In vitro effects of cantharidin on gilthead seabream (Sparus aurata) head-kidney leucocytes.

Cantharidin is a toxic vesicant terpene used in folk and traditional medicine due to its various therapeutic effects. Since there are no previous data on the effect of cantharidin in fish, this study aimed to investigate the in vitro related-inflammatory effects of cantharidin in gilthead seabream (Sparus aurata L.) head-kidney leucocytes (HKLs). In the first experiment, the HKLs were incubated with 0, 5 and 10 µg mL-1 of cantharidin for 24 h to delimit its possible toxic effects. In a second experiment, leucocytes were incubated with ranging concentrations from 0 to 10 µg mL-1 for 3, 6, or 12 h. Cell viability was higher in acidophilic granulocytes than in monocytes/macrophages and lymphocytes. Cantharidin caused apoptosis as was evidenced by transmission electron microscopy. In addition, cantharidin produced a time- and dose-dependent decrease of respiratory burst and phagocytic activities in HKLs, while their peroxidase activity was increased at 24 h of incubation with 5 and 10 µg mL-1 of cantharidin. Different changes in the gene expression were observed after incubation with cantharidin. While the gene expression of tnfa, il1b and crel was up-regulated in HKLs, the nfkb1 and igmh genes were down-regulated in comparison to the expression found in control HKLs. Present results offer a first view of the possible effects and action mechanisms of cantharidin in HKLs, as well as its implication in the inflammatory process, which could be of interest not only for basic research but also in the aquaculture sector.

Skin wound healing in gilthead seabream (Sparus aurata L.) fed diets supplemented with arginine.

Dietary administration of arginine on the wound healing process of gilthead seabream was studied. Two replicates of fish (n = 8) were fed with either a commercial diet [control diet (CON), no arginine added] and the CON diet supplemented with 1% arginine (ARG1) or with 2% arginine (ARG2) for 30 days. Afterward, half of the fish were sampled while the other half were injured and continued to be fed the same diet for an extra week. Results by image analysis showed that the wound closure rate was significantly improved in fish that were fed the ARG1 diet, compared with those in the CON group. After seven days of wound healing, the aminotransferase and creatine kinase levels in the serum and the protease and peroxidase activities in the skin mucus were down-regulated, while the immunoglobulin M level in the skin mucus was up-regulated in the ARG1 group after wounding and in the CON group before wounding. Compared with the CON diet, the ARG1 diet remarkedly depressed the gene expression of mpo, il-8, and tnf-α, and enhanced the gene expression of tgf-ß1, igf-1, pcna, krt2, mmp9, fn1α, and colIα and the antioxidant enzyme cat in the skin tissues after wounding. Furthermore, compared with both the ARG1 and the CON groups, negative effects of the ARG2 diet on wound healing were demonstrated. In conclusion, a 1% arginine supplementation facilitates skin wound healing and prevents a systemic inflammation reaction by alleviating the inflammatory response and enhancing the re-epithelialization and ECM biosynthesis in skin wound sites.

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.

Sterigmatomyces halophilus ß-glucan improves the immune response and bacterial resistance in Pacific red snapper (Lutjanus peru) peripheral blood leucocytes: In vitro study.

ß-Glucans are naturally occurring polysaccharides that are produced by bacteria, fungi and yeast. They are considered immunostimulants in fish acting on non-specific defense mechanism. Yeast-derived glucans from cell wall (Sterigmatomyces halophilus, ß-Gluc/Sh) have been used for this purpose in this study. Therefore, an in vitro assay using peripheral blood leucocytes (PBLs) from Pacific red snapper was performed to evaluate the stimulant effects of ß-Gluc/Sh and zymosan A (positive control) for 12 and 24 h and after bacterial challenge with Aeromonas hydrophila at 24 h. In addition, structural characterization of this marine yeast glucan was performed by proton nuclear magnetic resonance (NMR) revealing structures containing (1-6)-branched (1-3)-ß-D-glucan. PBLs responded positively to ß-Gluc/Sh where cell viability was higher than 80%. After challenge, ß-Gluc/Sh was able to inhibit cytotoxicity caused by A. hydrophila, highlighting that the PBLs incubated with ß-Gluc/Sh significantly increased the non-specific immune response, such as phagocytic activity, respiratory burst, nitric oxide and peroxidase activities followed by an increase in superoxide dismutase and catalase activities after 12 and 24 h post-stimulation and after challenge with the pathogen. Regarding induction of antioxidant gene expression, it was more pronounced in stimulated ß-Gluc/Sh leucocytes compared to other groups at all experimental times of the trial and after bacterial challenge. Indeed, our results clearly showed the ability of leucocytes to strongly react to ß-Gluc/Sh with an increase in cytokine gene expression, particularly the IL-1ß, IL-10 and IL-17 genes. These results confirm that S. halophilus yeast-derived ß-glucan, isolated from an extreme marine environment, is beneficial for increasing innate immune response and enhancing resistance against A. hydrophila in vitro.

