Gas Chromatographic-electroantennographic Detection Research Articles

Populations of the Gall Midge Dasineura oxycoccana on Cranberry and Blueberry Produce and Respond to Different Sex Pheromones

We identified and field-tested the sex pheromones of Dasineura oxycoccana (Johnson) (Diptera: Cecidomyiidae) midges collected from cranberry, Vaccinium macrocarpon Aiton, and from highbush blueberry, Vaccinium corymbosum L., commonly named cranberry tipworm (CTW) and blueberry gall midge (BGM), respectively. Coupled gas chromatographic-electroantennographic detection (GC-EAD) analyses of pheromone gland extract from the ovipositor of calling CTW females revealed one component (<10pg per ovipositor/pheromone gland) that elicited antennal responses from CTW males. Stepwise identification was based on its mass spectrum in a concentrated sample with 300 pheromone gland equivalents, retention indices (RI) on three GC columns (DB-5, DB-23, and DB 210), RI inter-column differentials, and RIs and double bond positions of other midge pheromones. These analyses indicated that (8Z)-2,14-diacetoxy-8-heptadecene (2,14-8Z-17) was the candidate pheromone of the CTW. GC-EAD analysis of pheromone gland extract from calling BGM females revealed two components that elicited antennal responses from BGM males. Retention times on the three GC columns were consistent with 2,14-8Z-17 and 2,14-17, indicating that these were candidate pheromone components of the BGM. The four stereoisomers of 2,14-8Z-17 were stereoselectively synthesized and field-tested in cranberry. Delta-type traps baited with SS-2,14-8Z-17 captured significantly more CTW males than did traps baited with any other single stereoisomer or with all four stereoisomers combined. In blueberry, delta-type traps baited with RR-2,14-8Z-17 captured significantly more BGM males than did traps baited with any other single stereoisomer or with all four stereoisomers combined. Subsequent field experiments demonstrated that RR-2,14-17 is the major pheromone component of BGM, and that RR-2,14-8Z-17 is a pheromone component that does not enhance attractiveness of RR-2,14-17. The BGM pheromone RR-2,14-17 has no antagonistic effect on the CTW pheromone SS-2,14-8Z-17 and vice versa. Our results substantiate the conclusion that populations of D. oxycoccana on cranberry and blueberry represent two cryptic species.

Identification of a sex pheromone component for Pennisetia marginata (Lepidoptera: Sesiidae)

AbstractRaspberry crown borer, Pennisetia marginata (Harris) (Lepidoptera: Sesiidae), is a native North American species and pest of many cane fruits. Using coupled gas chromatographic–electroantennographic detection analyses (GC-EAD) we identified candidate sex pheromone components in pheromone gland extracts and effluvia from calling females. Analysis of gland extracts on a Zebron-5 column revealed four components (A, B, C, and D) that elicited strong responses from male antennae. The two most EAD-active components A and B were hypothesised to be (3E,13Z)-octadecadienal [(3E,13Z)-18:Ald] and (3E,13Z)-octadecadienol [(3E,13Z)-18:OH], respectively. Their retention times on other GC columns matched those of authentic standards, thus supporting structural assignments. Synthetic (3E,13Z)-18:Ald, and its GC rearrangement product (2E,13Z)-octadecadienal [(2E,13Z)-18:Ald; component C], also induced antennal responses in GC-EAD analyses of female effluvia. Compounds D in pheromone gland extract, and E and F in female effluvia, elicited EAD responses but could not be identified. In field trapping experiments, (3E,13Z)-18:Ald was the only component that attracted male P. marginata when tested alone. Attractiveness of (3E,13Z)-18:Ald was reduced when combined in binary or ternary blends with any of the other identified components, suggesting one or all may act as pheromone antagonists. In Aldergrove, British Columbia, Canada, peak diel attraction of male P. marginata to (3E,13Z)-18:Ald occurred between 16:00 and 18:00 hours Pacific Daylight Time, at temperatures of 21–23°C, on 30 August 2010, 3 September 2010, and 13 September 2010. We conclude that (3E,13Z)-18:Ald is the major pheromone component of P. marginata.

