Integrated respirometry and metabolomics unveil circadian metabolic dynamics in Drosophila
- Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania, United States
- Chronobiology and Sleep Institute, University of Pennsylvania, United States
- Institute for Translational Medicine and Therapeutics, University of Pennsylvania, United States
- Howard Hughes Medical Institute, University of Pennsylvania, United States
- Department of Neuroscience, Perelman School of Medicine, University of Pennsylvania, United States
- Sable Systems International, United States
Figures
Figure 1 with 4 supplements
VCO2, VO2 and RQ profiles across teh circadian cycle in different genotypes.
(a–c) VCO2, VO2, and respiratory quotient (RQ) profiles across the 24 hr cycle in different genotypes. VCO2, VO2, and RQ (VCO2/VO2) were continuously recorded at 1 s intervals from Zeitgeber time (ZT) 0–24 and subsequently averaged into 5 min bins for analysis. Traces represent mean VCO2, VO2, and RQ values across the 24 hr recording period for wild-type flies under light-dark conditions (WT-LD), short-sleep mutants fumin (fmn) and sleepless (sss), the circadian clock mutant period01 (per01), and wild-type flies maintained in constant darkness (WT-DD). Source data are the continuous respirometry recordings. (d–f) Genotype-specific differences in VCO2, VO2, and RQ measured over a full 24 hr recording period. Boxplots show average values of (left to right) RQ, carbon dioxide production (VCO2), and oxygen consumption (VO2) across genotypes and lighting conditions. Measurements were taken continuously over a 24 hr period using a flow-through MAVEn system. Genotypes include WT-LD and WT-DD, short-sleep mutants fumin (fmn) and sleepless (sss), and circadian mutant per01. Group differences were assessed using the Kruskal-Wallis test, followed by Dunn’s multiple comparisons post hoc test. Significance is denoted as: p<0.05 (*), p<0.01 (**), p<0.001 (***); ns = not significant. (g–i) Body weight and normalized VCO2 and VO2 across different genotypes. Body weight (mg) and respiratory parameters (VCO2 and VO2) normalized to body weight (mg) were measured in wild-type (WT), fmn, sss, and per01 mutant flies. Statistical significance was assessed using one-way ANOVA followed by Dunnett’s multiple comparisons test against WT. p<0.05 (*), p<0.01 (**), p<0.001 (***); ns = not significant. For all panels with error bars or shaded error bands, values are presented as mean ± SEM. Data represent approximately 300 flies per genotype across 3 experimental days (25 flies/chamber, 4 chambers/experiment). The chamber was used as the experimental unit; n denotes the number of chambers.
Figure 1—figure supplement 1
VCO2 profiles across the 24 hr recording period in different genotypes.
(a–e) VCO2 was continuously measured at 1 s intervals from Zeitgeber time (ZT) 0–24 and averaged into 5 min bins for analysis. The traces display mean VCO2 values across the 24 hr recording period for the following genotypes: wild-type flies under light-dark conditions (WT-LD), short-sleep mutants fumin (fmn) and sleepless (sss), the circadian clock mutant period01 (per01), and wild-type flies in constant darkness (WT-DD). For all panels, traces are presented as mean ± SEM. Data represent approximately 300 flies per genotype across 3 experimental days (25 flies/chamber, 4 chambers/experiment). The chamber was used as the experimental unit; n denotes the number of chambers.
Figure 1—figure supplement 2
VO2 profiles across the 24 hr recording period in different genotypes.
(a–e) VO2 was continuously measured at 1 s intervals from Zeitgeber time (ZT) 0–24 and averaged into 5 min bins for analysis. The traces display mean VO2 values across the 24 hr recording period for the following genotypes: wild-type flies under light-dark conditions (WT-LD), short-sleep mutants fumin (fmn) and sleepless (sss), the circadian clock mutant period01 (per01), and wild-type flies in constant darkness (WT-DD). For all panels, traces are presented as mean ± SEM. Data represent approximately 300 flies per genotype across 3 experimental days (25 flies/chamber, 4 chambers/experiment). The chamber was used as the experimental unit; n denotes the number of chambers.
