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.

Genetic Contribution of the Adrenergic, Cholinergic, and Serotonergic Systems to Leiomyoma Development and Treatment.

The link between the autonomic nervous system and tumor biology is being unfold. We aim to study the contribution of genes of the adrenergic (ADBR2 - rs1042713, NM_000024.6:c.46G>A, NP_000015.2:p. Gly16Arg), cholinergic (CHRNA5 - rs16969968, NM_000745.3:c.1192G>A, NP_000736.2:p.Asp398Asn), and serotonergic systems (SLC6A4 - 5-HTTVNTR-intron2, HTR2A - rs6313, NM_000621.5:c.102C>T, NP_ 001365853 .1: p. Ser 34=) to gynecological tumorigenesis and their treatment by embolization. A total of 517 DNA samples from women were analyzed. Samples were genotyped by PCR, PCR-RFLP and EndPoint genotyping. Results show a statistically significant association between the AA genotype of the ADBR2 gene and GG genotype of the CHRNA5 gene with leiomyoma (OR = 2.311; p = 0.003 and OR = 2.165; p = 0.001, respectively), and the epistatic interaction between genotypes increases the risk (OR = 2.458; p= 0.043). The GG genotype (CHRNA5) shows a lower reduction of the volume of the main leiomyoma after treatment (p=0.015). Combination of the genotypes 12/12-AA (SLC6A4 - ADBR2) increases the risk to leiomyoma (OR = 2.540, p= 0.030). TT genotype of HTR2A gene in combination with any of the two risk genotypes (of ADBR2 or CHRNA5) increases substantially the risk (OR = 5.266, p = 0.006; OR = 6.364, p=0.007, respectively). We conclude that ADBR2 and CHRNA5 genes have a relevant role that is enhanced by the epistatic relationship with the genes HTR2A and SLC6A4. CHRNA5 gene may also be a modulator of the success of embolization. We confirm the contribution of the genetics of Autonomous Nervous System to tumor biology.

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.

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.

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.