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 Thereafter, ulvan was withdrawn from the diet and all juveniles were fed the basal diet for 30 days.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.

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.

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 pcDNATM6.2/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 CSTPP+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.

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.

The effect of dietary iron (Fe) and vitamin C (Ascorbic acid, AA) supplementation on development, gene expression and antioxidant status of Senegalese sole (Solea senegalensis Kaup, 1858) larvae was investigated. Larvae were reared from 7 to 28 days post hatching (dph) in triplicate using a recirculation system. Three experimental groups were established: The control group was fed Artemia enriched with the microalgae Tisochrysis lutea (T. lutea), the F group, T. lutea supplemented with Fe and the FP group, T. lutea supplemented with Fe and ascorbyl palmitate (AP). Enriched Artemia of F and FP groups displayed a significantly higher content of Fe than the control (850 mu g Fe g(-1) DW and 350 mu g Fe g(-1) DW, respectively). In addition, enriched Artemia of the FP group also showed a significantly higher content of AA than the control (3.5 mu g AA mg(-1) DW and 0.8 mu g AAmg(-1) DW, respectively). At 28 dph, Fe content in larvae from F and FP groups was 1.4- and 1.7-fold higher than the control, respectively. Moreover, larvae from FP group showed the highest AA content throughout the whole experiment (p < 0.05). Larvae from FP group grew faster and completed metamorphosis earlier than the other groups (p < 0.05). Moreover, antioxidant enzymatic activities, lipid peroxidation and total antioxidant content (TAC) at the end of the experiment (p < 0.05) indicated a positive iron-vitamin C interaction with better antioxidant status in FP group. Similar skeletal typology and histological pattern in the development of tissues was observed at 28 dph for larvae under all feeding regimes assayed. Nevertheless, an increasing collagen fibre was evidenced in larvae of FP group The gene expression pattern during the experimental period revealed that different levels of iron and vitamin C modified the expression levels of genes involved in stress (crh, pomca2 and hsc70-4l), glycolytic pathway (pkm), antioxidative defence (slc23a1), osmoregulation (aqp3a, aqp12 and nppc1) and iron homeostasis (hamp1, fpn, tf). The present study is the first report about the potential effect of iron and vitamin C during the first stages of development in S. senegalensis larvae.

Selenium (Se) is an essential nutrient and the chemical forms of Se present in food affect both its bioavailability and its toxicity. The aim of this study was to evaluate the effects of the rotifer enrichment with three sources of selenium supplementation: selenite (Se(IV)), selenomethionine (SeMet) and selenized yeast (SelPlex). For this purpose, rotifers were exposed to each source of selenium at two concentrations during 6 days (2 culture cycles of 3 days). The toxicity order for the three sources of Se was SelPlex > SeMet > Se(IV). Modelling calculations showed that SeMet and Selplex uptake followed a first-order kinetic model. Nevertheless, uptake of Se(IV) displayed a linear pattern with increasing exposure time. Speciation data revealed that the predominant form in rotifers was SeMet. Indeed, rotifers metabolized and chemically transformed Se(IV) into SeMet. If the source of Se assayed is Se(IV), rotifers with Se levels similar to those in copepods and high content of SeMet can be accomplished when they are exposed to 2 mg of Se per million rotifers for 12 h. For SeMet and SelPlex treatments, 0.2 mg of Se per million rotifers in 3 and 6 h, respectively, were required.

