Genetic structure of crucian carp (Cypriniformes, Cyprinidae, Carassius L. 1758) populations of middle-Dnieper basin

Comparative analysis of Middle Dnieper crucian carp's population structure was made by means of biochemical gene marking, cytometry and biological analysis. As a result the structure was found, which may by expounded like association of the genetically related species and forms. This association consists of diploid C. auratus (which predominates in population), C. carassius (which was found in the some of north-eastern reservoirs only) and triploid C. gibelio. In Ukraine the homogeneous populations of C. gibelio can be found on the north-east of Eastern Ukraine only. Though some of individuals maybe present with diploid C. auratus. Besides were found hybrid form: diploid C. auratus-C. carassius and presumably tetraploid C. auratus-C. gibelio and C. gibelio-C. carassius. However in the tetraploid's spectra were absent a number of specific alleles, that marking C. species-one of the parental species of triploid C. gibelio. Therefore presumptive tetraploids have not difference from C. auratus and diploid C. auratus-C. carassius on the gene markers level. Nevertheless they are neatly diagnose by the erythrocytes which have good difference and are bigger then diploid species erythrocytes in 40% (C. auratus-C. gibelio) and 100% (C. gibelio-C. carassius).     

Genetic analysis of offspring from intra‐ and interspecific crosses of Carassius auratus gibelio by chromosome and RAPD analysis

The ploidy of silver crucian carp Carassius auratus gibelio individuals, originating from nine natural habitats of Hungary, was estimated by erythrocyte nucleus area analysis. On the basis of DNA polymorphism, the genetic homogeneity or heterogeneity and the chromosome number of different offspring derived from the crossing of triploid and diploid populations and of two types of silver crucian carp females with other cyprinid males ( Cyprinus carpio, Carassius carassius, Carassius auratus and Barbus conchonius ) were determined. The results of chromosome and RAPD analysis demonstrated that diploid females could reproduce sexually with silver crucian carp and other cyprinid males and that the offspring of intra‐ and interspecific crosses contained the paternal DNA. Triploid females usually reproduced by gynogenesis and their offspring were clones, however, in very rare cases paternal genes were actually transmitted ( i.e . paternal leakage) to the offspring and the progeny were triploid interspecific hybrids. RAPD analysis showed that while the paternal DNA appeared in the offspring, the maternal phenotype was strongly expressed.     

Hybridization of crucian carp <Emphasis Type="Italic">Carassius carassius</Emphasis> (Linnaeus, 1758) in Ukrainian reservoirs and the genetic structure of hybrids

The hybridization of crucian carp Carassius carassius inhabiting polyspecific carp populations of Ukraine’s water bodies and the genetic structure of its hybrids were studied using biochemical gene marking and cytometry procedures. The widespread hybridization between C. auratus and C. carassius was justified by a large number of hybrids, which could form populations only represented by hybrid specimens. The hybrids of C. auratus × C. carassius were diploid, triploid, and, in exceptional cases, tetraploid. Females and males were presented in equal numbers and were most likely breeding by hybridogenesis. Alongside with that, some clonal hybrids of C. carassius × C. gibelio-1 were represented by tetraploid females, and one triploid female of C. carassius × Tinca tinca was found. Arguments are presented in favour of hybridization of alien C. auratus and endemic C. carassius and that this hybridization may appear as one of the mechanisms of replacement and suppression of the C. carassius species.     

Towards the conservation of crucian carp Carassius carassius: understanding the extent and causes of decline within part of its native English range

The extent and causes of crucian carp Carassius carassius decline were assessed during an initial study of c. 25 ponds in north Norfolk, eastern England, U.K., which was then replicated (a validation study) on another c. 25 ponds in an adjacent area. Of these ponds, c. 40 are known to have contained C. carassius during the 1970s-1980s. In the initial and validation studies, C. carassius were found in only 11 of these ponds, yielding declines of 76% (five of 21 ponds) and 68% (six of 19 ponds), respectively (72% decline overall). Non-native cyprinids, including goldfish Carassius auratus and common carp Cyprinus carpio and their hybrids with C. carassius, were observed in 20% of the ponds. Causes of C. carassius local extinction from 21 ponds were confidently determined as desiccation due to drought, terrestrialization and habitat deterioration, hybridization and competition with non-native cyprinids, agricultural land reclamation and predation (after the introduction of pike Esox lucius). This study led to C. carassius being designated as a Biodiversity Action Plan (BAP) species in the county of Norfolk, the first formal conservation designation for the species in the U.K. The C. carassius BAP plan aims to halt the decline of this much overlooked species through reintroductions and selective stocking of suitable ponds within the native range of the species.     

