【作者】 魏丹丹；

【导师】 王进军；

【作者基本信息】 西南大学 ， 农业昆虫与害虫防治， 2012， 博士

【摘要】 书虱(booklice)隶属于啮虫目(Psocoptera)、书虱科(Liposcelididae)、书虱属(Liposcelis),是世界范围内广泛发生并造成严重经济损失的一类储藏物害虫。书虱还能携带、传播病菌和引起过敏反应,直接威胁人们的身体健康。由于书虱具有取食范围广、活动能力强、繁殖率高和繁殖方式多样的特点；在防治上长期不合理地使用化学药剂,使书虱对多种储粮保护剂及熏蒸剂产生了较高的忍耐力或抗药性,其防控难度很大。常见的书虱种常混合发生,因而对书虱进行准确的物种鉴定是书虱有效防控及开展后续相关研究的前提。目前,国内外书虱的研究主要集中在基础生物学、与环境胁迫相关酶系的生理生化及分子生物学、以及应用防治等方面,而对书虱种群遗传与进化的研究非常少见。深入研究书虱种群遗传结构,有利于从分子水平上理解书虱的生态适应及成灾机制,为制定该类害虫的综合防控策略提供遗传学信息。此外,在啮总目中,书虱与寄生虱亲缘关系的界定一直存在争议,用于书虱分子系统发育关系研究的分子标记都局限于个别基因片段的联合或单个基因序列,说服力不强,而基于线粒体基因组序列的系统发育关系的研究,其结果更为准确、可靠。迄今为止,在300多种已完成线粒体基因组测序的昆虫中,仅有一种皮虱属于啮虫目,而关于其它啮虫目昆虫的线粒体基因组至今未见报道。因此,测定书虱线粒体基因组序列,不仅可以丰富昆虫线粒体基因组数量,推动昆虫比较基因组学和分子系统学研究的发展,而且还可阐明啮虫目和虱目之间的亲缘关系,有利于认识和理解两侧对称动物线粒体基因组进化的特征。本学位论文紧密围绕书虱种群遗传与进化这一主线,开展了一系列研究。在建立书虱常见种快速鉴定的分子技术的基础之上,重点解析书虱的种群遗传结构,测序获得书虱线粒体基因组序列,分析书虱线粒体基因组的进化属性和特点,重建基于线粒体基因组序列的啮总目昆虫系统发生关系,并构建书虱微卫星富集文库。本论文的主要研究结果如下：1、在全国范围内采集获得了嗜卷书虱Liposcelis bostrychophila Badonnel、嗜虫书虱L. entomophila (Enderlein)、小眼书虱L. paeta Pearman、三色书虱L. tricolor Badonnel、无色书虱L. decolor (Pearman)和云南书虱L. yunnaniensis Li&Li等6种书虱的20个地理种群。对6种书虱100头个体的核糖体转录内间隔区ITS(ITS1-5.8S-ITS2)基因进行克隆测序发现,6种书虱ITS1序列的长度变异范围在349-390bp之间,而ITS2序列的长度变异范围在198～418bp之间。基于ITS数据集计算的6种书虱种间水平的核苷酸歧异度(0.4167～0.7143)远远高于其他昆虫属内的种间水平,甚至达到某些昆虫科内的属级水平。鉴于ITS2基因的长度变异更加剧烈,本研究以ITS2作为书虱属内种间鉴定的DNA条形码,建立了一种基于多重PCR技术的快速鉴定这6种书虱的分子方法。利用ITS数据集,采用最大似然法(ML)和邻接法(NJ)构建的系统发育树显示,6种书虱可以很好地分为3支,这与传统形态分类学的结果相一致,说明ITS基因序列也适宜作为书虱分子系统发育关系研究的分子标记。2、为揭示书虱种内遗传多样性水平以及基因流的范围和方式,利用线粒体Cytb基因部分序列和核糖体ITS全序列对嗜卷书虱和嗜虫书虱共15个地理种群的种群遗传结构进行了比较研究。(1)基于Cytb数据集,共测序获得了103头嗜卷书虱和148头嗜虫书虱的Cytb基因片段,分别得到40个和39个单倍型。综合贝叶斯系统发育树和单倍型网络进化图的分析结果发现,嗜卷书虱的40个单倍型分为两支,它与两大动物地理区划西南区(SWCR)和华中区(CCR)的分布相吻合。对于39个嗜虫书虱单倍型,贝叶斯系统发育树与网络进化图均显示,来自同一种群或同一地理区域的单倍型并未聚在一起,即无种群或区域特异支。多层次的分子变异(AMOVA)分析表明,两种书虱种群在地理区划间存在显著的遗传结构。在进行书虱种群历史动态分析时,中性检验和误配分析参数表明,书虱在近期经历了种群扩张事件。两种书虱成对种群间的固定指数FST值多数大于0.33,说明两种书虱的种群遗传分化程度很高。然而基因流的计算结果显示,在这些种群之间存在着较高水平的基因流。这种看似矛盾的现象是由于书虱独特的生活习性所致,因为书虱种群中的个体趋于与近邻的个体相交配,这是导致其在同一分布区域的两端种群之间的基因流显著降低的重要原因。(2)基于ITS数据集,共测序获得了74头嗜卷书虱和124头嗜虫书虱的ITS序列,分别检测到59个和77个单倍型。贝叶斯系统发育树和单倍型网络进化图的分析结果均显示两种书虱无种群或地理区域特异支。AMOVA分析的结果表明,两种书虱在地理区划间不存在显著的遗传结构,这一结果与利用Cytb基因的研究有所不同,其差异的原因很可能是由于线粒体Cytb基因和核糖体ITS基因序列的进化速率不同,以及取样分布和样本量大小不一致所造成的。两种书虱成对种群间的固定指数FST值显示,高水平的种群遗传分化在两种书虱种群间普遍存在。(3)综合Cytb和ITS两种数据集的结果可以得出以下结论,两种不同生殖模式的书虱均具有很高的遗传多样性,且营专性孤雌生殖的嗜卷书虱遗传多样性高于两性生殖的嗜虫书虱遗传多样性,这一研究结果为解释书虱具有极强的环境适应能力提供了遗传信息上的证据。两种书虱种群间具有显著的遗传分化,但基因流相对较高,这就佐证了书虱具有易于随人类活动而传播和容易产生抗药性的特点。通过中性检验和误配分布检验的分析发现,书虱在近期经历了种群扩张事件,说明书虱即使在经历种群萎缩后,仍可以由极少数个体迅速建立起庞大的种群。Mantel检测表明,种群间的遗传距离与地理距离无相关性,即地理隔离并不是造成遗传分化的主要原因。因而,书虱种群间的遗传分化可能更多的是由随机遗传漂变、近交效应或防治策略所导致。3、对书虱属代表种嗜卷书虱的线粒体基因组序列进行测定和分析。结果发现,嗜卷书虱的线粒体基因组从典型动物的一个环状DNA分子裂化成了两个较小的环状DNA分子,分别命名为mt chromosome Ⅰ (8,530bp)和mt chromosome Ⅱ (7,933bp), GenBank登录号分别为JN645275和JN645276。其中mt chromosome Ⅰ包含22个基因,mt chromosome Ⅱ包含16个基因。此外,两环同时拥有一段长度为945bp的序列,且此序列包含了3个tRNA基因,因而,两环状染色体只包括典型动物线粒体基因组的35个基因,丢失了两个tRNA基因(trnH和trnN)。在两个环状线粒体基因组上均出现了蛋白质假基因,它们的存在说明线粒体小环是典型线粒体基因组在基因删除作用下进化而来的。定量PCR结果显示,mt chromosome Ⅰ和mt chromosome Ⅱ具有不相等的拷贝数,且前者是后者的两倍左右。PCR检测发现,这种特殊的线粒体基因组裂化方式在嗜卷书虱不同地理种群中稳定存在。基于线粒体基因组序列重建的啮总目系统发育树的结果表明,啮虫目和虱目(Phthiraptera)中的细角亚目(Ischnocera)均为并系群,且书虱和整个虱目互为姊妹群。在阐述两侧对称动物线粒体基因组进化趋势时,本论文引入了线粒体核型(mitochondrial karyotypes)的概念。分析发现两侧对称动物线粒体染色体有减小的趋势,其中线粒体基因组的裂化是这种趋势体现的重要策略之一。裂化后的环状线粒体基因组具有以下共同特征：1)不同的小环之间拥有一段相似度很高的序列作为转录和复制的信息位点；2)每个小环必须具有至少一个完整功能的基因。只有具备这样的条件,小环才能逐步取代典型的线粒体染色体大环。