Mutation Report for: G262V_musdo-ACHE

The house fly (Musca domestica Linnaeus) is an important disease vector. Insecticide resistance is an obstacle to effective house fly control. Previous studies have demonstrated that point mutations in acetylcholinesterase (Ace), carboxylesterase (MdalphaE7) and voltage-sensitive sodium channel (Vssc), and over-expression of CYP6D1v1 confer insecticide resistance in the house fly. However, information about the status and underlying mechanisms of insecticide resistance in Kazakhstani house flies is lacking. In this study, we investigated the occurrence of genetic mutations associated with insecticide resistance in field house flies collected at six different locations in southern Kazakhstan. Four mutations (V260L, G342A/V, and F407Y) in Ace and three mutations (G137D and W251L/S) in MdalphaE7 were detected with appreciable frequencies. Notably, haplotypes carrying triple-loci mutations in Ace and double mutations in MdalphaE7 were found in Kazakhstan. The L1014H and L1014F mutations in Vssc, and CYP6D1v1 resistance allele were detected at a low frequency in some of the six investigated house fly populations. Phylogenetic analyses of haplotypes supported multiple origins of resistance mutations in Ace and MdalphaE7. These observations suggest that house flies in southern Kazakhstan may exhibit significant resistance to organophosphates and carbamates. Regular monitoring of insecticide resistance is recommended to achieve effective house fly control by chemical agents in southern Kazakhstan.

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 propoxur-selected 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 (Ki) 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.

Acetylcholinesterase (AChE), encoded by the Ace gene, is the primary target of organophosphates (OPs) and carbamates (CBs) in insects. Ace mutations have been identified in OP and CB resistant strains of Musca domestica. In this study, the Ace gene was partially amplified and sequenced at amino acid positions 260, 342, and 407 to determine the frequencies of these mutations in housefly samples collected from farms and garbage disposal sites of 16 provinces in the Aegean and Mediterranean regions of Turkey. In addition, the percent remaining AChE activities in these samples were assayed by using three OPs (malaoxon, paraoxon, and dichlorvos) and one CB (carbaryl) compound as inhibitors. In all the analyzed samples, 13 different combinations at the three amino acid positions were identified and the L/V260-A/G342-F/Y407 combination was found in the highest frequency. No susceptible individual was detected. The highest mean percent remaining AChE activities were detected in the individuals having the L260-A/G342-F/Y407 genotype when malaoxon and paraoxon were used as inhibitors and in the individuals with the L260-A342-F/Y407 combination when dichlorvos and carbaryl were used as inhibitors. The obtained data were heterogeneous and there was no exact correlation between the molecular genetic background and the resistance phenotypes of the flies. The findings of this study at the molecular and biochemical levels indicate the presence of significant control problems in the field.

The cDNA of AChE in the housefly, Musca domestica, was sequenced and individual flies were genotyped by this gene in an inhibition assay of AChE activity with an organophaspate, fenitroxon. Mutations at Gly(342) and Tyr(407), which are reportedly conserved in resistant strains of Drosophila, were associated with the insensitivity to fenitroxon. Two other mutations, Ile(162) and Val(260), did not have an apparent effect on insensitivity. However, the four mutations are located in the active site of the enzyme, and therefore the non-neutral mutations in this gene are considered to cause the insensitivity of AChE in the development of insecticide resistance of the housefly.