We would like to thank members of the Tessier lab for critical reading of this manuscript and helpful suggestions during the preparation of this protocol.

1. Starvation
<ol>
	<li>Prepare fly starvation vials by saturating a cotton ball with 18.2 M&#937; water at the bottom of a standard fly vial. Alternatively, similarly saturate a small strip of filter paper with 18.2 M&#937; water and place at an angle within the vial.</li>
	<li>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.</li>
	<li>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 &#176;C, and the humidity above 70%. Leave vials untouched for 24 hr.</li>
</ol>
2. Taste Preference Assay
<ol>
	<li>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.
	<ol>
		<li>Prepare control tastant (1 mM sucrose) by combining 10 &#181;l of 100 mM sucrose solution, 13 &#181;l of red food coloring, and 977 &#181;l of 18.2 M&#937; water.</li>
		<li>Prepare experimental tastant (5 mM sucrose) by combining 50 &#181;l of 100 mM sucrose solution, 10 &#181;l of blue food coloring, and 940 &#181;l of 18.2 M&#937; water.</li>
	</ol>
	</li>
	<li>Make assay chambers using a standard 100 mm x 15 mm plastic petri dish prepared in the following manner:
	<ol>
		<li>Place three 10 &#181;l drops of control tastant nearest the edge of the plate at 12 o&#39;clock and another 3 drops at 6 o&#39;clock. Ensure that the spacing between drops is similar.</li>
		<li>Place three 10 &#181;l drops of experimental tastant nearest the edge of the plate at 3 o&#39;clock and another 3 drops at 9 o&#39;clock. Ensure that the spacing between drops is similar.</li>
		<li>Repeat steps 2.2.1 and 2.2.2 for as many replicates as desired.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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.</li>
	<li>Place the entire setup (assay chamber contained within cardboard box from step 2.4) into a 25 &#176;C incubator with at least 70% humidity for 2 hr.</li>
	<li>Repeat steps 2.3 through 2.5 for all replicates.</li>
	<li>After 2 hours, place the assay chambers, still contained within cardboard boxes, directly into a -20 &#176;C freezer until ready for quantitation.</li>
</ol>
3. Taste Preference Assay Quantification
<ol>
	<li>Allow a single assay chamber to warm up to room temperature (approximately 5 min).</li>
	<li>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).</li>
	<li>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.</li>
	<li>Calculate the preference index according to one of the following equations:
	<ol>
		<li>If the experimental tastant of interest is added to the red dye, then use (Nred +0.5Npurple)/(Nred +Nblue + Npurple).</li>
		<li>If the experimental tastant is added to the blue dye, then adjust the equation to (Nblue +0.5Npurple)/(Nblue +Nred + Npurple).</li>
	</ol>
	</li>
	<li>Repeat the calculations for all experimental conditions and replicates.</li>
</ol>
4. Optimization of Taste Preference Assay
<ol>
	<li>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:
	<ol>
		<li>Prepare 4 tastants using the same base compound (e.g. 5 mM sucrose) as indicated in step 2.1, but omit the food coloring.</li>
		<li>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%).</li>
		<li>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.</li>
		<li>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.</li>
		<li>Analyze all conditions to be tested with the same optimized food coloring concentrations.</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

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 splicing8,9. However, other proteins such as ionotropic glutamate re...

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 level3-6. 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&#39; taste preference. The ease and flexibility of this assay makes it a useful paradigm for understanding the nature of gustatory perception in Drosophila.

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

taste preference assay for adult drosophila

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.

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...

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Taste Preference Assay for Adult Drosophila

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.

Taste Preference Assay for Adult Drosophila

Olfactory and gustatory perception of the environment is vital for animal survival.  The most obvious application of these chemosenses is to be able to ...

taste-preference-assay-for-adult-drosophila

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9.6K Views.  Indiana University School of Medicine-South Bend. 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.

