Alarm pheromone production by two honeybee (Apis mellifera) types

Of 12 alarm pheromones assayed in European and Africanized honeybees, nine were found in larger quantities in the Africanized population. Isopentyl and 2-heptanone levels were similar in both; 2-methylbutanol-1 was greater in European workers. These differences were not due to age or geographical location. Significant positive correlations between alarm pheromone levels and defensive behavior, especially numbers of stings, were observed.     

Nasonov pheromone of the honeybee.Apis mellifera L. (Hymenoptera,Apidae)

Electroantennogram (EAG) responses from worker honeybee antennae were obtained for each Nasonov component. Response amplitudes to 10 g of components correlated well with reported relative abilities to attract foragers in the field. EAG responses of worker, queen, and drone antennae to natural pheromone were consistently greater than to synthetic pheromone, a difference only partly explained by enzymic conversion of geraniol to (E)-citral during preparation of natural extracts.     

Honeybee (Apis mellifera L.) queen feces: Source of a pheromone that repels worker bees

When placed in a small observation arena with workers, most young virgin honeybee queens released fecal (hindgut) material during agonistic interactions with workers and with each other. On release of this material, workers moved to the sides of the arena and groomed themselves. Bioassays of virgin queen fecal material demonstrated that it contains pheromone that repels workers and stimulates grooming behavior. Pheromone was present only in the feces of virgin queens that were more than 24 hr old and less than 2 weeks old. Feces of 2- to 4-day-old workers and virgin queens more than 2 weeks old did not elicit an avoidance response by workers. Moreover, the feces of young virgin queens had a strong fragrance, while that of older queens had a rancid odor and that of young workers had no detectable odor.     

Variability of chemosensory stimuli within honeybee (Apis mellifera) colonies: Differential conditioning assay for discrimination cues

Differential training of honeybee workers using the proboscis extension reflex is applied to the problem of evaluating compounds that may potentially provide cues for kin recognition in the honeybeeApis mellifera. These cues were obtained by contaminating glass rods and steel needles with different materials found in the hive. In particular it is shown that workers discriminate between: cuticular waxes from different adult workers; eggs from the same and different hives; similar aged larvae within the same hive; and needles contaminated with the Nasonov gland secretions of different adult workers. It appears that some of these differences are due to phenotypic variation among individuals that cannot be directly attributed to environmental factors.     

Recognition of Complex Odors by Restrained and Free-Flying Honeybees, Apis mellifera

Complex odor recognition in the honeybee was investigated using two behavioral assays: (1) the conditioning of the proboscis extension (CPE) with restrained individuals, and (2) the observation of foragers visiting an artificial feeder in a flight room. Nine compounds, previously identified as oilseed rape flower volatiles, were tested either individually or in mixtures. Different sets of experiments were done to determine: (1) the acquisition rate of the nine compounds in the CPE assay, and (2) the discrimination of the individual compounds after conditioning to a mixture, using the CPE assay and free-flying foragers. After conditioning to a complex mixture, honeybees established a hierarchy among the components, with some of them accounting for a major part of the behavioral activity of the mixture. Both behavioral assays led to the same classification of compounds, indicating good agreement between discriminating abilities of restrained individuals and of a population of foragers. The key compounds for recognition of these mixtures were those that were well learned when presented individually. However, the recognition of some compounds was affected by the other components of the mixture, with the activity of some compounds being either enhanced or reduced.     

