Treating hop plants with (Z)-jasmone increases colonization by Phorodon humuli (Hemiptera: Aphididae) spring migrants.

Hop plants were sprayed with (Z)-jasmone, at a rate of 50 g ha(-1), during the spring migration of the damson-hop aphid Phorodon humuli (Schrank) in 2002 and 2003. Numbers of P. humuli spring migrants colonizing hop plants, Humulus lupulus L., 2-6 and 7-11 days after applying this treatment were assessed in both years. During the first five-day period, significantly more spring migrants were found on hop plants treated with (Z)-jasmone, in comparison with control plants, in 2002. By contrast, no significant difference was evident in the second five-day period. Although the migration in 2003 was much lighter than in 2002, greater numbers of migrants were again removed from treated plants. Indeed, more spring migrants were removed from plants sprayed with (Z)-jasmone in this year during both five-day periods (11 and 44%, respectively) compared with the 23% greater numbers removed in the first five-day period in 2002. Therefore, unlike some other species of aphid, where numbers were consistently lower on plots sprayed with (Z)-jasmone, spraying the secondary host of P. humuli with this compound appears to increase colonization by spring migrants.

Electrophysiological responses of the lepidopterous stemborers Chilo partellus and Busseola fusca to volatiles from wild and cultivated host plants.

The stemborers Chilo partellus and Busseola fusca are major pests of subsistence cereal farming in Africa. Volatiles released by two cultivated hosts, sorghum and maize (Sorghum bicolor and Zea mays), and two wild grass hosts, Pennisetum purpureum and Hyparrhenia tamba, were collected by air entrainment. Electrophysiologically active components in these samples were detected by coupled gas chromatography-electroantennography (GC-EAG), and the active peaks identified by gas chromatography-mass spectrometry. A total of 41 compounds were identified from the four plant species, all of which, as well as two unidentified compounds, elicited an electrophysiological response from one or both of the stemborers. The compounds included a number of green leaf volatiles and other aliphatic aldehydes, ketones, and esters, mono- and sesquiterpenoids, and some aromatic compounds. EAG studies with authentic samples, conducted at two discriminating doses for all compounds, and dose-response curves for 14 of the most highly EAG-active compounds, showed significant differences in relative responses between species. The compounds that elicited large responses in both species of moths included linalool, acetophenone, and 4-allylanisole, while a number of compounds such as the aliphatic aldehydes octanal, nonanal, and decanal elicited a large response in B. fusca, but a significantly smaller response in C. partellus. Furthermore, the wild hosts produced higher levels of physiologically active compounds compared with either of the cultivated hosts. These differences are discussed in relation to the differential attraction/oviposition of the two stemborers observed in the field and, particularly for eastern African small-scale farming systems, in the context of using a push-pull strategy for their control.

Diel periodicity in the production of green leaf volatiles by wild and cultivated host plants of stemborer moths, Chilo partellus and Busseola fusca.

The volatile chemicals produced by four poaceous plant species, blue thatching grass, Hyparrhenia tamba, Napier grass, Pennisetum purpureum, sorghum, Sorghum bicolor, and maize, Zea mays, which are host plants for the lepidopterous stemborers, Chilo partellus and Busseola fusca, were collected by air entrainment and analyzed by gas chromatography. The total quantities of volatiles collected hourly, over a 9-hr period, from P. purpureum and H. tamba showed an approximately hundredfold increase in the first hour of the scotophase. Thereafter, the amount decreased rapidly to levels present during photophase. Although onset of scotophase also triggered an increase in quantities of volatiles collected from two cultivars of S. bicolor and two out of three cultivars of Z. mays, these increases were less dramatic than in the two wild grasses, being only up to 10 times as much as in the last hour of photophase. Analysis showed that up to 95% of the increase in volatiles at the onset of the scotophase was due to just four compounds, the green leaf volatiles hexanal, (E)-2-hexenal, (Z)-3-hexen-1-ol, and (Z)-3-hexen-1-yl acetate, with the latter dominating the volatile profile. Volatiles from P. purpureum were also collected at 10-min intervals for 70 min spanning the transition from light to dark. The vast increase in production of the green leaf volatiles in this species occurs in the first 10 min of the scotophase followed by a rapid decline within the next 20 min. The relevance of these results to the control of stemborers in a "push-pull" strategy is discussed.

