Substrate Report for: Permethrin

Carboxylesterases (CarEs) belong to a super family of multifunctional enzymes associated with the degradation of endogenous and exogenous compounds. Many insect CarEs are known to play important roles in catalyzing the hydrolysis of organophosphates (OPs), carbamates, and synthetic pyrethroids (SPs). The elevation of esterase activity through gene amplification and overexpression of estalpha2 and estbeta2 genes contributes to the development of resistance to OP insecticides in the mosquito Culex quinquefasciatus. Three additional CarE genes are upregulated in permethrin-resistant Cx. quinquefasciatus according to an RNA-seq analysis, but their function remains unknown. In this study, we, for the first time, characterized the function of these three novel genes using in vitro protein expression, an insecticide metabolism study and molecular docking analysis. All three CarE genes were significantly overexpressed in resistant mosquito larvae, but not adults, compared to susceptible strain. No gene copy differences in these three genes were found in the mosquitoes tested. In vitro high-performance liquid chromatography (HPLC) revealed that CPIJ018231, CPIJ018232, and CPIJ018233 metabolized 30.4% +/- 2.9%, 34.7% +/- 6.8%, and 23.2% +/- 2.2% of the permethrin, respectively. No mutations in resistant strains might significantly affect their CarE hydrolysis ability. A docking analysis further confirmed that these three CarEs from resistant strain all potentially metabolize permethrin. Taken together, these three carboxylesterase genes could play important roles in the development of permethrin resistance in Cx. quinquefasciatus larvae through transcriptional overexpression, metabolism, and detoxification.

The history of insecticide resistance in the horn fly, Haematobia irritans, and the relationship between the characteristics of horn fly biology and insecticide use on resistance development is discussed. Colonies of susceptible horn flies were selected for resistance with six insecticide treatment regimens: continuous single use of permethrin, diazinon and ivermectin: permethrin-diazinon (1:2) mixture; and permethrin-diazinon and permethrin-ivermectin rotation (4-month cycle). Under laboratory conditions, resistance developed during generations 21, 31 and 30 to permethrin, diazinon and ivermectin, respectively. The magnitude of resistance ranged from < 3-fold with ivermectin to 1470-fold with permethrin. Field studies demonstrated that use of a single class of insecticidal ear tag during the horn-fly season resulted in product failure within 3-4 years for pyrethroids and organophosphates, respectively. In laboratory studies, use of alternating insecticides or a mixture of insecticides delayed the onset of resistance for up to 12 generations and reduced the magnitude of pyrethroid resistance. In field studies, yearly alternated use of pyrethroids and organophosphates did not slow or reverse pyrethroid resistance (Barros et al., unpublished data), while a 2-year alternated use with organophosphates resulted in partial reversion of pyrethroid resistance. When pyrethroid and organophosphate ear tags were used in a mosaic strategy at two different locations, efficacy of products did not change during a 3-year period.

Susceptibility to six organophosphate (OP), two pyrethroid (PY), and one carbamate (C) insecticides was investigated in Culex pipiens quinquefasciatus Say, Aedes aegypti (L.), and Aedes polynesiensis Marks larvae from the island of Tahiti. Cx. p. quinquefasciatus and Ae. aegypti were compared with susceptible reference strains treated simultaneously. A low, but significant, resistance to bromophos (4.6x), chlorpyrifos (5.7x), fenthion (2.4x), fenitrothion (5.0x), temephos (4.3x) and permethrin (2.1x) was found in Cx. p. quinquefasciatus, and to malathion (1.5x), temephos (2.3x), permethrin (1.8x) and propoxur (1.7x) in Ae. aegypti. Cx. p. quinquefasciatus was shown to possess over-produced esterases A2 and B2, which are known to be involved in resistance to OPs in other countries. Ae. polynesiensis was less resistant than the Ae. aegypti reference strain to all insecticides except temephos (1.8x) and permethrin (6.7x). To determine whether Ae. polynesiensis had developed resistance to these insecticides in Tahiti, a geographical survey covering 12 islands of the Society, Tuamotu, Tubuai, Marquesas, and Gambier archipelagoes was undertaken with three insecticides (temephos, deltamethrin, and permethrin). Two- to threefold variations in LC50S were observed among collections. Results are discussed in relationship to the level of insecticide exposure on the different islands.

