Aquaculture facility-specific microbiota shape the zebrafish gut microbiome

Environmental microbiomes in recirculating aquaculture systems (RAS) can significantly shape host-associated microbial communities, and in zebrafish (Danio rerio) research, these interactions introduce uncontrolled sources of variation that may confound experimental outcomes across facilities. Despite widespread zebrafish use in microbiome studies, few have characterized microbial composition across multiple independent facilities to evaluate consequences of environmental microbiome variation on the host microbiome. We compared water and zebrafish gut microbiomes across five aquaculture facilities (two in the United States and three in Norway) using nested sampling and 16S rRNA gene sequencing, finding that alpha diversity was consistently higher in tank water than in fish guts, and beta diversity metrics revealed distinct clustering by sample type, facility, and location, with significant differences in microbial community composition across facilities and facility-specific profiles in both water and fish samples. Similarity Percentage analysis identified taxonomic drivers of these differences, while Fast Expectation-Maximization for Microbial Source Tracking detected measurable contributions of tank water microbiota to zebrafish gut communities, with Bray-Curtis dissimilarity values lowest between fish and water from the same tank and increasing with geographic and facility distance. These findings demonstrate that zebrafish aquaculture systems harbor unique microbial communities shaped by environmental and geographic factors, with tank water acting as a potential source of gut-associated microbes, and underscore the importance of incorporating environmental microbiome assessments into zebrafish experimental design to account for uncontrolled variation that may affect microbiome composition and reduce cross-study reproducibility. Standardized reporting of environmental conditions and microbial composition across facilities will be critical for strengthening reproducibility and interpretation in zebrafish microbiome research.