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As one of short-chain fatty acids, butyrate is an important metabolite of dietary fiber by the fermentation of gut commensals. Our recent study uncovered that butyrate promoted IL-22 production in fish macrophages to augment the host defense. In the current study, we further explored the underlying signaling pathways in butyrate-induced IL-22 production in fish macrophages. Our results showed that butyrate augmented the IL-22 expression in head kidney macrophages (HKMs) of turbot through binding to G-protein receptor 41 (GPR41) and GPR43. Moreover, histone deacetylase 3 (HDAC3) inhibition apparently up-regulated the butyrate-enhanced IL-22 generation, indicating HDACs were engaged in butyrate-regulated IL-22 secretion. In addition, butyrate triggered the STAT3/HIF-1α signaling to elevate the IL-22 expression in HKMs. Importantly, the evidence in vitro and in vivo was provided that butyrate activated autophagy in fish macrophages via IL-22 signaling, which contributing to the elimination of invading bacteria. In conclusion, we clarified in the current study that butyrate induced STAT3/HIF-1α/IL-22 signaling pathway via GPCR binding and HDAC3 inhibition in fish macrophages to activate autophagy that was involved in pathogen clearance in fish macrophages., Competing Interests: Conflict of interest disclosure The authors have no relevant financial or non-financial interests to disclose., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Recently, Vibrio anguillarum, a Gram-negative pathogenic bacterium, has been becoming a major constraint on the development of the turbot aquaculture industry because of its characteristics of worldwide distribution, broad host range and potentially devastating impacts. Although the functions of protein-coding mRNAs in the immune response against bacterial infection have been reported, as well as several non-coding RNAs (ncRNAs), such as circular RNAs (circRNAs) and microRNAs (miRNAs), the relationships between mRNAs and ncRNAs in the immune system of turbot liver are still limited during bacterial infection. In present study, the comprehensive analyses of whole-transcriptome sequencing were conducted in turbot liver infected by V. anguillarum. The differential expression was analyzed in the data of circRNAs, miRNAs, and mRNAs. The interactions of miRNA-circRNA pairs and miRNA-mRNA pairs were predicted basing on the negative regulatory relationships between miRNAs and their target circRNAs\mRNAs. The circRNA-related ceRNA regulatory networks were constructed for the analyses of regulated mechanism in turbot immune system. Subsequently, the RT-qPCR was carried out to verify the results of sequencing. Finally, we identified 31 circRNAs, 53 miRNAs and 948 mRNAs with differential expression. Gene set enrichment analyses using Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways showed that innate immunity was principally activated at the early stages of infection, while adaptive immunity was activated after 24 h. Finally, 65 circRNA-miRNA-mRNA pathways were constructed, based on the hypothesis of ceRNA regulatory networks. In conclusion, our findings provide new insights on the underlying immune response to bacterial infection and identify novel target genes for the prevention and control of disease in turbot., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

It has been known that vitamin D 3 (VD 3 ) not only plays an important role in regulating calcium and phosphorus metabolism in animals, but also has extensive effects on immune functions. In this study, the mechanism how VD 3 influences bactericidal ability in turbot was explored. The transcriptomic analysis identified that dietary VD 3 significantly upregulated the gene expression of C-type lectin receptors (CLRs), including mannose receptors (mrc1, mrc2, pla2r1) and collectins (collectin 11 and collectin 12) in turbot intestine. Further results obtained from in vitro experiments confirmed that the gene expression of mannose receptors and collectins in head-kidney macrophages (HKMs) of turbot was induced after the cells were incubated with different concentrations of VD 3 (0, 1, 10 nM) or 1,25(OH) 2 D 3 (0, 10, 100 pM). Meanwhile, both phagocytosis and bactericidal functions of HKMs were significantly improved in VD 3 or 1,25(OH) 2 D 3 -incubated HKMs. Furthermore, phagocytosis and bacterial killing of HKMs decreased after collectin 11 was knocked down. Moreover, VD 3 -enhanced antibacterial activities diminished in collectin 11-interfered cells. Interestingly, the evidence was provided in the present study that inactive VD 3 could be metabolized into active 1,25(OH) 2 D 3 via hydroxylases encoded by cyp27a1 and cyp27b1 in fish macrophages. In conclusion, VD 3 could be metabolized to 1,25(OH) 2 D 3 in HKMs, which promoted the expression of CLRs in macrophages, leading to enhanced bacterial clearance., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Galectins, a family of evolutionary conserved β-galactoside-binding proteins, have been characterized in a wide range of species. Many reports have indicated vital roles of galectins in innate immunity, especially in the mucosal tissues against infection. However, the systematic identification of galectin gene family is still lacking in teleost. Here, we characterized the galectin gene family and investigated their expression profiles post bacterial challenge in turbot (Scophthalmus maximus L.). In this study, a total of 13 galectin genes were characterized in turbot, phylogenetic analyses revealed their strong relationships to half smooth tongue sole and puffer fish, and syntenic analyses confirmed the orthology suggested by the phylogenetic analysis. In addition, the copy number of galectin genes is similar across a broad spectrum of species from fish to amphibians, birds, and mammals, ranging from 8 to 16 genes. Furthermore, the galectin genes were widely expressed in all the examined turbot tissues, and most of the galectin genes were strongly expressed in mucosal tissues (skin, gill and intestine). Moreover, majority of the galectin genes were significantly regulated after Vibrio anguillarum infection in the intestine, gill and skin, suggesting that galectins were involved in the mucosal immune response to V. anguillarum infection in turbot. In addition, subcellular localization analysis showed lgals3a was distributed in the cytoplasm and nucleus. However, the knowledge of galectins are still limited in teleost species, further studies should be carried out to better characterize its detailed roles in teleost mucosal immunity., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Extracellular ATP (eATP) is a potent singling molecule in activation of fish innate immunity while the molecular determinants for eATP release in fish were not completely understood. Connexin32 (Cx32) is a member of gap junction protein family that plays important immunological functions in mammals. However, the immune relevance of Cx32 and its role in ATP release in fish has not been investigated. Here, we identified, characterized three Cx32 isoform genes (Cx32.2, Cx32.2x and Cx32.7) from the Japanese flounder Paralichthys olivaceus, and investigated their role in inflammation-induced ATP release in fish. Expression analysis revealed that even though all the three Cx32 genes are constitutively expressed in all examined Japanese flounder tissues, Cx32.2 and Cx32.2x are dominantly expressed in liver, and Cx32.7 is highly expressed in intestine and head kidney macrophages. In addition, we showed that gene expression of all the three Cx32 isoforms was modulated by cAMP stimulation and inflammatory challenges. Furthermore, we revealed that Cx32 expression was upregulated in TNF-alpha overexpressed Japanese flounder FG-9307 cells. Moreover, overexpression of the three Cx32 isoforms significantly reduced the gene expression level of LPS-induced pro-inflammatory cytokine IL-8 and TNF-alpha, indicating that Cx32 is involved in modulating inflammatory response in fish. Finally, we showed that inflammation-induced ATP release was significantly increased in Cx32-overexpressed Japanese flounder FG-9307 cells, and this increased ATP release could be attenuated by pre-incubation with gap junction protein blocker carbenoxolone. Taken together, we for the first time reported the involvement of Cx32 in fish immunity. Our findings suggested that in addition to Cx43 and pannexin1 channels, Cx32 also plays a role in inflammation-induced ATP release in fish., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Paralichthys olivaceus (mean weight, 280.1 ± 10.5 g; mean length, 28.37 ± 2.3 cm) was reared in bio-floc and seawater for 6 months to determine the toxic effects of waterborne nitrite exposure (0, 25, 50, 100, and 200 mg/L) for 1 week, compared to those observed with bio-floc and seawater only. The effects on antioxidant activity, immune responses, and acetylcholinesterase activity were measured. Following nitrite exposure, superoxide dismutase activity in the liver and gills was significantly elevated and catalase activity was significantly increased, except for in the gills of P. olivaceus reared in bio-floc. Further, glutathione S-transferase activity was significantly elevated in the liver and gills, and glutathione was significantly lower. Meanwhile, acetylcholinesterase activity in the liver and gills was significantly inhibited and plasma lysozyme activity and immunoglobulin M were considerably elevated., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

