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．     

河南不同地区棉蚜种群共生菌Wolbachia的检测及系统发育分析

【目的】本研究旨在揭示河南省不同地区棉蚜Aphis gossypii种群共生菌Wolbachia的感染情况,明确Wolbachia的感染类型及分类地位。【方法】2016-2017年间采集河南省不同地区的13个棉蚜种群,通过扩增COI基因片段对其进行种类鉴定;通过扩增棉蚜种群中Wolbachia的wsp基因片段对其进行Wolbachia感染率的检测,应用neighbor-joining法构建系统进化树进行棉蚜种群中Wolbachia的系统发育分析。【结果】对河南省内不同地区采集的13个棉蚜种群的Wolbachia感染率而言,郑州(ZZ)种群最高(46.67%),信阳2(XY2)种群最低(6.67%),13个种群Wolbachia的感染率范围为6.67%～46.67%,平均感染率为28.35%。基于wsp基因构建的系统发育树表明,安阳和信阳的棉蚜种群感染的Wolbachia株系属于B大组,其余地区棉蚜种群感染的Wolbachia株系属于A大组。【结论】河南省不同地区的棉蚜种群Wolbachia感染率差别较大,且不同种群感染的Wolbachia株系分别属于A大组或B大组。     

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。     

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。     

我国桃蛀螟四个地理种群感染Wolbachia的研究

Wolbachia是一类广泛分布于节肢动物体内的细胞内共生菌,能参与调控寄主的多种生殖活动.桃蛀螟Conogethes punctiferalis (Guenée)可危害多种粮食作物和经济作物,近年来已成为我国玉米生产的重要害虫之一.本研究对通过对16S rRNA、fts以及wsp基因片断的PCR扩增,对我国桃蛀螟4个地理种群进行了Wolbachia感染检测,并对感染个体中wsp基因片断克隆测序,初步探究了我国桃蛀螟种群中Wolbachia的感染率和感染类群.研究结果表明我国4个桃蛀螟种群中Wolbachia感染率在2.0%～8.0%,平均感染率为4.5%.通过对不同种群感染的Wolbachia中wsp序列比对及系统树构建分析,在桃蛀螟中发现了5种Wolbachia亚型,分别命名为wPun1-wPun5,分属于A组的Uni、Dro亚组,以及B组的Div、Con亚组.此外,本研究在采自河北保定的桃蛀螟种群中发现了Wolbachia的超感染现象.     

沟眶象体内感染的Wolbachia的wsp基因克隆与序列分析

Wolbachia是广泛分布于昆虫细胞内的一类共生菌,为明确Wolbachia在沟眶象Eucryptorrhynchus chinensis(Olivier)体内的感染情况和分类地位,采用Wolbachia的wsp基因片段对沟眶象5个地理种群(宁夏灵武、中宁、中卫、安徽淮北、山东聊城)共100个样本,进行分子检测。经过克隆和测序得到wsp基因片段593 bp(Gen Bank number:KP713759),明确了沟眶象体内存在Wolbachia的感染,并且其在不同地理种群内的感染率均为100%;通过wsp通用引物检测发现沟眶象体内感染了1种Wolbachia,属于A群Dro亚群。     

首次发现沃尔巴克氏体Wolbachia对我国南亚果实蝇的感染现象

沃尔巴克氏体Wolbachia是广泛分布于节肢动物体生殖组织内的一类共生细菌,泰国等已发现Wolbachia感染南亚果实蝇Bactrocera(Zeugodacus)tau(Walker)的现象。利用沃尔巴克氏体wsp基因的1对通用引物(81F,691R)从我国南亚果实蝇的总DNA中扩增到1段500 bp左右的wsp基因片段,并克隆得到了Wolbachia相关序列,首次发现了沃尔巴克氏体对我国南亚果实蝇的感染现象。     

应用巢式PCR-DGGE技术分析稻虱缨小蜂体内Wolbachia的多样性

以采集自中国杭州和菲律宾的稻虱缨小蜂Anagrus nilaparvatae为研究对象,采用巢式PCR扩增Wolbachia的16S rDNA和wsp基因片段,并用DGGE分析稻虱缨小蜂体内Wolbachia的多样性.基于16S rDNA基因的分析结果准确地检测到稻虱缨小蜂体内细菌主要是Acinetobacter sp...     

