Search

The hedgehog signaling pathway plays an important role in early development and growth of most vertebrates. Sonic hedgehog (shh) gene is a critical regulator of embryonic development in many species, including humans. However, it is not clear what roles shh can play in the development of fish. In this paper, shh gene was cloned from Pseudopleuronectes yokohamae. The full-length complementary DNA (cDNA) of P. yokohamae sonic hedgehog gene (Pyshh) comprises 3194 bp, with a 1317-bp open reading frame (ORF) that encodes a polypeptide of 438 amino acids with a typical HH-signal domain and Hint-N domain. The conserved sequences of the protein among species were predicted by using multiple sequence comparison. The phylogenetic tree construction showed that PySHH is clustered in a branch of Pleuronectidae. To explore the expression of Pyshh gene in various tissues of P. yokohamae, we used real-time fluorescence quantitative PCR technology to detect it. The results showed that Pyshh gene is widely distributed in various tissues of P. yokohamae juveniles, different tissues of adult males and females, and is particularly expressed in immune organs. The Pyshh gene expression was higher in the muscle and brain of juvenile fish, and higher in bone, gill, and skin of male fish than that of female fish, suggesting that Pyshh might be involved in the formation of immune organs of P. yokohamae. The expression of Pyshh gene significantly upregulated from the gastrula stage to the hatching stage. Western blotting of the expression levels of PySHH during different embryonic development stages revealed that PySHH levels increased gradually during development stages from oosperm stage to hatching stage. These results indicate that Pyshh is highly conserved among species and plays a critical role in the complex process of embryonic development. Its precise regulation is essential for the proper formation of many organs and tissues in the body, and disruptions in its function may have serious consequences for the formation of immune organs in fish., (© 2024 Fisheries Society of the British Isles.)

Senegalese sole, Solea senegalensis, is a flatfish of high commercial value in the world. It has been identified as an interesting and promising species for marine commercial aquaculture diversification in Europe for at least four decades and was introduced to China in 2003. Early ontogenesis from embryo to juvenile stages in S. senegalensis was analysed under controlled laboratory conditions to provide morphological information for aquaculture. From 0 to 59 days post hatching (dph), 10-20 larvae were sampled and measured each day (0-17 dph) or every 2-6 days (17-59 dph). Morphological characteristics from the egg to the juvenile stage were described. The eggs were separate and spherical with multiple oil globules. After 3 dph, the yolk sac was completely absorbed, mouth and anus were open, a swim bladder appeared, and larvae began feeding on rotifers (Brachionus plicatilis). The larvae began metamorphosis as the notochord flexed upward and the left eye migrated upward after 10 dph. The left eye migrated to the dorsal midline at 15 dph. At 19 dph, the left eye was translocated to the right-ocular side, and the juveniles adopted a benthic lifestyle. The swim bladder degenerated, and the juveniles completed metamorphosis at 23 dph. The growth patterns of some parameters (TL, SL, BH, BW) during larval and juvenile development stages were identified. The inflection points, which are slopes of growth changes, were calculated in growth curves. Three inflection points occurring in the growth curves of larvae and juveniles were found to be associated with metamorphosis, weaning, and transitions in feeding habits. The basic information of embryo development and ontogenesis in this study represents a valuable contribution to the S. senegalensis industry, especially in artificial breeding and rearing techniques., (© 2024 Fisheries Society of the British Isles.)

Desert hedgehog (dhh) is a gene that is crucial for spermatogenesis and Leydig cell differentiation, but little is known regarding its influence on gonadal differentiation and development in fish. To understand its function, we cloned and characterized the dhh gene from Cynoglossus semilaevis (csdhh). The full length csdhh cDNA was 2473 bp, including a 1386 bp open reading frame (ORF), a 475 bp 5'-UTR, and a 612 bp 3'-UTR, encoding a predicted protein of 461 amino acid residues. Phylogenetic analysis showed that the putative protein belongs to the hedgehog (HH) family, and contains typical HH-N and HH-C domains. Amino acid sequence analysis revealed that CsDhh shares many features with Dhh analogues in other teleost species. Real-time quantitative PCR showed that csdhh was detected in eight different tissues in male and female tongue sole. During early embryonic development, the relative expression of the csdhh was significantly higher in the neural stage than in other embryonic developmental stages (P < 0.05). csdhh was detected at 20 days after hatching (dah) and at the critical period of male gonadal differentiation (80-95 dah), the relative expression of the csdhh was significantly higher in the male gonads than the female gonads. In 5, 8, and 12 month old gonads, the relative expression of the csdhh was significantly higher in male and pseudo-male than in female fish. The in situ hybridization (ISH) results showed that the hybridization signal was strongly expressed in primary and secondary spermatocytes, spermatids, and sertoli cells of the 1-year-old fish testis, with only weak signal expression in the corresponding ovarian tissue. These results suggest that csdhh is highly conserved in evolution and plays an important role in spermatogenesis in males and pseudo-males., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Most teleosts undergo a thyroid hormone (TH) regulated larval to juvenile transition known as metamorphosis. In Pleuronectiformes (flatfish), metamorphosis is most dramatic, and one eye of the symmetric pelagic larvae migrates to the opposite side of the head, giving rise to an asymmetric benthic juvenile with both eyes on the same side of the body. Asymmetric development occurs mostly in the head. To understand the genetic mechanisms underlying this developmental change we have generated a Solea senegalensis metamorphosing flatfish head transcriptome. Our results reveal that THs acting as integrative factors direct a stepwise genetic program that initiates a specific organismal level response followed by cell specific responses that lead to the long-term changes that characterise the post-metamorphic identity and physiology of the head. Flatfish head asymmetric development during metamorphosis and its TH dependency is conserved thus we consider the findings in sole most likely representative of all flatfish species., (Copyright © 2019. Published by Elsevier Inc.)

Harmful effects of triclosan (TCS) have been reported on several organisms; however, effects on early life stages of marine vertebrates are limited. Therefore, the objective of this work was to assess the effects of TCS during early development of the flatfish Solea senegalensis after initial characterization of cholinesterases (ChEs) and determination of selected biochemical markers baseline levels. Characterization of ChEs and determination of biochemical markers baseline levels of cholinergic activity, energy metabolism and oxidative stress were analysed in sole at 3 days after hatching (dah) and at the onset and end of metamorphosis. To assess TCS effects, fish were exposed during 96h to 30-500 μg L -1 TCS until 3 dah. Fish at 13 dah were exposed during 48h to 200-1,500 μg L -1 TCS and maintained until complete metamorphosis. Effects on survival, malformations, length, metamorphosis progression and biochemical markers were evaluated. The main ChE active form present in sole early life stages is acetylcholinesterase and baseline levels of oxidative stress and energy metabolism biomarkers changed according to fish developmental stage. Triclosan induced malformations (EC 50  = 180 μg L -1 at 3 dah), decreased growth (95 μg L -1 at 3 dah; 548 μg L -1 at 24 dah) and affected metamorphosis progression (391 μg L -1 at 17 dah). Impairment of antioxidant system was observed, with TCS affecting catalase at the end of metamorphosis test, however, no oxidative damage on lipids was detected. Glutathione S-transferase was the most sensitive endpoint during early larval test (LOEC = 30 μg L -1 ). Exposure to TCS affected S. senegalensis at individual and sub-individual levels, both at early larval stage and during the critical period of metamorphosis., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The receptor responsible for maternofetal transmission of immunoglobulin (Igs) in the teleosts is not clear. Polymeric immunoglobulin receptor (pIgR) specifically binds with IgA and IgM and mediates the transcytosis of intracellular polymeric immunoglobulins (pIgs) at the mucosal surface to protect against pathogens. Hence there is a possibility that it may be involved in the transmission of maternal Igs. The aim of the present study was to detect the expression and localization of pIgR during embryonal development in turbot (Scophthalmus maximus). pIgR gene was first cloned from eggs and embryos of turbot with or without parent immunization. The expression and distribution of pIgR in unfertilized egg and in embryos ranging from day 1 to day 5 after fertilization were analyzed using reverse transcriptase quantitative polymerase chain reaction and in situ hybridization. pIgR gene was detected in all eggs and embryos at different stages of development, with the highest level detected on the 5th day. pIgR mRNA was observed to be first located in the whole blastoderm and enveloped the yolk sac. Later, it was located around entoderm including primary digestive tract and pronephric tubule tract, and finally it was located at the joint of abdomen and vitelline membrane. Then, Eukaryotic expression plasmid carrying pIgR gene was constructed and transfected into HEK293T cells. Results showed mature pIgR protein located on the cellular membrane, and could bound IgM in vitro. Our findings provide information for studying the involvement of pIgR in maternal Igs transportation in turbot., (Copyright © 2019. Published by Elsevier Ltd.)

