淡色库蚊嗜血习性的嗅觉生理研究

发布时间：2018-07-04 11:13

本文选题：淡色库蚊 + 骚扰库蚊　；参考：《中国人民解放军军事医学科学院》2008年博士论文

【摘要】： 蚊虫搜寻宿主行为主要受宿主气味物质调控。目前已知淡色库蚊对鸡、鸭、鸽子等禽类宿主偏好,嗜血习性存在分化;骚扰库蚊在分类地位上和淡色库蚊同为尖音库蚊复合组,但是骚扰库蚊发展为自育蚊虫。本研究的主要目的是尝试将野生的不同嗜血习性淡色库蚊进行分离,以获得稳定的、有嗜血偏好的蚊株,为今后进行深入的嗜血习性分化机制研究奠定基础;分析和测试宿主动物的气味物质及影响蚊虫搜寻宿主行为的化学物质,研究结果有助于发展新的蚊虫引诱剂和驱避剂,用于蚊虫控制和种群监测。此外,通过测试淡色库蚊和骚扰库蚊对宿主动物气味化合物的行为反应差异,为揭示蚊虫与宿主互作机制提供理论依据,为发展新的防治方法干扰蚊虫与宿主之间的化学联系从而阻断虫媒病的流行提供了新的思路和途径。本研究主要结果如下: 1.偏好鸽子和偏好鼠的淡色库蚊株的建立过去的不少研究表明:淡色库蚊是有宿主偏好性差异的蚊种,对禽类的偏好胜过对哺乳类的偏好,但是也有部分研究显示,有部分环境采集的淡色库蚊更偏好哺乳动物。这表明自然界淡色库蚊的宿主偏好存在分化。通过宿主筛选可能将野生种群区分为不同嗜血习性的蚊株。据此,本研究从北京西郊采集淡色库蚊,用鸽子和昆明小鼠作为宿主,通过筛选和多次继代纯化,将野生淡色库蚊分为偏好鸽子和偏好鼠的蚊株。实验结果表明:初次筛选的野生种群中选择鸽子的蚊虫仅为27%,选择鼠的蚊虫有16%;继代筛选能逐步提高这两个蚊株选择其偏好宿主的比例。经过6次继代筛选和纯化后,偏好鸽子的蚊株选择鸽子的比例为55%左右,选择鼠的比例为10%左右;偏好鼠的蚊株选择鸽子的比例为31%左右,选择鼠的比例维持在34%左右。χ2检验F0代至鸽(或鼠)F7代的选择率之间具有统计学意义(P0.0001)。到第六代后,这两个蚊株选择鸽子和鼠的比例变化不大,这显示已经将野生种群分为两个不同宿主偏好蚊株。尽管本研究尚未得到专一性选择鸽子和老鼠的蚊株,但是,分离获得不同宿主偏好的蚊株也为今后宿主选择的遗传学研究提供了较好的材料。 2.鸽子、家鸡和昆明小鼠的气味物质组成为了进一步揭示其中影响淡色库蚊寻找宿主行为的化学成分,本研究对宿主动物气味物质组成进行了分析。本研究采用吸附剂和羽毛浸提方法收集气味物质,应用GC/MS方法分析化学物质组成。结果:(1)从GDX-101吸附鸽子气味物质中检测出42个化合物,烷烃类和芳香类化合物约占总数的81%,醛酮类、羧酸类化合物占13%,其它约为6%;二氯甲烷羽毛浸提液检测出59个化合物,其中烷烃及芳香类约占总数的60%,醛酮、羧酸类、醇酯类化合物占总数的32%,其它物质占8%。(2)从GDX-101吸附家鸡气味物质中检测出69个化合物,包含烷烃类、醛酮类、羧酸类、杂环芳香类以及萜类化合物。其中醛类化合物为优势成分(69个化合物中有12个醛类化合物,占17.4%),吲哚和对甲苯酚含量高(分别占总峰面积的10.77%和10.67%);家鸡羽毛浸提液检测出61个化合物,包含烷烃类(32个占总数的53%,)、羧酸酯类(占总数的13%)、杂环芳香类、烯烃类和胆固醇类(含量较高,占总峰面积的41.26%)。(3)从GDX-101吸附昆明小鼠气味物质中检测出30个化合物,主要包含烷烯烃类(11个,占总数的33.3%)、杂环芳香类(8个)、醛酮醇类(7个)和羧酸类(3个)等化合物。从鼠体毛浸提物中共鉴定出45个化合物,其中以烷烯烃类化合物为主(21个占总数的46.7%),其次醛酮类7个、羧酸和酯类6个、杂环芳香类化合物3个、胆固醇类4个、其它4个。胆甾醇和固醇类化合物含量较高,共占总峰面积的41.99%,为体毛提取物的主要成分。由于没有使用标准化合物,本文所有化合物的鉴定都是暂时鉴定。 3.影响淡色库蚊搜寻宿主行为的气味物质的鉴定结合上述3种宿主动物气味物质分析结果和已有的人体气味物质的报道,使用触角电生理实验和行为实验对影响淡色库蚊搜寻宿主行为的化学物质进行鉴定。触角电生理实验选用乳酸、氨水、α-蒎烯和苯甲醛等4种化合物测试了4个浓度(0.01, 0.1, 1和10μL/mL),实验结果表明:乳酸、苯甲醛和氨水引发了淡色库蚊雌蚊明显的触角电位(EAG)反应。乳酸在1μL/mL剂量时引发了最大的EAG反应,当浓度增加到10μL/mL,触角电位峰值反而下降。氨水在10μL/mL剂量时引发了最大的EAG反应。