High levels of soybean meal (SBM) in aquafeed leads to detrimental inflammatory response and oxidative stress in fish. In the present study, fermentation with Aspergillus awamori was conducted to explore the potential effects on improving the nutritional quality of soybean meal and the health status of turbot. A 63-day feeding trial (initial weight 8.53 ± 0.11 g) was carried out to evaluate the utilization of fermented soybean meal (FSM) by juvenile turbot. 0% (FM, control), 30% (S30, F30), 45% (S45, F45), and 60% (S60, F60) of fish meal were replaced with SBM or FSM, respectively. As the results showed, fermentation significantly reduced the contents of anti-nutritional factors in SBM, including raffinose (-98.8%), glycinin (-98.5%), β-conglycinin (-97.4%), trypsin inhibitors (-80%) and stachyose (-80%). A depression of fish growth performance and activities of superoxide dismutase and lysozyme were observed in S45 and S60 groups, while these inferiorities were only observed in F60 group. Meanwhile, fermentation also improved the heights of enterocytes and microvillus significantly in the F45 and F60 groups compared with those in SBM. An induced expression of anti-inflammatory cytokine transforming growth factor-β and depression of pro-inflammatory cytokines tumor necrosis factor-α and interleukin-1β in the distal intestine were observed in the F45 and F60 groups. Taken together, this study indicated that fermentation with Aspergillus awamori could improve the replacement level with soybean meal from 30% to 45% in turbot., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

A 12-week feeding trial was conducted to evaluate the effects of multi-strain probiotics (MSP) in a low fish meal (FM) diet on overall performance, gut microbiota, selected non-specific immune responses and antioxidant enzyme activities of olive flounder (Paralichthys olivaceus) juveniles. A total of 225 healthy olive flounders (initial mean body weight, 13.5 ± 0.01 g) were randomly separated into 3 groups of 75 fish, each group having three replicates of 25 fish; first group was fed with a FM-based control diet (Con), 2nd group was fed with a low-FM diet containing a blend of plant and animal protein meals replacing 30% of the FM protein (FM30), and 3rd group was fed with the FM30 diet supplemented with 10 8 -10 9  CFU kg -1 of the MSP (Pro). With the exception of lipid retention, which was significantly lower in fish fed the FM30 diet compared to the other two treatments, no other statistically significant differences were recorded with respect to any of the other growth and nutrient utilization parameters. Myeloperoxidase and lysozyme activities of fish fed the Pro diet were much higher and significantly different than those of fish fed the FM30 diet. Glutathione peroxidase activity was significantly higher in Pro- than in Con-fed fish, which, in turn, was significantly higher than FM30-fed fish. Expression of immune-related genes including IL-1β, IL-6, and TNF-α was markedly upregulated in livers of the fish fed Pro diet compared to those fed the Con and FM30 diets. Furthermore, supplementation of MSP in FM30 diet enriched the Lactobacillus abundance in the fish gut as well as predictive gene functions in relation to lipid and carbohydrate metabolisms. These data suggested that the MSP could reduce the potential adverse effects of the low-FM diet and might be used as a healthy immunostimulant for olive flounder., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In this study, potential immunological and hematological effects of different concentrations (0, 1, 10, and 50 μg L -l ) of waterborne zinc pyrithione (ZnPT) were studied in the blood of the olive flounder Paralichthys olivaceus over 30 days. Reduced alternative complement activity (ACH50) and lysozyme activity were measured in fish exposed to 10 and/or 50 μg L -l of ZnPT for 20 days. Decreased levels of total Ig were also observed in response to 10 and/or 50 μg L -l ZnPT during the exposure period. Levels of cortisol, a marker of stress, were significantly increased by 10 and 50 μg L -l ZnPT from day 10, and by 1 μg L -l exposure on day 30. The levels of red blood cells (RBCs) and white blood cells (WBCs) decreased following exposure to 10 and/or 50 μg L -l ZnPT, while no significant change was observed in hemoglobin level. Concentrations of total protein and albumin were significantly reduced with 50 μg L -l ZnPT at day 20. Alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase activities were significantly increased following exposure to 10 and/or 50 μg L -l ZnPT. Lipid peroxidation was induced by ZnPT, and higher concentrations (10 and 50 μg L -l ) significantly increased intracellular malondialdehyde levels during exposure. Regarding the subsequent antioxidant response, intracellular glutathione levels increased significantly in response to 10 and 50 μg L -l ZnPT on days 20 and 30. Similarly, catalase and superoxide dismutase activity was significantly increased in response to 10 and 50 μg L -l ZnPT after day 10. Taken together, changes in the studied parameters suggested the immunotoxicity of ZnPT, with modulations observed in hematological homeostasis and oxidative stress induction in the blood of olive flounder., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Adding immunopotentiators to plant protein based diets has been a feasible way to improve fish growth performance and healthy status. In this study, an 8-week trial was carried out to explore the effects of resveratrol, a natural polyphenolic compound, on growth performance, anti-oxidative capacity and immune responses in turbot fed soybean meal based diet. As the results showed, replacement 45% fish meal with soybean meal (SBM) significantly depressed the fish growth, feed utilization and the heights of villi and microvilli in distal intestine. The mRNA levels of hepatic antioxidant enzymes, including superoxide dismutase (sod), glutathione peroxidase (gsh-px) and peroxiredoxin 6 (prx 6), were highly inhibited in SBM group. The inflammation related genes in intestine were also responsive to soybean meal. Supplying resveratrol showed no effects on fish growth performance but significantly restored the intestinal morphology and improved the mRNA levels of hepatic antioxidant enzymes as well as the activity of SOD. Meanwhile, resveratrol significantly improved the mRNA levels of anti-inflammatory cytokine transforming growth factor-β and inhibited the expression of pro-inflammatory cytokines tumor necrosis factor-α (tnf-ɑ), interleukin-1β (il-1β) and interleukin-8 (il-8). The results indicate that resveratrol could attenuate the oxidative stress and inflammatory response induced by soybean meal in turbot. This study shows resveratrol is an effective immunopotentiator to carnivorous fishes fed plant protein sources., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Light is a key environmental factor that synchronizes various life stages from embryo development to sexual maturation in fish. For turbot, light spectra have the most influence at the larval and juvenile stages. In the current study, differences in the development of embryos and the performance of newly hatched turbot larvae exposed to five different spectra: full spectrum (LDF), blue (LDB, peak at 450 nm), green (LDG, peak at 533 nm), orange (LDO, peak at 595 nm) and red (LDR, peak at 629 nm), were examined. At 62.8 h post fertilization, a higher number of embryos exposed to short-wavelengths (LDG and LDB) had developed a heartbeat in comparison with embryos exposed to other wavelengths. Larvae exposed to the green spectrum had higher malformation rates than larvae exposed to the other spectra, indicating that larvae exposed to green light may have significantly reduced survival rates. The results of non-specific immunity parameters showed that the mRNA expression levels of cathepsin D (CTSD), cathepsin F (CTSF), catalase (CAT) and metallothionein (MT) in larvae exposed to LDB were significantly higher than those exposed to other spectra, but CAT activity in larvae exposed to LDB was significantly lower than larvae exposed to the other spectra. There was no significant difference in MT activity in larvae exposed to the five different spectra. The mRNA expression level of lysozyme (LZM) in larvae exposed to LDR was significantly higher than other spectra, while there was no significant difference in LZM activity observed in larvae exposed to LDR, LDG, LDB and LDF. The difference of the enzyme activity of total superoxide dismutase (T-SOD) was not significant among larvae exposed to the five spectra. mRNA expression of the heat shock protein 70 (HSP70) was significantly higher in newly hatched larvae exposed to LDB, LDR and LDG, indicating that larvae exposed to LDB, LDG and LDR exhibited a stress response. The mRNA expression level of the insulin-like growth factor-1 (IGF-1) and growth parameters in the newly hatched larvae exposed to the different spectra were not significantly different. The results of the present study indicate that LDO and LDF should be used for embryo incubation and newly hatched larvae when rearing turbot. This study provides a theoretical basis for optimizing the incubation light environment for fertilized turbot eggs, promoting immunity and reducing stress responses in newly hatched larvae., (Copyright © 2019. Published by Elsevier Ltd.)

