PROJECT SUMMARY/ABSTRACT (PROJECT 1) The gastrointestinal microbiota of humans and other animals, composed of vast numbers of individual cells representing many different species, serves as a virtual organ contributing to a wide range of physiological processes. More than most organs, it is subject to strong and frequent perturbations due to its connectedness to the outside world and the inter-species competition inherent in its nature as a multi-species consortium. These perturbations may be chemical, e.g. from antibiotic drugs, or biological, e.g. from invasion by new species, and may couple to physical characteristics of microbes such as motility and spatial distribution. It is likely that the large variability in human microbiome composition, both within individuals over time and between individuals in populations, is due in part to responses to variations in externally driven stimuli. Despite this, our understanding of how specific perturbations influence gut microbial communities, and whether generic features might unify diverse responses, remains minimal. We propose a series of experiments that will address this, using larval zebrafish as a model host-microbe system. The amenability of larval zebrafish to gnotobiotic manipulation enables controlled experiments with large replicate populations, and their optical transparency allows imaging-based quantifications of microbial abundances, spatial distributions, and dynamics. We focus on two important classes of perturbations: invasion by new species and exposure to antibiotics. The introduction of new species is a constant feature of the intestinal environment, and behaviors such as motility and chemotaxis likely play important roles in interactions between resident and invasive microbes. Building on advances in synthetic biology, we will engineer genetic switches to report on and control these behaviors in situ, using the control afforded by gnotobiotic zebrafish to manipulate intestinal communities. Antibiotic use is known from metagenomic studies to dramatically affect the gastrointestinal microbiota, even at low (sub- lethal) doses, for reasons that remain to be discovered. Building on preliminary data, we hypothesize that even weak antibiotic perturbations can dramatically alter bacterial behaviors, spatial distributions, and persistence within the gut, altering both species abundances and dispersal to new hosts. Our proposed experiments will provide new insights into how common perturbations influence intestinal microbiota dynamics and will establish a foundation for understanding the dynamics of human microbiomes during both health and disease.