General esterase,malathion carboxylesterase,and malathion resistance in Culex tarsalis

The role of esterases in malathion resistance in Culex tarsalis has been investigated. When larvae of a resistant and a sensitive strain were placed in water containing [¹⁴C]malathion, malathion penetrated to give initially similar internal levels. With resistant mosquitoes, after 15 min the internal malathion concentration decreased to low levels while the monoacid degradation products accumulated in the larvae and were excreted into the surrounding water, whereas in susceptible larvae the internal malathion level stayed high and was lethal. It is suggested that the decrease in internal malathion and the resulting resistance were caused by an active malathion carboxylesterase in the resistant strain. A specific assay for malathion carboxylesterase with [¹⁴C]malathion showed 55 times more activity in resistant than in susceptible larvae, whereas when general esterase activity was assayed with α-naphthyl acetate only 1.7 times the activity was found. Analyses by starch gel electrophoresis showed a peak of malathion carboxylesterase, 60-fold higher from resistant than from susceptible larvae, in a gel zone which did not stain for general esterase activity. General esterases that did not hydrolyze malathion showed different electrophoretic patterns in the two populations, which are likely due to the nonisogenic character of the strains. These results show that use of a specific assay and the demonstration of degradation of malathion in vivo are essential for assessment of the contribution of esterase activity to the malathion-resistant phenotype in mosquito populations.     

The biochemical basis of malathion resistance in the sheep blowfly,Lucilia cuprina

The in vivo and in vitro metabolism of [¹⁴C]malathion was studied in susceptible (LS) and malathion resistant (RM) strains of the sheep blowfly, Lucilia cuprina (Wiedemann). No difference was found between strains in the penetration, excretion, storage, or inhibitory potency of the insecticide. However, RM degraded malathion to its α- and β-monocarboxylic acid metabolites more rapidly than LS, both in vivo and in vitro. This enhanced degradation of [¹⁴C]malathion occurred in vitro in both mitochondrial and microsomal fractions of resistant flies. Kinetic analysis revealed that these fractions degraded malathion by discrete mechanisms. The enzymes from the mitochondria of both strains had the same K_m, whereas the microsomal enzyme from the RM strain had a fivefold higher K_m than that from the LS strain. Studies of esterase activities and the effect of enzyme inhibitors showed that both the mitochondrial and microsomal resistance mechanisms were the result of enhanced carboxylesterase activity. It was concluded that increased carboxylesterase detoxification of malathion adequately explained the high level of malathion resistance in RM if rate-limiting factors such as cuticular penetration were taken into account.     

The biochemical nature of malathion resistance in Anopheles stephensi from Pakistan

Malathion resistance in Anopheles stephensi from Pakistan was synergized by triphenyl phosphate, primarily a carboxylesterase inhibitor. There was a slight degree of antagonism with piperonyl butoxide. The major metabolite of malathion in larvae of both the resistant and susceptible strains was malathion monocarboxylic acid. Resistant larvae produced about twice as much of this product as the susceptible larvae. This suggests that a qualitative or a quantitative change in a carboxylesterase enzyme may be the basis of malathion resistance in this strain. Analysis of general esterase levels to α- and β-naphthyl acetate showed that there was no quantitative change in the amount of carboxylesterase enzyme present in the resistant strain as compared to the susceptible.     

Changes in malathion resistance with age in Anopheles stephensi from Pakistan

Resistance to malathion in Anopheles stephensi from Pakistan was measured at intervals during the first week of adult life. LT₅₀ values for homozygous resistant females decreased four-fold during the first 7 days of adulthood. A decrease in resistance with age also occurred in heterozygotes; the LT₅₀ values of males and females fell sevenfold during the first 5 days of adulthood. The sensitivity to malathion of a susceptible strain increased with age. A biochemical basis for the declining resistance levels was investigated. Resistant and susceptible adults were homogenized at intervals during the first week of adulthood and soluble extracts were incubated with [¹⁴C]malathion. The rate of malathion metabolism to mono- and dicarboxylic acids was faster in resistant than in susceptible mosquitoes. The rate of malathion metabolism decreased with age in both strains. A decrease in carboxylesterase activity with age in resistant and susceptible mosquitoes is thus responsible for the increasing sensitivity to malathion. Implications for the monitoring of resistance in the field by diagnostic dosages and for the future use of malathion in mosquito control are discussed.     

