球形芽孢杆菌对按蚊传疟能力的影响及机制研究

Malaria is a severe infectious parasitic disease. Anopheles dirus is one of the important vector transmitting malaria in China. Vector control is an effectual way to block the transmission.Being highly effective against mosquito larvae at very low doses and safe to other non-target organisms, Bacillus shpaericus(Bs) has become the predominant non-chemical means employed for control of mosquito larvae. Some anopheles mosquitoes can survive from Bs treatment because of nonstandard using.Vector competence of the survived mosquitoes will affect the prevalence and control of malaria. We firstly found that the vector competence of the survived mosquitoes from sublethal dose of Bs using was significantly increased and the melanization was also affected. The mechanism is is still unknown.As we know, anopheles' innate immunity and gut microflora play key roles in vector competence of mosquito's. So, this study will be working on the role and mechanism of anopheles' innate immunity and melanization related molecules in the impact of Bs treatment on mosquitoes's vector competence to malaria parasites, using the techniques of Realtime PCR and RNAi. Then,metagenomics methods based on high throughput sequencing will be used to study the role of gut microflora in the impact of Bs treatment on vector competence. Then, antimicrobial peptides(AMPs) will be also studied using techniques mentioned before to see the role of the crosstalk of innate immunity and gut microflora in the impact of Bs on vector competence of mosquitioes. This study will be helpful to understand the impact and mechanism of Bs treatment on anopheles' vector competence to malaria. It will provide a basis to wipe out malaria using vector control.

疟疾是一种严重的传染病，蚊媒控制是阻断疟疾传播的有效措施。球形芽孢杆菌(Bacillus sphaericus,Bs)作为一种生物杀虫剂越来越受到关注。我们前期研究首次发现，经亚致死剂量Bs处理而存活的按蚊传疟能力增强，按蚊对疟原虫的黑化也发生了变化，这将对疟疾的流行和防治带来新问题。其机制目前尚不清楚。研究显示，按蚊天然免疫和肠道菌群是影响其传疟能力的重要因素。本研究拟在已有研究基础上，以"大劣按蚊-约氏疟原虫-Bs"为研究模型，利用Realtime PCR、RNAi和宏基因组学等研究技术，通过对大劣按蚊抗疟及黑化关键分子（TEP1、PPOs等）、抗菌肽（CECs、DEFs等）和肠道菌群的研究，探讨Bs处理后按蚊传疟能力改变与按蚊天然免疫、肠道菌群及此二者相互作用的关系。本研究将有利于明确生物杀虫剂Bs的应用对按蚊传疟能力的影响及其分子机制，为通过媒介防治消灭疟疾奠定基础。

疟疾依然是危害人类健康的一种传染病，大劣按蚊和斯氏按蚊是我国和东南亚地区重要的传疟媒介，蚊媒控制是阻断疟疾传播的重要措施。随着蚊对化学杀虫剂的耐药性日益严重及化学杀虫剂存在的对人畜有害性和对环境的污染滞留等问题，生物杀虫剂的使用越来越广泛。球形芽孢杆菌（Bacillus sphaericus, Bs）和苏云金杆菌（Bacillus thuringiensis israelensis, Bti）是目前最为常用的两种生物杀虫剂，对蚊幼虫具有高效杀伤作用，且对非靶生物安全无害，不污染环境。然而，对生物杀虫剂Bs的应用基础研究还远远不够，尤其对生物杀虫剂使用的后续效应研究较少（如Bs的使用对存活蚊虫传病能力和繁殖能力的影响）。本课题首先利用Probit analysis的生物测定方法，检测明确了Bs和Bti对大劣按蚊和斯氏按蚊等的杀伤效应和合理剂量及影响因素。在此基础上，利用亚致死剂量Bs处理大劣按蚊，探讨了耐药性产生规律和Bs对大劣按蚊繁殖能力的影响，结果发现亚致死剂量Bs使用后存活按蚊的蚊卵产出受到抑制（对雌蚊受孕及之后的幼虫孵化、成蛹和羽化均无影响），但同时不合理的亚致死剂量使用极易导致大劣按蚊对Bs产生耐药性。继而，利用不易感和易感动物模型分别比较研究了Bs对大劣按蚊和斯氏按蚊传疟能力的影响，结果显示Bs能够降低大劣按蚊对约氏疟原虫的易感性，却能增强斯氏按蚊对约氏疟原虫的传疟能力。然后，本课题组从按蚊天然免疫和肠道菌群两个方面探讨了Bs对按蚊传疟能力影响的分子机制，阐明了Bs导致的传疟能力改变可能与TEP1、Rel2和Caudal参与的Imd信号通路有关；并利用基于高通量测序的宏基因组学方法比较分析了Bs处理前后大劣按蚊肠道菌群组成的异同，同时检测了抗菌肽在Bs处理前后的转录水平变化，按蚊肠道菌群组成的变化可能参与了Bs对按蚊传疟能力的影响。本研究为生物杀虫剂Bs等在现场的标准化推广应用及耐药性监测及制定相关对策提供了重要参考，明确Bs的后续效应（如Bs对按蚊繁殖能力、传疟能力的影响）则为更加合理地应用生物杀虫剂和避免对阻断疟疾传播不利的因素具有一定的指导意义。