Intestinal epithelial cells of Japanese flounder (Paralichthys olivaceus) as an in vitro model for studying intestine immune function based on transcriptome analysis.

Japanese flounder (Paralichthys olivaceus) is an economically crucial marine species, but diseases like hemorrhagic septicemia caused by Edwardsiella tarda have resulted in significant economic losses. E. tarda infects various hosts, and its pathogenicity in fish is not fully understood. Lipopolysaccharides (LPS) are components of the outer membrane of Gram-negative bacteria and are representative of typical PAMP molecules that cause activation of the immune system. The PoIEC cell line is a newly established intestinal epithelial cell line from P. olivaceus. In order to investigate whether it can be used as an in vitro model for studying the pathogenesis of E. tarda and LPS stimulation, we conducted RNA-seq experiments for the PoIECs model of E. tarda infection and LPS stimulation. In this study, transcriptome sequencing was carried out in the PoIEC cell line after treatment with LPS and E. tarda. A total of 62.52G of high-quality data from transcriptome sequencing results were obtained in nine libraries, of which an average of 87.96% data could be aligned to the P. olivaceus genome. Data analysis showed that 283 and 414 differentially expressed genes (DEGs) in the LPS versus Control (LPS-vs-Con) and E. tarda versus Control groups (Et-vs-Con), respectively, of which 60 DEGs were shared in two comparation groups. The GO terms were predominantly enriched in the extracellular space, inflammatory response, and cytokine activity in the LPS-vs-Con group, whereas GO terms were predominantly enriched in nucleus and positive regulation of transcription by RNA polymerase II in the Et-vs-Con group. KEGG analysis revealed that three immune-related pathways were co-enriched in both comparison groups, including the Toll-like receptor signaling pathway, C-type lectin receptor signaling pathway, and Cytokine-cytokine receptor interaction. Five genes were randomly screened to confirm the validity and accuracy of the transcriptome data. These results suggest that PoIEC cell line can be an ideal in vitro model for studies of marine fish gut immunity and pathogenesis of Edwardsiellosis.

Efficacy of bivalent vaccine against Aeromonas salmonicida and Edwardsiella tarda infections in turbot.

In recent years, more than one pathogenic organism has usually been isolated from diseased turbot Scophthalmus maximus, creating a pressing need for the development of combination vaccines to prevent fish diseases brought on simultaneously by various infections. In this study, the inactivated bivalent vaccine of Aeromonas salmonicida and Edwardsiella tarda was prepared by the formalin inactivation method. After challenge with A. salmonicida and E. tarda at 4 weeks post-vaccination in turbot, the relative percentage survival (RPS) of the inactivated bivalent vaccine was 77.1%. In addition, we assessed the effects of the inactivated bivalent vaccine and evaluated the immunological processes after immunization in a turbot model. Serum antibody titer and lysozyme activity of the vaccinated group were both upregulated and higher than that in control group after vaccination. The expression levels of genes (TLR2, IL-1ß, CD4, MHCI, MHCⅡ) that related to antigen recognition, processing and presentation were also studied in the liver, spleen and kidney tissues of vaccinated turbot. All the detected genes in the vaccinated group had a significant upward trend, and most of them reached the maximum value at 3-4 weeks, which had significant differences from the control group, suggesting that antigen recognition, processing and presentation pathway was activated by the inactivated bivalent vaccine. Our study provides a basis for further application of the killed bivalent vaccine against A. salmonicida and E. tarda in turbot, making it good potential that can be applied in aquaculture.

Transcriptome analysis of turbot (Scophthalmus maximus) kidney responses to inactivated bivalent vaccine against Aeromonas salmonicida and Edwardsiella tarda.

