圆口铜鱼亲本与子代肝脏转录组特征及对比分析

通过Illumina HiseqTM 2000测序平台首次开展了圆口铜鱼(Coreius guichenoti)亲本与子代肝脏转录组测序并对比分析其测序结果。对转录组进行拼接和组装, 共获得80688个Unigene, Unigene的长度主要分布在401—600 bp, 占总Unigene的43.56%。与Nr、GO、COG、KEGG等公共数据库比对并进行功能注释, 经分析圆口铜鱼亲本与子代差异表达基因共2701个, 其中上调1240个, 下调1461个。差异基因表达模式聚类热图显示, 同一样本不同重复基因表达相似。将差异基因映射到代谢通路KEGG数据库进行富集分析, 并对37个KEGG显著富集的代谢通路(P<0.05)构建其基因共表达网络, 结果显示丙酸盐代谢、丙酮酸代谢、原核生物固碳途径、乙醛和二羧酸代谢、脂肪酸代谢、萜类骨架生物合成是KEGG差异表达基因的核心代谢途径, 且这些基因在圆口铜鱼子代中表达量均显著上调。该结果丰富了圆口铜鱼基因组数据, 并首次从分子水平比较了圆口铜鱼亲本和子代的代谢差异, 同时解释了在同一循环水系统中子代不易患病的现象。     

长江宜宾江段铜鱼属鱼类种间食物关系

采用碳、氮稳定同位素技术对2012年4-5月长江宜宾江段铜鱼和圆口铜鱼的食物组成进行了分析,并利用多元统计分析方法探讨了铜鱼和圆口铜鱼种间食物关系,包括饵料相似性指数、重叠系数及2种鱼的摄食器官形态差异对食物组成的影响.结果表明:1)铜鱼的δ13C和δ15N变化范围分别为-21.15‰～-20.31‰和9.67‰～ 10.21‰,为偏动物性的杂食性鱼类;圆口铜鱼的δ13C和δ15N分别为-23.30‰～-21.18‰和7.40‰～ 9.21‰,相对铜鱼为偏植物性的杂食性鱼类,铜鱼和圆口铜鱼均与传统肠含物分析的结果存在一定的差异;2)两种鱼的食物相似性指数和食物重叠系数分别为78.7％和55.6％;3)主成分分析(PCA)表明,铜鱼和圆口铜鱼摄食器官形态特征存在一定的差异.长江宜宾江段铜鱼和圆口铜鱼种间竞争不激烈,作为向家坝蓄水前最后一次对长江宜宾江段铜鱼和圆口铜鱼食性及种间食物关系的调查,研究结果可为分析向家坝蓄水对宜宾江段鱼类营养结构的影响提供数据参考.     

三峡水库蓄水后铜鱼和圆口铜鱼肌肉和肝脏中重金属水平

为了解三峡水库蓄水后鱼体重金属富集现状及其潜在的生态风险和食品安全, 测定了三峡水库上、中、下游不同年龄组铜鱼(Coreius heterodon)和圆口铜鱼(C. guichenoti)肌肉和肝脏中重金属含量。利用等离子吸收光谱法、石墨炉原子吸收光谱法、原子荧光光谱法检测样品中Cu、Zn、Cr、Pb、Cd、Hg、As 等 7种重金属含量。结果表明: 7 种重金属在铜鱼和圆口铜鱼体内的含量水平基本一致, 重金属在铜鱼和圆口铜鱼肌肉中含量大小均为Zn Cu Cr Hg AsPb Cd, 在铜鱼肝脏中含量大小为Zn Cu Pb Cd Cr As Hg, 而在圆口铜鱼肝脏中含量大小为Zn Cu Pb Cr Cd Hg As。铜鱼和圆口铜鱼肝脏中重金属含量显著高于肌肉(P0.05)。两种鱼类间大部分重金属在库区上、中、下游无显著差异(P 0.05)。铜鱼和圆口铜鱼肌肉(可食用部分)中7 种重金属含量均未超过国家食品安全卫生标准, 属于安全食用范围;肝脏中除Cd 和Pb 外的其他元素含量均未超过国家标准。相关结果反映了三峡水库175 m 蓄水后底栖土著经济鱼类重金属的污染状况, 对了解该地区水产品质量安全状况及水产品安全评价提供了参考依据。       