Defensive Compounds and Male-Produced Sex Pheromone of the Stink Bug, Agroecus griseus

Agroecus griseus is a serious corn pest in Brazil. Contents of the dorsal abdominal glands (DAGs) in nymphs, and the metathoracic gland (MTG) in adults of this species were characterized and quantified. Compounds found were similar to those of other Pentatomidae species and included aliphatic hydrocarbons, aldehydes, oxo-alkenals, and esters. However, two compounds were found in the MTG that have not been described previously for this family. Mass spectrometry, infrared spectroscopy, and gas chromatographic analysis using coinjection with authentic standards confirmed the identities of the compounds as enantiopure (S)-2-methylbutyl acetate and 3-methyl-2-butenyl acetate. The five nymphal instars showed significantly different ratios of components, mainly between those of the first and later instars. No significant differences were detected in MTG contents between sexes. Gas chromatography (GC) analysis of aeration extracts of males and females showed the presence of a compound released exclusively by males. Gas chromatography - electroantennographic detection (GC-EAD) assays indicated that the male-specific compound is bioactive in females, suggesting the presence of an attractant pheromone. The mass spectrum and infrared data for this compound matched with methyl 2,6,10-trimethyltridecanoate, a sex pheromone component previously detected in the stink bugs, Euschistus heros and E. obscurus. The synthetic standard coeluted with the natural pheromone on two different GC stationary phases, confirming the proposed structure. Y-tube olfactometer assays showed that the synthetic standard was strongly attractive to females, and GC-EAD tests produced responses with antennae from females similar to those of the natural pheromone.

Male-Produced Sex Pheromone of the Stink Bug Edessa meditabunda

Edessa meditabunda is a secondary pest within the piercing-sucking stink bug complex that attacks soybean crops in Brazil. The behavioral responses of males and females to aeration extracts from conspecifics suggested the presence of a male-produced sex pheromone. Gas chromatographic (GC) analysis of male and female aeration extracts revealed the presence of two male-specific compounds in a ratio of 92:8. Gas chromatographic -electroantennographic detection (GC-EAD) assays indicated that the major component is bioactive for females, supporting the behavioral data. Analysis of the mass and infrared spectra of the male-specific compounds suggested that they were both methyl-branched long-chain methyl esters. On the basis of the mass spectra of the respective hydrocarbons obtained by micro derivatizations, the structures of these methyl esters were proposed to be methyl 4,8,12-trimethylpentadecanoate (major) and methyl 4,8,12-trimethyltetradecanoate (minor). An 11 step synthetic route that was based on a sequence of Grignard reactions, starting from cyclopropyl methyl ketone, was developed to obtain synthetic standards with a 7.9 % overall yield for the major compound and a 9.9 % yield for the minor. The synthetic standards co-eluted with the natural pheromones on three different GC stationary phases. Y-tube olfactometer assays showed that the synthetic standards, including the major compound alone and a mixture of the major and minor compounds in the proportion found in natural extracts, were strongly attractive to females.

Ambrosia Beetle (Coleoptera: Curculionidae) Responses to Volatile Emissions Associated With Ethanol-InjectedMagnolia virginiana

Xylosandrus germanus (Blandford) and other species of ambrosia beetles are key pests of ornamental nursery trees. A variety of laboratory- and field-based experiments were conducted in pursuit of improved monitoring strategies and to develop a trap tree strategy for ambrosia beetles. Traps baited with bolts prepared from Magnolia virginiana L. injected with ethanol caught five times more X. germanus than ethanol-baited traps. Basal stem injections of ethanol into M. virginiana induced more ambrosia beetle attacks than irrigating or baiting with ethanol, and no attacks occurred on water-injected trees. A positive correlation was also detected between concentration of injected ethanol and cumulative attacks. Solid phase microextraction-gas chromatography-mass spectrometry characterized bark emissions from ethanol- and water-injected M. virginiana at 1, 2, 10, and 16 d after treatment. Ethanol emission from injected trees steadily declined from 1 to 16 d after treatment, but was not emitted from water-injected trees. A variety of monoterpenes were also emitted in trace amounts from the ethanol- and water-injected trees. Antennal responses of X. germanus via gas chromatography-electroantennographic detection to volatiles from ethanol-injected M. virginiana occurred for ethanol, but not the various monoterpenes. X. germanus and other ambrosia beetles were also equally attracted to traps baited with ethanol alone compared with a synthetic mixture of ethanol plus various monoterpenes formulated to mimic ethanol-injected M. virginiana. Injecting concentrated solutions of ethanol into trees may be useful for establishing odor-based trap trees, which could aid with monitoring programs and/or potentially deflect ambrosia beetles away from valuable nursery stock.

