Microsatellite DNA Marker Analysis of Genetic Diversity in Wild Common Carp (Cyprinus carpio L.) Populations

Thirty microsatellite loci were used for analyzing six wild populations of common carp （Cyprinus carpio L.）. Observed （Ho） and expected （He） heterozygosity values, polymorphic information content （PIC）, and number of effective alleles （Ae） were all detected. Genetic similarity index and genetic distance were computed based on the allele frequency. The Hardy-Weinberg Equilibrium was checked according to the test of χ^2. Genetic differentiation and hierarchical partition of genetic diversity were evaluated by FST and Nm. A clustering dendrograrn was made based on the results of UPGMA methods using the PHYLIP software package （version 3.63）. There were totally 8,136 fragments ranging from 125 bp to 414 bp in length. Three to thirteen alleles were amplified in 30 loci and 210 alleles in all six populations. The average number of alleles in each locus was seven. The result showed that 1） the level of genetic variability was moderate in the six populations. Polymorphic information contents of the six wild common carp populations were 0.44, 0.52, 0.53, 0.57, 0.63, and 0.64 respectively. Effective alleles were from 1.04 to 4.72, the average numbers in each population were 2.19, 2.60, 2.42, 2.43, 2.45, and 2.33. The average expected heterozygosity values were 0.50, 0.59, 0.56, 0.56, 0.57, and 0.54 respectively; 2） the highest genetic similarity index that came from the populations of BR and ZL was 0.8511 and the lowest index was 0.6688, and it came from the populations of BR and HN. There was a correlation between the clustering result and the geographical distribution.

Rates and patterns of microsatellite mutations in common carp (Cyprinus carpio L.)

During genotyping 150 microsatellites in a F1 family of common carp, six mutations were found at five microsatellite loci. The overall mutation rate of common carp microsatellites was 2.53×10-4 per locus per generation. At five loci, mutations increased the length of alleles by at least one repeat unit, suggesting mutations at microsatellite loci in common carp do not follow strict stepwise mutation model. The data on mutation rates and patterns can facilitate population genetics studies, and provide useful parameters for estimating a long-term effective population size of common carp.

Successful Production of an All-Female Common Carp (Cyprinus carpio L.) Population Using cyp17a1-Deficient Neomale Carp

Due to sexual dimorphism in the growth of certain cultured fish species,the production of monosex fishes is desirable for the aquaculture industry.Nowadays,the most widely practiced technique available for the mass production of monosex fish populations is sex steroid-induced sex reversal.Here,a novel strategy for the successful production of all-female(AF)common carp(Cyprinus carpio L.),to take advantage of the sexual dimorphism in growth documented in this species,has been developed using genetic engineering via single gene-targeting manipulation without any exogenous hormone treatments.Male and female heterozygous cyp17a1-deficient common carp were first obtained using the clustered regularly interspaced short palindromic repeats/CRISPR-associated endonuclease 9(CRISPR/Cas9)technique.An all-male phenotype for homozygous cyp17a1-deficient carp,regardless of the individuals’sexdetermination genotypes(XY or XX),has been observed.A male-specific DNA marker newly identified in our laboratory was used to screen the neomale carp population with the XX genotype from the cyp17a1-deficient carp.These neomale carp develop a normal testis structure with normal spermatogenesis and sperm capacity.The neomale common carp were then mated with wild-type(WT)females(cyp17a1^(+/+)XX genotype)using artificial fertilization.All the AF offspring sample fish from the neomale-WT female mating were confirmed as having the cyp17a^(1+/-)XX genotype,and normal development of gonads to ovaries was observed in 100.00%of this group at eight months post-fertilization(mpf).A total of 1000 carp fingerlings,500 from the WT male and female and 500 from the neomale and WT female mating,were mixed and reared in the same pond.The average body weight of cyp17a1^(+/-)XX females was higher by 6.60%(8 mpf)and 32.66%(12 mpf)than that of the control common carp.Our study demonstrates the first successful production of a monosex teleost population with the advantages of sexual dimorphism in growth using genetic manipulation targeting a single locus.

