Graham D Moores

Identification of mutations conferring insecticide-insensitive AChE in the cotton-melon aphid, Aphis gossypii Glover

We have identified two mutations in the ace1 gene of Aphis gossypii that are associated with insensitivity of acetylcholinesterase (AChE) to carbamate and organophosphate insecticides. The first of these, S431F (equivalent to F331 in Torpedo californica), is associated with insensitivity to the carbamate insecticide pirimicarb in a range of A. gossypii clones. The S431F mutation is also found in the peach‐potato aphid, Myzus persicae (Sulzer), and a rapid RFLP diagnostic allows the identification of individuals of both aphid species with a resistant genotype. This diagnostic further revealed the presence of S431 in several other pirimicarb‐susceptible aphid species. The serine at this position in the wild‐type enzyme has only been reported for aphids and provides a molecular explanation of why pirimicarb has a specific aphicidal action. A less specific insensitivity to a wide range of carbamates and organophosphates is associated with a second mutation, A302S (A201 in T. californica).

Overexpression of a cytochrome P450 monooxygenase, CYP6ER1, is associated with resistance to imidacloprid in the brown planthopper, Nilaparvata lugens

The brown planthopper, Nilaparvata lugens, is an economically significant pest of rice throughout Asia and has evolved resistance to many insecticides including the neonicotinoid imidacloprid. The resistance of field populations of N.u2003lugens to imidacloprid has been attributed to enhanced detoxification by cytochrome P450 monooxygenases (P450s), although, to date, the causative P450(s) has (have) not been identified. In the present study, biochemical assays using the model substrate 7‐ethoxycoumarin showed enhanced P450 activity in several resistant N.u2003lugens field strains when compared with a susceptible reference strain. Thirty three cDNA sequences encoding tentative unique P450s were identified from two recent sequencing projects and by degenerate PCR. The mRNA expression level of 32 of these was examined in susceptible, moderately resistant and highly resistant N.u2003lugens strains using quantitative real‐time PCR. A single P450 gene (CYP6ER1) was highly overexpressed in all resistant strains (up to 40‐fold) and the level of expression observed in the different N.u2003lugens strains was significantly correlated with the resistance phenotype. These results provide strong evidence for a role of CYP6ER1 in the resistance of N.u2003lugens to imidacloprid.

Metabolic enzyme(s) confer imidacloprid resistance in a clone of Myzus persicae (Sulzer) (Hemiptera: Aphididae) from Greece

BACKGROUNDnPrevious studies have reported varying levels of resistance against imidacloprid in several insect species, including populations of the peach-potato aphid, Myzus persicae (Sulzer). These cases of resistance have been attributed to either target-site resistance or enhanced detoxification. In this study, a clone of M. persicae originating from Greece revealed a 60-fold resistance factor to imidacloprid.nnnRESULTSnThe Greek clone is compared in terms of metabolic enzyme activity and synergism profiles with other M. persicae clones showing lower imidacloprid resistance.nnnCONCLUSIONnA combination of in vitro biochemical assays and in vivo differential synergism studies using PBO and a close analogue EN 16/5-1 suggests that the mechanism conferring increased resistance in this clone is primarily due to enhanced oxidase activity.

Cross‐resistance between a Bacillus thuringiensis Cry toxin and non‐Bt insecticides in the diamondback moth

BACKGROUNDnBacillus thuringiensis Berliner (Bt) crystal (Cry) toxins are expressed in various transgenic crops and are also used as sprays in integrated pest management and organic agricultural systems. The diamondback moth (Plutella xylostella L.) is a major worldwide pest of crucifer crops and one that has readily acquired field resistance to a broad range of insecticides.nnnRESULTSnSelection of a subpopulation of the P. xylostella SERD4 population with the pyrethroid deltamethrin increased resistance to both deltamethrin and Cry1Ac relative to an unselected subpopulation. Selection of a second subpopulation with the Bt toxin Cry1Ac also increased resistance to both Cry1Ac and deltamethrin. A complementation test between the Cry1Ac-selected and deltamethrin-selected subpopulations suggested the presence of a common genetic locus or loci that control resistance to both insecticides. A piperonyl butoxide analogue with potent inhibitory activity against insect esterases significantly increased the toxicity of Cry1Ac and deltamethrin against the respective resistant subpopulations, but showed no such synergism with the unselected subpopulation of SERD4.nnnCONCLUSIONnSelection of one resistance phenotype resulted in the simultaneous selection of the other. This phenomenon could be due to a single mechanism acting against both classes of insecticide or to genetically linked, but separate, mechanisms.

An analogue of piperonyl butoxide facilitates the characterisation of metabolic resistance.

BACKGROUNDnPrevious 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.nnnRESULTSnThis 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.nnnCONCLUSIONnUsing 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.

BACKGROUNDnConventional 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.nnnRESULTSnThe 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.nnnCONCLUSIONnThe 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.