Identification and field evaluation of sex pheromones in two hawk moths Deilephila elpenor lewisii and Theretra oldenlandiae oldenlandiae (Lepidoptera: Sphingidae)

The sex pheromones of two species of hawk moth, Deilephila elpenor lewisii (Butler) and Theretra oldenlandiae oldenlandiae (Fabricius), were analyzed using gas chromatography–electroantennographic detection (GC–EAD) and GC–mass spectrometry (GC–MS). Two and three EAD-active components were found in D. elpenor lewisii and T. oldenlandiae oldenlandiae, respectively. GC–MS analyses using authentic compounds and extracts derivatized by dimethyl disulfide and 4-methyl-1,2,4-triazoline-3,5-dione identified the two components in D. elpenor lewisii as (E)-11-hexadecenal (E11–16:Ald) and (10E,12E)-10,12-hexadecadienal (E10,E12–16:Ald), and the three in T. oldenlandiae oldenlandiae as E11–16:Ald, E10,E12–16:Ald, and (10E,12Z)-10,12-hexadecadienal (E10,Z12–16:Ald). In field-trap tests, no males of either species were attracted to any single components. Male moths of D. elpenor lewisii were specifically attracted to a binary blend of E11–16:Ald and E10,E12–16:Ald at a ratio of 85:15, whereas males of T. oldenlandiae oldenlandiae were attracted to a ternary blend of E11–16:Ald, E10,Z12–16:Ald and E10,E12–16:Ald at a ratio of 30:40:30. We therefore conclude that the sex pheromone of D. elpenor lewisii is a mixture of E11–16:Ald and E10,E12–16:Ald and that of T. oldenlandiae oldenlandiae is E11–16:Ald, E10,Z12–16:Ald and E10,E12–16:Ald.

Geographic variation in fruit volatiles emitted by the hawthorn Crataegus mollis and its consequences for host race formation in the apple maggot fly, Rhagoletis pomonella

AbstractThe shift of the apple (AP) maggot fly, Rhagoletis pomonella (Walsh) (Diptera: Tephritidae), from its ancestral host downy hawthorn, Crataegus mollis (DH) (Torr. &amp; A. Gray) Scheele, to introduced domesticated AP, Malus domestica Borkh. (both Rosaceae), is a model for ecological divergence and incipient sympatric speciation with gene flow. However, a portion of the variation contributing to the sympatric host shift from DH to AP appears to have a different biogeographic history, pre‐dating the shift. One potential source of standing variation may trace to a number of different native hawthorn species infested by R. pomonella in the southern USA, where the AP‐attacking race is absent. Herein, we investigate this possibility for the southern red hawthorn (SR) endemic to Texas, Crataegus mollis var. texana (Buckl.), which has been described as a member of the Molles series that includes the more northern distributed DH. We report results from chemical analyses of host fruit volatiles, fly behavioural responses to synthetic fruit blends, and microsatellite surveys of fly populations, implying that R. pomonella infesting SR may behaviourally and genetically represent a native host race differing from the DH‐infesting fly. No fly reared from SR responded to AP fruit volatiles in flight tunnel assays. However, coupled gas chromatographic‐electroantennographic detection (GC‐EAD) profiles for SR fruit contain all five of the component esters that comprise the standard AP volatile blend inducing behavioural orientation for AP‐infesting flies, compounds that appear to be largely missing from volatile profiles for DH fruit. Thus, SR‐infesting flies do not represent a source for a preassembled AP‐accepting phenotype. However, they may help explain why the ancestral DH race that shifted to AP in the northeastern USA had the ability to recognize AP fruit esters, potentially enabling the shift to AP. Our results highlight how categorizing speciation into different geographic modes may not adequately describe the evolutionary origins of important genetic variation fuelling adaptive radiation and the genesis of new biodiversity.

