Absorption of protein macromolecules by the enterocytes of the carp (Cyprinus carpio L.)

Summary In the adult carp, the ultrastructure of the enterocytes of the distal segment of the medium intestine is similar to that of the intestinal cells of certain mammals during the neonatal period. Frequent aspects of pinocytosis are visible at the base of a plateau of already developed microvillosities. The apical cytoplasm shows the presence of a dense tubulo-vesicular network. The vacuoles which separate off run together to form a voluminous supranuclear body. Their P.A.S. positive and orthochromatic contents are rich in alkaline phosphatases. The permeability of this epithelium to macromolecules is demonstrated by the administration of “Horseradish Peroxidase” (HRP). This protein penetrates by pinocytosis into the apical tubulo-vesicular system and reaches the blood circulation via the extra-cellular spaces. The presence of certain structures involved in protein transport following a period of experimental fasting of six months reflects their independence with regard to exogenous supplies. This study has been carried out thanks to a grant by the C.N.R.S. (R.C.P. No 250), and with the electron microscope of the department of plant Biology (Professor Leredde) kindly placed at our disposal by Mr. B. Lugardon. We are also grateful to Mrs A. Rup for her technical assistance.     

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.     

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.     

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     

鲤鱼肌肉生长抑制素基因(MSTN)的克隆及其组织表达特征

肌肉生长抑制素(Myostatin,MSTN)是动物肌肉发育和生长过程中的负调控因子,对MSTN的研究将有助于促进动物生产。鲤鱼是我国的主要淡水养殖对象之一。因此,我们采用RT-PCR方法克隆了鲤鱼MSTN cDNA(No.EF551058)的部分序列,长度为921bp,编码306个氨基酸残基。鲤鱼MSTN具有MSTN的共同特征,有蛋白酶水解位点RIRR和9个保守的半胱氨酸残基。多重序列比较发现其与斑马鱼GDF8有极近的亲缘关系,96.7%的氨基酸序列同源。不同组织的RT-PCR分析发现鲤鱼MSTN主要在肌肉和脑部表达,而其他所检测组织未见表达。鲤鱼MSTN不仅在肌肉生长发育中发挥作用,可能在神经系统发育中也有其作用。     

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.     

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。     

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.     

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.     

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.     

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.     

An in vitro study of long-term potentiation in the carp (Cyprinus carpio L.) olfactory bulb

Long-term potentiation (LTP) of synaptic transmission is considered a cellular mechanism for neural plasticity and memory formation. Previously, we showed that in the carp olfactory bulb, LTP occurs at the dendrodendritic mitral-to-granule cell synapse following tetanic electrical stimulation applied to the olfactory tract, and suggested that it is involved in the process of olfactory memory formation. As a first step towards understanding mechanisms underlying plasticity at this synapse, we examined the effects of various drugs (glutamate and GABA receptor agonists and antagonists, noradrenaline, and drugs affecting cAMP signaling) on dendrodendritic mitral-to-granule cell synaptic transmission in an in vitro preparation. Two forms of LTP are involved: a postsynaptic form (tetanus-evoked LTP) and a presynaptic form. The postsynaptic form is evoked at the granule cell dendrite following tetanic olfactory tract stimulation and is suppressed by the NMDA receptor antagonist, D-AP5, enhanced by noradrenaline, and occluded by the metabotropic glutamate receptor agonist, trans-ACPD. The presynaptic form occurs at the mitral cell dendrite following blockade of the GABA_A receptor by picrotoxin and bicuculline, or via activation of cAMP signaling by forskolin and 8-Br-cAMP.     

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.     

Use of common carp (Cyprinus carpio L.) in increasing the fertilizer value of phospate rock

Thirty-two glass jars (3 L each) in the laboratory and outdoor tanks (300 L) were used to examine the influence of common carp ( Cyprinus carpio L. ) in increasing the fertilizer value of phoshate rock in eight treatment combinations in quadruplicate. Input of water soluble hosphate was determined to quantify the effects of bioturbation, fish excrements and soil. The level of orthophosphate in water was always lowest in the control series. Introduction of common carp fry resulted in a net increase of 0.1 to 0.114 mg. L^-1 of orthophosphate attributable to the effect of fish excrement. Bioturbation caused by common carp resulted in as high as a 72 to 100 % influx of phosphate from bottom soil in the presence of phosphate rock but only about 7 to 8 % in the absence of phosphate rock.

Zusammenfassung

Die Bedeutung eingesetzter Karpfen (Cyprinus carpio L.) zur Steigerung des Düngerwertes von Phosphatgestein
32 Glasgefäße (3 L Volumen) wurden im Labor und 4 Behälter (300 L) im Freien aufgestellt urn den Einfluß zunehmender Düngung durch Phosphatgestein auf Karpfen ( Cyprinus carpio L. ) in 8 unterschiedlichen Applikationen mit jeweils 4 Replikaten zu testen. Der Eintrag wasserloslichen Phosphats wurde bestimmt, urn die Wirkung von Bioturbation, Fischexkrementen und Bodensubstrat zu prüfen. Die Konzentration an Orthophosphat war in den Kontrollen am niedrigsten. Das Einsetzen von Karpfen-Jungfischen erhöhte die Nertozunahme des Orrhophosphats auf 0,1 bis 0,114 mg. ^-1 und diese Zunahme muß den Ausscheidungen der Fische zugeschrieben werden. Die durch die Karpfen verursachte Bioturbation resultiene in einer 72 bis 100% igen Zunahme des Influx des Phosphats aus dem Bodensubstrat wenn Phosphatgestein dem Boden zugegeben wurde, wahrend dieser Wert nur 7–8%) ohne Phosphatgestein betrug.     

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.     

Characterisation of cDNAs of key genes involved in apoptosis in common carp (Cyprinus carpio L.)

Apoptosis is a morphologically and biochemically distinct form of eukaryotic cell death that occurs under a variety of physiological and pathological conditions. Many of the cell deaths that occur during normal embryological development and during normal tissue turnover display the morphological hallmarks of apoptosis. In the last 20 or so years a better biochemical picture of how the process occurs has been produced, at least in higher vertebrates. It is now widely accepted that many of the proteolytic cleavages occurring during apoptosis are mediated by caspases, the activation of which, in turn, has been found to be tightly regulated. The current study focuses on the sequencing and analysis of key genes involved in the apoptotic process, based on sequence similarity to known apoptosis genes from genetic models such as zebrafish (Danio rerio) and other vertebrates. The present study identifies key components of the apoptotic process in common carp (Cyprinus carpio L.), which in turn can be used, for example, to monitor the fate of the cellular components of the immune system after an immune challenge.