2.4.1. Dihydroethidium (DHE)

Flight muscles were dissected quickly and placed in DHE (molecular probes) in PBS (1:1000 dilution) for 3 min and then washed in PBS three times for 3 min each. The samples were then fixed in 4% (*w*/*v*) PFA for 3 min and then washed again in PBS twice for 2 min each time. Flight muscles were then mounted in PBS and immediately photographed under a Zeiss confocal microscope (n = 5 independent experiments).

## 2.4.2. Spectrophotometric Analysis

Flies were homogenized using a pestle, and ROS generation was detected from isolated mitochondria by amplex red using fluorescence spectrophotometer (Hitachi F-2710) described previously [18]. Briefly, 5 μg horseradish peroxidase (Sigma-Aldrich, St. Louis, MO, USA) was added to the ROS buffer (mmol/L, 20 Tris-HCl, 250 sucrose, 1 EGTA-Na4, 1 EDTA-Na2, pH 7.4 at 37 ◦C) and the baseline fluorescence was obtained (excitation at 560 nm and emission at 590 nm) for 30 min (n = 4 independent experiments with 100 flies each to isolate mitochondria). The protein concentration was used to normalize the amount of mitochondria from the same extracts.

## *2.5. ATP Measurement*

ATP was measured from five individual groups of 2-week old females flies (n = 5 independent experiments with 20 flies each), using Roche ATP bioluminescence assay kit CLS II according to manufacturer's instructions. Briefly, flies were homogenized in the lysis buffer and incubated for 5 min at room temperature. The extract was spun down at 10,000 *g* and the supernatant was transferred into a microwell plate. Upon addition of luciferase reagent, the luminescence was measured using a luminometer. The ATP measurements were normalized to protein from the same extract.
