Tongue Sole Research Articles

Two Nipped-B-Like Protein A (Nipbla) Gametologs in Chinese Tongue Sole (Cynoglossus semilaevis): The Identification of Alternative Splicing, Expression Pattern, and Promoter Activity Analysis

In mammals, the mutation of nipped-B-like protein (nipbl) leads to Cornelia de Lange Syndrome (CdLS), characterized by low birth weight, short stature, and structural abnormalities of the skeleton, heart, and gut. In Chinese tongue sole (Cynoglossus semilaevis), a typical marine fish exhibiting sexual size dimorphism, the nipbl homolog gene (nipped-B-like protein A (nipbla)) was also screened with female higher expression level by somatotropic and reproductive tissues’ transcriptomic analysis. In this study, two nipbla genes, namely, nipbla-w and nipbla-z, were identified from the W and Z chromosomes of C. semilaevis, respectively. Similar to other mammalian and fish species nipbl, the two homolog proteins of C. semilaevis contained two conserved domains, namely, cohesion_HEAT and Nipped-B_C. The phylogenetic tree analysis showed that these two nipbla gametolog proteins were first clustered together and then grouped with other fish species. At least two types of alternative splicing sites were observed in exon 12 of the nipbla-z gene, which produced nipbla-z-tv1 and nipbla-z-tv2. Also, the sex-biased expression patterns of different nipbla-w and nipbla-z transcripts in female and male tissues were revealed by quantitative PCR (qPCR). The highest expression level of nipbla-w was observed in female gonad. While nipbla-z-tv1 exhibited relatively high expression in the muscle, liver, gonad, and brain, nipbla-z-tv2 only showed its expression superiority in the muscle of male individuals. The promoter regions of nipbla genes were amplified, and their transcription activity was successfully verified by a dual-luciferase reporter system. After nipbla-w and nipbla-z knockdown in the brain cell lines by RNA interference, a series of growth-related genes were influenced, including Bone Morphogenetic Protein 4 (bmp4), Wnt Family Member 11 (wnt11), and Sprouty Related EVH1 Domain Containing 2 (spred2). The prediction of transcription factors suggested that c-Jun, sex-determining region Y (SRY), POU Class 1 Homeobox 1 (POU1F1a), myogenic differentiation antigen (MyoD), signal transducer and activator of transcription 5a (STAT5A), and nuclear factor I C (Nfic) might be the putative upstream regulatory factors for nipbla; among them, c-Jun has been verified to effectively regulate the transcriptional activity of nipbla. The identification of two nipbla genes provided important data for interpreting the sexual size dimorphism in C. semilaevis.

Identification of lncRNA-miRNA-mRNA Network Involved in Sexual Size Dimorphism of Chinese Tongue Sole (Cynoglossus semilaevis)

Chinese tongue sole (Cynoglossus semilaevis) is a flatfish species unique to Northeast Asia, exhibiting the typical female-biased sexual size dimorphism (SSD). To explore the possible regulatory roles of non-coding RNAs (ncRNAs) on this phenomenon, whole transcriptomic analysis was performed by using female, male, and pseudomale C. semilaevis to identify differentially expressed (DE) long ncRNAs (DE lncRNAs), microRNA (DE miRNAs), and differentially expressed genes (DEGs) from the brain, gonad, liver, and muscle tissues. Most of them were concentrated in the gonad and muscle, and the gene expression patterns of pseudomale individuals were similar to male individuals. The association of DE lncRNAs and target messenger RNAs (mRNAs) was predicted based on antisense, cis-, and trans-regulatory mechanisms, with enriched protein digestion and absorption, cyclic adenosine monophosphate (cAMP) signaling pathway, sulfur metabolism, cell cycle, and splicesome (p < 0.05). Furthermore, weighted gene co-expression network analysis (WGCNA) was employed to cluster the expression patterns of DE lncRNA, and two modules (greenyellow and blue) had the highest positive and negative correlations with growth traits, respectively. Importantly, the female-biased expression in the greenyellow module and the male- and pseudomale-biased expression in the blue module were observed in the gonad. The target gene analysis for DE miRNA revealed 3,034 mRNA-miRNA pairs with the opposite expression patterns. Finally, the lncRNA-miRNA-mRNA network, including 385 DE lncRNAs, 138 DE miRNAs, and 456 DEGs, was constructed. Among which, 78 DE lncRNAs, 12 DE miRNAs, and 13 DEGs involved in cell growth and death pathway were related to the SSD of C. semilaevis. This study described the lncRNA-miRNA-mRNA regulatory network in the SSD of C. semilaevis for the first time. The functional prediction analysis suggested that these DE lncRNAs and DE miRNAs might be involved in flatfish SSD by regulating several potential growth-related pathways (e.g., cell cycle, cAMP signaling, and Rap1 signaling). Further studies related to these ncRNAs will enlarge our understanding of the regulatory effects of ncRNAs on fish SSD.

