Assessing the role of ulvan as immunonutrient in Solea senegalensis.

Immunonutrition is a promising and viable strategy for the development of prophylactic measures in aquaculture. Ulvan, a sulphated marine polysaccharide from green seaweeds, has many biological activities including the immunomodulatory ones. The aim of this study was to assess the short and long-term effects of an ulvan-rich extract obtained from U. ohnoi as immunonutrient in Senegalese sole juveniles. In this work, an ulvan-rich extract from Ulva ohnoi has been obtained by the hot water method and isolated by ethanol precipitation. The FTIR analysis revealed that the ulvan-rich extact had very similar characteristics to previously published ulvan spectra. The total sulfate and protein content was 24.85 ± 3.98 and 0.91 ± 0.04 %, respectively. In vitro assays performed in Senegalese sole (Solea senegalensis) macrophages showed that the ulvan obtained in this study did not compromise the cell viability at concentrations up to 1 mg ml-1 and expression levels of lyg, irf1, il6, il10, c7, tf and txn were significantly upregulated in a concentration dependent-manner. Finally, S. senegalensis juveniles were fed basal diets and diets supplemented with the ulvan-rich extract at ratios 1 and 2 % for 30 days and then, challenged with Photobacterium damselae subsp. piscicida (Phdp). Thereafter, ulvan was withdrawn from the diet and all juveniles were fed the basal diet for 30 days. At 30 days post withdrawal (dpw), juveniles were challenged with Phdp. The expression profiles of a set of genes related to the immune system in spleen were evaluated as well as the lysozyme, peroxidase and bactericidal activity in plasma. Dietary effects of 1 % ulvan resulted in a boost of the immune response and increased disease resistance at short-term whereas juveniles fed diets supplemented with 2 % ulvan showed a significant decrease in the bactericidal activity and lack of protection against Phdp. At long-term (30 days after the withdrawal of ulvan), an improved response was observed in juveniles previously fed 1 % ulvan.

Evaluation of an oral DNA nanovaccine against photobacteriosis in Solea senegalensis.

Infectious diseases are one of the main causes of social and economical losses in world aquaculture. Senegalese sole (Solea senegalensis) is an important species for aquaculture in southern Europe, whose production is affected by the appearance of bacterial diseases such as photobacteriosis, a septicemia caused by Photobacterium damselae subsp. piscicida (Phdp). The aim of this study was to obtain an oral DNA nanovaccine and to evaluate its efficacy against Phdp in S. senegalensis juveniles. For this purpose, the amplified product corresponding to the protein inosine-5'-monophophate dehydrogenase (IMPDH) from Phdp, was cloned into the expression vector pcDNA™6.2/C-EmGFP-GW obtaining the DNA vaccine named as pPDPimpdh. The correct transcription and protein expression was verified at 48 h post tansfection in HEK293 cells. Chitosan nanoparticles (CS-TPP NPs) were prepared by ionotropic gelation and their features were appropriate for use as oral delivery system. Therefore, pPDPimpdh was protected with chitosan CS-TPP NPs throughout complex coacervation method giving as a result a DNA nanovaccine referred as CS-TPP+pPDPimpdh NPs. Sole juveniles were vaccinated orally with CS-TPP NPs, pPDPimpdh and CS-TPP+pPDPimpdh NPs followed by a challenge with Phdp at 30 days post vaccination (dpv). The relative percentage survival (RPS) for pPDPimpdh vaccinated groups was 6.25%, probably due to its degradation in the digestive tract. RPS value obtained for CS-TPP NPs and CS-TPP+pPDPimpdh NPs was 40% and antibodies were observed in both cases. However, a delay in mortality was observed in sole juveniles vaccinated orally with CS-TPP+pPDPimpdh NPs. In fact, an upregulation of tf, mhcII, cd8a and igm in the posterior gut and c3, hamp1, tf and cd4 in spleen was observed in juveniles vaccinated with CS-TPP+pPDPimpdh NPs. After challenge, a modulation of cd8a and cd4 expression levels in the posterior gut and c3, tf, lyg, cd4, igm and igt expression levels in spleen was observed. Moreover, the concentration of lysozyme in skin mucus significantly increased in fish vaccinated orally with CS-TPP+pPDPimpdh NPs at 11 dpc. These data indicate that oral vaccination with CS-TPP+pPDPimpdh NPs could be acting through the non-specific immune responses as well as the specific humoral and cell mediated immunity and provide the first step toward a development of an oral DNA nanovaccine against Phdp in sole.

