

44.90 ± 40.41 ± 38.81 ± 27.74 ± 26.92 ± 26. 15 ± 22.05 ± 22.87 ± 19.81 ±

10.87 5.08 6.98 6.52 6.86 6.72 3.93 4.27 3.93

(±SE)

a a b b be bcd bcde bcde cde cde cde de e e

Chemicals (E)-2-Hexenal (E)-2-Hexenol Nonanal (+)-Terpinen-4-ol Heptanal P. nigrispinus (synthetic) Benzyl alcohol Hexanal (±)-a-Terpineol Benzaldehyde (±)-Linalool Nerol n-tridecane trans-Piperitol

Mean EAGsb 82.81 80.61 41.96 38.68 36.50

± 14.03 ± 13.64 ± 5.45 ± 8.74 ±4.11

34.89 26.94 25.27 24.6 24.58 19.73 19.24 17.94 17.45

± ± ± ± ± ± ± ± ±

3.61 3.79 4 3 5.45 3.56 2.45 2.96 2.74

(±SE)

a a b b b b b b b b b b b b

"Numbers followed by different letters are significantly different within sex (P < 0.05; Duncan's multiple-range test). b EAGs are represented as percent response to the standard = 1-hexanol (50 /tg). cResponse to chemicals in bold type differ significantly between sexes.

Antennal Chemoreceptive Sensitivity. Podisus maculiventris dose-response curves with (E)-2-hexenal (Figure 1A) and (E)-2-hexenol (Figure 1B) showed receptors for both sexes to be equally sensitive to these compounds (threshold = 50 /ig), Responses of males to the 50-jug dose were significantly greater than responses of females. Both sexes were less sensitive to (E)-2-hexenal or (E)-2hexenol than to the remaining compounds, which reached threshold at the ca. 5-fj.g dose. EAGs of males to (±)-linalool were significantly greater than those of females (Figure 1F). In Podisus nigrispinus threshold responses for (E)-2-hexenal (Figure 2A) and (E)-2-hexenol (Figure 2B) were similar for those obtained for SSB. However, threshold responses of BSSB to the other compounds occurred between the 0.5-jug and 5-/:*g stimulus loads (Figure 2C-F). Interspecific Comparison of Antennal Sensitivity. Overall, slopes of doseresponse curves for the selected odorants in SSB were similar to those in BSSB. Usually, responses increased from the lowest to the highest dose presented to

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the antennae. Dose-response curves for (E)-2-hexenal (Figure 2A) and (E)-2hexenol (Figure 2B) were nearly identical for both species. Receptors for nonanal, (±)-a-terpineol, benzyl alcohol, and (±)-linalool in SSB were less sensitive than those in BSSB (Figures 1C-F, 2C-F). Localization of Antennal Receptors. In general, extirpation of one or two antennal segments resulted in decreased EAGs to the plant odor, 1-hexanol, and two pheromone components, (+)-a-terpineol (E)-2-hexenal; with one exception, responses of BSSB males to (+)-a-terpineol with the distal segment removed did not differ from responses from intact antennae (Figure 3). EAGs recorded from the three proximal segments were smaller than those recorded from the whole antenna and the antenna with distal segment extirpated. DISCUSSION

Antennal Chemoreceptive Selectivity. Generally, (E)-2-hexenal and (E)-2hexenol elicited the greatest EAGs for both SSB and BSSB. Hexan-1-ol, (E)2-hexenol, (Z)-3-hexenol, and their derivatives, e.g., (E)-2-hexenal (also a pheromone component), are components of green odor and are referred to as green leaf volatiles (GLVs) (Visser et al., 1979). Since the release of GLVs increases after insect feeding (Whitman and Eller, 1990; Dicke et al., 1990), the presence of these volatiles in the air could be used by predators as a cue to find prey that are feeding on plants. Responses of P. maculiventris and P. nigrispinus to (E)-2-hexenol and (E)-2-hexenal could be based in two aspects. First, the compounds have relatively low molecular weights and high volatilities based on Kovats relation indices (Table 1). However, this may not be the main factor for such high responses since hexanal is more volatile and EAGs elicited by it were smaller. Second, (E)-2-hexenal is a component of the pheromonal attractant for both SSB and BSSB, and GLVs, in general, may be attractants both to other phytophagous insects (Visser and Ave, 1978) and may enhance activity of insect pheromones (Dickens, 1989; Dickens et al., 1990, 1993; Light et al., 1993). Thus, specific sensitivity to these odorants would facilitate location of prey by Podisus through deciphering attractant semiochemical messages from them as shown for an insect parasitoid (Whitman, 1988; Whitman and Eller, 1990). The lack of response of antennal receptors in predaceous bugs to larval washes of lepidopterous larvae, such as H. virescens in our current study, might indicate the use of plant volatiles for location of prey habitat by these generalist predators. Once in the area of potential prey, the bug may use other senses to pinpoint its prey. For example, substrate vibrations produced by prey feeding may serve as directional cues for P. maculiventris in search of prey (Pfannenstiel et al., 1995). Another method is used by the predaceous clerid beetle, Thana-

