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Insulin of carp (Cyprinus carpio) was isolated and crystallized. The insulin was biologically active in two tests; it decreased the blood glucose level and stimulated 14CO2-formation from glucose. The chemical properties are similar to those of insulins from other species. The insulins of carp and of mammals differ greatly immunologically. Antibodies against carp insulin crossreact with carp proinsulin.
In carp Cyprinus carpio L. preimmunized with a protein antigen, human gamma globulin (HGG), rapid localization of antigen occurred in the splenic ellipsoids within 1 day of a secondary immunization given 8 weeks later. Furthermore, after injection of immune complexes or heat-aggregated HGG, antigen was trapped more quickly in the pronephros than when antigen was injected alone. These results support the conclusion that in fish, as in homoiotherms, this form of antigen trapping is antibody-dependent.
Transgenic common carp, Cyprinus carpio, possessing the long terminal repeat (LTR) sequence of avian Rous sarcoma virus (RSV) fused to the rainbow trout (rt) growth hormone (GH1) complementary DNA (cDNA) were produced by microinjection. Initial studies showed that the transgenic common carp transmitted the foreign DNA to a significant fraction of their progeny in three of four crosses of transgenic males with control females. These progeny grew 20 to 40% faster than their nontransgenic full siblings. In this study, additional experiments were conducted to evaluate inheritance and expression of the foreign GH gene in transgenic common carp, and the growth performance of these transgenic fish. Four P1 (parental generation produced by microinjection) x nontransgenic controls, four P1 x P1, and one P1 x F1 matings of transgenic carp containing RSVLTR-rtGH1 cDNA were made. The percentages of transgenic progeny resulting from these matings were: 0, 32, 42, 100 (4 progeny only), 21, 21, 31, 30, and 23%, respectively. All crosses except 1 siblot (control x P1) exhibited progeny ratios below the expected 50 or 75% transgenic. These results indicate that most of these transgenic P1 had the foreign gene in their germ line but were mosaics, and at least one transgenic individual did not have the RSVLTR-rtGH1 cDNA in the gonadal tissue. Both P1 and F1 transgenic fish produce trout growth hormone mRNA and polypeptide as determined by reverse transcription polymerase chain reaction amplification, RNA dot-blot hybridization, and radio-immunobinding assay. Growth response by families of F1 transgenic fish to the addition of rtGH1 cDNA varied widely.(ABSTRACT TRUNCATED AT 250 WORDS)
As a part of observation on the suitability as a second intermediate host of Clonorchis sinensis, various infection experiments were tried to Cyprinus carpio nudus. The results obtained were summarized as follows: 1. The cercariae which attempted to contact with Cyprinus carpio nudus in the water were observed under the stereomicroscope. After contact, cercariae began to separate its tails from the bodies at 4 minutes increasingly, and then the number ranged to 80 percent at 13 minutes after the encounter. But very few cercariae could actually invade into the epidermis of the fish. 2. When Cyprinus carpio nudus were exposed to a number of cercariae in the beaker, only a few cercariae could invade through the epidermis. Most of the invaded cercariae were killed before forming the cyst. Also rare encysted cercariae were found to be dead within 48 hours. 3. Cyprinus carpio nudus were reared with Parafossarulus manchouricus shedding numerous cercariae in the indoor aquarium. When observed after 12 days, to determine the susceptibility, no metacercaria was found infected. 4. Cyprinus carpio nudus were reared with Parafossarulus manchouricus shedding numerous cercariae in Kimhae lake for 2 months in summer of 1982, in order to check the natural infection. No metacercaria of Clonorchis sinensis was found from the fish. 5. In the cases of control fish, Pseudorasbora parva, numerous cercariae were inspected on the course of invasion through epidermis of the fish under the stereomicroscope. And many metacercariae were also found from the fish whenever they were kept in beaker, indoor aquarium, or the lake for natural infection. The results suggested that Cyprinus carpio nudus might not be proper intermediate host of Clonorchis sinensis.
Cyprinus carpio fish (carp), exposed to elemental or metallic mercury (Hg0) at concentrations of 2.0, 20.0, and 200.0 mg per liter of water, were kept in concrete tanks for 159 days. Ten fish were used for each concentration level. Thirteen samples of peripheral blood were collected from each animal through gill puncture, 12 during the first 90 days of the experiment, and the last one at the end of the experiment. The micronucleus test (MNT) was designed to study dose and time yield effects of mercury after indirect exposure in vivo. The results indicated that for a concentration of 2.0 mg Hg0/l, there was no significant increase in frequency of micronuclei (MN), but at higher concentrations (20.0 and 200.0 mg Hg0/l) there was a significant increase in MN frequencies. This effect was higher after 31 days of exposure, followed by slight stabilization and gradual decrease.
