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- How tryptophan levels in plant-based aquafeeds affect fish physiology, metabolism and proteomePublication . Cerqueira, Marco; Schrama, Denise; Silva, Tome S.; Colen, R.; Engrola, Sofia; Conceicao, Luis E. C.; Rodrigues, Pedro; Farinha, Ana PaulaFish 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.
- Protein changes as robust signatures of fish chronic stress: a proteomics approach to fish welfare researchPublication . Raposo de Magalhães, Cláudia; Schrama, Denise; Farinha, Ana Paula; Revets, Dominique; Kuehn, Annette; Planchon, Sébastien; Rodrigues, Pedro; Cerqueira, MarcoBackground 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.
- Cognitive appraisal in fish: stressor predictability modulates the physiological and neurobehavioural stress response in sea bassPublication . Cerqueira, Marco; Millot, S.; Felix, A.; Silva, T.; Oliveira, G. A.; Oliveira, Catarina; Rey, S.; MacKenzie, S.; Oliveira, R.The role of cognitive factors in triggering the stress response is well established in humans and mammals (aka cognitive appraisal theory) but very seldom studied in other vertebrate taxa. Predictability is a key factor of the cognitive evaluation of stimuli. In this study, we tested the effects of stressor predictability on behavioral, physiological and neuromolecular responses in the European sea bass (Dicentrarchus labrax). Groups of four fish were exposed to a predictable (signalled) or unpredictable (unsignalled) stressor. Stressor predictability elicited a lower behavioural response and reduced cortisol levels. Using the expression of immediate early genes (c-fos, egr-1, bdnf and npas4) as markers of neuronal activity, we monitored the activity of three sea bass brain regions known to be implicated in stressor appraisal: the dorsomedian telencephalon, Dm (putative homologue of the pallial amygdala); and the dorsal (Dld) and ventral (Dlv) subareas of the dorsolateral telencephalon (putative homologue of the hippocampus). The activity of both the Dm and Dlv significantly responded to stressor predictability, suggesting an evolutionarily conserved role of these two brain regions in information processing related to stressor appraisal. These results indicate that stressor predictability plays a key role in the activation of the stress response in a teleost fish, hence highlighting the role of cognitive processes in fish stress.
- Fish pathology research and diagnosis in aquaculture of farmed fish; a proteomics perspectivePublication . Moreira, Márcio; Schrama, Denise; Farinha, Ana Paula; Cerqueira, Marco; Raposo de Magalhães, Cláudia; Carrilho, Raquel; Rodrigues, PedroOne of the main constraints in aquaculture production is farmed fish vulnerability to diseases due to husbandry practices or external factors like pollution, climate changes, or even the alterations in the dynamic of product transactions in this industry. It is though important to better understand and characterize the intervenients in the process of a disease outbreak as these lead to huge economical losses in aquaculture industries. High-throughput technologies like proteomics can be an important characterization tool especially in pathogen identification and the virulence mechanisms related to host-pathogen interactions on disease research and diagnostics that will help to control, prevent, and treat diseases in farmed fish. Proteomics important role is also maximized by its holistic approach to understanding pathogenesis processes and fish responses to external factors like stress or temperature making it one of the most promising tools for fish pathology research.