Variation in the load of the horn fly, Haematobia irritans, in cattle herds is determined by the presence or absence of individual heifers.

The distribution of horn flies, Haematobia irritans (L.) (Diptera: Muscidae), in herds of Danish Holstein-Friesian cattle was investigated in two studies conducted during two field seasons. In the first study, highly significant differences in fly distribution between the most and the least fly-susceptible heifers were observed. In one herd, the mean difference between the most fly-susceptible and the most fly-resistant heifers was 268 Ha. irritans specimens. The highest ratio between upper and lower mean fly number was 64.1:1, whereas the lowest was 3.1:1. In the second year, it was demonstrated that the heifers kept their rank in fly attraction over time. The trial clearly demonstrated that some heifers were attracting flies, whereas others, even in the same herd, only carried a few. In the second study, heifers were moved in and out of herds in an attempt to manipulate fly loads in the herds. In year 1, one herd (herd A) received four fly-resistant heifers from another herd (herd B), resulting in a drop in the mean number of flies, whereas herd B received four fly-susceptible heifers from herd A, resulting in an elevation of the mean number of flies. In year 2, a similar pattern emerged using herds C and D, and when the cattle were later returned to their original herds, the fly loads returned to their original distribution. The data presented here show unequivocally that, for horn flies, there can be considerable differences in fly loads for individual heifers within the Holstein-Friesian breed. Furthermore, the overall fly load within herds can be manipulated, and can be reversed. Thus, the distribution in the number of flies within a herd appears to depend on the number of fly-resistant or fly-susceptible heifers. The possible role of chemical factors emitted by heifers, i.e. volatile semiochemicals, in determining differences in fly loads is discussed, whereby attractants are emitted by fly-susceptible heifers and enable flies to locate their host, and repellents are emitted by fly-resistant heifers such that the flies are actively repelled from the herd.

The role of volatile semiochemicals in mediating host location and selection by nuisance and disease-transmitting cattle flies.

The role of volatile semiochemicals in mediating the location and selection within herds of Holstein-Friesian heifers by nuisance and disease-transmitting cattle flies was investigated using coupled gas chromatography-electrophysiology (GC-EAG), coupled gas chromatography-mass spectrometry (GC-MS), electrophysiology (EAG), laboratory behaviour and field studies. Using volatile extracts collected by air entrainment from heifers in the Netherlands, a number of active peaks were located by coupled GC-EAG for Musca autumnalis (de Geer) (Diptera: Muscidae) and Haematobia irritans (L.) (Diptera: Muscidae). Volatile samples were also collected from two heifers in Denmark shown in previous counting experiments to differ significantly in their fly loads. Coupled GC-EAG using Ha. irritans antennae revealed differences in the EAG response to the samples, with additional EAG activity in the sample collected from the heifer with the lower fly load. To identify more EAG active compounds, volatiles were also collected from 48-h-old urine by air entrainment. In total, 23 compounds were located and identified by coupled GC-EAG and GC-MS. Further electrophysiological testing of these compounds with five fly species [M. autumnalis, Ha. irritans, Hydrotaea irritans (L.) (Diptera: Muscidae), Stomoxys calcitrans (L.) (Diptera: Musicidae) and Wohlfahrtia magnifica (Schiner) (Diptera: Sarcophagidae)] showed that only some of the compounds were physiologically active across the range of flies tested. These included 1-octen-3-ol, 6-methyl-5-hepten-2-one, (Z)-3-hexen-1-ol, naphthalene, and all EAG active compounds identified from urine. Compounds showing significant EAG activity were tested for behavioural activity using a wind-tunnel designed for measuring upwind flight behaviour. At certain concentrations, 1-octen-3-ol, 6-methyl-5-hepten-2-one and 3-octanol increased upwind flight, whereas naphthalene, propyl butanoate and linalool reduced upwind flight. In field studies using small herds of heifers ranked according to their fly load, individual slow-release formulations of 1-octen-3-ol and 6-methyl-5-hepten-2-one, when applied to low and high fly loading heifers, reduced fly loads on these individuals. This study provides evidence for the hypothesis that the natural differential attractiveness within herds of Holstein-Freisian heifers, i.e. a single host species, for cattle flies is partly due to differences in volatile semiochemicals emitted from the host. It is suggested that this phenomenon applies to other vertebrate host species and their associated insect pests.

