Characterizations of general esterases in relation to malathion susceptibility in two field populations of the oriental migratory locust, Locusta migratoria manilensis (Meyen)

General esterases were characterized and compared from two populations of the oriental migratory locust, Locusta migratoria manilensis, collected from Huanghua and Pingshan Counties, Hebei Province, China. General esterases were most concentrated in the thorax and abdomen, which contained 46.1 and 36.1% of total esterase activity in females, and 42.7 and 36.0% in males, respectively, when α-naphthyl acetate was used as a substrate. There was no distinct difference in esterase banding patterns in different body regions for the substrates α-naphthyl acetate and β-naphthyl acetate on non-denaturing polyacrylamide gel electrophoresis (PAGE). However, the general esterase activities in the Huanghua population were 1.8-fold higher than those in the Pingshan population in both females and males. Increased esterase activity in the Huanghua population appeared to be mainly due to several additional esterase bands detected on non-denaturing PAGE. Inhibition studies of general esterases using four inhibitors, including paraoxon, malaoxon, eserine, and carbaryl, indicated that most general esterases in the two populations were B-type. The increased esterase activity in the Huanghua population appeared to be associated with a 1.8-fold decreased susceptibility to malathion. Such differences may attribute to the difference in control practices for the locust between Huanghua and Pingshan Counties.     

Increased activity and reduced sensitivity of acetylcholinesterase associated with malathion resistance in a field population of the oriental migratory locust, Locusta migratoria manilensis (Meyen)

The susceptibility to malathion, and the activity and sensitivity of acetylcholinesterase (AChE, EC 1.1.1.7) were compared between two populations of the oriental migratory locust, Locusta migratoria manilensis (Meyen) collected from Wudi County of Shandong Province in East China and Huangliu County of Hainan Province in South China. Huangliu population showed 8.5-fold resistance to malathion compared with Wudi population. AChE from Huangliu population showed 4.8-fold higher activity than that from Wudi population toward the model substrate acetylthiocholine (ATC). Kinetic studies indicated that AChE from Huangliu population had 2.6-fold lower affinity, but 5.0-fold higher catalytic activity toward ATC than AChE from Wudi population. Significantly increased activity of AChE in Huangliu population was also confirmed by non-denaturing polyacrylamide gel electrophoresis. Inhibition kinetics revealed that AChE from Huangliu population was 9.8-, 2.4-, 8.0- and 7.7-fold less sensitive to inhibition by paraoxon, malaoxon, chlopyrifos oxon, demeton-S-methyl, respectively, than that from Wudi population. Our studies revealed that a mild resistance to malathion in Huangliu population was associated with reduced sensitivity and increased catalytic activity of AChE. Our results suggest that alterations of AChE may play an important role conferring or contribute to malathion resistance in Huangliu population of the locust.     

Variability in resistance-related enzyme activities in field populations of the tarnished plant bug, Lygus lineolaris

Widespread use of Bt crops for control of lepidopterous pests has reduced insecticide use and provided the tarnished plant bug the opportunity to become a serious pest on mid-South cotton. Organophosphate insecticides have predominantly been used against plant bugs in recent years due to the reduced efficacy of other insecticides. In this study, a biochemical approach was developed to survey enzymatic levels associated with organophosphate resistance levels in field populations of the tarnished plant bug. Forty-three populations were collected from the delta areas of Arkansas, Louisiana, and Mississippi. Three esterase substrates and one substrate each of glutathione S-transferase (GST) and acetylcholinesterase (AChE) were used to determine corresponding detoxification enzyme activities in different populations. Compared to a laboratory susceptible colony, increases up to 5.29-fold for esterase, 1.96-fold for GST, and 1.97-fold for AChE activities were detected in the field populations. In addition to the survey of enzyme activities among the populations, we also examined the susceptibility of major detoxification enzymes to several inhibitors which could be used in formulations to synergize insecticide toxicity against the target pests. As much as 52-76% of esterase, 72-98% of GST, and 93% of AChE activities were inhibited in vitro. Revealing variable esterase and GST activities among field populations may lead to a better understanding of resistance mechanisms in the tarnished plant bug. This study also reports effective suppression of detoxification enzymes which may be useful in future insecticide resistance management program for the tarnished plant bug and other Heteropteran pests on Bt crops.     

