Comparison of the flower scent of the sexually deceptive orchid <Emphasis Type="Italic">Ophrys iricolor</Emphasis> and the female sex pheromone of its pollinator <Emphasis Type="Italic">Andrena morio</Emphasis>

Summary. Ophrys flowers mimic the female produced sex pheromone of their pollinator species to attract males for pollination. The males try to copulate with the putative female and thereby pollinate the flower. Using electrophysiological and chemical analyses, floral volatiles released by O. iricolor as well as the female sex pheromone of its pollinator species, Andrena morio are investigated. Overall, 38 peaks comprising 41 chemical compounds, were found to release reactions in the antennae of male A. morio bees. Analyses using coupled gas chromatography-mass spectrometry revealed the presence of alkanes and alkenes with 20 to 29 carbon atoms, aldehydes (C9 to C24) and two esters. Almost all of those compounds were found in similar proportions in both, the floral extracts of O. iricolor and cuticle surface extracts of A. morio females. The pattern of biologically active volatiles described here is very similar to that used by other Ophrys species pollinated by Andrena males.     

Behavioural and electrophysiological responses of the olive fruit fly, Bactrocera oleae (Rossi) (Diptera: Tephritidae), to male- and female-borne sex attractants

In the olive fruit fly, Bactrocera oleae, females attract males by producing 1,7-dioxaspiro[5.5]undecane (olean), the main component of the sex pheromone secreted by rectal glands. It has been recently demonstrated that males are able to produce (Z)-9-tricosene (muscalure) in rectal glands, a compound that selectively attracts females. In this study, a male grooming reaction that may transfer the male-borne compounds from rectal to urotergal glands was observed, suggesting that urotergal glands could be involved in B. oleae sexual communication. GC/MS, EAG, GC/EAD analyses and behavioural assays were carried out to compare the role of male rectal and urotergal glands during courtship. In both male glands, olean and muscalure amounts were age dependent. Extracts of rectal glands contained higher amounts of olean and/or muscalure than urotergal ones. Extracts of rectal and urotergal glands of males and females elicited EAG responses in both sexes. GC/EAD showed that female EAG response to male rectal extracts was mainly due to olean and muscalure. Synthetic compounds evoked EAG dose-dependent responses in both sexes, and the EAG response to muscalure was higher as compared to olean. Rectal and urotergal glands from old males were able to attract females, while urotergal glands from young males attracted only males. Overall, our results add knowledge to the mating system of B. oleae, giving first evidences on the electrophysiological activity of muscalure towards both sexes, as well as on the involvement of male urotergal glands in the chemical sexual communication of this pest.     

Identification of sex and parasitism via pheromones by the Ozark zigzag salamander

Chemical communication appears to be the primary mode of information transfer among woodland salamanders of the genus Plethodon. These pheromones have been shown to convey a wide variety of information, and are used to establish territorial boundaries. Responses to pheromones can include changes in both behavior and energy consumption. The ability of Ozark zigzag salamanders, Plethodon angusticlavius, to detect the sex of a conspecific through chemical substrate markings has not been assessed. In addition, some populations of P. angusticlavius are naturally infected with the ectoparasitic mite Hannemania eltoni. Parasitism can impact both the behavior and metabolism of the host as well as the behavior of others towards the parasitized individual. We examined whether sex and parasitism of the pheromone donor and receiver influenced behavioral and metabolic responses in P. angusticlavius. In laboratory trials, behavior of P. angusticlavius was influenced by the sex and parasite load of the pheromone donors and receivers. Behavioral responses were also influenced by whether the receiver was in its own territory or in the territory of the pheromone donor. Metabolic responses were not affected by the sex or parasite load of the pheromone donor, but males had higher metabolic rates than females, even after adjusting for differences in body size. Overall, our study indicates that both sex and parasitism can be detected via pheromones in this species, and the nature of the response depends on residency status, sex, and parasite load of the receiver.     

Mating disruption of the honeybee mite Varroa destructor under laboratory and field conditions

Mating in the mite Varroa destructor, an ectoparasite of the honeybee, takes place within the sealed brood cell of its host and is triggered by a female sex pheromone consisting of three fatty acids and their respective ethyl esters. In a laboratory bioassay, we observed interactions among offspring of a Varroa female at 11 days after host cell capping in the absence and in the presence of the sex pheromone and clearly demonstrated that male mites were not able to distinguish between receptive daughters and either older or immature and unreceptive females when exposed to the pheromone. In addition, mating attempts in the presence of the pheromone with otherwise receptive females were clearly of shorter duration and males often failed to select a receptive female. In order to evaluate the effect of pheromone exposure on successful copulations and the number of spermatozoa transferred under field conditions, we sprayed one of the pheromone components, oleic acid, on an empty brood comb before host egg-laying and Varroa infestation and counted the spermatozoa of daughter mites taken from this comb. We could show that the number of spermatozoa was indeed reduced, and 20 % of females lacked spermatozoa. Our results open up new possibilities and represent a promising step toward biological control of Varroa mites in beehives.     

