The Role of microRNA-133 in Hemocyte Proliferation and Innate Immunity of Scylla paramamosain.

MicroRNAs (miRNAs) are important signaling regulators that are involved in regulating the innate immunity of crustacean. However, few studies focus on the role of crustacean miRNAs in the cellular immunity have been reported. In this study, we showed that the expression of miR-133 was significantly up-regulated in the mud crab Scylla paramamosain after infection by white spot syndrome virus (WSSV) or Vibrio parahaemolyticus. The anti-miRNA oligonucleotide AMO-miR-133 was used to knock down miR-133 expression in S. paramamosain. The number of WSSV copies increased significantly in WSSV-infected crabs after miR-133 knockdown. Knockdown of miR-133 also enhanced the mortality rates of WSSV-infected and V. parahaemolyticus-infected mud crabs, and it significantly enhanced the expression of the astakine, which was confirmed by real-time quantitative PCR and western blot analysis. The data also indicate that miR-133 may affect hemocyte proliferation in S. paramamosain by regulating astakine expression. miR-133 Knockdown enhanced the apoptosis or phagocytosis of crab hemocytes, and increased the mortality of mud crabs after WSSV or V. parahaemolyticus infection. These results indicate that miR-133 is involved in the host immune response to WSSV and V. parahaemolyticus infection in mud crabs. Taken together, our research provides new insights for the control of viral or vibrio diseases in S. paramamosain.

Differential expression of microRNAs in mud crab Scylla paramamosain in response to white spot syndrome virus (WSSV) infection.

Till date numerous microRNAs (miRNAs) have been discovered from various organisms, including fish, shellfish and crustaceans. The miRNAs are known to regulate immune functions in crustaceans, but little is known about the role of miRNAs against viral infection in crab. We performed small RNA sequencing to characterize the differentially expressed miRNAs in WSSV infected Scylla paramamosain, in comparison to that in uninfected crab, at 2 h and 12 h post infection. In total, 24 host miRNAs were up-regulated and 25 host miRNAs were down-regulated in response to WSSV infection at 2 h post infection. And 27 host miRNAs were up-regulated and 30 host miRNAs were down-regulated in response to WSSV infection at 12 h post infection. Further, the gene ontology analysis revealed that many signaling pathways were mediated by these miRNAs. The integral component of membrane is the most important biological process and endocytosis pathway is the most important pathway, which indicates that endocytosis is very important for WSSV infection. This study is one important attempt at characterizing crab miRNAs that response to WSSV infection, and will help unravel the miRNA pathways involved in antiviral immunity of crab.

WSSV proteins and DNA genome released by ultrasonic rupture can infect crayfish as effectively as intact virions.

Proteins and nucleic acids from ultrasonically ruptured white spot syndrome virus (WSSV) can infect crayfish and cause death as effectively as intact WSSV virions. In this study, ultrasound was used to rupture the virus and the resulting suspension was filtered through a 50 nm membrane. Analysis by PCR and SDS-PAGE showed that both viral genes (VP19, VP26, VP28 and DNA polymerase) and proteins (VP15, VP19, VP26 and VP28) were present in the filtered solution. Electron microscopy showed that there were no intact virions in the filtered solution. When crayfish were injected with the filtered solution or with intact WSSV, the mortality in each group was 100 %. The same result was seen when crayfish were challenged orally with the filtered solution and intact WSSV. The filtered solution of ultrasonically ruptured virus, which contains viral proteins and residual DNA genome, can thus infect the host as effectively as intact virions. When the solution of viral proteins and residual DNA genome was digested with DNase I and then injected into crayfish, the survival rate was 100 %. We also found that, although viral proteins (except VP15) in the solution of ruptured virus were destroyed by treatment with DNase I, DNase I did not destroy the structural proteins of intact virions. A remaining viral protein in the DNase I-treated solution protects the DNA genome from degradation and we concluded that this protein is VP15, which is a DNA-binding protein. Our study highlights the extreme danger in producing vaccines from proteins obtained by ultrasonic rupture of viruses sincethe viral DNA genome is difficult to degrade and, if present, will lead to viral infection.

Dietary Bacillus amyloliquefaciens enhance survival of white spot syndrome virus infected crayfish.

Bacillus amyloliquefaciens, which is closely related to Bacillus subtilis, produces a series of metabolites that can inhibit the growth of fungi and bacteria. Here, we investigated the effect of B. amyloliquefaciens used as a probiotic on the innate immunity of the crayfish Procambarus clarkii when challenged with white spot syndrome virus (WSSV). Dietary B. amyloliquefaciens supplement significantly reduced the mortality of WSSV-challenged crayfish and reduced copy numbers of WSSV. The quantitative reverse transcription-polymerase chain reaction results showed that B. amyloliquefaciens supplement increased the expression of several immune-related genes, including Toll-like receptor, NF-κB and C-type-lectin. Further analysis showed that B. amyloliquefaciens supplement also had an effect on three immune parameters, including total hemocyte count, phenoloxidase activity and superoxide dismutase activity. In both infected and uninfected crayfish, B. amyloliquefaciens supplement significantly decreased hemocyte apoptosis. Our results showed that B. amyloliquefaciens can regulate innate immunity of crayfish and reduce the mortality following WSSV challenge. This study provides a novel insight into the potential for therapeutic or prophylactic intervention with B. amyloliquefaciens to regulate crayfish immunity and protect against WSSV infection, and also provides a theoretical basis for the use of probiotics as aquatic feed additives.

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.