Electrophysiological Responses of Trissolcus japonicus, T. basalis, and T. oenone (Hymenoptera: Scelionidae) to Volatile Compounds Associated with New Zealand Stink Bugs (Hemiptera: Pentatomidae).

Parasitoid biological control agents rely heavily on olfaction to locate their hosts. Chemical cues associated with hosts and non-hosts are known to influence the expression of host preferences and host-specificity. A better understanding of how and why parasitoids attack some species and not others, based on volatile organic compounds associated with potential hosts, can provide key information on the parasitoid's host preferences, which could be applied to pre-release risk assessments for classical biological control agents. Electrophysiological techniques such as electroantennography (EAG) and GC-EAD (gas chromatography coupled with electroantennographic detection) are widely used to identify bioactive semiochemicals. But the application of these techniques to understanding how chemical ecological cues mediate parasitoid host specificity has not been as thoroughly explored. We conducted GC-EAD and EAG studies to identify olfactory-active compounds associated with adult females of nine stink bug species from Aotearoa/New Zealand on the antennae of three closely related parasitoid species: Trissolcus japonicus Ashmead, a pre-emptively (= proactively) approved biocontrol agent against brown marmorated stink bug; T. basalis (Wollaston), a biocontrol agent introduced against Nezara viridula L. in 1949; and T. oenone Johnson, a native Australasian pentatomid parasitoid. Eight compounds associated with stink bugs elicited antennal responses from all three parasitoids, and we were able to identify seven of these. (E)-2-hexenal, (E)-4-oxo-2-hexenal, (E)-2-octenal and (E)-2-decenal generally elicited stronger responses in the three parasitoids, while n-tridecane, n-dodecane, and (E)-2-decenyl acetate elicited weaker responses. We discuss how and why the results from electrophysiological experiments can be applied to non-target risk assessments within biological control programmes.

Molecular evidence for multiple origins and high genetic differentiation of non-native winter crane fly, Trichocera maculipennis (Diptera: Trichoceridae), in the maritime Antarctic.

Native biodiversity and ecosystems of Antarctica safeguarded from biological invasion face recent threats from non-native species, accelerated by increasing human activities and climate changes. Over two decades ago, the winter crane fly, Trichocera maculipennis, was first detected on King George Island. It has now successfully colonized several research stations across King George Island. To understand the origin, genetic diversity, and population structure of this Holarctic species, we conducted mitochondrial DNA cytochrome c oxidase subunit I (COI) sequence analysis across both its native and invasive ranges. In parallel, we performed microsatellite loci analysis within the invasive ranges, utilizing 12 polymorphic microsatellite markers. Furthermore, we compared body sizes among adult males and females collected from three different locations of King George Island. Our COI sequence analysis exhibited two different lineages present on King George Island. Lineage I was linked to Arctic Svalbard and Polish cave populations and Lineage II was related to Canadian Terra Nova National Park populations, implying multiple origins. Microsatellite analysis further exhibited high levels of genetic diversity and significant levels of genetic differentiation among invasive populations. Body sizes of adult T. maculipennis were significantly different among invasive populations but were not attributed to genetics. This significant genetic diversity likely facilitated the rapid colonization and establishment of T. maculipennis on King George Island, contributing to their successful invasion. Molecular analysis results revealed a substantial amount of genetic variation within invasive populations, which can serve as management units for invasive species control. Furthermore, the genetic markers we developed in the study will be invaluable tools for tracking impending invasion events and the travel routes of new individuals. Taken together, these findings illustrate the highly invasive and adaptable characteristics of T. maculipennis. Therefore, immediate action is necessary to mitigate their ongoing invasion and facilitate their eradication.

Synthesis and Biological Testing of Ester Pheromone Analogues for Two Fruitworm Moths (Carposinidae).

