Figure 3. Comparison of the density (Wolbachia genomes/host genome) of multiple Wolbachia strains cultured in the ex vivo setting. Wolbachia strains: wAlbB (checkered) and wMelPop (solid) in ovaries of An. gambiae, An. stephensi, C. tarsalis Ae. aegypti, and D. melanogaster 2 days post infection. Ovaries naturally infected with wMelPop from D. melanogaster were cultured ex vivo for 2 days (yellow hatched) and are compared to the density in the fly (yellow horizontal lines). As the density of Wolbachia used as the inoculum for uninfected ovaries differed between strains, the final Wolbachia genomes:host genomes value were normalized to 106 bacterial cells to compare between strains. The wMelPop strain infects D. melanogaster ovaries approximately 500 to 3000 fold higher, while wAlbB infects Ae. aegypti ovaries 1000 to 7000 fold greater than the other insect species. Asterisk(s) denote significance (** P,0.01, * P,0.05). There was no significant difference in wMelPop titer after two days of ex vivo culture compared to in vivo. For each insect species and Wolbachia strain, five pools, each containing five pairs of ovaries, were evaluated for Wolbachia density. Error bars indicated SEM. doi:10.1371/journal.pone.0036277.g003 normalizing the densities of Wolbachia in the inoculum, the wAlbB strain infected the Anopheles germline at a greater density (24 times for An. gambiae (P,0.008) and 34 times for An. stephensi (P,0.008) than the wMelPop strain (Figure 3). Wolbachia infects, but does not replicate in, cultured germline tissue To determine if Wolbachia can replicate in cultured ovaries, we assayed bacterial density in the cultured host tissues at various times post-infection. For some species, we saw an initial drop in titer after the removal of Wolbachia from the media, but in all species there was an apparent increase in the ratio of Wolbachia to host genome copies by six days post inoculation (Figure 4). However, these differences were not statistically significant. Further examination of the results showed that the elevated density at latter time points was due mainly to a reduction in the number of host genomes rather than an increase in Wolbachia genomes. To confirm this, we completed another two experiments. First, we cultured naturally infected ovaries from 1–3 day old D. melanogaster and measured the changes in Wolbachia density over time. The variation in the S7 gene had a major bearing on the ratio of Wolbachia genomes to host genomes (Figure 4F). Second, we cultured host tissue for 6 days prior to addition of Wolbachia and compared the observed densities to those of ovaries inoculated with the same titer of Wolbachia immediately after dissection. Delaying the inoculation resulted in significantly higher Wolba- chia:host gene ratios for, Ae. aegypti (P,0.007) (Figure 5), while in three other species there was a general trend towards higher values, indicating that for most species, and particularly Aedes, host degradation was responsible for the increase in relative Wolbachia Wolbachia Infection in Cultured Insect Germlines density observed in the time course experiments. Taken together, these data suggest Wolbachia is not significantly replicating in ovaries in an ex vivo environment. Discussion In order to be maternally transmitted after horizontal transfer, Wolbachia must first invade the host ovarian tissues. Previously, we reported that Wolbachia is capable of replicating within Anopheles cells in vitro and profoundly influences host gene expression [11,12]. After microinjection of the wMelPop strain Wolbachia into adult female Anopheles mosquitoes, the bacteria was noticeably absent from the ovarian tissue, despite dissemination to and replication in numerous other host tissues [10]. Here, we show that Anopheles and other insect germlines are amenable to Wolbachia infection in an ex vivo organ culture system. After two days of co-culture, ovaries became infected with Wolbachia, predominantly in the ovarian follicular epithelium, though there was evidence of Wolbachia residing in the An. gambiae oocyte as well. It is possible that, in order to migrate through follicular epithelium cells, Wolbachia may require the cells to be metabolically active, which may not occur under ex vivo culture conditions. After injection of wMel into uninfected Drosophila, the bacteria were seen to infect the follicular cells at approximately 6– 8 days and the germline tissue after approximately 15 days [7]. Our observation of shorter invasion times most likely results from the ability of Wolbachia to directly access the germline tissue without the need to migrate through somatic tissue or contend with the host immune response. We then compared the infection properties of two divergent strains of Wolbachia (wAlbB from Ae. albopictus and wMelPop from D. melanogaster). The ability of Wolbachia to invade it’s native host (wMelPop and D. melanogaster) and closely related hosts (wAlbB and Ae. aegypti) at a higher density suggests that these relationships have developed in close association and are more stable than more disparate associations. Additionally, levels of wMelPop from naturally infected ovaries cultured in the ex vivo system for 2 days were similar to both the density of wMelPop in naturally-infected fly ovaries and the density of wMelPop that had invaded uninfected fly ovaries in the ex vivo system, suggesting that the density of infection in the ex vivo system has biological relevance. Taken together, these results validate the ex vivo culturing technique as a tool to assess Wolbachia-host associations. To ascertain if ex vivo culture can be used to adapt Wolbachia to the Anopheles germline environment, we assayed Wolbachia density over time to determine if the bacteria replicates in cultured ovarian tissue. Although the ratio of Wolbachia to host genomes increased in all species over time, further analysis of the number of copies of Wolbachia and host reference genes implicated degradation of the host cells as the main cause of the observed increase in Wolbachia density. In some species, there is an initial decline in Wolbachia 2 dpi. This may indicate that bacteria that were bound to the ovary at 0 dpi failed to enter and consequently, later time points had lower Wolbachia titers. Culturing naturally infected ovaries gave similar results to infection ex vivo in that the host gene markedly influenced the density (ratio of Wolbachia genomes to host genomes). Delaying addition of the Wolbachia inoculum to the cultured Ae. aegypti ovaries significantly increased the titer, suggesting for this species that host degradation is responsible for the increase in Wolbachia density over time, rather than replication of the bacteria in the germline tissue. While it has been reported that ovaries can be cultured using similar techniques for over 2 months [17], it is likely that the tissue is not metabolically active. Host derived factors may therefore be absent which could be PLoS ONE | www.plosone.org 4 April 2012 | Volume 7 | Issue 4 | e36277