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Japanese flounder (Paralichthys olivaceus) is one of the most economically important marine fish cultured in north Asia. Vibrio anguillarum is a severe bacterial pathogen to Japanese flounder and many other aquaculture species. In order to understand the immune response of flounder during bacterial infection, we systematically examined the transcriptome profiles of flounder spleen at three time points after V. anguillarum challenge. More than one billion high quality reads were obtained, approximately 80.70% of which were successfully mapped to the reference genome of flounder. A total of 6060, 4688 and 4235 differentially expressed genes (DEGs) were captured at 6, 12 and 24-h post-infection, respectively. The DEGs exhibited dynamic changes in expression and were assigned into four different profiles based on expression trend. GO and KEGG analysis showed that the DEGs were enriched in various immune-related terms, including response to stimulation, immune system and pathways of cytokine-cytokine receptor interaction, Jak-STAT signaling and Toll-like receptor signaling. Furthermore, a network of highly interactive DEGs involved in 11 immune-related pathways was detected by utilizing the weighted co-expressing network analysis (WGCNA). Accordingly, 26 hub genes were discovered that constituted an elaborate immune regulatory network and functioned mainly in pathogen recognition, antigen processing, and molecular signaling. The results of this study provided the first systematical transcriptome profile of flounder in association with V. anguillarum infection and can serve as a valuable resource of target genes for future studies on the molecular mechanisms underlying the immune defense of flounder against bacterial infection. • Vibrio anguillarum -induced transcriptome profiles of flounder were investigated. • A total of 8923 differentially expressed genes (DEGs) were found at 6, 12 and 24 hpi. • Co-expression networks of immune relevant DEGs were constructed. • Twenty-six hub genes with multiple interactions were identified. • The hub genes represented mainly genes involved in immune defense against pathogens. [ABSTRACT FROM AUTHOR]

Abstract: Japanese flounder (Paralichthys olivaceus) is an important economic fish species cultured worldwide. In this report, we compared the potentials of ten housekeeping genes as quantitative real time RT-PCR (qRT-PCR) references for the study of gene expression in Japanese flounder under normal physiological conditions and during bacterial infection. For this purpose, the expression of the ten genes in eight flounder tissues (liver, spleen, kidney, heart, muscle, brain, gill, and intestine) was determined by qRT-PCR before and after bacterial infection. The expression levels of the housekeeping genes were then compared and evaluated with geNorm and NormFinder algorithms. The results showed that before bacterial infection, the tested genes exhibited tissue-specific expressions to various degrees, with β-actin and ubiquitin-conjugating enzyme being ranked as the most stable genes across tissue types. Following bacterial challenge, all the tested genes varied in expression levels in tissue-dependent manners and no cross-all-tissue type reference gene was identified among the examined panel of housekeeping genes; however, α-tubulin was recognized as the most stable gene in four (spleen, heart, muscle, and gill) of the eight examined tissues. These results indicate that for qRT-PCR analysis of gene expression in Japanese flounder as a function of bacterial infection, the choice of reference genes should be made according to tissue type. [Copyright &y& Elsevier]

In this study, we examined the function of a Japanese flounder (Paralichthys olivaceus) microRNA (miRNA), pol-miR-363-3p. We found that pol-miR-363-3p targets an ubiquitin-specific protease (USP), USP32. USP is a family of deubiquitinating enzymes essential to the functioning of the ubiquitin proteasome system. In mammals, USP32 is known to be associated with cancer and immunity. In fish, the function of USP32 is unknown. We found that flounder USP32 (PoUSP32) expression was detected in the major tissues of flounder, particularly intestine. In vitro and in vivo studies showed that pol-miR-363-3p directly regulated PoUSP32 in a negative manner by interaction with the 3′UTR of PoUSP32. Overexpression of pol-miR-363-3p or interference with PoUSP32 expression in flounder cells significantly blocked Streptococcus iniae infection. Consistently, in vivo knockdown of pol-miR-363-3p or overexpression of PoUSP32 enhanced dissemination of S. iniae in flounder tissues, whereas in vivo knockdown of PoUSP32 inhibited S. iniae dissemination. In addition, pol-miR-363-3p knockdown also significantly promoted the tissue dissemination of the viral pathogen megalocytivirus, which, as well as S. iniae , regulated pol-miR-363-3p expression. Together these results revealed an important role of pol-miR-363-3p in flounder immune defense against bacterial and viral infection. • PoUSP32 was a target gene of pol-miR-363-3p and regulated negatively by the latter. • pol-miR-363-3p expression was regulated by Streptococcus iniae and megalocytivirus. • PoUSP32 knockdown/pol-miR-363-3p overexpression blocked S. iniae infection in vitro. • In vivo pol-miR-363-3p knockdown promoted S. iniae and megalocytivirus infection. • In vivo PoUSP32 overexpression/knockdown affected S. iniae invasion in flounder. [ABSTRACT FROM AUTHOR]

