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Identification of Proliferating Cells in Hard Clams.

Rhea Hanselmann [1]

Daniel Gibson [1]

The origin of hemocytes, the circulating "blood cells" of bivalve molluscs, including hard clams (Mercenaria mercenaria) has not been identified (1, 2). Proliferation of hemocytes, however, can be recognized through their increased numbers in diseased animals. Proliferating cell nuclear antigen (PCNA), or cyclin, is a protein produced during the late G1 and S phases of the cell cycle (3, 4). Using antibodies that recognize PCNA in mice, we attempted to identify the origin of hemocytes in the hard shell clams. Quahog parasite unknown (QPX) is a protist that causes severe inflammation and mortality in infected clams (5, 6). We attempted to induce hemocyte proliferation by exposing clams to QPX in a 10-1 water column in which 12 ml of undiluted QPX culture (at a concentration of 7*[10.sup.6] cells/ml) were added every 10 days; by injecting QPX between the membranous mantles and the right valves, 3 cm ventral anterior to the siphon and into the pericardial cavities (0.25 ml of undiluted QPX culture); and by inje cting an inert particle (India ink, 1:10 dilution in sterile seawater [7, 8]) into the pericardial cavities. The controls consisted of two groups of clams. One group was injected with sterile seawater in the pericardial cavities; the other was untreated. Groups were sampled at 24 h, and at 1, 4, and 8 weeks after the start of the experiment. At sampling, the animals were shucked, fixed in 10% neutral buffered formalin (NBF) for 24 h, and embedded in paraffin. Sections were cut (4-6 [micro]m), mounted onto positively charged slides (Fisher-brand, Superfrost/Plus and ProbeOn Plus slides) and stained either with hematoxylin and eosin (H&E) (9) or with anti-PCNA with a hematoxylin counterstain (Zymed, PCNA Staining Kit).

Clams injected with QPX in the pericardial cavities showed mild focal inflammation associated with viable and necrotic QPX organisms. At 2 months post-injection, viable QPX organisms were no longer identified. QPX organisms and associated inflammation were not observed in clams injected in the mantle cavity. After 2 months of water column exposure, only very rare infection by QPX organs with minimal inflammation was observed in mantle tissue. India ink injection caused a minimal inflammatory response. Pools of injected ink in the tissues and vascular spaces were either engulfed by individual hemocytes or surrounded and sequestered by hemocytes (encapsulation), forming thin-walled granulomas (6, 10). Numerous individual hemocytes containing India ink were eliminated from the clams by diapedesis over lumenal epithelial surfaces (Fig. 1A). Thick-walled granulomas (6, 10) were also identified in the gills, pericardial sacs, and other organs of saline-injected animals, indicating that the injection may not have b een sterile.

Using the anti-mouse PCNA, areas of abundant PCNA staining (black-stained nuclei), indicating areas of marked cell proliferation, were identified in the reserve cells of the digestive gland (Fig. 1B), the proliferative epithelial cells of the gill base (2) (Fig. 1C), and in early proliferative phases of reproductive epithelium. As expected, cells of other tissues throughout the body also stained positive for PCNA (i.e., epithelium of the intestine, foot and body wall, and gill plical epithelium), but in much lower numbers. Proliferating hemocytes were identified in the inflammatory cells forming the thick-walled granulomas (Fig. 1D) and rarely in adjacent non-inflammatory cells. In no other areas examined were proliferating hemocytes identified.

These results demonstrate that the epitopes associated with PCNA are conserved between the clam and the mouse, as shown by the positive staining of known proliferative cells in the clam body. Previous studies have shown that hemocytes appear to migrate to areas of infection in bivalves (2, 6). In diseased bivalves, hemocyte numbers appear to increase; the site of origin of these hemocytes has never been determined (2). This study provides evidence that the hemocytes of the hard clam proliferate directly at the inflammatory site, as opposed to a possible bone marrow-like area in the body of the clam, with subsequent migration of hemocytes to sites of infections, as seen in vertebrates.

This work was supported, in part, by a NOAA Sea Grant Award to the Woods Hole Oceanographic Institution Sea Grant Program, Grant No. NA86RG0075, Project Number R/A-39.

(1.) Worcester Polytechnic Institute, Worcester, MA.

Literature Cited

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(2.) Cheng, T. C. 1996. Pp. 631-642 and 299-333 in The Eastern Oyster, Crassostrea virginica. V. S. Kennedy, R. I. E. Newell, and A. F. Eble, eds. Maryland Sea Grant College Publication UM-SG-TS-96-01.

(3.) Waseem, N. H., and D. P. Lane. 1990. J. Cell. Sci. 96: 121.

(4.) Ogata, K., Y. Ogata, R. M. Nakamura, and E. M. Tan. 1985. J. Immunol. 135: 2623.

(5.) Kleinschuster, S. J., R. Smolowitz, and J. Parent. 1998. J. Shellfish Res. 17: 75-78.

(6.) Smolowitz, R., D. Leavitt, and Frank Perkins. 1998. J. lnvertebr. Pathol. 71: 9-25.

(7.) Stauber, L. A. 1950. Biol. Bull. 98: 227-241.

(8.) Tripp, M. R. 1960. Biol. Bull. 119: 273-282.

(9.) Humanson, G. L. 1997. Pp. 101-118 in Humanson's Animal Tissue Techniques, 5th ed. J. K. Presnell and M. P. Schreibman, eds. The John Hopkins University Press, Baltimore, MD.

(10.) Eble, A. F., and R. Scro. 1996. Pp. 25-30 in The Eastern Oyster, Crassostrea virginica. V. S. Kennedy, R. I. E. Newell, and A. F. Eble, eds. Maryland Sea Grant College Publication UM-SG-TS-96-01.
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Author:Hanselmann, Rhea; Smolowitz, Roxanna; Gibson, Daniel
Publication:The Biological Bulletin
Geographic Code:1USA
Date:Oct 1, 2000
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