Here we investigated the ecological and biological drivers of bacterial diversity associated with fish mucosal sites (gill, skin, midgut, and hindgut) across 101 fish species from the EPO along with gill samples from an additional 17 species (15 unique) from the Western Atlantic. We curated a list of both categorical and continuous metadata types to describe the biology of the fish including habitat, diet, and swim performance. In addition we tested the effects of host phylogeny on microbial diversity. Lastly we performed an extensive meta-analysis of gamma diversity of marine fishes as compared to other vertebrate classes and show the importance of including multiple body sites along with microbial biomass in measures of diversity.
Of all the factors tested across the 101 fish species, the ‘body site’ was the strongest predictor of microbial community composition followed by general depth, habitat benthic substrate, and fish microbial biomass. Body site is frequently one of the strongest predictors when comparing single fish species including Atlantic salmon 14, Pacific Chub Mackerel 7,14, Yellowtail Kingfish 15, and Southern Bluefin Tuna 16, but this was the first to demonstrate the effect across a range of fish species representing a diverse phylogenetic sampling. This suggests that there are conserved aspects of the body sites across fish lineages, which select for or enrich certain microbial communities. We hypothesize this could be due to body site differences in microbial exposure, immune function, mucus chemistries, morphology, and host anatomy and physiology 17. Microbial biomass, habitat depth 1 (shallow ‘neritic’, mid water ‘mesopelagic’, or deep sea ‘bathypelagic or abyssal’), and substrata group (pelagic, soft bottom, rock associated, deep water benthic) were also top predictors of beta diversity across the entire dataset suggesting that habitat had an influence on the microbiome of fishes.
Each body site had specific associations with the various ecological and biological parameters. We observed decreasing microbial diversity in the gills of larger fishes and hypothesize that high performance swimming fishes may have adaptations related to keeping gills clear of microbial fouling to maintain respiratory performance. Fish skin primarily functions as a protective barrier by preventing invasion of pathogens, but in some species the skin can have additional physiological roles such as a source of gas exchange 18. Here, skin microbiomes were primarily explained by the type of bottom structure of the habitat from which the fish lives. For shallow environments, the benthic substrate (e.g. mud, sediment, rocky reef) will likely have a stronger contribution of microbes directly to the water column as compared to deeper water systems and therefore may explain how sediment types can influence external microbiomes of fishes 19,20. The gastrointestinal tract can vary in morphology and function across fishes with many differing types of stomachs, lengths, and enzyme productions 21–23.
Several unique observations with the gill microbiomes in regards to the ecology of fishes suggests a novel evolutionary role. Fishes that were morphologically built for high acceleration (higher dorsal length to total length ratio), had more sea water associated microbes contributing to the gill, skin, and hindgut as compared to sediment microbes. Fishes with a higher microbial biomass in the gills were associated with a higher proportion of sediment sourced microbes as compared to seawater. For neritic fishes, we observed that gill microbial biomass was negatively associated with increasing distance from shore. Combined, we hypothesize that fishes that live closer to shore, such as in the intertidal or subtidal zones as opposed to pelagic fishes, will have lower physiological requirements for swim performance.Therefore, as a potential tradeoff, these fishes will have higher microbial biomass accumulating in the gills as a result of their closer proximity to or in contact with marine sediments. Gills are the source of both gas exchange and nitrogenous waste excretion in fishes and are composed of a generalized conserved morphology with gill arches, filaments, and lamellae. Understanding how microbes may enhance or disrupt these physiological processes will be important areas of research in the future; especially as it relates to aquaculture production of pelagics 24.
Contrary to expectations, we did not observe a direct association between trophic level and alpha diversity. We estimated trophic level by two primary methods. First, we estimated the trophic level by using previously documented diet data derived from the literature. In addition, we used the ratio of ‘relative intestinal length : total body length < RIL:TL>” as an indicator of trophic level25. Fishes that are more herbivorous will have a higher RIL greater than 1 and upwards of 5–30 whereas carnivorous fishes will generally be much lower < 1 26. Previous work in mammals and fishes has shown that broad trophic levels are generally associated with hindgut microbiome diversity 12,27,28. In temperate marine ecosystems, herbivorous marine fishes are rare and therefore it is possible that our limited sampling of the lower trophic extremes could have led to a lack of signal in our study. We did however show that beta diversity between the midgut and hindgut was generally smaller (more similar) in fishes of lower trophic level based on the diet data. This would suggest that microbial differentiation is greater at the start vs. end of the intestine in more carnivorous fishes. Although we did not measure stomach content or relative intestinal content, it is possible that the higher trophic fishes have lower feeding frequencies and thus higher rates of fasting in the wild which has been shown to be a strong predictor of gut microbial communities 29. It’s also possible that herbivorous fishes, which feed at a higher frequency rate 30, may have overall more similar microbial communities at the proximal and terminal ends of the gut for enhanced nutrient digestion 31. Our study did attempt to collect mostly adult sized fish but it's possible that age could be a confounding variable as herbivorous fish when juveniles are known to have a higher trophic diet 32.
