SUMMARY Male insects may increase their chance of successful reproduction by releasing pheromones that attract females or elicit sexual acceptance. In parasitic wasps, male pheromones have been suggested for a few species but no chemicals have been identified so far. Here we report the first identification of a male sex pheromone in parasitic Hymenoptera. In abdomens of male jewel wasps, Nasonia vitripennis Walker, we found a mixture of(4R,5R)- and(4R,5S)-5-hydroxy-4-decanolide (HDL), which was released intermittently and attracted virgin females, but no males, in an olfactometer bioassay. However, only a few minutes after copulation mated females avoided the male-derived pheromone. Neither preference nor avoidance was shown by mated females after 24 h and even after they had been allowed to oviposit for 6 days. Nasonia vitripennis females normally mate only once. Thus,their variable response to the sex attractant depending on the mating status makes sense from an evolutionary perspective. Firstly, it increases the chance of virgins to be inseminated. Secondly, by terminating the response or even avoiding the male pheromone, mated females decrease the probability of encountering males and being disturbed by their courtship activities when searching for new oviposition sites.

The rare European click beetle, Elater ferrugineus L., develops exclusively in old, hollow deciduous trees. As a result of massive habitat loss caused by modern forestry, it is threatened throughout its entire distribution range and regarded as an indicator species for undamaged natural forests. As E. ferrugineus lives cryptically and its populations are frequently overlooked, we investigated its sex pheromone to develop a reliable detection tool. Pheromone gland extracts of single female E. ferrugineus were examined by using gas chromatography-mass spectrometry (GC-MS). All samples contained 7-methyloctyl 5-methylhexanoate, 7-methyloctyl octanoate, 7-methyloctyl 7-methyloctanoate, and 7-methyloctyl (Z)-4-decenoate in a ratio of approximately 1:1:3:3. Structures of all four esters, which have not been reported as pheromone compounds before, were confirmed by synthesis. A blend of the components was tested in the field and proved to be attractive for E. ferrugineus males, which were found to swarm exclusively during the day. This blend provides a noninvasive and effective monitoring method for this cryptic species, promising future collection records of E. ferrugineus in regions where it exists below the limit of detection by conventional collecting methods.

Based on analysis of pheromone gland extracts, highly attractive new baits have been developed for three click beetle pests. That for Agriotes brevis is a mixture of geranyl butanoate and (E,E)-farnesyl butanoate, and that for A rufipalpis and A sordidus contains geranyl hexanoate alone. From known data from species populating Russia, optimized bait compositions for species in Central and Western Europe were developed as follows: geranyl octanoate + geranyl butanoate for A lineatus, geranyl isovalerate for A litigiosus, geranyl hexanoate + geranyl octanoate for A obscurus, geranyl butanoate alone for A sputator and (E,E)-farnesyl acetate alone for A ustulatus. Although slight differences were found in gland contents with A litigiosus var laichartingi and fenotypus typicus, nevertheless there were no differences in response to the optimum bait. There were no differences in pheromone composition or response to the optimized bait between the two morphological forms ('black' and 'red') of A ustulatus. As a result of these studies, highly effective pheromone baits are now available for monitoring and population reduction in all important pest click beetle species in Central and Western Europe.

Polyploidy has played a key role in plant evolution and diversification. Despite this, the processes governing reproductive isolation among cytotypes growing in mixedploidy populations are still largely unknown. Theoretically, coexistence of diploid and polyploid individuals in sympatric populations is unlikely unless cytotypes are prezygotically isolated through assortative pollination. Here, we investigated the pre-mating barriers involved in the maintenance of three co-occurring cytotypes from the genus Gymnadenia (Orchidaceae): tetraploid and octoploid G. conopsea and tetraploid G. densiflora. We assessed differences in flowering phenology, floral morphology, and visual and olfactory cues, which could lead to assortative mating. Gas chromatography coupled with Electronic supplementary material The online version of this article (electroantennographic detection was used to identify scent compounds with physiological activity in the two main pollinators, Deilephila porcellus and Autographa gamma. The importance of olfactory cues was also assessed in the field by analysing the moths' responses to the olfactory display of the plants, and by following the pollinator's behaviour on artificial arrays. Our complex approach demonstrated that the coexistence of Gymnadenia cytotypes in mixed-ploidy populations was only partly explained by differences in floral phenology, as cytotypes with overlapping flowering (i.e., octoploid G. conopsea and tetraploid G. densiflora) might freely exchange pollen due to only 1 mm differences in spur lengths and the lack of assortative behaviour of pollinators. While floral colour among the cytotypes was similar, floral scent differed significantly. Though both pollinator species seemed to physiologically detect these differences, and the floral scent alone was sufficient to attract them, pollinators did not use this cue to discriminate the cytotypes in the field. The absence of premating barriers among cytotypes, except partial temporal segregation, suggests the existence of other mechanisms involved in the cytotypes' coexistence. The genetic differences in ITS sequences among cytotypes were used to discuss the cytotype's origin.

