热应激对大菱鲆心肌损伤及细胞凋亡的影响

为解析热应激对大菱鲆心脏损伤及其机制,实验从组织形态、生理生化反应及凋亡基因表达等多个水平,分别使用H.E染色法、电镜观察法、酶活性检测法、qPCR检测基因表达法开展了本研究。结果显示,随着温度升高,心肌纤维肿胀,断裂,间质宽度增加,炎性细胞浸润,线粒体结构破坏等组织损伤现象加重,但在24°C-24 h时组织损伤明显减轻;CK活性随着热应激加剧显著升高;LDH、SOD活性,MDA含量在24°C时达到峰值,表明大菱鲆遭受到热应激,心肌防御酶发挥抵抗作用,维持机体稳态。qPCR显示,大菱鲆心肌细胞Bax基因和Caspase-3基因变化趋势一致,随着热应激的加剧,表达量降低,而Bcl-2基因逐渐升高。表明在热应激程度较轻时,大菱鲆心肌通过降低Bax、Caspase-3基因表达,促进抗凋亡基因Bcl-2的表达,减少心肌细胞丢失来减少热应激损伤。当热应激加剧至28°C时,热应激超过自身生理调节阈值,损伤加重,机体防御系统自身也受损,造成大菱鲆心脏结构严重损伤甚至机体死亡。研究表明,随着温度升高,大菱鲆心肌损伤加重,机体通过调节心肌防御酶活性以及使细胞凋亡,最大限度维持稳态,减少组织损伤。超过24...     

低氧胁迫和恢复对长吻鮠脑组织低氧应答基因、生理生化指标和食欲的影响

为了解长吻鮠脑组织应对低氧胁迫的调控机制，实验运用酶活性测定、H.E染色、qRT-PCR和TUNEL检测等方法，分析比较了低氧胁迫[(0.8±0.1) mg/L] 0、2、4、6 h(标示为H0、H2、H4和H6)和恢复[(7.3±0.5) mg/L]2、4、6 h (标示为R2、R4和R6)下长吻鮠脑组织低氧应答基因、生理生化指标和食欲基因的变化。结果显示，在低氧胁迫和恢复下，长吻鮠脑组织氧传感蛋白相关基因(HIFs、PHDs和Vhl)表达量整体呈现出先上升后下降趋势；呼吸代谢酶(HK、PK和LDH)活性在H0时显著升高，SDH和MDH活性在H6时显著降低，恢复溶解氧后，代谢模式由无氧呼吸逐渐转变为有氧呼吸；抗氧化酶(GSH-Px、CAT和SOD)和应激指标(MDA和LPO)在低氧2 h后逐渐升高，恢复溶解氧后氧化应激现象仍然存在。观察脑组织形态发现，在低氧胁迫下长吻鮠脑组织出现了神经细胞肿胀、空泡等受损现象，恢复溶解氧6 h后脑组织受损并未得到有效改善。随着低氧时间延长，脑组织细胞凋亡程度不断增加，凋亡相关基因(Bax、Caspase-3和p53)表达量显著升高，而Bcl-2基因表...     

低氧胁迫下鲢miR-17a-5p及其靶基因分析

为了探究miR-17a-5p在鲢低氧胁迫下的功能,在前期鲢small RNA测序的基础上对鲢miR-17a-5p进行靶基因预测及功能富集分析,通过双荧光素酶活性验证其与HIF-1α的靶向关系,并检测低氧胁迫下miR-17a-5p和其靶基因在鲢肝脏、脑、心脏和鳃四个组织中的动态表达特征。结果显示,鲢miR-17a-5p在不同物种间高度保守,预测出381个miR-17a-5p的潜在靶基因显著富集在硫代谢、mTOR信号通路以及萜类骨架的生物合成3个KEGG信号通路上。miR-17a-5p可与HIF-1α mRNA的3′UTR结合,并降低HIF-1α mRNA水平,低氧胁迫下miR-17a-5p的表达呈下降趋势,而HIF-1α表达则呈上升趋势;3个显著富集KEGG通路中的11个miR-17a-5p潜在靶基因在低氧胁迫过程中的不同组织中的表达,尤其是肝脏中的表达,除SGK1外,均呈显著上升趋势。研究表明,低氧胁迫下鲢各组织中miR-17a-5p的表达下调减弱了其对靶基因的抑制作用,进而导致HIF-1α、ddit4和Lrp5等响应低氧胁迫的基因表达上调。本研究为低氧胁迫下鲢miRNA的表达与调控机...     

