BMC Genomics 2008, 9:200 http://www.biomedcentral.com/1471-2164/9/200 ? Edhazardia aedis ? Ty Antonospora locustae 5.4 Brachiola algerae 15-20 Ty ETyncephalitozoon cuniculi 2.9 Encephalitozoon hellem 2.5 Encephalitozoon intestinalis 2.3 Nosema bombycis 15.3 Ty Nosema furnacalis 10.2 Nosema pyrausta 10.2 Vairimorpha sp. 10.5 Spraguea lophii 6.2 Ty Vavraia oncoperae (ex grass grub) 10.2 Vavraia oncoperae (ex. porina) 8.0 Glugea atherinae 19.5 Ty tSFicoighneusmhreiapts3ic consensus of microsporidian phylogenetic rela- Schematic consensus of microsporidian phylogenetic relationships. Microsporidian relationships from a consen- sus of published SSU phylogenies [28, 30, 40] and concate- nated tubulin genes (unpublished data). Genome sizes are labeled and the reported presence of Gypsy/Ty transposons is indicated by (Ty). many such elements have been found in N. bombycis [22] and now B. algerae and E. aedis. However, the majority of these elements are closely related members of the Ty3/ gypsy family. Therefore genome expansion cannot be entirely due to an invasion since the elements must have existed in the common ancestor and been purged from E. cuniculi and possibly other compacted microsporidian genomes. It also remains to be seen if these genomes are substantially heterogeneous. It is possible that many genes do exist in relatively compact regions while other regions are dominated by non-coding sequence. An extreme version of such a situation is seen in the small and compacted genome of the picoplankton Ostreococcus tauri. Here most chromosomes in the genome show a high gene density but 2 chromosomes out of 20 contain 77% of the transposons identified in the genome [34]. A second factor that has been hypothesized to affect genome size is cell size. A correlation between genome size and cell size has been observed in eukaryotes gener- ally [35] and microsporidia specifically [36]. However in the microsporidia, variation in cell size in the different life stages can confound correlations between genome size and cell size. As a rough correlation though, the genome size of B. algerae is estimated at 15–20 Mb and that of A. locustae is reported to be 5.4 Mb [37], whereas the spores of both B. algerae and A. locustae are of comparable sizes [38,39] suggesting, in this case, that cell size is not neces- sarily a factor. A further consideration is whether the com- plexity of the life cycles of different microsporidia is reflected in genome size and gene number. Both A. locus- tae and E. cuniculi have a simple life cycle with monomor- phic spores and are restricted to a narrow host range. Edhazardia aedis has a more complex life cycle with multi- ple spore types and must be adapted to both the larval and adult stage of the mosquito. Brachiola algerae, known to have a larger genome, has a simple life cycle with mono- morphic spores, but has a broad host range and can infect both mammals and insects. Conclusion The E. cuniculi genome is a model for compacted nuclear genomes, but potentially not a good model for micro- sporidian genomes generally. We have shown that the genomes of B. algerae and E. aedis are structured very dif- ferently: they have large proportions of non-coding sequence and many transposable elements, resulting in a very low and perhaps variable gene-density compared with E. cuniculi. The sample of identifiable genes found in the surveys, and the proportions of these genes shared with E. cuniculi both suggest that the complexity of the proteome is not the major factor contributing to genome size variation. The phylogeny of microsporidia suggests multiple events of compaction and/or expansion, which raises interesting questions about what forces the genomes to compact so severely, and why such a force would then cease to operate on the genome. Methods Microsporidia, genomic DNA extraction, and genomic library construction 6.1 × 107 uninucleate Edhazardia aedis spores harvested from Aedes aegypti larvae were ruptured by glass bead- beating, and spores were examined for breakage via light microscopy. E. aedis genomic DNA was purified by the standard phenol-chloroform method and served as tem- plate for whole genome rolling-circle amplification using Genomiphi (Amersham). 4.5 μg of amplified E. aedis genomic DNA was sheared, blunt end-repaired, and cloned into pCR4Blunt-TOPO (Invitrogen) according to the manufacturer's specifications. 182 different E. aedis clones with an average length of 1,283 bp were end- sequenced using ABI Big Dye 3.1 chemistry. Six different E. aedis library clones containing coding, non-coding, or transposable segments were checked for chimeric sequence by PCR of non-Genomiphi-treated E. aedis genomic DNA. From this, successful amplification of frag- ments between 250 and 450 bp did not support the idea of chimeras being present in the Genomiphi-created E. aedis genomic library. Page 7 of 9 (page number not for citation purposes)