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Genetic Characterization of Microsporidia from European Bombus

Before the Pollinator Parasites project, the only genetic marker available with which to study Nosema bombi was based on ribosomal RNA. The Pollinator Parasites project has demonstrated that the entire sequence of this gene from eleven microsporidian infections of eight species of bumble bee revealed subtle variation at this locus (Tay et al. 2005).

This variation could not be partitioned among populations of N. bombi found in different host species, but indicated a single panmictic species exhibiting little genetic variability. Ribosomal RNA therefore lacks the resolution necessary to investigate the population genetic processes and phylogeographic patterns within this parasite species, though it does allow for species differentiation (Klee et al. 2006).

In order to improve upon this dearth of genetic markers, a partial genomic library was created from N. bombi DNA to provide a number of new genomic regions, which could subsequently be tested for their usefulness as population genetic and phylogeographic markers.

We generated a genomic library of N. bombi and sequenced 38 clones containing DNA from anonymous regions of its genome. PCR primers designed on only 14 of these clones could successfully amplify N. bombi infected hosts but not uninfected hosts.

Three of these 14 loci revealed considerable intraspecific variability in DNA sequence that could allow some separation of the microsporidia based on host species. These are the first genetic markers of their kind for microsporidia providing sufficient resolution to differentiate among genetic variants that display host species specificity.

European bumble bee (Bombus spp.) species has revealed the presence of only
one microsporidian species, N. bombi as revealed by Pollinator Parasites project results (Tay, OÕMahony and Paxton 2005). Yet significant sequence polymorphism was revealed even within isolates from one host individual.

To understand the source of the multiple DNA sequence variants of N. bombi rRNA found in a single host, we PCR amplified, cloned and sequenced from many clones the rRNA from two individual spores, isolated by laser microdissection from one host. At least two rRNA variants were found per spore, which were also found in a DNA extract of multiple spores from the same host.

Two possible explanations for this degree of polymorphism are that single bees can be infected with multiple ÔstrainsÕ of N. bombi and that different copies of the rRNA gene co-exist in one N. bombi genome. The reults provide evidence from single spore analyses that an individual N. bombi spore harbours multiple rRNA copies that are not homologous in sequence. This is the first time that DNA sequences have been obtained from single binucleate microsporidia, and they demonstrate that concerted evolution has not homogenized the sequences of all rRNA copies within a single N. bombi spore.

The results presented represent major progress in the understanding of Microsporidia molecular genetics in general and of N. bombi in particular. All major results are either published in scientific medie , submitted for publication in scientific media, or in manuscript to be published.

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