Endocrine patterns associated with ovarian development in female Pacific halibut (Hippoglossus stenolepis).

The Pacific halibut (Hippoglossus stenolepis) is a large migratory demersal flatfish species that occupies a top trophic role in the North Pacific Ocean and Bering Sea ecosystems, where it also supports various fisheries. As a first attempt to characterize the endocrine mechanisms driving sexual maturation in this important species, we collected pituitary, ovarian and blood samples from Pacific halibut females captured in the wild that were classified histologically into various female developmental stages. We conducted gene expression analyses of gonadotropin beta subunits in the pituitary and observed that mRNA expression levels of fshb gradually increased throughout vitellogenesis, remained elevated until before ovulation and declined after spawning. In contrast, the mRNA expression levels of lhb markedly increased during oocyte maturation and remained elevated until after spawning. Ovarian mRNA expression levels of the gonadotropin receptor genes fshr and lhr peaked during oocyte maturation and before spawning, respectively, immediately following the developmental stage at which pituitary fshb and lhb mRNA expression first reached maximum levels. The ovarian gene expression patterns of steroidogenic enzyme genes cyp19a1 and hsd20b2 paralleled those of fshr and lhr, respectively. Testosterone and 17ß-estradiol (E2) plasma levels increased concomitantly with fshr and cyp19a1 mRNA expression levels, and vitellogenin plasma levels increased throughout vitellogenesis and reached maximum levels prior to spawning. These results are consistent with the notion that in female Pacific halibut, as in other teleosts, vitellogenesis and oocyte maturation and ovulation are likely under the control of pituitary gonadotropic hormones Fsh and Lh, respectively.

Generation of a chromosome-level genome assembly for Pacific halibut (Hippoglossus stenolepis) and characterization of its sex-determining genomic region.

The Pacific halibut (Hippoglossus stenolepis) is a key species in the North Pacific Ocean and Bering Sea ecosystems, where it also supports important fisheries. However, the lack of genomic resources limits our understanding of evolutionary, environmental and anthropogenic forces affecting key life history characteristics of Pacific halibut and prevents the application of genomic tools in fisheries management and conservation efforts. In the present study, we report on the first generation of a high-quality chromosome-level assembly of the Pacific halibut genome, with an estimated size of 602 Mb, 24 chromosome-length scaffolds that contain 99.8% of the assembly and a N50 scaffold length of 27.3 Mb. In the first application of this important resource, we conducted genome-wide analyses of sex-specific genetic variation by pool sequencing and characterized a potential sex-determining region in chromosome 9 with a high density of female-specific SNPs. Within this region, we identified the bmpr1ba gene as a potential candidate for master sex-determining (MSD) gene. bmpr1ba is a member of the TGF-ß family that in teleosts has provided the largest number of MSD genes, including a paralogue of this gene in Atlantic herring. The genome assembly constitutes an essential resource for future studies on Pacific halibut population structure and dynamics, evolutionary history and responses to environmental and anthropogenic influences. Furthermore, the genomic location of the sex-determining region in Pacific halibut has been identified and a putative candidate MSD gene has been proposed, providing further support for the rapid evolution of sex-determining mechanisms in teleost fish.

Controlled experiments to explore the use of a multi-tissue approach to characterizing stress in wild-caught Pacific halibut (Hippoglossus stenolepis).

The integration of multiple tissues in physiological and ecological analyses can enhance methodological approaches, increase applications for data and extend interpretation of results. Previous investigations of the stress response in fish have focused primarily on cortisol levels in a single matrix-blood plasma-which confines interpretations of cortisol levels to a short temporal frame. Epidermal mucus has been proposed as an alternative or complement to plasma that may provide a view to cortisol levels over a different temporal window allowing comparative assessment. Here, we explore the potential for multi-tissue cortisol analysis using both plasma and epidermal mucus in Pacific halibut (Hippoglossus stenolepis). The relative timing at which cortisol increased and decreased in the two matrices as well as cortisol concentrations at estimated peak levels were compared in two trials after (i) inducing cortisol synthesis by adrenocorticotropic hormone (ACTH1-24) administration and (ii) inducing cortisol elimination using cortisol (hydrocortisone, 98%) injection. The ACTH treatment elicited a peak plasma cortisol response approximately 12 hours post-injection, while mucus cortisol concentrations peaked later at approximately 62 hours post-injection. Exogenous cortisol treatments suggested relatively little transfer of cortisol from plasma to mucus, potentially reflecting differential effects of endogenous and exogenous cortisol. Our results suggest the potential utility of mucus as a sampling matrix that provides an extended window for detection of the stress response as compared to plasma. Results also suggest the utility of a multi-tissue approach to cortisol analysis with potential applications to applied fisheries research. Increased understanding of the relative scale of the cortisol response to stress (e.g. capture) will allow researchers and managers to better interpret the physiological condition and survival outcome of fish subjected to regulatory discard.