Existence of Microsatellites in Expressed Sequence Tags of Common Carp (Cyprinus carpio L.) Available in GenBank dbEST Database

Common carp expressed sequence tags （ESTs） were analyzed for the existence of microsatellites, or simple sequence repeats （SSRs）. In the NCBI dbEST database, a total of 10612 sequences were registered before December 31, 2004. A complete search of 2-6 nucleotide microsatellites resulted in the identification of 513 SSR-containing ESTs, accounting for 4.8% of the total. Cluster analysis indicated that 73 sequences of SSR-containing ESTs fell into 27 groups and the remaining 440 ESTs were independent. A total of 467 unique SSR-containing ESTs were identified. These EST-SSRs contained a vari- ety of simple sequence types, and di- and tri-nucleotide repeats were the most abundant, accounting for 42.1% and 27.9% of the whole, respectively. Of the dinucleotide repeats, CA/TG was the most abundant, followed by GA/TC. BLASTx search showed that 38.1% of the SSR loci could be associated with genes or proteins of known or unknown function. BLASTx searches of SSR-containing ESTs also showed high frequencies （98/179） of hits on zebrafish sequences.

Genome evolution trend of common carp (Cyprinus carpio L.) as revealed by the analysis of microsatellite loci in a gynogentic family

Genome evolution arises from two main ways of duplication and reduction. Fish specific genome duplication （FSGD） may have occurred before the radiation of the teleosts. Common carp （Cyprinus carpio L.） has been considered to be a tetraploid species, because of its chromosome numbers （2n=100） and its high DNA content. Using 69 microsatellite primer pairs, the variations were studied to better understand the genome evolution （genome duplication and diploidization） of common carp from a gynogenetic family. About 48% of primer pairs were estimated to amplify duplicates based on the number of PCR amplification per individual. Segregation patterns in the family suggested a partially duplicated genome structure and disomic inheritance. This indicates that the common carp is tetraploid and polyploidy occurred by allotetraploidy. Two primer pairs （HLJ021 and HLJ332） were estimated to amplify reduction based on the number of PCR amplification per individual. One allele in HLJ002 locus and HLJ332 locus was clearly lost in the gynogenetic family and the same as in six wild populations. Segregation patterns in the family suggested a partially diplodization genome structure. A hypothesis transition （dynamic） and equilibrium （static） were proposed to explain the common carp genome evolution between genome duplication and diploidization.

The Use of Enzyme-digested Soybean Residue For Feeding Common Carp

This experiment was an attempt to use the soybean residue derived from the production of 'soy milk'. The residue contains about 18% protein, 70% caf bohydrates and 7.5% lipid as fish feed for reafing common carp, Cyrinus carpto. There were 4 types of diets: (1)soybean residue, (2 ) soybean residue digested with Papain, (3) soybean residue (64%)mixed with beef liver (34%) and (4) same mixture as (3) but digested with Papain. The results indicate that the percentage increase in weight and length of fish feeding with beef liver supplemented diets was higher than those feeding with soybean residue alone. This was possibly due to the fact that beef liver was able to supplement the nutrient deficiency in soybean. The two types of feeds (2 and 4) digested with Papain also yielded significantly better fish growth in terms of weight and length gains, than their counterparts without digestion. Furthermore, the water turbidity of the tanks added with digested feeds was significantly less, as papain was able to hyrolyse the protein substrates suspended in the water, and thus lowered the turbidity

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-transgeniccommon carp were extensively studied. Here we show that each F4 transgenic fish contained about 200 copies of thepMThGH-transgene and the transgenes were integrated into the host genome generally with concatemers in a head-to-tail arrangement at 4-5 insertion sites. By using a method of plasmid rescue, four hundred copies of transgenes fromtwo individuals of F4 transgenic fish, A and B, were recovered and clarified into 6 classes. All classes of recoveredtransgenes 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, indicatingthat most transgenes were faithfully inherited during the four generations of reproduction. The other five classes weredifferent from the original configuration in both molecular weight and restriction map, indicating that a few transgeneshad undergone mutation, rearrangement or deletion during integration and germline transmission. In the five types ofaberrant transgenes, three flanking sequences of the host genome were analyzed. These sequences were common carpβ-actin gene, common carp DNA sequences homologous to mouse phosphoglycerate kinase-1 and human epidermalkeratin 14, respectively.

