(<Emphasis Type="Italic">E,E</Emphasis>)-α-Farnesene,an Alarm Pheromone of the Termite <Emphasis Type="Italic">Prorhinotermes canalifrons</Emphasis>

The behavioral and electroantennographic responses of Prorhinotermes canalifrons to its soldier frontal gland secretion, and two separated major components of the secretion, (E)-1-nitropentadec-1-ene and (E,E)-α-farnesene, were studied in laboratory experiments. Behavioral experiments showed that both the frontal gland secretion and (E,E)-α-farnesene triggered alarm reactions in P. canalifrons, whereas (E)-1-nitropentadec-1-ene did not affect the behavior of termite groups. The alarm reactions were characterized by rapid walking of activated termites and efforts to alert and activate other members of the group. Behavioral responses to alarm pheromone differed between homogeneous and mixed groups, suggesting complex interactions. Antennae of both soldiers and pseudergates were sensitive to the frontal gland secretion and to (E,E)-α-farnesene, but soldiers showed stronger responses. The dose responses to (E,E)-α-farnesene were identical for both soldiers and pseudergates, suggesting that both castes use similar receptors to perceive (E,E)-α-farnesene. Our data confirm (E,E)-α-farnesene as an alarm pheromone of P. canalifrons.     

Isolation of a Pyrazine Alarm Pheromone Component from the Fire Ant, <Emphasis Type="Italic">Solenopsis invicta</Emphasis>

Alarm pheromones in social insects are an essential part of a complex of pheromone interactions that contribute to the maintenance of colony integrity and sociality. The alarm pheromones of ants were among the first examples of animal pheromones identified, primarily because of the large amount of chemical produced and the distinctive responses of ants to the pheromone. However, the alarm pheromone of the fire ant, Solenopsis invicta, eluded identification for over four decades. We identified 2-ethyl-3,6-dimethylpyrazine as an alarm pheromone component of S. invicta. Worker fire ants detect the pyrazine alarm pheromone at 30 pg/ml, which is comparable to alarm pheromone sensitivities reported for other ant species. The source of this alarm pheromone are the mandibular glands, which, in fire ants, are not well developed and contain only about 300 pg of the compound, much less than the microgram quantities of alarm pheromones reported for several other ant species. Female and male sexuals and workers produce the pyrazine, which suggests that it may be involved in fire ant mating flight initiation, as well as the typical worker alarm response. This is the first report of 2-ethyl-3,6-dimethylpyrazine from a Solenopsis species and the first example of this alkaloid functioning as an alarm pheromone.     

Isolation of the trail recruitment pheromone ofSolenopsis invicta

TheSolenopsis invicta trail pheromone is synthesized by the Dufour's gland and is released through the sting apparatus. The recruitment subcategory of theS. invicta trail pheromone was shown to be composed of a mixture of the orientation pheromone, (Z,E)--farnesene and an unidentified homosesquiterpene consisting of three rings and one double bond (C-1). C-1 is present in worker Dufour's glands at only 75 pg per worker equivalent. This is the first report that demonstrates that different exocrine products from the same gland control different subcategories of behavior related to mass recruitment.     

Trail Pheromone Disruption of Argentine Ant Trail Formation and Foraging

Trail pheromone disruption of invasive ants is a novel tactic that builds on the development of pheromone-based pest management in other insects. Argentine ant trail pheromone, (Z)-9-hexadecenal, was formulated as a micro-encapsulated sprayable particle and applied against Argentine ant populations in 400 m² field plots in Hawai‘i Volcanoes National Park. A widely dispersed point source strategy for trail pheromone disruption was used. Traffic rates of ants in bioassays of treated filter paper, protected from rainfall and sunlight, indicated the presence of behaviorally significant quantities of pheromone being released from the formulation for up to 59 days. The proportion of plots, under trade wind conditions (2–3 m s⁻¹), with visible trails was reduced for up to 14 days following treatment, and the number of foraging ants at randomly placed tuna-bait cards was similarly reduced. The success of these trail pheromone disruption trials in a natural ecosystem highlights the potential of this method for control of invasive ant species in this and other environments.     

