Discovery of 3-methyl-2-buten-1-yl acetate,a new alarm component in the sting apparatus of Africanized honeybees

We analyzed the alarm pheromone components from five colonies of Africanized honeybees and three colonies of European honeybees collected in Mexico. Analyses revealed a novel alarm pheromone component that was only present in appreciable quantities in the Africanized bee samples. Analysis of the mass spectrum and subsequent synthesis confirmed that this compound is 3-methyl-2-buten-1-yl acetate (3M2BA), an unsaturated derivative of IPA. In Africanized honeybees, sampling from stings of guards showed that 3M2BA was present at levels of 0–38% the amount of isoamyl acetate (IPA). Behavioral assays from three colonies each of Africanized and European bees showed that 3M2BA recruited worker bees from hives of both Africanized bees and European bees at least as efficiently as isopentyl acetate IPA, a compound widely reported to have the highest activity for releasing alarm and stinging behavior in honeybees. However, a mixture of of 3M2BA and IPA (1:2) recruited bees more efficiently than either of the compounds alone. None of the compounds differed in their efficacy for inducing bees to pursue the observers.     

Genetic Relatedness and Chemical Profiles in an Unusually Peaceful Eusocial Bee

Colonies of the stingless bee Tetragonilla collina frequently occur in unusually high densities and in direct neighborhood (nest aggregations), in rainforests of Southeast Asia. To investigate whether close relatedness and/or similar chemical profiles facilitate the co-occurrence of multiple T. collina colonies, we investigated aggressive behavior, genetic relatedness and cuticular hydrocarbon (CHC) profiles within and between colonies and nest aggregations. Although 17 out of 19 colonies within aggregations were largely unrelated, intraspecific aggression between different colonies was basically absent both within and among aggregations. This lack of aggression should favor social parasitism and hence the occurrence of unrelated individuals within a colony. However, low within-colony relatedness was found in only five out of 19 colonies where it may be explained by queen turnover or the occurrence of foreign workers. CHC profiles of colonies within and among aggregations were statistically different. However, many workers could chemically not be assigned to their maternal colony, indicating considerable overlap among colonies in odor profiles of workers. Moreover, odor profiles tended to be more similar within than among aggregations, although most colonies were unrelated. Thus, CHC profiles were a poor indicator of relatedness in T. collina. The lack of correlation between relatedness and chemical similarity in T. collina may be explained by the incorporation of resin-derived terpenes in their CHC profiles. The composition of these terpenes was highly similar among colonies, particularly within aggregations, hence potentially decreasing chemical distinctiveness and increasing behavioral tolerance.     

Intranidal worker reactions to volatile compounds identified from cephalic secretions in the stingless bee,Scaptotrigona postica (Hymenoptera,Meliponinae)

From pentane extracts of worker heads of the stingless bee (Scaptotrigona postica), 70 volatile compounds were identified by combined gas chromatographic-mass spectroscopic analyses. A bioassay was developed to evaluate intranidal reactions of workers to synthetic volatiles. Thirty-six of the cephalic volatiles were tested. Thirteen types of behavioral reactions were recorded in a semiquantitative manner. The test was run in the brood nest where mainly young nurse bees are present and also in the storage area of the nest with old foragers traffic. The results obtained were compared and discussed in order to understand the chemical communication system of this species. Especially in the dark interior of the nest, which in nature is found in hollow tree cavities, chemical messages obviously play a particularly important role in the communication systems of the bees.     

The Chemical Basis of Host-Plant Recognition in a Specialized Bee Pollinator

Many pollinators specialize on a few plants as food sources and rely on flower scents to recognize their hosts. However, the specific compounds mediating this recognition are mostly unknown. We investigated the chemical basis of host location/recognition in the Campanula-specialist bee Chelostoma rapunculi using chemical, electrophysiological, and behavioral approaches. Our findings show that Ca. trachelium flowers emit a weak scent consisting of both widespread and rare (i.e., spiroacetals) volatiles. In electroantennographic analyses, the antennae of bees responded to aliphatics, terpenes, aromatics, and spiroacetals; however, the bioassays revealed a more complex response picture. Spiroacetals attracted host-naive bees, whereas spiroacetals together with aliphatics and terpenes were used for host finding by host-experienced bees. On the intrafloral level, different flower parts of Ca. trachelium showed differences in the absolute and relative amounts of scent, including spiroacetals. Scent from pollen-presenting flower parts elicited more feeding responses in host-naive bees as compared to a scentless control, whereas host-experienced bees responded more to the nectar-presenting parts. Our study demonstrates the occurrence of learning (i.e., change in the bee’s innate chemical search-image) after bees gain foraging experience on host flowers. We conclude that highly specific floral volatiles play a key role in host-flower recognition by this pollen-specialist bee, and discuss our findings into the broader context of host-recognition in oligolectic bees.     