The aim of this study was to evaluate the short- and long-term effects of diets consisting in rotifers enriched with different oil emulsions (differing mainly in triacylglycerols levels) and provided during the first week of life. Survival, growth, metamorphosis progress, lipid profile and molecular regulatory pathways were evaluated. For comparison purposes, a diet used routinely in the aquaculture industry based on microalgae was also carried out. The present study shows that dietary triacylglycerols (TAG) levels and fatty acid composition modulate larval survival and growth when supplied during larval pelagic stages. Histological observation and gene expression pattern showed that early larvae regulated intestinal lipid transport, showing a coordinate activation of apolipo-proteins transcripts. Multivariate analyses identified the major contribution of apoEa to discriminate samples by dietary treatments, pointing out the importance of this apolipoprotein as key molecular marker for intestinal lipid mobilization. However, our data indicate that transport capacity is limited in early larvae, increasing in anterior intestine as intestine maturates with larval development, while the increase of fat deposits in the posterior intestine in older larvae supports the limited capacity of this gut section for lipid transport. The accumulation of TAG, phosphocholine (PC) and oleic acid (OA) along pre- and early metamorphosis indicates that these lipids influences positively daily mortality rate, eye-migration progress and growth. Moreover, larval TAG amounts correlate highly with apoA-NBa3 mRNA levels, indicating an active role of this apolipoprotein in endogenous lipid mobilization during development. These data provide new evidence that TAG, PC and OA dietary levels are important for larval development and complement the available information about energy management in sole larvae reinforcing the rotifer feeding period as critical to produce high-quality larvae.Statement of relevance: This paper provides new clues about the mechanisms involved in lipid management in larvae and their consequences during their life-cycle to produce high-quality fry and optimize husbandry procedures. (C) 2016 Elsevier B.V. All rights reserved.

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.

The aim of this work was to assess the effects of a dietary supplement of vitamin C (as ascorbyl palmitate, AP) delivered through new and optimized alginate microcapsules (MA) on larval performance, antioxidant status and gene expression profiles. For this purpose, three MA (referred to as MA-1, MA-2 and MA-3) with increasing AP content ranging from 1700 to 5700μg AP g−1 DW were assayed. Sole larvae were fed the three MA from first feeding to 7days post-hatch (dph) and later replaced by Artemia from 7 to 28 dph. The experimental groups were performed in triplicate using a recirculation system. The efficiency of AP inclusion into MA was 55%. At 7 dph, larval ascorbic acid (AA) content was proportional to the AP concentration included in MA (p<0.05). Larval growth until 7 dph was low but significantly higher in larvae fed MA-2 and MA-3. After Artemia supplying, a compensatory growth was detected in the three experimental groups whereas a faster metamorphic process and a higher weight at 28 dph in larvae fed MA with a higher AP content (MA-2 and MA-3) was observed (p<0.05). Moreover, Artemia feeding substantially increased the larval AA content during development peaking at 18 dph and decreasing at the end of metamorphosis with significantly lower AA levels in larvae fed MA-3. This reduction coincided with a higher total antioxidant capacity and lipid peroxidation whereas no differences in catalase or total glutathione peroxidase were detected. Expression data revealed that MA diets significantly modified the transcript levels of 16 genes involved in antioxidant defence (gpx1), tissue structure (col1a1 col1a2 and col1a3), stress (hsc70-1, hsc70-2, hsc70-3, gr1, gr2, pomcb), glycolytic pathway (gapdh1 and pkm), osmoregulation (cftr, nkcc2 and nkcc1) and pigmentation (mc1r). All these data reveal novel new insights about role of vitamin C during early stages of S. senegalensis to modulate growth, stress and antioxidative status in later developmental stages.

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.

To date, most research on larval lipid nutrition has been centered on essential fatty acid requirements. However, less is known about effects of dietary fatty acid (FA) composition on lipid absorption and metabolism, as addressed in this study. Senegalese sole were fed live preys enriched with different oils: cod liver oil (CLO), linseed oil (LSO), soybean oil (SBO), and olive oil (OO), that are rich in long-chain polyunsaturated FAs (LC-PUFA), n-3 PUFA, n-6 PUFA or monounsaturated FAs (MUFA), respectively. Larvae reared on CLO showed significantly improved growth and survival, faster onset of metamorphosis and maturation of the intestine, lower lipid accumulation in liver after metamorphosis and an up-regulation of genes involved in lipid transport and phospholipid metabolism, while key lipogenesis genes were down-regulated. From the remaining treatments, the LSO diet induced the closest performance to CLO, with larvae completing metamorphosis at a similar time and having the second best growth and survival by the end of the experiment. They were also grouped closer to the CLO treatment than the remaining vegetable oil treatments, based on the patterns of gene expression. These results showed that oil sources rich in LC-PUFA and n-3 PUFA were superior to those having high n-6 PUFA or MUFA levels in the larval nutrition of Senegalese sole and indicate that this effect might be at least partly explained by an up-regulation of phospholipid metabolism and apolipoprotein synthesis, likely leading to enhanced lipid transport and mobilization, as well as tissue growth and remodeling.Statement of relevance: The nutritional and physiological roles of dietary C18 PUFA in the absence of LC-PUFA have rarely been investigated in fish, particularly in larvae, as it is unfeasible to culture most marine species in such extreme dietary conditions. Senegalese sole (Solea senegalensis), a marine species of high commercial aquaculture importance in southern Europe, has relatively low LC-PUFA requirements during the larval stage compared to other marine finfish, making it a good model species to investigate this subject. A holistic approach was implemented to determine the effects these FAs may have on fish performance and metabolism, by analyzing growth, development, intestinal maturation, lipid and FA composition, hepatic and intestinal lipid accumulation, and expression of genes involved in lipid metabolism, absorption and transport (qPCR). (C) 2015 Elsevier B.V. All rights reserved.

