【作者】 王宁新；

【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， 2009， 博士

【摘要】 Wolbachia是一种广泛存在于节肢动物体内的胞内共生菌,早期研究表明节肢动物中约16%的物种被感染。近来根据Long-PCR等检测手段发现约76%的物种感染Wolbachia,同时发现不少寄主感染多株系的Wolbachia,因此推断Wolbachia可能是目前分布最广、丰度最大的胞内共生微生物类群。母系遗传的胞内共生菌Wolbachia能够操纵寄主的生殖活动,如诱导精卵不容、孤雌生殖、雌性化、杀雄作用等。此外,研究表明Wolbachia在物种形成过程中起到重要作用。动物的线粒体基因(mtDNA)具有许多独特的优点:母系遗传;基因组较小;重组率低;变异速度快(一般认为线粒体基因是核基因进化速率的4-10倍)等。线粒体基因逐步成为重要的分子标记,广泛应用于种群遗传学、生物进化和系统发育等相关研究。mtDNA长期以来被认为是一种中性进化的分子标记,然而近来一些研究者发现跟其它基因组基因相比,整个线粒体实际上处于非常强烈的选择压力之下,接受着来自线粒体本身的直接选择和其它母系遗传因素的间接选择,以一种“非中性”的速率进化。任何母系遗传的相关因素都有可能影响到线粒体基因的变异,胞内共生菌Wolbachia就是一个典型例子。Wolbachia能够影响寄主线粒体基因的变异,因此可以通过寄主线粒体基因的变异来研究Wolbachia的感染情况。Hurst总结前人研究结果表明内共生菌对寄主的线粒体基因(mtDNA)能够产生的影响,对已有的例子进行分析,可以分为四类:(1)内共生菌驱动的mtDNA多样性的降低;(2)内共生菌驱动的多样性的增加;(3)内共生菌驱动的空间范围内mtDNA的变异;(4)内共生菌连带mtDNA并系的产生。根据前人对不同地区榕小蜂感染率的研究发现,榕小蜂几乎是感染Wolbachia最多的昆虫之一,达到59%-67%。榕小蜂长期生活在密闭的果内,果内小蜂的感染情况以及对寄主线粒体基因可能的影响引起我们极大的研究兴趣。在本文中,我们主要探讨了榕树Ficus hispida中四种榕小蜂体内Wolbachia感染率以及对不同寄主mtDNA的影响。感染率的初步研究结果表明,在Ficus hispida中,传粉榕小蜂Ceratosolen solmsi和非传粉小蜂Philotrypesis pilosa感染Wolbachia,而另外两种非传粉小蜂Philotrypesis sp.和Apocrypta backeri则完全未感染Wolbachia。并且,两个感染Wolbachia物种中的感染率也不同,C. solmsi是89.3%,而P. pilosa的感染率是100%。进一步通过Wolbachia的两个特异基因wsp和ftsZ序列得到结论:传粉榕小蜂C. solmsi只感染一个Wolbachia株系,而非传粉榕小蜂P. pilosa感染多株系Wolbachia,其中两个株系是相对比较稳定的,并且其中一个株系与感染C. solmsi的株系只有一个碱基的差异。这说明Wolbachia除了垂直传播以外,在这两个物种当中很可能还存在水平传播,尽管传播途径还不是很清楚。从聚类树上可以看出,所有的C. solmsi个体依据它们的感染状态被分成两大支,并且两个线粒体分子标记(COI和Cytb)得出相似的拓扑结构,意味着它们有着相似的变化趋势。分别对比感染和未感染Wolbachia个体间的线粒体基因序列(COI和Cytb),我们发现二者差异明显:一方面,对于COI和Cytb,感染和未感染类群间的核苷酸差异分别达到9.2%和15.3%;另一方面,同样对于这两个线粒体基因,感染和未感染类群内部核苷酸的差异均小于1%。进一步比较发现这两个线粒体基因,感染个体较未感染个体显示出明显降低的多样性。P. pilosa和P. sp.的COI和Cytb两个基因的拓扑结构存在一定差异。对于COI,P. pilosa和P. sp.均出现非常多的单体型;而对于Cytb,P. pilosa种内依然出现较多不同的单体型,而P. sp.中出现的单体型则相对较少。数据表明,对于COI,这两个姐妹种在核苷酸多样性等方面都非常相似,差异不大;对于Cytb,与COI不同,两姐妹种间差异较大。使用三个不同的中性检验方法对C. solmsi,P. pilosa和P. sp.进行检测发现,只有C. solmsi中感染Wolbachia个体的COI序列明显偏离中性进化,而所有个体的Cytb基因,以及未感染Wolbachia的C. solmsi的COI基因均符合中性进化。核基因结果也非常有意思,对于ITS2片段来说,种内个体的序列几乎完全一致,从分子数据上确定了我们所取的标本来自同一物种,并且排除了其它因素的作用,证明是Wolbachia的存在对寄主产生了影响。对于EF1-α,姐妹种P. pilosa和P. sp.的序列非常混乱,很难将两个种区分开来,可能是由于我们所取的基因片段非常保守的缘故;另一方面,在传粉榕小蜂中,感染和未感染个体的EF1-α存在三个固定位点碱基的变异,尽管这三个碱基的变化并没有引起任何氨基酸水平的变异,目前还不能对此做出一个满意的解释。通过对Wolbachia株系的研究,推测在对叶榕中,Wolbachia的两种传播方式并存,或许水平传播方式也是大量榕小蜂感染Wolbachia的一个可能原因。通过对wsp基因序列研究发现,wsp存在明显的基因重组现象。基因重组现象是细菌新物种形成的一个重要方式。对Wolbachia基因组大量基因的研究发现,存在许多重组的蛋白基因,这也从另一个方面解释了大量不同株系存在的原因。在研究Wolbachia对线粒体基因的影响的同时,还在两个姐妹种P. pilosa和P. sp.的COI中发现大量的线粒体假基因,而Cytb中未出现,同时在对叶榕中另外两种小蜂的线粒体基因中均未发现线粒体假基因的存在。这很可能是两姐妹种的COI基因在进化中受到特殊的压力造成的。在这些假基因中,最明显的特征就是大量终止密码子的提前出现。姐妹种中很多个体都同时得到两个或多个不同的线粒体假基因序列。进一步研究发现,这些假基因都是在两个姐妹种发生分离之后产生的;并且可能是通过独立的从线粒体到核基因组的转移和转移后复制事件两种方式产生的。在对F. hispida四种小蜂的研究过程中发现,内共生菌Wolbachia和线粒体假基因的存在都对线粒体基因的使用产生不同程度的影响。这对我们一直使用的线粒体分子标记产生影响,尤其是对感染Wolbachia的节肢动物的系统进化、种群结构等研究提出了严峻考验,提醒我们在使用线粒体分子标记时一定要慎重,以防得到的结论有偏差。一个如此奇妙的小小的榕果,带给我们如此多的研究结果和未知谜团,等待进一步深入挖掘。更多还原