Turbot (Scophthalmus maximus) is a commercially important marine flatfish for global aquaculture. With intensive farming, turbot production is limited by several diseases, in which Aeromonas salmonicida and Edwardsiella tarda are two main causative agents. Vaccination is an effective and safe alternative to disease prevention compared to antibiotic treatment. In the previous study, we developed an inactivated bivalent vaccine against A. salmonicida and E. tarda with relative percent survival (RPS) of 77.1 %. To understand the protection mechanism in molecular basis of the inactivated bivalent vaccine against A. salmonicida and E. tarda, we use RNA-seq to analyze the transcriptomic profile of the kidney tissue after immunization. A total of 391,721,176 clean reads were generated in nine libraries by RNA-seq, and 96.35 % of the clean reads were mapped to the reference genome of S. maximus. 1458 (866 upregulated and 592 downregulated) and 2220 (1131 upregulated and 1089 downregulated) differentially expressed genes (DEGs) were obtained at 2 and 4 weeks post-vaccination, respectively. The DEGs were enriched in several important immune-related GO terms, including cytokine activity, immune response, and defense response. In addition, the analysis of several immune-related genes showed upregulation and downregulation, including pattern recognition receptors, complement system, cytokines, chemokines and immune cell surface markers. Eight DEGs (ccr10, calr, casr, mybpha, cd28, thr18, cd20a.3 and c5) were randomly selected for qRT-PCR analysis, which confirmed the validity of the RNA-seq. Our results provide valuable insight into the immune mechanism of inactivated bivalent vaccine against A. salmonicida and E. tarda in Scophthalmus maximus.

Immune responses to Vibrio vulnificus formalin-killed vaccine and ghost vaccine in Scophthalmus maximus.

In this research, Vibrio vulnificus formalin-killed (FKCs) vaccine and ghost (VVGs) vaccine were successfully developed, and shown to prevent vibriosis of Scophthalmus maximus resulting from V. vulnificus. The antibody titre of FKCs and VVGs vaccine was 1: 28 and 1: 211 . The RPS of FKCs and VVGs vaccine was 60% and 80%. In order to improve the understanding of vaccine protection mechanism, transcriptome data was used to analyse the immune response of S. maximus infected with V. vulnificus after vaccination with FKCs and VVGs vaccine. In the SmCon and SmIV groups, a series of innate immune-related genes were upregulated (such as, TLR5, Tp12, AP-1 and IL-1ß) or downregulated (such as, CASP6 and CASP8), which suggested that the immune protection mechanism induced by inactivated vaccine was similar to that of autoimmune response. In the SmIV and SmGho group, a number of innate and adaptive immune-related genes (such as, STAT1, IFN-γ and MHC Ia) were activated, in which the expression of these genes was higher in SmGho, and VVGs vaccine induced stronger innate and acquired immune responses. In conclusion, the results lay a foundation for further study on the molecular mechanisms of immune protection induced by VVGs vaccine and FKCs vaccine.

Molecular characterization, expression analysis and immune effect of Galectin-8 from Japanese flounder (Paralichthys olivaceus).

Galectin-8 gene belongs to the agglutinin family, which can specifically recognize ß-galactoside bonds and play essential roles in many biological processes. In this study, we researched the sequence characteristics and immune-related function of Galectin-8 gene in Japanese flounder Paralichthys olivaceus, named PoGalectin-8. The results showed that the open reading frame of PoGalectin-8 was 891 bp, which encoding a protein with 296 amino acid residues and containing typical HXNPR and WGXEE motifs in the N-terminal and C-terminal CRD domains. Sequence alignment showed that PoGalectin-8 was conserved in different aquatic animals and exhibited the highest similarity (95.27%) with Seriola dumerili. PoGalectin-8 expressed in all detected tissues and exhibited the highest expression level in spleen, followed by skin and kidney. After infected by Edwardsiella tarda, the expression of PoGalectin-8 was down-regulated in the spleen and skin tissues of P. olivaceus. Further to study its immune-related functions, the recombinant PoGalectin-8 (rPoGalectin-8) was expressed and purified. The rPoGalectin-8 can specifically bind to lipopolysaccharide and peptidoglycan, the main components of cell walls from Gram-negative and Gram-positive bacteria. Bacteria binding and the microbial agglutinating experiments showed that the rPoGalectin-8 could bind and agglutinate all examined Gram-positive and Gram-negative bacteria. This study implied that PoGalectin-8, as a pattern recognition receptor, may play important roles during immune responses against bacterial infection, which laid a foundation for further functional identification of Galectin-8 in aquatic animal immunity.

