-
Jie Du,
Huanxi Zhu,
Peng Liu,
Jing Chen,
Yunji Xiu,
Wei Yao,
Ting Wu,
Qian Ren,
Qingguo Meng,
Wei Gu,
Wen Wang
[show abstract]
[hide abstract]
ABSTRACT: Freshwater prawn Macrobrachium rosenbergii inoculated with 100 μl novel pathogen spiroplasma strain MR-1008 in logarithmic phase (10(8) spiroplasmas ml(-1)) were examined for alkaline phosphatase (AKP) activity, acid phosphatase (ACP) activity, superoxide dismutase (SOD) activity, catalase (CAT) activity, as well as expressions of 7 immune related genes in hepatopancreas after 1-28 d. Hematoxylin-eosin (HE) staining showed obvious pathological features in hepatopancreas connective and epithelial tissue. Enzyme activity analyse showed that hepatopancreas AKP and ACP activity increased markedly (P < 0.05) when inoculated with spiroplasma MR-1008 after 5 d and 10 d, respectively. SOD enzyme activity changed less obviously and slightly increased at 1 day post- inoculation, but CAT activity decreased significantly after 5 d inoculation. The expression levels of lipopolysaccharide-and β-1,3-glucan-binding protein (LGBP), peroxinectin (PE), α2-macroglobulin (α2M), AKP, ACP, CAT, and copper/zinc SOD (Cu, Zn-SOD) genes in the hepatopancreas were examined by Real Time-PCR (qRT-PCR) and the results demonstrated that these immune-related genes were induced by challenge with spiroplasma MR-1008. The results suggested that the prawn immune responses could be activated or inhibited by spiroplasma MR-1008, and that the hepatopancreas also plays key roles in innate immunity for defense against the pathogen.
Fish & Shellfish Immunology 11/2012; · 3.32 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Flow cytometry provides rapid and reproducible methods for analyzing crustacean cellular immune responses to pathogens. We used this method to investigate the hemocytes sub-populations of freshwater prawn Macrobrachium rosenbergii and their immune responses to a novel pathogen spiroplasma MR-1008. M. rosenbergii inoculated with 100 μl spiroplasma strain MR-1008 in logarithmic phase (10(8) spiroplasmas ml(-1)) were examined for total hemocytes count (THC) and changes in differential involvement of hemocytes sub-populations during 1-28 d after inoculation. The results showed that THC was dramatically lowered 1 d after inoculation, and it obviously increased at the 5 d after inoculation; thereafter, a high level of THC was maintained to 15 d. Three morphologically distinct hemocytes sub-populations including granular cells (GC), semigranular cells (SGC) and hyaline cells (HC) could be identified by flow cytometry, and the proportions of the 3 kinds of cell categories varied obviously during the infection of spiroplasma suggesting differential involvement according to the pathogen. The flow cytometry used in this study confirmed that the semigranular cells were the main hemocytes involved in the cellular defense against spiroplasma in the M. rosenbergii.
Fish & Shellfish Immunology 07/2012; 33(4):795-800. · 3.32 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Anti-lipopolysaccharide factor (ALF) is a type of basic protein and an important antimicrobial peptide that can bind and neutralize lipopolysaccharides (LPS). This protein shows a broad spectrum of antimicrobial activity. In this study, three forms of ALF designated as MrALF5, MrALF6, and MrALF7 were identified from giant freshwater prawn, Macrobrachium rosenbergii. MrALF5, MrALF6, and MrALF7 genes encode 133, 121, and 120 amino acids of the corresponding proteins, respectively. All these ALF proteins contain LPS-binding domain with two conserved cysteine residues. The genomic sequences of MrALF5 and MrALF7 were amplified. The genomic structures of MrALF5 and MrALF7 comprise three exons interrupted by two introns. Phylogenetic analysis showed that MrALF5, MrALF6, and MrALF7 were clustered into clade II. Evolutionary analysis showed that ALF genes from M. rosenbergii may suffer a rapid evolution. MrALF5 was expressed mainly in the hepatopancreas, gills, and heart. MrALF6 was mainly distributed in the intestine and hepatopancreas. The highest expression level of MrALF7 was detected in the hepatopancreas. MrALF6, as well as MrALF7, was downregulated by Escherichia coli challenge, and all three ALF genes were upregulated by Vibrio or white spot syndrome virus challenge. MrALF6 was also upregulated by Staphylococcus aureus challenge. In summary, the three isoforms of ALF genes may participate in the innate immune response against bacteria and virus infecting the giant fresh water prawn.
