Preliminary Study Of CpG ODNs Stimulating Hemocyte Autophagy In Chinese Mitten Crab(Eriocheir Sinensis)

Autophagy is a highly conserved intracellular homeostatic process involved in numerous responses in both vertebrate and invertebrate. In the present study, we detected autophagy in hemocytes of Chinese mitten crab Eriocheir sinensis by Western-blot and immunofluorescence assay, and investigated its induction by CpG oligodeoxynucleotides(ODNs).The increase of LC3-conversion(LC3-II/LC3-I) and LC3-puncta formation were observed in hemocytes of crabs after both rapamycin and LPS injection. And the ratio of LC3-conversion and the percentage of LC3-puncta formation were also significantly increased after CpG ODNs stimulation, and reached the highest values at 24 h post-injection. Moreover, the mRNA expression levels of autophagy-related genes, Esgabarap and Esatg7, both dramatically increased after CpG ODNs injection, and reached the peak at 6 h post-injection and lasted till 24 h post-injection.The generation of ROS in hemocytes was induced and reached peak at 6 h post-injection in CpG-pUC57 group. The increased ROS generation and activated autophagy triggered by CpG ODNs were weakened after the treatment of the ROS scavenger, N-Acetyl-L-cysteine(NAC).In summary, autophagy existed in E.sinensis, and it could be detected by Western-blot and immunofluorescence assay after in vivo rapamycin or LPS stimulation. The increase of LC3-conversion, LC3-puncta formation and expression levels of autophagy-related genes suggested that CpG ODNs could induce autophagy in crabs. Additional treatment of NAC with CpG ODNs could weaken the ROS generation and autophagy, indicating that CpG ODNs induced autophagy via the activation of ROS generation in the hemocytes. The results provided useful information to understand autophagy in crab, and they were also helpful for the application of CpG ODNs as the novel immune stimulants in aquaculture.