Chemical Ecology of Stingless Bees

Stingless bees (Hymenoptera, Apidae: Meliponini) represent a highly diverse group of social bees confined to the world’s tropics and subtropics. They show a striking diversity of structural and behavioral adaptations and are important pollinators of tropical plants. Despite their diversity and functional importance, their ecology, and especially chemical ecology, has received relatively little attention, particularly compared to their relative the honeybee, Apis mellifera. Here, I review various aspects of the chemical ecology of stingless bees, from communication over resource allocation to defense. I list examples in which functions of specific compounds (or compound groups) have been demonstrated by behavioral experiments, and show that many aspects (e.g., queen-worker interactions, host-parasite interactions, neuronal processing etc.) remain little studied. This review further reveals that the vast majority of studies on the chemical ecology of stingless bees have been conducted in the New World, whereas studies on Old World stingless bees are still comparatively rare. Given the diversity of species, behaviors and, apparently, chemical compounds used, I suggest that stingless bees provide an ideal subject for studying how functional context and the need for species specificity may interact to shape pheromone diversification in social insects.     

Intraspecific Geographic Variation of Fragrances Acquired by Orchid Bees in Native and Introduced Populations

Male orchid bees collect volatiles, from both floral and non-floral sources, that they expose as pheromone analogues (perfumes) during courtship display. The chemical profile of these perfumes, which includes terpenes and aromatic compounds, is both species-specific and divergent among closely related lineages. Thus, fragrance composition is thought to play an important role in prezygotic reproductive isolation in euglossine bees. However, because orchid bees acquire fragrances entirely from exogenous sources, the chemical composition of male perfumes is prone to variation due to environmental heterogeneity across habitats. We used Gas Chromatography/Mass Spectrometry (GC/MS) to characterize the perfumes of 114 individuals of the green orchid bee (Euglossa aff. viridissima) sampled from five native populations in Mesoamerica and two naturalized populations in the southeastern United States. We recorded a total of 292 fragrance compounds from hind-leg extracts, and found that overall perfume composition was different for each population. We detected a pronounced chemical dissimilarity between native (Mesoamerica) and naturalized (U.S.) populations that was driven both by proportional differences of common compounds as well as the presence of a few chemicals unique to each population group. Despite these differences, our data also revealed remarkable qualitative consistency in the presence of several major fragrance compounds across distant populations from dissimilar habitats. In addition, we demonstrate that naturalized bees are attracted to and collect large quantities of triclopyr 2-butoxyethyl ester, the active ingredient of several commercially available herbicides. By comparing incidence values and consistency indices across populations, we identify putative functional compounds that may play an important role in courtship signaling in this species of orchid bee.     

Chemical Signals in the Stingless Bee, <Emphasis Type="Italic">Frieseomelitta varia</Emphasis>, Indicate Caste,Gender, Age,and Reproductive Status

Chemical compounds on the cuticle are a rich source of information used during interactions among social insects. Despite the multitude of studies on these substances and their function in ants, wasps, and honeybees, little is known about this subject in stingless bees (Hymenoptera: Apidae, Meliponini). We studied the chemical composition of the cuticle of the stingless bee, Frieseomelitta varia, by gas chromatography-mass spectrometry (GC-MS), to investigate potential chemical variation among castes, gender, age, and reproductive status. We found differences in the cuticular hydrocarbon composition among workers, males, and queens, recording both qualitative and quantitative differences among individuals of different ages and gender. The cuticle of physogastric queens presented a chemical profile that was distinct from all other groups in the analysis, with high relative abundances of alkenes and alkadienes with 27, 29, and 31 carbon atoms. We discuss the possibility that these compounds signal a queen’s presence to the colony, thereby initiating all vital worker-queen interactions.     

