Genetic Evidence for Gonochoristic Reproduction in Gynogenetic Silver Crucian Carp (Carassius auratus gibelio Bloch) as Revealed by RAPD Assays

Sex evolution has been a debating focus in evolutionary genetics. In lower vertebrates of reptiles, amphibians, and fish, a species or a bioform reproduces either sexually or asexually but never both. A few species were found to consist of all females in fish. These all-female species can propagate by asexual reproduction modes, such as gynogenesis and hybridogenesis. However, the coexistence of sexuality and asexuality in a single species was recently noted only in a cyprinid fish silver crucian carp, Carassius auratus gibelio. This fish had been demonstrated to be capable of gynogenesis stimulated by sperm from other related species. Surprisingly, natural populations of this fish consist of a minor but significant portion (approx. 20%) of males. As different clones with specific phenotypic and genetic characteristics have been found, and RAPD markers specific to each clone have recently been identified, this fish offers many advantages for analyzing whether or not genetic recombination occurs between different clones. In this study, artificial propagation was performed in clone F and clone D. Ovulated eggs from clone F were divided into two parts and respectively inseminated with sperm from a clone D male and from a red common carp (Cyprinus carpio) male. The control clone D individuals were selected from gynogenetic offspring of clone D activated by sperm of red common carp. The phenotype and sex ratio in the experimental groups were also observed. Using RAPD molecular markers, which allow for reliable discrimination and genetic analysis of different clones, we have revealed direct molecular evidence for gonochoristic reproduction in the gynogenetic silver crucian carp and confirmed a previous hypothesis that the silver crucian carp might reproduce both gynogenetically and gonochoristically. Therefore, we conclude that the silver crucian carp possesses two reproductive modes, i.e., gynogenetic and gonochoristic reproduction. The response mechanism of two reproductive development modes may be the first discovery in vertebrates. Additionally, we discuss the evolutionary implication between gynogenetic and gonochoristic reproduction modes and the contribution of the minor proportion of males to genetic flexibility in the gynogenetic silver crucian carp. Received: 5 January 2000 / Accepted: 3 August 2000     

Genetic Diversity in Crucian Carp (Carassius auratus)

A survey of restriction fragment polymorphism in mitochondrial DNA of three subspecies of Carassius auratus throughout four provinces in China was undertaken using 17 restriction enzymes. Two carp, Cyprinus carpio rubbrofuscus and Cyprinus carpio carpio, were included as the outgroup. A total of 16 haplotypes was observed: 5 in tetraploids of C. auratus auratus; 8 in hexaploids of C. auratus auratus; and 2 in C. auratus gibelio and C. auratus cuvieri, respectively. The tetraploids and hexaploids share three common haplotypes as I, V, and VI. C. a. Cuvieri may have diverged first among the three subspecies. Interestingly, C. a. auratus and C. a. cuvieri did not form monophyletic clades, which indicated that the classification of Carassius auratus required further studies. The current hypothesis, that hexaploids originated from tetraploids by a polyploidy event, is less favorable, based on the distribution of haplotypes and the lower diversity in tetraploids than in hexaploids. Our data also indicate that divergence of hexaploids and tetraploids might be recent and mtDNA polymorphism existed before the divergence. Meanwhile, genetic isolation exists between the hexaploids and the tetraploids.     

Genetic Diversity among Different Clones of the Gynogenetic Silver Crucian Carp, Carassius auratus gibelio, Revealed by Transferrin and Isozyme Markers

Genetic diversity among four clones (A, D, E, F) of gynogenetic silver crucian carp was studied using transferrin and isozymes in the blood as markers. Of the five proteins investigated, three (transferrin, esterase and superoxide dismutase) indicated polymorphism and eight polymorphic loci were detected. These loci were probably encoded by codominant alleles and their inheritance patterns were analyzed. Intraclonal homogeneity and interclonal heterogeneity were observed in these clones, which allowed us to infer the clonal nature and evolutionary relationship between them. Clonal diversity in this population of silver crucian carp in China was also compared with data reported from gynogenetic crucian carp in Germany.     

