Héctor Cordero

Dietary administration of β-1,3/1,6-glucan and probiotic strain Shewanella putrefaciens, single or combined, on gilthead seabream growth, immune responses and gene expression

It is widely known that β-glucans and probiotic bacteria are good immunostimulants for fish. In the present work we have evaluated the dietary effect of β-1,3/1,6-glucan (isolated from Laminarina digitata) and Pdp 11 (Shewanella putrefaciens, probiotic isolated from gilthead seabream skin), single or combined, on growth, humoural (seric level of total IgM antibodies and peroxidase and antiprotease activities) and cellular innate immune response (peroxidase and phagocytic activities of head-kidney leucocytes), as well as the expression of immune-related genes in gilthead seabream (Sparus aurata). Four treatment groups were established: control (non-supplemented diet), Pdp 11 (10(9) cfu g(-1)), β-1,3/1,6-glucan (0.1%) and β-1,3/1,6-glucan + Pdp 11 (0.1% and 10(9) cfu g(-1), respectively). Fish were sampled after 1, 2 and 4 weeks of feeding. Interestingly, all supplemented diets produced increments in the seabream growth rates, mainly the Pdp 11-suplemented diet. Overall, Pdp 11 dietary administration resulted in decreased serum IgM levels and peroxidase activity. However, the seric antiprotease activity was increased in fish fed with both supplements together. Furthermore, β-1,3/1,6-glucan and combined diet increased phagocytic activity after 2 or 4 weeks. At gene level, IL-1β and INFγ transcripts were always up-regulated in HK but only the interleukin reached significance after 4 weeks in the group fed with β-glucan. On the contrary, IgM gene expression tended to be down-regulated being significant after 1 week in seabream specimens fed with β-glucan or β-glucan plus Pdp 11. These results suggest that β-1,3/1,6-glucan and Pdp 11 modulate the immune response and stimulates growth of the gilthead seabream, one of the species with the highest rate of production in Mediterranean aquaculture.

Effect of dietary supplementation of probiotics and palm fruits extracts on the antioxidant enzyme gene expression in the mucosae of gilthead seabream (Sparus aurata L.)

Antioxidant activity is particularly important, since oxidation is an unavoidable reaction in all living bodies. At present, natural antioxidants to be used on food as an alternative to synthetic ones are being sought. Gilthead seabream (Sparus aurata L.) specimens were fed for 4 weeks with diets enriched with bacterial probiotics (Shewanella putrefaciens Pdp11 and Bacillus sp), single or in combination with Tunisian dates palm fruit extracts. The expression of the main antioxidant enzyme genes (superoxide dismutase, catalase and glutathione reductase) in the mucosae (gut, skin and gill) was evaluated after 2 and 4 weeks. Previously, free radical scavenging and several antioxidant assays were developed to know the antioxidant properties present on the palm fruits extracts. The results demonstrated that experimental diets alter the expression of the studied antioxidant genes, primarily in the gill and skin. Furthermore, the tested probiotics and mainly, the aqueous date palm fruits extracts had significant antioxidant properties based on their protective effect against the levels of intracellular reactive oxygen species, especially when administering during 4 weeks. For this reason, probiotics and date palm fruit extracts may serve as good natural antioxidants and could potentially be considered as a functional food ingredient for fish in farms.

The treatment with the probiotic Shewanella putrefaciens Pdp11 of specimens of Solea senegalensis exposed to high stocking densities to enhance their resistance to disease.

Aquaculture industry exposes fish to acute stress events, such as high stocking density, and a link between stress and higher susceptibility to diseases has been concluded. Several studies have demonstrated increased stress tolerance of fish treated with probiotics, but the mechanisms involved have not been elucidated. Shewanella putrefaciens Pdp11 is a strain isolated from healthy gilthead seabream (Sparus aurata L.) and it is considered as probiotics. The aim of this study was to evaluate the effect of the dietary administration of this probiotics on the stress tolerance of Solea senegalensis specimens farmed under high stocking density (PHD) compared to a group fed a commercial diet and farmed under the same conditions (CHD). In addition, during the experiment, a natural infectious outbreak due to Vibrio species affected fish farmed under crowding conditions. Changes in the microbiota and histology of intestine and in the transcription of immune response genes were evaluated at 19 and 30 days of the experiment. Mortality was observed after 9 days of the beginning of the experiment in CHD and PHD groups, it being higher in the CHD group. Fish farmed under crowding stress showed reduced expression of genes at 19 day probiotic feeding. On the contrary, a significant increase in immune related gene expression was detected in CHD fish at 30 day, whereas the gene expression in fish from PHD group was very similar to that showed in specimens fed and farmed with the conventional conditions. In addition, the dietary administration of S. putrefaciens Pdp11 produced an important modulation of the intestinal microbiota, which was significantly correlated with the high number of goblet cells detected in fish fed the probiotic diet.

