Bacteriophages can influence the level of virulence of BGB324 mouse bacterial pathogens [16] and can change the phenotypic properties of closely related strains of bacteria. In Wolbachia-infected Drosophila, Culex, Nasonia and other insects, WO prophages appear to be temperate, that is, they have an integrated prophage form and can also generate virions which result in bacterial lysis [6, 11, 15, 17] and [18]. In the parasitoid wasp, N. vitripennis, Bordenstein et al used a quantitative PCR

assay to demonstrate that Wolbachia titer, which correlates with CI intensity, is inversely related to copy number of temperate WOVitA [15]. This relationship, known as the Phage Density Model, predicts that low CI strains of Wolbachia will have a high number of phage particles, and, conversely, high CI strains of Wolbachia

will have low titers this website of phage particles [15, 19]. In Drosophila, however, it is not known which of the diverse prophage elements give rise to lytic viruses, how their lytic properties are regulated, or the effect of lysis on host phenotype. Although most tailed bacteriophages have evolved a temperate lifestyle, it is not yet known if the prophage elements in wRi are functional, defective, satellite phages, or agents of gene transfer [20]. Typically, mature WO phage particles are detected using primers specific to the open reading frame encoding a putative minor capsid protein C (ORF7) [5]. In wRi of D. simulans, however, ORF7 is present in all four prophage insertions [WRi_005560], [WRi_007170], [WRi_010220], and [WRi_012630] and so the presence of ORF7 is not a specific indicator of which phage is active. In this paper we measure the relative copy number of mature, active WORiC phage particles in whole flies and tissues

of D. simulans and determine variations in Wolbachia and WO copy number between individual larval hosts by quantitative PCR. A comparison of the genome architecture of known active phages WOVitA1 and WOCauB2 to WORiC identifies modules for head assembly and DNA packaging as well as tail morphogenesis that are conserved in all known active WO phages. Methods Strains Fenbendazole and media D. simulans (Riverside) (DSR) stocks were maintained at room temperature on a standard diet of cornmeal, dextrose and yeast. Stocks were stably infected with a single Wolbachia strain (wRi) and have been maintained at the University of Alberta laboratory for approximately 6 years. The presence of Wolbachia was confirmed at regular intervals using 81F and 691R wsp primer pairs [21]. DNA extractions DNA from whole flies and gonads was extracted from animals that were less than 5 days post eclosion. Newly eclosed flies were separated by sex and allowed to develop to the appropriate age. Gonad DNA samples were obtained from 4 groups of 100-150 testes each and 4 groups of 75-150 ovaries each. Whole fly DNA was taken from 4 groups of 15 flies each.