Specificity of laboratory trail following by the argentine ant,Iridomyrmex humilis (Mayr), to (Z)-9-hexadecenal,analogs, and gaster extract

In laboratory trail-following bioassays of Argentine ant workers,Iridomyrmex humilis (Mayr), the geometric isomer, (E)-9-hexadecenal, of the trail pheromone component (Z)-9-hexadecenal elicited insignificant trail following as did the potentially more stable formate analogs, (Z)-7-tetradecenyl formate, (E)-7-tetradecenyl formate, and tetradecyl formate. Further, in direct choice tests, workers showed no preference for gaster extract trails (0.002 ant equiv/cm) over trails of (Z)-9-hexadecenal (0.2 ng/cm). Moreover, a 10-fold increase in synthetic trail concentration to 2.0 ng/cm caused (Z)-9-hexadecenal trails to be significantly preferred over gaster extract trails by trail-following ants.     

Pheromone Disruption of Argentine Ant Trail Integrity

Disruption of Argentine ant trail following and reduced ability to forage (measured by bait location success) was achieved after presentation of an oversupply of trail pheromone, (Z)-9-hexadecenal. Experiments tested single pheromone point sources and dispersion of a formulation in small field plots. Ant walking behavior was recorded and digitized by using video tracking, before and after presentation of trail pheromone. Ants showed changes in three parameters within seconds of treatment: (1) Ants on trails normally showed a unimodal frequency distribution of walking track angles, but this pattern disappeared after presentation of the trail pheromone; (2) ants showed initial high trail integrity on a range of untreated substrates from painted walls to wooden or concrete floors, but this was significantly reduced following presentation of a point source of pheromone; (3) the number of ants in the pheromone-treated area increased over time, as recruitment apparently exceeded departures. To test trail disruption in small outdoor plots, the trail pheromone was formulated with carnuba wax-coated quartz laboratory sand (1 g quartz sand/0.2 g wax/1 mg pheromone). The pheromone formulation, with a half-life of 30 h, was applied by rotary spreader at four rates (0, 2.5, 7.5, and 25 mg pheromone/m²) to 1- and 4-m² plots in Volcanoes National Park, Hawaii. Ant counts at bait cards in treated plots were significantly reduced compared to controls on the day of treatment, and there was a significant reduction in ant foraging for 2 days. These results show that trail pheromone disruption of Argentine ants is possible, but a much more durable formulation is needed before nest-level impacts can be expected. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Argentine Ant Trail Pheromone Disruption is Mediated by Trail Concentration

Argentine ant trail pheromone disruption, using continuous release of the trail pheromone compound (Z)-9-hexadecanal, reduces the incidence of trails and foraging rates of field populations. However, little is known about the concentrations of pheromone required for successful disruption. We hypothesized that higher pheromone quantities would be necessary to disrupt larger ant populations. To test this, we laid a 30-cm long base trail of (Z)-9-hexadecanal on a glass surface at low and high rates (1 and 100 pg/cm) (Trail 1), and laid a second, shorter trail (Trail 2, 10 cm long, located 1.5 cm upwind) near the middle of Trail 1 at six rates (1, 10, 100, 1,000, 10,000, and 100,000 pg/cm). We then recorded and digitized movements of individual ants following Trail 1, and derived a regression statistic, r ², as an index of trail integrity, and also recorded arrival success at the other end of the trail (30 cm) near a food supply. Disruption of trails required 100 fold more pheromone upwind, independent of base-trail concentration. This implies that in the field, trail disruption is likely to be less successful against high ant-trail densities (greater concentration of trail pheromone), and more successful against newly formed or weak trails, as could be expected along invasion fronts.     

The Tea Weevil, <Emphasis Type="Italic">Myllocerinus aurolineatus</Emphasis>, is Attracted to Volatiles Induced by Conspecifics

The tea weevil, Myllocerinus aurolineatus (Voss) (Coleoptera: Curculionidae), is a leaf-feeding pest of Camellia sinensis (O.Ktze.) with aggregative behaviors that can seriously reduce tea yield and quality. Although herbivore-induced host plant volatiles have been shown to attract conspecific individuals of some beetle pests, especially members of the Chrysomelidae family, little is known about the volatiles emitted from tea plants infested by M. aurolineatus adults and their roles in mediating interactions between conspecifics. The results of behavioral bioassays revealed that volatile compounds emitted from tea plants infested by M. aurolineatus were attractive to conspecific weevils. Volatile analyses showed that infestations dramatically increased the emission of volatiles, (Z)-3-hexenal, (Z)-3-hexenol, (E)-β-ocimene, linalool, phenylethyl alcohol, benzyl nitrile, indole, (E, E)-α-farnesene, (E)-nerolidol, and 31 other compounds. Among the induced volatiles, 12 chemicals, including γ-terpinene, benzyl alcohol, (Z)-3-hexenyl acetate, myrcene, benzaldehyde, (Z)-3-hexenal, and (E, E)-α-farnesene, elicited antennal responses from both sexes of the herbivore, whereas (E)-β-ocimene elicited antennal responses only from males. Using a Y-tube olfactometer, we found that six of the 13 chemicals, γ-terpinene, benzyl alcohol, (Z)-3-hexenyl acetate, myrcene, benzaldehyde, and (Z)-3-hexenal, were attractive to both males and females; two chemicals, (E/Z)-β-ocimene and (E, E)-α-farnesene, were attractive only to males; and four chemicals, (E)-4,8-dimethyl-1,3,7-nonatriene, phenylethyl alcohol, linalool, and (Z)-3-hexenol, were attractive only to females. The findings provide new insights into the interactions between tea plants and their herbivores, and may help scientists develop new strategies for controlling the herbivore.     

