Amino acid substitution in Ace paralogous acetylcholinesterase accompanied by organophosphate resistance in the spider mite Tetranychus kanzawai

Insecticide resistant strains of the kanzawa spider mite, Tetranychus kanzawai, with insensitive AChE have spread widely throughout Japan. To clarify the molecular mechanism of this insensitivity, acetylcholinesterase (AChE) cDNA of the resistant strains of T. kanzawai was determined based on the AChE cDNA sequence of Tetranychus urticae and the sequences compared between the two spider mite species. The cDNA encoded 687 amino acids of AChE primary structure showing high homology to T. urticae. Amino acid homology indicated that the AChE is an Ace paralogous type of insect AChE. There were only three substitutions of amino acid residues between the AChEs of the two species. In the AChE of the resistant strain of T. kanzawai, one of the three amino acid substitutions was Phe439Trp, which lines the acyl pocket of the enzyme active site. Considering that the same substitution was found at the equivalent position of Ace paralogous AChE in the resistant strain of Culex tritaeniorhynchus, Phe439Trp substitution likely plays an important role in the insecticide insensitivity of the mite AChE.     

Analysis of two acetylcholinesterase genes in Bombyx mori

Two acetylcholinesterases (AChE, EC 3.1.1.7) cDNAs were identified and cloned from silkworm, Bombyx mori. One of those, BmAChE-o cDNA, is comprised of 3197 nucleotides which encode 638 amino acids, having an amino acid sequence homology of 72% with Drosophila melanogaster Ace-orthologous AChE (AO-AChE). In some species, another AChE group based on the sequence, Drosophila Ace-paralogous AChE (AP-AChE) has been recognized in relation to organophosphate- or carbamate-resistance, but there have been few reports of AP-AChE among lepidopteran species. However, we isolated the AP-AChE from lepidopteran silkworm, and cloned full ORF as BmAChE-p, which cDNA consisted of 2465 nucleotides that encode 683 amino acids. The homologies with other AP-AChEs were over 60% when compared. Although silkworm is not a target of pesticides, the genomic information obtained in this study will contribute to insecticide-resistance study on lepidopteran pest species.     

Cloning of the acetylcholinesterase 1 gene and identification of point mutations putatively associated with carbofuran resistance in Nilaparvata lugens

Molecular mechanisms of carbofuran resistance in the brown planthopper, Nilaparvata lugens Stål, were investigated. A carbofuran-resistant strain (CAS) showed approximately 45.5- and 15.1-fold resistance compared with a susceptible strain (SUS) and a non-selected field strain (FM), respectively. Activities of the esterase and mixed-function oxidase were approximately 2.8- and 1.6-fold higher, respectively, in the CAS strain than in the SUS strain, suggesting that these enzymes play a minor role in carbofuran resistance. Interestingly, the insensitivity of acetylcholinesterase (AChE) to carbofuran was approximately 5.5- and 3.7-fold higher in the CAS strain compared to the SUS and FM strains, respectively, indicating that AChE insensitivity is associated with carbofuran resistance. Western blot analysis identified two kinds of AChEs, of which the type-1 AChE (encoded from Nlace1, which is paralogous to the Drosophila AChE gene) was determined to be the major catalytic AChE in N. lugens. The open reading frame of Nlace1 is composed of 1989 bp (approximately 74 kD) and revealed 52.5% and 24.3% amino acid sequence identities to those of Nephotettix cincticeps and Drosophila melanogaster, respectively. Screening of point mutations identified four amino acid substitutions (G119A, F/Y330S, F331H and H332L) in the CAS strain that likely contribute to AChE insensitivity. The frequencies of these mutations were well correlated with resistance levels, confirming that they are associated with reduced sensitivity to carbofuran in N. lugens. These point mutations can be useful as genetic markers for monitoring resistance levels in field populations of N. lugens.     

