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.

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.

Genetic evidence for gonochoristic reproduction in gynogenetic silver crucian carp (Carassius auratus gibelio bloch) as revealed by RAPD assays

Sex evolution has been a debating focus in evolutionary genetics. In lower vertebrates of reptiles, amphibians, and fish, a species or a bioform reproduces either sexually or asexually but never both. A few species were found to consist of all females in fish. These all-female species can propagate by asexual reproduction modes, such as gynogenesis and hybridogenesis. However, the coexistence of sexuality and asexuality in a single species was recently noted only in a cyprinid fish silver crucian carp, Carassius auratus gibelio. This fish had been demonstrated to be capable of gynogenesis stimulated by sperm from other related species. Surprisingly, natural populations of this fish consist of a minor but significant portion (approx. 20%) of males. As different clones with specific phenotypic and genetic characteristics have been found, and RAPD markers specific to each clone have recently been identified, this fish offers many advantages for analyzing whether or not genetic recombination occurs between different clones. In this study, artificial propagation was performed in clone F and clone D. Ovulated eggs from clone F were divided into two parts and respectively inseminated with sperm from a clone D male and from a red common carp (Cyprinus carpio) male. The control clone D individuals were selected from gynogenetic offspring of clone D activated by sperm of red common carp. The phenotype and sex ratio in the experimental groups were also observed. Using RAPD molecular markers, which allow for reliable discrimination and genetic analysis of different clones, we have revealed direct molecular evidence for gonochoristic reproduction in the gynogenetic silver crucian carp and confirmed a previous hypothesis that the silver crucian carp might reproduce both gynogenetically and gonochoristically. Therefore, we conclude that the silver crucian carp possesses two reproductive modes, i.e., gynogenetic and gonochoristic reproduction. The response mechanism of two reproductive development modes may be the first discovery in vertebrates. Additionally, we discuss the evolutionary implication between gynogenetic and gonochoristic reproduction modes and the contribution of the minor proportion of males to genetic flexibility in the gynogenetic silver crucian carp.

The innate immune response to grass carp hemorrhagic virus (GCHV) in cultured Carassius auratus blastulae (CAB) cells

Virus infection of mammalian cells activates an innate antiviral immune response characterized by production of interferon (IFN) and the subsequent transcriptional upregulation of IFN-stimulated genes (ISGs) by the JAK-STAT signaling pathway. Here, we report that a fish cell line, crucian carp (Carassius auratus L.) blastulae embryonic (CAB) cells, can produce IFN activity and then form an antiviral state after infection with UV-inactivated grass carp hemorrhagic virus (GCHV), a double-stranded (ds) RNA virus. From UV-inactivated GCHV-infected CAB cells, 15 pivotal genes were cloned and sequenced, and all of them were shown to be involved in IFN antiviral innate immune response. These IFN system genes include the dsRNA signal sensing factor TLR3, IFN, IFN signal transduction factor STAT1, IFN regulatory factor IRF7, putative IFN antiviral effectors Mx1, Mx2, PKR-like, Viperin, IFI56, and other IFN stimulated genes (ISGs) IFI58, ISG15-1, ISG15-2, USP18, Gig1 and Gig2. The identified fish IFN system genes were highly induced by active GCHV, UV-inactivated GCHV, CAB IFN or poly(I).poly(C), and showed similar expression patterns to mammals. The data indicate that an IFN antiviral innate immune response similar to that in mammals exists in the UV-inactivated GCHV-infected CAB cells, and the IFN response contributes to the formation of an antiviral state probably through JAK-STAT signaling pathway. This study provides strong evidence for existence of IFN antiviral innate immune response in fish, and will assist in elucidating the origin and evolution of vertebrate IFN system. (c) 2006 Elsevier Ltd. All rights reserved.

