PoIL8-L, a teleost interleukin-8 like, enhances leukocyte cellular vitality and host defense against bacterial infections in Japanese flounder (Paralichthys olivaceus).

Interleukin-8 (IL-8), a CXC chemokine, exerts pivotal effect on cell migration, inflammatory response, and immune regulation. In this study, we examined the immunological characteristics of an IL-8 like homologue (PoIL8-L) in Japanese flounder (Paralichthys olivaceus). PoIL8-L contains a conserved chemokine CXC domain and 105 amino acid residues. PoIL8-L expression in tissues was constitutive, and significantly regulated by V. havieri or E. tarda infection. In vitro, rPoIL8-L could bind to eight tested bacteria, exhibited bacteriostatic and bactericidal effects against certain bacteria, and could bind to the targeted bacterial Ⅳ pilin protein rPilA of E. tarda. Furthermore, rPoIL8-L could attach to peripheral blood leukocytes, and enhance their immune genes expression, respiratory burst, chemotaxis, proliferation, acid phosphatase activity, and phagocytic activity. Additionally, rPoIL8-L induce neutrophils to extrude neutrophil extracellular traps. In vivo, rPoIL8-L could promote host resistance to E. tarda infection. In summary, these findings provide fresh perspectives on the immunological antibacterial properties of IL-8 in teleost.

IL-18 signaling is regulated by caspase 6/8 and IL-18BP in turbot (Scophthalmus maximus).

Interleukin (IL)-18 is synthesized as a precursor that requires intracellular processing to become functionally active. In human, IL-18 is processed by caspase 1 (CASP1). In teleost, the maturation and signal transduction mechanisms of IL-18 are unknown. We identified two IL-18 variants, IL-18a and IL-18b, in turbot. IL-18a, but not IL-18b, was processed by CASP6/8 cleavage. Mature IL-18a bound specifically to IL-18 receptor (IL-18R) α-expressing cells and induced IL-18Rα-IL-18Rß association. Bacterial infection promoted IL-18a maturation in a manner that required CASP6 activation and correlated with gasdermin E activation. The mature IL-18a induced proinflammatory cytokine expression and enhanced bacterial clearance. IL-18a-mediated immune response was suppressed by IL-18 binding protein (IL-18BP), which functioned as a decoy receptor for IL-18a. IL-18BP also functioned as a pathogen pattern recognition receptor and directly inhibited pathogen infection. Our findings revealed unique mechanism of IL-18 maturation and conserved mechanism of IL-18 signaling and regulation in turbot, and provided new insights into the regulation and function of IL-18 related immune signaling.

Immune and regulative characterization of complement-related gene cfhl5 in response to Vibrio harveyi challenge in Cynoglossus semilaevis.

Complement factor H-related protein (CFHR) plays an important role in regulating complement activation and defensive responses. The function of CFHR2 (complement factor H related 2), a member of the CFHR family, in fish remains unclear. Here, we report the genetic relationship, expression characteristics and regulatory mechanism of cfhl5 (complement factor H like 5) gene, which encodes CFHR2 in Chinese tongue sole. We observed that the cfhl5 gene was widely expressed in several tissues, such as brain, heart and immune organs, and was most abundantly expressed in liver. After injection with Vibrio harveyi, the expression of cfhl5 was up-regulated significantly in liver, spleen and kidney at 12 or 24 hours post infection (hpi), suggesting an involvement of this gene in the acute immune response. Knockdown of cfhl5 in liver cells significantly up-regulated the expression of the pro-inflammatory cytokines tnf-α (tumor necrosis factor-alpha) and il1ß (interleukin-1beta), the immunomodulatory factor il10 (interleukin-10) and the lectin complement pathway gene masp1 (MBL-associated serine protease 1), and down-regulated the expression of complement components c3 (complement 3) and cfi (complement factor I). In our previous work, we found that cfhl5 gene was significantly higher methylated and lower expressed in the resistant family compared with the susceptible family. Therefore, we used dual-luciferase reporter system to determine the effect of DNA methylation on this gene and found that DNA methylation could inhibit the promoter activity to reduce its expression. These results demonstrated that the expression of cfhl5 is regulated by DNA methylation, and this gene might play an important role in the immune response by regulating the expression of cytokines and complement components genes in Chinese tongue sole.

