Identification of human-derived volatile chemicals that interfere with attraction of the Scottish biting midge and their potential use as repellents.

The Scottish biting midge, Culicoides impunctatus (Diptera: Ceratopogonidae), is a major pest in Scotland, causing a significant impact to the Scottish tourist and forestry industries. C. impunctatus is a generalist feeder, preferring to feed on large mammals, and is notorious for its attacks on humans. Until now, there was anecdotal evidence for differential attraction of female host-seeking C. impunctatus to individual human hosts, and the mechanism for this phenomenon was unknown. Using extracts of human odor collected by air entrainment, electroantennogram recordings to identify the physiologically active components, followed by behavioral assays, we show, for the first time, the differential attraction of female C. impunctatus to human odors and the chemical basis for this phenomenon. Certain chemicals, found in greater amounts in extracts that cause low attractiveness to midges, elicit a repellent effect in laboratory assays and repellency trials in the field. Differences in the production of these natural human-derived compounds could help to explain differential "attractiveness" between different human hosts. A mixture of two compounds in particular, 6-methyl-5-hepten-2-one and geranylacetone [(E)-6,10-dimethylundeca-5,9-dien-2-one], showed significant repellency (87, 77.4, 74.2, and 31.6% at hours 0, 1, 2, and 3, respectively) in the field and have the potential to be developed as novel repellents.

Dolichodial: a new aphid sex pheromone component?

The sex pheromones of many aphid species from the subfamily Aphididae comprise a mixture of the iridoids (cyclopentanoids) (1R,4aS,7S,7aR)-nepetalactol and (4aS,7S,7aR)-nepetalactone. In this paper, we investigate whether other chemicals, in addition to nepetalactol and nepetalactone, are released from Dysaphis plantaginea (rosy apple aphid) oviparae as part of their sex pheromone. Four compounds present in an air entrainment sample collected from D. plantaginea oviparae feeding on apple (Malus silvestris c.v. Braburn) elicited electrophysiological responses from male D. plantaginea. Active peaks were tentatively identified by gas chromatography (GC) coupled with mass spectrometry, with identification confirmed by peak enhancement with authentic compounds on GC columns of different polarities. The electroantennography-active chemicals were (1R,4aS,7S,7aR)-nepetalactol, (4aS,7S,7aR)-nepetalactone, (1S,2R,3S)-dolichodial, and phenylacetonitrile. (1S,2R,3S)-Dolichodial elicited a behavioral response from male D. plantaginea and naïve-mated female parasitoids, Aphidius ervi. This is the first report of electrophysiological and behavioral responses from any aphid morph to (1S,2R,3S)-dolichodial. Whether or not (1S,2R,3S)-dolichodial is a third component of the aphid sex pheromone is discussed.

Antiectoparasitic activity of the gum resin, gum haggar, from the East African plant, Commiphora holtziana.

The mechanism of ixodid tick (Acari: Ixodidae) repellency by gum haggar, a resin produced by Commiphora holtziana (Burseraceae), was investigated by evaluating activity against the cattle tick, Boophilus microplus. In an arena bioassay, a hexane extract of the resin of C. holtziana exhibited a repellent effect lasting up to 5h. The hydrocarbon fraction of the resin extract was shown to account for the repellent activity, and was analysed by coupled gas chromatography-mass spectrometry (GC-MS). Major sesquiterpene hydrocarbons were tentatively identified as germacrene-D, delta-elemene and beta-bourbonene. The identity and stereochemistry of the former compound was confirmed as the (+)-isomer by peak enhancement using enantioselective GC, whereas the latter 2 compounds, which are most likely degradation products of germacrene-type precursors, were identified through isolation by preparative gas chromatography followed by microprobe-NMR spectroscopy. GC comparison of gum haggar with another resin, C. myrrha, which was inactive in the tick bioassay, showed that the latter contained much lower levels of these hydrocarbons. To assess the suitability of the gum haggar resin as a general acarine repellent, further tests were made on a major acarine pest of European and US animal husbandry systems, the red poultry mite, Dermanyssus gallinae (Acari: Dermanyssidae). Gum haggar extract, and the isolated hydrocarbon fraction, showed strong repellent effects in an olfactometer assay, and again gum myrrh showed no effect. These findings provide a scientific basis for the observed anti-tick properties of gum haggar, and demonstrate the potential for its development as a general acarine repellent for use in animal husbandry systems.