The formation of the polyploid hybrids from different subfamily fish crossings and its evolutionary significance

This study provides genetic evidences at the chromosome, DNA content, DNA fragment and sequence, and morphological levels to support the successful establishment of the polyploid hybrids of red crucian carp x blunt snout bream, which belonged to a different subfamily of fish (Cyprininae subfamily and Cultrinae subfamily) in the catalog. We successfully obtained the sterile triploid hybrids and bisexual fertile tetraploid hybrids of red crucian carp (RCC) (female symbol) x blunt snout bream (BSB) (male symbol) as well as their pentaploid hybrids. The triploid hybrids possessed 124 chromosomes with two sets from RCC and one set from BSB; the tetraploid hybrids had 148 chromosomes with two sets from RCC and two sets from BSB. The females of tetraploid hybrids produced unreduced tetraploid eggs that were fertilized with the haploid sperm of BSB to generate pentaploid hybrids with 172 chromosomes with three sets from BSB and two sets from RCC. The ploidy levels of triploid, tetraploid, and pentaploid hybrids were confirmed by counting chromosomal number, forming chromosomal karyotype, and measuring DNA content and erythrocyte nuclear volume. The similar and different DNA fragments were PCR amplified and sequenced in triploid, tetraploid hybrids, and their parents, indicating their molecular genetic relationship and genetic markers. In addition, this study also presents results about the phenotypes and feeding habits of polyploid hybrids and discusses the formation mechanism of the polyploid hybrids. It is the first report on the formation of the triploid, tetraploid, and pentaploid hybrids by crossing parents with a different chromosome number in vertebrates. The formation of the polyploid hybrids is potentially interesting in both evolution and fish genetic breeding.     

四类不同倍性鲫鱼在胚胎发育期间同工酶基因表达的比较分析

Isozyme zymograms of esterase (EST), lactate dehydrogenase (LDH), malate dehydrogenase (MDH) and superoxide dismutase (SOD) were analysed by polyacrylamide gradient gel electrophoresis at different developmental stages of embryogenesis in 4 types of various ploidy crucian carp embryos, including haploids, diploids, natural triploids, and multiple tetraploids, and 2 types of haploid and diploid common carp embryos. Haploid embryos of crucian carp (Carassius auratus) and common carp (Cyprinus carpio) were produced by treating eggs with UV-irradiated milt from blunt snout bream (Megalobrama amblycephala). Natural triploid embryos were obtained from the eggs of gynogenetic silver crucian carp (Carassius auratus gibelio) inseminated with milt from red common carp. Multiple tetraploid embryos were also produced by gynogenesis from eggs of the newly discovered multiple tetraploid females inseminated with milt from red common carp. Gradient gel electrophoresis indicated that the band types and staining intensity of 4 isozymes expressed in haploid embryos of crucian carp and red common carp were similar to that in the correlative diploid embryos. In natural triploid silver crucian carp embryos, the zymograms of MDH and SOD isozymes were identical with that of diploid crucian carp embryos, but the EST and LDH isozymes manifested more new enzyme bands in comparison with diploid embryos. The corresponding expressed products of some bands in the triploid embryos, such as EST5 and EST6, could be observed also in red common carp embryos, which provided evidence for hybrid origin about the gynogenetic fish. The multiple tetraploids incorporated one foreign genome of red common carp, therefore, the effects of genes from the foreign genome could be observed in the multiple tetraploid embryos. Gene expression of the isozymes in the tetraploid embryos was somewhat similar to that in hybrids. Owing to interaction of triploid silver crucian carp genomes and common carp haploid genome, some isozyme bands, such as EST5 and EST6, changed in quantity, and some bands increased, such as s-SOD1, s-SOD2, s-SOD3 and s-SOD4 in the tetraploid embryos. Moreover, the heterogeneity was revealed among embryos developed from gynogenetic eggs of 3 different multiple tetraploid individuals.     

