洪湖碘泡虫的再描述及其近缘种的鉴别性研究

研究基于形态特征和18S rDNA序列相似度、遗传距离、变异位点、GC含量和系统发育比较分析,对采自河南龙湖的寄生于异育银鲫鳃部的一种黏孢子虫以及相似性极高且易混淆的黏孢子虫种类(洪湖碘泡 Myxobolus honghuensis Liu,et al. 2012、瓶囊碘泡虫Myxobolus ampullicapsulatus Zhao,et al. 2008、咽碘泡虫Myxobolus pharynae Lu,et al. 2012和吴李碘泡虫Myxobolus wulii (Wu Li,1986) 进行了系统的鉴别研究。研究结果显示: 河南龙湖异育银鲫鳃部所检获的黏孢子虫为洪湖碘泡虫,该种群对所寄生的异育银鲫未造成疾病症状; 咽碘泡虫与洪湖碘泡虫各种群在形态上极相似,两者间18S rDNA序列相似度为99%-100%,遗传距离为0-0.0013,GC含量均为44.31%,变异位点为2个,表明咽碘泡虫与洪湖碘泡虫应为同一物种。     

Genetic structure of crucian carp (Cypriniformes, Cyprinidae, Carassius L. 1758) populations of middle-Dnieper basin

Comparative analysis of Middle Dnieper crucian carp's population structure was made by means of biochemical gene marking, cytometry and biological analysis. As a result the structure was found, which may by expounded like association of the genetically related species and forms. This association consists of diploid C. auratus (which predominates in population), C. carassius (which was found in the some of north-eastern reservoirs only) and triploid C. gibelio. In Ukraine the homogeneous populations of C. gibelio can be found on the north-east of Eastern Ukraine only. Though some of individuals maybe present with diploid C. auratus. Besides were found hybrid form: diploid C. auratus-C. carassius and presumably tetraploid C. auratus-C. gibelio and C. gibelio-C. carassius. However in the tetraploid's spectra were absent a number of specific alleles, that marking C. species-one of the parental species of triploid C. gibelio. Therefore presumptive tetraploids have not difference from C. auratus and diploid C. auratus-C. carassius on the gene markers level. Nevertheless they are neatly diagnose by the erythrocytes which have good difference and are bigger then diploid species erythrocytes in 40% (C. auratus-C. gibelio) and 100% (C. gibelio-C. carassius).     

江苏盐城地区异育银鲫大红鳃疾病病原学研究及病理学观察

为鉴定近期江苏盐城地区养殖异育银鲫大红鳃疾病病原,研究从患病濒死鱼中分离到两株致病菌并对其理化特性、分子生物学、感染病理学进行了研究,同时开展了鉴别诊断和回归试验以及药敏检测。结果显示,两分离株理化特性与温和气单胞菌（A.sobria）特征相符,与GenBank中A.sobria的gyrB基因同源性达99%以上,在系统发育树上均与A.sobria聚为一族。同时与鲤科疱疹病毒2型（CyHV-2）鉴别诊断结果表明,患病鱼组织中CyHV-2检测为阴性。组织学上,鳃和肾组织具有典型病理变化。人工感染两株A.sobria后,鱼体出现与自然发病相似的大红鳃症状,并能从发病鱼组织中再分离到相同病原菌;而人工感染CyHV-2组织匀浆后的鲫虽然出现死亡,但无大红鳃症状出现。综上,确认此次大红鳃疾病的病原为A.sobria。药敏实验表明,分离菌株对诺氟沙星、氧氟沙星、氯霉素三种药物敏感。研究为临床上更好的预防异育银鲫大红鳃疾病奠定了基础。     

Description of a new species of Myxobolus (Myxozoa: myxobolidae) based on morphological and molecular data

Myxobolus ampullicapsulatus n. sp. was isolated from the gills of Carassius auratus auratus (L., 1758) in Chongqing, China. Myxospores were pyriform, measuring 16.5-19.5 microm long x 8.5-10.0 microm wide x 7.0 microm thick. Two equal polar capsules were ampullaceous, measuring 7.0-10.0 microm long x 2.5-4.0 microm wide, containing polar filaments coiled 9-10 turns. Spore length of this species exceeds that of the majority of other Myxobolus spp., and those overlapping in this dimension can be differentially diagnosed by other characters. Furthermore, the small subunit ribosomal DNA (SSU rDNA) of M. ampullicapsulatus n. sp. is unique among myxozoans sequenced to date. Phylogenetic analyses of the SSU rDNA gene sequence placed this species in a clade composed exclusively of gill parasites, most closely related to Myxobolus longisporus, which also infects the gills of cyprinid fishes in China.     

