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Proteinograms, a semiquantitative analytical method that separates proteins into multiple bands, have not been explored in teleosts for diagnostic or prognostic purposes. This study aimed to establish reference values for proteinograms in the serum of gilthead seabream (Sparus aurata) and European sea bass (Dicentrarchus labrax), two important farmed fish species in the Mediterranean region. Serum proteins were studied using SDS-PAGE, electropherogram, and HPLC-mass spectrometry. SDS-PAGE analysis revealed four major bands of proteins around 11, 25, 70, and 100 kDa in the serum of gilthead seabream and European sea bass. Electropherogram results showed that a protein with a molecular weight of 76.8 kDa was the most abundant protein in the serum of gilthead seabream, while a peak of 75.5 kDa was the most abundant in European sea bass. HPLC-mass spectrometry detected 87 proteins and 119 proteins in the serum of gilthead seabream and European sea bass, respectively, including α1-globulins, α2-globulins, β-globulins, and γ-globulins. Notably, the albumin sequence was not detected in either of the two species. These results help to characterize the serum protein profile and to establish reference proteinograms for these two fish species. They also provide a basis for the development of novel approaches for the rapid detection of loss of haemostasis due to stress, health disorders or disease in farmed fish., Competing Interests: Declaration of competing interest All authors declare that they have no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Total protein levels in fish are widely used in health and welfare studies, providing a simple and accessible measure. However, the multifaceted role of blood proteins makes it sometimes challenging to link total protein content to specific health issues, while specific protein fractions may offer more precise insights into fish biology and health, particularly in farmed fish species where such data is often lacking. Data were gathered from two experiments involving Dicentrarchus labrax and Sparus aurata, key species in European marine aquaculture. The aim was (1) to assess how different globulin fractions contribute to total protein content in blood and (2) how these contributions vary across different sampling times in healthy animals. In D. labrax, the beta1 globulin fraction emerged as the major contributor (34.16%), followed by albumin and alpha2 globulins (18.24% and 16.41%, respectively). In contrast, pre-albumins and alpha1 fractions had the least contribution (5.49% and 7.71%). S. aurata exhibited albumin as the primary contributor (23.39%), followed by beta1 and alpha2 globulins (19.71% and 19.15%, respectively), with gamma and alpha1 fractions contributing the least (5.34% and 8.63%). Notably, the study revealed relatively stable contributions of globulin fractions to total proteins within both species, albeit with minor variations over time, potentially linked to environmental and individual factors. Furthermore, larger fish displayed higher total protein levels. This research underscores the need for further investigation into the diverse factors influencing globulin contributions to total proteins, ultimately enhancing health and welfare monitoring for farmed fish species., (© 2024. The Author(s).)

Photobacteriosis is a septicaemic bacterial disease affecting several marine species around the globe, resulting in significant economic losses. Although many studies have been performed related to the pathogen virulence and resistance factors, information regarding the host defence mechanisms activated once an infection takes place is still scarce. The present study was designed to understand innate immune responses of farmed juvenile gilthead seabream ( Sparus aurata ) after Photobacterium damselae subsp. piscicida ( Phdp ) infection. Therefore, two groups of seabream juveniles were intraperitoneally injected with 100 µL of PBS (placebo) or 100 µL of exponentially growing Phdp (1 × 10 6 CFU/mL; infected). The blood, plasma, liver, and head kidney of six fish from each treatment were sampled immediately before infection and 3, 6, 9, 24 and 48 h after infection for the broad screening of fish immune and oxidative stress responses. Infected animals presented marked anaemia, neutrophilia and monocytosis, conditions that are correlated with an increased expression of genes related to inflammation and phagocytic activity. Similar studies with different fish species and bacteria can be useful for the definition of health biomarkers that might help fish farmers to prevent the occurrence of such diseases.

Cultured fish can be induced to swim, although the suitability and benefits remain to be tested. Sustained swimming exercise (SSE) training and detraining (DET) were applied in juvenile gilthead sea bream (Sparus aurata) and the metabolic rates were investigated. Fish with a total body mass of 80.5 ± 1.5 g and total length 17.2 ± 0.1 cm were maintained untrained (spontaneously swimming activity, UNT), swim-trained (induced sustained swimming activity, SSE) at 1 BL s -1 for 28 days, or detrained (28 days of swimming followed by 10 days of untraining, DET). Standard metabolic rate (SMR), maximum metabolic rate (MMR), and excess post-exercise oxygen consumption (EPOC) were assessed (n = 10). In addition, the effects of SSE training (51 days) on blood and plasma parameters were investigated before and immediately after applying a high-intensity swimming (HIS) protocol. SMR, MMR, and EPOC values were not different between SSE, UNT, or DET fish (143.2, 465.5 mg O 2 kg -1  h -1 , and 459.1 mg O 2 kg -1 , respectively). Spite the lack of differences between treatments, the dispersion in the residuals for SMR, MMR, and absolute aerobic scope (AAS) values followed the order UNT > DET > SSE, indicating that swim training decreases the individual variation of these metabolic parameters. Haematological parameters, plasma glucose, lactate, and cortisol levels were similar between SSE and UNT groups before HIS. Plasma glucose and lactate levels increased in both groups after HIS, being higher in the SSE group. Plasma cortisol levels were similar between both groups after HIS. Results suggest that SSE training improves energy use and reduces individual variation in SMR and MMR, an effect that declines with detraining., (Copyright © 2021 Elsevier Inc. All rights reserved.)

This study aimed to find the relation of Sparus aurata (gilthead seabream) reproductive activities to some blood parameters as complete blood count, liver enzymes, some hormones related to reproduction process and microscopic findings of gonads, as well as expression of Bromodomain testis-specific gene. Eighty-eight sexually mature seabream were collected and investigated through the four seasons. Red blood cells were higher in autumn and spring. Hemoglobin was high in summer, MCV highest values ​​were seen in winter and summer, while MCHC was highest in summer. The values ​​of white blood cells increased significantly in spring, summer, and autumn compared with winter. The highest value of lymphocytes was recorded in spring and autumn. Eosinophil was recorded the highest value in the spring. The highest value of segmented neutrophils was recorded in summer. The highest value of band neutrophil was recorded in summer and winter. Alanine aminotransferase and aspartate aminotransferase showed high values in the winter. Luteinizing hormone (LH) was higher in females, males, and hermaphrodites during winter. Follicle-stimulating hormone (FSH) was higher in females during spring. The highest value of estradiol 17-β and progesterone was recorded in summer. The highest value of total testosterone was recorded in spring. Microscopically, ovaries were immature and inactive during spring and summer but well developed in autumn and winter. During spring and summer, testes were immature and began spermatogenesis process but well developed with the appearance of spermatids and spermatozoa during autumn and winter. The expression of Brdt was higher in testes than ovary. Brdt recorded high expression in autumn and spring than in summer and winter., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

We evaluated the efficacy of mozuku fucoidan supplementation to alternative dietary proteins used in fish meal (FM) replacement to enhance growth, immunity, and stress resistance of Pagrus major. Seven isonitrogenous (45% protein) experimental diets were formulated where diet 1 (D1) was FM-based control diet. Diets 2 to 7 were formulated by replacing 25, 50, and 75% of FM protein with soy protein isolate (SPI) protein, and each replacement level was supplemented without or with fucoidan at 0.4% for diet groups D2 (FM25), D3 (FM25Fu), D4 (FM50), D5 (FM50Fu), D6 (FM75), and D7(FM75Fu), respectively. Each diet was randomly allocated to triplicate groups of fish (4.1 g) for 56 days. Significantly higher weight gain and specific growth rate were observed in fish fed FM50Fu diet group, and it was not differed (P > 0.05) with fish fed FM25Fu diet group. FM-based control diet showed intermediate value, and it was not differed (P > 0.05) with or without fucoidan-supplemented ≤ 50% FM replacement groups and FM75Fu diet group. Significantly lower growth performances were observed in FM75 diet group. At each replacement level, fucoidan-supplemented groups showed nonsignificant improvement of feed utilization performances. Fish fed fucoidan-supplemented diets showed best condition of oxidative and freshwater stress resistance. Lysozyme activity, NBT, and peroxidase activity showed higher (P > 0.05) values in fucoidan-supplemented groups compared with the non-supplemented groups. Catalase activity was significantly lower in FM75Fu diet group. Catalase activity is significantly influenced by the interaction effects of fucoidan and FM replacement level. In conclusion, fucoidan supplementation could increase the efficiency of utilizing SPI (≥ 75%) without any adverse effects on red sea bream performance.

