Neuroethological bases of pheromonal sex communication in male honeybees (drones), Apis mellifera
Final Report Abstract
Honeybees, "Apis mellifera", are a main-stream animal model for the study of ethology, neurobiology, and animal cognition. Yet, for all the knowledge acquired on this model organism, crucial aspects of its reproductive behavior still remain elusive. 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. Drones compete for the queen in flight, scrambling for a good position to approach her and mate. Within 15-30 minutes the queen mates in mid-air with typically 10-20 drones, who die after copulation. 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 its existence was never confirmed conclusively. In this project, my collaborators and I investigated the pheromonal sex communication system of honeybee drones in an integrative research approach combining behavioral, neuroanatomical, and neurophysiological experiments. The low accessibility of congregation areas high up in the air is a major hurdle and precise control of experimental conditions remains often 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. Drones are an organism specially adapted for mating and their olfactory system is tuned for sex communication. Although the general layout of the drone antennal lobe (AL), the primary olfactory center of the insect brain, is known, a thorough, quantitative description of its organization is still lacking. We used confocal microscopy and 3D reconstructions aiming to build a morphological atlas of the drone AL. The drone AL, has been reported to contain 103 functional units, the glomeruli, 4 of which are hypertrophied macroglomeruli (MG). In contrast, our current results show a consistently higher number of glomeruli (~ 117). MG2, MG1, and MG4 are always the largest glomeruli and consistently at the same position. For "MG3", however, neither size nor position are consistent in different ALs. Furthermore, we found other glomeruli with a similar size as "MG3", which challenges the current status of "MG3" as a macroglomerulus. Our latest results cast into doubt, whether honeybee drones indeed have 4 macroglomeruli, whereas for other bee species only 2 have been described so far. Recently, the largest macroglomerulus, MG2, has been shown to respond specifically to 9-ODA. Behavioral experiments on free-flying drones and in our walking simulator suggest a combinatorial processing of 9-ODA and other queen-derived components. It is neither known how odors involved in sex communication are processed in the AL of drones nor to which odors the other macroglomeruli respond to. Using "in-vivo" calcium imaging, we studied neuronal responses of AL output neurons to a wide panel of honeybee pheromones. Comparing our output data with results from an earlier study on the AL input will help us understanding how the drone olfactory system processes odors that are crucial for sex communication and mating. Our walking simulator proved to be a powerful experimental tool for investigating insect behavior under controlled laboratory conditions while at the same time allowing the animals a high degree of behavioral freedom as they were allowed to freely walk in any direction. Building on the strengths of this experimental approach, we expanded the anticipated range of our behavioral experiments studying not only innate but also learned behavioral responses, and in a spin-off project we investigated aversive conditioning in honeybee workers. Our walking simulator allowed for the first time to study classical and operant conditioning in honeybees in the same experimental setup under controlled laboratory conditions. Workers were able to learn to associate both a light stimulus (color illumination; classical conditioning) as well as their own behavior (turning to the left or to the right; operant conditioning) with heat punishment. If given the choice between a classical and an operant learning rule, workers significantly avoided 1) the punished color and 2) turning into the punished direction at group level. Individual bees, however, were rarely using both learning rules at the same time: most bees avoided either the punished color or turning into the punished direction and, hence, relied on either the classical or the operant learning rule. In a next step, we are planning to investigate, whether there is a genetic basis favoring classical or operant learning ability in honeybees, e.g. with respect to different patrilines in honeybee colonies.
Publications
- Honeybee drones are attracted by groups of consexuals in a walking simulator. Journal of Experimental Biology 2014, 217: 1278-1285
Brandstaetter, Bastin, Sandoz
(See online at https://doi.org/10.1242/jeb.094292)