Electrophysiological and Behavioral Responses of the Multicolored Asian Lady Beetle, <Emphasis Type="Italic">Harmonia axyridis</Emphasis> Pallas,to Sesquiterpene Semiochemicals

The role of two volatile sesquiterpenes, (E)-β-farnesene and (−)-β-caryophyllene, in the chemical ecology of the multicolored Asian lady beetle, Harmonia axyridis Pallas, was investigated by using both electrophysiological and behavioral techniques. (E)-β-Farnesene is the major component of the alarm pheromone of most aphid species, which are preyed on by H. axyridis. (−)-β-Caryophyllene was previously isolated from the headspace volatiles above overwintering and aggregated H. axyridis females. These sesquiterpenes elicited significant electroantennogram (EAG) activity from both H. axyridis male and female antennae. In a four-arm olfactometer, male and female H. axyridis were highly attracted toward (E)-β-farnesene, whereas only males were attracted to (−)-β-caryophyllene. In a bioassay technique that used a passively ventilated plastic box, both male and female H. axyridis aggregated in the (−)-β-caryophyllene-treated side of the box. These results support the potential usefulness of (E)-β-farnesene and (−)-β-caryophyllene in push–pull strategies that use H. axyridis as a biological control agent in aphid-infested sites or to control this new urban pest in residential structures.     

Semiochemicals released by electrically stimulated red imported fire ants,Solenopsis invicta

The red imported fire ant Solenopsis invicta Buren, has evolved sophisticated chemical communication systems that regulate the activities of the colony. Among these are recruitment pheromones that effectively attract and stimulate workers to follow a trail to food or alternative nesting sites. Alarm pheromones alert, activate, and attract workers to intruders or other disturbances. The attraction and accumulation of fire ant workers in electrical equipment may be explained by their release of pheromones that draw additional worker ants into the electrical contacts. We used chemical analysis and behavioral bioassays to investigate if semiochemicals were released by electrically shocked fire ants. Workers were subjected to a 120 V, alternating-current power source. In all cases, electrically stimulated workers released venom alkaloids as revealed by gas chromatography. We also demonstrated the release of alarm pheromones and recruitment pheromones that elicited attraction and orientation. Arrestant behavior was observed with the workers not electrically stimulated but near those that were, indicating release of unkown behavior-modifying substances from the electrically stimulated ants. It appears that fire ants respond to electrical stimulus by generally releasing exocrine gland products. The behaviors associated with these products support the hypothesis that the accumulation of fire ants in electrical equipment is the result of a foraging worker finding and closing electrical contacts, then releasing exocrine gland products that attract other workers to the site, who in turn are electrically stimulated.     

Plant Volatiles Influence Electrophysiological and Behavioral Responses of <Emphasis Type="Italic">Lygus hesperus</Emphasis>