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

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

Stereoisomeric composition of the sex pheromone of the pine sawfly <Emphasis Type="Italic">Microdiprion pallipes</Emphasis> (Hym., Diprionidae)

Summary. The basic chemical structure of the sex pheromone of the pine sawfly Microdiprion pallipes (Fallén) has earlier been identified as the propionate ester of (2S,3S,7R/S,11R/S)/(2R,3R,7R/S,11R/S)-3,7,11- trimethyl-2-tridecanol. We now report the results from further investigations on the male response to individual stereoisomers and to blends of stereoisomers, both in electroantennographic (EAG) recordings and in field trapping experiments. We also present our attempts to determine the stereochemistry of the compounds present in females of M. pallipes. By comparing gas chromatograms and mass spectra obtained from natural extracts with those from synthetic compounds it was found that the females contain one or more of the four (2S,3S,7R/S,11R/S)-3,7,11-trimethyl-2- tridecanol isomers (SS++-1). The active pheromone component is the corresponding propionate ester 2. In EAG experiments, males responded most strongly to five propionate ester samples, namely two four-isomer blends: SS++-2 and SR++-2, and three individual stereoisomers: SSSR-, SRRR- and SRSR-2. In a series of field trapping experiments it was found that males were attracted to the SR++-2 four-isomer blend and to the individual isomer SSSR-2. Based on the EAG-recordings and field responses of males and the stereoisomers found in the females, we suggest that the propionate ester of (2S,3S,7S,11R)-3,7,11-trimethyl-2- tridecanol (SSSR-2) is used as a main component of the sex pheromone in M. pallipes. Apparently the males react to other stereoisomers in addition to that or those produced by the females.     

Two phylogenetically distinct species of sexually deceptive orchids mimic the sex pheromone of their single common pollinator, the cuckoo bumblebee Bombus vestalis

Sexually deceptive orchids of the genus Ophrys attract male insects for pollination. Pollinator attraction is achieved by mimicking sex pheromones of virgin females of their pollinators, mostly bee species. In earlier investigations, we showed that the phylogenetically distinct Ophrys species O. chestermanii and O. normanii on Sardinia attract their pollinator, males of the cuckoo bumblebee B. vestalis, with the same bouquets of relatively polar volatile compounds. In this investigation, we studied the sex pheromone of virgin females of B. vestalis with the aim of identifying male-attracting compounds and of comparing them with labellum extracts of the two orchids, which were found to release male-attracting compounds in earlier investigations (G?gler et al. 2009). In bioassays, shock-frozen females, cuticle extracts and polar fractions of cuticle extracts of virgin females stimulated mating behaviour in the males. Using gas chromatography coupled with electroantennography (GC-EAD) and gas chromatography coupled with mass spectrometry (GC-MS), we detected in polar fractions of cuticle extracts of B. vestalis females the same electrophysiologically active compounds as in labellum extracts of both orchid species, including aldehydes, esters, fatty acids and alcohols. Since statistical comparisons of the relative proportions of esters showed strong similarities between virgin females and orchids, our results support the hypotheses that this highly specialized Ophrys–pollinator relationship represents another case of chemical mimicry and that esters play a key role in male attraction.     