Wolbachia在北京地区小菜蛾种群中的感染

Wolbachia是一类广泛分布于节肢动物体内,可以引起其寄主生殖行为改变的细胞内共生细菌。小菜蛾Plutella xylostella(L.)是常见的鳞翅目害虫,严重危害十字花科植物。通过对Wolbahcia的wsp基因的PCR扩增及克隆测序,明确Wolbachia在北京地区小菜蛾野生种群内及其个体体内的分布情况。研究发现,在北京地区小菜蛾的野生种群中,感染了一种Wolbahcia,将其命名为wXyl,并在GenBank中进行注册;在小菜蛾成虫的个体体内,其胸部,腹部及足内都有Wolbachia分布,而且所感染的Wolbachia完全相同。wXyl属于Wolbachia B组中的Pip亚组。研究首次发现在我国小菜蛾的野生种群内有Wolbachia感染的现象。     

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.     

Revisiting <Emphasis Type="Italic">Wolbachia</Emphasis> Supergroup Typing Based on WSP: Spurious Lineages and Discordance with MLST

The obligate intracellular bacteria Wolbachia are taxonomically subdivided into eight supergroups (named A-H). Supergroup typing of strains has been mostly based on phylogenetic inference of the Wolbachia surface protein (wsp), a gene that recently has been shown to experience high rates of recombination. This brings into question its suitability not only for microtaxonomy, but also for supergroup classification of the genus. A Multilocus Sequence Typing (MLST) scheme for Wolbachia has recently been developed that types strains based on five conserved genes, thus providing a rigorous supergroup annotation of strains. Here we report striking discrepancies in supergroup designation between MLST and wsp inferences, and propose a revision of current methods for Wolbachia supergroup typing. Transfer of whole wsp gene sequences between supergroups A and B has occurred. Furthermore, as a result of intragenic recombination, wsp phylogeny creates spurious basal lineages that are not supported by MLST. For example, the proposed supergroup G, based upon wsp alone, likely represents only a wsp recombinant clade. Removal of supergroup G is advised until and unless the existence of this lineage is substantiated by other sequence information (e.g., MLST). We recommend a full characterization MLST for a correct strain typing, while, based on the current data set, use of a single MLST gene can be effective for supergroup designation of A and B strains. Finally, we note that the sharing of wsp sequences between A and B strains indicates a strong genetic cohesiveness of Wolbachia strains, supporting designation of these bacteria within the same species, W. pipientis.     

Addition of wsp Sequences to the Wolbachia Phylogenetic Tree and Stability of the Classification

Wolbachia are symbiotic bacteria altering reproductive characters of numerous arthropods. Their most recent phylogeny and classification are based on sequences of the wsp gene. We sequenced wsp gene from six Wolbachia strains infecting six Trichogramma species that live as egg parasitoids on many insects. This allows us to test the effect of the addition of sequences on the Wolbachia phylogeny and to check the classification of Wolbachia infecting Trichogramma. The six Wolbachia studied are classified in the B supergroup. They confirm the monophyletic structure of the B Wolbachia in Trichogramma but introduce small differences in the Wolbachia classification. Modifications include the definition of a new group, Sem, for Wolbachia of T. semblidis and the merging of the two closely related groups, Sib and Kay. Specific primers were determined and tested for the Sem group.     

Phylogeny and PCR-based classification of Wolbachia strains using wsp gene sequences.

