Identification of a Spindlin homolog in gibel carp (Carassius auratus gibelio)

Spindlin has been suggested to play an important role during the transition from oocyte maturation to embryo development in mouse, but its homolog similar to the mouse Spindlin in molecular and expression characterization has not been identified up to now in other vertebrates. In this study, a full length of cDNA sequence is cloned and sequenced from the gibel carp (Carassius auratus gibelio). It contains 1240 nucleotides with an open reading frame of 771 nt encoding 257 amino acids. Based on its amino acid sequence alignment and comparison analysis with the known Spin family proteins, the newly cloned Spin is named Carassius auratus gibelio Spindlin (CagSpin). Its product could be detected from mature eggs to blastula embryos, but its content decreased from the two-cell stage, and could not be detected after the gastrula stage. It suggests that the CagSpin should be a maternal protein that is expressed during oocyte maturation, and plays a crucial role in early cleavage of embryogenesis. CagSpin is the first homolog similar to mouse spindlin identified in fish, and also in other vertebrates. GST pull-down assay reveals the first biochemical evidence for the association of CagSpin and beta-tubulin, the microtubule component. Therefore, CagSpin may play important functions by interacting with beta-tubulin and other spindle proteins during oocyte maturation and egg fertilization.     

Utilization of several plant proteins by gibel carp (Carassius auratus gibelio)

Six isonitrogenous (gross protein content 35%) and isoenergetic (gross energy content 17 kJ g⁻¹) diets were formulated to investigate the effects of inclusion of plant proteins on the gibel carp ( Carassius auratus gibelio L.). The plant proteins tested were: soybean cake (SBC), potato protein concentrate (PPC), peanut cake (PNC), cottonseed cake (CSC) and rapeseed cake (RSC). Fish meal (FM) was used as control. In each diet, 27% of the protein was supplied by fish meal, and the rest supplied by the plant protein tested. Each diet was fed to three groups of gibel carp for 8 weeks in a recirculation system. Specific growth rate (SGR) in fish fed the control diet was significantly higher than those in the other groups, and SGR in fish fed the PPC was significantly lower than in fish fed other plant proteins. There was no significant difference in SGR among the other groups. Feeding rates were ranked in the order: RSC > CSC > FM > PNC > SBC > PPC. Conversion efficiency was highest in groups fed FM, SBC and PNC, followed by groups fed CSC and RSC, and was lowest in the group fed PPC. The fish fed PPC showed lower protein retention than those fed FM and SBC. FM showed highest energy retention while PPC showed lowest. There was no significant relationship between SGR and intake of digestible protein (g g⁻¹ day⁻¹), digestible lysine (g g⁻¹ day⁻¹), digestible methionine (g g⁻¹ day⁻¹) or digestible total essential amino acids (g g⁻¹ day⁻¹), suggesting that the differences in SGR could not alone account for any of these variables.     

Effect of feeding frequency on growth, feed utilization, and size variation of juvenile gibel carp (Carassius auratus gibelio)

Juvenile (3.0 ± 0.2 g) gibel carp (Carassius auratus gibelio) were fed to satiation for 8 weeks to investigate the effect of feeding frequency on growth, feed utilization and size variation. Five feeding frequencies were tested: two meals per day (M2), three meals per day (M3), four meals per day (M4), 12 meals per day (M12) and 24 meals per day (M24). The results showed that daily food intake increased significantly with the increase in feeding frequency and there was no significant difference between daily food intakes in M12 and M24 treatments. Growth rate, feed efficiency increased significantly with increasing feeding frequencies. Size variation was not affected by feeding frequency. Apparent digestibility of dry matter was not influenced by feeding frequency, while apparent digestibility of protein and energy increased significantly at high feeding frequencies. The feeding frequency had no significant effect on the moisture, lipid, protein, or energy contents of gibel carp, while the ash content decreased with increased feeding frequency. It was recommended that 24 meals per day was the optimal feeding frequency for juvenile gibel carp.     

Karyotypic diversity in polyploid gibel carp,Carassius auratus gibelio Bloch

Polyploid gibel carp, Carassius auratus gibelio, is an excellent model system for evolutionary genetics owing to its specific genetic background and reproductive modes. Comparative karyotype studies were performed in three cultured clones, one artificially manipulated group, and one mated group between two clones. Both the clones A and P had 156 chromosomes in their karyotypes, with 36 metacentric, 54 submetacentric, 36 subtelocentric, 24 acrocentric, and six small chromosomes. The karyotype of clone D contained 162 chromosomes, with 42 metacentric, 54 submetacentric, 36 subtelocentric, 24 acrocentric, and six small chromosomes. All the three clones had six small chromosomes in common. Group G, being originated from the clone D by artificial manipulation, showed supernumerary microchromosomes or chromosomal fragments, in addition to the normal chromosome complement that was identical to the clone D. The offspring from mating between clones D and A had 159 chromosomes. Comparing with the clone A, the DA offspring showed three extra metacentric chromosomes. In addition, variable RAPD fingerprint patterns and unusual SCAR marker inheritance were, respectively, detected among individuals of artificial group G and in the mated DA offspring. Both the chromosome and molecular findings suggest that genome reshuffling might have occurred by manipulation or mating of the clones.     

