Following the two rounds of whole-genome duplication (WGD) during deuterosome evolution, a third genome duplication occurred in the ray-fined fish lineage and is considered to be responsible for the teleost-specific lineage diversification and regulation mechanisms. As a receptor-regulated SMAD (R-SMAD), the function of SMAD3 was widely studied in mammals. However, limited information of its role or putative paralogs is available in ray-finned fishes. In this study, two SMAD3 paralogs were first identified in the transcriptome and genome of Japanese flounder (Paralichthys olivaceus). We also explored SMAD3 duplication in other selected species. Following identification, genomic structure, phylogenetic reconstruction, and synteny analyses performed by MrBayes and online bioinformatic tools confirmed that smad3a/3b most likely originated from the teleost-specific WGD. Additionally, selection pressure analysis and expression pattern of the two genes performed by PAML and quantitative real-time PCR (qRT-PCR) revealed evidence of subfunctionalization of the two SMAD3 paralogs in teleost. Our results indicate that two SMAD3 genes originate from teleost-specific WGD, remain transcriptionally active, and may have likely undergone subfunctionalization. This study provides novel insights to the evolution fates of smad3a/3b and draws attentions to future function analysis of SMAD3 gene family.

Sex reversal induced by temperature change is a common feature in fish. Usually, the sex ratio shift occurs when temperature deviates too much from normal during embryogenesis or sex differentiation stages. Despite decades of work, the mechanism of how temperature functions during early development and sex reversal remains mysterious. In this study, we used Chinese tongue sole as a model to identify features from gonad transcriptomic and epigenetic mechanisms involved in temperature induced masculinization. Some of genetic females reversed to pseudomales after high temperature treatment which caused the sex ratio imbalance. RNA-seq data showed that the expression profiles of females and males were significantly different, and set of genes showed sexually dimorphic expression. The general transcriptomic feature of pesudomales was similar with males, but the genes involved in spermatogenesis and energy metabolism were differentially expressed. In gonads, the methylation level of cyp19a1a promoter was higher in females than in males and pseudomales. Furthermore, high-temperature treatment increased the cyp19a1a promoter methylation levels of females. We observed a significant negative correlation between methylation levels and expression of cyp19ala. In vitro study showed that CpG within the cAMP response element (CRE) of the cyp19a1a promoter was hypermethylated, and DNA methylation decreased the basal and forskolin-induced activities of cyp19a1a promoter. These results suggested that epigenetic change, i.e., DNA methylation, which regulate the expression of cyp19a1a might be the mechanism for the temperature induced masculinization in tongue sole. It may be a common mechanism in teleost that can be induced sex reversal by temperature.

Following the two rounds of whole-genome duplication that occurred during deuterostome evolution, a third genome duplication event occurred in the stem lineage of ray-finned fishes. This teleost-specific genome duplication is thought to be responsible for the biological diversification of ray-finned fishes. DEAD-box polypeptide 3 (DDX3) belongs to the DEAD-box RNA helicase family. Although their functions in humans have been well studied, limited information is available regarding their function in teleosts. In this study, two teleost Ddx3 genes were first identified in the transcriptome of Japanese flounder (Paralichthys olivaceus). We confirmed that the two genes originated from teleost-specific genome duplication through synteny and phylogenetic analysis. Additionally, comparative analysis of genome structure, molecular evolution rate, and expression pattern of the two genes in Japanese flounder revealed evidence of subfunctionalization of the duplicated Ddx3 genes in teleosts. Thus, the results of this study reveal novel insights into the evolution of the teleost Ddx3 genes and constitute important groundwork for further research on this gene family.

