Development of novel EST-SSR markers in common carp by data mining from public EST sequences

Expressed sequence tags (ESTs) are a source for microsatellite development. In the present study, EST-derived microsatelltes (EST-SSRs) were generated and characterized in the common carp (Cyprinus carpio) by data mining from updated public EST databases and by subsequent testing for polymorphism. About 5.5% (555) of 10,088 ESTs contain repeat motifs of various types and lengths with CA being the most abundant dinucleotide one. Out of the 60 EST-SSRs for which PCR primers were designed, 25 loci showed polymorphism in a common carp population with the alleles per locus ranging from 3 to 17 (mean 7). The observed (H-O) and expected (HE) heterozygosities of these EST-SSRs were 0.13-1.00 and 0.12-0.91, respectively. Six EST-SSR loci significantly deviated from the Hardy-Weinberg equilibrium (HWE) expectation, and the remaining 19 loci were in HWE. Of the 60 primer sets, the rates of polymorphic EST-SSRs were 42% in common carp, 17% in crucian carp (Carassius auratus), and 5% in silver carp (Hypophthalmichthys molitrix), respectively. These new EST-SSR markers would provide sufficient polymorphism for population genetic studies and genome mapping of the common carp and its closely related fishes. (c) 2007 Published by Elsevier B.V.

Morphological and genetic differences of Trypanosoma in some Chinese freshwater fishes: difficulties of species identification

Blood smears and purified trypanosome from freshwater fishes yellow catfish (Pseudobagras fulvidraco) and common carp (Cyprinus carpio) captured from Niushan Lake, Hubei Province were examined to determine whether all of their trypanosomes were Trypanosoma pseudobagri, a species of supposed host specificity and widespread existence across China. Trypanosomes occurred in 16/16 blood smears, and morphometric character analysis of trypanosomes from these smears showed that there were three morphospecies, Trypanosoma sp Carpio, T. sp Pseudobagri, and T. sp. 18S rDNA sequences of trypanosomes from 16 samples revealed three genetic groups among these fish trypanosomes. Group 1 was from C. carpio containing T. sp Carpio; groups 2 and 3 were from P. fulvidraco containing T. sp Pseudobagri and T. sp, respectively. The high similarity of morphometric characters and 18S rDNA sequences showed that T. sp Carpio and T. siniperca probably were the same species. T. sp Pseudobagri was the first occurrence in China. Sequence comparison showed that T. sp Pseudobagri sequence was most similar to that of clone Marv, whereas T. sp sequence differ from those of T. sp Carpio and T. sp Pseudobagri by 5.4 and 5.8%, respectively, and tentatively identified as T. pseudobagri. It was concluded that three species of trypanosomes, at least three genotypes occur in Niushan Lake fishes, and P. fulvidraco in this region appear to contain both types, although the identification of T. pseudobagri remains a problem.

Rapid growth cost in “all-fish” growth hormone gene transgenic carp: Reduced critical swimming speed

Evidence has accumulated that there is a trade-off between benefits and costs associated with rapid growth. A trade-off between growth rates and critical. swimming speed (U-crit) had been also reported to be common in teleost fish. We hypothesize that growth acceleration in the F-3 generation of "all-fish" growth hormone gene (GH) transgenic common carp (Cyprinus carpio L.) would reduce the swimming abilities. Growth and swimming performance between transgenic fish and non-transgenic controls were) compared. The results showed that transgenic fish had a mean body weight 1.4-1.9-fold heavier, and a mean specific growth rate (SGR) value 6%-10% higher than the controls. Transgenic fish, however, had a mean absolute U-crit (cm/s) value 22% or mean relative Ucrit (BL/s) value 24% lower than the controls. It suggested that fast-growing "all-fish" GH-transgenic carp were inferior swimmers. It is also supported that there was a trade-off between growth rates and swimming performance, i.e. faster-growing individuals had lower critical swimming speed.

Identification of genome organization in the unusual allotetraploid form of Carassius auratus gibelio

The unusual allotetraploid form with unequal contribution of chromosome sets was discovered from the gynogenetic offspring of Carassius auratus gibelio stimulated by red common carp sperm. In this study, genomic in situ hybridization (GISH) and fluorescent in situ hybridization (FISH) with 45S rDNA probe are used. The GISH results lead to the identification of species-specific chromosomes, which permits to demonstrate the origin and genome organization in the allotetraploid form. Moreover, chromosome localization of 45S rDNA and co-localizations of 45S rDNA and Cyprinus carpio genomic DNA further confirm that one extra 45S rDNA positive chromosome in the allotetraploid form originates from the paternal haploid genome of C carpio, and other 5 45S rDNA-containing chromosomes are from the maternal genome of Carassius auratus gibelio. And, the correlation between 45 rDNA and the nucleolar organizer regions (NORs) is confirmed by silver nitrate staining. The data provide direct experiment evidence that the allotetraploid actually contains three chromosome sets of Carassius auratus gibelio and one chromosome set of C carpio, and will be a useful genetic material for both basic research and breeding practice. (c) 2006 Elsevier B.V. All rights reserved.