Molecular characterization of two acetylcholinesterase genes from the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae)

Acetylcholinesterase (AChE), which is encoded by the ace gene, catalyzes the hydrolysis of the neurotransmitter acetylcholine to terminate nerve impulses at the postsynaptic membrane. AChE is a primary target of many insecticides including organophosphates (OP) and carbamates (CB). In this study, full-length cDNA sequences of two ace genes (Nlace1 and Nlace2) were sequenced from the brown planthopper (BPH) Nilaparvata lugens, the most destructive insect pest of rice crops. Nlace1 cDNA is 2842 nucleotides long and contains an ORF potentially encoding a 790 amino acid peptide. Nlace2 cDNA is 2852 bp in length and contains an ORF that potentially encodes a 672 amino acid peptide. NlAChE1 has an identity of 40% with NlAChE2 at the amino acid sequence level. Phylogenetic analysis of 59 AChEs from 32 animal species showed that NlAChE1 is most closely related to AChE1s from Blattella germanica and Nephotettix cincticeps, while NlAChE2 is most closely related to AChE2 from N. cincticeps. Quantitative RT-PCR analysis showed that Nlace1 is expressed at a much higher level than Nlace2 in all developmental stages and tissues, demonstrating that NlAChE1 may be the dominant AChE form of the two enzymes. This result will help reveal the resistance mechanism of N. lugens to organophosphorous and carbamate insecticides and promote development of more selective insecticides targeting the main NlAChE1.     

Comparison of catalytic properties and inhibition kinetics of two acetylcholinesterases from a lepidopteran insect

Acetylcholinesterase (AChE) is the primary target of organophosphate (OP) and carbamate (CB) insecticides. Many insect species have been shown to have two different AChE genes. The amino acid identity between the two lepidopteran AChEs is lower than 40%, and potential differences in enzymatic function have not been characterized. In this study, the cDNAs encoding two AChEs (Boma-AChE1 and Boma-AChE2) from Bombyx mandarina were sequenced, and the corresponding proteins were heterologously expressed to compare their enzymatic properties and interactions with insecticides in vitro. Both of these enzymes had high specific activities for acetylthiocholine iodide. Studies on substrate and inhibitor specificities confirmed that both enzymes belong to AChE. Insecticide inhibition assays indicated that Boma-AChE1 was more sensitive than Boma-AChE2 to eight of the nine insecticides tested. However, Boma-AChE2 was more sensitive than Boma-AChE1 to one of the OP insecticides, heptenophos. The results suggested that two AChEs from a lepidopteran insect have distinct catalytic properties and responses to different inhibitors.     

Frequencies and evolution of organophosphate insensitive acetylcholinesterase alleles in laboratory and field populations of the house fly, Musca domestica L.

Resistance to organophosphate (OP) and/or carbamate insecticides can be due to mutations in the acetylcholinesterase gene (Ace). Genotypes of house fly, Musca domestica L., Ace were determined in twelve laboratory maintained strains (originally from North America, Europe and Asia) and two field collected populations from New York and Florida. There were 15 Ace alleles found and 11 of the alleles coded for a susceptible form of the enzyme (i.e., V260, A316, G342 and F407). Three of the four resistance alleles were previously described, while one is new. Phylogenetic analysis of the alleles suggests multiple origins of the F407Y mutation and multiple origins of the G342A mutation that confer OP resistance. Genotyping of field collected house flies from New York and Florida populations revealed the presence of only one resistance allele, Acev10 (containing the non-synonymous mutations for A342 and Y407). All other alleles detected from the field-collected flies coded for a susceptible AChE. Thus, we were able to categorize individual flies as having homozygous susceptible (Ace^S/Ace^S), homozygous insensitive (Ace^I/Ace^I or Acev10/Acev10) or heterozygous AChE. The frequencies of Ace^S and Ace^I were not different between the NY2002 and FL2002 populations. Both populations were out of Castle-Hardy-Weinberg equilibrium, having an excess of Ace^S/Ace^I individuals and very few Ace^S/Ace^S individuals. Comparison of Ace, Vssc and CYP6D1 genotypes indicates individual house flies commonly have resistance alleles at multiple loci. Comparison of genotype data with bioassays, as well as the use of genotype data in resistance studies is discussed.     

