Unveiling a new hemocyte subpopulation in white shrimp<i> (Penaeus</i><i> vannamei)</i> and the characterization of immune response in hemocyte subpopulation

Abstract

Hemocytes are the primary cellular components of the shrimp immune system, playing a crucial role in host defense. However, a comprehensive understanding of their subpopulations and specific functions remains incomplete. In this study, four hemocyte subpopulations, designated as H1, H2, H3, and H4, were identified in Penaeus vannamei using transmission electron microscopy (TEM) and flow cytometry. The H1 subpopulation was the most abundant cells, the smallest in size, lacked granules, and had the highest nucleus-to-cytoplasm (N:C) ratio, identifying it as prohemocytes (immature cells). The H2 subpopulation fits the criteria of hyaline hemocytes. They are relatively small, have a large nucleus, and contain no or very few cytoplasmic granules. The H3 subpopulation was the least abundant cell. These cells are larger than HCs, have a moderate N:C ratio, and contain a few granules, identifying them as semi-granulocytes. The H4 subpopulation, representing granulocytes, had the largest cell size and the lowest N:C ratio and was characterized by the presence of large granules in the cytoplasm. Non-specific immune responses were investigated through various parameters and gene expression profiling. Each hemocyte subpopulation exhibited distinct immune functions. Prohemocytes strongly expressed notch-1, suggesting a role in hemocyte proliferation. Hyalinocytes exhibited strong phagocytic activity and produced superoxide anions. Semigranulocytes exhibited high expression of lysozyme and anti-lipopolysaccharide factor. Granulocytes showed high expression of propo-1, propo-2, and antimicrobial peptide genes. Following Vibrio parahaemolyticus injection, the H1 subpopulation significantly increased at 6 h post-infection before returning to baseline levels, whereas the H4 subpopulation followed an opposite trend. These findings suggest that both H1 and H4 hemocytes play critical roles in the immune response against V. parahaemolyticus.