Phylogenetic relationships of silver crucian carp Carassius auratus gibelio, C. auratus cuvieri, crucian carp C. carassius, and common carp Cyprinus carpio as inferred from mitochondrial DNA variation

PCR-RFLP analysis of the ND3/ND4L/ND4 and 12S/16S rRNA regions and nucleotide sequence variation of the cytochrome b gene were used to study the mtDNA divergence in species of the family Cyprinidae, to examine the phylogenetic relationships of the species, and to identify their taxonomic status. The results indicated that an ancestral form diverged into silver crucian carp and crucian carp after its separation from the common carp lineage. The divergence of continental Carassius auratus gibelio and Japanese C. auratus cuvieri occurred more recently. Two well distinguishable mtDNA phylogroups, suggesting divergent evolution, were observed in continental C. auratus gibelio populations. The divergence was possibly related to the formation of two silver crucian carp groups with different types of reproduction, triploid gynogenetic and diploid gonochoric. At the same time, the results supported the high probability of current genetic exchange between the forms. In view of these findings and high morphological similarity of the two forms, they were not considered to be separate species.     

Mitochondrial DNA variation in diploid and triploid forms of silver crucian carp Carassius auratus gibelio

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 differed in at least 2.5% of nucleotide substitutions and fall into two phylogroups. 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.     

Inheritance of microsatellite DNA in bisexual gynogenesis complex of Fangzheng silver crucian carp,Carassius auratus gibelio (Bloch)

Five gynogenetic progeny groups of silver crucian carp Carassius auratus gibelio were produced and sex ratios (males:total progeny) of each of the progeny groups were analysed. About 110 males and 366 females were genotyped at 15 microsatellite loci for comparison with their parents to (1) verify the gynogenesis status of Fangzheng C. auratus gibelio, (2) detect the incorporation of paternal genetic material into the offspring and (3) study the possible association of genetic exchange at microsatellite loci with the existence of sex. The sex ratios in progenies of five groups were highly variable, but all had significant female bias. The sex ratio ranged from 0 to 0·37. Significant differences in the sex ratio within and between groups were also found. Microsatellite genotyping at 15 loci showed that 100 and 97% of the progeny shared the same genotype with the mother in four groups and in one group, respectively, confirming that gynogenesis is the general mechanism of reproduction in C. auratus gibelio. However, 0·63% of all offspring did show incorporation of paternal genetic material. No single loci tested were associated with the occurrence of male progeny, indicating unknown genetic mechanisms for sex determination in C. auratus gibelio.     

Isolation and characterization of microsatellite markers from Fangzheng silver crucian carp,Carassius auratus gibelio (Bloch), and cross‐amplification in the closely related species crucian carp,Carassius auratus auratus (Linnaeus)

We report the development of 20 microsatellite markers for Fangzheng silver crucian carp, Carassius auratus gibelio (Bloch). Nineteen out of 20 showed polymorphism with alleles ranging from two to 14. These loci were screened to amplify the closely related species crucian carp, Carassius auratus auratus (Linnaeus) and all of them can amplify DNA with the size similar to the former. The origin of silver crucian carp is in issue and the population genetic structure is still unclear. Microsatellite markers isolated from the silver crucian carp and their utility in the crucian carp will be useful for these researches.     

The ploidy and reproductive mechanism of crucian carp, Carassius auratus gibelio

With the method of Feulgen staining, through cytophotometric measurements, the DNA contents of sex-reversed male (genotypic female) crucian carp (2n = 150 ±) had average values of 2.75 and 1.36 A.U., respectively, in erythrocytes and spermatozoa. The ratio of the erythrocyte and spermatozoon DNA contents of the 'male' crucian carp is 2.01:1 (2C: 1C) and is the same as in the normal male, 2.02:1. The crucian carp has a DNA value of 4.1 pg, which is 68% of that in humans. The results of the study suggest that both sex-reversal males and normal males have the ability to complete the meiotic division during their gametogenesis, and that the population has diploidized over a long term of evolution.     

Genetic diversity analysis of crucian carp (Carassius auratus) based on cyt b and D-loop-containing region around Hongze Lake

Environmental Biology of Fishes - Crucian carp (Carassius auratus) is an important freshwater aquaculture species in China. The study of the genetic diversity of native crucian carp will provide...     

