浙江B型与非B型（China-ZHJ-1）烟粉虱种群共生细菌的检测及系统发育分析

烟粉虱体内存在共生细菌,包括初生共生细菌（primary endosymbiont）和次生共生细菌（secondary endosymbiont）。本项研究应用PCR技术检测了烟粉虱浙江B型和非B型China-ZHJ-1种群中共生细菌的分布。结果表明,烟粉虱B型和非B型体内均存在初生共生细菌,而两者次生共生细菌的组成存在差异。一种肠杆菌科次生共生细菌仅在B型烟粉虱中发现,而另两种次生共生细菌Wolbachia和杀雄菌Arsenophonus仅在非B型中发现。初生共生细菌的系统发育分析表明,B型是入侵生物型,而浙江非B型是本地生物型。     

烟粉虱及其优势寄生蜂内共生菌的种类及系统发育分析

为了研究入侵我国的2个主要烟粉虱隐种Bemisia tabaci MEAM1和MED及其3种优势寄生蜂(浅黄恩蚜小蜂Encarsia sophia、丽蚜小蜂E.formosa、海氏桨角蚜小蜂Eretmocerus hayati)体内感染内共生菌的种类丰度,并进一步探讨其系统发育关系,本文利用分子生物学手段对昆虫体内细菌的16S rRNA基因序列进行扩增、测序和分析,并采用邻接法(Neighbor-Joining,NJ)和最大似然法(MaximumLikehood,ML)分别构建优势内共生菌的系统发育树。结果表明,烟粉虱2个隐种内共生菌的种类丰度大于其3种优势寄生蜂,3种优势寄生蜂中丽蚜小蜂内共生菌的种类丰度最高;同源性分析发现烟粉虱和寄生蜂所携带的Rickettsia基因同源性达到99%,属于Rickettsia bellii种,进一步的进化树分析也发现所研究物种的Rickettsia和Hamiltonella均可各自聚为同一进化分支。烟粉虱及其优势寄生蜂体内含有种类丰富的内共生菌,其中优势内共生菌Rickettsia和Hamiltonella各自亲缘关系很近,说明内共生菌在烟粉虱和寄生蜂间可能进行水平传播。     

共生菌Cardinium在宿主体内的分布、检测及其生殖调控作用

内共生菌是存在于宿主细胞内,与宿主存在互利共生关系的一类细菌。内共生菌在节肢动物的生长和繁殖过程中起着十分重要的作用, 包括向宿主提供食物中缺乏的营养物质、调控宿主生殖方式,增强宿主适应性等。Cardinium属于拟杆菌门Bacteroidetes,通过细胞质遗传,具有诱导宿主细胞质不亲和、产雌孤雌生殖、雌性化等生殖异常现象,并且还可影响宿主的适合度,以利于其本身在宿主不同种群和世代间的分布和传播。由于Cardinium是近几年人们新发现的一类新型内共生菌,目前对其在节肢动物宿主体内的分布、其生物学生态学功能等尚不完全明确。本文在综合国内外文献资料的基础上,从Cardinium在节肢动物体内的分布、其在昆虫种群的传播规律及其检测手段、其对节肢动物宿主的生殖调控作用等几个方面进行了综述,并对其应用前景进行了展望,以期引起更多的学者关注该胞内共生菌,推动人们对该内共生菌的研究及利用。     

三种抗生素对B型和Q型烟粉虱内共生菌的去除效果比较研究

利用烟粉虱Bemisia tabaci(Gennadius)内共生菌特异性引物,研究了内共生菌在B、Q型烟粉虱种群中的分布和感染率,同时评价了3种不同的抗生素利福平、氨苄青霉素和硫酸卡那霉素分别在3种不同的浓度下(100.0、50.0及25.0μg/mL)对烟粉虱内共生菌的去除效果。结果表明:B、Q型烟粉虱原生内共生细菌Portiera的带菌率均为100.0%;B、Q型烟粉虱次生内共生菌Hamiltonella的带菌率分别为91.7%和100.0%;B型烟粉虱次生内共生菌Rickettsia的带菌率为87.5%,Q型为0;其它次生内共生菌在B、Q型烟粉虱中均未检测到。利福平、氨苄青霉素和硫酸卡那霉素在3种不同的浓度下均不能去除B、Q型烟粉虱Portiera;利福平、氨苄青霉素在3种不同的浓度下均能完全去除B型烟粉虱Rickettsia,硫酸卡那霉素在不同浓度下去除Rickettsia的效果不同;3种抗生素去除Hamiltonella的能力受抗生素种类以及浓度的影响。同一抗生素在不同浓度下去除Hamiltonella的效果均是100.0μg/mL>50.0μg/mL>25.0μg/mL;不同浓度的抗生素去除Hamiltonella的效果均是利福平>氨苄青霉素>硫酸卡那霉素,各浓度与各抗生素之间的去除Hamiltonella的效果均具有显著性差异。     

