Dietary restriction and the transcription factor clock delay eye aging to extend lifespan in Drosophila Melanogaster –

Experimental materials

A full detailed description of the materials, primer sequences, software packages, and commercial assays used in this study are reported in Supplementary Data 7. Data was collected with Microsoft Excel (version 16.58) and figures and statistics were generated in GraphPad Prism software (version 9).

Fly stocks

The genotypes of the Drosophila Melanogaster lines used in this study are listed in Supplementary Table 2. The following lines were obtained from the Bloomington Drosophila Stock Center: Oregon R. (25125), GMR-GAL4 (1104), Elav-GS-GAL4 (43642), Trpl-GAL4 (52274), clkout (56754), UAS-csChrimson (55134), UAS-CLK-∆1 (36318), UAS-CLK-∆2 (36319), Gβ76c-RNAi (28507), tsp42Ej/sun-RNAi (29392), retinin-RNAi (57389), ATPα-RNAi (28073), nrv2-RNAi (28666), nrv3-RNAi (60367), and mCherry-RNAi (Bloomington RNAi-cnt, 35785). The following lines were obtained from the Vienna Drosophila Resource Center: arr1-RNAi (22196), RNAi-cnt (empty vector, 60100). The UAS-Clk line was a gift from Paul Hardin’s laboratory at Texas A&M University. The following lines were outcrossed to w1118 for this manuscript: UAS-CLK-∆1OC and Canton-SOC. The Trpl-GAL4 line was recombined with GAL80 for this manuscript: Trpl-GAL4; GAL80ts. Ethical approval was not requested for the experiments performed in this study given the exclusive use of fruit flies.

Age of fly strains used in this study

Canton-S and tim01 mutant flies (11 days old) were used for the time-course microarray experiment. nCLK-∆1 flies (11 days old) were used for the RNA-Seq. experiment. The following strains (6, 10, 14, 18, and 22 days old) were used in phototaxis experiments: nCLK-∆1, prCLK-cnt, prCLK-∆1, prCLK-OE, GMR-GAL4 > RNAi-cnt, GMR-GAL4 > Gbeta76c-RNAi, GMR-GAL4 > retinin-RNAi, GMR-GAL4 > sun-RNAi, Canton-S, Oregon-R, clkout, nCLK-∆2, cry01, cry02, cryB, GMR-GAL4 > ATPα-RNAi, GMR-GAL4 > arr1-RNAi, GMR-GAL4 > nrv2-RNAi, GMR > GAL4 > nrv3-RNAi, Spa-GAL4 > RNAi-cnt, Spa-GAL4 > ATPα-RNAi. The follow strains (6, 10, 14, 18, and 25 days old) were used in ERG experiments: nCLK-∆1, prCLK-cnt, prCLK-∆1, prCLK-OE. The follow strains (6 and 14 days old) were used in tangential eye sections experiments: prCLK-cnt, prCLK-∆1, prCLK-OE. The following strains (11 days old) were used in RT-PCR experiments: nCLK-∆1, nCLK-∆2, w1118, ninaE17, rh32, rh41, rh6G. The following strains (6-days old to death) were used for lifespan analyses: nCLK-∆1, nCLK-∆2, w1118, ninaE17, rh32, rh41, rh6G, Gq1, prCLK-cnt, prCLK-∆1, prCLK-OE, GMR-GAL4 > RNAi, GMR-GAL4 > Gbeta76c-RNAi, GMR-GAL4 > -retinin-RNAi, GMR-GAL4 > sun-RNAi, Canton-S, csChrimson, TRP365, GMR-GAL4 > ATPα-RNAi, GMR-GAL4 > arr1-RNAi, GMR-GAL4 > nrv2-RNAi, GMR > GAL4 > nrv3-RNAi, Spa-GAL4 > RNAi-cnt, Spa-GAL4 > ATPα-RNAi. nCLK-∆1 flies at 18 days of age were used in the hemolymph mass-spectrometry experiment.

