Molecular characterization, expression profiling, and functional analysis of prohibitin 1 in red seabream, Pagrus major.

Prohibitin 1 (PHB1) is ubiquitously expressed in multiple compartments within cells and is involved in the cell cycle, cell signaling, apoptosis, transcriptional regulation, and mitochondrial biogenesis at the cellular level and in the inflammation-associated and immunological functions of B and T lymphocytes. PHB1 is an important protein that performs antioxidant regulation and immune functions inside and outside cells but has not been sufficiently studied in teleost fish. Our study aimed to elucidate the functional properties and gain new insights into the biological processes and immune system of red seabream (Pagrus major), a commercially important fish cultured in South Korea and East Asia. PHB1 mRNA was most abundantly expressed in the head kidney of healthy red seabream, and significant changes in its expression were observed after artificial infection with bacteria and viruses. On analysis, reporter gene was also significantly upregulated by polyinosinic-polycytidylic acid, lipopolysaccharides, and hydrogen peroxide. Consequent to the functional characterization of PHB1 in cells via recombinant protein preparation, the activity of leukocytes was enhanced and the reactive oxygen species-induced stress in red blood cells was reduced. The results reveal the functional characteristics of PHB1 and provide new insights into the biological processes and immune system of P. major, with beneficial implications in the study of stress responses.

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.

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.

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.

Two IFNa3s mediate the regulation of IRF9 in the process of infection with Streptococcus iniae in yellowfin seabream, Acanthopagrus latus (Hottuyn, 1782).

IRF9 can play an antibacterial role by regulating the type I interferon (IFN) pathway. Streptococcus iniae can cause many deaths of yellowfin seabream, Acanthopagrus latus in pond farming. Nevertheless, the regulatory mechanism of type I IFN signalling by A. latus IRF9 (AlIRF9) against S. iniae remains elucidated. In our study, AlIRF9 has a total cDNA length of 3200 bp and contains a 1311 bp ORF encoding a presumed 436 amino acids (aa). The genomic DNA sequence of AlIRF9 has nine exons and eight introns, and AlIRF9 was expressed in various tissues, containing the stomach, spleen, brain, skin, and liver, among which the highest expression was in the spleen. Moreover, AlIRF9 transcriptions in the spleen, liver, kidney, and brain were increased by S. iniae infection. By overexpression of AlIRF9, AlIRF9 is shown as a whole-cell distribution, mainly concentrated in the nucleus. Moreover, the promoter fragments of -415 to +192 bp and -311 to +196 bp were regarded as core sequences from two AlIFNa3s. The point mutation analyses verified that AlIFNa3 and AlIFNa3-like transcriptions are dependent on both M3 sites with AlIRF9. In addition, AlIRF9 could greatly reduce two AlIFNa3s and interferon signalling factors expressions. These results showed that in A. latus, both AlIFNa3 and AlIFNa3-like can mediate the regulation of AlIRF9 in the process of infection with S. iniae.

Exploring the Integrated Role of miRNAs and lncRNAs in Regulating the Transcriptional Response to Amino Acids and Insulin-like Growth Factor 1 in Gilthead Sea Bream (Sparus aurata) Myoblasts.

In this study, gilthead sea bream (Sparus aurata) fast muscle myoblasts were stimulated with two pro-growth treatments, amino acids (AA) and insulin-like growth factor 1 (Igf-1), to analyze the transcriptional response of mRNAs, microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) and to explore their possible regulatory network using bioinformatic approaches. AA had a higher impact on transcription (1795 mRNAs changed) compared to Igf-1 (385 mRNAs changed). Both treatments stimulated the transcription of mRNAs related to muscle differentiation (GO:0042692) and sarcomere (GO:0030017), while AA strongly stimulated DNA replication and cell division (GO:0007049). Both pro-growth treatments altered the transcription of over 100 miRNAs, including muscle-specific miRNAs (myomiRs), such as miR-133a/b, miR-206, miR-499, miR-1, and miR-27a. Among 111 detected lncRNAs (>1 FPKM), only 30 were significantly changed by AA and 11 by Igf-1. Eight lncRNAs exhibited strong negative correlations with several mRNAs, suggesting a possible regulation, while 30 lncRNAs showed strong correlations and interactions with several miRNAs, suggesting a role as sponges. This work is the first step in the identification of the ncRNAs network controlling muscle development and growth in gilthead sea bream, pointing out potential regulatory mechanisms in response to pro-growth signals.

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.

Synthetic Antimicrobial Peptides Fail to Induce Leucocyte Innate Immune Functions but Elicit Opposing Transcriptomic Profiles in European Sea Bass and Gilthead Seabream.

