Positive and negative controls for FISH experiments targeting the gfp gene. The presence and distribution of Gfp-tagged Asaia in tissues of donor insects (positive controls) and of individuals submitted to transmission trials in absence of the tagged strain (negative controls) have been evaluated by FISH with the FITC-labelled Eu338 eubacterial probe (green), the Cy3-labelled Asaia-specific probes (magenta) and the Cy5.5-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 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.