朱砂叶螨体内感染的Wolbachia的wsp基因序列测定与分析

应用Wolbachia的wsp基因特异引物,通过PCR扩增法对我国朱砂叶螨Tetranychus cinnabarinus7个地理种群进行了检测。在采自黑龙江佳木斯、安徽安庆、江苏镇江和浙江慈溪的4个地理种群中扩增出了596bp左右的Wolbachia的wsp基因片段,而在河北威县、山东滨州和湖北赤壁3个地理种群中未发现这个Wolbachia特征基因片段,表明 Wolbachia在我国朱砂叶螨中的侵染较为普遍。通过对我国朱砂叶螨体内感染的 Wolbachia的wsp基因序列进行系统发育分析,得出它们全部与B大组的Ori组的Wolbachia株十分相近或完全相同,提示它们可能是相近或相同的株。     

我国尖音库蚊复合组蚊虫的杂交及其与Wolbachia感染的关系

为了了解我国尖音库蚊复合组蚊虫间杂交卵的不孵化现象和明确该现象与共生微生物Wolbachia感染的关系,对该复合组实验室种群4个亚种进行了笼内杂交和抗生素处理后的杂交。试验表明: 在复合组蚊虫中骚扰库蚊Culex pipiens molestus与淡色库蚊Cx. Pipiens pallens、致倦库蚊Cx. Ipiens quinquefasciatus与尖音库蚊Cx. Pipiens pipiens之间存在有单向胞质不融合现象,骚扰库蚊的雄虫与尖音库蚊、致倦库蚊和淡色库蚊的雌虫杂交卵的孵化率分别为0.06%、0.46%和0.19%;该胞质不融合现象可以通过抗生素处理而消除,处理后骚扰库蚊雄虫与其余3个亚种雌虫F₃杂交卵的孵化率均有提高,分别为89.49%（t=3.90×10^-28＜t_0.01=2.704）、23.39%（t=9.15×10^-7＜t_0.01=2.660_）和22.27%（t=5.08×10^-4＜t_0.01=2.750）,并可因抗生素处理而产生新的不融合类型。     

Wolbachia在我国广赤眼蜂种群内的感染

Wolbachia是广泛分布于节肢动物生殖组织内的一类细胞内共生菌,它属于原细菌的α亚类,能够通过调控寄主的生殖活动而促进其在寄主种群中的扩散。通过对wsp 基因的克隆及PCR-RFLP分析确定了Wolbachia在我国广赤眼蜂种群内的存在,并发现有2种Wolbachia 菌系的感染,命名为wEvaA和wEvaB。经过克隆分离得到了这2种Wolbachia的wsp基因序列,在GenBank的登录号为AY390279和 AY390280 ,并由基于wsp基因的聚类树中发现,这两种Wolbachia菌系均属于A组。     

几种蚊虫线粒体DNA-16SrRNA序列及其相互关系的研究

测定我国尖音库蚊复合组4亚种（尖音库蚊、淡色库蚊、致倦库蚊和骚扰库蚊）、三带喙库蚊、白纹伊蚊和中华按蚊的线粒体DNA 16S rRNA基因（mtDNA-16S rRNA)序列,发现我国尖音库蚊复合组4亚种mtDNA-16S rRNA序列基本一致,长度为555bp(554bp),GC含量为25．41％。该序列与其他3种蚊虫在种间存在差异,与三带喙库蚊的种间差异为0．54％;与白纹伊蚊的种间差异为5．77％;与中华按蚊的种间差异为9．62％。分子系统关系分析表明该序列与传统分类系统的同属或同亚科种类近似性相一致。     

不同生物型烟粉虱体内Wolbachia共生菌的检测及其系统树分析

Wolbachia是广泛分布于节肢动物体内一类共生细菌,它能够通过多种机制调控宿主的生殖方式。近年来的研究表明,Wolbachia与许多外来生物的成功入侵相关。本文利用长PCR的方法特异扩增了不同生物型烟粉虱（共24个种群）体内Wolbachia的wsp基因,结果发现B型和Q型烟粉虱入侵种群体内均未检测到Wolbachia,而在非B/Q型的浙江种群和肯尼亚种群体内检测到了Wolbachia。对该wsp基因进行测序并和已知序列进行同源性分析发现,浙江烟粉虱种群的Wolbachia属于B组Con/Rug亚种群,而肯尼亚种群属于B组Btab1亚种群。Wolbachia的存在与否可能与烟粉虱的成功入侵有一定的关系。图2表2参23     

