Coupled Gas Chromatography-electroantennography Research Articles

Characterisation of aphid antixenosis in aphid-resistant ancestor wheat, Triticum monococcum.

Due to the increasing presence of insecticide resistance across cereal aphid populations, new aphid management strategies, including the engineering of host resistance to aphids into commercial wheat varieties, are required. Previous studies have identified ancestor wheat, Triticum monococcum accessions MDR045 and MDR049, with resistance against the grain aphid, Sitobion avenae. To test the hypothesis that resistance can be accounted for by antixenosis (reduced attractiveness of host plants) via the release of repellent volatile organic compounds (VOCs), we explored the response of S. avenae to MDR045 and MDR049 following S. avenae herbivory, using behaviour and electrophysiology experiments. In four-arm olfactometry assays, alate S. avenae showed aphid density-dependent reduced preference to VOC extracts from T. monococcum MDR045 and MDR049. By contrast, alate S. avenae showed aphid density-dependent increased preference to extracts from aphid-susceptible hexaploid wheat, Triticum aestivum var. Solstice and T. monococcum MDR037. Coupled gas chromatography-electroantennography (GC-EAG), using the antennae of alate S. avenae, located 24 electrophysiologically active compounds across all tested accessions. Synthetic blends created from 21 identified EAG-active compounds confirmed bioactivity of corresponding VOC extracts in four-arm olfactometry assays against alate S. avenae. Our data suggest that resistance of T. monococcum MDR045 and MDR049 to S. avenae can be at least partially accounted for by antixenosis through antennal perception of specific repellent VOC blends induced by S. avenae feeding behaviour. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Effects of root inoculation of entomopathogenic fungi on olfactory-mediated behavior and life-history traits of the parasitoid Aphidius ervi (Haliday) (Hymenoptera: Braconidae).

Although most biological control programs use multiple biological agents to manage pest species, to date only a few programs have combined the use of agents from different guilds. Using sweet pepper (Capsicum annuum L.), the entomopathogenic fungus Akanthomyces muscarius ARSEF 5128, the tobacco peach aphid Myzus persicae var. nicotianae and the aphid parasitoid Aphidius ervi as the experimental model, we explored whether root inoculation with an entomopathogenic fungus is compatible with parasitoid wasps for enhanced biocontrol of aphids. In dual-choice behavior experiments, A. ervi was significantly attracted to the odor of M. persicae-infested C. annuum plants that had been inoculated with A. muscarius, compared to noninoculated infested plants. There was no significant difference in attraction to the odor of uninfested plants. Myzus persicae-infested plants inoculated with A. muscarius emitted significantly higher amounts of indole, (E)-nerolidol, (3E,7E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene and one unidentified terpene compared to noninoculated infested plants. Coupled gas chromatography-electroantennography, using the antennae of A. ervi, confirmed the physiological activity of these elevated compounds. Inoculation of plants with A. muscarius did not affect parasitism rate nor parasitoid longevity, but significantly increased the speed of mummy formation in parasitized aphids on fungus-inoculated plants. Our data suggest that root inoculation of C. annuum with A. muscarius ARSEF 5128 alters the olfactory-mediated behavior of parasitoids, but has little effect on parasitism efficiency or life-history parameters. However, increased attraction of parasitoids towards M. persicae-infested plants when inoculated by entomopathogenic fungi can accelerate host localization and hence improve biocontrol efficacy. © 2023 Society of Chemical Industry.