A range of ester pheromone analogues for carposinid moths were synthesized and evaluated for biological activity. The analogues aimed to take advantage of the structural commonality of (7Z)-alken-11-ones found in this family. Analogues were tested on two pest species: Heterocrossa rubophaga and Coscinoptycha improbana. Two of the analogues, (2Z)-nonenyl nonanoate and (4Z)-heptyl undecenoate, elicited significant electroantennogram responses. Only (4Z)-heptyl undecenoate gave consistent responses with both moth species in single sensillum recording. Field trapping trials were conducted with these two analogues both individually and in combination with the pheromone of each of the two moth species. No attraction was observed to either of the analogues alone, by either moth species. However, when (4Z)-heptyl undecenoate was coupled with the pheromone, it produced a strong inhibitory effect in H. rubophaga, reducing male moth trap catch by over 95%. No inhibitory effect on male moth trap catch was observed in C. improbana.

Olfactory Receptor Neurons for Plant Volatiles and Pheromone Compounds in the Lucerne Weevil, Sitona discoideus.

Antennal olfactory receptor neurons (ORNs) for pheromone-related and plant volatile compounds were identified and characterized in the lucerne weevil, Sitona discoideus (Gyllenhal), using the single sensillum recording technique. Our study using five pheromone-related compounds and 42 plant volatile compounds indicates that S. discoideus have highly specialized ORNs for pheromone and plant volatile compounds. Different groups of ORNs present in both males and females of S. discoideus were highly sensitive to 4-methylheptane-3,5-dione (diketone) and four isomers (RR, RS, SR and SS) of 5-hydroxy-4-methylheptan-3-one, respectively. Our results also indicate that male S. discoideus, using the sensory input from antennal ORNs, can distinguish both diketone and the RR-isomer from others, and RS- and SS-isomers from others, although it was unclear if they can distinguish between RS-isomer and SS-isomer, or between diketone and the SR-isomer. It also appeared that female S. discoideus could distinguish between RS-isomer and SS-isomers. The antennae of S. discoideus thus contain sex-specific sets of ORNs for host- and non-host plant volatile compounds. Both sexes of S. discoideus have highly sensitive and selective ORNs for some green-leaf volatiles, such as (Z)-3-hexenol and (E)-2-hexenal. In contrast, male antennae of S. discoideus house three distinct groups of ORNs specialized for myrcene and (E)-ß-ocimene, 2-phenylethanol, and phenylacetaldehyde, respectively, whereas female antennae contain three groups of ORNs specialized for (±)-linalool and (±)-α-terpineol, myrcene and (E)-ß-ocimene, (±)-1-octen-3-ol, and 3-octanone. Our results suggest that S. discoideus use a multi-component pheromone communication system, and a sex-specific set of ORNs with a narrow range of response spectra for host-plant location.

Sex-biased gene expression in antennae of Drosophila suzukii.

Drosophila suzukii differs from other members of the genus Drosophila in its host preference and oviposition behavior. The flies are attracted to ripening fruits, and females have a serrated ovipositor enabling eggs to be laid inside the fruit. In addition to its huge economic impact, its unique chemoecological, morphological, and physiological characteristics have garnered considerable research interests. In this study, we analyzed D. suzukii antennal transcriptomes to identify sex-biased genes by comparison of differential gene expressions between male antennae (MA) and female antennae (FA). Among 13,583 total genes of the fly genome, 11,787 genes were expressed in either MA or FA. There are only 132 genes (9 in MA, 7 in FA, and 116 in both, FPKM >1) were expressed in antennae exclusively, and 2,570 genes (9 in MA, 0 in FA, and 2,561 in both) were enriched in antennae containing 185 and 113 sex-biased genes in MA and FA, respectively. Interestingly, many immune-related genes were highly expressed in MA, whereas several chemosensory genes were at high rank in FA. We identified 27 sex-biased chemosensory genes including odorant and gustatory receptors, odorant-binding proteins, chemosensory proteins, ionotropic receptors, and cytochrome P450s, and validated the gene expressions using quantitative real-time PCR. The highly expressed sex-biased genes in antennae are likely involved in the fly specific mating, host-finding behaviors, or sex-specific functions. The molecular results demonstrated here will facilitate to find the unique chemoreception of D. suzukii, as well as on the development of new management strategies for this pest.

