Polymorphic microsatellites from silver crucian carp (Carassius auratus gibelio Bloch) and cross‐amplification in common carp (Cyprinus carpio L.)

Fifteen polymorphic microsatellites were isolated from the genome of silver crucian carp (Carassius auratus gibelio Bloch). Allele numbers ranged from two to four with an average of 2.7/locus, and the proportion of tri‐ and diallelic heterozygotes was 99.3%. The individuals tested seem to have originated from two different clonal lines: 14 of 16 showed the same genotype at all loci tested, whereas the remaining two were also identical, but different from the former ones. Eleven out of 15 primer pairs cross‐amplified products from the genome of common carp, whereas only five from that of zebrafish.     

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.     

基于G-SSR和EST-SSR标记的鲫6个群体遗传结构分析

研究同时利用非编码区和编码区微卫星标记(G-SSR和EST-SSR)分析黑龙江、长江、奉化江及淮河水系共6个野生鲫(Carassius auratus)群体的遗传多样性及遗传结构, 并比较2类不同来源SSR用于鲫群体遗传多样性分析的差异。8个G-SSR标记在6个鲫群体中检测到173个等位基因, 平均N_a、N_e、H_o、H_e以及PIC分别为22、12.9、0.769、0.893和0.879, 群体间F_st值介于0.008—0.085, 其中来自黑龙江水系的2个群体与其余水系的所有群体均达到或接近于中等程度的遗传分化, 而长江、奉化江和淮河水系4个群体间的遗传分化程度不明显。Nei’s遗传距离介于0.203—0.701; 根据遗传距离所绘制的UPGMA聚类图将6个鲫群体划分为2个大分支, 其中来自黑龙江水系的2个群体聚为一枝, 其余水系群体聚为另一枝。贝叶斯分析也支持这一结果, 将6个鲫群体划分为2个最佳理论群。利用8个EST-SSR标记在6个鲫群体中共检测到155个等位基因, 平均N_a、N_e、H_o、H_e以及PIC分别为19、9.5、0.728、0.870和0.855; 群体间F_st值和Nei’s遗传距离分别介于0.005—0.084和0.117—0.683; 基于EST-SSR标记的UPGMA聚类分析和贝叶斯分析也将6个鲫群体划为两大类群: 黑龙江水系群体; 长江、奉化江和淮河水系群体。G-SSR和EST-SSR标记检测6个鲫群体的平均多态信息含量(PIC)分别为0.786—0.864和0.761—0.833。研究结果显示: 6个野生鲫群体均具有较高的遗传多样性, 但黑龙江水系群体多样性低于其他水系群体; 尽管EST-SSR标记的多态性略小于G-SSR标记, 但是2类微卫星标记均揭示了相似的鲫群体遗传结构和分化格局。研究结果对鲫种质资源的保护和EST-SSR标记在鱼类群体遗传学研究价值的评价提供了新的信息。     

A molecular approach to detect hybridisation between crucian carp (Carassius carassius) and non-indigenous carp species (Carassius spp. and Cyprinus carpio)

1. Releases of non‐native fish into the wild is an increasing problem posing considerable ecological and genetic threats through direct competition and hybridisation. 2. We employed six microsatellite markers to identify first generation hybrids and backcrosses between native crucian carp (Carassius carassius) and introduced goldfish (C. auratus) and common carp (Cyprinus carpio) in the U.K. We also investigated the genetic characteristics of the taxonomically controversial gibel carp (Carassius spp.) from sites across Europe. 3. Natural hybridisation between goldfish and crucian carp occurs frequently, although hybrids between all other species pairs were observed. Only 62% of British crucian carp populations (n = 21) consisted exclusively of pure crucian carp. In some populations hybrids were so frequent, that no pure crucian carp were caught, indicating a high competitive ability of hybrids. 4. Most hybrids belonged to the F1 generation but backcrossing was evident at a low frequency in goldfish × crucian carp hybrids and goldfish × common carp hybrids. Furthermore, some local populations had high frequencies of backcrosses, raising the opportunity for introgression. 5. Gibel carp from Germany and Italy belonged to two triploid clonal lineages that were genetically closely related to goldfish, whereas all individuals identified from British populations proved to be crucian carp × goldfish hybrids. 6. Our study suggests that the release of closely related exotic cyprinids not only poses a threat to the genetic integrity and associated local adaptations of native species, but may also contribute to shifts in community structure through competitive interactions.     

