Metagenomic and metabolomic analysis of the early gut microbiota development in Nipponia nippon

Abstract

The crested ibis (Nipponia nippon) is an endangered bird whose early gut microbiota development remains poorly understood. To address this gap, this study integrated metagenomics and metabolomics to investigate the dynamic succession of gut microbiota and metabolism in naturally hatched chicks during early development. Fecal samples were collected from naturally hatched crested ibis chicks aged 3 to 33 days and divided into groups Z3, Z8, Z13, Z18, Z23, Z28, and Z33. Integrated metagenomics and metabolomics were used to assess microbial gene richness, alpha and beta diversity, taxonomic composition, functional profiles, differentially expressed metabolites, pathway enrichment, and microbe-metabolite correlations. Gut microbial gene richness and alpha diversity increased from Z3 to Z13, then fluctuated and stabilized, with Z13-Z18 representing a critical colonization window. Beta diversity analysis revealed progressive structural remodeling with age. At the phylum level, Pseudomonadota dominated early stages, while Bacillota increased over time. At the genus level, Plesiomonas, Clostridium, and Escherichia were prevalent; Enterobacter and Ligilactobacillus decreased by Z33, whereas Campylobacter, Cetobacterium and Proteus increased. Functional profiling indicated that Z3 exhibited high metabolic activity, including carbohydrate, amino acid, and energy metabolism, while Z33 showed reduced but more specialized functions. Metabolomics identified numerous differentially expressed metabolites across age groups, with pathway enrichment in cofactor biosynthesis, nucleotide metabolism, and amino acid metabolism. Correlation analysis revealed opposite metabolic associations: Corynebacterium was positively correlated with most metabolites, whereas Enterobacter, Citrobacter, and Klebsiella showed negative correlations. These findings reveal dynamic succession of gut microbiota and metabolism during early development in crested ibises. The identification of a critical colonization window and age-related microbial and metabolic shifts provides fundamental insights for improving captive breeding and conservation of this endangered species.