Rapid establishment of pure lines of silver carp (Hypophthalmichthys molirix) and bighead carp (Aristichthys nobilis)

The diversity of gynogenetic, artificial sex reversal and natural silver carp and bighead carp is examined using randomly amplified polymorphic DNA (RAPD) method. All of the 187 bands are obtained and 19 (10.16%) of them are polymorphic in gynogenetic silver carp. Meanwhile 32 (15.61%) out of 205 bands are polymorphic in control group. In gynogenetic bighead carp a total of 232 bands are identified and 11 (4.74%) out of them are polymorphic, while 25 (10.37%) out of 241 bands are polymorphic in control group. The genetic distance of four populations is calculated and it is 0.102 and 0.023 for gynogenetic silver carp and gynogenetic bighead carp respectively. The values of natural silver carp and bighead carp are 0.161 and 0.104. From the UPGMA trees constructed based on genetic distance, the sex reversal individuals that match with the gynogenetic female individuals are picked out. A new breeding process of establishing a pure line is developed.

Identification and expression analysis of two IFN-inducible genes in crucian carp (Carassius auratus L.)

Interferon (IFN) exerts its antiviral effects mainly through activation of a subset of IFN-stimulated genes (ISG), but relatively few of fish ISGs have been isolated and characterized so far. Here, we report two fish ISGs, termed CaIF158 and CaIF156, cloned from a subtractive cDNA library constructed with mRNAs obtained from crucian carp (Carassius auratus L.) blastulae embryonic (CAB) cells infected by UV-inactivated GCHV and mock-infected cells. Database search revealed that both ISGs had a high-level homology with all members of a well conserved gene family with multiple tetratricopeptide repeat (TPR) motifs, including human IF160, IF158, IF156, IFI54 and their homologues in some other mammalian species. The transcripts of CaIF158 and CaIF156 were undetectable in CAB cells but could be induced by active GCHV, UV-inactivated GCHV or CAB IFN. Analysis of expression difference between them and IFN signal factors, CaSTAT1 and CaIRF7, indicated that their transcriptions were mediated possibly through JAK-STAT signal pathway, which was further supported by the induction analysis in UV-inactivated GCHV infected, IFN-treated and untreated cells in the presence or absence of cycloheximide (CHX), a potent inhibitor of protein synthesis. In addition, a pufferfish (Fugu rubrides) DNA sequence representing putative FrIFI56 was also revealed when CalF158 and CalF156 were used to search the pufferfish genome database. Phylogenetic analysis showed that these fish ISGs form a unique clad independent of mammalian homologues, reflecting a distant evolutionary relationship from mammals. These studies identified the first teleost IFI56 and IFI58 orthologues. (C) 2003 Elsevier B.V All rights reserved.

Dynamics of microcystins-LR and -RR in the phytoplanktivorous silver carp in a sub-chronic toxicity experiment

A sub-chronic toxicity experiment was conducted to examine tissue distribution and depuration of two microcystins (microcystin-LR and microcystin -RR) in the phytoplanktivorous filter-feeding silver carp during a course of 80 days. Two large tanks (A, B) were used, and in Tank A, the fish were fed naturally with fresh Microcystis viridis cells (collected from a eutrophic pond) throughout the experiment, while in Tank B, the food of the fish were M. viridis cells for the first 40 days and then changed to artificial carp feed. High Performance Liquid Chromatography (HPLC) was used to measure MC-LR and MC-RR in the M. viridis cells, the seston, and the intestine, blood, liver and muscle tissue of silver carp at an interval of 20 days. MC-RR and MC-LR in the collected Microcystis cells varied between 268-580 and 110-292 mug g(-1) DW, respectively. In Tank A, MC-RR and MC-LR varied between 41.5-99.5 and 6.9-15.8 mug g(-1) DW in the seston, respectively. The maximum MC-RR in the blood, liver and muscle of the fish was 49.7, 17.8 and 1.77 mug g(-1) DW, respectively. No MC-LR was detectable in the muscle and blood samples of the silver carp in spite of the abundant presence of this toxin in the intestines (for the liver, there was only one case when a relatively minor quantity was detected). These findings contrast with previous experimental results on rainbow trout. Perhaps silver carp has a mechanism to degrade MC-LR actively and to inhibit MC-LR transportation across the intestines. The depuration of MC-RR concentrations occurred slowly than uptakes in blood, liver and muscle, and the depuration rate was in the order of blood > liver > muscle. The grazing ability of silver carp on toxic cyanobacteria suggests an applicability of using phytoplanktivorous fish to counteract cyanotoxin contamination in eutrophic waters. (C) 2003 Elsevier Ltd. All rights reserved.

