Dynamic impacts of short-term bath administration of enrofloxacin on juvenile black seabream Acanthopagrus schlegelii.

Dynamic impacts of short-term enrofloxacin (ENR) exposure on juvenile marine fish are not well understood, and the underlying mechanisms remain unclear. We therefore investigated the accumulation and elimination of ENR in the liver of juvenile black seabream Acanthopagrus schlegelii. Meanwhile, the dynamic alterations of biochemical parameters and liver transcriptomes after short-term bath immersion and withdrawal treatment were explored. The results indicated that the contents of ENR in the liver were significantly increased after bath administration for 24 h, and then quickly declined to very low concentrations along with the decontamination time increasing. Judging from the changes in biochemical indicators and liver transcriptomic alterations, 0.5 and 1 mg/L ENR exposure for 24 h triggered oxidative stress, impairment of immune system, as well as aberrant lipid metabolism via differential molecular pathways. Interestingly, biochemical and transcriptome analysis as well as integrated biomarker response (IBR) values showed that more significant changes appeared in 1 mg/L ENR group at decontamination periods, which indicated that the impact of high dose ENR on juvenile A. schlegelii may persist even after depuration for 7 days. These results revealed that the risk of short-term bath of 1 mg/L ENR should not be overlooked even after depuration period. Therefore, attention should be paid to the dosage control when administering the drug to juvenile A. schlegelii, and the restoration of physiological disturbance may be an important factor in formulating a reasonable treatment plan.

The Expansion of Sirtuin Gene Family in Gilthead Sea Bream (Sparus aurata)-Phylogenetic, Syntenic, and Functional Insights across the Vertebrate/Fish Lineage.

The Sirtuin (SIRT1-7) family comprises seven evolutionary-conserved enzymes that couple cellular NAD availability with health, nutrition and welfare status in vertebrates. This study re-annotated the sirt3/5 branch in the gilthead sea bream, revealing three paralogues of sirt3 (sirt3.1a/sirt3.1b/sirt3.2) and two of sirt5 (sirt5a/sirt5b) in this Perciform fish. The phylogeny and synteny analyses unveiled that the Sirt3.1/Sirt3.2 dichotomy was retained in teleosts and aquatic-living Sarcopterygian after early vertebrate 2R whole genome duplication (WGD). Additionally, only certain percomorphaceae and gilthead sea bream showed a conserved tandem-duplicated synteny block involving the mammalian-clustered sirt3.1 gene (psmd13-sirt3.1a/b-drd4-cdhr5-ctsd). Conversely, the expansion of the Sirt5 branch was shaped by the teleost-specific 3R WGD. As extensively reviewed in the literature, human-orthologues (sirt3.1/sirt5a) showed a high, conserved expression in skeletal muscle that increased as development advanced. However, recent sirt3.2 and sirt5b suffered an overall muscle transcriptional silencing across life, as well as an enhanced expression on immune-relevant tissues and gills. These findings fill gaps in the ontogeny and differentiation of Sirt genes in the environmentally adaptable gilthead sea bream, becoming a good starting point to advance towards a full understanding of its neo-functionalization. The mechanisms originating from these new paralogs also open new perspectives in the study of cellular energy sensing processes in vertebrates.

Subchronic oral exposure to polystyrene microplastics affects hepatic lipid metabolism, inflammation, and oxidative balance in gilthead seabream (Sparus aurata).

Microplastics (MPs) pose a clear threat to aquatic organisms affecting their health. Their impact on liver homeostasis, as well as on the potential onset of nonalcoholic fatty liver disease (NAFLD), is still poorly investigated and remains almost unknown. The aim of this study was to evaluate the outcomes of subchronic exposure to polystyrene MPs (PS-MPs; 1-20 µm; 0, 25, or 250 mg/kg b.w./day) on lipid metabolism, inflammation, and oxidative balance in the liver of gilthead seabreams (Sparus aurata Linnaeus, 1758) exposed for 21 days via contaminated food. PS-MPs induced an up-regulation of mRNA levels of crucial genes associated with lipid synthesis and storage (i.e., PPARy, Srebp1, Fasn) without modifications of genes involved in lipid catabolism (i.e., PPARα, HL, Pla2) or transport and metabolism (Fabp1) in the liver. The increase of CSF1R and pro-inflammatory cytokines gene expression (i.e., TNF-α and IL-1ß) was also observed in exposed fish in a dose-dependent manner. These findings were confirmed by hepatic histological evaluations reporting evidence of lipid accumulation, inflammation, and necrosis. Moreover, PS-MPs caused the impairment of the hepatic antioxidant defense system through the alteration of its enzymatic (catalase, superoxide dismutase, and glutathione reductase) and non-enzymatic (glutathione) components, resulting in the increased production of reactive oxygen species (ROS) and malondialdehyde (MDA), as biomarkers of oxidative damage. The alteration of detoxifying enzymes was inferred by the decreased Ethoxyresorufin-O-deethylase (EROD) activity and the increased activity of glutathione-S-transferase (GST) at the highest PS-MP dose. The study suggests that PS-MPs affect the liver health of gilthead seabream. The liver dysfunction and damage caused by exposure to PS-MPs result from a detrimental interplay of inflammation, oxidative damage, and antioxidant and detoxifying enzymatic systems modifications, altering the gut-liver axis homeostasis. This scenario is suggestive of the involvement of MP-induced effects in the onset and progression of hepatic lipid dysfunction in gilthead seabream.

