title: Resistance mutation conserved between insects and mites unravels the benzoylurea insecticide mode of action on chitin biosynthesis.
abstract: Despite the major role of chitin biosynthesis inhibitors such as benzoylureas (BPUs) in the control of pests in agricultural and public health for almost four decades, their molecular mode of action (MoA) has in most cases remained elusive. BPUs interfere with chitin biosynthesis and were thought to interact with sulfonylurea receptors that mediate chitin vesicle transport. Here, we uncover a mutation (I1042M) in the chitin synthase 1 (CHS1) gene of BPU-resistant Plutella xylostella at the same position as the I1017F mutation reported in spider mites that confers etoxazole resistance. Using a genome-editing CRISPR/Cas9 approach coupled with homology-directed repair (HDR) in Drosophila melanogaster, we introduced both substitutions (I1056M/F) in the corresponding fly CHS1 gene (kkv). Homozygous lines bearing either of these mutations were highly resistant to etoxazole and all tested BPUs, as well as buprofezin-an important hemipteran chitin biosynthesis inhibitor. This provides compelling evidence that BPUs, etoxazole, and buprofezin share in fact the same molecular MoA and directly interact with CHS. This finding has immediate effects on resistance management strategies of major agricultural pests but also on mosquito vectors of serious human diseases such as Dengue and Zika, as diflubenzuron, the standard BPU, is one of the few effective larvicides in use. The study elaborates on how genome editing can directly, rapidly, and convincingly elucidate the MoA of bioactive molecules, especially when target sites are complex and hard to reconstitute in vitro.
authors.affiliation: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas, GR-70013 Heraklion, Crete, Greece.|Laboratory of Molecular Entomology, Department of Biology, University of Crete, GR-70013 Heraklion, Crete, Greece.
authors.forename: Vassilis
authors.identifier: 0000-0003-4608-7482
authors.initials: V
authors.lastname: Douris
authors.affiliation: Bayer CropScience AG, R&D Pest Control Biology, D-40789 Mannheim, Germany.|Developmental Biology, Department of Biology, Martin-Luther University Halle-Wittenberg, 06120 Halle, Germany.
authors.forename: Denise
authors.initials: D
authors.lastname: Steinbach
authors.affiliation: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas, GR-70013 Heraklion, Crete, Greece.|Laboratory of Molecular Entomology, Department of Biology, University of Crete, GR-70013 Heraklion, Crete, Greece.
authors.forename: Rafaela
authors.initials: R
authors.lastname: Panteleri
authors.affiliation: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas, GR-70013 Heraklion, Crete, Greece.
authors.forename: Ioannis
authors.initials: I
authors.lastname: Livadaras
authors.affiliation: Department of Biological Chemistry and Crop Protection, Rothamsted Research, Hertfordshire, Harpenden AL5 2JQ, United Kingdom; john.pickett@rothamsted.ac.uk vontas@imbb.forth.gr ralf.nauen@bayer.com.
authors.forename: John Anthony
authors.identifier: 0000-0002-8386-3770
authors.initials: JA
authors.lastname: Pickett
authors.affiliation: Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, 1098 XH Amsterdam, The Netherlands.|Laboratory of Agrozoology, Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, 9000 Ghent, Belgium.
authors.forename: Thomas
authors.initials: T
authors.lastname: Van Leeuwen
authors.affiliation: Bayer CropScience AG, R&D Pest Control Biology, D-40789 Mannheim, Germany; john.pickett@rothamsted.ac.uk vontas@imbb.forth.gr ralf.nauen@bayer.com.
authors.forename: Ralf
authors.initials: R
authors.lastname: Nauen
authors.affiliation: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas, GR-70013 Heraklion, Crete, Greece; john.pickett@rothamsted.ac.uk vontas@imbb.forth.gr ralf.nauen@bayer.com.|Laboratory of Pesticide Science, Department of Crop Science, Agricultural University of Athens, GR-11855 Athens, Greece.
authors.forename: John
authors.initials: J
authors.lastname: Vontas
mesh_terms: D000383:Agriculture; D000818:Animals; D001681:Biological Assay; D064113:CRISPR-Cas Systems; D002686:Chitin / Q000737:chemistry*; D005091:Exons; D000072669:Gene Editing; D049750:Genome, Insect; D005838:Genotype; D006720:Homozygote; D007313:Insecta / Q000235:genetics*; D007305:Insecticide Resistance / Q000235:genetics*; D007306:Insecticides; D007814:Larva; D008925:Mites / Q000235:genetics*; D009154:Mutation; D009711:Nucleotides; D010080:Oxazoles / Q000737:chemistry; D010802:Phylogeny; D017422:Sequence Analysis, DNA; D064233:Sulfonylurea Receptors; D013829:Thiadiazines / Q000737:chemistry; D014508:Urea / Q000737:chemistry*
chemical_list: D007306:Insecticides; D009711:Nucleotides; D010080:Oxazoles; D064233:Sulfonylurea Receptors; D013829:Thiadiazines; D002686:Chitin; C083997:buprofezin; D014508:Urea; C508136:etoxazole
keywords: CRISPR/Cas9; benzoylureas; insecticide resistance; mosquito control; resistance management