Verification of protein sparing by feeding carbohydrate to common carp Cyprinus carpio

A 9-week feeding trial in floating freshwater cages(1.0 m×1.0 m×2.0 m) was conducted to study the effects of different dietary levels of protein and starch on growth,body composition,and gene expression of enzymes in common carp,Cyprinus carpio(mean body weight,36.12±1.18 g) to evaluate the protein-sparing effect of dietary carbohydrate.Four diets were formulated with corn starch as the carbohydrate source to obtain corn starch levels of 6.5%,13%,19.5%,or 26%and protein levels of 30.5%,28.2%,26.4%,and 24.2%.The results showed no differences in growth performance of fish fed the diets with different protein and corn starch levels,but body composition and glucose metabolic enzyme activity of carp were significantly affected by the different diets(P<0.05).Weight gain,specific growth rate,and the feed conversion ratio were not different in fish fed the different dietary treatments.Protein efficiency ratio increased significantly as corn starch level increased(P<0.05).Whole-body crude lipid composition increased with increasing dietary corn starch level(P<0.05).Glucokinase(GK),hexokinase,and pyruvate kinase(PK) activities increased significantly with increasing dietary corn starch level(P<0.05),whereas glucose-6-phosphate(G6Pase) activity decreased with increasing dietary corn starch level(P<0.05).GK gene expression was significantly higher in fish fed the high-corn starch diet than those fed the low-corn starch diet(P<0.05).G6 pase gene expression tended to decrease with increasing starch level(P>0.05).In summary,the results indicate a protein-sparing effect by substituting carbohydrate in the diet of common carp.

Effect of Certain Toxicants on Gonadotropin-Induced Ovarian Non-Esterified Cholesterol Depletion and Steroidogenic Enzyme Stimulation of the Common Carp Cyprinus carpio in vitro

Isolated ovarian tissues from the common carp, Cyprinus carpio were incubated in vitro to obtain a discrete effect of four common toxicants of industrial origin, namely phenol, sulfide, mercuric chloride and cadmium chloride, on gonadotropin-induced alteration of nonesterified and esterified cholesterol and steroidogenic enzymes, △5-3β-HSD and 17β-HSD activity. Stage II ovarian tissue containing 30-40% mature oocytes were shown to be most responsive to gonadotropins in depleting only nonesterified cholesterol moiety and stimulating the activity of both. Safe doses of above mentioned toxicants when added separately to stage II ovarian tissue with oLH (1 μg/incubation) gonadotropin-induced depletion of nonesterified cholesterol and gonadotropin-induced stimulation of the activity of both enzymes was significantly inhibited. Esterified cholesterol remained almost unaltered. Findings clearly indicate the impairment of gonadotropin induced fish ovarian steroidogenesis by the four toxicants separately.

Effect of Water Temperature on Feeding Rhythm in Common Carp (Cyprinus carpio haematopterus Temminck et Schlegel)

Feeding rhythm of common carp was investigated from 4℃ to 34℃. The results indicated that there was a diel feeding rhythm for both adult （630-850 g） and youth （61-91 g） at all tested temperatures. There were two main activity peaks at 8：00-11：00 a.m. and 19：00-23：00 p.m., during which feeding quantities were 10.68%-32.53% and 16.25%-33.41% of the daily intake, respectively. When water temperature dropped to below 10℃, the feeding peak concentrated at 8：00-11：00 a.m. and 19：00 p.m. to 4：00 a.m. At 6℃, though both adult and youth would still feed, the feeding quantities were only 0.01% and 0.35% of body mass. Daily feeding rate of adult and youth reached 1.21% and 2.63% at 14℃, respectively. Both adult and youth carps reached the maximum daily feeding rate at 28℃, being 2.84% and 12.06% of body mass, respectively. The daily feeding rate of adult and youth reduced suddenly after at 34℃, and the daily feeding rate was only 0.74% and 9.45% of body mass, respectively. There was significant difference in daily feeding rate at different water temperatures （P〈0.05）.