Studies on hydrolases in various house fly strains and their role in malathion resistance

Aliesterase, carboxylesterase, and phosphorotriester hydrolase activities in six house fly strains were studied in relation to malathion resistance. Selection of two susceptible strains with malathion for three generations resulted in an increase in both carboxylesterase activity and LD₅₀ of malathion, indicating that the increased detoxication by the enzyme was the major mechanism selected for malathion resistance. With the highly resistant strains, however, the carboxylesterase activity alone was not sufficient to explain the resistance level, and the involvement of additional mechanisms, including phosphorotriester hydrolase activity, was suggested. The E₁ strain, which had high phosphorotriester hydrolase activity but normal or low carboxylesterase activity, showed a moderate level, i.e., sevenfold resistance. Upon DEAE-cellulose chromatography, two or three esterase peaks were resolved from susceptible, moderately resistant, and highly resistant strains. The substrate specificity, the sensitivity to paraoxon inhibition, and the $\text{α}\text{β}$ ratio of malathion hydrolysis were studied for each esterase peak from the different strains. The results suggested the existence of multiple forms of esterases with overlapping substrate specificity in the house fly.     

Esterase-mediated malathion resistance in the human head louse, Pediculus capitis (Anoplura: Pediculidae)

Resistance in a dual malathion- and permethrin-resistant head louse strain (BR-HL) was studied. BR-HL was 3.6- and 3.7-fold more resistant to malathion and permethrin, respectively, compared to insecticide-susceptible EC-HL. S,S,S-Tributylphosphorotrithioate synergized malathion toxicity by 2.1-fold but not permethrin toxicity in BR-HL. Piperonyl butoxide did not synergize malathion or permethrin toxicity. Malathion carboxylesterase (MCE) activity was 13.3-fold and general esterase activity was 3.9-fold higher in BR-HL versus EC-HL. There were no significant differences in phosphotriesterase, glutathione S-transferase, and acetylcholinesterase activities between strains. There was no differential sensitivity in acetylcholinesterase inhibition by malaoxon. Esterases from BR-HL had higher affinities and hydrolysis efficiencies versus EC-HL using various naphthyl-substituted esters. Protein content of BR-HL females and males was 1.6- and 1.3-fold higher, respectively, versus EC-HL adults. Electrophoresis revealed two esterases with increased intensity and a unique esterase associated with BR-HL. Thus, increased MCE activity and over-expressed esterases appear to be involved in malathion resistance in the head louse.     

The effect of pretreatment with S,S,S-tributyl phosphorotrithioate on deltamethrin resistance and carboxylesterase activity in Aphis gossypii (Glover) (Homoptera: Aphididae)

The synergism of S,S,S-tributyl phosphorotrithioate (DEF) and its effect on carboxylesterase activity were investigated in deltamethrin-selected resistant (DRR) and susceptible (DSS) strains of cotton aphids, Aphis gossypii (Glover). Compared to the DSS strain, the DRR strain showed 23,900-fold resistance to deltamethrin, and 7560- and 99-fold cross-resistance to bifenthrin and ethofenprox, respectively. The synergist, DEF, increased the toxicity of both deltamethrin and bifenthrin, but not of ethofenprox when DEF was pretreated of 15 h. DEF exhibited significant inhibition on the carboxylesterase activity in the DRR strain, but no significant effect on that of the DSS strain in vitro. After the cotton aphids exposing to DEF, the carboxylesterase activity decreased gradually until 15 h and then gradually recovered until 24 h in the DRR strain, which fluctuated according to the effect of DEF on the deltamethrin toxicity detected using DEF pretreatment in the DRR strain. Therefore, our studies suggested that the effect of DEF on carboxylesterase was associated with deltamethrin resistance in the DRR strain.     

