Drosophila Fly Stocks and Rearing Protocol

The Drosophila melanogaster Oregon R (modENCODE, FBst0025211) and Phs-hidY (FBst0024638; Grether et al. 1995 (link)) fly stocks were obtained from the Bloomington Drosophila Stock center (Cook et al. 2010 (link)). The Drosophila melanogaster Canton S flies were obtained from the Bloomington Drosophila Stock center (RRID:BDSC_64349). Flies were grown on either Cornmeal, Sucrose, and Yeast Media (LabExpress fly media) or Glucose, Active Yeast, and proprionic acid media (10:5 fly media) at 25 ${}^{\circ }$ C, 60% RH. 5 females and 4 males were placed per vial for Oregon R and approximately 10 female and male flies per vial for Canton S. Oregon R flies were transferred to fresh vials daily and progeny from previous vials were collected for experiments. Canton S flies were transferred to fresh vials every 3–4 days and progeny were selected for experiments. An Oregon R stock with a Y chromosome carrying a Phs-hid insertion was generated (http://flystocks.bio.indiana.edu/Browse/misc-browse/hs-hid˙method.html), by crossing double balancer females with Y-lethal males crossed with Y-lethal males (FBst0024638, Grether et al. 1995 (link)). We introduced the Y chromosome into the Oregon R background using double balanced 2nd and 3rd chromosomes. The 4th chromosome was not tracked. For our experiments we used Oregon R (FBst0025211) males and virgin females obtained from the Oregon R Y-lethal stock after 37 ${}^{\circ }$ C heat shock using a water bath for 2 h during third instar. Canton S flies sex were sorted after mating, for female only experimentation. We verified the absence of males during collection of individual flies. When males were present in the vial, none of those flies were used for experiments. This occurred in about 1 out of every 10 heat shocked vials. Newly enclosed flies were placed in batches of 35 per vial for use in experimental assays. Vials contained either CDF (Lee and Micchelli 2013 (link)), LabExpress fly media, or 10:5 fly media . Flies were aged for 3–4 days at 25 ${}^{\circ }$ C 60% RH. After aging on solid food, flies were loaded in the WAFFL (as described below) to proceed with the defined assays.