The roles of alpha- and beta-adrenoceptors in the chronotropic responses to norepinephrine in carp heart (Cyprinus carpio)

1. The chronotropic effect of norepinephrine was studied in isolated spontaneously beating atrial preparations of carp (Cyprinus carpio) heart. 2. Norepinephrine, 0.1 microM, caused a positive chronotropic effect, while at 1 microM it caused either a positive or a negative chronotropic effect. The positive chronotropic effect, observed in 13 preparations, was potentiated by phentolamine and almost completely blocked by propranolol. 3. The negative chronotropic effect observed in the other 5 preparations was greater in the presence of propranolol, reduced by phentolamine and not affected by atropine. 4. These results indicate that alpha- and beta-adrenoceptors may coexist, mediating the negative and positive chronotropic effects, respectively, in isolated atrial preparations of carp heart.     

鲤鱼生长激素在毕赤酵母中的表达

 将编码鲤鱼 (Cyprinuscarpio )生长激素 (GH)成熟肽的cDNA克隆到毕赤酵母 (P .pastoris)胞内表达载体pHIL D2中 ,构建重组表达质粒pHIL D2 GH .转化组氨酸缺陷型酵母GS115,获得表达鲤鱼GH的酵母工程菌 .经甲醇诱导 ,SDS PAGE和Western印迹检测表明 ,鲤鱼GH在酵母中得到表达 ,表达产物在胞内以可溶状态存在 ,具有鲤鱼GH的免疫活性 .用诱导后的酵母投喂罗非鱼 ,实验结果证实所构建的工程菌具有明显的促生长作用     

应用微卫星多态分析四个鲤鱼群体的遗传多样性

选择12个斑马鱼功能基因的微卫星标记和12个鲤鱼微卫星标记,检测黑龙江鲤(Cyprinus carpiohaematopterusTemminck et Schlegel)、散鳞镜鲤(C.carpio)、荷包红鲤抗寒品系(C.carpiovar.wuyuanensis)、松浦鲤(C.carpioSongpu carp)的群体遗传多样性。共检测到3882个扩增片段,长度为126～489bp,在群体间扩增出1～5个等位基因不等,共计59个等位基因,平均等位基因2.46个。数据经PHYLIP V3.6软件估算和MEGA3软件作图,确立4个群体间的亲缘关系。并应用Bootstrap检验估计系统树中节点的自引导值,并进行了系统发生分析。结果表明①4个群体检测的有效等位基因数都在55个以上,平均观测杂合度为0.36～0.43,平均期望杂合度为0.49～0.53,平均多态信息含量为0.21～0.25,说明这几个群体多态性属于中度偏高水平,遗传多样性较高;②群体间相似系数都在0.84以上,遗传距离较近,为0.067～0.170,与前人研究结果一致。聚类分析显示,松浦鲤与散鳞镜鲤亲缘关系最近,荷包红鲤抗寒品系与它们的亲缘关系较黑龙江鲤更近;③在3个斑马鱼功能基因相关的微卫星位点上,黑龙江鲤缺失特异扩增条带,这可能与几种鱼类的育成史有关。     

Genetic divergence between Cyprinus carpio carpio and Cyprinus carpio haematopterus as assessed by mitochondrial DNA analysis,with emphasis on origin of European domestic carp

Although common carp is the major fish species in Asian and European aquaculture and many domestic varieties have occurred, there is a controversy about the origination of European domestic common carp. Some scientists affirmed that the ancestor of European domestic common carp was Danube River wild common carp, but others considered it might be Asian common carp. For elucidating origination of European domestic common carp, we chose two representative European domestic common carp strains (German mirror carp and Russian scattered scaled mirror carp) and one wild common carp strain of Cyprinus carpio carpio subspecies (Volga River wild common carp) and two Asian common carp strains, the Yangtze River wild common carp (Cyprinus carpio haematopterus) and traditionally domestic Xingguo red common carp, as experimental materials. ND5–ND6 and D-loop segments of mitochondrial DNA were amplified by polymerase chain reaction and analyzed through restriction fragment length polymorphism (RFLP) and sequencing respectively. The results revealed that HaeIII and DdeI digestion patterns of ND5–ND6 segment and sequences of control region were different between European subspecies C. carpio carpio and Asian subspecies C. carpio haematopterus. Phylogenetic analysis showed that German mirror carp and Russian scattered scaled mirror carp belonged to two subspecies, C. carpio carpio and C. carpio haematopterus, respectively. Therefore, there were different ancestors for domestic carp in Europe: German mirror carp was domesticated from European subspecies C. carpio carpio and Russian scattered scaled mirror carp originated from Asian subspecies C. carpio haematopterus.     