Hypoxia is an increasing threat to aquatic ecosystems and its impact on economically and ecologically important marine fish species needs to be studied. Especially, the consequences of hypoxia when occurring along with harmful algal blooms (HABs) are currently not well documented. In this study, we investigated the effect of constant and intermittent (daily and weekly) hypoxia on respiration, immunity, hematological parameters, and oxidative status of red seabream for 2, 4, and 6 weeks. Under constant and daily intermittent hypoxia, respiration rate significantly increased in 2 weeks compared to the control. Constant and daily intermittent hypoxia caused significant decreases in the activity of alternative complement pathway, lysozyme, and the level of total immunoglobulin (Ig), as well as significant increases in the concentrations of cortisol, hemoglobin, red blood cells, and white blood cells. A significantly higher level of malondialdehyde was measured for all hypoxia-exposed groups, indicating lipid peroxidation and oxidative stress. At 4 and 6 week, the level of glutathione and enzymatic activities of glutathione reductase and glutathione peroxidase were significantly decreased after constant and daily intermittent hypoxia challenge. The enzymatic activities of superoxide dismutase and catalase were significantly increased at 2 and 4 weeks, but they were decreased after 6 weeks by constant and daily intermittent hypoxia. Constant and daily intermittent hypoxia with subsequent non-toxin producing dinoflagellate Cochlodinium polykrikoides treatment significantly reduced the respiration rate in 3 and 24 h exposure and survival rate of red seabream. Taken together, the red seabream can be vulnerable to HABs under hypoxia condition through inhibition of immunity and antioxidant defense ability. Our findings are helpful in better understanding of molecular and physiological effects of hypoxia, which can be used in aquaculture and fisheries management., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

In this study, the effect of ammonia derived from different stocking densities on immunological, hematological, and oxidative stress parameters was analyzed in the blood or liver of red seabream. Density- and time-dependent increases in inorganic nitrogen compounds were measured for 20 days by analyzing the three major inorganic nitrogen compounds, total ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen. Three immunity parameters, alternative complement activity, lysozyme activity, and total immunoglobulin content were significantly decreased in the blood at the highest stocking density (10 kg m -3 ). The concentrations of hemoglobin and white blood cells were significantly decreased at 10 kg m -3 , while there was no significant change in red blood cells. The significant increases in cortisol level and the enzymatic activities of alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase at 10 kg m -3 clearly supported inorganic nitrogen compounds-triggered stress. A significant elevation of lipid peroxidation value and depletion of intracellular glutathione were observed at 5 and/or 10 kg m -3 in the liver tissue. The hepatic enzymatic activities of antioxidant defense enzymes, catalase and superoxide dismutase were also significantly increased. When a protein skimmer removes the inorganic nitrogen compounds at the highest density, most parameters showed no significant change. Taken together, these results suggest that accumulated inorganic nitrogen compounds at the highest stocking density inhibit innate immunity and induce oxidative stress in red seabream. This information will be helpful to maintain homeostasis of red seabream by controlling immunity and oxidative status through inorganic nitrogen compounds removal in intensive culture condition., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Several amino acids (AA) are known to regulate key metabolic pathways that are crucial for immune responses. In particular, arginine (ARG) appears to have important roles regarding immune modulation since it is required for macrophage responses and lymphocyte development. Moreover, citrulline (CIT) is a precursor of arginine, and it was reported as an alternative to ARG for improving macrophage function in mammals. The present study aimed to explore the effects of dietary ARG and CIT supplementation on the gilthead seabream ( Sparus aurata ) immune status. Triplicate groups of fish (23.1 ± 0.4 g) were either fed a control diet (CTRL) with a balanced AA profile, or the CTRL diet supplemented with graded levels of ARG or CIT (i.e., 0.5 and 1% of feed; ARG1, CIT1, ARG2, and CIT2, respectively). After 2 and 4 weeks of feeding, fish were euthanized and blood was collected for blood smears, plasma for humoral immune parameters and shotgun proteomics, and head-kidney tissue for the measurement of health-related transcripts. A total of 94 proteins were identified in the plasma of all treatments. Among them, components of the complement system, apolipoproteins, as well as some glycoproteins were found to be highly abundant. After performing a PLS of the expressed proteins, differences between the two sampling points were observed. In this regard, component 1 (61%) was correlated with the effect of sampling time, whereas component 2 (18%) seemed associated to individual variability within diet. Gilthead seabream fed ARG2 and CIT2 at 4 weeks were more distant than fish fed all dietary treatments at 2 weeks and fish fed the CTRL diet at 4 weeks. Therefore, data suggest that the modulatory effects of AA supplementation at the proteome level were more effective after 4 weeks of feeding and at the higher inclusion level (i.e., 1% of feed). The bactericidal activity increased in fish fed the highest supplementation level of both AAs after 4 weeks. Peripheral monocyte numbers correlated positively with nitric oxide, which showed an increasing trend in a dose-dependent manner. The colony-stimulating factor 1 receptor tended to be up-regulated at the final sampling point regardless of dietary treatments. Data from this study point to an immunostimulatory effect of dietary ARG or CIT supplementation after 4 weeks of feeding in the gilthead seabream, particularly when supplemented at a 1% inclusion level., (Copyright © 2020 Ramos-Pinto, Azeredo, Silva, Conceição, Dias, Montero, Torrecillas, Silva and Costas.)

Background: Aquaculture is a fast-growing industry and therefore welfare and environmental impact have become of utmost importance. Preventing stress associated to common aquaculture practices and optimizing the fish stress response by quantification of the stress level, are important steps towards the improvement of welfare standards. Stress is characterized by a cascade of physiological responses that, in-turn, induce further changes at the whole-animal level. These can either increase fitness or impair welfare. Nevertheless, monitorization of this dynamic process has, up until now, relied on indicators that are only a snapshot of the stress level experienced. Promising technological tools, such as proteomics, allow an unbiased approach for the discovery of potential biomarkers for stress monitoring. Within this scope, using Gilthead seabream (Sparus aurata) as a model, three chronic stress conditions, namely overcrowding, handling and hypoxia, were employed to evaluate the potential of the fish protein-based adaptations as reliable signatures of chronic stress, in contrast with the commonly used hormonal and metabolic indicators., Results: A broad spectrum of biological variation regarding cortisol and glucose levels was observed, the values of which rose higher in net-handled fish. In this sense, a potential pattern of stressor-specificity was clear, as the level of response varied markedly between a persistent (crowding) and a repetitive stressor (handling). Gel-based proteomics analysis of the plasma proteome also revealed that net-handled fish had the highest number of differential proteins, compared to the other trials. Mass spectrometric analysis, followed by gene ontology enrichment and protein-protein interaction analyses, characterized those as humoral components of the innate immune system and key elements of the response to stimulus., Conclusions: Overall, this study represents the first screening of more reliable signatures of physiological adaptation to chronic stress in fish, allowing the future development of novel biomarker models to monitor fish welfare.

Sea bream (Sparus aurata Linneaus) was acclimated to three salinity concentrations, viz. 5 (LSW), 38 (SW) and 55psμ (HSW) and three water temperatures regimes (12, 19 and 26 °C) for five weeks. Osmoregulatory capacity parameters (plasma osmolality, sodium, chloride, cortisol, and branchial and renal Na + ,K + -ATPase activities) were also assessed. Salinity and temperature affected all of the parameters tested. Our results indicate that environmental temperature modulates capacity in sea bream, independent of environmental salinity, and set points of plasma osmolality and ion concentrations depend on both ambient salinity and temperature. Acclimation to extreme salinity resulted in stress, indicated by elevated basal plasma cortisol levels. Response to salinity was affected by ambient temperature. A comparison between branchial and renal Na + ,K + -ATPase activities appears instrumental in explaining salinity and temperature responses. Sea bream regulate branchial enzyme copy numbers (V max ) in hyperosmotic media (SW and HSW) to deal with ambient temperature effects on activity; combinations of high temperatures and salinity may exceed the adaptive capacity of sea bream. Salinity compromises the branchial enzyme capacity (compared to basal activity at a set salinity) when temperature is elevated and the scope for temperature adaptation becomes smaller at increasing salinity. Renal Na + ,K + -ATPase capacity appears fixed and activity appears to be determined by temperature., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

