The gut microbiota of the marine herbivorous fish Kyphosus sydneyanus are thought to play an important role in host nutrition by supplying short-chain fatty acids (SCFAs) through fermentation of dietary red and brown macroalgae. Here, using 645 metagenome-assembled genomes (MAGs) from wild fish, we determined the capacity of different bacterial taxa to degrade seaweed carbohydrates along the gut. Most bacteria (99%) were unclassified at the species level. Gut communities and CAZyme-related transcriptional activity were dominated by Bacteroidia and Clostridia. Both classes possess genes CAZymes acting on internal polysaccharide bonds, suggesting their role initiating glycan depolymerization, followed by rarer Gammaproteobacteria and Verrucomicrobiae. Results indicate that Bacteroidia utilize substrates in both brown and red algae, whereas other taxa, namely, Clostridia, Bacilli, and Verrucomicrobiae, utilize mainly brown algae. Bacteroidia had the highest CAZyme gene densities overall, and Alistipes were especially enriched in CAZyme gene clusters (n = 73 versus just 62 distributed across all other taxa), pointing to an enhanced capacity for macroalgal polysaccharide utilization (e.g., alginate, laminarin, and sulfated polysaccharides). Pairwise correlations of MAG relative abundances and encoded CAZyme compositions provide evidence of potential inter-species collaborations. Co-abundant MAGs exhibited complementary degradative capacities for specific substrates, and flexibility in their capacity to source carbon (e.g., glucose- or galactose-rich glycans), possibly facilitating coexistence via niche partitioning. Results indicate the potential for collaborative microbial carbohydrate metabolism in the K. sydneyanus gut, that a greater variety of taxa contribute to the breakdown of brown versus red dietary algae, and that Bacteroidia encompass specialized macroalgae degraders.

BACKGROUND: Biogeography has been linked to differences in gut microbiota in several animals. However, the existence of such a relationship in fish is not clear yet. So far, it seems to depend on the fish species studied. However, most studies of fish gut microbiotas are based on single populations. In this study, we investigated the gut microbiota of fish from three wild populations of the two-banded sea bream Diplodus vulgaris (Geoffroy Saint-Hilaire, 1817) to determine whether its diversity, structure and potential functionality reflect the geographic origin of the fish, at large and small geographical scale. Additionally, we explored the host- and environmental-related factors explaining this relationship.
RESULTS: We showed that the taxonomy and potential functionality of the mucosa-associated gut microbiota of Diplodus vulgaris differ to varying degrees depending on the spatial scale considered. At large scale, we observed that both the taxonomical structure and the potential functionality of the fish microbiota differed significantly between populations. In contrast, the taxonomical diversity of the microbial community displayed a significant relationship with factors other than the geographic origin of the fish (i.e. sampling date). On the other hand, at small scale, the different composition and diversity of the microbiota differ according to the characteristics of the habitat occupied by the fish. Specifically, we identified the presence of Posidonia oceanica in the benthic habitat as predictor of both the microbiota composition and diversity. Lastly, we reported the enrichment of functions related to the metabolism of xenobiotics (i.e. drugs and 4-aminobenzoate) in a population and we indicated it as a potential target of future monitoring.
CONCLUSIONS: With this study, we confirmed the importance of investigating the gut microbiota of wild fish species using multiple populations, taking into account the different habitats occupied by the individuals. Furthermore, we underscored the use of the biodegradation potential of the gut microbiota as an alternative means of monitoring emerging contaminants in Mediterranean fish.