Cloning and expression of an atrazine inducible cytochrome P450, CYP4G33, from Chironomus tentans (Diptera: Chironomidae)

Previous studies performed in our laboratory have measured the effect of atrazine exposure on cytochrome P450-dependent monooxygenase activity and have found increased activity in midge larvae (Chironomus tentans) as a result of atrazine exposure (1-10 ppm). Here we report the cloning and expression of a specific C. tentans CYP4 gene that is responsive to atrazine induction with an open reading frame of 1678 bp which encodes a putative protein of 559 amino acid residues. Alignments of deduced amino acid sequences with other insect P450 genes and phylogenetic analysis indicated a high degree of similarity to other insect CYP4 genes. Northern blotting analysis employing a fragment of 1200 bp from the CYP4 gene as a probe indicated that the CYP4 gene was expressed in all developmental stages, but was expressed at highest levels in late instar larvae. Additionally, over-expression of CYP4 in C. tentans exposed to atrazine (10 mg/l) confirms the ability of atrazine to induce specific P450 genes and provides insight into potential consequences of atrazine exposure in aquatic organisms.     

Resistance mechanisms and associated mutations in acetylcholinesterase genes in Sitobion avenae (Fabricius)

Wheat aphid, Sitobion avenae (fabricius), is one of the most important wheat pests and has been reported to be resistant to commonly used insecticides in China. To determine the resistance mechanism, the resistant and susceptible strains were developed in laboratory and comparably studied. A bioassay revealed that the resistant strain showed high resistance to pirimicarb (RR: 161.8), moderate reistance to omethoate (32.5) and monocrotophos (33.5), and low resistance to deltamethrin (6.3) and thiodicarb (5.5). A biochemistry analysis showed that both strains had similar glutathione-S-transferase (GST) activity, but the resistant strain had 3.8-fold higher esterase activity, and its AChE was insensitive to this treatment. The I₅₀ increased by 25.8-, 10.7-, and 10.4-folds for pirimicarb, omethoate, and monocrotophos, respectively, demonstrating that GST had not been involved in the resistance of S. avenae. The enhanced esterase contributed to low level resistance to all the insecticides tested, whereas higher resistance to pirimicarb, omethoate, and monocrotophos mainly depended on AChE insensitivity. However, the AChE of the resistant strain was still sensitive to thiodicarb (1.7-fold). Thus, thiodicarb could be used as substitute for control of the resistant S. avenae in this case. Furthermore, the two different AChE genes cloned from different resistant and susceptible individuals were also compared. Two mutations, L436(336)S in Sa.Ace1 and W516(435)R in Sa.Ace2, were found consistently associated with the insensitivity of AChE. They were thought to be the possible resistance mutations, but further work is needed to confirm this hypothesis.     

与昆虫抗药性相关的乙酰胆碱酯酶基因突变研究进展

有机磷和氨基甲酸酯类杀虫剂的广泛使用导致许多害虫产生了明显的抗药性。害虫对这些杀虫剂产生抗性的一个重要原因是其乙酰胆碱酯酶 (AChE)基因发生突变,从而导致AChE敏感度下降。简要概述了AChE基因发生突变的昆虫种类,介绍了AChE基因突变对其结构与功能的影响、变构AChE的特性、AChE基因突变对适合度的影响以及AChE突变不同组合对抗性的影响。这些突变可为设计新颖的反抗性化合物开辟新的途径。     

The clone of laccase gene and its potential function in cuticular penetration resistance of Culex pipiens pallens to fenvalerate

Decreased insecticides cuticular penetration, as one of resistant mechanisms in insect, has been extensively documented. Laccases, are enzymes with p-diphenol oxidase activity, was related to the cuticular tanning in insect. In this study, one laccase 2 gene (CpLac2) was cloned from Culex pipiens pallens. The CpLac2 contains an open reading frame (ORF) of 2289 bp and encodes a putative 762 amino acid protein. The deduced protein of CpLac2 was more similar to laccase 2 than other insect laccases, and shared the highest identity with laccases from the same family mosquito, Aedes aegypti and Anopheles gambiae. The developmental expression model of CpLac2 in C. pipiens pallens was measured by RT-PCR. The result showed the CpLaC2 was abundantly expressed in egg, the 4th instar larva and pupa, which suggested the role of CpLac2 for egg chorion tanning and cuticular sclerotization. Meanwhile, the expression of CpLac2 in fenvalerate-susceptible and -resistant strains of C. pipiens pallens was measured by real-time PCR. The result revealed the CpLac2 was significant higher expressed in resistant strain than in susceptible strain. The overexpression of CpLac2 in resistant strain suggested that resistance could derive from reinforcement of the cuticle, which decreased the penetration of insecticide in cuticle.     

