Resistance-associated Esterases Research Articles

The interactions between piperonyl butoxide and E4, a resistance‐associated esterase from the peach‐potato aphid, Myzus persicae Sulzer (Hemiptera: Aphididae)

It has been reported previously that piperonyl butoxide (PBO) can inhibit both P450 and esterase activity. Although the method by which PBO combines with cytochrome P450 has been identified, the way in which it acts as an esterase inhibitor has not been established. This paper characterises the interactions between PBO and the resistance-associated esterase in Myzus persicae, E4. After incubation with PBO/analogues, hydrolysis of 1-naphthyl acetate by E4 is increased, but sequestration of azamethiphos is reduced. Rudimentary in silico modelling suggests PBO docks at the lip of the aromatic gorge. PBO binds with E4 to accelerate small substrates to the active-site triad, while acting as a blockade to larger, insecticidal molecules. Structure-activity studies with analogues of PBO also reveal the essential chemical moieties present in the molecule.

Esterase inhibition by synergists in the western flower thrips Frankliniella occidentalis

Western flower thrips (WFT), Frankliniella occidentalis (Pergande), is among the most important crop pests in the south-eastern region of Spain. Its increasing resistance to insecticides constitutes a serious problem, and understanding the mechanisms involved is therefore of great interest. Use of synergists to inhibit the enzymes involved in insecticide detoxification is widely used to determine their responsibility for insecticide resistance. However, they do not always act as intended or expected, and caution must be exercised when interpreting synergist results. Laboratory-selected strains of WFT were used to analyse the effects of the synergists piperonyl butoxide (PBO), S,S,S-tributyl phosphorotrithioate (DEF) and methiocarb on total esterase activity. Significant differences were found, indicating esterase activity inhibition by DEF, a lower effect for methiocarb and a small inhibition of the activity by PBO. Esterase isoenzyme inhibition by these compounds showed a similar result; this assay revealed an extreme sensitivity of Triplet A (resistance-associated esterases) to DEF. In an in vivo assay carried out with these compounds at different incubation times, only DEF caused posterior in vitro esterase activity inhibition, with a maximum effect 1 h after treatment. In this work, only DEF shows true synergistic inhibition of WFT esterases.

An analogue of piperonyl butoxide facilitates the characterisation of metabolic resistance

Previous work has demonstrated that piperonyl butoxide (PBO) not only inhibits microsomal oxidases but also resistance-associated esterases. The ability to inhibit both major metabolic resistance enzymes makes it an ideal synergist to enhance xenobiotics but negates the ability to differentiate which enzyme group is responsible for conferring resistance. This study examines an analogue that retains the ability to inhibit esterases but is restricted in its ability to act on microsomal oxidases, thus allowing an informed decision on resistance enzymes to be made when used in conjunction with the parent molecule. Using examples of resistant insects with well-characterised resistance mechanisms, a combination of PBO and analogue allows identification of the metabolic mechanism responsible for conferring resistance. The relative potency of PBO as both an esterase inhibitor and an oxidase inhibitor is also discussed.

A novel assay reveals the blockade of esterases by piperonyl butoxide

Conventional in vitro assays sometimes fail to reveal esterase inhibition by piperonyl butoxide (PBO), although synergism studies suggest loss of esterase-mediated sequestration of insecticide does take place. A new in vitro assay has been devised that routinely reveals binding between PBO and these esterases. The new 'esterase interference' assay detects the blockade of resistance-associated esterases in a species, Myzus persicae Sülzer, where this has not previously been seen. The assay also demonstrates directly the protective effect esterases may confer to target sites of insecticides. The new assay reveals esterase blockade by PBO and thus has the potential to be used as a high-throughput screening method for other potential synergists.