Sox3, which belongs to the SoxB1 subgroup, plays major roles in neural and gonadal development. In the present study, Japanese flounder Paralichthys olivaceus sox3 gene (Posox3) and its promoter sequence were isolated and characterized. The deduced PoSox3 protein contained 298 amino acids with a characteristic HMG-box domain. Alignment and phylogenetic analyses indicated that PoSox3 shares highly identical sequence with Sox3 homologues from different species. The promoter region of Posox3 has many potential transcription factor (TF) binding sites. The expression profiles of Posox3 in different developmental stages and diverse adult tissues were analyzed by quantitative real-time RT-PCR (qRT-PCR). Posox3 mRNA was maternally inherited, and maintained at a considerably high expression level between the blastula stage and the hatching stage during embryonic development. Posox3 was abundantly expressed in the adult brain and showed sexually dimorphic expression pattern. In situ hybridization (ISH) was carried out to investigate the cellular distribution of Posox3 in the ovary, and results showed the uniform distribution of Posox3 throughout the cytoplasm of oogonia and stage I-III oocytes. These results indicate that Posox3 has potentially vital roles in embryonic and neural development and may be involved in the oogenesis process. Our work provides a fundamental understanding of the structure and potential functions of Sox3 in Paralichthys olivaceus.

R-spondin 1 (Rspo1) is a potential female-determining gene in mammals that could regulate the Wnt/β-catenin signaling pathway. The deletion of Rspo1 causes sex reversal in females. To investigate sexual determination and differentiation, we cloned and analyzed the Rspo1 gene in Cynoglossus semilaevis. Phylogenetic and gene structure analyses revealed that Rspo1 gene exhibited high sequence conservation and contained an N-terminal signal peptide, two furin-like cysteine-rich domains (FU1 and FU2), a thrombospondin type 1 repeat, and a C-terminal region enriched with basic charged amino acids. qRT-PCR revealed that Rspo1 expressed sexual dimorphism in gonad, with higher expression levels in the ovary than in the testis, thus, suggesting the involvement of Rspo1 in gonad differentiation. In situ hybridization results demonstrated that Rspo1 was expressed in premature germ cells, including spermatogonia and spermatocytes in the testis and stage II and stage III oocytes in the ovary. The methylation levels in two CpG sites of Rspo1 promoter significantly differed among females, males, and pseudomales. After 30days of exposure to high temperature, the expression of Rspo1 significantly decreased in female individuals, some of which were prone to males. However, no difference of Rspo1 gene expression was observed between the control group and high-temperature group in males. These preliminary findings suggested that Rspo1 played a crucial role in sex determination and development. This study laid the groundwork for further sex control breeding techniques in C. semilaevis.

GATA4 is a well-known transcription factor of the GATA family implicated in regulation of sex determination and gonadal development in mammals. In this study, we cloned the full-length cDNA of Paralichthys olivaceus gata4 (Po-gata4). Phylogenetic, gene structure, and synteny analysis showed that Po-GATA4 is homologous to GATA4 of teleost and tetrapod. Po-gata4 transcripts were detected in Sertoli cells, spermatogonia, oogonia and oocytes, with higher transcript levels overall in the testis than the ovary. The promoter region of P. olivaceus R-spondin1was found to contain a GATA4-binding motif. Results of CBA (cleaved amplified polymorphic sequence-based binding assay) indicated that GATA4 could indeed bind to the promoter sequence of R-spondin1. Moreover, human GATA4 recombinant protein could upregulate R-spondin1 in P. olivaceus ovary cells and FBCs (flounder brain cell line). In FBCs, overexpression of Po-gata4 resulted in elevated transcript levels of R-spondin1. Taken together, our results indicate that Po-GATA4 is involved in gonadal development by regulating R-spondin1 expression.

Tudor domain containing protein 1 (tdrd1) is a member of the Tudor family and has shown essential functions during embryogenesis and gametogenesis. In this study, we cloned the full length cDNA of Paralichthys olivaceus tdrd1 (Potdrd1). PoTDRD1 is a multidomain protein with an N-terminal MYND zinc finger domain, followed by four tandem extended Tudor domains. Sequence comparison, genomic structure, phylogenetic analyses and synteny analyses showed that Potdrd1 was homologous to those of other teleosts. In adult individuals, the expression of Potdrd1 was higher in testis than in ovary, demonstrating a sexually dimorphic gene expression pattern. In situ hybridization (ISH) showed that Potdrd1 mRNA was detected in oogonia and oocytes of ovary as well as in spermatogonia and spermatocytes of testis. In juveniles during gonad differentiation its expression level increased rapidly from 30 dph to 100 dph and showed obvious sexual dimorphism that was in accordance with the expression of anti-Mullerian hormone (amh). Potdrd1 mRNA was consistently detected during embryogenesis, and its level was higher from unfertilzed eggs to the blastula stage and subsequently decreased until hatching. When chimeric RNA containing green fluorescent protein (GFP) and 3' untranslated regions (UTR) of Potdrd1 was microinjected into zebrafish fertilized eggs, the green fluorescence could be visualized only in putative PGCs. These results indicated that Potdrd1 is a germline specific and sexually dimorphic factor that potentially functionate in germline development and gametogenesis in Japanese flounder.