Electrophysiological and behavioral responses of Helicoverpa assulta (Lepidoptera: Noctuidae) to tobacco volatiles

The Oriental tobacco budworm moth, Helicoverpa assulta (Guenee) (Lepidoptera: Noctuidae), is a serious pest on tobacco in China. The flowering stage of the host plant is one of the most attractive stages to H. assulta for feeding and oviposition. Nine electrophysiologically active compounds in tobacco headspace at flower stage were detected by gas chromatography–electroantennographic detection (GC–EAD). These compounds were subsequently identified by gas chromatography-mass spectrometry (GC–MS) as (E)-β-ocimene, octanal, (Z)-3-hexenyl acetate, (Z)-3-hexen-1-ol, nonanal, (Z)-3-hexenyl-2-methyl butyrate, decanal, linalool, and (E)-β-caryophyllene. The synthetic blend containing nine of the above compounds attracted mated H. assulta females from a distance by upwind oriented flight. Selected subtraction assays showed that the 4-component mixture of (E)-β-ocimene, (Z)-3-hexenyl acetate, nonanal, and (E)-β-caryophyllene elicited equivalent levels of attraction as the 9-component mixture. The removal of any of the four compounds from the 4-component blend resulted in a significant decrease in female upwind flight behavior.

Chopper-stabilized gas chromatography-electroantennography: Part I. Background, signal processing and example

A new method that can improve gas-chromatography-electroantennographic detection (GC-EAD) by orders of magnitude through a technique known as chopper stabilization combined with matched filtering in colored noise is presented. The EAD is a physiological recording from the antenna of an insect which can be used to find compounds in the GC effluent that the antenna is able to detect, having important applications for pest control and understanding of chemical communication in nature. The new method is demonstrated with whole-animal male Helicoverpa zea antennal preparations for detection of major pheromone component (cis-11-hexadecenal) and compared to results obtained using traditional EAD recording techniques. Results indicate that chopper stabilization under these circumstances can increase odorant detection performance by a factor of approximately 104 over traditional methods. The time course of the response of the antenna is also better resolved under chopped conditions. Although the degree of improvement is expected to vary with insect species, odor, and insect preparation, under most circumstances a more sensitive and robust GC-EAD instrument will result from the application of this technique.

Sex pheromone of the African armyworm Spodoptera exempta (Lepidoptera: Noctuidae): identification of components of the Okinawan population and formulation for population monitoring

In 2010, abrupt outbreaks of the African armyworm, Spodoptera exempta (Walker), occured on the Tarama, Iriomote and Kikai Islands in southwestern Japan. Analysis by gas chromatography-electroantennographic detection (GC-EAD) revealed two EAG-active compounds on male antenna in crude extract of virgin females. These compounds were identified as (9Z)-9-tetradecenyl acetate (Z9-14:Ac) and (9Z,12E)-9,12-tetradecadienyl acetate (Z9E12-14:Ac) in ca. 90:10 ratio by subsequent GC-MS analyses. (11Z)-11-Hexadecenyl acetate (Z11-16:Ac), which had previously been identified as a third component in the Kenyan population, was not detected. Binary blends of Z9-14:Ac and Z9E12-14:Ac at ratios between 99:1 and 90:10 showed a potent attractiveness in the field, superior to that of virgin females and comparable to that of the three-component formulation determined in Kenya. For the population survey, a 98:2 blend was used. In Tarama, only a few moths of S. exempta were captured with a light trap during the night when more than 600 males were captured with synthetic sex pheromone; more S. exempta captures with a light trap had been reported than with sex-pheromone traps in Kenya. This indicates that the Okinawan population has different properties from the Kenyan population in pheromone composition and behavioral response to light.

New Sex Pheromone Blend for the Lightbrown Apple Moth, Epiphyas postvittana

The composition of the sex pheromone gland of the lightbrown apple moth, Epiphyas postvittana (Walker) was re-investigated. In addition to the two previously identified compounds, (E)-11-tetradecenyl acetate (E11-14Ac) and (E,E)-9,11-tetradecadienyl acetate (E9E11-14Ac), seven additional candidate pheromone compounds were identified: (E)-11-tetradecen-1-ol (E11-14OH), tetradecyl acetate, hexadecanal, (E)-11-hexadecenyl acetate (E11-16Ac), hexadecyl acetate, octadecanal, and octadecyl acetate. Gas chromatographic-electroantennographic detection analysis showed that only four (of the nine) compounds (E11-14OH, E11-14Ac, E9E11-14Ac, and E11-16Ac) elicited electrophysiological responses. When either E11-14OH or E11-16Ac were tested at various ratios in three-component blends with both E11-14Ac and E9E11-14Ac, no increase in trap catch was observed compared to that to the previously identified binary blend. However, when these two compounds (1% E11-14OH and 0.5% E11-16Ac) were both added to E11-14Ac and E9E11-14Ac, trap catch was roughly double that to the previously identified binary blend alone. The new four-component blend should be more sensitive for detecting this economically important insect, especially in low population areas (i.e., in newly invaded habitats). In addition, this new blend may enhance pheromone control of this pest, through approaches such as mating disruption, lure and kill, and mass trapping.

