Characterization of esterases in malathion-resistant and susceptible strains of the pteromalid parasitoid Anisopteromalus calandrae

General esterase, malathion-specific carboxylesterase, phosphotriesterase, glutathione S-transferase, cytochrome P-450-dependent monooxygenase activity, and target site sensitivity were compared in malathion-resistant (R) and malathion-susceptible (S) strains of the parasitoid Anisopteromalus calandrae (Howard) (Hymenoptera: Pteromalidae). Activity against -naphthyl acetate was not significantly different in male and female wasps for either strain. General esterase activity ranged from 1.2-fold to 2.5-fold higher in the R strain compared with the S strain, but these differences between strains were not consistent. Based on V_max/K_m ratios estimated for a number of analogs of four substrates (-naphthyl acetate, β-naphthyl acetate, 4-methylumbelliferyl acetate, and p-nitrophenyl acetate) there was no evidence that general esterase activity was elevated or reduced in the R strain. Malathion-specific carboxylesterase (MCE) activity, determined by using 2,3-¹⁴C-malathion as substrate, was 10- to 30-fold higher in the R strain compared with that in the S strain. The MCE has a pH optima at about pH 7, is cytosolic, and is labile upon storage at −80°C. MCE activity could be recovered from native 10% PAGE gels and IEF–PAGE gels (pI=5.2), but the peak of MCE activity also contained the major peak of activity against -naphthyl acetate. There was no evidence for major involvement of phosphotriesterase, glutathione S-transferase, monooxygenase, or altered acetylcholinesterase in the resistance. These data suggest that an increased activity of a MCE in the R strain is the probable major mechanism conferring resistance to malathion in A. calandrae. This study provides the first characterization of a biochemical resistance mechanism in a parasitoid with a high level of resistance to an organophosphate insecticide.     

Biochemical characterization of hydrolytic and oxidative enzymes in insecticide resistant and susceptible strains of the German cockroach (Dictyoptera: Blattellidae).

We have identified resistance mechanisms in the German cockroach, Blattella germanica (L.), for propoxur and chlorpyrifos in strains of cockroaches that display multiresistance to several organophosphate and carbamate insecticides. The resistance mechanisms involve the combined effects of increased oxidative and hydrolytic metabolism and both strains are resistant to chlorpyrifos and propoxur. Experiments designed to test for similarity in metabolic enzymes suggest that, although the mechanisms involve similar processes, the enzymes responsible for insecticide detoxification are different in the two strains. Both resistant strains exhibited enhanced activity toward alpha-naphtholic esters relative to a standard susceptible strain; however, analysis of the progeny from resistant X susceptible crosses suggests that this general esterase activity is inherited differently than propoxur or chlorpyrifos resistance. Hybrids of the propoxur-resistant strain displayed the highest activity of all cockroaches tested, in contrast to hybrids of the chlorpyrifos-resistant strain, which were similar to the susceptible strain. Native gel electrophoresis of cytosolic preparations provided further evidence for differences in the pattern of hydrolytic enzymes and inheritance of resistance in the two strains. Analysis of components of the cytochrome P450-dependent monooxygenase system and activities toward model substrates indicate that the two resistance mechanisms also involve different oxidative processes. The propoxur-resistant strain displayed significantly higher levels of total cytochrome P450, but no other components were correlated with resistance. In contrast with the chlopyrifos-resistant strain, which was similar to the susceptible strain in all parameters measured, activity toward model substrates was higher in the propoxur-resistant strain than in any of the other strains and hybrids tested.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alterations in esterases are associated with malathion resistance in Habrobracon hebetor (Hymenoptera: Braconidae)

