Fluorescence in situ hybridization of polytene chromosomes. Polytene chromosomes were hybridized with a Wolbachia-specific WRi_004290 probe amplified directly from genomic DNA from the Wb- Hawaii line and visualized at 40X magnification. A single band is observed and not the 8–10 bands expected (Panel A). Polytene chromosomes were again hybridized with the WRi_004290 probe amplified directly from genomic DNA from the Wb- Hawaii line and visualized at 20X (Panel B) and 40X (Panel C). A single band is observed and not the 8–10 bands expected. Polytene chromosomes were simultaneously hybridized with differentially-labelled probes for the Wolbachia-specific WRi_004290 (Panel D, pink) and a D. ananassae actin gene (Panels E &F, green) that were generated from sequence-verified plasmids and visualized at 40X. While hybridization by the plasmid-derived WRi_004290 probe is not detected, hybridization by the plasmid-derived actin probe is detected. This suggests that the previous detection with Wolbachia probes amplified directly from genomic DNA may be detecting hybridization of spurious amplification products and that the lateral gene transfer is heterochromatic.