Flow cytometric measurement of hemocyte viability and phagocytic activity in the clam, Ruditapes philippinarum

The assessment of blood cell viability and defense abilities is a major concern in the study of pathological processes. In this work, we devised and validated flow cytometric assays to measure viability and phagocytic activity of hemocytes from the clam Ruditapes philippinarum, a species susceptible to the bacteria-caused Brown Ring Disease (BRD). Validated assays were subsequently used to measure hemocyte parameters following experimental contamination with BRD's etiologic agent Vibrio tapetis. Results show that clams that developed BRD symptoms had lower phagocytic rates and a higher percentage of dead hemocytes than those that did not. In vitro interactions between hemocytes and V. tapetis demonstrated that clam hemocytes are able to phagocytose formalin-fixed V. tapetis, but at lower rates than latex beads. Live V. tapetis were able to kill clam hemocytes in vitro. The in vitro assay also showed that phagocytosis increased with increasing temperature from 8° to 21°C. This work demonstrated the efficiency of flow cytometry for measuring molluscan blood cell activities during host/pathogen interactions and points the way for further experiments using this analytical tool.