The effects of Bacillus subtilis on the immunity, mucosal tissue morphology, immune-related gene transcriptions, and intestinal microbiota in flounder (Paralichthys olivaceus) with two feeding methods: Continuous versus discontinuous feeding.

Probiotics as dietary additives can improve weight gain, feed efficiency, and disease resistance in cultured fish. In this research, we evaluated and compared the effects of Bacillus subtilis on immunity, mucosal tissue morphology, immune-related gene transcriptions, and intestinal microbiota in flounder (Paralichthys olivaceus) by a 30-day feeding experiment based on a continuous feeding schedule (E1) and a discontinuous feeding schedule (E2). As a result, the use of B. subtilis exerted the best positive effects on survival rate, enzyme activity, mucosal tissue morphology, immune-related gene transcriptions, and intestinal microbiota in flounders. Alkaline phosphatase (AKP), lysozyme (LZM), and superoxide dismutase (SOD) activities in the liver of E2 were higher than those of E1 (P < 0.05). Furthermore, the villi length in the intestinal tract and the fold length in the stomach of E2 were also higher than in E1 (P < 0.05). The il-1 expression levels in the spleen were significantly increased in E2 (P < 0.05) compared to E1. We performed 16 S rRNA sequencing analysis to find that Bacillus in E1 (1.06%) and E2 (1.01%) had higher relative abundances than in E0 (0.053%) at the end of the experiments, indicating that short-term application of B. subtilis with the continuous or discontinuous feeding method can allow both the adaptation of the ecosystem to the presence of probiotics by the establishment of new species in the gut microbiota and the ability these new probiotic species to perform corresponding functions. No significant differences in the ability of probiotic establishment were observed between E1 and E2. Our findings provided a unique perspective to explore the mechanism of immune enhancement with probiotics and to screen the optimal administration strategy in aquaculture application for probiotic use. Together, these results point to some level of enhancement in immune status by continuous and discontinuous feeding after a short-term feeding period, which could be used as a prophylactic strategy for flounder health management.

Exosomes derived from olive flounders infected with Streptococcus parauberis: Proteomic analysis, immunomodulation, and disease resistance capacity.

Multidrug-resistant Streptococcus parauberis causes high fish mortality in aquaculture, necessitating an urgent need for innovative control strategies. This study aimed to develop an immunizing agent against S. parauberis using exosomes isolated from the plasma of olive flounders infected experimentally with S. parauberis (Sp-Exo). Initially, we tested the in vitro immunomodulatory effect of Sp-Exo in murine macrophage RAW264.7 cells and compared it to that of exosomes isolated from naïve fish (PBS-Exo-treated). Notably, Sp-Exo treatment significantly (p < 0.05) upregulated pro-and anti-inflammatory cytokines (Il1ß, Tnfα, and Il10), antimicrobial peptide, defensin isoforms (Def-rs2 and Def-ps1), and antiviral (Ifnß1 and Isg15) genes. In vivo studies in larval and adult zebrafish revealed similar patterns of immunomodulation. Furthermore, larval and adult zebrafish exhibited significantly (p < 0.05) enhanced resistance to S. parauberis infection following treatment with Sp-Exo compared to that with PBS-Exo. Proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ) approach revealed the presence of 77 upregulated and 94 downregulated differentially expressed proteins (DEPs) in Sp-Exo, with 22 and 37 significantly (p < 0.05) upregulated and downregulated DEPs, respectively. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and Search Tool for the Retrieval of Interacting Genes/Proteins analyses revealed that these genes are associated with key pathways, such as innate immune responses, complement system, acute phase responses, phospholipid efflux, and chylomicron remodeling. In conclusion, Sp-Exo demonstrated superior immunomodulatory activity and significant resistance against S. parauberis infection relative to that on treatment with PBS-Exo. Proteomic analysis further verified that most DEPs in Sp-Exo were associated with immune induction or modulation. These findings highlight the potential of Sp-Exo as a promising vaccine candidate against S. parauberis and other bacterial infections in olive flounder.