Carboxylesterases (CarEs) belong to a super family of multifunctional enzymes associated with the degradation of endogenous and exogenous compounds. Many insect CarEs are known to play important roles in catalyzing the hydrolysis of organophosphates (OPs), carbamates, and synthetic pyrethroids (SPs). The elevation of esterase activity through gene amplification and overexpression of estalpha2 and estbeta2 genes contributes to the development of resistance to OP insecticides in the mosquito Culex quinquefasciatus. Three additional CarE genes are upregulated in permethrin-resistant Cx. quinquefasciatus according to an RNA-seq analysis, but their function remains unknown. In this study, we, for the first time, characterized the function of these three novel genes using in vitro protein expression, an insecticide metabolism study and molecular docking analysis. All three CarE genes were significantly overexpressed in resistant mosquito larvae, but not adults, compared to susceptible strain. No gene copy differences in these three genes were found in the mosquitoes tested. In vitro high-performance liquid chromatography (HPLC) revealed that CPIJ018231, CPIJ018232, and CPIJ018233 metabolized 30.4% +/- 2.9%, 34.7% +/- 6.8%, and 23.2% +/- 2.2% of the permethrin, respectively. No mutations in resistant strains might significantly affect their CarE hydrolysis ability. A docking analysis further confirmed that these three CarEs from resistant strain all potentially metabolize permethrin. Taken together, these three carboxylesterase genes could play important roles in the development of permethrin resistance in Cx. quinquefasciatus larvae through transcriptional overexpression, metabolism, and detoxification.

To address concerns around age-related sensitivity to pyrethroids, a life-stage physiologically based pharmacokinetic (PBPK) model, supported by in vitro to in vivo extrapolation (IVIVE) was developed. The model was used to predict age-dependent changes in target tissue exposure of 8 pyrethroids; deltamethrin (DLM), cis-permethrin (CPM), trans-permethrin, esfenvalerate, cyphenothrin, cyhalothrin, cyfluthrin, and bifenthrin. A single model structure was used based on previous work in the rat. Intrinsic clearance (CLint) of each individual cytochrome P450 or carboxylesterase (CES) enzyme that are active for a given pyrethroid were measured in vitro, then biologically scaled to obtain in vivo age-specific total hepatic CLint. These IVIVE results indicate that, except for bifenthrin, CES enzymes are largely responsible for human hepatic metabolism (>50% contribution). Given the high efficiency and rapid maturation of CESs, clearance of the pyrethroids is very efficient across ages, leading to a blood flow-limited metabolism. Together with age-specific physiological parameters, in particular liver blood flow, the efficient metabolic clearance of pyrethroids across ages results in comparable to or even lower internal exposure in the target tissue (brain) in children than that in adults in response to the same level of exposure to a given pyrethroid (Cmax ratio in brain between 1- and 25-year old = 0.69, 0.93, and 0.94 for DLM, bifenthrin, and CPM, respectively). Our study demonstrated that a life-stage PBPK modeling approach, coupled with IVIVE, provides a robust framework for evaluating age-related differences in pharmacokinetics and internal target tissue exposure in humans for the pyrethroid class of chemicals.

1. The metabolism of deltamethrin (DLM), cis-permethrin (CPM) and trans-permethrin (TPM) was studied in liver microsomes, liver cytosol and plasma from male Sprague-Dawley rats aged 15, 21 and 90 days and from adult humans. 2. DLM and CPM were metabolised by rat hepatic microsomal cytochrome P450 (CYP) enzymes and to a lesser extent by microsomal and cytosolic carboxylesterase (CES) enzymes, whereas TPM was metabolised to a greater extent by CES enzymes. 3. In human liver, DLM and TPM were mainly metabolised by CES enzymes, whereas CPM was metabolised by CYP and CES enzymes. 4. The metabolism of pyrethroids by cytosolic CES enzymes contributes to the overall hepatic clearance of these compounds. 5. DLM, CPM and TPM were metabolised by rat, but not human, plasma CES enzymes. 6. This study demonstrates that the ability of male rats to metabolise DLM, CPM and TPM by hepatic CYP and CES enzymes and plasma CES enzymes increases with age. In all instances, apparent intrinsic clearance values were lower in 15 than in 90 day old rats. As pyrethroid-induced neurotoxicity is due to the parent compound, these results suggest that DLM, CPM and TPM may be more neurotoxic to juvenile than to adult rats.