Volatiles from whitefly-infested plants elicit a host-locating response in the parasitoid, Encarsia formosa.

The blend of volatile compounds emitted by bean plants (Phaseolus vulgaris) infested with greenhouse whitefly (Trialeurodes vaporariorum) has been studied comparatively with undamaged plants and whiteflies themselves. Collection of the volatiles and analysis by gas chromatography revealed more than 20 compounds produced by plants infested with whitefly. Of these, 4 compounds, (Z)-3-hexen-1-ol, 4,8-dimethyl-1,3,7-nonatriene, 3-octanone, and one unidentified compound were emitted at higher levels than from the undamaged control plants. Synthetic (Z)-3-hexen-1-ol, 4,8-dimethyl-1,3,7-nonatriene, or 3-octanone all elicited a significant increase in oriented flight and landing on the source by the parasitoid, Encarsia formosa, in wind tunnel bioassays. Two-component mixtures of the compounds and the three-component mixture all elicited a similar or, in most cases, a better response by the parasitoid, the most effective being a mixture of (Z)-3-hexen-1-ol and 3-octanone. These results demonstrate that E. formosa uses volatiles from the plant-host complex as olfactory cues for host location.

Asymmetrical generalisation between pheromonal and floral odours in appetitive olfactory conditioning of the honey bee (Apis mellifera L.).

The capacity to generalise between similar but not identical olfactory stimuli is crucial for honey bees, allowing them to find rewarding food sources with varying volatile emissions. We studied bees' generalisation behaviour with odours having different biological values: typical floral odours or alarm compounds. Bees' behavioural and peripheral electrophysiological responses were investigated using a combined proboscis extension response conditioning-electroantennogram assay. Bees were conditioned to pure linalool (floral) or to pure isoamyl acetate (alarm) and were tested with different concentrations of both compounds. Electrophysiological responses were not influenced by conditioning, suggesting that the learning of individual compounds does not rely on modulations of peripheral sensitivity. Behaviourally, generalisation responses of bees conditioned to the alarm compound were much higher than those of bees conditioned to the floral odour. We further demonstrated such asymmetrical generalisation between alarm and floral odours by using differential conditioning procedures. Conditioning to alarm compounds (isoamyl acetate or 2-heptanone) consistently induced more generalisation than conditioning to floral compounds (linalool or phenylacetaldehyde). Interestingly, generalisation between the two alarm compounds, which are otherwise chemically different, was extremely high. These results are discussed in relation to the neural representation of compounds with different biological significance for bees.

Response of the ladybird parasitoid Dinocampus coccinellae to toxic alkaloids from the seven-spot ladybird, Coccinella septempunctata.

Electrophysiological and behavioral responses of the ladybird parasitoid Dinocampus coccinellae to volatiles from the seven-spot ladybird, Coccinella septempunctata, were investigated to identify semiochemicals involved in host location. Coupled gas chromatography-electroantennography (GC-EAG) with D. coccinellae located a small peak of prominent activity in an extract of volatiles from adult C. septempunctata. The active compound was identified by coupled GC-mass spectrometry and by comparison with an authentic sample as the free-base alkaloid precoccinelline, which forms part of the toxic defense of this ladybird. Behavioral studies in an olfactometer showed that D. coccinellae was significantly attracted to the volatile extract and also to the alkaloid. Myrrhine, a stereoisomer of precoccinelline found in low amounts in C. septempunctata and in other ladybird species, was shown to be electrophysiologically active and significantly attractive. Perception of ladybird alkaloids by D. coccinellae is a rare example of toxicants acting as aerially transmitted cues for interactions between the third and fourth trophic levels.

