Research on eosinophilic and basophilic granulocytes in bivalve species has focused primarily on their morphologies and dye affinities. Few studies have investigated the immune functions of these granulocytes because of limitations in experimental methods. These methods must meet two criteria: (1) the studied granulocytes must survive and (2) eosinophilic granulocytes must be distinguishable from basophilic granulocytes. Unfortunately, few current methods have met both criteria. In this study, neutral red, a low-toxicity dye, was used to stain the hemocytes in three bivalve species (the clam Paphia undulata, the scallop Chlamys farreri, and the oyster Ostrea plicatula). The results show that the granulocytes in these three bivalve species are classified into 2 categories: eosinophilic granulocytes with a colorless or light-yellow cytoplasm and basophilic granulocytes with a deep-red cytoplasm. In addition to the difference in tinctorial property, the population of eosinophilic granulocytes was also significantly less than that of basophilic granulocytes (P < 0.05). The observed percentages of total hemocytes were 16.2 ± 2.28% versus 50.5 ± 5.34% in P. undulata, 7.8 ± 2.15% versus 51.5 ± 4.24% in C. farreri, and 22.3 ± 1.92% versus 40.2 ± 3.84% in O. plicatula. With respect to dimension parameters such as cell diameter, nucleus diameter, and nucleus diameter/cell diameter between eosinophilic and basophilic granulocytes, no significant differences were observed in either P. undulata or O. plicatula. These results suggest that neutral red staining can be used to distinguish eosinophilic from basophilic granulocytes. In addition, because of its nontoxic properties, neutral red staining is a promising method for the study of the immune function of these two types of granulocytes.
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Vol. 32 • No. 3