Morphological and neurophysiological characterization of olfactory sensory organs in the house cricket

House crickets, Acheta domesticus (L.), (Orthoptera Gryllidae) are known serious pests of wheat, grain, oil seed, and millet. These insects have paired antennae that are endowed with many sensory organs (sensilla) which allow them to acquire information pertaining to smell, taste, and mechanical stimuli about their environment. Each long antenna has many segments (~200) called annuli. Each annulus bears as many as five types of sensilla. There are three types of olfactory sensilla that are used to detect odor molecules. Two types (type-I and type II) resemble short hair-like structures or pegs called basiconic sensilla. Another type resembles a short peg within a pit (coeloconic sensilla). There is one type of mechanosensory sensillum (trichoid sensillum) that resembles a long hair, responsible for detecting mechanical stimulation. One type of gustatory sensillum bears a single terminal pore and is responsible for detecting taste molecules. Results thus far, using electroantennograms, reveal that house crickets respond to complex odorant mixtures in fruits, such as cherry, peach, and banana. Gas chromatography-electroantennographic detection with coupled mass spectrometry techniques (GC-EAD/MS) will be used to determine single component volatiles responsible for these responses.

General Food Semiochemicals Attract Omnivorous German Cockroaches, Blattella germanica

Stale beer and peanut butter are effective baits for the German cockroach (GCRs), Blattella germanica (L.) (Dictyoptera: Blattellidae). In still-air arena olfactometer experiments it was previously shown that headspace volatile extracts of peanut butter and solvent extract of beer attract male GCRs. The objective of this study was to identify the semiochemicals that mediate attraction of GCRs to these sources. Coupled gas chromatographic-electroantennographic detection (GC-EAD) and GC-mass spectrometric (MS) analyses of these attractive extracts, or fractions thereof, and of synthetic standards revealed many candidate semiochemicals. Elaborate olfactometer experiments determined that 1-hexanol from peanut butter, and ethanol and 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP) from beer, are the key semiochemicals of these food sources. 1-Hexanol is a well-known headspace volatile of decomposing lipids, ethanol conveys food fermentation, and DDMP with a caramel-type flavor has been found in several types of food. By responding to these rather general food-derived compounds, the omnivorous GCRs appear to exploit semiochemicals that indicate the presence of various food components, such as lipids and carbohydrates. Synthetic equivalents of these semiochemicals may be formulated as baits or be added to, and thus enhance the attractiveness of, natural food sources as trap or insecticidal baits.

Forty-two compounds in eleven essential oils elicit antennal responses from Aedes aegypti

Essential oils of various plants can be effective at repelling mosquitoes. The repellent properties are often ascribed to their dominant constituents. Our objective was to analyse several essential oils by coupled gas chromatographic-electroantennographic detection (GC-EAD) on the premise that those compounds that are detected by the antennae of the yellow fever mosquito, Aedes aegypti L. (Diptera: Culicidae), are candidate repellents even though they may be minor constituents and thus be overlooked in GC-mass spectrometric analyses of essential oils. In the essential oils of catnip, cinnamon, citronella, cumin, eucalyptus, geranium, ginger, melissa, peppermint, rosemary, and thyme, 42 components induced antennal responses, most commonly β-caryophyllene, linalool, 1,8-cineole, geraniol, and geranial. Some of these 42 components are known insect repellents, indicating that GC-EAD screening of essential oils is a viable analytical technique to detect quantitatively minor constituents, which could be potent repellents when tested at an appropriate dose.

A Deceptive Pollination System Targeting Drosophilids through Olfactory Mimicry of Yeast

In deceptive pollination, insects are bamboozled into performing nonrewarded pollination. A prerequisite for the evolutionary stability in such systems is that the plants manage to generate a perfect sensory impression of a desirable object in the insect nervous system [1]. The study of these plants can provide important insights into sensory preference of their visiting insects. Here, we present the firstdescription of a deceptive pollination system that specifically targets drosophilid flies. We show that the examined plant (Arum palaestinum) accomplishes its deception through olfactory mimicry of fermentation, a strategy that represents a novel pollination syndrome. The lily odor is composed of volatiles characteristic of yeast, and produces in Drosophila melanogaster an antennal detection pattern similar to that elicited by a range of fermentation products. By functional imaging, we show that the lily odors target a specific subset of odorant receptors (ORs), which include the most conserved OR genes in the drosophilid olfactome. Furthermore, seven of eight visiting drosophilid species show a congruent olfactory response pattern to the lily, in spite of comprising species pairs separated by ∼40 million years [2], showing that the lily targets a basal function ofthe fly nose, shared by species with similar ecological preference.