Impacts of Date Palm Seeds （Phoenix dactyliferous L.） on Growth Indices and Nutrient Utilization of Common Carp Cyprinus carpio L,

This study was carried out to study the effect of adding different levels of date palm seed （Phoentix dactyliJProus） powder in the diet of common carp （Cyprinus carpio） at fish laboratory of Animal Production Department, College of Agricultural Sciences, Sulaimani University, Sulaimani, Iraq. The actual experimental feeding trials were conducted for 70 d and the adaptation period for 21 d. A total of 81 common carp fingerlings weighted 62 ± 2 g were divided into three experimental groups. The control treatment T1 was with 0 g date palm seed, T2 with 2.5 g date palm seed per kg diet and T3 with 5 g date palm seed per kg diet. Each treatment was in three replicates in which three common carp fingerlings were stocked in each aquarium which fed the experimental diets twice daily. The results indicated that weight gain （WG） and specific growth rate （SGR） in the third treatment （T3） group were significantly higher than those of fish fed control diets. T2 consumed more diet than other treatments with 306.940 g with higher feed conversion ratio （FCR）; T3 was higher significantly in food efficiency. No significant （P 〈 0.05） differences obtained from consumed protein while the protein efficiency ratio was significantly higher in T3 with 319.323. Control was higher significantly in flesh weight without viscera with the third treatment; in flesh weight without head and viscera each the control and the second treatment was significantly higher than the third treatment. As a conclusion, the date palm seeds powder have a positive effect on growth performance of common carp.

Fermentative Biohydrogen Production with Enteric Bacteria Isolated from the Intestine of Wild Common Carp Dwelling in Tarim River Basin

The biological hydrogen generating from fermentation of low-cost lignocellulosic feedstocks by hydrogen-producing bacteria has attracted many attentions in recent years. In the present investigation, ten hydrogen-producing bacteria were newly isolated from the intestine of wild common carp (Cyprinus carpio L.), and identified belonging to the genera of Enterobacter and Klebsiella based on analysis of the 16S rDNA gene sequence and examination of the physiological and biochemical characteristics. All the isolates inherently owned the ability to metabolize xylose especially the cotton stalk hydrolysate for hydrogen production with hydrogen yield (HY) higher than 100 mL·L-1. In particular, two isolates, WL1306 and WL1305 obtained higher HY, hydrogen production rate (HPR), and hydrogen production potential (HPP) using cotton stalk hydrolysate as sugar substrate than the mixed sugar of glucose & xylose, which obtained the HY of 249.5 ± 29.0, 397.0 ± 36.7 mL·L-1, HPR of 10.4 ± 1.2, 16.5 ± 1.5 mL·L-1·h-1, HPP of 19.5 ± 2.3, 31.0 ± 2.8 mL·L-1·g-1sugar, separately. The generation of soluble metabolites, such as the lactate, formate, acetate, succinate and ethanol reflected the mixed acid fermentation properties of the hydrogen production pathway.