Fly husbandry and survival analyses

All flies were maintained at 25 ± 1 °C, 60% humidity under a 12 h:12 h LD cycle (~750lux, as measured with a Digital Lux Meter, Dr. Meter Model LX1330B) unless otherwise indicated. Fly stocks and crosses were maintained on a standard fly media which consisted of 1.5% yeast extract, 5% sucrose, 0.46% agar, 8.5% of cornmeal, and 1% acid mix (a 1:1 mix of 10% propionic acid and 83.6% phosphoric acid). Fly bottles were seeded with live yeast prior to collecting virgins or setting up crosses. Mated adult progeny were then transferred to ad libitum (AL) or dietary restriction (DR) media within three days of eclosion. Adult female flies used in experiments were transferred to fresh media every 48 h at which point deaths were recorded for survival analysis. AL and DR fly media differed only in their percentage of yeast extract, respectively containing 5% or 0.5% (Yeast Extract, B.D. Bacto, Thermo Scientific 212720, Cat no. 90000-722). Optogenetic experiments: For experiments using the csChrimson channel rhodopsin48, adult flies were transferred to media supplemented with 50 μM all-trans-retinal (Sigma–Aldrich, R2500-1G) or drug vehicle (100% ethanol), and maintained under a 12 h:12 h red light:dark cycle, with ~10lux of red light (~590 nm) during the light phase. Elav-GeneSwitch flies: GeneSwitch62, adult flies were transferred to media supplemented with 200 μM RU486 (Mifepristone, United States Biological), indicated as either AL+ or DR+, for post-developmental induction of transgenic elements; isogenic control flies were transferred to food supplemented with a corresponding concentration of drug vehicle (100% ethanol), indicated as either AL- or DR-. prCLK-Δ1 experiments: GAL80 temperature-sensitive crosses were set in bottles at 25 °C, 60% humidity under a 12 h:12 h LD cycle for 4 days. Parental flies were removed, and the bottles were transferred to 18 °C for ~3 weeks to suppress GAL4 activity throughout development. After ecolsion, the F1 generations were sorted onto AL or DR food the flies were maintained at 30 °C to de-repress GAL80 and activate GAL4 (60% humidity under a 12 h:12 h LD cycle) for the remainder of their lifespans. The F1 generations for these experiments share the same genetic background, as both the UAS-CLK-Δ1 and the Canton-S control lines were fully outcrossed to the same w1118 strain prior to setting up the cross with Trpl-GAL4; GAL80ts.

Circadian time-course expression analysis

Mated Canton-S and Tim01 females were reared on AL or DR diets for seven days at 25 ± 1 °C, under a 12:12 h light-dark (LD) regimen. Beginning on the seventh day, four independent biological replicates (per diet/timepoint) of approximately 35 female flies (approximately 11 days old) were collected on dry ice every 4 h for 20 h starting at ZT 0 (six total timepoints, 48 total samples). RNA extraction, DNA amplification/labeling, and gene expression arrays were performed following the same protocols as in ref. 63. In summary, RNA was isolated from whole-fly lysates with Qiagen’s RNeasey Lipid Tissue Mini Kit (74804), and RNA quantity and quality were accessed with a Nanodrop and Agilent’s bioanalyzer (RNA 600 Nano Kit (5067-15811)). DNA amplification from total RNA was performed using Sigma’s TransPlex Complete Whole Transcriptome Amplification Kit (WTA2) and purified with Qiagen’s QIAquick PCR Purification Kit (28104). Gene expression labeling was performed with NimbleGen One-Color DNA Labeling Kit (05223555001) and hybridized to NimbleGen 12-Plex gene expression arrays. Arrays were quantitated with NimbleGen’s NimbleScan2 software (version 2.6), and downstream expression analyses were conducted in R (version 3.2.4) ( with the LIMMA package 3.34.5. Transcript-level expressions from the four independent biological replicates were averaged for each timepoint.

