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Abstract: The effect of diet on larval growth, anionic trypsinogen gene expression (ssetryp1), and trypsin (EC 3.4.21.4) and chymotrypsin (EC 3.4.21.1) activities was assessed in Solea senegalensis. Changes in larval carbon stable isotope (δ13C) composition were used to estimate carbon assimilation. Diets were supplied for 20days to fish held in larval rearing tanks and consisted of live rotifers, Artemia sp. nauplii, rotifers followed by Artemia sp., rotifers co-fed with inert diet and inert diet alone. Growth was significantly faster in larvae fed only Artemia and those fed rotifers and Artemia (k =0.381–0.387day−1). Trypsin and chymotrypsin activities increased from 3 to 4days after hatching (DAH) in all dietary treatments, while ssetryp1 transcripts increased at 4–5 DAH only in larvae fed live prey. ssetryp1 gene expression was activated later in larvae fed only Artemia and this corresponded with Artemia δ13C values being reflected in larval tissue. Larval δ13C values also indicated greater selection and/or assimilation of rotifers in relation to the inert diet. Results demonstrate that during early larval development of sole, diet modulates ssetryp1 gene expression. The rapid and intense response to diets that promoted different growth and survival suggests the suitability of this biomarker as a nutritional status indicator in early sole larvae. [Copyright &y& Elsevier]

Abstract: We describe the cloning of a cDNA encoding TRH in Solea senegalensis. Phylogenetic analysis clustered it with other fish counterparts. Eight conserved TRH progenitors and highly divergent spacers were identified. Expression profiles of TRH were analyzed in juvenile tissues, during larval development, and in post-larvae grew under four photoperiods (12L:12D, 12D:12L, 24L:0D and 24D:0L) during four weeks using a real-time PCR approach. In juvenile fish, TRH mRNA was expressed ubiquitously although with the highest transcript levels in brain. During larval development, TRH mRNA levels were higher at 2–3days after hatching decreasing progressively until metamorphosis. Hormonal treatments using thiourea and T4 showed no regulation at transcriptional level by thyroid hormones. Moreover, no significant changes in mRNA levels between photoperiods at any time were detected. Overall, these results indicate that TRH do not participate in the regulation of HPT nor adaptation to photoperiod in sole. [Copyright &y& Elsevier]

Abstract: Stocking density and ration size are two major factors influencing aquaculture production. To evaluate their effects on growth and immune system in Senegalese sole (Solea senegalensis) juveniles, a 2 × 2 experimental design using two rations (1.0% and 0.25% of the total fish biomass) and two different initial stocking densities (7 and 30 kg m−2) was performed throughout a 60 days culture period. Soles fed 1.0% showed a higher specific growth rate (SGR) than those fed 0.25% (3.3-fold). No differences in SGR at 60 days were found between densities in spite of reduced values were detected at high density after 20 days (soles fed 0.25%) and 40 days (soles fed 1%) suggesting a compensatory growth. Physiologically, plasma cortisol levels were elevated in soles at high density (45-fold higher than at 7 kg m−2) whereas no differences associated to the feeding ration were observed. To assess the effects at a molecular level, the mRNA levels of genes involved in cellular stress (heat shock proteins HSP70 and HSP90), growth (insulin-like growth factors IGF-I, the spliced variants IGF-Ia and IGFI-b, and IGF-II) and innate immune system (g-type lysozyme and hepcidin (HAMP1)) were quantified. No differences in HSP90 expression were detected between densities or rations. In contrast, IGF-I, IGF-Ia and IGF-II showed reduced transcript levels in liver and HSP70 in liver and kidney at high density. Finally, g-type lysozyme and HAMP1 expression was greatly affected by both factors exhibiting an important reduction in the transcript levels at high density and low ration. Overall, our results show that S. senegalensis juveniles might exhibit satisfactory SGR at high density although the high plasma cortisol levels indicate a crowding stress that could negatively affect the expression levels of some of the genes studied. [Copyright &y& Elsevier]

