Insecticides of the tetronic/tetramic acid family (cyclic ketoenols) are widely used to control sucking pests such as whiteflies, aphids and mites. They act as inhibitors of acetyl-CoA carboxylase (ACC), a key enzyme for lipid biosynthesis across taxa. While it is well documented that plant ACCs targeted by herbicides have developed resistance associated with mutations at the carboxyltransferase (CT) domain, resistance to ketoenols in invertebrate pests has been previously associated either with metabolic resistance or with non-validated candidate mutations in different ACC domains. A recent study revealed high levels of spiromesifen and spirotetramat resistance in Spanish field populations of the whitefly Bemisia tabaci that was not thought to be associated with metabolic resistance. We confirm the presence of high resistance levels (up to >640-fold) against ketoenol insecticides in both Spanish and Australian B. tabaci strains of the MED and MEAM1 species, respectively. RNAseq analysis revealed the presence of an ACC variant bearing a mutation that results in an amino acid substitution, A2083V, in a highly conserved region of the CT domain. F1 progeny resulting from reciprocal crosses between susceptible and resistant lines are almost fully resistant, suggesting an autosomal dominant mode of inheritance. In order to functionally investigate the contribution of this mutation and other candidate mutations previously reported in resistance phenotypes, we used CRISPR/Cas9 to generate genome modified Drosophila lines. Toxicity bioassays using multiple transgenic fly lines confirmed that A2083V causes high levels of resistance to commercial ketoenols. We therefore developed a pyrosequencing-based diagnostic assay to map the spread of the resistance alleles in field-collected samples from Spain. Our screening confirmed the presence of target-site resistance in numerous field-populations collected in Sevilla, Murcia and Almeria. This emphasizes the importance of implementing appropriate resistance management strategies to prevent or slow the spread of resistance through global whitefly populations.

中文翻译：

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烟粉虱中与酮烯醇抗性相关的新型乙酰辅酶A羧化酶突变的鉴定和功能表征

tetronic/tetramic acid 家族的杀虫剂（环状酮烯醇）被广泛用于控制吸吮害虫，如粉虱、蚜虫和螨虫。它们充当乙酰辅酶 A 羧化酶 (ACC) 的抑制剂，ACC 是跨类群脂质生物合成的关键酶。虽然有充分证据表明，除草剂靶向的植物 ACC 已经产生了与羧基转移酶 (CT) 域突变相关的抗性，但无脊椎动物害虫对酮烯醇的抗性先前已与代谢抗性或不同 ACC 域中未经验证的候选突变相关. 最近的一项研究表明，西班牙粉虱 Bemisia 烟粉虱的西班牙田间种群对螺蛉和螺虫乙酯具有高水平的抗性，这被认为与代谢抗性无关。我们确认存在高阻力水平（高达 > 640 倍）分别对抗西班牙和澳大利亚烟粉虱 MED 和 MEAM1 物种中的酮烯醇杀虫剂。RNAseq 分析揭示了 ACC 变异体的存在，该变异体在 CT 结构域的高度保守区域中存在导致氨基酸取代 A2083V 的突变。易感品系和抗性品系之间相互杂交产生的 F1 后代几乎完全具有抗性，这表明是常染色体显性遗传模式。为了从功能上研究该突变和先前在抗性表型中报告的其他候选突变的贡献，我们使用 CRISPR/Cas9 生成基因组修饰的果蝇系。使用多个转基因果蝇系的毒性生物测定证实 A2083V 会导致对商业酮烯醇的高水平抗性。因此，我们开发了一种基于焦磷酸测序的诊断分析，以绘制西班牙现场采集样本中抗性等位基因的分布图。我们的筛选证实了在塞维利亚、穆尔西亚和阿尔梅里亚收集的众多田间种群中存在目标位点抗性。这强调了实施适当的抗药性管理策略以防止或减缓抗药性在全球粉虱种群中传播的重要性。