Alan L. Devonshire

A carboxylesterase with broad substrate specificity causes organophosphorus, carbamate and pyrethroid resistance in peach-potato aphids (Myzus persicae)

Abstract Carboxylesterase E4, the enzyme previously shown to cause resistance to organophosphorus insecticides in peach-potato aphids, Myzus persicae, was purified and characterized by electrophoretic and enzyme kinetic techniques. Its insecticidal substrate specificity, determined by following the rate of recovery of esterase activity after inhibition by a range of acylating inhibitors, included a variety of carbamates and organophosphates, although the catalytic center activity for these substrates was low. Radiometric measurement of hydrolysis of the pyrethroid, permethrin, showed that E4, whether purified or in crude aphid homogenates, hydrolyzed the (1S)trans enantiomer rapidly but hydrolysis of the other three isomers could not be detected. Such absolute specificity for one enantiomer of a pyrethroid is rare. The rates of hydrolysis of the various insecticidal classes correlated well with the relative degrees of resistance to them, and no other resistance mechanisms have been detected. Although the enzyme is relatively inefficient in degrading insecticidal esters, it is produced in very large quantity, accounting for approximately 3% of the total protein in very resistant aphids. Its effect is thus mediated not only by hydrolysis but also by sequestering a substantial proportion of a toxic dose of insecticide. It is effective in this respect because the molar amount present is similar to that of a lethal dose of insecticide. These results support earlier indirect evidence for “overproduction” of E4, probably because of structural gene duplication or amplification and have direct implications for strategies to delay the buildup of resistance or for developing synergists to overcome resistance.

A sodium channel point mutation is associated with resistance to DDT and pyrethroid insecticides in the peach-potato aphid, Myzus persicae (Sulzer) (Hemiptera: Aphididae)

The voltage‐gated sodium channel is the primary target site of DDT and pyrethroid insecticides, and point mutations in the domain II region of the channel protein have been implicated in the knockdown resistant (kdr ) phenotype of several insect species. Here, we report that one of these mutations, a leucine‐to‐phenylalanine replacement in transmembrane segment IIS6, is also found in certain insecticide‐resistant clones of the peach‐potato aphid, Myzus persicae. The mutation was present in four clones with amplified E4 esterase genes, but was absent from both susceptible clones and those with amplified FE4 genes. The inferred presence of kdr‐type resistance in the four E4 clones was subsequently confirmed by bioassays that showed this to be the primary mechanism of resistance to deltamethrin and DDT, although the esterase‐based mechanism also contributes to the overall level of deltamethrin resistance. The kdr mutation on its own conferred 35‐fold resistance to deltamethrin and this was enhanced up to 540‐fold when it was present in a high (E4) esterase background. The esterase (FE4) mechanism was far less effective without the kdr mutation, conferring just 3–4‐fold resistance to deltamethrin. These findings, and the linkage disequilibrium of the kdr mutation within clones overproducing the E4 esterase, have important implications for the evolution of resistance in this insect and for the use of pyrethroid sprays in the management of M. persicae populations in the field.

Chromosomal location of the amplified esterase genes conferring resistance to insecticides in Myzus persicae (Homoptera: Aphididae)

A genomic probe encompassing most of an esterase gene (E4) that is amplified in insecticide-resistant Myzus persicae was hybridized in situ to mitotic and meiotic chromosome preparations of aphid clones of known esterase type and resistance level. Binding, which was detected using the biotin-avidin system located both known types of amplified esterase sequences (E4 and FE4). All except one of the E4-producing clones had a single amplified site, on autosome 3 near the breakpoint of an autosomal 1,3 translocation which previous work had shown to be genetically linked to insecticide resistance. The exceptional clone had two other E4-encoding sites. The most resistant FE4-producing clone (800F) had amplified sequences at five sites (three loci: two homozygous and one heterozygous). Altogether, amplified E4 and/or FE4 sequences were found on four of the five autosome pairs of M. persicae. Possible origins of these multiple loci are discussed.

Different forms of insensitive acetylcholinesterase in insecticide-resistant house flies (Musca domestica)

Abstract A new form of acetylcholinesterase insensitive to organophosphorus inhibitors was identified in house flies collected from animal farms in the U.K. Its degree of insensitivity to a number of inhibitors ranged from 4-fold (omethoate) to >40-fold (tetrachlorvinphos and dichlorvos) compared with our standard susceptible enzyme, and its spectrum of response to inhibitors differed widely from that of a previously studied insensitive form. In addition, the form of acetylcholinesterase reported here is unusual in having a 3.4-fold greater affinity (lower Km) for acetylthiocholine than the normal enzyme. The possible toxicological significance of this is discussed.

Changes in the methylation of amplified esterase DNA during loss and reselection of insecticide resistance in peach-potato aphids, Myzus persicae

Insecticide resistance in peach-potato aphids, Myzus persicae, results from the amplification of genes encoding an esterase that hydrolyses and sequesters insecticides. Resistance is normally stable, but highly resistant aphid clones sometimes lose resistance when insecticidal selection pressure is removed. This loss of resistance, termed reversion, arises from a loss of elevated esterase enzyme through transcriptional control, i.e. without loss of the amplified esterase DNA sequences. We have shown that loss of the elevated enzyme occurred simultaneously with loss of methylation at CCGG sites in the amplified DNA sequences. During reselection of resistance in these revertant clones, enzyme levels increased, but there was no corresponding return of methylation to DNA sequences. Thus, although DNA methylation is closely correlated with expression of the amplified esterase genes during reversion, it may not be a factor in the reverse process.