Abstract: Hepcidin antimicrobial peptides (HAMPs) are key molecules of the innate immune system against bacterial infections and in iron metabolism. In this study we report the molecular cloning and genomic characterization of four HAMP genes (referred to as HAMP1, HAMP2, HAMP3 and HAMP4) in the redbanded seabream (Pagrus auriga). All these genes possessed the eight characteristic cysteine residues involved in protein folding. No canonical sequence for convertase-mediated processing of the HAMP3 propeptide was identified. At the genomic level, all four HAMP genes consisted of two introns and three exons. Phylogenetic analysis revealed that HAMPs could group in two main clusters with HAMP2, HAMP3 and HAMP4 belonging to the more complex and diversified HAMP2-like group of acanthopterygians. Quantitation of mRNA levels in adult tissues showed that HAMP1 was ubiquitously expressed, HAMP2 mainly in kidney, spleen and intestine, whereas HAMP3 and HAMP4 in liver. During development, HAMP2 and HAMP3 were expressed at a high level in embryos. Moreover, the expression levels of the four HAMP genes increased between 5 and 15days after hatching when larvae started external feeding. Induction experiments with lipopolysaccharide revealed significant changes in gene expression of the four HAMP genes in kidney, liver and spleen. However, expression profiles differed in magnitude and time course response. HAMP1 mRNAs increased rapidly in kidney at 1h p.i. whereas HAMP2 did later at 24h. Moreover, HAMP4 transcripts increased more than 5000-fold in liver whereas HAMP2 mRNAs dropped significantly in spleen at 3h p.i. All these data suggest that HAMPs are involved in the response against bacterial infections although additional functions in iron regulation and embryogenesis in fish should be considered. [Copyright &y& Elsevier]

Abstract: Insulin-like growth factors (IGFs) I and II (IGF-I and IGF-II) play important roles in fish growth and development. The present study was aimed at isolating cDNAs encoding both IGF-I and IGF-II in the redbanded seabream (Pagrus auriga), and at measuring relative gene expression levels in different organs and during larval development. A fragment of 1321 nucleotides coding for IGF-I was cloned from liver using 3′ and 5′ RACE techniques. It included an open reading frame of 558 nucleotides, encoding a 185-amino acid preproIGF-I. With respect to IGF-II, a fragment of 1544 nucleotides was cloned as well. The open reading frame spanned 648 nucleotides, rendering a 215-amino acid preproIGF-II. The deduced mature 67-amino acid IGF-I and 70-amino acid IGF-II exhibited high sequence identities with their corresponding fish counterparts, ranging between 88.6-100% and 79.1–98.5%, respectively. Real-time PCR showed the highest IGF-I transcripts in liver (∼200-fold higher than head-kidney). In contrast, the highest IGF-II mRNAs were detected in gills and heart (∼16-fold higher than head-kidney). In addition, both IGFs exhibited different gene expression patterns during larval development suggesting that their expression is developmentally regulated. IGF-I reached the highest expression levels at 18 days after hatching (11.6-fold higher than 1 day after hatching), whereas IGF-II expression did not change significantly. Both hepatic IGF-I and IGF-II mRNA levels increased sharply (3.1- and 19-fold higher than control, respectively) 3 h after injection of porcine growth hormone, but remained unchanged from 6 to 24 h after treatment. Our results are discussed in relation to those previously reported for other bony fish. [Copyright &y& Elsevier]

Abstract: Lysozymes are key proteins of the innate immune system against bacterial infections. In this study we report the molecular cloning and characterization of the c-type and g-type lysozymes in brill (Scophthalmus rhombus). Catalytic and other conserved residues required for functionality were identified. Phylogenetic analysis revealed distinct evolutionary histories for each lysozyme type. Expression profiles of both lysozyme genes were studied in juvenile tissues using a real-time PCR approach. c-Type lysozyme was expressed mainly in stomach and liver, whereas the g-type was detected in all tissues with highest mRNA levels observed in the spleen. Induction experiments revealed that g-type transcripts increased significantly in head kidney after lipopolysaccharide (25- and 23-fold at 12 and 24h, respectively) and Photobacterium damselae subsp. piscicida (17-fold at 24h) treatments. In contrast, no induction was observed for c-type lysozyme. All these data suggest that g-type lysozyme is involved in the response against bacterial infections, whereas c-type lysozyme may also play a role in digestion. [Copyright &y& Elsevier]

Abstract: The transcription of Mx mRNA after poly I:C induction and sole aquabirnavirus infection has been analysed in SAF-1 and TV-1 cells (derived from gilt-head seabream and turbot, respectively). Both cell lines were stimulated with 10μgml−1 poly I:C and Mx mRNA was analysed by a specific RT-PCR at several times post-induction. The results showed a high level of Mx expression from 12 to 120h after induction in SAF-1 cells, whereas in TV-1 cells Mx mRNA was only detected at 12 and 24h. The treatment with different concentrations of poly I:C showed that TV-1 cells are less sensitive to this inductor than the SAF-1 cell line. The antiviral activity derived from poly I:C induction has been clearly demonstrated against sole aquabirnavirus on both cell lines. The inoculation of sole aquabirnavirus resulted in the Mx mRNA transcription at 48, 72, and 96h post-infection (p.i.) in SAF-1 cells. On the contrary, inoculated TV-1 cells only showed a faint Mx mRNA band at 24 and 48h p.i. This study has established different patterns of Mx expression in both cells under study as a consequence of the poly I:C induction and sole aquabirnavirus infection, and it shows that gilt-head seabream and turbot Mx inhibit sole aquabirnavirus replication. [Copyright &y& Elsevier]

