【作者】 杨明伟；

【导师】 董双林；

【作者基本信息】 南京农业大学 ， 农业昆虫与害虫防治， 2008， 博士

【摘要】 化学防治的推广使得害虫对杀虫剂产生了严重的抗性,防治效果显著下降,同时造成天敌杀伤和环境污染等问题。利用昆虫性信息素来防治害虫因为具有灵敏度高、选择性强、对天敌无害、不造成环境污染等优点,逐渐成为了一种具有发展前景的绿色防治技术。因此,针对重要的农业害虫,深入开展两性间信息素通讯系统的研究,全面了解两性间信息素通讯系统的复杂性,不仅有助于阐明昆虫两性间的化学通讯机制,而且对于进一步开发更为高效的性信息素防治技术具有重大的应用价值。本文以甜菜夜蛾(Spodoptera exigua Hubner)和斜纹夜蛾(Spodoptera litura Fabricius)为对象,针对当前蛾类昆虫两性间信息素通讯系统研究的一些薄弱环节,如雄性信息素及其功能、雌性信息素对同种雌蛾的影响、以及雄蛾对雌性信息素电生理反应的昼夜节律等,进行了研究。主要结果如下：1甜菜夜蛾和斜纹夜蛾雄性信息素的存在证据甜菜夜蛾和斜纹夜蛾在两性共同放置时,其交配行为均可分为求偶萌动期、求偶兴奋期、预交配期和成功交配期4个阶段。在雌雄蛾分开放置时,雌蛾的求偶行为没有变化,但雄蛾只能观察到求偶萌动期和兴奋期两个阶段,几乎不能进入到打开味刷的预交配期,说明雌性信息素的存在对于雄蛾打开味刷起决定性作用。两种夜蛾在雄蛾触角切除后两性间几乎不能成功交配；雌蛾触角切除后对交配率有明显影响,在甜菜夜蛾和斜纹夜蛾中使交配率分别下降了31%和28%；而当雄蛾味刷去除后,甜菜夜蛾和斜纹夜蛾的交配率分别下降30%和27%左右,与雌蛾切除触角的下降程度相似,间接证明了味刷中雄性性信息素的存在。2甜菜夜蛾和斜纹夜蛾雄蛾味刷组分的化学鉴定分别用正己烷、二氯甲烷和甲醇对两种夜蛾的雄蛾味刷进行浸提,并用1个味刷当量的提取液进行电生理测定,结果发现只有甲醇提取液能够引起雌、雄蛾显著的EAG反应,并且甲醇提取液在两种夜蛾间存在交叉活性。进一步对甲醇提取液的气谱-触角电位联用(GC-EAD)分析,在两种提取液中都发现一个活性峰。该活性峰的保留时间很短,且在两种夜蛾提取液间非常接近,推测可能为同一种物质。由于该组分极性较强、热稳定性差,气质联用(GC-MS)分析未能得到定性结果。对甜菜夜蛾味刷正己烷提取液的GC-MS分析表明,其主要组分为棕榈酸(16：0)、亚油酸(18：2)、油酸(18：1)和硬脂酸(18：0)四种有机酸；根据GC图谱对比,推测斜纹夜蛾味刷同样含有这4种组分,该4种有机酸是否具有雄性信息素活性或作为信息素的前体物质,有待进一步研究。此外,从正己烷提取液中还鉴定出3种直链烷烃,推测其为味刷表皮的碳氢化合物。3甜菜夜蛾和斜纹夜蛾雄蛾味刷中有机酸组分的滴度、比例及日动态变化利用GC定量分析了两种夜蛾3日龄雄蛾味刷中4种有机酸的滴度以及甜菜夜蛾不同日龄味刷中各组分滴度的动态变化。结果表明：3日龄甜菜夜蛾味刷中棕榈酸、油酸、亚油酸和硬脂酸滴度分别为27.80、24.58、15.40和5.10 ng/ME,相对比例为38.14、33.73、21.13和7.00%；斜纹夜蛾的滴度分别为24.36、20.02、15.87、和6.02 ng/ME,相对比例为36.75、30.21、23.95和9.08%,与甜菜夜蛾的情况相似。甜菜夜蛾雄蛾味刷中,4种有机酸在刚羽化(0日龄)雄蛾中的滴度很低,随后快速上升,2日龄后稳定下来,不再有显著变化。4甜菜夜蛾和斜纹夜蛾雌蛾对同种雌性信息素的电生理和行为反应EAG电生理测定表明,甜菜夜蛾和斜纹夜蛾雌蛾对自身释放的雌性信息素也能产生明显的触角电位,尽管反应强度较同种雄蛾的明显要弱。进一步的求偶行为观察表明,当甜菜夜蛾雌蛾暴露在雌性信息素中,其求偶行为发生了明显的改变,表现为求偶率较对照明显降低,求偶高峰期发生后移,求偶时间延长到光期开始后2小时；但暴露在雌性信息素下的雌蛾腺体内的性信息素滴度没有改变。此外,趋避实验结果表明,雌蛾在嗅觉仪中对雌性信息素也没有明显的趋避反应。求偶行为的改变有利于雌蛾在时间上避开其他雌蛾个体,从而减轻竞争并获得更多的与雄蛾的交配机会。5甜菜夜蛾和斜纹夜蛾成虫对雌性信息素EAG反应的时动态及其与PBP表达量的关系EAG测定表明,斜纹夜蛾和甜菜夜蛾雄蛾触角对雌性信息素的敏感性没有明显的昼夜节律,在光期依然表现出了与暗期相似的敏感性。为探讨其机制,利用实时荧光定量PCR初步测定了斜纹夜蛾雄蛾触角内的两个信息素结合蛋白(PBP)的表达量,表明光期的表达量较暗期明显降低。两者间的不一致暗示,除PBP外可能还有其他蛋白参与了对性信息素分子的运输,从而在光期维持较高的触角敏感性;或者PBP担负暗期相关的多种功能,而在光期PBP表达量明显降低后仍足以维持相当的触角敏感性。更多还原

【Abstract】 Due to the wide and intensive use of chemical insecticides in fields, insect pests have developed serious resistances to most varieties of insecticides, resulting in unsatisfied efficacies and even failures in pest control. Insect sex pheromone, as an alternative method to chemical insecticides, has many advantages such as high sensitivity, species-specificity and safety to environment, and therefore has been considered to be one of the prosperous green pest control methods. Obviously, thoroughly understanding the sex pheromone communication system between male and female insects would be very important not only for the clarification of the mechanisms of sex communication but also for the development of more efficient pest control method targeting on sex pheromone communication system. Here in the present study, some less-studied aspects of sex pheromone communication system in Spodoptera exigua and S. litura were explored, which included chemical identification of male sex pheromone, electrophysiological and