Grass carp HB-EGFa enhances resistance to Aeromonas hydrophila infection by fortifying gut mucosal barrier function

Grass carp (Ctenopharyngodon idella) is a globally important aquaculture species, yet its production is severely threatened by pathogenic infections. The gut mucosal barrier plays a pivotal role in defending against pathogens, and heparin-binding EGF-like growth factor (HB-EGF) is known to regulate epithelial barrier integrity in mammals. However, the molecular characteristics and functional roles of HB-EGF in grass carp remain unclear. Here, we identified grass carp HB-EGFa and its receptor EGF receptor (EGFR), which showed high sequence conservation with cyprinid orthologs. Under normal conditions, HB-EGFa was widely expressed across tissues, with the highest levels in muscle and gill, while EGFR was most abundant in the liver. During Aeromonas hydrophila infection, both genes were upregulated in immune tissues (spleen, liver, kidney and gut). Functional assays with recombinant HB-EGFa revealed it enhanced gut barrier integrity in challenged fish by preserving villus architecture, increasing goblet cells, and modulating tight junctions (upregulating ZO-1, Occludin, Claudin-1; downregulating Claudin-2). It also stimulated epithelial proliferation and suppressed apoptosis, while upregulating antimicrobial effectors (MUC2, LEAP-2, Lyz1 and Hepcidin) and reducing gut bacterial load. These results demonstrate that grass carp HB-EGFa strengthens mucosal barrier function and innate immunity against A. hydrophila, highlighting its potential as a target for improving aquaculture disease resistance.