Proceedings of the Third World Fisheries Congress: Feeding the World with Fish in the Next Millenium—The Balance between Production and Environment
Hemocyte Type of Chinese Scallop Chlamys farreri
Jing Xing, Wenbin Zhan
Culture of Chinese scallop Chlamys farreri in Shandong Province is well known for both its culture area and yield. It has become a dominant industry in marine aquaculture since the decline of shrimp culture: the culture area was 200 million m2 more, yield was nearly 1 million tons (including shells), and productive values surpassed 3 billion yuan. However, the mortality rate of scallop has been high since 1997. In Yantai, Weihai, Rizhao, and Qingdao regions, the mortality rate was about 50%; the highest rate was more than 80%. Therefore, in the process of exploring effective and practical preventive measures, we try to find a new way to improve the disease resistance and immune competency of scallops.
Bivalve hemolymph cells perform several functions, including wound and shell repair, transport and digestion of nutrients, and internal defense (Bayne 1990; Karp 1990). To understand these functions, it is important to distinguish the hemocyte populations and characterize them. Two basic cell types are recognized among bivalve hemocytes: agranular (hyalinocytes) and granular (granulocytes) (Carballal et al. 1997). Studies on mussel and oyster hemocyte types have been carried out by several authors (Auffret 1989; Carballal et al. 1997), but there are few reports on the Chinese scallop hemocyte.
Because the relationship between hemocyte type and defense function was not known, the aim of this study was to characterize the hemocyte of Chinese scallop, as a preliminary step toward understanding their defense mechanisms. To this end, morphological, gradient-density centrifugation, and flow cytometric technique criteria were used. The method of morphological criteria is generally used in identifying hemocyte types, cell separation techniques are often used to obtain pure or enriched fractions of hemocyte populations, and flow cytometry can classify hemocytes according to the size and interior structure of the cell.
Chinese scallops (shell length 5–10 cm) were collected from a culture farm in Qingdao and kept in a circulating seawater system in the laboratory until the next day. Hemolymph was obtained from the posterior adductor muscle sinus with a sterile hypodermic needle (23G) and a 2-mL syringe.
Monolayers of hemocytes were prepared by allowing cells to settle onto glass slides (spontaneous monolayers) or by adhering cells to glass slides using a smear. To prepare spontaneous monolayers, hemolymph was withdrawn directly (1:4) into filtered seawater (0.22 µm). A drop of this suspension was deposited on a clean slide and kept in a wet chamber for 30 min. To prepare smears, hemolymph was withdrawn and simultaneously diluted (1:3) in anti-aggregant modified Alsever’s solution (glucose, 20.8 g; sodium citrate, 8 g; EDTA → ethylenediamine tetraacetic acid [EDTA], 3.36 g; NaCl, 22.3 g; H2O, 1,000 mL). The slides were subsequently fixed in methanol and stained with Giemsa.
Hemocyte dimensions were estimated in a smear. A total of 110 stained cells were measured under a light microscope using an ocular micrometer. Cell size was determined by measuring the longest axis excluding pseudopod. Nuclear size was estimated by measuring the longest nuclear diameter.
Dimensions of cells, fixed in suspension, were also determined. For this purpose, the hemolymph was withdrawn directly (1:1) in Alsever’s solution containing 6% formalin, fixed for 15 min, then resuspended in Alsever’s solution; then, cells were stained by adding 100 µL of Trypan blue for 30 min. A drop of the solution was deposited on a glass slide and observed under a light microscope using an ocular micrometer. Differences in dimensions among hemocyte types were analyzed by using SPSS software.