Cloning and expression analysis of GATA1 gene in Carassius auratus red var.

BACKGROUND: GATA1 is a key transcription factor in the GATA family, and promotes the differentiation and maturation of red blood cell, which is essential for normal hematopoiesis. RESULTS: Our results showed that the cDNA sequence of GATA1 was 2730 bp long encoding 443 amino acids. qRT-PCR analysis demonstrated that GATA1 had the highest expression in testis (T), followed by pituitary (P) and spleen (S). GATA1 gene expression in C. auratus red var. embryo from the neuroblast stage (N) to the embryo hatching (H) changes continuously; and the gene expression levels of nonylphenol (NP)-treated and those of control embryos were significantly different. Moreover, Methylation levels of GATA1 gene in NP-treated embryos were higher than those in control embryos, indicating that NP affected GATA1 methylation. CONCLUSIONS: Our study provides cues for further studying the roles of GATA1 gene in fish development, and suggested a potential molecular mechanism by which NP leads to abnormal development of fish embryos.

Evolutionary dynamics of 18S and 5S rDNA in autotriploid Carassius auratus.

The Carassius auratus (crucian carp) complex of the Dongting water system exhibits coexistence of diploid and triploid forms. As reported, triploid C. auratus is autotriploid origin. Ribosomal DNA (rDNA) with evolutionary conservation is widely used to study polyploidization. Here, we investigated genomic and transcribed rDNA sequences (18S and 5S) in diploid (2nCC, 2n = 100) and triploid (3nCC, 3n = 150) C. auratus. The results showed that the genetic traits and expression of 18S and 5S rDNA from 2nCC individuals were identified in 3nCC individuals. Moreover, pseudogenization of rDNA (18S and 5S) sequences were also observed in both 2nCC and 3nCC individuals, but expression of these variants was not detected. Based on the transcribed rDNA consensus sequence between 2nCC and 3nCC individuals, the functional secondary structures of 18S rRNA (expansion segments, ES6S) and 5S rRNA were predicted. These data demonstrated that complex evolutionary dynamics existed in the rDNA family of C. auratus. The evolutionary conservation of rDNA revealed that autotriploidization could not induce the divergence in Carassius taxa of the Dongting water system. These observations will expand our knowledge of the evolutionary dynamics of the rDNA family in vertebrates.

Evidence for the paternal mitochondrial DNA in the crucian carp-like fish lineage with hybrid origin.

In terms of taxonomic status, common carp (Cyprinus carpio, Cyprininae) and crucian carp (Carassius auratus, Cyprininae) are different species; however, in this study, a newborn homodiploid crucian carp-like fish (2n=100) (2nNCRC) lineage (F1-F3) was established from the interspecific hybridization of female common carp (2n=100)×male blunt snout bream (Megalobrama amblycephala, Cultrinae, 2n=48). The phenotypes and genotypes of 2nNCRC differed from those of its parents but were closely related to those of the existing diploid crucian carp. We further sequenced the whole mitochondrial (mt) genomes of the 2nNCRC lineage from F1 to F3. The paternal mtDNA fragments were stably embedded in the mt-genomes of F1-F3 generations of 2nNCRC to form chimeric DNA fragments. Along with this chimeric process, numerous base sites of F1-F3 generations of 2nNCRC underwent mutations. Most of these mutation sites were consistent with the existing diploid crucian carp. Moreover, the mtDNA organization and nucleotide composition of 2nNCRC were more similar to those of the existing diploid crucian carp than those of the parents. The inheritable chimeric DNA fragments and mutant loci in the mt-genomes of different generations of 2nNCRC provided important evidence of the mtDNA change process in the newborn lineage derived from hybridization of different species. Our findings demonstrated for the first time that the paternal mtDNA were transmitted into the mt-genomes of homodiploid lineage, which provided new insights into the existence of paternal mtDNA in the mtDNA inheritance.

Hox genes reveal genomic DNA variation in tetraploid hybrids derived from Carassius auratus red var. (female) × Megalobrama amblycephala (male).

