Alternative splicing of histone demethylase Kdm6bb mediates temperature-induced sex reversal in the Nile tilapia.

Sex determination in many fish species is remarkably plastic and temperature sensitive. Nile tilapia display a genetic sex-determination system (XX/XY). However, high-temperature treatment during critical thermosensitive periods can induce XX females into XXm pseudo-males, and this phenomenon is termed temperature-induced sex reversal (TISR). To investigate the molecular mechanism of TISR in Nile tilapia, we performed Iso-seq analysis and found a dramatic effect of high temperature on gene alternative splicing (AS). Kdm6bb histone demethylase showed a novel AS at intron 5 that generates Kdm6bb_tv1 transcripts without intron 5 and Kdm6bb_tv2 with intron 5. Kdm6bb_tv1 encodes a full-length protein while Kdm6bb_tv2 encodes a truncated protein. Expression analysis revealed that intron 5 splicing of Kdm6bb is male and gonad biased at larval stage, and only gonad biased at adult stage. High-temperature treatment induced intron 5 splicing in the gonads of XX and XY fish, resulting in increased Kdm6bb_tv1 expression. To directly test the role of Kdm6bb_tv1 in Nile tilapia TISR, we knocked out expression of Kdm6bb_tv1. However, Kdm6bb_tv1-/- homozygous mutants showed embryonic lethality. Overexpression of Kdm6bb_tv1, but not Kdm6bb_tv2, induced sex reversal of XX females into pseudo-males. Overexpression of Kdm6bb_tv1, as with high-temperature treatment, modified the promotor region of Gsdf and Dmrt1 by demethylating the trimethylated lysine 27 of histone 3 (H3K27me3), thereby increasing expression. Collectively, these studies demonstrate that AS of Kdm6bb intron 5 increases the expression of Kdm6bb_tv1, which acts as a direct link between high temperature and activation of Gsdf and Dmrt1 expression, leading to male sex determination.

Expression changes of non-specific cytotoxic cell receptor (NCCRP1) and proliferation and migration of NCCs post-Nocardia seriolae infection in Northern Snakehead.

Non-specific cytotoxic cells (NCCs) are essential to the cytotoxic cell-mediated immune response in teleost. The fish non-specific cytotoxic cell receptor protein 1 (NCCRP1) plays an important role as a membrane protein in the recognition of target cells and the activation of NCC. However, the roles of fish NCCs during pathogen infection require comprehensive studies. In this study, the coding sequence of northern snakehead (Channa argus) nccrp1 (Canccrp1) was cloned. Canccrp1 contains an open reading frame of 690 bp, encoding a peptide of 229 amino acids with a conserved F-box-associated domain (FBA) and proline-rich motifs (PRMs). Transcriptional expression analysis revealed that the constitutive expression of Canccrp1 was higher in the immune-related organs, such as liver, kidneys, and spleen. Moreover, mRNA and protein expression of Canccrp1 gradually increased in the spleen at 1-6 days post infection (dpi) with Nocardia seriolae, in addition to reaching peak expression in both the kidneys and liver at 2 dpi. A polyclonal antibody prepared against recombinant CaNCCRP1 effectively labeled NCCs in peripheral blood and different tissues. Then, immunofluorescence (IF) staining showed that the number of NCCs was significantly increased and showed a scattered distribution in the early stages of N. seriolae infection (2 and 4 dpi) before the forming of granulomas. At the late stages of N. seriolae infection (6 dpi), more NCCs migrated to preexisting granulomas, showing significant coaccumulation with N. seriolae. All these results clearly indicate the expression changes of CaNCCRP1, and the number and localization changes of NCCs post-N. seriolae infection, implying potential roles for fish NCCs in the antimicrobial infection process in fish.

Transcriptome analysis in the spleen of Northern Snakehead (Channa argus) challenged with Nocardia seriolae.

