Flow Cytometric Analysis of Defense Functions of Hemocytes from the Penaeid Shrimp, Penaeus vannamei

Morphology and functions of hemocytes from Penaeus vannamei were analyzed by flow cytometry (FCM) on the basis of cellular structures and incorporation of fluorescent markers. In shrimps there were three types of circulating hemocytes (hyalinocytes, semi‐granulocytes, and granulocytes) identified by FCM and light microscopy. Analysis of hemocytes after propidium iodide staining indicated that the majority of hemocytes were alive after been collected in the physiological anticoagulant. The phagocytic activity level of hemocytes was analyzed using fluorescent beads and the shrimp hemocytes showed different phagocytic activity that could be inhibited by sodium azide. Respiratory burst activity, which was measured using 2′,7′‐dichlorofluorescein diacetate, can be stimulated by phorbol‐12‐myristate‐13‐acetate. Mitochondrial membrane potential was probed according to the accumulation of 3′,3′‐dihexyloxacarbocyanine iodide in the mitochondrial matrix. The data showed more granular cells displayed higher fluorescence intensity than less granular hemocytes. FCM can rapidly, accurately, and directly quantify the morphology and function of a large number of individual cells, and will lead to a better understanding of the shrimp immune system. In addition, such parameters can be used in later experiments to measure defense mechanisms and the modulation of shrimp response to pathogenic agents and environmental stress.