Cracking the honeybee olfactory code: from sexual communication to social behavior – Bee-o-CHOC

The honeybee Apis mellifera is the most important pollinator for agriculture worldwide. Its pollination service is gravely endangered by colony losses, which dramatically increased recently. To promote and maintain the health and genetic diversity of our honeybee populations, a better understanding of their reproductive and social behaviors is crucial. Honeybees’ behaviors strongly rely on olfaction both outside and within the hive. Many studies have described the neural structures involved in olfactory perception and learning, while neurophysiological recordings have provided extensive knowledge about olfactory processing in the bee brain. Surprisingly, however, little is known about processes at the periphery of the bee olfactory system, i.e. at the level of olfactory receptor neurons housed in the antennae. The key molecular actors involved in the detection of odorants are the olfactory receptors (ORs) expressed at the olfactory receptor neurons membrane. While the honeybee genome has been sequenced 10 years ago and ~170 genes encoding candidate ORs have been annotated, only 3 ORs are now tentatively deorphanized (i.e. the odor ligand for this receptor has been identified). Understanding the odor ligands that activate honeybees’ olfactory receptor neurons will open new research opportunities for understanding their remarkable olfactory abilities. In this project, we will express individual honeybee ORs in heterologous expression systems (Drosophila ORNs and Xenopus oocytes), and characterize their response spectra by screening ecologically-relevant odorants. More specifically, we will deorphanize ORs which are overexpressed in male bees (drones) and in workers, thus putatively involved in mating and in social behaviors like nestmate discrimination, respectively. The project proposes three aims:

AIM 1 addresses sexual communication in honey bees and endeavors to crack their sex code. We will aim to find the ligands for the three orphan ORs that are overexpressed in males and are thought to detect queen- and drone-produced pheromones. Individual ORs will be expressed in Drosophila olfactory receptor neurons, and receptor activation will be evaluated electrophysiologically by screening queen- and drone-emitted volatiles. The use of Drosophila ORNs is especially well adapted for deorphanizing other insects’ ORs because it provides a typical olfactory sensillar/cellular environment. We will confirm the activation of the drone brain (macroglomeruli) by candidate ligands using in vivo optical imaging. Their effect on drones’ behavior will be assessed in the lab using a locomotion compensator and within drone congregation areas using odor-laden baits.

AIM 2 addresses the language of bees’ social interactions, aiming to cracking their social code. The honeybee genome contains ~44 ORs of the 9-exon clade, which are more strongly expressed in female (worker) antennae. They are thought to detect cuticular hydrocarbons involved in honeybee nestmate discrimination, but this hypothesis has not been demonstrated yet. We will perform a comprehensive study of 9-exon ORs of the honeybee, implementing a high-throughput deorphanization method based on robot recordings from Xenopus oocytes. Learning and dyadic encounter (aggression) experiments will test their implication in nestmate recognition.

AIM 3 will map the honeybee olfactory repertoire in their brain. For all deorphanized ORs, we will thus study the coding of main and secondary ligands in the antennal lobe using in vivo optical imaging and our recent preparation for accessing hidden regions of this structure. Then, using multi-dimensional scaling techniques, we will use co-clustering of the odor-response spectra of individual ORs and of identified AL glomeruli to map OR representation in the AL.

The results shall stimulate both future fundamental research and the development of new honeybee protection and conservation strategies, by manipulating their mating and social behaviors.