Taste Preference Assay for Adult Drosophila

Summary

Taste is an important sensory process which facilitates attraction to beneficial substances and avoidance of toxic substances. This protocol describes a simple ingestion assay for determining Drosophila gustatory preference for a given chemical compound.

Abstract

Olfactory and gustatory perception of the environment is vital for animal survival. The most obvious application of these chemosenses is to be able to distinguish good food sources from potentially dangerous food sources. Gustation requires physical contact with a chemical compound which is able to signal through taste receptors that are expressed on the surface of neurons. In insects, these gustatory neurons can be located across the animal's body allowing taste to play an important role in many different behaviors. Insects typically prefer compounds containing sugars, while compounds that are considered bitter tasting are avoided. Given the basic biological importance of taste, there is intense interest in understanding the molecular mechanisms underlying this sensory modality. We describe an adult Drosophila taste assay which reflects the preference of the animals for a given tastant compound. This assay may be applied to animals of any genetic background to examine the taste preference for a desired soluble compound.

Introduction

Animals use chemosensation to distinguish advantageous conditions apart from disadvantageous conditions. This perception can be critical for such things as determining the best food source, avoiding toxic substances or determining the best mating partner¹. Chemosensation is often divided into two sensory components: olfactory senses and gustatory senses. A main distinguishing characteristic of these senses is that olfaction (smell) is used to sample the surrounding gaseous chemical environment while gustation (taste) requires physical contact with a nonvolatile substrate. Both sensory modalities stimulate neurological responses which are processed and decoded in the brain to produce the appropriate attractive or repulsive behavior². These senses are therefore critical for animal survival.

The fruit fly Drosophila melanogaster is a model organism which continues to grow in popularity for use in understanding how insects perceive smell and taste. Fruit flies offer tremendous advantages over other model systems due to the wealth of genetic tools available for the dissection of molecular, cellular, and behavioral pathways. Work over the last 15 years has been particularly instrumental in characterizing the specific cellular identities, neuronal receptors, and signaling mechanisms involved in both smell and taste. Now, the power of Drosophila genetics is being used to further elucidate how these processes are coded at the single neuron and single circuit level^3-6. Therefore, assays which provide easily scored readouts of alterations to sensory pathways are vital to the continuing advance of these fields.

While a great deal is known about how olfactory signals are coded and processed in the brain, much less is understood about similar mechanisms in the gustatory pathway. We describe here a protocol which can be used to ascertain taste preference in Drosophila. Drosophila, like mammals, generally prefer sweet tasting compounds as opposed to bitter tasting compounds. Any combination of these food sources can be utilized in this experimental design to determine how known genetic alterations affect taste choice. In addition, pharmacological intervention strategies can similarly be assessed for their effects on animals' taste preference. The ease and flexibility of this assay makes it a useful paradigm for understanding the nature of gustatory perception in Drosophila.

Protocol

1. Starvation

1. Prepare fly starvation vials by saturating a cotton ball with 18.2 MΩ water at the bottom of a standard fly vial. Alternatively, similarly saturate a small strip of filter paper with 18.2 MΩ water and place at an angle within the vial.
2. Collect flies into sets of ~100 animals on a CO₂ pad and then add the flies to a prepared vial.
   Note: Best results are obtained with animals that are less than 5 days old. However, the exact age of the animals can be controlled as an experimental variable to determine changes in taste preference over time.
3. Use a cotton ball or foam stopper to secure the vials closed. Place vials on their side in an environmentally controlled incubator. Maintain the temperature at 25 °C, and the humidity above 70%. Leave vials untouched for 24 hr.