Benefits of a predtor-induced morphology in crucian carp

Crucian carp (Carassius carassius) develop a deeper body in response to chemical cues from piscivores. This change in body morphology has been suggested to be a predator-induced defence. Here we investigate the possible benefits of the induced body morphology in laboratory experiments. Pike foraging behaviour when feeding on crucian carp of different body depths was recorded using video. Further, in a preference experiment pike were allowed to choose between shallow-bodied and deep-bodied crucian carp of similar lengths. Crucian carp body morphology did not affect predatory behaviours (activity, searching, following, observing, capture success) in northern pike, but an increase in crucian carp body depth led to an increase in handling time in pike. In the preference experiment, pike preferred shallow-bodied crucian carp over deep-bodied. Thus, a change in body morphology, induced by the presence of piscivores, benefits crucian carp by increasing piscivore handling times and an avoidance of the deep-bodied phenotype.     

Difference in the hypoxia tolerance of the round crucian carp and largemouth bass: implications for physiological refugia in the macrophyte zone

The hypoxia tolerance of larval and juvenile round crucian carp, Carassius auratus grandoculis, and largemouth bass, Micropterus salmoides, was determined using respirometry to examine the potential of hypoxic areas in the macrophyte zone as physiological refugia for round crucian carp. The tolerance, which was measured as the critical oxygen concentration (Pc), was 1.32 mg O₂/l in the round crucian carp and 1.93 mg O₂/l in the largemouth bass. As the round crucian carp tolerated hypoxia better than the largemouth bass, hypoxic areas in the macrophyte zone might function as physiological refugia for round crucian carp.     

三种鲫鱼品系同工酶比较研究

采用聚丙烯酰胺凝胶垂直板电泳技术,对彭泽鲫、银鲫D系以及野鲫三种鲫鱼品系的心、肝和肾脏组织的乳酸脱氢酶(LDH)、苹果酸脱氢酶(MDH)、苹果酸酶(ME)、酯酶(EST)和超氧化物歧化酶(SOD)同工酶表型进行了比较研究.结果表明彭泽鲫乳酸脱氢酶同工酶比银鲫D系在肝组织多出二条酶带(LDH7'和LDH8');超氧化物歧化酶在心和肾组织中分别多出一条酶带(SOD12'),表明彭泽鲫和银鲫已在生化水平产生明显的分化,推测它们可能起源于不同的地区,由不同的祖先,独立演化而形成.此外,彭泽鲫和银鲫D系的同工酶电泳图谱都包含野鲫的基本酶带,而彭泽鲫和野鲫的酯酶同工酶电泳图谱尤为相似,推测彭泽鲫和银鲫可能起源于野鲫,而彭泽鲫和野鲫的关系较近,银鲫和野鲫的关系较远.     

Consumption of crucian carp (Carassius carassius L., 1758) by restocked pike (Esox lucius L., 1758) in a lake with frequent winter hypoxia