To study the effect of Edwardsiella tarda infection on miRNAs expression profile in Japanese flounder, fish were injected intraperitoneally with E. tarda. The miRNAs involved in regulating immune responses were analyzed by high-throughput sequencing. A total of 164 mature miRNAs were identified, of which 17 miRNAs were differentially expressed (DE miRNAs) after E. tarda infection, indicating that they were immune-related miRNAs. To further examine the relationship between the miRNAs and their predicted target mRNAs, a total of 22 predicted target mRNAs, mainly related to endocytic signaling pathway, NF-κB signaling pathway, and p53 signaling pathway, were detected with miRNA mimics in HEK-293T cells by dual-luciferase reporter experiments. Finally, we confirmed that insulin receptor substrate-2 (IRS2a and IRS2b) were regulated by miR-7a. And the target sites of the 3' untranslated region (UTR) of IRS2a and IRS2b were verified by dual-luciferase reporter experiments. Furthermore, we found that the E. tarda and LPS significantly increased host miR-7a expression. In vivo and in vitro studies revealed that IRS2-mediated PI3K/AKT/GSK3β signaling pathway was suppressed. Taken together, these results implied that miR-7a might be a key regulator of PI3K/AKT/GSK3β signaling pathway via suppressing the IRS2a and IRS2b genes., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The receptor responsible for maternofetal transmission of immunoglobulin (Igs) in the teleosts is not clear. Polymeric immunoglobulin receptor (pIgR) specifically binds with IgA and IgM and mediates the transcytosis of intracellular polymeric immunoglobulins (pIgs) at the mucosal surface to protect against pathogens. Hence there is a possibility that it may be involved in the transmission of maternal Igs. The aim of the present study was to detect the expression and localization of pIgR during embryonal development in turbot (Scophthalmus maximus). pIgR gene was first cloned from eggs and embryos of turbot with or without parent immunization. The expression and distribution of pIgR in unfertilized egg and in embryos ranging from day 1 to day 5 after fertilization were analyzed using reverse transcriptase quantitative polymerase chain reaction and in situ hybridization. pIgR gene was detected in all eggs and embryos at different stages of development, with the highest level detected on the 5th day. pIgR mRNA was observed to be first located in the whole blastoderm and enveloped the yolk sac. Later, it was located around entoderm including primary digestive tract and pronephric tubule tract, and finally it was located at the joint of abdomen and vitelline membrane. Then, Eukaryotic expression plasmid carrying pIgR gene was constructed and transfected into HEK293T cells. Results showed mature pIgR protein located on the cellular membrane, and could bound IgM in vitro. Our findings provide information for studying the involvement of pIgR in maternal Igs transportation in turbot., (Copyright © 2019. Published by Elsevier Ltd.)

Bacillus sp. SJ-10 (BSJ-10) was identified from traditional Korean fermented fish, the previously recognized prebiotic β-glucooligosaccharides (BGO), and their combination as a synbiotic were prepared to evaluate their individual and synergistic effects in olive flounder (Paralichthys olivaceus). Four diets (one control and three treatments) were formulated containing neither BSJ-10 nor BGO (control), 1 × 10 8  CFU g -1 BSJ-10 (BSJ-10), 0.1% BGO (BGO), and 1 × 10 8  CFU g -1 BSJ-10 + 0.1% BGO (BSJ-10 + BGO). Triplicates of 15 fish (weight 10 ± 0.25 g) were randomly allocated to the four diet groups and fed one of the diets for 8 weeks. At the end of the experiment, fish weight gain (WG), specific growth rate (SGR), feed conversion ratio, and protein efficiency ratio in BSJ-10, BGO and BSJ-10 + BGO diets were positively modulated (P < 0.05) compared with control. Specially, WG and SGR were significantly (P < 0.05) higher in BSJ-10 + BGO than that of BSJ-10 and BGO (individual component). The innate immune parameters such as respiratory burst, superoxide dismutase, and lysozyme activity (LSZ) of fish fed BSJ-10 and BSJ-10 + BGO (both groups) were significantly (P < 0.05) higher than the control. Moreover, myeloperoxidase activity (MPO) and LSZ of fish fed BSJ-10 + BGO were significantly higher compared with individual component. Compared with control, intestinal BSJ-10 content, expression of interleukin (IL)-1β in liver and kidney, and tumor necrosis factor (TNF)-α in liver were higher in both groups, but microvillus length was increased (P < 0.05) only in BSJ-10 + BGO. During in vivo challenge experiment with Streptococcus iniae (1 × 10 8  CFU ml -1 ), survival rate of fish was significantly higher in all treatment groups versus control. Moreover, in BSJ-10 + BGO, protection against S. iniae infection and transcription of TNF-α and IL-6 in gill were significantly (P < 0.05) higher than the individual component. Collectively, an improved WG, SGR, MPO, LSZ, transcription of IL-6 and TNF-α, and cumulative survival rate against streptococcosis clearly demonstrates a synergistic outcome of diet BSJ-10 + BGO as synbiotic in olive flounder., (Copyright © 2018. Published by Elsevier Ltd.)

Viral hemorrhagic septicemia virus (VHSV) is one of the most serious viral pathogen that infects farmed fish. In this study, we measured the replication of VHSV increased steadily at 9, 24, 72, and 120 h after infection and progression of necrosis was observed at 72 hpi. We performed transcriptomic and metabolomics profiling of kidney tissues collected at each infection time using Illumina HiSeq2000 and ultra-performance liquid chromatography/quadrupole time-of-flight mass spectroscopy to investigate the mechanisms of VHSV infection in the kidneys of olive flounder. A total of 13,862 mRNA molecules and 72 metabolites were selected to identify the mechanisms of infection and they were integrated using KEGG pathway database. Six KEGG metabolic pathways, including carbohydrate metabolism, amino acid metabolism, lipid metabolism, transport and catabolism, metabolism of cofactors and vitamins, and energy metabolism, were significantly suppressed, whereas the immune system was activated by VHSV infection. A decrease in levels of amino acids such as valine, leucine, and isoleucine, as well as in their derivative carnitines, was observed after VHSV infection. In addition, an increase in arachidonic acid level was noted. Integrated analysis of transcriptome and metabolome using KEGG pathway database revealed four types of responses in the kidneys of olive flounder to VHSV infection. Among these, the mechanisms related to the immune system and protein synthesis were activated, whereas ATP synthesis and the antioxidant system activity were suppressed. This is the first study describing the mechanisms of metabolic responses to VHSV infection in olive flounder. The results suggest that the suppression of ATP synthesis and antioxidant systems, such as glutathione and peroxisome signaling, could be the cause of necrosis, whereas the activation of the immune system could result in the inflammation of kidney tissue in VHSV-infected olive flounder., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

In this study, we investigated the immunostimulatory and protective effects of CpG motif oligonucleotides (CpG-ODNs) against Edwardsiella tarda infection in olive flounder (Paralichthys olivaceus). Groups of fish injected with CpG-ODNs (1585, 1668, and 2007) or PBS (control) showed varying mortality rates in response to challenge with E. tarda. The survival rates of fish treated with CpG-ODN 1668 and 2007, which belonged to the same class type B, were 45% and 60%, respectively, with CpG-ODN 2007 showing the highest survival rate. Further analysis showed that the respiratory burst and bactericidal activities induced by CpG-ODN 2007 were higher than those in the control group (induced by non-CpG-ODNs) or in the group of fish induced by CpG-ODN 1585, which belonged to class type A. Additionally, the respiratory burst activity induced by CpG-ODN 2007 was higher than that induced by CpG-ODN 1668, despite similar bactericidal activity titers. In vivo experiments showed that CpG-ODN 2007 stimulation resulted in higher survival rates than CpG-ODN 1668 stimulation, possibly owing to differences in respiratory burst activity. In summary, we demonstrated that differences in CpG-motif or class type altered respiratory burst and bactericidal activities, resulting in differences in survival rates against E. tarda challenge in the olive flounder. Therefore, it is necessary to use CpG-ODNs optimized against E. tarda infection in olive flounder, because different CpG motifs belonging to the same class type have different effects., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

