cDNA cloning and characterization of a novel SNX gene differentially expressed in previtellogenic oocytes of gibel carp

To understand the molecular events governing fish oogenesis, a multiple technique was used to identify the genes differentially expressed at different phases during fish oogenesis. This technique is a combination of suppression subtractive hybridization, SMART cDNA synthesis and RACE-PCR. Here we report the cDNA cloning and expression characterization of a novel SNX gene based on its differential transcription between previtellogenic and fully mature oocytes in naturally gynogenetic gibel carp. First, a cDNA fragment selectively expressed in previtellogenic oocytes was identified and used to screen a SMART cDNA library prepared from the same mRNA sample by RACE-PCR for cloning fully length cDNA. The full length cDNA was 1392-bp long and coded for a novel SNX protein with 225 amino acids. The 5' UTR had 72 bp and 3' UTR had 642 bp. Unlike most of maternal genes that are transcribed after vitellogenesis and stored in oocytes, this gene is expressed at a higher level in the previtellogenic oocytes and at a much lower level in fully matured oocytes. However, RT-PCR analysis of tissues showed it was ubiquitous transcription. The novel gene is named fish sorting nexin (fSNX), because it contains a conserved PX domain. The fact which major expression of the gene occurs in the previtellogenic oocytes suggests that it might have an important function in the oogenesis. (C) 2003 Elsevier Inc. All rights reserved.

Genetic heterogeneity and ploidy level analysis among different gynogenetic clones of the polyploid gibel carp

Background: Some triploid and tetraploid clones have been identified in the gynogenetic gibel carp, Carassius auratus gibelio Bloch, by karyotypic and cytologic analyses over many years. Further, 5-20% males and karyotypic diversity have been found among their natural and artificial populations. However, the DNA contents and the relation to their ploidy level and chromosome numbers have not been ascertained, and whether normal meiosis occurs in spermatogenesis needs to be determined in the different clones. Methods: The sampled blood cells or sperms were mixed with blood cells from chicken or individual gibel carp and fixed in 70% pre-cooled ethanol overnight at 4degreesC. The mixed cell pellets were washed 2-3 times in 1x phosphate buffered saline and then resuspended in the solution containing 0.5% pepsin and 0.1 M HCl. DNA was stained with propidium iodide solution (40 mug/mL) containing 4 kU/ml RNase. The measurements of DNA contents were performed with Phoenix Flow Systems. Results: Triploid clones A, E, F, and P had almost equal DNA content, but triploid clone D had greater DNA content than did the other four triploid clones. DNA content of clone M (7.01 +/- 0.15 pg/nucleus) was almost equal to the DNA content of clone D (5-38 +/- 0.06 pg/nucleus) plus the DNA content of common carp sperm (1.64 +/- 0.02 pg/nucleus). The DNA contents of sperms from clones A, P, and D were half of their blood cells, suggesting that normal meiosis occurs in spermatogenesis. Conclusions: Flow cytometry is a powerful method to analyze genetic heterogeneity and ploidy level among different gynogenetic clones of polyploid gibel carp. Through this study, four questions have been answered. (a) The DNA content correlation among the five triploid clones and one multiple tetraploid clone was revealed in the gibel carp, and the contents increased with not only the ploidy level but also the chromosome number. (b) Mean DNA content was 0.052 pg in six extra chromosomes of clone D, which was higher than that of each chromosome in clones A, E, F, and P (about 0.032 pg/ chromosome). This means that the six extra chromosomes are larger chromosomes. (c) Normal meiosis occurred during spermatogenesis of the gibel carp, because DNA contents of the sperms from clones A, P, and D were almost half of that in their blood cells. (d) Multiple tetraploid clone M (7.01 +/- 0.15 pg/nucleus) contained the complete genome of clone D (5.38 +/- 0.06 pg/nucleus) and the genome of common carp sperm (1.64 +/- 0.02 pg/nucleus). Cytometry Part A 56A:46-52, 2003. (C) 2003 Wiley-Liss, Inc.

Molecular cytogenetic detection of paternal chromosome fragments in allogynogenetic gibel carp, Carassius auratus gibelio Bloch

In gynogenesis, sperm from related species activates egg and embryonic development, but normally does not contribute genetically to the offspring. In gibel carp, Carassius auratus gibelio Bloch, however, gynogenetic offspring often show some phenotypes apparently derived from the heterologous sperm donor. This paternal effect of allogynogenesis is outstanding in an artificial clone F produced by cold treatment of clone E eggs after insemination with blunt-nose black bream (Megaloabrama amblycephala Yin) sperm. Karyotype analysis revealed 5-15 supernumerary microchromosomes in different individuals of clone F in addition to 156 normal chromosomes inherited from the maternal clone E. A painting probe was prepared from the microdissected microchromosomes, and used to investigate the origin of these microchromosomes. Strong positive signals were detected on each microchromosomes of clone F and on 4 pairs of chromosomes in blunt-nose black bream, whereas no signals were detected on the chromosomes of clone E. This result indicates that some paternal chromosome fragments of blunt-nose black bream have been incorporated into the artificial clone F. Therefore, the manipulation of allogynogenesis may provide a unique method to transfer DNA between diverse species for fish breeding.

