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

Abstract

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.

Author Summary

M. persicae is the most economically important aphid pest in many regions of the world due to its large host range and the damage it causes through direct feeding and through the transmission of plant viruses. This species has developed resistance to most classes of insecticide; and although, to date, the neonicotinoids have remained an effective control measure, recent reports of resistance in M. persicae represent a threat to the long-term effectiveness of this chemical class. Work on other insect species has shown that resistance can arise through increased production of metabolic enzymes (such as cytochrome P450s) that detoxify the insecticide. In this study we made use of recent advances in genomics to quantify the expression of all genes encoding detoxification enzymes in a resistant strain of M. persicae. We found resistance correlated with the over-expression of a single P450 gene, and we show that this is due to gene amplification. We also showed that over-expression of cuticular proteins and reduced penetration of insecticide through the cuticle may also play a role in resistance. These findings will influence the design and monitoring of management strategies that aim to slow or prevent the development of resistance.