After the determination of gfp and 16S rRNA gene copies of Gfp-tagged Asaia, total Asaia, and bacteria, the following ratios were calculated: Gfp-labelled Asaia to total Asaia ratio,

Gfp-labelled Asaia to bacteria ratio (GfpABR), and Asaia to bacteria 16S rRNA gene copy ratio (ABR), the latter according to Favia et al. [6]. These ratios were used to estimate the relative abundances of the introduced strain within total Asaia population in S. titanus individuals and of Gfp-labelled Asaia and Asaia sp. in the bacterial community associated with the insect samples. Statistical analyses To compare the Gfp Asaia density detected in co-feeding or venereal transmission experiments for every tested period, q-PCR data relative to the gfp gene VRT752271 ic50 concentration were log-transformed, after YH25448 mw adding the constant 10, and PX-478 analyzed by one-way analysis of variance (ANOVA). In addition, means were separated by Tukey test (P<0.05) when variance homogeneity was satisfied (Levene test, P<0.05). Fluorescent in situ hybridization Fluorescent in situ hybridization analysis was carried out on organs dissected in a sterile saline solution from donor and recipient S. titanus individuals that were not used for Real time PCR experiments. The dissected organs were fixed for 2 min at 4°C in 4% paraformaldehyde and washed in

PBS. All hybridization experiment steps were performed as previously described [4] using specific and universal fluorescent probes. For detection of Gfp-labelled Asaia, probes gfp540 (5’-CCTTCGGGCATGGCACTCTT-3’) and gfp875 (5’-GGTAAAAGGACAGGGCCATCGCC-3’) were labelled with Cy5.5 (indodicarbocyanine, absorption/emission at 675-694 nm). Probes Asaia1 and Asaia2, labelled with Cy3 (indocarbocyanine, absorption/emission at 550/570 nm), were used to observe the total

Asaia population hosted by S. titanus individuals [6]. As a positive control for the hybridization experiment, a universal bacterial probe EUB388 labelled with fluorescein isothiocyanate (FITC, absorption/emission at 494/520 nm) was also used [32]. After hybridization, the samples were mounted in antifading medium and then observed in a laser scanning confocal until microscope SP2- AOBS (Leica). Authors’ contributions EG designed and performed most of the experiments, analyzed data and wrote the manuscript. EC and AR provided the Asaia strain SF2.1(cGfp) and designed the experiments, MM designed FISH experiments and performed confocal microscopy observations. GF gave suggestions and contributed to data analysis. AA and DD designed and supervised all the experiments. All authors have read and approved the final manuscript. Acknowledgements We are grateful to Greg Hurst for English editing of the manuscript.