Comparative studies on in vitro sperm decondensation and pronucleus formation in egg extracts between gynogenetic and bisexual fish

A cell-free system based upon the egg extracts from gynogenetic gibel carp (Carassius auratus gibelio) or bisexual red common carp (Cyprinus carpio red variety) was developed to investigate developmental behaviors of the demembranated sperm nuclei. Both red common carp and gibel carp sperm nuclei could decondense fully and form pronuclei in the red common carp egg extracts. Gibel carp sperm nuclei could also decondense fully and form pronuclei in the gibel carp egg extracts, but red common carp sperm nuclei could not decondense sufficiently in the same extracts. The significant differences of morphological changes were further confirmed by ultrastructural. observation of transmission electron microscopy. The data further offer cytological evidence for gonochoristic reproduction in the gynogenetically reproducing gibel carp. In addition, the sperm nuclei in vitro decondensation is dependent on the pH in the extracts, and the decondensed efficiency is optimal at pH 7. However, no DNA replication was observed in the two kinds of egg extracts during the incubation period of the sperm nuclei. It is suggested that the egg extracts prepared from the gynogenetic gibel carp should be a valid in vitro system for studying molecular mechanism on gynogenesis and reproduction mode diversity in fish.

Cyclin A2 is differentially expressed during oocyte maturation between gynogenetic silver crucian carp and gonochoristic color crucian carp

Silver crucian carp (Carassius auratus gibelio) is a unique gynogenetic fish. Because of its specific genetic background and reproduction mode, it is an intriguing model system for understanding regulatory mechanism of oocyte maturation division. It keeps its chromosomal integrity by inhibiting the first meiotic division (no extrusion of the first pole body). The spindle behavior during oocyte maturation is significantly different from that in gonochoristic fish. The chromosomes are first arranged in a tripolar spindle, and then they turn around and are reunited mutually to form a normal bipolar spindle. A new member of the fish A-type cyclin gene, cyclin A2, has been isolated by suppression of subtractive hybridization on the basis of its differential transcription in fully-grown oocytes between the gynogenetic silver crucian carp and gonochoristic color crucian carp. There are 18 differing amino acids in the total 428 residues of cyclin A2 between the two forms of crucian carps. In addition, cDNAs of cyclin A1 and cyclin B have also been cloned from them. Thus two members of A-type cyclins, cyclin A1 and cyclin A2, are demonstrated to exist in fish, just as in frog, humans, and mouse. Northern blotting reveals that cyclin A2 mRNA is more than 20-fold and cyclin A1 mRNA is about 2-fold in fully grown oocytes of gynogenetic silver crucian carp compared to gonochoristic color crucian carp. However, cyclin B does not show such a difference between them. Western blot analysis also shows that the cyclin A2 protein stockpiled in fully grown oocytes of gynogenetic crucian carp is much more abundant than in gonochoristic crucian carp. Moreover, two different cyclin A2 expression patterns during oocyte maturation have been revealed in the two closely related crucian carps. For color crucian carp, cyclin A2 protein is translated only after hormone stimulation. For silver crucian carp, cyclin A2 protein can be detected throughout the process of maturation division. The different expression of cyclin A2 may be a clue to understanding the special maturation division of gynogenetic silver crucian carp.

Identification of antiviral-relevant genes in the cultured fish cells induced by UV-inactivated virus

UV-inactivated grass carp hemorrhage virus (GCHV) can induce high titer of interferon in cultured CAB (crucian carp (Carassius auratus L.) blastulae) cells, and thus defend host cells against the virus invasion. The mechanism is proposed that an antiviral state should be established in the host cells by activating expression of a set of antiviral-relevant genes. In this study, suppressive subtractive hybridization is applied to constructing a subtracted cDNA library with mRNAs isolated from UV-inactivated GCHV infected and mock-infected CAB cells. 272 differential cDNA fragments are identified by both PCR and dot blot from the subtractive cDNA library. Sequencing analysis reveals 69 genes, including 46 known gene homologues, and 23 unknown putative genes. The known genes include the genes involved in interferon signaling pathways, such as Stat1 and Jak1, the antiviral genes, such as Mx and Viperin, and a set of interferon-stimulated genes observed in mammalian cells. Most of the unknown putative genes contain AU-rich element in their sequences. Differential expressions of these genes are further confirmed by virtual Northern blot and RT-PCR. The data imply that UV-inactivated GCHV is not only able to induce production of interferon in the infected CAB cells, but also leads to the expression of a series of antiviral-relevant genes or immune-relevant genes, and therefore reveals that the signaling pathway of interferon system and antiviral mechanism in fish are similar to those in mammals.