Dietary dehydrated lemon peel improves the immune but not the antioxidant status of gilthead seabream (Sparus aurata L.).

Lemon (Citrus limon) is the third most important species of citrus in the world, while Spain is the major producer in Europe. Numerous beneficial effects of lemon are known, which explains their use in traditional medicine. The paper describes the effect of dietary dehydrated lemon peel (a sub-product of the lemon industry) on the growth, immune and antioxidant status of gilthead seabream (Sparus aurata L.) over a period of 30 days. Fish fed diets enriched with dehydrated lemon peel (1.5% and 3%) for 15 days showed improved growth and both humoral (seric immunoglobulin M) and cellular (peroxidase activity and phagocytic ability of head kidney leucocytes) immunity, as well as the expression of some immune-related genes (nkefa, il1ß, igth and csfr1). However, decreases growth promotion was observed after thirty days of trial. Neither the anti-oxidant enzymes activity nor the expression of several anti-oxidants and anti-stress genes in liver was improved by the diet. The possible inclusion of dehydrated lemon peel in fish diets for its immunostimulant effects is discussed.

Influence of waterborne arsenic on nutritive and potentially harmful elements in gilthead seabream (Sparus aurata).

Fish are an important source of nutrients in human nutrition. Although arsenic (As) is considered potentially carcinogenic for human being, very little is known about its toxicity in fish biology. To increase our knowledge of the effect of exposure to waterborne As on fish, gilthead seabream (Sparus aurata) were exposed to 5 µM As2O3 and the bioaccumulation of macronutrients (Ca, K, Mg, Na, P), micronutrients (Fe, Mn, Zn) and Potentially Harmful Elements (As, Cd) was determined using spectrometric techniques. All elements were determined in the muscle and liver of non-exposed fish and those exposed to As for 2, 10 or 30 days. The concentrations of K, Na, Mg, Mn and Zn (in muscle) and Fe and Mn (in liver) of control (non-exposed) fish were higher than those determined in exposed fish. Furthermore, neither As nor Cd accumulated in the edible part (muscle) of seabream and were only evident in liver after 30 days of continuous exposure to As, but both concentrations remained below legally established limits.

Long-term intake of white tea prevents oxidative damage caused by adriamycin in kidney of rats.

BACKGROUND: White tea infusion (Camelia sinensis) has antioxidants properties. The infusion contains polyphenols that have been proposed to induce antioxidant response element (ARE) response via nuclear factor E2-related factor 2 (NRF2). Adriamycin (ADR) has antitumour properties and oxidative effects. Oxidative stress is related to a variety of kidney diseases. Prevention of the oxidative stress through long-term intake of white tea and the study of the molecular mechanisms involved in protection could be of great interest. Rats were given distilled water, 0.015 or 0.045 g of solid white tea extract kg(-1) body weight for 12 months. Animals received an injection of ADR. In kidney, oxidative stress parameters were measured, the expressions of nuclear factor E2-related factor 2 gene (Nrf2), and detoxifying and antioxidants genes were analysed, and the activities of catalase (CAT), superoxide dismutase (SOD) and glutathione reductase (GR) were measured. RESULTS: ADR administration increased oxidative parameters and decreased the antioxidant activity; significantly increased the expression of analysed genes and the activity of CAT and SOD and decreased GR activity. The highest white tea dose protected redox status and inhibited ARE response. CONCLUSION: Long-term intake of white tea protected kidney against the oxidative stress. ADR activated the ARE response but in animals treated with the highest dose of white tea, this response was inhibited, probably for antioxidant protection. © 2015 Society of Chemical Industry.

