Wolbachia Infection in Cultured Insect Germlines Figure 4. Density of Wolbachia in insect ovaries measured over time. The left axis indicates the ratio of Wolbachia genomes/host genome (red line). The right axis indicates the fold change for Wolbachia (green) and host (blue) single copy gene for each species: An. gambiae (A; Ag), An. stephensi (B; As), C. tarsalis (C; Ct) Ae. aegypti (D; Aa) and D. melanogaster (E; Dm) infected with wAlbB. D. melanogaster ovaries naturally infected with wMelPop were also cultured (F; Dm). For each insect species infected with wAlbB, three pools, each containing five pairs of ovaries, were evaluated for Wolbachia density. Four pools each containing five pairs of ovaries were completed for naturally infected D. melanogaster ovaries. Error bars indicate SEM. doi:10.1371/journal.pone.0036277.g004 critical for Wolbachia replication. In naturally infected Drosophila oocytes, Wolbachia replication has been observed during the period of ovarian development approximately mid-oogenesis [22]. The lack of an active cell cycle may be a reason for the observed lack of replication in cultured ovaries. As the bacteria do not seem to be replicating in the host tissue, we presume that this approach is not suitable to pre-adapt Wolbachia to the Anopheles germline environ- ment. If these ex vivo results are representative of the live mosquito, they suggest that Wolbachia is able to successfully infect the ovaries of Anopheles. However, stable introduction of Wolbachia into these important vectors has not been accomplished, nor has infection PLoS ONE | www.plosone.org 5 April 2012 | Volume 7 | Issue 4 | e36277