Dietary Histidine, Threonine, or Taurine Supplementation Affects Gilthead Seabream (Sparus aurata) Immune Status.

AAs have become interesting feed ingredients to be used in functional fish feeds as not only are they protein building blocks, but they also participate in several other key metabolic processes. In the present study, a comprehensive analysis of transcriptomics, hematology, and humoral immune parameters (plasma and skin mucus) were measured twice over the course of the feeding trial (four weeks). Plasma antiprotease activity increased in fish fed Thr compared to those fed the CTRL and Tau treatments, regardless of sampling time. The bactericidal activity in skin mucus decreased in fish fed Tau and His treatments compared to those fed the CTRL diet after two weeks. The membrane IgT (mIgT) was upregulated in fish fed Tau after four weeks, while C-type lectin domain family domain 10 member (clec10a) was downregulated in fish fed Thr after two weeks of feeding. By comparing the molecular signatures of head-kidney by means of a PLS-DA, it is possible to visualize that the main difference is between the two sampling points, regardless of diet. Altogether, these results suggest that dietary supplementation with these AAs at the tested levels causes mild immune-modulation effects in gilthead seabream, which should be further studied under disease challenge conditions.

Short-Term Supplementation of Dietary Arginine and Citrulline Modulates Gilthead Seabream (Sparus aurata) Immune Status.

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.

How tryptophan levels in plant-based aquafeeds affect fish physiology, metabolism and proteome.

Fish meal replacement by plant-protein sources is a priority in aquaculture feeds. Within this framework, dietary supplementation with essential amino acids (EAA), as tryptophan (TRP), is strategic to ensure that the individual nutritional needs are met, besides promoting enhanced immunological status. The purpose of this study was to examine the beneficial effects of TRP incorporation in plant-protein source diets on fish growth performance and nutritional status. We tested diets with 20% lower (LTRP) and 27% higher (HTRP) of the putative requirements of TRP for seabream (Sparus aurata) and assessed its impact on fish physiology and liver metabolism and proteome. After 12 weeks, growth performance, body proximate, hepatic composition and liver metabolic profiling were similar between diets. Nevertheless, liver proteome analysis indicated a higher accumulation of proteins involved in acute-phase responses, typically triggered by infection, inflammation or trauma, in fish fed with HTRP diet as compared with those fed with LTRP. The overall results obtained suggest a potential beneficial effect of TRP supplementation in terms of immune stimulation, without compromising growth or feed intake. Moreover, proteomics and metabolic profiling demonstrate to be valuable tools in this endeavour. SIGNIFICANCE: Nutritional needs are hard to assess in aquaculture fisheries, and many times controversial depending on the methodology employed. The estimated amino acid requirements depend on both fish species and stage development, making it extremely hard to standardise. On the other hand, the substitution of fish-based to plant-based protein sources diets towards a sustainable aquaculture, may imbalance these requirements, being necessary further studies to assess the impact on fish growth and development. Finally, the incorporation of crystalized amino acids such as TRP into diets aims global better performance both at fish health/immune condition and growth development. This work focused on the potential beneficial effects of TRP supplementation into diets with a plant-based protein source, addressing the effects on the liver metabolism and proteome, and on growth performance of Gilthead seabream juveniles, a species with special relevance and economical importance in the Mediterranean region. The present study by employing proteomics together with metabolic profiling shows that TRP supplementation at the tested doses, does not compromise growth performance, and seems to stimulate the immune system. Our findings can contribute to the development of new feed formulations for Gilthead seabream species, therefore, reinforcing the resilience and competitiveness of the on-growing aquaculture industry and impact directly the sustainability of living resources with the decrease of the fisheries needs to fulfil the human search for quality proteins consume.

Dietary tryptophan supplementation induces a transient immune enhancement of gilthead seabream (Sparus aurata) juveniles fed fishmeal-free diets.

