Metagenomic sequencing reveals the dynamic changes of pig gut fungal composition following the ages and identifies fungal species associated with diarrhea in piglets.

Fungi are crucial components of the pig gut microbiome, influencing host immunity and metabolism. However, the investigation about gut fungi via metagenomic sequencing remains challenging due to analytical complexity. Here, we characterized pig gut fungal profiles using 750 metagenomes collected from public repositories and our previous datasets based on a comprehensive collection of fungal reference genomes, and revealed dynamic compositional changes of pig gut fungi from birth to market (7d, 14d, 21d, 28d, 35d, 70d, and 140d). Weaning significantly shaped the gut fungal community, affecting key fungi like Lachancea kluyveri and Kazachstania slooffiae. Inter-kingdom interaction analysis revealed significant correlations between fungi and bacteria, such as between L. kluyveri and Lactobacillus amylovorus (r = -0.48) and between K. slooffiae and Lactobacillus johnsonii (r = 0.75). We identified 87 diarrhea-associated fungal species at the significance threshold of LDA > 2.0 in three experimental piglet cohorts. In antibiotic-free piglets, fungal species from Saccharomyces and Aspergillus, some of which have been considered as potential probiotics, were enriched in healthy individuals, whereas in antibiotic-treated groups, Saccharomyces spp. and K. slooffiae were higher in diarrheal piglets. Notably, K. slooffiae were negatively correlated with the pathogen M. circinelloides, suggesting a protective role during gut dysbiosis. This study provides a foundation for developing fungal-based interventions to improve pig health.