To assess the effects of stocked pike (Esox lucius L.) on crucian carp (Carassius carassius L.) biomasses, the annual consumption of pike was estimated and compared with removal fishery catches. The studied lake, Lake Savijärvi, is a small (40 ha), shallow and eutrophicated lake in southern Finland with frequent algal blooms during summers and fish kills during winters. Until the 1980s, the fish fauna consisted of pike, perch (Perca fluviatilis L.), roach [Rutilus rutilus (L.)], crucian carp, and tench (Tinca tinca L.). Since 2003, when crucian carp were abundant in the extreme and with only a few roach and tench individuals, the lake has been biomanipulated by removal seining. To enhance the effects of seining, piscivorous pike were restocked in the spring of 2008. During the 3‐year study, the catch of crucian carp decreased from 243 to 136 kg while the catch of pike increased from 0.1 to 5.7 kg per seining hectare due to their spawning in the lake as of 2009. The total crucian carp consumption by captured pike was 588 kg during the 2008–2010 study period. When applying the number of pike estimated with the mark‐recapture method, consumption estimate increased to 917 kg, or 22.9 kg per lake hectare in 2010. Thus, pike consumption of crucian carp was about 17% of the removal catch in 2010.     

不同捕食者暴露对鲫幼鱼行为、运动和形态特征的影响

自然界中鱼类的捕食者种类繁多,为验证猎物鱼针对不同捕食者是否会做出相异的反捕食策略选择,选取鲫（Carassius auratus）幼鱼为猎物鱼,乌鳢（Channa argus）和胡子鲶（Clarias fuscus）为捕食者,将鲫幼鱼分别暴露于空白（对照）、乌鳢、胡子鲶和双捕食者（同时存在乌鳢和胡子鲶）环境中持续2个月,随后考查不同组鲫幼鱼的生长、行为特征、形态和运动能力等指标之间的差异。结果表明：经过2个月的捕食胁迫处理,鲫幼鱼的自发运动、隐蔽场所利用率和勇敢性等行为指标在4个组之间均未表现出显著性差异。相比对照组,乌鳢、胡子鲶和双捕食者暴露组鲫幼鱼的快速启动反应时滞显著缩短（P<0.05）;但所有处理组中仅乌鳢暴露组鲫幼鱼的快速启动120ms移动距离（S_120ms）和体高（BD）显著高于对照组（P<0.05）。另外鲫幼鱼的临界游泳速度（U_crit）和活跃代谢率（M_O2active）在各组之间也未表现出显著差异。乌鳢暴露组鲫幼鱼快速启动能力的提升可能与形态上体高的提高有关;另外相比临界游泳能力,快速启动能力可能在鱼类逃避捕食者过程中更为关键;鲫幼鱼表现出对乌鳢的反应最为明显,可能与乌鳢口裂较大,给鲫幼鱼带来的威胁更大有关。整体而言,应对捕食胁迫时,鲫幼鱼仅表现出形态和快速启动能力的反捕食响应,其行为特征的保守性可能是对生长作出的妥协,这对于鱼类适应生境中长期存在的捕食胁迫十分关键。     

Development and application of environmental DNA surveillance for the threatened crucian carp (Carassius carassius)

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A comparative study of aldehyde dehydrogenase and alcohol dehydrogenase activities in crucian carp and three other vertebrates: apparent adaptations to ethanol production

Summary In the final step of the pathway producing ethanol in anoxic crucian carp (Carassius carassius L.), acetaldehyde is reduced to ethanol by alcohol dehydrogenase. The presence of aldehyde dehydrogenase in the tissues responsible for ethanol production could cause an undesired oxidation of acetaldehyde to acetate coupled with a reduction of NAD⁺ to NADH. Moreover, acetaldehyde could competitively inhibit the oxidation of reactive biogenic aldehydes. In the present study, the distribution of aldehyde dehydrogenase (measured with a biogenic aldehyde) and alcohol dehydrogenase (measured with acetaldehyde) were studied in organs of crucian carp, common carp (Cyprinus carpio L.), rainbow trout (Salmo gairdneri Richardson), and Norwegian rat (Rattus norvegicus Berkenhout). The results showed that alcohol dehydrogenase and aldehyde dehydrogenase activities were almost completely spatially separated in the crucian carp. These enzymes occurred together in the other three vertebrates. In the crucian carp, alcohol dehydrogenase was only found in red and white skeletal muscle, while these tissues contained exceptionally low aldehyde dehydrogenase activities. Moreover, the low aldehyde dehydrogenase activity found in crucian carp red muscle was about 1000 times less sensitive to inhibition by acetaldehyde than that found in other tissues and other species. The results are interpreted as demonstrating adaptations to avoid a depletion of ethanol production, and possibly inhibition of biogenic aldehyde metabolism.Abbreviations ADH alcohol dehydrogenase - ALDH aldehyde dehydrogenase - DOPAL 3,4-dihydroxyphenylacetaldehyde - MAO monoamine oxidase - PCA perchloric acid     

