Regional asymmetry of metabolic and antioxidant profile in the sciaenid fish shi drum (Umbrina cirrosa) white muscle. Response to starvation and refeeding.

The objective of the present study is to characterize the metabolic and antioxidant profile of white muscle of shi drum in two sites of the body, anterior dorsal (AM) and posterior dorsal (PM) portions. In addition, it will be analyzed the possible effect of starvation and a subsequent refeeding, with two different protocols, pair feeding and ad libitum. Activities of key enzymes of intermediary metabolism and of antioxidant enzymes, as well as lipid peroxidation, as an index of oxidative stress, were evaluated. The results indicate the existence of a regional asymmetry of the metabolic capacities of the white muscle of shi drum, which is likely related to the different contribution to swimming of the body regions examined. Starvation induces a metabolic depression that is more marked in those activities that support burst swimming in PM, while those activities supporting maintenance requirements are conserved. The greatest energy demands during starvation appear to lie in AM, which showed the highest oxidative metabolism rate. The increased use of fatty acids as energy source for AM leads to oxidative stress. A period of more than four weeks of refeeding for full restoration of metabolic capacities in AM is needed, probably related to the higher muscle mass located in this region. On the contrary, all enzyme activities in PM returned to control levels in both refeeding protocols, but pair feeding seems to be advantageous since compensatory growth has been taking place without signs of oxidative stress. This work was addressed to gain knowledge on the physiology of a promising fish species in aquaculture like shi drum. The results displayed here show how the starving and further re-feeding events could generate oxidative stress situations characterized by high lipid peroxidation levels which may influence negatively on the quality of the edible part of the fish. This study opens an interesting field on this fish species which deserves being investigated in the future.

Dietary carbohydrates improve oxidative status of common dentex (Dentex dentex) juveniles, a carnivorous fish species.

Common dentex (Dentex dentex) is an appreciated carnivorous fish with high growth rate and life cycle adaptable to existing farming techniques. Since the use of carbohydrates is an economic and sustainable alternative for a protein-sparing effect, the study of how this macronutrient affects the welfare of carnivorous species must be studied. The aim of the present study was to evaluate the effects of different types and levels of carbohydrates on common dentex oxidative status. Nine isonitrogenous (43%) and isoenergetic (22MJkg-1) diets were formulated combining three types (pregelatinized starch-PS, dextrin-Dx and maltodextrin-Mx) and three levels (12, 18 and 24%) of carbohydrates. The activities of catalase-CAT, superoxide dismutase-SOD, glutathione peroxidase-GPX, glutathione reductase-GR and glucose 6-phosphate dehydrogenase-G6PDH, SOD isoenzymatic profile, lipid peroxidation-LPO and protein oxidation-PO were determined in liver and white muscle. SOD and CAT were not affected. GPX in liver and white muscle and GR in liver increased at higher inclusion carbohydrates levels. The lowest levels of GR and G6PDH in both tissues and LPO in liver were observed in maltodextrin groups. No significant effects by carbohydrate source were observed in liver PO and white muscle LPO. Regarding carbohydrate level effect, 18% and 24% dietary inclusion level decreased LPO in white muscle and PO in liver. LPO in liver was also decreased at 24% inclusion level. Altogether, results indicate the use of carbohydrates as an alternative energy source does not produce negative effects on oxidative status of common dentex, on the contrary, even contribute to their oxidative protection.

Nutritional and metabolic responses in common dentex (Dentex dentex) fed on different types and levels of carbohydrates.

The present study was aimed to evaluate the capacity of common dentex (Dentex dentex) to efficiently use dietary carbohydrates. So, the effects of different type and levels of carbohydrates on growth performance, feed utilization, fish composition, plasma metabolites and key metabolic pathways in liver and white muscle of common dentex are presented. Nine isonitrogenous (43%) and isoenergetic (22 MJ kg(-1)) diets were formulated combining three types, pregelatinized starch (PS), dextrin (Dx) and maltodextrin (Mx), and three levels (12, 18 and 24%) of carbohydrates. Growth performance was not significantly influenced by treatments. The best feed utilization was observed in 18% Mx group. Higher hepatic lipid content was found in fish fed lower dietary carbohydrate levels. PS induced higher liver and white muscle hexokinase and pyruvate kinase activities compared to the lower values observed for Mx. Malic enzyme and glucose 6-phosphate dehydrogenase in liver and white muscle were lower in Mx group. The influence of dietary carbohydrates source was more noticeable than those induced by the carbohydrate level, when glycolysis and lipogenesis pathways were considered. Common dentex is able to use properly dietary carbohydrates, although optimal dietary inclusion levels are below 24%. The greater protein-sparing effect was promoted by the less complex carbohydrate (maltodextrin) and the best feed utilization indices were obtained at intermediate levels of inclusion (18%).