Previous laboratory studies have shown that the mirid Lygus hesperus is attracted to volatiles emitted from alfalfa; feeding damage increases the amounts of several of these volatiles, and visual cues can enhance attraction further. The present study tested single plant volatiles in electrophysiological and behavioral trials with L. hesperus. Electroantennogram (EAG) analyses indicated that antennae responded to most plant volatiles included in the test, and that when gender differences were observed, males usually were more responsive than females. Antennal responses to the alcohols ((E)-3-hexenol, (Z)-3-hexenol, 1-hexanol), the acetate (E)-2-hexenyl acetate, and the aldehyde (E)-2-hexenal were among the strongest. Moderate responses were observed for (E)-β-ocimene, (E,E)-α-farnesene, (±)-linalool, and methyl salicylate. A dose dependent response was not observed for several terpenes (β-myrcene, β-caryophyllene, (+)-limonene, or both (R)-(+)- and (S)-(−)-α-pinenes). EAG responses, however, were not always consistent with behavioral assays. In Y-tube bioassays, males did not exhibit a positive behavioral response to any of the compounds tested. Instead, males were repelled by (E)-2-hexenyl acetate, (±)-linalool, (E,E)-α-farnesene, and methyl salicylate. In contrast, female L. hesperus moved upwind towards (R)-(+)-α-pinene, (E)-β-ocimene, and (E,E)-α-farnesene, and showed a negative response towards (Z)-3-hexen-1-ol, (S)-(−)-α-pinene, and methyl salicylate. This study emphasizes the use of multiple approaches to better understand host plant finding in the generalist herbivore L. hesperus.     

Stereochemistry-dependent inhibition of RAS farnesylation by farnesyl phosphonic acids

This investigation compares the effects of three farnesyl pyrophosphate analogs on selected aspects of isoprenoid metabolism. E,E-α-Hydroxyfarnesylphosphonate was prepared by an improved variation on a literature synthesis, which also gave access to the new Z,E-α-hydroxyfarnesyl- and α-hydroxygeranylphosphonates. A striking find is that only E,E-α-hydroxyfarnesylphosphonate induces alteration of RAS processing in intact human-derived leukemia cells and inhibits farnesyl protein transferase in enzyme assays, while the Z,E-α-farnesyl- and geranylphosphonates are inactive. The inhibitory activity of E,E-α-hydroxyfarnesylphosphonate is greater in enzyme than intact cell assays. This active compound does not significantly inhibit geranylgeranyl protein transferase I or squalene synthase, nor does it diminish cholesterol synthesis. These results indicate that the length of the terpenoid chain and olefin stereochemistry allow selective inhibition of critical enzymes of terpenoid metabolism. Discrimination was observed between inhibition of farnesyl protein transferase and squalene synthase by E,E-α-hydroxyfarnesylphosphonate, even though both enzymes utilize farnesyl pyrophosphate as their natural substrate.     

Biochemical and behavioral evidence foe hybridization between fire ants,Solenopsis invicta andSolenopsis richteri (Hymenoptera: Formicidae)

Behavioral and biochemical evidence is presented for hybridization between the fire ants,Solenopsis richteri andS. invicta. The response of the two species to extracts of their trail pheromones presented as a point source is clearly species-specific; however, hybrid workers responded to parental Dufour's gland extracts and parental workers responded to Dufour's gland extracts of the hybrid. The behavioral evidence for hybridization was confirmed by gas Chromatograph comparison of the Dufour's gland extracts of the three fire ant forms, which showed a pattern for the hybrid that was intermediate to the two parental species.     

Plant Volatiles Enhance Behavioral Responses of Grapevine Moth Males, Lobesia botrana to Sex Pheromone

Plant volatiles play an important role in the lives of phytophagous insects, by guiding them to oviposition, feeding and mating sites. We tested the effects of different host-plant volatiles on attraction of Lobesia botrana males to the female-produced sex pheromone, in a wind tunnel. Addition of volatile emissions from grapevines or individual plant volatiles to pheromone increased the behavioral responses of L. botrana males over those to pheromone alone. At a low release rate (under-dosed) of pheromone, addition of (E)-β-caryophyllene, (Z)-3-hexenyl acetate, 1-hexanol, or 1-octen-3-ol increased all behavioral responses, from activation to pheromone source contact, while addition of (E)-4,8-dimethyl-1,3,7-nonatriene, (E)-β-farnesene, (Z)-3-hexenol, or methyl salicylate affected only the initial behavioral responses. Dose–response experiments suggested an optimal release ratio of 1:1000 (sex pheromone: host plant volatile). Our results highlight the role of plant volatiles in the sensory ecology of L. botrana.     