Identification of Potential Blind-Side Hypermelanosis-Related lncRNA-miRNA-mRNA Regulatory Network in a Flatfish Species, Chinese Tongue Sole (Cynoglossus semilaevis).

Blind-side hypermelanosis has emerged as a major concern in commercial rearing environments of the flatfish aquaculture industry. To date, the underlying molecular mechanisms are not well understood. To fill this gap, in this study, whole transcriptomic sequencing and analyses were performed using normal skins and hypermelanic skins of the blind side of Chinese tongue sole (Cynoglossus semilaevis). Differentially expressed long non-coding RNAs (DElncRNAs), miRNAs (DEmiRNAs), and differentially expressed genes as well as their competing endogenous RNA (ceRNA) networks were identified. A total of 34 DElncRNAs, 226 DEmiRNAs, and 610 DEGs were identified. Finally, lncRNA–miRNA–mRNA regulatory networks (involving 29 DElncRNAs, 106 DEmiRNAs, and 162 DEGs) associated with blind-side hypermelanosis were constructed. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of 162 DEGs in ceRNA networks identified DEGs (e.g., oca2, mc1r, and ihhb) in pigmentation-related biological processes and DEGs (e.g., ca4, glul, and fut9) in nitrogen metabolism, glycosphingolipid biosynthesis, and folate biosynthesis pathways, as well as their corresponding DElncRNAs and DEmiRNAs to potentially play key regulatory roles in blind-side hypermelanosis. In conclusion, this is the first study on the ceRNA regulatory network associated with blind-side hypermelanosis in flatfish. These new findings expand the spectrum of non-coding regulatory mechanisms underpinning blind-side hypermelanosis, which facilitates the further exploration of molecular regulatory mechanisms of malpigmentation in flatfish.

Immune role of the complement component 6 gene and its associated novel miRNA, miR-727, in half-smooth tongue sole (Cynoglossus semilaevis)

The complement component 6 (C6) gene is a component of the membrane attack complex (MAC), which causes rapid lytic destruction of bacteria. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene stability, including that of immune genes. However, current research on the function of C6 and its regulation by miRNAs is lacking. In the present study, we identified and characterized C6 and a novel miRNA, miR-727 (designated CsC6 and Cse-miR-727, respectively), of the half-smooth tongue sole (Cynoglossus semilaevis) that responded to infection with Vibrio anguillarum, a Gram-negative pathogen of marine fish. The full-length cDNA of CsC6 contained a 256 bp 5′ untranslated region (5′-UTR), a 2820 bp open reading frame (ORF) encoding 939 amino acids, and a 205 bp 3′-UTR. SMART analysis showed that CsC6 contains typical C6 domains, including three TSP1 domains, one LDLa domain, one MACPF domain, two CCP domains and two FIMAC domains. CsC6 and Cse-miR-727 are widely expressed in the 13 tissues of half-smooth tongue sole, and their expression in immune tissues is significantly changed after V. anguillarum infection, generally showing an inverse trend. We confirmed that CsC6 was the target gene of Cse-miR-727 using the dual luciferase reporter assay and that Cse-miR-727 regulated CsC6 at the protein level using quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. The hepatic expression levels of not only the MAC components C7, C8α, C8β, C8γ and C9 but also the MAPKs, NF-κβ, AP-1, IL1β, IL6 and TNFα, which are involved in many signaling pathways, changed significantly in half-smooth tongue sole following stimulation with the Cse-miR-727 agomir and inhibitor. This evidence suggested that CsC6 could be mediated by Cse-miR-727 to affect MAC assembly and immune signaling pathways in half-smooth tongue soles. To our best knowledge, this study is the first to investigate the regulatory mechanism and immune response of complement genes mediated by miRNAs in fish.