Intraspecific Scaling of the Resting and Maximum Metabolic Rates of the Crucian Carp (Carassius auratus)

The question of how the scaling of metabolic rate with body mass (M) is achieved in animals is unresolved. Here, we tested the cell metabolism hypothesis and the organ size hypothesis by assessing the mass scaling of the resting metabolic rate (RMR), maximum metabolic rate (MMR), erythrocyte size, and the masses of metabolically active organs in the crucian carp (Carassius auratus). The M of the crucian carp ranged from 4.5 to 323.9 g, representing an approximately 72-fold difference. The RMR and MMR increased with M according to the allometric equations RMR = 0.212M ^0.776 and MMR = 0.753M ^0.785. The scaling exponents for RMR (b _r) and MMR (b _m) obtained in crucian carp were close to each other. Thus, the factorial aerobic scope remained almost constant with increasing M. Although erythrocyte size was negatively correlated with both mass-specific RMR and absolute RMR adjusted to M, it and all other hematological parameters showed no significant relationship with M. These data demonstrate that the cell metabolism hypothesis does not describe metabolic scaling in the crucian carp, suggesting that erythrocyte size may not represent the general size of other cell types in this fish and the metabolic activity of cells may decrease as fish grows. The mass scaling exponents of active organs was lower than 1 while that of inactive organs was greater than 1, which suggests that the mass scaling of the RMR can be partly due to variance in the proportion of active/inactive organs in crucian carp. Furthermore, our results provide additional evidence supporting the correlation between locomotor capacity and metabolic scaling.     

彭泽鲫两个雌核发育克隆的染色体组型分析

采用PHA和秋水仙素体内注射法直接制作肾细胞染色体标本,对彭泽鲫种群内两个不同雌核发育克隆的亲本进行染色体数目及组型分析。结果表明,彭泽鲫种群内的两个不同克隆存在染色体数目及组型差异,其中克隆H包含6条超数染色体在内的染色体众数是156,150条基本染色体的组型公式为：42M 36SM 39ST 33T,NF=228;克隆L包含12条超数染色体在内的染色体众数是162,150条基本染色体的组型公式为：36M 45SM 33ST 36T,NF=231。两个不同雌核发育克隆的发现及其染色体的差异说明彭泽鲫种群内同样存在着类似银鲫种群内的遗传多样性。     

鲫鱼遗传多样性的初步研究

用１７种限制性内切酶对鲫属普通鲫鱼低背型,高背型,异育银鲫,日本白鲫及华南鲤的变种红鲤共１２４个个体的线粒体ＤＮＡ进行了ＲＦＬＰ分析,１４种酶具多态,共计４３种限制性态型,１１种单倍型。     

人工诱导雌核发育日本白鲫

分别用遗传失活的散鳞镜鲤(Cyprinuscarpio L.)、团头鲂(Megalobrama amblycephala)精子诱导日本白鲫(Carassiuscuvieri)进行雌核发育.未经冷休克处理,用UV照射过的镜鲤的精子(其最佳UV照射剂量为4200 mJ/cm2)与日本白鲫卵子受精获得(32.4±3.3)%雌核发育单倍体和(0.7±0.3)%杂交二倍体,而照射过的团头鲂精子(其最佳UV照射剂量为3600mJ/cm2)与日本白鲫卵子受精得到了(33.8±1.4)%雌核发育单倍体和(0.5-±0.3)%杂交二倍体.日本白鲫卵子分别经灭活的镜鲤、团头鲂的精子激活(精子经各自最佳UV剂量处理),施以冷休克处理(受精后2min,0-4℃,40min),其孵化率分别为(27.8±2.1)%和(29.4±3.3)%,发育到摄食期,其正常的雌核发育二倍体鱼苗分别为(15.7±3.4)%和(23.6±4.1)%,在镜鲤实验组中,其正常的雌核发育二倍体鱼苗占孵化鱼苗总数的56%,这比团头鲂实验组中正常雌核发育二倍体鱼苗的比率(达到80%)低,结果表明诱导日本白鲫雌核发育,与母本遗传关系远的团头鲂精子较镜鲤的更有效.从染色体数目、外形特征和性腺结构等方面证实雌核发育后代的生物学特性.雌核发育日本白鲫全为雌性,这表明其雌性是同配XX型.雌核发育日本白鲫在生产上将有着潜在的价值,如比普通日本白鲫长得更快,抗病能力强等.所有的雌核发育日本白鲫的染色体数目都是100,而所有的团头鲂与日本白鲫的杂交后代都是三倍体(3n=124),新三倍体鱼在鱼类养殖和理论研究中将存在潜在的价值.     

