Search

Abstract Background Genes in the major histocompatibility complex (MHC) have a critical role in both the innate and adaptive immune responses because of their involvement in presenting foreign peptides to T cells. However, the nature has remained largely unknown. Results We examined the genetic variation in MHC class IIB in half-smooth tongue sole (Cynoglossus semilaevis) after challenge with vibrio anguillarum. Two thousand and four hundred fry from 12 half-smooth tongue sole families were challenged with Vibrio anguillarum. To determine any association between alleles and resistance or susceptibility to V. anguillarum, 160 individuals from four high-resistance (HR, < 40.55% mortality) families and four low-resistance (LR, > 73.27% mortality) families were selected for MHC IIB exon2 gene sequence analysis. The MHC IIB exon2 genes of tongue sole displayed a high level of polymorphism and were discovered at least four loci. Meanwhile, the dN/dS [the ratio of non-synonymous (dN) substitutions to synonymous (dS) substitutions] in the peptide-binding region (PBR) was higher than that in the non-peptide-binding region (non-PBR). Eighty-eight alleles were discovered among 160 individuals, and 13 out of 88 alleles were used to analyze the distribution pattern between the resistant and susceptible families. Certain alleles presented in HR and LR with a different frequency, while other alleles were discovered in only the HR or LR families, not both. Five alleles, Cyse-DBB*6501, Cyse-DBB*4002, Cyse-DBB*6102, Cyse-DBB*5601 and Cyse-DBB*2801, were found to be associated with susceptibility to V. anguillarum with a frequency of 1.25%, 1.25%, 1.25%, 1.25% and 2.5% in the HR families, and 35%, 33.75%, 27.5%, 16.25%, 15% in the LR families (p < 0.01, 0.01, 0.01, 0.01, 0.01), respectively. Four alleles, Cyse-DBB*3301, Cyse-DBB*4701, Cyse-DBB*6801 and Cyse-DBB*5901, were found to be associated with resistance to V. anguillarum, with a frequency of 13.75%, 11.25%, 11.25%, 8.75% in the HR families and 1.25%, 1.25%, 1.25%, 1.25% and 1.25% in the LR families (p < 0.01, 0.05, 0.05 and p = 0.064), respectively. Conclusions Elucidation of the role of MHC II B genes in half-smooth tongue sole should prove to be helpful to the in-depth development of marker-assisted selective breeding in half-smooth tongue sole.

Abstract: 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 5months 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 9months. 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 5months. However, GH mRNA levels of the immature females were significantly higher compared with those of the mature males at age 9months (P<0.05). At age 11months, 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. [Copyright &y& Elsevier]

Abstract: 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 7months (P <0.05) and at 18months the mean body weight of the females (771.0±44.3g) was as much as 4.9 times higher than that of males (130.6±6.0g). 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 8months of age. However, between 9 and 12months 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 12months of age. The present results provide important clues for understanding the sexual growth dimorphism mechanisms in half-smooth tongue sole. [ABSTRACT FROM AUTHOR]

Abstract: Cytochrome P450 aromatase (P450arom) is an enzyme responsible for the conversion of androgen to estrogen. The P450arom plays an important role in gonadal differentiation and sex determination in teleosts. To study the role of gonadal cytochrome P450 aromatase (P450aromA) in sex differentiation, the cDNA was isolated from the ovary of the half-smooth tongue-sole, Cynoglossus semilaevis. The deduced amino acid sequence exhibited high homology with P450arom of other teleosts. Alignment and phylogenetic analysis of the P450arom showed that the tongue sole P450aromA belonged to the gonadal P450arom subfamily. The 5''-flanking region of the P450aromA gene contains two Ad4 binding motifs and a half site of the palindrome sequence of the estrogen-responsive element (the ERE half). P450aromA transcripts were highly abundant in the ovary, less abundant in the testis, but not present in the brain, liver, spleen, kidney, muscle, skin, heart, head kidney, gill, intestine, or eyes by RT-PCR. The P450aromA mRNA increased with gonad development, but was less abundant in the testis compare to the ovary in all phases. Highly abundant P450aromA was detected in the female ovary, but was less abundant in the testis in neo-males obtained by methyltestosterone or temperature treatment. These results imply that P450aromA may be involved in gonadal development and sex differentiation in the tongue sole. [Copyright &y& Elsevier]

