METHOD DETAILS

Drosophila lines listed in the key resources table without a ‘Source’ entry were generated for this study. Of these, MiMIC (MI) and CRiMIC (CR) alleles designated ‘TG4’ were converted to T2A-GAL4 containing alleles via Recombinase Mediated Cassette Exchange (RMCE) as previously described.⁷⁸ Correctly oriented and framed RMCE events were confirmed via UAS-YFP expression.^85,86 Alleles designated ‘KO-kG4’ (Kozak-GAL4) were generated as previously described.²² The sgRNA and homology arm sequences and PCR verification details for each line can be found at https://flypush.research.bcm.edu/pscreen/crimic/crimic.php. For CRISPR/Cas9-induced point mutations, we crossed transgenically-expressed guide RNA lines targeting Hn and Hmgcl coding sequences to a maternal germline Cas9 source and isolated stable mutant lines bearing early frameshift mutations. All mutant lines used were first outcrossed to w¹¹¹⁸ for removal of y¹ and then maintained over fluorescently marked balancer chromosomes.

Chemically defined media used in this study were prepared as described in Piper et al.,¹⁵ with modifications to support larval development. Briefly, sucrose, agar, low solubility amino acids (L-isoleucine, L-leucine, and L-tyrosine), and stock solutions of buffer, metal ions and cholesterol were combined with MilliQ water using a magnetic stirrer. After autoclaving at 120°C for 15 min, the solution was cooled at room temperature with stirring to ~65°C. Stock solutions for amino acids, vitamins, nucleosides, choline, inositol, and preservatives were then added. Liquid food was dispensed into sterile vials. The chemically defined diet recipes, including modifications used for targeted nutrigenomics and dose-response assays, as well as the standard sugar-yeast medium are listed in the supplemental information (Table S2).

To obtain large numbers of developmentally synchronized first instar larvae, embryos were collected from population cages containing approximately 200 females and 50 males (5–10 days old) that were allowed to lay for 6 h on apple juice agar plates supplemented with yeast paste. Embryos were collected and washed with distilled water and then transferred to a new plate (yeast paste-free) and incubated at 25°C for 24 h to hatch. Homozygous individuals (AAD/AAD) were collected from cages comprising heterozygous adults (the allele of interest and a fluorescently marked balancer chromosome). To collect AAD/+ individuals (controls), cages comprised heterozygous males and virgin w¹¹¹⁸ (+/+) females. Where the gene of interest was X-linked, w¹¹¹⁸ were used as a control.

First instar larvae were selected based on the absence of the fluorescent balancer. Using a blunt metal probe, larvae were transferred to the required medium. For the larval diet-switching experiment, larval locomotor assay, electrophysiology and metabolomic analysis, larvae at the desired developmental stage were collected by floating in 20% sucrose solution.⁸⁷ Briefly, 20% w/v sucrose solution was poured into each vial. The top layer of food was then gently disrupted with a metal probe in order to release the larvae. The solution with floating larvae was poured through a fine cloth mesh to isolate the larvae. Larvae were then rinsed three times with Phosphate Buffer Saline (PBS) and transferred to fresh media or microtubes tubes using a fine paintbrush.

Twenty first instar larvae were transferred to a vial containing the appropriate diet and monitored every 12 h for 25 days for the presence of pupae and adults. This was replicated 5 times per genotype per diet. The same approach was used to assess shop mutants and controls raised on modified synthetic media with 0% cysteine, 0% tyrosine, 50% phenylalanine and 75% phenylalanine.

Large numbers of developmentally synchronized embryos obtained from multiple cages containing shop/FM7a,sChFP females were collected and washed in distilled water, and transferred to 15 mL tubes containing PBS. Once the embryos had settled (1 min), excess PBS was removed. Using a micropipette set to 5 μL and a tip cut to widen the bore, small volumes of embryos were aspirated and then dispensed into vials onto the medium surface. This was performed for each of the 75 modified synthetic diets tested with varied levels of cysteine, serine, and methionine replicated 5 times (Table S1B). Vials were checked daily for 20 days and the total number of adult flies were counted with the survival proportion of shop (shop/total flies) calculated. The total number of adults per vial ranged from 10 to 80 individuals (mean = 44.17, s.d. = 9.40).