Identification of human-derived volatile chemicals that interfere with attraction of Aedes aegypti mosquitoes.

It is known that human individuals show different levels of attractiveness to mosquitoes. In this study, we investigated the chemical basis for low attractiveness. We recorded behaviors of Aedes aegypti toward the hands of human volunteers and toward the volatile chemicals produced by their bodies. Some individuals, and their corresponding volatiles, elicited low upwind flight, relative attraction, and probing activity. Analyzing the components by gas chromatography coupled to electrophysiological recordings from the antennae of Aedes aegypti, enabled the location of 33 physiologically relevant compounds. The results indicated that higher levels of specific compounds may be responsible for decreased "attractiveness." In behavioral experiments, five of the compounds caused a significant reduction in upwind flight of Aedes aegypti to attractive human hands. Thus, unattractiveness of individuals may result from a repellent, or attractant "masking," mechanism.

Host recognition by the specialist hoverfly Microdon mutabilis, a social parasite of the ant Formica lemani.

The larva of the hoverfly Microdon mutabilis is a specialist social parasite of the ant Formica lemani that is adapted to local groups of F. lemani colonies but mal-adapted to colonies of the same species situated only a few hundred meters away. At a study site in Ireland, F. lemani shares its habitat with four other ant species. All nest under stones, making the oviposition choice by M. mutabilis females crucial to offspring survival. In this study, we tested the hypothesis that, as an extreme specialist, M. mutabilis should respond to cues derived from its host rather than from its microenvironment, a phenomenon that has hitherto only been addressed in the context of herbivorous insects and their parasitoids. In behavioral assays, M. mutabilis females reacted to volatiles from F. lemani colonies by extending their ovipositors, presumably probing for an oviposition substrate. This behavior was not observed toward negative controls or volatiles from colonies of Myrmica scabrinodis, the host ant of the closely related Microdon myrmicae. Coupled gas chromatography-electroantennography (GC-EAG) that used antennal preparations of M. mutabilis located a single physiologically active compound within an extract of heads of F. lemani workers. Coupled GC-mass spectrometry (GC-MS) tentatively identified the compound as a methylated methylsalicylate. GC co-injection of the extract with authentic samples showed that of the four possible isomers (methyl 3-, 4-, 5-, and 6-methylsalicylate), only methyl 6-methylsalicylate co-eluted with the EAG-active peak. Furthermore, the response to methyl 6-methylsalicylate was four times higher than to those of the other isomers. Coupled GC-EAG and GC-MS also revealed physiological responses to two constituents, 3-octanone and 3-octanol, of the M. scabrinodis alarm pheromone. However, the behavioral trials did not reveal any behavior that could be attributed to these compounds. Results are discussed in the context of four phases of host location behavior, and of the characteristics, which volatile cues should provide to be useful for an extreme specialist such as M. mutabilis.

Interactions between host-plant volatiles and the sex pheromones of the bird cherry-oat aphid, Rhopalosiphum padi and the damson-hop aphid, Phorodon humuli.

The bird cherry-oat aphid, Rhopalosiphum padi (L.), and the damson-hop aphid, Phorodon humuli (Schrank), migrate at the same time of year and colonize closely related Prunus spp. as primary hosts, but utilize (1R,4aS,7S,7aR)-nepetalactol and (1RS,4aR,7S,7aS)-nepetalactol, respectively, as sex pheromones. Interactions between these sex pheromones and benzaldehyde and methyl salicylate, plant volatiles common to primary hosts of both species, were investigated to assess whether they confer reproductive isolation between these species. Female autumn migrants (gynoparae) and males of these two species were caught in the field with water traps baited with their respective sex pheromones. Rhopalosiphum padi gynoparae and males also responded positively to benzaldehyde. Release of either benzaldehyde or methyl salicylate with the conspecific sex pheromone increased catches of both species of aphid. However, releasing both plant volatiles with the sex pheromone of R. padi increased catches of gynoparae and males, but reduced those with the sex pheromone of P. humuli. These results support the hypothesis that specific plant volatiles synergize responses of autumn migrating aphids to their sex pheromone. Because these interactions are species-specific, they may be important in allowing males to discriminate between conspecific sexual females (oviparae) and those of other aphid species.

