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- Fish allergenicity modulation using tailored enriched diets—Where are we?Publication . Schrama, Denise; Czolk, Rebecca; Raposo de Magalhães, Cláudia; Kuehn, Annette; Rodrigues, PedroFood allergy is an abnormal immune response to specific proteins in a certain food. The chronicity, prevalence, and the potential fatality of food allergy, make it a serious socio-economic problem. Fish is considered the third most allergenic food in the world, affecting part of the world population with a higher incidence in children and adolescents. The main allergen in fish, responsible for the large majority of fish-allergic reactions in sensitized patients, is a small and stable calcium-binding muscle protein named beta-parvalbumin. Targeting the expression or/and the 3D conformation of this protein by adding specific molecules to fish diets has been the innovative strategy of some researchers in the fields of fish allergies and nutrition. This has shown promising results, namely when the apo-form of beta-parvalbumin is induced, leading in the case of gilthead seabream to a 50% reduction of IgE-reactivity in fish allergic patients.
- 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.
- 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.
- Amyloodiniosis in aquaculture: A reviewPublication . Moreira, Márcio; Costas, Benjamín; Rodrigues, Pedro; Lourenço‐Marques, Cátia; Sousa, Rui; Schrama, Denise; Raposo de Magalhães, Cláudia; Farinha, Ana Paula; Soares, FlorbelaFish ectoparasites are one of the pathogens groups that pose great concern to the aquaculture industry. The dinoflagellate Amyloodinium ocellatum is responsible for amyloodiniosis, a parasitological disease with a strong economic impact in temperate and warm water aquaculture, mainly in earthen pond semi-intensive systems. Amyloodiniosis represents one of the most important bottlenecks for aquaculture and, with the predictable expansion of the area of influence of this parasite to higher latitudes due to global warming it might also be a threat to other aquaculture species that are not yet parasitized by A. ocellatum. This review made a compilation of the existing knowledge about this parasite and the disease associated with it. It was noticed that, except from the life cycle characterisation, detection methods, histopathological analysis, and treatments, there are still a lot of areas that need a further investment in research. Areas like parasite-host interactions, epidemiological models, taxonomy, host physiological responses to parasitism, and genome sequencing, amongst others, can contribute to a better understanding of this disease. These proposed approaches and routes of investigation will enhance and contribute to a more standardised knowledge, creating the opportunity for a better understanding of amyloodiniosis impacts on fish and contributing for the development of new tools against A. ocellatum, that may reduce fish mortality in aquaculture production due to amyloodiniosis outbreaks.
- 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.
- Effect of EDTA enriched diets on farmed fish allergenicity and muscle quality; a proteomics approachPublication . Raposo de Magalhães, Cláudia; Schrama, Denise; Fonseca, Flavio; Kuehn, Annette; Morisset, Martine; Ferreira, Sara; Goncalves, Amparo; Rodrigues, PedroFish is one of the most common elicitors of food-allergic reactions worldwide. These reactions are triggered by the calcium-binding muscle protein beta-parvalbumin, which was shown to have reduced immunoglobulin E (IgE)-binding capacity upon calcium depletion. This work aimed to reduce gilthead seabream allergenicity using diets supplemented with a calcium chelator. Three experimental feeds were tested, differing in ethylenediaminetetraacetic acid (EDTA) supplementation, and its effects on muscle and parvalbumin's IgE-reactivity were analyzed. Chromatographic determination of EDTA showed no accumulation in the muscle and sensory results demonstrated that the lowest concentration did not affect fish quality as edible fish. Proteomics revealed one protein related to muscle contraction with significantly different relative abundance. Immunoblot assays performed with fish-allergic patients sera indicated a 50% reduction in IgE-reactivity upon EDTA presence. These preliminary results provide the basis for the further development of a non-GMO approach to modulate fish allergenicity and improve safety of aquaculture fish.
- 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.
- Improvement of the cryopreservation protocols for the dusky grouper, Epinephelus marginatusPublication . F Riesco, Marta; Raposo de Magalhães, Cláudia; Engrola, Sofia; Martinez-Paramo, Sonia; Mira, Sara; Cunha, Maria Emilia; Cabrita, ElsaThe dusky grouper, Epinephelus marginatus, is a potential species for aquaculture production although the limited number of males kept in captivity has been the cause of some constraints in its production. Sperm cryopreservation emerged as a solution for this problem. However, spermatozoa can be severely affected by freezing/thawing processes and poor sperm quality is a limiting factor in reproduction success. The present study aimed at evaluating two main aspects in the design of a cryopreservation protocol-extender additives (taurine, glucose, cholesterol, BSA) and sperm containers (0.5 mL straws, 2 mL cryovials and 5 mL macrotubes). Sperm quality was assessed through the evaluation of the percentage of motile cells, viable cells, DNA fragmentation, lipid peroxidation and apoptosis. Some specific techniques, such as Caspase 3/7 detection, which provides information on membrane integrity and cell death, detecting early and late apoptotic and necrotic events, were required to establish an optimized cryopreservation protocol for this species. Taurine was the most suitable cryopreservation additive in terms of viable cells and cholesterol presented the highest percentage of necrotic cells in this study. Caspase 3/7 assay enabled us to detect necrotic damage induced by cryopreservation. Statement of relevance: The development of reproductive tools in dusky grouper, a potential species for aquaculture production, emerges as important tool to decrease the number of wild males maintained in captivity. A cryopreservation protocol was previously described for this species although several constraints in terms of sperm quality were detected. Our work provided new evidences that cryopreservation protocols can be improved through the addition of certain additives and use of appropriate sperm containers. Specific sperm analysis was crucial to identify and establish the most suitable conditions for breeders management and species conservation purposes. (C) 2016 Elsevier B.V. All rights reserved.