The impact of silver carp (Hypophthalmichthys molitrix) on the rotifer community in a eutrophic subtropical Chinese lake

From 20 April to 25 June in 1999, an enclosure experiment was conducted in Lake Donghu to assess the impact of planktivorous silver carp on the planktonic rotifer community. We set up four treatments with silver carp biomass at 0, 116, 176, and 316 g m(-2). Total rotifer density was significantly higher in the no-fish enclosure than in fish-present enclosures. Fish predation on the rotifers alleviated zooplankton competition and resulted in dominance of small zooplankton species (Anureaopsis fissa, Trichocerca pusilla and Moina micrura) in fish-present enclosures. However, some relatively larger species (Polyarthra vulgaris, Brachionus angularis, Brachionus calyciflorus, and Asplanchna spp.) showed higher densities in the no-fish enclosure than in fish-present enclosures.

Molecular cloning and characterization of crucian carp (Carassius auratus L.) interferon regulatory factor 7

Interferon (IFN) can induce an antiviral state via interferon-regulatory transcription factors (IRFs), which bind to and control genes directed by the interferon-stimulated response element (ISRE). Here we describe a fish IRF, termed CaIRF7, cloned from a subtractive cDNA library which is constructed with mRNAs obtained from crucian carp (Carassius auratus L.) blastulae embryonic (CAB) cells infected by UV-inactivated GCHV and mock-infected cells. CaIRF7 cDNA was found to be 1816 bp in length, with a 42 bp 5' UTR and a 508 bp 3' UTR. The open reading frame translates into 421 amino acids in which a DNA-binding domain (DBD) containing the repeated tryptophan motif and IRFs association domain have been identified. Like chicken GgIRF3, CaIRF7 was most similar to mammalian IRF7 with 27 to 30% identity overall and some 37% identity in their DBDs. A single transcript of 1.9 kb was detected in virally induced CAB cells by virtual Northern blotting. RT-PCR analysis revealed a wide tissue distribution of CaIRF7 constitutive expression, with detectable transcript in non-infected CAB cells and various tissues of healthy crucian carp. In addition, CaIRF7 expression was differentially increased by stimulation of the CAB cells with active GCHV, UV-inactivated GCHV or CAB IFN, indicating that the activation of CaIRF7 was directly regulated by IFN. (C) 2003 Published by Elsevier Ltd.

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.

Silver carp, Hypophthalmichthys molitrix, in the Poyang Lake belong to the Ganjiang River population rather than the Changjiang River population

The Poyang Lake is the largest lake and the main nursery area in the middle basin of the Changjiang (Yangtze) River. We compared molecular genetic markers of silver carp among populations of the Changjiang River, the Ganjiang River and the Poyang Lake using the ND5/6 region of mtDNA. Analysis of restriction fragment length polymorphisms (RFLPs) of this region revealed distinct variation between the Ganjiang River and the Changjiang River populations. The Poyang Lake is linked with the Ganjiang River and the Changjiang River. Shared RFLP fragments between the Ganjiang River population and the Poyang Lake population are as high as 61.4%. The value is 47.74% between the populations of the Changjiang River and that of the Poyang Lake. Frequencies of bands peculiar to the Ganjiang River population are the same as in the Poyang Lake population. We conclude that the Poyang Lake silver carp population consists mainly of the Ganjiang River population. The water level of the Poyang Lake outlet, which is higher than that of the Changjiang River in the silver carp spawning season, supports this conclusion.

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.

