淡色库蚊(Culex pipiens pallens)微卫星DNA位点的克隆

本研究利用地高辛标记的寡核苷酸(GT)8、(CAC)5、(GATA)4为探针,通过与基因组文库中的重组质粒DNA进行杂交,从686个重组质粒中筛选到8条淡色库蚊(Culex pipiens pallens)的微卫星(Microsatellite)DNA序列,已在GenBank数据库注册(Accession numberAY573280-AY573287).     

荧光定量PCR快速检测淡色库蚊击倒抗性相关钠通道基因突变(L1014F)

本研究的目的是采用荧光定量PCR扩增技术,建立快速检测淡色库蚊Culex pipiens pallens钠离子通道L1014位点击倒抗性(Knockdown resistance,kdr)相关点突变方法,该方法的原理是以突变位点为3′端设计两条特异性上游引物,和一条非特异下游引物组成两对引物平行双管法,用荧光定量PCR...     

中国库蚊属9个亚属的支序分析

本研究以我国已知的库蚊属 9个亚属的形态学和生态学资料为基础 ,应用 Hennig86程序软件进行支序分析 ,探讨我国库蚊属 ( Culex)各个亚属间的系统发育关系 ,支序图分析结果表明 :我国库蚊属 9个亚属均为独立的自然类群 ,可稳定地分为两个同源类群 :第一类群为真黑蚊亚属 ( Eum)和族角蚊亚属 ( Lop) ,具有胸侧板无鳞簇、幼虫孳生环境单一等典型的祖征性状和雄蚊触须短等新征 ,属于比较古老的类群 ;第二类群包括新库蚊亚属 ( Nex)梅蚊亚属 ( Mai)路蚊亚属 ( Lut) ,库状蚊亚属 ( Cui)泰蚊亚属 ( Tha) ,包蚊亚属 ( Bar)和库蚊亚属 ( Cux)等 7个亚属 ,与第一个类群互为姐妹群 ,其中包蚊亚属和库蚊亚属具有雄蚊尾器构造高度复杂化的共有新征 ,属于最进化的类群。此外 ,文中还对真黑蚊亚属、新库蚊亚属、梅蚊亚属、库状蚊亚属和泰蚊亚属的分类地位进行了评述和讨论。     

诱导毕赤酵母分泌表达致倦库蚊防御素Defensin成熟肽

目的诱导毕赤酵母(Pichia pastoris)分泌表达表达致倦库蚊(Culex quinquefasciatus)防御素(defensin)成熟肽,为进一步研究致倦库蚊defensin的抗病原作用、机理及其应用奠定基础。方法利用生物信息和RT-PCR技术克隆了编码致倦库蚊防御素的成熟肽全基因序列,并克隆到毕赤酵母甲醇诱导型分泌表达载体pPICZα-A的α-factor信号肽序列的下游,构建成pPICZα-A-defensin重组表达载体,表达载体经SacⅠ线性化处理后电击转化毕赤酵母X-33感受态细胞,转化子经Zeocin抗性筛选,菌落PCR,Mut表型鉴定和Tricine-SDS-PAGE分析,成功获得了能够有效表达重组defensin的pPICZα-A-Defensin/X-33甲醇利用表型工程菌。结果在甲醇诱导24 h后pPICZα-A-Defensin/X-33的工程菌分泌表达了预期Mr8 200大小的致倦库蚊防御素成熟重组肽。结论致倦库蚊防御素可以在毕赤酵母中诱导分泌表达。     

徐州地区三带喙库蚊胃血来源分析

应用PCR技术检测江苏徐州地区三带喙库蚊Culex tritaeniorhynchus蚊胃血来源,研究其吸血习性.根据三带喙库蚊可能吸血对象(猪、人、牛、羊、鼠、鸡)的线粒体DNA细胞色素b序列的差异,设计种特异引物,建立聚合酶链反应(PCR)体系鉴定三带喙库蚊胃血来源.同时,用该方法检测猪血、人血、牛血、羊血、鼠血、鸡血中提取的DNA,验证其特异性;对吸饲人血后不同时间(1、12、24、36、48、60h)的中华按蚊进行检测,测试该方法的敏感性.该方法可从已知动物血样提取的DNA中分别扩增得到689(人)、561(牛)、453(猪)、225(羊)、485(鼠)、266 bp(鸡)大小的特异性条带;吸人血36 h内的中华按蚊均能扩增出特异性条带,在吸人血后48、60 h的中华按蚊中,均未扩出特异性条带.共检测24只现场三带喙库蚊,血源来自猪、人、鸡、牛、鼠、羊分别为7、6、3,2、2、1只,其中5只蚊虫兼吸吸血.建立的PCR检测方法可鉴定三带喙库蚊蚊胃血来源,结果稳定可靠;徐州地区三带喙库蚊不仅嗜吸猪血和人血,也可兼吸其他动物血.     