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.

Screen for stage-specific expression genes between tail bud stage and heartbeat beginning stage in embryogenesis of gynogenetic silver crucian carp

A systemic study was initiated to identify stage-specific expression genes in fish embryogenesis by using suppression subtractive hybridization (SSH) technique. In this study, we presented a preliminary result on screen for stage-specific expression genes between tail bud stage (TBS) and heartbeat beginning stage (HBS) in gynogenetic silver crucian carp (Carassius auratus gibelio). Two SSH plasmid libraries specific for TBS embryos and HBS embryos were constructed, and stage-specific expression genes were screened between the two stages. 1963 TBS positive clones and 2466 HBS positive clones were sampled to PCR amplification, and 1373 TBS and 1809 HBS PCR positive clones were selected to carry out dot blots. 169 TBS dot blot positive clones and 272 HBS dot blot positive clones were sequenced. Searching GenBank by using these nucleotide sequences indicated that most of the TBS dot blot positive clones could not be found homologous sequences in the database, while known genes were mainly detected from HBS dot blot positive clones. Of the 79 known genes, 20 were enzymes or kinases involved in important metabolism of embryonic development. Moreover, specific expressions of partial genes were further confirmed by virtual northern blots. This study is the first step for making a large attempt to study temporal and spatial control of gene expression in the gynogenetic fish embryogenesis.

Mortalities induced by the copepod Sinergasilus polycolpus in farmed silver and bighead carp in a reservoir

The frequency distributions of the parasitic copepod Sinergasilus polycolpus were examined in silver carp Hypophthalmichthys molitrix and bighead carp Aristichthys nobilis during a disease outbreak of the 2 species of fish in a reservoir in China. The mean abundance of the copepod was positively related with host length and age, and the overdispersion of the copepod in both silver and bighead carp was fitted well with negative binomial distribution. Although parasite-induced host mortality was observed, a peaked age-parasite abundance curve was not detected in the present parasite-host system. It is also proposed that this peaked age-abundance curve is unlikely to be observed in its natural host populations.

A conserved family of doublesex-related genes from fishes

The sex-determining gene Mab-3 of C. elegans and the doublesex gene of Drosophila each contain a common DM domain and share a similar role. Human doublesex-related gene DMRT1 also encodes a conserved DM-related DNA-binding domain. We present here the amplification of a broad range of DM domain sequences from three fish species using degenerate PCR. Our results reveal unexpected complexity of the DM domain gene family in vertebrates. (C) 2002 Wiley-Liss, Inc.

Characterization of an iridovirus from the cultured pig frog Rana grylio with lethal syndrome

Three virus isolates, RGV-9506, RGV-9807 and RGV-9808, were obtained from cultured pig frogs Rana grylio undergoing lethal infections. Previously, the first isolate, RGV-9506, was shown to be an iridovirus based on ultrastructural and morphological studies. In the present study, the original isolate, along with 2 recent ones, were more extensively characterized by experimental infection studies, histopathology, electron microscopy, serological reactivity, gel electrophoresis of viral polypeptides and DNA restriction fragments, PCR amplification, and nucleic acid sequence analysis of the major capsid protein (MCP) gene. The 3 isolates were shown to be identical to each other, and very similar to FV3, the type species of the genus Ranavirus (family Iridoviridae). These results suggest that RGV should be considered a strain of FV3, and indicate that FV3-like iridoviruses are capable of causing widespread, severe disease among cultured frogs.

Differential expression and characterization analysis of a new gene with WD domains in fish oogenesis

A new gene with WD domains is cloned and characterized according to its differential transcription and expression between previtellogenic oocytes (phase I oocytes) and fully-grown oocytes (phase V oocytes) from natural gynogenetic silver crucian carp (Carassius auratus gibelio) by using the combinative methods of suppressive subtraction hybridization, SMART cDNA synthesis and RACE-PCR. The full-length cDNA is 1870 bp. Its 5 ' untranslated region is 210 bp, followed by an open reading frame of 990 bp, which has the typical vertebrate initiator codon of ANNATG. The open reading frame encodes a protein with 329 amino acids. It has 670 bp of 3 ' untranslated region and an AATAAA polyadenylation signal. Because it has 92% homology to STRAP (serine-threonine kinase receptor-associated protein), a recently reported gene, we named it FSTRAP (fish STRAP). Virtual Northern blotting indicated that the FSTRAP was transcribed in fully-grown oocytes (phase V oocytes), but not in previtellogenic oocytes (phase I oocytes). RT-PCR analysis showed that FSTRAP was transcribed in brain, heart, kidney, muscle, ovary, spleen and testis, but not in liver. And its mRNA could be detected in the oocytes from phase II to phase V. Western blotting also showed that FSTRAP protein could be detected in brain, heart, kidney, muscle, ovary, spleen and testis except liver. Results of Western blotting on various oocytes were also similar to the RT-PCR data. FSTRAP protein was not expressed in the previtellogenic oocytes. Its expression initiated from phase II oocytes after vitellogenesis, and was consistent with the mRNA transcription.