1. The metabolism of the pyrethroids deltamethrin (DLM), cis-permethrin (CPM) and trans-permethrin (TPM) was studied in human expressed cytochrome P450 (CYP) and carboxylesterase (CES) enzymes. 2. DLM, CPM and TPM were metabolised by human CYP2B6 and CYP2C19, with the highest apparent intrinsic clearance (CLint) values for pyrethroid metabolism being observed with CYP2C19. Other CYP enzymes contributing to the metabolism of one or more of the three pyrethroids were CYP1A2, CYP2C8, CYP2C9*1, CYP2D6*1, CYP3A4 and CYP3A5. None of the pyrethroids were metabolised by CYP2A6, CYP2E1, CYP3A7 or CYP4A11. 3. DLM, CPM and TPM were metabolised by both human CES1 and CES2 enzymes. 4. Apparent CLint values for pyrethroid metabolism by CYP and CES enzymes were scaled to per gram of adult human liver using abundance values for microsomal CYP enzymes and for CES enzymes in liver microsomes and cytosol. TPM had the highest and CPM the lowest apparent CLint values for total metabolism (CYP and CES enzymes) per gram of adult human liver. 5. Due to their higher abundance, all three pyrethroids were extensively metabolised by CES enzymes in adult human liver, with CYP enzymes only accounting for 2, 10 and 1% of total metabolism for DLM, CPM and TPM, respectively.

The use of pyrethroids has increased throughout the world over the past few decades, as organophosphate, carbamate and organochlorine insecticides are being phased out. Permethrin is widely used in the USA for crops treatment, at concentrations around 750 x 103 mug/L. In our study 3.6 mug/L permethrin decreases the fission-rate and the fruiting bodies formation of slime mould Dictyostelium discoideum. Whereas 3.6 x 104 mug/L kills the 100% of amoebae, showing a 24 h-LC50 = 96.6 mug/L. This concentration induces an increase in the pseudocholinesterase activity as well as in both butyrylcholinesterase and heat-shock-protein 70 presence. Our results highlight the high sensitivity of Dictyostelium to permethrin, at concentration of about 105 lesser than what used for agricultural pest control. If we match our results on 6 days of exposure, with the permethrin relatively slow permanence (30 days) in the aerobic soil, as well as the higher effect of permethrin than organophosphate, carbamate and organochlorine pesticides on D. discoideum, the damage on the dictyostelids community, by use of permethrin, is clear. Our data suggest that, if the sustainable agriculture implementation is a topic of the modern "industrial" farming, the permethrin cannot represent a reliable alternative to organochlorine, organophosphate or carbamate pesticides, in implementing Integrated Pest Management programmes.

Carboxylesterases hydrolyze chemicals containing such functional groups as a carboxylic acid ester, amide and thioester. The liver contains the highest carboxylesterase activity and expresses two major carboxylesterases: HCE1 and HCE2. In this study, we analyzed 104 individual liver samples for the expression patterns of both carboxylesterases. These samples were divided into three age groups: adults (>or= 18 years of age), children (0 days-10 years) and fetuses (82-224 gestation days). In general, the adult group expressed significantly higher HCE1 and HCE2 than the child group, which expressed significantly higher than the fetal group. The age-related expression was confirmed by RT-qPCR and Western immunoblotting. To determine whether the expression patterns reflected the hydrolytic activity, liver microsomes were pooled from each group and tested for the hydrolysis of drugs such as oseltamivir and insecticides such as deltamethrin. Consistent with the expression patterns, adult microsomes were approximately 4 times as active as child microsomes and 10 times as active as fetal microsomes in hydrolyzing these chemicals. Within the same age group, particularly in the fetal and child groups, a large inter-individual variability was detected in mRNA (430-fold), protein (100-fold) and hydrolytic activity (127-fold). Carboxylesterases are recognized to play critical roles in drug metabolism and insecticide detoxication. The findings on the large variability among different age groups or even within the same age group have important pharmacological and toxicological implications, particularly in relation to pharmacokinetic alterations of ester drugs in children and vulnerability of fetuses and children to pyrethroid insecticides.