Attraction of reproductive honey bee swarms to artificial nests by Nasonov pheromone

A crossover experimental design was established to test the attractancy of Nasonov pheromone to reproductive swarms of honey bees. Nineteen swarms were attracted to artificial nest cavities containing a slow-release blend of the Nasonov components citral, geraniol, and nerolic + geranic acids, and only four swarms were attracted to pheromone-free artificial nests. The results indicate that Nasonov pheromone plays a key role in the attraction of honey bee swarms to nest cavities.     

Qualitative Analysis of Red Imported Fire Ant Nests Constructed in Silica Gel

Red imported fire ants, Solenopsis invicta Buren, build nests by excavating soil. Incorporation of ant-derived chemicals in nesting material has long been known; however, only a few chemicals have been identified. This paucity of identified ant-derived chemicals may be due to the interference from soil-borne compounds in chemical analysis. In the laboratory, red imported fire ants were able to build their nest using moistened silica gel as the only building material. This provided an opportunity to establish a profile of ant-derived chemicals in nest material without the presence of any soil-borne artifacts. A new method for profiling ant-derived chemicals in nest material by using GC-MS was developed. All nests contained cuticular hydrocarbons and venom alkaloids. Phosphoric acid, glycerol, lactic acid, and malonic acid also were identified from samples collected from the silica gel nest.     

Odorants that Induce Hygienic Behavior in Honeybees: Identification of Volatile Compounds in Chalkbrood-Infected Honeybee Larvae

Social insects that live in large colonies are vulnerable to disease transmission due to relatively high genetic relatedness among individuals and high rates of contact within and across generations. While individual insects rely on innate immune responses, groups of individuals also have evolved social immunity. Hygienic behavior, in which individual honeybees detect chemical stimuli from diseased larvae and subsequently remove the diseased brood from the nest, is one type of social immunity that reduces pathogen transmission. Three volatile compounds, collected from larvae infected with the fungal pathogen Ascosphaera apis and detected by adult honey bees, were identified by coupled gas chromatography-electroantennographic detection and gas chromatography-mass spectrometry. These three compounds, phenethyl acetate, 2-phenylethanol, and benzyl alcohol, were present in volatile collections from infected larvae but were absent from collections from healthy larvae. Two field bioassays revealed that one of the compounds, phenethyl acetate is a key compound associated with Ascosphaera apis-infected larvae that induces hygienic behavior.     

Robber bees (Lestrimelitta limao) and their host chemical and visual cues in nest defense byTrigona (Tetragonisca) angustula (Apidae: Meliponinae)

The nest of the stingless bee,Trigona (Tetragonisca) angustula, is guarded by bees positioned in the nest entrance and others hovering in front of it. Hovering guard bees track returning foragers sideways along the last 10 cm in front of the nest, but intercept and incapacitate nest intruders by clinging with mandibles to wings and legs. When attacked by the cleptobiotic stingless beeLestrimelitta limao, the colony strengthens its aerial defense with hundreds of additional hoverers. To test our hypothesis that this reaction is due to interspecific chemical communication based on kairomone effects, we presented synthetic cephalic volatiles of both species at the nest entrance and counted the number of bees leaving the nest and taking up hovering positions. We conclude that guard bees recognizeL. limao by the major terpenoids of their volatile cephalic secretions, geranial, neral (=citral) and 6-methyl-5-hepten-2-one; other components may fine-tune this recognition. The effect of chemical stimuli is not significantly enhanced by combination with a dummy ofL. limao. Guard bees, we hypothesize, respond to this kairomone by secreting a species specific alarm pheromone; a major component of this pheromone, benzaldehyde, recruits additional bees to defend the nest.     

The Effects of Nectar–Nicotine on Colony Fitness of Caged Honeybees

Nectar of many bee flowers contains secondary compounds, which are considered toxic for honeybees on repeated exposure. Although many anecdotal reports indicate the toxicity of secondary compounds to bees, only a few studies have tested the extent of toxicity at different honeybee ages, especially at the larval stages. Honeybees encounter nicotine at trace concentrations (between 0.1 and 5 ppm) in floral nectar of a few Nicotiana spp. and in Tilia cordata. Adult honeybee workers tolerate these nicotine concentrations. In controlled nonchoice feeding experiments with caged bees, we investigated the effect of nicotine on hatching success and larval and forager survival. Naturally occurring concentrations of nectar–nicotine did not affect hatching success of larvae or their survival, but the latter was negatively affected by higher concentrations of nicotine (50 ppm). Concentrations of nicotine in fresh honey samples from the hives were 90% lower than the concentrations in the offered experimental sucrose solutions. Our results indicate that honeybees can cope with naturally occurring concentrations of nicotine, without notable mortality, even when consumed in large quantities for more than 3 weeks.     

