Herbivorous Fish Microbiome Adaptations to Sulfated Dietary Polysaccharides.

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.

Nutritional Attributes, Substitutability, Scalability, and Environmental Intensity of an Illustrative Subset of Current and Future Protein Sources for Aquaculture Feeds: Joint Consideration of Potential Synergies and Trade-offs.

Aquaculture is anticipated to play an increasingly important role in global food security because it may represent one of the best opportunities to increase the availability of healthy animal protein in the context of resource and environmental constraints. However, the growth and sustainability of the aquaculture industry faces important bottlenecks with respect to feed resources, which may be derived from diverse sources. Here, using a small but representative subset of potential aquafeed inputs (which we selected to highlight a range of relevant attributes), we review a core suite of considerations that need to be accommodated in concert in order to overcome key bottlenecks to the continued development and expansion of the aquaculture industry. Specifically, we evaluate the nutritional attributes, substitutability, scalability, and resource and environmental intensity of each input. On this basis, we illustrate a range of potential synergies and trade-offs within and across attributes that are characteristic of ingredient types. We posit that the recognition and management of such synergies and trade-offs is imperative to satisfying the multi-objective decision-making associated with sustainable increases in future aquaculture production.

Towards the development of a sustainable soya bean-based feedstock for aquaculture.

Soya bean (Glycine max (L.) Merr.) is sought after for both its oil and protein components. Genetic approaches to add value to either component are ongoing efforts in soya bean breeding and molecular biology programmes. The former is the primary vegetable oil consumed in the world. Hence, its primary usage is in direct human consumption. As a means to increase its utility in feed applications, thereby expanding the market of soya bean coproducts, we investigated the simultaneous displacement of marine ingredients in aquafeeds with soya bean-based protein and a high Omega-3 fatty acid soya bean oil, enriched with alpha-linolenic and stearidonic acids, in both steelhead trout (Oncorhynchus mykiss) and Kampachi (Seriola rivoliana). Communicated herein are aquafeed formulations with major reduction in marine ingredients that translates to more total Omega-3 fatty acids in harvested flesh. Building off of these findings, subsequent efforts were directed towards a genetic strategy that would translate to a prototype design of an optimal identity-preserved soya bean-based feedstock for aquaculture, whereby a multigene stack approach for the targeted synthesis of two value-added output traits, eicosapentaenoic acid and the ketocarotenoid, astaxanthin, were introduced into the crop. To this end, the systematic introduction of seven transgenic cassettes into soya bean, and the molecular and phenotypic evaluation of the derived novel events are described.

Enrichable consortia of microbial symbionts degrade macroalgal polysaccharides in Kyphosus 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.

Tropical Red Macroalgae Cultivation with a Focus on Compositional Analysis.

To create carbon efficient sources of bioenergy feedstocks and feedstuff for aquaculture and terrestrial livestock, it is critical to develop and commercialize the most efficient seaweed cultivation approach with a sustainable nutrient input supply. Here, we present data for a novel, onshore tropical macroalgae cultivation system, based on influent deep seawater as the nutrient and carbon sources. Two red algal species were selected, Agardhiella subulata and Halymenia hawaiiana, as the basis for growth optimization. Highest productivity in small-scale cultivation was demonstrated with A. subulata in the 10% deep seawater (64.7 µg N L-1) treatment, growing at up to 26% specific growth rate day-1 with highest yields observed at 247.5 g m-2 day-1 fresh weight. The highest yields for H. hawaiiana were measured with the addition of 10% deep seawater up to 8.8% specific growth rate day-1 and yields at 63.3 g fresh weight m-2 day-1 equivalent. Biomass should be culled weekly or biweekly to avoid density limitations, which likely contributed to a decrease in SGR over time. With a measured 30-40% carbon content of the ash-free dry weight (20-30% of the dry weight) biomass, this translates to an almost 1:1 CO2 capture to biomass ratio. The compositional fingerprint of the high carbohydrate content of both Agardhiella and Halymenia makes for an attractive feedstock for downstream biorefinery applications. By focusing on scaling and optimizing seaweed farming technologies for large-scale onshore farms, the opportunities for yield potential, adaptability to cultivation conditions, and meeting global sustainability goals through novel, carbon-negative biomass sources such as seaweed can be realized.

Enrichable consortia of microbial symbionts degrade macroalgal polysaccharides in Kyphosus fish.

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.

Fine scale transitions of the microbiota and metabolome along the gastrointestinal tract of herbivorous fishes.

BACKGROUND: Gut microorganisms aid in the digestion of food by providing exogenous metabolic pathways to break down organic compounds. An integration of longitudinal microbial and chemical data is necessary to illuminate how gut microorganisms supplement the energetic and nutritional requirements of animals. Although mammalian gut systems are well-studied in this capacity, the role of microbes in the breakdown and utilization of recalcitrant marine macroalgae in herbivorous fish is relatively understudied and an emerging priority for bioproduct extraction. Here we use a comprehensive survey of the marine herbivorous fish gut microbial ecosystem via parallel 16S rRNA gene amplicon profiling (microbiota) and untargeted tandem mass spectrometry (metabolomes) to demonstrate consistent transitions among 8 gut subsections across five fish of the genus of Kyphosus. RESULTS: Integration of microbial phylogenetic and chemical diversity data reveals that microbial communities and metabolomes covaried and differentiated continuously from stomach to hindgut, with the midgut containing multiple distinct and previously uncharacterized microenvironments and a distinct hindgut community dominated by obligate anaerobes. This differentiation was driven primarily by anaerobic gut endosymbionts of the classes Bacteroidia and Clostridia changing in concert with bile acids, small peptides, and phospholipids: bile acid deconjugation associated with early midgut microbiota, small peptide production associated with midgut microbiota, and phospholipid production associated with hindgut microbiota. CONCLUSIONS: The combination of microbial and untargeted metabolomic data at high spatial resolution provides a new view of the diverse fish gut microenvironment and serves as a foundation to understand functional partitioning of microbial activities that contribute to the digestion of complex macroalgae in herbivorous marine fish.