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.

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.

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.

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.

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.

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.

Fueling the engine: induction of AMP-activated protein kinase in trout skeletal muscle by swimming.

AMP-activated protein kinase (AMPK) is well known to be induced by exercise and to mediate important metabolic changes in the skeletal muscle of mammals. Despite the physiological importance of exercise as a modulator of energy use by locomotory muscle, the regulation of this enzyme by swimming has not been investigated in fish. We found that sustained swimming (40 days at 0.75 body lengths s(-1)) increased AMPK activity in red and white trout skeletal muscle (3.9- and 2.2-fold, respectively) as well as the expression of AMPK target genes involved in energy use: lipoprotein lipase and citrate synthase in red and white muscle and CPT1ß1b and PGC-1α in red muscle. Furthermore, electrical pulse stimulation of cultured trout myotubes increased AMPK activity and glucose uptake (1.9- and 1.2-fold, respectively) in an AMPK-dependent manner. These results suggest that AMPK may play an important mediatory role in the metabolic adaptation to swimming in fish skeletal muscle.

A combined strategy involving Sanger and 454 pyrosequencing increases genomic resources to aid in the management of reproduction, disease control and genetic selection in the turbot (Scophthalmus maximus).

BACKGROUND: Genomic resources for plant and animal species that are under exploitation primarily for human consumption are increasingly important, among other things, for understanding physiological processes and for establishing adequate genetic selection programs. Current available techniques for high-throughput sequencing have been implemented in a number of species, including fish, to obtain a proper description of the transcriptome. The objective of this study was to generate a comprehensive transcriptomic database in turbot, a highly priced farmed fish species in Europe, with potential expansion to other areas of the world, for which there are unsolved production bottlenecks, to understand better reproductive- and immune-related functions. This information is essential to implement marker assisted selection programs useful for the turbot industry. RESULTS: Expressed sequence tags were generated by Sanger sequencing of cDNA libraries from different immune-related tissues after several parasitic challenges. The resulting database ("Turbot 2 database") was enlarged with sequences generated from a 454 sequencing run of brain-hypophysis-gonadal axis-derived RNA obtained from turbot at different development stages. The assembly of Sanger and 454 sequences generated 52,427 consensus sequences ("Turbot 3 database"), of which 23,661 were successfully annotated. A total of 1,410 sequences were confirmed to be related to reproduction and key genes involved in sex differentiation and maturation were identified for the first time in turbot (AR, AMH, SRY-related genes, CYP19A, ZPGs, STAR FSHR, etc.). Similarly, 2,241 sequences were related to the immune system and several novel key immune genes were identified (BCL, TRAF, NCK, CD28 and TOLLIP, among others). The number of genes of many relevant reproduction- and immune-related pathways present in the database was 50-90% of the total gene count of each pathway. In addition, 1,237 microsatellites and 7,362 single nucleotide polymorphisms (SNPs) were also compiled. Further, 2,976 putative natural antisense transcripts (NATs) including microRNAs were also identified. CONCLUSIONS: The combined sequencing strategies employed here significantly increased the turbot genomic resources available, including 34,400 novel sequences. The generated database contains a larger number of genes relevant for reproduction- and immune-associated studies, with an excellent coverage of most genes present in many relevant physiological pathways. This database also allowed the identification of many microsatellites and SNP markers that will be very useful for population and genome screening and a valuable aid in marker assisted selection programs.

Transcriptional regulation of the gilthead seabream (Sparus aurata) interleukin-6 gene promoter.

