Table 1_Transcriptomic characterization of mucosal hemocytes in the eastern oyster (Crassostrea virginica) underlines their potential role as immune sentinels.xlsx

Substantial progress has been made in understanding hemocytes within the hemolymph (herein referred to as circulatory hemocytes), which are essential for immune defense and various physiological functions in bivalve mollusks. Yet, our knowledge of peripheral immunity, particularly the role of hemocytes associated with mucosal surfaces covering pallial (gills, mantle) organs (herein referred to as mucosal hemocytes), remains limited. While mucosal and circulatory hemocytes share similar morphologies, they exhibit distinct functional profiles and cell surface epitopes. The molecular mechanisms underlying these functional differences remain poorly understood. To address this knowledge gap, we characterized gene expression profiles (using RNA sequencing) in granulocytes and agranulocytes isolated via flow cytometry from hemolymph and mucus covering pallial tissues of the eastern oyster, Crassostrea virginica. Mucosal hemocytes showed overexpression of genes related to cell motility, cytokine activity, signaling, and cell adhesion, supporting the hypothesis that they may have sentinel functions. Despite a consistent dichotomy in gene expression between granulocytes and agranulocytes across both body fluids, it was more pronounced in circulatory hemocytes. Circulatory granulocytes showed functions linked to phagocytosis and pathogen killing, whereas circulatory agranulocytes overexpressed genes associated with mitosis and early inflammation compared to their mucosal counterparts. To our knowledge, this is the first study combining flow cytometry sorting and transcriptomic methods to characterize hemocytes from different body fluids in a marine invertebrate. Results underline the potential role of mucosal hemocytes as immune sentinels, although more studies, possibly using single-cell transcriptomic methods and functional assays associated with pathogen challenge experiments, are needed to probe their specific functions.