Molecular analysis of silver crucian carp (Carassius auratus gibelio Bloch) clones by SCAR markers

Random amplified polymorphic DNA (RAPD) molecular markers specific for one, two or three clones have been identified from five gynogenetic clones of silver crucian carp (Carassius auratus gibelio Bloch) using RAPD markers developed earlier. In this study, three RAPD markers (RA1-PA, RA2-EF and RA4-D) produced by Opj-1, and two RAPD DNA fragments (RA3-PAD and RA5-D) produced by Opj-7, were selected for molecular cloning and sequencing. Sequence data indicated that there were identical 801-bp nucleotide sequences in the shared marker RA1-PA cloned respectively from clones P and A, and the shared marker RA2-EF (which was cloned from clones E and F), were also of identical 958-by nucleotide sequences. The nucleotide sequences of the shared marker RA3-PAD fragments were also similar for 1181 by among clones P, A and D. The specific fragment RA4-D was composed of 628 bp, and the fragment RA5-D from clone D contained 385 nucleotides. According to the nucleotide sequences, we designed and synthesized five pairs of sequence characterized amplified regions (SCAR) primers to identify the specific fragments in these gynogenetic clones of silver crucian carp. Only individuals from clones P and A amplified a specific band using a pair of SCI-PA primers synthesized according to the marker RA1-PA sequences, whereas no products were detected in individuals from clones D, E and F. The PCR products amplified using SC2-EF and SC3-PAD primers were as expected. Furthermore, the pair of SC4-D primers amplified specific bands only in individuals from clone D, although weak bands could be produced in all individuals of the five clones when lower annealing temperatures were used. However, an additional pair of SC5-D primers designed from the RA5-D marker sequences could amplify a DNA band in individuals from clones P, A and D, and the same weak band was produced in clone E, whereas no products were detected in individuals from clone F. Searches in GenBank revealed that the 385-bp DNA fragment from RA5-D was homologous to the 5' end of gonadotropin I beta subunit 2 gene and growth hormone gene. No homologous sequences were found for other markers in GenBank. The SCAR markers identified in this study will offer a powerful, easy, and rapid method for discrimination of different clones and for genetic analyses that examine their origins and unique reproductive modes in crucian carp. Furthermore, they will likely benefit future selective breeding programs as reliable and reproducible molecular markers. (C) 2001 Elsevier Science B.V. All rights reserved.

Growth and feed utilization in two strains of gibel carp, Carassius auratus gibelio: paternal effects in a gynogenetic fish

Gibel carp (Carassius auratus gibelio Bloch) is a natural gynogenetic fish which requires sperm of the same or related species to activate egg development. The eggs of one gibel carp were divided into two batches. One batch was 'fertilized' with sperm from gibel carp (strain DD), and the other 'fertilized' with sperm from red common carp (Cyprinus carpio red variety) (strain DR). The juveniles were transferred to the laboratory 36 days post-hatch. Triplicate groups of each strain were fed a formulated diet at either 3% or satiation ration for 8 weeks. At both the restricted and satiation rations, specific growth rate was significantly higher in strain DR than in strain DD. At the 3% ration, there was no significant difference in feeding rate or feed conversion efficiency between the two strains. At the satiation ration, strain DR had a significantly lower feeding rate but higher feed conversion efficiency than strain DD. At the satiation ration, strain DR had a significantly lower intake protein, but higher recovered protein than strain DD. There was no significant difference in faecal protein loss between the two strains. At the 3% ration, strain had no significant effects on intake protein, faecal protein or recovered protein. Neither faecal energy loss nor recovered energy was affected by strain or ration. At both the 3% and satiation ration, final body contents of dry matter and lipid were significantly lower in strain DR than strain DD, while there was no significant difference in protein and energy content between the two strains at either ration level. The results suggested that gibel carp 'fertilized' with sperm of common carp grew faster than those 'fertilized' with sperm of gibel carp through increased feed conversion efficiency and protein retention.

Genetic diversity among different clones of the gynogenetic silver crucian carp, Carassius auratus gibelio, revealed by transferrin and isozyme markers

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