Development of a pheromone trap monitoring system for orange wheat blossom midge, Sitodiplosis mosellana, in the UK.

Field-trapping experiments with synthetic 2,7-nonadiyl dibutyrate, the female-produced sex pheromone of the orange wheat blossom midge, Sitodiplosis mosellana (Géhin), demonstrated that pheromone traps were highly attractive to males and caught very few non-target organisms. Different formulations of pheromone were tested to identify the optimum release rate and dispenser type for use in pheromone traps in the UK. Key findings were that racemic pheromone was as effective as enantiomerically pure (2S,7R)-2,7-nonadiyl dibutyrate, that release rates higher than 0.5 microg day(-1) were not necessary and that the optimal formulation was a 1 mg pheromone loading in a rubber septum. Pheromone traps gave a reliable indication of peak midge emergence, onset of flight and abundance of midges throughout the season. A strong correlation between maximum trap catch and crop infestation levels was obtained.

Aphid alarm pheromone produced by transgenic plants affects aphid and parasitoid behavior.

The alarm pheromone for many species of aphids, which causes dispersion in response to attack by predators or parasitoids, consists of the sesquiterpene (E)-beta-farnesene (Ebetaf). We used high levels of expression in Arabidopsis thaliana plants of an Ebetaf synthase gene cloned from Mentha x piperita to cause emission of pure Ebetaf. These plants elicited potent effects on behavior of the aphid Myzus persicae (alarm and repellent responses) and its parasitoid Diaeretiella rapae (an arrestant response). Here, we report the transformation of a plant to produce an insect pheromone and demonstrate that the resulting emission affects behavioral responses at two trophic levels.

Response of economically important aphids to components of Hemizygia petiolata essential oil.

The essential oil of Hemizygia petiolata Ashby (Lamiaceae) contains high levels (>70%) of the sesquiterpene (E)-beta-farnesene, the alarm pheromone for many economically important aphid species. In order to test the suitability of H. petiolata oil as a source of (E)-beta-farnesene for use in new integrated aphid control strategies, behavioural responses of pest aphid species were studied in laboratory and field experiments. In alarm pheromone assays the peach-potato aphid, Myzus persicae Sulzer, and the pea aphid, Acyrthosiphon pisum (Harr), showed a lower level of response to the oil than expected given the high levels of (E)-beta-farnesene. It was shown that minor components in the oil, (+)-bicyclogermacrene and (-)-germacrene D, caused inhibition of the alarm response for M. persicae and A. pisum respectively. Nevertheless, in olfactometer studies the oil was directly repellent to A. pisum and the grain aphid, Sitobion avenae F. Sitobion avenae was not only repelled by (E)-beta-farnesene but also by (-)-germacrene D. Furthermore, although it was not directly repellent to M. persicae, the oil interfered with its attraction to host plant stimuli. In field plot experiments, numbers of A. pisum were significantly reduced in plots treated with a slow release formulation of the oil, when compared with control plots.

Antennal electrophysiological responses of three parasitic wasps to caterpillar-induced volatiles from maize (Zea mays mays), cotton (Gossypium herbaceum), and cowpea (Vigna unguiculata).

Many parasitic wasps are attracted to volatiles that are released by plants when attacked by potential hosts. The attractiveness of these semiochemicals from damaged plants has been demonstrated in many tritrophic systems, but the physiological mechanisms underlying the insect responses are poorly understood. We recorded the antennal perception by three parasitoids (Cotesia marginiventris, Microplitis rufiventris, and Campoletis sonorensis) to volatiles emitted by maize, cowpea, and cotton plants after attack by the common caterpillar pest Spodoptera littoralis. Gas chromatography-electroantennography (GC-EAG) recordings showed that wasps responded to many, but not all, of the compounds present at the physiologically relevant levels tested. Interestingly, some minor compounds, still unidentified, elicited strong responses from the wasps. These results indicate that wasps are able to detect many odorant compounds released by the plants. It remains to be determined how this information is processed and leads to the specific behavior of the parasitoids.

Insect host location: a volatile situation.