粪便收集时间和饲料中肉骨粉含量对异育银鲫(Carassius auratus gibelio) 消化率的影响

本研究探讨了通过收集器不同粪便收集时间和饲料中肉骨粉(MBM)含量对异育银鲫干物质、蛋白、能量、磷的表观消化率 (ADC) 影响.不同梯度(0, 20, 40, 60, 80 ,100%)的肉骨粉替代鱼粉(FM) 蛋白配制成六种等氮 (粗蛋白: 410 g/kg) 等能 (总能: 18 kJ/g) 的饲料,通过11周的饲养实验,实验开始后2周开始收集粪便,收集时间分别是:排粪后 1 min, 投喂后4h和16h. 结果表明,干物质、蛋白、能量、磷的消化率明显随粪便收集时间增加而升高(p<0.05),但在高肉骨粉替代饲料中差异不显著(p>0.05). 干物质、蛋白、能量、磷的消化率随饲料中肉骨粉含量的增加呈线性或近线性下降.因此,在消化率测定中应该尽快收集排出的粪便以保证消化率的真实性.消化率是影响肉骨粉利用一个因素.     

Effect of inclusion of blue-green algae meal on growth and accumulation of microcystins in gibel carp (Carassius auratus gibelio)

Six isonitrogenous (crude protein content: 38%) and isoenergetic (gross energy content: 17 kJ g^?1) diets were formulated to investigate the effects of inclusion of blue‐green algae meal on gibel carp (Carassius auratus gibelio). In each diet, 15% of the protein was supplied by fishmeal; the remainder was supplied by soybean meal and blue‐green algae meal. Diet 1 was used as control with no blue‐green algae meal whereas the content in diets 2–6 was 15.15, 29.79, 44.69, 59.58 and 74.48%, respectively. Each diet was fed to five groups of gibel carp for 12 weeks in a flow‐through system. Final body weight and specific growth rate (SGR) of fish fed diet 5 were significantly lower than the control diet (P < 0.05). Mortality of gibel carp increased with increase in algae meal inclusion (P < 0.05), but there was no significant difference between fish fed diets 3–6 (P > 0.05). Feed conversion efficiency (FCE) decreased with the increase in algae meal inclusion (P < 0.05). Fish‐fed diet 6 showed the highest feeding rate (P < 0.05), while there were no significant differences among the other groups (P > 0.05). Apparent digestibility coefficient of dry matter, protein, and energy decreased with increasing algae meal inclusion in the diets (P < 0.05). Aspartate aminotransferase (GOT) activity in the liver was not significantly different among groups (P > 0.05). Liver alanine aminotransferase (GPT) activity of fish‐fed diets 4, 5 and 6 was significantly lower than the control diet (diet 1; P < 0.05). Microcystins in the muscle, liver, gallbladder, and spleen increased with increasing algae inclusion (P < 0.05).     

饲料中豆粕替代鱼粉蛋白对异育银鲫生长、代谢及免疫功能的影响

本实验评价了饲料中豆粕替代鱼粉蛋白后对异育银鲫的生长、饲料利用、氮代谢和鱼体免疫力等的影响。实验设计4种等氮等能的饲料,每种3个重复,分别以豆粕替代饲料中鱼粉蛋白的0%（对照,D1）、20%（D2）、80%（D3）和100%（D4）。实验在半循环水养殖系统持续16周,鱼的初重约2.32g,实验期间水温23－30℃。结果表明,随着饲料中豆粕含量的升高,摄食率显著升高(P＜0.05),特定生长率、饲料转化效率、蛋白沉积率和能量沉积率显著降低(P＜0.05);蛋白表观消化率显著升高,干物质和能量表观消化率则显著降低(P＜0.05);总氮摄入量、表观氮摄入量、粪氮排出量、非粪氮排泄量、总氮沉积率均随着饲料中豆粕含量的升高呈显著降低到的趋势(P< 0.05),生产每千克鱼的氮排放量则随着饲料中豆粕含量的升高显著升高(P< 0.05);血清葡萄糖和甘油三酯的含量显著升高,而胆固醇的含量显著降低(P＜0.05);血清的溶菌酶显著降低,超氧化物岐化酶逐渐升高(P＜0.05)。     