The Efficacy of Chromium as a Growth Enhancer for Mirror Carp (Cyprinus carpio L): An Integrated Study Using Biochemical, Genetic, and Histological Responses

A growth trial was conducted on juvenile mirror carp (Cyprinus carpio L.) for 8 weeks to compare the efficacy of three chromium (Cr) compounds (Cr chloride, Cr picolinate, and Cr yeast) at a level 0.5 mg/kg as a potential growth enhancer. In addition, a high level of Cr (2.0 mg/kg) as Cr chloride has also been added in parallel for comparison. All Cr fortified diets at a level 0.5 mg/kg produced superior growth for carp compared to the control group and the group fed the high level of Cr chloride (2.0 mg/kg). Metabolic indicators measured included two of the key liver enzymes (hexokinase, HK) and (glucose-6-phosphate dehydrogenase, G6PD) activity. The results validated the positive effect of Cr at a level 0.5 mg/kg on enzyme activity and carbohydrate utilization producing significantly better growth performance for mirror carp. The study also included measurement of DNA strand breaks in the erythrocytes using the comet assay which revealed significantly (P < 0.05) increased DNA damage in fish fed on high level of Cr chloride (2.0 mg/kg) but the other treatments were not significantly different (P > 0.05) from the control groups. The concentration of Cr in the liver, gut, and whole fish tissues increased with increasing dietary Cr supplementation. Overall, Cr supplementation at a level 0.5 mg/kg from different sources may affect growth performance in carp by activation of some key liver enzymes (HK and G6PD).     

Molecular phylogeny of three subspecies of common carp Cyprinus carpio, based on sequence analysis of cytochrome b and control region of mtDNA

The complete cytochrome b and the control region of mtDNA (about 2070 bp in total) of 10 strains belonging to three subspecies of the common carp, including three wild subspecies (the Yangtze River wild common carp – Cyprinus carpio haematopterus , Yuanjiang River wild common carp – Cyprinus carpio rubrofuscus and Volga River wild common carp – Cyprinus carpio carpio ) and seven domestic strains (Xingguo red carp, Russian scattered scaled mirror carp, Qingtian carp, Japanese Koi carp, purse red carp, Big-belly carp, German mirror carp) were sequenced. Phylogenetic analysis indicated that the 10 strains form three distinct clades, corresponding to C. c. haematopterus , C. c. rubrofuscus and C. c. carpio respectively. Purse red carp, an endemic domestic strain in Jiangxi province of China, showed a higher evolution rate in comparison with the other strains of C. c. haematopterus , most probably because of intensive selection and a long history of domestication. Base variation ratios among the three subspecies varied from 0.78% (between C. c. haematopterus and C. c. rubrofuscus ) to 1.47%(between C. c. carpio and C. c. rubrofuscus ). The topography of the phylogenetic tree and the geographic distribution of three subspecies closely resemble each other. The divergence time between C. c. carpio and the other two subspecies was estimated to be about 0.9 Myr and about 0.5 Myr between C. c. haematopterus and C. c. rubrofuscus . Based on phylogenetic analysis, C. c. rubrofuscus might have diverged from C. c. haematopterus .     

基于线粒体D-loop区和Cyt b基因分析广西禾花鲤三个群体遗传结构

研究基于线粒体D-loop区和Cyt b基因部分序列, 分析了广西全州、融水、环江三地禾花鲤(Cyprinus carpio)的遗传结构。在3个群体中共鉴定到19种D-loop与Cyt b序列的组合单倍型, 总的单倍型多样性(H_d)和核苷酸多样性(π)分别为0.916±0.010和0.008±0.004, 禾花鲤线粒体DNA具有单倍型多样性高和核苷酸多样性低的特点。环江群体单倍型多样性最低, 但是核苷酸多样性却最高, 反映了环江群体存在最明显的谱系混杂。分子方差分析(AMOVA)显示遗传变异主要来源于群体内部(71.54%), 禾花鲤三个群体间有显著的遗传分化(F_st=0.285; P<0.01)。系统发育分析表明, 多数样本的线粒体单倍型属于华南鲤(C. carpio rubrefusus)类型, 同时三地禾花鲤中均检测到一定频率的西鲤(C. carpio carpio)或远东鲤(C. carpio haematopterus)类型的线粒体单倍型, 提示禾花鲤可能受到鲤养殖品种的杂交渐渗。研究初步揭示了广西禾花鲤的种质资源现状, 为禾花鲤这一地方特色“稻鱼共作”品种的选育和苗种管理提供了必要的依据。     

