Identification of Queen Sex Pheromone Components of the Bumblebee Bombus terrestris

We investigated the origin and chemical composition of the queen sex pheromone of the primitively eusocial bumblebee, Bombus terrestris (Apidae). Physiologically and behaviorally active compounds were identified by coupled gas chromatography electroantennography (GC-EAD), gas chromatography-mass spectrometry (GC-MS), and laboratory behavioral tests. In the behavioral assays, virgin queens frozen previously at −20°C were highly attractive to males. Dummies impregnated with surface and cephalic extracts obtained from virgin queens that had been frozen at −50°C were more attractive to males than odorless dummies. Male mating behavior was stimulated by components of cephalic secretions that are smeared onto the cuticle surface by the queen. Overall, 21 compounds present in surface and cephalic extracts evoked electroantennographic responses in male antennae. These included saturated and unsaturated fatty acids, ethyl- and methyl esters of the fatty acids, heptacosene, 2-nonanone, and geranyl geraniol. A blend of synthetic versions of these compounds elicited typical male mating behavior. Since solvent-impregnated dummies were approached by the males, but did not release copulatory behavior, visual cues may be important in the initial step of stimulating male mating behavior. Close-range olfactory signals are more important for releasing male mating behavior as well as for species recognition. In further behavioral assays, the attractiveness of a frozen virgin queen decreased as the storage time at −20°C increased from 2 hr to 1 d. Therefore, the chemical composition of the sex pheromone may change during freezing as behaviorally active compounds may decompose.     

Comparison of Age-dependent Quantitative Changes in the Male Labial Gland Secretion of <Emphasis Type="Italic">Bombus Terrestris</Emphasis> and <Emphasis Type="Italic">Bombus Lucorum</Emphasis>

Age-related changes of antennal-active components of male labial gland extracts were studied in two closely related bumblebee species, Bombus terrestris and B. lucorum. In B. terrestris, compounds eliciting electroantennogram (EAG) responses of virgin queens were ethyl dodecanoate, 2,3-dihydrofarnesal, 2,3-dihydrofarnesol, hexadecan-1-ol, octadeca-9,12,15-trien-1-ol, and geranylcitronellol. Compounds that elicited EAG responses from queens of B. lucorum were ethyl dodecanoate, ethyl tetradec-7-enoate, ethyl tetradec-9-enoate, ethyl hexadec-9-enoate, hexadecan-1-ol, hexadec-7-enal, octadeca-9,12-dien-1-ol, octadeca-9,12,15-trien-1-ol, and octadecan-1-ol. Quantities of these compounds in the labial glands changed significantly over the lifetime of the respective males of the two species. In both species, concentrations of the respective compounds reached their maximum within seven days after eclosion. Subsequently, a rapid decrease in the amount of EAG-active compounds occurred in B. terrestris, whereas in B. lucorum the amount of active compounds stayed approximately constant or decreased at a slow rate. Microscopy showed that in B. terrestris secretory cells of the labial glands undergo apoptosis from the fifth to the tenth day of life, whilst in B. lucorum labial gland cells remain unchanged throughout the life of the males.     

Comparative study by electrophysiology of olfactory responses in bumblebees (Bombus hypnorum andBombus terrestris)

Electrophysiological data (EAG) were recorded on adult bumblebees stimulated with floral and/or pheromonal pure odorants at different concentrations. The responses of queen, worker, and male bees are compared and the sensitivities of these insects to the pure odorants tested are discussed.     

Premating Behavior of Bombus Confusus Males and Analysis of Their Labial Gland Secretion

Premating behavior in the bumblebee Bombus confusus was studied. Visual searching for females is not the only premating strategy of this species, as was believed earlier. Males of B. confusus have a normally developed labial gland and its secretion is used to mark a perch from which they visually search for females. The labial gland secretion contains geranylcitronellol and (Z)-9-octadecenyl acetate as the main components.     

