Pathogen-specific immune responses in Cornu aspersum maxima reveal hematopoietic activity of the hepatopancreas and innate lymphoid-like functions of hyalinocytes.

Snails depend entirely on innate immunity, primarily mediated by hemolymph amoebocytes and hyalinocytes, which are responsible for pathogen recognition, phagocytosis and immune signaling. While amoebocyte functions are well established, the role of hyalinocytes and the origin of hemolymph cells remain unclear. Hyalinocytes proliferate rapidly during pathogenic infections associated with increased mortality and inflammation. This study investigated snail dysbiosis following oral administration and hemocoel injection of the intracellular Listeria monocytogenes SN3 and the extracellular Pseudomonas baetica 7A snail pathogens and the symbiotic effect of the probiotic Lactiplantibacillus plantarum Sgs14. Chemotaxis and necrosis assays revealed that snail pathogens increased amoebocyte and hyalinocyte numbers and induced cell necrosis, whereas the probiotic elicited the so-called "normal" inflammatory response without cell necrosis. Histological analysis showed increased cell accumulation within hepatopancreatic cavities without significant connective tissue damage. Immunohistochemistry indicated elevated expression of the LGR5 receptor homologue in hepatopancreatic cavity cells following pathogenic treatments, while no expression was detected in hemolymph cells. Hemocoel injection of bacteria or hepatopancreatic cell suspensions altered hemolymph cell populations, causing short-term increase in amoebocytes, hyalinocytes, and dead cells. In vitro and in vivo assays demonstrated that L. monocytogenes SN3 strain-activated hyalinocytes adhered to infected amoebocytes and induced necrosis, highlighting their potential cytotoxic role. Flow cytometry and immunofluorescence analysis revealed upregulation of immune marker homologues (CD16, CD49b, IFN-γ, IL-17A), mainly in hyalinocytes, following pathogenic challenge. These findings identify the hepatopancreas as a primary hemopoietic organ in snails and demonstrate pathogen-specific immune modulation, elucidating the mechanisms underlying snail responses and the functional activation of hyalinocytes as cytotoxic effectors.