Northern snakehead (Channa argus) is an indigenous fish species and is one of popularly cultured snakeheads in China and other Asian countries. Unfortunately, Nocardia seriolae infections have caused considerable losses in the snakehead aquaculture industry. However, the infectivity and the immune response induced by N. seriolae in snakehead are unclear. In order to better understand the immune response of Northern snakehead in a series of time points after N. seriolae challenge, we conducted the transcriptomic comparison in snakehead spleen at 48, 96, and 144 h after the challenge of N. seriola against their control counterparts. Gene annotation and pathway analysis of differentially expressed genes (DEGs) were carried out to understand the functions of the DEGs. Additionally, protein-protein interaction networks were conducted to obtain the interaction relationships of immune-related DEGs. These results revealed the expression changes of multiple DEGs and signaling pathways involved in immunity during N. seriolae infection, which will facilitate our comprehensive understanding of the mechanisms involved in the immune response to bacterial infection in the northern snakehead.

Global expression response of genes in sex-undifferentiated Nile tilapia gonads after exposure to trace letrozole.

The aromatase inhibitor letrozole can be found in rivers, effluents, and even drinking water. Studies have demonstrated that letrozole affects various metabolic pathways and may cause reproductive toxicity, especially in fish exposed during development. However, studies on the effect of a low concentration of letrozole at the whole-gonad transcriptomic level in the early stage of fish sexual development have not been investigated. The aim of our study was to explore the potential effects of a low concentration of letrozole on the gonad transcriptome of Nile tilapia at an early stage of sexual development. In this study, 9 dpf (days postfertilization) Nile tilapia were exposed to trace letrozole for 12 days. Letrozole exposure from 9 dpf to 21 dpf persistently altered phenotypic sex development and induced the male-biased sex ratio. The transcriptome results showed that 1173 differentially expressed genes (DEGs) were present in the female control vs 1.5 µg/L letrozole-treated female comparison group and that 1576 DEGs were present in the 1.5 µg/L letrozole-treated female vs male control comparison group. Differentially expressed gene enrichment analysis revealed several crucial pathways, including the drug metabolism-cytochrome P450 pathway, the ErbB-PI3K/Akt/mTOR pathway, and the calcium signalling pathway. Further analysis of these identified DEGs indicated that some key genes correlated with metabolism and epigenetic regulation were significantly affected by letrozole, such as UDP-glucuronosyltransferase (Ugt), glutathione S-transferase omega-1 (Gsto1), lysine-specific demethylase 6bb (Kdm6bb, original name is Kdm6a), jumonji and AT-rich interaction domain containing 2 (Jarid2b, original name is Jarid2), growth arrest and DNA damage inducible gamma (Gadd45g), and chromobox protein 7 (Cbx7). The qRT-PCR validation results for twelve DEGs showed that the Pearson's correlation of the log10fold change values between the qPCR and RNA-Seq results was 0.90, indicating the accuracy and reliability of the RNA-Seq results. Our study is the first to report the effect of letrozole on the transcriptome of gonads from fish during early-stage sexual development. These findings will be useful for understanding the toxic effects and molecular mechanisms of letrozole exposure at the early stage of gonad development on the sexual development of aquatic organisms.

Transcriptome Profiling and Analysis of Genes Associated with High Temperature-Induced Masculinization in Sex-Undifferentiated Nile Tilapia Gonad.

Artificially high temperatures during critical thermosensitive periods (TSPs) can induce the sex reversal of Nile tilapia (Oreochromis niloticus) females into pseudomales; Nile tilapia is a GSD + TE (genotypic plus temperature effects) fish species. Previous studies have shown that water temperature affects the expression levels of many genes in the gonad or brain in various teleost species. However, few studies on the effect of temperature at the whole-gonad transcriptomic level in the early stage of sex differentiation have been reported in fish species exhibiting GSD + TE. In this study, RNA-Seq was performed to characterize the transcriptomic profile and identify genes exhibiting temperature- and sex-biased expressions in the Nile tilapia gonad at 21 dpf. A total of 42 genes were found to be associated with both high-temperature treatment and sex development, as the expression levels of these genes differed in both FC (female control) vs MC (male control) and FC vs FT (high temperature-treated females in the TSP). Among these genes, the transcriptional alterations of many male sex determination and differentiation genes, such as Dmrt1, Gsdf, and the DNA damage-inducible protein GADD45 alpha, suggested that the male pathway is initiated after high-temperature treatment and that its initiation may play a role in high temperature-induced masculinization in Nile tilapia. The qRT-PCR validation results for thirteen differentially expressed genes showed that the Pearson's correlation of the log10 fold change values between the qPCR and RNA-Seq results was 0.70 (p < 0.001), indicating the accuracy and reliability of the RNA-Seq results. Our study provides insights into how high-temperature treatment induces the sex reversal of Nile tilapia females.

