THE EFFECT of HIGH AMMONIA CONCENTRATION ON GILL STRUCTURE ALTERNATION AND EXPRESSION of SOD AND HSP90 GENES IN GRASS CARP,CTENOPHARYNGODON IDELLA

实验通过测定草鱼的24H半致死浓度,鳃的细胞结构以及SOd和HSP90的表达模式研究了草鱼在组织学和分子生物学水平上对高浓度氨氮暴露的响应。经过半致死实验确定的氨氮24H lC50为243 Mg/l试验中草鱼被置于5个浓度的处理组中(50、72、104、151、220 Mg/l),之后取鳃组织进行组织切片分析,取肝脏、肠和鳃来测定SOd和HSP90的表达情况。经过高浓度的氨氮暴露处理,鳃组织的细胞排列和结构产生了明显的变化,并且SOd和HSP90的表达受到了显著的上调。这些结果表明,高浓度的氨氮能够损害鳃部的细胞结构并且诱导应激蛋白的表达。这个结果同样显示出,SOd和HSP90可以作为评估草鱼氨氮暴露水平的良好指标。Histological and molecular responses of grass carp Ctenopharyngodon idella to high concentration ammonia exposure were studied in this study by determining the lethal concentration,gill cellular structure,and expression patterns of sod and hsp90 genes.Grass carps were treated at five ammonia concentrations(50,72,104,151 and 220 mg/L) for 24h,and then the gill tissues were collected for histological analysis.In addition,three tissues(gill,intestine and liver) were collected to measure the expression of sod and hsp90 genes.After exposure to high concentration of ammonia,the arrangement and structures of gill cells changed dramatically.Edema and fusion of secondary lamellae were observed,and the edge of cell and nucleus could not be clearly defined.Meanwhile,the two genes(sod and hsp90) in different tissues were unregulated significantly,indicating that high concentration of ammonia could impair the cellular structures and induce the expression of stress proteins.These results also suggested that sod and hsp90 were suitable biomarkers for ammonia exposure assessment in grass carp.FoundsoftheNationalProgramonKeyBasicResearchProject(2009CB118706

Effect of rapid temperature change on expression of hsp70 and hsp90 in grass carp(Ctenopharyngodon idella)

为研究不同温度条件下草鱼热休克蛋白(HSP70和HSP90)的表达模式,了解草鱼对温度的耐受和适应机理,实验将在20℃下驯化的草鱼在7个实验温度(22,24,26,28,30,32和34℃)中热激3 H,然后20℃恢复2 H,取肝脏,肌肉和鳃测定HSP70和HSP90表达。结果表明,HSP70和HSP90表达量随着温度升高而上升,当温度达到34℃时,肌肉与鳃热休克蛋白基因表达量显著下降,草鱼耗氧率、热休克蛋白表达和死亡率之间的关系符合氧限制热耐受理论(OClTT)。基于OClTT理论,草鱼生理临界温度为28℃,当温度超过28℃,热休克蛋白表达所需能量主要由无氧代谢提供,进而导致体内氧自由基和变性蛋白的增加,影响草鱼的生长和存活。Grass carp(Ctenopharyngodon idella)is one of the most important cultured fishes in China.During transportation in summer,massive mortality often occurs.As molecular chaperones,Hsps assist cells in their recovery from stress and promote cytoprotection.Limited reports described the expression of Hsps in grass carp at high temperatures,and the relationship between oxygen consumption,expression of heat shock protein and mortality needs to be investigated based on the oxygen-and capability-limited thermal tolerance theory(OCLTT)to further elucidate the thermal adaptation of this species to high temperature in view of integrated biology.In the present study,the expressions of hsp70 and hsp90 after rapid temperature increase were studied.Grass carps were acclimated at 20 ℃ and then exposed to designated temperatures(20,22,24,26,28,30,32,34 ℃)for 3 h heat shock.After 2 h-recovery at 20 ℃,liver,muscle and gill of tested fish were sampled and hsp70 and hsp90 were determined using semi-quantitive real-time PCR.Expression of hsps increased with temperature increase until 32 ℃ in muscle and gill,and then decreased at 34 ℃.In liver,expression of hsps kept increasing in all temperature treatments.These results indicated that hsp70 and hsp90 were sensitive to temperature increase in grass carp.Analyzed heat shock proteins data with previous results of oxygen consumption and lethal temperatures of grass carp,the physiological adaptations of grass carp could be explained by oxygen-and capability-limited thermal tolerance theory(OCLTT),which pointed out that the imbalance between oxygen demand and tissue oxygen supply ability was the primary factor which limited organisms' thermal tolerance and the ability of aerobic metabolism greatly limited the survival of organism beyond critical temperatures.With temperature increase,the maximum value of oxygen consumption in grass carp occurred at 28 ℃,and then the oxygen consumption decreased when temperature was beyond 28 ℃,indicating a transformation from aerobic metabolism to anaerobic metabolism at this critical temperature.The expressions of heat shock proteins kept increasing until 32 ℃ in muscle and gill in grass carp,and provided defense against high temperature.However,this protection of heat shock proteins was limited and then death occurred when temperature was beyond 32 ℃.The change of metabolism,increase of oxygen free radicals and the consumption of energy due to stress proteins synthesis of grass carp were closely related to the decrease of capability of immune response when water temperature was beyond 28 ℃.In general,high water temperature could alter the metabolic pathway,induce heat shock protein expression,reduce immune response,and then cause large-scale mortality.Therefore,appropriate ways should be applied to maintain water temperature under 28 ℃ to avoid large-scale mortality during transportation.国家“九七三”重点基础研究发展计划(2009CB118706

