For food supply, capsules were provided with grapevine leaves whose petiole

was placed inside an Eppendorf tube containing a nutritive solution [27] and sealed with parafilm to maintain leaf turgor during the experiments. At the end of the mating period, individuals mated with infected S. titanus learn more were fed on sterile sugar diets for different periods (24 to 96 hours), in order to permit the insect’s body colonization by the bacteria acquired during mating. After the incubation periods, both insects and diets were collected and conserved as described above. To control whether the Gfp Asaia transfer really took place by mating, rather than by co-feeding while the two individuals remained in the same capsule, co-housing

trials were set up. Further 12 males and 14 females, after the acquisition of the Gfp-marked bacterium, were placed in Petri dishes together with an uninfected individual of the same sex, under the same conditions of the venereal transfer experiments. After 2 days (without copulation), both the specimens were fed on sterile sugar diets for different periods (24 to 96 hours), like for the other trials. For each co-feeding experiment, other 56 individuals fed on sterile sugar diets were used as donors in trials designed as negative control; similarly, for each venereal transmission experiment, 56 individuals fed on sterile solution were mated with PLX3397 solubility dmso specimens of the opposite sex as negative control (Table 3). After mating of negative control individuals, receiving specimens were maintained singularly on sugar diets for periods varying from 24 to 96 hours to simulate the transmission trials. selleck screening library Quantitative real-time PCR for the Gfp-tagged Asaia Subsequent to the transmission trials, S. titanus individuals and sugar diets for molecular analyses were submitted to total DNA isolation. Nucleic acids extraction was performed by sodium dodecyl sulfate-proteinase K-cethyltrimethyl ammonium bromide treatment [28], which for insects was modified as described in Raddadi et al. [29]. The precipitated DNA was resuspended in 50 µl (insect samples) or in 20 µl (diet samples)

of TE buffer, pH 8 and kept at -20°C until use. Quantitative real-time PCR was performed on a Chromo4 real-time detector (Bio-Rad, Milan, Italy) to measure the presence and concentration of Gfp-tagged Asaia in insects and diets. The reactions were performed with IQTM SYBR® Green Supermix (Bio-Rad), using primers targeting the gfp cassette (GFP540F / GFP875R) [30] and the insect’s 18S rRNA gene (MqFw / MqRv) [31]. The latter were used to normalize the gfp concentration values for the total DNA amount of each sample. To calculate the relative abundance of Gfp-labelled Asaia respect to the total Asaia cells and the whole bacterial community, Asaia-specific and eubacterial primers were used also, according to Favia et al. [6]. To construct standard curves, the gfp gene of Asaia strain SF2.