【作者】 牛丽华；

【导师】 黄大卫；

【作者基本信息】 山东农业大学 ， 农业昆虫与害虫防治， 2013， 博士

【摘要】 昆虫作为陆地上最成功和最庞大的生物类群，对多种生态环境都具有高度的适应性。昆虫的这种适应性除了与自身的生理构造和机能相关外，很大程度上还得益于微生物共生菌的帮助。昆虫宿主相关的微生物共生体参与宿主的多种生命过程，包括营养、免疫、生存适度、繁殖以及与昆虫宿主的共进化和共物种形成等。榕果-传粉榕小蜂共生体系是阐释昆虫和植物之间共生和协同进化的重要经典模型。经过上百万年的进化，传粉榕小蜂和榕树之间几乎形成了严格的“一对一”模式。在研究两种大生物的共生过程中，已经有越来越多的生态学家开始关注细菌或真菌，这类第三方参与者在互利共生过程中发挥的重要作用。但是到目前为止，除了知道Wolbachia在榕小蜂中具有非常高的发生和分布率外，我们对榕果-榕小蜂互利共生系统中的其他微生物群体了解的还非常少。Janzen通过观察和论证榕果环境的特殊性，推测认为榕果果腔应当是无菌环境。Miller采用培养法从无花果中培养到真菌和细菌各一株。近期，Martinsin采用非培养的方法从六种榕树和传粉榕小蜂中检测到大量真菌。我们推测榕树榕小蜂系统中应当还有大量的细菌微生物的存在，这些细菌微生物在榕树榕小蜂的共生过程中可能扮演者非常重要的角色。根据环境选择假说和元基因组理论，在特殊的果内环境选择压力下，榕果榕小蜂相关微生物群体组成或者基因进化等应当先于榕小蜂发生变化，以帮助榕小蜂适应榕果的选择压力。这为验证微生物的环境选择假说和元基因组进化理论提供了良好的模型。本研究中，我们以16S rDNA克隆文库和传粉榕小蜂基因组为数据基础，调查了对叶榕榕果子房和四种榕小蜂相关细菌微生物的多样性。对此，我们希望可以解决如下几个问题：1）榕果环境是否为前人所认为的无细菌环境？2）本系统的细菌群体和开放或者半开放环境中的是否不同？3）榕小蜂相关的细菌群体来源于榕果、亲代小蜂还是其它渠道？4）同一微环境中密切联系的四种榕小蜂是否具有相似的细菌群体？5）是否还存在其它的类似Wolbachia的垂直传播的共生细菌存在？通过对对叶榕四种榕小蜂(Ceratosolen solmsi, Philotypesis pilosa, P. sp. andApocrypta bakeri)相关细菌微生物的13个16S rDNA克隆文库的研究发现：1）对叶榕榕小蜂相关的细菌微生物具有多样性；2）在高级分类水平上，榕小蜂和果外环境中的昆虫具有类似的多样性；在低级分类水平上，榕小蜂表现出特有的细菌群体结构；3）不同种类的榕树及其榕小蜂可能具有不同的细菌群体多样性。由于榕小蜂种类或者生活习性的不同，存在于同一微型果腔内的四种榕小蜂表现出不同的细菌微生物结构特征：1）不同种类榕小蜂相关细菌群体表现为种内差异小和种间差异大；2）四种榕小蜂的相关细菌多样性可以分为三种完全不同的组成，即C. solmsi组、P. pilosa组、A. bakeri和P. sp.组；3）传粉榕小蜂C. solmsi的细菌多样性具有典型的植食性昆虫的特征，以Enterobacteriaceae科为最主要的优势菌群；4)同科不同属的非传粉榕小蜂A. bakeri和P. sp.具有相同的拟寄生寄主，菌群组成结构几乎完全一致，以Tepidimonas属为优势菌群；5）P. pilosa的菌群组成既有别于同科同属的P. sp.也不同于幼虫前期拟寄居的C. solmsi，体现了其潜在的杂食性特征；6）四种榕小蜂的菌群分布格局超越了榕小蜂亲缘关系的范围，表现出和榕小蜂的生态位或食性的完美对应。本研究首次采用磁珠细胞破碎和化学提取相结合的方法提取了对叶榕AB期榕果子房的细菌DNA模板；然后通过设计榕果子房相关细菌微生物的部分16S rDNA序列的特异性引物，在PCR过程中成功排除了叶绿体DNA的干扰，构建了4个C. solmsi产卵前后的16S rDNA克隆文库。通过对AB期对叶榕榕果子房的细菌微生物研究发现：1）密闭果腔中的AB期子房具有细菌多样性，打破了子房“无细菌”的假说；2）传粉榕小蜂C. solmsi产卵前后，总体上子房细菌结构相对稳定，存在部分菌群的微小变化；3）榕果子房具有独特的细菌群体组成，以Delftia属和Acinetobacter属等为主要优势菌群。C. solmsi在子房中产卵后，子房中Acinetobacter属出现Accs支系，通过分析我们推测其是经过C. solmsi产卵发生垂直传播。1）产卵前，Accs在子房中未被检测到，但产卵后，Accs在子房中的丰度约为12%，是Acfh的3~4倍；2）Accs在所有的C. solmsi克隆文库中都稳定存在，丰度和胞内共生菌Wolbachia相近，约2.2%；3）Accs在NCBI序列的最高相似度仅为97%，在系统树上聚为单独一个支系；4）Accs支系和Acfh支系的序列在18个位点存在稳定的碱基差异。C. solmsi基因组数据类似于一个hologenome，包含了大量的微生物信息。通过对细菌、真菌和病毒等的序列数据统计分析发现：1）未组装基因组中微生物多样性的规模极大的超过克隆文库的多样性，可以更加全面的反应相关微生物的组成信息；2）完成组装的基因组中的微生物信息具有偏好性，主要基本上只包含高丰度菌群的序列信息，与克隆文库的研究发现相似；3）基因组中存在部分特殊真菌和病毒如Candida属和茧蜂病毒等。密闭果腔内榕小蜂相关的细菌微生物组成结构与榕小蜂的生态位密切关系为深入研究微生物与宿主的相关性，尤其是holobiont理论提供了良好的研究模型。另外，本研究中所发现的多种特殊的细菌、真菌和病毒等也为研究为进一步深入探索榕小蜂与微生物的共生关系开辟了新的契机。更多还原

【Abstract】 Insects, as the most successful and largest population in the terrestrial, have high fitnessfor lots of inhabits, that partially benefits from their microbial symbionts. Host-associatedmicrobial symbionts take part in many life process of macroorganism, including nutrition,immunization, fitness, and reproduction, and have played significant roles on the cospeciationand coevolution with their host insects. Fig-pollinating wasp mutualism is widely regarded asone of the most classical models to explain mutualisms and coevolution between insect andplant host. Hundreds of species of pollinating fig wasps (PFW), keeping extreme “one to one”pattern, distinguish one responsible fig species especially from others to finish pollination andoviposition in the past millions of years.Ecologists have