CpG ODNs induced autophagy via reactive oxygen species (ROS) 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, autophagy in hemocytes of Chinese mitten crab Eriocheir sinensis was observed by Western-blot and immunofluorescence assay, and its induction by CpG oligodeoxynucleotides (ODNs) was investigated. The increase of LC3-conversion (LC3-II/LC3-I) and LC3-puncta formation were observed in hemocytes of crabs after rapamycin injection. And the ratio of LC3-conversion and the percentage of LC3-puncta formation were also significantly increased after CpG ODNs stimulation, and the highest values were 1.89-fold and 3.77-fold compared to that in pUC57 group 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, which were 2.66- and 2.82-fold (P <0.01) for EsGabarap, and 6.16-fold and 6.10-fold (P <0.01) for EsAtg7 compared to saline and pUC57 groups, respectively. The generation of ROS in hemocytes was induced and reached peak at 6 h post-injection in CpG-pUC57 group, which was 1.30-fold (P <0.01) and 1.66-fold (P <0.01) of that in saline and pUC57 group, respectively. The increased ROS generation and autophagy triggered by CpG ODNs were abolished after the treatment of the ROS scavenger, N-acetyl-L-cysteine (NAC). It was suggested that CpG ODNs could induce autophagy and up-regulate the expression levels of autophagy-related genes in crabs 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.