Biochemical mechanisms of malathion resistance were investigated in a malathion-resistant strain of the parasitoid Habrobracon hebetor Say collected from a farm storage in Kansas. General esterase activities were significantly lower in the resistant strain compared with those in a susceptible strain. However, no significant differences were found in activities of malathion specific carboxylesterase (MCE), glutathione S-transferase and cytochrome P450 dependent O-demethylase activities, cytochrome P450 contents, and sensitivity of acetylcholinesterase to inhibition by malaoxon between the 2 strains. Because MCE was not elevated in the resistant strain, the weak malathion resistance in H. hebetor may result from a different mechanism compared with that hypothesized for some insect species in which reduced general esterase activity is accompanied by an elevated MCE. Decreased esterase activity in the resistant strain suggested that null alleles of some esterases were associated with the resistance. Indeed, E1 and E2, major esterases in the susceptible strain, were not present in the resistant strain on polyacrylamide gels that were stained for esterase activity using the model substrate 1-naphthyl acetate. In contrast, the activity of esterase E3 on the gels was much higher in the resistant strain as compared with that of the susceptible strain. These findings indicate that malathion resistance in H. hebetor is associated with both an increased activity of the esterase E3 and null alleles of the esterases E1 and E2.     

有机磷对抗拟除虫菊酯家蝇的毒力

测定敏感、抗溴氰菊酯(Del-R)、抗氯菊酯(2Cl-R)的家蝇品系对有机磷杀虫剂敌敌畏、辛硫磷及马拉硫磷的LD₅₀,α-乙酸萘酯（α-NA）酯酶动力学,酯酶的活性和酯酶的抑制作用。Del-R和2Cl-R的家蝇品系对三种有机磷杀虫剂的抗性倍数为0.966～7.190倍,均为低抗水平。三个家蝇品系的羧酸酯酶活性水平与抑制中浓度存在正相关性,说明羧酸酯酶在抗拟除虫菊酯家蝇对有机磷杀虫剂的抗性中起一定的作用。     

A cluster of esterase genes on chromosome 3R ofDrosophila melanogaster includes homologues of esterase genes conferring insecticide resistance inLucilia cuprina

We identify an esterase isozyme inDrosophila melanogaster, EST 23, which shares biochemical, physiological, and genetic properties with esterase E3, which is involved in resistance to organophosphate insecticides inLucilia cuprina. Like E3, theD. melanogaster EST 23 is a membrane-bound -esterase which migrates slowly toward the anode at pH 6.8. Both enzymes have similar preferences for substrates with shorter acid side chain lengths. Furthermore, on the basis of their high sensitivity to inhibition by paraoxon and their insensitivity to inhibition by eserine sulfate, both enzymes were classified as subclass I carboxylesterases. The activity of each enzyme peaks early in development and, again, in the adult stage. Both enzymes are found in the male reproductive system and larval and adult digestive tissues, the latter being consistent with a role for these enzymes in organophosphate resistance. Fine structure deficiency mapping localizedEst 23 to cytological region 84D3 to E1-2 on the right arm of chromosome 3. Moreover, we show that the genes encoding three other esterase phenotypes also map to the same region; these phenotypes involve allozymic differences in EST 9 (formerly EST C), ali-esterase activity, defined by the hydrolysis of methyl butyrate, and malathion carboxylesterase activity, defined by hydrolysis of the organophosphate malathion. This cluster corresponds closely to that encompassing E3 and malathion carboxylesterase on chromosome 4 inL. cuprina, the homologue of chromosome 3R inD. melanogaster.     

Enhanced esterase gene expression and activity in a malathion-resistant strain of the tarnished plant bug, Lygus lineolaris

