Wolbachia strains and Anopheles species should be assessed to determine which pairing is most likely to be successful. Establishing stable Wolbachia infections in novel hosts by embryonic or adult microinjection is a very labor-intensive process with a high failure rate. The development of a rapid assay to assess which Wolbachia strains are most effective at invading the insect germline would greatly facilitate the generation of novel stable Wolbachia infections in insect hosts of interest. Various mosquito tissues have been cultured in an ex vivo setting using cell culture media [16,17]. Fat body and ovarian culture has been used to dissect mosquito physiological responses [18,19,20]. As such, ex vivo mosquito organ culture provides a controlled tractable system to assess specific experimental treatments on insect tissues. Here, we utilized ex vivo tissue culture to develop a simple, rapid, assay to assess germline infectivity in novel Wolbachia-host combinations. Results Wolbachia infects the germline of multiple insect species in culture The host species chosen for Wolbachia invasion experiments varied in their natural Wolbachia infection status; naturally infected and cured (D. melanogaster), uninfected yet empirically susceptible to transinfection (Ae. aegypti), or uninfected (An. gambiae, An. stephensi, C. tarsalis). qPCR analysis comparing the number of Wolbachia genomes normalized to host genome copy number found that both the male and female germline tissues of all five insect species were susceptible to Wolbachia infection. There was a positive correlation between inoculum titer and Wolbachia germline infection density, indicating that ex vivo infection occurred in a density-dependent manner. In five of the ten infection experi- ments, significantly higher Wolbachia densities were observed in ovaries inoculated with a higher titer of bacteria. In each of the five other cases, there was a trend towards higher bacterial densities when a greater amount of Wolbachia was used to inoculate the germline tissue, though the differences were not statistically significant (Figure 1). To examine the localization of the infection and confirm that qPCR results were not due to surface contamination by Wolbachia, Wolbachia Infection in Cultured Insect Germlines we performed fluorescence in situ hybridization (FISH) on wAlbB- infected ovaries followed by confocal microscopy. Wolbachia was visually detected in the ovaries of all five species (Figure 2, Movies S1, S2, S3, S4, and S5). Most of the infection was observed in the ovarian follicular epithelium, however fluorescent signal was also seen within the An. gambiae ovarian follicle (Figure S1). Corrob- orating the qPCR results, a much more intense wAlbB infection was seen in Ae. aegypti ovaries compared to the other four insect species (Figure 2). Wolbachia reaches higher infection titer in native hosts and close relatives Analysis by qPCR shows that both the ovaries and testes of Ae. aegypti are far more susceptible to infection with wAlbB (from the closely related Ae. albopictus) than the other species tested (Figure 1), reaching titers 1000–7000 fold higher than other species (Figure 3) (Kruskal-Wallis test P,0.02, Dunn’s test shown in Table S1). Similarly, the wMelPop strain from D. melanogaster infected fly ovaries at a density approximately 500 to 3000 fold higher than the ovaries of other mosquito species (Figure 3) (Kruskal-Wallis P,0.02, Dunn’s test shown in Table S1). The wMelPop strain, which has been transinfected into Ae. aegypti mosquitoes [21], infects the Ae. aegypti germline in the ex vivo assay at a higher density than both Anopheles mosquito species (2.4 times greater than An. gambiae (P,0.03); 4.2 times greater than An. stephensi (P,0.03)) (Figure 3). We also cultured ovaries from a naturally wMelPop- infected host using the ex vivo system. Infected ovaries from 20-day- old D. melanogaster were cultured and ovarian Wolbachia titer was compared to levels in ovaries immediately dissected from the fly. There was no significant difference in the wMelPop titer in vivo compared to ex vivo, nor between Wolbachia titer of naturally infected ovaries after ex vivo culture and titer in fly ovaries which were infected using the ex vivo system (Figure 3). wAlbB infects Anopheles germline more efficiently than wMelPop Our data suggest that Anopheles ovaries are more susceptible to infection with the wAlbB strain than the wMelPop strain. After Figure 1. Wolbachia density (Wolbachia genomes/host genome) in the germline (ovaries and testes) of An. gambiae, An. stephensi, C. tarsalis, Ae. aegypti and D. melanogaster 2 days post infection. Two replicate experiments were completed to infect the germline of insects with wAlbB 8.56104 live cells inoculated into each well for replicate 1 (R1), while 5.56105 live cells were added to each well in replicate 2 (R2). For each replicate, five pools, each containing five pairs of ovaries or testes, were evaluated for Wolbachia density. Asterisk(s) denote significance (** P,0.01, * P,0.05). Error bars indicated SEM. doi:10.1371/journal.pone.0036277.g001 PLoS ONE | www.plosone.org 2 April 2012 | Volume 7 | Issue 4 | e36277