Remnant Emily J, Morton Craig J, Daborn Phillip J, Lumb Christopher, Yang Ying Ting, Ng Hooi Ling, Parker Michael W, Batterham Philip
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.
Australian Cancer Research Foundation Rational Drug Discovery Centre, St Vincent's Institute of Medical Research, Fitzroy, VIC 3056, Australia.
Insect Biochem Mol Biol. 2014 Nov;54:11-21. doi: 10.1016/j.ibmb.2014.08.008. Epub 2014 Sep 1.
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.
广泛使用老一代杀虫剂可能导致对作用于同一靶位点的新型化学类别预先存在交叉抗性。苯基吡唑类杀虫剂通过作用于配体门控氯离子通道（LGCCs）来阻断昆虫体内的抑制性神经传递。苯基吡唑类是广泛用于农业和家庭害虫防治的广谱杀虫剂。到目前为止，所有已确定的对苯基吡唑类靶位点抗性的案例都是基于Rdl（对狄氏剂抗性）LGCC亚基的突变，Rdl是环二烯类杀虫剂的主要靶位点。我们研究了Rdl突变在黑腹果蝇对苯基吡唑类抗性中的作用，探索自然发生的变异，并通过诱变产生预测的抗性突变。黑腹果蝇近交系中Rdl位点的自然变异包括基因重复，以及在拟果蝇的一个高抗性品系中发现的含有两个Rdl突变的品系。这些突变对暴露于两种苯基吡唑类杀虫剂氟虫腈和吡丙醚后的存活率有中等程度的影响。同源建模表明，Rdl氯离子通道孔包含与氟虫腈和吡丙醚结合的关键残基。对这些位点进行诱变并评估转基因品系体内的抗性，结果表明Ala(301)位点的氨基酸同一性影响抗性水平，甘氨酸替代丝氨酸时存活率更高。我们证实，Rdl 301位点的点突变在黑腹果蝇中对苯基吡唑类提供中等抗性。我们还强调了在整个生物体中测试预测突变以验证候选基因方法的有益方面。
