Genetic polymorphism and isoenzyme patterns of lactate dehydrogenase in tench (Tinca tinca), crucian carp (Carassius carassius) and carp (Cyprinus carpio)

Isoenzyme patterns and the polymorphism of lactate dehydrogenase (LDH) were investigated in 3 fish species of family Cyprinidae, i.e. tench (Tinca tinca), crucian carp (Carassius carassius) and carp (Cyprinus carpio). The isoenzyme patterns were tissue and species specific. In crucian carp subunits with different electrophoretic mobility are present, which are genetically controlled from the B1, B2, A1, A2 and C loci, while the set of loci in carp is B1, B2, A, C1 and C2 and in tench B, A, C. The locus B of LDH in tench, the locus B2 in crucian carp, and the loci B1, C1 and C2 in carp are polymorphic and have two different alleles in each case. The polymorphism did not affect the total LDH activity in the tissues. All the populations investigated were in Hardy-Weinberg equilibrium. The genetic control of the polymorphism in B1 and C1 loci in carp was proved by test matings. The polymorphism in B loci tested in erythrocytes may be utilized as genetic markers in the fish breeding.     

A molecular approach to detect hybridisation between crucian carp (Carassius carassius) and non-indigenous carp species (Carassius spp. and Cyprinus carpio)

1. Releases of non‐native fish into the wild is an increasing problem posing considerable ecological and genetic threats through direct competition and hybridisation. 2. We employed six microsatellite markers to identify first generation hybrids and backcrosses between native crucian carp (Carassius carassius) and introduced goldfish (C. auratus) and common carp (Cyprinus carpio) in the U.K. We also investigated the genetic characteristics of the taxonomically controversial gibel carp (Carassius spp.) from sites across Europe. 3. Natural hybridisation between goldfish and crucian carp occurs frequently, although hybrids between all other species pairs were observed. Only 62% of British crucian carp populations (n = 21) consisted exclusively of pure crucian carp. In some populations hybrids were so frequent, that no pure crucian carp were caught, indicating a high competitive ability of hybrids. 4. Most hybrids belonged to the F1 generation but backcrossing was evident at a low frequency in goldfish × crucian carp hybrids and goldfish × common carp hybrids. Furthermore, some local populations had high frequencies of backcrosses, raising the opportunity for introgression. 5. Gibel carp from Germany and Italy belonged to two triploid clonal lineages that were genetically closely related to goldfish, whereas all individuals identified from British populations proved to be crucian carp × goldfish hybrids. 6. Our study suggests that the release of closely related exotic cyprinids not only poses a threat to the genetic integrity and associated local adaptations of native species, but may also contribute to shifts in community structure through competitive interactions.     

Concentrations of a Koi herpesvirus (KHV) in tissues of experimentally infected Cyprinus carpio koi as assessed by real-time TaqMan PCR

The Koi herpesvirus (KHV) is a herpes-like virus now recognized as a worldwide cause of mortality among populations of koi Cyprinus carpio koi and common carp Cyprinus carpio carpio. Temperature is a key factor influencing virus replication both in cell culture and in the tissues of experimentally infected fish. Genomic DNA sequences were used to optimize a rapid real-time TaqMan PCR assay to detect and quantify KHV DNA as found in the tissues of virus-exposed fish. The assay allowed analytical enumeration of target KHV genome copies ranging from 10(1) to 10(7) molecules as present in infected cell lines or fish tissues. The new assay was specific for KHV and did not detect DNA from 3 related herpes-like viruses found in fish, the Cyprinid herpesvirus 1 (CyHV-1), Cyprinid herpesvirus 2 (CyHV-2), Ictalurid herpesvirus 1 (IcHV-1) or the KF-1 cell line used for virus growth. Concentrations of KHV DNA were evaluated in 7 different tissues of replicate groups of virus-exposed koi held at water temperatures of 13, 18, 23 and 28 degrees C. Viral DNA was detected among virus-exposed koi at all 4 water temperatures but mortality was only observed among fish at 18, 23, and 28 degrees C. Time and temperature and the interactions between them affected concentrations of viral DNA detected in tissues of koi exposed to KHV. Although there were no recognized patterns to viral DNA concentrations as found in different tissues over time, KHV genome copies for all tissues increased with time post virus exposure and with water temperature. The remarkably rapid and systemic spread of the virus was demonstrated by the presence of viral DNA in multiple tissues 1 d post virus exposure. The greatest DNA concentrations found were in the gill, kidney and spleen, with virus genome equivalents consistently from 10(8) to 10(9) per 10(6) host cells. High levels of KHV DNA were also found in the mucus, liver, gut, and brain. Koi surviving infection at 62 to 64 d post virus exposure contained lower KHV genome copies (up to 1.99 x 10(2) per 10(6) host cells) as present in gill, kidney or brain tissues.     

