New binding sites of nicotinic acetylcholine receptors from Myzus persicae

The green peach aphid, Myzus persicae, is the most serious agricultural pest worldwide , its control relies mainly on insecticides such as neonicotinoids, due to their broad spectrum. Nicotinic acetylcholine receptors (nAChRs) are the target genes of neonicotinoids, , better knowledge of their toxicological mechanisms is crucial for developing new specific neonicotinoids, especially for non-model insects. In this study, we successfully cloned four nAChR alpha subunits in M. persicae and coexpressed them with Rattus norvegicus beta 2 in the Xenopus oocyte system. Screening results showed that Mp alpha 1 and Mp alpha 8 were narrowly tuned to sulfoxaflor and thiamethoxam, with EC50 values of 1.117 x 10-4 M and 9.681 x 10-5 M, respectively. Molecular modeling and docking results showed that a combination of positive (Lys) and aromatic (Trp) residues or positive (Lys) and polar (Ser) residues located at the interface of the corresponding alpha and beta subunits of nAChR in Mp alpha 1/rat beta 2 or Mp alpha 8/rat beta 2 was the main contributor to ligand binding. Therefore, three residues (W143A, K144A and F260A) of Mp alpha 1 and four residues (K63A, K134A, S146A and V290A) of Mp alpha 8 were mutated to validate their contributions to the binding affinity of the corresponding ligand. In Mp alpha 1, W143A resulted in a significantly lower sensitivity to sulfoxaflor than the other three mutations, and in Mp alpha 8, S146A significantly reduced the response to thia-methoxam, suggesting crucial roles of these predicted hydrogen bonding sites in channel activation. These results provide evidence of the neonicotinoid toxicological mechanism in M. persicae and reveal new gene targets and binding sites for future pesticide design.