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. • PoIL-34 expression occurred in multi-tissues and responded to bacterial infection. • rPoIL-34 bound to and promoted the activation and bacterial resistance of PBLs. • rPoIL-34 regulated the expressions of inflammatory cytokines and specific JAK/STAT. • In vivo PoIL-34 overexpession affected the expressions of inflammatory cytokines. • PoIL-34 overexpession reduced bacterial dissemination in fish tissues. [ABSTRACT FROM AUTHOR]

Megalocytivirus is a serious viral pathogen to many farmed fish including Japanese flounder (Paralichthys olivaceus). In this study, in order to systematically identify host immune genes induced by megalocytivirus infection, we examined the transcription profiles of flounder infected by megalocytivirus for 2, 6, and 8 days. Compared with uninfected fish, virus-infected fish exhibited 1242 differentially expressed genes (DEGs), with 225, 275, and 877 DEGs occurring at 2, 6, and 8 days post infection, respectively. Of these DEGs, 728 were upregulated and 659 were downregulated. The majority of DEGs were time-specific and formed four distinct expression profiles well correlated with the time of infection. The DEGs were classified into diverse Gene Ontology (GO) functional terms and enriched in 27 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, approximately one third of which were related to immunity. Weighted co-expression network analysis (WGCNA) was used to identify 16 key immune DEGs belonging to seven immune pathways (RIG-I-like receptor signaling pathway, JAK-STAT signaling pathway, TLR signaling pathway, cytokine-cytokine receptor interaction, phagosome, apoptosis, and p53 signaling pathway). These pathways interacted extensively and formed complicated networks. This study provided a global picture of megalocytivirus-induced gene expression profiles of flounder at the transcriptome level and uncovered a set of key immune genes and pathways closely linked to megalocytivirus infection. These results provided a set of targets for future delineation of the key factors implicated in the anti-megalocytivirus immunity of flounder. • A total of 1242 differentially expressed genes were induced by megalocytivirus. • The expressions of the DEGs were highly time-dependent and clustered into 4 groups. • Sixteen key immune genes forming complex networks with each other were identified. • The interactive key genes belonged to 7 immune pathways vital to antiviral immunity. [ABSTRACT FROM AUTHOR]

Interleukin (IL)-21 is a pleiotropic cytokine and plays a vital role in immunity. In the current study, we examined the immune function of Japanese flounder Paralichthys olivaceus IL-21 (PoIL-21). PoIL-21 shares moderate (25.17%–46.25%) sequence identities with other teleost IL-21. PoIL-21 expression occurred in multiple tissues, especially intestine, and was regulated by bacterial infection in a time dependent manner. PoIL-21 was secreted by peripheral blood leukocytes (PBL) upon LPS stimulation. Recombinant PoIL-21 (rPoIL-21) bound to a wide range of Gram-negative and Gram-positive bacteria and inhibited the growth of the fish bacterial pathogen Streptococcus iniae. rPoIL-21 also interacted with PBL, resulting in enhanced cell proliferation, ROS production, and expression of IL-1β, TNF-α, CD8β, T-bet, PoIL-21, PoIL-21 receptor, and STAT. Consequently, the presence of rPoIL-21 significantly reduced bacterial infection in PBL. In vivo study showed that rPoIL-21 upregulated the expression of inflammatory cytokines and PoIL-21. Taken together, these results indicate that PoIL-21 is an inducible, secreted cytokine with a broad range of binding capacities and plays an important role in the regulation of anti-bacterial immunity. • PoIL-21 was secreted by peripheral blood leukocytes (PBL) upon LPS stimulation. • rPoIL-21 bound to various bacteria and inhibited the growth of bacterial pathogen. • rPoIL-21 bound to PBL and enhanced cell proliferation and ROS production..► rPoIL-21 significantly promoted the resistance of PBL against bacterial infection • rPoIL-21 enhanced the expression of CD8β, T-bet, IL-1, TNF-α, PoIL-21R, and STAT. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are involved in many biological activities including immune defense against pathogens. In this study, we applied high-throughput sequencing technology to examine miRNAs in Japanese flounder (Paralichthys olivaceus) infected with Streptococcus iniae at different times. A total of 1038 miRNAs were identified, of which, 249 were novel miRNAs, and 81 showed differential expression (named DEmiRNAs) after S. iniae infection. Of the 81 DEmiRNAs identified, 34 and 58 occurred at 6 h and 24 h post-infection, respectively; most DEmiRNAs were strongly time-specific, and only 13.6% of the DEmiRNAs were shared between the two time points. A total of 9582 target genes were predicted for the 81 DEmiRNAs. The putative target genes were enriched in various GO and KEGG pathways of biological processes and molecular/cellular functions, in particular endocytosis, regulation of transcription, lysososme, and the signaling pathways of MAPK, ErbB, and AMPK. One of the DEmiRNAs, pol-3p-10740_175, was found to target dual specificity phosphatase 6 (Dusp6) and repress the expression of the latter. Transfection of flounder FG cells with pol-3p-10740_175 caused a significant inhibition on S. iniae invasion. The results of this study provided the first S. iniae -induced miRNA profile in Japanese flounder and indicated that flounder miRNAs play an important role in antibacterial immunity. • Streptococcus iniae infection altered the expression of 81 miRNAs in flounder. • There were 34 and 58 differentially expressed miRNAs at 6 and 24 hpi, respectively. • The putative target genes of the miRNAs were enriched in various GO/KEGG pathways. • One miRNA, pol-3p-10740_175, negatively regulated dual specificity phosphatase 6. • Overexpression of pol-3p-10740_175 significantly inhibited S. iniae invasion. [ABSTRACT FROM AUTHOR]

