Identification of the female sex pheromone of Bastilla arctotaenia (Lepidoptera: Erebidae).

(3Z,6Z,9Z)-3,6,9-henicosatriene was identified as a major component of female sex pheromone of Bastilla arctotaenia (Lepidoptera: Erebidae), a pest of cultivated roses, by gas chromatograph-electroantennographic detector( GC-EAD) and gas chromatograph/mass spectrometry (GC/MS) analyses. The single (3Z,6Z,9Z)-3,6,9-henicosatriene (1.0 mg/lure) successfully attracted B. arctotaenia males in the field.

Non-targeted metabolomics aids in sex pheromone identification: a proof-of-concept study with the triangulate cobweb spider, Steatoda triangulosa.

Targeted metabolomics has been widely used in pheromone research but may miss pheromone components in study organisms that produce pheromones in trace amount and/or lack bio-detectors (e.g., antennae) to readily locate them in complex samples. Here, we used non-targeted metabolomics-together with high-performance liquid chromatography-mass spectrometry (HPLC-MS), gas chromatography-MS, and behavioral bioassays-to unravel the sex pheromone of the triangulate cobweb spider, Steatoda triangulosa. A ternary blend of three contact pheromone components [N-4-methylvaleroyl-O-isobutyroyl-L-serine (5), N-3-methylbutyryl-O-isobutyroyl-L-serine (11), and N-3-methylbutyryl-O-butyroyl-L-serine (12)] elicited courtship by S. triangulosa males as effectively as female web extract. Hydrolysis of 5, 11 and 12 at the ester bond gave rise to two mate-attractant pheromone components [butyric acid (7) and isobutyric acid (8)] which attracted S. triangulosa males as effectively as female webs. Pheromone components 11 and 12 are reported in spiders for the first time, and were discovered only through the use of non-targeted metabolomics and GC-MS. All compounds resemble pheromone components previously identified in widow spiders. Our study provides impetus to apply non-targeted metabolomics for pheromone research in a wide range of animal taxa.

Decoding the genetic and chemical basis of sexual attractiveness in parasitic wasps.

Attracting and securing potential mating partners is of fundamental importance for reproduction. Therefore, signaling sexual attractiveness is expected to be tightly coordinated in communication systems synchronizing senders and receivers. Chemical signaling has permeated through all taxa of life as the earliest and most widespread form of communication and is particularly prevalent in insects. However, it has been notoriously difficult to decipher how exactly information related to sexual signaling is encoded in complex chemical profiles. Similarly, our knowledge of the genetic basis of sexual signaling is very limited and usually restricted to a few case studies with comparably simple pheromonal communication mechanisms. The present study jointly addresses these two knowledge gaps by characterizing two fatty acid synthase genes that most likely evolved by tandem gene duplication and that simultaneously impact sexual attractiveness and complex chemical surface profiles in parasitic wasps. Gene knockdown in female wasps dramatically reduces their sexual attractiveness coinciding with a drastic decrease in male courtship and copulation behavior. Concordantly, we found a striking shift of methyl-branching patterns in the female surface pheromonal compounds, which we subsequently demonstrate to be the main cause for the greatly reduced male mating response. Intriguingly, this suggests a potential coding mechanism for sexual attractiveness mediated by specific methyl-branching patterns in complex cuticular hydrocarbon (CHC) profiles. So far, the genetic underpinnings of methyl-branched CHCs are not well understood despite their high potential for encoding information. Our study sheds light on how biologically relevant information can be encoded in complex chemical profiles and on the genetic basis of sexual attractiveness.

