Faculty of Life and Environmental Sciences, Shimane University, Matsue, Shimane 690-8504, Japan; Interdisciplinary Institute for Science Research, Organization for Research and Academic Information, Shimane University, Matsue, Shimane 690-8504, Japan.
Agrochemicals Research Center, Mitsui Chemicals Crop and Life Solutions, Inc, Mobara, Chiba 297-0017, Japan.
Pestic Biochem Physiol. 2024 Feb;199:105776. doi: 10.1016/j.pestbp.2024.105776. Epub 2024 Jan 11.
γ-Aminobutyric acid receptors (GABARs) are crucial targets for pest control chemicals, including meta-diamide and isoxazoline insecticides, which act as negative allosteric modulators of insect GABARs. Previous cell-based assays have indicated that amino acid residues in the transmembrane cavity between adjacent subunits of Drosophila RDL GABAR (i.e., Ile276, Leu280, and Gly335) are involved in mediating the action of meta-diamides. In this study, to confirm this result at the organismal level, we employed CRISPR/Cas9-mediated genome editing, generated six transgenic Drosophila strains carrying substitutions in these amino acid residues, and investigated their sensitivity to broflanilide and isocycloseram. Flies homozygous for the I276F mutation did not exhibit any change in sensitivity to the tested insecticides compared to the control flies. Conversely, I276C homozygosity was lethal, and heterozygous flies exhibited ∼2-fold lower sensitivity to broflanilide than the control flies. Flies homozygous for the L280C mutation survived into adulthood but exhibited infertility. Both heterozygous and homozygous L280C flies exhibited ∼3- and ∼20-fold lower sensitivities to broflanilide and isocycloseram, respectively, than the control flies. The reduction in sensitivity to isocycloseram in L280C flies diminished to ∼3-fold when treated with piperonyl butoxide. Flies homozygous for the G335A mutation reached the adult stage. However, they were sterile, had small bodies, and exhibited reduced locomotion, indicating the critical role of Gly335 in RDL function. These flies exhibited markedly increased tolerance to topically applied broflanilide and isocycloseram, demonstrating that the conserved Gly335 is the target of the insecticidal actions of broflanilide and isocycloseram. Considering the significant fitness costs, the Gly335 mutation may not pose a serious risk for the development of resistance in field populations of insect pests. However, more careful studies using insect pests are needed to investigate whether our perspective applies to resistance development under field conditions.
γ-氨基丁酸受体（GABARs）是害虫防治化学品的重要靶标，包括甲脒类和异噁唑啉类杀虫剂，它们作为昆虫 GABARs 的负变构调节剂。先前的基于细胞的测定表明，果蝇 RDL GABAR 相邻亚基之间跨膜腔中的氨基酸残基（即 Ile276、Leu280 和 Gly335）参与了甲脒类药物的作用。在这项研究中，为了在机体水平上证实这一结果，我们采用了 CRISPR/Cas9 介导的基因组编辑，生成了六个携带这些氨基酸残基取代的转基因果蝇品系，并研究了它们对溴氟虫酰胺和异噁唑啉的敏感性。与对照果蝇相比，I276F 突变的纯合子对测试杀虫剂的敏感性没有任何变化。相反，I276C 纯合子是致命的，杂合子对溴氟虫酰胺的敏感性比对照果蝇低约 2 倍。L280C 突变的纯合子果蝇存活至成年，但表现出不育。杂合子和纯合子 L280C 果蝇对溴氟虫酰胺和异噁唑啉的敏感性分别比对照果蝇低约 3 倍和 20 倍。L280C 果蝇对异噁唑啉的敏感性降低到约 3 倍，当用增效醚处理时。G335A 突变的纯合子果蝇达到了成年阶段。然而，它们不育，体型较小，运动能力降低，表明 Gly335 在 RDL 功能中起关键作用。这些果蝇对局部施用的溴氟虫酰胺和异噁唑啉表现出明显增加的耐受性，表明保守的 Gly335 是溴氟虫酰胺和异噁唑啉杀虫作用的靶标。考虑到显著的适应性成本，Gly335 突变可能不会对田间害虫种群产生抗药性的发展构成严重威胁。然而，需要使用昆虫进行更仔细的研究，以调查我们的观点是否适用于田间条件下的抗药性发展。