Effects of the sulfated polysaccharide ulvan from Ulva ohnoi on the modulation of the immune response in Senegalese sole (Solea senegalensis).

Sulfated polysaccharides derived from green seaweeds exhibit many beneficial biological activities and have great potential to be used as nutraceutical in aquaculture. In this work, we evaluated the effects of the sulfated polysaccharide ulvan from Ulva ohnoi on Senegalese sole (Solea senegalensis) juveniles at the transcriptomic level. Cytotoxicity assay performed in liver primary cell cultures from sole determined that the different ulvan concentrations assayed did not impair cell viability. Juveniles were intraperitoneally (IP) injected with ulvan (0.5 mg/fish) followed by a challenge with Photobacterium damselae subsp. piscicida (Phdp) at 7 days. RNASeq analyses at 2 days post injection (dpi) revealed that 402 transcripts were differentially expressed in liver between ulvan IP injected and control groups before the challenge. Genes related to bacterial and antiviral defence, complement system, chemokines, proteasomes and antigen presentation were upregulated in ulvan treated groups. A detailed expression analysis of sixteen genes related to innate and adaptive immune system was performed in two systemic tissues: liver and spleen. Ulvan injection provoked the upregulation of tlr22 and a transient inflammatory response was initiated in both liver and spleen at 2 dpi. As consequence, expression of acute phase proteins, antimicrobial peptides and complement genes was induced. Moreover, expression of mhcI, mhcII, psmb10 and bcl6 was also induced 2 dpi. At 2 dpi with Phdp, inflammatory cytokines and genes related to bacterial and antiviral defense, iron metabolism, complement system and antigen presentation were differentially modulated in survival juveniles previously IP injected with ulvan. Moreover, mortality was retarded in ulvan treated juveniles. These results provide new evidence about the role of ulvan as a bioactive compound with immunomodulatory activity in Senegalese sole as well as its possible use as vaccine adjuvant against Phdp. This is the first published study that evaluates the transcriptomic response of Senegalese sole IP injected with ulvan.

Synthesis of thieno[2,3-h]-/[3,2-h]quinolines and thieno[2,3-f] quinolines by Brønsted acid mediated cycloisomerisation.

Several thieno[2,3-h]-/[3,2-h]- and [2,3-f]quinolines have been synthesised from 2,3-dihalogenated pyridines or -quinolines by site-selective Pd catalysed cross-coupling reactions and Brønsted acid mediated cycloisomerisations as the final key step. This newly developed synthetic strategy is used in a modular way to synthesize diverse regioisomeric derivatives, tolerates various functional groups, and proceeds with high selectivity, and the desired final products have been isolated in high overall yields.

Dietary vegetable oils: effects on the expression of immune-related genes in Senegalese sole (Solea senegalensis) intestine.