Peripheral modulation of pheromone response by inhibitory host compound in a beetle

We identified several compounds, by gas chromatographic-electroantennographic detection (GC-EAD), that were antennally active in the bark beetle Ips typographus and also abundant in beetle-attacked spruce trees. One of them, 1,8-cineole (Ci), strongly inhibited the attraction to pheromone in the field. Single-sensillum recordings (SSRs) previously showed olfactory receptor neurons (ORNs) on I. typographus antennae selectively responding to Ci. All Ci neurons were found within sensilla co-inhabited by a pheromone neuron responding to cis-verbenol (cV); however, in other sensilla, the cV neuron was paired with a neuron not responding to any test odorant. We hypothesized that the colocalization of ORNs had a functional and ecological relevance. We show by SSR that Ci inhibited spontaneous activity of the cV neuron only in sensilla in which the Ci neuron was also present. Using mixtures of cV and Ci, we further show that responses to low doses (1-10 ng) of cV were significantly reduced when the colocalized Ci neuron simultaneously responded to high doses (1-10 μg) of Ci. This indicated that the response of the Ci neuron, rather than ligand-receptor interactions in the cV neuron, caused the inhibition. Moreover, cV neurons paired with Ci neurons were more sensitive to cV alone than the ones paired with the non-responding ORN. Our observations question the traditional view that ORNs within a sensillum function as independent units. The colocalization of ORNs might sharpen adaptive responses to blends of semiochemicals with different ecological significance in the olfactory landscape.

Organosulphur constituents in garlic oil elicit antennal and behavioural responses from the yellow fever mosquito

Garlic (Allium sativum) and its essential oil have long been used for their distinct flavour, therapeutic effects and as a topical and systemic insect repellent. We tested the hypothesis that the yellow fever mosquito, Aedes aegypti L. (Diptera: Culicidae), responds electrophysiologically and behaviourally to specific components of the steam-distilled essential oil of garlic. In coupled gas chromatographic-electroantennographic detection analyses of garlic oil, antennae of female Ae. aegypti responded to 14 compounds. Seven of them [diallyl disulphide, diallyl trisulphide, diallyl tetrasulphide, 2-(2,3-dithia-5-hexenyl)-3,4-dihydro-2H-thiopyran, 3-(2,3-dithia-5-hexenyl)-3,4-dihydro-2H-thiopyran, 6-methyl-4,5,8,9-tetrathiadodeca-1,11-diene and 4,5,9,10-tetrathiatrideca-1,12-diene] were isolated or synthesized and tested for their ability to repel host-seeking female Ae. aegypti. A solution of diallyl trisulphide and diallyl tetrasulphide applied to a human forearm provided protection from female mosquitoes significantly longer than the paraffin oil control. All compounds had mean protection times significantly shorter than an equivalent dose of the ‘gold standard’N,N-diethyl-3-methylbenzamide. Understanding the common moiety in organosulfur compounds that causes repellence could lead to the design of analogues that are more effective than their natural counterparts in repelling mosquitoes.

2,3-Dihydrohomofarnesal: Female Sex Attractant Pheromone Component of Callosobruchus rhodesianus (Pic)

Callosobruchus rhodesianus (Pic) (Coleoptera: Chrysomelidae: Bruchinae) is a pest of stored legumes through the Afro-tropical region. In laboratory bioassays, males of C. rhodesianus were attracted to volatiles collected from virgin females. Collections were purified by various chromatographic techniques, and the biologically active component isolated using gas chromatographic-electroantennographic detection analysis. Gas chromatography-mass spectrometry and NMR analyses suggested that the active compound was 2,3-dihydrohomofarnesal, i.e., 7-ethyl-3,11-dimethyl-6,10-dodecadienal. The structure was confirmed by non-stereoselective and enantioselective total synthesis. Using chiral gas chromatography, the absolute configuration of the natural compound was confirmed as (3S,6E)-7-ethyl-3,11-dimethyl-6,10-dodecadienal. Y-tube olfactomter assays showed that only the (S)-enantiomer attracted males of C. rhodesianus. The (R)-enantiomer and racemate did not attract males, suggesting that the (R)-enantiomer inhibits the activity of the natural compound. In combination with previous reports about sex attractant pheromones of congeners, we suggest that a saltational shift of the pheromone structure arose within the genus Callosobruchus.