A comprehensive description of oocyte developmental stages in Pacific halibut, Hippoglossus stenolepis.

Accurate characterization of oocyte development is essential to understanding foundational aspects of reproductive biology and successful management of Pacific halibut (Hippoglossus stenolepis). Here this study provides complete histological descriptions for eight oocyte developmental stages in addition to postovulatory follicles and demonstrates the potential for oocyte size frequency distribution to act as a proxy for ovarian developmental stage and future maturity assessments. Importantly, it provides the first histological evidence that Pacific halibut have a group-synchronous ovarian developmental pattern with determinate fecundity and support for their batch-spawning strategy.

Physiological Responses to Swimming-Induced Exercise in the Adult Zebrafish Regenerating Heart.

Exercise promotes a set of physiological responses known to provide long-term health benefits and it can play an important role in cardioprotection. In the present study, we examined cardiac responses to exercise training in the adult zebrafish and in the context of cardiac regeneration. We found that swimming-induced exercise increased cardiomyocyte proliferation and that this response was also found under regenerating conditions, when exercise was performed either prior to and after ventricular cryoinjury (CI). Exercise prior to CI resulted in a mild improvement in cardiac function and lesion recovery over the non-exercise condition. Transcriptomic profiling of regenerating ventricles in cryoinjured fish subjected to exercise identified genes possibly involved in the cardioprotective effects of exercise and that could represent potential targets for heart regeneration strategies. Taken together, our results suggest that exercise constitutes a physiological stimulus that may help promote cardiomyogenic mechanisms of the vertebrate heart through the induction of cardiomyocyte proliferation. The zebrafish exercise model may be useful for investigating the potential cardioprotective effects of exercise in teleost fish and to contribute to further identify and develop novel avenues in basic research to promote heart regeneration.

Swimming exercise to control precocious maturation in male seabass (Dicentrarchus labrax).

BACKGROUND: Male European seabass, already predominant (~ 70%) in cultured stocks, show a high incidence (20-30%) of precocious sexual maturation under current aquaculture practices, leading to important economic losses for the industry. In view of the known modulation of reproductive development by swimming exercise in other teleost species, we aimed at investigating the effects of sustained swimming on reproductive development in seabass males during the first year of life in order to determine if swimming could potentially reduce precocious sexual maturation. METHODS: Pre-pubertal seabass (3.91 ± 0.22 g of body weight (BW)) were subjected to a 10 week swimming regime at their optimal swimming speed (Uopt) in an oval-shaped Brett-type flume or kept at rest during this period. Using Blazka-type swim tunnels, Uopt was determined three times during the course of the experiment: 0.66 m s- 1 at 19 ± 1 g BW, 10.2 ± 0.2 cm of standard length (SL) (week 1); 0.69 m s- 1 at 38 ± 3 g BW, 12.7 ± 0.3 cm SL (week 5), and also 0.69 m s- 1 at 77 ± 7 g BW, 15.7 ± 0.5 cm SL (week 9). Every 2 weeks, size and gonadal weight were monitored in the exercised (N = 15) and non-exercised fish (N = 15). After 10 weeks, exercised and non-exercised males were sampled to determine plasma 11-ketotestosterone levels, testicular mRNA expression levels of genes involved in steroidogenesis and gametogenesis by qPCR, as well as the relative abundance of germ cells representing the different spermatogenic stages by histological examination. RESULTS: Our results indicate that sustained swimming exercise at Uopt delays testicular development in male European seabass as evidenced by decreased gonado-somatic index, slower progression of testicular development and by reduced mRNA expression levels of follicle stimulating hormone receptor (fshR), 3-beta-hydroxysteroid dehydrogenase (3ßhsd), 11-beta hydroxysteroid dehydrogenase (11ßhsd), estrogen receptor-beta (erß2), anti-mullerian hormone (amh), structural maintenance of chromosomes protein 1B (smc1ß), inhibin beta A (inhba) and gonado-somal derived factor 1 (gsdf1) in exercised males as compared with the non-exercised males. CONCLUSIONS: Swimming exercise may represent a natural and non-invasive tool to reduce the incidence of sexually precocious males in seabass aquaculture.

