Molecular and functional analysis of mutations that result in insecticide resistance in Colorado potato beetle
The functional aspects of specific mutations, R30K, S291G, and I392T, found associated with AChE gene of OP- and carbamate-resistant Colorado potato beetle (CPB) were determined using recombinant AChEs. From the previous studies, azinphosmethyl resistant CPB strain (AZ-R) possesses S291G and R30K mutations and their AChE was 16-fold less sensitive to azinphosmethyl than that of insecticide-susceptible (SS) strain, in terms of biomolecular rate constant (ki). A carbamate-resistant strain (BERTS) showed high resistance to carbofuran (12-fold), but was relatively less resistant to azinphosmethyl (1.35-fold) than SS CPB. The AZ-R strain was 2.6-fold more resistant to carbofuran and 8-fold more resistant to azinphosmethyl than SS CPB, respectively, in a discriminating dose bioassay. A substrain of BERTS, BERTS-R, possessing only the S291 G mutation, elicited high resistance to carbofuran and moderate resistance to azinphosmethyl. The BERTS-S substrain, possessing S291G and I392T mutations, was susceptible to both azinphosmethyl and carbofuran.
In this study, enzymatic properties of altered AChEs from CPB were examined using recombinant AChEs obtained from baculovirus expression system. The S291G mutation increased hydrolysis activity of larger substrates (e.g. BTC) and increased sensitivity to inhibition by larger inhibitors (e.g. paraoxon, DFP, and N-propyl carbofuran) in the altered recombinant AChEs. The R30K mutation caused further increasement of hydrolysis activity of larger substrates and the sensitivity to inhibition by larger inhibitors in combination with the S291G mutation. The 1392T mutation compensated the effect of S291G. Thus, the altered recombinant AChE with both S291G and I392T mutations elicited a substrate specificity and inhibitory properties more similar to the susceptible form of ACNE without mutations.
Two rapid molecular diagnostic systems were successfully developed for monitoring of OP and pyrethroid resistances by determining resistant allele frequencies. These techniques were used to genotyping pyrethroid resistance in 16 field populations of CPB by detection of the kdr mutation. The predicted resistant levels of populations by genotyping were closely correlated to the esfenvalerate bioassay results by the rank-correlation coefficient of a non-parametric rank correlation test for independence. These two DNA based diagnostic procedures are reliable, accurate and affordable techniques for monitoring field population of CPB for resistance.
0307: Molecular biology