莽山部分蜘蛛Wolbachia的感染及其wsp基因的序列分析

沃尔巴克氏体(Wolbachia)是一类广泛存在于节肢动物和线虫体内的胞内共生菌,能调控宿主的生殖方式。现用PCR方法检测了莽山自然保护区跳蛛科(Salticidae)、漏斗蛛科(Agelenidae)和狼蛛科(Lycosidae)部分蜘蛛种类的Wolbachia感染状况。结果证实跳蛛科蓝翠蛛(Siler cupreus)、狼蛛科小雾豹蛛(Pardosa mionebulosa)和漏斗蛛科指形龙角蛛(Draconarius digitusiformis)受Wolbachia感染,其感染率分别为3.7%、8.6%和40.0%。同时,基于Wolbachia的wsp基因序列构建系统发育树,结果表明:D.digitusiformis感染的Wolbachia属于A群,P.mionebulosa感染的Wolbachia属于B群,S.cupreus感染的Wolbachia属于G群;而且同一科的蜘蛛感染的Wolbachia亲缘关系较远,而不同科蜘蛛感染的Wolbachia系统发育关系较近,这可能与Wolbachia的水平传播有关。     

黄金肥蛛体内Wolbachia的感染检测及wsp基因序列分析

细胞质共生细菌Wolbachia能引起节肢动物的胞质不亲和、孤雌生殖、遗传雄性的雌性化和杀雄等。本研究采用Wolbachia的wsp基因的通用引物,通过PCR扩增法检测了7种蜘蛛体内Wolbachia的感染情况,发现在黄金肥蛛体内存在Wolbachia感染。对黄金肥蛛体内的wsp基因进行了克隆与测序,并利用所测序列与其他已发表的wsp基因序列建立系统树,结果表明其体内Wolbachia属于A大组．     

显微注射青霉素G获得单感染Wolbachia和单感染Cardinium白背飞虱品系

Wolbachia和Cardinium都是广泛存在于节肢动物体内的一类母系遗传的共生细菌, 可以通过不同方式操纵寄主的生殖行为。Wolbachia和Cardinium感染同一寄主在自然界比较常见, 但是在某些可以同时感染Wolbachia和Cardinium的寄主中其单感染品系较难发现。本研究检测了云南文山(YN)、 海南三亚(HN)这2个不同地理种群中Wolbachia和Cardinium的感染情况; 以双感染Wolbachia和Cardinium的白背飞虱Sogatella furcifera海南种群为实验材料, 运用显微注射方法对双感染Wolbachia和Cardinium的白背飞虱若虫注射不同浓度青霉素G以获得单感染品系。结果表明： 白背飞虱自然种群中单感染Wolbachia比率极低, 本实验用到的海南种群未检测到单感染Wolbachia成虫; 通过显微注射青霉素G的方法可以从白背飞虱双感染品系中筛选获得单感染品系, 当青霉素G注射浓度为0.2%(w/v), 注射龄期为5龄时得到单感染品系效率最高; F5代的检测结果显示显微注射得到的单感染品系可以稳定遗传。本研究为获得单感染品系白背飞虱提供了快捷方法, 同时为其他双感染Wolbachia和Cardinium节肢动物不同感染品系的筛选提供参考。     

共生菌Wolbachia在中国二斑叶螨种群中的扩散规律

共生菌Wolbachia在中国二斑叶螨Tetranychus urticae Koch中分布广泛,所有的地理种群中均感染Wolbachia。以二斑叶螨湖南长沙(HN),辽宁兴城(LN)和江苏徐州(JS)3个地理种群为实验材料,经筛选获得100%感染和不感染Wolbachia的品系后,人工设置Wolbachia感染率为50%的品系,通过PCR技术检测二斑叶螨连续世代Wolbachia感染率动态变化,研究Wolbachia在二斑叶螨种群中的扩散规律。结果表明:3个地理种群的垂直传播效率都为100%;HN种群Wolbachia感染率上升速度最快,F7代达到100%感染;LN种群F12达到100%感染;而JS地理种群中Wolbachia感染率速度上升最慢,在F20代达到100%感染,其后感染率均能稳定在100%。LN种群Wolbachia通过诱导胞质不亲和的策略,JS种群的Wolbachia通过提高寄主适合度的策略,而HN种群Wolbachia则通过诱导胞质不亲和与提高寄主适合度两者相结合的策略,最终达到在二斑叶螨中维持感染状态并扩散传播的目的。本研究结果为今后利用Wolbachia的扩散规律控制有害生物及疾病传播提供了基础。     

