Gene amplification and microsatellite polymorphism underlie a recent insect host shift

Host plant shifts of herbivorous insects may be a first step toward sympatric speciation and can create new pests of agriculturally important crops; however, the molecular mechanisms that mediate this process are poorly understood. Certain races of the polyphagous aphid Myzus persicae have recently adapted to feed on tobacco (Myzus persicae nicotianae) and show a reduced sensitivity to the plant alkaloid nicotine and cross-resistance to neonicotinoids a class of synthetic insecticides widely used for control. Here we show constitutive overexpression of a cytochrome P450 (CYP6CY3) allows tobacco-adapted races of M. persicae to efficiently detoxify nicotine and has preadapted them to resist neonicotinoid insecticides. CYP6CY3, is highly overexpressed in M. persicae nicotianae clones from three continents compared with M. persicae s.s. and expression level is significantly correlated with tolerance to nicotine. CYP6CY3 is highly efficient (compared with the primary human nicotine-metabolizing P450) at metabolizing nicotine and neonicotinoids to less toxic metabolites in vitro and generation of transgenic Drosophila expressing CYP6CY3 demonstrate that it confers resistance to both compounds in vivo. Overexpression of CYP6CY3 results from the expansion of a dinucleotide microsatellite in the promoter region and a recent gene amplification, with some aphid clones carrying up to 100 copies. We conclude that the mutations leading to overexpression of CYP6CY3 were a prerequisite for the host shift of M. persicae to tobacco and that gene amplification and microsatellite polymorphism are evolutionary drivers in insect host adaptation

Molecular and functional characterization of CYP6BQ23, a cytochrome P450 conferring resistance to pyrethroids in European populations of pollen beetle, Meligethes aeneus

The pollen beetle (Meligethes aeneus F.) is widespread throughout much of Europe where it is a major coleopteran pest of oilseed rape (Brassica napus). The reliance on synthetic insecticides for control, particularly the pyrethroid class, has led to the development of populations with high levels of resistance. Resistance to pyrethroids is now widespread throughout Europe and is thought to be mediated by enhanced detoxification by cytochrome P450ś and/or mutation of the pyrethroid target-site, the voltage-gated sodium channel. However, in the case of cytochrome P450 mediated detoxification, the specific enzyme(s) involved has (have) not yet been identified. In this study a degenerate PCR approach was used to identify ten partial P450 gene sequences from pollen beetle. Quantitative PCR was then used to examine the level of expression of these genes in a range of pollen beetle populations that showed differing levels of resistance to pyrethroids in bioassays. The study revealed a single P450 gene, CYP6BQ23, which is significantly and highly overexpressed (up to ∼900-fold) in adults and larvae of pyrethroid resistant strains compared to susceptible strains. CYP6BQ23 overexpression is significantly correlated with both the level of resistance and with the rate of deltamethrin metabolism in microsomal preparations of these populations. Functional recombinant expression of full length CYP6BQ23 along with cytochrome P450 reductase in an insect (Sf9) cell line showed that it is able to efficiently metabolise deltamethrin to 4-hydroxy deltamethrin. Furthermore we demonstrated by detection of 4-hydroxy tau-fluvalinate using ESI-TOF MS/MS that functionally expressed CYP6BQ23 also metabolizes tau-fluvalinate. A protein model was generated and subsequent docking simulations revealed the predicted substrate-binding mode of both deltamethrin and tau-fluvalinate to CYP6BQ23. Taken together these results strongly suggest that the overexpression of CYP6BQ23 is the primary mechanism conferring pyrethroid resistance in pollen beetle populations throughout much of Europe