Molecular cloning of the complement regulatory factor I isotypes from the common carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis>)

Factor I is a novel serine protease that regulates complement activation. Here we report the complete primary structure of two isotypic factor Is isolated from the common carp ( Cyprinus carpio), a pseudotetraploid teleost. A carp hepatopancreas cDNA library was screened using two RT-PCR-amplified cDNA fragments encoding part of the carp factor I-like serine protease domain. Two distinct cDNA clones, designated FI-A and FI-B, were isolated. Their deduced amino acid sequences share 75.2% identity with each other. FI-A has a typical factor I-like domain organization composed of two disulfide-linked polypeptides (H-chain and L-chain). On the other hand, FI-B contains a novel sequence of 115 amino acids inserted at the N-terminus of the H-chain. Genomic Southern hybridization suggests that FI-A and FI-B are encoded by distinct genes in the carp genome. Expression analysis by RT-PCR revealed that the major site of FI-A expression is the ovary, whereas FI-B expression is detected mainly in the hepatopancreas at a level higher than that of FI-A. The present data, taken together, suggest that carp have duplicated genes coding for factor I, and FI-B with the novel insertion plays a dominant role in the complement system. In addition, homology search of the fugu genome database using the carp FI-A and FI-B sequences identified a putative fugu factor I gene, which has an exon/intron organization different from that of the human orthologue.     

Hypoxia tolerance of hybrids of carp <Emphasis Type="Italic">Cyprinus carpio</Emphasis> and golden carp <Emphasis Type="Italic">Carassius auratus</Emphasis>

Hypoxia tolerance of diploid gynogenetic and triploid backcross hybrids between golden carp Carassius auratus and carp Cyprinus carpio was investigated in comparison with carp. The more that heredity of golden carp was in their genotype, the better the hybrids survived under hypoxia conditions.     

Fertility of triploid hybrids of Prussian carp (<Emphasis Type="Italic">Carassius gibelio</Emphasis>) with common carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis> L.)

Fertility of backcross triploid hybrids containing one genome of Prussian carp and two genomes of common carp is investigated. The females of hybrids of Prussian carp and common carp (Prussian × common carp) are prolific and produce diploid gametes. Since males of such hybrids are sterile, their reproduction is realized by means of induced gynogenesis. Triploid progeny is obtained by backcrossing female Prussian × common carp with carp males. Among triploids obtained from hybrids F₁ and among hybrids of the first gynogenetic generation, there were no prolific specimens. However, in reproduction of diploid hybrids by means of gynogenesis during six generations, the female fertility in the backcross progeny is restored. From backcross triploid females (daughters of Prussian × common carp of the sixth gynogenetic generation), a viable triploid gynogenetic progeny and a tetraploid backcross (by carp) progeny are obtained. The obtained data may be considered as the experimental proof of the hypothesis of reticular speciation.     

Mutation rate and pattern of microsatellites in common carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis> L.)

Microsatellites are popular molecular markers in genetic and evolutionary studies. Their mutational dynamics have been extensively studied in humans and fruit flies, but few data were available in fish. By genotyping 55 individuals of a F1 pedigree, we investigated the mutation rates and patterns of 49 microsatellites in one of the most important fresh water fish species, the common carp (Cyprinus carpio L.). The overall mutation rate of the 49 loci was 5.56×10⁻⁴/locus/generation (95% confidence interval 1.52×10⁻⁴ and 1.63×10⁻³). The change of allele size was between +2 to −5 repeat units, assuming that the mutation allele arose from the parental allele most similar in size to the mutant.     

Polymorphism of microsatellite markers in Russian common carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis> L.) breeds

Using five microsatellite loci, genotyping and genetic diversity estimates were obtained for nine samples representing seven common carp breeds most widespread in Russia. For comparison, the samples of Amur wild common carp (Cyprinus carpio haematopterus) and a sample of European Hungarian carp were used. In the samples examined (n = 148) a total of 78 alleles were revealed. The highest mean allele number per locus (7.3) was identified in Amur wild common carp, while the lowest number was found in Cherepets carps (4.0). In different breeds, the observed heterozygosities varied from 0.819 (Altai carp) to 0.651 (Cherepets scaly carp). Three out of five microsatellite loci (MFW-24, MFW-28, and MFW-19) revealed a high level of population differentiation. In the dendrogram of genetic differences, all breeds clustered into two groups. One of these groups was composed of the two strains of Ropsha carp, Stavropol carp, Amur wild common carp, and the two samples of Cherepets carp. The second cluster included Altai carp (Priobskii and Chumysh populations), two Angelinskii carp breeds (mirror and scaly), and Hungarian carp. The pairs of breeds/populations/strains, having common origin, were differentiated. Specifically, these were two populations of Altai carp, two strains of Ropsha carp, as well as the breeds of Angelinskii and Cherepets carps. The reasons for genetic differentiation of Russian common carp breeds, as well as the concordance of the evolutionary histories of these breeds, some of which originated from the European breeds, while the others contain substantial admixture of the Amur wild common carp, are discussed.     