安荔赤眼蜂在中国野外首次发现及其体内Wolbachia的检测

【目的】为丰富赤眼蜂Trichogramma的种类资源,明确野外新采集的一种赤眼蜂的种类,探明该赤眼蜂所感染Wolbachia的类型。【方法】采用挂米蛾Corcyra cephalonica卵卡法在华南农业大学树木园诱集到两批赤眼蜂,通过形态鉴定和PCR扩增ITS2序列并测序分析的分子鉴定手段对野外采集的赤眼蜂材料进行种类鉴定;通过PCR扩增Wolbachia的外膜蛋白基因(wsp)序列,检测赤眼蜂体内Wolbachia的感染情况;通过PCR扩增wsp序列和多位点序列分型(multilocus sequence typing, MLST)对检测到的赤眼蜂体内的Wolbachia进行同源性分析。【结果】所诱集到的两批赤眼蜂均被鉴定为安荔赤眼蜂Trichogramma oleae Voegelé & Pointel,体内Wolbachia的感染率达100%。该Wolbachia株系与安荔赤眼蜂(前南斯拉夫品系)、短管赤眼蜂T. pretiosum(乌拉圭品系)以及T. deion(荷兰品系)体内Wolbachia亲缘关系较近,属于B超组Sib亚组,对应MLST序列型为ST486。【结论】安荔赤眼蜂T. oleae为中国野外首次发现,是完全感染Wolbachia的产雌孤雌生殖品系。本研究为害虫生物防治提供了一种新的天敌种类资源,并为进一步探明Wolbachia与赤眼蜂的互作提供了研究材料。     

铃木氏果蝇不同地理种群中Wolbachia的检测和系统发育分析

铃木氏果蝇Drosophila suzukii是原产于东南亚地区的重要果树害虫, 近年来传入北美和欧洲等地区造成严重的危害。本研究利用Wolbachia的16S rDNA和wsp基因特异引物（分别为16S-F/16S-R和81F/691R）对铃木氏果蝇7个地理种群（中国的5个种群、 韩国的1个种群和美国的1个种群）的Wolbachia进行了PCR检测并对检测结果进行了比较; 对感染个体体内Wolbachia的16S rDNA基因片段进行测序, 确定了我国铃木氏果蝇体内Wolbachia的分类地位。基于Wolbachia的16S rDNA基因特异引物检测结果发现, 我国5个铃木氏果蝇种群广泛感染Wolbachia（感染率36.7%~80.0%）, 而韩国和美国2个种群均未检测到该菌的感染。而利用wsp基因特异引物无法检测到该菌。基于Wolbachia的16S rDNA基因构建系统发育树表明, 我国铃木氏果蝇种群感染的Wolbachia全部属于A组。这些结果为研究Wolbachia感染对铃木氏果蝇生物学及生态学的影响奠定了基础。     