- Effect of creatine and EDTA supplemented diets on European seabass (Dicentrarchus labrax) allergenicity, fish muscle quality and omics fingerprintPublication . Schrama, Denise; Raposo de Magalhães, Cláudia; Cerqueira, Marco; Carrilho, Raquel; Farinha, Ana Paula; Costa, Ana Rosa da; Gonçalves, Amparo; Kuehn, Annette; Revets, Dominique; Planchon, Sébastien; Engrola, Sofia; Rodrigues, PedroThe relatively easy access to fish worldwide, alongside the increase of aquaculture production contributes to increased fish consumption which result in higher prevalence of respective allergies. Allergies to fish constitute a significant concern worldwide. beta-parvalbumin is the main elicitor for IgE-mediated reactions. Creatine, involved in the muscle energy metabolism, and ethylenediamine tetraacetic acid (EDTA), a calcium chelator, are potential molecules to modulate parvalbumin. The purpose of this study was to test creatine (2, 5 and 8%) and EDTA (1.5, 3 and 4.5%) supplementation in fish diets to modulate beta-parvalbumin expression and structure and its allergenicity in farmed European seabass (Dicentrarchus labrax) while assessing its effects on the end-product quality. Fish welfare and muscle quality parameters were evaluated by plasma metabolites, rigor mortis, muscle pH and sensory and texture analysis. Proteomics was used to assess alterations in muscle proteome profile and metabolic fingerprinting by Fourier transform infrared spectroscopy was used to assess the liver metabolic profile. In addition, IgE-reactivity to parvalbumin was analysed using fish allergic patient sera. Metabolic fingerprinting of liver tissue revealed no major alterations in infrared spectra with creatine supplementation, while with EDTA, only absorption bands characteristic of lipids were altered. Comparative proteomics showed up regulation of (tropo) myosin and phosphoglycerate mutase 2 with Creatine supplementation. In the case of EDTA proteomics showed up regulation of proteins involved in cellular and ion homeostasis. Allergenicity seems not to be modulated with creatine or EDTA supplementation as no decreased expression levels were found and IgE-binding reactivity showed no quantitative differences.
- A Proteomics and other Omics approach in the context of farmed fish welfare and biomarker discoveryPublication . Raposo de Magalhães, Cláudia; Cerqueira, Marco; Schrama, Denise; Moreira, Márcio; Boonanuntanasarn, Surintorn; Rodrigues, PedroThe rapid and intensive growth of aquaculture over the last decade, poses a tremendous challenge to this industry in order to comply with the latest guidelines, established to minimise its negative effects on the environment, animal welfare and public health. Farmed fish welfare has become one of the main priorities towards sustainable aquaculture production with several initiatives launched by the European Union within the framework of the 2030 agenda. It is clear that an unbiased and reliable way to access farmed fish welfare needs to be implemented due to the lack of reliable indicators and standardised methods that are used at present. In this review, we start by addressing the status quo of animal and fish welfare definition in particular, describing the methods and assays currently used to measure it. We then explain why we believe these methods are unreliable and why there is a need to establish new ones that will promote productivity and consumer's acceptance of farmed fish. The establishment of a new type of welfare biomarkers using cutting-edge technologies like proteomics and other omics technologies is proposed as a solution to this issue. Therefore, we provide a brief description of these new methodologies, describing for each one how they can improve our scientific knowledge and the role they can play in farmed fish welfare biomarker discovery.
- Dietary creatine supplementation in gilthead seabream (Sparus aurata): comparative proteomics analysis on fish allergens, muscle quality, and liverPublication . Schrama, Denise; Cerqueira, Marco; Raposo de Magalhães, Cláudia; Rosa Da Costa, Ana; Wulff, Tune; Gonçalves, Amparo; Camacho, Carolina; Colen, R.; Fonseca, Flávio; Rodrigues, PedroThe quality of fish flesh depends on the skeletal muscle's energetic state and delaying energy depletion through diets supplementation could contribute to the preservation of muscle's quality traits and modulation of fish allergens. Food allergies represent a serious public health problem worldwide with fish being one of the top eight more allergenic foods. Parvalbumins, have been identified as the main fish allergen. In this study, we attempted to produce a low allergenic farmed fish with improved muscle quality in controlled artificial conditions by supplementing a commercial fish diet with different creatine percentages. The supplementation of fish diets with specific nutrients, aimed at reducing the expression of parvalbumin, can be considered of higher interest and beneficial in terms of food safety and human health. The effects of these supplemented diets on fish growth, physiological stress, fish muscle status, and parvalbumin modulation were investigated. Data from zootechnical parameters were used to evaluate fish growth, food conversion ratios and hepatosomatic index. Physiological stress responses were assessed by measuring cortisol releases and muscle quality analyzed by rigor mortis and pH. Parvalbumin, creatine, and glycogen concentrations in muscle were also determined. Comparative proteomics was used to look into changes in muscle and liver tissues at protein level. Our results suggest that the supplementation of commercial fish diets with creatine does not affect farmed fish productivity parameters, or either muscle quality. Additionally, the effect of higher concentrations of creatine supplementation revealed a minor influence in fish physiological welfare. Differences at the proteome level were detected among fish fed with different diets. Differential muscle proteins expression was identified as tropomyosins, beta enolase, and creatine kinase among others, whether in liver several proteins involved in the immune system, cellular processes, stress, and inflammation response were modulated. Regarding parvalbumin modulation, the tested creatine percentages added to the commercial diet had also no effect in the expression of this protein. The use of proteomics tools showed to be sensitive to infer about changes of the underlying molecular mechanisms regarding fish responses to external stimulus, providing a holistic and unbiased view on fish allergens and muscle quality.