Abstract: Half-smooth tongue sole females grow larger and faster than males. An all-female population would be of significant benefit for tongue sole aquaculture. In the present study, a female-specific AFLP marker (CseF305) was isolated from female genomic DNA of the tongue sole and sequenced. One pair of SCAR primers was designed based on the sequences of the female-specific marker. A PCR method for identifying genetic sex of the sole was developed. PCR amplification of genomic DNA from tongue sole adults using the SCAR primers resulted in a specific fragment in 30 female individuals, but not in 30 males. Secondly, effects of methyltestosterone treatment on the female:male sex ratio of tongue sole fry were examined. Methyltestosterone at a concentration of 20–100 μg/L·H2O can induce genetic females to reverse to phenotypic males in juvenile tongue sole and produce a high proportion of males (97–100% males). Phenotypic males with female genotype, that is, neo-males, were detected by using female-specific SCAR primers. The neo-males were cultured and matured, and used to mate with normal females to produce progeny. 130,000 fry were produced by using sperm from neo-males. Genetic sex identification demonstrated that 73% of the neo-male progeny fry contained female-specific DNA markers. Three combinations of sex chromosomes (ZZ, ZW and WW) were observed in the neo-male progeny. WW super-female individuals containing 2 huge heteromorphic chromosomes were found in some fry. [Copyright &y& Elsevier]

Abstract: A half-smooth tongue-sole, Cynoglossus semilaevis Sox10 (Accession no.: EU070763) was isolated from brain of tongue sole by using homologous cloning and RACE method. The complete cDNA of the tongue sole Sox10 contains a 35 bp 5′UTR, a 1338 bp open reading frame (ORF) encoding 445 amino acids and a 1155 bp 3′UTR. A condensed phylogenetic tree was constructed based on the amino acid sequences of tongue sole Sox10 and other well-defined vertebrate Sox. The overall topology of the tree showed the tongue sole Sox10 clusters with all Sox10. Alignment of amino acid residues of the tongue sole Sox10 gene with those from other vertebrate indicated high level conservation of amino acid sequence. The RT-PCR analysis demonstrated that the tongue sole Sox10 was highly expressed in brain, gills, skin and eyes, intermediately in spleen, heart, head–kidney and muscles, weakly expressed in kidneys and intestine and no expression in liver and gonad. The Sox10 was also expressed weakly in germ cell and zygote. We cannot detect the expression of the Sox10 in 8-cells stage. However it resumed expression weakly from blastula stage to middle of gastrula. And it expressed highly from neurula stage to 25 dah (day after hatching). It suggested that the Sox10 was involved in the development of embryos and larvae in tongue sole. [Copyright &y& Elsevier]

Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) play crucial roles as signaling mediators for the TNF receptor (TNFR) superfamily and the interleukin-1 receptor/Toll-like receptor (IL-1R/TLR) superfamily. TRAFs collectively play important roles in multiple biological processes and organismal immunity. However, systematic identification of the TRAF gene family in teleost fish has not yet been reported, and there is little available information about its roles in innate immunity in Chinese tongue sole (Cynoglossus semilaevis), an aquaculture fish of high economic value. In the present study, we identified and characterized seven TRAF genes, namely, CsTRAF2a , CsTRAF2b , CsTRAF3 , CsTRAF4 , CsTRAF5 , CsTRAF6 and CsTRAF7, in Chinese tongue sole, and the complete ORFs of the CsTRAFs were cloned. Sequence analysis revealed various genomic structures of the CsTRAFs and showed that they contain typical conserved domains compared with mammalian TRAFs. Phylogenetic analysis indicated the evolutionary relationships of TRAF family members in teleost fish and revealed an absence of TRAF1 in most species and TRAF5 in some species of teleosts. Analysis of the gene structures and motifs showed the diversity and distribution of exon-intron structures and conserved motifs in Chinese tongue sole and several other teleost species. Real-time quantitative PCR was used to investigate the expression patterns of CsTRAF genes in tissues of healthy fish and in the gills, livers and spleens of fish after bacterial infection with Vibrio harveyi. The results indicate that only CsTRAF2a is relatively highly expressed in the brain and that the other CsTRAFs are highly expressed in immune-related tissues and may participate in the immune response after infection with pathogenic bacteria. Functional analysis of CsTRAF3 , CsTRAF4 and CsTRAF6 revealed that only CsTRAF6 could strongly activate the NF-кB pathway after overexpression of CsTRAF3 , CsTRAF4 and CsTRAF6 in HEK-293T cells. This systematic analysis provided valuable information about the diverse roles of TRAFs in the innate immune response to pathogenic bacterial infection in teleost fish and will contribute to the functional characterization of CsTRAF genes in further research. • We identified seven TRAF genes in Chinese tongue sole, subsequently cloned the complete ORFs of CsTRAFs. • Bioinformatic analysis confirmed the identities and nomenclature. • The CsTRAFs are highly expressed in immune-related tissues and participate in the immune response after V. harveyi infection. • Only CsTRAF6 could strongly activated NF-кB pathway after co-transfection with NF-кB reporter plasmid in HEK-293T cells. [ABSTRACT FROM AUTHOR]

