Primary culture of hemocytes from Eriocheir sinensis and their immune effects to the novel crustacean pathogen Spiroplasma eriocheiris
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To investigate the interaction between Chinese mitten crab Eriocheir sinensis hemocytes and the pathogen Spiroplasma eriocheiris, a system for in vitro culture of E. sinensis hemocytes with high viability was developed. Following optimization of conditions, hemocytes survived for >35 days. After challenge with the novel crustacean pathogen S. eriocheiris, E. sinensis hemocytes began to develop vacuoles, and then they began to die (within 60 h). Real-time RT-PCR analysis of S. eriocheiris infected hemocytes identified increased expression levels of anti-lipopolysaccharide factor (ALF), peroxinectin (Pox) and clip domain serine protease (cSP) genes. The expression levels of ALF, Pox, and cSP genes in hemocytes of E. sinensis demonstrated that all three immune genes were significantly induced by challenge with S. eriocheiris. Increases in Pox mRNA levels were highest (up to 36-fold) and peaked at 24–48 h post-challenge (pc) (P < 0.05) and lesser increases were evident with ALF and cSP, peaking at 24 h and at 12–48 h pc, respectively. The hemocytes culture method described herein provides a feasible in vitro research model of E. sinensis that can be used to study its immune reactions against various crab pathogens.
KeywordsEriocheir sinensis Cell culture Defense response Spiroplasma eriocheiris Real-time PCR Immune-related gene
We appreciate Professor O. Roger Anderson’s assistance in editing the manuscript. This work was supported by grants from the National Natural Sciences Foundation of China (NSFC No. 31000072; 31170120), Postdoctoral Foundation of Jiangsu Province (No. 1001043C), Project for aquaculture in Jiangsu Province (No. J2009-43; No. PJ2011-65), A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and the Cultivation Plan for Excellent Doctoral Dissertations of Nanjing Normal University (No. 1243211601031).
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