三氯生对雄性斑马鱼鳃组织凋亡相关基因表达的影响

为探究三氯生(TCS)对于硬骨鱼类的凋亡相关基因表达的影响,以雄性斑马鱼(Danio Rerio)为试验动物,采用半静态水体接触染毒法,将斑马鱼分别置于质量浓度为17、34和68μg/L的TCS溶液中,并设计空白对照组,42 d后,对鳃组织进行采样,利用普通PCR和RT-qPCR技术分析雄性斑马鱼鳃组织凋亡基因Bcl-2、Bax、MDM2、p53的表达量。研究发现,与空白对照组相比,Bax和p53基因在TCS处理组中的表达量都出现了下调趋势,且在68μg/L的TCS处理组中表达水平显著下调;Bcl-2在各TCS处理组中无显著变化;MDM2基因在34μg/L和68μg/L TCS处理浓度组中表现为极显著上调。研究结果表明:高浓度TCS对雄性斑马鱼鳃组织凋亡相关基因的表达具有一定的影响。     

三氯生对斑马鱼卵巢抗氧化和凋亡相关基因表达的影响

为了探讨三氯生(TCS)对硬骨鱼类性腺损伤作用的分子机制,通过半静态水体暴露法对斑马鱼(Danio rerio)进行染毒,将斑马鱼暴露在2种不同TCS浓度(0.034 mg/L和0.068 mg/L)溶液中,同时设置空白对照组,42 d后,利用实时荧光定量PCR(RT-qPCR)技术,探究TCS对斑马鱼卵巢抗氧化及凋亡相关基因表达的影响。结果发现:与空白对照相比,SOD、CAT和GPx1a基因在0.068 mg/L TCS处理组表达水平显著下调,GPx1和MT-2基因在0.034 mg/L浓度组表达显著上调;Bcl-2基因在0.068 mg/L浓度组表达显著下调,p53基因在0.034 mg/L浓度组表达极显著上调,Bax在TCS处理组中均显著上调。以上研究结果表明:较高浓度TCS显著影响了斑马鱼卵巢抗氧化和凋亡相关基因的表达,从而产生了氧化损伤和加速细胞凋亡的发生。     

急性高温胁迫对大口黑鲈"优鲈3号"肝脏凋亡相关酶活性和基因表达的影响

为了探究急性高温胁迫对大口黑鲈“优鲈3号”凋亡相关酶活性和基因表达的影响,将25℃下暂养的大口黑鲈“优鲈3号”直接放入37℃水环境中,探究急性高温胁迫后幼鱼肝脏组织Caspase3、Caspase9和基因酶活的变化。结果显示:37℃高温胁迫后,肝脏Caspase3和Caspase9酶活均显著升高,且随着胁迫时间的延长呈升高趋势,在48 h达到峰值。基因Caspase3和Caspase9的表达量均呈现先升高再下降的趋势,在6 h的表达量达到峰值,随之下降,并在48 h与对照组无显著性差异。BCL-2的表达量于1 h达到峰值,之后显著下降,48 h时BCL-2的表达量仅为对照组的0.04倍。结果表明,高温胁迫会使大口黑鲈“优鲈3号”幼鱼发生凋亡甚至细胞坏死。     

低氧胁迫对大口黑鲈“优鲈3号”hif-1α、glut-1及caspases基因的影响

为研究低氧胁迫对大口黑鲈"优鲈3号"呼吸代谢和细胞凋亡的影响,在溶解氧(DO)为(0.70±0.05) mg/L的条件下,测定了试验鱼肝脏及脑组织hif-1α、glut-1和caspases基因的表达。结果显示:与DO为(7.50±0.50)mg/L的常氧对照组相比,低氧胁迫能引起大口黑鲈"优鲈3号"肝脏及脑组织hif-1α、glut-1表达水平的显著上调,同时也可诱导caspase 3和caspase 9基因的表达,致使细胞发生凋亡;复氧24 h后,hif-1α、glut-1基因在试验鱼肝脏及脑组织中的表达量均恢复至常氧水平,caspases基因在脑组织中的表达量极显著高于对照组(P0.01),caspase 3在肝组织中的表达量显著高于对照组(P0.05),caspase 9则恢复至常氧水平。试验结果初步表明,hif-1α和glut-1是大口黑鲈"优鲈3号"低氧信号传导途径中的重要组成部分,低氧胁迫可能会激活线粒体凋亡通路,从而可能会诱导细胞凋亡的发生。     