Antibiotic Susceptibility of Bacterial Pathogens That Infect Olive Flounder (Paralichthys olivaceus) Cultivated in Korea.

Paralichthys olivaceus (olive flounder) is widely cultivated in Korea. However, data on the antibiotic susceptibility of bacterial pathogens that infect olive flounders in Korea are limited. The susceptibility of 84 strains of 3 pathogenic bacteria (Streptococcus spp., Vibrio spp., and Edwardsiella piscicida) to 18 antibiotics was tested using the minimum inhibitory concentration (MIC) panels, and the distribution of the MIC values for each species was confirmed. Among the panel antibiotics, nine commonly used antibiotics were selected, and the multiple antibiotic resistance (MAR) index and antibiotic resistance pattern were indicated using the disk diffusion method. It was confirmed that most of the isolates had a MAR index greater than 0.2, indicating a high-risk source. The distribution patterns of the MIC values and resistance pattern between gram-positive and gram-negative bacteria showed slightly different results. Ampicillin, erythromycin, and clindamycin were more effective against gram-positive bacteria than gram-negative bacteria. However, the MIC values of flumequine for gram-positive bacteria were higher than those of gram-negative bacteria. Through the distribution patterns of the MIC values and resistance patterns presented in this study, the need for monitoring the multidrug-resistant bacteria in aquaculture is emphasised.

Downregulated pol-miR-140-3p induces the expression of the kinesin family member 5A against Streptococcus parauberis infection in olive flounder.

MicroRNAs (miRNAs) are small non-coding RNAs that participate in various biological and cellular processes by regulating target gene expression. miRNAs are also known to play vital roles in the pathogenesis of various diseases, including infections, as well as the disease progression and defense responses. In this study, we examined the expression levels of pol-miR-140-3p and its target gene, kinesin family member 5A (KIF5A), in association with the Streptococcus parauberis (S. parauberis) infection, a major bacterial pathogen that causes streptococcosis in olive flounder (Paralichthys olivaceus). KIF5A is a heavy chain isoform of kinesin-1, which is known to be brain-specific, and this study is the first examination of KIF5A expression related to the regulation of miRNA in olive flounder (named PoKIF5A). There were significant differences in expression levels between infected and healthy olive flounder as the expression of pol-miR-140-3p in the infected fish was lower than that in the control, while the expression of PoKIF5A was higher in the infected fish than in the healthy controls. These contradictory results suggest that downregulated pol-miR-140-3p induces the expression of PoKIF5A against S. parauberis infection in olive flounder.

Vitellogenin-derived fragment in embryos of Japanese flounder Paralichthys olivaceus with binding and bactericidal activities against an infectious bacterium via an interaction with saccharides.

Thirty- and 90-kDa proteins with binding ability to Edwardsiella tarda, a causative bacterium of Edwardsiellosis in fish, were purified from the embryo of Japanese flounder Paralichthys olivaceus. The proteins were isolated with affinity chromatography, in which the bacterium was used as a ligand and galactose, mannose, and ethylenediaminetetraacetic acid (EDTA) were used as elution agents, followed by gel filtration chromatography. N-terminal amino acid sequencing and liquid chromatography with quadrupole time-of-flight tandem mass spectrometry (LC/Q-TOF-MS) analysis revealed that the 90-kDa protein was lipovitellin heavy-chain (LvH), which is one of the proteolytically cleaved products of maternal vitellogenin (Vg) and represents the main precursor of the egg yolk in teleosts, and the 30-kDa protein was an N-terminal bit of LvH. On the other hand, Vg in the serum of the mother fish did not bind to E. tarda. While the 90-kDa protein did not show anti-bacterial activity, the 30-kDa protein strongly exhibited activity toward E. tarda, with a minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) below 0.06 µM, suggesting that the latter protein plays an important role during embryogenesis in the flounder. This is the first report showing that Vg-derived products have monosaccharides-binding activity and a fragment derived from LvH exhibits bactericidal activity.

