Fish allergenicity modulation using tailored enriched diets—Where are we?

Food 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.info:eu-repo/semantics/publishedVersio

Protein changes as robust signatures of fish chronic stress: a proteomics approach to fish welfare research

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.This study received Portuguese national funds from FCT - Foundation for Science and Technology through project UIDB/04326/2020 and project WELFISH (Refª 16–02-05-FMP-12, “Establishment of Welfare Biomarkers in farmed fish using a proteomics approach”) financed by Mar2020, in the framework of the program Portugal 2020. Cláudia Raposo de Magalhães acknowledges an FCT PhD scholarship, Refª SFRH/BD/138884/2018. Denise Schrama acknowledges an FCT PhD scholarship, Refª SFRH/BD/136319/2018.info:eu-repo/semantics/publishedVersio

Mid-infrared spectroscopic screening of metabolic alterations in stress-exposed gilthead seabream (Sparus aurata)

Stress triggers a battery of physiological responses in fish, including the activation of metabolic pathways involved in energy production, which helps the animal to cope with the adverse situation. Prolonged exposure to stressful farming conditions may induce adverse effects at the whole-animal level, impairing welfare. Fourier transform infrared (FTIR) spectroscopy is a rapid biochemical fingerprinting technique, that, combined with chemometrics, was applied to disclose the metabolic alterations in the fish liver as a result of exposure to standard stressful practices in aquaculture. Gilthead seabream (Sparus aurata) adults exposed to different stressors were used as model species. Spectra were preprocessed before multivariate statistical analysis. Principal components analysis (PCA) was used for pattern recognition and identification of the most discriminatory wavenumbers. Key spectral features were selected and used for classification using the k-nearest neighbour (KNN) algorithm to evaluate whether the spectral changes allowed for the reliable discrimination between experimental groups. PCA loadings suggested that major variations in the hepatic infrared spectra responsible for the discrimination between the experimental groups were due to differences in the intensity of absorption bands associated with proteins, lipids and carbohydrates. This broad-range technique can thus be useful in an exploratory approach before any targeted analysis.FCT: SFRH/BD/138884/2018/ SFRH/BD/136319/2018/ UIDB/04326/2020/ 16-02-05-FMP-12info:eu-repo/semantics/publishedVersio

Fish pathology research and diagnosis in aquaculture of farmed fish; a proteomics perspective

One 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.This work received national funding through the Foundation for Science and Technology (FCT) through project UID/Multi/04326/2020 and projects WELFISH (Refª 16-02-05-FMP-12, “Establishment of Welfare Biomarkers in farmed fish using a proteomics approach”), ALLYFISH (Refª 16-02-01-FMP-0014, “Development of a farmed fish with reduced allergenic potential”) and project SAÚDE & AQUA (MAR-02.05.01-FEAMP-0009), both financed by Mar2020, in the framework of the program Portugal 2020. Márcio Moreira, Cláudia Raposo de Magalhães, and Denise Schrama acknowledge FCT PhD fellowships SFRH/BD/118601/2016, SFRH/BD/138884/2018, and SFRH/BD/136319/2018, respectivelyinfo:eu-repo/semantics/publishedVersio

Fish processing and digestion affect parvalbumins detectability in Gilthead Seabream and European seabass

Consumption of aquatic food, including fish, accounts for 17% of animal protein intake. However, fish consumption might also result in several side-effects such as sneezing, swelling and anaphylaxis in sensitized consumers. Fish allergy is an immune reaction to allergenic proteins in the fish muscle, for instance parvalbumin (PV), considered the major fish allergen. In this study, we characterize PV in two economically important fish species for southern European aquaculture, namely gilthead seabream and European seabass, to understand its stability during in vitro digestion and fish processing. This information is crucial for future studies on the allergenicity of processed fish products. PVs were extracted from fish muscles, identified by mass spectrometry (MS), and detected by sandwich enzyme-linked immunosorbent assay (ELISA) after simulated digestion and various food processing treatments. Secondary structures were determined by circular dichroism (CD) after purification by anion exchange and gel filtration chromatography. In both species, PVs presented as α-helical and β-sheet structures, at room temperature, were shown to unfold at boiling temperatures. In European seabass, PV detectability decreased during the simulated digestion and after 240 min (intestinal phase) no detection was observed, while steaming showed a decrease (p < 0.05) in PVs detectability in comparison to raw muscle samples, for both species. Additionally, freezing (−20 °C) for up to 12 months continued to reduce the detectability of PV in tested processing techniques. We concluded that PVs from both species are susceptible to digestion and processing techniques such as steaming and freezing. Our study obtained preliminary results for further research on the allergenic potential of PV after digestion and processing