Biochemical determination of acetylcholinesterase genotypes conferring resistance to the organophosphate insecticide chlorpyriphos in field populations of Bemisia tabaci from Benin, West Africa

Resistance to chlorpyriphos insecticide in Bemisia tabaci from a field population collected in Benin, West Africa was suggested with bioassay showing the presence of two sub-populations. Patterns of acetylcholinesterase (AChE) inhibition by the organophosphate chlorpyriphos-oxon were analyzed to estimate the number of possible genotypes with different sensitivity expected in three B. tabaci field populations collected in Benin. The analysis of inhibition patterns in these populations compared with four laboratory strains of B. tabaci using chlorpyriphos-oxon allowed the differentiation of three possible genotypes. In the reference strain SUD-S we detected two different acetylcholinesterases with different sensitivity to chlorpyriphos oxon suggesting the presence of two genes ace 1 and ace 2. The proportion of the insensitive enzyme (ace 2) was estimated to be 31%. In field populations we can detect two alleles at the same gene locus ace 1: one susceptible ace1S and one resistant ace1R. Both strains called Arizona University and Mexico-S2 have lost sensitive ace1S but the field populations from Benin clearly contained at least three genotypes confirming heterogeneous populations not completely resistant.     

麦穗鱼脑乙酰胆碱酯酶(AChE)的纯化及其比较性研究

用PEG2000双水相萃取、DEAE-Sephadex A-50和Sephadex G-200方法分离纯化麦穗鱼Pseudorasbora parva脑中的乙酰胆碱酯酶(AChE),然后比较分析纯酶液与粗酶液中鱼脑AChE 的动力学特性和抗抑制性,以便更直接地了解底物与酶以及毒剂与酶的反应关系。研究结果表明,经过一系列步骤的纯化,最后所得的AChE是纯度较高的酶液;通过对AChE的动力学研究发现,纯化后的麦穗鱼脑AChE与底物之间的亲和力和其在粗酶状态时没有显著的差别,而且纯化后的鱼脑AChE对底物抑制作用更敏感;抗抑制性研究发现,纯化后的麦穗鱼脑AChE对经溴水氧化的马拉硫磷(malathion)和三唑磷(triazophos)的敏感性显著高于粗酶状态的AChE,酶更易受抑制。     

Partial purification and characterization of a methyl-parathion resistance-associated general esterase in Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae)

Increased hydrolytic metabolism of organophosphate insecticides has been associated with resistance among Nebraska western corn rootworm populations. In this study, resistance-associated esterases were partially purified by differential centrifugation, ion exchange, and hydroxyapatite column chromatography, with a final purification factor of 100-fold and recovery of approximately 10%. Kinetic analysis of the partially purified enzyme indicated that the K_m of the group II esterases was identical for the two populations, although V_max was consistently threefold higher in the resistant population. A putative esterase, DvvII, was further purified to homogeneity by preparative polyacrylamide gel electrophoresis. DvvII is a monomer with a molecular weight of approximately 66 kDa, although three distinct isoforms with similar pIs were evident based on isoelectric focusing gel electrophoresis. Immunoassays with the Myzus persicae E4 antiserum indicated that group II esterases from D. v. virgifera were cross-reactive and expressed at much higher titers in the resistant population relative to the susceptible counterpart. These results suggest that the resistance is likely associated with overproduction of an esterase isozyme in resistant D. v. virgifera populations.     