nCLK-Δ1 RNA-seq analyses

nCLK-Δ1 (Elav-GS-GAL4 > UAS-nCLK-Δ1) adult female flies were developed on standard stock food (1.5% yeast-extract) for four days. Three independent biological replicates of 100 mated female flies were then reared on AL or DR diets treated with RU486 or vehicle control at 25 ± 1 °C, under a 12:12 h LD regimen. Diets were changed approximately every 48 h, until the seventh day at which point flies were flash-frozen (~11 days old) on dry ice at ZT 0 and ZT 12 (lights-on and -off, respectively). See Supplementary Fig. 2a for RNA-seq. experimental design. RNA extraction: Frozen flies were vortexed to remove heads and mRNA from each biological replicate of pooled heads was isolated with the Quick-RNA MiniPrep Kit (Zymo Research #11-328), per manufacturers’ instructions. Fragment library preparation and deep sequencing: Library preparation was performed by the Functional Genomics Laboratory (FGL), a QB3-Berkeley Core Research Facility at University of California, Berkeley. cDNA libraries were produced from the low-input RNA using the Takara SMART-Seq v4 Ultra-low-input RNA kit. An S220 Focused-Ultrasonicator (Covaris®) was used to fragment the DNA, and library preparation was performed using the KAPA hyper prep kit for DNA (KK8504). Truncated universal stub adapters were used for ligation, and indexed primers were used during PCR amplification to complete the adapters and to enrich the libraries for adapter-ligated fragments. Samples were checked for quality on an AATI (now Agilent) Fragment Analyzer. Samples were then transferred to the Vincent J. Coates Genomics Sequencing Laboratory (GSL), another QB3-Berkeley Core Research Facility at UC Berkeley, where Illumina sequencing library molarity was measured with quantitative PCR with the Kapa Biosystems Illumina Quant qPCR Kits on a BioRad CFX Connect thermal cycler. Libraries were then pooled evenly by molarity and sequenced on an Illumina NovaSeq6000 150PE S4 flowcell, generating 25 M read pairs per sample. Raw sequencing data were converted into fastq format, sample-specific files using the Illumina bcl2fastq2 (v2.20) software on the sequencing center local linux server system. Read alignment and differential expression analyses: Raw fastq reads were filtered by the Trimmomatic software64 (Trimmomatic-0.36) to remove Illumina-specific adapter sequences and the minimal length was set to 36 (MINLEN) for trimming sequences. The paired-end filtered reads were then aligned to the D. Melanogaster dm6 genome (BDGP Release 6 + ISO1 MT/dm6) by HISAT2 (Galaxy Version 2.2.1 + galaxy0)65 to generate BAM files with the specific strand information set to “Reverse”. Count files were then generated by featureCounts (Galaxy Version 2.0.1 + galaxy2)66 and the D. Melanogaster reference genome was utilized as the gene annotation file with specific strand information set to “stranded (Reverse)”. The resulting count files (tabular format) were then analyzed with DESeq2 (Galaxy Version + galaxy1)67 with fit-type set to “local”, and P-values of less than 0.05 (adjusted for multiple testing) were considered differentially expressed between factor levels. Normalized count reads were outputted for visualization of expression (heatmaps), and Supplementary Data Files 3a contains normalized count reads across all experimental samples. UCSC genome browser visualization: The makeUCSCfile software package from HOMER (v4.11) was utilized to generate bedGraph files for visualizing changes in tag density at exon 2 of clk comparing nCLK-Δ1 and control samples (Supplementary Fig. 2b).

Heatmap visualizations

We employed the heatmap2 (Galaxy Version 3.0.1) function from R ggplot2 package to visualize bioinformatics data. Data were not clustered, and data were scaled by row for normalization across timepoints.

