Figure 1.

An ex vivo assay allows for rapid quantification of Wolbachia elimination in adult female worm ovaries near the distal tip cell due to compound treatment. Here, effects of a 3-day doxycycline treatment on female adult B. pahangi worms is shown. Worms are extracted from jirds and treated in 24-well plates, with one worm per well and usually 2 worms per treatment condition (with total of four ovaries analyzed). Worms are fixed, their ovaries dissected out, and stained with Wolbachia-specific 16S rRNA fluorescent in situ hybridization (FISH) (yellow). The stained ovaries are mounted on slides with DAPI-containing mounting medium to stain DNA (blue) and their distal ends imaged using a confocal microscope. The Wolbachia-specific 16S rRNA FISH is quantified by high content image analysis and normalized to DMSO control samples (percent elimination indicated here for each displayed ovary). Panels on the right are the enlarged sections demarcated with a white box in the ovary images. Wolbachia wBp is indicated with arrows and the distal tip cell nucleus with an arrowhead. Scale bar = 10 µm.