New roles for cis-jasmone as an insect semiochemical and in plant defense.

cis-jasmone, or (Z)-jasmone, is well known as a component of plant volatiles, and its release can be induced by damage, for example during insect herbivory. Using the olfactory system of the lettuce aphid to investigate volatiles from plants avoided by this insect, (Z)-jasmone was found to be electrophysiologically active and also to be repellent in laboratory choice tests. In field studies, repellency from traps was demonstrated for the damson-hop aphid, and with cereal aphids numbers were reduced in plots of winter wheat treated with (Z)-jasmone. In contrast, attractant activity was found in laboratory and wind tunnel tests for insects acting antagonistically to aphids, namely the seven-spot ladybird and an aphid parasitoid. When applied in the vapor phase to intact bean plants, (Z)-jasmone induced the production of volatile compounds, including the monoterpene (E)-beta-ocimene, which affect plant defense, for example by stimulating the activity of parasitic insects. These plants were more attractive to the aphid parasitoid in the wind tunnel when tested 48 h after exposure to (Z)-jasmone had ceased. This possible signaling role of (Z)-jasmone is qualitatively different from that of the biosynthetically related methyl jasmonate and gives a long-lasting effect after removal of the stimulus. Differential display was used to compare mRNA populations in bean leaves exposed to the vapor of (Z)-jasmone and methyl jasmonate. One differentially displayed fragment was cloned and shown by Northern blotting to be up-regulated in leaf tissue by (Z)-jasmone. This sequence was identified by homology as being derived from a gene encoding an alpha-tubulin isoform.

Aphids, predators and parasitoids.

A number of studies have demonstrated the role of herbivore-induced release of plant volatiles in mediating foraging behaviour of aphid parasitoids, particularly with the parasitoid Aphidius ervi, its aphid host Acyrthosiphon pisum and the aphid food plant Vicia faba. These studies have shown that feeding by the aphid alters the composition of volatiles released by the plant and that these compounds act as synomones for the foraging parasitoid. Of particular interest is the species-specificity of the herbivore-induced synomones associated with different aphids feeding on V. faba. Aphids employ various pheromones that mediate behaviour, particularly mating and alarm responses. These pheromones play important roles in reproduction and defence against predation and parasitism. Many species of aphids reproduce sexually on their primary hosts during the autumn and the sexual females produce a pheromone that attracts males. The sex pheromones for a number of aphid species have been identified and laboratory and field studies have shown that synthetic material can act as a kairomone in attracting predators and parasitoids. The aphid alarm pheromone is released from the cornicles of aphids when they are attacked by predators or parasitoids. The activity of the main alarm pheromone component, (E)-beta-farnesene, is inhibited by the related sesquiterpene hydrocarbon beta-caryophyllene, which is reported to attract the lacewing Chrysoperla carnea. In addition, electrophysiological studies have shown that the seven-spot ladybird, Coccinella septempunctata, possesses specific olfactory receptors for (E)-beta-farnesene and beta-caryophyllene. Laboratory studies show these compounds to have behavioural activity with C. septempunctata, suggesting that they may be involved in prey location.

Identification of the gland secreting oviposition-deterring pheromone in the cabbage seed weevil, Ceutorhynchus assimilis, and the mechanism of pheromone deposition.

