We thank Rachelle Kanippayoor for her help in identifying the new wild-type strain as being of the melanogaster species. R.W.F, O.F. and A.F.S were responsible for research design; R.W.F, M.N. and O.F. performed the experiments. R.W.F, M.N., O.F. and A.F.S. analyzed the data; R.W.F., I.S.M. and A.F.S. wrote the manuscript. 
This work was supported by PSC-CUNY research awards, jointly funded by The Professional Staff Congress and The City University of New York to A.F.S.; by internal funding from Western University to A.F.S. and I.S.M.; by a training support from the National Alliance for Hispanic Health&#8217;s Alliance/Merck Ciencia (Science) Hispanic Scholars Program and a University Fellowship from the Yale Graduate School of Arts and Sciences to R.W.F.

1. Equipment and Reagents Created In-house (See List of Material for Others)
<ol>
	<li>Prepare a Drosophila cold anesthesia apparatus to perform fly work.
	<ol>
		<li>Cut a porous polyethylene sheet to cover a small plastic box (12.7 cm long, 10.2 cm wide), typically the top cover of a pipette tips box (12.7 cm long, 10.2 cm wide, 3.8 cm deep).</li>
		<li>Fill the box with crushed ice, cover with the porous polyethylene sheet.&#160;</li>
	</ol>
	</li>
	<li>Prepare a T-maze apparatus; adapt the apparatus previously described in details 28-31.</li>
	<li>Prepare a fly aspirator as described 32.</li>
	<li>Prepare a phototaxis response apparatus to displace flies in and out of test vials without stress:
	<ol>
		<li>Use a countercurrent apparatus, as described by29,33.</li>
	</ol>
	</li>
</ol>
NOTE: The T-maze can also be used for other purposes, as described by28.
<ol start="5">
	<li>Ensure homogeneous lighting conditions: perform the experiment on a bench covered with a white bench cover, and in front of a white board. 
	NOTE: For these experiments, the fly strains used were Drosophila melanogaster wild-types: Canton-S, Oregon and Samarkand flies from our laboratory stocks34; Elwood flies were collected in Fall 2011 in the Elwood neighborhood of Huntington, on Long Island, New York, USA.</li>
</ol>
2. Preparing the Flies before the Experiment
1-2 days before performing the experiment:&#160;
<ol>
	<li>Maintain flies on standard cornmeal-agar-molasses-yeast medium at 25 &#176;C on a 12 hr light/dark cycle.</li>
	<li>Collect the responder flies under cold anesthesia 1-2 days prior to performing the experiment
	<ol>
		<li>Transfer the flies from the bottles in which they have been raised into 50 ml Falcon tubes, using a funnel.</li>
		<li>Place the falcon tube with the flies under ice level, into an insulated ice bucket.</li>
		<li>Place the cold anesthesia apparatus with crushed ice at -4 &#176;C to chill.</li>
		<li>Wait 5 min for the flies to enter a chill coma.</li>
		<li>Transfer the flies onto the porous polyethylene sheet of the cold anesthesia apparatus.</li>
		<li>Collect the responder flies on the cold anesthesia apparatus. At RT, under a stereomicroscope, separate 3-7 days old male from female flies and maintain these flies in groups of 40.</li>
	</ol>
	</li>
	<li>Collect the emitter flies 1-2 days prior to performing the experiment.
	<ol>
		<li>Gently collect Canton-S mixed-sex flies from their bottles, using a mouth aspirator, controlling for their number.</li>
		<li>Maintain these flies in group of 60-100, as they will be the emitters.</li>
		<li>Prepare as many samples of emitters as data points needed.</li>
	</ol>
	</li>
	<li>Allow at least a night of recovery after collection of the responders and emitters, to minimize any confounding effect of the cold anesthesia and mouth aspirator collection on behavior.</li>
	<li>Open the bag of test vials, to allow for the stale plastic-smelling air in the bag to be replaced by fresh air, at least 1 day prior to the experiment.</li>
</ol>
2 hr before performing the experiment:
<ol start="6">
	<li>Ensure that the temperature in the room in which the experiment is performed is around 23-25 &#176;C, with even light, and humidity above 30%.</li>
	<li>Transfer the responders and emitters fruit flies into fresh food vials to ensure that these flies are not starved.</li>
	<li>Let the fruit flies adjust to the environment for 2 hr, on the bench on which the experiment will be performed.</li>
</ol>
3. Performing the Social Avoidance Experiment
<ol>
