This research was supported by grants from the National Institute of General Medical Sciences (GM099883 and GM074675) to E.A.K. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank the people from our machine shop at Harvard Medical School for the design of the behavioral apparatus (contact: <a href="http://mikesmachine.com">http://mikesmachine.com</a>).
Author contributions: S.T., B.C. designed the behavioral apparatus. S.T. designed and optimized the experimental protocol. S.T. and B.C. performed and analyzed the experiments. S.T., E.A.K. and B.C. wrote the paper.

Note: The following is a step-by-step description of the experimental protocol we use to allow reliable triggering of aggression between pairs of male fruit flies (D. melanogaster). This procedure induces the formation of a &#34;loser&#34; effect. Using this protocol, a short-term &#34;winner&#34; effect also has been recently reported 5,6. Figure 1 shows the timeline of the behavioral experiments.
<img alt="Figure 1" src="/files/ftp_upload/53395/53395fig1.jpg" />
Figure 1.&#160;Timeline of aggression experiment. On day 1, late-stage male pupae (when wings become black) are isolated in freshly prepared isolation vials. On day 5 after emerging as adults, 4 day-old male flies are anesthetized with CO2 and painted with different colors for identification purposes. On day 7, food cups and fighting chambers are prepared to set up the behavioral experiments. Two 6-7 day-old adult males are loaded into the chambers and are able to interact. <a href="https://www.jove.com/files/ftp_upload/53395/53395fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
1. Day 1: Social Isolation of Pupae
<ol>
	<li>Raise and maintain fly stocks in an environmentally controlled 12 hr light/12 hr dark cycle incubator at 25 &#176;C and 50% relative humidity.</li>
	<li>Prepare isolation vials: Heat fresh fly food until melted. Using a Pasteur pipette, transfer approximately 1.5 ml of melted fly food into single empty glass test tubes (1.6 x 10 cm). Allow the food to solidify.</li>
	<li>Isolate pupae: With a fine paintbrush, gently remove a late-stage male pupa (when wings become black) from the stock vial and place it on the side of a freshly prepared food containing test tube. Close isolation vials with cotton (Figure 2).</li>
	<li>Place isolation vials containing pupae into the 12 hr light/12 hr dark incubator at 25 &#176;C and 50% relative humidity.</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/53395/53395fig2.jpg" />
Figure 2.&#160;Social Isolation. One late-stage male pupa is placed on the side on a freshly prepared isolation vial. Isolation vials containing pupa are placed in the 12 hr light/12 hr dark incubator at 25 &#176;C and 50% relative humidity. <a href="https://www.jove.com/files/ftp_upload/53395/53395fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Day 5: Painting the Flies for Individual Identification Purposes
<ol>
	<li>Perform this step at day 5 with 4 day-old male flies and two days prior the behavioral experiments to minimize the negative effect of CO2 anesthesia.</li>
	<li>Anesthetize fly with CO2.</li>
	<li>With a fine toothpick, place a small dot of acrylic paint on the thorax. Avoid placing paint on the head, wings, abdomen or legs (Figure 3).</li>
	<li>Let paint dry for approximately 10 sec, and gently transfer flies back to their original isolation vials.</li>
	<li>Return isolation vials to the 12 hr light/12 hr dark incubator at 25 &#176;C and 50% relative humidity.</li>
</ol>
<img alt="Figure 3" src="/files/ftp_upload/53395/53395fig3.jpg" />
Figure 3.&#160;Individual identification. After anesthetizing a fly with CO2, a small drop of acrylic paint is applied on the thorax. For individual identification purpose, different colors are used (white and blue here). See discussion below about use of CO2 at this step. <a href="https://www.jove.com/files/ftp_upload/53395/53395fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Day 7: Preparing the Apparatus
<ol>
	<li>Prepare Food Cups:
	<ol>
		<li>Heat fresh fly food until it melts.</li>
		<li>Using a Pasteur pipette, transfer approximately 0.8 ml food to the lid of a screw cap vial (1.5 cm diameter, 1 cm height) and allow to cool. Avoid air bubbles.</li>
		<li>Make fresh yeast paste by mixing dry yeast with a few drops of water. When thick, apply a small drop of yeast paste to the center of the food cup surface with a toothpick (Figure 4).</li>
	</ol>
	</li>
</ol>
<img alt="Figure 4" src="/files/ftp_upload/53395/53395fig4.jpg" />
Figure 4.&#160;Preparing the food cup. A food cup is prepared with fresh fly food. After the food solidifies, a dot of yeast paste is placed in the center of the food cup surface. <a href="https://www.jove.com/files/ftp_upload/53395/53395fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Prepare Individual Fighting Chambers:
	<ol>
		<li>Thoroughly wash the apparatus and individual fighting chambers with water before use each time. Dry with paper towel before use.</li>
		<li>Place a food cup in the center of each fighting chamber and fix in place with a drop of putty.</li>
		<li>Cover fighting chambers with the apparatus lid (Figure 5).</li>
		<li>Place the apparatus into a high-humidity environment (60%) at 25 &#176;C.</li>
		<li>Place a light source above the apparatus to illuminate the fighting chambers and set a video camera for recording from above.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 5" src="/files/ftp_upload/53395/53395fig5.jpg" />
Figure 5.&#160;Preparing the individual fighting chamber. The freshly prepared food cup with a dot of yeast paste on the surface is fixed into washed individual fighting chambers. Chambers are closed with the two-piece cover allowing insertion of an opaque plastic divider into individual fighting chambers. <a href="https://www.jove.com/files/ftp_upload/53395/53395fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Day 7: Behavioral Experiment
<ol>
	<li>Perform the behavioral experiments with 6-7 day-old male flies.</li>
	<li>Insert plastic dividers into each fighting chamber.</li>
	<li>Remove the cotton plug from the isolation tube and position the tube below an open hole on the side of the apparatus.</li>
	<li>Let the fly enter into the fighting chamber by negative geotaxis, usually within a few seconds.</li>
	<li>Close the sliding wall after the fly has left the tube and entered the chamber.</li>
	<li>Repeat the procedure to introduce the second fly into the fight chamber on the other side of the apparatus.</li>
	<li>Place the apparatus back in the illuminated videotaping position.</li>
	<li>Begin video recording and remove the plastic divider to allow flies to interact.
