Giardia lamblia is microaerophilic protozoan parasite of humans and animals that causes significant diarrhealdisease worldwide. Ingested cysts transform into motile trophozoites as they pass into the gastrointestinaltract. As microaerophiles, non-encysting trophozoites have a fermentative metabolism with unique endproducts. Encysting trophozoites have a dramatically different metabolism, defined by metabolic shifts fromglycolysis to the GlcNAc pathway, arginine fermentation by the ADiHP pathway, and upregulation of lipidbiosynthesis during the production of the cyst wall. Giardia produces no known toxin, and colonization does notelicit a robust inflammatory reaction. Giardia colonization of the small intestine occurs in a niche alreadyinhabited by commensal microbiota, yet in vivo Giardia-microbiome interactions have largely been ignored inmodels of pathogenesis. We recently showed that Giardia infection of mice results in a dysbiosis at the primarysite of colonization in the small intestine and other dysbioses extend to other regions of the gut. Usingbioluminescent imaging (BLI) methods developed in our lab, we discovered that trophozoites rapidly encyst infoci of infection within days of inoculation, primarily in the small intestine. Using BLI-mediated precise samplingof these encysting foci, we demonstrated that encysting trophozoites have a unique in vivo transcriptionalsignature defined by significant upregulation of encystation and redox-response. We thus hypothesize thatencysting trophozoites have a specific and differential impact on small intestine dysbiosis ascompared to non-encysting trophozoites. This proposed work evaluates and quantifies the effect ofencysting and non-encysting trophozoite metabolic activity on host microbiome metabolic activity. InAim 1, we compare the differential impacts of encysting and non-encysting Giardia trophozoites on the smallintestinal 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 ofencysting and non-encysting trophozoites during the course of infection. We define microbiome communitystructure and metabolism using total community metagenomic and metabolomic analyses and key immuneresponses using BLI-mediated precision ex vivo sampling of sites with encysting and non-encystingtrophozoites. In Aim 2, we further explore the metabolic interactions between encysting or non-encystingtrophozoites and the microbiome by infecting animals with a dual-reporter encystation-defective CWP1 nullmutant that is unable to generate viable cysts. Defining exactly how the microbiome is affected in Giardiainfections is a first and necessary step toward designing microbiome-mediated therapies. Our detailedanalyses of Giardia-microbiome interactions provide a foundation for future studies of the impacts ofcolonizing Giardia trophozoites (encysting and non-encysting) and the host microbiome on overall gutecological health.