Microbial Ecology of the Zebrafish Intestine

描述（由申请人提供）：所有动物都与微生物群落密切相关，微生物群落在动物发育和适应中起着重要作用。与脊椎动物相关的数量最多的微生物群落是肠道微生物的组合，或肠道微生物群。与动物相关的微生物群落在宿主体内聚集和持续存在的机制尚不清楚。关于与动物相关的微生物群落生态学的基本未解问题包括这些群落通过宿主选择或随机过程聚集的程度。如果宿主选择很重要，那么这种选择是通过什么机制发生的？选择性力是否会随着宿主发育阶段的变化而变化？影响社区聚集的宿主选择压力是否也有助于社区的稳健性？了解这些群落的组装原理对于开始设计治疗策略以安全有效地促进有益微生物群落和预防或纠正病原微生物群落是必不可少的。我们建议应用生态学理论和建模来理解模型脊椎动物斑马鱼肠道微生物群的组装和持久性。我们已经建立了在无菌条件下培养斑马鱼的方法（“无菌”），并将它们与确定的微生物群落联系起来。斑马鱼为这些研究提供了许多优势。它们的繁殖力和快速发展使我们能够一次培养数千个无菌个体，并设计具有高度复制性的实验。斑马鱼及其相关细菌的遗传易感性，允许从相互作用的双方对宿主-微生物群关联进行实验操作。开发斑马鱼的透明度使我们能够监测活体动物肠道的细菌定植，并研究肠道微生物群组装的空间和时间模式。本研究的总体目标是通过对无菌营养和养殖的改进和标准化，以及对斑马鱼相关微生物群落的定义进行简化分析，从而推进无菌斑马鱼模型，使该模型可以被微生物生态学家和发育遗传学家广泛用于研究动物相关微生物群落的动态。在Aim 1中，我们建议使用生态学理论来计算宿主选择和随机过程在斑马鱼肠道微生物群组装中的相对重要性。我们将询问宿主选择是否随宿主发育阶段和微生物分类而变化。在目标2中，我们将确定饮食和免疫系统在斑马鱼微生物群宿主选择中的相对重要性。为了解决饮食的作用，我们将研究不同饮食饲养的鱼的肠道微生物群聚集。为了解决免疫系统的作用，我们将分析先天或适应性免疫系统缺陷的斑马鱼突变体以及接种肠道菌群疫苗的宿主的肠道菌群组装。在目标3中，我们将研究宿主选择的机制。我们将使用已定义的微生物群落来确定初始定植种群的大小。我们将使用体内成像来检查肠道定植的空间和时间模式。最后，我们将通过监测已建立的微生物群作为这些宿主因素的功能的入侵，确定饮食和免疫系统如何促进社区稳健性。总的来说，这些研究将为宿主相关微生物群落的建立和维持提供更深入的机制理解。这些知识对于合理设计益生元、益生菌和抗生素来治疗与微生物群失衡相关的人类疾病（如炎症性肠病和糖尿病）至关重要。

DESCRIPTION (provided by applicant): All animals exist in intimate associations with communities of microorganisms, which play important roles in animal development and fitness. The most numerically abundant microbial communities associated with vertebrate animals are the assemblages of microbes in the gut, or the gut microbiota. The mechanisms by which animal-associated microbial communities assemble and persist inside the host are poorly understood. Fundamental unanswered questions about the ecology of animal-associated microbial communities include the extent to which these communities are assembled by host selection or by stochastic processes. If host selection is important, what mechanisms by which this selection occurs? Do selective forces change as a function of host developmental stage? Do the host selective pressures that influence community assembly also contribute to community robustness? An understanding of the assembly principles of these communities is essential for beginning to design therapeutic strategies for humans to safely and effectively promote beneficial microbial communities and prevent or correct pathogenic ones. We propose to apply ecological theory and modeling to understand the assembly and persistence of the gut microbiota in the model vertebrate zebrafish. We have established methods to rear zebrafish under sterile conditions ("germ-free") and associate them with defined microbial communities. The zebrafish offers many advantages for these studies. Their fecundity and rapid development allows us to rear thousands of germ-free individuals at a time and design experiments with a high degree of replication. The genetic tractability of zebrafish and their associated bacteria, permit experimental manipulation of host-microbiota associations from both sides of the interaction. The transparency of developing zebrafish allows us to monitor bacterial colonization of the gut in live animals and study spatial and temporal patterns of gut microbiota assembly. An overarching goal of this proposal is to advance the germ-free zebrafish model, through the improvement and standardization of germ-free nutrition and husbandry, and the development of defined zebrafish-associated microbial communities for reductionist analyses, such that this model can become widely used by microbial ecologists and developmental geneticists to study the dynamics of animal-associated microbial communities. In Aim 1, we propose to use ecological theory to calculate the relative importance of host selection and stochastic processes in the assembly of the zebrafish gut microbiota. We will ask whether host selection varies as a function of host developmental stage and microbial taxonomy. In Aim 2, we will determine the relative importance of diet and the immune system in host selection of the zebrafish microbiota. To address the role of diet, we will investigate gut microbiota assembly in fish reared on different diets. To address the role of the immune system, we will analyze gut microbiota assembly in zebrafish mutants with defect innate or adaptive immune systems, and in hosts vaccinated against members of their gut microbiota. In Aim 3 we will investigate the mechanisms that underlie host selection. We will use defined microbial communities to determine the size of the initial colonizing population. We will use in vivo imaging to examine the spatial and temporal patterns of gut colonization. Finally we will determine how diet and immune system contribute to community robustness by monitoring invasion of established microbiota as a function of these host factors. Collectively these studies will provide a deeper mechanistic understanding of how host- associated microbial communities are established and sustained. Such knowledge is essential for the rational design of prebiotics, probiotics, and antibiotics to treat human pathologies associated with imbalances of the microbiota such as inflammatory bowel diseases and diabetes.