Analysis of SSRs Derived from an EST Library of Half-Smooth Tongue Sole Cynoglossus semilaevis

A complementary DNA (cDNA) library was constructed from half-smooth tongue sole spleen. A long-read expressed sequence tag (EST) database was generated, containing 3100 cDNA clones, of which 220 clones were fully sequenced. A total of 1060 non-redundant simple sequence repeats (SSRs) were obtained from the cDNA library. An average of 5 kb sequence generates 1 SSR in the half-smooth tongue sole spleen cDNA library. The proportion of the SSR unit size was different in the cDNA library. The monomeric repeats (51.4%) are the most abundant class of SSR in the dataset. The dimeric, trimeric, tetrameric and hexameric re- peats are represented in decreasing proportions of 27.2%, 16.0%, 2.8% and 1.9%, respectively. The frequency of pentameric repeats was observed the least (only 0.7%). Most of the monomeric and dimeric repeats are distributed in 3' and 5' un-translation region. If translation regions are considered merely, trimeric repeats are the highest, accounting for 57% of the total microsatellites.     

Analysis of SSRs derived from an EST library of half-smooth tongue sole <Emphasis Type="Italic">Cynoglossus semilaevis</Emphasis>

A complementary DNA (cDNA) library was constructed from half-smooth tongue sole spleen. A long-read expressed sequence tag (EST) database was generated, containing 3100 cDNA clones, of which 220 clones were fully sequenced. A total of 1060 non-redundant simple sequence repeats (SSRs) were obtained from the cDNA library. An average of 5 kb sequence generates 1 SSR in the half-smooth tongue sole spleen cDNA library. The proportion of the SSR unit size was different in the cDNA library. The monomeric repeats (51.4%) are the most abundant class of SSR in the dataset. The dimeric, trimeric, tetrameric and hexameric repeats are represented in decreasing proportions of 27.2%, 16.0%, 2.8% and 1.9%, respectively. The frequency of pentameric repeats was observed the least (only 0.7%). Most of the monomeric and dimeric repeats are distributed in 3′ and 5′ un-translation region. If translation regions are considered merely, trimeric repeats are the highest, accounting for 57% of the total microsatellites.     

Effects of BPA and E2 on expression profiles of genes related to hypothalamic-pituitary-gonadal axis of half-smooth tongue sole Cynoglossus semilaevis

Endocrine disrupting chemicals (EDCs) are increasingly viewed as persistent pollutants, similar to natural hormones in function. This paper describes the expression profiles of 7 genes (DMRT, VTG, GnRHR, FSHR, CYP17A, CYP19A, and CYP19B) involved in sex steroid synthesis and action as well as sexual development in adult male and female Cynoglossus semilaevis, after exposure to different concentrations of Bisphenol A (BPA) and 17β-estradiol (E2). Both BPA (1, 10, 50, 125, and 250 mg/kg) and E2 (0.5, 5, and 10 mg/kg) induced changes in target gene expression, although the estrogenic effects of E2 as a model estrogen were stronger. Among the 7 genes, VTG, CYP17A and CYP19 responded strongly to BPA or E2 exposure and can thus serve as reference biomarkers for estrogenic EDCs exposure in marine teleosts. These data will provide a window to establish a hypothalamic-pituitary-gonadal model in C. semilaevis to better understand the effect pathways of EDCs.     

Application of AFLP markers for population genetic study on half-smooth tongue sole Cynoglossus semilaevis

The genetic diversity of wild and hatchery populations of half-smooth tongue sole Cynoglossus semilaevis, based on observation of amplified fragment length polymorphism (AFLP) was described. Two hundred individuals from four wild populations, Laizhou (LZ), Weihai (WH), Qingdao (QD), Rizhao (RZ), and one hatchery population, Mingbo (MB), were screened using eight different AFLP primer combinations. A total of 384 loci were screened in the five studied populations. 48.4%, 51.3%, 50.7%, 49.3% and 45.8% of these loci were polymorphic among the individuals tested in the LZ, WH, QD, RZ and MB populations, respectively. The number of polymorphic loci detected by single primer combinations ranged from 17 to 35. The average heterozygosity of the LZ, WH, QD, RZ and MB populations was 0.072, 0.093, 0.092, 0.090 and 0.063, respectively. The WH population showed the highest genetic diversity in terms of total number of AFLP bands, total number of polymorphic bands, average heterozygosity and percentage of low frequency (0-0.2) polymorphic loci among all the populations, while the LZ population was the lowest among the wild populations. Compared with the wild populations, the hatchery population showed a low genetic viability.     

