【作者】 李建勋；

【导师】 王进军；

【作者基本信息】 西南大学 ， 农业昆虫与害虫防治， 2008， 博士

【摘要】 80年代中后期以来,甜菜夜蛾Spodoptera exigua(Hübner)在我国逐渐成为一种危害农作物,特别是多种蔬菜的重要害虫,经常暴发成灾,给农业生产造成严重损失。由于我国农业生产长期形成的用药习惯,造成甜菜夜蛾对化学防治的抗性上升较快,防治十分困难。因此,运用化学生态学原理,探索研究甜菜夜蛾化学感受机制,阐明甜菜夜蛾与寄主植物之间的相互关系,对于甜菜夜蛾的可持续控制具有重要的理论和实践意义。本学位论文基于昆虫化学生态学的理论与方法,利用光学显微镜、扫描电镜、触角电位仪、PCR等技术和方法,在改进甜菜夜蛾室内饲养方法的基础上,系统研究了甜菜夜蛾成虫头部的化学感器的主要形态、特征、数量和分布,成虫生物学习性以及幼虫头部化学感器;进一步从化学感受的分子基础研究了甜菜夜蛾触角气味结合蛋白;运用触角电位技术研究了甜菜夜蛾的化学感受的电生理反应,并对甜菜夜蛾化学感受的机理进行了探讨,通过研究获得了以下主要结果。1.首先改进了甜菜夜蛾的室内饲养技术,对甜菜夜蛾蛹的保存技术及定量饲养进行了研究。实验结果表明,在冰箱冷藏室(4℃)条件下甜菜夜蛾的蛹基本上不能有效保存;而在人工气候箱内16℃条件下,甜菜夜蛾的蛹可以较好地保,羽化率达到49.81%,虽然与未处理的蛹羽化率差异显著,但对成虫的生殖力无显著的影响,可以满足后续研究的需求。饲养空间对甜菜夜蛾种群动态的影响研究结果表明,不同的生存空间对甜菜夜蛾种群增长有明显的影响。生存空间在一定范围内的变化直接影响着甜菜夜蛾的种群增长。采用一次性塑料杯室内饲养甜菜夜蛾时,在每杯饲养15头的密度下种群具有较高的繁殖指数,但考虑到在饲养过程中发现高龄幼虫有自残现象,因此提出每杯饲养以10头为宜。2.在室内观察了甜菜夜蛾化蛹、羽化、求偶、交配、产卵等的日节律及时辰节律。结果表明,甜菜夜蛾化蛹的性比和羽化的性比均接近1:1;甜菜夜蛾羽化具有明显的昼夜节律,其中晚上00:00-8:00羽化率最高,达到总羽化数量的一半以上,且和其它时间段差异显著,而且羽化集中在蛹期第4-8d(占95%左右),时间上雄蛹有滞后现象;甜菜夜蛾成虫交配具有显著昼夜节律,交配主要发生在02:00-07:00时之间,发生的概率超过85%;单次交配持续时间40-100min的发生率为70.44%;甜菜夜蛾产卵集中在夜晚,其中00:00-5:00所产卵量占总产卵数的65.46%,该段时间是甜菜夜蛾集中产卵时段。3.通过扫描电镜观察了甜菜夜蛾成虫触角及头部感器。结果表明,甜菜夜蛾雌虫触角显著长于雄虫,但鞭节数却显著少于雄虫,说明雄虫鞭节较为粗短;在触角表面共观察到8种感器,分别是毛形感器、刺形感器、锥形感器、腔锥形感器、耳形感器、鳞形感器、柱锥形感器和花形感器。其中毛形感器数量最多,花形感器最少;在喙及下唇须上共观察到3种感器,分别是刺形感器、锥形感器和花形感器。4.甜菜夜蛾幼虫头部感器扫描电镜的观察结果表明,幼虫头部感器较为丰富,其头部毛序即为一种刺形感器。相对于成虫触角,幼虫触角感器无论是种类、还是数量要少于成虫,幼虫触角上仅有1个栓状感器、5个锥状感器及2根毛形感器。幼虫口器跗肢下颚须上有8个丹锥感受器,排列为3-2-3;外颚叶有2个较大栓锥形感器及1个丹锥感受器;负须节有4根毛形感器。下唇须有栓锥形感受器和锥形感受器各1个。5.采用RT-PCR等克隆技术,通过提取甜菜夜蛾成虫触角总RNA,根据鳞翅目气味结合蛋白记忆序列设计引物,克隆获得了甜菜夜蛾PBP1和PBP2的基因序列。并以此推导出其氨基酸序列,并进行了序列比对。结果表明,甜菜夜蛾PBP1的cDNA具有一个492bp的开放阅读框,编码164个氨基酸,前23个氨基酸残基具有真核生物分泌蛋白信号肽的典型特征。甜菜夜蛾PBP1前体和成熟蛋白分别包括164个氨基酸残基和141个氨基酸残基,预测的分子量分别为18.9kD和16.4kD,预测的等电点分别为5.56和5.27。甜菜夜蛾PBP2序的cDNA具有一个510bp的开放阅读框,编码170个氨基酸,前27个氨基酸残基具有真核生物分泌蛋白信号肽的典型特征。甜菜夜蛾PBP2前体和成熟蛋白分别包括170个氨基酸残基和143个氨基酸残基,预测的分子量分别为18.9kD和16.1kD,预测的等电点分别为5.6和4.98。PBP1和PBP2蛋白均具有真核生物信息素结合肽的共同特征,序列中含有6个半胱氨酸位点,形成两个二硫键。最后通过构建系统发育树分析了其同源性。6.利用触角电位(EAG)电生理技术测定了甜菜夜蛾对植物挥发性气味物质的电生理反应。结果表明,甜菜夜峨对葱的6种气味物质有不同程度的EAG峰值,雄蛾较雌蛾反应强烈,雌蛾引发触角电位的反应强度依次为:甲基丙基二硫醚＞二烯丙基二硫醚＞二丙基二硫醚＞2-甲基-2-戊烯醛＞2-十三酮＞2-十一酮;雄蛾引发触角电位的反应强度依次为:2-甲基-2-戊烯醛＞二烯丙基二硫醚＞二丙基二硫醚＞甲基丙基二硫醚＞2-十一酮＞2-十三酮,且雌雄蛾间差异较大。总体来看,本学位论文首次从昆虫生理学、分子生物学、电生理学等方面较为全面地研究了甜菜夜蛾的化学感受机理,这不仅丰富和发展了昆虫化学生态学研究内容,为阐明害虫行为反应的本质做了有益探讨,而且对于农业害虫的可持续治理具有指导意义。更多还原

【Abstract】 The beet armyworm Spodoptera exigua(Hübner),belongs to Lepidoptera and Noctuidae,has become one of serious pests in China since 1980’s and caused severe losses in agricultural production,especially for vegetables.However the traditional usage in chemical pesticide management has resulted in serious resistance development of S.exigua.As a result,to explore the chemical sensor mechanism of S.exigua and clarify the relationship between beet armyworm and its host plants possess important theoretical and practical significance by means of chemical ecology.Based on the theory of insect chemical ecology,the main morphology,characteristics,quantity and distribution of sensilla on the head of both adult and larvae of beet armyworm were systematically studied in this study via