This study investigated the effect of the dietary supplementation of bovine lactoferrin (LF) on growth performance, hematological and immunological parameters, antioxidant enzymes activity and disease resistance against Vibrio harveyi in yellowfin sea bream (Acanthopagrus latus) fingerling. The fish with initial body weight 10 ± 0.3 g were randomly distributed at 10 fish per each 250 L fiberglass tank, and fed with four experimental diets (a control basal diet and three supplemented diets with 400, 800 and 1200 mg LF kg -1 diet) for 8 weeks. The obtained results showed that fish fed with LF supplemented diets had significantly higher final body weight as compared to control diet (P < 0.05). There were no significant differences between LF-treatments and the control group in white blood cell counts, red blood cell counts, hemoglobin and hematocrit. Total protein and complement activity (ACH50) in the serum of yellowfin sea bream were enhanced with increasing the dietary LF supplementation level (P < 0.05). The mucus lysozyme activity in fish fed on 800 and 1200 mg LF kg -1 was significantly higher than those fed on 400 mg LF kg -1 and control fish (P < 0.05). None of the antioxidant enzymes (catalase, glutathione reductase, glutathione S-transferase) was affected by LF supplementation (P > 0.05). Fish fed with dietary LF had a significantly higher survival rate than those fed with the control diet after challenge with Vibrio harveyi (P < 0.05). These results revealed that diet supplementation in A. latus especially with 1200 mg LF kg -1 improve fish growth performance and immune parameters, as well as survival rate against Vibrio harveyi., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Due to their diverse applications, gold nanoparticles (AuNPs) are expected to increase of in the environment, although few studies are available on their mode of action in aquatic organisms. The genotoxicity of AuNPs, alone or combined with the human pharmaceutical gemfibrozil (GEM), an environmental contaminant frequently detected in aquatic systems, including in marine ecosystems, was examined using gilthead seabream erythrocytes as a model system. Fish were exposed for 96 h to 4, 80 and 1600 μg L -1 of 40 nm AuNPs with two coatings - citrate or polyvinylpyrrolidone; GEM (150 μg L -1 ); and a combination of AuNPs and GEM (80 μg L -1 AuNPs + 150 μg L -1 GEM). AuNPs induced DNA damage and increased nuclear abnormalities levels, with coating showing an important role in the toxicity of AuNPs to fish. The combined exposures of AuNPs and GEM produced an antagonistic response, with observed toxic effects in the mixtures being lower than the predicted. The results raise concern about the safety of AuNPs and demonstrate interactions between them and other contaminants., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

We determined the supplementation effects of dietary fucoidan on growth, immune responses, blood characteristics, and oxidative stress resistance of juvenile red sea bream. A fishmeal (FM)-based basal diet supplemented with 0% (D1, control), 0.05% (D2), 0.1% (D3), 0.2% (D4), 0.4% (D5), and 0.8% (D6) mozuku fucoidan to formulate six experimental diets. Each diet was randomly allocated to triplicate groups of fish (3.8 g) for 60 days. Results showed that fish-fed diet D5 showed significantly higher (P < 0.05) growth performance compared to the control (D1). Diet groups D2 to D4 also showed intermediate values compared to D1. Feed conversion efficiency and protein efficiency ratio were significantly higher in diet group D5, which was not significantly different with D3. Fucoidan supplementation increased whole-body lipid, which was significantly higher in the D5 group. Condition factor (CF) was significantly higher in fish fed ≥ 0.2% fucoidan-supplemented diet groups. Diet group D5 and D4 showed significantly lower blood urea nitrogen (BUN) and aspartate aminotransferase (AST) level, respectively. Dietary fucoidan reduced the oxidative stress of fish. Among the measured nonspecific immune parameters, only peroxidase activity (PA) and total serum protein (TSP) were significantly influenced by dietary supplementation and it was higher in D4 group. Fucoidan supplementation reduces thiobarbituric acid reactive substance (TBARS) values numerically and it was lowest in fish-fed diet group D5. Under the present experimental condition, finally, we concluded that 0.3-0.4% dietary fucoidan supplementation enhanced the growth and health performance of red sea bream by increasing growth, immune response, blood characteristics, and oxidative stress resistance.

Both inosine (INO) and vitamin C (l-ascorbic acid, AsA) play important roles in growth performance, feed utilization and health status of fish. Therefore, a 56 days feeding trial was conducted to determine the interactive effects of dietary INO and AsA on growth performance, oxidative status, innate and adaptive immune responses of red sea bream. Fish growth performance and fed utilization parameters were significantly affected by dietary INO supplementation but not by AsA. Fish fed diets with INO at 4 g kg -1 diet in combination of high and low levels of AsA (3.1 g kg-1 and 9.3 g kg-1) produced the highest growth and feed utilization performances. In terms of growth and feed utilization performances no significant interaction effects were observed between INO and AsA. Dietary INO significantly influenced hematocrit, glucose and glutamyl oxaloacetic transaminase (GOT) content of red sea bream meanwhile AsA also significantly influenced hematocrit, glucose, total cholesterol, blood urea nitrogen (BUN) and glutamic-pyruvate transaminase (GPT) content of the test fish. No significant interaction effects was also observed between INO and AsA on measured hematological parameters. Reactive oxygen metabolites (d-ROMs) significantly influenced by both INO and AsA. Fish fed diet groups D1, D4 and D6 showed best oxidative stress resistance. Only INO was a significant factor on nitro-blue-tetrazolium activity (NBT) and bactericidal activity (BA). Neither INO nor AsA was a significant factor on serum catalase activity (CAT), total serum protein (TSP), peroxidase activity (PA) and lysozyme activity (LA). No significant interaction effects was observed between INO and AsA on measured innate immune parameters. Agglutination antibody titer was significantly influenced by dietary supplementation, after 15 days of vaccination but not in 21 days. In the day 15th fish and diet group D3 and D5 showed significantly higher values compared to diet groups control and D1. INO was the only significant factor of increasing agglutination antibody titer in 15 t h day. While AsA was not a significant factor on agglutination antibody titer values in day 15 t h, there was an interaction between dietary INO and AsA levels. Finally under the experimental conditions, fish fed high INO and low AsA levels (4 g kg -1 and 0.31 g kg -1 diet, respectively) showed best growth and feed utilization performance. Simultaneously, low level of INO and high level of AsA (2 g kg -1 and 0.93 g kg -1 diet, respectively) improved blood chemistry and immunological parameters. Furthermore, combined use of INO and AsA is possible to improve hemato-immunological responses of red sea bream., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The effects of dietary administration of Bacillus subtilis on the growth, digestive enzyme activity, blood chemistry, oxidative status and immune response of red sea bream (Pagrus major) were evaluated in the current study. Fish fed five different levels of B. subtilis at 0 (BS0), 1 × 10 4 (BS1), 1 × 10 6 (BS2), 1 × 10 8 (BS3) and 1 × 10 10 (BS4) CFU kg -1 diet for 60 days. The obtained results showed that B. subtilis supplementation significantly improved growth performance (FBW, WG and SGR), feed utilization (FI, FCE, PER and PG) and whole-body protein content when compared to the control group (P < 0.05). Furthermore, the specific activities of amylase, protease and lipase enzymes up regulated significantly upon B. subtilis incorporation in red sea bream diets (P < 0.05). No changes have been reported on blood biochemical variables except for the plasma total protein, which increased significantly in fish fed BS3 diet when compared with the control diet (P < 0.05). Hematocrit, hemoglobin and the nitro blue tetrazolium values also reported the highest values significantly in fish fed B. subtilis, especially in case of BS3 and BS4 diets (P < 0.05). Serum bactericidal activity enhanced significantly in BS2, BS3 and BS4 groups (P < 0.05), while mucus bactericidal activity showed no significant activity among tested groups (P > 0.05). Serum lysozyme activity exhibited higher values in case of BS3 and BS4 groups than BS0 group (P < 0.05), while mucus lysozyme activity increased only in BS3 group. Also, serum peroxidase activity enhanced significantly in fish fed BS2 and BS3 diets (P < 0.05), however, no activities were observed in the collected mucus. All groups reflexed high tolerance ability against oxidative stress except for BS0 and BS1 groups. Additionally, catalase activity increased significantly in all B. subtilis fed groups when compared to BS0 group (P < 0.05). Considering the obtained results, the supplementation of B. subtilis in the diet of red sea bream at 1 × 10 8 and 1 × 10 10  CFU kg -1 diet could improve the growth, feed utilization, health condition and immune response., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Organotin compounds, such as tributyltin (TBT) and triphenyltin (TPT), have been widely used to control marine fouling. Here, we show that organotin stimulation reduces the hormone levels in the plasma of two economically important aquaculture fish. Blood plasma samples were collected from juvenile red seabream and black rockfish exposed to environmentally realistic concentrations of TBT and TPT for 14 days. The levels of two plasma biomarkers, namely the yolk protein precursor vitellogenin (VTG) and the sex steroid 17β-estradiol (E2), were measured to determine the endocrine disrupting potential of the organotin compounds. Both organotin compounds were dose-dependently accumulated in the blood of two fish. Exposure to waterborne TBT and TBT significantly decreased the plasma VTG levels in both the juvenile fish in a dose-dependent manner. In contrast, the treatment with E2, a well-known VTG inducer, significantly increased the plasma VTG levels in both the fish. In addition, the mRNA levels of vtg were also downregulated in the liver tissues of both the fish at 100 and/or 1000 ng L -1 of TBT or TPT exposure. The plasma E2 titers were significantly suppressed at 100 and/or 1000 ng L -1 of TBT or TPT exposure for 14 days compared to their titer in the control. Since estrogen directly regulates vtg gene expression and VTG synthesis, our results reveal the endocrine disrupting potential of organotin compounds, and subsequently the endocrine modulation at early stage of fish can trigger further fluctuations in sexual differentiation, maturation, sex ration or egg production. In addition, the results demonstrate their effects on non-target organisms, particularly on animals reared in aquaculture and fisheries., (Copyright © 2018 Elsevier Inc. All rights reserved.)