Location of Vibrio anguillarum resistance-associated trait loci in half-smooth tongue sole Cynoglossus semilaevis at its microsatellite linkage map

A cultured female half-smooth tongue sole(C ynoglossus semilaevis) was crossed with a wild male, yielding the fi rst fi lial generation of pseudo-testcrossing from which 200 fi sh were randomly selected to locate the V ibrio anguillarum resistance trait in half-smooth tongue sole at its microsatellite linkage map. In total, 129 microsatellites were arrayed into 18 linkage groups, ≥4 each. The map reconstructed was 852.85 c M in length with an average spacing of 7.68 c M, covering 72.07% of that expected(1 183.35 cM). The V. anguillarum resistance trait was a composite rather than a unit trait, which was tentatively partitioned into Survival time in Hours After V. anguillarum Infection(SHAVI) and Immunity of V. A nguillarum Infection(IVAI). Above a logarithm of the odds(LOD) threshold of 2.5, 18 loci relative to SHAVI and 3 relative to IVAI were identifi ed. The 3 loci relative to IVAI explained 18.78%, 5.87% and 6.50% of the total phenotypic variation in immunity. The microsatellites bounding the 3 quantitative trait loci(QTLs) of IVAI may in future aid to the selection of V. anguillarum-immune half-smooth tongue sole varieties, and facilitate cloning the gene(s) controlling such immunity.     

Genome-wide identification and prediction of long non-coding RNAs in half-smooth tongue sole Cynoglossus semilaevis

Journal of Oceanology and Limnology - At present, the function and profile of long noncoding RNAs (IncRNAs) in half-smooth tongue soles Cynoglossus semilaevis remain poorly understood. Lherefore,...     

Isolation,characterization and expression analysis of TRPV4 in half-smooth tongue sole Cynoglossus semilaevis

Journal of Oceanology and Limnology - The transient receptor potential vanilloid 4 (TRPV4), another Ca2+ entry channel, belongs to the vanilloid subfamily and responds to a number of different...     

Sensory bases and the mode of feeding behavior of tongue sole <Emphasis Type="Italic">Cynoglossus semilaevis</Emphasis>

We conducted behavior experiments on the roles of vision, olfaction, and the lateral line in feeding in tongue sole Cynoglossus semilaevis Günter. Chemical cues from the prey alone could not elicit any searching or attacking behavior by the fish; only when moving foods were presented, the chemical nature of the foods affected feeding. In addition, we found that normal tongue sole did not accurately recognize moving artificial prey of different shapes. Neither chemosense nor vision plays an important role in prey detection. Reactions to moving objects and the response of the oropharyngeal cavity to different foods showed that the (eyeless) tongue sole use mainly the lateral line for prey detection and capture. Gustation in the oropharyngeal cavity is essential for the fish to swallow its prey.     

Identification of two major histocompatibility (MH) class II A genes and their association to Vibrio anguillarum infection in half-smooth tongue sole (Cynoglossus semilaevis)