In Vivo Effects of Lipopolysaccharide on Peroxisome Proliferator-Activated Receptor Expression in Juvenile Gilthead Seabream (Sparus Aurata).

Fish are constantly exposed to microorganisms in the aquatic environment, many of which are bacterial pathogens. Bacterial pathogens activate the innate immune response in fish involving the production of pro-inflammatory molecules that, in addition to their immune-related role, can affect non-immune tissues. In the present study, we aimed at investigating how inflammatory responses can affect metabolic homeostasis in the gilthead seabream (Sparus aurata), a teleost of considerable economic importance in Southern European countries. Specifically, we mimicked a bacterial infection by in vivo administration of lipopolysaccharide (LPS, 6 mg/kg body weight) and measured metabolic parameters in the blood and, importantly, the mRNA expression levels of the three isotypes of peroxisome proliferator activated receptors (PPARα, ß, and γ) in metabolically-relevant tissues in seabream. PPARs are nuclear receptors that are important for lipid and carbohydrate metabolism in mammals and that act as biological sensors of altered lipid metabolism. We show here that LPS-induced inflammatory responses result in the modulation of triglyceride plasma levels that are accompanied most notably by a decrease in the hepatic mRNA expression levels of PPARα, ß, and γ and by the up-regulation of PPARγ expression only in adipose tissue and the anterior intestine. In addition, LPS-induced inflammation results in an increase in the hepatic mRNA expression and protein activity levels of members of the mitogen-activated protein kinase (MAPK) family, known in mammals to regulate the transcription and activity of PPARs. Our results provide evidence for the involvement of PPARs in the metabolic response to inflammatory stimuli in seabream and offer insights into the molecular mechanisms underlying the redirection of metabolic activities under inflammatory conditions in vertebrates.

Transcriptomic features of Pecten maximus oocyte quality and maturation.

The king scallop Pecten maximus is a high valuable species of great interest in Europe for both fishery and aquaculture. Notably, there has been an increased investment to produce seed for enhancement programmes of wild scallop populations. However, hatchery production is a relatively new industry and it is still underdeveloped. Major hurdles are spawning control and gamete quality. In the present study, a total of 14 scallops were sampled in the bay of Brest (Brittany, France) to compare transcriptomic profiles of mature oocytes collected by spawning induction or by stripping. To reach such a goal, a microarray analysis was performed by using a custom 8x60K oligonucleotide microarray representing 45,488 unique scallop contigs. First we identified genes that were differentially expressed depending on oocyte quality, estimated as the potential to produce D-larvae. Secondly, we investigated the transcriptional features of both stripped and spawned oocytes. Genes coding for proteins involved in cytoskeletal dynamics, serine/threonine kinases signalling pathway, mRNA processing, response to DNA damage, apoptosis and cell-cycle appeared to be of crucial importance for both oocyte maturation and developmental competence. This study allowed us to dramatically increase the knowledge about transcriptional features of oocyte quality and maturation, as well as to propose for the first time putative molecular markers to solve a major bottleneck in scallop aquaculture.

Extending Immunological Profiling in the Gilthead Sea Bream, Sparus aurata, by Enriched cDNA Library Analysis, Microarray Design and Initial Studies upon the Inflammatory Response to PAMPs.