Half-smooth tongue sole (Cynoglossus semilaevis) is a valuable fish for aquaculture in China. This fish exhibits sexual dimorphism, particularly different growth rates and body sizes between two genders. Thus, C. semilaevis is a good model that can be used to investigate mechanisms responsible for such dimorphism, this model can also be utilized to answer fundamental questions in evolution and applied fields of aquaculture. Hence, advances in second-generation sequencing technology, such as 454 pyrosequencing, could provide a robust tool to study the genome characteristics of non-model species.

Members of major histocompatibility complex (MHC) family are important in immune systems. Great efforts have been made to reveal their complicated gene structures. But many existing studies focus on partial sequences of MHC genes. In this study, by gene cloning and sequencing, we identified cDNA sequences and DNA sequences of the MHC class II B in two flatfishes, stone flounder (Kareius bicoloratus) and homozygous diploid Japanese flounder (Paralichthys olivaceus). Eleven cDNA sequences were acquired from eight stone flounder individuals, and most of the polymorphic sites distributed in exons 2 and 3. Twenty-eight alleles were identified from the DNA fragments in these eight individuals. It could be deduced from their Bayesian inference phylogenetic tree that at least four loci of MHC class II B exist in stone flounder. The detailed whole-length DNA sequences in one individual were analyzed, revealing that the intron length varied among different loci. Four different cDNA sequences were identified from one homozygous diploid Japanese flounder individual, implying the existence of at least four loci. Comparison of the cDNA sequences to the DNA sequence confirmed that six exons existed in this gene of Japanese flounder, which was a common feature shared by Pleuronectiformes fishes. Our results proved the multi-locus feature of MHC class II B. The sequences we obtained would provide detailed and systematic data for further research.

Quantitative real time RT-PCR has been described as the most sensitive method for the detection of low abundance mRNA. To date, no reference genes have been screened in the half-smooth tongue sole (Cynoglossus semilaevis). The aim of this study was to select the most stable genes for quantitative real-time RT-PCR. Eight housekeeping genes (18S, TUBA, B2M, ACTB, EF1A, GAPDH, RPL17 and UBCE) were tested at different developmental stages, in different tissues, and following exposure to the drug SB-431542. Using geNorm, BestKeeper and NormFinder software, GAPDH/B2M, GAPDH/18S and UBCE/GAPDH were identified as the most suitable genes from samples taken of different developmental stages while 18S/RPL17 were consistently ranked as the best reference genes for different tissue types. Furthermore, TUBA/B2M, TUBA/UBCE and B2M/TUBA were found to be the most suitable genes in samples treated with the drug, SB-431542 by geNorm, BestKeeper and NormFinder respectively. Across both different developmental stages and tissue types, the combination of 18S and GAPDH was the most stable reference gene analyzed by Ref-Finder. To test and verify the screened reference genes, the expression profiles of LEFTY-normalized to the combination of GAPDH/18S and ACTB were presented. These results will be useful for future gene-expression studies in the half-smooth tongue sole.

Teleost fishes exhibit extraordinary diversity, plasticity and adaptability with their sex determination and sexual development, and there is growing evidence that non-coding RNAs (ncRNAs) are emerging as critical regulators of reproduction. Japanese flounder (Paralichthys olivaceus) is an important marine cultured fish that presents significant sexual dimorphism with bigger females, in which gynogenesis has been applied for aquaculture industry. In order to reveal the regulatory mechanisms of sexual development in gynogenetic female and sex-reversed neo-male P. olivaceus, the lncRNA-miRNA-mRNA interactions were investigated using high-throughput sequencing. A total of 6772 differentially expressed mRNAs (DEmRNAs), 2284 DElncRNAs, and 244 DEmiRNAs were obtained between gynogenetic female ovaries and sex-reversed neo-male testes. Genes in the steroid hormone biosynthesis and secretion pathway were enriched and mostly significantly upregulated in neo-male testes. Subsequently, network analysis uncovered high functional specificity for gynogenetic P. olivaceus sperm motility, as co-expressed DEmRNAs were significantly enriched in microtubule and cytoskeleton-related biological processes. Clustered miRNAs were characterized in the P. olivaceus genome with examples of the largest conserved let-7 clusters. The 20 let-7 members are distributed in 11 clusters and may not transcribe together with their neighboring miR-125b, with let-7 repressing cyp11a and miR-125b repressing esr2b, both as key steroidogenesis pathway genes. In summary, this study provides comprehensive insights into the mRNA-miRNA-lncRNA functional crosstalk in teleost sexual development and gametogenesis and will expand our understanding of ncRNA biology in teleost gynogenesis.

