We thank Ravikumar Boyapati for helping with setting up the arena. This work is funded by Wellcome trust DBT Intermediate India Alliance grant (Grant number IA/I/15/2/502074) to P.K. M.S. was funded by a fellowship by the Indian Council of Medical Research (ICMR). A.B. was funded by NCBS-TIFR institutional funds (No. 12P4167) and the Department of Atomic Energy, Government of India (No. 12-R&#38;amp; D-TFR-5.04-0800 and 12-R&#38;amp; D-TFR-5.04-0900).

Adult male flies of the Drosophila melanogaster Canton-S (CS) wild-type strain were&#160;used for the present study.
1. Experimental preparation
<ol>
	<li>Fly rearing
	<ol>
		<li>Collect adult male flies emerging within a 12 h period and maintain them on standard fly media (prepared in house) for 48 h at 25 &#176;C with 75% relative humidity in a 12 h light/dark cycle. 
		NOTE: The Drosophila media composition used was (for 1 L of media) corn flour (80 g), D-glucose (20 g), sugar (40 g), agar (8 g), yeast powder (15 g), propionic acid (4 mL), methyl 4-hydroxybenzoate (1.25 g in 3 mL of ethanol), and orthophosphoric acid (600 &#181;L) (see Table of Materials). The initial feeding period ensures that the flies have access to sufficient food and nutrients before the starvation phase. While both male and female flies elicit the search behavior, but starvation time is more consistent among male flies. Additionally, female flies change their feeding preference after mating11.</li>
	</ol>
	</li>
	<li>Starvation procedure
	<ol>
		<li>After the feeding period, starve the flies of food but with access to water. 
		NOTE: To standardize the hunger state across trials and strains, it is suggested to determine the duration of 90% survival of the population under food-starved conditions. Based on this result, CS flies were starved for 28 h (Figure 1A) as circadian rhythms and other factors can impact the search behavior12. Circadian changes of fly searching activities at different times of a day were also measured (Figure 1B). We noticed reduced relative search activity at night compared to light phase in flies.</li>
		<li>Calculate the food-starvation tolerance by depriving two- day old flies of food. Place 15-20 flies in a vial with soaked tissue paper at the bottom. This serves as a substrate and ensure that the flies have access to water throughout the starvation period.</li>
		<li>Count the number of flies during the food-starvation period at regular intervals of 1 h. The duration at which 90% of the starved flies survived was used as the starvation period (Figure 1A). Use multiple replicates (3-4 for each strain) to minimize the impact of individual variations and provide a more reliable assessment of starvation tolerance. 
		NOTE: The food-starvation tolerance of each strain was determined in order to establish a standardized hunger state among different strains and experiments. By counting the surviving flies at these intervals, one can monitor the survival rate over time and determine how long each strain could tolerate food deprivation before succumbing to starvation.</li>
	</ol>
	</li>
	<li>Procedure for recording the behavior
	<ol>
		<li>Transfer individual starved flies in small tubes (Figure 2A). Do this in batches of 5-6 flies to minimize isolation time. By testing single flies, isolating each fly, any observed changes or actions can be attributed to the specific fly being observed, rather than being influenced by interactions with other flies.</li>
		<li>Use 90 mm Petri dishes as an arena for behavioral assays (bigger petri dishes can be used. There was no difference in behavior in Petri dishes with larger diameter). Illuminate the arena from the bottom by a panel of surface-mounted cool white LEDs (Figure 2B,C). To maintain a uniform visual environment and minimize external distraction, surround the experimental arena by a white polyvinyl chloride pipe (51.5 mm height, 114 mm inner diameter). 
		NOTE: This pipe acts as a barrier, preventing any visual stimuli from outside the arena from influencing the fly&#39;s behavior. By reducing external distractions, one can focus solely on the fly&#39;s interaction with the food source, maintaining consistency throughout the experiment.</li>
		<li>Use light intensity of 320 lux at the center of the arena.</li>
		<li>Position 0.2 &#181;L sugar solution in the center of the arena. 500 mM sucrose solution was used in the reported experiments but this can be varied. Introduce the fly to the arena using a 2 mL micro centrifuge tube (inner diameter 8.7 mm, length reduced to 5 mm by stuffing cotton at the bottom; Figure 2A) housing a single fly inverted over the sugar drop.</li>
