Biochemistry of Esterases Associated with Organophosphate Resistance in Lucilia cuprina with Comparisons to Putative Orthologues in Other Diptera

Esterase activities associated with organophosphate insecticide resistance in the Australian sheep blowfly, Lucilia cuprina, are compared with similar activities in other Diptera. The enzymes making the major contribution to methyl butyrate hydrolysis (ali-esterase) in L. cuprina, M. domestica, and D. melanogaster comigrate during electrophoresis. The enzymes in L. cuprina and D. melanogaster correspond to the naphthyl acetate hydrolyzing E3 and EST23 isozymes of those species. These and previously published data suggest that the ali-esterases of all three species are orthologous. Strains of L. cuprina fall into four groups on the basis of quantitative determinations of their ali-estesterase, OP hydrolase, and malathion carboxylesterase activities and these groups correspond to their status with respect to two types of OP resistance. Strains susceptible to OPs have high ali-esterase, low OP hydrolase, and intermediate MCE activities; those resistant to malathion but not diazinon have low ali-esterase, intermediate OP hydrolase, and high MCE activities; those resistant to diazinon but not malathion have low ali-esterase, high OP hydrolase, and low MCE activities; those resistant to both OPs have low ali-esterase, high OP hydrolase, and high MCE activities. The correlated changes among the three biochemical and two resistance phenotypes suggest that they are all properties of one gene/enzyme system; three major allelic variants of that system explain OP susceptibility and the two types of OP resistance. Models are proposed to explain the joint contribution of OP hydrolase and MCE activities to malathion resistance and the invariant association of low ali-esterase and elevated OP hydrolase activities in either type of resistance.     

Hydrolysis of pyrethroids by carboxylesterases from Lucilia cuprina and Drosophila melanogaster with active sites modified by in vitro mutagenesis

The cloned genes encoding carboxylesterase E3 in the blowfly Lucilia cuprina and its orthologue in Drosophila melanogaster were expressed in Sf9 cells transfected with recombinant baculovirus. Resistance of L. cuprina to organophosphorus insecticides is due to mutations in the E3 gene that enhance the enzyme's ability to hydrolyse insecticides. Previous in vitro mutagenesis and expression of these modifications (G137D, in the oxyanion hole and W251L, in the acyl pocket) have confirmed their functional significance. We have systematically substituted these and nearby amino acids by others expected to affect the hydrolysis of pyrethroid insecticides. Most mutations of G137 markedly decreased pyrethroid hydrolysis. W251L was the most effective of five substitutions at this position. It increased activity with trans permethrin 10-fold, and the more insecticidal cis permethrin >130-fold, thereby decreasing the trans:cis hydrolysis ratio to only 2, compared with >25 in the wild-type enzyme. Other mutations near the bottom of the catalytic cleft generally enhanced pyrethroid hydrolysis, the most effective being F309L, also in the presumptive acyl binding pocket, which enhanced trans permethrin hydrolysis even more than W251L. In these assays with racemic 1RS cis and 1RS trans permethrin, two phases were apparent, one being much faster suggesting preferential hydrolysis of one enantiomer in each pair as found previously with other esterases. Complementary assays with individual enantiomers of deltamethrin and the dibromo analogue of cis permethrin showed that the wild type and most mutants showed a marked preference for the least insecticidal 1S configuration, but this was reversed by the F309L substitution. The W251L/F309L double mutant was best overall in hydrolysing the most insecticidal 1R cis isomers. The results are discussed in relation to likely steric effects on enzyme-substrate interactions, cross-resistance between pyrethroids and malathion, and the potential for bioremediation of pyrethroid residues.     

Two single mutations commonly cause qualitative change of nonspecific carboxylesterases in insects

Carboxylesterases provide key mechanisms of resistance to insecticides, particularly organophosphates (OPs), in insects. One resistance mechanism is a qualitative change in the properties of a carboxylesterase. Two mutant forms, G151D and W271L, have been observed, mostly in dipteran species, to affect substrate specificity of enzymes. But whether these two single mutations can commonly change character of insect carboxylesterases is unknown. In our study carboxylesterase genes from seven insects distributed among four orders were cloned, mutated at position 151 or 271 and expressed in Escherichia coli. The kinetics of the purified recombinant proteins was examined towards an artificial carboxylester and two OP insecticides. The G/A151D and W271L mutation significantly reduced carboxylesterase activity in 87.5% and 100% cases, respectively, and at the same time conferred OP hydrolase activities in 62.5% and 87.5% cases, respectively. Thus, the change at position 271 is more effective to influence substrate specificity than that at position 151. These results may suggest that these two mutations have the potential to cause insecticide resistance broadly in insects.     