Genome-Wide Identification and Interaction Analysis of Turbot Heat Shock Protein 40 and 70 Families Suggest the Mechanism of Chaperone Proteins Involved in Immune Response after Bacterial Infection.

Hsp40-Hsp70 typically function in concert as molecular chaperones, and their roles in post-infection immune responses are increasingly recognized. However, in the economically important fish species Scophthalmus maximus (turbot), there is still a lack in the systematic identification, interaction models, and binding site analysis of these proteins. Herein, 62 Hsp40 genes and 16 Hsp70 genes were identified in the turbot at a genome-wide level and were unevenly distributed on 22 chromosomes through chromosomal distribution analysis. Phylogenetic and syntenic analysis provided strong evidence in supporting the orthologies and paralogies of these HSPs. Protein-protein interaction and expression analysis was conducted to predict the expression profile after challenging with Aeromonas salmonicida. dnajb1b and hspa1a were found to have a co-expression trend under infection stresses. Molecular docking was performed using Auto-Dock Tool and PyMOL for this pair of chaperone proteins. It was discovered that in addition to the interaction sites in the J domain, the carboxyl-terminal domain of Hsp40 also plays a crucial role in its interaction with Hsp70. This is important for the mechanistic understanding of the Hsp40-Hsp70 chaperone system, providing a theoretical basis for turbot disease resistance breeding, and effective value for the prevention of certain diseases in turbot.

A bacterial ghost vaccine against Aeromonas salmonicida infection in turbot (Scophthalmus maximus).

Aeromonas salmonicida is one of the most prevalent pathogens that causes huge economic losses to aquaculture. Effective vaccination is the first choice for preventing infection. Bacterial ghost (BG), an empty bacterial shell devoid of cytoplasm, is a promising vaccine antigen with distinct advantages. Herein, we established strategies for producing a substantial yield of A. salmonicida ghost (ASG) and investigated the immune-protective properties of it. As a result, 2.84 mg/ml NaOH was discovered to be capable of inducing considerable amounts of ASG. Furthermore, the ASG vaccine elicited adaptive immunity in turbots after rapid activation of innate immunity. Even though formalin-killed cells (FKC) produced a few more antibodies than ASG, ASG ultimately provided a much stronger immune protection effect because it strengthened cellular immunity, with a relative percentage survival (RPS) of 50.1 % compared to FKC. These findings demonstrated that ASG effectively activated cell-mediated immunity, which helped get rid of microorganisms inside cells. Therefore, this study presented novel perspectives for future research on furunculosis vaccine products based on ASG as an antigen.

The extracellular matrix protein EFEMP2 is involved in the response to VHSV infection in the olive flounder Paralichthys olivaceus.

The EGF-containing fibulin-like extracellular matrix protein 2 (EFEMP2) is involved in connective tissue development, elastic fiber formation, and tumor growth. In this study, we characterized the cDNA of EFEMP2 (PoEFEMP2), a member of the fibulin family of ECM proteins, in the olive flounder Paralichthys olivaceus. The coding region of PoEFEMP2 encodes a protein that contains six calcium-binding EGF-like (EGF-CA) domains and four complement Clr-like EGF-like (cEGF) domains. PoEFEMP2 shows 67.51-96.77 % similarities to orthologs in a variety of fish species. PoEFEMP2 mRNA was detected in all tissues examined; the highest levels of PoEFEMP2 mRNA expression were observed in the heart, testis, ovary and muscle. The PoEFEMP2 mRNA level increases during early development. In addition, the PoEFEMP2 mRNA level increased at 3 h post-infection (hpi) and decreased from 6 to 48 hpi in flounder Hirame natural embryo (HINAE) cells infected with viral hemorrhagic septicemia virus (VHSV). Disruption of PoEFEMP2 using the clustered regularly interspaced short palindromic repeats/CRISPR-associated-9 (CRISPR/Cas9) system resulted in a significant upregulation of VHSV G mRNA levels and immune-related genes expression in knockout cells. These findings implicate PoEFEMP2 in antiviral responses in P. olivaceus.

Manipulated C5aR1 over/down-expression associates with IL-6 expression during bacterial inflammation in half-smooth tongue sole (Cynoglossus semilaevis).