This study was conducted to examine the effects of vitamin E on growth performance, oxidative stress and non-specific immunity of turbot (Scophthalmus maximus) fed with high-fat diet. Results showed that high-fat diet significantly increased hepatosomatic index, viscerosomatic index, hepatic malondialdehyde level and decreased catalase and superoxide dismutase activities, whereas final weight, specific growth rate and survival rate remained unchanged. Meanwhile, nitro blue tetrazolium positive leucocytes of head kidney, respiratory burst activity in head-kidney macrophage, phagocytic index and serum lysozyme activity were significantly reduced after feeding with high-fat diet. Furthermore, fish fed with high-fat diet promoted higher expression of heat shock protein (hsp70, hsp90), and inhibited expression of complement component 3 (c3) in the liver and tumor necrosis factor-α (tnf-α), interleukine 1β (il-1β), toll like receptor 22 (tlr-22) in the spleen and head-kidney, respectively. However, simultaneous supplementation with 480 mg kg -1 vitamin E protected turbot against high-fat diet-induced hepatic oxidative stress, hypoimmunity through attenuating lipid peroxidation, renewing antioxidant enzymes activities and nonspecific immune responses, and modulating the expression of stress protein (hsp70, hsp90) and immune-related genes (c3, tnf-α, il-1β, tlr-22). In conclusion, the obtained results indicate the vitamin E as a wildly used functional feed additive contributes potentially to alleviate high-fat diet-induced hepatic oxidative stress and hypoimmunity, maintain the health, and improve the broodstock management for turbot., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Two novel type I interferon genes (JfIFN3 and JfIFN4) have been identified in Japanese flounder Paralichthys olivaceus. Open reading frames of JfIFN3 and JfIFN4 were 555bp and 528bp, encoding 184aa and 175aa, respectively. The genomic structures of JfIFN3 and JfIFN4 are composed of 5 exons and 4 introns. JfIFN4 has 2 conserved cysteine residues, while JfIFN3 has 4. JfIFN3 and JfIFN4 showed the highest amino acid sequence identities to turbot IFN1 (74%) and IFN2 (62%), respectively. Interestingly, JfIFN3 and JfIFN4 were clustered in distinct branches with JfIFN1 and JfIFN2, which have reported so far. The mRNA levels of JfIFN4 were apparently increased in the kidney and spleen at 3 h after ployI:C injection, while JfIFN1-3 were not detected by RT-PCR., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Vitronectin (Vtn) is a multifunctional protein that plays significant roles in cell adhesion, migration, spreading and survival, and in the regulation of membrane attack complex formation and the terminal pathway of complement activation in innate immune response. However, the expression and immune significance of Vtn in fish remains largely unknown. In order to understand the involvement of Vtn in fish innate immune response, here we cloned and characterized a full-length Vtn ortholog cDNA, termed PoVtn, from Japanese flounder Paralichthys olivaceus. The deduced PoVtn protein is comprised of 438 amino acids with a 19-amino-acid signal peptide sequence ( 1 Met- 19 Ala) at the N-terminus. Protein domain analysis revealed that PoVtn possesses a conserved N-terminal somatomedin B domain followed by a conserved RGD motif and four haemopexin-like domains. Sequence analysis revealed that PoVtn has two potential glycosylation sites and shares 44-74% sequence identity with other teleost Vtn proteins. PoVtn mRNA was ubiquitously distributed in all examined normal tissues and showed the highest expression in Japanese flounder hepatopancreas tissue. PoVtn expression was induced by LPS and poly(I:C) challenges in the Japanese flounder head kidney macrophages (HKMs) and peripheral blood leucocytes (PBLs) and shows a pathogen-associated molecular pattern- and cell type-dependent manner. The expression of PoVtn was also modulated by bacterial challenge with Edwardsiella tarda in Japanese flounder immune-related tissues including head kidney, gill and spleen. Furthermore, overexpression of PoVtn in Japanese flounder FG-9307 cells significantly attenuated the LPS- and poly(I:C)-induced proinflammatory cytokines IL-1beta and TNF-alpha gene expression. Taken our findings together, we for the first time characterized Vtn gene expression in response to inflammatory stimuli in fish. Our results suggested a potential role of PoVtn in regulating fish innate immunity., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Immunoglobulin D (IgD) is considered to be an enigmatic Ig molecule because of the lack understanding of its immunological functions. In the present study, a partial δ region of the flounder IgD was recombinantly expressed, purified and used as an immunogen to produce monoclonal antibodies (MAbs) against the H chain of flounder IgD. After fusion, a total of 97 hybridomas were generated and observed under an inverted microscope One of the hybridomas, designated 5G7, gave strong positive results in an indirect enzyme-linked immunosorbent assay (ELISA) and was cloned and subcloned by limiting dilution. Western blot analysis showed that MAb 5G7 could specifically recognize a 118 kDa protein from peripheral blood lymphocytes (PBLs), which was identified to be the H chain of flounder IgD by mass spectrometric analysis. Indirect immunofluorescence assay tests (IIFAT) showed that specific fluorescence signals were observed on the membranes of the PBLs, which suggests that MAb 5G7 could recognize the membrane-bound IgD molecule. Moreover, only the subset of IgD+/IgM + B cells were observed in the PBLs of healthy flounder when tested by flow cytometry analysis. Consistent with the results of flow cytometry, a double immunofluorescence assay test (DIFAT) showed that the positive lymphocytes were stained with both green and red fluorescence signals, which represent the IgM+/IgD + lymphocytes subset. These results demonstrate that the produced MAb 5G7 could specifically recognize the flounder IgD, which provides a useful tool to study the functions of flounder IgD., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Suppressor of cytokine signaling (SOCS) family members are key regulators of the immune system, particularly cytokine action, and have now been discovered in a number of fish species. Here we identified eight SOCS proteins (CISH, SOCS1a, SOCS1b, SOCS3a, SOCS3b, SOCS5, SOCS6 and SOCS9) in the Japanese flounder and analyzed their mRNA expressions after injection of poly (I:C) and formalin-killed cells (FKC) of Edwardsiella tarda. The expressions of all eight SOCS genes were detected in all the tissues examined. Stimulation of Japanese flounder reared at 15 or 25 °C with poly (I:C) affected the gene expressions of CISH, SOCS1a, SOCS1b and SOCS3a. All SOCS genes mRNA levels were significantly changed after FKC injection. Significant up-regulation of SOCS1a, SOCS1b, SOCS3a and SOCS3b genes was detected at 3, 12 and 24 hpi. SOCS5 and SOCS6 genes were significantly down-regulated at 3 hpi. SOCS9 gene was significantly up-regulated at 12 hpi. These results suggest that all eight of the SOCS genes are involved in immune responses, and that the CISH, SOCS1 and SOCS3 genes have functions distinct from those of the other SOCS members., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Apoptosis-associated speck-like protein containing a CARD domain (ASC) is a critical adaptor molecule in multiple inflammasome protein complexes that mediate inflammation and host defense. However, few studies have been performed in lower vertebrates such as in teleost. Here we identified and characterized a novel ASC gene (namely PoASC) from Japanese flounder Paralichthys olivaceus. The complete cDNA sequence of PoASC contains a 22 bp 5'-untranslated sequence, a 612 bp open reading frame, and a 438 bp 3'-untranslated sequence. The deduced PoASC protein is comprised of 203 amino acids with a conserved N-terminal PYD domain and a C-terminal CARD domain and shows 35-62% sequence identity with other vertebrate ASC proteins. PoASC mRNA transcripts was detected in various Japanese flounder tissues and is dominantly expressed in hepatopancreas. Oligomeric speck-like structures were observed when PoASC was exogenously expressed in Japanese flounder FG-9307 cells. Immune challenge experiments revealed that PoASC gene expression was significantly induced in the Japanese flounder head kidney macrophages and peripheral blood leukocytes by the canonical TLR ligands LPS, Poly(I:C) and zymosan stimulations. In addition, the induction of PoASC was also observed in Edwardsiella tarda challenged head kidney and gill tissues. Furthermore, we for the first time showed that extracellular ATP, an important signaling molecule in triggering innate immune response and activation of NLR inflammasome, significantly up-regulates PoASC expression in the Japanese flounder head kidney macrophages in a dose-dependent manner. Together, these findings addressed the involvement of PoASC in TLR and extracellular ATP-mediated innate immune signaling in the Japanese flounders., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

