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Metabolic resistance to synthetic pyrethroids in Helicoverpa armigera has recently been associated with the chimeric cytochrome P450 enyzme CYP337B3. One variant of the latter, CYP337B3v1, accounts for 40–50 fold resistance to fenvalerate in an Australian population while a second variant, CYP337B3v2, has been associated with ~7 fold resistance to cypermethrin in a Pakistani population. Here we show that ~250–1200 fold resistance to fenvalerate in populations of the species from northern and northwestern China is largely due to P450-based metabolism, and that CYP337B3v2 is also at high frequency in these populations but absent in a susceptible control strain. However we find little correlation between the level of resistance and CYP337B3v2 frequency, either across the resistant populations studied, or over time within them. While there is variation between populations in the levels of CYP337B3v2 expression, this is not correlated with the level of resistance either. These data suggest that much of the variation in the level of fenvalerate resistance in China is explained by P450s other than CYP337B3. We also find that both the level of resistance and CYP337B3v2 frequency decline in field populations transferred to the laboratory and remained there without fenvalerate exposure, suggesting a fitness cost to both characters in the absence of the pesticide pressure. However the declines in the two characters are not well correlated across populations, again consistent with a large contribution to the variation in resistance levels from factors other than CYP337B3.

We have obtained a Helicoverpa armigera strain (MonoR) showing high level resistance to monocrotophos by 15 generations of selection on a weakly resistant field-caught strain. Bioassays using various synergists show a major role for esterases in the resistance, and little if any role for cytochrome P450s or glutathione S-transferases. Biochemical data also show elevated levels of esterase activity in the resistant line. There were also minor differences among our strains in the kinetics of acetylcholinesterase activity and in its susceptibility to monocrotophos inhibition, and sequence comparisons revealed one amino acid substitution in AChE1 but none in AChE2 in the MonoR strain. However the AChE1 substitution does not match any others linked to resistance in this or other species and would be located well away from the enzyme active site, so it may not be causally involved with resistance. Native PAGE shows several individual esterase isozymes are more intensely staining in the resistant line and native Western analysis with an antibody against Clade 1 esterases show that several of these belong to Clade 1 and that their greater staining intensity in MonoR is due to greater amounts of the respective enzymes. Proteomic analysis of gel slices also matches the overexpressed bands with up to six Clade 1 enzymes, with some involvement also from esterases in three other Clades. Two of the isozymes that are overexpressed in MonoR are also overexpressed in a fenvalerate resistant line and proteomic analysis of that line bears out previous proteomics of another fenvalerate resistant line which also implicates Clade 1 isozymes.

Interspecific comparisons of bioassay and biochemical data suggest two major patterns of target site resistance to carbamates and organophosphates. Pattern I resistance, which is generally more effective for carbamates, has been shown in two sub-species of mosquitoes to be due to a particular Gly-Ser mutation in the oxyanion hole within the active site of one of their two acetylcholinesterase enzymes. Intriguingly, different substitutions at the equivalent site confer organophosphate hydrolytic ability on other esterases responsible for metabolic resistance in some other species. In the case of the aphid, Myzus persicae, Pattern I resistance is due to a Ser-Phe mutation in the vicinity of the acyl pocket of acetylcholinesterase. Pattern II resistance is at least as effective for organophosphates as it is for carbamates and may even be specific to organophosphates in some cases. Molecular studies on this pattern of resistance in three higher Diptera show that it is due to changes that constrict the acetylcholinesterase active site gorge and limit binding of the insecticide to the catalytic residues at the base of the gorge. One case of Pattern II resistance in the mosquito, Culex tritaeniorhynchus, involves the same site near the acyl pocket of acetylcholinesterase, albeit a different substitution, as that involved in Pattern I resistance in M. persicae.

Resistance to organophosphorus (OP) insecticides in Lucilia cuprina arises from two mutations in carboxylesterase E3 that enable it to hydrolyse the phosphate ester of various organophosphates, plus the carboxlyester in the leaving group in the case of malathion. These mutations are not found naturally in the orthologous EST23 enzyme in Drosophila melanogaster. We have introduced the two mutations (G137D and W251L) into cloned genes encoding E3 and EST23 from susceptible L. cuprina and D. melanogaster and expressed them in vitro with the baculovirus system. The ability of the resultant enzymes to hydrolyse the phosphate ester of diethyl and dimethyl organophosphates was studied by a novel fluorometric assay, which also provided a sensitive titration technique for the molar amount of esterase regardless of its ability to hydrolyse the fluorogenic substrate used. Malathion carboxylesterase activity was also measured. The G137D mutation markedly enhanced (>30-fold) hydrolysis of both classes of phosphate ester by E3 but only had a similar effect on the hydrolysis of dimethyl organophosphate in EST23. Introduction of the W251L mutation into either gene enhanced dimethyl (23-30-fold) more than diethyl (6-10-fold) organophosphate hydrolysis and slightly improved (2-4-fold) malathion carboxylesterase activity, but only at high substrate concentration.

