Microsatellite markers in common carp (Cyprinus carpio L.)

Microsatellite markers of the poly (CA) type in common carp ( Cyprinus carpio L.) are described. Clones containing a (CA) repeat were isolated from a common carp genomic library and sequenced. The number of repeats found was high compared to mammals but comparable with other teleost fishes. Classification of the repeats (perfect, imperfect and compound) are compared with the Atlantic cod ( Gadus morhua L.), rainbow trout ( Oncorhynchus mykiss ), and Atlantic salmon ( Salmo salar L.). A total of 41 primer sets were designed and tested for polymorphism on a test panel of eight animals (derived from outbred lines, inbred lines and gynogenetic clones). Thirty-two markers were found to be polymorphic. The heterozygosity in the outbred animals was 60·4%, 51·1% in the inbred animals and 0% in the gynogenetic clones. The average number of alleles among the eight animals was 4·7 per marker. Six markers (18·8%) gave an additional polymorphic amplification product besides the polymorphic amplification product in the expected size range. The possibility that these loci are tetraploid is discussed. The polymorphic loci described for common carp will be valuable as genetic markers for use in population, breeding, and evolutionary studies.     

Soybean meal induces intestinal inflammation in common carp (Cyprinus carpio L.)

The development of soybean meal (SBM) induced enteritis in the hindgut of the omnivorous common carp (Cyprinus carpio L.). The developed condition was assessed when carp, continuously fed on animal protein, were transferred to a diet in which 20% of the protein was replaced by SBM. After week 1, most of the inflammation parameters were already present, but at week 3, a strong aggravation of the condition was observed which included a shortening of the mucosal folds, the disappearance of the supranuclear vacuoles, an increased number of goblet cells, a thickened lamina propria and sub-epithelial mucosa with increased numbers of basophilic granulocytes as well as a decreased uptake capacity of enterocytes (impaired endocytosis and microvilli). Contrary to previous observations made with respect to Atlantic salmon, common carp start to recover from the fourth to the fifth week after switching to SBM feeding. At this stage, the supranuclear vacuoles refill and most of the parameters revert to basal levels. During the enteritis process, a real-time quantitative PCR analysis was conducted to measure the expression of inflammatory and anti-inflammatory cytokine genes in the isolated intraepithelial lymphocytes (IEL). The pro-inflammatory interleukin 1 beta (IL-1 beta) and tumour necrosis factor alpha1 (TNF-alpha1) genes were up-regulated during the inflammation process while the anti-inflammatory interleukin 10 (IL-10) was down-regulated after an initial up-regulation at week 1. Transforming growth factor beta (TGF-beta) expression showed an up-regulation from week 3 onwards despite the high Ct value and the low primer efficiency shown. This study confirms the contribution of IEL (mainly T-like cells) and basophils in the enteritis process. In addition, the results show a clear involvement of up- and down-regulated cytokine genes in both the onset and recovery of the SBM-induced enteritis in the hindgut of carp.     

Monoclonal antibodies against spermatozoa of the common carp (Cyprinus carpio L.)

Summary Eleven monoclonal antibodies that recognize membrane determinants on spermatozoa of the carp Cyprinus carpio L. have been produced. Indirect immunofluorescence revealed that these determinants are uniformly distributed on the surface of head and midpiece. Most of them are also present on the outer membrane of precursor sperm cells. Although none of the monoclonal antibodies reacted with carp somatic tissue, five monoclonal antibodies were positive for surface membrane determinants of oogonia and early prophase oocytes in carp ovary. Preliminary analysis of the testis and ovary of three other species of fish showed that some carp determinants are shared with germ cells from Barbus conchonius, Clarias lazera, or Salmo gairdneri.Abbreviation WCS Wageningen Carp Sperm antibody     

Response of common carp (Cyprinus carpio L.) leucocytes to hormone-induced stress

The influence of cortisone on leucocytes composition in the blood of common carp (Cyprinus carpio L.) is studied. Following the hormone injection, the relative number of leucocytes decreased and the number of neutrophils and blast-form cells increased in the leucocyte spectrum of experimental fish.     