向家坝和溪洛渡蓄水对圆口铜鱼不同年龄个体下行移动的影响

拦河筑坝所导致的物理阻隔和生态阻隔是影响鱼类迁徙的关键因素。圆口铜鱼Coreius guichenoti(SauvageDabry de Thiersant,1874)作为长江上游重要的河流性洄游鱼类,其迁徙不可避免受到金沙江下游向家坝和溪洛渡大型水电站蓄水的影响。关于向家坝和溪洛渡蓄水是否会显著阻隔圆口铜鱼个体的下行以及不同年龄个体之间受阻隔的影响是否存在差异,迄今为止,还未有相关报道。本文拟通过2012—2014年对长江上游永善江段和宜宾江段圆口铜鱼年龄结构变动的研究,探讨向家坝和溪洛渡蓄水对圆口铜鱼不同年龄个体下行阻隔的影响程度。结果表明:1)向家坝和溪洛渡蓄水对圆口铜鱼下行产生了明显的阻隔效应;2)向家坝和溪洛渡蓄水对1龄个体的下行阻隔最明显且最严重;3)与单一的大型水库和大坝相比,多个梯级的大型水库和大坝会对圆口铜鱼的下行产生更为严重的阻隔效应。本文拟为金沙江下游圆口铜鱼的资源保护提供基础数据支持。     

养殖施氏鲟的性腺转录组特征分析

鲟是目前世界上最古老的软骨硬鳞鱼类之一, 雌雄个体之间无明显的第二性征。为了解人工养殖下鲟性腺发育的分子特征, 研究以人工养殖2龄施氏鲟(Acipenser schrenckii Brandt)为研究对象, 对其精巢与卵巢进行转录组测序分析。结果发现, 雌雄性腺中共有19690个差异表达基因转录本, 其中与性别分化相关基因包括转录因子Dmrt1、Sox9、Foxl2等和生长转化因子Amh、Bmp15、Gdf9等。另外, 通过差异表达基因KEGG代谢通路富集分析发现了4条与卵巢发育相关的通路, 分别为黄体酮介导的卵母细胞成熟、卵母细胞减数分裂、卵巢类固醇合成、促性腺激素释放激素信号通路。其中, 卵巢类固醇合成通路中18个差异表达基因的表达模式暗示了2龄施氏鲟限制卵巢雌激素的合成, 但精巢中雄激素的合成未受影响。研究结果为研究鲟性腺分化和发育机制以及今后在mRNA表达水平上鉴定鲟性别提供了基础。     

鱼类生长激素生物活性和定量免疫测定技术的研究进展

生长激素(growth hormone, GH)是调节鱼类生长、发育和代谢的一种重要激素.为了弄清鱼体内GH水平与鱼体生长的关系, 以及研究外源GH基因在受体鱼体内的表达部位、表达效率和表达调控等问题, 首先必须建立鱼类GH灵敏、特异的微量检测技术.此外, 在进行鱼类GH分离纯化中, 很重要的一点就是鉴定所获得的GH制品是否具有生物活性, 同样也需要建立鱼类GH灵敏、特异的生物活性检测技术.     

蓝太阳鱼第一次性周期性腺发育的组织学

蓝太阳鱼(Lepomis cyanellus)是广东省于1997年从北美引进的小型淡水鱼类,本文采用组织切片技术对蓝太阳鱼的性腺发育进行了研究。蓝太阳鱼的性腺发育程序可以分为5个时期。在1月龄,卵巢和精巢处于第Ⅰ期;2~3月龄时,卵巢和精巢发育到第Ⅱ期;4月龄时发育到第Ⅲ期;5~6月龄时发育到第Ⅳ期;7~8月龄时达到性成熟第Ⅴ期。过冬时,卵巢退化到第Ⅱ~Ⅲ期,而精巢仍停留在第Ⅴ期。成熟卵巢的成熟系数为2.80%~8.10%,成熟精巢的成熟系数为1.45%~3.45%。精巢和卵巢发育都为不完全同步型,且精巢发育比卵巢稍快。蓝太阳鱼的繁殖期在广州地区为3~11月,为多次产卵类型。本文从生殖和生长的关系上对蓝太阳鱼生长缓慢的原因进行了初步探讨,并将蓝太阳鱼作为一种鱼类实验动物的可行性进行了分析。     

