Full‐length <scp>coding</scp> sequence analysis of the voltage‐gated sodium channel and acetylcholinesterase genes reveals target‐site mutations and acetylcholinesterase gene duplication in housefly ( <i>Musca domestica</i> ) populations in Japanese

Houseflies (Musca domestica L.), a widespread pest in livestock facilities, have developed resistance to commonly utilized insecticides, including pyrethroids, organophosphates and carbamates. However, the genetic status of insecticide resistance in Japanese housefly populations remains largely elusive. We used hybridization probe capture and next-generation sequencing (NGS) to examine the mutations across the entire coding sequence of two insecticide target genes, voltage-gated sodium channel (VGSC) and acetylcholinesterase (AChE), from 240 individual M. domestica collected from 14 livestock facilities in Japan between 2013 and 2024. This study is the first to utilize these molecular methods to examine insecticide resistance in houseflies collected from the field. Resistance-related mutations in VGSC (e.g., super-kdr [M918T + L1014F], kdr-His [L1014H] and L1014F) were detected in most wild populations, with differing allele frequencies. Notably, super-kdr was widespread (12/15 populations) with frequencies ranging from 0.06 to 1.00, whereas kdr-His was observed in 8/15 populations with 0.03-0.70 frequencies. For AChE, three known resistance mutations (V260L, G342A/V and F407Y) were observed in all populations, with at least one resistance allele present at 0.65-1.00 frequencies. An elevation in NGS read counts for AChE was noted in several populations, along with the presence of three distinct alleles at amino acid position 342, suggesting AChE gene duplication. These duplications may help in mitigating the fitness costs linked to AChE resistance. Our results show the widespread presence and complexity of insecticide resistance mutations in Japanese M. domestica populations and underscore the use of NGS-based probe capture techniques for monitoring the evolution of resistance.