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.

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 ß-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.

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.

Stressor controllability modulates the stress response in fish.

BACKGROUND: In humans the stress response is known to be modulated to a great extent by psychological factors, particularly by the predictability and the perceived control that the subject has of the stressor. This psychological dimension of the stress response has also been demonstrated in animals phylogenetically closer to humans (i.e. mammals). However, its occurrence in fish, which represent a divergent vertebrate evolutionary lineage from that of mammals, has not been established yet, and, if present, would indicate a deep evolutionary origin of these mechanisms across vertebrates. Moreover, the fact that psychological modulation of stress is implemented in mammals by a brain cortical top-down inhibitory control over subcortical stress-responsive structures, and the absence of a brain cortex in fish, has been used as an argument against the possibility of psychological stress in fish, with implications for the assessment of fish sentience and welfare. Here, we have investigated the occurrence of psychological stress in fish by assessing how stressor controllability modulates the stress response in European seabass (Dicentrarchus labrax). RESULTS: Fish were exposed to either a controllable or an uncontrollable stressor (i.e. possibility or impossibility to escape a signaled stressor). The effect of loss of control (possibility to escape followed by impossibility to escape) was also assessed. Both behavioral and circulating cortisol data indicates that the perception of control reduces the response to the stressor, when compared to the uncontrollable situation. Losing control had the most detrimental effect. The brain activity of the teleost homologues to the sensory cortex (Dld) and hippocampus (Dlv) parallels the uncontrolled and loss of control stressors, respectively, whereas the activity of the lateral septum (Vv) homologue responds in different ways depending on the gene marker of brain activity used. CONCLUSIONS: These results suggest the psychological modulation of the stress response to be evolutionary conserved across vertebrates, despite being implemented by different brain circuits in mammals (pre-frontal cortex) and fish (Dld-Dlv).

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.

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.

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.

Dietary Creatine Supplementation in Gilthead Seabream (Sparus aurata): Comparative Proteomics Analysis on Fish Allergens, Muscle Quality, and Liver.

The 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.

Thermal preference predicts animal personality in Nile tilapia Oreochromis niloticus.

Environmental temperature gradients provide habitat structure in which fish orientate and individual thermal choice may reflect an essential integrated response to the environment. The use of subtle thermal gradients likely impacts upon specific physiological and behavioural processes reflected as a suite of traits described by animal personality. In this study, we examine the relationship between thermal choice, animal personality and the impact of infection upon this interaction. We predicted that thermal choice in Nile tilapia Oreochromis niloticus reflects distinct personality traits and that under a challenge individuals exhibit differential thermal distribution. Nile tilapia were screened following two different protocols: 1) a suite of individual behavioural tests to screen for personality and 2) thermal choice in a custom-built tank with a thermal gradient (TCH tank) ranging from 21 to 33 °C. A first set of fish were screened for behaviour and then thermal preference, and a second set were tested in the opposite fashion: thermal then behaviour. The final thermal distribution of the fish after 48 h was assessed reflecting final thermal preferendum. Additionally, fish were then challenged using a bacterial Streptococcus iniae model infection to assess the behavioural fever response of proactive and reactive fish. Results showed that individuals with preference for higher temperatures were also classified as proactive with behavioural tests and reactive contemporaries chose significantly lower water temperatures. All groups exhibited behavioural fever recovering personality-specific thermal preferences after 5 days. Our results show that thermal preference can be used as a proxy to assess personality traits in Nile tilapia and it is a central factor to understand the adaptive meaning of animal personality within a population. Importantly, response to infection by expressing behavioural fever overrides personality-related thermal choice.

Are coping styles consistent in the teleost fish Sparus aurata through sexual maturation and sex reversal?

Individual differences in behaviour and physiological responses to stress are associated with evolutionary adaptive variation and thus raw material for evolution. In farmed animals, the interest in consistent trait associations, i.e. coping styles, has increased dramatically over the last years. However, one of limitations of the available knowledge, regarding the temporal consistency, is that it refers always to short-term consistency (usually few weeks). The present study used an escape response during a net restraining test, previously shown to be an indicative of coping styles in seabream, to investigate long-term consistency of coping styles both over time and during different life history stages. Results showed both short-term (14 days) consistency and long-term (8 months) consistency of escape response. However, we did not found consistency in the same behaviour after sexual maturation when the restraining test was repeated 16, 22 and 23 months after the first test was performed. In conclusion, this study showed consistent behaviour traits in seabream when juveniles, and a loss of this behavioural traits when adults. Therefore, these results underline that adding a life story approach to data interpretation as an essential step forward towards coping styles foreground. Furthermore, a fine-tuning of aquaculture rearing strategies to adapt to different coping strategies may need to be adjusted differently at early stages of development and adults to improve the welfare of farmed fish.

Behavioural stress responses predict environmental perception in European sea bass (Dicentrarchus labrax).

Individual variation in the response to environmental challenges depends partly on innate reaction norms, partly on experience-based cognitive/emotional evaluations that individuals make of the situation. The goal of this study was to investigate whether pre-existing differences in behaviour predict the outcome of such assessment of environmental cues, using a conditioned place preference/avoidance (CPP/CPA) paradigm. A comparative vertebrate model (European sea bass, Dicentrarchus labrax) was used, and ninety juvenile individuals were initially screened for behavioural reactivity using a net restraining test. Thereafter each individual was tested in a choice tank using net chasing as aversive stimulus or exposure to familiar conspecifics as appetitive stimulus in the preferred or non preferred side respectively (called hereafter stimulation side). Locomotor behaviour (i.e. time spent, distance travelled and swimming speed in each tank side) of each individual was recorded and analysed with video software. The results showed that fish which were previously exposed to appetitive stimulus increased significantly the time spent on the stimulation side, while aversive stimulus led to a strong decrease in time spent on the stimulation side. Moreover, this study showed clearly that proactive fish were characterised by a stronger preference for the social stimulus and when placed in a putative aversive environment showed a lower physiological stress responses than reactive fish. In conclusion, this study showed for the first time in sea bass, that the CPP/CPA paradigm can be used to assess the valence (positive vs. negative) that fish attribute to different stimuli and that individual behavioural traits is predictive of how stimuli are perceived and thus of the magnitude of preference or avoidance behaviour.

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