Comparative studies on in vitro sperm decondensation and pronucleus formation in egg extracts between gynogenetic and bisexual fish

A cell-free system based upon the egg extracts from gynogenetic gibel carp (Carassius auratus gibelio) or bisexual red common carp (Cyprinus carpio red variety) was developed to investigate developmental behaviors of the demembranated sperm nuclei. Both red common carp and gibel carp sperm nuclei could decondense fully and form pronuclei in the red common carp egg extracts. Gibel carp sperm nuclei could also decondense fully and form pronuclei in the gibel carp egg extracts, but red common carp sperm nuclei could not decondense sufficiently in the same extracts. The significant differences of morphological changes were further confirmed by ultrastructural. observation of transmission electron microscopy. The data further offer cytological evidence for gonochoristic reproduction in the gynogenetically reproducing gibel carp. In addition, the sperm nuclei in vitro decondensation is dependent on the pH in the extracts, and the decondensed efficiency is optimal at pH 7. However, no DNA replication was observed in the two kinds of egg extracts during the incubation period of the sperm nuclei. It is suggested that the egg extracts prepared from the gynogenetic gibel carp should be a valid in vitro system for studying molecular mechanism on gynogenesis and reproduction mode diversity in fish.

The onset of foreign gene transcription in nuclear-transferred embryos of fish

The transcriptional onset of hGH-transgene in fish was studied in the following three cases: the first is in MThGH-transgenic F-4 common carp (Cyprinus carpio) embryos, the second is in nuclear-transferred embryos supported by the transgenic F-4 embryonic nuclei, and the third is in nuclear-transferred embryos supported by the transgenic F-4 tail-fin nuclei. RT-PCR results show that the hGH-transgene initiates its transcriptional activity from early-gastrula stage, the early blastula stage and even 16-cell stage in the first, second and third cases, respectively. it looks like that fish egg cytoplasm could just offer a very restricted reprogramming on transcriptional activity of specific gene in differentiated cell nuclei by nuclear transplantation.

Time-Dependent Oxidative Stress Responses of Crucian Carp (Carassius auratus) to Intraperitoneal Injection of Extracted Microcystins

This study was conducted to investigate time-dependent changes in oxidative enzymes in liver of crucian carp after intraperitoneally injection with extracted microcystins 600 and 150 mu g kg(-1) body weight. The results showed that activities of antioxidant enzymes, including superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase generally exhibited a rapid increase in early phase (1-3 h post injection), but gradually decreased afterwards (12-48 h) compared with the control, with an evident time-dependent effect. These zigzag changes over time contributed a better understanding on oxidative stress caused by microcystins in fish.

Identification of immune genes in grass carp Ctenopharyngodon idella in response to infection of the parasitic copepod Sinergasilus major

The parasitic copepod Sinergasilus major is an important pathogen of grass carp Ctenopharyngodon idella. To understand the immune response of grass carp to the copepod infection, suppression subtractive hybridization method was employed to characterize genes up-regulation during the copepod infection in liver and gills of the fish. One hundred and twenty-two dot blot positive clones from infected subtracted library were sequenced. Searching available databases by using these nucleotide sequences revealed that 23 genes are immune-related, including known acute-phase reactants, and four novel genes encoding proteins such as source of immunodominant MHC-associated peptides (SIMP), TNF receptor-associated factor 2 binding protein (T2BP), poliovirus receptor-related protein 1 precursor, glycoprotein A repetitions predominant (GARP). The differential expression of seven immune genes, i.e. GARP, alpha-2-macroglobulin, MHC class I, C3, SIMP, T2BP, transferrin, as a result of infection was further confirmed by RT-PCR, with the up-regulation of alpha-2-macroglobulin, MHC class I, C3, SIMP and T2BP in the liver of infected fish, and down-regulation of SIMP in the gills of infected fish. The present study provides foundation for understanding grass carp immune response and candidate genes for further analysis.

Cytological and biochemical alterations in Carassius auratus hepatocytes from exposure to sediment containing dioxins and related compounds

Cytological and biochemical alterations of crucial carp (Carassius auratus) hepatocytes were characterized after exposure to sediments from a lake contaminated with dioxins and other industrial chemicals. Carp were exposed in 20 L water containing 25, 50, or 100 g of contaminated sediment for 2 and 4 weeks. Ultrastructural changes in the liver were characterized by severe enlargement of hepatocytes. Alterations in the cell. included formation of condensed and irregular cell nucleus, polynuclei, dispersed heterochromatin, enlargement of the nucleolus, and degeneration of the nucleus. Mitochondrial numbers were reduced and cristae were deformed. Myelin figures and lysosomes were increased, and sometimes cell organelles and cell matrix were totally lost after 4 weeks of exposure. The ultrastructural alterations were correlated with exposure time and sediment concentrations. Hepatosometic index was significantly increased in experimental groups at 2 and 4 weeks as compared with the control group. EROD enzyme activities were strongly induced in liver. A trend from rough endoplasmic reticulum (RER) to SER was observed. Our results suggest that the dioxin-like compounds bound by sediment were bioavailable to C. auratus and cause sublethal effects.