Determination of malathion and diazinon resistance by sequencing the Md alpha E7 gene from Guatemala, Colombia, Manhattan, and Thailand housefly (Musca domestica L.) strains

Organophosphate (OP) insecticides (parathion/diazinon) resistance in housefly (Musca domestica L.) is associated with the change in carboxylesterase activity. The product of alpha E7 gene, which is a member of alpha-esterase gene cluster, is probably playing a role in detoxification of the xenobiotic esters. In parathion/diazinon resistant M. domestica species Gly137 to Asp substitution was found in the active center of the product of alpha E7 gene. In malathion (an OP) resistant M. domestica strains Trp251 to Ser substitution was identified in the active center of the Md alpha E7. In our research, to understand the allelic diversity of the Md alpha E7, the gene was partially sequenced from four different housefly strains from different localities (Guatemala, Manhattan (USA), Colombia (USA) and Thailand). It was found out that; in Thailand strain one allele has Cys residue at the position of 251, the other allele contains a Trp for the same site. In Colombia strain, one allele has Asp137, the other allele contains a Gly residue at this point. The Manhattan and Guatemala strains have Asp137 and Trp251 residues on their both alleles at these two different positions.     

The genetic basis of malathion resistance in housefly (Musca domestica L.) strains from Turkey

Organophosphate insecticide (parathion/diazinon) resistance in housefly (Musca domestica L.) is associated with the change in carboxylesterase activity. The product of MdalphaE7 gene is probably playing a role in detoxification of xenebiotic esters. In our research, we have isolated, cloned and sequenced the MdalphaE7 gene from 5 different Turkish housefly strains. High doses of malathion (600 microg/fly) were applied in a laboratory environment for one year to Ceyhan1, Ceyhan2, Adana and Ankara strains while no insecticide treatment was performed in the laboratory to Kirazli strain. Trp251 --> Ser substitution was found in the product of MdalphaE7 gene in all malathion resistant and Kirazli stocks. In addition, we checked the malathion carboxylesterase (MCE), percent remaining activities in acetylcholinesterase (AChE), glutathion-S-transferase (GST), and general esterase activities in all 5 strains used in this study. In comparing with universal standard sensitive control WHO, a high level of MCE and GST activities were observed while lower level of general esterase activities was detected in the tested strains. In addition, a higher percent remaining activities in AChE than WHO susceptible strain were observed in all malathion resistant strains.     

The same amino acid substitution in orthologous esterases confers organophosphate resistance on the house fly and a blowfly.

Organophosphate (OP) insecticide resistance in certain strains of Musca domestica is associated with reduction in the carboxylesterase activity of a particular esterase isozyme. This has been attributed to a 'mutant ali-esterase hypothesis', which invokes a structural mutation to an ali-esterase resulting in the loss of its carboxylesterase activity but acquisition of OP hydrolase activity. It has been shown that the mutation in Lucilia cuprina is a Gly137-->Asp substitution in the active site of an esterase encoded by the Lc alpha E7 gene (Newcomb, R.D., Campbell, P.M., Ollis, D.L., Cheah, E., Russell, R.J., Oakeshott, J.G., 1997. A single amino acid substitution converts a carboxylesterase to an organophosphate hydrolase and confers insecticide resistance on a blowfly. Proc. Natl. Acad. Sci. USA 94, 7464-7468). We now report the cloning and characterisation of the orthologous M. domestica Md alpha E7 gene, including the sequencing of cDNAs from the OP resistant Rutgers and OP susceptible sbo and WHO strains. The Md alpha E7 gene has the same intron structure as Lc alpha E7 and encodes a protein with 76% amino acid identity to Lc alpha E7. Comparisons between susceptible and resistance alleles show resistance in M. domestica is associated with the same Gly137-->Asp mutation as in L. cuprina. Bacterial expression of the Rutgers allele shows its product has OP hydrolase activity. The data indicate identical catalytic mechanisms have evolved in orthologous Md alpha E7 and Lc alpha E7 molecules to endow diazinon-type resistance on the two species of higher Diptera.     