鲤鱼微卫星突变速率和模式(英文)

利用150个微卫星分子标记在F1代家系的基因型分析过程中,共有27600个等位基因从亲本向子代传递,其中在5个微卫星座位上检测到6个突变的等位基因。对突变的等位基因数目进行统计分析后得出:鲤鱼平均每个世代每个微卫星座位的突变速率为2.53×10-4。在发现突变的5个位点中,经测序发现,突变序列中插入1个以上的重复单元就导致了突变的发生。这些突变表明,鲤鱼的微卫星突变没有遵循严格的渐变突变模型(stepwise mutation model,SMM)。该文关于鲤鱼微卫星突变速率和模式的研究将会对统计鲤鱼有效群体的统计提供有效参数。     

Physiological compartmentation in gonadal tissue of the common carp (Cyprinus carpio L.)

Summary Physiological compartmentation in carp (Cyprinus carpio L.) gonads was investigated after intracardial injection of horseradish peroxidase (HRP) and two mouse anti-carp-sperm monoclonal antibodies.Immunohistochemistry revealed that a physiological barrier exists in carp testis for HRP and mouse IgG monoclonal antibody around the central lumina of the tubules in which the spermatozoa are located, but not around the cysts containing the precursor germ cells. The results with HRP were confirmed by electron microscopy. Mouse IgM monoclonal antibody did not penetrate the spermatogenic cysts. Probably because of its large size, it was almost exclusively located inside blood capillaries and only sparsely in the interstitial tissue.In the ovary, HRP was regularly distributed in the gonadal tissue, whereas the IgG antibody was predominantly localised on oogonia and early prophase oocytes. The results indicate that in contrast with the testis, no barrier around germ cells exists in the carp ovary.     

Corticosteroids affect the testicular androgen production in male common carp (Cyprinus carpio L.).

Our previous experiments to study the effect of stress adaptation on pubertal development in carp showed that repeated temperature stress and prolonged feeding with cortisol-containing food pellets, which mimics the endocrine stress effects, retarded the first waves of spermatogenesis and decreased 11-ketotestosterone (11KT) plasma levels. The objective of the present study was to investigate whether the decrease in plasma 11KT is caused by a direct effect of cortisol on the steroid-producing capacity of the testis or by an indirect effect, such as a decrease in plasma LH. Pubertal and adolescent isogenic male common carp (Cyprinus carpio L.) were fed with either cortisol-containing food pellets or control food pellets over a prolonged period. Our results indicate that cortisol has a direct inhibitory effect on the testicular androgen secretion independent of the LH secretion. Furthermore, the pubertal period is critical to the influence of cortisol regarding testicular androgen secretion, because the effect is no longer observed at adolescence.     

Differential macrophage polarisation during parasitic infections in common carp (Cyprinus carpio L.)

In many parasitic infections both classically activated macrophages (caMF) and alternatively activated macrophages (aaMF) play a pivotal role. To investigate if both types of macrophages also play an important role during parasitic infections in fish, we infected carp with either Trypanoplasma borreli or Trypanosoma carassii and determined the activation state of the head kidney leukocytes (HKL). Nitrite production was used as read-out for caMF and arginase activity as read-out for aaMF. Basal nitrite production and arginase activity of HKL were moderately different between the two infections. Differences were observed, however, after ex vivo re-stimulation of HKL. Re-stimulation with LPS and T. borreli lysates increased nitrite production by HKL of T. borreli-infected fish. Re-stimulation with cAMP increased arginase activity in HKL of T. carassii-infected fish. Our results indicate that T. borreli-infected carp are more prone to increase nitrite production by caMF while T. carassii-infected fish are more prone to increase arginase activity by aaMF.     

Polymorphism of the cytochrome oxidase b gene (cyt b) in Russian populations of common carp and wild common carp (Cyprinus carpio L.)

