Sulfated polysaccharides are known to be immune-stimulators in mammals and can be used as food additives to enhance immunity. In this study, the immune-stimulating activity of water-soluble anionic macromolecules F2 fractionation isolated from Codium fragile using ion-exchange chromatography was tested in olive flounder, Paralichythys olivaceus, in vitro and in vivo. The gene expression of interleukin (IL)-1β was adopted to check the immune-affection. As a result, in vitro study revealed that the expression of IL-1β was significantly upregulated in head kidney cells by 1 and 5 μg/ml of polysaccharides 4 h and by 5 μg/ml of polysaccharides at 24 h. In vivo, IL-1β gene expression in head kidney was significantly upregulated by 20 and 100 μg of the polysaccharides at day 1 post-i.p. injection, while downregulated at day 3 but not significant. Meanwhile, in peritoneal cells, it was upregulated by 20 μg of the polysaccharides at day 1 but the upregulation was sustained until day 3 though it was not significant. These results indicate that the sulfated polysaccharides from C. fragile are an immune-stimulator and might be potential feed additives for olive flounder.

Introduction
Materials and Methods
    1. Polysaccharides
    2. Fish
    3. In vitro analysis of gene expression in headkidney
    4. In vivo analysis of gene expression in peritonealcells and head kidney
    5. Total RNA extraction and cDNA synthesis
    6. Real-time PCR assay
    7. Statistical analysis
Results
    1. In vitro analysis of gene expression in headkidney
    2. In vivo analysis of gene expression in headkidney and peritoneal cells
Discussion

• Yong Yang(Department of Marine Biotechnology, Gangneung-Wonju National University, Gangneung 25457, Korea)
• Sang Guan You(Department of Marine Food Science and Technology, Gangneung-Wonju National University, Gangneung 25457, Korea)
• Suhee Hong(Department of Marine Biotechnology, Gangneung-Wonju National University, Gangneung 25457, Korea) Corresponding author