异源四倍体鲫鲤、三倍体湘云鲫和它们父母本线粒体DNA 12S rRNA基因遗传变异的分析

采用质粒克隆测序方法,获得了异源四倍体鲫鲤5个个体、异源四倍体鲫鲤雌核发育二倍体后代2个个体、三倍体湘云鲫2个个体及红鲫、湘江野鲤和日本白鲫各1个个体的线粒体DNA 12S rRNA基因的全序列。经对比发现,异源四倍体5个个体共享2种单元型,异源四倍体鲫鲤雌核发育二倍体后代2个个体、三倍体湘云鲫2个个体以及红鲫、湘江野鲤和日本白鲫各1个个体分别共享1种单元型。用MEGA 1.0 软件分析了它们的碱基组成和核苷酸序列差异,用邻接法构建系统进化树。它们间的序列同源性在95%~99%之间,异源四倍体鲫鲤、三倍体湘云鲫和它们母本（分别为红鲫和日本白鲫）之间的序列同源性大于异源四倍体鲫鲤、三倍体湘云鲫和它们父本（分别为湘江野鲤和异源四倍体鲫鲤）之间的序列同源性,结果表明：异源四倍体鲫鲤和三倍体湘云鲫在线粒体DNA 12S rRNA基因上具有母性遗传特征。本研究另一值得注意地方的是异源四倍体鲫鲤经过9代(F3-F11)繁殖后,在5个个体中发现了2种单元型,说明在四倍体基因库中存在遗传多样性,为四倍体基因库的繁殖、保护和种群复壮提供了一些有价值的信息。     

银鲫与彩鲫卵母细胞cDNA文库构建及周期蛋白A1的cDNA克隆

分别取行天然雌核发育繁殖的银鲫和两性生殖的彩鲫的卵母细胞为材料,提取总RNA,分离mRNA,进而反转录合成cDNA并定向插入λgtll Sfi-Not克隆载体,经体外包装构建了银鲫与彩鲫卵母细胞的表达型cDNA文库。测试结果表明库容量分别达到3.1×106(银鲫)和1.6×106(彩鲫)。进一步人工合成Cyclin A1保守引物,采用PCR扩增文库的方法,克隆了银鲫(1616bp)与彩鲫(1626bp)的Cyclin A1全长cDNA。序列分析结果表明:两种鱼编码区长度均为1173bp,起始于一个包含在脊椎动物起始密码子ANNATG基元内ATG的单一开放读码框,编码391个氨基酸;5'-端非编码区长度也同为70bp,3-'端非编码区长度略有不同,银鲫为373bp,而彩鲫则为383bp;二者3'-端均带有AATAAA的Poly(A)加尾信号以及24bp(银鲫)和27bp(彩鲫)的Poly(A)尾巴。比较银鲫、彩鲫和金鱼与人、爪蟾Cyclin A1氨基酸序列同源性的结果表明,Cyclin A1在人、爪蟾与鱼类之间具有较高同源性;而在银鲫、彩鲫和金鱼之间,Cyclin A1仅在周期蛋白框外存在5个氨基酸的差异,且这些差异均是由个别碱基的变异造成的。       