Orthologous E3 and EST23 carboxylesterases have been enriched over 200-fold from organophosphate (OP) susceptible strains of Lucilia cuprina and Drosophila melanogaster, respectively. Mutants of E3 are associated with OP resistance but no resistance mutations of EST23 are known. The behaviours of the two enzymes were very similar during purification which involved differential centrifugation followed by three or four ion exchange and gel filtration chromatographic steps. Nondenaturing polyacrylamide gel electrophoresis and histochemical staining for esterase activity revealed no other esterases in the enriched material. Two-dimensional polyacrylamide gel electrophoresis (native followed by denaturing) showed that a major 70-kDa component of each preparation comigrates with E3 and EST23 activities, respectively. Kinetic properties of the enzymes are also very similar. Estimates of K m , K cat , and K cat / K m for α-naphthyl acetate are 42 ± 18 μ M, 19 sec −1 , and 4.6 × 10 5 M −1 sec −1 , respectively, for E3, and 62 ± 25 μ M, 23 sec −1 , and 3.7 × 10 5 M −1 sec −1 , for EST23. Both enzymes are potently inhibited by dibrom and less potently by another OP, diisopropylflurophosphate. E3 is also potently inhibited by paraoxon, whereas EST23 is at least 8-fold less susceptible to inhibition by paraoxon. This supports previous analyses of crude homogenates which showed that E3 is more susceptible to inhibition by paraoxon and fenitrooxon than is EST23 or the target site for OP action, acetylcholinesterase. It is proposed that the unusual affinity of E3 for such OPs is a necessary precondition for mutations that enable it to confer OP resistance on L. cuprina.

Three malathion carboxylesterase (MCE) phenotypes have been described previously in Lucilia cuprina adults: low, intermediate, and high. The MCE specific activities of adults with the intermediate and high phenotypes are 21- and 33-fold higher than those with the low phenotype, but the high phenotype is almost 1000-fold more resistant to malathion than either of the other phenotypes. Here we show that MCE activity also peaks in Day 4 larvae from three lines representative of these phenotypes. The MCE activity of the low-line larvae is only 2.7- and 4-fold lower than those of the intermediate and high-line larvae, respectively. The relatively high MCE activity of the low-line larvae is largely explained by their crop activity. Assays in the presence of esterase inhibitors reveal three distinct types of MCE activity across the three lines and two developmental stages. Activity in the low-line adults is not completely inhibited by paraoxon and is classified as a subclass II carboxylesterase. The MCE activities in the larvae of the low and intermediate lines and adults from the intermediate line are inhibited by low concentrations of paraoxon, classifying them as subclass I carboxylesterases. The MCE activities in the high-line larvae and adults are also classified as subclass I carboxylesterases, but they are more sensitive to inhibition by triphenyl phosphate than those in the other two lines. These data suggest that MCE in the malathion-resistant high line may be structurally different from the MCEs in the malathion-susceptible intermediate and low lines.

Eight esterases identified using native polyacrylamide gel electrophoresis were characterized in a susceptible and an organophosphate-resistant strain of Lucilia cuprina. Developmental profiles and tissue distributions were determined for all eight esterases and substrate and inhibitor specificities were also determined for six of them. Esterases E1 and E2 were isozymes of acetylcholinesterase, with E1 present throughout development and E2 largely confined to adults. Esterases E8, which was mainly confined to the neuromuscular and digestive tissues of early larvae, and E7, which was only recovered from 2- to 3-day-old adults, were not abundant enough for biochemical analysis. E4 and E13, classified as carboxylesterases, were only moderately sensitive to inhibition by organophosphates and found mainly in hemolymph. E3 and E9 were also identified as carboxylesterases but were highly sensitive to inhibition by organophosphates. They were the only enzymes found to differ between the two strains, both being detected only in the organophosphate-susceptible strain. In this strain they were both abundant in pupae, and E3 was also abundant in larvae and sexually mature adults, localized mainly in digestive tissues and the adult reproductive tract. The inhibitor, developmental, and tissue specificities of E3 all support genetic data reported previously that E3 is encoded by the major organophosphate resistance locus, ROP-1. While genetic data are not available for E9, its concentration in pupae suggests that it is unlikely to confer major gene resistance in feeding stages of L. cuprina.