We thank the Duke Physics Shop, especially Phil Lewis, for helping us build the behavioral apparatus and attachments and creating the drawing. This work is funded by the National Institutes of Health under award number R01GM100027.

1. Preparing Flies to be Assayed
<ol>
	<li>Culture flies on standard molasses/cornmeal media at incubator set at 25 &#176;C and 65% humidity. Take care to not overcrowd the vials. For example, put 8 females and 6 males into a narrow food vial. 
	Note: The &#34;narrow food vial&#34; used here has an inner diameter of 2.3 cm. We typically dispensed about 10 ml of fly food into each vial. The fly food recipe we used is described here: http://flystocks.bio.indiana.edu/Fly_Work/media-recipes/molassesfood.html.</li>
	<li>2 - 3 days after females eclosion, prepare vials with fresh yeast paste as shown in Figure 1E. Make fresh yeast paste by mixing 6 g of active yeast with 10 ml of 0.5 % proprionic acid. Use a spatula to apply the yeast paste onto the sidewall of the food vials. Collect 30 - 35 eclosed females together with 20 - 25 males into the vials.
	<ol>
		<li>In general, collect the males from the same vials where the females are collected (so as to save time and effort). However, if there is concern about the fertility of males, use WT males instead. Also, yeast paste is important for stimulating egg production therefore prepare it fresh daily.</li>
	</ol>
	</li>
	<li>Maintain the collected females/males at 25 &#176;C, 65% humidity, unless the flies are temperature sensitive.</li>
	<li>After ~4 - 5 days, check the vials to see if females are ready for egg-laying experiments. They are ready when the surface of the food medium is wet from larvae actively burrowing in the food (see Figure 1E - F). 
	Note: Females typically withhold laying eggs once the food surface becomes wet and crawling with larvae. This step ensures that females would be ready to lay many eggs when they encounter substrates with desired texture (e.g., ~1% agarose).</li>
</ol>
2. Chamber Construction, Assembly, Assay Setup
<ol>
	<li>Have a machine shop build acrylic egg-laying chambers (Figure 1A - D). The engineering drawing of different pieces were shown in Supplementary Figure 1 - 3. High resolution pictures can also be found here (http://www.rebeccayang.org/pdf/chamber%20design.pdf).</li>
	<li>Insert plastic sheets into loading (top) piece of the chamber as shown in Figure 1C. This serves as the bottom surface while loading flies into individual egg-laying arenas.</li>
	<li>Anesthetize females on a CO2 pad and load them individually into each egg-laying arena. Allow ~30 min for flies to recover from the CO2 and to become acclimated to the new environment.</li>
	<li>Prepare the agarose substrates.
	<ol>
		<li>For convenience, keep a premade bottle of melted 1% agarose in a 55 &#176;C water bath.</li>
		<li>Add the desired amount of stock sucrose solution (2 M) into a 50 ml conical tube and mixing it with appropriate amount of agarose. For example, to prepare the 150 mM sucrose substrate, place 750 &#181;l of 2 M sucrose solution into the tube and then fill the tube with agarose to the 10 ml mark.</li>
		<li>Prepare the plain substrate in the same manner but add distilled water instead of sucrose solution. 
		Note: The final concentration of agarose in this protocol is slightly less than 1%. In our experience, the exact concentration of agarose does not matter so long as it is controlled to be within ~0.9 - 1.1% and that the two substrates are of the same agarose concentration.</li>
	</ol>
	</li>
	<li>Take the substrate (bottom) piece of the chamber and pipette 1,000 &#181;l of agarose substrate into each trough as seen in Figure 1D.</li>
	<li>Allow agarose to solidify for ~30 min.</li>
	<li>Once the agarose substrates and flies are ready, assemble all three pieces of the egg-laying chamber and then take out the plastic sheets.</li>
	<li>Place the chambers in fly incubators. 
	Note: Length of egg-laying experiments can vary depending on experimental needs. We typically run the experiment O/N (14 - 16 hr). Also, no significant impact of circadian timing on egg-laying preferences was observed.</li>
	<li>Anesthetize females by injecting CO2 into the chamber. Disassemble the chamber, discard the anesthetized flies into fly morgue (i.e., an empty coffee can filled with some corn oil). Take pictures of the results for recordkeeping (see Figure 2).</li>
	<li>Count the number of eggs manually and calculate preference indices for analysis. Calculate preference index as (Na - Nb)/(Na + Nb) where Na and Nb represent number of eggs on site a vs. site b, respectively.</li>
</ol>

