Mutation in ace1 associated with an insecticide resistant population of Plutella xylostella

Insensitive acetylcholinesterase (AChE) was determined to be involved in an EPN-resistant (ER) strain and a contaminated susceptible (CS) strain of diamondback moth (DBM, Plutella xylostella L.), as estimated by AChE inhibition assay using DDVP as a inhibitor in a nondenaturing electrophoresis gel. The ER strain exhibited very high AChE insensitivity, high resistance ratio, and two point mutations (G324A, A298S) in ace1-type AChE gene (Pxace1). The CS strain showed low AChE insensitivity, low resistance ratio, and it has only one point mutation (G324A). These findings suggest that the A298S mutation, along with reported G324A mutation (Baek et al, 2005), can be important in the development of organophosphate resistance. These results also suggest that the A298S mutation could be a good candidate for a molecular diagnosis marker for resistance monitoring. Three molecular diagnosis methods (Quantitative Sequencing; QS, PCR amplification of specific alleles; PASA and restriction fragment length polymorphism; RFLP) were developed which successfully detected specific resistance associated point mutations. Seven local population DBMs were surveyed and showed high insecticide resistance levels and a A298S mutation in Pxace1. These methods can be used to monitor the resistance allele in field population of DBMs and resistance management strategy.     

Determination of organophosphate and carbamate resistance allele frequency in diamondback moth populations by quantitative sequencing and inhibition tests

A quantitative sequencing (QS) protocol was established for predicting the frequencies of the A298S and G324A mutations in the diamondback moth (Plutella xylostella) type-1 acetylcholinesterase (AChE) locus, putatively involved in organophosphate (OP) and carbamate (CB) insecticide resistance. The nucleotide resistant signal ratio at each mutation site was generated from sequencing chromatograms and plotted against the corresponding resistance allele frequency. Frequency prediction equations were generated from the plots by linear regression, and the signal ratios were highly correlated with resistance allele frequencies (r² > 0.987). QS analysis of 15 representative regional field populations of DBM in Korea revealed that the allele frequencies of both A298S and G324A were over 70% in most field populations, implying the prevalent state of these resistance-associated mutations. In the AChE inhibition assay, all populations showed reduced sensitivity to paraoxon, DDVP, carbaryl, and carbofuran, supporting the notion that DBM resistance to OPs and CBs is widespread in Korea.     

Identification and Molecular Characterization of Two Acetylcholinesterases from the Salmon Louse,Lepeophtheirus salmonis

Acetylcholinesterase (AChE) is an important enzyme in cholinergic synapses. Most arthropods have two genes (ace1 and ace2), but only one encodes the predominant synaptic AChE, the main target for organophosphates. Resistance towards organophosphates is widespread in the marine arthropod Lepeophtheirus salmonis. To understand this trait, it is essential to characterize the gene(s) coding for AChE(s). The full length cDNA sequences encoding two AChEs in L. salmonis were molecularly characterized in this study. The two ace genes were highly similar (83.5% similarity at protein level). Alignment to the L. salmonis genome revealed that both genes were located close to each other (separated by just 26.4 kbp on the L. salmonis genome), resulting from a recent gene duplication. Both proteins had all the typical features of functional AChE and clustered together with AChE-type 1 proteins in other species, an observation that has not been described in other arthropods. We therefore concluded the presence of two versions of ace1 gene in L. salmonis, named ace1a and ace1b. Ace1a was predominantly expressed in different developmental stages compared to ace1b and was possibly active in the cephalothorax, indicating that ace1a is more likely to play the major role in cholinergic synaptic transmission. The study is essential to understand the role of AChEs in resistance against organophosphates in L. salmonis.     

Molecular characterization of two acetylcholinesterase genes from the oriental tobacco budworm, Helicoverpa assulta (Guenée)

Acetylcholinesterase (AChE) has been known to be the target of organophosphorous and carbamate insecticides. Only a single AChE, however, existed in insects and was involved in insecticide resistance, recently another AChE is reported in mosquitoes and aphids. We have cloned cDNAs encoding two ace genes, designated as Ha-ace1 and Ha-ace2 by a combined degenerate PCR and RACE strategy from adult heads of the oriental tobacco budworm, Helicoverpa assulta. The Ha-ace1 and Ha-ace2 genes encode 664 and 647 amino acids, respectively and have conserved motifs including a catalytic triad, a choline-binding site and an acyl pocket. Both Ha-AChEs were determined to be secretory proteins based on the existence of a signal peptide. The Ha-ace1 gene, the first reported ace1 in lepidopterans, belongs to the ace1 subfamily whereas the Ha-ace2 gene showed high similarity to those in the ace2 subfamily. Phylogenetic analysis showed that the Ha-ace1 gene was completely diverged from the Ha-ace2, suggesting that the Ha-ace genes are duplicated. Quantitative real time-PCR revealed that expression level of the Ha-ace1 gene was much higher than that of the Ha-ace2 in all body parts examined. The biochemical properties of purified proteins by affinity chromatography showed substrate specificity for acetylthiocholine iodide, and inhibitor specificity for BW284C51 and eserine and their peptide sequences partially identified by a MALDI-TOF mass spectrometer demonstrated that two Ha-AChEs were expressed in vivo.     