The complement component 5a/complement component 5 receptor 1 (C5a/C5aR1) pathway plays a crucial role in the onset and development of inflammation, but relevant studies in fish are lacking. In this study, we successfully characterized the relationship between half-smooth tongue sole (Cynoglossus semilaevis) C5aR1 (CsC5aR1) and bacterial inflammation. First, we showed that the overexpression of CsC5aR1 significantly increased bacterial pathological damage in the liver and intestine, whereas inhibition attenuated the damage. The in vitro experiments suggested that CsC5aR1 was able to positively regulate the phagocytic activity and respiratory burst of tongue sole macrophages. In terms of both transcriptional and translational levels, overexpression/inhibition of CsC5aR1 was followed by a highly consistent up-regulation/decrease of its downstream canonical inflammatory factor interleukin-6 (CsIL-6). Furthermore, we stimulated macrophages by lipopolysaccharide (LPS) and lipoteichoic acid (LTA) and found a broad-spectrum response to bacterial infections by the C5a/C5aR1 complement pathway together with the downstream inflammatory factor CsIL-6. Subsequently, we directly elucidated that CsIL-6 is an indicator of C5a/C5aR1-mediated inflammation at different infection concentrations, different infectious bacteria (Vibrio anguillarum and Mycobacterium marinum), and different detection levels. These results might provide a new inflammation bio-marker for early warning of bacteria-induced hyperinflammation leading to fish mortality and a promising target for the treatment of bacterial inflammation in teleost.

Molecular characterization, expression and immune functional analysis of cystatin 10 in turbot.

BACKGROUND: Cystatin is a protease inhibitor that also regulates genes expression linked to inflammation and plays a role in defense and regulation. METHODS AND RESULTS: Cystatin 10 (Smcys10) was cloned from Scophthalmus maximus and encodes a 145 amino acid polypeptide. The results of qRT-PCR showed that Smcys10 exhibited tissue-specific expression patterns, and its expression was significantly higher in the skin than in other tissues. The expression level of Smcys10 was significantly different in the skin, gill, head kidney, spleen and macrophages after Vibrio anguillarum infection, indicating that Smcys10 may play an important role in resistance to V. anguillarum infection. The recombinant Smcys10 protein showed binding and agglutinating activity in a Ca2+-dependent manner against bacteria. rSmcys10 treatment upregulated the expression of IL-10, TNF-α and TGF-ß in macrophages of turbot and hindered the release of lactate dehydrogenase (LDH) from macrophages after V. anguillarum infection, which confirmed that rSmcys10 reduced the damage to macrophages by V. anguillarum. The NF-κB pathway was suppressed by Smcys10, as demonstrated by dual-luciferase analysis. CONCLUSIONS: These results indicated that Smcys10 is involved in the host antibacterial immune response.

Differentially expressed proteins and microbial communities of the skin regulate disease resistance to Chinese tongue sole (Cynoglossus semilaevis).

Vibriosis, caused by Vibrio, seriously affects the health of fish, shellfish, and shrimps, causing large economic losses. Teleosts are represent the first bony vertebrates with both innate and adaptive immune responses against pathogens. Aquatic animals encounter hydraulic pressure and more pathogens, compared to terrestrial animals. The skin is the first line of defense in fish, constituting the skin-associated lymphoid tissue (SALT), which belongs to the main mucosa-associated lymphoid tissues (MALT). However, little is known about the function of immunity related proteins in fish. Therefore, this study used iTRAQ (isobaric tags for relative and absolute quantitation) to compare the skin proteome between the resistant and susceptible families of Cynoglossus semilaevis. The protein integrin beta-2, the alpha-enolase isoform X1, subunit B of V-type proton ATPase, eukaryotic translation initiation factor 6, and ubiquitin-like protein ISG15, were highly expressed in the resistant family. The 16S sequencing of the skin tissues of the resistant and susceptible families showed significant differences in the microbial communities of the two families. The protein-microbial interaction identified ten proteins associated with skin microbes, including immunoglobulin heavy chain gene (IGH), B-cell lymphoma/leukemia 10 (BCL10) and pre-B-cell leukemia transcription factor 1 isoform X2 (PBX2). This study highlights the interaction between skin proteins and the microbial compositions of C. semilaevis and provides new insights into understanding aquaculture breeding research.

miR-144 affects the immune response and activation of inflammatory responses in Cynoglossus semilaevis by regulating the expression of CsMAPK6.