鲤神经球蛋白基因序列与低氧表达分析

为深入了解鲤珠蛋白家族的基因组成和表达模式,及其与低氧适应能力的相关性,本实验通过鲤基因组框架图比对和全长cDNA文库筛查,获得鲤神经球蛋白(neuroglobin,Ngb)基因完整序列,证实鲤不仅具有独特的脑组织特异表达的II型肌红蛋白(myoglobin-2,Mb-2)基因,也具有Ngb珠蛋白基因,实时荧光定量PCR实验显示,该基因在脑组织特异性表达,并呈现出低氧应答特征。基因结构和系统发生分析表明,鱼类Ngb基因高度保守,鲤Ngb蛋白可能与斑马鱼直系同源蛋白具有相似的结构及功能,而与鲤Mb-2存在明显差异。鲤Ngb基因表达量在两个品系间存在显著差异,耐低氧能力强的散鳞镜鲤Ngb基因表达量高于荷包红鲤抗寒品系。研究表明,鲤Ngb基因可能以与Mb-2基因分工协作的方式共同实现脑组织供氧,执行应对低氧胁迫的神经保护功能,在鲤低氧适应中具有重要作用。     

大黄鱼冷诱导结合蛋白(CIRP)基因cDNA克隆及低温胁迫对其时空表达的影响

为研究冷诱导结合蛋白(CIRP)在大黄鱼低温适应过程中的作用,采用同源克隆技术获得了大黄鱼CIRP基因,并通过实时荧光定量PCR(qRT-PCR)技术检测了低温胁迫下大黄鱼各组织中CIRP基因的表达变化。结果显示,大黄鱼CIRP基因cDNA序列全长1211 bp,推测编码一个由182个氨基酸组成、分子量为19 ku的蛋白。序列比对显示硬骨鱼类CIRP基因序列较为保守,特别是N端的RNA识别基序(RRM)高度保守。系统进化树分析显示大黄鱼CIRP与银鳕相似性最高(81.5%),所有硬骨鱼类聚为一个大支。慢性低温胁迫中(12℃缓降至6℃),皮肤和肌肉中CIRP基因的表达量呈随温度降低而升高的趋势,6℃时表达量分别为12℃时的11.56倍和9.03倍;鳃、脑和心脏中CIRP基因表达量均呈先显著上升后显著下降的趋势,其峰值均出现在8℃时,表达量分别为12℃时的5.07、7.69和2.83倍;肠、肾脏、肝脏和脾脏中的表达量随温度降低而下降,6℃时的表达量最低,与12℃时相比降幅分别为80.0%、65.6%、91.2%、55.6%。急性低温胁迫(由12℃骤降至8℃并胁迫4 h)条件下,鳃、皮肤、脑、肝脏、心脏中CIRP基因表达量均呈先升高后降低的变化,反映了机体对低温胁迫的应激、适应过程;肌肉和肠中的表达量则始终显著低于胁迫前。慢性和急性低温胁迫中各组织CIRP表达量变化的差异提示,大黄鱼机体在低温胁迫响应和适应中有着多种调节机制。研究表明,CIRP基因参与了大黄鱼应对低温胁迫的过程。     

