Generating Wolbachia-free Drosophila Lines

We generated a Wolbachia uninfected line using antibiotics. Freshly-laid eggs were surface-sterilized in 2.7% sodium hypochlorite (Sigma-Aldrich, St. Louis, MO) for 2 min and twice in 70% ethanol for 2 min. Following two 1 min rinses in distilled water, they were raised on a normal cornmeal, molasses, and yeast food containing 50 µg/mL tetracycline hydrochloride (Wako Pure Chemical Industries, Osaka, Japan). After tetracycline treatment for three generations, a single female was transferred to a vial to establish isofemale lines. After laying eggs, each female was assayed for the presence or absence of Wolbachia infections by PCR (see Supplementary Methods). After selection of the vial derived from an uninfected female, flies were maintained on tetracycline-free normal food. Because antibiotic treatment can eliminate other fly microorganisms besides Wolbachia such as beneficial gut-associated microbes, these were restored by backcrossing Wolbachia uninfected tetracycline-treated females with control infected males as described previously^{33 (link)}.