Bovine brain acetylcholinesterase primary sequence involved in intersubunit disulfide linkages

Three distinct classes of membrane-bound acetylcholinesterases (AChEs) have been identified. A12 AChE is composed of 12 catalytic subunits that are linked to noncatalytic collagen-like subunits through intersubunit disulfide bonds. G2 AChE is localized in membranes by a glycoinositol phospholipid covalently linked to the C-terminal amino acid. Brain G4 AChE involves two catalytic subunits linked by a direct intersubunit disulfide bond while the other two are disulfide-linked to a membrane-binding 20-kDa noncatalytic subunit. Molecular cloning studies have so far failed to find evidence of more than one AChE gene in any organism although alternative splicing of torpedo AChE mRNA results in different C-terminal sequences for the A12 and G2 AChE forms. Support for a single bovine AChE gene is provided in this report by amino acid sequencing of the N-terminal domains from the G2 erythrocyte, G4 fetal serum, and G4 brain AChE. Comparison of the 38-amino acid sequences reveals virtually complete identity among the three AChE forms. Additional extensive identity between the fetal serum and brain AChEs was demonstrated by sequencing several brain AChE peptides isolated by high performance liquid chromatography after trypsin digestion of nitrocellulose blots of brain AChE catalytic subunits. Cysteines involved in intersubunit disulfide linkages in brain AChE were reduced selectively with dithiothreitol in the absence of denaturants and radioalkylated with iodoacetamide. The observed sequence of the major radiolabeled tryptic peptide was C*SDL, where C* was the radioalkylated cysteine residue. This sequence is precisely the same as that observed at the C terminus of fetal bovine serum AChE and shows close homology to the C-terminal sequence of torpedo A12 AChE. We conclude that the mammalian brain G4 AChEs utilize the same exon splicing pattern as the A12 AChEs and that factors other than the primary sequence of the AChE catalytic subunits dictate assembly with either the collagen-like or the 20-kDa noncatalytic subunits.     

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.     

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.     

Characterization of trimeric acetylcholinesterase from a legume plant, <Emphasis Type="Italic">Macroptilium atropurpureum</Emphasis> Urb.

We recently identified plant acetylcholinesterases (E.C.3.1.1.7; AChEs) homologous to the AChE purified from a monocotyledon, maize, that are distinct from the animal AChE family. In this study, we purified, cloned and characterized an AChE from a dicotyledon, siratro. The full-length cDNA of siratro AChE is 1,441 nucleotides, encoding a 382-residue protein that includes a signal peptide. This AChE is a disulfide-linked 125-kDa homotrimer consisting of 41–42 kDa subunits, in contrast to the maize AChE, which exists as a mixture of disulfide and non-covalently linked 88-kDa homodimers. The plant AChEs apparently consist of various quaternary structures, depending on the plant species, similar to the animal AChEs. We compared the enzymatic properties of the dimeric maize and trimeric siratro AChEs. Similar to electric eel AChE, both plant AChEs hydrolyzed acetylthiocholine (or acetylcholine) and propionylthiocholine (or propionylcholine), but not butyrylthiocholine (or butyrylcholine), and their specificity constant was highest against acetylcholine. There was no significant difference between the enzymatic properties of trimeric and dimeric AChEs, although two plant AChEs had low substrate turnover numbers compared with electric eel AChE. The two plant AChE activities were not inhibited by excess substrate concentrations. Thus, similar to some plant AChEs, siratro and maize AChEs showed enzymatic properties of both animal AChE and animal BChE. On the other hand, both siratro and maize AChEs exhibited low sensitivity to the AChE-specific inhibitor neostigmine bromide, dissimilar to other plant AChEs. These differences in enzymatic properties of plant AChEs may reflect the phylogenetic evolution of AChEs. Kosuke Yamamoto and Yoshie S. Momonoki contributed equally to this work.     

