Fly stocks and maintenance
Wild-type fruit fly Drosophila melanogaster w1118 strains were utilized in all experiments and sourced from the Bloomington Stock Center. The flies were maintained under standardized conditions, a temperature of 25°C and relative humidity of 60%, with a 12-hour light/12-hour dark photoperiod. Flies were collected as experimental within 8 hours post-eclosion, with 25 individuals per vial. Each vial diet was replaced with fresh provisions every 2–3 days.
Diet
The cornmeal diet was obtained from Genesee Scientific (# 66–112), while the yeast-based diet was modified from previously published recipes30, 31. Chemically Defined Diet (CDD) was prepared and modified based on previous research14–17. Variations of the CDD, including methionine-restricted diets, nutrient-enriched diets, a sulfa quinoxaline-added diet, and isotope tracing diets (U-13C5-Methionine and U-13C6-Sucrose (Fructose)) were also developed. Diets can be stored at 4°C for several weeks (after which they will shrink due to loss of water and pull away from the side of the vial).
Egg counting and egg development time
Mated female flies were transferred to fresh diet vials every day to allow for observation of egg production and egg development time. Egg counting from the 3rd day after mating lasts for 30 days.
Climbing activity and body weight
Fly climbing activities were assessed referring to a previously described32. Shortly, 10 single-sex flies, were transferred to an empty standard 23 × 95 mm plastic vial and then gently tapped to the bottom 3 times. The number of flies that reached the top vial within 20 seconds was then scored as climbing. Consecutive trials were separated by 1 minute of rest, and each experiment was performed on a minimum of 3 vials of 10 flies per condition repeated 3 times. Test every 5–10 days and follow up for 2 months.
For body weight, 50 flies were collected, weighed every 10 days, and followed up for 2 months. Each experiment was performed on at least 150 flies (3 vials of 50 flies per condition) and repeated 3 times.
Lifespan assessment
Flies were collected within 8 hours after eclosion and sorted under CO2 anesthesia. The individuals were then randomly assigned to various feeding regimes, with 25 single-sex flies placed in each vial. Throughout the experiment, the flies were transferred to a fresh vial containing the corresponding diet every 2–3 days. Mortality was monitored daily by checking for dead flies in the vials and recording the number of deaths.
Folic acid concentration assay
Diets or 100 flies were prepared and quantified according to the Folic Acid Concentration Assay Kit (Novus Biologicals NBP2-82433).
Tissue preparation
Separation of the male reproductive system, which consists of the testes, seminal vesicles, accessory glands, and ejaculatory duct. Separation of the female reproductive system, which consists of the bursa, spermatheca, seminal receptacle, and ovaries. Tissues were observed or stored at -80°C for future experiments.
Sperm was isolated as in previous studies27–29. The seminal vesicle and ejaculatory duct (90 minutes after mating) were separated from the male reproductive system and torn using a dissecting needle. The tissue was placed into a centrifuge tube containing 300 µl of ddH2O and mixed for 10 minutes at room temperature (RT), seminal vesicles and ejaculatory duct were discarded, and the sperm was centrifuged at 3000 rpm for 5 minutes at RT. Save the pelleted sperm for mass spectrometry or store it at -80°C for future use.
Testes staining
The male reproductive system was dissected, and removed the seminal vesicles, glands, and ejaculatory duct. W1118 male testes were harvested and dissected in PBS and fixed in 5% formaldehyde for 20 minutes, followed by washing in PBS + 0.1% Triton X-100 for 15 minutes. The samples were stained as described26, with rhodamine-phalloidin (0.1 µM, Sigma-Aldrich, P1951) and DAPI (10 µM, Invitrogen D1306) for 20 minutes. The images were captured using a Zeiss LSM510 confocal motorized inverted microscope with an XM-10 monochrome camera.