Profiling expression changes of genes associated with temperature and sex during high temperature-induced masculinization in the Nile tilapia brain.

Fish sex-determining mechanisms can be classified as genotypic (GSD), temperature (TSD), or genotypic plus temperature effects (GSD+TE). Previous studies have shown that culturing water temperature during thermosensitive periods (TSP) could affect the expression of many genes in the gonad in some fish. However, few studies have focused on gene expression changes in the brain after temperature treatment during TSP in fish species. In this study, three families were developed by crossing XX neomales with XX females and one of them was used for transcriptome analysis. The results showed that a total of 105, 3164 and 4666 DEGs were respectively obtained in FC (female control) vs. FT (high temperature-treated females at TSP), FC vs. MC (male control), and MC vs. FT comparison groups. By profiling analysis, we show that the mRNA expression levels of 16 differentially expressed genes (DEGs) exhibited significant downregulation or upregulation after high temperature treatment and reached a similar level as that in MC. Among the 16 DEGs, LOC100699848 (lysine specific demethylase 6A) and Jarid2 contained JmjC domain, showing the possible important role of JmjC domain in response to temperature treatment in Nile tilapia. Kdm6b (lysine demethylase 6B) and Jarid2 have been shown to play important roles in reptile TSD, showing the relative conservation of underlying regulation mechanisms between TSD in reptile and TSD or GSD+TE in fish species. Finally, the transcriptome profiling was validated by quantitative real-time PCR in nine selected genes. These results provide a direction for investigating the GSD+TE molecular mechanism in fish species.

Gonad Transcriptome Analysis of High-Temperature-Treated Females and High-Temperature-Induced Sex-Reversed Neomales in Nile Tilapia.

BACKGROUND: Nowadays, the molecular mechanisms governing TSD (temperature-dependent sex determination) or GSD + TE (genotypic sex determination + temperature effects) remain a mystery in fish. METHODS: We developed three all-female families of Nile tilapia (Oreochromis niloticus), and the family with the highest male ratio after high-temperature treatment was used for transcriptome analysis. RESULTS: First, gonadal histology analysis indicated that the histological morphology of control females (CF) was not significantly different from that of high-temperature-treated females (TF) at various development stages. However, the high-temperature treatment caused a lag of spermatogenesis in high-temperature-induced neomales (IM). Next, we sequenced the transcriptome of CF, TF, and IM Nile tilapia. 79, 11,117, and 11,000 differentially expressed genes (DEGs) were detected in the CF-TF, CF-IM, and TF-IM comparisons, respectively, and 44 DEGs showed identical expression changes in the CF-TF and CF-IM comparisons. Principal component analysis (PCA) indicated that three individuals in CF and three individuals in TF formed a cluster, and three individuals in IM formed a distinct cluster, which confirmed that the gonad transcriptome profile of TF was similar to that of CF and different from that of IM. Finally, six sex-related genes were validated by qRT-PCR. CONCLUSIONS: This study identifies a number of genes that may be involved in GSD + TE, which will be useful for investigating the molecular mechanisms of TSD or GSD + TE in fish.

Characterization of 2-Cys peroxiredoxin 3 and 4 in common carp and the immune response against bacterial infection.

Accumulating evidence suggests that peroxiredoxins (Prxs) eliminate excessive cellular H2O2 and are important factors in redox signaling pathways. In this study, we cloned the full-length cDNAs and genomic sequences of Prx3 and Prx4 from common carp. The common carp Prx3 and Prx4 open reading frames were 753 base pairs (bp) and 783bp in length, respectively, and contained seven exons and six introns. Multiple sequence alignment and phylogenetic analyses revealed that the common carp Prx1-4 proteins share high identities and similar characteristics with other known animal Prxs. Prx3 and Prx4 mRNA were constitutively expressed in all tissues, and the highest Prx3 and Prx4 transcript abundances occurred in head kidney. Although the highest Prx4 protein and mRNA expression were also observed in head kidney, many differences were detected between Prx4 mRNA and protein expression levels in other tissues. Prx3 expression increased significantly in the head kidney 12h after an Aeromonas hydrophila challenge. The A. hydrophila challenge upregulated Prx3 mRNA expression in liver and spleen, increased Prx4 mRNA expression levels in liver and spleen excluding at 36h in spleen, but decreased Prx4 mRNA expression level in the head kidney. The mature Prx4 peptide was recombinantly expressed and purified using Dextrin Beads 6FF and it exhibited thioredoxin (Trx)-dependent peroxidase activity. These data suggest that Prx3 and Prx4 are constitutive and inducible proteins that might play important roles in innate immune function. The Trx-dependent peroxidase activity analysis of recombinant Prx4 further verified the important role of Prxs in the redox system of fish.

