A Bioactive Extract Rich in Triterpenic Acid and Polyphenols from Olea europaea Promotes Systemic Immunity and Protects Atlantic Salmon Smolts Against Furunculosis.

In the present study, the modulation of the transcriptional immune response (microarray analysis) in the head kidney (HK) of the anadromous fish Atlantic salmon (Salmo salar) fed a diet supplemented with an olive fruit extract (AQUOLIVE®) was evaluated. At the end of the trial (133 days), in order to investigate the immunomodulatory properties of the phytogenic tested against a bacterial infection, an in vivo challenge with Aeromonas salmonicida was performed. A total number of 1,027 differentially expressed genes (DEGs) (805 up- and 222 downregulated) were found when comparing the transcriptomic profiling of the HK from fish fed the control and AQUOLIVE® diets. The HK transcripteractome revealed an expression profile that mainly favored biological processes related to immunity. Particularly, the signaling of i-kappa B kinase/NF-kappa and the activation of leukocytes, such as granulocytes and neutrophils degranulation, were suggested to be the primary actors of the innate immune response promoted by the tested functional feed additive in the HK. Moreover, the bacterial challenge with A. salmonicida that lasted 12 days showed that the cumulative survival was higher in fish fed the AQUOLIVE® diet (96.9 ± 6.4%) than the control group (60.7 ± 13.5%). These results indicate that the dietary supplementation of AQUOLIVE® at the level of 0.15% enhanced the systemic immune response and reduced the A. salmonicida cumulative mortality in Atlantic salmon smolts.

Efficacy of ulvan on immune response and immuno-antioxidant gene modulation in Labeo rohita against columnaris disease.

This study reports the effect of ulvan enriched diet on the influence of growth, changes in hemato-biochemical indices, improvement of antioxidant system, enhancement of innate-adaptive immunity and modification of immuno-antioxidant genes expression in Labeo rohita against Flavobacterium columnaris. The weight gain (WG) was significantly high (P > 0.05) in unchallenged normal and challenged fish fed with diets enriched with 25 and 50 mg kg-1 ulvan; the FCR was better (P > 0.05) when fed with 50 mg kg-1 enriched diet. In normal fish fed with or without ulvan supplementation was noted 100% survival rate (SR). In both groups, the red blood cell (RBC) and while blood cell (WBC) counts increased significantly (P > 0.05) when fed with 50 mg kg-1 ulvan diet whereas the hemoglobin (Hb) level increased significantly on being fed with 25 and 50 mg kg-1 ulvan diets. The SOD activity was enhanced significantly in both groups fed with any dose of ulvan diets whereas the MDA and GPx activity increased only with 25 and 50 mg kg-1 ulvan diets. The phagocytic (PC) activity significantly increased with any enriched diet and control diet groups while the respiratory burst (RB) activity increased only with 50 mg kg-1 ulvan diet. The alternate complement pathway (ACP), activity of lysozyme (Lyz), and immunoglobuline M (IgM) were better in both groups fed with 50 mg kg-1 ulvan diet. The SOD and GPx antioxidant gene expression were significantly high in both groups fed with any ulvan diet while the Nrf2 gene expression was high with 50 mg kg-1 ulvan diet. The IL-1ß, TNFα, hepcidin, Lyz, and IgM cytokines or proteins mRNA expression were significant in both groups fed with all ulvan supplement diet whereas the ß-2M expression was significant only with 50 mg kg-1 ulvan diet. The present research indicates that both L. rohita groups fed with 50 mg kg-1 ulvan diet significantly improved growth, antioxidant system, immune defense system, and immuno-antioxidant related gene expression against F. columnaris.

Hot water extract of Pleurotus pulmonarius stalk waste enhances innate immune response and immune-related gene expression in red hybrid tilapia Oreochromis sp. following challenge with pathogen-associated molecular patterns.