Detection and quantitative analysis of polychlorinated biphenyls in tilapia from Hawaiian waters

The purpose of this study was to investigate polychlorinated biphenyls (PCBs) contamination in tilapia (Oreochromis mossambicus) collected from the Manna stream and Ala Wai Canal of O'ahu, an island of the geographically isolated Hawaiian archipelago. Our results show that the average concentrations of PCBs varied from 51.90 to 89.42 ng g(-1) lipid weight for the sampling sites. Relative toxic potencies (RTPs) and toxic equivalencies (TEQs) were determined to be 20.38-40.60 ng TCDD g(-1) lipid weight and 2.89-4.17 ng TEQ g(-1) lipid weight by 7-ethoxy-resorufin-O-deethylase (EROD) activity analysis and calculation of PCB concentrations based on toxic equivalency factors (TEFs), respectively. Penta-chlorinated congeners were found to be predominant, which revealed that Aroclor 1254 was a possible major source of PCBs in our fish samples. PCB 118, an indicator PCBs, constituted more than 55% and 30% of the total PCBs and TEQs, respectively. In addition, PCB 118 was found to have a linear correlation to the total PCBs (R = 0.975) and TEQs (R = 0.782). Detection of concentrated PCBs in Hawaiian waters suggests a potentially adverse impact of this pollutant on human health, as well as ecological systems, and suggests the necessity of environmental monitoring and hazard assessment of PCBs within the Hawaiian Islands. (c) 2008 Published by Elsevier Ltd.

Identification of genome organization in the unusual allotetraploid form of Carassius auratus gibelio

The unusual allotetraploid form with unequal contribution of chromosome sets was discovered from the gynogenetic offspring of Carassius auratus gibelio stimulated by red common carp sperm. In this study, genomic in situ hybridization (GISH) and fluorescent in situ hybridization (FISH) with 45S rDNA probe are used. The GISH results lead to the identification of species-specific chromosomes, which permits to demonstrate the origin and genome organization in the allotetraploid form. Moreover, chromosome localization of 45S rDNA and co-localizations of 45S rDNA and Cyprinus carpio genomic DNA further confirm that one extra 45S rDNA positive chromosome in the allotetraploid form originates from the paternal haploid genome of C carpio, and other 5 45S rDNA-containing chromosomes are from the maternal genome of Carassius auratus gibelio. And, the correlation between 45 rDNA and the nucleolar organizer regions (NORs) is confirmed by silver nitrate staining. The data provide direct experiment evidence that the allotetraploid actually contains three chromosome sets of Carassius auratus gibelio and one chromosome set of C carpio, and will be a useful genetic material for both basic research and breeding practice. (c) 2006 Elsevier B.V. All rights reserved.

Immunization of rainbow trout fry with Ichthyophthirius multifiliis sonicate: protection of host and immunological changes

Rainbow trout fry (10 weeks post hatch) were immunized (injection or immersion) with sonicated formalin-killed trophonts of the fish parasitic ciliate Ichthyophthirius multifiliis. Challenge infections 22 days after immunization showed a relative protection represented by significantly fewer established parasites and lower prevalence in the immunized groups compared to the controls. Associations between the obtained protection and changes in differential leukocyte counts, haematocrit values, anti Ichthyophthirius multifiliis antibodies, mucous cell density and some epidermal cell markers were investigated. No changes in antibody titers, haematocrit values and mucous cell counts were associated with the response; however, a minor change in peripheral blood neutrophils and epidermal cell markers were found.

云南鲤属（Cyprinus）鱼类的分类整理

关于鲤属鱼类的分类，过去已有两次比较系统的研究，但对亚属的划分、种了阶元的判定等仍存在争议。原在是分类所循外形性状变异较大、互相重叠。本文试图从骨骼比较入手，寻找新的分类依据。经比较分析，鲤属鱼类的第一脊椎横突、上颌骨、额骨、枕骨突、犂骨、下咽骨和下颌骨等存在明显差异，可以作为分种的鉴别特征。据此重新分类整理，认为Mesocyprinus亚属仍能成立，产于洱海的厚唇鲤Cyprinus crassilabris Chen et Hwang这个名称无效，首次把它归入杞麓鲤Cyprinus carpio chilia Wu et al.的同物异名；Cyprinus pellegrini pellegrini Tchang、Cyprinus pellegrini barbatus Chen et Hwang、Cyprinus yunnanensis yunnanensis Tchang和Cyprinus yunanensis daliensis Chen et Hwang在骨骼上有明显分化，由亚种提升为和自独立的种。整理后统计去南共有鲤属鱼类12个种和亚种。以骨骼特征做出检索表。

