Field-evolved Resistance to Neonicotinoids in the Mosquito, , is Associated with Downregulation and Mutations of Nicotinic Acetylcholine Receptor Subunits Combined with Cytochrome P450-mediated Detoxification

Neonicotinoid insecticides act selectively on their nicotinic receptor targets leading to variable sensitivity among arthropods. This study aimed to investigate the molecular mechanisms underlying contrasting susceptibility to neonicotinoids observed in wild populations of two mosquito sibling species. Bioassays and a synergism test revealed that the sister taxa, and , from Yaounde, Cameroon, rely on cytochrome P450s to detoxify neonicotinoids and develop resistance. However, contrary to , populations are evolving stronger resistance to several active ingredients facilitated by mutations and reduced expression of nicotinic acetylcholine receptors. Six mutations were detected in coding sequences of the β1 and α6 subunits, including two substitutions in one of the loops that modulate ligand binding and sensitivity. Allele frequencies were strongly correlated with a susceptibility gradient between and suggesting that the mutations may play a key role in sensitivity. Messenger RNA expression levels of the β1, α3, and α7 subunits decreased dramatically, on average by 23.27, 17.50, 15.80-fold, respectively, in wild populations compared to a susceptible insectary colony. By contrast, only the β2 and α9-1 subunits were moderately downregulated (5.28 and 2.67-fold change, respectively) in field-collected adults relative to susceptible colonized mosquitoes. Our findings provide critical information for the application and resistance management of neonicotinoids in malaria prevention.