In aquaculture, commercial fish such as red hybrid tilapia are usually raised at high density to boost the production within a short period of time. This overcrowded environment, however, may cause stress to the cultured fish and increase susceptibility to infectious diseases. Antibiotics and chemotherapeutics are used by fish farmers to overcome these challenges, but this may increase the production cost. Studies have reported on the potential of mushroom polysaccharides that can act as immunostimulants to enhance the immune response and disease resistance in fish. In the current study, hot water extract (HWE) from mushroom stalk waste (MSW) was used to formulate fish feed and hence administered to red hybrid tilapia to observe the activation of immune system. Upon 30 days of feeding, the fish were challenged with pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharides (LPS) and polyinosinic:polycytidylic acid (poly (I:C)) to mimic bacterial and viral infection, respectively. HWE supplementation promoted better feed utilisation in red hybrid tilapia although it did not increase the body weight gain and specific growth rate compared to the control diet. The innate immunological parameters such as phagocytic activity and respiratory burst activity were significantly higher in HWE-supplemented group than that of the control group following PAMPs challenges. HWE-supplemented diet also resulted in higher mRNA transcription of il1b and tnfa in midgut, spleen and head kidney at 1-day post PAMPs injection. Tlr3 exhibited the highest upregulation in the HWE fed fish injected with poly (I:C). At 3-days post PAMPs injection, both ighm and tcrb expression were upregulated significantly in the spleen and head kidney. Results showed that HWE supplementation enhances the immune responses of red hybrid tilapia and induced a higher serum bactericidal activity against S. agalactiae.

Effects of aqueous and ethanolic leaf extracts from drumstick tree (Moringa oleifera) on gilthead seabream (Sparus aurata L.) leucocytes, and their cytotoxic, antitumor, bactericidal and antioxidant activities.

Aqueous and ethanolic extracts of drumstick, Moringa oleifera, leaves were evaluated in vitro to ascertain their principal active components and determine their immunostimulant, cytotoxic, antitumoral, bactericidal and antioxidant activities. Phytochemical screening of M. oleifera leaf extracts showed a greater abundance of phenolic and cyanogenic glycosides in aqueous than in ethanolic extracts, characterized by several flavonoids, condensed tannins and saponins. No significant effects on gilthead seabream (Sparus aurata) head-kidney leucocyte activities (phagocytic ability and capacity, respiratory burst and peroxidase) were detected after incubation for 24 h with different concentrations (0.001/1 mg mL-1) of either extract. In addition, the aqueous extract showed a marked cytotoxic effect on both SAF-1 (at doses above 0.01 mg mL-1) and PLHC-1 (at doses above 0.25 mg mL-1) cell lines. The ethanolic extract improved the viability of SAF-1 cells and decreased the viability of PLHC-1 cells when used at higher concentrations. Both the ethanolic and, particularly, the aqueous extracts showed significant bactericidal activity on pathogenic Vibrio anguillarum and Photobacterium damselae strains. The antiradical activity of M. oleifera, as determined by the ABTS assay, increased in a linear dose-response with increasing extract concentrations. The results as a whole for the cytotoxic, bactericidal and antioxidant activities of M. oleifera leaf extracts point to their possible use as additives in functional diets for farmed fish.

Identification of novel long non-coding RNAs involved in bisphenol A induced immunotoxicity in fish primary macrophages.

Bisphenol A (BPA), a well-known environmental endocrine-disrupting chemical (EDC), could pose a great toxicity risk to aquatic organisms. The present study aimed to evaluate the underlying role of long non-coding RNAs (lncRNAs) in BPA-induced immunotoxicity in head kidney (HK) macrophages of the red common carp (Cyprinus carpio), using lncRNA-RNA sequencing (RNA-Seq). In BPA-exposed HK macrophages group, 2,095 and 1,138 differentially expressed mRNAs (DEGs) and lncRNAs (DE-lncRNAs) were obtained, respectively, compared with controls. The qRT-PCR validation results of DEGs and DE-lncRNAs were similar to the RNA-Seq results. The KEGG analysis of DEGs and target genes of DE-lncRNAs have shown that some immune-related signaling pathways, including NF-kappa B, Toll-like receptor, B-cell receptor, Jak-STAT, and Hippo signaling pathways, were severely disrupted by BPA exposure. Moreover, we observed the synergic regulation of some mRNAs involved in immune response such as two hub genes traf6 and mapk1/3 and their upstream lncRNAs in HK macrophages upon the BPA exposure or its analogue bisphenol S (BPS) exposure. This suggested the dysregulation of lncRNAs by BPA or BPS may lead to a change in the expression of hub genes, which affects the cross-talk of various signaling pathways by interaction with other network genes. In conclusion, the present study demonstrates the potential role of lncRNAs in immunotoxicity of bisphenol compounds in red common carp HK macrophages, and our results provide evidence for further exploring lncRNA's role in EDC-induced toxicity in aquatic organisms.

Changes in the immune function of rainbow trout (Oncorhynchus mykiss) provide insights into strategies against BDE-47 stress.