Major histocompatibility complex class II antigens are important in vertebrate immune system.In the present study,the full cDNA sequence of class II A gene was synthesized by RACE-PCR from half-smooth tongue sole(Cynoglossus semilaevis),and its open reading frame(ORF) polymorphism was studied.The whole cDNA sequence was 992 bp in length,including the ORF with 717 bp.Twenty-five alleles were identified and clustered into two distinct groups according to the specific nucleotides/amino acids in specific positions.Eleven alleles belonged to Cyse-DAA while fourteen alleles belonged to Cyse-DBA.Four Cyse-DAA alleles were observed in one individual,and three to five Cyse-DBA alleles were observed in each of the three detected individuals,which indicated that at least two loci existed in each gene.Moreover,in order to study the function of the alleles in resistance to infection,200 individuals were intraperitoneally injected with Vibrio anguillarum and the first 20 dead individuals and 20 surviving ones were selected for genotype analysis.Fifty-six alleles were identified among the 40 individuals.Twenty-nine alleles belonged to Cyse-DAA and the other 27 alleles belonged to Cyse-DBA.Eighteen alleles were selected for studying their function in resistance to infection.Alleles Cyse-DAA*0201,Cyse-DAA*1101,Cyse-DBA*0401,Cyse-DBA*1102,Cyse-DBA*1801 and Cyse-DBA*2201 were identi-fied only in surviving individuals,while alleles Cyse-DAA*0901,Cyse-DBA*1101 and Cyse-DBA*1401 occurred more frequently in dead individuals.This study confirmed the existence and polymorphism of two class II A genes as well as the relationship between alleles of class II A genes and disease susceptibility/resistance in half-smooth tongue sole.     

Sequence polymorphism of two major histocompatibility (MH) class II B genes and their association with Vibrio anguillarum infection in half-smooth tongue sole (Cynoglossus semilaevis)

Major histocompatibility complex (MHC) class Ⅱ B molecules play an important role in the adaptive immune response in fish. Previous study has reported that two highly polymorphic class ⅡB genes, Cyse-DAB and Cyse-DBB exist in half-smooth tongue sole (Cynoglossus semilaevis). In this study, the polymorphism within exon 2 of the class Ⅱ B genes following bacterial challenge was evaluated. Two hundred C. semilaevis individuals were injected intraperitoneally with Vibrio anguillarum. Muscle tissue from the first 20 dead and 20 of the survivors was collected for genotyping. Sixty alleles from the 40 individuals were isolated, of which 32 belonged to Cyse-DAB and 28 belonged to Cyse-DBB. The rate of dN (non-synonymous substitution) was higher than that of dS (synonymous substitution) in the PBRs (peptide binding residues) of both class Ⅱ B genes. Conversely, the rate of dS was higher than dN in the non-PBRs and the complete exon 2 sequence. Thus, the results suggest that positive selection has occurred in the PBRs and purifying selection in the non-PBRs and exon 2. Thirteen class Ⅱ B alleles were used to study the association between alleles and resistance to infection. Though not significant, alleles Cyse-DAB*0601, Cyse-DAB*0706, and Cyse-DBB*0101, Cyse-DBB*1301 were only found in surviving individuals and may represent alleles that have resistance against V. anguillarum infection. Alleles Cyse-DAB*0701 and Cyse-DAB*1301 were significantly more prevalent in dead individuals than in surviving ones and may represent alleles that are associated with increased susceptibility to V. anguillarum infection.     

Half Smooth Tongue Sole(Cynoglossus semilaevis) Under Low Salinity Stress Can Change Hepatic igf2 Expression Through DNA Methylation

Salinity is a crucial environmental stress that severely affects fish growth and survival.Under environmental stress,DNA methylation plays an important role in gene expression and genome function.To better understand the epigenetic regulation mechanism of igf2 under low salinity stress,we analyzed the DNA methylation at 5’UTR,exon1,intron1,and exon2,and analyzed the relationship of DNA methylation with mRNA abundance as well as the special single CpG sites methylation patterns of igf2 in the liver of half smooth tongue sole under low salinity(15)for 0,7,and 60 d.When exposed to low salinity,DNA methylation at 5’UTR and exon2 remained stable,while it was up-regulated firstly and then down-regulated at exon1 and intron1.Some single CpG sites of igf2 associated with low salinity,and most of these sites with significantly changed methylation levels(P<0.05)are located in intron1 area.The discrepant variation of single CpG sites methylation levels and igf2 expression further revealed that females and males showed different response to low salinity.Remarkably,the 38-CpG site of intron1 servers as a sexual marker.Additionally,our integrative analysis demonstrated that regional DNA of igf2 methylation had highly complex interplay on gene expression.The single CpG sites in intron1 were indispensable epigenetic markers under external environmental stress.Above all,to resist the low salinity stress,half smooth tongue sole liver can regulate the expression of igf2 through methylation of CpG sites in intron1.     