		<li>Once the fly starts ingesting the droplet, remove the fly container, giving the fly unrestricted access to the food source.</li>
		<li>Film the 2D position of the arena with an overhead camera. 
		NOTE: Flea3 (Point Grey, 1214 mm lens, see Table of Materials) was used for the present study and recorded at 40 frames per second (fps). However, any camera that provides good contrast with the background can be used. One can record at 30-60 fps, depending on the nature of the experiment. Record in .avi format since it is compatible with the tracking software.</li>
		<li>Record the trial until the time the fly escaped from the arena. The flies were freely walking and there was no lid on the arena.</li>
		<li>By allowing the fly to decide when to stop searching, fly away, or walk to the periphery of the arena, observe the fly&#39;s natural behavior and food search strategy.Wipe the Petri dishes with 70% ethanol between the trials and dry them completely or else use a new Petri dish. 
		NOTE: It is important to conduct all experiments between 2 h and 6 h after lights are on when the flies exhibit consistently high activity levels. This time frame ensures that the flies are in an active state, maximizing the chances of observing their natural food searching behavior and reducing the impact of other factors that may affect their behavior, such as circadian rhythms. The behavioral set-up was housed inside a temperature and humidity- controlled room. The arena was&#160;placed on a vibration- free table. This experimental setup involved isolating and testing individual flies for their response to sugar. A sugar drop was provided to the starved flies and their behavior was video recorded (Figure 2B,C, Video 1). The experiments were conducted during a specific time frame when the flies exhibited consistent high activity levels.</li>
	</ol>
	</li>
	<li>Analyze the trajectories following step 2 to determine the search behavior.</li>
</ol>
2. Analysis of trajectories for local search
<ol>
	<li>Analyze the recorded videos using Ctrax software11 (see Table of Materials). 
	NOTE: The software tracks and converts the fly&#39;s position in the video into x, y coordinates, enabling precise tracking and analysis of its movements. Refer to Supplementary File 1 for details on how to use Ctrax.</li>
	<li>Divide the trajectories into two phases: the initial feeding phase and the search phase. Define the end of feeding and start of walking as flies moving at a speed &#62;4 mm s-1 in three consecutive frames. 
	NOTE: After the feeding was over, the rest of the trajectory was used as the search response of the flies. This study used VirtualDub (see Table of Materials) to remove the feeding phase from the videos before tracking.</li>
	<li>Quantify the search by the following parameters:
	<ol>
		<li>Path length: This parameter represents the distance walked by the fly from its starting point during the food search (in mm).</li>
		<li>Stay time: The time spent by the flies walking during the search (in s). It indicates the duration of the search and fly&#39;s persistence in searching for food.</li>
		<li>Meander: Calculate this as a ratio by dividing the beeline (the distance between the first and the last point of the path) of the path to the total path length, and subtracting from 1. High values of meander indicate more tortuosity in the trajectory.</li>
		<li>Number of returns: Use the developed algorithm to identify and count the number of returns using two concentric circles. 
		NOTE: An inner circle indicating the origin of search, Rin (2.5 mm) and the outer circle indicating the minimum distance Rout (4 mm) that the fly had to move away from the origin. A return was defined as a movement out of the outer circle (Rout) and then coming back into the inner circle (Rin).</li>
		<li>Activity rate: Expressed as a percentage, calculate the activity rate by determining the time during food searching when the fly&#39;s walking speed exceeds 2 mm/s. This parameter reflects the fly&#39;s level of activity and engagement in the food search. It distinguishes active foragers from less active individuals. 
		NOTE: MATLAB and Python were used in the present study for further analysis of the trajectories. The scripts can be accessed here: https://github.com/eagermeagre/sugar_elicited_search. The analysis procedure involves using the Ctrax software to track the fly&#39;s movements and determine parameters such as the duration of food search, total path length, search time, meander, number of returns and activity rate. These parameters provide valuable insights into the fly&#39;s behavior and foraging efficiency during the recorded food searching experiments.</li>
	</ol>
	</li>
</ol>