behavioral responses of females to the conspecific female sex pheromone, and diel rhythm of electrophysiological response of males to female sex pheromone. The main results are as follows:1. Evidence for the presence of male sex pheromone in S. exigua and S. lituraThe copulation behaviors of S. exigua and S. litura were investigated by direct observation. For the two moth species, when male and female were placed together in a container, the copulation behavior of both sexes could be divided into four sequential periods:precalling period, calling period, precopulation period and copulation period. However, when male and female placed separately in different container, the females presented the same four periods, but males only presented the first two periods with the third and fourth periods hardly observed, indicating that the presence of female sex pheromone was crucial for males to display the hairpencils. Hairpencil and antennal excision assays were carried out to confirm the effect of the hairpencils on the mating success. Males with antennal excision resulted in almost no mating success; while females with no antennae caused a reduction in mating success rate by 37% in S. exigua and 28% in S. litura. In the case of males with hairpencil excision, the mating rate was decreased by 30% in S. exigua and 27% in S. litura. Taken together the results of antennal excision and hairpencil excision assays, it was suggested that the male sex pheromone was released from the hairpencil, and had a significant influence on mating success between female and male moths.2. Chemical identification of compounds produced by male hairpencil glands from S. exigua and S. lituraElectroantennogram (EAG) was conducted to investigate the effects of male hairpencil extracts on EAG responses of male and female S. exigua and S. litura. Hexane, Methylenechloride and methanol were used to extract the hairpencils, but only the extract by methanol could elicit a significant EAG response in both male and female moths, and a cross-response was found between two moth species for both hairpencils. The further GC-EAD assay led a finding of an active compound but the chemical identification of this compound failed by GC-MS possibly due to the heat instability and strong polarity of the compound. Moreover, hexane extract of hairpencils from male S. exigua were analyzed by GC-MS, and four chemical components were identified as palmitic(C16:0), linoleic(C18:2), oleic(C18:1), and stearic acid(C18:0). Hexane extract of hairpencils from male S. litura was considered to contain the same four acids based on comparing of gas chromatograms from the both moth species. Whether these four organic acids acted as male sex pheromone components or not needed to be further confirmed.3. Quantitative analysis of compounds produced by male hairpencil glands from S. exigua and S. lituraGC quantitative analysis of the hairpencil extracts with hexane from three old moths showed a similar titers and relative proportions of four components between the two moth species. The