This study describes the development and validation of an enriched oligonucleotide-microarray platform for Sparus aurata (SAQ) to provide a platform for transcriptomic studies in this species. A transcriptome database was constructed by assembly of gilthead sea bream sequences derived from public repositories of mRNA together with reads from a large collection of expressed sequence tags (EST) from two extensive targeted cDNA libraries characterizing mRNA transcripts regulated by both bacterial and viral challenge. The developed microarray was further validated by analysing monocyte/macrophage activation profiles after challenge with two Gram-negative bacterial pathogen-associated molecular patterns (PAMPs; lipopolysaccharide (LPS) and peptidoglycan (PGN)). Of the approximately 10,000 EST sequenced, we obtained a total of 6837 EST longer than 100 nt, with 3778 and 3059 EST obtained from the bacterial-primed and from the viral-primed cDNA libraries, respectively. Functional classification of contigs from the bacterial- and viral-primed cDNA libraries by Gene Ontology (GO) showed that the top five represented categories were equally represented in the two libraries: metabolism (approximately 24% of the total number of contigs), carrier proteins/membrane transport (approximately 15%), effectors/modulators and cell communication (approximately 11%), nucleoside, nucleotide and nucleic acid metabolism (approximately 7.5%) and intracellular transducers/signal transduction (approximately 5%). Transcriptome analyses using this enriched oligonucleotide platform identified differential shifts in the response to PGN and LPS in macrophage-like cells, highlighting responsive gene-cassettes tightly related to PAMP host recognition. As observed in other fish species, PGN is a powerful activator of the inflammatory response in S. aurata macrophage-like cells. We have developed and validated an oligonucleotide microarray (SAQ) that provides a platform enriched for the study of gene expression in S. aurata with an emphasis upon immunity and the immune response.

Exercise-Induced Hypertrophic and Oxidative Signaling Pathways and Myokine Expression in Fast Muscle of Adult Zebrafish.

Skeletal muscle is a plastic tissue that undergoes cellular and metabolic adaptations under conditions of increased contractile activity such as exercise. Using adult zebrafish as an exercise model, we previously demonstrated that swimming training stimulates hypertrophy and vascularization of fast muscle fibers, consistent with the known muscle growth-promoting effects of exercise and with the resulting increased aerobic capacity of this tissue. Here we investigated the potential involvement of factors and signaling mechanisms that could be responsible for exercise-induced fast muscle remodeling in adult zebrafish. By subjecting zebrafish to swimming-induced exercise, we observed an increase in the activity of mammalian target of rapamycin (mTOR) and Mef2 protein levels in fast muscle. We also observed an increase in the protein levels of the mitotic marker phosphorylated histone H3 that correlated with an increase in the protein expression levels of Pax7, a satellite-like cell marker. Furthermore, the activity of AMP-activated protein kinase (AMPK) was also increased by exercise, in parallel with an increase in the mRNA expression levels of pgc1α and also of pparda, a ß-oxidation marker. Changes in the mRNA expression levels of slow and fast myosin markers further supported the notion of an exercise-induced aerobic phenotype in zebrafish fast muscle. The mRNA expression levels of il6, il6r, apln, aplnra and aplnrb, sparc, decorin and igf1, myokines known in mammals to be produced in response to exercise and to signal through mTOR/AMPK pathways, among others, were increased in fast muscle of exercised zebrafish. These results support the notion that exercise increases skeletal muscle growth and myogenesis in adult zebrafish through the coordinated activation of the mTOR-MEF2 and AMPK-PGC1α signaling pathways. These results, coupled with altered expression of markers for oxidative metabolism and fast-to-slow fiber-type switch, also suggest improved aerobic capacity as a result of swimming-induced exercise. Finally, the induction of myokine expression by swimming-induced exercise support the hypothesis that these myokines may have been produced and secreted by the exercised zebrafish muscle and acted on fast muscle cells to promote metabolic remodeling. These results support the use of zebrafish as a suitable model for studies on muscle remodeling in vertebrates, including humans.

Construction of an Ostrea edulis database from genomic and expressed sequence tags (ESTs) obtained from Bonamia ostreae infected haemocytes: Development of an immune-enriched oligo-microarray.