MicroRNAs are increasingly recognized for their pivotal role in the immune system, yet the specific regulatory functions of fish-derived microRNAs remain largely unexplored. In this research, we discovered a novel miRNA, Cse-miR-144, in the Chinese tongue sole (Cynoglossus semilaevis), characterized by a 73-base pair precursor and a 21-nucleotide mature sequence. Our findings revealed that the expression of Cse-miR-144 was notably inhibited by various Vibrio species. Utilizing bioinformatics and dual-luciferase assay techniques, we established that the pro-inflammatory cytokine gene CsMAPK6 is a direct target of Cse-miR-144. Subsequent in vitro and in vivo western blotting analyses confirmed that Cse-miR-144 can effectively reduce the protein levels of CsMAPK6 post-transcriptionally. Moreover, CsMAPK6 is known to be involved in the activation of the Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-kB). Additional investigations using qPCR and ELISA demonstrated that suppression of Cse-miR-144 leads to an upsurge in the liver mRNA levels of various immune genes (including MYD88, TRAF6, NF-κB, TRAF2, TRAF3, and TNF), alongside a marked increase in the production and secretion of pro-inflammatory cytokines (IL-1ß, IL-6, and IL-8) in the bloodstream of C. semilaevis. These findings collectively underscore the potential of Cse-miR-144 as a key inhibitor of CsMAPK and its crucial role in modulating the immune and inflammatory responses in teleost fish. Compared to the siRNA, miRNA is a better tool in controlling the expression of target gene with a lower cost.

Oral Administration of Antimicrobial Peptide NZ2114 Through the Microalgal Bait Tetraselmis subcordiformis (Wille) Butcher for Improving the Immunity and Gut Health in Turbot (Scophthalmus maximus L.).

Antibiotics are widely used in aquaculture to treat the bacterial diseases. However, the improper use of antibiotics could lead to environmental pollution and development of resistance. As a safe and eco-friendly alternative, antimicrobial peptides (AMPs) are commonly explored as therapeutic agents. In this study, a mutant strain of Tetraselmis subcordiformis containing AMP NZ2114 was developed and used as an oral drug delivery system to reduce the use of antibiotics in turbot (Scophthalmus maximus) aquaculture. The gut, kidney, and liver immune-related genes and their effects on gut digestion and bacterial communities in turbot fed with NZ2114 were evaluated in an 11-day feeding experiment. The results showed that compared with the group fed with wild-type T. subcordiformis, the group fed with T. subcordiformis transformants containing NZ2114 was revealed with decreased levels of both pro-inflammatory factors (TNF-α and IL-1ß), inhibitory effect on Staphylococcus aureus, Vibrio parahaemolyticus, and Vibrio splendidus demonstrated by the in vitro simulation experiments, and increased richness and diversity of the gut microbiota of turbot. In conclusion, our study provided a novel, beneficial, and low-cost method for controlling bacteria in turbot culture through the oral drug delivery systems.

Water-in-oil adjuvant challenges in fish vaccination: An experimental inactivated adjuvanted vaccine against betanodavirus infection in Senegalese sole.

The extensive growth of intensive fish farming has led to a massive spread of infectious diseases. Nervous necrosis virus (NNV) is the causative agent of the viral encephalo- and retinopathy disease which has become a major threat for fish farming all over the globe. The devastating mortality rates recorded in disease outbreaks, especially when infected specimens are at early stages of development, have a high economic impact on the sector. Currently, vaccines are the most cost-effective preventing tool in the fight against viruses. Inactivated vaccines have the advantage of simplicity in their development at the same time as present the antigen in a similar manner than the natural infection in the host. Nevertheless, they usually trigger weaker immune responses needing adjuvants to boost their effectiveness. In this work, we have intraperitoneally vaccinated Senegalese sole juveniles (Solea senegalensis) with a previously designed inactivated vaccine against NNV based on binary ethylenimine (BEI), mixed or not with an oil-adjuvant. Our results demonstrated the potential activation of different immune pathways when the vaccine was administered alone compared to the oil-adjuvanted vaccine, both resulting in an equivalent partial improvement in survival following a NNV challenge. However, whilst the vaccine alone led to a significant increase in specific antibodies, in the adjuvanted version those antibodies were kept basal although with a slight improvement in their neutralization capacity. At transcriptional level, neither vaccine (adjuvanted or not) triggered the immune system activation during the vaccination period. However, after NNV infection, the BEI-inactivated vaccines alone and oil-adjuvanted both elicited the stimulation of antiviral responsive genes (rtp3, herc4), antigen presentation molecules (mhcii) and T-cell markers (cd8a) in the head-kidney. Additionally, the oil-adjuvanted vaccine appears to stimulate mediator cytokines (il6) and B-cell markers (ight and ighm). Surprisingly, when the adjuvant was administered alone, fish showed the highest survival rates concomitantly with a lack of NNV-IgM production, pointing to the possible induction of different immune pathways than the B-cell responses via antibodies by the adjuvant. Since this combined vaccine did not succeed in the full extension of protection against the pathogen, further studies should be performed focusing on unravelling the molecular mechanisms through which adjuvants trigger the immune response, both independently and when added to a vaccine antigen.

