Abstract:

The functional roles of animal-associated microbiota have traditionally been inferred from correlative sequencing studies, leaving causal relationships between specific symbionts and host phenotypes largely unresolved. Here, we present an integrative framework combining shotgun metagenomics, targeted cultivation, whole-genome sequencing, and gnotobiotic experimentation to validate the ecological functions of microbial symbionts across 20 animal species spanning vertebrates and invertebrates. Metagenomic profiling using PICRUSt2 and HUMAnN3 revealed high functional redundancy (85% of metabolic pathways shared across 80% of samples), with key pathways including nitrogen fixation in terrestrial hosts (2.5%), vitamin B12 biosynthesis in vertebrate guts (1.8%), antimicrobial production in marine systems (1.2%), and butyrate synthesis (3.2%). From 120 cultivated isolates recovered across 12 specialised media, we prioritised 100 strains for draft genome sequencing, confirming the presence of nifH nitrogenase genes in Azotobacter spp. from Lumbricus terrestris, cobS cobalamin biosynthesis genes in Lactobacillus spp. from Mus musculus, and polyketide synthase clusters in Vibrio spp. from Salmo salar. In vitro assays validated nitrogen fixation (15 µmol/L ammonia production), antimicrobial activity (10–15 mm inhibition zones against Escherichia coli), and short-chain fatty acid production. Gnotobiotic experiments in germ-free Mus musculus and Danio rerio demonstrated that mono-association with Lactobacillus spp. rescued 80% of growth deficits and normalised vitamin B12 levels (110 pg/mL versus 50 pg/mL in germ-free controls, p < 0.05), while multi-species consortia (Lactobacillus + Clostridium) enhanced phenotypic recovery to 90% through metabolic cross-feeding. These findings transition microbiome research from correlation to causation, establishing a robust pipeline for symbiont validation with direct applications in conservation probiotics and sustainable agriculture.

Keywords:

Functional symbionts, gnotobiotic validation, metagenomics, nitrogen fixation, vitamin B12

Aim/Objectives

Methods