Ferroptosis Triggered by Gill Dysbiosis Mediates Immune Failure in Grass Carp Overwintering Syndrome

Overwintering syndrome (OWS) causes massive mortality in grass carp ( Ctenopharyngodon idella ) during early spring, yet its pathogenesis beyond the "low-temperature–pathogen" paradigm remains unclear. This study investigates the cascade mechanism through integrated multi-omics analyses on gill tissues of healthy and OWS-affected grass carp. Gill histopathology revealed progressive necrotizing inflammation in OWS-affected grass carp. Transcriptomics identified 962 differentially expressed genes, highlighting upregulation of cytokine signaling, porphyrin metabolism, and ferroptosis alongside downregulation of adrenergic pathways. Ferroptosis was confirmed through the suppression of glutathione peroxidase 4 (GPX4) and the accumulation of iron in necrotic lamellae. Mucous cell depletion and eosinophil disintegration compromised the mucosal barriers of the gills. Metagenomic analysis first revealed a state of microbial dysbiosis, marked by bacterial dominance comprising 92.21% of the community, along with an enrichment of biofilm and exotoxin genes and a reduction in immune modulation, all exacerbating barrier dysfunction. Subsequent integrated correlation analysis linked the specifically enriched bacteria taxa, including Caldilinea sp. CFX5 and Ilumatobacter sp., to the upregulation of metabolic and virulence pathways, while also revealing significant microbiota–gene interactions that modulate gill immune and metabolic functions. Although previously reported pathogens like Flavobacterium and Aeromonas showed increased abundance in OWS-affected fish, their overall abundance remained low. Based on these findings, we propose a "Dysbiosis–Ferroptosis–Collapse" axis, in which microbiota-driven barrier disruption promotes iron overload and the suppression of GPX4, triggering non-resolving inflammation and ion dyshomeostasis. Targeting this cascade provides a novel theoretical framework for intervening in and developing new treatments for OWS.