PROJECT SUMMARY Background: Biological processes emerge from molecular interactions within and between organisms, including hosts and their associated microbiota. Understanding molecular interactions between the host immune system and microbial communities is a major goal of microbiome research. The immune system co- exists with beneficial microbes, while simultaneously identifying and eliminating invaders. Successful interactions between the host immune system and microbiota are essential for maintaining host health, while failed interactions can cause detrimental effects such as human diseases like inflammatory bowel disease (IBD) and Crohn's Disease. The microbiota can also influence health phenotypes including obesity and diabetes. Host genetics has been demonstrated to play a role in establishing host immune-microbe relationships and can impact composition of the microbiota. Despite our knowledge of the existence of these relationships, the immune gene variants interacting with the microbiome, particularly in the context of diverse diets and environments, are still unknown. Hypothesis: variation in immune genes, particularly those in the adaptive immune system MAP-kinase pathway, are involved in host ?associated microbial communities. Specific Aims: (1) Use divergent stickleback populations to identify natural genetic variants affecting host- associated microbiome composition, as well host immune gene expression in response to targeted manipulations of the microbiota. (2) Use CRISPR/Cas9 manipulations of key immune genes in divergent threespine stickleback populations to test hypotheses regarding the functional relationship between variation in host immune response and microbiota composition. Study Design: To determine the effects of natural genetic variants associated with variance in microbiota composition, I will raise stickleback families from both freshwater and marine laboratory lines in a single environment. I will then assess variance microbial composition using 16S sequencing. To map natural genetic variants affecting host transcriptional state and microbiome composition, I will use crosses of divergent stickleback populations, measure influences on host immune response, host gene expression, and microbial composition. These data will be combined in QTL/eQTL mapping studies. Finally, to determine how immune system disturbance varies across genetically divergent populations, I will use the CRISPR-Cas9 system to abrogate function of variants and exchange alleles between marine and freshwater populations, then measure outcomes on host immune response, gene expression, and microbial composition. .