Morphological and functional heterogeneity in olfactory perception between antennae and maxillary palps in the pumpkin fruit fly, Bactrocera depressa.

The morphology and ultrastructure of the olfactory sensilla on the antennae and maxillary palps were investigated through scanning electron microscopy (SEM) and transmission electron microscopy (TEM), and their responses to five volatile compounds were measured using electroantenogram (EAG) and electropalpogram (EPG) techniques in the pumpkin fruit fly, Bactrocera depressa (Shiraki; Diptera: Tephritidae). Male and female B. depressa displayed distinct morphological types of olfactory sensilla in the antennae and maxillary palps, with predominant populations of trichoid, basiconic, and coeloconic sensilla. Basiconic sensilla, the most abundant type of olfactory sensilla in the antennae, could be further classified into two different types. In contrast, the maxillary palps exhibited predominant populations of a single type of curved basiconic sensilla. High-resolution SEM observation revealed the presence of multiple nanoscale wall-pores on the cuticular surface of trichoid and basiconic sensilla, indicating that their primary function is olfactory. In contrast, coeloconic sensilla displayed several longitudinal grooves around the sensillum peg. The TEM observation of individual antennal olfactory sensilla indicates that the basiconic sensilla are thin-walled, while the trichoid sensilla are thick-walled. The profile of EAG responses of male B. depressa was different from their EPG response profile, indicating that the olfactory function of maxillary palps is different from that of antennae in this species. The structural and functional variation in the olfactory sensilla between antennae and maxillary palps suggests that each plays an independent role in the perception of olfactory signals in B. depressa.

Olfactory attraction mediated by the maxillary palps in the striped fruit fly, Bactrocera scutellata: Electrophysiological and behavioral study.

Here, we report that the olfactory attraction of the striped fruit fly, Bactrocera scutellata (Hendel; Diptera: Tephritidae), a serious pest of pumpkin and other cucurbitaceae plants, to cue lure and raspberry ketone is mediated by the maxillary palps. The antennae, bearing three morphological types (basiconic, trichoid, and coeloconic) of olfactory sensilla, in male and female B. scutellata exhibited significant electroantennogram (EAG) responses to a plant volatile compound, 3-octanone, and methyl eugenol, whereas cue lure, raspberry ketone, and zingerone that are known to attract several other species of Bactrocera fruit flies elicited no significant EAG responses from both sexes. In contrast, maxillary palps, housing one morphological type of basiconic sensilla, displayed the largest electropalpogram (EPG) responses to cue lure followed by raspberry ketone among the five compounds tested in male and female B. scutellata, with only minor EPG responses to 3-octanone, which indicates that the maxillary palps are responsible for detecting cue lure and raspberry ketone in this species. In field trapping experiments, significant number of male B. scutellata were captured in the traps baited with cue lure or raspberry ketone, in which the attractiveness of cue lure was significantly higher than that of raspberry ketone. Methyl eugenol and zingerone were not behaviorally attractive to B. scutellata although they elicited significant EPG responses. Our study indicates that the behavioral attraction of B. scutellata to cue lure and raspberry ketone is mediated by the olfactory sensory neurons present in the maxillary palps.

Species- and sex-specific distribution of antennal olfactory sensilla in two tortricid moths, Epiphyas postvittana and Planotortrix octo.