The decreased availability of fish oil, traditionally used as oil source in marine aquafeeds, has lead to the search for alternatives oils. Vegetable oils (VO) are being extensively used as lipid sources in marine fish diets, inducing an imbalance on certain dietary fatty acids. Alteration on the dietary ratio of w-6/w-3 has been described to have detrimental effects on fish immunity. Senegalese sole has high susceptibility to stress and diseases, and little is known on the effects of dietary VO on its immunity. In this study, Senegalese sole juveniles were fed diets (56% crude protein, 12% crude lipid) containing linseed (100LO), soybean (100SO) or fish (100FO) oils as unique oil source. Growth, cortisol and intestinal fatty acid composition were determined after 90 days. Moreover, at the final of the experiment a stress test (5 min of net chasing) was carried out. To evaluate the effect of diets and stress on intestine immunology, expression profiles of a set of 53 immune-related genes using RT-qPCR was also performed. The use of VO did not induced changes in fish growth, but affected fatty acid profile of intestine and expression of immune-related genes. The use of SO (rich in n-6 fatty acids) induced an over-expression of those genes related to complement pathway, recognizing pathogen associated to molecular patterns, defensive response against bacteria, defensive response against viruses, antigen differentiation, cytokines and their receptors. This general over-expression could indicate an activation of inflammatory processes in fish gut. When a stress was applied, a decrease of mRNA levels of different immune-related genes with respect to the unstressed control could be observed in fish fed 100FO. However, fish fed 100LO, with a higher ALA/LA ratio, seemed to ameliorate the effects of combined effects of FO substitution plus stressful situation whereas fish fed 100SO did not show this type of response.

De novo assembly, characterization and functional annotation of Senegalese sole (Solea senegalensis) and common sole (Solea solea) transcriptomes: integration in a database and design of a microarray.

BACKGROUND: Senegalese sole (Solea senegalensis) and common sole (S. solea) are two economically and evolutionary important flatfish species both in fisheries and aquaculture. Although some genomic resources and tools were recently described in these species, further sequencing efforts are required to establish a complete transcriptome, and to identify new molecular markers. Moreover, the comparative analysis of transcriptomes will be useful to understand flatfish evolution. RESULTS: A comprehensive characterization of the transcriptome for each species was carried out using a large set of Illumina data (more than 1,800 millions reads for each sole species) and 454 reads (more than 5 millions reads only in S. senegalensis), providing coverages ranging from 1,384x to 2,543x. After a de novo assembly, 45,063 and 38,402 different transcripts were obtained, comprising 18,738 and 22,683 full-length cDNAs in S. senegalensis and S. solea, respectively. A reference transcriptome with the longest unique transcripts and putative non-redundant new transcripts was established for each species. A subset of 11,953 reference transcripts was qualified as highly reliable orthologs (>97% identity) between both species. A small subset of putative species-specific, lineage-specific and flatfish-specific transcripts were also identified. Furthermore, transcriptome data permitted the identification of single nucleotide polymorphisms and simple-sequence repeats confirmed by FISH to be used in further genetic and expression studies. Moreover, evidences on the retention of crystallins crybb1, crybb1-like and crybb3 in the two species of soles are also presented. Transcriptome information was applied to the design of a microarray tool in S. senegalensis that was successfully tested and validated by qPCR. Finally, transcriptomic data were hosted and structured at SoleaDB. CONCLUSIONS: Transcriptomes and molecular markers identified in this study represent a valuable source for future genomic studies in these economically important species. Orthology analysis provided new clues regarding sole genome evolution indicating a divergent evolution of crystallins in flatfish. The design of a microarray and establishment of a reference transcriptome will be useful for large-scale gene expression studies. Moreover, the integration of transcriptomic data in the SoleaDB will facilitate the management of genomic information in these important species.

Anti-Bacterial and Immunostimulatory Properties of Ulvan-Loaded Chitosan Nanoparticles for Use in Aquaculture.