R-spondin2 (Rspo2) is a member of the R-spondin family, which plays important roles in cell proliferation, cell fate determination and organogenesis. Rspo2 exhibits important functions during embryonic development and muscle maintenance in adult human, mouse and Xenopus. In the present study, the tongue sole Cynoglossus semilaevis Rspo2 (CsRspo2) gene was isolated and characterized, and its role in muscle development during embryogenesis was studied. Our results showed that CsRspo2 expression was abundant during gastrulation and significantly high during somite formation, but then decreased markedly after hatching. CsRspo2 expression was high in brain and gill, moderate in heart, ovary and testis, and almost undetectable in muscle and other tissues. Moreover, the potential involvement of Rspo2 in muscle development was investigated. We found that overexpression of CsRspo2 mRNA in zebrafish embryos resulted in slow development and abnormal muscle formation at the embryonic stage. Our work provides a fundamental understanding of the structure and potential functions of CsRspo2 during muscle development.

The apolipoprotein E (ApoE) is a key component of several lipoproteins involved in lipid homeostasis. In this study, two cDNA sequences encoding ApoE (referred to as apoEa and apoEb) were characterized in the flatfish Solea senegalensis. The predicted peptides contained conserved structural blocks related with their capacity for lipid binding and lipoprotein receptor interaction. At genomic level, both genes contained five exons and four introns and they were organized into two tandem arrays with apoA-IV gene copies. The phylogenetic analysis clearly separated them into two well-supported clusters that matched with their organization in the genome of teleosts. Whole-mount in situ hybridization located the apoEa signal in the yolk syncytial layer (YSL) of lecitothrophic larval stages (0dph) and in the anterior intestine of exotrophic larvae and benthic fish. In the case of apoEb, hybridization signals were located in the YSL, tail bud, eyes and mouth at 0dph and in the otic vesicle, hindbrain, eyes, pharynx, mouth, heart and intestine at 1dph. In exotrophic larvae, apoEb was ubiquitously expressed in several tissues such as taste buds, brain, mouth, nostril, gills, intestine, liver and around the neuromasts and eyes. Quantification of mRNA levels in pools of whole larvae confirmed distinct expression patterns with a significant reduction of apoEa and an increase of apoEb mRNA levels throughout larval development. Moreover, only apoEa transcripts increased in response to food supply suggesting that this paralog mostly participates in the absorption and transport of dietary lipids and the apoEb in the redistribution of endogenous lipids as well as in neural tissue regeneration., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The objective of the study was to clarify the effects of initiation time on chromosome set doubling induced by hydrostatic pressure shock through nuclear phase fluorescent microscopy in turbot Scophthalmus maximus. The ratio of developmentally delayed embryo and chromosome counting was used to assess induction efficiency. For the embryos subjected to a pressure of 67.5 MPa for 6 min at prometaphase (A group), chromosomes recovered to the pre-treatment condition after 11-min recovering. The first nuclear division and cytokinesis proceeded normally. During the second cell cycle, chromosomes did not enter into metaphase after prometaphase, but spread around for about 13 min, then assembled together and formed a large nucleus without anaphase separation; the second nuclear division and cytokinesis was inhibited. The ratio of developmentally delayed embryo showed that the second mitosis of 78% A group embryo was inhibited. The result of chromosome counting showed that the tetraploidization rate of A group was 72%. For the embryos subjected to a pressure of 67.5 MPa for 6 min at anaphase (B group), chromosomes recovered to the pre-treatment condition after about 31-min recovering. Afterwards, one telophase nucleus formed without anaphase separation; the first nuclear division was inhibited. The time of the first cleavage furrow occurrence of B group embryos delayed 27 min compared with that of A group embryos. With the first cytokinesis proceeding normally, 81.3% B group embryos were at two-cell stage around the middle of the second cell cycle after treatment. Those embryos were one of the two blastomeres containing DNA and the other without DNA. The first nuclear division of those embryos was inhibited. During the third cell cycle after treatment, 65.2% of those abovementioned embryos were at four-cell stage, cytokinesis occurred in both blastomeres, and nuclear division only occurred in the blastomere containing DNA. Of those abovementioned embryos, 14.0% were at three-cell stage and cytokinesis only occurred in the blastomere containing DNA. The result of chromosome counting showed that the tetraploidization rate of B group was only 7%. To summarize what had been mentioned above, mechanisms on chromosome set doubling of tetraploid induction would be different with different initiation time of hydrostatic pressure treatment. Chromosome set doubling was mainly due to inhibition of the second mitosis when hydrostatic pressure treatment was performed at prometaphase. Otherwise, chromosome set doubling was mainly due to inhibition of the first nuclear division when hydrostatic pressure treatment was performed at anaphase. Induction efficiency of tetraploidization resulted from inhibition of the second cleavage was higher than which resulted from inhibition of the first nuclear division. This study was the first to reveal biological mechanisms on the two viewpoints of chromosome set doubling through effect of initiation time of hydrostatic pressure treatment on chromosome set doubling in tetraploid induction.

Background: The identification of DNA methyltransferases (Dnmt) expression patterns during development and their regulation is important to understand the epigenetic mechanisms that modulate larval plasticity in marine fish. In this study, dnmt1 and dnmt3 paralogs were identified in the flatfish Solea senegalensis and expression patterns in early developmental stages and juveniles were determined. Additionally, the regulation of Dnmt transcription by a specific inhibitor (5-aza-2'-deoxycytidine) and temperature was evaluated., Results: Five paralog genes of dnmt3, namely dnmt3aa, dnmt3ab, dnmt3ba, dnmt3bb.1 and dnmt3bb.2 and one gene for dnmt1 were identified. Phylogenetic analysis revealed that the dnmt gene family was highly conserved in teleosts and three fish-specific genes, dnmt3aa, dnmt3ba and dnmt3bb.2 have evolved. The spatio-temporal expression patterns of four dnmts (dnmt1, dnmt3aa, dnmt3ab and dnmt3bb.1) were different in early larval stages although all of them reduced expression with the age and were detected in neural organs and dnmt3aa appeared specific to somites. In juveniles, the four dnmt genes were expressed in brain and hematopoietic tissues such as kidney, spleen and gills. Treatment of sole embryos with 5-aza-2'-deoxycytidine down-regulated dntm1 and up-regulated dntm3aa. Moreover, in lecithotrophic larval stages, dnmt3aa and dnmt3ab were temperature sensitive and their expression was higher in larvae incubated at 16 °C relative to 20 °C., Conclusion: Five dnmt3 and one dnmt1 paralog were identified in sole and their distinct developmental and tissue-specific expression patterns indicate that they may have different roles during development. The inhibitor 5-aza-2'-deoxycytidine modified the transcript abundance of dntm1 and dntm3aa in embryos, which suggests that a regulatory feedback mechanism exists for these genes. The impact of thermal regime on expression levels of dnmt3aa and dnmt3ab in lecithotrophic larval stages suggests that these paralogs might be involved in thermal programing.

