Mutations in the acetylcholinesterase gene of Bactrocera dorsalis associated with resistance to organophosphorus insecticides

Mutations in the gene encoding the enzyme acetylcholinesterase (AChE) of the oriental fruit fly, Bactrocera dorsalis, associated with resistance to an organophosphorus insecticide have been characterized. Three point mutations producing nonsynonymous changes in the predicted amino acid sequence of the product of the B. dorsalis ace gene in resistant vs. susceptible flies have been identified. One of these changes is unique to B. dorsalis while the other two occur at sites that are identical to mutations previously described for another Bactrocera species. Although the precise role of the third mutation is not clearly established, the independent origin of two identical alterations in these two species strongly supports the idea proposed previously that molecular changes associated with insecticide resistance in key genes and enzymes such as AChE are largely constrained to a limited number of sites. The results obtained here also suggest that the widespread use of organophosphorus insecticides will likely lead to a predictable acquisition of resistance in wild populations of B. dorsalis as well as other pest species. For surveys of B. dorsalis populations that may develop resistance, diagnostic tests using PCR-RFLP based methods for detecting the presence of all three mutations in individual flies are described.     

A small deletion in the olive fly acetylcholinesterase gene associated with high levels of organophosphate resistance

Organophosphate resistance in the olive fly was previously shown to associate with two point mutations in the ace gene. The frequency of these mutations was monitored in Bactrocera oleae individuals of increasing resistance. In spite of the difference in resistance among the individuals, there was no correlation between mutation frequencies and resistance level, indicating that other factors may contribute to this variation. The search for additional mutations in the ace gene of highly resistant insects revealed a small deletion at the carboxyl terminal of the protein (termed Delta3Q). Significant correlation was shown between the mutation frequency and resistance level in natural populations. In addition, remaining activity of acetylcholinesterase enzyme (AChE) after dimethoate inhibition was higher in genotypes carrying the mutation. These results strongly suggest a role of Delta3Q in high levels of organophosphate (OP) resistance. Interestingly, the carboxyl terminal of AChE is normally cleaved and substituted by a glycosylphosphatidylinositol (GPI) anchor. We hypothesize that Delta3Q may improve GPI anchoring, thus increasing the amount of AChE that reaches the synaptic cleft. In this way, despite the presence of insecticide, enough enzyme would remain in the cleft for its normal role of acetylcholine hydrolysis, allowing the insect to survive. This provides a previously un-described mechanism of resistance.     

昆虫乙酰胆碱酯酶基因研究进展

对昆虫乙酰胆碱酯酶（acetylcholinesterase, AChE,EC 3.1.1.7）的基因结构和表达等方面的研究进展进行了综述。分析了昆虫乙酰胆碱酯酶基因的结构,包括10个外显子的特征。对已经报道的昆虫AChE基因进行了系统归纳,并基于已知全序列的昆虫AChE基因,进行了昆虫AChE基因的分子进化分析。对昆虫AChE基因的结构特点及其功能,以及昆虫AChE基因的活性位点、AChE的变构与昆虫抗药性的关系进行了探讨。最后对昆虫AChE基因研究中存在的问题和前景进行了分析和展望。     

Resistance and synergistic effects of insecticides in Bactrocera dorsalis (Diptera: Tephritidae) in Taiwan