Antimicrobial peptides (AMPs) are promising molecules in diverse fields, including aquaculture. AMPs possess lytic effects on a wide range of pathogens, resulting in a potential replacement for traditional antimicrobials in aquaculture. In addition, they also have modulatory effects on host immune responses. Thus, the objective of this work was to evaluate the immunomodulatory capability of three known synthetic AMPs derived from European sea bass, NK-lysin (Nkl), hepcidin (Hamp), and dicentracin (Dic), in head-kidney cell suspensions from European sea bass and gilthead seabream. The tested peptides were neither cytotoxic for European sea bass nor gilthead seabream cells and failed to modulate the respiratory burst and phagocytosis activities. However, they modified the pattern of transcription of immune-related genes differently in both species. Peptides were able to promote the expression of marker genes for anti-inflammatory (il10), antiviral (mx, irf3), cell-mediated cytotoxicity (nccrp1, gzmb), and antibody responses (ighm) in European sea bass, with the Nkl peptide being the most effective. Contrary to this, the effects of those peptides on gilthead seabream mainly resulted in the suppression of immune responses. To conclude, European sea bass-derived peptides can be postulated as potential tools for immunostimulation in European sea bass fish farms, but more efforts are required for their universal use in other species.

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.

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.

Functional Characterization of the Almstn2 Gene and Its Association with Growth Traits in the Yellowfin Seabream Acanthopagrus latus (Hottuyn, 1782).

Myostatin (mstn), also known as GDF8, is a growth and differentiation factor of the transforming growth factor-ß (TGF-ß) superfamily and plays a key inhibitory effect in the regulation of skeletal muscle development and growth in vertebrates. In the present study, to comprehend the role of the mstn2 gene of the yellowfin seabream Acanthopagrus latus (Almstn2b), the genomic sequence of Almstn2b is 2359 bp, which encodes 360 amino acids and is composed of three exons and two introns, was obtained. Two typical regions, a TGF-ß propeptide and TGF-ß domain, constitute Almstn2b. The topology indicated that Almstn2 was grouped together with other Perciformes, such as the gilthead seabream Sparus aurata. Moreover, Almstn2b was mainly expressed in the brain, fins, and spleen. Furthermore, five SNPs, one in the exons and four in the introns, were identified in the Almstn2b gene. The allele and genotype frequencies of SNP-Almstn2b +1885 A/G were significantly related to the total weight, interorbital distance, stem length, tail length, caudal length, caudal height, body length, and total length (p < 0.05). The allele and genotype frequencies of SNP-Almstn2b +1888 A/G were significantly related to the weight, interorbital distance, long head behind the eyes, body height, tail length, caudal length, and body length. Additionally, the relationship between the SNP-Almstn2b +1915 A/G locus and weight and long head behind the eyes was significant (p < 0.05). Furthermore, the other two SNPs were not significantly associated with any traits. Thus, the SNPs identified in this study could be utilized as candidate SNPs for breeding and marker-assisted selection in A. latus.

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.

Skeletal Morphogenesis and Anomalies in Gilthead Seabream: A Comprehensive Review.

The gilthead seabream, one of the most important species in Mediterranean aquaculture, with an increasing status of exploitation in terms of production volume and aquafarming technologies, has become an important research topic over the years. The accumulation of knowledge from several studies conducted during recent decades on their functional and biological characteristics has significantly improved their aquacultural aspects, namely their reproductive success, survival, and growth. Despite the remarkable progress in the aquaculture industry, hatchery conditions are still far from ideal, resulting in frequent abnormalities at the beginning of intensive culture, entailing significant economic losses. Those deformities are induced during the embryonic and post-embryonic periods of life, and their development is still poorly understood. In the present review, we created a comprehensive synthesis that covers the various aspects of skeletal morphogenesis and anomalies in the gilthead seabream, highlighting the genetic, environmental, and nutritional factors contributing to bone deformities and emphasized the potential of the gilthead seabream as a model organism for understanding bone morphogenesis in both aquaculture and translational biological research. This review article addresses the existing lack in the literature regarding gilthead seabream bone deformities, as there are currently no comprehensive reviews on this subject.

Molecular cloning and characterization of endoplasmic reticulum stress related genes grp78 and atf6α from black seabream (Acanthopagrus schlegelii) and their expressions in response to nutritional regulation.

Glucose-regulated protein 78 (grp78) and activating transcription factor 6α (atf6α) are considered vital endoplasmic reticulum (ER) molecular chaperones and ER stress (ERS) sensors, respectively. In the present study, the full cDNA sequences of these two ERS-related genes were first cloned and characterized from black seabream (Acanthopagrus schlegelii). The grp78 cDNA sequence is 2606 base pair (bp) encoding a protein of 654 amino acids (aa). The atf6α cDNA sequence is 2168 base pair (bp) encoding a protein of 645 aa. The predicted aa sequences of A. schlegelii grp78 and atf6α indicated that the proteins contain all the structural features, which were characteristic of the two genes in other species. Tissues transcript abundance analysis revealed that the mRNAs of grp78 and atf6α were expressed in all measured tissues, but the highest expression of these two genes was all recorded in the gill followed by liver/ brain. Moreover, in vivo experiment found that fish intake of a high lipid diet (HLD) can trigger ERS by activating grp78/Grp78 and atf6α/Atf6α. However, it can be alleviated by dietary betaine supplementation, similar results were also obtained by in vitro experiment using primary hepatocytes of A. schlegelii. These findings will be beneficial for us to evaluate the regulator effects of HLD supplemented with betaine on ERS at the molecular level, and thus provide some novel insights into the functions of betaine in marine fish fed with an HLD.