Bone morphogenetic proteins (BMPs) play crucial roles in vertebrate developmental process and are associated with the mechanisms which drive early skeletal development. As a first approach to elucidating the role of BMPs in regulating fish bone formation and growth, we describe the cloning, expression profiling and promoter functional analysis of bmp6 and bmp7 in tongue sole (Cynoglossus semilaevis). The full length of bmp6 and bmp7 cDNA sequences is 1939 and 1836 bp, which encodes a protein of 428 and 427 amino acids, respectively. Tissue expression distribution of bmp6 and bmp7 was examined in 14 tissues of mature individuals by quantitative real-time PCR (qRT-PCR). The results revealed that bmp6 was predominantly expressed in the gonad, and bmp7 exhibited the highest expression level in the dorsal fin. Further comparison of bmp6 expression levels between female and male gonads showed that the expression in the ovary was significantly higher than in the testis. Moreover, bmp6 and bmp7 expression levels were detected at 15 sampling time points of early developmental stages (egg, larva, juvenile and fingerling stages). The highest expression level of bmp6 was observed in the egg stage (multi-cell and gastrula stage); while bmp7 exhibited the highest expression in the larva stage (1-4 days old). The high expression levels of BMP6 in the ovary as well as at early embryonic stages indicated that the maternally stored transcripts of bmp6 might play a role in early embryonic development. Whole-mount in situ hybridization showed that bmp6 and bmp7 exhibited similar spatial expression patterns. Both bmp6 and bmp7 signals were first detected in the head and anterior regions in newly hatched larvae, and then, the mRNAs appeared in the crown-like larval fin, jaw, operculum and fins (pectoral, dorsal, pelvic and anal) along with early development. Subsequently, we characterized the 5'-flanking regions of bmp6 and bmp7 by testing the promoter activity by luciferase reporter assays. Positive regulatory regions were, respectively, detected at the location of -272 to +28 and -740 to -396 in bmp6 and bmp7 gene. The predicted transcription factor binding sites (CREB, AP1 and methyl-CpG-binding protein) in the regions might participate in the transcriptional regulation of these two genes.

GATA-binding protein 6 (GATA6), a transcription factor of the GATA family, plays an important role in gonadal cell proliferation, differentiation, and endoderm development. In this study, the full-length coding sequence of tongue sole (Cynoglossus semilaevis) GATA6 was identified. The sequence consisted of 1494 nucleotides encoding a peptide of 497 amino acids, which included two conserved zinc finger domains. Phylogenetic, gene structure, and synteny analysis showed that C. semilaevis GATA6 was homologous to teleost and tetrapod GATA6. C. semilaevis GATA6 mRNA exhibited high expression in heart, intestine, liver, kidney, and gonad. Embryonic development expression profiles revealed that GATA6 is involved in morphogenesis because its expression increased at the blastula stage. The in situ hybridization results showed strong GATA6 signals in spermatogonia, spermatocytes, and Sertoli cells of the testis. The signals were also detected in the oogonia and oocytes of the ovary. The expression of C. semilaevis GATA6 was sexually dimorphic, and the methylation pattern in the promoter region varied among males, females, and pseudomales. These results suggested that GATA6 might influence the gonad development and reproduction of C. semilaevis. This study provides the groundwork for further development of breeding techniques in C. semilaevis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The GATA family of transcription factors is characterized by two zinc finger domains and is involved in different cellular processes. GATA4 is a highly conserved transcription factor that regulates embryonic morphogenesis and cellular differentiation. GATA4 in vertebrates regulates its target genes to influence genital ridge differentiation. In this study, the GATA4 from tongue sole (Cynoglossus semilaevis) was characterized to understand the function of this transcription factor in sex differentiation. The full-length cDNA of C. semilaevis GATA4 comprised 2031bp, encoding a predicted polypeptide consisting of 402 amino acids with two conserved zinc finger domains. Phylogenetic, gene structure, and synteny analyses showed that C. semilaevis GATA4 was homologous to tetrapod GATA4. The mRNA transcript of C. semilaevis GATA4 exhibited high expression in the heart, liver, and gonad. GATA4 expression is dimorphic in the male and female gonads. Embryonic development expression profiles revealed the possible involvement of C. semilaevis GATA4 in morphogenesis. In situ hybridization results showed strong GATA4 signals in the spermatogonia and spermatocytes of the testis and in the oogonia, primary oocytes, and secondary oocytes of the ovary. The expression of C. semilaevis GATA4 in the male, pseudomale, and female gonads showed significantly different methylation levels of the two CpG sites (-2738 and -2647) among the three genders. Basing on these results, we speculated that GATA4 plays a potential role in sex differentiation. This study lays the groundwork for further sex control breeding techniques in C. semilaevis., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Trypsin is an important serine protease that is considered to be involved in digestion of protein in teleost fish. Nevertheless, studies on trypsin/trypsinogen in fish embryos are very limited. In this study, the trypsinogen of turbot (Scophthalmus maximus) (tTG) was identified and the expression patterns and activity of trypsinogen/trypsin were investigated. The results showed that the tTG mRNA was evenly distributed in the oocytes and was also expressed along the yolk periphery in early embryos. At later embryo stages and 1 days after hatching (dph), the tTG mRNA concentrated at the alimentary tract and head. Quantitative expression analysis showed that the tTG transcripts decreased after fertilization until the gastrula stage, then increased with the embryo and larvae development. This result was also confirmed by the specific activity analysis of trypsin and in-situ-hybridization (ISH). All of the results indicated that tTG in early embryo stages was maternally derived and expressed by itself after gastrula stages. Additionally, location of tTG mRNA in embryos and larvae was investigated; we considered that trypsin may have multiple functions during the embryo development process. Based on our results regarding trypsinogen in embryos and early development, we concluded that the trypsin/trypsinogen in turbot embryos was inherited from a maternal source and we suggested that trypsin in early development has multiple functions in the process of development., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The ontogenesis of the saccus vasculosus (SV) of turbot Scophthalmus maximus is described using histological and immunohistochemical methods to assess the general morphology, as well as the distribution of proliferative cells and several calcium-binding proteins (CaBP). The results reveal that the SV begins to differentiate on hatching, when immature coronet cells are morphologically distinguishable. Further morphogenesis involves the formation of a tubular avascular SV, which remains until premetamorphic larval stages. Folding and vascularization of the SV occurs mostly during metamorphosis, when S. maximus settle down on the bottom. Proliferative cells were placed within the SV itself and in the neighbouring infundibular hypothalamus. Their putative relationship with the growth of the SV is discussed. The CaBPs analysed are expressed in coronet cells. Parvalbumin is expressed in these cells from the beginning of their differentiation, while calretinin expression arises in the tubular SV and becomes more widespread over time. These data emphasize the importance of calcium buffering in the function of coronet cells., (© 2015 The Fisheries Society of the British Isles.)

Melatonin actions are mediated through G protein-coupled transmembrane receptors. Recently, mt1, mt2, and mel1c melatonin receptors were cloned in the Senegalese sole. Here, their day-night and developmental expressions were analyzed by quantitative PCR. These results revealed distinct expression patterns of each receptor through development. mel1c transcripts were more abundant in unfertilized ovulated oocytes and declined during embryonic development. mt1 and mt2 expression was higher at the earliest stages (2-6 days post-fertilization), decreasing before (mt2) or during (mt1) metamorphosis. Only mt1 and mel1c expression exhibited day-night variations, with higher nocturnal mRNA levels. These results suggest different roles and transcriptional regulation of these melatonin receptors during flatfish development and metamorphosis.

The vasa gene encodes an ATP-dependent RNA helicase of the DEAD box protein family that functions in a broad range of molecular events involving duplex RNA. In most species, the germline specific expression of vasa becomes a molecular marker widely used in the visualization and labeling of primordial germ cells (PGCs) and a tool in surrogate broodstock production through PGC transplantation. The vasa gene from tongue sole (Cynoglossus semilaevis) was characterized to promote the development of genetic breeding techniques in this species. Three C. semilaevis vasa transcripts were isolated, namely vas-l, vas-m, and vas-s. Quantitative real-time PCR results showed that C. semilaevis vasa transcripts were prevalently expressed in gonads, with very weak expression of vas-s in other tissues. Embryonic development expression profiles revealed the onset of zygotic transcription of vasa mRNAs and the maternal deposit of the three transcripts. The genetic ZW female juvenile fish was discriminated from genetic ZZ males by a pair of female specific primers. Only the expression of vas-s can be observed in both sexes during early gonadal differentiation. Before PGCs started mitosis, there was sexually dimorphic expression of vas-s with the ovary showing higher levels and downward trend. The results demonstrated the benefits of vasa as a germline specific marker for PGCs during embryonic development and gonadal differentiation. This study lays the groundwork for further application of C. semilaevis PGCs in fish breeding.