Several microbial disease outbreaks in farm stocks of newly cultured sparid fish species, such as common seabream, redbanded seabream, and white seabream, were recorded from 2004 to 2006. This study describes the isolation and characterization of the potential causative agents, either bacteria or viruses, of these outbreaks. The isolated bacterial strains were characterized according to traditional taxonomical analyses and sequencing of a 16S rDNA fragment. Most bacteria were identified as Vibrio spp. and Photobacterium damselae subsp. damselae. The development of cytopathic effects (CPE) on different fish cell lines, the application of specific nested-PCR tests for infectious pancreatic necrosis virus (IPNV), viral nervous necrosis virus (VNNV) and viral hemorrhagic septicemia virus (VHSV), and subsequent sequence analyses were used for virus detection and identification. VNNV, related to the striped jack neural necrosis virus (SJNNV) genotype, and VHSV, related to the genotype Ia, were the only viruses detected. VNNV was isolated from the three fish species under study in five different outbreaks, whereas VHSV was isolated from common seabream and white seabream during two of these outbreaks. IPNV was not detected in any case. [ABSTRACT FROM AUTHOR]

Abstract: A multiplex-PCR assay for the authentication of the Atlantic mackerel Scomber scombrus in commercial canned products has been developed. This novel method consists of a S. scombrus-specific fragment (123bp) corresponding to the mitochondrial NADH dehydrogenase subunit 5, and a positive amplification control corresponding to the small rRNA 12S subunit (188bp). The system was assayed using six different canned products labeled as S. scombrus. We observed a positive identification in all samples, revealing this methodology as a potential molecular tool for direct application in the authentication of S. scombrus canned products. [Copyright &y& Elsevier]

Breeding programs are essential for aquaculture. The present study was conducted to investigate the genetic parameters of four growth traits in Senegalese sole. Families were produced by mass spawning (n = 2171 offspring) from an industrial broodstock. Weight, total area, standard length and width (the two latter measured directly on the fish and by image analysis) were determined before the on-growing stage (age ~ 400 d) and at harvest (~800 d) in RAS. Phenotypic data are presented and discussed. Females grew faster than males and a high variation of sex ratios by family was observed. Heritabilities were high for all traits ranging from 0.568 to 0.609 at 400 d and from 0.424 to 0.500 at 800d. The genetic correlations between traits were very high (>0.94) at both ages. Genetic estimates for traits measured in vivo and by digital image analysis were similar. To investigate genomic regions associated with these growth traits, a low-density array using a set of 60 single nucleotide polymorphisms (SNPs) distributed in 17 linkage groups was designed. Using samples from wild fish, a total of 49 assays for SNP analysis were validated. The association analysis was carried out using two fast-growth families and two slow-growth families (n = 279). Two significantly-associated SNPs with most of the traits at both ages were detected. They were located in the general transcription factor 3C polypeptide 4 (gtf3c4) and the mitochondrial fission process protein 1 (mtfp1). Results are of high relevance for genetic breeding programs in this species. • Genetic estimates for four growth traits before on-growing and at harvest in Senegalese sole were determined. • Genetic correlations between growth traits at different ages and between ages were high and positive. • Genetic estimates on image-measured traits are highly consistent with those directly measured on fish. • A low-density array with 49 SNPs widely distributed in the genome was validated. • GWAS analysis identified two markers significantly associated with growth. [ABSTRACT FROM AUTHOR]

Meagre ( Argyrosomus regius ) is a fast-growing species currently produced in aquaculture. This species is highly sensitive to low environmental temperatures which results in high mortality events during production cycles. In this study, the effects of dietary phospholipids (PLs) on growth and cold tolerance were evaluated. For this purpose, control (CTRL) and PL-enriched diets (three-fold higher levels than CTRL) were supplied to meagre juveniles (12.9 ± 2.5 g) for 60 days, and growth was determined using a longitudinal approach. Weight gaining and SGR reduction were significantly different between dietary treatments. Animals fed with the PL-enriched diet were 4.1% heavier and grew 3.2% faster than those fed with the CTRL diet. Survival was higher than 98% in both groups. After finishing the growth trial, animals were submitted to two cold challenges and cold tolerance was evaluated as temperature at death (Tdeath), risk to death and lethal doses (LD) 50 and 90 using the cumulative degree cooling hours 6 h (CD6H). Tdeath ranged between 7.54 and 7.91 °C without statistical differences between dietary treatments. However, risk to death was significantly smaller (0.91-fold lower) and LD50 and LD90 were higher in animals fed with the PL-enriched than those supplied the CTRL diet. To assess the fatty acid (FA) composition of liver and brain in animals fed both diets after a cold challenge, FA profiles were determined in juveniles maintained at 14 °C and challenged at 7 °C. FA amounts increased in the liver of animals challenged at 7 °C. In contrast, several FAs reduced their levels in the PL-enriched diet with respect to CTRL indicating that these animals were able to mobilize efficiently lipids from this organ mitigating the negative effects of lipid accumulation during the cold challenge. In brain, the PL-enriched diet increased DHA level during the cold shock indicating a role in maintaining of brain functions. These results open a new research line that could improve the cold tolerance of meagre through dietary supplementation before winter.

