| The unicellular and eukaryotic microsporidia, are a large group of obligated intracellular microbial pathogens, known as the most successful parasites in nature, shows an extremely diversity in the Microsporidia during the long history of evolution. Researchers have studied on microsporidia for a long time since 1857, in the time of Nosema bombycis was firstly identified by Carl Nageli. Currently reported microsporidia comprise>1500 species in more than 187 genera. Some reports supported the hypothesis that the numbers of microsporidian species may be very close to that of the species of animal. Microsporidia have a broad host range including almost the entire vertebrate (including human) and invertebrate (particularly insects). So far, the microsporidian infection of a lot of hosts including Daphnia magna, nematodes, insects (including locustae, honey bee and silkworm), fish, rabbit and human were confirmed and these microsporidia genomes were sequenced which provide a global insight to Microsporidia biology. It is reasonable to deduce that there are still a large amount of unknown microsporidia species, which remains to be for further research.Papilio machaon Linnaeus, an insect of Lepidoptera, is often used as ornamental butterfly due to its beautiful wings, and considerable important medicinal resource. A microsporidian which is similar to N. bombycis in ultrastructure and genome sequence was isolated from P. machaon in 2012. The genomics study of this pathogen is of great importance for the research on microsporidiosis of P. machaon, and also for the origin and evolution of N. bombycis. Based on the whole genome sequencing data, the prediction and annotation of protein-coding sequences were performed. Gene duplication, genome structure and transposons were analyzed by comparing with the genome of the microsporidia and N. bombycis.The major results were listed below:1. Isolation and identification of the pathogen from Papilio machaon LinnaeusA microsporidian was isolated from Papilio machaon Linnaeus. It was firstly identified morphologically and then 16S rDNA were sequenced. We found the pathogen has a close phylogenetic relationship with N. bombycis. The mature spore was 3.22±0.46 μm × 1.96±0.30 μm in size. Dual-nuclei structures and 10-13 polar filament coils inside cytoplasm were observed. And no parasitophorous vacuole were found in host cells. The complete sequences of rDNA is 4286 bp (KM190863), which has a 98% similarity with N. bombycis. Silkworm infection analysis showed the spores isolate from P. machaon can infect silkworm. The phylogenetic trees of rRNA genes show that it belong to Nosema, and was a sister to the clade comprising N. bombycis, Nosema spodopterae, and Nosema sp. PX1, belong to Nosema. We named this microsporidia as Nosema sp. PM-1.2. Annotation analysis of genome sequence of Nosema sp. PM-12.605 protein coding sequences (CDSs) were predicted with Glimmer3 software from the TEs pre-masked genome sequences composed of 188 scaffolds, with the average length of 795 bp, accounting for 26.9% of the whole genome (7.7 Mb). The gene number of Nosema sp. PM-1 was only the half of N. bombycis, with the similar protein length distribution compared with that of N. bombycis. Nr, SwissProt, KEGG and GO database were used for gene annotation and functional classification. A total of 2,264 genes were annotated,963 of which were classified by Gene Ontology annotation and 1,344 genes owned functional annotations from Nr database.2,182 genes with functional domain was found in Nosema sp. PM-1, using the SMART DB. And 385 of them contain transmembrane domain, which may contribute to the parasitic life cycle.3. The analysis of gene families in Nosema sp. PM-1Paralogous predictions were performed for all CDSs of Nosema sp. PM-1 and TV. bombycis.291 multi gene families were built from 944 Nosema sp. PM-1 CDSs, covering 36.2% of total genes. For N. bombycis,1,077 paralogous families were clustered from 3,639 CDSs, covering 71.0% of total genes. The paralogous analysis suggested that, after speciation from the last common ancestor, Nosema sp. PM-1 and N. bombycis experienced distinct diverse genome evolution. The events of gene duplication were occurred for only 36.2% genes of Nosema sp. PM-1, while in N. bombycis, this value arrived at 71.0%. This conclusion should contribute to explain the difference of genome size between these two species. In order to adapt to different parasitic environment, microsporidians suffer from different selection pressures, and experienced diverse gene duplications. N. bombycis may undergo larger gene duplications compared with Nosema sp. PM-1.1,756 orthologous clusters were identified from all 7,733 proteins of Nosema sp. PM-1 and N. bombycis, based on the software OtrhoMCL. Tor Nosema sp. PM-1,1,878 (72.1%) proteins are homologous to 2,490 (48.6%) N. bombycis proteins. Such high percentage of orthologous confirmed the close relationship between the two species. Functions of the common genes shared among the two microsporidia principally related to some fundamental pathways, such as