Sperm number and sperm viability
Put the isolated seminal vesicles or seminal receptacle in 5–10 µl of PBS. The tissue was punctured using a dissecting needle and allowed to rest for 10 minutes to release the sperm. Stain with DAPI or SYBR28, 29. DAPI staining resulted in the blue staining of the sperm heads, while SYBR staining produced green fluorescence in live sperm and red fluorescence in membrane-compromised sperm when used in conjunction with propidium iodide. The SYBR staining procedure was carried out as follows: Sperm samples were diluted in a live-cell imaging solution that contained 10% BSA. A stock solution of SYBR 14 dye was created by adding 900 µl of DMSO. Then, 1 µl of the SYBR 14 stock solution and 5 µl of propidium iodide solution were mixed with the diluted sperm sample. The mixture was incubated at 37°C for 5–10 minutes, following which the sample was observed using a confocal microscope.
Metabolite extraction
10 flies or 30 (young)/90 (old) flies’ sperm were collected under light CO2 anesthesia and rapidly frozen in liquid nitrogen. The frozen flies were then ground using a CryoMill. Metabolite extraction was performed as described in the previous study33. The supernatant was transferred to a new Eppendorf tube and dried in a vacuum concentrator at RT. The dry pellets were reconstituted into 30 µl sample solvent (15 µl water and then 15 µl methanol/acetonitrile (1:1 v/v)) and 3 µl was further analyzed by liquid chromatography coupled to high resolution mass spectrometry. Mobile phase A is water with 5 mM ammonium acetate, pH 6.9, and mobile phase B is 100% acetonitrile.
An Ultimate 3000 UHPLC (Dionex) was coupled to Q Exactive-Mass Spectrometer (QE-MS, Thermo Scientific) for metabolite separation and detection. For metabolite analysis, used a HILIC method, with an Xbridge amide column (100 × 2.1 mm internal diameter, 3.5 µm; Waters), for compound separation at RT. The mobile phase and gradient information have previously been described33. The gradient is linear as follows: 0 minute, 85% B; 1.5 minutes, 85% B; 5.5 minutes, 35% B; 10 minutes, 35% B; 10.5 minutes, 35% B; 10.6 minutes, 10% B; 12.5 minutes, 10% B; 13.5 minutes, 85% B; and 20 minutes, 85% B. The flow rate is 0.15 ml/minute from 0 to 5.5 minutes, 0.17 ml/minute from 6.9 to 10.5 minutes, 0.3 ml/minute from 10.6 to 17.9 minutes, and 0.15 ml/minute from 18 to 20 minutes. The QE-MS is equipped with a heated electrospray ionization probe, and the relevant parameters are listed: evaporation temperature, 120°C; sheath gas, 30; auxiliary gas, 10; sweep gas, 3; spray voltage, 3.6 kV for positive mode and 2.5 kV for negative mode. The capillary temperature was set at 320°C, and S-lens was 55. A full scan ranges from 70 to 900 (m/z) was used. The resolution was set at 70,000. The maximum injection time was 200 ms. Automated gain control was targeted at 3 x 106 ions.
Polyamine extraction
Metabolite extraction was performed as described in previous study34. The supernatant was transferred to a new Eppendorf tube and dried in vacuum concentrator at RT. The dry pellets were reconstituted into 30 µl sample solvent (water: initial gradient, 1:4, v/v) and 3 µl was further analyzed by LC-MS. Initial gradient is 99% A (2 mM ammonium acetate in 10% (v/v) acetonitrile) plus 1% B (0.4% (v/v) acetic acid in 10% (v/v) acetonitrile).