The flat oyster, Ostrea edulis, is one of the main farmed oysters, not only in Europe but also in the United States and Canada. Bonamiosis due to the parasite Bonamia ostreae has been associated with high mortality episodes in this species. This parasite is an intracellular protozoan that infects haemocytes, the main cells involved in oyster defence. Due to the economical and ecological importance of flat oyster, genomic data are badly needed for genetic improvement of the species, but they are still very scarce. The objective of this study is to develop a sequence database, OedulisDB, with new genomic and transcriptomic resources, providing new data and convenient tools to improve our knowledge of the oyster's immune mechanisms. Transcriptomic and genomic sequences were obtained using 454 pyrosequencing and compiled into an O. edulis database, OedulisDB, consisting of two sets of 10,318 and 7159 unique sequences that represent the oyster's genome (WG) and de novo haemocyte transcriptome (HT), respectively. The flat oyster transcriptome was obtained from two strains (naïve and tolerant) challenged with B. ostreae, and from their corresponding non-challenged controls. Approximately 78.5% of 5619 HT unique sequences were successfully annotated by Blast search using public databases. A total of 984 sequences were identified as being related to immune response and several key immune genes were identified for the first time in flat oyster. Additionally, transcriptome information was used to design and validate the first oligo-microarray in flat oyster enriched with immune sequences from haemocytes. Our transcriptomic and genomic sequencing and subsequent annotation have largely increased the scarce resources available for this economically important species and have enabled us to develop an OedulisDB database and accompanying tools for gene expression analysis. This study represents the first attempt to characterize in depth the O. edulis haemocyte transcriptome in response to B. ostreae through massively sequencing and has aided to improve our knowledge of the immune mechanisms of flat oyster. The validated oligo-microarray and the establishment of a reference transcriptome will be useful for large-scale gene expression studies in this species.

Luteinizing hormone induces ovulation via tumor necrosis factor α-dependent increases in prostaglandin F2α in a nonmammalian vertebrate.

Ovulation is induced by the preovulatory surge of luteinizing hormone (LH) that acts on the ovary and triggers the rupture of the preovulatory ovarian follicle by stimulating proteolysis and apoptosis in the follicle wall, causing the release of the mature oocyte. The pro-inflammatory cytokine tumor necrosis factor α (TNFα) and prostaglandin (PG) F2α (PGF2α) are involved in the control of ovulation but their role mediating the pro-ovulatory actions of LH is not well established. Here we show that Lh induces PGF2α synthesis through its stimulation of Tnfα production in trout, a primitive teleost fish. Recombinant trout Tnfα (rTnfα) and PGF2α recapitulate the stimulatory in vitro effects of salmon Lh (sLh) on contraction, proteolysis and loss of cell viability in the preovulatory follicle wall and, finally, ovulation. Furthermore, all pro-ovulatory actions of sLh are blocked by inhibition of Tnfα secretion or PG synthesis and all actions of rTnfα are blocked by PG synthesis inhibitors. Therefore, we provide evidence that the Tnfα-dependent increase in PGF2α production is necessary for the pro-ovulatory actions of Lh. The results from this study shed light onto the mechanisms underlying the pro-ovulatory actions of LH in vertebrates and may prove important in clinical assessments of female infertility.

The Olfactory Transcriptome and Progression of Sexual Maturation in Homing Chum Salmon Oncorhynchus keta.

Reproductive homing migration of salmonids requires accurate interaction between the reception of external olfactory cues for navigation to the spawning grounds and the regulation of sexual maturation processes. This study aimed at providing insights into the hypothesized functional link between olfactory sensing of the spawning ground and final sexual maturation. We have therefore assessed the presence and expression levels of olfactory genes by RNA sequencing (RNAseq) of the olfactory rosettes in homing chum salmon Oncorhynchus keta Walbaum from the coastal sea to 75 km upstream the rivers at the pre-spawning ground. The progression of sexual maturation along the brain-pituitary-gonadal axis was assessed through determination of plasma steroid levels by time-resolved fluoroimmunoassays (TR-FIA), pituitary gonadotropin subunit expression and salmon gonadotropin-releasing hormone (sgnrh) expression in the brain by quantitative real-time PCR. RNAseq revealed the expression of 75 known and 27 unknown salmonid olfactory genes of which 13 genes were differentially expressed between fish from the pre-spawning area and from the coastal area, suggesting an important role of these genes in homing. A clear progression towards final maturation was characterised by higher plasma 17α,20ß-dihydroxy-4-pregnen-3-one (DHP) levels, increased pituitary luteinizing hormone ß subunit (lhß) expression and sgnrh expression in the post brain, and lower plasma testosterone (T) and 17ß-estradiol (E2) levels. Olfactomedins and ependymin are candidates among the differentially expressed genes that may connect olfactory reception to the expression of sgnrh to regulate final maturation.