Effects of Salinity Fluctuation on the Expression of hsp70 and hsp90s Genes in Sea Cucumber,Apostichopus japonicus Selenka

为研究夏季暴雨导致的养殖池塘盐度变化对仿刺参(APOSTICHOPuS JAPOnICuS)生理的影响,在实验室内模拟野外盐度变化,采用半定量rT-PCr的方法研究了其HSP70、HSP90A及HSP90b基因的表达。实验中,盐度先以每6H降2.5的速度由30降至20;保持96H后,再以同样的速度由20升至30,随后在盐度为30条件下保持96H。在盐度变化过程中随机取样。HSP70和HSP90b基因在盐度降至20后表达量开始显著升高(P<0.05),HSP90A基因在盐度降至22.5时表达量开始显著升高(P<0.05);但保持在盐度为20条件下,3个基因的表达量均逐渐降低至初始值。在之后的盐度升高及恢复阶段,3个基因表达量均有先升高后降低的趋势,表明HSP70、HSP90A和HSP90b基因是仿刺参在盐度胁迫下的重要响应因子。The expression of hsp70,hsp90a and hsp90b genes in sea cucumber(apostichopus japonicus Selenka) were studied in a simulated field salinity decrease after a heavy rainfall in summer.Salinity firstly decreased gradually from 30 to 20 at a rate of 2.5 every 6 h,and then was maintained at 20.After 96 h at the low salinity(20),salinity was increased gradually to 30 at a rate of 2.5 every 6 h,and then was maintained at 30 for 96 h.Results showed that expressions of hsp70,hsp90a and hsp90b genes all increased after salinity decrease,led to the maximum levels at the lowest salinity,and then dropped to the original level after 72 h at low-salinity exposure.Similarly,the expression of these three genes in sea cucumber increased with the salinity increase,and decreased to the original level after 72 h at low-salinity exposure.Compared to the second salinity fluctuation,the gene expressions were lower than those of the first salinity fluctuation,which might be caused by energy budget.In this study the expression of hsp90a and hsp90b genes was similar to hsp70 gene's expression,and the upregulated expression of hsp90s also could be regarded as the physiologic adaptation of Apostichopus japonicus to environmental stresses.国家自然科学基金项目(30400333);国家科技支撑计划项目(2006BAD09A01);山东省优秀中青年科学家科研奖励基金计划项目(BS2009NY019)资

Effects of Heat-Shock in Pelagic Stages on Survival and Hsp70 Expression of Juvenile Sea Cucumber,Apostichopus japonicus Selenka

设计不同温度(21.5,26,28和30℃),对刺参(APOSTICHOPuS JAPOnICuS)囊胚期、原肠期和耳状幼体期幼体进行热激处理,热激45 MIn后,将幼体在21.5℃条件下继续培养至耳状幼体后期,计算刺参幼体的存活率,并用蛋白杂交方法测定幼体HSP70的表达量。结果表明,高温刺激对幼体的存活有显著性影响,各时期幼体的存活率均随热激温度的升高而降低,原肠期的存活率要高于囊胚期和耳状幼体时期。原肠期幼体HSP70表达量要比囊胚期和耳状幼体时期高,这表明在发育过程中HSP70表达量与幼体存活率具有相关性。The effects of heat-shock in pelagic stages on survival and Hsp70s expression of juvenile sea cucumber,Apostichopus japonicus Selenka were investigated.Larvae at the stage of blastula,gastrula and auricularia were heat-shocked at selected temperatures(21.5,26,28 and 30 ℃) for 45 min and returned to 21.5 ℃ for continuous rearing.After the heat-shock,there were significant differences in survival among larvae in the four heat-shocked treatments.The survival rate of larvae decreased with the increasing of heat-shock temperature,and the rate was higher in the stage of gastrula than those in blastula and auricularia.The Hsp70s expression of larvae in the stage of gastrula was higher than those in the stages of blastula and auricularia,suggesting that the survival of larvae partly correlates with the expression of Hsp70s.国家科技支撑计划项目(2006BAD09A01);山东省优秀中青年科学家科研奖励基金计划项目(BS2009NY019)资

Microeological Relationship Between Microalgal Virus and Host

病毒感染、杀死和裂解微藻是海洋生态系统中的普遍现象,在自然海洋环境中,有多种因素可以导致浮游植物细胞的损失,其中微藻的自然死亡(即细胞裂解)率是导致微藻损失的一个重要因素。由病毒介导的宿主死亡不仅可影响藻类物种的种间演替,也可能会影响种内演替、藻类群落的丰度及多样性等。越来越多的证据表明,病毒可以通过减少宿主种群数量或防止藻类宿主种群数量达到高峰的方式来控制浮游植物动力学指标。因此,藻类病毒与宿主之间的相互作用在赤潮动力学和感染的传播中发挥了重要作用。同时,病毒具有高度特异性宿主范围的发现拓展了我们对微藻种群动力学过程的认识。本文从病毒-微藻稳定感染系统模型、病毒对微藻种群动力学的调节、病毒介导的微藻死亡、宿主对病毒侵染的防御以及影响病毒与宿主相互作用的环境因素等方面综述微藻类病毒与宿主的相互作用关系。Algal viruses can infect,kill and lyse phytoplankton in marine ecosystems.Natural mortality(also known as cell lysis) of phytoplankton has been recognized as an important factor that caused the loss of microalgae.Viral-mediated mortality may not only affect algal species succession,but also affect intraspecies succession,the abundance and diversity of host cell communities in natural marine environments.More and more evidence showed that viruses can change phytoplankton dynamics through reduction of host populations,or by preventing algal host populations from reaching high levels.Therefore,algal virus-host interactions play important roles in bloom dynamics and the propagation of infection.At the same time,the identification of highly specific host ranges of viruses is changing our understanding of population dynamics.This paper reviews progress has been made in virus-host relationships,which involves the Virus-phytoplankton systems,viral control of phytoplankton dynamics,viral-mediated mortality of phytoplankton,host defense to viral offence,effect of viruses on algal population and important environmental factors affecting virus-host interactions.“十一五”国家科技支撑计划重大项目子课题(2006BAD09A06);福建省自然科学基金项目(D0710020);厦门大学近海海洋环境科学国家重点实验室开放基金项目(MEL0608