The growth arrest and DNA-damage-inducible protein 45 gamma (Gadd45g) is known to play a major role in embryonic development and sex determination. In this study, two Gadd45g genes were isolated from half-smooth tongue sole ( Cynoglossus semilaevis ). Using chromosomal fluorescence in situ hybridization (FISH), Gadd45g1 and Gadd45g 2 were located on the W and Z chromosomes, respectively. The full-length cDNA sequences of Gadd45g1 (1270 bp) and Gadd45g2 (1181 bp) were predicted to contain a 480-bp coding sequence that could encode a protein of 159 amino acids residues. A phylogenetic tree showed that the predicted Gadd45g1 and Gadd45g2 amino acid sequences clustered closely in one branch. It is proposed that Gadd45g1 and Gadd45g2 are paralogous genes derived from the divergence of the sex chromosome. Ka/Ks ratios indicated that Gadd45g1 and Gadd45g2 may have undergone a high number of mutations and have a divergence time of only about 68,000 years, although Gadd45g homologs are highly conserved. The qRT-PCR demonstrated that Gadd45g1 and Gadd45g2 were highly expressed in ovary, and negligibly expressed in testis of male and neo-male. During development of the ovary (from 80 to 150 days), the expression levels of both genes reached high levels. Gadd45g1 was also highly expressed at 50 days, the stage just before gonad differentiation in C. semilaevis . All these findings imply functional divergence of the two Gadd45g homologs; Gadd45g1 may be necessary for sex differentiation in the early stage of gonad development, and then Gadd45g1 and Gadd45g2 maintain ovary development and the female character of half-smooth tongue sole. [ABSTRACT FROM AUTHOR]

Abstract: Lysozymes are considered to be potent innate immune molecules against the invasion of bacterial pathogens. The goose-type lysozyme is one of the three major distinct lysozyme types identified in the animal kingdom including teleosts. In this report, we identified, sequenced, and characterized the goose-type lysozyme gene (CsGLys) from half-smooth tongue sole (Cynoglossus semilaevis). The full-length cDNA of CsGLys is 1191 bp in length from the transcription start site to polyadenylation site, including a 91 bp 5′-terminal untranslated region (UTR), a 452 bp 3′-terminal UTR and a 648 bp open reading frame (ORF) of encoding a polypeptide with 215 amino acids. The deduced amino acid sequence of CsGLys possesses a Goose Egg White Lysozyme (GEWL) domain with three conserved residues (E91, D104 and D121) essential for catalytic activity. The CsGLys gene consisting of 2535 bp, was similar to those of other teleost species such as Japanese flounder and large yellow croaker with five exons interrupted by four introns. The 5′-flanking region of CsGLys gene shows several transcriptional factor binding sites related to immune response. Tissue expression profile analysis by quantitative real-time reverse transcription PCR showed that CsGLys mRNA was constitutively expressed in all examined tissues with the predominant expression in skin and the weakest expression in heart. The expression of CsGLys after challenged with bacteria Vibrio anguillarum was up-regulated in blood, head kidney, liver and spleen at 12 h post-infection and it reached the peak level at the same time point with a 19.89-, 4.21-, 14.45- and 10.37-fold increase, respectively, while the CsGLys expression was down-regulated to lower level than the normal level in each tested tissues except in liver from the 48 h until 96 h. These results suggest that CsGLys might play an important role in half-smooth tongue sole host defense against the bacteria infection. [Copyright &y& Elsevier]

Abstract: Here we present a comparison of the different chromosome preparation methods, including the method of the single embryo or larva, the method of juvenile-swimming and the method of phytohemagglutinin injection, used for the different developmental stages of the half-smooth tongue sole, Cynoglossus semilaevis. The mean index of mitosis for the three methods was 0.79%, 0.09% and 0.15%, respectively. From the well-spread metaphase chromosomes obtained, it was determined that C. semilaevis has a diploid chromosome number of 42 and heterotypic sex chromosomes. Also, the metaphase chromosomes produced could be used for karyotype preparation and banding studies. The three methods are also successfully used for chromosome preparation in Japanese flounder (Paralichthys olivaceus) and summer flounder (Paralichthys dentatus). [Copyright &y& Elsevier]