Bacterial infection induces pyroptotic signaling-mediated neutrophil extracellular traps (NETs) formation in turbot (Scophthalmus maximus).

Neutrophils can capture and kill pathogens by releasing neutrophils extracellular traps (NETs), which play critical roles in anti-microbial infection in mammals; however, the mechanisms involved in NETs formation and its role in anti-bacterial infection in teleost fish remains largely unknown. In this study, to explore the function of NETs in turbot, we established an in vitro bacterial infection model in head kidney derived neutrophils, and found that the haemolysin over-expressed Edwardsiella piscicida (ethA+) could induce a robust phenotype of NETs, compared with that in wild type or ethA mutant (ethA+ -ΔethA) strains. Besides, the NETosis was mediated by ethA+ -induced pyroptosis, and arms the ability of bacterial killing in neutrophils of turbot. Moreover, we found that neutrophils elastase (NE) might involves in this pyroptotic signaling, rather than inflammatory Smcaspase. Taken together, this study reveals the important role of pyroptosis in NETs formation in turbot neutrophils, suggesting that NETs formation is a critical immune response during bacterial infection in teleost fish.

Characterization and the potential immune role of class A scavenger receptor member 4 (SCARA4) in bacterial infection in turbot (Scophthalmus maximus L.).

The class A scavenger receptors play important roles in innate immunity and are distributed on plasma membrane of macrophages and other cell types. Notably, the class A scavenger receptor 4 (SCARA4) contains a typical C-type (calcium-dependent) lectin domain, which belongs to the collectin family of pattern recognition receptors and is involved in the immune response against infection. Here, one turbot SCARA4 gene was identified with a 2,292 bp open reading frame (ORF) encoding 763 amino acid residues. Multiple sequence analysis and phylogenetic analysis confirmed that SmSCARA4 gene was more close to that of P. olivaceus. Gene structure and syntenic analysis showed conserved exon/intron organization pattern and syntenic pattern across selected vertebrate species. Tissue distribution analysis showed SmSCARA4 was expressed in all the tested healthy tissues with the relative high expression levels in skin, gill and spleen. Following both E. tarda and V. anguillarum challenge in vivo, SmSCARA4 was significantly repressed in gill and intestine. Remarkably, SmSCARA4 showed the strongest binding ability to LPS and strongest upregulation in turbot head kidney macrophages in response to LPS. Knockdown and overexpression of SmSCARA4 revealed its interactions with the two pro-inflammatory cytokines, TNF-α and IL-1ß. Finally, repression of SmSCARA4 via combined treatment of LPS and overexpression of SmSCARA4 construct in turbot head kidney macrophages further indicated an inhibitory role of SmSCARA4 in LPS-stimulated inflammation. Taken together, turbot SmSCARA4 plays an important role in turbot immunity, especially in the mucosa-related systems; SmSCARA4 possesses strong binding specificity to LPS, and exerts protective roles in response to LPS infection by reducing the release of pro-inflammatory cytokines. The mechanisms of inhibitory role of SmSCARA4 in LPS-elicited inflammation await further investigation.

Genome-wide characterization of gap junction (connexins and pannexins) genes in turbot (Scophthalmus maximus L.): evolution and immune response following Vibrio anguillarum infection.

