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.

Rapid Visual Detection of Spiroplasma eriocheiris by Loop-Mediated Isothermal Amplification with Hydroxynaphthol Blue Dye.

In recent years, a new type of Spiroplasma has been found that can cause "tremor disease" of the Chinese mitten crab Eriocheir sinensis. The outbreak of epidemic tremor disease has caused a serious setback in the Chinese mitten crab farming industry, with an incidence rate of more than 30% and mortality rates of 80-100%. Therefore, finding a sensitive method to detect tremor disease in E. sinensis has become a current research focus. In this research, a loop-mediated isothermal amplification detection method coupled with hydroxynaphthol blue dye (LAMP-HNB) was developed and used to rapidly detect Spiroplasma eriocheiris. First, we designed and synthesized specific outer primers, inner primers and loop primers based on the 16S ribosomal RNA gene of S. eriocheiris. Second, the LAMP-HNB detection method for S. eriocheiris was successfully established by screening the primers, adjusting the temperature and time of the reaction, and optimizing the concentrations of Mg2+ and dNTPs. In the specific tests, only samples infected with S. eriocheiris showed positive results, and other infections caused by bacteria and parasites tested negative, proving that the test has high specificity. Moreover, the detection limit was 2.5 × 10-6 ng/µL, indicating high sensitivity. This method for detecting S. eriocheiris provides rapid visual output based on LAMP-HNB detection and is a simple, fast, sensitive, and inexpensive method that can be applied to a wide range of field investigations.

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.

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.

Immunity of turbot Induced by inactivated vaccine of Aeromonas salmonicida from the perspective of DNA methylation.

Introduction: DNA methylation was one of the most important modification in epigenetics and played an important role in immune response. Since the introduction of Scophthalmus maximus, the scale of breeding has continued to expand, during which diseases caused by various bacteria, viruses and parasites have become increasingly serious. Therefore, the inactivated vaccines have been widely researched and used in the field of aquatic products with its unique advantages. However, the immune mechanism that occurred in turbot after immunization with inactivated vaccine of Aeromonas salmonicida was not clear. Methods: In this study, differentially methylated regions (DMRs) were screened by Whole Genome Bisulfite Sequencing (WGBS) and significantly differentially expressed genes (DEGs) were screened by Transcriptome sequencing. Double luciferase report assay and DNA pull-down assay were further verified the DNA methylation state of the gene promoter region affected genes transcriptional activity after immunization with inactivated vaccine of Aeromonas salmonicida. Results: A total of 8149 differentially methylated regions (DMRs) were screened, in which there were many immune-related genes with altered DNA methylation status. Meanwhile, 386 significantly differentially expressed genes (DEGs) were identified, many of which were significantly enriched in Toll-like receptor signaling pathway, NOD-like receptor signaling pathway and C-type lectin receptor signaling pathway. Combined analysis of WGBS results and RNA-seq results, a total of 9 DMRs of negatively regulated genes are located in the promoter region, including 2 hypermethylated genes with lower expression and 7 hypomethylated genes with higher expression. Then, two immune-related genes C5a anaphylatoxin chemotactic receptor 1-like (C5ar1-Like) and Eosinophil peroxidase-like (EPX-Like), were screened to explore the regulation mechanism of DNA methylation modification on their expression level. Moreover, the DNA methylation state of the gene promoter region affected genes transcriptional activity by inhibiting the binding of transcription factors, which lead to changes in the expression level of the gene. Discussion: We jointly analyzed WGBS and RNA-seq results and revealed the immune mechanism that occurred in turbot after immunized with inactivated vaccine of A. salmonicida from the perspective of DNA methylation.

Development of Two Recombinase Polymerase Amplification EXO (RPA-EXO) and Lateral Flow Dipstick (RPA-LFD) Techniques for the Rapid Visual Detection of Aeromonas salmonicida.

Aeromonas salmonicida is the pathogen underlying furunculosis, causing a septicemic infection that influences both salmonid and non-salmonid fish. Early diagnosis of these contagions is essential for disease surveillance and prevention, so a rapid and sensitive approach is needed. Herein, a recombinase polymerase amplification EXO (RPA-EXO) assay and RPA with a lateral flow dipstick (RPA-LFD) were produced for testing A. salmonicida. The RPA-EXO and RPA-LFD primer sets were devised based on the conserved fragment sequence of the vapA gene. Then, RPA-EXO and RPA-LFD reaction systems were established, and the reaction temperature and time were optimized. After optimization, the RPA-EXO method was capable of testing A. salmonicida within 10 min, and the RPA-LFD method could detect A. salmonicida in only 5 min. The RPA-EXO and RPA-LFD methods exhibited high specificity with no cross-reaction with other strains. To assess sensitivity, a partial vapA gene was cloned, and serial plasmid dilutions were created ranging from 1 × 106 to 1 × 10-1 copies/µL. The detection limit of RPA-EXO was 1 × 102 copies/µL, and the detection limit of RPA-LFD was 1 copy/µL. For spiked turbot tissue samples, the sensitivity detection of A. salmonicida was 1.2 × 101 CFU/mL and 1.2 CFU/mL by RPA-EXO and RPA-LFD, respectively. In comparative analyses of clinical samples, the diagnostic results of RPA-EXO and RPA-LFD were compared with those of the standard conventional PCR test and showed nearly 100% consistency. Therefore, our RPA-EXO and RPA-LFD assays exhibited excellent specificity and sensitivity, which provided two simple, fast and dependable methods to conduct large-scale field investigations of A. salmonicida in resource-limited settings.

