Comprehensive Non-Specific Immune and Gene Expression Profiling of FACS-Sorted Hemocyte Subpopulations in Litopenaeus vannamei After Vibrio parahaemolyticus (AHPND Strain) Infection

Vibrio parahaemolyticus is the causative agent of acute hepatopancreatic necrosis disease (AHPND) in white shrimp aquaculture, which often causes high mortality. Circulating hemocytes play a central role during vibriosis infection, however, the hemocyte subpopulation specific responses to VP-AHPND infection remain poorly understood. This study used flow cytometry and fluorescence-activated cell sorting (FACS) to characterize four hemocyte subpopulations (H1–H4), including prohemocyte (H1), hyalinocyte (H2), semi-granulocyte (H3), and granulocyte (H4) based on non-specific immune functions and gene-expression profiles after VP-AHPND infection. Differentially hemocyte counts changed dynamically over 48 h, revealing hemocyte subpopulation specific responses to infection, which are H1 peaked during 6 h post infection, H2 increased for most of the period, whereas H3 and H4 declined at 24 and 48 h, respectively. Gene set variation analysis revealed marked early activation (6 hpi) of coagulation and notch-1 in the H1 subpopulation. The H2 subpopulation showed enrichment of pattern-recognition and coagulation genes, which mostly enhanced in the 12 hpi. Meanwhile, genes associated with prophenoloxidase and antimicrobial peptides (AMPs) were strongly upregulated especially in the late sampling time point (24 and 48 h), coinciding with a decrease in the percentages of their corresponding hemocytes (H3 and H4 subpopulation). These findings reveal that VP infection triggers a coordinated, subpopulation-specific innate immune response instead of a uniform reaction across all hemocytes. By integrating the relative proportions of hemocyte subpopulations, non-specific immune, and gene-expression, this study offers new mechanistic insights into shrimp cellular immunity and highlights key hemocyte subpopulations and pathways as potential targets for enhancing disease resistance in white shrimp aquaculture.