Polybrominated diphenyl ethers (PBDEs) are ubiquitous in marine ecosystems and have been suggested to bioaccumulate in aquatic food webs, with potentially negative impacts on marine organism. In this study, a 21-day experiment was performed under controlled laboratory conditions, in which 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), the most biotoxic PBDE in the marine environment, was fed to rainbow trout (Oncorhynchus mykiss) at concentrations of 50 and 500 ng g-1 in the diet. BDE-47 significantly decreased the specific growth rate of O. mykiss and was highly concentrated in the liver and head kidney, as evidenced by increased bioaccumulation factor (BAF) values. Tissue observation revealed impairment of the microstructure of the head kidney. Important immune factors in the skin, blood and head kidney were significantly inhibited by BDE-47 treatment (p < 0.05), whereas the respiratory burst activity of macrophages was enhanced. Additionally, immune-related genes were strongly downregulated following BDE-47 exposure (p < 0.05). In a bacterial challenge, the treatment groups had much higher mortality than did the control group (p < 0.05). BDE-47 accumulated and impaired immune organs, and the hierarchy of immune responses was impaired, consequently reducing O. mykiss resistance to pathogen invasion.

Effects of an exopolysaccharide from Lactococcus lactis Z-2 on innate immune response, antioxidant activity, and disease resistance against Aeromonas hydrophila in Cyprinus carpio L.

Microbial exopolysaccharides (EPS) from Lactococcus have been found to have an important role in the probiotic activity of this bacterium; however, the immunomodulatory and antioxidant activities have not been fully explored in aquaculture. In the present study, we investigated EPS-2 from Lactococcus lactis Z-2, isolated from healthy common carp, for its immunomodulatory and antioxidant effects and disease resistance against Aeromonas hydrophila in Cyprinus carpio L. We found that the molecular weight of EPS-2 was 18.65 KDa. The monosaccharide composition of this polymer was rhamnose, xylose, mannose, glucose, and galactose at a molar percentage of 13.3%, 14.1%, 18.5%, 27.4%, and 26.7%, respectively. EPS-2 treatment could modulate the immune responses in vitro and in vivo. In vitro tests showed that EPS-2 could significantly enhance the proliferation and phagocytosis activities (P < 0.05) as well as induce the production of nitic oxide (NO), pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6), and anti-inflammatory cytokines (IL-10, TGF-ß) (P < 0.05) in head kidney cells. When the fish were gavaged with three different concentrations of EPS-2 (250, 500, 1000 µg/mL) for 7 days and infected with A. hydrophila, different expression patterns of the NO, cytokines, lysozyme (LZM), and alkaline phosphatase (AKP) in the serum and of antioxidants (T-AOC, SOD, CAT, GSH, GSH-Px and MDA) in hepatopancreas were observed. Before infection with A. hydrophila, EPS-2 supplementation significantly up-regulated the NO production, protein levels of pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6), LZM and AKP activities, and levels of antioxidant molecules compared to those in the negative (G1) group (P < 0.05), whereas levels of NO and pro-inflammatory cytokines and LZM and AKP activities were significantly lower than those in the positive (G2) group after infection (P < 0.05). However, whether infected or not, the expression levels of anti-inflammatory cytokines (IL-10, TGF-ß) were significantly increased in the EPS-2 treatment groups (P < 0.05). These results indicate that EPS-2 has immunomodulatory and antioxidant effects on common carp, both in vitro and/or in vivo, and can be applied as a common carp feed supplement to enhance fish immunity and disease resistance against A. hydrophila.

Melamine and curcumin enriched diets modulate the haemato-immune response, growth performance, oxidative stress, disease resistance, and cytokine production in oreochromis niloticus.

Currently, feed adulteration and contamination with melamine (MEL) are considered one of the serious issues in the aquatic industry. With the limited studies of MEL exposure alone in fish, its adverse impacts on fish cannot be evaluated well. Accordingly, this study aimed to investigate the effects of MEL containing diets on the immune response, disease resistance to Aeromonas hydrophila, growth performance, chemical composition, immune-related genes expression, and histopathology of both spleen and head kidneys. Also, the efficacy of curcumin (CUR) dietary supplementation to alleviate MEL negative impacts were evaluated. A total of 180 apparently healthy Oreochromis niloticus (O. niloticus) were divided into four groups with three replicates fed the basal diet only, basal diet fortified with 200 mg/kg CUR, basal diet containing 1 % MEL, or a basal diet containing CUR + MEL. The results displayed that MEL significantly reduced growth performance indices and body crude lipid contents. Anemic, leukopenic, lymphocytopenic, heterocytopenic, esonipenic, hypoproteinemic and hypoalbuminic conditions were apparent. Moreover, depleted immune and antioxidant indicators including lysozyme activity, nitric oxide, immunoglobulin M, complement 3, glutathione peroxidase, and superoxide dismutase enzyme activity were recorded. Also, MEL reduced the disease resistance of O. niloticus to bacterial infection. Furthermore, MEL induced downregulation of mRNA levels of interleukin 1ß and tumor necrosis factor α in the spleen together with obvious pathological perturbations in both spleen and head kidneys. The CUR addition resulted in a significant enhancement in most indices. These results may conclude that MEL could alter both innate and adaptive immune responses via the negative transcriptional effect on immune-related genes together with the oxidative damage of the immune organs. Furthermore, CUR dietary supplements could be advantageous for mitigating MEL negative impacts, thus offering a favorable aquafeed additive for O. niloticus.