In Vivo Molecular Responses of Fast and Slow Muscle Fibers to Lipopolysaccharide in a Teleost Fish, the Rainbow Trout (Oncorhynchus mykiss).

The physiological consequences of the activation of the immune system in skeletal muscle in fish are not completely understood. To study the consequences of the activation of the immune system by bacterial pathogens on skeletal muscle function, we administered lipopolysaccharide (LPS), an active component of Gram-negative bacteria, in rainbow trout and performed transcriptomic and proteomic analyses in skeletal muscle. We examined changes in gene expression in fast and slow skeletal muscle in rainbow trout at 24 and 72 h after LPS treatment (8 mg/kg) by microarray analysis. At the transcriptional level, we observed important changes in metabolic, mitochondrial and structural genes in fast and slow skeletal muscle. In slow skeletal muscle, LPS caused marked changes in the expression of genes related to oxidative phosphorylation, while in fast skeletal muscle LPS administration caused major changes in the expression of genes coding for glycolytic enzymes. We also evaluated the effects of LPS administration on the fast skeletal muscle proteome and identified 14 proteins that were differentially induced in LPS-treated trout, primarily corresponding to glycolytic enzymes. Our results evidence a robust and tissue-specific response of skeletal muscle to an acute inflammatory challenge, affecting energy utilization and possibly growth in rainbow trout.

GLUT2-mediated glucose uptake and availability are required for embryonic brain development in zebrafish.

Glucose transporter 2 (GLUT2; gene name SLC2A2) has a key role in the regulation of glucose dynamics in organs central to metabolism. Although GLUT2 has been studied in the context of its participation in peripheral and central glucose sensing, its role in the brain is not well understood. To decipher the role of GLUT2 in brain development, we knocked down slc2a2 (glut2), the functional ortholog of human GLUT2, in zebrafish. Abrogation of glut2 led to defective brain organogenesis, reduced glucose uptake and increased programmed cell death in the brain. Coinciding with the observed localization of glut2 expression in the zebrafish hindbrain, glut2 deficiency affected the development of neural progenitor cells expressing the proneural genes atoh1b and ptf1a but not those expressing neurod. Specificity of the morphant phenotype was demonstrated by the restoration of brain organogenesis, whole-embryo glucose uptake, brain apoptosis, and expression of proneural markers in rescue experiments. These results indicate that glut2 has an essential role during brain development by facilitating the uptake and availability of glucose and support the involvement of glut2 in brain glucose sensing.

Swimming-induced exercise promotes hypertrophy and vascularization of fast skeletal muscle fibres and activation of myogenic and angiogenic transcriptional programs in adult zebrafish.

BACKGROUND: The adult skeletal muscle is a plastic tissue with a remarkable ability to adapt to different levels of activity by altering its excitability, its contractile and metabolic phenotype and its mass. We previously reported on the potential of adult zebrafish as a tractable experimental model for exercise physiology, established its optimal swimming speed and showed that swimming-induced contractile activity potentiated somatic growth. Given that the underlying exercise-induced transcriptional mechanisms regulating muscle mass in vertebrates are not fully understood, here we investigated the cellular and molecular adaptive mechanisms taking place in fast skeletal muscle of adult zebrafish in response to swimming. RESULTS: Fish were trained at low swimming speed (0.1 m/s; non-exercised) or at their optimal swimming speed (0.4 m/s; exercised). A significant increase in fibre cross-sectional area (1.290±88 vs. 1.665±106 µm2) and vascularization (298±23 vs. 458±38 capillaries/mm2) was found in exercised over non-exercised fish. Gene expression profiling by microarray analysis evidenced the activation of a series of complex transcriptional networks of extracellular and intracellular signaling molecules and pathways involved in the regulation of muscle mass (e.g. IGF-1/PI3K/mTOR, BMP, MSTN), myogenesis and satellite cell activation (e.g. PAX3, FGF, Notch, Wnt, MEF2, Hh, EphrinB2) and angiogenesis (e.g. VEGF, HIF, Notch, EphrinB2, KLF2), some of which had not been previously associated with exercise-induced contractile activity. CONCLUSIONS: The results from the present study show that exercise-induced contractile activity in adult zebrafish promotes a coordinated adaptive response in fast muscle that leads to increased muscle mass by hypertrophy and increased vascularization by angiogenesis. We propose that these phenotypic adaptations are the result of extensive transcriptional changes induced by exercise. Analysis of the transcriptional networks that are activated in response to exercise in the adult zebrafish fast muscle resulted in the identification of key signaling pathways and factors for the regulation of skeletal muscle mass, myogenesis and angiogenesis that have been remarkably conserved during evolution from fish to mammals. These results further support the validity of the adult zebrafish as an exercise model to decipher the complex molecular and cellular mechanisms governing skeletal muscle mass and function in vertebrates.

