title: Cytochrome P450 associated with insecticide resistance catalyzes cuticular hydrocarbon production in Anopheles gambiae.
abstract: The role of cuticle changes in insecticide resistance in the major malaria vector Anopheles gambiae was assessed. The rate of internalization of (14)C deltamethrin was significantly slower in a resistant strain than in a susceptible strain. Topical application of an acetone insecticide formulation to circumvent lipid-based uptake barriers decreased the resistance ratio by ∼50%. Cuticle analysis by electron microscopy and characterization of lipid extracts indicated that resistant mosquitoes had a thicker epicuticular layer and a significant increase in cuticular hydrocarbon (CHC) content (∼29%). However, the CHC profile and relative distribution were similar in resistant and susceptible insects. The cellular localization and in vitro activity of two P450 enzymes, CYP4G16 and CYP4G17, whose genes are frequently overexpressed in resistant Anopheles mosquitoes, were analyzed. These enzymes are potential orthologs of the CYP4G1/2 enzymes that catalyze the final step of CHC biosynthesis in Drosophila and Musca domestica, respectively. Immunostaining indicated that both CYP4G16 and CYP4G17 are highly abundant in oenocytes, the insect cell type thought to secrete hydrocarbons. However, an intriguing difference was indicated; CYP4G17 occurs throughout the cell, as expected for a microsomal P450, but CYP4G16 localizes to the periphery of the cell and lies on the cytoplasmic side of the cell membrane, a unique position for a P450 enzyme. CYP4G16 and CYP4G17 were functionally expressed in insect cells. CYP4G16 produced hydrocarbons from a C18 aldehyde substrate and thus has bona fide decarbonylase activity similar to that of dmCYP4G1/2. The data support the hypothesis that the coevolution of multiple mechanisms, including cuticular barriers, has occurred in highly pyrethroid-resistant An gambiae.
authors.affiliation: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Heraklion 70013, Greece; Department of Biology, University of Crete, Vassilika Vouton, Heraklion 70013, Greece;
authors.forename: Vasileia
authors.initials: V
authors.lastname: Balabanidou
authors.affiliation: Department of Biology, University of Crete, Vassilika Vouton, Heraklion 70013, Greece;
authors.forename: Anastasia
authors.initials: A
authors.lastname: Kampouraki
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Nevada, Reno, NV 89557;
authors.forename: Marina
authors.initials: M
authors.lastname: MacLean
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Nevada, Reno, NV 89557;
authors.forename: Gary J
authors.initials: GJ
authors.lastname: Blomquist
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Nevada, Reno, NV 89557;
authors.forename: Claus
authors.initials: C
authors.lastname: Tittiger
authors.affiliation: Instituto de Investigaciones Bioquímicas de La Plata, Centro Científico Tecnológico La Plata, Consejo Nacional de Investigaciones Científicas y Técnicas - Facultad de Ciencias Médicas, Universidad Nacional de La Plata, La Plata 1900, Argentina;
authors.forename: M Patricia
authors.initials: MP
authors.lastname: Juárez
authors.affiliation: Instituto de Investigaciones Bioquímicas de La Plata, Centro Científico Tecnológico La Plata, Consejo Nacional de Investigaciones Científicas y Técnicas - Facultad de Ciencias Médicas, Universidad Nacional de La Plata, La Plata 1900, Argentina;
authors.forename: Sergio J
authors.initials: SJ
authors.lastname: Mijailovsky
authors.affiliation: Department of Biology, University of Crete, Vassilika Vouton, Heraklion 70013, Greece;
authors.forename: George
authors.initials: G
authors.lastname: Chalepakis
authors.affiliation: Department of Biology, University of Crete, Vassilika Vouton, Heraklion 70013, Greece;
authors.forename: Amalia
authors.initials: A
authors.lastname: Anthousi
authors.affiliation: Department of Vector Biology, Liverpool School of Tropical Medicine, Liverpool L3 5QA, United Kingdom;
authors.forename: Amy
authors.initials: A
authors.lastname: Lynd
authors.affiliation: Department of Vector Biology, Liverpool School of Tropical Medicine, Liverpool L3 5QA, United Kingdom;
authors.forename: Sanou
authors.initials: S
authors.lastname: Antoine
authors.affiliation: Department of Vector Biology, Liverpool School of Tropical Medicine, Liverpool L3 5QA, United Kingdom; janet.hemingway@lstmed.ac.uk vontas@imbb.forth.gr.
authors.forename: Janet
authors.initials: J
authors.lastname: Hemingway
authors.affiliation: Department of Vector Biology, Liverpool School of Tropical Medicine, Liverpool L3 5QA, United Kingdom;
authors.forename: Hilary
authors.initials: H
authors.lastname: Ranson
authors.affiliation: Department of Vector Biology, Liverpool School of Tropical Medicine, Liverpool L3 5QA, United Kingdom;
authors.forename: Gareth J
authors.initials: GJ
authors.lastname: Lycett
authors.affiliation: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Heraklion 70013, Greece; Pesticide Science Laboratory, Department of Crop Science, Agricultural University of Athens, 11855 Athens, Greece janet.hemingway@lstmed.ac.uk vontas@imbb.forth.gr.
authors.forename: John
authors.initials: J
authors.lastname: Vontas
mesh_terms: D000818:Animals; D000852:Anopheles / Q000378:metabolism*; D002384:Catalysis; D003577:Cytochrome P-450 Enzyme System / Q000502:physiology*; D005260:Female; D006838:Hydrocarbons / Q000378:metabolism*; D007305:Insecticide Resistance; D009570:Nitriles / Q000493:pharmacokinetics; D011722:Pyrethrins / Q000493:pharmacokinetics
chemical_list: D006838:Hydrocarbons; D009570:Nitriles; D011722:Pyrethrins; C017180:decamethrin; D003577:Cytochrome P-450 Enzyme System
keywords: cytochrome P450; hydrocarbons; insecticide resistance; malaria; mosquito cuticle