异源精子在银鲫雌核发育子代中的生物学效应

黑龙江省方正县双凤水库的两性型银鲫群体是三倍体雌核发育种群。异源精子不仅能刺激银鲫卵雌核发育,而且还能影响雌核发育子代的某些性状,如对于子代的生长、性比、体色和肝脏LDH同工酶等都产生了影响。为区别于原有术语“雌核发育gynogenesis”,我们把这种表现了异源精子生物学效应的雌核发育称之为“异精雌核发育allogynogenesis”,发育的子代称之为“异育银鲫”。异育银鲫已以其明显的生长优势在生产上显示了优良的经济性状。       

Composition of leucocytes of the head kidney of the crucian carp (<Emphasis Type="Italic">Carassius auratus gibelio</Emphasis>, Cypriniformes: Cyprinidae) as affected by invasion of cestode <Emphasis Type="Italic">Digramma interrupta</Emphasis> (Cestoda; Pseudophyllidea)

The composition of leucocytes of the head kidney is studied in the crucian carps (Carassius auratus) either contaminated or uncontaminated with Digramma interrupta. The composition of leucocytes in the pronephros of the crucian carp from Lake Baikal basin has a lymphoid character. Compared to the crucian carp from the European part of Russia, in the fish from Baikal the granulocytopoetic processes are more pronounced. This is proved by the high content of young forms of granulocytes. In the fish infected with digramma, the immune suppression of proliferation of blasts and young forms of eosinophils was revealed. On the other hand, the inflammatory and humoral specific immune reactions are enhanced. Partial suppression of the immune response of C. auratus to invasion by D. interrupta facilitates development of the parasite.     

Comparative studies on histological and ultra-structure of the pituitary of different ploidy level fishes

The histological and ultra-structure of the pituitary in diploid red crucian carp (Carassius auratus red var.), triploid crucian carp and allotetraploid hybrids within and after the breeding season were comparatively studied. The result showed that there were six endocrine cell types in the pituitary of these three kinds of fishes, and there was an obvious difference in cell size among different ploidy level fishes. As for the same type of pituitary cells, the cell size was increased gradually with the increasing ploidy level. In the breeding season, the allotetraploid hybrids had higher proportion of gonadotropin cells (GTH) than triploids, and the trploids had higher proportion of GTH than diploids. The results were related to the earlier sexual maturity of allotetraploid hybrids and sterility of triploid crucian carp. On the other hand, among the three kinds of fishes, the proportion of somatotropin (STH) cells in triploids crucian carp was the highest, whereas that in allotetraploid hybrids was the lowest. The results might be connected with the faster growth rate of triploids and slower growth rate of allotetraploid hybrids. In addition, in GTH cells of meso-adenohypophysis after the breeding season, there were many endocrine particles in triploids, while those endocrine particles were released from the cells in allotetraploids and diploids. This result showed that the sterility of triploid crucian carp might be related to the hormone which was not released from the GTH cells. In a word, the present study indicated that the differences in the structure of pituitary among different ploidy level fishes contributed to their difference in the growth rate and gonadal development.     