BACKGROUND: Allotetraploid F1 hybrids (4nF1) (AABB, 4n = 148) were generated from the distant hybridization of Carassius auratus red var. (RCC) (AA, 2n = 100) (♀) × Megalobrama amblycephala (BSB) (BB, 2n = 48) (♂). It has been reported that Hox gene clusters are highly conserved among plants and vertebrates. In this study, we investigated the genomic organization of Hox gene clusters in the allotetraploid F1 hybrids and their parents to investigate the polyploidization process. RESULTS: There were three copies of Hox genes in the 4nF1 hybrids, two copies in RCC and one copy in BSB. In addition, obvious variation and pseudogenization were observed in some Hox genes from 4nF1. CONCLUSION: Our results reveal the influence of polyploidization on the organization and evolution of Hox gene clusters in fish and also clarify some aspects of vertebrate genome evolution.

Anatomical features for the adequate choice of experimental animal models in biomedicine: I. Fishes.

Fish constitute the oldest and most diverse class of vertebrates, and are widely used in basic research due to a number of advantages (e.g., rapid development ex-utero, large-scale genetic screening of human disease). They represent excellent experimental models for addressing studies on development, morphology, physiology and behavior function in other related species, as well as informative analysis of conservation and diversity. Although less complex, fish share many anatomical and physiological features with mammals, including humans, which make them an important complement to research in mammalian models. In this review we describe and compare the most relevant anatomical features of the most used teleostean species in research, to be taken into consideration when selecting an animal model: zebrafish (Danio rerio), medaka (Oryzias latypes), the turquoise killifish (Nothobranchius furzeri), and goldfish (Carassius auratus). Zebrafish and medaka are the mainstream models for genetic manipulability and studies on developmental biology; the turquoise killifish is an excellent model for aging research; goldfish has been largely employed for neuroendocrine studies.

Molecular characterization and expressing analysis of the c-type and g-type lysozymes in Qihe crucian carp Carassius auratus.

Lysozyme as an important nonspecific immune factor, can kill bacteria by hydrolyzing ß-1,4-glycosidic linkages of peptidoglycan layer, and plays an important role in innate immune response against pathogen infection. In the present study, we report molecular cloning, tissue distribution and functional characterization of the c-type and g-type lysozymes in Qihe crucian carp Carassius auratus (designated as Ca-clys and Ca-glys, respectively). The full-length of Ca-clys and Ca-glys cDNA were cloned using RT-PCR and RACE methods. Catalytic and other conserved residues, required for functionality, were identified by multiple sequence alignment and structure predicted. The findings indicating the Ca-clys with signal peptide sequence, while the Ca-glys without, imply that the two isozymes function in different sites of cell. Phylogenetic analysis revealed that Ca-clys and Ca-glys genes evolve at different rates. Moreover, spatial expression analysis showed that Ca-clys transcript was most abundant in kidney and least in gill. However, the expression level of Ca-glys was significantly lower compared with Ca-clys in various tissues, which was the most abundant in spleen and least in brain. After intraperitoneal injection with A. hydrophila and lipopolysaccharide (LPS), the mRNA levels of Ca-clys and Ca-glys were generally up-regulated in liver and gill, but indicated the different expression changes in spleen, kidney and head kidney. With regard to the lysozyme activity, it was showed that the total enzyme activities generally increased in liver, gill, spleen, and head kidney after stimulation. These results confirmed that both Ca-clys and Ca-glys play an important role in non-specific immunity after A. hydrophila invasion. In this study, it was speculated that expressions of Ca-clys and Ca-glys were regulated in different patterns against pathogens.

Allopolyploidization is not so simple: evidence from the origin of the tribe Cyprinini (Teleostei: Cypriniformes).