Locating a host plant is crucial for a phytophagous (herbivorous) insect to fulfill its nutritional requirements and to find suitable oviposition sites. Insects can locate their hosts even though the host plants are often hidden among an array of other plants. Plant volatiles play an important role in this host-location process. The recognition of a host plant by these olfactory signals could occur by using either species-specific compounds or specific ratios of ubiquitous compounds. Currently, most studies favor the second scenario, with strong evidence that plant discrimination is due to central processing of olfactory signals by the insect, rather than their initial detection. Furthermore, paired or clustered olfactory receptor neurons might enable fine-scale spatio-temporal resolution of the complex signals encountered when ubiquitous compounds are used.

Electroantennogram responses of the three migratory forms of the damson-hop aphid, Phorodon humuli, to aphid pheromones and plant volatiles.

Electroantennogram (EAG) responses were recorded from alate fundatrigeniae (spring migrants), gynoparae (the winged female form that produces sexual females) and males, the three migratory forms of the damson-hop aphid, Phorodon humuli (Schrank). EAG responses of gynoparae and males showed typical dose response characteristics to (E)-2-hexenal, (-)-R-carvone, hexanenitrile and (1RS,4aR,7S,7aS)-nepetalactol, the sex pheromone of this species. The 34 plant volatiles elicited broadly similar EAG response profiles in the three migratory forms. Green leaf volatiles produced large responses in all forms; however, the relative order of responsiveness varied. EAG responses to isomers of the monoterpene carvone differed between forms, with males being most, and spring migrants least, responsive. The hop-plant volatile and aphid alarm pheromone, (E)-beta-farnesene, evoked similar EAG responses in all forms. By contrast, males were most responsive to the three sex pheromone components, (-)-(4aS,7S,7aR)-nepetalactol, (+)-(4aS,7S,7aR)-nepetalactone and (1RS,4aR,7S,7aS)-nepetalactol. Males were no more responsive to their own sex pheromone, (1RS,4aR,7S,7aS)-nepetalactol, than to the other aphid sex pheromone components tested. Spring migrants and gynoparae also responded to the three sex pheromone components. This study indicates that migratory forms of P. humuli detect a wide range of volatile compounds, and that they are equally well-adapted for the detection of volatiles associated with host and non-host plants and with other species of aphid.

Responses of female orange wheat Blossom midge, Sitodiplosis mosellana, to wheat panicle volatiles.

Air entrainment samples of volatiles from panicles of intact wheat, Triticum aestivum, cultivar 'Lynx' were collected at the ear emergence/early anthesis growth stage. In an olfactometer bioassay, both freshly cut panicles and an air entrainment sample were found to attract female orange wheat blossom midge adults, Sitodiplosis mosellana. Coupled gas chromatography-electroantennography (GC-EAG) analyses of panicle volatiles located six electrophysiologically active components. These were identified by coupled gas chromatography-mass spectrometry and coinjection with authentic standards, on polar and nonpolar GC columns, as acetophenone, (Z)-3-hexenyl acetate, 3-carene, 2-tridecanone, 2-ethyl-1-hexanol, and 1-octen-3-ol. Although none of these was active when presented individually at the levels present in the entrainment sample, acetophenone, (Z)-3-hexenyl acetate, and 3-carene were active in the olfactometer when presented at a higher dose of 100 ng on filter paper. However, the six-component blend and a blend of acetophenone, (Z)-3-hexenyl acetate, and 3-carene, in the same ratio and concentration as in a natural sample, was as attractive to female S. mosellana as the whole air entrainment sample.

Responses of the aphids Phorodon humuli and Rhopalosiphum padi to sex pheromone stereochemistry in the field.

