Molecular cloning,characterization and expression analysis of Sox9a and Foxl2 genes in half-smooth tongue sole(Cynoglossussemilaevis)

Two transcription factors, Sox9a and Foxl2 were cloned from half-smooth tongue sole （Cynoglossus semilaevis）. Sox9a is a new duplication of C.semilaevis Sox9 gene. The complete cDNA of Sox9a gene was 1 842 bp long coding for 487 amino acids and Foxl2 gene was 1 817 bp coding for 308 amino acids. Sox9a was expressed higher in male brain, pituitary and gonad and Foxl2 were higher in female brain, pituitary and gonad. The expression of Sox9a gene in gonads of neo-males was higher than that of normal females. Sox9a and Foxl2 were expressed higher in gastrula stage than in other stages. In the period of sex differentiation, the expression of Sox9a was first going up and then going down and Foxl2 was higher expressed at 37 dph. The highest expressions of Sox9a and Foxl2 genes occurred in nine-month and 12-month old gonad tissues, respectively. Sox9a gene was considered to have inevitable links with sex reversal, sex differentiation and cell differentiation of embryos and formation of spermatogenic ceils. Foxl2 was considered to play a role in sex differentiation, cell differentiation of embryos but not to be necessary for sex determination and sex reversal.

Phenotypic and genetic parameter estimation of juvenile growth and bottom color traits in half-smooth tongue sole,Cynoglossus semilaevis

Half-smooth tongue sole（Cynoglossus semilaevis） is a commercially important fish species. There were no reports about accurate genetic parameters of growth traits in C. semilaevis, despite efforts aimed at the culture of this species. Therefore, the main goal of this study was to estimate the parameters for the early growth traits（total length, body depth and body weight） of C. semilaevis based on the data obtained from 80 families. The trait of fish with pure white bottom or not（named as the trait of bottom color） was also analyzed. All the heritabilities estimated for the traits were medium, which varied from 0.210 to 0.362. Genetic correlations among growth traits were highly positive, varying from 0.913 to 0.959, indicating that selection of one of the three traits would result in correlated increase in the others. Also, genetic correlations between bottom color and growth traits were positive and varying from 0.241 to 0.353, suggesting that selection for the bottom color can be utilized to enhance the selection of growth traits. In addition, fish from the top performing 16 full-sib families were selected as broodstock, based on the above analysis and the comparison of family breeding value. This research provides an important basic material to implement selective breeding in C. semilaevis.

Establishment and characterization of an astroglial cell line derived from the brain of half-smooth tongue sole(Cynoglossus semilaevis)

An astroglial cell line was established from the brain of half smooth tongue sole （Cynoglossus semilaevis） and was designated as CSAC. CSAC shows the morphological homogeneity of epithelial cells. The cell identity was tested by the presence of glial fibrillary acidic protein （GFAP）, which was revealed by RT-PCR and immunofluorescence. The cell line was optimally maintained at 24 ℃ in minimum essential medium supplemented with HEPES, antibiotics, 20% fetal bovine serum, 2- Mercaptoethanol （2-Me） and basic fibroblast growth factor. Chromosome analysis revealed that the CSAC cells maintained a normal diploid chromosome number （2n=42）. The fluorescent signals were observed in CSAC after the cells were transfected with green fluorescent protein （GFP） reporter plasmids. The CSAC cell line may serve as a valuable tool for studies on the potential functions of fish astroglial cells.

Molecular characterization and expression analysis of Patched 1 gene in the half-smooth tongue sole(Cynoglossus semilaevis)

The signaling molecule hedgehog（Hh） is essential for cellular signaling required for body pattern formation in embryonic and individual development in vertebrate and invertebrates.Patched 1（Ptc1）,a receptor of Hh,mediates the activity of Hh by binging the Hh ligands.In this study,the Ptc1 of half-smooth tongue sole Cynoglossus semilaevis（CsPtc1） was cloned and characterized.The full-length of CsPtc1 cDNA is 5 212 nucleotides（nt）,and encodes a protein with 1 543 amino acid residues.CsPtc1 shared many features with Ptc1 of other telesot species.Real-time quantitative PCR revealed CsPtc1 mRNA expressed in brain,liver,heart,gill,intestines,spleen,gonad and kidney,with expression level in testis significantly higher than in ovary.In testis,hybridization signals were mainly detected in primary spermatocytes,secondary spermatocytes,and sertoli cells,while weak signals were found in oocytes.Correspondingly,the degree of methylation is higher in female than in male and pseudomale.Results indicate that CsPtc1 may be involved in Desert Hedgehog（DHH） maintenance of the male and pseudo-male germ line and spermatogenesis.