Fish & Shellfish Immunology 07/2012; 33(4):766-74. · 3.32 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: To investigate the interaction between Chinese mitten crab Eriocheir sinensis hemocytes and the pathogen Spiroplasma eriocheiris, a system for in vitro culture of E. sinensis hemocytes with high viability was developed. Following optimization of conditions, hemocytes survived for >35 days. After challenge with the novel crustacean pathogen S. eriocheiris, E. sinensis hemocytes began to develop vacuoles, and then they began to die (within 60 h). Real-time RT-PCR analysis of S. eriocheiris infected hemocytes identified increased expression levels of anti-lipopolysaccharide factor (ALF), peroxinectin (Pox) and clip domain serine protease (cSP) genes. The expression levels of ALF, Pox, and cSP genes in hemocytes of E. sinensis demonstrated that all three immune genes were significantly induced by challenge with S. eriocheiris. Increases in Pox mRNA levels were highest (up to 36-fold) and peaked at 24-48 h post-challenge (pc) (P < 0.05) and lesser increases were evident with ALF and cSP, peaking at 24 h and at 12-48 h pc, respectively. The hemocytes culture method described herein provides a feasible in vitro research model of E. sinensis that can be used to study its immune reactions against various crab pathogens.
Molecular Biology Reports 06/2012; 39(10):9747-54. · 2.93 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: C-type lectins (CTLs) are believed to play important roles in the innate immunity of invertebrates and serve as pattern recognition receptors, opsonins, or effector molecules. In this study, the full-lengths cDNA of 4 CTL genes from giant freshwater prawn Macrobrachium rosenbergii were cloned and designated as MrLec1, MrLec2, MrLec3, and MrLec4. All of these 4 lectin cDNAs encode proteins with 2 carbohydrate recognition domains (CRDs). While MrLec1, MrLec3, and MrLec4 had signal peptides, no signal peptide was detected in MrLec2. Two carbohydrate recognition motifs within two CRDs of each lectin were predicted (QPE, EPG in MrLec1; EPT, EPA in MrLec2; QPT, NPR in MrLec3; KPN, EPD in MrLec4). Phylogenetic analysis showed that MrLec4 belongs to group A whereas MrLec1, MrLec2, and MrLec3 belong to group B. Positive selection in dual-CRD lectins suggested their probable roles in innate immunity, and positively selected induced amino acid diversity of lectins may confer their ability to recognize a broad range of microbes. The qRT-PCR analysis in adult prawns showed that MrLec1 is mainly expressed in the hepatopancreas, gills, and stomach, MrLec2 and MrLec4 are mainly distributed in the hepatopancreas, and MrLec3 is mainly expressed in the hepatopancreas and stomach. Time-course analysis using qRT-PCR showed that MrLec1 to MrLec4 are all upregulated by the Vibrio anguillarum challenge. MrLec1 is upregulated after 2, 12, and 24 h of white spot syndrome virus (WSSV) challenge. The expression of MrLec2 increases after 12 and 24 h of WSSV challenge, and the transcript of MrLec3 and MrLec4 are downregulated after 2 h of WSSV challenge. The results suggest the potential roles of dual-CRD lectins in the innate immunity of M. rosenbergii.