Reproductive and developmental defects in a malathion-resistant,laboratory-selected population of Drosophila melanogaster

We have examined the chromosomal basis for reproductive and developmental defects that are associated with malathion resistance in a laboratory-selected population of Drosophila melanogaster. Strains homozygous for second or third chromosomes from this population were more resistant to malathion and had greater mixed-function oxidase activity, decreased fertility, and lower egg production when compared with first chromosome or susceptible strains. Some of the strains carrying resistant third chromosomes were developmentally delayed and required a significantly longer time to pupate. Delayed pupation was not associated with increased in vitro degradation of ecdysone by larvae having increased mixed-function oxidase activity, nor could it be reversed by feeding larvae ecdysone. Differences in mixed function oxidase activity among strains homozygous for second or third chromosomes were strongly correlated with malathion resistance but not with fitness. Although both second and third chromosome strains had high mixed-function oxidase activity, only fly extracts from the third chromosome strains oxidatively degraded [³H]juvenile hormone in vitro to a significant extent. A deficit of vitellogenic oocytes and increased egg laying by females in response to topically applied juvenile hormone-I supported the hypothesis that juvenile hormone titer was lower than normal in these strains. The results indicate that different polygenic systems control malathion resistance and associated fitness defects in this selected population of D. melanogaster. Although these systems are partly independent, they overlap due to pleiotropic effects of third chromosomal genes controlling mixed-function oxidase activity on female reproduction.     

抗性甜菜夜蛾Na-K-ATP酶、Ca-ATP酶和Ca-Mg-ATP酶对高效氯氟氰菊酯的敏感性

分别测定了甜菜夜蛾Spodoptera exigua敏感和抗高效氯氟氰菊酯品系神经系统Na-K-ATP酶、Ca-ATP酶和Ca-Mg-ATP酶的活力。结果表明,敏感和抗性品系Na-K-ATP酶活力差异不显著,而抗性品系Ca-ATP酶和Ca-Mg-ATP酶活力明显低于敏感品系。在浓度为1.0×10-8~1.0×10-3 mol/L时,高效氯氟氰菊酯对敏感和抗性品系Na-K-ATP酶、Ca-ATP酶和Ca-Mg-ATP酶的活力均有抑制,并且对敏感品系的抑制作用高于对抗性品系。高效氯氟氰菊酯浓度为1.0×10-4 mol/L 时,对敏感品系Na-K-ATP酶活力的抑制率为29.6%,对抗性品系的为21.8%,对敏感品系Ca-ATP酶活力的抑制率为34.3%,对抗性品系为21.9%,对敏感品系Ca-Mg-ATP酶活力的抑制率为22.3%,对抗性品系为16.9%,存在显著差异。表明甜菜夜蛾抗性品系上述3种ATP酶对高效氯氟氰菊酯的敏感性已明显下降。     

in vitro metabolism of azinphosmethyl in susceptible and resistant houseflies

The in vitro metabolism of [¹⁴C-methoxy] or [³²P]azinphosmethyl by subcellular fractions of abdomens from a resistant and a susceptible strain of houseflies was studied. The degradative activity in both strains was associated with the microsomal and soluble fractions and required NADPH and glutathione, respectively. The resistant strain possessed higher activity for both the mixed-function oxidases and the glutathione transferase than the susceptible strain, and both systems appear to be important in the resistance mechanism. The mixed-function oxidases were involved in the oxidative desulfuration as well as the dearylation of azinphosmethyl. A glutathione transferase located in the soluble fraction catalyzed the formation of desmethyl azinphosmethyl and methyl glutathione. This enzyme also demethylated azinphosmethyl oxygen analog. Although the soluble fraction exhibited both glutathione S-alkyltransferase and S-aryltransferase activity against noninsecticidal substrates, no evidence of the transfer of the benzazimide moiety from azinphosmethyl to glutathione was obtained. Sephadex G-100 chromatography of the soluble enzymes revealed a common eluting fraction responsible for both types of transferase activity.     

Fipronil resistance mechanisms in the rice stem borer, Chilo suppressalis Walker

Fipronil resistance mechanisms were studied between the laboratory susceptible strain and the selective field population of rice stem borer, Chilo suppressalis Walker in the laboratory. The borer population was collected from Wenzhou county, Zhejiang province. After five generations of selection, fipronil resistance ratio was 45.3-fold compared to the susceptible strain. Synergism experiments showed that the synergistic ratios of PBO, TPP and DEF on fipronil in susceptible and resistant strains of C. suppressalis were 7.55-, 1.93- and 2.91-fold, respectively, and DEM showed no obvious synergistic action on fipronil. Activities of carboxylesterase and microsomal-O-demethylase in the resistant strain were 1.89- and 1.36-fold higher that in susceptible strain, and no significant difference of glutathione-S-transferase activity was found between the resistant and susceptible strains. The K_m and V_max experiments also demonstrated that fipronil resistance of C. suppressalis was closely relative to the enhanced activities of carboxylesterase and microsomal-O-demethylase. Moreover, cross-resistance between fipronil and other conventional insecticides and the multiple resistant properties of the original Wenzhou’s population were also discussed.     