A Proteomics and other Omics approach in the context of farmed fish welfare and biomarker discovery

The 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.project WELFISH [16-02-05-FMP-12]FCTPortuguese Foundation for Science and TechnologyEuropean Commission [SFRH/BD/118601/2016]info:eu-repo/semantics/publishedVersio

Improvement of the cryopreservation protocols for the dusky grouper, Epinephelus marginatus

The 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.European Social Fund [IF/00482/2014/CP1217/CT0005]Operational Programme Human PotentialFoundation for Science and Technology of Portugal (FCT)POCTEP programme under the European territorial cooperation objective [0251_ECOAQUA_5_E]European Regional Development Fund (ERDF) Portugal Spain[CCMAR/SC/BPD/09/2016

Gilthead Seabream Liver Integrative Proteomics and Metabolomics Analysis Reveals Regulation by Different Prosurvival Pathways in the Metabolic Adaptation to Stress

The 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

Effect of creatine and EDTA supplemented diets on European seabass (Dicentrarchus labrax) allergenicity, fish muscle quality and omics fingerprint

The 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.info:eu-repo/semantics/publishedVersio

Resíduos da indústria de biscoitos e massas em dietas para tilápia-do-nilo

The objectives of this work were to determine the apparent digestibility coefficients (ADC) of dry matter (DM), crude protein (CP) and gross energy (GE) of coconut biscuit residues (CBR), sweet biscuits (SBR) and noodles (NR) for Nile tilapia and evaluate the performance of fingerlings fed with diets containing the residues with the highest nutritional value. In the digestibility study, 72 juveniles (with an average weight of 70.5 ± 2.7 g) were organized in a completely randomized design with three treatments and three replications (12 fish per replication). ADC was determined using an indirect method in which 0.10% chromium oxide (Cr2O3) acted as an indicator. To evaluate performance, 225 fingerlings (with an average weight of 3.4 ± 0.5 g) were distributed across 15 tanks (150 L), creating a completely randomized design with five treatments and three replications. The test diets contained 0, 4, 8, 12 or 16% CBR. ADCDM, ADCCP and ADCGE for each of the different residues were, respectively: 85, 88 and 86% for CBR; 32, 51 and 46% for SBR; 24, 31 and 26% for NR. The inclusion of CBR significantly affected final weight of the fish, specific growth rate and protein retention rate, as well as the carcass composition values for dry matter, mineral matter, gross energy and ethereal extract. CBR presents better digestibility than the other tested residues and it can be used as a substitute for maize meal in quantities up to 11.62%, thus improving fish performance.Objetivou-se determinar os coeficientes de digestibilidade aparente da matéria seca (CDAMS), proteína bruta (CDAPB) e energia bruta (CDAEB) dos resíduos de biscoito de coco (RBC), biscoito doce (RBD) e macarrão (RM) para a tilápia-do-nilo e avaliar o desempenho destes alevinos alimentados com dietas contendo o resíduo com o maior valor nutritivo. No estudo de digestibilidade 72 juvenis (70,5±2,7g) foram distribuídos em um delineamento inteiramente casualizado com três tratamentos e três repetições (12 peixes por repetição). A determinação dos CDA foi realizada pelo método indireto, com a utilização de 0,10% de óxido crômico (Cr2O3) como indicador. Para avaliação do desempenho 225 alevinos (3,4±0,5g) foram distribuídos em 15 tanques (150 L), compondo um delineamento inteiramente casualizado com cinco tratamentos e três repetições. As dietas testes continham níveis de inclusão de 0, 4, 8, 12 e 16% do RBC. Os CDAMS, CDAPB, CDAEB para os resíduos foram respectivamente: 85, 88 e 86% para RBC; 32, 51 e 46% para RBD e 24, 31 e 26%, para RM. A inclusão do RBC afetou significativamente as variáveis: peso final, taxa de crescimento específico e taxa de retenção proteica, assim como os valores da composição da carcaça para matéria seca, matéria mineral, energia bruta e extrato etéreo. O RBC apresenta melhor digestibilidade em relação aos demais resíduos testados, podendo substituir o farelo de milho em até 11,62%, melhorando o desempenho dos peixes