title: The role of Rdl in resistance to phenylpyrazoles in Drosophila melanogaster.
abstract: Extensive use of older generation insecticides may result in pre-existing cross-resistance to new chemical classes acting at the same target site. Phenylpyrazole insecticides block inhibitory neurotransmission in insects via their action on ligand-gated chloride channels (LGCCs). Phenylpyrazoles are broad-spectrum insecticides widely used in agriculture and domestic pest control. So far, all identified cases of target site resistance to phenylpyrazoles are based on mutations in the Rdl (Resistance to dieldrin) LGCC subunit, the major target site for cyclodiene insecticides. We examined the role that mutations in Rdl have on phenylpyrazole resistance in Drosophila melanogaster, exploring naturally occurring variation, and generating predicted resistance mutations by mutagenesis. Natural variation at the Rdl locus in inbred strains of D. melanogaster included gene duplication, and a line containing two Rdl mutations found in a highly resistant line of Drosophila simulans. These mutations had a moderate impact on survival following exposure to two phenylpyrazoles, fipronil and pyriprole. Homology modelling suggested that the Rdl chloride channel pore contains key residues for binding fipronil and pyriprole. Mutagenesis of these sites and assessment of resistance in vivo in transgenic lines showed that amino acid identity at the Ala(301) site influenced resistance levels, with glycine showing greater survival than serine replacement. We confirm that point mutations at the Rdl 301 site provide moderate resistance to phenylpyrazoles in D. melanogaster. We also emphasize the beneficial aspects of testing predicted mutations in a whole organism to validate a candidate gene approach.
authors.affiliation: Department of Genetics and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, VIC 3052, Australia; School of Biological Sciences, University of Sydney, Sydney, NSW 2006, Australia. Electronic address: emily.remnant@sydney.edu.au.
authors.forename: Emily J
authors.initials: EJ
authors.lastname: Remnant
authors.affiliation: Australian Cancer Research Foundation Rational Drug Discovery Centre, St Vincent's Institute of Medical Research, Fitzroy, VIC 3056, Australia.
authors.forename: Craig J
authors.initials: CJ
authors.lastname: Morton
authors.affiliation: Department of Genetics and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, VIC 3052, Australia.
authors.forename: Phillip J
authors.initials: PJ
authors.lastname: Daborn
authors.affiliation: Department of Genetics and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, VIC 3052, Australia.
authors.forename: Christopher
authors.initials: C
authors.lastname: Lumb
authors.affiliation: Department of Genetics and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, VIC 3052, Australia.
authors.forename: Ying Ting
authors.initials: YT
authors.lastname: Yang
authors.affiliation: Australian Cancer Research Foundation Rational Drug Discovery Centre, St Vincent's Institute of Medical Research, Fitzroy, VIC 3056, Australia.
authors.forename: Hooi Ling
authors.initials: HL
authors.lastname: Ng
authors.affiliation: Australian Cancer Research Foundation Rational Drug Discovery Centre, St Vincent's Institute of Medical Research, Fitzroy, VIC 3056, Australia; Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, VIC 3052, Australia.
authors.forename: Michael W
authors.initials: MW
authors.lastname: Parker
authors.affiliation: Department of Genetics and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, VIC 3052, Australia.
authors.forename: Philip
authors.initials: P
authors.lastname: Batterham
mesh_terms: D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D018118:Chloride Channels; D004026:Dieldrin; D029721:Drosophila Proteins / Q000235:genetics* / Q000378:metabolism; D004331:Drosophila melanogaster / Q000187:drug effects* / Q000235:genetics*; D020440:Gene Duplication; D007305:Insecticide Resistance / Q000235:genetics*; D007306:Insecticides / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009154:Mutation / Q000187:drug effects; D017354:Point Mutation; D011720:Pyrazoles / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D011963:Receptors, GABA-A / Q000235:genetics* / Q000378:metabolism
chemical_list: D018118:Chloride Channels; D029721:Drosophila Proteins; D007306:Insecticides; D011720:Pyrazoles; D011725:Pyridines; C082030:Rdl protein, Drosophila; D011963:Receptors, GABA-A; C532951:pyriprole; D004026:Dieldrin; D000409:Alanine; C082360:fipronil
keywords: Drosophila melanogaster; Fipronil; Insecticide resistance; Ligand gated chloride channel; Pyriprole; Rdl; Transgenic; homology modelling