蚜虫初级内共生菌Buchnera aphidicola研究进展

动物和细菌的内共生关系一直是生物学关注的热点,相关研究不但对于了解动物宿主的生长、繁殖等有重要意义,也有助于探讨生命起源和进化等生命现象。蚜虫类昆虫体内存在一类专性的胞内共生菌Buchnera,它对于蚜虫营养代谢和正常发育至关重要,被称为蚜虫的初级内共生菌。由于两者间具有专性共生关系,使其成为内共生关系研究的理想模型。本文将从Buchnera的基本特征、Buchnera与蚜虫进化关系、Buchnera在共生关系中的作用及Buchnera基因组学等方面对Buchnera研究现状进行综述,并对未来的研究热点进行展望。     

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。     

麦长管蚜脱巴克纳氏共生菌的分子验证

蚜虫脱共生效果的鉴定对蚜虫-巴克纳氏菌共生体系的研究至关重要.采用光学显微镜观察及特异性PCR扩增技术,对麦长管蚜(Sitobion aoenae)脱巴克纳氏共生菌的效果进行了研究.结果表明:用利福平处理5 d和10 d后的麦长管蚜,PCR检测发现有巴克纳氏菌(Buchnera)特异性扩增条带,说明共生菌仍然存在;在利福平处理 15 d后,PCR检测没有发现特异性扩增条带,说明麦长管蚜胞内共生细菌的核酸已经被降解;光学显微镜观察与PCR检测结果相一致.     

蚜虫与其胞内共生细菌的相互作用

蚜虫—巴克纳氏菌之间是一种典型的互利共生关系,两者相互依存,缺少一方,另一方便不能生存。研究表明,共生细菌能为寄主蚜虫提供必需氨基酸和维生索,并对寄主具有一些非营养功能,如促进蚜虫传播循环性病毒等。寄主蚜虫则是为共生菌提供一个合适的生存场所,并对共生菌的生长和繁殖进行调控。现代分子生物学技术从基因水平证明了蚜虫与共生菌的相互依赖性。     

蚜虫与其初级内共生菌进化关系: 假说及演化机理

蚜虫是半翅目(Hemiptera)中一类取食植物韧皮部汁液的昆虫, 由于具有一些独特的生物学特征, 是研究重要适应进化问题的理想模型。蚜虫体内存在一类专性的胞内共生菌Buchnera, 对于蚜虫营养代谢和正常发育有重要贡献, 被称为蚜虫的初级内共生菌。蚜虫-Buchnera是研究共生关系的理想模型, 两者系统发育格局的研究有助于理解生物间专性共生关系的演化。本文系统综述了两者在不同分类水平（高级阶元、 低级阶元）上的系统发育关系研究。现有证据暗示： 两者在低级阶元水平上具有系统发育一致性, 而在高级阶元水平上可能没有平行演化关系, 这些对早期研究提出的平行演化假说提出了质疑。在现有研究的基础上, 本文建议从增加取样类群、 增加基因数目和数据量、 系统发育一致性检验等几个方面开展更深入的系统发育研究, 并开展Buchnera的转移实验, 从而检验Buchnera的横向转移及其基因在不同蚜虫支系中的进化速率一致性, 以便更客观地揭示蚜虫-Buchnera的进化关系。     

培菌白蚁菌圃和粪便微生物多样性分析

【背景】培菌白蚁是属于白蚁科的一类与鸡枞菌属真菌共生的高等白蚁,其与体内肠道微生物和体外菌圃微生物形成三维共生体系。【目的】分析培菌白蚁菌圃和粪便的微生物多样性,并与肠道微生物进行比较。【方法】通过Illumina MiSeq高通量测序方法对培菌白蚁菌圃和粪便样品进行细菌16S rRNA基因和真菌ITS测序分析。【结果】高通量测序获得培菌白蚁菌圃和粪便样品细菌和真菌的有效序列和OTU数目。5个样品细菌OTU数目在90-199之间,而真菌OTU在10-58之间,细菌的种类多样性明显大于真菌。不论是细菌还是真菌,粪便样品的OTU数目多于菌圃样品。经物种分类分析,菌圃样品主要优势细菌是变形菌门(Proteobacteria),其相对含量超过82.4%;其次是拟杆菌门(Bacteroidetes)和厚壁菌门(Firmicutes);粪便样品中优势细菌为拟杆菌门,其次是变形菌门,粪便优势菌属为别样杆菌属和营发酵单胞菌属,这与培菌白蚁肠道菌多样性组成一致。培菌白蚁菌圃和粪便样品共生真菌主要为担子菌门(Basidiomycota)和子囊菌门(Ascomycota)。菌圃优势真菌为鸡枞菌属(Termitomyces),相对含量在51.83%以上,菌圃中还鉴定到炭角菌属(1%,Xylaria)。【结论】为今后培菌白蚁-体内外微生物共生关系研究以及微生物的分离培养提供了依据和参考。     

