Molecular characterization of shrimp harbinger transposase derived 1 (HARBI1)-like and its role in white spot syndrome virus and Vibrio alginolyticus infection.

The role of the nuclease, HARBI1-like protein (mjHARBI1-like) in the innate immunity of Marsupenaeus japonicus was explored in this study. The 1361 bp cDNA sequence of mjHARBI1-like was cloned from M. japonicus using RACE. RT-qPCR analysis results showed that the gills and hepatopancreas of M. japonicus were the main tissues where mjHARBI1-like is expressed. In addition, it was also found that white spot syndrome virus (WSSV) or Vibrio alginolyticus challenge could stimulate mjHARBI1-like expression. After mjHARBI1-likewas inhibited, expression of immune genes such as toll, p53, myosin, and proPO were significantly downregulated (P < 0.01). However, in shrimp hemocytes, hemocyanin and tumor necrosis factor-α (TNF-α) were up-regulated significantly (P < 0.01). This study demonstrated that mjHARBI1-like plays a key role in the progression of WSSV and V. alginolyticus infection. Specifically, the cumulative mortality of WSSV-infected and V. alginolyticus-infected shrimp was significantly advanced by double-strand RNA interference (dsRNAi) of mjHARBI1-like. Apoptosis studies indicated that mjHARBI1-dsRNA treatment caused a reduction in hemocyte apoptosis in bacterial and viral groups. In addition, phagocytosis experiments illustrated that mjHARBI1-dsRNA treatment led to a lower phagocytosis rate in hemocytes of V. alginolyticus-challenged shrimp. It was also found that knockdown of mjHARBI1-like inhibited shrimp phenoloxidase (PO) activity, superoxide dismutase (SOD) activity, and total hemocyte count (THC) after WSSV or V. alginolyticus infection. These data indicate a regulative role of mjHARBI1-likein the immunity of shrimp in response to pathogen infection. Resultantly, it was concluded that mjHARBI1-like might have a positive effect on the anti-WSSV immune response of shrimp by regulating apoptosis, THC, PO activity, and SOD activity. Additionally, mjHARBI1-like might promote anti-V. alginolyticus infection by participating in regulating phagocytosis, apoptosis, SOD activity, PO activity, and THC.

Sun B ，Qian X ，Zhu F 《-》

Molecular cloning of Kuruma shrimp Marsupenaeus japonicus endonuclease-reverse transcriptase and its positive role in white spot syndrome virus and Vibrio alginolyticus infection.

This study investigated the function of endonuclease-reverse transcriptase (mjERT) in Marsupenaeus japonicus. The 1129 bp cDNA sequence of mjERT was cloned from M. japonicus using rapid amplification of cDNA ends (RACE) PCR, and RT-qPCR analysis indicated that mjERT was highly expressed in the gills and hepatopancreas of M. japonicus. We also found that white spot syndrome virus (WSSV) or Vibrio alginolyticus challenge could enhance the expression of mjERT. When mjERT was inhibited, immune genes such as toll, p53, hemocyanin and tumor necrosis factor-α (TNF-α) were significantly down-regulated (P < .01) in the hemocytes of shrimp, while myosin was significantly up-regulated (P < .01). We demonstrated that mjERT is very important for the progression of WSSV infection and that the cumulative mortality of WSSV-infected and V. alginolyticus-infected shrimps was significantly increased following mjERT RNA interfere (RNAi). Apoptosis data provided information to suggest that mjERT-dsRNA challenge caused less apoptosis in hemocytes in both the disease-free and viral group. We also revealed that mjERT-dsRNA treatment resulted in a lower phagocytosis rate in the hemocytes of V. alginolyticus-challenged shrimp. Finally, we found that the absence of mjERT had an significantly negative impact upon shrimp phenoloxidase (PO) activity, superoxide dismutase (SOD) activity and total hemocyte count (THC) following WSSV or V. alginolyticus infection, indicating a regulative role for mjERT in the innate immunity of shrimp in response to pathogenic infection. In summary, we concluded that mjERT might promote the anti-WSSV immune response of shrimp by regulating apoptosis, PO activity, THC and SOD activity, and also exert a positive role in the immune response against V. alginolyticus by regulating phagocytosis, SOD activity, PO activity and THC.

Ma X ，Sun B ，Zhu F 《-》

The crustin-like peptide plays opposite role in shrimp immune response to Vibrio alginolyticus and white spot syndrome virus (WSSV) infection.

