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>
	<l...

<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 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 ...

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 border="0" cellpadding="0" cellspacing="0" width="696">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">UltraPure Agarose&#160;</td>
			<td style="width:104px;">Invitrogen</td>
			<td style="width:119px;">16500-500</td>
			<td style="width:204px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Sucrose</td>
			<td style="width:104px;">Sigma</td>
			<td style="width:119px;">S0389</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Water bath&#160;</td>
			<td style="width:104px;">Fisher</td>
			<td style="width:119px;">15-462-6Q</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">LifeCam Cinema webcam</td>
			<td style="width:104px;">Microsoft</td>
			<td style="width:119px;">H5D-00013</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:269px;">Red LEDs</td>
			<td style="width:104px;">Cree</td>
			<td style="width:119px;">C503B-RAN-CA0B0AA1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Egg-laying chambers</td>
			<td style="width:104px;">Custom Built</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Camera holders</td>
			<td style="width:104px;">Custom Built</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">LED holders</td>
			<td style="width:104px;">Custom Built</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Fly vials (narrow)</td>
			<td style="width:104px;">Genesee</td>
			<td style="width:119px;">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 p...

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. ...

The overall goal of this high throughput assay is to provide a simple, efficient, and scalable platform to identify genes and neurons that control a simple choice behavior in Drosophila. The main advantage of this method is actually you can infectioning as an eggening choice, and a single animal in a highly suitable manner. To begin the experiment, place eight females and six males into a narrow food vial filled with standard fly media.Then use the female progenies grown from this vial for the egg-laying experiments. Two to three days after female eclosion, make fresh yeast paste by mixing six grams of active yeast with 10 milliliters of 0.5%propionic acid. Use a spatula to apply the yeast paste onto the side wall of the food vials.Next, place 30 previously collected eclosed females and 20 males into the vials. After four days, check the surface of the food medium. If the surface is wet from the larvae actively burrowing in the food, then the female flies are ready for egg-laying experiments.Continue with the experiment by obtaining previously constructed acrylic egg-laying chambers. Insert plastic sheets into the top, loading a piece of the chamber to serve as the bottom surface. Next, anesthetize the females on a carbon dioxide pad and load them individually into each egg-laying arena.Allow the flies 30 minutes to recover from the carbon dioxide and to acclimate to the new environment. While the flies are adjusting, fill a tube with agarose up to the 10 milliliter mark, then prepare a 150 milliliter sucrose substrate by placing 750 microliters of two molar sucrose solution into the tube. Keep a bottle of melted 1%agrose in a water bath.Prepare the plain substrate in the same manner, but replace the sucrose solution with distilled water. Next, obtain the substrate piece of the chamber and pipette 1, 000 microliters of the agarose substrate into each trough. Set the agarose aside to solidify for 30 minutes.Once the agarose substrates and flies are ready, assemble all three pieces of the egg-laying chamber and then take out the plastic sheets. Place the chambers in fly incubators overnight. Anesthetize females by injecting carbon dioxide into the chamber.Disassemble the chamber, and discard the anesthetized flies. Finally, take pictures of the eggs for record keeping. The preference index, or PI, was calculated as the number of eggs laid by female flies when given the choice between a plain substrate and a sucrose containing substrate.Females robustly preferred the plain substrate for egg-laying. Following this procedure, other methods like radio recording are opportunities genetics can be performed in order to answer other questions like how the flies behave prior depositing each egg.

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7.8K Views.  Duke University. The overall goal of this high throughput assay is to provide a simple, efficient, and scalable platform to identify genes and neurons that control a simple choice behavior in Drosophila. The main advantage of this method is actually you can infectioning as an eggening choice, and a single animal in a highly suitable manner. To begin the experiment, place eight females and six males into a narrow food vial filled with standard fly media.Then use the female progenies grown from this vial...