Molecular identification of hybrids of the invasive gibel carp Carassius auratus gibelio and crucian carp Carassius carassius in Swedish waters

Both mitochondrial DNA sequence and two nuclear microsatellite markers were used to confirm the identity of the first record of Carassius auratus gibelio in the western (Swedish) Baltic Sea region. A total of 49 fishes were analysed, where 22 were from three Swedish sites connected to the Baltic Sea. The D-loop mitochondrial DNA sequences showed that 16 of 22 Swedish fishes were related to C. a. gibelio. The phylogenetic analysis of these sequences showed that these fish are probably not native, but represent different lineages of C. a. gibelio from China, Japan and Russia. All except three of these 16 fishes had microsatellite alleles suggesting hybridization with Carassius carassius. These findings suggest that a cryptic invasion of C. a. gibelio might be in progress.     

异育银鲫养殖环境典型病原微生物检测和细菌群落解析

【背景】水产养殖病害是限制淡水渔业发展的一个重要因素,养殖环境的微生物状况与鱼类健康紧密相关,已引起人们广泛关注。【目的】阐明养殖水体和沉积物中病原微生物丰度和细菌群落多样性变化特征。【方法】利用荧光定量PCR方法和末端限制性片段长度多态性(Terminal restriction fragment length polymorphism,T-RFLP)技术,对异育银鲫养殖环境水体和沉积物中的典型病原微生物进行定量检测,以及细菌群落多样性分析。【结果】养殖过程中病原菌丰度呈现不同的变化趋势,嗜水气单胞菌(Aeromonas hydrophila)、温和气单胞菌(A. sobria)和维氏气单胞菌(A. veronii)与鲤疱疹病毒Ⅱ型丰度变化呈显著正相关。水体中病原菌丰度受pH、温度和溶解氧等环境因子的影响显著。T-RFLP分析表明沉积物样品较水体样品细菌群落组成复杂,并且沉积物细菌群落动态变化幅度高于水体。水体和沉积物中细菌T-RFs数量范围分别在11-29和20-32之间,Shannon-wiener指数在1.44-2.87和2.44-3.25之间。【结论】养殖水体中的病原菌丰度高于沉积物,而沉积物细菌群落丰富度和多样性高于水体。病原菌丰度和细菌群落结构变化与环境因子密切相关,明确养殖池塘环境中病原微生物的生态分布和丰度变化,将为指导养殖过程中病原性疾病发生早期预警提供理论依据。     

Intestinal microbiota of gibel carp (Carassius auratus gibelio) and its origin as revealed by 454 pyrosequencing

The intestinal microbiota has received increasing attention, as it influences growth, feed conversion, epithelial development, immunity as well as the intrusion of pathogenic microorganisms in the intestinal tract. In this study, pyrosequencing was used to explore the bacterial community of the intestine in gibel carp (Carassius auratus gibelio), and the origin of these microorganisms. The results disclosed great bacterial diversities in the carp intestines and cultured environments. The gibel carp harbored characteristic intestinal microbiota, where Proteobacteria were predominant, followed by Firmicutes. The analysis on the 10 most abundant bacterial operational taxonomic units (OTUs) revealed a majority of Firmicutes in the intestinal content (by decreasing order: Veilonella sp., Lachnospiraceae, Lactobacillales, Streptococcus sp., and Lactobacillus sp.). The second most abundant OTU was Rothia sp. (Actinobacteria). The most likely potential probiotics (Lactobacillus sp., and Bacillus sp.) and opportunists (Aeromonas sp., and Acinetobacter sp.) were not much abundant. Bacterial community comparisons showed that the intestinal community was closely related to that of the sediment, indicating the importance of sediment as source of gut bacteria in gibel carp. However, 37.95 % of the OTUs detected in feed were retrieved in the intestine, suggesting that food may influence markedly the microbiota of gibel carp, and therefore may be exploited for oral administration of probiotics.     