Epigenetic control of cyp19a1a expression is critical for high temperature induced Nile tilapia masculinization.

In fish species with temperature-dependent sex determination (TSD) or genotypic sex determination plus temperature effects (GSD + TE), temperature can either affect sex differentiation or determine the sex. However, it is unknown if epigenetic control of cyp19a1a expression is critical for high temperature induced masculinization in the freshwater fish Nile tilapia. We analyzed the cyp19a1a DNA methylation levels in three age groups and found that they were lower in females than in males. At 8 months of age, males had DNA methylation levels of the cyp19a1a promoter that were almost twice as high as those of females. Exposure to high temperatures increased the cyp19a1a promoter DNA methylation levels from 30.87 ± 4.56% to 48.34 ± 0.92% (P = 0.035) in females and from 50.33 ± 7.38% to 51.66 ± 4.75% in males (P = 0.867). The increases in the cyp19a1a promoter DNA methylation levels were associated with the mRNA expression levels and might play a role in promoting gonadal differentiation in high temperature induced group females toward the male pathway. Western blot analysis revealed that the cyp19a1a protein expression levels in females significantly declined after high temperature treatment; only a slight decline was recorded in male fish. These results reveal that epigenetic control of cyp19a1a mRNA and protein expression is related to the environmental temperature and sex ratios in fish with TSD or GSD + TE.

Global DNA Methylation Changes in Nile Tilapia Gonads during High Temperature-Induced Masculinization.

In some fish species, high or low temperature can switch the sex determination mechanisms and induce fish sex reversal when the gonads are undifferentiated. During this high or low temperature-induced sex reversal, the expressions of many genes are altered. However, genome-wide DNA methylation changes in fish gonads after high or low temperature treatment are unclear. Herein, we compared the global DNA methylation changes in the gonads from control females (CF), control males (CM), high temperature-treated females (TF), and high temperature-induced males (IM) from the F8 family of Nile tilapia (Oreochromis niloticus) using methylated DNA immunoprecipitation sequencing. The DNA methylation level in CF was higher than that in CM for various chromosomes. Both females and males showed an increase in methylation levels on various chromosomes after high-temperature induction. We identified 64,438 (CF/CM), 63,437 (TF/IM), 98,675 (TF/CF), 235,270 (IM/CM) and 119,958 (IM/CF) differentially methylated regions (DMRs) in Nile tilapia gonads, representing approximately 0.70% (CF/CM), 0.69% (TF/IM), 1.07% (TF/CF), 2.56% (IM/CM), and 1.30% (IM/CF)of the length of the genome. A total of 89 and 65 genes that exhibited DMRs in their gene bodies and promoters were mapped to the Nile tilapia genome. Furthermore, more than half of the genes with DMRs in the gene body in CF/CM were also included in the IM/CM, TF/CF, TF/IM, and IM/CF groups. Additionally, many important pathways, including neuroactive ligand-receptor interaction, extracellular matrix-receptor interaction, and biosynthesis of unsaturated fatty acids were identified. This study provided an important foundation to investigate the molecular mechanism of high temperature-induced sex reversal in fish species.

Differential expression analysis of genes involved in high-temperature induced sex differentiation in Nile tilapia.

