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.     

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。     

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.     

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.     

Genetic differences in natural antibody levels in common carp (Cyprinus carpio L.)

In mammals, natural antibodies (Nabs) are mostly of the IgM isotype and can bind to a particular antigen or pathogen even if the host has never been exposed. Despite their early detection and abundance, the exact role and genetic control of Nabs remain unclear. We have used an indirect ELISA with three different antigens (keyhole limpet haemocyanin, chicken ovalbumin and bovine serum albumin) to demonstrate the ubiquitous presence of Nabs in common carp. Serum levels of Nabs increased with age, i.e. 10-month-old fish showed higher levels than 4-month-old fish. Also, fish grown in earth ponds showed higher levels of Nabs than fish grown in a clean environment of UV-treated water. Furthermore, we show that Nabs are present in different levels in the serum of carp lines with a different genetic background, suggestive of a genetic control. These genetic differences were independent of antigen, age and environment. Genetic differences in levels of Nabs could not unequivocally be related to differences in survival under farmed conditions. The possibilities for using levels of Nabs as marker criterion for selection for genetic disease resistance are discussed.     

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.     

Studies on the effect of paraquat on glycogen mobilization in liver of common carp (Cyprinus carpio L.)

1. A herbicide, paraquat (1,1'dimethyl-4,4'-bipyridilium-dichloride) was administered to carp in 0.5-10.0 ppm concentrations, respectively, and blood sugar level, glucose-6-phosphatase and glycogen phosphorylase activities of liver were determined. 2. Paraquat treatment caused an increase of blood sugar level and enhanced phosphorylase and glucose-6-phosphatase activities. 3. Paraquat can induce alterations in endoplasmic reticulum that might contribute to the changes in glucose-6-phosphatase activity, resulting in an increase of blood glucose level and/or all the effects can be attributed to a high level of circulating epinephrine produced by paraquat toxicosis.     

The effect of oral immuno-stimulation in juvenile carp (Cyprinus carpio L.)

The effect of a 2-week period of oral immuno-stimulation from the age of 2 or 6 weeks post-fertilisation (wpf; before and after reaching the ability to produce antibodies) onwards was investigated on various immune functions of the common carp, Cyprinus carpio. The immuno-stimulants Aeromonas salmonicida lipopolysaccharide, Yeast DNA (containing unmethylated CpG motifs) or high-M alginate (an extract of algae containing poly-mannuronic acid) were used. The effect of this treatment was studied on the kinetics of B cells in head kidney and peripheral blood leucocytes using flow cytometry, on the total plasma IgM level using ELISA, on cytokine and inducible nitric oxide synthase (iNOS) expression in the intestine, and acute phase protein expression in the liver, using real time quantitative PCR, and on exposure to Vibrio anguillarum. Oral administration of immuno-stimulants from 6 wpf resulted in decreased WCI12(+) (B) cell percentages in PBL (only after administration of LPS) and head kidney (all test groups), and a decreased total IgM level in plasma, suggesting that suppressive effects are strongly indicative of oral or juvenile tolerance. After administration from 2 wpf, the effects on WCI12(+) (B) cell percentages were less pronounced: the group fed with Yeast DNA showed higher percentages compared to the control group at 6 wpf, but lower percentages at 8 wpf. No changes were observed in the cytokine or iNOS expression levels in the intestine or acute phase protein expression in the liver. A challenge with V. anguillarum resulted in an initially higher cumulative mortality in the group fed with LPS, but lower mortality in the groups fed with Yeast DNA or high-M alginate compared to the control group, providing a provisional warning especially for the use of pathogen-derived immuno-stimulants, such as A. salmonicida LPS, in larval and juvenile fish.     

The effect of various ambient ammonia concentrations on the nitrogen metabolism of carp fry (Cyprinus carpio L.)

Authors studied how 125, 375 and 625 micrograms/l amounts of NH4Cl added to the water influenced the ammonia excretion of carp fry with an average weight of 2.4 g, compared to the control. During the course of the experiments the NH4N concentration, the pH and the temperature were measured for three days, twice daily. On the basis of our results the threshold concentration exerting harmful effects on the ammonia household of carp fry (disturbing the normal rate of metabolism in the fishes and decreasing their growth rate) is between 125 and 375 micrograms/l. NH4H (50-100 micrograms/l NH3). The ammonia concentrations exceeding 375 micrograms/l NH4+ (and 100 micrograms/l NH3, respectively) can be regarded as undesirable and harmful in frybreeding fish ponds. With regard to the ambient ammonia, a daily cycle developed in the excretion of ammonia: contrary to the control, a minimum in ammonia excretion was measured in the morning, while a maximum was measured in the afternoon.     

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.     

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.     

Salt stress and resistance to hypoxic challenges in the common carp (Cyprinus carpio L.)

Long term exposure to brackish water (171 mm NaCl) affected the capacity of common carp Cyprinus carpio to deal with hypoxic conditions and the critical oxygen concentrations for oxygen consumption increased. In addition, regulation of ammonia excretion was lost. The cytosolic phosphorylation potential (the index of the energy status of a cell in terms of potential transferable phosphate groups) in the lateral muscle on the other hand remained relatively unaffected, indicating that oxygen transport to the tissues was not severely compromised. It appears that exposure to brackish water reduces the capacity of common carp to cope with hypoxic conditions mainly because of the high energetic cost of hyperventilation under conditions where energy stores are depleted, and not because of any impeded oxygen transport mechanisms.     

Phenotypic characteristics and pathogenicity of Aeromonas genomospecies isolated from common carp (Cyprinus carpio L.)

AIMS: To evaluate the relationship between the genomospecies, phenotypic profile and pathogenicity for carp of 37 motile Aeromonas strains. METHODS AND RESULTS: Aeromonas strains were identified to genomospecies level by the 16S rDNA restriction fragment length polymorphism (RFLP) method and characterized phenotypically by the API 20E and API Zym systems and by conventional tube or plate methods. 16S rDNA RFLP analysis showed that the strains belonged to five species, Aeromonas bestiarum (5), Aerom. salmonicida (13), Aerom. veronii (11), Aerom. sobria (6) and Aerom. encheleia (2). Most strains of Aerom. bestiarum (80%) and Aerom. salmonicida (85%) could be separated by growth at 4 and 42 degrees C, autoagglutination after boiling, reaction for lipase (C14) and naphthol-AS-BI-phosphohydrolase. All strains of Aerom. veronii corresponded to Aerom. veronii biotype sobria and could be separated from Aerom. sobria by citrate utilization, growth at 37 and 42 degrees C, amygdalin and cellobiose fermentation. All strains of Aerom. bestiarum and most strains of Aerom. salmonicida (76.9%) and Aerom. veronii (63.6%) were pathogenic for carp. CONCLUSIONS: The biochemical identification of carp Aeromonas strains is not entirely clear. Some association between Aeromonas species, phenotypic profile and specific disease signs was observed. SIGNIFICANCE AND IMPACT OF THE STUDY: The results will be useful for ichthyopathology laboratories in the diagnosis of motile aeromonad septicaemia in carp.     

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.