24.1
Lifespan
After sorting into vials of cornmeal medium, fresh food was provided every two days by tipping flies, under light CO2 anaesthesia into new vials of cornmeal medium. Deaths were scored when the flies were tipped to new vials, a fly being recorded as dead if it remained immobile after agitation of the vial. Living flies that were stuck to the food or walls of the vial were rescued using a paintbrush, any that couldn't be rescued were censored from the data.
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24.1.1 Generation 3
For each family, mated male flies were sorted into 5 vials at a density of 20 flies per vial (n=100).
24.1.2 Generation 5
For each line, mated male flies were sorted into 10 vials at a density of 20 flies per vial (n=200).
24.2
Development Time, Sex Ratio and Viability
Five males and 5 females per line were placed into bottles fitted with a small dish of laying medium. After 6 hours the plates were removed and left under standard conditions for 12 hours for the eggs to hatch. The first instar larvae were then picked using a syringe and placed in cornmeal vials at a controlled density. The larvae were then reared under standard conditions, until the first eclosion, at which point the newly eclosed flies were removed from the vials at regular intervals, counted and the sex of each fly recorded.
Data were analysed by performing
24.2.1 Generation 3
Each family produced 5 vials of 20 larvae.
24.2.2 Generation 5
Each line produced 10 vials of 30 larvae.
24.3
Wet Weight, Dry Weight and Water Content
Twenty males and 20 females of age 5 days were collected from each line. They were placed individually into microfuge tubes, and wet weight was recorded by weighing the live flies on a 5- point balance. The flies were knocked out prior to weighing by cooling them in an ice bucket. The flies were then killed by freezing overnight at -20°C, before being dried for 24 hours at 45°C. The dried flies were then weighed, and their dry weight recorded. Water content in µg could then be determined as the difference between wet and dry weight and water content as a percentage of total wet weight could be calculated.
24.4
Early Fecundity
Three-day old virgin female flies, 20 per line, were placed individually into vials containing charcoal medium (Table 16) sprinkled lightly with live yeast. Two virgin male W- Dahomey flies were then added to each vial, and the flies were allowed to mate for 24 hours. At four, five and six days old the flies were tipped onto fresh charcoal medium sprinkled with live yeast and the used vials retained and frozen. Eggs were counted in each of the three vials and an average taken.
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25
Stress Assays
25.1
Heat Stress
Flies were sorted, at a density of 10 flies per vial, into vials of cornmeal medium under light CO2 anaesthesia and allowed to rest for 24 hours before the experiment began. For the experiment, the flies were transferred without aneasthesia into vials containing a damp circle of filter paper but no food, which were then plugged, arranged randomly in a rack, and submerged in a 38°C water bath. The vials were checked after 15 minutes and dead flies scored by briefly raising the rack out of the water and agitating each vial, a fly being recorded as dead if it showed no movement on agitation. After this initial check, the flies were then checked for deaths every 5 minutes until all flies were dead.
25.1.1 Generation 3
Ten vials of 10 flies, aged 6 days old, were sorted from each family. Five of these vials were tested, and the other five were kept at room temperature as controls.
25.1.2 Generation 5
Six vials of 10 flies, aged 5 days old, were sorted for each line. All vials were tested for this
generation, since the results from generation three showed that the disc of wet paper was sufficient to keep the flies active for the duration of the experiment at room temperature.
25.2
Oxidative Stress
Flies were sorted at a density of 20 flies per vial. The vials contained stock medium, with a further 1ml of stock medium, spiked with 20mM paraquat, layered on top. The food was spiked by
dissolving the paraquat in a small amount of dH2O before mixing it into the food once it had cooled to 55°C. Stock medium was used instead of cornmeal because it was smoother and made applying a thin layer easier and more economical. Deaths were scored every day and flies were tipped onto fresh paraquat food every three days. To conserve paraquat, vials were combined when the number of flies in them dropped low enough that the combined density would not be greater than 20 flies per vial.
25.2.1 Generation 5
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25.3
Desiccation
Flies were sorted into vials with no food or water. The vials were stored under standard conditions and checked for deaths twice daily until the first death was recorded, at which point they were checked at narrower regular intervals.
25.3.1 Generation 3
Flies were sorted into 5 vials (where possible) of 10, aged 15 days old. The vials were stoppered with cheesecloth, and once the first death was recorded further deaths were scored at 30-minute
intervals until all the flies had died.
25.3.2 Generation 5
Flies were sorted into 5 vials of 20, aged 6 days old. The vials were stoppered with sponge bungs, and once the first death was recorded further deaths were scored at 4-hour intervals until all the flies had died.
25.4
Starvation
Flies were sorted into vials containing starvation medium, an agar-based medium containing only essential minerals and the amino acid glutamine, but no further nutrients. The vials were kept under standard conditions until the first death was recorded, at which point deaths were scored every 12 hours until all flies had died. Every three days the flies were tipped without anaesthesia onto fresh starvation medium.
25.4.1 Generation 3
Flies were sorted into 5 vials of 10, aged 7 days old.
25.4.2 Generation 5
Flies were sorted into 5 vials of 20, aged 6 days old.