Effects of sustained predation by fast‐growing transgenic common carp (Cyprinus carpio Linnaeus, 1758) on gastropods in artificial environments

The present study addressed the effects of growth hormone‐transgenic and non‐transgenic common carp (Cyprinus carpio L.) predation on the community and populations of four gastropod species cultured in artificial environments. After a 110‐day population‐based predation experiment (three replicate pools [16 m²] for each genotype with one fish [total length 45.0~51.4 cm] and 150 Bellamya aeruginosa (Reeve 1863) per pool), there were no significant effects of predation by either transgenic or non‐transgenic carp on the biomass, number, or size selectivity of the population. Following a 10‐day community‐based predation experiment (three replicate pools [16 m²] for each genotype with one fish [total length 45.0~50.5 cm] and 150 Alocinma longicornis (Benson 1842), 100 Parafossarulus sinensis (Neunayr 1883), 55 B. aeruginosa, and 33 Radix auricularia L. per pool), the consumption rate and the number of gastropods predated by transgenic carp were 2.60 times and 2.85 times higher, respectively, than those of non‐transgenic carp. Furthermore, the biomass of A. longicornis, P. sinensis and B. aeruginosa consumed by transgenic carp was significantly (p < .05) higher than that by non‐transgenic carp. There was a significant difference in the type selection of the four gastropods by the transgenic and non‐transgenic carp, but both predators preferred R. auricularia and avoided B. aeruginosa. Compared with the non‐transgenic carp, predation by transgenic carp resulted in a significant decrease in A. longicornis (p < .05) and an increase in B. aeruginosa (p < .05). These results indicate that the effects of predation by both transgenic and non‐transgenic carp on the B. aeruginosa population tend to be similar, but their effects on the community composed of the four gastropods were significant different. This information may be useful for assessing the environmental risk of transgenic carp.     

Hybridization of crucian carp, Carassius carassius (Linnaeus, 1758), in Ukrainian reservoirs and genetic structure of hybrids

Hybridization of crucian carps Carassius carassius in polyspecific crucian populations of reservoirs of Ukraine and genetic structure of the hybrids were investigated using biochemical gene marking and cytometric procedure. The fact of wide hybridization between C. auratus and C. carassius was proved to be true by large number of hybrids which can form populations consisting only from hybrid individuals. Hybrids C. auratus x C. carassius were diploid, tryploid and in exceptional cases tetraploid; females and males which most likely breed by hybridogenesis. Besides, some clonal hybrids C. carassius x C. gibelio-1 appearing as tetraploid females, and one triploid female C. carassius x Tinca tinca were revealed. It is supported that hybridization of alien C. auratus with endemic C. carassius became one of mechanisms of replacement and depressions of populations of the last.     

Effect of age,size and digestive tract development on weaning effectiveness in crucian carp,Carassius carassius (Linnaeus, 1758)

The study aim was to determine the optimum age, wet body weight (WBW) and total length (TL) of the crucian carp, Carassius carassius (L.), to ensure the effectiveness of weaning directly without a gradual transfer from live food to a compound feed. Moreover, the state of development of the digestive tract was analyzed histologically based on the height of enterocytes. Experimental rearing was conducted between days 5 and 45 post hatch (DPH). Initial WBW of fish was 2.2 ± 0.6 (n = 30) mg and TL 6.1 ± 0.1 (n = 30) mm. Rearing was carried out at 27 ± 0.5°C, with fish divided into six groups: one control (C) fed with Artemia sp. nauplii, and five groups initially fed with Artemia sp. but later replaced by a compound feed. Weaning with the compound diet started at 15, 20, 25, 30 and 35 DPH in groups labeled F15, F20, F25, F30, F35, respectively. Larvae were fed three times per day (08.00 h, 13.00 h, 18.00 h) in equal portions (4% of larvae biomass per day, converted to the dry matter of the feed). Daily biomass growth was adopted as 15%. Each group was triplicated (n = 50 individuals per replicate). Highest values of TL 42.1 ± 0.7 (n = 30) mm and WBW 905.3 ± 50.3 (n = 30) mg were recorded in the control group at 45 DPH; lowest survival rate of 45 DPH was in group F15 (90.7 ± 1.2%, n = 30). The highest value of the enterocyte epithelial length was observed in individuals within groups F30, 34.8 ± 1.2 μm (n = 30) and F35, 35.4 ± 3.6 μm (n = 30), respectively, 30 and 35 DPH; highest percentage of deformations on the final day of the experiment was in group F15 (100 ± 0.0%, n = 30). The results indicate that an effective direct transfer from live food to prepared diets (with no gradual transfer) cannot be performed with crucian carp larvae before 30 DPH at 27°C, when the fish have reached TL = 31.1 ± 0.4 mm (n = 30) and WBW = 436.9 ± 13.7 mg (n = 30).     

