本文選題：淡色庫蚊 + 騷擾庫蚊�。� 參考：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2008年博士論文

【摘要】： 蚊蟲搜尋宿主行為主要受宿主氣味物質(zhì)調(diào)控。目前已知淡色庫蚊對雞、鴨、鴿子等禽類宿主偏好,嗜血習(xí)性存在分化;騷擾庫蚊在分類地位上和淡色庫蚊同為尖音庫蚊復(fù)合組,但是騷擾庫蚊發(fā)展為自育蚊蟲。本研究的主要目的是嘗試將野生的不同嗜血習(xí)性淡色庫蚊進行分離,以獲得穩(wěn)定的、有嗜血偏好的蚊株,為今后進行深入的嗜血習(xí)性分化機制研究奠定基礎(chǔ);分析和測試宿主動物的氣味物質(zhì)及影響蚊蟲搜尋宿主行為的化學(xué)物質(zhì),研究結(jié)果有助于發(fā)展新的蚊蟲引誘劑和驅(qū)避劑,用于蚊蟲控制和種群監(jiān)測。此外,通過測試淡色庫蚊和騷擾庫蚊對宿主動物氣味化合物的行為反應(yīng)差異,為揭示蚊蟲與宿主互作機制提供理論依據(jù),為發(fā)展新的防治方法干擾蚊蟲與宿主之間的化學(xué)聯(lián)系從而阻斷蟲媒病的流行提供了新的思路和途徑。本研究主要結(jié)果如下: 1.偏好鴿子和偏好鼠的淡色庫蚊株的建立 過去的不少研究表明:淡色庫蚊是有宿主偏好性差異的蚊種,對禽類的偏好勝過對哺乳類的偏好,但是也有部分研究顯示,有部分環(huán)境采集的淡色庫蚊更偏好哺乳動物。這表明自然界淡色庫蚊的宿主偏好存在分化。通過宿主篩選可能將野生種群區(qū)分為不同嗜血習(xí)性的蚊株。據(jù)此,本研究從北京西郊采集淡色庫蚊,用鴿子和昆明小鼠作為宿主,通過篩選和多次繼代純化,將野生淡色庫蚊分為偏好鴿子和偏好鼠的蚊株。實驗結(jié)果表明:初次篩選的野生種群中選擇鴿子的蚊蟲僅為27%,選擇鼠的蚊蟲有16%;繼代篩選能逐步提高這兩個蚊株選擇其偏好宿主的比例。經(jīng)過6次繼代篩選和純化后,偏好鴿子的蚊株選擇鴿子的比例為55%左右,選擇鼠的比例為10%左右;偏好鼠的蚊株選擇鴿子的比例為31%左右,選擇鼠的比例維持在34%左右。χ2檢驗F0代至鴿(或鼠)F7代的選擇率之間具有統(tǒng)計學(xué)意義(P0.0001)。到第六代后,這兩個蚊株選擇鴿子和鼠的比例變化不大,這顯示已經(jīng)將野生種群分為兩個不同宿主偏好蚊株。盡管本研究尚未得到專一性選擇鴿子和老鼠的蚊株,但是,分離獲得不同宿主偏好的蚊株也為今后宿主選擇的遺傳學(xué)研究提供了較好的材料。 2.鴿子、家雞和昆明小鼠的氣味物質(zhì)組成 為了進一步揭示其中影響淡色庫蚊尋找宿主行為的化學(xué)成分,本研究對宿主動物氣味物質(zhì)組成進行了分析。本研究采用吸附劑和羽毛浸提方法收集氣味物質(zhì),應(yīng)用GC/MS方法分析化學(xué)物質(zhì)組成。結(jié)果:(1)從GDX-101吸附鴿子氣味物質(zhì)中檢測出42個化合物,烷烴類和芳香類化合物約占總數(shù)的81%,醛酮類、羧酸類化合物占13%,其它約為6%;二氯甲烷羽毛浸提液檢測出59個化合物,其中烷烴及芳香類約占總數(shù)的60%,醛酮、羧酸類、醇酯類化合物占總數(shù)的32%,其它物質(zhì)占8%。(2)從GDX-101吸附家雞氣味物質(zhì)中檢測出69個化合物,包含烷烴類、醛酮類、羧酸類、雜環(huán)芳香類以及萜類化合物。其中醛類化合物為優(yōu)勢成分(69個化合物中有12個醛類化合物,占17.4%),吲哚和對甲苯酚含量高(分別占總峰面積的10.77%和10.67%);家雞羽毛浸提液檢測出61個化合物,包含烷烴類(32個占總數(shù)的53%,)、羧酸酯類(占總數(shù)的13%)、雜環(huán)芳香類、烯烴類和膽固醇類(含量較高,占總峰面積的41.26%)。(3)從GDX-101吸附昆明小鼠氣味物質(zhì)中檢測出30個化合物,主要包含烷烯烴類(11個,占總數(shù)的33.3%)、雜環(huán)芳香類(8個)、醛酮醇類(7個)和羧酸類(3個)等化合物。從鼠體毛浸提物中共鑒定出45個化合物,其中以烷烯烴類化合物為主(21個占總數(shù)的46.7%),其次醛酮類7個、羧酸和酯類6個、雜環(huán)芳香類化合物3個、膽固醇類4個、其它4個。膽甾醇和固醇類化合物含量較高,共占總峰面積的41.99%,為體毛提取物的主要成分。由于沒有使用標準化合物,本文所有化合物的鑒定都是暫時鑒定。 3.影響淡色庫蚊搜尋宿主行為的氣味物質(zhì)的鑒定 結(jié)合上述3種宿主動物氣味物質(zhì)分析結(jié)果和已有的人體氣味物質(zhì)的報道,使用觸角電生理實驗和行為實驗對影響淡色庫蚊搜尋宿主行為的化學(xué)物質(zhì)進行鑒定。觸角電生理實驗選用乳酸、氨水、α-蒎烯和苯甲醛等4種化合物測試了4個濃度(0.01, 0.1, 1和10μL/mL),實驗結(jié)果表明:乳酸、苯甲醛和氨水引發(fā)了淡色庫蚊雌蚊明顯的觸角電位(EAG)反應(yīng)。乳酸在1μL/mL劑量時引發(fā)了最大的EAG反應(yīng),當濃度增加到10μL/mL,觸角電位峰值反而下降。氨水在10μL/mL劑量時引發(fā)了最大的EAG反應(yīng)。淡色庫蚊雌蚊對苯甲醛和氨水的EAG反應(yīng)與劑量正相關(guān),即隨著劑量的增加反應(yīng)值增大。對α-蒎烯四個劑量的刺激都沒有產(chǎn)生明顯的EAG反應(yīng),說明該雌蚊可能對α-蒎烯不敏感。實驗結(jié)果表明宿主氣味物質(zhì)中存在能明顯引起該蚊蟲EAG反應(yīng)的物質(zhì),進一步證實淡色庫蚊與宿主存在化學(xué)聯(lián)系。行為實驗采用“Y”型嗅覺儀測定部分宿主氣味物質(zhì)對偏好鴿子淡色庫蚊株的引誘作用。