The skin of carp, Cyprinus carpio, was studied at the ultrastructural level after exposure of the fish to low and high concentrations of cadmium in the water (22 and 560 micrograms/L, respectively) for different periods. The effects of the low concentration of cadmium were similar to those of the high concentration, although they appeared later. The basal lamina and the skin surface became highly undulating. Chloride cells appeared between the pavement cells. Necrotic pavement cells were seen from the first day on, while apoptotic pavement cells appeared after several days. Filament cells contained many electron-transparent and electron-dense secretory vesicles. Mitotic cells were commonly seen, mainly in cells adjacent to club cells or close to the epidermal surface. Mucous cells differentiated close to the skin surface. They became elongated and synthesized highly electron-dense mucosomes. The epidermis became infiltrated by many leucocytes. As the experiment progressed, many leucocytes degenerated, and their remnants were found within macrophages and club cells. Fibroblasts displayed intense synthesis and, in fish from the low cadmium concentration, deposited a dense network of collagen fibers in the dermis. Melanosomes were located in the extensions of melanocytes. In these cells aggregation of melanosomes and apoptotic processes were common. Several of these changes were observed earlier under the impact of stressors other than cadmium. Some changes, such as the appearance of tumorlike bodies at the skin surface, the appearance of Merkel cells throughout the epidermis, and the coupling of leucocytes, may be specific for cadmium.
Methallibure treatment of mature Cyprinus carpio causes a marked reduction in the steroidogenesis, as indicated by the fall in 3beta-HSD activity in the gonads. Significance of these results is discussed.
Carp Cyprinus carpio macrophages were depleted by intraperitoneal (i.p.) injection of clodronate-liposomes for the in vivo study of the effect of macrophage depletion on the resistance of carp to infection with blood flagellate parasites. Clodronate released inside the cell induces apoptosis of (murine) macrophages. Following i.p. injection of carp with liposomes alone, but not with Trypanoplasma borreli, neutrophilic granulocytes rapidly migrated from the head kidney to the peritoneal cavity. The majority of liposomes in the peritoneal cavity were not taken up by newly arrived neutrophilic granulocytes, however, but by resident macrophages. After 2 i.p. injections of clodronate-liposomes, the percentage of macrophages present in the peritoneal cavity was significantly reduced, as evaluated by flow cytometry. Macrophage-depleted carp that were infected i.p. with T. borreli suffered from high mortality. However, these fish did not show lethal parasitaemia but did show clear bacteraemia. Macrophage-depleted carp that were infected i.p. with Trypanosoma carassii showed a minor increase in parasitaemia. In addition, macrophage-depleted carp, immune to T. borreli as a result of having survived a prior infection, remained immune to i.p. reinfection with T. borreli. Succesful depletion of peritoneal macrophages seemed to have a minor effect on the resistance of carp against blood flagellates. However, carp macrophages are essential as a first line of defence against (bacterial) infection.
Gonadal function in fish, Cyprinus carpio was significantly affected by sublethal doses of mercuric chloride (HgCl2) and cadmium chloride (CdCl2) in chronic (45 days) exposure. Parameters investigated were nonesterified (NE) and esterified (E) cholesterol of ovary, liver and serum and ovarian 3 beta-Hydroxysteroid and 17 beta-Hydroxysteroid dehydrogenase enzyme activity and serum and pituitary gonadotropin (GtH) levels. Both the pollutants were able to reduce the hypothalamic extract (HE) or gonadotropin releasing hormone (GnRH) induced pituitary GtH release in vitro. Short term (96h) exposure of the fish to the pollutants had no significant effect on the gonadal function. In addition to the deleterious effect of pollutants on the gonadal steroidogenesis and pituitary gonadotropin release, using [4-14C] cholesterol as a tracer it was found that for 45 days exposure, HgCl2 had an adverse effect on the transport of cholesterol from circulation to ovary.
