Stephen P Foster

Amplification of a Cytochrome P450 Gene Is Associated with Resistance to Neonicotinoid Insecticides in the Aphid Myzus persicae

The aphid Myzus persicae is a globally significant crop pest that has evolved high levels of resistance to almost all classes of insecticide. To date, the neonicotinoids, an economically important class of insecticides that target nicotinic acetylcholine receptors (nAChRs), have remained an effective control measure; however, recent reports of resistance in M. persicae represent a threat to the long-term efficacy of this chemical class. In this study, the mechanisms underlying resistance to the neonicotinoid insecticides were investigated using biological, biochemical, and genomic approaches. Bioassays on a resistant M. persicae clone (5191A) suggested that P450-mediated detoxification plays a primary role in resistance, although additional mechanism(s) may also contribute. Microarray analysis, using an array populated with probes corresponding to all known detoxification genes in M. persicae, revealed constitutive over-expression (22-fold) of a single P450 gene (CYP6CY3); and quantitative PCR showed that the over-expression is due, at least in part, to gene amplification. This is the first report of a P450 gene amplification event associated with insecticide resistance in an agriculturally important insect pest. The microarray analysis also showed over-expression of several gene sequences that encode cuticular proteins (2–16-fold), and artificial feeding assays and in vivo penetration assays using radiolabeled insecticide provided direct evidence of a role for reduced cuticular penetration in neonicotinoid resistance. Conversely, receptor radioligand binding studies and nucleotide sequencing of nAChR subunit genes suggest that target-site changes are unlikely to contribute to resistance to neonicotinoid insecticides in M. persicae.

Mutation of a nicotinic acetylcholine receptor β subunit is associated with resistance to neonicotinoid insecticides in the aphid Myzus persicae

BackgroundMyzus persicae is a globally important aphid pest with a history of developing resistance to insecticides. Unusually, neonicotinoids have remained highly effective as control agents despite nearly two decades of steadily increasing use. In this study, a clone of M. persicae collected from southern France was found, for the first time, to exhibit sufficiently strong resistance to result in loss of the field effectiveness of neonicotinoids.ResultsBioassays, metabolism and gene expression studies implied the presence of two resistance mechanisms in the resistant clone, one based on enhanced detoxification by cytochrome P450 monooxygenases, and another unaffected by a synergist that inhibits detoxifying enzymes. Binding of radiolabeled imidacloprid (a neonicotinoid) to whole body membrane preparations showed that the high affinity [3H]-imidacloprid binding site present in susceptible M. persicae is lost in the resistant clone and the remaining lower affinity site is altered compared to susceptible clones. This confers a significant overall reduction in binding affinity to the neonicotinoid target: the nicotinic acetylcholine receptor (nAChR). Comparison of the nucleotide sequence of six nAChR subunit (Mpα1-5 and Mpβ1) genes from resistant and susceptible aphid clones revealed a single point mutation in the loop D region of the nAChR β1 subunit of the resistant clone, causing an arginine to threonine substitution (R81T).ConclusionPrevious studies have shown that the amino acid at this position within loop D is a key determinant of neonicotinoid binding to nAChRs and this amino acid change confers a vertebrate-like character to the insect nAChR receptor and results in reduced sensitivity to neonicotinoids. The discovery of the mutation at this position and its association with the reduced affinity of the nAChR for imidacloprid is the first example of field-evolved target-site resistance to neonicotinoid insecticides and also provides further validation of exisiting models of neonicotinoid binding and selectivity for insect nAChRs.

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.

The ups and downs of insecticide resistance in peach-potato aphids (Myzus persicae) in the UK

Abstract The peach-potato aphid, Myzus persicae, provides an excellent demonstration of how genetic and ecological factors can interact to determine the dynamics of resistance and influence success of resistance management. The diversity of resistance mechanisms, based on both enhanced detoxification of insecticides and modifications to their target sites, that this species shows is now well understood at the genotypic, biochemical and molecular levels. These developments have yielded rapid and precise methods for detecting individual mechanisms, and for monitoring frequency changes in the presence and absence of insecticide selection. One factor influencing the prediction of these changes in the UK is the occurrence of prolonged periods of parthenogenesis in M. persicae, which results in non-random associations between particular resistance genes through the absence of genetic recombination. This has important implications for the rate at which resistance genes can accumulate in populations under selection by insecticides, and also for the speed at which they will decline when selection is relaxed. Although it is becoming apparent that resistance can be associated with various fitness costs, the relative contribution of individual resistance genes to these effects is still under investigation. However, the overall effect of the `ups’ and `downs’ of resistance in M. persicae, in the UK at least, has been to help maintain resistance phenotypes at manageable levels in most seasons. Unfortunately, the increasing genetic diversity of resistance in this species is progressively eroding the supply of effective insecticides. This in turn highlights the importance of ensuring sufficient chemical diversity to combat existing resistance mechanisms and to restrict the use of novel insecticides in order to sustain their effectiveness.