Oxidative cleavage of a carboxyester bond as a mechanism of resistance to malaoxon in houseflies

The synergistic effect of triphenyl phosphate (a carboxyesterase inhibitor), sesamex (inhibitor of microsomal oxidation) and O,O-diethyl O-phenyl phosphorothioate on the toxicity of malathion and malaoxon for one susceptible and two resistant strains of housefly was studies. It was found that in the resistant strain G (characterized by high carboxyesterase activity) both malathion and malaoxon were synergized by triphenyl phosphate, but only malaoxon (and not malathion) by sesamex. The other resistant strain E 1, moderately tolerant for malathion but highly resistant to malaoxon, differed from strain G in that triphenyl phosphate had no effect; its response to sesamex was similar to that of strain G. The third synergist, O,O-diethyl O-phenyl phosphorothioate, combined the properties of triphenyl phosphate and sesamex. It was found to be the best of the three compounds used.Biochemical in vitro studies showed that both resistant strains could degrade malaoxon oxidatively at a rate at least 10 × higher than that of the susceptible strain. This oxidation could be inhibited by very low concentrations of the thiono analogue; a malaoxon to malathion ratio of 10:1 gave an inhibition of about 70% at a malaoxon concentration of 5 × 10^?6M. The product of this oxidation is malaoxon β-monocarboxylic acid. This metabolite was also found 1 hr after application of malaoxon in vivo.The results mentioned in this paper indicate that houseflies may become resistant to malaoxon by an increased rate of oxidative carboxyester bond cleavage.     

In vitro metabolism of insecticides during midgut penetration

A study of the metabolism of ¹⁴C-labeled dieldrin, DDT, malathion, and carbaryl during penetration of the isolated midgut of two insects (Blaberus discoidalis and Manduca sexta) and a section of the intestine of a mammal (Mus musculus) is reported. There was appreciable metabolism of malathion during penetration, including differences in the activation reaction to malaoxon, between insects and mammals. Metabolism was relatively slow during penetration of carbaryl and the chlorinated hydrocarbon insecticides, and little difference in metabolic patterns was noted among the three species. The penetration studies were supported by experiments in which insecticides were incubated with intact and homogenized midgut preparations.     

Evidence that the elevated carboxylesterase (esterase 2) in organophosphorus-resistant Myzus persicae (Sulz.) is identical with the organophosphate-hydrolyzing enzyme

An enzyme hydrolyzing methylparaoxon in vitro in an organophosphorus-resistant strain of the peach potato aphid (Myzus persicae Sulz.) is present in the same electrophoretic fraction as a carboxylesterase (esterase 2) which has previously been shown to have characteristically increased activity in organophosphorus resistant strains of this aphid.No in vitro organophosphate hydrolysis was found in a susceptible strain with low carboxyl-esterase-2 activity. Carboxylesterase-2 and the methylparaoxon-hydrolyzing enzyme are both inhibited by n-propylparaoxon but not by methyl- and ethylparaoxon. This indicates that the two enzymes are identical.     

Role of a microsomal carboxylesterase in reducing the action of malathion in eggs of Triatoma infestans

A microsomal malathion carboxylesterase present in Triatoma infestans eggs was active from the first day of embryonic development. This microsomal egg malathionase (MEM) showed a unique band in polyacrilamide gel electrophoresis (PAGE) when malathion was used as substrate. In vivo metabolism of [¹⁴C]malathion during all embryonic development showed a 10% degradation due to carboxylesterases. The in vitro evaluation of the same metabolic pathway catalyzed by the microsomal fraction of T. infestans eggs showed partial inhibition by paraoxon. α- and β-malathion monoacids were identified as the main metabolites of the in vivo and in vitro metabolic pathways. The carboxylesterase band that appeared in PAGE (MEM) from the first day of embryonic development could be the main cause of malathion tolerance in T. infestans eggs.     