长江上游圆口铜鱼生长方程的分析

根据2005～2007年在长江上游鱼类资源调查中采集到的476尾圆口铜鱼Coreius guichenoti Sauvage et Dabry标本的数据,对其生长方程进行了分析.圆口铜鱼鳞片半径-体长的函数关系在雌雄群体间不存在显著性差异并符合方程L=85.429S0.8125 (R2=0.9635),推算体长表现出Lee氏现象;运用非线性混合模型方法,对基于4种备选生长方程(von Bertalanffy生长方程(VBG)、Richards生长方程、Gompertz生长方程和Robertson生长方程)的36种不同模型分别以生长推算数据进行拟合,并根据AIC选择最优生长模型,得到不同备选方程对圆口铜鱼生长描述的优劣顺序为:VBG、Richards、Gompertz、Robertson方程,其中最优生长模型为VBG Model 6:Lt=602.9(1-e-0.1693(t+0.024)) (雌雄群体间在0.01水平上不存在显著性差异).圆口铜鱼的生长特征指数φ=4.7891±0.01612.对于存在Lee氏现象的鱼类,在使用生长推算数据构建生长方程时,建议使用非线性混合模型以对参数值进行较合理的估计.本文为圆口铜鱼种群动态及资源保护提供了基础资料,并为不同种鱼类、同种鱼类不同生活史阶段表现出的不同生长模式的识别提供了一个范例.     

长江上游江津和宜宾江段圆口铜鱼资源量估算

为了解长江上游特有鱼类圆口铜鱼（Coreius guichenoti）的种群数量及分布,2007~2009年对江津和宜宾江段的渔业捕捞情况和渔获物组成进行了调查,根据单位捕捞努力量渔获量对该江段的年渔获量进行了统计分析,采用FISATⅡ渔业评估软件中体长股分析模块对圆口铜鱼的资源量进行了估算。2007、2008和2009年,江津江段鱼类年总渔获量分别为267 850、689 975和586 575尾,宜宾江段鱼类年总渔获量分别为485 100、108 075和169 950尾;其中,江津江段圆口铜鱼的年渔获量分别为56 932、98 323和100 620尾,宜宾江段圆口铜鱼的年渔获量分别为66 099、16 952和32 812尾。渔获个体主要分布在80~220mm体长组。估算出2007~2009年江津江段圆口铜鱼年资源量分别为319 416、905 715和1124 470尾,年均值为783 200尾;以质量计,年资源量分别为25.33、129.72和244.48 t,年均值为133.18 t。宜宾江段圆口铜鱼年资源量分别为469 395、191 207和200 197尾,年均值为286 933尾;以质量计,年资源量分别为60.18、43.11和15.14 t,年均值为39.48 t。以上结果表明,长江上游江津江段圆口铜鱼年均资源量大于宜宾江段;2007~2009年宜宾江段圆口铜鱼资源量呈下降趋势,而江津江段圆口铜鱼资源量呈上升趋势。     

厚颌鲂和圆口铜鱼耗氧率与窒息点的测定

用封闭静水式装置测定了体重2.3-4.7g厚颌鲂幼鱼的耗氧率和窒息点,用封闭静水式和封闭流水式装置测定了体重9.9-55.1g圆口铜鱼的耗氧率和窒息点。结果表明:在15-27℃条件下,厚颌鲂的耗氧率随着温度的升高而升高,耗氧率与水温呈线性关系;在水温24.8℃时厚颌鲂的窒息点为(0.91±0.08)mg/L。在水温23-27℃、封闭静水实验条件下,圆口铜鱼的耗氧率随体重增加而降低,两者呈指数关系;圆口铜鱼耗氧率昼夜变化明显,夜间耗氧率大于白天,推测圆口铜鱼夜间活动较多。在水温24.5-26.0℃条件下,体重21.8-46.3g圆口铜鱼的窒息点变幅较小,平均(1.14±0.23)mg/L。研究表明两种鱼都为耗氧率和窒息点较高的鱼类。       

Genetic diversity in population of largemouth bronze gudgeon (Coreius guichenoti Sauvage et Dabry) from Yangtze River determined by microsatellite DNA analysis

Largemouth bronze gudgeon (Coreius guichenoti Sauvage et Dabry 1874), one of the endemic fish species in the upper reaches of the Yangtze River in China, is a benthic and potamodromous fish that is typically found in rivers with torrential flow. Three dams in the Yangtze River, Ertan Dam, Three Gorges Dam and Gezhouba Dam, may have had vital impacts on the habitat and spawning behaviors of largemouth bronze gudgeon, and could ultimately threaten the survival of this fish. We studied the population genetic diversity of C. guichenoti samples collected at seven sites (JH, GLP, BX, HJ, MD, SDP and XB) within the Yangtze River and one of its tributaries, the Yalong River. Genetic diversity patterns were determined by analyzing genetic data from 11 polymorphic microsatellite loci. A high genetic diversity among these largemouth bronze gudgeon populations was indicated by the number of microsatellite alleles (A) and the expected heterozygosity (HE). No significant population variation occurred among GLP, BX, HJ and MD populations, but dramatic population differentiation was observed among JH and XB, two dam-blocked populations, versus other populations. Tests for bottlenecks did not indicate recent dramatic population declines and concurrent losses of genetic diversity in any largemouth bronze gudgeon populations. To the contrary, we found that dams accelerated the population differentiation of this fish.     