Figure 1—figure supplement 3
Respiratory quotient (RQ) profiles across the 24 hr recording period in different genotypes.
(a–e) RQ (VCO2/VO2) was continuously recorded at 1 s intervals from Zeitgeber time (ZT) 0–24 and subsequently averaged into 5 min bins for analysis. Traces represent mean RQ values across the 24 hr recording period for wild-type flies under light-dark conditions (WT-LD), short-sleep mutants fumin (fmn) and sleepless (sss), the circadian clock mutant period01 (per01), and wild-type flies maintained in constant darkness (WT-DD). For all panels, traces are presented as mean ± SEM. Data represent approximately 300 flies per genotype across 3 experimental days (25 flies/chamber, 4 chambers/experiment). The chamber was used as the experimental unit; n denotes the number of chambers.
Figure 1—figure supplement 4
Day-night differences in genotype-specific metabolic rates.
Boxplots depict day (Zeitgeber time [Z]T0–12) and night (ZT12–24) averages plotted side-by-side for each genotype to facilitate direct day-night comparison. Panels show genotype-specific differences in (A) carbon dioxide production (VCO2), (B) oxygen consumption (VO2), and (C) respiratory quotient (RQ). Genotypes include wild-type flies under light-dark conditions (WT-LD), wild-type flies maintained in constant darkness (WT-DD), short-sleep mutants fumin (fmn) and sleepless (sss), and the circadian mutant period01 (per01). Measurements were acquired continuously using a flow-through MAVEn system and binned into day and night intervals. Group differences were assessed using the Kruskal-Wallis test followed by Dunn’s multiple comparisons post hoc test. Significance is denoted as: p<0.05 (*), p<0.01 (**), p<0.001 (***); ns = not significant. Source data are the same continuous respirometry recordings shown in Figure 1. Data represent approximately 300 flies per genotype across 3 experimental days (25 flies/chamber, 4 chambers/experiment). The chamber was used as the experimental unit; n denotes the number of chambers.
Figure 2
Twenty-four-hr time course of respiratory parameters across genotypes.
Normalized temporal profiles of carbon dioxide production (VCO2) (top), oxygen consumption (VO2) (middle), and respiratory quotient (RQ) (bottom) plotted over a 24 hr cycle (Zeitgeber time [Z]T0–24) for genotypes: wild-type in light-dark (WT-LD) and constant darkness (WT-DD), short-sleep mutants fumin (fmn) and sleepless (sss), and circadian mutant per01. Curves reveal genotype-specific rhythmicity and metabolic dynamics across the 24 hr recording period. Significant genotype×time interactions were observed for all variables (VCO2, VO2, RQ), indicating genotype-dependent alterations in respiratory rhythmicity. p<0.05 considered significant. For all panels with error bars or shaded error bands, values are presented as mean ± SEM. Source data are the same continuous respirometry recordings shown in Figure 1; Figure 2 presents rhythmicity analysis of VCO2, VO2, and RQ across the 24 hr cycle. Data represent approximately 300 flies per genotype across 3 experimental days (25 flies/chamber, 4 chambers/experiment). The chamber was used as the experimental unit; n denotes the number of chambers.
Figure 3
Phase distribution of respiratory rhythms across genotypes.
Polar plots depicting the phase (peak timing) of rhythmic expression for carbon dioxide production (VCO2), oxygen consumption (VO2), and respiratory quotient (RQ) over a 24 hr cycle (Zeitgeber time [ZT]0–24) for each genotype. Genotypes include wild-type in light-dark (WT-LD) and constant darkness (WT-DD), short-sleep mutants fumin (fmn) and sleepless (sss), and circadian mutant per01. Statistical significance of rhythmicity was determined using the RAIN algorithm: darker-colored bars indicate significant rhythms (p<0.05), and lighter-colored bars indicate non-significant rhythms (p>0.05). Where applicable, plotted values are presented as mean ± SEM. Source data are the same continuous respirometry recordings shown in Figure 1; Figure 3 presents phase/peak-timing analysis derived from the rhythmicity analysis.