In Silico and In Vitro multiple analysis approach for screening naturally derived ligands for red seabream aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (AHR) is a key ligand-dependent transcription factor that mediates the toxic effects of compounds such as dioxin. Recently, natural ligands of AHR, including flavonoids, have been attracting physiological and toxicological attention as they have been reported to regulate major biological functions such as inflammation and anti-cancer by reducing the toxic effects of dioxin. Additionally, it is known that natural AHR ligands can accumulate in wildlife tissues, such as fish. However, studies in fish have investigated only a few ligands in experimental fish species, and the AHR response of marine fish to natural AHR ligands of various other structures has not been thoroughly investigated. To explore various natural AHR ligands in marine fish, which make up the most fish, it is necessary to develop new screening methods that consider the specificity of marine fish. In this study, we investigated the response of natural ligands by constructing in vitro and in silico experimental systems using red seabream as a model species. We attempted to develop a new predictive model to screen potential ligands that can induce transcriptional activation of red seabream AHR1 and AHR2 (rsAHR1 and rsAHR2). This was achieved through multiple analyses using in silico/ in vitro data and Tox21 big data. First, we constructed an in vitro reporter gene assay of rsAHR1 and rsAHR2 and measured the response of 10 representatives natural AHR ligands in COS-7 cells. The results showed that FICZ, Genistein, Daidzein, I3C, DIM, Quercetin and Baicalin induced the transcriptional activity of rsAHR1 and rsAHR2, while Resveratrol and Retinol did not induce the transcriptional activity of rsAHR isoforms. Comparing the EC50 values of the respective compounds in rsAHR1 and rsAHR2, FICZ, Genistein, and Daidzein exhibited similar isoform responses, but I3C, Baicalin, DIM and Quercetin show the isoform-specific responses. These results suggest that natural AHR ligands have specific profiling and transcriptional activity for each rsAHR isoform. In silico analysis, we constructed homology models of the ligand binding domains (LBDs) of rsAHR1 and rsAHR2 and calculated the docking energies (U_dock values) of natural ligands with measured in vitro transcriptional activity and dioxins reported in previous studies. The results showed a significant correlation (R2=0.74(rsAHR1), R2=0.83(rsAHR2)) between docking energy and transcriptional activity (EC50) value, suggesting that the homology model of rsAHR1 and rsAHR2 can be utilized to predict the potential transactivation of ligands. To broaden the applicability of the homology model to diverse compound structures and validate the correlation with transcriptional activity, we conducted additional analyses utilizing Tox21 big data. We calculated the docking energy values for 1860 chemicals in both rsAHR1 and rsAHR2, which were tested for transcriptional activation in Tox21 data against human AHR. By comparing the U_dock energy values between 775 active compounds and 1085 inactive compounds, a significant difference (p<0.001) was observed between the U_dock energy values in the two groups, suggesting that the U_dock value can be applied to distinguish the activation of compounds. Furthermore, we observed a significant correlation (R2=0.45) between the AC50 of Tox21 database and U_dock values of human AHR model. In conclusion, we calculated equations to translate the results of an in silico prediction model for ligand screening of rsAHR1 and rsAHR2 transactivation. This ligand screening model can be a powerful tool to quantitatively estimate AHR transactivation of major marine agents to which red seabream may be exposed. The study introduces a new screening approach for potential natural AHR ligands in marine fish, based on homology model-docking energy values of rsAHR1 and rsAHR2, with implications for future agonist development and applications bridging in silico and in vitro data.