In aquaculture, animals can be continually exposed to environmental stress factors that put their health and even survival at risk. Two experiments were performed to evaluate the impact of different stress conditions (acute crowding and anaesthetic) on the natural haemolytic complement activity in serum and skin mucus of gilthead seabream (Sparus aurata L.). In the first experiment, fish were subjected to 10 kg m -3 (low density, control group) and 50 kg m -3 (high density, crowding group) during 2, 24 and 48 h. In the second experiment, fish were unexposed (control) or exposed to 40 ppm of MS-222 or 5 ppm or 10 ppm of clove oil for 1 h. In fish maintained in acute crowding conditions only an increase of the haemolytic complement activity was observed in the skin mucus after 24 h of exposure. However, a similar statistically significant increase was observed in serum and skin mucus of fish exposed for 1 h to the lowest concentration of clove oil (5 ppm) tested. The results point to a new and alternative way to assess stress in farmed fish by using skin mucus instead of blood serum and confirm that the measurement of natural haemolytic complement activity serves as an indicator of stress in fish., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Seasonal temperature changes may take organisms to the upper and lower limit of their thermal range, with respective variations in their biochemical and metabolic profile. To elucidate these traits, we investigated metabolic and antioxidant patterns in tissues of sea bream Sparus aurata during seasonal acclimatization for 1 yr in the field. Metabolic patterns were assessed by determining lactate dehydrogenase, citrate synthase, and β-hydroxyacyl CoA dehydrogenase activities, their kinetic properties and plasma levels of glucose, lactate, and triglycerides and tissue succinate levels. Oxidative stress was assessed by determining antioxidant enzymes superoxide dismutase, catalase, and glutathione reductase activities and levels of thiobarbituric acid reactive substances. Xanthine oxidase (XO) activity was determined as another source of reactive oxygen species (ROS) production. Furthermore, we studied the antiapoptotic protein indicator Bcl-2 and the apoptotic protein indicators Bax, Bad, ubiquitin, and caspase as well as indexes of autophagy (LC3B II/LC3B I and SQSTM1/p62) in the liver and the heart to identify possible relationships between oxidative stress and cell death. The results indicate clear seasonal metabolic patterns involving oxidative stress during summer as well as winter. During cold acclimatization, lipid oxidation is induced, while during increased temperatures, warm-induced metabolic activation and carbohydrate oxidation are observed. Thus, oxidative stress seems to be more prominent during warming because of the increased aerobic metabolism. The seasonal profile of apoptosis and XO as another source of ROS matches the results obtained in the laboratory and are interpreted within the framework of oxygen- and capacity-limited thermal tolerance.

This study evaluated the effects of dietary substitution of fishmeal by graded levels of a blend composed of Aspergillus oryzae fermented rapeseed meal [0% (RM0), 25% (RM25), 50% (RM50), 75% (RM75) and 100% (RM100)] on growth performance, haemato-immunological responses and antioxidative status of Pagrus major (average weight 5.5 ± 0.02 g). After 56 days, growth performances were significantly improved in fish fed RM25 diet compared to control (P < 0.05). Meanwhile, up to 50% replacement of fishmeal did not affect growth performance, feed conversion efficiency, protein efficiency ratio, protein apparent digestibility, protease activity, fish somatic indices and survival compared to control. While blood hematocrit and plasma protein were significantly enhanced in groups fed RM0 and RM25 diets, most of the hematological parameters did not change through the trial except glutamic pyruvate transaminase which was significantly increased in RM75 and RM100 groups and blood cholesterol which was gradually decreased with the increasing level of the blend. Interestingly, feeding fish with RM25 and RM50 diets significantly showed enhanced lysozyme, bactericidal and peroxidase activities and fish fed the same diets showed high resistance against oxidative stress (biological antioxidant potential and reactive oxygen metabolites). Additionally, catalase activity and tolerance against low salinity seawater were higher in fish fed RM25 diet. These findings suggested that, at a moderate level (25% and 50%), substitution of fishmeal by the fermented rapeseed meal promoted growth, nutrient utilization, and exerted immune responses and anti-oxidative effects in red sea bream., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Bisphenol A (BPA) is a known environmental endocrine-disrupting chemical that is widely used in plastics manufacturing. BPA enters in the aquatic environment mainly through urban and industrial sewage effluents, thereby posing a potential threat to organisms living in these ecosystems. This study was conducted to investigate the effect of BPA on VTG production with direct (sodium dodecyl sulfate-polyarylamide gel electrophoresis) and indirect (alkali-labile phosphate (ALP), total plasma calcium and protein) methods in immature male yellowfin seabream ( Acanthopagrus latus) as a marine fish model. Fish were randomly distributed into seven groups that were administered 1, 10, 50, and 100 µg g -1 week -1 of BPA and 2 µg g -1 week -1 of 17β-estradiol (E2) over a period of 2 weeks. Solvent controls received olive oil, whereas controls were not injected. The fish were sampled on days 0, 7, and 14, and their blood plasma and liver were obtained. The results showed that the hepatosomatic index of all treated fish was elevated in comparison with controls. Direct and indirect indicators showed that fish VTG protein was induced by BPA and E2 exposure. The protein was found to have two bands with molecular weights around 210 and 190 KDa. ALP, total plasma calcium and protein levels were increased in dose- and time-dependent manners. The results of this study demonstrated that short-term exposure of yellowfin seabream to BPA induced adverse effects in the reproductive system of hermaphrodite fish.

This study aimed to evaluate to what extent diel water temperature oscillations over a short period of time (41 h) affected haematologic and plasmatic parameters of gilthead sea bream. Sea bream juveniles (160 fish; 177.2 ± 20.9 g) were divided in two homogenous groups, experimental and control. The experimental group was exposed to water at 22.3 ± 0.6 °C between 5 a.m. and 5 p.m. and to colder water from 5 p.m. to 5 a.m. (temperature variation of 8.6 ± 0.3 °C) during 41h period, whereas for the control group, water temperature was maintained constant (22.4 ± 0.7 °C) for the same period. Fish were fed three times per day (10:30 a.m., 2 p.m., 4 p.m.). Blood was collected from fish caudal veins (n = 6) at different time points (10 a.m., 1 p.m., 5 p.m., 6:30 p.m., 8 p.m. and 10 a.m. of the following day) from each group, for haematologic (haematocrit and haemoglobin) and plasmatic (glucose, cortisol, triglycerides and total protein) determinations. All parameters studied tended to vary throughout the study period, both for experimental and control groups. Significant differences were observed between the two groups for all the studied parameters at different sampling points. These differences were observed near the influence of the cold-water period or during the cooling-water period. Results suggested that colder water temperature cycles depressed fish metabolic activity and affected the levels of the analysed blood-circulating metabolites. Overall, results suggested that stress response was similar for experimental and control groups and that gilthead sea bream were able to cope with diel water temperature variations.

The influence of chronic stress, induced by food deprivation (FD) and/or high stocking density (HSD), was assessed on stress, vasotocinergic and isotocinergic pathways of the gilthead sea bream (Sparus aurata). Fish were randomly assigned to one of the following treatments: (1) fed at low stocking density (LSD-F; 5kg·m -3 ); (2) fed at high stocking density (HSD-F, 40kg·m -3 ); (3) food-deprived at LSD (LSD-FD); and (4) food-deprived at HSD (HSD-FD). After 21days, samples from plasma, liver, hypothalamus, pituitary and head-kidney were collected. Both stressors (FD and HSD) induced a chronic stress situation, as indicated by the elevated cortisol levels, the enhancement in corticotrophin releasing hormone (crh) expression and the down-regulation in corticotrophin releasing hormone binding protein (crhbp) expression. Changes in plasma and liver metabolites confirmed a metabolic adjustment to cope with energy demand imposed by stressors. Changes in avt and it gene expression, as well as in their specific receptors (avtrv1a, avtrv2 and itr) at central (hypothalamus and pituitary) and peripheral (liver and head-kidney) levels, showed that vasotocinergic and isotocinergic pathways are involved in physiological changes induced by FD or HSD, suggesting that different stressors are handled through different stress pathways in S. aurata., (Copyright © 2017 Elsevier Inc. All rights reserved.)

