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Microsporidia are a diverse group of spore-forming obligate endoparasitic fungi that have over 1,300 named species. Microsporidian spores are ubiquitous in the environment, but species diversity remains vastly unexplored, as the total number of species is suspected to equal that of their hosts. Microsporidian parasites are also model organisms for the study of genome reduction since they possess some of the smallest eukaryotic genomes known to date.To examine the diversity of microsporidian parasites in the environment, I screened for the presence of microsporidian parasites in poorly sampled reservoirs: soil, sand, and compost. I amplified ribosomal DNA (rDNA) sequences for 23 undescribed and three described microsporidian species that were highly diverse phylogenetically, including representatives from four of the five major microsporidian clades. Molecular screening for the hosts revealed that one undescribed microsporidian infected a free-living marine nematode (Odontophora rectangula). I characterized the infection and ultrastructure of the parasite by transmission electron microscopy and fluorescent in situ hybridization. It is a novel microsporidian that I named Sporanauta perivermis (“marine spore of roundworms”). S. perivermis infects the hypodermal, muscle, and reproductive tissues of adult O. rectangula. However, the infection pattern differed between genders where only reproductive tissues were infected in adult females (uteri and eggs), suggesting that S. perivermis is transmitted vertically. Juvenile hosts showed similar infection patterns to adults, and infection pattern allowed prediction of host gender prior to adulthood.Phylogenetic analyses revealed that S. perivermis is sister to a clade containing the Daphnia-infecting microsporidian Ordospora colligata and the Encephalitozoon lineage (five species), which contains three species that infect humans. A genomic survey of S. perivermis indicated high levels of similarity in gene content (over 90%), gene length, and synteny between S. perivermis, O. colligata and Encephalitozoon. S. perivermis and O. colligata shared chromosomal arrangements that were not present in Encephalitozoon genomes, including chromosomal rearrangements that could be linked to genome reduction mechanisms. The genome size of S. perivermis is at least 2.2 Mbp, but is likely larger since its intergenic regions were longer, on average, than those of relatives with genome sizes of 2.3-3.0 Mbp.
RNA-processing encompasses several critical steps in the regulation of gene expression. Both transcription and pre-mRNA splicing are important for the formation of mature RNA. Most eukaryotic genes are interrupted by introns, the removal of which is catalyzed by the spliceosome. The spliceosome is a large molecular machine comprised of five small nuclear RNAs (snRNAs) and up to two hundred proteins. In addition to constitutive removal of introns, alternative splicing increases transcriptome complexity, as it allows for the formation of multiple transcript isoforms from a single pre-mRNA. Although these processes are well-studied in model systems, relatively little is known about their evolution in unicellular eukaryotes. To investigate RNA-processing in reduced systems, I examined the transcriptomes of the microsporidian parasite Encephalitozoon cuniculi, and the red alga Cyanidioschyzon merolae. E. cuniculi and C. merolae harbour reduced genomes of 2.9Mbp and 16.5Mbp, respectively. Both genomes were annotated with fewer than 30 spliceosomal introns, and both have undergone reduction in spliceosomal components, including the loss of the U1 snRNA. Illumina RNAseq was used to sequence the transcriptomes of E. cuniculi at three time-points during its intracellular stage, and C. merolae under light and dark phases of its growth cycle. I found extremely low levels of pre-mRNA splicing for nearly all intron-containing genes in both organisms, under all conditions examined. These levels of splicing appear to be lower than in any other eukaryote examined, suggesting that reduction in unrelated spliceosomes reveals a common evolutionary trend: decreased splicing efficiency.In addition to intron-retention, I found examples of other types of alternative splicing in these two reduced systems. C. merolae displayed all major types of alternative splicing, and some events occurred at relatively high frequencies. The presence of few or no alternative splicing regulatory protein-coding genes in C. merolae and E. cuniculi, respectively, made this finding especially surprising. Also, I found high levels of antisense transcription in C. merolae, with the potential to play a regulatory role in gene expression.
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