A Rapid Food-Preference Assay in Drosophila

Summary

We present a protocol for a two-choice feeding assay for flies. This feeding assay is fast and easy to run and is suitable not only for small-scale laboratory research, but also for high-throughput behavioral screens in flies.

Abstract

To select food with nutritional value while avoiding the consumption of harmful agents, animals need a sophisticated and robust taste system to evaluate their food environment. The fruit fly, Drosophila melanogaster, is a genetically tractable model organism that is widely used to decipher the molecular, cellular, and neural underpinnings of food preference. To analyze fly food preference, a robust feeding method is needed. Described here is a two-choice feeding assay, which is rigorous, cost-saving, and fast. The assay is Petri-dish-based and involves the addition of two different foods supplemented with blue or red dye to the two halves of the dish. Then, ~70 prestarved, 2-4-day-old flies are placed in the dish and allowed to choose between blue and red foods in the dark for about 90 min. Examination of the abdomen of each fly is followed by the calculation of the preference index. In contrast to multiwell plates, each Petri dish takes only ~20 s to fill and saves time and effort. This feeding assay can be employed to quickly determine whether flies like or dislike a particular food.

Introduction

Despite dramatic differences in the anatomical structure of taste organs between flies and mammals, the flies' behavioral responses to many tastant substances are strikingly similar to those of mammals. For example, flies prefer sugar^{1,2,3,4,5,6,7,8}, amino acids^9,10, and low salt¹¹, which indicate nutrients, but reject bitter foods^12,13,14,15 that are unpalatable or toxic. Over the past two decades, flies have proven to be a highly valuable model organism for advancing the understanding of many fundamental questions related to taste sensation and food consumption, including tastant detection, taste transduction, taste plasticity, and feeding regulation^{16,17,18,19,20}. Remarkably, a number of studies have demonstrated that the taste transduction and neural circuit mechanisms underlying taste perception are analogous between fruit flies and mammals. Therefore, the fruit fly serves as an ideal experimental organism, enabling researchers to uncover evolutionarily conserved concepts and principles that govern food detection and consumption in the animal kingdom.

To investigate taste sensation in flies, it is critical to establish a fast and rigorous assay to objectively measure food preference. Over the years, various feeding methods, such as dye-based assays^{11,12,13,21,22,23}, the fly proboscis extension response assay²⁴, the Capillary Feeder (CAFE) assay^25,26, the Fly Liquid-Food Interaction Counter (FLIC) assay²⁷, and other combinatorial methods have been developed to quantitatively measure food preference and/or food intake for fruit flies^28,29,30,31. One of the popular feeding paradigms is the dye-based two-choice feeding assay using either a multiwell microtiter plate^12,21,32 or, as described here, a small Petri dish^11,22 as the feeding chamber. This assay is designed based on the transparency of the fly's abdomen. During this assay, flies are placed into the feeding chamber and presented with two food options mixed with either red dye or blue dye. Once the assay is complete, fly abdomens appear red or blue depending on which food they have consumed.

Both the Petri-dish and the multiwell-plate dye-based feeding assays are highly robust and yield approximately the same results. Using these two assays, numerous important discoveries and breakthroughs have been made toward deciphering the highly diversified receptors and cells responsible for sensing food tastes and food texture^{11,12,21,22,32,33}. In the dye-based assay, one experimental step requiring considerable time and effort is preparing and loading food into the feeding chamber. To reduce the food preparation and loading time, this assay was modified by replacing the multiwell microtiter plate with a small Petri dish, which is divided into two equal compartments. In the Petri-dish-based assay, two different foods supplemented with blue or red dye are added to the two halves of the dish. Then, ~70 prestarved, 2-4-day-old flies are placed in the dish and allowed to choose between blue and red foods in the dark for about 90 min. The abdomen of each fly is then examined, and the preference index (PI) is calculated.