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     

基于16S rDNA序列的Wolbachia的检测及分型

Wolbachia是广泛分布于节肢动物体内的一类共生细菌。采用16S rDNA特异片段的PCR-RFLP方法对烟粉虱Bemisia tabaci （Gennadius）不同生物型及米蛾Corcyra cephalonica （Stainton）共生菌Wolbachia进行了检测与分型分析。基于wsp基因对烟粉虱共生菌B组Wolbachia以及米蛾共生菌Wolbachia进行了系统树分析,并对相应的Wolbachia16S rDNA特异片段进行了克隆、测序以及序列比对。结果表明:16S rDNA的特异片段经NheⅠ酶切后RFLP图谱可有效检测与鉴别Wolbachia。烟粉虱共生菌Wolbachia的16S rDNA特异片段经VspⅠ酶切后可得到预期RFLP图谱,而米蛾共生菌B组Wolbachia （基于wsp序列分析为B组）则产生不同的RFLP图谱。序列分析表明,Nauru型烟粉虱体内B组Wolbachia的16S rDNA片段序列与已知B组Wolbachia对应序列（DQ278884）同源性为100%;米蛾体内B组Wolbachia 16S rDNA特异片段有碱基变异,并存在于VspⅠ识别位点内,这是导致VspⅠ酶切后RFLP图谱不同的原因。结果提示,B组Wolbachia 16S rDNA特异片段经VspⅠ酶切的RFLP图谱存在多态性。本研究结果可为今后Wolbachia的检测与分型提供借鉴。     

褐飞虱不同致害性种群体内共生菌18S rDNA部分序列比较

分离纯化了褐飞虱3种不同致害性种群体内类酵母共生菌 (yeast-like symbionts, YLS),并对其18S rDNA基因序列进行了比较。结果表明,褐飞虱3种不同致害性种群体内类酵母共生菌18S rDNA均扩增出600 bp左右的片断。依据获得的18S rDNA特异性序列,结合已知真菌的18S rDNA部分序列,构建了不同宿主的YLS分子系统树。结果显示, 褐飞虱3种不同致害性种群体内的YLS同属子囊菌亚门（Ascomycotina）的核菌纲（Pyrenomycetes）,并与此纲中的Hypomyces chrysospermus亲缘关系相对最近。     

福建省烟粉虱自然种群Wolbachia感染特点

Wolbachia是专性的细胞内细菌,广泛存在于节肢动物生殖组织。已有的研究结果表明,节肢动物中存在A组和B组Wolbachia,而烟粉虱Bemisia tabaci中主要检测到了B组Wolbachia。本研究从福建省采集到17个不同烟粉虱地理种群,首先通过rDNA-ITS1克隆测序鉴定了不同烟粉虱地理种群的生物型,然后采用Wolbachia 16S rDNA的特异引物,并通过PCR-RFLP技术分析了不同烟粉虱地理种群中Wolbachia的感染特点。结果表明：从福建省闽侯、平潭、南平、来舟、漳平和沙县采集到的烟粉虱自然种群属于非B型,而非B型烟粉虱种群中存在广泛的超感染现象,即单个非B型烟粉虱个体中同时感染了不同型Wolbachia。相反,B型烟粉虱自然种群的个体中只感染A组Wolbachia。该研究依据密集采样的数据进一步证实了Wolbachia在烟粉虱自然种群中的分布确实与宿主的生物型密切相关,提示Wolbachia可能在烟粉虱的种群分化中发挥作用。     