測試物質(zhì)選用羧酸類、醛類、烷烯烴類、萜烯類、雜環(huán)芳香類及氨水等5類共19種化合物,測定了3個濃度(0.1, 1和10μL/mL)對淡色庫蚊的引誘作用。結(jié)果表明:羧酸類化合物中,辛酸和肉豆蔻酸表現(xiàn)引誘活性,且引誘活性隨著濃度升高而增大。特別是辛酸濃度達到10μL/mL時,對淡色庫蚊引誘作用超過80%(P=0.0031)。10μL/mL乳酸對淡色庫蚊有微弱引誘作用(57.27±4.49%,P=0.042),0.1μL/mL和1μL/mL濃度引誘作用與對照之間沒有統(tǒng)計學(xué)意義(P=0.362)。苯甲酸和壬酸在測試的三個劑量都沒有明顯引誘作用(P=1.133,P=0.675)。4種醛類物質(zhì)對淡色庫蚊沒有明顯引誘作用。壬醛、苯甲醛、己醛濃度超過1μL/mL,對淡色庫蚊引誘率明顯下降,幾乎不到15%,這顯示這幾種醛可能存在潛在的驅(qū)避作用,需要進一步的驅(qū)避實驗驗證。5種烷烯烴類物質(zhì)對淡色庫蚊都沒有明顯的引誘作用(P0.05);3種雜環(huán)芳香類物質(zhì)中,吲哚和對甲苯酚表現(xiàn)出明顯引誘活性,0.1μL/mL和1μL/mL吲哚對淡色庫蚊引誘率超過80%(P=0.0047,P=0.0072)。0.1μL/mL對甲苯酚的引誘率最高(87.31±5.11%,P=0.0096),濃度升高引誘率下降,濃度10μL/mL時引誘率為64.38±6.34%,仍然表現(xiàn)引誘活性(P=0.023)。3個濃度的萘都不顯示明顯的引誘活性(P0.05);高濃度的氨水(10μL/mL)對淡色庫蚊表現(xiàn)明顯引誘活性,引誘率達到73%左右(P=0.0385)。淡色庫蚊對a-蒎烯不表現(xiàn)明顯的趨向行為(P0.05)。選用辛酸、氨水、對甲苯酚、壬醛、β-月桂烯等5種化合物,研究對兩個不同宿主偏好蚊株的引誘作用。結(jié)果顯示:兩個不同宿主偏好的蚊株對上述5種化合物反應(yīng)沒有明顯差異,只10μL/mL辛酸和0.1μL/mL對甲苯酚引起的引誘反應(yīng)在兩個蚊株之間有差異(P=0.007, P=0.024),辛酸和對甲苯酚對偏好鴿子的蚊株的引誘作用大于對偏好鼠的蚊株的引誘作用。其余對偏好鴿子蚊株有明顯引誘活性的化合物,對偏好鼠的蚊株同樣表現(xiàn)為引誘活性,只是引誘率有所差別。只在昆明小鼠氣味物質(zhì)中檢測出來的氨對兩個蚊株都表現(xiàn)為或多或少的引誘作用。 4.騷擾庫蚊對動物及氣味物質(zhì)的行為反應(yīng) 雖然自育蚊蟲在第一次生殖周期無需吸血可完成產(chǎn)卵,但是否會有搜尋宿主行為卻值得關(guān)注,本研究就騷擾庫蚊在第一次生殖周期是否表現(xiàn)搜尋宿主行為進行了初步的研究和探討。使用篩選籠和”Y”型嗅覺儀,測試了處于第一次生殖周期的騷擾庫蚊對鴿子和昆明小鼠以及5種氣味化合物的行為反應(yīng),結(jié)果該蚊蟲對活體動物和化合物都表現(xiàn)出行為反應(yīng),飛向氣味陷阱中。動物選擇實驗中,選擇鴿子的比例為7.7%,選擇鼠的比例為8.3%,選擇率明顯低于淡色庫蚊�；衔镆T實驗中,辛酸、對甲苯酚和氨水對騷擾庫蚊具引誘作用,辛酸和氨水在10μL/mL時產(chǎn)生最大引誘效果(72.02±1.20%,P=0.00012;70.57±1.44%,P=0.00034);對甲苯酚在0.1μL/mL濃度產(chǎn)生最大引誘率(76.45±1.99%,P=0.00028)。β-月桂烯在三個濃度下沒有產(chǎn)生明顯引誘作用,同對照之間沒有顯著差異(P=0.92)。壬醛在1μL/mL和10μL/mL時,引誘率分別為17.97±1.15% (P=0.00012)和12.58±1.49% (P=0.00013),顯示潛在的驅(qū)避作用。本結(jié)果說明,騷擾庫蚊在自育產(chǎn)卵期間也可表現(xiàn)搜尋宿主行為,這暗示騷擾庫蚊的嗅覺受體可能在羽化時即已成熟了。通過對自育和非自育蚊蟲搜尋宿主行為進行比較研究,將有利于揭示蚊蟲搜尋宿主行為發(fā)生或抑制的機理。這對通過控制蚊蟲刺叮行為來控制蚊蟲危害,將是蚊蟲控制的新途徑和新思路。
[Abstract]:The behavior of mosquitoes searching host is mainly regulated by the host odorant. At present, it is known that the Culex pipiens pallens is preferred to chickens, ducks, and dove, and the habit of bloodthirsty is differentiated, and the harassing Culex pipiens in the classified position and Culex pipiens pallens is the compound of Culex pipiens pipiens. The isolation of Culex pipiens pallens of different bloodthirsty habits to obtain stable and bloodthirsty mosquitoes will lay the foundation for further study on the mechanism of blood addiction differentiation in the future; to analyze and test the odor substances in the host animals and the chemical substances that