Differential expression of microRNAs in giant freshwater prawn (Macrobrachium rosenbergii) during the infection of Vibrio parahaemolyticus.

MicroRNAs (miRNAs) are a category of small non-coding RNAs regarded as vital regulatory factors in various biological processes, especially immune regulation. The differently expressed miRNAs in Macrobrachium rosenbergii after the challenge of Vibrio parahaemolyticus were identified using high-throughput sequencing. A total of 18 known as well as 12 novel miRNAs were markedly differently expressed during the bacterial infection. The results of the target gene prediction and enrichment analysis indicated that a total of 230 target genes involved in a large variety of signaling pathways and biological processes were mediated by the miRNAs identified in the current research. Additionally, the effects of novel-miR-56, a representative differentially expressed miRNA identified in the previous infection experiment, on the immune-related gene expression in M. rosenbergii were explored. The expression of the immune-related genes including Spätzle1(Spz1), Spz4, Toll-like receptor 1 (TLR1), TLR2, TLR3, immune deficiency (IMD), myeloid differentiation factor 88 (MyD88), anti-lipopolysaccharide factor 1 (ALF1), crustin1, as well as prophenoloxidase (proPO) was significantly repressed in the novel-miR-56-overexpressed prawns. The expression of these genes tested in the novel-miR-56-overexpressed M. rosenbergii was still signally lower than the control in the subsequent V. parahaemolyticus challenge, despite the gene expression in each treatment increased significantly after the infection. Additionally, the cumulative mortality of the agomiR-56-treated prawns was significantly higher than the other treatments post the bacterial challenge. These results suggested that novel-miR-56 might function as a negative regulator of the immune-related gene expression of M. rosenbergii in the innate immune defense against V. parahaemolyticus.

Transcriptomics-based analysis of Macrobrachium rosenbergii growth retardation.

Macrobrachium rosenbergii is an economically important crustacean in many parts of the world, but in recent years, growth retardation has become an increasingly serious issue. While the underlying causes remain unclear, this has inevitably impacted on aquaculture and production outputs. In this study, gill, hepatopancreas, and muscle tissue samples from M. rosenbergii, with distinct growth differences, underwent transcriptome sequencing and bioinformatics analyses using high-throughput sequencing. In total, 59,796 unigenes were annotated. Differential expression analyses showed that the most differentially expressed genes (DEGs) were screened in gill tissue (1790 DEGs). In muscle and hepatopancreas tissues, 696 and 598 DEGs were screened, respectively. These DEGs were annotated to Kyoto Encyclopedia of Genes and Genomes pathways, which identified several significantly enriched pathways related to growth metabolism, such as PI3K-AKT, glycolysis/gluconeogenesis, and starch and sucrose metabolism. These results suggest that low growth metabolism levels may be one cause of M. rosenbergii growth retardation. Our data provide support for further investigations into the causes and molecular mechanisms underpinning growth retardation in M. rosenbergii.

Identification and characterization of TOR in Macrobrachium rosenbergii and its role in muscle protein and lipid production.

The recent scarcity of fishmeal and other resources means that studies on the intrinsic mechanisms of nutrients in the growth and development of aquatic animals at the molecular level have received widespread attention. The target of rapamycin (TOR) pathway has been reported to receive signals from nutrients and environmental stresses, and regulates cellular anabolism and catabolism to achieve precise regulation of cell growth and physiological activities. In this study, we cloned and characterized the full-length cDNA sequence of the TOR gene of Macrobrachium rosenbergii (MrTOR). MrTOR was expressed in all tissues, with higher expression in heart and muscle tissues. In situ hybridization also indicated that MrTOR was expressed in muscle, mainly around the nucleus. RNA interference decreased the expression levels of MrTOR and downstream protein synthesis-related genes (S6K, eIF4E, and eIF4B) (P < 0.05) and the expression and enzyme activity of the lipid synthesis-related enzyme, fatty acid synthase (FAS), and increased enzyme activity of the lipolysis-related enzyme, lipase (LPS). In addition, amino acid injection significantly increased the transcript levels of MrTOR and downstream related genes (S6K, eIF4E, eIF4B, and FAS), as well as triglyceride and total cholesterol tissue levels and FAS activity. Starvation significantly increased transcript levels and enzyme activities of adenylate-activated protein kinase and LPS and decreased transcript levels and enzyme activities of FAS, as well as transcript levels of MrTOR and its downstream genes (P < 0.05), whereas amino acid injection alleviated the starvation-induced decreases in transcript levels of these genes. These results suggested that arginine and leucine activated the TOR signaling pathway, promoted protein and lipid syntheses, and alleviated the pathway changes induced by starvation.