	<li>Perform each experiment at the same time of the day, in a range of 3&#8211;4 hr in the afternoon between Zeitgeber time ZT5 and ZT9.</li>
	<li>Place the T-maze on the pounding pad, and tighten the screw clip so the elevator is stable.</li>
	<li>Transfer the responder flies into a fresh test vial. Snap this vial containing the responder flies onto the top part of the T-maze.</li>
	<li>Slant the T-maze apparatus and tap it on the pounding mat so the fruit flies fall in the elevator.</li>
	<li>Move the top part of the elevator down so the flies are between the top and bottom section of the T-maze, but pay attention to keep the elevator above the choice point &#8211; to prevent the flies from escaping.</li>
	<li>Obtain vials with dSO (see Section 5 to generate dSO-free vials that were previously occupied by flies).
	<ol>
		<li>While the responder flies are adjusting to this new environment, place the emitter flies in a test vial.</li>
		<li>Place a piece of cotton to close the vial so the fruit flies do not escape.</li>
		<li>Mechanically agitate this vial on a mini-vortex as follows: vortex for 15 sec, remove the vial from the vortex for 5 sec. Repeat 3 times for a total of 55 sec.</li>
		<li>Remove the emitter flies out of the fresh vial &#8212; by transferring in a food vial &#8212; and quickly place this dSO filled vial into one of the two sides of the T-maze apparatus.</li>
		<li>Alternate the side of placement for the dSO filled vial in each new run.</li>
	</ol>
	</li>
	<li>Place a fresh test vial on the other side of the T-maze.</li>
	<li>Bring the elevator completely down, so the fruit flies can chose between the fresh test vial and the test vial with dSO, and start the timer.</li>
	<li>Let the flies choose between the fresh vial and the dSO vial, for 1 min, unless noted otherwise.</li>
	<li>After 1 min, move the elevator up to separate the flies in the fresh vial, dSO vial, and those stuck in the elevator.</li>
	<li>Count the number of flies in each vial, and in the elevator.</li>
	<li>Repeat this for each genotype/condition.</li>
</ol>
4. Analyzing the Social Avoidance Data
<ol>
	<li>Count the number of fruit flies in each vial for each genotype, and transfer the data to a spreadsheet.</li>
	<li>Using a spreadsheet program, calculate the Performance Index (PI) for each genotype by subtracting the number of flies in the dSO vial from the number of flies in the dSO-free vial, and then dividing by the total number of flies.</li>
	<li>Compare the PIs&#8217; means using statistical analysis software for analysis of the results.
	<ol>
		<li>Ideally, prepare 2-3 internal repeats for each experiment, and 3 independent repeats, performed on different days, with different bottles or crosses30.</li>
		<li>Use Gaussian distribution statistical tests, as the data follow a normal distribution.</li>
		<li>When testing one effect (i.e., density &#8212; Figure 2), use a one-way ANOVA, with post-test for multiple comparison (column by column), or a simple t-test to compare each result to a control.</li>
		<li>When group experiments are performed, test both conditions and genders (empty vials, stressed and un-stressed flies; and gender effect &#8212; Figure 1), using a two-way ANOVA.</li>
	</ol>
	</li>
</ol>
5. Generate Vials Previously Occupied by Unstressed Flies &#8212; Perform Instead of 3.5 to 3.6
<ol>
	<li>Use phototaxis to prepare vials occupied by flies that have not been stressed, as a control.</li>
	<li>Transfer the flies in a test vial, and snap it into the countercurrent apparatus, with fresh empty vials in the opposite location.</li>
	<li>Lay the apparatus horizontally with the distal vial directly in front of a 15 W fluorescent cool-white light, and cover with a black cloth.</li>
	<li>Let the flies gently move in the fresh vial. This might take several minutes. Once most of the flies are walking toward the light in the distal vial, start the timer for 1 min.</li>
	<li>Gently remove the black cloth, switch the location of the light, recover with the black cloth, and allow the flies to leave the test vial.</li>
	<li>At that time, initiate the choice experiment as in 3.2 to 3.5. Skip 3.6 and 3.7.</li>
	<li>Move the slider of the apparatus, to separate the flies that vacated the test vial from the other side.</li>
	<li>Use the vacated test vial in the T-maze right away for the choice experiment, as in 3.7.</li>
</ol>