额尔齐斯河银鲫繁殖生物学研究

2013年4—10月在额尔齐斯河中国段下游采集了546尾银鲫(Carassius auratus gibelio)样本, 依据传统生物学和组织学方法, 对额尔齐斯河银鲫的繁殖生物学特性进行了分析和研究。统计表明, 额尔齐斯河银鲫的性比(雌/雄)为10.84﹕1。根据卵母细胞和精细胞发育及比例情况, 将银鲫卵巢和精巢发育分别划分为6期。依据不同性腺发育期比例、性体指数和卵径频率分布, 推测银鲫为同步产卵鱼类, 繁殖期为5—7月, 6月为高峰期。采用Logistic回归方程获得其初次性成熟体长(SL₅₀)和年龄(A₅₀)分别为: 雌鱼161 mm和2.3龄; 雄鱼135 mm和1.9龄。绝对繁殖力为(42453±28205)粒, 相对繁殖力为(98.19±34.6)粒/g。绝对繁殖力与体长线性相关性较低, 与体重呈幂指数相关, 但与年龄及卵巢重的相关性不显著。研究进一步丰富了额尔齐斯河银鲫生物学研究资料, 为其资源养护和可持续利用提供科学依据。     

一种新的异育银鲫病原———腐败希瓦氏菌

【目的】江苏盐城一家养殖场的异育银鲫暴发疾病,通过对病原进行研究,旨在为该病的防治提供理论依据和参考。【方法】从病鱼体表病灶和内脏中分离出优势菌株,经人工感染试验证实为病原菌。采用传统的形态、生理生化表型鉴定与16S rDNA序列分析相结合的方法确定菌株的分类地位。运用K-B琼脂法对病原菌株进行药物敏感性测定。【结果】综合菌株形态、生理生化表型以及16S rDNA序列分析的结果,确定该分离株为腐败希瓦氏菌(Shewanella putrefaciens)。回接感染试验证实腐败希瓦氏菌即是导致此次异育银鲫发病死亡的致病原,其半数致死量(LD50)为2.1×103cfu/g。该株腐败希瓦氏菌对吡哌酸、萘啶酸、氟哌酸、氟啶酸、氟苯尼考、利福平、美满霉素、氟罗沙星、恩诺沙星、复达欣、菌必治、先锋Ⅳ、罗红霉素和左氟沙星等抗生素敏感。【结论】首次报道了异育银鲫一种新的病原,说明腐败希瓦氏菌作为一种潜在的新病原也可能会对异育银鲫的养殖造成威胁。     

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 .     

异育银鲫源杀鲑气单胞菌杀鲑亚种的分离鉴定

【目的】分离鉴定江苏省扬州市养殖场异育银鲫患病病原。【方法】采用常规的理化特性和分子生物学的方法,对从濒死异育银鲫肝脏处分离到的菌株YZ-1进行表型生物学、分子生物学及药敏试验的系统研究。【结果】该菌株16S r RNA基因(序列长度1 446 bp,Gen Bank登录号为JX164202)与其它杀鲑气单胞菌16S r RNA基因一致性在99%-100%之间,构建发育树确定该菌株为杀鲑气单胞菌杀鲑亚种(Aeromonas salmonicida subsp.salmonicida)。人工回感可导致异育银鲫死亡。药敏试验结果显示:对头孢呋辛、复方新诺明、恩诺沙星等23种抗生素敏感;对阿米卡星、四环素、大观霉素、头孢拉定等11种抗生素中度敏感;对青霉素G、链霉素、庆大霉素、氟苯尼考、万古霉素等10种抗生素耐药。【结论】研究结果证实引起异育银鲫死亡的病原为杀鲑气单胞菌杀鲑亚种。     

Morphological and mtDNA sequence studies on three crucian carps (Carassius: Cyprinidae) including a new stock from the Ob River system, Kazakhstan

Three morphologically and genetically distinct forms of the genus Carassius were collected from the Ob River system, Kazakhstan, Central Asia; Carassius carassius , Carassius gibelio gibelio and an unknown stock tentatively referred to as Carassius gibelio sub-species M. The last mentioned had 33–41 gill rakers, being intermediate between the other two forms (23–27 in C. carassius and 44–49 in C. g. gibelio ), and five scales in the upper transverse series, less than in the others. It also had a relatively larger erythrocyte suggesting triploidy and an mtDNA haplotype distinct from all other known crucian carps. Comparative mtDNA phylogenetic analysis suggested that C. gibelio gibelio in the Ob River system was introduced from China and the Amur River, the same possibly being true for European C. gibelio gibelio based on published haplotypes. C. gibelio sub-species M is thought to be more widely distributed in central Asia, probably extending as far west as European Russia.     