Large-Scale Screening of Growth-Related Variants in Chinese Tongue Sole (Cynoglossus semilaevis)

Growth is the most valuable economic trait for improving aquaculture fish species, since fast growth can reduce labor cost and make more economic benefits. However, the knowledge about how many and which genes are related to the growth of Chinese tongue sole is limited. High-throughput sequencing screening of variants is a fast, economical and accurate assay to identify genes related to growth in crops, livestock and other aquaculture products. In the present study, genome-wide resequencing of 30 female Chinese tongue sole individuals from large and small groups to screen growth trait-related variations (SNPs, InDels) was carried out. In total, 6545735 SNPs and 1016745 InDels were detected, while 31 genes related to growth traits were identified. Their functions were mainly involved in muscle architecture, post-embryonic development, neurosensory development and hormone regulation. Furthermore, 18 of the 31 genes related to growth trait are located on W chromosome, indicating that W chromosome plays an important role in regulating the body size of female tongue soles. The markers and genes identified in our study can be applied to selective breeding of tongue sole and thus will promote the aquaculture industry and increase economic efficiency.

DNA Methylation of pacap Gene Is Involved in Growth Superiority of Female Half Smooth Tongue Sole (Cynoglossus semilaevis) in Different Salinities

Pituitary adenylate cyclase activating polypeptide (PACAP) and growth hormone-releasing hormone (GHRH) play important roles in the GH/IGF growth axis in fishes. To determine whether epigenetic change is involved in the regulation of pacap and ghrh responses to low salinity stress in Cynoglossus semilaevis, the correlation between growth traits, DNA methylation status and gene expression level in low salinity (15, S15) and optimal salinity (30, S30) at day 7 (D7) and day 60 (D60) were analyzed. Results showed that exposure to low salinity for 60 days attenuated C. semilaevis growth rate. Under low salinity, DNA methylation level of pacap promoter increased in females and decreased in males at day 7, but inverted at day 60. Additionally, pacap expression was up-regulated in both males and females. The pacap promoter methylation level was higher and expression level was lower in female than in male. The results suggest that pacap promoter methylation level is negatively correlated to mRNA level and positively correlated to body weight, while gene expression level is negatively related with body weight. With the decrease of salinity, DNA methylation level of ghrh promoter and exon1, as well as its gene expression displayed minor changes. Overall, pacap gene seems to play an important role in fish growth, contributing to female growth superiority, while ghrh gene seems not pertinent under salinity stress. The results indicate that low salinity potentially affects fish growth through regulating DNA methylation in pacap promoter. This study expands the understanding of the molecular mechanism of how salinity modulates fish growth from the epigenetic perspective.

Transcriptome Profiling Reveals the Sexual Dimorphism of Gene Expression Patterns during Gonad Differentiation in the Half-Smooth Tongue Sole (Cynoglossus semilaevis).

The half-smooth tongue sole (Cynoglossus semilaevis), one of the most economically-important fish species in China, exhibits sexually dimorphic growth. An understanding of sex-related gene expression patterns in the tongue sole may inform sex regulation and breeding processes that increase fish production. However, the gene expression patterns during gonad development in the tongue sole remain unknown. In this study, transcriptome sequencing analyses were performed during gonad differentiation in the tongue sole, namely, at 62days post-hatching (dph), 100dph, 120dph, and 150dph. Differentially expressed genes associated with sex differentiation and gonad development were identified at each time point. Trend analysis showed that gene expression patterns varied over time. These expression patterns either explained common, non-sexually-dimorphic features or indicated significant sexual dimorphism. Transcript structure analyses identified both sex and time differences among samples. This study investigated the time-dependent expression patterns of several sex-related genes, including Dmrt1, Amh, Foxl2, aromatase encoding gene, Esr, and the Sox gene family, during gonad differentiation in the tongue sole. These results might clarify the significant sexual differences during early development in the tongue sole and might provide insight into the mechanisms controlling sex differentiation and development.