中国部分地区柑桔木虱体内感染Wolbachia的检测及其系统发育分析

Wolbachia是一种广泛分布于节肢动物体内的共生细菌, 该共生菌经寄主卵的细胞质传播并参与多种寄主生殖活动调控, 对节肢动物的物种进化有着重要意义并可能应用于害虫的生物防治。本研究以柑桔黄龙病（Huanglongbing, HLB）的主要传播媒介——柑桔木虱Diaphorina citri为研究对象, 通过Wolbachia的16S rDNA、 细胞分裂基因（ftsZ）、 表面蛋白基因（wsp）的特异引物对来自于中国广西北海、 广西柳州、 广东深圳、 广东阳春、 福建福州5个地区的柑桔木虱进行PCR扩增, 并对扩增产物进行克隆测序, 与GenBank中相关序列进行比对分析, 建立了柑桔木虱共生Wolbachia的系统发育树。结果显示上述5个地区的柑桔木虱均存在Wolbachia感染, 通过Wolbachia的16S rDNA, ftsZ基因和wsp基因DNA序列的系统发育分析表明, 5个地区的柑桔木虱体内的Wolbachia均属于B组; 基于wsp基因的系统发育分析进一步表明5个地区的亚洲柑桔木虱体内的Wolbachia属于B组的Con亚组, 且不同地区柑桔木虱体内的Wolbachia的16S rDNA, ftsZ基因和wsp基因序列差异不明显。研究结果为认识柑桔黄龙病传播媒介昆虫的进化及其综合防治提供了重要的参考依据。     

尖音库蚊复合组杂交子一代4龄幼虫形态性状的研究

对我国尖音库蚊复合组（Culex pipiens complex）中的尖音库蚊Cx. Pipiens pipiens、淡色库蚊Cx. Pipiens pallens和致倦库蚊Cx. Pipiens quinquefasciatus之间杂交的F₁代幼虫形态进行了方差和典型变量分析,结果发现杂交组F₁代4龄幼虫在呼吸管指数、1-S第1对毛和第2对毛的分枝数3个性状上与亲本有明显的差异,而且介于两亲本之间。     

椿叶花椒果实精油对两种蚊虫的生物活性及成分分析

为筛选环保型的植物源灭蚊活性物质, 研究了椿叶花椒 Zanthoxylum ailanthoides 果实精油对白纹伊蚊Aedes albopictus和致倦库蚊Culex pipiens quinquefasciatus成蚊的熏杀活性, 并测试了精油对这两种蚊幼虫的毒杀效果。此外, 采用GC-MS分析了该精油的化学成分。 结果表明：在102.145 μg·cm^-3熏杀剂量下, 精油对白纹伊蚊和致倦库蚊成蚊的KT50值分别为2.788和3.505 min, 24 h致死率分别为100%和97.53%。该精油对白纹伊蚊和致倦库蚊4龄幼虫的LC50值分别为45.237和20.759 mg·L^-1。从椿叶花椒果实精油中共鉴定出14种化合物, 其中2-十一酮为主要成分, 相对含量为89.86%。结果说明椿叶花椒果实精油对两种蚊虫均有明显的杀虫活性, 具有开发成植物源灭蚊剂的潜力。     

二斑叶螨体内感染的Wolbachia的wsp基因序列测定与分析

应用Wolbachia的wsp基因特异引物, 通过PCR扩增法检测了二斑叶螨Tetranychus urticae Koch 12个地理种群（辽宁兴城、河北昌黎、湖南长沙、河南郑州、江苏徐州、甘肃兰州、云南昆明、山东泰安、新疆石河子、宁夏银川、陕西眉县和上海）感染Wolbachia的状况。结果表明,12个地理种群全部感染了Wolbachia。克隆分离得到了它们的wsp基因序列,在GenBank的登录号依此为： AY585714、 AY585712、AY785376、AY785375、AY585713、AY785374、AY785371、AY712954、AY712955、AY785372、AY785377和AY785373。对序列进行系统发育分析,发现它们全部与B大组Ori组的Wolbachia株系十分相近或完全相同。总体来看,它们的聚类关系基本上呈平行交叉分布,在不同种群之间没有构成明显的地理分布聚集性。     