四类不同倍性鲫鱼在胚胎发育期间同工酶基因表达的比较分析

用聚丙烯酰胺梯度凝胶电泳比较分析了单倍体、二倍体、三倍体和复合四倍体4类不同倍性鲫鱼以及单倍体和二倍体鲤鱼在胚胎发育时期4种同工酶(EST,LDH,MDH,SOD)酶谱。结果表明,单倍体鲫鱼和单倍体鲤鱼胚胎与各自的二倍体胚胎相比,同工酶酶谱看不出差异;天然三倍体银鲫胚胎的MDH和SOD同工酶酶谱与二倍体鲫相似,但EST和LDH同工酶比二倍体增多了酶带,有的酶带如EST5和EST6还可在鲤鱼胚胎中找到相应的表达产物,提供了天然雌核发育三倍体银鲫杂交起源的证据;复合四倍体由于含有鲤鱼的一个外来基因组,其胚胎的基因表达有些与杂种类似,在所分析的4种同工酶酶谱中,都可观察到来自鲤鱼基因的影响。此外,在由源于不同复合四倍体个体的卵子发育形成的胚胎间,还观察到同工酶基因表达的异质性。     

Identification of natural hybrids of gibel carp Carassius auratus gibelio (Bloch) and crucian carp Carassius carassius (L.) from lower Dyje River floodplain (Czech Republic)

Morphometric and genetic variation were examined in parental Carassius auratus gibelio and Carassius carassius individuals, and their natural hybrids. Of meristic traits, only the number of gill rakers clearly distinguished hybrids (39·4 ± 1·4) from parental C. a. gibelio (48·3 ± 0·7) and C. carassius (28·6 ± 1·1). MtDNA sequences showed that the hybrids were descendants of female C. a. gibelio . Microsatellite analysis confirmed the presence in hybrids of variants typical of C. a. gibelio and C. carassius .     

Massive production of all-female diploids and triploids in the crucian carp

In many species of aquaculture importance, all-female and sterile populations possess superior productivity due to faster growth and a relatively homogenous size of individuals. However, the production of all-female and sterile fish in a large scale for aquaculture is a challenge in practice, because treatments necessary for gynogenesis induction usually cause massive embryonic and larval mortality, and the number of induced gynogens is too small for their direct use in aquaculture. Here we report the massive production of all-female triploid crucian carp by combining artificial gynogenesis, sex reversal and diploid-tetraploid hybridization. Previously, we have obtained an allotetraploid carp population (4n = 200) by hybridization between red crucian carp (Carassius auratus red var; ♀) and common carp (Cyprinus carpio; ♂). We induced all-female diploid gynogens of the Japanese crucian carp (Carassius cuvieri; 2n = 100). We also generated male diploid gynogens of the same species treated gynogenetic fry with 17-α-methyltestosterone, leading to the production of sex-revered gynogenetic males. Finally, these males were used to cross with the female diploid Japanese crucian carp gynogens and the allotetraploid females, resulting in the production of fertile all-female diploid Japanese crucian carp (2n=100) and sterile all-female triploid hybrids (3n = 150), respectively. Therefore, diploid crucian carp gynogenetic females and sex-reversed male together with an allotetraploid line provide an opportunity to produce all-female triploid populations in a large scale to meet demands in aquaculture industry.     

Organization and variation analysis of 5S rDNA in different ploidy-level hybrids of red crucian carp × topmouth culter

Through distant crossing, diploid, triploid and tetraploid hybrids of red crucian carp (Carassius auratus red var., RCC♀, Cyprininae, 2n = 100) × topmouth culter (Erythroculter ilishaeformis Bleeker, TC♂, Cultrinae, 2n = 48) were successfully produced. Diploid hybrids possessed 74 chromosomes with one set from RCC and one set from TC; triploid hybrids harbored 124 chromosomes with two sets from RCC and one set from TC; tetraploid hybrids had 148 chromosomes with two sets from RCC and two sets from TC. The 5S rDNA of the three different ploidy-level hybrids and their parents were sequenced and analyzed. There were three monomeric 5S rDNA classes (designated class I: 203 bp; class II: 340 bp; and class III: 477 bp) in RCC and two monomeric 5S rDNA classes (designated class IV: 188 bp, and class V: 286 bp) in TC. In the hybrid offspring, diploid hybrids inherited three 5S rDNA classes from their female parent (RCC) and only class IV from their male parent (TC). Triploid hybrids inherited class II and class III from their female parent (RCC) and class IV from their male parent (TC). Tetraploid hybrids gained class II and class III from their female parent (RCC), and generated a new 5S rDNA sequence (designated class I-N). The specific paternal 5S rDNA sequence of class V was not found in the hybrid offspring. Sequence analysis of 5S rDNA revealed the influence of hybridization and polyploidization on the organization and variation of 5S rDNA in fish. This is the first report on the coexistence in vertebrates of viable diploid, triploid and tetraploid hybrids produced by crossing parents with different chromosome numbers, and these new hybrids are novel specimens for studying the genomic variation in the first generation of interspecific hybrids, which has significance for evolution and fish genetics.     