During early development, most organisms display rhythmic physiological processes that are shaped by daily changes in their surrounding environment (i.e., light and temperature cycles). In fish, the effects of daily photocycles and their interaction with temperature during early developmental stages remain largely unexplored. We investigated the existence of circadian rhythms in embryonic development and hatching of three teleost species with different daily patterns of behavior: diurnal (zebrafish), nocturnal (Senegalese sole), and blind, not entrained by light (Somalian cavefish). To this end, fertilized eggs were exposed to three light regimes: 12 h of light: 12 h of darkness cycle (LD), continuous light (LL), or continuous darkness (DD); and three species-appropriate temperature treatments: 24°C, 28°C, or 32°C for zebrafish and cavefish and 18°C, 21°C, or 24°C for sole. The results pointed to the existence of daily rhythms of embryonic development and hatching synchronized to the LD cycle, with different acrophases, depending on the species: zebrafish embryos advanced their developmental stage during the light phase, whereas sole did so during the dark phase. In cavefish, embryogenesis occurred within 24 h post fertilization (hpf) at the same pace during day or night. The hatching rhythms appeared to be controlled by a clock mechanism that restricted or "gated" hatching to a particular time of day/night (window), so that embryos that reached a certain developmental state by that time hatch, whereas those that have not wait until the next available window. Under LL and DD conditions, hatching rhythms and the gating phenomenon persisted in cavefish, in zebrafish they split into ultradian bouts of hatching occurring at 12-18-h intervals, whereas in sole DD and LL produced a 24-h delay and advance, respectively. Hatching rates were best under the LD cycle and the reported optimal temperature for each species (95.2±2.7% of the zebrafish and 83.3±0.1% of the cavefish embryos hatched at 28°C, and 93.1±2.9% of the sole embryos hatched at 21°C). In summary, these results revealed that hatching rhythms in fish are endogenously driven by a time-keeping mechanism, so that the day and time of hatching are determined by the interplay between the developmental state (temperature-sensitive) and the circadian clock (temperature-compensated), with the particular phasing being determined by the diurnal/nocturnal behavior of the species.

The small ubiquitin-like modifier (SUMO) pathway is an essential biological process in eukaryote, and Ubc9 is an important E2 conjugating enzyme (UBE2) for SUMO pathway and plays a critical role in cellular differentiation, development and sex modification in various species. However, the relationship between Ubc9 and sex modification and development in fish remains elusive. To elucidate the impact of Ubc9 on sex modification and development, the full length of the cDNA and genomic sequence was cloned from half-smooth tongue sole, Cynoglossus semilaevis. Real-time quantitative RT-PCR demonstrated that ubc9 was ubiquitously expressed in different tissues, and the expression levels varied in the different stages of embryonic and gonadal development. In addition, the expression level was significantly higher in the temperature-treated females than the normal females and males. Moreover, the PET-32-Ubc9 plasmid was constructed and the recombinant protein was expressed in Escherichia coli. Follistatin gene expression was initially up-regulated and FSE genes (cyp19a1a, ctnnb1, foxl2) were initially down-regulated after the injection of Ubc9 protein, prior to 96 h eventually recovered to normal levels. Taken together, the results show that Ubc9 is involved in embryogenesis, gametogenesis and sex modification, and exerts an effect on gene expression., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The morphogenesis and cell proliferation in the retina of turbot (Psetta maxima, Pleuronectiformes: Teleostei) from embryo through metamorphosis have been examined by using proliferating cell nuclear antigen (PCNA)-immunohistochemistry and general histological procedures. In the embryonic retina, cell proliferation and spatial cell reorganization form the anlage of the pigment epithelium and neural retina. Neurogenesis begins around hatching in the temporal retina, dorsal to the optic nerve exit, and then a wave of cell differentiation spreads to the nasal retina to yield a laminated retina by the end of the prolarval turbot stage. Germinal zones in the differentiated retina persist as a rim at the retinal margin, as well as surrounding the optic fissure in premetamorphic and metamorphic turbot larvae. In these zones, progenitor cells with different morphologies show a similar spatial arrangement, which suggests that they have a similar retinogenic potential. During metamorphosis, asymmetric proliferative activity in turbot germinal zones is associated with a marked expansion of the retinal tissue. Scattered stem cells in the laminated retina, related to the lineage of rod photoreceptors, were also observed both in large premetamorphic larvae and metamorphic turbots. The proliferative activity of these cells increases considerably during metamorphosis, when rod photoreceptors become morphologically differentiated., (Copyright © 2013 Wiley Periodicals, Inc.)

Fertilised eggs of Senegalese sole were incubated at 15, 18 or 21 °C, and after hatching all larvae were reared at 21 °C until 30 days post-hatch. By this point larvae from the 18 or 21 °C temperature groups had 11 and 9% more muscle fibres than those from 15 °C, respectively. Hyperplastic growth during metamorphosis was higher in larvae from 18 °C. Embryonic temperature induced gene expression changes, albeit with a variable pattern throughout development. Myf5, myod2, myHC and fst mRNA levels were significantly higher at several stages prior to hatching in embryos incubated at 21 °C, whereas hsp90AB and hsp70 transcripts were present at higher levels in the 15 °C group. Myf5, myod1, myod2, pax7, myog, fst, igf-II, igf1r, hsp90AA and hsp90AB were expressed at higher levels during early development, particularly during somitogenesis. In contrast, mrf4, myHC, mylc2, igf-I, mstn1 and hsp70 were up-regulated at later stages of larval development, namely during and after metamorphosis. This study is the first example of thermal plasticity of myogenesis with prolonged effect in a flatfish., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Specification of primordial germ cells during early embryogenesis is a critical biological issue in reproduction and development. Yet, little is known in marine economic fish species. Vasa, a component of germ plasm, is the most-documented germ cell marker in teleosts. We isolated a full-length vasa cDNA (Smvas) from turbot (Scophthalmus maximus), a marine Euteleostei species, and investigated its expression patterns by RT-PCR and in situ hybridization during embryogenesis and gametogenesis to identify the germ cell lineage in this species. The deduced amino acid sequence of the isolated cDNA shared typical characteristics of Vasa protein and high identity to Vasa homologues in medaka (76.9%) and zebrafish (68.5%). The Smvas transcripts were exclusively detected in germ cells of testis and ovary, and exhibited an interesting dynamic localization pattern during oogenesis. The distribution pattern of Smvas during embyogenesis in this Euteleostei closely resembled the pattern observed in zebrafish (belonging to Osteriophysans) rather than medaka (belonging to Euteleostei). Thus, it is concluded that Smvas isolated in this study is a germ cell specific molecular marker in turbot. Furthermore, we hypothesize that Euteleostei could localize vasa mRNA by a special mode. The results not only facilitate the germ cell manipulation of the turbot, but also improve our understanding of germline development and evolution of vasa localization in teleost., (Copyright © 2012 Wiley Periodicals, Inc.)

Circadian rhythms are established very early during vertebrate development. In fish, environmental cues can influence the initiation and synchronization of different rhythmic processes. Previous studies in zebrafish and rainbow trout have shown that circadian oscillation of clock genes represents one of the earliest detectable rhythms in the developing embryo, suggesting their significance in regulating the coordination of developmental processes. In this study, we analyzed the daily expression of the core clock components Per1, Per2, Per3, and Clock during the first several days of Senegalese sole development (0-4 d post fertilization or dpf) under different lighting regimes, with the aim of addressing when the molecular clock first emerges in this species and how it is affected by different photoperiods. Rhythmic expression of the above genes was detected from 0 to 1 dpf, being markedly affected in the next few days by both constant light (LL) and dark (DD) conditions. A gradual entrainment of the clock machinery was observed only under light-dark (LD) cycles, and robust rhythms with increased amplitudes were established by 4 dpf for all clock genes currently studied. Our results show the existence of an embryonic molecular clock from the 1st d of development in Senegalese sole and emphasize the significance of cycling LD conditions when raising embryos and early larvae.