Interleukin-6 (IL-6) has been identified and characterized from several fish species and its mRNA expression is induced by pathogen-associated molecular patterns (PAMPs) and cytokines in immune cells and tissues. However, the transcriptional regulation of the IL-6 gene in fish is not well understood. In the present study, we have cloned and sequenced a 1028 bp 5'-flanking DNA region from the IL-6 gene in seabream (Sparus aurata). Sequence analysis of the seabream IL-6 promoter (sbIL-6P) evidenced the presence of a conserved TATA motif and conserved response elements for NF-κB, C/EBPß (NF-IL6), AP-1 and GRE, similar to other vertebrate IL-6 promoters. Functional characterization of sbIL-6P was performed by cloning sbIL-6P into a luciferase expression vector and by transfecting it into L6 muscle cells, a mammalian cell line shown previously to express IL-6 in response to pro-inflammatory stimuli. We show here that the activity of sbIL-6P was significantly induced by pro-inflammatory cytokines such as tumor necrosis factor alpha (TNFα), IL-6 and IL-2, as well as by lipopolysaccharide (LPS), but significantly repressed by dexamethasone. In addition, the stimulatory effects of TNFα on sbIL-6P activity appeared to be mediated by the NF-κB, p38 MAPK and JNK signaling pathways. Deletion analyses of sbIL-6P suggested that activation of sbIL-6P by TNFα and IL-6 required the presence of binding motifs present in the proximal promoter (-171 to -84) whereas activation by IL-2 required binding motifs present in the distal promoter (-1024 to -864). The results from this study indicate, for the first time in fish, that pro-inflammatory cytokines, LPS and glucocorticoids can regulate the activity of the IL-6 gene at a transcriptional level and identify important regions in its response to cytokines.

Luteinizing hormone stimulation of in vitro ovulation in brook trout (Salvelinus fontinalis) involves follicle contraction and activation of proteolytic genes.

Luteinizing hormone (LH) is an essential hormone for the stimulation of the ovulatory process in vertebrates. However, little is known in fish regarding the different mechanisms induced by LH during ovulation that facilitate the rupture of the follicle wall and the subsequent expulsion of the mature oocyte. In this study, the effects of salmon LH (sLH) on in vitro ovulation were investigated in brook trout (Salvelinus fontinalis) isolated follicles. sLH significantly stimulated in vitro ovulation and contraction of brook trout preovulatory follicles. In order to investigate the possible involvement of proteolytic events in the ovulatory action of LH, the expression of genes known to have a crucial role in the degradation of follicle wall structure was examined. Our results show that sLH clearly stimulated the mRNA expression levels of matrix metalloproteinases (MMPs; including mmp2 and mmp19) and other enzymes with proteolytic action during ovulation, such as a disintegrin and metalloproteinase with thrombospondin-like motifs 1 (adamts1) and plasminogen (plg), in brook trout preovulatory follicles. In addition, the expression of mmp2, adamts1 and plg increased in brook trout follicles during the progression of LH-induced ovulation. Interestingly, the expression of tissue inhibitor of matrix metalloproteinase 2 (timp2), a known regulator of MMP2 activity, paralleled that of mmp2, suggesting the existence of a controlled mechanism of MMP2 action. Therefore, the known increase in proteolytic activity during ovulation in fish could be the result of the stimulation of the expression of proteolytic enzymes by LH in preovulatory follicles. We propose that LH may stimulate ovulation in brook trout follicles by stimulating proteolysis of the follicle wall and by stimulating follicle contraction.

Stage-specific gene expression during spermatogenesis in the Senegalese sole (Solea senegalensis), a fish with semi-cystic type of spermatogenesis, as assessed by laser capture microdissection and absolute quantitative PCR.

