Robert Twele

An Inhibitory Sex Pheromone Tastes Bitter for Drosophila Males

Sexual behavior requires animals to distinguish between the sexes and to respond appropriately to each of them. In Drosophila melanogaster, as in many insects, cuticular hydrocarbons are thought to be involved in sex recognition and in mating behavior, but there is no direct neuronal evidence of their pheromonal effect. Using behavioral and electrophysiological measures of responses to natural and synthetic compounds, we show that Z-7-tricosene, a Drosophila male cuticular hydrocarbon, acts as a sex pheromone and inhibits male-male courtship. These data provide the first direct demonstration that an insect cuticular hydrocarbon is detected as a sex pheromone. Intriguingly, we show that a particular type of gustatory neurons of the labial palps respond both to Z-7-tricosene and to bitter stimuli. Cross-adaptation between Z-7-tricosene and bitter stimuli further indicates that these two very different substances are processed by the same neural pathways. Furthermore, the two substances induced similar behavioral responses both in courtship and feeding tests. We conclude that the inhibitory pheromone tastes bitter to the fly.

Orchids Mimic Green-Leaf Volatiles to Attract Prey-Hunting Wasps for Pollination

An outstanding feature of orchids is the diversity of their pollination systems [1]. Most remarkable are those species that employ chemical deceit for the attraction of pollinators [2]. The orchid Epipactis helleborine is a typical wasp flower, exhibiting physiological and morphological adaptations for the attraction of pollinating social wasps [3]. As noted by Darwin [1], this species is almost entirely overlooked by other potential pollinators, despite a large nectar reward. Therefore, the mechanism for the attraction of pollinating social wasps was something of a mystery. By using a combination of behavioral experiments, electrophysiological investigations, and chemical analyses, we demonstrate for the first time that the flowers of E. helleborine and E. purpurata emit green-leaf volatiles (GLVs), which are attractive to foragers of the social wasps Vespula germanica and V. vulgaris. GLVs, emitted by damaged plant tissues, are known to guide parasitic wasps to their hosts [4]. Several E. helleborine GLVs that induced response in the antennae of wasps were also emitted by cabbage leaves infested with caterpillars (Pieris brassicae), which are common prey items for wasps [5]. This is the first example in which GLVs have been implicated in chemical mimicry for the attraction of pollinating insects.

Orchid mimics honey bee alarm pheromone in order to attract hornets for pollination.

Approximately one-third of the worlds estimated 30,000 orchid species are deceptive and do not reward their pollinators with nectar or pollen. Most of these deceptive orchids imitate the scent of rewarding flowers or potential mates. In this study, we investigated the floral scent involved in pollinator attraction to the rewardless orchid Dendrobium sinense, a species endemic to the Chinese island Hainan that is pollinated by the hornet Vespa bicolor. Via chemical analyses and electrophysiological methods, we demonstrate that the flowers of D. sinense produce (Z)-11-eicosen-1-ol and that the pollinator can smell this compound. This is a major compound in the alarm pheromones of both Asian (Apis cerana) and European (Apis mellifera) honey bees and is also exploited by the European beewolf (Philanthus triangulum) to locate its prey. This is the first time that (Z)-11-eicosen-1-ol has been identified as a floral volatile. In behavioral experiments, we demonstrate that the floral scent of D. sinense and synthetic (Z)-11-eicosen-1-ol are both attractive to hornets. Because hornets frequently capture honey bees to feed to their larvae, we suggest that the flowers of D. sinense mimic the alarm pheromone of honey bees in order to attract prey-hunting hornets for pollination.

The discovery of 2,5-dialkylcyclohexan-1,3-diones as a new class of natural products.