We investigated the morphology and distribution of antennal sensilla in males and females of two tortricid moths, Epiphyas postvittana and Planotortrix octo, by scanning electron microscopy. The number and overall length of flagellomeres were significantly greater in females than in males in both species. The antennae of each species bearing six morphological types of sensilla (trichodea, basiconica, coeloconica, auricillica, chaetica, and styloconica), with different numbers and distributions along the antennae. Among these sensilla, four types (trichodea, basiconica, coeloconica, and auricillica) displayed multi-porous cuticular surfaces, indicating that their primary sensory function is olfactory. Each of these four types of sensilla could be further classified into subtypes according to their size, shape, and surface structure. Both E. postvittana and P. octo exhibited sexual dimorphism of the profiles of antennal olfactory sensilla. Trichoid sensilla were the most abundant type in both species. Subtype I trichoid sensilla were male-specific in both species, indicating that they are responsible for the perception of conspecific female sex pheromone. By contrast, subtype II trichoid sensilla were more abundant in female antennae in both species, suggesting that some subtype II trichoid sensilla are involved in female-specific behaviors, such as oviposition. Chaetic and styloconic sensilla displayed relatively even distributions along the antennae. Our results indicate that the antennae of E. postvittana and P. octo have species-specific and sex-specific profiles of olfactory sensilla. The morphological information obtained in our study provides a basis for electrophysiological and behavioral studies of the olfactory sensory function of each morphological type of sensilla.

Morphology and distribution of antennal sensilla of two tortricid moths, Cydia pomonella and C. succedana (Lepidoptera).

Morphology of antennal sensilla and their distributions were investigated in male and female adults of two tortricid moths, Cydia pomonella and C. succedana using scanning electron microscopy. The antennae of both sexes of the two species were filiform, and the overall lengths of the antennae and the number of consisting segments were greater in males than in females. Six types of sensilla (s.) were identified from the antennae of both sexes in the two species: s. trichodea, s. basiconica, s. coeloconica, s. auricillica, s. chaetica, and s. styloconica, in varying numbers and distribution along the antennae. Among them, surface of four sensilla types (s. trichodea, s. basiconica, s. coeloconica, s. auricillica) were multiporous in the two species, indicating that the primary function of these sensilla is olfactory. The s. trichodea were the most numerous on the antennae in both sexes of the two species. Male C. pomonella has a greater number of s. trichodea than the female. The four sensilla types were further divided into different subtypes in the two species; s. trichodea into three subtypes, s. basiconica into two subtypes, s. coeloconica into two subtypes in C. pomonella and one subtype in C. succedana, and s. auricillica into two subtypes. Sexual dimorphism was observed in the subtypes of s. trichodea. The long subtype of s. trichodea occurs only on male antennae, whereas the short subtypes mainly on female antennae. These findings would be helpful for further studies on detailed chemo-receptive functions of each subtype of the antennal sensilla.

Tarsi of Male Heliothine Moths Contain Aldehydes and Butyrate Esters as Potential Pheromone Components.

The Noctuidae are one of the most speciose moth families and include the genera Helicoverpa and Heliothis. Females use (Z)-11-hexadecenal as the major component of their sex pheromones except for Helicoverpa assulta and Helicoverpa gelotopoeon, both of which utilize (Z)-9-hexadecenal. The minor compounds found in heliothine sex pheromone glands vary with species, but hexadecanal has been found in the pheromone gland of almost all heliothine females so far investigated. In this study, we found a large amount (0.5-1.5 µg) of hexadecanal and octadecanal on the legs of males of four heliothine species, Helicoverpa zea, Helicoverpa armigera, H. assulta, and Heliothis virescens. The hexadecanal was found on and released from the tarsi, and was in much lower levels or not detected on the remaining parts of the leg (tibia, femur, trochanter, and coxa). Lower amounts (0.05-0.5 µg) of hexadecanal were found on female tarsi. This is the first known sex pheromone compound to be identified from the legs of nocturnal moths. Large amounts of butyrate esters (about 16 µg) also were found on tarsi of males with lower amounts on female tarsi. Males deposited the butyrate esters while walking on a glass surface. Decapitation did not reduce the levels of hexadecanal on the tarsi of H. zea males, indicating that hexadecanal production is not under the same neuroendocrine regulation system as the production of female sex pheromone. Based on electroantennogram studies, female antennae had a relatively high response to hexadecanal compared to male antennae. We consider the possible role of aldehydes and butyrate esters as courtship signals in heliothine moths.

Morphology and Distribution of Antennal Sensilla of the Bean Bug Riptortus pedestris (Hemiptera: Alydidae).