<ol>
	<li>Fry, S. N., Rohrseitz, N., Straw, A. D., 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=TrackFly:+virtual+reality+for+a+behavioral+system+analysis+in+free-flying+fruit+flies.">TrackFly: virtual reality for a behavioral system analysis in free-flying fruit flies.</a> J Neurosci Methods. 171, 110-117 (2008).</li><li>Slawson, J. B., Kim, E. Z., Griffith, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+video+tracking+of+locomotion+in+adult+Drosophila+melanogaster.">High-resolution video tracking of locomotion in adult Drosophila melanogaster.</a> J Vis Exp. 24, 1096 (2009).</li><li>Dankert, H., Wang, L., Hoopfer, E. D., Anderson, D. J., Perona, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+monitoring+and+analysis+of+social+behavior+in+Drosophila.">Automated monitoring and analysis of social behavior in Drosophila.</a> Nat Methods. 6, 297-303 (2009).</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>Simon, J. C., 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=A+new+chamber+for+studying+the+behavior+of+Drosophila.">A new chamber for studying the behavior of Drosophila.</a> PLoS One. 5, 8793 (2010).</li><li>Wang, L., Dankert, H., Perona, P., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inaugural+Article:+A+common+genetic+target+for+environmental+and+heritable+influences+on+aggressiveness.">Inaugural Article: A common genetic target for environmental and heritable influences on aggressiveness.</a> in Drosophila. Proceedings of the National Academy of Sciences. 105, 5657-5663 (2008).</li><li>Schneider, J., Dickinson, M. H., Levine, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Social+structures+depend+on+innate+determinants+and+chemosensory+processing+in+Drosophila.">Social structures depend on innate determinants and chemosensory processing in Drosophila.</a> Proceedings of the National Academy of Sciences. 2, 17174-17179 (2012).</li><li>Miyamoto, T., Amrein, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+male+courtship+by+a+Drosophila+pheromone+receptor.">Suppression of male courtship by a Drosophila pheromone receptor.</a> Nat Neurosci. 11, 874-876 (2008).</li><li>Villella, A., Hall, J. C., Jeffrey, 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=Chapter+3+Neurogenetics+of+Courtship+and+Mating+in+Drosophila.+.">Chapter 3 Neurogenetics of Courtship and Mating in Drosophila. .</a> Advances in Genetics. 62, 67-184 (2008).</li><li>Ejima, A., Griffith, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Courtship+Initiation+Is+Stimulated+by+Acoustic+Signals+in+Drosophila+melanogaster.">Courtship Initiation Is Stimulated by Acoustic Signals in Drosophila melanogaster.</a> PLoS ONE. 3, 3246 (2008).</li><li>Mery, F., 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=Public+Versus+Personal+Information+for+Mate+Copying+in+an+Invertebrate.">Public Versus Personal Information for Mate Copying in an Invertebrate.</a> Current Biology. 19, 730-734 (2009).</li><li>Montell, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+taste+of+the+Drosophila+gustatory+receptors.">A taste of the Drosophila gustatory receptors.</a> Curr. Opin. Neurobiol. 19, 345-353 (2009).</li><li>Billeter, J. -. C., Atallah, J., Krupp, J. J., Millar, J. G., Levine, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specialized+cells+tag+sexual+and+species+identity+in+Drosophila+melanogaster.">Specialized cells tag sexual and species identity in Drosophila melanogaster.</a> Nature. 461, 987-991 (2009).</li><li>Krupp, J. J., 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=Social+experience+modifies+pheromone+expression+and+mating+behavior+in+male+Drosophila+melanogaster.">Social experience modifies pheromone expression and mating behavior in male Drosophila melanogaster.</a> Curr Biol. 18, 1373-1383 (2008).</li><li>Kent, C., Azanchi, R., Smith, B., Formosa, A., Levine, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Social+context+influences+chemical+communication+in+D.+melanogaster+males.">Social context influences chemical communication in D. melanogaster males.</a> Curr Biol. 18, 1384-1389 (2008).</li><li>Levine, J. D., Funes, P., Dowse, H. B., Hall, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resetting+the+Circadian+Clock+by+Social+Experience+in+Drosophila+melanogaster.">Resetting the Circadian Clock by Social Experience in Drosophila melanogaster.</a> Science. 298, 2010-2012 (2002).</li><li>Ganguly-Fitzgerald, I., Donlea, J., Shaw, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Waking+Experience+Affects+Sleep+Need+in+Drosophila.">Waking Experience Affects Sleep Need in Drosophila.</a> Science. 313, 1775-1781 (2006).</li><li>Billeter, J. -. C., Levine, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Who+is+he+and+what+is+he+to+you?+Recognition+in+Drosophila+melanogaster.">Who is he and what is he to you? Recognition in Drosophila melanogaster.