European aquaculture is an industry with a high sustainability profile contributing to the supply of safe seafood. However, several diseases can affect farmed fish and it is imperative to find alternatives for chemotherapeutic treatments when disease outbreaks occur. Maintenance of health through nutrition is well-establish in modern animal farming, and amino acids (AA) are promising candidates as functional additives to improve fish health. Therefore, the goal of this research is to provide a better understanding of the influence of tryptophan supplementation on nutritional condition and immune mechanisms in fish. Triplicate groups of fish (13.3 ±â€¯0.3g) previously fed with a fishmeal-based diet were either fed a control diet with an extreme formulation (0% fishmeal) but meeting the AA requirements (CTRL), or the SUP diet, formulated as the CTRL with an increase in tryptophan (TRP) content. After 2 and 13 weeks of feeding, head-kidney (HK), liver (L) and white skeletal muscle (WSM) were collected for gene expression, whereas plasma was suited for humoral immune parameters. A holistic approach using transcriptomic, humoral and zootechnical parameters was undertaken. The expression of 29-31 genes for WSM, L or HK confirms an effect due to the treatment across time. A two-way ANOVA analysis revealed that 15-24 genes varied significantly depending on the tissue, and the multivariate analysis by means of PLS-DA explained (R2) and predicted (Q2) with four components up to 93% and 78% of total variance, respectively. Component 1 (R2 = 50.06%) represented the time effects, whereas components 2 (24.36%) and 3 (13.89%) grouped fish on the basis of dietary treatment, at early sampling. The HK results in particular suggest that fish fed SUP diet displayed an immunostimulated state at 2 weeks. No major differences were observed in plasma humoral parameters, despite an increase in antiprotease and peroxidase activities after 13 weeks regardless of dietary treatment. These results suggest that tryptophan supplementation may improve the seabream immune status after 2 weeks. Hence, the use of functional feeds is especially relevant during a short-term feeding period before a predictable stressful event or disease outbreak, considering that these putative advantageous effects seem to disappear after a 13 weeks feeding period.

STC1 and PTHrP Modify Carbohydrate and Lipid Metabolism in Liver of a Teleost Fish.

Stanniocalcin 1 (STC1) and parathyroid hormone-related protein (PTHrP) are calciotropic hormones in vertebrates. Here, a recently hypothesized metabolic role for these hormones is tested on European sea bass treated with: (i) teleost PTHrP(1-34), (ii) PTHrP(1-34) and anti-STC1 serum (pro-PTHrP groups), (iii) a PTHrP antagonist PTHrP(7-34) or (iv) PTHrP(7-34) and STC1 (pro-STC1 groups). Livers were analysed using untargeted metabolic profiling based on proton nuclear magnetic resonance (1H-NMR) spectroscopy. Concentrations of branched-chain amino acid (BCAA), alanine, glutamine and glutamate increased in pro-STC1 groups suggesting their mobilization from the muscle to the liver for degradation and gluconeogenesis from alanine and glutamine. In addition, only STC1 treatment decreased the concentrations of succinate, fumarate and acetate, indicating slowing of the citric acid cycle. In the pro-PTHrP groups the concentrations of glucose, erythritol and lactate decreased, indicative of gluconeogenesis from lactate. Taurine, trimethylamine, trimethylamine N-oxide and carnitine changed in opposite directions in the pro-STC1 versus the pro-PTHrP groups, suggesting opposite effects, with STC1 stimulating lipogenesis and PTHrP activating lipolysis/ß-oxidation of fatty acids. These findings suggest a role for STC1 and PTHrP related to strategic energy mechanisms that involve the production of glucose and safeguard of liver glycogen reserves for stressful situations.

Enhanced dietary formulation to mitigate winter thermal stress in gilthead sea bream (Sparus aurata): a 2D-DIGE plasma proteome study.

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.

Nutritional mitigation of winter thermal stress in gilthead seabream: Associated metabolic pathways and potential indicators of nutritional state.

