Marked interspecific differences in the neuroanatomy of the male olfactory system of honey bees (genus Apis).

All honey bee species (genus Apis) display a striking mating behavior with the formation of male (drone) congregations, in which virgin queens mate with many drones. Bees' mating behavior relies on olfactory communication involving queen-but also drone pheromones. To explore the evolution of olfactory communication in Apis, we analyzed the neuroanatomical organization of the antennal lobe (primary olfactory center) in the drones of five species from the three main lineages (open-air nesting species: dwarf honey bees Apis florea and giant honey bees Apis dorsata; cavity-nesting species: Apis mellifera, Apis kochevnikovi, and Apis cerana) and from three populations of A. cerana (Borneo, Thailand, and Japan). In addition to differences in the overall number of morphological units, the glomeruli, our data reveal marked differences in the number and position of macroglomeruli, enlarged units putatively dedicated to sex pheromone processing. Dwarf and giant honey bee species possess two macroglomeruli while cavity-nesting bees present three or four macroglomeruli, suggesting an increase in the complexity of sex communication during evolution in the genus Apis. The three A. cerana populations showed differing absolute numbers of glomeruli but the same three macroglomeruli. Overall, we identified six different macroglomeruli in the genus Apis. One of these (called MGb), which is dedicated to the detection of the major queen compound 9-ODA in A. mellifera, was conserved in all species. We discuss the implications of these results for our understanding of sex communication in honey bees and propose a putative scenario of antennal lobe evolution in the Apis genus.

Age-specific olfactory attraction between Western honey bee drones (Apis mellifera) and its chemical basis.

During the mating season, drones (males) of the Western honey bee (Apis mellifera) form congregations numbering thousands high in the air. Virgin queens arrive at these congregations after they have formed and mate on the fly with 15-20 drones. To explain the formation of drone congregations, a drone-produced aggregation pheromone has been proposed many years ago but due to the low accessibility of natural mating sites in bees, its study has progressed slowly. Recently, we used a walking simulator in controlled laboratory conditions to show that drones are indeed attracted by groups of other drones. Since these previous experiments were carried out with drones captured when flying out of the hive, it is currently unclear if this olfactory attraction behaviour is related to the drones' sexual maturity (usually reached between 9 and 12 days) and may thus be indicative of a possible role in congregation formation, or if it is observed at any age and may represent in-hive aggregation. We thus assessed here the dependency of drone olfactory attraction on their age. First, we performed behavioural experiments in the walking simulator to measure olfactory preferences of drones in three age groups from 2-3 to 12-15 days. Then, we performed chemical analyses in the same age groups to evaluate whether chemical substances produced by the drones may explain age differences in olfactory attraction. We show that honey bee drones are attracted by conspecifics of the same age when they are sexually mature (12-15 days old) but not when they are younger (2-3 and 7-8 days old). In parallel, our data show that drones' chemical profile changes with age, including its most volatile fraction. These results are discussed in the context of drone mutual attraction both within the hive and at drone congregations.

Virgin queen attraction toward males in honey bees.

Although the honeybee is a crucial agricultural agent and a prominent scientific model organism, crucial aspects of its reproductive behaviour are still unknown. During the mating season, honeybee males, the drones, gather in congregations 10-40 m above ground. Converging evidence suggests that drones emit a pheromone that can attract other drones, thereby increasing the size of the congregation. Virgin queens join the vicinity of the congregation after it has formed, and mate with as many as 20 males in mid-air. It is still unclear which sensory cues help virgin queens find drone congregations in the first place. Beside visual cues for long-range orientation, queens may use olfactory cues. We thus tested virgin queens' olfactory orientation on a walking simulator in which they have full control over odour stimulation. We show that sexually-mature virgin queens are attracted to the odour bouquet from a group of living drones. They are not attracted to the bouquet from a group of workers. In addition, non-sexually receptive females (workers) of the same age are not attracted to the drone odour bouquet. Interpreted in the context of mating, these results may suggest that virgin queens use volatile olfactory cues from the drones to find the congregations.

Honeybee drones are attracted by groups of consexuals in a walking simulator.

During the mating season, honeybee males, the drones, gather in congregation areas 10-40 m above ground. When a receptive female, a queen, enters the congregation, drones are attracted to her by queen-produced pheromones and visual cues and attempt to mate with the queen in mid-air. It is still unclear how drones and queens find the congregations. Visual cues on the horizon are most probably used for long-range orientation. For shorter-range orientation, however, attraction by a drone-produced aggregation pheromone has been proposed, yet so far its existence has not been confirmed conclusively. The low accessibility of congregation areas high up in the air is a major hurdle and precise control of experimental conditions often remains unsatisfactory in field studies. Here, we used a locomotion compensator-based walking simulator to investigate drones' innate odor preferences under controlled laboratory conditions. We tested behavioral responses of drones to 9-oxo-2-decenoic acid (9-ODA), the major queen-produced sexual attractant, and to queen mandibular pheromone (QMP), an artificial blend of 9-ODA and several other queen-derived components. While 9-ODA strongly dominates the odor bouquet of virgin queens, QMP rather resembles the bouquet of mated queens. In our assay, drones were attracted by 9-ODA, but not by QMP. We also investigated the potential attractiveness of male-derived odors by testing drones' orientation responses to the odor bouquet of groups of 10 living drones or workers. Our results demonstrate that honeybee drones are attracted by groups of other drones (but not by workers), which may indicate a role of drone-emitted cues for the formation of congregations.