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.

Long-term changes of Copepoda community (1957-1996) in a subtropical Chinese lake stocked densely with planktivorous filter-feeding silver and bighead carp

In contrast to the relatively well documented impact of particulate-feeding fish on zooplankton communities, little attention has been devoted to the impact of filter-feeding fish. Filter-feeding silver and bighead carp are the most intensively cultured fish species in Asia and comprise much of the production of Chinese aquaculture. However, little information is known about the impact of either fish on the zooplankton community. Long-term changes in the Copepoda community (1957-1996) were studied at two sampling stations of a subtropical Chinese lake (Lake Donghu) dominated by silver and bighead carp. For both calanoids and cyclopoids, the littoral station (I) was much more resource profitable than the pelagic station (II). There has been a tremendous increase in the annual fish catch over the past 30 years due to the increased stocking with fingerlings of the two carp species. There was a notably higher fish density at Station I than at Station II. Cyclopoid abundance was notably higher at Station I than at Station II during the 1950s to the 1980s, while the reverse became true in the 1990s. This is probably because when fish abundance increased to an extremely high level, the impact of fish predation on the cyclopoids became more important than that of food resources at the littoral station. At both stations, cyclopoid abundance was relatively low in spite of the presence of abundant prey. Similarly, calanoid density did not differ significantly between the two stations in the 1950s and 1960s, but was significantly lower at Station I than at Station II during the 1980s and 1990s. Such changes are attributed to the gradient of fish predation between the stations and an increasing predation pressure by the fish. The increased fish predation also correlated with a shift in summer-dominant calanoids from larger species to smaller ones. In conclusion, the predaceous cyclopoids are affected by fish predation to a much lesser extent than the herbivorous calanoids, and therefore increased predation by filter-feeding fish results in a definite increase in the cyclopoid/calanoid ratio. Predation by filter-feeding fish has been a driving force in shaping the copepod community structure of Lake Donghu during the past decades.

Applications of factor-criteria system reconstruction analysis in the reproduction research on grass carp, black carp, silver carp and bighead in the Yangtze River

The natural reproduction of grass carp, black carp, silver carp, and bighead will be affected adversely by the Three Gorges Project in the Yangtze River. One of the methods to save the fish is to regulate the water levels, keeping them suited for the species to spawn. Nine factors associated with the scale of larvae-flood of the four species are classified into five levels, and the ranges of these factors producing larvae-floods are given by using the "factor-criteria system reconstruction analysis" method. Moderate beginning water levels and flow, with high daily increases in the rate of water level and flow, and a long duration of water level rising are important for the production of a large larvae-flood.

Differential gene expression of protein kinases in oocytes between natural gynogenetic silver crucian carp and amphimictic crucian carp

A modified mRNA differential display method has been applied to studying differential expression of protein kinase genes in oocytes between natural gynogenetic silver crucian carp and amphimictic crucian carp. Total RNA was reverse transcribed using downstream 3' primers T(12)MA, T(12)MG and T12MC respectively. Then the reverse transcription products were amplified using upstream 5' kinase-specific primer designed according to protein kinase conserved sequence. The PCR products had different patterns and numbers of: cDNA bands on polyacrylamide:gel. Totally 21 cDNAs fragments were recovered and cloned. Two of them were confirmed to be particularly expressed in oocytes of amphimictic crucian carp, and another was specific for gynogenetic silver crucian carp.

Gut contents of silver carp, Hypophthalmichthys molitrix, and the disruption of a centric diatom, Cyclotella, on passage through the esophagus and intestine

The digestibility of algae by stomachless filter-feeding fish has been debated for decades. Results from gut contents and digestive enzyme analysis suggest poor utilization, while the measurement of food assimilation using radiolabeled isotope techniques indicate reasonable assimilation efficiency. Phytoplankton in the gut contents of the planktivorous filter-feeding silver carp were studied during March-May. The fish were cultured in a large net cage in a shallow, hypertrophic subtropical Chinese lake, Lake Donghu. In terms of biomass, the dominant phytoplankton in the fore-gut contents were Cyclotella (average 77.8%, range 69.7-93.5%) and Cryptomonas (average 9.57%, range 0-20.4%). The Ivlev's electivity index E of silver carp was much higher for Cyclotella (1.54) than for Cryptomonas (0.56). The majority of the phytoplankton found in the intestines of silver carp were 8-20 mu m, but they were also able to collect particles as small as 4.5-10 mu m, smaller than their filtering net meshes, suggesting that the secretion of mucus may play an important role in collecting such small particles. We conclude that disruption of cell walls is principally by the pharyngeal teeth, explaining previous contradictory conclusions. (C) 1999 Elsevier Science B.V. All rights reserved.

Influence of dioxin and metal-contaminated sediment on phase I and II biotransformation enzymes in silver crucian carp

Several biochemical responses were measured in silver crucian carp (Carassius auratus gibelio) after exposure to sediments obtained from contaminated Ya-Er Lake, No, 1 pond, and an unpolluted reference site, Honglian Lake. After 1 week of exposure, a significant induction of the phase I biotransformation enzyme (ethoxylresorufin-o-deethylase, EROD) was found (83-fold of control), whereas the phase II biotransformation enzyme (glutathione S-transferase, GST) exhibited a slight, but significant induction (1,4-fold of control) after 4 weeks of exposure. The level of cellular glutathione in the liver was also slightly elevated after 4 weeks of exposure. The delayed response of GST to the contaminants indicates that the phase I and phase II biotransformation enzymes are regulated differently in fish. The results suggest that EROD is a sensitive bioindicator to assess the toxicity of dioxin-contamined sediment in the laboratory, (C) 1998 Academic Press.

Effects of silver carp density on zooplankton and water quality: Implications for eutrophic lakes in China

We examined the responses of zooplankton community, water transparency, chlorophyll a and nutrients to manipulation of density of silver carp (Hypophthyalmichthys molitrix) in an one-way factorial experiment using enclosures placed in Donghu (East Lake, 30 degrees 33' N, 114 degrees 23' E), located in Wuhan, P. R. China. Enclosures (18.75 m(3)) were treated with four silver carp densities, 0, 81, 225, 485 g/m(2). Total zooplankton abundance (excluding nauplii and rotifers except for Asplanchna sp.) and the mean size of dominant cladoceran species were significantly greater in enclosures with 0 and 81 fish densities than those in enclosures with 225 and 485 fish densities. Water transparency also improved significantly when silver carp densities were 0 or 81 g/m(2). We did not find significant effects of silver carp density on chlorophyll a, total phosphorus, or total nitrogen concentrations. We conclude that by reducing planktivorous fish to below the current density (190 g/m(2)), the zooplankton community can be shifted from the dominance of small-bodied Moina sp. to dominance of large-bodied Daphnia sp. Further, the water clarity can be increased.