Sex Pheromone of the Alfalfa Plant Bug, Adelphocoris lineolatus: Pheromone Composition and Antagonistic Effect of 1-Hexanol (Hemiptera: Miridae)

The sex pheromone composition of alfalfa plant bugs, Adelphocoris lineolatus (Goeze), from Central Europe was investigated to test the hypothesis that insect species across a wide geographical area can vary in pheromone composition. Potential interactions between the pheromone and a known attractant, (E)-cinnamaldehyde, were also assessed. Coupled gas chromatography-electroantennography (GC-EAG) using male antennae and volatile extracts collected from females, previously shown to attract males in field experiments, revealed the presence of three physiologically active compounds. These were identified by coupled GC/mass spectrometry (GC/MS) and peak enhancement as hexyl butyrate, (E)-2-hexenyl butyrate and (E)-4-oxo-2-hexenal. A ternary blend of these compounds in a 5.4:9.0:1.0 ratio attracted male A. lineolatus in field trials in Hungary. Omission of either (E)-2-hexenyl-butyrate or (E)-4-oxo-2-hexenal from the ternary blend or substitution of (E)-4-oxo-2-hexenal by (E)-2-hexenal resulted in loss of activity. These results indicate that this Central European population is similar in pheromone composition to that previously reported for an East Asian population. Interestingly, another EAG-active compound, 1-hexanol, was also present in female extract. When 1-hexanol was tested in combination with the ternary pheromone blend, male catches were reduced. This compound showed a dose-response effect with small doses showing a strong behavioral effect, suggesting that 1-hexanol may act as a sex pheromone antagonist in A. lineolatus. Furthermore, when (E)-cinnamaldehyde was field tested in combination with the sex pheromone, there was no increase in male catch, but the combination attracted both males and females. Prospects for practical application are discussed.

Identification and application of bacterial volatiles to attract a generalist aphid parasitoid: from laboratory to greenhouse assays.

Recent studies have shown that microorganisms emit volatile compounds that affect insect behaviour. However, it remains largely unclear whether microbes can be exploited as a source of attractants to improve biological control of insect pests. In this study, we used a combination of coupled gas chromatography-electroantennography (GC-EAG) and Y-tube olfactometer bioassays to identify attractive compounds in the volatile extracts of three bacterial strains that are associated with the habitat of the generalist aphid parasitoid Aphidius colemani, and to create mixtures of synthetic compounds to find attractive blends for A. colemani. Subsequently, the most attractive blend was evaluated in two-choice cage experiments under greenhouse conditions. GC-EAG analysis revealed 20 compounds that were linked to behaviourally attractive bacterial strains. A mixture of two EAG-active compounds, styrene and benzaldehyde applied at a respective dose of 1 μg and 10 ng, was more attractive than the single compounds or the culture medium of the bacteria in Y-tube olfactometer bioassays. Application of this synthetic mixture under greenhouse conditions resulted in significant attraction of the parasitoids, and outperformed application of the bacterial culture medium. Compounds isolated from bacterial blends were capable of attracting parasitoids both in laboratory and greenhouse assays, indicating that microbial cultures are an effective source of insect attractants. This opens new opportunities to attract and retain natural enemies of pest species and to enhance biological pest control.

Electrophysiologically and behaviourally active semiochemicals identified from bed bug refuge substrate

Bed bugs are pests of public health importance due to their relentless biting habits that can lead to allergies, secondary infections and mental health issues. When not feeding on human blood bed bugs aggregate in refuges close to human hosts. This aggregation behaviour could be exploited to lure bed bugs into traps for surveillance, treatment efficacy monitoring and mass trapping efforts, if the responsible cues are identified. The aim of this study was to identify and quantify the bed bug aggregation pheromone. Volatile chemicals were collected from bed bug-exposed papers, which are known to induce aggregation behaviour, by air entrainment. This extract was tested for behavioural and electrophysiological activity using a still-air olfactometer and electroantennography, respectively. Coupled gas chromatography-electroantennography (GC-EAG) was used to screen the extract and the GC-EAG-active chemicals, benzaldehyde, hexanal, (E)-2-octenal, octanal, nonanal, decanal, heptanal, (R,S)-1-octen-3-ol, 3-carene, β-phellandrene, (3E,5E)-octadien-2-one, (E)-2-nonenal, 2-decanone, dodecane, nonanoic acid, 2-(2-butoxyethoxy)ethyl acetate, (E)-2-undecanal and (S)-germacrene D, were identified by GC-mass spectrometry and quantified by GC. Synthetic blends, comprising 6, 16, and 18 compounds, at natural ratios, were then tested in the still-air olfactometer to determine behavioural activity. These aggregation chemicals can be manufactured into a lure that could be used to improve bed bug management.