三种库蚊核糖体DNA第二内转录间隔区(ITS2)序列测定与系统进化分析

对广东省野外采集的库蚊属中2个亚属中的3种库蚊,褐尾库蚊Culex fuscanus(Lutzia、路蚊亚属)、二带喙库蚊Cx.bitaeniorhynchus和致倦库蚊Cx.pipiens quinquefasciatus(Culex、库蚊亚属)进行核糖体DNA第二内转录间隔区(ITS2)序列测定,并与GenBank中已知库蚊属中路蚊亚属、库蚊亚属、新库蚊亚属和黑蚊亚属的11种和伊蚊属1种蚊虫的ITS2进行序列分析。结果表明:褐尾库蚊与同亚属的非洲蚊种(Culex tigripes)亲缘关系最近,基因同源性为65.27%;库蚊亚属的二带喙库蚊与伪杂鳞库蚊基因同源性为75.87%;致倦库蚊与尖音库蚊复合组内淡色库蚊的基因同源性97.13%。在所选库蚊中,黑蚊亚属与伊蚊最接近,其次是新库蚊亚属和路蚊亚属的蚊虫。在库蚊属4个亚属中,路蚊亚属与库蚊亚属有更近的亲缘关系,这也印证了库蚊亚属某些幼虫与路蚊亚属幼虫具有相似习性的遗传基础。系统进化关系分析结果与经典分类学分类系统接近一致,但库蚊亚属的进化可能更具多样性。     

应用高分辨率熔解曲线方法鉴定三带喙库蚊(双翅目:蚊科)

本研究旨在建立高分辨率熔解曲线准确、快速、特异鉴定三带喙库蚊Culex tritaeniorhynchus的方法。设计三带喙库蚊线粒体基因组COⅢ基因的特异性引物,在普通PCR基础上,进行高分辨率熔解曲线及其重复性分析。结果显示,普通PCR扩增出262 bp的COⅢ基因片段,经基因测序和序列分析,与形态学鉴定结果一致;三带喙库蚊有明显的高分辨率熔解曲线,对照蚊种和阴性对照均无熔解曲线;COⅢ基因熔解曲线走势差异较小,具良好的重复性。结果表明,基于COⅢ基因的高分辨率熔解曲线能准确、快速、特异的鉴定三带喙库蚊,可作为三带喙库蚊相关标本特异性鉴定的手段。     

淡色库蚊(Culex pipiens Pallens)与击倒抗性(kdr)相关的钠通道基因突变

本研究采用RT-PCR技术,使用简并引物分别从淡色库蚊(Culex pipiens pallens)敏感品系和抗溴氰菊酯品系中扩增出钠通道ⅡS4～ⅡS6区域的基因片段,长度为359bp.该基因片段所编码的氨基酸与黑尾果蝇(Drosophila melanogaster)、家蝇(Musca domestica)、埃及伊蚊(Aedes aegypti)、冈比亚按蚊(Anopheles gambiae)及德国小蠊(Blattella germanica)等昆虫相应区域的氨基酸序列具有较高的同源性,其同源性分别为95.8%,95.0%,100.0%,98.3%和95.0%.经序列比对,确认抗溴氰菊酯品系淡色库蚊钠通道基因在1014位点发生了突变该位点的碱基"A"突变为"T",其对应氨基酸由亮氨酸(L)变为苯丙氨酸(F),该突变(L1014F型)在多种昆虫中较为常见.     

苏丹瓦乌地区蚊媒密度监测及其影响因素分析

为明确任务区蚊媒种类、密度及其与环境因素的关系,笔者采用人诱法和容器指数法对苏丹瓦乌地区蚊媒的成虫和幼虫开展了密度监测。结果表明,在瓦乌维和任务区,主要蚊种为淡色库蚊、冈比亚按蚊和阿拉伯按蚊,由旱季进入雨季,蚊虫组成由单一的淡色库蚊变化为3种蚊虫共存的状态。其中阿拉伯按蚊偏好于干燥环境,而冈比亚按蚊则选择湿润环境。同时研究发现,按蚊幼虫选择弱酸性、低盐度、较清澈的积水孳生,水藻和植物有利于其孳生繁衍。库蚊则选择酸性、较浑浊的积水进行孳生。按蚊幼虫的孳生特点和成虫的活动生态特点,为开展任务区疟疾防治工作提供了基础资料和技术支持。     