Wolbachia引起黑腹果蝇产生“修饰-挽救”因子的初步探究

沃尔巴克氏体(Wolbachia)是一种广泛分布于昆虫体内的共生细菌,对宿主的生殖具有多种调控作用,其中,最常见的调控方式称为细胞质不亲和(Cytoplasmic Incompatibility,CI),即感染了Wolbachia的雄性宿主与未感染或感染不同品系Wolbachia的雌性宿主交配后,胚胎在发育早期死亡。关于CI产生的原因,目前人们认为是一种"精子修饰-卵子挽救"机制,本研究初步探索了黑腹果蝇中参与"修饰-挽救"机制的重要因子。本研究采用定量RT-PCR方法,检测了3种基因:yem-α(yemanuclein)、uif(uninflatable)、Prosα4T1(proteasomeα4 subunit,Testis-specific 1)在感染和未感染果蝇中的表达差异。结果发现,Wolbachia感染能够显著上调雌蝇中yem-α和uif的表达,并且显著下调雄蝇中yem-α的表达。尽管Prosα4T1在雄性果蝇体内特异表达,但是Wolbachia感染对Prosα4T1表达无明显影响。因此,yem-α可能是Wolbachia产生"修饰-挽救"机制的重要因子之一,Wolbachia可能通过调控果蝇体内yem-α的表达,影响卵子发生和精子发生进程,导致CI的产生。     

我国三地区黑腹果蝇中Wolbachia的系统发育关系及其对宿主生殖的影响

【目的】Wolbachia 是广泛存在于节肢动物和丝状线虫体内的一类共生菌, 能够以多种方式对宿主产生影响。精卵细胞质不亲和（CI）是其引起的最普遍的表型, 即感染Wolbachia的雄性宿主与未感染或感染不同品系的雌性宿主交配后, 不能产生后代或后代极少, 而感染同品系Wolbachia的雌雄宿主交配后则能正常产生后代。我们前期研究发现, 湖北武汉、 云南六库和天津3个地区黑腹果蝇Drosophila melanogaster被Wolbachia感染。本研究旨在明确这3个地区黑腹果蝇中Wolbachia的系统发育关系及其对宿主生殖的影响。【方法】利用Clustal X软件对Wolbachia的wsp基因序列进行比对, 利用MEGA软件构建系统发育树。采用多位点序列分型（MLST）的方法对Wolbachia进行分型。通过区内交配和区之间杂交的方式研究不同地区黑腹果蝇体内Wolbachia 的关系及其对果蝇生殖的影响。【结果】湖北武汉、 云南六库和天津3个地区黑腹果蝇中感染的Wolbachia都是属于A大组的Mel亚群。这3个地区果蝇感染的Wolbachia的序列类型（ST）不同, Wolbachia之间存在一定的差异。湖北武汉和天津果蝇中的Wolbachia能引起强烈的CI表型, 而云南六库果蝇中的Wolbachia引起的CI强度相对较弱。武汉果蝇中Wolbachia不能完全挽救天津果蝇中Wolbachia引起的CI表型, 而天津果蝇中Wolbachia也不能完全挽救武汉果蝇中Wolbachia引起的CI表型。【结论】武汉和天津地区黑腹果蝇中的Wolbachia可能距离较远。Wolbachia的长期共生可能对黑腹果蝇的进化产生了一定的影响, 湖北武汉与云南六库的黑腹果蝇中感染的Wolbachia属于不同的序列类型, 这2个地区的黑腹果蝇已发生了一定的分歧, 产生了一定的生殖隔离。     

雌性截形叶螨中Wolbachia抑制Spiroplasma

【目的】探究截形叶螨中Wolbachia与Spiroplasma这两种共生菌之间的竞争关系,评估叶螨交配类型对其寿命的影响。【方法】对不同共生菌感染类型的雌螨在不同交配类型以及不同日龄的情况下其体内共生菌滴度进行定量PCR检测,对不同交配类型的雌螨寿命进行记录,利用SPSS19.00(IBM)分析交配类型、成螨日龄对共生菌滴度的影响,以及叶螨交配类型对寿命的影响。【结果】不同交配类型以及不同日龄的雌螨,其体内共生菌滴度存在显著差异;对于Wolbachia滴度,单感染Wolbachia雌螨与双感染在4日龄、10日龄有差异;对于Spiroplasma滴度,单感染Spiroplasma雌螨均显著高于双感染;双感染中Wolbachia滴度显著高于Spiroplasma;双感染雌螨与同一感染或不感染品系雄螨交配后的存活时间长于与单感染Spiroplasma雄螨交配,单感染Spiroplasma雌螨与同一感染或不感染品系雄螨交配后的存活时间长于与双感染雄螨交配。【结论】交配类型和成螨日龄影响共生菌滴度;Wolbachia的存在抑制Spiroplasma;交配类型会对雌螨寿命产生影响。     