Immunohistological Biomarkers of Toxicity by a Pharmaceutical Antidepressant in the Freshwater Cichlid Fish Cichlasoma dimerus (Teleostei, Cichliformes).

Melano-macrophage centers (MMCs) are nodular clusters of pigmented macrophages, implicated in homeostasis and destruction and recycling of endogenous and exogenous material. They can increase in size and/or frequency under environmental stress resulting in immunohistological biomarkers of water quality. Fluoxetine (FLX), a commonly prescribed antidepressant, can cause neuroendocrine, behavioral and reproductive alterations in teleost fish. In the present study, we analyzed the effects of a 2-week 50 µg/L FLX exposure on MMCs in histological sections of spleen and head-kidney (HK) of the cichlid fish Cichlasoma dimerus. In the spleen, FLX caused an increase in the area and a decrease in the number of MMCs. An increase in the proportion of the HK occupied by MMCs was observed in FLX-exposed fish, due to an increase in their number but not their area. The deposition rate of MMCs varies according to the hemolymphopoietic organ and would be the result of a differential response to FLX on homeostatic functions (elimination of cellular debris, iron processing and immune response).

Diphenyl diselenide dietary supplementation protects against methylmercury-chloride-induced immunotoxicity in the head kidney and spleen of grass carp (Ctenopharyngodon idella) via regulation of purinergic signaling and the NLRP3 inflammasome.

This study aimed to evaluate whether dietary supplementation with diphenyl diselenide (Ph2Se2) would prevent the impaired immune and inflammatory responses elicited by methylmercury chloride (CH3HgCl) via protective effects on purinergic signaling in fish immune organs. Tissue and lymphocytic nucleoside triphosphate diphosphohydrolase (NTPDase) activity for adenosine triphosphate (ATP) and adenosine diphosphate (ADP) was downregulated in the head kidney and spleen of grass carp (Ctenopharyngodon idella) exposed to CH3HgCl. Concomitantly, adenosine deaminase (ADA) activity was upregulated. Further, nucleotide-binding oligomerization domain-like receptor (NLRP3) inflammasome gene expression was upregulated in the spleen and head kidney of CH3HgCl-exposed grass carp. Dietary supplementation with Ph2Se2 ameliorated these CH3HgCl-mediated alterations on purinergic enzymes, and their activities returned to baseline levels (except NTPDase activity for ADP). Based on these results, purinergic signaling in immune organs and lymphocytes can be considered a pathway linked to pro-inflammatory effects during exposure to environmental CH3HgCl concentrations, which may contribute to mortality of the affected fish. Since dietary supplementation with 3 mg Ph2Se2/kg in the feed prevented the CH3HgCl-induced alterations, it can be considered a potential suitable treatment to prevent impaired immune and inflammatory responses caused by Hg.

Effects of aloe-emodin on innate immunity, antioxidant and immune cytokines mechanisms in the head kidney leucocytes of Labeo rohita against Aphanomyces invadans.

The effect of aloe-emodin incorporated diets on innate immune response, disease resistance, pro and/or anti-inflammatory cytokine gene transcription in Labeo rohita against Aphanomyces invadans is reported for the first time. In healthy and infected groups fed with 5 mg aloe-emodin enriched diet the white blood cell (WBC) count increased significantly (p > 0.05) after 6th week. In both groups fed with any enriched diet the biochemical parameters such as albumin, globulin, and albumin/globulin ratio did not vary significantly; however with 5 mg aloe-emodin diet the albumin and globulin levels increased significantly (p > 0.05) after 6th week. The serum phagocytic activity (PA), respiratory burst activity (RBA), serum complement C3 (CC3), and lysozyme activity (LA) did not increase with any diet between weeks 2 and 4, whereas with 5 mg aloe-emodin diet increased significantly in both groups after 6th week. The pro-inflammatory cytokines such as IL-1ß, IL-8, TNF-α, and iNOS significantly modulated the expression in both groups on being fed with 5 mg aloe-emodin incorporation diet on 8th week. Healthy fish fed with any aloe-emodin diet did not suffer mortality. However, the infected fish fed with 1, 5, and 10 mg kg-1 aloe-emodin diets registered 5%, 10%, and 15% mortality. The present study indicates that healthy and infected L. rohita exhibited enhanced innate immune response, disease resistance, pro and/or anti-inflamatory cytokine gene transcription levels against A. invadans.