急性氨氮暴露对大弹涂鱼炎性反应相关基因表达的影响

为研究急性氨氮胁迫对大弹涂鱼炎性反应相关基因表达的影响,实验挑选初始体质量为(15.14±0.05) g的健康大弹涂鱼幼鱼180尾,进行96 h的急性氨氮胁迫实验。结果显示,大弹涂鱼96 h氨氮半致死浓度为8.99 mg/L总氨氮(0.11 mg/L非离子氨,T-AN);氨氮胁迫后TNF基因的mRNA表达量分别于12和96 h时显著上调,96 h时表达量达到0 h时的2倍;IL-1基因的mRNA表达量12 h时显著上调,为0 h时表达量的6倍;氨氮胁迫后IL-6基因的mRNA表达量分别于12和96 h时显著上调,表达量达到0 h时的1.5倍;氨氮胁迫后IL-8基因的mRNA表达量在24 h时出现显著下调。研究表明,大弹涂鱼96 h氨氮半致死浓度为8.99 mg/L总氨氮;半致死浓度的氨氮胁迫48 h后,TNF、IL-1、IL-6和IL-8基因的mRNA表达量持续升高,推测过度炎性应激可能是导致鱼类氨中毒死亡的原因之一。     

二氧化碳麻醉对鲢生理生化指标及神经调控相关基因表达的影响

为研究CO 2麻醉对鲢(Hypophthalmichthys molitrix)的生理效应及其作用机制,设置充CO 2、充O 2实验组,并以持续充空气作为对照组,分别测定鲢的生理生化指标、神经递质含量和神经调控相关基因表达变化。结果显示:与对照组比较,CO 2麻醉处理后,鲢呼吸频率降低37%,血清K+浓度显著升高4倍,肝糖原含量降低86.96%,血清中乳酸、尿素氮浓度分别升高1.08和1.57倍。以上指标在充O 2处理下变化趋势与充CO 2处理下相似,但是测定值高于充CO 2处理组。CO 2处理组血清皮质醇水平无显著变化,但充O 2处理组皮质醇显著升高2.41倍;CO 2处理组的脑组织多巴胺和5-羟色胺水平下降。qPCR结果显示,CO 2麻醉后脑组织双孔K+通道蛋白kcnk3a、kcnk9和缺氧相关hif-1α和epo基因表达量均升高。结果表明,CO 2麻醉鲢可以有效降低新陈代谢和应激反应,是一种安全、有效的麻醉方法。     

不同水流速度对中华倒刺鲃幼鱼自由基代谢的影响

在(25±0.5) ℃下, 设置4个水流速度实验组, 分别为静水对照组0 bl/s(体长/秒)、1.0 bl/s组、2.0 bl/s组和4.0 bl/s组, 研究不同水流速度对中华倒刺鲃(Spinibarbus sinensis)幼鱼肌肉自由基代谢的影响, 训练周期为8周。结果显示, 肌肉超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-P_X)的活性随着水流速度的增加而升高, 训练组过氧化氢酶(CAT)活性出现不同程度的下降, 而抗氧化物谷胱甘肽(GSH)的含量未发生明显变化(P>0.05); 训练组肌肉总抗氧化能力(T-AOC)均高于对照组, 2.0 bl/s组最高, 4.0 bl/s组次之, 1.0 bl/s组最低, 且2.0 bl/s和4.0 bl/s组T-AOC与对照组有显著性差异(P<0.05)。8周训练后, 2.0 bl/s组丙二醛(MDA)含量显著低于其他3组(P<0.05), 而H₂O₂含量随着水流速度增加呈上升趋势。8周运动训练对骨骼肌B细胞淋巴瘤因子-2 (Bcl-2)、Bcl-2相关X蛋白(Bax)的含量及血清肌酸激酶(CK)活性无显著影响(P>0.05), 但2.0 bl/s组Bax和Bax/ Bcl-2显著低于4.0 bl/s组(P<0.05)。结果表明, 2.0 bl/s的水流速度可提高中华倒刺鲃肌肉的抗氧化能力, 降低脂质过氧化水平, 并在一定程度上抑制肌细胞凋亡。

     

Molecular responses differ between sensitive silver carp and tolerant bighead carp and bigmouth buffalo exposed to rotenone