基于wsp基因的叶螨体内Wolbachia株系的多样性与重组分析

胞内共生菌Wolbachia能对多种叶螨产生生殖调控作用.为更好地筛选有潜在应用价值的Wolbachia株系,本研究应用PCR技术对自然种群的截形叶螨Tetranychus truncatus、二斑叶螨T.urticae、神泽叶螨T.kanzawai 和山楂叶螨Amphitetranychus viennensis体内的Wolbachia感染情况进行检测,并对Wolbachia的wsp基因进行序列分析和基因重组检测.结果表明,叶螨中的Wolbachia株系具有较高的遗传多样性,其中截形叶螨感染两种分化较大的株系.不同叶螨感染特有的Wolbachia株系说明Wolbachia与其宿主存在一定的协同进化关系.二斑叶螨和截形叶螨感染同一株系的Wolbachia,可能由于水平传播造成.同时,不同株系Wolbachia的wsp基因间普遍存在着基因重组现象.     

三色书虱体内共生微生物Wolbachia的wsp基因的分子检测

采用常规PCR和巢式PCR方法对三色书虱Liposcelis tricolor体内的共生微生物Wolbachia的wsp基因进行分子检测;通过Wolbachia的通用引物以及A、B亚群引物分别比较了常规PCR和巢式PCR对wsp基因扩增的灵敏性。从三色书虱体内扩增出了610bp的Wolbachia的wsp基因片段,500bp的WolbachiaA亚群的wsp基因片段和450bp的Wolbachia B亚群的wsp基因片段。扩增结果说明三色书虱被A和B两个亚群的Wolbachia混合感染;巢式PCR比常规PCR更为灵敏。     

苹果蠹蛾体内wolbachia wsp基因克隆与序列分析

【目的】对苹果蠹蛾Cydia pomonella L.体内共生菌Wolbachia进行分子生物学鉴定,确定该虫体内Wolbachia的进化位置,为进一步探讨Wolbachia对其生殖作用的调控机制提供理论依据。【方法】应用Wolbachia的wsp基因特异引物,通过PCR扩增法检测了苹果蠹蛾10个地理种群(新疆伊犁、吐鲁番、和田、石河子、奎屯、哈密、库尔勒、阿拉尔、喀什、和甘肃张掖)感染Wolbachia的状况,并对阿拉尔种群体内的Wolbachia的wsp基因进行测序和序列分析。【结果】苹果蠹蛾10个地理种群全部感染了tWolbachia,利用wsp基因的特异性引物从阿拉尔种群体内扩增出了617 bp的Wolbachia的wsp基因片段(GenBank登录号为KC832324),系统发育分析结果表明,苹果蠹蛾体内感染的Wolbachia属于A群Dor亚群,与锤角细蜂亲缘关系较近。【结论】苹果蠹蛾体内普遍感染了Wolbachia,属于A群Dor亚群。     

Prevailing triple infection with Wolbachia in Callosobruchus chinensis (Coleoptera: Bruchidae)

Prevailing triple infection with three distinct Wolbachia strains was identified in Japanese populations of the adzuki bean beetle, Callosobruchus chinensis. When a polymerase chain reaction (PCR) assay was conducted using universal primers for ftsZ and wsp, Wolbachia was detected in all the individuals examined, 288 males and 334 females from nine Japanese populations. PCR-restriction fragment length polymorphism (RFLP) analysis of cloned wsp gene fragments from single insects revealed that three types of wsp sequences coexist in the insects. Molecular phylogenetic analysis of the wsp sequences unequivocally demonstrated that C. chinensis harbours three phylogenetically distinct Wolbachia, tentatively designated as wBruCon, wBruOri and wBruAus, respectively. Diagnostic PCR analysis using specific primers demonstrated that, of 175 males and 235 females from nine local populations, infection frequencies with wBruCon, wBruOri and wBruAus were 100%, 96.3% and 97.0%, respectively. As for the infection status of individuals, triple infection (93.7%) dominated over double infection (6.1%) and single infection (0.2%). The amounts of wBruCon, wBruOri and wBruAus in field-collected adult insects were analysed by using a quantitative PCR technique in terms of wsp gene copies per individual insect. Irrespective of original populations, wBruCon and wBruOri (107 -108 wsp copies/insect) were consistently greater in amount than wBruAus (106 -107 wsp copies/insect), suggesting that the population sizes of the three Wolbachia strains are controlled, although the mechanism is unknown. Mating experiments suggested that the three Wolbachia cause cytoplasmic incompatibility at different levels of intensity.     