Oriental fruit flies, Bactrocera dorsalis (Hendel), were treated with 10 insecticides, including six organophosphates (naled, trichlorfon, fenitrothion, fenthion, formothion, and malathion), one carbamate (methomyl), and three pyrethroids (cyfluthrin, cypermethrin, and fenvalerate), by a topical application assay under laboratory conditions. Subparental lines of each generation treated with the same insecticide were selected for 30 generations and were designated as x-r lines (x, insecticide; r, resistant). The parent colony was maintained as the susceptible colony. The line treated with naled exhibited the lowest increase in resistance (4.7-fold), whereas the line treated with formothion exhibited the highest increase in resistance (up to 594-fold) compared with the susceptible colony. Synergism bioassays also were carried out. Based on this, S,S,S-tributyl phosphorotrithioate displayed a synergistic effect for naled, trichlorfon, and malathion resistance, whereas piperonyl butoxide displayed a synergistic effect for pyrethroid resistance. All 10 resistant lines also exhibited some cross-resistance to other insecticides, not only to the same chemical class of insecticides but also to other classes. However, none of the organophosphate-resistant or the methomyl-resistant lines exhibited cross-resistance to two of the pyrethroids (cypermethrin and fenvalerate). Overall, the laboratory resistance and cross-resistance data developed here should provide useful tools and information for designing an insecticide management strategy for controlling this fruit fly in the field.     

Cloning,mutagenesis, and expression of the acetylcholinesterase gene from a strain of Musca domestica; the change from a drug-resistant to a sensitive enzyme

Insect acetylcholinesterase (AChE) is known to be a primary target of organophosphorus and carbamate insecticides. However chronic exposure to these chemicals has led to resistance to applied insecticides, due usually to mutation of the AChE gene. Analysis of the AChE gene (hm) of Musca domestica (the housefly), which is cloned in this report, reveals the relationship between mutation and insecticide resistance. The 2,076 bp hm encodes a mature protein of 612 amino acids (67 kDa), and an 80 residue signal peptide. Unlike the enzyme of 'sensitive' strains, the AChE used in this study was resistant to the organophosphorus insecticide, trichlorphon. DNA sequencing showed that this AChE is identical to that of the sensitive strains with the exception of three amino acids Met-82, Ala-262, and Tyr-327. Site-directed mutagenesis of the Ala-262 and Tyr-327 residues largely restored sensitivity to the insecticide, suggesting that these two residues are the key structural elements controlling sensitivity. In addition to these residues, Glu-234 and Ala-236 in the conserved sequence FGESAG are thought to play a role in modulating sensitivity to organophosphorus insecticides.     

Development of resistance to spinosad in oriental fruit fly (Diptera: Tephritidae) in laboratory selection and cross-resistance

In this study, we assessed the potential for the development of resistance to the insecticide spinosad in a laboratory colony of the oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae). Resistance was selected by using topical applications of spinosad. After eight generations of selection, the LD50 of the selected line was 408 times greater compared with that of the untreated parental colony. This spinosad-resistant line did not exhibit cross-resistance to 10 other insecticides tested, including six organophosphates (naled, trichlorfon, fenitrothion. fenthion, formothion, and malathion) one carbamate (methomyl), and three pyrethroids (cyfluthrin, cypermethrin, and fenvalerate). However, using lines previously selected for resistance to these same insecticides, two of the 10 lines tested (naled- and malathion-resistant) did show some cross-resistance to spinosad. Also, oriental fruit flies from different field collections where naled and malathion have been used for control purposes displayed some resistance to spinosad. In addition, the effects of direct ingestion of spinosad through dietary supplementation also were tested. Overall, the laboratory resistance and cross-resistance data developed in this study provide new information that will be useful for managing the development of resistance when spinosad is used to control B. dorsalis in the field.     

Which acetylcholinesterase functions as the main catalytic enzyme in the Class Insecta?