【Abstract】 Wolbachia are widespread intracellular bacteria that are found in arthropods and nematodes. Some earlier studies showed that about 16% of the arthropods were infected with Wolbachia, however, a recent meta-analysis estimated that more than 76% of insect species harbor Wolbachia, and some hosts harbor multiple strains of Wolbachia, which probably made it the most abundant intracellular bacteria genus so far discovered.This maternally inherited endosymbiont could manipulate a lot of reproductive processes in invertebrates, including sperm-egg incompatibility (cytoplasmic incompatibility), parthenogenesis, feminization and male killing; meanwhile, it is proposed that Wolbachia also play important roles in speciation.Mitochondria possess many ideal properties: maternally inherited; smaller genome size; higher evolutionary rate and lower recombination rate than nuclear genes, and so on. mtDNA has therefore remained the marker of choice in many populations, biogeographic and phylogenetic studies.mtDNA were regarded as neutrally evolutionary markers for a long time, however, when compared with other genomes, the whole mitochondrial genome evolves under great pressures, not only from the mitochondria itself (direct) but also from other maternally inherited factors (indirect) influences. Any maternally inherited factor could influence its host mitochondrial variations, and endosymbiont Wolbachia is a splendid case, so the host mtDNA no doubt reflects their long evolutionary history with intracellular bacteria. Hurst (2005) summarized earlier relevant studies and classified into four categories: (1) symbiont-driven reduction in mtDNA diversity, (2) symbiont-driven increases in diversity, (3) symbiont-driven changes in mtDNA variation over space and (4) symbiont-associated paraphyly of mtDNA.Based on some Wolbachia prevalence investigation, fig wasps has been proved almost to be the highest infected insects, up to 59%-67%. Fig wasps live in an airtight fig fruit most of their lifetime, so the infection status and the effects of Wolbachia on different hosts attracted our great interests. In this study, we made a survey of the infection status and unveiled the effects of Wolbachia on different hosts of four fig wasps species associated with Ficus hispida.The pollinating fig wasp Ceratosolen solmsi and one of the non-pollinating fig wasps, Philotrypesis pilosa, were infected with Wolbachia, and the other two non-pollinating fig wasps, Philotrypesis sp. and Apocrypta backeri, were uninfected. Moreover, the incidences of the two infected species were different, and C. solmsi was 89.3% while P. pilosa was 100%. Based on two Wolbachia specific gene, wsp and ftsZ, all C. solmsi individuals harbored one strain of Wolbachia, while P. pilosa individuals harbored different strains of Wolbachia. Two of the P. pilosa strains were comparatively stable and one strain shared with C. solmsi except one base variation, which suggesting that Wolbachia from the two species may have horizontal transmission besides vertical transmission, although the routes were unknown.All the C. solmsi individuals were divided into two groups, according to their infection statuses, based on mtDNA neighbor-joining trees. And the two mitochondrial markers (COI and Cytb) got similar topologies. The nucleotide divergence of COI and Cytb between infected and uninfected were up to 9.2% and 15.3% respectively, meanwhile, the