Modulation of immunity and gut microbiota after dietary administration of alginate encapsulated Shewanella putrefaciens Pdp11 to gilthead seabream (Sparus aurata L.).

The potential benefits of probiotics when administering to fish could improve aquaculture production. The objective of this study was to examine the modulation of immune status and gut microbiota of gilthead seabream (Sparus aurata L.) specimens by a probiotic when administered encapsulated. Commercial diet was enriched with Shewanella putrefaciens Pdp11 (SpPdp11, at a concentration of 10(8) cfu g(-1)) before being encapsulated in calcium alginate beads. Fish were fed non-supplemented (control) or supplemented diet for 4 weeks. After 1, 2 and 4 weeks the main humoral and cellular immune parameters were determined. Furthermore, gene expression profile of five immune relevant genes (il1ß, bd, mhcIIα, ighm and tcrß) was studied by qPCR in head kidney. On the other hand, intestinal microbiota of fish was analysed at 7 and 30 days by DGGE. Results demonstrated that administration of alginate encapsulated SpPdp11 has immunostimulant properties on humoral parameters (IgM level and serum peroxidase activity). Although no immunostimulant effects were detected on leucocyte activities, significant increases were detected in the level of mRNA of head-kidney leucocytes for mhcIIα and tcrß after 4 weeks of feeding the encapsulated-probiotic diet. The administration of SpPdp11 encapsulated in alginate beads produced important changes in the DGGE patterns corresponding to the intestinal microbiota. Predominant bands related to lactic acid bacteria, such as Lactococcus and Lactobacillus strains, were sequenced from the DGGE patterns of fish fed the probiotic diet, whereas they were not sequenced from fish receiving the control diet. The convenience or not of probiotic encapsulation is discussed.

Increases in immune parameters by inulin and Bacillus subtilis dietary administration to gilthead seabream (Sparus aurata L.) did not correlate with disease resistance to Photobacterium damselae.

The present work evaluates the effects of inulin and Bacillus subtilis, single or combined, on immune parameters, immune-related gene expression and protection against Photobacterium damselae subsp. piscicida in gilthead seabream (Sparus aurata). Three trials were conducted. In the first trial, different concentrations of inulin (10, 15 and 30 g kg(-1)) (as a prebiotic) were administered to determine the optimal concentration for stimulating the seabream's immune system. In the second trial, the optimum concentration of inulin (10 g kg(-1)) was combined with B. subtilis (as a probiotic). Following two and four weeks of the treatment, the main immune parameters, as well as the expression of seven immune-related genes, were measured. In the final trial, fish fed the same diet as in the second trial were challenged intraperitoneally with P. damselae subsp. piscicida (10(9) cfu g(-1)). Treatment groups for the second and third trial were control (non-supplemented diet), inulin (10 g kg(-1)), B. subtilis (10(7) cfu g(-1)) and inulin + B. subtilis (10 g kg(-1) and 10(7) cfu g(-1) respectively). Dietary administration of inulin or B. subtilis for two weeks stimulated the serum complement activity and the IgM level, as well as leucocyte phagocytic activity; furthermore, inulin stimulated leucocyte respiratory burst activity. When inulin and B. subtilis were administered together (as a synbiotic), only the serum complement activity and the IgM level increased in a statistically significant manner. Furthermore, the complement activity showed a significant increase in fish fed the three experimental diets for four weeks. The challenge experiment showed that the fish fed inulin or the synbiotic diet had non-significantly lower or significantly higher cumulative mortality, respectively, compared with the control group (non-supplemented diet). These results suggest that inulin and B. subtilis modulate the immune response of the gilthead seabream, although the combined administration increases susceptibility to infection by P. damselae subsp. piscicida.