Alternative prophylactic strategies to limit farm animal infection are needed in order to avoid the use of antibiotics. Anti-bacterial and immunostimulatory properties of bioactive compounds are of great interest in aquaculture. Marine derived polysaccharides, such as chitosan and ulvan, together with nanotechnology, have become the focus of attention in the scientific community due to their wide range of biological properties. In this work, chitosan and ulvan-loaded chitosan nanoparticles (referred as CS-TPP NPs and CS-UL-TPP NPs, respectively), obtained by the ionotropic gelation method, had round shape, and the mean sizes were 137.00 ± 5.44 and 325.50 ± 4.95 nm, respectively. No study about the anti-bacterial activity of both types of NPs against Photobacterium damselae subsp. piscicida, an important fish pathogen, has been reported so far. Furthermore, the potential immunostimulatory effects of CS-UL-TPP NPs after oral administration in fish have not yet been evaluated. The percentage of bacterial inhibition against P. damselae subsp. piscicida was determined through in vitro assays, and it was significantly higher in CS-UL-TPP NPs than in CS-TPP NPs at concentrations below 0.03 mg mL-1. The effects on the immune system of CS-TPP and CS-UL-TPP NPs were evaluated in Solea senegalensis juveniles at 30 days after oral administration. Lysozyme activity as well as gene expression levels of il1b, il6, hamp1, tf and c3 was significantly higher in CS-UL-TPP NP-treated groups than in the controls, and no significant differences were observed in CS-TPP NP-treated groups. Thus, ulvan extracted from the macroalgae Ulva ohnoi could improve anti-bacterial and immunostimulant properties of CS-TPP NPs thereby making them suitable to be used as vaccine adjuvant or as immunostimulant.

Genomic characterization, phylogeny and gene regulation of g-type lysozyme in sole (Solea senegalensis).

The g-type lysozyme is a key protein of the innate immune system to fight bacterial infections. In this study we cloned and characterized the gene encoding for g-type lysozyme in Senegalese sole (Solea senegalensis). The deduced amino acid sequence comprised 195 residues containing the three conserved catalytic residues and two cysteines. A BAC analysis revealed that the gene is structured in 5 exons and 4 introns. Also, two polyadenylation signals that generate two cDNAs differing in 3'-UTR length were detected. Promoter analysis showed the presence of the main cis-acting elements involved in the transcriptional regulation of the gene. At genomic level, the g-type lysozyme was associated with mucolipin 1 and the peptidoglycan recognition protein 2 conforming a cluster of antidefensive genes with a well-conserved synteny across Percomorpha. FISH analysis using the BAC clone revealed a single hybridization signal located in an acrocentric chromosome pair. The phylogenetic analysis confirmed that the g-type lysozyme represents a complex group in fish that has been shaped by gene duplications and diversification with several positions under Darwinian selection. Expression analysis in juvenile tissues indicated that transcript levels were higher in gills, spleen and heart. During development, gene expression activated just at the beginning of metamorphosis, increasing progressively until climax. Hormonal treatments demonstrated that this gene was regulated positively by thyroid hormones during development and negatively by dexamethasone. In contrast, no response was observed after all-trans retinoic acid or 4-diethylaminobenzaldehyde treatments. Finally, treatments using lipopolysaccharide, lipoteichoic acid, peptidoglycan, zymosan and poly(I:C) activated gene expression in a time- and tissue-specific manner. Taken together, data indicate that g-type lysozyme is a high evolutionary conserved gene that diversified to adapt to changing environment and pathogen conditions. Gene expression can be activated by diverse pathogen stimuli and modulated by physiological factors with important consequences for the aquaculture of this species.

Molecular characterization and gene expression of thyrotropin receptor (TSHR) and a truncated TSHR-like in Senegalese sole.

