Herbivorous Fish Microbiome Adaptations to Sulfated Dietary Polysaccharides

Abstract

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.

Keywords: fish gut microbiome; kyphosid; macroalgal digestion; polysaccharide utilization; sulfatase.

FIG 1

Relative abundance of fish gut-associated microbial taxa. Relative abundances are based on Kraken2 analysis of unassembled Illumina NovaSeq reads. Sample ID numbers indicate fish numbers (Table S1), followed by gut region code, in progressively distal order. Abbreviations: GI, midgut; HG, hindgut.

FIG 2

Single-copy rpoB gene bacterial taxonomy. Predicted protein sequences from this study are shown in black, identified by fish number, gut compartment, and Prokka annotation number. Sequences in red are from GenBank nr, with selected species known to digest sulfated macroalgal polysaccharides highlighted in boldface as outgroups. Blue text indicates predicted rpoB sequences from terrestrial ruminant MAGs (not currently included in GenBank as protein sequences) prefixed with GenBank genome identifier codes from reference , followed by Prokka annotation numbers from this study (e.g., RUGXXXXX). Additional information about these sequences is provided in Table S2.

FIG 3

Relative abundance of dietary algal taxa. Relative abundances of algal taxa were calculated based Kraken2 analysis of the same sets of unassembled Illumina NovaSeq reads shown in Fig. 1.

FIG 4

Polysaccharide hydrolase family enrichment. (A) Text colors denote predicted substrates based on CAZy annotations: red, red algae; brown, brown algae; green, green algae; purple, nonalgae; gray, ambiguous. Abbreviations: norm, normalized per 1 million predicted metagenomic proteins; HG, hindgut; rum, ruminant. Full data and significance calculations are provided in Data Set S2. (B) Taxonomic distribution of families enriched in fish relative to terrestrial ruminants, based on the top BLAST matches to database relatives. (Note that not all candidates had database matches.) (C) Intra-CAZy family diversity within fish gut metagenomes. The top of the box indicates the 75% percentile boundary, the bottom of the box indicates the 25% percentile boundary, the horizontal line indicates the median, X indicates the mean value, the whisker indicates the standard deviation, and dots indicate the outlier points.

FIG 5

(A) SulfAtlas enzyme family enrichment. Abbreviations: avg norm, group average normalized per 1 million predicted metagenomic proteins; HG, hindgut; rum, ruminant. Full data are provided in Data Set S2. (B) Taxonomic assignments of fish sulfatase class genes matching GenBank nr sequences with minimum blastp E value scores of <1e−5 and alignments covering >30% of database protein length; (C) Intra-SulfAtlas family diversity within fish gut metagenomes. Box-whisker plot elements are defined in the legend to Fig. 4C.

FIG 6

SulfAtlas and CAZy family colocalization networks. Enzyme classes enriched in fish relative to terrestrial ruminants are shown as nodes, with edges denoting colocalization on the same contig within an intervening distance of 25 or fewer genes. Node size is proportional to the total number of edges and edge thickness to connection frequency. Self-loops have been omitted to simplify visualization. (A) Full network diagram for families described in Fig. 4 and 5; (B) subnetwork including CAZy families only; (C) subnetwork including SulfAtlas families only. Raw data tables are provided in Data Set S3.

FIG 7

Colocalization network diagrams for individual fish-enriched CAZy families. Text colors denote predicted macroalgal substrate sources, as defined in the legend to Fig. 4. Numbers in parentheses indicate relative gene frequencies per 1 million proteins in adult fish metagenomes. The maximum intervening distance between nodes is 25 genes.

FIG 8

Colocalization network diagrams for individual fish-enriched SulfAtlas families. Numbers in parentheses indicate relative gene frequencies per 1 million proteins in adult fish metagenomes. The maximum intervening distance between nodes is 25 genes.

FIG 9

Colocalization frequencies for CAZy and SulfAtlas enzyme families. (A) Colocalization frequencies between SulfAtlas and CAZy family members; (B) self-colocalization frequencies within SulfAtlas families; (C) self-colocalization frequencies within CAZy families. Darker colors indicate higher values for the number of times each intersecting family pair or self-pair occurred on the same metagenomic contig with an intervening distance of 25 genes or less. Text colors for CAZy families denote predicted macroalgal sources, as defined in the legend to Fig. 4.