Characterization of acephate resistance in the diamondback moth Plutella xylostella

Decreased acetylcholinesterase (AChE) sensitivity and metabolic detoxification mediated by glutathione S-transferases (GSTs) were examined for their involvement in resistance to acephate in the diamondback moth, Plutella xylostella. The resistant strain showed 47.5-fold higher acephate resistance than the susceptible strain had. However, the resistant strain was only 2.3-fold more resistant to prothiofos than the susceptible strain. The resistant strain included insects having the A298S and G324A mutations in AChE1, which are reportedly involved in prothiofos resistance in P. xylostella, showing reduced AChE sensitivity to inhibition by methamidophos, suggesting that decreased AChE1 sensitivity is one factor conferring acephate resistance. However, allele frequencies at both mutation sites in the resistant strain were low (only 26%). These results suggest that other factors such as GSTs are involved in acephate resistance. Expression of GST genes available in P. xylostella to date was examined using the resistant and susceptible strains, revealing no significant correlation between the expression and resistance levels.     

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。     

Triazophos resistance mechanisms in the rice stem borer (Chilo suppressalis Walker)

A field population of the rice stem borer (Chilo suppressalis Walker) with 203.3-fold resistance to triazophos was collected. After 8-generation of continuous selection with triazophos in laboratory, resistance increased to 787.2-fold, and at the same time, the resistance to isocarbophos and methamidophos was also enhanced by 1.9- and 1.4-fold, respectively, implying some cross-resistance between triazophos and these two organophosphate insecticides. Resistance to abamectin was slightly enhanced by triazophos selection, and fipronil and methomyl decreased. Synergism experiments in vivo with TPP, PBO, and DEM were performed to gain a potential indication of roles of detoxicating enzymes in triazophos resistance. The synergism results revealed that TPP (SR, 1.92) and PBO (SR 1.63) had significant synergistic effects on triazophos in resistant rice borers. While DEM (SR 0.83) showed no effects. Assays of enzyme activity in vitro demonstrated that the resistant strain had higher activity of esterase and microsomal O-demethylase than the susceptible strain (1.20- and 1.30-fold, respectively). For glutathione S-transferase activity, no difference was found between the resistant and the susceptible strain when DCNB was used as substrate. However, 1.28-fold higher activity was observed in the resistant strain when CDNB was used. These results showed that esterase and microsomal-O-demethylase play some roles in the resistance. Some iso-enzyme of glutathione S-transferase may involve in the resistance to other insecticides, for this resistant strain was selected from a field population with multiple resistance background. Acetylcholinesterase as the triazophos target was also compared. The results revealed significant differences between the resistant and susceptible strain. The V_max and K_m of the enzyme in resistant strain was only 32 and 65% that in the susceptible strain, respectively. Inhibition tests in vitro showed that I₅₀ of triazophos on AChE of the resistant strain was 2.52-fold higher. Therefore, insensitive AChE may also involved in triazophos resistance mechanism of rice stem borer.     

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.     

Toxicological and biochemical characterization of coumaphos resistance in the San Roman strain of Boophilus microplus (Acari: Ixodidae)

The San Roman strain of the southern cattle tick, Boophilus microplus, collected from Mexico was previously reported to have a high level of resistance to the organophosphate acaricide coumaphos. An oxidative detoxification mechanism was suspected to contribute to coumaphos resistance in this tick strain, as coumaphos bioassay with piperonyl butoxide (PBO) on larvae of this resistant strain resulted in enhanced coumaphos toxicity, while coumaphos assays with PBO resulted in reduced toxicity of coumaphos in a susceptible reference strain. In this study, we further analyzed the mechanism of oxidative metabolic detoxification with synergist bioassays of coroxon, the toxic metabolite of coumaphos, and the mechanism of target-site insensitivity with acetylcholinesterase (AChE) inhibition kinetics assays. Bioassays of coroxon with PBO resulted in synergism of coroxon toxicity in both the San Roman and the susceptible reference strains. The synergism ratio of PBO on coroxon in the resistant strain was 4.5 times that of the susceptible strain. The results suggested that the cytP450-based metabolic detoxification existed in both resistant and susceptible strains, but its activity was significantly enhanced in the resistant strain. Comparisons of AChE activity and inhibition kinetics by coroxon in both susceptible and resistant strains revealed that the resistant San Roman strain had an insensitive AChE, with a reduced phosphorylation rate, resulting in a reduced bimolecular reaction constant. These data indicate a mechanism of coumaphos resistance in the San Roman strain that involves both insensitive AChE and enhanced cytP450-based metabolic detoxification.     