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.

Identification of Antimicrobial Peptide Genes in Black Rockfish Sebastes schlegelii and Their Responsive Mechanisms to Edwardsiella tarda Infection.

The black rockfish, Sebastes schlegelii, is a typical viviparous teleost, which belongs to the family Scorpaenidae. Due to its high economic and ecological values, S. schlegelii has been widely cultured in East Asian countries. With the enlargement of cultivation scale, bacterial and viral diseases have become the main threats to the farming industry of S. schlegelii, which have resulted in significant economic losses. In this study, Illumina shotgun sequencing, single-molecule real-time (SMRT) sequencing, 10× genomics and high-throughput chromosome conformation capture (Hi-C) technologies were collectively applied to assemble the genome of S. schlegelii. Then, we identified the antimicrobial peptide genes (AMPs) in the S. schlegelii genome. In total, 214 AMPs were identified in the S. schlegelii genome, which can be divided into 33 classes according to the annotation and cataloging of the Antimicrobial Peptides Database (APD3). Among these AMPs, thrombin-derived C-terminal peptide (TCP) was the dominant type, followed by RegIIIgamma and chemokine. The amino acid sequences of the TCP, cgUbiquitin, RegIIIalpha, RegIIIgamma, chemokine shared 32.55%, 42.63%, 29.87%, 28.09%, and 32.15% similarities among the same type in S. schlegelii. Meanwhile, the expression patterns of these AMPs in nine healthy tissues and at different infection time points in intestine were investigated. The results showed that the numbers and types of AMPs that responded to Edwardsiella tarda infection gradually increased as the infection progressed. In addition, we analyzed the phylogenetic relationships of hepcidins in teleost. The identification of AMPs based on the whole genome could provide a comprehensive database of potential AMPs, and benefit for the understanding of the molecular mechanisms of immune responses to E. tarda infection in S. schlegelii. This would further offer insights into an accurate and effective design and development of AMP for aquaculture therapy in the future.

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.

Identification and Characterization of Long Non-coding RNAs in the Intestine of Olive Flounder (Paralichthys olivaceus) During Edwardsiella tarda Infection.

Long non-coding RNAs (lncRNAs) play widespread roles in fundamental biological processes, including immune responses. The olive flounder (Paralichthys olivaceus), an important economical flatfish widely cultured in Japan, Korea, and China, is threatened by infectious pathogens, including bacteria, viruses, and parasites. However, the role of lncRNAs in the immune responses of this species against pathogen infections is not well-understood. Therefore, in this study, we aimed to identify lncRNAs in the intestine of olive flounder and evaluate their differential expression profiles during Edwardsiella tarda infection, which is an important zoonotic and intestinal pathogen. A total of 4,445 putative lncRNAs were identified, including 3,975 novel lncRNAs and 470 annotated lncRNAs. These lncRNAs had shorter lengths and fewer exons compared with mRNAs. In total, 115 differentially expressed lncRNAs (DE-lncRNAs) were identified during E. tarda infection. To validate the expression pattern of lncRNAs, six DE-lncRNAs were randomly selected for quantitative real-time PCR. The co-located and co-expressed mRNAs of DE-lncRNAs were predicted, which were used to conduct the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. The target genes of DE-lncRNAs enriched numerous immune-related processes and exhibited a strong correlation with immune-related signaling pathways. To better understand the extensive regulatory functions of lncRNAs, the lncRNA-miRNA-mRNA regulatory networks were constructed, and two potential competing endogenous RNA (ceRNA) networks, LNC_001979-novel_171-Potusc2 and LNC_001979-novel_171-Podad1, were preliminarily identified from the intestine of olive flounders for the first time. In conclusion, this study provides an invaluable annotation and expression profile of lncRNAs in the intestine of olive flounder infected with E. tarda; this forms a basis for further studies on the regulatory function of lncRNAs in the intestinal mucosal immune responses of olive flounder.

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.

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.

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.

Differences in DNA Methylation Between Disease-Resistant and Disease-Susceptible Chinese Tongue Sole (Cynoglossus semilaevis) Families.