Nowadays, high temperature effects on the molecular pathways during sex differentiation in teleosts need to be deciphered. In this study, a systematic differential expression analysis of genes involved in high temperature-induced sex differentiation was done in the Nile tilapia gonad and brain. Our results showed that high temperature caused significant down-regulation of CYP19A1A in the gonad of both sexes in induction group, and FOXL2 in the ovary of the induction group. The expressions of GTHα, LHß and ERα were also significantly down-regulated in the brain of both sexes in the induction and recovery groups. On the contrary, the expression of CYP11B2 was significantly up-regulated in the ovary, but not in the testis in both groups. Spearman rank correlation analysis showed that there are significant correlations between the expressions of CYP19A1A, FOXL2, or DMRT1 in the gonads and the expression of some genes in the brain. Another result in this study showed that high temperature up-regulated the expression level of DNMT1 in the testis of the induction group, and DNMT1 and DNMT3A in the female brain of both groups. The expression and correlation analysis of HSPs showed that high temperature action on tilapia HSPs might indirectly induce the expression changes of sex differentiation genes in the gonads. These findings provide new insights on TSD and suggest that sex differentiation related genes, heat shock proteins, and DNA methylation genes are new candidates for studying TSD in fish species.

Molecular cloning, characterization and expression of cathepsin D from grass carp (Ctenopharyngodon idella).

Cathepsin D is a lysosomal aspartic proteinase which participates in various degradation functions within the cell. In this current study, we cloned and characterized the complete cDNA of grass carp cathepsin D through 5'- and 3'-RACE. The cathepsin D contained a 56 bp 5' terminal untranslated region (5'-UTR), a 1197 bp open reading frame encoding 398 amino acids, and a 394 bp 3'-UTR. Grass carp cathepsin D shared high similarity with those from other species, and showed the highest amino acid identity of 91% to Danio rerio. Unlike many other organisms, the grass carp cathepsin D contains only one N-glycosylation site closest to the N-terminal. Real-time quantitative RT-PCR demonstrated that Cathepsin D expressed in all twelve tissues (bladder, brain, liver, heart, gill, muscle, fin, eye, intestines, spleen, gonad and head kidney). The relative expression levels of Cathepsin D in gonad and liver were 26.58 and 24.95 times as much as those in fin, respectively. The expression level of Cathepsin D in muscle approximately 16-fold higher, in intestines and spleen were 12-fold higher. The cathepsin D expression showed an upward trend during embryonic development. After challenged with Aeromonas hydrophil, the expression of grass carp cathepsin D gene showed significant changes in the four test tissues (liver, head kidney, spleen and intestines). The fact that the bacterial infection can obviously improve the cathepsin D expression in immune-related organs, may suggest that cathepsin D plays an important role in the innate immune response of grass carp.

Induction of mitogynogenetic diploids and identification of WW super-female using sex-specific SSR markers in half-smooth tongue sole (Cynoglossus semilaevis).

The half-smooth tongue sole (Cynoglossus semilaevis) is an important cultured marine fish as well as a promising model fish for the study of sex determination mechanisms. In the present study, a protocol for artificial induction of mitogynogenesis by hydrostatic pressure using heterologous sperm was developed in half-smooth tongue sole in order to assess homozygosity of gynogens and to identify WW super-female. The optimal initiation time for pressure shock of mitogynogenetic embryos was determined to be 21.5 min after insemination when water temperature is at 22-23°C, while the optimal pressure and treatment duration were determined to be 70 MPa for 4 min. About 1,500 mitogynogenetic diploid larvae were obtained. Ten tongue sole microsatellite markers were used for homozygosity analysis of 24 mitogynogenetic larvae. Among the 24 larvae, the percentage of homozygosity ranged from 73.91% to 87.50% with an average homozygosity of 80.54%. Sex-specific simple sequence repeat (SSR) markers, CseF-SSR1, were isolated and used for identifying WW super-female mitogynogens in the tongue sole. The amplification of genomic DNA using the sex-specific SSR marker produced one DNA band of 206 bp in ZZ males, two DNA bands of 206 and 218 bp in ZW females, and one DNA band of 218 bp in WW super-females. Four WW "super-female" gynogens were observed in 39 mitogynogenetic diploids, indicating a ZW sex determination mechanism in the tongue sole. Thus, a protocol for the induction of artificial mitogynogenesis has been developed for the first time in half-smooth tongue sole, and the WW super-female diploids were identified in the mitogynogens by sex-specific SSR markers. These findings lay the foundation and provide important tool for the elaboration of sex determination mechanism, generation of WW super-females, and development of clone line and breeding of all-female stock in the half-smooth tongue sole.

[Isolation of microsatellite markers for Lateolabrax japonicus and polymorphic analysis].

