Wolbachia as an Agent for Population Replacement in Anopheles Gambiae

描述(由申请人提供)：传统方法控制疟疾的失败刺激了创造转基因蚊子用于疟疾控制的努力。转基因策略要想取得成功，就必须积极推动转基因进入自然按蚊种群，使其频率更高。沃尔巴克氏菌是一种母系遗传的内共生菌，与细胞质不亲和性(CI)有关，即当感染的雄性与未感染的雌性交配时，卵孵化减少。无论男性的感染状态如何，受感染的女性的交配都是可生育的。CI为受感染的雌性赋予生殖优势，并允许沃尔巴克氏菌通过昆虫种群快速传播。与沃尔巴克氏菌紧密连锁的转基因特征预计将通过基因搭便车进入种群，用不易寄生虫传播的种群取代自然种群。虽然沃尔巴克氏菌感染在蚊子中很常见，但从未在任何种类的按蚊中观察到过。因此，基于沃尔巴克氏菌的疟疾控制战略需要人工将感染转移到导致寄生虫传播的按蚊物种中。人工沃尔巴克氏菌转基因已经在几个医学上重要的伊蚊物种中取得了成功，但尚未在按蚊身上取得成功。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菌感染，因此，以前未成功的按蚊转基因尝试很可能是由于技术原因，而不是内在的感染遗传障碍。我们将使用最近发展的胚胎和成体注射技术来建立冈比亚按蚊的沃尔巴克氏菌感染，并在表型、遗传和种群水平上研究沃尔巴克氏菌和按蚊之间的相互作用。在拟议的研究结束时，我们将1)用沃尔巴克氏菌人工转导冈比亚按蚊，2)确定沃尔巴克氏菌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，以及3)评估沃尔巴克氏菌将母系遗传的转基因驱动到冈比亚按蚊笼子种群的潜力。这些结果将为成功部署转基因蚊子控制疟疾奠定基础。控制疟疾的转基因战略需要主动传播引入的转基因，使其在自然按蚊种群中出现频率较高。从理论上讲，沃尔巴克氏菌内共生菌可以作为一种机制将基因传播到种群中。我们建议评估将沃尔巴克氏菌感染转移到冈比亚按蚊中，并评估共生体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.