Combining of transcriptomic and proteomic data to mine immune-related genes and proteins in the liver of Cynoglossus semilaevis challenged with Vibrio anguillarum.

The liver is a multi-functional organ including metabolism, substance synthesis, detoxification, and various immune functions, and its role in immunity has attracted more and more attention. However, research on the liver immune response of fish infected by pathogenic bacteria is currently lacking. In this study, the transcriptomics and proteomics of the liver of Cynoglossus semilaevis infected with Vibrio anguillarum were analyzed. A total of 1470 genes and 497 proteins were differentially expressed in the pairwise comparison of obvious symptoms of infection (HOSG), no obvious symptoms of infection (NOSG) and PBS treatment (CG). Gene ontology and KEGG enrichment pathways analysis showed that differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) were mainly enriched in toll-like receptors (TLRs), complement and coagulation cascades, nucleotide oligomerization domain (NOD)-like receptors (NLRs), mitogen-activated protein kinase (MAPK) and phagosome signaling pathways, which suggested the combined action of the five pathways were significant to enhance the liver immune defense. The combination of transcriptomic and proteomic analysis showed that ITGß1, C3, C5 and MRC1 were significantly up-regulated, which might play an important role in the liver immune response to the recognition of V. anguillarum, inflammatory response and phagocytosis. The transcriptome and proteome data we obtained provide information on some key genes and proteins for further study of the mechanism of liver immune response.

Transcriptome analysis unveils survival strategies of Streptococcus parauberis against fish serum.

Streptococcus parauberis is an important bacterial fish pathogen that causes streptococcosis in a variety of fish species including the olive flounder. Despite its importance in the aquaculture industry, little is known about the survival strategy of S. parauberis in the host. Therefore, the objective of this study was to produce genome-wide transcriptome data and identify key factors for the survival of S. parauberis SPOF3K in its host. To this end, S. parauberis SPOF3K was incubated in olive flounder serum and nutrient-enriched media as a control. Although S. parauberis SPOF3K proliferated in both culture conditions, the transcriptomic patterns of the two groups were very different. Interestingly, the expression levels of genes responsible for the replication of an S. parauberis plasmid in the presence of olive flounder serum were higher than those in the absence of olive flounder serum, indicating that this plasmid may play an important role in the survival and proliferation of S. parauberis in the host. Several ATP-binding cassette transporters known to transport organic substrates (e.g., biotin and osmoprotectants) that are vital for bacterial survival in the host were significantly up-regulated in S. parauberis cultured in serum. In addition, groEL, dnaK operon, and members of the clp protease family, which are known to play important roles in response to various stressors, were up-regulated in S. parauberis incubated in serum, thus limiting damage and facilitating cellular recovery. Moreover, important virulence factors including the hyaluronic acid capsule (has operon), sortase A (srtA), C5a peptidase (scp), and peptidoglycan O-acetyltransferase (oatA) were significantly upregulated in S. paraubers in serum. These results indicate that S. paraubers can resist and evade the humoral immune responses of fish. The transcriptomic data obtained in this study provide a better understanding of the mode of action of S. parauberis in fish.

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.

Differential proteome profile of gill and spleen in three pathogen-infected Paralichthys olivaceus.