The regulation of immune responses against white spot syndrome virus or Vibrio alginolyticus in toll-like receptors silenced giant freshwater prawn (Macrobrachium rosenbergii).

Toll-like receptors, which are a class of cell-surface proteins, have been regarded as the most important pattern recognition receptors in the innate immunity and play a vital role in multiple innate immune responses against pathogen invasion. The full-length cDNA of a novel Toll-like receptor (MrToll3) was identified from Macrobrachium rosenbergii in the current research. The nucleotide sequence of MrToll3 is 4481 bp long and contains a 3726-bp open reading frame encoding a putative protein of 1241 amino acids. MrToll3 was constitutively expressed in all the examined tissues, and high expression of MrToll3 was detected in gill, heart, and ganglion. The result of RNA interference assay revealed that silencing of MrToll1 remarkably suppressed the prophenoloxidase (proPO) expression and phenoloxidase (PO) activities while enhancing MrToll2 expression in the prawns. Furthermore, the expression of myeloid differentiation factor 88 (MyD88), anti-lipopolysaccharide factor (ALF) and crustin was remarkably down-regulated in the MrToll1-silenced prawns after white spot syndrome virus (WSSV) or Vibrio alginolyticus challenge. MrToll2-silenced prawns exhibited the significant decline of ALF and crustin expression post the pathogen challenges, and silencing of MrToll3 obviously improved the immune deficiency (IMD) expression during the whole RNA interference assay. Additionally, higher mortality was observed in MrToll1-or MrToll2-silenced prawns after V. alginolyticus challenge, and the MrToll1-silenced prawns also showed the obviously enhanced susceptibility to WSSV. These results suggested that MrToll1, 2, and 3 were involved in the innate immune responses against WSSV and V. alginolyticus in M. rosenbergii.

Effects of dietary inulin and mannan oligosaccharide on immune related genes expression and disease resistance of Pacific white shrimp, Litopenaeus vannamei.

The effects of inulin and mannan oligosaccharide (MOS) at different doses (2.5, 4 and 10 mg/g) in singular or combined diet on growth rate, immune related genes expression, and resistance to white spot syndrome virus (WSSV) and Vibrio alginolyticus in Pacific white shrimp (Litopenaeus vannamei) were investigated. At the end of 28-day singular feeding experiment, the highest values of specific growth rate (SGR) and the expression of toll-like receptor1, 2 and 3 (TLR1, 2, 3), signal transducer and activator of transcription (STAT), crustin, anti-lipopolysaccharide factor (ALF) as well as prophenoloxidase (proPO) were observed in shrimp individually fed with 5 mg/g dietary inulin or MOS, respectively. Compared with individual treatments, diet containing combined prebiotics (5 mg/g inulin and MOS) significantly improved the expression of TLRs, STAT, proPO, crustin and ALF in L. vannamei after four-week feeding. Additionally, Pacific white shrimp fed with combined dietary prebiotics showed significantly higher expression of immune related genes and lower cumulative mortality in WSSV and Vibrio alginolyticus challenges, compared to the singular feeding groups and control. These results in the present study demonstrated that the combined supplementation of inulin (5 mg/g) and MOS (5 mg/g) remarkably enhanced innate immune response and pathogen resistance of shrimp, and should be considered as a promising immunostimulatory additive for the culture of Pacific white shrimp.

A Vibrio owensii strain as the causative agent of AHPND in cultured shrimp, Litopenaeus vannamei.

The causative agent of shrimp AHPND was identified as specific Vibrio parahaemolyticus strains, which harbor a virulent plasmid that contains the toxic genes pirA and B (pirAB). Herein, a Vibrio bacterium was isolated from shrimp in Shanghai. This bacterium was identified as Vibrio owensii using 16S rRNA gene phylogeny, whole genome sequencing and comparative analysis. The V. owensii cells are rod-shape (1.86 ±â€¯0.15 µm) with a single polar flagellum (4 µm). In addition, V. owensii form mauve colonies with jagged edges on CHROMagar plates. The pirAB genes on the plasmid revealed 100% sequence similarity to that of AHPND V. parahaemolyticus, and the encoded proteins were detected in the culture media. Subculture of V. owensii showed that the pirAB genes are unstable, and their loss rate is approximately 22% and reaches a dynamic equilibrium after the fifth generation. Upon immersion bioassay, the cumulative mortality of V. owensii (pirAB+)-infected shrimp was up to 100% within 4 days, and typical AHPND clinical signs were observed. Approximately 105 CFU/hepatopancreas of V. owensii cells were observed in the pirAB+-infected shrimp based on both culture-dependent and -independent assay. Our results indicate that the expression of pirAB in the V. owensii strain is responsible for AHPND.