这也暗示,动物线粒体主要功能基因的“丢失”,很可能是由于它位于具有上述特征的其他线粒体染色体小环上,只是未被检测到而已。嗜卷书虱线粒体基因组的裂化现象,有助于理解动物线粒体的起源和进化。从其他书虱种(如小眼书虱)的线粒体基因组测序结果看,线粒体基因组的裂化在书虱属中可能是一种普遍的现象。书虱线粒体基因组的裂化和虱目中吸虱类线粒体基因组的多裂化现象很可能是相互独立进化的遗传学事件。4、为了以后更为全面地解析书虱的种群遗传结构,对嗜卷书虱和嗜虫书虱基因组中微卫星位点进行了筛选。利用链霉亲和素与生物素之间的强亲和性原理,将链霉亲和素偶联的磁珠与生物素标记的微卫星探针(AC)12、(TC)12、(ATC)8、(ATG)8、(AAC)8、(ATAC)6及(GATA)6结合后,再退火杂交含接头和微卫星序列的单链书虱基因组DNA限制性酶切片段,经PCR扩增形成双链后进行克隆、建库。本论文成功构建了6个嗜卷书虱微卫星富集文库和7个嗜虫书虱微卫星富集文库；两种书虱13个文库中共随机检测5,218个克隆,其中嗜卷书虱2,542个克隆；嗜虫书虱2,676个克隆,其阳性克隆率在9.38%至100%之间。经检测发现了两种书虱的260条微卫星DNA序列。本研究的微卫星富集文库构建过程是FIASCO法和Bloor的微卫星磁珠富集法的结合。该方法省略了酶切产物胶回收纯化步骤,提高了微卫星富集的效率。对微卫星序列进行分析发现,书虱两碱基微卫星主要以多拷贝微卫星位点存在,且其中有相当一部分是以基因家族的形式存在(不同位点的一侧侧翼序列极为相似),这与鳞翅目昆虫的微卫星家族极为类似。书虱三碱基重复微卫星在最终得到的单拷贝多态性位点中,其所占比例要远高于二碱基微卫星,并具有较高的优化率。这两种书虱微卫星富集文库的建立,将为嗜卷书虱和嗜虫书虱的种群遗传与进化、基因连锁图谱构建和分子系统发育等后续研究提供大量分子遗传标记。综上所述,本论文对6种书虱种间和2种书虱种内两个层次水平的遗传进化规律进行了深入分析；克隆得到了6种书虱的ITS全序列,并由此建立了一套快速鉴定常见书虱种的分子方法,为书虱DNA条形码的开发和应用提供了借鉴,同时也为书虱种间进化关系的研究提供了分子标记；联合使用线粒体基因Cytb和核基因ITS对两种书虱种群遗传结构进行比较解析,系统阐明了书虱现实的种群遗传多样性、基因流的范围和方式、遗传分化程度以及种群历史动态,为解释书虱极强的抗胁迫能力和环境适应能力提供了基础的遗传学信息；获得了嗜卷书虱的全线粒体基因组序列,为啮总目系统发生关系和两侧对称动物线粒体基因组进化模式提供了全新的视角；构建了两种书虱的13个微卫星富集文库,为后续多态性微卫星位点的筛选和书虱种群遗传结构研究奠定了坚实的基础。本研究结果,充实了比较基因组学生物进化的研究内容,从线粒体基因组序列阐明了书虱物种多样性的形成进化机制和书虱与环境之间的相互作用机制,也为书虱抗药性治理方案的制定提供了丰富的遗传学信息,具有重要的理论意义和实践价值。更多还原

【Abstract】 The psocids (booklice), Liposcelis spp.(Psocoptera:Liposcelididae), are major pests of stored grain and commonly occur on a wide range of stored products. Increasingly, the genus of Liposcelis has gained recognition of their importance due to their feeding on stored grains, contaminating food, and agricultural commodities as well as transmitting harmful microorganisms, including fungi and bacteria. Intensive use of chemical insecticides for pest control has facilitated resistance development in psocids. Control of these pests has proven difficult because they do not respond to management tactics that have been developed for other stored-product pests. The psocids have close morphological similarities and often commix occur at the same ecosystems. Therefore, a first step necessary to further implement population study of one Liposcelis species, species-discriminating criteria based on molecular techniques are needed. Previous research has focused on psocid bionomics, physiology and biochemistry, molecular biology, and control measures in grain storage systems. However, the population genetic structure and gene flow of psocids have not been well categorized, which may be useful to understand the formation of resistance mechanisms of psocids to insecticides and helpful to plan appropriate strategies for the control of these pests. Therefore, a detailed understanding of psocid gene flow patterns, determining the geographical origins and dispersal of source populations and the resultant genetic structure among populations within China is also needed. Liposcelididae species has a very special position in the phylogeny of Psocodea. Thus far, complete mt genome sequences have been determined for more than300species of insects. Only one of these species, a barklouse, Lepidopsocid sp., however, is from the order Psocoptera and no booklice has been sequenced for complete mitochondrial genomes. It is necessary to reconstruct the phylogenetic relationships among the major lineages of Psocodea using mitochondrial genome sequences, and the characteristic of mitochondrial genome