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.     

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.     

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.     

Effects of triazophos on biochemical substances of transgenic Bt rice and its nontarget pest Nilaparvata lugens Stål under elevated CO2

The brown planthopper (BPH), Nilaparvata lugens Stål, is a serious rice pest throughout Asia. Recent outbreaks of N. lugens populations were mainly associated with the overuse of pesticides and resistance to insecticides. Warmer global temperatures that are associated with anthropogenic climate change are likely to have marked ecological effects on terrestrial ecosystems. However, the effects of elevated CO₂ concentrations on the biochemical, physiological and nutrient quality of transgenic Bt rice that has been treated with pesticides and on the control efficacy of the pesticides are not understood. The present study investigated changes in soluble sugar content, free amino acid levels, oxalic acid levels, flavonoids levels, and triazophos residues in transgenic Bt rice (TT51) and the control efficacy of triazophos for N. lugens following triazophos foliar spray under conditions of elevated CO₂ (eCO₂). Our findings showed that the soluble sugar content of TT51 treated with triazophos under eCO₂ was significantly higher than that under ambient CO₂ (aCO₂) and also higher than that of the non-transgenic parent (MH63) under aCO₂. However, the results for free amino acid levels were the opposite of those for soluble sugar levels. The oxalic acid and flavonoid contents of rice plants significantly decreased with increases in triazophos concentration, CO₂ concentration, and days after treatment (DAT). The oxalic acid and flavonoid contents of TT51 treated with triazophos under eCO₂ were significantly lower than those under aCO₂ and also lower than those of MH63 under aCO₂. The residue concentration of triazophos varied with CO₂ concentration, rice variety, and DAT. The residues in TT51 treated with 80 ppm of trizaopos under eCO₂ were significantly lower than those under aCO₂ and those in MH63 under aCO₂. The survival rate of nymphs N. lugens in TT51 under eCO₂ was significantly higher than that under aCO₂ and that in MH63 under aCO₂ at 1 DAT or 15 DAT after the release of 2nd instars nymphs. These findings indicated that (1) for TT51, triazophos reduced the resistance of rice plants to N. lugens with an elevated CO₂ concentration, as N. lugens consumed more phloem sap on TT51 plants; (2) triazophos dissipation in TT51 under eCO₂ was significantly faster than that under aCO₂ and that in MH63 under aCO₂; (3) the control efficacy of triazophos for N. lugens significantly decreased under eCO₂. The present findings provide important information for integrated pest management among transgenic varieties.     

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.     

Genotyping of acetylcholinesterase in insects

To investigate the genotyping criteria for the insect acetylcholinesterase gene (ace), we cloned two types of ace genes in domestic (Bombyx mori) and wild silkworm (Bombyxmandarina) through RT-PCR. The cloned genes were named Bm-ace1, Bm-ace2, Bmm-ace1 and Bmm-ace2, respectively. The ORFs of Bm-ace1 and Bmm-ace1 contained 2025 base pairs, encoding 683 amino acid residues (AA’s). The predicted protein has a molecular weight (MW) of 76.955 kDa and an isoelectric point (pI) of 6.36. The Bm-ace2 and Bm-ace2 genes contained 1917 bp nucleotides, encoding 638 AA’s. The predicted protein has a MW of 71.675 kDa and a pI of 5.49. Both ace1 and ace2 contain signature domains of acetylcholinesterases. Homology analysis of 18 NCBI downloaded insect AChEs peptide sequences and the 4 AChEs deducted in this study revealed that type 1 and type 2 insect AChEs had significant differences. Type 2 sequence is more conserved than type 1. Near the active centers of both types of AChEs, 48 strictly conserved AA’s (336-384) are present, and homology of these two peptide fragments was only 54.16%. Meanwhile, at AA positions 280-297, type 1 and type 2 AChEs both have conserved sequences with the similarity of the two being 52.94%. In type 2 AChEs, a uniquely conserved peptide sequence is found at positions 226-239 (QHLRVRHHQDKPL). We propose to use the above mentioned three conserved regions as criteria for insect acetylcholinesterases gene genotyping. This will benefit the genotyping of other acetylcholinesterase genes and the study of their functions.     

