本文選題：淡色庫(kù)蚊 + 糖餐行為　； 參考：《浙江大學(xué)》2016年博士論文

【摘要】：淡色庫(kù)蚊是我國(guó)北方最常見(jiàn)的蚊種,同時(shí)也是乙型腦炎和絲蟲(chóng)病的重要傳播媒介。長(zhǎng)期以來(lái)蚊蟲(chóng)的防治工作大量依賴(lài)化學(xué)殺蟲(chóng)劑,產(chǎn)生環(huán)境污染、抗藥性不斷增強(qiáng)、對(duì)人畜具有潛在危害等問(wèn)題。攝糖行為在蚊蟲(chóng)生活史中普遍存在糖餐對(duì)于維持成蚊生命活動(dòng)所需能量必不可少。此外,運(yùn)用成蚊攝糖習(xí)性控制成蚊數(shù)量在蚊蟲(chóng)防治工作中得到廣泛應(yīng)用。然而,成蚊如何定位糖餐植物尚不明確,而成蚊的趨糖習(xí)性、適合度、生理狀況與對(duì)糖餐植物偏好性差異之間的關(guān)系也有待研究。本論文以淡色庫(kù)蚊及其糖餐植物為研究對(duì)象,對(duì)成蚊的攝糖趨性、成蚊適合度以及生理狀況與糖餐植物偏好性之間的關(guān)系、成蚊對(duì)糖餐植物揮發(fā)物行為反應(yīng)、偏好性糖餐植物糯米條揮發(fā)物解析、淡色庫(kù)蚊嗅覺(jué)相關(guān)基因等方面進(jìn)行了研究,主要包括以下內(nèi)容：(1)通過(guò)定量控制淡色庫(kù)蚊幼蟲(chóng)的食物豐富度,對(duì)羽化后成蚊翅長(zhǎng)、體重以及生理狀態(tài)進(jìn)行分析,并利用糖染色法研究了不同幼蟲(chóng)期食物營(yíng)養(yǎng)條件下的淡色庫(kù)蚊雌、雄蚊對(duì)于糖餐趨性的時(shí)間變化。結(jié)果表明幼蟲(chóng)期的食物豐富度不僅能夠影響成蚊的體型,同樣對(duì)其生理狀態(tài)及能量?jī)?chǔ)備具有明顯影響。而幼蟲(chóng)期的食物越豐富,其羽化后早期成蚊對(duì)糖趨性越強(qiáng)。相同幼蟲(chóng)期食物營(yíng)養(yǎng)條件下雌蚊在同一時(shí)間的攝食個(gè)體比例一直高于雄蚊攝糖比例。(2)室內(nèi)條件下向新羽化的淡色庫(kù)蚊提供偏好程度不同的開(kāi)花植物以及果實(shí)作為糖源,研究了攝食不同偏好程度的糖餐植物對(duì)淡色庫(kù)蚊壽命以及繁殖力的影響。結(jié)果表明大多數(shù)情況下攝食偏好的糖餐植物的成蚊其壽命及產(chǎn)卵量均顯著高于其他組的成蚊；對(duì)成蚊攝糖個(gè)體的比例時(shí)間變化研究發(fā)現(xiàn),喂食偏好的糖餐植物的成蚊其攝糖比例顯著高于其他組,且壽命的差異可能與攝糖量有天。(3)利用Y型嗅覺(jué)儀測(cè)定淡色庫(kù)蚊雌蚊對(duì)三種開(kāi)花植物(小葉女貞、糯米條、夾竹桃)揮發(fā)物的行為反應(yīng),并以三種開(kāi)花植物作為糖餐植物喂食成蚊,研究攝食不同糖餐植物對(duì)成蚊自身生理狀態(tài)及能量?jī)?chǔ)備的影響。結(jié)果表明淡色庫(kù)蚊成蚊能夠依據(jù)植物揮發(fā)物氣味對(duì)不同的開(kāi)花植物做出選擇,其偏好程度與前期試驗(yàn)結(jié)果相一致；不同組的成蚊生理狀態(tài)之間存在一定差異；攝食其偏好的糖餐植物后成蚊的能量?jī)?chǔ)備高于其他組成蚊的能量?jī)?chǔ)備。(4)室內(nèi)條件下利用Y型嗅覺(jué)儀測(cè)試了新羽化的淡色庫(kù)蚊雌蚊對(duì)18種常見(jiàn)的開(kāi)花植物揮發(fā)物的行為反應(yīng),并依據(jù)不同濃度下各種化合物的行為引誘活性配制了全組分混合物及由6種化合物組成的簡(jiǎn)化組分混合物,并對(duì)兩種化合物混合物的引誘效果進(jìn)行比較。