Identification of the sex pheromone of Conogethes pluto: a pest of Alpinia

In recent years, Conogethes pluto (Lepidoptera: Crambidae) has become a major pest of Alpinia and other ornamental gingers in the Northern Territory and Queensland, Australia. This pest damages the flowers and bores into the stems, causing substantial losses to production. Currently, no synthetic sex pheromone is available to monitor or control this pest. This work aims at the identification of the sex pheromone of this pest. Analysis of the sex pheromone gland of female C. pluto by gas chromatography/electroantennogram detector revealed the presence of seven candidate pheromone compounds that elicited electroantennogram responses. Using gas chromatography/mass spectrometry analysis and micro-derivatization reactions, six compounds were identified as (E)-10-hexadecenal, as the main pheromone compound, (Z)-10-hexadecenal, hexadecanal, (E)-10-hexadecen-1-ol, (10E,12E)-hexadeca-10,12-dienal and (3Z,6Z,9Z)-tricosa-3,6,9-triene as minor pheromone compounds. In two-field trapping experiments, C. pluto responded to the six-component blend, and three of six compounds, i.e., (E)-10-hexadecenal, (3Z,6Z,9Z)-tricosa-3,6,9-triene, and (10E,12E)-hexadeca-10,12-dienal were shown to be necessary for attraction. In a subsequent experiment testing various doses (i.e., 0.01, 0.1, and 1 mg) of the six-component blend, the largest number of males was captured in traps baited with a lure loading of 1 mg. The availability of the sex pheromone of C. pluto will enable monitoring and provides the basis for additional control options for this pest.     

Population differences in male responses to chemical mating cues in the white-spotted longicorn beetle, Anoplophora malasiaca

Adult Anoplophora malasiaca beetles were collected from field populations inhabiting three different host plants: Citrus, Salix and Vaccinium. Male orientation responses towards odours of wounded branches of each host plant were examined. Males of all three populations were attracted most frequently to odours of their original hosts, indicating that male orientation behaviour was induced by the odours of the plants from which they were originally collected. We also observed the mating responses of the males to elytra extracts from young and mature females of the three populations and chemically analysed those extracts. Citrus males were highly responsive to female extracts, regardless of origin. Salix males were less reactive than Citrus males, but responded moderately to female extracts of all populations. Vaccinium males were never attracted to Citrus female extracts and responded more to extracts from mature Vaccinium and Salix females, indicating that males might distinguish female origin and maturity. All eight hydrocarbons, four ketones, and three lactones that were previously identified as female contact sex pheromones were found in the extracts of three populations and both ages. Higher amounts of lactones on mature females likely caused greater male responses. These results suggested that males would be more likely to approach a female feeding on the same host plant and subsequently recognise her sexual maturity based on the profile of contact sex pheromones on her elytra.     

Mating expenditures reduced via female sex pheromone modulation in the primitively eusocial halictine bee, Lasioglossum (Evylaeus) malachurum (Hymenoptera: Halictidae)

The present paper reports on behavioral experiments and gas chromatographic analysis of chemical communication in the mating biology of the primitively eusocial sweat bee Lasioglossum (Evylaeus) malachurum. In a dual-choice experiment, a female made odorless was significantly less attractive than an untreated one. Attraction in L. (Evylaeus) malachurum is therefore mediated by a female-produced sex pheromone. Further bioassays showed that unmated gynes are more attractive to males than mated ones. Males are able to differentiate between the two groups of females as little as 3 h after mating. Biotests with different samples obtained from attractive gynes showed surface extracts to be most attractive. Behavioral tests with synthetic copies of the compounds identified as cuticular constituents of virgin gynes were highly attractive to males; the volatile bouquets consisting of n-alkanes, n-alkenes and iso- pentenyl esters of unsaturated fatty acids were the most attractive samples. Isopentenyl esters of unsaturated fatty acids were the key compounds in inducing male inspections as well as stimulating pounces and copulatory attempts. Virgin and nesting gynes differed clearly in the relative and absolute amounts of the volatiles on the cuticle. The total amount of volatiles was significantly higher in virgin gynes and decreased in breeding queens. Hydrocarbons were the dominant group of compounds in both groups of females. The relative amounts of the wax-type ester, hexadecyl oleate, iso- pentenyl esters, and a hitherto unidentified steroid were higher in attractive virgin gynes, while the relative proportions of hydrocarbons and lactones dominated in nesting queens. The site of sex pheromone production in attractive young L. (Evylaeus) malachurum gynes remains unknown. Head glands or Dufour's gland secretions may be involved. Another possible source of the `active principle' found among the cuticular lipids could be glandular cells of the epidermis. The significance of modulation of female sex pheromone composition is discussed in terms of a reduction in mating expenditures. Received: 30 April 1998 / Accepted after revision: 24 July 1998     

Communication ecology of webbing clothes moth: 4. Identification of male- and female-produced pheromones

