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.     

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     

半滑舌鳎性逆转的遗传特性研究

半滑舌鳎雌雄个体生长差异悬殊,由于性逆转而造成的其群体中雌性比例过低大大制约了养殖效率。性逆转是鱼类及两栖类生物性别决定事件中有趣的生物学问题,其发生的遗传机制鲜有研究。在该研究中,分别利用雄鱼和伪雄鱼组建10个半同胞家系,对这10个半同胞家系中的子代雌雄比进行研究,结果发现,2个伪雄鱼的家系,其后代个体中遗传雌性鱼全部逆转为生理雄鱼;在另外8个雄鱼家系中其性逆转比呈连续分布,表现为典型的数量性状特征;半滑舌鳎性逆转的遗传力较低,仅为0.058。以上结果表明,伪雄鱼作为父本的遗传可能为完全的父本效应遗传,性逆转由于其较低的遗传力不适合于做家系选育而适合于做家系内的选育或结合分子标记的遗传评估,以提高雌性比的遗传进展,半滑舌鳎逆转比的数量遗传特征说明其性别决定是多基因作用的结果。     

半滑舌鳎FTZ-F1cDNA克隆及表达分析

为研究性别相关基因FTZ-F1在半滑舌鳎鱼中的表达特征,采用同源克隆策略,从其精巢分离了3143bp长的半滑舌鳎FTZ-F1(hsFTZ-F1)的全长cDNA,该序列包含1458bp开放阅读框,66bp长的5'末端非编码区(UTR),1619bp长的3'末端UTR。mRNA的组织分布、氨基酸序列和系统发生分析表明:hsFTZ-F1属于SF-1/Ad4BP类群。RT-PCR分析表明:hsFTZ-F1mRNA的分布广泛,几乎在所有组织都有表达,但在性腺、肾脏、脑和头肾组织中表达最强,其他组织表达较弱,雌鱼脑和头肾中的表达量明显高于雄性。胚胎发育过程中表达量都高于孵化后仔鱼的表达量,表明hsFTZ-F1可能参与了半滑舌鳎的器官形成过程。     

半滑舌鳎精子发生和精子形成的超微结构

用电子显微镜对半滑舌鳎(Cynoglossus semilaevis)精子发生的过程及精子的超微结构进行了观察。半滑舌鳎精巢属于小叶型,精小叶由各期生精细胞和支持细胞构成。半滑舌鳎的精子发生经历了初级精原细胞、次级精原细胞、初级精母细胞、次级精母细胞和精子细胞,再经过精子形成过程发育成为精子。初级精母细胞成熟分裂的前期Ⅰ,同源染色体经历了联会复合体形成和解聚的变化。在精子形成的过程中,精细胞大致经历了核质浓缩、线粒体迁移及鞭毛的发生等过程。核质浓缩时,精细胞核内位于植入窝周围的染色质首先由细颗粒状浓缩成粗大颗粒状,然后细胞核其他部位的染色质也逐渐浓缩成粗大颗粒状。这些已浓缩成粗大颗粒状的染色质再进一步浓缩为电子密度高的均匀状物质。随着核质的浓缩,核外膜与核内膜之间的间隙增大形成核膜间隙,核内一些没有参与染色质浓缩的物质通过出芽形成囊泡,先排入核膜间隙,然后再外排到细胞质中。核浓缩过程中细胞核的体积和表面积都大大缩小;鞭毛的形成与细胞核的浓缩是同步进行的,当一对中心粒移近细胞核时,核膜凹陷形成植入窝,其周围染色质浓缩的同时,远端中心粒(基体)逐渐向后产生轴丝。成熟精子无顶体,头细长,主要为核占据,核凹窝发达,线粒体4-5个环绕在鞭毛基部形成袖套,尾细长,具侧鳍,尾部轴丝为"9 2"结构。     

半滑舌鳎幼鱼消化酶同功酶谱初步研究

以半滑舌鳎幼鱼为研究材料,利用聚丙烯酰胺非变性电泳和同功酶活性染色对半滑舌鳎消化酶进行了初步分离。蛋白酶采用明胶原位消化法,肝脏蛋白酶检测出3条酶带,其中1条强带,2条弱带;后肠蛋白酶出现了2条酶带,其中有1条强带,1条弱带,并且与肝脏蛋白酶的前2条酶带分别在同一分子量水平上;前肠和中肠蛋白酶均只出现了1条弱带,且在同一分子量水平上。半滑舌鳎肠道各部分的淀粉酶带都在相似分子量附近出现了一条弱酶带,而肝脏在不同的分子量水平出现了1条酶带。肝脏、前肠和后肠脂肪酶均出现了4条酶带,其中,肝脏和前肠均分别有1条强带,1条中强带,2条弱带;后肠脂肪酶则有1条中强带,3条弱带;中肠脂肪酶出现了3条弱带。肝脏和肠道各部分脂肪酶的第一条酶带都在同一分子量水平上,而且活性相似。     