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.     

池养条件下银鲫与鲫鱼的生物学特性比较及其在生产上的意义

银鲫在我国分布于黑龙江流域和新疆额尔齐斯河等水域中。在四川省池养条件下,比较了银鲫与本地鲫的形态、食性、生长、繁殖、含肉量、鱼肉营养成分和起水率等生物学特性。形态差别主要在于银鲫体型较高,头略短,侧线鳞和脊椎骨数稍多。食性相似。银鲫生长较本地鲫约快150%。银鲫在2足龄左右第一次性成熟,产卵盛期较本地鲫略晚1个月,性比为(?)34.1%(体长6.1—14.5厘米)—(?)5.95%(体长15.4—18.5厘米)。银鲫的肥满度,含肉量,鱼肉含脂量以及起水率均较本地鲫高。初步讨论了银鲫的某些生物学特性在其养殖和选育上的经济意义。       

流式细胞术用于粘孢子虫分类学的探讨

利用流式细胞仪测定了2种粘孢子虫的散射光(包括FSC和SSC)和自发荧光等物理特征参数;采用Cellquest软件对所获参数进行相关对比研究分析.结果表明,FCM可对单个物种分析乃至多个物种的区分鉴定,因此对不同种类的粘孢子虫的区分具有较明显的应用前景;本研究也将为粘孢子虫流式细胞数据库的建立奠定技术基础.     

滇池高背鲫和方正银鲫酯酶、乳酸脱氢酶同工酶的比较研究

本研究以方正银鲫(Carassius auratus gibelio Bloch)普通鲫(Carassius auratus)和滇池高背鲫(Carassius sp.)的各种组织器官为材料,进行酯酶(Esterase)和乳酸脱氢酶(LDH)同工酶电泳图谱的分析比较。结果表明:9种不同组织中酯酶同工酶谱带各不相同,有明显的组织特异性。滇池高背鲫的酯酶谱图有3种表型。方正银鲫和滇池高背鲫同一组织的LDH同工酶酶谱也有明显差异。等电聚焦凝胶电泳(T=7.5％,C=5％)的结果又表明这二种鱼的肝脏、脑、卵的酯酶同工酶酶谱及电泳扫描图亦有差异。这些结果揭示滇池高背鲫与方正银鲫至少在生化水平上已有明显的分化,很可能起源于不同的地区,由不同的祖先,独立演化而形成。滇池高背鲫与云南普通鲫的LDH酶谱较为接近,这说明滇池高背鲫最可能起源于云南本地的普通鲫。     

四倍体异育银鲫新品种长丰鲫肌肉品质和营养成分分析

研究对长丰鲫、彭泽鲫、异育银鲫D系以及红鲫的肌肉品质和营养成分进行了对比分析。结果显示:肌纤维密度方面, 单位面积肌纤维密度长丰鲫最高[(184.2624.11) fiber/mm2], 其次为彭泽鲫[(149.7212.51)fiber/mm2]、异育银鲫D系[(134.4515.96) fiber/mm2]和红鲫[(119.8522.86) fiber/mm2]。粗蛋白含量、总氨基酸和呈味氨基酸方面, 长丰鲫、彭泽鲫和异育银鲫D系无明显区别。高度不饱和脂肪酸中(n3), 长丰鲫含量最高(24.2%), 依次为彭泽鲫(14.4%), 异育银鲫(11.3%)和红鲫(8.3%)。在高度不饱和脂肪酸中, 二十二碳六烯酸(DHA)长丰鲫含量最高(10.3%), 依次为红鲫(4.9%)、彭泽鲫(4.5%)和银鲫(2.9%); 花生四烯酸(AA)长丰鲫含量最高(8.3%), 依次为彭泽鲫(5.1%)、银鲫(3.8%)和红鲫(0.6%)。结果表明, 长丰鲫新品种在所检测的肌肉品质和营养成分指标方面, 优于彭泽鲫、异育银鲫D系和红鲫。     