中国尖音库蚊复合组蚊虫的抗性动态和遗传多样性研究

本文通过采用生物测定、蛋白质电泳和等位酶分析等方法对4个地理种群尖音库蚊复合组蚊虫(Culex pipiens comples)的抗性水平、群体中酯酶基因表型分布和种群遗传多样性进行了研究.抗性检测结果表明,4个库蚊种群对敌敌畏、对硫磷、氯菊酯和溴氰菊酯的抗性较高,对残杀威、巴沙和胺菊酯的抗性较低.4个种群抗性大小是:山东淄川>湖北沙市>广东茂名>北京沙河,淄川种群蚊虫对敌敌畏抗性为66.7倍.酯酶电泳结果中,沙市和茂名2个库蚊种群酯酶表型多态性最高,淄川库蚊种群多态性最低,过量表达的B1比率占100%.种群遗传多样性研究表明,每位点平均等位基因数(A)为1.92,平均多态位点百分率(P)为52.78%,平均预期杂合度(He)为0.130,群体间遗传分化系数(Fst)值为0.368,平均基因流Nm=1.52,说明4个种群有较丰富的遗传多样性,群体之间存在相当多的遗传多样性,种群间的基因交流较低,遗传分化较大,表明与地理位置存在一定对应关系.     

上海市中心城区成蚊种群动态及新变化研究

为全面掌握上海中心城区蚊虫种群动态及新变化,选取城区蚊虫较易出现的公园和绿化区域环境类型（人民广场及人民公园地区）,采用CO：诱捕法进行连续超过220d的成蚊动态监测研究。结果显示,2012年224d共捕捉到蚊虫20826头,包括3属6种;淡色库蚊和白纹伊蚊为上海中心城区优势蚊种（构成比分别为71．8％和19．7％）;不同蚊种季节分布差异性显著,淡色库蚊和白纹伊蚊高峰期分别位于5～6月和7～8月,且整体上淡色库蚊密度高于白纹伊蚊（除8月下旬外,其余均P〈0．05）,三带喙库蚊在7月中下旬显著上升（分别为681和580头／旬）;蚊虫13变化趋势波动幅度较大,在相同的月份或季节亦出现较大日变化差异;将2008～2011年历史监测数据与2012年进行趋势比较,仅个别年份间有显著相关性：不同年份间的旬分布亦存在统计学差异。本研究提示,上海市中心城区的优势蚊种仍为淡色库蚊和白纹伊蚊,该两种蚊种的季节分布与某些文献报道存在差异,主要由上海城区的气候特点决定;上海城区的蚊媒防治工作仍应针对这两种蚊虫的季节分布采取针对性的有效防控措施,此外三带喙库蚊对城市环境的适应性不断提高,为上海城区的蚊媒防治工作提出了新的挑战。     

上海市城区中心地带三带喙库蚊种群新动态及应对措施的研究

为全面掌握上海中心城区三带喙库蚊Culextritaeniorhynchus种群动态新变化并开展相应的科学有效的防治,选取城区成蚊较容易出现的公园和绿化区域环境类型（人民广场及人民公园地区）,采用24h连续性成蚊动态监测与蚊幼孳生地调查相结合的方法,对上海市中心城区三带喙库蚊种群动态变化进行研究。研究结果显示三带喙库蚊高峰出现在7月份;人民广场及人民公园地区的三带喙库蚊构成比最高达到32．79％,人民公园2号监测点的三带喙库蚊构成比达到52．6％,并在人民公园1号监测点周边发现2处三带喙库蚊孳生地,而历年监测中三带喙库蚊间断性捕获,多出现在8月份。本研究在上海中心城区首次报道三带喙库蚊的孳生地,且该蚊种已成为公园和绿化区域的优势蚊种。随着气候变化与城市环境的改变,三带喙库蚊对城市环境的适应性不断提高,除淡色库蚊和白纹伊蚊外,三带喙库蚊也应成为上海市城区的重点防治蚊种。     

