Real-time polymerase chain reaction
cDNA was prepared from 7 to 10 whole, day-old male flies. Total RNA was extracted using Trizol (Invitrogen, Carlsbad, CA, USA) for each genotype. At least three independent RNA extractions were prepared for each sample. Relative message abundance was determined by amplification and staining with SYBR Green I using an ABI 7000 SDS (Applied Biosystems, Carlsbad, CA, USA). Expression of Rp49 and corresponding control y1w67c23 flies were used for normalization. Differences between genotypes were assessed by t-test or nested anova.
Primer sequences are listed below.
- Forward: 5′-AGAAACACCGAGTGCGTCTG-3′
- Reverse: 5′-CCACCGTGTTGTCATGATTC-3′
- Forward: 5′-AAACCGACAGTTCTGGAAG-3′
- Reverse: 5′-GCAGGAACTGACGATTGATG-3′
- Forward: 5′-CCGCTGGTTAAATTCGAGTC-3′
- Reverse 5′-ATCCGTAGATCCTCGACAGC-3′
- Forward: 5′-CTGGGTGGGTGATTATCTGG-3′
- Reverse: 5′-AGCCTGCTTGTTGAAATGGT-3′
- Forward: 5′-ACTCAATGGATACTGCCCAAGA-3′
- Reverse: 5′-CAAGGTGTCCCACTAATGCATA-3′
- dFatp insertion
- Forward: 5′-TGAACCTCGCACTCGGCTGAT-3′
- Reverse: 5′-TTGGACAACTATGCGAACAGC-3′
Following collection, treatment, and aging, five female or eight male flies were weighed and homogenized in 500 μL of 0.05% PBS/Triton X buffer. Following lipase inactivation and centrifugation, the supernatant was added to 10 volumes of preheated (37 °C) Thermo Infinity Reagent (#TR22321; Thermo Electron Corp, Waltham, MA, USA). Absorbance of 520 nm was determined following 10 min of incubation with agitation at 37 °C. Resulting triglyceride measures were normalized per fly or per μg total protein. Each data point is based on at least three replicate measures.
After collection and mating as described in fly stocks and maintenance, 10-day-old adult flies were housed in vials of standard food (10% sucrose/10% yeast). Flies were separated into vials of five and left to equilibrate for 1 h. The percentage of flies engaged in proboscis extension in each vial was scored as in Wong et al. (2009)
. Each vial was observed 10 times in a double-blind fashion. To assess the amount of food consumed, 10-day-old adult flies were fed standard food supplemented with 0.5% (w/v) FD&C Blue #1 (Spectrum Chemical Mfg. Corp., Gardena, CA, USA). Following exposure to dyed food, five female or eight male flies were homogenized in 200 μL PBS and cleared by centrifugation at 15 700 g
. After centrifugation, the absorbance of the supernantant was measured at 625 nm. Having established blue-dye feeding assays as a control, data collected from proboscis extension methods were analyzed by Student’s t
After collection and mating as described, 30 male or female flies were weighed and homogenized in 200 μL PBS/0.05 × Triton X. Homogenate was cleared by spinning for 2 min at 13 000 rpm. Twenty microlitre of fly homogenate was added to 1 μL amyloglucosidase at 0.1 U μL−1. Sample was incubated with agitation at 37 °C for 1 h. Absorbance of 340 nm was determined within 30 min following incubation. Resulting glycogen was normalized per milligram dry weight. Data were analyzed using Student’s t-test.
Free FA were assessed from the hemolymph of 25–30 male flies per genotype. Hemolymph was obtained by puncturing cuticle with a sterile needle and then ‘pulse’ centrifuging flies in an Eppendorf with a mesh screen to separate solid material. One microliter of hemolymph sample was combined with 49 μL BioVision Free Fatty Acid reagent (BioVision, Mountain View, CA, USA). Reagents were mixed according to manufacturer’s instructions following standardization preparation and acyl-coA synthesis. Reaction was allowed to incubate, protected from light, for 30 min at 37 °C. Absorbance of 570 nm was determined following reaction completion. Data were analyzed using Student’s t-test.
Background genotypes utilized in this study frequently displayed unusually high early-life mortality. Because this early mortality is unrelated to aging, we have plotted all survival assays except for stress assays with deaths from the first 20 days censored. Differences in all survival curves were assessed following censoring using log-rank analysis, applied only to post-20-day data. To provide a clear illustration of age-dependent mortality differences, we also plotted age-specific mortality for all curves in their entirety (Supporting Information.
