Aquaculture facility-specific microbiota shape the zebrafish gut microbiome

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.

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 analyses revealed significant clustering by sample type, facility, and geographic location, with facility identity explaining the largest proportion of compositional variance. Multivariate dispersion also differed significantly across facilities, indicating that observed compositional differences reflect both shifts in community composition and differences in within-facility variability. Each facility harbored a distinct microbial community in both water and fish gut samples, with geographic location further structuring community composition between Oregon and Norwegian facilities. Similarity Percentage analysis identified key taxa driving facility differences, including Cetobacterium, Vibrio, and Aeromonas in fish gut microbiomes and Pseudomonas and Rheinheimera in tank water. Microbial source tracking using FEAST revealed that facility-level tank water contributed measurably to fish gut microbiome composition in most facilities, though unknown sources dominated estimates across all facilities (71-99%) and the strength of fish-water microbiome association varied substantially across facilities.

This study demonstrates that zebrafish aquaculture facilities harbor unique microbial communities shaped by both environmental and geographic factors. While tank water microbiomes show associations with zebrafish gut microbiome composition, the dominant contribution of unknown sources to gut microbiome composition suggests that factors beyond the immediate tank water environment- including diet, host physiology, and other facility-specific conditions- are primary drivers of gut microbiome variation. The strength of this association varied considerably across facilities and appeared related to fish domestication history, a pattern that warrants direct experimental investigation. 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.