As part of NEURO-SWARM, I have been working at the host institution the Ear Institute at University College London between April 2017- March 2019. As part of the lab of Joerg Albert, I have received excellent training in mechanical and electrophysiological analysis to study mosquito hearing. I have also contributed with my molecular and cellular expertise to expand the array of techniques used by the lab.
During the first six months, we performed RNA-Seq analysis of mosquito ears and heads in male and female mosquitoes at six different circadian time points along the day (one of them coinciding with the circadian time point when mosquitoes swarm). We wanted to investigate how the patterns of expression of octopamine and serotonin receptors change along the day. In malaria mosquitoes, swarming behaviour occurs only at sunset. I expected a link between the action of octopamine and serotonin in modulating mosquito hearing and the acoustic interactions that take place within the swarm. This should be reflected in the temporal pattern of receptor expression. Our results show that two octopamine and two serotonin receptors are expressed in the mosquito ear and their expression is temporally modulated. Interestingly, one octopamine receptor shows a peak of expression at swarming time. By contrast, the expression of one serotonin receptor is lowest during swarming (Fig. 1). These results suggest octopamine and serotonin as putative candidates for modulating mosquito acoustic communication during swarming.
The next part of the project investigated how the observed molecular data link to the physiology of the malaria mosquito ear. I first aimed at analyzing whether auditory response properties followed the same temporal pattern as that observed for octopamine and serotonin receptors. Mechanical and electrical measurements of the male and female mosquito ears were assessed at the same circadian time points as the RNA-Seq analysis. Although a difference was found in the spontaneous tuning of the ear in male mosquitoes in the absence of sound stimuli, which is shifted towards higher frequencies at swarming time (reflecting changes in the properties of the auditory neurons), the ears showed the same electrical and mechanical responses to sound along the day both in males and females. These results are both surprising and interesting and further analyses are required to interpret them.
The last part of the project investigated the direct effect of octopamine and serotonin in mosquito auditory physiology and swarming behaviour. I artificially activated their receptors by injecting octopamine and serotonin into the mosquito (Fig. 2). Octopamine injection has two major effects. First, it causes the erection of the small fibrillae that cover the mosquito antenna (the sound receiver), a phenomenon that in nature is observed only when mosquitoes swarm. Second, it has a direct effect on auditory physiology causing a shift of both the mechanical and electrical tuning of the ear towards higher frequencies. This mimics the effect observed for the spontaneous tuning of the ear during swarming, suggesting a direct role of octopamine in modulating the auditory physiology of swarming mosquitoes. Serotonin injection seems to affect mostly the electrical responses of the ear, but does not cause the erection of the antennal fibrillae. To assess the effect of blocking the action of octopamine and serotonin in mosquito hearing, we are currently developing octopamine and serotonin receptor mutants in collaboration with the European Commission infrastructure project Infravec2. These mutants will allow us to distinguish the role of single octopamine and serotonin receptors in mosquito hearing to unravel this fascinating system. Moreover, mutant mosquitoes will be tested in the swarming facilities at the University of Perugia (Italy) following protocols that we are currently developing to identify any defects in malaria mosquito swarming behaviour.