The scavenger receptors (SRs) gene family, as one of pattern recognition receptors, participates in the innate immune response in diverse lineages. However, the systematic identification, characteristics and functions of SRs family are lacking in teleost. Here, we identified all 19 SRs family members in Japanese flounder (Paralichthys olivaceus) based on the genome and transcriptome data. Phylogenetic and Ka/Ks analysis demonstrated that these SRs genes were divided into five classes and all exhibited pronounced purified selection pressures. Whole genome duplication event was found in colec12, scarb2, and lamp1. Gene structure, functional domain and motif distribution analyses indicated that SRs within the different subfamilies are severely conservative. SRs genes showed diverse expression patterns in the embryogenesis and unchanged tissues. The regulations of 14 SRs genes in blood, gill and kidney after E. tarda infection suggested their roles in innate immune response. Meanwhile, ten SRs genes were differentially expressed after E. tarda stimulation in macrophages in vitro. Then we proved that PoSCARA3 could suppress the activity of NF-κB and AP-1 in HEK 293T cells by dual-luciferase assays. In summary, this study provided valuable basis for further functional characterization and immune functions of SRs genes in P. olivaceus.

Myosins are important eukaryotic motor proteins that bind actin and utilize the energy of ATP hydrolysis to perform a broad range of functions such as muscle contraction, cell migration, cytokinesis, and intracellular trafficking. However, the characterization and function of myosin is poorly studied in teleost fish. In this study, we identified 60 myosin family genes in a marine teleost, black rockfish (Sebastes schlegelii), and further characterized their expression patterns. myosin showed divergent expression patterns in adult tissues, indicating they are involved in different types and compositions of muscle fibers. Among 12 subfamilies, S. schlegelii myo2 subfamily was significantly expanded, which was driven by tandem duplication events. The up-regulation of five representative genes of myo2 in the skeletal muscle during fast-growth stages of juvenile and adult S. schlegelii revealed their active role in skeletal muscle fiber synthesis. Moreover, the expression regulation of myosin during the process of myoblast differentiation in vitro suggested that they contribute to skeletal muscle growth by involvement of both myoblast proliferation and differentiation. Taken together, our work characterized myosin genes systemically and demonstrated their diverse functions in a marine teleost species. This lays foundation for the further studies of muscle growth regulation and molecular mechanisms of indeterminate skeletal muscle growth of large teleost fishes.

Myostatin (MSTN) as a negative regulator of muscle growth has been identified in Japanese flounder. Yet, most fish experienced the teleost specific genome duplication and possess at least two mstn genes. In current study, the second mstn gene named Pomstna is identified in Japanese flounder. Pomstna is clustered with other mstn2 of teleosts and owned highly conserved TGF-beta domain. In addition to muscle, Pomstna also highly expressed in brain and spleen. Using the primarily cultured muscle cells of Japanese flounder, we found that Pomstna could inhibit the proliferation and differentiation of muscle cells in vitro. As a ligand of TGF-beta signaling pathway, Pomstnb could regulate the expression of p21 and myod by activating the TGF-beta signaling pathway. Different from the function of Pomstnb, Pomstna could not activate the TGF-beta signaling pathway in vitro. During the differentiation of PoM cells, the expression of Pomstnb decreased significantly but the expression of Pomstna showed no change. Our study suggests that Pomstna could negatively regulate the growth and differentiation of muscle like Pomstnb yet through a different regulatory mechanism than Pomstnb. The present study suggests that muscle proliferation and differentiation were regulated by mstn not only through the TGF-beta signaling pathway but also other unknown mechanisms.