Super-infections of Wolbachia in byturid beetles and evidence for genetic transfer between A and B super-groups of Wolbachia

Wolbachia are maternally inherited bacteria responsible for altering host reproduction. The two main groups found in insects, A and B, are based on molecular characterization using ribosomal, ftsZ, wsp (Wolbachia surface protein) or groE genes. We have used the wsp and ftsZ genes to study Wolbachia in byturid beetles. Byturus affinis contained a single copy of the ftsZ gene which grouped with A ftsZ sequences and a single copy of the wsp gene which grouped with B wsp sequences. This suggests that genetic exchange between A and B groups has occurred in the Wolbachia of this beetle. FtsZ and wsp sequences that were identical or nearly identical to those of B. affinis were found in B. tomentosus, suggesting that it also contains the same recombinant Wolbachia genotype. Most other byturids had more than one wsp sequence with at least one from the A and B groups, suggesting multiple copies of bacterial genes or multiple infections. B. ochraceus and B. unicolor both had four distinct wsp gene sequences. All the byturids had a closely related A wsp sequence and most a closely related B wsp sequence. Therefore, there appears to be an association between specific A and B wsp types.     

Diversity and phylogeny of Wolbachia infecting Bactrocera dorsalis (Diptera: Tephritidae) populations from China

Wolbachia are a common and widespread group of symbiotic bacteria found in the reproductive tissues of arthropods. Bactrocera dorsalis (Hendel) is an important pest causing considerable economic losses of fruits and vegetables in several southern provinces of China. In this study, polymerase chain reaction (PCR) with general Wolbachia surface protein (wsp) primers was used to test the presence of Wolbachia in 1,500 individuals of B. dorsalis from five geographical populations of China. We detected 19 individuals of B. dorsalis infected by Wolbachia, and the infection rates of different populations varied. Comparison of wsp gene sequences from 19 individuals and search of the GenBank identified four new sequences, probably representing four Wolbachia strains. Sequence comparison showed that the four Wolbachia strains from B. dorsalis in China belonged to three groups (Kue, Mel, and Cuc). Phylogenetic analysis of the wsp sequences suggests that geographical isolation of Wolbachia exists among the populations of B. dorsalis in China, and gene flow of Wolbachia might have occurred between B. dorsalis populations of China and Thailand. Phylogenetic analysis performed on the host mitochondrial cytochrome oxidase I (COI) gene and wsp gene suggests that host has coevolved with Wolbachia.     

小麦和大豆蚜虫中内共生菌Wolbachia的感染检测和系统发育分析

Wolbachia是一类在节肢动物中广泛感染的胞内共生菌。为了了解其在我国蚜虫中的感染情况, 本研究通过扩增wsp基因片段对采集自我国多个地区的3种小麦蚜虫（荻草谷网蚜Sitobion miscanthi、 麦二叉蚜Schizaphis graminum和禾谷缢管蚜Rhopalosiphum padi）和1种大豆蚜虫（大豆蚜Aphis glycines）样品进行了内共生菌Wolbachia的感染检测。结果显示： 3种小麦蚜虫中均未检测出Wolabchia。大豆蚜也仅在采集自北京和杭州的种群中发现了Wolbachia的感染, 感染率分别为95.8%和22.9%, 并且所检测的个体均为单株系感染。wsp基因序列的比对分析显示, 大豆蚜感染的Wolbachia株系与多个亲缘关系较远的昆虫物种中所感染的Wolbachia株系间具有高度一致的基因序列。wsp基因序列构建的系统发育关系和序列一致性均表明大豆蚜感染的Wolbachia株系属于B大组CauB组。本研究为今后探讨Wolbachia在我国蚜虫中的寄主范围和株系多样性提供了数据支持。     