The effects of pesticides on blood characteristics and histological changes in erythrocytes of the fish species Cyprinus carpio and Puntius ticto were studied. The fishes were exposed to sub lethal concentrations of different chlorinated pesticides namely aldrin, dieldrin, DDT, BHC and chlordane for 10, 20 and 30 d in continuous flow-through test. The LC50 values were calculated based on acute toxicity tests and the sublethal doses were arrived at for chronic bioassay studies. Results showed an increase in haemoglobin content of both Cyprinus carpio and Puntius ticto in case of aldrin and dieldrin. Haemoglobin content reduced from an initial 13 g/100 ml to 8.07 and 10.15 g/100 ml in case of Cyprinus at the end of ten days exposure to aldrin and dieldrin respectively, and gradually increased to 8.7 g/100 ml and 10.15 g/100 ml after 20 d of exposure. The haemoglobin content after 30 d exposure to aldrin and dieldrin was 10.15 g/100 ml and 11.6 g/100 ml respectively. In case of Puntius ticto, the haemoglobin content in control fishes recorded was 12.8 g/100 ml while in case of fish exposed to aldrin, the haemoglobin content reduced initially on ten days exposure to 10.15 g/100 ml and increased to 11.6 g/100 ml and 13.0 g/100 ml during twenty days and thirty days exposure respectively. This trend was also observed with dieldrin in both the fishes studied. Red blood cells were also counted in case of all the pesticides and exposure periods with respect to Cyprinus carpio and Puntius ticto. Irrespective of the species and pesticide, the RBC counts uniformly showed decreasing trend with the increase in exposure period, while packed cell volume, PCV(%) showed increasing trend with respect to increase in exposure period in case of aldrin and dieldrin in both the fishes. But DDT, BHC and chlordane showed decreasing trend in PCV(%) values with increasing periods of exposure.
The effects of zinc and copper salts on the survival of the two species of freshwater fish, common carp, Cyprinus carpio, and grass carp, Ctenopharyngodon idellus, were investigated. It was discovered that the levels of tolerance to the concentration of the metals were species-specific. Cyprinus carpio was found to be more susceptible to copper, whereas Ctenopharyngodon idellus was more susceptible to zinc. In general, copper was more toxic than zinc, as revealed by the survival times. The body and the gills of dead fish seemed to be covered by a veil-like film which looked like coagulated mucus and which was formed by the heavy-metal ions reacting with some constituents of the mucus and which was formed by the heavy-metal ions reacting with some constituents of the mucus secreted by the gill. The histopathological assessment of the gill and liver of Cyprinus carpio was also carried out. Particles were observed around the gills of the dead fish treated with zinc and copper salts, although no other major changes were found in the gill. Several histopathological changes were observed in the livers, including the presence of particles. The symptoms of the liver suggested that the internal injury was also an important feature of the intoxicants.
The normal blood coagulation in Cyprinus carpio and Oreochromis mossambicus was investigated by thrombelastography and standard thrombelastographs were obtained for each species. The effect of stress on blood coagulation was thereafter determined. The thrombelastograms indicated hypercoagulability under stress conditions. A prominent observation is the differences in the elasticity of the clot under various conditions. Fibrinolysis occurred in all cases, except in the case of Cyprinus carpio after severe agitation for 30 min. Relevant values for various parameters are given.
The mitochondrial genome from Cyprinus carpio oocytes is a 10.5 megadalton, circular DNA molecule. The carp mitochondrial DNA was cloned in pBR325. Three recombinant plasmids accounted for the entire genome. Mapping of this DNA using 11 different restriction endonucleases is reported here. Both the large and small rRNA genes were then localized using Southern blot analysis. The subunit I of the cytochrome oxidase, the cytochrome b, the tRNAGlu and the URF 4 genes were localized by nucleotide sequence analysis and homology studies with human mtDNA. Our results suggest that a similar gene order has been maintained in the mitochondrial genomes of Chordata and support the hypothesis of a common ancestor for all vertebrate organelle genomes. This study constitutes the first report on the genome organization of a fish mtDNA and provides information for further investigation in connection with sequence determination, replication, and gene expression in carp mitochondria.
The ontogeny of carp (Cyprinus carpio L.) immune cells was studied in mucosal organs (intestine, gills and skin) using the monoclonal antibodies WCL38 (intraepithelial lymphocytes), WCL15 (monocytes/macrophages) and WCI12 (B cells). In addition, recombination activating gene 1 expression was examined in the intestine with real time quantitative PCR and in situ hybridization to investigate extrathymic generation of lymphocytes. WCL38(+) intraepithelial lymphocytes (putative T cells) appeared in the intestine at 3 days post-fertilization (dpf), which is shortly after hatching but before feeding, implying an important function at early age. These lymphoid cells appear in the intestine before the observation of the first thymocytes at 3-4 dpf, and together with the expression of recombination activating gene 1 in the intestine, suggests that similar to mammals at least part of these cells are generated in the intestine. WCL15(+)monocytes/macrophages appeared in the lamina propria of the intestine at 7 dpf, but considerably later in the epithelium, while WCI12(+) (B) cells appeared in intestine and gills at 6-7 weeks. From these results it can be concluded that putative T cells occur much earlier than B cells, and that B cells appear much later in the mucosae than in other internal lymphoid organs (2 wpf).