Japanese flounder (Paralichthys olivaceus) is an important economic fish species farmed in China and other countries. It is susceptible to infection by Edwardsiella tarda , a severe fish pathogen with a broad host range. In this study, we employed high-throughput deep sequencing technology to identify, in a global scale, flounder kidney microRNAs (miRNAs) induced by E. tarda at different stages of infection. Differentially expressed miRNAs (DEmiRNAs) and mRNAs (DEmRNAs) exhibiting significantly altered expression levels before and after E. tarda infection were examined. A total of 96 DEmiRNAs were identified, for which 2779 target genes were predicted. Eighty-seven miRNA–mRNA pairs, involving 29 DEmiRNAs and 86 DEmRNAs, showed negative correlations in their expression patterns. GO and KEGG enrichment analysis revealed that the putative target genes of the DEmiRNAs were associated with diverse biological processes, cellular components, and molecular functions. One of the DEmiRNAs, pol-miR-182-5p, was demonstrated to regulate sphingosine-1-phosphate receptor 1 (PoS1PR1) negatively in a manner that depended on the specific interaction between the seed sequence of pol-miR-182-5p and the 3'-UTR of PoS1PR1. Overexpression of pol-miR-182-5p in flounder cells promoted apoptosis and inhibited cellular viability. Knockdown of PoS1PR1 in flounder enhanced E. tarda invasion and dissemination in fish tissues. These results provide new insights into miRNA-mediated anti-bacterial immunity in flounder. • Time-dependent profiles of flounder kidney miRNAs induced by E. tarda was obtained. • Ninety-six differentially expressed miRNAs (DEmiRNAs) were identified. • One DEmiRNA, pol-miR-182-5p, promoted apoptosis and death of flounder cells. • Sphingosine-1-phosphate receptor 1 (PoS1PR1) is a target of pol-miR-182-5p. • PoS1PR1 is required for inhibition of E. tarda dissemination in fish tissues. [ABSTRACT FROM AUTHOR]

Abstract: Iridoviruses are large double-stranded DNA viruses with icosahedral capsid. The Iridoviridae family contains five genera, one of which is Megalocytivirus. Megalocytivirus has emerged in recent years as an important pathogen to a wide range of marine and freshwater fish. In this study, we aimed at developing effective genetic vaccines against megalocytivirus affecting farmed fish in China. For this purpose, we constructed seven DNA vaccines based on seven genes of rock bream iridovirus isolate 1 from China (RBIV-C1), a megalocytivirus with a host range that includes Japanese flounder (Paralichthys olivaceus) and turbot (Scophthalmus maximus). The protective potentials of these vaccines were examined in a turbot model. The results showed that after vaccination via intramuscular injection, the vaccine plasmids were distributed in spleen, kidney, muscle, and liver, and transcription of the vaccine genes and production of the vaccine proteins were detected in these tissues. Following challenge with a lethal-dose of RBIV-C1, fish vaccinated with four of the seven DNA vaccines exhibited significantly higher levels of survival compared to control fish. Of these four protective DNA vaccines, pCN86, which is a plasmid that expresses an 86-residue viral protein, induced the highest protection. Immunological analysis showed that pCN86 was able to (i) stimulate the respiratory burst of head kidney macrophages at 14 d, 21 d, and 28 d post-vaccination, (ii) upregulate the expression of immune relevant genes involved in innate and adaptive immunity, and (iii) induce production of serum antibodies that, when incubated with RBIV-C1 before infection, significantly reduced viral loads in kidney and spleen following viral infection of turbot. Taken together, these results indicate that pCN86 is an effective DNA vaccine that may be used in the control of megalocytivirus-associated diseases in aquaculture. [Copyright &y& Elsevier]