Phylosymbiosis occurs when the “microbiomes recapitulate the phylogeny of the host” and is primarily studied in guts of invertebrates and mammals 33. Our study showed that the hindgut, gill, and skin microbiomes are more similar in fishes that are more genetically similar. To our knowledge, this is the first study in vertebrates to comprehensively evaluate and show phylosymbiosis, in the context of branching length, occurring across multiple body sites. In addition, the discovery of possible phylosymbiosis occurring in the fish gill has not been shown previously. A positive association between the microbiome and host phylogeny is an important finding for guiding future probiotic discovery as most current probiotics used in aquaculture are derived from terrestrial livestock. For vertebrates, phylosymbiosis has primarily been investigated in mammals with a focus on the ‘internal’ gut microbiome 33–35. Only a few studies have investigated phylosymbiosis on animal surfaces such as mammal skin 36 (38 species, 10 orders) 13, bird feathers (7 species, 1 order) 37, Chondrichthyes vs Osteichthyes skin (9 species, 9 orders) 38, tropical reef fish skin (44 species, 5 orders) 39. It is likely that previous attempts to evaluate phylosymbiosis in fishes have been limited due to limited sampling across evolutionary time scales. In addition, since habitat is an important driver of the fish microbiome, it is important to account for this by having enough samples across diverse habitats as well. For fish, our results suggest that phylosymbiosis is strongest in the hindgut with the gill and skin following in importance in the context of weighted measures. For gut comparisons, future studies should aim to investigate the importance of reproductive strategies in fishes (viviparity vs ovoviviparity vs. oviparity) to determine if phylosymbiosis and potentially co-evolution is stronger in fishes which utilize viviparity. Another important aspect to focus on in future analyses is how microbial diversity corresponds with hosts with high species radiation but shallow overall branching length “low genetic divergence” such as some freshwater cichlids 40.
Microbial source tracking analyses showed that sea water contributes more microbes to the fish mucosal environment as compared to sediment. Across body sites, midgut overall had the most microbial sources identified whereas the hindgut and gill had the least (most unknowns). For all body sites however, the majority of microbes were of unknown origin which suggests that further research needs to be conducted to establish a holistic microbial library of the entire marine ecosystem from this California Current Ecosystem region. This sampling effort should include sampling of sediments from deeper depths, sea water from bays and offshore environments, representatives from the thousands of marine invertebrate species, and hundreds of macroalgae species.
Understanding the factors that shape the microbial ecology across vertebrates and specifically fishes remains challenging. Our study showed that amongst vertebrates, fishes have the most unique assemblage of total microbial diversity, which we hypothesized corresponds to the diverse evolutionary history and habitat types exhibited by fishes. For gut samples, 92% of ASVs found in fishes were not found in other classes such as mammals, birds, reptiles, and amphibians despite mammals and reptiles having higher gamma diversity at a 50 species cross-section. Some caveats of our design however was that although all of our fish samples were wild, many of the mammal, bird, reptile, and amphibian samples were from zoo collections 4. Zoo and wild samples may differ in diversity due to restrictions in diet but also in feeding frequency 41. In addition, the actual sampling of organisms was not random across the tree but instead was opportunistic based on available data, thus future studies should revisit these comparisons when higher species representation is obtained especially from wild samples across a large home range. We demonstrated across one of the largest samplings of marine fishes species to date, that mucosal diversity is greater in body sites outside of the hindgut, particularly the midgut, gill, and skin. This is in contrast to mammals and specifically humans that seem to have the highest proportion of microbial diversity concentrated in the hindgut ‘stool’42. Few studies look at the foregut of mammals compared to the hindgut making it difficult to speculate how selection may differ across vertebrate classes. For fish however, because of this drastic difference in midgut to hindgut diversity, it's possible that the majority of midgut microbes are simply from diet and ingested seawater, representing a reservoir of microbes for intestinal colonization. Our study suggested that mammals may have expanded diversity in the hindgut as compared to other vertebrates, including fishes. Whereas fishes, may have higher cumulative diversity spread across other body sites. However, one major caveat to gamma diversity comparisons in vertebrates is the morphological differences in body sites. Fishes and to some extent amphibians have gills whereas mammals, birds, and reptiles have lungs for breathing. External surfaces such as the ‘skin’ also differ widely. The ideal comparison would be to process and extract the entire animal at once, but typically this isn’t feasible due to size limitations. In order to do broad gamma diversity comparisons across vertebrates, one may need to focus on conserved body sites including reproductive organs, oral cavity, anus with the understanding that one would be excluding diversity elsewhere. Higher hindgut diversity in mammals could be explained by the higher occurrence of herbivory in mammals 43. Our study did not include tropical marine fishes from coral reef ecosystems as we concentrated on the California Current marine ecosystem ‘CCE’. The CCE does have tropical fish, but they are associated with rocky reef benthic habitat. The addition of gut microbiomes from coral reef fishes, which are primarily localized in tropical environments, may influence this outcome as herbivory is higher in that habitat for fishes 44. In mammals, one of the most important drivers of hindgut microbial diversity is the complexity and physiology of the gut, namely if hindgut fermentation occurs 45. The adaptation of herbivory may have led to expanded physiological and morphological attributes in the foregut and hindgut leading to a novel ecological niche for microbial colonization and symbiosis of fermentative taxa 5.
Because few vertebrate microbiome studies are inclusive of multiple body sites, it is difficult to compare cumulative gamma diversity of the fishes here to other vertebrates aside from our observation of higher microbial diversity in external body sites such as the gill and skin in fishes. Our attempts to estimate total microbial diversity across body sites extrapolated to 35,000 fish species demonstrates that despite an incredibly rich dataset with a large range of fish diversity, our investigations of microbial diversity in fishes are superficial. We suspect the expanded diversity in external sites in fishes may be explained by an evolutionary pressure of a high exposure rate to microbes in the aquatic environment. This may have led to the diversification of the immune system including mucosal site specific lymphoid associated tissue and mucus production 46–48. Microbiome diversity in the hindgut but not external sites is partially associated with immune system complexity of the hosts 49. A follow up study would be to compare the immune components (e.g. gene expression, protein, or metabolome profiles) across these different body sites (gill, skin, gut) within an individual fish to determine the extent the host immune system influences (permits or excludes) microbial diversity at a local body site level. In addition, fishes that are naturally exposed to higher microbial diversity in their life history (oligotrophic vs eutrophic or pelagic vs. benthic) may exhibit differences in the evolution of their immune system.