Attracting a mate is critical in all species that sexually reproduce. Most animals, particularly insects, do this using chemical compounds called pheromones which can be sensed by potential mates. But how these vast range of different compounds encode and convey the information needed to secure a partner is not fully understood, and the genes that drive this complex communication mechanism are largely unknown. To address this knowledge gap, Sun et al. studied the parasitic wasp Nasonia vitripennis. Like other insects, female N. vitripennis contain a wide range of chemical compounds on their cuticle, the outer waxy layer coating their surface. Sun et al. set out to find exactly which of these compounds, known as cuticular hydrocarbons, are involved in sexual communication. They did this by simultaneously inactivating two related genes that they hypothesized to be responsible for synthesizing and maintaining chemical compounds on the cuticle of insects. The genetic modification altered the pattern of chemicals on the surface of the female wasps by specifically up- and down-regulating compounds with similar branching structures. The mutant females were also much less sexually attractive to male wasps. These findings suggest that the chemical pattern identified by Sun et al. is responsible for communicating and maintaining sexual attractiveness in N. vitripennis female wasps. This is a significant stepping stone towards unravelling how sexual attractiveness can be encoded in complex mixtures of pheromones. The results also have important implications for agriculture, as this parasitic wasp species is routinely used to exterminate particular fly populations that cause agricultural damage. The work by Sun et al. provides new insights into how these wasps sexually communicate, which may help scientists improve their rearing conditions and sustain them over multiple generations. This could contribute to a wider application of this more sustainable, eco-friendly alternative to destructive agricultural pesticides.

Identification of a Female-Produced Sex Attractant Pheromone of the Winter Firefly, Photinus corruscus Linnaeus (Coleoptera: Lampyridae).

Firefly flashes are well-known visual signals used by these insects to find, identify, and choose mates. However, many firefly species have lost the ability to produce light as adults. These "unlighted" species generally lack developed adult light organs, are diurnal rather than nocturnal, and are believed to use volatile pheromones acting over a distance to locate mates. While cuticular hydrocarbons, which may function in mate recognition at close range, have been examined for a handful of the over 2000 extant firefly species, no volatile pheromone has ever been identified. In this study, using coupled gas chromatography - electroantennographic detection, we detected a single female-emitted compound that elicited antennal responses from wild-caught male winter fireflies, Photinus corruscus. The compound was identified as (1S)-exo-3-hydroxycamphor (hydroxycamphor). In field trials at two sites across the species' eastern North American range, large numbers of male P. corruscus were attracted to synthesized hydroxycamphor, verifying its function as a volatile sex attractant pheromone. Males spent more time in contact with lures treated with synthesized hydroxycamphor than those treated with solvent only in laboratory two-choice assays. Further, using single sensillum recordings, we characterized a pheromone-sensitive odorant receptor neuron in a specific olfactory sensillum on male P. corruscus antennae and demonstrated its sensitivity to hydroxycamphor. Thus, this study has identified the first volatile pheromone and its corresponding sensory neuron for any firefly species, and provides a tool for monitoring P. corruscus populations for conservation and further inquiry into the chemical and cellular bases for sexual communication among fireflies.

Field Confirmation of (Z)-9-Heptacosene and (3Z,6Z,9Z)-Tricosatriene as Key Sex Pheromone Components of Korean Conogethes punctiferalis Guenée (Lepidoptera: Crambidae).

Recently, insufficient attractiveness of Conogethes punctiferalis Guenée adult males to sex pheromone, -(E)-10-hexadecenal and (Z)-10-hexadecenal, has been reported. To identify the other essential components of sex pheromone, male and female body extracts were analyzed. Two hydrocarbon components, (Z)-9-heptacosene (Z9-27:HC) and (3Z,6Z,9Z)-tricosatriene (Z3,Z6,Z9-23:HC), were identified from only female body extract. There was a significant difference in the electroantennogram (EAG) response of male antennae to Z3,Z6,Z9-23:HC and Z9-27:HC at all test concentrations compared to the response to the hexane control. In field attraction testing, the addition of Z9-27:HC and Z3,Z6,Z9-23:HC to binary aldehyde pheromones significantly increased trap catches of C. punctiferalis male adults. Based on the female and male body extract analysis and field attraction test, Z9-27:HC and Z3,Z6,Z9-23:HC were determined to be other essential sex pheromone components of the Korean C. punctiferalis population. No significant difference was observed in the number of male captures between the bucket trap and delta trap. Pheromone traps with a color close to yellow shade attracted more male adults than traps with a color close to blue shade.

Identification of the Sex Pheromone of the Pink Grass Worm, Tmetolophota atristriga, Reveals Possible Population Differences in Male Response to Sex Pheromone.