Genotypes, allele frequencies and dynamic distribution on resistance-associated esterase genes of Culex pipiens complex in Hangzhou

To investigate the genotypes , allele frequencies and dynamic distribution on resistance associated esterase genes of Culex pipiens complex in Hangzhou. The PCR-restriction fragment length polymorphism (PCR-RFLP) assay was applied to type the resistance associated esterase genes, and dynamic surveillance on frequencies of the resistance associated esterase gene of natural population of Culex pipiens complex in Hangzhou during 2003-2005, and phenotype of the resistance associated esterase genes were detected by esterase starch gel electrophoresis technique. The PCR-RFLP assay of esterase allele genes for three consecutive years disclosed four esterase genotypes, namely, the world-wide highly active homozygous Est beta 1(1) (50%-54%), homozygous Est beta 2 (29%-34%), heterozygous Est beta 1(1)/beta 2 (5%-10%) and Est beta N (3.13%) of a new homozygous genotype. The research of the resistance associated esterase genes phenotype in natural population of Culex pipiens complex in Hangzhou in 2005 with esterase starch gel electrophoresis technique revealed four major types, namely, Est beta 1(1) (61%), Est alpha 2/beta 2 (12%), Est alpha 8/beta 8 (7%) and sensitive phenotype (29%). There should be various resistance associated esterase genotypes in natural population of Culex pipiens complex in Hangzhou. During the period of 2003-2005, Est beta 1(1) was the major type; Est alpha 2/beta 2 was the second. Est beta N was a new esterase genotype detected in 2005 only with a mere percentage of 3.13%. As for its resistance to the new insecticide, a follow-up study should be needed. The molecular typing of the amplified esterase gene should be consistent with the resistance associated esterase genes phenotype.

Effect of pretreatment with piperonyl butoxide on pyrethroid efficacy against insecticide‐resistant Helicoverpa armigera (Lepidoptera: Noctuidae) and Bemisia tabaci (Sternorrhyncha: Aleyrodidae)

Pyrethroid resistance in B-type Bemisia tabaci Gennadius and Australian Helicoverpa armigera Hübner field populations is primarily conferred by esterase isoenzymes which metabolise and sequester pyrethroid insecticides. It has been shown previously that pyrethroid resistance-associated esterases in H. armigera are inhibited by the insecticide synergist piperonyl butoxide (PBO) over a 22-h period. It is demonstrated here that similar inhibition can be obtained against B-type B. tabaci. Small-scale field trials showed excellent levels of pyrethroid control when insects were pretreated with PBO and then dosed with pyrethroid during the time of maximum esterase inhibition. These results demonstrate that PBO can restore pyrethroid efficacy in the field against both B-type B. tabaci and resistant H. armigera.

Immunological assessment of an insecticide resistance-associated esterase in the Western corn rootworm

In previous investigations, we have determined that organophosphate resistance in the western corn rootworm, Diabrotica virgifera virgifera, is at least partially attributed to a group of non-specific carboxylesterases referred to as group II. Antiserum raised against a purified 66-kDa group II esterase is specific for the denatured enzyme. This antiserum reacts similarly with both beetle homogenates from resistant and susceptible populations, although there is much higher signal intensity in immunoblots of resistant relative to susceptible beetles. These results suggest that overproduction of group II esterases is the underlying basis of esterase-mediated resistance in D. v. virgifera by demonstrating that (1) group II esterases are immunologically indistinguishable between the resistant and susceptible populations, and (2) the intensity differences are due to increased group II esterase proteins in the resistant population. The diagnostic potential of immunological-based assays was tested with a traditional diagnostic concentration bioassay and a biochemical-based native PAGE assay. Significant correlations were observed among all three diagnostic assays (regression coefficients ranging from 0.95 to 0.96). These results demonstrate the importance of the 66-kDa protein as a resistance-associated biochemical marker, thus emphasizing the potential for 66-kDa protein-targeted immunoassays in resistance monitoring programs.

Bioremediation of organophosphorus pesticides by surface-expressed carboxylesterase from mosquito on Escherichia coli.

