Single-cell transcriptomics reveals transcriptional diversity of sea cucumber perivisceral fluid coelomocytes

Echinoderms possess a complex immune system, primarily relying on coelomocytes – immune cells circulating in coelomic fluids. Over the last decades, various coelomocytes have been described based on morphological features, with holothuroids exhibiting the greatest diversity of cell morphotypes among echinoderm classes. However, while the overall immune function of these cells is broadly accepted, their respective functions remain unclear, and molecular data specific to the different cell types are still limited in the literature. In recent years, the advent of single-cell RNA sequencing (scRNA-seq) has enabled the uncovering of cellular heterogeneity in an unprecedented way. However, this powerful tool remains largely underutilised in studies of non-model organisms, especially in invertebrates. In this study, we address the gap in functional information and molecular data on sea cucumber coelomocytes using scRNA-seq on coelomocytes from the perivisceral fluid of Holothuria forskali . We identified 10 distinct clusters, each assumed to correspond to a distinct transcriptional coelomocyte population. Among these, cluster 0 occupies a central position relative to the others, suggesting it may represent “undifferentiated cells”, whereas cluster 6 is markedly divergent from all other clusters. Functional enrichment analyses as well as identification of immune marker genes revealed that some clusters ensure key immune functions, including pathogen recognition, phagocytosis, complement activation and redox balance regulation, providing some clues about their identity. In addition, examination of the processed samples under a microscope confirms the presence of a small proportion of recently discovered carotenocytes in the perivisceral fluid, a cell type rich in carotenoids. By using transcriptomics data previously obtained for this cell type by bulk RNA sequencing (bRNA-seq), it was possible to confidently identify cluster 6 as carotenocytes and provide further insights into their gene expression. Coelomocytes display a wide transcriptional diversity, with carotenocytes exhibiting a clearly distinct expression in comparison to all other clusters. While further analyses are needed to link other clusters to the different morphotypes previously described in the literature, this pioneer study provides preliminary data on the functional diversity of holothuroid coelomocytes, which could be of broad interest for a better understanding of holothuroid immunity and interpreting stress response, as well as for the study of immune cell lineage evolution across deuterostomes.