长江上游网箱养殖圆口铜鱼杀鲑气单胞菌杀鲑亚种的分离与鉴定

研究首次报道了圆口铜鱼(Coreius guichenoti)疥疮病, 从患病圆口铜鱼的肝脏中分离到优势菌株YTL1, 并运用形态学观察、生理生化检测、16S rRNA和6个管家基因的系统发育分析等对分离菌株进行鉴定。基于以上实验结果, YTL1被最终鉴定为杀鲑气单胞菌杀鲑亚种(Aeromonas salmonicida subsp. salmonicida)。通过标准Kirby-Bauer纸片扩散法进行抗菌药物敏感性试验, 筛选治疗该暴发病的有效药物, 结果显示YTL1对氟苯尼考, 诺氟沙星和氨苄青霉素等13种抗生素敏感, 对6种抗生素如杆菌肽, 链霉素和卡那霉素有耐药性, 对红霉素具有中等敏感性。因此, 氟苯尼考被建议用来伴饵投喂, 并取得了较好的疾病控制效果。草鱼幼鱼(Ctenopharyngodon idella)和斑马鱼(Danio rerio)的人工感染试验结果显示, 经腹腔注射7.6×106—7.6×108 CFU/mL的YTL1菌液后, 感染鱼的症状与患病圆口铜鱼症状相似。研究证明基于6个管家基因的多序列位点分型是鉴定杀鲑气单胞菌至亚种水平的一种有效方法, 杀鲑气单胞菌是圆口铜鱼人工养殖的最大威胁之一, 并发现鲤科鱼类, 如草鱼和斑马鱼均是杀鲑气单胞菌杀鲑亚种的易感宿主。     

Isolation and characterization of polymorphic microsatellites in a Yangtze River fish,brass gudgeon (Coreius heterodon Bleeker)

The brass gudgeon (Coreius heterodon) is a fish of economic importance in the Yangtze River. From a (GATA)_n‐enriched genomic library, 25 microsatellites were developed by employing the fast isolation by AFLP of sequences containing repeats (FIASCO) protocol. Nine loci exhibited polymorphism with two to 12 alleles (mean 3.9 alleles/locus) in a test population, and observed heterozygosity ranged from 0.1111 to 0.9630 (mean 0.4426). Three of the nine loci showed polymorphism in a congeneric species, the largemouth bronze gudgeon Coreius guichenoti. These loci should provide sufficient level of genetic diversity to evaluate the fine‐scale population structure of C. heterodon.     

Physiological responses to swimming fatigue in juvenile largemouth bronze gudgeon Coreius guichenoti

Stepped velocity tests were conducted on juvenile largemouth bronze gudgeon Coreius guichenoti in a swim tunnel respirometer, and oxygen consumption increased with swimming speed to fatigue and then decreased during recovery. Serum levels of total protein, glucose and triglycerides initially decreased, increased at fatigue and then decreased during recovery. Levels stabilized after 120 min, corresponding to the time necessary to recover from fatigue.     

金沙江中游圆口铜鱼早期资源现状

2010年6月至7月在金沙江中游攀枝花格里坪金沙滩(26°35°28.96″N,101°31°52.01″E)设置同定采样断面,开展了金沙江中游圆口铜鱼早期资源现状的调查.结果表明,金沙江中游有金安桥、朵美、皮拉海、灰拉古、观音岩5个圆口铜鱼产卵场,产卵总量为3078.2万粒.圆口铜鱼的产卵行为和产卵量受江水的温度、流量、涨落水持续时间等多个因素的影响.调查期间圆口铜鱼的产卵初始时间为6月7日,盛期在6月下旬至7月上旬.近几年来,圆口铜鱼的产卵规模呈现出逐年下降的趋势,产卵场的位置也有所改变.通过了解金沙江中游江段圆口铜鱼早期资源分布状况,为金沙江中游圆口铜鱼资源保护提供了基础资料.     