Selective Behaviour of Honeybees in Acquiring European Propolis Plant Precursors

Honey bees harvest resins from various plant species and use them in the hive as propolis. While there have been a number of studies concerning the chemical composition of this antimicrobial product, little is known about selective behavior and bee preference when different potential plant sources of resin are available. The main objective of this paper was to investigate some aspects of behavioral patterns of honeybees in the context of resin acquisition. Samples of propolis originating from temperate zones of Europe and the supposed botanical precursors of the product were analyzed. Taxonomical markers of bud resins of two white birch species, aspen, black poplar, horse-chestnut, black alder, and Scots pine were determined through GC-MS analysis. All these trees have been reported as sources of propolis, but comparisons of the chemical composition of their bud resins with the compositions of propolis samples from seven European countries have demonstrated the presence of taxonomical markers only from black poplar, aspen, and one species of birch. This suggests selective behavior during the collection of bud resins by honeybees. To examine the causes of such selectivity, the antimicrobial properties of bud resins were determined. Horse-chestnut resins had lower antimicrobial activity than the other resins which did not differ significantly.     

Identification of Floral Volatiles and Pollinator Responses in Kiwifruit Cultivars, <Emphasis Type="Italic">Actinidia chinensis</Emphasis> var. <Emphasis Type="Italic">chinensis</Emphasis>

Volatiles emitted from unpollinated in situ flowers were collected from two male cultivars, ‘M33’, ‘M91’, and one female cultivar ‘Zesy002’ (Gold3) of kiwifruit (Actinidia chinensis var. chinensis). The samples were found to contain 48 compounds across the three cultivars with terpenes and straight chain alkenes dominating the headspace. Electrophysiological responses of honey bees (Apis mellifera) and bumble bees (Bombus terrestris) to the headspace of the kiwifruit flowers were recorded. Honey bees consistently responded to 11 floral volatiles from Gold3 pistillate flowers while bumble bees consistently responded to only five compounds from the pistillate flowers. Nonanal, 2-phenylethanol, 4-oxoisophorone and (3E,6E)-α-farnesene from pistillate flowers elicited responses from both bee species. Overall, honey bees were more sensitive to the straight chain hydrocarbons of the kiwifruit flowers than the bumble bees, which represented one of the main differences between the responses of the two bee species. The floral volatiles from staminate flowers of the male cultivars ‘M33’ and ‘M91’ varied greatly from those of the pistillate flowers of the female cultivar Gold3, with most of the bee active compounds significantly different from those in the Gold3 flower headspace. The total floral emissions of ‘M33’ flowers were significantly less than those of the Gold3 flowers, while the total floral emissions of the ‘M91’ flowers were significantly greater than those of the Gold3 flowers.     

Predator deterrence by mandibular gland secretions of bees (Hymenoptera: Apoidea)

Volatile lipids from the mandibular gland secretions of bees (Hymenoptera: Apoidea) are potent olfactory repellents of foraging ants (Formica, Crematogaster) in biologically relevant contexts and quantities. In contrast, differential success in capture of bee and fly prey by predatory asilid flies (Efferia), reduviid bugs (Apiomerus), and arachnids (Agelenopsis, Argiope) is better explained by prey size than by chemical repellence, aposematism, or possession of a sting. Supernormal doses of some allomones, applied to worker honeybees (Apis mellifera) that were fed toArgiope aurantia spiders, elicted more frequent preenvenomation pauses following ensnarement but did not significantly increase other prey-handling times. These pauses merely delayed the bee's demise. Mandibular gland secretions of solitary bees augment their other secondary defenses in at least two contexts:

1. during intranest encounters when repelling intruding ants, and
2. retaliation delivered to their arthropodan predators which, if the bee is nearly too large for the predator to handle, may allow the bee to escape.