The insecticide resistance-associated esterase, carboxylesterase B1 (CaE B1), from mosquito was used to degrade the organophosphorus compounds. To eradicate the need for enzyme purification and minimize the resistance to mass transport of the substrate and product across the cell membranes, the CaE B1 was displayed on the cell surface of Escherichia coli fused to the C-terminus of the ice nucleation protein (INP). The presence of CaE B1 on the bacterial cell surface was verified by SDS-PAGE, Western blotting analysis, and immunofluorescence microscopy. More than 50% of active CaE B1 is exported across the membrane and anchored onto the cell surface as determined by proteinase accessibility and cell fractionation experiments. In contrast, only a 6% drop in activity for proteinase K-treated cells was detected from E.coli cells containing pET-B1. From the degradation experiment, more than 80% of the malathion was degraded by whole cells containing plasmid pUC-NC-B1. Constitutive expression of CaE B1 on the surface using INPNC resulted in no cell lysis, and the suspended cultures also exhibited good stability. Because of their high biodegradation activity and superior stability, these "live biocatalysts" are promising for detoxification of organophosphorus pesticides.

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

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

Molecular characterization of the amplified aldehyde oxidase from insecticide resistant Culex quinquefasciatus.

Primary structural information including the complete nucleotide sequence of the first insect aldehyde oxidase (AO) was obtained from the common house mosquito Culex quinquefasciatus (Say) through cloning and sequencing of both genomic DNA and cDNA. The deduced amino-acid sequence encodes a 150-kDa protein of 1266 amino-acid residues, which is consistent with the expected monomeric subunit size of AO. The Culex AO sequence contains a molybdopterin cofactor binding domain and two iron-sulfur centres. A comparison of the partial sequences of AO from insecticide resistant and susceptible strains of C. quinquefasciatus shows two distinct alleles of this enzyme, one of which is amplified in the insecticide resistant strain on a 30-kb DNA amplicon alongside two resistance-associated esterases. The amplified AO gene results in elevated AO activity in all life stages, but activity is highest in 3rd instar larvae. The elevated enzyme can be seen as a separate band on polyacrylamide gel electrophoresis. The role of AO in xenobiotic oxidation in mammals and the partial inhibition of elevated AO activity by a range of insecticides in Culex, suggest that this AO may play a role in insecticide resistance.

Novel approaches for remediation of pesticide pollutants

The use of synthetic pesticides has become an indispensable tool in agriculture for the control of pests. Therefore, the search for remedies and techniques for decontamination and detoxification of a pesticide-contaminated environment has become an important part of the research. Currently, bioremediation seems to be one of the most environmentally safe and cost-effective methods. In nature, the existence of abundant material resources can be used to degrade the environmental pesticide pollutants. At present, a number of microorganisms, capable of degrading pesticides, have been isolated and characterised. For insects, insecticide resistance-associated esterases have been purified and characterised from several insect species, and a new family of cytochrome P450 apparently associated with insecticide resistance in the tobacco budworm, was discovered. Generally, two bioremediation approaches have been used one directly based on microorganisms, and the other involved in isolated enzymes. For the former, in addition to using natural microorganism strains, with genetic techniques certain desirable biodegradation pathways from different organisms are brought together in a single host. However, because of their own limits, especially problems associated with releasing genetically altered microorganisms into the environment, the strategy based on enzymes seems more feasible. In the long term, collaborations between microbiologists, biochemists, and engineers will become increasingly important to efficiently dispose of the pesticide pollutants.

Polarized Ketone Inhibition of 1-Naphthyl Acetate Esterase in Azinphosmethyl-Resistant and -Susceptible Tufted Apple Bud Moths, Platynota idaeusalis (Walker): Novel Insecticide Synergists