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.

The aim of this work was to evaluate the genomic responses of premetamorphic sole larvae (9 days post-hatching, dph) fed diets with different lipid and triacylglycerol (TAG) content. For this purpose, two diets with high (rotifers enriched with a fish oil-based emulsion; referred to as HTAG) and low (rotifers enriched with a krill oil-based emulsion; LTAG) levels of total lipids and TAG were evaluated. Lipid class and fatty acid (FA) profiles, histological characterization of intestine, liver and pancreas and expression patterns using RNA-seq were determined. Discriminant analysis results showed that larvae could be clearly differentiated on the basis of their FA profile as a function of the diet supplied until 9dph although no difference in growth was observed. RNA-seq analysis showed that larvae fed HTAG activated coordinately the transcription of apolipoproteins (apob, apoa4, apoc2, apoe, and apobec2) and other related transcripts involved in chylomicron formation, likely to facilitate proper lipid absorption and delivery. In contrast, larvae fed LTAG showed higher mRNA levels of several pancreatic enzymes (try1a, try2, cela1, cela3, cela4, chym1, chym2, amy2a and pnlip) and appetite modulators (agrp1) and some intra- and extracellular lipases. Moreover, KEGG analysis also showed that several transcripts related to lipid metabolism and glycolysis were differentially expressed with a higher abundance in larvae fed LTAG diet. All these data suggest that early larvae were able to establish compensatory mechanisms for energy homeostasis regulating key molecules for FA and TAG biosynthesis, FA uptake and intracellular management of TAG and FA to warrant optimal growth rates.

In the present work, seven genes encoding Na(+),K(+)-ATPase (NKA) β-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: β1 (atp1b1a and atp1b1b), β2 (atp1b2a and atp1b2b), β3 (atp1b3a and atp1b3b) and β4 (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 β1-type transcripts were more abundant in pituitary than the other β 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 β subunits in tissues, during development and osmoregulation with β1 subunits involved in the adaptation to hyperosmotic conditions and β3 subunits to hypoosmotic environments.

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.

This article presents the first physical mapping carried out in the Senegalese sole (Solea senegalensis), an important marine fish species of Southern Europe. Eight probes were designated to pick up genes of interest in aquaculture (candidate genes) from a bacterial artificial chromosome (BAC) library using a method of rapid screening based on a 4-dimension PCR. Seven known and 3 unknown clones were isolated and labeled. The 10 BAC clones were used as probes to map the karyotype of the species by fluorescence in situ hybridization (FISH). Nine out of the 10 clones were localized in only 1 chromosome pair, whereas the remaining one hybridized on 2 chromosome pairs. The 2-color FISH experiments showed colocation of 4 probes in 2 chromosome pairs. In addition, 2-color FISH was carried out both with 5S rDNA and the BAC containing the lysozyme gene published previously. This first genetic map of the Senegalese sole represents a starting point for future studies of the sole genome. In addition, 7 out of the 10 BAC clones were sequenced using next-generation sequencing, and bioinformatic characterization of the sequences was carried out. Hence the anchoring of the sequences to specific chromosomes or chromosome arms is now possible, leading to an initial scaffold of the Senegalese sole genome.

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.

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 tissuespecific 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. (C) 2011 Elsevier Ltd. All rights reserved.