Comparative analysis of the humoral immunity of skin mucus from several marine teleost fish.

Fish skin mucus contains several immune substances that provide the first line of defence against a broad spectrum of pathogens although they are poorly studied to date. Terminal carbohydrate composition and levels of total IgM antibodies, several immune-related enzymes (lysozyme, peroxidase, alkaline phosphatase, esterases, proteases and antiproteases) as well as the bactericidal activity (against fish pathogenic Vibrio harveyi, Vibrio angillarum, Photobacterium damselae and non-pathogenic bacteria Escherichia coli, Bacillus subtilis, Shewanella putrefaciens) were identified and measured in the skin mucus of five marine teleosts: gilthead seabream (Sparus aurata), European sea bass (Dicentrarchus labrax), shi drum (Umbrina cirrosa), common dentex (Dentex dentex) and dusky grouper (Epinephelus marginatus). First, lectin binding results suggests that skin mucus contain, in order of abundance, N-acetylneuraminic acid, glucose, N-acetyl-glucosamine, N-acetyl-galactosamine, galactose and fucose residues. Second, results showed that while some immune activities were very similar in the studied fish (e.g. IgM and lysozyme activity) other such as protease, antiprotease, alkaline phosphatase, esterase and peroxidase activities varied depending on the fish species. High levels of peroxidase and protease activity were found in U. cirrosa respect to the values obtained in the other species while E. marginatus and S. aurata showed the highest levels of alkaline phosphatase and esterase activities, respectively. Moreover, skin mucus of S. aurata revealed higher bactericidal activity against pathogenic bacteria, contrarily, to what happened with non-pathogenic bacteria (E. coli, B. subtilis). Thus, study of the variations in the carbohydrate profile and immune-related components of the fish skin mucus could help to understand the fish resistance as well as the presence and distribution of pathogens and magnitude of infections, aspects that are of major importance for the aquaculture industry.

Identification and functional characterization of a new IL-1 family member, IL-1Fm2, in most evolutionarily advanced fish.

The IL-1 family consists of 11 members that play an important role as key mediators in inflammation and immunity. Here, we report the identification of a new member of the IL-1 family (IL-1Fm2) that is present in species belonging to the most evolutionarily advanced group of teleost fish (Series Percomorpha), including Perciformes, Beloniformes, Gasterosteiformes, Cyprinodontiformes and Pleuronectiformes. However, IL-1Fm2 seems to be absent in Tetraodontiformes, which also belong to the Percomorpha. The expression pattern of gilthead seabream IL-1Fm2 revealed that although it was hardly induced by PAMPs, the combination of PAMPs and recombinant IL-1Fm2 synergistically induced its expression in macrophages and granulocytes. In addition, recombinant IL-1Fm2 was able to activate the respiratory burst of seabream phagocytes and to synergistically induce the expression of IL-1ß, TNF-α, IL-8 and IL-10 when combined with PAMPs. Finally, although gilthead seabream IL-1Fm2 did not show a conserved caspase-1 processing site, macrophages processed IL-1Fm2 before being released. However, both pan-caspase and caspase-1 inhibitors failed to inhibit the processing and release of IL-1Fm2. These results demonstrate an important role of IL-1Fm2 in the regulation of fish immune responses, shed light on the evolution of the IL-1 family in vertebrates and point to the complexity of this cytokine family.

Estrogen signaling through the G protein-coupled estrogen receptor regulates granulocyte activation in fish.

Neutrophils are major participants in innate host responses. It is well known that estrogens have an immune-modulatory role, and some evidence exists that neutrophil physiology can be altered by these molecules. Traditionally, estrogens act via classical nuclear estrogen receptors, but the identification of a G protein-coupled estrogen receptor (GPER), a membrane estrogen receptor that binds estradiol and other estrogens, has opened up the possibility of exploring additional estrogen-mediated effects. However, information on the importance of GPER for immunity, especially, in neutrophils is scant. In this study, we report that gilthead seabream (Sparus aurata L.) acidophilic granulocytes, which are the functional equivalent of mammalian neutrophils, express GPER at both mRNA and protein levels. By using a GPER selective agonist, G1, it was found that GPER activation in vitro slightly reduced the respiratory burst of acidophilic granulocytes and drastically altered the expression profile of several genes encoding major pro- and anti-inflammatory mediators. In addition, GPER signaling in vivo modulated adaptive immunity. Finally, a cAMP analog mimicked the effects of G1 in the induction of the gene coding for PG-endoperoxide synthase 2 and in the induction of CREB phosphorylation, whereas pharmacological inhibition of protein kinase A superinduced PG-endoperoxide synthase 2. Taken together, our results demonstrate for the first time, to our knowledge, that estrogens are able to modulate vertebrate granulocyte functions through a GPER/cAMP/protein kinase A/CREB signaling pathway and could establish therapeutic targets for several immune disorders in which estrogens play a prominent role.

