DIETS (Direct Intake Estimation and longitudinal Tracking of Solid food consumption) is a sensitive assay that can measure food consumption in flies over days

(A) Schematic outline of the DIETS assay. (B) Comparison of 24 h control diet (CD) consumption in males and females of increasing group sizes. Females consistently eat a greater amount of food than males, regardless of the group size. Mean consumption by both sexes increased with group size. (C) CD consumption by groups of 50 male flies was measured over 24 h at different temperatures. An increase in the amount of food consumed was observed with an increase in temperature. (D) Feeding by groups of 50 male flies on varying concentrations of CD was measured. Flies show compensatory feeding upon diluting or concentrating CD. (E) Comparing the sensitivity of DIETS, CAFE, and EX-Q assays in resolving food intake difference between 1X, 2X, and 4X concentrations of 2.5 % sucrose - 2.5 % yeast extract (2.5 % SY) diet. DIETS show comparable resolution to CAFE and EX-Q. (F) Longitudinal measurement of CD consumption over days 1, 4, 7, 14, 21, and 28 for 50 male flies. Consumption mostly remains unaltered, except for a dip in feeding by the end of 28 days, possibly due to the mortality observed. Flies start dying between the 7th and the 14th day. Scatter plots of raw data with mean ± 95 % CI are shown. Δ ‘effect size’ plots below each raw data graph depict mean differences (black dots; values labeled) between relevant groups or standardized mean difference, hedge’s g for Fig. 1E. The 95 % CI (black lines) and distribution of the mean differences (curve), generated are also shown. See Materials and Methods for further details.

DIETS for short-term feeding measurements and food choice

(A) Comparing the sensitivity of DIETS with blue dye uptake in measuring short-term feeding. 30 minutes of feeding on CD supplemented with Brilliant blue FCF in differentially starved male flies was measured using DIETS. Flies showed an increase in feeding with increased starvation (left panel). A simultaneous dye accumulation measurement was conducted by crushing the flies after feeding (right panel). Hedge’s g is effect size, in this case, the difference in mean of two groups, standardized over the pooled standard deviation of the two groups. DIETS assay yields larger ‘g’ values compared to blue dye estimation for comparison of feeding between the same groups. (B) Continuous 2 h feeding measurements in sated male and female flies measured over 2 days. Both sexes show a distinct evening feeding peak just around the lights-off time at ZT = 11.5-12.5. (C) 24 h, food preferences between CD and CD mixed with quinine. Flies show an increasing dose-dependent aversion towards CD adulterated with quinine. (D) 2 h food preferences between CD and CD adulterated with 15 mM quinine in 24 h starved flies. Flies showed a strong aversion towards quinine-laced food. Scatter plots of raw data with mean ± 95 % CI are shown. Δ ‘effect size’ plots below each raw data graph depict mean differences (black dots; values labeled), between the two relevant groups being compared. The 95 % CI (black lines) and distribution of the mean differences (curve) generated by bootstrap resampling of the data are also shown. In (A) standardized effect size, Hedge’s g has been plotted, and MOEs have been reported. See Materials and Methods for further details.

DIETS is well suited to study longitudinal feeding on high-fat diets and associated transcriptional and physiological changes

(A) In a longitudinal feeding study, flies were fed 5%, 10% and 20% saturated high-fat diets made with coconut oil (sHFD), and feeding measurements were conducted on days 1, 4, and 7. While consumption remained mostly constant up to 10 % extra coconut oil in the diet, feeding decreased noticeably with the addition of 20 % coconut oil. (B) Differential expression of takeout (to), Cytochrome P450-4e3 (Cyp4e3), Odorant-binding protein 83a (Obp83a), and Turandot A (TotA) was examined by qPCR from heads of flies that have been fed with 20 % sHFD for 1, 4, and 7 days, with respect to control flies fed CD. These genes were earlier found to be some of the most differentially expressed genes from an earlier study with a similar design. The direction and magnitude of differential expression of the above candidate genes on day 4 and 7 were comparable to that in day 7 samples from the previous study (Stobdan et al. 2019), as shown. (C) Energy intake was determined over 24 h periods on days 1, 4, and 7 in groups of flies fed 30 % HSD or isocaloric 10 % sHFD. The corresponding mean feeding values are represented above inside pink boxes. Flies fed on 10 % sHFD showed increased energy intake compared to the CD and HSD fed groups on all days. (D) The same flies were transferred to 0.75 % agar vials after 7 days of dietary treatment and the number of dead flies recorded every 6 - 12 hours. Survival time (left panel), and percent survival under starvation (right panel) of flies are shown. While HSD treatment increases starvation resistance, sHFD treatment does not. Scatter plots of raw data with mean ± 95 % CI are shown. For Figure 4C starvation resistance was plotted as Kaplan-Meier graph. Δ ‘effect size’ plots below each raw data graph depict mean differences (black dots; values labeled), between the two relevant groups being compared. The 95 % CI

DIETS is well suited to study longitudinal feeding on high-fat diets and associated transcriptional and physiological changes

(A) In a longitudinal feeding study, flies were fed 5%, 10% and 20% saturated high-fat diets made with coconut oil (sHFD), and feeding measurements were conducted on days 1, 4, and 7. While consumption remained mostly constant up to 10 % extra coconut oil in the diet, feeding decreased noticeably with the addition of 20 % coconut oil. (B) Differential expression of takeout (to), Cytochrome P450-4e3 (Cyp4e3), Odorant-binding protein 83a (Obp83a), and Turandot A (TotA) was examined by qPCR from heads of flies that have been fed with 20 % sHFD for 1, 4, and 7 days, with respect to control flies fed CD. These genes were earlier found to be some of the most differentially expressed genes from an earlier study with a similar design. The direction and magnitude of differential expression of the above candidate genes on day 4 and 7 were comparable to that in day 7 samples from the previous study (Stobdan et al. 2019), as shown. (C) Energy intake was determined over 24 h periods on days 1, 4, and 7 in groups of flies fed 30 % HSD or isocaloric 10 % sHFD. The corresponding mean feeding values are represented above inside pink boxes. Flies fed on 10 % sHFD showed increased energy intake compared to the CD and HSD fed groups on all days. (D) The same flies were transferred to 0.75 % agar vials after 7 days of dietary treatment and the number of dead flies recorded every 6 - 12 hours. Survival time (left panel), and percent survival under starvation (right panel) of flies are shown. While HSD treatment increases starvation resistance, sHFD treatment does not. Scatter plots of raw data with mean ± 95 % CI are shown. For Figure 4C starvation resistance was plotted as Kaplan-Meier graph. Δ ‘effect size’ plots below each raw data graph depict mean differences (black dots; values labeled), between the two relevant groups being compared. The 95 % CI (black lines) and distribution of the mean differences (curve) generated by bootstrap resampling of the data are also shown. See Materials and Methods for further details.