The olfactory mode of action of mosquito insect repellents

Mosquitoes use their sense of smell to find human hosts, and disrupting mosquito olfaction should be an effective strategy to reduce host-seeking. Indeed, commonly used mosquito repellents, such as DEET, IR3535, picaridin, and oil of lemon eucalyptus presumably function by disrupting mosquito chemosensory signaling. However, how these repellents directly target the Anopheles gambiae olfactory system is not well understood. We developed transgenic Anopheles mosquitoes that express the calcium indicator GCaMP6f in most olfactory neurons. This allows direct monitoring of the activity of Anopheles mosquito olfactory neurons towards mosquito repellents. We found synthetic repellents DEET, IR3535, and picaridin did not directly activate Anopheles olfactory neurons, but instead masked the ability of olfactory neurons to respond to attractive odorants. A simple behavioral assay developed to examine the direct response of mosquitoes towards these odors found they did not directly repel Anopheles mosquitoes. This suggests the olfactory mode of action of man-made repellents is to render the host “odor-invisible” to the Anopheles mosquito. Interestingly, DEET odors could directly repel Aedes and Culex mosquitoes, suggesting a divergence of responses among mosquitoes. Natural repellents could directly repel Anopheles mosquitoes. We found natural repellents, like oil of lemon eucalyptus, activated small and reproducible subsets of olfactory neurons. Using a combination of microdissection, RNA-seq, and functional testing of Anopheles odorant receptors in a heterologous system, we identified the odorant receptors directly activated by oil of lemon eucalyptus. These odorant receptors can act as a guide as we investigate how odor repellents drive avoidance behaviors.