Cartilage acidic protein 1A (hCRTAC1-A) is an extracellular matrix protein (ECM) of human hard and soft tissue that is associated with matrix disorders. The central role of fibroblasts in tissue integrity and ECM health made primary human dermal fibroblasts (NHDF) the model for the present study, which aimed to provide new insight into the molecular function of hCRTAC1-A. Specifically, we explored the differential expression patterns of specific genes associated with the presence of hCRTAC1-A by RNA-seq and RT-qPCR analysis. Functional enrichment analysis demonstrated, for the very first time, that hCRTAC1-A is involved in extracellular matrix organization and development, through its regulatory effect on asporin, decorin, and complement activity, in cell proliferation, regeneration, wound healing, and collagen degradation. This work provides a better understanding of putative hCRTAC1-A actions in human fibroblasts and a fundamental insight into its function in tissue biology., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Plant extracts are known for their high content and diversity of polyphenols, which can improve fish oxidative status. A growth trial with Senegalese sole postlarvae (45 days after hatching) fed with one of four experimental diets-control (CTRL), and supplemented with curcumin (CC), green tea (GT), and grape seed (GS) extracts-was performed to assess if supplementation could improve growth performance and oxidative status. At the end of the growth trial, postlarvae were submitted to a thermal stress to assess their robustness. Sole growth was improved by CC and GS diets when compared to those fed the CTRL. CC and CTRL postlarvae presented the lowest oxidative damage (lipid peroxidation and protein carbonylation values). Stress-related biomarkers (heat shock protein 70 and glutathione-S-transferase) decreased in CC fish compared to those fed the CTRL diet, which might be due to a direct antioxidant capacity. In contrast, oxidative damage increased in GT and GS sole reared in standard conditions. However, after a thermal stress, GT and GS diets prevented the increase of protein carbonylation content and the decrease of antioxidant glutathione, depending on exposure time. Overall, dietary supplementation with natural extracts modulated oxidative status and stress response after a short/long-term exposure to temperature.

Shape quality is very important in flatfish aquaculture due to the impact on commercialization. The Senegalese sole ( Solea senegalensis ) is a valuable flatfish with a highly elliptic body that slightly changes with age and size, and it is prone to accumulating malformations during the production cycle. The present study aims to investigate the genetic parameters of two growth traits (weight and standard length) and six shape quality predictors (ellipticity, three body heights (body height at the pectoral fin base [BHP], body maximum height [BMH] and caudal peduncle height [CPH]) and two ratios (BMH/BHP and BMH/CPH)). These traits were measured before the on-growing stage (age ~400 days (d)) and at harvest (~800 d). Phenotypic data, heritabilities and genetic and phenotypic correlations between the traits are presented and discussed. High or very high heritabilities (0.433-0.774) were found for growth traits, body heights and ellipticity and they were higher at 400 than 800 d. In contrast, the ratios of BMH/BHP and BMH/CPH were less heritable (0.144-0.306). Positive and very high (>0.95) correlations between growth traits and the three heights were found and decreased with age. In contrast, ellipticity had negative and medium-high genetic correlations with growth traits and heights, indicating fish selected for bigger size would also become rounder. The ratio of BMH/CPH showed low genetic correlations with all traits and provided complementary information to ellipticity for a better fitting to the expected lanceolate body morphology of sole. The genetic correlations for all traits at both ages were very high, indicating that selection before entering the growth-out stage in recirculation aquaculture systems is recommended to accelerate genetic gains.