Some species of fish are more tolerant of rotenone, a commonly used non-specific piscicide, than others. This species-specific tolerance to rotenone has been thought to be associated with the uptake and the efficiency at which the chemical is detoxified. However, rotenone stimulates oxidative stress and superoxides, which are also toxic. Understanding the modes in which fish physiologically respond to rotenone is important in developing improved protocols for its application in controlling aquatic nuisance species. Using a molecular approach, we investigated the physiological and molecular mechanisms of rotenone resistance. Species-specific responses were observed when rotenone-sensitive silver, Hypophthalmichthys molitrix, and both rotenone-resistant bighead carp, Hypophthalmichthys nobilis, and bigmouth buffalo, Ictiobus cyprinellus, were exposed to rotenone. Rotenone levels in plasma were highest 90?min after exposure in both silver carp and bigmouth buffalo, but bigmouth buffalo tolerated over twice the burden (ng?mL(-1)?g(-1)) than silver carp. Expression of genes related with detoxification (cyp1a and gst) increased in silver carp, but either decreased or remained the same in bighead carp. Genes linked with oxidative stress in the cytosol (gpx, cat and sod1) and hsp70 increased only in silver carp after a 6-h exposure. Expression of genes associated with oxidative stress in the mitochondria (sod2 and ucp2) differed between silver carp and bighead carp. Expression of sod2 changed minimally in bighead carp, but expression of ucp2 linearly increased to nearly 85-fold of the level prior to exposure. Expression of sod2 and ucp2 did not change until 6?h in silver carp. Use of sod1 and sod2 to combat oxidative stress results in hydrogen peroxide production, while use of ucp2 produces nitric oxide, a chemical known to inhibit apoptosis. We conclude that the mechanism at which a fish handles oxidative stress plays an important role in the tolerance to rotenone.     

Cloning and characterization of <Emphasis Type="Italic">Bax1</Emphasis> and <Emphasis Type="Italic">Bax2</Emphasis> genes of <Emphasis Type="Italic">Ctenopharyngodon idellus</Emphasis> and evaluation of transcript expression in response to grass carp reovirus infection

Multidomain proapoptotic Bcl-2-associated X (Bax) protein is an essential effector responsible for mitochondrial outer membrane permeabilization, resulting in cell death via apoptosis. In this study, two Bax genes of grass carp (Ctenopharyngodon idellus), designated as CiBax1 and CiBax2, were isolated and analyzed. The obtained CiBax1 cDNA is 2058 bp long, with a 579 bp open reading frame (ORF) coding a protein of 192 amino acid residues. The full-length cDNA of CiBax2 is 1161 bp, with a 618 bp ORF coding 205 amino acids. Both CiBax1 and CiBax2 are typical members of Bcl-2 family containing conserved Bcl and C-terminal domains, and they share conserved synteny with zebrafish Bax genes despite the grass carp Bax mapping to different linkage groups. Phylogenetic analysis showed that CiBax1 was clustered with Bax from most teleost fish, and CiBax2 was close to Bax2 from teleost fish but far separated from that of Salmo salar. Quantitative real-time PCR analysis revealed broad expression of CiBax1 and CiBax2 in tissues from healthy grass carp, but the relative expression level differed. The mRNA expression of CiBax1 and CiBax2 was both upregulated significantly and peaked in all examined tissues at days 5 or 6 post-infection with grass carp reovirus. Subcellular localization indicated that CiBax1 protein was localized in both nucleus and cytosol, while CiBax2 protein only in cytosol. Moreover, CiBax2, but not CiBax1 was colocalized with mitochondrion under normal condition. Taken together, the findings would be helpful for further understanding of the function of Bax in teleost fish.     

Invasive silver carp may compete with unionid mussels for algae: First experimental evidence

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The effects of partially substituting Indian carps or adding silver carp on polycultures including small indigenous fish species (SIS)

