Characterization of feeding micro-behaviors in flies.

(A) Cartoon schematic of setup for filming flies on the FLIC. Frame capture is triggered by interactions of flies with the food source. (B) Frequency of all five main categories of observed feeding micro-behaviors: other (O), interaction (I), fast (F), long (L), long/quick (LQ). Each data point represents the total number of times an event occurred normalized to all observed events for each fly. N=8 flies, 528 events. (C) Percentage of flies from (B) that engaged in each feeding micro-behavior. (D) Representative temporal plots of sated (N=4 flies, 171 events) and starved (N=4 flies, 357 events) feeding micro-behaviors (left panel) and frequency of each behavior (right panel, two-tailed t-tests). Each row in left panel represents one fly, and frequencies are relative to all behaviors (two-tailed t-tests). (E) Heat maps of starved (left) and sated (right) transitional probabilities of observed behaviors from (D) (transitional probabilities are generated by dividing the observed count for each event pair by the total occurrences of the given event. P-values are determined based on Z-scores, as described in (Blumstein & Daniel, 2007)). Long events contain both L and LQ events. (F) Frequency of each feeding micro-behavior during meal (7AM/PM-10AM/PM) vs. non-meal times of day (one-tailed t-tests). N=9 flies, 693 events, frequency is relative to total interactions + feeding events.

Library of feeding micro-behaviors

FLIC signal characteristics distinguish between event-types.

(A-C) Representative FLIC signal generated during a visually identified interaction (I), fast feeding (F), or long feeding (L/LQ) event. (D-I) Signal characteristics generated by the FLIC during visually identified I, F, or L/LQ events. Each data point represents one event from one fly (one-way ANOVA with Tukey’s post-hoc).

Feeding event durations are increased by hedonic food environments.

(A) Cartoon schematic of event number and event duration (top). Cartoon schematic of hedonic and control food choice environments, and comparisons used to define metrics for homeostatic and hedonic feeding (bottom). (B) Total number of events with sucrose or yeast in the control food choice environment from sated or 24-hr starved female Canton-S flies (two-way ANOVA with Tukey’s post-hoc). (C) Total number of events with sucrose or yeast in the control food choice environment from sated female or male Canton-S flies (two-way ANOVA with Tukey’s post-hoc). (D-E) Median event durations with sucrose (D) or yeast (E) in the control vs. hedonic food choice environments from sated female and male Canton-S flies (two-way ANOVA). (F-G) Total number of events with sucrose (F) or yeast (G) in the control vs. hedonic food choice environments from sated female and male Canton-S flies (two-way ANOVA). (H-I) Median event durations (H) and total number of events (I) with sugar in the indicated food choice environments from sated female Canton-S flies (one-way ANOVA with Tukey’s post-hoc). (J-K) Median event durations (J) and total number of events (K) with sugar in the indicated food choice environment from sated female Canton-S flies (one-way ANOVA with Tukey’s post-hoc). (L-M) Median event durations (L) and total number of events (M) with sucrose in the indicated food choice environments (one-way ANOVA).

Hedonic feeding is modulated by homeostatic hunger.

(A) Total number of events with sucrose or yeast in the control food choice environment from sated or 24-hr starved female Canton-S flies (two-way ANOVA with Tukey’s post-hoc). (B) Total number of events with sucrose or yeast in the control food choice environment from 24-hr vs. 48-hr starved female Canton-S flies (two-way ANOVA with Tukey’s post-hoc). (C) Median event durations with sucrose in the control vs. hedonic food environments from sated vs. 24-hr starved female Canton-S flies (two-way ANOVA). (D) Linear regression of interactions with yeast (licks) versus mean event durations with indicated sugar. Each data point represents one fly (yeast licks are expressed as a transformation to the 0.25 power and event durations are expressed as a log transformation to achieve normality, call = lm(SucroseDurations∼YeastLicks*FoodEnvironment), two-way ANOVA, adjusted R2=0.1835).

Hedonic hunger requires distinct mushroom body lobes.

(A) Representative maximum intensity projections from brains of flies expressing Nsyb-GAL4>UAS-CaMPARI exposed to control or hedonic food environments, quantified at right (scale bar = 100 uM, two-tailed t-test). (B) Representative maximum intensity projections from brains of flies expressing MB238Y-GAL4>UAS-CaMPARI, quantified at right (scale bar = 10 uM, one-tailed t-test). For both (A) and (B), green represents unconverted CaMPARI and magenta represents converted CaMPARI. (C-E) Median event durations with sucrose in control vs. hedonic food environments from sated female flies expressing UAS-GtACR driven by either (C) MB131B-GAL4 (γ(d);γ(m)), (D) MB008B-GAL4 (α/β(c);α/β(p);α/β(s)), or (E) MB461B-GAL4 (α’/β’(ap);α’β’(m)) (two-way ANOVAs with Tukey’s post-hoc). (F) Median event durations with sucrose in control vs. hedonic food environments from sated female flies expressing PAM-GAL4>UAS-GtACR (two-way ANOVA with Tukey’s post-hoc). (G) Median event durations with sucrose in control vs. hedonic food environments from 24-hr starved female flies expressing MB461B-GAL4>UAS-GtACR (two-way ANOVA).

Flies exhibit distinct morning and evening feeding peaks.

(A) Representative temporal plot of food interactions on 2% sucrose FLIC from female Canton-S flies monitored over 24 hrs. Each data point represents the binned average number of events from a population of 38 flies in the previous 30-minute interval. Shaded regions indicate the times of day designated as “meal-times”.

Hedonic food environments do not elicit increases in total number of events or event durations with yeast but do promote increased volumetric intake.

(A) Con-Ex measurement of volumetric sucrose consumptions (left panel) and total mass consumed (right panel) in the control vs. hedonic food environments from female Canton-S flies. (one-way ANOVAs). (B) Total number of events with sugar or yeast in indicated food choice environments from sated female Canton-S flies (two-way ANOVA). (C) Median event durations with yeast in the indicated food choice environments from sated female Canton-S flies (one-way ANOVA).

Mushroom body lobes are not required for homeostatic hunger.

(A-C) Total number of events with sucrose or yeast in the control food choice environment from 24-hr starved female flies expressing UAS-GtACR driven by either (A) MB131B-GAL4 (γ(d);γ(m)), (B) MB008B-GAL4 (α/β(c);α/β(p);α/β(s)), or (C) MB461B-GAL4 (α’/β’(ap);α’β’(m)) (two-way ANOVAs).