Absence of protein polymorphism attributable to insecticide-insensitivity of acetylcholinesterase in the green rice leafhopper, Nephotettix cincticeps

The cDNA sequence of acetylcholinesterase (AChE) from the green rice leafhopper, Nephotettix cincticeps, was amplified, based on conserved peptide sequences of AChEs. A 2.3 kb contiguous sequence, containing an ORF encoding an AChE precursor with 677 amino acid residues was obtained. The deduced protein sequence showed the most similarity to that of AChE in the Colorado potato beetle, having common features in the primary AChE structure. cDNA sequences of individual leafhoppers from an insecticide susceptible strain and the resistant strain Nakagawara, whose methylcarbamate-insensitive AChEs show 10(2) or more I(50) ratio for propoxur, were compared. No fixed inter-strain difference was identified in the protein sequence, though amino acid substitution polymorphism was found at one position in the susceptible strain. Insecticide-insensitivity of leafhopper AChE does not result from changes in the protein primary structure that is encoded by the AChE gene sequence isolated in this study.     

An amino acid substitution on the second acetylcholinesterase in the pirimicarb-resistant strains of the peach potato aphid,Myzus persicae

cDNAs encoding two acetylcholinesterases (AChEs) were isolated from the peach potato aphid, Myzus persicae. MpAChE1 was orthologous and MpAChE2 was paralogous with the ace of Drosophila melanogaster. The deduced amino acid sequence of MpAChE1 cDNA was identical between the pirimicarb susceptible and resistant strains. However, a single amino acid substitution of Ser431Phe on MpAchE2 was found in the pirimicarb resistant strains. This substitution was located in the acyl pocket of the enzyme and was thought to alter the ligand specificity.     

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.     

Mutation in acetylcholinesterase1 associated with triazophos resistance in rice stem borer, Chilo suppressalis (Lepidoptera: Pyralidae)

Two full-length genes encoding different acetylcholinesterases (AChEs), designated as Ch-ace1 and Ch-ace2, were cloned from strains of the rice stem borer (Chilo suppressalis) susceptible and resistant to the organophosphate insecticide triazophos. Sequence analysis found an amino acid mutation A314S in Ch-ace1 (corresponding to A201 in Torpedo californica AChE) that was consistently associated with the occurrence of resistance. This mutation removed an MspA1 I restriction site from the wild type allele. An assay based on restriction fragment length polymorphism (RFLP) analysis was developed to diagnose A314S genotypes in field populations. Results showed a strong correlation between frequencies of the mutation and phenotypic levels of resistance to triazophos. The assay offers a prospect for rapid monitoring of resistance and assisting with the appropriate choice of insecticide for combating damage caused by C. suppressalis.     

Comparative inhibition kinetics for acetylcholinesterases extracted from organophosphate resistant and susceptible strains of Boophilus microplus (Acari: Ixodidae)

In this study, acetylcholinesterases (AChEs) were extracted from two Mexican Boophilus microplus strains that demonstrated resistance to the organophosphate (OP) acaricide, coumaphos, in bioassay. The rate of inhibition of the extracted AChEs by the diethyl-OP paraoxon was determined for two resistant strains and two susceptible strains of B. microplus. The time to inhibition of 50% AChE activity was approximately two-fold greater for the resistant strains. Kinetic analysis of the interaction of the resistant AChEs with paraoxon revealed reduced bimolecular reaction constants (ki). Apparent conformational changes in the AChE of the resistant strains were reflected in reduced Km and Vmax values. The bimolecular reaction constants (ki) of the resistant strains were most affected by a slower rate of enzyme phosphorylation (k2).     

Existence of Two Acetylcholinesterases in the Mosquito Culex pipiens (Diptera: Culicidae)

Abstract: Two acetylcholinesterases (AChEs), AChE1 and AChE2, differing in substrate specificity and in some aspects of inhibitor sensitivity, have been characterized in the mosquito Culex pipiens . The results of ultracentrifugation in sucrose gradients and nondenaturing gel electrophoresis of AChE activity peak fractions show that each AChE is present as two molecular forms: one amphiphilic dimer possessing a glycolipid anchor and one hydrophilic dimer that does not interact with nondenaturing detergents. Treatment by phosphatidylinositol-specific phospholipase C converts each type of amphiphilic dimer into the corresponding hydrophilic dimer. Molecular forms of AChE1 have a lower electrophoretic mobility than those of AChE2. However, amphiphilic dimers and hydrophilic dimers have similar sedimentation coefficients (5.5S and 6.5S, respectively). AChE1 and AChE2 dimers, amphiphilic or hydrophilic, resist dithiothreitol reduction under conditions that allow reduction of Drosophila AChE dimers. In the insecticide-susceptible strain S-LAB, AChE1 is inhibited by 5 × 10⁻⁴ M propoxur (a carbamate insecticide), whereas AChE2 is resistant. All animals are killed by this concentration of propoxur, indicating that only AChE1 fulfills the physiological function of neurotransmitter hydrolysis at synapses. In the insecticide-resistant strain, MSE, there is no mortality after exposure to 5 × 10⁻⁴ M propoxur: AChE2 sensitivity to propoxur is unchanged, whereas AChE1 is now resistant to 5 × 10⁻⁴ M propoxur. The possibility that AChE1 and AChE2 are products of tissue-specific posttranslational modifications of a single gene is discussed, but we suggest, based on recent results obtained at the molecular level in mosquitoes, that they are encoded by two different genes.     