Molecular characterization and functional study of a tandem-repeat Galectin-9 from Japanese flounder (Paralichthys olivaceus).

Galectin-9 is a ß-galactoside-binding lectin which could modulate a variety of biological functions including recognition, aggregation and clearance of pathogen. In this study, one Galectin-9 (named PoGalectin-9) was identified from Japanese flounder Paralichthys olivaceus. PoGalectin-9 belongs to the tandem-repeat type, containing one 127-amino acids CRD domain within N terminal and one 122-amino acids CRD domain within C-terminal. The open reading frame of PoGalectin-9 cDNA was 921 bp encoding 306 amino acids. Sequence similarity comparison confirmed that PoGalectin-9 shared high homology with other Galectin-9. The tissue distribution and expression profiles after bacterial infection were also investigated. PoGalectin-9 was widely distributed in all of the examined tissues of Japanese flounder but was predominantly expressed in the spleen, kidney and intestine. After Edwardsiella tarda challenge, the expression of PoGalectin-9 was up-regulated in spleen and down regulated in kidney. ELISA experiment showed that recombinant PoGalectin-9 (rPoGalectin-9) exhibit binding capacity to lipopolysaccharide (LPS) and peptidoglycan (PGN), which is significantly correlated with the concentration of rPoGalectin-9. Meanwhile, the rPoGalectin-9 protein showed strong agglutinating activities against both Gram-negative bacteria and Gram-positive bacteria. Bacterial binding experiments showed that rPoGalectin-9 could bind all examined bacteria. In conclusion, the present study indicate that PoGalectin-9 might play important roles during the immune responses of Japanese flounder against bacterial pathogens.

Rapid visual detection of Aeromonas salmonicida by loop-mediated isothermal amplification with hydroxynaphthol blue dye.

To make crucial prevention, reduce fish losses and minimize the economic damage of diseases on the fish farm owners, a rapid detection of fish pathogens is mandatory. In this study, a loop-mediated isothermal amplification assay combined with hydroxynaphthol blue dye (LAMP-HNB) was developed and used for the rapid detection of Aeromonas salmonicida that caused significant economic losses in fish farming. Firstly, a pair of outer and inner primers specific for conserved fragment of vapA gene in A. salmonicida were designed and synthesized. Secondly, by optimizing the reaction conditions including reaction temperature, time, Mg2+ concentration, dNTP concentration and primer ratio, a LAMP-HNB assay was successfully established for the detection of A. salmoncida. Thirdly, the assay showed good specificity with no false-positive and false-negative results, and good sensitivity with the detection limit of 3.077 × 10-6  ng/µl, which was 102 times more sensitive than the conventional PCR. Finally, the LAMP-HNB assay was validated by the fish samples inoculated with different concentrations of A. salmoncida. This is the first development of rapid visual detection of A. salmonicida based on LAMP-HNB assay, which has great application prospect and market for diagnostic testing, health certification and active surveillance programmers.

Full-length transcriptome sequencing from multiple immune-related tissues of Paralichthys olivaceus.

Olive flounder (Paralichthys olivaceus) is an important economical flatfish in Japan, Korea and China, but its production has been greatly threatened by various of diseases. Although RNA-seq has provided valuable insights into the host-pathogen interaction, there are still some disadvantages, such as a short sequencing length, the incomplete or inaccurate splicing. Therefore, we generated a full-length transcriptome using mixed immune-related tissues of P. olivaceus with PacBio Sequel platform. In this study, 379,671 full-length non-chimeric (flnc) reads were generated with average length of 2482 bp, which is longer than any previously reported in P. olivaceus. A total of 66,420 isoforms of transcript were identified, 46,850 of which were novel isoforms of known genes accounting for 70.54%. In addition, 7720 novel genes, 12,540 alternative splicing (AS) events, 9296 alternative polyadenylation (APA) events, 2298 transcription factors (TFs), 10,270 lncRNAs and 5400 fusion transcripts were identified. Furthermore, functional annotation showed that most of the full-length transcripts were enriched in immune-related signaling pathways. Otherwise, the mRNA-miRNA interacting networks confirmed that 28.5% of mRNAs were predicted to be targeted by more than one miRNA. These results facilitate the understanding of gene structure, post-transcriptional regulatory networks, and subsequently proteomic diversity. In conclusion, our study provides the full-length transcriptome from multiple immune-related tissues of P. olivaceus, which is valuable for exploring its immune responses.