Polymorphism of the cytochrome oxidase b gene (<Emphasis Type="Italic">cyt b</Emphasis>) in Russian populations of common carp and wild common carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis> 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 (π) and haplotype (h) diversity was detected in two strains of Ropsha common carp (MM, π = 0.67%, h = 0.7; and BB, π = 0.21%, h = 0.9) and in one population (Am) of Amur wild common carp (π = 0.26%; h = 0.6). The second population of Amur wild common carp (Ac) and Hungarian common carp were characterized by lower variation estimates (π = 0.035%, h = 0.4; and π = 0.09%, h = 0.7, respectively). Genetic homogeneity was demonstrated for the populations of Volga and Don wild common carp (π = 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.     

cDNA sequence and tissues expression analysis of lipoprotein lipase from common carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis> Var. Jian)

A full-length cDNA coding lipoprotein lipase (LPL) was cloned from liver of adult common carp (Cyprinus carpio Var. Jian) by RT-PCR and rapid amplification of cDNA ends (RACE) approaches. The cDNA obtained was 2,411 bp long with a 1,524 bp open reading frame (ORF) encoding 507 amino acids. This amino acid sequence contains two structural regions: N-terminus (24–354 residues) and C-terminus (355–507 residues). Before N-terminus, 1–23 residues is signal peptide, 6–23 residues is transmembrance helix. At N-terminus, some conversed functional sites were found, including two N-linked glycosylation sites Asn⁴¹ and Asn⁸⁸; one catalytic triad Ser¹⁷⁴, Asp¹⁹⁸ and His²⁸³; one conserved heparin-binding site Arg³²¹ to Arg³²⁴ (RKNR); eight cysteines residues Cys⁶⁹ and Cys⁸², Cys²⁵⁸ and Cys²⁸¹, Cys³⁰⁶ and Cys³²⁵, Cys³¹⁷ and Cys³²⁰ which are involved in four disulfide bridges; one polypeptide “lid” that participates in substrate specificity. At C-terminus, Asn⁴⁰¹ is another N-linked glycosylation site, and Trp⁴³⁴ and Trp⁴³⁵ (WW) is lipid-binding site. The amino acid sequence has a high similarity, and shows similar structural features to LPL of other species. Tissue distribution of LPL mRNA in liver, head kidney, mesenteric adipose tissue, heart and white muscle of common carp was analyzed by semi-quantitative RT-PCR method using β-actin gene as internal control. The result showed that the expressions of LPL mRNA were detected in all examined tissues of common carp. The expression levels of LPL in the mesenteric adipose tissue was highest among these tissues, following in liver and head kidney, and the lowest expression was found in heart and white muscle.     

Quantitative trait loci for morphometric traits in multiple families of common carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis>)

Quantitative trait locus (QTL) mapping is frequently used to understand the genetic architecture of quantitative traits. Herein, we performed a genome scan for QTL affecting the morphometric characters in eight full-sib families containing 522 individuals using different statistical methods (Sib-pair and half-sib model). A total of 194 QTLs were detected in 25 different regions on 10 linkage groups (LGs). Among them, 37 QTLs on five LGs (eight, 13, 24, 40 and 45) were significant (5% genome-wide level), while the remaining 40 (1% chromosome-wide level) and 117 (5% chromosome-wide level) indicated suggestive effect on those traits. Heritabilities for most morphometric traits were moderate to high, ranging from 0.21 to 0.66, with generally strong phenotypic and genetic correlations between the traits. A large number of QTLs for morphometric traits were co-located, consistent with their high correlations, and may reflect pleiotropic effect on the same genes. Biological pathways were mapped for possible candidate genes on QTL regions. One significantly enriched pathway was identified on LG45, which had a P-value of 0.04 and corresponded to the “regulation of actin cytoskeleton pathway”. The results are expected to be useful in marker-assisted selection (MAS) and provide valuable information for the study of gene pathway for morphometric and growth traits of the common carp.     

cDNA cloning and expression analysis of two distinct <Emphasis Type="Italic">Sox8</Emphasis> genes in <Emphasis Type="Italic">Paramisgurnus dabryanus</Emphasis> (Cypriniformes)