Spermatogenesis is a complex process where hormonal signals regulate the interaction of different cell types in a tight spatial and temporal fashion. The Senegalese sole (Solea senegalensis) is a marine flatfish that, in contrast to many fish, exhibits a semi-cystic, asynchronous pattern of spermatogenesis progression. This pattern is characterized by the release of spermatids into the tubule lumen, where they transform into spermatozoa. In this study, we used laser capture microdissection (LCM) to isolate cells from cysts containing spermatogonia, spermatocytes, spermatids or spermatozoa in order to investigate developmental patterns of gene expression. Furthermore, we also analyzed the stage-specific expression of the same set of genes throughout spermatogenesis (early-mid, late and maturing spermatogenic stages) in tissue fragments of the Senegalese sole testis. Genes analyzed by absolute qPCR in cysts isolated by LCM and stage-specific testis samples included genes involved in steroid synthesis and action (3ß-hsd, 17ß-hsd, 20ß-hsd, star, star-like, progesterone receptor), gonadotropin action (fshr, lhr), the kisspeptin system (kiss2, kiss2r) and other genes important for the production of mature gametes (zona pellucida 2.2, claudin and clusterin). Our results show that, in general, steroidogenesis-related genes tended to increase with spermatogenesis progression and that 3ß-hsd and 20ß-hsd were expressed in germ cells but 17ß-hsd was not. Our results also show that fshr is expressed in most testicular cell types, including germ cells. In contrast, lhr is expressed only in late spermatogenesis and is not expressed in any of the germ cell types examined, indicating that, in contrast to fshr, lhr may be primarily expressed in non-germinal cells (e.g. Leydig cells). Furthermore, kisspeptin and its receptor were expressed in all germ cell types examined and, as expected, gamete maturation-related genes were more expressed in mature stages. These results illustrate that key factors that participate in the hormonal regulation of spermatogenesis in the Senegalese sole testis show complex cell type- and stage-specific patterns of gene expression.

Effects of sustained swimming on the red and white muscle transcriptome of rainbow trout (Oncorhynchus mykiss) fed a carbohydrate-rich diet.

Training at sustainable swimming speeds can produce changes in fish skeletal muscle that are important for aquaculture due to their growth-potentiating effects. Such changes may be even more relevant when fish are fed diets containing an increasing proportion of carbohydrates as an energy source. We evaluated the effects of moderate-intensity sustained swimming on the transcriptomic response of red and white muscle in rainbow trout fed a carbohydrate-rich diet using microarray and qPCR. Analysis of the red and white muscle transcriptome in resting or swimming (1.3 body lengths/s) fish for 30days revealed significant changes in the expression of a large number of genes (395 and 597, respectively), with a total of 218 differentially expressed genes (DEGs) common for both muscles. A large number of the genes involved in glucose use and energy generation, contraction, development, synthesis and catabolism of proteins were up-regulated in red and white muscle. Additionally, DEGs in both muscles were involved in processes of defense response and apoptosis. Skeletal muscle contraction activates a transcriptional program required for the successful adaptation of both muscles to the changing demands imposed by swimming conditions. Future studies should further clarify the mechanisms involved in the adaptation of both tissues to exercise and assess possible benefits of such conditions for cultured fish.

Tumor necrosis factor alpha may act as an intraovarian mediator of luteinizing hormone-induced oocyte maturation in trout.

In fish, like in other vertebrates, luteinizing hormone (Lh) is an essential hormone for the completion of oocyte maturation. In salmonid fish (i.e., salmon and trout), oocyte maturation is induced by Lh through its stimulation of the production of the maturation-inducing steroid, 17alpha,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P). In mammals, several factors, including ovarian cytokines and growth factors, have been reported to contribute to the regulation of oocyte maturation. In fish, growing evidence suggests that tumor necrosis factor alpha (hereafter referred to as Tnf) could play multiple physiological roles in the control of ovarian function. In the present study, we have investigated the possible involvement of Tnf in the regulation of oocyte maturation in brown trout (Salmo trutta). Our results show that in vitro treatment of brown trout preovulatory follicles with coho salmon (Oncorhynchus kisutch) Lh (sLh) significantly increased oocyte maturation, as assessed by germinal vesicle breakdown (GVBD), and that this effect was blocked by TAPI-1 (an inhibitor of Tnf-converting enzyme or Tace/Adam17). Furthermore, treatment of preovulatory follicles with sLh increased the expression of tnf and tace/adam17 as well as the secretion of the Tnf protein. Importantly, recombinant trout Tnf (rtTnf) significantly increased GVBD in vitro. Our results also show that the stimulatory effects of rtTnf on oocyte maturation may be the result of the direct involvement of rtTnf in stimulating the production of the maturation-inducing steroid as evidenced, first, by the stimulatory effects of rtTnf on 17,20beta-P production in vitro and on the expression of cholesterol side-chain cleavage P450 cytochrome (p450scc) and 20beta-hydroxysteroid dehydrogenase/carbonyl reductase 1 (cbr1), the enzyme responsible for the production of 17,20beta-P, and, second, by the ability of TAPI-1 to block the stimulatory effects of sLh on 17,20beta-P production and cbr1 expression. Furthermore, sLh and rtTnf increased the expression of the Lh receptor (lhr) and decreased the expression of aromatase (cyp19a1), and TAPI-1 completely blocked the effects of sLh. These results strongly suggest that Tnf may contribute to the regulation of oocyte maturation by Lh in trout.

