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.     

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.     

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.     

Phylogenetic relationships of silver crucian carp in Carassius auratus complex based on mtDNA analysis

The phylogenetic relationships of Carassius genus subspecies were investigated based on the data of the variability of nucleotide sequences of the mtDNA cytochrome b (cyt b) and control region (CR). Dendrograms constructed based on the BA, ML, NJ, and MP methods revealed five clusters of the congruent topologies that substantially corresponded to geographical localities and taxonomic conception of the C. auratus complex. An analysis of two mtDNA fragment topologies demonstrated that the island forms of Japanese crucian carps C. cuvieri and C. auratus langsdorfii diverged later compared to the divergence of continental C. auratus forms (4.0?C4.5 mln years ago, by molecular calibration). Among the continental silver crucian carps, C. a. gibelio forms two clusters corresponding to two phylogroups with a mean uncorrected genetic distance p = 0.044. The genealogical combination of haplotypes with the first C. a. gibelio phylogroup was observed in C. auratus clade. According to the data of mtDNA analysis, these subspecies represent sister lineages with a level of intergroup divergence of p = 0.022?C0.036. No genetic differences were observed between diploid (except for the two C. a. gibelio phylogroups) and polyploid C a. auratus, as well as monophyly in polyploid forms. New approaches based on a comparative study of the nuclear markers might help to unravel the origin of gynogenetic forms and phylogenetic relationships within the C. auratus complex.     

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.     

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.     

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.     

Direct antibacterial activity of CD8+/CD4+ T-cells in ginbuna crucian carp,Carassius auratus langsdorfii

Cytotoxic T cells (CTLs) constitute an important component of the specific effector mechanism in killing against microbial-infected or transformed cells. In addition to these activities, recent studies in mammals have suggested that CTLs can exhibit direct antimicrobial activity. Therefore, the present investigation was conducted to find out the microbicidal activity of CD8α⁺ T cells of ginbuna crucian carp, Carassius auratus langsdorfii. The CD8α⁺ T cells from immunised ginbuna exhibited the antibacterial activity against both facultative intracellular bacteria and extracellular bacteria. The maximum reduction of viable count of pathogens was recorded with effector (sensitized) cells and target (bacteria) ratio of 10:1 co-incubated for a period of 1–2 h at 26 °C when effector cells were derived from ginbuna 7 days after one booster dose at 15th day of primary sensitization/immunisation. Sensitized CD8α⁺ T cells are found to kill 92.1 and 98.9% of Lactococcus garvieae and Edwardsiella tarda, respectively. No significant difference in the bacterial killing activity could be recorded against facultative intracellular bacteria and extracellular bacteria. The specificity study indicated the non-specific killing of bacteria. CD8α⁺ T cells from E. tarda immunised ginbuna exhibited 40% of non-specific killing activity against L. garvieae and those from L. garvieae immunised ginbuna showed 42.7% of non-specific killing activity against E. tarda. Furthermore, CD4⁺ T cells also killed 88% and 95.7% of L. garvieae and E. tarda, respectively. In addition to T cell subsets, surface IgM⁺ cells also killed both types of pathogens. Therefore, the present study demonstrated the direct antibacterial activity of CD8α⁺, CD4⁺ T-cells and surface IgM⁺ cells in fish.     

Inhibition of CYP450 1A and 3A by berberine in crucian carp Carassius auratus gibelio

Berberine has long been considered as an antibiotic candidate in aquaculture. However, studies regarding its effects on drug-metabolizing enzymes in fish are still limited. In the present study, the effects of berberine on cytochrome P4501A (CYP1A) and CYP3A in crucian carp were investigated. Injection of different concentrations of berberine (0, 5, 25, 50, and 100 mg/kg) inhibited the CYP1A mRNA expression, thereby inhibiting further the catalytic activity of CYP1A-related ethoxyresorufin-O-deethylase (EROD). Furthermore, both CYP1A expression and EROD activity were further inhibited with increasing berberine concentrations. In addition, the CYP3A expressions at both the mRNA and the protein levels were downregulated by higher berberine concentrations. The catalytic activity of CYP3A-related erythromycin N-demethylase (ERND) was also inhibited by berberine at a dose of no less than 25 mg/kg. Moreover, at the berberine concentration exceeding 25 mg/kg, the inhibition of CYP3A expression and ERND activity increased with increasing berberine concentrations. In vitro experiments were also performed. When berberine was pre-incubated with the crucian carp liver microsomes, it competitively inhibited the corresponding EROD activity with the IC₅₀ of 11.7 μM. However, the ERND activity was slightly inhibited by berberine with the IC₅₀ of 206.4 μM. These results suggest that, in crucian carp, berberine may be a potent inhibitor to CYP1A, whereas the CYP3A inhibition needs a higher concentration of berberine.     

