Comparative genomics of nicotinic acetylcholine receptors reveals early divergence of the α6 subunit in cryptic Anopheles species.

BACKGROUND: Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels that mediate fast cholinergic transmission in the insect central nervous system and serve as targets for several widely used classes of insecticides. nAChR-targeting formulations are being deployed in malaria vector control programs to mitigate widespread resistance to pyrethroids. However, the extent of genetic variation at insecticide target sites that may facilitate evolutionary responses to chemical exposure among vector species remains poorly understood. RESULTS: Here, we used whole-genome and Sanger sequencing to examine amino acid substitutions across all 11 nAChR subunits in sibling species of the Anopheles gambiae complex in Africa. The gene family is highly constrained, with only 33 nonsynonymous mutations detected at very low frequency in wild populations throughout the continent. No substitutions were observed at canonical ligand-binding residues within orthosteric domains, suggesting that acetylcholine and neonicotinoid binding affinities are unlikely to differ between the cryptic species. However, despite the evolutionary constraint, we identified two linked amino acid substitutions in the α6 subunit indicating early divergence of the receptor between species. CONCLUSIONS: These findings show that relaxed constraint can enable subtle amino acid changes in specific subunits of a conserved receptor family. The role of these mutations in shaping heterogeneous responses to insecticide exposure among mosquito species warrants further investigation.