Tracing metabolic routes of dietary carbohydrate and protein in rainbow trout (Oncorhynchus mykiss) using stable isotopes ([¹³C]starch and [¹5N]protein): effects of gelatinisation of starches and sustained swimming.

Here we examined the use of stable isotopes, [¹³C]starch and [¹5N]protein, as dietary tracers to study carbohydrate assimilation and distribution and protein utilisation, respectively, by rainbow trout (Oncorhynchus mykiss). The capacity of glucose uptake and use by tissues was studied, first, by varying the digestibility of carbohydrate-rich diets (30 % carbohydrate), using raw starch and gelatinised starch (GS) and, second, by observing the effects of two regimens of activity (voluntary swimming, control; sustained swimming at 1·3 body lengths/s, exercise) on the GS diet. Isotopic ratio enrichment (¹³C and ¹5N) of the various tissue components (protein, lipid and glycogen) was measured in the liver, muscles, viscera and the rest of the fish at 11 and 24 h after a forced meal. A level of 30 % of digestible carbohydrates in the food exceeded the capacity of rainbow trout to use this nutrient, causing long-lasting hyperglycaemia that raises glucose uptake by tissues, and the synthesis of glycogen and lipid in liver. Total 13C recovered 24 h post-feeding in the GS group was lower than at 11 h, indicating a proportional increase in glucose oxidation, although the deposition of lipids in white muscle (WM) increased. Prolonged hyperglycaemia was prevented by exercise, since sustained swimming enhances the use of dietary carbohydrates, mainly through conversion to lipids in liver and oxidation in muscles, especially in red muscle (RM). Higher recoveries of total 15N for exercised fish at 24 h, mainly into the protein fraction of both RM and WM, provide evidence that sustained swimming improves protein deposition, resulting in an enhancement of the protein-sparing effect.

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.

Transcriptional and proteomic profiling of flatfish (Solea senegalensis) spermatogenesis.

The Senegalese sole (Solea senegalensis) is a marine flatfish of high economic value and a target species for aquaculture. The efforts to reproduce this species in captivity have been hampered by the fact that farmed males (F1) often show lower sperm production and fertilization capacity than wild-type males (F0). Our knowledge on spermatogenesis is however limited to a few studies. In a previous work, we identified by 2-D DIGE several potential protein markers in testis for the poor reproductive performance of F1 males. Therefore, the objectives of the present study were, first, to investigate changes in genes and proteins expressed in the testis throughout spermatogenesis in F0 males by using a combination of transcriptomic and proteomic approaches and, second, to further compare the testis proteome between late spermatogenic stages of F0 and F1 fish to identify potential indicators of hampered reproductive performance in F1 fish. We identified approximately 400 genes and 49 proteins that are differentially expressed during the progression of spermatogenesis and that participate in processes such as transcriptional activation, the ubiquitin-proteasome system, sperm maturation and motility or cytoskeletal remodeling. Interestingly, a number of these proteins differed in abundance between F0 and F1 fish, pointing toward alterations in cytoskeleton, sperm motility, the ubiquitin-proteasome system and the redox state during spermiogenesis as possible causes for the decreased fertility of F1 fish.

Direct involvement of tumor necrosis factor-α in the regulation of glucose uptake in rainbow trout muscle cells.