济南市蚊类调查报告

1.本文报告了济南市蚊虫种类,孳生地,栖息场所,幼虫发现季节,尖音库蚊淡色变种成虫、幼虫及白纹伊蚊幼虫的密度调查结果。 2.过去在济南曾有人作过蚊类的初步观察,而此次调查较为普遍。共采获蚊类标本3属11种。其中吉浦伊蚊、拟态库蚊、二带喙库蚊、褐尾库蚊4种为济南市新发现之蚊种。其中褐尾库蚊在山东省为首次发现。 3.3属蚊虫中以尖音库蚊淡色变种为最普遍,其与居民之关系亦最为密切;次之为白纹伊蚊与中华按蚊。 4.尖音库蚊淡色变种之幼虫从5月上旬开始出现,到8月中旬达最高峯,9、10月份渐少,到11月下旬绝迹。白纹伊蚊之幼虫,8月上旬密度最高,9月中旬绝迹。各种蚊幼 之密度随雨量之增减而消长。     

竹叶花椒果实精油对两种蚊虫的毒杀活性研究

采用浸液法测试了竹叶花椒果实的水蒸汽蒸馏精油对白纹伊蚊和致倦库蚊幼虫的毒杀效果,并用三角瓶密闭熏蒸法研究了精油对这两种成蚊的熏杀活性;此外,采用GC-MS分析了该精油的化学成分。研究结果:(1)精油对白纹伊蚊和致倦库蚊的Ⅰ、Ⅱ、Ⅲ、Ⅳ龄期幼虫及蛹的LC50值分别为25.634/61.472、31.763/76.431、52.356/110.172、258.497/121.884和198.263/162.048mg.L-1;(2)精油对白纹伊蚊和致倦库蚊成蚊的LC50值分别为24.957和29.517μg.cm-3;(3)在147.52μg.cm-3熏杀剂量下,精油对白纹伊蚊和致倦库蚊成蚊的KT50值分别为3.493和2.993min,24h致死率均为100%;(4)共鉴定出18种化合物,其中萜烯类10种,占精油总量的67.122%,为竹叶花椒果实精油的主要成分。竹叶花椒果实精油对白纹伊蚊和致倦库蚊均有明显的致死作用,且作用速度快,具有开发为植物源杀蚊剂的潜力。     

致倦库蚊对登革Ⅱ型病毒的中肠感染屏障作用

为探讨致倦库蚊对登革Ⅱ型病毒的中肠感染屏障作用,通过病毒分离、逆转录聚合酶链反应、透射电镜等技术进行了相关研究。结果表明：吸食感染性血液后,登革Ⅱ型病毒能侵染白纹伊蚊中肠上皮细胞并大量复制,但不能侵染致倦库蚊中肠上皮细胞。以上研究证明致倦库蚊对登革Ⅱ型病毒存在中肠感染屏障。     

Results of a survey to detect the mosquito Aedes albopictus in the French Riviera.

A programme of surveillance was initiated in 1992, in the French Riviera, to detect a possible introduction of Aedes albopictus from Italy and to prevent nuisances caused by mosquitoes in the touristic localities of the C?te d'Azur. In five years, numerous mosquito breeding places have been located. Nine species have been collected: Anopheles claviger, An. plumbeus, Aedes geniculatus, Ae. vittatus, Culex hortensis, Cx. impudicus, Cx. pipiens, Culiseta fumipennis, Cs. longiareolata but no Ae. albopictus was found. Nuisances were mainly due to hypogean populations of Cx. pipiens. Breeding places in urban sites have been controlled or suppressed. The discovery of an important larval population of An. plumbeus in an urban area might further stress the importance of this species already suspected to transmit indigenous malaria in cities.     