Identification of a Spindlin homolog in gibel carp (Carassius auratus gibelio)

Spindlin has been suggested to play an important role during the transition from oocyte maturation to embryo development in mouse, but its homolog similar to the mouse Spindlin in molecular and expression characterization has not been identified up to now in other vertebrates. In this study, a full length of cDNA sequence is cloned and sequenced from the gibel carp (Carassius auratus gibelio). It contains 1240 nucleotides with an open reading frame of 771 nt encoding 257 amino acids. Based on its amino acid sequence alignment and comparison analysis with the known Spin family proteins, the newly cloned Spin is named Carassius auratus gibelio Spindlin (CagSpin). Its product could be detected from mature eggs to blastula embryos, but its content decreased from the two-cell stage, and could not be detected after the gastrula stage. It suggests that the CagSpin should be a maternal protein that is expressed during oocyte maturation, and plays a crucial role in early cleavage of embryogenesis. CagSpin is the first homolog similar to mouse spindlin identified in fish, and also in other vertebrates. GST pull-down assay reveals the first biochemical evidence for the association of CagSpin and beta-tubulin, the microtubule component. Therefore, CagSpin may play important functions by interacting with beta-tubulin and other spindle proteins during oocyte maturation and egg fertilization.     

雌核发育银鲫原肠期胚胎和尾芽期胚胎差异表达基因的呈现

构建了雌核发育银鲫原肠期胚胎和尾芽期胚胎间的抑制性差减杂交cDNA质粒文库。对原肠期’739个和尾芽期816个PCR阳性克隆进行斑点杂交,得到72个原肠期和98个尾芽期斑点杂交阳性克隆。测序和基因数据库比对结果表明：72个原肠期斑点杂交阳性克隆中,包括19个已知基因的cDNA片段和31个没有同源性的cDNA片段;98个尾芽期斑点杂交阳性克隆中,包括52个已知基因的cDNA片段和37个没有同源性的cDNA片段。采用虚拟Northern杂交和RT-PCR证实了部分基因在银鲫胚胎发育过程中的差异表达。这些差异表达基因的呈现为进一步研究银鲫胚胎发育的分子机制奠定了基础。     

Utilization of several plant proteins by gibel carp (Carassius auratus gibelio)

Six isonitrogenous (gross protein content 35%) and isoenergetic (gross energy content 17 kJ g⁻¹) diets were formulated to investigate the effects of inclusion of plant proteins on the gibel carp ( Carassius auratus gibelio L.). The plant proteins tested were: soybean cake (SBC), potato protein concentrate (PPC), peanut cake (PNC), cottonseed cake (CSC) and rapeseed cake (RSC). Fish meal (FM) was used as control. In each diet, 27% of the protein was supplied by fish meal, and the rest supplied by the plant protein tested. Each diet was fed to three groups of gibel carp for 8 weeks in a recirculation system. Specific growth rate (SGR) in fish fed the control diet was significantly higher than those in the other groups, and SGR in fish fed the PPC was significantly lower than in fish fed other plant proteins. There was no significant difference in SGR among the other groups. Feeding rates were ranked in the order: RSC > CSC > FM > PNC > SBC > PPC. Conversion efficiency was highest in groups fed FM, SBC and PNC, followed by groups fed CSC and RSC, and was lowest in the group fed PPC. The fish fed PPC showed lower protein retention than those fed FM and SBC. FM showed highest energy retention while PPC showed lowest. There was no significant relationship between SGR and intake of digestible protein (g g⁻¹ day⁻¹), digestible lysine (g g⁻¹ day⁻¹), digestible methionine (g g⁻¹ day⁻¹) or digestible total essential amino acids (g g⁻¹ day⁻¹), suggesting that the differences in SGR could not alone account for any of these variables.     

市售鲫鱼肠道中沙门菌的调查研究

目的 为摸清市场中出售鲫鱼沙门菌的污染情况.方法 以徐州市某市场上出售的鲫鱼为材料,主要采用SS琼脂平板对120份肠道样品中的沙门菌进行初步鉴定和培养计数.结果 在120份鲫鱼肠道中共检出沙门菌32份,阳性率为26.7％ (32/120),菌群数量分布在(0.32 ～9.12)×103 CFU/g.结论 本研究对徐州地区市售鱼类沙门菌检疫及疾病防治等提供重要参考.     