</a> Curr. Opin. Neurobiol. 23, 17-23 (2013).</li><li>Suh, G. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+population+of+olfactory+sensory+neurons+mediates+an+innate+avoidance+behaviour+in+Drosophila.">A single population of olfactory sensory neurons mediates an innate avoidance behaviour in Drosophila.</a> Nature. 431, 854-859 (2004).</li><li>Bonini, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Tribute+to+Seymour+Benzer+1921-2007.">A Tribute to Seymour Benzer 1921-2007.</a> 180, 1265-1273 (2008).</li><li>Castermans, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neurobeachin+gene+is+disrupted+by+a+translocation+in+a+patient+with+idiopathic+autism.">The neurobeachin gene is disrupted by a translocation in a patient with idiopathic autism.</a> Journal of Medical Genetics. 40, 352-356 (2003).</li><li>Medrihan, L., 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=Neurobeachin,+a+protein+implicated+in+membrane+protein+traffic+and+autism,+is+required+for+the+formation+and+functioning+of+central+synapses.">Neurobeachin, a protein implicated in membrane protein traffic and autism, is required for the formation and functioning of central synapses.</a> J Physiol. 587, 5095-5106 (2009).</li><li>Simon, A. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+assay+to+study+social+behavior+in+Drosophila:+measurement+of+social+space+within+a+group.">A simple assay to study social behavior in Drosophila: measurement of social space within a group.</a> Genes Brain Behav. 11, 243-252 (2012).</li><li>Venkatesh, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cold Spring Harbor Meeting: From Molecules to Circuit Behavior. , (2013).</li><li>Hamilton, P. J., 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=De+novo+mutation+in+the+dopamine+transporter+gene+associates+dopamine+dysfunction+with+autism+spectrum+disorder.">De novo mutation in the dopamine transporter gene associates dopamine dysfunction with autism spectrum disorder.</a> Mol Psychiatry. 18, 1315-1323 (2013).</li><li>Gadow, K. D., 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=Association+of+dopamine+gene+variants,+emotion+dysregulation+and+ADHD+in+autism+spectrum+disorder.">Association of dopamine gene variants, emotion dysregulation and ADHD in autism spectrum disorder.</a> Research in Developmental Disabilities. 35, 1658-1665 (2014).</li><li>Fernandez, R. W., Akinleye, A. A., Nurilov, M., Rouzyi, Z., Simon, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> , (2014).</li><li>Ali, Y. O., Escala, W., Ruan, K., Zhai, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assaying+Locomotor,+Learning,+and+Memory+Deficits+in+Drosophila+Models+of+Neurodegeneration.">Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration.</a> J Vis Exp. , 2504 (2011).</li><li>Connolly, J. B., Tully, T., Roberts, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila: A Practical Approach. 1, 265-317 (1998).</li><li>Tully, T., Quinn, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classical-conditioning+and+retention+in+normal+and+mutant+Drosophila+melanogaster.">Classical-conditioning and retention in normal and mutant Drosophila melanogaster.</a> J Comp Physiol [A]. 157, 263-277 (1985).</li><li>Krashes, M. J., 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=Drosophila+Aversive+Olfactory+Conditioning.">Drosophila Aversive Olfactory Conditioning.</a> Cold Spring Harbor Protocols. 2011, (2011).</li><li>Ejima, A., Griffith, L. C., Zhang, B., Freeman, M. R., 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=Ch.+30.">Ch. 30.</a> Drosophila Neurobiology, A Laboratory Manual. , 475-481 (2010).</li><li>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=Behavioral+mutants+of+Drosophila+melanogaster+isolated+by+countercurrent+distribution.">Behavioral mutants of Drosophila melanogaster isolated by countercurrent distribution.</a> PNAS. 58, 1112-1119 (1967).</li><li>Simon, A. F., Shih, C., Mack, A., 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=Steroid+control+of+longevity+in+Drosophila+melanogaster.">Steroid control of longevity in Drosophila melanogaster.</a> Science. 299, 1407-1410 (2003).</li><li>Vaux, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Research+methods:+Know+when+your+numbers+are+significant.">Research methods: Know when your numbers are significant.</a> Nature. 492, 180-181 (2012).</li><li>Stowers, L., Logan, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexual+dimorphism+in+olfactory+signaling.">Sexual dimorphism in olfactory signaling.</a> Curr. Opin. Neurobiol. 20, 770-775 (2010).</li><li>Simon, A. F., Liang, D. T., Krantz, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+decline+in+behavioral+performance+of+Drosophila+melanogaster+with+age.">Differential decline in behavioral performance of Drosophila melanogaster with age.</a> Mechanisms of Ageing and Development. , 127-647 (2006).</li></ol>