Polymorphism of the mitochondrial cyt b gene was examined in 35 individuals of common carp and wild common carp (Cyprinus carpio L.). The fish examined represented two natural populations from Khabarovsk krai (Ac and Am), Volga wild common carp, Don wild common carp, and two common carp breeds, Ropsha (strains BB and MM) common carp and Hungarian common carp. The highest level of nucleotide (pi) and haplotype (h) diversity was detected in two strains of Ropsha common carp (MM, pi = 0.67%, h = 0.7; and BB, pi = 0.21%, h = 0.9) and in one population (Am) of Amur wild common carp (pi = 0.26%; h = 0.6). The second population of Amur wild common carp (Ac) and Hungarian common carp were characterized by lower variation estimates (pi = 0.035%, h = 0.4; and pi = 0.09%, h = 0.7, respectively). Genetic homogeneity was demonstrated for the populations of Volga and Don wild common carp (pi = 0, h = 0). In the sample of the cyt b sequences examined, three lineages were identified. Lineages I and II united all haplotypes of the Am Amur wild common carp along with two haplotypes of Ropsha common carp, strain MM. The third lineage (III) was formed by the haplotypes of three individuals of Ropsha common carp strain MM, all representatives of Ropsha common carp strain BB, Hungarian common carp, Ac Amur wild common carp, and Don and Volga wild common carps. Statistically significant amino acid differences were observed only for the sequences, corresponding to haplotypes of lineage III, and the sum of sequences of lineages I and II. Effectiveness of different types of markers to differentiate the two subspecies of European and Amur wild common carp (C. c. carpio and C. c. haematopterus) is discussed, as well as the issues of the origin and dispersal of Russian common carp and wild common carp breeds.     

Genetic control of glucosephosphate isomerase (GPI) in common carp (Cyprinus carpio L.)

In common carp, a freshwater fish species of tetraploid origin, GPI enzymes are present in two variants: GPI-A and GPI-B. GPI-A is coded by two loci segregating for two (GPI-A 1*) and six (GPI-A2*) alleles. Experimental crosses of the ornamental (Koi) variety of common carp revealed that GPI-B is coded by only one locus (GPI-B*). Another GPI-B* locus must have been silenced in the process of functional diploidization. It was also shown that the GPI-A2* locus segregated independently from the GPI-B* locus, demonstrating that the loci are located on different chromosomes.     

Divergent selection for antibody production in common carp (Cyprinus carpio L.) using gynogenesis

A base population (n = 101) of carp, consisting of a single hybrid cross, was immunized with the hapten-carrier complex DNP-KLH. to perform a divergent selection for antibody response. Measurement of the DNP-specific antibody response at 12 and 21 days postimmunization, allowed the classification of a low number of individual carp as early/high (10%) or late/low (13%) responders. Three individuals defined as early/high and three defined as late/low responding, were gynogenetically reproduced to obtain corresponding homozygous progenies within one generation only. Upon immunization with DNP-KLH, the antibody response was found to be significantly higher in the early/high responder homozygous offspring. Although the homozygosity of the offspring apparently caused a (s)lower antibody response (compared with the base population), the differences between the high and low responder offspring do indicate a genetic influence on the antibody response. The realized heritability (h2) for antibody production was estimated at 0.37 ± 0.36. The present study provides the basis for a divergent selection of homozygous inbred carp lines with a genetically controlled difference in antibody response. These inbred lines will allow us to investigate relationship(s) between immune responsiveness and resistance to infectious diseases in fish.     

