Single-cell RNA sequencing reveals IgM⁺ B cell differentiation and protective responses after DNA vaccination and Vibrio anguillarum challenge in flounder (Paralichthys olivaceus)

B cells are central mediators of vaccine-induced protective immunity in vertebrates. However, cellular programs through which teleost B cells contribute to vaccine-mediated protection remain poorly elucidated. To assess these responses, flounder were intramuscularly immunized with a Vibrio anguillarum DNA vaccine and challenged 35 days after immunization. Head kidney IgM⁺ B cells were isolated using a previously established monoclonal antibody and subjected to single-cell RNA sequencing at 7 days post-challenge. Seven IgM⁺ B cell subsets were resolved: innate B cells, CXCR4⁺ B cells, mature B cells, plasmablasts, TIRAP⁺ B cells, CD22⁺ B cells, and CD44⁺ B cells. Trajectory reconstruction identified three differentiation routes, with CXCR4⁺ B cells and mature B cells representing transitional states closely linked to B cell-mediated protective immunity. Innate B cells and plasmablasts showed prominent enrichment of phagocytic and antibody-secretion programs, respectively. Vaccination expanded both CXCR4⁺ and mature B cell subsets and enhanced pathways associated with T cell interactions, indicating increased capacity to coordinate T cell-dependent immune responses. Plasmablasts also underwent a transcriptional shift from effector activation toward immune homeostasis, suggesting a sustained contribution to long-term protection. These findings demonstrate the functional heterogeneity and differentiation architecture of teleost IgM⁺ B cells and identify T cell-associated B cell subsets as critical cellular mediators of DNA vaccine-induced protection. This study provides new insight into B cell differentiation and vaccine-responsive adaptive immunity in teleosts and broadens understanding of conserved and lineage-specific mechanisms of vertebrate adaptive immunity.