Hydrolytic metabolism of pyrethroid insecticides in humans is one of the major catabolic pathways that clear these compounds from the body. Rodent models are often used to determine the disposition and clearance rates of these esterified compounds. In this study the distribution and activities of esterases that catalyze pyrethroid metabolism have been investigated in vitro using several human and rat tissues, including small intestine, liver and serum. The major esterase in human intestine is carboxylesterase 2 (hCE2). We found that the pyrethroid trans-permethrin is effectively hydrolyzed by a sample of pooled human intestinal microsomes (5 individuals), while deltamethrin and bioresmethrin are not. This result correlates well with the substrate specificity of recombinant hCE2 enzyme. In contrast, a sample of pooled rat intestinal microsomes (5 animals) hydrolyze trans-permethrin 4.5-fold slower than the sample of human intestinal microsomes. Furthermore, it is demonstrated that pooled samples of cytosol from human or rat liver are approximately 2-fold less hydrolytically active (normalized per mg protein) than the corresponding microsomal fraction toward pyrethroid substrates; however, the cytosolic fractions do have significant amounts (approximately 40%) of the total esteratic activity. Moreover, a 6-fold interindividual variation in carboxylesterase 1 protein expression in human hepatic cytosols was observed. Human serum was shown to lack pyrethroid hydrolytic activity, but rat serum has hydrolytic activity that is attributed to a single CE isozyme. We purified the serum CE enzyme to homogeneity to determine its contribution to pyrethroid metabolism in the rat. Both trans-permethrin and bioresmethrin were effectively cleaved by this serum CE, but deltamethrin, esfenvalerate, alpha-cypermethrin and cis-permethrin were slowly hydrolyzed. Lastly, two model lipase enzymes were examined for their ability to hydrolyze pyrethroids. However, no hydrolysis products could be detected. Together, these results demonstrate that extrahepatic esterolytic metabolism of specific pyrethroids may be significant. Moreover, hepatic cytosolic and microsomal hydrolytic metabolism should each be considered during the development of pharmacokinetic models that predict the disposition of pyrethroids and other esterified compounds.

The in vitro metabolism of permethrin and its hydrolysis products in rats was investigated. Cis- and trans-permethrin were mainly hydrolyzed by liver microsomes, and also by small-intestinal microsomes of rats. trans-Permethrin was much more effectively hydrolyzed than the cis-isomer. When NADPH was added to the incubation mixture of the liver microsomes, three metabolites, 3-phenoxybenzyl alcohol (PBAlc), 3-phenoxybenzaldehyde (PBAld) and 3-phenoxybenzoic acid (PBAcid), were formed. However, only PBAlc was formed by rat liver microsomes in the absence of cofactors. The microsomal activities of rat liver and small intestine were inhibited by bis-p-nitrophenyl phosphate, an inhibitor of carboxylesterase (CES). ES-3 and ES-10, isoforms of the CES 1 family, exhibited significant hydrolytic activities toward trans-permethrin. When PBAlc was incubated with rat liver microsomes in the presence of NADPH, PBAld and PBAcid were formed. The NADPH-linked oxidizing activity was inhibited by SKF 525-A. Rat recombinant cytochrome P450, CYP 2C6 and 3A1, exhibited significant oxidase activities with NADPH. When PBAld was incubated with the microsomes in the presence of NADPH, PBAcid was formed. CYP 1A2, 2B1, 2C6, 2D1 and 3A1 exhibited significant oxidase activities in this reaction. Thus, permethrin was hydrolyzed by CES, and PBAlc formed was oxidized to PBAld and PBAcid by the cytochrome P450 system in rats.

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.

Carboxylesterases hydrolyze a large array of endogenous and exogenous ester-containing compounds, including pyrethroid insecticides. Herein, we report the specific activities and kinetic parameters of human carboxylesterase (hCE)-1 and hCE-2 using authentic pyrethroids and pyrethroid-like, fluorescent surrogates. Both hCE-1 and hCE-2 hydrolyzed type I and II pyrethroids with strong stereoselectivity. For example, the trans-isomers of permethrin and cypermethrin were hydrolyzed much faster than corresponding cis-counterparts by both enzymes. Kinetic values of hCE-1 and hCE-2 were determined using cypermethrin and 11 stereoisomers of the pyrethroid-like, fluorescent surrogates. K(m) values for the authentic pyrethroids and fluorescent surrogates were in general lower than those for other ester-containing substrates of hCEs. The pyrethroid-like, fluorescent surrogates were hydrolyzed at rates similar to the authentic pyrethroids by both enzymes, suggesting the potential of these compounds as tools for high throughput screening of esterases that hydrolyze pyrethroids.