Gynoparous female and male damson-hop aphids, Phorodon humuli (Schrank), were caught in the field by water traps that were releasing the sex pheromone of this species, (1RS,4aR,7S,7aS)-nepetalactol. No behavioral activity was elicited by (4aS,7S,7aR)-nepetalactone, the major sex pheromone component of other aphid species such as Megoura viciae Buckton, even though olfactory cells were found in the secondary rhinaria on the third antennal segment of P. humuli that responded strongly to this compound. Gynoparous female P. humuli in the field responded less strongly to (1R,4aS,7S,7aR)-nepetalactol, the sex pheromone of the bird cherry-oat aphid, Rhopalosiphum padi (L.), than they did to the (4aR,7S,7aS)-nepetalactols, but males responded only to the latter. The (4aR,7S,7aS)-nepetalactone showed no electrophysiological activity so was not used in field trials. Releasing either the (4aS,7S,7aR)-nepetalactone or the (1R,4aS,7S,7aR)-nepetalactol with the (4aR,7S,7aS)-nepetalactols did not inhibit the response of P. humuli gynoparous females and males to the latter. Males of R. padi responded as strongly to the (4aR,7S,7aS)-nepetalactols as they did to (1R,4aS,7S,7aR)-nepetalactol. Males of P. humuli and R. padi responded positively to an increased concentration of the (4aR,7S,7aS)-nepetalactols released from two vials compared with that from a single vial, as did P. humuli (in one of two experiments) and R. padi to the (1RS,4aR,7S,7aS)- and (1R,4aS,7S,7aR)-nepetalactols when released together.

cis-Jasmone treatment induces resistance in wheat plants against the grain aphid, Sitobion avenae (Fabricius) (Homoptera: Aphididae).

cis-Jasmone is a plant volatile known to have roles as an insect semiochemical and in inducing plant defence. It was evaluated in laboratory and field trials for control of cereal aphids. In an olfactometer bioassay cis-jasmone was repellent to alatae of the grain aphid, Sitobion avenae (Fabricius) (Homoptera: Aphididae). Moreover, wheat, Triticum aestivum (L), seedlings sprayed with formulated cis-jasmone 24 h previously were less susceptible to attack by S. avenae than control plants. In field simulator studies, significantly fewer alate S. avenae settled on cis-jasmone-treated plants over a 24-h period. In addition, the intrinsic rate of population increase, r(m), of S. avenae apterae was reduced on cis-jasmone treated seedlings. In a series of small-plot experiments conducted over four years, cis-jasmone applications reduced cereal aphid populations infesting wheat in the field.

Isoflavanones from the allelopathic aqueous root exudate of Desmodium uncinatum.

Three isoflavanones, 5,7,2',4'-tetrahydroxy-6-(3-methylbut-2-enyl)isoflavanone (1), 4",5"-dihydro-5,2',4'-trihydroxy-5"-isopropenylfurano-(2",3";7,6)-isoflavanone (2) and 4",5"-dihydro-2'-methoxy-5,4'-dihydroxy-5"-isopropenylfurano-(2",3";7,6)-isoflavanone (3) and a previously known isoflavone 5,7,4'-trihydroxyisoflavone [genistein (4)] were isolated and characterised spectroscopically from the root exudate of the legume Desmodium uncinatum (Jacq.) DC. We propose the names uncinanone A, B, and C for compounds 1, 2 and 3, respectively. Isolated fractions containing uncinanone B (2) induced germination of seeds from the parasitic weed Striga hermonthica (Del.) Benth. and fractions containing uncinanone C (3) moderately inhibited radical growth, the first example of a newly identified potential allelopathic mechanism to prevent S. hermonthica parasitism.

Role of semiochemicals in mate location by parasitic sea louse, Lepeophtheirus salmonis.

The role of olfaction and diffusible pheromones in mate location behavior of sea lice, Lepeophtheirus salmonis, was assessed with Y-tube behavioral bioassays. The pheromone "emitting" animals were located in a chamber in one arm of a Y-tube arena, with artificial seawater flowing through both arms. Adult male sea lice displayed both activation and directional responses to seawater conditioned with preadult II virgin females, but were only activated by mated adult female conditioned water. Further, when males were given the choice of preadult II virgin females or mated adult females, a significant number of males chose the arm with the preadult II virgin females. Adult males showed activation responses when presented with water conditioned with adult males but were not attracted to them. When presented with adult males, preadult II virgin females showed only directional responses, but not activation responses. Preadult II virgin female conditioned water was extracted using solid-phase extraction (SPE) protocols pioneered for semiochemical isolation. Adult male sea lice showed significant directional responses to the preadult II virgin female SPE extract. Distillation under vacuum was performed on the extract to give a distillate comprising components with a molecular weight range and physical properties comparable to those of compounds utilized as volatile semiochemicals by terrestrial organisms and a residue comprising components with higher molecular weight range comparable to those utilized as involatile semiochemicals. Adult males were found to be both significantly activated and attracted to the distillate, but not to the residue. This research provides evidence that small, lipophilic organic molecules are used by sea lice as sex pheromone signals to locate a member of the opposite sex.