温度对黑豆蚜体内共生菌胞数量及宿主体型大小的影响

为了明确饲养温度对黑豆蚜Aphis fabae 内共生菌和宿主蚜虫体型大小的影响,对在室内不同温度下饲养的黑豆蚜内共生菌胞数量和宿主蚜虫体型大小进行了观察和统计分析。结果表明,温度对同一发育时期蚜虫内共生菌胞数量的影响在不同温度范围内有所不同,1龄若蚜体内的菌胞数量除在25℃与35℃间有显著差异外,在其余各温度间没有显著差异; 其余时期的蚜虫内共生菌胞数量在高温（> 30℃）下显著低于较低温度下的菌胞数量,存在负直线相关性。温度对菌胞数量随宿主发育到产仔前的变化趋势有不同程度的影响,在较低温度（15℃、20℃和25℃）下,菌胞数量随虫体发育显著增加; 但在高温（30℃和35℃）下,蚜虫体内菌胞数逐渐增加直到3龄达到最高,然后略有下降（30℃）或显著下降（35℃）。除1龄若蚜外,蚜虫体型大小总体呈现随温度升高而降低的格局,但随其内共生菌数量增多而增大（35℃下除外）。据此认为,温度可能通过作用于蚜虫内共生菌胞数量而影响蚜虫体型的大小。     

桃蚜自然种群初级和次级共生菌的分子鉴定

蚜虫不仅带有专性初级内共生菌,还常常带有不同类群的兼性次级共生菌。本研究采用真细菌16S rDNA的通用引物,从桃蚜Myzus persicae (Sulzer)烟草种群体内扩增出1.5 kb的共迁移目的条带,然后将其克隆,并在阳性克隆筛选时进行了RFLP分析。当采用EcoRⅠ进行单酶切分析时,目的片段被切割成～650 bp和～850 bp两个小片段;当采用EcoRⅠ和HindⅢ进行双酶切分析时,该蚜群共生菌酶切谱带被分成2组,其中1组(GroupⅠ)只具有一个EcoRⅠ酶切位点,而另1组(Group Ⅱ)不仅具有一个EcoRⅠ酶切位点,还具有一个HindⅢ酶切位点,而且GroupⅠ为优势共生菌群。在此基础上分别对2组共生菌的16S rDNA全序列（～1.5 kb）进行了测定,结果表明：GroupⅠ属于泛菌属Pantoea,与成团泛菌Pantoea agglomerans亲缘关系最近（同源性达99.70%）,而Group Ⅱ属于蚜虫的专性内共生菌,即Buchnera aphidicola（同源性达99.50%）。这是在蚜虫体内存在泛菌次级共生菌的首次报道。     

烟粉虱内共生菌16S rDNA的变异与系统发生

对 5年连续饲养在不同种寄主植物上的B型烟粉虱北京种群的内共生菌 1 6SrDNA基因进行了PCR扩增和测序。结合已知序列 ,构建了不同寄主植物烟粉虱初生内共生菌约 1 0 0 0bp的 1 6SrDNA及次生内共生菌约1 2 5 0bp的 1 6SrDNA的分子系统树。结果表明 ,中国北京不同寄主植物的B型烟粉虱内共生菌及世界其它地区烟粉虱内共生菌可能是同一种的不同生态型 ,内共生菌在其宿主分化后进行了选择 ,之后与其宿主长期共同进化、共同适应 ,为宿主对不同生境的适应提供了一定的基础     

Wolbachia infections of the whitefly Bemisia tabaci

We report the first systematic survey for the presence of Wolbachia endosymbionts in aphids and whiteflies, particularly different populations and biotypes of Bemisia tabaci. Additional agriculturally important species included were predator species, leafhoppers, and lepidopterans. We used a polymerase chain reaction (PCR)-based detection assay with ribosomal 16S rDNA and Wolbachia cell surface protein (wsp) gene primers. Wolbachia were detected in a number of whitefly populations and species, whitefly predators, and one leafhopper species; however, none of the aphid species tested were found infected. Single, double, and triple infections were detected in some of the B. tabaci populations. PCR and phylogenetic analysis of wsp gene sequences indicated that all Wolbachia strains found belong to group B. Topologies of the optimal tree derived by maximum likelihood (ML) and a ML tree in which Wolbachia sequences from B. tabaci are constrained to be monophyletic are significantly different. Our results indicate that there have been at least four independent Wolbachia infection events in B. tabaci. The importance of the presence of Wolbachia infections in B. tabaci is discussed.     

