Sole Metamorphosis Research Articles

Flatfish metamorphosis: A hypothalamic independent process?

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

Differential effects of transient constant light-dark conditions on daily rhythms of Period and Clock transcripts during Senegalese sole metamorphosis

Studies on the developmental onset of the teleost circadian clock have been carried out in zebrafish and, recently, in rainbow trout and Senegalese sole, where rhythms of clock gene expression entrained by light-dark (LD) cycles have been reported from the first days post fertilization. However, investigations of molecular clock rhythms during crucial developmental phases such as metamorphosis are absent in vertebrates. In this study, we documented the daily expression profile of Per1, Per2, Per3, and Clock during Senegalese sole pre-, early-, middle-, and post-metamorphic stages under LD 14:10 cycles (LD group), as well as under transient exposure to constant light (LL-LD group) or constant dark (DD-LD group) conditions. Our results revealed that robust rhythms of clock genes were maintained along the metamorphic process, although with declining amplitudes and expression levels. All daily profiles were affected by transient constant conditions, in particular Per1, Per3, and Clock amplitudes and Per2 acrophase. Rhythm parameters were progressively restored upon reversion to LD cycles but even after 9 d under cycling conditions, a prolonged effect on clock function was observed, especially in the LL-LD group. These results reflect the differential sensitivity of clock machinery of sole to transitory light cues, being Per1 and Per3 predominantly clock regulated and supporting the role of Per2 as part of the light input pathway. Interestingly, there is no reversal in the phase of clock gene rhythms between pre- and post-metamorphic animals that would be coincident with the switch from diurnal to nocturnal locomotor activity, which occurs in this species just before the beginning of this process. Whether specialized central pacemakers dictate the phase of locomotor activity or this control is exerted outside of the core clock mechanism remains to be elucidated. Our results emphasize the importance of maintaining cycling light-dark conditions in aquaculture practices during ontogeny of Senegalese sole. (Author correspondence: munoz.cueto@uca.es or carlos.pendon@uca.es)

Exploring the larval transcriptome of the common sole (Solea solea L.)

BackgroundThe common sole (Solea solea) is a promising candidate for European aquaculture; however, the limited knowledge of the physiological mechanisms underlying larval development in this species has hampered the establishment of successful flatfish aquaculture. Although the fact that genomic tools and resources are available for some flatfish species, common sole genomics remains a mostly unexplored field. Here, we report, for the first time, the sequencing and characterisation of the transcriptome of S. solea and its application for the study of molecular mechanisms underlying physiological and morphological changes during larval-to-juvenile transition.ResultsThe S. solea transcriptome was generated from whole larvae and adult tissues using the Roche 454 platform. The assembly process produced a set of 22,223 Isotigs with an average size of 726 nt, 29 contigs and a total of 203,692 singletons. Of the assembled sequences, 75.2% were annotated with at least one known transcript/protein; these transcripts were then used to develop a custom oligo-DNA microarray. A total of 14,674 oligonucleotide probes (60 nt), representing 12,836 transcripts, were in situ synthesised onto the array using Agilent non-contact ink-jet technology. The microarray platform was used to investigate the gene expression profiles of sole larvae from hatching to the juvenile form. Genes involved in the ontogenesis of the visual system are up-regulated during the early stages of larval development, while muscle development and anaerobic energy pathways increase in expression over time. The gene expression profiles of key transcripts of the thyroid hormones (TH) cascade and the temporal regulation of the GH/IGF1 (growth hormone/insulin-like growth factor I) system suggest a pivotal role of these pathways in fish growth and initiation of metamorphosis. Pre-metamorphic larvae display a distinctive transcriptomic landscape compared to previous and later stages. Our findings highlighted the up-regulation of gene pathways involved in the development of the gastrointestinal system as well as biological processes related to folic acid and retinol metabolism. Additional evidence led to the formation of the hypothesis that molecular mechanisms of cell motility and ECM adhesion may play a role in tissue rearrangement during common sole metamorphosis.ConclusionsNext-generation sequencing provided a good representation of the sole transcriptome, and the combination of different approaches led to the annotation of a high number of transcripts. The construction of a microarray platform for the characterisation of the larval sole transcriptome permitted the definition of the main processes involved in organogenesis and larval growth.

