Structure and function of haemocytes in two marine gastropods, Megathura crenulata and Aplysia californica

Aplysia californica is an important mollusc for neurobiological research and Megathura crenulata is becom- ing valuable as the source of keyhole limpet haemocyanin (KLH) which shows promise in treating cancer, allergy and immunosuppression. The culture of these animals provides opportunities to assess their physiological responses to stress and disease. Molluscan haemocytes are known to be involved in a variety of physiological responses, yet the haemocytes of this opisthobranch and vetigastropod have not been characterized. The purpose of this study is to describe the morphology of these cells and summarize their functions based on a suite of assays previously developed on other species of mol- luscs. Using morphology, differential centrifugation and staining reactions, we identify a single type of circulating haemocyte in the blood of both animals. All haemocytes lack granules and contain glycogen and vesicles that react as lysosomes. In A. californica, three to four wing-like lamellipodia extend from the ovoid cell body, whereas in M. crenulata haemocytes are simple ovoid cells. The haemocytes are actively phagocytic and rates of phagocytosis are higher when the assays are performed in the presence of plasma, relative to tests with washed cells. When haemocytes engulf yeast, peroxidase and superoxides are produced. Phenoloxidase activity was not detected. When blood is removed from these molluscs, the plasma does not clot and the haemocytes rapidly adhere to one another in suspension, or settle on sub- strates, migrate and form nodules. Cell spreading and aggregation involves microfilaments and micro- tubules, and can be inhibited by EDTA, cytochalasin B, caffeine and, to a lesser extent, RGD and colchicine. All cells show immunoreactivity against a polyclonal antibody to ACTH which is consistent with previous studies suggesting molluscan haemocytes contain molecules similar to those involved with vertebrate stress responses. These results should be useful in future studies evaluating the physiological status of these animals in the wild and in culture.