异育银鲫c型溶菌酶全长cDNA序列的生物信息学分析及其组织表达分析

利用RACE及克隆等方法获得了异育银鲫（Carassius auratus gibelio）c型溶菌酶基因全长cDNA序列．序列分析表明,所克隆的异育银鲫溶菌酶的cDNA全长751 bp,包括溶菌酶基因开放阅读框（ORF）438 bp,5′ 非编码区（UTR）为109 bp和3′ UTR为204 bp．438 bp ORF共编码146个氨基酸,其成熟肽的分子量预测值为14　543.6,理论等电点为8.86．通过ClustalW软件,将异育银鲫和其它多个物种c型溶菌酶的氨基酸序列进行多序列比对发现,所克隆的异育银鲫溶菌酶编码的氨基酸序列中存在c型溶菌酶的活性中心（Glu53和Asp69）,且与活性位点相邻的氨基酸序列高度保守．同时,8个保守的半胱氨酸残基也与其它物种的c型溶菌酶相一致．结合BLASTN分析的结果,可以确认所获得的异育银鲫溶菌酶cDNA序列属于c型溶菌酶．异育银鲫c型溶菌酶和人c型溶菌酶（pdb 1at6_）在蛋白质序列上有50%相似性,其三维（3-D）结构非常类似．通过氨基酸空间位置比较发现,两者具有类似的酶活中心,异育银鲫c型溶菌酶只能形成3个二硫键,比人少1个．荧光定量RT-PCR检测和溶菌酶活性测定显示,异育银鲫头肾和脾脏c型溶菌酶mRNA的表达量约为肝胰脏的2.9 倍和1.7 倍,异育银鲫头肾和脾脏的溶菌酶活性约为肝胰脏的6.2 倍和4倍．     

异育银鲫幼鱼对饲料中赖氨酸的利用及需要量研究

以添加晶体氨基酸的半精制饲料饲喂异育银鲫幼鱼,通过69d的生长实验来确定其赖氨酸需要量。饲料以白鱼粉为主要蛋白源,饲料中的总赖氨酸含量分别为1.82%、2.32%、2.82%、3.32%3、.82%、4.32%和4.82%7个水平。实验在室内循环水养殖系统中进行,每种饲料随机3个重复。实验结果表明,异育银鲫能够利用饲料中的晶体赖氨酸、蛋氨酸。在投喂后3h,其血浆中的游离赖氨酸、蛋氨酸含量最高。当饲料中赖氨酸含量为3.32%时,异育银鲫的终末尾均重、特定生长率和鱼空壳占体重的百分比最高,肝体指数最低。当饲料中赖氨酸含量为3.82%时,异育银鲫的干物质表观消化率显著高于其他组(PPP>0.05)。血红蛋白含量以赖氨酸含量为2.82%的饲料组最高,4.82%组最低;随着饲料中赖氨酸含量的升高,异育银鲫红细胞数下降,血清脲氮含量升高,且血清脲氮含量具有组间显著性差异(P<0.05)。根据折线法,由异育银鲫的特定生长率同饲料中赖氨酸水平的相关性得出其赖氨酸需要量为3.27%,占饲料蛋白的8.52%。       

Effect of feeding fungal endophyte (Acremonium coenophialum )-infected tall fescue seed on reproductive performance in CD-1 mice through continuous breeding

This study was designed to assess the effect of endophyte-infected (Acremonium coenophialum ) tall fescue (KY-31) seed (80% infected) on reproductive performance in CD-1 mice by continuous breeding. Twenty-four pairs of 70-d-old CD-1 mice were randomly allocated to four diets: 1) mouse chow ad libitum; 2) 40% infected fescue seed and 60% chow (w/w); 3) reduced intake (100% chow) similar to the intake, adjusted daily, in Diet 2; and 4) 60% infected fescue seed and 40% chow. Males and females were randomly paired (six pairs/treatment) and placed on the above diets. The mice were fed the corresponding diets for 80 d, although the pairs were separated on Day 60 (prior to the birth of the 3rd litter) and the females were monitored for one additional gestation period (20 d). The pregnancy data (litters produced) among the four treatments were 100.0 (18), 77.8 (14), 100.0 (18) and 80.0% (12) respectively. Similarly, the average number of pups born per litter among the four treatments was 11.8, 9.3, 10.1, and 9.8. When the chow treatment (1 and 3) and the fescue treatments (2 and 4) were pooled and compared, the percent pregnancy was 100.0 (n = 36) and 78.8 (n = 26), and the pups born per litter (means +/- SEM) were 11.0 +/- 0.5 and 9.5 +/- 0.6, respectively. Also the intervals between the three litters born during the 60-d cohabitation period were 21.6 +/- 1.1 and 24.5 +/- 0.9 d for the chow and fescue treatments, respectively. The results point out that 40 and 60% infected fescue seed in the diet of mice does influence (P < 0.05) their reproductive capacity as measured by percent pregnancy and litter size.     