<ol>
	<li>Perisse, E., Burke, C., Huetteroth, W., Waddell, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shocking+revelations+and+saccharin+sweetness+in+the+study+of+Drosophila+olfactory+memory.">Shocking revelations and saccharin sweetness in the study of Drosophila olfactory memory.</a> Curr. Biol. 23 (17), 752-763 (2013).</li><li>Yamamoto, N. C., Koganezawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+and+circuits+of+courtship+behaviour+in+Drosophila+males.">Genes and circuits of courtship behaviour in Drosophila males.</a> Nat. Rev. Neurosci. 14 (10), 681-692 (2013).</li><li>Zwarts, L., Versteven, M., Callaerts, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+and+neurobiology+of+aggression+in+Drosophila.">Genetics and neurobiology of aggression in Drosophila.</a> Fly. 6 (1), 35-48 (2012).</li><li>Joseph, R. M., Devineni, A. V., King, I. F., Heberlein, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oviposition+preference+for+and+positional+avoidance+of+acetic+acid+provide+a+model+for+competing+behavioral+drives+in+Drosophila.">Oviposition preference for and positional avoidance of acetic acid provide a model for competing behavioral drives in Drosophila.</a> Proc. Natl. Acad. Sci. 106 (27), 11352-11357 (2009).</li><li>Miller, P. M., Saltz, J. B., Cochrane, V. A., Marcinkowski, C. M., Mobin, R., Turner, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+variation+in+decision-making+behavior+in+Drosophila+melanogaster.">Natural variation in decision-making behavior in Drosophila melanogaster.</a> PLoS One. 6 (1), 16436 (2011).</li><li>Schwartz, N. U., Zhong, L., Bellemer, A., Tracey, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Egg-laying+decisions+in+Drosophila+are+consistent+with+foraging+costs+of+larval+progeny.">Egg-laying decisions in Drosophila are consistent with foraging costs of larval progeny.</a> PloS One. 7 (5), 37910 (2012).</li><li>Yang, C. H., Belawat, P., Hafen, E., Jan, L. Y., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+egg-laying+site+selection+a+system+to+study+simple+decision-making+processes.">Drosophila egg-laying site selection a system to study simple decision-making processes.</a> Science. 319 (5870), 1679-1683 (2008).</li><li>Kacsoh, B. Z., Lynch, Z. R., Mortimer, N. T., Schlenke, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fruit+flies+medicate+offspring+after+seeing+parasites.">Fruit flies medicate offspring after seeing parasites.</a> Science. 339 (6122), 947-950 (2013).</li><li>Wu, C. -. L., Fu, T. -. F., Chou, Y. -. Y., Yeh, S. -. 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+Single+Pair+of+Neurons+Modulates+Egg-Laying+Decisions+in+Drosophila.">A Single Pair of Neurons Modulates Egg-Laying Decisions in Drosophila.</a> PloS one. 10 (3), e0121335 (2015).</li><li>Yang, C. H., He, R., Stern, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+and+circuit+basis+of+sucrose+rejection+by+Drosophila+females+in+a+simple+decision-making+task.">Behavioral and circuit basis of sucrose rejection by Drosophila females in a simple decision-making task.</a> J. Neurosci. 35 (4), 1396-1410 (2015).</li><li>Gou, B., Liu, Y., Guntur, A. R., Stern, U., Yang, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanosensitive+neurons+on+the+internal+reproductive+tract+contribute+to+egg-laying+induced+acetic+acid+attraction+in+Drosophila.">Mechanosensitive neurons on the internal reproductive tract contribute to egg-laying induced acetic acid attraction in Drosophila.</a> Cell Rep. 9 (2), 522-530 (2014).</li><li>Stern, U., Zhu, E. Y., He, R., Yang, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-duration+animal+tracking+in+difficult+lighting+conditions.">Long-duration animal tracking in difficult lighting conditions.</a> Sci. Rep. 5, 10432 (2015).</li><li>Zhu, E. Y., Guntur, A. R., He, R., Stern, U., Yang, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Egg-laying+demand+induces+aversion+of+UV+light+in+Drosophila+females.">Egg-laying demand induces aversion of UV light in Drosophila females.</a> Curr. Biol. 24 (23), (2014).</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, 451-457 (2009).</li><li>Klatpoetke, N. C., 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=Independent+Optical+Excitation+of+Distinct+Neural+Populations.">Independent Optical Excitation of Distinct Neural Populations.</a> Nat. Methods. 11 (3), 338-346 (2014).</li></ol>