Volatiles from a Mite-Infested Spruce Clone and Their Effects on Pine Weevil Behavior

Induced responses by Norway spruce (Picea abies) seedlings to feeding damage by two mite species were studied by analyzing the volatiles emitted during infestation. Four specimens of a Norway spruce (Picea abies L.) clone were infested with mites of Nalepella sp., another four with Oligonychus ununguis, and four were kept mite-free as controls. After a year of infestation, spruce volatiles were collected, analyzed, and identified using SPME-GC-MS. In addition, enantiomers of chiral limonene and linalool were separated by two-dimensional GC. Methyl salicylate (MeSA), (-)-linalool, (E)-β-farnesene, and (E,E)-α-farnesene were the main volatiles induced by both species of mites, albeit in different proportions. The ability of the main compounds emitted by the mite-infested spruces to attract or repel the pine weevil, Hylobius abietis (L.), was tested. (E)-β-farnesene was found to be attractive in the absence of spruce odor, whereas methyl salicylate had a deterrent effect in combination with attractive spruce odor. The other tested compounds had no significant effects on the behavior of the weevils.     

Trail pheromone of ponerine ant Gnamptogenys striatula: 4-methylgeranyl esters from Dufour's gland

Dufour's gland is the origin of the trail pheromone of Gnamptogenys striatula. Chemical analysis of the glandular extracts revealed a series of new natural products, especially esters of (2E)-3,4,7-trimethyl-2,6-octadien-1-ol (4-methylgeraniol), and (2E)-3,4,7-trimethyl-2,6-nonadien-1-ol (a bishomogeraniol isomer) with medium-chain fatty acids. Bioassays with synthetic racemates of the esters revealed that the 4-methylgeranyl esters are highly active as trail pheromones, while the bishomogeranyl esters are either marginally active or not active at all. Assays with the individual 4-methylgeranyl esters showed each of them to be inferior to the glandular secretion in eliciting trail following. However, the mixture of racemic 4-methylgeranyl octanoate and the corresponding decanoate and dodecanoate, the main Dufour's volatile constituents, is as active as the natural secretion at similar concentration. We conclude that the trail pheromone constitutes a mixture of at least the 4-methylgeranyl esters identified in the gland. Since G. striatula generally preys on small arthropods rather than monopolizing large resources, we assume that trails are rarely used during foraging, but more often during nest migration. Production of new societies in this species is generally performed by budding, a period of considerable predation risk. Utilizing trails for efficient displacement in this context is, therefore, highly adaptive. This behavioral repertoire may also provide the ants with additional means of food resource exploitation.     

From Terpenoids to Aliphatic Acids: Further Evidence for Late-Instar Switch in Osmeterial Defense as a Characteristic Trait of Swallowtail Butterflies in the Tribe Papilionini

We compared the chemical compositions of the osmeterial secretions of fourth and fifth (last) instars of eight swallowtail species of the tribe Papilionini. Four species (Papilio demoleus, P. polytes, P. paris, and P. macilentus) are Asian Rutaceae-feeding swallowtails. The other four (Chilasa epicydes, C. agestor, P. troilus, and P. glaucus) represent more distant clades within the Papilionini and species with larval hosts in other plant families. We conducted a quantitative analysis for six species, but only qualitative analysis for P. glaucus and C. agestor. In all eight species, regardless of larval host plant, secretions of the fourth instar principally consisted of mono- and sesquiterpene hydrocarbons, whereas those of the fifth instar comprised aliphatic acids and their esters. Consistent with earlier findings, our results suggest that this “heterogeneous” pattern of osmeterial chemistry, not seen in other tribes, may characterize the Papilionini as a whole. Unlike those of most Papilio species, the fourth and fifth instars of Chilasa species resemble each other in body coloration. Thus, the heterogeneous osmeterial pattern is not necessarily associated with color change in papilionid larvae. The major terpenoids identified in fourth instar larval secretions from the six species were α-pinene, sabinene, β-myrcene, limonene, β-phellandrene, (Z)-β-ocimene, (E)-β-ocimene, p-mentha-1,4(8)-diene, β-elemene, β-caryophyllene, (E)-β-farnesene, (3Z,6E)-α-farnesene, (Z)-α-bisabolene, germacrene-A, (E)-α-bisabolene, and germacrene-B. The profiles for individual species differed both qualitatively and quantitatively from one another, and certain species also secreted methyl 3-hydroxy-n-butyrate and oxygenated sesquiterpenes in relatively large proportions. Secretions from fifth instars were composed of varying proportions of isobutyric, 2-methylbutyric, and acetic acids, and methyl and ethyl (minor) esters of both isobutyric and 2-methylbutyric acids. The heterogeneity of osmeterial chemistry in the tribe Papilionini may represent fine-tuning of chemical defense in response to shifting predation pressures as the larvae age and grow.     