Summary. We investigated the hypothesis that aggregation signals produced by male webbing clothes moths (WCM), Tineola bisselliella (Hum.) (Lepidoptera: Tineidae), and close-range male attractant signals produced by females have a pheromonal basis, at least in part. Gas chromatographic-electroantennographic detection (GC-EAD) and GC-mass spectrometric analyses of bioactive methanolic extracts of male WCM disclosed three candidate pheromone components: hexadecanoic acid methyl ester (16:Ester), (Z)-9-hexadecenoic acid methyl ester (Z9—16:Ester), and octadecanoic acid methyl ester (18:Ester). In bioassay experiments in a large Plexiglas™ arena, a blend of synthetic 16:Ester plus Z9—16:Ester was attractive to male and virgin (but not mated) female WCM; the 18:Ester was inactive. GC-EAD analyses of pheromone gland extracts from female WCM revealed (E,Z)-2,13-octadecadienal (E2Z13—18:Ald) and (E,Z)-2,13-octadecadienol (E2Z13—18:OH) as candidate sex pheromone components. In arena bioassay experiments, 1—5 female equivalents of synthetic E2Z13—18:Ald (0.2 ng) and E2Z13—18:OH (0.1 ng) were more attractive to male WCM than were two virgin female WCM. We anticipate that the combination of aggregation and sex pheromones, male-produced sonic aggregation signals, and habitat-derived semiochemicals will be highly effective in attracting male and female WCM to commercial traps. Received 12 January 2001; accepted 8 June 2001.     

Population differentiation in female sex pheromone and male preferences in a solitary bee

Population differentiation in female mating signals and associated male preferences can drive reproductive isolation among segregated populations. We tested this assumption by investigating intraspecific variation in female sex pheromone and associated male odour preferences among distant populations in the solitary bee Colletes cunicularius (L.) by using quantitative gas chromatography and by performing field bioassays with synthetic blends of key sex pheromone compounds. We found significant differences in sex pheromone blends among the bee populations, and the divergence in odour blends correlated positively with geographic distance, suggesting that genetic divergence among distant populations can affect sex pheromone chemistry. Our behavioural experiments, however, demonstrate that synthetic copies of allopatric female sex pheromones were cross-attractive to patrolling males from distant populations, making reproductive isolation by non-recognition of mating signals among populations unlikely. Our data also show that patrolling male bees from different populations preferred odour types from allopatric populations at the two sites of bioassays. These male preferences are not expected to select for changes in the female sex pheromone, but may influence the evolution of floral odour in sexually deceptive orchids of the genus Ophrys that are pollinated by C. cunicularius males.     

Identification, synthesis and activity of sex pheromone gland components of the autumn gum moth (Lepidoptera: Geometridae), a defoliator of Eucalyptus

Summary. The autumn gum moth, Mnesampela privata (Guenée) (Lepidoptera: Geometridae), is native to Australia and can be a pest of plantation eucalypts. Field-collected and laboratory-reared female autumn gum moths were dissected to remove glands likely to contain components of the sex pheromone. Using gas chromatography (GC) and combined gas chromatography–mass spectrometry (GC-MS), three compounds were identified from female extracts, namely (3Z,6 Z,9 Z)-3,6,9-nonadecatriene, 1-hexadecanol and 1-octadecanol (confirmed by comparison with synthetic samples). Nonadecatriene elicited an antennal response in male autumn gum moth during gas chromatographic analyses combined with electroantennographic detection (GC-EAD). In electroantennogram (EAG) recording male M. privata antennae responded to the nonadecatriene. Nonadecatriene was synthesised via Kolbe electrolysis, starting with (9Z,12Z,15Z)-octadeca-9,12,15-trienoic acid (linolenic acid) and propanoic acid or via an alternative four-step method also starting from linolenic acid. In field trials (3Z,6Z,9Z)-3,6,9-nonadecatriene proved attractive to male moths. Thus, we conclude that (3Z,6Z,9Z)-3,6,9- nonadecatriene is a sex pheromone component of autumn gum moth. This component has been identified in extracts from other geometrids in the same subfamily, Ennominae. However, to our knowledge this is the first example where (3Z,6Z,9Z)-3,6,9-nonadecatriene has been found in females and also proved attractive to male moths when presented on its own. Our results are discussed in relation to other geometrid pheromones.     

Sex attractant of an arctiid moth (Utetheisa ornatrix): A pulsed chemical signal

Summary The sex attractant pheromone produced by the female of the moth Utetheisa ornatrix was shown to contain Z, Z, Z-3, 6, 9-heneicosatriene. The compound, whose structure was confirmed by synthesis, proved active in electroantennogram and field bioassays. Pheromone emission occurs discontinuously, in the form of short pulses (pulse repetition rate=1.5±0.2 pulses/s). It is argued that such temporal patterning — which had not previously been demonstrated for an airborne chemical signal — can provide close-range orientation cues to the male moth as it seeks out the female.     

