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.

Isolated presynaptic nerve terminals (synaptosomes) prepared from rat brain were used to evaluate the action of a classic T-syndrome (cismethrin) and CS-syndrome (deltamethrin) pyrethroid on voltage-sensitive calcium channels by measuring calcium influx and neurotransmitter release with multiple fluorescent assays. Both cismethrin and deltamethrin stimulated calcium influx in a stereospecific manner; however, they did so by different mechanisms. Neurotransmitter released occurred only with synaptosomes treated with deltamethrin. This release was stereospecific, stimulated by depolarization, unaltered by tetrodotoxin, but blocked by ω-conotoxin MVIIC. Electrophysiological experiments with Cav 2.2 expressed in Xenopus oocytes validated the interaction of deltamethrin with this N-type calcium channel. Thus, cismethrin and deltamethrin elicit different actions at presynaptic nerve terminals. The modification of Cav 2.2 voltage-sensitive calcium channels by deltamethrin is consistent with enhanced neurotransmitter release, a physiological response that has been observed during acute neurotoxicity of CS syndrome pyrethroids. Rat brain synaptosomes were also used to evaluate the action of 11 pyrethroids (bifenthrin, bioallethrin, cismethrin, cyfluthrin, cyhalothrin, cypermethrin, deltamethrin, esfenvalerate, fenpropathrin, permethrin, and tefluthrin) by measuring calcium uptake, membrane depolarization, and neurotransmitter release as before. Our results indicate that only a subset of the commercially available pyrethroids act as voltage-sensitive calcium channels agonists (permethrin, cyfluthrin, cyhalothrin, cypermethrin, deltamethrin, and esfenvalerate). Pyrethroids that significantly enhanced calcium influx were also more potent in releasing neurotransmitter. Increased glutamate release appears to be due to an agonistic action of these compounds on Cav 2.1 and Cav 2.2 voltage-sensitive calcium channels, most associated with neurotransmitter release. Biochemical and electrophysiological experiments provide functional evidence for a distinct mechanism of action for some of the pyrethroids on voltage-sensitive calcium channels at the presynaptic nerve terminal that results in enhanced neurotransmitter release, a response that is consistent with the observed symptomology of CS-syndrome pyrethroids. Such results will allow a more complete understanding of the molecular and cellular nature of pyrethroid-induced neurotoxicity and expand our knowledge of the structure-activity relationships of pyrethroids in regards to the voltage-sensitive calcium channel.

The purpose of this dissertation is to purify and characterize the carboxylesterase(s) associated with permethrin resistance in the permethrin-resistant (PE-R) strain of Colorado potato beetle (CPB), Leptinotarsa decemlineata, and to develop an immunoassay system for the detection of resistance in field populations of CPB. Most carboxylesterase (CbE) activity is found in the hemolymph and the soluble fraction of body tissue. Among a number of charged forms of CbE identified from hemolymph, the pI 4.5-4.9 CbEs are quantitatively elevated and are most responsible for permethrin resistance in the PE-R strain. Permethrin CbEs (i.e., pI 4.2-4.8 CbEs) have been purified from the hemolymph of the PE-R strain through several chromatographic procedures. The pI 4.8 CbE is a 46-48 kDa monomeric protein. The pi 4.5 CbE is likely a 57-59 kDa dimeric protein. All pI 4.5-4.8 forms are glycoproteins but the charge heterogeneity is not associated with N-glycan moieties. Biochemical properties of the pI 4.2-4.5 CbEs have been comparatively characterized through substrate kinetic analyses, specific inhibition studies, and pH-temperature experiments. The pI 4.8 and 4.5 CbEs share a number of similarities in their biochemical properties and functional role in resistance despite of their distinct molecular properties. The kinetics of inhibition of the pI 4.5-4.8 CbEs by permethrin and DDT are best described by a mixed-noncompetitive type and a noncompetitive type inhibition, respectively. The kinetic analyses indicate the presence of hydrophobic non-catalytic site(s) as well as hydrophobic catalytic site(s) that are available for the binding to hydrophobic insecticides. Along with a low level of permethrin hydrolysis, the hydrophobic binding nature of the pI 4.5-4.8 CbEs suggests that permethrin resistance is mainly conferred by sequestration rather than rapid hydrolysis of permethrin. The nonspecific sequestration by the pI 4.5-4.8 CbEs appears to be associated with the cross-resistance of the PE-R strain to other hydrophobic insecticides such as other pyrethroids, DDT, and abamectin. Polyclonal antisera have been generated against the 30, 48, and 60 kDa denatured CbE immunogens. A high degree of cross-reactivities of the antisera to different immunogens indicate that all CbE immunogens share a high level of structural similarity. An antibody capture immunoassay using denatured CPB hemolymph is shown to be effective in detecting the different levels of permethrin CbE in permethrin-resistant and -susceptible populations of CPB.