Responses of the putative trachoma vector, Musca sorbens, to volatile semiochemicals from human faeces.

The putative vector of trachoma, Musca sorbens, prefers to lay its eggs on human faeces on the ground. This study sought to determine whether M. sorbens females were attracted to volatile odours from human faeces in preference to odours from the faeces of other animals, and to determine whether specific volatile semiochemicals mediate selection of the faeces. Traps baited with the faeces of humans and local domestic animals were used to catch flies at two trachoma-endemic locations in The Gambia and one in Ethiopia. At all locations, traps baited with faeces caught more female M. sorbens than control traps baited with soil, and human faeces was the most successful bait compared with soil (mean rate ratios 44.40, 61.40, 10.50 [P<0.001]; 8.17 for child faeces [P = 0.004]). Odours from human faeces were sampled by air entrainment, then extracts of the volatiles were tested by coupled gas chromatography-electroantennography with laboratory-reared female M. sorbens. Twelve compounds were electrophysiologically active and tentatively identified by coupled mass spectrometry-gas chromatography, these included cresol, indole, 2-methylpropanoic acid, butanoic acid, pentanoic acid and hexanoic acid. It is possible that some of these volatiles govern the strong attraction of M. sorbens flies to human faeces. If so, a synthetic blend of these chemicals, at the correct ratios, may prove to be a highly attractive lure. This could be used in odour-baited traps for monitoring or control of this species in trachoma-endemic regions.

Common Cerambycid Pheromone Components as Attractants for Longhorn Beetles (Cerambycidae) Breeding in Ephemeral Oak Substrates in Northern Europe

Longhorn beetles are ecologically important insects in forest ecosystems as decomposers of woody substrates, microhabitat engineers, and as components of forest food webs. These species can be greatly affected both positively and negatively by modern forestry management practices, and should be monitored accordingly. Through headspace sampling, coupled gas chromatography-electroantennography, gas chromatography-mass spectrometry, and field bioassays, we identified two compounds, 2-methyl-1-butanol and 3-hydroxy-2-hexanone, that constitute aggregation-sex pheromone attractants of three cerambycid species which breed primarily in different types of fresh, recently dead oak wood in Northern Europe: Pyrrhidium sanguineum (L.), Phymatodes alni ssp. alni (L.), and Phymatodes testaceus (L.) (Cerambycinae: Callidiini). Analyses of headspace volatiles collected from live insects indicated that the male-produced aggregation-sex pheromone of P. sanguineum is a 1–15:100 blend of (R)-2-methyl-1-butanol and (R)-3-hydroxy-2-hexanone, whereas the corresponding ratios for P. alni were 70–110:100. In field bioassays, adult P. sanguineum and P. alni were significantly attracted to multiple blends with varying ratios of the two compounds. When tested individually, the compounds were minimally attractive. In contrast, adult P. testaceus exhibited nonspecific attraction to both of the individual compounds and to different blends, despite the hydroxyketone not being part of its pheromone, which consists of (R)-2-methyl-1-butanol alone. Overall, our results suggest that a blend of 50:100 of racemic 2-methyl-1-butanol and 3-hydroxy-2-hexanone is appropriate for parallel, cost-efficient pheromone-based monitoring of all three species. In particular, these species could serve as useful indicators of how modern forestry practices affect a whole guild of saproxylic insects that require ephemeral deadwood substrates for successful breeding.

Attractiveness of host banana leaf materials to the banana weevil, Cosmopolites sordidus in Ghana for development of field management strategies.

