Isolation and characterization of polymorphic microsatellite loci from RAPD product in half-smooth tongue sole (Cynoglossus semilaevis) and a test of cross-species amplification

Eleven polymorphic microsatellite loci have been isolated and characterized from random amplified polymorphic DNA product in half-smooth tongue sole, Cynoglossus semilaevis. Twenty-one microsatellites were selected for designing microsatellite primers, of which 11 gave working primer pairs. They had between three and 12 alleles. Observed and expected heterozygosities varied from 0.53 to 0.93, and from 0.52 to 0.80, respectively. Five additional fish species assessed for cross-species amplification revealed between one and three positive amplifications and between zero and three polymorphic loci per species.     

半滑舌鳎早期形态及发育特征

半滑舌鳎卵子为分离的球形浮性卵 ,卵径 1 18-1 3 1mm。卵膜薄、光滑、透明 ,具弹性。多油球 ,一般为 97-12 5个 ,多数在 10 0个左右 ,油球径 0 0 4-0 11mm。在培养水温为 2 0 5-2 2 8℃的条件下 ,卵子授精后 3 7h仔鱼孵出 ,3 0min后仔鱼全部孵出。初孵仔鱼全长 2 56-2 68mm。 1日龄仔鱼 ,出现胸鳍芽。 1 5日龄仔鱼 ,巡游模式基本建立。 2日龄仔鱼 ,逐渐建立外源性摄食关系。 3日龄仔鱼 ,出现鳔泡 ,个体发育进入后期仔鱼期。 18日龄 ,个体发育进入稚鱼期。 2 5日龄稚鱼 ,右眼开始向上移动。 2 7日龄稚鱼 ,右眼已转到头顶。 2 9日龄稚鱼 ,右眼完全转到左侧 ,胸鳍退化 ,各鳍鳍条发育完全。 57日龄 ,个体发育进入幼鱼期。 79日龄幼鱼 ,鳔退化、鳞片发育完全 ,侧线 3条。研究结果表明 :前期仔鱼培育期间 ,除了严格控制适宜的培养水温外 ,仔鱼开口后就应及时投喂一定密度的适口饵料 ,这是苗种培育中不可忽视的重要环节和技术措施之一 ;稚鱼变态期间加强鲜活饵料的投喂是提高稚鱼变态成活率的关键所在。仔鱼孵化后出现的管状感觉器官以及背、臀鳍膜上的泡状结构在早期发育阶段的生态和生理作用尚不清楚。半滑舌鳎成鱼无鳔和无胸鳍 ,而在早期发育期间具有鳔泡和胸鳍 ,这是生物个体发育史中祖先特征的重演     

Identification of differential genes in the ovary relative to the testis and their expression patterns in half-smooth tongue sole (Cynoglossus semilaevis)

Half-smooth tongue sole (Cynoglossus semilaevis) is a rare marine flatfish whose mature ovary and testis greatly differ in volume and weight. The length and weight of mature females are over twice greater than those of mature males. To obtain sufficient information on gonad differentiation and the relationship between gonad development and growth in the fish, we compared gene expression between the ovary and testis using suppression subtractive hybridization (SSH). Testis cDNAs are subtracted from...     

渤海南部半滑舌鳎的食性及摄食的季节性变化

本文研究了渤海南部半滑鳎的食性及其摄食的季节性变化,胃食物分析结果表明,半滑舌鳎为底栖生物食性鱼类,以虾类,蟹类,双壳类及部分中下层小型鱼类为主要食物,兼食一些多毛类,头足类,腹足类,棘皮动物及海葵,半滑舌鳎终年摄食,其摄食强度的周年变化不大,半滑舌鳎常年捕食鲜明鼓虾,日本鼓虾,口虾蛄,隆线强蟹及泥古隆背蟹等。夏季加强捕食小刀蛏,凸壳肌蛤等双壳类,降春季外还捕食一些以虾虎鱼为主的中下层小型鱼类。     

Polymorphic dinucleotide microsatellites in tongue sole (Cynoglossus semilaevis)

The tongue sole, Cynoglossus semilaevis, is a rare marine flatfish distributed in Chinese coastal waters. From a (GT)_n‐enriched genomic library, 57 microsatellites were isolated and characterized. Seventeen of these loci were polymorphic in a test population with alleles ranging from three to 13, and observed and expected heterozygosities from 0.1613 to 1.0000 and from 0.2126 to 0.8983, respectively. Five loci deviated from the Hardy–Weinberg equilibrium in the sampled population, and linkage disequilibrium between two loci was significant after applying Bonferroni correction. Three additional fish species assessed for cross‐species amplification revealed that only one locus was also polymorphic in one species. These polymorphic microsatellite loci should provide sufficient level of genetic diversity to evaluate the breeding strategy and investigate the fine‐scale population structure in C. semilaevis.     