银鲫肌酸激酶M3-CK cDNA的克隆及其表达特征

用抑制性差减杂交结合SMART cDNA合成和RACE—PCR技术克隆到雌核发育银鲫(Carassius auratus gibelio)肌酸激酶M3-CK基因的全长cDNA。银鲫M3-CK cDNA全长1551bp，编码380个氨基酸，与普通鲤鱼(cyprinus carpio)M3-CK的氨基酸序列同源性高达95％。种系分析表明，银鲫M3-CK与其它脊椎动物的肌肉型肌酸激酶聚为较近的一支，与鲤鱼的M3-CK聚在一起，与脑特异型肌酸激酶及线粒体型肌酸激酶分歧较大。虚拟Northern杂交显示银鲫M3-CK基因在胚胎发育中差异表达。RT—PCR表明，银鲫M3-CK基因在成熟卵母细胞和胚胎发育早期可检测到少量的转录产物，在胚胎发育期间从肌肉效应期开始转录，并一直持续表达。组织RT—PCR表明，银鲫M3-CK基因只在心脏和肌肉表达。     

Identification of natural hybrids of gibel carp Carassius auratus gibelio (Bloch) and crucian carp Carassius carassius (L.) from lower Dyje River floodplain (Czech Republic)

Morphometric and genetic variation were examined in parental Carassius auratus gibelio and Carassius carassius individuals, and their natural hybrids. Of meristic traits, only the number of gill rakers clearly distinguished hybrids (39·4 ± 1·4) from parental C. a. gibelio (48·3 ± 0·7) and C. carassius (28·6 ± 1·1). MtDNA sequences showed that the hybrids were descendants of female C. a. gibelio . Microsatellite analysis confirmed the presence in hybrids of variants typical of C. a. gibelio and C. carassius .     

Characterization of bmp15 and its regulation by human chorionic gonadotropin in the follicle of gibel carp (Carassius auratus gibelio)

Bone morphogenetic protein (BMP15) is a member of the transforming growth factor β (TGF-β) superfamily with a key role in regulating follicle development in mammals and birds. However, potential ovarian roles of BMPs remain unexplored in teleosts. In this study, the full-length sequences of bmp15 were obtained using rapid-amplification of cDNA ends (RACE). The full-length cDNA sequence of bmp15 is 2217 bp which contained 214 bp 5'-UTR and 845 bp 3'-UTR. The open reading frame (ORF) sequence of bmp15 is 1158 bp, encoding a predicted protein of 385 amino acid residues. BMP15 has a specific RXXR protease cleavage site of TGF-β superfamily (is RIRR) and six conserved cysteine residues. Using real-time quantitative PCR revealed that bmp15 mRNA was largely expressed in the ovary and testis and mostly in oocytes within the follicle, slightly expressed in muscle, liver and pituitary. BMP15 is mainly present at stage I follicles by real-time quantitative PCR and immunohistochemistry. Phylogenetic analysis showed that gibel carp bmp15 was similar to bmp15 of zebrafish and other fish species. Treatment with human chorionic gonadotropin (hCG) in isolated follicles of gibel carp in vitro showed altered bmp15 mRNA expression: when treated with 10 ng/mL hCG for 10h, the expression level of bmp15 was significantly increased. However, with proceeding cultivation, the expression level of BMP15 mRNA decreased. The results of this study indicate that bmp15 may play a key role during development of follicles in gibel carp, especially in early stage follicles.     

Temporal changes in body mass, body composition and metabolism of gibel carp Carassius auratus gibelio during food deprivation

To investigate the response to starvation, gibel carp Carassius auratus gibelio [12.5 ± 0.03 g (mean ± SE, n = 24)] were deprived of food at 25.8 ± 0.2°C (mean ± SE, n = 56) for 56 days. Body mass, proximate composition in whole body and muscle, and respiration were measured at 7‐day intervals. Body mass decreased with prolongation of deprivation, with a significant decline recorded after 7 days deprivation. Fish lost 22% of their fresh mass and 34% of dry mass after 56 days. Fish lost 38% of the body lipid over the first 7 days, and lost body lipid at a rate of 0–11% per week over the remaining 49 days. Body protein was lost at 1–5% per week throughout deprivation. Compared with the initial composition, body lipid concentration was lower and ash concentration higher on day 7. Water as a percentage of body mass was higher after 28 days, and protein concentration lower after 42 days, than at the start of deprivation. Muscle lipid and protein concentration was lower, and % water higher, after 7 days than at the start of deprivation, whereas muscle ash concentration was relatively constant during deprivation. After 56 days, fish lost body water by 18%, body lipid by 84%, body protein by 30%, and body energy by 45%. Oxygen consumption rate dropped from day 1 to day 3, increased from day 4 to day 14, gradually decreased from day 15 to day 35, and maintained a relatively constant level from day 36 to day 56. Results of the present experiment reveal that gibel carp utilize body lipid as a major energy source in the first 7 days of food deprivation, then turn to body protein as an energy fuel when lipid reserves are heavily depleted. Oxygen consumption is maintained at a relatively low and constant level when most lipid reserves are exhausted.     