The authors have nothing to disclose.

The chambers and protocols described here have several improvements over previous egg-laying assays. First, they increase the throughput of assaying preferences of single animals significantly. Each chamber can assay 30 single females and it takes less than an hour to set up. Second, they increase the consistency of the egg-laying preferences over previous methods. The standardization of the dimensions of the arena, size of the egg-laying substrates, and distance between substrates makes it easier to compare the results between different experimental days and from different research groups. Third, the chambers can be fitted with attachments to accommodate additional analysis one wants to pursue (Supplementary Figure 4). For example, for behavioral analysis, video cameras can be attached to the top of the chamber to record the flies7,11-13. Most importantly, this approach is scalable. These chambers are relatively inexpensive to produce with the help of a machine shop. Also, it typically takes a skilled worker less than 2 hr to set up 5 chambers (150 single females) worth of egg-laying assays.
One of the most critical factors when studying egg-laying preference of single females is to ensure that each female is primed to lay many eggs. (A preference of &#34;50 vs. 0 egg&#34; is much more convincing than a preference of &#34;1 vs. 0 egg&#34;). This protocol, when properly executed, should allow regular wild-type flies (Canton S and w1118) to lay at least 50 eggs/per female O/N. Several factors contribute to producing females with high egg-laying rate. First, one should take care to not overpopulate the vials/bottles when culturing the females to be assayed. Overcrowding often produces smaller larvae, which would grow into smaller females that lay fewer eggs. Second, one should make sure that females to be assayed have access to plenty of yeast paste when mixed together with males in the food vials. It is recommended that 0.5% proprionic acid instead of water be used to prepare yeast paste because proprionic acid not only increases egg-laying but also reduces fungal infection in vials. Third, one should not assay the females until the larvae/food surface of the food vials look like the picture shown in Figure 1F; if not sufficiently egg-laying deprived, females would not lay as many eggs when assayed in the chambers. Also, note that the food vials used here are the &#34;narrow vials&#34;. If one uses vials with larger diameter, one needs to increase the number of females/males to be placed in the vial so that the females would be ready to be assayed in 4 - 5 days.
While the protocol described here specifically focuses on examining females&#39; preferences between sucrose and plain substrates, it can be adapted to study females&#39; preference in other conditions. For example, these chambers can be used to assay flies&#39; preference when choosing between harder and softer agarose (e.g., 1% vs. 1.5% agarose), as well as substrates that differ in other chemosensory cues (e.g., 3% acetic acid). If fitted with lid that contains LED to illuminate one or both substrates, the chamber may also provide an efficient platform to conduct optogenetic studies (Supplementary Figure 4).
Finally, it is worth noting that although this setup allows higher throughput for analyzing egg-laying preferences of Drosophila, it has some important limitations. First, the fixed dimension of the chambers limits the flexibility of the behavioral assay. For example, one will need to make new chambers in order to test how other parameters (e.g., distance between substrates) may affect the egg-laying preferences of flies. In addition, this system does not capture the complex environment Drosophila encounter in the wild. It is very rare that flies will have to decide between a pure sucrose substrate vs. a pure plain substrate for egg-laying in nature. Thus while our assay is efficient in studying the neural basis of a simple decision task, one needs to be aware that the specific &#34;decisions&#34; females make in our chambers may not be ethologically relevant.

