title: Characterization of the circulating hemocytes in mud crab (Scylla olivacea) revealed phenoloxidase activity.
abstract: This study focused on an isolation and characterization of the circulating hemocytes in mud crab, Scylla olivacea. Isolation of specific cell types of hemocytes from crab hemolymph was accomplished by using 60% Percoll density gradient centrifugation. Four separated bands of the hemocytes were successfully obtained. Characterization of these isolated hemocytes by light microscope using trypan blue-rose bengal staining, rose bengal-hematoxilin staining, and phase contrast revealed four distinct types of hemocyte cells. Using their specific morphology and granularity, they were identified as hyaline cell (HC), small granular cell (SGC), large granular cell (LGC) and mixed granular cell (MGC). Transmission electron microscopy (TEM) revealed more details on specific cell size, size of cytoplasmic granule, and nuclear to cytoplasmic ratio, and confirmed the classification. Relative abundance of these cells types in the hemolymph of an adult crab were 15.50±8.22% for HC, 55.50±7.15% for SGC, 13.50±5.28% for LGC, and 15.50±3.50% for MGC. Proteomic analysis of protein expression for each specific cell types by two-dimensional electrophoresis identified two highly abundant proteins, prophenoloxidase (ProPO) and peroxinectin in LGC. Determination of phenoloxidase (PO) activity in each isolated cell types using in vitro and in situ chemical assays confirmed the presence of PO activity only in LGC. Based on an increased PO activity of crab hemolymph during the course of White Spot Syndrome Virus (WSSV) infection, these results suggest that prophenoloxidase pathway was employed for host defense mechanism against WSSV and it may link to the role of large granular hemocyte.
authors: Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakhonpathom 73170, Thailand.
authors: Seksan
authors: S
authors: Mangkalanan
authors: Center of Excellence for Shrimp Molecular Biology and Biotechnology, Faculty of Science, Mahidol University, Rama VI Rd., Bangkok 10400, Thailand.
authors: Piyachat
authors: P
authors: Sanguanrat
authors: Center of Excellence for Shrimp Molecular Biology and Biotechnology, Faculty of Science, Mahidol University, Rama VI Rd., Bangkok 10400, Thailand; Shrimp-Virus Interaction Laboratory, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Thailand Science Park, KlongLuang, Pathumthani 12120, Thailand; Department of Biotechnology, Faculty of Science, Mahidol University, Rama VI Rd., Bangkok 10400, Thailand.
authors: Tanatchaporn
authors: T
authors: Utairangsri
authors: Center of Excellence for Shrimp Molecular Biology and Biotechnology, Faculty of Science, Mahidol University, Rama VI Rd., Bangkok 10400, Thailand; Shrimp-Virus Interaction Laboratory, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Thailand Science Park, KlongLuang, Pathumthani 12120, Thailand; Department of Biotechnology, Faculty of Science, Mahidol University, Rama VI Rd., Bangkok 10400, Thailand.
authors: Kallaya
authors: K
authors: Sritunyalucksana
authors: Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakhonpathom 73170, Thailand. Electronic address: chartchai.kri@mahidol.ac.th.
authors: Chartchai
authors: C
authors: Krittanai
mesh_terms: D000818:Animals; D001775:Blood Circulation; D003386:Brachyura / Q000276:immunology*; D003594:Cytoplasmic Granules / Q000276:immunology* / Q000648:ultrastructure; D004266:DNA Virus Infections / Q000276:immunology*; D006434:Hemocytes / Q000276:immunology* / Q000648:ultrastructure; D046529:Microscopy, Electron, Transmission; D014442:Monophenol Monooxygenase / Q000378:metabolism*; D040901:Proteomics; D046848:White spot syndrome virus 1 / Q000276:immunology*
chemical_list: D014442:Monophenol Monooxygenase
keywords: Hemocyte; Mud crab; Prophenoloxidase; Scylla olivacea