Prior to all experiments, fly cultures were maintained at a constant density for at least two generations. Fifteen virgin females and five males were mated in 300-mL bottles with 50 mL standard 10% sucrose 10% yeast unless otherwise described. Adult progeny were synchronized by collecting within 2 h of eclosion, over a 24-h time period. Groups of 20 age- and sex-matched flies were immediately transferred into narrow polypropylene vials containing 5 mL of appropriate food medium. Food vials were changed every second day at which time dead flies were removed and counted. Flies were housed in a 25 °C incubator on a 12:12 light/dark cycle at 50% relative humidity.
For starvation resistance, flies were collected and housed as described and placed on agar-only food. Survival measurements were recorded twice per day in 12-h intervals. To assess survival on high-nutrient diet, standard 10% sucrose/yeast food was supplemented with an additional 10% weight/volume brewer’s yeast, dry palmitic acid, or dry linoleic acid. Survival measurements were recorded every second day.
Oxidative stress was induced by introducing 40 mm methyl viologen dichloride hydrate (Sigma-Aldrich, St. Louis, MO, USA) dissolved in 5% sucrose. Deaths were recorded three times per day in 8-h intervals. Temperature stress was assessed at either 4 or 28 °C. Cold survival was assessed twice per day in 12-h intervals. Before recording deaths from flies under cold stress, flies were given 1 h to recover movement at room temperature. Heat stress was recorded every second day.
CO2 production was measured using a flow-through respirometry system. Twenty-eight-day-old exercised and unexercised flies were immobilized 24 h before measurement by CO2 gas and separated into groups of five flies per sample. Samples were assayed in random order to evenly distribute variance between measurements. For each measurement, seven samples were transferred into 2-mL glass measurement chambers in a room kept under constant light at 25 °C, and one chamber was left empty as a blank reference. Chambers were consecutively flushed for 150 s at a flow rate of 90 mL min−1 with CO2-free, water-saturated room air through an 8-channel MUX flow multiplexer (Sable Systems International, Las Vegas, NV, USA). Flushing of all samples was consecutively repeated four times per measurement, resulting in 20 min intervals during which the chambers were sealed before the second, third, and fourth flushing. Integrated CO2 concentration over time was measured for all samples during the fourth flushing using a Li-7000 CO2/H2O Analyzer (Sable Systems International) and used to calculate CO2 output over time per fly. All n values were between 19 and 46. Results were analyzed using a two-tailed t-test (Prism; GraphPad Software, San Diego, CA, USA).
Adult flies separated by age, genotype, and/or treatment were dissected, ventral side up, in room temperature PBS. Having exposed the heart and fat bodies, partially dissected flies were rinsed 3 × in fresh PBS. Lysotracker green (Molecular Probes, Eugene, OR, USA) was diluted to 0.01 μm in PBS and applied to dissected preps for 1 min. Samples were washed three additional times in PBS. Stained hearts and fat bodies were subsequently removed and mounted in one drop of antifade reagent (Molecular Probes). Slides were imaged on an Olympus BX41 compound fluorescence microscope (Olympus, Center Valley, PA, USA) using a 40 × objective. Images were analyzed using Image (National Institutes of Health, Bethesda, MD, USA). A minimum of five samples were analyzed for each tissue type. Data were subjected to Student’s t-test following quantification.
Oil Red O
Adult flies were dissected similarly to lysotracker staining with the following changes. Dissections were performed in room temperature PBS/0.05% Triton X and fixed in 4% paraformaldehyde/PBS for 10 min. Following fixation, samples were washed 3 × in PBS. Oil Red O (Sigma-Aldrich) was diluted 1:100 in isopropanol and applied to partially dissected preps for 20 min at room temperature. Following staining, samples were washed 3 × in ddH2O. Hearts were removed and mounted as previously described. Imaging and analysis were identical to lysotracker protocols. A minimum of 10 fly hearts were analyzed for each genotype/treatment. Baseline stains (before exercise) were performed in both male and female flies and exhibited similar cardiac Oil Red O phenotypes compared to wild-type controls. All exercise experiments were performed on males.
Data for triglycerides, total protein, feeding rate, glycogen, free FA, fluorescent stains, and single-time-point cardiac analyses were analyzed by Student’s t-test. Survival analyses were conducted using log-rank test. Negative geotaxis, cardiac, and metabolic analyses traced over time were analyzed by two-way anova. Bonferroni pos t-test s were used to compare significantly different values. For exercise treatment, multivariate regression was used to assess treatment by age. All statistics were generated using GraphPad Prism software version 5 (La Jolla, CA, USA).