Summary The response of the forest cockchafer, Melolontha hippocastani F. (Coleoptera, Scarabaeidae), towards volatiles emitted by different host plants and conspecifics was tested in field experiments during the flight period at dusk. Funnel traps containing artificially damaged leaves from the host plants Carpinus betulus L. and Quercus rubra L., as well as from the non‐host plant Prunus serotina Ehrh. caught significantly more beetles than empty control traps. On the other hand, traps baited with undamaged leaves from Q. rubra did not catch significantly more beetles than empty controls. Leaves from C. betulus damaged by beetle feeding did not attract more beetles than artificially damaged leaves. By use of gas chromatography coupled with electroantennographic detection (GC‐EAD) electrophysiological responses of males and females were shown for 18 typical plant volatiles. A synthetic mixture of selected typical green plant volatiles was also highly attractive in the field. A total of 9982 beetles was caught during the field experiments, among them only 33 females. This suggests that attraction to damaged foliage during flight period at dusk is male‐specific. Field experiments testing the attractiveness of female M. hippocastani towards conspecific males by employing caged beetles and beetle extracts indicated that males of M. hippocastani use a female‐derived sex pheromone for mate location. On wired cages containing either unmated feeding females, or unmated females without access to foliage, or feeding males in combination with extracts from unmated females, significantly more males landed during the flight period than on comparable control cages containing feeding males or male extracts. A possible scenario of mate location in M. hippocastani involving feeding‐induced plant volatiles and a female‐derived sex pheromone is discussed.

Ceropegia species (Apocynaceae) have deceptive pitfall flowers and exploit small flies as pollinators, supposedly by chemical mimicry. Only preliminary data on the composition of flower scents are available for a single species so far, and the mimicry system is not yet understood in any species. We collected data on basic pollination aspects of C. dolichophylla, analyzed floral scent by gas chromatography linked to mass spectrometry (GC/MS), identified electrophysiologically active scent components by gas chromatography coupled with electroantennographic detection (GC/EAD), and determined compounds responsible for pollinator attraction in bioassays. We found that flowers of C. dolichophylla are visited by small flies of several taxa. Only Milichiidae and Chloropidae carried pollinaria and are, thus, pollinators. The pollen transfer efficiency (PTE) at two different sites was 2% and 4%, respectively. The floral scent was dominated by spiroacetals, mainly (2S,6R,8S)-8-methyl-2-propyl-1,7-dioxaspiro[5.5]undecane, n-tridecane, and N-(3-methylbutyl)acetamide. This spiroacetal and the acetamide elicited the most intense electrophysiological responses in fly antennae, and bioassays confirmed the capability of the spiroacetal in eliciting behavioral responses in pollinators. Most flies, determined as pollinators of C. dolichophylla, are kleptoparasites. They exploit insect prey of predatory arthropods as food source to which they are attracted by volatiles. undecane and N-(3-methylbutyl)acetamide have not been identified before as volatiles of other plants, however, they are known as insect volatiles. Both compounds occur in the venom glands of paper wasps, a potential food source for the pollinators of C. dolichophylla. We propose that C. dolichophylla shows a kleptomyiophilous pollination strategy. It mimics insect related odors to exploit the food-seeking behavior of its kleptoparasitic pollinators.

Mate finding in the forest cockchafer, Melolontha hippocastani, occurs during a spectacular swarming period at dusk. The swarming flights are mainly performed by males whereas most of the females stay within the host trees and continue feeding. Males orientate towards damage-induced green leaf volatiles (GLV) allowing location of mechanically damaged foliage. In order to distinguish between unspecific leaf damage and damage caused by feeding females, male cockchafers orientate by a sex attractant. Here we show this compound to be 1,4-benzoquinone, which has been known for more than 40 years as a highly effective defence compound used by numerous arthropod species to repel enemies. 1,4-benzoquinone synergistically increased the number of males caught in GLV-baited funnel traps during the swarming period. Significantly more males landed on wire cages baited with a combination of 1,4-benzoquinone and GLV than on cages baited with only GLV. The results suggest that the sex pheromone of M. hippocastani might have evolved from a primary role as a defence compound.

Analyses of pentane extracts of frass, whole beetles, and volatiles trapped on Porapak-Q from Pityophthorus Eichhoff spp. fed on Pinus radiata D. Don demonstrated that (E)-pityol [2-(1-hydroxy-1-methylethyl)-5-methyltetrahydrofuran] was produced by male Pityophthorus carmeli Swaine, female Pityophthorus nitidulus (Mannerheim), and female Pityophthorus setosus Blackman. (E)-(−)-Conophthorin) [(5S,7S)-(−)-7-methyl-1,6-dioxaspiro[4.5]decane] was produced by male P. carmeli and male P. nitidulus. Only the (2R,5S)-(+) stereoisomer of (E)-pityol was produced by male P. carmeli and female P. setosus. In field bioassays in central coastal California, P. setosus was attracted to (E)-(+)-pityol, whereas P. carmeli responded only to a combination of (E)-(−)-conophthorin and (E)-(+)-pityol. Male P. setosus and female P. carmeli responded to these treatments with larger numbers than opposite-sex conspecifics. (E)-(−)-Conophthorin alone did not attract species of Pityophthorus but significantly reduced catches of P. setosus to (E)-(+)-pityol. Lasconotus pertenuis Casey (Coleoptera: Colydiidae) and Ips mexicanus (Hopkins) (Coleoptera: Scolytidae) were attracted to a combination of (E)-(−)-conophthorin and (E)-(+)-pityol, and showed a trend for attraction to all (E)-(−)-conophthorin-containing treatments. (E)-(−)-Pityol was neither attractive nor interruptive for any taxon. (E)-(+)-Pityol is shown to be an aggregation pheromone component for P. carmeli and P. setosus. (E)-(−)-Conophthorin functions as a pheromone component for P. carmeli and may also function as a synomone that decreases competition of P. carmeli and P. nitidulus with P. setosus and as a kairomone for L. pertenuis. These semiochemicals have been useful in studying relationships among twig insects and the pathogen Fusarium circinatum (Nirenberg and O’Donnell), causal agent of pitch canker disease in P. radiata.