Extensive use of insecticides on cotton in the mid-South has prompted resistance development in the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois). A field population of tarnished plant bugs in Mississippi with 11-fold higher resistance to malathion was used to examine how gene regulation conferred resistance to this organophosphate insecticide. In laboratory bioassays, synergism by the esterase inhibitors S,S,S,-tributylphosphorotrithioate (DEF) and triphenylphosphate (TPP) effectively abolished resistance and increased malathion toxicity by more than 80%. Esterase activities were compared in vitro between malathion susceptible and resistant (selected) strains. More than 6-, 3- and 10-fold higher activities were obtained with the resistant strain using alpha-naphthyl acetate, beta-naphthyl acetate, and p-nitrophenyl acetate, respectively. Up to 95% and 89% of the esterase activity in the susceptible and resistant strains, respectively, was inhibited by 1 mM DEF. Inhibition of esterase activity up to 75% and 85% in the susceptible and resistant strains, respectively, was obtained with 0.03 mM TPP. Esterase activities in field populations increased by up to 5.4-fold during the fall season. The increase was synchronized with movement of the insect into cotton where exposure to pesticides occurred. Esterase cDNA was cloned and sequenced from both malathion susceptible and resistant strains. The 1818-nucleotide cDNA contained a 1710-bp open reading frame coding a 570 amino acid protein which was similar to many insect esterases conferring organophosphate resistance. No amino acid substitution was observed between susceptible and resistant strains, indicating that esterase gene mutation was not involved in resistance development in the resistant strain in Mississippi. Further examination of esterase gene expression levels using quantitative RT-PCR revealed that the resistant strain had a 5.1-fold higher level of esterase mRNA than the susceptible strain. The results of this study indicated that up-regulation of the esterase gene appeared to be related to the development of resistance in the tarnished plant bug.     

Genetic variability in esterases and the insecticide resistance in brazilian strains of Oryzaephilus mercator and Oryzaephilus surinamensis (Coleoptera: Silvanidae)

Oryzaephilus mercator and O. surinamensis are stored grains and processed food pests, the latter being responsible for major economical losses. Polyacrylamide gel electrophoresis was used to analyse esterase patterns during insect development. Seven esterases, three cholinesterases, two carboxylesterases and two acetylesterases, were identified in O. mercator, one of which was proper to adults. Five esterases, of which four were cholinesterases, occurred in O. surinamensis. Strains of O. mercator and O. surinamensis were also exposed to malathion and chlorpyrifos-methyl. According to the LC50 estimates, OmLC-M and OmLA strains of O. mercator and OsLB strain of O. surinamensis were the most resistant to both insecticides. However, higher sensitivity to malathion and chlorpyrifos-methyl has also been verified in some of its esterases. Cholinesterases OmEST-1 and OsEST-5 seem to be involved in this resistance. These results suggest that organophosphate tolerance may be related to genetic variability in esterase isoenzymes.     

Mechanisms of organophosphate resistance in a field population of oriental migratory locust, Locusta migratoria manilensis (Meyen)

The susceptibilities to three organophosphate (OP) insecticides (malathion, chlorpyrifos, and phoxim), responses to three metabolic synergists [triphenyl phosphate (TPP), piperonyl butoxide (PBO), and diethyl maleate (DEM)], activities of major detoxification enzymes [general esterases (ESTs), glutathione S-transferases (GSTs), and cytochrome P450 monooxygenases (P450s)], and sensitivity of the target enzyme acetylcholinesterase (AChE) were compared between a laboratory-susceptible strain (LS) and a field-resistant population (FR) of the oriental migratory locust, Locusta migratoria manilensis (Meyen). The FR was significantly resistant to malathion (57.5-fold), but marginally resistant to chlorpyrifos (5.4) and phoxim (2.9). The malathion resistance of the FR was significantly diminished by TPP (synergism ratio: 16.2) and DEM (3.3), but was unchanged by PBO. In contrast, none of these synergists significantly affected the toxicity of malathion in the LS. Biochemical studies indicated that EST and GST activities in the FR were 2.1- to 3.2-fold and 1.2- to 2.0-fold, respectively, higher than those in the LS, but there was no significant difference in P450 activity between the LS and FR. Furthermore, AChE from the FR showed 4.0-fold higher activity but was 3.2-, 2.2-, and 1.1-fold less sensitive to inhibition by malaoxon, chlorpyrifos-oxon, and phoxim, respectively, than that from the LS. All these results clearly indicated that the observed malathion resistance in the FR was conferred by multiple mechanisms, including increased detoxification by ESTs and GSTs, and increased activity and reduced sensitivity of AChE to OP inhibition.     

Anti-P450lpr antiserum inhibits specific monooxygenase activities in LPR house fly microsomes.