脱共生作用对黑豆蚜氨基酸代谢的影响(英文)

为了解共生菌对黑豆蚜蛋白质、氨基酸代谢的影响 ,用利福平处理黑豆蚜以除去其细胞内共生细菌 ,产生脱共生蚜虫。结果表明 ,被脱去共生菌的蚜虫与未经抗生素处理的正常蚜虫相比 ,7日龄时 ,脱共生蚜虫每毫克鲜重的总蛋白含量降低了 2 9% ,每毫克鲜重的游离氨基酸含量提高了 17%。对黑豆蚜取食的蚕豆苗韧皮部组织中必需氨基酸所占的比例进行分析后发现 ,蚕豆苗韧皮部组织中的必需氨基酸含量仅占 2 0 % ,而有共生菌的黑豆蚜组织中必需氨基酸已达到 4 4% ,脱共生后降低到 37% ,这些结果证明了黑豆蚜的胞内共生菌为其寄主提供了部分必需氨基酸。通过对游离氨基酸组成的分析发现 ,在测定的 17种氨基酸中 ,必需氨基酸中的苏氨酸在共生蚜虫中所占的比例为 2 1 6 % ,在脱共生蚜虫中仅为 16 7%。同样 ,非必需氨基酸中的酪氨酸和丝氨酸 ,在共生蚜虫中分别占总游离氨基酸的 8 9%和 5 6 % ,而在脱共生蚜虫中却分别升高到 2 1 1%和 13 6 %。这些结果表明 ,各种氨基酸比例的失调 ,造成了脱共生蚜虫蛋白质合成受阻和部分游离氨基酸的积累 ,并因此导致蚜虫发育和繁殖的失调。     

毛蚜属Chaitophorus蚜虫(半翅目:蚜科:毛蚜亚科)与初级内共生菌Buchnera的演化关系

【目的】蚜虫体内共生菌种类丰富,二者关系十分密切。几乎所有蚜虫都具有一类专性的初级内共生菌Buchnera aphidicola,二者的专性共生关系使蚜虫-Buchnera成为研究共生关系演化的理想模型。本研究对蚜虫-Buchnera在低级阶元水平上的"平行演化假说"进行了验证。【方法】选取在杨属Populus或柳属Salix植物上营同寄主全周期生活的毛蚜属Chaitophorus蚜虫作为研究对象,基于不同来源的分子标记(蚜虫线粒体基因、核基因和内共生菌基因),运用最大似然法和贝叶斯法重建蚜虫和Buchnera的系统树,并利用Tree Map、Jane和Para Fit检验二者是否具有协同系统发生关系。【结果】Tree Map和Jane分析检测到毛蚜属蚜虫与Buchnera具有显著的共成种信号,Para Fit分析结果表明二者的总体关联极为显著。【结论】毛蚜属蚜虫与其初级内共生菌Buchnera在种级及以下水平上符合"平行演化假说",并且二者的演化关系不会受到寄主植物差异的影响。     

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。     

刺吸式昆虫次生内共生菌的研究进展

蚜虫作为典型的刺吸式昆虫,需要以取食植物韧皮部汁液来补充营养,几乎所有蚜虫均带有一种能为其提供植物韧皮部缺失营养物质的初生共生菌Buchnera aphidicola。此外,蚜虫还可携带一种或多种次生内共生菌。在众多共生菌—寄主系统中,蚜虫与其所带内共生菌间的互作研究最为透彻。虽然次生内共生菌对寄主的存活和生殖影响并不显著,但其在寄主对环境耐受力、天敌防御能力等方面作用明显。本文在查阅大量蚜虫次生内共生菌相关文献的基础上,着重对蚜虫次生内共生菌的种类及传播规律、次生内共生菌对蚜虫表型的影响、蚜虫次生内共生菌基因组学等方面的研究现状进行综述,以求为刺吸式昆虫次生内共生菌的研究提供参考。     

Endosymbiosis of Aphids with Microorganisms: a Model Case of Dynamic Endosymbiotic Evolution