Pax3 and Pax7 are members of the Pax gene family which are essential for embryo and organ development. Both genes have been proved to be markers of muscle satellite cells and play key roles in the process of muscle growth and repair. Here, we identified two Pax3 genes (SsPax3a and SsPax3b) and two Pax7 genes (SsPax7a and SsPax7b) in a marine teleost, black rockfish (Sebastes schlegelii). Our results showed SsPax3 and SsPax7 marked distinct populations of muscle satellite cells, which originated from the multi-cell stage and somite stage, respectively. In addition, we constructed a muscle injury model to explore the function of these four genes during muscle repair. Hematoxylin-eosin (H-E) of injured muscle sections showed new-formed myofibers occurred at 16 days post-injury (dpi). ISH (in situ hybridization) analysis demonstrated that the expression level of SsPax3a and two SsPax7 genes increased gradually during 0-16 dpi and peaked at 16 dpi. Interestingly, SsPax3b showed no significant differences during the injury repair process, indicating that the satellite cells labeled by SsPax3b were not involved in muscle repair. These results imply that the muscle stem cell populations in teleosts are more complicated than in mammals. This lays the foundation for future studies on the molecular mechanism of indeterminant growth and muscle repair of large fish species.

The spotted knifejaw (Oplegnathus punctatus) is a newly emerging fishery species inhabiting the Pacific Ocean around Hawaii, China, Japan and Korean Peninsula. Little information on the mechanism of gonadal development and gametogenesis in this species could be used for research and breeding work. In this study, RNA-seq technology was applied to generate a deep-coverage sequencing data of spotted knifejaw testis and ovary. A total of 262,392,754 reads (ovary 133,403,270, testis 128,989,484) were generated from the cDNA library. After filtering and assembling, a total of 113,794 unigenes were obtained with the N50 of 1658bp. Unigenes were annotated with multiple public databases, including non-redundant protein databases (NR) (42,460, 37.31%), Swiss-Prot (33,632, 29.56%), eukaryotic Orthologus Groups (KOG) (26,195, 23.02%), Kyoto Encyclopedia of Genes and Genomes (KEGG) (10,978, 9.65%), and Gene Ontology (GO) (30,514, 26.82%). By comparing ovary and testis, 4496 differentially expressed unigenes (1986 in female, 2510 in male) were identified, in which 469 were specially expressed in females and 859 in males. The expression levels of 12 unigenes were confirmed by qRT-PCR. In addition, 35,054 simple sequence repeats were identified. By GO and KEGG analyses, a set of unigenes related to gonadal development and gametogenesis were filtered. foxl2 was deduced to be a key regulator for gonadal development and gametogenesis in females and dmrt1 in males. bmp15, nanos3, sox9 and amh were likely to function in the regulation of gonad physiology and germ line cells maintenance in ovary and testis. Interestingly, p53, apoptosis, Jak-STAT and neuroactive ligand-receptor interaction pathways were found to be pivotal in regulating gonadal development and gametogenesis of spotted knifejaw in various aspects. This study provides a fundamental support for further research in reproduction biology, population genetics and functional genomics in spotted knifejaw.

Ringer's or phosphate buffer saline (PBS) solution buffer usually was used as dilution butter in intraperitoneal injection. Stress could activate immune response, inflammatory response and glycogen metabolic process. The impact of solution buffer as a stressor to immune system was ignored in immunostimulatory experiment. In this report, we tested the hypothesis that the innate immune response and glycogen metabolic process were altered when it were challenged with Ringer's in Japanese flounder (Paralichthys olivaceus). RNA-seq was performed after challenge with Ringer's at 8 h and 48 h. The data revealed that the expression profiles of blood, gill, and kidney were significantly changed. Differentially expressed genes (DEGs) were identified, and energy metabolic and immune-related genes were up-regulated or down-regulated obviously. GO and KEGG analyses showed that DEGs were mainly enriched in innate immune terms and pathways. Weighted gene co-expression networks analysis (WGCNA) also indicated the highest association module with stress. A total of 16 genes were detected in the gray module, which were immune-related and metabolic-related genes. These results provided fundamental information on intraperitoneal injection with solution buffer. It offered useful clues to further explore the functional mechanism of stress and immunity.