The pink grass worm, Tmetolophota atristriga (Walker), is an endemic New Zealand noctuid moth species that is abundant throughout the North and South Islands. The larvae are minor defoliators of agricultural pasture. We investigated the sex pheromone of this species. Analysis of extract of the female sex pheromone gland identified six compounds: two monounsaturated compounds, (Z)-11-hexadecenal (Z11-16:Ald) and (Z)-11-hexadecenyl acetate (Z11-16:Ac), three saturated compounds, hexadecanal (16:Ald), hexadecyl acetate (16:Ac) and octadecan-1-ol (18:OH), and a triene hydrocarbon, (3Z,6Z,9Z)-tricosatriene (Z3Z6Z9-23:Hy). Several field-trapping experiments testing combinations of the six compounds were conducted. Results suggested that males of two different populations of T. atristriga responded differently to different blends of the compounds. Males of one population responded equally to a two-component blend as to other blends, including the one with all six compounds. By contrast, males of the second population responded only to the six-component blend or a ternary blend of Z11-16:Ald, Z11-16:Ac and Z3Z6Z9-23:Hy. In experiments testing different doses of Z11-16:Ald and Z11-16:Ac in a binary or a six-component blend, a 1 mg dose of the binary blend gave the greatest male catch for both populations. This is the second sex pheromone identification of a New Zealand species of Noctuidae and is the first reported occurrence of Z3Z6Z9-23:Hy as a sex pheromone component of any noctuid species.

Identification and Bioassays of Sex-Specific Compounds From a Nuisance Net-Spinning Caddisfly Smicridea fasciatella (Trichoptera: Hydropsychidae).

Municipalities in Arizona and Nevada along the Colorado River are subject to seasonal mass emergences of a nuisance net-spinning caddisfly, Smicridea fasciatella McLachlan (Trichoptera: Hydropsychidae). Here, we describe the characterization and field testing of S. fasciatella extracts to evaluate their potential as lures in baited traps. Solvent extracts of external (i.e., full body-cuticular hydrocarbon, abdominal hexane washes) and internal (i.e., crushed abdomen) parts of adult S. fasciatella were prepared from both sexes, and analyzed by coupled gas chromatography-mass spectrometry (GCMS). Several sex-specific compounds were identified, including (6Z,9Z)-6,9-nonadecadiene and (3Z,6Z,9Z)-3,6,9-nonadecatriene from males, and 2-undecanone, 2-tridecanone, and a heptadecene isomer from females. Extracts from both sexes were also analyzed by coupled gas chromatography-electroantennographic detection (EAD) using antennae of males for detection. Antennae of males weakly responded to 2-undecanone and 2-tridecanone, and to their corresponding alcohols, 2-undecanol and 2-tridecanol, which were included in field tests. Extracts of adult males did not elicit a response from male antennae, suggesting that males do not produce aggregation pheromones attractive to other males. The synchronized, dense populations of lekking males and other possible mating signals (e.g., visual recognition) may have contributed to the minimal attraction seen to test lures deployed in PHEROCON 1C traps. Overall, our results suggest that for this species, attractant pheromones have minimal or no role in bringing the sexes together for mating.

The Sex Pheromone of the Pine Brown-Tail Moth, Euproctis terminalis (Lepidoptera: Erebidae).

The pine brown tail moth, Euproctis terminalis (Walker 1855), is a periodic pest in pine plantations in South Africa. The larvae feed on pine needles and can cause severe defoliation when population densities are high. Population densities fluctuate temporally and spatially, complicating the prediction of potential growth loss and tree mortality. The aim of this study was to identify the sex pheromone of the pine brown tail moth to provide stakeholders with a tool for monitoring it. Gas chromatography-electroantennogram detection and gas chromatography/mass spectrometry analyses of female pheromone gland extracts identified the major component as (Z,Z,Z,Z)-7,13,16,19-docosatetraen-1-ol isobutyrate. Traps baited with (Z,Z,Z,Z)-7,13,16,19-docosatetraen-1-ol isobutyrate caught more males than unbaited traps. A delta trap was shown to be a superior design compared to a bucket funnel trap. This pheromone can now be used for monitoring E. terminalis in pine plantations.