Cadmium disrupts melanocortin 2 receptor signaling in rainbow trout.

Cadmium is an endocrine disruptor and inhibits corticosteroid production, but the mechanisms are far from clear. We tested the hypothesis that sublethal exposure to environmentally realistic levels of cadmium impairs cortisol production by disrupting the melanocortin 2 receptor (MC2R) signaling in rainbow trout (Oncorhynchus mykiss). Fish were exposed to sublethal concentrations of cadmium (0.75 or 2.0 µg/L) in a flow-through system for 7 d and subjected to an acute secondary stressor to evoke a cortisol response. Cadmium exposure for 7 d did not affect plasma cortisol concentrations, but head kidney mc2r mRNA levels were higher than in control fish. The cortisol stress performance to a secondary-stressor was attenuated in the cadmium groups, and this corresponded with transient reduction in transcript abundance of mc2r and the gene encoding its accessory protein MRAP1 but not MRAP2 in the head kidney. Furthermore, in vivo cadmium exposure attenuated the adrenocorticotropic hormone (ACTH)-, but not 8-br-cAMP-stimulated cortisol production in head kidney slices ex vivo. This corresponded with reduced transcript abundance of mc2r and mrap1, but not mrap2 in these tissue slices. Also, reporter assays with CHO cells transiently transfected with rainbow trout mc2r and zebrafish mrap1 revealed a dose-independent inhibition in ACTH-stimulated luciferase activity by cadmium. Collectively, waterborne exposure to environmentally realistic concentration of cadmium compromises the stressor-induced cortisol response, and a mode of action involves the disruption of MC2R signaling in rainbow trout.

Stress hormones modulate lipopolysaccharide stimulation of head kidney interleukin-6 production in the catfish Heteropneustes fossilis: In vivo and in vitro studies.

Interleukin-6 (IL-6) is a pleiotropic cytokine secreted by immune tissues such as monocytes/macrophages and have pro-inflammatory/anti-inflammatory and neuroendocrine actions. In this study, we report the modulatory effects of stress hormones, the cortisol agonist dexamethasone and catecholamines on lipopolysaccharide (LPS) - induced stimulation of head kidney IL-6 in the catfish Heteropneustes fossilis. In the in vivo study, the intraperitoneal administration of LPS stimulated, and dexamethasone time-dependently inhibited IL-6 level. In the in vitro study, the incubation of macrophage cultures with LPS stimulated IL-6 level significantly in all incubation times. Dexamethasone did not alter the basal IL-6 level but inhibited time-dependently the LPS-induced stimulation. Likewise, catecholamines did not alter the basal level of IL-6. Both epinephrine and norepinephrine inhibited the LPS-induced stimulation of IL-6. Dopamine, on the other hand, was ineffective. The results indicate that IL-6 is a useful marker of head kidney macrophage activity for studying endocrine-immune interactions in the catfish.

Molecular characterization of grass carp interleukin-6 receptor and the agonistic activity of its soluble form in head kidney leucocytes.

Interleukin (IL)-6 receptor (IL-6R) can specifically bind to IL-6 and the complex subsequently recruits a transmembrane signal transducer, gp130, to trigger the intracellular signal transduction. IL-6R exists in two forms, a transmembrane IL-6R and a soluble IL-6R (sIL-6R), leading to different signal transduction mechanisms as classic signaling and trans-signaling, respectively. There is now a general consensus that these two modes of signal transduction can mediate anti-inflammatory and pro-inflammatory activities of IL-6. The study on Il-6r is limited although Il-6 has been well studied in teleost. In the present study, a cDNA encoding grass carp Il-6r (gcIl-6r) was isolated. An in-silico analysis showed that gcIl-6r shared the same functional domains and conserved gene synteny at its loci with mouse homologue, and its amino acid sequence was conserved in fish species. A tissue distribution assay demonstrated that gcil6r mRNA was expressed with high levels in immune tissues including spleen and head kidney, and its expression was induced by LPS and Poly I:C in grass carp head kidney leucocytes (HKLs). An in vitro binding assay showed that recombinant soluble gcIl-6r (rgcsIl-6r) could specifically bind to recombinant gcIl-6 (rgcIl-6) protein. Moreover, rgcIl-6 stimulated suppressor of cytokine signaling 3 (socs3)'s mRNA expression in grass carp HKLs and it combined with rgcsIl-6r increased socs3 mRNA expression in CIK cells with gp130 but without Il-6r expression. In HKLs, rgcIl-6 stimulated the mRNA levels of both pro-inflammatory (tnfa and il1b) and anti-inflammatory (il10) cytokines, and rgcsIl-6r could augment these stimulatory effects of gcIl-6. Taken these data together, gcsIl-6r can mediate the immuno-regulatory functions of gcIl-6 and has an agonistic property in these actions of Il-6 in grass carp.

