Wolbachia as an Agent for Population Replacement in Anopheles Gambiae

描述（由申请人提供）：控制疟疾的传统方法的失败刺激了创造用于控制疟疾的转基因蚊子的努力。要使转基因策略成功，必须将转基因基因以高频率主动导入自然按蚊种群。沃尔巴克氏体是母体遗传的内共生菌，与细胞质不相容（CI）有关，即当受感染的雄性与未感染的雌性交配时，卵孵化减少。受感染的雌性交配是可生育的，无论雄性是否受感染。CI为受感染的雌性提供了繁殖优势，并使沃尔巴克氏体在昆虫种群中迅速传播，传播频率很高。与沃尔巴克氏体密切相关的转基因性状有望通过基因搭便车的方式进入种群，用对寄生虫传播有抵抗力的种群取代自然种群。虽然沃尔巴克氏体感染在蚊子中很常见，但从未在任何种类的按蚊中观察到。因此，基于沃尔巴克氏体的疟疾控制策略需要将感染人工转移到负责寄生虫传播的按蚊物种中。人工转染沃尔巴克氏体已经成功地在几种医学上重要的伊蚊中获得成功，但尚未成功地在按蚊中获得成功。我们的体外研究表明，冈比亚按蚊的遗传背景能够容纳至少3种不同的沃尔巴克氏体感染，因此，以前不成功的按蚊转染尝试可能是由于技术而不是感染的内在遗传障碍。我们将使用最近开发的胚胎和成体注射技术在冈比亚按蚊中建立沃尔巴克氏体感染，并在表型、遗传和种群水平上研究沃尔巴克氏体和按蚊之间的相互作用。在研究的最后，我们将1)人工转染冈比亚按蚊Wolbachia， 2)确定Wolbachia感染对先前通过细胞系实验确定的候选按蚊基因表达的影响，3)评估Wolbachia将母体遗传的转基因基因驱动到冈比亚按蚊笼群中的潜力。这些结果将为成功利用转基因蚊子控制疟疾奠定基础。疟疾控制的转基因策略需要在自然按蚊种群中高频率地主动传播引入的转基因。沃尔巴克氏体内共生菌理论上可以作为一种将基因传播到种群中的机制。我们建议评估将沃尔巴克氏体感染转移到冈比亚按蚊中，并评估其作为疟疾控制的候选转基因驱动系统。

DESCRIPTION (provided by applicant): Failure of traditional methods to control malaria has stimulated efforts to create transgenic mosquitoes for malaria control. Transgenes must be actively driven into natural Anopheles populations to high frequency for transgenic strategies to be successful. Wolbachia are maternally inherited endosymbionts associated with cytoplasmic incompatibility (CI) i.e., reduced egg hatch when an infected male mates with an uninfected female. Matings of infected females are fertile regardless of the infection status of the male. CI confers a reproductive advantage to infected females and allows Wolbachia to spread rapidly through insect populations to high frequency. Transgenic traits tightly linked to Wolbachia are expected to be driven into the population by genetic hitchhiking, replacing the natural population with one that is refractory to parasite transmission. While Wolbachia infections are common in mosquitoes, they have never been observed in any species of Anopheles. Thus, Wolbachia-based strategies for malaria control require the artificial transfer of infection into Anopheles species responsible for parasite transmission. Artificial Wolbachia transfections have succeeded in several medically-important Aedes species but have not yet succeeded in Anopheline mosquitoes. Our in vitro studies show that the Anopheles gambiae genetic background is competent to harbor at least 3 diverse Wolbachia infections, and thus, previous unsuccessful Anopheles transfection attempts are likely due to technique rather than an intrinsic genetic block to infection. We will use recently-developed embryonic and adult injection techniques to establish Wolbachia infections in Anopheles gambiae and examine the interaction between Wolbachia and Anopheles at the phenotypic, genetic and population levels. By the end of the proposed research, we will 1) artificially transfect Anopheles gambiae with Wolbachia, 2) determine the effect of Wolbachia infection on expression of candidate Anopheles genes previously identified by cell line experiments and 3) evaluate the potential for Wolbachia to drive maternally-inherited transgenes into Anopheles gambiae cage populations. These results will lay the foundation for the successful deployment of genetically-modified mosquitoes for malaria control. Transgenic strategies for malaria control require the active spread of introduced transgenes to high frequency in natural Anopheles populations. Wolbachia endosymbionts can theoretically act as a mechanism to spread genes into populations. We propose to evaluate transfer Wolbachia infection into Anopheles gambiae and evaluate the symbiont as a candidate transgene drive system for malaria control.