Haematophagous insects are frequently carriers of parasitic diseases, including malaria. The mosquito Anopheles gambiae is the major vector of malaria in sub-Saharan Africa and is thus responsible for thousands of deaths daily. Although the role of olfaction in A. gambiae host detection has been demonstrated, little is known about the combinations of ligands and odorant binding proteins (OBPs) that can produce specific odor-related responses in vivo. We identified a ligand, indole, for an A. gambiae odorant binding protein, AgamOBP1, modeled the interaction in silico and confirmed the interaction using biochemical assays. RNAi-mediated gene silencing coupled with electrophysiological analyses confirmed that AgamOBP1 binds indole in A. gambiae and that the antennal receptor cells do not respond to indole in the absence of AgamOBP1. This case represents the first documented instance of a specific A. gambiae OBP–ligand pairing combination, demonstrates the significance of OBPs in odor recognition, and can be expanded to the identification of other ligands for OBPs of Anopheles and other medically important insects.

Significance: An old enigma in insect toxicology, the mode of action (MoA) of selective chitin biosynthesis inhibitors in arthropods, is resolved. Benzoylureas, buprofezin, and etoxazole share a MoA by directly interacting with chitin synthase 1. The finding that a single mutation confers striking levels of insecticide resistance against three putative different MoAs has important ramifications on resistance management strategies and rational use of insecticides against major agricultural pestsand vectors of human diseases. Our results also show that CRISPR/Cas9-mediated gain-of-function mutations in single-copy genes of highly conserved target sites in arthropods provide opportunities for comprehensive insecticide resistance investigations across species boundaries and against several insecticide classes.