Monooxygenase activity in microsomes from the LPR strain of house fly (Musca domestica L.) was inhibited by anti-P450lpr, and antiserum specific for house fly cytochrome P450lpr. Anti-P450lpr did not inhibit house fly cytochrome P450 reductase or rat cytochrome P450 monooxygenase assays, consistent with specific inhibition of P450lpr. Anti-P450lpr inhibited the ability of cytochrome P450 reductase to reduce carbon monoxide treated LPR microsomal cytochrome P450, up to 49% of the total, showing that inhibition of cytochrome P450 reduction is the major mechanism of inhibition. Anti-P450lpr inhibited 98% of methoxyresorufin-O-demethylase activity and all the benzo(a)pyrene hydroxylase activity in LPR microsomes, but none of the pentoxyresorufin-O-dealkylase activity. The antiserum partially inhibited ethoxyresorufin-O-dealkylase and ethoxycoumarin-O-dealkylase activity. These results demonstrate that methoxyresourfin-O-demethylase activity and benzo(a)pyrene hydroxylase activity are characteristic substrates for P450lpr activity in the LPR strain of house fly.     

细胞色素P450介导抗性的进化可塑性

细胞色素P450是超基因家族,由其介导的杀虫剂代谢解毒的增强是昆虫产生抗药性的普遍而主要的机制。近年的研究表明,细胞色素P450介导的代谢抗性表现出一定程度的进化可塑性:即使是同种昆虫的不同种群在相同种类杀虫剂的胁迫下,进化选择出的抗性相关的细胞色素P450也有所不同,抗性的产生也可以是几种不同细胞色素P450协同作用或控制P450表达的调控因子的不同。     

Polymorphisms in a carboxylesterase gene between organophosphate-resistant and -susceptible Aphis gossypii (Homoptera: Aphididae)

Resistance to omethoate was suppressible by the hydrolytic enzyme inhibitor SSS-tributyl phosphorotrithioate in a laboratory-selected resistant cotton aphid, Aphis gossypii Glover, strain, suggesting the involvement of hydrolytic enzymes in the detoxification process. The kinetic properties of carboxylesterases from both resistant and susceptible cotton aphids were characterized by four acyl ester substrates: alpha-naphthyl acetate (alpha-NA), alpha-naphthyl butyrate (alpha-NB), alpha-naphthyl phosphate (alpha-NP), and beta-naphthyl phosphate (beta-NP). No significant differences of carboxylesterase activity were found between resistant and susceptible strains by using either alpha-NP or beta-NP as substrates. In contrast, the susceptible A. gossypii exhibited significantly higher activity compared with resistant aphids with either alpha-NA or alpha-NB as substrates. To understand the molecular basis of this esterase-mediated resistance, carboxylesterase genes from both strains were cloned. Two genes share 99.4% identity at the nucleic acid level and 99.2% identity at the amino acid level. The full length of the cDNA opening reading frame is 1581 bp, encoding 526 amino acids. Four amino acid substitutions, Thr210 --> Met210, Asn294 --> Lys294, Gly408 --> Asp408, and Ser441 --> Phe441, were identified in the resistant strain. Probing of Southern blots with the 0.5 kb esterase fragment showed the same banding patterns and intensities with genomic DNA extracts from both resistant and susceptible A. gossypii. Furthermore, the MspI and HpaII fragments are the same in both strains, indicating there is no methylation of sequences detected by the probe. The combined results suggest that the structural gene substitution is likely the molecular basis of the organophosphate resistance in this laboratory-selected cotton aphid strain.     