affect the behavior of the mosquitoes in search of the host. The results are helpful for the development of new mosquito attractants. And repellent, used in mosquito control and population monitoring. In addition, by testing the behavioral responses of Culex pipiens pallens and mosquitoes to host animal odorant compounds, it provides a theoretical basis for revealing the interaction mechanism of mosquitoes and hosts, and the development of new control methods to interfere with the chemical connections between mosquitoes and hosts and thus block the epidemic of insect vector diseases. New ideas and approaches are provided. The main findings of this study are as follows:
1. establishment of Culex pipiens pallens with preference for dove and preference mice
A number of previous studies have shown that Culex pipiens pallens is a species with a preference for host preference over fowl preference to mammals, but some studies have shown that some of the Culex pipiens pallens are preferred to mammals. This suggests that the host preference of Culex pipiens pallens is differentiated. In this study, the wild Culex pipiens pallens was collected from the western suburb of Beijing, with dove and Kunming mice as the host. The wild Culex pipiens pipiens pallens was divided into the preference of dove and the preferred mouse by screening and subculture. The results showed that the selection of dove's mosquitoes in the first selected wild population. Only 27% of the insects and 16% of the mosquitoes were selected in the mouse, and the proportion of the two mosquitoes could be gradually improved by the stepwise selection. After 6 subculture screening and purification, the proportion of the selected dove in dove was about 55%, and the ratio of the selected mice was about 10%; the ratio of the selected mosquitoes selected to dove was 31%, and the ratio of the mice was selected. The cases maintained at about 34%. The selection rate of the F0 generation to the F7 generation of the pigeon (or rat) was statistically significant (P0.0001). After the sixth generation, the two mosquitoes chose not to change the proportion of dove and mice. This shows that the wild population has been divided into two different host preferences. Although this study has not yet obtained the exclusive choice of dove and mice However, the isolation of mosquito species with different host preference also provides a good material for genetic research of host selection in the future.