The cloned genes encoding carboxylesterase E3 in the blowfly Lucilia cuprina and its orthologue in Drosophila melanogaster were expressed in Sf9 cells transfected with recombinant baculovirus. Resistance of L. cuprina to organophosphorus insecticides is due to mutations in the E3 gene that enhance the enzyme's ability to hydrolyse insecticides. Previous in vitro mutagenesis and expression of these modifications (G137D, in the oxyanion hole and W251L, in the acyl pocket) have confirmed their functional significance. We have systematically substituted these and nearby amino acids by others expected to affect the hydrolysis of pyrethroid insecticides. Most mutations of G137 markedly decreased pyrethroid hydrolysis. W251L was the most effective of five substitutions at this position. It increased activity with trans permethrin 10-fold, and the more insecticidal cis permethrin >130-fold, thereby decreasing the trans:cis hydrolysis ratio to only 2, compared with >25 in the wild-type enzyme. Other mutations near the bottom of the catalytic cleft generally enhanced pyrethroid hydrolysis, the most effective being F309L, also in the presumptive acyl binding pocket, which enhanced trans permethrin hydrolysis even more than W251L. In these assays with racemic 1RS cis and 1RS trans permethrin, two phases were apparent, one being much faster suggesting preferential hydrolysis of one enantiomer in each pair as found previously with other esterases. Complementary assays with individual enantiomers of deltamethrin and the dibromo analogue of cis permethrin showed that the wild type and most mutants showed a marked preference for the least insecticidal 1S configuration, but this was reversed by the F309L substitution. The W251L/F309L double mutant was best overall in hydrolysing the most insecticidal 1R cis isomers. The results are discussed in relation to likely steric effects on enzyme-substrate interactions, cross-resistance between pyrethroids and malathion, and the potential for bioremediation of pyrethroid residues.

Carboxylesterases are enzymes that catalyze the hydrolysis of a wide range of ester-containing endogenous and xenobiotic compounds. Although the use of pyrethroids is increasing, the specific enzymes involved in the hydrolysis of these insecticides have yet to be identified. A pyrethroid-hydrolyzing enzyme was partially purified from mouse liver microsomes using a fluorescent reporter similar in structure to cypermethrin (Shan, G., and Hammock, B. D. (2001) Anal. Biochem. 299, 54-62 and Wheelock, C. E., Wheelock, A. M., Zhang, R., Stok, J. E., Morisseau, C., Le Valley, S. E., Green, C. E., and Hammock, B. D. (2003) Anal. Biochem. 315, 208-222) and subsequently identified as a carboxylesterase (NCBI accession number BAC36707). The expressed sequence tag was then cloned, expressed in baculovirus, and purified to homogeneity. Kinetic constants for a large number of both type I and type II pyrethroid or pyrethroid-like substrates were determined. This esterase possesses similar kinetic constants for cypermethrin and its fluorescent-surrogate (k(cat) = 0.12 +/- 0.03 versus 0.11 +/- 0.01 s(-1)). Compared with their cis- counterparts, trans-permethrin and cypermethrin were hydrolyzed 22- and 4-fold faster, respectively. Of the four fenvalerate isomers the (2R)(alphaR)-isomer was hydrolyzed at least 1 order of magnitude faster than any other isomer. However, it is unlikely that this enzyme accounts for the total pyrethroid hydrolysis in the microsomes because both isoelectrofocusing and native PAGE indicate the presence of a second region of cypermethrin-metabolizing enzymes. A second carboxylesterase gene (NCBI accession number NM_133960), isolated during a cDNA mouse liver library screening, was also found to hydrolyze pyrethroids. Both these enzymes could be used as preliminary tools in establishing the relative toxicity of new pyrethroids.

A strain of Mexican Boophilus microplus (Cz) collected near Coatzacoalcos, Veracruz, Mexico, exhibits a moderate, but significant, level of permethrin resistance. Unlike other highly permethrin resistant strains, the Cz strain does not have a mutation within the sodium channel gene that results in target-site insensitivity. However, the Cz strain possesses a substantial increase in general and permethrin esterase activity relative to highly permethrin resistant and control strains suggesting the involvement of a metabolic esterase(s) in the expression of permethrin resistance. We report the isolation of a 62.8 kDa protein from Cz strain larvae that we think is the esterase previously reported as Cz EST9. In addition, internal amino acid sequence data obtained from the 62.8 kDa protein suggest that it is the gene product of a previously reported B. microplus carboxylesterase cDNA. We propose that the 62.8 kDa protein (Cz EST9) has permethrin hydrolytic activity and as a result plays an important role in Cz strain resistance to permethrin.