Detection of Myxobolus rotundus (Myxozoa: Myxosporea) in skin mucus of crucian carp Carassius auratus auratus using a monoclonal antibody

Diagnosis of myxosporean Myxobolus rotundus infection was conducted by examining skin mucus from the infected crucian carp Carassius auratus auratus with a monoclonal antibody, MAb 2D12, raised previously against the parasite. A positive reaction was observed in skin mucus collected from infected fish, and spores and pre-spore stages of the parasite were identified by the MAb 2D12. It was also demonstrated that M. rotundus infection can be successfully detected by a simple method, enzyme-linked immunosorbent assay (ELISA), and that skin mucus collected from infected fish skin had a significantly higher optical density (OD) value than that from uninfected fish.     

Identification of a novel C1q family member in color crucian carp (Carassius auratus) ovary

Potential roles of C1q/tumor necrosis factor (TNF) superfamily proteins have been observed in vertebrate oogenesis and oocyte maturation, but no ovary-specific member has been identified so far. In this study, we have cloned and identified a novel member of C1q family with a C1q domain in the C-terminal from fully grown oocyte cDNA library of color crucian carp and demonstrated that the gene might be specifically expressed in ovary and therefore designated as Carassius auratus ovary-specific C1q-like factor, CaOC1q-like factor. It encodes a 213 amino acid protein with a 17 amino acid signal peptide. There is only one protein band of about 24.5 kDa in the extracts from phase I to phase IV oocytes, but two positive protein bands are detected in the extracts of mature eggs and fertilized eggs. Furthermore, the mobility shift of the smaller target protein band cannot be eliminated by phosphatase treatment, but the larger protein band increases its mobility on the gel after phosphatase treatment, suggesting that the larger protein might be a phosphorylated form. Immunofluorescence localization indicates that the CaOC1q-like proteins localize in cytoplasm, cytoplasm membrane and egg envelope of the oocytes at cortical granule stage and vitellogenesis stage, whereas they were compressed to cytoplasm margin in ovulated mature eggs and discharged into perivitelline space between cytoplasm membrane and egg envelope after egg fertilization. Further studies on distribution and translocation mechanism of the CaOC1q-like factor will be benefit to elucidate the unique function in oogenesis, oocyte maturation and egg fertilization.     

New cell types of the pancreatic islets in the crucian carp,Carassius carassius

Summary The pancreatic islets ofCarassius carassius have been studied by electron microscopy. 1. Besides A-, B- and D-cells, two new cell types, the fourth and the fifth, have been identified. The fourth cell type is numerous; it occurs interposed among the other types of islet cells or in small clusters. The secretory granules (90–280 mg in diameter) are round or oval and usually with much lower electron density than α- and δ-granules. The secretory granules of the fifth type of cell (approximately 140–240 mμ in diameter) contain finely granular material and an electron dense core that is round or often tetra- or hexagonal. 2. The islet cells with clear cytoplasmic matrix generally contain large numbers of fine, agranular and cored vesicles 400–680 ? in diameter. They appear, in bead-like chains, or randomly scattered throughout the cytoplasm, or often clustered in aggregates close to the secretory granules and show evidence of incorporation into the secretory granules. The two types of vesicles may be formed by constriction or pinching-off of the tubular smooth endoplasmic reticulum.     

Karyotypic diversity in polyploid gibel carp,Carassius auratus gibelio Bloch

Polyploid gibel carp, Carassius auratus gibelio, is an excellent model system for evolutionary genetics owing to its specific genetic background and reproductive modes. Comparative karyotype studies were performed in three cultured clones, one artificially manipulated group, and one mated group between two clones. Both the clones A and P had 156 chromosomes in their karyotypes, with 36 metacentric, 54 submetacentric, 36 subtelocentric, 24 acrocentric, and six small chromosomes. The karyotype of clone D contained 162 chromosomes, with 42 metacentric, 54 submetacentric, 36 subtelocentric, 24 acrocentric, and six small chromosomes. All the three clones had six small chromosomes in common. Group G, being originated from the clone D by artificial manipulation, showed supernumerary microchromosomes or chromosomal fragments, in addition to the normal chromosome complement that was identical to the clone D. The offspring from mating between clones D and A had 159 chromosomes. Comparing with the clone A, the DA offspring showed three extra metacentric chromosomes. In addition, variable RAPD fingerprint patterns and unusual SCAR marker inheritance were, respectively, detected among individuals of artificial group G and in the mated DA offspring. Both the chromosome and molecular findings suggest that genome reshuffling might have occurred by manipulation or mating of the clones.     

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].     