Using cell culture systems from the Persian Gulf Arabian yellowfin sea bream, Acanthopagrus arabicus, to assess the effects of dexamethasone on gonad and brain aromatase activity and steroid production.

Dexamethasone (DEX) is a synthetic glucocorticoid widely used as pharmaceutical and usually exists in effluents with varying degrees of concentrations. In this study, cultivated Brain, ovary and testis cells from Arabian Sea bream, Acanthopagrus arabicus, were treated by DEX at concentrations of 0, 0.3, 3.0, 30.0 and 300.0 µg/ml for 48 h. The aromatase activity and steroid (17-ß-estradiol (E2), progesterone (P) and testosterone (T)) production by cells were measured at 12, 24 and 48 h of the experiment. The results showed that the sensitivity of cultivated ovarian, testicular and brain cells to DEX increased dose dependently. DEX was potent inhibitor of aromatase activity at specially 30.0 and 300.0 µg/ml in the cultivated ovarian and testicular cells at different sampling time. On the other hand, DEX was found to stimulate the aromatase activity of fish brain. DEX also decreased E2, P and T production by cultivated ovarian and testicular cells during the experiment. While, DEX caused an increase in the production of E2 and P by brain cells, which seems logical considering the stimulating effect of this drug on brain aromatase activity. In conclusion, results highlight that DEX is able to change the activity of aromatase, and disrupt the biosynthesis of estrogens and thus affect reproduction in fish.

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.

Ecotoxicological responses of juvenile Sparus aurata to BDE-99 and BPA exposure: A multi-biomarker approach integrating immune, endocrine and oxidative endpoints.

Pentabromodiphenyl ether (BDE-99) and bisphenol A (BPA) are synthetic organic compounds present in several daily use products. Due to their physicochemical properties, they are ubiquitously present in aquatic ecosystems and considered highly persistent. Recent evidence has confirmed that both emerging compounds are toxic to humans and terrestrial mammals eliciting a wide range of detrimental effects at endocrine and immune levels. However, the ecotoxicological responses that they can trigger in vertebrate marine species have not yet been established. Hence, this study aimed to investigate the ecotoxicological responses of juvenile Sparus aurata upon chronic (28 days) dietary exposure to BDE-99 and BPA (alone and combined) following an integrated multi-biomarker approach that combined fitness indicators (Fulton's K and splenosomatic indexes) with endocrine [cortisol, 17ß-estradiol (E2), 11-ketotestosterone (11-KT) concentrations] and immune (peroxidase and antiprotease activities) endpoints in fish plasma, and oxidative stress [superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST) activities, and lipid peroxidation (LPO)] endpoints in the fish spleen. The mixture of BDE-99 and BPA yielded the highest IBR index value in both plasma and spleen biomarkers, therefore, suggesting that the effects of these compounds are more severe when they act together. Endocrine biomarkers were the most responsive in the three contaminated treatments. Fitness indicators were not affected by the individual nor the interactive effects of BDE-99 and BPA. These findings highlight the relevance of accounting for the interactive effects of emerging chemical contaminants and integrating responses associated with distinct biological pathways when investigating their impacts on marine life, as such a multi-biomarker approach provides a broader, more realistic and adequate perspective of challenges faced by fish in a contaminated environment.

Participation of Hepcidins in the Inflammatory Response Triggered by λ-Carrageenin in Gilthead Seabream (Sparus aurata).

The role of hepcidins, antimicrobial peptides involved in iron metabolism, immunity, and inflammation, is studied. First, gilthead seabream (Sparus aurata L.) head-kidney leucocytes (HKLs) were incubated with λ-carrageenin to study the expression of hepcidin and iron metabolism-related genes. While the expression of most of the genes studied was upregulated, the expression of ferroportin gene (slc40a) was downregulated. In the second part of the study, seabream specimens were injected intramuscularly with λ-carrageenin or buffer (control). The expression of the same genes was evaluated in the head kidney, liver, and skin at different time points after injection. The expression of Hamp1m, ferritin b, and ferroportin genes (hamp1, fthb, and slc40a) was upregulated in the head kidney of fish from the λ-carrageenin-injected group, while the expression of Hamp2C and Hamp2E genes (hamp2.3 and hamp2.7) was downregulated. In the liver, the expression of hamp1, ferritin a (ftha), slc40a, Hamp2J, and Hamp2D (hamp2.5/6) genes was downregulated in the λ-carrageenin-injected group. In the skin, the expression of hamp1 and (Hamp2A Hamp2C) hamp2.1/3/4 genes was upregulated in the λ-carrageenin-injected group. A bioinformatic analysis was performed to predict the presence of transcription factor binding sites in the promoter region of hepcidins. The primary sequence of hepcidin was conserved among the different mature peptides, although changes in specific amino acid residues were identified. These changes affected the charge, hydrophobicity, and probability of hepcidins being antimicrobial peptides. This study sheds light on the poorly understood roles of hepcidins in fish. The results provide insight into the regulatory mechanisms of inflammation in fish and could contribute to the development of new strategies for treat inflammation in farm animals.