Drosophila melanogaster is a powerful genetic model organism to study the neural basis of behaviors. The rapid developments of genetic tools to manipulate neurons in a targeted manner and the emergence of sophisticated behavioral analysis tools have significantly improved our ability to dissect the circuit mechanisms that underlie the sensory-motor transformation processes of several innate and learned behaviors1-3.
Drosophila egg-laying is a suitable model to study the neural basis of simple decision making processes. In particular, Drosophila females have been shown to possess the ability to compare and rank their options before "committing" to depositing an egg onto a given option4-8. For example, when given only a plain (sucrose-free) substrate or only a sucrose-containing substrate, females readily accept either option for egg-laying. However, when presented with both options, females robust reject the sucrose substrate in some contexts7,9,10. Relatively little is known about the neural mechanisms that allow females to "choose the greater of two goods", however. A major obstacle has been the lack of an efficient method to assay egg-laying preferences such that one can use a systematic genetic screening approach to study this problem.
In this report, we describe the protocol we developed that allows egg-laying preferences of females to be assayed at single-animal resolution and with substantially improved throughput and consistency over previous methods. Specifically, we provide the blueprints for constructing the chambers we designed, the protocol for preparing the females so that each is primed to lay many eggs, and the protocol for using the chambers.

<table><tbody><tr><td>UltraPure Agarose&amp;nbsp;</td><td>Invitrogen</td><td>16500-500</td><td></td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S0389</td><td></td></tr><tr><td>Water bath&amp;nbsp;</td><td>Fisher</td><td>15-462-6Q</td><td></td></tr><tr><td>LifeCam Cinema webcam</td><td>Microsoft</td><td>H5D-00013</td><td></td></tr><tr><td>Red LEDs</td><td>Cree</td><td>C503B-RAN-CA0B0AA1</td><td></td></tr><tr><td>Egg-laying chambers</td><td>Custom Built</td><td></td><td></td></tr><tr><td>Camera holders</td><td>Custom Built</td><td></td><td></td></tr><tr><td>LED holders</td><td>Custom Built</td><td></td><td></td></tr><tr><td>Fly vials (narrow)</td><td>Genesee</td><td>32-116BC</td><td></td></tr></tbody></table>

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.