Characterization and functional study of Galectin3 from Japanese flounder (Paralichthys olivaceus).

Galectins belong to the ß-galactoside binding protein family and participate in both innate and acquired immunity. In this study, we described the molecular characteristics of Galectin3 gene from Japanese flounder (Paralichthys olivaceus), designed as PoGalectin3. Its open reading frame was 1128 bp, encoding a protein composed of 375 amino acids. PoGalectin3 belongs to chimeric galactose agglutinin, which contains a C-terminal carbohydrate recognition domain (CRD) (L250-P372), and its N-terminal is rich in proline (P) and glycine (G). Multiple sequence alignment and phylogenetic tree showed that PoGalectin3 was conservative in different aquatic animals. Tissue distribution confirmed that PoGalectin3 showed significantly highest expression in brain, moderate expression in liver, intestine and muscle. PoGalectin3 was significantly increased post infection with Edwardsiella tarda from intestine tissue of P. olivaceus. In order to investigate the binding ability of PoGalectin3 to pathogen-associated molecular patterns, the recombinant PoGalectin3 protein (rPoGalectin3) was successfully expressed and purified, and an Enzyme linked immunosorbent assay (ELISA) experiment was performed. ELISA refers to the qualitative and quantitative detection method of immune response by combining soluble antigen or antibody with solid-phase carrier. It was confirmed that rPoGalectin3 exhibited high affinity to lipopolysaccharide and peptidoglycan. The rPoGalectin3 also exhibited a concentration dependent binding capacity with Gram-positive bacteria (Bacillus pumilus, Bacillus subtilis, Bacillus cereus) and Gram-negative bacteria (Aeromonas salmonicida, E. tarda, Vibrio vulnificus). In addition, the results of microbial agglutination experiment showed that rPoGalectin3 could agglutinate Gram-positive bacteria (B. pumilus, B. subtilis) and Gram-negative bacteria (A. salmonicida, E. tarda) in the presence of Ca2+. In conclusion, this research laid an important foundation for the specific function analysis of PoGalectin3, which provide theoretical basis for the prevention and control of aquatic diseases.

Molecular identification and expression analysis of four Lysin motif (LysM) domain-containing proteins from turbot (Scophthalmus maximus).

Lysin motif (LysM) is involved in chitin, peptidoglycan and other structurally-related oligosaccharides recognition and binding, and it is important for the biological processes of responsing to bacterial and viral infections and pathogen defense. LysM is also a widely spread protein, ranging from prokaryotes to eukaryotes, including bacteria, plants and mammals. However, research of LysM in teleosts especially in marine fish was rarely scarce. In the present study, four novel LysM domain-containing proteins in turbot (Scophthalmus maximus), named as SmLysMd1, SmLysMd2, SmLysMd3, and SmLysMd4, were cloned and identified firstly. The full-length cDNA of SmLysMd1 was 1235 bp with a 678 bp ORF, capable of encoding a peptide of 225 amino acids. The complete cDNA sequence of SmLysMd2 was 1273 bp, and contained a 675 bp ORF, encoding a predicted protein of 224 amino acids. The full-length of SmLysMd3 cDNA was 2132 bp, containing a ORF of 987 bp, with a ORF of encoding 328 amino acids. The full-length SmLysMd4 cDNA was 1115 bp contained a 888 bp ORF, encoding 295 amino acids. And all the four predicated proteins contained a specific LYSM domain. Moreover, SmLysMd1 and SmLysMd2 belong to the intracellular non-secretory types, and SmLysMd3 and SmLysMd4 belong to the anchored transmembrane types. In addition, the four SmLysMd were ubiquitously expressed in all the examined tissues. Moreover, the SmLysMds levels were up-regulated in muscle and liver, and had a reduce tendency immediately in different degree following Vibrio vulnificus challenge, indicating that the turbot LysM could be participant in the immune responses to bacterial infections. The present result of LysM in turbot for the first time in a marine fish will provide foundation knowledge for the functions studies of LysM in immune responses. Further studies should be carried out to better understand their immune mechanism in turbot and other teleosts.

