Wolbachia pipientis is an obligate bacterial endosymbiont of insects with a wide distribution. It is a member of the order Rickettsiales and is closely related to the insect vectored mammalian pathogens Anaplasma and Ehrlichia. Ten supergroups of Wolbachia have been identified within the species W. pipientis . Supergroups A and B are common insect symbionts which probably diverged from one another 50-60 MYA . The rapid spread of Wolbachia through insect populations is enhanced by symbiont-driven modifications to normal host reproductive patterns which are manifested as cytoplasmic incompatibility (CI), parthenogenesis, male killing and feminization (reviewed in ).
The possibility of genetically transforming fastidious obligate intracellular bacteria and targeting them to insect vectors of human disease has stimulated renewed interest in Wolbachia's bacteriophage WO. The Wolbachia of Drosophila simulans, wRi, has acquired four prophage elements that are integrated into the bacterial genome as 18- to 77-kb sequences, termed wRi-WO-A, wRi-WO-B (two identical copies) and wRi-WO-C . In contrast wMel, found in Drosophila melanogaster, has one WO-A, one WO-B and a small pyocin-like element. All of these prophage elements are integrated into the Wolbachia chromosome at unique sites. Masui et al  were the first to demonstrate the existence of the prophage WO in Wolbachia of the cricket Teleogryllus taiwanemma and later in D. simulans (wCof, wRi), the moths Ephestia kuehniella (wCauB, wCauA, wKue, wSca) and Corcyra cepharonica (wCep)  by electron microscopy and PCR. The WO prophages from Wolbachia infecting D. simulans, D. melanogaster, Culex pipiens, T. taiwanemma, Nasonia vitripennis and E. kuehniella have been sequenced [4, 6–12]. WO phage genome sequences from wRi, wMel, and wPip are inferred from their respective bacterial chromosome genome sequencing projects. WOcauB2 and WOcauB3 are two strains of WO phages infecting Wolbachia of E. kuehniella that have been sequenced from the lytic phase . WOcauB2 has a genome of 43,016 bp encoding 47 predicted open reading frames (ORFs), whereas WOcauB3 has a genome of 45,078 bp and 46 predicted ORFs. With respect to WO phages, little is known about their gene expression, lytic activity, or influence on the phenotypic properties of their hosts.
The nomenclature surrounding the WO phages from different Wolbachia strains varies. Originally, the phage found in wKue was tentatively named WO , irrespective of how many types of integrated prophages were present. When wMel was sequenced , the two prophage inserts were named WO-A and WO-B respective to the origin of replication. Two phage types in wRi, WO-A and WO-B, were named based on sequence homology to the wMel phages, with the addition of one more phage type, WO-C . WOPip is present as five integrated copies in the Wolbachia of C. pipiens and these are designated WOPip1 through 5 . They have been reported to be more closely related to WO-B of wMel than WO-A of wMel .
Bacteriophages are believed to be the mobile genetic elements responsible for the high level of genetic diversity in Wolbachia [10, 13] and  through lateral transfer between co-infecting strains. As in other prokaryotes, prophage integration and transformation in Wolbachia appear to be major sources of lateral gene acquisition . A group of genes present in the wRi and wMel prophage WO-B genome is most similar to genes found in Rickettsia , suggesting that interspecies horizontal transfer mediated by phages has also occurred in an insect harboring both bacteria.
Bacteriophages can influence the level of virulence of bacterial pathogens  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 . 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 . 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 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 . Typically, mature WO phage particles are detected using primers specific to the open reading frame encoding a putative minor capsid protein C (ORF7) . 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.