Most insects possess two different acetylcholinesterases (AChEs) (i.e., AChE1 and AChE2; encoded by ace1 and ace2 genes, respectively). Between the two AChEs, AChE1 has been proposed as a major catalytic enzyme based on its higher expression level and frequently observed point mutations associated with insecticide resistance. To investigate the evolutionary distribution of AChE1 and AChE2, we determined which AChE had a central catalytic function in several insect species across 18 orders. The main catalytic activity in heads was determined by native polyacrylamide gel electrophoresis in conjunction with Western blotting using AChE1- and AChE2-specific antibodies. Of the 100 insect species examined, 67 species showed higher AChE1 activity; thus, AChE1 was considered as the main catalytic enzyme. In the remaining 33 species, ranging from Palaeoptera to Hymenoptera, however, AChE2 was predominantly expressed as the main catalytic enzyme. These findings challenge the common notion that AChE1 is the only main catalytic enzyme in insects with the exception of Cyclorrhapha, and further demonstrate that the specialization of AChE2 as the main enzyme or the replacement of AChE1 function with AChE2 were rather common events, having multiple independent origins during insect evolution. It was hypothesized that the generation of multiple AChE2 isoforms by alternative splicing allowed the loss of ace1 during the process of functional replacement of AChE1 with AChE2 in Cyclorrhapha. However, the presence of AChE2 as the main catalytic enzyme in higher social Hymenoptera provides a case for the functional replacement of AChE1 with AChE2 without the loss of ace1. The current study will provide valuable insights into the evolution of AChE: which AChE has been specialized as the main catalytic enzyme and to become the main target for insecticides in different insect species.     

Rapid microtitre plate test distinguishes insecticide resistant acetylcholinesterase genotypes in the mosquitoes Anopheles albimanus, An.nigerrimus and Culex pipiens

A rapid method of distinguishing insecticide insensitive acetylcholinesterase (AChE) genotypes was applied to three species of mosquitoes. This relies on comparing rates of an AChE mediated reaction in the presence and absence of insecticides which are inhibitors, using a kinetic microtitre plate reader. Clearer and more rapid resolution between genotypes was achieved than with previous assays which measure the amount of product formed at a fixed end-point. Results are presented for the F1s from crossing resistant and susceptible Anopheles albimanus Wiedemann and Culex pipiens L., for a strain of An. albimanus with a translocation linking the AChE gene to the Y chromosome and for field collected An. nigerrimus Giles. Propoxur and malaoxon were used as inhibitors. In all three species the enzyme was more insensitive to propoxur than malaoxon. Susceptible enzymes in all species also showed higher uninhibited AChE activity than their resistant counterparts. Presentation of both inhibited and uninhibited activities side by side may be useful to identify insects likely to be misclassified due to abnormally low AChE activities. Estimated frequencies of the three resistance genotypes in field populations of An. nigerrimus conformed to Hardy-Weinberg ratios. The implications of this technique for laboratory and field studies on insects are discussed.     

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.     

Determination of organophosphorous and carbamate insecticides by flow injection analysis.

A flow injection system, incorporating an acetylcholinesterase (AChE) single bead string reactor (SBSR), for the determination of some organophosphorous (azinphos-ethyl, azinphos-methyl, bromophos-methyl, dichlorovos, fenitrothion, malathion, paraoxon, parathion-ethyl and parathion-methyl) and carbamate insecticides (carbofuran and carbaryl) is presented. The detector is a simple pH electrode with a wall-jet entry. Variations in enzyme activity due to inhibition are measured from pH changes when the substrate (acetylcholine) is injected before and after the passage of the solution containing the insecticide. The percentage inhibition of enzyme activity is correlated to the insecticide concentration. Several parameters influencing the performance of the system are studied and discussed. The detection limits of the insecticides ranged from 0.5 to 275 ppb. The determination of these compounds was conducted in Hepes buffer and a synthetic sea water preparation. The enzyme reactor can be regenerated after inhibition with a dilute solution of 2-PAM and be reused for analysis. The immobilized enzyme did not lose any activity up to 12 weeks when stored at 4 degrees C.     

High‐throughput genotyping of single‐nucleotide polymorphisms in ace‐1 gene of mosquitoes using MALDI‐TOF mass spectrometry