Somatic growth is a balance between protein synthesis and degradation, and it is largely influenced by nutritional clues. Antioxidants levels play a key role in protein turnover by reducing the oxidative damage in the skeletal muscle, and hence promoting growth performance in the long-term. In the present study, Senegalese sole postlarvae (45 days after hatching, DAH) were fed with three experimental diets, a control (CTRL) and two supplemented with natural antioxidants: curcumin (CC) and grape seed (GS). Trial spanned for 25 days and growth performance, muscle cellularity and the expression of muscle growth related genes were assessed at the end of the experiment (70 DAH). The diets CC and GS significantly improved growth performance of fish compared to the CTRL diet. This enhanced growth was associated with larger muscle cross sectional area, with fish fed CC being significantly different from those fed the CTRL. Sole fed the CC diet had the highest number of muscle fibers, indicating that this diet promoted muscle hyperplastic growth. Although the mean fiber diameter did not differ significantly amongst treatments, the proportion of large-sized fibers (>25 μm) was also higher in fish fed the CC diet suggesting increased hypertrophic growth. Such differences in the phenotype were associated with a significant up-regulation of the myogenic differentiation 2 ( myod2 ) and the myomaker ( mymk ) transcripts involved in myocyte differentiation and fusion, respectively, during larval development. The inclusion of grape seed extract (GS diet) resulted in a significant increase in the expression of myostatin1 . These results demonstrate that both diets (CC and GS) can positively modulate muscle development and promote growth in sole postlarvae. This effect is more prominent in CC fed fish, where increased hyperplastic and hypertrophic growth of the muscle was associated with an upregulation of myod2 and mymk genes., (Copyright © 2020 Xavier, Engrola, Conceição, Manchado, Carballo, Gonçalves, Colen, Figueiredo and Valente.)

Phospholipids (PL) are essential molecules for larval growth and development. In this study, growth, lipid metabolism and gene expression responses associated with different dietary PL levels in pelagic sole larvae were evaluated. In a first trial, the long-term effects on growth and survival of two experimental microdiets (MD) containing high (High-PL) or low (Low-PL) PL levels were tested and compared to a diet based on live prey (rotifers). The MD were supplied from 3 to 10 days post-hatch (dph) and Artemia from day 8 to 29 dph. High-PL fed larvae had higher dry mass (1.2-fold) than Low-PL fed larvae at 8 dph and both MD were smaller (2.9-fold) than larvae fed live preys. However, a compensatory growth (33% between 8 and 20 dph) occurred when MD were substituted by Artemia and by the end of the trial no significant differences in mass or survival occurred between the dietary treatments. In a second trial, growth, lipid metabolism and gene expression profiles of larvae fed with MD up to 8 dph were analyzed. Growth data confirmed that mass of larvae fed with High-PL was higher (1.3-fold) than the those fed Low-PL and they had lower levels of triacylglycerol (2.8-fold), cholesterol (1.2-fold) and cetoleic acid (1.7-fold). Histological analysis indicated an excess of lipid vacuoles in larvae fed with Low-PL and the expression analysis revealed a coordinated response to enhance lipid mobilization since the expression of genes involved in PL intermediate synthesis, PL remodeling as well as eight apolipoprotein was up-regulated. The down-regulation of apolipoprotein apob2 in larvae fed with Low-PL indicated a specific regulation by PL levels. The present work provides insight into the responses associated with dietary PL in early fish larvae, which will be of use for future studies aimed as designing effective larval sole diets., (Copyright © 2020 Hachero-Cruzado, Rodriguez-Rua, Torrent, Roman-Padilla and Manchado.)

Investigating gene flow between closely related species and its variation across the genome is important to understand how reproductive barriers shape genome divergence before speciation is complete. An efficient way to characterize differential gene flow is to study how the genetic interactions that take place in hybrid zones selectively filter gene exchange between species, leading to heterogeneous genome divergence. In the present study, genome-wide divergence and introgression patterns were investigated between two sole species, Solea senegalensis and Solea aegyptiaca, using restriction-associated DNA sequencing (RAD-Seq) to analyze samples taken from a transect spanning the hybrid zone. An integrative approach combining geographic and genomic clines methods with an analysis of individual locus introgression accounting for the demographic history of divergence was conducted. Our results showed that the two sole species have come into secondary contact postglacially, after experiencing a prolonged period (ca. 1.1 to 1.8 Myrs) of allopatric separation. Secondary contact resulted in the formation of a tension zone characterized by strong reproductive isolation, which only allowed introgression in a limited fraction of the genome. We found multiple evidence for a preferential direction of introgression in the S. aegyptiaca genetic background, indicating a possible recent or ongoing movement of the hybrid zone. Deviant introgression signals found in the opposite direction suggested that S. senegalensis could have possibly undergone adaptive introgression that has not yet spread throughout the entire species range. Our study thus illustrates the varied outcomes of genetic interactions between divergent gene pools that recently met after a long history of divergence.