Orchids employing sexual deceit attract males of their pollinator species through specific volatile signals that mimic female-released sex pheromones. One of these signals proved to be 2-ethyl-5-propylcyclohexan-1,3-dione (chiloglottone1), a new natural product that was shown to be most important in the relations between orchids of the genus Chiloglottis, native to Australia, and corresponding pollinator species. Systematic investigations on the mass spectrometric fragmentation pattern of 2,5-dialkylcyclohexan-1,3-diones identified key ions providing information about the structures of the substituents at positions 2 and 5. Results enabled us to identify 2-ethyl-5-pentylcyclohexan-1,3-dione (chiloglottone2) and 2-butyl-5-methylcyclohexan-1,3-dione (chiloglottone3) as new natural products that play a decisive role in the pollination syndrome of some Chiloglottis species. During field bioassays, pure synthetic samples of chiloglottone1–3 or mixtures thereof proved to be attractive to the corresponding orchid pollinators. Because of their likely biogenesis from ubiquitous fatty acid precursors, 2,5-dialkylcyclohexan-1,3-diones may represent a hitherto overlooked, widespread class of natural products.

Spitting out information: Trigona bees deposit saliva to signal resource locations

Stingless bees of the species Trigona spinipes (Fabricius 1793) use their saliva to lay scent trails communicating the location of profitable food sources. Extracts of the cephalic labial glands of the salivary system (not the mandibular glands, however) contain a large amount (approx. 74%) of octyl octanoate. This ester is also found on the scent-marked substrates at the feeding site. We demonstrate octyl octanoate to be a single compound pheromone which induces full trail following behaviour. The identification of the trail pheromone in this widely distributed bee makes it an ideal organism for studying the mechanism of trail following in a day flying insect.

Fertility signals in the bumblebee Bombus terrestris (Hymenoptera: Apidae)

In eusocial Hymenoptera, queen control over workers is probably inseparable from the mechanism of queen recognition. In primitively eusocial bumblebees (Bombus), worker reproduction is controlled not only by the presence or absence of a dominant queen but also by other dominant workers. Furthermore, it was shown that the queen dominance is maintained by pheromonal cues. We investigated whether there is a similar odor signal released by egg-laying queens and workers that may have a function as a fertility signal. We collected cuticular surface extracts from nest-searching and breeding Bombus terrestris queens and workers that were characterized by their ovarian stages. In chemical analyses, we identified 61 compounds consisting of aldehydes, alkanes, alkenes, and fatty acid esters. Nest-searching queens and all groups of breeding females differed significantly in their odor bouquets. Furthermore, workers before the competition point (time point of colony development where workers start to develop ovaries and lay eggs) differed largely from queens and all other groups of workers. Breeding queens showed a unique bouquet of chemical compounds and certain queen-specific compounds, and the differences toward workers decrease with an increasing development of the workers’ ovaries, hinting the presence of a reliable fertility signal. Among the worker groups, the smallest differences were found after the competition point. Egg-laying females contained higher total amounts of chemical compounds and of relative proportions of wax-type esters and aldehydes than nest-searching queens and workers before the competition point. Therefore, these compounds may have a function as a fertility signal present in queens and workers.

Reproductive competition in the bumble-bee Bombus terrestris: do workers advertise sterility?

Reproductive competition in social insects is generally mediated through specific fertility pheromones. By analysing Dufours gland secretion in queens and workers of Bombus terrestris under varying social conditions, we demonstrate here that the volatile constituents of the secretion exhibit a context-dependent composition. The secretion of egg-laying queens is composed of a series of aliphatic hydrocarbons (alkanes and alkenes), while that of sterile workers contains in addition octyl esters, dominated by octyl hexadecanoate and octyl oleate. These esters disappear in workers with developed ovaries, whether queenright (QR) or queenless (QL), rendering their secretion queen-like. This constitutes an unusual case in which the sterile caste, rather than the fertile one, possesses extra components. Individually isolated (socially deprived) workers developed ovaries successfully, but failed to oviposit, and still possessed the octyl esters. Thus, whereas social interactions are not needed in order to develop ovaries, they appear essential for oviposition and compositional changes in Dufours gland secretion (ester disappearance). The apparent link between high ester levels and an inability to lay eggs lends credence to the hypothesis that these esters signal functional sterility. We hypothesize that by producing a sterility-specific secretion, workers signal that ‘I am out of the competition’, and therefore are not attacked, either by the queen or by the reproductive workers. This enables proper colony function and brood care, in particular sexual brood, even under the chaotic conditions of the competition phase.