Cloning, tissue and ontogenetic expression of the taurine transporter in the flatfish Senegalese sole (Solea senegalensis)

Flatfish species seem to require dietary taurine for normal growth and development. Although dietary taurine supplementation has been recommended for flatfish, little is known about the mechanisms of taurine absorption in the digestive tract of flatfish throughout ontogeny. This study described the cloning and ontogenetic expression of the taurine transporter (TauT) in the flatfish Senegalese sole (Solea senegalensis). Results showed a high similarity between TauT in Senegalese sole and other vertebrates, but a change in TauT amino acid sequences indicates that taurine transport may differ between mammals and fish, reptiles or birds. Moreover, results showed that Senegalese sole metamorphosis is an important developmental trigger to promote taurine transport in larvae, especially in muscle tissues, which may be important for larval growth. Results also indicated that the capacity to uptake dietary taurine in the digestive tract is already established in larvae at the onset of metamorphosis. In Senegalese sole juveniles, TauT expression was highest in brain, heart and eye. These are organs where taurine is usually found in high concentrations and is believed to play important biological roles. In the digestive tract of juveniles, TauT was more expressed in stomach and hindgut, indicating that dietary taurine is quickly absorbed when digestion begins and taurine endogenously used for bile salt conjugation may be recycled at the posterior end of the digestive tract. Therefore, these results suggest an enterohepatic recycling pathway for taurine in Senegalese sole, a process that may be important for maintenance of the taurine body levels in flatfish species.Electronic supplementary materialThe online version of this article (doi:10.1007/s00726-010-0826-6) contains supplementary material, which is available to authorized users.

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

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

Iodothyronine deiodinases and thyroid hormone receptors regulation during flatfish (Solea senegalensis) metamorphosis

Thyroid hormone-induced metamorphosis seems to represent an ancestral feature of chrordates (urochordates, cephalochordates and vertebrates), but also of nonchordate animals. Although thyroid hormones and thyroid hormone receptor profiles during metamorphosis have been analyzed in different vertebrate taxa, including fish, developmental expression and activity of type 2 (dio2, D2) and type 3 (dio3, D3) iodothyronine deiodinases, two key enzymes in anuran metamorphosis, remain unknown in any fish species. The aim of this work was to investigate the development of thyroid hormone system during the metamorphosis of a flatfish species, the Senegalese sole, focusing on the deiodinases developmental profile. We have cloned sole D2 and D3 and analyzed several parameters of thyroid hormones system in pre-, early-, middle-, and late-metamorphic larvae. Both deiodinases contain in their catalytic centers an UGA triplet encoding for a selenocystein (Sec) residue as expected. Left eye migration and rotation in body position were associated with a significant increase in both thyroid hormones and thyroid hormone receptors at the middle-late metamorphic stages. Although dio2 expression slightly increased during metamorphosis, D2 activity augmentation was much more significant. Sole dio3 expression declined only slightly, whereas the D3 activity clearly decreased at mid-late metamorphic period. This developmental profile of deiodinases sustained the rise of thyroid hormones levels observed during sole metamorphosis. No clear cut daily rhythms were observed in the parameters analyzed although it seemed that thyroid hormone system was more active during daytime, in particular at late metamorphic stages. These developmental changes point out the importance not only of thyroid hormones and their receptors but also of dio2 and dio3 in mediating flatfish metamorphosis, as it has been described in amphibians.

Molecular characterization, gene expression and transcriptional regulation of thyroid hormone receptors in Senegalese sole

Thyroid hormones (THs) play a key role in larval development, growth and metamorphosis in flatfish. Their genomic effects are mediated by thyroid hormone receptors (TRs). In this study, cDNAs encoding for TRαA, TRαB, and TRβ have been sequenced in Senegalese sole ( Solea senegalensis). Main domains and conserved motifs were identified. Also, a truncated TRαB isoform (referred to as TRαBtr) and a spliced TRβ variant (referred to as TRβv) were detected. A phylogenetic analysis grouped both TRα and TRβ genes into two separate clusters with their fish and mammalian counterparts. Expression profiles during larval development and in juvenile tissues were analyzed using a real-time PCR approach. In juvenile fish, TRαA, TRαB, TRβv, and TRβ showed distinct transcript levels in tissues. During metamorphosis, only TRβv and TRβ modified their mRNA levels in a similar way to the T4 contents. To evaluate the possible regulation of TRs by their cognate ligand T4 during sole metamorphosis, larvae were exposed to the goitrogen thiourea (TU). TRβ transcripts decreased significantly at 11 and 15 days after treatment. Moreover, adding exogenous T4 hormone to TU-treated larvae restored the steady-state levels or even increased TRβ and TRβv mRNAs with respect to the untreated control. Overall, these results demonstrate that TRβ transcription is up-regulated by THs playing a major role during metamorphosis in Senegalese sole.