cDNA cloning, characterization, and expression analysis of the Rac1 and Rac2 genes from Cynoglossus semilaevis.

Rac1 and Rac2, belonging to the small Rho GTPase family, play an important role during the immune responses. In this study, a Rac1 homolog (CsRac1) and a Rac2 homolog (CsRac2) were cloned from the Cynoglossus semilaevis. The full-length of CsRac1 and CsRac2 cDNA was 1219 bp and 1047 bp, respectively. Both CsRac1 and CsRac2 contain a 579 bp open reading frame (ORF) which encoding a 192 amino acids putative protein. The predicted molecular weight of CsRac1 and CsRac2 was 21.41 kDa and 21.35 kDa, and their theoretical pI was 8.50 and 7.91, respectively. Sequence analysis showed that the conserved RHO domain was detected both from amino acid of CsRac1 and CsRac2. Homologous analysis showed that CsRac1 and CsRac2 share high conservation with other counterparts from different species. The CsRac1 and CsRac2 transcript showed wide tissue distribution, in which CsRac1 and CsRac2 exhibit the highest expression level in liver and gill, respectively. The expression level of CsRac1 and CsRac2 fluctuated in the liver and gill tissues at different time points after challenged by Vibrio harveyi. Specifically, CsRac1 and CsRac2 were significantly up-regulated at 48 h and 96 h post injection. Moreover, the knocking down of CsRac1 and CsRac2 in cell line (TSHKC) reduced the expression of CsPAK1, CsIL1-ß and CsTNF-α. The present data suggests that CsRac1 and CsRac2 might play important roles in the innate immunity of half-smooth tongue sole.

Molecular identification and function analysis of bactericidal permeability-increasing protein/LPS-binding protein 1 (BPI/LBP1) from turbot (Scophthalmus maximus).

Bactericidal permeability-increasing protein (BPI) and lipopolysaccharide-binding protein (LBP) play important roles in host antimicrobial defense. In the present study, we identified one isoform of BPI/LBP gene from turbot (Scophthalmus maximus), designated as SmBPI/LBP1. The full-length cDNA sequence of SmBPI/LBP1 was 1826 bp, which encoding one secreted protein with 480 amino acid residues. Structurally, the SmBPI/LBP1 showed high similarity to its homologs from other vertebrates or invertebrates, which all contained a signal peptide, a BPI/LBP/CETP N-terminal with a LPS-binding domain, and a BPI/LBP/CETP C-terminal domain. The deduced amino acid sequences of SmBPI/LBP1 shared significant similarity to BPI/LBP of Seriola lalandi dorsalis (71%) and Paralichthys olivaceus (69%). Phylogentic analysis further supported that SmBPI/LBP1 act as a new member of vertebrate BPI/LBP family. SmBPI/LBP1 was ubiquitously expressed in all tested tissues, with the highest expression level in spleen tissue. The mRNA expression of SmBPI/LBP1 in spleen and kidney were significantly up-regulated after Vibrio vulnificus challenge. Finally, the recombinant SmBPI/LBP1 showed high affinity to lipopolysaccharide, followed by peptidoglycan and lipoteichoic acid, which is the ubiquitous component of Gram-negative or Gram-positive bacteria. These results indicated that SmBPI/LBP1 probably played important roles in immune response against bacteria infection.

Identification and expression of complement component C8α, C8ß and C8γ gene in half-smooth tongue sole (Cynoglossus semilaevis) and C8α recombinant protein antibacterial activity analysis.