Comparison of kinetic properties of a hydrophilic form of acetylcholinesterase purified from strains susceptible and resistant to carbamate and organophosphorus insecticides of green rice leafhopper (Nephotettix cincticeps Uhler)

A hydrophilic form of acetylcholinesterase (AChE) was purified from N-methyl carbamate susceptible (SA) and highly N-methyl carbamate-resistant (N3D) strains of the green rice leafhopper (GRLH), Nephotettix cincticeps Uhler. Both of purified AChE from SA and N3D strains displayed the highest activities toward acetylthiocholine (ATCh) at pH 8.5. In the SA strain, the optimum concentrations for ATCh, propionylthiocholine (PTCh), and butyrylthiocholine (BTCh) were about 1 × 10⁻³, 2.5 × 10⁻³, and 1 × 10⁻³ M, respectively. However, in the N3D strain, substrate inhibition was not identified for ATCh, PTCh, and BTCh to 1 × 10⁻² M. The K_m value in the SA strain was 51.1, 39.1, and 41.6 μM and that in the N3D strain was 91.8, 88.1, and 85.2 μM for ATCh, PTCh, and BTCh, respectively. The K_m value in the N3D strain indicated about 1.80-, 2.25-, and 2.05-fold lower affinity than that of the SA strain for ATCh, PTCh, and BTCh, respectively. The V_max value in the SA strain was 70.2, 30.5, and 4.6 U/mg protein and that in the N3D strain was 123.0, 27.0, and 14.5 U/mg protein for ATCh, PTCh, and BTCh, respectively. The V_max value in the N3D strain was 1.75- and 3.15-fold higher for ATCh and BTCh than that in the N3D strain. However, it was 1.13-fold lower for PTCh. The increased activity of AChE in the N3D strain is due to the qualitatively modified enzyme with a higher catalytic efficiency. The bimolecular rate constant (k_i) for propoxur was 27.1 × 10⁴ and 0.51 × 10⁴ M⁻¹ min⁻¹ in the SA and N3D strain and that for monocrotophos was 0.031 × 10⁴ and 2.0 × 10⁴ M⁻¹ min⁻¹ in the SA and N3D strain. AChE from the N3D strain was 53-fold less sensitive than SA strain to inhibition by propoxur. In contrast, AChE from the N3D strain was 65-fold more sensitive to inhibition by monocrotophos than AChE from the SA strain. This indicated negatively correlated cross-insensitivity of AChE to propoxur and monocrotophos.     

Insensitivity of acetylcholinesterase in a field strain of the fall armyworm, Spodoptera frugiperda (J.E. Smith)

Acetylcholinesterase (AChE) was purified from adult heads of the fall armyworm (Spodoptera frugiperda) by using a two-step procedure involving gel filtration on a Sephadex G-200 column and affinity chromatography on a procainamide-ECH Sephadex 4B column. Both susceptible and field strains possessed two AChE isozymes, namely, AChE-1 and AChE-2, with subunit molecular weights of 63.7 and 66.1 kDa. The purified AChE had an apparent K_m value of 33.5 μM and a V_max of 7.07 μmol/min/mg protein in the susceptible strain. The apparent K_m and the V_max were 2.2- and 2.0-fold higher, respectively, in the field strain than in the susceptible strain. The purified AChE from the field strain was 17- to 345-fold less sensitive than that from the susceptible strain to inhibition by carbamates (carbaryl, eserine, methomyl, and bendiocarb) and organophosphates (methyl paraoxon and paraoxon), insensitivity being highest toward carbaryl. The results further support the notion that insensitive AChE played an important role in the insecticide resistance observed in the field strain.     

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.     