After laying an egg into a pod of Brassica napus, the female cabbage seed weevil, Ceutorhynchus assimilis, brushes the caudal setae of the eighth abdominal tergite (VIII UT) on the host pod as she walks along it, depositing oviposition-deterring pheromone (ODP). The VIII UT is periodically extended and withdrawn, thus repeatedly rubbing against the posterior fold of the seventh urotergite (VII UT) which bears the individual outlets of glandular epidermal cells. In post-diapause, sexually mature, gravid (i.e. oviferous) females (virgin or mated) the cells of this VII UT gland were hypertrophic, showing intense secretory activity. Extracts of VII UT from these individuals elicited strong electrophysiological responses from antennal club gustatory sensilla and deterred oviposition. In pre-diapause (sexually immature) females, the cells of the VII UT gland were neither hypertrophic nor active and an extract of their VII UT elicited no significant electrophysiological or behavioural response. Extract of female rectum was a less potent oviposition deterrent than VII UT extract and elicited an electrophysiological response similar to male rectum extract. An extract of ovarian calyces and ovaries elicited no behavioural response. We conclude that ODP is secreted by the epidermal cells of the VII UT posterior fold.

Effect of conditioning on discrimination of oilseed rape volatiles by the honeybee: use of a combined gas chromatography-proboscis extension behavioural assay.

The coupled gas chromatography-proboscis extension assay technique was used on restrained worker bees to study responses to components of an extract of oilseed rape floral volatiles. Bees were stimulated with the effluent from the gas chromatograph after either paired or unpaired conditioning to the extract, or after a control treatment. Proboscis extension activity was elicited in six areas of the chromatogram. However, the number of bees responding in two of these areas were too low to be considered in the present study. One significant area of activity was associated with the major component, (E,E)-alpha-farnesene, whilst the others were associated with several minor components. Although all three groups of bees, irrespective of the treatments applied, showed some responses to the components eluting from the GC column, only bees subjected to paired conditioning consistently responded when re-tested to the mixture. In addition, paired conditioning increased the responsiveness of individuals in terms of the number of bees responding at least once to the effluent from the gas chromatograph. This work confirmed the occurrence of key compounds in floral volatile mixtures. Possible synergistic/inhibitory effects between components, relating to olfactory experience, are discussed.

Sex pheromone of aphid parasitoidPraon volucre (Hymenoptera, Braconidae).

Mating in the aphid parasitoidPraon volucre is mediated by a female-produced sex pheromone, which laboratory behavioral studies suggest is released from the abdomen. Gas chromatography coupled with electrophysiological and behavioral assays on males showed one area of activity in an air entrainment sample of virgin females. However, levels of pheromone production are very low and this has so far precluded its identification.

Antennal perception of oilseed rape,Brassica napus (Brassicaceae), volatiles by the cabbage seed weevilCeutorhynchus assimilis (Coleoptera, Curculionidae).

The response of theCeutorhynchus assimilis antenna to volatiles in air entrainment-derived extracts of oilseed rape,Brassica napus, was studied using coupled gas chromatography (GC)-electroantennography (EAG) and coupled GC-single cell recording (SCR). By means of these techniques and coupled gas chromatography-mass spectrometry (GC-MS), 25 active compounds were identified, including isoprenoids and compounds derived from fatty acids and amino acids. Some of the latter, the isothiocyanates and goitrin, and probably indole and benzyl cyanide, are catabolites of glucosinolates. The electrophysiological activity of the identified compounds was confirmed by EAG using a physiologically discriminating dose, and by SCR studies. The importance of the combined use of the EAG and SCR techniques was demonstrated, since specific olfactory cells were located for five compounds that did not elicit significant EAG responses. The majority of the olfactory cells from which single cell recordings were obtained showed very high specificity, and in numerous recordings there were consistent pairings of specific cell types.

Winter host component reduces colonization by bird-cherry-oat aphid,Rhopalosiphum padi (L.) (homoptera, aphididae), and other aphids in cereal fields.

Methyl salicylate, a volatile component ofPrunus padus, the winter host ofRhopalosiphum padi, was found to reduce colonization of the summer host by this aphid. The compound was identified by gas chromatographic analysis coupled with recordings from cells in the primary rhinarium on the sixth antennal segment of the aphid. Methyl salicylate eliminated the attractancy of oat leaves to spring migrants in olfactometer tests. In Sweden, this compound significantly decreased colonization of field grown cereals byR. padi and in the U.K., populations ofSitobion avenae andMetopolophium dirhodum were significantly lower on treated plots.

