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Pheromones play an important role in the mating behavior of many insects, especially in bees, and it has recently been shown that female Osmia bicornis use the odor bouquet found on male antennae in female choice. Nevertheless, the glandular origin of the substances found on the male antennae is so far unknown. In this study, we investigated if antennal glands exist in males and if they are the glandular source of substances on the antennae’s cuticle, as has been suggested by previous studies. Since electron and light microscopy revealed no such glands, we investigated further possible places of origin for the antennal compounds by conducting chemical analyses of solvent extracts of the male abdominal surface and the antennae, as well as of two glands found in the bee’s head, the labial and mandibular gland. Our results clearly show that the substances found on the abdominal surface and in solvent extracts of the antennae differ significantly in relative amounts. Therefore, the compounds on the antennae must have additional sources. Since the relative amounts of compounds on the surface of antennae are different from the blends on the mandibular gland or the labial gland, neither one of them can be the sole source of the antennal compounds. Hence we believe another glandular source is also involved in the production of the bouquet on the antennae. Especially interesting were high amounts of sterols on the antennae, which also play a role in population differences. This sheds new light on the importance of the antennal compounds and also the importance sterols might play in communication.

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 (Gogler 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.

Bumblebees of the subgenus Psithyrus are obligate social parasites of Bombus colonies. Parasitic females enter host colonies and replace the host queen. The offspring of the parasite is reared by the host workers. Females locate host colonies by nest searching flights and recognition of species-specific nest odours at the entrance. We investigated inter- and intraspecific odour variation of 45 hydrocarbons of nests of potential hosts by coupled gas chromatography/mass spectrometry and tested the preferences of the parasite females of B. bohemicus and B. rupestris for these mixtures in Y-olfactometer choice tests. Interspecific and intracolonial differences in the odour bouquets of the host species were found to be predominantly due to different patterns of alkenes. Furthermore, we found intercolonial differences within the single species. In behavioural assays, females of the two species showed different preferences for the offered nest odours, implicating different host spectra. Bombus rupestris showed a clear preference for the scent of its host, B. lapidarius. Bombus bohemicus females were attracted by B. terrestris, B. lucorum, and B. cryptarum in a similar manner. The results show that volatile signals enable parasite females to discriminate between potential host species.

Foragers of several species of stingless bees deposit pheromone spots in the vegetation to guide recruited nestmates to a rich food source. Recent studies have shown that Trigona and Scaptotrigona workers secrete these pheromones from their labial glands. An earlier report stated that species within the genus Geotrigona use citral from their mandibular glands for scent marking. Since convincing experimental proof for this conjecture is lacking, we studied the glandular origin of the trail pheromone of Geotrigona mombuca. In field bioassays, newly recruited bees were diverted by artificial scent trails that branched off from the natural scent trail deposited by their nestmates only when they were baited with extracts from the foragers’ labial glands. Compounds extracted from the mandibular glands, however, did not release trail following behavior. This demonstrates that the trail pheromone of G. mombuca is produced in the labial glands, as in Trigona and Scaptotrigona. Furthermore, in chemical analyses citral was identified exclusively in the foragers’ mandibular glands, which disproves its supposed role as a trail pheromone. The labial glands contained a series of terpene- and wax type esters, with farnesyl butanoate as major constituent. We, therefore, postulate that the trail pheromone of G. mombuca is composed of a blend of esters.

In the bumble bee, Bombus terrestris L., we investigated whether a chemical signal corresponding to the egg-marking in honey bees occurs, and whether the pattern of volatiles encodes caste-, colony- or even individual-specific information. Volatile compounds were obtained by surface extraction of individual eggs with pentane. The resulting complex mixtures were analysed by gas chromatography and mass spectrometry. Major classes of identified compounds were n-alkanes, n-alkenes and n-alkadienes comprising approximately 95% of the extracted volatile material. In addition, small amounts of methyl-branched alkanes, ethyl esters, methyl esters, wax type esters and acetates were present. Eggs produced by queens and individual workers showed significantly different compositions of volatiles. Furthermore, the volatile patterns were found to be colony-specific. Egg-odours contained all compounds identified in the Dufour’s glands and some additional components. Therefore, females very likely mark eggs with excretions of Dufour’s gland, however, other glands may also be involved.

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.