三倍体和二倍体银鲫的精子发生

应用透射电子显微镜观察了行雌核发育生殖的三倍体(体细胞具有150±染色体)和二倍体 (2n=100)银鲫(Carassius auratus gibelio Bloch)的精子发生过程.两种倍性的雄性的精子发生过程中,染色体数和细胞核的体积均呈3∶2的关系;而精巢的结构、细胞的形态和细胞器的构成均无明显的区别.其精子发生经历了精原细胞、初级精母细胞、次级精母细胞和精子细胞几个阶段,精子细胞再经过精子形成过程成为精子;三倍体银鲫的精子发生与二倍体一样能正常完成减数分裂,形成形态和功能正常的雄性配子.正常的精子发生过程证明,体细胞具有150±条染色体的黑龙江银鲫是已经初步完成二倍化的三倍体,雄性个体在生殖群体中起着重要的作用;推测黑龙江银鲫的二倍体实际可能是四倍体,则三倍体的黑龙江银鲫为偶倍性的六倍体,因此其减数分裂可以正常进行,同时又经历了一定程度的二倍化,所以雄性可育,且参与繁殖后代 [动物学报 54(3):467-474,2008].     

Effect of Adrenaline on Amylolytic Activity of the Intestinal Mucosa, Glycemia Level, and Glycogen Concentration in Tissues of the Golden Carp Carassius auratus and Crucian Carp Carassius carassius

Effects of adrenaline on amylolytic activity in the intestinal mucosa, glycemia level and glycogen concentration in the carp hepatopancreas and muscles was studies under in vivo conditions. There was a considerable similarity revealed between dynamics of the glycogen concentration and amylolytic activity, which indicates participation of adrenaline in regulation not only of metabolism, but also of processes of hydrolysis of polysaccharides in the fish intestine.     

日本白鲫IGF-1基因全长cDNA克隆及组织表达分析

通过已获得的多个物种IGF-1基因的cDNA序列,设计合成简并引物,用日本白鲫的肝脏总RNA反转录获得的cDNA做模板,克隆获得了IGF-1中间序列.在此基础上,通过SMART RACE,获得了IGF-1全长cDNA序列.经过序列对比分析,所得到的序列是日本白鲫(Carassius cuvieri)IGF-1 cDNA全长,其中开放阅读框为486 bp,编码含161个氨基酸的蛋白质,该蛋白质含有保守的信号肽、B、C、A、D和E结构域和6个半胱氨酸残基.同时,系统进化分析表明,在进化过程中IGF-1基因在鱼类中保持着高度保守的进化特征.IGF-1在日本白鲫中广泛表达于多个组织,尤其在肝脏中表达量最高.在垂体、心脏、肾脏和肌肉中有较高的表达.而在脑、脾脏、精巢和卵巢中的表达量较低.     

Mitochondrial DNA Variation in Diploid and Triploid Forms of Silver Crucian Carp <Emphasis Type="Italic">Carassius auratus gibelio</Emphasis>

Variation of two mtDNA fragments amplified in polymerase chain reaction was compared by the RFLP method in a population of the sympatric bisexual (diploid) and gynogenetic (triploid) forms of silver crucian carp. The mtDNA haplotypes of all individuals was shown to be divided into two phylogenetic groups differing by at least 2.5% of nucleotide substitutions in their sequences. All gynogenetic individuals have haplotypes of a single phylogroup, whereas haplotypes of diploid fish belonged to both mtDNA phylogroups. This testifies to a possibility of transformation of gynogenetic individuals into a bisexual form, whereas the reverse process is either extremely rare or impossible.__________Translated from Genetika, Vol. 41, No. 6, 2005, pp. 811–816.Original Russian Text Copyright © 2005 by Brykov, Apalikova, Eliseikina, Kovalev.     