Food habits and seasonal variation of stomach contents of tongue soleCynoglossus semilaevis (Günther) in the Bohai Sea

Examination of the food habits and seasonal variation of the stomach contents of adult tongue sole,Cynoglossus semilaevis (Günther) taken in July 1982–July 1983 from Laizhou Bay and the Huanghe River estuary of the Bohai Sea showed crustacea, bivalvia and small fishes comprised the main prey. Invertebrates such as polychaeta, cephalopoda, gastropoda, echinodermata and actiniaria were also intermittently found in them. They intensively fed all the year found (monthly feeding rate of over 80%). The main food items wereAlpheus japonica, Alpheus distinguendus. Oratosquilla oratoria, Eucrate crenata andCarcinoplax vestitus, etc. In summer and autumn, the portion of bivalvia such asCultellus attenuatus andMusculus senhousei increased steadily. From summer to winter, a stable proportion of small fishes such asRhinogobius pflaumi andSetipinna taty was in the diet. Contribution No. 1637 from the Institute of Oceanology, Academia Sinica.     

Identification and characterization of 43 microsatellite markers derived from expressed sequence tags of the sea cucumber (Apostichopus japonicus)

The sea cucumber Apostichopus japonicus is a commercially and ecologically important species in China. A total of 3056 potential unigenes were generated after assembling 7597 A. japonicus expressed sequence tags (ESTs) downloaded from Gen-Bank. Two hundred and fifty microsatellite-containing ESTs (8.18%) and 299 simple sequence repeats (SSRs) were detected. The average density of SSRs was 1 per 7.403 kb of EST after redundancy elimination. Di-nucleotide repeat motifs appeared to be the most abundant type with a percentage of 69.90%. Of the 126 primer pairs designed, 90 amplified the expected products and 43 showed polymorphism in 30 individuals tested. The number of alleles per locus ranged from 2 to 26 with an average of 7.0 alleles, and the observed and expected heterozygosities varied from 0.067 to 1.000 and from 0.066 to 0.959, respectively. These new EST-derived microsatellite markers would provide sufficient polymorphism for population genetic studies and genome mapping of this sea cucumber species.     

Location of <Emphasis Type="Italic">Vibrio anguillarum</Emphasis> resistance-associated trait loci in half-smooth tongue sole <Emphasis Type="Italic">Cynoglossus semilaevis</Emphasis> at its microsatellite linkage map

A cultured female half-smooth tongue sole (Cynoglossus semilaevis) was crossed with a wild male, yielding the first filial generation of pseudo-testcrossing from which 200 fish were randomly selected to locate the Vibrio anguillarum resistance trait in half-smooth tongue sole at its microsatellite linkage map. In total, 129 microsatellites were arrayed into 18 linkage groups, ≥4 each. The map reconstructed was 852.85 cM in length with an average spacing of 7.68 cM, covering 72.07% of that expected (1 183.35 cM). The V. anguillarum resistance trait was a composite rather than a unit trait, which was tentatively partitioned into Survival time in Hours After V. anguillarum Infection (SHAVI) and Immunity of V. Anguillarum Infection (IVAI). Above a logarithm of the odds (LOD) threshold of 2.5, 18 loci relative to SHAVI and 3 relative to IVAI were identified. The 3 loci relative to IVAI explained 18.78%, 5.87% and 6.50% of the total phenotypic variation in immunity. The microsatellites bounding the 3 quantitative trait loci (QTLs) of IVAI may in future aid to the selection of V. anguillarum-immune half-smooth tongue sole varieties, and facilitate cloning the gene(s) controlling such immunity.     

Characterization of genic microsatellite markers derived from expressed sequence tags in Pacific abalone (Haliotis discus hannai)

Simple sequence repeat (SSR) markers were developed from the expressed sequence tags (ESTs) of Pacific abalone (Haliotis discus hannai).Repeat motifs were found in 4.95% of the ESTs at a frequency of one repeat every 10.04 kb of EST sequences,after redundancy elimination.Seventeen polymorphic EST-SSRs were developed.The number of alleles per locus varied from 2-17,with an average of 6.8 alleles per locus.The expected and observed heterozygosities ranged from 0.159 to 0.928 and from 0.132 to 0.922,respective...     

