Characterization of sensory neurons in Anopheles mosquito appendages

As vectors of Plasmodium parasites, Anopheles mosquitoes are a threat to global human health. By following different sensory cues, females use neurons in their chemosensory appendages to locate hosts for blood-feeding. However, a comprehensive characterization of the neurons innervating appendages of Anopheles mosquitoes has been lacking. To gain genetic access to all Anopheles coluzzii mosquito neurons, we created a knock-in of the pan-neuronal gene bruchpilot (brp) using the Homology Assisted CRISPR Knock-in (HACK) approach. Using this new genetic reagent, we visualized and quantified all neurons in female sensory appendages of the head (antennae, maxillary palps, and labella) and body (tarsi and ovipositor). For consistent and efficient 3-dimentional neuron counting, we developed and optimized a semi-automatic image processing pipeline. We compared neuron numbers in our pan-neuronal mosquito line to those in a mosquito line with only odorant receptor (OR) neurons labeled. We found that 36%, 38%, 73%, and 100% of neurons in antennae, palps, labella, and legs, respectively, did not express any odorant receptors, suggesting that those additional neurons likely express ionotropic or gustatory gene families. Our pan-neuronal line allows genetic access and identification of new chemosensory gene targets for mutational and functional analyses of mosquito responses to important odors involved in host-seeking and blood-feeding, including attractants and repellents.