Muscle development and body growth in larvae and early post-larvae of shi drum, Umbrina cirrosa L., reared under different larval photoperiod: muscle structural and ultrastructural study.

Shi drum specimens were maintained under four different photoperiod regimes: a natural photoperiod regime (16L:8D), constant light (24L), equal durations of light and dark (12L:12D) and a reduced number of daylight hours (6L:18D) from hatching until the end of larval metamorphosis. Specimens were then kept under natural photoperiod conditions until 111 days post-hatching. Muscle and body parameters were studied. During the vitelline phase, there was little muscle growth and no photoperiod effects were reported; however, a monolayer of red muscle and immature white muscle fibres were observed in the myotome. At hatching, external cells (presumptive myogenic cells) were already present on the surface of the red muscle. At the mouth opening, some presumptive myogenic cells appeared between the red and white muscles. At 20 days, new germinal areas were observed in the apical extremes of the myotome. At this stage, the 16L:8D group (followed by the 24L group) had the longest body length, the largest cross-sectional area of white muscle and the largest white muscle fibres. Conversely, white muscle hyperplasia was most pronounced in the 24L group. Metamorphosis was complete at 33 days in the 24L and 12L:12D groups. At this moment, both groups showed numerous myogenic precursors on the surface of the myotome as well as among the adult muscle fibres (mosaic hyperplastic growth). The 16L:8D group completed metamorphosis at 50 days, showing a similar degree of structural maturity in the myotome to that described in the 24L and 12L:12D groups at 33 days. When comparing muscle growth at the end of the larval period, hypertrophy was highest in the 16L:8D group, whereas hyperplasia was higher in the 24L and 16L:8D groups. At 111 days, all groups showed the adult muscle pattern typical of teleosts; however, the cross-sectional area of white muscle, white muscle fibre hyperplasia, body length and body weight were highest in the 24L group, followed by the 12L:12D group; white muscle hypertrophy was similar in all groups. Larval survival was higher under natural photoperiod conditions compared to all the other light regimes.

Antioxidant enzymatic defenses and oxidative damage in Dentex dentex fed on different dietary macronutrient levels.

A wide range of antioxidant mechanisms are present in fish maintaining an adequate "oxidative balance". When this balance tilts in favor of the oxidant agents "oxidative stress" arises with detrimental effects in molecules of great biological importance. Little has been reported about the influence of different dietary energy sources on antioxidant defenses in fish. The influence of different dietary macronutrient combinations on the key antioxidant enzyme activity, the oxidative damage to lipids and proteins and the possible modifications in the SOD isoenzymatic pattern were evaluated in liver, white muscle, heart and erythrocytes of common dentex (Dentex dentex). Four experimental diets with different protein:lipid:carbohydrate ratios (43/16/28; 43/24/4; 38/19/28 and 38/24/13) were formulated. In general, neither different dietary macronutrient levels nor the interaction among them induces substantial modifications in enzymatic antioxidant defense mechanisms. Two constitutive SOD isoforms, CuZn-SOD I and Mn-SOD, were detected in the tissues analyzed in all experimental groups, independently of diet formulation, but, a third SOD isoenzyme, CuZn-SOD II seems to be induced in white muscle by higher dietary protein levels. Densitometric analyses of western blotting membranes revealed higher CuZn-SOD expression in the heart of dentex fed on lower dietary protein levels, although these differences did not correlate with the SOD activity. Finally, a direct relation exists between the lipid or protein intake level and occurrence of oxidative damage in different tissue components.

Digestive enzymatic profile of Dentex dentex and response to different dietary formulations.

