Hydrolysis of organophosphorus insecticides by in vitro modified carboxylesterase E3 from Lucilia cuprina

Resistance of the blowfly, Lucilia cuprina, to organophosphorus (OP) insecticides is due to mutations in LcalphaE7, the gene encoding carboxylesterase E3, that enhance the enzyme's ability to hydrolyse insecticides. Two mutations occur naturally, G137D in the oxyanion hole of the esterase, and W251L in the acyl binding pocket. Previous in vitro mutagenesis and expression of these modifications to the cloned gene have confirmed their functional significance. G137D enhances hydrolysis of diethyl and dimethyl phosphates by 55- and 33-fold, respectively. W251L increases dimethyl phosphate hydrolysis similarly, but only 10-fold for the diethyl homolog; unlike G137D however, it also retains ability to hydrolyse carboxylesters in the leaving group of malathion (malathion carboxylesterase, MCE), conferring strong resistance to this compound. In the present work, we substituted these and nearby amino acids by others expected to affect the efficiency of the enzyme. Changing G137 to glutamate or histidine was less effective than aspartate in improving OP hydrolase activity and like G137D, it diminished MCE activity, primarily through increases in Km. Various substitutions of W251 to other smaller residues had a broadly similar effect to W251L on OP hydrolase and MCE activities, but at least two were quantitatively better in kinetic parameters relating to malathion resistance. One, W251G, which occurs naturally in a malathion resistant hymenopterous parasitoid, improved MCE activity more than 20-fold. Mutations at other sites near the bottom of the catalytic cleft generally diminished OP hydrolase and MCE activities but one, F309L, also yielded some improvements in OP hydrolase activities. The results are discussed in relation to likely steric effects on enzyme-substrate interactions and future evolution of this gene.     

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.     

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.     

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.     

Progress towards the development of a transgenic strain of the Australian sheep blowfly (Lucilia cuprina) suitable for a male-only sterile release program

The Australian sheep blowfly Lucilia cuprina is the most important pest species involved in cutaneous myiasis (flystrike) of sheep in Australia and New Zealand. In New Zealand L. cuprina is primarily controlled through the application of insecticides. However, there is an increased interest in biological methods of control of this species. We have proposed to develop a genetically modified strain of L. cuprina that would be ideal for a male-only sterile release program. To that end we have developed a method for making transgenic L. cuprina using a piggyBac vector and an EGFP marker gene. We have also developed in Drosophila melanogaster a 2-component genetic system for controlling female viability. Females carrying both components of the system die unless fed a diet that contains tetracycline. We anticipate that the female-killing system will need to be optimised for L. cuprina in order to make a strain with the properties required for a male-only release program.     

Asymmetry – where evolutionary and developmental genetics meet

The mechanisms responsible for the fine tuning of development, where the wildtype phenotype is reproduced with high fidelity, are not well understood. The difficulty in approaching this problem is the identification of mutant phenotypes indicative of a defect in these fine-tuning control mechanisms. Evolutionary biologists have used asymmetry as a measure of developmental homeostasis. The rationale for this was that, since the same genome controls the development of the left and right sides of a bilaterally symmetrical organism, departures from symmetry can be used to measure genetic or environmental perturbations. This paper examines the relationship between asymmtry and resistance to organophosphorous insecticides in the Australian sheep blowfly, Lucilia cuprina. A resistance gene, Rop-1, which encodes a carboxylesterase enzyme, also confers a significant increase in asymmetry. Continued exposure of resistant populations to insecticide has selected a dominant suppressor of the asymmetry phenotype. Genetic evidence indicates that the modifier is the L. cuprina Notch homologue.     

