Abstract Insect nicotinic acetylcholine receptors (nAChRs) are ligand gated ion channels mainly expressed in the central nervous system of insects. They are the directed targets of nicotinic modulation insecticides including neonicotinoids, the most widely used insecticides in the world. However, the resistance development from pests and the negative impacts on the pollinators affect their applications and create demand for the alternatives. Thus, it is very important to understand the mode of action of these insecticides at the molecular level, which is actually unclear for more than 30 years. In this study, we systematically examined the susceptibility of ten Drosophila melanogaster nAChR subunits mutants against eleven nicotinic modulation insecticides. Our results showed that there are several subtypes of nAChRs with distinct subunits compositions that are responsible for the toxicity of different insecticides, respectively. At least three of them are the major molecular targets of seven structurally similar neonicotinoids in vivo . On the other hand, the spinosyns may exclusively act on the α6 homomeric nAChR but not any other heteromeric pentamers. Behavioral assays using thermogenetic tools further confirmed the bioassay results and support the idea that receptor activation rather than inhibition leads to the insecticidal effects of neonicotinoids. The present findings reveal native nAChR subunit interactions with various insecticides and have important implications for resistance management and the development of novel insecticides targeting this important ion channels. Author Summary The neonicotinoids and spinosyns make up about 27% of the insecticides by world market value. Novel insecticides like sulfoxaflor, flupyradifurone and triflumezopyrim are developed as alternatives due to the negative effects of neonicotinoids on pollinators. Although all act via insect nicotinic acetylcholine receptors, the mode of action is unclear. Our work shows that these insecticides act on diverse receptor subtypes with distinct subunit compositions. This finding could lead to the development of more selective insecticides to control pests with minimal effects on beneficial insects.

Nicotinic modulation insecticides act on diverse receptor subtypes with distinct subunit compositions