Wolbachia are a group of intracellular inherited bacteria that infect a wide range of arthropods. They are associated with a number of different reproductive phenotypes in their hosts, such as cytoplasmic incompatibility, parthenogenesis and feminization. While it is known that the bacterial strains responsible for these different host phenotypes form a single clade within the alpha-Proteobacteria, until now it has not been possible to resolve the evolutionary relationships between different Wolbachia strains. To address this issue we have cloned and sequenced a gene encoding a surface protein of Wolbachia (wsp) from a representative sample of 28 Wolbachia strains. The sequences from this gene were highly variable and could be used to resolve the phylogenetic relationships of different Wolbachia strains. Based on the sequence of the wsp gene from different Wolbachia isolates we propose that the Wolbachia pipientis clade be initially divided into 12 groups. As more sequence information becomes available we expect the number of such groups to increase. In addition, we present a method of Wolbachia classification based on the use of group-specific wsp polymerase chain reaction (PGR) primers which will allow Wolbachia isolates to be typed without the need to clone and sequence individual Wolbachia genes. This system should facilitate future studies investigating the distribution and biology of Wolbachia strains from large samples of different host species.     

Mosaic nature of the wolbachia surface protein

Lateral gene transfer and recombination play important roles in the evolution of many parasitic bacteria. Here we investigate intragenic recombination in Wolbachia bacteria, considered among the most abundant intracellular bacteria on earth. We conduct a detailed analysis of the patterns of variation and recombination within the Wolbachia surface protein, utilizing an extensive set of published and new sequences from five main supergroups of Wolbachia. Analysis of nucleotide and amino acid sequence variations confirms four hypervariable regions (HVRs), separated by regions under strong conservation. Comparison of shared polymorphisms reveals a complex mosaic structure of the gene, characterized by a clear intragenic recombining of segments among several distinct strains, whose major recombination effect is shuffling of a relatively conserved set of amino acid motifs within each of the four HVRs. Exchanges occurred both within and between the arthropod supergroups. Analyses based on phylogenetic methods and a specific recombination detection program (MAXCHI) significantly support this complex partitioning of the gene, indicating a chimeric origin of wsp. Although wsp has been widely used to define macro- and microtaxonomy among Wolbachia strains, these results clearly show that it is not suitable for this purpose. The role of wsp in bacterium-host interactions is currently unknown, but results presented here indicate that exchanges of HVR motifs are favored by natural selection. Identifying host proteins that interact with wsp variants should help reveal how these widespread bacterial parasites affect and evolve in response to the cellular environments of their invertebrate hosts.     

我国麦蚜体内的沃尔巴克氏体(Wolbachia)的检测

沃尔巴克氏体是广泛分布于节肢动物体内的一类共生细菌 ,它们参与多种调控其寄主生殖活动的机制。本研究通过wsp基因的特异性扩增证实了沃尔巴克氏体在麦长管蚜Sitobionavenae(Fabricius)体内的共生。而且在麦长管蚜的两种体色型 (绿色型和红色型 )中都检测到了沃尔巴克氏体的共生。     

Wolbachia在玉米螟赤眼蜂内的三重感染

Wolbachia是一类广泛存在于节肢动物体内的共生菌。玉米螟赤眼蜂Trichogramma ostriniae是我国玉米田间的优势赤眼蜂种, 据报道, 赤眼蜂种内有Wolbachia感染。本文利用Wolbachia的16s rDNA和wsp基因引物通过PCR方法对玉米螟赤眼蜂的野生种群进行了调查, 发现以wsp基因为鉴定依据, 检测的所有个体都感染了3种Wolbachia ［wOstGDAa (GenBank accession no. EU157103), wOstGDAb (GenBank accession no. EU157104) 和 wOstGDB (GenBank accession no. EU157105)］。本文首次报道了野生赤眼蜂种群内Wolbachia的三重感染率几乎为100%。根据本研究的结果, 可以推测当不同种赤眼蜂寄生同一寄主时, Wolbachia可能会在不同赤眼蜂种间进行横向传播。