Figure 4
Temporal profiling of respiratory quotient (RQ) in wild-type (WT) flies under light-dark conditions.
RQ was extracted every 2 hr across a 24 hr light-dark (LD) cycle in WT flies. Lag analyses were performed by advancing or delaying the RQ data by −120, –60, −30, –15, −5, +5, +15, +30, +60-, and +120 min relative to Zeitgeber time (ZT). Each panel represents the RQ profile corresponding to a specific time shift, illustrating phase-dependent respiratory dynamics across the 24 hr LD cycle. Data represent approximately 300 flies per genotype across 3 experimental days (25 flies/chamber, 4 chambers/experiment). The chamber was used as the experimental unit; n denotes the number of chambers. RQ rhythmicity itself was assessed from the continuous respirometry time series (Table 1); Figure 4 shows the day-night RQ pattern used as the reference for the lag-based metabolite correlations, to which the metabolomics data contribute.
Figure 5
Exemplar temporal relationships between metabolites and respirometry.
(a) Rank-based correlation between respiratory quotient (RQ) and selected metabolites. Scatter plots depict the rank-order relationships between RQ and (A) hydroxyhexadecenoylcarnitine and (B) quinolinate. Each point represents a timepoint after aligning data with respective time shifts. Spearman rank correlation coefficient (ρ) and corresponding p-values are indicated on each panel. A positive lag (+120 min for hydroxyhexadecenoylcarnitine) or negative lag (−120 min for quinolinate) denotes the temporal shift in RQ relative to the metabolite dataset. Statistical significance was defined as p<0.05. (b) Temporal alignment of metabolite with RQ in wild-type flies under light-dark cycles (WT-LD) flies. Z-scored time series of RQ, hydroxyhexadecenoylcarnitine, and quinolinate across a 24 hr light-dark cycle. Top panel: hydroxyhexadecenoylcarnitine shows a positive lag of +120 min and strong positive correlation with RQ (ρ=+0.78), suggesting its rise after changes in RQ. Bottom panel: quinolinate shows a negative lag of –120 min and strong negative correlation with RQ (ρ=−0.77), indicating it precedes changes in RQ.
Figure 6
Metabolite-respiratory quotient (RQ) correlations and pathway enrichment in wild-type flies under light-dark cycles (WT-LD).
(A) Heatmap depicting Spearman correlations (|ρ|>0.7, p<0.05) between RQ and metabolites across the 24 hr LD cycle in wild-type flies maintained under light-dark (LD) conditions. Metabolites were clustered based on correlation patterns, highlighting groups with similar temporal associations with respiratory activity. (B) Pathway enrichment analysis of significantly correlated metabolites, illustrating metabolic pathways most closely linked to respiratory dynamics. Pathways with a p-value less than 0.05 were considered statistically significant.
Figure 7
Correlation of metabolites with respiratory quotient (RQ) and pathway enrichment in short-sleep mutants.
(A, C) Heatmaps showing Spearman correlations (|ρ|>0.7, p<0.05) between RQ and metabolites in the short-sleep mutants fmn (A) and sss (C). (B, D) Pathway enrichment analyses of metabolites significantly correlated with RQ in fmn (B) and sss (D). Pathways with p-values less than 0.05 were considered statistically significant.
Figure 8
Correlation of metabolites with respiratory quotient (RQ) and pathway enrichment in circadian mutant and wild-type flies in constant darkness.
(A, C) Heatmaps displaying Spearman correlations (|ρ|>0.7, p<0.05) between RQ and metabolites in the circadian mutant per01 (A) and wild-type flies maintained in constant darkness (WT-DD) (C). (B, D) Pathway enrichment analyses of metabolites significantly correlated with RQ in per01 (B) and WT-DD (D). Pathways with p-values less than 0.05 were considered statistically significant.
Figure 9
Baseline oxygen consumption in isolated gut tissue from fmn and per01 mutants.