Coastal herbivorous fishes consume macroalgae, which is then degraded by microbes along their digestive tract. However, there is scarce genomic information about the microbiota that perform this degradation. This study explores the potential of Kyphosus gastrointestinal microbial symbionts to collaboratively degrade and ferment polysaccharides from red, green, and brown macroalgae through in silico study of carbohydrate-active enzyme and sulfatase sequences. Recovery of metagenome-assembled genomes (MAGs) from previously described Kyphosus gut metagenomes and newly sequenced bioreactor enrichments reveals differences in enzymatic capabilities between the major microbial taxa in Kyphosus guts. The most versatile of the recovered MAGs were from the Bacteroidota phylum, whose MAGs house enzyme collections able to decompose a variety of algal polysaccharides. Unique enzymes and predicted degradative capacities of genomes from the Bacillota (genus Vallitalea) and Verrucomicrobiota (order Kiritimatiellales) highlight the importance of metabolic contributions from multiple phyla to broaden polysaccharide degradation capabilities. Few genomes contain the required enzymes to fully degrade any complex sulfated algal polysaccharide alone. The distribution of suitable enzymes between MAGs originating from different taxa, along with the widespread detection of signal peptides in candidate enzymes, is consistent with cooperative extracellular degradation of these carbohydrates. This study leverages genomic evidence to reveal an untapped diversity at the enzyme and strain level among Kyphosus symbionts and their contributions to macroalgae decomposition. Bioreactor enrichments provide a genomic foundation for degradative and fermentative processes central to translating the knowledge gained from this system to the aquaculture and bioenergy sectors.IMPORTANCESeaweed has long been considered a promising source of sustainable biomass for bioenergy and aquaculture feed, but scalable industrial methods for decomposing terrestrial compounds can struggle to break down seaweed polysaccharides efficiently due to their unique sulfated structures. Fish of the genus Kyphosus feed on seaweed by leveraging gastrointestinal bacteria to degrade algal polysaccharides into simple sugars. This study reconstructs metagenome-assembled genomes for these gastrointestinal bacteria to enhance our understanding of herbivorous fish digestion and fermentation of algal sugars. Investigations at the gene level identify Kyphosus guts as an untapped source of seaweed-degrading enzymes ripe for further characterization. These discoveries set the stage for future work incorporating marine enzymes and microbial communities in the industrial degradation of algal polysaccharides.

Coastal herbivorous fishes consume macroalgae, which is then degraded by microbes along their digestive tract. However, there is scarce foundational genomic work on the microbiota that perform this degradation. This study explores the potential of Kyphosus gastrointestinal microbial symbionts to collaboratively degrade and ferment polysaccharides from red, green, and brown macroalgae through in silico study of carbohydrate-active enzyme and sulfatase sequences. Recovery of metagenome-assembled genomes (MAGs) reveals differences in enzymatic capabilities between the major microbial taxa in Kyphosus guts. The most versatile of the recovered MAGs were from the Bacteroidota phylum, whose MAGs house enzymes able to decompose a variety of algal polysaccharides. Unique enzymes and predicted degradative capacities of genomes from the Bacillota (genus Vallitalea) and Verrucomicrobiota (order Kiritimatiellales) suggest the potential for microbial transfer between marine sediment and Kyphosus digestive tracts. Few genomes contain the required enzymes to fully degrade any complex sulfated algal polysaccharide alone. The distribution of suitable enzymes between MAGs originating from different taxa, along with the widespread detection of signal peptides in candidate enzymes, is consistent with cooperative extracellular degradation of these carbohydrates. This study leverages genomic evidence to reveal an untapped diversity at the enzyme and strain level among Kyphosus symbionts and their contributions to macroalgae decomposition. Bioreactor enrichments provide a genomic foundation for degradative and fermentative processes central to translating the knowledge gained from this system to the aquaculture and bioenergy sectors.

Given their role in lipid digestion, feed supplementation with bile salts could be an economic and sustainable solution to alterations in adiposity and intestinal inflammation generated by some strategies currently used in aquaculture. An important part of the metabolism of bile salts takes place in the intestine, where the microbiota transforms them into more toxic forms. Consequently, we aimed to evaluate the gut immune response and microbial populations in gilthead seabream (Sparus aurata) fed a diet supplemented with a blend of bile salts with proven background as a regulator of lipid metabolism and fat content. After the 90-day feeding trial, a differential modulation of the microbiota between the anterior and posterior intestine was observed. While in the anterior intestine the relative abundance of Desulfobacterota doubled, in the posterior intestine, the levels of Firmicutes increased and Proteobacteria, Actinobacteriota, and Campylobacterota were reduced when supplementing the diet with bile salts. Even so, only in the anterior intestine, there was a decrease in estimated richness (Chao1 and ACE indices) in presence of dietary bile salts. No significant differences were displayed in alpha (Shannon and Simpson indices) nor beta-diversity, showing that bile sales did not have a great impact on the intestinal microbiota. Regarding the gene expression profile in 2 h postprandial-fish, several changes were observed in the analyzed biomarkers of epithelial integrity, nutrient transport, mucus production, interleukins, cell markers, immunoglobulin production and pathogen recognition receptors. These results may indicate the development of an intestinal immune-protective status to tackle future threats. This work also suggests that this immune response is not only regulated by the presence of the dietary bile salts in the intestine, but also by the microbial populations that are in turn modulated by bile salts. After a fasting period of 2 days, the overall gene expression profile was stabilized with respect to fish fed the unsupplemented diet, indicating that the effect of bile salts was transient after short periods of fasting. On the balance, bile salts can be used as a dietary supplement to enhance S. aurata farming and production without compromising their intestinal health.

