Preliminary Study On The Immunosuppression And Growth-promoting Induced By CpG ODNs In Aquatic Crustaceans

Crustacean is one of most economically important aquatic animals in China. The outbreak of the disease has caused the decline of crustacean productions and huge economic losses. CpG oligodeoxynucleotides（CpG ODNs） have been widely used as a novel vaccine adjuvant in mammals due to its immune protection in some diseases induced by infection, tumor and virus. In recent years, its immunoenhancement effect has also been observed in some aquatic animals. But most of the studies mainly focus on its immune stimulation mechanism as well as its short-term effects. Our study mianly discussed its long-term immnue effects and its promotion of the growth in the Chinese mitten crab, Eriocheir sinensis, and the immune response induced by different types of CpG ODNs in shrimp, Litopenaeus vannamei.Crabs were fed CpG ODNs diets（40 mg/kg） with interval（4 days’ CpG ODNs and 3 days’ basic diets） and continuous（CpG ODNs diets continuously） feeding strategy for 21 days. In the CI group, the mRNA transcripts of EsTolls（EsToll1 and EsToll2） and EsMyD88（adaptor molecule） in hepatopancreas maintained at a significantly higher level（p < 0.01） compared with the CC group after 7 and 14 days’ feeding, while there was no significant difference between them at the 21 st day. Moreover, after a significant increase at 7th day, the expression level of EsLITAF mRNA [Lipopolysaccharide-induced tumor necrosis factor（TNF）-α] in CC group decreased at 14th-21 st day, while that in CI group kept increasing at 14 th day, followed by a decrease at 21 st day. The TNF-α in plasma of CC group was abnormally increased at the 21 st day（p < 0.01） in CC group compared with the significant raise at 7th-14 th in CI group. Moreover, the phagocytic activity and reactive oxygen（ROS） level of hemocytes in continuous CpG ODNs feeding crabs were significantly lower than those in interval feeding crabs. These results indicated that long-term and continuous CpG ODNs stimulation could reduce the activation of pro-inflammatory cytokine production, hemocyte phagocytosis and ROS generation, displaying immunosuppressive effects on the immune system of crabs.After juvenile crabs were fed with the CpG ODNs supplemented diets for 28 day, the percentage of weight gain and the ecdysis rate in the modified interval feeding group（3 days CpG ODNs dites and 4 days basic diets） were significantly up-regulated compared with those in the continuous and basic feeding groups. Meanwhile, the interval CpG ODNs stimulation could also enhance the concentration of ecdysone and Ca²⁺ in plasma, and promote the accumulation of glucose in plasma and glycogen in hepatopancreas at different time. Moreover, the secretion of amylas, pepsin and alkaline phosphatase activity were also significantly upregulated followed the changes of ecdysone. The paraffin sections and scanning electron microscopy of hindgut of juvenile crabs showed that the interval CpG ODNs stimulation could increase the intestinal absorption area by increasing the numbers of intestinal wall fold and microvilli. These results indicated that interval feeding strategy could enhance the growth of juvenile crabs following ecdysis rate increase by promoting the accumulation of energy substance induced by improving the environment of hindgut of juvenile crabs.The binding ability of Lv Toll1, LvToll2 and LvToll3 of L. vannamei to various CpG ODNs were determined by surface plasmon resonance（SPR） technology. Compared with the control（negative and blank）, LvToll1, LvToll2 and LvToll3 exhibited the strongest binding ability to A-type CpG ODN. However, LvToll1 almost had no binding activity to B, C, and P-type CpG ODN. The binding activity of LvToll2 and LvToll3 to C, B and P-type CpG ODN were weakened change in order. The experiment in vivo showed that A-type CpG ODN could significantly induce the mRNA expression of three Toll receptors at 24 h. In addition, the P-type CpG ODN could also activate the mRNA expression of LvToll1-3 at 12 h.In conclusion, the interval CpG ODNs stimulation could not only dramatically decrease the immunosuppressive, but also promote the growth by increasing the ecdysis, maintaining the energy balance between immune and growth in Eriocheir sinensis, which could be serve as a valid and safe transportation way. Moreover, LvToll1-3 showed the strongest binding ability to A-type CpG ODN than other CpG ODN types in vitro of L. vannamei. However, both A and P-type were strongly activate LvToll1-3 mRNA expression than other types of CpG ODN in vivo. In consequence, the reconstruction of A and P-type CpG ODN would be employed in future experiment for the better immune effects and safety.