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.

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.

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

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）.

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 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.

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

表示顺序标注的普通鲤鱼(EST ) 为存在 ofmicrosatellites，或简单顺序重复(SSR ) 被分析。在 NCBI dbEST 数据库， 10612sequences 的一个总数在 2004 年 12 月 31 日前被登记。2-6 nucleotidemicrosatellites 的完全的搜索导致了 513 包含 SSR EST 的鉴定，为 4.8% 总数的财务。聚合分析显示包含 SSR EST 的 73 个序列掉进 27 个组并且留下 440 EST 是独立的。467 唯一的包含 SSR EST 的一个总数被识别。这些 EST-SSRs 包含了许多简单顺序类型，并且 di-nucleotiderepeats 和 tri-nucleotiderepeats 是最丰富的，为 42.1% 全部和 27.9% 的财务分别地。thedinucleotide 重复， CA/TG 是最丰富的，由 GA/TC 列在后面。BLASTx 搜索证明 SSR loci 的 that38.1% 能与基因或蛋白质被联系知道或包含 SSR EST 的未知 function.BLASTx 搜索也在 zebrafishsequences 上显示出点击的高频率(98/179 ) 。

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.

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。

Enzyme-linked immunosorbent assay of changes in serum levels of growth hormone(cGH)in common carps(Cyprinus carpio)

The aim of the present study was to purify the common native carp growth hormone (ncGH), produce monoclonal antibodies (mAbs) to common native carp growth hormone (ncGH), and further enhance the sensitivity of enzyme-linked immunosorbent assays (ELISA) for ncGH. Additionally, we investigated changes in serum ncGH levels in carps raised in different environmental conditions. The recombinant grass carp (Ctenopharyngodon idella) growth hormone was purified and used as antigen to immunize the rabbit. The natural ncGH was isolated from the pituitaries of common carp. SDS-PAGE and Western blot utilizing the polyclonal anti-rgcGH antibody confirmed the purification of ncGH from pituitaries. Purified ncGH was then used as an immunogen in the B lymphocyte hybridoma technique. A total of 14 hybridoma cell lines (FMU-cGH 1-14) were established that were able to stably secrete mAbs against ncGH. Among them, eight clones (FMU-cGH1-6, 12 and 13) were successfully used for Western blot while nine clones (FMU-cGH 1-7, 9 and 10) were used in fluorescent staining and immunohistochemistry. Epitope mapping by competitive ELISA demonstrated that these mAbs recognized five different epitopes. A sensitive sandwich ELISA for detection of ncGH was developed using FMU-cGH12 as the coating mAb and FMU-cGH6 as the enzyme labeled mAb. This detection system was found to be highly stable and sensitive, with detection levels of 70 pg/mL. Additionally, we found that serum ncGH levels in restricted food group and in the net cage group increased 6.9-and 5.8-fold, respectively, when compared to controls, demonstrating differences in the GH stress response in common carp under different living conditions.

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.

Acute and chronic un-ionized ammonia toxicity to ‘all-fish’ growth hormone transgenic common carp(Cyprinus carpio L.)

Ammonia is toxic to fish in natural and artificial waters.We evaluated the acute(96 h) and chronic(21 d) toxicity of un-ionized ammonia to GH transgenic common carp(Cyprinus carpio L.) and non-transgenic common carp using a static-renewal bioassay.The 24,48,72 and 96 h median lethal concentrations(LC50) of un-ionized ammonia were slightly lower in transgenic carp(2.64,2.44,2.28 and 2.16 mg N/L,respectively) than in non-transgenic carp(2.70,2.64,2.52 and 2.33 mg N/L,respectively).Similarly,the median lethal time(LT50) was significantly shorter for transgenic carp(1.41,7.91 and 117.42 h) than for non-transgenic common carp(2.53,14.06 and 150.44 h) following exposure to 3.86,3.29,or 2.09 mg N/L,respectively.Moreover,the mortality of transgenic carp was significantly higher than that of non-transgenic carp at all un-ionized ammonia concentrations((0.91 ± 0.12),(0.48 ± 0.06) and(0.12 ± 0.01) mg N/L) during the 21 d chronic toxicity test.Our results suggest that GH transgenic carp are less tolerant of unionized ammonia than non-transgenic carp.Our data are useful for evaluating potential environmental risk,optimizing stocking density in intensive aquaculture and establishing water quality criteria for ammonia in aquaculture.

A consensus linkage map of common carp (Cyprinus carpio L.) to compare the distribution and variation of QTLs associated with growth traits

The ability to detect and identify quantitative trait loci (QTLs) in a single population is often limited. Analyzing multiple populations in QTL analysis improves the power of detecting QTLs and provides a better understanding of their functional allelic variation and distribution. In this study, a consensus map of the common carp was constructed, based on four populations, to compare the distribution and variation of QTLs. The consensus map spans 2371.6 cM across the 42 linkage groups and comprises 257 microsatellites and 421 SNPs, with a mean marker interval of 3.7 cM/marker. Sixty-seven QTLs affecting four growth traits from the four populations were mapped to the consensus map. Only one QTL was common to three populations, and nine QTLs were detected in two populations. However, no QTL was common to all four populations. The results of the QTL comparison suggest that the QTLs are responsible for the phenotypic variability observed for these traits in a broad array of common carp germplasms. The study also reveals the different genetic performances between major and minor genes in different populations.