title: Molecular and functional characterization of CYP6BQ23, a cytochrome P450 conferring resistance to pyrethroids in European populations of pollen beetle, Meligethes aeneus.
abstract: 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.
authors: Bayer CropScience AG, R&D, Pest Control Biology, Monheim, Germany; University of Hohenheim, Institute of Phytomedicine, Dept. Applied Entomology, Stuttgart, Germany; Rothamsted Research, Biological Chemistry and Crop Protection Dept., Harpenden, Herts, UK.
authors: Christoph T
authors: CT
authors: Zimmer
authors: Rothamsted Research, Biological Chemistry and Crop Protection Dept., Harpenden, Herts, UK.
authors: Chris
authors: C
authors: Bass
authors: Rothamsted Research, Biological Chemistry and Crop Protection Dept., Harpenden, Herts, UK.
authors: Martin S
authors: MS
authors: Williamson
authors: Bayer CropScience AG, R&D, Research Technologies, Monheim, Germany.
authors: Martin
authors: M
authors: Kaussmann
authors: Bayer CropScience AG, R&D, Pest Control Biology, Monheim, Germany.
authors: Katharina
authors: K
authors: Wölfel
authors: Bayer CropScience AG, R&D, Research Technologies, Monheim, Germany.
authors: Oliver
authors: O
authors: Gutbrod
authors: Bayer CropScience AG, R&D, Pest Control Biology, Monheim, Germany. Electronic address: ralf.nauen@bayer.com.
authors: Ralf
authors: R
authors: Nauen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001517:Coleoptera / Q000235:genetics*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D018628:Gene Dosage; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D007305:Insecticide Resistance / Q000235:genetics*; D007306:Insecticides / Q000737:chemistry; D008969:Molecular Sequence Data; D011722:Pyrethrins / Q000737:chemistry; D020539:Sequence Analysis, Protein
chemical_list: D019476:Insect Proteins; D007306:Insecticides; D011722:Pyrethrins; D003577:Cytochrome P-450 Enzyme System
keywords: Coleoptera; Cytochrome P450; Meligethes; Pollen beetle; Pyrethroids; Resistance