Molecular cloning and mRNA expression of a ryanodine receptor gene in the cotton bollworm, Helicoverpa armigera

Ryanodine receptors (RyRs) are the targets of novel diamide insecticides. The cotton bollworm, Helicoverpa armigera, is one of the most important cotton pests in the world. In this study, we report the full-length RyR cDNA sequence (named as HaRyR) of H. armigera. The 16,083-bp contiguous sequence encoded 5, 142 amino acid residues, which shares 80% and 78% overall identities with its homologues in Nilaparvata lugens (NlRyR) and Drosophila melanogaster (DmRyR), respectively. All hallmarks of RyR proteins are conserved in the HaRyR, including the GXRXGGGXGD motif conserved in the Ca²⁺ release channels and four copies of RyR domain unique to RyR channels. The previously identified seven lepidopteran-specific RyR residues were also found in HaRyR (N⁴⁹⁷⁷, N⁴⁹⁷⁹, N⁴⁹⁹⁰, L⁵⁰⁰⁵, L⁵⁰³⁶, N⁵⁰⁶⁸ and T⁵¹¹⁹). An amino acid sequence alignment showed that the N-terminal region of HaRyR (residues 188–295) shared high sequence identity with NlRyR (94%) and DmRyR (92%), and moderate sequence identity (47–50%) with three rabbit RyR isoforms, while the short segment of the C-terminal transmembrane region of HaRyR (residues 4632–4676) exhibited moderate sequence identity with NlRyR (69%) and DmRyR (67%), and low sequence identity (19–28%) with three rabbit RyR isoforms. In addition, expression analysis of HaRyR revealed that the mRNA expression level in eggs was significantly lower than in third instar larvae, pupae and adults, and anatomical regulation of HaRyR expression was also observed with the highest expression level in head compared with thorax and abdomen. Our results lay a foundation for comprehensive structural and functional characterization of HaRyR and for understanding of the molecular mechanisms of toxicity selectivity of diamide insecticides among different species.     

Amino acid substitutions conferring insecticide insensitivity in Ace-paralogous acetylcholinesterase

Since insecticide insensitivity of acetylcholinesterase (AChE) was, found about 40 years ago, a cause of the resistance to organophosphates in the spider mite, more than 30 insect and Acarus species have added to the instance. Based on the 3-dimensional analysis of Torpedo AChE structure and sequencing of Drosophila AChE gene (Ace), amino acid substitutions conferring the insensitivity have been found in Drosophila melanogaster. However, no amino acid substitution responsible for the AChE insensitivity had been found in insects and Acari except Brachicera flies until the second type of AChE paralogous to Ace was discovered in Schizaphis graminus and Anopheles gambiae. Sequencing of Ace-paralogous AChE cDNAs has been followed in insect species of various orders. Now, various amino acid substitutions are found and correspond to different biochemical properties of insensitive AChEs in relation to the function of substituted amino acids in the 3-dimensional structure. Existence of two AChE genes raises questions about differentiation of the two genes, site of gene expression, and function of each enzyme.     

Molecular and biochemical mechanisms of organophosphate resistance in laboratory-selected lines of the oriental fruit fly (Bactrocera dorsalis)

Genetic and biochemical factors leading to resistance to various organophosphate (OP) based insecticides were studied in lines selected for OP-resistance in the oriental fruit fly Bactrocera dorsalis. Lines were separately selected for resistance to naled, trichlorfon, fenitrothion, fenthion, formothion, and malathion. Overall, these lines showed increased resistance ratios ranging from 13.7- to 814-fold relative to a susceptible (S) line. Also, in these newly selected lines the same three point mutations in the ace gene, previously identified in resistance studies and designated as I214V, G488S and Q643R, were found. As expected, the enzyme from the resistant lines showed lower overall activity and reduced sensitivity to inhibition by fenitrothion, methyl-paraoxon and paraoxon compared to the wild type acetylcholinesterase (AChE) enzyme. The apparent V_max values for esterase from the resistant lines were 1.2-3.69 times higher than that of the S line. Although only the naled-, trichlorfon- and fenthion-r lines showed lower esterase affinities (based on apparent K_m values) compared with the S line, all of the V_max/K_m ratios were higher in the resistant lines compared to that of the S line. The OP-resistant lines also displayed an overall similar pattern of isozyme expression, except for one additional band found only in the naled-r line and one band that was absent in the trichlorfon-, malathion-, and fenthion-r lines. Our results also show that overall, multiple examples of high OP resistance in selected lines of B. dorsalis exhibiting the same genetic alterations in the ace gene seen previously resulted in different effects on esterase enzyme activity in relation to various OP compounds.     