Effect of Chia Seed on Physicochemical and Sensory Characteristics of Common Carp Restructured as Functional Food

Physicochemical and sensory characteristics of restructured meat of common carp （Cyprinus carpio） fortified with 0-8 g/100 g of chia seed flour （CSF） was evaluated. It had a higher nutritional value （higher fibre content and protein retention] （p 〈 0.05） and better cooking characteristics （higher cooking yield and moisture retention） （p 〈 0.05） than the control. The colour （a＊, b＊） increased; lightness and whiteness index decrease （p 〈 0.05）. Hardness increase （p 〈 0.05） occurred because of CSF addition. Differential scanning calorimetry showed that fibre fortification did not interfere with the thermal transitions of the restructured meat. No significant differences were detected with the preference test scores of 4% or 8% CSF compared with the control. Restructured （4%-8% CSF） had a higher content of fibre and fat, which could be linoleic and linolenic acid, and an increase in the content of protein compared with those of commercial products, among had 1.62 and 2.25 mg AGE/g. Therefore, the restructured properties of common carp were governed by CSF addition.

Effect of Mercuric Chloride and Cadmium Chloride on Gonadal Function and Its Regulation in SexuallyMature Common Carp Cyprinus carpio

Gonadal function in fish, CJprinus 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) cholestcrol of ovary,liver and serum and ovarian 3β-Hydroxysteroid and 17β-Hydroxysteroid dehyrogenaseenzyme activity and serum and pituitary gonadotropin (GtH) levels. Both the pollutantswere 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 tothe pollutants had no significant effect on the gonadal function. ln addition to thedeleterious 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 expeure, HgCl2'had an adverse effect on the transport of cholesterol from circulation to ovary.

Parasitic Copepods on Common Carp(Cyprinus carpio,L.1758)from Gradche Reservoir(Macedonia)

During the parasitological examinations of the common carp(Cyprinus carpio,L.1758)from the Gradche Reservoir(Macedonia),a total of 126 fish samples were examined,from which parasite infestation was determined in 87 fish(69.05%).In this research,the following parasitic copepods were identified:Ergasilus sieboldi,Ergasilus briani,and Lernaea cyprinacea.Ergasilus sieboldi was found on gills of common carp,with a prevalence of 1.461%,while the mean intensity was 2.357.Ergasilus briani was found on fins of common carp,with a prevalence of 1.879%,while the mean intensity was 38.274.Lernaea cyprinacea was found on the fins of common carp,with a prevalence of 0.552%,while mean intensity was 2,000.Our findings of Ergasilus sieboldi,Ergasilus briani,and Lernaea cyprinacea in common carp are considered as first records for Macedonia.At the same time,common carp is a new host for Ergasilus briani in Macedonian waters.

Effects of solid-state fermentation product of yeast supplementation on liver and intestinal health,and resistance of common carp(Cyprinus carpio)against spring viraemia carp virus

This study aimed to investigate the effects of solid-state fermentation products of yeast(SFPY)on liver and intestinal health and disease resistance of common carp(Cyprinus carpio).A total of 200 common carp with an initial average weight of 2.55±0.004 g were divided into 5 groups(4 replications per group and 10 fish per replication),and were fed with one of five diets,including a control diet and 4 diets supplemented with 2‰(Y2),3‰(Y3),4‰(Y4),or 5‰(Y5)SFPY,respectively,for 8 weeks.Results indicated that,the addition of SFPY to the diet of common carp did not affect the growth performance or survival rate of fish(P=0.253).Interestingly,with the addition of SFPY,the triacylglycerol(TAG)content of the liver presented a linear decreasing tendency(P=0.004),with significantly decreased in Y4 and Y5 groups(P=0.035)compared with control.Serum lipopolysaccharide(LPS)content and diamine oxidase(DAO)activity presented a negative linear relationship with the addition of SFPY(P=0.015,P=0.030),while serum lipopolysaccharide binding protein(LBP)content first decreased and then increased(P<0.001).The total antioxidant capacity(T-AOC)in the intestine of fish increased continuously with increasing SFPY supplementation(P=0.026),reaching the highest level in Y5 group.The villus height in all experimental groups were significantly higher than that in the control group(P<0.001).Furthermore,compared to the control,adding 3‰SFPY to the control diet of common carp significantly increased the relative abundance of Fusobacteria(P=0.018)and decreased that of Proteobacteria(P=0.039)at phylum level,and increased the relative abundance of Cetobacterium(P=0.018)and decreased that of Shewanella(P=0.013)at genus level.Compared with the control,the relative mRNA expression level of spring viraemia of carp virus N protein(SVCV-n)in the kidney was lower than that of the control group without significance and bottomed out in Y4 group(P=0.138).In conclusion,dietary SFPY enhanced the SVCV resistance capacity of common carp by improving liver and intestinal health and modulating the gut microbiota.Thus,SFPY is a potential feed additive to be used in aquaculture to reduce the huge economic loss of common carp due to SVCV disease.Based on liver TAG content and intestinal villus height,the optimal addition level of SFPY was 3.02‰and 2.72‰,respectively.