BACKGROUNDThe banana weevil, Cosmopolites sordidus, has been frequently cited as the most challenging constraint to banana and plantain production, particularly in small‐scale (smallholder) farming. For the development of a new, low‐cost weevil management technology based on attractive host plant material, we previously identified (2R,5S)‐theaspirane as the active component of attractive senesced banana leaves. In this new study, we used behavioural (olfactometer) bioassays with adult weevils to compare the attractiveness of four different developmental stages of banana leaves, i.e. unfolding (pale green), matured green (deep green), matured yellowing and senesced, to determine which leaf developmental stage would be most appropriate for use in weevil management. We also investigated the attractiveness of senesced leaf extracts prepared using different solvents to determine which solvent would be most appropriate for local production of leaf extracts. Coupled gas chromatography–electroantennography (GC–EAG) was then used with adult weevils to confirm the presence of (2R,5S)‐theaspirane in attractive leaf extracts.RESULTSOf the leaf materials tested, only the odour of senesced leaf material was significantly attractive to adult weevils (P < 0.005). Furthermore, an extract of senesced material prepared using palm wine alcohol was significantly attractive (P < 0.05). Using coupled GC–EAG with weevil antennae, (2R,5S)‐theaspirane was identified as a minor component with strong EAG activity within the palm wine alcohol extract.CONCLUSIONThe results suggest that palm wine alcohol extracts of senesced banana leaf material could be used to lure adult C. sordidus to traps in the field, as part of an ethnobotanical‐based approach for C. sordidus management on smallholder farms. © 2018 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Isolation and identification of floral attractants from a nectar plant for the dried bean beetle, Acanthoscelides obtectus (Coleoptera: Chrysomelidae, Bruchinae).

The response of virgin females of the legume pest Acanthoscelides obtectus (Coleoptera: Bruchidae) to headspace extracts of volatiles collected from flowers of a nectar plant, Daucus carota, was investigated using behaviour (four-arm olfactometry) and coupled gas chromatography-electroantennography (GC-EAG). Odours from inflorescences were significantly more attractive to virgin female beetles than clean air. Similarly, a sample of volatile organic compounds (VOCs) collected by air entrainment (dynamic headspace collection) was more attractive to beetles than a solvent control. In coupled GC-EAG experiments with beetle antennae and the VOC extract, six components showed EAG activity. Using coupled GC-mass spectrometry (GC-MS) and GC peak enhancement with authentic standards, the components were identified as α-pinene (S:R 16:1), sabinene, myrcene, limonene (S:R 1:3), terpinolene and (S)-bornyl acetate. Females preferred the synthetic blend of D. carota EAG-active volatiles to the solvent control in bioassays. When compared directly, odours of D. carota inflorescences elicited stronger positive behaviour than the synthetic blend. This is the first report of behaviourally active volatiles linked to pollen location for A. obtectus, and development of the six-component blend is being pursued, which could underpin the design of semiochemical-based field management approaches against this major pest of stored products. © 2018 Society of Chemical Industry.

Conspecific and Heterogeneric Lacewings Respond to (Z)-4-Tridecene Identified from Chrysopa formosa (Neuroptera: Chrysopidae).

Green lacewings (Chrysopidae) are predators of soft-bodied pest insects and are among the most important biological control agents in crop protection. Chrysopa spp. are of special importance since, unlike most green lacewing species, adults are also predatory. The current study was undertaken in search of Chrysopa formosa compounds with semiochemical activity. Using coupled gas chromatography-electroantennography (GC-EAG), head and thorax extracts of C. formosa elicited EAG responses to a compound subsequently identified by coupled GC/mass spectrometry, microchemistry, chemical synthesis and GC peak enhancement as (Z)-4-tridecene. In field experiments, this compound decreased attraction of adult C. formosa to (1R,4aS,7S,7aR)-nepetalactol and that of Chrysoperla carnea species-complex to a ternary floral lure, with the inhibitory effect found to be dose-dependent. Our results suggest that (Z)-4-tridecene may serve as a general warning signal among multiple green lacewing species. Perspectives for potential practical applications are discussed.