Mechanisms of organophosphate resistance in a field population of oriental migratory locust, Locusta migratoria manilensis (Meyen)

The susceptibilities to three organophosphate (OP) insecticides (malathion, chlorpyrifos, and phoxim), responses to three metabolic synergists [triphenyl phosphate (TPP), piperonyl butoxide (PBO), and diethyl maleate (DEM)], activities of major detoxification enzymes [general esterases (ESTs), glutathione S-transferases (GSTs), and cytochrome P450 monooxygenases (P450s)], and sensitivity of the target enzyme acetylcholinesterase (AChE) were compared between a laboratory-susceptible strain (LS) and a field-resistant population (FR) of the oriental migratory locust, Locusta migratoria manilensis (Meyen). The FR was significantly resistant to malathion (57.5-fold), but marginally resistant to chlorpyrifos (5.4) and phoxim (2.9). The malathion resistance of the FR was significantly diminished by TPP (synergism ratio: 16.2) and DEM (3.3), but was unchanged by PBO. In contrast, none of these synergists significantly affected the toxicity of malathion in the LS. Biochemical studies indicated that EST and GST activities in the FR were 2.1- to 3.2-fold and 1.2- to 2.0-fold, respectively, higher than those in the LS, but there was no significant difference in P450 activity between the LS and FR. Furthermore, AChE from the FR showed 4.0-fold higher activity but was 3.2-, 2.2-, and 1.1-fold less sensitive to inhibition by malaoxon, chlorpyrifos-oxon, and phoxim, respectively, than that from the LS. All these results clearly indicated that the observed malathion resistance in the FR was conferred by multiple mechanisms, including increased detoxification by ESTs and GSTs, and increased activity and reduced sensitivity of AChE to OP inhibition.     

A cluster of at least three esterase genes inLucilia cuprina includes malathion carboxylesterase and two other esterases implicated in resistance to organophosphates

Three distinct malathion carboxylesterase (MCE) phenotypes have been identified among strains ofLucilia cuprina. The high-activity phenotype shows 1.6-and 33-fold more MCE specific activity than the intermediate- and low-activity phenotypes, respectively. Flies with high MCE activity are 1000-fold more resistant to malathion than flies with either low or intermediate MCE phenotypes, which are equally susceptible. High and low MCE specific activity are allelic and encoded by theRmal gene on chromosome 4.Rmal is clustered within one map unit of two other esterase genes,Rop1 andE9, which are implicated in resistance to other organophosphate insecticides. Intermediate MCE specific activity is also inherited within the cluster, although its allelism toRmal, Rop1, orE9 is unclear. The cluster does not contain the gene for the hemolymph esterase E4, which maps 6.1 map units fromRop1, on the other side of thebubbled wing marker. The cluster appears to be homologous to part of a tandem array of 11 esterase genes on chromosome 3R ofDrosophila melanogaster.     

两个单点突变对昆虫羧酸酯酶降解马拉硫磷的影响

马拉硫磷是一种高效低毒的有机磷杀虫剂, 分子量大且结构特殊, 广泛用于农业害虫的防治。羧酸酯酶突变是昆虫对有机磷类杀虫剂产生代谢抗性的重要机制之一。本实验室前期已从棉蚜Aphis gossypii、 褐飞虱Nilaparvata lugens、 斜纹夜蛾Spodoptera litura、 家蚕Bombyx mori、 异色瓢虫Harmonia axyridis、 赤拟谷盗Tribolium castaneum和西方蜜蜂Apis mellifera中各克隆了一个非特异性羧酸酯酶基因, 通过体外定点突变构建了G/A151D和W271L两种突变体, 并进行了原核细胞表达和纯化。本实验在体外测定了这7种昆虫野生型和两种突变型羧酸酯酶对马拉硫磷的降解。结果显示： 棉蚜、 西方蜜蜂、 斜纹夜蛾、 赤拟谷盗的野生型羧酸酯酶能够降解马拉硫磷, 两个突变并不能提高它们的降解活性, 而家蚕、 异色瓢虫和褐飞虱的野生型羧酸酯酶不能降解马拉硫磷, G/A151D和/或W271L突变能使这些酯酶获得马拉硫磷羧酸酯酶（MCE）的活性, 有可能使这些昆虫对马拉硫磷产生抗性。不同物种的MCE活性相差较大, 斜纹夜蛾的MCE活性最高, 其k_cat/K_m值为1.8~1.9 L/μmol·min, 其次是赤拟谷盗, 其K_cat/K_m值为0.87~0.95 L/μmol·min, 其他昆虫的MCE活性相对较低, 相差可高达10倍。     