Abstract Aliphatic and aromatic polarized trifluoromethylketones were synthesized as inhibitors of 1-naphthyl acetate (NA) esterase and potential insecticide synergists for azinphosmethyl-resistant tufted apple bud moths. A 1-NA resistance-associated esterase (RAE) was isolated from whole body homogenates of azinphosmethyl-resistant adult females. A second susceptible-associated 1-NA esterase (SE) was purified from azinphosmethyl-susceptible adult females of the tufted apple bud moth. In structure-activity studies, octylthio-1,1,1-trifluoromethyl-2-propanone (OTFP) was the most potent inhibitor of whole body 1-NA esterase from resistant and susceptible adult females. The I 50 s for inhibition of purified RAE and SE were 1 × 10 −8.5 and 1 × 10 −8.6 M, respectively. Longer and shorter n -alkyl groups or a S -phenyl substitution reduced inhibitory activity. These inhibitors were also found to compete with 1-NA substrate for the esteratic active site. OTFP alone had minimal toxicity toward third and fifth instars of the tufted apple bud moth but synergized azinphosmethyl toxicity in resistant insects. Although novel esterases were found in resistant bud moth larvae and adults, there was no evidence that the resistance-associated esterases had a unique function different from that found in susceptible insects.

Aldehyde oxidase is coamplified with the World's most common Culex mosquito insecticide resistance-associated esterases.

The evolution and spread of insecticide resistance is an important factor in human disease prevention and crop protection. The mosquito Culex quinquefasciatus is the main vector of the disease filariasis and a member of a species complex which is a common biting nuisance worldwide. The common insecticide resistance mechanism in this species involves germline amplification of the esterases estalpha21 and estbeta21. This amplification has arisen once and rapidly spread worldwide. Less common and more variable resistance phenotypes involve coamplification of estalpha3 and estbeta1, or individual amplification of a single estbeta1, different alleles of the same estalpha and estbeta gene loci. Estalpha21 and estbeta21 are on the same large fragment of amplified DNA (amplicon) 2.7 kb apart. We have now shown that this amplicon contains another full-length gene immediately 5' of estalpha21 which codes for a molybdenum-containing hydroxylase, with highest homology to aldehyde oxidase (AO) from other organisms. The full-length putative AO gene is not present on the estalpha3/estbeta1 or estbeta1 amplicons, but multiple truncated 5' ends of this gene are present around the presumed estalpha3/estbeta1 amplicon breakpoint. Polymerase chain reaction (PCR) analysis of insecticide-susceptible genomic DNA demonstrated that a different allele of the putative AO gene in its non-amplified form is immediately 5' of estalpha. The 'AO' gene on the estalpha21/estbeta21 amplicon is expressed and resistant insects have greater AO activity. This AO activity is sensitive to inhibition by an aldehyde-containing herbicide and pesticide. This enzyme may confer a selective advantage to these insects in the presence of insecticide, as AO in mammals is believed to be important in the detoxification process of several environmental pollutants.

Esterase Inhibitors Synergise the Toxicity of Pyrethroids in AustralianHelicoverpa armigera(Hübner) (Lepidoptera: Noctuidae)

Pyrethroid resistance in AustralianHelicoverpa armigerafield populations is primarily a consequence of the overproduction of esterase isoenzymes which metabolise and possibly sequester pyrethroid insecticides. Biochemical studies show that pyrethroid resistance-associated esterases inH. armigeraare inhibited by organophosphorous compounds, such as ethion, chlorpyrifos and its oxon, profenofos, and acephate. The organophosphates bind to the active site of the enzyme, thus preventing pyrethroid detoxification. Esterase inhibition by organophosphates does not occur immediately after dosage, but occurs rapidly, with maximum enzyme inhibition from 2 to 24 h after dosage, depending on the inhibitor used. These enzyme inhibition studies are supported by bioassay data using nontoxic doses of organophosphate as synergists for pyrethroids. The data show excellent levels of pyrethroid synergism against resistantH. armigera.One hundred percent mortality could be achieved whenH. armigerawere pretreated with some organophosphates and then dosed with pyrethroid, during the time of maximum enzyme inhibition. However, even when synergists and pyrethroids were applied together, up to 90;pc mortality was observed with more effective pyrethroids. Use of organophosphate synergists in the field may have the potential to restore some pyrethroid susceptibility in AustralianH. armigera.