A maize landrace that emits defense volatiles in response toherbivore eggs possesses a strongly inducible terpene synthase gene.

Maize (Zea mays) emits volatile terpenes in response to insect feeding and egg deposition to defend itself against harmful pests. However, maize cultivars differ strongly in their ability to produce the defense signal. To further understand the agroecological role and underlying genetic mechanisms for variation in terpene emission among maize cultivars, we studied the production of an important signaling component (E)‐caryophyllene in a South American maize landrace Braz1006 possessing stemborer Chilo partellus egg inducible defense trait, in comparison with the European maize line Delprim and North American inbred line B73. The (E) ‐caryophyllene production level and transcript abundance of TPS23, terpene synthase responsible for (E) ‐caryophyllene formation, were compared between Braz1006, Delprim, and B73 after mimicked herbivory. Braz1006–TPS23 was heterologously expressed in E. coli, and amino acid sequences were determined. Furthermore, electrophysiological and behavioral responses of a key parasitic wasp Cotesia sesamiae to C. partellus egg‐induced Braz1006 volatiles were determined using coupled gas chromatography electroantennography and olfactometer bioassay studies. After elicitor treatment, Braz1006 released eightfold higher (E)‐caryophyllene than Delprim, whereas no (E)‐caryophyllene was detected in B73. The superior (E)‐caryophyllene production by Braz1006 was positively correlated with high transcript levels of TPS23 in the landrace compared to Delprim. TPS23 alleles from Braz1006 showed dissimilarities at different sequence positions with Delprim and B73 and encodes an active enzyme. Cotesia sesamiae was attracted to egg‐induced volatiles from Braz1006 and synthetic (E)‐caryophyllene. The variation in (E)‐caryophyllene emission between Braz1006 and Delprim is positively correlated with induced levels of TPS23 transcripts. The enhanced TPS23 activity and corresponding (E)‐caryophyllene production by the maize landrace could be attributed to the differences in amino acid sequence with the other maize lines. This study suggested that the same analogous genes could have contrasting expression patterns in different maize genetic backgrounds. The current findings provide valuable insight not only into genetic mechanisms underlying variation in defense signal production but also the prospect of introgressing the novel defense traits into elite maize varieties for effective and ecologically sound protection of crops against damaging insect pests.

Electrophysiological and behavioral responses of female African rice gall midge, Orseolia oryzivora Harris and Gagné, to host plant volatiles.

African rice gall midge, Orseolia oryzivora Harris and Gagné, is a major pest of rice in Africa. Depsite its economic importance, its chemical ecology is not well understood. Here, we assessed behavioral and electrophysiological responses of O. oryzivora to host plant volatiles. In olfactometer bioassays, mated female O. oryzivora were attracted to volatiles emitted from intact rice plants but were repelled by volatiles collected from plants infested by conspecifics. In a choice test, there was a preference for volatiles from uninfested plants over those from infested plants. Coupled gas chromatography-electroantennography analyses of panicle volatiles isolated four electrophysiologically active components: (S)-linalool, 4,8-dimethyl-1,3,7-nonatriene, (E)-caryophyllene, and (R/S)-(E)-nerolidol. A synthetic blend of volatiles at the same concentration and ratio as that from an intact plant was attractive to mated females, whereas a blend based on the ratio of volatiles from an infested plant was repellent. This suggests that O. oryzivora uses olfaction for host plant recognition. The identification of blends of volatiles emitted by plants that can both attract and repel O. oryzivora may aid the development of sustainable control measures.

Responses of the two-spotted oak buprestid, Agrilus biguttatus (Coleoptera: Buprestidae), to host tree volatiles.