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simus dubious, which has receptors tuned to the aggregation pheromone of its prey, the southern pine beetle, Dendroctonus frontalis (Payne et al., 1984). The second most active class of compounds in our study, the aldehydes heptanal, hexanal, and nonanal, are known plant volatiles (Hedin et al., 1976; Light et al., 1988). These compounds could be used by predators, such as SSB and BSSB, and parasites as cues for prey habitat location. Antennal receptors of some parasitoids respond to these aldehydes and their alcohol derivatives (Microplitis demolitor, Ramachandran and Norris, 1991; Campoletis sonorensis, Baehrecke et al., 1989; and Microplitis croceipes, Li et al., 1992). Alternatively, the high sensitivity to plant odors could also be explained by the fact that many of these compounds are the same as or closely related to pheromones, and, thus, may stimulate some pheromone receptors. Antennae were more responsive to (±)-a-terpineol and benzyl alcohol than to related monoterpenes and benzenoid compounds (Table 1). Significant responses to these compounds reflect their importance as pheromone components for both SSB and BSSB (Aldrich et al., 1984a, 1991). Sexually dimorphic responses to compounds such as (±)-a-terpineol may be explained by differences in the number and distribution of functional sensillar and/or receptor neuron types. While sexual differences in the number and distribution of sensilla do occur (Dickens and Sant'Ana, unpublished), receptor neurons associated with them have not been characterized. Variations in responses to the same compound by SSB and BSSB may be related to functional adaptations with regard to the relative importance of certain odorants in chemical communication schemes for SSB and BSSB. Similarities in EAGs of SSB and BSSB to plant odors and most pheromonal compounds may be explained by similar acceptor populations in both species. Antennal Chemoreceptive Sensitivity. Both SSB and BSSB showed significant sexual differences in sensitivities to plant volatiles and pheromonal compounds. Dose-response curves for (E)-2-hexenal and (E)-2-hexenol for SSB did not differ from those observed for BSSB, thus indicating similar detection mechanisms for these compounds. Thresholds for each compound were the same for both species (50-sg stimulus load). EAGs peaked at the 50-/xg stimulus load before decreasing significantly at the 500-sg stimulus load. Such a decrease in response may be due to decreased volatility at the higher dose or to unexplained physiological phenomena. SSB and BSSB males and females were, in general, more sensitive to (±)a-terpineol, benzyl alcohol, (±)-linalool, and nonanal (Figures 1 and 2), which are part of the pheromone blend released by males to attract females and other males (Aldrich et al., 1984a). Nonanal is present in small amounts in the dorsal abdominal gland of female SSB, but a behavioral role has not been assigned to it (Aldrich et al., 1984b). Sensitivity of both sexes of SSB and BSSB to nonanal observed here indicate its potential role as a chemical messenger.

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Differences in relative saturation levels and sensitivities in dose-response curves in SSB and BSSB for (E)-2-hexenol and (E)-2-hexanal (a pheromone component) (Figures 1A and B, 2A and B) compared to the pheromone components, (±)-a-terpineol, benzyl alcohol, and (±)-linalool are striking (Figures 1D and E, 2D and E). These differences may be due to differences in relative volatilities [(±)-a-terpineol, (±)-linalool and benzaldehyde are less volatile than (E)-2-hexenol and (E)-2-hexanal] or in numbers of receptive sensilla. Since pheromone components are of critical importance to species propagation, a few sensilla, sensitive and specifically tuned to them, would effectively enhance detection of these important signals in an inherently noisy environment. Localization of Receptors for Plant Volatiles and Pheromones. Directly proportional relationships between EAG amplitudes and the length of antenna exposed to the stimulus (or in this case number of antennal segments remaining) indicates that receptors for each of these odorants are distributed over each of the terminal two segments (Figure 3). Similar results have been found for several other insects including: Argyrotaenia velutinana (Roelofs and Comeau, 1971), Trichoplusia ni (Mayer etal., 1984), Oryzaephilus surinatnensis (White, 1991), and Acyrthosiphon pisum (Giessen et al., 1994). Although EAGs continued to decrease after removing the two distal segments, small, but significant, EAGs could still be recorded for each of the chemicals tested indicating that receptors for these odorants are present on segments 1-3 (Figure 3). In the tarnished plant bug, Lygus lineolaris (Miridae), EAGs to a plant odor (1-hexanol) and an insect-produced odor [(E)-2-hexenyl butyrate] did not decrease after removal of the distal antennal segment (Dickens et al., 1995). This suggested that receptors for both odorants tested were housed principally on the second and third segments of the four-segmented antenna, as was shown in behavioral bioassays of sexual attraction in a closely related species, L. hesperus (Graham, 1988). In conclusion, receptor selectivity and sensitivity of SSB and BSSB to environmental volatiles and pheromone compounds are similar. GLVs and other plant volatiles are used by SSB and BSSB together with the male-produced aggregation pheromone (which has components that are also present in plant emissions) as cues for mating, location of hosts, feeding, and oviposition. Chemical attractants for predators would be useful in programs of integrated pest management (IPM) using natural enemies (Sant'Ana et al., 1997). Such selective use of semiochemicals to assemble and keep SSB in potato fields could lessen damage by the Colorado potato beetle (Hough-Goldstein and McPherson, 1996). Acknowledgments—-We thank Aida Ester Sztein for the comments on the early version of the manuscript. We also thank Dr. Jeffrey R. Aldrich and Evaldo F. Vilela for making possible the exchange program between the United States and Brazil. This study was part of a doctoral thesis by Josue Sant'Ana and was supported by a grant from CNPq, Brazil. The authors are grateful for

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critical reviews provided by Dr. J. R. Aldrich, USDA-ARS, Insect Chemical Ecology Laboratory, Beltsville Agricultural Research Center, Beltsville, Maryland, and Professor Dr. F. E. Hanson, Department of Biological Sciences, University of Maryland-Baltimore County, Catonsville, Maryland.

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