Effects of body mass and temperature on routine metabolic rate of juvenile largemouth bronze gudgeon Coreius guichenoti

The effects of body mass (M) and temperature (T) on routine metabolic rate (m(R) ) were assessed in the largemouth bronze gudgeon Coreius guichenoti, from Three Gorges Reservoir, Yangtze River, China. The m(R) increased with increasing M by factors (b-value in the equation m(R) = aM(b) ) of 0·843, 0·800, 0·767, 0·788 and 0·822 at 10, 15, 20, 25 and 30° C, respectively. A significant interaction between M and T on m(R) was observed. The variation in the b-value at different T suggests that the b-values were not consistent with the universal allometric exponent 0·75. After controlling for M, the relationship between the normalized standard metabolic rate (m(S), mg O(2) kg(-1) h(-1)) and T was described by an exponential equation: m(S) = 9·89e((0·093T)) . The results indicate that the effects of M on m(R) depend on T. The increased water temperature induced by dam construction on the Yangtze River may cause a marked increase in energy demand by this species, with potential ecological consequences.     

Genetic diversity in the bronze gudgeon, <Emphasis Type="Italic">Coreius heterodon</Emphasis>, from the Yangtze River system based on mtDNA sequences of the control region

The bronze gudgeon, Coreius heterodon (Bleeker), is an economically important species, which only inhabits the Yangtze River. The stock declined drastically in recent years due to dam construction, over-fishing, and pollution. Little is known about its population genetic structure. In this study, the sequences of the mitochondrial DNA (mtDNA) control regions of natural bronze gudgeon was determined for fish collected from four sites in the Yangtze River (n = 102). The molecular data were used to estimate the genetic diversity and differentiation of the bronze gudgeon. The results showed that 28 haplotypes and 22 variable sites were found, and the haplotype diversity (π) and nucleotide diversity (h) were 0.849 and 0.00257, respectively. A low level of genetic diversity exists in the bronze gudgeon. Analysis of molecular variance (AMOVA) suggests that 98.8% of the genetic variability occurred within the populations; the site of collection had little influence on diversity. Future research should focus on investigating the genetic divergence of populations in different tributaries and using additional polymorphic markers, such as microsatellite DNA, to verify the results and improve interpretation.     

In vivo induction of oocyte maturation and ovulation in zebrafish

The maturation of fish oocytes is a well-characterized system induced by progestins via non-genomic actions. In a previous study, we demonstrated that diethylstilbestrol (DES), a non-steroidal estrogen, induces fish oocyte maturation via the membrane progestin receptor (mPR). Here, we attempted to evaluate the effect of DES as an environmental endocrine disrupting chemical (EDC) upon fish oocyte maturation using live zebrafish. DES triggered oocyte maturation within several hours in vivo when administrated directly into the surrounding water. The natural teleost maturation-inducing hormone, 17alpha, 20beta-dihydroxy-4-pregnen-3-one (17,20beta-DHP) also induced oocyte maturation in vivo. Steroids such as testosterone, progesterone or 17alpha-hydroxyprogesterone were also effective in vivo. Further studies indicated that externally applied 17,20beta-DHP even induced ovulation. In contrast to 17,20beta -DHP, DES induced maturation but not ovulation. Theoretically this assay system provides a means to distinguish pathways involved in the induction of ovulation, which are known to be induced by genomic actions from the pathway normally involved in the induction of oocyte maturation, a typical non-genomic action-dependent pathway. In summary, we have demonstrated the effect of EDCs on fish oocyte maturation in vivo. To address the effects, we have explored a conceptually new approach to distinguish between the genomic and non-genomic actions induced by steroids. The assay can be applied to screens of progestin-like effects upon oocyte maturation and ovulation for small molecules of pharmacological agents or EDCs.     

Microsatellite DNA variation among samples of bronze gudgeon, Coreius heterodon, in the mainstem of the Yangtze River, China

Bronze gudgeon, Coreius heterodon, is an endemic and economically important fish in the Yangtze River, whose abundance has declined dramatically because of dam construction, overfishing, and water pollution. The Gezhouba and Three Gorges dams block connection of the bronze gudgeon populations above and below the dams. We collected bronze gudgeon from four sites in the mainstem of the Yangtze River, with one site above the dams and three sites below the dams, and studied genetic structure within and among the samples using 12 microsatellite DNA markers. Differences in indexes of genetic diversity were not significant among all the samples. No recent dramatic decrease of effective population size was inferred for all the samples using the population bottleneck test. Overall and pairwise genetic differentiation showed no significant genetic differentiation. Membership proportions of three genetic clusters inferred using assignment analysis were not significantly different among the samples. These results suggested that the genetic diversity and structure of bronze gudgeon were uniform across the samples. However, the Hardy–Weinberg equilibrium test, fixation index and linkage disequilibrium test indicated genetic subdivision of bronze gudgeon in the upper reach of the Three Gorges Dam. The present study and future studies including tributary samples will provide an important baseline of genetic diversity and population structure of bronze gudgeon in the Yangtze River, which is critical for monitoring and evaluating impacts of the large-scale dams on this species.