Molecular identification of hybrids of the invasive gibel carp Carassius auratus gibelio and crucian carp Carassius carassius in Swedish waters

Both mitochondrial DNA sequence and two nuclear microsatellite markers were used to confirm the identity of the first record of Carassius auratus gibelio in the western (Swedish) Baltic Sea region. A total of 49 fishes were analysed, where 22 were from three Swedish sites connected to the Baltic Sea. The D-loop mitochondrial DNA sequences showed that 16 of 22 Swedish fishes were related to C. a. gibelio. The phylogenetic analysis of these sequences showed that these fish are probably not native, but represent different lineages of C. a. gibelio from China, Japan and Russia. All except three of these 16 fishes had microsatellite alleles suggesting hybridization with Carassius carassius. These findings suggest that a cryptic invasion of C. a. gibelio might be in progress.     

温度驯化对鲫偏好游泳行为的影响

温度变化通常会引起鱼类的生理功能和游泳行为产生适应性变化。为考察温度驯化对鲫偏好游泳速度的影响,本研究以鲫(Carassius auratus)为实验对象,分别在(15±1)℃和(25±1)℃条件下将单尾实验鱼置于梯度流速选择仪(流速范围为11.86~65.45 cm·s-1,等距离划分为5个流速区域,从第一到第五流速区域的流速连续增加)中拍摄1 h,采用Ethovision XT9软件分析视频资料并计算实验鱼在不同流速区域平均进入频次(F,次)、单次进入停留时间(T,s·次-1)和平均停留时间百分比(Pt,%)等流速选择行为指标。结果表明:25℃下,随着流速的上升、F值显著增加以及水流速度过高,鲫无法在最高流速区域停留较长时间而导致鲫在第四流速区域的Pt达到47%,显著高于其他流速区域(P0.05);15℃下,鲫第一到第三流速区域的F值相近,而第四和第五流速区域F显著下降;同时其T值的最大值也出现在最低流速区域,导致该温度组的最低流速区域的Pt达到最大(37%),显著高于其他流速区域(P0.05);在第一至第三速流速区域25℃组的F显著低于15℃组,且25℃组实验鱼在第四流速区域的T值显著大于15℃组(P0.05),导致25℃组在第一、第二速流速区域的Pt显著低于15℃组,而25℃组在第四、第五速流速区域的Pt显著高于15℃组(P0.05);实验鱼在25和15℃下其偏好游泳速度分别为27.91~41.30和11.86~15.18 cm·s-1;温度下降导致鲫的偏好游泳速度明显降低,与低温环境下鲫的整体生理功能受限有关。     

Analysis of genetic heterogeneity among five gynogenetic clones of silver crucian carp, Carassius auratus gibelio Bloch, based on detection of RAPD molecular markers

The gynogenetic silver crucian carp, Carassius auratus gibelio, is a unique model system for studying evolutionary genetics and selective breeding, owing to its specific genetic background and reproductive modes. Five gynogenetic clones were analyzed by the random amplified polymorphic DNA (RAPD) technique, using 30 10-nucleotide-long primers. Twenty-six primers produced well-amplified DNA fragments with reproducible banding patterns, and 24 primers were polymorphic. Nearly identical banding patterns were observed among individuals within each clone, suggesting that each clone might possess a specific pattern owing to its gynogenesis. In contrast, the RAPD patterns of the five clones differed from each other. A phylogenetic tree was constructed using UPGMA cluster analysis based on a total of 3,744 distinguishable fragments (156 per individual). Average genetic distances within and among the five clones clearly indicated their intraclonal homogeneity, interclonal heterogeneity, and phylogenetic relationships. Clones A and P were the most closely related, whereas the most divergence was seen between clone D and clone E or F. A total of 88 polymorphic fragments were scored from 24 primers after excluding bands that were monomorphic for the five clones. Most primers corresponding to the polymorphic fragments amplified reproducible markers specific for one clone or that were shared by two, three, or four clones. Several primers (e.g., Opj-1, Opj-7, and Opp-10) produced abundant banding patterns that could be used to discriminate between the five clones. Markers specific for one or two clones were also identified. The RAPD markers identified in this study will likely benefit evolutionary genetics and selective breeding studies.     

