Neural and molecular mechanisms underlying the olfactory modulation of aggression in honeybees

Abstract

Although honeybees were domesticated over 7000 years ago, finding ways to manage their defensive responses against intruders, including humans, is still a current challenge. This is in part due to the complexity of this behaviour, which starts with the detection of the threat by a few specialized individuals and culminates into a generalized, collective attack triggered by the release of an alarm pheromone. Numerous studies have investigated honeybee aggression and stinging behaviour both in the laboratory and field, including the sensory triggers and the potential regulatory mechanisms. However the specific neural and molecular mechanisms regulating this behaviour are still unknown. In my PhD thesis, I investigated the role of olfactory signals and brain biogenic amines in modulating aggression in honeybees, integrating behavioural, physiological, and pharmacological experiments. Using a novel assay to measure the stinging behaviour of individual bees under controlled conditions, I first explored whether a range of plant odours could modulate aggression, in particular by interacting with the alarm pheromone released by aroused bees. I identified two floral compounds, linalool and 2-phenylethanol, that block the recruitment elicited by the alarm pheromone. These odours do not prevent the bees from perceiving the alarm pheromone. Instead, this blocking effect appears to correlate with the appetitive value of these odours. This suggests that a complex sensory integration takes place when honeybees are faced with the decision of engaging or not into defensive tasks. Furthermore, a field test demonstrated that linalool could also be used to manage aggressive colonies, highlighting the practical application of these findings. To gain a better understanding of the neuronal mechanism underlying this effect of floral odours on honeybee aggression, I next investigated how these floral compounds affect the representation of the alarm pheromone in the primary olfactory center of the honeybee brain, the antennal lobe, using in vivo calcium-imaging to monitor the activity of neurons in this area. The antennal lobes are structured into functional units called glomeruli, and an odour identity and concentration is encoded within the pattern and intensity of activated glomeruli. We expected that the representation of the mixture of an appetitive floral odour with the alarm pheromone may not be linearly obtained from the representation of each compound, thus revealing the neuronal mechanisms at play during our previous aggression assays. However, analysis of the data suggests no such mechanism, which could be a clue that this processing is happening in higher brain centers.Finally, I investigated the role of brain biogenic amines in honeybee aggression. Biogenic amines are important neuromodulators and have been implicated in the regulation of the aggressive behavior of a number of species. However, their potential role in regulating the honeybee’s stinging behavior had not been investigated so far. My experiments showed that bees from aggressive colonies have higher serotonin levels in their central brain than bees from gentle colonies. In this region, bees exposed to the alarm pheromone during an aggression test also had more dopamine and serotonin than control bees. Serotonin levels were also higher in the optic lobes of aggressive bees, and in the subesophageal zone of bees responding to the alarm pheromone. Pharmacologically increasing serotonin and dopamine levels induced higher aggressiveness in bees, while decreasing them reduced aggressiveness. This confirms for the first time that serotonin and dopamine play a key role in regulating honeybee aggression. This thesis is the first integrated study of the molecular and neural mechanisms underlying aggressive behaviour in honeybees. Importantly, this body of work does not only increase our understanding of honeybee behaviour, it will also find application in the development of novel, olfactory-based methods to control honeybee aggression.