To investigate population structure and marker assisted breeding, fast isolation by AFLP of sequences containing repeats (FIASCO) and GenBank database mining were used to develop novel microsatellite markers for sea perch (Lateolabrax japonicus). Genomic DNA fragments containing SSR sequences were captured by hybridization to (GT)(13) biotin-labeled probe and were ligated to PMD18-T vector. Among 150 randomly chosen clones from the SSR-enriched library, 66 sequences contained microsatellite motif over five repeats. In addition, 540 cDNA sequences and 132 ESTs of Lateolabrax japonicus were downloaded from GenBank and screened for di-, tri- and tetra-nucleotide repeats, while 22 sequences were found to contain microsatellites. As a result, 15 microsatellite loci were shown to be polymorphic in 30 Lateolabrax japonicus individuals, with the alleles ranging from two to ten, the observed heterozygosities from 0.6000-1.0000, and the expected heterozygosities from 0.5079-0.8890. Four loci (SP17, SP52, SP94 and SP468) were deviated from HWE in the sampled population after Bonferroni's correction, and no linkage disequilibrium was found among all loci (P<0.003), whereas null alleles were detected at locus SP52 (P<0.05). Among 15 polymorphic loci, the PIC values, which can be used for related population genetics analysis, were all above 0.5, with the exception of SP17 and SP468.

Growth differences and dimorphic expression of growth hormone (GH) in female and male Cynoglossus semilaevis after male sexual maturation.

Half-smooth tongue sole, Cynoglossus semilaevis, is an ideal model to investigate the regulatory mechanisms of sexual growth dimorphism in fish species. The aim of the study was to investigate the effect of differential age of sexual maturity for females and males on growth and GH mRNA expression in C. semilaevis. The body weight differences between the sexes were not significant in C. semilaevis at age 5 months when females and males were all immature. Significant differences in body weight between the sexes were found after early sexual maturation of males at the age of 9 months. The body weight of 21-month-old females (621.4 ± 86.4g), still not immature, was even 3.28 times higher than that of the males (189.7 ± 14.4g). The cDNAs encoding GH in C. semilaevis was cloned. The GH gene is 2924bp long and consists of six exons and five introns. The results of qRT-PCR showed that GH mRNA levels of the immature females were not significantly different from that of immature males at age 5 months. However, GH mRNA levels of the immature females were significantly higher compared with those of the mature males at age 9 months (P<0.05). At age 11 months, GH mRNA levels of females were even 6.4-fold higher than that of males. In conclusion, for the first time we show that early sexual maturity of males is the main cause of sexual growth dimorphism in C. semilaevis and exert significant effect on GH mRNA expression.

Growth differences and differential expression analysis of pituitary adenylate cyclase activating polypeptide (PACAP) and growth hormone-releasing hormone (GHRH) between the sexes in half-smooth tongue sole Cynoglossus semilaevis.

Pituitary adenylate cyclase activating polypeptide (PACAP) and growth hormone-releasing hormone (GHRH) are regulators of growth hormone secretion. In this article, we examined the difference in growth and mRNA expression of PACAP and GHRH between the sexes in half-smooth tongue sole, an important cultured fish species indicating sexually growth dimorphism in China. Firstly, a significant body weight difference between females and males was first observed at 7 months (P<0.05) and at 18 onths the mean body weight of the females (771.0±44.3 g) was as much as 4.9 times higher than that of males (130.6±6.0 g). As a result, half-smooth tongue sole, Cynoglossus semilaevis, is a good model to investigate the effects of growth-related genes expression on sexual growth dimorphism. Secondly, the cDNAs encoding PRP/PACAP and GHRH were isolated. Two differently processed mRNA transcripts of PRP/PACAP (PRP-encoding and PRP splice variant) were found. PACAP and GHRH mRNA was highly abundant in brain and less abundant in other tissues. However, PACAP mRNA was expressed in most brain regions, and was lower in the cerebellum. GHRH mRNA was predominantly expressed in the hypothalamus and weakly expressed in all areas of the brain examined. Ontogenetic expression analysis indicated that PACAP and GHRH mRNA was detected in the early stages of embryogenesis. Finally, differential expression showed that there was no significant difference of the expression level of PACAP or GHRH between the sexes before 8 months of age. However, between 9 and 12 months of age, the GHRH mRNA expression level in males was significantly higher than in females (P<0.05), which might be associated with GH deficiency in males. In contrast, the male PACAP mRNA expression level was not significantly higher than that in females even at 9 and 12 months of age. The present results provide important clues for understanding the sexual growth dimorphism mechanisms in half-smooth tongue sole.

