Identification and characterization of mutations in housefly (Musca domestica) acetylcholinesterase involved in insecticide resistance

Abstract

Acetylcholinesterase (AChE) insensitive to organophosphate and carbamate insecticides has been identified as a major resistance mechanism in numerous arthropod species. However, the associated genetic changes have been reported in the AChE genes from only three insect species; their role in conferring insecticide insensitivity has been confirmed, using functional expression, only for those in Drosophila melanogaster. The housefly, Musca domestica, was one of the first insects shown to have this mechanism; here we report the occurrence of five mutations (Val-180-->Leu, Gly-262-->Ala, Gly-262-->Val, Phe-327-->Tyr and Gly-365-->Ala) in the AChE gene of this species that, either singly or in combination, confer different spectra of insecticide resistance. The baculovirus expression of wild-type and mutated housefly AChE proteins has confirmed that the mutations each confer relatively modest levels of insecticide insensitivity except the novel Gly-262-->Val mutation, which results in much stronger resistance (up to 100-fold) to certain compounds. In all cases the effects of mutation combinations are additive. The mutations introduce amino acid substitutions that are larger than the corresponding wild-type residues and are located within the active site of the enzyme, close to the catalytic triad. The likely influence of these substitutions on the accessibility of the different types of inhibitor and the orientation of key catalytic residues are discussed in the light of the three-dimensional structures of the AChE protein from Torpedo californica and D. melanogaster.

Mechanisms of resistance to malathion in the medfly Ceratitis capitata

“…Sequence analysis showed a single amino acid substitution Gly328Ala (Torpedo numbering) when the C and Table 4 Amino acid substitutions found in the comparison of the predicted CcaE7 protein of different individuals from the C and the W strains of C. capitata W strains were compared. This mutation is adjacent to the glutamate of the catalytic triad in D. melanogaster (Walsh et al, 2001), and has been associated with insecticide resistance in populations of D. melanogaster, M. domestica and Plutella xylostella (Walsh et al, 2001;Menozzi et al, 2004;Baek et al, 2005;Lee et al, 2007). Individuals with the WR phenotype were homozygous for this mutation, whereas individuals with the WS phenotype were either heterozygous or wild type at this position.…”

“…A total of 14 mutations associated to resistance to insecticides have been reported in AChE in higher Diptera (Mutero et al, 1994;Kozaki et al, 2001;Vontas et al, 2001Vontas et al, , 2002Walsh et al, 2001;Hsu et al, 2006). To investigate whether these or other mutations were present in individuals of the resistant strain, we obtained a coding sequence of the AChE gene for C. capitata (Ccace) from three individuals of each of the three phenotypes investigated, WR, WS and C. Phylogenetic analysis showed that the Ccace gene described here may be designated as an ace2 ortholog according to the nomenclature of Weill et al (2003) as indeed higher Diptera have a single ace gene.…”

“…Changes in the gene's regulation to produce more AChE to overcome the effect of the insecticide has been reported for Drosophila melanogaster (Charpentier and Fournier, 2001) and Aonidiella aurantii (Levitin and Cohen, 1998). However, point mutations in the AChE gene (ace2 for higher Diptera and ace1 for other insects) that make the enzyme less sensitive to inhibition by the insecticides, have more often been identified as being responsible for insecticide resistance (Mutero et al, 1994;Walsh et al, 2001;Vontas et al, 2002;Weill et al, 2003;Baek et al, 2005;Hsu et al, 2006).…”

Expression of the housefly acetylcholinesterase in a bioreactor and its potential application in the detection of pesticide residues

“…The purified AChE from a susceptible housefly was shown to be sensitive to pesticides (Shi et al 2001), and the expressed AChE showed the similar properties with the AChE purified from the body of housefly, therefore, the expression of the ace gene cloned from a housefly strain susceptible to insecticides provided a good choice for isolating AChE. The acetylcholinesterase gene from a wild strain of M. domestica has been expressed in Sf cells, the FLAG-AChE showing enzyme activity, however, the K i of the recombinant enzyme to dichlorovos was only 1.34 lM -1 min -1 (Walsh et al 2001). The K i of our expressed AChE to dichlorovos is 838.6 lM -1 min -1 , much higher than the previously reported FLAG-AChE, suggesting our enzyme is more sensitive to insecticides.…”

“…The Trichoplusia ni cell line Tn-5B1-4 has been shown to produce especially high yields of recombinant protein and was even capable of producing small amounts of galactose-terminated N-glycans (Altmann et al 1999). In another previous report (Walsh et al 2001), to reveal the underlying resistance mechanism, the ACheE gene from wild and resistant strains of housefly (Musca domestica) were successfully expressed in insect cell line Sf, and the expressed fusion protein FLAG-AChE showed strong enzyme activity, suggesting that insect cells are suitable for expressing biologically active the housefly AChE.…”