CCR6 is an important binding receptor of CCL20 and beta-defensins, and has multiple functions in the innate and acquired immune responses. In this study, we cloned the PoCCR6A and PoCCR6B genes of the Japanese flounder and studied the gene structure and expression patterns of these two genes in bacterial infection. The full-length PoCCR6A cDNA is 1415 bp and the open reading frame (ORF) is 1113 bp, encoding a 370-amino-acid peptide. The full-length PoCCR6B cDNA is 2193 bp and the ORF is 1029 bp, encoding a 363-amino-acid peptide. The structures of PoCCR6A and PoCCR6B indicate that they are single-exon genes. The predicted proteins encoded by PoCCR6A and PoCCR6B have the typical G-protein-coupled receptor (GPCR) family signature of seven transmembrane domains and several conserved structural features. A tissue distribution analysis showed that PoCCR6A is predominately expressed in the intestine, gill, and blood, and PoCCR6B in the gill, spleen, and liver. The expression patterns of the two chemokine receptors were analyzed during bacterial infection. In spleen and kidney, the expression of PoCCR6A was significantly upregulated at 24 h after infection, whereas the expression of PoCCR6B was steady at these time points. While in intestine, both of them were upregulated at 6 h-12 h after infection, and in gill the expression levels of them were upregulated at 24 h. The patterns of expression suggested that PoCCR6A and PoCCR6B play an important role in the immune response of the Japanese flounder, especially in the mucosal tissues., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The mRNA level of the endonuclease domain-containing 1 gene (Jf&#95;ENDOD1) in Japanese flounder Paralichthys olivaceus kidney was significantly increased after injection of formalin-killed bacteria cells (FKC) in the previous microarray study. ENDOD1 is a member of the DNA/RNA non-specific nucleases family, and its role in fish immunity has not been reported. The open reading frame of Jf&#95;ENDOD1 cDNA was 912 bp, encoding 303 amino acids. The first 27 amino acids were predicted to be a signal peptide and the mature Jf&#95;ENDOD1 was calculated as 32 kDa. The amino acid sequence of Jf&#95;ENDOD1 showed 76% identity to that of large yellow croaker Larimichthys crocea. Transcripts of Jf&#95;ENDOD1 were marginally detected in all sampled tissues from healthy fish, while they were significantly detected in brain, kidney, spleen and intestine at 6 h post FKC injection. Jf&#95;ENDOD1 recombinant protein produced in Escherichia coli showed DNase activity. Furthermore, to evaluate the DNase activities in vivo, total proteins from Japanese flounder kidney and spleen were extracted at 12, 24 and 72 h post Edwardsiella tarda FKC injection. The DNase activity of extracted protein was higher in treated fish than in untreated fish. Since the mRNA levels were significantly up-regulated after the FKC treatment, Jf&#95;ENDOD1 might be responsible for the activities., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Reflecting the natural biology of mass spawning fish aquaculture production of fish larvae is often hampered by high and unpredictable mortality rates. The present study aimed to enhance larval performance and immunity via the oral administration of an immunomodulator, β-glucan (MacroGard(®)) in turbot (Scophthalmus maximus). Rotifers (Brachionus plicatilis) were incubated with or without yeast β-1,3/1,6-glucan in form of MacroGard(®) at a concentration of 0.5 g/L. Rotifers were fed to first feeding turbot larvae once a day. From day 13 dph onwards all tanks were additionally fed untreated Artemia sp. nauplii (1 nauplius ml/L). Daily mortality was monitored and larvae were sampled at 11 and 24 dph for expression of 30 genes, microbiota analysis, trypsin activity and size measurements. Along with the feeding of β-glucan daily mortality was significantly reduced by ca. 15% and an alteration of the larval microbiota was observed. At 11 dph gene expression of trypsin and chymotrypsin was elevated in the MacroGard(®) fed fish, which resulted in heightened tryptic enzyme activity. No effect on genes encoding antioxidative proteins was observed, whilst the immune response was clearly modulated by β-glucan. At 11 dph complement component c3 was elevated whilst cytokines, antimicrobial peptides, toll like receptor 3 and heat shock protein 70 were not affected. At the later time point (24 dph) an anti-inflammatory effect in form of a down-regulation of hsp 70, tnf-α and il-1β was observed. We conclude that the administration of MacroGard(®) induced an immunomodulatory response and could be used as an effective measure to increase survival in rearing of turbot., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Nascent polypeptide-associated complex (NAC) is a conserved heterodimeric protein consisting of alpha and beta subunits. In addition to acting as a protein translation chaperone by forming a heterodimer with the beta subunit, NAC alpha (NACA) also shows important immune significance independent of NAC beta in mammalian cells. In lower vertebrates, however, the immunological relevance of NACA has not been revealed yet. In the present study, we identified and characterized a NACA gene (termed poNACA) involved in innate immune response in Japanese flounder Paralichthys olivaceus. poNACA encodes a 215-amino-acid protein, with an apparent molecular weight of 23.5 kDa and an isoelectric point of 4.51. Tissue distribution analysis revealed that poNACA gene was constitutively expressed in all examined tissues and showed dominant expression in hepatopancreas and gonad tissues. In enriched Japanese flounder head kidney macrophages and peripheral blood leucocytes, the expression of poNACA mRNA transcript was significantly induced by LPS, Poly(I:C) and zymosan stimulations. In vivo experiments further revealed that poNACA gene expression was up-regulated in head kidney, gill and spleen tissues in response to Edwardsiella tarda challenges. Furthermore, overexpression of poNACA in Japanese flounder FG-9307 cells resulted in increased gene expression of IL-1beta, IL-11 and TNF-alpha, and myxovirus resistance (Mx). Taken together, our findings indicate that an immune response gene, poNACA, involved in innate immune regulation in P. olivaceus has been identified., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The interferon stimulated gene 15 (ISG15) is strongly induced in many cell types by double-stranded RNA (polyinosinic: polycytidylic acid, poly I:C) and viral infection. In this study, we described the nucleotide, mRNA tissue distribution and regulation of an ISG15 gene from turbot, Scophthalmus maximus (SmISG15). SmISG15 gene is 862 bp in length, composed of two exons and one intron, and encodes 158 amino acids. The deduced protein exhibits the highest homology (44.7-71.2% identity) with ISG15s from other fishes and possesses two conserved tandem ubiquitin-like (UBL) domains and a C-terminal RLRGG conjugating motif known to be important for the functions of ISG15s in vertebrates. Phylogenetic analysis grouped SmISG15 into fish ISG15. SmISG15 mRNA was constitutively expressed in all tissues examined, with higher levels observed in immune organs. Gene expression analysis was performed for SmISG15 in the spleen, head kidney, gills and muscle of turbots challenged with poly I:C or turbot reddish body iridovirus (TRBIV) over a 7-day time course. The result showed that SmISG15 was upregulated by both stimuli in all four tissues, with induction by poly I:C apparently stronger and initiated more quickly. A two-wave induced expression of SmISG15 was seen in the spleen, head kidney and gills, suggesting an induction of SmISG15 either by IFN-dependent or -independent pathway. These results provide insights into the roles of fish ISG15 in antiviral immunity., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Gene expression profiling of poly (I:C)-treated Japanese flounder, Paralichthys olivaceus, under different temperatures was investigated using microarray analysis. The response was analyzed in spleen tissue at 3 and 24 h post injection (hpi) at 15 °C and 25 °C. A large number of genes in fish treated with poly (I:C) at 25 °C were expressed at 3 hpi, whereas the expression profiles at 24 hpi appeared to be similar to those of the controls. Cluster analysis of the different expression profiles showed three distinct groups of up-regulated genes in fish reared at 15 °C. These were early (3 hpi), early-to-late (3 and 24 hpi), and late (24 hpi) up-regulated genes. These genes included type I IFN-related genes and inflammatory genes. Among the up-regulated genes, most of the type I IFN-related genes played early-to-late- and late-responding genes at 15 °C but early-responding genes at 25 °C. Thus, several up-regulated genes in these groups from the microarray result were further verified by qPCR. These results indicate that the type I IFN gene expressions of P. olivaceus treated with poly (I:C) can be regulated in a temperature-dependent manner., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Interferon regulatory factors (IRFs) act as transcription mediators in virus-, bacteria-, and interferon (IFN)-induced signaling pathways and play diverse functions in antimicrobial defense, immune modulation, hematopoietic differentiation, and cell apoptosis. In this study, we described for the first time eleven IRFs (IRF1, IRF1L, IRF2X1, IRF3, IRF4a, IRF4b, IRF5, IRF6, IRF7, IRF8, and IRF9) from half-smooth tongue sole (Cynoglossus semilaevis) and examined their tissue distributions and expression patterns under different conditions. The deduced protein sequences of these IRFs (except IRF1) share high identities (71.8-86.6%) with other corresponding IRFs in other teleosts, whereas the sequence identity of IRF1 with the corresponding IRF1 in other teleosts is only 58.1%. A conserved N-terminal DNA binding domain (DBD), which is characterized by a winged type helix-loop-helix motif with four to six tryptophan repeats, is present in all IRFs. Another conserved IRF associated domain (IAD), which mediates the interactions in the C-terminal part of the protein, is present in all IRFs except IRF1 and IRF2X1, which instead contain the IAD2 domain. Several special domains also were found, including a serine-rich domain (SRD) in IRF3, IRF4a, IRF4b, and IRF7; a proline-rich domain (PRD) in IRF9; nuclear localization signals (NLSs) in IRF5, IRF8, and IRF9; and a virus activated domain (VAD) in IRF5. Quantitative real time RT-PCR (qRT-PCR) analysis showed that expression of all IRFs occurred in multiple tissues. IRF1, IRF2X1, IRF4a, IRF5, IRF7, and IRF8 exhibited relatively high levels of expression in immune organs, whereas the other five IRFs displayed high levels of expression in non-immune organs. Infection with extracellular and intracellular bacterial pathogens and virus upregulated the expression of IRFs in a manner that depended on tissue type, pathogen, and infection stage. Specifically, IRF1 and IRF2X1 were highly induced by bacterial and viral pathogens; IRF1L and IRF6 responded mainly to extracellular and intracellular bacterial pathogens; IRF3, IRF5, IRF7, IRF8, and IRF9 were markedly induced by intracellular bacterial pathogen and virus; IRF4a and IRF4b were mainly induced by virus and intracellular bacterial pathogen respectively. These results indicate that the IRFs of C. semilaevis can be categorized into several groups which exhibit different expression patterns in response to the infection of different microbial pathogens. These results provide new insights into the roles of teleost IRFs in antimicrobial immunity., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Chemokines are small cytokines secreted by various cell types. They not only function in cell activation, differentiation and trafficking, but they also have influences on many biological processes. In this study, three novel CC chemokine genes Paol-SCYA105, 106 and 107 in Japanese flounder (Paralichthys olivaceus) were cloned and characterized. Paol-SCYA105 was mainly detected in gill, kidney and spleen, Paol-SCYA106 was detected in all tissues examined and Paol-SCYA107 was mainly detected in the spleen and kidney. Paol-SCYA105 and Paol-SCYA106 gene expressions peaked in kidney at day 3 after viral hemorrhagic septicemia virus infection and decreased at day 6, but Paol-SCYA106 still remained at a high level at day 6. Paol-SCYA107 gene expression was significantly up-regulated in kidney at day 6 after viral hemorrhagic septicemia virus infection. In response to infection by Gram-negative Edwardsiella tarda and Gram-positive Streptococcus iniae in kidney, only Paol-SCYA106 gene expression significantly increased. Together, these results indicate that these three novel CC chemokines are involved in the immune response against pathogen infections., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

