The need for high-resolution gut microbiome characterization to design efficient strategies for sustainable aquaculture production

The study addresses how microbiome-directed fibers (MDFs) might modulate the gut microbiome of Atlantic salmon to improve aquaculture sustainability and production. With rising global seafood demand and the need for sustainable feeds, MDFs that selectively stimulate beneficial gut microbes are attractive. Prior work in terrestrial animals and in vitro salmon models suggested mannans (α-MOS and β-mannans) could shift microbial communities and enhance beneficial metabolites. However, functional evaluation in salmon has been limited by sparse genomic resources. The authors sought to: (i) mechanistically connect salmon gut microbes, their metabolic functions, and host physiology, and (ii) evaluate α- and β-mannans as MDFs in vivo across salmon life stages using integrated multi-omics. The overarching hypothesis was that mannan MDFs would selectively activate beneficial salmon gut microbes, yielding measurable host physiological benefits.

The paper reviews MDFs as targeted fibers whose structures match microbial enzymatic capabilities, citing success in monogastric models (e.g., Norway spruce galactoglucomannan selectively enriching Roseburia intestinalis and cross-feeding Faecalibacterium prausnitzii with butyrate shifts). Salmon studies reported growth effects from carbohydrate supplements such as fructo-oligosaccharides and α-MOS, and in vitro work (SalmoSim) indicated α-MOS increased Carnobacterium and certain acids linked to host benefits. Despite taxonomic detection of taxa like Carnobacterium, Roseburia, and Faecalibacterium in salmon, functional inference was hampered by limited genome data. The authors reference their Salmon Microbial Genome Atlas (SMGA), comprising 211 closed genomes and high/medium-quality MAGs from salmon gut, enabling genome-resolved functional omics to discern true diet-microbe interactions beyond feed DNA carry-over biases seen in amplicon-only studies.

Two feeding trials were conducted. - Low-dose mannan trial: Atlantic salmon were fed either a control diet or diets supplemented with 0.2% (w/w) of MDFs: two α-mannans (MC1, MC2) and an acetylated β-galactoglucomannan (MN3). Fish were reared in freshwater then seawater over 110 days, sampled at four timepoints: T0 (parr), T1 (pre-smolts), T2 (smolts), T3 (post-smolts). Phenotypes (growth, organ integrity, welfare indicators) and omics layers were collected: 16S rRNA gene profiling (V4; Illumina MiSeq; QIIME2/DADA2; SILVA 138 taxonomy; decontam filtering; diversity and PERMANOVA in R), host transcriptomics (hindgut tissue RNA-Seq; TruSeq Stranded mRNA; NovaSeq; STAR alignment to Salmo salar GCF_905237065.1; featureCounts; DESeq2), and metatranscriptomics (rRNA/tRNA depletion; host read removal; quantification against SMGA genomes and de novo assemblies with kallisto; DESeq2). - High-dose β-mannan trial: Freshwater salmon were fed control or 4% MN3 (Norway spruce β-galactoglucomannan) for 28 days in triplicate tanks. Sampling included hindgut and pyloric caeca for 16S rRNA profiling, host transcriptomics (hindgut and pyloric caeca RNA-Seq), metatranscriptomics (hindgut), and targeted SCFA metabolomics (GC–MS; acetate, formate, etc.; statistical analysis with t-tests FDR<0.05). Bioinformatics and statistics: Amplicon data processed with cutadapt, QIIME2/DADA2; diversity analyses with phyloseq/vegan; transcriptomics with STAR/featureCounts/DESeq2; metatranscriptomics with fastp, SortMeRNA, STAR host subtraction, kallisto quantification against SMGA and de novo Megahit assemblies, DRAM functional annotation, DESeq2 for differential expression; SCFA analysis via GC–MS and MetaboAnalyst. Statistical significance thresholds used FDR-corrected p<0.05. Experimental design details note acclimation periods, feeding regimes, and standardized welfare/biometric assessments.