Purification and characterization of a carboxylesterase involved in malathion-specific resistance from Tribolium castaneum (Coleoptera: Tenebrionidae)

Specific resistance to malathion in a strain of Tribolium castaneum is due to a 44-fold increase in malathion carboxylesterase (MCE) activity relative to a susceptible strain, whereas non-specific esterase levels are slightly lower. Unlike the overproduced esterase of some mosquito and aphid species, MCE in Tribolium castaneum accounts for only a small fraction (0.033-0.045%) of the total extractable protein respectively in resistant and susceptible strains. The enzyme was purified to apparent homogeneity from these two strains and has a similar molecular weight of 62,000. However, preparative isoelectricfocusing indicated that resistant insects possess one MCE with pI of 7.3, while susceptible insects possess a MCE with a pI of 6.6. Purified MCE from both populations had different K(m) and V(m) values for hydrolysis of malathion as well as for alpha-naphthyl acetate. The kinetic analysis suggests that MCE of resistant insects hydrolyses malathion faster than the purified carboxylesterase from susceptible beetles and that this enzyme has greater affinity for malathion than for naphthyl esters. Malathion-specific resistance is due to the presence of a qualitatively different esterase in the resistant strain.     

Identification of an opd (organophosphate degradation) gene in an Agrobacterium isolate

We isolated a bacterial strain, Agrobacterium radiobacter P230, which can hydrolyze a wide range of organophosphate (OP) insecticides. A gene encoding a protein involved in OP hydrolysis was cloned from A. radiobacter P230 and sequenced. This gene (called opdA) had sequence similarity to opd, a gene previously shown to encode an OP-hydrolyzing enzyme in Flavobacterium sp. strain ATCC 27551 and Brevundimonas diminuta MG. Insertional mutation of the opdA gene produced a strain lacking the ability to hydrolyze OPs, suggesting that this is the only gene encoding an OP-hydrolyzing enzyme in A. radiobacter P230. The OPH and OpdA proteins, encoded by opd and opdA, respectively, were overexpressed and purified as maltose-binding proteins, and the maltose-binding protein moiety was cleaved and removed. Neither protein was able to hydrolyze the aliphatic OP malathion. The kinetics of the two proteins for diethyl OPs were comparable. For dimethyl OPs, OpdA had a higher k(cat) than OPH. It was also capable of hydrolyzing the dimethyl OPs phosmet and fenthion, which were not hydrolyzed at detectable levels by OPH.     

The acetylcholinesterase gene and organophosphorus resistance in the Australian sheep blowfly, Lucilia cuprina

Acetylcholinesterase (AChE), encoded by the Ace gene, is the primary target of organophosphorous (OP) and carbamate insecticides. Ace mutations have been identified in OP resistants strains of Drosophila melanogaster. However, in the Australian sheep blowfly, Lucilia cuprina, resistance in field and laboratory generated strains is determined by point mutations in the Rop-1 gene, which encodes a carboxylesterase, E3. To investigate the apparent bias for the Rop-1/E3 mechanism in the evolution of OP resistance in L. cuprina, we have cloned the Ace gene from this species and characterized its product. Southern hybridization indicates the existence of a single Ace gene in L. cuprina. The amino acid sequence of L. cuprina AChE shares 85.3% identity with D. melanogaster and 92.4% with Musca domestica AChE. Five point mutations in Ace associated with reduced sensitivity to OP insecticides have been previously detected in resistant strains of D. melanogaster. These residues are identical in susceptible strains of D. melanogaster and L. cuprina, although different codons are used. Each of the amino acid substitutions that confer OP resistance in D. melanogaster could also occur in L. cuprina by a single non-synonymous substitution. These data suggest that the resistance mechanism used in L. cuprina is determined by factors other than codon bias. The same point mutations, singly and in combination, were introduced into the Ace gene of L. cuprina by site-directed mutagenesis and the resulting AChE enzymes expressed using a baculovirus system to characterise their kinetic properties and interactions with OP insecticides. The K(m) of wild type AChE for acetylthiocholine (ASCh) is 23.13 microM and the point mutations change the affinity to the substrate. The turnover number of Lucilia AChE for ASCh was estimated to be 1.27x10(3) min(-1), similar to Drosophila or housefly AChE. The single amino acid replacements reduce the affinities of the AChE for OPs and give up to 8.7-fold OP insensitivity, while combined mutations give up to 35-fold insensitivity. However, other published studies indicate these same mutations yield higher levels of OP insensitivity in D. melanogaster and A. aegypti. The inhibition data indicate that the wild type form of AChE of L. cuprina is 12.4-fold less sensitive to OP inhibition than the susceptible form of E3, suggesting that the carboxylesterases may have a role in the protection of AChE via a sequestration mechanism. This provides a possible explanation for the bias towards the evolution of resistance via the Rop-1/E3 mechanism in L. cuprina.     