Complement component C8, which mediates membrane attack complex formation and bacterial lysis, plays important roles in the complement system. The cDNA sequences of the C8α, C8ß and C8γ genes were cloned from half-smooth tongue sole (Cynoglossus semilaevis). Full-length cDNA of CsC8α (C8α of C. semilaevis), CsC8ß and CsC8γ was 1990, 2219 and 886 bp, respectively, which contained open reading frames of 1797, 1749 and 666 bp, encoding 598, 582 and 221 amino acids, respectively. The deduced proteins of CsC8α, CsC8ß and CsC8γ showed the closest amino acid similarity to C8α (73%) of Siniperca chuatsi, C8ß (76%) of Oryzias latipes and C8γ (72%) of Takifugu rubripes, respectively. The highest expression level of CsC8α, CsC8ß and CsC8γ among the 13 normal tissues was observed in liver tissue, followed by much lower levels in other tissues. After infection with Vibrio anguillarum, CsC8α, CsC8ß and CsC8γ were significantly up-regulated in all of the detected tissues, including the intestine, liver, gill, head kidney, blood and spleen. Then, a recombinant expression plasmid was constructed, and the recombinant CsC8α protein was expressed in GS115 pichia pastoris yeast. Furthermore, to investigate the biological functions of recombinant CsC8α, an antibacterial assay was performed, and the results showed that recombinant CsC8α obviously inhibited growth of V. anguillarum, Edwardsiella tarda and Vibrio parahaemolyticus. Taken together, these results suggest that CsC8α, CsC8ß and CsC8γ may play important roles in the immune defense of C. semilaevis.

Molecular characterization and expression analysis of a novel r-spondin member (rspo2l) in Chinese tongue sole (Cynoglossus semilaevis).

Numerous studies suggest R-spondins (Rspos) plays a role in mammalian sex development and differentiation by activating WNT signaling pathways. However, Rspos are frequently less reported in teleosts. In this study, a molecular characterization and expression analysis was conducted with a new rspondin member in the Chinese tongue sole, rspondin2-like (rspo2l). The length of rspo2l cDNA is 1251 bp with 732 bp of coding sequence. A qRT-PCR analysis revealed that the transcription of rspo2l was distributed in various tissues, with high transcription levels in the liver, skin, and gills which might indicate a possible role in immunity. We next examined a time-course of transcription levels in four immune tissues (gill, liver, spleen, and kidney) after Vibrio harveyi challenge. It was found that rspo2l was up-regulated in the gills, spleen, and kidney and down-regulated in the liver, and the greatest responses occurred at 24 and 48 h after bacterial challenge. An assessment of ß-catenin, the key regulator of the canonical WNT signaling pathway, at different time points in four immune organs revealed that its transcription profile was similar to that of rspo2l after bacterial challenge. The results suggest that tongue sole rspo2l might play a role in immune responses after bacterial challenge, while the potential link with the WNT signaling pathway still requires further investigation. This is the first report about the involvement of rspondins in fish immune responses.

Integrated analysis of mRNA-seq in the haemocytes of Eriocheir sinensis in response to Spiroplasma eriocheiris infection.