Crustin is an antimicrobial peptide (AMP) that plays a key role in innate immunity of crustaceans. In this study, we cloned the entire 660 bp crustin-like sequence with a 507 bp open reading frame encoding a 168 amino acid from Marsupenaeus japonicus. The crustin-like gene was primarily expressed in gills and over-expressed in shrimp hemocytes after challenge with WSSV or Vibrio alginolyticus. After knockdown crustin-like gene using specific double-stranded RNA (CRU-dsRNA), IMD, Rab7, L-lectin, mitogen-activated protein kinase, p53, prophenoloxidase and Rho were down-regulated and nitric oxide synthase, myosin and tumor necrosis factor-α were up-regulated in hemocytes at 24 h post dsRNA treatment. After WSSV challenge, The mortality, WSSV copy number and expressions of WSSV immediate early genes (IE1, IE2, DNA polymerase, VP28) were both decreased but the apoptosis rate was increased in CRU-dsRNA-treated shrimps, indicating that WSSV may take advantage of crustin-like to benefit its replication. After silenced the crustin-like, the results of phagocytosis showed that the phagocytic rate of shrimp hemocytes on WSSV decreased significantly. In contrast, the absence of crustin-like in shrimps increased the mortality following V. alginolyticus challenge, indicating that crustin-like may play a positive role in the antibacterial process. The phagocytosis experiment showed there was a higher phagocytosis rate of hemocytes after CRU-dsRNA treatment. The result indicated that V. alginolyticus may be able to use crustin-like to avoid phagocytosis of shrimp hemocytes. These results further added to our understanding of the function of crustin-like peptide and also provided its potential role in innate immunity in shrimp.

Sun B ，Wang Z ，Zhu F 《-》

Molecular characterization of diphthamide biosynthesis protein 7 in Marsupenaeus japonicus and its role in white spot syndrome virus infection.

Diphthamide biosynthesis protein 7 (Dph7) is a vital protein for diphthamide biosynthesis in archaea and eukaryotes. The 1143 bp cDNA sequence of Dph7 was cloned from the gills of Marsupenaeus japonicus using RT-PCR and RACE. Data showed that Dph7 was highly expressed in the gills and digestive gland of M. japonicus. Furthermore, the expression of dph7 was induced by infection with white spot syndrome virus (WSSV). When Dph7 was knocked down, immune genes such as toll, prophenoloxidase (proPO), p53, tumor necrosis factor-α (TNF-α) and signal transducer and activator of transcription (STAT) were significantly down-regulated (P < 0.01) in hemocytes. First, we demonstrated that Dph7 is very important in the progression of WSSV infection and that the time of death for WSSV-infected shrimp was significantly advanced following RNAi targeting of Dph7. We also investigated the effect of Dph7 on apoptosis rate in M. japonicas and found that Dph7-dsRNA treatment caused lower levels of apoptosis in hemocytes, both in the disease-free group and the WSSV group. Knock-down of Dph7 affected the activity of both phenoloxidase (PO) and superoxide dismutase (SOD), and total hemocyte count (THC) after infection with WSSV, indicating that Dph7 plays a regulatory role in the immunological reaction of shrimp in response to WSSV infection. Thus, we conclude that Dph7 may promote the anti-WSSV immune response of shrimp by regulating apoptosis, SOD and PO activity, and can influence the progression of WSSV infection.

Wang Z ，Sun B ，Zhu F 《-》

Molecular characterization of carboxypeptidase B-like (CPB) in Scylla paramamosain and its role in white spot syndrome virus and Vibrio alginolyticus infection.

Carboxypeptidase plays an important physiological role in the tissues and organs of animals. In this study, we cloned an entire 2316 bp carboxypeptidase B-like (CPB) sequence with a 1302 bp open reading frame encoding a 434 amino acid peptide from Scylla paramamosain. The CPB gene was expressed highly in hepatopancreas and decreased in crab hemocytes after challenges with white spot syndrome virus (WSSV) or Vibrio alginolyticus. After CPB gene knockdown using double-stranded RNA (CPB-dsRNA), the expression of JAK, STAT, C-type lectin, crustin antimicrobial peptide, Toll-like receptors, prophenoloxidase, and myosin II essential light chain-like protein were down-regulated in hemocytes at 24 h post dsRNA treatment. CPB knockdown decreases total hemocyte count in crabs indicated that CPB may negatively regulate crab hemocyte proliferation in crabs. CPB showed an inhibitory effect on hemocyte apoptosis in crabs infected with WSSV or V. alginolyticus. The phagocytosis rate of WSSV by hemocytes was increased after CPB-dsRNA treatment. After WSSV challenge, the mortality and WSSV copy number were both decreased but the rate of hemocyte apoptosis was increased in CPB-dsRNA-treated crabs. The results indicate that the antiviral activity of the crabs was enhanced when CPB was knocked down, indicating WSSV may take advantage of CPB to benefit its replication. In contrast, the absence of CPB in crabs increased mortality following the V. alginolyticus challenge. The phagocytosis rate of V. alginolyticus by hemocytes was increased after CPB-dsRNA treatment. It was revealed that CPB may play a positive role in the immune response to V. alginolyticus through increasing the phagocytosis rate of V. alginolyticus. This research further adds to our understanding of the CPB and identifies its potential role in the innate immunity of crabs.

Qian X ，Lai Y ，Zhu F 《-》