Selective sweeps at the organophosphorus insecticide resistance locus, Rop-1, have affected variation across and beyond the α-esterase gene cluster in the Australian sheep blowfly, Lucilia cuprina

A major theoretical consequence of selection at a locus is the genetic hitchhiking of linked sites (selective sweep). The extent of hitchhiking around a gene is related to the strength of selection and the rate of recombination, with its impact diminishing with distance from the selected site. At the Rop-1 locus of the sheep blowfly, Lucilia cuprina, polymorphisms at two different sites within the LcαE7 gene encode forms of the protein that confer organophosphorus insecticide resistance. To assess the impact of selection at these two sites on variation around LcαE7, we sequenced regions within six other genes along chromosome IV across isogenic (IV) strains of L. cuprina. High levels of linkage disequilibrium, characterized by low haplotype number (K) and diversity (H), and significant R(2) values were observed for two genes, LcαE1 and LcαE10, both members of the same α-esterase gene cluster as LcαE7. A significant R(2) value was also observed for a gene predicted to be the next closest to LcαE7, AL03, but not for any of the other genes, LcRpL13a, Lcdsx, or LcAce. Skews in the site frequency spectra toward high-frequency variants were significant for LcαE1 (Fay and Wu's H = -2.91), LcαE10 (H = -1.85), and Lcdsx (H = -2.00). Since the selective sweeps, two forms of likely returning variation were observed, including variation in microsatellites in an intron of LcαE10 and a recombination event between LcαE7 and LcαE10. These data suggest that two incomplete soft sweeps have occurred at LcαE7 that have significantly affected variation across, and beyond, the α-esterase gene cluster of L. cuprina. The speed and impact of these selective sweeps on surrounding genomic variation and the ability of L. cuprina to respond to future environmental challenges are discussed.     

Mutations in acetylcholinesterase genes of Rhopalosiphum padi resistant to organophosphate and carbamate insecticides.

Apple grain aphid, Rhopalosiphum padi (Linnaeus), is an important wheat pest. In China, it has been reported that R. padi has developed high resistance to carbamate and organophosphate insecticides. Previous work cloned from this aphid 2 different genes encoding acetylcholinesterase (AChE), which is the target enzyme for carbamate and organophosphate insecticides, and its insensitive alteration has been proven to be an important mechanism for insecticide resistance in other insects. In this study, both resistant and susceptible strains of R, padi were developed, and their AChEs were compared to determine whether resistance resulted from this mechanism and whether these 2 genes both play a role in resistance. Bioassays showed that the resistant strain used was highly or moderately resistant to pirimicarb, omethoate, and monocrotophos (resistance ratio, 263.8, 53.8, and 17.5, respectively), and showed little resistance to deltamethrin or thiodicarb (resistance ratio, 5.2 and 3.4, respectively). Correspondingly, biochemistry analysis found that AChE from resistant aphids was very insensitive to the first 3 insecticides (I50 increased 43.0-, 15.2-, and 8.8-fold, respectively), but not to thiodicarb (I50 increased 1.1-fold). Enzyme kinetics tests showed that resistant and susceptible strains had different AChEs. Sequence analysis of the 2 AChE genes cloned from resistant and susceptible aphids revealed that 2 mutations in Ace2 and 1 in Ace1 were consistently associated with resistance. Mutation F368(290)L in Ace2 localized at the same position as a previously proven resistance mutation site in other insects. The other 2 mutations, S329(228)P in Ace1 and V435(356)A in Ace2, were also found to affect the enzyme structure. These findings indicate that resistance in this aphid is mainly the result of insensistive AChE alteration, that the 3 mutations found might contribute to resistance, and that the AChEs encoded by both genes could serve as targets of insecticides.     

Molecular characterization of esterase E3 gene associated with organophosphorus insecticide resistance in the New World screwworm fly, Cochliomyia hominivorax

Abstract.  The New World screwworm, Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae), is one of the most important myiasis-causing flies in South America. It is responsible for severe economic losses to livestock producers, mainly because it causes mortality in newborn calves and reductions in the quality of leather and in the production of milk and meat. The economic losses caused by myiasis, along with those caused by other internal and external parasites, are the main factors limiting meat production. In Brazil, C. hominivorax has been controlled by applying insecticides, particularly organophosphate (OP)-based compounds. However, the improper and continuous use of these chemicals can lead to the selection of OP-resistant strains. This, associated with the fast development of OP resistance in other myiasis-causing flies, shows the importance of investigating resistance in C. hominivorax. Based on the findings of previous studies, the objective of the current work was to isolate and sequence the E3 gene in C. hominivorax. Mutations at the positions (Gly137 and Trp251) responsible for conferring OP resistance in Lucilia cuprina and Musca domestica L. (Muscidae) were identified in C. hominivorax . In addition, the orthologous region in C. hominivorax contained motifs that are highly conserved among carboxyl/cholinesterases and contribute to the catalytic mechanism of the active site. The characterization of this gene in natural populations of New World screwworm can be an important tool for monitoring resistance to insecticides throughout its current geographic distribution. This will provide information for the selection and implementation of more effective pest management programmes.     