The biochemical basis of resistance to organophosphorus insecticides in the sheep blowfly, Lucilia cuprina

Abstract The metabolism in vivo and in vitro of [ 14 C]parathion and [ 14 C]paraoxon was studied in a susceptible (LS) and an organophosphorus-resistant (Q) strain of the sheep blowfly, Lucilia cuprina . Both strains detoxified the insecticides in vivo via a number of pathways, but the resistant strain produced more of the metabolites diethyl phosphate and diethyl phosphorothionate. No difference was found between strains in the rate of penetration of the compounds used. Also, in vitro studies showed no difference between strains in the sensitivity of head acetylcholinesterase to inhibition by paraoxon. Both the microsomal and the 100,000g supernatant fractions degraded paraoxon, but resistance in Q could be explained by the eightfold greater rate of diethyl phosphate production with or without added NADPH. Parathion was also degraded to diethyl phosphorothionate by an NADPH-requiring enzyme in microsomal preparations from both strains. However, Q produced significantly more diethyl phosphorothionate in vivo than LS. It was concluded that organophosphorus resistance in Q was due mainly to a microsomal phosphatase hydrolyzing phosphate but not phosphorothionate esters, probably enhanced by a microsomal oxidase detoxifying the latter.

Cloning, heterologous expression and co-assembly of Mpβ1, a nicotinic acetylcholine receptor subunit from the aphid Myzus persicae

Nicotinic acetylcholine receptors (nAChRs) play a major role in excitatory synaptic transmission in insects and are also the target site for chloronicotinyl insecticides such as imidacloprid. Here we report the cloning and characterization of a novel nAChR beta subunit, Mpbeta1, from the aphid Myzus persicae, an economically important pest species. Sequence analysis has identified an open reading frame of 509 amino acids with features typical of nAChR subunits. The Mpbeta1 gene is expressed as a single major transcript of 4.6 kb, considerably larger than the predicted length of the Mpbeta1 open reading frame (1527 bp). By heterologous expression in Drosophila S2 cells, the Mpbeta1 subunit has been shown to co-assemble with the previously cloned nAChR subunits Mpalpha1 and Mpalpha2. In contrast, no co-assembly of Mpbeta1 could be detected with either Mpalpha3 or Mpalpha4. With the aim of gaining a clearer insight into the influence of subunit composition upon assembly, the ability of M. persicae nAChR subunits to co-assemble with vertebrate nAChR subunits has also been examined.

Characterisation of insecticide-insensitive acetylcholinesterase: Microcomputer-based analysis of enzyme inhibition in homogenates of individual house fly (Musca domestica) heads

Abstract A spectrophotometer interfaced with a microcomputer to collect and analyse data automatically was used to study the inhibition kinetics of house fly acetylcholinesterase by malaoxon and dichlorvos in the presence of substrate. This system simply and unequivocally identified insecticide-insensitive enzymes from single fly heads, even when these enzymes were hitherto uncharacterised. As well as identifying individual insects homozygous for a particular form of acetylcholinesterase, the method also recognised heterozygotes because the mixture of enzymes then present gave a heterogeneous response to inhibitors. In the latter case, each component enzyme present could be identified. The technique is valuable for characterising heterogeneous insect populations collected from the field, for establishing homozygous strains, and for more detailed biochemical study.

Inheritance of the amplified esterase genes responsible for insecticide resistance in Myzus persicae (Homoptera: Aphididae)

Insecticide-resistant and susceptible clones of Myzus persicae were induced to produce sexual morphs and crossed in the laboratory. Progeny clones were analysed for karyotype, esterase (E4, FE4 or S) gene type and activity, and amplified E4 and FE4 genes were located on their chromosomes by fluorescence in situ hybridization (FISH). Amplified FE4 genes of resistant parent clones (800F and French R) were inherited according to expectations. Chromosomal locations of these genes (on autosomes 1 and 3 in 800F, and on 1 and 2 in French R) were confirmed by FISH analysis of progeny that had inherited an autosome 2 marker (a dissociation) from the susceptible parent (DS). Inheritance of amplified E4 genes could not be studied directly as none of the available clones was able to produce mating females. Males from two clones with amplified E4 genes (and with the A1,3 translocation that is common to all E4-producing genotypes) were therefore mated with females from clones with amplified FE4 genes at known chromosomal locations. Progeny were obtained with both E4 and FE4 genes, a combination not yet found in nature. Analysis of F1 and subsequent generations confirmed that the amplified E4 site on autosome 3T is close to the translocation breakpoint, and apparently coallelic with the amplified FE4 site on the normal autosome 3 inherited from 800F. One of the translocated parent clones (4156) had two additional E4 sites, unlinked to the translocation, which were inherited according to expectation. Esterase activities of progeny clones, measured by immunoassay, mostly corresponded to the number of amplified sites inherited, with some discrepancies which could be attributed to copy number differences between sites, inheritance of partially methylated genes from French R, or position effect variegation at the site on 3T. Inheritance of the A1,3 translocation in two crosses differed markedly from expectation.

Biochemical and Molecular Characterisation of Insecticide Insensitive Acetylcholinesterase in Resistant Insects

Over half of the world’s insecticide market depends on organophosphorus (OP) and carbamate insecticides acting on acetylcholinesterase (AChE). Their extensive use over many years has led to the development of resistance in many insect species, one of the most important mechanisms being target site insensitivity. A range of AChE variants can be found within a species, differing markedly in their catalytic efficiency and insecticide insensitivity profiles. This is illustrated by our studies of the AChE from resistant strains of housefly (Musca domestica) (1), whitefly (Bemisia tabaci) (2) and the aphids, Myzus persicae (3) and Aphis gossypii (4).