药剂对小菜蛾抗性及敏感品系乙酰胆碱酯酶抑制作用比较

采用浸叶法测定了云南通海、元谋和澜沧的小菜蛾plutella xylostella田间种群对常用杀虫剂的抗药性。结果表明,云南上述地区小菜蛾田间种群对各类杀虫剂均产生了不同程度的抗性。对有机磷类药剂的抗药性为1.74~31.1倍;对菊酯类药剂的抗药性为7.41~764倍;对阿维菌素类药剂则产生了 5.60~4.06×104倍的抗性。通过离体和活体试验测定了药剂对小菜蛾头部乙酰胆碱酯酶(AChE)的抑制作用。敌敌畏和灭多威对通海抗性品系AChE离体和活体内的抑制中浓度(I50)分别是敏感品系的209、26.5倍和2.21、2.16倍;敌敌畏对通海小菜蛾种群的离体和活体内抑制中时间(IT50)小于敏感品系,分别是敏感品系的0.32和0.17倍;而灭多威对通海小菜蛾种群的离体和活体内抑制中时间(IT50)则大于敏感品系,分别是敏感品系的1.37和1.74倍。     

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.     

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.     

Influence of three diets on susceptibility of selected insecticides and activities of detoxification esterases of Helicoverpa assulta (Lepidoptera: Noctuidae)

The oriental tobacco worm, Helicoverpa assulta Guenée, is one of the most destructive pests of tobacco and peppers in China. We determined the susceptibility of H. assulta reared on an artificial diet, chili pepper and tobacco to four insecticides (fenvalerate, phoxim, methomyl, indoxacarb) under laboratory conditions associated with the activities of acetylcholinesterase (AChE), carboxylesterase (CarE) and glutathione S-transferase (GST) in its larvae. H. assulta larvae that were fed with chili pepper were more susceptible to fenvalerate, indoxacarb, and phoxim than those that were fed with tobacco and the artificial diet, but not to methomyl. The larvae that were fed with chili pepper were 3.65-, 2.49-, 1.92- and 2.44-fold more susceptible to fenvalerate, phoxim, methomyl, and indoxacarb than those fed with tobacco, respectively. The AChE activities of H. assulta larvae that were fed with chili pepper and tobacco were 2.12 and 1.07 μmol mg⁻¹ 15 min⁻¹, respectively, almost 2-fold difference. The CarE activity of H. assulta larvae that were fed with chili pepper, tobacco and the artificial diet was 4.12, 7.40 and 7.12 μmol mg⁻¹ 30 min⁻¹, respectively. Similarly, the GST activities of H. assulta larvae that were fed with chili pepper, tobacco and the artificial diet was 52.02, 79.37 and 80.02 μmol mg⁻¹ min⁻¹, respectively. H. assulta larvae that were fed with chili pepper were more resistance to the tested insecticides. The low activities of AChE and the high activities of CarE and GST lead to H. assulta become more susceptible to the tested insecticides.     