Scent Marking in Male Premating Behavior of Bombus confusus

Premating behavior in the species Bombus confusus Schenck, 1859 was studied. The marking habits of bumblebee males were revealed and the use of their labial gland secretion in scent marks was proved chemically. Identical compounds were present both in the labial gland secretion and on male-marked objects. This chemical proof is reported for the first time from natural conditions of a B. confusus locality. Males' flight activities, site tenacity, and male–male interactions are described in detail. Contrary to literature reports, male B. confusus behavior does not basically differ from that of already known perching bumblebee species.     

Unusual Fatty Acids in the Fat Body of the Early Nesting Bumblebee, <Emphasis Type="Italic">Bombus pratorum</Emphasis>

Unusual fatty acids with 24, 26, and 28 carbon atoms were found in triacylglycerols (TAGs) isolated from fat body tissue of bumblebee Bombus pratorum. The most abundant one was (Z,Z)-9,19-hexacosadienoic acid. Its structure was determined by mass spectrometry after derivatization with dimethyl disulfide and by infrared spectroscopy. ECL (equivalent chain length) values of its methyl ester were determined on both DB-1 and DB-WAX capillary columns. (Z,Z)-9,19-Hexacosadienoic acid is quite rare in nature. So far it has been identified only in marine sponges, and this work is the first evidence of its occurrence in a terrestrial organism. HPLC/MS analysis of the bumblebee TAGs showed that (Z,Z)-9,19-hexacosadienoic acid is present in one third of all TAG molecular species. As it was found in all sn-TAG positions, it is likely that (Z,Z)-9,19-hexacosadienoic acid is transported to tissues. Interestingly, labial gland secretion of B. pratorum was found to contain (Z,Z)-7,17-pentacosadiene, a hydrocarbon with markedly similar double bond positions and geometry. Possible biosynthetic relationships between these two compounds are discussed.     

Chemistry of the Cephalic Labial Gland Secretions of Male Bombus morrisoni and B. rufocinctus, Two North American Bumblebee Males with Perching Behavior

The labial gland secretions from males of the North American bumblebees Bombus morrisoni Cresson and B. rufocinctus Cresson were analyzed by gas chromatography/mass spectrometry. In both species, 3,7,11,15-tetramethyl-2,6,10,14-hexadecatetraenyl acetate was found as the major compound of a complex mixture of alkenols, acetates, hydrocarbons, and wax-type esters. In addition to a mixture of saturated and mono-unsaturated straight chain hydrocarbons, the labial gland of both species contained the isoprenoid hydrocarbons (6E, 10E)-3,7,15-trimethyl-3-methylene-hexadeca-1,6,10,14-tetraene [β-springene], three isomers of 3,7,11,15-tetramethyl-hexadeca-1,3,6,10,14-pentaene [α-springene], and two further unidentified cyclic diterpenes. In B. morrisoni, 3,7,11-trimethyl-2,6,10-dodecatrien-1-ol and 3,7,11,15-tetramethyl-6,10,14-hexadecatrien-1-ol were detected as characteristic alcohols, as well as small amounts of 9-hexadecenol and hexadecanol. Furthermore, a large peak of hexadecyl dodecanoate and minor amounts of 9-hexadecenyl, 9-octadecenyl, and eicosenyl 9-tetradecenoate were found as typical esters in this species. In B. rufocinctus, 9-hexadecenol, hexadecanol, and 9-octadecenol were present in considerable amounts, with their acetates and 9-tetradecenoic, tetradecanoic, hexadecanoic, and 11-octadecenoic acids. The chemical composition of cephalic labial glands in male bumblebees with perching behavior is discussed.     

Exocrine glands ofPolyrhachis simplex: Chemistry and function

The mandibular glands of the Israeli weaver ant,Polyrhachis simplex, contain a mixture of 4-heptanone, 6-methyl-5-hepten-2-one and 6-methyl-5-hepten-2-ol;its Dufour's gland secretion consists mainly ofn-tridecane. The significance of these glandular secretions in the biology of the weaver ant is discussed.     

