Reference: Biol. Bull. 199: 199 –200. (October 2000) Identification of Proliferating Cells in Hard Clams Rhea Hanselmann 1 , Roxanna Smolowitz (Marine Biological Laboratory, Woods Hole, Massachusetts), and 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 inflamma- tion and mortality in infected clams (5, 6). We attempted to induce hemocyte proliferation by exposing clams to QPX in a 10-l water column in which 12 ml of undiluted QPX culture (at a concentra- tion of 710 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 injecting an inert particle (India ink, 1:10 dilution in sterile seawater [7, 8]) into the peri- cardial 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 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 inflam- mation 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 man- tle 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 1 Worcester Polytechnic Institute, Worcester, MA. Figure 1. Proliferating epithelial cells and hemocytes of Mercenaria mercenaria stained with anti-Mouse Proliferating Cell Nuclear Antigen (PCNA). (A) India ink is phagocytized and eliminated by diapedesis of India ink-filled hemocytes (arrow) into the renal tubular lumens (H&E, bar = 13 m). (B) Nuclei of proliferating reserve cells of the digestive gland’s tubular epithelia stained black (arrow) with anti-PCNA. PCNA negative nuclei stain blue (arrowhead) (hematoxylin counterstain, bar = 13 m). (C) Strong PCNA nuclear staining is present in the proliferating nuclear epithelial cells at the base of the gills (arrows). PCNA negative nuclei stain blue (arrowhead) (hematoxylin counterstain, bar = 13 m). (D) Nuclei of some hemocytes in thick-walled granulomas in the kidney tissue of clams are positive for PCNA stain (arrows) and present evidence for the proliferation of hemocytes directly at the inflammatory site (hematoxylin counterstain, bar = 13 m). 199 PHYSIOLOGY AND BIOCHEMISTRY