Single-cell transcriptomics unveils leukocyte heterogeneity in the gills of Larimichthys crocea in response to parasitic infection

Background Fish gills serve as critical immune interfaces against aquatic pathogens, yet their leukocyte heterogeneity in response to parasitic infections remains poorly understood. Methods Single-cell RNA sequencing was employed to elucidate leukocyte responses in the gills of Larimichthys crocea during Cryptocaryon irritans infection. Results A total of 13,070 leukocytes from the gills under steady-state and infected conditions were profiled and classified into eight principal lineages: T cells (> 70% of total immune cells), ILC2-like cells, NK-like cells, neutrophils, cpa5 + granulocytes, B cells, macrophages, and dendritic cells. Following infection, T cell subsets exhibited distinct responses: Regulatory T cells expanded and demonstrated immunoregulatory capacity; CD8 + T cells exhibited cytotoxic responses; CD4 - CD8 - T cells displayed Th17-like functions; and γδ T cells showed Th2-like activity. ILC2-like cells significantly increased in abundance and upregulated type 2 cytokine expression, whereas cytotoxic NK-like cells enhanced chemokine signaling and cytotoxicity. Neutrophils increased in number and oxidative activity, while cpa5 + granulocytes highlighted immunomodulatory functions. Macrophages, dendritic cells, and B cells exhibited compartmentalized activation states, upregulating gene modules associated with pathogen recognition, antigen processing/presentation, chemotactic activity, and antibody defenses. Conclusions These findings describe a multi-layered immune cell defense strategy in the gills of teleosts against parasitic infection, showing conserved and fish-specific adaptations. Understanding gill immunity provides viable targets for enhancing parasite resistance in aquaculture, such as modulating ILC2/Treg pathways to prevent infections.