Production of sexuals in a fission-performing ant: dual effects of queen pheromones and colony size

Models based on the kin selection theory predict that in social hymenopterans, queens may favor a lower investment in the production of sexuals than workers. However, in perennial colonies, this conflict may be tuned down by colony-level selection because of the trade off between colony survival and reproductive allocation. In this study, we present a survey of sexual production in colonies of Aphaenogaster senilis, a common species of ant in the Iberian Peninsula. Similar to most species that reproduce by fission, males were found in large excess compared to gynes (172:1). Sexuals were more likely to be found in queenless than in queenright (QR) field colonies. However, we also found a few gynes and numerous males in very large QR colonies. We compared these data with those available in the literature for A. rudis, a congeneric species from North America that has independent colony founding. The sex ratio in this species was only five males for each female, and sexuals were mostly found in QR nests, irrespective of colony size. We confirmed queen inhibition of sexual production in A. senilis in laboratory experiments and provide evidence that this inhibition is mediated by a nonvolatile pheromone. To seek the potential source of such a queen pheromone, we analyzed the secretions of two conspicuous exocrine glands, the Dufour’s and postpharyngeal glands (DG and PPG, respectively) in both queens and workers. Both secretions were composed of hydrocarbons, but that of DG also contained small quantities of tetradecanal and hexadecanal. The hydrocarbon profile of the DG and PPG showed notable caste specificity suggesting a role in caste-related behavior. The PPG secretions also differed between colonies suggesting its role in colony-level recognition. We suggest that in A. senilis, there are two modes of colony fission: First, in very large colonies, gynes are produced, probably because of the dilution of the queen pheromone, and consequently one or more gynes leave the mother colony with workers and brood to found a new nest. This is beneficial at the colony level because it avoids the production of costly sexuals in small colonies. However, because the queen and workers have different optima for sexual production, we hypothesize that queens tend to overproduce the pheromone to delay their production. This in turn may drive workers to leave the mother colony during nest relocation and to produce sexuals once they are away from the queen’s influence, creating a second mode of colony fission.

An olfactory shift is associated with male perfume differentiation and species divergence in orchid bees.

Saltational changes may underlie the diversification of pheromone communication systems in insects, which are normally under stabilizing selection favoring high specificity in signals and signal perception. In orchid bees (Euglossini), the production of male signals depends on the sense of smell: males collect complex blends of volatiles (perfumes) from their environment, which are later emitted as pheromone analogs at mating sites. We analyzed the behavioral and antennal response to perfume components in two male morphotypes of Euglossa cf. viridissima from Mexico, which differ in the number of mandibular teeth. Tridentate males collected 2-hydroxy-6-nona-1,3-dienyl-benzaldehyde (HNDB) as the dominant component of their perfume. In bidentate males, blends were broadly similar but lacked HNDB. Population genetic analysis revealed that tri- and bidentate males belong to two reproductively isolated lineages. Electroantennogram tests (EAG and GC-EAD) showed substantially lower antennal responses to HNDB in bidentate versus tridentate males, revealing for the first time a mechanism by which closely related species acquire different chemical compounds from their habitat. The component-specific differences in perfume perception and collection in males of two sibling species are in agreement with a saltational, olfaction-driven mode of signal perfume evolution. However, the response of females to the diverged signals remains unknown.

Enfleurage, lipid recycling and the origin of perfume collection in orchid bees

Enfleurage, the extraction of elusive floral scents with the help of a lipophilic carrier (grease), is widely used in the perfume industry. Male neotropical orchid bees (Euglossini), which accumulate exogenous fragrances as pheromone analogues, use a similar technique. To collect fragrances, the bees apply large amounts of straight-chain lipids to odoriferous surfaces from their cephalic labial glands, which dissolve the volatiles, and the mixture is then transferred to voluminous hind-leg pockets. Here, we show that males do in fact operate a lipid conveyor belt to accumulate and concentrate their perfume. From the hind-leg pockets of caged male Euglossa viridissima, deuterated derivatives of carrier lipids were consecutively sequestered, shuttled back to the labial glands and reused on consecutive bouts of fragrance collection. Such lipid cycling is instrumental in creating complex perfume bouquets. Furthermore, we found that labial glands of male orchid bees are strikingly similar to those of scent-marking male bumblebees in terms of size, form and structure. This, and a prominent overlap in secretory products, led us to propose that perfume collection evolved from scent-marking in ancestral corbiculate bees.