Male flies were exposed to the choice or fixed diet for 20 days (food was changed every 2–3 days) and then switched to the other diet (experimental groups) or the same diet (control groups). Heads were removed via vortexing and manually separated from the bodies. Heads or bodies were then homogenized for 20 s in 200 μl of a 1:4 (v:v) water:MeOH solvent mixture using the Fast Prep 24 (MP Biomedicals). Following the addition of 800 μl of methanol, the samples were incubated for 30 min on dry ice, then homogenized again. The mixture was spun at 13,000 RPM for 5 min at 4°C, and the soluble extract was collected into vials. This extract was then dried in a speedvac at 30°C for approximately 3 hr. Using a liquid chromatography triple quadrupole tandem mass spectrometry (LC–QQQ–MS) machine in the multiple reaction monitoring (MRM) mode, we targeted 205 metabolites in 25 important metabolic pathways, in both positive MS and negative MS modes.

After removing any metabolites missing from more than 8 of 76 head samples (10.5%) and 8 of 78 body samples (10.3%), we were left with 103 head metabolites and 125 body metabolites. Metabolite abundance for remaining missing values in this data set were log-transformed and imputed using the k-nearest neighbor algorithm with the impute package of R Bioconductor (http://www.bioconductor.org). We then normalized the data to the standard normal distribution (μ = 0, σ2 = 1). PCA was performed using the made4 package of R Bioconductor.

Our PCA results suggest the choice diet can elicit two processes: (1) a 5-HT2A-dependent, fast-responding metabolic network that is associated with aging despite only accounting for a small number of variation in the metabolomes (2.0–2.2%) and (2) a 5-HT2A-independent, stable metabolic component that accounts for a significant amount of metabolome variation (10.2–11.2%). To distinguish these two groups of metabolites, we used a linear model to analyze the variance attributed to diet, genotype, and interaction of the two for each metabolite:

Metabolite abundance (Y) = Diet + Genotype + Diet * Genotype.

We found 18 of 103 total metabolites in the head and 19 of 125 total metabolites in the body are modulated by choice diet in a 5-HT2A-dependent manner (Figure 4A,B, ANOVA, pDiet*Genotype≤0.05). To identify 5-HT2A independent change, we searched for metabolites that have a diet effect (pDiet≤ 0.05) but did not show a diet–genotype interaction (pDiet*Genotype>0.05). In doing so, we found 45 and 46 metabolites in the head and body, respectively. We plotted a heatmap to illustrate the expression changes of the metabolite upon nutrient presentation using the R package gplots. The metabolites were mapped to biochemical pathways using MetaboAnalystR 3.0 (Pang et al., 2020). Because the small number of detectable compounds in our targeted metabolomics panel (N = 103 in heads or 125 in bodies) limits the power of the enrichment analysis, candidate pathways were therefore identified as those with the number of hits greater than 3.

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