Digestive physiology of on-growing common dentex (Dentex dentex), including protease, amylase and lipase activity in stomach, pyloric caeca, anterior and posterior intestine, was evaluated. The influence of dietary macronutrient balance on these digestive processes was also assessed. Four experimental diets with different protein:lipid:carbohydrate ratios (43/16/28; 43/24/4; 38/19/28 and 38/24/13) were formulated. The highest activity for acid proteases was located in the stomach at pH 1.5. Alkaline proteolytic activities showed the highest values in the pyloric caeca and posterior intestine at pH 8.5-9.0. Dentex showed substantial amylase activity in the pyloric caeca and posterior intestine. Lipase activity was higher in the pyloric caeca, anterior and posterior intestine and was not detected in the stomach. Feed composition influenced alkaline protease activity in the anterior and posterior intestine and was higher for the diet with less protein and more carbohydrates. Enhanced amylase activity was observed in the pyloric caeca and posterior intestine in those groups fed on higher carbohydrate and lower lipid level diets. High dietary carbohydrate levels produced the highest lipase activity but this only occurred in the anterior intestine. We can conclude that the digestive tract of dentex adapts well to protein digestion and possesses a high potential for digesting the other dietary macronutrients, too. Dietary carbohydrate content seems to induce changes in protease, amylase and lipase activity.

Early presence of immune cells in the developing gonad of the gilthead seabream (Sparus aurata Linnaeus, 1758).

A great deal is known regarding the process of sex differentiation in fish. However, little is known about the presence of immune cells and cytokines in this process. In the gilthead seabream, both immune cells and cytokines play an important role in the tissue reorganization of the gonads during the adult reproductive cycle. We have studied, using light microscopy and immunocytochemistry, the ontogenetic development of the gilthead seabream gonads, focusing on the presence of immune cells and cytokines. We show that the testicular area is quickly differentiated and becomes functional in specimens less than a year old, while the ovarian area differentiates later and continues to develop during the first two years of life. Throughout the morphogenesis process, acidophilic granulocytes were present in the gonad. Interleukin-1b (Il1b) is produced in the testicular area in juveniles and male fish, but not in the ovarian area. Macrophage-colony stimulating factor receptor (Mcsfr) is not produced in the undifferentiated gonad and is only found once the testicular area is well developed.

Use of different combinations of macronutrients in diets for dentex (Dentex dentex): effects on intermediary metabolism.

The influence of the dietary macronutrient balance on the intermediary metabolism of common dentex (Dentex dentex L.) was evaluated. Four experimental diets combining high and low levels of macronutrients were formulated. Dentex fed on 43% protein had higher liver and muscle lipid content, corresponding with an increased hepatic G6PDH activity. This "excess" of hepatic lipids at higher protein levels could be used to obtain energy as would be reflected by hepatic HOAD. In the liver, 43% of dietary protein induced higher AlaAT and FBPase activities. Similarly, dentex fed on the P(43)C(28) and P(38)C(28) diets showed an increased hepatic and muscular gluconeogenic pathways (higher FBPase activity) from amino acids (elevated AlaAT) and/or glycerol (elevated GK). However, changes in glycemia were not observed among dietary treatments. At coronary level, the use of lower dietary protein induced an increase in the activity of glycolytic (PK and HK-IV) and lipolytic (HOAD) enzymes. Considering the overall results and the experimental conditions, it could be suggested that dietary protein could be reduced until 38% without affecting negatively the normal physiology of dentex. Moreover, high dietary carbohydrate levels could not be used efficiently by dentex given that gluconeogenesis occurs.

Oxidative stress and antioxidant defenses after prolonged starvation in Dentex dentex liver.

The aim of this work was to evaluate the effects of prolonged starvation and refeeding on antioxidant status and some metabolic-related parameters in common dentex (Dentex dentex) liver. Fish deprived of food for 5 weeks showed a significant increase in lipid peroxidation, measured as malondialdehyde (MDA) levels. The activity of the antioxidative enzymes superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPX) in starved fish significantly increased (by 42%, 22%, and 52%, respectively), whereas glutathione reductase (GR) activity was significantly depressed by 53% compared to controls. No qualitative changes in the SOD isoenzymatic pattern were detected by nondenaturing PAGE analysis, but the isoforms corresponding to CuZn-SOD I and II were enhanced in starved fish. The activity of the enzymes indicative of oxidative metabolism, beta-hydroxyacyl CoA dehydrogenase (HOAD) and citrate synthase (CS), significantly increased (by 123% and 28%, respectively), and that of glucose-6-phosphate dehydrogenase (G6PDH) was inhibited by 56%. Oxidative damage under these circumstances is reversible since all biomarkers assayed returned to control values after refeeding. Our results show that prolonged starvation leads to a pro-oxidant situation and oxidative stress despite activation of antioxidant defense mechanisms, and that inhibition of G6PDH activity might be responsible for this failure in cellular antioxidant defenses.