Electroretinogram assays

ERGs were performed and analyzed in two independent laboratories. ERGs were recorded for eight nCLK-Δ1 female flies reared on AL or DR diets supplemented with vehicle or RU486 at day 14 (18 days of age) at the Baylor College of Medicine (BCM), and at day 21 (25 days of age) at the University of California, Santa Barbara (UCSB). ERGs were recorded for prCLK-Δ1 and prCLK-OE female flies at UCSB reared on AL or DR and maintained at either 18 °C or 30 °C at ages 6, 10, 14, and 18 days old. BCM: ERG recordings were performed as in ref. 68. Flies were glued on a glass slide. A recording electrode was placed on the eye and a reference electrode was inserted into the back of the fly head. Electrodes were filled with 0.1 M NaCl. During the recording, a 1 s pulse of light stimulation was given. The ERG traces of at least eight flies per genotype/diet were recorded and analyzed by LabChart8 software (AD Instruments). UCSB: Mated female flies (nCLK-Δ1 and prCLK-Δ1) were reared on AL or DR diets starting at 4 days old with and without RU486. ERGs were recorded at ages 6, 10, 14, and 18 days old. ERG recordings were performed as in ref. 69. Two glass electrodes were filled with Ringer’s solution and electrode cream was applied to immobilized flies. A reference electrode was placed on the thorax, while the recording electrode was placed on the eyes. Flies were then exposed to a 10 s pulse of ~200lux white light, a light intensity that is comparable to the phototaxis assay. An EI-210 amplifier (Warner Instruments) was used for amplifying the electrical signal from the eye after light stimulation, and the data were recorded using a Powerlab 4/30 device along with the LabChart 6 software (AD Instruments). Raw data were then uploaded into R-statistical software for plotting and statistical analysis. All electroretinograms were performed between ZT4-8 or ZT12-14.

Positive phototaxis assay

Positive phototaxis was performed using an adapted protocol from ref. 70. Phototaxis measurements were recorded longitudinally on populations of female flies aged (6, 10, 14, 18, and 22 days old) on either AL or DR food (with or without 200 μM RU486 when indicated) at a density of 10–25 flies per tube prior to and after phototaxis measurements. Approximately 160–480 flies were used in each phototaxis experiment. On the day of phototaxis recording, eight groups of flies (four AL and four DR groups) were placed in separate 2.5 × 20 cm tubes (created from three enjoined narrow fly vials [Genesee Scientific]) and dark-adapted for 15 min prior to light exposure (no food was available in the vials during phototaxis assays). Flies were then gently tapped to the bottom of the tube, placed horizontally, and exposed to white light from an LED strip (Ustellar, UT33301-DW-NF). A gradient of light intensity was created, with 500lux at the nearest point in the fly tube to the light source and 150lux at the furthest point. Phototaxis activity was recorded by video at 4 K resolution (GoPro, Hero5 black). Positive phototaxis was scored manually as the percentage of flies that had traveled >19 cm toward the light source in three 15 s intervals (15 s, 30 s, and 45 s). “Phototaxis index” was calculated by averaging the percent of positive phototaxis for each vial at the three 15 s intervals. To control for light-independent wandering activity, a phototaxis index was also calculated when the light source was placed in parallel to the fly tube, such that all parts of the tube were equally illuminated with 500lux. We accessed positive phototaxis behavior from the following fly strains in this study: Canton-S, clkout, cry01, cry02, cryB, nCLK-∆1, nCLK-∆2, GMR-GAL4 > mCherry-RNAi (cnt), GMR-GAL4 > arr1-RNAi, GMR-GAL4 > ATPα-RNAi, GMR-GAL4 > retinin-RNAi, GMR-GAL4 > sun-RNAi, GMR-GAL4 > nrv2-RNAi, GMR-GAL4 > nrv3-RNAi, prCLK-∆1, prCLK-OE, Spa-GAL4 > ATPα-RNAi, and Spa-GAL4 > RNAi-cnt.