The authors declare that they have no competing financial interests.

This protocol describes a detailed procedure for the social avoidance assay. Canton-S will only avoid a vial in which flies have previously been mechanically stressed, and that sex and number of responders does not affect that social avoidance performance. However, genetic background of the responders has a major influence.
The following are several critical steps for performing this experiment successfully: 1) always transfer the flies 2 hr before the experiment and make sure not to disturb t...

The goal of this method is to easily quantify a new aspect of simple social behavior in Drosophila melanogaster, independent from courtship and aggression.
Social interactions are crucial to the proper development and health of individuals within a society, as well as the functionality of a social group as a whole. The high complexity of these interactions necessitates large sample sizes and a system that allows for simplification of the behavior as the genetic and neural bases of social behavior are still poorly understood. Drosophila melanogaster is a powerful genetic model that can be used to identify the genetic and neural bases of social interactions. Indeed, D. melanogaster has a repertoire of complex social behaviors and some direct measurements of socialization have already been done1-7. However, most of these efforts have been focused on relatively complex social behaviors, such as aggressive interactions3,6, various aspects of courtship3,8-12, and how social experience affects other behaviors such as learning, or circadian rhythm13-17. In addition, many of these assays rely on analyzing complex interaction patterns of groups of flies, using video tracking and computer software to analyze the resulting abundance of data. Such analyses are invaluable, and lead to important new insights such as the dynamic of fly-fly interactions in groups7. One limitation, however, is the inaccessibility of these assays to the community at large, and the limited knowledge of the mechanisms underlying recognition of others. In other words, the basis of the emission of a signal by one individual and its recognition by another is still poorly understood18.
In contrast, flies also exhibit a simple behavior, social avoidance, where individuals move away from a signal emitted by stressed flies: the D. melanogaster Stress Odorant or dSO19. In a high-throughput assay, this behavior can be quantified as the avoidance of a stress signal emitted by other flies, or social avoidance19. Flies are placed in a T-maze apparatus and given the choice to avoid a vial containing dSO. Using this assay, CO2 was shown to be a component of the dSO, and part of the neural circuitry necessary to respond to CO2 was dissected19.
The social avoidance assay presented here is similar conceptually to the simple behavioral assays developed in Seymour Benzer&#8217;s laboratory that enabled generations of researchers to dissect complex behaviours20. Analysis of social avoidance can be carried out cost-effectively using the T-maze assay, allowing for more widespread study of social behavior. For example, using this assay we recently demonstrated that different genetic risks for autism have contrasting effects in social behavior assays. Mutants for a candidate gene for autism &#8212; neurobeachin21,22 &#8212; present deficiencies both in social space (described elsewhere23) and social avoidance24. Abnormal dopaminergic signaling is also proposed to play a role in the etiology of autism in humans25,26. In contrast to the results obtained with neurobeachin, we found that social avoidance performance was unaffected by increased or decreased levels of the Drosophila Vesicular Monoamine Transporter (VMAT) in dopaminergic cells, although social space was directly correlated to these levels of VMAT27. The contrasting results obtained with neurobeachin and VMAT underscore the possibility of identifying various forms of asocial behavior, and thus the different underlying neural circuitries modulating the response to others.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Stereo Zoom Microscope&#160;</td>
			<td>Nikon&#160;&#160;</td>
			<td>SMZ-645</td>
			<td>Any other standard scope for fly handling would work</td>
		</tr>
		<tr>
			<td>Small paint brushes&#160;</td>
			<td></td>
			<td></td>
			<td>for pushing flies</td>
		</tr>
		<tr>
			<td>Porous Polyethylene, 12&#34; x 12&#34; Sheet</td>
			<td>Flystuff - Genesee</td>
			<td>46-100</td>
			<td>http://www.flystuff.com/ProductInfo.php?productID=46-100 
			Porous Plastic sheet for the cold anesthesia box</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-Alarm Timer/Stopwatch</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Sharpie pens</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive Tape</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mini vortex</td>
			<td>Fisher</td>
			<td>14-955-151&#160;</td>
			<td>http://www.fishersci.com/ecomm/servlet/itemdetail 
			For mechanical agitation of the flies - any vortex would work.</td>
		</tr>
		<tr>
			<td>Corning Life Sciences DL No.:352017, Falcon test tube; round bottom; disposable; no closure, 14mL;&#160; 17 x 100mm</td>
			<td>Fisher</td>
			<td>14-959-8 &#160;&#160;</td>
			<td>http://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&#38;langId=-1&#38;catalogId=29104&#38;productId=2771811&#38;distype=0&#38;highlightProductsItemsFlag=Y&#38;fromSearch=1&#38;searchType=PROD&#38;hasPromo=0 
			These snap in place in the in-house made T-maze and counter-current apparatus (see text)</td>
		</tr>
		<tr>
			<td>cotton balls</td>
			<td></td>
			<td></td>
			<td>to close the vials after the experiment.</td>
		</tr>
		<tr>
			<td>trifold board and white bench cover</td>
			<td></td>
			<td></td>
			<td>to provide a white background, and a homogeneous light.</td>
		</tr>
		<tr>
			<td>white bench cover</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pounding pad</td>
			<td></td>
			<td></td>
			<td>any mouse pad works.</td>
		</tr>
		<tr>
			<td>large black cloth</td>
			<td></td>
			<td></td>
			<td>to cover the counter-current apparatus in phototaxis response.</td>
		</tr>
		<tr>
			<td>cool-white light&#160;</td>
			<td>Home Depot</td>
			<td>1000516563</td>
			<td>http://www.homedepot.ca/product/illume-26-fluorescent-plug-in-linear/911423 
			any similar linear light with fluorescent light bulb cool-white at 13-15W would work</td>
		</tr>
	</tbody>
</table>