BACKGROUND: Olive flounder (Paralichthys olivaceus) is one of the major cultured fish species in Asia including Korea. However, the mass mortality of olive flounder caused by various pathogens leads to huge economic loss. The pathogens that lead to fish mortality include parasites, bacteria, and viruses that can cause various kinds of diseases. OBJECTIVE: The purpose of this study was to investigate the protein expression patterns in the gills and spleens of olive flounder after artificial infection. We hypothesized that proteomics levels in gills and spleen may be differentially expressed depending on infectious agents. METHODS: To investigate the expression pattern of proteins in gills and spleens, olive flounders were experimentally infected with VHSV (virus), S. parauberis (bacteria), or M. avidus (pathogenic ciliate). Proteins were extracted from the gills and spleens of infected olive flounder. We used 2-DE analysis with LC-MS/MS to investigate proteome changes in infected olive flounders. RESULTS: The results of the LC-MS/MS analyses showed different protein expression profiles depending on pathogenic sources and target organs. Proteins related to cytoskeletal structure like keratin, calmodulin and actin were mostly expressed in the infected gills. Proteins involved in the metabolism pathway like glycolysis were expressed mainly in the spleens. The protein profiles of S. parauberis and VHSV infection groups had many similarities, but the profile of the M. avidus infection group was greatly different in the gill and spleen. CONCLUSION: Our results indicate that measures according to the characteristics of each pathogen are necessary for disease prevention and treatment of farmed fish.

Probiotic Potential of the Farmed Olive Flounder, Paralichthys olivaceus, Autochthonous Gut Microbiota.

In recent years, considerable and growing attention has been given to the application of host-associated microorganisms as a more suitable source of probiotics in aquaculture sector. Herein, we isolated and screened the olive flounder gut microbiota for beneficial bacterial strains that might serve as potential probiotics in a low fishmeal extruded aquafeed. Among the ten identified isolates, Bacillus amyloliquefaciens SK4079 and B. subtilis SK4082 were screened out based on their heat-resistant ability as well as enzymatic and non-hemolytic activities. Although both strains were well able to utilize carboxymethyl cellulose (CMC), xylan, and soybean meal (SBM) as a single carbon source in the minimal nutrient M9 medium, B. subtilis exhibited significantly higher cellulase, xylanase, and protease activities than B. amyloliquefaciens. The two selected strains were well able to degrade the undesirable anti-nutritional component of the SBM, which would limit its utilization as protein source in aquafeed industry. Significantly higher biofilm formation capacity and notably stronger adhesive interactions with the flounder's skin mucus were detected in B. subtilis than B. amyloliquefaciens. Immobilization of the spores from the selected strains, in a SBM complex carrier, remarkably enhances their thermal resistance at 120 °C for 5 min and different drying conditions. It was also interesting to learn that the B. subtilis spores could survive and remain viable after being sprayed onto extruded low-fish meal feed pellets for as long as 6 months. Overall, the findings of the present study could help the food/feed industries achieve their goal of developing cost-effective yet efficient products.

Molecular characterization and expression analysis of Japanese flounder (Paralichthys olivaceus) chemokine receptor CXCR2 in comparison with CXCR1.

Chemokines are categorized into five families; one of the families is the CXC chemokines, which are critical in the pro-inflammatory process. CXC chemokines transmit signals and mediate a cell's biological activities by binding to cell surface receptors known as chemokine receptors (CXCRs). In this study, the CXCR2 from Japanese flounder (Paralichthys olivaceus) (JfCXCR2) was identified and characterized at the molecular level. The JfCXCR2 gene has a 1077 bp open reading frame that encodes a protein of 359 amino acid residues with seven transmembrane domains. Phylogenetic analysis of JfCXCR2 revealed that it belonged to the fish CXCR2 subfamily. Furthermore, JfCXCR2 was compared with the previously identified Japanese flounder CXCR1 (JfCXCR1). The expression analysis of uninfected Japanese flounder showed that JfCXCR1 and JfCXCR2 were expressed in all the tissues and organs tested but mainly in immune-related organs, including the kidney and spleen. Infection by Streptococcus iniae significantly increased the level of JfCXCR1 and JfCXCR2 mRNA in the kidney at days 1 and 3 post-infection. On the other hand, VHSV (viral hemorrhagic septicemia virus) and Edwardsiella tarda infection significantly increased JfCXCR2 mRNA levels in the kidney at days 3 and 6 post-infection, respectively. Conversely, JfCXCR1 expression was not significantly changed by either E. tarda or VHSV infection. Additionally, the peripheral blood leukocytes (PBLs) stimulated by recombinant proteins rCXCL8_L1a and rCXCL8_L1b were found to have significantly increased levels of JfCXCR1 and JfCXCR2 mRNA. Interestingly, even higher levels of JfCXCR1 and JfCXCR2 expression were observed in PBLs stimulated with rCXCL8_L1a and rCXCL8_L1b than in PBLs stimulated with either recombinant protein. These data suggest that bacterial infections may activate JfCXCR1. By contrast, JfCXCR2 may be activated by both bacterial and viral infection to mediate the immune response. These data can contribute to further understanding the functions of CXCR1 and CXCR2 in the fish immune system.