Efficacy of nutritional selenium nanoparticles on growth performance, immune response, antioxidant capacity, expression of growth and immune-related genes, and post-stress recovery in juvenile Sobaity seabream (Sparidentex hasta).

This study evaluated the impacts of nano-Se on the growth, immunity, antioxidant capacity, physiological parameters, gene expression, and stress resistance of fingerling Sobaity seabream (Sparidentex hasta). The fish with an average weight of 21.5 ± 0.1 g were divided into four treatment groups in triplicates that received one of the test diets supplemented with varying levels of nano-Se: 0 (control), 0.5 (Se-0.5), 1 (Se-1), and 2 (Se-2) mg/Kg for 60 days. The results showed that final weight, weight gain rate, specific growth rate, feed intake, and feed conversion ratio improved with significant linear and quadratic trends (P < 0.05) in response to nano-Se-supplemented diets, and the best values were measured in the Se-2 group. Superoxide dismutase activity level remained unaffected among the four groups (P > 0.05). Catalase activity increased in nano-Se-supplemented groups, with the highest level measured in fish fed the Se-0.5 diet. Glutathione peroxidase activity levels were not significantly different between the control and nano-Se groups, but the lowest malondialdehyde concentration was detected in the Se-2 group. Nano-Se had no marked effect on total plasma Ig levels; however, the highest lysozyme activity and alternative complement activity (ACH50) were observed in the Se-0.5 and Se-2 groups, respectively. No significant differences (P > 0.05) were observed in plasma total protein, albumin, globulin, triglyceride, and thyroid hormone (T3 and T4) contents among the groups. However, the lowest cholesterol and low-density lipoprotein values and the highest high-density lipoprotein concentration were measured in the Se-2 group. The Se-0.5 and Se-1 groups exhibited significantly lower levels of aspartate aminotransferase activity, and the lowest alkaline phosphatase activity level was detected in the Se-1 group. The expression level of insulin-like growth factor I gene in all nano-Se-fed groups was significantly higher than the control. Also, the expression of interleukin-1ß and lysozyme genes was significantly upregulated in nano-Se-supplemented groups, with the highest values in the Se-2 group. Following acute crowding stress, plasma cortisol and lactate levels at all post-stress time intervals were not significantly different among the experimental groups. Fish fed the Se-0.5 and Se-2 diets tended to have lower plasma glucose concentrations than other groups. In conclusion, dietary nano-Se at 2 mg/kg is recommended to promote growth performance and enhance antioxidant and immune parameters in Sobaity juveniles.

Identification of two MEF2s and their role in inhibiting the transcription of the mstn2a gene in the yellowfin seabream, Acanthopagrus latus (Hottuyn, 1782).

Myocyte-specific enhancer binding factor 2 (MEF2), which belongs to the MADS superfamily, is a pivotal and conserved transcription factor that combines with the E-box motif to control the expression of muscle genes. Myostatin (mstn), a muscle growth inhibitor, is a vital member of the TGF-ß superfamily. Currently, an understanding of the mechanisms of A. latus mstn (Almstn) transcriptional regulation mediated by MEF2 in fish muscle development is lacking. In the present study, two AlMEF2s (AlMEF2A and AlMEF2B) and Almstn2a were characterized from Acanthopagrus latus. AlMEF2A and AlMEF2B had 456 and 315 amino acid (aa) residues, respectively. Two typical regions, a MADS-box, MEF2, and transcriptionally activated (TAD) domains, are present in both AlMEF2s. The expression profiles of the two AlMEF2 genes were similar. The AlMEF2 genes were mainly expressed in the brain, white muscle, and liver, while Almstn2a expression was higher in the brain than in other tissues. Moreover, the expression trends of AlMEF2s and Almstn2a were significantly changed after starvation and refeeding in the five groups. Additionally, truncation experiments showed that -987 to +168 and -105 to +168 were core promoters of Almstn2a that responded to AlMEF2A and AlMEF2B, respectively. The point mutation experiment confirmed that Almstn2a transcription relies on the mutation binding sites 1 or 5 (M1/5) and mutation binding sites 4 or 5 (M4/5) for AlMEF2A and AlMEF2B regulation, respectively. The electrophoretic mobile shift assay (EMSA) further verified that M1 (-527 to -512) was a pivotal site where AlMEF2A acted on the Almstn2a gene. Furthermore, a siRNA interference gene expression experiment showed that reduced levels of AlMEF2A or AlMEF2B could prominently increase Almstn2a transcription. These results provide new information about the regulation of Almstn2a transcriptional activity by AlMEF2s and a theoretical basis for the regulatory mechanisms involved in muscle development in fish.

