Abstract
BACKGROUND: Environmental microbiomes, such as those in recirculating aquaculture systems (RAS), can play a key role in shaping host-associated microbial communities. In zebrafish (Danio rerio) research, these interactions can introduce uncontrolled sources of variation, potentially confounding experimental outcomes across multiple facilities. Despite widespread zebrafish use in microbiome studies, few have characterized the microbial composition of both tank water and fish across multiple independent facilities to evaluate the consequences of environmental microbiome variation on the host microbiome. RESULTS: We compared water and zebrafish gut microbiomes across five aquaculture facilities-two in the United States and three in Norway-using a nested sampling design and 16S rRNA gene sequencing. 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. Differences in microbial community composition were significant across facilities, with both water and fish samples exhibiting facility-specific profiles. Similarity Percentage analysis identified taxonomic groups driving these differences, while Fast Expectation-Maximization for Microbial Source Tracking detected measurable contributions of tank water microbiota to zebrafish gut communities. Bray-Curtis dissimilarity values were lowest between fish and water from the same tank and increased with geographic and facility distance, indicating local microbial overlap. Relative abundance patterns and ordination plots further supported distinct and structured microbial assemblages across systems. CONCLUSIONS: This study demonstrates that zebrafish aquaculture systems harbor unique microbial communities shaped by both environmental and geographic factors, with tank water acting as a potential source of gut-associated microbes. These findings underscore the importance of incorporating environmental microbiome assessments into zebrafish experimental design, particularly for studies focused on host-microbe interactions. Without such consideration, unaccounted variation in environmental microbiota may affect microbiome composition and reduce cross-study reproducibility. Moving forward, standardized reporting of environmental conditions and microbial composition across facilities will be critical for strengthening reproducibility and interpretation in zebrafish microbiome research.