Abstract Acetylcholinesterase (AChE) plays a vital role in the nervous system of insects and other animal species and serves as the target for many chemical agents such as organophosphate and carbamate insecticides. The mosquito, Culex pipiens complex, a vector of human disease, has evolved to be resistant to insecticides by a limited number of amino acid substitutions in AChE1, which is encoded by the ace‐1 gene. The aims of this study are to identify single nucleotide polymorphism (SNP) sites in the ace‐1 gene of the C. pipiens complex and explore an economical high‐throughput method to differentiate the genotypes of these sites in mosquitoes collected in the field. We identified 22 SNP sites in exon regions of the ace‐1 gene. Four of them led to non‐synonymous mutations, that is, Y163C, G247S, C677S and T682A. We used matrix‐assisted laser desorption ionization – time‐of‐flight mass spectrometry for genotyping at these four sites and another site F416V, which was relevant to insecticide resistance, in 150 mosquitoes collected from 15 field populations. We were able to synchronize analysis of the five SNP sites in each well of a 384‐well plate for each individual mosquito, thus decreasing the cost to one‐fifth of the routine analysis. Heterozygous genotypes at Y163C and G247S sites were observed in one mosquito. The possible influence of the five SNP sites on the activity or function of the enzyme is discussed based on the predicted tertiary structure of the enzyme.     

Mutations of acetylcholinesterase1 contribute to prothiofos-resistance in Plutella xylostella (L.)

Insensitive acetylcholinesterase (AChE) is involved in the resistance of organophosphorous and carbamate insecticides. We cloned a novel full-length AChE cDNA encoding ace1 gene from adult heads of the diamondback moth (DBM, Plutella xylostella). The ace1 gene encoding 679 amino acids has conserved motifs including catalytic triad, choline-binding site and acyl pocket. Northern blot analysis revealed that the ace1 gene was expressed much higher than the ace2 in all examined body parts. The biochemical properties of expressed AChEs showed substrate specificity for acetylthiocholine iodide and inhibitor specificity for BW284C51 and eserine. Three mutations of AChE1 (D229G, A298S, and G324A) were identified in the prothiofos-resistant strain, two of which (A298S and G324A) were expected to be involved in the prothiofos-resistance through three-dimensional modeling. In vitro functional expression of AChEs in Sf9 cells revealed that only resistant AChE1 is less inhibited with paraoxon, suggesting that resistant AChE1 is responsible for prothiofos-resistance.     

Ace2, rather than ace1, is the major acetylcholinesterase in the silkworm, Bombyx mori

Abstract  Two acetylcholinesterase ( ace ) genes have been reported in many insect species. In pests such as Helicoverpa assulta and Plutella xylostellas , ace 1 gene encodes the predominant synaptic enzyme that is the main target of organophosphorus (OP) and carbamate pesticides. It has been reported that pesticide selection has an impact on the ace gene evolution. The domesticated silkworm, Bombyx mori , also has two ace genes. We studied ace gene expression and enzyme activities in silkworm as this has not faced pesticide selection over the past decades. The expression levels of two ace genes, Bm- ace 1 and Bm- ace 2, were estimated by quantitative real-time polymerase chain reaction. Bm- ace 2 was expressed more highly than Bm- ace 1 in all tested samples of different developmental stages or tissues, suggesting ace 2, rather than ace 1, is the major type of acetylcholinesterase (AChE) in Bombyx mori . This is inconsistent with the aforementioned lepidopterons agricultural pests, partly be due to the widespread use of pesticides that may induce high expression of the ace 1 gene in these pests. Besides high expression in the head, Bm- ace 1 also expresses highly in the silk glands and Bm- ace 2 is abundant in the germline, implying both ace genes may have potential non-hydrolytic roles in development. Furthermore, we found that the mRNA levels of two ace genes and their ratios ( ace 2/ ace 1) change day to day in the first and third instars. This challenges the conventional method of estimating enzymatic activity using crude extract as an enzyme solution, as it is a mixture of AChE1 and AChE2. An efficient and simple method for separating different AChEs is necessary for reliable toxicological analyses.     

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.     