Cloning, characterization and promoter analysis of common carp hairy/Enhancer-of-split-related gene, her6

Some members of hairy/Enhancer-of-split-related gene (HES) family have important effects on axial mesoderm segmentation and the establishment and maintenance of the somite fringe. In fishes. the her6 gene, a member of the HES family, is the homologue Of heS1 in mammals and chicken. In this study, the her6 gene and its full-length cDNA from the common carp (Cyprinus carpio) were isolated and characterized. The genomic sequence of common carp her6 is approximately 1.7 kb. with four exons and three introns, and the full-length cDNA of 1314 bp encodes a Putative polypeptide of 271 amino acids. To analyse the promoter sequence of common carp her6. sequences of various lengths upstream from the transcription initiation site of her6 were fused to enhanced green fluorescent. protein gene (eGFP) and introduced into zebrafish embryos by microinjection to generate transgenic embryos. Our results show that the upstream sequence of 500 bp can direct highly efficient and tissue-specific expression of eGFP in zebrafish embryos. whereas a fragment of 200 bp containing the TATA box and a partial suppressor of hairless paired site sequence (SPS) is not sufficient to drive eGFP expression in zebrafish embryos.

Growth and energy budget of F-2 'all-fish' growth hormone gene transgenic common carp

The growth and energy budget for F-2 'all-fish' growth hormone gene transgenic common carp Cyprinus carpio of two body sizes were investigated at 29.2 degrees C for 21 days. Specific growth rate, feed intake, feed efficiency, digestibility coefficients of dry matter and protein, gross energy intake (I-E), and the proportion of I-E utilized for heat production (H-E) were significantly higher in the transgenics than in the controls. The proportion of I-E directed to waste products [faecal energy (F-E) and excretory energy loss (Z(E) + U-E) where Z(E) is through the gills and U-E through the kidney], and the proportion of metabolizable energy (M-E) for recovered energy (R-E) were significantly lower in the transgenics than in the controls. The average energy budget equation of transgenic fish was as follows: 100 I-E = 19.3 F-E + 6.0 (Z(E) + U-E) + 45.2 H-E + 29.5 R-E or 100 M-E = 60.5 H-E + 39.5 R-E. The average energy budget equation of the controls was: 100 I-E = 25.2 F-E + 7.4 (Z(E) + U-E) + 35.5 H-E + 31.9 R-E or 100 M-E = 52.7 H-E + 47.3 R-E. These findings indicate that the high growth rate of 'all-fish' transgenic common carp relative to their non-transgenic counterparts was due to their increased feed intake, reduced lose of waste productions and improved feed efficiency. The benefit of the increased energy intake by transgenic fish, however, was diminished by their increased metabolism.

Methylmercury, total mercury and total selenium in four common freshwater fish species from Ya-Er Lake, China

Snakehead fish (Ophiocephalus argus cantor), silver carp (Hypophthalmichthys molitrtix), crucian carp (Carassius carassius), and common carp (Cyprinus carpio) are four common freshwater fish species in China. In this study, the level of methylmercury (MeHg), total mercury (T-Hg), and total selenium (T-Se) in muscle samples of these four fish species from Ya-Er Lake, China, were analyzed using atomic fluorescence spectrometry coupled with high-performance liquid chromatography, and inductively coupled plasma mass spectrometry. The concentrations of MeHg in all the fish species were significantly correlated with those of T-Hg. Higher T-Hg and MeHg concentrations had accumulated in the snakehead fish, which is a strongly predatory fish, than in the other three species. The concentration ratios of MeHg and T-Hg in the muscles of these four fish species were almost equal. Conversely, there was negative correlation between the concentrations of T-Hg and T-Se, which implies that there is a competition between these two elements with respect to bioaccumulation. It is noteworthy that of all the muscle samples tested, the level of T-Hg exceeded the maximum allowable limit in fish [0.4 mg kg(-1) (w/w) recommended by the World Health Organization] in 38.46% of those of the silver carp, 18.18% of those of the crucian carp, and 100% of those of snakehead fish. These results show that the consumption of contaminated fish is a potential threat to human health and that necessary preventive measures to safeguard public health should be emphasized.