Pol-miR-150 regulates anti-bacterial and viral infection in Japanese flounder (Paralichthys olivaceus) via the lysosomal protein LMP2L.

MiR-150 is a microRNA (miRNA) present in a number of teleost species, but its target and regulation mechanism are unknown. Similarly, lysosome membrane protein 2-like (LMP2L) is a gene identified in fish but with unknown function. In this study, we examined the regulation mechanism and function of flounder miR-150 (named pol-miR-150) and its target gene LMP2L (named PoLMP2L) in association with bacterial and viral infection. We found that pol-miR-150 expression was not only modulated by the bacterial pathogen Streptococcus iniae but also by the viral pathogen megalocytivirus. Pol-miR-150 targeted PoLMP2L by binding to the 3'-untranslated region (3'-UTR) of PoLMP2L and inhibited PoLMP2L expression in vitro and in vivo. PoLMP2L is a member of the CD36 superfamily of scavenger receptors and homologous to but phylogenetically distinct from lysosomal integral membrane protein type 2 (LIMP2). PoLMP2L was localized mainly in the lysosomes and expressed in multiple organs of flounder. In vivo knockdown and overexpression of PoLMP2L enhanced and suppressed, respectively, S. iniae dissemination in flounder tissues, whereas in vivo knockdown and overexpression of pol-miR-150 produced the opposite effects on S. iniae dissemination. In addition, pol-miR-150 knockdown also significantly inhibited the replication of megalocytivirus. The results of this study revealed the regulation mechanism and immune functions of fish miR-150 and LMP2L, and indicated that LMP2L and miR-150 play an important role in the antimicrobial immunity of fish.

A deep-sea pathogenic Bacillus subtilis isolate employs different strategies to escape the killing of teleost and murine complements.

Bacillus subtilis subsp. subtilis G7 was isolated from a deep-sea hydrothermal vent and is pathogenic to pathogenic to fish (Japanese flounder) and mice. G7 is able to survive in host sera and phagocytes. In this study, we investigated the underlying mechanism of G7 serum resistance. We found that (i) the remaining complement activity was very low in G7-incubated flounder serum but high in G7-incubated mouse serum; (ii) cleaved C3 and C5 components were detected on flounder serum-incubated G7 but not on mouse serum-incubated G7; (iii) abundant uncleaved C5 was localized in G7-incubated mouse, but not flounder, serum; (iv) G7-incubated flounder, but not mouse, serum exhibited strong chemotactic activity; (v) pre-treatment with low-dose lysozyme abolished the serum resistance of G7. Hence, G7 activates flounder complement but is protected from complement-mediated destruction by its cell wall structure, while G7 prevents the activation of mouse complement. These results indicate that G7 employs different mechanisms to avoid the complement killing of different hosts.

Regulatory Role of Fc Receptor in mIgM+ B Lymphocyte Phagocytosis in Flounder (Paralichthys olivaceus).