The identification of allopolyploidization events benefits from molecular dating and divergence assessments of progenitor genomes. Information on gene duplications only, either orthologs or paralogs, provides incomplete information. Analyses of mitochondrial DNA yield insights into matrilineal history, which may differ from patrilineal evolution. Two important food and pet fishes, the common carp (Cyprinus carpio) and goldfish (Carassius sp.), appear to have experienced allotetraploidization sometime from 12 to 20 million years ago (Ma). However, much work is necessary to detail the initial polyploidization event. Herein, we use this group of fishes as a model system to investigate competing scenarios for allopolyploidization. We analyze both the nuclear genes encoding growth hormone (GH), recombination activating protein 1 (RAG1) and HOXA2B gene, and the maternal heredited 12 concatenated mitochondrial protein-coding gene in 19 species of cyprinids and use two species in Balitoridae as outgroup taxa. Our analyses clarify the phylogenetic position of the paternal and maternal ancestors for the common carp and goldfish. The estimation of matrilineal divergence (10.71-12.42 Ma) is significantly younger than the dates of the parental ancestor divergedthat obtained by nuclear genes (16.62-19.64 Ma). Analyses of both genomes date the allopolyploidization event of the common ancestor of Cy. carpio and Ca sp. to about 10.71-12.42 Ma, which is most likely represented by maternal divergent time. The divergence of the two copies of the nuclear genes which was more ancient than the maternal markers might have been included the divergence of the progenitors' genome divergence when the allopolyploidization event occurred. Thus, the scenarios of allopolyploidzation for this group of fish can be suggested as the following: the matrilineal common ancestor of species in tribe Cyprinini might have doubled its genome by mating with a paternal ancestor in the subfamily Cyprininae, which was a sister-group that diverged around 4.20-8.93 Ma. Our work provides new evidence for the divergence dates of allopolyploidization within the Cyprinini, and documents the necessity of considering both matrilineal and patrilineal histories when investigating allopolyploidization.

Inheritance of the complete mitochondrial genomes in three different ploidy fishes.

Diploid natural gynogenetic goldfish (2nGRCG), triploid hybrids (3nRB) and tetraploid hybrids (4nRB) are generated by distant hybridization of red common goldfish (RCG, Carassius auratus red var.) and blunt snout bream (BSB, Megalobrama amblycephala). In the present study, we obtained the complete mitochondrial DNA (mtDNA) sequences of the hybrid offspring and compared them with the homologous sequences of RCG and BSB. All mtDNA sequences of these hybrids were 16,580bp in length, and the genes number, size, and order were quite similar to that of RCG. Genetic analysis revealed that the mtDNA sequences of these hybrids had high similarity (>99%) and low divergence (<2%) to their maternal RCG, yet lower similarities (84%) and higher divergences (16%) to their paternal BSB. The phylogenetic analysis also showed that the sequences of 2nGRCG, 3nRB and 4nRB were clustered with RCG rather than with BSB. These results indicate that the mitochondrial genomes of 2nGRCG, 3nRB and 4nRB remain maternally inherited after hybridization and polyploidization. Moreover, clade separation of hybrid offspring from their paternal BSB in the phylogenetic tree implies that phylogenetic analysis of mtDNA is incomplete for elucidating the true relationships between different species, particularly when they have undergone hybridization or allopolyploidization. Our study provides significant information for both evolution and genetic studies of mtDNA for hybrid species and allopolyploidization species.

Partial cloning, tissue distribution and effects of epigallocatechin gallate on hepatic 3-hydroxy-3-methylglutaryl-CoA reductase mRNA transcripts in goldfish (Carassius auratus).

Epigallocatechin gallate (EGCG), the major active component of the green tea, has recently been found to inhibit 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCoAR) activity in vitro and to modulate lipogenesis in vivo. In this study we have evaluated the effects of short-term in vivo exposure to EGCG (6 µg g(-1) BW or 9 µg g(-1) BW) on hepatic HMGCoAR gene expression of goldfish (Carassius auratus). We initially characterized a partial sequence of goldfish HMGCoAR suggesting that the obtained fragment shares high similarity (>92%) with other fish HMGCoAR sequences. Further, the HMGCoAR transcript was detected in all goldfish tissues (except muscle) but primarily in liver, brain and gonads; on the contrary, low expression levels were found in intestine, heart, gill, and kidney. Both EGCG doses significantly decreased hepatic HMGCoAR mRNA levels 180 min post-injection. HMGCoAR was also significantly down-regulated at 90 min after injection in fish treated with the highest dose of EGCG. Our results demonstrate that hepatic HMGCoAR gene expression is acutely responsive to short-term EGCG exposure in goldfish. This finding suggests a potential role of EGCG in transcriptional regulation of the rate-limiting enzyme in cholesterol synthesis.

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.

[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 (4.0-4.5 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 = 0.022-0.036. 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.