Characterization of the cyp19a1a gene from a BAC sequence in half-smooth tongue sole (Cynoglossus semilaevis) and analysis of its conservation among teleosts

The cyp19ala gene encodes an aromatase that plays a key role in sex differentiation of the gonad. The first bacterial artificial chromosome （BAC） sequence of half-smooth tongue sole （Cynoglossus sernilaeuis） containing the intact cyp19ala gene was reported and the conservation and synteny of the cyp19ala gene among teleosts were analyzed in the study. The BAC is 107 367 bp in size, with an overall guanine-cytosine （GC） content of 43.44%, and contains 4.38% transposable elements. Nine genes were predicted, including seven functional genes and two hypothetical genes. The cyp19ala gene of all tested teleosts has nine exons and eight introns, and potential binding sites flanking the transcriptional start site are conserved. The ex- pression pattern among teleosts is also similar during ovarian differentiation. Synteny analysis revealed a conserved gene cluster PKH4B-SL9A5-FHOD3-CEBPG-CEBPA among teleosts. These findings suggest that, among teleosts, cyp19ala genes not only have similar genomic structures, but also have conserved function- s. The genomic environment of cyp19ala in tongue sole is not universal in teleosts, reflecting the particular evolution of tongue sole cyp19ala after it diverged from the other teleosts.

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.

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.

Development of 57 Novel Polymorphic Microsatellite Markers in Half-Smooth Tongue Sole(Cynoglossus semilaevis)

Half-smooth tongue sole (Cynoglossus semilaevis) is a promising species for aquaculture in China.The wild population of C. semilaevis is under threat from environmental factors. Microsatellite markers are very suitable for assessing genetic diversity. Four microsatellite-enriched libraries of half smooth tongue sole (Cynoglossus semilaevis) were constructed,from which 57 polymorphic microsatellites were isolated and characterized.The polymorphism of these microsatellites was assessed by genotyping in 30 individual fish.The number of alleles ranged from 2 to 11, with an average of 4.614 alleles per locus.The values of observed and expected heterozygosities ranged from 0.1000 to 1.0000 and from 0.0966 to 0.8847 respectively. Polymorphism information content (PIC) ranged from 0.0905 to 0.862.These markers would be useful for population structure assessment,genetic linkage map construction and parentage analysis for this species.

Development and characterization of a cell line from the embryos of half smooth tongue sole (Cynoglossus semilaevis)

A new cell line, CSEC, has been successfully established from embryos at gastrula stage of a cultured marine fish, half smooth tongue sole （Cynoglossus sernilaevis）. CSEC cells grow actively and stably more than 50 passages for over 200 d in DMEM medium supplemented with 15% FBS （fetal bovine serum）, 2.5 ng/cm^3 bFGF （basic fibroblast growth factor）, 1 ng/cm^3 LIF （leukemia inhibitory factor） and 50 mmol/dm^3 2-ME （2-mecaptoethanol）. The cells grew well in the temperature range of 24-30 ℃ and the optimal growth temperature was 24 ℃. FBS and bFGF concentrations are the two key components for CSEC cells proliferation. Chromosome analysis reveals that CSEC cells have a normal diploid karyotype with 2n=42t. The significant fluorescent signals were observed in CSEC after transfection with the GFP reporter gene, suggesting that the CSEC cell line can be used as a useful tool for transgenic and genetic manipulation studies. CSEC cells showed the cytopathic effect （CPE） after infection with lymphosystis disease virus （LCDV） in 2 d. Moreover, the LCDV particles can be observed in the cytoplasm of virus-infected cells by electron microscopy. It suggests that CSEC could be potentially used for the study of aquatic virus.

Analysis of DNA Methylation Level by Methylation-Sensitive Amplification Polymorphism in Half Smooth Tongue Sole(Cynoglossus semilaevis)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.

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.

Isolation and Identification of Photobacterium damselae subsp.damselae(PDD) from Tongue Sole

In May 2016,an epizootic occured among cultured tongue soles caused mass deaths in a fish farm in Qinhuangdao,China.In order to find out the etiological agent,a bacterial strain was isolated from ascites and other tissues of sick tongue sole aseptically collected.The isolate was identified as Photobacterium damselae subsp.damsela（PDD） by isolation culture,Gram staining,physiological identification,morohological observation,biochemical identification and 16S rDNA sequence analysis.The results showed that the isolate shared 99.6% homology with the reference strain in GenBank.The animal regression test displayed that the isolate had very strong pathogenicity to tongue sole.The LD（50） was 3.1 × 10~4 CFU/mL,and it showed pathogenicity to mammals.The antimicrobial susceptibility test showed the isolate was highly sensitive to nrofloxacin,Norfloxacin,Ciprofloxacin,Mequindox;moderately sensitive to Cefradine,Doxycycline;and insensitive to Gentamicin,Ceftriaxone,Tilmicosin,etc..