Early life stage mechanisms of an active fish species to cope with ocean warming and hypoxia as interacting stressors.

Ocean's characteristics are rapidly changing, modifying environmental suitability for early life stages of fish. We assessed whether the chronic effects of warming (24 °C) and hypoxia (<2-2.5 mg L-1) will be amplified by the combination of these stressors on mortality, growth, behaviour, metabolism and oxidative stress of early stages of the white seabream Diplodus sargus. Combined warming and hypoxia synergistically increased larval mortality by >51%. Warming induced faster growth in length and slower gains in weight when compared to other treatments. Boldness and exploration were not directly affected, but swimming activity increased under all test treatments. Under the combination of warming and hypoxia, routine metabolic rate (RMR) significantly decreases when compared to other treatments and shows a negative thermal dependence. Superoxide dismutase and catalase activities increased under warming and were maintained similar to control levels under hypoxia or under combined stressors. Under hypoxia, the enzymatic activities were not enough to prevent oxidative damages as lipid peroxidation and DNA damage increased above control levels. Hypoxia reduced electron transport system activity (cellular respiration) and isocitrate dehydrogenase activity (aerobic metabolism) below control levels. However, lactate dehydrogenase activity (anaerobic metabolism) did not differ among treatments. A Redundancy Analysis showed that ∼99% of the variability in mortality, growth, behaviour and RMR among treatments can be explained by molecular responses. Mortality and growth are highly influenced by oxidative stress and energy metabolism, exhibiting a positive relationship with reactive oxygen species and a negative relationship with aerobic metabolism, regardless of treatment. Under hypoxic condition, RMR, boldness and swimming activity have a positive relationship with anaerobic metabolism regardless of temperature. Thus, seabreams may use anaerobic reliance to counterbalance the effects of the stressors on RMR, activity and growth. The outcomes suggests that early life stages of white seabream overcame the single and combined effects of hypoxia and warming.

Seascapes Shaped the Local Adaptation and Population Structure of South China Coast Yellowfin Seabream (Acanthopagrus latus).

Understanding the genetic composition and regional adaptation of marine species under environmental heterogeneity and fishing pressure is crucial for responsible management. In order to understand the genetic diversity and adaptability of yellowfin seabream (Acanthopagrus latus) along southern China coast, this study was conducted a seascape genome analysis on yellowfin seabream from the ecologically diverse coast, spanning over 1600 km. A total of 92 yellowfin seabream individuals from 15 sites were performed whole-genome resequencing, and 4,383,564 high-quality single nucleotide polymorphisms (SNPs) were called. By conducting a genotype-environment association analysis, 29,951 adaptive and 4,328,299 neutral SNPs were identified. The yellowfin seabream exhibited two distinct population structures, despite high gene flow between sites. The seascape genome analysis revealed that genetic structure was influenced by a variety of factors including salinity gradients, habitat distance, and ocean currents. The frequency of allelic variation at the candidate loci changed with the salinity gradient. Annotation of these loci revealed that most of the genes are associated with osmoregulation, such as kcnab2a, kcnk5a, and slc47a1. These genes are significantly enriched in pathways associated with ion transport including G protein-coupled receptor activity, transmembrane signaling receptor activity, and transporter activity. Overall, our findings provide insights into how seascape heterogeneity affects adaptive evolution, while providing important information for regional management in yellowfin seabream populations.

Broodstock nutritional programming differentially affects the hepatic transcriptome and genome-wide DNA methylome of farmed gilthead sea bream (Sparus aurata) depending on genetic background.