Genetic diversity of common carp from two largest Chinese lakes and the Yangtze River revealed by microsatellite markers

Six polymorphic microsatellites (eight loci) were used to study the genetic diversity and population structure of common carp from Dongting Lake (DTC), Poyang Lake (PYC), and the Yangtze River (YZC) in China. The gene diversity was high among populations with values close to 1. The number of alleles per locus ranged from 2 to 11, and the average number of alleles among 3 populations ranged from 6.5 to 7.9. The mean observed (H (O)) and expected (H (E)) heterozygosity ranged from 0.4888 to 0.5162 and from 0.7679 to 0.7708, respectively. Significant deviations from Hardy-Weinberg Equilibrium expectation were found at majority of the loci and in all three populations in which heterozygote deficits were apparent. The analysis of molecular variance (AMOVA) indicated that the percent of variance among populations and within populations were 3.03 and 96.97, respectively. The Fst values between populations indicated that there were significant genetic differentiations for the common carp populations from the Yangtze River and two largest Chinese freshwater lakes. The factors that may result in genetic divergence and significant reduction of the observed heterozygosity were discussed.

An enzyme-linked immunosorbent assay for rare minnow (Gobiocypris rarus) vitellogenin and comparison of vitellogenin responses in rare minnow and zebrafish (Danio rerio)

A competitive enzyme-linked immunosorbent assay (ELISA) was developed to determine vitellogenin (Vtg) in rare minnow (Gobiocypris rarus) based on the separation and purification of rare minnow Vtg (r-Vtg) as well as the production of polyclonal antibody against r-Vtg in rabbits. Three different ELISAs for measuring r-Vtg were then compared: (1) indirect ELISA with the antibody against carp (Cyprinus carpio) Vtg (c-Vtg) (IC-ELISA); (2) competitive ELISA with the antibody against c-Vtg, and using r-Vtg for coating the plates and preparing standard curve (CC-ELISA); (3) competitive ELISA with the antibody against r-Vtg, and using r-Vtg for coating the plates and preparing standard curve (CR-ELISA). The result showed that the homologous CR-ELISA was the most sensitive among the three assays for quantifying r-Vtg. The sensitivities to 17 alpha-ethinylestradiol (EE2) Of rare minnow and zebrafish (Danio rerio) were compared upon the establishment of homologous competitive ELISA. The lowest observed effect concentrations (LOECs) to induce Vtg were found to be 0.8 ng EE2 l(-1) for rare minnow and 4 ng EE2 l(-1) for zebrafish respectively. Afterwards, CR-ELISA was applied to measure Vtg concentration in whole body homogenate (WBH) of juvenile rare minnow fed by three diets (tubifex from wastewater treatment plant, Artemia nauplii and commercial pellet food), and the agreements between bioassay and GC-MS analysis demonstrated that rare minnow was a sensitive fish model for assessing estrogenic effects of endocrine disrupting compounds in aquatic environment. (c) 2006 Elsevier B.V. All rights reserved.

Comparison of evolutionary rates in the mitochondrial DNA cytochrome b gene and control region and their implications for phylogeny of the Cobitoidea (Teleostei : Cypriniformes)

It is widely accepted that mitochondrial DNA (mtDNA) control region evolves faster than protein encoding genes with few exceptions. In the present study, we sequenced the mitochondrial cytochrome b gene (cyt b) and control region (CR) and compared their rates in 93 specimens representing 67 species of loaches and some related taxa in the Cobitoidea (Order Cypriniformes). The results showed that sequence divergences of the CR were broadly higher than those of the cyt b (about 1.83 times). However, in considering only closely related species, CR sequence evolution was slower than that of cyt b gene (ratio of CR/cyt b is 0.78), a pattern that is found to be very common in Cypriniformes. Combined data of the cyt b and CR were used to estimate the phylogenetic relationship of the Cobitoidea by maximum parsimony, neighbor-joining, and Bayesian methods. With Cyprinus carpio and Danio rerio as outgroups, three analyses identified the same four lineages representing four subfamilies of loaches, with Botiinae on the basal-most clade. The phylogenctic relationship of the Cobitoidea was ((Catostomidae + Gyrinocheilidae) + (Botiinae + (Balitorinae + (Cobitinae + Nemacheilinae)))), which indicated that Sawada's Cobitidae (including Cobitinae and Botiinae) was not monophyletic. Our molecular phylogenetic analyses are in very close agreement with the phylogenetic results based on the morphological data proposed by Nalbant and Bianco, wherein these four subfamilies were elevated to the family level as Botiidae, Balitoridae, Cobitidae, and Nemacheilidae. (c) 2005 Elsevier Inc. All rights reserved.

