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Fish are the most diverse and widely distributed vertebrates, yet little is known about the microbial ecology of fishes nor the biological and environmental factors that influence the fish microbiome. The microbiota from 101 species of Southern California marine fishes, spanning 22 orders, 55 families, and 83 genera representing ~ 25% of local marine fish diversity, was analyzed to identify patterns that explain microbial diversity patterns in a geographical subset of marine fish biodiversity. We compared fish microbiomes (gill, skin, midgut, and hindgut) using alpha, beta, and gamma diversity along with establishing a novel method to estimate microbial biomass (Qiime2 plugin katharoseq). For oceanic fishes from the neritic zone, host size and distance from shore were negatively associated with microbial biomass densities and diversity in the gills. Body site was the strongest driver for beta diversity with strong evidence of phylosymbiosis observed across the gill, skin, and hindgut, but not midgut. The majority of microbes from all fish body sites were of unknown origin but overall sea water generally contributes more microbes to fish mucus compared to marine sediment. In a meta-analysis of vertebrate hindguts (569 species), mammals had the highest gamma diversity when controlling for host species number while fishes had the highest percent of unique microbial taxa (92%). In fishes, the midgut, gill, and skin contains the majority of microbial diversity which collectively can be 5.5 times higher than the hindgut. The composite dataset will be useful to vertebrate microbiome researchers and fish biologists interested in microbial ecology with applications in aquaculture and fisheries management.

BackgroundMicrobial succession in vertebrates has primarily focused on vertical transmission and ontogenetic development in the mammalian gut. Teleosts comprise the majority of vertebrate diversity, yet little is known about how the microbiome develops in fish, particularly when vertical transmission is limited or absent for broadcast spawners. Biological factors such as diet, age, phylogeny, and trophic level along with environmental factors such as water salinity, temperature, and depth have been shown to influence the mucosal microbiomes of fish. Here we investigate how various microbial-rich surfaces from the built environment ‘BE’ influence the development of the mucosal microbiome (gill, skin, and digesta) of an economically important marine fish, yellowtail kingfish, Seriola lalandi, over time.ResultsFor the first experiment, we sampled gill and skin microbiomes from 36 fish reared in three tank conditions, and demonstrate that the gill is more influenced by the surrounding environment than the skin. In a second experiment, fish microbiomes (gill, skin, and digesta) and the BE (tank side, water, inlet pipe, airstones, and air diffusers) were sampled from indoor reared fish at three ages (43 dph, 137 dph, 430 dph; n=12 per age). At 430 dph, 20 additional fish were sampled from an outdoor ocean net pen. A total of 304 samples were processed for 16S rRNA gene sequencing. Gill and skin alpha diversity increased while gut diversity decreased with age. Diversity was much lower in fish from the ocean net pen compared to indoor fish. We quantified the change in community dynamics driven by the BE and show that the gill and skin are most influenced by the BE early in development, with aeration equipment having more impact in later ages, while the gut microbiome becomes increasingly differentiated from the environment over time.ConclusionsOur findings suggest that fish mucosal microbiomes are differentially influenced by the built environment with a high turnover and rapid succession occurring in the gill and skin while the gut microbiome is more stable. We demonstrate how individual components of a hatchery system, especially aeration equipment, may contribute directly to microbiome development in a marine fish. In addition, results demonstrate how early life (larval) exposure to stressors in the rearing environment may influence fish microbiome development which is important for animal health and aquaculture production.

Next Generation Sequencing technologies have enabled many advances across biology with microbial ecology benefiting primarily through expanded sample sizes. Although the cost of running sequencing instruments has decreased substantially over time, the price of library preparation methods has largely remained unchanged. We developed a low cost, miniaturized (5 µL), high-throughput (384-sample), amplicon library preparation method with the acoustic liquid handler, Echo 550. Our method reduces costs of library preparation to $1.42 USD per sample, a 58% reduction compared to existing automated methods and a 21-fold reduction from commercial kits, without compromising sequencing success or distorting the microbial community composition analysis. The cost savings of implementing the miniaturized library preparation (going from triplicate 25 µL reactions to triplicate 5 µL reactions) are large enough to cover a MiSeq sequencing run for 768 samples, while preserving accurate microbiome measurements.