从江及周边地区稻田鲤的遗传结构及系统发育关系

为探究贵州从江及邻近的榕江、黎平和锦屏以及广西三江、湖南的通道和靖州等地稻田鲤Cyprinus carpio Linnaeus的遗传结构及系统关系, 研究基于mtDNA Cyt b基因测序技术对6个地理群体进行了分析。447尾稻田鲤中共界定出29种Cyt b基因序列单倍型, 结合NCBI中鲤同源序列的系统发育和序列比对分析表明, 从江及邻近苗侗族地区稻田鲤是一个含欧亚2大谱系、3个鲤亚种和多个母系血统成分的混合群体, 主体类群是华南鲤C. carpio rubrofuscus, 同时混有欧洲鲤C. carpio carpio和远东鲤C. carpio haematopterus, 遭受国内外鲤养殖品种的遗传渗析。遗传多样性分析显示, 欧洲鲤亚种群体为低H_d低π, 遗传多样性贫乏, 远东鲤和华南鲤2个亚种群体呈高H_d低π特点。华南鲤6个稻田群体除锦屏为低H_d低π外, 其余都为高H_d低π, 从江的H_d最高, 黎平的π最高。遗传分化和分子方差(AMOVA)分析显示, 谱系间和亚种间存在显著的遗传分化(P<0.001), 锦屏和三江与其他群体存在中等-高度的分化等级, 其余群体间分化较弱, 群体间变异贡献率为16.69%, 群体内为81.14%。综合分析表明, 从江及邻近地区稻田鲤群体间存在有限的遗传交流, 群体内和群体间有不同的遗传结构。研究丰富了该地区稻田鲤遗传背景资料, 可为湘黔桂交界苗侗族地区稻田鲤的种质资源鉴定与保护提供理论依据。     

用PCR-DGGE对岩原鲤×锦鲤杂交F1代进行分子鉴定

运用变性梯度凝胶电泳(DGGE)技术,选用核基因光间受体视黄类物质结合蛋白(IRBP)作为分子标记,成功鉴定了岩原鲤Procypris rabaudi×锦鲤Cyprinus carpio的杂交F1代.DGGE胶图显示,杂交个体IRBP基因的等位基因条带分别与岩原鲤和锦鲤的IRBP基因条带相对应.对各条带切胶回收后的测序表明,岩原鲤等位基因的相似性为99.8％,锦鲤等位基因的相似性为98.8％,而杂交个体等位基因的相似性为95.8％ ～96.7％,与岩原鲤和锦鲤间基因的相似性(95.5％～96.9％)接近.这一方法将有助于鱼类的杂种鉴定和种质研究.     

Protection against experimental Aeromonas salmonicida infection in carp by oral immunisation with bacterial antigen entrapped liposomes

Liposome-entrapped atypical Aeromonas salmonicida antigen was prepared to investigate the potential protective efficacy for A. salmonicida infection. Carp (Cyprinus carpio) were immunised orally with liposome-entrapped A. salmonicida antigen. After immunisation, significantly higher antigen-specific antibodies were detected in serum, intestinal mucus and bile than non-immunised control group. Furthermore, immunised carp were challenged by immersion with 1 x 10(6) cfu ml(-1) of A. salmonicida for 60 min. Of the eight non-immunised carp, three carp died (62.5% survival), whereas five out of six (83.5%) immunised survived. Furthermore, the development of skin ulcers was significantly inhibited in carp immunised with liposomes containing A. salmonicida antigen. These results suggest that liposomes containing A. salmonicida antigen have the potential for the induction of a protective immune response against atypical A. salmonicida infection and also suggest the possibility of developing a vaccine that may ultimately be used for the prevention of fish diseases.     