Marine herbivorous fish that feed primarily on macroalgae, such as those from the genus Kyphosus, are essential for maintaining coral health and abundance on tropical reefs. Here, deep metagenomic sequencing and assembly of gut compartment-specific samples from three sympatric, macroalgivorous Hawaiian kyphosid species have been used to connect host gut microbial taxa with predicted protein functional capacities likely to contribute to efficient macroalgal digestion. Bacterial community compositions, algal dietary sources, and predicted enzyme functionalities were analyzed in parallel for 16 metagenomes spanning the mid- and hindgut digestive regions of wild-caught fishes. Gene colocalization patterns of expanded carbohydrate (CAZy) and sulfatase (SulfAtlas) digestive enzyme families on assembled contigs were used to identify likely polysaccharide utilization locus associations and to visualize potential cooperative networks of extracellularly exported proteins targeting complex sulfated polysaccharides. These insights into the gut microbiota of herbivorous marine fish and their functional capabilities improve our understanding of the enzymes and microorganisms involved in digesting complex macroalgal sulfated polysaccharides. IMPORTANCE This work connects specific uncultured bacterial taxa with distinct polysaccharide digestion capabilities lacking in their marine vertebrate hosts, providing fresh insights into poorly understood processes for deconstructing complex sulfated polysaccharides and potential evolutionary mechanisms for microbial acquisition of expanded macroalgal utilization gene functions. Several thousand new marine-specific candidate enzyme sequences for polysaccharide utilization have been identified. These data provide foundational resources for future investigations into suppression of coral reef macroalgal overgrowth, fish host physiology, the use of macroalgal feedstocks in terrestrial and aquaculture animal feeds, and the bioconversion of macroalgae biomass into value-added commercial fuel and chemical products.

Natural waterways near urban areas are heavily impacted by anthropogenic activities, including their microbial communities. A contaminant of growing public health concern in rivers is antibiotic resistant genes (ARGs), which can spread between neighboring bacteria and increase the potential for transmission of AR bacteria to animals and humans. To identify the matrices of most concern for AR, we compared ARG burdens and microbial community structures between sample types from the Scioto River Watershed, Ohio, the United States, from 2017 to 2018. Five environmental matrices (water, sediment, periphyton, detritus, and fish gut) were collected from 26 river sites. Due to our focus on clinically relevant ARGs, three carbapenem resistance genes (blaKPC, blaNDM, and blaOXA-48) were quantified via DropletDigital™ PCR. At a subset of nine urbanized sites, we conducted16S rRNA gene sequencing and functional gene predictions. Carbapenem resistance genes were quantified from all matrices, with blaKPC being the most detected (88 % of samples), followed by blaNDM (64 %) and blaOXA-48 (23 %). Fish gut samples showed higher concentrations of blaKPC and blaNDM than any other matrix, indicating potential ARG bioaccumulation, and risk of broader dissemination through aquatic and nearshore food webs. Periphyton had higher concentrations of blaNDM than water, sediment, or detritus. Microbial community analysis identified differences by sample type in community diversity and structure. Sediment samples had the most diverse microbial communities, and detritus, the least. Spearman correlations did not reveal significant relationships between the concentrations of the monitored ARGs and microbial community diversity. However, several differentially abundant taxa and microbial functions were identified by sample type that is definitive of these matrices\' roles in the river ecosystem and habitat type. In summary, the fish gut and periphyton are a concern as AR reservoirs due to their relatively high concentration of carbapenem resistance genes, diverse microbial communities, and natural functions that promote AR.

