Morphological Studies Of Hemocyte And Cloning Of Anti-disease Genes Of Sea Scallop Chlamys Farreri

Sea scallop Chlamys farreri is a commercially important species in aquaculture. In recent years, severe diseases caused mass mortality of this scallop and brought great loss to local economy in northern China. Hemocyte and non-specific anti-infectious factors are known as the main line of internal defense. In order to explore the mechanisms involved in the anti-infectious process of scallop C. farreri, studies on morphology and classification of hemocytes as well as molecular cloning of humoral anti-infectious factors were carried out in this study.Microscopic and flow cytometric studies of C. farreri were carried out. Three distinct cell types: I, II and III, were identified based on transmission electron microscopy. Type I hemocyte had abundant cytoplasmic vesicles, well developed mitochondria, Golgi apparatus and rough endoplasmic reticulum. Type II was more electron-lucent, harboring relatively fewer vesicles and organelles in the cytoplasm. Type III was small with a relatively big nucleus but fewer vesicles and organelles. When stimulated with yeast cells, the proportion and morphology of hemocytes underwent manifest changes. The number of Type I hemocyte increased remarkably; the cytoplasm became electron-denser. Vesicles and orgaheu'es as well as electron-density of Type II increased slightly. Granulocyte, reported in many other bivalve mollusks, was not identified in this scallop. Flow cytometry data on light scanter pattern (FSC v. SSC) showed clearly three separate cell populations: 1) large cell with high granularity, 2) medium cell with medium granularity and 3) small cell with low granularity. The average cell number in hemolymph was 2.03 ?0.43 X107 ml-1. The three populations represented 58.0%, 24.1% and 17.9% respectively. Afterspreading against glass slide, live cells showed three different patterns, which werenamed as Type A, B and C respectively. Type A hemocyte was bright; lobopodia stretched out usually from one side of cell body. Type B was dark; filopodia stretchedout from any direction. Type C was dark and no obvious spreading. The features of spreading, flow cytometry and ultrastructure were concordance with each other. It confirmed the three-type classification.Alpha2-macroglobulin (a2M) is an important innate immune factor found in many animals. Acting as a proteinase inhibitor, 2M protects tissues from injury by excess proteinase. It can also combine with various cytokins, lectins and endotoxin of bacteria, and in turn regulate various immune reactions. The structural similarity between a2M and complement factors C3, C4 and C5, suggests a2M may play a certain roll in hemolysis. Proteinase protection assay was employed to test the a2M activity in this study. For the first time, a2M activity was detected in the hemolymph from C. farreri and abalone (Hatiotis discus hannai Ino.). Then, based on homologue of a2M in different animals, degenerate RT-PCR and 5' or 3' RACE were employed to clone the cDNA encoding 2M in C. farreri. The cDNA spans 6408bp, which contains an open reading frame of 5157bp, 5' untranslated region of 18bp, 3' untranslated region of 1235bp (Accession No. AY395573). The open reading frame encodes a protein of 1719 amino acid residules. The first 16 arnino acids in the N-terminus were recognizedas signal peptide. The relative molecular weight of the deduce protein is 192 kDa, isoelectric point is 4.85. Blast analysis showed the protein has highest similarity (39% identity) with 2M from Limuluspolyphemus. Typical a2M architectural domains were found. Phylogenic analysis showed human complent factors C3, C4 and C5 are closer to invertebrate 2Ms than vertebrate 2Ms. The three dimensional structure of scallop a2M was constructed with SWISS-MODEL. Six alpha-helixes form an inner alpha-helix bucket, and another six alpha-helixes form an outer bucket. The two-layered alpha-helix buckets consist of the core structure of the molecule. Beta-plated sheets, beta-turns and loops are on the outer surface. Expression of 2M in different tissues of C. farreri was determined...