Oestrogen differentially modulates lymphoid and myeloid cells of the European sea bass in vitro by specifically regulating their redox biology.

Besides their obvious role in sex determination and reproduction, oestrogens display a prominent and complex immunomodulatory role across all vertebrates. To date, our knowledge on the oestrogenic immunomodulation in non-mammalian species is, however, scarce. In both teleosts and mammals, the direct immunomodulatory function of oestrogen is underscored by the presence of multiple oestrogen receptor subtypes in the various immune cells. For a better understanding of the regulatory processes, we investigated the oestrogen receptor expression in two major lymphoid organs of European sea bass: the head-kidney and the spleen. All oestrogen receptor subtypes, including nuclear and membrane oestrogen receptors, were present in both immune organs as well as in the isolated leucocytes. The same findings have been previously made for the thymus. To determine the oestrogen responsiveness of the different immune cell populations and to evaluate the importance of non-genomic and genomic pathways, we assessed the kinetics and the concentration dependent effects of 17ß-oestradiol on isolated leucocytes from the head-kidney, the spleen and the thymus in vitro. Given the importance of reactive oxygen species as signalling and defence components in mammalian immune cells, the oxidative burst capacity, the redox status and the viability of both lymphoid and myeloid cells were measured by flow cytometry. The treatment with 17ß-oestradiol specifically modulated these parameters depending on (1) the time kinetic, (2) the concentration of 17ß-oestradiol, (3) the immune cell population (lymphoid and myeloid cells) as well as (4) the lymphoid organs from which they originated. The observed in vitro oestrogenic effects as well the presence of various oestrogen receptor subtypes in the immune cells of sea bass suggest a complex and direct oestrogenic action via multiple interconnected oestrogen-signalling pathways. Additionally, our study suggests that the oestrogenic regulation of the sea bass immune function involves a direct and tissue specific modulation of the immune cell redox biology comprising redox signalling, NADPH-oxidase activity and H2O2-permeability, thus changing oxidative burst capacity and immature T cell fate because oestrogen impacted thymocyte viability. Importantly, immune cells from both primary and secondary lymphoid organs have shown specific in vitro oestrogen-responsiveness. As established in mammals, oestrogen is likely to be specifically and directly involved in immature T cell differentiation and mature immunocompetent cell function in sea bass too.

Potential ability for metallothionein and vitamin E protection against cadmium immunotoxicity in head kidney and spleen of grass carp (Ctenopharyngodon idellus).

Cadmium (Cd) pollution is an important issue affecting the food safety of aquatic products. Cd can impair the immune system and cause irreversible damage to fish and other aquatic organisms. The immunoprotection activities of exogenous metallothionein (MT) and vitamin E (VE) were investigated in Cd poisoned grass carp, Ctenopharyngodon idellus, in the present study. C. idellus were divided into three groups: Cd+phosphate-buffered saline (PBS) group; Cd+MT; and Cd+VE. All fish were injected with cadmium chloride (CdCl2) on the first day and then treated with PBS, MT or VE four days post-injection. Fish not injected with Cd were used as a negative control. Cd exposure caused severe head-kidney and splenic injury in C. idellus, mainly expressed as an increase in Cd content, histological damage, percentage of head-kidney and splenic cells apoptosis and decreases in immune-related gene mRNA transcript expression. However, MT and VE treatments protected against Cd-induced immunotoxicity in C. idellus by decreasing Cd contents, lessening histological damage, reducing the percentage of apoptosis and recovering immune-related mRNA transcript expression. Our results demonstrate that MT and VE can alleviate Cd-induced immunotoxicity and that MT has a more powerful effect than VE, indicating that MT could be a potential antidote in cases of Cd poisoning.