半滑舌鳎仔、稚鱼视网膜结构与视觉特性

对1-50d半滑舌鳎仔、稚鱼视网膜和全长50mm的半滑舌鳎幼鱼视网膜结构和视觉特性进行了研究。结果表明:(1)3d仔鱼色素层形成,15d仔鱼没有显著的视网膜运动反应,25d时具有正常感受自然光的明视功能,43d半滑舌鳎稚鱼适应自然光的功能丧失;(2)半滑舌鳎仔鱼阶段感受细胞主要为高密度的单锥,视杆细胞和双锥细胞出现的较晚;单锥融合成双锥时,由于半滑舌鳎视锥细胞椭圆体细长,融合程度较差,尽管在视网膜横切面上能够看到双锥,但在切向切面上仍呈现单锥排列方式;随其生长发育,视锥和神经节细胞密度降低,视杆细胞密度增加,31d后视杆细胞数量显著增加;同时,外核层细胞核与神经节细胞的比值增大,网络会聚程度提高;相关数据表明,20-31d是视网膜结构和视觉特性发生明显变化的过渡时期,这是与半滑舌鳎从浮游生活到底栖生活生态环境的变化相适应的;(3)半滑舌鳎内核层结构特殊,50mm时只有1层水平细胞,属感光系统不发达类型,双极细胞和无长突细胞共4-5层,但不可分辨;内核层细胞层数的减少,基本上没有分化的水平细胞、双极细胞和无长突细胞,说明半滑舌鳎视网膜的光敏感性不高;(4)半滑舌鳎仔鱼浮游生活阶段视敏度较高,视觉在捕食行为中具有重要意义;底栖生活后,视敏度和光敏感性都较差,视觉在捕食行为中不可能具有重要作用     

半滑舌鳎仔鱼的摄食与生长

在水温23.4-24.0℃、盐度33.00、pH7.78-8.02的培养条件下,半滑舌鳎初孵仔鱼全长为3.55±0.161mm(n=60),依照公式4/3π.R/2.(r/2)2计算其卵黄囊体积为0.606±0.106mm3(n=60)。1日龄仔鱼,全长为4.99±0.211mm(n=10),卵黄囊体积较初孵仔鱼缩小近10倍,约为0.066±0.008mm3(n=10);2日龄,仔鱼的巡游模式建立,仔鱼全长为5.61±0.069mm,卵黄囊体积为0.030±0.002mm3(n=10),口初开;2.5日龄,口完全裂开,口裂0.24±0.024mm(n=10),仔鱼进入摄食期;3日龄,仔鱼的外源性摄食关系初步建立;4日龄,仔鱼的摄食率达100%,完成了内源性营养向外源性营养的转换;5日龄,仔鱼的卵黄完全被吸收,仅剩聚成一团的小油球,仔鱼的混合营养期持续2.5天时间;21日龄,稚鱼全长为12.96±0.611mm(n=11),仍有40%的个体残余的油球还没有完全被吸收,其体积仅为0.0000005±0.000003mm3(n=11)。仔鱼发育过程中,其长度的生长存在内源性营养阶段的快速生长、混合营养阶段的慢速生长以及饥饿期间的负生长三个生长期相,平均增长率为0.45mm/d,依照TL=aD3 bD2 cD d方程式对仔鱼的全长与日龄进行回归,其生长模型为TL(mm)=0.0026D3-0.0704D2 0.7993D 3.55(R2=0.9811,n=324)。仔鱼耐受饥饿的时间临界点发生在孵化后第10天(即9日龄)。仔鱼具有摄食能力的时间约6天,不可逆转饥饿期的时间约3天。残余的油球较长时间的存在,相对地延长了仔鱼混合营养期的时间,对仔鱼的发育、生长和存活有着至关重要的作用。5-20日龄的个体都具有胸角这个明显的形态学特征,只是饥饿个体和不可逆转饥饿期个体的胸角比摄食个体更为明显和尖锐,胸角不能作为区分健康仔鱼和饥饿仔鱼的形态学依据之一[动物学报51(6)1023-1033,2005]。     

半滑舌鳎脑芳香化酶基因cDNA克隆及表达分析

为研究脑型芳香化酶(P450aromB)在半滑舌鳎性别分化中的作用,采用同源克隆策略,从半滑舌鳎脑分离了2184bp长的脑型芳香化酶的全长cDNA,该基因编码498个氨基酸。氨基酸序列和系统发育分析表明,P450aromB属于脑型P450arom,P450aromB的氨基酸序列与其他鱼类脑型P450arom的同源性较高(48.3%-66.1%),与性腺型P450arom的同源性较低(34.2%-49.9%),与自身的性腺型芳香化酶同源性为45.1%。RT-PCR分析表明:P450aromB mRNA的表达具有明显组织特异性,P450aromB只在性腺、脑、鳃和皮肤中表达,且脑中表达量远高于性腺,而在雌雄鱼的其他组织中都不表达。经过甲基睾酮浸浴处理和高温诱导半滑舌鳎由雌性性反转为雄性后,脑中P450aromB的表达量降低,这些结果表明P450aromB参与了半滑舌鳎的性腺分化和性别决定过程。     