increasingly pay attention to a third-party, Bacterial or fungi which havebeen known to play very important roles in more and more mutualism interaction, to interpretand reveal these previously unexplored components of even the most classic two-partnerassociations. To date, we have known little knowledge about the microbial communityassociated with fig and fig wasps, except the inheritable endosymbiont, Wolbachia, which hashigh prevalence across fig wasp species. Janzen have reviewed “fig pseudolocule sterility”and indicated that microbial or fungal clones were never found growing in the pseudoloculeof undamaged developing figs and female wasps remain relatively intact for many weeks afterdying in syconium. Miller et al. cultured a single bacterial species, Serratia plymuthica(Lehmann and Neumann) Bergey et al., and a single yeast species, Candida guilliermondii(Castellani), from healthy fig and associated PFW Blastophaga psenes. In contrast, a largefungal microflora was revealed from six species of developing and healthy figs in Panama,using culture-free methods. We supposed that there should also be a specific bacteriacommunity in the fig-fig wasp symbiosis system, and the bacterial microflora may even playsome important roles in uncovering lots of unexplored components of the classic model. Inthe other word, the genes or composition of microbial community associated with fig and figwasps should evolve faster than fig wasps under the constraint of syconia. And the mutualismof fig and fig wasp would be an ideal model to verify the hypothesis “microbe are everywhereand environment select” and the theory of hologenome.In the present study, we have screened the microbial communities of the four species offig wasps (PFW: Ceratosolen solmsi; NPFWs:(Apocrypta bakeri, Philotrypesis pilosa andPhilotrypesis sp.) and the fig ovaries associated with Ficus hispida using culture-free methodsincluding16S rDNA clone libraries and high throughput database from genome. Herein, we asked five questions:(1) whether the bacterial communities associated with the fig-fig waspsymbiosis system are as simple as the “sterile” syconium, consistent with previous studies?(2)Is the bacterial community of the system, different from the other habit?(3) Where does thebacteria community of fig wasps come from, fig fruit, mother wasp or some other uncertainvariables?(4) Whether the bacterial communities of the four species of wasps varied, due totheir different ecological niches in the small house?(5) Is there any other endosymbiontexisting within fig wasps, response to the regulation of wasps’ reproduction, in addition toWolbachia?We have reached to the following conclusions with our research: Firstly, the bacterialcommunities associated with the four fig species of Ficus hispida present three characters:1)Fig wasps contain large bacterial communities much more than previous estimation.2) Figwasps and other insects living outside fig share similar bacterial diversity on the level ofphylum, however, they distinguish with each other on lower level with taxa.3) Different figspecies and their fig wasp probable contain different bacterial diversity.Secondly, the four fig wasp species with different taxonomic status