Cross-species single-cell atlas of hemocyte conservation and diversification in crustaceans

Abstract

Crustacean immunity plays a crucial role in aquaculture disease management, while a systematic comparison of hemocyte heterogeneity, developmental trajectories, and immune response plasticity across ecologically divergent crustacean species remains largely unexplored. This study employed cross-species single-cell RNA sequencing analysis to conduct a comprehensive comparative of hemocytes from four economically important crustacean species: Procambarus clarkii (21,623 cells), Penaeus japonicus (43,054 cells), Penaeus monodon (30,913 cells), and Penaeus vannamei (145,094 cells). Conserved hemocyte populations, including hematopoietic progenitors (prohemocytes) and mature effector cells (e.g., proPO and VEGF/ALF clusters), were identified. Trajectory analysis revealed developmental relationships between hemocyte subsets, with prohemocytes consistently positioned at the start of differentiation pathways across species. Pseudotime analysis revealed two conserved differentiation trajectories: from prohemocytes to proPO lineage (melanization/antimicrobial synthesis) and from prohemocytes to VEGF/ALF lineage (phagocytosis/antimicrobial synthesis). Species-specific variations in cluster composition and gene expression reflect adaptations to distinct ecological niches. Comparative analysis of immune-related genes expression, including prophenoloxidase system components and antimicrobial peptides, uncovered both conserved and species-specific patterns, reflecting evolutionary adaptations to diverse aquatic environments. Differential responses to immune challenges (LPS, WSSV, and ammonia stress) underscore the complexity and adaptability of crustacean immune systems. These findings offer novel insights into crustacean hematopoiesis and immune cell diversity, offering a valuable resource for invertebrate immunity and potential applications in aquaculture disease management.