Analysis of Sugar-Induced Foraging Behavior in Drosophila

<ol>
	<li>Jander, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecological+aspects+of+spatial+orientation.">Ecological aspects of spatial orientation.</a> Annu Rev Ecol Evol Syst. 6 (1), 171-188 (1975).</li><li>Dethier, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Communication+by+insects:+Physiology+of+dancing.">Communication by insects: Physiology of dancing.</a> Science. 125 (3243), 331-336 (1957).</li><li>White, J., Tobin, T. R., Bell, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+search+in+the+housefly+Musca+domestica+after+feeding+on+sucrose.">Local search in the housefly Musca domestica after feeding on sucrose.</a> J. Insect Physiol. 30 (6), 477-487 (1984).</li><li>Bell, W. J., Cathy, T., Roggero, R. J., Kipp, L. R., Tobin, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sucrose+stimulated+searching+behaviour+of+Drosophila+melanogaster+in+a+uniform+habitat:+modulation+by+period+of+deprivation.">Sucrose stimulated searching behaviour of Drosophila melanogaster in a uniform habitat: modulation by period of deprivation.</a> Animal Behav. 33, 436-448 (1985).</li><li>Dethier, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscopic+Brains.">Microscopic Brains.</a> Science. 143 (3611), 1138-1145 (1964).</li><li>Bell, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Searching+behavior+patterns+in+insects.">Searching behavior patterns in insects.</a> Annu Rev Entomol. 35 (1), 447-467 (1990).</li><li>Brockmann, 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=Sugar+intake+elicits+intelligent+searching+behavior+in+flies+and+honey+bees.">Sugar intake elicits intelligent searching behavior in flies and honey bees.</a> Front Behav Neurosci. 12, 280 (2018).</li><li>Shakeel, M., Brockmann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+effects+of+sugar+intake+on+fly+local+search+and+honey+bee+dance+behaviour.">Temporal effects of sugar intake on fly local search and honey bee dance behaviour.</a> J Comp Physiol A Neuroethol Sens Neural Behav Physiol. , (2023).</li><li>Murata, S., Brockmann, A., 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=Pharyngeal+stimulation+with+sugar+triggers+local+searching+behavior+in+Drosophila.">Pharyngeal stimulation with sugar triggers local searching behavior in Drosophila.</a> J Exp Biol. 220 (Pt 8), 3231-3237 (2017).</li><li>Nishijo, H., Ono, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+mechanisms+of+feeding+behavior+and+its+disorders+new+insights+into+metabolic+syndrome.">Neural mechanisms of feeding behavior and its disorders new insights into metabolic syndrome.</a> IntechOpen. , (2021).</li><li>Carvalho, G. B., Kapahi, P., Anderson, D. J., Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allocrine+modulation+of+feeding+behavior+by+the+sex+peptide+of+Drosophila.">Allocrine modulation of feeding behavior by the sex peptide of Drosophila.</a> Curr Biol. 16 (7), 692-696 (2006).</li><li>Xu, K., Zheng, X., Sehgal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+feeding+and+metabolism+by+neuronal+and+peripheral+clocks+in+Drosophila.">Regulation of feeding and metabolism by neuronal and peripheral clocks in Drosophila.</a> Cell Metab. 8 (4), 289-300 (2008).</li><li>Branson, K., Robie, A. A., Bender, J., Perona, P., Dickinson, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+ethomics+in+large+groups+of+Drosophila.">High-throughput ethomics in large groups of Drosophila.</a> Nat Methods. 6 (6), 451-457 (2009).</li><li>Corfas, R. A., Sharma, T., Dickinson, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+food-sensing+neurons+trigger+idiothetic+local+search+in+Drosophila.">Diverse food-sensing neurons trigger idiothetic local search in Drosophila.</a> Curr Biol. 29 (10), 1660-1668.e4 (2019).</li><li>Behbahani, A. H., Palmer, E. H., Corfas, R. A., Dickinson, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+re-zero+their+path+integrator+at+the+center+of+a+fictive+food+patch.">Drosophila re-zero their path integrator at the center of a fictive food patch.</a> Curr Biol. 31 (20), 4534-4546.e5 (2021).</li><li>Titova, A. V., 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=Displacement+experiments+provide+evidence+for+path+integration+in+Drosophila.">Displacement experiments provide evidence for path integration in Drosophila.</a> J Exp Biol. 226 (12), jeb245289 (2023).</li><li>Stocker, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+organization+of+the+chemosensory+system+in+Drosophila+melanogaster:+a+review.">The organization of the chemosensory system in Drosophila melanogaster: a review.</a> Cell Tissue Res. 275 (1), 3-26 (1994).</li><li>LeDue, E. E., Chen, Y. C., Jung, A. Y., Dahanukar, A., Gordon, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharyngeal+sense+organs+drive+robust+sugar+consumption+in+Drosophila.">Pharyngeal sense organs drive robust sugar consumption in Drosophila.</a> Nat Commun. 6, 6667 (2015).</li><li>Gelperin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abdominal+sensory+neurons+providing+negative+feedback+to+the+feeding+behavior+of+the+blowfly.">Abdominal sensory neurons providing negative feedback to the feeding behavior of the blowfly.</a> Zeitschrift f&#252;r vergleichende Physiologie. 72 (1), 17-31 (1971).</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><li>DeAngelis, B. D., Zavatone-Veth, J. A., Gonzalez-Suarez, A. D., Clark, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporally+precise+optogenetic+activation+of+sensory+neurons+in+freely+walking+Drosophila.">Spatiotemporally precise optogenetic activation of sensory neurons in freely walking Drosophila.</a> Elife. 9, e54183 (2020).</li><li>Grover, D., Katsuki, T., Li, J., Dawkins, T. J., Greenspan, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+brain+activity+during+complex+social+behaviors+in+Drosophila+with+Flyception2.">Imaging brain activity during complex social behaviors in Drosophila with Flyception2.</a> Nat Commun. 11 (1), 623 (2020).</li></ol>