The Chinese mitten crab Eriocheir sinensis is an important economic crustacean that has been exposed to various diseases. Spiroplasma eriocheiris, isolated from tremor-diseased E. sinensis, was first identified as a lethal pathogen of freshwater crustaceans. To understand the pathogenesis of S. eriocheiris to E. sinensis, the transcriptomic profiles of haemocytes in the experimental and control groups at 1 d and 7 d post-injection were obtained using Illumina HiSeq 2500. These results showed that 40,358,724, 44,462,112, 45,516,576 and 37,713,728 paired-end clean reads were obtained from the cDNA libraries of DZ1 (the control group at 1 d), DZ7 (the control group at 7 d), SY1 (the experimental group at 1 d) and SY7 (the experimental group at 7 d), respectively. In total, 106,641 unique transcript fragments (unigenes) were assembled, with an average length of 710 bp. On the first day of stimulation, 33,084 up-regulated transcripts and 19,208 down-regulated transcripts were found in the experimental group compared with those in the control group. On the seventh day of stimulation, 40,198 up-regulated transcripts and 12,032 down-regulated transcripts were found in the experimental group compared with those in the control group. Some canonical immune-related pathways were identified via KEGG pathway analysis, including complement and coagulation cascades, the VEGF signalling pathway, the Wnt signalling pathway, natural killer cell-mediated cytotoxicity, the MAPK signalling pathway, neuroactive ligand-receptor interactions, and the Lysosome pathway. We found important immune-related genes (GNPTAB, MASP2, F7, F5, NFATC, TRAF6, MAP3K5, and TRa) in the KEGG pathway, and those genes were confirmed by qRT-PCR analysis. In addition, the significantly enriched neuroactive ligand-receptor interaction pathway was associated with intense paroxysmal tremors of infected crabs. Our results provide valuable information for the further analysis of the mechanisms of E. sinensis defence against S. eriocheiris invasion.

Identification and function analysis of ras-related nuclear protein from Macrobrachium rosenbergii involved in Spiroplasma eriocheiris infection.

A ras-related nuclear protein (Ran) protein was obtained from Macrobrachium rosenbergii, named MrRan. Phylogenetic analysis results showed that MrRan was clustered in one group together with other crustaceans. Tissue distribution analysis revealed that MrRan was expressed mainly in gill, intestine and stomach, and expressed weakly in muscle. The MrRan expression levels in gill and hemocyte of prawns were significantly up-regulated after challenged by Spiroplasma eriocheiris. The copy number of S. eriocheiris in MrRan dsRNA injection group was significantly less than control groups during infection. Meanwhile, silencing MrRan obviously increased the survival rate of prawns. The subcellular localization experiment suggested that recombinant MrRan was mainly located in the nucleus, and relatively weak in the cytoplasm. Finally, over-expression in Drosophila S2 cell indicated that MrRan could increase copies of S. eriocheiris and decrease of cell viability. The present study suggested that MrRan participated in regulating the phagocytosis of S. eriocheiris in M. rosenbergii.

Interaction of heat shock protein 60 (HSP60) with microRNA in Chinese mitten crab during Spiroplasma eriocheiris infection.

Heat shock protein 60 from the Chinese mitten crab Eriocheir sinensis (EsHSP60) was previously identified in relation to Spiroplasma eriocheiris infection by isobaric tags for relative and absolute quantitation labelling followed by liquid chromatography-tandem mass spectrometry. In the present study, to validate the immune function of this protein, the cDNA of the EsHSP60 gene was cloned. Various crab tissues were assessed using real-time PCR, which showed that EsHSP60 transcription occurred in all tissues examined. The expression profiles of EsHSP60 in haemolymph at transcription and protein levels when infected with S. eriocheiris were investigated by real-time PCR and Western blot analysis, respectively. A significant increase of EsHSP60 transcription and protein expression appeared post-injection in response to S. eriocheiris infection when compared to the control group. The double-luciferase reporter gene assay showed that the microRNA PC-533-3p interacted with the 3'-untranslated region of EsHSP60 and inhibited the translation of EsHSP60. The expression profiles of PC-533-3p during S. eriocheiris infection were also investigated by real-time PCR. However, the change tendency of PC-533-3p was opposite to that of the EsHSP60 after S. eriocheiris challenge. These data indicate that the EsHSP60 proteins may play an important role in mediating the immune responses of E. sinensis to an S. eriocheiris challenge.

A novel C-type lectin is involved in the innate immunity of Macrobrachium nipponense.