λ-cyhalothrin and cypermethrin induced in vivo alterations in the activity of acetylcholinesterase in a freshwater fish, Channa punctatus (Bloch)

In the present study, the in vivo effects of λ-cyhalothrin and cypermethrin on the activity of acetylcholinesterase (AChE, EC 3.1.1.7) were evaluated for 96 h in brain, muscle and gills of Channa punctatus. Both compounds exhibited tissue specific as well as dose dependent decrease in the activity of AChE. The treated fish showed a significant decrease in the activity of AChE in brain and a lesser inhibition in muscle and gills in response to the increasing concentrations of λ-cyhalothrin as well as cypermethrin. Our results indicated that the brain was the main target organ for both insecticides, followed by muscle and gills, as determined by AChE inhibition study. However, these organs showed variations in the degree of AChE inhibition for separate treatments of both insecticides. The λ-cyhalothrin was a more potent AChE inhibitor as compared to cypermethrin. These findings indicated that apart from the established mechanism of delayed closure of sodium ion channels, these pyrethroids inhibit the activity of AChE in C. punctatus which could further aggravate their neurotoxic effects.     

Ace and ace-like genes of invasive redlegged earth mite: copy number variation,target-site mutations,and their associations with organophosphate insensitivity

Background

Invasive Australian populations of redlegged earth mite, Halotydeus destructor (Tucker), are evolving increasing organophosphate resistance. In addition to the canonical ace gene, the target gene of organophosphates, the H. destructor genome contains many radiated ace-like genes that vary in copy number and amino acid sequence. In this work, we characterise copy number and target-site mutation variation at the canonical ace and ace-like genes and test for potential associations with organophosphate insensitivity. This was achieved through comparisons of whole-genome pool-seq data from alive and dead mites following organophosphate exposure.

Results

A combination of increased copy number and target-site mutations at the canonical ace was associated with organophosphate insensitivity in H. destructor. Resistant populations were segregating for G119S, A201S, F331Y at the canonical ace. A subset of populations also had copy numbers of canonical ace > 2, which potentially helps overexpress proteins carrying these target-site mutations. Haplotypes possessing different copy numbers and target-site mutations of the canonical ace gene may be under selection across H. destructor populations. We also detected some evidence that increases in copy number of radiated ace-like genes are associated with organophosphate insensitivity, which might suggest potential roles in sequestration or breakdown of organophosphates.

Conclusion

Different combinations of target-site mutations and (or) copy number variation in the canonical ace and ace-like genes may provide non-convergent ways for H. destructor to respond to organophosphate selection. However, these changes may only play a partial role in organophosphate insensitivity, which appears to have a polygenic architecture. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.     

Mating pair combinations of insecticide-treated male and female Nilaparvata lugens Stål (Hemiptera: Delphacidae) planthoppers influence protein content in the male accessory glands (MAGs) and vitellin content in both fat bodies and ovaries of adult females

The brown planthopper, Nilaparvata lugens (Stål) (Hemiptera: Delphacidae), is an insect pest in which offspring are produced by the mating of adult males with adult females. This species is a classic case in which pest resurgence is induced by insecticides. In the past, studies of resurgence mechanisms have focused on insecticide-induced stimulation of reproduction in adult females. To date, however, the role that males play in the resurgence mechanisms of N. lugens has not been investigated. The aim of the present study is to examine changes in protein levels in male accessory glands (MAGs) induced by the insecticides triazophos and deltamethrin and to determine their relationship with vitellin content in the fat bodies and ovaries of adult females in the context of mating pairs. Our results show that protein content in MAGs is significantly affected by male mating status, insecticide type, and insecticide concentration. Insecticide application induced increased protein levels in MAGs. A greater quantity of MAG products was transferred to females via mating. Thus, protein levels in MAGs significantly decreased after mating. Experimental matings indicate that vitellin content in both fat bodies and ovaries of adult females in mating pairs consisting of a treated male and an untreated female (♂_t × ♀_ck) is significantly greater than that of females in pairs consisting of an untreated male and an untreated female (♂_ck × ♀_ck). Under various concentrations of the two insecticides, vitellin levels are highest in mating pairs consisting of a treated male and a treated female (♂_t × ♀_t), followed by mating pairs consisting of an untreated male with a treated female (♂_ck × ♀_t). These findings demonstrate that (1) insecticides have an effect on males; (2) insecticide effect can be transferred to females; and (3) the reproductive effect of insecticides is strongest in mating pairs in which both the males and females are treated compared to pairs in which only one individual is treated. These findings provide valuable information about the role of males in pesticide-induced resurgence of N. lugens.