Fc receptor (FcR) is an important opsonin receptor on the surface of immune cells, playing an important role in antibody-dependent cell-mediated immunity. Our previous work found that the FcR of flounder showed a marked expression response in phagocytizing IgM+ B cell, which suggested that FcR might participate in regulating Ig-opsonized phagocytosis. In this paper, in order to elucidate the potential role of FcR in mediating phagocytosis of IgM+ B cell, flounder anti-E. tarda serum was prepared and complement-inactivated for the use of E. tarda opsonization, and the sera of healthy flounder were used as control. Flow cytometric analysis showed that the phagocytosis rates of antiserum-opsonized E. tarda in peripheral blood mIgM+ B lymphocytes were significantly higher than the control group, and higher phagocytosis rates of mIgM+ B lymphocyte could be detected with an increasing incubation time ranging from 1 to 5 h. The phagocytosis rates of antiserum-opsonized E. tarda by mIgM+ B lymphocyte for an incubation time of 1, 3 or 5 h were 51.1, 63.0, and 77.5% respectively, which were significantly higher than the phagocytosis rates in the control groups with 40.2, 50.9, and 63.8%, respectively. While the Fc fragment of IgM on the surface of opsonized E. tarda was blocked by rabbit anti-flounder IgM polyclonal antibodies, phagocytosis rates of mIgM+ B lymphocyte decreased significantly compared with the unblocked group. Moreover, the proportion of mIgM+ B lymphocytes with higher intracellular reactive oxygen species (ROS) levels rose to 32.1% from the control level of 23.0% after phagocytosis of antiserum-opsonized E. tarda. FcγRII and Syk were found to be significantly upregulated, while FcγRIII was significantly downregulated in the mIgM+ B lymphocytes post phagocytosis. Furthermore, when FcγRII of mIgM+ B lymphocytes was blocked by the prepared antibodies, their phagocytosis rate of antiserum-opsonized E. tarda was 39.0%, which was significantly lower than the unblocked group of 54.0%. These results demonstrate that FcR plays a critical role in mediating phagocytosis and bactericidal activity of mIgM+ B lymphocytes, which would facilitate an improved understanding of the regulatory roles of FcR in phagocytosis of teleost B lymphocytes.

Phosphatase and Tensin Homolog (PTEN) of Japanese Flounder-Its Regulation by miRNA and Role in Autophagy, Apoptosis and Pathogen Infection.

MicroRNAs (miRNAs) are small non-coding RNAs with important roles in diverse biological processes including immunity. Japanese flounder (Paralichthys olivaceus) is an aquaculture fish species susceptible to the infection of bacterial and viral pathogens including Edwardsiella tarda. In a previous study, pol-miR-novel_547, a novel miRNA of flounder with unknown function, was found to be induced by E. tarda. In the present study, we investigated the regulation and function of pol-miR-novel_547 and its target gene. We found that pol-miR-novel_547 was regulated differently by E. tarda and the viral pathogen megalocytivirus, and pol-miR-novel_547 repressed the expression of PTEN (phosphatase and tensin homolog) of flounder (PoPTEN). PoPTEN is ubiquitously expressed in multiple tissues of flounder and responded to bacterial and viral infections. Interference with PoPTEN expression in flounder cells directly or via pol-miR-novel_547 promoted E. tarda invasion. Consistently, in vivo knockdown of PoPTEN enhanced E. tarda dissemination in flounder tissues, whereas in vivo overexpression of PoPTEN attenuated E. tarda dissemination but facilitated megalocytivirus replication. Further in vitro and in vivo studies showed that PoPTEN affected autophagy activation via the AKT/mTOR pathway and also modulated the process of apoptosis. Together these results reveal for the first time a critical role of fish PTEN and its regulatory miRNA in pathogen infection, autophagy, and apoptosis.

Skin proteome profiling of tongue sole (Cynoglossus semilaevis) challenged with Vibrio vulnificus.

