Isolation of Wolbachia from Infected Females

Wolbachia was isolated from the ovaries of wAlbB-infected females according to [24 (link)] with the following modifications. Ovaries from 200 wAlbB-infected females were dissected on ice and suspended in 50 μL of ice-cold Schneiders media (Sigma-Aldrich) in a 1.5 mL eppendorf tube. The ovaries were crushed briefly using a small plastic pestle after which one 3 mm glass bead was added and the suspension vortexed for 2 min. One mL of ice-cold Schneiders media was added to the homogenised and the solution were centrifuged at 4°C for 5 min at 2000 x g. The supernatant was subsequently sequentially filtered through 5 μM and 1.2 μM syringe filters. The resulting filtrate was centrifuged for 4°C for 10 min at 12000 x g. The supernatant was discarded and the pellet resuspended in 50 μL of ice-cold Schneiders media until use. The extraction was repeated with ovaries from W.T. females for use in control injections. Total bacterial counts were estimated using the LIVE/DEAD staining kit (Thermofisher) and counting the live stained bacteria in a hemocytometer.