Genetics of esterase-mediated insecticide resistance in the aphid Schizaphis graminum

The genetics of organophosphate resistance, including the pattern of inheritance and the underlying genetic mechanism, were investigated in the aphid Schizaphis graminum (the greenbug). Resistant greenbugs with pattern 1 (R1) and pattern 2 (R2) esterase and susceptible (S) greenbugs were induced into the sexual cycle and crossed. Each resistance-associated esterase pattern in the greenbug was inherited in a Mendelian fashion as a single gene. An aphid clone that displayed both resistance-associated esterase patterns was produced in the F1 generation of a cross between R2 and R1 insects, and was designated pattern 3 (R3). DNA from different R1 aphids and the R3 aphid clone displayed a restriction fragment pattern different from that of R2 and S clones, and about a twofold increase in hybridization compared with S aphids. DNA from R2 clones showed no restriction fragment differences or increased hybridization signal compared to S insects. These findings suggest that the mechanism behind the R1 esterase activity may be gene amplification, but that the R2 elevated esterase activity may result from a different genetic mechanism.

Genetics of esterase-mediated insecticide resistance in the aphid Schizaphis graminum

The genetics of organophosphate resistance, including the pattern of inheritance and the underlying genetic mechanism, were investigated in the aphid Schizaphis graminum (the greenbug). Resistant greenbugs with pattern 1 (R1) and pattern 2 (R2) esterase and susceptible (S) greenbugs were induced into the sexual cycle and crossed. Each resistance-associated esterase pattern in the greenbug was inherited in a Mendelian fashion as a single gene. An aphid clone that displayed both resistance-associated esterase patterns was produced in the F1 generation of a cross between R2 and R1 insects, and was designated pattern 3 (R3). DNA from different R1 aphids and the R3 aphid clone displayed a restriction fragment pattern different from that of R2 and S clones, and about a twofold increase in hybridization compared with S aphids. DNA from R2 clones showed no restriction fragment differences or increased hybridization signal compared to S insects. These findings suggest that the mechanism behind the R1 esterase activity may be gene amplification, but that the R2 elevated esterase activity may result from a different genetic mechanism.

Purification and Characterization of a Resistance-Associated Esterase from the Colorado Potato Beetle, Leptinotarsa decemlineata (Say)

Two strains of the Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae), were found to be resistant to carbofuran and azinphosmethyl when compared to a susceptible strain in bioassays using a discriminating insecticide dose. The percentage of mortalities for carbofuran were 115.1- and 70.8-fold lower in the resistant New York (NY) and Hot Spot (HS) strains, respectively, than in susceptible beetles. When treated with azinphosmethyl, the percentages of mortalities for the NY and HS populations were 65.1- and 4.4-fold lower, respectively, than the susceptible strain. The synergist S,S,S-tributyl phosphorotrithioate (DEF) increased azinphosmethyl toxicity, suggesting esterase involvement in azinphosmethyl resistance. Although 1-naphthyl acetate esterase activities in individual fourth instars of resistant strains were not significantly different, a novel resistance-associated esterase (RAE) (p I = 6.23) was discovered in the first and fourth instars and adults of NY and HS CPBs, but not in the susceptible strain. The RAE was purified from fourth stadium NY CPBs by Rotofor followed by polyacrylamide gel isoelectric focusing. Methyl paraoxon was the most potent inhibitor of the purified RAE with an I 50 of 0.1 μ M. The esterase inhibitors octylihio-1,1,1-trifluoropropan-2-one (OTFP), DEF, and azinphosmethyl had I 50s of 1.1, 5.5, and 9.8 μ M, respectively. Carbofuran and eserine hemisulfate were poor inhibitors, with I 50s greater than 100 μ M. In substrate competition assays with the RAE, methyl paraoxon and OTFP were competitive inhibitors.

