Flow cytometric characterization of the hemocytes of sea hares from tidal pools in Jeju Island off the south coast of Korea

Abstract

The members in the family Aplysidae known as sea hares play a crucial role as a grazer in small tidal pools or shallow subtidal hard bottoms. Like other marine gastropods, hemocyte types and functions of sea hares are limitedly known. This study identified and characterized the hemocytes of common Aplysia species Aplysia kurodai, A. juliana, and A. oculifera in rocky tidal pools in Jeju Island off the south coast of Korea using flow cytometry and light microscopy. The flow cytometry identified three different hemocytes in the hemolymph of the three sea hare species: granulocytes, hyalinocytes, and blast-like cells. The granulocytes exhibited pseudopodia on the cell surface and granules in the cytoplasm. Morphology of the hyalinocyte was similar to that of the granulocytes, while they lack cytoplasmic granules. The blast-like cells were small and round, with very thin cytoplasm. The hyalinocytes were the most abundant in the hemolymph, accounting for 89.8–92.6% of the total hemocytes. Flow cytometry indicated that the granulocytes and blast-like cells were less than 5.6% and 5.4% of the total hemocyte populations. Flow cytometry also revealed that the granulocytes and hyalinocytes are engaged in cellular defensive activities such as intra-cellular lysosomal content, phagocytosis, and ROS production. The mean lysosomal contents of the granulocytes (0.4 × 105–0.2 × 105 A U.) were 2–3 times higher than that of hyalinocytes (0.2 × 105–0.6 × 105 A U.). In addition, the ROS production of the granulocytes (0.98 × 106–1.95 × 106 A U.) was about twice higher than that of the hyalinocytes (0.62 × 106–1.14 × 106 A U.). Of the three species of sea hares, the granulocytes showed comparatively higher phagocytosis capacity (70.4–92.3%) than that of the hyalinocytes (34.8–46.0%). Flow cytometry and microscopy indicated that the hemocyte types and their functions were identical, regardless of the species.