A trial was carried out with gilthead seabream juveniles, aiming to investigate the ability of an enhanced dietary formulation (diet Winter Feed, WF, containing a higher proportion of marine-derived protein sources and supplemented in phospholipids, vitamin C, vitamin E and taurine) to assist fish in coping with winter thermal stress, compared to a low-cost commercial diet (diet CTRL). In order to identify the metabolic pathways affected by WF diet, a comparative two dimensional differential in-gel electrophoresis (2D-DIGE) analysis of fish liver proteome (pH 4­7) was undertaken at the end of winter. A total of 404 protein spots, out of 1637 detected, were differentially expressed between the two groups of fish. Mass spectrometry analysis of selected spots suggested that WF diet improved oxidative stress defense, reduced endoplasmic reticulum stress, enhanced metabolic flux through methionine cycle and phenylalanine/tyrosine catabolism, and induced higher aerobic metabolism and gluconeogenesis. Results support the notion that WF diet had a positive effect on fish nutritional state by partially counteracting the effect of thermal stress and underlined the sensitivity of proteome data for nutritional and metabolic profiling purposes. Intragroup variability and co-measured information were also used to pinpoint which proteins displayed a stronger relation with fish nutritional state. SIGNIFICANCE: Winter low water temperature is a critical factor for gilthead seabream farming in the Mediterranean region, leading to a reduction of feed intake, which often results in metabolic and immunological disorders and stagnation of growth performances. In a recent trial, we investigated the ability of an enhanced dietary formulation (diet WF) to assist gilthead seabream in coping with winter thermal stress, compared to a standard commercial diet (diet CTRL). Within this context, in the present work, we identified metabolic processes that are involved in the stress-mitigating effect observed with diet WF, by undertaking a comparative analysis of fish liver proteome at the end of winter. This study brings information relative to biological processes that are involved in gilthead seabream winter thermal stress and shows that these can be mitigated through a nutritional strategy, assisting gilthead seabream to deal better with winter thermal conditions. Furthermore, the results show that proteomic information not only clearly distinguishes the two dietary groups from each other, but also captures heterogeneities that reflect intra-group differences in nutritional state. This was exploited in this work to refine the variable selection strategy so that protein spots displaying a stronger correlation with "nutritional state" could be identified as possible indicators of gilthead seabream metabolic and nutritional state. Finally, this study shows that gel-based proteomics seems to provide more reliable information than transmissive FT-IR spectroscopy, for the purposes of nutritional and metabolic profiling.

Visualization and Differential Analysis of Protein Expression Data Using R.

Data analysis is essential to derive meaningful conclusions from proteomic data. This chapter describes ways of performing common data visualization and differential analysis tasks on gel-based proteomic datasets using a freely available statistical software package (R). A workflow followed is illustrated using a synthetic dataset as example.

Metabolic fingerprinting of gilthead seabream (Sparus aurata) liver to track interactions between dietary factors and seasonal temperature variations.

Farmed gilthead seabream is sometimes affected by a metabolic syndrome, known as the "winter disease", which has a significant economic impact in the Mediterranean region. It is caused, among other factors, by the thermal variations that occur during colder months and there are signs that an improved nutritional status can mitigate the effects of this thermal stress. For this reason, a trial was undertaken where we assessed the effect of two different diets on gilthead seabream physiology and nutritional state, through metabolic fingerprinting of hepatic tissue. For this trial, four groups of 25 adult gilthead seabream were reared for 8 months, being fed either with a control diet (CTRL, low-cost commercial formulation) or with a diet called "Winter Feed" (WF, high-cost improved formulation). Fish were sampled at two time-points (at the end of winter and at the end of spring), with liver tissue being taken for FT-IR spectroscopy. Results have shown that seasonal temperature variations constitute a metabolic challenge for gilthead seabream, with hepatic carbohydrate stores being consumed over the course of the inter-sampling period. Regarding the WF diet, results point towards a positive effect in terms of performance and improved nutritional status. This diet seems to have a mitigating effect on the deleterious impact of thermal shifts, confirming the hypothesis that nutritional factors can affect the capacity of gilthead seabream to cope with seasonal thermal variations and possibly contribute to prevent the onset of "winter disease".

