title: Functional characterization of the Tetranychus urticae CYP392A11, a cytochrome P450 that hydroxylates the METI acaricides cyenopyrafen and fenpyroximate.
abstract: Cyenopyrafen is a Mitochondrial Electron Transport Inhibitor (METI) acaricide with a novel mode of action at complex II, which has been recently developed for the control of the spider mite Tetranychus urticae, a pest of eminent importance globally. However, some populations of T. urticae are cross-resistant to this molecule, and cyenopyrafen resistance can be readily selected in the lab. The cytochrome P450s genes CYP392A11 and CYP392A12 have been strongly associated with the phenotype. We expressed the CYP392A11 and the CYP392A12 genes with T. urticae cytochrome P450 reductase (CPR) in Escherichia coli. CYP392A12 was expressed predominately as an inactive form, witnessed by a peak at P420, despite optimization efforts on expression conditions. However, expression of CYP392A11 produced a functional enzyme, with high activity and preference for the substrates Luciferin-ME EGE and ethoxycoumarin. CYP392A11 catalyses the conversion of cyenopyrafen to a hydroxylated analogue (kcat = 2.37 pmol/min/pmol P450), as well as the hydroxylation of fenpyroximate (kcat = 1.85 pmol/min/pmol P450). In addition, transgenic expression of CYP392A11 in Drosophila melanogaster, in conjunction with TuCPR, confers significant levels of fenpyroximate resistance. The overexpression of CYP392A11 in multi-resistant T. urticae strains, not previously exposed to cyenopyrafen, which had been indicated by microarray studies, was confirmed by qPCR, and it was correlated with significant levels of cyenopyrafen and fenpyroximate cross-resistance. The implications of our findings for insecticide resistance management strategies are discussed.
authors.affiliation: Institute of Molecular Biology & Biotechnology, Foundation for Research & Technology Hellas, 100 N. Plastira Street, GR-700 13 Heraklion, Crete, Greece; Department of Biology, University of Crete, 71409 Heraklion, Greece.
authors.forename: M
authors.initials: M
authors.lastname: Riga
authors.affiliation: Environmental Chemical Processes Laboratory (ECPL), Department of Chemistry, University of Crete, 71003 Heraklion, Greece.
authors.forename: A
authors.initials: A
authors.lastname: Myridakis
authors.affiliation: Institute of Molecular Biology & Biotechnology, Foundation for Research & Technology Hellas, 100 N. Plastira Street, GR-700 13 Heraklion, Crete, Greece; Department of Biology, University of Crete, 71409 Heraklion, Greece.
authors.forename: D
authors.initials: D
authors.lastname: Tsakireli
authors.affiliation: Institute of Molecular Biology & Biotechnology, Foundation for Research & Technology Hellas, 100 N. Plastira Street, GR-700 13 Heraklion, Crete, Greece; Department of Biology, University of Crete, 71409 Heraklion, Greece.
authors.forename: E
authors.initials: E
authors.lastname: Morou
authors.affiliation: Environmental Chemical Processes Laboratory (ECPL), Department of Chemistry, University of Crete, 71003 Heraklion, Greece.
authors.forename: E G
authors.initials: EG
authors.lastname: Stephanou
authors.affiliation: Bayer CropScience AG, R&D Pest Control Biology, Alfred Nobel Str. 50, D-40789 Monheim, Germany.
authors.forename: R
authors.initials: R
authors.lastname: Nauen
authors.affiliation: Institute for Biodiversity and Ecosystem Dynamics (IBED), University of Amsterdam (UvA), Science Park 904, 1098 XH Amsterdam, The Netherlands; Laboratory of Agrozoology, Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, 9000 Ghent, Belgium.
authors.forename: T
authors.initials: T
authors.lastname: Van Leeuwen
authors.affiliation: Institute of Molecular Biology & Biotechnology, Foundation for Research & Technology Hellas, 100 N. Plastira Street, GR-700 13 Heraklion, Crete, Greece; Department of Biology, University of Crete, 71409 Heraklion, Greece.
authors.forename: V
authors.initials: V
authors.lastname: Douris
authors.affiliation: Institute of Molecular Biology & Biotechnology, Foundation for Research & Technology Hellas, 100 N. Plastira Street, GR-700 13 Heraklion, Crete, Greece; Laboratory of Pesticide Science, Department of Crop Science, Agricultural University of Athens, 75 Iera Odos Street, GR-11855 Athens, Greece. Electronic address: vontas@imbb.forth.gr.
authors.forename: J
authors.initials: J
authors.lastname: Vontas
mesh_terms: D056810:Acaricides / Q000378:metabolism* / Q000494:pharmacology; D000181:Acrylonitrile / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D060829:Arthropod Proteins / Q000235:genetics / Q000378:metabolism*; D001565:Benzoates / Q000378:metabolism* / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000187:drug effects; D008658:Inactivation, Metabolic; D007305:Insecticide Resistance; D011720:Pyrazoles / Q000378:metabolism* / Q000494:pharmacology; D040102:Tetranychidae / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics
chemical_list: D056810:Acaricides; D060829:Arthropod Proteins; D001565:Benzoates; D011720:Pyrazoles; C000591892:cyenopyrafen; D003577:Cytochrome P-450 Enzyme System; C415144:fenpyroximate; D000181:Acrylonitrile
keywords: Cyenopyrafen; Cytochrome P450; Detoxification; Diagnostic; Insecticide resistance; Transgenic drosophila