a series of techniques and methods such as optical microscope,scanning electron microscopy,EAG,PCR and improvement of indoors breeding;the odorant binding proteins from antenna of beet armyworm were carried out at molecular levels;the electrophysiological responses by EAG were also conducted.Through several years’ work,the chemical sensor mechanism of the beet armyworm was systematically studied and the main results were as follows:1.Firstly,the basic laboratory rearing techniques for beet armyworm were improved and the preservation of pupae of beet armyworm was investigated.The results showed that the beet armyworm pupae can not survive in refrigerator(4℃);however under the condition of artificial incubator(16℃) pupae live well and the emergence rote amount to 49.81%t,which was although significantly low from the counterpart of untreated pupae but no effects for the further studies.Besides,the effects of breeding space on population dynamics of the beet armyworm were studied.The results showed that there were significant effects of space on population development of the beet armyworm.Disposable plastic cups were adopted in indoor breeding for beet armyworm and there is a high population trend index with a total of 15 beet armyworms in one cup.Considering that self-mutilation phenomenon exists among old larvae,it is recommended that 10 armyworms should be raised per cup.2.Daily rhythm and circadian rhythm of pupation,emergence,courtship,mating and oviposition for the beet armyworm were observed indoors.The results showed that the sex ratios of both pupation and emergence were close to 1:1;there was an obvious circadian rhythm for emergence,highest emergence rate concentrated between zero to eight o’clock in the morning amounting to over 50 percent of total emergence quantity and significantly different from other time quantum;pupation concentrated during 4- to 8-d pupae(accounting for 95%or so) and there was hysteresis phenomenon for male pupae;most matings occurred after 23:00 in the evening and there was an occurrence rate of 70.44 percent for an mating duration between 40 to 100 min;the eggs laid between zero to five o’clock in the morning amounted to 65.46 percent of the total eggs.3.Sensors on the head of beet armyworm were observed though scanning electron microscope. The results showed that there were longer antenna for female beet armyworm while the quantity of flagellum was more for males;a total of 8 types of sensors were observed in antenna,those incuding Sensilla trichodea,Sensilla chaetica,Sensilla basiconicum,Sensilla coeloconica,Sensilla auricillica, Sensilla squamiformia,Sensilla styloconica and Sensilla ecfloresco Among these sensors,the number of Sensilla trichodea was largest and there was a smallest number of Sensilla ecfloresco.On proboscis and labipalp three types of sensors were observed,Sensilla chaetica,Sensilla basiconicum and Sensilla ecfloresco.4.The sensors on the head of beet armyworm larvae are also abundant and chaetotaxy on the head is one type of Sensilla