Differential expression of two Carassius auratus Mx genes in cultured CAB cells induced by grass carp hemorrhage virus and interferon

UV-inactivated GCHV (grass carp hemorrhage virus) is able to induce an antiviral state in cultured CAB cells (crucian carp Carassius auratus blastulae embryonic cells) via the production of interferon (IFN). In the current work, the full-length cDNAs of two Mx genes, termed CaMx1 and CaMx2, have been cloned and sequenced from UV-inactivated GCHV-infected and still IFN-producing CAB cells by suppression subtractive hybridization. Their putative proteins show the characteristically structural features of mammalian IFN-induced Mx proteins, including GTP-binding motif, dynamin family signature and leucine zipper motif. CaMx1 exhibits 85% sequence identity to zebrafish MxA and 72-74% to three Atlantic salmon Mx proteins. CaMx2 is most similar to zebrafish MxE, with 80% identity, and then rainbow trout Mx3, with 52%. Constitutive expression was detected by RT-PCR for CaMx1, but not for CaMx2, in normal CAB cells, but their up-regulations could be induced after treatment with active GCHV, UV-inactivated GCHV and CAB IFN. Distinct kinetics of expression was observed for either CaMx1 or CaMx2 corresponding to the three stimuli, and even between CaMx1 and CaMx2, corresponding to the same stimulus. Upon virus infection, the transcriptional induction was strongly blocked for CaMx2 by cycloheximide (CHX), whereas almost nothing was observed for CaMx1. By contrast, following treatment with CAB IFN, CHX did not inhibit either gene transcription. Collectively, these results suggest that there are very distinct mechanisms for modulating the expression of both CaMx1 and CaMx2 in normal and GCHV-infected CAB cells.

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.

Discovery of a male-biased mutant family and identification of a male-specific SCAR marker in gynogenetic gibel carp Carassius auratus gibelio

Gibel carp ( Carassius auratus gibelio) is a uniquely gynogenetic species with a minor ratio of males in natural habitats, but its male origin and sex determination mechanisms have been unknown. In this study, a male-biased mutant family was discovered from the gynogenetic gibel carp, and a male-specific SCAR marker was identified from the mutant family. Normal spermatogenesis was observed in the male testes by immuno. fluorescence histochemistry. Nearly identical AFLP profiles were observed between males and females, but a male-specific 86 bp AFLP fragment was screened by sex-pool bulked segregant analysis and individual screening. Based on the male-specific AFLP fragment, a total of 579 bp sequences were cloned by genome walking. Subsequently, a male-specific SCAR marker was designed, and the male-specific DNA fragment was confirmed to be steadily transmitted to the next generation and consistently detected only in males. (C) 2009 National Natural Science Foundation of China and Chinese Academy of Sciences. Published by Elsevier Limited and Science in China Press. All rights reserved.

The effect of different dietary iron levels on growth and hepatic iron concentration in juvenile gibel carp (Carassius auratus gibelio)

P>An 83-day growth trial was conducted using a flow-through system to examine the effects of different dietary iron levels on growth and hepatic iron concentration in juvenile gibel carp (Carassius auratus gibelio). Six purified diets supplemented with different levels of iron (0, 10, 30, 60, 100 and 200 mg kg(-1)) (as ferrous sulfate) were fed to triplicate groups of fish (initial weight 2.12 +/- 0.00 g per fish). The results showed that the addition of iron to the basal diet did not significantly affect the specific growth rate (SGR), feed efficiency (FE), survival, red blood cell amount (RBC), hemoglobin content (Hb), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) or mean corpuscular hemoglobin concentration (MCHC). Hepatic iron concentration and hematocrit (Hct) were significantly influenced by dietary iron level (P < 0.05). On the basis of the iron concentration for the maintenance of optimum hepatic iron concentration and Hct, it was concluded that the dietary iron concentration of juvenile gibel carp should be not less than 202 mg Fe kg(-1) diet.

Effect of high dietary starch levels on the growth performance, blood chemistry and body composition of gibel carp (Carassius auratus var. gibelio)

An 8-week growth trial was carried out in a semi-recirculation system to investigate the effect of high dietary starch levels on the growth performance, blood chemistry, starch utilization and body composition of gibel carp (Carassius auratus var. gibelio). Five isonitrogenous and isocarloric experimental diets were formulated to contain different starch levels (24%, 28%, 32%, 36% and 40% respectively). Triplicate groups of fish (24 fish per tank with an average body weight, of 8.5 g) were assigned to each diet. The results showed that dietary carbohydrate levels significantly affected the growth performance, hepatopancreatic lipid content, pyruvate kinase (PK) activity and whole-body lipid content. Growth performance, body crude lipid and plasma glucose concentrations showed a decreasing trend with an increase in dietary starch from 24% to 40%. Pyruvate kinase activities and hepatopancreatic lipid content showed an increasing trend with the dietary starch increasing from 24% to 32%, and then a decreasing trend with the dietary starch increasing from 32% to 40%. No significant difference in the hepatopancreatic hexokinase (HK) activity, plasma triglyceride contents, body crude protein, ash and calcium (Ca) and phosphorus (P) contents was observed between different treatments. In conclusion, higher dietary starch levels (32-40%) significantly (P < 0.05) decreased the growth of gibel carp in the present study.