Hero Turned Villain: Identification of Components of the Sex Pheromone of the Tomato Bug, Nesidiocoris tenuis.

Nesidiocoris tenuis (Reuter) (Heteroptera: Miridae) is a tropical mirid bug used as a biocontrol agent in protected crops, including tomatoes. Although N. tenuis predates important insect pests, especially whitefly, it also causes damage by feeding on tomato plants when prey populations decline, resulting in significant economic losses for growers. The pest is now established in some all-year-round tomato crops in Europe and control measures involve the application of pesticides which are incompatible with current IPM programs. As part of future IPM strategies, the pheromone of N. tenuis was investigated. Volatile collections were made from groups and individuals of mated and unmated, females and males. In analyses of these collections by gas chromatography coupled with electroantennographic (EAG) recording from antennae of male bugs, two EAG-active components were detected and identified as 1-octanol and octyl hexanoate. Unlike other mirids, both male and female N. tenuis produced the two compounds, before and after mating, and both sexes gave EAG responses to both compounds. Furthermore, only octyl hexanoate was detected in whole body solvent washes from both sexes. These compounds are not related to the derivatives of 3-hydroxybutyrate esters found as pheromone components in other members of the Bryocrinae sub-family, and the latter could not be detected in volatiles from N. tenuis and did not elicit EAG responses. Nevertheless, experiments carried out in commercial glasshouses showed that traps baited with a blend of the synthetic pheromone components caught essentially only male N. tenuis, and significantly more than traps baited with octyl hexanoate alone. The latter caught significantly more N. tenuis than unbaited traps which generally caught very few bugs. Traps at plant height caught more N. tenuis males than traps 1 m above or at the base of the plants. The trap catches provided an indication of population levels of N. tenuis and were greatly reduced following an application of insecticide.

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.

Detection of heliothine sex pheromone components in the Australian budworm moth, Helicoverpa punctigera: electrophysiology, neuroanatomy, and behavior.

This study examined electrophysiological responses of the Australian budworm moth Helicoverpa punctigera, to heliothine sex pheromone components, via single sensillum recordings (SSR), and examined male neuroanatomy using confocal microscopy and 3D imaging tools. We found that male H. punctigera have three distinct regions of the macroglomerular complex (MGC) in the antennal lobe. Male antennae have only two functional types of sensilla trichoidea (A and C) and type A sensilla contain an olfactory sensory neuron (OSN) that responds to the major sex pheromone component (Z)-11-hexadecenal (Z11-16:Ald) with axons projecting to the cumulus of the macroglomerular complex (MGC) in the antennal lobe. Type C sensilla contained large-spiking receptor neurons which responded primarily to (Z)-9-tetradecenal (Z9-14:Ald) and to a lesser degree to (Z)-11-hexadecenol (Z11-16:OH). These were co-compartmentalized with small-spiking receptor neurons in type C sensilla which responded strongly to Z9-14:Ald and (Z)-9-hexadecenal (Z9-16:Ald), and to a lesser degree to (Z)-11-hexadecenyl acetate (Z11-16:OAc) and Z11-16:OH. Axons from the two co-localized neurons in Type C sensilla projected to the two small MGC units, the dorsomedial anterior and dorsomedial posterior, respectively. In wind tunnel assays, the addition of Z9-16:Ald to an otherwise attractive blend completely shut down male H. punctigera upwind flight.

Analyses of Skin Secretions of Vipera ammodytes (Linnaeus, 1758) (Reptilia: Serpentes), with Focus on the Complex Compounds and Their Possible Role in the Chemical Communication.