The Sox9 gene attracts a lot of attention because of its connection with gonadal development and differentiation. However, Sox8, belonging to the same subgroup SoxE, has rarely been studied. To investigate the function as well as the evolutionary origin of SOXE subgroup, we amplified the genomic DNA of Paramisgurnus dabryanu using a pair of degenerate primers. Using rapid amplification of the cDNA ends (RACE), it was discovered that P. dabryanu has two duplicates: Sox8a and Sox8b. Each has an intron of different length in the conserved HMG-box region. The overall sequence similarity of the deduced amino acid of PdSox8a and PdSox8b was 46.26%, and only two amino acids changed in the HMG-box. This is the first evidence showing that there are two distinct duplications of Sox8 genes in Cypriniformes. Southern blot analysis showed only one hybrid band, with lengths 7.4 or 9.2 kb. Both semi-quantitative RT-PCR and real-time quantitative PCR assay displayed that both PdSox8a and PdSox8b are downregulated during early embryonic development. In adult tissues, the two Sox8 genes expressed ubiquitously, and expression levels are particularly high in the gonads and brain. In gonads, both PdSox8a and PdSox8b are expressed at a higher level in the tesis than in the ovary. PdSox8a and PdSox8b may have functional overlaps and are essential for the neuronal development and differentiation of gonads.     

The effect of food odor background on gustatory preferences and gustatory behavior of carp <Emphasis Type="Italic">Cyprinus carpio</Emphasis> and cod <Emphasis Type="Italic">Gadus morhua</Emphasis>

It was shown that stimulation by food odor (aquatic extract of food organisms, 10^?2 and 10^?3 g/l) does not cause shifts in gustatory preferences in carp Cyprinus carpio and cod Gadus morhua but modifies gustatory behavior. The level of consumption by carp of control granules and granules with attractive, by taste, L-proline (0.1 M) or deterrent L-lysine (0.1 M) (item by item presentation of granules) and by cod of control granules and granules with indifferent, to it, L-asparagine (0.1 M) (presentation of 10 granules simultaneously) is similar prior to and during olfactory stimulation. In the presence of food odor, the duration of taste testing for most types of granules, as well as the number of repeated graspings of granules with an attractive taste do not change in fish. At the same time, granules with indifferent or repulsive gustatory properties are rejected and repeatedly grasped by fish against the background of food odor more frequently than in water without odor. Olfactory stimulation leads to a considerable increase in the average number of graspings per one grasped granule with an indifferent or repulsive taste. Such behavior manifested by fish in the presence of food odor in response to granules with unattractive gustatory properties is apparently caused by the contradiction between the information coming via different chemosensory canals—olfactory and gustatory. The obtained results indicate that food stimulation caused by food odor in nature can lead to an increase in the actual consumption of only those accessible food items that have an attractive taste for fish.     

Ontogenetic shift in dietary preference and low dietary overlap in rohu (<Emphasis Type="Italic">Labeo rohita</Emphasis>) and common carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis>) in semi-intensive polyculture ponds

In order to investigate ontogenetic changes in diet and diet overlap between rohu (Labeo rohita) and common carp (Cyprinus carpio) in polyculture ponds, food preferences of different size classes of these fishes were quantified. Rohu diet consisted of both phytoplankton and zooplankton, and there was a distinct ontogenetic shift in the relative importance of these food items. Zooplankton was the dominant food for rohu up to 20.6 cm total length (TL) and then gradually decreased in importance as fish grew. Phytoplankton was initially a minor component of rohu diet but gradually increased in importance and became the dominant food for rohu at 24.2 cm TL. Phytoplankton biovolume in rohu guts was positively correlated with fish size (TL). Chesson’s α indicated that rohu of all sizes preferentially selected Cladocera and avoided Cyanophyceae and Euglenophyceae. Young rohu initially preferred Rotifera and Copepoda but gradually switched to Bacillariophyceae and Chlorophyceae. Common carp diet consisted of phytoplankton, zooplankton, and benthic macroinvertebrates, but was dominated by benthic macroinvertebrates (63–92% of total diet). As common carp grew, the proportion of zooplankton ingested decreased and the proportion of benthic macroinvertebrates increased. Benthic macroinvertebrate biovolume in common carp guts was positively correlated with fish size. Common carp of up to 15.4 cm TL preferentially selected zooplankton, but common carp larger than 18.9 cm TL avoided this food item. Common carp of all sizes avoided phytoplankton. A low dietary overlap was found between rohu and common carp (Schoener overlap index: 0.08–0.35), probably due to ingestion of smaller quantities of zooplankton by the latter. Dietary overlap also decreased with increasing rohu and common carp size because of divergent ontogenetic shifts in dietary preferences of the two species.     