刘氏中剑水蚤防治蚊幼虫的研究

作者在济宁市各种水域中广泛采集了中剑水蚤，经过培养后一种鉴定为刘氏中剑水蚤（Ｍｅｓｏｃｙｃｌｏｐｓｌｅｕｃｋａｒｔｉ），在实验室及现场中进行了系统评价。在室内，刘氏中剑水蚤对三种蚊幼虫均有较高的捕食力，２０２０比例下，中剑水蚤对白纹伊蚊Ⅰ龄幼虫在２４、４８和７２ｈ捕食率分别为９６％、１００％和１００％，对淡色库蚊幼虫分别为６５％、７５％和８０％，对中华按蚊分别为７５％、９０％和９０％。蚊笼模拟试验表明，中剑水蚤可分别于８、９和１０周内消灭白纹伊蚊、淡色库蚊和中华按蚊的幼虫种群，成蚊于２周后消失。大池培养经济易行，获得成功。现场试验证明，刘氏中剑水蚤在蚊虫孳生地可成功地建立自己的种群，存活时间长于蚊虫季节，因此可长期防治蚊幼虫。中剑水蚤防治白纹伊蚊的效果好于淡色库蚊。应用中剑水蚤的长持效和Ｂ．ｓ．及Ｂ．ｔ．ｉ．的速效，在防治淡色库蚊和白纹伊蚊方面取得了更明显的效果，幼虫密度降低率９０％以上，持效整个蚊虫季节。因此二者合用可作为蚊虫综合防治的一个组成部分。     

Culex tritaeniorhynchus and Aedes albopictus (Diptera: Culicidae) as natural vectors of Dirofilaria immitis (Spirurida: Filariidae) in Miki City, Japan

Natural infections with Dirofilaria immitis (Leidy) in a population of mosquitoes and dogs and its antibodies in a human population were surveyed in Miki City, a rural area of Japan, to reveal ecological features of this arthropod-borne zo?notic parasite. Microfilaremic and amicrofilaremic infection rates in 190 dogs were 52.6 and 9.0%, respectively, as determined by the hematocrit centrifuge method and the indirect fluorescent antibody test. An enzyme-linked immunosorbent assay (ELISA) test for antibodies to D. immitis showed that almost all of 917 inhabitants had antibodies in varying degrees, indicating frequent exposure of this parasite to the human population. Among the six species of wild mosquitoes sampled from 1985 through 1987 by attraction with dry ice, Culex tritaeniorhynchus Giles was the most abundant in two sites and Aedes albopictus (Skuse) in another two sites. Filarial infections were found every year in Cx. tritaeniorhynchus and Ae. albopictus collected between July and September, as examined by ELISA for detecting larval antigens in mosquito homogenate, suggesting that these species are important natural vectors in this survey area.     

Induction of oogenesis in mosquitoes (Diptera: Culicidae) by infusion of the hemocoel with amino acids

As done previously with adult females of Culex pipiens pallens Coquillett, a mixture of 17 amino acids was infused into the hemocoel of females of seven anautogenous and one autogenous mosquito species belonging to three genera. In Culex. p. quinquefasciatus Say, Cx. tritaeniorhynchus Giles, Cx. kyotoensis Yamaguti & LaCasse, Aedes albopictus (Skuse), Armigeres subalbatus (Coquillett), and Cx. p. molestus Forskal, which previously had laid autogenously matured first batch of eggs, ovarian development was stimulated and frequently continued to maturity. In most mosquitoes, the number of mature follicles nearly doubled when the period of infusion was extended from 24 to 48 h. Therefore, the two previously indicated roles of amino acids, one to initiate ovarian development and the other to regulate the number of maturing oocytes, were confirmed in these species. In Cx. halifaxii Theobald and Ae. japonicus (Theobald), however, the frequency of activation and maturation of ovaries was low compared with the other species, indicating that those species may require some factors other than an increase in amino acids for normal ovarian development after a blood meal.     

Susceptibility of Aedes albopictus and Culex tritaeniorhynchus (Diptera: Culicidae) collected in Miki City, Japan, to Dirofilaria immitis (Spirurida: Filariidae)

Female Aedes albopictus (Skuse) and Culex tritaeniorhynchus Giles from Miki City, Japan, were examined for susceptibility to infection by Dirofilaria immitis. Third-stage larvae were detected from day 7 in 10-40% of Ae. albopictus and from day 10 in 20-60% of Cx. tritaeniorhynchus, irrespective of microfilarial intake, which varied from 500 to 15,000 microfilariae per ml. The number of third-stage larvae in the labium usually varied from one to three per mosquito up to day 21, confirming the ability of these mosquitoes to transmit D. immitis.     