RNA extraction and cDNA preparation

Adult female flies (strains nCLK-∆1, nCLK-∆2, w1118; ninaE17, rh32, rh41, rh6G, GMR-GAL4 > ATPα-RNAi, GMR-GAL4 > mCherry-RNAi) were maintained on AL or DR for, then flash-frozen on dry ice (11 days old). Heads were separated from bodies (thorax and abdomen) by vigorous shaking. Flies were then ground using a hand-held homogenizer at room temperature following MiniPrep instructions. Total RNA was isolated using the Quick-RNA MiniPrep Kit (Zymo Research, 11-328). RNA was collected into 30 μl DNAse/RNAse-free water and quantified using the NanoDrop 1000 Spectrophotometer (Thermo Scientific). For each experiment, 120–180 age-, genotype-, and diet-matched flies were collected, and three independent RNA extractions were performed. To extract RNA from heads, 40–60 flies were used; to extract RNA from bodies, 20–30 flies were used. cDNA preparation: The iScript Reverse Transcription Supermix for RT-qPCR (Bio-Rad, 1708841) was used to generate cDNA from RNA extracted from heads and bodies. For each group, 1 μg of total RNA was placed in a volume of 4 μl iScript master mix, then brought to 20 μl with DNAse/RNAse-free water. A T1000 thermocycler (BioRad) was used for first-strand RT-PCR reaction following iScript manufacturers’ instructions—priming step (5 min at 25 °C), reverse transcription (30 min at 42 °C), and inactivation of the reaction (5 min at 85 °C).

Real-time quantitative PCR

Reactions were performed in a 384-well plate. Each reaction contained 2 μl of 1:20 diluted cDNA, 1 μl of primers (forward and reverse at 10 μM), 5 μl SensiFAST SYBR Green No-ROX Kit (BIOLINE, BIO-98020), and 2 μl of DNAse/RNAse-free water. The qPCR reactions were performed with a Light Cycler 480 Real-Time PCR machine (Roche Applied Science) with the following run protocol: pre-incubation (95 °C for 2 min), forty PCR cycles of denaturing (95 °C for 5 s, ramp rate 4.8 °C/s), and annealing and extension (60 °C for 20 s, ramp rate 2.5 °C/s). The PCR primer sequences (forward and reverse) are in Supplementary Data 7.

Hemolymph mass spectrometry

Proteomic sample preparation: nCLK-Δ1 female flies (Elav-GeneSwitch-GAL4 > UAS-nCLK-Δ1) were reared on AL diet plus RU486 or vehicle control (N = 300 flies per biological replicate, n = 3 biological replicates). On day 14 (18 days old), flies were snap-frozen on dry ice and transferred to prechilled vials. The vials were vortexed for 5–10 s to remove heads and the frozen bodies were transferred to room temperature vials fitted with 40 µm filters. Headless bodies were thawed at room temperature for 5 min and spun at 2000 × g for 10 min at 4 °C. Following the spin, hemolymph collected at the bottom of each vial, and the bodies remained within the filters. Digestion: A Bicinchoninic Acid protein assay (BCA) was performed for each of the hemolymph samples and a 100 µg aliquot was used for tryptic digestion for each of the six samples. Protein samples were added to a lysis buffer containing a final concentration of 5% SDS and 50 mM triethylammonium bicarbonate (TEAB), pH ~7.55. The samples were reduced to 20 mM dithiothreitol (DTT) for 10 min at 50 °C, subsequently cooled at room temperature for 10 min, and then alkylated with 40 mM iodoacetamide (IAA) for 30 min at room temperature in the dark. Samples were acidified with a final concentration of 1.2% phosphoric acid, resulting in a visible protein colloid. 90% methanol in 100 mM TEAB was added at a volume of seven times the acidified lysate volume. Samples were vortexed until the protein colloid was thoroughly dissolved in the 90% methanol. The entire volume of the samples was spun through the micro S-Trap columns (Protifi) in a flow-through Eppendorf tube. Samples were spun through in 200 µL aliquots for 20 s at 4000 × g. Subsequently, the S-Trap columns were washed with 200 µL of 90% methanol in 100 mM TEAB (pH ~7.1) twice for 20 s each at 4000 × g. S-Trap columns were placed in a clean elution tube and incubated for 1 h at 47 °C with 125 µL of trypsin digestion buffer (50 mM TEAB, pH ~8) at a 1:25 ratio (protease:protein, wt:wt). The same mixture of trypsin digestion buffer was added again for overnight incubation at 37 °C.

