segment of the actin gene were used for C. tarsalis [31]. D. melanogaster S7 primers were designed using Primer3 [32]. Primer sequences are found in table S2. All qPCR was performed on a Rotor Gene Q (Qiagen) using the Rotor Gene SYBR Green PCR Kit (Qiagen) using manufacturers recommended settings. Each reaction contained DNA extracted from five ovaries or testes and was performed in duplicate. A third technical replicated was completed when there was a discrepancy between the replicates. Wolbachia to host genome ratios were calculated using Qgene [33]. Changes in individual Wolbachia or host gene values were estimated by raising the reaction efficiency to the exponent power of the negative threshold cycle (E2Ct) as previously described [27]. For single comparisons between infected ovaries, data were analyzed using a Mann-Whitney U test, while multiple compar- isons were performed using a Kruskal-Wallace test followed by a Dunn’s Post-Hoc test. Fluorescence in situ hybridization In order to ensure that qPCR results were not due to Wolbachia contaminating the surface of cultured organs, we used fluorescence in situ hybridization (FISH) to assess Wolbachia infection in mosquito tissues. After two days in culture, FISH was completed on ovary tissue according to previously published procedures [10] with slight modifications. Briefly, Schneider’s media was removed from culture wells and ovaries were washed thoroughly three times with fresh Schneider’s media, fixed in 4% paraformaldehyde in PBS for 20 minutes then moved to 6% hydrogen peroxide in ethanol for 4 days to minimize autofluorescence. Ovaries were allowed to hybridize overnight in 1 ml of FISH hybridization buffer (50% formamide, 56 SSC, 200 g/liter dextran sulfate, 250 mg/liter poly(A), 250 mg/liter salmon sperm DNA, 250 mg/ liter tRNA, 0.1 M dithiothreitol (DTT), 0.56 Denhardt’s solution) with Wolbachia specific probes W1 and W2 labeled with a 5-prime rhodamine fluorophore [34]. After hybridization, ovaries under- went three successive washes in 16 SSC, 10 mM DTT and three times in 0.56 SSC, 10 mM DTT. Tissues were mounted on a slide with SlowFade Gold antifade reagent (Invitrogen) and counterstained with DAPI (Roche). Images were captured on an LSM 510 META confocal microscope (Zeiss) and compared to no probe controls. Images were processed using LSM image browsers (Zeiss) and Photoshop 7.0 (Adobe). Supporting Information Wolbachia Infection in Cultured Insect Germlines Movie S2 Fluorescence in situ hybridization of Wolba- chia infection in An. stephensi ovaries. Serial confocal images of the Z-axis (Z-stack) of 15 sections (0.45 mm step size). Red is Wolbachia, blue is host nuclei (DAPI). (MOV) Movie S3 Fluorescence in situ hybridization of Wolba- chia infection in C. tarsalis ovaries. Serial confocal images of the Z-axis (Z-stack) of 57 sections (0.49 mm step size). Red is Wolbachia, blue is host nuclei (DAPI). (MOV) Movie S4 Fluorescence in situ hybridization of Wolba- chia infection in Ae. aegypti ovaries. Serial confocal images of the Z-axis (Z-stack) of 43 sections (0.49 mm step size). Red is Wolbachia, blue is host nuclei (DAPI). (MOV) Movie S5 Fluorescence in situ hybridization of Wolba- chia infection in D. melanogaster ovaries. Serial confocal images of the Z-axis (Z-stack) of 25 sections (0.48 mm step size). Red is Wolbachia, blue is host nuclei (DAPI). (MOV) Figure S1 Magnified image from a Z stack of An. gambiae (Movie S1) showing Wolbachia infection with the ovarian follicle. Red is Wolbachia, blue is host nuclei (DAPI). The scale bar represents 10 mm. (TIF) Table S1 Dunn’s test for pairwise significance after a Kruskal-Wallis test comparing Wolbachia densities between all species (Figure 3). Asterisks indicate significance after correcting for multiple comparisons using a false discovery rate of less than 5%. (DOCX) Table S2 List of primers used for qPCR to estimate Wolbachia density. (DOCX) Acknowledgments We thank the Ross Confocal Microscopy Facility for confocal microscope access and John Gibas for assistance with confocal microscopy. We are grateful to the Johns Hopkins mosquito core facility, Katie Provost-Javier, Jose Ramirez and Shuzhen Sim for help providing mosquitoes. Movie S1 Fluorescence in situ hybridization of Wolba- chia infection in An. gambiae ovaries. Serial confocal images of the Z-axis (Z-stack) of 23 sections (0.48 mm step size). Red is Wolbachia, blue is host nuclei (DAPI). (MOV) Author Contributions Conceived and designed the experiments: GLH ADP JLR. Performed the experiments: GLH ADP PX. Analyzed the data: GLH ADP JLR. Contributed reagents/materials/analysis tools: GLH JLR. Wrote the paper: GLH ADP JLR. References 1. Jeyaprakash A, Hoy MA (2000) Long PCR improves Wolbachia DNA amplification: wsp sequences found in 76% of sixty-three arthropod species. Insect Mol Biol 9: 393–405. 2. Hilgenboecker K, Hammerstein P, Schlattmann P, Telschow A, Werren JH (2008) How many species are infected with Wolbachia?–A statistical analysis of current data. FEMS Microbiol Lett 281: 215–220. 3. Werren JH, Baldo L, Clark ME (2008) Wolbachia: master manipulators of invertebrate biology. Nat Rev Microbiol 6: 741–751. 4. Vavre F, Fleury F, Lepetit D, Fouillet P, Bouletreau M (1999) Phylogenetic evidence for horizontal transmission of Wolbachia in host-parasitoid associations. Mol Biol Evol 16: 1711–1723. 5. O’Neill SL, Giordano R, Colbert AM, Karr TL, Robertson HM (1992) 16S rRNA phylogenetic analysis of the bacterial endosymbionts associated with cytoplasmic incompatibility in insects. Proc Natl Acad Sci USA 89: 2699–2702. 6. Werren JH, Zhang W, Guo LR (1995) Evolution and phylogeny of Wolbachia: reproductive parasites of arthropods. Proc Biol Sci 261: 55–63. 7. Frydman HM, Li JM, Robson DN, Wieschaus E (2006) Somatic stem cell niche tropism in Wolbachia. Nature 441: 509–512. 8. Rigaud T, Juchault P (1995) Success and failure of horizontal transfers of feminizing Wolbachia endosymbionts in woodlice. J Evol Biol 8: 249–255. 9. Huigens ME, de Almeida RP, Boons PAH, Luck RF, Stouthamer R (2004) Natural interspecific and intraspecific horizontal transfer of parthenogenesis- inducing Wolbachia in Trichogramma wasps. Proc Biol Sci 271: 509–515. 10. Hughes GL, Koga R, Xue P, Fukastu T, Rasgon JL (2011) Wolbachia infections are virulent and inhibit the human malaria parasite Plasmodium falciparum in Anopheles gambiae. PLoS Pathog 7: e1002043. 11. Rasgon JL, Ren X, Petridis M (2006) Can Anopheles gambiae be infected with Wolbachia pipientis? Insights from an in vitro system. Appl Environ Microbiol 72: 7718–7722. 12. Hughes GL, Ren X, Ramirez JL, Sakamoto JM, Bailey JA, et al. (2011) Wolbachia infections in Anopheles gambiae cells: transcriptomic characterization of a novel host-symbiont interaction. PLoS Pathog 7: e1001296. PLoS ONE | www.plosone.org 7 April 2012 | Volume 7 | Issue 4 | e36277