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 (ß(2)m) (caauß2m-1a, caauß2m-1b, caauß2m-2, caauß2m-3a and caauß2m-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 ß(2)m-1 and ß(2)m-2 isoforms are clustered with those of other cyprinid fishes, while ß(2)m-3 isoforms make a cluster that is separated from a common ancestor of salmonid and cyprinid fishes. This finding suggests that the ß(2)m isoforms of ginbuna cruician carp comprise two lineages and may possess different functions. The MHC class I and ß(2)m sequences from one clonal strain will facilitate our understanding of the interaction of MHC class I with ß(2)m in teleosts.

Recent invasion and low level of divergence between diploid and triploid forms of Carassius auratus complex in Croatia.

Carassius auratus is an invasive species in European waters, comprising a complex of diploid and polyploid forms with different modes of reproduction. However, the evolutionary history and relationships between the diploids and polyploids are still unresolved. In this study, 51.5% diploids and 48.5% triploids, including four triploid males, were discovered among the 363 individuals sampled in Croatia. We used eight microsatellite loci and mitochondrial displacement loop sequences to analyze the structure and origin of populations; and to attempt to infer the evolutionary history of the two different forms in Croatia. Microsatellite analyses revealed high allelic and clonal diversity, corroborating that high propagule vectors can compensate for the negative effects of genetic bottlenecks in successful invasive species. The absence of significant population structuring confirmed recent origin and rapid spreading of populations. No evidence was found for the existence of native European populations. Distances between individuals using both nuclear and mtDNA markers revealed the absence of substantial clustering on the ploidy level, while the split between the different ploidies on population level was only partial, suggesting that the reproductive isolation between the two forms is either of a very recent origin, or that there exists uni-, or bidirectional gene flow between the diploid and triploid forms.

Clonal diversity and genealogical relationships of gibel carp in four hatcheries.

To conserve and utilize the genetic pool of gynogenetic gibel carp (Carassius auratus gibelio), the Fangzheng and Qihe stock hatcheries have been established in China. However, little information is available on the amount of genetic variation within and between these populations. In this study, clonal diversity in 101 fish from these two stock hatcheries and 35 fish from two other hatcheries in Wuhan and Pengze respectively was analysed for variation in serum transferrin. Thirteen clones were found in Fangzheng and Qihe, of which 12 were novel. Six clones were specific to Fangzheng and three specific to Qihe, whereas four were shared among the Fangzheng and Qihe fish. To obtain more knowledge on genetic diversity and genealogical relationships within gibel carp, the complete mitochondrial DNA (mtDNA) control region (approximately 920 bp) was sequenced in 64 individuals representing all 14 clones identified in the four hatcheries. Differences in the mtDNA sequences varied remarkably among hatcheries, with the Fangzheng and Qihe lines demonstrating high diversity and Wuhan and Pengze showing no variation. The Fangzheng and Qihe lines might represent two distinct matrilineal sources. One of the Qihe samples carried the haplotype shared by a most widely cultivated Fangzheng clone, indicating that a Fangzheng clone escaped from cultivated ponds and moved into the Qihe hatchery. Four Fangzheng samples clustered within the lineage formed mainly by Qihe samples, most likely reflecting historical gene flow from Qihe to Fangzheng. It is suggested that clones in Wuhan originated from Fangzheng, consistent with their introduction history, supporting the hypothesis that gibel carp in Pengze were domesticated from individuals in the Fangzheng hatchery.

Molecular cloning and characterization of two types of CD8alpha from ginbuna crucian carp, Carassius auratus langsdorfii.

We cloned and sequenced full-length cDNAs for two types of CD8alpha from the S3n strain of ginbuna crucian carp (Carassius auratus langsdorfii) and quantified the expression of CD8alpha genes after sensitization by scale grafting, employing a model system of clonal triploid ginbuna and tetraploid ginbuna-goldfish hybrids. RT-PCR yielded four different fragments of CD8alpha homologue from the S3n strain of ginbuna and these sequences were classified into two groups. The two types of ginbuna CD8alpha (gbCD8alpha) were also found in other strains of triploid ginbuna and goldfish, which are a subspecies of ginbuna. The gbCD8alpha chains consisted of a signal peptide, Ig superfamily (IgSf) V-like domain, hinge, transmembrane domain, and cytoplasmic domain similar to other known CD8alpha. Phylogenetic analysis indicated that both types of gbCD8alpha are closely related to CD8alpha from other vertebrates. Expression of both types of gbCD8alpha mRNA was detected in the gill, thymus, head kidney, posterior kidney, spleen, intestine and peripheral blood leucocytes. In addition, quantitative real-time PCR analysis demonstrated that copy numbers of both gbCD8alpha gene products in kidney cells increased significantly following grafting with allogeneic but not isogeneic scales, and that regulation of expression correlated with that of TCRbeta. Expression of both gbCD8alpha genes after second scale allografting was elevated compared to that after the first set of grafting. These results suggest that expression analysis of these two gbCD8alpha sequences provides a useful tool to address the involvement of cytotoxic T-lymphocytes during the cell-meditated immune response in fish.

