淡色库蚊摄糖习性及嗅觉相关基因研究

发布时间：2018-06-22 17:59

本文选题：淡色库蚊 + 糖餐行为　；参考：《浙江大学》2016年博士论文

【摘要】：淡色库蚊是我国北方最常见的蚊种,同时也是乙型脑炎和丝虫病的重要传播媒介。长期以来蚊虫的防治工作大量依赖化学杀虫剂,产生环境污染、抗药性不断增强、对人畜具有潜在危害等问题。摄糖行为在蚊虫生活史中普遍存在糖餐对于维持成蚊生命活动所需能量必不可少。此外,运用成蚊摄糖习性控制成蚊数量在蚊虫防治工作中得到广泛应用。然而,成蚊如何定位糖餐植物尚不明确,而成蚊的趋糖习性、适合度、生理状况与对糖餐植物偏好性差异之间的关系也有待研究。本论文以淡色库蚊及其糖餐植物为研究对象,对成蚊的摄糖趋性、成蚊适合度以及生理状况与糖餐植物偏好性之间的关系、成蚊对糖餐植物挥发物行为反应、偏好性糖餐植物糯米条挥发物解析、淡色库蚊嗅觉相关基因等方面进行了研究,主要包括以下内容：(1)通过定量控制淡色库蚊幼虫的食物丰富度,对羽化后成蚊翅长、体重以及生理状态进行分析,并利用糖染色法研究了不同幼虫期食物营养条件下的淡色库蚊雌、雄蚊对于糖餐趋性的时间变化。结果表明幼虫期的食物丰富度不仅能够影响成蚊的体型,同样对其生理状态及能量储备具有明显影响。而幼虫期的食物越丰富,其羽化后早期成蚊对糖趋性越强。相同幼虫期食物营养条件下雌蚊在同一时间的摄食个体比例一直高于雄蚊摄糖比例。(2)室内条件下向新羽化的淡色库蚊提供偏好程度不同的开花植物以及果实作为糖源,研究了摄食不同偏好程度的糖餐植物对淡色库蚊寿命以及繁殖力的影响。结果表明大多数情况下摄食偏好的糖餐植物的成蚊其寿命及产卵量均显著高于其他组的成蚊；对成蚊摄糖个体的比例时间变化研究发现,喂食偏好的糖餐植物的成蚊其摄糖比例显著高于其他组,且寿命的差异可能与摄糖量有天。(3)利用Y型嗅觉仪测定淡色库蚊雌蚊对三种开花植物(小叶女贞、糯米条、夹竹桃)挥发物的行为反应,并以三种开花植物作为糖餐植物喂食成蚊,研究摄食不同糖餐植物对成蚊自身生理状态及能量储备的影响。结果表明淡色库蚊成蚊能够依据植物挥发物气味对不同的开花植物做出选择,其偏好程度与前期试验结果相一致；不同组的成蚊生理状态之间存在一定差异；摄食其偏好的糖餐植物后成蚊的能量储备高于其他组成蚊的能量储备。(4)室内条件下利用Y型嗅觉仪测试了新羽化的淡色库蚊雌蚊对18种常见的开花植物挥发物的行为反应,并依据不同浓度下各种化合物的行为引诱活性配制了全组分混合物及由6种化合物组成的简化组分混合物,并对两种化合物混合物的引诱效果进行比较。结果表明(E)-β-罗勒烯、苯甲醛、己醇、苯乙醇、(Z)-3-己烯-1-醇和芳樟醇等15种化合物以及全组分混合物和简化组分混合物对淡色库蚊均表现出明显的引诱效果,而两种混合物引诱力之间无显著差异。(5)利用固相微萃取技术对糯米条的挥发性气味进行收集,并利用气相色谱-质谱联用技术对挥发物主要成分进行分析。结果显示糯米条主要挥发性化合物包括苯甲醛、α-蒎烯、苯乙醛、苯甲醇、苯乙醇等芳香族化合物；糯米条浸提物及相关化合物混合物在Y型嗅觉仪试验中对淡色库蚊均表现出显著的引诱效果。(6)从淡色库蚊成蚊触角中克隆出10个编码气味结合蛋白的基因和1个编码嗅觉辅助受体的基因,并获得这些基因的cDNA序列全长序列。实时荧光定量PCR结果表明编码气味结合蛋白的基因主要集中在触角表达,而编码嗅觉辅助受体的基因在触角及雌蚊头部均有表达。将气味结合蛋白CpalOBP2和 CpalOBP3的cDNA序列连入pET-22b载体中,并转化至大肠杆菌BL21(DE3)pLysS菌株中进行诱导原核表达,成功得到了两个基因编码的蛋白条带。综上所述,本文研究了淡色库蚊幼虫期食物营养对成蚊体型及摄糖比例的影响,探究摄食不同糖餐植物对成蚊摄糖比例、存活时间、产卵量以及生理状况的影响,测定了淡色库蚊雌蚊对常见植物挥发物的行为反应,对偏好的糖餐植物糯米条挥发性化合物进行鉴定,并对淡色库蚊嗅觉相关基因进行了初步研究。研究结果表明淡色库蚊幼虫期食物营养对成蚊体型、生理状态、摄糖个体比例均有影响；摄食不同糖餐植物对成蚊摄糖个体比例、寿命、产卵量、能量储备均有显著影响；不同种类的植物挥发物对淡色库蚊雌蚊引诱效果不同,且引诱效果与化合物浓度密切相关；糯米条挥发物主要由芳香族化合物组成,其戊烷浸提物和化合物混合物对淡色库蚊具有引诱活性；克隆出10个编码气味结合蛋白的基因和1个编码嗅觉辅助受体的基因。相关研究结果将为研究“成蚊-糖餐植物”关系提供相关数据支撑,本研究中筛选出的对成蚊具有引诱活性的糖餐植物及其挥发性活性化合物将对利用蚊虫摄糖习性防治成蚊、开发安全、高效、环保的的蚊虫气味引诱剂和含毒糖诱剂提供必要的理论基础和参考信息。
[Abstract]:Culex pipiens pallens is the most common mosquito in the north of our country. It is also an important medium for Japanese encephalitis and filariasis. The prevention and control of mosquitoes has long been dependent on chemical insecticides, causing environmental pollution, increasing resistance and potential harm to human and animal. Sugar eating behavior is common in the life of mosquitoes. It is essential to maintain the energy required for the mosquito's life activities. In addition, the number of adult mosquitoes used to control the mosquito control is widely used in the mosquito control work. However, it is not clear how the adult mosquitoes locate the sugar meal plants, and the relationship between the saccharide habit, fitness, physiological status and the preference difference between the sugar meal plants is also needed. In this paper, the study of Culex pipiens pallens and its sugar meal plants, the relationship between the sugar uptake, the fitness of the adult and the preference of the sugar meal plant, the behavior response of the mosquito to the sugar meal plant volatiles, the parsing of the volatiles of the