The egg-laying chambers are composed of several pieces: a substrate (bottom) piece, a divider (middle) piece, a loading (top) piece, and 2 sliding doors (Figure 1A - D). These pieces are used to independently setup flies and substrates before egg-laying experiments. Figure 1F shows how vials should look when female flies are ready for egg-laying. When flies are given a choice between a plain substrate and a sucrose-containing substrate, females robustly preferred the plain substrate for egg-laying as shown in Figure 2.
<img alt="Figure 1" src="/files/ftp_upload/53716/53716fig1.jpg" /> 
Figure 1. Egg-laying Chambers and Egg-laying Protocol. (A) Fully assembled egg -laying chamber. (B) Disassembled pieces. a: the loading piece (top), b: the divider piece (middle), c: the substrate (bottom) piece. Engineering drawings of these pieces are shown in Supplementary Figure 1 - 3. (C) The loading piece of the chamber. a&#39;: sliding doors, a&#34;: rails. The plastic sheets are inserted into the loading piece to serve as a floor to keep the loaded flies in place. We typically put color tapes onto the edge of the plastic sheets (red arrow). (D) The substrate (bottom) and the divider (middle) pieces of the chamber. Agarose is deposited into individual troughs to serve as egg-laying substrates (arrows). Quadrilateral outlines the egg-laying arena for a single fly. (E) Day 0 of collected females/males in a yeasted vial. (F) Day 4 - 5 of collected females/males in a yeasted vial. Note that larvae and adult females have eaten most of the yeast and the surface of the food has become occupied by larvae. The wet surface food crawling with larvae prevents females from laying more eggs in the vial. (G) Schematic depicting the protocol for setting up egg-laying behavior experiments. <a href="https://www.jove.com/files/ftp_upload/53716/53716fig1large.jpg" target="_blank">(Please click here to view a larger version of this figure)</a>
<img alt="Figure 2" src="/files/ftp_upload/53716/53716fig2a.jpg" /> 
Figure 2. Representative Egg-laying Results for Flies Choosing between Sucrose and Plain Substrates. (A) Side view of egg-laying results of wild type flies when they were given a choice between sucrose (150 mM) and plain substrates. White box outlines one egg-laying arena for a single fly. (B) Top view of egg-laying results. White box outlines one egg-laying arena for a single fly. (C) Preference index (PI) of wild type flies when asked to choose between a sucrose-containing substrate and a plain substrate. PI for each female is calculated as follows: (number of eggs on sucrose substrate - number of eggs on the plain substrates)/total number of eggs. Error bar indicates SEM. <a href="https://www.jove.com/files/ftp_upload/53716/53716fig2large.jpg" target="_blank">(Please click here to view a larger version of this figure)</a>
<img alt="Supplemental Figure 1" src="/files/ftp_upload/53716/53716supfig1.jpg" /> 
Supplementary Figure S1. Engineering Drawings for the Divider and the Substrate Piece of the Chamber. (A - A&#34;) Different views of the substrate piece of the chamber. (B - B&#39;) Different views of the middle divider piece of the chamber. <a href="https://www.jove.com/files/ftp_upload/53716/Supplemental1.jpg" target="_blank">(Please click here to download this file)</a>
<img alt="Supplemental Figure 2" src="/files/ftp_upload/53716/53716supfig2.jpg" /> 
Supplementary Figure S2. Engineering Drawings for the Loading Piece of the Chamber. (A - A&#39;) Different views of the upper layer of the loading piece of the chamber. (B - B&#39;) Different views of the bottom layer of the loading piece of the chamber. <a href="https://www.jove.com/files/ftp_upload/53716/Supplemental2.jpg" target="_blank">(Please click here to download this file)</a>
<img alt="Supplemental Figure 3" src="/files/ftp_upload/53716/53716supfig3.jpg" /> 
Supplementary Figure S3. Engineering Drawing for the Sliding Door and the Rail on the Chamber. (A) The rail fixed onto the top piece of the chamber. (B) The sliding door for the chamber. (<a href="https://www.jove.com/files/ftp_upload/53716/Supplemental3.jpg" target="_blank">Please click here to download this file)</a>
<img alt="Supplemental Figure 4" src="/files/ftp_upload/53716/53716supfig4.jpg" /> 
Supplementary Figure S4. Accessories and Customization of Egg-laying Chambers. (A) Egg-laying chambers equipped with cameras to video record flies during egg-laying experiments13. Ctrax14 with extension12 is used to track the flies and custom MATLAB code is used to plot the trajectories. (B) Setup to illuminate egg-laying substrates with red light. LEDs (1) are connected to a LED driver (2). LED intensity is controlled by microcontroller (3). For more information about the camera, light delivery system and tracking animals in abnormal lighting conditions, see Zhu et al. and Stern et al. (C) Red LEDs (light off) attached to top of egg-laying chambers. (D) Red LEDs (light on) attached to top of egg-laying chambers. The LED intensity of red LEDs is typically set to be around 7 - 10 &#181;W/mm when CsChrimson15 is used to activate the neurons. (E) Egg-laying chambers equipped with cameras and LEDs to video record flies during experiments with red light (light off). (F) Egg-laying chambers equipped with cameras and LEDs to video record flies during experiments with red light (light on). (<a href="https://www.jove.com/files/ftp_upload/53716/Supplemental4.jpg" target="_blank">Please click here to download this file)</a>

Watch this Scientific Journal Video about High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster at JoVE.com

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.

