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An organophosphate (OP)-resistant clone (OR-0) of the greenbug, Schizaphis graminum, showed 27.2-, 19.9-, 65.8-, 44.5-, 18.2-, 4.8-, and 3.1-fold greater resistance to dimethoate, omethoate, disulfoton, demeton-S-methyl, parathion, methyl parathion, and chlorpyrifos, respectively, than an OP-susceptible clone (OSS). General esterase and glutathione S-transferase activities were similar in the OSS and OR-0 clones, whereas cytochrome P450 O-demethylase activities were below the detection limit in both greenbug clones. In contrast, acetylcholinesterase (AChE) activity in the OR-0) clone was 2.3-fold higher than that in the OSS clone. Kinetic studies also revealed that AChE from the OR-0 clone was 2.1-, 2.1-, 1.6-, 4.4-, and 1.3-fold less sensitive to inhibition by methyl paraoxon, paraoxon, demeton-S-methyl, omethoate, and demeton S,respectively, than AChE from the OSS clone (P< 0.05). However, Northern blot analysis of mRNA, using a 296-bp AChE cDNA fragment generated by seminested PCR as a homologous probe, indicated that the increased activity of AChE in the OR-0 clone was not due to the overexpression of the AChE gene. Our overall results suggest that the increased AChE activity in the OR-0 clone most likely is due to structural modifications of AChE, leading to both an increased catalytic activity with the model substrate acetylthiocholine and a reduced sensitivity to inhibition by OP compounds.

The profiles of esterase activity in an organophosphate (OP)-susceptible (OSS) clone and an OP-resistant (OR-2) clone of the greenbug (Schizaphis graminum) were compared using nondenaturing polyacrylamide gel electrophoresis (PAGE) coupled with esterase assays after gel fractionations. A distinct peak of esterase activity was found in the esterase profiles of the OR-2 clone but not in the OSS clone. The peak represents the elevated esterases identified previously and constitutes a major biochemical difference between the OSS and the OR-2 clones. The esterases within that peak hydrolyzed five substrates, including phenyl acetate (PA), β-naphthyl acetate (β-NA), α-naphthyl butyrate (α-NB), p-nitrophenyl acetate (p-NA), and α-naphthyl acetate (α-NA). The most preferred substrate was PA followed by β-NA, α-NB, p-NA, and α-NA. Nondenaturing PAGE revealed that the major esterase peak was contributed by three different esterase bands. These esterases showed a similar affinity to β-NA and were highly sensitive to inhibition by both paraoxon and p-hydroxymecuribenzoic acid. In addition, the enzyme activity was slightly to moderately activated by Ca2+, but significantly inhibited by Triton X-100. These characteristics suggested that the elevated esterases in the OR-2 clone were arylesterases or arylesterase-like esterases with certain biochemical properties resembling phosphoric triester hydrolase. However, these arylesterase-like esterases were not able to efficiently utilize paraoxon and chlorpyrifos-oxon as their substrates. Thus, the elevated esterases identified in the OR-2 clone appeared to contribute to OP resistance by sequestrating OP molecules.

The midgut proteinases from a Bacillus thuringiensis-susceptible (IA-S) and four laboratory-selected resistant strains (KS-SC, KS-NE, IA-1, and IA-3) of European corn borer (Ostrinia nubilalis) were characterized using three synthetic substrates, Nα-benzoyl-L-arginine p-nitroanilide (BApNA) for trypsin-like, N-succinyl-ala-ala-pro-phe p-nitroanilide (SAAPFpNA) for chymotrypsin-like, and N-succinyl-ala-ala-pro-leu p-nitroanilide (SAAPLpNA) for elastase-like proteinase activities. The hydrolyzing efficiency of trypsin-like proteinases, determined by Vmax, decreased 35% in the KS-SC resistant strain compared with the susceptible strain. There were no significant differences in the Michaelis constant (Km) among the five strains for the same substrate. When the purified B. thuringiensisCry1Ab protoxin was used as the substrate, there was a detectable reduction in the hydrolysis of protoxin that was mediated by midgut proteinases from the KS-SC strain compared with the IA-S strain. Thus, the reduced trypsin-like proteinase activity appeared to lead to the reduced activation of the B. thuringiensisprotoxins. This may confer or contribute to B. thuringiensisresistance in this strain. However, no significant difference was found in trypsin activity between the IA-S strain and the three other resistant strains (i.e., KS-NE, IA-1, and IA-3) and in chymotrypsin activity among all strains examined. These results suggest that other resistance mechanisms are responsible for the B. thuringiensisresistance in the KS-NE, IA-1, IA-2, and IA-3 strains of European corn borer.

The organophosphate-resistant strains, OR-0, OR-1, and OR-2, of the greenbug (Schizaphis graminum) showed 1.6-, 32-, and 42-fold resistance, respectively, to parathion compared with a susceptible (OSS) strain. Enzyme kinetics revealed that esterase activities in the OR-1 and OR-2 strains were 1.9- and 2.4-fold higher, respectively, than that of the OSS strain when α-naphthyl acetate (αNA) was used as substrate. A good correlation between the αNA-hydrolyzing esterase level and parathion resistance among these strains suggested that these esterases played a predominate role in conferring organophosphate resistance. Microassays of the αNA-hydrolyzing esterase activity in individual greenbugs indicated that the OR-1 and OR-2 strains were less homogenous than the OSS strain. In contrast, there was no significant difference in the enzyme activity among the OSS, OR-0, and OR-1 strains when phenyl acetate (PA) was used as substrate. However, the PA-hydrolyzing esterase activity was approximately 12-fold higher in the OR-2 strain than in the OSS strain. Approximately 90% of the PA-hydrolyzing esterase activity was inhibited by 1 μM of paraoxon, but only 10% of the activity was inhibited byp-hydroxymercuribenzoic acid at the same concentration. The PA-hydrolyzing esterase(s) appeared to have characteristics of both type A (based on the substrate specificity) and type B (based on the inhibitor specificity) esterases and could be a key factor conferring organophosphate resistance in the OR-2 strain.