Municipal wastewater treatment plant (WWTP) effluent contains pharmaceuticals and personal care products known to affect fish health and reproduction. The microbiome is a community of bacteria integral in maintaining host health and is influenced by species, diet, and environment. This study investigated changes in the diversity and composition of the gut content microbiome of rainbow darter (Etheostoma caeruleum) at ten sites on the Grand River, Ontario, Canada. Gut contents were collected in fall 2018 from these fish at sites upstream and downstream of two municipal wastewater treatment plants (WWTPs; Waterloo and Kitchener). 16S rRNA genes were sequenced to determine the composition and diversity (alpha and beta) of microbial taxa present. Gut content bacterial alpha diversity increased downstream of both WWTP outfalls; dominance of bacterial amplicon sequence variants decreased compared to upstream fish. Fish collected at different sites had distinct bacterial communities, with upstream samples dominant in Proteobacteria and Firmicutes, and downstream samples increasingly abundant in Proteobacteria and Cyanobacteria. In mammals, increased abundance of Proteobacteria is indicative of microbial dysbiosis and has been linked to altered health outcomes, but this is not yet known for fish. This research indicates that the fish gut content microbiome was altered downstream of WWTP effluent outfalls and could lead to negative health outcomes.

Herbivorous fishes play important ecological roles in coral reefs by consuming algae that can otherwise outcompete corals, but we know little about the gut microbiota that facilitates this process. This study focussed on the gut microbiota of an ecologically important coral reef fish, the convict surgeonfish Acanthurus triostegus. We sought to understand how the microbiome of this species varies along its gastrointestinal tract and how it varies between juvenile and adult fish. Further, we examined if the bacteria associated with the diet consumed by juveniles contribute to the gut microbiota. 16S rRNA gene amplicon sequencing showed that bacterial communities associated with the midgut and hindgut regions were distinct between adults and juveniles; however, no significant differences were seen for gut wall samples. The microbiota associated with the epilithic algal food source was similar to that of the juvenile midgut and gut wall but differed from the microbiome of the hindgut. A core bacterial community including members of taxa Epulopiscium and Brevinemataceae was observed across all gastrointestinal and diet samples, suggesting that these bacterial symbionts can be acquired by juvenile convict surgeonfish horizontally via their diet and then are retained into adulthood.

The expansion of offshore oil exploration increases the risk of marine species being exposed to oil pollution in currently pristine areas. The adverse effects of oil exposure through toxic properties of polycyclic aromatic hydrocarbons (PAHs) have been well studied in Atlantic cod (Gadus morhua). Nevertheless, the fate of conjugated metabolites in the intestinal tract and their effect on the diversity of intestinal microbial community in fish is less understood. Here, we investigated the intestinal microbial community composition of Atlantic cod after 28 days of exposure to crude oil (concentration range 0.0-0.1 mg/L).
Analysis of PAH metabolites in bile samples confirmed that uptake and biotransformation of oil compounds occurred as a result of the exposure. Various evidence for altered microbial communities was found in fish exposed to high (0.1 mg/L) and medium (0.05 mg/L) concentrations of oil when compared to fish exposed to low oil concentration (0.01 mg/L) or no oil (control). First, altered banding patterns were observed on denaturing gradient gel electrophoresis for samples pooled from each treatment group. Secondly, based on 16S rRNA sequences, higher levels of oil exposure were associated with a loss of overall diversity of the gut microbial communities. Furthermore, 8 operational taxonomic units (OTUs) were found to have significantly different relative abundances in samples from fishes exposed to high and medium oil concentrations when compared to samples from the control group and low oil concentration. Among these, only one OTU, a Deferribacterales, had increased relative abundance in samples from fish exposed to high oil concentration.
The results presented herein contribute to a better understanding of the effects of oil contamination on the gut microbial community changes in fish and highlight the importance of further studies into the area. Our findings suggest that increased relative abundance of bacteria belonging to the order Deferribacterales may be indicative of exposure to oil at concentrations higher than 0.05 mg/L.