透明鲫—一个适合活体研究的鱼类模型

目的了解鲫成体透明的原因,探讨该性状的应用特性,为透明鲫作为水生实验动物材料系统开发提供基础。方法对透明鲫进行繁殖,并观察其后代性状,了解透明性状的遗传规律;体视镜观察透明鲫色素细胞的种类与分布,并与鲫比较;组织切片和压片确认微孢子虫对透明鲫的感染,并观察感染症状的变化。结果鲫的透明性状可以遗传,大部分后代表现为通体透明,心、肝、肾、肠、鳔、鳃、脊椎等组织器官肉眼清晰可见。与正常鲫比较,透明鲫的主要色素细胞为黄色素细胞,并未发现虹彩色素细胞,黑色素细胞的数量也大为减少。微孢子虫对鱼体的感染过程可直观观察,病原的扩散和空间分布能实时获得,具普通鱼类无法比拟的应用优势。结论虹彩色素细胞的缺失是鲫透明突变的结构基础。由于透明鲫内部器官可直接观测,无需依靠解剖或复杂仪器系统,在同一动物身上可能获得一系列的动态试验数据,或可作为模型材料广泛应用于生命科学不同领域。     

彭泽鲫两个雌核发育克隆的 RAPD分析

采用随机扩增多态DNA技术,对彭泽鲫种群内两个不同雌核发育克隆进行比较分析.在使用的30个随机引物中有28个引物的扩增效果良好,两个克隆各5个样本共产生清晰可辨的扩增带2154条(其中克隆H为1088条,克隆L为1066条).克隆H群体内的平均遗传距离为0.018070±0.015489,克隆L群体内的平均遗传距离为0.017827±0.015203,两个克隆群体间的平均遗传距离为0.352511±0.009692,群体内和群体间的遗传距离清晰地表明了同一雌核发育克隆群体的遗传相似性和不同克隆群体间的遗传异质性.由NJ聚类法构建的分支树状图也很好地反映了两个雌核发育克隆群体及个体间的相互关系.在28个引物对两个克隆的扩增带中,一共可找到91条多态性片段,一些引物如Opj15、Opp8、Opq11和Opq14等,产生了非常丰富、稳定的多态性标记,可以作为两个雌核发育克隆的有效鉴别指标.彭泽鲫两个雌核发育克隆在分子水平上的差异进一步证实了彭泽鲫种群的遗传多样性,与银鲫相类似,彭泽鲫种群的遗传多样性对单性脊椎动物的进化理论研究和彭泽鲫的选种育种实践都具有重要的意义.     

Phylogenetic relationships of silver crusian carp Carassius auratus gibelio, C. auratus cuvieri, crucian carp Carassius carassius, and common carp Cyprinus carpio as inferred from mitochondrial DNA variation

PCR-RFLP analysis of the ND3/ND4L/ND4 and 12S/16S rRNA regions and nucleotide sequence variation of the cytochrome b gene were used to study the mtDNA divergence in species of the family Cyprinidae, to examine the phylogenetic relationships of the species, and to identify their taxonomic status. The results indicated that an ancestral form diverged into silver crucian carp and crucian carp after its separation from the common carp lineage. The divergence of continental Carassius auratus gibelio and Japanese C. auratus cuvieri occurred more recently. Two well distinguishable mtDNA phylogroups, suggesting divergent evolution, were observed in continental C. auratus gibelio populations. The divergence was possibly related to the formation of two silver crucian carp groups with different types of reproduction, triploid gynogenetic and diploid gonochoric. At the same time, the results supported the high probability of current genetic exchange between the forms. In view of these findings and high morphological similarity of the two forms, they were not considered to be separate species.     

