| 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.
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