catalytic, binding, cellular process and metabolic process, which defined a category of core gene families as the gene basis to construct the microsporidia.Some important genes, such as polar tube protein, cell wall protein, Ricin B-lectin protein, serine protease inhibitor, were identified in Nosema sp. PM-1 genome. Using the annotation information and blastp result of the proteins from Nosema sp. PM 1 and N. bombycis,103 differential genes specific to N. bombycis were identified among 2605 genes of Nosema sp. PM-1. Accordingly, among 5128 genes of N. bombycis,423 differential genes specific to Nosema sp. PM-1 were identified.4. Gene duplications in Nosema sp. PM-1Segmental duplications, tandem duplications and interspersed repeat were identified in Nosema sp. PM-1, based on the blast result of genome sequence and protein sequence against themselves. There were differences in types and copies of gene duplications in the two microsporidia. In Nosema sp. PM-1,250 segmental duplications (10.9% of whole genome) were identified; 238 genes enclosed in 106 tandem duplication segment (7.4% of whole genome); and 534 interspersed duplication genes with the length of 116 kb (1.5% of whole genome). In N. bombycis,993 segmental duplications (21.6% of whole genome),86 segmental duplications (186 genes,2.7% of whole genome) and 1333 interspersed duplication genes (3.2% of whole genome) were found. The data suggested that Nosema sp. PM-1 prefer to tandem duplication, while N. bombycis prefer to segmental duplication.5. The genomic synteny analysis between Nosema sp. PM-1 and//, bombycis443 synteny blocks were identified based on the location of orthologs in each genome, with the 4.1 Mb (53%) sequences in Nosema sp. PM-1 and 4.2 Mb (27%) sequences in N. bombycis. The gene sequences in synteny blocks were quite conservative, while the non coding sequences were much more variable. Synteny map based on scaffolds in Nosema sp. PM-1 shows that one scaffold in Nosema sp. PM-1 usually matched with several scaffolds in TV. bombycis, and the total of 78 scaffolds of Nosema sp. PM-1 matches 271 scaffolds of N. bombycis, which may suggest that the genome structure of Nosema sp. PM-1 and Nosema bombycis were quite different.The longest synteny block between Nosema sp. PM-1 and A;. bombycis with 39 pairs of homologous genes was more than 100 kb, containing 48 genes from Nosema sp. PM-1 and 66 genes from N. bombycis respectively. Sequences with high identity were identified within this synteny block. However, the delete\insert of genes and gene duplication, gene reverstion also locate in it. The high identity of genome sequence indicate the close relationship within the two species, while the differences indicate that the two microsporidia experienced diverse genome evolution.6. Identification and phylogenetic analysis of transposonsBy searching the Repbase with RepeatMasker,670 transposons were identified in Nosema sp. PM-1 genome, with the total length of 381 kb. In Nosema sp. PM-1, the percentage of transposons in whole genome was 4.95%, much smaller than that of 38.57% in N. bombycis. Gypsy was the largest family of transposon in Nosema sp. PM-1, with 101 copies and a total length of 141 kb, occupied 1.83% of whole genome size.The structure-based software MITE-Hunter was applied to search for MITEs in the Nosema sp. PM-1 genome.21 families of MITEs were characterized and named NpmME1-NpmME21. Thirteen of 21 MITEs families are completely novel based on the nucleotide composition of target site duplication (TSD) and terminal inverted repeats (TIR). Five of 21 NpmMEs belong to Stowaway-like family, one belongs to Tourist-like family and two in Pegasus-like family. By comparison of N. bombycis MITEs, NpmME20 were the same as NbME1, with the copy number up to 30 times than the latter, indicating that the family might undergo a burst expansion after species divergence.The pairwise distance within each family was calculated, and the results showed that distance of NpmMEl and NpmME8 were obviously larger than NpmME10, NpmME13 and NpmME16, suggested that the first two families were potentially older. NpmMEs were widely, nonrandom, distributed in the genome. Most NpmMEs preferentially inserted into flanking region rather than into gene region. The structural characteristics and the distribution of NpmMEs copies in the Nosema sp. PM-1 genome were investigated, and it was found that NpmMEs might associate with gene sections and change the gene structure.By studying the microsporidia isolated from Papilio machaon Linnaeus, we identified a new species of Nosema, Nosema sp. PM-1. The morphological characteristics were observed, and integration of Bombyx mori was confirmed, which provide useful material to the epidemiology of Silkworm Pebrine. Taxonomic and comparative genomics study showed that Nosema sp. PM-1 and N. bombycis were closely related, but different. The predicted genes and information of transposons provide much useful data for the study of functional genomics and genome evolution of Nosema genus. |
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