Genetics and Drosophila strains
Drosophila melanogaster stocks and crosses were maintained on standard cornmeal agar media at 25°C. The strains used were pink1B9 (a kind gift from A. Whitworth, MRC, Centre for Developmental and Biomedical Genetics, University of Sheffield, Sheffield, UK) and w1118 (Bloomington Stock Centre, Indiana University, Bloomington, IN). All analyses were performed using male flies.
FA (Sigma) and FiA (Sigma) were incorporated into the liquid fly food at 4 mM and left to set. This concentration was used because 4 mM FA was previously shown to have beneficial effects on pink1B9 mutant phenotypes when administered during development. Two different treatment protocols (Figure 1F) were used based on the objectives of the study: (i) To test the effect of FiA and FA treatment throughout development, crosses between pink1B9 and w1118 were established on normal food and transferred to FiA- or FA-containing food after 2 days. Larvae were treated with FiA or FA throughout development. The adult flies were kept on drug-containing food throughout their lifespan. (ii) To test the effect of FiA treatment beginning at early to middle stages of adulthood, newly hatched adult male flies were collected from normal food and transferred to FiA-containing food at 1 or 10 days after eclosion. In both treatment conditions, adult flies were transferred to vials with fresh food every 2-3 days.
Defective thorax analysis
The depression in the flies’ upper thorax (defective or crushed thorax) was visually assessed as a binary assay on 1-day-old males. A chi-squared test was performed to determine whether the degree (percentage) of defective thorax was significant in each population under analysis.
Analysis of dopaminergic neurons
Brains of 30-day-old files (unless otherwise stated) were dissected and stained using anti-tyrosine hydroxylase (Immunostar, WI) to detect PPL1 cluster neurons as described previously. Brain samples were placed in PBS plus 0.1% Triton X-100 in a coverslip clamp chamber (ALA Scientific Instruments Inc., NY), positioned using a harp-shaped construction of platinum wire and nylon string and imaged using confocal microscopy. Tyrosine hydroxylase-positive PPL1 cluster neurons were counted in each brain hemisphere. Data acquired for the assessment of each genotype were obtained as a single experimental set before statistical analysis.
Statistical analyses were performed using GraphPad Prism 7 (www.graphpad.com). Data are presented as mean values with error bars indicating ±SD. The number of biological replicates per experimental variable (n) is indicated in the bars. Statistical significance was assessed using one-way ANOVA, two-way ANOVA, or a two-tailed paired t-test. The significance is indicated as * for p <0.05, ** for p <0.01 and *** for p <0.001.