Genetic variability comparison of cultured Israeli carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis>) from Korea using microsatellites

In aquaculture, cultured fish often undergo continuous cross-fertilization without any inflow of new broodstock. This lowers genetic diversity, leading to increased disease rates and decreased survival rates. To improve the mass production and easy culture of Israeli carp, it is essential to investigate the population structure and genetic diversity of these fish. However, such a survey has not yet been performed on Korean Israeli carp. In this study, we used seven microsatellite markers to analyze the genetic diversity and association of cultured Israeli carp from Korea and China. The average numbers of alleles per locus (N _A ) for two Korean (KorA and KorB) and two Chinese (ChA and ChB) populations were as follows: KorA (10.42), KorB (14.43), ChA (20.57) and ChB (20.71). The expected heterozygosity (H _e ) ranged from 0.672 to 0.897 and from 0.827 to 0.938 in the Korean sample and Chinese sample respectively. The genetic diversity of the Korean Israeli carp was about half that of the Chinese carp. The diversity of the Korean Israeli carp was very low, suggesting that the immunity of this population could be weak, and that diversity–recovery studies are urgently needed. Therefore, our results may therefore form the foundation for future research efforts towards genetic monitoring and selective breeding, continuous research needs to be conducted in order to recover the genetic diversity of the Korean Israeli carp.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Age determination in common carp <Emphasis Type="Italic">Cyprinus carpio</Emphasis>: history,relative utility of ageing structures,precision and accuracy

The common carp Cyprinus carpio is one of the most widely-distributed freshwater fishes in the world. Due to its value for conservation and fisheries in several native/translocated areas of distribution and its detrimental effects on the aquatic ecosystem in most invasive areas, robust age-based population dynamics models are required for successful management of this species. The present study provides a global review of age determination in carp, including a historical account of ageing methods, an assessment of the relative utility of ageing structures, and an evaluation of precision and accuracy (i.e. validation) of age estimates. Historically, scales were by far the most widely-employed structure, followed by the operculum, otolith, dorsal spine, vertebra and fin ray. However, in countries where carp is categorised as ‘high risk’ of impact, use of alternative structures to the scale was predominant. Causal criteria analysis showed scales and opercula to provide inconsistent evidence for successful annulus identification/counting, whereas consistent evidence was found for otoliths, dorsal spines, vertebrae and (pectoral) fin rays. Precision was always above reference thresholds for scales, whereas for otoliths, dorsal spines and fin rays was in several cases below. Accuracy was addressed sporadically and mostly in high-risk countries. It is suggested that dorsal spines or pectoral fin rays should be used in lieu of scales as non-lethal ageing structures, and otoliths (or vertebrae, pending more research) otherwise, and that validation should always be attempted as part of the set-up of more appropriate ageing protocols and use of correct terminology.     

Influence of feeding regime and of food biochemical composition upon feeding behavior in common carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis>)

It is revealed that long-term starvation considerably increases the speed of reaction to food and ration size in common carp (Cyprinis carpio) under conditions simulating the benthic type of feeding. The presence of food in the intestine and the digestion stage considerably affect the fish feeding behavior. Six hours postfeeding irrespective of the feeding regime and food composition, the ration size decreases statistically significantly, while the latent time of feeding increases. The forage composition has a less pronounced effect upon fish feeding behavior than the process of digestion. However, the former factor considerably effects the glycemia level. The most pronounced decrease in the reaction to food and in the ration size coincide with an increase in the glycemia level and perhaps with an increase in the amino acid levels in the fish blood.     

Reaction of osmoregulatory system in juveniles of Russian sturgeon <Emphasis Type="Italic">Acipenser gueldenstaedtii</Emphasis> (Acipenseridae) on influence of comatotropin of carp <Emphasis Type="Italic">Cyprinus carpio</Emphasis>

For the first time by the example of juveniles of Russian sturgeon Acipenser gueldenstaedtii Brandt, the role of growth hormone of hypophysis in the osmotic regulation of acipenserids was studied, also the process of transformation of reserve chloride was described. In Russian sturgeon juveniles, administration of growth hormone of carp Cyprinus carpio at a dose of 1 mg per 1 g body weight causes changes in the fine structure of numerous reserve (undifferentiated) gill epithelial cells and thus stimulates their transformation into mature chloride cells involved in transport of monovalent ions. Increase in the number of chloride cells, on account of reserve cells, was accompanied by higher enzymatic activity of Na⁺/K⁺-ATPase in gill homogenates of the injected fish adapting to a hyperosmotic environment of salinity 12.5‰ (403 mosm/l) than in the intact fish under the same conditions but without hormonal treatment. Morphophysiological changes observed in the injected juveniles provided the more efficient dynamics of blood serum osmolarity under saline exposure than that in the intact fish. Their serum osmolality during the maximum rise (24 hours after the transfer of fish into the water of 12.5‰ salinity) was lower (p <0.01), than that of the intact fish. The inclusion of growth hormone in the osmoregulatory process was established.