Polymorphism in the acetylcholinesterase gene of Musca domestica L. field populations in Turkey

Acetylcholinesterase (AChE), encoded by the Ace gene, is the primary target of organophosphates (OPs) and carbamates (CBs) in insects. Ace mutations have been identified in OP and CB resistant strains of Musca domestica. In this study, the Ace gene was partially amplified and sequenced at amino acid positions 260, 342, and 407 to determine the frequencies of these mutations in housefly samples collected from farms and garbage disposal sites of 16 provinces in the Aegean and Mediterranean regions of Turkey. In addition, the percent remaining AChE activities in these samples were assayed by using three OPs (malaoxon, paraoxon, and dichlorvos) and one CB (carbaryl) compound as inhibitors. In all the analyzed samples, 13 different combinations at the three amino acid positions were identified and the L/V260-A/G342-F/Y407 combination was found in the highest frequency. No susceptible individual was detected. The highest mean percent remaining AChE activities were detected in the individuals having the L260-A/G342-F/Y407 genotype when malaoxon and paraoxon were used as inhibitors and in the individuals with the L260-A342-F/Y407 combination when dichlorvos and carbaryl were used as inhibitors. The obtained data were heterogeneous and there was no exact correlation between the molecular genetic background and the resistance phenotypes of the flies. The findings of this study at the molecular and biochemical levels indicate the presence of significant control problems in the field.     

A novel acetylcholinesterase gene in mosquitoes codes for the insecticide target and is non-homologous to the ace gene in Drosophila

Acetylcholinesterase (AChE) is the target of two major insecticide families, organophosphates (OPs) and carbamates. AChE insensitivity is a frequent resistance mechanism in insects and responsible mutations in the ace gene were identified in two Diptera, Drosophila melanogaster and Musca domestica. However, for other insects, the ace gene cloned by homology with Drosophila does not code for the insensitive AChE in resistant individuals, indicating the existence of a second ace locus. We identified two AChE loci in the genome of Anopheles gambiae, one (ace-1) being a new locus and the other (ace-2) being homologous to the gene previously described in Drosophila. The gene ace-1 has no obvious homologue in the Drosophila genome and was found in 15 mosquito species investigated. In An. gambiae, ace-1 and ace-2 display 53% similarity at the amino acid level and an overall phylogeny indicates that they probably diverged before the differentiation of insects. Thus, both genes are likely to be present in the majority of insects and the absence of ace-1 in Drosophila is probably due to a secondary loss. In one mosquito (Culex pipiens), ace-1 was found to be tightly linked with insecticide resistance and probably encodes the AChE OP target. These results have important implications for the design of new insecticides, as the target AChE is thus encoded by distinct genes in different insect groups, even within the Diptera: ace-2 in at least the Drosophilidae and Muscidae and ace-1 in at least the Culicidae. Evolutionary scenarios leading to such a peculiar situation are discussed.     

杀虫药剂抗性家蝇品系乙酰胆碱酯酶基因的特征分析

乙酰胆碱酯酶(AChE)是有机磷和氨基甲酸酯类杀虫药剂的作用靶标,这两大类杀虫药剂的广泛应用导致了昆虫对抗性的选择。靶标的修饰是某些昆虫产生抗性的分于机理,这种抗性是和AChE的变更型相关的,这些变更型的酶显示出对杀虫药剂的不被感性。利用RT-PCR和Streptavidin偶联磁珠技术从两种抗性家蝇(Musca domestica)品系D3和Kash中分别分离了AChE基因并测定了其按苷酸颅序。eDNA的可读框长2082bp．由此推导出了AChE的氨基酸顺序,通过与敏感家蝇品系Cooper的比较,发现了一些核苷酸顺序差异和4个氨基酸点突变,其中3个替代可能与杀虫药剂不敏感性有关。这一结果表明D3和Kash均属于CH₂抗性类型。     