The ploidy and reproductive mechanism of crucian carp, Carassius auratus gibelio

With the method of Feulgen staining, through cytophotometric measurements, the DNA contents of sex-reversed male (genotypic female) crucian carp (2n = 150 ±) had average values of 2.75 and 1.36 A.U., respectively, in erythrocytes and spermatozoa. The ratio of the erythrocyte and spermatozoon DNA contents of the 'male' crucian carp is 2.01:1 (2C: 1C) and is the same as in the normal male, 2.02:1. The crucian carp has a DNA value of 4.1 pg, which is 68% of that in humans. The results of the study suggest that both sex-reversal males and normal males have the ability to complete the meiotic division during their gametogenesis, and that the population has diploidized over a long term of evolution.     

新疆额尔齐斯河水系银鲫克隆多样性研究

银鲫（Carassius auratus gibelio Bloch）由于其独特的遗传背景和繁殖方式而成为研究进化遗传学和选择育种的一个独特的模式生物。到目前为止,我们对新疆额尔齐斯河水系银鲫群体的多样性状况一直知之甚少。为了更好地了解额尔齐斯河水系银鲫群体的克隆多样性状况,本研究中,我们采集了来自新疆额尔齐斯河水系的4个鲫鱼群体。通过流式细胞术分析血细胞样品,结果证实这些鲫鱼均为三倍体银鲫。通过血清转铁蛋白电泳表型分析,我们从这些银鲫群体中鉴定出总共8个不同的克隆。在这些鉴定的克隆中,有4个克隆(克隆A、J、M和S)同于以前鉴定的克隆,而另外的4个克隆是新发现的。克隆A和M分布最广,出现在所调查的4个群体中;克隆J出现在2个不同的群体中;其余的5个克隆中每个克隆均为单个群体所拥有。不同克隆在群体中的这种分布谱式可能反映了银鲫的各个克隆可在不同水体之间迁移以及同一克隆在不同水体中生存能力存在有差异。在取样的银鲫群体中,发现有一个群体的克隆多样性水平明显低于其他3个群体,而这后3个群体的克隆多样性水平是与已报道过的银鲫群体相似的。这一结果可能暗示着修建水利工程和过度捕捞等人类活动的不利影响。本研究所揭示的克隆多样性将有助于进化遗传学和选择育种研究。同时,也反映了保护额尔齐斯河水系银鲫克隆多样性的重要性。     

Growth and feed utilization in two strains of gibel carp, Carassius auratus gibelio: paternal effects in a gynogenetic fish

Gibel carp ( Carassius auratus gibelio Bloch) is a natural gynogenetic fish which requires sperm of the same or related species to activate egg development. The eggs of one gibel carp were divided into two batches. One batch was 'fertilized' with sperm from gibel carp (strain DD), and the other 'fertilized' with sperm from red common carp ( Cyprinus carpio red variety) (strain DR). The juveniles were transferred to the laboratory 36 days post-hatch. Triplicate groups of each strain were fed a formulated diet at either 3% or satiation ration for 8 weeks. At both the restricted and satiation rations, specific growth rate was significantly higher in strain DR than in strain DD. At the 3% ration, there was no significant difference in feeding rate or feed conversion efficiency between the two strains. At the satiation ration, strain DR had a significantly lower feeding rate but higher feed conversion efficiency than strain DD. At the satiation ration, strain DR had a significantly lower intake protein, but higher recovered protein than strain DD. There was no significant difference in faecal protein loss between the two strains. At the 3% ration, strain had no significant effects on intake protein, faecal protein or recovered protein. Neither faecal energy loss nor recovered energy was affected by strain or ration. At both the 3% and satiation ration, final body contents of dry matter and lipid were significantly lower in strain DR than strain DD, while there was no significant difference in protein and energy content between the two strains at either ration level. The results suggested that gibel carp 'fertilized' with sperm of common carp grew faster than those 'fertilized' with sperm of gibel carp through increased feed conversion efficiency and protein retention.     

Isolation and characterization of microsatellite markers from Fangzheng silver crucian carp,Carassius auratus gibelio (Bloch), and cross‐amplification in the closely related species crucian carp,Carassius auratus auratus (Linnaeus)

We report the development of 20 microsatellite markers for Fangzheng silver crucian carp, Carassius auratus gibelio (Bloch). Nineteen out of 20 showed polymorphism with alleles ranging from two to 14. These loci were screened to amplify the closely related species crucian carp, Carassius auratus auratus (Linnaeus) and all of them can amplify DNA with the size similar to the former. The origin of silver crucian carp is in issue and the population genetic structure is still unclear. Microsatellite markers isolated from the silver crucian carp and their utility in the crucian carp will be useful for these researches.