Composition of odorous substances in 2. dove, domestic chicken and Kunming mice
In order to further reveal the chemical constituents that affect the search for host behavior of Culex pipiens pallens, this study analyses the composition of odorant substances in the host animals. This study uses adsorbents and feathers to collect odor substances and analyze the composition of chemical substances by GC/MS method. Results: (1) it was detected from the GDX-101 adsorption of dove odor substances. 42 compounds, alkanes and aromatic compounds accounted for about 81% of the total, aldehydes and ketones, carboxylic acids accounted for 13%, and other 6%, and dichloromethane feathers detected 59 compounds, of which alkanes and aromatics accounted for 60% of the total, aldehydes, ketones, carboxylic acids and alcohol esters accounted for 32% of the total, and other substances accounted for 8%. (2) from GDX-101. 69 compounds were detected in chicken odor substances, including alkanes, aldehydes and ketones, carboxylic acids, heterocyclic aromatics and terpenoids. Among them, aldehydes were dominant (12 aldehydes in 69 compounds, 17.4%), indoles and presol (10.77% and 10.67% of the total peak area), and chicken feather extraction solution. 61 compounds were detected, including alkanes (32 accounting for 53% of the total), carboxylic esters (13%), heterocyclic aromatics, olefins and cholesterol (41.26%). (3) 30 compounds were detected from GDX-101 adsorbed Kunming mice odor substances, including alkenes (11, 33.3% of the total), heterocyclic aromatic compounds. Aromatic compounds (8), aldehydes and ketones (7) and carboxylic acids (3) have been identified as 45 compounds, including alkenes (21, 46.7%), followed by 7 aldehydes and ketones, 6 carboxylic acids and esters, 3 heterocyclic aromatic compounds, 4 cholesterol, 4, cholesterol and sterols. A high content of 41.99% of the total peak area is the main component of the body hair extract. The identification of all the compounds in this article is a temporary identification because of the lack of standard compounds.
3. identification of odorous substances affecting Culex pipiens pallens searching for host behavior
Combined with the results of the analysis of the odorant substances of the 3 species of host animals and the reports of the existing human odor substances, the chemical substances that affect the search for the host behavior of Culex pipiens pallens were identified by electric antennae and behavior experiments. The electroantennae electrophysiological experiments used lactic acid, ammonia, alpha pinene and benzaldehyde to test 4 concentrations. 0.01, 0.1, 1 and 10 mu L/mL), the experimental results showed that lactic acid, benzaldehyde and ammonia caused the obvious antennae potential (EAG) reaction of the female mosquito of Culex pipiens pipiens pallens. The maximum EAG reaction caused by lactic acid at 1 u L/mL dose, when the concentration increased to 10 mu L/mL, the peak of the antennae potential decreased. The maximum EAG reaction caused by the ammonia water at 10 um L/mL dose. The EAG response of female mosquitoes to benzaldehyde and ammonia was positively correlated with the dose, that is, the response value increased with the increase of the dose. There was no obvious EAG response to the four doses of alpha pinene, indicating that the female mosquito could not be sensitive to alpha pinene. The experimental results showed that the host odor substance could cause the mosquitoes' EAG reaction obviously. Substance, further confirmed the chemical connection between the Culex pipiens pallens and the host. The behavior experiment used the "Y" type olfactory detector to determine the attractant effect of some host odorants on the preferred dove pipiens pipiens pallens strain. The test material selected 5 kinds of 19 compounds, carboxylic acids, aldehydes, alkenes, terpenes, heterocyclic aromatics and ammonia water, and determined 3 concentrations. The induction of Culex pipiens pallens by degree (0.1, 1 and 10 mu L/mL). The results showed that in carboxylic acids, octanoic acid and myristic acid showed the inducement activity, and the inducement activity increased with the concentration. Especially when the octanic acid concentration reached 10 mu, the lure of Culex pipiens pallens was over 80% (P=0.0031).10 mu L/mL lactic acid to Culex pipiens pallens. The inducement (57.27 + 4.49%, P=0.042), 