In the present work, five genes encoding different Na(+),K(+) ATPase (NKA) α-isoforms in the teleost Solea senegalensis are described for the first time. Sequence analysis of predicted polypeptides revealed a high degree of conservation across teleosts and mammals. Phylogenetic analysis clustered the five genes into three main clades: α1 (designated atp1a1a and atp1a1b), α2 (designated atp1a2) and α3 (designated atp1a3a and atp1a3b) isoforms. Transcriptional analysis in larvae showed distinct expression profiles during development. In juvenile tissues, the atp1a1a gene was highly expressed in osmoregulatory organs, atp1a2 in skeletal muscle, atp1a1b in brain and heart and atp1a3a and atp1a3b mainly in brain. Quantification of mRNA abundance after a salinity challenge showed that atp1a1a transcript levels increased significantly in the gill of soles transferred to high salinity water (60 ppt). In contrast, atp1a3a transcripts increased at low salinity (5 ppt). In situ hybridization (ISH) analysis revealed that the number of ionocytes expressing atp1a1a transcripts in the primary gill filaments was higher at 35 and 60 ppt than at 5 ppt and remained undetectable or at very low levels in the lamellae at 5 and 35 ppt but increased at 60 ppt. Immunohistochemistry showed a higher number of positive cells in the lamellae. Whole-mount analysis of atp1a1a mRNA in young sole larvae revealed that it was localized in gut, pronephric tubule, gill, otic vesicle, yolk sac ionocytes and chordacentrum. Moreover, atp1a1a mRNAs increased at mouth opening (3 DPH) in larvae incubated at 36 ppt with a greater signal in gills., (Copyright © 2014 Elsevier Inc. All rights reserved.)

To study the substitution of fish oil by vegetable oils in fish diets, juveniles Senegalese sole (Solea senegalensis) were fed diets (56 % crude protein, 12 % crude lipid) containing either linseed (100LO) or soybean (100SO) oils in comparison with a 100 % fish oil-based diet (100FO) for 90 days. Samples of muscle, liver, and intestine were collected for biochemical analysis and for glucocorticoid receptor-related genes, including GR1 and GR2, and the associated heat shock proteins HSP70, HSP90AA, and HSP90AB. Besides, basal levels of plasma cortisol were also determined. After the feeding period, a stress test, consisting on 5 min of net chasing, was applied to a selected population of each dietary group. Total replacement of fish oil by vegetable oils did not induced changes in fish growth and performance, but affected fatty acid profile of muscle, liver, and intestine, reflecting those tissues the characteristic fatty acids of each type of dietary oil. A tendency to conserve the ARA/EPA ratio could be observed in the different tissues, despite of the level of these fatty acids in diet. Chasing stress induced an increase of muscle GR1 and a reduction in intestinal GR2 relative expressions at any of the experimental diets assayed. In liver, chasing stress induced an increase in both GR1 and GR2 gene expression in fish fed fish oil diets. Similarly, chasing stress induced an increase of muscle HSP70 and decrease of HSP90AB in liver at any of the experimental diet assayed. Besides, vegetable oils decreased the expression of HSP70 in intestine, being the relative expression of liver HSP90AA increased by the inclusion of linseed oil in the diet, at any of the experimental conditions assayed.

Fish aquaculture is considered to be one of the most sustainable sources of protein for humans. Many different species are cultured worldwide, but among them, marine flatfishes comprise a group of teleosts of high commercial interest because of their highly prized white flesh. However, the aquaculture of these fishes is seriously hampered by the scarce knowledge on their biology. In recent years, various experimental 'omics' approaches have been applied to farmed flatfishes to increment the genomic resources available. These tools are beginning to identify genetic markers associated with traits of commercial interest, and to unravel the molecular basis of different physiological processes. This article summarizes recent advances in flatfish genomics research in Europe. We focus on the new generation sequencing technologies, which can produce a massive amount of DNA sequencing data, and discuss their potentials and applications for de novo genome sequencing and transcriptome analysis. The relevance of these methods in nutrigenomics and foodomics approaches for the production of healthy animals, as well as high quality and safety products for the consumer, is also briefly discussed.