Secretory expression and characterization of the recombinant myofibril-bound serine proteinase of crucian carp (Carassius auratus) in Pichia pastoris

The myofibril-bound serine proteinase (MBSP) is effective in the degradation of myofibrillar proteins, including myosin heavy chain (MHC), α-actinin, actin, and tropomyosin and was thus regarded as an important proteinase responsible for the metabolism of fish muscle in vivo. In order to better understand the characteristic differences between native MBSP and recombinant MBSP (rMBSP) and to obtain large quantity of MBSP for its application in protein science study, the crucian carp MBSP gene was cloned (669 bp) and expressed in Pichia pastoris (P. pastoris). The recombinant P. pastoris strain was cultured in shake flasks, and 66.85 mg rMBSP/L in the fermentation supernatant was obtained. SDS-polyacrylamide gel electrophoresis (PAGE) showed a main protein band with molecular weight of approximately 36 kDa. Substrate specificity analysis revealed that the rMBSP specifically cleaved substrates at the carboxyl side of lysine residue which differed from native MBSP that cleaved substrates at the carboxyl side of arginine and lysine residues. The optimum temperature and optimum pH range of the rMBSP were 55 °C and pH 7.5, respectively. Furthermore, similar to native MBSP, the rMBSP also revealed high thermostability and pH stability and is effective in degradation of myofibrillar proteins from the skeletal muscle of crucian carp.     

Molecular characterization of MHC class I and beta-2 microglobulin in a clonal strain of ginbuna crucian carp, Carassius auratus langsdorfii

Clonal ginbuna crucian carp is, a naturally gynogenetic fish, and is a useful model animal for studying T-cell-mediated immunity. To gain molecular information on MHC class I molecules from this species, we have identified four types of MHC class I (caauUA-S3n, caauUF-S3n, caauZE-S3n, and caauZB-S3n) and five beta 2-microglobulin (β(2)m) (caauβ2m-1a, caauβ2m-1b, caauβ2m-2, caauβ2m-3a and caauβ2m-3b) by an expressed sequence tag (EST) analysis and using homology cloning with degenerated primers. Like UA class I genes in other cyprinid fish, the caauUA-S3n shows features of classical MHC class I, such as conservation of all key amino acids interacting with antigenic peptides, and ubiquitous tissue expression. A phylogenetic analysis shows that the β(2)m-1 and β(2)m-2 isoforms are clustered with those of other cyprinid fishes, while β(2)m-3 isoforms make a cluster that is separated from a common ancestor of salmonid and cyprinid fishes. This finding suggests that the β(2)m isoforms of ginbuna cruician carp comprise two lineages and may possess different functions. The MHC class I and β(2)m sequences from one clonal strain will facilitate our understanding of the interaction of MHC class I with β(2)m in teleosts.     

Seasonal feeding activity and ontogenetic dietary shifts in crucian carp,Carassius carassius

Synopsis A pronounced seasonal cycle in the activity of the intestinal alkaline phosphatase indicated a 6 month pause in feeding by crucian carp in an anoxic pond during winter. Zero enzyme activity was not reached until ice cover of the pond was complete, although even in the preceding two months no food was found in the guts of the fish. The summer diets of four size classes of fish consisted of varying proportions of planktonic and benthic arthropods, including Odonata which were consumed exclusively by the largest size class of crucian carp. Chironomid larvae were consumed even by the smallest fish investigated, although planktonic Cladocera were preferred by this size class. Benthic Cladocera were almost totally absent in the diet of fish> 10 cm. Habitat segregation between size classes developed synchronously with the shift to benthic food. The stunting of crucian carp populations in small ponds is interpreted as resulting from limited resources.     

Clonal diversity in the Japanese silver crucian carp,Carassius langsdorfii inferred from genetic markers

The Japanese silver crucian carp, Carassius langsdorfii, is a naturally polyploid fish that reproduces by gynogenesis, resulting in a geographically clustered clonal population. To determine the amount of clonal diversity in this species, we used genetic markers to compare fish from several Japanese locations. Samples were collected from Lake Kasumigaura (Ibaraki Pref.), Lake Imba (Chiba Pref.), Lake Fukushimagata (Niigata Pref.), Lake Biwa (Shiga Pref.), Lake Koyama (Tottori Pref.), and the Niyodo and Monobe Rivers (Kochi Pref.). The genetic markers used were the microsatellite loci GF1(*), GF17(*), and GF29(*), as well as isozyme (creatinekinase CK(*)) and mtDNA (D-loop region) RFLP. In the seven locations sampled, 61 clonal lines were identified by combining the genotypes of the three microsatellite loci. Populations at all locations consisted of six to 28 clonal lines. Fourteen common clonal lines were detected in two or more locations, and 13 of these were observed in Lake Biwa. We suggest that these clonal lines have been widely distributed in Japanese freshwaters through the intentional stocking of fish species such as "gengorobuna" (C. cuvieri) and "ayu" (Plecoglossus altivelis altivelis) obtained from Lake Biwa and its adjacent waters. The CK(*) genotypes and mtDNA haplotypes of fish from Lake Kasumigaura differed from those of other locations.     

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...