Effects of Montmorillonite on Alleviating Dietary Cd-Induced Oxidative Damage in Carp (Carassius auratus)

The present study was designed to investigate the effects of montmorillonite (MMT) on dietary Cd-induced oxidative damage in liver and kidney of carp (Carassius auratus). One hundred eighty carp were randomly divided into four groups and fed with a basal diet, a basal diet supplemented with 0.5% MMT, Cd-comtaminated basal diet (120 mg Cd/kg dry weight) and Cd-contaminated basal diet supplemented with 0.5% MMT, respectively. After 60 days, fish were sacrificed to measure malondialdehyde (MDA) content and antioxidative indices in liver and kidney. The results showed that the exposure of carp to dietary Cd caused decreases in glutathione peroxidase activity, catalase activity, superoxide dismutase activity, glutathione content and total antioxidant capacity level, while MMT supplemented in diet compensated Cd-induced decreases in above antioxidant indices to some extent in liver and kidney. As compared with the control group, increases in MDA content were observed in both measured tissues of carp exposed to dietary Cd, while MDA content decreased in carp exposed to Cd-contaminated basal diet supplemented with MMT in comparison with the Cd-contaminated group. It was suggested that MMT, when co-administered with Cd in diet, could alleviate dietary Cd-induced oxidative damage in liver and kidney of carp.     

Isolation and Characterization of Polymorphic Microsatellite Markers for the Masked Palm Civet (Paguma larvata)

The masked palm civet (Paguma larvata) has been suspected to be the host of a SARS-like CoV virus that causes severe acute respiratory syndrome in humans. In China, the palm civet lives wild and is farmed, but even though the species is a potential carrier of the virus, its geographic distribution and genetic diversity have never been studied. We report the isolation and characterization of six polymorphic microsatellite markers for P. larvata. To characterize each locus, two farmed masked palm civet populations from Shanxi and Guangxi provinces in China were genotyped. The number of alleles per locus ranged from 3 to 15, and the observed heterozygosity for these populations was 47.1 and 68.7%, respectively.     

Natural Hybridization between Diploid Crucian Carp Species and Genetic Independence of Triploid Crucian Carp Elucidated by DNA Markers

The population structure of genus Carassius in Lake Koyama, southeast Japan, was analyzed by genetic markers as microsatellite DNA, mtDNA RFLP and isozymes. Based on the ploidy level and morphological analysis, four Carassius groups were detected. The triploid group was identified as Ginbuna (C. langsdorfii). In the diploid group, Nagabuna (C. burugeri sp) and Gengoroubuna, (C. cuvieri) were identified. Remaining diploid individuals had morphological traits that were intermediate between Nagabuna and Gengoroubuna. These were considered as hybrids and their descendants. From the results of mtDNA RFLP and isozyme patterns, the triploid population was considered to be independent from the gene pools of diploid. The hybrids had the mtDNA haplotypes which were common to Gengoroubuna and Nagabuna populations. Based on the three microsatellite loci, Ginbuna was classified into six clonal lines. In the diploid population, substitution of the major alleles of Nagabuna and Gengoroubuna were found. The hybrids had alleles that were common in Nagabuna and Gengoroubuna. The values of the hybrid index (IH) which are ranged from 0.771 to 0.964 in Nagabuna, from 0.102 to 0.806 in the hybrids and from 0.068 to 0.157 in Gengoroubuna. The hybrid population was verified to be derived from crossbreeding between the Gengoroubuna and Nagabuna populations. Evidence of backcrossing in nature by microsatellite DNA markers was also obtained in the diploid populations.     

Karyotype and Resource Protection of the Wild Crucian Carps ( Carassius auratus)in the Dongting Lake Water Svstem

鲫( Carassius auratus)是洞庭湖水系一种重要的经济鱼类.为了解洞庭湖水系野鲫的细胞遗传背景,采用PHA和秋水仙素活体注射法,对沅水和澧水采集的野鲫样本逐一进行肾细胞染色体制片及组型分析.结果发现,在两条河流的野鲫群体中均检测出染色体数为100和基本染色体数为150的两种不同倍性个体,其中,两条河流染色体数为100的二倍体鲫组型公式为2N=28M+ 22SM+ 28ST +22T,NF=150;基本染色体数为150的三倍体鲫组型公式为3N =42M +33SM +42ST +33T,NF=225.在沅水和澧水不同采样点随机采集的共100尾野鲫中,检测出的三倍体比例(85％)远高于二倍体(15％),且二倍体与三倍体鲫个体在形态特征上不存在明显差异(P＞0.05).两种不同倍性鲫在同一水体的共存对于鲫的遗传进化与选育具有一定的理论和实践意义,而二倍体鲫种群的大量减少,则提示我们应该从染色体组遗传多样性角度加强对洞庭湖水系二倍体野鲫资源的保护.