Cholinesterase (ChE) inhibition in pumpkinseed (Lepomis gibbosus) as environmental biomarker: ChE characterization and potential neurotoxic effects of xenobiotics

Inhibition of cholinesterases (ChEs) has been widely used as an environmental biomarker of exposure to organophosphates (OP) and carbamate (CB) pesticides. More recently, this biomarker has been suggested as a putative biomarker for exposure to detergents. The use of cholinesterase inhibition as effect criterion in Ecotoxicology requires the previous characterization of the specific enzymatic forms that may be present in different tissues or organs. Different ChEs isoforms may be present in the same tissue and may exhibit distinct sensitivities towards environmental contaminants. This work intended to characterize the soluble ChEs present in pumpkinseed sunfish (Lepomis gibbosus) total head and dorsal muscle homogenates, through the use of different substrates and selective inhibitors of cholinesterasic activity. Also, the in vitro effects of sodium dodecylsulphate (SDS - anionic detergent) and chlorfenvinphos (organophosphate pesticide) on the enzymatic activity of the mentioned species were investigated. In general terms, the predominant cholinesterasic form present in both tissues was acetylcholinesterase. Chlorfenvinphos was responsible for inhibitory effects on AChE activity, while SDS did not cause any significant effect. These results suggest that in environmental monitoring programs, L. gibbosus head and dorsal muscle AChE can be an adequate diagnostic tool for exposure to OP pesticides; this conclusion however is not applicable to detergent residues. We also discuss the usefulness of L. gibbosus as an alternative model system and valuable option for freshwater ecotoxicological monitoring programs.     

Combined detoxification mechanisms and target mutation fail to confer a high level of resistance to organophosphates in Cydia pomonella (L.) (Lepidoptera: Tortricidae)

Despite the frequent and widespread applications of organophosphates against Cydia pomonella this species has developed low levels of resistance to this chemical group. Investigations concerning the mechanisms involved in resistance are scarce, and usually consider only one of the potential mechanisms. With the aim of a better understanding the resistance mechanisms and their possible interaction, four of these mechanisms were investigated simultaneously in one sensitive (Sv) and two resistant strains (Raz and Rdfb) of this insect. Resistant strains displayed an increased mixed function oxidase activity, whereas carboxylesterase activity varied upon the substrate used. The three strains had similar β-naphtyl acetate activity, and the hydrolysis of α-naphthyl acetate and p-nitrophenyl valerate was higher in the Sv strain. The p-nitrophenyl acetate activity was highest in the resistant strains and was strongly inhibited by azinphos and DEF. The Raz strain has a modified acetylcholinesterase (AChE), which resulted in a 0.7-, 3.2- and 21.2-fold decrease in the susceptibility to chlorpyriphos-ethyl-oxon, azinphos-methyl-oxon, and paraoxon-methyl, respectively. These combined resistance mechanisms only conferred to Raz a 0.6-, 7.9- and 3.1-fold resistance to the related insecticides. Organophosphates resistance in C. pomonella results from a combination of mechanisms including modified affinities to carboxylesterase substrates, and increased metabolisation of the insecticide. The apparent antagonism between increased functionalisation and reduced sensitivity of the AChE target is discussed.     

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.     

Enzyme inhibition by molluscicidal component of Areca catechu and Carica papaya in the nervous tissue of vector snail Lymnaea acuminata

In the present study toxic effects of active molluscicidal component of Areca catechu and Carica papaya was studied on certain enzymes in the nervous tissue of freshwater snail Lymnaea acuminata. In in vivo and in vitro exposure of arecoline (active component of Areca catechu seed) and papain (C. papaya latex and seed) significantly inhibited the acetylcholinesterase (AChE), acid and alkaline phosphatase (ACP/ALP) activity in the nervous tissue of L. acuminata. The inhibition kinetics of these enzymes indicate that arecoline and papain caused competitive and uncompetitive inhibition of AChE, respectively, whereas arecoline caused competitive-non-competitive inhibition of ACP/ALP and papain caused non-competitive inhibition of ACP/ALP. Thus the inhibition of AChE, ACP and ALP by arecoline and papain in the nervous tissue of L. acuminata may be the cause of molluscicidal activity of A. catechu and C. papaya, respectively.