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Stingless bees, like honeybees, live in highly organized, perennial colonies. Their eusocial way of life, which includes division of labor, implies that only a fraction of the workers leave the nest to forage for food. To ensure a sufficient food supply for all colony members, stingless bees have evolved different mechanisms to recruit workers to foraging or even to communicate the location of particular food sites. In some species, foragers deposit pheromone marks between food sources and their nest, which are used by recruited workers to locate the food. To date, pheromone compounds have only been described for 3 species. We have identified the trail pheromone of a further species by means of chemical and electrophysiological analyses and with bioassays testing natural gland extracts and synthetic compounds. The pheromone is a blend of wax type and terpene esters. The relative proportions of the single components showed significant differences in the pheromones of foragers form 3 different colonies. This is the first report on a trail pheromone comprised of esters of 2 different biogenetic origins proving variability of the system. Pheromone specificity may serve to avoid confusions between the trails deposited by foragers of different nests and, thus, to decrease competition at food sources.

Reproductive division of labor in advanced eusocial honey bees and stingless bees is based on the ability of totipotent female larvae to develop into either workers or queens. In nearly all species, caste is determined by larval nutrition. However, the mechanism that triggers queen development in Melipona bees is still unresolved. Several hypotheses have been proposed, ranging from the proximate (a genetic determination of caste development) to the ultimate (a model in which larvae have complete control over their own caste fate). Here, we showed that the addition of geraniol, the main compound in labial gland secretions of nurse workers, to the larval food significantly increases the number of larvae that develop into queens. Interestingly, the proportion of queens in treated brood exactly matched the value (25%) predicted by the two-locus, two-allele model of genetic queen determination, in which only females that are heterozygous at both loci are capable of developing into queens. We conclude that labial gland secretions, added to the food of some cells by nurse bees, trigger queen development, provided that the larvae are genetically predisposed towards this developmental pathway. In Melipona beecheii, geraniol acts as a primer pheromone representing the first caste determination substance identified to date.

In the sexually deceptive orchid genus Ophrys, reproductive isolation is based on the specific attraction of males of a single pollinator species by mimicking the female species-specific sex pheromone. Changes in the odor composition can lead to hybridization and speciation by the attraction of a new pollinator that acts as an isolation barrier toward other sympatrically occurring Ophrys species. On Sardinia, we investigated the evolutionary origin of two sympatrically occurring endemic species, Ophrys chestermanii and O. normanii, which are both pollinated by males of the cuckoo bumblebee Bombus vestalis. Chemical and electrophysiological analyses of floral scent and genetic analyses with amplified fragment length polymorphisms and plastid-markers clearly showed that O. normanii is neither a hybrid nor a hybrid species. The two species evolved from different ancestors, viz. O. normanii from O. tenthredinifera and O. chestermanii from O. annae, and converged to the same pollinator attracted by the same bouquet of polar compounds. In spite of sympatry, pollinator sharing and overlapping blooming periods, no evidence has been obtained for gene flow between O. chestermanii and O. normanii indicating an unusual case among sexually deceptive orchids in which postmating rather than premating reproductive isolation mechanisms strongly prevent interspecific gene flow.

Approximately one-third of the world's 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.

Reproductive competition in social insects is generally mediated through specific fertility pheromones. By analysing Dufour's 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 Dufour's 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.

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.

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.

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.

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.