Morphological and molecular phylogenetic analysis of two Saprolegnia sp (Oomycetes) isolated from silver crucian carp and zebra fish

Two Saprolegnia isolates, JY isolated from silver crucian carp (Carassius auratus gibelio Bloch) and BMY isolated from zebra fish (Brachydanio rerio Hamilton) came from infections occurring concurrently in different locations in China. To confirm whether the two isolates were from the same Saprolegnia clone, comparative studies have been carried out based on their morphological, physiological and molecular characteristics. Observations showed that morphologically (both asexual and sexual organs) the two isolates were broadly similar and both isolates under-went repeated zoospore emergence. Comparing 704 base pairs of internal transcribed spacer (ITS) region and the 5.8S rDNA, we found isolates JY and BMY shared an identical ITS sequence with a minor variation (99.6 % similarity). Forty available sequences for representatives Saprolegnia spp. belonged to four phylogenetically separate clades. The two studied isolates fell within clade I that comprised a group of isolates which showed almost an identical ITS sequence but had been identified as a number of different morphological species. our findings suggest that isolates JY and BMY appear to belong to the S. ferax clade and this clade (1) contains a number of closely related phylogenetic species. This is distinct from the more common fish pathogenic isolates, which belong to the S. parasitica clade (III) and are characterized by having cysts decorated by bundles of long hooked hairs and two further clades (II and IV) containing largely saprotrophic or soil born species. (C) 2009 Published by Elsevier Ltd on behalf of The British Mycological Society.

Molecular characterization and expression pattern of fetuin-B in gibel carp (Carassius auratus gibelio)

Fetuin-B has recently been cloned and identified from rats, mice, and humans; their expression patterns, however, have not been elucidated yet. In this study, Cagfetuin-B has been cloned in gibel carp. RT-PCR and Western blot detection revealed that Cagfetuin-B is first transcribed from the blastula stage and at a relatively stable level afterward during embryogenesis and the larval stage. Cagfetuin-B transcripts are predominantly distributed over the yolk syncytial layer in the early embryos and later restricted to the cells of liver and brain in newly hatched larvae. Moreover, a dynamic distribution of Cagfetuin-B protein was observed in brain, kidney, liver, and skin during morphogenesis. In adult fish, Cagfetuin-B transcripts are restricted in liver and ovary. Our work, for the first time, revealed the extra-hepatic transcription and a dynamic distribution of fetuin-B during embryogenesis and in adults, which indicates the potential roles of fetuin-B in fish organogenesis.

Effects of dietary manganese on growth and tissue manganese concentrations of juvenile gibel carp, Carassius auratus gibelio

A 68-day growth trial was conducted in a flow-through system to determine the effect of dietary manganese levels on growth and tissue manganese concentration of juvenile gibel carp (Carassius auratus gibelio). Seven purified diets containing 7.21, 8.46, 9.50, 10.50, 13.03, 19.72 and 22.17 mg manganese (as manganic sulfate) per kilogram diet were fed to triplicate groups of fish (initial weight 3.21 +/- 0.01 g). The results showed that dietary manganese levels did not significantly affect feed intake of the fish. Specific growth rate, feed efficiency, total hepatic superoxide dismutase activity, carcass and skeletal manganese concentration increased significantly with increased dietary manganese(P < 0.05) while condition factor decreased significantly(P < 0.05). It was concluded that dietary requirement of manganese was 13.77 mg Mn per kilogram diet. Carcass and skeletal manganese concentration could also be used to evaluate the manganese requirement. Total hepatic superoxide dismulase activity was not a sensitive indicator for dietary requirement.