Cellular and morphological plasticity of Holothuria grisea in response to acute osmotic stress and evidence for crystal cell physiological function

Although echinoderms have traditionally been characterized as stenohaline osmoconformers lacking specialized osmoregulatory organs, they retain the capacity to regulate solute concentrations and utilize tube feet and body walls for water and salt exchange. In holothurians, specifically, it has been speculated that the immune cells, known as crystal cells, may be involved in osmoregulation. The present study, therefore, aimed to elucidate the response mechanisms of the sea cucumber Holothuria grisea to short-term exposure (24 and 48 h) to hypersaline (45 ppt) and hyposaline (25 ppt) water by analyzing its morphometrical, physiological, and immunological responses, with a special focus on the role of crystal cells. To do that, we analyzed morphometrical parameters (body weight, length, and width) and physiological indices, including coelomocyte counts (total, differential, and viability) and biochemical analyses of the cell-free coelomic fluid (CFCF) and coelomocyte lysate (CL). The latter included total protein and glucose concentrations, and the activities of esterase, alkaline phosphatase, and peroxidase. The results demonstrated that body weight and width were notably affected by osmotic stress (primarily hypersalinity), independent of the exposure time. Most alterations in coelomocyte profiles occurred after hypersaline exposure, critically including a significant increase in crystal cell numbers. Biochemical parameters in both CFCF and CL were significantly modulated by salinity and time, showing substance-specific increases or decreases. Overall, H. grisea exhibits physiological plasticity, integrating rapid morphological adjustments with specific, time-dependent cellular and humoral responses. These findings provide novel experimental evidence supporting the involvement of crystal cells in osmoregulation, thereby significantly enhancing our understanding of acute stress response mechanisms in Holothuroidea. A comprehensive understanding of these physiological responses is essential for defining the species' ecophysiological limits and informing its distribution within dynamic coastal environments. Furthermore, the identified cellular and humoral modulations provide sensitive biomarkers for environmental monitoring, enabling early detection of osmotic stress in natural populations driven by climate change or anthropogenic alterations in salinity regimes.