The author(s) declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

The current study introduces a straightforward paradigm for investigating sugar-elicited search behavior in Drosophila, first described by Dethier2. This innate behavior allows the flies to engage in a local search for additional food resources following the encounter of a food reward. The most crucial aspect of the experimental protocol involves appropriately motivating the flies. Firstly, the flies must be in a state of hunger, having been deprived of food while still having access to water, to ensure sugar ingestion. In order to have the uniform hunger state across experimental trials the duration at which 90% of the population survives was used as the starvation period. Crucially, the induction of a search response post-feeding necessitates the provision of a food stimulus that is of sufficient quality but not enough to fully satiate the flies. Therefore, standardizing the concentration and amount of sugar, and starvation duration might be time-consuming but is imperative for robust and reliable behavior.
In this study, a 500 mM, 0.2 &#181;L sucrose solution was employed as the stimulus for starved flies. Sugar intake evokes a characteristic local search behavior characterized by increased turning behavior and frequent returns to the location of the sugar drop (Video 1). Conversely, hungry flies that are not provided with any sugar fail to exhibit a search response. Notably, all the behavior-related parameters including path length, stay time, meander, and number of returns were significantly lower in unfed flies. We have previously demonstrated that the ingestion of water alone does not elicit a search response9.
This setup offers a cost-effective and low-maintenance approach for studying this innate behavior. While a backlit arena is used in this study, top lighting can also be employed as long as there is sufficient contrast between the fly and the background. The tracking software used relies on the detection of fly movement against a static background13. The camera and resolution settings can be adjusted based on the specific scale of behavior under investigation. Importantly, this methodology enables the study of various components of foraging behavior, including sensory cue attention during foraging, food- engagement and feeding, locomotor control of search, and decision-making processes associated with exploitation and exploration, among others. Moreover, this paradigm facilitates the investigation of local search, a behavior that is commonly observed across diverse taxa in various ecological contexts6. Studying this behavior in Drosophila, opens avenues for scientific inquiry aimed at understanding the neural pathways involved in foraging. We have studied local search in honey bees and have shown that the behavior bears resemblance to flies7,8.
Recent studies have demonstrated that local search behavior can be triggered by optogenetic activation of various sugar sensory neurons in flies14,15,16. However, it remains unclear to what extent the local searches observed in these studies accurately represent the natural behavior of flies in response to actual sugar intake. Feeding behavior is tightly regulated in flies, and these findings indicate that the activation of pharyngeal sugar receptors initiates the search behavior. Tarsal taste sensilla are responsible for detecting sugar and inducing the proboscis extension reflex, while pharyngeal taste neurons determine whether feeding should proceed17,18. Once ingested, the sugar solution travels through the esophagus to the proventriculus and enters the crop, with its expansion monitored by a recurrent nerve19. Additionally, it is worth noting that some of the aforementioned studies involved the harnessing or confinement of flies, whereas this method allows the animals to freely walk throughout the experiment. The flies in our experiments were sufficiently motivated to stay and search within the arena without the imposition of a lid.
Understanding the intricate interplay between neural pathways, genetic factors, and environmental cues that govern search behavior in flies can shed light on the fundamental principles of information processing, learning, and memory formation. Furthermore, dysregulation of foraging behavior has been implicated in various human disorders, including eating disorders and obesity. The extensive array of neurogenetic tools available in Drosophila provides a valuable resource for investigating the sugar-elicited search behavior and unravelling the neural and genetic mechanisms underlying foraging. In combination with optogenetic manipulation and functional imaging, this paradigm presents a powerful and promising approach20,21,22. However, modifying the setup for real-time manipulation of neuronal activity with optogenetics might be challenging. To monitor the neuronal activity in the brain while a fly is performing the searching behavior a different set-up will be needed such as the tethered fly on a tread ball. Many aspects of foraging behavior, such as feeding regulation and decision-making processes, are highly conserved across species. Therefore, insights gained from studying the neural mechanisms of foraging in flies can provide valuable insights into similar processes in other organisms, including humans.