This study describes the responses of the vasotocinergic and isotocinergic systems to food deprivation and re-feeding processes in immature gilthead sea bream (Sparus aurata). The animals were subjected to the following experimental treatments: (1) normal feeding (control), (2) food deprivation for 21 days; and (3) re-feeding for 7 days, beginning 14 days after starvation. The animals were sampled at 0, 7, 14 and 21 days from the beginning of the trial. The pituitary and plasma arginine vasotocin (AVT) and isotocin (IT) levels and the hypothalamic pro-vasotocin and pro-isotocin mRNA expression levels were measured. In addition, the mRNA levels of three receptors, avtr v1, avtr v2 and itr, were analyzed in target organs associated with (1) the integration and control of different physiological pathways related to stress and food intake (i.e., the hypothalamus), (2) hormonal release into the bloodstream (i.e., the pituitary), and (3) metabolism and its control (i.e., the liver). The metabolic parameters in the liver were also determined. The hepatosomatic index decreased, and hepatic metabolites were mobilized beginning in the early stages of starvation. Moreover, an over-compensation of these parameters occurred when the fish were re-fed after starvation. In terms of the vasotocinergic and isotocinergic systems, feed restriction induced a clear time-dependent regulation among metabolic organization, stress regulation and orexigenic processes in the mature hormone concentration and pro-peptide and receptor mRNA expression. Our results reveal the important role of the AVT/IT endocrine systems in the orchestration of fish physiology during starvation and re-feeding and indicate their involvement in both central and peripheral organs.

Widespread use of pharmaceuticals and suboptimal wastewater treatment have led to increased levels of these substances in aquatic ecosystems. Lipid-lowering drugs such as gemfibrozil, which are among the most abundant human pharmaceuticals in the environment, may have deleterious effects on aquatic organisms. We examined the genotoxicity of gemfibrozil in a fish species, the gilthead seabream (Sparus aurata), which is commercially important in southern Europe. Following 96-h waterborne exposure, molecular (erythrocyte DNA strand breaks) and cytogenetic (micronuclei and other nuclear abnormalities in cells) endpoints were measured. Gemfibrozil was positive in both endpoints, at environmentally relevant concentrations, a result that raises concerns about the potential genotoxic effects of the drug in recipient waters., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The impact of thermal imprinting on the plasticity of the hypothalamic-pituitary-interrenal (HPI) axis and stress response in an adult ectotherm, the gilthead sea bream ( Sparus aurata , L.), during its development was assessed. Fish were reared under 4 thermal regimes, and the resulting adults exposed to acute confinement stress and plasma cortisol levels and genes of the HPI axis were monitored. Changes in immune function, a common result of stress, were also evaluated using histomorphometric measurements of melanomacrophages centers (MMCs) in the head kidney and by monitoring macrophage-related transcripts. Thermal history significantly modified the HPI responsiveness in adult sea bream when eggs and larvae were reared at a higher than optimal temperature (HT, 22°C), and they had a reduced amplitude in their cortisol response and significantly upregulated pituitary pomc and head kidney star transcripts. Additionally, after an acute stress challenge, immune function was modified and the head kidney of adult fish reared during development at high temperatures (HT and LHT, 18-22°C) had a decreased number of MMCs and a significant downregulation of dopachrome tautomerase. Thermal imprinting during development influenced adult sea bream physiology and increased plasma levels of glucose and sodium even in the absence of an acute stress in fish reared under a high-low thermal regime (HLT, 22-18°C). Overall, the results demonstrate that temperature during early development influences the adult HPI axis and immune function in a teleost fish., (© 2017 Society for Endocrinology.)

The characterization of cholinesterase activity in brain and muscle of gilthead seabream was carried out using four specific substrates and three selective inhibitors. In addition, K m and V max were calculated from the Michaelis-Menten equation for ASCh and BSCh substrates. Finally, the in vitro sensitivity of brain and muscle cholinesterases to three organophosphates (OPs) was also investigated by estimating inhibition kinetics. The results indicate that AChE is the enzyme present in the brain, whereas in muscle, a typical AChE form is present along with an atypical form of BChE. Very low ChE activity was found in plasma with all substrates used. The inhibitory potency of the studied OPs on brain and muscle AChEs based on bimolecular inhibition constants (k i ) was: omethoate < dichlorvos < azinphosmethyl-oxon. Furthermore, muscle BChE was found to be several orders of magnitude (from 2 to 4) more sensitive than brain and muscle AChE inhibition by dichlorvos and omethoate.

Low water temperatures during winter are common in farming of gilthead sea bream in the Mediterranean. This causes metabolic disorders that in extreme cases can lead to a syndrome called "winter disease." An improved immunostimulatory nutritional status might mitigate the effects of this thermal metabolic stress. A trial was set up to assess the effects of two different diets on gilthead sea bream physiology and nutritional state through plasma proteome and metabolites. Four groups of 25 adult gilthead sea bream were reared during winter months, being fed either with a control diet (CTRL) or with a diet called "winter feed" (WF). Proteome results show a slightly higher number of proteins upregulated in plasma of fish fed the WF. These proteins are mostly involved in the immune system and cell protection mechanisms. Lipid metabolism was also affected, as shown both by plasma proteome and by the cholesterol plasma levels. Overall, the winter feed diet tested seems to have positive effects in terms of fish condition and nutritional status, reducing the metabolic effects of thermal stress.

Environmental stressors caused by inadequate aquaculture management strategies suppress the immune response of fish and make them more susceptible to diseases. Therefore, efforts have been made to relieve stress in fish by using various functional feed additives in the diet, including probiotics. The present work evaluates the effects of Lactobacillus rhamnosus (LR) on physiological stress response, blood chemistry and mucus secretion of red sea bream (Pagrus major) under low salinity stress. Fish were fed four diets supplemented with LR at [0 (LR0), 1 × 10 2 (LR1), 1 × 10 4 (LR2) and 1 × 10 6 (LR3) cells g -1 ] for 56 days. Before stress, blood cortisol, urea nitrogen (BUN) and total bilirubin (T-BIL) showed no significant difference (P > 0.05), whereas plasma glucose and triglyceride (TG) of fish-fed LR2 and LR3 diets were significantly lower (P < 0.05) than those of the other groups. Plasma total cholesterol (T-CHO) of fish-fed LR3 diet was significantly (P < 0.05) lower than that of the other groups. Furthermore, total plasma protein, mucus myeloperoxidase activity and the amount of mucus secretion were significantly enhanced in LR-supplemented groups when compared with the control group (P < 0.05). After the application of the low salinity stress test, plasma cortisol, glucose, T-CHO and TG contents in all groups showed an increased trend significantly (P < 0.01) compared to the fish before the stress challenge. However, plasma total protein and the amount of secreted mucus showed a decreased trend in all groups. On the other hand, BUN, T-BIL and mucus myeloperoxidase activity showed no significant difference after exposure to the low salinity stress (P > 0.05). In addition, the fish that received LR-supplemented diets showed significantly higher tolerance against low salinity stress than the fish-fed LR-free diet (P < 0.05). The physiological status and the detected immune responses, including total plasma protein and mucus myeloperoxidase activity in red sea bream, will provide a more comprehensive outlook of the effects of probiotics to relieve stress in fish.

Individual differences in behaviour and physiological responses to stress are associated with evolutionary adaptive variation and thus raw material for evolution. In farmed animals, the interest in consistent trait associations, i.e. coping styles, has increased dramatically over the last years. However, one of limitations of the available knowledge, regarding the temporal consistency, is that it refers always to short-term consistency (usually few weeks). The present study used an escape response during a net restraining test, previously shown to be an indicative of coping styles in seabream, to investigate long-term consistency of coping styles both over time and during different life history stages. Results showed both short-term (14 days) consistency and long-term (8 months) consistency of escape response. However, we did not found consistency in the same behaviour after sexual maturation when the restraining test was repeated 16, 22 and 23 months after the first test was performed. In conclusion, this study showed consistent behaviour traits in seabream when juveniles, and a loss of this behavioural traits when adults. Therefore, these results underline that adding a life story approach to data interpretation as an essential step forward towards coping styles foreground. Furthermore, a fine-tuning of aquaculture rearing strategies to adapt to different coping strategies may need to be adjusted differently at early stages of development and adults to improve the welfare of farmed fish.