Characterising metabolic resistance in pyrethroid-insensitive pollen beetle (Meligethes aeneus F.) from Poland and Switzerland.

BACKGROUNDnPollen beetle (Meligethes aeneus F.) has become the most important pest of oilseed rape in Europe, but its control has been greatly hindered by pyrethroid resistance. Target-site resistance has been implicated previously, and, whilst synergism has been found with piperonyl butoxide (PBO), the exact nature of metabolic resistance has remained unknown. The use of PBO, in conjunction with its analogue EN 16/5-1, has allowed the characterisation of metabolic resistance.nnnRESULTSnIn vitro assays in combination with in vivo studies using PBO and EN 16/5-1 showed that high synergism of pyrethroids was primarily correlated with an oxidative mechanism, although a limited contribution by esterases was implicated in one population.nnnCONCLUSIONnDifferential synergism has enabled the characterisation of pyrethroid resistance in populations of M. aeneus. It was found to be principally due to an oxidative-based mechanism, and, if a synergist were to be used to inhibit this enzyme group, renewed control against resistant pests could be achieved.

Repellents Inhibit P450 Enzymes in Stegomyia (Aedes) aegypti

The primary defence against mosquitoes and other disease vectors is often the application of a repellent. Despite their common use, the mechanism(s) underlying the activity of repellents is not fully understood, with even the mode of action of DEET having been reported to be via different mechanisms; e.g. interference with olfactory receptor neurones or actively detected by olfactory receptor neurones on the antennae or maxillary palps. In this study, we discuss a novel mechanism for repellence, one of P450 inhibition. Thirteen essential oil extracts from Colombian plants were assayed for potency as P450 inhibitors, using a kinetic fluorometric assay, and for repellency using a modified World Health Organisation Pesticide Evaluations Scheme (WHOPES) arm-in cage assay with Stegomyia (Aedes) aegypti mosquitoes. Bootstrap analysis on the inhibition analysis revealed a significant correlation between P450-inhibition and repellent activity of the oils.

Esterase isoenzymes and insecticide resistance in Frankliniella occidentalis populations from the south‐east region of Spain

BACKGROUNDnFrankliniella occidentalis (Pergande) is among the most important crop pests in the south-east region of Spain; its increasing resistance to insecticides constitutes a serious problem, and understanding the mechanisms involved is therefore of great interest. To this end, F. occidentalis populations, collected from the field at different locations in south-east Spain, were studied in terms of total esterase activity and esterase isoenzyme pattern.nnnRESULTSnIndividual thrips extracts were analysed by native polyacrylamide gel electrophoresis (PAGE) and stained for esterase activity with the model substrate alpha-naphthyl acetate. Significant correlations were found between resistance to the insecticides acrinathrin and methiocarb and the presence of a group of three intensely stained bands, named Triplet A. For each individual thrips extract, total esterase activity towards the substrates alpha-naphthyl acetate and alpha-naphthyl butyrate was also measured in a microplate reader. Insects possessing Triplet A showed a significantly higher alpha-naphthyl acetate specific activity and alpha-naphthyl acetate/alpha-naphthyl butyrate activity ratio. This observation allowed a reliable classification of susceptible or resistant insects either by PAGE analysis or by total esterase activity determination.nnnCONCLUSIONnThe PAGE and microplate assays described can be used as a monitoring technique for detecting acrinathrin- and methiocarb-resistant individuals among F. occidentalis field populations.

Investigating the potential of selected natural compounds to increase the potency of pyrethrum against houseflies Muscadomestica (Diptera: Muscidae)

BACKGROUNDnA study was undertaken to determine the efficacy of seven natural compounds compared with piperonyl butoxide (PBO) in synergising pyrethrum, with the intention of formulating an effective natural synergist with pyrethrum for use in the organic crop market.nnnRESULTSnDiscriminating dose bioassays showed PBO to be significantly more effective at synergising pyrethrum in houseflies than the seven natural compounds tested, causing 100% mortality in insecticide-susceptible WHO and resistant 381zb strains of housefly. The most effective natural synergists against WHO houseflies were dillapiole oil, grapefruit oil and parsley seed oil, with 59, 50 and 41% mortality respectively, compared with 18% mortality with unsynergised pyrethrum. Against 381zb houseflies, the most effective natural synergists were parsley seed oil and dillapiole oil. Esterase inhibition by the natural compounds and PBO in vitro showed no correlation with pyrethrum synergism in vivo, whereas the inhibition of oxidases in vitro more closely correlated with pyrethrum synergism in vivo.nnnCONCLUSIONnDillapiole oil and parsley seed oil showed the greatest potential as pyrethrum synergists. PBO remained the most effective synergist, possibly owing to its surfactant properties, enhancing penetration of pyrethrins. The results suggest the involvement of oxidases in pyrethroid resistance in houseflies, with the efficacy of synergists showing a high correlation with inhibition of oxidases.