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. ...

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Neuroscience

7.9K Views.  Duke University. 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.

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

Methods to Assay Drosophila Behavior

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

Methods to Assay Drosophila Behavior

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

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

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 ...

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous system and protects neural tissue from toxins and pathogens. Defects in the formation of this barrier have been correlated with neuropathies, and the breakdown of this barrier has been observed in many neurodegenerative diseases. Therefore, it is critical to identify the genes that regulate the formation and maintenance of the blood-brain barrier to identify potential therapeutic targets. In order to understand the exact roles these genes play in neural development, it is necessary to assay the effects of altered gene expression on the integrity of the blood-brain barrier. Many of the molecules that function in the establishment of the blood-brain barrier have been found to be conserved across eukaryotic species, including the fruit fly, Drosophila melanogaster. Fruit flies have proven to be an excellent model system for examining the molecular mechanisms regulating nervous system development and function. This protocol describes a step-by-step procedure to assay for blood-brain barrier integrity during the embryonic and larval stages of D. melanogaster development.

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Blood-brain barrier integrity is critical for nervous system function. In Drosophila melanogaster, the blood-brain barrier is formed by glial cells during late embryogenesis. This protocol describes methods to assay for blood-brain barrier formation and maintenance in D. melanogaster embryos and third instar larvae.

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous ...

Developmental Biology

Measuring and Altering Mating Drive in Male Drosophila melanogaster

Despite decades of investigation, the neuronal and molecular bases of motivational states remain mysterious. We have recently developed a novel, reductionist, and scalable system for in-depth investigation of motivation using the mating drive of male Drosophila&#160;melanogaster (Drosophila), the methods for which we detail here. The behavioral paradigm centers on the finding that male mating drive decreases alongside fertility over the course of repeated copulations and recovers over ~3 d. In this system, the powerful neurogenetic tools available in the fly converge with the genetic accessibility and putative wiring diagram available for sexual behavior. This convergence allows rapid isolation and interrogation of small neuronal populations with specific motivational functions. Here we detail the design and execution of the satiety assay that is used to measure and alter courtship motivation in the male fly. Using this assay, we also demonstrate that low male mating drive can be overcome by stimulating dopaminergic neurons. The satiety assay is simple, affordable, and robust to influences of genetic background. We expect the satiety assay to generate many new insights into the neurobiology of motivational states.

Measuring and Altering Mating Drive in Male Drosophila melanogaster

This article describes a behavioral assay that uses male mating drive in Drosophila melanogaster to study motivation. Using this method, researchers can utilize advanced fly neurogenetic techniques to uncover the genetic, molecular, and cellular mechanisms that underlie this motivation.

Despite decades of investigation, the neuronal and molecular bases of motivational states remain mysterious. We have recently developed a novel, ...

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which enables them to seek out their preferred thermal landscape. To define the temperature preferences of larvae over a defined linear range, we developed an assay using a temperature gradient. To establish a single-directional gradient, two aluminum blocks are connected to independent water baths, each of which controls the temperature of individual blocks. The two blocks set the lower and upper limits of the gradient. The temperature gradient is established by placing an agarose-coated aluminum plate over the two water-controlled blocks so that the plate spans the distance between them. The ends of the aluminum plate that is set on the top of the water blocks defines the minimum and maximum temperatures, and the regions in-between the two blocks form a linear temperature gradient. The gradient assay can be applied to&#160;larvae of different ages and can be used to identify mutants that exhibit phenotypes, such as those with mutations affecting genes encoding TRP channels and opsins, which are required for temperature discrimination.

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Here, we present a protocol to determine the preferred environmental temperature of Drosophila larvae using a continuous thermal gradient.

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which ...