of psocid will also give us invaluable insights into the evolution of mitochondrial genomes in bilateral animals.In this thesis, we focused on the scientific issue on population genetic and evolution of major psocid species and the research include establishing the molecular technology for quickly identification of Liposcelis spp.; studying of population genetics of two psocid species; sequencing the whole mitochondrial genome of representative psocid species; analyzing the new evolutionary properties and patterns of mitochondrial genome, and constructing the microsatellite enriched libraries of L. bostrychophila and L. entomophila. The main results as follows:1. Nucleotide sequences of the nuclear rDNA internal transcribed spacer (ITS) region were determined in100individuals of six Liposcelis species (L. bostrychophila Badonnel, L. entomophila (Enderlein), L. paeta Pearman, L. tricolor Badonnel, L. decolor (Pearman) and L. yunnaniensis Li&Li) from16locations (20populations) of China. For these six psocid species, the lengths of ITS1sequences ranged from349to390bp and the lengths of ITS2sequences from198to418bp. We also calculate the nucleotide divergence for these six psocid species using the ITS sequence alignments according to p-distances and the result showed that the inter-specific nucleotide divergence (0.4167to0.7143) of these six Liposcelis species was much higher than other insects at the same level and it almost reach the inter-genus nucleotide divergence of other insects. The molecular phylogenetic tree of six Liposcelis species was constructed inferred from the ITS-5.8S-ITS2sequencetypes and was generated from Maximum Likelihood (ML) and Neighbor-joining (NJ) methods. From both the ML and NJ phylogenetic trees, these six species were well divided into three clades which are consistent to the accepted morphological classification. We also developed a molecular identification method for six Liposcelis species based on ITS2sequence. The six reverse primers was designed to bind to one of these six species only and worked with the same forward primer. The identification method involves a single PCR reaction using DNA from a psocid and the special reverse primer was only able to generate a PCR product from its complementary species. Above results demonstrate that ITS1-5.8S-ITS2sequences are useful tools for phylogeny estimation of Liposcelis species and the ITS2sequence can be an excellent tool for species discrimination to facilitate entomological studies of the six species and developed multiplex PCR method here is proved to be reliable when tested through different geographical populations. This study showed that ITS (ITS1and ITS2) sequences displayed considerable differentiation both in length and base composition within psocid species and which suggest that great genetic differentiation existed within these species.2. The two psocids, L. bostrychophila and L. entomophila were sampled in15localities in China and analyzed for polymorphism at mitochondrial DNA (Cytb) and ITS (ITS1-5.8S-ITS2) regions to study genetic diversity in psocid populations at intra-specific levels.