- Gilthead seabream liver integrative proteomics and metabolomics analysis reveals regulation by different prosurvival pathways in the metabolic adaptation to stressPublication . Raposo de Magalhães, Cláudia; Farinha, Ana Paula; Blackburn, Gavin; Whitfield, Phillip D.; Carrilho, Raquel; Schrama, Denise; Cerqueira, Marco; Rodrigues, PedroThe study of the molecular mechanisms of stress appraisal on farmed fish is paramount to ensuring a sustainable aquaculture. Stress exposure can either culminate in the organism’s adaptation or aggravate into a metabolic shutdown, characterized by irreversible cellular damage and deleterious effects on fish performance, welfare, and survival. Multiomics can improve our understanding of the complex stressed phenotype in fish and the molecular mediators that regulate the underlying processes of the molecular stress response. We profiled the stress proteome and metabolome of Sparus aurata responding to different challenges common to aquaculture production, characterizing the disturbed pathways in the fish liver, i.e., the central organ in mounting the stress response. Label-free shotgun proteomics and untargeted metabolomics analyses identified 1738 proteins and 120 metabolites, separately. Mass spectrometry data have been made fully accessible via ProteomeXchange, with the identifier PXD036392, and via MetaboLights, with the identifier MTBLS5940. Integrative multivariate statistical analysis, performed with data integration analysis for biomarker discovery using latent components (DIABLO), depicted the 10 most-relevant features. Functional analysis of these selected features revealed an intricate network of regulatory components, modulating different signaling pathways related to cellular stress, e.g., the mTORC1 pathway, the unfolded protein response, endocytosis, and autophagy to different extents according to the stress nature. These results shed light on the dynamics and extent of this species’ metabolic reprogramming under chronic stress, supporting future studies on stress markers’ discovery and fish welfare research.
- Habituation and conditioning in gilthead sea bream (Sparus aurata): effects of aversive stimuli, reward and social hierarchiesPublication . Folkedal, Ole; Ferno, Anders; Nederlof, Marit A. J.; Fosseidengen, Jan E.; Cerqueira, Marco; Olsen, Rolf E.; Nilsson, JonatanTo tailor the farming environment to a fish species, we should understand the species-specific responses to stimuli, including the degree of adaption and learning. Groups of gilthead sea bream were given a delay Pavlovian conditioning regime using a conditioning stimulus (CS) of light flashes signalling arrival of food. Controls were exposed to light flashes unrelated to feeding. Fish in both treatments showed an initial fear response of moving away from the CS combined with reduced swimming speed. In subsequent trials, the Control fish largely habituated the fleeing response but sustained to respond by reducing the swimming speed. The Conditioning fish also stopped to escape from the CS, but opposed to the Control group they gradually increased their swimming speed in response to the CS. In addition, the number of fish in the feeding/CS area increased and became similar to basal level after around 16 trials. A small and variable proportion of the fish displayed black vertical bands on their body and territorial behaviour, and a social hierarchy could interfere with the processes of habituation and conditioning. The swimming speed of the fish increased with number of dark individuals, but this was not found during the CS and the light stimulus thus seemed to overrule the effect of territorial behaviour. The persistent negative response to light flashes in the Control suggests that fish seemingly adapted to repetitive stressors are still in a state of alertness. The change in the response to light shows the potential for rewarding aversive stimuli to reduce stress.
- Evaluating the impact of methionine-enriched diets in the liver of European seabass through label-free shotgun proteomicsPublication . Farinha, Ana Paula; Schrama, Denise; Silva, Tome; Conceicao, Luis E. C.; Colen, Rita; Engrola, Sofia; Rodrigues, Pedro; Cerqueira, MarcoPlant 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.