The proinflammatory cytokine TNF-α is known to have a direct action on skeletal muscle in mammals. However, little is known regarding the potential effects of cytokines on nonimmune tissues, particularly in skeletal muscle, in fish. The aim of this study was to investigate the effects of recombinant trout TNF-α (rtTNF-α) on skeletal muscle carbohydrate metabolism in rainbow trout (Oncorhynchus mykiss). We used a primary cell culture of muscle cells from rainbow trout to show that rtTNF-α stimulates glucose uptake in myoblasts and myotubes at concentrations that do not affect the viability of the cells, requiring de novo protein synthesis as shown by the impairment of rtTNF-α-stimulated glucose uptake by cycloheximide. With the use of specific inhibitors, we show that rtTNF-α-stimulated glucose uptake is mediated by the p38MAPK, NF-κB, and JNK pathways. Additionally, we provide evidence that the stimulatory effects of rtTNF-α on glucose uptake in trout skeletal muscle cells may be caused, at least in part, by an increase in the amount of GLUT4 at the plasma membrane. Incubation of trout muscle cells with conditioned medium from LPS-stimulated trout macrophages, enriched in TNF-α, increased glucose uptake. Our results indicate that recombinant, as well as native trout TNF-α, directly stimulates glucose uptake in trout muscle cells and provide evidence, for the first time in nonmammalian vertebrates, for a potential regulatory role of TNF-α in skeletal muscle metabolism.

Molecular identification of genes involved in testicular steroid synthesis and characterization of the response to gonadotropic stimulation in the Senegalese sole (Solea senegalensis) testis.

In male teleosts, testicular steroids are essential hormones for the regulation of spermatogenesis and their production is regulated by pituitary gonadotropins. In the Senegalese sole (Solea senegalensis), an economically important flatfish with semi-cystic and asynchronous spermatogenesis, the gonadotropic regulation of spermatogenesis, particularly regarding the production and regulation of testicular steroids, are not well understood. For this reason, we first cloned and characterized the response of several key genes for the production and action of testicular steroids to the in vivo administration of human chorionic gonadotropin (hCG) and, second, we investigated the transcriptomic effects of hCG in the Senegalese sole testis. We succeeded in cloning the full-length cDNAs for Steroidogenic Acute Regulatory protein (StAR), 3ß-hydroxysteroid dehydrogenase (3ß-HSD), 17ß-HSD and 20ß-HSD and a partial cDNA for the nuclear progesterone receptor. In this study we also identified a transcript encoding a protein with homology to StAR, which we named StAR-like, that could represent a new member of the StAR-related lipid transfer (START) family. All the cloned genes were expressed in the testis and their expression levels were significantly increased by the in vivo administration of hCG. The plasma levels of testosterone and 11-ketotestosterone also increased in response to hCG administration, likely as a result of the induction of the expression of steroidogenic enzymes by hCG. Furthermore, gene expression analysis by microarray identified 90 differentially expressed genes in the testis in response to hCG administration, including genes potentially involved in steroidogenesis, progression of spermatogenesis and germ cell maturation and cytoskeletal organization. Our results have identified for the first time a number of key genes involved in the regulation of steroid production and spermatogenesis in the Senegalese sole testis that are under gonadotropic control.

Fish under exercise.

Improved knowledge on the swimming physiology of fish and its application to fisheries science and aquaculture (i.e., farming a fitter fish) is currently needed in the face of global environmental changes, high fishing pressures, increased aquaculture production as well as increased concern on fish well-being. Here, we review existing data on teleost fish that indicate that sustained exercise at optimal speeds enhances muscle growth and has consequences for flesh quality. Potential added benefits of sustained exercise may be delay of ovarian development and stimulation of immune status. Exercise could represent a natural, noninvasive, and economical approach to improve growth, flesh quality as well as welfare of aquacultured fish: a FitFish for a healthy consumer. All these issues are important for setting directions for policy decisions and future studies in this area. For this purpose, the FitFish workshop on the Swimming Physiology of Fish ( http://www.ub.edu/fitfish2010 ) was organized to bring together a multidisciplinary group of scientists using exercise models, industrial partners, and policy makers. Sixteen international experts from Europe, North America, and Japan were invited to present their work and view on migration of fishes in their natural environment, beneficial effects of exercise, and applications for sustainable aquaculture. Eighty-eight participants from 19 different countries contributed through a poster session and round table discussion. Eight papers from invited speakers at the workshop have been contributed to this special issue on The Swimming Physiology of Fish.