Surplus arginine reduced lipopolysaccharide induced transcription of proinflammatory genes in Atlantic salmon head kidney cells.

In aquaculture production, studies of salmon health and interaction between pathogens and nutrition are of high importance. This study aimed to compare genes and pathways involved in salmon head kidney cells and liver cells, isolated from the same fish, towards polyinosinic acid: polycytidylic acid (poly I:C) and lipopolysaccharide (LPS), with and without addition of surplus arginine. Selected transcriptional responses of genes involved in inflammation, polyamine synthesis, oxidation and apoptosis were elucidated. For the genes related to inflammation, viperin, Mx and Toll like receptor 3 (TLR3), transcription were significantly upregulated by poly I:C in head kidney cells, while viperin was upregulated in liver cells. Surplus arginine did not affect poly I:C induced responses with the exception of reducing poly I:C induced Mx transcription in head kidney cells. Gene transcription of Interleukin 1ß (IL-1ß), Interleukin-8 (IL-8) and cyclooxygenase 2 (Cox2) were elevated during LPS treatment in all liver and head kidney cell cultures. In addition, LPS induced significantly, CD83 transcription in liver cells and TNF-α transcription in head kidney cells. Surplus arginine significantly reduced IL-8, Cox2 and TNF-α transcription in head kidney cells. LPS upregulated arginase in head kidney cells while poly I:C upregulated S-adenosyl methionine decarboxylase (SAMdc) transcription in liver cells. This suggests that LPS and poly I:C modulates genes involved in polyamine synthesis. In addition, in head kidney cells, surplus arginine, when cultured together with LPS, increased the transcription of ornithine decarboxylase (ODC) the limiting enzyme of polyamine synthesis. The genes involved with oxidation and apoptosis were not affect by any of the treatments in liver cells, while LPS decreased caspase 3 transcription in head kidney cells. In liver cells, protein expression of catalase was reduced by surplus arginine alone and when challenged with poly I:C. Both liver cells and head kidney cells isolated from the same individual fish responded to LPS and poly I:C, depending on the gene analyzed. Additionally, arginine could modulate transcription of pro-inflammatory genes induced by LPS in salmon immune cells, thus affecting salmon immunity.

In vitro effects of Origanum vulgare leaf extracts on gilthead seabream (Sparus aurata L.) leucocytes, cytotoxic, bactericidal and antioxidant activities.

Origanum vulgare is a well-known medicinal plant that has been used since ancient times as an additive in foods and cosmetic preparations. The possible application of O. vulgare extracts in fish was assessed by using gilthead seabream (Sparus aurata) as a marine fish model due to its importance in aquaculture. The in vitro effects of aqueous and ethanolic leaf extracts of O. vulgare were tested in order to observe any immunostimulant, cytotoxic, bactericidal or antioxidant properties. The results showed that medium or high concentration of aqueous extracts and low concentrations of ethanolic extract, increased head kidney leucocyte activities as well as the number of SAF-1 cells. However, moderate to high concentrations of ethanolic extracts decreased both leucocyte activities and the number of viable SAF-1 cells, suggesting some possible toxic effect towards them. Only the highest concentration of the aqueous extract and medium to high concentrations of the ethanolic extracts showed cytotoxic activity against the tumor PLHC-1 cell line. Bactericidal activity was only detected against Vibrio harveyi, V. anguillarum and Photobacterium damselae when using the highest concentration of aqueous extract and moderate to high concentrations of ethanolic extract. Finally, both plant extracts presented antioxidant activity particularly the aqueous extract. Overall, the results suggest that both extracts (when used at the appropriate concentration) have immunostimulant, cytotoxic, bactericidal and antioxidant properties, making O. vulgare an interesting candidate for incorporation as additive in functional diets for farmed fish.

Effects of dietary arginine on antioxidant status and immunity involved in AMPK-NO signaling pathway in juvenile blunt snout bream.