Baseline oxygen consumption rate (OCR, fmol/mm2/s) measured by tissue respirometry in individual dissected guts from the short-sleep mutant fmn (A) and the circadian-clock mutant per01 (B) relative to iso31 genetic-background controls. For each gut, OCR was averaged over a 24 hr window beginning ~12 hr after loading, once tissue respiration had stabilized (12–36 hr). To combine independent runs, OCR was normalized to the median of the iso31 controls within each run and log2(x+10)-transformed. One iso31 gut in the third run of the per01 experiment was excluded as an outlier; no other values were removed. Baseline OCR differed significantly between iso31 and each mutant (Mann-Whitney test, p<0.05; pooled across 3 independent runs; fmn vs iso31, n=12 vs 12; per01 vs iso31, n=12 vs 11 after the iso31 outlier exclusion).
Tables
Table 1
Rhythmicity metrics for respiratory parameters across genotypes.
| Genotype | RQ JTK lag | RQ JTK period | RQ RAIN p-value | VCO2 JTK lag | VCO2 JTK period | VCO2 RAIN p-value | VO2 JTK lag | VO2 JTK period | VO2 RAIN p-value |
|---|---|---|---|---|---|---|---|---|---|
| WT-LD | 19.75 | 20 | 0.12 | 4 | 24 | 1.1143e-05 | 4 | 24 | 0.001 |
| fumin | 4.25 | 22.5 | 9.54e-08 | 3.25 | 24 | 0.0002 | 14 | 20 | 0.69 |
| sss | 12.5 | 20 | 1.41e-07 | 2 | 24 | 1.33e-09 | 1.25 | 24 | 1.14e-10 |
| per01 | 2 | 20 | 0.006 | 3 | 24 | 1.05e-13 | 6 | 21.5 | 1.82e-07 |
| WT-DD | 19.25 | 23 | 0.02 | 7.5 | 24 | 0.0002 | 7 | 24 | 9.4145e-06 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Drosophila melanogaster) | Isogenic control (iso/iso31) | Laboratory background strain | iso/iso31 | Wild-type isogenic control; used as WT-LD/WT-DD and as genetic-background control for gut OCR experiments. |
| Genetic reagent (D. melanogaster) | Fumin (fmn) | Kume et al., 2005 | Dat[fmn] (fumin) | Dopamine transporter mutant; short-sleep/hyperactive phenotype. |
| Genetic reagent (D. melanogaster) | Sleepless (sss) | Koh et al., 2008; Chen et al., 2015; Wu et al., 2010 | sss | Short-sleep mutant lacking functional Sleepless protein. |
| Genetic reagent (D. melanogaster) | Period null (per01) | Konopka and Benzer, 1971 | per[01] | Circadian clock mutant lacking a functional molecular clock. |
| Other | MAVEn flow-through respirometry system | Sable Systems International | MAVEn by Sable Systems International (Multiple animal versatile energetics flow-through system) | Instrument. Continuous whole-fly respirometry. The MAVEn splits airflow into 16 fly-chamber channels plus one dedicated baseline channel (Materials and methods – Respirometry chambers and MAVEn system’s airflow management). Each experiment used all 16 chambers: 4 chambers×4 genotypes, 25 flies/chamber. |
| Other | RC respirometry chambers | Sable Systems International | RC chambers, 70 mm × 20 mm | Instrument. 70 mm × 20 mm borosilicate glass chambers; used as the 16 fly-chamber channels of the MAVEn system. |
| Other | Side-Trak 840 Series mass-flow controller | Sierra Instruments, Inc | Side-Trak 840 Series | Instrument. Reference-airflow control. |
| Other | LI-7000 CO2 analyzer | LI-COR Biosciences | LI-7000 | Instrument. Measurement of CO2 production. |
| Other | Oxzilla II differential O2 analyzer | Sable Systems International | Oxzilla II | Instrument. Measurement of O2 consumption. |
| Other | Resipher System | Lucid Scientific (GA, USA) | Resipher | Instrument. Continuous OCR measurement in dissected gut tissue. |
| Chemical compound, drug | Drierite/desiccant | Drierite | Cat. no. 26800; CAS: 7779-18-9 | Used in CO2 scrubbing/drying columns as described in Materials and methods. |
| Chemical compound, drug | Ascarite | Acros Organics | CAS: 81133-20-2 | CO2 scrubbing material. |