Peptides were eluted from the S-Trap column the following morning in the same elution tube as follows: 80 µL of 50 mM TEAB was spun through for 1 min at 1000 × g. Eighty microliters of 0.5% formic acid was spun through next for 1 min at 1000 × g. Finally, 80 µL of 50% acetonitrile in 0.5% formic acid was spun through the S-Trap column for 1 min at 4000 × g. These pooled elution solutions were dried in a speed vac and then resuspended in 0.2% formic acid. Desalting: The resuspended peptide samples were desalted with stage tips containing a C18 disk, concentrated, and resuspended in aqueous 0.2% formic acid containing “Hyper Reaction Monitoring” indexed retention time peptide standards (iRT, Biognosys). Mass-spectrometry system: Briefly, samples were analyzed by reverse-phase HPLC-ESI-MS/MS using an Eksigent Ultra Plus nano-LC 2D HPLC system (Dublin, CA) with a cHiPLC system (Eksigent) which was directly connected to a quadrupole time-of-flight (QqTOF) TripleTOF 6600 mass spectrometer (SCIEX, Concord, CAN). After injection, peptide mixtures were loaded onto a C18 precolumn chip (200 µm × 0.4 mm ChromXP C18-CL chip, 3 µm, 120 Å, SCIEX) and washed at 2 µl/min for 10 min with the loading solvent (H2O/0.1% formic acid) for desalting. Subsequently, peptides were transferred to the 75 µm × 15 cm ChromXP C18-CL chip, 3 µm, 120 Å, (SCIEX), and eluted at a flow rate of 300 nL/min with a 3 h gradient using aqueous and acetonitrile solvent buffers. Data-dependent acquisitions (for spectral library building): For peptide and protein identifications the mass spectrometer was operated in data-dependent acquisition53 mode, where the 30 most abundant precursor ions from the survey MS1 scan (250 msec) were isolated at 1 m/z resolution for collision-induced dissociation tandem mass spectrometry (CID-MS/MS, 100 msec per MS/MS, ‘high sensitivity’ product ion scan mode) using the Analyst 1.7 (build 96) software with a total cycle time of 3.3 s as previously described71. Data-independent acquisitions: For quantification, all peptide samples were analyzed by data-independent acquisition (DIA, e.g., SWATH, SWAG), using 64 variable-width isolation windows72,73. The variable window width is adjusted according to the complexity of the typical MS1 ion current observed within a certain m/z range using a DIA ‘variable window method’ algorithm (more narrow windows were chosen in ‘busy’ m/z ranges, wide windows in m/z ranges with few eluting precursor ions). DIA acquisitions produce complex MS/MS spectra, which are a composite of all the analytes within each selected Q1 m/z window. The DIA cycle time of 3.2 s included a 250 msec precursor ion scan followed by 45 msec accumulation time for each of the 64 variable SWATH segments.

Identification of photoreceptor-enriched CLK-output genes

Diagram of bioinformatics steps reported in Supplementary Fig. 5A. Gene lists are reported in Supplementary Data 11. We identified the top 1000 photoreceptor-enriched genes from ref. 74 (GSE93782). We then filtered this list for genes that oscillate in a circadian fashion, and that are downregulated with age from ref. 16 (GSE81100). Approximately 1/3 of the photoreceptor-enriched genes (366 genes) were expressed in a circadian fashion in young wild-type heads and approximately one-half of these (172 genes) displayed a significant loss in expression with age (5- vs 55-day old heads). We further analyzed the remaining gene lists to identify those that are significantly upregulated on DR compared to AL at either ZT 0 or ZT 12 from control (vehicle-treated) samples from our nCLK-Δ1 RNA-Seq analyses. Transcripts with a DESeq2 P ≤ 0.05 (non-adjusted) were considered differentially expressed. To increase the chance of including false negatives we utilized the raw P-values instead of the adjusted P-values (multiple testing correction) and performed additional experiments to validate these downstream targets. For the final filtering step, we analyzed the genes that were significantly downregulated in nCLK-Δ1 on DR (RU486 vs vehicle-treated controls), resulting in the identification of Gβ76c, retinin, and sun.