(1) In the Cytb dataset, A433bp fragment of the Cytb genes were aligned and analyzed from103L. bostrychophila and148L. entomophila individuals. Totally,39haplotypes were detected in L. entomophila populations and40haplotypes in L. bostrychophila populations. When the Bayesian tree and median-joining network for haplotypes were combined, the40haplotypes of L. bostiychophila were divided into two clades that coincided with our predefined regions (SWCR and CCR region). However, no population-specific clustering pattern was revealed by the Bayesian and median-joining network analysis for the39haplotypes of L. entomophila. AMOVA results showed that there was significant genetic differentiation of L. bostiychophila and L. entomophila populations at various hierarchical levels (among regions, among populations within regions, and within populations), and comprehensive analysis of demographic history changes for L. bostrychophila and L. entomophila populations using two neutrality tests and mismatch distributions indicate a rapid demographic expansion for both species. For L. bostrychophila and L. entomophila, most of the pairwise FST values were statistically significant, which indicate that there was widespread population genetic differentiation within and between two species populations. However, the high levels of gene flow were also detected among these populations and this paradoxical phenomenon could be explained using the specific life habit of psocids.(2) In the ITS dataset,74individuals of L. bostrychophila were sequenced and59haplotypes were detected whereas124individuals of L. entomophila were sequenced and77haplotypes were detected. The phylogenetic analyses and median-joining networks did not show clear pattern of structure for both species according to the same sampling localities, or predefined geographic region. In contrast to the AMOVAs result inferred from the Cytb dataset, several AMOVAs at different hierarchical levels inferred from ITS gene showed that there were no genetic structures of L. bostrychophila and L. entomophila populations according to our predefined regions, however pairwise estimates of FST among L. bostrychophila and L. entomophila populations revealed that the genetic differentiation existed among a large portion of populations. The neutrality tests and mismatch distribution test statistics for both psocid species populations indicated a rapid historically demographic expansion in the past.(3) From above analysis, both Cytb and ITS sequences show high genetic diversity in these two species, and significant genetic structure and population differentiation were also detected. The high genetic diversity of L. bostrychophila and L. entomophila might explain why these species has a broad tolerance to environmental and habitat stresses and the fast mutational processes inherent in individuals, as well as populations, can enable these two Liposcelis species to successfully adapt to complex environments. Compared to sexually reproducing L. entomophila, asexually reproducing L. bostrychophila has a higher genetic diversity. The Mantel test indicated that for both species there was no evidence for isolation-by-distance (IBD). Therefore, the significant population differentiation detected in both psocids may be mainly due to other factors, such as genetic drift, inbreeding or control practices, and less by geographic distance. The neutrality test and mismatch distribution statistics revealed that the two species might have undergone population expansions in the past in the