Identification of Floral Volatiles Involved in Recognition of Oilseed Rape Flowers, Brassica napus by Honeybees, Apis mellifera

Volatiles from oilseed rape, Brassica napus, flowers were sampled by air entrainment and their relevance to the natural odor profile of the flowers was confirmed by conditioned proboscis extension (CPE) assays with honeybee, Apis mellifera L., foragers. Coupled gas chromatography (GC)-CPE analysis of the air entrainment samples was used to locate key compounds involved in the recognition of B. napus flowers, and the compounds were then identified using coupled gas chromatography-mass spectrometry and comparison with authentic samples. Six regions of the gas chromatograms elicited CPE responses from bees previously conditioned to the total extract, and from these areas 16 compounds were identified that elicited CPE activity from conditioned bees when tested with synthetic samples. Eight of the 16, -pinene, phenylacetaldehyde, p-cymene, -terpinene, linalool, 2-phenyl-ethanol, (E,E)--farnesene, and 3-carene, gave the highest responses. When the bees were conditioned to the total extract of flower volatiles, a mixture of the eight components elicited responses from 83% of the individuals, suggesting that the eight-component mixture accounted for a major part of the CPE activity of the total extract. In addition, a mixture of the three most active compounds, phenylacetaldehyde, linalool, and (E,E,)--farnesene, evoked responses from 85% of the bees after the latter had been conditioned to the eight-component mixture. Thus, these three compounds appear to play a key role in the recognition of the eight component mixture and, by inference, of oilseed rape flowers.     

Influence of Social Environment on Genetically Based Subfamily Signature in the Honeybee

In honeybees, the cuticular hydrocarbon profiles are partly genetically based and differ between subfamilies, which suggests that they might be used by the workers as labels for subfamily recognition. This ability could potentially form the basis for nepotistic conflicts between subfamilies that would be detrimental to the inclusive fitness of the colony. Here we have compared the subfamily hydrocarbon profiles of 5-day-old workers maintained in isolation with those kept in their parental colony. We demonstrate that the cuticular hydrocarbon profiles tend to be less distant between most subfamilies within the hive compared with those held in isolation. The main consequence of this partial homogenization of the majority of subfamily signatures may result in a reduction of the number of recognizable subfamilies in the colony. Nevertheless, a few subfamilies retain very distinct cuticular hydrocarbon profiles.     

Mandibular gland components of european and africanized honey bee queens (Apis mellifera L.)

The composition of the five-component honey bee queen mandibular gland pheromone (QMP) of mated European honey bee queens was compared to those of virgin and drone-laying (i.e., laying only haploid unfertilized eggs that develop into males), European queens and Africanized mated queens. QMP of mated European queens showed significantly greater quantities of individual components than all queen types compared, except for a significantly greater quantity of 9-hydroxy-(E)-2-decenoic acid (9-HDA) found in Africanized queens. Glands of European drone-laying queens contained quantities intermediate between virgin and mated queens, reflecting their intermediate reproductive state and age. QMP ontogeny shifts from a high proportion of 9-keto-(E)-2-decenoic acid (ODA) in young unmated queens to roughly equal proportions of ODA and 9-HDA in mated queens. A biosynthetic shift occurs after mating that results in a greater proportion of 9-HDA, methylp-hydroxybenzoate (HOB), and 4-hydroxy-3-methoxyphenylethanol (HVA) production, accompanied by a decreased proportion of ODA. Africanized QMP proportions of ODA and 9-HDA were significantly different from European queens. A quantitative definition of a queen equivalent of QMP is proposed for the various queen types, and a standard queen equivalent for mated European honeybee queen mandibular gland pheromone is adopted as 200µg ODA, 80µg 9-HDA, 20µg HOB, and 2 µg HVA.     

Honeybee nestmate recognition: Effects of queen fecal pheromones

Previous work has shown that queen honeybees,Apis mellifera, produce waxy esters composed of 8–14 carbon acids and 6–14 carbon alcohols in their feces. We tested these esters for effects on nestmate recognition; 11 of the 12 esters tested significantly modified the recognition characteristics of worker honeybees. Pairwise tests showed that workers can discriminate between at least some pairs of queen esters and that workers can discriminate between a queen ester and hexadecane (another known nestmate recognition cue). These results suggest that a queen may use the esters to enable workers to recognize her or to scent-mark her colony.     