Data visualization and feature selection methods in gel-based proteomics.

Despite the increasing popularity of gel-free proteomic strategies, two-dimensional gel electrophoresis (2DE) is still the most widely used approach in top-down proteomic studies, for all sorts of biological models. In order to achieve meaningful biological insight using 2DE approaches, importance must be given not only to ensure proper experimental design, experimental practice and 2DE technical performance, but also a valid approach for data acquisition, processing and analysis. This paper reviews and illustrates several different aspects of data analysis within the context of gel-based proteomics, summarizing the current state of research within this field. Particular focus is given on discussing the usefulness of available multivariate analysis tools both for data visualization and feature selection purposes. Visual examples are given using a real gel-based proteomic dataset as basis.

Dietary tools to modulate glycogen storage in gilthead seabream muscle: glycerol supplementation.

The quality and shelf life of fish meat products depend on the skeletal muscle's energetic state at slaughter, as meat decomposition processes can be exacerbated by energy depletion. In this study, we tested dietary glycerol as a way of replenishing muscle glycogen reserves of farmed gilthead seabream. Two diets were tested in duplicate (n = 42/tank). Results show 5% inclusion of crude glycerol in gilthead seabream diets induces increased muscle glycogen, ATP levels and firmness, with no deleterious effects in terms of growth, proximate composition, fatty acid profile, oxidative state, and organoleptic properties (aroma and color). Proteomic analysis showed a low impact of glycerol-supplementation on muscle metabolism, with most changes probably reflecting increased stress coping capacity in glycerol-fed fish. This suggests inclusion of crude glycerol in gilthead seabream diets (particularly in the finishing phase) seems like a viable strategy to increase glycogen deposition in muscle without negatively impacting fish welfare and quality.

Effects of preslaughter stress levels on the post-mortem sarcoplasmic proteomic profile of gilthead seabream muscle.

Fish welfare is an important concern in aquaculture, not only due to the ethical implications but also for productivity and quality-related reasons. The purpose of this study was to track soluble proteome expression in post-mortem gilthead seabream muscle and to observe how preslaughter stress affects these post-mortem processes. For the experiment, two groups of gilthead seabream (n = 5) were subjected to distinct levels of preslaughter stress, with three muscle samples being taken from each fish. Proteins were extracted from the muscle samples, fractionated, and separated by 2DE. Protein identification was performed by MALDI-TOF-TOF MS. Analysis of the results indicates changes on several cellular pathways, with some of these changes being attributable to oxidative and proteolytic activity on sarcoplasmic proteins, together with leaking of myofibrillar proteins. These processes appear to have been hastened by preslaughter stress, confirming that it induces clear post-mortem changes in the muscle proteome of gilthead seabream.

Identification of proteins with potential osteogenic activity present in the water-soluble matrix proteins from Crassostrea gigas nacre using a proteomic approach.

Nacre, when implanted in vivo in bones of dogs, sheep, mice, and humans, induces a biological response that includes integration and osteogenic activity on the host tissue that seems to be activated by a set of proteins present in the nacre water-soluble matrix (WSM). We describe here an experimental approach that can accurately identify the proteins present in the WSM of shell mollusk nacre. Four proteins (three gigasin-2 isoforms and a cystatin A2) were for the first time identified in WSM of Crassostrea gigas nacre using 2DE and LC-MS/MS for protein identification. These proteins are thought to be involved in bone remodeling processes and could be responsible for the biocompatibility shown between bone and nacre grafts. These results represent a contribution to the study of shell biomineralization process and opens new perspectives for the development of new nacre biomaterials for orthopedic applications.

PROTEOMICS in aquaculture: applications and trends.