Effect of feeding frequency on growth, feed utilization, and size variation of juvenile gibel carp (Carassius auratus gibelio)

Juvenile (3.0 +/- 0.2 g) gibel carp (Carassius auratus gibelio ) were fed to satiation for 8 weeks to investigate the effect of feeding frequency on growth, feed utilization and size variation. Five feeding frequencies were tested: two meals per day (M2), three meals per day (M3), four meals per day (M4), 12 meals per day (M12) and 24 meals per day (M24). The results showed that daily food intake increased significantly with the increase in feeding frequency and there was no significant difference between daily food intakes in M12 and M24 treatments. Growth rate, feed efficiency increased significantly with increasing feeding frequencies. Size variation was not affected by feeding frequency. Apparent digestibility of dry matter was not influenced by feeding frequency, while apparent digestibility of protein and energy increased significantly at high feeding frequencies. The feeding frequency had no significant effect on the moisture, lipid, protein, or energy contents of gibel carp, while the ash content decreased with increased feeding frequency. It was recommended that 24 meals per day was the optimal feeding frequency for juvenile gibel carp.

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.

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.

Spawning areas and early development of long spiky-head carp (Luciobrama macrocephalus) in the Yangtze River and Pearl River, China

The spawning areas and early development of long spiky-head carp, Luciobrama macrocephalus (Lacepede), an endemic fish species in China, were investigated in the Yangtze River and Pearl River of central and southeastern China between 1961 and 1993. The potamodromous fish migrated upstream to spawn between May and July as the floodwater began to rise. The water-hardened eggs drifted down the river, and the embryos and larvae developed in the course of drifting. The spawning areas of the fish were widely found in the upper and middle main channels and large tributaries. Two large dams (Gezhouba dam and Danjiangkou dam) did not significantly impact on the reproduction of the fish. Fifty stages of the early development from one cell to the juvenile with fully formed fins were observed and characterized pictorially. The larvae of long spiky-head carp could be distinguished from the larvae of other co-occurring species by counting the number of somites and comparing the proportion of sizes of eye to otic capsule.

Experimental study of trophic cascade effect of silver carp (Hypophthalmichthys molitrixon) in a subtropical lake, Lake Donghu: on plankton community and underlying mechanisms of changes of crustacean community

An enclosure experiment was carried out to test trophic cascade effect of filter-feeding fish on the ecosystem: growth of crustacean zooplankton, and possible mechanism of changes of crustacean community structure. Four fish biomass levels were set as follows: 0, 116, 176 and 316 g m(-2), and lake water ( containing ca. 190 g m(-2) of filter-feeding fishes) was comparatively monitored. Nutrient levels were high in all treatments during the experiment. Lowest algal biomass were measured in fishless treatment. Algal biomass decreased during days 21-56 as a function of fish biomass in treatments of low (LF), medium (MF) and high (HF) fish biomass. Crustaceans biomass decreased with increasing fish biomass. Small-bodied cladocerans, Moina micrura, Diaphanosoma brachyurum and Scapholeberis kingii survived when fish biomass was high whilst, large-bodied cladocerans Daphnia spp. and the cyclopoids Theromcyclops taihokuensis, T. brevifuratus, Mescyclops notius and Cyclops vicinus were abundant only in NF enclosures. Evasive calanoid Sinodiaptomus sarsi was significantly enhanced in LF, but decreased significantly with further increase of fish biomass. Demographic data indicated that M. micrura was well developed in all treatments. Our study indicates that algal biomass might be controlled by silver carp biomass in eutrophic environment. Changes of crustacean community are probably affected by the age of the first generation of species. Species with short generation time were dominant and species with long generation time survived less with high fish biomass. Evasive calanoids hardly developed in treatments with high fish biomass because of the ( bottle neck) effect of nauplii. Species abundance were positively related to fish predation avoidance. Other than direct predation, zooplankton might also be suppressed by filter-feeding fish via competition.

Myxobolus lentisuturalis sp n. (Myxozoa : Myxobolidae), a new muscle-infecting species from the Prussian carp, Carassius gibelio from China

A new highly pathogenic muscle-infecting species of the genus Myxobolus Butschli, 1882 is described from the Prussian carp, Carassius gibelio (Bloch, 1782) using spore morphology and SSU rDNA sequence data. Phylogenetic analyses elucidated relationship of the newly described Myxobolus lentisuturalis to other Myxobolus species and supported its position of an independent species.