The bean bug, Riptortus pedestris is a major pest of bean pods and some tree fruits in north-east Asian countries. Scanning electron microscopy was conducted to investigate the morphology and distribution of antennal sensilla of R. pedestris to help in understanding the sensory mechanisms of the bug. Average antennal lengths of male and female R. pedestris were 11.00 mm and 9.84 mm, respectively, consisting of four distinct segments, scape, pedicel, basiflagellum, and distiflagellum. Based on the gross appearance, the antennal sensilla were classified into four major types (trichodea, basiconica, chaetica, and coeloconica), which could be further classified into four trichoid, three basiconic, four chaotic, and two coeloconic subtypes, based on their size, tip shape, presence of socket, and surface structure. Among them, two subtypes of trichoid sensilla, all three subtypes of basiconic sensilla, four subtypes of chaetic sensilla and two subtypes of coeloconic sensilla had numerous pores along the surface, suggesting their olfactory function. Nine subtypes (2 trichoid, 1 basiconic, 4 chaetic, and 2 coeloconic subtypes) showed a distinct socket structure at the base. Among the four antennal segments, the distiflagellum possessed highest number of sensilla. Trichoid sensilla were most abundant, followed by basiconic, chaetic, and coeloconic sensilla. Each subtype of sensilla exhibited distinct distribution profile along the four antennal segments. Two subtypes of trichoid sensilla, one subtype of basiconic sensilla and one subtype of chaetica sensilla were distributed on scape, pedicel, and basiflagellum, whereas distribution of other subtypes of sensilla was confined to basiflagellum and distiflagellum. Microsc. Res. Tech. 79:501-511, 2016. © 2016 Wiley Periodicals, Inc.

Identification of olfactory receptor neurons in Uraba lugens (Lepidoptera: Nolidae) and its implications for host range.

Phytophagous insects detect volatile compounds produced by host and non-host plants, using species-specific sets of olfactory receptor neurons (ORNs). To investigate the relationship between the range of host plants and the profile of ORNs, single sensillum recordings were carried out to identify ORNs and corresponding active compounds in female Uraba lugens (Lepidoptera: Nolidae), an oligophagous eucalypt feeder. Based on the response profiles to 39 plant volatile compounds, 13 classes of sensilla containing 40 classes of ORNs were identified in female U. lugens. More than 95% (163 out of 171) of these sensilla contained 16 classes of ORNs with narrow response spectra, and 62.6% (107 out of 171) 18 classes of ORNs with broad response spectra. Among the specialized ORNs, seven classes of ORNs exhibited high specificity to 1,8-cineole, (±)-citronellal, myrcene, (±)-linalool and (E)-ß-caryophyllene, major volatiles produced by eucalypts, while nine other classes of ORNs showed highly specialized responses to green leaf volatiles, germacrene D, (E)-ß-farnesene and geranyl acetate that are not produced by most eucalypts. We hypothesize that female U. lugens can recognize their host plants by detecting key host volatile compounds, using a set of ORNs tuned to host volatiles, and discriminate them from non-host plants using another set of ORNs specialized for non-host volatiles. The ORNs with broad response spectra may enhance the discrimination between host and non-host plants by adding moderately selective sensitivity. Based on our finding, it is suggested that phytophagous insects use the combinational input from both host-specific and non-host specific ORNs for locating their host plants, and the electrophysiological characterization of ORN profiles would be useful in predicting the range of host plants in phytophagous insects.

Characterization of olfactory receptor neurons for pheromone candidate and plant volatile compounds in the clover root weevil, Sitona lepidus.