Effects of date palm fruit extracts on skin mucosal immunity, immune related genes expression and growth performance of common carp (Cyprinus carpio) fry.

The aim of this study was to investigate the effects of date palm fruit extracts (DPFE) on skin mucosal immunity, immune related genes expression and growth performance of fry common carp (Cyprinus carpio). One hundred and twenty specimens (4.06 ± 0.13 g) were supplied and allocated into six aquaria; specimens in three aquaria were fed non-supplemented diet (control) while the fish in the other 3 aquaria were fed with DPFE at 200 ml kg(-1). At the end of feeding trial (8 weeks) skin mucus immune parameters (total immunoglobulins, lysozyme, protease and alkaline phosphatase activity) and immune related gene expression (tumor necrosis factor α [tnfa], lysozyme [ly] and interleukin-1-beta, [il1b]) in the head-kidney were studied. The results revealed that feeding carp fry with 200 ml kg(-1) DPFE remarkably elevated the three skin mucus immune parameters tested (P < 0.05). However, evaluation of immune related gene expression demonstrated that the expression of tnfa and il1b was considerably decreased (P < 0.05) in fish fed DPFE diet, while the expression of ly remained similar (P > 0.05) compared to control fish (fed control diet). Furthermore, growth performance parameters were significantly improved in fry fed DPFE (P < 0.05). More studies are needed to understand different aspects of DPFE administration in fry mucosal immunity.

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 (il1β, bd, mhcIIα, ighm and tcrβ) 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 tcrβ 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.

In vitro immunotoxicological effects of heavy metals on European sea bass (Dicentrarchus labrax L.) head-kidney leucocytes

The knowledge about the direct effects of heavy metals on fish leucocytes is still limited. We investigate the in vitro effects of heavy metals (Cd, Hg, Pb or As) on oxidative stress, viability and innate immune parameters of head-kidney leucocytes (HKLs) from European sea bass (Dicentrarchus labrax). Production of free oxygen radicals was induced by Cd, Hg and As, mainly after 30 min of exposure. Cd and Hg promoted both apoptosis and necrosis cell death while Pb and As did only apoptosis, in all cases in a concentration-dependent manner. Moreover, expression of genes related to oxidative stress and apoptosis was significantly induced by Hg and Pb but down-regulated by As. In addition, the expression of the metallothionein A gene was up-regulated by Cd and Pb exposure though this transcript, as well as the heat shock protein 70, was down-regulated by Hg. Cd, methylmercury (MeHg) and As reduced the phagocytic ability, whereas Hg and Pb increased it. Interestingly, all the heavy metals decreased the phagocytic capacity (the number of ingested particles per cell). Leucocyte respiratory burst changed depending on the metal exposure, usually in a time- and dose-manner. Interestingly, the expression of immune-related genes was slightly affected by Cd, MeHg, As or Pb being Hg the form producing the greatest alterations, which included down-regulation of immunoglobulin M and hepcidin, as well as the up-regulation of interleukin-1 beta mRNA levels. This study provides an in vitro approach for elucidating the heavy metals toxicity, and particularly the immunotoxicity, in fish leucocytes.

Toxicological in vitro effects of heavy metals on gilthead seabream (Sparus aurata L.) head–kidney leucocytes

Heavy metals provoke toxicological effects on aquatic animal species, including fish, though their effects on fish leucocytes and immunotoxicology are still limited. In the present work the effects of heavy metals (Cd, Hg, Pb or As) on viability, oxidative stress and innate immune parameters of isolated head-kidney leucocytes from gilthead seabream (Sparus aurata) are studied. Cytotoxicity results indicated that short exposures (30 min or 2h) to Hg promoted both apoptosis and necrosis cell death of leucocytes whilst Cd, Pb and As did only by apoptosis, in all cases in a concentration- and time-dependent manner. In addition, production of free oxygen radicals was induced by Cd, Hg and As heavy metals. Cd failed to change phagocytosis but Hg and As increased the percentage of phagocytic cells but decreased the number of ingested particles per cell whilst Pb increased both phagocytic parameters. On the other hand, respiratory burst activity was significantly reduced by incubation with Cd, Hg and As but increased with Pb. Furthermore, the gene expression profiles partly support the functional finding of this work. This study provides an in vitro approach for elucidating the heavy metals toxicity, and particularly the immunotoxicity, in fish leucocytes.