Video: Taste Preference Assay for Adult Drosophila

Appetitive Associative Olfactory Learning in Drosophila Larvae

In the following we describe the methodological details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with genetic interference, provides a handle to analyze the neuronal and molecular fundamentals of specifically associative learning in a simple larval brain.
Organisms can use past experience to adjust present behavior. Such acquisition of behavioral potential can be defined as learning, and the physical bases of these potentials as memory traces1-4. Neuroscientists try to understand how these processes are organized in terms of molecular and neuronal changes in the brain by using a variety of methods in model organisms ranging from insects to vertebrates5,6. For such endeavors it is helpful to use model systems that are simple and experimentally accessible. The Drosophila larva has turned out to satisfy these demands based on the availability of robust behavioral assays, the existence of a variety of transgenic techniques and the elementary organization of the nervous system comprising only about 10,000 neurons (albeit with some concessions: cognitive limitations, few behavioral options, and richness of experience questionable)7-10.
Drosophila larvae can form associations between odors and appetitive gustatory reinforcement like sugar11-14. In a standard assay, established in the lab of B. Gerber, animals receive a two-odor reciprocal training: A first group of larvae is exposed to an odor A together with a gustatory reinforcer (sugar reward) and is subsequently exposed to an odor B without reinforcement 9. Meanwhile a second group of larvae receives reciprocal training while experiencing odor A without reinforcement and subsequently being exposed to odor B with reinforcement (sugar reward). In the following both groups are tested for their preference between the two odors. Relatively higher preferences for the rewarded odor reflect associative learning - presented as a performance index (PI). The conclusion regarding the associative nature of the performance index is compelling, because apart from the contingency between odors and tastants, other parameters, such as odor and reward exposure, passage of time and handling do not differ between the two groups9.

Appetitive Associative Olfactory Learning in Drosophila Larvae

Drosophila larvae are able to associate odor stimuli with gustatory reward. Here we describe a simple behavioral paradigm that allows the analysis of appetitive associative olfactory learning.

In the following we describe the methodological  details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with ...

A Single-fly Assay for Foraging Behavior in Drosophila

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster. In a light-tight box illuminated by red light that is invisible to fruit flies, a camera linked to custom data acquisition software monitors the position of six flies simultaneously. Each fly is confined to walk in individual arenas containing a food odor at the center. The testing arenas rest on a porous floor that functions to prevent odor accumulation. Latency to locate the odor source, a metric that reflects olfactory sensitivity under different physiological states, is determined by software analysis. Here, we discuss the critical mechanics of running this behavioral paradigm and cover specific issues regarding fly loading, odor contamination, assay temperature, data quality, and statistical analysis.

A Single-fly Assay for Foraging Behavior in Drosophila

In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster.

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video ...

Testing Drosophila Olfaction with a Y-maze Assay

Detecting signals from the environment is essential for animals to ensure their survival. To this aim, they use environmental cues such as vision, mechanoreception, hearing, and chemoperception through taste, via direct contact or through olfaction, which represents the response to a volatile molecule acting at longer range. Volatile chemical molecules are very important signals for most animals in the detection of danger, a source of food, or to communicate between individuals. Drosophila melanogaster is one of the most common biological models for scientists to explore the cellular and molecular basis of olfaction. In order to highlight olfactory abilities of this small insect, we describe a modified choice protocol based on the Y-maze test classically used with mice. Data obtained with Y-mazes give valuable information to better understand how animals deal with their perpetually changing environment. We introduce a step-by-step protocol to study the impact of odorants on fly exploratory response using this Y-maze assay.

Testing Drosophila Olfaction with a Y-maze Assay

We present a choice test to reveal the influence of odorants on Drosophila behavior using a Y-maze assay.

Detecting signals from the environment is essential for animals to ensure their survival. To this aim, they use environmental cues such as vision, ...

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. When selecting sites to deposit their eggs, female flies are capable of ranking the relative attractiveness of their options and choosing the &#34;greater of two goods.&#34; However, most egg-laying preference assays are not practical if one wants to take a systematic genetic screening approach to search for the circuit basis underlying this simple decision-making process, as they are population-based and laborious to set up. To increase the throughput of studying of egg-laying preferences of single females, we developed custom chambers that each can simultaneously assay egg-laying preferences of up to thirty individual flies as well as a protocol that ensures each female has a high egg-laying rate (so that their preference is readily discernable and more convincing). Our approach is simple to execute and produces very consistent results. Additionally, these chambers can be equipped with different attachments to allow video recording the egg-laying animals and to deliver light for optogenetics studies. This article provides the blueprints for fabricating these chambers and the procedure for preparing the flies to be assayed in these chambers.