Vibrio vulnificus is a major pathogen of cultured Cynoglossus semilaevis and results in skin ulceration and haemorrhage, but the proteomic mechanism of skin immunity against V. vulnificus remains unclear. In this study, we investigated the histopathology and skin immune response in C. semilaevis with V. vulnificus infection at the protein levels, the differential proteomic profiling of its skin was examined by using iTRAQ and LC-MS/MS analyses. A total of 951 proteins were identified in skin, in which 134 and 102 DEPs were screened at 12 and 36 hpi, respectively. Selected eleven immune-related DEPs (pvß, Hsp71, MLC1, F2, α2ML, HCII, C3, C5, C8ß, C9 and CD59) were verified for their immune roles in the V. vulnificus infection via using qRT-PCR assay. KEGG enrichment analysis revealed that most of the identified immune proteins were significantly associated with complement and coagulation cascades, antigen processing and presentation, salivary secretion and phagosome pathways. To our knowledge, this study is the first to describe the proteome response of C. semilaevis skin against V. vulnificus infection. The outcome of this study contributed to provide a new perspective for understanding the molecular mechanism of local skin mucosal immunity, and facilitating the development of novel mucosal vaccination strategies in fish.

Prediction of genomic breeding values based on pre-selected SNPs using ssGBLUP, WssGBLUP and BayesB for Edwardsiellosis resistance in Japanese flounder.

BACKGROUND: Edwardsiella tarda causes acute symptoms with ascites in Japanese flounder (Paralichthys olivaceus) and is a major problem for China's aquaculture sector. Genomic selection (GS) has been widely adopted in breeding industries because it shortens generation intervals and results in the selection of individuals that have great breeding potential with high accuracy. Based on an artificial challenge test and re-sequenced data of 1099 flounders, the aims of this study were to estimate the genetic parameters of resistance to E. tarda in Japanese flounder and to evaluate the accuracy of single-step GBLUP (ssGBLUP), weighted ssGBLUP (WssGBLUP), and BayesB for improving resistance to E. tarda by using three subsets of pre-selected single nucleotide polymorphisms (SNPs). In addition, SNPs that are associated with this trait were identified using a single-SNP genome-wide association study (GWAS) and WssGBLUP. RESULTS: We estimated a heritability of 0.13 ± 0.02 for resistance to E. tarda in Japanese flounder. One million SNPs at fixed intervals were selected from 4,978,724 SNPs that passed quality controls. GWAS identified significant SNPs on chromosomes 14 and 24. WssGBLUP revealed that the putative quantitative trait loci on chromosomes 1 and 14 contained SNPs that explained more than 1% of the genetic variance. Three 50 k-SNP subsets were pre-selected based on different criteria. Compared with pedigree-based prediction (ABLUP), the three genomic methods evaluated resulted in at least 7.7% greater accuracy of predictions. The accuracy of these genomic prediction methods was almost unchanged when pre-selected trait-related SNPs were used for prediction. CONCLUSIONS: Resistance to E. tarda in Japanese flounder has a low heritability. GWAS and WssGBLUP revealed that the genetic architecture of this trait is polygenic. Genomic prediction of breeding values performed better than ABLUP. It is feasible to implement genomic selection to increase resistance to E. tarda in Japanese flounder with 50 k SNPs. Based on the criteria used here, pre-selection of SNPs was not beneficial and other criteria for pre-selection should be considered.

Micro-Transcriptome Analysis Reveals Immune-Related MicroRNA Regulatory Networks of Paralichthys olivaceus Induced by Vibrio anguillarum Infection.

