Preliminary Study On Immune Protective Mechanism And Possible Application Of CpG ODNs In Crustacean Aquaculture

During the last few decades, the worldwide crustacean culture is beset withfrequent occurred diseases and suffered huge economic losses. To develop novelimmunostimulants may bring new ideas for disease control in aquaculture. CpGoligodeoxynucleotides (CpG ODNs) with unmethylated CpG dinucleotides in theircertain flanking sequences (CpG motif) can activate various immune responses andare considered as a kind of novel immunostimulants. In the present study, the immuneprotective mechanism of CpG ODNs was investigated and the possible application inthe aquaculture was also evaluated.The TNF-α activities and the level of NO were significantly enhanced post thestimulations of CpG ODN2395. LvTLR1and LvTLR3could bind with CpG ODN2395according to their functional basis and the binding activities were verified by theELISA and dual luciferase assays. Moreover, the induced TNF-α activity and NO byCpG ODN2395were abolished after the treatment of chloroquine (CQ). Combiningthe localization of CpG ODN2395in the cytoplasma of haemocytes, these resultssuggest that TLR pathways could sense CpG ODNs in shrimp Litopenaeus vannamei,resulting in the boosted immune responses.CpG-rich plasmid pUC57-CpG was constructed and employed to stimulate theshrimp L. vannamei. It showed that pUC57-CpG could promote the apoptosis andregeneration of circulating hemocytes, and enhance the phagocytosis and reactiveoxygen species (ROS) production, which might also contribute to the boostedimmunity against the infection of pathogens.CpG ODNs were employed as supplements in diets to fed crab Eriocheir sinensisto evaluate their possible application in aquaculture. Twenty-eight days after fed with CpG ODNs containing diets, the activities of lysozyme and catalase were significantlyincreased. When challenged with Aeromonas Hydrophila, the cumulative mortalitiesof crabs fed with CpG ODNs containing diets significantly declined compared withothors. Interestingly, the final weights of crabs were also increased after four weeks’feeding with CpG ODNs. Moreover, CpG ODNs could be internalized directly bycrab haemocytes and mostly located in the late endosome. The CpG ODNs suppliedin diets presented the enhancements of immunoprotection efficiency and growth rateby which might depend on the uptaking and locating processes, and they could beused as a potential immunostimulant for the crab farming.In conclusion, as a novel immunostimulant, CpG ODNs could trigger the immuneresponses of crustaceans, probably through the internalization of hemocytes andactivating the Toll like receptor signaling pathways directly. And, CpG ODNs as thesupplements in diets might be a possible way to be used in the crustacean aquaculture.