Study On Ontogenesis And Phagocytosis Of Chinese Shrimp Haemocytes And Its White Spot Syndrome Virus Receptor

In this thesis, monoclonal antibodies apecifically against haemocytes of Chinese shrimp (Fenneropenaeus chinensis) were generated. Employing these Mabs as probes, the appearance of Chinese shrimp haemocytes were detected to be at the limb bud stage and the appearance of granular haemocytes were at the embryonic-nauplius stage. Utilizing flow cytometry technique, the phagocytic percentage of Chinese shrimp haemocytes against injected fluorescence microbeads were observed to rise rapidly after injection and peak at 12.58% in 6 hours. The phagocyted microbeads accumulated in the lymph organ of shrimp. The White Spot Syndrome Virus (WSSV) infection stimulated the phagocytosis at the early stage but impaired it soon after. Furthermore, an in vitro combination system of Digoxigenin (DIG) labeled WSSV and isolated haemocytes plasma membrane (HPM) was established as a selecting system basing on the ELISA and Dot-blot. Using this system, monoclonal antibody R2H1 was screened and identified to impair the in vitro combination of WSSV-DIG and HPM.Ⅰ. Mab 2A3 against all haemocytes and Mab 1H11 specific to cytoplasmic granules in granular haemocytes were generated and employed to study the ontogenesis of haemocytes by IFAT in Chinese shrimp embryos of blastula, gastrula, limb bud stage, embryonic-nauplius and nauplius I stages. With Mab 2A3, the first positive cells were detected at the limb bud stage of the embryos. Distinguishable positive haemocytes with a diameter of about 7μm appeared in embryonic-nauplius. At the following nauplius I stage, configuration of haemocytes became more integrated and clear. Their diameters were almost similar to those of haemocytes from adult shrimp. With Mab 1H11, the first positive granular haemocytes were identified at the stage of embryonic-nauplius which existed in clusters with positive punctate fluorescence of granules distributed around the periphery of granular haemocytes. Increased number of both total and granular haemocytes at nauplius I stage impressed a possible enhancement of the immune system as the embryogenesis progressed.Ⅱ. Flow cytometry technology was employed to investigate the phagocytosis of Chinese shrimp haemocytes against injected fluorescence microbeads. The phagocytic percentage of normal Chinese shrimp haemocytes increased rapidly after injection and reached the peak of 12.58% in 6 hours. After that, the percentage decrease slowly. Microscopy observation of peripheral haemocytes and lymph organ showed that the main phagocytic haemocytes subgroup was semi-granular haemocytes and the phagocyted microbeads accumulated in lymph organ. As a response to WSSV infection, the phagocytic percentage at 1 day post infection (14.42%) was slightly higher than control value. However, the percentage then decreased and maintained at about 50% of the control value after that until the percentage increased again to 11.23% at 6 days post infection just before most of the shrimp died.Ⅲ. Chinese shrimp HPM isolated by differential centrifugation were incubated with Digoxingenin labeled WSSV (WSSV-DIG) at 4°C for 4h and detected by antibodies against DIG. Both ELISA and Dot-Blot assay showed positive results of the WSSV-DIG and HPM in vitro combination. When polyclonal antibody against HPM of Chinese shrimp was allowed to bind to the HPM at 37°C for 1h before the incubation with WSSV-DIG, the in vitro combination was blocked in both Dot-Blot and ELISA assay. This result proved that the ELISA and Dot-Blot in vitro combination was a practicable screening system for WSSV receptor antibodies. Using the ELISA system, Mab R2H1 was identified to significantly impair the combination of WSSV and HPM. When confirmed by Dot-blot assay, the R2H1 blocked group showed a significantly lighter color comparing with the unblocked group. It can be deduced that Mab R2H1 recognizes one of the WSSV receptors on HPM, the decrease of WSSV combination was due to the occupation of WSSV binding site by Mab R2H1.