BACKGROUND Agrilus bigutattus (Fabricius) is a forest pest of increasing importance in the United Kingdom. The larvae damage weakened native oaks and are thought to contribute to premature tree death. Suspected links with acute oak decline (AOD) are not yet confirmed, but AOD‐predisposed trees appear to become more susceptible to A. biguttatus attack. Thus, management may be necessary for control of this insect. To explore the possibility of monitoring beetle populations by baited traps, the host tree volatiles regulating A. biguttatus–oak interactions were studied.RESULTSBiologically active volatile organic compounds in dynamic headspace extracts of oak foliage and bark were identified initially by coupled gas chromatography–electroantennography (GC‐EAG) and GC–mass spectrometry (GC‐MS), and the structures were confirmed by GC coinjection with authentic compounds. Of two synthetic blends of these compounds comprising the active leaf volatiles, the simpler one containing three components evoked strongly positive behavioural responses in four‐arm olfactometer tests with virgin females and males, although fresh leaf material was more efficient than the blend. The other blend, comprising a five‐component mixture made up of bark volatiles, proved to be as behaviourally active for gravid females as bark tissue.CONCLUSIONSThese initial results on A. biguttatus chemical ecology reveal aspects of the role of attractive tree volatiles in the host‐finding of beetles and underpin the development of semiochemically based surveillance strategies for this forest insect. © 2015 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Identification of Host-Plant Volatiles and Characterization of Two Novel General Odorant-Binding Proteins from the Legume Pod Borer, Maruca vitrata Fabricius (Lepidoptera: Crambidae).

Chemoreception is a key feature in selection of host plant by phytophagous insects, and odorant-binding proteins (OBPs) are involved in chemical communication of both insects and vertebrates. The legume pod borer, Maruca vitrata Fabricius (Lepidoptera: Crambidae) is one of the key pest species of cowpea and widely distributed throughout tropical and subtropical regions, causing up to 80% of yield loss. In this study, we investigated the electrophysiological responses of female M. vitrata to floral volatiles from V. unguiculata. Seventeen electroantennogram-active compounds were identified from floral volatiles of V. unguiculata by coupled gas chromatography-electroantennography (GC-EAD) and gas chromatography-mass spectrometry (GC-MS). Then, we cloned two novel full-length GOBP genes (MvitGOBP1 and MvitGOBP2) from the antennae of M. vitrata using reverse transcription PCR. Protein sequence analysis indicated that they shared high sequence similarity with other Pyralididae insect GOBPs and had the typical six-cysteine signature. Real-time PCR analysis indicated that MvitGOBP1-2 mRNA was highly expressed in the antennae of female adult with several thousands-fold difference compare to other tissue. Next, the recombinant MvitGOBP1-2 was expressed in Escherichia coli and purified using Ni ion affinity chromatography. Fluorescence binding assays demonstrated that MvitGOBP1-2 had different binding affinities with 17 volatile odorant molecules including butanoic acid butyl ester, limonene, 4-ethylpropiophenone, 1H-indol-4-ol, butanoic acid octyl ester and 2-methyl-3-phenylpropanal. In the field trapping experiment, these six floral volatiles could effectively attract female moths and showed significant difference compared with the blank lure. These results suggested that MvitGOBPs and the seventeen floral volatiles are likely to function in the olfactory behavior response of female moths, which may have played crucial roles in the selection of oviposition sites. The six compounds that we have identified from the volatiles of V. unguiculata may provide useful information for exploring efficiency monitoring and integrated pest management strategies of this legume pod borer in the field.

Volatile fragrances associated with flowers mediate host plant alternation of a polyphagous mirid bug.