Snakes rely heavily on chemical cues when foraging, searching for mates, etc. Snakes' sex attractiveness pheromones comprise mainly heavy, semi-volatile compounds such as ketones. Here we investigated the composition of skin secretions of adult Vipera ammodytes (Linnaeus, 1758) individuals. The samples were analyzed by gas chromatography/mass spectrometry and the identification of the compounds was performed using commercial mass spectral libraries and retention times. The relative concentrations of all detected compounds were tested for significant differences between (1) male vs. female live individuals, (2) shed skin vs. live individuals, and (3) pre-reproductive vs. reproductive live individuals. We detected fifty-nine compounds of which six were ketones. Two ketones (2-pentacosanone and 2-heptacosanone) were present in many of the samples and thus may have an important role in the V. ammodytes chemical communication. We did not find significant differences between the relative concentrations of the compounds between male and female individuals (only three compounds are exceptions). Significant differences were found between extracts from shed skins and live individuals and between live pre-reproductive individuals and live reproductive individuals. The results of the study suggest that chemical communication in V. ammodytes involves less compounds in comparison to the known literature data for other species.

Morphological, chemical and electrophysiological investigations of Telchin licus (Lepidoptera: Castniidae).

The giant sugarcane borer Telchin licus (Drury, 1773) (Lepidoptera: Castniidae) is a day-flying moth pest of sugarcane, pineapples and bananas. To better understand the chemical communication in this species, we examined the morphology of its olfactory system and the chemical composition of its body parts. The ventral surface of the clubbed antennae of T. licus has six morphological types of sensilla: sensilla trichodea, basiconica, chaetica, squamiforma, coeloconica, and auricillica. The telescopic ovipositor shows no evidence of a sexual gland, or female-specific compounds. On the other hand, the midleg basitarsus of males releases (E,Z)-2,13-octadecadienol and (Z,E)-2,13-octadecadienol, which are electroantennographically active in both sexes. These compounds are known female sex pheromones in the Sesiidae family and are male-specific compounds in another castniid moth, although further investigations are necessary to elucidate their ecological role in the Castniidae family.

Attraction and Electrophysiological Response to Identified Rectal Gland Volatiles in Bactrocera frauenfeldi (Schiner).

Bactrocera frauenfeldi (Schiner) (Diptera: Tephritidae) is a polyphagous fruit fly pest species that is endemic to Papua New Guinea and has become established in several Pacific Islands and Australia. Despite its economic importance for many crops and the key role of chemical-mediated sexual communication in the reproductive biology of tephritid fruit flies, as well as the potential application of pheromones as attractants, there have been no studies investigating the identity or activity of rectal gland secretions or emission profiles of this species. The present study (1) identifies the chemical profile of volatile compounds produced in rectal glands and released by B. frauenfeldi, (2) investigates which of the volatile compounds elicit an electroantennographic or electropalpographic response, and (3) investigates the potential function of glandular emissions as mate-attracting sex pheromones. Rectal gland extracts and headspace collections from sexually mature males and females of B. frauenfeldi were analysed by gas chromatography-mass spectrometry. Male rectal glands contained (E,E)-2-ethyl-8-methyl-1,7-dioxaspiro [5.5]undecane as a major component and (E,E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane as a moderate component. Minor components included palmitoleic acid, palmitic acid, and ethyl oleate. In contrast, female rectal glands contained (E,E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane and ethyl laurate as major components, ethyl myristate and ethyl palmitoleate as moderate components, and 18 minor compounds including amides, esters, and spiroacetals. Although fewer compounds were detected from the headspace collections of both males and females than from the gland extractions, most of the abundant chemicals in the rectal gland extracts were also detected in the headspace collections. Gas chromatography coupled electroantennographic detection found responses to (E,E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane from the antennae of both male and female B. frauenfeldi. Responses to (E,E)-2-ethyl-8-methyl-1,7-dioxaspiro[5.5]undecane were elicited from the antennae of females but not males. The two spiroacetals also elicited electropalpographic responses from both male and female B. frauenfeldi. Ethyl caprate and methyl laurate, found in female rectal glands, elicited responses in female antennae and palps, respectively. Y-maze bioassays showed that females were attracted to the volatiles from male rectal glands but males were not. Neither males nor females were attracted to the volatiles from female rectal glands. Our findings suggest (E,E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane and (E,E)-2-ethyl-8-methyl-1,7-dioxaspiro[5.5]undecane as components of a sex-attracting pheromone in B. frauenfeldi.