2. Taste Preference Assay

1. Prepare all tastants for the assay on the same day as testing.
   Note: The exact tastants to be used will vary depending on experimental question being asked. The following are example tastants used in this protocol. See section 4 for optimizations.
   1. Prepare control tastant (1 mM sucrose) by combining 10 µl of 100 mM sucrose solution, 13 µl of red food coloring, and 977 µl of 18.2 MΩ water.
   2. Prepare experimental tastant (5 mM sucrose) by combining 50 µl of 100 mM sucrose solution, 10 µl of blue food coloring, and 940 µl of 18.2 MΩ water.
2. Make assay chambers using a standard 100 mm x 15 mm plastic petri dish prepared in the following manner:
   1. Place three 10 µl drops of control tastant nearest the edge of the plate at 12 o'clock and another 3 drops at 6 o'clock. Ensure that the spacing between drops is similar.
   2. Place three 10 µl drops of experimental tastant nearest the edge of the plate at 3 o'clock and another 3 drops at 9 o'clock. Ensure that the spacing between drops is similar.
   3. Repeat steps 2.2.1 and 2.2.2 for as many replicates as desired.
3. Empty 1 vial of ~100 starved flies onto a CO₂ pad just long enough to anesthetize all animals (approximately 10 sec). Brush the animals into the middle of a prepared assay chamber and cover with the dish lid.
   Note: Longer periods of CO₂ exposure should be avoided to improve recovery time and limit interference with the feeding behavior. Exposure to ice (~5 min) may be used for anesthetizing to avoid CO₂ behavioral effects that may arise from even limited exposure.
4. Place the assay chamber in an opaque cardboard box. Be sure to label the outside of the box with the condition and genotype being tested.
5. Place the entire setup (assay chamber contained within cardboard box from step 2.4) into a 25 °C incubator with at least 70% humidity for 2 hr.
6. Repeat steps 2.3 through 2.5 for all replicates.
7. After 2 hours, place the assay chambers, still contained within cardboard boxes, directly into a -20 °C freezer until ready for quantitation.

3. Taste Preference Assay Quantification

1. Allow a single assay chamber to warm up to room temperature (approximately 5 min).
2. Under a dissection microscope, using a brush or pair of forceps, group animals based on the color of their abdomen: red, blue, purple or clear (Figure 1).
3. Record the number of animals in each grouping. Consider clear animals to have not participated in the assay and therefore do not include them in any calculations.
4. Calculate the preference index according to one of the following equations:
   1. If the experimental tastant of interest is added to the red dye, then use (N_red +0.5N_purple)/(N_red +N_blue + N_purple).
   2. If the experimental tastant is added to the blue dye, then adjust the equation to (N_blue +0.5N_purple)/(N_blue +N_red + N_purple).
5. Repeat the calculations for all experimental conditions and replicates.

4. Optimization of Taste Preference Assay

1. Empirically determine the concentration of food coloring indicators to be used so food coloring does not affect the outcome of the taste assay, as follows:
   1. Prepare 4 tastants using the same base compound (e.g. 5 mM sucrose) as indicated in step 2.1, but omit the food coloring.
   2. Add 1.3% red food coloring to one of the tastants. Make the remaining 3 tastants with blue food coloring of varying concentrations in each tube (e.g. 0.6%, 1%, and 1.3%).
   3. Complete protocol steps 2.2 through 3.4 for each tastant pair: 1.3% red vs. 0.6% blue; 1.3% red vs. 1% blue and 1.3% red vs. 1.3% blue.
   4. Repeat step 4.1.1-4.1.3 with different percentages of blue food coloring until the preference index averages a value of 0 (Figure 2).
      Note: As a starting point, 1.3% red food coloring coupled with 1% blue food coloring typically yields good results. If no satisfactory concentration of blue food coloring can be matched to 1.3% dye, then step 4.1.1 through 4.1.3 can be repeated with varying concentrations of red coloring and a constant concentration of blue food coloring.
   5. Analyze all conditions to be tested with the same optimized food coloring concentrations.

Results

Some typical results from taste preference assays are shown below. In most experiments some variation in intensity of abdominal coloring will be seen (Figure 1). Any coloring in the abdomen whether intense or weak is considered a positive ingestion. It is therefore advisable for researchers to score animals while blind to the experimental condition so as to limit any potential biases.

It is also important to cho...

Discussion

We have described a simple but effective protocol for determining taste preference in Drosophila. Versions of this assay are routinely used in experiments to determine the contributions of gustatory receptors (GRs) to perceiving the different qualities (bitter, sweet, sour, salty, and umami) of taste compounds. The Drosophila genome contains approximately 60 genes which encode 68 identified gustatory receptors by alternative splicing^8,9. However, other proteins such as ionotropic glutamate re...

Materials

Name	Company	Catalog Number	Comments
Blue Food Coloring (Water, Propylene Glycol, FD&C Blue 1 and Red 40, Propylparaben)	McCormick	N/A
Cryo/Freezer Boxes w/o Dividers	Fisher	03-395-455
Dumont #5 Forceps	Fine Science Tools	11251-20
Glacial Acetic Acid	Fisher	BP2401-500
Leica S6 E Stereozoom 0.63X-4.0X microscope	W. Nuhsbaum, Inc.	10446294
Petri dish (100 mm x 15 mm)	BD Falcon	351029	Reuseable if thoroughly washed and dried
Quick-Snap Microtubes	Alkali Scientific Inc.	C3017
Red Food Coloring (Water, Propylene Glycol, FD&C Reds 40 and 3, Propylparaben)	McCormick	N/A
Sucrose	IBI Scientific	IB37160