BACKGROUND: Broodstock nutritional programming improves the offspring utilization of plant-based diets in gilthead sea bream through changes in hepatic metabolism. Attention was initially focused on fatty acid desaturases, but it can involve a wide range of processes that remain largely unexplored. How all this can be driven by a different genetic background is hardly underlined, and the present study aimed to assess how broodstock nutrition affects differentially the transcriptome and genome-wide DNA methylome of reference and genetically selected fish within the PROGENSA® selection program. RESULTS: After the stimulus phase with a low fish oil diet, two offspring subsets of each genetic background received a control or a FUTURE-based diet. This highlighted a different hepatic transcriptome (RNA-seq) and genome-wide DNA methylation (MBD-seq) pattern depending on the genetic background. The number of differentially expressed transcripts following the challenge phase varied from 323 in reference fish to 2,009 in genetically selected fish. The number of discriminant transcripts, and associated enriched functions, were also markedly higher in selected fish. Moreover, correlation analysis depicted a hyper-methylated and down-regulated gene expression state in selected fish with the FUTURE diet, whereas the opposite pattern appeared in reference fish. After filtering for highly represented functions in selected fish, 115 epigenetic markers were retrieved in this group. Among them, lipid metabolism genes (23) were the most reactive following ordering by fold-change in expression, rendering a final list of 10 top markers with a key role on hepatic lipogenesis and fatty acid metabolism (cd36, pitpna, cidea, fasn, g6pd, lipt1, scd1a, acsbg2, acsl14, acsbg2). CONCLUSIONS: Gene expression profiles and methylation signatures were dependent on genetic background in our experimental model. Such assumption affected the magnitude, but also the type and direction of change. Thus, the resulting epigenetic clock of reference fish might depict an older phenotype with a lower methylation for the epigenetically responsive genes with a negative methylation-expression pattern. Therefore, epigenetic markers will be specific of each genetic lineage, serving the broodstock programming in our selected fish to prevent and mitigate later in life the risk of hepatic steatosis through changes in hepatic lipogenesis and fatty acid metabolism.

High-fat-diet induced inflammation and apoptosis via activation of Ire1α in liver and hepatocytes of black seabream (Acanthopagrus schlegelii).

The present study aimed to reveal the role of inositol-requiring enzyme 1α (Ire1α) in mediating high-fat-diet (HFD) induced inflammation and apoptosis in fish and elucidate underling mechanisms of action. In experiment 1, black seabream juveniles were fed a control diet (Control, 12 % dietary lipid) or a high fat diet (HFD, 19 % dietary lipid) for eight weeks. In experiment 2, primary hepatocytes were isolated from black seabream juveniles and treated with oleic acid (OA, 200 µmol/L), OA + transfection with non-silencing control siRNA (negative control) (OA + NC), and OA + transfection with ire1α-small interfering RNA (OA + siire1α) for 48 h versus untreated (Control). Results indicated that fish fed HFD increased lipid deposition in the liver and caused hepatic steatosis. HFD group had significantly higher ire1α/Ire1α mRNA and phosphorylated protein expression and endoplasmic reticulum stress (ERS) related genes expression compared to the Control group, indicating that ERS was triggered. Meanwhile, feeding HFD induced inflammation and apoptosis by evaluated nuclear factor kappa B (nf-κb) mRNA and phosphorylated Nf-κb p65 protein expression, and c-Jun N-terminal kinase (jnk) mRNA and protein expression. However, knock down of ire1α (OA + siire1α) in primary hepatocytes alleviated OA-induced increased expression of ire1α/Ire1α mRNA and protein expression, nf-κb/Nf-κb p65 mRNA and phosphorylated protein expression, and jnk/Jnk mRNA and phosphorylated protein expression. These findings revealed the underling mechanism of action of HFD in fish, confirming that HFD increased ESR stress and Ire1α that, in turn, activated Nf-κb and Jnk pathways in hepatocytes and liver mediating HFD-induced inflammation and apoptosis.

Chitosan-Based Sustained Expression of Sterol Regulatory Element-Binding Protein 1a Stimulates Hepatic Glucose Oxidation and Growth in Sparus aurata.