Acetylcholinesterase point mutations putatively associated with monocrotophos resistance in the two-spotted spider mite

Molecular mechanisms of monocrotophos resistance in the two-spotted spider mite (TSSM), Tetranychus urticae Koch, were investigated. A monocrotophos-resistant strain (AD) showed ca. 3568- and 47.6-fold resistance compared to a susceptible strain (UD) and a moderately resistant strain (PyriF), respectively. No significant differences in detoxification enzyme activities, except for the cytochrome P450 monooxygenase activity, were found among the three strains. The sensitivity of acetylcholinesterase (AChE) to monocrotophos, however, was 90.6- and 41.9-fold less in AD strain compared to the UD and PyriF strains, respectively, indicating that AChE insensitivity mechanism plays a major role in monocrotophos resistance. When AChE gene (Tuace) sequences were compared, three point mutations (G228S, A391T and F439W) were identified in Tuace from the AD strain that likely contribute to the AChE insensitivity as predicted by structure analysis. Frequencies of the three mutations in field populations were predicted by quantitative sequencing (QS). Correlation analysis between the mutation frequency and actual resistance levels (LC₅₀) of nine field populations suggested that the G228S mutation plays a more crucial role in resistance (r² = 0.712) compared to the F439W mutation (r² = 0.419). When correlated together, however, the correlation coefficient was substantially enhanced (r² = 0.865), indicating that both the F439W and G228S mutations may work synergistically. The A391T mutation was homogeneously present in all field populations examined, suggesting that it may confer a basal level of resistance.     

Expression of Ace-paralogous acetylcholinesterase of Culex tritaeniorhynchus with an amino acid substitution conferring insecticide insensitivity in baculovirus-insect cell system

In Ace paralogous acetylcholinesterase (AP-AChE) of Culex tritaeniorhynchus, an amino acid substitution, Phe455Trp, is accompanied by the insecticide insensitivity. To confirm the responsibility of the substitution to the insensitivity, AP-AChE cDNA with and without a Phe455Trp substitution and Ace orthologous AChE (AO-AChE) cDNA were expressed in a baculovirus-insect cell system and the biochemical properties of AChEs (AP-CxTI, AP-CxTS, and AO-CxT, respectively) determined. AP-CxTI which has the same level of affinity to ACh, the natural substrate, showed a drastic decline in affinity to the artificial substrates composed of a longer moiety. The sensitivity of AP-CxTI to inhibitors was extremely reduced when compared with AP-CxTS. The insensitivity to tested organophosphates was greater than to monomethyl carbamates. AO-AChE showed similar substrate specificity and a slightly higher sensitivity to inhibitors when compared with AP-CxTS. Taking the position of Phe455Trp in the acyl pocket of the active site into account, the dimensions of the acyl pocket appear to became smaller by the substitution and insensitive to inhibitors.     

Susceptibilities to methamidophos and enzymatic characteristics in 18 species of pest insects and their natural enemies in crucifer vegetable crops

The susceptibilities to methamidophos as well as the kinetic and inhibitory parameter of acetylcholinesterases (AChE) and the activities of carboxyestsrases (CarE) and glutathione-S-transferases (GST) were studied in 18 species field populations of insects collected in Fuzhou, China during April and May 2000 and 2001. The insect species included five hymenopteran endoparasitoids, one hymenopteran exoparasitoid, one hymenopteran hyperparasitoid, one dipteran predator, four coleopteran predator ladybirds, six herbivorous pest insects of lepidoptera, diptera, homoptera, and coleoptera, respectively. There existed significant correlations between the susceptibility to methamidophos and the k_i values of AChE to methamidophos, dichlorvos, and carbofuran and between the k_i and V_max values of AChE among 18 species of insects. The six herbivorous pests and four ladybirds showed significantly low k_i and V_max values of AChE compared to the seven parasitoids and predator Epistrophe balteate. It was difficult to correlate the susceptibility to methamidophos or the k_i values with the K_m values of AChE, or with the activity of CarE and GST. The activities of CarE and GST varied depending on the different insect species. Significant synergisms of piperonyl butoxide (PB), triphenyl phosphate (TPP), and diethyl maleate (DEM) with methamidophos were observed in 14 pest insects and their natural enemies. Synergisms of PB were found to be the greatest. Reduced k_i values suggested that insensitive AChE might play a critical role in the tolerance to methamidophos in the 18 insect species. The detoxification enzymes, mixed-function oxidase (MFO), CarE, and GST, were believed to be involved in the tolerance to methamidophos. MFO might play the most important role, and CarE or GST might be important in the tolerance in some insect species. Different models of tolerance to methamidophos and enzymatic potential were existed in parasitoids, predators, and herbivores based on the different selection of insecticide pressure (either directly by exposing to the spray in the field, or indirectly by the insecticides penetrated into the body of host insects) as well as different ecological and biological habitats.     