Thyroid hormones (THs) play a key role in larval development, growth and metamorphosis in flatfish. Their synthesis is tightly regulated by the hypothalamic-pituitary-thyroid axis. Thyroid-stimulating hormone receptor (TSHR) is a key protein in the control of thyroid function stimulating TH synthesis after binding its ligand, the thyrotropin. In teleost fish, numerous reports have associated the TSHR with gametogenesis. However, little information about its role during larval development is available. In this study, we report the cloning of two different cDNAs with high similarity to TSHR. Phylogenetic analysis clustered both cDNAs separately. One of them (referred to TSHR) grouped with TSHR orthologs in tetrapods and teleost fish and possessed the three typical conserved domains and regulatory motifs. The second receptor (referred to as TSHRtr-like) represented a novel truncated cDNA bearing the extracellular and part of the transmembrane domain. TSHRtr-like orthologs were only found in teleosts, which suggests that it could have appeared after fish-specific 3R genome duplication. Expression profiles of both genes are analyzed in juvenile tissues and during larval development using a real-time PCR approach. In juvenile fish, TSHR and TSHRtr-like are expressed ubiquitously although transcript levels varied between organs. In both cases, the highest mRNAs levels are detected in brain. During larval development, both genes are expressed to a high level during the first stages (2-3days after hatching) reducing progressively their abundance in the whole larvae during metamorphosis. This reduction in mRNA abundance is more accentuated for the TSHRtr-like gene. To evaluate the possible regulation of both receptors by T4 during sole metamorphosis, larvae are exposed to the goitrogen thiourea (TU). Only TSHRtr-like modifies its expression, increasing its transcripts at 11days after treatment. Moreover, adding exogenous T4 hormone to TU-treated larvae restores the TSHRtr-like steady-state levels similar to the untreated control. Overall, these results demonstrate the existence of two thyrotropin receptors differentially regulated by THs in teleosts.

Complete mitochondrial genome of the blackspot seabream, Pagellus bogaraveo (Perciformes: Sparidae), with high levels of length heteroplasmy in the WANCY region.

The complete mitochondrial genome sequence of the blackspot seabream, Pagellus bogaraveo, was obtained using the long PCR/cloning method. The total length of the mitogenome was 16,941 bp, and had a gene content (13 protein-coding, two ribosomal RNAs, and 22 transfer RNAs) and organization similar to those observed in most other vertebrates. Nevertheless, two main features in the WANCY region revealed as unique in P. bogaraveo mitogenome. First, the O(L) was disrupted by the insertion of a 66 bp long element that was determined to be a tRNA(Cys) pseudogene; second, the existence of high levels of length heteroplasmy, both intra-and inter-individuals, as a result of sequence duplications and deletions. The tandem duplication and random loss (TDRL) model as well as recombination are proposed to account for the length heteroplasmy and gene rearrangements in the P. bogaraveo WANCY region.

Molecular characterization, gene expression and transcriptional regulation of cytosolic HSP90 genes in the flatfish Senegalese sole (Solea senegalensis Kaup).

HSP90 proteins are chaperones that play a pivotal role in controlling multiple regulatory pathways such as stress defense, hormone signalling, cell cycle control, cell proliferation and differentiation, and apoptosis. In this study, two cDNAs encoding for cytosolic HSP90, referred to as HSP90AA and HSP90AB, have been sequenced. Main features and sequence identities with other fish and mammals are described. Phylogenetic analysis grouped both 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, HSP90AB was constitutively expressed with lower transcript levels in skeletal muscle. In contrast, HSP90AA was mainly expressed in heart, skeletal muscle and skin. During metamorphosis, HSP90AB mRNA levels did not change whereas HSP90AA transcripts decreased significantly at the beginning of metamorphosis with the lowest mRNA levels at the metamorphosis climax. Due to the role of thyroid hormones (THs) on sole metamorphosis, the transcriptional regulation of HSP90 genes by THs was evaluated. Larvae exposed to the goitrogen thiourea (TU) exhibited higher HSP90AA mRNA levels than untreated control. Moreover, adding exogenous T4 hormone to TU-treated larvae restored the steady-state levels with respect to the untreated control. Unlike HSP90AA, the transcript levels of HSP90AB did not vary under any treatments. The response of both HSP90 genes to thermal stress in post-metamorphic individuals was also studied. A heat shock treatment (+7.9 degrees C for 1 h) rapidly activated HSP90AA (but not HSP90AB) transcription, reaching a peak after 30 min and declining expression levels progressively in the following 24 h. No significant changes in HSP90AA or HSP90AB transcript levels after a cold shock (-10 degrees C for 1 h) were observed. Overall, these results demonstrate that HSP90AA transcription is down-regulated by THs and up-regulated after a heat shock in Senegalese sole.

