| Mud crab Scylla serrata (Forskal) is the commercially important marine crab species in China. In recent years, serious diseases occurred in major mud crab culture regions in southeast of China. PCR detection of white spot syndrome virus (WSSV) in diseased mud crabs collected from Zhejiang Province during 2006-2008 showed WSSV prevalence was 34.82%, indicating that WSSV could be closely related to the diseases of mud crabs.To further study on the pathogenicity of WSSV to mud crab, healthy mud crabs was injected intramuscularly with serial 10-fold dilutions of WSSV inoculum. The cumulative mortalities in groups challenged with 10-1, 10-2, 10-3 and 10-4 dilutions were 100%, 100%, 66.7% and 38.9% at 10 days post-injection, respectively. All moribund and dead mud crabs except control group were positive for WSSV by PCR. Based on viral load of WSSV inoculum by quantitative real-time PCR, the median lethal dose (LD50) of WSSV in S. serrata was calculated as 1.10×106 virus copies/crab, or 7.34×103 virus copies/g crab weight by the Muench-Reed method.The activities of phenoloxidase (PO), peroxidase (POD) and superoxide dismutase (SOD) in hemolymph of WSSV-infected moribund crabs, were significantly lower than control group, whereas alkaline phosphatase (ALP), glutamate-pyruvate transaminase (GPT) and glutamic-oxaloacetic transaminase (GOT) were higher than control group.WSSV was mainly distributed in gills, subcuticular epithelia, heart, intestine and stomach by immunohistochemical analysis with Mabs against WSSV. The epithelial cells of infected gill showed hypertrophied nuclei with basophilic inclusions. Numerous bacilliform virus particles were observaed in nuclei of infected gill cells by transmission electron microscopy. Based on the above results, it could be concluded that WSSV is a major pathogen of mud crab with high pathogenicity.To better understand the molecular pathogenesis of WSSV in mud crab, two-dimensional electrophoresis (2-DE) was used to study on protein expression profiles in hemolymph of WSSV-infected and control crabs. 22 significantly differentially expressed protein spots (12 showed overexpression and 10 showed underexpression in WSSV-infected mud crabs compared with control crabs) were successfully identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis. Overexpressed proteins included hemocyanin subunit 1, hemocyanin subunit, alpha-2 macroglobulin (A2M), heat shock proteins 70 (Hsp70), cytosolic manganese superoxide dismutase (MnSOD), catalase (CAT), cryptocyanin, glutamine synthetase, arginine kinase and ribosomal protein S28. Underexpressed proteins included prophenoloxidase (proPO), heat shock protein 90-2 (Hsp90-2), cryptocyanin 1, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin, tropomyosin, synaptosomal-associated protein (SNAP) and transposase. These differentially expressed proteins were mainly involved in immune defense, antioxidant defense, stress response, amino acid synthesis and glycolytic metabolism. The data may help us to better understanding of the mechanism of molecular responses of mud crab to WSSV infection. |