Mutations in acetylcholinesterase associated with insecticide resistance in the cotton aphid, Aphis gossypii Glover

Two acetylcholinesterase genes, Ace1 and Ace2, have been fully cloned and sequenced from both organophosphate-resistant and susceptible clones of cotton aphid. Comparison of both nucleic acid and deduced amino acid sequences revealed considerable nucleotide polymorphisms. Further study found that two mutations occurred consistently in all resistant aphids. The mutation F139L in Ace2 corresponding to F115S in Drosophila acetylcholinesterase might reduce the enzyme sensitivity and result in insecticide resistance. The other mutation A302S in Ace1 abutting the conserved catalytic triad might affect the activity and insecticide sensitivity of the enzyme. Phylogenetic analysis showed that insect acetylcholinesterases fall into two subgroups, of which Ace1 is the paralogous gene whereas Ace2 is the orthologous gene of Drosophila AChE. Both subgroups contain resistance-associated AChE genes. To avoid confusion in the future work, a nomenclature of insect AChE is also suggested in the paper.     

小菜蛾抗性个体不敏感乙酰胆碱酯酶的鉴定

乙酰胆碱酯酶(AChE)敏感性降低是小菜蛾对有机磷和氨基甲酸酯产生抗性的重要机制之一,已得到广泛的承认和报道。一种用硝酸纤维素膜的斑点法鉴定个体小菜蛾的抗性AChE不敏感性的应用技术,提供了早期侦测和随后监测田间种群抗性的可能性。此法操作简便灵敏。小菜蛾抗性品系(GBR)和田间种群成虫头部AChE活力,在残杀威抑制时,抑制率分别为50.97%和43.96%,有对氧磷存在时,分别为63.78%和35.87%,较敏感品系的AChE为不敏感。     

Fenitroxon insensitive acetylcholinesterases of the housefly, Musca domestica associated with point mutations

The cDNA of AChE in the housefly, Musca domestica, was sequenced and individual flies were genotyped by this gene in an inhibition assay of AChE activity with an organophaspate, fenitroxon. Mutations at Gly(342) and Tyr(407), which are reportedly conserved in resistant strains of Drosophila, were associated with the insensitivity to fenitroxon. Two other mutations, Ile(162) and Val(260), did not have an apparent effect on insensitivity. However, the four mutations are located in the active site of the enzyme, and therefore the non-neutral mutations in this gene are considered to cause the insensitivity of AChE in the development of insecticide resistance of the housefly.     

The Ace locus of Drosophila melanogaster: structural gene for acetylcholinesterase with an unusual 5'' leader.

The Ace locus of Drosophila melanogaster has been mapped at the molecular level. cDNA clones from the locus have been isolated and their sequence determined, confirming that Ace forms the structural gene for acetylcholinesterase (AChE). The cDNAs have a 1950 nucleotide open reading frame from which the complete amino acid sequence of AChE has been deduced. The Drosophila enzyme is found to have extensive homology to the known sequence of Torpedo AChE. Ace cDNAs have an unusual structure with a long 5' leader and several short upstream open reading frames.     

Comparison of Anopheles gambiae and Culex pipiens acetycholinesterase 1 biochemical properties

Selection of insensitive acetycholinesterase 1 (AChE1) has occurred in several mosquito species controlled with carbamate (CX) and organophosphate (OP) insecticides. In case of pyrethroid resistance, these insecticides represent an alternative for disease vector control program. Their heavy use in agriculture has selected resistant populations of Anopheles gambiae in West Africa. The evolution of resistance has to be studied to prevent, or at least slow down, the spread of resistant mosquito in wild populations. An. gambiae shares the same resistance mechanism to CX and OP insecticides as Culex pipiens, which was attributed to the G119S substitution in the AChE1 enzyme. By comparing resistant AChE1 from both species, we show here that similar resistance levels are obtained toward 10 insecticides of both classes. Moreover, similar AChE1 activity levels are recorded between either susceptible or resistant mosquitoes of both species. Enzymes belonging to both species seem thus to share identical properties. Consequently, we hypothesize that fitness cost associated with AChE1 insensitivity in C. pipiens mosquitoes should be similar in An. gambiae and thus be used in strategies to control resistant populations where malaria is prevalent.