Further insights into the strange role of bacterial endosymbionts in whitefly, Bemisia tabaci: comparison of secondary symbionts from biotypes B and Q in China

The percentage infection of secondary symbionts (SS) (Wolbachia, Arsenophonus, Rickettsia, Hamiltonella, Fritschea and Cardinium) in the exotic Bemisia tabaci (Genn.) invaders, commonly known as biotypes B and Q from China, were determined by PCR. In total, 373 biotype B and 1830 biotype Q individuals were screened for the presence of SS. Biotype B was more abundant than biotype Q from 2005 to 2006, and biotype Q was more abundant from 2007 to 2009. Each of the SS, with the exception of Fritschea, was detected in both biotypes B and Q; Fritschea was found in none of the samples examined. For biotype B, the percentage infection of Hamiltonella was the highest (92.0%) followed by Rickettsia (70.2%). For biotype Q, the percentage infection of Hamiltonella was again the highest (73.3%). Arsenophonus was the least common of the SS observed in both biotypes B and Q. The percentage infection of Wolbachia, Rickettsia and Hamiltonella in biotype B was each significantly higher than in biotype Q, whereas the percentage infection of Cardinium in biotype B was significantly lower than in biotype Q. The percentage infection of SS in biotypes B and Q varied from year to year over the period 2005-2009. Furthermore, within biotype Q, two distinct subgroups were identified which differ from each other in terms of their SS complement. We discuss these results in the light of the potentially influential factors and roles of the SS.     

Arsenophonus GroEL Interacts with CLCuV and Is Localized in Midgut and Salivary Gland of Whitefly B. tabaci

Cotton leaf curl virus (CLCuV) (Gemininiviridae: Begomovirus) is the causative agent of leaf curl disease in cotton plants (Gossypium hirsutum). CLCuV is exclusively transmitted by the whitefly species B. tabaci (Gennadius) (Hemiptera: Alerodidae). B. tabaci contains several biotypes which harbor dissimilar bacterial endo-symbiotic community. It is reported that these bacterial endosymbionts produce a 63 kDa chaperon GroEL protein which binds to geminivirus particles and protects them from rapid degradation in gut and haemolymph. In biotype B, GroEL protein of Hamiltonella has been shown to interact with Tomato yellow leaf curl virus (TYLCV). The present study was initiated to find out whether endosymbionts of B. tabaci are similarly involved in CLCuV transmission in Sriganganagar (Rajasthan), an area endemic with cotton leaf curl disease. Biotype and endosymbiont diversity of B. tabaci were identified using MtCO1 and 16S rDNA genes respectively. Analysis of our results indicated that the collected B. tabaci population belong to AsiaII genetic group and harbor the primary endosymbiont Portiera and the secondary endosymbiont Arsenophonus. The GroEL proteins of Portiera and Arsenophonus were purified and in-vitro interaction studies were carried out using pull down and co-immunoprecipitation assays. In-vivo interaction was confirmed using yeast two hybrid system. In both in-vitro and in-vivo studies, the GroEL protein of Arsenophonus was found to be interacting with the CLCuV coat protein. Further, we also localized the presence of Arsenophonus in the salivary glands and the midgut of B. tabaci besides the already reported bacteriocytes. These results suggest the involvement of Arsenophonus in the transmission of CLCuV in AsiaII genetic group of B. tabaci.     

Phylogenetic congruence of armored scale insects (Hemiptera: Diaspididae) and their primary endosymbionts from the phylum Bacteroidetes