A newly isolated Japanese flounder (Paralichthys olivaceus) β-actin promoter and its derivative compact construct Poβ-actinΔ−1080/−801Δ−500/−201 have recently been demonstrated to promote ectopic gene expression in cell lines. Different Poβ-actin promoter deletion mutants were constructed and functionally characterized. Mutational analyses by dual-luciferase detected that three regulatory elements, including one enhancer (−1399/−1081) and two silencers (−1080/−801, −500/−201) in the first intron. The sequence located at −1399/−1081 was determined to significantly affect promoter activity. Additionally, the first exon (−1489/−1400) could also remarkably promote the β-actin promoter activity. In the following transduction application, we removed the two silencers and generated a compact reconstruct promoter/enhancer (Poβ-actinΔ−1080/−801Δ−500/−201), which exhibited relatively stronger promoter activity compared with Poβ-actin. Furthermore, the green fluorescent protein (GFP) transgenic stable flounder cell line was obtained by the reconstructed Poβ-actinΔ−1080/−801Δ−500/−201 promoter. Our study provided the potential application of Japanese flounder β-actin, particularly Poβ-actinΔ−1080/−801Δ−500/−201, in ectopic gene expression in the future.

Members of transforming growth factor-β (TGF-β) superfamily are vital regulators during the development of fish ovary. However, its intraovarian functions in teleost are still unclear. As members of the TGF-β superfamily, gdf9 and bmp15 are necessary for follicle formation and granulosa cell proliferation. Here in Japanese flounder, quantitative real-time polymerase chain reaction (qRT-PCR) and in situ hybridization (ISH) analysis showed that gdf9 and bmp15 were mainly expressed in oogonia and oocytes, whereas weakly expressed in non-ovarian tissues. Overexpression of single gdf9 and the co-overexpression with bmp15 could up-regulate the expression of most steroidogenic genes, while the overexpression of single bmp15 could down-regulate the expression of most steroidogenic genes. These findings demonstrate that single gdf9 and the combination with bmp15 may act as "activator", while single bmp15 may act as "inhibitor" in the process of steroidogenesis in flounder. This was also verified in negative feedback regulation of gdf9 and bmp15 during hormone treatment. High concentration of human chorionic gonadotropin (hCG) could down-regulate gdf9 and up-regulate bmp15, which were beneficial for the homeostasis of hCG hormone. Besides, knockdown of either gdf9 or bmp15 could significantly down-regulate most steroidogenic genes. This indicated that heterodimer of GDF9:BMP15 might be the most bioactive ligand in gonad development of flounder. Taken together, our study provided a novel recognition that gdf9 and bmp15 could regulate steroidogenesis in teleost through mechanism different from that in mammals.

Fish-egg lectins (FELs) have been identified in several teleost species and have been proved to play important roles in innate immune system against pathogen infection. In this study a novel fish-egg lectin (OppFEL) was identified from spotted knifejaw (Oplegnathus punctatus), and the expression patterns against bacterial infection was characterized. The amino acid sequence is highly homologous to other teleost FELs, containing five repeats of the conserved TECPR domain. Expression of OppFEL was widely observed in examined tissues, with the most abundant transcripts observed in gill, showing a pattern of tissue specific expression. The OppFEL expression was significantly up-regulated following a Gram-negative bacterium (Vibrio anguillarum) challenge in vivo, suggesting participation in host antibacterial immune responses. Recombinant OppFEL protein (rOppFEL) possessed calcium dependent binding capacities and agglutination to four Gram-negative bacterium and two Gram-positive bacterium. Sugar binding assay revealed that rOppFEL specifically bound to insoluble lipopolysaccharide and peptidoglycan. In addition, rOppFEL was also proved to have hemagglutinating activity against erythrocytes from Mus musculus, O. punctatus, Sebastes schlegelii and Paralichthys olivaceus. Dual-luciferase analysis showed that overexpression of OppFEL could suppress the activity of NF-κB in a dose dependent manner. Taken together, these results suggest that OppFEL is a unique fish-egg lectin that possesses apparent immunomodulating property and is involved in host defense against pathogens invasion.