Name: a rapid food-preference assay in drosophila
Uploaded: 2021-02-11T12:30:00
Description: Watch this Scientific Journal Video about A Rapid Food-Preference Assay in Drosophila at JoVE.com

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 le...

<ol>
	<li>Jiao, Y., Moon, S. J., Montell, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Drosophila+gustatory+receptor+required+for+the+responses+to+sucrose,+glucose,+and+maltose+identified+by+mRNA+tagging.">A Drosophila gustatory receptor required for the responses to sucrose, glucose, and maltose identified by mRNA tagging.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (35), 14110-14115 (2007).</li><li>Dahanukar, A., Foster, K., van der Goes van Naters, W. M., Carlson, J. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amino+acids,+taste+circuits,+and+feeding+behavior+in+Drosophila:+towards+understanding+the+psychology+of+feeding+in+flies+and+man.">Amino acids, taste circuits, and feeding behavior in Drosophila: towards understanding the psychology of feeding in flies and man.</a> Journal of Endocrinology. 192 (3), 467-472 (2007).</li><li>Zhang, Y. V., Ni, J., Montell, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+basis+for+attractive+salt-taste+coding+in+Drosophila.">The molecular basis for attractive salt-taste coding in Drosophila.</a> Science. 340 (6138), 1334-1338 (2013).</li><li>Weiss, L. A., Dahanukar, A., Kwon, J. Y., Banerjee, D., Carlson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+and+cellular+basis+of+bitter+taste+in+Drosophila.">The molecular and cellular basis of bitter taste in Drosophila.</a> Neuron. 69 (2), 258-272 (2011).</li><li>Moon, S. J., Kottgen, M., Jiao, Y., Xu, H., Montell, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+taste+receptor+required+for+the+caffeine+response+in+vivo.">A taste receptor required for the caffeine response in vivo.</a> Current Biology. 16 (18), 1812-1817 (2006).</li><li>Dweck, H. K. M., Carlson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+logic+and+evolution+of+bitter+taste+in+Drosophila.">Molecular logic and evolution of bitter taste in Drosophila.</a> Current Biology. 30 (1), 17-30 (2020).</li><li>Lee, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gustatory+receptors+required+for+avoiding+the+insecticide+L-canavanine.">Gustatory receptors required for avoiding the insecticide L-canavanine.</a> Journal of Neuroscience. 32 (4), 1429-1435 (2012).</li><li>Montell, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+taste+of+the+Drosophila+gustatory+receptors.">A taste of the Drosophila gustatory receptors.</a> Current Opinion in Neurobiology. 19 (4), 345-353 (2009).</li><li>Clyne, P. J., Warr, C. G., Carlson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candidate+taste+receptors+in+Drosophila.">Candidate taste receptors in Drosophila.</a> Science. 287 (5459), 1830-1834 (2000).</li><li>Liman, E. R., Zhang, Y. V., Montell, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+coding+of+taste.">Peripheral coding of taste.</a> Neuron. 81 (5), 984-1000 (2014).</li><li>Scott, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gustatory+processing+in+Drosophila+melanogaster.">Gustatory processing in Drosophila melanogaster.</a> Annual Review of Entomology. 63, 15-30 (2018).</li><li>Freeman, E. G., Dahanukar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+neurobiology+of+Drosophila+taste.">Molecular neurobiology of Drosophila taste.</a> Current Opinion in Neurobiology. 34, 140-148 (2015).</li><li>Tanimura, T., Isono, K., Yamamoto, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taste+sensitivity+to+trehalose+and+its+alteration+by+gene+dosage+in+Drosophila+melanogaster.">Taste sensitivity to trehalose and its alteration by gene dosage in Drosophila melanogaster.</a> Genetics. 119 (2), 399-406 (1988).</li><li>Zhang, Y. V., Raghuwanshi, R. P., Shen, W. L., Montell, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Food+experience-induced+taste+desensitization+modulated+by+the+Drosophila+TRPL+channel.">Food experience-induced taste desensitization modulated by the Drosophila TRPL channel.</a> Nature Neuroscience. 16 (10), 1468-1476 (2013).</li><li>Bantel, A. P., Tessier, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taste+preference+assay+for+adult+Drosophila.">Taste preference assay for adult Drosophila.</a> Journal of Visualized Experiments: JoVE. (115), e54403 (2016).</li><li>Shiraiwa, T., Carlson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proboscis+extension+response+(PER)+assay+in+Drosophila.">Proboscis extension response (PER) assay in Drosophila.</a> Journal of Visualized Experiments: JoVE. (3), e193 (2007).</li><li>Ja, W. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prandiology+of+Drosophila+and+the+CAFE+assay.">Prandiology of Drosophila and the CAFE assay.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (20), 8253-8256 (2007).</li><li>Diegelmann, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+CApillary+FEeder+assay+measures+food+intake+in+Drosophila+melanogaster.">The CApillary FEeder assay measures food intake in Drosophila melanogaster.</a> Journal of Visualized Experiments: JoVE. (121), e55024 (2017).</li><li>Ro, J., Harvanek, Z. M., Pletcher, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FLIC:+high-throughput,+continuous+analysis+of+feeding+behaviors+in+Drosophila.">FLIC: high-throughput, continuous analysis of feeding behaviors in Drosophila.</a> PLoS One. 9 (6), 101107 (2014).</li><li>Yoshihara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+recording+of+calcium+signals+from+identified+neurons+and+feeding+behavior+of+Drosophila+melanogaster.">Simultaneous recording of calcium signals from identified neurons and feeding behavior of Drosophila melanogaster.</a> Journal of Visualized Experiments: JoVE. (62), e3625 (2012).</li><li>Deshpande, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+Drosophila+food+intake:+comparative+analysis+of+current+methodology.">Quantifying Drosophila food intake: comparative analysis of current methodology.</a> Nature Methods. 11 (5), 535-540 (2014).</li><li>Yapici, N., Cohn, R., Schusterreiter, C., Ruta, V., Vosshall, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+taste+circuit+that+regulates+ingestion+by+integrating+food+and+hunger+signals.">A taste circuit that regulates ingestion by integrating food and hunger signals.</a> Cell. 165 (3), 715-729 (2016).</li><li>Jiang, L., Zhan, Y., Zhu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+quantitative+food-intake+assays+and+forcibly+activating+neurons+to+study+appetite+in+Drosophila.">Combining quantitative food-intake assays and forcibly activating neurons to study appetite in Drosophila.</a> Journal of Visualized Experiments: JoVE. (134), e56900 (2018).</li><li>Moon, S. J., Lee, Y., Jiao, Y., Montell, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Drosophila+gustatory+receptor+essential+for+aversive+taste+and+inhibiting+male-to-male+courtship.">A Drosophila gustatory receptor essential for aversive taste and inhibiting male-to-male courtship.</a> Current Biology. 19 (19), 1623-1627 (2009).</li><li>Zhang, Y. V., Aikin, T. J., Li, Z., Montell, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+basis+of+food+texture+sensation+in+Drosophila.">The basis of food texture sensation in Drosophila.</a> Neuron. 91 (4), 863-877 (2016).</li><li>Itskov, P. 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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 ...

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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 ...

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

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 ...

Taste Preference Assay for Adult Drosophila

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 ...

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

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 ...

Novel Assay for Cold Nociception in Drosophila Larvae

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 ...