The appearance pattern of tooth germs in the round crucian carp,Carassius auratus grandoculis

Cyprinid fishes generally replace their teeth alternately and cephalad. The larvae ofCarassius auratus grandoculis also replace their teeth alternately and cephalad, in a pattern of 4-2-3-1-. However, adults ofCarassius species replace their teeth from anterior to posterior, in a pattern of 1-2-3-4-1-. So I analyzed the appearance pattern of tooth germs in larvae and juveniles inCarassius auratus grandoculis. At stage 5 of the post-larval period, developmental difference is made between both sides. In the pharyngeal dentition on one side developing poorly, the anterior tooth on the fifth replacement wave, tooth₄[An2] appeared later than the central teeth on following replacement wave, tooth₅[Pol]. Moreover, the anterior tooth on the seventh replacement wave, tooth₆[An2], appeared later than the central teeth on the following replacement wave, tooth₇[Pol], on both sides. The reverse of tooth germ appearance between anterior teeth and central teeth makes a change of replacement pattern from 4-2-3-1-4- to 1-2-3-4-1-. The change of replacement pattern is caused by the confusion of tooth germs of anterior teeth on both sides.Mylopharyngodon piceus andCyprinus carpio make a change of replacement patterns in the early juvenile period, too. This change of replacement pattern may be a specialized character among the subfamily Cyprininae.     

雌核发育银鲫子代中微卫星特异序列分析

雌核发育个体的基因型基本上完全与母本相同,这是源于卵子发生过程中没有经过减数分裂.父本的遗传物质是在随机水平、亚基因组水平或基因组水平参与到子代的遗传重组过程,从而对长期突变积累的雌核发育生物基因组进行补偿,一直是遗传学家关注的问题.本文对雌核发育银鲫特异个体及父母本5个微卫星位点的扩增条带进行了克隆测序,相似性比对结果显示,特异个体所表现的父咎匾霥NA条带,序列结果与父本完全相同或相似(SCM4、SCM9、YJ5),并在某些位点上保留了母本的特异条带(YJ5),而个体本身特异的DNA条带与父母本的相似性均较高(SCM13).同时,所检测到的个体经越冬后查验为雌性个体,进一步进行同源繁育,研究变异条带在繁殖中的命运.连续2代的繁育检测结果表明,融合了父本特异性条带的银鲫个体在繁殖过程中仍行雌核发育的生殖方式,变异来的条带能够传递给子代,进一步证实同源雌核发育银鲫通过小概率两性融合事件丰富银鲫种群的遗传多样性[动物学报 53(3):537-544,2007].     

Studies on resistance characteristic and cDNA sequence conservation of transferrin from crucian carp, Carassius auratus

Transferrin (Tf) is a kind of non-heme β-globulin with two iron ions (Fe³⁺)-binding sites. To prove Tf’s physiological functions, Fe³⁺-proteins, serum iron contents, and total iron-binding capabilities were tested for Tfs of crucian carps (Carassius auratus) and sliver carps (Hypophthalmichthys molitrix). The above results demonstrated that sliver carps shared 1/3 Tf alleles with crucian carps; Tf of crucian carps had stronger Fe³⁺-binding ability and transportation ability in plasma than that of sliver carps. In addition, the results of oxygen consumption experiments indicated that crucian carps had the higher oxygen utility rate than sliver carps. For acute hypoxia exposure assay, normoxic gas mixture, hypoxic gas mixture A, and hypoxic gas mixture B were used to induce oxygen-regulated gene expression of crucian carps in acute hypoxia. The results of quantitative real-time PCR revealed that mRNA levels of Tf gene, Tfr gene and ATPase gene were down-regulated in acute hypoxia but mRNA level of LDHa gene was up-regulated in acute hypoxia. The results of crucian carp Tf-cDNA sequence analysis showed that cDNA regions of two Fe³⁺-binding sites were T₇₄₇–T₁₀₂₆ and T₁₇₃₇–A₁₈₈₄ based on the principle of bioinformatics. The sequence conservation of two Fe³⁺-binding sites was higher than that of the other five regions, which were confirmed according to the subregion model of Tf-cDNA sequence.     

Polymorphic microsatellites from silver crucian carp (Carassius auratus gibelio Bloch) and cross‐amplification in common carp (Cyprinus carpio L.)

Fifteen polymorphic microsatellites were isolated from the genome of silver crucian carp (Carassius auratus gibelio Bloch). Allele numbers ranged from two to four with an average of 2.7/locus, and the proportion of tri‐ and diallelic heterozygotes was 99.3%. The individuals tested seem to have originated from two different clonal lines: 14 of 16 showed the same genotype at all loci tested, whereas the remaining two were also identical, but different from the former ones. Eleven out of 15 primer pairs cross‐amplified products from the genome of common carp, whereas only five from that of zebrafish.     

Meiotic chromosome behaviour in male triploid transparent coloured crucian carp, Carassius auratus L.