Characterization of a novel esterase conferring insecticide resistance in the mosquito Culex tarsalis

Resistance to the organophosphate insecticide, malathion, in a strain of Culex tarsalis mosquitoes is due to increased activity of a malathion carboxylesterase (MCE). To determine whether resistance was due to a qualitative or quantitative change in the MCE, the enzyme was purified from both malathion-resistant and -susceptible mosquitoes. Enzyme kinetic measurements revealed that the two strains have one MCE in common, but resistant mosquitoes also have a unique MCE which hydrolyses malathion 18 times faster. Interestingly, this MCE does not hydrolyse α-naphthyl acetate, a substrate commonly used to detect increased levels of esterases in other organophosphate-resistant insects. Unlike the over-produced esterase of some related mosquito species, each MCE in C. tarsalis accounts for only a small fraction (0.015%) of the total extractable protein in either strain. Therefore, resistance in these insects is due to the presence of a qualitatively different enzyme, and not to a quantitative increase of a non-specific esterase. This study therefore demonstrates that the underlying biochemical mechanisms of insecticide resistance in one insect cannot necessarily be predicted from those of another, even closely related species. © 1995 Wiley-Liss, Inc.     

Insecticide resistance and malathion carboxylesterase in the sheep blowfly,Lucilia cuprina

Resistance to the organophosphorus insecticide malathion in genetically related strains of the Australian sheep blowflyLucilia curprina was examined. Separate lines of blowflies were established by homozygosis of the fourth chromosome of the parental RM strain. Both the RM and the derived resistant (der-R) strains are approximately 100 times more resistant to malathion than the related susceptible der-S strain, resistance being correlated with a 45- to 50-fold increase in a malathion carboxylesterase (MCE) activity. MCE has a pH optimum ranging between 6.6 and 8.0 and is strongly inhibited by the carboxylesterase inhibitors triphenyl phosphate, paraoxon, and diiospropylfluorophosphate. Subcellular fractionation revealed that MCE was localized predominantly to the cytosol and mitochondria in both resistant and susceptible blowflies. A single MCE was purified to homogeneity from RM blowflies. It has a pI of 5.5, is a monomer of 60.5 kDa, and hydrolyzes malathion with aV _max of 755 nmol/min/mg protein and aK _m of 11.0 µM. L. cuprina have thus evolved a remarkable MCE which is faster and more efficient at hydrolyzing a specific insecticide than any other insect esterase yet described.     

Determination of Malathion and Diazinon Resistance by Sequencing the MdαE7 Gene from Guatemala,Colombia, Manhattan,and Thailand Housefly (Musca domestica L.) Strains

Organophosphate (OP) insecticides (parathion/diazinon) resistance in housefly (Musca domestica L.) is associated with the change in carboxylesterase activity. The product of E7 gene, which is a member of -esterase gene cluster, is probably playing a role in detoxyfication of the xenobiotic esters. In parathion/diazinon resistant M. domestica species Gly¹³⁷ to Asp substitution was found in the active center of the product of E7 gene. In malathion (an OP) resistant M. domestica strains Trp²⁵¹ to Ser substitution was identified in the active center of the MdE7. In our research, to understand the allelic diversity of the MdE7, the gene was partially sequenced from four different housefly strains from different localities (Guatemala, Manhattan (USA), Colombia (USA), and Thailand). It was found out that; in Thailand strain one allele has Cys residue at the position of 251, the other allele contains a Trp for the same site. In Colombia strain, one allele has Asp¹³⁷, the other allele contains a Gly residue at this point. The Manhattan and Guatemala strains have Asp¹³⁷ and Trp²⁵¹ residues on their both alleles at these two different positions.     

