Transcriptomic changes behind Sparus aurata hepatic response to different aquaculture challenges: An RNA-seq study and multiomics integration.

Gilthead seabream (Sparus aurata) is an important species in Mediterranean aquaculture. Rapid intensification of its production and sub-optimal husbandry practices can cause stress, impairing overall fish performance and raising issues related to sustainability, animal welfare, and food safety. The advent of next-generation sequencing technologies has greatly revolutionized the study of fish stress biology, allowing a deeper understanding of the molecular stress responses. Here, we characterized for the first time, using RNA-seq, the different hepatic transcriptome responses of gilthead seabream to common aquaculture challenges, namely overcrowding, net handling, and hypoxia, further integrating them with the liver proteome and metabolome responses. After reference-guided transcriptome assembly, annotation, and differential gene expression analysis, 7, 343, and 654 genes were differentially expressed (adjusted p-value < 0.01, log2|fold-change| >1) in the fish from the overcrowding, net handling, and hypoxia challenged groups, respectively. Gene set enrichment analysis (FDR < 0.05) suggested a scenario of challenge-specific responses, that is, net handling induced ribosomal assembly stress, whereas hypoxia induced DNA replication stress in gilthead seabream hepatocytes, consistent with proteomics and metabolomics' results. However, both responses converged upon the downregulation of insulin growth factor signalling and induction of endoplasmic reticulum stress. These results demonstrate the high phenotypic plasticity of this species and its differential responses to distinct challenging environments at the transcriptomic level. Furthermore, it provides significant resources for characterizing and identifying potentially novel genes that are important for gilthead seabream resilience and aquaculture production efficiency with regard to fish welfare.

A new window into fish welfare: A proteomic discovery study of stress biomarkers in the skin mucus of gilthead seabream (Sparus aurata).

Fish skin mucus is a dynamic external mucosal layer that acts as the first line of defense in the innate immune system. Skin mucus' exudation and composition change severely under stress, making it a valuable biofluid to search for minimally invasive stress markers. This study focused on the skin mucus proteome response to repetitive handling, overcrowding, and hypoxia, using Sparus aurata, an important species in the Mediterranean aquaculture, as a model. Biomarker discovery analysis was performed using label-free shotgun proteomics coupled with bioinformatics to unveil the most predictive proteins for the stressed phenotype. A mean of 2166 proteins were identified at a < 0.2% false discovery rate, from which the differentially abundant proteins (DAPs) were mainly involved in the immune system and protein metabolism. A sparse partial least squares regression analysis revealed a high correlation between DAPs and plasma physiological stress indicators. Feature selection, performed by recursive feature elimination followed by logistic regression analysis of the selected proteins, disclosed 28 candidate biomarkers with values of area under the curve >0.75. These minimally invasive biomarkers could be used in forthcoming species-specific stress management protocols to improve fish welfare and promote farmed fish safety, positive societal outcomes, and business sustainability. SIGNIFICANCE: The fish skin mucus holds a great promise into fish welfare, as a valuable source of minimally invasive biomarkers for stress assessment. In this shotgun proteomics discovery study, we have identified 28 candidate biomarkers by combining a comprehensive functional analysis of the stress regulated proteome with predictive modeling, supported by a significant correlation (p < 0.01) with physiological stress indicators (cortisol, lactate and glucose). The candidate biomarkers showed a good predictive value in the testing set (AUC > 0.75), paving the way for the next step in their validation by targeted proteomics. An early and timely assessment of fish stressful events, by using minimally invasive biomarkers, as those that can be found in the fish skin mucus, can contribute to promote fish health/welfare in the aquaculture sector and its sustainability. The adoption of preventive and surveillance measures based on proteomics approaches can therefore help to avoid unnecessary adverse outcomes with a negative impact on this primordial food sector.

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. ß-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 ß-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.

Data on European seabass fed with methionine-enriched diets obtained through label free shotgun proteomics.