- At 0.2% inclusion of two α-mannans (MC1, MC2) and one acetylated β-galactoglucomannan (MN3): - No effects on growth KPIs (weight, length) or organ integrity. - 16S profiling detected 839 genera (44 phyla) overall; no MDF-driven structural changes (alpha/beta diversity) and no diet effect by PERMANOVA; temporal shifts reflected life stage transitions only. - Metatranscriptomics: 117,261 microbial genes detected across samples; activity profiles varied with life stage but did not cluster by diet; only 208 DEGs across all life stages vs control (8–36 per stage), none linked to mannan metabolism. - Host transcriptomics: strong life-stage effects but no significant differences between MDF diets and control. - At 4% β-mannan (MN3): - Significant microbiome compositional changes: Shannon diversity (Wilcoxon p=0.045 hindgut), PERMANOVA diet effect (hindgut p=0.013; pyloric caeca p=0.0035). - Differential genus abundance: increased Burkholderia-Caballeronia-Paraburkholderia (BCP; p<0.001) and Pseudomonas (hindgut p<0.001; pyloric caeca p<0.05); decreased Limosilactobacillus (p<0.001). - No significant changes in host phenotype (Wilcoxon p=0.72 for body weight) or host transcriptome clustering (hindgut PCA) compared to control. - Host DEGs minimal and not linked to mannan: hindgut 6/47,563 genes; pyloric caeca 2/47,433 genes. - Hindgut metatranscriptomics: expression of 17,094 bacterial genes; no significant clustering between diets; only 5 significant DEGs unrelated to mannan processing. - Targeted metabolomics: acetate increased (hindgut t-test p=1.39e-7; pyloric caeca p=3.216e-4), without corresponding upregulation of fermentation pathway genes; no detection of endomannanases or mannan-specific acetyl esterases (CE2/CE17) indicative of galactoglucomannan depolymerization. - Across trials, only one BCP population showed consistent abundance shifts, with no evidence of β-mannan utilization or production of beneficial metabolites from transcript data. - Genome-resolved metatranscriptomics highlighted active pathways for pectic galactans, xylans, chitin, beta-glucans, xyloglucans, mucin derivatives, and undecorated manno-oligosaccharides in selected salmon gut microbes. Host transporters for SCFAs/organic acids (e.g., SLC16A1/MCT1, SLC13 family) were expressed, suggesting capacity to absorb microbial fermentation products. - Lactic acid bacteria (Lactobacillus, Limosilactobacillus) expressed xylosidases, glucosidases, and galactosidases (e.g., GH42, GH43), indicating potential responsiveness to MDFs based on pectin-derived galactans or (arabino)xyloligosaccharides rather than mannans used here.

The multi-omics evaluation demonstrates that α- and β-mannan MDFs, at a realistic industry inclusion (0.2%) and even at a high inclusion (4%), did not deliver meaningful functional activation of the salmon gut microbiome nor measurable host physiological benefits under normal rearing conditions. The modest microbiome compositional shifts at 4% did not translate to functional gene expression changes related to mannan metabolism or host responses. This addresses the initial hypothesis by showing that these specific mannans are poorly matched to the enzymatic capabilities of prevalent salmon gut microbes. The findings underscore the limitations of inferring function from amplicon data alone and highlight the necessity of host-specific genome resources (SMGA) to design selective MDFs. The data instead point toward endogenous LAB (Lactobacillus, Limosilactobacillus) as actionable targets, given their active carbohydrate-degrading enzyme expression aligned with pectin-derived galactans and (arabino)xyloligosaccharides. Strategically aligning MDF chemical structures with the enzymatic toolbox of naturally occurring beneficial taxa could more effectively modulate the salmon gut microbiome toward health-relevant metabolites (e.g., lactate, succinate, acetate) that the host can absorb via expressed transporters.

This study provides an integrated, high-resolution assessment of MDF effects in Atlantic salmon, showing that α- and β-mannan-derived MDFs have negligible impacts on gut microbial function and host physiology at 0.2% inclusion and only limited, non-functional compositional shifts at 4%. The work validates the importance of genome-resolved, host-specific microbiome resources (SMGA) for rational MDF design and cautions against relying on taxonomic signals to predict function. It identifies endogenous LAB as promising targets and suggests that MDFs based on pectin-derived galactans or (arabino)xyloligosaccharides tailored to LAB enzymatic capacities may yield clearer microbiome and host benefits. Future research should apply precision MDFs matched to salmon gut microbiome capabilities, and assess interventions under stress or disease states to broaden potential targets and evaluate functional outcomes.

- Experimental randomization/blinding: The experiments were not randomized and investigators were not blinded to group allocation or outcomes. - Sample size determination: No statistical methods were used to predetermine sample sizes. - Environmental context: Trials were conducted under normal, healthy rearing conditions without pathogenic or environmental stressors; effects under stress/disease may differ. - Ingredient characterization: Exact structural composition of proprietary α-mannans (MC1, MC2) was unavailable, potentially obscuring structure-function interpretation. - Functional capacity: The salmon gut microbiome showed scarce mannan-degrading capabilities (e.g., lack of detected endomannanases/CE2/CE17), limiting the capacity to test mannan MDF hypotheses. - Amplicon biases: While addressed via SMGA and multi-omics, amplicon-based results can be influenced by feed DNA carry-over. - Generalizability: Findings pertain to tested life stages, diets, and rearing conditions; results may not generalize to other aquaculture settings, species, or diet formulations.