Food-seeking and foraging behavior is a fundamental survival strategy exhibited by organisms across taxa. Two types of foraging behaviors have been identified in insects: hunger-induced search for food and post-meal local search1. When hungry, insects rely on sensory cues to locate food sources. Upon encountering and consuming a small food patch, they initiate a local search behavior characterized by convoluted paths and circling around the food location.
Sugar-elicited search behavior, a particular form of local search, was first studied over 60 years ago, by American biologist Vincent Dethier in blowflies2. When starved, flies are presented with a small amount of sugar such that it does not satiate them, they begin a local search. Typical search behavior is characterized by a highly tortuous walk with low locomotory and high turning rate and making returns to the location of the sugar drop. Subsequent studies had investigated this behavior in house flies and fruit flies3,4. The initiation, intensity, and duration of the search is regulated by the internal state of the animal (e.g., deprivation and motivation) as well as external factors such as resource availability and quality1,5,6.
Advancements in tracking technologies have provided researchers with valuable tools to capture and analyze behavior within controlled arenas. Here, we present a behavioral paradigm for tracking freely walking flies after sugar ingestion. This simple setup enables the study of sugar-elicited search behavior in Drosophila by capturing and analyzing the fly&#39;s motion in response to concentrated sugar solution provided in an arena. Employing advanced tracking technology and data analysis techniques, the locomotor patterns, spatial exploration, and response dynamics to sugar stimuli have been successfully quantified.
Using this assay, it has been experimentally demonstrated that the sugar-elicited search involves the use of path integration and can be spatially-temporally separated from sugar intake7,8. Furthermore, it has been shown that the behavior can be triggered by the activation of pharyngeal taste neurons9. Recent results show that sugar stimulus is not an innate releasing mechanism but also modulatory, and controls the initiation of the behavior temporally8. Using this paradigm, we have also studied this behavior in honey bees (Apis mellifera)7,8.
The eventual aim of this research is to unravel neural circuits and novel genetic components involved in regulating search behavior through targeted genetic manipulations and neuroimaging techniques. Food search behaviors have proven to be highly effective experimental paradigms for studying navigation and spatial memory in insects. These behaviors provide a unique opportunity to investigate the sensory perception, decision-making processes, and motor coordination involved in the search for rewarding food sources in flies. Additionally, the findings from these studies have broader implications for understanding feeding behaviors in other organisms, including humans, as many fundamental genetic and neural mechanisms are evolutionarily conserved. Dysregulation of feeding behaviors is associated with various neurological and metabolic disorders10. Therefore, the neural and genetic mechanisms underlying search behavior in flies may offer new avenues for understanding and potentially addressing these complex human health challenges.

<table><tbody><tr><td>2 mL Eppendorf tube</td><td>Sigma Aldrich</td><td>BR780546</td><td>Used to introduce the fly to the sugar drop</td></tr><tr><td>Agar</td><td>SRL</td><td>9002-18-0</td><td></td></tr><tr><td>Azure lens</td><td>https://www.rmaelectronics.com/azure-photonics-azure-1214mm/</td><td></td><td></td></tr><tr><td>Camera</td><td>Logicool, Japan</td><td></td><td></td></tr><tr><td>Corn flour</td><td>locally available</td><td></td><td></td></tr><tr><td>Ctrax software</td><td>https://ctrax.sourceforge.net/</td><td></td><td></td></tr><tr><td>D-glucose</td><td>SRL</td><td>50-99-7</td><td></td></tr><tr><td>Flea3</td><td>Sony</td><td></td><td>https://www.flir.com/products/flea3-usb3/?vertical=machine+vision&amp;amp;segment=iis</td></tr><tr><td>glass tube</td><td>Borosil</td><td></td><td>Used to house the flies individually&amp;nbsp;</td></tr><tr><td>Kimwipe</td><td>Kimberly-Clark</td><td>34155</td><td>Used to provide access to water for flies during food starvation</td></tr><tr><td>LED light panel</td><td></td><td></td><td>custom-made in the workshop</td></tr><tr><td>Light Meter</td><td>TENMARS&amp;nbsp;</td><td>&amp;nbsp;TM-203</td><td></td></tr><tr><td>Methyl 4-hydroxybenzoate</td><td>Fisher Scientific</td><td>99-76-3</td><td></td></tr><tr><td>Orthophosphotic acid</td><td>SRL</td><td>7664-38-2&amp;nbsp;</td><td></td></tr><tr><td>Petri dish (90 mm)</td><td>Tarsons</td><td>460090</td><td></td></tr><tr><td>Propionic acid</td><td>SRL</td><td>79-09-4</td><td></td></tr><tr><td>Sucrose</td><td>Qualigens</td><td>Q28105</td><td></td></tr><tr><td>Sugar</td><td>locally available</td><td></td><td></td></tr><tr><td>VirtualDub&amp;nbsp;</td><td>https://www.virtualdub.org/</td><td></td><td></td></tr><tr><td>White polyvinyl chloride pipe (67 mm inner diameter &amp;times; 100 mm height)</td><td></td><td></td><td>custom-made in the workshop</td></tr><tr><td>Yeast powder</td><td>SRL</td><td>REF-34266</td><td></td></tr></tbody></table>

