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>

<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. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Gr+receptor+is+required+for+response+to+the+sugar+trehalose+in+taste+neurons+of+Drosophila.">A Gr receptor is required for response to the sugar trehalose in taste neurons of Drosophila.</a> Nature Neuroscience. 4 (12), 1182-1186 (2001).</li><li>Ueno, K., 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=Trehalose+sensitivity+in+Drosophila+correlates+with+mutations+in+and+expression+of+the+gustatory+receptor+gene+Gr5a.">Trehalose sensitivity in Drosophila correlates with mutations in and expression of the gustatory receptor gene Gr5a.</a> Current Biology. 11 (18), 1451-1455 (2001).</li><li>Fujii, 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=Drosophila+sugar+receptors+in+sweet+taste+perception,+olfaction,+and+internal+nutrient+sensing.">Drosophila sugar receptors in sweet taste perception, olfaction, and internal nutrient sensing.</a> Current Biology. 25 (5), 621-627 (2015).</li><li>Wang, Z., Singhvi, A., Kong, P., 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=Taste+representations+in+the+Drosophila+brain.">Taste representations in the Drosophila brain.</a> Cell. 117 (7), 981-991 (2004).</li><li>Thorne, N., Chromey, C., Bray, S., Amrein, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taste+perception+and+coding+in+Drosophila.">Taste perception and coding in Drosophila.</a> Current Biology. 14 (12), 1065-1079 (2004).</li><li>Slone, J., Daniels, J., Amrein, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sugar+receptors+in+Drosophila.">Sugar receptors in Drosophila.</a> Current Biology. 17 (20), 1809-1816 (2007).</li><li>Dus, M., 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=Nutrient+sensor+in+the+brain+directs+the+action+of+the+brain-gut+axis+in+Drosophila.">Nutrient sensor in the brain directs the action of the brain-gut axis in Drosophila.</a> Neuron. 87 (1), 139-151 (2015).</li><li>Toshima, N., Tanimura, 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+preference+for+amino+acids+is+dependent+on+internal+nutritional+state+in+Drosophila+melanogaster.">Taste preference for amino acids is dependent on internal nutritional state in Drosophila melanogaster.</a> Journal of Experimental Biology. 215 (16), 2827-2832 (2012).</li><li>Melcher, C., Bader, R., Pankratz, M. 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. M., 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=Automated+monitoring+and+quantitative+analysis+of+feeding+behaviour+in+Drosophila.">Automated monitoring and quantitative analysis of feeding behaviour in Drosophila.</a> Nature Communications. 5, 4560 (2014).</li><li>Qi, 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=A+quantitative+feeding+assay+in+adult+Drosophila+reveals+rapid+modulation+of+food+ingestion+by+its+nutritional+value.">A quantitative feeding assay in adult Drosophila reveals rapid modulation of food ingestion by its nutritional value.</a> Molecular Brain. 8, 87 (2015).</li><li>Simpson, J. H., Looger, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+imaging+and+optogenetics+in+Drosophila.">Functional imaging and optogenetics in Drosophila.</a> Genetics. 208 (4), 1291-1309 (2018).</li></ol>

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><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 CO&lt;sub&gt;2&lt;/sub&gt; 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...

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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

6.1K Views.  Monell Chemical Senses Center. Ons protocol is ontworpen om een fijne test uit te voeren voor de fruitvlieg, een krachtig modelorganisme dat het mogelijk maakt om te ontwerpen voor alle genen, receptoren, neuronen en een neuropsycholoog die betrokken zijn bij testsensatie en voedselconsumptie. Zoals mensen vliegen als voedsel dat smaakt: zoet, licht zout, lichtzure of afwijzende voedsel dat bitter, te zout of te zuur smaakt. De kracht van deze techniek komt voort uit de snelheid en het gemak.U kunt veel verschillend...

Video: A Rapid Food-Preference Assay in Drosophila

Combining Quantitative Food-intake Assays and Forcibly Activating Neurons to Study Appetite in Drosophila

Food consumption is under the tight control of the brain, which integrates the physiological status, palatability, and nutritional contents of the food, and issues commands to start or stop feeding. Deciphering the processes underlying the decision-making of timely and moderate feeding carries major implications in our understanding of physiological and psychological disorders related to feeding control. Simple, quantitative, and robust methods are required to measure the food ingestion of animals after experimental manipulation, such as forcibly increasing the activities of certain target neurons. Here, we introduced dye-labeling-based feeding assays to facilitate the neurogenetic study of feeding control in adult fruit flies. We review available feeding assays, and then describe our methods step-by-step from setup to analysis, which combine thermogenetic and optogenetic manipulation of neurons controlling feeding motivation with dye-labeled food intake assay. We also discuss the advantages and limitations of our methods, compared with other feeding assays, to help readers choose an appropriate assay.

Combining Quantitative Food-intake Assays and Forcibly Activating Neurons to Study Appetite in Drosophila

Quantitative food-intake assays with dyed food provide a robust and high-throughput means to evaluate feeding motivation. Combining the food consumption assay with thermogenetic and optogenetic screens is a powerful approach to investigate the neural circuits underlying appetite in adult Drosophila melanogaster.

Food consumption is under the tight control of the brain, which integrates the physiological status, palatability, and nutritional contents of the food, ...

Behavior

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer basic questions on how light information is processed and shared between rapid and circadian behaviors. Drosophila larvae display a stereotypical avoidance behavior when exposed to light. To investigate light dependent behaviors comparably simple light-dark preference tests can be applied. In vertebrates and arthropods the neural pathways involved in sensing and processing visual inputs partially overlap with those processing photic circadian information. The fascinating question of how the light sensing system and the circadian system interact to keep behavioral outputs coordinated remains largely unexplored. Drosophila is an impacting biological model to approach these questions, due to a small number of neurons in the brain and the availability of genetic tools for neuronal manipulation. The presented light-dark preference assay allows the investigation of a range of visual behaviors including circadian control of phototaxis.

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Here we describe a light-dark preference test for Drosophila larva. This assay provides information about innate and circadian regulation of light sensing and processing photobehavior.

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer ...

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 ...

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. ...

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 ...

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 ...

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).

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

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.

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

The CApillary FEeder (CAFE) Assay: A Method to Track Food Consumption and Preference in Drosophila

Source: Diegelmann, S., et al. <a href="https://www.jove.com/video/55024/" target="_blank">The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster</a>. J. Vis. Exp. (2017).
This video describes the CApillary FEeder, or CAFE, assay, a behavioral method that measures food intake and preference of unrestrained fruit flies. The featured protocol shows how to perform the assay with minimal evaporation from the liquid-filled capillaries used to deliver the food and track its intake.

Source: Diegelmann, S., et al. The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster. J. Vis. Exp. (2017).
This video describes the ...

A Rapid Food-Preference Assay in Drosophila

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