A modified method to detect the phagocytic ability of eosinophilic and basophilic haemocytes in the oyster Crassostrea plicatula

The immune defence system of bivalve species largely depends on haemocytes. Haemocytes are generally classified as hyalinocytes (H) or granulocytes (G), and each cell type is further sub-classified as eosinophilic (E) or basophilic (B) haemocytes. Until recently, research on eosinophilic and basophilic haemocytes has primarily focused on their morphologies, dye affinities and intracellular components. Few studies have investigated their phagocytic ability because of the absence of appropriate experimental methods. In this study, we introduce a modified method suitable to detect the phagocytic ability of eosinophilic and basophilic haemocytes. This modified method involves neutral red staining by employing fluorescent microspheres as the phagocytosed medium. Specifically, haemocytes are incubated with fluorescent microspheres and then stained with neutral red. Next, the stained haemocytes are fixed by acetone and are counterstained by propidium iodide. Finally, the haemocytes are observed under a multifunctional microscope to analyse the phagocytic ability by counting the number of eosinophilic or basophilic haemocytes involved in phagocytosis (calculation for phagocytic rate, PR) and the number of phagocytosed microspheres by each eosinophilic or basophilic haemocyte (calculation for phagocytic index, PI). By employing this modified method in the oyster Crassostrea plicatula, we found that the PRs of G and H were very similar to the data obtained by another method, flow cytometry, indicating that this modified method has high accuracy. Additionally, we also found that the PR and PI in E-G were 70.9 ± 7.3% and 1.0 ± 0.2, respectively, which were both significantly higher than those in B-G (53.1 ± 6.4% and 0.7 ± 0.1). The PR and PI in E-H were 16.3 ± 2.8% and 0.2 ± 0.1, respectively, while in B-H, the PR and PI were 13.3 ± 3.6% and 0.2 ± 0.1, respectively, with no significant difference observed. Based on this result, eosinophilic granulocytes are more active in phagocytosis than basophilic granulocytes in the oyster immune defence system.