褐飞虱乙酰胆碱酯酶基因全长cDNA的克隆及序列分析

利用反转录聚合酶链式反应(RT_PCR)结合快速扩增cDNA末端(RACE)技术克隆了褐飞虱Nilaparvata lugens 乙酰胆碱酯酶基因cDNA。该cDNA全长2 467 bp,包含一个1 938 bp的开放阅读框(GenBank登录号AJ852420); 在推导出的646个氨基酸残基的前体蛋白中, N端的前30个氨基酸残基为信号肽,随后的616个氨基酸残基是成熟的乙酰胆碱酯酶序列,其预测的分子量为69 418 D。在一级结构中,形成催化活性中心的3个氨基酸残基(Ser242,Glu371和His485),以及在亚基内形成二硫键的6个半胱氨酸完全保守; 位于催化功能域的14个芳香族氨基酸中有10 个完全保守。该酶的氨基酸序列与黑尾叶蝉的同源性最高,达83%。对来自23种昆虫（包括褐飞虱）的30个乙酰胆碱酯酶的聚类分析显示,褐飞虱的乙酰胆碱酯酶与其中6个Ⅱ型乙酰胆碱酯酶（AChE2）同属一个支系; 此外,只存在于昆虫AChE2中的超变区及特异的氨基酸残基,也存在于褐飞虱的乙酰胆碱酯酶中。以上结果表明,所克隆的褐飞虱的乙酰胆碱酯酶基因是一个与黑腹果蝇的orthologous型基因同源的AChE2基因。     

Acetylcholinesterases of the nematode Steinernema carpocapsae. Characterization of two types of amphiphilic forms differing in their mode of membrane association.

We analyzed the molecular forms of acetylcholinesterase (AChE) in the nematode Steinernema carpocapsae. Two major AChEs are involved in acetylcholine hydrolysis. The first class of AChE is highly sensitive to eserine (IC50 = 0.05 microM). The corresponding molecular forms are: an amphiphilic 14S form converted into a hydrophilic 14.5S form by mild proteolysis and two hydrophilic 12S and 7S forms. Reduction of the amphiphilic 14S form with 10 mM dithiothreitol produces hydrophilic 7S and 4S forms, indicating that it is an oligomer of hydrophilic catalytic subunits linked by disulfide bond(s) to a hydrophobic structural element that confers the amphiphilicity to the complex. Sedimentation coefficients suggest that 4S, 7S, 12S forms correspond to hydrophilic monomer, dimer, tetramer and that the 14S form is also a tetramer linked to one structural element. The second class of AChE is less sensitive to eserine (IC50 = 0.1 mM). Corresponding molecular forms are hydrophilic and amphiphilic 4S forms (monomers) and a major amphiphilic 7S form converted into a hydrophilic dimer by Bacillus thuringiensis phosphatidylinositol-specific phospholipase C. This amphiphilic 7S form thus possesses a glycolipid anchor. It appears that Steinernema (a very primitive invertebrate) presents AChEs with two types of membrane association that closely resemble those described for amphiphilic G2 and G4 forms of AChE in more evolved animals.     

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.     

Invertebrate acetylcholinesterases: Insights into their evolution and non-classical functions

Acetylcholinesterase (AChE) plays a pivotal role in synaptic transmission by hydrolyzing the neurotransmitter acetylcholine. In addition to the classical function of AChE in synaptic transmission, various non-classical functions have been elucidated. Unlike vertebrates possessing a single AChE gene (ace), invertebrates (nematodes, arachnids, and insects) have multiple ace loci, encoding diverse AChEs with a range of different functions. In the field of toxicology, AChE with synaptic function has long been exploited as the target of organophosphorus and cabarmate pesticides to control invertebrate pests for the past several decades. However, many aspects of the evolution and non-classical roles of invertebrate AChEs are still unclear. Although currently available information on invertebrate AChEs is fragmented, we reviewed the recent findings on their evolutionary status, molecular/biochemical properties, and deduced non-classical (non-neuronal) functions.