Molecular mechanisms conferring asymmetrical cross-resistance between tebufenozide and abamectin in Plutella xylostella

Based on the confirmation of asymmetrical cross-resistance between abamectin and tebufenozide in Plutella xylostella, the present work proved that the cytochrome P450 monooxygenase plays a decisive role in cross-resistance, and the expression of various cytochrome P450 (CYP450) genes in different strains was surveyed to elucidate the molecular basis of the underlying mechanisms. Enzyme analysis showed the activity of cytochrome P450 monooxygenase was notable enhanced in the strains resistant to both tebufenozide (3.07-fold) and abamectin (3.37-fold), suggesting that the enhancement of cytochrome P450 monooxygenase is the main detoxification mechanism responsible for the cross-resistance. CYP4M7 (64.58-fold) and CYP6K1 (41.97-fold) had extremely high expression levels in the Teb-R strain, selected using tebufenozide, which was highly resistant to tebufenozide (RR 185.5) and moderately cross-resistant to abamectin (RR 41.0). When this strain was subjected to further selection using abamectin, the resultant Aba-R strain showed a higher expression of CYP6K1 (60.32-fold). However, the expression of CYP4M7 was reduced (10.62-fold). Correspondingly, the Aba-R strain became more resistant to abamectin (RR 593.8) and less resistant to tebufenozide (RR 28.0). Therefore, we concluded that the over expression of CYP4M7 was the main cause for tebufenozide resistance, and that CYP6K1 mainly conferred abamectin resistance. The asymmetrical cross-resistance occurred because tebufenozide selection not only enhanced the expression of CYP4M7, but also that of CYP6K1. This is the first report on the molecular mechanism of asymmetrical cross-resistance between insecticides.     

Cytochrome P450 MA expression in insecticide-resistant German cockroaches (Dictyoptera: Blattellidae).

Cytochrome P450 monooxygenases are detoxification enzymes commonly involved in insecticide resistance by insects. Recently, an overexpressed form of this enzyme, P450 MA, was purified from an insecticide-resistant strain of German cockroach, Blattella germanica (L.), and polyclonal antisera (anti-P450 MA) was produced. To test hypotheses that the overexpressed condition of P450 MA has evolved in > 1 geographic location and that P450 MA might be involved in insecticide resistance to specific insecticides, investigations were conducted using 4 insecticide-resistant and 1 susceptible German cockroach strains. In western blots that used anti-P450 MA antiserum as a probe, substantial differences in expression of P450 MA were observed. Strains showing the highest P450 MA expression had both the highest tolerance to the organophosphate insecticide chlorpyrifos and cytochrome P450-mediated demethylation activity. Results support the hypothesis that cytochrome P450 MA is potentially overexpressed in insecticide-resistant populations on a global scale.     

两个单点突变对昆虫羧酸酯酶降解马拉硫磷的影响

马拉硫磷是一种高效低毒的有机磷杀虫剂, 分子量大且结构特殊, 广泛用于农业害虫的防治。羧酸酯酶突变是昆虫对有机磷类杀虫剂产生代谢抗性的重要机制之一。本实验室前期已从棉蚜Aphis gossypii、 褐飞虱Nilaparvata lugens、 斜纹夜蛾Spodoptera litura、 家蚕Bombyx mori、 异色瓢虫Harmonia axyridis、 赤拟谷盗Tribolium castaneum和西方蜜蜂Apis mellifera中各克隆了一个非特异性羧酸酯酶基因, 通过体外定点突变构建了G/A151D和W271L两种突变体, 并进行了原核细胞表达和纯化。本实验在体外测定了这7种昆虫野生型和两种突变型羧酸酯酶对马拉硫磷的降解。结果显示： 棉蚜、 西方蜜蜂、 斜纹夜蛾、 赤拟谷盗的野生型羧酸酯酶能够降解马拉硫磷, 两个突变并不能提高它们的降解活性, 而家蚕、 异色瓢虫和褐飞虱的野生型羧酸酯酶不能降解马拉硫磷, G/A151D和/或W271L突变能使这些酯酶获得马拉硫磷羧酸酯酶（MCE）的活性, 有可能使这些昆虫对马拉硫磷产生抗性。不同物种的MCE活性相差较大, 斜纹夜蛾的MCE活性最高, 其k_cat/K_m值为1.8~1.9 L/μmol·min, 其次是赤拟谷盗, 其K_cat/K_m值为0.87~0.95 L/μmol·min, 其他昆虫的MCE活性相对较低, 相差可高达10倍。     