straightforward assay for quantification of social avoidance in drosophila melanogaster

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by stressed conspecifics due to an odorant released during stress known as the Drosophila stress odorant (dSO). Through the use of the T-maze apparatus, one can quantify the avoidance of the dSO by responder flies in a very affordable and robust assay. Conditions necessary to obtain a strong performance are presented here. A stressful experience is necessary for the flies to emit dSO, as well as enough emitter flies to cause a robust avoidance response to the presence of dSO. Genetic background, but not their group size, strongly altered the avoidance of the dSO by the responder flies. Canton-S and Elwood display a higher performance in avoiding the dSO than Oregon and Samarkand strains. This behavioral assay will allow identification of mechanisms underlying this social behavior, and the assessment of the influence of genes and environmental conditions on both emission and avoidance of the dSO. Such an assay can be included in batteries of simple diagnostic tests used to identify social deficiencies of mutants or environmental conditions of interest.

The social avoidance assay is a robust test quantifying the ability of Drosophila melanogaster to recognize a stress signal (dSO) emitted by other flies, and thus assessing one aspect of social interactions. The assay is performed using an apparatus commonly used in various behavior assays known as a T-maze, which present the flies with a choice between two different options &#8212; left or right19,28-31. In this case, the efficiency at which the flies avoid the dSO is quantified by calculating and co...

Watch this Scientific Journal Video about Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster at JoVE.com

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Here, we present a protocol to quantify the avoidance of stressed individuals. This paradigm is powerful yet user-friendly and can be used to assess the influence of genes and environment on one kind of social interaction in Drosophila melanogaster.

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by ...

straightforward-assay-for-quantification-social-avoidance-drosophila

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

11.2K Views.  Yale University. Here, we present a protocol to quantify the avoidance of stressed individuals. This paradigm is powerful yet user-friendly and can be used to assess the influence of genes and environment on one kind of social interaction in Drosophila melanogaster.

Video: Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Combining Behavioral Endocrinology and Experimental Economics: Testosterone and Social Decision Making

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in rodents have shown a direct link between testosterone and aggressive behavior1 and folk wisdom adapts these findings to humans, suggesting that testosterone induces antisocial, egoistic or even aggressive behavior2. However, many researchers doubt a direct testosterone-aggression link in humans, arguing instead that testosterone is primarily involved in status-related behavior3,4. As a high status can also be achieved by aggressive and antisocial means it can be difficult to distinguish between anti-social and status seeking behavior.
We therefore set up an experimental environment, in which status can only be achieved by prosocial means. In a double-blind and placebo-controlled experiment, we administered a single sublingual dose of 0.5 mg of testosterone (with a hydroxypropyl-&beta;-cyclodextrin carrier) to 121 women and investigated their social interaction behavior in an economic bargaining paradigm. Real monetary incentives are at stake in this paradigm; every player A receives a certain amount of money and has to make an offer to another player B on how to share the money. If B accepts, she gets what was offered and player A keeps the rest. If B refuses the offer, nobody gets anything. A status seeking player A is expected to avoid being rejected by behaving in a prosocial way, i.e. by making higher offers.
The results show that if expectations about the hormone are controlled for, testosterone administration leads to a significant increase in fair bargaining offers compared to placebo. The role of expectations is reflected in the fact that subjects who report that they believe to have received testosterone make lower offers than those who say they believe that they were treated with a placebo. These findings suggest that the experimental economics approach is sensitive for detecting neurobiological effects as subtle as those achieved by administration of hormones. Moreover, the findings point towards the importance of both psychosocial as well as neuroendocrine factors in determining the influence of testosterone on human social behavior.

The procedure described in this protocol shows that testosterone administration and folk beliefs about testosterone may be associated with directly opposed social behaviors.

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in ...