Fish & Shellfish Immunology 03/2012; 33(2):155-67. · 3.32 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The Chinese mitten crab Eriocheir sinensis is one of the most important freshwater aquaculture crustacean species in China. MicroRNAs (miRNAs) are small non-coding RNAs that are important effectors in the intricate host-pathogen interaction network. To increase the repertoire of miRNAs characterized in crustaceans and to examine the relationship between host miRNA expression and pathogen infection, we used the Illumina/Solexa deep sequencing technology to sequence two small RNA libraries prepared from haemocytes of E. sinensis under normal conditions and during infection with Spiroplasma eriocheiris. The high-throughput sequencing resulted in approximately 30,975,151 and 30,826,277 raw reads corresponding to 12,077,088 and 16,271,545 high-quality mappable reads for the normal and infected haemocyte samples, respectively. Bioinformatic analyses identified 735 unique miRNAs, including 36 that are conserved in crustaceans, 134 that are novel to crabs but are present in other arthropods (PN-type), and 565 that are completely new (PC-type). Two hundred twenty-eight unique miRNAs displayed significant differential expression between the normal and infected haemocyte samples (p < 0.0001). Of these, 133 (58%) were significantly up-regulated and 95 (42%) were significantly down-regulated upon challenge with S. eriocheiris. Real-time quantitative PCR (RT-qPCR) experiments were preformed for 10 miRNAs of the two samples, and agreement was found between the sequencing and RT-qPCR data. To our knowledge, this is the first report of comprehensive identification of E. sinensis miRNAs and of expression analysis of E. sinensis miRNAs after exposure to S. eriocheiris. Many miRNAs were differentially regulated when exposed to the pathogen, and these findings support the hypothesis that certain miRNAs might be essential in host-pathogen interactions. Our results suggest that elucidation of the molecular mechanisms responsible for miRNA regulation of the host's innate immune system should help with the development of new control strategies to prevent or treat S. eriocheiris infections in crustaceans.
Fish & Shellfish Immunology 12/2011; 32(2):345-52. · 3.32 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: An extracellular copper–zinc superoxide dismutase (ecCuZnSOD) gene was cloned from hemocytes of Chinese mitten crab, Eriocheir sinensis. The ecCuZnSOD cDNA consists of 891 bp with an open reading frame (ORF) of 624 bp that encodes 208 amino acids (aa) with 24 residues of a signal peptide sequence. The predicted molecular mass of the mature protein (184 aa) is 21.76 kDa with an estimated pI of 6.02. Amino acids are responsible for binding Cu (His90, 92, 109, and 171) and Zn (His109, 117, and 126, and Asp 129), two cysteines (95 and 189) that form a disulfide bond, two CuZnSOD signatures (GFHVHEWGVDGS) from 88 to 99 and (GNAGGRVACCTI) from 189 to 200. The deduced amino acid sequence of E. sinensis ecCuZnSOD showed similarity of 75% and 58.7% with the ecCuZnSOD of giant freshwater prawn Macrobrachium rosenbergii and mud crab Scylla serrata, respectively. Spiroplasma eriocheiris is a novel pathogen of tremor disease of E. sinensis. This paper also studied the relationship between E. sinensis ecCuZnSOD and S. eriocheiris. The ecCuZnSOD transcript in hemocytes significantly increased in 1 h, had returned to the original value by 24 h, and increased after 192 h by the S. eriocheiris inoculation. The ecCuZnSOD transcript increased significantly at 1 h after S. eriocheiris injection indicating induction of the immune system of the crab within a short time. After 192 h the ecCuZnSOD transcript increased, meanwhile the tremor symptom of the crab also appeared, induction of the ecCuZnSOD has relation with the immune response of hosts to resist S. eriocheiris.Highlights► We cloned extracellular copper/zinc superoxide dismutase from Eriocheir sinensis. ► We analyzed the characterisation of ecCuZnSOD from Eriocheir sinensis. ► ecCuZnSOD expressed in all organ of Eriocheir sinensis. ► ecCuZnSOD transcript is relation with the S. eriocheiris injection.
Aquaculture. 320:56-61.