Apolygus lucorum (Hemiptera: Miridae) is an important insect pest of cotton and fruit trees in China. The adults prefer host plants at the flowering stage, and their populations track flowering plants both spatially and temporally. In this study, we examine whether flower preference of its adults is mediated by plant volatiles, and which volatile compositions play an important role in attracting them. In olfactometer tests with 18 key host species, the adults preferred flowering plants over non-flowering plants of each species. Coupled gas chromatography-electroantennography revealed the presence of seven electrophysiologically active compounds from flowering plants. Although the adults responded to all seven synthetic plant volatiles in electroantennography tests, only four (m-xylene, butyl acrylate, butyl propionate and butyl butyrate) elicited positive behavioral responses in Y-tube olfactometer bioassays. The adults were strongly attracted to these four active volatiles in multi-year laboratory and field trials. Our results suggest that these four fragrant volatiles, which are emitted in greater amounts once plants begin to flower, mediate A. lucorum’s preference to flowering host plants. We proved that the use of commonly occurring plant volatiles to recognize a large range of plant species can facilitate host selection and preference of polyphagous insect herbivore.

Semiochemical modulation of host preference of Callosobruchus maculatus on legume seeds

Host preference of Callosobruchus maculatus (F.) on seeds of three legume cultivars, Ife-brown and black-eyed cowpeas [Vigna unguiculata L. (Walp)], and soybean (Glycine max L.), was investigated. Mated female C. maculatus showed high (90–95%) attraction to the three legume cultivars in Y-tube bioassays. However, the weevils discriminated among the cultivars in four-choice tests and showed greater attraction to Ife-brown cowpea (50%) than to soybean (30%) and black-eyed cowpea (15%). Coupled gas chromatography-electroantennography (GC-EAD) and GC–MS analyses of the headspace volatile organic compounds (VOCs) emitted by the legume seeds identified 2-ethyl hexanol as the principal EAD active component. Emission of 2-ethyl hexanol was two-fold greater in Ife-brown cowpea (∼0.54 μg g−1 seeds) compared with black-eyed cowpea (∼0.23 μg g−1 seeds) and soybean (∼0.21 μg g−1 seeds). Synthetic 2-ethyl hexanol attracted 68% of female C. maculatus at 0.01 μg dose in Y-tube bioassays. These results demonstrated that host preference in C. maculatus is odor-mediated, and identified 2-ethyl hexanol as a potential attractant for C. maculatus.

Novel use of stir bar sorptive extraction (SBSE) as a tool for isolation of oviposition site attractants for gravid Culex quinquefasciatus

Mosquitoes such as Culex quinquefasciatus Say (Diptera: Culicidae) are important vectors of organisms that cause disease in humans. Research into the development of effective standardized odour baits for blood-fed females (oviposition attractants), to enable entomological monitoring of vector populations, is hampered by complex protocols for extraction of physiologically active volatile chemicals from natural breeding site water samples, which have produced inconsistent results. Air entrainment and solvent extraction are technically demanding methods and are impractical for use in resource poor environments where mosquito-borne disease is most prevalent. This study reports the first use of a simple, robust extraction technique, stir bar sorptive extraction (SBSE), to extract behaviourally active small lipophilic molecules (SLMs) present in water samples collected from Cx. quinquefasciatus breeding sites in Tanzania. Extracts from a pit latrine and from a cess pool breeding site attracted more gravid Cx. quinquefasciatus in pair choice bioassays than control extracts, and coupled gas chromatography-electroantennography (GC-EAG) allowed tentative identification of 15 electrophysiologically active chemicals, including the known oviposition attractant, skatole (3-methylindole). Here, we have demonstrated, using simple pair choice bioassays in controlled laboratory conditions, that SBSE is effective for the extraction of behaviourally and electrophysiologically active semiochemicals from mosquito breeding site waters. Further research is required to confirm that SBSE is an appropriate technique for use in field surveys in the search for oviposition cues for Cx. quinquefasciatus.