Abstract: Heat shock protein (Hsp) 70 is a molecular chaperone that plays an important role in protein folding and transport. In addition, Hsp70 is also involved in regulation of innate and adaptive immune response. In this study, we examined the biological activity and the immunomodulatory property of an Hsp70 homologue, PoHsp70, from Japanese flounder (Paralichthys olivaceus). Recombinant PoHsp70 purified from Escherichia coli exhibits apparent ATPase activity; however, a mutant PoHsp70, PoHsp70M, that bears mutation of the ATPase-associated domain, was completely abolished in activity. Expression of PoHsp70 was upregulated in a time-dependent manner by vaccination of flounder with a DNA vaccine, pSia10, that expresses a Streptococcus iniae antigen, Sia10. To examine whether PoHsp70 possessed any adjuvant potential, the DNA vaccine plasmids pSia10Hsp70 and pSia10Hsp70M were constructed. pSia10Hsp70 co-expresses Sia10 and PoHsp70, while pSia10Hsp70M co-expresses Sia10 and PoHsp70M. Following vaccination of flounder, production of Sia10 plus PoHsp70 and Sia10 plus PoHsp70M was detected in pSia10Hsp70- and pSia10Hsp70M-vaccinated fish respectively. At one month post-vaccination, comparable levels of serum antibodies were detected in fish vaccinated with pSia10Hsp70, pSia10Hsp70M, and pSia10. Subsequent protection analysis showed that, following S. iniae challenge, pSia10Hsp70 induced a survival rate that was significantly higher than that induced by pSia10, while pSia10Hsp70M induced a survival rate similar to that induced by pSia10. These results indicate that PoHsp70 is an effective adjuvant and that the adjuvanticity of PoHsp70 requires the intrinsic ATPase activity. [Copyright &y& Elsevier]

Abstract: Bacterial sialidases are a group of glycohydrolases that are known to play an important role in invasion of host cells and tissues. In this study, we examined in a model of Japanese flounder (Paralichthys olivaceus) the potential function of NanA, a sialidase from the fish pathogen Edwardsiella tarda. NanA is composed of 670 residues and shares low sequence identities with known bacterial sialidases. In silico analysis indicated that NanA possesses a sialidase domain and an autotransporter domain, the former containing five Asp-boxes, a RIP motif, and the conserved catalytic site of bacterial sialidases. Purified recombinant NanA (rNanA) corresponding to the sialidase domain exhibited glycohydrolase activity against sialic acid substrate in a manner that is pH and temperature dependent. Immunofluorescence microscopy showed binding of anti-rNanA antibodies to E. tarda, suggesting that NanA was localized on cell surface. Mutation of nanA caused drastic attenuation in the ability of E. tarda to disseminate into and colonize fish tissues and to induce mortality in infected fish. Likewise, cellular study showed that the nanA mutant was significantly impaired in the infectivity against cultured flounder cells. Immunoprotective analysis showed that rNanA in the form of a subunit vaccine conferred effective protection upon flounder against lethal E. tarda challenge. rNanA vaccination induced the production of specific serum antibodies, which enhanced complement-mediated bactericidal activity and reduced infection of E. tarda into flounder cells. Together these results indicate that NanA plays an important role in the pathogenesis of E. tarda and may be exploited for the control of E. tarda infection in aquaculture. [Copyright &y& Elsevier]

Abstract: CpG-containing oligodeoxynucleotides (ODNs) are known to be immunostimulatory in vertebrate systems and can activate both innate and adaptive immune responses. In this report, we described the selection, identification, and analysis of CpG motifs with immunoprotective effects in Japanese flounder. Sixteen CpG ODNs were synthesized and examined for the ability to inhibit bacterial dissemination in Japanese flounder blood. Four ODNs with the strongest inhibitory effects were selected and mixed to form ODNs 4M. In addition, a plasmid, pCN6, was constructed that contains the sequences of the four selected ODNs. When administered into Japanese flounder via intraperitoneal injection, both ODNs 4M and pCN6 could, in dose and time dependent manners, afford short-term protection against the infections of two different bacterial pathogens. Immunological analyses showed that ODNs 4M and, especially, pCN6 activated head kidney macrophages and enhanced serum bactericidal activity via probably the alternative pathway of complement activation. When used as a DNA vaccine to immunize Japanese flounder, pCN6 conferred apparent protections (42.9% and 52.6%, respectively, in terms of relative percent survival) against the challenges of two different fish pathogens at 4-week post-vaccination. Transcriptional analysis showed that vaccination with pCN6 upregulated the expression of the genes encoding NKEF, MHC IIα, IL-1β, Mx, and MHC Iα. These results demonstrate that ODNs 4M and pCN6 are immunostimulatory in Japanese flounder and can induce short- and long-term nonspecific protections against bacterial infections. [Copyright &y& Elsevier]