Thyroid hormones, in particular 3,5,3'-triiodothyronine or T3, are involved in multiple physiological processes in mammals such as protein, fat and carbohydrate metabolism. However, the metabolic actions of T3 in fish are still not fully elucidated. We therefore tested the effects of T3 on Sparus aurata energy metabolism and osmoregulatory system, a hyperthyroid-induced model that was chosen. Fish were implanted with coconut oil depots (containing 0, 2.5, 5.0 and 10.0μg T3/g body weight) and sampled at day 3 and 6 post-implantation. Plasma levels of free T3 as well as glucose, lactate and triglyceride values increased with increasing doses of T3 at days 3 and 6 post-implantation. Changes in plasma and organ metabolite levels (glucose, glycogen, triglycerides, lactate and total α amino acid) and enzyme activities related to carbohydrate, lactate, amino acid and lipid pathways were detected in organs involved in metabolism (liver) and osmoregulation (gills and kidney). Our data implicate that the liver uses amino acids as an energy source in response to the T3 treatment, increasing protein catabolism and gluconeogenic pathways. The gills, the most important extruder of ammonia, are fuelled not only by amino acids, but also by lactate. The kidney differs significantly in its substrate preference from the gills, as it obtained metabolic energy from lactate but also from lipid oxidation processes. We conclude that in S. aurata lipid catabolism and protein turnover are increased as a consequence of experimentally induced hyperthyroidism, with secondary osmoregulatory effects., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Fish growth is strongly influenced by environmental and nutritional factors and changing culture conditions can help optimize it. The importance of early-life experience on the muscle phenotype later in life is well known. Here, we study the effects of 5 weeks of moderate and sustained swimming activity (5 BL s(-1)) in gilthead sea bream during early development. We analysed growth and body indexes, plasma IGF-I and GH levels, feed conversion, composition [proximate and isotopic ((15)N/(13)C)] and metabolic key enzymes (COX, CS, LDH, HOAD, HK, ALAT, ASAT) of white muscle. Moderate and continuous exercise in fingerlings of gilthead sea bream increased plasma IGF-I, whereas it reduced plasma GH. Under these conditions, growth rate improved without any modification to feed intake through an increase in muscle mass and a reduction in mesenteric fat deposits. There were no changes in the content and turnover of muscle proteins and lipid reserves. Glycogen stores were maintained, but glycogen turnover was higher in white muscle of exercised fish. A lower LDH/CS ratio demonstrated an improvement in the aerobic capacity of white muscle, while a reduction in the COX/CS ratio possibly indicated a functional adaptation of mitochondria to adjust to the tissue-specific energy demand and metabolic fuel availability in exercised fish. We discuss the synergistic effects of dietary nutrients and sustained exercise on the different mitochondrial responses.

The aim of this study was to evaluate the effects of IMUNO-2865(®) on hematological and antioxidative parameters in sea bream. Total of 640 sea bream were fed with diets containing 0 (Group 1), 1 (Group 2), 10 (Group 3) and 25 (Group 4) g of IMUNO-2865(®) kg(-1) feed during 90 days. Samples were taken each month and three months after the supplementation. A significant heterophils increase was observed in group 4 compared to group 1 after two months, and an increase in monocytes number was observed in group 4 compared to the other groups after one month. Glutathione peroxidase (GSH-Px) and paraoxonase-1 (PON1) were significantly increased in groups 3 and 4 compared to the control group three months into the experiment. Superoxide dismutase (SOD) was increased in group 4 compared to the control group from day 60 until the end of the experiment, and in groups 2 and 3 compared to the control after three months. Based on the differences in the cellular immunity and oxidative stress parameters, with an overall absence of mortality, the results of this study suggest that the use of IMUNO-2865(®) in aquaculture is safe and possess a cumulative immunostimulatory effect on sea bream., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Gilthead seabream (Sparus aurata) were fed a basal (control) diet and four experimental diets (R600, R1200, R1800 and R2400), containing 600, 1200, 1800 and 2400 mg kg(-1), respectively, of rosemary extract (Rosmarinus officinalis L.). At 4 and 12 weeks from the beginning of the ongrowing period, the fish were sacrificed, blood was drawn to obtain plasma and the liver and intestines were dissected. Growth and feed intake were unaffected by rosemary extract addition. A histological examination of the intestine revealed no differences among the dosages, while the liver showed a sharp decrease in hepatic steatosis in diets supplemented with rosemary extract. Furthermore, plasma alanine aminotransferase was lower with these diets at the end of the ongrowing period. Rosemary extract reduced the plasma levels of glucose and triglycerides on week 4 and glucose and HDL/LDL cholesterol ratio on week 12, suggesting better transport and energy metabolism of the lipids. Overall, the most evident effect of rosemary extract was observed with the 600 mg kg(-1) dose.

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., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

All functions in animals rely on daily rhythms, and mealtime can act as a rhythm-marker of nutrients assimilation and use. The effects of meal timing and food composition on carbohydrate use and protein retention of gilthead sea bream were studied. Three groups were fed twice a day (10am and at 5pm) for two months with two alternating diets: a commercial diet (Cd) and a high-carbohydrate, low-protein diet (Ed). The Ed/Cd group received the Ed diet in the morning and the Cd diet in the afternoon, and the Cd/Ed group received these diets in the reverse order. The Cd/Cd group only received the commercial diet (control group). After 56days, two force-feeding experiments (PF1 and PF2) measured for all three groups the fate of a single meal labelled with (15)N-protein and (13)C-starch through the retention of both isotopes in the main organs and tissue reserves. In PF1 fish were fed at 10am (morning mealtime), and in PF2 at 5pm (afternoon mealtime). Fish were sampled at the next two mealtimes (PF1: 7 and 24h post-feeding, PF2: 17 and 24h post-feeding). Nutrients recovery differed according to, first, the dietary regime, and second, the last meal received (Cd or Ed). Daily lower protein intake was compensated with higher protein retention combined with more use of carbohydrates for energy. Nevertheless, carbohydrates from the morning meal were used more efficiently. So, the use of carbohydrate for energy production and protein for growth can be improved by adjusting diet composition and mealtime., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The hypothalamus-pituitary-interrenal (HPI) axis, involved in the regulation of the neuroendocrine stress responses, presents important players such as corticotropin-releasing hormone (CRH, generally considered as the initiator of this pathway) and CRH-binding protein (CRH-BP, considered as an antagonist of CRH function). CRH and CRH-BP full-length cDNA sequences were obtained from Sparus aurata by screening a brain cDNA library, and their phylogenetic analysis as well as their roles during acute and chronic stress responses were assessed. mRNA expression levels and plasma cortisol concentrations were measured by RT qPCR and ELISA, respectively, in S. aurata juveniles submitted to: i) different environmental salinities in a short-time course response; and ii) food deprivation during 21 days. In addition, osmoregulatory and metabolic parameters in plasma corroborated a clear reorganization depending on the stress source/period. Salinity transfer induced stress as indicated by enhanced plasma cortisol levels, as well as by up-regulated CRH and down-regulated CRH-BP expression values. On the other hand, food deprivation did not affect both expression levels, although plasma cortisol concentrations were enhanced. These results suggest that different stressors are handled through different stress pathways in S. aurata., (Copyright © 2014 Elsevier Inc. All rights reserved.)

