FLIC: High-Throughput, Continuous Analysis of Feeding Behaviors in <i>Drosophila</i>

<div><p>We present a complete hardware and software system for collecting and quantifying continuous measures of feeding behaviors in the fruit fly, <i>Drosophila melanogaster</i>. The FLIC (<u>F</u>ly <u>L</u>iquid-Food <u>I</u>nteraction <u>C</u>ounter) detects analog electronic signals as brief as 50 µs that occur when a fly makes physical contact with liquid food. Signal characteristics effectively distinguish between different types of behaviors, such as feeding and tasting events. The FLIC system performs as well or better than popular methods for simple assays, and it provides an unprecedented opportunity to study novel components of feeding behavior, such as time-dependent changes in food preference and individual levels of motivation and hunger. Furthermore, FLIC experiments can persist indefinitely without disturbance, and we highlight this ability by establishing a detailed picture of circadian feeding behaviors in the fly. We believe that the FLIC system will work hand-in-hand with modern molecular techniques to facilitate mechanistic studies of feeding behaviors in <i>Drosophila</i> using modern, high-throughput technologies.</p></div

New types of behavioral inference from the FLIC system.

<p>(A) Flies spent 10% of their time in behaviors we categorized as tasting two foods prior to making their first meal choice. Fraction of time is calculated based on “total time spent tasting/time until the first meal”. (B) A greater fraction of tasting events were directed toward the food the flies choose to consume (mean Tasting PI = 0.35). A Tasting PI = 1 implies a fly tasted a single food before ultimately consuming that food. A Tasting PI = −1 implies that a fly tasted a single food before ultimately consuming the opposite food. (C) While a cumulative preference index (left panel) is effective at portraying overall feeding tendencies, time-dependent preference indices (right panel) reveal subtle differences in behavioral choices as the experiment progresses. Flies exhibited a strong preference toward 10% sucrose in the first 30 min, which was attenuated in later time periods then returned to a strong preference (N = 34; the size of the symbol is proportional to the sample size contributed to calculate PI in a given period). (D) Flies with increased feeding motivation (through longer periods of starvation) experienced their first meal earlier than control flies. Flies starved for increasing periods (0 hr, 24 hr, or 48 hr) exhibited reduced latencies until their first feeding event. Latency curves were found to be significantly different via log-rank test. (E) Flies with increased hunger (through longer periods of starvation) exhibited meals that were of significantly longer duration than control flies (One-way ANOVA followed by post-hoc test using a Bonferroni correction). (F) Taste input plays a role in motivation by decreasing latency to the first meal. Flies with loss of function in the trehalose receptor, <i>ΔGr5a</i>, were significantly delayed in their first meal of a liquid trehalose food compared with control animals (log-rank test). **P≤0.01; ***P≤0.001</p

Comparison between traditional food choice assays and the FLIC system.

<p>(A) The analog signals from feeding (left) and tasting (right) behaviors have distinct characteristics. (B) When presented with identical food in both food wells, male and female flies do not exhibit a preference, which rules out systematic bias in the FLIC system (open symbol, male; closed symbol, female; pooled paired randomization test, P = 0.97). (C) Flies exhibited strong preference in favor of 10% sucrose over 100 µM denatonium when measured using both two-dye and FLIC assays (Box charts represent mean, standard error of mean, and 10–90% quantile whiskers). (D) Flies demonstrated strong preference toward 10% sucrose over 1% sucrose when measured using both the CAFE and FLIC assays (Box charts represent mean, standard error of mean, and 10–90% quantile whiskers). (E) Estimates of food consumption using the CAFE and FLIC assays. Longer starvation resulted in increased food consumption as well as total feeding time (linear regression, P<1×10⁻⁵ for both assays). Changes in food volume in the capillary tubes was undetectable when fully fed flies were used, and only FLIC data are presented for that treatment. *P≤0.05; **P≤0.01; ***P≤0.001.</p

Illustration of the FLIC system.

<p>(A) Cartoon of the <i>Drosophila</i> Feeding Monitor (DFM) from the top- and side-view along with a flowchart of data collection and processing. Analog signals from all DFMs are collected by the Master Control Unit (MCU), which relays the information to the PC where the signals are visualized and recorded by the real-time monitoring software. (B) Representative signals from each of two feeding wells within a single feeding arena taken from a 90 min subset of a 24-hour feeding measurement. Close-up signal patterns representative of two distinct classes of feeding behavior events are presented as insets. (C) Histograms representing the distribution of durations for individual feeding behavior events (an event is a set of contiguous signals above baseline) over a 24 hr measurement period. Each plot represents values from a single fly, and distributions for three flies are presented. N represents the number of behavior events observed. (D) Histograms representing the size of the intervals between successive feeding behaviors over a 24 hr measurement period. Each plot represents values from a single fly, and distributions for the same three flies as in panel C are presented. (E) Among-fly variability in the average feeding duration and average time between feeding events. Each point represents the average value over a 24 hr period (N = 21). (F) Event-time distribution that represents the fraction of the population that has experienced at least one feeding at a given elapsed time from a randomized point between 12pm-2pm (N = 21). It took roughly 197 min for 50% of the population to feed at least once during this time of the day.</p