The viral nervous necrosis virus (VNNV) is the causal agent of viral encephalopathy and retinopathy (VER), a worldwide fish disease that is responsible for high mortality in both marine and freshwater species. Infected fish suffer from encephalitis, which leads to abnormal swimming behavior and extensive cellular vacuolation and neuronal degeneration in the central nervous system (CNS) and retina. The marine fish gilthead seabream (Sparus aurata) does not develop VER but it is an asymptomatic carrier of VNNV. In this study, we report that VNNV was able to replicate and persist for up to 3 months in the CNS of the gilthead seabream without causing any neural damage. In addition, we found an early inflammatory response in the CNS that was characterized by the induction of genes encoding pro-inflammatory cytokines, a delayed but persistent induction of anti-inflammatory cytokines, and the infiltration of IgM(+) B lymphocytes, suggesting that local adaptive immunity played a major role in the control of VNNV in the CNS of this species., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Thyroid hormones (THs) play a key role in larval development, growth and metamorphosis in flatfish. Their genomic effects are mediated by thyroid hormone receptors (TRs). In this study, cDNAs encoding for TRalphaA, TRalphaB, and TRbeta have been sequenced in Senegalese sole (Soleasenegalensis). Main domains and conserved motifs were identified. Also, a truncated TRalphaB isoform (referred to as TRalphaBtr) and a spliced TRbeta variant (referred to as TRbetav) were detected. A phylogenetic analysis grouped both TRalpha and TRbeta genes into two separate clusters with their fish and mammalian counterparts. Expression profiles during larval development and in juvenile tissues were analyzed using a real-time PCR approach. In juvenile fish, TRalphaA, TRalphaB, TRbetav, and TRbeta showed distinct transcript levels in tissues. During metamorphosis, only TRbetav and TRbeta modified their mRNA levels in a similar way to the T4 contents. To evaluate the possible regulation of TRs by their cognate ligand T4 during sole metamorphosis, larvae were exposed to the goitrogen thiourea (TU). TRbeta transcripts decreased significantly at 11 and 15 days after treatment. Moreover, adding exogenous T4 hormone to TU-treated larvae restored the steady-state levels or even increased TRbeta and TRbetav mRNAs with respect to the untreated control. Overall, these results demonstrate that TRbeta transcription is up-regulated by THs playing a major role during metamorphosis in Senegalese sole.

Ribosomal proteins (RPs) comprise a large set of highly evolutionarily conserved proteins that are often over-represented in complementary DNA libraries. They have become very useful markers in comparative genomics, genome evolution, and phylogenetic studies across taxa. In this study, we report the sequences of the complete set of 60S RPs in Senegalese sole (Solea senegalensis) and Atlantic halibut (Hippoglossus hippoglossus), two commercially important flatfish species. Amino-acid sequence comparisons of the encoded proteins showed a high similarity both between these two flatfish species and with respect to other fish and human counterparts. Expressed sequence tag analysis revealed the existence of paralogous genes for RPL3, RPL7, RPL41, and RPLP2 in Atlantic halibut and RPL13a in Senegalese sole as well as RPL19 and RPL22 in both species. Phylogenetic analysis of paralogs revealed distinct evolutionary histories for each RP in agreement with three rounds of genome duplications and lineage-specific duplications during flatfish evolution. Steady-state transcript levels for RPL19 and RPL22 RPs were quantitated during larval development and in different tissues of sole and halibut using a real-time polymerase chain reaction approach. All paralogs were expressed ubiquitously although at different levels in different tissues. Most RP transcripts increased coordinately after larval first-feeding in both species but decreased progressively during the metamorphic process. In all cases, expression profiles and transcript levels of orthologous genes in Senegalese sole and Atlantic halibut were highly congruent. The genomic resources and knowledge developed in this survey will be useful for the study of Pleuronectiformes evolution.

The application of large-scale genomics to Senegalese sole (Solea senegalensis) has allowed for the identification of six different trypsinogen genes. The catalytic triad (His-57, Asp-102, and Ser-195) and other residues required for trypsin functionality were conserved across all trypsinogens. Sequence identities, charges and phylogenetic analysis allowed them to be classified into three groups: group I or anionic trypsinogens (ssetryp1a, ssetryp1b and ssetryp1c), group II or cationic trypsinogen (ssetryp2) and group III or psychrophilic trypsinogens (ssetryp3 and ssetrypY). The expression profiles of these genes were studied in juvenile tissues and during larval development using a real-time PCR approach. In juvenile fish, trypsinogens were expressed mainly in the intestine. Transcripts of ssetryp1c were the highest in all tissues except in brain where those of ssetryp2 were the most abundant. During larval development, ssetryp1 variants and ssetryp2 transcript levels increased from 2 to 6 days after hatching, and decreased thereafter. In contrast, transcripts of group III trypsinogens increased slightly or not significantly in premetamorphosis and decreased at metamorphosis. The expression levels ssetryp3 and ssetrypY were the lowest in larvae (from 172- to 1391-fold lower than ssetryp1 and ssetryp2). In contrast, they were expressed at a similar level as ssetryp2, although lower than ssetryp1, in juvenile tissues.