sampled areas.3. In this study, we sequenced the mitochondrial (mt) genome of L. bostrychophila and found that the typical single mt chromosome of bilateral animals has fragmented into and been replaced by two medium-sized chromosomes; each of these chromosomes has about half of the genes of the typical mt chromosome of bilateral animals. These mt chromosomes are8,530bp (mt chromosome I) and7,933bp (mt chromosome II) in size. For mt chromosome I, six protein-coding genes and five tRNA genes were on one strand whereas one protein-coding gene, one rRNA gene and nine tRNA genes were on the other strand. For mt chromosome II, five protein-coding genes and four tRNA genes were on one strand whereas one protein-coding gene, one rRNA gene and five tRNA genes were on the other strand. There is an identical region (945bp) on both of the two mt chromosomes, and three tRNA genes and an extensive non-coding region that contains putative D-loop and control sequences were included in this region. Intriguingly, qPCR test indicated that mt chromosome I is twice as abundant as chromosome II. PCR test of multipartite mt genome are successful in other strains of L. bostrychophila, indicating that the2-chromosome structure is stable but with product size slightly change, which possibly due to deletion of some of non-coding regions. The mitochondrial gene order of L. bostrychophila differs radically from Lepidopsocidae sp.(Psocoptera) and the parasitic lice which are closest relatives of Liposcelididae. Totally, four putative pseudogenes on the two mitochondrial chromosomes demonstrated the formation of mini-circles was proceeding step by step with degeneration of the functional protein genes, and they are also powerful proofed that the recombination were happened between these two chromosomes. It appears that the selection pressure for compact mt genomes in bilateral animals favors small mt chromosomes when small mt chromosomes co-exist with the typical large mt chromosomes. Thus, small mt chromosomes may have selective advantages over large mt chromosomes in bilateral animals. Phylogenetic analyses of mt genome sequences of Psocodea (i.e. Psocoptera plus Phthiraptera) indicate that:1) the order Psocoptera (booklice and barklice) is paraphyletic; and2) the order Phthiraptera (the parasitic lice) is monophyletic. Within parasitic lice, however, the suborder Ischnocera is paraphyletic; this differs from the traditional view that each suborder of parasitic lice is monophyletic. From this study, we summarized that the mini-circle mt chromosomes which can replaced the typical animal single circle mt must have at least one functional gene and one non-coding region which has a high similarity across different mini-circles. We also speculated that the mt chromosomes in bilateral animals have the tendency of reducing their size and the fragmentation of mt genome is one of important strategies of processing of mt genome streaming. According to result of sequencing the mt genome of other Liposcelis species, we intend to conclude that multipartite mt genomes are common in Liposcelis species and the multipartite mt genome of Liposcelis species also give us invaluable insights into the origin and evolution of mitochondria in bilateral animals.4. In the present paper, microsatellite-enriched libraries of L. bostrychophila and