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。     

Molecular characterization of Cynoglossus semilaevis CD28

T lymphocyte activation requires a combination of signals, one of which is provided by interaction between CD28 and its ligands on antigen presenting cells. Although CD28-like sequences have been identified in a few teleosts, the function of fish CD28 is virtually unknown. In this study, we cloned and analyzed a CD28 gene, CsCD28, from half-smooth tongue sole (Cynoglossus semilaevis). The deduced amino acid sequence of CsCD28 contains 229 residues and shares 20.2%-40.3% overall sequence identities with known fish CD28 sequences. CsCD28 possesses structural features conserved in mammalian and teleost CD28, which include the MYPPPY motif in the extracellular immunoglobulin-like domain and the YXN motif in the cytoplasmic domain. The CsCD28 gene is 2746 bp and composed of four exons and three introns, which in organization resemble those of mammalian and trout CD28. Quantitative real time RT-PCR analysis showed that CsCD28 expression occurred predominately in kidney, spleen, gut, and gill. CsCD28 is localized on the surface of head kidney lymphocytes, and antibody ligation of CsCD28 induced significant levels of cellular proliferation. Taken together, these results indicate that CsCD28 is similar to mammalian CD28 in genetic and protein structures and possibly plays a role in T cell activation.     

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.     

Interleukin-8 of Cynoglossus semilaevis is a chemoattractant with immunoregulatory property

Interleukin-8 (IL-8), or CXCL8, is a member of the CXC chemokine family that in mammals is known to mediate inflammatory response. In this study, we identified and analyzed an IL-8 orthologue, CsIL8, from half-smooth tongue sole (Cynoglossus semilaevis). The deduced amino acid sequence of CsIL8 contains 100 residues and is closely related to the lineage 1 IL-8 of a number of fish species. In silico analysis identified in CsIL8 a CXC chemokine domain that contains four conserved cysteine residues, two of which form the CXC signature motif of CXC chemokines. Expression of CsIL8 as determined by quantitative real time RT-PCR was detected in a wide range of tissues under normal physiological conditions and was upregulated by bacterial challenge and by vaccination with a subunit vaccine. Purified recombinant CsIL8 (rCsIL8) induced migration of peripheral blood leukocytes and head kidney (HK) lymphocytes and stimulated the proliferation of these cells in a dose-dependent manner. When rCsIL8 was added to the cell culture of HK lymphocytes, it upregulated the expression of interleukin-1β and CsIL8 in a time-dependent fashion. These results indicate that CsIL8 is a biologically active CXC chemokine with immunoregulatory activity and that CsIL8 is involved in pathogen-induced inflammatory response.     

Comparative Mucous miRomics in Cynoglossus semilaevis Related to Vibrio harveyi Caused Infection

Marine Biotechnology - Epidermal mucus is an important barrier and regulating mediator in fish. MicroRNAs (miRNAs) are proved to be involved in various biological processes, also as promising...     

A patched1 gene homologue participates in female differentiation of Cynoglossus semilaevis

Patched (Ptch) is a receptor in the hedgehog signaling pathway, essential for animal development. Our previous study showed that ptch1 gene participates in the maintenance of the male germline and spermatogenesis in Cynoglossus semilaevis (csptch1). In this study, we identified a patched1 gene homolog (csptch1 x1). The csptch1 x1 gene is 5761 bp long, with a 4638 bp coding sequence that encodes 1545 amino acids. The Csptch1 x1 protein has 12 transmembrane regions and sterol-sensing domains and is highly homologous to the csptch1 (91 amino acids difference). Expression pattern analysis showed that csptch1 x1 is expressed in eight different tissues of adult tongue sole, and the expression is significantly higher in tissues of female than that in male tissues. The expression pattern in developmental stages was also analyzed. csptch1 x1 could be detected at the 1-cell stage and was highly expressed at the blastocyst, somite, and blastopore closing stages, implying that it participates in cell differentiation. In ovarian development, the expression of csptch1 x1 was initiated at 20 days after hatching (dah) and was significantly high at 35–50 and 95–150 dah. In situ hybridization showed that csptch1 x1 was predominantly expressed in primordial germ cells, oocytes, and follicular cells, but the expression of the gene was lower in the testis. These results suggest that csptch1 x1 may be mainly involved in female differentiation and ovarian development, different from the role of csptch1 in spermatogenesis.