Isolation of Female-Specific AFLP Markers and Molecular Identification of Genetic Sex in Half-Smooth Tongue Sole (<Emphasis Type="Italic">Cynoglossus semilaevis</Emphasis>)

The sex-specific molecular marker is a useful gene resource for studying sex- determining mechanisms and controlling fish sex. Artificially produced male and female half-smooth tongue sole (Cynoglossus semilaevis) were used to screen sex-specific amplified fragment length polymorphism (AFLPs) molecular markers. The phenotypic sex of 28 tongue soles was determined by histological sectioning of gonads. The AFLP analysis of 15 females and 13 males via 64 primer combinations produced a total of 4681 scorable bands, of which 42.11% and 43.39% of bands were polymorphic in females and males, respectively. Seven female-specific AFLP markers were identified and designated as CseF382, CseF575, CseF783, CseF464, CseF136, CseF618, and CseF305, respectively. One female-specific AFLP marker (CseF382) was amplified, recovered from the gels, cloned, and sequenced (accession no. DQ487760). This female-specific AFLP marker was converted into a single-locus polymerase-chain reaction (PCR) marker of a sequence-characterized amplified region (SCAR). A simple PCR method of using the specific primers was developed for identifying genetic sex of half-smooth tongue sole. PCR products demonstrated that the initial 15 females produced the female-specific band of about 350 bp, but the initial 13 male individuals failed to produce the band. We also investigated the applicability of the PCR primers in other tongue sole individuals. The same female-specific fragment of about 350 bp was found in the additional 59 female individuals, but not in the additional 58 male individuals. This AFLP-based molecular sexing technique may have great application potential in elucidation of sex determination mechanisms and sex control in half-smooth tongue sole.     

半滑舌鳎胚胎发育中后期卵黄囊超微结构观察

在胚胎发育中期,半滑舌鳎胚胎由胚体、卵黄囊和卵周液构成.对半滑舌鳎胚胎发育中后期的卵黄囊进行超微结构观察.结果表明,卵黄囊是由卵黄囊膜和包裹其内的卵黄物质组成.在半滑舌鳎胚胎发育过程中,卵黄囊内的卵黄物质逐渐消耗产生低分子量的卵黄磷蛋白分裂小泡.分裂小泡转移到卵黄囊内部消黄细胞中,在消黄细胞的作用下分裂小泡转化成卵黄颗粒.随后卵黄颗粒在卵黄囊内表面聚集成囊状结构并转移运输到卵黄囊膜内部,最后把卵黄物质从卵黄囊膜转移并释放到卵周液中,为胚胎发育提供营养.     

Cathepsin B of Cynoglossus semilaevis: identification, expression, and activity analysis

Cathepsin B (EC 3.4.22.1) is a member of the papain family cysteine protease and in mammals is known to be involved in protein degradation and other biological functions. However, very little is known about the function of cathepsin B in fish. In this study, we identified and analyzed a cathepsin B homologue (CsCatB) from tongue sole (Cynoglossus semilaevis, Pleuronectiformes), an economic fish species cultured in China. CsCatB is composed of 322 amino acid residues and shares 70-81.3% overall sequence identities with its counterpart in teleosts and humans. CsCatB possesses typical cathepsin B structural features including the propeptide region and the papain family cysteine protease domain, the latter containing the four catalytic residues (Q101, C107, H277, and N297) that are conserved in lower and higher vertebrates. Quantitative real time RT-PCR analysis showed that CsCatB expression occurred in multiple tissues and was positively regulated by bacterial infection and by immunization with a subunit vaccine. Recombinant CsCatB purified from Escherichia coli exhibited apparent protease activity, which was optimal at 35 °C and pH 5.5. In contrast, a mutant CsCatB bearing glutamic acid substitution at H277 was dramatically reduced in proteolytic activity. These results indicate that CsCatB is a biologically active protease that is likely to be involved in host immune response during bacterial infection and vaccination.