L. entomophila were constructed utilizing methodologies that exploit the strong affinity between biotin and the protein streptavidin. We propose a fast and easy protocol which is combination of two different published methods. Briefly, high-quality genomic DNA was digested by the restriction enzyme Msel and then ligated to designed adaptors.250-700bp microsatellite-containing DNA fragments were captured by streptavidin-coated magnetic beads. The beads affinity capture of microsatellite repeats using biotinylated oligonucleotide probes (AC)12,(TC)12,(ATC)8,(ATG)8,(AAC)8,(ATAC)6, and (GATA)6Subsequently, PCR was used to amplify the captured molecules for transferring single strand DNA to double strand DNA. The PCR products (enriched microsatellite DNA fragments) were ligated to pGEM-T Easy vector and transformed into Trans5a competent cells. In total,13microsatellite enriched libraries were constructed; six for L. bostrychophila, and seven for L. entomophila. A total of5,218clones were PCR screened for microsatellite content. The clones of L. bostiychophila and L. entomophila were2,542and2,676, respectively. The percentage of the microsatellite positive clones ranged9.38%-100%. The number of microsatellites detected for these two species was260. Comparative analysis of microsatellite sequences for these two psocids revealed that the microsatellite sequences exist in multiple copies in the psocid genome. In addition, they were found to have similar or almost identical flanking regions. From our present study, the tri-nucleotide libraries yielded greater results for determining gene flow.In summary, in the present study, the genetic evolution patterns of psocids at both the inter-and intra-specific levels were analyzed systematically. Internal transcribed spacers (ITS) regions were cloned from six psocid species. Based on great variation of the ITS2sequences, a molecular identification method for six Liposcelis species was developed. The study here will provide potential molecular markers to investigate the evolutionary relationship among the species of genus Liposcelis and also will provide the DNA barcoding tools in psocids species. Population genetics of two asexually and sexually reproducing psocids, L. bostrychophila and L. entomophila were comparatively analyzed using the mitochondrial Cytb and nuclear ITS gene sequences and a thorough understanding of these two psocids population genetics, including gene flow patterns, genetic diversities and the resultant genetic structure among populations within China is useful for proposing successful integrated pest management tactics for these pests in grain storage systems. The completed mt genome of L. bostrychophila was sequenced and this multipartite mt genome provided new insights into the phylogeny of psocodea and the evolution of mitochondrial chromosomes in bilateral animals. We constructed13microsatellite enriched libraries for two psocids. Accumulation of multiple polymorphic microsatellite loci will greatly facilitate studies of individual identification and they are crucial to investigations of population genetics and evolution, gene linkage mapping, and elucidating the molecular phylogeny of psocids in China and internationally. Therefore, the studies included in this thesis have important theoretical and practical significance.更多还原