Tangential retinal sectioning and imaging

Control, prCLK-OE, and prCLK-Δ1 female flies were reared on AL or DR food for 2 or 10 days. Flies were then decapitated (6 and 14 days old) and whole-fly heads were fixed in 2.5% glutaraldehyde overnight and then transferred into 2.0% osminium tetraoxide for approximately 4 h. The heads were then dehydrated in 100% ethanol and embedded in Epon. Tangential retinal sections (~0.5 micron slices) were stained with 0.1% Toluine Blue. Image capture was performed with a Nikon Ni-E upright microscope with a motorized stage, MQA18000 DS-Fi3 Microscope Camera controlled by the NIS Elements 5.20 software. The microscope was set to ‘brightfield’, auto-exposure was set to ‘continuous’, power at 100%, and auto-white was selected for each image taken with either a ×20 and ×40 objective.

Identification of differential gene expression in flies reared in LD vs DD

Gene expression changes in heads of y.w. flies housed in 12:12 LD vs constant darkness (DD) were accessed from ref. (GSE3842)13. Fold-changes in response to light were calculated by averaging the changes in expression at each timepoint from a circadian time-course microarray and comparing expression between flies housed in LD vs. DD. Individual genes were scored as significantly differentially expressed by performing a Student’s t-test (paired, two-tailed) with P ≤ 0.05.

Statistics and reproducibility

The individual biological replicates “n” and the number of individual flies “N” is denoted in each figure legend along with the particular statistical test utilized. The P-value statistics are included in each figure. All error bars are represented as the standard error of the mean (SEM), and all graphs were generated in PRISM 9 (GraphPad). The experiments in this manuscript were performed with populations of female flies (i.e., typically greater than 20 flies per biological replicate).

Figure 3b: The number of biologically independent flies used are as follows (Day 2, 6, 10, 14): AL cnt 10, 14, 14, 10 flies, AL prCLK-Δ1 4, 5, 4, 0 flies, AL prCLK-OE 10, 10, 9, 10 flies, DR cnt 10, 10, 10, 10 flies, DR prCLK-Δ1 5, 6, 7, 0 flies, DR prCLK-OE 10, 10, 12, 10 flies. The ERG amplitudes for the prCLK-Δ1 flies on day 14 were non-responsive (flat) so we did not include those data in the graph. The ERG amplitudes were collected from flies examined over two independent experiments.

Figure 3c: The number of biologically independent flies with similar observable phenotypes to the main figure are as follows (Day 2, 10): AL cnt 4, 2 flies, AL prCLK-Δ1 2, 3 flies, AL prCLK-OE 2, 2 flies, DR cnt 3, 6 flies, DR prCLK-Δ1 3, 1 flies, DR prCLK-OE 4, 3 flies.

Figure 5: The number of flies used for lifespan analyses are as follows: (a) LD housed flies: AL N = 560, DR N = 584; DD housed flies: AL N = 460, DR N = 462. (b-g) w1118; ninaE17 flies: AL N = 514, DR N = 511; w1118; rh32 flies: AL N = 543, DR N = 597; w1118; rh41 flies: AL N = 550, DR N = 593; w1118; rh6G flies: AL N = 533, DR N = 563; w1118; Gqα1 flies: AL N = 403, DR N = 400. (h) RNAi control flies: AL N = 365, DR N = 351; arr1-RNAi flies: AL N = 333, DR N = 322. (i) Retinal-treated flies: AL N = 289, DR N = 236; Vehicle-treated flies: AL N = 256, DR N = 126. (j) RNAi control flies: AL N = 493, DR N = 490; ATPα RNAi flies: AL N = 510, DR N = 535. Survival data is plotted as an average of three independent lifespan repeats.