从ATPase8-6基因研究杂交多倍体鱼线粒体母性遗传

异源四倍体鲫鲤是世界上首例人工培育的两性可育并形成群体的且能自然繁殖的四倍体鱼。本文采用质粒克隆测序法测定了红鲫、异源四倍体鲫鲤、三倍体湘云鲫和三倍体湘云鲤的ATPase8和ATPase6基因全序列 ,结合鲤鱼、日本白鲫和斑马鱼的同源序列 ,对不同倍性水平鲤科鱼类的ATPase8和ATPase6基因进行了比较 ,分析了碱基组成、变异情况以及核苷酸和氨基酸序列差异。红鲫、鲤鱼、异源四倍体鲫鲤、日本白鲫、三倍体湘云鲫和三倍体湘云鲤之间的序列差异为 0 0 % - 1 3 4 % ,它们与外群斑马鱼之间的序列差异为 2 7 9% -31 0 %。用MEGA软件中的MP法、ME法、NJ法和UPGMA法构建分子系统树 ,得到了相似的拓扑结构。结果分析表明 ,人工杂交多倍体异源四倍体鲫鲤、三倍体湘云鲫和三倍体湘云鲤在线粒体ATPase8和ATPase6基因上具有严格的母性遗传特征。值得注意的是 ,异源四倍体鲫鲤经过 1 1代的繁育后 ,与其原始母本红鲫仍然保持了非常高的同源性 ,说明了新的异源四倍体基因库在线粒体ATPase8和ATPase6基因上拥有稳定的遗传特性。对不同倍性鲤科鱼类线粒体ATPase8和ATPase6基因的研究表明 ,ATPase8和ATPase6基因是杂交鱼后代遗传变异研究的一个很好的分子标记     

Evidence for recombination of mitochondrial DNA in triploid crucian carp

In this study, we report the complete mitochondrial DNA (mtDNA) sequences of the allotetraploid and triploid crucian carp and compare the complete mtDNA sequences between the triploid crucian carp and its female parent Japanese crucian carp and between the triploid crucian carp and its male parent allotetraploid. Our results indicate that the complete mtDNA nucleotide identity (98%) between the triploid crucian carp and its male parent allotetraploid was higher than that (93%) between the triploid crucian carp and its female parent Japanese crucian carp. Moreover, the presence of a pattern of identity and difference at synonymous sites of mitochondrial genomes between the triploid crucian carp and its parents provides direct evidence that triploid crucian carp possessed the recombination mtDNA fragment (12,759 bp) derived from the paternal fish. These results suggest that mtDNA recombination was derived from the fusion of the maternal and paternal mtDNAs. Compared with the haploid egg with one set of genome from the Japanese crucian carp, the diploid sperm with two sets of genomes from the allotetraploid could more easily make its mtDNA fuse with the mtDNA of the haploid egg. In addition, the triple hybrid nature of the triploid crucian carp probably allowed its better mtDNA recombination. In summary, our results provide the first evidence of mtDNA combination in polyploid fish.     

A laboratory study of the effects of shelter availability and invasive crayfish on the growth of native stream fish

1. The alteration of stream habitats by urbanisation reduces the availability of shelter. Reduced shelter availability may increase both predation risks and metabolic costs, negatively affecting the growth performance of stream fish. Although urbanised streams often allow the establishment of invasive species, the additive or interactive effects of shelter availability and invasive species are rarely explored. The invasive red swamp crayfish (Procambarus clarkii) is a strong shelter competitor and predator in streams. 2. We conducted a stream‐channel experiment to investigate how shelter availability and this invasive crayfish species affect the growth performance (measured as the loss in body mass under starved conditions) of two native benthic species, Japanese crucian carp (Carassius auratus complex) and an endangered cobitid fish (Lefua echigonia). 3. We found that both the absence of shelter and the presence of crayfish increased body mass loss in the crucian carp. However, the growth performance of the crucian carp was improved by the presence of shelter except in the presence of crayfish, in which case the shelter available did not ameliorate the negative effect of the crayfish on growth performance (i.e. an interactive effect). This result suggests that crayfish may affect the growth performance of the crucian carp through chemical cues as well as through shelter occupancy. 4. In contrast, shelter availability is the primary factor affecting the body mass loss of the cobitid fish. However, the growth performance of these fish was unaffected by the presence of crayfish. The change in the growth performance of the cobitid fish in response to the absence of shelter was greater than that of the crucian carp. This finding suggests that the cobitid fish may be more vulnerable than the crucian carp to a reduction in shelter availability. 5. Our study demonstrates that reduced shelter availability and/or crayfish invasion can have significant, indirect negative effects on the growth performance of native fish, but whether those effects are interactive might vary depending on the species. Our findings have clear implications for stream restoration and habitat assessment.