Aflatoxin Contamination of Preharvest Corn as Influenced by Timing and Method of Inoculation

Four corn (Zea mays L.) hybrids were grown in 1977 and 1978 and inoculated with Aspergillus flavus Link 20 or 40 days after silking. Inoculation methods included needle, knife, and multiple-puncture injury to the kernels. The level of aflatoxin contamination, insect damage to the ear, and the percentage of ears having visible greenish A. flavus Link-type mold were determined. Differences among hybrids were not significant for any of the three characteristics measured, although aflatoxin levels of the early-maturing, loose-husked hybrids were approximately twice as high as those of two later-maturing, tight-husked types. Differences among treatments for insect damage rating were not statistically significant. Delaying inoculation until 40 days after silking significantly reduced the aflatoxin contamination level of samples harvested at maturity. Fewer than one-half the ears inoculated at 40 days after silking (35.3%) exhibited visible signs of infection compared with ears inoculated 20 days after silking (82.9%). The needle inoculations were less effective in eliciting aflatoxin production (163 μg/kg and 45.1% visibly infected ears) than were knife (202 μg/kg and 61.8% visibly infected ears) and multiple puncture (305 μg/kg and 70.4% visibly infected ears) methods of inoculation.     

额尔齐斯河几种鱼类寄生三代虫种类鉴定及系统发育

对采自额尔齐斯河的银鲫(Carassius auratus gibelio Bloch)、东方欧鳊(Abramis brama orientalis Berg)、黏鲈(Gymnocephalus cernua Linnaeus)、金鳟(Oncorhynchus mykiss Walbaum)感染的三代虫(Gyrodactylus sp.)进行了研究, 通过几丁质结构的形态测量与比较, 初步鉴定寄生于银鲫的为细锚三代(G. sprostonae), 寄生东方欧鳊的为秀丽三代虫(G. elegans), 寄生黏鲈的为普氏三代虫(G. prostate), 寄生金鳟的为细鳞鲑三代虫(G. brachymystacis)。同时将测定的三代虫的ITS序列, 与GenBank上三代虫序列进行比对分析, 发现上述4种三代虫分别与细锚三代虫、秀丽三代虫、细鳞鲑三代虫、普氏三代虫的ITS同源性都在99.3%以上, 进一步验证了形态学的鉴定结果。系统发育分析显示细鳞鲑三代虫和细锚三代虫位于G. (Limnonephrotus)亚属的一枝, 秀丽三代虫和普氏三代虫位于G. (Gyrodactylus)亚属的一枝, 而此两亚属亲缘关系较远。     

双氟沙星对异育银鲫血脑屏障渗透性及消除规律

以异育银鲫（Carassais auratus gibebio）为研究对象,采用组织匀浆法和高效液相色谱法,研究了双氟沙星（Difloxacin,DIF）通过异育银鲫血脑屏障情况,并比较分析了大脑和外周组织中DIF消除差异。结果显示,根据DIF 96h 半数致死剂量（2840 mg/kg b.W）给药后,第96h时异育银鲫大脑组织匀浆中DIF的含量为（10.490.35） g/g;同时在临床推荐用药剂量（20 mg/kg）给药后的15个时间点（0960h）上均能从大脑组织匀浆中检测出DIF。上述结果表明DIF能渗透通过血脑屏障而进入异育银鲫大脑组织。另外,在大脑和外周组织消除过程上,以大脑组织中的DIF消除过程最为平缓（按照20 mg/kg给药）。到试验第960h,大脑组织中DIF含量最高,为（0.3920.007） g/g,且大脑中的消除半衰期最长,为1157.713h。因此,异育银鲫大脑组织可作为DIF药物残留分析的靶组织。另根据欧盟关于食品中DIF最大残留限量（MRL）之规定,实验条件下DIF休药期至少为25d。结果为研究鱼类血脑屏障作用,DIF神经毒性及其在水产养殖上的临床应用提供了参考。