不同电学参数和水流条件下电栅对鲤(Cyprinus carpio)的阻拦效果

脉冲直流电导阻鱼技术已被广泛应用于阻止入侵物种扩散、驱赶鱼类远离危险区域并将其引导至有利区域,如鱼道。但是脉冲直流电的有效性受多方面因素的影响,其主导因素目前尚未定论。以鲤为研究对象,在静水条件下采用4因素3水平正交实验方法分析了脉冲电压、脉冲宽度、脉冲频率和电极摆放方式的电栅拦鱼效果及其对鱼类受伤尾数的主次影响,然后选择最优组合开展不同流速下(0,0.3,0.5 m/s)的驱鱼实验,分析流速对拦鱼效果的影响。结果表明:在静水工况下,影响拦鱼电栅效率的主要因素为电极布置方式>脉冲宽度>脉冲频率>脉冲电压,电极布置为方式1(垂直水流间距80 cm)、脉冲电压140 V、脉冲频率10 Hz、脉冲宽度10 ms是拦鱼效果最佳的组合。电学参数对鲤的伤害无显著性,平均受伤尾数不超过实验鱼总数的15%。流水条件下,三种流速工况下拦鱼电栅的阻拦率无显著差异,但是随着流速的增高阻拦率呈现先降低后增加的趋势。该研究将为保护鱼类资源和防止入侵鱼类跨区域传播提供依据。

Cloning and expression analysis in mature individuals of two chicken type-Ⅱ GnRH (cGnRH-Ⅱ) genes in common carp (Cyprinus carpio)

Gonadotropin-releasing hormone (GnRH) is a conservative neurodecapeptide fam-ily, which plays a crucial role in regulating the gonad development and in controlling the final sexual maturation in vertebrate. Two differing cGnRH-II cDNAs of common carp, namely cGnRH-II cDNA1 and cDNA2, were firstly cloned from the brain by rapid amplification of cDNA end (RACE) and reverse transcription- polymerase chain reaction (RT-PCR). The length of cGnRH-II cDNA1 and cDNA2 was 622 and 578 base pairs (bp), respectively. The cGnRH-II pre-cursors encoded by two cDNAs consisted of 86 amino acids, including a signal peptide, cGnRH-II decapeptide and a GnRH-associated peptide (GAP) linked by a Gly-Lys-Arg proteolytic site. The results of intron trapping and Southern blot showed that two differing cGnRH-II genes in common carp genome were further identified, and that two genes might exist as a single copy. The multi-gene coding of common carp cGnRH-II gene offered novel evidence for gene duplica-tion hypothesis. Using semi-quantitative RT-PCR, expression and relative expression levels of cGnRH-II genes were detected in five dissected brain regions, pituitary and gonad of common carp. With the exception of no mRNA2 in ovary, two cGnRH-II genes could be expressed in all the detected tissues. However, expression levels showed an apparent difference in different brain regions, pituitary and gonad. According to the expression characterization of cGnRH-II genes in brain areas, it was presumed that cGnRH-II might mainly work as the neurotransmitter and neuromodulator and also operate in the regulation for the GnRH releasing. Then, the ex-pression of cGnRH-II genes in pituitary and gonad suggested that cGnRH-II might act as the autocrine or paracrine regulator.