Antennal olfactory receptor neurons (ORNs) for pheromone and plant volatile compounds were identified and characterized in male and female clover root weevil, Sitona lepidus (Gyllenhal), using the single sensillum recording technique with five pheromone-related compounds, and 40 host and non-host plant volatile compounds. Overall, seven different types of olfactory sensilla containing specialized ORNs were identified in each sex of S. lepidus. Among them, three different types of sensilla in the males and two types in the females housed ORNs specialized for pheromone-related compounds. The ORNs in males were specialized for 4-methyl-3,5-heptanedione or one or more of four stereoisomers of 5-hydroxy-4-methyl-3-heptanone. In contrast, female sensilla did not contain ORNs sensitive to 4-methyl-3,5-heptanedione while they contained ORNs sensitive to and specialized for the stereoisomers of (4S,5S)-5-hydroxy-4-methyl-3-heptanone. In addition to the pheromone-related ORNs, four types of olfactory sensilla contained ORNs responsive to plant volatile compounds in male S. lepidus, and five types in females. Most of the ORNs identified in S. lepidus showed a high degree of specificity to specific volatile compounds although some of the active compounds showed overlapping response spectra in the ORNs across different types of sensilla. The most active plant volatile compounds were the four green leaf volatile compounds, (E)-2-hexenol, (Z)-2-hexenol, (Z)-3-hexenol and (E)-2-hexenal, and isomers of two monoterpenols, (±)-linalool and (±)-α-terpineol, all eliciting strong responses from relatively large numbers of ORNs in male and female S. lepidus. Our study indicates that S. lepidus has a set of highly sensitive and selective ORNs for pheromone and plant volatile compounds. Further work is needed to elucidate the behavioral implications of these findings.

Repellent effect of santalol from sandalwood oil against Tetranychus urticae (Acari: Tetranychidae).

Thirty-four essential oils were screened for their repellent activities against the twospotted spider mite, Tetranychus urticae Koch (Acarina: Tetranychidae), at 0.1% concentration level using choice and no-choice laboratory bioassays. Of these, 20 essential oils showed significant repellencies against T. urticae in the choice tests. In subsequent no-choice tests using these 20 essential oils, only sandalwood oil showed significant repellency against T. urticae. Total number of eggs oviposited by T. urticae was significantly lower than controls in the choice tests when the kidney bean leaves were treated with 1 of 14 essential oils. The significant repellency of sandalwood oil against T. urticae lasted at least for 5 h at the 0.1% concentration level. Our GC-MS analysis indicated that the major components of the sandalwood oil were alpha-santalol (45.8%), beta-santalol (20.6%), beta-sinensal (9.4%), and epi-beta-santalol (3.3%). Santanol, a mixture of the two main components in the sandalwood oil, appears to be responsible for the repellency of sandalwood oil against T. urticae.

Moth olfactory trichoid sensilla exhibit nanoscale-level heterogeneity in surface lipid properties.

Chemical force microscopy (CFM) based on tapping mode Atomic force microscopy (AFM) utilized with topographic and phase-shift analyses was used to investigate the topography and surface chemical properties, respectively, of the long trichoid sensilla on the antennae of male Helicoverpa zea. AFM topographic imaging revealed regular series of step-ridges along nearly the entire length of each sensillum, except for the basal ca. 1/3 portions, which were devoid of such ridges. Inter-ridge regions were flat, with regularly spaced pores, ca. 30 nm in diameter populating these planar areas. Many pores exhibited a raised dome that often nearly completely spanned the depression, with only the edges of the depressed portion of the pore still visible. Some pores were observed also along the bases of the ridges. CFM probing of the surface for chemical interactions with the SiO(2) hydrophilic tip revealed consistently diminished hydrogen bonding of the ridge edge areas with the tip than along the flat planar inter-ridge regions. Surfaces of domes over the pores also tended to have less hydrogen bonding with the tip than the planar surfaces. Functionalizing the CFM tip by bonding octadecyl-hydrocarbon to it eliminated these surface chemical-CFM tip interactions and no differences in tip interaction with the sensillar surfaces were observed. Trichoid sensilla from the male antennae of a second species, Utethesia ornatrix, did not exhibit similar heterogeneity between ridge edges versus planar areas with regard to hydrogen bonding with the SiO(2) hydrophilic tip. Pores on U. ornatrix sensilla occurred only along the bases of ridges on their trichoid sensilla. We suggest that the surface lipids of the H. zea sensilla are distributed in a chemically heterogeneous fashion to aid adsorption and transport of aldehyde pheromone component molecules through the pores into the sensillum lumen, possibly through solubilization in an epicuticular lipid layer. The trichoid sensilla of U. ornatrix do not exhibit such surface chemical heterogeneity, and this species-difference may be due to the usage by U. ornatrix of hydrocarbon molecules rather than aldehydes for their sex pheromone components.