Structure,organization and expression of common carp (Cyprinus carpio L.) NKEF-B gene

Natural killer (NK) cell enhancing factor (NKEF) belongs to the newly defined peroxiredoxin (Prx) family. Its functions are to enhance NK cell cytotoxicity and to protect DNA and proteins from oxidative damage. In this study, a partial cDNA sequence of carp NKEF-B was isolated from thymus cDNA library. Subsequently, the full-length cDNA of carp NKEF-B was obtained by means of 3′ and 5′ RACE, respectively. The full-length cDNA of carp NKEF-B was 1022 bp, consisting of a 73 bp 5′-terminal untranslated region (UTR), a 355 bp 3′-terminal UTR, and a 594 bp open reading frame coding for a protein of 197 amino acids. Carp NKEF-B contained two consensus Val-Cys-Pro (VCP) motifs and three consensus cysteine (Cys-51, Cys-70 and Cys-172) residues. Sequence comparison showed that the deduced amino acid sequence of carp NKEF-B had an overall similarity of 74–96% to that of other species homologues. Phylogenetic analysis revealed that carp NKEF-B forms a cluster with other known teleost NKEF-Bs. Then, by PCR we obtained a 5.1-k long genomic DNA of carp NKEF-B containing six exons and five introns. Real-time RT-PCR results showed that carp NKEF-B gene was predominantly detected in kidney and head kidney under un-infected conditions. Whereas under SVCV-infection condition, the expression of NKEF-B gene was significantly increased in blood cells, gill, intestine and spleen, but maintained in liver, and decreased significantly in kidney and head kidney. Finally, the rNKEF-B was constructed and expressed in Escherichia coli. By using an antibody against carp rNKEF-B, immunohistochemical study further indicated that NKEF-B positive cells are mainly some RBCs and a few epithelial cells in gill and intestine, and that under SVCV-infection condition, these positive cells or positive products in their cytoplasm were mainly increased in gill and spleen sections of carp. The results obtained in the present study will help to understand the function of NKEF-B in teleost innate immunity.     

Characterization of transgene integration pattern in F4 hGH-transgenic common carp （Cyprinus carpio L.）

The integration pattern and adjacent host sequences of the inserted pMThGH-transgene in the F4 hGH-transgenic common carp were extensively studied. Here we show that each F4 transgenic fish contained about 200 copies of the pMThGH-transgene and the transgenes were integrated into the host genome generally with concatemers in a head-totail arrangement at 4-5 insertion sites. By using a method of plasmid rescue, four hundred copies of transgenes from two individuals of F4 transgenic fish, A and B, were recovered and clarified into 6 classes. All classes of recovered transgenes contained either complete or partial pMThGH sequences. The class I, which comprised 83% and 84.5% respectively of the recovered transgene copies from fish A and B, had maintained the original configuration, indicating that most transgenes were faithfully inherited during the four generations of reproduction. The other five classes were different from the original configuration in both molecular weight and restriction map, indicating that a few transgenes had undergone mutation, rearrangement or deletion during integration and germline transmission. In the five types of aberrant transgenes, three flanking sequences of the host genome were analyzed. These sequences were common carp β-actin gene, common carp DNA sequences homologous to mouse phosphoglycerate kinase-1 and human epidermal keratin 14, respectively.     

Seasonal short-term effects of naltrexone on LH secretion in male carp (Cyprinus carpio L.)

In order to evaluate the influence of the season (the stage of gonad maturity) on the modulatory role of endogenous opioid peptides in LH secretion in fish, sexually mature male carp (Cyprinus carpio L.) were intravenously injected with naltrexone-opioid receptor antagonist (5 or 50 microg kg(-1)) in the period of natural spawning (June) or gonad recrudescence (December). Moreover, the possible involvement of the dopaminergic system was studied in fish pre-treated with pimozide (dopamine receptor antagonist) and in intact fish. Blood samples were taken every minute, up to 10 min after naltrexone injection. In June, naltrexone significantly lowered LH levels in comparison to saline injected males. In December, there were no differences between saline and naltrexone-injected carps. In fish pre-treated with pimozide, neither in June nor in December were any significant differences in LH levels between control group and the groups injected with naltrexone found. The results showed that, in male carp, LH secretion under the influence of naltrexone depends on the stage of gonad maturity what suggests that the feedback of gonadal steroids on LH release could be mediated by the endogenous opioids. The role of dopamine in these processes is also discussed.     