Microbial cleavage of various organophosphorus insecticides.

Bacteria able to utilize Aspon, Azodrin, Dasanit, diazinon, malathion, Orthene, parathion, Trithion, dimethoate, Dylox, methyl parathion, and Vapona as sole phosphorus sources were isolated from soil and sewage. Individual isolates used from 3 to 10 of these insecticides as sole phosphorus sources. The extent of growth of two Pseudomonas strains in media containing diazinon and malathion was in the range expected from the amount of insecticide supplied, and their proliferation resulted in disappearance of the chemical. Resting cells of the pseudomonads derived from cultures grown on diazinon or malathion but not orthophosphate caused extensive destruction of these two organophosphates in the presence or absence of chloramphenicol. Extracts of the two bacteria derived from organophosphate-grown cultures catalyzed the disappearance of Aspon, Azodrin, Dasanit, diazinon, malathion, Orthene, parathion, and Trithion but not dimethoate, Dylox, methyl parathion, and Vapona. Results from gas chromatographic analysis suggested that the extracts formed dimethyl phosphate from azodrin, dimethyl phosphorodithioate from malathion, diethyl phosphorodithioate from Trithion, and diethyl phosphorothioate from Dasanit, diazinon, and parathion. Dimethyl phosphate, dimethyl phosphorothioate , dimethyl phosphorodithioate, diethyl phosphate, and diethyl phosphorothioate were not used by the pseudomonads as sole phosphorus sources.     

Microbial cleavage of various organophosphorus insecticides.

Bacteria able to utilize Aspon, Azodrin, Dasanit, diazinon, malathion, Orthene, parathion, Trithion, dimethoate, Dylox, methyl parathion, and Vapona as sole phosphorus sources were isolated from soil and sewage. Individual isolates used from 3 to 10 of these insecticides as sole phosphorus sources. The extent of growth of two Pseudomonas strains in media containing diazinon and malathion was in the range expected from the amount of insecticide supplied, and their proliferation resulted in disappearance of the chemical. Resting cells of the pseudomonads derived from cultures grown on diazinon or malathion but not orthophosphate caused extensive destruction of these two organophosphates in the presence or absence of chloramphenicol. Extracts of the two bacteria derived from organophosphate-grown cultures catalyzed the disappearance of Aspon, Azodrin, Dasanit, diazinon, malathion, Orthene, parathion, and Trithion but not dimethoate, Dylox, methyl parathion, and Vapona. Results from gas chromatographic analysis suggested that the extracts formed dimethyl phosphate from azodrin, dimethyl phosphorodithioate from malathion, diethyl phosphorodithioate from Trithion, and diethyl phosphorothioate from Dasanit, diazinon, and parathion. Dimethyl phosphate, dimethyl phosphorothioate , dimethyl phosphorodithioate, diethyl phosphate, and diethyl phosphorothioate were not used by the pseudomonads as sole phosphorus sources.     

Structural characterization and reversal of the natural organophosphate resistance of a D-type esterase, Saccharomyces cerevisiae S-formylglutathione hydrolase

Saccharomyces cerevisiae expresses a 67.8 kDa homodimeric serine thioesterase, S-formylglutathione hydrolase (SFGH), that is 39.9% identical with human esterase D. Both enzymes possess significant carboxylesterase and S-formylglutathione thioesterase activity but are unusually resistant to organophosphate (OP) inhibitors. We determined the X-ray crystal structure of yeast (y) SFGH to 2.3 A resolution by multiwavelength anomalous dispersion and used the structure to guide site-specific mutagenesis experiments addressing substrate and inhibitor reactivity. Our results demonstrate a steric mechanism of OP resistance mediated by a single indole ring (W197) located in an enzyme "acyl pocket". The W197I substitution enhances ySFGH reactivity with paraoxon by >1000-fold ( k i (W197I) = 16 +/- 2 mM (-1) h (-1)), thereby overcoming natural OP resistance. W197I increases the rate of OP inhibition under pseudo-first-order conditions but does not accelerate OP hydrolysis. The structure of the paraoxon-inhibited W197I variant was determined by molecular replacement (2.2 A); it revealed a stabilized sulfenic acid at Cys60. Wild-type (WT) ySFGH is inhibited by thiol reactive compounds and is sensitive to oxidation; thus, the cysteine sulfenic acid may play a role in the regulation of a "D-type" esterase. The structure of the W197I variant is the first reported cysteine sulfenic acid in a serine esterase. We constructed five Cys60/W197I variants and show that introducing a positive charge near the oxyanion hole, W197I/C60R or W197I/C60K, results in a further enhancement of the rates of phosphorylation with paraoxon ( k i = 42 or 80 mM (-1) h (-1), respectively) but does not affect the dephosphorylation of the enzyme. We also characterized three histidine substitutions near the oxyanion hole, G57H, L58H, and M162H, which significantly decrease esterase activity.     