Cloning and Characterization of Cholesterol 25-Hydroxylase (ch25h) From a Marine Teleost, Chinese Tongue Sole (Cynoglossus semilaevis), and Its Gene Expressions in Response to Dietary Arachidonic Acid

Our previous studies have shown that ARA regulated the gonadal steroidogenesis and the synthesis of gonadotrophin-releasing hormone (GnRH) in the brain in a gender-dependent manner in Chinese tongue sole, a marine teleost with typical sex dimorphism. As a following up-study, the current study aimed to clone and characterize a gene responding to dietary ARA differently between males and females, cholesterol 25-hydroxylase (ch25h), which has important roles in cholesterol and lipid metabolism of mammals, but is rarely investigated in fish. The full-length cDNA of Chinese tongue sole ch25h was cloned and characterized, and its transcription in the gonads, brain and liver of both males and females in response to dietary ARA levels (0.34%, 2.53%, and 9.63% of TFA) was investigated. The Chinese tongue sole Ch25h, which putatively is Ch25h subtype B, shared moderate identity to its known orthologs of other teleost and lower identity to human Ch25h. It has high transcription levels in the gonads, followed by skin and muscle, but low levels in the intestine, spleen, and kidney. High ARA levels significantly increased the ch25h transcription in the gonads and brain of male fish, but did not affect the transcription in the females. However, the effect of dietary ARA on ch25h transcription in the liver showed an opposite gender-difference pattern to those in the gonads and brain. To our knowledge, this is the first study in marine teleost investigating the nutritional regulation of ch25h expression.

Assessment of Polycyclic Aromatic Hydrocarbon Concentrations and Heavy Metal Profile of Traditional Drum-Smoked and Convective Kiln-Smoked Bonga Shad and Nigerian Tongue Sole

This study assessed the polycyclic aromatic hydrocarbon (PAH) concentrations and heavy metal profile of traditional drum-smoked and convective kiln-smoked Bonga shad and Nigerian tongue sole. Forty samples of fresh Bonga shad and Nigerian tongue sole were purposively collected from twenty processing centers in Lagos State. The samples were divided into two batches with each batch smoked using traditional drum and kiln methods. Chemical PAHs and heavy metals (lead, mercury, cadmium, and chromium) were analyzed. Results showed that the concentrations of major PAHs fluorene, anthracene, benzo(b)fluoranthene, benzo(a)anthracene, benzo(a)pyrene, and benzo(ghi)perylene were as follows: for Bonga shad, 0.54 ± 0.03, 0.56 ± 0.07, 0.59 ± 0.07, 0.54 ± 0.04, 0.53 ± 0.04, and 0.57 ± 0.01, respectively and for Nigerian tongue sole, 0.48 ± 0.06, 0.62 ± 0.03, 0.57 ± 0.03, 0.56 ± 0.06, 0.62 ± 0.08, and 0.61 ± 0.08 mg kg−1, respectively for drum-smoked samples while for kiln-smoked samples, the values were: for Bonga shad, 0.43 ± 0.13, 0.52 ± 0.08, 0.37 ± 0.11, 0.36 ± 0.13, 0.34 ± 0.20, and 0.38 ± 0.23 and for Nigerian tongue sole, 0.41 ± 0.18, 0.49 ± 0.06, 0.37 ± 0.11, 0.37 ± 0.10, 0.28 ± 0.07, and 0.34 ± 0.13 mg kg−1, respectively. The values for drum-smoked fish samples exceeded the maximum permissible level of 0.5 mg kg−1 (Regulation EU 1272/2013). Lead, mercury, cadmium, and chromium contents of drum-smoked Bonga shad and Nigerian tongue sole were 0.12 ± 0.03, 0.20 ± 0.04, 0.22 ± 0.05, and 0.10 ± 0.03, respectively and 0.15 ± 0.04, 0.13 ± 0.04, 0.19 ± 0.05, and 0.12 ± 0.04 mg kg−1, respectively while the values for kiln-smoked Bonga shad and Nigerian tongue sole were 0.16 ± 0.05, 0.19 ± 0.05, 0.24 ± 0.05, and 0.12 ± 0.03, respectively and 0.18 ± 0.04, 0.15 ± 0.04, 0.19 ± 0.05, and 0.16 ± 0.04 mg kg−1, respectively which were within safety limits of World Health Organization. The smoking methods and fish types had significant difference on PAHs (t = 8.326, df = 5.0, p = 0.0002). In conclusion, the study showed that drum-smoked fish had higher chemical contaminants when compared with kiln-smoked fish and could pose health risks to consumers if not appropriately monitored.

Transcriptome of Gonads From High Temperature Induced Sex Reversal During Sex Determination and Differentiation in Chinese Tongue Sole, Cynoglossus semilaevis.

