Resistance pattern and point mutations of insensitive acetylcholinesterase in a carbamate-resistant strain of housefly ( Musca domestica)

Abstract

To examine the resistance pattern and point mutations present in the carbamate-resistant strain of housefly (SH-CBR), Musca domestica, from Shanghai, China, the fly’s resistance spectrum and k i ratios to organophosphate (OP) and carbamate (CB) insecticides were determined. The cDNA encoding acetylcholinesterase (AChE) from both a susceptible control strain (SH-S) and the SH-CBR strain of housefly were cloned and sequenced using RT-PCR. Based on two major patterns of target site resistance suggested by Russell et al. [R. J. Russell, C. Claudianos, P. M. Campbell, I. Horne, T. D. Sutherland, J. G. Oakeshott, Two major classes of target site insensitivity mutations confer resistance to organophosphate and carbamate, Pestic. Biochem. Physiol. 79 (2004), 84–93.], the present results indicate a Pattern I resistance: resistance to CBs was greater than that to OPs; the SH-S/SH-CBR k i ratios were also greater for CBs than for OPs. The cDNA was 2079 nucleotides long, encoding a protein of 693 amino acids. The cDNA deduced amino acid sequence of the housefly had a high degree of identity with previously described insect AChEs and exhibited the same structural feature as vertebrate TcAChE. Three mutations in the region of the active site, V261L, G343A, and F408Y, which were identical to those identified in the 77M and YBOL housefly strains, were identified in the AChE from the SH-CBR strain. Additionally, a novel mutation, D422L, was found at the outer surface of the protein where the residue presumably could not interact directly with amino acid residues lining the active site gorge. Homologous modelling of housefly AChE, based on the high resolution crystal structure of fruit fly ( Drosophila melanogaster) AChE, indicated that D422 may be involved in a salt bridge with H341. The distance between D422 and H341 is predicted to be 3.8 Å, and modification of the charge on the side chain of D422 (D to V) would likely affect the profile of the acyl pocket via the Y339 component. Such a structural change in the acyl pocket in the mutant may underlie the decreased affinity of AChE for CBs.