Male primer endocrine responses to preovulatory female cyprinids under natural conditions in Sweden

This study investigated two related aspects of male-female reproductive interactions in the family Cyprinidae: (1) whether ovulating female rudd Scardinius erythrophthalmus (subfamily Leuciscinae) induce endocrine and gonadal priming responses in conspecific males, a phenomenon which has been described only in species from the subfamily Cyprininae such as goldfish, Carassius auratus, crucian carp Carassius carassius and common carp, Cyprinus carpio and (2) whether the stimuli mediating these responses are species-specific. Field studies of three sympatric European cyprinids, two leuciscins (S. erythrophthalmus and white bream Blicca bjoerkna) and one cyprinin (C. carassius), were conducted on fishes captured in Sweden in the spawning season and held in net pens under natural conditions. As previously reported in C. carassius, male S. erythrophthalmus increased milt (sperm and seminal fluid) volume and plasma concentrations of the sperm maturation hormone 4-pregnen-17,20β-diol-3-one (17,20β-P) when they were held with female S. erythrophthalmus induced to ovulate by injection of Ovaprim (GnRH analogue plus dopamine antagonist). Male S. erythrophthalmus had larger milt volumes than male C. carassius prior to and following exposure to ovulatory conspecifics, but exhibited a smaller proportional milt increase in response to stimulation, suggesting species differences in sperm allocation at spawning. The presence of female S. erythrophthalmus and B. bjoerkna did not affect milt volumes of C. carassius under two experimental conditions: (1) ovulating S. erythrophthalmus and B. bjoerkna did not increase the milt volumes of C. carassius and (2) S. erythrophthalmus and B. bjoerkna did not interfere with the milt volume increase induced in male C. carassius by ovulating conspecifics. These results suggest that, as in C. auratus, C. carassius and C. carpio (subfamily Cyprininae), female S. erythrophthalmus (subfamily Leuciscinae) release a preovulatory pheromone that exerts priming effects on male hormones and sperm allocation. The findings also indicate that C. carassius discriminate between the reproductive odours of conspecifics and heterospecifics.     

复合四倍体异育银鲫两种不同生殖方式的细胞学观察

在复合四倍体异育银鲫（）×银鲫（）的受精过程中,精子入卵后经过解凝、核化,最终形成雄性原核,并可与卵子的雌性原核融合,证明了复合四倍体异育银鲫卵子具有与两性融合生殖极为相似的拟两性融合生殖的能力;而在复合四倍体异育银鲫（）×兴国红鲤（）的组合中,精子入卵后以固缩状态存在,又表现出典型的雌核发育型生殖行为。因此我们认为复合四倍体异育银鲫具有两种不同的生殖发育机制。此外,我们还观察到在第一次有丝分裂中期有核物质被排斥到纺锤体之外的现象。本文就复合四倍体异育银鲫生殖发育的机制进行了初步的探讨。     

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.     

三种鲫鱼品系同工酶比较研究

采用聚丙烯酰胺凝胶垂直板电泳技术,对彭泽鲫、银鲫D系以及野鲫三种鲫鱼品系的心、肝和肾脏组织的乳酸脱氢酶(LDH)、苹果酸脱氢酶(MDH)、苹果酸酶(ME)、酯酶(EST)和超氧化物歧化酶(SOD)同工酶表型进行了比较研究.结果表明彭泽鲫乳酸脱氢酶同工酶比银鲫D系在肝组织多出二条酶带(LDH7'和LDH8');超氧化物歧化酶在心和肾组织中分别多出一条酶带(SOD12'),表明彭泽鲫和银鲫已在生化水平产生明显的分化,推测它们可能起源于不同的地区,由不同的祖先,独立演化而形成.此外,彭泽鲫和银鲫D系的同工酶电泳图谱都包含野鲫的基本酶带,而彭泽鲫和野鲫的酯酶同工酶电泳图谱尤为相似,推测彭泽鲫和银鲫可能起源于野鲫,而彭泽鲫和野鲫的关系较近,银鲫和野鲫的关系较远.     

Morphological, cytogenetic, and molecular evidence for introgressive hybridization in birch.

