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.

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.
<ol>
	<li>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. 
	&#8203;NOTE: After assembly, reuse this apparatus as long as the seal holds.</li>
</ol>
2.&#160;Preparing starvation vials
<ol>
	<li>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.</li>
	<li>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.</li>
</ol>
3. Wet starvation of flies prior to the experiment
<ol>
	<li>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.</li>
</ol>
4. Reagent setup
<ol>
	<li>Preparation of dyes 
	&#8203;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.
	<ol>
		<li>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 &#181;M and tested against a series of red dye concentrations. Based on the red dye dosage curve, the optimal red dye concentration was 210 &#181;M, 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 &#181;M, as differences of this magnitude and greater can affect experimental outcomes.</li>
	</ol>
	</li>
	<li>Preparation of 1% agarose
	<ol>
		<li>Combine 0.5 g agarose and 50 mL of pure water (or some multiple&#160;thereof) in a microwave-safe vessel. Microwave the agarose solution until dissolved, stirring it as needed.</li>
	</ol>
	</li>
	<li>Preparation of other food components
	<ol>
		<li>Dissolve each food component, including sucrose and any experimental compounds, in water at a 100-fold or higher concentration of the final tested concentration. 
		&#8203;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.</li>
	</ol>
	</li>
	<li>Preparation of food media
	<ol>
		<li>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 &#176;C water bath while not in use to prevent the agarose from hardening before being distributed into dishes.</li>
	</ol>
	</li>
	<li>Preparing dishes for the experiment 
	NOTE: Ensure that all dishes are completely dry before starting.
	<ol>
		<li>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. 
		&#8203;NOTE: Make sure all dishes are fully set before beginning the experiment. Use the dishes within 30 min.</li>
	</ol>
	</li>
</ol>
5. Initiating the two-way feeding assay
<ol>
	<li>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&#39;s physiology and health and should therefore be avoided.</li>
	<li>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. 
	&#8203;NOTE: A dark environment minimizes the influence of the fly&#39;s visual pathway on feeding behavior and removes any environmental cues from outside the dish.</li>
</ol>
6. Terminating the two-way feeding assay
<ol>
	<li>After 90 min have elapsed, transfer the chambers to a -20 &#176;C freezer to sacrifice the flies. After ~1 h, count the flies. 
	&#8203;NOTE: Invert each Petri dish before placing the dish in the freezer to ensure that no flies will be frozen onto the food.</li>
</ol>
7. Assigning a preference index (PI) to determine food preference
<ol>
	<li>Under a standard dissection microscope, examine the flies&#39; 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).</li>
	<li>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):</li>
</ol>
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 &#62; 0 indicates a preference for the experimental compound, PI &#60; 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
<ol>
	<li>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.</li>
</ol>

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The authors declare no conflicts of interest or competing financial interests.

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&#39; physiological condition. If too many flies fail to survive the prolonged starvation, en...

Despite dramatic differences in the anatomical structure of taste organs between flies and mammals, the flies&#39; 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&#39;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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>35 mm Petri dish</td>
			<td>Fisher Scientific</td>
			<td>08-772E</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Thomas Scientific</td>
			<td>C756P56</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear adhesive</td>
			<td>Fisher Scientific</td>
			<td>NC9884114</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical centrifuge tubes</td>
			<td>Fisher Scientific</td>
			<td>05-527-90</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection microscope</td>
			<td>Amscope</td>
			<td>SM-2T-6WB-V331</td>
			<td></td>
		</tr>
		<tr>
			<td>FCF Brilliant Blue</td>
			<td>Wako Chemical</td>
			<td>3844-45-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Fly CO2 anesthesia setup</td>
			<td>Genesee Scientfic</td>
			<td>59-114/54-104M</td>
			<td></td>
		</tr>
		<tr>
			<td>Fly incubator with programmable day/night cycle</td>
			<td>Powers Scientific Inc.</td>
			<td>IS33SD</td>
			<td></td>
		</tr>
		<tr>
			<td>Fly lines</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass dish (microwave-safe)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Fisher Scientific</td>
			<td>06-666A</td>
			<td></td>
		</tr>
		<tr>
			<td>Media storage bottle</td>
			<td>Fisher Scientific</td>
			<td>50-192-9998</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic divider cut to fit the dish from a sheet no thicker than 5 mm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic fly vials</td>
			<td>Genesee Scientific</td>
			<td>32-116</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Millipore Sigma</td>
			<td>S9378</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulforhodamine B</td>
			<td>Millipore Sigma</td>
			<td>S9012</td>
			<td></td>
		</tr>
		<tr>
			<td>Tastant compound of interest</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>Benchmark Scientific</td>
			<td>BV1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Fisher Scientific</td>
			<td>FSGPD05</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

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 &#34;0&#34; 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...

Watch this Scientific Journal Video about A Rapid Food-Preference Assay in Drosophila at JoVE.com

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.

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 ...

a-rapid-food-preference-assay-in-drosophila

Research

JoVE Journal

Neuroscience

6.0K Views.  Monell Chemical Senses Center. 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.

Video: A Rapid Food-Preference Assay in Drosophila

Methods to Assay Drosophila Behavior

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases1. We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials2-4. The rapid iterative negative geotaxis (RING) assay5 has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously using large number of animals, with the high-throughput approach making it more amenable for screening experiments.