Spectral differentiation of blue opsins between phylogenetically close but ecologically distant goldfish and zebrafish.

Zebrafish and goldfish are both diurnal freshwater fish species belonging to the same family, Cyprinidae, but their visual ecological surroundings considerably differ. Zebrafish are surface swimmers in conditions of broad and shortwave-dominated background spectra and goldfish are generalized swimmers whose light environment extends to a depth of elevated short wavelength absorbance with turbidity. The peak absorption spectrum (lambdamax) of the zebrafish blue (SWS2) visual pigment is consistently shifted to short wavelength (416 nm) compared with that of the goldfish SWS2 (443 nm). Among the amino acid differences between the two pigments, only one (alanine in zebrafish and serine in goldfish at residue 94) was previously known to cause a difference in absorption spectrum (14-nm lambdamax shift in newt SWS2). In this study, we reconstructed the ancestral SWS2 pigment of the two species by applying likelihood-based Bayesian statistics and performing site-directed mutagenesis. The reconstituted ancestral photopigment had a lambdamax of 430 nm, indicating that zebrafish and goldfish achieved short wavelength (-14 nm) and long wavelength (+13 nm) spectral shifts, respectively, from the ancestor. Unexpectedly, the S94A mutation resulted in only a -3-nm spectral shift when introduced into the goldfish SWS2 pigment. Nearly half of the long wavelength shift toward the goldfish pigment was achieved instead by T116L (6 nm). The S295C mutation toward zebrafish SWS2 contributed to creating a ridge of absorbance around 400 nm and broadening its spectral sensitivity in the short wavelength direction. These results indicate that the evolutionary engineering approach is very effective in deciphering the process of functional divergence of visual pigments.

Characterization of repetitive DNA sequences carrying 5S rDNA of the triploid ginbuna (Japanese silver crucian carp, Carassius auratus langsdorfi).

Repetitive DNA sequences (Hi-b; 209 bp in length) were isolated from the HindIII digests of the genomic DNA of the triploid ginbuna, Carassius auratus langsdorfi. Sequence analyses revealed that the Hi-b repetitive units were comprised of the complete coding regions of 5S rDNA (120 bp in size) and their 5'flanking regions. The sequences of the Hi-b units from the same individual were highly homogeneous. Southern blot hybridization to the Hi-b probe displayed intricate patterns that represented the presence of other repetitive units containing the Hi-b related sequences. A major family of repetitive sequences related to the Hi-b was then obtained by the polymerase chain reaction using asymmetry primers for the 5S coding regions. These 331-bp sequences (AZ5S's) contained 5S pseudogenes as well as the almost entire Hi-b sequences, and seemed to be the true 5S rDNAs. The tandem arrangements of the AZ5S sequences explained most of the complex results of Southern blots. Another class of intriguing repeat units (Hi-b-beta and Hi-b-gamma) were also isolated. Fluorescence in situ hybridization data revealed two major signals on a pair of homologous chromosomes and several minor signals on other chromosomes in the triploid ginbuna, indicating the existence of the 5S related sequences as several separate clusters. The major spots were shared with the tetraploid ginbuna and goldfish, but not with the diploid ginbuna. When the genomic organization of the Hi-b related sequences in other cyprinid fishes was examined, the hybridization patterns of the ginbuna were very similar to those of the goldfish, but were clearly different from those of the gengorobuna. The carp genome showed less complex patterns. Thus, the present 5S rDNA-related sequences could be candidates for phylogenetic molecular markers for the crucian carp.