Giardia lamblia is microaerophilic protozoan parasite of humans and animals that causes significant diarrheal disease worldwide. Ingested cysts transform into motile trophozoites as they pass into the gastrointestinal tract. As microaerophiles, non-encysting trophozoites have a fermentative metabolism with unique end products. Encysting trophozoites have a dramatically different metabolism, defined by metabolic shifts from glycolysis to the GlcNAc pathway, arginine fermentation by the ADiHP pathway, and upregulation of lipid biosynthesis during the production of the cyst wall. Giardia produces no known toxin, and colonization does not elicit a robust inflammatory reaction. Giardia colonization of the small intestine occurs in a niche already inhabited by commensal microbiota, yet in vivo Giardia-microbiome interactions have largely been ignored in models of pathogenesis. We recently showed that Giardia infection of mice results in a dysbiosis at the primary site of colonization in the small intestine and other dysbioses extend to other regions of the gut. Using bioluminescent imaging (BLI) methods developed in our lab, we discovered that trophozoites rapidly encyst in foci of infection within days of inoculation, primarily in the small intestine. Using BLI-mediated precise sampling of these encysting foci, we demonstrated that encysting trophozoites have a unique in vivo transcriptional signature defined by significant upregulation of encystation and redox-response. We thus hypothesize that encysting trophozoites have a specific and differential impact on small intestine dysbiosis as compared to non-encysting trophozoites. This proposed work evaluates and quantifies the effect of encysting and non-encysting trophozoite metabolic activity on host microbiome metabolic activity. In Aim 1, we compare the differential impacts of encysting and non-encysting Giardia trophozoites on the small intestinal microbiome metabolism and on known host immune responses. We will infect animals with a dual- tagged (constitutive and encystation-specific) reporter strain to facilitate non-invasive in vivo quantification of encysting and non-encysting trophozoites during the course of infection. We define microbiome community structure and metabolism using total community metagenomic and metabolomic analyses and key immune responses using BLI-mediated precision ex vivo sampling of sites with encysting and non-encysting trophozoites. In Aim 2, we further explore the metabolic interactions between encysting or non-encysting trophozoites and the microbiome by infecting animals with a dual-reporter encystation-defective CWP1 null mutant that is unable to generate viable cysts. Defining exactly how the microbiome is affected in Giardia infections is a first and necessary step toward designing microbiome-mediated therapies. Our detailed analyses of Giardia-microbiome interactions provide a foundation for future studies of the impacts of colonizing Giardia trophozoites (encys...