Development of novel microsatellite markers for Holothurian scabra (Holothuriidae), Apostichopus japonicas (Stichopodidae) and cross-species testing in other sea cucumbers

Thirty-five new microsatellite loci from the sea cucumbers H olothurian scabra(Jaeger, 1833) and Apostichopus japonicas(Selenka, 1867) were screened and characterized using the method of magnetic bead enrichment. Of the twenty-four polymorphic loci tested, eighteen were consistent with Hardy-Weinberg equilibrium after a modified false discovery rate(B-Y FDR) correction, whereas six showed statistically significant deviations(CHS2 and CHS11: P 0.014 790; FCS1, FCS6, FCS8 and FCS14: P 0.015 377). Furthermore, four species of plesiomorphous and related sea cucumbers(Holothurian scabra, Holothuria leucospilota, Stichopus horrens and Apostichopus japonicas) were tested for mutual cross-amplification using a total of ninety microsatellite loci. Although transferability and universality of all loci were generally low, the results of the cross-species study showed that the markers can be applied to identify individuals to species according to the presence or absence of specific microsatellite alleles. The microsatellite markers reported here will contribute to the study of genetic diversity, assisted breeding, and population conservation in sea cucumbers, as well as allow for the identification of individuals to closely related species.     

Sequences characterization of microsatellite DNA sequences in Pacific abalone (Haliotis discus hannai)

The microsatellite-enriched library was constructed using magnetic bead hybridization selection method, and the microsatellite DNA sequences were analyzed in Pacific abalone Haliotis discus hannai. Three hundred and fifty white colonies were screened using PCR-based technique, and 84 clones were identified to potentially contain microsatellite repeat motif. The 84 clones were sequenced, and 42 microsatellites and 4 minisatellites with a minimum of five repeats were found (13.1% of white colonies screened). Besides the motif of CA contained in the oligoprobe, we also found other 16 types of microsatellite repeats including a dinucleotide repeat, two tetranucleotide repeats, twelve pentanucleotide repeats and a hexanucleotide repeat. According to Weber(1990), the microsatellite sequences obtained could be categorized structurally into perfect repeats (73.3%), imperfect repeats(13.3%), and compound repeats (13.4%). Among the microsatellite repeats, relatively short arrays (＜ 20 repeats) were most abundant,accounting for 75.0%. The largest length of microsatellites was 48 repeats, and the average number of repeats was 13.4. The data on the composition and length distribution of microsatellites obtained in the present study can be useful for choosing the repeat motifs for microsatetlite isolation in other abalone species.     

Analysis of DNA methylation level by methylation-sensitive amplification polymorphism in half smooth tongue sole (<Emphasis Type="Italic">Cynoglossus semilaevis</Emphasis>) subjected to salinity stress

Increasingly arisen environmental constraints may contribute to heritable phenotypic variation including methylation changes, which can help the animals with development, growth and survival. In this study, we assessed the DNA methylation levels in three tissues (gonad, kidney and gill) of half smooth tongue sole under the salinity stress. The methylation-sensitive amplification polymorphism (MSAP) technique was applied to illustrate the regulation of epigenetic mechanism in environmental stimuli. Fish were subjected to 15 salinity treatment for 7 and 60 days, respectively. A total of 11259 fragments were amplified with 8 pairs of selective primers. The levels of methylated DNA in different tissues of females and males without salinity stress were analyzed, which were 32.76% and 47.32% in gonad; 38.13% and 37.69% in kidney; 37.58% and 34.96% in gill, respectively. In addition, the significant difference was observed in gonad between females and males, indicating that discrepant regulation in gonadal development and differentiation may involve sex-related genes. Further analysis showed that total and hemi-methylation were significantly decreased under 15 salinity for 7 days, probably resulting in up-regulating salt-tolerance genes expression to adjust salt changing. With the adjustment for 60 days, total and hemi-methylation prominently went back to its normal levels to obtain equilibrium. Particularly, full methylation levels were steady along with salinity stress to maintain the stability of gene expression. Additionally, the data showed that gonads in females and gills in males were superior in adaptability. As a result, DNA methylation regulates tissue- specific epiloci, and may respond to salinity stress by regulating gene expression to maintain animal survival and activity.