tracking sugar-elicited local searching behavior in drosophila

Foraging behavior is essential for the survival of organisms as it enables them to locate and acquire essential food resources. In Drosophila, hunger triggers a distinct search behavior following the consumption of small quantities of a sugar solution. This report presents a simple experimental setup to study sugar-elicited search behavior with the aim of uncovering the underlying mechanisms. Minute quantities of concentrated sugar solution elicit sustained searching behavior in flies. The involvement of path integration in this behavior has been established, as flies utilize their trajectory to return to the sugar location. The most recent findings provide evidence of temporal modulation in the initiation and intensity of the search behavior after sugar intake. We have also used this setup for artificial activation of specific taste-receptor neurons in the pharynx, which elicits the search behavior. The Drosophila neurogenetic toolkit offers a diverse array of tools and techniques that can be combined with the sugar-elicited search behavior paradigm to study the neural and genetic mechanisms underlying foraging. Understanding the neural basis of hunger-driven searching behavior in flies contributes to the field of neurobiology as a whole, offering insights into the regulatory mechanisms that govern feeding behaviors not only in other organisms but also in humans.

The flies needed to be starved for the period estimated by the food-starvation tolerance and the response to sugar was individually tested (Figure 1A and Figure 2A). The behavior was recorded inside a temperature and humidity-controlled room. 0.2 &#181;L of 500 mM sucrose solution was used for the reported experiments. The sugar drop was positioned in the center of the arena and the flies were introduced to the sugar (Figure 2B). The behavior was recorded until flies escaped from the arena. The videos were analyzed to extract x,y coordinates and trajectories of the flies. Several parameters were used to quantify the behavioral response: path length, stay time, meander, number of returns and activity rate.
Sugar intake in starved flies leads to a local search with a meandering path and loops (Figure 3A,C). As a negative control, starved flies were recorded that were not presented with sugar8. These flies when introduced to and empty arena, did not initiate a search and escaped the arena (Figure 3B,D). This group is referred as unfed flies. The parameters of search: path length, stay time, meander and number of returns were significantly lower in unfed flies compared to the flies that were given a sugar drop (Figure 4A-D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65955/65955fig01.jpg" /> 
Figure 1: Starvation curves of wildtype strain CS. (A)&#160;Starvation curve showing survival rate for wild-type CS male and female flies. (B) Circadian changes of fly searching activities at different times of a day. Error bars represent S.E.M. (n = 10). <a href="https://www.jove.com/files/ftp_upload/65955/65955fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65955/65955fig02.jpg" /> 
Figure 2: Experimental setup including starvation tolerance calculation, behavior recording procedures, and trajectory analysis. (A) Food-starvation tolerance was determined to establish a standardized hunger state. For behavior recording, individual flies were isolated in small tubes before the experiment. The behavioral arena, a 90 mm Petri dish, was uniformly illuminated from the bottom. A drop of sugar solution was placed in the center and flies were given unrestricted access to the food source. (B) The behavior was recorded until flies escaped from the arena. Video analysis used Ctrax software for tracking and MATLAB/Python for trajectory analysis. (C) Photograph of the experimental setup. <a href="https://www.jove.com/files/ftp_upload/65955/65955fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65955/65955fig03.jpg" /> 
Figure 3: Necessity of the sugar intake for initiating local search. (A) Individual trajectories of flies fed 500 mM, 0.2 &#181;L sugar solution. (B) Individual trajectories of flies that were given no sugar. (C) Overlay of search trajectories flies from the control group (n = 11). (D) Overlay of the paths of the flies (n = 11) which were given no sugar. All the trajectories are normalized to the starting point of walking. This figure is adapted from Shakeel and Brockmann8. <a href="https://www.jove.com/files/ftp_upload/65955/65955fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65955/65955fig04.jpg" /> 
Figure 4: Reduction of behavioral parameters in unfed flies. (A-D) Path length, stay time, meander and number of returns were smaller for hungry flies that were not given sugar reward compared to control flies that were given stimulated with sugar. **p &#60; 0.001, ***p &#60; 0.0001, ****p &#60; 0.00001, Wilcoxon Rank Sum Test. This figure is adapted from Shakeel and Brockmann8. <a href="https://www.jove.com/files/ftp_upload/65955/65955fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Video 1: Search behavior of a fly with real-time trajectory of the path. <a href="https://www.jove.com/files/ftp_upload/65955/Video 1.zip" target="_blank">Please click here to download this Video.</a>
Supplementary File 1: Step-wise instructions for tracking the video file using CTRAX. <a href="https://www.jove.com/files/ftp_upload/65955/Manal et al supplementary 1 ctrax_instructions.zip" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Author Spotlight: Exploring Behavioral Pathways Through Cross-Species Insights in Foraging and Communication at JoVE.com