Mediation of host selection by cuticular hydrocarbons in the honeybee tracheal MiteAcarapis woodi (Rennie)

Using a simple two-choice bioassay and video analysis of individual locomotory tracks, it was determined that a preference for young-bee hosts over old-bee hosts in female honeybee tracheal mites,Acarapis woodi (Rennie), is chemically mediated. When presented with a choice of cuticular extracts from 5-day-old and <1-day-old adult bees, mites showed a significant preference for the young-bee extract in three of four bee colony sources. This discrimination was due apparently to a greater positive response elicited by the young-bee extract rather than a deterrent effect of old-bee extract, as the extract of old bees evoked a significantly higher response than a hexane control. A bioassay of silica-gel fractions of young-bee extract indicated that cuticular hydrocarbons alone were responsible for tracheal mite response. Further fractionation of a hydrocarbon fraction by argentation chromatography demonstrated that both saturated and unsaturated hydrocarbons were involved in the mite response, but when presented in opposition, mites showed a stronger response to the saturated than to the unsaturated components. Mites placed in zones treated with cuticular extract of young bees exhibited higher angular velocities than those placed on hexane, causing them to remain in the extract-treated zones for extended periods. These results point to a possible control strategy whereby migration of tracheal mites to young-bee hosts could be disrupted by artificially altering the chemical profile of the honeybee cuticle.     

Eclectic chemisociality of the honeybee

A dazzling variety of honeybee behaviors are triggered by pheromones produced by disparate exocrine glands. A multiplicity of chemical releasers of social behavior has been demonstrated to regulate a diversity of societal interactions, and many of these compounds are synthesized with great caste specificity. Recent investigations have resulted in the identification of a host of new compounds that are products of either worker or queen honeybees. This report fractionates these newly identified exocrine products according to their glandular proveniences and focuses on both the structural and behavioral eclecticism that characterizes these chemical signaling agents.     

Isolation of 24-methylenecholesterol-derived oxidation products from queen honeybee ovaries (Apis mellifica L.)

The bioecological situation of 24-methylenecholesterol, the main sterol of many pollens and previously isolated from larvae, workers, and queens of the honeybeeApis mellifica L., is reviewed. A search for steroids in queen honeybee ovaries has led to the isolation of three compounds occurring in minute amounts: 24-methylenecholest-4-en-3-one (1), 24-oxochoiesterol (2), and 24-oxocholest-4-en-3-one (3), which were identified by direct comparison with authentic samples prepared from 24-methyienechoiesterol. These three substances have not been detected in lipids from other parts of queen or from worker honeybees, and their presence may reflect a particular oxidizing capability of the ovaries. The results so far observed are discussed in relationship with previous observations reported on honeybees.     

The influence of a queen-produced substance, 9HDA,on swarm clustering behavior in the honeybeeApis mellifera L.

The effect of enantiomers of the queen-produced substance, 9-hydroxy-(E)-2-decenoic acid (9HDA) on swarm clustering behavior of the honeybeeApis mellifera was studied. Caged queens were removed from the swarms at the start of each test and were replaced with small Petri dishes containing one of the following treatments: 100 gS(+) enantiomer of 9HDA, 100 gR(-) enantiomer of 9HDA, 200 g racemic (R, S) 9HDA, and a vehicle-treated control. Each swarm was considered to have dispersed when it had lost 50% of its starting weight. All treatments with 9HDA resulted in significantly longer swarm aggregation when compared with the control. Enantiomers were not shown to have different effects at theP 0.05 level of significance. However, observations on swarm behavior indicated that theR(-) enantiomer was the most active in retarding swarm dispersal.     

(Z)-11-eicosen-1-ol,an important new pheromonal component from the sting of the honey bee,Apis mellifera L. (Hymenoptera,Apidae.)

(Z)-11-Eicosen-1-ol was identified by GC-MS and microchemical methods as a major volatile component, ca. 5 g per insect, secreted by the sting apparatus of the worker honey bee. When presented on moving lures at the hive entrance, (Z)-11-eicosen-1-ol, like isopentyl acetate already known as an alarm pheromone, elicited stinging, and together these two compounds were as active as the natural pheromone from the sting. On stationary lures, (Z)-11-eicosen-1-ol prolonged the effectiveness of isopentyl acetate.     