The present study assessed the effects of dietary arginine on antioxidant status and immunity involved in AMPK-NO signaling pathway in juvenile blunt snout bream. Fish were fed six practical diets with graded arginine levels ranging from 0.87% to 2.70% for 8 weeks. The results showed that compared with the control group (0.87% dietary arginine level), significantly higher mRNA levels of adenosine monophosphate activated protein kinase (AMPK) and nitric oxide synthetase (NOS), activities of total nitric oxide synthetase (T-NOS) and nitric oxide synthetase (iNOS), and plasma nitric oxide (NO) contents were observed in fish fed with 1.62%-2.70% dietary arginine levels. Significantly higher levels of NOS and iNOS were observed in fish fed with 1.62%-2.70% dietary arginine levels in enzyme-linked immune sorbent assay. At dietary arginine levels of 1.22%-2.70%, the mRNA levels of iNOS were significantly improved. Dietary arginine also significantly influenced plasma interleukin 8 (IL-8) and tumour necrosis factor-α (TNF-α) contents. Furthermore, dietary arginine significantly affected the activity and mRNA level of glutathione peroxidase (GPx), the mRNA levels of pro-inflammatory factor including IL-8 and TNF-α and plasma malondialdehyde (MDA) content. However, total superoxide dismutase (T-SOD) activity, plasma complement component 3 (C3) content, plasma immunoglobulin M (IgM) content, plasma interleukin 1ß (IL-1ß) content and the mRNA levels of copperzinc superoxide dismutase (Cu/Zn-SOD), manganese superoxide dismutase (Mn-SOD) and IL-1ß were not significantly affected by dietary arginine. After Aeromonas hydrophila challenge, the death rate was significantly lowered in fish fed with 1.62%-1.96% dietary arginine levels. Furthermore, the mRNA levels of AMPK, NOS and iNOS, plasma NO content and the activities of T-NOS and iNOS showed an upward trend with increasing dietary arginine levels. Significantly higher levels of NOS and iNOS were observed in fish fed with 1.62%-2.70% dietary arginine levels in enzyme-linked immune sorbent assay. At dietary arginine levels of 1.96%-2.31%, T-SOD activities were significantly improved. Significantly higher GPx activities were observed in fish fed with 1.22%-2.70% dietary arginine levels. At dietary arginine levels of 1.22%-2.31%, the plasma TNF-α and IL-8 contents were significantly decreased. Significantly lower plasma IL-1ß contents were observed in fish fed 1.62%-1.96% dietary arginine levels. Dietary arginine significantly influenced the mRNA levels of antioxidant and pro-inflammatory genes including Cu/Zn-SOD, Mn-SOD, GPx, IL-8, TNF-α and IL-1ß. Significantly higher plasma C3 contents and significantly lower plasma MDA contents were observed in fish fed with 1.62%-1.96% arginine levels. Furthermore, plasma IgM contents were significantly improved at dietary arginine levels of 1.62%-2.31%. However, high dietary arginine group (2.70%) significantly improved the mRNA levels of pro-inflammatory genes including IL-8, TNF-α and IL-1ß and plasma MDA, IL-8, TNF-α and IL-1ß contents as compared with optimal dietary arginine levels (1.62% and 1.96%). The present results indicate that optimal arginine level (1.62% and 1.96%) could improve antioxidant capacity, immune response and weaken tissues inflammatory involved in arginine-AMPK-NO signaling pathway, while high arginine level resulted in excessive NO production, leading to increase oxidative stress damage and inflammatory response in juvenile blunt snout bream.

Effect of glyphosate-based herbicide on hematological and hemopoietic parameters in common carp (Cyprinus carpio L).

The effects of Roundup (glyphosate-based herbicide) on peripheral blood parameters and hematopoietic tissue cellular composition and activity in common carp juveniles were evaluated. The fish were exposed for 7 days at 0.0, 0.1, 0.5, or 5.0 mg/L of glyphosate. In fish exposed to Roundup, hematological alterations were observed; however, most of them were not directly related to the herbicide concentration. An increase in Ht and MCV, and decrease in Hb, MCH, and MCHC compared to the control were observed. Fish exposed to Roundup showed also a reduction in WBC and oxidative metabolic activity of phagocytes (NBT) compared to the control. The fish exposed to 0.1 and 5.0 mg/L showed increased glucose values, whereas in those subjected to 0.5 mg/L blood glucose concentration declined compared to the control. Cholesterol significantly increased at 0.1 mg/L and decreased at 5.0 mg/L. Analysis of head kidney hematopoietic tissue revealed that Roundup at concentrations 0.5 and 5.0 mg/L caused a significant increase in the rate of cell proliferation accompanied by an increase in frequency of early blast cells. No significant differences occurred in percentages of most cell lineages but the frequency of monocytoid, eosinophilic, and basophilic lineage cells significantly increased in the herbicide-exposed fish compared to the control. The obtained results revealed that sublethal concentrations of Roundup that may occur in polluted natural waters caused a slight anemic and significant immunosuppressive response in common carp juveniles. On the other hand, they indicate a considerable compensatory potential of carp hematopoietic system.