Toxicokinetics of Malathion in Larval Stages of the Toad Bufo arenarum (Hensel): Effect of Exogenous Spermidine

The aim of this work was to study the absorption, biotransformation, and excretion of malathion (¹⁴C-methoxy) and its metabolites in larval stages of the toad Bufo arenarum (Hensel). Also, changes in malathion metabolization by the action of the exogenous polyamine spermidine were studied. Malathion clearance from the media was uniexponential, and spermidine reduced the uptake in the larvae, causing an increase in the apparent half-life of the toxicant. Concomitant with this effect, spermidine increased the level of induction of mixed-function oxidases due to malathion and caused a progressively higher malaoxon/malathion ratio. As a consequence of the higher conversion to the active metabolite malaoxon, spermidine also provoked a significant enhancement in the inhibitory effect of Malathion on acetylcholinesterase activity. [methoxy¹⁴C]malathion metabolites, such as carboxylesterase and glutathione S-transferase products, were detected in the toad larvae and in the media. The excreted products of carboxylesterase activity were about 70% of the total radioactivity, and the glutathione S-transferase products (methyl glutathione) were 20–30% of the total radioactivity. No significant variations in the levels of excreted products due to the action of exogenous spermidine were detected. Malathion inhibited carboxylesterase activity, independent of the presence of spermidine in the media. In turn, glutathione S-transferase activity was induced by spermidine, but was not affected by the exposure to low concentrations of malathion for 48 h. We conclude that the presence of spermidine in the medium modifies malathion toxicokinetics, increasing its toxicity in B. arenarum larvae.     

Kinetic efficiency of mutant carboxylesterases implicated in organophosphate insecticide resistance

Resistance to organophosphorus (OP) insecticides in Lucilia cuprina arises from two mutations in carboxylesterase E3 that enable it to hydrolyse the phosphate ester of various organophosphates, plus the carboxlyester in the leaving group in the case of malathion. These mutations are not found naturally in the orthologous EST23 enzyme in Drosophila melanogaster. We have introduced the two mutations (G137D and W251L) into cloned genes encoding E3 and EST23 from susceptible L. cuprina and D. melanogaster and expressed them in vitro with the baculovirus system. The ability of the resultant enzymes to hydrolyse the phosphate ester of diethyl and dimethyl organophosphates was studied by a novel fluorometric assay, which also provided a sensitive titration technique for the molar amount of esterase regardless of its ability to hydrolyse the fluorogenic substrate used. Malathion carboxylesterase activity was also measured. The G137D mutation markedly enhanced (>30-fold) hydrolysis of both classes of phosphate ester by E3 but only had a similar effect on the hydrolysis of dimethyl organophosphate in EST23. Introduction of the W251L mutation into either gene enhanced dimethyl (23-30-fold) more than diethyl (6-10-fold) organophosphate hydrolysis and slightly improved (2-4-fold) malathion carboxylesterase activity, but only at high substrate concentration.     

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.     

Beta-cypermethrin resistance associated with high carboxylesterase activities in a strain of house fly, Musca domestica (Diptera: Muscidae)

A housefly strain, originally collected in 1998 from a dump in Beijing, was selected with beta-cypermethrin to generate a resistant strain (CRR) in order to characterize the resistance and identify the possible mechanisms involved in the pyrethroid resistance. The resistance was increased from 2.56- to 4419.07-fold in the CRR strain after 25 consecutive generations of selection compared to a laboratory susceptible strain (CSS). The CRR strain also developed different levels of cross-resistance to various insecticides within and outside the pyrethroid group such as abamectin. Synergists, piperonyl butoxide (PBO) and S,S,S-tributyl phosphorotrithioate (DEF), increased beta-cypermethrin toxicity 21.88- and 364.29-fold in the CRR strain as compared to 15.33- and 2.35-fold in the CSS strain, respectively. Results of biochemical assays revealed that carboxylesterase activities and maximal velocities to five naphthyl-substituted substrates in the CRR strain were significantly higher than that in the CSS strain, however, there was no significant difference in glutathione S-transferase activity and the level of total cytochrome P450 between the CRR and CSS strains. Therefore, our studies suggested that carboxylesterase play an important role in beta-cypermethrin resistance in the CRR strain.