白纹伊蚊和埃及伊蚊对基孔肯雅病毒的易感性和传播性的研究

用４株基孔肯雅病毒经口感染白纹伊蚊和埃及伊蚊,进行了易感性和传播性的研究。结果表明,这两种蚊虫对基孔肯雅病毒易感。无论白纹伊蚊或埃及伊蚊,感染后第５－６天即可通过吸血将病毒传播给乳鼠,至第８－１３天,传播率可高达５５．５５％－１００％。感染蚊亦可经叮咬将病毒传播给小鸡。埃及伊蚊的易感性和传播率高于白纹伊蚊。实验还发现,不同来源毒株之间存在一定差异,如分离自云南白纹伊蚊的Ｍ８１株的感染率和传播率均高于其它毒株。这些结果表明,白纹伊蚊和埃及伊蚊在基孔肯雅病毒的保存和传播中起重要作用。     

Wolbachia and cytoplasmic incompatibility in the California Culex pipiens mosquito species complex: parameter estimates and infection dynamics in natural populations

Before maternally inherited bacterial symbionts like Wolbachia, which cause cytoplasmic incompatibility (CI; reduced hatch rate) when infected males mate with uninfected females, can be used in a program to control vector-borne diseases it is essential to understand their dynamics of infection in natural arthropod vector populations. Our study had four goals: (1) quantify the number of Wolbachia strains circulating in the California Culex pipiens species complex, (2) investigate Wolbachia infection frequencies and distribution in natural California populations, (3) estimate the parameters that govern Wolbachia spread among Cx. pipiens under laboratory and field conditions, and (4) use these values to estimate equilibrium levels and compare predicted infection prevalence levels to those observed in nature. Strain-specific PCR, wsp gene sequencing, and crossing experiments indicated that a single Wolbachia strain infects Californian Cx. pipiens. Infection frequency was near or at fixation in all populations sampled for 2 years along a >1000-km north-south transect. The combined statewide infection frequency was 99.4%. Incompatible crosses were 100% sterile under laboratory and field conditions. Sterility decreased negligibly with male age in the laboratory. Infection had no significant effect on female fecundity under laboratory or field conditions. Vertical transmission was >99% in the laboratory and approximately 98.6% in the field. Using field data, models predicted that Wolbachia will spread to fixation if infection exceeds an unstable equilibrium point above 1.4%. Our estimates accurately predicted infection frequencies in natural populations. If certain technical hurdles can be overcome, our data indicate that Wolbachia can invade vector populations as part of an applied transgenic strategy for vector-borne disease reduction.     

Susceptibility of Aedes aegypti and Aedes albopictus larvae to Ascogregarina culicis and Ascogregarina taiwanensis (Apicomplexa: Lecudinidae) from Florida

The susceptibility of Aedes aegypti to Ascogregarina culicis and Aedes albopictus to Ascogregarina taiwanensis was examined with mosquito and parasite strains from Tampa, FL. When each host was bioassayed with its natural gregarine, the infection intensity indicated that Ae. aegypti was 59% more susceptible to A. culicis (87 gamonts/larva) than Ae. albopictus to A. taiwanensis (47 gamonts/larva). Infections in single and mixed host populations exposed to 100 oocysts/larva of one and both parasites demonstrated that Ae. aegypti harbors higher A. culicis gamont loads than Ae. albopictus of A. taiwanensis. In dual gregarine exposures of single host populations, the A. culicis infection intensity in Ae. aegypti was reduced by approximately 50%. A. taiwanensis exhibited the same capability of infecting Ae. albopictus in single and dual exposures. In mixed host populations there were no cross infections, but A. taiwanensis in Ae. albopictus produced an infection intensity of approximately 70% lower than that of A. culicis in Ae. aegypti.     