This Petri-dish-based two-choice feeding assay is affordable, simple, and fast. One multiwell plate requires approximately 110 s to fill, whereas each Petri dish takes only ~20 s. In addition, the multiwell plate requires pipetting small volumes of food into a large number of small wells (e.g., 60 or more wells per plate), which demands considerable precision and attention. Conversely, the Petri-dish-based assay requires only two actions per plate. As the feeding assay can involve a large number of replicates, the Petri-dish-based assay saves a nontrivial amount of time and effort. This assay gives results equivalent to those from the multiwell-based assay and has proven successful in addressing many fundamental questions in taste sensation, including salt taste coding¹¹, taste plasticity modified by food experience²², and the molecular basis of food texture sensation³³. In summary, this Petri-dish-based two-choice assay is a powerful tool to investigate how flies perceive external and internal nutrient milieus to elicit appropriate feeding behavior.

Protocol

1. Assembling the assay chambers

NOTE: While this protocol describes the use of a 35 mm Petri dish (Figure 1A), the desired effect can be achieved using any watertight, smooth-bottomed vessel that can be bisected and covered.

1. First, bisect a lidded 35 mm Petri dish by fixing a length of plastic (5 mm in width and 3 mm in height) down the midline with waterproof adhesive, forming two watertight compartments. Confirm that the seal is complete to avoid leakage that can lead to mixing of the two food substrates being assayed.
   ​NOTE: After assembly, reuse this apparatus as long as the seal holds.

2. Preparing starvation vials

1. Prepare a sufficient number of empty plastic fly vials; then, loosely compact a piece of tissue paper at the bottom. Compress the tissue paper enough that it fills the space, but not so much that it forms a dense mass.
   NOTE: Make sure that there are no deep crevices or folds in the tissue, as this can lead to flies getting trapped.
2. Add ~3 mL pure water to the vial so that the tissue is completely saturated, but there is no standing water. Ensure that there are no large droplets of excess water on the wall of the vial. Alternatively, substitute agarose for the soaked paper by preparing a 1% w/v agar solution (without sucrose) by adding 5 mL of 1% agarose to each empty vial and allowing the agarose to solidify at room temperature.

3. Wet starvation of flies prior to the experiment

1. Initiate starvation 24 h before the time of the experiment. Under CO₂ anesthesia, sort groups of ~70, 2-4-day-old flies into the prepared starvation vials, labeling each vial with the genotype and time of starvation.

4. Reagent setup

1. Preparation of dyes
   ​NOTE: Prior to performing any experiments, it is important to perform a preliminary control assay to determine the correct concentrations of red and blue dyes to use.
   1. For the control assay, prepare a range of dilutions for each dye, and perform the feeding assay with the same food with a different dye color. Use the results to identify two-dye concentrations (one red, one blue) that yield a PI of ~0 when no experimental compound is added (see section 7).
      NOTE: For example, the final blue dye concentration was fixed at 50 µM and tested against a series of red dye concentrations. Based on the red dye dosage curve, the optimal red dye concentration was 210 µM, which gave minimal dye bias (Figure 1B). A higher red dye concentration drives flies to prefer red food, whereas a lower concentration drives flies to prefer blue food. Carefully refine blue or red dye concentrations in increments of 1 µM, as differences of this magnitude and greater can affect experimental outcomes.
2. Preparation of 1% agarose
   1. Combine 0.5 g agarose and 50 mL of pure water (or some multiple thereof) in a microwave-safe vessel. Microwave the agarose solution until dissolved, stirring it as needed.
3. Preparation of other food components
   1. Dissolve each food component, including sucrose and any experimental compounds, in water at a 100-fold or higher concentration of the final tested concentration.
      ​NOTE: The total volume of each food ingredient added to 1% agar should not exceed 1 mL per 10 mL molten agar. Otherwise, the agarose may be too dilute and will not solidify appropriately.
4. Preparation of food media
   1. Mix agar, dye, and the desired experimental compound in conical polypropylene centrifuge tubes (15 or 50 mL); use water instead of the experimental tastant in the control food. Do this while the agar is still completely liquid and mix thoroughly using a vortex mixer. Keep the tubes in a 60 °C water bath while not in use to prevent the agarose from hardening before being distributed into dishes.
5. Preparing dishes for the experiment
   NOTE: Ensure that all dishes are completely dry before starting.
   1. Pipette 1 mL of red experimental food medium into one side of the assay dish (Figure 1A); repeat for the desired number of dishes. Allow the agarose to cool until firm (3-5 min), and then pipette 1 mL of blue control food into the other side of the dishes (Figure 1A). Repeat this process with the control red/experimental blue pair.
      ​NOTE: Make sure all dishes are fully set before beginning the experiment. Use the dishes within 30 min.