Cytological features of serotonin-containing neurons and their processes in the retina of the carp (Cyprinus carpio). An immunohistochemical study using flat-mount preparations

The morphological features of serotonin-containing neurons (SCNs) and their processes in the retina of the carp (Cyprinus carpio) were immunohistochemically studied by applying a modified peroxidase-antiperoxidase technique using flat-mount preparations. The somata of immunoreactive SCNs were mostly located in the innermost part of the inner nuclear layer (INL). These cells were distributed at a density of 64.8 cells/mm2, this being similar to the density of dopamine-containing interplexiform cells. The processes of the SCNs ramified successively into two finer branches, eventually forming a broad, extensive network in the thin layer just subjacent to the plane of the somata of the SCNs. Processes originating from neighboring SCNs exhibited cytoplasmatic continuity with each other at the lightmicroscope level. Due to their location and cytological features, these SCNs appeared to correspond to amacrine cells.     

Koi herpesvirus disease

Koi herpesvirus (KHV) disease emerged at the late 1990s, and has rapidly spread to the world. In Japan, KHV disease first occurred at October 2003. The disease resulted in mass mortality of wild carp as well as cultured carp. Until now, KHV-infected carp were found in 42 out of 47 prefectures in Japan. Only carp Cyprinus carpio is susceptible to KHV, while goldfish, closely-related species to carp, is not. The affected carp swim lethargically. Sunken eyes and gill necrosis are frequently noticed, but no marked internal signs are observed. Optimal water temperature for the disease is 18-23 degrees C. Under 13 degrees C or over 28 degrees C, no death occurs. Keep at over 30 degrees C cures KHV disease, but can make the fish latent carriers. Because the fish do not get acquired immunity against KHV disease under low water temperature, the disease recurs with increase of water temperature. Isolation of KHV is difficult. KHV disease is diagnosed through epidemiological investigation, disease signs and PCR detection of KHV DNA. Vaccine development is ongoing for restart of culturing carp at KHV-contaminated places.     

Genetic diversity and population structure inferred from the partially duplicated genome of domesticated carp,Cyprinus carpio L.

Genetic relationships among eight populations of domesticated carp (Cyprinus carpio L.), a species with a partially duplicated genome, were studied using 12 microsatellites and 505 AFLP bands. The populations included three aquacultured carp strains and five ornamental carp (koi) variants. Grass carp (Ctenopharyngodon idella) was used as an outgroup. AFLP-based gene diversity varied from 5% (grass carp) to 32% (koi) and reflected the reasonably well understood histories and breeding practices of the populations. A large fraction of the molecular variance was due to differences between aquacultured and ornamental carps. Further analyses based on microsatellite data, including cluster analysis and neighbor-joining trees, supported the genetic distinctiveness of aquacultured and ornamental carps, despite the recent divergence of the two groups. In contrast to what was observed for AFLP-based diversity, the frequency of heterozygotes based on microsatellites was comparable among all populations. This discrepancy can potentially be explained by duplication of some loci in Cyprinus carpio L., and a model that shows how duplication can increase heterozygosity estimates for microsatellites but not for AFLP loci is discussed. Our analyses in carp can help in understanding the consequences of genotyping duplicated loci and in interpreting discrepancies between dominant and co-dominant markers in species with recent genome duplication.     

Formation of muscle proteins during freezing

The effect of different conditions on the formation and properties of cryogels prepared by the freezing-thawing procedure from suspensions and solutions of the carp (Cyprinus carpio) myofibrillar proteins was studied. The freezing of water solutions and suspensions of the native myofibrillar proteins resulted in the formation of the structures mainly stabilized by non-covalent bonds. When muscle proteins were denatured prior to the freezing they formed the structures stabilized by both non-covalent and covalent disulfide bonds.     

The shift from C-19 to C-21 steroid synthesis in spawning male common carp, Cyprinus carpio, is regulated by the inhibition of androgen production by progestogens produced by spermatozoa

There is a rapid shift in the steroidogenic pathway from androgen to progestogen production in spawning male common carp, Cyprinus carpio. Experiments were conducted to determine the mechanism regulating this shift using in vitro cultures of testicular fragments and isolated sperm of spermiating male carp. The levels of 11-ketotestosterone (11-KT) continually increased for 48 h with or without gonadotropin (GtH) stimulation, suggesting that 11-KT is the principal androgen produced by carp testes. Ovine prolactin (oPRL) enhanced GtH-stimulated 11-KT production, but by itself had no effect. Gonadotropin, carp pituitary extract, and pregnenolone all enhanced the production of 11-KT, testosterone (T), and 17 alpha-hydroxyprogesterone (17-P) in a dose-dependent manner. No 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-P) was detected in response to any of these agents; 17 alpha,20 alpha-dihydroxy-4-pregnen-3-one (17,20 alpha-P) was not measured. Both 17,20 beta-P and 17,20 alpha-P inhibited 11-KT production in a dose-dependent manner in the presence of either GtH, 17-P, or T. Isolated sperm and testicular fragment preparations both produced 17,20 beta-P and approximately tenfold more 17,20 alpha-P when incubated with 17-P. Only testicular fragments, however, produced 11-KT. We conclude that androgen synthesis occurs only within somatic cells of common carp testes. GtH, and perhaps PRL, stimulates the production of steroid precursors that, under normal physiological conditions, are metabolized to androgens.(ABSTRACT TRUNCATED AT 250 WORDS)     

