Harnessing midgut mitochondrial dynamics to enhance Anopheline mosquito fitness

描述（由申请方提供）：在综合疟疾控制策略中使用转基因蚊子将要求蚊子阻断寄生虫发育并保持与野生蚊子种群的竞争力。操纵调节免疫和健身的关键信号级联，如胰岛素/胰岛素生长因子信号（IIS）级联，代表了实现这一目标的新方法。在蚊子和其他模式无脊椎动物中，中肠作为IIS控制免疫、寿命、代谢和繁殖的中心发挥作用。中肠IIS的作用主要通过线粒体动力学介导，线粒体动力学定义为线粒体生物发生和通过线粒体自噬清除受损线粒体的总和。在无脊椎动物和哺乳动物中，IIS依赖性线粒体动力学以及因此线粒体代谢调节广泛的重要生理学，包括上皮屏障完整性、干细胞维持和分化、寿命和免疫，表明这种调节在活生物体中是基本的。通过我们对斯氏按蚊的研究，我们发现中肠IIS的操纵改变了线粒体生物发生和线粒体自噬的关键平衡，导致蚊子对恶性疟原虫感染的抗性以及蚊子寿命和繁殖的表型变化。因此，我们提出，IIS依赖的线粒体动力学控制A。stephensi“中肠健康”，这是IIS对免疫力，寿命，代谢和生殖的影响的基础。为了确定IIS依赖的线粒体动力学如何调节这些重要的表型，我们将使用五种不同的处理（Akt转基因（TG）、PTEN TG、胰岛素喂养、IGF 1喂养和操纵A. stephensi胰岛素样肽（AsILP））来“推拉”中肠中的线粒体动力学。这将使我们能够识别和操纵IIS级联下游的特定基因靶点，这些靶点保留疟原虫抗性，同时增强中肠健康和整体蚊子健康。为了实现这一点，我们将首先定义中肠IIS如何通过线粒体自噬调节线粒体生物发生和清除，以及这些过程对能量稳态，干细胞维持和分化，上皮完整性和细胞死亡过程的影响。由于我们的四种外源性治疗（Akt TG、PTEN TG、胰岛素喂养、IGF 1喂养）以可预测的模式影响中肠中AsILP转录物表达，AsILP可能作为IIS依赖性中肠线粒体动力学的天然介导物起作用。因此，我们将操纵AsILP来改变中肠线粒体动力学，并进一步阐明这种与恶性疟原虫感染的IIS依赖性控制的关联。基于IIS依赖的线粒体动力学与蚊子适应性和对寄生虫感染的抗性的关联，我们将确定最佳控制中肠线粒体动力学以特异性增强这些表型的候选基因。为此，我们将使用各种工具在中肠中过表达或破坏候选基因的表达，最终目标是产生稳定转化的合适的A。对恶性疟原虫感染具有完全抗性。

DESCRIPTION (provided by applicant): The use of transgenic mosquitoes in an integrated malaria control strategy will require mosquitoes that block parasite development and remain competitive with wild mosquito populations. Manipulation of key signaling cascades that regulate both immunity and fitness, such as the insulin/insulin growth factor signaling (IIS) cascade, represent a novel approach to achieving this goal. In mosquitoes and other model invertebrates the midgut functions as a center for IIS control of immunity, lifespan, metabolism, and reproduction. The effects of midgut IIS are largely mediated through mitochondrial dynamics, defined as the sum of mitochondrial biogenesis and clearance of damaged mitochondria through mitophagy. In invertebrates and mammals, IIS- dependent mitochondrial dynamics and, hence, mitochondrial metabolism regulate a wide range of important physiologies, including epithelial barrier integrity, stem cell maintenance and differentiation, lifespan and immunity, indicating that this regulation is fundamental in living organisms. Through our work with Anopheles stephensi, we have discovered that manipulation of IIS in the midgut alters the critical balance of mitochondrial biogenesis and mitophagy, resulting in phenotypic changes to mosquito resistance to Plasmodium falciparum infection, as well as mosquito lifespan and reproduction. Thus, we propose that IIS-dependent mitochondrial dynamics control A. stephensi "midgut health," which underlies the effects of IIS on immunity, lifespan, metabolism, and reproduction. To define how IIS-dependent mitochondrial dynamics regulate these important phenotypes, we will use five distinct treatments (Akt transgenic (TG), PTEN TG, insulin-fed, IGF1-fed, and manipulation of A. stephensi insulin-like peptides (AsILPs)) to "push and pull" mitochondrial dynamics in the midgut. This will allow us to identify and manipulate specific gene targets downstream of the IIS cascade that retain malaria parasite resistance while concurrently enhancing midgut health and overall mosquito fitness. To accomplish this, we will first define how midgut IIS regulates mitochondrial biogenesis and clearance through mitophagy and the impacts of these processes on energy homeostasis, stem cell maintenance and differentiation, epithelial integrity, and cell death processes. Since our four exogenous treatments (Akt TG, PTEN TG, insulin-fed, IGF1-fed) impact AsILP transcript expression in the midgut in predictable patterns, AsILPs likely function as natural mediators of IIS-dependent midgut mitochondrial dynamics. As such, we will manipulate AsILPs to alter midgut mitochondrial dynamics and to further clarify this association with IIS- dependent control of P. falciparum infection. Based on associations of IIS-dependent mitochondrial dynamics with mosquito fitness and resistance to parasite infection, we will identify candidate genes that optimally control midgut mitochondrial dynamics to specifically enhance these phenotypes. To this end, we will overexpress or disrupt expression of candidate genes in the midgut using a variety of tools, with the ultimate goal of generating stably transformed, fit A. stephensi that ar completely resistant to P. falciparum infection.