The Cross-Reactivity Spectrum of a Polyclonal Antiserum Raised against the Native Amplified A 2 Esterase Involved in Insecticide Resistance

Broad-spectrum insecticide resistance in the mosquito Culex quinquefasciatus is usually due to the overproduction of A- and B-type carboxylesterases (EC 3.1.1.1). Antiserum was raised against the carboxylesterase A 2 purified from an organophosphate-resistant Culex strain. This antiserum was used to show that, contrary to earlier reports, the A(2) esterase is immunologically related to other A- and B-type carboxylesterases from resistant and susceptible Culex strains. Dot-blot immunoassays revealed that the purified esterase B 2 is about 50-fold less reactive with the antiserum than the purified esterase A 2. A strong immunological relationship was observed between the A 2 antiserum and the organophosphate and carbamate target site acetylcholinesterase. A lower cross-reactivity with Anopheles stephensi esterases and no reactivity with resistance-associated esterases from grain beetles, planthoppers, and cockroaches were observed. The antiserum cross-reacted with some commercially available vertebrate esterases indicating its affinities with these enzymes. The observed cross-reactivity is not due to the antiserum cross-reacting with glycosylated residues, as the A 2 esterase to which the antiserum was raised is unglycosylated. The utility of this antiserum in future esterase cDNA cloning programs is discussed.

Isolation and Characterization of an Insecticide-Resistance-Associated Esterase in the Tobacco Budworm Heliothis virescens (F.)

1-Naphthyl acetate (1-NA) esterase activity was elevated in whole body homogenates of first, third, and fifth stadium tobacco budworms resistant to thiodicarb and cypermethrin compared to susceptible budworms at the same developmental stage. Increased esterase activity was attributed mostly to three esterases designated A1, B1, and C1 with isoelectric points of 4.8, 5.1, and 5.3, respectively, as determined by isoelectric focusing-polyacrylamide gel electrophoresis (IEF-PAGE). These three esterases were purified by preparative liquid column isoelectric focusing followed by slab IEF-PAGE and sodium dodecyl sulfate-PAGE. Specific antibody to the resistance-associated esterase of the house fly (anti-E1, N. Motoyama) and the green peach aphid (anti-E4, A. Devonshire) was immunoreactive on Western blots with purified esterase A1, while minimal or no reactivity was noted for esterases B1 and C1. Anti-E4 detected A1 in 10,000g supernatant from homogenates of resistant and susceptible tobacco budworms. Kinetic studies demonstrated that methyl paraoxon, cypermethrin, DDT, and thiodicarb were competitive inhibitors of the 1-NA esterase activity of A1, whereas eserine hemisulfate had no effect on activity. An amino-terminal sequence analysis revealed that residues Gly-12, Arg-15, and Gly-16 were conserved with Culex esterase B1 (CuB1), Drosophila melanogaster esterase-6 (DmE6) and acetylcholinesterase, H. virescens JH esterase, human butyrylcholinesterase, and rabbit liver esterase form 2, while Arg-15 was missing in E4 from Myzus persicae. A1 demonstrated the greatest sequence homology with that of the DmE6 of Drosophila and the resistance-associated esterase, CuB1, of Culex.

Organophosphate Resistance in the Tobacco Aphid (Homoptera: Aphididae): Purification and Characterization of a Resistance-Associated Esterase

A 1-naphthyl acetate esterase was identified by isoelectric focusing in organophosphate resistant (red) tobacco aphids, Myzus nicotianae Blackman, which apparently was lacking in susceptible (green) individuals of the same species. This resistance-associated esterase (RAE) was purified from the cytosol of whole body homogenates of adult tobacco aphids by gel permeation chromatography and two successive DEAE-Sephacel ion exchange columns. The enzyme eluted from the second DEAE column in 0.23 to 0.28 M NaCl and was ≍95% pure as determined by high-performance liquid chromatography (HPLC) on a BIO-SIL TSK-125 column. Purified RAE had an apparent molecular weight of 67,300 as determined by gel permeation HPLC and 66,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Wide range isoelectric focusing (pH 3.5-9.5) on polyacrylamide gel plates indicated that the isoelectric point was 4.61. Rabbit antiserum to the green peach aphid, M. persicae (Sulzer), esterase (E4) specifically cross-reacted with the tobacco aphid RAE, and the two esterases had identical molecular weights and isoelectric points as determined by immunoblotting. RAE protein was not detectable in susceptible tobacco aphids.