The history of insecticide resistance in the horn fly, Haematobia irritans, and the relationship between the characteristics of horn fly biology and insecticide use on resistance development is discussed. Colonies of susceptible horn flies were selected for resistance with six insecticide treatment regimens: continuous single use of permethrin, diazinon and ivermectin: permethrin-diazinon (1:2) mixture; and permethrin-diazinon and permethrin-ivermectin rotation (4-month cycle). Under laboratory conditions, resistance developed during generations 21, 31 and 30 to permethrin, diazinon and ivermectin, respectively. The magnitude of resistance ranged from < 3-fold with ivermectin to 1470-fold with permethrin. Field studies demonstrated that use of a single class of insecticidal ear tag during the horn-fly season resulted in product failure within 3-4 years for pyrethroids and organophosphates, respectively. In laboratory studies, use of alternating insecticides or a mixture of insecticides delayed the onset of resistance for up to 12 generations and reduced the magnitude of pyrethroid resistance. In field studies, yearly alternated use of pyrethroids and organophosphates did not slow or reverse pyrethroid resistance (Barros et al., unpublished data), while a 2-year alternated use with organophosphates resulted in partial reversion of pyrethroid resistance. When pyrethroid and organophosphate ear tags were used in a mosaic strategy at two different locations, efficacy of products did not change during a 3-year period.

The inhibition profiles of the pI4.8 and 4.5 carboxylesterases from the permethrin-resistant strain of Colorado potato beetle were evaluated for a variety of inhibitors and insecticides. Both carboxylesterases were determined to be serine-hydroxyl-type esterases with different inhibition specificities toward phenylmethylsulfonyl fluoride, a serine-hydroxyl proteinase inhibitor, and para-hydroxylmercuribenzoate, a cysteine-sulfhydryl group arylesterase inhibitor. The para-hydroxylmercuribenzoate-binding site appears separate from the catalytic binding site as determined by p-chloromercuribenzoate-agarose affinity chromatography of the purified pI4.5-4.8 carboxylesterases. The pI4.8 and 4.5 carboxylesterases, however, were not significantly different in their sensitivity to various insecticides. The levels of inhibition achieved by the insecticides and eserine showed a positive correlation with the hydrophobicity of the compounds, suggesting that the pI4.8 and 4.5 carboxylesterases can interact with a variety of hydrophobic compounds through nonspecific hydrophobic site(s). The kinetics of inhibition of the pI4.5-4.8 carboxylesterases elicited by permethrin and DDT are most similar to a mixed-noncompetitive type and a noncompetitive type of inhibition, respectively. Analysis of these kinetic differences indicates the presence of hydrophobic catalytic site(s) as well as hydrophobic noncatalytic site(s) that are available for the binding of esteratic (e.g., permethrin) and nonesteratic (e.g., DDT) hydrophobic insecticides, respectively. Along with a low level of permethrin hydrolysis, the hydrophobic binding nature of the pI4.5-4.8 carboxylesterases suggests that resistance to permethrin is mainly conferred by sequestration. Sequestration of insecticides by the pI4.5-4.8 carboxylesterases appears to be nonspecific and associated with the cross-resistance of the PE-R strain to other hydrophobic insecticides, such as other pyrethroids, DDT, and abamectin.

Susceptibility to six organophosphate (OP), two pyrethroid (PY), and one carbamate (C) insecticides was investigated in Culex pipiens quinquefasciatus Say, Aedes aegypti (L.), and Aedes polynesiensis Marks larvae from the island of Tahiti. Cx. p. quinquefasciatus and Ae. aegypti were compared with susceptible reference strains treated simultaneously. A low, but significant, resistance to bromophos (4.6x), chlorpyrifos (5.7x), fenthion (2.4x), fenitrothion (5.0x), temephos (4.3x) and permethrin (2.1x) was found in Cx. p. quinquefasciatus, and to malathion (1.5x), temephos (2.3x), permethrin (1.8x) and propoxur (1.7x) in Ae. aegypti. Cx. p. quinquefasciatus was shown to possess over-produced esterases A2 and B2, which are known to be involved in resistance to OPs in other countries. Ae. polynesiensis was less resistant than the Ae. aegypti reference strain to all insecticides except temephos (1.8x) and permethrin (6.7x). To determine whether Ae. polynesiensis had developed resistance to these insecticides in Tahiti, a geographical survey covering 12 islands of the Society, Tuamotu, Tubuai, Marquesas, and Gambier archipelagoes was undertaken with three insecticides (temephos, deltamethrin, and permethrin). Two- to threefold variations in LC50S were observed among collections. Results are discussed in relationship to the level of insecticide exposure on the different islands.