Differential mRNA expression levels and gene sequences of carboxylesterase in both deltamethrin resistant and susceptible strains of the cotton aphid, Aphis gossypii

Extensive use of insecticides on cotton has prompted resistance development in the cotton aphid, Aphis gossypii (Glover) in China. A deltamethrin‐selected population of cotton aphids from Xinjiang Uygur Autonomous Region, China with 228.59‐fold higher resistance to deltamethrin was used to examine how carboxylesterase conferred resistance to this pyrethroid insecticide. The carboxylesterase activity in the deltamethrin‐resistant strain was 3.67‐, 2.02‐ and 1.16‐fold of the susceptible strain when using α‐naphthyl acetate (α‐NA), β‐naphthyl acetate (β‐NA) and α‐naphthyl butyrate (α‐NB) as substrates, respectively. Carboxylesterase cDNA was cloned and sequenced from both deltamethrin‐resistant and susceptible strains. The cDNA contained 1581 bp open reading frames (ORFs) coding a 526 amino acid protein. Only one amino acid substitution (Val⁸⁷‐Ala) was observed between deltamethrin‐resistant and susceptible strains but it is not genetically linked to resistance by the catalytic triad and signature motif analysis. The real‐time polymerase chain reaction analysis indicated that the resistant strain had a 6.61‐fold higher level of carboxylesterase mRNA than the susceptible strain. The results revealed that up‐regulation of the carboxylesterase gene, not modified gene structure, may be responsible for the development of resistance in cotton aphids to deltamethrin.     

Characterization of a novel esterase conferring insecticide resistance in the mosquito Culex tarsalis

Resistance to the organophosphate insecticide, malathion, in a strain of Culex tarsalis mosquitoes is due to increased activity of a malathion carboxylesterase (MCE). To determine whether resistance was due to a qualitative or quantitative change in the MCE, the enzyme was purified from both malathion-resistant and -susceptible mosquitoes. Enzyme kinetic measurements revealed that the two strains have one MCE in common, but resistant mosquitoes also have a unique MCE which hydrolyses malathion 18 times faster. Interestingly, this MCE does not hydrolyse α-naphthyl acetate, a substrate commonly used to detect increased levels of esterases in other organophosphate-resistant insects. Unlike the over-produced esterase of some related mosquito species, each MCE in C. tarsalis accounts for only a small fraction (0.015%) of the total extractable protein in either strain. Therefore, resistance in these insects is due to the presence of a qualitatively different enzyme, and not to a quantitative increase of a non-specific esterase. This study therefore demonstrates that the underlying biochemical mechanisms of insecticide resistance in one insect cannot necessarily be predicted from those of another, even closely related species. © 1995 Wiley-Liss, Inc.     

Purification and characterization of a carboxylesterase involved in malathion-specific resistance from Tribolium castaneum (Coleoptera: Tenebrionidae)

Specific resistance to malathion in a strain of Tribolium castaneum is due to a 44-fold increase in malathion carboxylesterase (MCE) activity relative to a susceptible strain, whereas non-specific esterase levels are slightly lower. Unlike the overproduced esterase of some mosquito and aphid species, MCE in Tribolium castaneum accounts for only a small fraction (0.033-0.045%) of the total extractable protein respectively in resistant and susceptible strains. The enzyme was purified to apparent homogeneity from these two strains and has a similar molecular weight of 62,000. However, preparative isoelectricfocusing indicated that resistant insects possess one MCE with pI of 7.3, while susceptible insects possess a MCE with a pI of 6.6. Purified MCE from both populations had different K(m) and V(m) values for hydrolysis of malathion as well as for alpha-naphthyl acetate. The kinetic analysis suggests that MCE of resistant insects hydrolyses malathion faster than the purified carboxylesterase from susceptible beetles and that this enzyme has greater affinity for malathion than for naphthyl esters. Malathion-specific resistance is due to the presence of a qualitatively different esterase in the resistant strain.