A sustainable semi-intensive pond aquaculture technology including major carp species (Indian, Chinese and common carp) as cash-crop and small indigenous fish species (SIS) as food for the farmers' families is being optimized in Bangladesh. Silver carp inclusion in the polyculture is now being considered, because this very efficient filter feeder has a strong impact on pond ecology and also on the farmers' family nutrition because it is a cheap fish that the family can afford to eat instead of selling. The present paper is centered on the reduction of silver carp negative effects on other species while keeping the advantages of increased total yield and income due to silver carp stocking. It presents the results of two experiments, one on-station and one on-farm, in which 3–5 silver carp/100 m² were added or partially substituted major carp filter feeders. The basic stocking density was 100 carps (rohu, catla and a bottom feeder, either mrigal or common carp, at a 1:1:1 ratio) and 250 SIS (punti and mola) per 100 m². In the on-station experiment silver carp density was 3 and 5 fish/100 m² and the large carp bottom feeder was common carp. In the on-farm experiment silver carp density was 5 fish/100 m² and the bottom feeder was either common carp or mrigal.Most of the water quality and fish performance parameters tested were not affected by the polyculture composition. Adding 3–5% silver carp or substituting 3–5% of the herbivorous fish species by this highly efficient filter feeder increased grazing pressure on the phytoplankton, which led to a 25–40% reduction of the chlorophyll concentration in the water column. The increased grazing pressure was not enough to affect other water quality parameters and fewer effects on the availability of food for the other fish species occurred than when the silver carp addition was 10% of the polyculture, as reported in a previous work. The strong negative effects of silver carp on the other species of the polyculture and the higher total yields and income recorded in previous experiments with the addition of 10 silver carp/100 m² were much weaker and their expression depended on other pond conditions when 3 or 5 silver carp/100 m² were added or substituted the same number of rohu or catla, either when the bottom feeder was mrigal or common carp. It was concluded that stocking 3 silver carp/100 m² over the usual 100 large carp and 250 SIS /100 m² can be considered a ‘no effect’ stocking density in relation to the control without silver carp, while stocking 10 silver carp/100 m² should be preferred by farmers to keep the option of selling or consuming the silver carp.     

三角帆蚌钩介幼虫在5种寄主鱼体寄生效果的比较

选用黄颡鱼、草鱼、鲢、鳙和鲫作为三角帆蚌(Hyriopsis cumingii)的寄主鱼,分析了寄苗悬液钩介幼虫密度和寄苗时间对5种鱼初始寄苗量的影响,并对寄生在这5种鱼上的钩介幼虫发育情况进行了观察。结果表明:无论高密度寄苗还是低密度寄苗,长时间寄苗还是短时间寄苗,黄颡鱼和草鱼的寄苗量都明显高于鲢、鳙和鲫。提高寄苗悬液钩介幼虫密度,寄主鱼的寄苗量显著增加,但增加寄苗时间,寄主鱼的寄苗量未见显著提高。黄颡鱼和草鱼的稚蚌脱苗率显著高于鲢、鳙和鲫,寄生在鲢、鳙和鲫鳃上的钩介幼虫约80%未发育至稚蚌便提前脱落。不论在寄苗量方面,还是在稚蚌脱苗率方面,黄颡鱼和草鱼均是三角帆蚌钩介幼虫的最佳寄主。     

The effect of fish density and species combination on growth and utilization of natural food in ponds

Abstract. Interactions between bottom-feeding fish (common carp, Cyprinus carpio L., and hybrid tilapia) and a filter feeder (silver carp, Hypophthalmichthys molitrix (Valenciennes)) in polyculture were studied in fertilized ponds with no supplementary feeding. The silver carp were stocked at two densities: 1300 and 2600/ha. Growth rate and yield of each of the species were also compared with those in polyculture of bottom feeders alone, and in a monoculture of silver carp alone, at the same densities. The consumption of natural feed as melabolizable energy (ME) was calculated by the energy balance (requirement for maintenance and growth divided by the utilization efficiency).
Increasing the density of silver carp reduced proportionally their own growth rate, due to the limited amount of food. Above a density of about 1000/ha, silver carp inhibit the growth rate of common carp and tilapia. This inhibition was small at 1300 silver carp/ha, but considerable at 2600/ha. However, the presence of bottom feeders increased the growth rate of silver carp at both densities. Calculated natural food consumption showed that growth interactions between species were brought about through the availability of food. The synergistic effect of the bottom-feeding fish on the silver carp is assumed to be due to the upwelling of bottom nutrients to the upper layers of water by the burrowing of the fish in the mud. Combined yield of all species in the polyculture was highest at the density of 1300 silver carp/ha (2116 kg/ha in 156 days). At 2600 silver carp/ha the inhibition of growth rate of silver carp itself, reduced total yield as compared with the lower density.