Larvae were reared on the 0% cysteine diet under constant density (20 per vial with 5 vials for shop and 10 vials for controls) and adult eclosion monitored daily. Adults of the same genotype that eclosed on the same day were combined and transferred evenly to either the 100% diet or 0% cysteine diet. Since shop is X-linked and shop hemizygous males are sterile,⁹ only male flies (shop and control) were included in the longevity experiment. Flies were given fresh media and scored for survival three days per week. Escapees were censored from the analysis. Eight vials each containing 1 to 9 flies were tracked for shop per diet. Six vials each containing 1 to 14 flies were tracked for the control per diet.

Embryos were collected and dispensed on to the complete synthetic diet (100% cysteine) as per the multi-nutrient array. On days 2, 4, 6, and 8 post-hatching, larvae from 10 replicate vials were floated in 20% sucrose, washed, and transferred to fresh vials containing the 0% cysteine diet. An additional group of 10 vials were kept on the complete media as a control. Numbers of adult shop males and control (FM7a, sChFP) males were scored for 20 days post egg-lay. The total number of adults per replicate ranged from 29 to 97 individuals (mean = 56.8, s.d. = 15.9).

Five replicates of 10 five-day-old larvae from each genotype and diet condition were collected, washed in PBS, blotted dried, and weighed. Samples were then transferred to 1.5 mL safe lock microtubes tubes (Eppendorf), flash frozen in liquid nitrogen, and stored at −80°C. Thawed larvae were homogenized using a disposable pestle in 20 μL of ice-cold extraction solvent consisting of 2:6:1 chloroform:methanol:water with 2 μM of (CHAPS, CAPS, PIPES and TRIS) acting as internal standards. Once ground additional solvent was added to a final ratio of 20 μL of extraction solvent per mg of larvae. Samples were vortexed for 30 s and then sonicated in an ice-water bath for 10 min and centrifuged at 4°C (22,000 ×g for 10 min). The supernatant was then transferred to a glass vial for LC-MS based metabolomic analysis. 20 μL of each extract were combined to make a pooled quality control sample.

LC-MS was performed using a Dionex Ultimate 3000 UHPLC coupled to an QExactive Plus mass spectrometer (Thermo Scientific). Samples were analyzed by hydrophilic interaction liquid chromatography (HILIC) following a previously published method.⁸⁸ The chromatography utilized a ZIC-p(HILIC) column 5μm 150 × 4.6 mm with a 20 × 2.1 mm ZIC-pHILIC guard column (both Merck Millipore, Australia) (25°C). A gradient elution of 20 mM ammonium carbonate (A) and acetonitrile (B) (linear gradient time-%B: 0 min-80%, 15 min-50%, 18 min-5%, 21 min-5%, 24 min-80%, 32 min-80%) was utilized. Flow rate was maintained at 300 μL/min. Samples were stored in the autosampler (6°C) and 10 μL was injected for analysis. MS was performed at 70,000 resolution operating in rapid switching positive (4 kV) and negative (−3.5 kV) mode electrospray ionization (capillary temperature 300°C; sheath gas flow rate 50; auxiliary gas flow rate 20; sweep gas 2; probe temp 120°C). Samples were randomized and processed in a single batch with intermittent analysis of pooled quality-control samples to ensure reproducibility and minimize variation. For accurate metabolite identification, a standard library of ~500 metabolites were analyzed before sample testing and accurate retention time for each standard was recorded. This standard library also forms the basis of a retention time prediction model used to provide putative identification of metabolites not contained within the standard library.⁸⁹

Locomotor assay modified from Brooks et al.⁹⁰ Five replicates of 5-day-old larvae for each genotype and diet condition were collected, washed in PBS and blotted dry then transferred using a blunt metal probe to a Petri dish containing SY medium with blue food dye (Queen) and allowed to acclimate for 3 min. The plate was placed on a uniform black background to reduce aberrant reflections and filmed for 3 min with a webcam (Logitech). The movie file was converted into avi format (ffmpeg), and analyzed in ImageJ using the wrMTrck plugin⁹¹ (Table S1F). For each larva, the total length of movement (mm) was calculated. For each genotype and diet, 5 replicate plates (25 individuals total) were tracked.