結(jié)果表明(E)-β-羅勒烯、苯甲醛、己醇、苯乙醇、(Z)-3-己烯-1-醇和芳樟醇等15種化合物以及全組分混合物和簡(jiǎn)化組分混合物對(duì)淡色庫(kù)蚊均表現(xiàn)出明顯的引誘效果,而兩種混合物引誘力之間無(wú)顯著差異。(5)利用固相微萃取技術(shù)對(duì)糯米條的揮發(fā)性氣味進(jìn)行收集,并利用氣相色譜-質(zhì)譜聯(lián)用技術(shù)對(duì)揮發(fā)物主要成分進(jìn)行分析。結(jié)果顯示糯米條主要揮發(fā)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；糯米條浸提物及相關(guān)化合物混合物在Y型嗅覺(jué)儀試驗(yàn)中對(duì)淡色庫(kù)蚊均表現(xiàn)出顯著的引誘效果。(6)從淡色庫(kù)蚊成蚊觸角中克隆出10個(gè)編碼氣味結(jié)合蛋白的基因和1個(gè)編碼嗅覺(jué)輔助受體的基因,并獲得這些基因的cDNA序列全長(zhǎng)序列。實(shí)時(shí)熒光定量PCR結(jié)果表明編碼氣味結(jié)合蛋白的基因主要集中在觸角表達(dá),而編碼嗅覺(jué)輔助受體的基因在觸角及雌蚊頭部均有表達(dá)。將氣味結(jié)合蛋白CpalOBP2和 CpalOBP3的cDNA序列連入pET-22b載體中,并轉(zhuǎn)化至大腸桿菌BL21(DE3)pLysS菌株中進(jìn)行誘導(dǎo)原核表達(dá),成功得到了兩個(gè)基因編碼的蛋白條帶。綜上所述,本文研究了淡色庫(kù)蚊幼蟲(chóng)期食物營(yíng)養(yǎng)對(duì)成蚊體型及攝糖比例的影響,探究攝食不同糖餐植物對(duì)成蚊攝糖比例、存活時(shí)間、產(chǎn)卵量以及生理狀況的影響,測(cè)定了淡色庫(kù)蚊雌蚊對(duì)常見(jiàn)植物揮發(fā)物的行為反應(yīng),對(duì)偏好的糖餐植物糯米條揮發(fā)性化合物進(jìn)行鑒定,并對(duì)淡色庫(kù)蚊嗅覺(jué)相關(guān)基因進(jìn)行了初步研究。研究結(jié)果表明淡色庫(kù)蚊幼蟲(chóng)期食物營(yíng)養(yǎng)對(duì)成蚊體型、生理狀態(tài)、攝糖個(gè)體比例均有影響；攝食不同糖餐植物對(duì)成蚊攝糖個(gè)體比例、壽命、產(chǎn)卵量、能量?jī)?chǔ)備均有顯著影響；不同種類(lèi)的植物揮發(fā)物對(duì)淡色庫(kù)蚊雌蚊引誘效果不同,且引誘效果與化合物濃度密切相關(guān)；糯米條揮發(fā)物主要由芳香族化合物組成,其戊烷浸提物和化合物混合物對(duì)淡色庫(kù)蚊具有引誘活性；克隆出10個(gè)編碼氣味結(jié)合蛋白的基因和1個(gè)編碼嗅覺(jué)輔助受體的基因。相關(guān)研究結(jié)果將為研究“成蚊-糖餐植物”關(guān)系提供相關(guān)數(shù)據(jù)支撐,本研究中篩選出的對(duì)成蚊具有引誘活性的糖餐植物及其揮發(fā)性活性化合物將對(duì)利用蚊蟲(chóng)攝糖習(xí)性防治成蚊、開(kāi)發(fā)安全、高效、環(huán)保的的蚊蟲(chóng)氣味引誘劑和含毒糖誘劑提供必要的理論基礎(chǔ)和參考信息。
[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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R384.1

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