0.1 mu L/mL and 1 micron L/mL had no statistical significance between the control and the control (P=0.362). The three doses of benzoic acid and NONYLIC acid had no obvious inducement (P=1.133, P=0.675).4 aldehydes had no obvious inducement to Culex pipiens pallens. Nonylaldehyde, benzaldehyde, and aldehydes were more than 1 u L/m. L, the lure rate of Culex pipiens pallens decreased significantly, almost less than 15%, which shows that these aldehydes may have potential repellents. Further repellent experiments are needed to verify that.5 alkenes have no obvious attractant to Culex pipiens pallens (P0.05); of the 3 heterocyclic aromatics, indole and presol show obvious attractant activity. The inducement rate of 0.1 mu L/mL and 1 L/mL indole to Culex pipiens pallens was higher than 80% (P=0.0047, P=0.0072).0.1 mu L/mL (87.31 + 5.11%, P=0.0096), the concentration increased and the attractant rate was 64.38 + 6.34% at the concentration of 10 mu L/mL, and the naphthalene, which was lure alive (P=0.023).3 concentration, did not show obvious attractant activity (P0.). 05); high concentration of ammonia (10 L/mL) showed an obvious attractant to Culex pipiens pallens (10). The inducement rate was about 73% (P=0.0385). The Culex pipiens pallens did not exhibit obvious tendency to a- pinene (P0.05). The inducement of octanoic acid, ammonia, methol, nonaldehyde, and beta laurene was selected to study the inducement of two different hosts. The results showed that there was no significant difference in the reaction between the two species of the two species with different host preferences. Only 10 mu L/mL octanoic acid and 0.1 L/mL p-cresol induced the difference between the two mosquitoes (P=0.007, P=0.024). The inducement of octanoic acid and p-cresol to the preferred dove mosquitoes was greater than that of the preferred mosquitoes. The other compounds that had obvious attractants to the dove mosquitoes were also attracted to the mosquitoes, only the attractants were different. The ammonia detected only in the odor substances in Kunming mice showed more or less attraction to the two mosquitoes.
4. behavioral responses of Culex pipiens to animal and odorous substances
Although there is no need of blood sucking to complete spawning in the first reproductive cycle, it is worth paying attention to whether there will be a search for the host behavior in the first reproductive period. This study harassed the mosquito in the first reproductive cycle to perform the search for the host behavior. The first reproduction was tested using the screening cage and the "Y" olfactory apparatus. The response of Culex mosquitoes to dove and Kunming mice and 5 odorant compounds showed that the mosquitoes showed behavioral responses to both living animals and compounds and flew to the odor trap. In animal selection experiments, the proportion of selected dove was 7.7%, the proportion of selected mice was 8.3%, and the selection rate was significantly lower than the Culex pipiens pipiens pallens. In the experiment, occult acid, PN and ammonia have a lure effect on harassing Culex mosquitoes. When octane and ammonia water are at 10 L/mL, the maximum attractant effect (72.02 + 1.20%, P=0.00012; 70.57 + 1.44%, P=0.00034); the maximum inducement rate (76.45 + 1.99%, P=0.00028) in the concentration of methenol at 0.1 mu (76.45 +, P=0.00028). There was no significant difference between the inducement and the control (P=0.92). The induced rates were 17.97 + 1.15% (P=0.00012) and 12.58 + 1.49% (P=0.00013) at 1 and 10 mu L/mL, respectively, showing potential repellent. The results showed that the harassing Culex pipiens could also search for the host behavior during the self breeding and spawning, which suggested harassing the olfactory of Culex mosquitoes. The body may be mature at the time of emergence. Through a comparative study of the search for host behavior of self fertility and non fertile mosquitoes, it will be helpful to reveal the mechanism of mosquitoes searching for the occurrence or inhibition of host behavior. This will be a new approach and new idea for controlling mosquitoes by controlling mosquito sting behavior.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2008
【分類號】：R384

【引證文獻】

相關(guān)碩士學(xué)位論文 前1條

1 林杰;三種蚤對宿主動物的氣味選擇性及其光學(xué)組織結(jié)構(gòu)的研究[D];中國疾病預(yù)防控制中心;2011年

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本文編號：2095885