| Other | Glass wool | Fisher Scientific | 11-388 | Laboratory material. Separator material in scrubbing columns. |
| Other | Nafion tubing | Perma Pure, LLC | TT-070 | Laboratory material. Used to re-humidify scrubbed air. |
| Other | Bev-A-Line nonpermeable tubing | United States Plastic Corp. | 56280 | Laboratory material. Tubing used to connect respirometry components. |
| Chemical compound, drug | Magnesium perchlorate | Fisher Scientific | M54 | Water-vapor scrubbing before O2 analysis. |
| Other | 10 mL syringe body | Fisher Scientific | 14955459 | Laboratory material. Housing for small water-vapor scrubbing columns. |
| Other | Rubber stoppers | Fisher Scientific | 14-135E | Laboratory material. Used to secure tubing to scrubbing columns. |
| Other | Schneider’s Media | Gibco/Life Technologies (Thermo Fisher Scientific) | Cat. no. 21720024 | Cell culture medium. Used for Drosophila gut dissection and Resipher OCR assay (300 µL per well; Materials and methods – Gut tissue respirometry). |
| Chemical compound, drug | Poly-D-lysine | Gibco/Life Technologies (Thermo Fisher Scientific) | Cat. no. A3890401 | 96-Well plates coated with 50 µL for 30 min to promote gut tissue adhesion (Materials and methods – Gut tissue respirometry). |
| Software, algorithm | MAVEn Controller software | Sable Systems International | System control and respirometry data acquisition. | |
| Software, algorithm | GraphPad Prism | GraphPad Software | Version 10; RRID:SCR_002798 | Statistical analysis and outlier identification for gut OCR analysis. |
| Software, algorithm | Nitecap | Brooks et al., 2022 | https://nitecap.org | Exploratory circadian/rhythmicity analysis framework. |
| Software, algorithm | RAIN | Thaben and Westermark, 2014 | RAIN via Nitecap | Rhythmicity detection with FDR-adjusted p-values. |
| Software, algorithm | JTK_CYCLE | Hughes et al., 2010 | JTK_CYCLE via Nitecap | Period and peak-phase/lag estimation. |
| Software, algorithm | MetaboAnalyst | MetaboAnalyst | Version 5.0; RRID:SCR_015539 | Metabolic pathway enrichment and topology analysis. |
| Other | Human Metabolome Database (HMDB) | HMDB | HMDB; RRID:SCR_007712; https://hmdb.ca | Database. Metabolite identifiers used for pathway analysis. |
| Other | KEGG Drosophila melanogaster pathway library | KEGG | KEGG; RRID:SCR_012773; Drosophila melanogaster pathway library | Database. Pathway library used in MetaboAnalyst topology/enrichment analysis. |
| Other | Positive/negative ion-switching targeted LC-MS metabolomics platform | Yuan et al., 2012; Malik et al., 2024; Malik et al., 2018 | Ion-switching LC-MS as described | Method. Previously published LC-MS workflow used for the metabolomics dataset integrated with respirometry. |
Additional files
-
Supplementary file 1
Supplementary tables.
(A) Metabolites showing strong lag-dependent correlations with respiratory quotient (RQ) across genotypes and lighting conditions, with best lag direction, lag time, Spearman ρ at the strongest lag, nominal p-value, and Benjamini-Hochberg FDR. (B) Metabolite set enrichment analysis (MSEA over-representation analysis against KEGG pathways) for each genotype/condition, corresponding to the enrichment panels in Figures 6—8. (C) Experimental unit, sample size, and error representation for the respirometry-based figures (Figures 1—3, Figure 1—figure supplements 1–4).
- https://cdn.elifesciences.org/articles/108681/elife-108681-supp1-v1.docx
-
MDAR checklist
- https://cdn.elifesciences.org/articles/108681/elife-108681-mdarchecklist1-v1.docx
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Integrated respirometry and metabolomics unveil circadian metabolic dynamics in Drosophila
eLife 14:RP108681.
https://doi.org/10.7554/eLife.108681.3