differences within infected and uninfected individuals were both less than 1% for either mitochondrial gene. Compared with uninfected individuals, the infected ones showed reduction in mtDNA diversity.Based on the COI and Cytb clustering trees, P. pilosa and P. sp. differed a lot in their topologies. For COI, both of the sister species owned a lot of different haplotypes and they shared a lot in nucleotide diversity; for Cytb, P. pilosa still owned many haplotypes, while P. sp. had few haplotypes, and they differed a lot from each other in nucleotide diversity.Based on three tests of neutral evolution, only the infected individuals of C. solmsi evolved departing from neutrality, and all the Cytb genes and the infected individuals of C. solmsi evolved neutrally. And the results of nuclear markers were also very interesting. For ITS2, almost the same results were got from the either species, which confirmed that all our samples were selected from the same species and excluded some demographic factors influences based on the ITS2 data. As for EF1-α, the results of sister species P. pilosa and P. sp. were in chaos, and it was hard to differentiate the two species, probably because EF1-αis conserved and it is more suitable for classifying higher taxonomic level species; on the other hand, the infected and uninfected C. solmsi differed from each other by three fixed nucleotide of EF1-α, although they didn’t cause any variation at the amino acid level, and so far it has not got a satisfying answer.It was postulated that vertical and horizontal transmissions of Wolbachia co-existed in F. hispida. Probably the horizontal transmission is one of the reasons why so many fig wasps were infected with Wolbachia. wsp showed evident recombination, which is one way of bacteria speciation. Dipping into Wolbachia genomes, there was a lot of recombinant proteins, which could explain why there are so many different stains of Wolbachia.In the process of investigating the effect of Wolbachia on host mtDNA, we co-amplified many Numt (nuclear mitochondrial genes) of COI from two Philotrypesis sister species, while none were found in other two fig wasps. It may suggest that COI of the two Philotrypesis species received different pressures from other genes. The appearance of the stop codon was the most characteristic of these Numt. Many individuals possessed more than two Numt sequences. It was concluded that these transfer events happened after the divergence of the two sister species, and independent transfer from mitochondria into the nuclear and duplications of the transferred fragments were both involved in appearance of so many Numt.Based on our research, both the endosymbiont Wolbachia and Numt could influence the employment of authentic mtDNA, which make the results based on the mitochondrial marker unreliable, especially in the phylogeny reconstruction and population structure of infected arthropods. It reminded us to be more careful when using the mitochondrial markers, in case of getting false conclusions.Such a small fig fruit has brought us with so many interesting results and puzzles, more and more work needs to be done.更多还原