Jinshan Xu

Comparative genomics of parasitic silkworm microsporidia reveal an association between genome expansion and host adaptation

BackgroundMicrosporidian Nosema bombycis has received much attention because the pébrine disease of domesticated silkworms results in great economic losses in the silkworm industry. So far, no effective treatment could be found for pébrine. Compared to other known Nosema parasites, N. bombycis can unusually parasitize a broad range of hosts. To gain some insights into the underlying genetic mechanism of pathological ability and host range expansion in this parasite, a comparative genomic approach is conducted. The genome of two Nosema parasites, N. bombycis and N. antheraeae (an obligatory parasite to undomesticated silkworms Antheraea pernyi), were sequenced and compared with their distantly related species, N. ceranae (an obligatory parasite to honey bees).ResultsOur comparative genomics analysis show that the N. bombycis genome has greatly expanded due to the following three molecular mechanisms: 1) the proliferation of host-derived transposable elements, 2) the acquisition of many horizontally transferred genes from bacteria, and 3) the production of abundnant gene duplications. To our knowledge, duplicated genes derived not only from small-scale events (e.g., tandem duplications) but also from large-scale events (e.g., segmental duplications) have never been seen so abundant in any reported microsporidia genomes. Our relative dating analysis further indicated that these duplication events have arisen recently over very short evolutionary time. Furthermore, several duplicated genes involving in the cytotoxic metabolic pathway were found to undergo positive selection, suggestive of the role of duplicated genes on the adaptive evolution of pathogenic ability.ConclusionsGenome expansion is rarely considered as the evolutionary outcome acting on those highly reduced and compact parasitic microsporidian genomes. This study, for the first time, demonstrates that the parasitic genomes can expand, instead of shrink, through several common molecular mechanisms such as gene duplication, horizontal gene transfer, and transposable element expansion. We also showed that the duplicated genes can serve as raw materials for evolutionary innovations possibly contributing to the increase of pathologenic ability. Based on our research, we propose that duplicated genes of N. bombycis should be treated as primary targets for treatment designs against pébrine.

Complete Genome Sequences from Three Genetically Distinct Strains Reveal High Intraspecies Genetic Diversity in the Microsporidian Encephalitozoon cuniculi

ABSTRACT Microsporidia from the Encephalitozoonidae are obligate intracellular parasites with highly conserved and compacted nuclear genomes: they have few introns, short intergenic regions, and almost identical gene complements and chromosome arrangements. Comparative genomics of Encephalitozoon and microsporidia in general have focused largely on the genomic diversity between different species, and we know very little about the levels of genetic diversity within species. Polymorphism studies with Encephalitozoon are so far restricted to a small number of genes, and a few genetically distinct strains have been identified; most notably, three genotypes (ECI, ECII, and ECIII) of the model species E. cuniculi have been identified based on variable repeats in the rRNA internal transcribed spacer (ITS). To determine if E. cuniculi genotypes are genetically distinct lineages across the entire genome and at the same time to examine the question of intraspecies genetic diversity in microsporidia in general, we sequenced de novo genomes from each of the three genotypes and analyzed patterns of single nucleotide polymorphisms (SNPs) and insertions/deletions across the genomes. Although the strains have almost identical gene contents, they harbor large numbers of SNPs, including numerous nonsynonymous changes, indicating massive intraspecies variation within the Encephalitozoonidae. Based on this diversity, we conclude that the recognized genotypes are genetically distinct and propose new molecular markers for microsporidian genotyping.

Identification of NbME MITE families: Potential molecular markers in the microsporidia Nosema bombycis

Six novel families of miniature inverted-repeat transposable elements (MITEs) were characterized in the microsporidia Nosema bombycis and were named NbMEs. The structural characteristics and the distribution of NbME copies in the N. bombycis genome were investigated, and it was found that portions of NbMEs are associated with gene sections. Potential molecular markers for various N. bombycis strains were identified in this study through utilization of the MITE-AFLP technique. Three distinct pathogenic isolates collected from different areas were distinguished, and polymorphisms were detected using the NbME5 marker, thereby establishing this NbME as a potential marker for studying isolate variation in N. bombycis.

Genome-wide transcriptional response of silkworm (Bombyx mori) to infection by the microsporidian Nosema bombycis.

