5-labeled probes specific for the gfp gene (yellow). A) Superposition of a CLSM image after staining with DAPI over the interferential contrast microscopy picture of a salivary gland lobe of an individual used as donor during co-feeding trials (bar = 50 µm).

B,C) CLSM images after hybridization with the Cy3-tagged probes targeting the whole Asaia population (B), or with the Cy5.5-marked probes specific for the Gfp strain (C). In D-G) an ovariole of a female mated with a male which was not previously fed with the Gfp-tagged Asaia is shown. D) Interferential contrast micrograph showing the ovariole (bar = 150 µm). E-G) CLSM images of FISH with the FITC-labeled Mdm2 antagonist eubacterial probe (E), the Cy3-tagged probes targeting the whole Asaia population (F), and the Cy5.5-marked probes specific for the gfp gene (G). While the occurrence of bacteria (and Asaia in particular) is shown, no hybridization signal was observed with the gfp gene-specific probes. Co-feeding experiments Donor individuals previously exposed to gfp Asaia were allowed to feed on artificial diets, and ‘recipient’ individuals then exposed to this diet. There was a high frequency Selleck Bortezomib of transfer of Asaia to both the food source and to S. titanus during feeding, as indicated in Figure 1A. The occurrence of gfp gene-positive signals in sugar diets previously exposed to donor insects confirms the earlier indications of a release of Asaia

by S. titanus during feeding events [4]. The proportion of diets that assayed positive for Asaia showed a trend characterized by a peak corresponding to 48 hours post exposure to the donor (16 out of 19 positive samples; while 7 out of 10 samples were positive after 24 hours), followed by a decrease starting PRKD3 from the 72 hours acquisition (10 out of 14 positive samples; 4 out of 10 after 96 hours). The average concentration of the marked strain, calculated by the number

of gfp gene copies per ng of DNA of the diet sample, increased up to 48 hours after the end of the inoculation (3 × 103 gfp gene copies / ng DNA) and then started decreasing reaching a value of 3.9 × 102 gfp gene copies / ng DNA after 96 hours acquisition (Table 1). The proportion of the Gfp strain within the total Asaia population followed a similar trend, increasing up to 30% at 72 hours, and decreasing after 96 hours (Figure 2A). This decline could be attributed to the occurrence of other bacteria that can compete with Asaia for the nutrient sources. Beside the highly frequent release of both Gfp- and wild type Asaia into the diet, other bacteria were inoculated into the feeding medium by S. titanus, as the GfpABR with ABR of 6% and 36% respectively (Table 2). Other bacteria associated with the leafhopper could also be transmitted during feeding events, including the phytoplasma and possibly the endosymbiont “Candidatus Cardinium hertigii”, observed to reside in S. titanus salivary glands [25].