titers of palmitic, linoleic, oleic, and stearic acid were 27.80,24.58,15.40, and 5.10 ng/FM with a relative proportion of 38.14,33.73,21.13 and 7.00%, respectively, in S. exigua. While in S. litura, the corresponding titers were 24.36,20.02,15.87, and 6.02 ng/FM, and the relative proportion were 36.75,30.21,23.95%,9.08%, respectively. The dynamics measurements of extracts from different day old moths showed a sharp increase in the titers of the four acids from 0-day-old moth to two-day-old moth, and a stabilization of the titers was achieved since two-day-old moth on.4. Electrophysiological and behavioral responses of both sexes of S. exigua and S. litura to the conspecific female sex pheromoneElectroantennogram (EAG) recordings showed that female S. exigua and S. litura were indeed capable of perceiving their own sex pheromones (two single components and their mixture), and displayed a similar dose-response relation pattern to that of respective males, although intensities of female responses were much less than those of corresponding males. Furthermore, the female S. exigua calling behavior was apparently influenced by presence of the female sex pheromone. The proportions of calling females in the peak calling period were significantly reduced and the calling peak time postponed by 2.5h, but the calling duration was prolonged by 1.5h into 2h of photophase, compared with controls. However, the pheromone titers in the female moth glands were the same between treatment and the control, implicating a reduced pheromone biosynthesis in the glands of treated moths. In addition, olfactometer experiment exhibited no obvious tendency or escape behavior response of females to sex pheromone stimuli. Such modification in calling behavior in S. exigua was speculated to decrease the competition among female individuals and subsequently to obtain more chances to mate with males.5. Daily changes of EAG responses to the conspecific female sex pheromone in S. exigua and S. litura, and relation between EAG response and expression level of PBP in male S. lituraEAG response to conspecific female sex pheromone component by female and male S. litura and male S. exigua indicated no obvious diel rhythms in EAG responses. Males retained similar sensitivity between the scotophase and photophase. To explore the mechanisms of such high EAG sensitivity in photophase, the transcription levels of PBP genes in male S. litura antennae were measured by Real-time quantitive PCR in one scotophase time point and three photophase time points. The results showed a significant reduction from scotophase to photophase in the amounts of SlitPBPl and SlitPBP2 mRNA. As PBPs was thought to transport the sex pheromone molecules to the receptors localized on the dentrite membrane of olfactory neuron, the PBP expression level should positively correlate to the EAG responses. Therefore, such incongruity between the EAG sensitivity and PBP expression level might imply a more complicated situation regarding the PBP functions and sex pheromone transportations.更多还原