Histopathological indicators: a useful fish health monitoring tool in common carp (Cyprinus carpio Linnaeus, 1758) culture

In order to evaluate the relationship between water quality in ponds and indices of histopathological changes occurring in the vital organs of the common carp (Cyprinus carpio L., 1758), two six-month field experiments were carried out using two different water supplies: from the nearby stream and a tube well. The fish were fed supplemental feed: raw cereals, pelleted and extruded compound feed. Histopathological analysis, alteration frequencies, and semi-quantitative scoring of the changes were used to assess the health status of the fish. Ponds supplied by stream water were characterized by higher water hardness, dissolved oxygen and pH values, while those supplied by the tube well had higher electroconductivity, total ammonium and orthophosphates content. Fish survival rate and habitat suitability index were lower in ponds supplied by stream water, while the weight gain did not differ between the two water supplies. The use of stream water resulted in a higher level of histopathological changes in gills and liver. Among the water quality parameters, pH level had the strongest influence on fish. Differences in water supply produced greater influence on the level of histopathological changes than the type of feed applied. Gills were the most sensitive organ, while the kidney was the least responsive.     

Genetic polymorphism and isoenzyme patterns of lactate dehydrogenase in tench (Tinea tinea), crucian carp (Carassius carassius) and carp (Cyprinus carpio)

Isoenzyme patterns and the polymorphism of lactate dehydrogenase (LDH) were investigated in 3 fish species of family Cyprinidae, i.e. tench ( Tinea tinea ), crucian carp ( Carassius carassius ) and carp ( Cyprinus carpio). The isoenzyme patterns were tissue and species specific. In crucian carp subunits with different electro-phoretic mobility are present, which are genetically controlled from the B¹, B², A¹, A²and C loci, while the set of loci in carp is B¹, B², A, C¹and C²and in tench B, A, C. The locus B of LDH in tench, the locus B²in crucian carp, and the loci B¹, C¹and C²in carp are polymorphic and have two different alleles in each case. The polymorphism did not affect the total LDH activity in the tissues. All the populations investigated were in Hardy-Weinberg equilibrium. The genetic control of the polymorphism in B¹and C¹loci in carp was proved by test matings. The polymorphism in B loci tested in erythrocytes may be utilized as genetic markers in the fish breeding.     

Mark retention and fish survival associated with a low-cost marking technique for common carp Cyprinus carpio (Linnaeus, 1758)

Common carp (Cyprinus carpio, [Linnaeus, 1758]) have long been established in the United States and in some cases their populations can be very dense, leading biologists to expend significant effort towards reducing numbers of common carp in some populations. Estimating abundance of common carp prior to removal efforts would be useful in evaluating success of these removal efforts, but marking large numbers of fish can be expensive. Therefore, a low-cost tagging option is needed. In this study, we used tank and field experiments to determine the retention and longevity of hole-punch marks in the opercula of common carp. For the tank experiment, fish were double marked with a size-3 self-piercing tag and an operculum hole-punch mark (using a paper hole-punch tool with a hole diameter of 6.4 mm) on opposite sides of the fish. Over the entirety of the 180–day tank experiment, retention of the self-piercing tags and hole-punch marks was 100% and no marking mortality was observed. For the field experiment, 883 common carp were tagged at random in two groups, a double-marked group (n = 416, both self-piercing tags and hole-punch) and a single-mark group (n = 467, self-piercing tag). Fish were sampled monthly for 398 days. Because the length distribution sampled was bimodal, we evaluated tag retention of fish <330 mm TL (small fish, n = 273) and > 331 mm TL (large fish, n = 143), separately. Hole-punch mark retention was high for both size classes throughout most of the field experiment. For large fish, retention of hole-punch marks was 100% for the entire 398-day experiment. For small fish, retention of hole-punch marks was 100% through 184-day and remained above 93% through 328-days, but declined to 0% by day 398. Our results suggest that the operculum hole-punch mark is a valuable low-cost, long-term technique for tagging common carp.     