In this study the effect of diet supplementation with different levels of short-chain fructooligosaccharides (scFOS) on the hepatic oxidative status, hematology and innate immune parameters was evaluated in turbot reared at 15 °C and 20 °C. Four practical diets containing half of the protein provided by plant ingredients and the other half by fish meal were supplemented with scFOS at 0%, 0.5%, 1.0% and 2.0% and fed to turbot juveniles for 9 weeks. Independently of the rearing temperature, diet with 1% scFOS increased the haematocrit (Ht) while 2% scFOS augmented the mean corpuscular haemoglobin concentration (MCHC). Mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), white blood cells (%) and lysozyme were higher in fish reared at 15 °C, whereas red blood cells and neutrophil numbers increased in fish reared at 20 °C. Catalase (CAT) and glutathione peroxidase (GPX) activities were affected by rearing temperature being lower in fish reared at 20 °C. Compared to the control diet, at 15 °C, turbot fed 0.5 or 1% scFOS presented lower activities of CAT and glutathione reductase (GR). At 20 °C turbot fed the 2% scFOS diet presented lower activities of CAT and GPX. Lipid peroxidation (LPO) and glucose 6-phosphate dehydrogenase (G6PDH) activity were not affected by temperature nor dietary prebiotic incorporation. Results of this study suggest scFOS has no effect on innate immunology or hematology. High temperature (20 °C) does not induce turbot oxidative stress, but the recommended dietary scFOS incorporation level for counteracting oxidative stress may differ with other rearing temperature., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

In recent years, increasing diseases especially bacterial diseases have brought a host of losses with the expansive cultivation of turbot (Scophthalmus maximus). In order to do more research about the immune system of turbot for better understanding the mechanism of resisting diseases, the immunoglobulin genes related to secretory and membrane-bound IgM (s-IgM and m-IgM) of turbot were cloned using homology sequences cloning and SMART RACE PCR method. The heavy chain of s-IgM cDNA is 1900 bp in length including a leader region, a variable region, four constant regions (CH1, CH2, CH3 and CH4) and a C-terminal while the cDNA of m-IgM is 1795 bp with the same leader region, variable region, three constant regions (CH1, CH2 and CH3) and two transmembrane regions (TM1 and TM2). The sequence of IgM gene was also obtained and the structure consisted of V-CH1-CH2-CH3-CH4-TM1-TM2 is similar to other fishes. The highest level of s-IgM expression was observed in spleen, followed by kidney, gills, eyes, skin of the healthy turbot whereas the same profile of m-IgM expression is found with low level. And s-IgM takes up dominant proportion of total IgM expression. Also the relative expressions of s-IgM and m-IgM were analyzed in turbot vaccinated with the live attenuated vaccine Vibrio anguillarum. Not only the transcriptions of both s-IgM and m-IgM in liver, spleen and kidney of turbot injected with V. anguillarum MVAV6203 were up-regulated but also the expressions of s-IgM and m-IgM in spleen, kidney, gut, skin and gills of bath-vaccinated turbot were increased. Comparing the ratio changes of relative expression of m-IgM and s-IgM in vaccinated turbot, we found that the proportion of m-IgM were increasing in both administration routes, which probably indicated that the increasing expression of m-IgM strengthen the phagocytic ability of B cells., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The objective of this study was to locate the functional region responsible for the chemotaxis-inducing activity of flounder interleukin 8 (IL-8), which lacks the glutamic acid-leucine-arginine (ELR) motif essential for the induction of neutrophil migration by mammalian IL-8. Using a human cell line, we produced a secretory recombinant protein of flounder IL-8, and analyzed its chemotaxis-inducing activity on leukocytes collected from the flounder kidney. The recombinant IL-8 induced significant migration in neutrophils, which were morphologically and functionally characterized. Using the Edman degradation method, the N-terminal amino acid sequence of rIL-8 was identified as VSLRSLGV. To examine the significance of the N-terminal region for the bioactivity of flounder IL-8, we prepared several recombinant proteins that containing mutations at the N-terminus. Modification of three residues (residues 9-11: serine-leucine-histidine) corresponding in position to the ELR motif in mammalian IL-8 did not reduce its chemotaxis-inducing activity. However, deletion of the first six or more residues significantly reduced its chemotaxis-inducing activity. We propose that residue 6 (leucine) at the N-terminus is important for the chemotaxis-inducing activity of flounder IL-8., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Hepcidin is an antimicrobial peptide and a regulator of iron homeostasis. In turbot (Scophthalmus maximus), two types of hepcidins have been identified, which share approximately 50% sequence identity. In this study, we examined the antimicrobial potentials of the two hepcidins in the form of synthesized peptides, SmHep1P and SmHep2P. We found that SmHep1P and SmHep2P exhibited apparent bactericidal activities against both Gram-positive and Gram-negative bacteria in a dose-dependent manner. The bactericidal effect of SmHep1P was stronger against Gram-positive bacteria, while the bactericidal effect of SmHep2P was stronger against Gram-negative bacteria. Fluorescence and electron microscopy showed that both peptides were able to bind to the target bacterial cells and alter the surface structure of the cells. In vitro studies showed that SmHep1P and SmHep2P reduced bacterial invasion into cultured fish cells. In vivo studies showed that turbot administered with SmHep1P and SmHep2P exhibited significantly enhanced resistance against bacterial and viral infection. In both in vivo and in vitro studies, the antimicrobial activities of SmHep2P were in most cases significantly stronger than those of SmHep1P. Together these results indicate that the two hepcidins of turbot most likely possess antimicrobial properties and play a role in the innate immune defense against bacterial and viral pathogens., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Signal transducer and activator of transcription 2 (STAT2) is an important molecule involved in the type I interferon signalling pathway. To date, little STAT2 homologue is available in fish except Atlantic salmon and goldfish. In this paper, STAT2 was firstly cloned and characterized from turbot, a marine flatfish with high economic value. Briefly, turbot STAT2 cDNA is 3206 bp in length encoding a predicted protein of 793 amino acids. The phylogenetic tree shows that turbot STAT2 protein shared the closest relationship with Atlantic salmon. Analysis of subcellular distribution indicates that STAT2 is mainly present in the cytoplasm of TK cells. Stat2 mRNA is constitutively expressed in widespread tissues and induced by several folds in turbot tissues and TK cells after stimulation with Vibrio anguillarum and lymphocystis disease virus (LCDV). Unlike the higher vertebrate STAT2, turbot STAT2 nuclear export signal (NES) exists not in the C-terminal 79 amino acids but in N-terminal 137-312 amino acids (STAT&#95;alpha domain). The nuclear translocation of turbot STAT2 after Poly(I:C) treatment proved its transcription activity in TK cells. All these results suggested that STAT2 may be involved in the immune response in turbot as a transcription factor., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Nuclear factor 45 (NF45) is a component of the protein complex called nuclear factor of activated T-cells (NFAT), which in mammals regulates interleukin (IL)-2 expression. To date very little is known about fish NF45. In this study, we identified a NF45 (named CsNF45) from half smooth tongue sole Cynoglossus semilaevis and examined its gene organization, expression profile, and regulatory function. We found that CsNF45 is composed of 387 residues and shares 90.3%-97.9% overall sequence identities with the NF45 of human and teleosts. Genetic analysis showed that the genomic sequence of the ORF region of CsNF45 consists of 14 exons and 13 introns. Constitutive expression of CsNF45 occurred in multiple tissues including gill, muscle, brain, heart, liver, head kidney, spleen, and gut. Experimental infection with viral and bacterial pathogens upregulated the expression of CsNF45 in head kidney and spleen in a time-dependent manner. Transient transfection analysis showed that CsNF45 was localized in the nucleus and able to stimulate the activity of mouse IL-2 promoter. These results indicate that CsNF45 possesses immunoregulatory property and is possibly involved in host immune defense against bacterial and viral infection., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Antimicrobial peptide plays an important role in fish immunity. The small molecular antimicrobial peptide Hepcidin in turbot was studied and reported in this paper. The Ferroportin 1 (FPN1) and Transferrin Receptor (TFR) genes, which are related to Hepcidin, were cloned in turbot. The characteristics of Hepcidin and its related genes were studied, including an analysis of the expression patterns and cloning of the Hepcidin promoter, the relationship between Hepcidin and NF-κB and the regulation of iron-metabolism. The results showed that the promoter of SmHepcidin contains the binding sites of NF-κB, and NF-κB may directly or indirectly receive feedback signals from SmHepcidin. In the liver, spleen and kidney, in which there was an increased SmHepcidin expression level, SmFPN1 dramatically decreased and SmTFR was also either decreased or exhibited no obvious change after bacterial/viral infection and an injection of exogenous Hepcidin protein. RNAi experiments in turbot kidney cells confirmed the expression changes of these gene patterns. Furthermore, the administration of exogenous Hepcidin protein, which regulates the level of chelatable iron in cells, further confirmed the function of Hepcidin in iron metabolism. It is speculated that the rapidly increased expression of SmHepcidin may induce changes in the expression of related genes, and that the in vivo chelatable iron concentration which participates in the antibacterial process was also changed when exogenous pathogens are present in turbot. It is suggested that SmHepcidin plays a defensive role against pathogenic infection., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Pentraxins are a family of evolutionarily conserved proteins that play an important part in innate immunity. C-reactive protein (CRP) is a member of the pentraxin family and in humans is known to be the major acute phase protein. In this work, we report the identification and analysis of a CRP, CsCRP, from half-smooth tongue sole (Cynoglossus semilaevis). CsCRP is composed of 228 amino acid residues and possesses a Pentraxin/CRP domain. Expression of CsCRP occurred in a wide range of tissues and was upregulated by pathogen infection in kidney, spleen, blood, and, in particular, liver. Following bacterial infection, CsCRP level in blood rose rapidly within 12 h and was approximately 3.8 fold of that of the basal level. Purified recombinant CsCRP (rCsCRP) was able to interact with Gram-negative and Gram-positive bacteria including those of pathogenic nature in a dose-dependent manner. When peripheral blood leukocytes (PBL) were infected with bacterial pathogen in the presence of rCsCRP, the respiratory burst and phagocytic capacity of the cells were increased to significant extents. Taken together, these results indicate that CsCRP is an acute phase protein that plays a role in innate immune defense against bacterial infection., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