(Z,E)-α-farnesene: Major component of secretion from metathoracic scent gland of cotton seed bug,Oxycarenus hyalinipennis (Costa) (Heteroptera; Lygaeidae)

The isolation and identification of (Z,E)--farnesene[(3Z, 6E)-3,7,11-trimethyldodeca-1,3,6,10-tetraene] (III) as the major component of the secretion from the metathoracic scent gland of the cotton seed bug,Oxycarenus hyalinipennis, is reported. The compound was identified by a combination of [1H] NMR, [¹³C] NMR, UV, and mass spectral data and by comparison with a synthetic sample, prepared by dehydration of (E)-nerolidol. (Z,E)--Farnesene (III) has been recorded previously in insects (ants and termites), but this is the first report of its occurrence in a member of the Heteroptera. (E)2-Octenyl acetate (XIII) and 2-octenal (XIV) were identified as minor components of the secretion. In addition, three minor monoterpenoid and two other sesquiterpenoid components were detected and tentatively identified.     

Role of (3<Emphasis Type="Italic">Z</Emphasis>,6<Emphasis Type="Italic">Z</Emphasis>,8<Emphasis Type="Italic">E</Emphasis>)-Dodecatrien-1-ol in Trail Following,Feeding, and Mating Behavior of <Emphasis Type="Italic">Reticulitermes hesperus</Emphasis>

Trail pheromones mediate communication among western subterranean termites, Reticulitermes hesperus Banks. Repetitive passages of ≥28 termites were required to establish a pheromone trail and trails needed to be reinforced because they lasted <48 hr. The minimal threshold concentration for inducing responses from termite workers and secondary reproductives was between 0.01 and 0.1 fg/cm of (3Z,6Z,8E)-dodecatrien-1-ol (henceforth, dodecatrienol). Workers showed optimal trail-following behavior to dodecatrienol at a concentration of 10 fg/cm. Trails with concentrations >10 pg/cm were repellent to workers. Workers did not detect pheromone gradients, responding equally to increasing or decreasing gradients of dodecatrienol, and termite workers were not able to differentiate between different concentrations of dodecatrienol. Termites preferred dodecatrienol trails to 2-phenoxyethanol trails. Antennae played a key role in trail pheromone perception. Dodecatrienol acted as an arrestant for worker termites (10 fg/cm²) and male alates (5 ng/cm²), whereas sternal gland extracts from females attracted male alates. Workers and alates, upon contact with filter paper disks treated with higher doses (10 fg/cm² and 5 ng/cm², respectively) of dodecatrienol, were highly excited (increased antennation and palpation) and repeatedly returned to the treated disks. Dodecatrienol did not act as a phagostimulant when offered on a paper towel disk. Reticulitermes hesperus is highly responsive to dodecatrienol, and it may play an important role in orientation of workers and alates.     

Emission of Volatile Organic Compounds After Herbivory from <Emphasis Type="Italic">Trifolium pratense</Emphasis> (L.) Under Laboratory and Field Conditions

Plants emit a wide range of volatile organic compounds in response to damage by herbivores, and many of the compounds have been shown to attract the natural enemies of insect herbivores or serve for inter- and intra-plant communication. Most studies have focused on volatile emission in the laboratory while little is known about emission patterns in the field. We studied the emission of volatiles by Trifolium pratense (red clover) under both laboratory and field conditions. The emission of 24 compounds was quantified in the laboratory, of which eight showed increased emission rates after herbivory by Spodoptera littoralis caterpillars, including (E)-β-ocimene, the most abundant compound, (Z)-β-ocimene, linalool, (E)-β-caryophyllene, (E,E)-α-farnesene, 4,8-dimethyl-1,3,7-nonatriene (DMNT), 1-octen-3-ol, and methyl salicylate (MeSA). While most of these compounds have been reported as herbivore-induced volatiles from a wide range of plant taxa, 1-octen-3-ol seems to be a characteristic volatile of legumes. In the field, T. pratense plants with varying herbivore damage growing in established grassland communities emitted only 13 detectable compounds, and the correlation between herbivore damage and volatile release was more variable than in the laboratory. For example, the emission of (E)-β-ocimene, (Z)-β-ocimene, and DMNT actually declined with damage, while decanal exhibited increased emission with increasing herbivory. Elevated light and temperature increased the emission of many compounds, but the differences in light and temperature conditions between the laboratory and the field could not account for the differences in emission profiles. Our results indicate that the release of volatiles from T. pratense plants in the field is likely to be influenced by additional biotic and abiotic factors not measured in this study. The elucidation of these factors may be important in understanding the physiological and ecological functions of volatiles in plants.