Control of witchweed Striga hermonthica by intercropping with Desmodium spp., and the mechanism defined as allelopathic.

During investigations into the control of insect damage to maize crops in subsistence farming in Kenya, which involved intercropping with repellent plants, the fodder legumes silverleaf (Desmodium uncinatum) and greenleaf (D. intortum) were also found to reduce dramatically the infestation of maize by parasitic witchweeds such as Striga hermonthica. This effect was confirmed by further field testing and shown to be significantly greater than that observed with other legumes, e.g., cowpea, as were the concomitant yield increases. The mechanism was investigated, and although soil shading and addition of nitrogen fertilizer showed some benefits against S. hermonthica infestation, a putative allelopathic mechanism for D. uncinatum was observed. In screenhouse studies, a highly significant reduction in S. hermonthica infestation was obtained when an aqueous solution, eluting from pots in which D. uncinatum plants were growing, was used to irrigate pots of maize planted in soil seeded with high levels of S. hermonthica. Growth of the parasitic weed was almost completely suppressed. whereas extensive infestation occurred with the control eluate. Laboratory investigations into the allelopathic effect of D. uncinatum, using samples of water-soluble chemical components exuded from cleaned roots, demonstrated that this involved a germination stimulant for S. hermonthica and also an inhibitor for haustorial development.

Semiochemical strategies for sea louse control: host location cues.

The development of behavioural bioassays and electrophysiological recording techniques has enabled the role of semiochemicals to be investigated for the first time in Lepeophtheirus salmonis (Krøyer). Adult male sea lice L salmonis were activated by, and attracted to, salmon-conditioned water (SCW) and SCW extract. Non-host fish odours, turbot-conditioned water (TCW) and turbot-conditioned water extract (TCW extract), elicited activation but no attraction in the lice. Solid-phase extraction techniques were developed to extract low molecular weight components of fish odours, and biological activity was shown to be confined to these. Electrophysiological activity from aesthetascs in the first antennae was found and neural responses to odours were recorded from the antennal nerves. Preliminary experiments on individual chemicals (isophorone, 1-octen-3-ol) linked to salmon revealed behavioural activation and electrophysiological responses in adult male L salmonis. Isophorone was shown to be significantly attractive to sea lice when placed in a slow-release system in a perfused tank of seawater in a choice situation. Proof of concept has been established for successfully extracting odour cues from seawater, analysing their biological activity and applying these to slow-release technologies for field trapping of lice. Future work involving linked GC-MS techniques using behaviour, electrophysiological responses, and organolepsis will establish further host location cues specific to Atlantic salmon.

Encoding of host and non-host plant odours by receptor neurones in the eucalyptus woodborer, Phoracantha semipunctata (Coleoptera: Cerambycidae).

In the eucalyptus woodborer, Phoracantha semipunctata, responses from single receptor neurones to host ( Eucalyptus globulus) and non-host ( Pinus pinaster, Olea europeae) plant odours were examined, using gas chromatography linked with electrophysiological recordings. Thirty-two compounds elicited responses that appeared as increased firing rates correlating with the elution of the active components. A subset of neurones classified as type A ( n=17) responded to one compound, a second subset B ( n=15) responded to two compounds and a third subset C ( n=9) responded to three or more compounds. For each neurone of type B or C, the active chemicals were structurally related. Neurones responding to monoterpenes, sesquiterpenes and non-terpenoids, respectively, showed non-overlapping response spectra, whereas neurones within one chemical group showed some overlap. The results suggest that most neurones, which receive information about plant odours, are narrowly rather than broadly tuned, each specialised for the detection of one or two related compounds. Although most neurones responded to compounds released by host and non-host species, a few responded specifically to compounds of only one species. This suggests a chemical "fingerprint" mechanism for discrimination between odour blends of the different plants, where only a few compounds may be specific for a particular species or individual.