Acetylcholinesterase point mutations in European strains of Tetranychus urticae (Acari: Tetranychidae) resistant to organophosphates

BACKROUND: In Tetranychus urticae Koch, acetylcholinesterase insensitivity is often involved in organophosphate (OP) and carbamate (CARB) resistance. By combining toxicological, biochemical and molecular data from three reference laboratory and three OP selected strains (OP strains), the AChE1 mutations associated with resistance in T. urticae were characterised. RESULTS: The resistance ratios of the OP strains varied from 9 to 43 for pirimiphos‐methyl, from 78 to 586 for chlorpyrifos, from 8 to 333 for methomyl and from 137 to 4164 for dimethoate. The insecticide concentration needed to inhibit 50% of the AChE1 activity was, in the OP strains, at least 2.7, 55, 58 and 31 times higher for the OP pirimiphos‐methyl, chlorpyrifos oxon, paraoxon and omethoate respectively, and 87 times higher for the CARB carbaryl. By comparing the AChE1 sequence, four amino acid substitutions were detected in the OP strains: (1) F331W (Torpedo numbering) in all the three OP strains; (2) T280A found in the three OP strains but not in all clones; (3) G328A, found in two OP strains; (4) A201S found in only one OP strain. CONCLUSIONS: Four AChE1 mutations were found in resistant strains of T. urticae, and three of them, F331W, G328A and A201S, are possibly involved in resistance to OP and CARB insecticides. Among them, F331W is probably the most important and the most common in T. urticae. It can be easily detected by the diagnostic PCR‐RLFP assay developed in this study. Copyright © 2009 Society of Chemical Industry     

Sequence variation in the two-component histidine kinase gene of Botrytis cinerea associated with resistance to dicarboximide fungicides

The complete two-component histidine kinase gene (Bos1) was sequenced from eight dicarboximide-resistant (D^R) and six-sensitive (D^S) field isolates of Botrytis cinerea. Comparisons in the predicted amino acid sequences of Bos1 showed that each two D^R isolates had a single point mutation at amino acid position 365 from an isoleucine to serine (I365S) or to an asparagine (I365N). Three D^R isolates were characterized with a change from glutamine to proline at position 369 (Q369P) as well as an asparagine to serine at the position 373 (N373S). These mutations located within the 90-amino-acid repeats of Bos1 have been reported previously. One new mutation, however, was found in the D^R isolate 65-E8. In this isolate, a null mutation at the amino acid position 1040 in the Bos1 was detected. Inoculation tests showed that this isolate was nearly nonpathogenic to cucumber. After the Bos1 gene from the sensitive isolate 38B1 was transferred into the resistant isolate 65-E8, all transformants tested were sensitive to iprodione and capable of infecting cucumber. DNA fingerprint generated by micro-satellite primed-PCR showed that isolates were not clustered based on their sensitivity to iprodione. Results from this study indicate that mutations in the Bos1 gene associated with dicarboximide resistance are diverse in B. cinerea, and the Bos1 gene is associated with virulence of B. cinerea.     