In the last decades there have been several evidences that traditionally used live preys like rotifers and Artemia salina have nutritional deficiencies that result in a general decrease of fish health, causing anomalies in the development, in growth and in pigmentation. In this study a partial of total replacement of traditional live preys with preserved copepods that represent the natural food of the larvae was evaluated during Solea solea culture. In this study a positive effect of co-feeding preserved copepods in sole larviculture was observed since larvae fed this diet growth and survived better, showed a better tolerance to captive conditions and had a better response to the final thermal/density stress-test with respect to larvae fed a traditional diet. Morphometric data were fully supported by molecular and biochemical ones. Moreover, liver histological investigations, revealed that the inclusion of preserved copepods in the larval diet was able to improve lipid assimilation. In conclusion, preserved copepods may be considered a suitable food for sole when used as a supplement to the traditional diet based on rotifers and Artemia nauplii., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background, Aim and Scope: The environmental presence of polybrominated diphenyl ethers (PBDEs), among which BDE-47 and BDE-99 are particularly abundant, makes toxicity data necessary to assess the hazard risk posed by PBDE to aquatic organisms. This study examines the effects of BDE-47 and BDE-99 on embryo-larval stages of the marine flatfish turbot., Materials and Methods: The turbot embryos were exposed at nominal concentrations of BDE-47 and BDE-99 for 6 days. Selected dose levels were relevant for investigating sublethal and lethal effects., Results: Both tested compounds caused lethal toxicity as well as non-lethal malformations during embryo development. We found a high toxic potency of BDE-47 compared to BDE-99 (LC₅₀ values for embryos and larvae, respectively, BDE-47: 27.35 and 14.13 μg L⁻¹; BDE-99: 38.28 and 29.64 μg L⁻¹)., Discussion: The present study shows high sensitivity of fish early life stages (ELS) to PBDE compounds. Based on environmental concentrations of dissolved PBDEs from various aquatic ecosystems, waterborne BDE-47 and BDE-99 pose little risk of acute toxicity to marine fish at relevant environmental concentrations., Conclusions: Turbot fish ELS proved to be an excellent model for the study of ecotoxicity of contaminants in seawater. The results demonstrate harmful effects of PBDE on turbot ELS at concentrations in the range of parts per billion units., Recommendations and Perspectives: In the perspective of risk assessment, ELS endpoints provide rapid, cost-effective and ecologically relevant information, and links should be sought between these short-term tests and effects of long-term exposures in more realistic scenarios.

The existence of two arylalkylamine N-acetyltransferase 1 (Aanat1) genes in the genome of some teleosts has been reported recently by in silico analysis. However, there are no data concerning the similarities and/or differences between them and many questions remain to be answered, such as their expression sites, development, or kinetics. Here, we report the cloning of Aanat1a and Aanat1b cDNAs from the sole retina and show for the first time that at least three Aanat genes are expressed in a vertebrate species. Because melatonin is involved in fish ontogeny, we analyzed the developmental transcript levels of Aanat1a and Aanat1b by quantitative real-time PCR, showing their inverse and stage-specific expression patterns. Aanat1a was more abundant during early than late larval stages. Before metamorphosis, nocturnal expression was higher. At metamorphosis, Aanat1a expression decreased and lost these day-night variations. In contrast, the abundance of Aanat1b transcripts, low during early developing stages, rose significantly throughout metamorphosis. This situation seemed to apply to the adult because Aanat1a expression was lower than Aanat1b expression in the retina of adults, where the former did not exhibit day-night variations, while the latter did so with much higher nocturnal transcript levels. In situ hybridization analysis detected Aanat1a and Aanat1b messengers in the outer and inner nuclear layers of retina. The differences in abundance and distinct day-night expression patterns between Aanat1a and Aanat1b during sole development suggest different functions for these two enzymes as well as the existence of interactions between the melatoninergic and thyroid hormone systems during flatfish metamorphosis., (© 2011 John Wiley & Sons A/S.)

We have examined the histogenesis of the olfactory system during turbot development using histological and immunohistochemical methods. Proliferating cell nuclear antigen (PCNA) immunohistochemistry was used to detect dividing cells, whereas calretinin (CR) immunohistochemistry was used to distinguish some neuronal components of the olfactory system. Around hatching, the olfactory placode of embryos transforms into an olfactory pit, which enlarges progressively during development. In metamorphic turbots, the right olfactory organ moves to the tip of the head. Each olfactory chamber opens to the external medium by two nostrils and accessory nasal sacs develop during metamorphosis. The order of birth of olfactory receptor cells in the sensory epithelium follows the pattern of most teleosts: ciliated cells differentiate prior to microvillous cells in turbot larvae, and crypt cells are generated during metamorphosis. Axons of olfactory sensory neurons reach the rostral forebrain by hatching, and calretinin-immunoreactive (CR-ir) glomerular fields were apparent during the subsequent larval development. During metamorphosis olfactory bulbs become strongly distorted by head torsion and glomeruli acquire asymmetric organization. The spatio-temporal course of proliferation in the olfactory system reveals changes in the distribution of dividing cells in the sensory epithelium throughout the developmental period investigated. In the olfactory bulb, proliferative activity becomes restricted to the ventral periventricular zone in turbot larvae, as well as in metamorphic specimens., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The chemokines are a superfamily of chemotactic cytokines playing an important role in leukocyte chemotaxis. Here, a turbot head kidney cDNA library was constructed in which KC70 was identified as a CC chemokine. Unknown 5' and 3' parts of the cDNA were amplified by 5' and 3' rapid amplification of cDNA ends (RACE). The complete cDNA of KC70 contains a 59-bp 5' UTR, a 336-bp ORF, and a 152-bp 3' UTR. Four exons and three introns were identified in KC70. Phylogenetic analysis showed that KC70 was similar to CCL19. In normal turbot KC70 was expressed in all tissues except brain and skin. Infection of turbot with pathogenic bacteria significantly increased expression of KC70 in the liver. Expression of KC70 in head kidney first increased and then decreased after bacterial challenge. No significant change was observed in the spleen after bacterial challenge. During embryonic development, KC70 was highly expressed after the gastrula stage. These results indicated KC70 plays important and multiple roles in turbot immune response.

The effect of the dioxin-like PCB 126 (3,3',4,4',5-pentachlorobiphenyl) on the early development of the marine flatfish sole (Solea solea) was tested in a newly developed early life stage (ELS) test that includes the metamorphosis of the symmetric larvae into an asymmetrical flatfish. Early life stages of sole were exposed to a concentration series of PCB 126 in seawater until 4, 8, 10 and 15 days post fertilisation (dpf). Subsequently the development of the larvae was registered under further unexposed conditions. The LC50s at the start of the free-feeding stage (12 dpf) ranged between 39 and 83 ng PCB 126/l depending on exposure duration. After the fish had completed the metamorphosis, the LC50 values ranged between 1.7 and 3.7 ng PCB 126/l for the groups exposed for 4, 8 and 10 dpf, respectively. Thus exposure for only 4 days, covering only the egg stage, was sufficient to cause adverse effects during a critical developmental phase two weeks later. The internal dosages of these larvae, determined by means of an in vitro gene reporter assay as dioxin-equivalent values (TEQ), revealed a LD50 of 1ng TEQ/g lipid, which is within the same order of magnitude as TEQ levels found in fish from highly polluted areas. This study indicates that ELS fish tests that are terminated shortly after the fish becomes free-feeding, underestimate the toxic potential of compounds with low acute toxicity such as PCBs. Our prolonged ELS with this native marine flatfish suggests that reproductive success of fish populations at contaminated sites can be affected by persistent compounds that are accumulated by the female fish and passed on to the eggs.

Differential expression of genes is crucial to embryogenesis. The analysis of gene expression requires appropriate references that should be minimally regulated during the embryonic development. To select the most stable genes for gene normalization, the expression profiles of eight commonly used reference genes (ACTB, GAPDH, rpL17, alpha-Tub, EF1-alpha, UbcE, B2M, and 18S rRNA) were examined during Japanese flounder (Paralichthys olivaceus) embryonic development using quantitative real-time polymerase chain reaction. It was found that all seven mRNA genes appeared to be developmentally regulated and exhibited significant variation of expression. However, further analyses revealed the stage-specific expression stability. Hence when normalization using these mRNA genes, the differential and stage-related expression should be considered. 18S rRNA gene, on the other hand, showed the most stable expression and could be recommended as a suitable reference gene during all embryonic developmental stages in P. olivaceus. In summary, our results provided not only the appropriate reference gene for embryonic development research in P. olivaceus, but also possible guidance to reference gene selection for embryonic gene expression analyses in other fish species.