Insects in the sap-sucking hemipteran suborder Sternorrhyncha typically harbor maternally transmitted bacteria housed in a specialized organ, the bacteriome. In three of the four superfamilies of Sternorrhyncha (Aphidoidea, Aleyrodoidea, Psylloidea), the bacteriome-associated (primary) bacterial lineage is from the class Gammaproteobacteria (phylum Proteobacteria). The fourth superfamily, Coccoidea (scale insects), has a diverse array of bacterial endosymbionts whose affinities are largely unexplored. We have amplified fragments of two bacterial ribosomal genes from each of 68 species of armored scale insects (Diaspididae). In spite of initially using primers designed for Gammaproteobacteria, we consistently amplified sequences from a different bacterial phylum: Bacteroidetes. We use these sequences (16S and 23S, 2105 total base pairs), along with previously published sequences from the armored scale hosts (elongation factor 1alpha and 28S rDNA) to investigate phylogenetic congruence between the two clades. The Bayesian tree for the bacteria is roughly congruent with that of the hosts, with 67% of nodes identical. Partition homogeneity tests found no significant difference between the host and bacterial data sets. Of thirteen Shimodaira-Hasegawa tests, comparing the original Bayesian bacterial tree to bacterial trees with incongruent clades forced to match the host tree, 12 found no significant difference. A significant difference in topology was found only when the entire host tree was compared with the entire bacterial tree. For the bacterial data set, the treelengths of the most parsimonious host trees are only 1.8-2.4% longer than that of the most parsimonious bacterial trees. The high level of congruence between the topologies indicates that these Bacteroidetes are the primary endosymbionts of armored scale insects. To investigate the phylogenetic affinities of these endosymbionts, we aligned some of their 16S rDNA sequences with other known Bacteroidetes endosymbionts and with other similar sequences identified by BLAST searches. Although the endosymbionts of armored scales are only distantly related to the endosymbionts of the other sternorrhynchan insects, they are closely related to bacteria associated with eriococcid and margarodid scale insects, to cockroach and auchenorrynchan endosymbionts (Blattabacterium and Sulcia), and to male-killing endosymbionts of ladybird beetles. We propose the name "Candidatus Uzinura diaspidicola" for the primary endosymbionts of armored scale insects.     

Biotype-dependent secondary symbiont communities in sympatric populations of Bemisia tabaci

The sweet potato whitefly, Bemisia tabaci, harbors Portiera aleyrodidarum, an obligatory symbiotic bacterium, as well as several secondary symbionts including Rickettsia, Hamiltonella, Wolbachia, Arsenophonus, Cardinium and Fritschea, the function of which is unknown. Bemisia tabaci is a species complex composed of numerous biotypes, which may differ from each other both genetically and biologically. Only the B and Q biotypes have been reported from Israel. Secondary symbiont infection frequencies of Israeli laboratory and field populations of B. tabaci from various host plants were determined by PCR, in order to test for correlation between bacterial composition to biotype and host plant. Hamiltonella was detected only in populations of the B biotype, while Wolbachia and Arsenophonus were found only in the Q biotype (33% and 87% infection, respectively). Rickettsia was abundant in both biotypes. Cardinium and Fritschea were not found in any of the populations. No differences in secondary symbionts were found among host plants within the B biotype; but within the Q biotype, all whiteflies collected from sage harboured both Rickettsia and Arsenophonus, an infection frequency which was significantly higher than those found in association with all other host plants. The association found between whitefly biotypes and secondary symbionts suggests a possible contribution of these bacteria to host characteristics such as insecticide resistance, host range, virus transmission and speciation.     

Cospeciation of psyllids and their primary prokaryotic endosymbionts

Psyllids are plant sap-feeding insects that harbor prokaryotic endosymbionts in specialized cells within the body cavity. Four-kilobase DNA fragments containing 16S and 23S ribosomal DNA (rDNA) were amplified from the primary (P) endosymbiont of 32 species of psyllids representing three psyllid families and eight subfamilies. In addition, 0.54-kb fragments of the psyllid nuclear gene wingless were also amplified from 26 species. Phylogenetic trees derived from 16S-23S rDNA and from the host wingless gene are very similar, and tests of compatibility of the data sets show no significant conflict between host and endosymbiont phylogenies. This result is consistent with a single infection of a shared psyllid ancestor and subsequent cospeciation of the host and the endosymbiont. In addition, the phylogenies based on DNA sequences generally agreed with psyllid taxonomy based on morphology. The 3' end of the 16S rDNA of the P endosymbionts differs from that of other members of the domain Bacteria in the lack of a sequence complementary to the mRNA ribosome binding site. The rate of sequence change in the 16S-23S rDNA of the psyllid P endosymbiont was considerably higher than that of other bacteria, including other fast-evolving insect endosymbionts. The lineage consisting of the P endosymbionts of psyllids was given the designation Candidatus Carsonella (gen. nov.) with a single species, Candidatus Carsonella ruddii (sp. nov.).