DNA methylation, the most widely studied and most well-understood epigenetic modification, has been reported to play crucial roles in diverse processes. Although it has been found that DNA methylation can modulate the expression of immune-related genes in teleosts, a systemic analysis of epigenetic regulation on teleost immunity has rarely been performed. In this research, we employed whole-genome bisulfite sequencing to investigate the genome-wide DNA methylation profiles in select disease-resistant Cynoglossus semilaevis (DR-CS, family 14L006) and disease-susceptible C. semilaevis (DS-CS, family 14L104) against Vibrio harveyi infection. The results showed that following selective breeding, DR-CS had higher DNA methylation levels and different DNA methylation patterns, with 3,311 differentially methylated regions and 6,456 differentially methylated genes. Combining these data with the corresponding transcriptome data, we identified several immune-related genes that exhibited differential expression levels that were modulated by DNA methylation. Specifically, DNA methylation of tumor necrosis factor-like and lipopolysaccharide-binding protein-like was significantly correlated with their expression and significantly contributed to the disease resistance of the selected C. semilaevis family. In conclusion, we suggest that artificial selection for disease resistance in Chinese tongue sole causes changes in DNA methylation levels in important immune-related genes and that these epigenetic changes are potentially involved in multiple immune responses in Chinese tongue sole.

Identification of Potential Immune-Related circRNA-miRNA-mRNA Regulatory Network in Intestine of Paralichthys olivaceus During Edwardsiella tarda Infection.

Olive flounder (Paralichthys olivaceus) is an important economical flatfish in Japan, Korea, and China, but its production has been greatly threatened by disease outbreaks. In this research, we aimed to explore the immune responsive mechanism of P. olivaceus against Edwardsiella tarda infection by profiling the expression of circRNA, miRNA, and mRNA by RNA-seq and constructing a regulatory circular circRNA-miRNA-mRNA network. Illumina sequencing of samples from normal control (H0), 2 h (H2), 8 h (H8), and 12 h (H12) post-challenge was conducted. Differentially expressed (DE) circRNA (DE-circRNAs), miRNAs (DE-miRNAs), and mRNAs [differential expression genes (DEGs)] between challenge and control groups were identified, resulting in a total of 62 DE-circRNAs, 39 DE-miRNAs, and 3,011 DEGs. Based on the differentially expressed gene results, miRNA target interactions (circRNA-miRNA pairs and miRNA-mRNA pairs) were predicted by MiRanda software. Once these paired were combined, a preliminary circRNA-miRNA-mRNA network was generated with 198 circRNA-miRNA edges and 3,873 miRNA-mRNA edges, including 44 DE-circRNAs, 32 DE-miRNAs, and 1,774 DEGs. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was performed to evaluate the function of the DEGs in this network, and we focused and identified two important intestinal immune pathways (herpes simplex infection and intestinal immune network for IgA production) that showed statistical significance between the challenge and control groups. Furthermore, three critical DEGs (nectin2, MHC II α-chain, and MHC II ß-chain) were identified, mapped into the preliminary circRNA-miRNA-mRNA network, and new circRNA-miRNA-mRNA regulatory networks were constructed. In conclusion, we, for the first time, identified circRNA-miRNA-mRNA network from P. olivaceus in the pathogenesis of E. tarda and provided valuable resources for further analyses of the molecular mechanisms and signaling networks.

Spiroplasma eriocheiris Invasion Into Macrobrachium rosenbergii Hemocytes Is Mediated by Pathogen Enolase and Host Lipopolysaccharide and ß-1, 3-Glucan Binding Protein.

Spiroplasma eriocheiris is a crustacean pathogen, without a cell wall, that causes enormous economic loss. Macrobrachium rosenbergii hemocytes are the major targets during S. eriocheiris infection. As wall-less bacteria, S. eriocheiris, its membrane protein should interact with host membrane protein directly and firstly when invaded in host cell. In this investigation, six potential hemocyte receptor proteins were identified firstly that mediate interaction between S. eriocheiris and M. rosenbergii. Among these proteins, lipopolysaccharide and ß-1, 3-glucan binding protein (MrLGBP) demonstrated to bind to S. eriocheiris using bacterial binding assays and confocal microscopy. Four spiroplasma ligand proteins for MrLGBP were isolated and identified. But, competitive assessment demonstrated that only enolase of S. eriocheiris (SeEnolase) could be a candidate ligand for MrLGBP. Subsequently, the interaction between MrLGBP and SeEnolase was confirmed by co-immunoprecipitation and co-localization in vitro. After the interaction between MrLGBP and SeEnolase was inhibited by antibody neutralization test, the virulence ability of S. eriocheiris was effectively reduced. The quantity of S. eriocheiris decreased in Drosophila S2 cells after overexpression of MrLGBP, compared with the controls. In addition, RNA interference (RNAi) knockdown of MrLGBP made M. rosenbergii more sensitive to S. eriocheiris infection. Further studies found that the immune genes, including MrLGBP and prophenoloxidase (MrproPO), MrRab7A, and Mrintegrin α1 were significantly up-regulated by SeEnolase stimulation. After SeEnolase pre-stimulation, the ability of M. rosenbergii resistance to S. eriocheiris was significantly improved. Collectively, this investigation demonstrated that MrLGBP and pathogen SeEnolase involved in mediating S. eriocheiris invasion into M. rosenbergii hemocytes.

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.

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.

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.