Sociochemical alteration of honeybee hoarding behavior

Hoarding experiments were conducted with honeybees (Apis mellifera L.) in cages containing comb treated with either 2-heptanone, isopentyl acetate, citral, or geraniol. 2-Heptanone increased hoarding rates; isopentyl acetate decreased hoarding rates; citral and geraniol had no observed effect.     

Bioassay of compounds derived from the honeybee sting

Nine compounds identified from honeybee,Apis mettifera L., sting extracts and one compound identified from the honeybee mandibular gland were evaluated in a standardized laboratory test for their effectiveness in eliciting an alarm response from caged honeybees. Two,n-decyl acetate and benzyl alcohol, were judged ineffective as alarm pheromones. The remaining eight—2-nonanol, isopentyl acetate,n-butyl acetate,n-hexyl acetate, benzyl acetate, isopentyl alcohol, andn-octyl acetate from the sting and 2-heptanone from the mandibular gland-produced responses of similar frequency and strength.in cooperation with Louisiana Agricultural Experiment Station. Mention of a proprietory product does not constitute an endorsement by the USDA.     

Nasonov pheromone of the honey bee,Apis mellifera L. (Hymenoptera,Apidae). part III.

GC and GC-MS analyses of the multicomponent Nasonov pheromone of the honey bee, and of the air above insects releasing the pheromone, show that constant composition is maintained during release, despite differing volatilities of the components. The regulating mechanism may involve a specific enzyme process, detected in excised Nasonov glands, which converts the major component geraniol into the more volatile (E)-citral. Analysis of honey bees of known ages and at different times of year shows that maximum secretion occurs when foraging is most likely.     

The Nasonov pheromone of the honeybeeApis mellifera L. (Hymenoptera,Apidae). Part II. Bioassay of the components using foragers

The Nasonov pheromone of the honeybee comprises seven components, (Z)-citral, nerol, geraniol, nerolic acid, geranic acid, and (E,E)-farnesol. Bioassay of individual components showed each attracted foraging bees. A mixture of components in proportions present in the honeybee was as attractive as the natural secretion, and each component contributed to the attractiveness of the mixture. Honeybees responded anemotactically to the source of Nasonov odor. The presence of footprint pheromone enhanced the attractiveness of the synthetic Nasonov mixture. Nasonov and footprint pheromones may prove useful in attracting honeybees to crops needing pollination.     

Cuticle alkanes of honeybee larvae mediate arrestment of bee parasiteVarroa jacobsoni

The ectoparasitic miteVarroa jacobsoni invades worker brood cells of the honeybeeApis mellifera during the last 20 hr before the cells are sealed with a wax cap. Cuticle extracts of 8-day-old worker honeybee larvae occupying such brood cells have an arrestment effect on the mite. The mites run for prolonged periods on the extract, systematically returning onto the stimulus after touching the borders of the treated area. Mites increase walking speed and path straightness in response to increasing doses of a nonpolar fraction of the cuticle extract. Saturated straight-chain odd-numbered C₁₉–C₂₉ hydrocarbons were identified by thin-layer argentation chromatography and gas chromatography-mass spectrometry as the most active constituents, with branched alkanes also contributing to the arrestment effect of this active fraction. Analysis of the behavior responses to syntheticn-alkanes indicate that the response is probably based on a synergism between the different alkane components of the fraction rather than to an individual compound.     

Nasonov pheromone of the honey bee,Apis mellifera L. (Hymenoptera: Apidae)

Composition of the Nasonov pheromone of the honey bee has been reexamined using new procedures, including analysis of pheromone from single live insects by capillary column GC-MS. Two new components have been identified, nerol and (E,E)-farnesol, and the presence of components proposed previously has been confirmed. Absolute amounts or relative proportions of components in the pheromonal secretion have been determined.