glutinous rice strips, and the olfactory related genes of the Culex pipiens pipiens pallens. The main contents are as follows: (1) by quantitative control of the food richness of the larvae of Culex pipiens pallens, the length, body weight and physiological state of the adult mosquitoes were analyzed, and the time changes of the glycosylation time of the male Culex pipiens pallens and male mosquitoes under different larval stages of food nutrition were studied by sugar staining. The results showed that the young was young. The food richness of the insect stage not only affects the shape of the adult mosquito, but also has an obvious influence on its physiological state and energy reserve. The more the food is in the larval stage, the higher the sugar taxis of the early adult mosquitoes after the emergence of the larvae. The proportion of the female mosquito at the same time in the same larval stage is higher than that of the male mosquito. (2) the effects of different preference blooming plants and fruit as sugar sources on the life and fecundity of Culex pipiens pallens were studied under the indoor conditions of Culex pipiens pallens with different preferences. The results showed that in most cases, the life and fecundity of adult mosquitoes were both significant. The ratio of the adult to adult mosquitoes was higher than that of the adult mosquitoes in the other groups. It was found that the percentage of sugar uptake of adult mosquitoes in the feeding preference sugar meal plants was significantly higher than that of the other groups, and the difference in life expectancy might be the day of sugar uptake. (3) three species of flowering plants (Ligustrum lucidum, sticky rice, and oleander) were measured by the Y olfactory apparatus. The behavioral responses of the volatiles were fed to three flowering plants as a sugar meal plant, and the effects of different sugar feeding plants on the physiological state and energy reserve of adult mosquitoes were studied. The results showed that the adult Culex pipiens pipiens pallens could choose different flowering plants according to the odor of plant volatiles, and their preference degree and preliminary test results were found. The physiological states of the adult mosquitoes in different groups were different, and the energy reserve of the adult mosquitoes after feeding their preference was higher than the energy reserve of other mosquitoes. (4) under indoor conditions, the behavioral responses of the female mosquito of Culex pipiens pipiens pallens to 18 common flowering plant volatiles were measured by the Y type olfactory apparatus. The attractant activity of various compounds at different concentrations was made up of a full component mixture and a simplified mixture consisting of 6 compounds, and the inducement effect of the mixture of the two compounds was compared. The results showed that (E) - beta - alenene, benzaldehyde, hexanol, benzyl alcohol, (Z) -3- hexene -1- alcohol and linalool and other 15 compounds and all The mixture of components and the mixture of simplified components showed obvious attractant effect on the Culex pipiens