Size of blood meals of Aedes albopictus and Culex tritaeniorhynchus (Diptera: Culicidae) feeding on an unrestrained dog infected with Dirofilaria immitis (Spirurida: Filariidae)

Blood meal volume was determined for two species of mosquitoes, Aedes albopictus (Skuse) and Culex tritaeniorhynchus Giles, recently demonstrated to be natural vectors of canine heartworm in Japan. When fed on unrestrained dogs, volumes were from 1 nl to 6 microliters in both species, but percentages of engorged mosquitoes were 45.2-64.3% for Ae. albopictus and 11.9-22.2% for Cx. tritaeniorhynchus. Ae. albopictus preferred to feed on human bait versus canine bait when both were exposed concurrently.     

云南中缅边境蚊科昆虫群落的研究

为调查和了解云南省瑞丽市中缅边境蚊虫多样性状况,应用灯诱法和采集幼虫法对瑞丽市中缅边境上高丽村和周边方圆4 km原始森林内的蚊虫的优势种组成、蚊虫群落结构的集中性指数、多样性指数和均匀度指数进行分析.结果显示,在居民区共采获蚊虫3 305只,共5属13种,隶属于蚊科Culicidae中的伊蚊属Aedes、库蚊属Cule...     

Development of lymphatic filarial parasite Wuchereria bancrofti (Spirurida: Onchocercidae) in mosquito species (Diptera: Culicidae) fed artificially on microfilaremic blood

The efficiency of laboratory colonies of mosquitoes such as Anopheles stephensi Liston, Aedes aegypti (L.) Liverpool strain, Ae. aegypti wild type, Aedes albopictus (Skuse), Culex tritaeniorhynchus Giles, Culex sitiens Wiedemann, and Armigeres subalbatus Coquillett in supporting the development of Wuchereria bancrofti (Cobbold) (Spirurida: Onchocercidae) microfilariae to infective larvae was investigated. The mosquitoes were fed on heparinized microfilaremic human blood by using a membrane-feeding unit with Parafilm as membrane. The rate of infection, parasite development, and parasite burden were compared with that in the known vector mosquito Culex quinquefasciatus Say. Cx. quinquefasciatus showed the highest percentage of infection, followed by Ae. aegypti Liverpool strain and An. stephensi. The rate of development of the parasite was more or less similar in all the three species, and infective larvae were found on day 13. When the larvae were harvested on day 17, Cx. quinquefasciatus yielded the highest numbers, followed by Ae. aegypti Liverpool strain and An. stephensi. The percentage of infection was low, and the development was slow in Cx. tritaeniorhynchus compared with the other susceptible species. The parasite developed to second-stage larvae only by day 22 and to infective larvae by day 28. When 2-wk-old Cx. tritaeniorhynchus were fed on microfilaremic blood, they could develop the parasite to infective larvae by day 13 postfeeding. All other species of mosquitoes tested were found to be refractory to parasite development. It is shown that Cx. quinquefasciatus is the most suitable mosquito host for the production of infective larvae. However, Ae. aegypti Liverpool strain, which is commonly used for Brugia malayi filarial parasite, also can be used for generation of W. bancrofti infective larvae to circumvent the problem of maintaining two mosquito species.     

褐尾库蚊对白纹伊蚊和致倦库蚊捕食能力的研究

褐尾库蚊是白纹伊蚊、致倦库蚊的天敌,褐尾库蚊幼虫随虫龄的增大,各龄日捕食白纹伊蚊的数量增加,随着害蚊虫口密度的增加褐尾库蚊幼虫日捕食白纹伊蚊的数量增加;2、3、4龄褐尾库蚊幼虫日捕食白纹伊蚊的数量范围分别在4～8只,8～13只,10～19只之间。2、3、4龄褐尾库蚊幼虫对白纹伊蚊的捕食量分别呈函数关系y2=1.0615Ln（x）＋3.9494（R^2=0.9091）,y3=2.7116Ln（x）＋3.8863（R^2=0.8919）,y4=5.7767Ln（x）＋0.394（R^2=0.9722）,它们增幅大小的拐点分别是3、5、10只/100cm^3。4龄褐尾库蚊幼虫对致倦库蚊的捕食量呈函数关系y=1.4133Ln（x）＋0.5334（R^2=0.9520）,增幅大小的拐点是5只/100cm^3,害蚊单种种群时褐尾库蚊对白纹伊蚊幼虫的捕食量是致倦库蚊的4倍。2种害蚊混合种群时褐尾库蚊对白纹伊蚊的捕食量是致倦库蚊的15倍。