Subfunctionalization of POMC paralogues in Senegalese sole (Solea senegalensis).

The precursor protein proopiomelanocortin (POMC) gives rise to a variety of biologically active peptides through cell-specific posttranslational processing. Two transcripts of pomc were found in the flatfish Solea senegalensis (ssePOMC-A and ssePOMC-B), that most likely represent subfunctionalized paralogues: ssePOMC-A lacks the N-terminal cleavage site for ß-MSH, whereas ssePOMC-B cannot yield ACTH and completely lacks the opioid consensus sequence in the ß-END region. An analysis of nucleotide substitution rates shows that the POMC-derived peptides possess well-conserved regions under purifying selection, except the ß-END derived from POMC-B, which has undergone positive selection. The calculated K(s) values for ssePOMC-A versus ssePOMC-B and zebrafish POMCαversus zebrafish POMCß are 0.40 and 0.72, respectively, indicating that the zebrafish POMC paralogues started to evolve almost twice as early in evolution, and that the Solea POMC paralogues arose independently from the whole genome duplication event that gave rise to the zebrafish paralogues. This makes ssePOMC-B the first identified POMCα orthologue that lacks the opioid consensus. Furthermore, pomc-a expression is down-regulated in chronic stressed S. senegalensis juveniles, whereas pomc-b expression levels remain unaffected, indicating different physiological roles for both POMC paralogues. The distribution of functional POMC-derived peptide hormones over two pomc genes in S. senegalensis suggests subfunctionalization of the paralogues, a relevant notion when studying POMC function in endocrine responses.

Bone morphogenetic protein 15 and growth differentiation factor 9 expression in the ovary of European sea bass (Dicentrarchus labrax): cellular localization, developmental profiles, and response to unilateral ovariectomy.

Vertebrate oocytes actively contribute to follicle development by secreting a variety of growth factors, among which bone morphogenetic protein 15 (BMP15/Bmp15) and growth differentiation factor 9 (GDF9/Gdf9) have been paid particular attention. In the present study, we describe the cellular localization, the developmental profiles, and the response to unilateral ovariectomy (a procedure implying the surgical removal of one of the ovaries) of protein and mRNA steady-state levels of Bmp15 and Gdf9 in the ovary of European sea bass, an important fish species for marine aquaculture industry. In situ hybridization and immunohistochemistry demonstrated that the oocyte is the main production site of Bmp15 and Gdf9 in European sea bass ovary. During oocyte development, Bmp15 protein expression started to be detected only from the lipid vesicle stage onwards but not in primary pre-vitellogenic (i.e. perinucleolar) oocytes as the bmp15 mRNA already did. Gdf9 protein and gdf9 mRNA expression were both detected in primary perinucleolar oocytes and followed similar decreasing patterns thereafter. Unilateral ovariectomy induced a full compensatory growth of the remaining ovary in the 2-month period following surgery (Á. García-López, M.I. Sánchez-Amaya, C.R. Tyler, F. Prat 2011). The compensatory growth elicited different changes in the expression levels of mRNA and protein of both factors, although the involvement of Bmp15 and Gdf9 in the regulatory network orchestrating such process remains unclear at present. Altogether, our results establish a solid base for further studies focused on elucidating the specific functions of Bmp15 and Gdf9 during primary and secondary oocyte growth in European sea bass.

Chronic and acute stress responses in Senegalese sole (Solea senegalensis): the involvement of cortisol, CRH and CRH-BP.

The hypothalamus-pituitary-interrenal (HPI) axis is pivotal in the endocrine stress response of fish. Hypothalamic corticotropin-releasing hormone (CRH) initiates the endocrine stress response and stimulates the release of adrenocorticotropic hormone (ACTH) from the pituitary pars distalis, which in turn activates cortisol production and release by the interrenal cells of the head kidney. CRH activity depends on the levels of a specific CRH binding protein (CRH-BP). We have characterized the cDNAs coding for CRH and CRH-BP in Senegalese sole (Solea senegalensis) and investigated their mRNA expression in juveniles that were submitted to a protocol that involved exposure to a chronic stressor (viz. increased cultivation densities) followed by an acute stressor (viz. transfer to increased ambient salinity). Juveniles were cultivated at three densities (1.9, 4.7 and 9.8 kg/m(2)) for 33 days, and then exposed to an osmotic challenge that involved transfer from seawater (39‰ salinity, SW) to hypersaline seawater (55‰, HSW). The highest density imposed stress as indicated by elevated cortisol levels and CRH mRNA expression compared to fish stocked at low density. Fish kept at high density differentially responded to a posterior transfer to HSW; no cortisol or CRH response was seen, but osmoregulatory and metabolic parameters were affected. No differences in CRH-BP mRNA expression levels were found at different stocking densities; transfer to HSW enhanced expression in both low and high density stocked animals, suggesting that CRH-BP acts as a modulator of the acute stress response, not so of the chronic stress response. We conclude that stocking of Senegalese sole at high density is a stressful condition that may compromise the capacity to cope with subsequent stressors.