pallens, but there was no significant difference between the two mixtures. (5) the volatile odor of glutinous rice strips was collected by solid phase microextraction and the main components of the volatiles were analyzed by gas chromatography mass spectrometry. The results showed that the main components of the volatiles were analyzed by GC-MS. The main volatile compounds in the sticky rice strip include benzaldehyde, alpha pinene, phenylacetaldehyde, benzyl alcohol, benzol and other aromatic compounds. The glutinous rice extract and the mixture of related compounds showed significant attractant effect on the Culex pipiens pallens in the Y type olfactory apparatus test. (6) 10 coded odorant combined eggs were cloned from the antennae of Culex pipiens pallens. The genes of white and 1 genes encoding olfactory auxiliary receptors and the full length sequence of the cDNA sequences of these genes were obtained. Real time fluorescence quantitative PCR results showed that the genes encoding odorant binding proteins were mainly concentrated in the antennae, while the genes encoding the olfactory auxiliary receptors were expressed in the antennae and the head of the female mosquito. The odorant binding protein CpalOBP2 and the gene were expressed. The cDNA sequence of CpalOBP3 was incorporated into the pET-22b vector and transformed into the Escherichia coli BL21 (DE3) pLysS strain to induce the prokaryotic expression, and two genes encoded protein bands were successfully obtained. In summary, the effects of food nutrition on the body shape and sugar uptake ratio of Culex pipiens pallens were studied. The behavioral responses of the female mosquitoes of Culex pipiens pallens to common plant volatiles were measured, and the volatile compounds of the sticky rice strips of the preferred sugar meal plants were identified, and the olfactory related genes of Culex pipiens pallens were preliminarily studied. The results showed that the larvae of Culex pipiens pallens were the larvae of Culex pipiens pallens. The effects of food nutrition on the shape of mosquito, physiological state and the proportion of sugar uptake have significant influence on the proportion of individual sugar, life, egg production and energy reserve, and the effects of different kinds of plant volatiles on the female mosquito of Culex pipiens pallens are different, and the effect of the attractant is closely related to the concentration of the compound. The volatiles of sticky rice strips are mainly composed of aromatic compounds, whose pentane extract and compound mixture have lure activity to Culex pipiens pallens. 10 genes encoding odorant binding proteins and 1 genes encoding olfactory auxiliary receptors are cloned. The related research results will provide relevant data for the study of the relationship of "adult mosquito sugar meal". It will provide the necessary theoretical basis and information for the development of safe, efficient and environmentally friendly mosquito scent attractants and toxic lures.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R384.1

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