Abstract Almost all aphids harbor prokaryotic intracellular symbionts in the cytoplasm of mycetocytes, huge cells in the abdomen specialized for this purpose. The aphids and their intracellular symbionts are in close mutualistic association and unable to live without their partner. The intracellular symbionts of various aphids are of a single origin; they are descendants of a prokaryote that was acquired by the common ancestor of the present aphids. The date of establishment of the symbiotic association is estimated to be 160–280 million years ago using 16S rRNA molecular clock calibrated by aphid fossils. Molecular phylogeny indicates that the intracellular symbiont belongs to a group of gut bacteria, suggesting the possibility that it was derived from a gut microbe of aphids. While the in-tracellular symbionts are universal and highly conserved amongst aphids, other types of symbiotic microorganisms are also present. In various aphids, bacterial “secondary” intracellular symbionts are found in addition to the standard symbionts. They are thought to be acquired many times in various lineages independently. Some Cerataphidini aphids do not have intracellular symbiotic system but harbor yeast-like extracellular symbionts in the hemocoel. In a lineage of this group, symbiont replacement from intracellular prokaryote to extracellular yeast must have occurred. The diversity of the endosymbiotic system of aphids illuminates a dynamic aspect of endosymbiotic evolution.     

Buchnera aphidicola of the birch blister aphid,Hamamelistes betulinus (Horváth, 1896) (Insecta,Hemiptera, Aphididae: Hormaphidinae): molecular characterization,transmission between generations and its geographic significance

The birch blister aphid Hamamelistes betulinus, like most aphids, is host to obligate symbiotic bacterium Buchnera aphidicola. Ultrastructural and molecular analyses did not reveal the presence of secondary symbionts in the body of H. betulinus. The bacteria Buchnera aphidicola are transmitted to the next generation vertically (maternally). The bacteria released from the cytoplasm of the bacteriocyte to the haemolymph migrate to the embryo at the cellular blastoderm stage, through the opening at its posterior pole. Next, the bacteria enter the cytoplasm of newly formed bacteriocytes. The concept of the relationship between the geographic distribution of Hormaphidini aphids and the presence/absence of bacterium Buchnera aphidicola is discussed.     

Independent origins and horizontal transfer of bacterial symbionts of aphids

Many insect groups have obligate associations with primary endosymbionts: mutualistic bacteria that are maternally transmitted and derived from an ancient infection. Often, the same insects are hosts to 'secondary' bacterial symbionts which are maternally transmitted but relatively labile within host lineages. To explore the dynamics of secondary symbiont associations in aphids, we characterized bacteria infecting 15 species of macrosiphine aphids using DNA sequencing, diagnostic polymerase chain reaction (PCR), diagnostic restriction digests, phylogenetic analyses, and electron microscopy to examine aphids from nature and from laboratory colonies. Three types of bacteria besides Buchnera were found repeatedly; all three fall within the Enterobacteriaceae. The R-type has a 16S rDNA less than 0.1% different from that of the secondary symbiont previously reported from Acyrthosiphon pisum and is related to Serratia species. The T-type includes a symbiont previously reported from a whitefly; the U-type comprises a new cluster near the T-type. The T-type was found in every one of 40 Uroleucon ambrosiae clones collected throughout the United States. In contrast, A. pisum individuals were infected by any combination of the three symbiont types. Secondary symbionts were maternally transmitted for 11 months within laboratory-reared A. pisum clones and were present in sexually produced eggs. PCR screens for a bacteriophage, APSE-1, indicated its presence in both A. pisum and U. ambrosiae containing secondary symbionts. Electron microscopy of R-type and T-type bacteria in A. pisum and in U. ambrosiae revealed rod-shaped organisms that attain extremely high densities within a few bacteriocytes.     

