title: iTRAQ-based proteomic study of the effects of Spiroplasma eriocheiris on Chinese mitten crab Eriocheir sinensis hemocytes.
abstract: Spiroplasma eriocheiris is as a novel pathogen of Chinese mitten crab Eriocheir sinensis tremor disease. The hemocytes have been shown to be major target cells in S. eriocheiris infection. The aim of this study was to examine the hemocytes' immune response at the protein levels. The 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, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 1075 proteins were identified by LC-MS/MS and de novo sequencing data. Using a 1.2-fold change in expression as a physiologically significant benchmark, 76 differentially expressed proteins (7.07%) were reliably quantified by iTRAQ analysis. Thirty-five (3.26%) proteins were up-regulated and 41 (3.81%) proteins were down-regulated resulting from a S. eriocheiris infection. Approximately 20 differential proteins in hemocytes were involved in the stress and immune responses. Up-regulated proteins included alpha-2-macroglobulin (α2M), prostaglandin D synthase (GST), ferritin, and heat shock protein 60. Down-regulated proteins included two lectins (mannose-binding protein and hemocytin), three kinds of serine proteinase inhibitors (two serpins and pacifastin), three different kinds of serine proteases, mitogen-activated protein kinase kinase (MAPKK), and two thioredoxins (Trx), crustin, etc. Selected bioactive factors (α2M, 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. The prophenoloxidase-activating system, antimicrobial action and antioxidant system involved in the immune responses of E. sinensis is believed to be a resistance to S. eriocheiris infection. This is the first report of the proteome response of crab hemocytes against S. eriocheiris infection. These findings contribute to our understanding of tremor disease processes in crabs, and provide the first evidence to promote a search for potential biomarkers of the disease.
authors.affiliation: Jiangsu Key Laboratory for Biodiversity & Biotechnology and Jiangsu Key Laboratory for Aquatic Crustacean Diseases, College of Life Sciences, Nanjing Normal University, Nanjing 210023, China.
authors.forename: Qingguo
authors.initials: Q
authors.lastname: Meng
authors.affiliation: Jiangsu Key Laboratory for Biodiversity & Biotechnology and Jiangsu Key Laboratory for Aquatic Crustacean Diseases, College of Life Sciences, Nanjing Normal University, Nanjing 210023, China.
authors.forename: Libo
authors.initials: L
authors.lastname: Hou
authors.affiliation: Jiangsu Key Laboratory for Biodiversity & Biotechnology and Jiangsu Key Laboratory for Aquatic Crustacean Diseases, College of Life Sciences, Nanjing Normal University, Nanjing 210023, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: Jiangsu Key Laboratory for Biodiversity & Biotechnology and Jiangsu Key Laboratory for Aquatic Crustacean Diseases, College of Life Sciences, Nanjing Normal University, Nanjing 210023, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Huang
authors.affiliation: Key and Open Laboratory of Marine and Estuary Fisheries, Ministry of Agriculture of China, East China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Shanghai 200090, China.
authors.forename: Yanqing
authors.initials: Y
authors.lastname: Huang
authors.affiliation: College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China.
authors.forename: Siyao
authors.initials: S
authors.lastname: Xia
authors.affiliation: Jiangsu Key Laboratory for Biodiversity & Biotechnology and Jiangsu Key Laboratory for Aquatic Crustacean Diseases, College of Life Sciences, Nanjing Normal University, Nanjing 210023, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Gu
authors.affiliation: Jiangsu Key Laboratory for Biodiversity & Biotechnology and Jiangsu Key Laboratory for Aquatic Crustacean Diseases, College of Life Sciences, Nanjing Normal University, Nanjing 210023, China. Electronic address: njnuwang@263.net.
authors.forename: Wen
authors.initials: W
authors.lastname: Wang
mesh_terms: D000818:Animals; D060829:Arthropod Proteins / Q000235:genetics* / Q000378:metabolism; D003386:Brachyura / Q000235:genetics* / Q000378:metabolism / Q000382:microbiology*; D002853:Chromatography, Liquid; D005786:Gene Expression Regulation; D006434:Hemocytes / Q000378:metabolism / Q000382:microbiology; D007113:Immunity, Innate; D008969:Molecular Sequence Data; D020543:Proteome; D060888:Real-Time Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013149:Spiroplasma / Q000502:physiology*; D053719:Tandem Mass Spectrometry
chemical_list: D060829:Arthropod Proteins; D020543:Proteome
keywords: Eriocheir sinensis; Hemocytes; Proteomics; Spiroplasma eriocheiris; iTRAQ