A wide range of endocrine disrupter chemicals can mimic steroid hormones causing adverse health effects. Nonylphenol (NP) and t-octhylphenol (t-OP) are man-made alkylphenolic environmental contaminants possessing controversial endocrine disruption properties. This study has investigated the effects of NP and t-OP enriched diets on hepatic tissue and biotransformation activities in the liver. To this aim, sea bream juveniles were fed with commercial diet enriched with three different doses of NP (NP1: 5mg/kg bw, NP2: 50mg/kg bw and NP3: 100mg/kg bw) or t-OP (t-OP1: 5mg/kg bw, t-OP2: 50mg/kg bw and t-OP3: 100mg/kg bw) for 21 days. A significant increase of the hepatosomatic index was observed in NP1 and t-OP1. Alteration of liver morphology was observed in both NP and t-OP exposed juveniles although the most altered endpoints were observed in t-OP2 with 100% of tissue degeneration. Ethoxyresorufin-O-deethylase activity was significantly inhibited by NP and t-OP (p<0.05), while catalase activity was significantly induced, at both doses. A different pattern of protein expression of different isoforms of both vitellogenin and zona radiata protein was evidenced within the treatments. In addition, a significant increase in the abundance of the stress induced heat shock protein 70 gene in the liver of t-OP2 fish and a significant increase in the abundance of the estrogen induced cathepsin D gene in the liver of NP1 and t-OP2 fish, were observed. Finally, estradiol-17β (E2) and testosterone (T) plasma levels and E2/T showed significantly different patterns in NP and t-OP exposed against control fish., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The aim of the current study was to phenotype fish metabolism and the transcriptionally-mediated response of hepatic mitochondria of gilthead sea bream to intermittent and repetitive environmental stressors: (i) changes in water temperature (T-ST), (ii) changes in water level and chasing (C-ST) and (iii) multiple sensory perception stressors (M-ST). Gene expression profiling was done using a quantitative PCR array of 60 mitochondria-related genes, selected as markers of transcriptional regulation, oxidative metabolism, respiration uncoupling, antioxidant defense, protein import/folding/assembly, and mitochondrial dynamics and apoptosis. The mitochondrial phenotype mirrored changes in fish performance, haematology and lactate production. T-ST especially up-regulated transcriptional factors (PGC1α, NRF1, NRF2), rate limiting enzymes of fatty acid β-oxidation (CPT1A) and tricarboxylic acid cycle (CS), membrane translocases (Tim/TOM complex) and molecular chaperones (mtHsp10, mtHsp60, mtHsp70) to improve the oxidative capacity in a milieu of a reduced feed intake and impaired haematology. The lack of mitochondrial response, increased production of lactate and negligible effects on growth performance in C-ST fish were mostly considered as a switch from aerobic to anaerobic metabolism. A strong down-regulation of PGC1α, NRF1, NRF2, CPT1A, CS and markers of mitochondrial dynamics and apoptosis (BAX, BCLX, MFN2, MIRO2) occurred in M-ST fish in association with the greatest circulating cortisol concentration and a reduced lactate production and feed efficiency, which represents a metabolic condition with the highest allostatic load score. These findings evidence a high mitochondrial plasticity against stress stimuli, providing new insights to define the threshold level of stress condition in fish., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Nonylphenol (NP) is an endocrine disrupting chemical which has been shown to be able to modulate the endocrine system of various organisms by different mechanisms. The objective of this study was to investigate the potential effects of 4-NP on steroid and thyroid hormone levels in sexually immature male yellowfin seabream (Acanthopagrus latus), a protandrous hermaphrodite species. For this, the fish were injected with ascending doses (10, 50, 100, and 200 μg g(-1) body weight) of 4-nonylphenol (4-NP) or vehicle during 2 weeks. After 7 and 14 days the fish were anesthetized, blood sample were collected and plasma steroid and thyroid hormone concentrations were quantified by radioimmunoassay. The result showed that 4-NP induced a significant increase in 17β-etradiol levels at dose 10 μg g(-1) , while the levels of this hormone in the higher doses decreased compared with the control group. However, 4-NP treatment did not have any significant effect on plasma levels of testosterone. In addition, it was observed that 4-NP affect the level of thyroid hormones in fish. Plasma thyroxine levels increased in a dose-dependent manner after 7 and 14 days of the exposure. In contrast, a significant decrease in triiodothyronine levels was observed during the experiment period. Moreover, no significant change was detected for thyroid stimulating hormone levels in 4-NP-treated fish. These results indicated that 4-NP could lead to disturb the balance of steroid and thyroid hormones with potential consequences for sexually immature male yellowfin seabream., (Copyright © 2012 Wiley Periodicals, Inc.)

Androgens can induce complete spermatogenesis in immature or prepubertal teleost fish; however, many aspects of the role of androgens in adult teleost spermatogenesis remain elusive. We used the in situ forming microparticle (ISM) system containing 1mg of testosterone (T)/kg body weight (T-ISM) in a homogenous population of gilthead seabream at testicular involution stage to study in vivo the effects of T on the sex steroid hormone balance and on the physiology of the gilthead seabream gonad. The levels of T, 11-ketotestosterone (11KT) and 17β-estradiol (E2) in plasma, gonad and liver were determined in T-ISM implanted specimens after 7, 14, 21 and 28 days. The effect of T-ISM was evaluated on (i) de novo synthesis and metabolism of T in the gonad and liver by measuring the gene expression levels of the main steroidogenic proteins involved, (ii) the progress of spermatogenesis, (iii) the presence of different leukocyte cell types in the gonad, and (iv) the mRNA expression of some genes involved in the leukocyte migratory influx into the gonad and of some immune-relevant molecules. T-ISM implants promote an increase of T up to supra-physiological levels which induce a depletion of E2 levels and maintain the 11KT levels at physiological concentrations. The gene expression profile of some steroidogenic enzymes in gonad and liver ruled out the transformation of T into estrogenic compounds following T-ISM implantation. Moreover, androgens may also be involved in the leukocyte migratory influx, which occurred even when cytokine, chemokine and cell adhesion molecule gene expressions were down-regulated. Moreover, T-ISM implants block germ cell proliferation, although increased dmrt1 gene expression may prevent the complete depletion of germ cells in the gonad. Furthermore, T down-regulated the expression of several tlr genes, which may result in the inhibition of the immune response in the gonad through the impaired ability to recognize and respond to pathogens., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Herein, we studied whether sustained exercise positively affects growth of gilthead sea bream by alterations in a) plasma concentrations of insulin and IGF-I, b) signaling pathways in muscle, or c) regulation of lipid metabolism. Specifically, we evaluated the effects of moderated swimming (1.5 body lengths per second; BL/s) on the circulating concentrations of insulin and IGF-I, morphometric parameters, and expression of genes related to lipid metabolism in gilthead sea bream (80-90 g BW). Exercise increased the specific growth rate (P < 0.05) and reduced the hepatosomatic index (P = 0.006). Plasma IGF-I concentrations increased in exercised fish (P = 0.037), suggesting a role for this endocrine factor in the control of muscular growth and metabolic homeostasis during swimming. The observed decrease in plasma insulin concentrations (P = 0.016) could favor the mobilization of tissue reserves in exercised fish. In this sense, the increase in liver fatty acid content (P = 0.041) and the changes in expression of peroxisome proliferator-activated receptors PPARα (P = 0.017) and PPARγ (P = 0.033) indicated a hepatic lipid mobilization. Concentration of glycogen in both white and red muscles was decreased (P = 0.021 and P = 0.017, respectively) in exercised (n = 12) relative to control (n = 12) gilthead sea bream, whereas concentrations of glucose (P = 0.016) and lactate (P = 0.0007) were decreased only in red muscle, indicating the use of these substrates. No changes in the glucose transporter and in lipoprotein lipase mRNA expression were found in any of the tissues studied. Exercised sea bream had decreased content of PPARβ mRNA in white and red muscle relative to control sea bream expression (P = 0.001 and P = 0.049, respectively). Mitogen-activated protein kinase phosphorylation was significantly down-regulated in both white and red muscles of exercised sea bream (P = 0.0374 and P = 0.0371, respectively). Tumor necrosis factor-α expression of white muscle was down-regulated in exercised gilthead sea bream (P = 0.045). Collectively, these results contribute to the knowledge base about hormonal regulation of growth and lipid metabolism in exercised gilthead sea bream., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The processing of intestinal fluid, in addition to a high drinking rate, is essential for osmoregulation in marine fish. This study analyzed the long-term response of the sea bream (Sparus aurata L.) to relevant changes of external salinity (12, 35 and 55 p.p.t.), focusing on the anterior intestine and in the less-often studied rectum. Intestinal water absorption, epithelial HCO(3)(-) secretion and gene expression of the main molecular mechanisms (SLC26a6, SLC26a3, SLC4a4, atp6v1b, CFTR, NKCC1 and NKCC2) involved in Cl(-) and HCO(3)(-) movements were examined. The anion transporters SLC26a6 and SLC26a3 are expressed severalfold higher in the anterior intestine, while the expression of Atp6v1b (V-type H(+)-ATPase β-subunit) is severalfold higher in the rectum. Prolonged exposure to altered external salinity was without effect on water absorption but was associated with concomitant changes in intestinal fluid content, epithelial HCO(3)(-) secretion and salinity-dependent expression of SLC26a6, SLC26a3 and SLC4a4 in the anterior intestine. However, the most striking response to external salinity was obtained in the rectum, where a 4- to 5-fold increase in water absorption was paralleled by a 2- to 3-fold increase in HCO(3)(-) secretion in response to a salinity of 55 p.p.t. In addition, the rectum of high salinity-acclimated fish shows a sustained (and enhanced) secretory current (I(sc)), identified in vitro in Ussing chambers and confirmed by the higher expression of CFTR and NKCC1 and by immunohistochemical protein localization. Taken together, the present results suggest a functional anterior-posterior specialization with regard to intestinal fluid processing and subsequently to salinity adaptation of the sea bream. The rectum becomes more active at higher salinities and functions as the final controller of intestinal function in osmoregulation.

