Protein arginine methyltransferase 1 mediates regeneration in Dugesia japonica.

As a typical organism of platyhelminth, Dugesia japonica attracts more and more attention for its strong regenerative ability. Protein arginine methyltransferase (PRMT) family is composed of a class of enzymes with methyltransferase activities, which play fundamental roles in vivo in many important physiological processes. PRMT1 is a predominant type Ⅰ PRMT, which has been reported to be expressed in Schmidtea mediterranea. Nevertheless, the existence and the specific biological functions of PRMT1 in Dugesia japonica need further investigation. In this study, we acquired the full-length sequence of DjPRMT1 and confirmed it was a conserved protein. Thereafter, whole-mount in situ hybridization results showed DjPRMT1 was mainly expressed in neoblasts of adult worms, and obvious aggregation of DjPRMT1 was observed at the wound site in early stages of regeneration. Silencing of the DjPRMT1 gene retarded the movement of planarians with decreased DjPIWI-A expression, and DjPRMT1 knockdown also affected planarian regeneration with slightly attenuated proliferation around the blastema of posterior-facing wounds regeneration. In summary, these preliminary results demonstrated DjPRMT1 was involved in the regeneration of planarian.

Induction of hsp70, hsp90, and catalase activity in planarian Dugesia japonica exposed to cadmium.

The hsp70 and hsp90 expression patterns and catalase (CAT) activity in the freshwater planaria Dugesia japonica exposed to cadmium (Cd) under laboratory conditions were investigated. Planaria were exposed to a range of Cd concentrations (0-150 µg Cd/L) for 24 h. The expression levels of hsp70 and hsp90 were determined by relative quantitative real-time polymerase chain reaction. Within the overall dose range in the experiment, the expression level of hsp70 and the activity of CAT in D. japonica were altered significantly. Hsp70 was induced in D. japonica upon Cd exposure concentrations as low as 9.375 µg Cd/L. No significant effect on the expression level of hsp90 was observed. Our findings demonstrated that stress gene hsp70, but not hsp90, was responsive to Cd contamination in D. japonica CAT activity was significantly induced at concentrations of 18.75, 37.5, and 75 µg Cd/L after 24-h exposure. We recommend that the use of hsp70 as a biomarker should be complemented by evidence of changes in other parameters, such as CAT activity, in D. japonica.

Mortality and antioxidant responses in the planarian (Dugesia japonica) after exposure to copper.

The planarians (Dugesia japonica) are distributed widely in China, Japan, Korea, and southern Siberia. In this study, the acute toxicity of copper on D. japonica was evaluated using mortality and the activity of the enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), and reactive oxygen species (ROS) as endpoints. Acute toxicity tests were conducted according to the American Society for Testing and Materials guidelines. The 24-, 48-, 72-, and 96-h median lethal concentration that killed 50% of individuals (LC50) were calculated as 8.70, 6.31, 4.48, and 4.23 mg Cu²âº/L, respectively, based on measured copper concentrations. When compared with different phyla or classes of freshwater animals, the rank of D. japonica in species sensitivity was in the range of 25-26 for 96-h LC50. The antioxidant enzymes SOD and CAT were determined in D. japonica exposed to two copper concentrations (50 and 100 µg Cu²âº/L) with a short-term exposure (15 days). They all attained peak value and then reduced during the experimental period. The GPx activities were activated only for 100 µg/L treatments at days 3 and 6 and then renewed to the original level. Meanwhile, copper significantly increased the levels of ROS in D. japonica. Our study suggests that the adult D. japonica was less sensitive to copper than most other aquatic species. Copper may induce oxidative stress and interfere with the antioxidant defense system of the D. japonica, including SOD and CAT. GPx might be an insusceptible antioxidant enzyme in the metabolic detoxification processes in adult D. japonica.

Identification and characterization of Deoxyribonuclease II in planarian Dugesia japonica.

Deoxyribonuclease II (DNase II) has been found to regulate inflammation, autoimmunity and apoptosis in vertebrates and invertebrates. The strong capacity of degrading DNA makes DNase II play an important role in the immune process. Planarian has become one of the model references due to its strong immune system, the environment they live makes planarians face the threat of microorganisms and injury, the strong immune system can protect planarians from the threat of bacterial and infection. In this study, we found that there was DNase in the lysis buffer of planarians, then we acquired the sequence of DjDN2s (Dugesia japonica DNase2s) and confirmed the DjDN2s were conserved DNase IIs. The predicted structure showed the active sites and binding patterns of DjDN2s. Whole-mount in situ hybridization results showed DjDN2s mainly expressed in immune organs. Quantitative real-time PCR revealed that the expression of DjDN2s upregulated in varying degrees when got hurt and challenged with bacteria, and the knockdown of DjDN2s led to the slower repair of wound. The recombinant phages which take DjDN2 also had the ability to degrade DNA and clear young biofilm of Gram-negative bacteria. Collectively, DNase II of planarian might play a role in the antimicrobial response and wound-induced response.

Identification and characterization of an atypical RIG-I encoded by planarian Dugesia japonica and its essential role in the immune response.

Retinoic acid-inducible gene I (RIG-I), an RNA sensor with a conserved structure, activates the host interferon (IFN) system to produce IFNs and cytokines for eliminating pathogens upon recognizing PAMPs. However, the biological functions and the mechanism by which RIG-I regulates the innate immunity response in invertebrates are still unknown at present. Here we identified an atypical RIG-I in planarian Dugesia japonica. Sequence analysis, 3D structure modeling and phylogenetic analysis showed that this atypical protein was clustered into a single clade at the base of the tree in invertebrates, suggesting that DjRIG-I is an ancient and unique protein of the RIG-I-like receptors (RLRs). In situ hybridization analysis revealed that the DjRIG-I mRNAs were predominantly expressed in the pharynx and head of the adult and regenerative planarians. Stimulation with PAMPs induced the over-expression of DjRIG-I in planarians. The molecular simulation demonstrated that DjRIG-I formed a large hole-structure for the docking of dsRNAs, and the pull-down assay confirmed the interaction between DjRIG-I and viral analog poly(I:C). Importantly, some representative antiviral/antibacterial genes in the RIG-I-mediated IFN and P38 signaling pathway, TBK1, IRF-3, Mx, and P38, were significantly upregulated in planarians stimulated with PAMPs. Interference of the DjRIG-I expression by RNAi, inhibited the PAMPs-induced over-expression, suggesting that DjRIG-I is a key player for downstream signaling events. These results indicate that DjRIG-I triggered the intracellular signaling cascades independent of the classical CARD domains and played an essential role in the virus/bacteria-induced innate immunity of planarian.