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

A growing number of genetically encoded tools are becoming available that allow non-invasive manipulation of the neural activity of specific neurons in Drosophila melanogaster1. Chief among these are optogenetic tools, which enable the activation or silencing of specific neurons in the intact and freely moving animal using bright light. Channelrhodopsin (ChR2) is a light-activated cation channel that, when activated by blue light, causes depolarization of neurons that express it. ChR2 has been effective for identifying neurons critical for specific behaviors, such as CO2 avoidance, proboscis extension and giant-fiber mediated startle response2-4. However, as the intense light sources used to stimulate ChR2 also stimulate photoreceptors, these optogenetic techniques have not previously been used in the visual system. Here, we combine an optogenetic approach with a mutation that impairs phototransduction to demonstrate that activation of a cluster of loom-sensitive neurons in the fly's optic lobe, Foma-1 neurons, can drive an escape behavior used to avoid collision. We used a null allele of a critical component of the phototransduction cascade, phospholipase C-&beta;, encoded by the norpA gene, to render the flies blind and also use the Gal4-UAS transcriptional activator system to drive expression of ChR2 in the Foma-1 neurons. Individual flies are placed on a small platform surrounded by blue LEDs. When the LEDs are illuminated, the flies quickly take-off into flight, in a manner similar to visually driven loom-escape behavior. We believe that this technique can be easily adapted to examine other behaviors in freely moving flies.

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Genetically encoded optogenetic tools enable noninvasive manipulation of specific neurons in the Drosophila brain. Such tools can identify neurons whose activation is sufficient to elicit or suppress particular behaviors. Here we present a method for activating Channelrhodopsin2 that is expressed in targeted neurons in freely walking flies.

A  growing number of genetically encoded tools are becoming available that allow non-invasive  manipulation of the neural activity of specific neurons in ...

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such behavior is spontaneous locomotor activity. Here we describe our protocol that utilizes Drosophila population monitors and a tracking system that allows continuous monitoring of the spontaneous locomotor activity of flies for several days at a time. This method is simple, reliable, and objective and can be used to examine the effects of aging, sex, changes in caloric content of food, addition of drugs, or genetic manipulations that mimic human diseases.

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster are useful in studying genetic or environmental manipulations that affect behaviors such as spontaneous locomotor activity.&#160; Here we describe a protocol that utilizes monitors with infrared beams and data analysis software to quantify spontaneous locomotor activity.&#160;

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such ...

Conditions Affecting Social Space in Drosophila melanogaster

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a straightforward manner. As we previously demonstrated 1, in a two-dimensional chamber, we first force the flies to form a tight group, subsequently allowing them to take their preferred distance from each other. After the flies have settled, we measure the distance to the closest neighbor (or social space), processing a static picture with free online software (ImageJ). The analysis of the distance to the closest neighbor allows researchers to determine the effects of genetic and environmental factors on social interaction, while controlling for potential confounding factors. Diverse factors such as climbing ability, time of day, sex, and number of flies, can modify social spacing of flies. We thus propose a series of experimental controls to mitigate these confounding effects. This assay can be used for at least two purposes. First, researchers can determine how their favorite environmental shift (such as isolation, temperature, stress or toxins) will impact social spacing 1,2. Second, researchers can dissect the genetic and neural underpinnings of this basic form of social behavior 1,3. Specifically, we used it as a diagnostic tool to study the role of orthologous genes thought to be involved in social behavior in other organisms, such as candidate genes for autism in humans 4.

Conditions Affecting Social Space in Drosophila melanogaster

The effect of genes and environment on social space of Drosophila melanogaster can be quantified through a powerful but straightforward analytical paradigm. We show here different factors that can affect this social space, and thus need to be taken into consideration when designing experiments in this paradigm.

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a ...

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

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense organs provide an excellent model system for the study of mechanosensation. This protocol describes a simple touch behavior in fruit flies which can be used to identify mutations that interfere with mechanosensation. The tactile stimulation of a macrochaete bristle on the thorax of flies elicits a grooming reflex from either the first or third leg. Mutations that interfere with mechanotransduction (such as NOMPC), or with other aspects of the reflex arc, can inhibit the grooming response. A traditional screen of adult behaviors would have missed mutants that have essential roles during development. Instead, this protocol combines the touch screen with mosaic analysis with a repressible cell marker (MARCM) to allow for only limited regions of homozygous mutant cells to be generated and marked by the expression of green fluorescent protein (GFP). By testing MARCM clones for abnormal behavioral responses, it is possible to screen a collection of lethal p-element mutations to search for new genes involved in mechanosensation that would have been missed by more traditional methods.

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

In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal.

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense ...