Sex pheromone of the large aspen tortrix, Choristoneura conflictana(Lepidoptera: Tortricidae)

Summary. Three components that elicited antennal response from male Choristoneura conflictana were found from female gland extracts analyzed using a coupled gas chromatographic-electroantennographic detector system. The main component in gland extracts was (Z)-11-tetradecenal (Z11-14:Ald). Two minor components also elicited antennal response: (E)11-tetradecenal (E11-14:Ald) and (Z)-11- tetradecen-1-ol (Z11-14:OH). Analysis of effluvia indicated that calling virgin females release mostly Z11-14:Ald and trace amounts of Z11-14:OH. Field and wind tunnel behavioral studies showed that Z11-14:Ald alone attracted male moths in a dose response pattern. Tests comparing male response to blends of components detected in gland extracts showed that addition of 1.8% of E11-14:Ald to Z11-14:Ald did not influence male moths in the wind tunnel, but resulted in significantly lower trap captures in the field. The threecomponent blend [Z11-14:Ald (100), E11-14:Ald (1.8), Z11-14:OH (11)], was less attractive than Z11-14:Ald alone in both field and wind tunnel studies. Traps baited with two virgin female moths were equally attractive to males as traps baited with the three-component synthetic blend but less attractive than traps baited with Z11-14:Ald alone. Field tests of various blends of the two components (Z11-14:Ald, Z11-14:OH) detected in the females’ effluvia showed that the addition of 1–10% Z11-14:OH to Z11-14:Ald did not affect the males’ response to Z11-14:Ald. Our data demonstrate that female C. conflictana release sex pheromone components in a different ratio than they are stored in the pheromone gland. The sex pheromone is comprised of a single component, Z11-14:Ald, that can be used to monitor mated and virgin male C. conflictana throughout their flight period.     

Host-plant green-leaf volatiles synergize the synthetic sex pheromones of the corn earworm and codling moth (Lepidoptera)

Summary The capture of adult male moths in female sex pheromone traps of two key agricultural pests, the corn earworm (Helicoverpa zea) and the codling moth (Cydia pomonella), is enhanced or synergized by a certain group of host-plant volatiles, the green-leaf volatiles (GLVs). Since female adults of both species call and release their sex pheromones while perched upon the leaves of their host-plants, the volatile constituents from the leaves of a number of host-plants were compared. Sex pheromone traps containing one of the prominent leaf volatiles of certainH. zea hosts, (Z)-3-hexenyl acetate, not only significantly increased the capture ofH. zea males but were preferred over traps baited only with sex pheromone. Similarly, traps baited with synthetic sex pheromome ofC. pomonella plus a blend of GLVs captured significantly more males than traps baited only with sex pheromone. Since male moths are not captured in traps baited only with these GLVs, it appears that these GLVs act as pheromone synergists which increase or enhance the attraction or arrestment of male moths in pheromone traps.     

Female sex pheromone of Ostrinia orientalis – throwing a light on the relationship between O. orientalis and the European corn borer, O. nubilalis

Summary. The sex pheromone of Ostrinia orientalis (Lepidoptera: Crambidae) was analyzed by gas chromatography–electroantennographic detection (GC–EAD), GC–mass spectrometry and a series of bioassays. Three EAD-active compounds were detected in the female sex pheromone gland extract, and identified as tetradecyl acetate (14:OAc), (Z)-11-tetradecenyl acetate (Z11-14:OAc) and (E)-11-tetradecenyl acetate (E11-14:OAc). The titers (ratio) of 14:OAc, Z11-14:OAc and E11-14:OAc in 3-day-old virgin females were 0.49 ng (10), 4.86 ng (98) and 0.10 ng (2), respectively. In a wind-tunnel bioassay, the 98:2 blend of Z11- and E11-14:OAc, but not Z11-14:OAc alone, elicited the same male behavioral responses as virgin females and crude gland extracts. 14:OAc was inactive by itself, and did not show any synergistic effect on the binary blend. Field trapping experiments also confirmed the attractiveness of the binary blend to O. orientalis males. Based on these results, we concluded that the sex pheromone of O. orientalis is a 98:2 mixture of Z11-14:OAc and E11-14:OAc. This sex pheromone is very similar to that of the Z-type European corn borer, O. nubilalis. The present finding raises the question of whether O. orientalis , which is indistinguishable from O. nubilalis based on external morphology, is a biologically distinct species independent from O. nubilalis.     