     

All-trans-farnesyl hexanoate and geranyl octanoate in the dufour gland secretion ofAndrem (Hymenoptera: Apidae)

The volatile material which emanates from the Dufour gland of femaleAndrena bees has been examined in 13 species. In 11 of these, all-trans-farnesyl hexanoate is the dominating compound, whereas in two species geranyl octanoate is the largest component of the volatile secretion. Either of these two terpene esters were also found as major component in the cephalic secretion of some maleNomada bees. Bees of the genusNomada are nest parasites onAndrena. The chemical identification was carried out by capillary gas chromatography using a splitter-free intake system, alone and in combination with mass spectrometry. Some farnesyl and geranyl esters were prepared in the course of the work by reacting terpenol and acid with CDI (N, N′-carbonyldiimidazole). The secretion is thought to serve as a nest marking.     

Wax Lipids Signal Nest Identity in Bumblebee Colonies

The signalling functions of cuticular lipids, particularly cuticular hydrocarbons, have gained considerable attention in social insect communication. Information transfer between individuals by means of these substances has been examined extensively. However, communication with cuticular lipids is not limited to inter-individual recognition. Cuticular compounds can also have a signalling function in the nest environment. Workers of the bumblebee Bombus terrestris leave cuticular lipid traces, so-called footprints, that mark their nest entrance. In addition, there is evidence that bumblebees sense nesting material to identify their colony. In this study, we examined the signalling potential of bumblebee wax, and tested if bumblebee workers are able to identify their colony with the help of wax scent. Chemical analyses of wax extracts using coupled gas chromatography–mass spectrometry showed that wax from colonies of the bumblebee B. terrestris contained a complex blend of cuticular lipids, dominated by hydrocarbons and wax esters. Comparing the relative compound amounts of wax samples from different colonies, we found that wax scent patterns varied with nest identity. Olfactometer bioassays showed that bumblebees were able to discriminate between wax scents from their own and a foreign colony. Our findings suggest that wax emits characteristic olfactory profiles that are used by workers to recognize their colony.     

Surface lipids of social waspPolistes melricus say and its nest and nest pedicel and their relation to nestmate recognition

In eight replicate laboratory tests wherePolistes metricus adults were allowed to choose between their own nest, a second nest, and neither nest, they selected their own nest 66% of the observed time. When the surface hydrocarbons had been extracted from the nests, the wasps chose their own nest only 8% of the time, but after the hydrocarbons were reapplied to the surface of the respective nests, they selected their own nest 47% of the time. These changes are significant. The cuticular lipids were analyzed from individualP. metricus adult females collected from 13 colonies. Surface lipids were recovered from the paper and pedicels of their nests. Eighteen hydrocarbons were identified in these lipid fractions. The major components of the wasp cuticular lipids weren-heptacosane,n-nonacosane, methylhentriacontane, and methyltritriacontane. Factor analysis revealed that extracts of pedicels are all similar in composition, while cuticle and paper extracts vary, sometimes similarly according to colony identity.     

Chemical Changes Associated with the Invasion of a Melipona scutellaris Colony by Melipona rufiventris Workers

Wax constituents produced by worker bees and the chemistry of the nest batumen (mixture of wax, mud, and floral materials) in a Melipona scutellaris colony changed when it was invaded by Melipona rufiventris workers. Gas chromatography/mass spectrometry analyses showed that after invasion, the M. scutellaris workers of the invaded colony produced waxes with higher relative abundance of triacontanyl acetate and decreased the amounts of n-alkanes and n-9-alkenes. On the other hand, waxes from M. rufiventris workers displayed few changes. The change in the composition of the M. scutellaris waxes chemically differentiates that species from the M. rufiventris invader workers. Comparative analyses of batumens samples from pure and invaded colonies revealed greater amounts of terpenes and phenolic derivatives in the batumen from the invaded colony. This is the first report on the chemical characterization of batumens from stingless bees.     

Discriminant analysis of cuticular hydrocarbons of social waspPolistes exclamans viereck and surface hydrocarbons of its nest paper and pedicel

The articular lipids were analyzed from individualPolistes exclamans workers collected from 10 nests. Surface lipids were also recovered from the paper and pedicels of these nests. Twenty-two hydrocarbons were identified in these lipid fractions. The major components of the wasp cuticular lipids weren-heptacosane,n-nonacosane, methylhentriacontane, and methyltritriacontane. Discriminant analysis of the hydrocarbon profiles of the adult wasps showed that the wasps group together according to their respective colonies. Several colonies from the same geographical location clustered more closely together than colonies from diverse locations. The nest papers and pedicels did not group with the wasps from their respective colonies, but the nest papers clustered together separately, as did the nest pedicels.