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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.

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. European Union through FEDER [ALG-01-0247-FEDER3520] European Union through COMPETE 2020 [ALG-01-0247-FEDER3520] European Union through CRESC Algarve 2020 [ALG-01-0247-FEDER3520] Portuguese Foundation for Science and Technology (FCT)Portuguese Foundation for Science and Technology [UIDB/04326/2020] Portuguese Foundation for Science and Technology (FCT) through COMPETE Programme Portuguese Foundation for Science and Technology (FCT) through Operational Human Potential Programme info:eu-repo/semantics/publishedVersion

Plant protein sources play an essential role in aquaculture by reducing the use of fish meal to sustainable levels, although further supplementation is needed to fulfill fish nutritional requirements. This work addressed fish growth performance and proteome changes to dietary methionine in European seabass juveniles. A dose-dependent response to methionine (Met) was observed on fish growth consistent with proteomic analyses, suggesting Met requirement >= 0.9% (w/w). Fish fed at 0.77% (w/w) exhibited reduced growth and an enrichment in proteins involved in cellular homeostasis. Proteomics data suggest an optimal nutritional status at 1.36% Met (w/w), together with putative beneficial effects on the immune system up to 1.66% Met (w/w). The response to dietary Met involved the convergence of different metabolic and signalling pathways implicated in cell growth and immune response e.g., mTOR, Hedgehog or the T Cell receptor signalling, coupled with a fine-tuning regulation of amino acid metabolism and translation. European Union through FEDER, COMPETE 2020; CRESC Algarve 2020; Portuguese Foundation for Science and Technology (Ministry of Science and Higher Education, Portugal); European Social FundsEuropean Social Fund (ESF) [UIDB/04326/2020, IF/00482/2014/CP1217/CT0005]; [ALG-01-0247-FEDER-3520] ALG-01-0247-FEDER-3520 info:eu-repo/semantics/publishedVersion

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.

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 4-7) 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. (C) 2016 Elsevier B.V. All rights reserved.

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.

This study was partially supported by the projects SAARGO – POCI/MAR/6123/2004 and HYDRAA – PTDC/MAR/71685/2006, granted by Fundacao para a Ciencia e Tecnologia, Portugal. NR, M de V and TSS acknowledge financial support from Fundacao para a Ciencia e Tecnologia, Portugal, through grants SFRH/BDP/34888/2007, SFRH/BD/40698/2007 and SFRH/BD/41392/2007 respectively.

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.

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. Current Protein and Peptide Science, 15, 4-22. doi:10.2174/1389203715666140221112334

The response to tissue damage is a complex process, which involves the coordinated regulation of multiple proteins to ensure tissue repair. In order to investigate the effect of tissue damage in a lower vertebrate, samples were taken from rainbow trout (Oncorhynchus mykiss) at day 7 after damage and proteins were separated using 2DE. The experimental design included two groups of rainbow trout, which were fed organic feed either with or without astaxanthin. In total, 96 proteins were found to be affected by tissue damage, clearly demonstrating in this lower vertebrate the complexity and magnitude of the cellular response, in the context of a regenerative process. Using a bioinformatics approach, the main biological function of these proteins were assigned, showing the regulation of proteins involved in processes such as apoptosis, iron homeostasis, and regulation of muscular structure. Interestingly, it was established that exclusively within the astaxanthin feed group, three members of the annexin protein family (annexin IV, V, and VI) were regulated in response to tissue damage.

Background Expansion of aquaculture requires alternative feeds and breeding strategies to reduce dependency on fish oil (FO) and better utilization of dietary vegetable oil (VO). Despite the central role of intestine in maintaining body homeostasis and health, its molecular response to replacement of dietary FO by VO has been little investigated. This study employed transcriptomic and proteomic analyses to study effects of dietary VO in two family groups of Atlantic salmon selected for flesh lipid content, 'Lean' or 'Fat'. Results Metabolism, particularly of lipid and energy, was the functional category most affected by diet. Important effects were also measured in ribosomal proteins and signalling. The long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis pathway, assessed by fatty acid composition and gene expression, was influenced by genotype. Intestinal tissue contents of docosahexaenoic acid were equivalent in Lean salmon fed either a FO or VO diet and expression of LC-PUFA biosynthesis genes was up-regulated in VO-fed fish in Fat salmon. Dietary VO increased lipogenesis in Lean fish, assessed by expression of FAS, while no effect was observed on β-oxidation although transcripts of the mitochondrial respiratory chain were down-regulated, suggesting less active energetic metabolism in fish fed VO. In contrast, dietary VO up-regulated genes and proteins involved in detoxification, antioxidant defence and apoptosis, which could be associated with higher levels of polycyclic aromatic hydrocarbons in this diet. Regarding genotype, the following pathways were identified as being differentially affected: proteasomal proteolysis, response to oxidative and cellular stress (xenobiotic and oxidant metabolism and heat shock proteins), apoptosis and structural proteins particularly associated with tissue contractile properties. Genotype effects were accentuated by dietary VO. Conclusions Intestinal metabolism was affected by diet and genotype. Lean fish may have higher responsiveness to low dietary n-3 LC-PUFA, up-regulating the biosynthetic pathway when fed dietary VO. As global aquaculture searches for alternative oils for feeds, this study alerts to the potential of VO introducing contaminants and demonstrates the detoxifying role of intestine. Finally, data indicate genotype-specific responses in the intestinal transcriptome and proteome to dietary VO, including possibly structural properties of the intestinal layer and defence against cellular stress, with Lean fish being more susceptible to diet-induced oxidative stress.

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.

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, β-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-β-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.

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.

The aim of this study was to identify possible metabolic molecular indicators of chronic stress in gilthead sea bream Sparus aurata . Two potential stressful conditions were tested: repeated handling and crowding at high stocking density. Gilthead seabream kept under optimized rearing conditions were used as control fish. Cortisol was measured as primary stress indicator and the liver proteome of stressed fish was compared to that of control fish using comparative proteomics. Plasma cortisol levels in sea bream repeatedly handled and crowded at high stocking density were significantly higher than in undisturbed control fish. A total of 560 spots were detected and the statistical analysis revealed a differential expression in about 50% of all detected proteins. Spots with greater than 2-fold or lower than 0.5-fold changes were identified by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Proteins like fatty acid binding protein (lipid transport and antioxidant role), heat shock cognate protein (chaperoning), calmodulin (Ca 2+ signaling), mitochondrial porine — voltage-dependent anion channel (lipid oxidation), glutamine synthetase (ammonia metabolism), cofilin and beta-tubulin (cytoskeleton), hemoglobin and several other proteins involved in carbohydrate metabolism (triose-phosphate isomerase, pyruvate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, alfa-enolase) were differentially expressed in fish under chronic stress. Some of these proteins may be used in the future as chronic stress and/or part of a panel of welfare biomarkers, after validation studies using RT-PCR and ELISA assays.