chaetica.Compared to the counterparts of adult,there are less types and numbers of larvae antenna with only one Sensilla styloconica,five Sensilla basiconicum and 2 Sensilla chaetica.There are 8 sensillum styloconicum on larvae palpus,arranged 3-2-3;2 Sensilla styloconica and 1 Sensilla basiconicum were observed on galea and 4 Sensilla trichodea were founded on palpifer.One Sensilla styloconica and one Sensilla basiconicum were recorded on labipalp.5.PBP1 and PBP2 sequences were firstly obtained from beet armyworm through RT-PCR technique and sequence analysis was conducted subsequently.The sequence analysis showed that cDNA of the beet armyworm PBP1 possess an open reading frame of 492bp encoding 164 amino acids and the first 23 amino acid residues contains the typical characteristics of signal peptides of the secreted proteins in eukaryotes.PBP1 precursor and mature protein contains 164 and 141 amino acid residues,respectively,with an predicted molecular weight of 18.9kD and 16.4kD,an predicted isoelectric point of 5.56 and 5.27.The sequence analysis showed that cDNA of the beet armyworm PBP2 possess an open reading frame of 510bp encoding 170 amino acids and the first 27 amino acid residues contains the typical characteristics of signal peptides of the secreted proteins in eukaryotes. In addition,the common characteristics of eukaryotic pheromone binding peptide were also observed and 6 cysteine residues can form two disulfide bond.PBP2 precursor and mature protein contains 170 and 143 amino acid residues,respectively,with an predicted molecular weight of 18.9kD and 16.1kD,an predicted isoelectric point of 5.5 and 4.98.In addition,the common characteristics of eukaryotic pheromone binding peptide were also observed and 6 cysteine residues can form two disulfide bond.The phylogenetic tree was built and its homology was analyzed subsequently.6.The electrophysiological responses of the beet armyworm antenna to plant volatile substances were determined through EAG technique.The results suggested that EAG peak value of the beet armyworm fluctuated a lot to 6 ordors matter from onion with stonger response recorded in male moths.The response degree of female moths from highest to lowest was in the order of methyl-n-propyl disulfide＞C diene Disulfide-＞b-propyl disulfide＞2-methyl-2-E-aldehyde＞2-Thirteen-＞2-Eleven-one; and the status of male moths was in the order of 2-methyl-2-E-aldehyde＞diallyl disulfide＞n-propyl disulfide b＞methyl-n-propyl disulfide＞2- Eleven-＞2-Thirteen-one. It can be seen that significant difference exists between male and female moths.In general,a systematic study on chemical sensor of the beet armyworm was carried out via the combination of insect physiology,molecular biology and electron physiology.The results above have not only enriched and developed the study content of insect chemical ecology but also provided some useful explorations for clarifying the nature of pest insect behavior response,and they are the basis of application study on integrated pest management.更多还原