Microsporidia have attracted much attention because they infect a variety of species ranging from protists to mammals, including immunocompromised patients with AIDS or cancer. Aside from the study on Nosema ceranae, few works have focused on elucidating the mechanism in host response to microsporidia infection. Nosema bombycis is a pathogen of silkworm pébrine that causes great economic losses to the silkworm industry. Detailed understanding of the host (Bombyx mori) response to infection by N. bombycis is helpful for prevention of this disease. A genome-wide survey of the gene expression profile at 2, 4, 6 and 8 days post-infection by N. bombycis was performed and results showed that 64, 244, 1,328, 1,887 genes were induced, respectively. Up to 124 genes, which are involved in basal metabolism pathways, were modulated. Notably, B. mori genes that play a role in juvenile hormone synthesis and metabolism pathways were induced, suggesting that the host may accumulate JH as a response to infection. Interestingly, N. bombycis can inhibit the silkworm serine protease cascade melanization pathway in hemolymph, which may be due to the secretion of serpins in the microsporidia. N. bombycis also induced up-regulation of several cellular immune factors, in which CTL11 has been suggested to be involved in both spore recognition and immune signal transduction. Microarray and real-time PCR analysis indicated the activation of silkworm Toll and JAK/STAT pathways. The notable up-regulation of antimicrobial peptides, including gloverins, lebocins and moricins, strongly indicated that antimicrobial peptide defense mechanisms were triggered to resist the invasive microsporidia. An analysis of N. bombycis-specific response factors suggested their important roles in anti-microsporidia defense. Overall, this study primarily provides insight into the potential molecular mechanisms for the host-parasite interaction between B. mori and N. bombycis and may provide a foundation for further work on host-parasite interaction between insects and microsporidia.

Multiple rDNA units distributed on all chromosomes of Nosema bombycis

Among Microsporidia, Nosema bombycis has a novel arrangement of LSUrRNA, SSUrRNA, ITS, IGS and 5SrRNA. To determine the distribution of rDNA among the chromosomes, we performed genome-wide screening and Southern blotting with three probes (SSU, ITS and IGS). Southern blotting revealed that ribosomal RNA genes are distributed on all chromosomes of N. bombycis, which is contrary to the previous result, which concluded that the N. bombycis rRNA genes were limited to a single chromosome. This wide distribution is similar to that of the rDNA unit of Encephalitozoon cuniculi. Screening of the N. bombycis genome detected 53 LSUrRNA elements, 43 SSUrRNA elements and 36 5SrRNA elements. However, it is still difficult to determine their loci on the chromosomes as the genomic map is unfinished.

Identification, Diversity and Evolution of MITEs in the Genomes of Microsporidian Nosema Parasites.

Miniature inverted-repeat transposable elements (MITEs) are short, non-autonomous DNA transposons, which are widespread in most eukaryotic genomes. However, genome-wide identification, origin and evolution of MITEs remain largely obscure in microsporidia. In this study, we investigated structural features for de novo identification of MITEs in genomes of silkworm microsporidia Nosema bombycis and Nosema antheraeae, as well as a honeybee microsporidia Nosema ceranae. A total of 1490, 149 and 83 MITE-related sequences from 89, 17 and five families, respectively, were found in the genomes of the above-mentioned species. Species-specific MITEs are predominant in each genome of microsporidian Nosema, with the exception of three MITE families that were shared by N. bombycis and N. antheraeae. One or multiple rounds of amplification occurred for MITEs in N. bombycis after divergence between N. bombycis and the other two species, suggesting that the more abundant families in N. bombycis could be attributed to the recent amplification of new MITEs. Significantly, some MITEs that inserted into the homologous protein-coding region of N. bombycis were recruited as introns, indicating that gene expansion occurred during the evolution of microsporidia. NbS31 and NbS24 had polymorphisms in different geographical strains of N. bombycis, indicating that they could still be active. In addition, several small RNAs in the MITEs in N. bombycis are mainly produced from both ends of the MITEs sequence.

Natural selection maintains the transcribed LTR retrotransposons in Nosema bombycis.