The administration of a single dose of chitosan nanoparticles driving the expression of sterol regulatory element-binding protein 1a (SREBP1a) was recently associated with the enhanced conversion of carbohydrates into lipids. To address the effects of the long-lasting expression of SREBP1a on the growth and liver intermediary metabolism of carnivorous fish, chitosan-tripolyphosphate (TPP) nanoparticles complexed with a plasmid expressing the N terminal active domain of hamster SREBP1a (pSG5-SREBP1a) were injected intraperitoneally every 4 weeks (three doses in total) to gilthead sea bream (Sparus aurata) fed high-protein-low-carbohydrate and low-protein-high-carbohydrate diets. Following 70 days of treatment, chitosan-TPP-pSG5-SREBP1a nanoparticles led to the sustained upregulation of SREBP1a in the liver of S. aurata. Independently of the diet, SREBP1a overexpression significantly increased their weight gain, specific growth rate, and protein efficiency ratio but decreased their feed conversion ratio. In agreement with an improved conversion of dietary carbohydrates into lipids, SREBP1a expression increased serum triglycerides and cholesterol as well as hepatic glucose oxidation via glycolysis and the pentose phosphate pathway, while not affecting gluconeogenesis and transamination. Our findings support that the periodical administration of chitosan-TPP-DNA nanoparticles to overexpress SREBP1a in the liver enhanced the growth performance of S. aurata through a mechanism that enabled protein sparing by enhancing dietary carbohydrate metabolisation.

Combined evaluation of multi-biomarkers and metal bioaccumulations in two different fish species (Sparus aurata and Chelon labrosus) from Izmir Bay, Türkiye (Aegean Sea): Spatial, temporal and tissue-specific approaches.

Biomarkers were investigated to assess the effects of metal accumulation in Sparus aurata and Chelon labrosus in the Izmir Bay. Fish were collected from the Inner and Outer Bays in November 2020 and May 2021. According to the metal and biomarker measurements found in the organs, it was observed that the levels were higher in the liver and gill tissues than in the muscle tissues. Significant differences between tissues were found for all metals and biomarkers. In addition, biochemical biomarkers were found to be significant predictors of metal bioaccumulation. Histological changes were observed in liver and gills in each species and location. Although the metal levels determined according to the health risk assessment were below the threshold limits, the As levels for the lifetime cancer risk were within the limits to be considered. In conclusion, this study will provide robust results for its related study area.

The potential of a combination of pungent spices as a novel supplement in gilthead seabream (Sparus aurata) diets to aid in the strategic use of fish oil in aquafeeds: a holistic perspective.

This work studied the potential of a combination of pungent spices (capsicum, black pepper, ginger, and cinnamaldehyde) to be used as a supplement in diets of gilthead seabream (Sparus aurata; 44.1 ± 4.2 g). During 90 days, fish were fed three experimental diets with low inclusion of fish oil and containing poultry fat as the main source of lipids, supplemented with graded levels of the tested supplement: 0 (control), 0.1 (SPICY0.1%), and 0.15% (SPICY0.15%). As a result, the pungent spices enhanced the growth performance, the activity of the bile-salt-activated lipase in the intestine, and decreased fat deposit levels within enterocytes. The SPICY0.1% diet reduced the feed conversion ratio and the perivisceral fat index and lipid deposits in the liver. Moreover, the ratio of docosahexaenoic acid/eicosapentaenoic acid in fillet increased in fish fed the SPICY0.1% diet, while the hepatic levels of docosahexaenoic acid and total n-3 polyunsaturated fatty acids increased in fish fed the SPICY0.15% diet. Furthermore, there was an effect on the expression of some biomarkers related to lipid metabolism in 2-h postprandial fish (fasn, elovl6, scd1b, cyp7a1, lpl, and pparß), and in 48 h fasted-fish fed with the SPICY0.1% diet, a regulation of the intestinal immune response was indicated. However, no significant differences were found in lipid apparent digestibility and proximate macronutrient composition. The spices did not affect biomarkers of hepatic or oxidative stress. No differences in microbial diversity were found, except for an increase in Simpson's Index in the posterior intestine of fish fed the SPICY0.1% diet, reflected in the increased relative abundance of the phylum Chloroflexi and lower relative abundances of the genera Campylobacter, Corynebacterium, and Peptoniphilus. In conclusion, the supplementation of gilthead seabream diets with pungent spices at an inclusion of 0.1% was beneficial to enhance growth performance and feed utilization; reduce fat accumulation in the visceral cavity, liver, and intestine; and improve the fish health status and condition. Results suggest that the tested supplement can be used as part of a nutritional strategy to promote a more judicious use of fish oil in fish diets due to its decreasing availability and rising costs.