Quantitative genetic estimates of growth-related traits in the common carp (Cyprinus carpio L.): A review

The common carp (Cyrpinus carpio L.) is the oldest cultured and the most domesticated fish species, as well as one of the most important freshwater fishes in the world. However, scientific studies on evaluating the growth-related quantitative traits in this fish are limited. Heritability, the most important parameter in selective breeding programs, was extensively studied for the growth-related traits. The values varied widely among the experiments and methods used because of the existence of common environmental, dominance and maternal effects. However, correlations in phenotypic and genetic levels first evaluated several years ago were limited. On the other hand, heterosis was widely reported and easily obtained for growth-related traits in the common carp. Meanwhile, genotype environment interaction and predic-tion of breeding values have been studied recently, and are very important in conducting selective breeding programs. The developmental quantitative genetics of growth-related traits was first analyzed in the common carp for reasonable selection during ontogeny. It is expected that genetic improvement will be achieved by carrying out direct selective breeding in the common carp.     

鲤鱼体长性状的QTL定位及其遗传效应分析

以大头鲤/荷包红鲤抗寒品系的重组自交系群体及其遗传连锁图谱, 利用Windows Map Manager 2.0的标记回归法进行QTL单标记定位分析和复合区间作图法进行QTL区间检测, 通过置换实验(1 000次重复)确定连锁群显著性水平阈值。在体长性状的标记回归研究中, 共7个标记达到显著水平(P<0.01), 对性状的贡献率为14.00%～27.00%, 其中3个标记达到极显著水平(P<0.001)。HLJ534, HLJ319, HLJ370座位可能与影响鲤鱼体长性状的主效基因连锁。在体长性状的QTL区间定位研究中, 共6个QTL达到连锁群显著水平(P=0.05), 对性状的贡献率为11.33.%～23.12%, 其中2个达到连锁群极显著性水平(P=0.01), 它们的加性效应方向并不一致。HLJ190-HLJ497区间和HLJ479-HLJ483区间是影响鲤鱼体长性状的主效QTL区间。     

Gene transfer in common carp (Cyprinus carpio L.) by microinjection into the germinal disc

We report here on the microinjection of the human growth hormone gene (hGH) into the germinal disc of common carp one-cell embryos. For microinjection, a linear 4.1 kb EcoRi-DNA fragment containing the mouse metallothionein-I promoter (mMT-I), fused to a structural gene coding for the hGH, was used. Of the 14 injected embryos, 9 developed to an age of 60 d. Of these nine fish, two were found to be transgenic and had integrated two to four copies per cell of the hGH construct.     

Application of PCR-RF-SSCP to study major histocompatibility class II B polymorphism in common carp (Cyprinus carpio L.)

A variety of methods have been applied for the characterization of major histocompatibility (MH) polymorphism in fish. We optimized a technique designated polymerase chain reaction-restriction fragments-single strand conformation polymorphism (PCR-RF-SSCP) for screening a large number of individuals for the Cyca-DAB1 and Cyca-DAB2 genes polymorphism in common carp. The advantages of this technique are simplicity, high sensitivity and low costs. PCR-RF-SSCP analysis revealed different genotypes consisting of unique combinations of the Cyca-DAB1 and Cyca-DAB2 sequences with the number of SSCP bands clearly correlating with the degree of heterozygosity of the Cyca-DAB1 and Cyca-DAB2 genes. We found four alleles for Cyca-DAB1 (*02-*05) gene but only one allele for Cyca-DAB2 (*02) and noted that the Cyca-DAB2 gene was either homozygous or absent. PCR-RF-SSCP analysis of n=79 carp individuals challenged with Aeromonas hydrophila indicated that individuals bearing no Cyca-DAB2 gene showed higher cumulative mortality and lower bacterial agglutination titers during the experiment. We suggest that our PCR-RF-SSCP method can be used to study correlations of different MH class II B genotypes/alleles with resistance of common carp to specific pathogens.