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

This protocol describes a high throughput assay for testing egg-laying preferences of Drosophila melanogaster at single-animal resolution. This assay provides a simple, efficient, and scalable platform to identify genes and circuit components that control a simple decision-making process.

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of the right type and quantity of food therefore has a major influence on quality of life. Research on feeding behavior focuses on the underlying processes that ensure actual feeding and unravels the role of factors regulating internal energy homeostasis and the neuronal bases of decision-making. The model organism Drosophila melanogaster, with its great variety of genetically traceable tools for labeling and manipulating single neurons, allows mapping of neuronal networks and identification of molecular signaling cascades involved in the regulation of food intake. This report demonstrates the CApillary FEeder assay (CAFE) and shows how to measure food intake in a group of flies for time spans ranging from hours to days. This easy-to-use assay consists of glass capillaries filled with liquid food that flies can freely access and feed on. Food consumption in the assay is accurately determined using simple measurement tools. Herein we describe step-by-step the method from setup to successful execution of the CAFE assay, and provide practical examples to analyze the food intake of a group of flies under controlled conditions. The reader is guided through possible limitations of the assay, and advantages and disadvantages of the method compared to other feeding assays in D. melanogaster are evaluated.

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

The CApillary FEeder (CAFE) assay is a simple, budget-friendly, highly reliable method for investigating mechanisms underlying food intake. Used with the highly versatile genetic model organism Drosophila melanogaster, it provides a powerful means of gaining new insights into regulatory mechanisms of food intake.

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of ...

A Rapid Food-Preference Assay in Drosophila

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&#160;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.

A Rapid Food-Preference Assay in Drosophila

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 ...

Feeding Test: Assessment of Feeding Preferences in Drosophila melanogaster

Source: Bovier, T. F., et al. <a href="https://www.jove.com/video/59535/" target="_blank">Methods to Test Endocrine Disruption in Drosophila melanogaster</a>. J. Vis. Exp. (2019).
This video describes a feeding assay for fruit flies in which colored food is used to monitor feeding and the effect added compounds may have on feeding activity. The example protocol tests the effects of several endocrine disrupting chemicals (EDC).

Test żywieniowy: ocena preferencji żywieniowych u Drosophila melanogaster

Source: Bovier, T. F., et al. Methods to Test Endocrine Disruption in Drosophila melanogaster. J. Vis. Exp. (2019).
This video describes a feeding assay ...

JoVE Encyclopedia of Experiments

Biology

Drosophila melanogaster (fruit fly)

Adult

Temperature Gradient Assay: A Method to Test Temperature Preference in Drosophila Larvae

Source: Liu, J., et al. <a href="https://www.jove.com/video/57963/" target="_blank">A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae</a>. J. Vis. Exp. (2018).
Drosophila fruit flies can distinguish small changes in temperature and therefore select environments with favorable thermal conditions. This video describes a behavioral assay that tests the temperature preference of Drosophila larvae on a linear thermal gradient.

Test gradientu temperatury: metoda badania preferencji temperatury u larw Drosophila

Source: Liu, J., et al. A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae. J. Vis. Exp. (2018).
Drosophila fruit flies ...

Larva

Taste Preference Assay: A Method for Measuring Feeding Behavior in Drosophila

Source: Bantel, A. P. and Tessier, C. R. <a href="https://www.jove.com/video/54403/" target="_blank">Taste Preference Assay for Adult Drosophila</a>. J. Vis. Exp. (2016).
This video describes the taste preference assay, a behavioral method used to measure attraction or avoidance towards colored solutions that taste differently by assessing the fly&#39;s abdominal coloration after ingestion of the preferred substance. The featured protocol demonstrates the procedure used to measure flies&#39; preference towards solutions of varying sucrose concentrations.

Test preferencji smakowych: metoda pomiaru zachowań żywieniowych u Drosophila

Source: Bantel, A. P. and Tessier, C. R. Taste Preference Assay for Adult Drosophila. J. Vis. Exp. (2016).
This video describes the taste preference ...

Taste Preference Assay for Adult Drosophila

Podsumowanie

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A Rapid Food-Preference Assay in Drosophila

Test żywieniowy: ocena preferencji żywieniowych u Drosophila melanogaster

Test gradientu temperatury: metoda badania preferencji temperatury u larw Drosophila

Test preferencji smakowych: metoda pomiaru zachowań żywieniowych u Drosophila