Molecular cloning, genomic structure, polymorphism and expression analysis of major histocompatibility complex class IIA and IIB genes of half-smooth tongue sole (Cynoglossus semilaevis).

Major histocompatibility complex (MHC) genes play an important role in the immune response of vertebrates. Its function is to present foreign peptide to the T-cell. In order to study the function and molecular polymorphism of class II genes in teleost, the full lengths of MHC class IIA and IIB cDNA were cloned from half-smooth tongue sole by homology cloning and rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR). Genomic organizations, molecular polymorphism, and expression profiles of class IIA and IIB were examined to study the function in fish. As in other teleost, four exons and three introns were identified in half-smooth tongue sole class IIA gene, five exons and four introns were identified in class IIB gene. The deduced amino acid sequence of class IIA had 27.3-69.8% identity with those of mammal and teleost. Nine class IIA alleles were identified from four individuals. Four different alleles observed in a single individual may infer the existence of two loci at least. The deduced amino acid sequence of class IIB had 7.9-71.9% identity with those of other species. Fifteen class IIB alleles were identified. Six different alleles observed in a single individual may suggest that there are at least three loci in class IIB genes. Real-time quantitative RT-PCR demonstrated that the MHC class IIA and IIB were ubiquitously expressed in twelve normal tissues. Challenge of half-smooth tongue sole with the pathogenic bacteria, Vibrio anguillarum, resulted in significant changes in the expression of MHC IIA and IIB mRNA in three tissues.

Construction of a genetic linkage map and mapping of a female-specific DNA marker in half-smooth tongue sole (Cynoglossus semilaevis).

The half-smooth tongue sole (Cynoglossus semilaevis, hereafter, "tongue sole") is a marine flatfish with great commercial importance for fisheries and aquaculture in China. It has also been a promising model for the study of sex determination mechanisms in fish. Here, we report the construction of a genetic linkage map for the tongue sole, based on 137 markers including 103 AFLP markers, 33 microsatellite markers, and one female-specific DNA marker. Twenty-six linkage groups (LGs) were found. The total map length was 934.6 cM (Kosambi), with an average spacing of 8.4 cM, covering 64.4% of the estimated genome size. Furthermore, a female-specific SCAR marker, CseF-382, was mapped on LG5. This study represents the first genetic linkage map in the tongue sole. This map has great potential in the identification of quantitative traits loci and sex-related genes and marker-assisted selection in the tongue sole. Meanwhile, the new set of polymorphic microsatellite markers developed in this study is not only useful for genetic mapping but also of critical importance for studies on genetic diversity and broodstock management in tongue sole.

[Development of female-specific AFLP marker CseF783 and its application in genetic sex identification in half-smooth tongue sole (Cynoglossus semilaevis)].

Molecular sex identification is important in studying sex control, sex determination, and all-female breeding in half-smooth tongue sole (Cynoglossus semilaevis). In the present study, a female-specific AFLP marker was isolated from Cynoglossus semilaevis by AFLP technique using the selective primer combination E-ACT/M-CAA. This marker was re-amplified, recovered from the agarose gels, cloned and sequenced. Bioinformatic analysis indicated that the length of the product was 791 bp, and the sequence showed no similarity to any known sequences deposited in the GenBank database using BLASTn. According to the DNA sequence of the female-specific AFLP marker, specific PCR primers were designed and PCR amplification was performed on 100 sex-known individuals of C. semilaevis (50 females and 50 males each). A specific band 324 bp in length was present in all females but absent in all males (except for one male), indicating that the female-specific AFLP marker was successfully converted into female-specific SCAR (sequence characterized amplified regions) marker. The sex analysis of 3-day-old C. semilaevis individuals using this female-specific SCAR marker indicated that the female ratio was 41.7%. The female-specific SCAR marker developed in this study allowed simple, reliable, and rapid molecular sex identification using small amounts of fin tissue without sacrifice of C. semilaevis especially at early stage of development.