Over the last forty years global aquaculture presented a growth rate of 6.9% per annum with an amazing production of 52.5 million tonnes in 2008, and a contribution of 43% of aquatic animal food for human consumption. In order to meet the world's health requirements of fish protein, a continuous growth in production is still expected for decades to come. Aquaculture is, though, a very competitive market, and a global awareness regarding the use of scientific knowledge and emerging technologies to obtain a better farmed organism through a sustainable production has enhanced the importance of proteomics in seafood biology research. Proteomics, as a powerful comparative tool, has therefore been increasingly used over the last decade to address different questions in aquaculture, regarding welfare, nutrition, health, quality, and safety. In this paper we will give an overview of these biological questions and the role of proteomics in their investigation, outlining the advantages, disadvantages and future challenges. A brief description of the proteomics technical approaches will be presented. Special focus will be on the latest trends related to the aquaculture production of fish with defined nutritional, health or quality properties for functional foods and the integration of proteomics techniques in addressing this challenging issue.

Changes in liver proteome expression of Senegalese sole (Solea senegalensis) in response to repeated handling stress.

The Senegalese sole, a high-value flatfish, is a good candidate for aquaculture production. Nevertheless, there are still issues regarding this species' sensitivity to stress in captivity. We aimed to characterize the hepatic proteome expression for this species in response to repeated handling and identify potential molecular markers that indicate a physiological response to chronic stress. Two groups of fish were reared in duplicate for 28 days, one of them weekly exposed to handling stress (including hypoxia) for 3 min, and the other left undisturbed. Two-dimensional electrophoresis enabled the detection of 287 spots significantly affected by repeated handling stress (Wilcoxon-Mann-Whitney U test, p < 0.05), 33 of which could be reliably identified by peptide mass spectrometry. Chronic exposure to stress seems to have affected protein synthesis, folding and turnover (40S ribosomal protein S12, cathepsin B, disulfide-isomerase A3 precursor, cell-division cycle 48, and five distinct heat shock proteins), amino acid metabolism, urea cycle and methylation/folate pathways (methionine adenosyltransferase I α, phenylalanine hydroxylase, mitochondrial agmatinase, serine hydroxymethyltransferase, 3-hydroxyanthranilate 3,4-dioxygenase, and betaine homocysteine methyltransferase), cytoskeletal (40S ribosomal protein SA, α-actin, ß-actin, α-tubulin, and cytokeratin K18), aldehyde detoxification (aldehyde dehydrogenase 4A1 family and aldehyde dehydrogenase 7A1 family), carbohydrate metabolism and energy homeostasis (fatty acid-binding protein, enolase 3, enolase 1, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, aconitase 1, mitochondrial ATP synthase α-subunit, and electron-transfer flavoprotein α polypeptide), iron and selenium homeostasis (transferrin and selenium binding protein 1), steroid hormone metabolism (3-oxo-5-ß-steroid 4-dehydrogenase), and purine salvage (hypoxanthine phosphoribosyltransferase). Further characterization is required to fully assess the potential of these markers for the monitoring of fish stress response to chronic stressors of aquaculture environment.

Changes in the soluble bone proteome of reared white seabream (Diplodus sargus) with skeletal deformities.

One of the main constrains for commercial aquaculture production of white seabream (Diplodus sargus) is the high incidence of skeletal malformations in reared fish. The purpose of this study was to obtain a better understanding of the mechanisms involved in the development of these types of skeletal malformations by comparative proteomic analysis of the vertebral column of normal and deformed fish using 2DE for protein separation and MS for protein identification. We observed a 3.2 and 3.4-fold increase in the expression of two tropomyosin isoforms, one of which (tropomyosin-4) is essential for the motility and polarization cycles of osteoclasts. Furthermore, a 1.6, 1.7 and 1.8-fold increase in three parvalbumin spots was detected, suggesting a cellular response to increased intracellular Ca²(+) levels. These results can be interpreted as signs of increased cellular activity in the bone of white seabream with skeletal deformities coupled to a higher degree of calcium mobilization, which elicits further studies into the use of these proteins as indicators of skeletal metabolic state.