Identification and Synthesis of Putative Pheromone Components of the Threatened Salt Marsh Bagworm Moth, Whittleia retiella (Lepidoptera: Psychidae).

Whittleia retiella (Newman, 1847) is a threatened salt marsh species of the bagworm moth family Psychidae. For its preservation it is necessary to develop efficient tools to survey its distribution and habitat requirements in order to use appropriate conservation methods. Such tools may be pheromone-based monitoring systems, which have documented efficacy in establishing the occurrence of cryptic insect species in nature. By using gas chromatography combined with electroantennographic detection (GC-EAD), we found two compounds in female W. retiella headspace samples and whole-body extracts that elicited electrophysiological activity in male antennae. Gas chromatograpy coupled with mass spectrometry (GC-MS) operating in electron impact (EI) mode and comparison of the analytical data with those of synthetic reference compounds showed the chemical structures of these putative pheromone components to be (1S)-1-methylpropyl (5Z)-dec-5-enoate and 1-methylethyl (5Z)-dec-5-enoate. Field assays using baits loaded with synthetic compounds revealed that conspecific males were attracted to (1S)-1-methylpropyl (5Z)-dec-5-enoate alone or in combination with 1-methylethyl (5Z)-dec-5-enoate, whereas 1-methylethyl (5Z)-dec-5-enoate neither attracted nor repelled males in the field assays when tested alone. This study shows the potential of using (1S)-1-methylpropyl (5Z)-dec-5-enoate for monitoring W. retiella to gather more detailed information about the geographic distribution and habitat needs of this rare moth.

Effectiveness of the Sex Pheromone for Monitoring Stenoma catenifer (Lepidoptera: Elachistidae) in Avocado Orchards.

The sex pheromone of the avocado moth Stenoma catenifer Walsingham has potential use in the detection of S. catenifer males in the Antillean race avocado in Chiapas, Mexico, although the study of local parameters that influence its effectiveness is required. In this study, trap type, trap height, pheromone release rate and duration of pheromone activity were evaluated to determine the effectiveness of this pheromone in field conditions in the Antillean race avocado crops of the coast of Chiapas, Mexico. The results obtained indicate that the capture of moths with the traps Pherocon III (delta type) and Pherocon 1C (wing type) was higher than that with the boat-type traps. The frequency of capture of S. catenifer males was higher when the traps were placed 4 and 6 m above ground level than that when they were 2 m high. The septum releases 85% of the amount of pheromone during the first 4 weeks, and there were no differences between the number of moth captures during the first 3 weeks, only between the fourth and the first 2 weeks. Our results suggest that the efficiency of the pheromone in Antillean race avocado depends on trap placement height and type as well as on the time of use of the pheromone septum.

Identification of (Z)-8-Heptadecene and n-Pentadecane as Electrophysiologically Active Compounds in Ophrys insectifera and Its Argogorytes Pollinator.

Sexually deceptive orchids typically depend on specific insect species for pollination, which are lured by sex pheromone mimicry. European Ophrys orchids often exploit specific species of wasps or bees with carboxylic acid derivatives. Here, we identify the specific semiochemicals present in O. insectifera, and in females of one of its pollinator species, Argogorytes fargeii. Headspace volatile samples and solvent extracts were analysed by GC-MS and semiochemicals were structurally elucidated by microderivatisation experiments and synthesis. (Z)-8-Heptadecene and n-pentadecane were confirmed as present in both O. insectifera and A. fargeii female extracts, with both compounds being found to be electrophysiologically active to pollinators. The identified semiochemicals were compared with previously identified Ophrys pollinator attractants, such as (Z)-9 and (Z)-12-C27-C29 alkenes in O. sphegodes and (Z)-9-octadecenal, octadecanal, ethyl linoleate and ethyl oleate in O. speculum, to provide further insights into the biosynthesis of semiochemicals in this genus. We propose that all these currently identified Ophrys semiochemicals can be formed biosynthetically from the same activated carboxylic acid precursors, after a sequence of elongation and decarbonylation reactions in O. sphegodes and O. speculum, while in O. insectifera, possibly by decarbonylation without preceding elongation.