Biochemical genetics of altered acetylcholinesterase resistance to insecticides in the house fly

Resistance to the organophosphate insecticide tetrachlorvinphos was examined in a house fly (Musca domestica L.) strain with an altered acetylcholinesterase (AChE) of decreased sensitivity to inhibition by the insecticide. Genetic tests showed that both resistance and the altered AChE were controlled by semidominant gene(s) on chromosome II. The gene for resistance was five crossover units from the mutant marker stubby wing (stw). A house fly strain was prepared in which resistance was introduced in to a susceptible stw strain by recombination. Biochemical assays revealed that the altered AChE was introduced along with resistance. Assays of the AChE of resistant and susceptible stw strains by two independent methods showed that the enzyme from resistant flies was 30 times more slowly inhibited by tetrachlorvinphos than the enzyme from susceptible flies.This work was supported in part by NIH Grant ES 00901.Technical Article 13340, Texas Agricultural Experiment Station.     

Biochemical basis of organophosphate and carbamate resistance in Asian citrus psyllid

The Asian citrus psyllid, Diaphorina citri Kuwayama, is a worldwide pest of citrus, which vectors the putative causal pathogen of huanglongbing. Current management practices warrant continuous monitoring of field populations for insecticide resistance. Baseline activities of acetylcholinesterase (AChE), general esterase, and glutathione S-transferase as well as sensitivity of AChE to selected organophosphate and carbamate insecticides were established for a susceptible laboratory strain (Lab) and compared with several field populations of D. citri from Florida. The specific activity of AChE in various D. citri populations ranged from 0.77 to 1.29 microM min(-1) mg of protein(-1); the Lab strain was characterized by the highest activity. Although reduced AChE sensitivity was observed in the Lab strain compared with field populations, overlap of 95% confidence intervals of I50 values (concentration required for 50% AChE activity inhibition) suggests no significant difference in AChE sensitivity among all populations tested for a given insecticide. There was no significant evidence of target site insensitivity in field populations that were exposed to the selected organophosphate and carbamate insecticides tested. The specific activity of general esterase and glutathione S-transferase was lowest in the Lab strain and was generally comparable to that of the field populations evaluated. The current data provide a mode-of-action specific baseline for future monitoring of resistance to organophosphate and carbamate insecticides in populations of D. citri.     

Fitness costs of an insecticide resistance and their population dynamical consequences in the oriental fruit fly

Naled is a commonly used insecticide for controlling populations of the oriental fruit fly, Bactrocera dorsalis (Hendel), in Taiwan and other countries. B. dorsalis has developed resistance to the insecticide, and the resistance management is an important issue. Ecological effects (e.g., fitness costs) of the resistance, when fully understood, can be used for the resistance management. This study examined the effects of the insecticide resistance on important life history traits (i.e., survival rates, stage durations, and fecundity) of the oriental fruit fly by comparing the traits of insecticide resistant individuals and susceptible individuals. Population dynamical properties were also examined using a stage-structured matrix model that was parameterized with the empirical data. The results revealed that susceptible individuals had shorter stage durations (e.g., grew faster) and reproduced more than resistant individuals. The average longevity of sexually mature susceptible adults was longer than that of sexually mature resistant adults. The matrix population model predicted that a population of the susceptible individuals would grow faster than a population of the resistant individuals in the absence of the insecticide. The sensitivity analysis of the model suggests that the sexually immature adult stage is a good candidate for controlling B. dorsalis populations.     

无脊椎动物乙酰胆碱酯酶研究进展

乙酰胆碱酯酶(AChE)是生物体中一种十分重要的神经递质水解酶,也是有机磷和氨基甲酸酯类杀虫剂的作用靶标。AChE在不同生物中的性质显著不同,如编码基因个数、序列保守性、表达分布及生理功能等。作为杀虫剂的主要作用靶标之一,AChE不但可以通过单个点突变引起昆虫抗药性,还能够通过多个点突变联合作用、靶标表达量变化及基因复制等方式引起抗药性并且改变昆虫的适合度代价。本文主要从AChE的基因类型、分子进化、蛋白结构、生理功能、与昆虫的抗药性关系、同一物种中不同AChE的性质等6个方面对昆虫纲、蛛形纲和线虫等无脊椎动物AChE的研究进展作一综述。