Sampling the fish gut microbiota

Despite an immense increase in the number of microbiota focused studies in teleosts, only few have reported functional data on whole metagenomes as it has proven difficult to extract high biomass microbial DNA from fish intestinal samples. The zebrafish is a promising model organism in the field of functional microbiota research. However, studies on the functional landscape of the zebrafish gut microbiota through shotgun based metagenomics are also scarce. Thus, there is a lack of consensus regarding an appropriate sampling method that accurately represents the gut microbiota of zebrafish, or any other fish species. To address this, we sought to systematically test and evaluate four different methods of sampling the zebrafish gut microbiota: collection of feces from the tank, the whole gut, intestinal content and the application of ventral pressure to facilitate extrusion of gut material. In addition, we included water samples as an environmental control to address the potential influence of the environmental microbiota on data interpretation of each sample type. To compare these sampling methods in a context of microbiota-based studies we employed a combination of genome resolved metagenomics and 16S metabarcoding techniques. We observed differences among sample types on all levels including sampling, bioinformatic processing, metagenome co-assemblies, generation of metagenome-assembled genomes (MAGs), functional potential, MAG coverage and population level microdiversity. Furthermore, our comparison to the environmental control demonstrated the potential impact of the environmental contamination on data interpretation. While all sample types tested are informative about the zebrafish gut microbiota, the results show that optimal sample type for studying fish microbiomes depends on the specific objectives of the study, and here we provide a guide on what factors to consider for designing functional metagenome based studies on teleost microbiomes.