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

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

Molecular characterization and expression analysis of five different elongation factor 1 alpha genes in the flatfish Senegalese sole (Solea senegalensis Kaup): Differential gene expression and thyroid hormones dependence during metamorphosis

BackgroundEukaryotic elongation factor 1 alpha (eEF1A) is one of the four subunits composing eukaryotic translation elongation factor 1. It catalyzes the binding of aminoacyl-tRNA to the A-site of the ribosome in a GTP-dependent manner during protein synthesis, although it also seems to play a role in other non-translational processes. Currently, little information is still available about its expression profile and regulation during flatfish metamorphosis. With regard to this, Senegalese sole (Solea senegalensis) is a commercially important flatfish in which eEF1A gene remains to be characterized.ResultsThe development of large-scale genomics of Senegalese sole has facilitated the identification of five different eEF1A genes, referred to as SseEF1A1, SseEF1A2, SseEF1A3, SseEF1A4, and Sse42Sp50. Main characteristics and sequence identities with other fish and mammalian eEF1As are described. Phylogenetic and tissue expression analyses allowed for the identification of SseEF1A1 and SseEF1A2 as the Senegalese sole counterparts of mammalian eEF1A1 and eEF1A2, respectively, and of Sse42Sp50 as the ortholog of Xenopus laevis and teleost 42Sp50 gene. The other two elongation factors, SseEF1A3 and SseEF1A4, represent novel genes that are mainly expressed in gills and skin. The expression profile of the five genes was also studied during larval development, revealing different behaviours. To study the possible regulation of SseEF1A gene expressions by thyroid hormones (THs), larvae were exposed to the goitrogen thiourea (TU). TU-treated larvae exhibited lower SseEF1A4 mRNA levels than untreated controls at both 11 and 15 days after treatment, whereas transcripts of the other four genes remained relatively unchanged. Moreover, addition of exogenous T4 hormone to TU-treated larvae increased significantly the steady-state levels of SseEF1A4 with respect to untreated controls, demonstrating that its expression is up-regulated by THs.ConclusionWe have identified five different eEF1A genes in the Senegalese sole, referred to as SseEF1A1, SseEF1A2, SseEF1A3, SseEF1A4, and Sse42Sp50. The five genes exhibit different expression patterns in tissues and during larval development. TU and T4 treatments demonstrate that SseEF1A4 is up-regulated by THs, suggesting a role in the translational regulation of the factors involved in the dramatic changes that occurs during Senegalese sole metamorphosis.

Culture of Senegal Sole Larvae without the Need for Rotifers

Two groups of Senegal sole (Solea senegalensis) larvae were cultured. One used rotifers for the first 10 days after hatching (dph) and enriched Artemia metanauplii from 6 to 30 dph and the other without rotifers, using enriched Artemia metanauplii as the sole food source. The quantity of metanauplii used was the same (group A), twice (group A2) and three times (group A3) the quantity of live prey (in dry weight) of the group fed with rotifers (group R). At the end of the experiment, the growth, in terms of total length and individual dry weight was significantly higher for the larvae on group A2 whilst rotifer fed larvae (R) showed the poorest results. Eye migration was also delayed by approximately 2 days in the group R. No significant differences were found in survival rates. The influence of diet on Senegal sole metamorphosis and its relationship with the size rather than the age of the fish are discussed.

Nutritional condition of metamorphosing sole: spatial and temporal analyses

Sole Solea solea larvae were collected during five cruises in the Bay of Biscay in 1992 and 1993. The larval growth rate, triacylgycerol (TAG) and sterol (ST) content, and the width of the marginal otolith increments were determined on the same individual larva as indicators of nutritional condition. The TAG content increased during the ontogenetic larval development up to an elevated value corresponding to the end of metamorphosis. During metamorphosis, sole larvae continued to accumulate TAG and to increase in length. Seasonal and yearly variations of growth and nutritional condition of sole larvae at the onset of metamorphosis were more important than spatial variations. Analysis of larval growth showed that, during each cruise, the faster growing larvae were generally those with the highest TAG content (and TAG : ST ratio). In 1993, larvae began to metamorphose earlier and their growth (both larval growth and recent growth) were significantly higher than in 1992. Their relatively low nutritional condition, however, seemed to be the result of a low accumulation of TAG. Sole metamorphosis occurs without growth arrest and the majority of the larvae during this phase are judged to be in good nutritional condition (TAG : ST ratio > 1). In the Bay of Biscay, metamorphosis does not seem to be a critical period in the life cycle of the sole. It is suggested that larvae originating from different cohorts probably have the same chance of survival during or just after metamorphosis when they enter the benthic environment.