The osteogenic and mineralogenic potential of the microalgae Skeletonema costatum and Tetraselmis striata CTP4 in fish models.

Skeletal disorders are problematic aspects for the aquaculture industry as skeletal deformities, which affect most species of farmed fish, increase production costs and affect fish welfare. Following recent findings that show the presence of osteoactive compounds in marine organisms, we evaluated the osteogenic and mineralogenic potential of commercially available microalgae strains Skeletonema costatum and Tetraselmis striata CTP4 in several fish systems. Ethanolic extracts increased extracellular matrix mineralization in gilthead seabream (Sparus aurata) bone-derived cell cultures and promoted osteoblastic differentiation in zebrafish (Danio rerio) larvae. Long-term dietary exposure to both extracts increased bone mineralization in zebrafish and upregulated the expression of genes involved in bone formation (sp7, col1a1a, oc1, and oc2), bone remodeling (acp5a), and antioxidant defenses (cat, sod1). Extracts also improved the skeletal status of zebrafish juveniles by reducing the incidence of skeletal anomalies. Our results indicate that both strains of microalgae contain osteogenic and mineralogenic compounds, and that ethanolic extracts have the potential for an application in the aquaculture sector as dietary supplements to support fish bone health. Future studies should also identify osteoactive compounds and establish whether they can be used in human health to broaden the therapeutic options for bone erosive disorders such as osteoporosis.

Oral cortisol and dexamethasone intake: Differential physiology and transcriptional responses in the marine juvenile Sparus aurata.

This study approached the long-term oral administration of cortisol (F) and dexamethasone (DEX), two synthetic glucocorticoids, compared to a control group (CT) in the juveniles of a marine teleost, the gilthead seabream (Sparus aurata). Physiologically, DEX treatment impaired growth, which appears to be linked to carbohydrate allocation in muscle and liver, hepatic triglycerides depletion, and reduced hematocrit. Hypophyseal gh mRNA expression was 2-fold higher in DEX than in CT or F groups. Similarly, hypothalamic trh and hypophyseal pomcb followed this pattern. Plasma cortisol levels were significantly lower in DEX than in CT, while F presented intermediate levels. In the posterior intestine, measured short circuit-current (Isc) was more anion absorptive in CT and F compared to the DEX group, whereas Isc remained unaffected in the anterior intestine. The derived transepithelial electric resistance (TEER) significantly differed between intestinal regions in the DEX group. These results provide new insights to understand better potential targeted biomarkers indicative of the differential glucocorticoid or mineralocorticoid-receptors activation in fish.

Cysteamine improves growth and the GH/IGF axis in gilthead sea bream (Sparus aurata): in vivo and in vitro approaches.

Aquaculture is the fastest-growing food production sector and nowadays provides more food than extractive fishing. Studies focused on the understanding of how teleost growth is regulated are essential to improve fish production. Cysteamine (CSH) is a novel feed additive that can improve growth through the modulation of the GH/IGF axis; however, the underlying mechanisms and the interaction between tissues are not well understood. This study aimed to investigate the effects of CSH inclusion in diets at 1.65 g/kg of feed for 9 weeks and 1.65 g/kg or 3.3 g/kg for 9 weeks more, on growth performance and the GH/IGF-1 axis in plasma, liver, stomach, and white muscle in gilthead sea bream (Sparus aurata) fingerlings (1.8 ± 0.03 g) and juveniles (14.46 ± 0.68 g). Additionally, the effects of CSH stimulation in primary cultured muscle cells for 4 days on cell viability and GH/IGF axis relative gene expression were evaluated. Results showed that CSH-1.65 improved growth performance by 16% and 26.7% after 9 and 18 weeks, respectively, while CSH-3.3 improved 32.3% after 18 weeks compared to control diet (0 g/kg). However, no significant differences were found between both experimental doses. CSH reduced the plasma levels of GH after 18 weeks and increased the IGF-1 ones after 9 and 18 weeks. Gene expression analysis revealed a significant upregulation of the ghr-1, different igf-1 splice variants, igf-2 and the downregulation of the igf-1ra and b, depending on the tissue and dose. Myocytes stimulated with 200 µM of CSH showed higher cell viability and mRNA levels of ghr1, igf-1b, igf-2 and igf-1rb compared to control (0 µM) in a similar way to white muscle. Overall, CSH improves growth and modulates the GH/IGF-1 axis in vivo and in vitro toward an anabolic status through different synergic ways, revealing CSH as a feasible candidate to be included in fish feed.