BMC Genomics 2008, 9:200 http://www.biomedcentral.com/1471-2164/9/200 cuniculi (2.9 Mbp), the genome of which has been com- pletely sequenced and encodes 1,997 protein-coding genes [10], and the insect parasite Antonospora locustae (5.4 Mbp) for which two sequence surveys are available [11,12]. These two species revealed just how microsporid- ian genomes had become so small compared with those of other eukaryotes. There has been a severe reduction in the number of genes in the genome, most likely a reflec- tion of the fact that microsporidia are dependent on their hosts for many metabolic processes and import many compounds from their host. Furthermore the genes that remain are packed together very densely: intergenic spaces are minimal. In E. cuniculi there are no selfish elements and just 15 small introns. Genes in E. cuniculi are also shorter than their homologues in Saccharomyces cerevisiae, which is hypothesized to result from the small number of proteins within the cell, and a correspondingly smaller interaction network [10,13]. This extreme compaction appears to have resulted in a high level of gene order con- servation between different species of microsporidia [12,14-16] and an unusually high level of overlapping transcription between adjacent genes [17,18]. Because the smallest microsporidian genomes are so unu- sual, they have garnered the greatest attention, and to date no large-scale survey of any larger genomes is available. This is unfortunate, because the form and content of these larger microsporidian genomes could differ from the smaller ones in many potentially interesting ways. On one hand they may contain a great many more genes, and could therefore reflect a greater cellular or metabolic com- plexity than the microsporidian parasites we presently know best. On the other hand these genomes may encode a great deal more non-coding DNA, which would have interesting implications for genome evolution within the group, and for why the smaller genomes are so compact. In other eukaryotes, it appears that variation in genome size on a relatively short evolutionary time scale is due to increased or decreased proportions of transposable ele- ments in a genome [19]. There are hints that this may also be at least partially true in some microsporidian genomes where selfish elements have been found [20,21]. Most Table 1: Summary of the data compared to the genome of E. cuniculi interestingly, a number of TY3/Gypsy retrotransposons have recently been described from the 15.3 Mbp genome of Nosema bombycis [22]. These elements are apparently flanked by areas of compacted genes that share a high level of synteny with E. cuniculi, perhaps suggesting an invasion of transposable elements into a compacted genome. More recently, the previously unsequenced sub- telomeric areas of E. cuniculi have been investigated and found to contain a large family of proteins with at least 30 distinct members. This family of duplicated proteins is also found within other human infecting microsporidia [23], demonstrating that the expansion of gene families might be more common than previously thought. Here, we describe low-redundancy genome sequence sur- veys of two distantly related microsporidia, Brachiola alge- rae (recently proposed to be renamed Anncaliia algerae [24]) and Edhazardia aedis. Both of these species have mosquitoes as their type hosts with the former having a very broad host range, including man, and the latter restricted to mosquitoes [25]. The genome size of E. aedis is not known, whereas the genome size of B. algerae has been estimated to be 15–20 Mbp [23]. In both species, we show that gene density is very low compared to that of the better-studied species of microsporidia. Specifically, we have found a considerable proportion of repetitive ele- ments in both genomes, large stretches of non-coding DNA, and some evidence that the gene density may vary over the genome. These surveys open the possibility that microsporidian genomes are not universally compacted, large genomes do not necessarily encode significantly more genes than do the smaller genomes, and that dense genomes may sometimes revert to a gene-sparse state. Results and discussion General features of the sequence data Two short-insert genomic libraries were constructed from B. algerae and a total of 219 clones fully or partially sequenced to yield 203,748 bp of non-overlapping sequence. A single E. aedis library was constructed and 290 sequence reads from 182 clones yielded 233,509 bp of non-overlapping sequence. Comparing these with the Total sequenced coding hits ORFS Non-coding E. cuniculi [10] B. algerae E. aedis bp 2,497,519 2,180,498 1,320,216 860,274 317,029 % 87 61 39 13 bp 203,748 41153(67) 22260 (34) 18893 (33) 162595 % 20 54 46 80 bp 233,509 33,617 (46) 19,099 (25) 14,518 (21) 199,892 % 14 57 43 86 Numbers in brackets indicates the number of genes or gene fragments in each sample. The percentages refer to the proportion of each sequenced class relative to the total amount sequenced. Page 2 of 9 (page number not for citation purposes)