Methods to Assay Drosophila Behavior

Drosophila melanogaster is a genetically and behaviorally tractable model system that has been used to understand the molecular and cellular basis of many important biological processes for over a century 1. Drosophila has been well exploited to gain insights into the genetic basis of fly behavior.

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular ...

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, ...

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by stressed conspecifics due to an odorant released during stress known as the Drosophila stress odorant (dSO). Through the use of the T-maze apparatus, one can quantify the avoidance of the dSO by responder flies in a very affordable and robust assay. Conditions necessary to obtain a strong performance are presented here. A stressful experience is necessary for the flies to emit dSO, as well as enough emitter flies to cause a robust avoidance response to the presence of dSO. Genetic background, but not their group size, strongly altered the avoidance of the dSO by the responder flies. Canton-S and Elwood display a higher performance in avoiding the dSO than Oregon and Samarkand strains. This behavioral assay will allow identification of mechanisms underlying this social behavior, and the assessment of the influence of genes and environmental conditions on both emission and avoidance of the dSO. Such an assay can be included in batteries of simple diagnostic tests used to identify social deficiencies of mutants or environmental conditions of interest.

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Here, we present a protocol to quantify the avoidance of stressed individuals. This paradigm is powerful yet user-friendly and can be used to assess the influence of genes and environment on one kind of social interaction in Drosophila melanogaster.

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by ...

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in their etiology. The fruit fly, Drosophila melanogaster, is a powerful model for exploring molecular-genetic mechanisms underlying alcohol-related behaviors and therefore holds great promise for identifying and understanding the function of genes that influence AUD. The use of the Drosophila model for these types of studies depends on the availability of assays that reliably measure behavioral responses to ethanol. This report describes an assay suitable for assessing ethanol sensitivity and rapid tolerance in flies. Ethanol sensitivity measured in this assay is influenced by the volume and concentration of ethanol used, a variety of previously reported genetic manipulations, and also the length of time the flies are housed without food immediately prior to testing. In contrast, ethanol sensitivity measured in this assay is not affected by the vigor of fly handling, sex of the flies, and supplementation of growth medium with antibiotics or live yeast. Three different methods for quantitating ethanol sensitivity are described, all leading to essentially indistinguishable ethanol sensitivity results. The scalable nature of this assay, combined with its overall simplicity to set-up and relatively low expense, make it suitable for small and large scale genetic analysis of ethanol sensitivity and rapid tolerance in Drosophila.

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

Straightforward assays for measuring ethanol sensitivity and rapid tolerance in Drosophila facilitate the use of this model organism for investigating these important ethanol-related behaviors. Here, a relatively simple, scalable assay for measuring ethanol sensitivity and rapid tolerance in flies is described.

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in ...

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense organs provide an excellent model system for the study of mechanosensation. This protocol describes a simple touch behavior in fruit flies which can be used to identify mutations that interfere with mechanosensation. The tactile stimulation of a macrochaete bristle on the thorax of flies elicits a grooming reflex from either the first or third leg. Mutations that interfere with mechanotransduction (such as NOMPC), or with other aspects of the reflex arc, can inhibit the grooming response. A traditional screen of adult behaviors would have missed mutants that have essential roles during development. Instead, this protocol combines the touch screen with mosaic analysis with a repressible cell marker (MARCM) to allow for only limited regions of homozygous mutant cells to be generated and marked by the expression of green fluorescent protein (GFP). By testing MARCM clones for abnormal behavioral responses, it is possible to screen a collection of lethal p-element mutations to search for new genes involved in mechanosensation that would have been missed by more traditional methods.

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal.

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense ...

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.

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.

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

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 ...

Novel Assay for Cold Nociception in Drosophila Larvae

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory machinery, such as in patients experiencing peripheral neuropathy or injury-induced sensitization, are not well characterized. The genetically tractable Drosophila model has been used to study the cells and genes required for noxious heat detection, which has yielded multiple conserved genes of interest. Little is known however about the cells and receptors important for noxious cold sensing. Although, Drosophila does not survive prolonged exposure to cold temperatures (&#8804;10 &#186;C), and will avoid cool, preferring warmer temperatures in behavioral preference assays, how they sense and possibly avoid noxious cold stimuli has only recently been investigated.
Here we describe and characterize the first noxious cold (&#8804;10 &#186;C) behavioral assay in Drosophila. Using this tool and assay, we show an investigator how to qualitatively and quantitatively assess cold nociceptive behaviors. This can be done under normal/healthy culture conditions, or presumably in the context of disease, injury or sensitization. Further, this assay can be applied to larvae selected for desired genotypes, which might impact thermosensation, pain, or nociceptive sensitization. Given that pain is a highly conserved process, using this assay to further study&#160;thermal nociception will likely glean important understanding of pain processes in other species, including vertebrates.

Novel Assay for Cold Nociception in Drosophila Larvae

Here we demonstrate a novel assay to study cold nociception in Drosophila larvae. This assay utilizes a custom-built Peltier probe capable of applying a focal noxious cold stimulus and results in quantifiable cold-specific behaviors. This technique will allow further cellular and molecular dissection of cold nociception.

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory ...