【作者】 李鸿波；

【导师】 杜予州；

【作者基本信息】 扬州大学 ， 农业昆虫与害虫防治， 2013， 博士

【摘要】 西花蓟马是危害园艺作物的世界性害虫。该害虫于2003年首次在北京发现,近年来已迅速蔓延至我国大部分地区,危害日趋严重。西花蓟马在入侵过程中,在夏季常常遭遇夏季的高温和冬季的低温胁迫,但有关该害虫的温度耐受性及其调控机制还未见报道。本文采用生态学和分子生物学的方法,研究了温度胁迫对西花蓟马的存活能力、繁殖的影响及温度胁迫后西花蓟马热蛋白的表达变化等内容。其主要结果如下：研究了不同低温和不同暴露时间对西花蓟马存活率和繁殖的影响。结果表明,西花蓟马的存活率在暴露温度的降低和暴露时间的延长而逐渐降低。在1-6h范围内,杀死50%西花蓟马若虫所需的温度(Ltem50)分别为-13.6、-12.5、-11.2、-10.4℃,这些值要高于响应时间下杀死雌成虫的Ltem50,但低于杀死雄成虫的Ltem50。因此,西花蓟马不同发育时期的耐寒性强弱顺序可表示为：雌成虫>若虫>雄成虫。成虫和若虫暴露于-13℃和-13.5℃下2h后,其存活率分别为25%和27%。根据识别温度定义,这两个温度分别被定义为若虫和成虫的识别温度。将成、若虫在0或5℃驯化2h后,再置于各自识别温度下,其存活率都得到了明显提高。其中,在0℃下驯化2h后,若虫和雌雄成虫的存活率得到了最大幅度的地提高,分别达46%、54%、49%。西花蓟马若虫经不同低温处理后,其发育历期、羽化后的成虫寿命、产卵时间与对照相比无显著差异,但产卵量显著降低；成虫经过低温处理后,其寿命,产卵量和产卵时间明显降低。调查了不同高温(33、35、37、39、41℃),暴露时间(0.5、1、2、4h)以及发育时期对西花蓟马存活及繁殖的影响。结果表明,西花蓟马成虫(雌虫和雄虫)和若虫的存活率在高温下随暴露时间的延长而显著降低,杀死成虫和若虫50%个体的温度在在0.5到4h范围内随时间的延长而逐渐降低。因此,西花蓟马不同发育时期的耐热性强弱顺序可表示为：雌成虫>若虫>雄成虫。奇怪的是,成虫在31℃,若虫在33℃下预热处理2h后,显著提高了其在致死高温下的存活率。经过2h的高温暴露后,西花蓟马成虫的寿命和产卵量随温度的升高显著降低。高温下的交配实验表明所有交配处理(U♂+S♀S♂+U♀,S♂+S♀)的寿命和产卵量显著低于对照组(U♂+U♀)。U♂+S♀交配组合下的寿命与S♂+U♀组合无显著差异,但显著高于S♂+S♀组合；U♂+S♀组合下的产卵量显著高于S♂+U♀组合,但与S♂+S♀组合的产卵量相当。采用RT-PCR和RACE的方法从西花蓟马中克隆了5个热激蛋白基因,并分别命名为Fohsp90, Fohsc701, Fohsc702, Fohsp60和Fohop。Fohsp90, Fohsc701, Fohsc702, Fohsp60和Fohop的cDNA全长分别为2508,2195,3065,2489,2276bp,其开放阅读框分别为2169,1920,1983,1728和1659bp。5个开放阅读框分别编码722,639,660,575和552个氨基酸,推测的分子量大小分别为83.26,69.81,72.73,60.87和62.25KDa。序列分析发现5个热激蛋白与其他物种具有很高的相似性,并含有各自家族的保守基序。基因组和cDNA序列的比较分析发现,5个hsps分别含有3,4,7,5和10个内含子。此外,5个hsps内含子的数量和插入位置与其他物种相比存在高度差异。最后,5个Hsps的系统发育分析表明本研究所获得的5个Hsps为各自家族的同源物。采用Real-time PCR检测了5个西花蓟马热激蛋白基因(hsp90, hsc701,hsc702, hsp60, hop)在不同发育时期和不同温度胁迫下的表达模式。不同发育阶段的表达模式表明,5个hsps在若虫,蛹和成虫时期具有表达,其中hsp90, hsc701和hsp60随着发育的进行呈显著的上调表达,而hsc702和hop的表达水平在三个时期无显著差异。在成虫和若虫期,5个hsps对低温不十分敏感,但都能被高温诱导上调表达,而且经高温诱导后若虫期5个hsps的表达水平显著高于成虫。西花蓟马成虫和若虫经高温驯化后,其表达水平显著高于直接暴露致死温度下的表达水平。研究了温度的升高和亚致死剂量阿维菌素对西花蓟马的生物学参数、抗氧化酶和热激蛋白表达的影响。结果表明：温度和阿维菌素浓度对西花蓟马的生物学参数具有显著影响。存活率、寿命和产卵量随温度和阿维菌素浓度的升高而明显降低。温度和阿维菌素显著降低了SOD的活力,温度的升高对CAT的活力没有显著影响,但阿维菌素与之的相互作用显著提高了其活力。然而,温度和阿维菌素浓度及其交互作用显著提高了POD和GST的活力。hsps的表达模式表明,温度的升高仅能诱导hsp90、hsc702、hop的上调表达。然而,温度和阿维菌素的联合作用导致5个hsps的显著上调表达,其中hsp90、hsp701、hsc702和hop在33℃最大值,hsp60在21℃达最大值。综上所述,西花蓟马较强的温度耐受性和快速驯化能力可能是西花蓟马能迅速入侵扩散的重要因素,其内在机制(特别是在高温胁迫下)可能与分子伴侣热激蛋白的上调表达有关。更多还原

【Abstract】 The western flower thrips, Frankliniella occidentalis is a worldwide insect pest of horticultural crops. F.occidentalis has dispersed to many areas in China and the damage is becoming more and more serious since it was first reported in Beijing in2003. During spread of F.occidentalis, they often encounter heat stress in summer and cold stress in winter. Unfortunately, thermal tolerance and corresponding regulating mechanism in this pest have never been studied. In present study, tolerance and reproduction to cold and heat stress in F.occidentalis and its corresponding physiological mechanism were studied by ecology and molecular method. The main results are as follows:The present study examined the effects of different low temperatures, exposure duration on survival rate and fitness of F.occidentalis. Results indicated that survival rate of both adults and larvae significantly declined with decrease of temperature and extension of exposure duration. During the duration from1to6h, the temperature needed to kill50%individuals (Ltemso) for larvae was higher than that for adult females, but lower than that for adult males. Therefore, the order in cold hardness of different life stages was presented as:adult female> larvae> adult males. When larvae and adults of F. occidentalis were transferred from the normal rearing conditions at26±1℃, to cold temperatures at-13℃(for larvae) or-13.5℃(for adults) for2h, survivorship rate was only25%for larvae and27%for the adults. These two temperatures could be defined as the discriminating temperatures for the two insect stages, respectively. Survival rates of the thrips that were pretreated at0℃or5℃for2h before exposure to their discriminating temperatures increased significantly, and maximum increase in survivorships to the cold temperatures was achieved by pretreating larvae or adults at0℃for2h, which resulted in a survivorship rate of46%,54%,49%for larvae, adult females and males, respectively. When cold exposures occurred at the larval stage, there were no differences in development time, longevity and reproduction duration of enclosed adults relative to the non-treated control. However the number of eggs laid of the adults derived from cold-treated larvae was significantly less than that of the non-treated thrips. When adult thrips were exposed to the cold temperatures, the longevity, fecundity and reproduction duration all decreased significantly compared to the non-cold treated control.This study investigated the effects of high temperatures (33,35,37,39,41℃), exposure durations (0.5,1,2,4h) and life stages on survival and reproduction of the western flower thrips. Results suggested that the survival rate of adults (female and male) and larvae declined significantly with the increase in the exposure time at all high temperature examined. Therefore, the order in heat resistance of different life stages was presented as:adult female> larvae> adult males The lethal temperature needed to kill the50%of the adults and larvae decreased rapidly with the increase in the exposure time from0.5to4h. Surprisingly, preheat treatment at31(adults) or33℃for2h (larvae) significantly improved the survival rate of both stages after exposure to lethal high temperature. After2h exposure, longevity and reproduction of adults lowed remarkably with the increasing temperature. Mating experiment after exposure to high temperature demonstrated longevity and reproduction in all treatments (U(?)+S♀, S(?)+U♀, S(?)+S♀) were lower than those in control (U(?)+U♀). Longevity in U(?)+S♀treatment was not different from that in S(?)+U♀treatment, but was higher than that in S(?)+S♀treatment; while the reproduction in U(?)+S♀was significantly lower than that in S(?)+U♀, and relative to S(?)+S♀, suggesting that high temperature have adverse influence on adult female and male, but the effects on female was more serious than male.Five heat shock protein genes were cloned from F. occidentalis by RT-PCR and RACE, and were named Fohsp90, Fohsc701, Fohsc702, Fohsp60and Fohop, respectively. The complete cDNA of Fohsp90, Fohsc70, Fohsp60and Fohop are2508,2195,3065,2489,2276bp, respectively, and their respective opening reading frames are2169,1920,1983,1728,1659bp, which encode722,639,660,575and552amino acids with predicted molecular weights of83.26,69.81,72.73,60.87and62.25KDa, respectively. Sequence analysis suggested that the five Hsps shared high similarity with those of other species and contained conserved motifs found in their respective family of other species. Analysis of genomic DNA demonstrated that the five hsps contained3,4,7,5,10introns, respectively. In addition, the number and positions of introns of five hsps were highly different from those of other species. Finally, phylogenetic analysis suggested that the five Hsps were homologues of their respective family. Expression patterns of five heat shock protein genes (hsp90, hsc701, hsc702, hsp60, hop) in response to development and different temperatures were examined by Real-time PCR. Results showed that five hsps were expressed at larvae, pupae and adults stage. Among them, hsp90, hsc70and hsp60were significantly up-regulated with the process of development, while the expression level of hsc702and hop were not different among three stages. The five hsps were not very sensitive to cold, but were significantly induced up-regulation by heat, and the expression level of five hsps in larvae were higher than that in adult. In addition, pre-heat treatment lead to significant up-regulation of four genes compared to direct exposure to lethal high temperature (control).The combined effects of elevated temperature and sub-lethal dose of avermectins on biological parameters, antioxidant enzymes and heat shock protein mRNA level were studied. Results showed that temperature and avermectins concentrations significantly influenced the biological parameters. The survival, longevity and reproduction decreased with increased temperature and pesticide concentration. Elevated temperature and avermectins significantly decreased activity of SOD. Elevated temperature had no effect on activity of CAT, but its activitywas obviously improved by the combination of temperature and avermectins. Activities of POD and GST significantly increased after exposure to combination of temperature and avermectins. Expression patterns of Hsps mRNA showed that only three heat shock proteins (hsp90, hsc702and hop) was induced up-regulation by the elevated temperature, and shifted folds is very small. However, five hsps level was significantly up-regulated after exposure to conditions of the combination of elevated temperature and avermectins, and the highest level of hsp90, hsc701, hsc702and hop was reached at33℃, but the highest level of hsp60was observed at21℃.Taken together, relative strong tolerance to extreme temperature and rapid hardening response of the western flower thrips may be important factors that determine its rapid dispersal in china, and the underlying mechanism, especially under heat stress conditions, may be related to up-regulation of the molecular chaperon Hsps.更多还原