Eight intact LTR retrotransposons (Nbr1-Nbr8) have been previously characterized from the genome of Nosema bombycis, a eukaryotic parasite with a compact and reduced genome. Here we describe six novel transcribed Nbr elements (Nbr9-Nbr14) identified through either cDNA library or RT-PCR. Like previously determined ones, all of them belong to the Ty3/Gypsy superfamily. Retrotransposon diversity and incomplete domains with insertions (Nbr12), deletions (Nbr11) and in-frame stop codons in coding regions (Nbr9) were detected, suggesting that both defective and loss events of LTR retrotransposon have happened in N. bombycis genome. Analysis of selection showed that strong purifying selection acts on all elements except Nbr11. This implies that selective pressure keeps both these Nbrs and their functions in genome. Interestingly, Nbr11 is under positive selection and some positively selected codons were identified, indicating that new functionality might have evolved in the Nbr11 retrotransposon. Unlike other transposable elements, Nbr11 has integrated into a conserved syntenic block and probably resulted in the inversion of both flanking regions. This demonstrates that transposable element is an important factor for the reshuffling and evolution of their host genomes, and may be maintained under natural selection.

Characterization of a transcriptionally active Tc1-like transposon in the microsporidian Nosema bombycis

The Tc1 transposable element has been found in a wide variety of organisms including vertebrates, insects and fungi but has not been previously reported in Microsporidia. In this study we characterize an intact DNA transposon (NbTc1) from the microsporidian Nosema bombycis. This transposable element encodes a 337 amino acid transposase sequence, which contains the D,D34E functional motif required for transposition. A Southern blot of N. bombycis DNA separated by pulsedesis shows that copies of the NbTc1 transposon are present on 10 of the 14 chromosomes of N. bombycis. Amino acid sequence variation among copies of the NbTc1 is low, suggesting a conserved function for this transposon within N. bombycis. Phylogenetic analysis indicates that NbTc1 is a new member of the Tc1 family lineage, quite distinct from all previously described Tc1 elements, including those from fungi, indicating that NbTc1 forms a unique clade of the Tc1 superfamily. However, the Tc1 transposon is too divergent to resolve the major phylogenetic relationships among these superfamilies. Reverse transcriptase PCR and Solexa sequencing suggest that NbTc1 possesses transcriptional activity. Considering the interest in Microsporidia as biological control agents, the NbTc1 transposon may be a useful vector for the efficient transfection of these important parasites into host species.

A genome-wide analysis of simple sequence repeats in Apis cerana and its development as polymorphism markers.

The Asian honeybee (Apis cerana) is an important indigenous species that play an indispensable role in the ecological balance and biological diversity. Few studies have been conducted to characterize the simple sequence repeats (SSRs) derived from A. cerana, so, in this study, a genome-wide screening for SSRs were firstly performed in the genome of A. cerana by comparison with that in west honeybee (Apis mellifera). There were 20,9991 SSRs distributed throughout the genome of A. cerana (Korea strain) and di-nucleotides were the most frequent SSR type. Both total number and density of SSRs in A. cerana genome were smaller than that in A. mellifera genome. Through comparing length discrepancy of SSRs loci among several isolates based on sequence alignment, 218 potential polymorphic SSRs primers derived from A. cerana were presented. Five among these SSR markers were evaluated for amplification in twenty-eight colonies of Apis cerana cerana (Chinese honeybee), which showed highly polymorphic, with the value of Polymorphism information content (PIC) ranging from 0.47 to 0.61. All these results will contribute to further develop more effective SSRs markers derived from A. cerana, which can be used to study genetic structure and population polymorphism of Asian honeybee.

The Nuclear Apparatus and Chromosomal DNA of the Microsporidian Nosema antheraeae

ABSTRACT: The microsporidian Nosema antheraeae is a pathogen of the Chinese oak silkmoth Antheraea pernyi, the molecular karyotype of which is still poorly understood. Here the diplokaryon of N. antheraeae strain NP‐YY has been visualized both by fluorescence and electron microscopy. In addition, pulsed‐field gel electrophoresis (PFGE) showed that the haploid genome of N. antheraeae is approximately 9.3–9.5 million base pairs organized into 15 chromosomal bands. The mean fluorescence intensity of N. antheraeae and Nosema bombycis DNA measured by flow cytometry confirmed that the genome size of these two species was congruent with measurements obtained by PFGE. These initial results on the chromosome organization of N. antheraeae provide a foundation for the comparative genomics of N. antheraeae with other species of Nosema.