a-c. VCO₂, VO₂ and Respiratory quotient (RQ) profiles across the circadian cycle in different genotypes. VCO₂, VO₂ and RQ (VCO₂/VO₂) was continuously recorded at one-second intervals from Zeitgeber Time (ZT) 0 to 24 and subsequently averaged into 5-minute bins for analysis. Traces represent mean VCO₂, VO₂ and RQ values across the circadian cycle for wild-type flies under light-dark conditions (WT-LD), short-sleep mutants fumin (fmn) and sleepless (sss), circadian clock mutant period01 (per01), and wild-type flies maintained in constant darkness (WT-DD). d-f. Genotype-specific differences in VCO2, VO2 and RQ measured over a full circadian timecourse. Boxplots show average values of (left to right) respiratory quotient (RQ), carbon dioxide production (VCO₂), and oxygen consumption (VO₂) across genotypes and lighting conditions. Measurements were taken continuously over a 24-hour period using a flow-through MAVEn system. Genotypes include wild-type under light-dark (WT-LD) and constant darkness (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.

Circadian timecourse 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-hour circadian cycle (ZT0–24) 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 circadian time. Significant genotype × time interactions were observed for all variables (VCO₂, VO₂, RQ), indicating altered circadian rhythmicity in mutants. p < 0.05 considered significant.

Circadian 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-hour cycle (ZT0–24) 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).

Circadian rhythmicity metrics for respiratory parameters across genotypes

Temporal Profiling of Respiratory Quotient in Wild-Type Flies Under Light-Dark Conditions.

Respiratory quotient (RQ) was extracted every 2 h across a 24-hour 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-minutes relative to Zeitgeber Time (ZT). Each panel represents the RQ profile corresponding to a specific time shift, illustrating the phase-dependent dynamics of respiration across the circadian cycle.

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’s rank correlation coefficient (ρ) and corresponding p-values are indicated on each panel. A positive lag (+120 minutes for Hydroxyhexadecenoylcarnitine) or negative lag (−120 minutes 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 respiratory quotient (RQ) in WT-LD flies. Z-scored time series of RQ, Hydroxyhexadecenoylcarnitine, and Quinolinate across a 24-hour light-dark cycle. Top Panel: Hydroxyhexadecenoylcarnitine shows a positive lag of +120 minutes 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 minutes and strong negative correlation with RQ (ρ=-0.77), indicating it precedes changes in RQ.

Metabolite-Respiratory Quotient Correlations and Pathway Enrichment in WT-LD Flies.

(A) Heatmap depicting Spearman correlations (|ρ| > 0.7, p < 0.05) between respiratory quotient (RQ) and metabolite across the circadian 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.

Correlation of Metabolites with Respiratory Quotient and Pathway Enrichment in Short-Sleep Mutants.

(A, C) Heatmaps showing Spearman correlations (|ρ| > 0.7, p < 0.05) between respiratory quotient (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.

Correlation of Metabolites with Respiratory Quotient 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 respiratory quotient (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.