This data article is associated with the research article "Evaluating the impact of methionine-enriched diets in the liver of European seabass through label-free shotgun proteomics". Here it is described the data obtained from proteomic analysis of 36 European seabass juveniles (3 fish x 3 replicate tanks) after 18 days of feeding with experimental diets containing four inclusion levels of methionine (Met): 0.77%, 1%, 1.36% and 1.66% Met (w/w). We analysed this dataset and compared it with that obtained during the long-term feeding period i.e., 85 days. Fish liver proteins were digested with trypsin and purified peptides were analysed by LC-MS/MS. Proteins were identified with at least two peptides at 0.1% Decoy false discovery rate (FDR). In this dataset, we present the analysis of the differential abundant proteins (DAP) with significant differences across treatments after 18 days of feeding (One-Way ANOVA, p < 0.05). Treatment's comparisons were also performed between the 18- and 85-days feeding trials through Two-Way ANOVA (p < 0.05). MS/MS raw data are available via ProteomeXChange with identifiers PXD019610 and 10.6019/PXD019610 (18-days dataset); and PXD019622 and 10.6019/PXD019622 (85-days dataset). This dataset corresponds to fish sampled after 18-days of experimental trial and is made available to support the study conducted in the afore-mentioned article, by performing the analysis during a short-term period of feeding. The data presented may be further used in other nutritional studies e.g., addressing hepatic changes mediated by Met.

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.

Evaluating the impact of methionine-enriched diets in the liver of European seabass through label-free shotgun proteomics.

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.

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.

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.

Shotgun proteomics to unravel marine mussel (Mytilus edulis) response to long-term exposure to low salinity and propranolol in a Baltic Sea microcosm.

Pharmaceuticals, among them the ß-adrenoceptor blocker propranolol, are an important group of environmental contaminants reported in European waters. Laboratory exposure to pharmaceuticals on marine species has been performed without considering the input of the ecosystem flow. To unravel the ecosystem response to long-term exposure to propranolol we have performed long-term exposure to propranolol and low salinity in microcosms. We applied shotgun proteomic analysis to gills of Mytilus edulis from those Baltic Sea microcosms and identified 2071 proteins with a proteogenomic strategy. The proteome profiling patterns from the 587 highly reproductive proteins among groups define salinity as a key factor in the mussel's response to propranolol. Exposure at low salinity drives molecular mechanisms of adaptation based on a decrease in the abundance of several cytoskeletal proteins, signalling and intracellular membrane trafficking pathway combined with a response towards the maintenance of transcription and translation. The exposure to propranolol combined with low salinity modulates the expression of structural proteins including cilia functions and decreases the expression of membrane protein transporters. This study reinforces the environment concerns of the impact of low salinity in combination with anthropogenic pollutants and anticipates critical physiological conditions for the survival of the blue mussel in the northern areas. BIOLOGICAL SIGNIFICANCE: Applying shotgun proteomic analysis to M. edulis gills samples from a long-term microcosm exposure to propranolol and following a proteogenomic identification strategy, we have identified 2071 proteins. The proteomic analysis unrevealed which molecular mechanisms drive the adaptation to low salinity stress and how salinity modulates the effects of exposure to propranolol. These results reinforce the idea of the impact of low salinity in combination with anthropogenic pollutants and anticipate critical physiological condition.

Coping with abiotic stress: proteome changes for crop improvement.

Plant breeders need new and more precise tools to accelerate breeding programs that address the increasing needs for food, feed, energy and raw materials, while facing a changing environment in which high salinity and drought have major impacts on crop losses worldwide. This review covers the achievements and bottlenecks in the identification and validation of proteins with relevance in abiotic stress tolerance, also mentioning the unexpected consequences of the stress in allergen expression. While addressing the key pathways regulating abiotic stress plant adaptation, comprehensive data is presented on the proteins confirmed as relevant to confer tolerance. Promising candidates still to be confirmed are also highlighted, as well as the specific protein families and protein modifications for which detection and characterization is still a challenge. This article is part of a Special Issue entitled: Translational Plant Proteomics.