Rickettsia symbiont in the pea aphid Acyrthosiphon pisum: novel cellular tropism, effect on host fitness, and interaction with the essential symbiont Buchnera

In natural populations of the pea aphid Acyrthosiphon pisum, a facultative bacterial symbiont of the genus Rickettsia has been detected at considerable infection frequencies worldwide. We investigated the effects of the Rickettsia symbiont on the host aphid and also on the coexisting essential symbiont Buchnera. In situ hybridization revealed that the Rickettsia symbiont was specifically localized in two types of host cells specialized for endosymbiosis: secondary mycetocytes and sheath cells. Electron microscopy identified bacterial rods, about 2 mum long and 0.5 mum thick, in sheath cells of Rickettsia-infected aphids. Virus-like particles were sometimes observed in association with the bacterial cells. By an antibiotic treatment, we generated Rickettsia-infected and Rickettsia-eliminated aphid strains with an identical genetic background. Comparison of these strains revealed that Rickettsia infection negatively affected some components of the host fitness. Quantitative PCR analysis of the bacterial population dynamics identified a remarkable interaction between the coexisting symbionts: Buchnera population was significantly suppressed in the presence of Rickettsia, particularly at the young adult stage, when the aphid most actively reproduces. On the basis of these results, we discussed the possible mechanisms that enable the prevalence of Rickettsia infection in natural host populations in spite of the negative fitness effects observed in the laboratory.     

The secondary endosymbiotic bacterium of the pea aphid Acyrthosiphon pisum (Insecta: homoptera)

The secondary intracellular symbiotic bacterium (S-symbiont) of the pea aphid Acyrthosiphon pisum was investigated to determine its prevalence among strains, its phylogenetic position, its localization in the host insect, its ultrastructure, and the cytology of the endosymbiotic system. A total of 14 aphid strains were examined, and the S-symbiont was detected in 4 Japanese strains by diagnostic PCR. Two types of eubacterial 16S ribosomal DNA sequences were identified in disymbiotic strains; one of these types was obtained from the primary symbiont Buchnera sp., and the other was obtained from the S-symbiont. In situ hybridization and electron microscopy revealed that the S-symbiont was localized not only in the sheath cells but also in a novel type of cells, the secondary mycetocytes (S-mycetocytes), which have not been found previously in A. pisum. The size and shape of the S-symbiont cells were different when we compared the symbionts in the sheath cells and the symbionts in the S-mycetocytes, indicating that the S-symbiont is pleomorphic under different endosymbiotic conditions. Light microscopy, electron microscopy, and diagnostic PCR revealed unequivocally that the hemocoel is also a normal location for the S-symbiont. Occasional disordered localization of S-symbionts was also observed in adult aphids, suggesting that there has been imperfect host-symbiont coadaptation over the short history of coevolution of these organisms.     

Transmission of symbiotic bacteria Buchnera to parthenogenetic embryos in the aphid Acyrthosiphon pisum (Hemiptera: Aphidoidea)

All phloem-feeding aphids have an absolute requirement for their primary bacterial symbionts Buchnera sp. The bacteria are transmitted vertically to either embryos in the viviparous morph or to eggs in the oviparous morph, with the implication that the symbiont population regularly passes through a genetic 'bottleneck', i.e. only a small proportion of the maternal symbiont population is transmitted to offspring. In this paper, we visualise this process in viviparous aphids using a specific immunolabelling technique for Buchnera. The images show a stream of bacteria originating from a single mycetocyte and entering the embryo, possibly via a membranous conduit, and individual bacterial cells free in the haemocoel of the aphid. Staining within the embryo blastoderm suggests over expression of antigen, perhaps indicative of rapid bacterial division immediately following infection.     

Estimating population size and transmission bottlenecks in maternally transmitted endosymbiotic bacteria

Many species of bacterial endosymbionts are acquired by animal hosts before birth, through direct transmission from mothers to eggs or embryos. This vertical transmission imposes a reduction in numbers or "bottleneck," and the size of this bottleneck affects the population structure and evolution of symbiotic lineages. We have estimated the size of the transmission bottleneck in Buchnera, the bacterial symbiont of aphids, using basic light and electron microscopy techniques. By serial-sectioning whole aphid abdomens, their eggs, and embryos, we determined the following parameters: (i) The average size of a Buchnera cell is 2.9 mm in diameter. (ii) The total number of Buchnera in an Acyrthosiphon pisum embryo was around 36,700 whereas a first instar nymph contained more than 119,000. (iii) The number of symbionts per bacteriocyte was around 800 in an embryo and 3200 in a first instar nymph. (iv) The total number of Buchnera transmitted to each sexual egg ranged from 850 in Nasonovia to 1800 in A. pisum to more than 8000 in Uroleucon ambrosiae. (v) The total number of secondary endosymbionts in A. pisum was 12,170 for an embryo and 18,360 for a first instar nymph. Secondary symbionts were arranged both extracellularly and in clusters of 2000–8000 bacteria inside bacteriocytes. These numbers are consistent with the few previous estimates of symbiont population sizes based on counts of gene copies.