Ninety‐four putative G protein‐coupled receptors (GPCRs) were identified in the Musca domestica genome. They were annotated and compared with their homologues in Drosophila melanogaster. Phylogenetic analyses of the GPCRs from both species revealed that several family members shared a closer relationship based on the domain architecture. The expression profiles of these genes were examined by quantitative real‐time PCR amongst three strains of the house fly, a near‐isogenic line strain with imidacloprid resistance (N‐IRS), the corresponding susceptible strain (CSS) and another strain derived from field populations with imidacloprid resistance (IRS). We found that five GPCR genes were upregulated in the N‐IRS and eight GPCR genes were upregulated in the IRS strains compared to the CSS strain. The transgenic lines of D. melanogaster with the GPCR genes (LOC101899380 in the N‐IRS strain and LOC101895664 in the IRS strain) exhibited significantly increased tolerance to imidacloprid, and higher expression of cytochrome P450 genes. Bioinformatic analysis of LOC101899380 was carried out based on its full‐length nucleic acid sequence and putative amino acid sequence, and it was named Methuselah‐like10 (Mthl10) owing to its homology with D. melanogaster Mthl10. A cell‐base cell counting kit‐8 toxicity assay demonstrated that the expression of the GPCR gene LOC101899380 in Spodoptera frugiperda (Sf9) cells using a baculovirus‐mediated expression system can elevate the cell tolerance to imidacloprid, indirectly supporting the hypothesis that the GPCR gene LOC101899380 plays some role in imidacloprid resistance. These results should be useful for furthering understanding of the regulatory pathway by which house flies develop resistance.

Two unique housefly strains, PSS and N-PRS (near-isogenic line with the PSS), were used to clarify the mechanisms associated with propoxur resistance in the housefly, Musca domestica. The propoxurselected resistant (N-PRS) strain exhibited >1035-fold resistance to propoxur and 1.70-, 12.06-, 4.28-, 57.76-, and 57.54-fold cross-resistance to beta-cypermethrin, deltamethrin, bifenthrin, phoxim, and azamethiphos, respectively, compared to the susceptible (PSS) strain. We purified acetylcholinesterase (AChE) from the N-PRS and PSS strains using a procainamide affinity column and characterized the AChE. The sensitivity of AChE to propoxur based on the bimolecular rate constant (K i) was approximately 100-fold higher in the PSS strain compared to the N-PRS strain. The cDNA encoding Mdace from both the N-PRS strain and the PSS strain were cloned and sequenced using RT-PCR. The cDNA was 2073 nucleotides long and encoded a protein of 691 amino acids. A total of four single nucleotide polymorphisms (SNPs), I162M, V260L, G342A, and F407Y, were present in the region of the active site of AChE from the N-PRS strain. The transcription level and DNA copy number of Mdace were significantly higher in the resistant strain than in the susceptible strain. These results indicated that mutations combined with the up-regulation of Mdace might be essential in the housefly resistance to propoxur.

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BACKGROUND The control of the housefly, Musca domestica, heavily relies on the application of insecticides. Propoxur, a carbamate, has been widely used for vector control. The housefly populations with high propoxur resistance display point mutations and overexpression of acetylcholinesterase. However, the roles of cytochrome P450 monoxygenases (P450s), as important detoxification enzymes, remain poorly understand in the housefly resistant to propoxur. RESULTS P450s were implied to contribute to propoxur resistance based on the synergism of piperonyl butoxide (PBO) and the increase of P450 enzyme activity in the near‐isogenic line propoxur resistant strain (N‐PRS). Five P450 genes (CYP6G4, CYP6A25, CYP304A1, CYP6D3, and CYP6A1) by RNA‐sequencing comparison were significantly up‐regulated in the N‐PRS strain with >1035‐fold resistance to propoxur. A total of 13 non‐synonymous mutations of three P450 genes (CYP6G4, CYP6D3, and CYP6D8) were found in the N‐PRS strain. The amino acid substitutions of CYP6D3 and CYP6D8 were probably not resistance‐associated single nucleotide polymorphisms (SNPs) because they were also found in the aabys susceptible strain. However, CYP6G4 variant in the N‐PRS strain was not found in the aabys strain. The conjoint analysis of mutations and a series of genetic crosses exhibited that the housefly propoxur resistance was strongly associated with the mutations of CYP6G4 gene. CONCLUSION Our results suggested that a combination of up‐regulated transcript levels and mutations of CYP6G4 contributed to propoxur resistance in the housefly. © 2021 Society of Chemical Industry.

Both insects and mammals all exhibit a daily fluctuation of susceptibility to chemicals at different times of the day. However, this phenomenon has not been further studied in the house fly (Musca domestica L.) and a better understanding of the house fly on chronobiology should be useful for controlling this widespread disease vector. Here we explored diel time-of-day variations in insecticide susceptibility, enzyme activities, and xenobiotic-metabolizing enzyme gene expressions. The house fly was most tolerant to beta-cypermethrin in the late photophase at Zeitgeber time (ZT) 8 and 12 [i.e., 8 and 12 h after light is present in the light-dark cycle (LD)]. The activities of cytochrome P450, GST, and CarE enzymes were determined in the house flies collected at various time, indicating that rhythms occur in P450 and CarE activities. Subsequently, we observed diel rhythmic expression levels of detoxifying genes, and CYP6D1 and MdαE7 displayed similar expression patterns with enzyme activities in LD conditions, respectively. No diel rhythm was observed for CYP6D3 expression. These data demonstrated a diel rhythm of metabolic detoxification enzymes and insecticide susceptibility in M. domestica. In the future, the time-of-day insecticide efficacy could be considered into the management of the house fly.