Gap junction (GJ), a special intercellular junction between different cell types, directly connects the cytoplasm of adjacent cells, allows various molecules, ions and electrical impulses to pass through the intercellular regulatory gate, and plays vital roles in response to bacterial infection. Up to date, the information about the GJ in turbot (Scophthalmus maximus L.) is still limited. In current study, 43 gap junction genes were identified in turbot, phylogeny analysis suggested that gap junctions from turbot and other species were clustered into six groups, GJA, GJB, GJC, GJD, GJE and PANX, and turbot GJs together with respective GJs from Japanese flounder, half-smooth tongue sole and large yellow croaker, sharing same ancestors. In addition, these 43 GJ genes distributed in different chromosomes unevenly. According to gene structure and domain analysis, these genes (in GJA-GJE group) were highly conserved in that most of them contain the transmembrane area, connexin domain (CNX) and cysteine-rich domain (connexin CCC), while PANXs contain Pfam Innexin. Although only one tandem duplication was identified in turbot gap junction gene, 235 pairs of segmental duplications were identified in the turbot genome. To further investigate their evolutionary relationships, Ka/Ks was calculated, and results showed that most ratios were lower than 1, indicating they had undergone negative selection. Finally, expression analysis showed that gap junction genes were widely distributed in turbot tissues and significantly regulated after Vibrio anguillarum infection. Taken together, our research could provide valuable information for further exploration of the function of gap junction genes in teleost.

A teleost interleukin-16 is implicated in peripheral blood leukocytes recruitment and anti-bacterial immunity.

Interleukin-16 (IL-16), as a lymphocyte chemoattractant cytokine, plays a crucial role in regulating cellular activities and anti-pathogen immunity. In teleost, the information about the antibacterial effect of IL-16 is scarce. In our study, we examined the immune functions of an IL-16 homologue (CsIL-16) from tongue sole Cynoglossus semilaevis. The CsIL-16 precursor (proCsIL-16) is comprised of 1181 amino acid residues, sharing 21.1%-67.3% identities with IL-16 precursor from invertebrate and vertebrate. The C-terminal proCsIL-16 containing two PDZ domains was designated as mature CsIL-16 which was released into the supernatant of peripheral blood leukocytes (PBLs). CsIL-16 was expressed in various tissues and regulated by bacterial invasion. Recombinant CsIL-16 (rCsIL-16), as a homodimer, was able to bind to the membrane of PBLs and played essential roles in regulating chemotaxis and activation of PBLs, which in vitro inhibited intracellular survival of E. tarda. Under in vivo condition, rCsIL-16 could dramatically regulate the induction of inflammatory genes, and suppress the bacterial dissemination in fish tissues. Collectively, our results reveal that CsIL-16 plays positive roles in antibacterial immunity, and provide insights into the immune function of CsIL-16.

Characterization of the turbot (Scophthalmus maximus) interleukin-18: Identification of splicing variants, phylogeny, synteny and expression analysis.

Interleukin-18 (IL-18) is a pro-inflammatory cytokine that belongs to the interleukin-1 (IL-1) family of cytokines. As occurs with IL-1ß, it is synthetized as an inactive precursor peptide that is mainly processed by the cysteine protease caspase-1 in the inflammasome complex. In mammals, and in collaboration with IL-12, it has been described as an important cytokine controlling the Th1-mediated immune responses through the induction of IFN-γ. Although its function in mammals is well stablished, the activity of this cytokine in teleost remains to be elucidated. This could be due, among other things, to the absence of this gene in the fish model species zebrafish, but also to its complex regulation. As it was observed for rainbow trout and human, il18 splicing variants were also found in turbot, which could represent a regulatory mechanism of its bioactivity. In the case of turbot, three splicing variants were observed (SV1-3), and one of them showed an insertion of 10 amino acids in the middle of the potential caspase-1 cleavage position, reflecting that this is probably a form resistant to the processing by the inflammasome. Phylogenetic and three-dimensional analyses of turbot Il18 revealed that it is relatively well-conserved in vertebrates, although only a partial conservation of the gene synteny was observed between fish and mammals. As it was expected, turbot il18 splicing variants were mainly expressed in immune tissues under healthy conditions, and their expression was induced by a bacterial challenge, although certain inhibitions were observed after viral and parasitic infections. In the case of the viral challenge, il18 downregulations did not seem to be due to the effect of type I IFNs.