Distinct Roles of Cuticular Aldehydes as Pheromonal Cues in Two Cotesia Parasitoids.

Cuticular compounds (CCs) that cover the surface of insects primarily serve as protection against entomopathogens, harmful substances, and desiccation. However, CCs may also have secondary signaling functions. By studying the role of CCs in intraspecific interactions, we may advance our understanding of the evolution of pheromonal communication in insects. We previously found that the gregarious parasitoid, Cotesia glomerata (L.), uses heptanal as a repellent pheromone to help avoid mate competition among sibling males, whereas another cuticular aldehyde, nonanal, is part of the female-produced attractive sex pheromone. Here, we show that the same aldehydes have different pheromonal functions in a related solitary parasitoid, Cotesia marginiventris (Cresson). Heptanal enhances the attractiveness of the female's sex pheromone, whereas nonanal does not affect a female's attractiveness. Hence, these common aldehydes are differentially used by the two Cotesia species to mediate, synergistically, the attractiveness of the main constituents of their respective sex pheromones. The specificity of the complete sex pheromone blend is apparently regulated by two specific, less volatile compounds, which evoke strong electroantennographic (EAG) responses. This is the first demonstration that volatile CCs have evolved distinct pheromonal functions to aid divergent mating strategies in closely related species. We discuss the possibility that additional compounds are involved in attraction and that, like the aldehydes, they are likely oxidative products of unsaturated cuticular hydrocarbons.

Do Helicoverpa armigera Moths Signal Their Fecundity by Emission of an Antagonist?

Female Helicoverpa armigera emit a pheromone, comprised of a 98:2 ratio of (Z)-11-hexadecenal to (Z)-9-hexadecenal, to attract males. It has been proposed that "immature" female H. armigera modulate attraction of males by emitting an antagonist, (Z)-11-hexadecenol, along with pheromone during the first two nights of calling. However, it is unclear why females would call and simultaneously release pheromone and an antagonist. We conducted observations of female calling during the first five nights after adult emergence to determine periodicity. We also measured the relative abundance of (Z)-11-hexadecenol to the major component, (Z)-11-hexadecenal, on the surface of the gland of calling females and compared it to the ratio of these two compounds inside the gland over the first three nights after adult emergence to determine how much antagonist may be released. We found that young females (< 1-d-old) are unlikely to call and, based on the relative proportion of (Z)-11-hexadecenol on the gland surface, even if they did call would be unlikely to release sufficient (Z)-11-hexadecenol to diminish male attraction.

Calling Behavior and Sex Pheromone Release and Storage in the Moth Chloridea virescens.

Female moths release sex pheromone to attract mates. In most species, sex pheromone is produced in, and released from, a specific gland. In a previous study, we used empirical data and compartmental modeling to account for the major pheromone gland processes of female Chloridea virescens: synthesis, storage, catabolism and release; we found that females released little (20-30%) of their pheromone, with most catabolized. The recent publication of a new pheromone collection method led us to reinvestigate pheromone release and catabolism in C. virescens on the basis that our original study might have underestimated release rate (thereby overestimating catabolism) due to methodology and females not calling (releasing) continuously. Further we wished to compare pheromone storage/catabolism between calling and non-calling females. First, we observed calling intermittency of females. Then, using decapitated females, we used the new collection method, along with compartmental modeling, gland sampling and stable isotope labeling, to determine differences in pheromone release, catabolism and storage between (forced) simulated calling and non-calling females. We found, (i) intact 1 d females call intermittently; (ii) pheromone is released at a higher rate than previously determined, with simulations estimating that continuously calling females release ca. 70% of their pheromone (only 30% catabolized); (iii) extension (calling)/retraction of the ovipositor is a highly effective "on/off' mechanism for release; (iv) both calling and non-calling females store most pheromone on or near the gland surface, but calling females catabolize less pheromone; (v) females are capable of producing and releasing pheromone very rapidly. Thus, not only is the moth pheromone gland efficient, in terms of the proportion of pheromone released Vs. catabolized, but it is highly effective at shutting on/off a high flux of pheromone for release.