Time-course microarray analyses

Four independent biological replicates (per diet/timepoint) of approximately 35 Canton-S female flies were collected on dry ice every 4 h for 20 h starting at ZT 0 (six total timepoints, 48 total samples). Differential expression was determined by two-tailed Student’s t-test (paired) comparing the averaged transcript-level expression values between AL and DR samples across all timepoints, and P-values less than 0.05 were considered significant. The JTK_CYCLE algorithm75 (v3.0) was utilized to identify circadian transcripts from the AL and DR time-course expression arrays. Transcript-level expression values for each of the four biological replicates (per timepoint/diet) were used as input for JTK_CYCLE, and the period length was set to 24 h. We defined circadian transcripts as those displaying a JTK_CYCLE P-value of less than 0.05 (non-adjusted) in wild-type flies (Canton-S) while displaying a JTK_CYCLE P-value of greater than 0.05 (non-adjusted) in circadian mutant flies (tim01). Subsequent analyses compared diet-dependent changes in JTK_CYCLE outputs (phase and amplitude).


Three independent biological replicates of 100 mated female adult flies were utilized per genotype/diet/timepoint. DEseq2 software67 was utilized and P-values of less than 0.05 (adjusted for multiple testing) were considered differentially expressed between factor levels for gene-ontology enrichment analyses.

ERG responses

For ERG experiments we quantified responses from 6 to 15 individual flies per standard in the field. Statistical significance was determined by two-tailed Student’s t-test (unpaired), comparing ERG responses between diet and genotypes. Full ERG statistics are reported in Supplementary Data 6.

Survival analyses

The Log-rank (Mantel-Cox) test was used to determine statistical significance by comparing average lifespan curves from a minimum of two independent lifespan replicates. Hazard Ratios (log-rank) were also utilized to determine the probability of death across genotypes, lighting conditions, and diet. Detailed Log-rank and hazard ratios for each lifespan are reported in Supplementary Data 10.

Positive phototaxis assay

Statistical significance for the phototaxis index at each timepoint was calculated with the Student’s t-test (two-tailed, unpaired). Two-way ANOVA or mixed-effects models were performed to determine statistical significance between diet, genotype, or time interactions. Full statistical output (two-way ANOVA and t-test) for all phototaxis experiments are reported in Supplementary Data 5.

Real-time quantitative PCR

Fold-change in gene expression was calculated using the ΔΔCt method and the values were normalized using rp49 as an internal control. P-values were calculated with the pairwise Student’s t-test comparing Log2 fold-changes in expression.

Mass-spectrometric data processing, quantification, and bioinformatics

Mass-spectrometric data-dependent acquisitions53 were analyzed using the database search engine ProteinPilot (SCIEX 5.0 revision 4769) using the Paragon algorithm (,4767). Using these database search engine results a MS/MS spectral library was generated in Spectronaut 14.2.200619.47784 (Biognosys). The DIA/SWATH data was processed for relative quantification by comparing peptide peak areas from various different timepoints during the cell cycle. For the DIA/SWATH MS2 data sets quantification was based on XICs of 6-10 MS/MS fragment ions, typically y- and b-ions, matching to specific peptides present in the spectral libraries. Differential protein expression analysis was performed using a paired t-test and P-values were adjusted for multiple testing. Peptides were identified at Q ≤ 0.01%, and significantly changed proteins were accepted at a 5% FDR (Q ≤ 0.01).

Gene-ontology enrichment analysis

To identify enriched gene-ontology (i.e., bioprocess) categories with the resultant lists from bioinformatics approaches, we utilized the “” package from HOMER (v4.11). Full gene-ontology lists including enrichment statistics (calculated assuming hypergeometric distribution, no adjustment for multiple-hypothesis testing) and associated gene lists are reported in supplementary data files. A maximal limit of 200 gene identifiers per GO category was implemented to reduce the occurrence of large, over-represented terms that lack specificity (i.e., metabolism).

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Spread the love

Leave a Reply

Your email address will not be published.