Molecular characterization and gene expression analysis of insulin-like growth factors I and II in the redbanded seabream, Pagrus auriga: transcriptional regulation by growth hormone.

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 ( approximately 200-fold higher than head-kidney). In contrast, the highest IGF-II mRNAs were detected in gills and heart ( approximately 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.

The complete mitochondrial genome of the Senegal sole, Solea senegalensis Kaup. Comparative analysis of tandem repeats in the control region among soles.

The complete nucleotide sequence of the mitochondrial genome for the Senegal sole Solea senegalensis Kaup was determined. The mitochondrial DNA was 16,659 base pairs (bp) in length. Sequence features of the 13 protein-coding genes, two ribosomal RNAs and 22 transfer RNAs are described. The non-coding control region (1017 bp) was compared with those of the closely related soles Solea solea and Solea lascaris. The typical conservative blocks were identified. A cluster of 42 and 22 tandemly arrayed repeats was detected near the 3' end of control region in S. solea and S. lascaris, respectively. On the contrary, only two (93.8% of haplotypes) or three copies (6.2%) of an 8-bp repeated sequence motif was found in S. senegalensis. Phylogenetic analysis showed that 7 out of 9 of haplotypes bearing three copies grouped in a separate cluster. Possible mechanisms influencing the evolution of control region among soles are discussed.

Transcriptional regulation of genes involved in retinoic acid metabolism in Senegalese sole larvae.

The aim of this study was the characterization of transcriptional regulatory pathways mediated by retinoic acid (RA) in Senegalese sole larvae. For this purpose, pre-metamorphic larvae were treated with a low concentration of DEAB, an inhibitor of RALDH enzyme, until the end of metamorphosis. No differences in growth, eye migration or survival were observed. Nevertheless, gene expression analysis revealed a total of 20 transcripts differentially expressed during larval development and only six related with DEAB treatments directly involved in RA metabolism and actions (rdh10a, aldh1a2, crbp1, igf2r, rarg and cyp26a1) to adapt to a low-RA environment. In a second experiment, post-metamorphic larvae were exposed to the all-trans RA (atRA) observing an opposite regulation for those genes involved in RA synthesis and degradation (rdh10a, aldh1a2, crbp1 and cyp26a1) as well as other related with thyroid- (dio2) and IGF-axes (igfbp1, igf2r and igfbp5) to balance RA levels. In a third experiment, DEAB-pretreated post-metamorphic larvae were exposed to atRA and TTNPB (a specific RAR agonist). Both drugs down-regulated rdh10a and aldh1a2 and up-regulated cyp26a1 expression demonstrating their important role in RA homeostasis. Moreover, five retinoic receptors that mediate RA actions, the thyroid receptor thrb, and five IGF binding proteins changed differentially their expression. Overall, this study demonstrates that exogenous RA modulates the expression of some genes involved in the RA synthesis, degradation and cellular transport through RAR-mediated regulatory pathways establishing a negative feedback regulatory mechanism necessary to balance endogenous RA levels and gradients.

Molecular and functional characterization of seven Na+/K+-ATPase ß subunit paralogs in Senegalese sole (Solea senegalensis Kaup, 1858).