T lymphocyte activation requires a combination of signals, one of which is provided by interaction between CD28 and its ligands on antigen presenting cells. Although CD28-like sequences have been identified in a few teleosts, the function of fish CD28 is virtually unknown. In this study, we cloned and analyzed a CD28 gene, CsCD28, from half-smooth tongue sole (Cynoglossus semilaevis). The deduced amino acid sequence of CsCD28 contains 229 residues and shares 20.2%-40.3% overall sequence identities with known fish CD28 sequences. CsCD28 possesses structural features conserved in mammalian and teleost CD28, which include the MYPPPY motif in the extracellular immunoglobulin-like domain and the YXN motif in the cytoplasmic domain. The CsCD28 gene is 2746 bp and composed of four exons and three introns, which in organization resemble those of mammalian and trout CD28. Quantitative real time RT-PCR analysis showed that CsCD28 expression occurred predominately in kidney, spleen, gut, and gill. CsCD28 is localized on the surface of head kidney lymphocytes, and antibody ligation of CsCD28 induced significant levels of cellular proliferation. Taken together, these results indicate that CsCD28 is similar to mammalian CD28 in genetic and protein structures and possibly plays a role in T cell activation.

Lysozymes are considered to be potent innate immune molecules against the invasion of bacterial pathogens. The goose-type lysozyme is one of the three major distinct lysozyme types identified in the animal kingdom including teleosts. In this report, we identified, sequenced, and characterized the goose-type lysozyme gene (CsGLys) from half-smooth tongue sole (Cynoglossus semilaevis). The full-length cDNA of CsGLys is 1191 bp in length from the transcription start site to polyadenylation site, including a 91 bp 5'-terminal untranslated region (UTR), a 452 bp 3'-terminal UTR and a 648 bp open reading frame (ORF) of encoding a polypeptide with 215 amino acids. The deduced amino acid sequence of CsGLys possesses a Goose Egg White Lysozyme (GEWL) domain with three conserved residues (E91, D104 and D121) essential for catalytic activity. The CsGLys gene consisting of 2535 bp, was similar to those of other teleost species such as Japanese flounder and large yellow croaker with five exons interrupted by four introns. The 5'-flanking region of CsGLys gene shows several transcriptional factor binding sites related to immune response. Tissue expression profile analysis by quantitative real-time reverse transcription PCR showed that CsGLys mRNA was constitutively expressed in all examined tissues with the predominant expression in skin and the weakest expression in heart. The expression of CsGLys after challenged with bacteria Vibrio anguillarum was up-regulated in blood, head kidney, liver and spleen at 12 h post-infection and it reached the peak level at the same time point with a 19.89-, 4.21-, 14.45- and 10.37-fold increase, respectively, while the CsGLys expression was down-regulated to lower level than the normal level in each tested tissues except in liver from the 48 h until 96 h. These results suggest that CsGLys might play an important role in half-smooth tongue sole host defense against the bacteria infection., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

As a crucial component in TLR/IL-1R signaling pathways, IRAK-4 plays a central role in innate and adaptive immunity. In the present study, the cDNA of IRAK-4 was cloned for the first time from half-smooth tongue sole (Cynoglossus semilaevis). The full-length cDNA of csIRAK-4 was 2149 bp and contained a 168 bp 5' UTR, a 580 bp 3' UTR and a 1401 bp CDS. The predicted protein sequence of csIRAK-4 had two typical domains, a death domain (DD) at the N terminus and a serine/threonine/tyrosine protein kinase domain (STYKc) at the C terminus. RT-PCR showed that csIRAK-4 mRNA was detected in all tested tissues, especially in immune-related organs, gonads and brain. After injected with inactivated Vibrio anguillarum, the expressions of csIRAK-4 were up-regulated significantly (P<0.05) in spleen and head kidney. During development, csIRAK-4 was expressed at all selected stages and low-level expression was detected at metamorphosis. Taken together, the present study indicated that csIRAK-4 played a crucial role in immune responses and might be involved in the process of development., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Ferritin is a major intracellular iron storage protein in higher vertebrates and plays an important role in iron metabolism. In this study, we identified and analyzed the biological activity of a ferritin M subunit (CsFerM) from half-smooth tongue sole (Cynoglossus semilaevis). The open reading frame (ORF) of CsFerM is 534 bp and encodes a protein that shares 79.7-86.4% overall sequence identities with the ferritin M subunits of a number of teleosts. In silico analysis identified in CsFerM a eukaryotic ferritin domain with conserved ferroxidase diiron center and ferrihydrite nucleation center. Quantitative real time RT-PCR analysis showed that under normal physiological conditions, expression of CsFerM was highest in liver, moderate in gill, spleen, and muscle, and low in gut, heart, and brain. Following experimental challenge with bacterial pathogens, CsFerM expression was significantly upregulated in kidney, spleen, and liver in time-dependent manners. Biological activity analysis showed that recombinant CsFerM purified from Escherichia coli exhibited apparent iron-binding activity and, when present in the culture medium of six different species of fish bacterial pathogens, completely inhibited bacterial growth. In contrast, a mutant CsFerM that bears alanine substitution at two conserved residues of the ferroxidase diiron center and ferrihydrite nucleation center was abolished in both iron-binding and antimicrobial capacity. These results demonstrate that CsFerM is a biologically active iron chelator with broad-spectrum antibacterial activity, which suggests a role for CsFerM in not only iron storage but also innate immunity. These results also indicate the importance of the conserved iron uptake and mineralization sites to the function of CsFerM., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