Functional morphology of the olfactory organ of the tongue sole, Cynoglossus semilaevis

The morphology and structure of the olfactory organ of Cynoglossus semilaevis Günther are described. The oval olfactory sacs on both sides differ in size and in the number of lamellae, with those on the abocular side having smaller sacs and fewer lamellae than those on the ocular side. On the ocular side, the average ratio of sac length to eye diameter is 2.1 (i.e.>1) with an average of 91 lamellae, while on the abocular side, the values were 1.7 (i.e.>1) and 69, respectively. In addition, the surface morphology varies in different parts of the lamella. The frontal part, near the anterior nostril, is a non-sensory margin with cilia-free epidermal cells. Within this is an internal ciliated sensory area, which is intercalated with ciliated receptor cells and a few ciliated non-sensory cells. Additionally, some dense ciliated non-sensory cells make up a non-sensory area, which also contains cilia-free epidermal cells distributed in patches. In the rear of the olfactory sac near the posterior nostril, the lamellae differ in morphology from those of the frontal olfactory sac but are similar in having few ciliated receptor cells. In other words, the surface of the lamellae in the rear part of the olfactory sac is mainly non-sensory. At present, four types of lamellae (I, II, III and IV ) have been recognized in relation to the pattern of the sensory epithelium. In this study, the frontal and rear lamellae resembled types I and IV, respectively, but are referred to as types I' and IV' because they are slightly less developed. Data on the ratio of length of lamellae to eye diameter, number of lamellae and the type of surface pattern of the lamellae show that the development of the olfactory system of C. semilaevis facilitates prey capture.

Isolation and Characterization of Pathogenic Listonella anguillarum of Diseased Haft-Smooth Tongue Sole(Cynoglossus semilaevis Günther)

Studies were conducted to determine the cause of the acute mortality of half-smooth tongue sole Cynoglossus semilaevis Günther juveniles in a fish farm in Jimo,Shandong Province,China,in June 2006. Gross signs of the diseased tongue sole included several petechiae and ecchymoses on the body and fin necrosis and hemorrhagic lesion at the base of the fin. Bacteria were isolated from kidney,liver and hemorrhagic lesions of the diseased tongue sole. Among14 strains,SJ060621 was proved to be highly virulent to juvenile tongue sole with LD50 value of <1.0×105 colony forming units(CFU) mL-1,while the remaining 13 were avirulent. Among the 16 antibiotics tested,SJ060621 was sensitive to gentamicin and nitrofurantoin. It was identified as Listonella anguillarum with conventional plate and tube tests in combination with API 20E analysis. 16S rRNA gene and partial HSP60 gene sequenceing analysis revealed that the strain was highly homologous with L. anguillarum. Examination of the infected musculature by electron microscopy indicated numerous bacteria and lots of macrophages containing phagocytosed bacteria. Histopathological investigations revealed severe necrotic degenerative changes in the infected organs. Indirect immunofluorescence assay(IFA) was employed to detect the location of occurrence of bacteria,and bacteria were found in aggregations in the inflammatory areas in musculature.

半滑舌鳎(Cynoglossus semilaevis)早期发育阶段的摄食特性及生长研究

采用实验生态学方法 ,研究半滑舌鳎早期发育阶段的摄食特性及生长特性。结果表明 ,半滑舌鳎初孵仔鱼在 2 3℃水温条件下 ,2— 3日龄开口摄食 ,开口后投喂轮虫。孵化后 1 2日龄 ,全长达到 8— 9mm左右 ,可摄食卤虫无节幼体。随着仔稚鱼的发育及摄食强度的增强 ,表现出越来越明显的摄食节律。 6日龄仔鱼在 2 4h内均有不同程度的摄食 ,相对来说 9:0 0、1 2 :0 0、1 5 :0 0、1 8:0 0为其摄食率的高峰。 1 6日龄稚鱼的摄食节律已出现较明显趋势 ,9:0 0、1 5 :0 0为其摄食率的高峰。 2 6日龄营底栖生活后稚鱼 ,由浮游生活方式变为底栖生活方式 ,摄食率的高峰出现在 1 8:0 0、2 4 :0 0。半滑舌鳎的捕食能力随其生长逐渐增强 ,6日龄仔鱼的平均摄食强度在摄食率高峰之后的 2— 3h内达到最高 ,出现在 1 2 :0 0、1 8:0 0 ;1 6日龄稚鱼在 1 0 :0 0—1 7:0 0期间平均摄食强度相对较大。 2 6日龄稚鱼 ,捕食能力明显增强 ,伴随着摄食率高峰的出现 ,平均摄食强度也分别在 1 8:0 0和 2 4 :0 0达到高峰。随着生活方式的转变 ,半滑舌鳎摄食节律有着明显的变化 :早期的浮游生活阶段 ,以白天摄食为主 ,摄食高峰出现在白天 ;营底栖生活阶段半滑舌鳎转为夜间摄食 ,摄食高峰出现在夜间。半滑舌鳎不同发育阶段的日平均摄食率分别?

