Quantitative Analysis of Phagocytosis by Amebocytes of a Sea Cucumber, Holothuria leucospilota

Abstract

Of the five types of coelomocytes in the body cavity of Holothuria leucospilota, a common sea cucumber found in shallow waters of Hong Kong, the phagocytic amebocyte is the most abundant. This study was designed to quantify the phagocytic capacity of these amebocytes using a flow cytometer. Fluorescent latex beads (1 ,um in diameter) were used as experimental particles. The percentage of phagocytizing cells, the number of ingested beads per cell, and the total number of beads ingested were measured simultaneously at different bead/cell ratios (5:1, 10:1, 25:1, 50:1, 100:1, and 200:1). The percentage of phagocytizing cells and the total number of ingested beads were both positively correlated with bead/cell ratio; within 2 h, 96% of the amebocytes contained beads, and on an average, each amebocyte ingested 6 beads when the bead/cell ratio was 200:1. After ingesting beads, the phagocytizing amebocytes transformed from petaloid form to filopodial form. Interruption of microfilaments by cytochalasin B reduced the effectiveness of phagocytosis. Additional key words: coelomocyte, flow cytometry, cytochalasin B, Holothuroidea, Echinodermata All multicellular invertebrates have phagocytic cells, which are capable of engulfing foreign particles, debris, or microorganisms just as an ameba engulfs food particles; the engulfed material is digested by intracellular enzymes (Davey 1990). Phagocytosis is an important feature of the immune response throughout the animal kingdom; it represents the first line of internal defense (Greenberg 1989). Phagocytosis is usually initiated by the extension and fusion of pseudopodia around the target particle, followed by the formation of a phagosome, a membrane-bounded vesicle. Alternatively, the phagosome can be formed by invagination of the local phagocytic cell membrane (Bayne 1990). Echinoderms, like other benthic marine animals, manage to survive in a bacterium-rich marine environment, and this ability may largely depend on their native immune mechanisms, among which phagocytosis is paramount (see review by Chia & Xing 1996). In echinoderms, it has been well established that there are six types of blood cells: amebocytes, spherule cells, vibratile cells, crystal cells, progenitor cells, and haemocytes (see review by Smith 1981). Hyman (1955), Hezel (1963), Endean (1966), Fontaine & a Author for correspondence. E-mail: boxjx@usthk.ust.hk Lambert (1977), Canicatti et al. (1989), and Smiley (1994) made major contributions to the morphological study of echinoderm coelomocytes. Phagocytosis is carried out by amebocytes. These cells are very efficient in recognizing foreign particles including bacteria, cell debris, foreign cells, and inert particles in the body and clearing them out (see review by Chia & Xing 1996). In two sea urchin species, Strongylocentrotus purpuratus and S. franciscanus, and one sea cucumber, Stichopus tremulus, amebocytes can phagocytize Gram-positive bacteria (Johnson 1969; Johnson & Chapman 1971). Amebocytes of the sea cucumber Holothuria polii can phagocytize fixed sheep erythrocytes injected into the coelom (Canicatti 1989). There have been some quantitative studies of phagocytosis in echinoderms, both in vivo and in vitro. Determining clearance by the absence of free particles in coelomic fluid of a sample, Reinisch & Bang (1971) concluded that amebocytes in the sea star Asterias vulgaris could remove 2.6 x 107 sea urchin coelomocytes in 1 h if the sea urchin cells were injected into the body cavity of the sea star. Using latex beads as experimental particles, Beck et al. (1993) demonstrated that in vitro phagocytosis in the sea star Asterias forbesi was positively correlated with incubation time. Thus phagocytosis by amebocytes in echinoderms has This content downloaded from 157.55.39.110 on Wed, 14 Dec 2016 05:02:35 UTC All use subject to http://about.jstor.org/terms