Identification and biological activity of sex pheromone components from females of the plum moth Illiberis rotundata Jordan (Lepidoptera: Zygaenidae: Procridinae)

The plum moth, Illiberis rotundata Jordan (Lepidoptera: Zygaenidae: Procridinae), is a pest of orchards in Japan and China. Few chemical ecological studies have been directed towards the Zygaenidae and particularly the Procridinae. To investigate the sex pheromone of this species, extracts of pheromone glands from adult female I. rotundata were analyzed by coupled gas chromatography-electroantennography (GC-EAG) and coupled gas chromatography-mass spectrometry (GC-MS). Whilst GC-EAG on male moths showed an active peak, identified as 2-butyl (7Z)-dodecenoate, GC-MS also revealed the presence of the homologue 2-butyl (9Z)-tetradecenoate. Electroantennographic investigations, as well as field tests, strongly suggested the natural compounds to have the (R)-configuration at the stereogenic centre. Field results demonstrate 0.2 mg of a 1:1-mixture of (2R)-butyl (7Z)-dodecenoate and (2R)-butyl (9Z)-tetradecenoate to be a powerful lure that may be used in pest control measures against I. rotundata. The chemical structures of the new pheromone components show the same features as those of other zygaenid species: unsaturated fatty acids esterified with a short chain chiral alcohol. This is the first example of a two-component blend constituting the pheromone of a procridinid species.

Identification of Volatile Compounds Used in Host Location by the Black Bean Aphid, Aphis fabae

Behavioral and electrophysiological responses of winged Aphis fabae to volatiles of faba bean, Vicia faba (var. Sutton dwarf), plants were studied and semiochemicals used in host location were identified. In olfactometer bioassays, aphids spent significantly more time in the region of the olfactometer where V. faba volatiles from an intact plant were present than in control regions with clean air. This response also occurred when an air entrainment sample of a V. faba plant was used as the odor source. Coupled gas chromatography-electroantennography revealed the presence of 16 electrophysiologically active compounds in the air entrainment sample. Fifteen of these were identified as (Z)-3-hexen-1-ol, 1-hexanol, (E)-2-hexenal, benzaldehyde, 6-methyl-5-hepten-2-one, octanal, (Z)-3-hexen-1-yl acetate, (R)-(-)-linalool, methyl salicylate, decanal, undecanal, (E)-caryophyllene, (E)-beta-farnesene, (S)-(-)-germacrene D, and (E,E,)-4,8,12-trimethyl-1,3,7,11-tridecatetraene. An olfactometer response was observed to a 15-component synthetic blend that comprised all identified compounds at the same concentration and ratio as in the natural sample, with the aphids spending significantly more time in the treated regions of the olfactometer where volatiles were present than in the control regions. These data are discussed in the context of insect host location and crop protection.

Electrophysiological and behavioural responses of the pollen beetle, Meligethes aeneus, to volatiles from a non-host plant, lavender, Lavandula angustifolia (Lamiaceae)

A semiochemical based push-pull strategy for control of oilseed rape pests is being developed at Rothamsted Research. This strategy uses insect and plant derived semiochemicals to manipulate pests and their natural enemies. An important element within this strategy is an understanding of the importance of non-host plant cues for pest insects and how such signals could be used to manipulate their behaviour. Previous studies using a range of non-host plants have shown that, for the pollen beetle Meligethes aeneus (Coleoptera: Nitidulidae), the essential oil of lavender, Lavandula angustifolia (Lamiaceae), was the most repellent. The aim of this study was to identify the active components in L. angustifolia oil, and to investigate the behaviour of M. aeneus to these chemicals, to establish the most effective use of repellent stimuli to disrupt colonisation of oilseed rape crops. Coupled gas chromatography-electroantennography (GC-EAG) and gas chromatography-mass spectrometry (GC-MS) resulted in the identification of seven active compounds which were tested for behavioural activity using a 4-way olfactometer. Repellent responses were observed with (±)-linalool and (±)-linalyl acetate. The use of these chemicals within a push-pull pest control strategy is discussed.