Generation of a novel Wolbachia infection in Aedes albopictus (Asian tiger mosquito) via embryonic microinjection

Genetic strategies that reduce or block pathogen transmission by mosquitoes are being investigated as a means to augment current control measures. Strategies of vector suppression and replacement are based upon intracellular Wolbachia bacteria, which occur naturally in many insect populations. Maternally inherited Wolbachia have evolved diverse mechanisms to manipulate host insect reproduction and promote infection invasion. One mechanism is cytoplasmic incompatibility (CI) through which Wolbachia promotes infection spread by effectively sterilizing uninfected females. In a prior field test, releases of Wolbachia-infected males were used to suppress a field population of Culex pipiens. An additional strategy would employ Wolbachia as a vehicle to drive desired transgenes into vector populations (population replacement). Wolbachia-based population suppression and population replacement strategies require an ability to generate artificial Wolbachia associations in mosquitoes. Here, we demonstrate a technique for transferring Wolbachia (transfection) in a medically important mosquito species: Aedes albopictus (Asian tiger mosquito). Microinjection was used to transfer embryo cytoplasm from a double-infected Ae. albopictus line into an aposymbiotic line. The resulting mosquito line is single-infected with the wAlbB Wolbachia type. The artificially generated infection type is not known to occur naturally and displays a new CI crossing type and the first known example of bidirectional CI in Aedes mosquitoes. We discuss the results in relation to applied mosquito control strategies and the evolution of Wolbachia infections in Ae. albopictus.     

Wolbachia induces density-dependent inhibition to dengue virus in mosquito cells

Wolbachia is a maternal transmitted endosymbiotic bacterium that is estimated to infect up to 65% of insect species. The ability of Wolbachia to both induce viral interference and spread into mosquito vector population makes it possible to develop Wolbachia as a biological control agent for dengue control. While Wolbachia induces resistance to dengue virus in the transinfected Aedes aegypti mosquitoes, a similar effect was not observed in Aedes albopictus, which naturally carries Wolbachia infection but still serves as a dengue vector. In order to understand the mechanism of this lack of Wolbachia-mediated viral interference, we used both Ae. albopictus cell line (Aa23) and mosquitoes to characterize the impact of Wolbachia on dengue infection. A serial of sub-lethal doses of antibiotic treatment was used to partially remove Wolbachia in Aa23 cells and generate cell cultures with Wolbachia at different densities. We show that there is a strong negative linear correlation between the genome copy of Wolbachia and dengue virus with a dengue infection completely removed when Wolbacha density reaches a certain level. We then compared Wolbachia density between transinfected Ae. aegypti and naturally infected Ae. albopictus. The results show that Wolbachia density in midgut, fatbody and salivary gland of Ae. albopictus is 80-, 18-, and 24-fold less than that of Ae. aegypti, respectively. We provide evidence that Wolbachia density in somatic tissues of Ae. albopictus is too low to induce resistance to dengue virus. Our results will aid in understanding the mechanism of Wolbachia-mediated pathogen interference and developing novel methods to block disease transmission by mosquitoes carrying native Wolbachia infections.     

Hypervariable prophage WO sequences describe an unexpected high number of Wolbachia variants in the mosquito Culex pipiens

Wolbachia are maternally inherited endosymbiotic bacteria that infect many arthropod species and may induce cytoplasmic incompatibility (CI) resulting in abortive embryonic development. Among all the described host species, mosquitoes of the Culex pipiens complex display the highest variability of CI crossing types. Paradoxically, searches for polymorphism in Wolbachia infecting strains and field populations hitherto failed or produced very few markers. Here, we show that an abundant source of the long-sought polymorphism lies in WO prophage sequences present in multiple copies dispersed in the genome of Wolbachia infecting C. pipiens (wPip). We identified up to 66 different Wolbachia variants in C. pipiens strains and field populations and no occurrence of superinfection was observed. At least 49 different Wolbachia occurred in Southern Europe C. pipiens populations, and up to 10 different Wolbachia were even detected in a single population. This is in sharp contrast with North African and Cretan samples, which exhibited only six variants. The WO polymorphism appeared stable over time, and was exclusively transferred maternally. Interestingly, we found that the CI pattern previously described correlates with the variability of Gp15, a prophage protein similar to a bacterial virulence protein. WO prophage sequences thus represent variable markers that now open routes for approaching the molecular basis of CI, the host effects, the structure and dynamics of Wolbachia populations.