Subpopulation-specific apoptotic responses of hemocytes to decapod iridescent virus 1 in Macrobrachium rosenbergii

Decapod iridescent virus 1 (DIV1) is an emerging viral pathogen responsible for high mortality in cultured Macrobrachium rosenbergii. Despite the pivotal role of hemocytes in shrimp antiviral immunity, the subpopulation-specific apoptotic dynamics in response to DIV1 remain unclear. In this study, we integrated flow cytometry and single-cell RNA sequencing to dissect hemocyte responses during DIV1 infection. Morphological and cytometric analyses identified three major hemocyte subtypes: agranular cells (including hyaline cells and prohemocytes), semi-granular cells (SGCs), and granular cells (GCs). DIV1 infection triggered a significant increase in apoptosis, especially in agranular cells, with rates peaking at 24 h post-infection. Gene set variation analysis revealed marked activation of apoptosis-related pathways, including p53 signaling, DNA repair, and reactive oxygen species (ROS) pathways, particularly in hyaline cells. RNA velocity analysis indicated differentiation trajectories from SGCs and prohemocytes toward hyaline cells, suggesting a compensatory mechanism to maintain immune competence under viral challenge. These findings uncover key immunological functions and lineage plasticity of hemocyte subpopulations and provide new insights into apoptosis-driven antiviral defense strategies in crustaceans.