Background: Ribosomal proteins (RPs) are key components of ribosomes, the cellular organelle responsible for protein biosynthesis in cells. Their levels can vary as a function of organism growth and development; however, some RPs have been associated with other cellular processes or extraribosomal functions. Their high representation in cDNA libraries has resulted in the increase of RP sequences available from different organisms and their proposal as appropriate molecular markers for phylogenetic analysis., Results: The development of large-scale genomics of Senegalese sole (Solea senegalensis) and Atlantic halibut (Hippoglossus hippoglossus), two commercially important flatfish species, has made possible the identification and systematic analysis of the complete set of RP sequences for the small (40S) ribosome subunit. Amino acid sequence comparisons showed a high similarity both between these two flatfish species and with respect to other fish and human. EST analysis revealed the existence of two and four RPS27 genes in Senegalese sole and Atlantic halibut, respectively. Phylogenetic analysis clustered RPS27 in two separate clades with their fish and mammalian counterparts. Steady-state transcript levels for eight RPs (RPS2, RPS3a, RPS15, RPS27-1, RPS27-2, RPS27a, RPS28, and RPS29) in sole were quantitated during larval development and in tissues, using a real-time PCR approach. All eight RPs exhibited different expression patterns in tissues with the lowest levels in brain. On the contrary, RP transcripts increased co-ordinately after first larval feeding reducing progressively during the metamorphic process., Conclusion: The genomic resources and knowledge developed in this survey will provide new insights into the evolution of Pleuronectiformes. Expression data will contribute to a better understanding of RP functions in fish, especially the mechanisms that govern growth and development in larvae, with implications in aquaculture.

Melatonin biosynthesis from serotonin involves the sequential activation of the arylalkylamine N-acetyltransferase (AANAT) and hydroxyindole-O-methyltransferase (HIOMT). Photoperiod synchronizes a daily rhythm in pineal and retinal melatonin secretion through controlling AANAT activity. Teleost fish possess two Aanat, one expressed in the retina (AANAT1) and the other expressed in the pineal gland (AANAT2). We report here the full-length cloning of Aanat1, Aanat2, SmHiomt and Otx5 (orthodenticle homeobox homolog 5) in the turbot (Scophthalmus maximus, Sm), a flatfish belonging to an evolutionary recent group of Teleost. The temporal expression pattern of the genes investigated is consistent with the idea that OTX5 is needed for photoreceptor specification, and that the pineal gland differentiates before the retina. SmAanat2 expression remained pineal specific during the period of time investigated, whereas SmOtx5 and SmHiomt expressions were seen in both the retina and pineal gland. Our results do not support the existence of a second SmHiomt, as is the case for SmAanat. Neither SmAanat2 nor SmHiomt mRNAs displayed cyclic accumulation in the pineal organ of embryos and larvae maintained under a light-dark cycle from fertilization onward. This is in marked contrast with the situation observed with zebrafish Aanat2, indicating that the molecular mechanisms controlling the development of the pineal melatonin system have been modified during the evolution of Teleost.

Antimicrobial peptides (AMPs) are regarded as important components of the host innate immune system and play crucial roles in host defence against microbial invasion. A small number of hepcidin AMPs have been isolated from teleosts. Here, we report the isolation of a hepcidin gene from the liver of turbot (Scophthalmus maximus) (GenBank accession numbers: and ). In the 1037 bp-long genomic sequence, three exons and two introns were identified. The full-length cDNA is 778bp long and contains an ORF of 273bp encoding a prepropeptide of 90 amino acid residues. The predicted prepropeptide consists of three domains: a signal peptide (24 amino acids), a prodomain (40 amino acids) and a mature peptide (26 amino acids). RT-PCR demonstrated that hepcidin transcripts were highly abundant in liver, abundant in heart, head kidney, spleen, skin and gill, less abundant in blood cell, gonad and intestine, and undetectable level in muscle. The level of the hepcidin mRNA in embryos gradually increases during embryogenesis from 2 h (2 cell stage) to 95 h (larva stage) after fertilisation. Challenge of turbot with pathogenic bacteria, Listonella anguillarum, significantly elevated hepcidin mRNA levels in liver and spleen in a time-dependent fashion. The hepcidin transcripts were detected in turbot embryonic cell line (TEC). Challenge of TEC cells with the pathogenic bacteria significantly elevated hepcidin mRNA levels.

Insulin-like growth factors I and II (IGF-I and IGF-II) play an important role as modulators of development, growth, and reproduction. This study aimed to isolate the IGF-I and IGF-II cDNAs and determine their temporal expression pattern in different organs and throughout larval development in Senegal sole. The rapid amplification of cDNA ends (RACE) was used to obtain both full-length IGFs sequences. A high sequence similarity with other teleosts sequences was observed. Domains B and A revealed as the most evolutionary conserved. Steady-state copy numbers of IGF-I and IGF-II were also quantified in different Senegal sole tissues by real-time PCR. IGF-I and IGF-II expressed ubiquitously with the highest mRNA levels in liver (88 x 10(6) molecules/microg total RNA) and gills (14.0 x 10(6) molecules/microg total RNA) respectively. IGF-II mRNA levels were higher than IGF-I in prehatching embryos and premetamorphic larvae with a significant drop before the commencement of eye migration in metamorphosis. The abundance of IGF-II transcripts correlated positively with the growth rate during larval development. The putative role of IGF-II on metamorphosis and larval growth is discussed.

Heat shock proteins (Hsps) are a family of highly conserved cellular proteins present in all organisms, mediating a range of essential housekeeping and cytoprotective functions as well-known molecular chaperones and recently as regulators of the immune response. By subtractive suppression hybridization, three Hsp40 homologues have been identified in the flounder (Paralichthys olivaceus) embryonic cells (FEC) after treatment with UV-inactivated turbot (Scophthalmus maximus L.) rhabdovirus (SMRV), termed PoHsp40A4, PoHsp40B6 and PoHsp40B11, whose encoded proteins all possess the conserved DnaJ domain, a signature motif of the Hsp40 family. Based on different protein structure and phylogenetic analysis, they can be categorized into two subfamilies, PoHsp40A4 for Type I Hsp40, PoHsp40B6 and PoHsp40B11 for Type II Hsp40. Further expression analysis revealed two very different types of kinetics in response either to heat shock or to virus infection, with a marked induction for PoHsp40A4 and a weak one for both PoHsp40B6 and PoHsp40B11. A very distinct tissue distribution of mRNA was also revealed among the three genes, even between PoHsp40B6 and PoHsp40B11. This is the first report on the transcriptional induction of Hsp40 in virally stimulated fish cells, and the differential expressions might reflect their different roles in unstressed and stressed cells.

The first successful cryopreservation of fish embryos was reported in the Japanese flounder by vitrification [Chen and Tian, Theriogenology, 63, 1207-1219, 2005]. Since very high concentrations of cryoprotectants are needed for vitrification and fish embryos have a large volume, Japanese flounder embryos must have low sensitivity to cryoprotectant toxicity and high permeability to water and cryoprotectants. So, we investigated the sensitivity and the permeability of Japanese flounder embryos. In addition, we assessed the survival of flounder embryos after vitrification with solutions containing methanol and propylene glycol, following Chen and Tian's report. The embryos were relatively insensitive to the toxicity of individual cryoprotectants at lower concentrations, especially methanol and propylene glycol as their report. Although their permeability to water and cryoprotectants could not be measured from volume changes in cryoprotectant solutions, the embryos appeared to be permeable to methanol but less permeable to DMSO, ethylene glycol, and propylene glycol. Although vitrification solutions containing methanol and propylene glycol, which were used in Chen and Tian's report, were toxic to embryos, a small proportion of embryos did survived. However, when vitrified with the vitrification solutions, no embryos survived after warming. The embryos became opaque during cooling with liquid nitrogen, indicating the formation of intracellular ice during cooling. When embryos had been kept in vitrification solutions for 60 min after being treated with the vitrification solution, some remained transparent during cooling, but became opaque during warming. This suggests that dehydration and/or permeation by cryoprotectants were insufficient for vitrification of the embryos even after they had been over-treated with the vitrification solutions. Thus, Chen and Tian's cryopreservation method lacks general application to Japanese flounder embryos.