In the present work, seven genes encoding Na(+),K(+)-ATPase (NKA) ß-subunits in the teleost Solea senegalensis are described for the first time. Sequence analysis of the predicted polypeptides revealed a high degree of conservation with those of other vertebrate species and maintenance of important motifs involved in structure and function. Phylogenetic analysis clustered the seven genes into four main clades: ß1 (atp1b1a and atp1b1b), ß2 (atp1b2a and atp1b2b), ß3 (atp1b3a and atp1b3b) and ß4 (atp1b4). In juveniles, all paralogous transcripts were detected in the nine tissues examined albeit with different expression patterns. The most ubiquitous expressed gene was atp1b1a whereas atp1b1b was mainly detected in osmoregulatory organs (gill, kidney and intestine), and atp1b2a, atp1b2b, atp1b3a, atp1b3b and atp1b4 in brain. An expression analysis in three brain regions and pituitary revealed that ß1-type transcripts were more abundant in pituitary than the other ß paralogs with slight differences between brain regions. Quantification of mRNA abundance in gills after a salinity challenge showed an activation of atp1b1a and atp1b1b at high salinity water (60 ppt) and atp1b3a and atp1b3b in response to low salinity (5 ppt). Transcriptional analysis during larval development showed specific expression patterns for each paralog. Moreover, no differences in the expression profiles between larvae cultivated at 10 and 35 ppt were observed except for atp1b4 with higher mRNA levels at 10 than 35 ppt at 18 days post hatch. Whole-mount in situ hybridization analysis revealed that atp1b1b was mainly localized in gut, pronephric tubule, gill, otic vesicle, and chordacentrum of newly hatched larvae. All these data suggest distinct roles of NKA ß subunits in tissues, during development and osmoregulation with ß1 subunits involved in the adaptation to hyperosmotic conditions and ß3 subunits to hypoosmotic environments.

Flatfish metamorphosis: a hypothalamic independent process?

Anuran and flatfish metamorphosis are tightly regulated by thyroid hormones that are the necessary and sufficient factors that drive this developmental event. In the present study whole mount in situ hybridization (WISH) and quantitative PCR in sole are used to explore the central regulation of flatfish metamorphosis. Central regulation of the thyroid in vertebrates is mediated by the hypothalamus-pituitary-thyroid (HPT) axis. Teleosts diverge from other vertebrates as hypothalamic regulation in the HPT axis is proposed to be through hypothalamic inhibition although the regulatory factor remains enigmatic. The dynamics of the HPT axis during sole metamorphosis revealed integration between the activity of the thyrotrophes in the pituitary and the thyroid follicles. No evidence was found supporting a role for thyroid releasing hormone (trh) or corticotrophin releasing hormone (crh) in hypothalamic control of TH production during sole metamorphosis. Intriguingly the results of the present study suggest that neither hypothalamic trh nor crh expression changes during sole metamorphosis and raises questions about the role of these factors and the hypothalamus in regulation of thyrotrophs.

Novel method for the authentication of frigate tunas (Auxis thazard and Auxis rochei) in commercial canned products.

A novel procedure for the authentication of frigate tunas (Auxis thazard and Auxis rochei) in commercially canned products has been developed. Three mitochondrial regions were simultaneously amplified by multiplex-Polymerase Chain Reaction, one corresponding to the small rRNA 12S subunit as a positive amplification control and two species-specific fragments corresponding to cytochrome b for A. rochei and ATPase 6 for A. thazard, respectively. Testing of two different detection systems revealed the fluorescence-based approach as the most sensitive. The results demonstrate that this rapid, low-cost methodology is a reliable molecular tool for direct application in the authentication of canned products.

Molecular characterization and transcriptional regulation of the sodium-dependent vitamin C transporter genes (slc23a1 and slc23a2) in a teleost fish, the Senegalese sole (Solea senegalensis).

Vitamin C (ascorbic acid, AA) is an antioxidant that acts as a free radical scavenger and cofactor for several important enzymatic reactions, thus being important for normal cellular functions, growth and development. Accumulation of AA in cells depends on two types of sodium-dependent vitamin C transporters (SVCTs), designed as SVCT1 and SVCT2. In human, they are the products of SLC23A1 and SLC23A2 genes, respectively. In the present work, the molecular cloning of the cDNAs corresponding to slc23a1 and slc23a2 in a teleost fish, the Senegalese sole (Solea senegalensis Kaup, 1858) is first described. Sequence analysis of the predicted polypeptides revealed a conserved topology with those of mammals with important motifs involved in structure and function, being also present in svct1 and svct2. Phylogenetic analyses including a range of vertebrate SVCTs suggest that both transporters are the result of an ancient gene duplication event that occurred prior to the divergence of tetrapods and teleosts, which took place 450 million years ago. Expression profiles in juvenile tissues and during larval development were analyzed using a real-time PCR approach. In juvenile fish, slc23a1 was strongly expressed in intestine, whereas slc23a2 exhibited a widespread distribution in tissues. Transcripts of both genes were detected at early developmental stages, probably representing mRNAs of maternal origin. A possible regulation by their own substrate was detected after first uptakes of AA from diet in both genes. During metamorphosis, both slc23a1 and slc23a2 were down-regulated, the former in a thyroid hormone (TH) dependent way. This pattern coincided with a significant reduction in the AA content of larvae during metamorphosis. These results are interpreted in a physiological context of general reduction in the metabolism of metamorphic larvae. Data presented here provide the first step toward a better understanding of the physiological role of SVCTs in teleost fish.