In recent years, quantitative real time reverse transcriptase-PCR (qRT-PCR) has been used frequently in the study of gene expression in turbot (Scophthalmus maximus) in relation to bacterial infection. However, no investigations on appropriate qRT-PCR reference genes have been documented. In this report, we determined the potential of eight housekeeping genes, i.e. β-actin (ACTB), ribosomal protein L17 (RPL17), α-tubulin (TUBA), elongation factor-1-α(EF1A), β-2-Microglobulin (B2M), RNA polymerase II subunit D (RPSD), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and 18S ribosomal RNA (18S rRNA), as internal standards for qRT-PCR analysis of gene expression in turbot as a function of bacterial infection. For this purpose, the expression of the eight housekeeping genes in seven turbot tissues was determined by qRT-PCR before and after bacterial challenge, and the data were analyzed with the geNorm and NormFinder algorisms. The results showed that the expression of all the examined genes exhibited tissue-dependent variations both before and after bacterial challenge. Before bacterial challenge, geNorm and NormFinder identified RPSD as the gene that showed least tissue specific expression. At 12 h post-bacterial infection, geNorm ranked ACTB/GAPDH, 18S rRNA/ACTB, ACTB/GAPDH, 18S rRNA/ACTB, RPL17/TUBA, RPSD/GAPDH, and RPSD/B2M, respectively, as the most stably expressed genes in liver, spleen, kidney, gill, heart, muscle, and brain. Comparable ranking orders were produced by NormFinder. Similar results were obtained at 24 h post-bacterial infection. Taken together, these results indicate that RPSD is the most stable gene across tissue types under normal physiological conditions and that, during bacterial infection, ACTB might be used as an internal standard for the normalization of gene expression in immune relevant organs; however, no single gene or single pair of genes in the examined set of housekeeping genes can serve as a universal reference across all tissue types under the condition of bacterial infection., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Signal transducer and activator of transcription 3 (STAT3) acts as an important mediator in multiple biological processes induced by different cytokines. So far, little information is available in fish STAT3. In this study, turbot (Scophthalmus maximus) STAT3 gene was cloned and characterized for the first time. The turbot STAT3 full-length cDNA consists of 2355 nucleotides encoding a polypeptide of 784 amino acids with four conserved domains including STAT&#95;int, STAT&#95;alpha, STAT&#95;bind and SH2 domain. The phylogenetic tree showed that turbot STAT3 shared the closest relationship with mandarin fish (Siniperca chuatsi) STAT3. The autoactivation experiment in yeast proved that turbot STAT3 was a strong transcription factor. The quantitative RT-PCR experiment indicated that Stat3 mRNA was expressed in widespread tissues with the highest expression levels in the liver. And the further expression patterns analysis revealed that turbot Stat3 expression levels were increased in liver, spleen, kidney of fish infected with Vibrio anguillarum and liver of fish infected with LCDV. Meantime, hepcidin, one of STAT3 target gene, was also up-regulated in liver of fish infected with two pathogens. These results suggested that turbot Stat3 may involved in the immune defense process as a transcription factor., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Calcium-activated potassium channels on plasma membrane enable potassium influx into the cell with ensuing changes in plasma membrane potential and consequent effects on cellular metabolic functions. Recently, this potassium channel was reported to regulate the cellular responses of mammalian immune cells. We have postulated the presence of such a channel in fish immune cells and its potential role in immunoregulation in fish. Employing specific primers and RNA template, we cloned a segment of a novel gene from turbot blood sample and subsequently obtained a full cDNA sequence using RACE approaches. Bioinformatic analysis revealed structural and phylogenetic characteristics of a novel small conductance calcium-activated potassium channel gene, we called TSKCa, which exhibits homologous domains to other species particularly in the transmembrane regions. Full-length TSKCa cDNA is 1698 bp with a 1632 bp open reading frame encoding a protein of 544 amino acids. TSKCa gene is expressed in majority of the tested organs and tissues of turbot. To assess the postulated immune function of TSKCa, we infected turbot with the pathogen Vibrio anguillarum. Here, semi-quantitative RT-PCR analysis demonstrated increased mRNA expression of TSKCa in head kidney, spleen and blood, indicating an important role of TSKCa in these organ tissues that mediate the immune defense response of turbot. In contrast, there was less change in expression in the turbot intestines and liver which were less implicated in the immune response in present study.

Rodlet cells are an enigmatic cell type described in tissues of both marine and freshwater teleosts. Although their structure is well established, up to date their function remains subject of debate. However, there is consensus among the majority of researchers that rodlet cells play an important role within immune system, and this function is probably related with the release of rodlets due to contractile capability of their fibrous layer. Regulation of the contraction mechanism would require proteins that modulate Ca(++) intracellular concentration to be expressed in rodlet cells. We performed a morphological and immunohistochemical study at light and electron microscopy levels to assess S100 protein immunoreactivity in developing rodlet cells. Immature stages did not exhibit immunoreactive signal; however, immunoreactivity was observed in the fibrous layer of both transitional and mature rodlet cells. The latter stage also showed immunosignal within the rodlets. These findings suggest a clear association between S100 protein expression and rodlet cell development that could be linked to the regulation of rodlet activity and contractile property of their fibrous layer. Furthermore, S100 protein antibody constitutes a novel marker for rodlet cells that could be used in future studies of this particular cell type.

Lysozymes are key molecules of innate immunity and proved high bactericidal activity in fish, thus becoming attractive as tools for enhancing fish defences. In this study, a full-length c-type lysozyme cDNA from Senegalese sole (Solea senegalensis) has been cloned and characterized. The cDNA sequence was inferred from two overlapping fragments obtained by RACE-PCR and consisting on 631bp coding for 143 aminoacids. Catalytic and other conserved residues required for lysozyme activity were identified. Pair wise alignments showed the higher identities with c-type lysozyme from other flatfish. Expression patterns under various conditions showed a basal level and a clear upregulation mostly in hematopoietic organs after stimulation with LPS or infection with Photobacterium damselae. This study represents a first step on the genetics and function of the c-lysozyme of Senegalese sole, though disclosing g-DNA structure, allelic variability and antibacterial activity must be requirements prior its immunological properties might have biotechnological applications.

Lysozymes are key proteins of the innate immune system against bacterial infections. In this study we report the molecular cloning and characterization of the c-type and g-type lysozymes in brill (Scophthalmus rhombus). Catalytic and other conserved residues required for functionality were identified. Phylogenetic analysis revealed distinct evolutionary histories for each lysozyme type. Expression profiles of both lysozyme genes were studied in juvenile tissues using a real-time PCR approach. c-Type lysozyme was expressed mainly in stomach and liver, whereas the g-type was detected in all tissues with highest mRNA levels observed in the spleen. Induction experiments revealed that g-type transcripts increased significantly in head kidney after lipopolysaccharide (25- and 23-fold at 12 and 24h, respectively) and Photobacterium damselae subsp. piscicida (17-fold at 24h) treatments. In contrast, no induction was observed for c-type lysozyme. All these data suggest that g-type lysozyme is involved in the response against bacterial infections, whereas c-type lysozyme may also play a role in digestion.

Chemokines represent a superfamily of chemotactic cytokines playing an important role in leucocyte chemotaxis. Here we report a novel turbot CXC chemokine screened from a turbot spleen cDNA library. The complete cDNA of the turbot CXC chemokine contains an 81bp 5' UTR, a 414bp open reading frame (ORF) encoding 137 amino acids and a 449bp 3' UTR. Four exons and three introns are identified in the turbot CXC chemokine gene. Phylogenetic analysis showed that the turbot CXC chemokine clustered apart from all other CXC chemokines. RT-PCR demonstrated that turbot CXC chemokine was expressed highly in spleen and head kidney. During the early stages of embryo development after fertilization, it appears that low expression level of turbot CXC chemokine was firstly observed at somites stage. Interestingly, the turbot chemokine was highly and rapidly (5h) induced in liver, spleen and head kidney of turbot after challenge with Vibrio anguillarum. Furthermore, the expression of CXC chemokine was also dramatically increased after challenge in turbot embryonic cells (TECs). These results indicated that the turbot CXC chemokine played an important role in turbot immune response.