Author Spotlight: Exploring Behavioral Pathways Through Cross-Species Insights in Foraging and Communication

This protocol describes a behavioral assay for recording sugar-elicited search behavior using Drosophila melanogaster. The assay can be utilized to study feeding and foraging-related behaviors, as well as the underlying neuronal mechanisms.

Tracking Sugar-Elicited Local Searching Behavior in Drosophila

Foraging behavior is essential for the survival of organisms as it enables them to locate and acquire essential food resources. In Drosophila, hunger ...

tracking-sugar-elicited-local-searching-behavior-in-drosophila

Research

JoVE Journal

Behavior

1.0K Views.  National Centre for Biological Sciences. This protocol describes a behavioral assay for recording sugar-elicited search behavior using Drosophila melanogaster. The assay can be utilized to study feeding and foraging-related behaviors, as well as the underlying neuronal mechanisms.

Video: Author Spotlight: Exploring Behavioral Pathways Through Cross-Species Insights in Foraging and Communication

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral paradigm, the experimenter usually assumes that the rapid diffusion of odorant molecules from the source leads to the creation of a stable gradient in the dish. To establish a quantitative correlation between sensory inputs and behavioral responses, it is necessary to achieve a more thorough characterization of the odorant stimulus conditions. In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral ...

Biology

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

Neuroscience

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

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 ...

A Low-cost Method for Analyzing Seizure-like Activity and Movement in Drosophila

Video tracking systems have been used widely to analyze Drosophila melanogaster movement and detect various abnormalities in locomotive behavior. While these systems can provide a wealth of behavioral information, the cost and complexity of these systems can be prohibitive for many labs. We have developed a low-cost assay for measuring locomotive behavior and seizure movement in D. melanogaster. The system uses a web-cam to capture images that can be processed using a combination of inexpensive and free software to track the distance moved, the average velocity of movement and the duration of movement during a specified time-span. To demonstrate the utility of this system, we examined a group of D. melanogaster mutants, the Bang-sensitive (BS) paralytics, which are 3-10 times more susceptible to seizure-like activity (SLA) than wild type flies. Using this novel system, we were able to detect that the BS mutant bang senseless (bss) exhibits lower levels of exploratory locomotion in a novel environment than wild type flies. In addition, the system was used to identify that the drug metformin, which is commonly used to treat type II diabetes, reduces the intensity of SLA in the BS mutants.

A Low-cost Method for Analyzing Seizure-like Activity and Movement in Drosophila

Using a webcam and a combination of free and inexpensive software programs, locomotive patterns in Drosophila melanogaster can be analyzed to detect differences in the speed, distance, and time of various motor activities.

Video tracking systems have been used widely to analyze Drosophila melanogaster movement and detect various abnormalities in locomotive behavior. While ...