Histopathological and ultrastructural features of Koi herpesvirus (KHV)-infected carp Cyprinus carpio, and the morphology and morphogenesis of KHV

Epizootics of Koi herpesvirus (KHV) cause mass mortalities in koi carp and common carp worldwide. We used a newly developed 'per-gill infection' procedure with live KHV, and then conducted detailed histopathological and ultrastructural studies of KHV-infected cells including an examination of the morphology and morphogenesis of KHV. The primary target of KHV was respiratory epithelial cells of the gill lamellae, and release of virions from infected epithelial cells resulted in a systemic infection affecting the kidney, spleen, heart, brain and liver. The pathognomonic feature of infected cells was the formation of intranuclear inclusion bodies with marginal hyperchromatosis in the nucleus. Within the nucleus, assembly of capsids and nucleocapsids and an increase in filamentous nucleoproteins were evident. Enveloped nucleocapsids budded from the inner nuclear membrane into the perinuclear space. De-enveloped nucleocapsids were translocated in the cytoplasm to be embedded within inclusion bodies where tegumentation of the nucleocapsid occurred. Enveloped virions that had budded into intracytoplasmic vesicles and virions located extracellularly were composed of an electron-dense core, surrounded in turn by the capsid, the tegument and finally an envelope with projections. The morphology and morphogenesis of KHV were the same as those of viruses within the family Herpesviridae.     

Characterization and SNP variation of the interleukin‐1β gene of bighead carp (Cyprinus pellegrini Tchang, 1933) and five strains of common carp [Cyprinus carpio Linnaeus (1758)] in China

Single nucleotide polymorphisms of Interleukin‐1β (IL‐1β) have been reported as markers for susceptibility to infectious diseases in humans and livestock. The present study was to determine the genetic variation of this cytokine in six carp strains. Among the sampled individuals, a total of 13 SNPs, including eight in introns and five in coding regions, were identified at intron 5, exon 6, intron 6 and exon 7. Three positions of 1700, 1733 and 1934 resulted in variable amino acid changes with Phe to Tyr, Pro to Leu and Lys to Asn, respectively. Five positions with minor allele frequency (MAF) were larger than 0.05. Among 13 SNPs, eight positions of allele frequency and ten positions of genotypic frequency showed significant differences between some populations. The genotype distributions of the 13 SNPs were consistent with the assumption of the Hardy‐Weinberg equilibrium, with the exception of two positions in the Yibu and bighead carp (P < 0.05), so as to supply the genetic information for research on infectious diseases.     

The use of carp, Cyprinus carpio (Linnaeus, 1758), instead of rabbit as antibody donor in immunological research on fishes

Antisera produced by carp were tested in the differentiation between two cyprinid fishes, bream and roach, and between the lipovitellins of two grey mullets, Mugil cephalus and Liza ramada. These antisera were more specific in the recognition o! different fish species than those produced by rabbits.
An antiserum against carp lipovitellin was produced with male carp as antibody donor.     

Polymorphic microsatellites from silver crucian carp (Carassius auratus gibelio Bloch) and cross‐amplification in common carp (Cyprinus carpio L.)

Fifteen polymorphic microsatellites were isolated from the genome of silver crucian carp (Carassius auratus gibelio Bloch). Allele numbers ranged from two to four with an average of 2.7/locus, and the proportion of tri‐ and diallelic heterozygotes was 99.3%. The individuals tested seem to have originated from two different clonal lines: 14 of 16 showed the same genotype at all loci tested, whereas the remaining two were also identical, but different from the former ones. Eleven out of 15 primer pairs cross‐amplified products from the genome of common carp, whereas only five from that of zebrafish.     

The use of carp, Cyprinus carpio (Linnaeus, 1758), instead of rabbit as antibody donor in immunological research on fishes.

Antisera produced by carp were tested in the differentiation between two cyprinid fishes, bream and roach, and between the lipovitellins of two grey mullets, Mugil cephalus and Liza ramada. These antisera were more specific in the recognition of different fish species than those produced by rabbits. An antiserum against carp lipovitellin was produced with male carp as antibody donor.     

Gene transfer, expression and inheritance of pRSV-rainbow trout-GH cDNA in the common carp, Cyprinus carpio (Linnaeus)

A recombinant plasmid containing the Rous sarcoma virus-long terminal repeat (RSV-LTR) promoter linked to rainbow trout (Salmo gairdneri) growth hormone (GH) cDNA was microinjected into fertilized carp eggs. Genomic DNA extracted from pectoral fin of individual presumptive transgenic fish was analyzed by dot blot and Southern blot hybridization, using the RSV-LTR and/or the GH cDNA sequences as probes. Out of 365 presumptive transgenic fish analyzed, 20 individuals were found to contain pRSV-rtGH-cDNA sequence in the genomic DNA. Expression of the trout GH polypeptide was detected by immunobinding assay in the red blood cells of nine transgenic fish tested. The level of expression, however, varied among the transgenics and could not be correlated with exogenous DNA copy number. Although there was considerable variation in the sizes of the transgenic fish, those microinjected during the one-cell stage were (P less than 0.05) 22% larger, on the average, than their sibling controls. A randomly selected fraction of the progeny derived from crosses between transgenic males and non-transgenic females inherited the foreign DNA. These transgenic progeny grew faster (P less than 0.05) than their non-transgenic siblings.