Attraction and antennal response of the common wasp, Vespula vulgaris (L.), to selected synthetic chemicals in New Zealand beech forests.

BACKGROUND: The common wasp, Vespula vulgaris (L.), and the German wasp, Vespula germanica (F.), are significant problems in New Zealand beech forests (Nothofagus spp.), adversely affecting native birds and invertebrate biodiversity. This work was undertaken to develop synthetic attractants for these species to enable more efficient monitoring and management. RESULTS: Seven known wasp attractants (acetic acid, butyl butyrate, isobutanol, heptyl butyrate, octyl butyrate and 2,4-hexadienyl butyrate) were field tested, and only heptyl butyrate and octyl butyrate attracted significantly higher numbers of wasps than a non-baited trap. Accordingly, a series of straight-chain esters from methyl to decyl butyrate were prepared and field tested for attraction of social wasps. Peak biological activity occurred with hexyl butyrate, heptyl butyrate, octyl butyrate and nonyl butyrate. Polyethylene bags emitting approximately 18.4-22.6 mg day(-1) of heptyl butyrate were more attractive than polyethylene bags emitting approximately 14.7-16.8 mg day(-1) of heptyl butyrate in the field. Electroantennogram (EAG) studies indicated that queens and workers of V. vulgaris had olfactory receptor neurons responding to various aliphatic butyrates. CONCLUSION: These results are the first to be reported on the EAG response and the attraction of social wasps to synthetic chemicals in New Zealand beech forests and will enable monitoring of social wasp activity in beech forests.

Detection and discrimination of mixed odor strands in overlapping plumes using an insect-antenna-based chemosensor system.

Olfactory signals, a major means of communication in insects, travel in the form of turbulent odor plumes. In terrestrial environments, an odor blend emitted from a single point source exists in every strand of the plume, whereas, in confluent plumes from two different odor sources, the strands have some chance of being coincident and comprising a new third odor in those strands. Insects have the ability to detect and interpret necessary olfactory information from individual filamentous odor strands in complex multifilament odor plumes. However, behaviorists have had no way to measure the stimulus situations they are presenting to their temporally acute insect subjects when performing Y-tube olfactometer or confluent pheromone plume wind tunnel assays. We have successfully measured the degree of plume-strand mixing in confluent plumes in a wind tunnel by using a multichannel insect-antenna-based chemosensor. A PC-based computer algorithm to analyze antennal signals from the probe portion of the system performed real-time signal processing and, following a short training session, classified individual odorant/mixture strands at sub-second temporal resolution and a few tens of millimeters of spatial resolution. In our studies, the chemosensor classified a higher frequency of strands of two different odorants emitted from two closely spaced filter papers as being "mixed" when the sources were located only 1 or 2 cm apart than when the sources were 5 or 10 cm apart. These experiments demonstrate the chemosensor's potential to be used for measuring odor stimulus situations in more complex multiple-plume environments.

Differences in cuticular lipid composition of the antennae of Helicoverpa zea, Heliothis virescens, and Manduca sexta.

Analyses of the hexane washes of antennae, forelegs and whole bodies of Helicoverpa zea, Heliothis virescens, and Manduca sexta revealed notable differences in the components of the cuticular coatings of each species. Most striking were the differences between the cuticular coatings of male and female antennae of both H. zea and H. virescens. Novel esters of short-chain acids (C2-C4) and long-chain secondary alcohols (C25-C32) were identified in the hexane washes of the male antenna and forelegs of H. zea and H. virescens. These compounds were found in only small amounts or were completely absent on the female antennae of both species. In H. zea, butyrates of 7- and 8-pentacosanol and 8- and 9-heptacosanol were found, whereas, in the foreleg extracts of H. virescens, acetates and propionates were detected in addition to butyrates. While cholesterol is a major component of antennal washes (10-15%), only traces were found in the foreleg extracts. Although the composition of the cuticular coating of M. sexta differed greatly from that of the other two species, the extractable coatings of the antennae of male and female M. sexta were nearly identical.

