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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.
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.
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 sugar1,2,3,4,5,6,7,8, amino acids9,10, and low salt11, which indicate nutrients, but reject bitter foods12,13,14,15 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 regulation16,17,18,19,20. 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 assays11,12,13,21,22,23, the fly proboscis extension response assay24, the Capillary Feeder (CAFE) assay25,26, 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 flies28,29,30,31. One of the popular feeding paradigms is the dye-based two-choice feeding assay using either a multiwell microtiter plate12,21,32 or, as described here, a small Petri dish11,22 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 texture11,12,21,22,32,33. 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.
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.
2. Preparing starvation vials
3. Wet starvation of flies prior to the experiment
4. Reagent setup
5. Initiating the two-way feeding assay
6. Terminating the two-way feeding assay
7. Assigning a preference index (PI) to determine food preference
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
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...
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...
The authors declare no conflicts of interest or competing financial interests.
The authors would like to thank Dr. Tingwei Mi for helping them optimize the two-choice feeding assay. They would also like to thank Samuel Chan and Wyatt Koolmees for their comments on the manuscript. This project was funded by the National Institutes of Health grants R03 DC014787 (Y.V.Z.) and R01 DC018592 (Y.V.Z.) and by the Ambrose Monell Foundation.
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 |
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