The sex of Chinese tongue sole (Cynoglossus semilaevis) is determined by both genetic sex determination (GSD) and environmental sex determination (ESD), making it an ideal model to study the relationship between sex-determination and temperature. In the present study, transcriptomes of undifferentiated gonads from genetic females and males, as well as differentiated gonads from males, females, and pseudomales under high and normal temperature treatments were generated for comparative transcriptomic analysis. A mean of 68.24 M high-quality clean reads was obtained for each library. Differentially expressed genes (DEGs) between different sexes and environmental treatments were identified, revealing that the heat shock protein gene family was involved in the high temperature induced sex reversal. The Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways that were enriched in pseudomale and genetic female comparison included neuroactive ligand-receptor interaction, cortisol synthesis and secretion, and steroid hormone biosynthesis. Furthermore, weighted gene co-expression network analyses were conducted on all samples, and two modules were positive correlated with pseudomale under high temperature. An illustrated protein-protein interaction map of the module identified a hub gene, hsc70. These findings provide insights into the genetic network that is involved in sex determination and sexual differentiation, and improve our understanding of genes involved in sex reversal under high temperature.

Novel molecular markers for high-throughput sex characterization of Cynoglossus semilaevis

Male and female Chinese tongue soles (Cynoglossus semilaevis) exhibit a great difference in growth rates. Karyotype analysis has revealed that the species is heterogametic (ZW/ZZ), and sex-reversed “pseudo-males” exist under both natural and aquaculture conditions. In this study, we developed three female-specific molecular markers for the sex identification of C. semilaevis. The markers were an insertion-deletion (indel)-type structural variation and two single nucleotide polymorphism (SNP) loci on the ZW sex chromosome (SNP_chr_8936186_C_G and SNP_chr_8935925_C_T). The indel marker can be used for female-specific identification with traditional PCR and agarose gel electrophoresis. The detection and use of the SNPs are based on the penta-primer amplification refractory mutation system. The SNPs on the ZW chromosome were successfully identified with high-throughput quantitative PCR. The two types of molecular markers were successfully used to identify normal males and pseudo-males in a population of 60 C. semilaevis. When compared with currently used markers, the markers identified in this study have high accuracy and can help in high-throughput detection. Our findings could provide an effective method for detecting the gender of fish with the ZW karyotype.

Extraction Optimization and Characterization of Gelatin from Half-Smooth Tongue Sole (Cynoglossus semilaevis Gunther) Skin

ABSTRACTHalf-smooth tongue sole is a popular warm-water fish, and its skin may be an available resource for gelatin extraction. Response surface method was used to optimize the extraction of gelatin from the fish skin. The most suitable conditions for maximum gelatin yield (24.2%) were pretreatment with 1.9 g/L NaOH, followed by hot water extraction at 63.54ºC for 4.81 h. The obtained gelatin had a high imino acid content (185 residues/1,000 residues), with a high content of crude protein and low levels of ash, fat, and moisture. The predominant sodium dodecyl sulfate–polyacrylamide gel electrophoresis bands revealed that the gelatin contained low-molecular-weight peptides, α-, β-, and γ chains. As compared with bovine gelatin, the fish skin gelatin solution had lower values of strength and clarity and higher levels of L* and viscosity. Additionally, Fourier-transform infrared spectra showed that gel from the gelatin had four amide bands (amide A, amide I, amide II, and amide III) similar to commercial gelatin. The results suggest that half-smooth tongue sole skin could be a promising source of gelatin in view of the comparability between its gelatin and commercial gelatin in physicochemical properties.

Coating Effects of ε-Polylysine and Rosmarinic Acid Combined with Chitosan on the Storage Quality of Fresh Half-Smooth Tongue Sole (Cynoglossus semilaevis Günther) Fillets

The study was to evaluate the effects of chitosan-based coating combined with rosmarinic acid (RA) with different concentrations of ε-polylysine (ε-PL) on flavor retention and sensorial properties of half-smooth tongue sole fillets during storage at 4 °C. Results showed that chitosan-based coatings combined with ε-PL and RA contributed to the reduction of off-flavor compounds, such as total volatile base nitrogen (TVB-N), trimethylamine (TMA), and ATP-related compounds, and accumulation of free amino acids (FAAs). Nineteen volatile organic compounds were analyzed by gas chromatography-mass spectrometer (GC/MS) during storage, including seven alcohols, six aldehydes, and six ketones. The coating treated fresh half-smooth tongue sole (HTS) fillets significantly reduced the relative content of off-odor volatiles, such as 1-octen-3-ol, propanal, hexanal, and octanal. According to sensory evaluation results, chitosan-based coating combined with ε-PL and RA was an effective way to maintain quality of HTS fillets during refrigerated storage.