A Rapid Food-Preference Assay in Drosophila

To select food with nutritional value while avoiding the consumption of harmful agents, animals need a sophisticated and robust taste system to evaluate their food environment. The fruit fly, Drosophila melanogaster, is a genetically tractable model organism that is widely used to decipher the molecular, cellular, and neural underpinnings of food preference. To analyze fly food preference, a robust feeding method is needed. Described here is a two-choice feeding assay, which is rigorous, cost-saving, and fast. The assay is Petri-dish-based and involves the addition of two different foods supplemented with blue or red dye to the two halves of the dish. Then, ~70 prestarved, 2-4-day-old flies are placed in the dish and&#160;allowed to choose between blue and red foods in the dark for about 90 min. Examination of the abdomen of each fly is followed by the calculation of the preference index. In contrast to multiwell plates, each Petri dish takes only ~20 s to fill and saves time and effort. This feeding assay can be employed to quickly determine whether flies like or dislike a particular food.

A Rapid Food-Preference Assay in Drosophila

We present a protocol for a two-choice feeding assay for flies. This feeding assay is fast and easy to run and is suitable not only for small-scale laboratory research, but also for high-throughput behavioral screens in flies.

To select food with nutritional value while avoiding the consumption of harmful agents, animals need a sophisticated and robust taste system to evaluate ...

Fly Climbing Assay: A Method to Test General Locomotion in Drosophila melanogaster

Source: Gevedon, O., et al. <a href="https://www.jove.com/video/59720/in-vivo-forward-genetic-screen-to-identify-novel-neuroprotective">In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster</a>. J. Vis. Exp. (2019).
This video describes a simple method to quantify fly locomotion and shows the climbing assay performed.&#160; The fly climbing assay is a general test of motor function which relies on a negative geotaxis response.

Test wspinaczki muchowej: metoda testowania ogólnej lokomocji u Drosophila melanogaster

Source: Gevedon, O., et al. In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster. J. Vis. Exp. (2019).
This ...

JoVE Encyclopedia of Experiments

Biology

Drosophila melanogaster (fruit fly)

Adult

Egg Laying Assay: A Method to Quantify the Egg-Laying Behavior of C. elegans

Source: Lee, S. Y. and Kang, K. <a href="https://www.jove.com/video/60419/measuring-effects-bacteria-chemicals-on-intestinal-permeability">Measuring the Effect of Chemicals on the Growth and Reproduction of&#160;Caenorhabditis elegans</a>.&#160;J. Vis. Exp. (2017).
C. elegans egg-laying behavior can provide important information in developmental, reproductive, and anatomical studies.&#160; This video introduces the important steps of an egg laying assay and shows a sample experiment in which a drug is tested for reproductive toxicity.

Test składania jaj: metoda ilościowego określania zachowania C. elegans podczas składania jaj

Source: Lee, S. Y. and Kang, K. Measuring the Effect of Chemicals on the Growth and Reproduction of&#160;Caenorhabditis elegans.&#160;J. Vis. Exp. 
...

Caenorhabditis elegans (worm)

Basic Methods

Island Assay: A High-Throughput Method to Evaluate Drosophila Locomotor Behavior

Source: Eidhof, I., et al. <a href="https://www.jove.com/video/55892/" target="_blank">High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay</a>. J. Vis. Exp. (2017).
This video describes the island assay, a behavioral method used to study Drosophila locomotion. The sample protocol features a setup for the assay compatible with semi-automated image analysis.

Test wyspowy: wysokoprzepustowa metoda oceny zachowania lokomotorycznego Drosophila

Source: Eidhof, I., et al. High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay. J. Vis. Exp. (2017).
This video describes the ...

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

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Methods to Assay Drosophila Behavior

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

A Single-fly Assay for Foraging Behavior in Drosophila

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Measuring and Altering Mating Drive in Male Drosophila melanogaster

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

A Rapid Food-Preference Assay in Drosophila

Test wspinaczki muchowej: metoda testowania ogólnej lokomocji u Drosophila melanogaster

Test składania jaj: metoda ilościowego określania zachowania C. elegans podczas składania jaj

Test wyspowy: wysokoprzepustowa metoda oceny zachowania lokomotorycznego Drosophila