and life habitspresent different bacterial compositions:1) There are small differences with intraspecies andlarge differences with interspecies.2) There are three distinct bacterial compositions, one forC. solmsi, the second for P. pilosa, and the third for A. bakeri and P. sp.3) The bacterialdiversity of C. solmsi shows the typical characters of the bacterial communities withphytophagous insects, which are rich in family Enterobacteriaceae.4) A. bakeri and P. sp.which belong to two different genera within the same family displays almost same bacterialcompositions corresponding to their similar life habits, and they are especially rich in genusTepidimonas.5) The bacterial composition with P. pilosa is different with those of the otherthree fig wasp species, and implies its different feeding habit.6) The three different bacterialcompositions with four wasp species are corresponding to their different ecological niches butnot their genetic relationship.Thirdly, through the thoroughly DNA extraction of bacteria within fig by cell disruptionand chemical extraction and targeted detection using specific primers which could not bind tothe ctDNA, we have succeeded to detect the bacterial diversity of fig ovaries with Ficushispida. Though simpler than the complicated bacterial diversity of rhizosphere, there are alsokinds of bacteria existing in fig ovaries other than sterile. The dominant consortium whichtypically includes genera Delftia and Acinetobacter and so on, are not infected apparently bythe behavior of oviposition. However, Accs strain emerged from ovaries after oviposition, andthen we got four evidences to verify the vertical transmission for the Accs strain.1) Accs strain existed in every clone libraries of C. solmsi with average relative richness2.2%whichsimilar to that of Wolbachia.2) The relative richness of Accs within ovaries is about12%,3times more than Acfh.3) Accs strains cluster into one branch on the NJ tree, with97%similarity with the most similar sequence in NCBI.4) There are18ribonucleotides divergencebetween Accs and Acfh.The genome database of C. solmsi is equal to the hologemoe of C. solmsi, which includealmost all the genome messeges of microbe. Through statistial analysis with these sequencesof microbe within the genome database, we found that:1) The real diversity of microbeswithin the holobiont of C. solmsi is much more than the detected result though clone libraries.2) the assembled genome of C. solmsi only contain little microbial sequences whichcorrespond to those dominant bacteria within clone libraries.3) There are some potentiallyimportant fungi and viruses sequences exsiting in the database which provide new chance toexplore the extensive interaction between fig wasps and their microbes.The interesting concordence between bacterial compositions and ecological niches of figwasp species within the same inclosed syconia would play an important role on the furtherresearch on interaction between hosts and its symbionts. Moreover, this system provides anideal model for hologenome theory. Great amounts of microbes, including bacteria, fungi andviruses, were uncovered in this study, which would provide more pathways to mine fig and figwasp system.更多还原