| The Chinese mitten crab, Eriocheir sinensis, is an economically important freshwater species for aquaculture in China. The aquaculture of E. sinensis is a rapidly developing industry, with an annual output worth～US$4 billion, currently in Jiangsu province of China. However, the diseases of bacteria, viruses and parasites have flourished within booming E. sinensis cultures, threatened the sustainability of the aquaculture populations of E. sinensis, and severely impacted its commercial production. Tremor disease (TD) is one of the most devastating diseases of E. sinensis. A spiroplasma was previously identified as a novel causative pathogen of the disease. The preliminary researches showed that S. eriocheiris entered in crab body through the gills or body surface, invaded its target cells-hemocytes and propagated in it, then was carried to connective tissues as well as neural, muscle and other tissues by blood circulation. The pathogen caused the crab tremor at later stage of infection. As the target cells of S. eriocheiris, the hemocytes of crab paly an important role in the process of the crab against the infection of the bacteria. Therefore, to explore the mechanism of S. eriocheiris infection the crab and the host immune defense mechanism could help us to reduce the economic losses caused by S. eriocheiris. Firstly, the differential proteomes of the crab hemocytes were analyzed immediately prior to inoculation of the pathogen, and at 10 d post-injection by isobaric tags for relative and absolute quantization (iTRAQ) labeling, followed by liquid chromatographytandem mass spectrometry (LC-MS/MS). Proteomics was used to study the possible mechanism of S. eriocheiris infection and host defense. Secondly, we selected two differentially expressed proteins (Rab7 protein and 14-3-3 protein) to explore its role in the process of the crab immune reaction. These findings could serve as a basis for future studies on the proteins implicated in the susceptibility/resistance o f E. sinensis to S. eriocheiris, as well as contribute to our understanding of disease processes in crabs. This study includes the following three aspects:1. Identify different hemocytes proteins of E. sinensis with iTRAQ during spiroplasma infectionThe differential proteomes of the crab hemocytes were analyzed immediately prior to injection with the pathogen, and at 10 d post-injection by isobaric tags for relative and absolute quantization (iTRAQ) labeling. These different proteins could divided into several categories:immunologic proteins, physiologic proteins and cytoskeleton/extracellular proteins. Using a 1.2-fold change in expression as a physiologically significant benchmark,76 differentially expressed proteins were reliably quantified by iTRAQ analysis. Thirty-five proteins were up-regulated and 41 proteins were down-regulated resulting from S. eriocheiris infection. Approximately 20 differential proteins in hemocytes were involved in the immune responses. Up-regulated proteins included prostaglandin D synthase (GST), ferritin, and heat shock protein 60 et al. Down-regulated proteins included two lectins (mannosebinding protein and hemocytin), three kinds of serine proteinase inhibitors (two serpins and pacifastin), three different kinds of serine proteases et al. Selected bioactive factors (a2M, GST, ferritin, tubulin, crustin, thioredoxin, clip domain serine protease and serpin) are verified by their immune roles in the S. eriocheiris infection using Real-time PCR. The variation trend of immune gene’s mRNA expression is similar with the result of iTRAQ, except the tubulin.2. Full length cloning of Rab7 protein and function analysisRab7, a small GTPase of the Rab family. In recent years, more Rab proteins have also been found to be involved in crustacean innate immunity. In order to validate the immune function of this protein and its role in the immune responses of the crab against S. eriocheiris infection. We cloned the cDNA of Rab7 gene by the techniques of homology cloning and RACE, analysis of nucleotide sequence. The recombinant Rab7 proteins were expressed in E. coli BL21. The expression profiles of Rab7 mRNA under S. eriocheiris treatment were investigated by real-time PCR. The expression profiles of Rab7 protein in haemocytes at protein levels under S. eriocheiris treatment were investigated by Western blot analysis, similar with the result of iTRAQ. The above results suggest Rab7 palys an important role in the process of the crab respond to S. eriocheiris.3. Expression of 14-3-3 protein and function analysisThe 14-3-3 protein was a kind of acid soluble protein and widely distributed in eukaryotes. In recent study, more 14-3-3 proteins have been found to be involved in crustacean innate immunity. In order to validate the immune function of this protein and its role in the immune responses of the crab against S. eriocheiris infection. Firstly, we cloned the 14-3-3 gene by PCR, analysis of nucleotide sequence. Secondly, the recombinant 14-3-3 proteins were expressed in E. coli BL21 and its polyclonal antibody also preparation. The expression profiles of 14-3-3 mRNA under S. eriocheiris treatment were investigated by real-time PCR. The expression profiles of 14-3-3 protein in haemocytes at protein levels under S. eriocheiris treatment were investigated by Western blot analysis, similar with the result of iTRAQ. The above results suggest 14-3-3 paly an important role in the process of the crab respond to S. eriocheiris.
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