A Simple Technique to Assay Locomotor Activity in Drosophila

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and diverse behavioral features. Identifying behavioral deficiency in animal models is a crucial measure of disease severity, for example, in neurodegenerative diseases where patients often experience impairments in motor function. However, with the availability of various systems to track and assess motor deficits in fly models, such as drug-treated or transgenic individuals, an economical and user-friendly system for precise evaluation from multiple angles is still lacking. A method based on the AnimalTracker application programming interface (API) is developed here, which is compatible with the Fiji image processing program, to systematically evaluate the movement activities of both adult and larval individuals from recorded video, thus allowing for the analysis of their tracking behavior. This method requires only a high-definition camera and a computer peripheral hardware integration to record and analyze behavior, making it an affordable and effective approach for screening fly models with transgenic or environmental behavioral deficiencies. Examples of behavioral tests using pharmacologically treated flies are given to show how the techniques can detect behavioral changes in both adult flies and larvae in a highly repeatable manner.

A Simple Technique to Assay Locomotor Activity in Drosophila

The present protocol assesses the locomotor activity of Drosophila by tracking and analyzing the movement of flies in a hand-made arena using open-source software Fiji, compatible with plugins to segment pixels of each frame based on high-definition video recording to calculate parameters of speed, distance, etc.

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and ...

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To complicate matters even further, the odors detected by the insect nervous system usually are not single compounds but mixtures whose composition and concentration ratios vary. This leads to an almost infinite amount of different olfactory stimuli which have to be evaluated by the nervous system.
To understand which aspects of an odor stimulus determine its evaluation by the fly, it is therefore desirable to efficiently examine odor-guided behavior towards many odorants and odor mixtures. To directly correlate behavior to neuronal activity, behavior should be quantified in a comparable time frame and under identical stimulus conditions as in neurophysiological experiments. However, many currently used olfactory bioassays in Drosophila neuroethology are rather specialized either towards efficiency or towards resolution.
Flywalk, an automated odor delivery and tracking system, bridges the gap between efficiency and resolution. It allows the determination of exactly when an odor packet stimulated a freely walking fly, and to determine the animal&#180;s dynamic behavioral reaction.

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

The automated tracking system Flywalk is used for high-resolution quantification of odor-guided behavior in Drosophila melanogaster.

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. 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 ...

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a considerable amount of information has been generated regarding ORN responses to odorants, the role of specific ORNs in driving behavioral responses remains poorly understood. Complications in behavior analyses arise due to different volatilities of odorants that activate individual ORNs, multiple ORNs activated by single odorants, and the difficulty in replicating naturally observed temporal variations in olfactory stimuli using conventional odor-delivery methods in the laboratory. Here, we describe a protocol that analyzes Drosophila larval behavior in response to simultaneous optogenetic stimulation of its ORNs. The optogenetic technology used here allows for specificity of ORN activation and precise control of temporal patterns of ORN activation. Corresponding larval movement is tracked, digitally recorded, and analyzed using custom written software. By replacing odor stimuli with light stimuli, this method allows for a more precise control of individual ORN activation in order to study its impact on larval behavior. Our method could be further extended to study the impact of second-order projection neurons (PNs) as well as local neurons (LNs) on larval behavior. This method will thus enable a comprehensive dissection of olfactory circuit function and complement studies on how olfactory neuron activities translate in to behavior responses.

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

This protocol analyzes navigational behavior of Drosophila larva in response to simultaneous optogenetic stimulation of its olfactory neurons. Light of 630 nm wavelength is used to activate individual olfactory neurons expressing a red-shifted channel rhodopsin. Larval movement is simultaneously tracked, digitally recorded, and analyzed using custom-written software.

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a ...

Navigational Behavior of Drosophila Larvae in Response to Optogenetic Stimulation of Olfactory Sensory Neurons

Source: Clark, D. A., et al. <a href="https://app.jove.com/v/57353">Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons.</a> J. Vis. Exp. (2018).
This video demonstrates a study of the navigational behavior of Drosophila larvae in response to optogenetic stimulation of olfactory sensory neurons (ORNs). Transgenic Drosophila larvae expressing light-sensitive ion channels in the ORNs are placed on agarose within a behavior arena. Optogenetic stimulation with light opens the channels, causing a cation influx that triggers the transmission of the signal to the brain, and the navigation behavior is recorded.

1. Building a Behavior Arena and Preparing Hardware to Enable Optogenetic Stimulation in the Behavior Arena

	To build a light-deprived behavior arena, ...

Tracking Sugar-Elicited Local Searching Behavior in Drosophila

Summary

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Chapters in this video

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

A Single-fly Assay for Foraging Behavior in Drosophila

A Low-cost Method for Analyzing Seizure-like Activity and Movement in Drosophila

A Simple Technique to Assay Locomotor Activity in Drosophila

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

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

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

Navigational Behavior of Drosophila Larvae in Response to Optogenetic Stimulation of Olfactory Sensory Neurons