Specification and differentiation of skeletal muscle cells are driven by the activity of genes encoding members of the myogenic regulatory factors (MRFs). In vertebrates, the MRF family includes MyoD, Myf5, myogenin, and MRF4. The MRFs are capable of converting a variety of nonmuscle cells into myoblasts and myotubes. To better understand their roles in fish muscle development, we isolated the MyoD gene from flounder (Paralichthys olivaceus) and analyzed its structure and patterns of expression. Sequence analysis showed that flounder MyoD shared a structure similar to that of vertebrate MRFs with three exons and two introns, and its protein contained a highly conserved basic helix-loop-helix domain (bHLH). Comparison of sequences revealed that flounder MyoD was highly conserved with other fish MyoD genes. Sequence alignment and phylogenetic analysis indicated that flounder MyoD, seabream (Sparus aurata) MyoD1, takifugu (Takifugu rubripes) MyoD, and tilapia (Oreochromis aureus) MyoD were more likely to be homologous genes. Flounder MyoD expression was first detected as two rows of presomitic cells in the segmental plate. From somitogenesis, MyoD transcripts were present in the adaxial cells that give rise to slow muscles and the lateral somitic cells that give rise to fast muscles. After 30 somites formed, MyoD expression decreased in the somites except the caudal somites, coincident with somite maturation. In the hatching stage, MyoD was expressed in other muscle cells and caudal somites. It was detected only in muscle in the growing fish.

Vitrification, is the most promising option for the cryopreservation of fish embryos but requires high concentrations of potentially toxic cryoprotectants. In this study, embryos from Turbot and Zebrafish, each in two developmental stages, were submitted to a four stepwise cryoprotectant incorporation protocol. After incubation in the vitrificant solution (5M dimethyl sulfoxide, 2M methanol, 1M ethylen-glycol and 10% sucrose) embryos were loaded in straws and plunged into liquid nitrogen. The activity of two cytoplasmic enzymes, LDH and G6PDH, and the hatching rates were analyzed in control embryos, those subjected to the cryoprotectant solutions and in frozen/thawed embryos. Results showed that the cryoprotectants incorporation protocol did not have important effects on the analyzed enzymatic activities, which remained at similar levels to that in control embryos but significantly reduced the hatching rates. Turbot was less sensitive than Zebrafish to the toxic effect of the cryoprotectants, achieving hatching rates of 74.8% in comparison with fresh control embryos at G stage, whereas in Zebrafish only 17.7% of hatching was reported with five somites-treated embryos. In Turbot, G stage was more resistant to the cryoprotectants and thus more convenient for further vitrification studies. After vitrification no survival was recorded and enzymatic activities dropped significantly, particularly in Zebrafish, indicating cell damage and loss of cytoplasmic enzymes. Nevertheless, total cell lysis was not produced, and once again Turbot was more resistant to the effect of vitrification, particularly at the later stage. In that stage, Turbot embryos showed around 50% of G6PDH activity after vitrification, in comparison with the control, indicating the preservation of some cellular activity after freezing-thawing, despite the loss of developmental ability.

Thyroid hormones (TH) are pleiotropic factors important for many developmental and physiological functions in vertebrates and particularly in amphibian metamorphosis. Their effects are mediated by two specific receptors (TRalpha and TRbeta), which are ligand-dependent transcription factors, members of the nuclear hormone receptor superfamily. Besides their pivotal role in amphibian metamorphosis, TH are also critical for fish metamorphosis. As this later role of TH is less studied, we analyzed their action in the turbot (Scophtalmus maximus), a metamorphosing flat fish. We describe the isolation of sequences for the turbot orthologs of a number of Xenopus genes, which are induced during amphibian metamorphosis. Developmental expression of these genes during turbot metamorphosis was studied by several methods and the expression patterns of these genes compared with those in Xenopus and flounder. We find that the period between the onset and the end of eye migration (day 22 to day 30 post-hatching) most likely corresponds to the metamorphic climax with either high TRalpha or high TH levels. Our results show that in contrast to amphibians, it is TRalpha and not TRbeta mRNA that is up-regulated during metamorphosis. Our results highlight the notion that TH regulates, through a rise of TR expression, a genetic cascade during turbot metamorphosis. The fact that TH regulates metamorphosis in amphibian and teleost fishes suggests that TH-regulated metamorphosis is a post-embryonic process conserved in most vertebrates.

The particular characteristics of fish embryos require the development of specific methods for cryopreservation. One of the main obstacles is related to the presence of membranes and compartments with different water and cryoprotectant permeability. To assess dimethyl sulfoxide (Me2SO4) permeability, we exposed turbot embryos (Scophthalmus maximus) at F stage (tail bud) to the cryoprotectant solutions used in a vitrification protocol and then evaluated the Me2SO4 content inside the embryo using high-performance liquid chromatography (HPLC). The Me2SO4 influx was analyzed in normal embryos and in embryos treated with pronase (2mg/ml) in order to increase chorion permeability. The evaluation was made after each step of cryoprotectant incorporation and removal. Three embryo compartments were distinguished: the perivitelline space (PVS), the yolk sac (YS) and the cellular compartment (CC), and the relative volumes of each, estimated using stereoscopic microscopy imaging, were 11.37, 81.23 and 7.40%, respectively. The Me2SO4 concentration inside the embryos was calculated based on their entrance into one, two or three compartments. Results suggest high entrance of Me2SO4 into the PVS and a low concentration of this cryoprotectant inside the other compartments. Pronase did not significantly increase Me2SO4 influx, but facilitated its elimination during the washing steps.

Successful fish embryo cryopreservation is still far from being achieved. Vitrification is considered the most promising option. Many factors are involved in the success of the process. The choice of a proper vitrification solution, the enzymatic permeabilization of embryos to increase cryoprotectant permeability, the adequate container for embryo loading, and the temperature for thawing, were the parameters considered at different developmental stages in the present study. After vitrification, embryo morphology was evaluated under stereoscopic microscopy, establishing the percentage of intact embryos. Two of the studied parameters yielded differences in this percentage, the volume of straw used for embryo loading (1 ml straws were significantly better than 0.5 ml straws, with regard to post-thawed embryo morphologies), and the thawing temperature, achieving 49% of embryos with intact morphology after thawing at 0 degrees C. After thawing, the intact embryos were incubated and periodically observed to detect morphological changes. Changes in the perivitelline space, shrinkage of the yolk and chorion ruptures as well as a progressive whitening of the embryo and yolk were observed. After 8 h all embryos showed clear signs of degradation and during this incubation period no embryo showed any developmental ability.

The ontogeny and organization of the terminal nerve (TN) during turbot development was studied using an antiserum to neuropeptide Y. First immunoreactive cells were detected in the olfactory placode at hatching time. At 1 day after hatching, a loose group of labeled neurons form an extracranial primordial ganglion of the TN. During the subsequent larval development, more perikarya displaying increased immunoreactivity were found along the course of the olfactory nerve. Moreover, labeled cells cross the meninx of the forebrain gathering in the olfactory bulb of larval turbot. Projections from these cells, directed both to the caudal brain and to the retina, develop when the cells become established in the olfactory bulb. The generation of immunoreactive cells in the olfactory organ extends into the metamorphic period, when a pronounced asymmetry affects the turbot morphology. At this time, the topological location of the immunoreactive cells in the TN becomes distorted. This developmental pattern was compared with those found in other teleosts and in other vertebrates. Preabsorption experiments of anti-neuropeptide Y serum with neuropeptide Y and FMRF-amide suggests that immunoreactive material observed in TN cells was not neuropeptide Y, and raises the possibility that other peptides, e.g. FMRF-amide-like peptides, exist in this neural system.

Larval stages of two economically important flatfish, the sole (Solea solea) and turbot (Scophthalmus maximus) were exposed to ambient and elevated levels of UV-B. Sole larvae, which naturally occur in the plankton in early spring, demonstrated skin lesions at elevated levels of UV-B. Histopathology of the sole revealed cellular changes in the integument, characteristic of sunburn damage, with a reduction in the size of mucus-secreting cells and an increased epidermal thickening, especially at the highest doses of UV-B (2.15 KJ bio eff/m2). Pigmentation in the sole is restricted to a few isolated melanocytes. The integrity of the heavily pigmented skin of turbot appeared to be unaffected by comparable doses of UV-B. Both species have protective mechanisms, which minimize the effects of naturally-occurring levels of UV-B. However, sole appear to be poorly adapted to accommodate any further increase in solar radiation.

