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Efferent modulation of auditory sensitivity in mosquitoes: From auditory transduction to swarming behaviour

Periodic Reporting for period 1 - NEURO-SWARM (Efferent modulation of auditory sensitivity in mosquitoes: From auditory transduction to swarming behaviour)

Reporting period: 2017-04-01 to 2019-03-31

Malaria is a disease transmitted by mosquitoes that kills nearly half a million people each year, mostly young children in Sub-Saharan Africa. Mosquito control, or the reduction of mosquito populations, is essential to fight the disease. The emergence of insecticide resistance in recent years push for the development of novel vector control tools to reduce the impact of malaria worldwide. The malaria mosquito depends on their sense of hearing to mate as mating partners are recognized through the sound produced by their beating wings. These acoustic interactions leading to mating take place when mosquitoes come close to each other in aerial swarms. As hearing and swarming behaviour are essential for mosquito reproduction, they are prime targets for control interventions. However, they have been traditionally underexploited due to a lack of understanding of the biological mechanisms that orchestrate them. The project NEURO-SWARM has investigated novel molecular pathways that modulate mosquito hearing and swarming behaviour to target them as a novel mosquito control approach.
Previously, we had shown that mosquito audition is modulated by the action of the biogenic amines octopamine and serotonin. As octopamine and serotonin receptors are potential targets for the development of novel insecticides, they offer a promising alternative to disrupt mosquito hearing and therefore mosquito reproduction. The objectives of the Marie Curie IF project NEURO-SWARM were to thoroughly study the role of octopamine and serotonin in malaria mosquito hearing and to investigate their relation to swarming behaviour. By combining molecular, physiological and behavioural analysis we aimed to unravel this fascinating system and understand its potential as vector control target.
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.
Mosquito hearing and swarming behaviour are novel targets for mosquito control tools as they would have a drastic impact on mosquito reproduction. Although in the last decade significant progress has been made in understanding the mechanical and electrical properties of the mosquito ear, very little is known about the molecular mechanisms that coordinate mosquito auditory physiology. Likewise, the endogenous cues that induce mosquitoes to swarm are still elusive. This gap in knowledge has seriously hampered the exploitation of mosquito hearing and swarming behaviour with public health purposes.
In NEURO-SWARM, I have investigated a system based on aminergic neurotransmitters that reveals novel molecular targets to potentially disrupt these mechanisms. The results show that both octopamine and serotonin contribute to control the mosquito auditory physiology, and suggest that they facilitate the acoustic interactions, by which mosquito mating decisions are being negotiated within the context of the swarm. In follow-up experiments, we will investigate how disrupting octopamine and serotonin signalling affects the ability of mosquitoes to swarm. All these data will inform us on the translation of our findings into mosquito control approaches. Our goal will be to disrupt the ability of mosquitoes to recognize their mating partners thereby reducing mosquito reproduction rates. This will not only have a clear public health impact to diminish disease-transmitting mosquito numbers, but will add mosquito hearing and swarming behaviour to the spectrum of mechanisms targeted by control interventions.