Ultimate 3000 UHPLC is coupled to QE-MS for polyamine profiling. A pH gradient-liquid chromatography method employing a Scherzo SM-C18 column (50 x 2.0 mm i.d., 3 µm; Imtakt, Japan) at 30 oC is used for polyamine separation34. The linear gradient used is as follows: 0 min, 1% B; 1 minute, 5% B, 3 minutes, 5% B; 5 minutes, 100% B, 10 minutes, 100% B. The flow rate is 0.2 ml/minute. Then the system is washed with 100% B for 10 minutes at flow rate 0.2 ml/minute to wash polyamine carryover. Repeat this washing step 4 times, then equilibrate column for 10 minutes with 1% B before analyzing next polyamine sample. The equilibrium step right before sample analysis is crucial as initial condition significantly impacts polyamine separation. The QE-MS is equipped with a HESI probe with related parameters set as below: heater temperature, 120°C; sheath gas, 30; auxiliary gas, 10; sweep gas, 3; spray voltage, 3.0 kV at positive mode; capillary temperature, 320°C; S-lens, 55; A scan range (m/z) of 70 to 240 was used in positive mode from 0.5 to 9 minutes; resolution: 70000; automated gain control (AGC), 3 × 106 ions. Customized mass calibration was performed before data acquisition.
Metabolomics data processing and metabolic flux analysis (MFA)
Analytical LC-MS peak extraction and integration were performed using commercially available software Sieve 2.2 (Thermo Scientific). The integrated peak intensities were used for further data analysis. Natural abundance was corrected as previously described35 for tracing studies.
We constructed methionine and one-carbon metabolic flux analysis (MFA). By setting flux values, as weights to a group of flux values, predict the mass isotopomer distribution (MID) of target metabolites by an average of the MID of the precursors. Mode as:
$${\stackrel{\sim}{{M}}}_{i}=\frac{\sum _{\forall j}{v}_{ji}{{M}}_{ji}}{\sum _{\forall j}{v}_{ji}}$$
$${L}_{i}={D}_{\text{KL}}\left({\stackrel{\sim}{{M}}}_{i}\parallel {{M}}_{i}\right)=\sum _{j}\left({M}_{i,j}+{\epsilon }_{\text{log}}\right)\text{log}\frac{{\stackrel{\sim}{M}}_{i,j}+{\epsilon }_{\text{log}}}{{M}_{i,j}+{\epsilon }_{\text{log}}}$$
\({M}_{i,j}\) and \({\stackrel{\sim}{M}}_{i,j}\) are element \(j\) in vector \({{M}}_{i}\) and \({\stackrel{\sim}{{M}}}_{i}\), respectively. \({\epsilon }_{\text{log}}\) is a small number added to maintain numerical stability. Sum of \({L}_{i}\) for all target metabolites was regarded as the loss function \({L}_{\text{total}}\) to minimize. The optimization problem was defined as:
$$\underset{{v}}{\text{min}}{L}_{\text{total}}\left({v}\right), \text{s}.\text{t}. {A}\bullet {{v}}^{T}={b}, 0\le {{v}}_{\text{min}}\le {v}\le {{v}}_{\text{max}}$$
\({A}\bullet {{v}}^{T}={b}:\) represents flux balance requirement and other equality constraints. \({{v}}_{\text{min}}\) and \({{v}}_{\text{max}}\) are lower and upper bounds for composite vector \({v}\). The optimization method refers to previous publication36, 37.
To reduce random errors in batch preparation and measurement, MIDs in all biological repeats are averaged to be the target MID for MFA. Network diagrams with flux values are plotted with the Python package Matplotlib, the directions of a flux arrow are set by directions of net flux, and the transparency of flux arrows is set based on its value.
Statistical analysis and bioinformatics
Analysis of metabolites was carried out with software Metaboanalyst (http://www.metaboanalyst.ca/MetaboAnalyst/) and software GENE-E (https://software.broadinstitute.org/GENE-E/) using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database (http://www.genome.jp/kegg/). All data are represented as Mean ± s.e.m. as indicated. P values were calculated by a two-tailed Student’s t test unless otherwise noted. All experiments were performed in at least three biological replicates and repeated in two to three independent experiments. Prism 7 (GraphPad) was used to perform statistical analysis.