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.     

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.     

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 in vitro degradation of [14C]malathion by enzymatic extracts from resistant and susceptible strains of Drosophila melanogaster

Enzyme preparations from Drosophila melanogaster flies degraded [¹⁴C]malathion to α- and β-malathion monoacids and, hence, were considered to contain malathion carboxylesterase (ME) activity. Although ME- activity was stable during preincubation in the absence of malathion, it decreased dramatically during the course of the reaction, and could not be completely recovered by Sephadex G-25 chromatography. Furthermore, the protein fraction after chromatography still contained ¹⁴C, suggesting that the enzyme had become inhibited by a bound, ¹⁴C-labeled derivative. Extracts from a resistant (malathion-selected), an intermediate control, and the susceptible Canton S strains of D. melanogaster differed in the lability of ME activity during the reaction. This difference was partly attributed to the production of small amounts of malaoxon (2–8%) by the extracts from the more resistant strains. No consistent strain differences were found when the rate of malathion degradation was measured during the first minute of reaction, either with or without a microsomal oxidase inhibitor (metyrapone) present. These results, together with the cross-resistance of the malathion-selected strain to other insecticides and the lack of a synergistic effect of two carboxylesterase inhibitors (triphenyl phosphate and S,S,S-tributylphosphorotrithioate) suggested that malathion carboxylesterase does not contribute significantly to the observed differences in malathion resistance between strains.     

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.     

The detection and characterization of malathion resistance in field populations of Anopheles culicifacies B in Sri Lanka

Malathion resistance was first detected in Sri Lankan Anopheles culicifacies in limited regions of the island in 1982. The frequency of resistance has been increasing slowly since then, but is not yet high enough to be considered an operational problem. Malathion toxicity is synergised in the resistant population by triphenyl phosphate, and metabolism studies suggest the involvement of a carboxylesterase enzyme. The spread of general esterase activity in individuals in an area of the island where resistance is present is wider than that in a totally malathion-susceptible area. However, the frequency of individuals with high esterase activity does not correlate well with resistance in the two field populations studied in detail. This suggests that a qualitative rather than a quantitative change in esterase activity may be involved in this resistance. Extrapolation from similar qualitatively changed carboxylesterases in other anophelines leads us to predict that the resistance in A. culicifacies will be malathion specific and inherited as a single semidominant characteristic.     

The biochemistry of insecticide resistance in Anopheles sacharovi: Comparative studies with a range of insecticide susceptible and resistant Anopheles and Culex species

Fourth instar larvae, the progeny from wild-caught Anopheles sacharovi females, were subjected to a number of biochemical tests and the results were compared to those from similar tests on laboratory insecticide resistant and susceptible strains of anopheline and culicine mosquitoes. DDT resistance in An. sacharovi is associated with the ability to rapidly metabolise DDT to DDE. The organophosphorus and carbamate resistance was not associated with quantitative changes in esterases, multifunction oxidases, or glutathione S-transferase. The acetylcholinesterase was less sensitive to malaoxon and propoxur than laboratory susceptible An. albimanus, and plots of inhibition suggest that the population was polymorphic for more than one form of acetylcholinesterase. Metabolism studies on malathion and pirimiphos methyl did not indicate resistance due to increased metabolism. There was no evidence of penetration barriers contributing to resistance to either DDT or malathion, and there was no indication of any resistance to pirimiphos methyl in our tests.     

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.     

Studies on the acetylcholinesterase of Anopheles albimanus resistant and susceptible to organophosphate and carbamate insecticides

Acetylcholinesterase from fourth instar Anopheles albimanus larvae was studied in vitro. The acetylcholinesterase from both the resistant and susceptible strains behaved as a single enzyme “type,” with straight pseudo first-order insecticide inhibition lines which intersected the Y axis at 100%. The enzyme from resistant larvae was more slowly inhibited than the susceptible enzyme; bimolecular rate constants (k_i) differed by approximately 1.2- to 6-fold for a range of organophosphorous compounds and 17- to 1570-fold for the carbamates. There was a good correlation between the levels of resistance and the acetylcholinesterase inhibition rates.     

Mechanisms of resistance to the juvenoid methoprene in the house fly Musca domestica L

The mechanisms of resistance and cross resistance to the juvenoids methoprene and R-20458 in the house fly, Musca domestica, were examined. Radiolabeled methoprene was found to be metabolized faster in resistant and cross-resistant house fly larvae than in susceptible larvae, and methoprene and R-20458 penetrated more slowly into larvae of the resistant strain. In vivo and in vitro metabolism of methoprene was largely by oxidative pathways followed by conjugation in all strains examined, and little or no ester change of methoprene was noted in vitro. In vitro oxidative metabolism of methoprene, R-20458, juvenile hormone I, and several model substrates was higher in resistant and cross-resistant larvae than in susceptible larvae. Juvenoid functionalities susceptible to metabolic attack by resistant strains are indicated.     

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.     

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.     

Malathion carboxylesterase enzymes in Anopheles arabiensis from Sudan

Malathion resistance in Anopheles arabiensis from Sudan is monofactorially inherited and is expressed in the adults but not in the larvae. The resistance is suppressed by the esterase synergist, triphenylphosphate. Semipurification of the soluble esterase enzymes by Sephadex G-25 and Sephacryl S-200 gel filtration revealed no difference between the enzymes of the resistant and susceptible strains with α- or β-naphthylacetate (NA) with a fixed substrate concentration in either the adults or larvae. However, with the malathion-specific assay a second peak of activity was observed in the adult resistant strain which was not present in either the larvae of this strain or the larvae and adults of the susceptible strain. A corresponding threefold difference in the K_m value for α-NA was also observed in the resistant adults over the range of this second peak, but there was no change in the K_m with β-NA.     

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.     

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.     

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.     

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.     

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.