Structural and functional conservation of half-smooth tongue sole Cynoglossus semilaevis RIP3 in cell death signalling

Receptor interacting protein kinase 3 (RIP3) has emerged as an essential molecular regulator in human inflammation with accumulating evidence in vertebrates. However, the structure and functions of RIP3 in teleosts remains elusive. Here we identified one RIP3 homologue from half-smooth tongue sole Cynoglossus semilaevis, designated CsRIP3. The open reading frame (ORF) of CsRIP3 contained 1491 nucleotides and encoded a protein with 496 amino acids (Mw = 55.44 kDa). CsRIP3 expression was detected in various tissues in half-smooth tongue soles. CsRIP3 expression was up-regulated after pathogens i.e. Vibrio and poly (i:c) treatment in vivo, indicating its possible role in teleost immune response. Further analysis using human cells demonstrated that CsRIP3 exhibited highly conserved pro-apoptotic and pro-necroptotic functions in comparison with human RIP3. In conclusion, for the first time we reported the homologous structure and functions of CsRIP3 in inflammatory cell death, which provides novel perspectives on fish immunity studies in future.

Low-Field NMR and MRI to Analyze the Effect of Edible Coating Incorporated with MAP on Qualities of Half-Smooth Tongue Sole (Cynoglossus Semilaevis Günther) Fillets during Refrigerated Storage

Nondestructive and fast measurement and characterization of fish is highly desired during various processing treatments. This research investigated the effectiveness of low field LF-NMR and MRI as fast monitoring techniques to estimate the qualities of half-smooth tongue sole fillets treated with edible coating combined with modified atmosphere packaging during refrigeration. T2 relaxation spectra showed three peaks representing bound water (T21), immobile water (T22), and free water (T23), respectively. pT22 accounted for the largest proportion of three types of water, followed by pT23. The weighted MRI provided the internal structure information associated with different samples, indicting the combination of edible coating and MAP (70% CO2 + 30% N2) is the best performance in the maintenance of qualities and freshness of HTS fillets. All results demonstrated that the combination of LF-NMR and MRI as fast and nondestructive methods have great potential to monitor qualities deterioration and predict shelf life in of HTS fillets during refrigerated storage.

Quality Characteristics of Tongue Sole (Cynoglossus semilaevis) Jangajji with Different Types of Gochujang during Low Temperature Storage

Quality Characteristics of Tongue Sole (Cynoglossus semilaevis) Jangajji with Different Types of Gochujang during Low Temperature Storage

Effects of different dietary DHA:EPA ratios on gonadal steroidogenesis in the marine teleost, tongue sole (Cynoglossus semilaevis).

The present study was conducted to investigate the effects of dietary DHA and EPA on gonadal steroidogenesis in mature females and males, with a feeding trial on tongue sole, a typical marine teleost with sexual dimorphism. Three experimental diets differing basically in DHA:EPA ratio, that is, 0·68 (diet D:E-0·68), 1·09 (D:E-1·09) and 2·05 (D:E-2·05), were randomly assigned to nine tanks of 3-year-old tongue sole (ten females and fifteen males in each tank). The feeding trail lasted for 90 d before and during the spawning season. Fish were reared in a flowing seawater system and fed to apparent satiation twice daily. Compared with diet D:E-0·68, diet D:E-1·09 significantly enhanced the oestradiol production in females, whereas diet D:E-2·05 significantly enhanced the testosterone production in males. In ovaries, diet D:E-1·09 induced highest mRNA expression of follicle-stimulating hormone receptor (FSHR), steroidogenic acute regulatory protein, 17α-hydroxylase (P450c17) and 3β-hydroxysteroid dehydrogenase (3β-HSD). In testes, diet 2·05 resulted in highest mRNA expression of FSHR, cholesterol side-chain cleavage enzyme, P450c17 and 3β-HSD. Fatty acid profiles in fish tissues reflected closely those of diets. Female fish had more gonadal EPA content but less DHA content than male fish, whereas there was a reverse observation in liver. In conclusion, the dietary DHA:EPA ratio, possibly combined with the dietary EPA:arachidonic acid ratio, differentially regulated sex steroid hormone synthesis in mature female and male tongue soles. Females seemed to require more EPA but less DHA for the gonadal steroidogenesis than males. The results are beneficial to sex-specific nutritive strategies in domestic teleost.