The complete mitochondrial genomes of two species from Sinocyclocheilus (Cypriniformes: Cyprinidae) and a phylogenetic analysis within Cyprininae

We determined the complete mitochondrial DNA sequences for two species of surface- and cave-dwelling-cyprinid fishes, Sinocyclocheilus grahami and S. altishoulderus. Sequence comparison of 13 protein-coding genes shows that the mutation pattern of each single gene is quite similar to those of other vertebrate animal species. Analysis of the ratios of Ka/Ks at these loci between Sinocyclocheilus and two other cyprinid species (Cyprinus carpio and Procypris rabaudi) show that Ka/Ks ratios are differed, consistent with purifying selection and variation in functional constraint among genes. Bayesian analysis and maximum likelihood analysis of the concatenated mitochondrial protein sequences for 14 cyprinid taxa support the monophyly of the family Cyprininae, and further confirm the monophyly of the genus Sinocyclocheilus. The two Sinocyclocheilus species fall within the Cyprinion-Onychostoma lineage, including Cyprinus, Carassius, and Procypris, rather than among the Barbinae, as previously suggested on morphological grounds.

云南两种珍稀鱼类的形态度量学和分子系统学研究

鱇鱼良白鱼（Anabarilius grahami）、大头鲤（Cyprinus pellegrini）均隶 属于鲤形目（Cypriniformes）鲤科（Cyprinidae），是中国特有鱼类之一，仅在 云南省分布，是抚仙湖和星云湖各自的主要经济鱼类。 本研究首先基于线粒体控制区全序列来研究我国抚仙湖鱇鱼良白鱼自然种群 及人工驯养种群的遗传多样性，发现鱇鱼良白鱼人工驯养种群遗传多样性较高，种 群复壮程度良好；自然种群遗传多样性匮乏，可能种群经历过“建群者效应”和 “瓶颈效应”；并且各自然地理居群具有广泛的基因交流，不存在遗传分化，建 议应被当成一个管理单元进行整体保护；同时，对照其他已报道的鱼类控制区结 构，对鱇鱼良白鱼控制区结构进行了分析，识别了其终止序列区、中央保守区和保 守序列区，找到了终止相关的序列TAS 以及保守序列（CSB-F、CSB-D、CSB-1、 CSB-2、CSB-3）。 其次，采用主成分分析方法对大头鲤及与其相关的三个鲤鱼类群——杞麓鲤 （Cyprinus chilia）、华南鲤（Cyprinus rubrofuscus）及柏元鲤（Cyprinus carpio var. yuankiang×Cyprinus pellegrini）进行了可量性状分析，了解了 不同类群的外部差异；依据主成分分析结果，运用逐步判别方法从37 个可量性 状中筛选出能有效鉴定大头鲤的10 项形态指标：头长、吻长、吻宽、眼间距、 尾柄高、头背部末端到腹鳍起点距离、背鳍起点到腹鳍起点距离、背鳍起点到臀 鳍起点距离、背鳍末端到尾鳍背部起点距离、尾鳍背部起点到臀鳍起点距离，这 些形态指标可以简单易行的从不同鲤鱼群体中筛选出大头鲤个体；同时通过60 年代、80 年代及2007 年大头鲤的对比，发现大头鲤的外部形态已经发生了很大 变化总体趋势是头部更加宽大，身体更加延长。 最后，利用线粒体控制区序列，并结合Genbank 上的已发布的鲤鱼线粒体控 制区序列，对从星云湖采集的62 尾个体进行了分子系统学研究。结果显示：星 云湖土著鲤鱼已经被大规模非土著鲤鱼入侵，大头鲤仅以杂交品系存在，星云湖 土著鲤鱼面临严重危机，迫切需要进行有效保护。

一种中草药复方制剂对罗非鱼非特异免疫力的影响

将杜仲、当归、黄芪、枸杞等10味中草药均匀粉碎后配制成复方制剂,按质量分数为0(对照组)、1%(A组)、3%(B组)、6%(C组)添加到饲料中投喂罗非鱼Oreochromis niloticus,分别在饲养试验的第4、11、18、25、32天采样,检测其血清溶菌酶活性、头肾巨噬细胞吞噬活性、血清杀菌活性、红细胞比容等免疫指标的变化,并在最后一次采样中检测其脾脏脏器系数。用嗜水气单胞菌Aeromonas hydrophila对试验鱼进行人工感染试验,分析中草药对鱼体免疫功能及免疫保护率的增强作用。饲养结果表