MicroRNAs (miRNAs) are non-coding regulatory RNAs that play a vital part in the host immune response to pathogen infection. Japanese flounder (Paralichthys olivaceus) is an important aquaculture fish species that has suffered from bacterial diseases, including that caused by Vibrio anguillarum infection. In a previous study, we examined the messenger RNA (mRNA) expression profiles of flounder during V. anguillarum infection and identified 26 hub genes in the flounder immune response. In this study, we performed the micro-transcriptome analysis of flounder spleen in response to V. anguillarum infection at 3 different time points. Approximately 277 million reads were obtained, from which 1218 miRNAs were identified, including 740 known miRNAs and 478 novel miRNAs. Among the miRNAs, 206 were differentially expressed miRNAs (DEmiRs), and 104 of the 206 DEmiRs are novel miRNAs identified for the first time. Most of the DEmiRs were strongly time-dependent. A total of 1355 putative target genes of the DEmiRs (named DETGs) were identified based on integrated analysis of miRNA-mRNA expressions. The DETGs were enriched in multiple functional categories associated with immunity. Thirteen key DEmiRs and 66 immune DETGs formed an intricate regulatory network constituting 106 pairs of miRNAs and DETGs that span five immune pathways. Furthermore, seven of the previously identified hub genes were found to be targeted by 73 DEmiRs, and together they formed interlinking regulatory networks. These results indicate that V. anguillarum infection induces complicated miRNA response with extensive influences on immune gene expression in Japanese flounder.

IL-34 regulates the inflammatory response and anti-bacterial immune defense of Japanese flounder Paralichthys olivaceus.

Interleukin (IL)-34 is a relatively recently discovered cytokine with pleiotropic effects on various cellular activities, including immune response. In fish, the knowledge on the function of IL-34 is limited. In the present work, we investigated the function of Japanese flounder Paralichthys olivaceus IL-34 (PoIL-34) in association with inflammation and immune defense. PoIL-34 possesses the conserved structure of IL-34 superfamily and shares 21.52% sequence identity with murine IL-34. PoIL-34 expression was detected in a wide range of tissues of flounder, in particular intestine, and was regulated to a significant extent by bacterial infection in a time-dependent fashion. In vitro studies showed that recombinant PoIL-34 (rPoIL-34) bound peripheral blood leukocytes (PBLs) and promoted ROS production, acid phosphatase activity, and cellular resistance against bacterial infection. At the molecular level, rPoIL-34 enhanced the expressions of inflammatory cytokines and specific JAK and STAT genes. Similar stimulatory effects of rPoIL-34 were observed in vivo. When PoIL-34 was overexpressed in flounder, the expressions of pro- and anti-inflammatory mediators were significantly affected in a tissue-dependent manner, which correlated with an augmented ability of the fish to eliminate invading pathogens from tissues. Together, these results indicated that PoIL-34 regulated inflammatory response probably via specific JAK/STAT pathways and had a significant influence on the immune defense of flounder against bacterial infection.

Transcriptome Analysis of Paralichthys olivaceus Erythrocytes Reveals Profound Immune Responses Induced by Edwardsiella tarda Infection.

Unlike mammalian red blood cells (RBCs), fish RBCs are nucleated and thus capable of gene expression. Japanese flounder (Paralichthys olivaceus) is a species of marine fish with important economic values. Flounder are susceptible to Edwardsiella tarda, a severe bacterial pathogen that is able to infect and survive in flounder phagocytes. However, the infectivity of and the immune response induced by E. tarda in flounder RBCs are unclear. In the present research, we found that E. tarda was able to invade and replicate inside flounder RBCs in both in vitro and in vivo infections. To investigate the immune response induced by E. tarda in RBCs, transcriptome analysis of the spleen RBCs of flounder challenged with E. tarda was performed. Six sequencing libraries were constructed, and an average of 43 million clean reads per library were obtained, with 85% of the reads being successfully mapped to the genome of flounder. A total of 1720 differentially expressed genes (DEGs) were identified in E. tarda-infected fish. The DEGs were significantly enriched in diverse Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, especially those associated with immunity, disease, and infection. Ninety-one key DEGs involved in 12 immune-related pathways were found to form extensive interaction networks. Twenty-one genes that constituted the hub of the networks were further identified, which were highly regulated by E. tarda and involved in a number of immune processes, notably pathogen recognition and signal transduction, antigen processing and presentation, inflammation, and splicing. These results provide new insights into the immune role of flounder RBCs during bacterial infection.