四种鲤科鱼肠道中高血糖素免疫活性内分泌细胞的研究

应用过氧化物酶抗过氧化物酶(PAP)免疫组织化学技术,用4种哺乳动物中培育出的抗血清对草鱼、青鱼、鲤和翘嘴红鲌4种鲤科鱼的肠内分泌细胞进行免疫、组织化学的鉴别和定位.证实了高血糖素免疫活性内分泌细胞在草鱼整个肠道中均呈阳性反应;在鲤、青鱼前肠中仅有少量阳性反应;在翘嘴红鲌肠道中未见阳性反应。胃蛋白酶原(Pepsinogen)、凝乳酶(Prochymosin)和神经特异烯醇酶(Neuron specific enolase)3种抗血清在4种鱼的肠道中均未发现阳性反应.本文重点描述草鱼高血糖素免疫活性内分泌细胞在肠道各段的分布,密度及其形态学特征,还就其可能的生理功能与草食性哺乳动物胃肠道中该类内分泌细胞进行比较和讨论。     

Comparison of the effects of gonadotropic preparations of the carp and stellate sturgeon pituitaries on in vivo and in vitro oocyte maturation in the Siberian sturgeon Acipenser baeri brandt

Injections of 2.5 mg/kg of stellate sturgeon (Acipenser stellatus Pall.) pituitary extract and 5 mg/kg of carp (Cyprinus carpio L.) pituitary extract in Siberian sturgeon (Acipenser baeri Brandt) females did not reveal significant differences in the effects of these preparations. There were no differences in the percentage of females that responded by ovulation, duration of the period from injection to ovulation, rate of ovulation, or quality of mature eggs as estimated by the percentage of fertilization or percentage of normal embryos at the small yolk plug stage. Thus, an insufficient efficiency in the artificial reproduction of the Siberian sturgeon grown in captivity is not related to the use of the carp pituitary preparation as a stimulus. Estimation of the ratio of specific activities of the pituitary extracts and purified gonadotropins of the stellate sturgeon and carp by in vitro oocyte maturation has shown that it varies within wide limits as a function of the medium composition and physiological state of follicles. Hence, the ratio of activities of the gonadotropins of different species as determined by in vitro maturation of sturgeon oocytes may markedly differ from that upon injection of these preparations in breeders.     

Development of 10 tri- and tetranucleotide microsatellite loci for population studies in the common carp (Cyprinus carpio L.)

Ten tri- and tetranucleotide microsatellite DNA markers were isolated and characterized from common carp (Cyprinus carpio L.) to estimate genetic potential. These markers were tested in the samples from two closely related carp populations (Cyprinus carpio var. xingguonensis and Cyprinus carpio var. wananensis). The number of the alleles ranged from three to nine, and observed and expected hererozygosities varied from 0.207 to 1.000 and from 0.499 to 0.900 in each population, respectively. No evidence for linkage disequilibrium was found, indicating that these markers will be useful for population studies.     

Structural differences between the Ca2+-ATPase enzymes of sarcoplasmic reticulum membrane from rabbit and carp muscles

1. Structural features were compared in sarcoplasmic reticulum Ca2+-ATPase enzymes from carp (Cyprinus carpio L.) and rabbit muscles. 2. Both membrane preparations contained the 105,000 mol. wt Ca2+ pump protein in high local density. 3. The tryptic cleavage of the carp enzyme gave different peptide fragments from those observed from rabbit enzyme. 4. Addition of vanadate, Ca2+ or lanthanides did not cause two-dimensional Ca2+-ATPase crystal formation, in contrast to the rabbit enzyme, which forms extensive arrays under these conditions. 5. No differences were found in this respect between microsome preparations derived from warm and cold adapted fishes. 6. A different primary sequence as well as a different disposition of the enzyme in the membrane may stand behind the observed dissimilarities.