Isogenic Drosophila Lines for Wolbachia Research

D. melanogaster lines with Wolbachia are described in Table 1. Lines with Wolbachia variants described in Riegler et al.[40] (link) were kindly provided by Markus Riegler and Scott O'Neill. wMelCS_b source and DrosDel w¹¹¹⁸ isogenic background were described elsewhere [12] , [59] (link). wMel variants were introduced in the DrosDel w¹¹¹⁸ iso isogenic background by chromosomes replacement using a first and third double balancer line and a second chromosome balancer line. The crosses were performed with Wolbachia-infected females, ensuring endosymbiont transmission through the germline. The fourth chromosome was not isogenized. All the Wolbachia genotypes were confirmed by PCR, as described in Riegler et al.[40] (link) (data not shown).
The lines were cleaned of possible chronic viral infections as described elsewhere [12] , [60] .
In order to homogenize the gut microbiota, embryos from each line were sterilized with 2% sodium hypochlorite, followed by 70% ethanol and washed with sterile water. Embryos were placed in new food vials and 150 µl of a bacterial inoculum from a reference stock was added. The inoculum was produced by mixing 5 ml of sterile water with 2 g of food from 10 days old vials containing VF-0058–3 flies [12] , and filtering it to remove eggs and larvae.
Tetracycline-treated lines were cleaned of Wolbachia infection by raising them for two generations in ready-mix dried food (Philip Harris) with 0.05 mg/ml of tetracycline hydrochloride (Sigma). Experiments were performed on lines that were raised without antibiotics for at least 6 generations.
Drosophila lines were maintained on standard cornmeal diet at a constant temperature of 25°C. We focused the analysis on males in the assumption that Wolbachia levels would be more stable in these. Wolbachia is present in ovaries and the sizes of these vary greatly with mating status and physiology of the female.