Localization of hematopoietic tissue of scallop, Chlamys farreri

There is no immunoglobin in hemolymph of mollusc, and the internal defense system is dependent on circulating blood cells, which develop a number of functions including wound and shell repair, transport and digestion of nutrients. Haemocyte could not mitose and was renewed by hematopoietic tissue. In this paper hematopoietic tissues of juvenile scallop were studied by microstructual observation. Monoclonal antibodies are highly specific molecular probes, which allow identification based on molecular level and are very effective in characterizing cell types and functions in the vertebrate immune system. Monoclonal antibodies were produced against hemocyte of scallop (Chlamys farreri) (1E7, 1F12, 2C6, 2H5). Tested by IIFAT, immunoenzyme stain, Flow cytometry (FCM) and western-blotting, the four Mabs were specific for hemocyte of scallop. Here they were used to localize hematopoietic tissue of juvenile scallop. Paraffin method and immunohistochemistry were employed to observe distribution of hemocyte and localization of hematopoietic tissue in juvenile scallop. For paraffin method, the juvenile scallop (shell length 2 mm,4 mm,8 mm, 1 cm, 1.5 cm) were fixed in Davidson' s solution for 24 h, and dehydrated in ethanol, then embedded in paraffin seperately, then the paraffin embedded tissues were cut into 5 μm sections and stained with hematoxylin eosin. For immunohistochemical method, the visceral mass of the juvenile scallop were absorbed with sieve paper and embedded in Jung tissue freezing medium (Leica), then the frost embedded tissue were cut into 5μm sections. Crysections were airdried, fixed in acetone, and blocked with 10% albumin bovine for 1 h at room temperature, then washed three times for 5 min with PBS-T, after that the slices were incubated in primary antibody (mixture of Mabs 1E7,1F12,2C6,2H5) for 1 h at 37℃ and washed again as above. The Mabs binding was detected with goat anti-mouse Ig serum conjugated with AP (1:20 000) for 1 h at 37℃ and washed three times with AP buffer (100 mmol·L-1 NaCl, 100 mmol·L-1 Tris-Cl, 5 mmol·L-1 MgCl2, pH 9.5). The reaction was developed with freshly prepared substrate solution, 66 μL nitroblue tetrazolium (NBT) and 33μL 5-bromo-4-chloro-3-indolyphosphate (BCIP) (Sigma) in 10 mL AP buffer for 5 mm, and then observed color development. Paraffin method showed that haemocytes were distributed in mantle, gill, stomach, kidney, digestive gland, adductor etc. Heart was located between digestive gland and adductor, the vessel distributed in digestive, stomach and kidney. There were three main haemocyte sinuses full of haemocyte: one was between digestive gland and kidney, the other two were around adductor. Additionally, it was found that there were vesicle tissues laid aside adductor, and it was surrounded by connective tissue membrane, hyperplastic and dense haemocyte located around the inner wall, and it was like the nidus of haemocyte. Immunohistochemical results showed haemocyte in different forms-dissociative, in sinus or in some organ of tissue, were positive. Reactions in kidney and vesicle tissues were stronger because of dense haemocyte. Two methods got same results, and characters of the vesicle tissue are like the place that haemocytes occured. In conclusion, morphological and immune features were accordant, the vesicle tissues around adductor may be the hemoatopoiesis tissue in juvenile scallop.