Biochemical mechanisms of resistance in strains of Oryzaephilus surinamensis (Coleoptera: Silvanidae) resistant to malathion and chlorpyrifos-methyl.

The acetylcholinesterase, carboxylesterase, and cytochrome P450 monooxygenase activities of three strains of Oryzaephilus srinamensis (L.) were examined to better understand biochemical mechanisms of resistance. The three strains were VOS49 and VOSCM, selected for resistance to malathion and chlorpyrifos-methyl, respectively, and VOS48, a standard susceptible strain. Cross-resistance to malathion and chlorpyrifos-methyl was confirmed in VOS49 and VOSCM. Acetylcholinesterase activity was not correlated to resistance among these strains. VOS49 and VOSCM showed elevated levels of carboxylesterase activity based on p-nitrophenylacetate, alpha-naphthyl acetate, or beta-naphthyl acetate substrates. PAGE zymograms showed major differences in caboxylesterase isozyme banding among strains. VOSCM had one strongly staining isozyme band. A band having the same Rf-value was very faint in VOS48. The VOS49 carboxylesterase banding pattern was different from both VOSCM and VOS48. Cytochrome P450 monooxygenase activity was based on cytochrome P450 content, aldrin epoxidase activity, and oxidation of organophosphate insecticides, all elevated in resistant strains. The monooxygenase activity varied with insecticide substrate and resistant strain, suggesting specific cytochromes P450 may exist for different insecticides. The monooxygenase activity of the VOS49 strain was much higher with malathion than chlorpyrifos-methyl as substrates, whereas VOSCM monooxygenase activity was higher with malathion than chlorpyrifos-methyl as substrates. Results are discussed in the context of resistance mechanisms to organophosphate insecticides in O. surinamensis.     

Multiple Mutations and Gene Duplications Conferring Organophosphorus Insecticide Resistance Have Been Selected at the Rop-1 Locus of the Sheep Blowfly, Lucilia cuprina

Sequences of the esterase gene E7 were compared across 41 isogenic (IV) strains of the sheep blowfly, Lucilia cuprina, and one strain of the sibling species, L. sericata. The 1.2-kb region sequenced includes sites of two insecticide resistance mutations. Gly137Asp confers resistance to organophosphorus insecticides (OPs), particularly preferring diethyl OPs such as diazinon, while Trp251Leu prefers dimethyl OPs, and particularly malathion, with the additional presence of carboxylester moieties. We found that there are just eight haplotypes among the 41 chromosomes studied: two Gly137Asp containing haplotypes, two Trp251Leu containing haplotypes, and four susceptible haplotypes, including the L. sericata sequence. While phylogenetic analysis of these haplotypes suggests that the Asp137 and Leu251 mutations each arose at least twice, evidence for recombination was detected across the region, therefore single origins for these resistance mutations cannot be ruled out. Levels of linkage disequilibrium in the data are high and significant hitchhiking is indicated by Fay and Wu s H test but not the Tajima test. A test of haplotype diversity indicates a paucity of diversity compared with neutral expectations. Both these results are consistent with a very recent selective sweep at the LcE7 locus. Interestingly, gene duplications of three different combinations of OP resistant haplotypes were identified in seven of the isogenic (IV) strains. All three types of duplication involve an Asp137 and a Trp251 haplotype. To examine whether more haplotypes existed before the hypothesised selective sweep, fragments of E7 surrounding the resistance mutations were amplified from pinned material dating back to before OPs were used. Four new sequence haplotypes, not sampled in the survey of extant haplotypes, were obtained that are all associated with susceptibility. This is suggestive of a higher historical level of susceptible allelic diversity at this locus.Reviewing Editor: Dr. Rasmus Nielsen