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

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. When selecting sites to deposit their eggs, female flies are capable of ranking the relative attractiveness of their options and choosing the &#34;greater of two goods.&#34; However, most egg-laying preference assays are not practical if one wants to take a systematic genetic screening approach to search for the circuit basis underlying this simple decision-making process, as they are population-based and laborious to set up. To increase the throughput of studying of egg-laying preferences of single females, we developed custom chambers that each can simultaneously assay egg-laying preferences of up to thirty individual flies as well as a protocol that ensures each female has a high egg-laying rate (so that their preference is readily discernable and more convincing). Our approach is simple to execute and produces very consistent results. Additionally, these chambers can be equipped with different attachments to allow video recording the egg-laying animals and to deliver light for optogenetics studies. This article provides the blueprints for fabricating these chambers and the procedure for preparing the flies to be assayed in these chambers.

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

This protocol describes a high throughput assay for testing egg-laying preferences of Drosophila melanogaster at single-animal resolution. This assay provides a simple, efficient, and scalable platform to identify genes and circuit components that control a simple decision-making process.

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. ...

Foraging Path-length Protocol for Drosophila melanogaster Larvae

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral variation. The larval path-length assay was developed to measure individual differences in foraging behavior that were later linked to the foraging gene. Larval path-length is an easily scored trait that facilitates the collection of large sample sizes, at minimal cost, for genetic screens. Here we provide a detailed description of the current protocol for the larval path-length assay first used by Sokolowski. The protocol details how to reproducibly handle test animals, perform the behavioral assay and analyze the data. An example of how the assay can be used to measure behavioral plasticity in response to environmental change, by manipulating feeding environment prior to performing the assay, is also provided. Finally, appropriate test design as well as environmental factors that can modify larval path-length such as food quality, developmental age and day effects are discussed.

Foraging Path-length Protocol for Drosophila melanogaster Larvae

We provide a detailed protocol for a Drosophila melanogaster foraging path-length assay. We discuss the preparation and handling of test animals, how to perform the assay and analyze the data.

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral ...

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of the right type and quantity of food therefore has a major influence on quality of life. Research on feeding behavior focuses on the underlying processes that ensure actual feeding and unravels the role of factors regulating internal energy homeostasis and the neuronal bases of decision-making. The model organism Drosophila melanogaster, with its great variety of genetically traceable tools for labeling and manipulating single neurons, allows mapping of neuronal networks and identification of molecular signaling cascades involved in the regulation of food intake. This report demonstrates the CApillary FEeder assay (CAFE) and shows how to measure food intake in a group of flies for time spans ranging from hours to days. This easy-to-use assay consists of glass capillaries filled with liquid food that flies can freely access and feed on. Food consumption in the assay is accurately determined using simple measurement tools. Herein we describe step-by-step the method from setup to successful execution of the CAFE assay, and provide practical examples to analyze the food intake of a group of flies under controlled conditions. The reader is guided through possible limitations of the assay, and advantages and disadvantages of the method compared to other feeding assays in D. melanogaster are evaluated.

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

The CApillary FEeder (CAFE) assay is a simple, budget-friendly, highly reliable method for investigating mechanisms underlying food intake. Used with the highly versatile genetic model organism Drosophila melanogaster, it provides a powerful means of gaining new insights into regulatory mechanisms of food intake.

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of ...

Quantification of Drosophila Grooming Behavior

Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a grooming assay protocol and novel chamber design that is cost-efficient and scalable for either small or large-scale studies of Drosophila grooming. Flies are dusted all over their body with Brilliant Yellow dye and given time to remove the dye from their bodies within the chamber. Flies are then deposited in a set volume of ethanol to solubilize the dye. The relative spectral absorbance of dye-ethanol samples for groomed versus ungroomed animals are measured and recorded. The protocol yields quantitative data of dye accumulation for individual flies, which can be easily averaged and compared across samples. This allows experimental designs to easily evaluate grooming ability for mutant animal studies or circuit manipulations. This efficient procedure is both versatile and scalable. We show work-flow of the protocol and comparative data between WT animals and mutant animals for the Drosophila type I Dopamine Receptor (DopR).

This protocol describes a scalable individual grooming assay technique in Drosophila that yields robust, quantitative data to measure grooming behavior. The method is based on comparing the difference in dye accumulation on the bodies of un-groomed versus groomed animals over a set period of time.

Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a ...

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Summary

Explore More Videos

Chapters in this video

Combining Behavioral Endocrinology and Experimental Economics: Testosterone and Social Decision Making

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Conditions Affecting Social Space in Drosophila melanogaster

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

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

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

Foraging Path-length Protocol for Drosophila melanogaster Larvae

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

Quantification of Drosophila Grooming Behavior