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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.

The peach^potato aphid Myzus persicae (Sulzer) can resist a wide range of insecticides, but until recently (1990) the only mechanism identi¢ed was the increased production of carboxylesterases (E4 or FE4), which cause enhanced degradation and sequestration of insecticidal esters. We have now identi¢ed two forms of target-site resistance involving changes in the acetylcholinesterase (AChE) and sodium channel (kdr) genes. Biochemical and DNA diagnostic methods can be used to identify all three mechanisms in individual aphids, and thereby establish their spatial distributions and temporal dynamics. Ampli¢ed genes underlie the increased production of esterases, but their expression is modulated by DNA methylation. Ampli¢cation of the E4 gene is in strong linkage disequilibrium with the kdr mechanism. This may re£ect strong insecticidal selection favouring aphids with multiple mechanisms, tight chromosomal linkage and/or the prominence of parthenogenesis in many M. persicae populations. The decreased ¢tness of resistant aphids under winter conditions may be a consequence of the altered sodium-channel gene a¡ecting behaviour and/or the perception of external stimuli.

The brown planthopper, Nilaparvata lugens, is an economically significant pest of rice throughout Asia and has evolved resistance to many insecticides including the neonicotinoid imidacloprid. The resistance of field populations of N. lugens to imidacloprid has been attributed to enhanced detoxification by cytochrome P450 monooxygenases (P450s), although, to date, the causative P450(s) has (have) not been identified. In the present study, biochemical assays using the model substrate 7-ethoxycoumarin showed enhanced P450 activity in several resistant N. lugens field strains when compared with a susceptible reference strain. Thirty three cDNA sequences encoding tentative unique P450s were identified from two recent sequencing projects and by degenerate PCR. The mRNA expression level of 32 of these was examined in susceptible, moderately resistant and highly resistant N. lugens strains using quantitative real-time PCR. A single P450 gene (CYP6ER1) was highly overexpressed in all resistant strains (up to 40-fold) and the level of expression observed in the different N. lugens strains was significantly correlated with the resistance phenotype. These results provide strong evidence for a role of CYP6ER1 in the resistance of N. lugens to imidacloprid.

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