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.

The regulatory mechanisms of IRF7 mediated by the type I IFN signalling pathway against Streptococcus iniae in yellowfin seabream, Acanthopagrus latus (Hottuyn, 1782).

Interferon regulatory factor 7 (IRF7) regulates type I interferon (IFN) genes via combining to the ISRE region in the immune response against bacteria. Streptococcus iniae is one of the dominant pathogenic bacteria of yellowfin seabream, Acanthopagrus latus. However, the regulatory mechanisms of A. latus IRF7 (AlIRF7) mediated by the type I IFN signalling pathway against S. iniae was ambiguously. In the present study, IRF7, and two IFNa3s (IFNa3 and IFNa3-like) were authenticated from A. latus. The total length of AlIRF7 cDNA is 2142 bp, containing a 1314 bp open reading frame (ORF) encoding an inferred 437 amino acids (aa). Three typical regions, a serine-rich domain (SRD), a DNA-binding domain (DBD), and an IRF association domain (IAD), are conserved in AlIRF7. Furthermore, AlIRF7 is fundamentally expressed in various kinds of organs, with high levels in the spleen and liver. Additionally, S. iniae challenge promoted AlIRF7 expression in the spleen, liver, kidney, and brain. AlIRF7 is confirmed to be located at the nucleus and cytoplasm by overexpression of AlIRF7. Moreover, truncation mutation analyses shows that the regions, -821 bp to +192 bp and -928 bp to +196 bp, were known as core promoters from AlIFNa3 and AlIFNa3-like, respectively. The point mutation analyses and electrophoretic mobile shift assay (EMSA) verified that AlIFNa3 and AlIFNa3-like transcriptions are depended on the M2/5 and M2/3/4 binding sites with AlIRF7 regulation, respectively. Additionally, an overexpression experiment showed that AlIRF7 can dramatically decrease the mRNA levels of two AlIFNa3s and interferon signalling molecules. These results suggest that two IFNa3s may mediate the regulation of AlIRF7 in the immune responses of A. latus against S. iniae infection.

Viral nervous necrosis resistance in gilthead sea bream (Sparus aurata) at the larval stage: heritability and accuracy of genomic prediction with different training and testing settings.

BACKGROUND: The gilthead sea bream (Sparus aurata) has long been considered resistant to viral nervous necrosis (VNN), until recently, when significant mortalities caused by a reassortant nervous necrosis virus (NNV) strain were reported. Selective breeding to enhance resistance against NNV might be a preventive action. In this study, 972 sea bream larvae were subjected to a NNV challenge test and the symptomatology was recorded. All the experimental fish and their parents were genotyped using a genome-wide single nucleotide polymorphism (SNP) array consisting of over 26,000 markers. RESULTS: Estimates of pedigree-based and genomic heritabilities of VNN symptomatology were consistent with each other (0.21, highest posterior density interval at 95% (HPD95%): 0.1-0.4; 0.19, HPD95%: 0.1-0.3, respectively). The genome-wide association study suggested one genomic region, i.e., in linkage group (LG) 23 that might be involved in sea bream VNN resistance, although it was far from the genome-wide significance threshold. The accuracies (r) of the predicted estimated breeding values (EBV) provided by three Bayesian genomic regression models (Bayes B, Bayes C, and Ridge Regression) were consistent and on average were equal to 0.90 when assessed in a set of cross-validation (CV) procedures. When genomic relationships between training and testing sets were minimized, accuracy decreased greatly (r = 0.53 for a validation based on genomic clustering, r = 0.12 for a validation based on a leave-one-family-out approach focused on the parents of the challenged fish). Classification of the phenotype using the genomic predictions of the phenotype or using the genomic predictions of the pedigree-based, all data included, EBV as classifiers was moderately accurate (area under the ROC curve 0.60 and 0.66, respectively). CONCLUSIONS: The estimate of the heritability for VNN symptomatology indicates that it is feasible to implement selective breeding programs for increased resistance to VNN of sea bream larvae/juveniles. Exploiting genomic information offers the opportunity of developing prediction tools for VNN resistance, and genomic models can be trained on EBV using all data or phenotypes, with minimal differences in classification performance of the trait phenotype. In a long-term view, the weakening of the genomic ties between animals in the training and test sets leads to decreased genomic prediction accuracies, thus periodical update of the reference population with new data is mandatory.