Formic acid in caustic cephalic secretions of stingless bee,Oxytrigona (Hymenoptera: Apidae)

The cephalic extracts of two species in the stingless bee genusOxytrigona were analyzed. Extracts made in diethyl ether, and then derivatized with diphenyldiazomethane, revealed large quantities of formic acid, potentially a major defensive secretion of this stingless bee group. We additionally identify several hydrocarbons, aldehydes, ketones, and acetates. Novel diketones identified by others were detected in both species.     

Host recognition and the study of a chemical basis for attraction by cuckoo bumble bees (Hymenoptera: Apidae)

Species ofPsithyrus (Hymenoptera; Apidae) are obligate bumble bee social parasites. In this study, females ofP. vestalis andP. ashtoni were presented with pentane extracts prepared from different body parts of queens of their respective host species,Bombus terrestris andB. terricola. Parasites of both species were capable of distinguishing host bees from other bumble bee species using chemical cues contained within extracts. Among extracts of several body parts presented to parasites, the abdomen produced the greatest behavioral response, with Dufour's gland and terminal tergal segments eliciting the greatest response among abdominal regions. Extracts of these two body parts obtained fromB. terrestris queens shared a number of compounds, identified by GC-MS. Among the identified compounds are a number that have been reported to be of importance in bee sociochemistry.     

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.     

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.     

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.     

Sex Pheromone of Queens of the Slave-Making Ant, Polyergus breviceps

The sex attractant pheromone produced in mandibular glands of queens of the slave-making ant Polyergus breviceps has been identified as a blend of methyl 6-methylsalicylate and 3-ethyl-4-methylpentanol. In field trials, each compound alone was completely unattractive to males, whereas blends of the two compounds attracted hundreds of males within a couple of hours.     

The Male Abdominal Glands of <Emphasis Type="Italic">Leucophaea maderae</Emphasis>: Chemical Identification of the Volatile Secretion and Sex Pheromone Function

In Leucophaea maderae, male calling behavior involves the release of a sex pheromone from the abdominal sternal glands. An extract of sternal glands attracted conspecific females over a distance. The compounds present were identified as hydroxy-3-butan-2-one, (2R, 3R)-butanediol, senecioic acid, and (E)-2-octenoic acid. The same components are also present in male tergal glands. The identified compounds were tested on their own and in mixtures. Their biological function is discussed.     

Synthesis of carrier-free tritium-labeled queen bee pheromone

A short synthesis of [4,5-³H₂] (E)-9-oxo-2-decenoic acid (ODA), a high-specific-activity tritium-containing isotopomer of the queen bee pheromone, is described. Catalytic tritiation of the ketal of ethyl 9-oxo-4-decenoate introduces tritium into two positions, one of which is completely unactivated. Subsequent transformation by selenation, oxidation, and hydrolysis affords the labeled 9-ODA at >60 Ci/mmol. The material is suitable for biochemical studies of binding and catabolism in ovarian, antennal, and other target tissues.Fellow of the Alfred P. Sloan Foundation (1981–1985) and Camille and Henry Dreyfus Teacher-Scholar (1981–1986).     

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.     

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.     

Evidence for a multicomponent sex pheromone in the yellowheaded spruce sawfly

The existence of a female-produced sex pheromone in the yellowheaded spruce sawfly,Pikonema alaskensis (Rohwer) (Hymenoptera: Tenthredinidae) was demonstrated by field and greenhouse bioassays. Virgin females, their empty cocoons (with which they were confined during handling procedures), and the hexane extract of these cocoons were attractive in the field. The only Florisil fraction of this extract consistently attractive by itself was that eluted with hexane, but three, more polar fractions (eluted with 5%, 25%, and 50% ether in hexane) each synergized the hexane fraction, increasing bioassay responses 10–30 times. Fractions derived directly from virgin females yielded comparable results. The greenhouse data corroborated the field data, except that the 5% ether-hexane fraction, while very synergistic in the field, was consistently inactive in the greenhouse.     

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.