Duplication of calsequestrin genes in teleosts: molecular characterization in the Senegalese sole (Solea senegalensis).

Calsequestrin is a moderate-affinity, high-capacity Ca(2+) binding protein in the sarcoplasmic reticulum of skeletal and cardiac muscle that seems to act as an intralumenal Ca(2+) buffer. Two different isoforms have been described in mammals, the skeletal and cardiac isoforms, encoded by CASQ1 and CASQ2 genes, respectively. In this study, we present molecular phylogenetic evidence of a gene duplication event of both calsequestrin genes in teleosts, referred to as casq1a/casq1b and casq2a/casq2b. We obtained the entire cDNAs encoding the four genes in the Senegalese sole (Solea senegalensis Kaup). Main features and sequence identities with other fish and mammalian calsequestrins are described. Expression profiles during larval development and in juvenile tissues were analyzed using a real-time PCR approach. In juvenile fish, casq1a and casq1b were highly expressed in skeletal muscle, whereas the highest casq2a and casq2b transcript levels were detected in heart and brain, respectively. During metamorphosis, casq2a and casq1b expression remained unchanged. In contrast, casq1a and casq2b mRNAs exhibited a continuous increase from the beginning of metamorphosis until post-metamorphosis. Transcriptional regulation of casq1 and casq2 genes by thyroid hormones (THs) was also evaluated. Larvae exposed to the goitrogen thiourea (TU) exhibited higher casq1a mRNA levels than untreated control, whereas expression of the remaining genes did not vary significantly. Moreover, addition of exogenous T4 hormone to TU-treated larvae increased the casq1a steady-state levels with respect to the untreated control at metamorphosis climax. Comprehensively, these results demonstrate the existence of four calsequestrin genes in teleosts differentially regulated by THs.

Molecular characterization of a novel type II keratin gene (sseKer3) in the Senegalese sole (Solea senegalensis): Differential expression of keratin genes by salinity.

Keratins make up the largest subgroup of intermediate filaments and, in chordates, represent the most abundant proteins in epithelial cells. Senegalese sole (Solea senegalensis) is a commercially important flatfish in which only two type I keratin genes, sseKer1 and sseKer2, have been described. We obtained the entire cDNAs encoding for a novel type II keratin, referred to as sseKer3. Main features and sequence identities with other fish and mammalian type II keratins are described. Expression profiles during larval development and in juvenile tissues were analyzed using a real-time PCR approach. In juvenile fish, sseKer3 was strongly expressed in gills, intestine, skin and stomach. During metamorphosis climax a drop in sseKer3 expression was observed. Transcriptional regulation of sseKer3 by thyroid hormones (THs) was also evaluated. Larvae exposed to the goitrogen thiourea (TU) exhibited higher mRNA levels than untreated control. Moreover, adding exogenous T4 hormone to TU-treated larvae restored or even reduced the sseKer3 transcript levels with respect to the untreated control. The possible role of keratins in osmotic stress response was evaluated in juvenile gills exposed to three different water salinities (15, 37 and 60psu). Whereas no significant changes in sseKer2 expression were detected; sseKer1 was early (12h) up-regulated at hypo-osmotic conditions and sseKer3 was down-regulated both at low and high salinity after 24h and 48h. Their possible role is discussed.