5. Initiating the two-way feeding assay

1. Temporarily paralyze experimental fly lines on ice until no obvious motor activities such as flying and climbing are observed. Once the flies are immobilized, gently invert the vial, and tap to transfer all the flies into the assay chamber.
   NOTE: Cold shock takes ~3-5 min. Prolonged exposure to cold may affect the fly's physiology and health and should therefore be avoided.
2. Quickly place the cover on the chamber and set it aside. Once all the flies have been transferred, move all chambers to a dark, enclosed space. Allow the assay to run for 90 min.
   ​NOTE: A dark environment minimizes the influence of the fly's visual pathway on feeding behavior and removes any environmental cues from outside the dish.

6. Terminating the two-way feeding assay

1. After 90 min have elapsed, transfer the chambers to a -20 °C freezer to sacrifice the flies. After ~1 h, count the flies.
   ​NOTE: Invert each Petri dish before placing the dish in the freezer to ensure that no flies will be frozen onto the food.

7. Assigning a preference index (PI) to determine food preference

1. Under a standard dissection microscope, examine the flies' abdominal color in each individual dish. Count the flies as either red, blue, or purple according to the color of their abdomen (Figure 2A). Count the fly if its abdomen is more than 50% colored, indicating robust feeding (Figure 2B). Exclude the fly if its abdomen contains only a tiny food spot, indicating poor eating (Figure 2C).
2. After the numbers of flies eating blue, red, or both blue and red foods have been counted, use the following equation to assign each Petri dish a preference index (PI):

PI = (Number of flies eating experimental food) - (Number of flies eating control food) / (Number of flies eating experimental food) + (Number of flies eating control food) + (Number of flies eating both)

PI > 0 indicates a preference for the experimental compound, PI < 0 indicates an aversion to the experimental compound, and PI = 0 indicates no effect of the compound on feeding behavior.

8. Cleaning the assay chambers

1. Promptly clean the Petri dishes by scraping out the food substrate and rinsing them with unscented soap and water. Soak the Petri dishes overnight in distilled water. Check that the dividing seal in each dish is still watertight, then let the dish air dry.
   NOTE: After ensuring that there is no residual agarose or dye staining, the Petri dishes are ready to use again.

Results

In this assay, a 35 mm dish was divided into two equal feeding compartments, with each half of the dish containing agarose food coupled with either blue or red dye (Figure 1A). To exclude dye bias, the blue and red dye concentrations were carefully refined to yield an approximate "0" PI when only these two dyes were added (Figure 1B). Once the Petri dish was loaded with tested food, ~70 wet-starved, 2-4-day-old adult flies were transferred to the dish, a...

Discussion

This method involves several crucial steps where problems can occur. First, make sure flies ingest a sufficient amount of food to provide stable data. If flies eat poorly, ensure that the flies have been wet-starved for at least 24 h, and that the experimental media contains at least a minimal sucrose concentration (2 mM). To further stimulate food consumption, prolong the wet-starvation period beyond 24 h, depending on the flies' physiological condition. If too many flies fail to survive the prolonged starvation, en...

Materials

Name	Company	Catalog Number	Comments
35 mm Petri dish	Fisher Scientific	08-772E
Agarose	Thomas Scientific	C756P56
Clear adhesive	Fisher Scientific	NC9884114
Conical centrifuge tubes	Fisher Scientific	05-527-90
Dissection microscope	Amscope	SM-2T-6WB-V331
FCF Brilliant Blue	Wako Chemical	3844-45-9
Fly CO2 anesthesia setup	Genesee Scientfic	59-114/54-104M
Fly incubator with programmable day/night cycle	Powers Scientific Inc.	IS33SD
Fly lines
Glass dish (microwave-safe)
Kimwipes	Fisher Scientific	06-666A
Media storage bottle	Fisher Scientific	50-192-9998
Plastic divider cut to fit the dish from a sheet no thicker than 5 mm
Plastic fly vials	Genesee Scientific	32-116
Sucrose	Millipore Sigma	S9378
Sulforhodamine B	Millipore Sigma	S9012
Tastant compound of interest
Vortex mixer	Benchmark Scientific	BV1000
Water bath	Fisher Scientific	FSGPD05