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。     

几种常见桔园螨类Wolbachia基因检测与序列分析

应用共生菌沃尔巴克氏体Wolbachia的wsp、ftsZ和16SrDNA基因特异引物,通过PCR扩增法对采自江西省南昌、九江和赣州等3地桔园内的东方钝绥螨、江原钝绥螨、尼氏真绥螨、巴氏新小绥螨、桔全爪螨和酢浆草如叶螨等6种常见螨类进行了检测。结果表明,在采自南昌桔园内的酢浆草如叶螨体内检测到Wolbachia感染,其wsp、ftsZ和16SrDNA基因长度为593、758和896bp,Genbank登录号依次为GQ162482、GQ162483和GQ162484,而其他5种螨体内均未检测出Wolbachia的感染;同时通过对酢浆草如叶螨体内感染的Wolbachia的wsp基因序列进行系统发育分析,得出其与B大组的Epo组的Wolbachia株十分相近,可能是相近或相同的株。     

Determination of Wolbachia diversity in butterflies from Western Ghats, India, by a multigene approach

Members of the genus Wolbachia are intracellular bacteria that are widespread in arthropods and establish diverse symbiotic associations with their hosts, ranging from mutualism to parasitism. Here we present the first detailed analyses of Wolbachia in butterflies from India with screening of 56 species. Twenty-nine species (52%) representing five families were positive for Wolbachia. This is the first report of Wolbachia infection in 27 of the 29 species; the other two were reported previously. This study also provides the first evidence of infection in the family Papilionidae. A striking diversity was observed among Wolbachia strains in butterfly hosts based on five multilocus sequence typing (MLST) genes, with 15 different sequence types (STs). Thirteen STs are new to the MLST database, whereas ST41 and ST125 were reported earlier. Some of the same host species from this study carried distinctly different Wolbachia strains, whereas the same or different butterfly hosts also harbored closely related Wolbachia strains. Butterfly-associated STs in the Indian sample originated by recombination and point mutation, further supporting the role of both processes in generating Wolbachia diversity. Recombination was detected only among the STs in this study and not in those from the MLST database. Most of the strains were remarkably similar in their wsp genotype, despite divergence in MLST. Only two wsp alleles were found among 25 individuals with complete hypervariable region (HVR) peptide profiles. Although both wsp and MLST show variability, MLST gives better separation between the strains. Completely different STs were characterized for the individuals sharing the same wsp alleles.     

Molecular evidence for the endosymbiont <Emphasis Type="BoldItalic">Wolbachia</Emphasis> in a non-filaroid nematode, <Emphasis Type="BoldItalic">Angiostrongylus cantonensis</Emphasis>

Wolbachia harbored by most filarial parasites, is critical to both embryogenesis and microfilarial development, and may lead to inflammation and pathogenesis in infected hosts. Based on alignment of the sequences from the wsp, ftsZ, and 16S rRNA genes, Wolbachia was demonstrated to exist in Angiostrongylus cantonensis, a non-filaroid nematode. Although the wsp gene may not be the best candidate for evolutionary analysis of Wolbachia, this gene has been sequenced from a broader coverage of the host species, making it feasible to be used for phylogenetic analysis in this study. The results from both Neighbor-joining and Maximum parsimony methods showed that this novel Wolbachia does not belong to any of the known groups (C or D) of nematode-derived Wolbachia. In addition, the wsp gene sequence of this newly identified endosymbiont revealed a high degree of identity (98%) with that from Diaea circumlita c2, tentatively classified into the putative group G. This suggests that Wolbachia from A. cantonensis could represent a deeply branched lineage in Wolbachia evolution or the occurrence of horizontal transfer between infected hosts. In conclusion, the findings provide some insights into our understanding of the evolution of Wolbachia, particularly the isolate from A. cantonensis.