Sex pheromone of the tea aphid,Toxoptera aurantii (Boyer de Fonscolombe) (Hemiptera: Aphididae)

The tea aphid, Toxoptera aurantii, also called the “black citrus aphid”, is one of the most destructive insect pests in commercial tea plantations and gardens in southern China. In autumn, declining day length triggers production of winged T. aurantii sexuparae, which produce both winged males and wingless oviparae. Oviparous females then release sex pheromone that attracts potential mates. GC–MS analysis of volatile headspace extracts of T. aurantii oviparae revealed that they emit (4aS,7S,7aR)-nepetalactone (I) and (1R,4aS,7S,7aR)-nepetalactol (II) in a ratio of 4.3–4.9:1. Field-trapping experiments with synthetic I and II singly or as two-component blends of different doses and ratios showed significant attraction of T. aurantii males, as well as weak attraction of sexuparae. Identification of the T. aurantii sex pheromone provides a new opportunity for developing a pheromone-based monitoring and management strategy for the sexual phase of tea aphids and, possibly, the alate sexparous generation in late summer and fall.     

Aggregation pheromones of bark beetles, Pityogenes quadridens and P. bidentatus, colonizing Scotch pine: olfactory avoidance of interspecific mating and competition

The bark beetles Pityogenes bidentatus and Pityogenes quadridens (Coleoptera, Curculionidae, Scolytinae) are sibling species that feed and reproduce in bark areas on branches of Scotch pine, Pinus sylvestris. To identify aggregation pheromone components of both species, hindguts and head/thoraxes of males and females of both species feeding in hosts were extracted in pentane and analyzed by gas chromatography and mass spectrometry. Hindguts of male P. bidentatus contained grandisol as the major component along with small amounts of (4S)-cis-verbenol and other monoterpenes. Dose–response bioassays in the laboratory showed the components were attractive at 0.2 ng/min to walking beetles from a distance of ≥25 cm. In the field in southern Sweden, grandisol and (4S)-cis-verbenol were weakly attractive alone when released at rates of 0.05 and 0.5 mg/day, respectively, from a slow-rotating trap pair. Catch increased 3.6- to 13-fold when the two components were released together. The male proportion of the catch was 0.8 early in the flight period, declined to 0.5 on the peak flight day, and then declined further during the next 2 weeks to 0.2 on the last day of the flight period. Hindguts of male P. quadridens contained (2S,5R)- and (2S,5S)-chalcogran, as well as (E)-2-(3,3-dimethylcyclohexylidene)ethanol (E-grandlure 2) and E/Z-mixture of 2-(3,3-dimethylcyclohexylidene)acetaldehyde (grandlures 3 and 4), while female hindguts had only a trace amount of chalcogran. Laboratory studies proved E-grandlure 2 is an essential pheromone component for P. quadridens. Field bioassays with a slow-rotating trap pair in which the attractiveness of blends containing various candidate components were compared with that of less complete mixtures, showed that chalcogran and E-grandlure 2 were synergistic aggregation pheromone components of P. quadridens. Field tests also showed that grandisol (from P. bidentatus) reduced attraction of P. quadridens to its aggregation pheromone, whereas E-grandlure 2 and chalcogran (from P. quadridens) reduced response of P. bidentatus to its aggregation pheromone. Our results suggest that aggregation pheromone components from males of each species not only attract conspecifics but also aid individuals in avoiding interspecific mating and competition for food and spatial resources within the bark phloem layer.     

Dissecting the mechanisms underlying old male mating advantage in a butterfly

Selection is expected to maximize an individual’s own genetic reward regardless of the potential fitness consequences for its sexual partners, which may cause sexual conflict. Although performance in holometabolous insects typically diminishes with age, old male mating advantage has been documented in a few species. Whether this pattern arises from female preference for older males based on, e.g., pheromone blends (intersexual selection), or from increased eagerness to mate in older compared to younger males is currently debated. We explore the mechanistic basis of old male mating advantage, using a series of experiments including behavioral as well as manipulative approaches, in the tropical butterfly Bicyclus anynana. Consistent with the residual reproductive value hypothesis, old male mating advantage was associated with a greater eagerness to mate, evidenced by a two times higher flying and courting activity in older than in younger males. In contrast, we found only limited support for a contribution of female preference for older males based on pheromone composition, although male sex pheromones clearly do play a role in mating success. Our results suggest that male behavior may play a primary role in old male mating advantage, and that pheromones are likely of secondary importance only. Male mating success was related to higher overall pheromone titers rather than variation in a single component. A dominant importance of male behavior in determining mating success may result in sexual conflict.