Histological and cytological studies on the developing thymus of mandarin fish Siniperca chuatsi (Perciformes : Teleostei)

The thymus of the mandarin fish, Siniperca chuatsi, was examined by light and transmission electron microscopy to understand its formation and cellular composition. Larvae of the mandarin fish were collected and sectioned from 1 to 35 days post-hatching (dph). On dph 7 the thymus was packed with lymphocytes. From 12 dph onward, mucous cells were observed on the epithelial layer; from 23 dph, three zones could be differentiated in the thymic parenchyma. The thymus was connected with the extension of the third, fourth and fifth branchial pouches throughout early development, remaining in a superficial position in the adult S. chuatsi. In the thymus of the adult fish, thymic epithelial cells (TECs) characteristic of tonofilaments were observed, with limiting TECs (LECs) found in subcapsular, subseptal, perivascular and nurse-like TECs containing viable intact lymphocytes inside their vacuoles. In addition, three kinds of granulocytes were observed throughout the thymus, and an incomplete blood-thymus barrier was found in the inner zone. Other cell components such as cystic cells, macrophages and plasma cells, were also described in the thymus of the adult S. chuatsi. The thymus development in mandarin fish agrees, to some extent, with the ontogenetic patterns observed in other fish species.

Nonylphenol, bisphenol-A and DDTs in Lake Donghu, China

A preliminary survey was carried out in April 2003 to estimate the levels of nonylphenol (NP), bisphenol-A (BPA) and residual DDTs in Lake Donghu. After then, the sediments of some areas of the lake were moved out for clearing the lake by the local government. Therefore, the variance of NP and BPA after the clearance of sediment in the surface water was determined from December 2003 to May 2004. Sediments, surface water and fish were collected from four sub-lake areas and the analytes were qualified by GC/MS in SIM mode after concentration onto an Oasis solid phase extraction cartridge. NP and BPA values ranged between 5.46-119.10 and 0.9913.42 mg/kg dw, respectively, in sediments, 75.2-179.6 and 15.1-62.5 mu g/L in surface water. Meanwhile, the bioaccumulation factors (BCFs) of NP and BPA were calculated in fish livers. Plasma vitellogenin (VTG), a sensitive biomarker showing oestrogenicity was detected in the captured male Wuchang bream (Megalobrama amblycephala Yih) and common carp (Cyprinus carpio). After sediment clearance, the concentrations of NP and BPA decreased to 0.65-25.04 and 0.04-21.32 mu g/L. The results indicate the presence of high-dose xenoestrogenic NP and BPA, probably major contributors which associate with VTG induction in Lake Donghu male fish.

Scanning electron microscopy of Aspidogaster ijimai Kawamura, 1913 and A-conchicola Baer, 1827 (Aspidogastrea, Aspidogastridae) with reference to their fish definitive-host specificity

Two species of aspidogastreans, namely Aspidogaster ijimai and A. conchicola, were studied by scanning electron microscopy. In nine lakes and an old river course, the Tian'ezhou oxbow, investigated in the flood plain of the Yangtze River, A. ijimai was obtained from the common carp (Cyprinus carpio) in three lakes, and A. conchicola from the black carp Mylopharyngodon piceus in three lakes and the oxbow. In none of the localities, however, were the two species found together. It is suggested that A. ijimai may be considered as a specialist parasite for the common carp, at least in the flood-plain lakes of the Yangtze River. The two parasites were similar in many aspects of their morphology. Their bodies can both be separated into a dorsal part and a ventral disc, with the body surface of the dorsal part elevated by transverse folds, and the disc subdivided into alveoli by transverse and longitudinal septa, although the number of alveoli was different in the two species. The depression on the ventral surface of the neck region was prominent for both species, and their ventral disc was covered densely with non-ciliated bulbous papillae. The position of mouth, osmo-regulatory pore and marginal organ was also similar for A. ijimai and A. conchicola. However, microridges in the trough of the folds in the neck region and numerous small pits on the upper part of the septa were found exclusively in A. ijimai, but uniciliated sensory papillae in A. conchicola.

Growth hormone gene transfer in common carp

The first successful case of transgenic fish was achieved in 1984. It is in a model system that the integration and expression of recombinant human growth hormone (hGH) in host red common carp (Cyprinus carpio, red var.) have been thoroughly studied. Recently, the integration sites have been recovered and characterized. Compared with non-transgenic peers, hGH-transgenic fish are prior in dietary utilization and growth performance. In view of bio-safety and bio-ethics, an "all-fish" construct CAgcGH, grass carp growth hormone fused with common carp P-actin promoter, has been generated and transferred into Yellow River carp (C carpio, local strain in Yellow River) fertilized eggs. Under middle-scale trial, CAgcGH-transgenics show higher growth rate and food conversion efficiency than the controls, which is consistent to laboratory findings. To avoid the potential impact of transgenic fish on the environment, a sterile strain of transgenic triploid fish has been successfully produced. The "all-fish" transgenic common carp is also approved safe enough as daily food, according to a test based on the pathological principles of new medicines issued by the Ministry of Health of China. The "all-fish" transgenic common carp with growth enhancement is now ready for market, but looking for governmental authorization. (C) 2003 Editions scientifiques et medicales Elsevier SAS and Ifremer/IRD/Inra/Cemagref. All rights reserved.

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.