Thiosulfate determination assay was modified from.⁹² Five replicates of 20 7-day-old and 14-day-old adult flies, or 50 5-day-old larvae for each genotype and diet condition were collected and transferred to 1.7mL microtubes (Eppendorf), frozen with liquid nitrogen. Thawed flies were homogenized to powder using a disposable pestle in 0.05% tween 20. Samples were then homogenized by vortexing for 20 s followed by centrifuging at 5000rpm for 1 min. For each sample, 500 μL supernatant was transferred individually to a new 1.7mL Eppendorf tube and centrifuged for 3 min at 14000rpm. For final data normalization, protein concentration of each sample was measured using Pierce BCA protein assay kit #23227. For thiosulfate determination, 250 μL supernatant from each sample was transferred individually to new 1.7mL Eppendorf tubes, followed by adding 750 μL methylene blue solution (a stock solution was prepared by dissolving 3 mg methylene blue in 250 mL 5M HCl). Samples were incubated at room temperature for 30 min. A standard curve based on a 1 mM freshly prepared sodium thiosulfate solution was prepared by transferring 0, 1, 5, 10, 20, 50, 75 and 100 μL sodium thiosulfate solution (these values correspond to the same amount of thiosulfate in nM) to fresh 1.7mL Eppendorf tubes. The volume was made up to 250 μL with 0.05% tween 20, followed by adding 750 μL methylene blue solution and incubated at room temperature for 30 min. For each experimental samples as well as the standard curve, 100 μL of solution was transferred to a 96-well plate (triplicates were prepared for experimental samples). Absorption at 670 nm was determined spectrophotometrically against 0.05% tween 20 as the reference.

Twenty-five 6-day-old larvae for each genotype and diet condition were collected, washed in PBS and blotted dry. Samples were then and transferred to 1.7mL microtubes (Eppendorf) containing 50μL PBS. Larvae were dissected and intracellular membrane potentials were recorded as previously described.⁹³ Briefly, recordings were performed on muscle 6 in segment A3. The free segmental nerve end was drawn into a microelectrode using an injector and stimulated with a DS2A isolated voltage stimulator (Digitimer) at 0.1 Hz with a suprathreshold stimulating pulse and postsynaptic potentials were recorded with electrodes (20–50 MΩ) filled with 3 M KCl. All recordings were conducted at room temperature with a Multiclamp 700B amplifier (Molecular Devices, Sunnyvale, CA) in current clamp using AxoGraph X software v1.7.6 (AxoGraph, Australia). A minimum of 20 EJPs were recorded per muscle and the average amplitude used. The mEJP recordings were done over a 2-min period and events detected by analyzed by fitting to a template in AxoGraph X.

Three to five replicates of 40 five-day-old larvae from each genotype and diet condition were collected into microtubes containing 500 μL of TRIzol (Invitrogen). Total RNA was isolated on the same day following the manufacturer’s instructions. RNA purity and concentration were evaluated by spectrophotometry (NanoDrop ND-1000, NanoDrop Technologies). RNA samples were then diluted to 5 μg/μL and stored at −80°C. RNA samples were treated with the RQ1 RNase-Free DNase (Promega) following the manufacturer’s instructions to remove genomic DNA contamination and cDNAs were generated using the kit Tetro cDNA Synthesis (Bioline). RT-qPCR analysis was performed in a 7500 Real-Time PCR System (Applied Biosystems) using the SYBR Green Reagent protocol (Applied Biosystems). Oligos were created in Geneious Prime (v 2023.2) using Primer-BLAST (NCBI). The ribosomal protein L11 gene (RpL11) was used as reference (housekeeper) for normalizing the expression levels of the genes of interest. Expression fold-change was calculated using the 2^(−ΔΔCt) method, normalized to the level of expression of control flies reared in control diet (100% Cys). To avoid values between 0 and 1, results were log2 transformed. The list of selected genes for evaluation, primer sequence and Real Time qPCR data is shown in Table S1G.