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.

Fsh and Lh direct conserved and specific pathways during flatfish semicystic spermatogenesis.

The current view of the control of spermatogenesis by Fsh and Lh in non-mammalian vertebrates is largely based on studies carried out in teleosts with cystic and cyclic spermatogenesis. Much less is known concerning the specific actions of gonadotropins during semicystic germ cell development, a type of spermatogenesis in which germ cells are released into the tubular lumen where they transform into spermatozoa. In this study, using homologous gonadotropins and a candidate gene approach, for which the genes' testicular cell-type-specific expression was established, we investigated the regulatory effects of Fsh and Lh on gene expression during spermatogenesis in Senegalese sole (Solea senegalensis), a flatfish with asynchronous and semicystic germ cell development. During early spermatogenesis, Fsh and Lh upregulated steroidogenesis-related genes and nuclear steroid receptors, expressed in both somatic and germ cells, through steroid-dependent pathways, although Lh preferentially stimulated the expression of downstream genes involved in androgen and progestin syntheses. In addition, Lh specifically promoted the expression of spermatid-specific genes encoding spermatozoan flagellar proteins through direct interaction with the Lh receptor in these cells. Interestingly, at this spermatogenic stage, Fsh primarily regulated genes encoding Sertoli cell growth factors with potentially antagonistic effects on germ cell proliferation and differentiation through steroid mediation. During late spermatogenesis, fewer genes were regulated by Fsh or Lh, which was associated with a translational and posttranslational downregulation of the Fsh receptor in different testicular compartments. These results reveal that conserved and specialized gonadotropic pathways regulate semicystic spermatogenesis in flatfish, which may spatially adjust cell germ development to maintain a continuous reservoir of spermatids in the testis.

Transcriptional assessment by microarray analysis and large-scale meta-analysis of the metabolic capacity of cardiac and skeletal muscle tissues to cope with reduced nutrient availability in Gilthead Sea Bream (Sparus aurata L.).

The effects of nutrient availability on the transcriptome of cardiac and skeletal muscle tissues were assessed in juvenile gilthead sea bream fed with a standard diet at two feeding levels: (1) full ration size and (2) 70 % satiation followed by a finishing phase at the maintenance ration. Microarray analysis evidenced a characteristic transcriptomic profile for each muscle tissue following changes in oxidative capacity (heart > red skeletal muscle > white skeletal muscle). The transcriptome of heart and secondly that of red skeletal muscle were highly responsive to nutritional changes, whereas that of glycolytic white skeletal muscle showed less ability to respond. The highly expressed and nutritionally regulated genes of heart were mainly related to signal transduction and transcriptional regulation. In contrast, those of white muscle were enriched in gene ontology (GO) terms related to proteolysis and protein ubiquitination. Microarray meta-analysis using the bioinformatic tool Fish and Chips ( http://fishandchips.genouest.org/index.php ) showed the close association of a representative cluster of white skeletal muscle with some of cardiac and red skeletal muscle, and many GO terms related to mitochondrial function appeared to be common links between them. A second round of cluster comparisons revealed that mitochondria-related GOs also linked differentially expressed genes of heart with those of liver from cortisol-treated gilthead sea bream. These results show that mitochondria are among the first responders to environmental and nutritional stress stimuli in gilthead sea bream, and functional phenotyping of this cellular organelle is highly promising to obtain reliable markers of growth performance and well-being in this fish species.