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Microsattelite loci for Microsporidia

To develop molecular genetic markers so as to differentiate between strains of the bumble bee infecting microsporidian Nosema bombi, we generated three partial genomic libraries for the microsporidia N. bombi and Nosema apis using both a non-enriched library and two libraries enriched for microsatellite repeat motifs.

Our initial attempts to generate a partial genomic library of N. bombi (non-enriched library) were largely unsuccessful because extracted DNA was contaminated with host DNA. This is an unfortunate consequence of the fact that microsporidia, including N. bombi, are obligate intracellular parasites that are therefore closely associated with host tissue.

All attempts to clone microsporidia DNA from host tissue will be similarly compromised. Our subsequent attempts to rid microsporidian spore extracts from host DNA using a DNAse treatment seem to have been largely successful. This method can be recommended in future attempts to clone microsporidian DNA. Our attempts subsequently to enrich a partial genomic library of N. bombi and N. apis seem to have been successful in that we have identified DNA sequences containing repeat motifs that show little or no similarity to host or other contaminant DNA. We were able to generate one microsatellite locus for N. bombi.

There was a low number of copies of the microsatellite repeat motif at this locus. Such loci with low repeat copy number usually do not display variation. Indeed, as anticipated, the locus proved to be monomorphic in the tested samples. The density of repeat motifs in the enriched libraries for both N. bombi and N. apis was very low, possibly due to the highly reduced state of microsporidian genomes.

The density of microsatellite containing clones generated from the enriched libraries is nevertheless extremely low. To place the results in context, other genomic libraries have been developed concurrently in the same laboratory by colleagues using the same techniques. When using orchid bee and colletid bee DNA as templates for cloning, ca 30% of clones from an enriched library were positive and contained repeat motifs.

The low number of microsatellite motifs in microsporidian DNA may be a consequence of their highly reduced genomes. Indeed, we have been unable to detect any microsatellite repeat motifs in the microsporidian genomes: Encephalitozoon cuniculi and Antonospora locustae that are deposited in GenBank, suggesting that they are at a very reduced density in microsporidian genomes. Thus, the common practice to use polymorphic microsatellite repeat motifs for population getentic studies, may not be suitable for this group of organisms.

The results are being prepared for scientific publication and should be of interest to all pathologists working on Microsporidia molecular biology, epidemiology and population genetics.

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Queen's University of Belfast
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