Genomic resources for a commercial flatfish, the Senegalese sole (Solea senegalensis): EST sequencing, oligo microarray design, and development of the Soleamold bioinformatic platform.

BACKGROUND: The Senegalese sole, Solea senegalensis, is a highly prized flatfish of growing commercial interest for aquaculture in Southern Europe. However, despite the industrial production of Senegalese sole being hampered primarily by lack of information on the physiological mechanisms involved in reproduction, growth and immunity, very limited genomic information is available on this species. RESULTS: Sequencing of a S. senegalensis multi-tissue normalized cDNA library, from adult tissues (brain, stomach, intestine, liver, ovary, and testis), larval stages (pre-metamorphosis, metamorphosis), juvenile stages (post-metamorphosis, abnormal fish), and undifferentiated gonads, generated 10,185 expressed sequence tags (ESTs). Clones were sequenced from the 3'-end to identify isoform specific sequences. Assembly of the entire EST collection into contigs gave 5,208 unique sequences of which 1,769 (34%) had matches in GenBank, thus showing a low level of redundancy. The sequence of the 5,208 unigenes was used to design and validate an oligonucleotide microarray representing 5,087 unique Senegalese sole transcripts. Finally, a novel interactive bioinformatic platform, Soleamold, was developed for the Senegalese sole EST collection as well as microarray and ISH data. CONCLUSION: New genomic resources have been developed for S. senegalensis, an economically important fish in aquaculture, which include a collection of expressed genes, an oligonucleotide microarray, and a publicly available bioinformatic platform that can be used to study gene expression in this species. These resources will help elucidate transcriptional regulation in wild and captive Senegalese sole for optimization of its production under intensive culture conditions.

Transcriptomic characterization of the larval stage in gilthead seabream (Sparus aurata) by 454 pyrosequencing.

Gilthead seabream (Sparus aurata) is a teleost belonging to the family Sparidae with a high economical relevance in the Mediterranean countries. Although genomic tools have been developed in this species in order to investigate its physiology at the molecular level and consequently its culture, genomic information on post-embryonic development is still scarce. In this study, we have investigated the transcriptome of a marine teleost during the larval stage (from hatching to 60 days after hatching) by the use of 454 pyrosequencing technology. We obtained a total of 68,289 assembled contigs, representing putative transcripts, belonging to 54,606 different clusters. Comparison against all S. aurata expressed sequenced tags (ESTs) from the NCBI database revealed that up to 34,722 contigs, belonging to about 61% of gene clusters, are sequences previously not described. Contigs were annotated through an iterative Blast pipeline by comparison against databases such as NCBI RefSeq from Danio rerio, SwissProt or NCBI teleost ESTs. Our results indicate that we have enriched the number of annotated sequences for this species by more than 50% compared with previously existing databases for the gilthead seabream. Gene Ontology analysis of these novel sequences revealed that there is a statistically significant number of transcripts with key roles in larval development, differentiation, morphology, and growth. Finally, all information has been made available online through user-friendly interfaces such as GBrowse and a Blast server with a graphical frontend.

Molecular cloning of Senegalese sole (Solea senegalensis) follicle-stimulating hormone and luteinizing hormone subunits and expression pattern during spermatogenesis.

Pituitary gonadotropins (GTHs), follicle-stimulating hormone (FSH) and luteinizing hormone (LH), are key regulators of vertebrate reproduction. However, in teleosts with testis of semi-cystic type and asynchronous spermatogenesis, as the flatfish Senegalese sole (Solea senegalensis), the physiological roles of FSH and LH are still not well understood. To gain insight into this mechanism, full-length complementary DNAs (cDNAs) encoding Senegalese sole FSH beta and LH beta subunits, and the common glycoprotein alpha subunit (CG alpha), were cloned and sequenced. The three cDNAs consisted of 550, 582 and 744 nucleotides encoding peptides of 120, 148 and 132 amino acids, respectively. Comparison of the deduced amino acid sequences of sole FSH beta, LH beta and CG alpha with those from other teleosts indicated that cysteine residues and potential N-linked glycosylation sites were fully conserved with respect to other percomorphs and salmonids. However, the primary structure of FSH beta and LH beta in pleuronectiforms appeared to be highly divergent. In situ hybridization of mature male pituitaries showed that fshb, lhb and cga mRNAs were localized in the proximal pars distalis and in the periphery of pars intermedia. Real-time quantitative reverse transcription-polymerase chain reaction indicated that the levels of all three transcripts in the pituitary of males increased during winter and spring, at the time when plasma levels of androgens raised and testicular germ cell development and spermatozoa production were stimulated. These results suggest that FSH and LH may regulate spermatogenesis in Senegalese sole similarly to that described for other teleosts with testis of cystic type and synchronous germ cell development.