Whole Genome Sequencing Provides Information on the Genomic Architecture and Diversity of Cultivated Gilthead Seabream (Sparus aurata) Broodstock Nuclei.

The gilthead seabream (Sparus aurata) is a species of relevance for the Mediterranean aquaculture industry. Despite the advancement of genetic tools for the species, breeding programs still do not often include genomics. In this study, we designed a genomic strategy to identify signatures of selection and genomic regions of high differentiation among populations of farmed fish stocks. A comparative DNA pooling sequencing approach was applied to identify signatures of selection in gilthead seabream from the same hatchery and from different nuclei that had not been subjected to genetic selection. Identified genomic regions were further investigated to detect SNPs with predicted high impact. The analyses underlined major genomic differences in the proportion of fixed alleles among the investigated nuclei. Some of these differences highlighted genomic regions, including genes involved in general metabolism and development already detected in QTL for growth, size, skeletal deformity, and adaptation to variation of oxygen levels in other teleosts. The obtained results pointed out the need to control the genetic effect of breeding programs in this species to avoid the reduction of genetic variability within populations and the increase in inbreeding level that, in turn, might lead to an increased frequency of alleles with deleterious effects.

Microsatellite Genome-Wide Database Development for the Commercial Blackhead Seabream (Acanthopagrus schlegelii).

Simple sequence repeats (SSRs), the markers with the highest polymorphism and co-dominance degrees, offer a crucial genetic research resource. Limited SSR markers in blackhead seabream have been reported. The availability of the blackhead seabream genome assembly provided the opportunity to carry out genome-wide identification for all microsatellite markers, and bioinformatic analyses open the way for developing a microsatellite genome-wide database in blackhead seabream. In this study, a total of 412,381 SSRs were identified in the 688.08 Mb genome by Krait software. Whole-genome sequences (10×) of 42 samples were aligned against the reference genome and genotyped using the HipSTR tools by comparing and counting repeat number variation across the SSR loci. A total of 156,086 SSRs with a 2-4 bp repeat were genotyped by HipSTR tools, which accounted for 55.78% of the 2-4 bp SSRs in the reference genome. High accuracy of genotyping was observed by comparing HipSTR tools and PCR amplification. A set of 109,131 loci with a number of alleles ≥ 3 and with a number of genotyped individuals ≥ 6 were reserved to constitute the polymorphic SSR database. Fifty-one polymorphic SSR loci were identified through PCR amplification. This strategy to develop polymorphic SSR markers not only obtained a large set of polymorphic SSRs but also eliminated the need for laborious experimental screening. SSR markers developed in this study may facilitate blackhead seabream research, which lays a certain foundation for further gene tagging and genetic linkage analysis, such as marker-assisted selection, genetic mapping, as well as comparative genomic analysis.

Muscle regeneration in gilthead sea bream: Implications of endocrine and local regulatory factors and the crosstalk with bone.

Fish muscle regeneration is still a poorly known process. In the present study, an injury was done into the left anterior epaxial skeletal muscle of seventy 15 g gilthead sea bream (Sparus aurata) juveniles to evaluate at days 0, 1, 2, 4, 8, 16 and 30 post-wound, the expression of several muscle genes. Moreover, transcripts' expression in the bone (uninjured tissue) was also analyzed. Histology of the muscle showed the presence of dead tissue the first day after injury and how the damaged fibers were removed and replaced by new muscle fibers by day 16 that kept growing up to day 30. Gene expression results showed in muscle an early upregulation of igf-2 and a downregulation of ghr-1 and igf-1. Proteolytic systems expression increased with capn2 and ctsl peaking at 1 and 2 days post-injury, respectively and mafbx at day 8. A pattern of expression that fitted well with active myogenesis progression 16 days after the injury was then observed, with the recovery of igf-1, pax7, cmet, and cav1 expression; and later on, that of cav3 as well. Furthermore, the first days post-injury, the cytokines il-6 and il-15 were also upregulated confirming the tissue inflammation, while tnfα was only upregulated at days 16 and 30 to induce satellite cells recruitment; overall suggesting a possible role for these molecules as myokines. The results of the bone transcripts showed an upregulation first, of bmp2 and ctsk at days 1 and 2, respectively; then, ogn1 and ocn peaked at day 4 in parallel to mstn2 downregulation, and runx2 and ogn2 increased after 8 days of muscle injury, suggesting a possible tissue crosstalk during the regenerative process. Overall, the present model allows studying the sequential involvement of different regulatory molecules during muscle regeneration, as well as the potential relationship between muscle and other tissues such as bone to control musculoskeletal development and growth, pointing out an interesting new line of research in this group of vertebrates.