Biology and mechanisms of sulfonylurea resistance in Schoenoplectiella juncoides,a noxious sedge in the rice paddy fields of Japan

Schoenoplectiella juncoides is a noxious sedge weed in rice paddy fields that has evolved resistance to sulfonylurea (SU) herbicides. The molecular basis of resistance is amino acid substitutions at Pro₁₉₇, Trp₅₇₄ or Asp₃₇₆ in the acetolactate synthase (ALS) enzyme, which is the target of SUs. Schoenoplectiella juncoides has two ALS genes and resistant plants have point mutations that cause amino acid substitutions in either encoded protein. Single‐nucleotide substitutions at the codon for Pro₁₉₇ in the ALS genes can cause six types of amino acid substitutions and all of these substitutions have been found in both ALS genes among Japanese SU‐resistant biotypes. Whole‐plant herbicide responses differ among the amino acid substitution types. Furthermore, analyses of ALS activity in plant extracts show that the extracts’ responses to herbicides differ, depending on which ALS gene is mutated. The activity responses of the ALS extracts to the SU, imazosulfuron, showed double‐sigmoid curves with plateaus of ~30% inhibition for Pro₁₉₇ substitutions in ALS1 and ~70% for Pro₁₉₇ substitutions in ALS2. This indicates that ALS1 and ALS2 contribute to the responses with a proportion of 7:3. The double‐sigmoid curves can be reconstructed to show the responses of the resistant and susceptible enzymes separately by regression analysis. The resistance levels of the separate ALS1 or ALS2 mutated enzyme are highly correlated with the whole‐plant responses, with a relationship that the former is the square of the latter. This could provide a quantitative insight into the physiological basis of resistance.     

Toxicological and biochemical characterizations of AChE in Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelididae)

The toxicological and biochemical characteristics of acetylcholinesterases (AChE) in the resistant and susceptible strains (SS) of Liposcelis bostrychophila were investigated. The two resistant strains were the dichlorvos-resistant strain (DDVP-R) and the phosphine-resistant strain (PH₃-R) with resistance ratios of 22.36 and 4.51, respectively. Compared to their susceptible counterpart, the AChE activity per insect and the specific activity of AChE in DDVP-R and PH₃-R were significantly higher. There were also significant kinetic differences between DDVP-R and PH₃-R. The apparent Michaelis-Menten constant (K_m) for acetylthiocholine iodide (ATChI) was obviously lower in SS than that in PH₃-R, indicating a higher affinity to the substrate ATChI in the susceptible strains. The affinity for the substrate ATChI in DDVP-R and SS were not significantly different. The V_max value of the PH₃-R was significantly greater when compared to the V_max for the SS suggesting a possible over expression of AChE in this resistant strain. The inhibition of AChE to insecticide exposure in vitro revealed that all six insecticides were inhibitory for the extracted AChE’s. Based on the I₅₀ values, AChE of the SS were more sensitive to dichlorvos, paraoxon-ethyl, malaoxon and demeton-S-methyl than those of the two resistant strains. As for carbaryl and eserine, the PH₃-R suggested a significantly higher I₅₀s compared to the susceptible strain, while, no significant differences were found between SS and DDVP-R.     

Molecular studies of knockdown resistance to pyrethroids: cloning of domain II sodium channel gene sequences from insects

Knockdown resistance (kdr) is a target-site resistance mechanism that confers nerve insensitivity to DDT and pyrethroid insecticides. In the housefly, Musca domestica, molecular cloning of the para-type sodium channel gene has revealed two amino acid mutations that are associated with kdr and super-kdr resistance phenotypes. Both mutations are located in the domain II region of the channel; Leu1014 to Phe in the hydrophobic segment IIS6 and Met918 to Thr in the IIS4-IIS5 linker. To investigate whether these mutations also occur in other insects, we have designed degenerate primers based on conserved sequences in the domain II region of the sodium channel and used these to PCR amplify this region from insecticide-susceptible strains of eight diverse insect species representing four different insect Orders: Helicoverpa armigera, Plutella xylostella, Spodoptera littoralis (Lepidoptera), Blattella germanica (Dictyoptera), Tribolium castaneum (Coleoptera), Myzus persicae, Aphis gossypii and Phorodon humuli (Hemiptera). The primers amplified closely related para-type sodium channel sequences from each insect with a minimum of 85% amino acid identity between species. All of the sequences contained ‘susceptible’ Leu and Met residues at the positions associated with kdr and super-kdr resistance in the housefly. Recent results detailing the presence of a kdr-type Leu to Phe mutation in pyrethroid-resistant strains of two important agricultural pests, P. xylostella and M. persicae, are discussed. ©1997 SCI