温度、盐度对半滑舌鳎胚胎发育的影响

温度及盐度是鱼类苗种生产当中2个重要的因子。研究了在不同水温、盐度条件下，半滑舌鳎胚胎发育的情况。通过对孵化时间、孵化率、初孵仔鱼畸形率、活力的比较，发现温度对受精卵的孵化影响显著。受精卵在18℃以下及28℃以上均不能孵化出仔鱼，在18～28℃水温条件下，受精卵可以孵化，其适宜的孵化水温为22～23℃，且随着温度升高受精卵孵化时间逐渐缩短。在水温23℃下，受精卵在盐度16以下及48以上均不能孵化出仔鱼，在盐度18～46的范围内，受精卵均可以孵化出仔鱼，盐度38以上均为畸形，且孵化时间不受盐度的影响，其适宜孵化盐度为28～32。

野生及人工养殖半滑舌鳎肌肉营养成分分析研究

分析了野生及人工养殖条件下半滑舌鳎背部肌肉的一般营养成分、氨基酸、脂肪酸及常量和微量元素的组成。结果表明,野生与养殖半滑舌鳎的蛋白质含量差异较小,分别为17.20%和17.17%;野生与养殖半滑舌鳎的脂肪含量相对较低,分别为0.15%和2.41%。两者均富含常量和微量元素,其中野生半滑舌鳎的硒和锌含量分别为7.92%和6.29%,较养殖半滑舌鳎高。在氨基酸含量方面,养殖半滑舌鳎呈味氨基酸天冬氨酸、谷氨酸、甘氨酸和丙氨酸略低于野生半滑舌鳎。不饱和脂肪酸的总含量两者相似。但养殖半滑舌鳎C19∶1含量为20.41%,明显高于野生半滑舌鳎的含量(11.68%)。野生半滑舌鳎在C22∶6(DHA)的含量为14.28%,高于养殖半滑舌鳎的含量(10.39%)。

程序化设计的简并引物克隆半滑舌鳎CYP17基因

采用1种新的简并引物设计方法——CODEHOP法来克隆半滑舌鳎CYP17基因。该法结合NCBI、Blastp、BlockMaker、CODEHOP等生物信息学资源和DNAMAN、Oligo6.0等生物软件,程序性设计半滑舌鳎CYP17基因的CODEHOP引物,从半滑舌鳎卵巢组织中成功克隆了CYP17基因,该基因片段与其它物种的CYP17家族具有较高的同源性,经鉴定,该基因属于整个脊椎动物的CYP17基因群,尤其是鱼类亚群。实验结果表明,程序性设计的简并引物较传统简并引物特异性更高,假阳性率少,更有利于实验成功。

半滑舌鳎病原鳗利斯顿氏菌表型及分子特征研究

2008年12月,江苏赣榆某渔场养殖半滑舌鳎出现大量死亡,症状主要表现为:头部、鳃盖及鳍基出血,尾鳍腐烂,腹腔膨胀并积有大量腹水,部分病鱼肠管脱出肛外。从病鱼肝脏、腹水中分离出大量优势生长的细菌,人工感染试验证明其对半滑舌鳎有较强的致病性。对3株分离菌进行了形态特征、理化特性、胞外酶及溶血素活性等表型生物学性状检验;测定了代表菌株的16SrRNA和gyrB基因序列,分析了16 S rRNA和gyrB两种基因序列的同源性,并构建了系统发生树,比较了两种基因对分离菌的鉴定能力;结果表明gyrB基因用于细菌种间鉴定更具优越性;基于16S rRNA和gyrB基因的系统发育学分析表明分离菌与鳗利斯顿氏菌具有高同源性,根据分离菌的表型及分子特征,判定分离菌为利斯顿氏菌属(Listonella MacDonell and Colwell 1986)的鳗利斯顿氏菌[Listonella anguillarum (Bergeman 1909) MacDonell and Colwell 1986]。胞外酶活性及溶血活性检测表明3株分离菌均具有淀粉酶、蛋白酶、卵磷脂酶等胞外酶活性,在含7%家兔脱纤血液营养琼脂培养基上呈β型溶血,PCR检测3株供试菌均可扩增出大小约493 bp的溶血素基因和大小约248 bp的金属蛋白酶基因。分离菌的耐药谱测定结果显示,对供试49种抗菌药物中的青霉素G等13种药物耐药,对羧苄青霉素等6种药物存在敏感与耐药的株间差异。