淡色库蚊雌蚊对苯甲醛和氨水的EAG反应与剂量正相关,即随着剂量的增加反应值增大。对α-蒎烯四个剂量的刺激都没有产生明显的EAG反应,说明该雌蚊可能对α-蒎烯不敏感。实验结果表明宿主气味物质中存在能明显引起该蚊虫EAG反应的物质,进一步证实淡色库蚊与宿主存在化学联系。行为实验采用“Y”型嗅觉仪测定部分宿主气味物质对偏好鸽子淡色库蚊株的引诱作用。测试物质选用羧酸类、醛类、烷烯烃类、萜烯类、杂环芳香类及氨水等5类共19种化合物,测定了3个浓度(0.1, 1和10μL/mL)对淡色库蚊的引诱作用。结果表明:羧酸类化合物中,辛酸和肉豆蔻酸表现引诱活性,且引诱活性随着浓度升高而增大。特别是辛酸浓度达到10μL/mL时,对淡色库蚊引诱作用超过80%(P=0.0031)。10μL/mL乳酸对淡色库蚊有微弱引诱作用(57.27±4.49%,P=0.042),0.1μL/mL和1μL/mL浓度引诱作用与对照之间没有统计学意义(P=0.362)。苯甲酸和壬酸在测试的三个剂量都没有明显引诱作用(P=1.133,P=0.675)。4种醛类物质对淡色库蚊没有明显引诱作用。壬醛、苯甲醛、己醛浓度超过1μL/mL,对淡色库蚊引诱率明显下降,几乎不到15%,这显示这几种醛可能存在潜在的驱避作用,需要进一步的驱避实验验证。5种烷烯烃类物质对淡色库蚊都没有明显的引诱作用(P0.05);3种杂环芳香类物质中,吲哚和对甲苯酚表现出明显引诱活性,0.1μL/mL和1μL/mL吲哚对淡色库蚊引诱率超过80%(P=0.0047,P=0.0072)。0.1μL/mL对甲苯酚的引诱率最高(87.31±5.11%,P=0.0096),浓度升高引诱率下降,浓度10μL/mL时引诱率为64.38±6.34%,仍然表现引诱活性(P=0.023)。3个浓度的萘都不显示明显的引诱活性(P0.05);高浓度的氨水(10μL/mL)对淡色库蚊表现明显引诱活性,引诱率达到73%左右(P=0.0385)。淡色库蚊对a-蒎烯不表现明显的趋向行为(P0.05)。选用辛酸、氨水、对甲苯酚、壬醛、β-月桂烯等5种化合物,研究对两个不同宿主偏好蚊株的引诱作用。结果显示:两个不同宿主偏好的蚊株对上述5种化合物反应没有明显差异,只10μL/mL辛酸和0.1μL/mL对甲苯酚引起的引诱反应在两个蚊株之间有差异(P=0.007, P=0.024),辛酸和对甲苯酚对偏好鸽子的蚊株的引诱作用大于对偏好鼠的蚊株的引诱作用。其余对偏好鸽子蚊株有明显引诱活性的化合物,对偏好鼠的蚊株同样表现为引诱活性,只是引诱率有所差别。只在昆明小鼠气味物质中检测出来的氨对两个蚊株都表现为或多或少的引诱作用。 4.骚扰库蚊对动物及气味物质的行为反应虽然自育蚊虫在第一次生殖周期无需吸血可完成产卵,但是否会有搜寻宿主行为却值得关注,本研究就骚扰库蚊在第一次生殖周期是否表现搜寻宿主行为进行了初步的研究和探讨。使用筛选笼和”Y”型嗅觉仪,测试了处于第一次生殖周期的骚扰库蚊对鸽子和昆明小鼠以及5种气味化合物的行为反应,结果该蚊虫对活体动物和化合物都表现出行为反应,飞向气味陷阱中。动物选择实验中,选择鸽子的比例为7.7%,选择鼠的比例为8.3%,选择率明显低于淡色库蚊。化合物引诱实验中,辛酸、对甲苯酚和氨水对骚扰库蚊具引诱作用,辛酸和氨水在10μL/mL时产生最大引诱效果(72.02±1.20%,P=0.00012;70.57±1.44%,P=0.00034);对甲苯酚在0.1μL/mL浓度产生最大引诱率(76.45±1.99%,P=0.00028)。β-月桂烯在三个浓度下没有产生明显引诱作用,同对照之间没有显著差异(P=0.92)。壬醛在1μL/mL和10μL/mL时,引诱率分别为17.97±1.15% (P=0.00012)和12.58±1.49% (P=0.00013),显示潜在的驱避作用。本结果说明,骚扰库蚊在自育产卵期间也可表现搜寻宿主行为,这暗示骚扰库蚊的嗅觉受体可能在羽化时即已成熟了。通过对自育和非自育蚊虫搜寻宿主行为进行比较研究,将有利于揭示蚊虫搜寻宿主行为发生或抑制的机理。这对通过控制蚊虫刺叮行为来控制蚊虫危害,将是蚊虫控制的新途径和新思路。
[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.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R384

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