A glucose tolerance test was performed in white sea bream Diplodus sargus, juveniles to evaluate the effect of a glucose load on plasma glucose, insulin, triacylglyceride levels, and on liver glycogen storage in order to study the capability of glucose utilization by this species. After being fasted for 48 h, fish were intraperitoneally injected with either 1 g of glucose per kg body weight or a saline solution. Plasma glucose rose from a basal level of 4 to a peak of 18-19 mmol l(-1), 2-4 h after glucose injection and fish exhibited hyperglycemia for 9 h. An insulin peak (from 0.5 to 0.8 ng ml(-1)) was observed 2-6 h after glucose injection, and basal value was attained within 9 h. Liver glycogen peaked 6-12 h after the glucose load and thereafter decreased to the basal value which was attained 24 h after injection. Plasma triacylglycerides in glucose-injected fish were only significantly higher than the basal value 12 h after injection. Glucose-injected fish generally showed lower plasma triacylglyceride levels than control fish. Our results indicate that under these experimental conditions, glucose acts as an insulin secretagogue in white sea bream juveniles. Moreover, insulin may have contributed to restoring basal plasma glucose levels by enhancing glucose uptake in the liver. Further studies are needed to corroborate the lipolytic action of glucose. Clearance of glucose from the blood stream was fast, comparatively to other species, indicating that white sea bream has a good capability of glucose utilization.

Aquatic organisms may suffer from exposure to high Cu concentrations, since this metal is widely used in feed supplementation, in pesticide formulation and as antifouling. Chronic exposure to Cu, even at sub-lethal doses, may strongly affect fish physiology. To date, several biomarkers have been used to detect Cu exposure in fish producing contrasting results. Therefore, we used a proteomic approach to clarify how Cu exposure may affect the serum proteome of gilthead sea bream (Sparus aurata), since serum could be considered a good source of early-biomarkers of Cu toxicosis. For this purpose we exposed juvenile gilthead sea bream to waterborne Cu (0.5 mg/L). Our results indicate that fish tightly regulate circulating Cu levels, which are not affected by metal exposure. This homeostatic control is mainly achieved by the liver, able to excrete high amounts of the metal via bile. Cu exposure caused differential expression of several serum proteins, 10 of which were identified by Mascot and BLAST search. All these proteins, with the exception of growth hormone receptor and γ-glutamyl-carboxylase, can be related to: 1) Cu-induced hepatotoxicity (cytochrome oxidase subunit I, alanine aminotransferase, glutathione S-transferase); 2) potential immunosuppression due to interference of Cu with the inflammation/immunity network (α-1 antitrypsin, angiotensinogen, complement component C3, recombination-activating protein-1 and warm temperature acclimation-related 65 kDa protein)., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Growth, haematological (haematocrit), biochemical (serum cortisol and glucose), and non-specific immune (lysozyme, serum haemolytic and haemagglutinating activities, extracellular respiratory burst activity) parameters, were monitored in European sea bass Dicentrarchus labrax and blackspot sea bream Pagellus bogaraveo subjected to a 31 days starvation compared to fed fish, to assess the responses to feed deprivation of these health status indicators. While haematocrit, serum cortisol, glucose and haemolytic activity of both species did not undergo significant variation following starvation, probably due to the short period applied, some non-specific immune parameters were affected significantly. In the starved sea bass, mucus lysozyme content doubled (1.8 U/mL) compared to the initial value. Haemagglutinating activity was significantly lower in starved sea bass than in fed fish after 31 days. In blackspot sea bream, a slight, not significant, reduction in haemagglutinating activity occurred 11 days after starvation. Respiratory burst activity decreased significantly in the starved fish. In spite of the limited number of examined parameters, the opportunity to use a panel of several indicators to obtain a more complete picture of health status in fish was underlined., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The winter syndrome in the gilthead sea bream Sparus aurata indicates that the species is exposed to critically low temperatures in Mediterranean aquaculture in winter. The present study of metabolic patterns and molecular stress responses during cold exposure was carried out to investigate this "disease", in light of the recent concept of oxygen and capacity limited thermal tolerance. The metabolic profile of fuel oxidation was examined by determining the activities of the enzymes hexokinase (HK), aldolase (Ald), pyruvate kinase (PK), L-lactate dehydrogenase (L-LDH), citrate synthase (CS), malate dehydrogenase (MDH) and 3-hydroxyacyl CoA dehydrogenase (HOAD) in heart, red and white muscle after exposure to temperatures of 10, 14 and 18°C. Especially, the increase in LDH activity combined with the accumulation of L-lactate in tissues indicates that temperatures below 14°C are critical for Sparus aurata and stimulate the anaerobic component of metabolism. Increase in the activity of HOAD suggests that oxidation of free fatty acids might contribute to ATP turnover at low temperatures. The expression of Hsp70 and Hsp90 in all tissues examined revealed a cellular stress response during cooling below 18°C. In the light of winter temperatures in S. aurata cultures around 10°C, our data suggest that the fish are exposed to stressful conditions at the low end of their thermal tolerance window. These conditions likely impair the aerobic capacity of the fish, compromise the rates of growth and reproduction and may contribute to elicit pathological conditions.

Food availability is far from constant but tends to be cyclic, and fish therefore show a variety of circadian rhythms which can be entrained to feeding time. The aim of this study was to investigate the synchronization to mealtimes of behavioral (locomotor activity), metabolic (glucose) and endocrine (cortisol and thyroid hormones) daily rhythms in gilthead seabream. To this end, fish were reared under a 12:12 LD cycle and fed 1% of their body weight once a day either at mid-light (ML) or at mid-dark (MD) of the LD cycle. Fish synchronized their locomotor activity to the phase in which food was delivered, ML and MD fish displaying 86+/-3% and 81+/-1% of their total daily activity during daytime and nighttime, respectively. Daily variations of blood glucose were strongly synchronized to feeding time in both experimental groups, peaking 8h after the meal. A postprandial cortisol peak was observed in both groups. In fish fed at MD the cortisol values were high during the 8h following feeding, whereas in fish fed at ML cortisol levels returned to basal values within 4h. In addition, MD fish showed a higher average daily cortisol value (24.75+/-2.65 ng/ml) than ML fish (10.30+/-2.18 ng/ml). Feeding time affected the magnitude of daily variations in the thyroid hormones. When the time of feeding was delayed by 12h, a shift of the same magnitude could be observed in the glucose daily rhythm and a dramatic change in the cortisol levels of the ML-fed group compared with results mentioned above. In summary, gilthead seabream synchronized their locomotor activity to the phase when food was provided and showed different degrees of synchronization of their behavioral, metabolic and endocrine rhythms to feeding time. Since these parameters are used to evaluate stress responses and welfare in seabream, their daily rhythm and synchronization to light and feeding time should be taken into account., (Copyright 2010 Elsevier Inc. All rights reserved.)

The physiological responses of fish to underwater noise are poorly understood and further information is needed to evaluate any possible negative effects of sound exposure. We exposed European sea bass and gilthead sea bream to a 0.1-1 kHz linear sweep (150 dB(rms) re 1 microPa). This band frequency is perceptible by many species of fish and is mainly produced by vessel traffic. We assessed the noise-induced motility reaction (analysing the movements) and the haematological responses (measuring blood glucose and lactate, and haematocrit levels). The noise exposure produced a significant increase in motility as well as an increase in lactate and haematocrit levels in sea bream and sea bass. A significant decrease of glucose was only observed in sea bream. A linear correlation between blood parameters and motility in fish exposed to the noise was observed. The acoustic stimulus produced intense muscle activity., (Copyright 2009 Elsevier Ltd. All rights reserved.)

During winter, low temperatures induce a direct metabolic depression in gilthead sea bream, without any significant compensatory effect below 13 degrees C. The present study therefore focused on how to improve response to cold in these fish, looking specifically at the two factors of diet (high energy, HiE, and low energy, LoE) and activity (normal, -SW, and sustained activity, +SW) prior to exposure to cold. Following a preparatory period of 75 days water was adjusted to 10 degrees C and kept for 40 days. Enzymatic activities and store deposition revealed that the HiE-SW group had acquired an energy surplus whilst the LoE+SW group exhibited an energy deficit. Liver enzyme activities evidenced diet dependence: LoE groups showed greater glucose-6-phosphate dehydrogenase activity and HiE groups showed greater lipoprotein lipase and hepatic lipase activities. Moreover, the HiE-SW group's lower citrate synthase/cytochrome-c-oxidase ratio reflected the energy surplus available. Perivisceral fat mobilisation caused by cold stress affected liver integrity, resulting in a pre-steatotic condition for the HE-SW group. The differences in liver enzyme activities produced by pre-cold conditions disappeared at low temperatures and enzymatic activities did not compensate. Therefore any improvement that would enable gilthead sea bream to face up to winter must be achieved prior to the appearance of low temperatures., (Copyright 2009 Elsevier Inc. All rights reserved.)