Figure 5. The effect of culturing before inoculation of Wolbachia on ovary density. Ovaries were inoculated with Wolbachia immediately after culture (day 0) or 6 days post culture for An. gambiae (Ag), An. stephensi (As), C. tarsalis (Ct), Ae. aegypti (Aa) and D. melanogaster (Dm). Two days post-infection, the density of Wolbachia was compared in these two treatments. Density of Wolbachia in ovaries was significantly different (P,0.007) between the Ae. aegypti day 0 and day 6 treatments. For each insect species, five pools, each containing five pairs of ovaries, were evaluated for Wolbachia density. Error bars indicate SEM. doi:10.1371/journal.pone.0036277.g005 been observed in the wild. Previous studies have indicated that Wolbachia are able to both invade and reproduce in Anopheles cells in culture and adult somatic tissues, but have not reported invasion into reproductive tissues [10,11]. Here, we show that the reproductive tissues can be infected when other host factors are absent, but that Wolbachia do not reproduce under these conditions. Therefore, the question as to why Anopheles species remain uninfected with Wolbachia remains. Possible explanations include a lack of reproduction in Anopheles ovaries under natural conditions, negative interactions between the bacteria and the host, factors blocking invasion of the ovaries in adult mosquitoes and many others, requiring further elucidation if Wolbachia is to be used for malaria control. The infection of ex vivo cultured insect ovaries with Wolbachia can be used as a tool for the assessment of the suitability of various Wolbachia strains to infect a given species of interest. By testing the ability of different Wolbachia strains to invade the host germline in culture, the strain with the greatest ability to do so can be chosen for transinfection. Given the similar horizontal infection routes, data gleaned from this approach may be more useful for assessment of adult transinfections. However, in general, affinity for the germline is critical for any novel Wolbachia-host association regardless of whether the transinfection is by adult or embryonic microinjection. This system can also be used to investigate the interactions between multiple Wolbachia strains infecting the same host and to examine the interactions between naturally infected hosts and their Wolbachia. Finally, ex vivo culture provides a tractable method by which the effects of various treatments on the ability of Wolbachia to invade host germline tissues can be investigated. For Wolbachia to be used for vector control in novel hosts, stably transinfected lines must be generated, which requires that the bacteria successfully invade the host germline tissue. Here, we demonstrate a new method for quickly evaluating the suitability of a given Wolbachia strain to invade the reproductive tissues of an Wolbachia Infection in Cultured Insect Germlines insect host. Using this method, we show that Wolbachia is able to infect the ovaries of both An. gambiae and An. stephensi, two important vectors of human malaria parasites. Our results also indicate that Wolbachia invasion occurs in a density dependent manner and that Wolbachia are able to more efficiently infect the tissues of their natural host or closely related species than those of more distantly related insects, providing evidence for the adaptation of Wolbachia to their hosts during long term association. This study provides support for the possibility that Wolbachia can infect Anopheles mosquitoes, and therefore be useful in the control of malaria. Materials and Methods Ex vivo culture of insect germline tissues Germline tissues (ovaries and testes) were dissected from two to three days old Anopheles gambiae (Keele strain), An. stephensi (Liston strain), Culex tarsalis (KNWR strain), Aedes aegypti (Rockefeller strain) and Drosophila melanogaster (w1118 flies previously treated with tetracycline to remove the natural Wolbachia infection) in PBS using a dissecting microscope (Olympus). For culture experiments that used naturally infected ovaries, untreated D. melanogaster (w1118 infected with wMelPop) were used. Prior to dissection, insects were surface sterilized with 100% ethanol for five minutes and rinsed in sterile PBS. After dissection, ovaries or testes were briefly washed with 100% ethanol and rinsed in Schneider’s media with 10% fetal bovine serum (FBS) (Invitrogen), 50 mg/ml kanamycin (Sigma), 10 units/mL penicillin and 10 mg/ml streptomycin (Invitrogen). Wolbachia replication dynamics are not affected by kanamycin, penicillin or streptomycin [23,24]. Five ovaries or testes were placed within a single cell culture insert (Millipore). Five replicates were completed for infection experiments, while three replicates were done for time course (replication) assays. Under sterile conditions, germline tissues were washed three times with 1 mL of fresh Schneider’s media then incubated at room temperature in 1 mL of Schneider’s media with 10% FBS, 50 mg/ml kanamycin (Sigma), 10 units/mL penicillin and 10 mg/ml streptomycin (Invitrogen). Wolbachia infection The wAlbB and wMelPop strains of Wolbachia were purified from infected Sua5B [25] (provided by M. Jacobs-Lorena) and Mos55 [26] (provided by S. O’Neill) cells, respectively, according to previously published procedures [27]. Purified Wolbachia were stained using the Live/Dead BacLight Bacterial Viability Kit (Invitrogen) and counted using a hemocytometer under a fluorescence microscope (Olympus) to determine the density of viable cells. One microliter of purified Wolbachia, with a known density, was then added to each tissue culture well containing ovaries or testes. When naturally infected ovaries were assessed, purified Wolbachia was not added to the culture. Quantitative real-time PCR (qPCR) After two days in culture, germline tissues were removed from the culture media, washed thoroughly 3 times with fresh Schneider’s media before DNA was extracted using the QIAamp DNA Micro Kit (Qiagen). This DNA was used as template for qPCR to estimate the density of Wolbachia in cultured ovaries or testes. wAlbB was detected using primers GF and BR that amplify the wsp gene [10] while wMelPop was detected with primers amplifying the single copy WD_0550 gene [28]. Wolbachia densities were normalized to single-copy host genes. For An. gambiae, An. stephensi and Ae. aegypti, primers amplifying the S7 rRNA gene were used [29,30], while primers which amplify a PLoS ONE | www.plosone.org 6 April 2012 | Volume 7 | Issue 4 | e36277