Dietary bile acid supplementation reveals beneficial effects on intestinal healthy status of tongue sole (Cynoglossus semiliaevis).

The aim of this study was to investigate the effects of dietary bile acids (BAs) on intestinal healthy status of tongue sole in terms of immunity, antioxidant status, digestive ability, mucosal barrier-related genes expression and microbiota. Three experimental diets were prepared with BA levels at 0 mg/kg (CT), 300 mg/kg (BA1) and 900 mg/kg (BA2) in a commercial basal diet. Each diet was fed to three replicates with 120 fish (10.87 ± 0.32 g) in each tank. After an 8-week feeding trial, growth parameters were significantly enhanced in both BAs supplementary groups (P < 0.05), and compared with CT group, survival rate in BA2 group was significantly improved (P < 0.05). Intestinal lysozyme activity and contents of immunoglobulin M and complement 3 were significantly increased in both BAs supplementary groups (P < 0.05), suggesting an enhancement effect on the non-specific immune response. BAs inclusion also significantly improved intestinal antioxidant capabilities by increasing antioxidase activities and decreasing malondialdehyde levels. In addition, compared with CT group, intestinal digestive ability was substantially enhanced as indicated by the significantly increased lipase activity in BA2 group (P < 0.05) and significantly increased amylase activity in BA1 and BA2 groups (P < 0.05). Coincidentally, BAs inclusion significantly upregulated the relative expression of intestinal mucosal barrier-related genes (P < 0.05). Further, dietary BAs distinctly remodeled intestinal microbiota by decreased the abundance of some potential pathogenic bacteria. In conclusion, dietary BAs supplementation is an effective way to improve the intestinal healthy status of tongue sole.

Identification and characterization of a C-type lectin in turbot (Scophthalmus maximus) which functioning as a pattern recognition receptor that binds and agglutinates various bacteria.

C-type lectins (CTLs) are important pathogen pattern recognition receptors that recognize carbohydrate structures. In present study, a C-type lectin domain family 4 member E-like gene from turbot, which tentatively named SmCLEC4E-like (SmCLEC4EL), was identified, and the expressional and functional analyses were performed. In our results, SmCLEC4EL showed conserved synteny with CLEC4E-like genes from several fish species in genome, and possessed a typical type II transmembrane CTL architecture: an N-terminal intracellular region, a transmembrane domain and a C-terminal extracellular region which contained a predicted carbohydrate recognition domain (CRD). In addition, SmCLEC4EL exhibited the highest expression level in spleen in healthy fish, and showed significantly induced expression in mucosal tissues, intestine and skin, under bacteria challenge. Finally, the recombinant SmCLEC4EL protein combined with LPS, PGN, LTA and five different kinds of bacteria in a dose-dependent manner, and agglutinated these bacteria strains in the presence of calcium. These findings collectively demonstrated that SmCLEC4EL, a calcium-dependent CTL, could function as a pattern recognition receptor in pathogen recognition and participate in host anti-bacteria immunity.

Comparative analysis of the miRNA-mRNA regulation networks in turbot (Scophthalmus maximus L.) following Vibrio anguillarum infection.

MicroRNAs could not only regulate posttranscriptional silencing of target genes in eukaryotic organisms, but also have positive effect on their target genes as well. These microRNAs have been reported to be involved in mucosal immune responses to pathogen infection in teleost. Therefore, we constructed the immune-related miRNA-mRNA networks in turbot intestine following Vibrio anguillarum infection. In our results, 1550 differentially expressed (DE) genes and 167 DE miRNAs were identified. 113 DE miRNAs targeting 89 DE mRNAs related to immune response were used to construct miRNA-mRNA interaction networks. Functional analysis showed that target genes were associated with synthesis and degradation of ketone bodies, mucin type O-Glycan biosynthesis, homologous recombination, biotin metabolism, and intestinal immune network for IgA production that were equivalent to the function of IgT and IgM in fish intestine. Finally, 10 DE miRNAs and 7 DE mRNAs were selected for validating the accuracy of high-throughput sequencing results by qRT-PCR. The results of this study will provide valuable information for the elucidation of the regulation mechanisms of miRNA-mRNA interactions involved in disease resistance in teleost mucosal immune system.