吡虫啉抗性棉蚜对氟啶虫胺腈敏感的毒理学机制

Neonicotinoid, such as imidacloprid is a kind of important insecticides to control sucking insect pests. However, resistance to neonicotinoids has been identified in several pest species. Our early studies indicate that there is no cross resistance between sulfoxaflor and imidacloprid. The P450 single oxygenase activity is improved, the relative expression level is increased and the nAChR β1 subunit is mutated (R81T) in imidacloprid-resistant A. gossypii. But these changes show no influence to the insecticidal activity of sulfoxaflor. The project will express A. gossypii CYP6CY3 gene in vitro, detect the metabolic product of sulfoxaflor, analyze the metabolism of sulfoxaflor and imidacloprid and compare the metabolism ability to sulfoxaflor between susceptible and resistant A. gossypii. And the fluorescent labeling experiments was carried out to determine the effect of nAChR mutation on sulfoxaflor and imidacloprid binding ability. The implementation of this project will clarify the toxicological mechanism of sulfoxaflor antagonizing imidacloprid-resistance, and will provides the theoretical foundation for the pest resistance management.

吡虫啉等新烟碱杀虫剂是一类控制刺吸式口器害虫的重要杀虫剂，但是害虫对该类药剂的抗药性已普遍发生。我们对实验室所选育的吡虫啉抗性品系棉蚜的研究发现：氟啶虫胺腈与吡虫啉不存在交互抗性；该品系棉蚜P450单加氧酶活性的提高，抗性相关基因CYP6CY3表达量的上调以及nAChR β1亚基R81T突变并未降低氟啶虫胺腈的杀虫活性。基于此，本项目拟体外表达棉蚜CYP6CY3基因，检测氟啶虫胺腈的代谢产物，分析CYP6CY3对氟啶虫胺腈和吡虫啉的代谢水平，比较敏感棉蚜和吡虫啉抗性棉蚜对氟啶虫胺腈解毒代谢能力的差异；进一步利用荧光偏振法测定氟啶虫胺腈与敏感棉蚜和吡虫啉抗性棉蚜nAChR的亲和力和结合量，明确棉蚜nAChR β1亚基R81T突变对氟啶虫胺腈和吡虫啉靶标结合能力的影响。从而揭示吡虫啉抗性棉蚜对氟啶虫胺腈敏感的毒理学机制，为更有效的开展害虫抗药性治理提供理论基础。

前期研究结果发现氟啶虫胺腈与吡虫啉不存在交互抗性；吡虫啉抗性棉蚜P450单加氧酶活性的提高，抗性相关P450基因表达量的上调以及nAChR β1亚基R81T突变并未显著降低氟啶虫胺腈的杀虫活性。基于此本项目建立了棉蚜代谢酶对吡虫啉及氟啶虫胺腈离体代谢方法。结果表明：吡虫啉抗性棉蚜对吡虫啉的解毒代谢能力大大提高；同等时间吡虫啉抗性品系棉蚜对吡虫啉的代谢量显著高于敏感棉蚜，药后12h和24h，吡虫啉抗性棉蚜对吡虫啉的代谢量分别是敏感种群的3.9倍和4.4倍。然而，吡虫啉抗性棉蚜对氟啶虫胺腈的代谢能力却没有明显变化；药后1~24h，吡虫啉抗性品系棉蚜对氟啶虫胺腈的代谢量与敏感棉蚜没有显著差异。通过棉蚜nAChRs 3D模型构建及Autodock6分子对接，预测了吡虫啉和氟啶虫胺腈与野生型及R81T突变型棉蚜nAChRs的靶标结合。结果表明：R81T突变对吡虫啉与受体蛋白结合的影响更显著。从可视化的氢键数量来看，突变前吡虫啉与受体蛋白的氢键键长2.8，突变后氢键作用力消失。然而，该位点突变对氟啶虫胺腈R和S体的影响均较小，表现为氢键键长由3.0变为2.9或由2.6变为2.8。而且，药剂与靶标蛋白的结合作用研究结果表明：R81T突变造成受体蛋白与吡虫啉的结合能力下降，结合常数KD值由1.742×10-5M上升至1.761×10-5M；然而，该靶标突变并未造成氟啶虫胺腈与受体蛋白的结合能力下降，反而结合常数KD值从1.765×10-5M下降至1.703×10-5M；因此，R81T靶标突变尚不能对氟啶虫胺腈与受体蛋白的结合产生抑制作用。本研究从解毒代谢水平和靶标结合两个方面揭示了氟啶虫胺腈拮抗棉蚜对吡虫啉抗性的毒理学机制，为更有效的开展害虫抗药性治理提供了理论基础。