Sex pheromone chemistry and field trapping studies of the elm spanworm Ennomos subsignaria (Hübner) (Lepidoptera:Geometridae)

The elm spanworm, Ennomos subsignaria (Hübner), occurs throughout Canada and the eastern United States and can be a destructive forest pest on a wide range of deciduous trees. Gas chromatography/mass spectrometry (GC/MS) and coupled gas chromatographic-electroantennographic detection (GC/EAD) analysis of pheromone gland extracts, in combination with chemical synthesis and field trapping studies have identified (2S, 3R)-2-((Z)-oct-2'-enyl)-3-nonyl oxirane (hereafter Z6-9S, 10R-epoxy-19:H) as the female-produced sex pheromone. Significantly more male moths were captured between 1-100 microg loadings of this compound on red rubber septa in sticky traps compared to blank (unbaited) traps; catches then declined at higher dosages (500-1000 microg). The other isomeric enantiomer, (2R, 3S)-2-[(Z)-oct-2'-enyl]-3-nonyl oxirane (Z6-9R, 10S-epoxy-19:H), at a 10-microg dosage did not elicit trap capture. The likely precursor to the active epoxide, (Z, Z)-6, 9-nonadecadiene ((Z, Z)-6, 9-19:H), identified in virgin female sex pheromone glands, did not elicit trap capture either, and inhibited trap capture when combined with the active epoxide. Racemic 2-((Z)-oct-2'-enyl)-3-nonyl oxirane showed no significant difference in trap capture compared with Z6-9S, 10R-epoxy-19:H, indicating that the opposite enantiomer was not antagonistic. The addition of the EAD-active diene epoxide enantiomers (2S, 3R)-2-[(Z, Z)-octa-2', 5'-dienyl]-3-nonyl oxirane or (2R, 3S)-2-[(Z, Z)-octa-2', 5'-dienyl]-3-nonyl oxirane in admixture with Z6-9S, 10R-epoxy-19:H (at 10% of the latter) did not enhance or decrease trap capture compared to Z6-9S, 10R-epoxy-19:H oxirane alone, so they are not likely pheromone components. This pheromone, impregnated in rubber septa at less than 100-microg dosage, can now be used as a trap bait to develop detection and monitoring strategies for this insect.

Electrophysiological and Behavioral Responses ofMicroplitis mediator(Hymenoptera: Braconidae) to Caterpillar-Induced Volatiles From Cotton

Microplitis mediator Haliday (Hymenoptera: Braconidae) is an important larval endoparasitoid of various lepidopteran pests, including Helicoverpa armigera (Hübner). In China, H. armigera is a key pest of cotton and is currently the focus of several biological control efforts that use M. mediator as principal natural enemy of this pest. To improve the success of biological control efforts, behavioral studies are needed that shed light on the interaction between M. mediator and H. armigera. In this study, we determined M. mediator response to volatile compounds from undamaged, mechanically injured, or H. armigera--damaged plants and identified attractive volatiles. In Y-tube olfactometer assays, we found that mechanically damaged plants and/or plants treated with H. armigera oral secretions did not attract wasps. However, volatiles from H. armigera-damaged plants elicited a strong attraction of both M. mediator sexes. Headspace extracts from H. armigera-damaged cotton were analyzed by coupled gas chromatography-electroantennographic detection (GC-EAD), and a total of seven different compounds were found to elicit electroantennogram (EAG) responses, including an unknown compound. Six different EAD-active volatiles were identified from caterpillar-damaged cotton plants, of which 3, 7-dimethyl-1, 3, 6-octatriene and (Z)-3-hexenyl acetate were the principal compounds. Olfactometer assays indicated that individual synthetic compounds of 3, 7-dimethyl-1, 3, 6-octatriene, (Z)-3-hexenyl acetate, and nonanal were attractive to M. mediator. Field cage studies showed that parasitism of H. armigera larvae by M. mediator was higher on cotton plants to which 3,7-dimethyl-1,3, 6-octatriene was applied. Our results show that the combination of terpenoids and green leaf volatiles may not only facilitate host, mate, or food location but may also increase H. armigera parasitism by M. mediator.