Analogous pleiotropic effects of insecticide resistance genotypes in peach–potato aphids and houseflies

We show that single-point mutations conferring target-site resistance (kdr) to pyrethroids and DDT in aphids and houseflies, and gene amplification conferring metabolic resistance (carboxylesterase) to organophosphates and carbamates in aphids, can have deleterious pleiotropic effects on fitness. Behavioural studies on peach–potato aphids showed that a reduced response to alarm pheromone was associated with both gene amplification and the kdr target-site mutation. In this species, gene amplification was also associated with a decreased propensity to move from senescing leaves to fresh leaves at low temperature. Housefly genotypes possessing the identical kdr mutation were also shown to exhibit behavioural differences in comparison with susceptible insects. In this species, resistant individuals showed no positional preference along a temperature gradient while susceptible genotypes exhibited a strong preference for warmer temperatures.

Comparative survival of insecticide-susceptible and resistant peach-potato aphids, Myzus persicae (Sulzer) (Hemiptera: Aphididae), in low temperature field trials

The survival of Myzus persicae (Sulzer) clones, representing the full range of the recognized levels of insecticide resistance, was measured after 3–4 week exposure to the winter climate in the field. Ten separate trials were carried out using known numbers of first/second instar nymphs on unsprayed oilseed rape and groundsel. These predominantly showed a negative association between resistance level and the proportion of aphids recovered alive after exposure. The strength of the relationship correlated closely with three meteorological variables: the length of time that aphids spent below 2°C, mean rainfall and mean windspeed. We conclude that selection for resistance to insecticides is subject to counteracting selection by cold, wet and windy conditions in the UK winter. Revertant clones, that had spontaneously lost extreme resistance to insecticides despite retaining resistance genes, survived no better than aphids that had kept their high resistance, indicating that overproduction of esterase was not the reason for poor survival.

Reduced response of insecticide-resistant aphids and attraction of parasitoids to aphid alarm pheromone; a potential fitness trade-off

Response to the alarm pheromone, (E)-beta-farnesene, produced by many species of aphids, was assessed in laboratory bioassays using an aphid pest, Myzus persicae (Sulzer), and its primary endoparasitoid, Diaeretiella rapae (McIntosh). This was done in three separate studies, the first investigating responses of a large number of M. persicae clones carrying different combinations of metabolic (carboxylesterase) and target site (kdr) insecticide resistance mechanisms, and the other two investigating the responses of young virgin female adult parasitoids. In M. persicae, both insecticide resistance mechanisms were associated with reduced repellence suggesting that each has a pleiotropic effect on aphid behaviour. In contrast, D. rapae females were attracted to the alarm pheromone source. The implications of this apparent fitness trade-off for the evolution and dynamics of insecticide resistance, and the potential for using beneficial insects to combat resistance development are discussed.

Reduced alarm response by peach–potato aphids, Myzus persicae (Hemiptera: Aphididae), with knock-down resistance to insecticides ( kdr )may impose a fitness cost through increased vulnerability to natural enemies

Termites were sampled using randomized soil pits in 64 cropping plots, each 25 x 25 m, forming an experimental agrisilvicultural system in both a 6- and an 18-year-old Terminalia ivorensis plantation, in which canopy cover, crop, cropping system and land preparation were the principal treatment variables. The treatments were established in April 1995 and sampling was carried out in November 1995, February 1996 and July 1996. A total of 82 termite species were found, of which 67 were soil-feeders. Overall termite abundance and the abundance of soil-feeders increased between November 1995 and July 1996, reaching a mean of nearly 6000 m -2 . Pooling termite data from these sampling dates, in the old plantation, the high canopy cover treatment (192 stems ha -1 ) had a greater abundance of termites, compared with the low canopy cover treatment (64 stems ha -1 ) and this effect was independent of crop type (plantain or cocoyam), cropping system (single stands or mixed crops) and land preparation (mulch retained or burned, plantain only). The young tree plantation (same tree densities as in the old plantation) showed no significant difference in termite abundance between high and low canopy (levels of tree foliage) densities, though the high canopy sheltered a greater number of termites. Analysis of covariance showed that crop yield (both plantain and cocoyam) was not directly linked to the abundance of all termite populations, but that the cocoyam yield was positively correlated with the abundance of soil-feeding termites (the majority in the assemblage) in the young plantation. This may be due to the beneficial conditioning of soil resulting from the foraging and construction activities of soil-feeders.

Intensification of insecticide resistance in UK field populations of the peach-potato aphid, Myzus persicae (Hemiptera: Aphididae) in 1996

The well-established carboxylesterase-based resistance to insecticides in Myzus persicae Sulzer has recently been accentuated by the appearance of aphids with a modified acetylcholinesterase (MACE) insensitive to pirimicarb and the novel aphicide, triazamate. This target site resistance mechanism was found in M. persicae from crops in the UK for the first time in 1996, together with especially large proportions of aphids with R 2 and R 3 levels of carboxylesterases, a combination that was associated with serious insecticide failures. This paper describes the incidence of both mechanisms and discusses the implications for future recommendations for aphid control in the UK.

Micro‐evolutionary change in relation to insecticide resistance in the peach–potato aphid, Myzus persicae

1. Phenotypic diversity is the fuel that powers evolution.