Identification and biosynthetic studies of the hydrocarbon sex pheromone in Utetheisa ornatrix.

The type II class of sex pheromones found in moths is composed of polyene hydrocarbons and their epoxides. Analysis of Utetheisa ornatrix females by gas chromatography-mass spectrometry and measurement of responses of male moths by coupled gas chromatography-electroantennogram detection confirmed the presence of large amounts of (Z,Z,Z)-1,3,6,9-heneicosatetraene (1,3,6,9-21:Hy) and smaller amounts of (Z,Z, Z)-3,6,9-heneicosatriene (3,6,9-21:Hy). Both compounds were detected in pheromone glands of newly emerged adults, with low amounts found in the late pupal stage, indicating that sex pheromone biosynthesis started in the late pupal stage. In our population of females (several hundred sampled), approximately 90% produced the tetraene, 1,3,6,9-21:Hy, as the major component, while the other 10% produced only a large amount (1500-2000 ng) of 3,6,9-21:Hy, with no detectable amount of the tetraene. This result could indicate that two distinct populations are present in our original collection site in Florida. Decapitated female moths accumulated 3,6,9-21:Hy and 1,3,6,9-21:Hy compared to the same age normal females, indicating that female moths continuously produce pheromone. A pheromone biosynthesis activating neuropeptide (PBAN)-like neuropeptide did not affect sex pheromone production as indicated by injection of synthetic PBAN and decapitation of U. ornatrix female adults. When the labeled precursor, D4-9,12,15-18:acid, was injected into the early pupal stage, the most abundantly labeled hydrocarbons were 3,6,9-21:Hy and 1,3,6,9-21:Hy in the female adults. This result indicated that 3,6,9-21:Hy could be biosynthesized from linolenic acid through chain elongation and decarboxylation. To determine how 1,3,6,9-21:Hy is produced, D4-3,6,9-21:Hy was injected into pupae and monitored for incorporation of label. No label was incorporated into 1,3,6,9-21:Hy, although a large amount of triene, 3,6,9-21 :Hy, was recovered in the pheromone gland. This indicates that U. ornatrix females do not use 3,6,9-21:Hy to produce 1,3,6,9-21:Hy, and the terminal double bond is introduced earlier in the biosynthetic pathway.

Perception of conspecific female pheromone stimulates female calling in an arctiid moth, Utetheisa ornatrix.

Perception of the female sex pheromone in Utetheisa ornatrix (Lepidoptera: Arctiidae) is responsible for induction and adjustment of calling by females and the collective phenomenon termed "female pheromonal chorusing". We found five olfactory-active compounds in the U. ornatrix female gland. When females were exposed to the entire pheromone or to two of its (synthetically prepared) components, (Z,Z,Z)-3,6,9-eicosatriene and (Z,Z,Z)-3,6,9-heneicosatriene, they were more likely to call during a given night, begin calling earlier, and briefly increase signal frequency with which they extrude their abdomen, an observable indication of calling in this species. Some females even initiated calling during photophase when exposed to the pheromone components. In general, female U. ornatrix are more sensitive to the complete blend of pheromone than to its individual compounds. We also tested the hypotheses: 1) that abdominal extrusion per se increases the rate of pheromone release; and 2) that greater abdominal pumping rhythm increases pheromone release rate. Contrary to our expectations: 1) females did not respond more strongly to a pulsed pheromone stimulus than to the constant release of pheromone at the same average release rate; and 2) we did not find a relationship between the frequency of abdominal pumping and pheromone release rate. Possible explanations for these unexpected findings are discussed.