淡色库蚊嗅觉受体介导高效氯氰菊酯抗性机制的研究

Olfactory systems play critical roles in mediating a variety of important behaviors in mosquitoes, including host seeking, oviposition and avoiding adverse environment. Studies have shown that resistant mosquitoes can use olfactory systems identity and avoid pesticides, but the specific mechanism is still unknown. We discovered that there were differences in the types and expression of olfactory receptors (ORs) from beta-cypermethrin resistant strains and sensitive strains of Culex pipiens pallens. Moreover, point mutation occours in odorant receptor co-receptor (Orco) from wild, sensitive and beta-cypermethrin resistant strains. Thus, we propose that ORs are able to identify the pesticides volatiles and participate in the formation of resistance. This project is to: Screen of differentially expressed ORs by transcriptome profiles of beta-cypermethrin resistant and sensitive mosquitoes. Study the correlation between Orco genotype and resistant phenotype and its effects on signal transduction of insecticide identification. Analyze the interaction between odorant receptors (ORx) and Orco gene on the identification and transmission of odor signals in mosquitoes and two heterologous expression systems (HEK293 cells and transgenic Drosophila). This project will study on the mechanism of olfactory receptors mediated resistance of mosquitoes, which is helpful to clarify mosquito resistance mechanism and the function of olfactory receptors, and is a new method to interfere mosquito behaviors, thus providing a theoretical basis for the effective improvement of chemical prevention and control measures, and laying scientific evidence for the preventive measures.

蚊虫寻找宿主、产卵地、躲避不利环境等行为主要依赖于嗅觉系统，研究显示抗性蚊虫能利用嗅觉识别并回避杀虫剂，但具体机制不详。我们研究发现高效氯氰菊酯抗性品系和敏感品系的淡色库蚊嗅觉受体(ORs)的种类和表达量存在差异，且不同品系的嗅觉受体共表达受体（Orco）基因存在点突变。因此，我们提出疑问ORs是否能够识别杀虫剂的气味，参与抗药性的形成呢?本项目通过抗性与敏感品系淡色库蚊的转录组分析筛选差异表达的ORs；研究Orco基因型与抗性表型的相关性及其对杀虫剂识别信号传递的影响；从异源表达系统（细胞和转基因果蝇）以及蚊虫层面解析配体型嗅觉受体（ORx）与Orco基因共同作用识别传递气味信号，探讨蚊虫抗药性形成的机制。本项目通过嗅觉受体介导蚊虫抗药性产生机理的深入研究，将有助于明晰蚊虫抗药性机理和嗅觉受体的功能，为有效改进化学防控手段提供理论基础，为我国的媒介控制措施的制订提供科学依据。

蚊虫是一类重要的医学昆虫，叮咬人类的同时还可以传播多种疾病，如疟疾、丝虫病、流行性乙型脑炎、登革热和寨卡病毒病等，对人类健康造成了严重威胁。蚊媒传染病频频爆发流行，病媒防治是控制蚊媒病流行的重要手段，而化学防治是蚊媒综合治理的主要方法。然而，长期大量、连续使用杀虫剂导致蚊虫抗药性显著增加。拟除虫菊酯类杀虫剂使用仅仅十几年，已有抗性百倍、甚至千倍的报道，严重影响了虫媒病的防控。蚊虫寻找宿主、产卵地、躲避不利环境等行为主要依赖于嗅觉系统，研究显示抗性蚊虫能利用嗅觉识别并回避杀虫剂，但具体机制不详。我们研究发现高效氯氰菊酯抗性品系和敏感品系的淡色库蚊嗅觉受体(ORs)的种类和表达量存在差异，且不同品系的嗅觉受体共表达受体（Orco）基因存在点突变。因此，我们提出疑问ORs是否能够识别杀虫剂的气味，参与抗药性的形成呢?本项目通过抗性与敏感品系淡色库蚊的转录组分析筛选差异表达的ORs；研究Orco基因型与抗性表型的相关性及其对杀虫剂识别信号传递的影响；从异源表达系统（细胞和转基因果蝇）以及蚊虫层面解析配体型嗅觉受体（ORx）与Orco基因共同作用识别传递气味信号，探讨蚊虫抗药性形成的机制。结果表明：通过蚊敏感品系与抗性品系转录组测序比较，共预测淡色库蚊的ORs基因182条，其中高效氯氰菊酯抗性品系和敏感品系差异表达的ORs基因23条。qPCR检测发现OR58，OR142在敏感种群中表达量较高，其余21条在抗性品系中高表达。通过转基因果蝇实验发现Orco基因点突变S261N、I371M可能影响对蚊虫对杀虫剂识别信号的识别。通过细胞和转基因果蝇实验发现OR10、OR43、OR58、OR88等嗅觉受体可以识别杀虫剂与蚊虫抗药性密切相关。本项目通过嗅觉受体介导蚊虫抗药性产生机理的深入研究，将有助于明晰蚊虫抗药性机理和嗅觉受体的功能，为有效改进化学防控手段提供理论基础，为我国的媒介控制措施的制订提供科学依据。