During embryogenesis of teleost fish, the formation of a yolk syncytial layer (YSL) enables the resorption of the yolk reserves and development up to the larval stage. We have examined the changes of the yolk cell structure in relation to yolk and oil-globule lipid utilization during development of the turbot (Scophthalmus maximus). After encapsulation by the YSL, resorption of the single, large oil globule occurred predominantly after yolk resorption and was slower in fasting larvae. The YSL was in contact with an enlarged perisyncytial space, but no vascular network or red blood cells were present within the walls of the yolk sac. Intrasyncytial channels infiltrated by pigmented lining cells were observed in the YSL surrounding the oil globule. Apolipoprotein E (apoE) has a prominent role in lipid metabolism because of its ability to interact with lipoprotein receptors. We performed molecular cloning of the putative low-density lipoprotein-receptor binding domain of turbot apoE. In situ hybridization analysis revealed a very high level of apoE transcripts in the YSL, while no expression could be detected in the intestine. YSL apoE expression was correlated with the synthesis of very low density lipoprotein (VLDL) particles. An extraordinarily high number of VLDL particles were poured into the perisyncytial space, and intrasyncytial channels enabled the transfer of yolk- and oil globule-derived lipids to the developing embryo or larva. The pattern of apoE mRNA distribution in relation to YSL lipoprotein synthesis indicates that apoE expression is a suitable molecular marker for monitoring endogenous lipid nutrition during the endoexotrophic period of teleost fish development.

Radiolabelled bacterial lipopolysaccharide (3H-LPS) obtained from Aeromonas salmonicida subsp. salmonicida was added to the petri dishes containing yolk sac larvae of Atlantic halibut (Hippoglossus hippoglossus L.). The larvae were exposed either to 6.25, 12.5, 25, 50 or 100 micrograms 3H-LPS ml-1. The uptake was both dependent on the LPS concentration and the time of exposure. After 5 days of exposure, each larva contained 1.8-7.4 ng 3H-LPS dependent on the initial concentration. After 10 days of exposure each larva contained 7.0-12.4 ng LPS and after 15 days they contained 18.3-34.9 ng 3H-LPS. Fluorescence microscopic analysis of sections obtained from larvae exposed to FITC-LPS (25, 50 and 100 micrograms ml-1) for 5, 10 and 15 days, revealed fluorescence in intestinal epithelial cells, cells in the connective tissue adjacent to the intestine, in cells located between the integumental layer and yolk sac, and in some epithelial cells in the integument. By use of immunohistochemical techniques, LPS was confined to intestinal epithelial cells, lumen of excretory duct and in numerous cells in the epidermal layer. Control specimens did not contain fluorescence or were immunohistochemically negative for LPS. In groups of larvae exposed to 12.5, 25, 50 and 100 micrograms LPS ml-1, the survival was significantly increased after exposure to 50 and 100 micrograms LPS ml-1 from day 20 (96 d degree) and throughout the yolk sac period compared to untreated larvae.

The timing of hatching in the Atlantic halibut (Hippoglossus hippoglossus) has been suggested to be regulated by environmental light conditions. However, the photosensory organ that perceives the triggering light has not been identified. In the present study, we investigated the morphogenesis of the pineal organ and the neurochemical differentiation of photoreceptors in the pineal organ and the retina of the Atlantic halibut during embryonic development. Immunocytochemical techniques were used for detection of integral protein components of the phototransduction process: opsins, arrestin (S-antigen) and alpha-transducin. We also studied the expression of serotonin (5-HT), a precursor of the neurohormone melatonin known to be synthesized by pineal photoreceptors. In the pineal anlage, opsin immunoreactive (ir) cells appear at 11 days post-fertilization (pf), arrestin, alpha-transducin and serotonin ir cells appear at 14 days pf; hatching took place 15 days pf. The retina contained no immunoreactive cells in embryos or in newly hatched larva. During this period, the pineal anlage is morphologically discernible only as a wedge-shaped region in the diencephalic roof, where elongated cells are aligned with their long axes converging toward a centrally located presumptive pineal lumen. The results show that the pineal photoreceptors contain serotonin and molecules involved in the phototransduction cascade before hatching. We suggest that the pineal organ has the capacity to perceive and mediate photic information before hatching in halibut embryos, and may thereby influence the timing of hatching.

The ontogeny of endocrine cells and nerve fibers containing immunoreactivities for 12 regulatory peptides and serotonin was studied in the digestive tract of a flatfish, the turbot (Scophthalmus maximus), using antisera specific for mammalian and teleostean hormones. Transient insulin-immunoreactive (-IR) endocrine cells were detected from day 5 to day 10 in stomach and intestine I. Somatostatin (SOM)-IR cells appeared at day 8 in the stomach anlage and intestine I. In contrast to the islet cells, they reacted with antisera against mammalian (m) SOM-14 and salmon (s) SOM-25. Infrequent nerve fibers reacting only with anti-mSOM-14 appeared around day 24. Thus, different forms of SOM seem to be present in the gastro-entero-pancreatic system and the enteric nervous system. Neuropeptide Y (NPY)-, salmon pancreatic polypeptide (sPP)- and mPP-immunoreactivities coexisted throughout development. In entero-endocrine cells, NPY/PP-immunoreactivity was first observed at day 8 and around day 24 in enteric nerve fibers. Glucagon (GLUC)-IR entero-endocrine cells appeared at day 5. No coexistence of NPY/PP- and GLUC-immunoreactivities was observed. The first insulin-like growth factor I (IGF-I)-IR cells were identified around day 8. They seemed to contain none of the other peptides. Their number and distribution exhibited great interindividual differences. Vasoactive intestinal polypeptide (VIP)-IR entero-endocrine cells appeared as late as around day 24. The first VIP-IR nerve fibers, however, were identified at day 5. Infrequent neurotensin (NT)-IR cells appeared along the intestine around day 10 and NT-IR nerve fibers at day 17. The first serotonin (SER)-IR cells were observed in the stomach anlage around day 10 and SER-IR nerve fibers at day 15 throughout the gastro-intestinal tract. Gastrin (GAS)/cholecystokinin (CCK)-IR cells appeared around day 11 in stomach and intestine I. The first substance P (SP)-IR enteric nerve fibers were detected around day 8 and SP-IR endocrine cells at day 11. Pancreastatin (PST)-IR cells were identified in the stomach anlage and intestine I around day 8 and contained NT-, GAS/CCK- and SER-immunoreactivities in coexistence. Thus, several developmental phases can be distinguished: (1) at the onset of exogenous feeding only transient INS-IR cells and VIP-IR nerve fibers are present; (2) a differentiated entero-endocrine system establishes during the early phase of exogenous feeding; (3) before the final differentiation of stomach and gut GAS/CCK-IR cell appear; (4) after metamorphosis most of the different types of regulatory peptide-containing nerve fibers develop, probably setting up the fine regulation of gastro-intestinal blood flow and motility.

We have determined the complete sequence of the translation elongation factor EF1 subunit alpha (EF1 alpha) mRNA of zebrafish, and the 3'-untranslated sequence of EF1 alpha mRNA of halibut. The 5'-untranslated leader sequence of the EF1 alpha mRNA starts with a polypyrimidine tract. This feature is shared with the mRNAs for ribosomal proteins, where it affects the utilization of mRNA by ribosomes. However, the secondary structures of these leader sequences may differ. 5'-Polypyrimidine tracts of vertebrate EF1 alpha mRNAs participate in the formation of stable stem-loop structures, whereas those of 15 randomly chosen mRNAs for ribosomal proteins do not. This difference may provide a basis for differential control of translation for the two classes of mRNA. The 3'-untranslated sequences of vertebrate EF1 alpha mRNA have diverged little during evolution. Analyses of sequence and putative secondary structures suggest that both sequence-specific interactions and secondary structures may have contributed to sequence conservation.