Locus Mapping, Molecular Cloning, and Expression Analysis of rps6kb2, a Novel Metamorphosis-Related Gene in Chinese Tongue Sole (Cynoglossus semilaevis).

Flatfish metamorphosis denotes the extraordinary transformation of a symmetric pelagic larva into an asymmetric benthic juvenile. This unique process involves eye migration, a 90° rotation in posture, and asymmetrical pigmentation for adaptation to a benthic lifestyle. In the present study, we used genetics to map a metamorphosis-related locus (q-10M) in the male linkage group (LG10M), a small interval of 0.9cM corresponding to a 1.8M-bp physical area in chromosome 9 in the Chinese tongue sole (Cynoglossus semilaevis). Combined with single-marker analysis, ribosomal protein S6 kinase 2 (rps6kb2) a member of the family of AGC kinases was identified as a novel metamorphosis-related candidate gene. Its expression pattern during metamorphosis was determined by quantitative RT-PCR and whole-mount in situ hybridization analysis. rps6kb2 gene was significantly expressed in metamorphic climax stage larvae and distributed in all the tissues transforming during metamorphosis, including tail, jaw, eye and skin of larvae. The results suggest that rps6kb2 has a general role in tissue transformations during flatfish metamorphosis including tail changes, skull remodeling, eye migration, and asymmetrical pigmentation.

Sexually Dimorphic Gene Expression Associated with Growth and Reproduction of Tongue Sole (Cynoglossus semilaevis) Revealed by Brain Transcriptome Analysis.

In this study, we performed a comprehensive analysis of the transcriptome of one- and two-year-old male and female brains of Cynoglossus semilaevis by high-throughput Illumina sequencing. A total of 77,066 transcripts, corresponding to 21,475 unigenes, were obtained with a N50 value of 4349 bp. Of these unigenes, 33 genes were found to have significant differential expression and potentially associated with growth, from which 18 genes were down-regulated and 12 genes were up-regulated in two-year-old males, most of these genes had no significant differences in expression among one-year-old males and females and two-year-old females. A similar analysis was conducted to look for genes associated with reproduction; 25 genes were identified, among them, five genes were found to be down regulated and 20 genes up regulated in two-year-old males, again, most of the genes had no significant expression differences among the other three. The performance of up regulated genes in Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was significantly different between two-year-old males and females. Males had a high gene expression in genetic information processing, while female’s highly expressed genes were mainly enriched on organismal systems. Our work identified a set of sex-biased genes potentially associated with growth and reproduction that might be the candidate factors affecting sexual dimorphism of tongue sole, laying the foundation to understand the complex process of sex determination of this economic valuable species.

Origin and evolution of GATA2a and GATA2b in teleosts: insights from tongue sole, Cynoglossus semilaevis.

Background. Following the two rounds of whole-genome duplication that occurred during deuterostome evolution, a third genome duplication occurred in the lineage of teleost fish and is considered to be responsible for much of the biological diversification within the lineage. GATA2, a member of GATA family of transcription factors, is an important regulator of gene expression in hematopoietic cell in mammals, yet the role of this gene or its putative paralogs in ray-finned fishes remains relatively unknown.Methods. In this study, we attempted to identify GATA2 sequences from the transcriptomes and genomes of multiple teleosts using the bioinformatic tools MrBayes, MEME, and PAML. Following identification, comparative analysis of genome structure, molecular evolution rate, and expression by real-time qPCR were used to predict functional divergence of GATA2 paralogs and their relative transcription in organs of female and male tongue soles (Cynoglossus semilaevis).Results. Two teleost GATA2 genes were identified in the transcriptomes of tongue sole and Japanese flounder (Paralichthysolivaceus). Synteny and phylogenetic analysis confirmed that the two genes likely originated from the teleost-specific genome duplication . Additionally, selection pressure analysis predicted these gene duplicates to have undergone purifying selection and possible divergent new functions. This was supported by differential expression pattern of GATA2a and GATA2b observed in organs of female and male tongue soles.Discussion. Our results indicate that two GATA2 genes originating from the first teleost-specific genome duplication have remained transcriptionally active in some fish species and have likely undergone neofunctionalization. This knowledge provides novel insights into the evolution of the teleost GATA2 genes and constituted important groundwork for further research on the GATA gene family.