O-group carp (Cyprinus carpio) which had been immunized against Ichthyophthirius multifiliis by controlled infections were challenged by topical application of theronts to the caudal fin. The parasites which established were examined ultrastructurally, and host leucocyte responses were compared with those observed in primary infections. In the primary exposure group eosinophils and (to a lesser extent) basophils were the predominant cells infiltrating infection sites. In contrast, parasite development in immunized fish initiated localized leucocytic infiltrations which were dominated by eosinophilic granular cells (EGCs) and basophils. Greater localized phagocytosis was recorded in immunized fish by neutrophils, macrophages and resident epidermal filament cells. In vitro studies indicated that pronephric leucocytes from immunized fish displayed enhanced non-specific phagocytosis. In the skin, leucocytes were observed in close proximity to the trophozoite surface in both immunized and primary exposure fish, often undergoing lysis and release of cellular contents. However, there was no evidence of active cell adherence nor of any cell-mediated damage incurred to the parasite in either case. These observations are discussed in relation to the possible role of leucocytes in mediating pathogenesis and immune responses.
Purified cathepsin L from carp, Cyprinus carpio, consists of a 28 kDa single-chain form that is different from the 24 and 5 kDa mammalian two-chain form. We cloned cathepsin L from carp hepatopancreas. The sequence consisted of a 1490 bp cDNA and a 1014 bp open reading frame, encoding a deduced protein of 337 amino acids that is likely processed to an active enzyme (single-chain form) with 222 amino acids. Its similarity to other types of vertebrate cathepsin L is less than 69%. Mammalian cathepsin L is further processed to a two-chain form, but possibly this is not the case with carp cathepsin L: the P1 site where cleavage occurred in the two-chain form of mammalian cathepsin L contains a serine, while carp cathepsin L processes a valine. Therefore, carp cathepsin L may have a different mechanism of action from mammalian cathepsin L.
Enolase from carp (Cyprinus Carpio) muscle was modified by diethylpyrocarbonate, tetranitromethane, N-bromosuccinimide and 5,5'-dithiobis(2-nitrobenzoic acid). The extent and rate of modification and its effect on the enzyme activity were determined. Modification of histidine, tyrosine and tryptophan residues caused complete inactivation of the enzyme; Mg2+ as well as 2-phosphoglycerate markedly altered the rates of modification and inactivation. The above-mentioned amino acid residues seem to be essential for the functioning of muscle enolases. Modification of cysteine residues had no effect on the enolase activity.
Since 1998, episodes of mass mortality have occurred in populations of common carp Cyprinus carpio carpio in Israel and in populations of koi Cyprinus carpio koi in Israel and the USA. A herpesvirus isolated from infected fish has been shown in experimental studies to induce disease and mortality similar to those observed in outbreaks at infected farms. Initial characteristics of the virus show that it is clearly different from Herpesvirus cyprini (CHV), the most commonly known herpesvirus from cyprinid fish. The koi herpesvirus (KHV) has 31 virion polypeptides. Twelve of the virion polypeptides of KHV have similar molecular weights to those of CHV and 10 are similar to those of channel catfish virus (CCV). Both virion polypeptide and restriction fragment length polymorphism analyses of genomic DNA showed that the first KHV isolates from Israel and the USA were identical. In contrast, the genomic DNA restriction fragments clearly distinguish KHV from CHV and CCV. A polymerase chain reaction (PCR) assay to detect the virus in koi tissues was developed with sequences obtained from 1 restriction fragment of KHV DNA. The PCR assay effectively detected a 484 base pair sequence from KHV but did not amplify genomic DNA from either CHV or CCV. The PCR assay detected as little as 1 pg of KHV DNA mixed with 100 ng of host DNA. Viral sequences were amplified from koi obtained from field collections and from koi that were experimentally exposed to 10(2) TCID50 ml(-1) of KHV via the waterborne route. All KHV exposed fish dying of infection between 8 and 10 d post exposure or surviving to 14 d post exposure were found to be positive by PCR, while unexposed control koi were all negative. The assay also showed the presence of KHV DNA in tissues of koi obtained from farms in Israel. The PCR assay should assist virus isolation procedures and histologic and electron microscopic analyses now commonly used to detect KHV infection. Current studies are examining the possibility of using the PCR to detect KHV DNA in live fish and the relative sensitivity and specificity of the KHV PCR assay compared with other diagnostic tests.