Meiotic chromosome behaviour was investigated by surface-spreading, air-drying and thin sectioning testes for light and electron microscope examination in artificial triploid transparent coloured crucian carp ( Carassius auratus L.) produced by hydrostatic pressure shock. Unsynapsed univalents, synapsed bivalents and partially synapsed trivalents could be observed and distinguished from each other in the surface-spread spermatocytes. The pairing of the partially paired trivalents mainly occurred in the telomeric regions. Similarly, lateral elements of unsynapsed univalents, typical synapsed bivalents and triple pairing configurations having three lateral elements and two central elements in the trivalents were also observed in the thin sectioned pachytene spermatocytes. The metaphase I cells were mainly composed of univalents, bivalents and trivalents, but a few tetravalents, pentavalents and hexavalents were also found. The relationship between disturbed chromosome pairing and abnormal spermatogenesis is briefly discussed.     

Subcellular localization and functional characterization of a fish IRF9 from crucian carp Carassius auratus

Mammalian interferon (IFN) regulatory factor 9 (IRF-9) has long been recognized as the DNA sequence recognition subunit of IFN-stimulated gene factor 3 (ISGF3) complex, which is critical for type I IFN to induce the expression of IFN-stimulated genes (ISGs) against viral infection. Recent studies have shown that fish IFN exerts antiviral effects by induction of a number of ISGs and also of itself; however, little is known about the role of fish IRF9 in IFN signaling. Here we identify a fish IRF9 orthologue (CaIRF9) from IFN-producing cell line, crucian carp Carassius auratus blastulae embryonic (CAB) cells. Analysis of subcellular distribution of CaIRF9-green fluorescent protein indicates that CaIRF9 is constitutively present in the nucleus, which is driven by two nuclear localization signals (NLS), one locating within DNA-binding domain (DBD) of CaIRF9 and the other immediately behind DBD, although human IRF9 contains only one NLS analogous to the former of CaIRF9. Overexpression of CaIRF9 together with CaSTAT2 not only activates ISRE-containing promoter but also upregulates the expression of fish ISGs. Strikingly, CaIRF9 together with CaSTAT2 also exhibits an ability to activate crucian carp IFN promoter, and blockade of cellular CaIRF9 attenuates IFN itself-induced activation of crucian carp IFN promoter. Taken together, these data suggest that crucian carp IFN induces the expression of ISGs and also of itself possibly by the JAK-STAT signaling pathway that is conserved from fish to mammals.     

Coexistence of diploid, triploid and tetraploid crucian carp (Carassius auratus) in natural waters

Background

Crucian carp (abbreviated CC) belongs to the genus of Carassius within the family of Cyprinidae. It has been one of the most important freshwater species for Chinese aquaculture and is especially abundant in the Dongting water system of Hunan province. CC used to be considered as all diploid forms. However, coexistence of diploid (abbreviated 2nCC), triploid (abbreviated 3nCC) and tetraploid crucian carp (abbreviated 4nCC) population of the Dongting water system was first found by our recently researches.

Results

We examined the ploidy level and compared biological characteristics in different ploidy CC. In reproductive mode, 2nCC was bisexual generative and 4nCC generated all-female offspring by gynogenesis. However, 3nCC generated progenies in two different ways. 3nCC produced bisexual triploid offspring fertilized with 3nCC spermatozoa, while it produced all-female triploid offspring by gynogenesis when its ova were activated by heterogenous spermatozoa. The complete mitochondrial DNA of three different ploidy fishes was sequenced and analyzed, suggesting no significant differences. Interestingly, microchromosomes were found only in 3nCC, which were concluded to be the result of hybridization. Allogenetic DNA fragments of Sox genes were obtained in 3nCC and 4nCC, which were absent in 2nCC. Phylogenetics analysis based on Sox4 gene indicated 3nCC and 4nCC formed a separate group from 2nCC.

Conclusions

In summary, this is the first report of the co-existence of three types of different ploidy crucian carps in natural waters in China. It was proved that the coexistence of different ploidy CC was reproductively maintained. We further hypothesized that 3nCC and 4nCC were allopolyploids that resulted from hybridization. The different ploidy CC population we obtained in this study possesses great significance for the study of polyploidization and the evolution of vertebrates.