Methyl salicylate and (-)-(1R,5S)-myrtenal are plant-derived repellents for black bean aphid,Aphis fabae Scop. (Homoptera: Aphididae).

Methyl salicylate and (-)-(1R,5S)-myrtenal stimulate specific olfactory cells in the primary rhinaria on the sixth and fifth antennal segments, respectively, of the black bean aphid.Aphis fabae. In behavioral studies employing a linear track olfactometer, both compounds were repellent toA. fabae and also inhibited attraction to volatiles from its host, broad bean (Vicia faba). Methyl salicylate is associated with secondary metabolite-based defense in plants, and the monoterpenoid (-)-(1R,5S)-myrtenal is metabolically related to (-)-(1S,5S)-α-pinene, an abundant component of defensive resins produced by gymnosperms. It is argued that these two compounds are employed byA. fabae as indicators of nutritionally unsuitable or nonhost plants.

Discrimination of oilseed rape volatiles by honey bee: Novel combined gas chromatographic-electrophysiological behavioral assay.

A novel technique for the simultaneous monitoring of electroan-tennogram (EAG) and conditioned proboscis extension (CPE) responses of honey bees to the effluent from a gas chromatograph (GC) was developed to locate biologically active components in blends of plant volatiles and to investigate odor recognition at the peripheral and behavioral levels. A six-component mixture, comprising compounds previously identified as oilseed rape floral volatiles, was used as the stimulus. Standard CPE and EAG recordings were done as a reference. EAG responses were elicited from unconditioned bees by all the components presented either in the coupled or the standard mode. Conditioned bees gave larger EAG responses than unconditioned bees, suggesting that antennal sensitivity is enhanced by conditioning. At the behavioral level, in both the standard and the coupled modes, only conditioned bees showed the proboscis extension response, with the majority of individuals responding to linalool, 2-phenylethanol, and benzyl alcohol.

Spring migration of damson-hop aphid,Phorodon humuli (Homoptera, Aphididae), and summer host plant-derived semiochemicals released on feeding.

Behavioral studies using an olfactometer demonstrated that spring migrants of the damson-hop aphid,Phorodon humuli, respond to semiochemicals released by spring migrants feeding on hop leaves. Samples of the total volatiles released on feeding were analyzed by coupled gas chromatographysingle cell recording techniques and showed the presence of several active components. Three compounds were identified, using coupled gas chromatography-mass spectrometry, as methyl salicylate, (E)-2-hexenal andß-caryophyllene. These three compounds elicited responses from separate olfactory receptors on the antenna. In the olfactometer, both (E)-2-hexenal andß-caryophyllene gave positive responses from spring migrants, and a mixture of the two compounds in the natural ratio was more attractive than (E)-2-hexenal alone. Addition of methyl salicylate eliminated the response to the active binary mixture.

Behavioral and electrophysiological responses of Aphids to host and nonhost plant volatiles.

Alate and apterous virginoparae ofAphis fabae Scop, and alate virginoparae ofBrevicoryne brassicae (L.), walking in a linear track olfactometer, were attracted by odor from leaves of their host plants.A. fabae responded to odor from undamaged but not damaged bean leaves. Gynoparae (autumn migrants) ofA. fabae, however, did not respond to their host plant (spindle,Euonymus europaeus) odor. Odors of certain nonhost plants masked the attractiveness of the host plant leaves, but tansy (Tanacetum vulgare) and summer savory (Satureja hortensis) volatiles repelledB. brassicae andA. fabae, respectively. 3-Butenyl isothiocyanate attractedB. brassicae andLipaphis erysimi (Kalt.), the latter species being more sensitive in both behavioral and electrophysiological studies. Isothiocyanate receptors were found on the antennae ofA. fabae, which was repelled by these compounds, 4-pentenyl isothiocyanate being the most active.