Extensive morphological variation of tetraploid birch (Betula pubescens) in Iceland is believed to be due to gene flow from diploid dwarf birch (B. nana) by means of introgressive hybridization. A combined morphological and cytogenetic approach was used to investigate this phenomenon in two geographically separated populations of natural birch woodland in Iceland. The results not only confirmed introgressive hybridization in birch, but also revealed bidirectional gene flow between the two species via triploid interspecific hybrids. The populations showed continuous morphological variation connecting the species, but karyotypically they consisted of only three types of plants: diploids, triploids, and tetraploids. No aneuploids were found. Some of the tetraploid plants had B. pubescens morphology as expected, but most of them had intermediate characters. Most of the diploid plants were B. nana, but some were intermediates and a few had B. pubescens morphology. The triploid plants were either intermediates or they resembled one of the two species. Similar introgressive variation was observed among the diploid and triploid progeny of open-pollinated B. nana in a garden. Birch samples including field plants and artificial hybrids were further examined using a molecular method based on genomic Southern hybridization. The experiments verified introgression at the DNA level.     

鲫鲤杂种F2精子多态性的研究

本文利用扫描电镜和透射电镜对鲫鲤杂种F2精子的超微结构进行了系统的研究。F2精子由头部、中片和尾部组成,头部前端有液泡,没有顶体。研究发现,F2精子头部大小差别明显,最小的精子头径只有1．32μm,而最大的那个“超级精子”头径约18．39μm,但是绝大部分精子头径在1．85-2．15μm。另外,通过透射电镜还发现了双核精子和双尾精子。结果表明,F2精子中存在着明显的多态性,其中有正常的精子,即单倍体精子;也有异常的精子,包括二倍体精子、四倍体精子甚至更高倍性的精子,还有非整倍体精子。本研究结果为二倍体的精子和二倍体的卵子结合产生四倍体的机制提供了有利的证据。     

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.     

Detection of hybridization between two loach species (<Emphasis Type="Italic">Paramisgurnus dabryanus</Emphasis> and <Emphasis Type="Italic">Misgurnus anguillicaudatus</Emphasis>) in wild populations

Artificial interspecific hybrids between large scale loach P. dabryanus and tetraploid pond loach M. anguillicaudatus (Cobitidae, Cypriniformes) are viable. To detect the occurrence of possible natural hybridization, genetic analyses by using microsatellite markers were performed for natural populations of large scale loach and pond loach, the reciprocal laboratory hybrids, and “supposed hybrids” with ambiguous morphology. The fertility of the artificial hybrids was also tested. At one diagnostic microsatellite (Mac50), one out of 20 “supposed hybrids” was identified to be F₁ hybrid between the two loach species because it had the same genotype as that of the laboratory hybrids. The triploid hybrids between the two species were confirmed to be female-sterile. The results show that rare hybridization has occurred between diploid large scale loach and tetraploid pond loach in nature although it may have little effect in genetic introgression. This study is helpful for fish conservation and encourages further investigation on natural hybridization and introgression of loaches.     

Genetic Diversity in Populations of Silver Crucian Carp <Emphasis Type="Italic">Carassius auratus gibelio</Emphasis> (Cyprinidae,Cypriniformes), Depending on the Type of Reproduction and Reservoir Size

This paper presents data on the polymorphism of inter simple sequence repeats of DNA in the silver crucian carp Carassius auratus gibelio in water bodies in the southern part of Western Siberia. The share of polymorphic ISSR-PCR bands in silver crucian carp populations from different lakes varied from 40 to 70%, and Nei’s gene diversity index varied from 0.16 to 0.25. The genetic diversity indices were lower in the populations that were largely represented by females than in bisexual ones. The highest levels of genetic polymorphism were revealed in populations with diploid–triploid complexes. The gene diversity index of the silver crucian carp population is positively correlated with the reservoir size (r = 0.90, p = 0.015; Rs = 0.74, p = 0.036).     

Genetic polymorphism and isoenzyme patterns of lactate dehydrogenase in tench (Tinca tinca), crucian carp (Carassius carassius) and carp (Cyprinus carpio)

Isoenzyme patterns and the polymorphism of lactate dehydrogenase (LDH) were investigated in 3 fish species of family Cyprinidae, i.e. tench (Tinca tinca), crucian carp (Carassius carassius) and carp (Cyprinus carpio). The isoenzyme patterns were tissue and species specific. In crucian carp subunits with different electrophoretic mobility are present, which are genetically controlled from the B1, B2, A1, A2 and C loci, while the set of loci in carp is B1, B2, A, C1 and C2 and in tench B, A, C. The locus B of LDH in tench, the locus B2 in crucian carp, and the loci B1, C1 and C2 in carp are polymorphic and have two different alleles in each case. The polymorphism did not affect the total LDH activity in the tissues. All the populations investigated were in Hardy-Weinberg equilibrium. The genetic control of the polymorphism in B1 and C1 loci in carp was proved by test matings. The polymorphism in B loci tested in erythrocytes may be utilized as genetic markers in the fish breeding.