C-type lectins (CTLs) play important roles in invertebrate innate immunity by recognizing and eliminating pathogens. In the present study, a low-density lipoprotein receptor class A (LDLa) domain-containing CTL was identified from the oriental river prawn Macrobrachium nipponense, designated as MnCTLDcp1. The full-length cDNA of MnCTLDcp1 was composed of 1462 bp, with a 999-bp ORF encoding a 332-aa protein. An LDLa and a single C-type lectin-like domain (CTLD) were found. The mRNA transcripts of MnCTLDcp1 was expressed the highest in heart. After the prawns were challenged by Aeromonas hydrophila and Staphylococcus aureus, the expression level of MnCTLDcp1 in heart and hemocytes were all significantly up-regulated. Sugar binding assay revealed that the MnCTLDcp1 could bind to the glycoconjugates of bacteria surface, such as LPS, PGN and they can compete with bacterial as competitors. The recombinant MnCTLDcp1 agglutinates Gram-positive (S. aureus and Bacillus subtilis) and Gram-negative bacteria (A. hydrophila, Vibrio parahaemolyticus, Escherichia coli and Pseudomonas aeruginosa) in the presence of calcium and also could bind to these bacteria. These results clearly suggested that MnCTLDcp1 functions as a pattern-recognition receptor involved in the innate immunity of M. nipponense.

Isolation and characterization of two novel C-type lectins from the oriental river prawn, Macrobrachium nipponense.

C-type lectins are a family of calcium-dependent carbohydrate-binding proteins which are believed to play important roles in the innate immunity of invertebrates. This study identified two novel C-type lectins, designated as MnCTLDcp2 and MnCTLDcp3, from the oriental river prawn Macrobrachium nipponense. The full-length cDNA of MnCTLDcp2 was of 1582 bp with an open reading frame (ORF) of 972 bp encoding a polypeptide of 323 amino acids. The complete nucleotide sequence of MnCTLDcp3 cDNA was 583 bp, containing a 555 bp ORF encoding a putative protein of 184 deduced amino acids. The deduced MnCTLDcp2 and MnCTLDcp3 proteins both contained a single C-type lectin-like domain (CTLD). Besides, MnCTLDcp2 contains a signal peptide and an low-density lipoprotein receptor class A (LDLa) domain. Reverse transcription PCR showed that MnCTLDcp2 was expressed in the heart, gill, nerve hepatopancreas and intestine; MnCTLDcp3 was expressed in the hepatopancreas, heart, nerve, gill and muscle. Their expression in the heart tissue was regulated following challenge with bacteria. The microbial agglutination assay showed that both MnCTLDcp2 and MnCTLDcp3 could agglutinated bacteria in the presence of calcium. All these results suggested that MnCTLDcp2 and MnCTLDcp3 functioned as pattern recognition receptors in the immune system of M. nipponense.

Molecular cloning, characterization, and expression analysis of two different types of lectins from the oriental river prawn, Macrobrachium nipponense.

Lectins, which are widely expressed in invertebrates, play important roles in many biological processes, including protein trafficking, cell signaling, pathogen recognition, as effector molecules, and so on (Wang and Wang, 2013). This study identified one novel M-type lectin and one L-Type lectin, designated as MnMTL1 and MnLTL1, from the oriental river prawn Macrobrachium nipponense. The full-length cDNA of MnMTL1 was 2064 bp with a 1761 bp ORF encoding a putative protein of 586 deduced amino acids. The full-length cDNA of MnLTL1 was 1744 bp with a 972 bp ORF encoding a 323-amino acid peptide. The deduced MnMTL1 protein contained a putative type II transmembrane region and a 440-aa Glycoside hydrolase family 47 (GH47) domain. One luminal carbohydrate recognition domain and a 23-aa type I transmembrane region were identified from the MnLTL1. MnMTL1 shared 78% identity with Marsupenaeus japonicus M-type lectin and MnLTL1 shared 83% similarity with M. japonicus L-type lectin. RT-PCR analysis showed that MnMTL1 and MnLTL1 were expressed in all tested tissues. Quantitative real-time PCR analysis revealed that MnMTL1 and MnLTL1 are substantially fluctuant during Aeromonas hydrophila and Aeromonas veronii infections. Based on immune responses and previous literature, we assumed that MnMTL1 and MnLTL1 might be functioned as pattern recognition receptors and play important roles in the immune response of M. nipponense.