In vitro characterization of 6-Coumarin loaded solid lipid nanoparticles and their uptake by immunocompetent fish cells.

The primary aim of the present work was to evaluate the in vitro uptake of 6-Coumarin (6COUM) loaded solid lipid nanoparticles (SLN) by two gilthead seabream (Sparus aurata L.) cell types: an established cell line (SAF-1 cells) and the primary cultures of head-kidney (HK)-the main haemopoietic organ in fish, equivalent to mammalian bone marrow-leucocytes. For this purpose, after the physicochemical characterization of SLN, the uptake by those immunocompetent fish cells was evaluated using flow cytometry and confocal microscopy. Concomitantly, the uptake of 6-COUM loaded SLN was compared with that achieved with 6-COUM loaded pectin microparticles (MPs), which were selected as a competitor of the delivery carriers. After SLN and MP physicochemical characterization, the results demonstrated that SAF-1 cells were able to internalize high percentages of 6-COUM SLNs when incubated for 4, 8 and 24h, with the highest SLN concentration tested (10 μg/ml). The ability of HK leucocytes to internalize SLN was also found to vary depending on both incubation time and SLN concentration. The highest values of HK leucocytes internalizing SLN particles (around 16%) were detected at the maximum SLN concentration (20 μg/ml) at incubation times of 4 or 8h. Conversely, HK leucocytes were unable to internalize MPs at any tested concentration and incubation time. A possible mechanism explaining the uptake into cells is proposed. The present work constitutes the first approximation to consider SLN as nanocarriers for delivering biologically active substances to fish.

Effects of Shewanella putrefaciens on innate immunity and cytokine expression profile upon high stocking density of gilthead seabream specimens

High stocking density increases the number of emerging diseases triggering economic losses worldwide. Probiotics provide an effective and natural solution for preventing some diseases through an improvement of innate immune system among others. In the present work dietary administration of the probiotic Shewanella putrefaciens (known as Pdp11) was evaluated under stress by high stocking density after 2 and 4 weeks of administration to gilthead seabream (Sparus aurata) specimens. Results showed an increase in cellular peroxidase and respiratory burst activity as well as a modulation of cytokine profile when Pdp11 was administered to fish reared at high stocking density. Overall, our results showed how Pdp11 is not only able to improve to some extent the cellular and humoral immunity but also to increase the gene expression profile of pro-inflammatory cytokines such as il1b or il6 in response to high stocking density in gilthead seabream. These findings may support the potential use of this probiotic as functional feed against stress in fish farms.

Dorso-ventral skin characterization of the farmed fish gilthead seabream (Sparus aurata).

The skin is the first barrier of defence in fish, protecting against any external stressor and preserving the integrity and homeostasis of the fish body. The aim of this study was to characterise gilthead seabream skin by isolating cells and studying the cell cycle by flow cytometry, to study the skin histology by scanning electron microscopy and the transcription level of some immune-relevant genes by RT-PCR. Furthermore, the results obtained from samples taken from the dorsal and the ventral part of the specimens are compared. No differences were observed in the cell cycle of cells isolated from the dorsal and ventral zones of the skin or in the gene expression of the genes studied in both epidermal zones. However, the epidermis thickness of the ventral skin was higher than that of the dorsal skin, as demonstrated by image analysis using light microscopy. Besides, scanning electron microscopy pointed to a greater cell size and area of microridges in the apical part of the dorsal epidermal cells compared with ventral skin epidermal cells. This study represents a step forward in our knowledge of the skin structure of an important farmed teleost, gilthead seabream, one of the most commonly farmed fish worldwide. Furthermore, for functional characterization, experimental wounds were carried out comparing the wound healing rate between the dorsal and ventral regions of skin over the time. The results showed higher ratio of wound healing in the ventral region, whose wounds were closed after 15 days, compared to dorsal region of skin. Taking into account all together, this study represents a step forward in our knowledge of the skin structure and skin regeneration of an important farmed teleost, gilthead seabream, one of the most commonly farmed fish worldwide.