26
Molecular Assays
26.1
Protein Carbonyl Accumulation
26.1.1 Generation of Oxidised BSA Standards
The hypochlorite concentration of household bleach (Domestos) was determined by diluting 1:10 in 0.01M NaOH and measuring absorbance at 290nm. The molar extinction coefficient of hypochlorite can then be used to determine concentration in the bleach (ε=350M-1cm-1).
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50mg/ml Bovine serum albumin (BSA) was then reacted with 5mM HOCl for 48 hours at 37°C. An aliquot of the BSA was then diluted to 40mg/ml for calibration. The remaining BSA was split into 100µg aliquots for use in the dot blot, drying off the supernatant in a vacuum centrifuge. The dried standards were stored at -20°C.
The calibration aliquot was quantified using the spectrophotometric method outlined in Levine, et al. (1990). Briefly, the sample was reacted with 1ml of 10mM 2,4-Dinitrophenylhydrazine (DNPH), in 2M HCl for 15 minutes. The derivatized sample was then precipitated with 1ml 28% trichloroacetic acid (TCA), and washed three times with 2.5ml ethanol:ethylacetate (1:1), making sure to
mechanically break up the pellet during each washing step, as well as agitating in a ribolyser to ensure the whole sample was washed. A blank, to which no DNPH was added was also carried through the whole process. After the washes, the pellet was dissolved in 1ml 6M guanidine, 20mM potassium phosphate in 2M HCl, adjusted to pH 2.5. Absorbance was read at 275nm and the carbonyl concentration determined using the molar extinction coefficient of carbonyl (ε=22,000M- 1cm-1), the total protein remaining in the sample was also determined using a micro-BCA (outlined in section 26.1.3) assay and used to correct this carbonyl value for protein lost during the washing process. After correcting for lost protein, the carbonyl content of the standards was determined as 8.9nmol carbonyl/mg protein.
26.1.2 Dot Blot to Determine Carbonyl Content
Individual flies were placed in microfuge tubes) to which 30µl of homogenising solution (92.5% DMSO, 7.5% dH2O, acidified with 0.5% trifluoroacetic acid) was added. The flies were homogenised with a motorised pestle for 40 seconds and centrifuged at 13,000g for 5 minutes. 20µl supernatant was removed to a fresh microfuge tube from which a further 5µl was removed for protein
determination (Section 26.1.3).
The remaining 15µl of sample was then diluted by adding 35µl of derivatizing solution
(homogenizing solution with 20mM DNPH). The standard protein was derivatized at this stage by adding 50ul derivatizing solution. The samples and standards were then incubated for 15 minutes in the dark and under constant agitation. After derivatization, the samples were diluted 1 in 16 and the standards were diluted to give a standard curve. These derivatized samples and standards were then dotted onto a PVDF membrane (Immobilon®-FL Membrane, CAT# IPFL00010) in 1 µl triplicates and then allowed to dry for 45 minutes under a fume hood.
After drying, the membrane was washed twice for 2 minutes in glacial ethanoic acid to remove free DNPH, before being equilibrated in dH2O for 5 minutes. The membrane was then incubated in blocking buffer, 10% milk (Marvel) in tris-buffered saline with 0.1% tween (TBST), for 1 hour at which
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point the buffer was replaced with 10ml of fresh buffer spiked with 2ul of the primary antibody (Goat anti-DNP, Sigma Aldrich). The membrane was incubated on a shaker for 2 hours, before being washed for 15 minutes in PBST, followed by 2 further washes of 5 minutes. Finally, the membrane was incubated for a further 1 hour in 10ml blocking buffer spiked with 10µl secondary antibody (anti-Goat IgG-Cy3 antibody produced in rabbit, Sigma Aldrich), incubated in the dark on a shaker for a further 1 hour before a final three washes in PBST as before. The washed membrane was
equilibrated in dH2O for 5 minutes before being scanned in a Typhoon FLA9000 on the Cy3 setting.
26.1.3 Protein Determination
The 5µl samples taken for protein determination were diluted 1:20 in PBS, and 10µl was removed to a fresh tube. To this, 10µl of BCA standard working reagent was added and incubated at 37°C for 30 minutes. The samples and protein standards were then measured on a Nanodrop 2000
(ThermoFisher) using the BCA program.
26.2
Lipid Content
After measuring dry weight, male flies were placed individually into microfuge tubes. Flies were homogenised in 100µl saturated Na2SO4, using a motorised pestle and the lipids were separated from the carbohydrates by adding 1ml chloraform:methanol (1:1) and inverting several times. Samples were centrifuged for 5 minutes at 10,000g and the supernatant containing the lipids was removed to a fresh tube.
Lipid standards were prepared from soybean extract, diluted in chloraform:methanol (1:1) at concentrations of 920, 460, 230, 115, 57.5, 28.75, 14.375 and 0ug/ml. Solvent was evaporated from standards and samples by placing in a 75°C heat block, with lids open, for 1 hour. The residue was resuspended in 0.2ml concentrated H2SO4 and heated at 90°C for 10 minutes and then cooled on ice for 5 minutes. 1ml of vanillin reagent (1.2% vanillin in 68% orthophosphoric acid) was added to each tube and left for 5 minutes. Three replicates of 200µl from each sample were loaded onto a 96-well assay plate and read at 490nm on a Tecan Infinite 200 PRO microplate reader (Tecan).