Thyroid hormones (TH) play a critical role in flatfish metamorphosis. Their levels are regulated by the pituitary-thyroid axis. The expression profile of thyroid stimulating hormone (TSH) beta subunit and thyroglobulin (Tg) was investigated using a real-time PCR approach. Both genes exhibited different expression patterns during larval development in Senegalese sole. TSH beta mRNAs reduced progressively at the commencement of metamorphosis. On the contrary, Tg transcripts increased sharply at the onset of metamorphosis and dropped after the metamorphosis climax. T4 levels, as determined by radioimmunoassay, clearly resembled the Tg expression profile with a peak at the metamorphosis climax. To investigate if such expression profiles were regulated by TH, premetamorphic larvae were exposed to the goitrogen thiourea (TU). TU-treated larvae were not able to complete metamorphosis. However, the addition of exogenous T4 enabled to revert this effect. Expression analysis showed higher mRNA levels of both TSH beta and Tg in TU-treated larvae in comparison to control larvae. Moreover, the TU+T4 treated larvae exhibited similar or lower mRNA levels than in the control. Present results demonstrate that TH mediate metamorphosis and down-regulate TSH beta and Tg at transcriptional level in Senegalese sole.

In the classical taxonomy, three Scomber species are distinguished: S. scombrus, S. australasicus, and S. japonicus. Yet, some fish taxonomists have recently recognized Scomber colias, inhabiting the Atlantic Ocean, as a separate species from S. japonicus, distributed in the Pacific Ocean. Such proposal was based on significant mitochondrial DNA divergence as well as great phenotypic variation among individuals from these two ocean basins. However, in the absence of nuclear DNA data this issue remains still controversial. In this study, a phylogenetic analysis of nuclear 5S rDNA sequences was performed. A total of 30 individuals of S. colias collected in the Atlantic and 34 specimens of S. japonicus from the Pacific were characterized. Moreover, nine individuals of Pacific S. australasicus and eight of Atlantic S. scombrus were included. Maximum likelihood, maximum parsimony, and neighbor-joining analyses revealed the presence of two well-supported distinct clades corresponding to S. colias and S. japonicus, respectively. Altogether, morphologic and genetic data are in agreement with the recognition of two different species, S. colias in the Atlantic, and S. japonicus in the Pacific.

Some units of the 5S rDNA of Solea senegalensis were amplified by PCR and sequenced. Three main PCR products (227, 441, and 2166 bp) were identified. The 227- and 441-bp fragments were characterized by highly divergent nontranscribed spacer sequences (referred to as NTS-I and NTS-II) that were 109 and 324 bp long, respectively, yet their coding sequences were nearly identical. The 2166-bp 5S rDNA unit was composed of two 5S rRNA genes separated by NTS-I and followed by a 1721-bp spacer containing the U2, U5, and U1 small nuclear RNA genes (snRNAs). They were inverted and arranged in the transcriptional direction opposite that of the 5S rRNA gene. This simultaneous linkage of 3 different snRNAs had never been observed before. The PCR products were used as probes in fluorescence in situ hybridization experiments to locate the corresponding loci on the chromosomes of S. senegalensis. A major 5S rDNA chromosomal site was located along most of the short arm of a submetacentric pair, while a minor site was detected near the centromeric region of an acrocentric pair.

The entire sequence of the mitochondrial cytochrome b gene and 2 partial sequences of the ribosomal RNA12S and 16S genes have been used to study the molecular phylogeny in 10 species of soles belonging to the genera Solea, Monochirus, Microchirus, Dicologlossa, and Synaptura from the Atlantic waters of the Gulf of Cadiz (Spain). The results obtained by means of different phylogenetic analyses (maximum likelihood, maximum parsimony, and neighbor-joining) were quite similar, supporting the monophyly of the Solea species. Nevertheless, they favor the differentiation of Dicologlossa cuneata and Dicologlossa hexophthalma in 2 distinct genera, since the most closely related species to the last one is Microchirus azevia. The fact that M. azevia is also more closely linked to Monochirus hispidus than to its congeneric Microchirus boscanion argues in favor of a taxonomic reorganization of these genera.

Rob is regarded as a constitutively expressed protein, although little is known about how rob gene is regulated. We show here by reverse transcription-PCR that the transcriptional levels of rob are strongly down-regulated in response to the superoxide-generating agent paraquat (PQ). Repression reached a maximum of 20-fold after 10 min exposure at 10 microM PQ. The magnitude of rob repression was comparable to that of induction quantified for the most sensitive SoxS targets. beta-Galactosidase expression with the rob2::lacZ transcriptional fusion indicates that down-regulation of rob expression takes place, at least in part, at the level of transcription initiation. Moreover, ca. 50% of the rob mRNA was degraded in <1 min after the addition of rifampin to inhibit transcription. This intrinsic short half-life, which is of obvious benefit for a rapid down-regulation after transcription ceases, was unaffected by the addition of PQ. No repression was observed in a soxR-null strain, indicating that the rob transcript level might be negatively modulated by the intracellular amounts of SoxS protein. Gel retardation assays support the idea that in vivo SoxS would block rob transcription directly.