	<ol>
		<li>For Aggression Experiments:
		<ol>
			<li>In these experiments, record fights for at least 20 min to ensure the formation of strong dominance relationships.</li>
			<li>After the desired time, stop video recording.</li>
			<li>Remove flies from the fighting chambers by reversing the procedure described above.</li>
			<li>Thoroughly wash the behavioral apparatus with mild detergent and water after each use and rinse.</li>
		</ol>
		</li>
		<li>For &#34;Loser&#34; Effect Experiments: 
		Note: In these experiments, first fights are considered as the conditioning phase and second fights as the test phase. The period of rest between fights can differ but 10 min allows formation of a strong &#34;loser&#34; effect.
		<ol>
			<li>Follow the procedure described above to perform a first fight.</li>
			<li>CRITICAL STEP: To induce the formation of a &#34;loser&#34; mentality, ensure that first fights are at least 20 min long in order to establish strong dominance relationships.&#160; After 20 min, re-insert the plastic divider into fighting chambers to separate the flies.</li>
			<li>After a 10 min period of rest, gently remove the plastic divider to allow flies to interact for second fights.</li>
			<li>At the end of the second 20 min fight, stop the video recording.</li>
			<li>Remove flies from behavioral chambers as above.</li>
			<li>Once again, thoroughly clean the behavioral apparatus.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
5. Behavioral Analysis
<ol>
	<li>Transferring Movies
	<ol>
		<li>Connect cameras to the computer.</li>
		<li>Convert flies .MTS to .mov flies and join movies clips together, using video converting software and the option &#34;join selected clips&#34;.</li>
		<li>Observe and analyze each movie with video reader software.</li>
	</ol>
	</li>
	<li>Scoring Behavior 
	Note: this can be done manually or using behavioral analysis program software if available. The illustration below describes a possible manual analysis for a &#34;loser&#34; effect.
	<ol>
		<li>Score the time of the first encounter between the flies on the food cup (encounters are interactions between pairs of flies that last at least 2 sec) and the time of appearance of the first lunge (a behavioral pattern in which one fly rises high on its hind legs and snaps down on and attempts to grab the opponent). Then, score the latency to lunge (the interval between the first encounter and the first lunge).</li>
		<li>Count the total number of encounters and lunges during a fight. 
		Note: If desired, one also can score other behavioral patterns, like wing threat, boxing, fencing, retreat or the numbers of encounters before the first lunge, for example.</li>
		<li>Score the time of establishment of dominance (time between the first encounter and the time when one fly retreats for three consecutive times off the food cup after receiving lunges). Note which fly is the winner and which the loser of the fight.</li>
	</ol>
	</li>
	<li>Calculating a &#34;Loser&#34; Effect
	<ol>
		<li>Divide the number of fights in which previous losers lose 2nd fights (Losers that lose: LL) by the total number of fights, and multiply by 100. The percentage obtained represents the &#34;loser&#34; effect: 
		[(# flies LL) / (# total fight)] x 100</li>
		<li>Use a Chi-square test to compare whether the &#34;loser&#34; effect is statistically different from the expected value of 50%.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Chen, S., Lee, A. Y., Bowens, N. M., Huber, R., Kravitz, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fighting+fruit+flies:+a+model+system+for+the+study+of+aggression.">Fighting fruit flies: a model system for the study of aggression.</a> Proc Natl Acad Sci U S A. 99, 5664-5668 (2002).</li><li>Hsu, Y., Earley, R. L., Wolf, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+aggressive+behaviour+by+fighting+experience:+mechanisms+and+contest+outcomes.">Modulation of aggressive behaviour by fighting experience: mechanisms and contest outcomes.</a> Biol Rev Camb Philos Soc. 81, 33-74 (2006).</li><li>Yurkovic, A., Wang, O., Basu, A. C., Kravitz, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learning+and+memory+associated+with+aggression+in+Drosophila+melanogaster.">Learning and memory associated with aggression in Drosophila melanogaster.</a> Proc Natl Acad Sci U S A. 103, 17519-17524 (2006).</li><li>Penn, J. K., Zito, M. F., Kravitz, E. A. <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+social+defeat+reduces+aggression+in+a+highly+aggressive+strain+of+Drosophila.">A single social defeat reduces aggression in a highly aggressive strain of Drosophila.</a> Proc Natl Acad Sci U S A. 107, 12682-12686 (2010).</li><li>Trannoy, S., Chowdhury, B., Kravitz, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Handling+alters+aggression+and+'loser'+effect+formation+in+Drosophila+melanogaster.">Handling alters aggression and 'loser' effect formation in Drosophila melanogaster.</a> Learn Mem. 22, 64-68 (2015).</li><li>Trannoy, S., Kravitz, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learning+and+memory+during+aggression+in+Drosophila:+handling+affects+aggression+and+the+formation+of+a+''loser''+effect.">Learning and memory during aggression in Drosophila: handling affects aggression and the formation of a ''loser'' effect.</a> Journal of Nature and Science. 1 (3), e56 (2015).</li><li>Alekseyenko, O. V., Chan, Y. B., Li, R., Kravitz, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+dopaminergic+neurons+that+modulate+aggression+in+Drosophila.">Single dopaminergic neurons that modulate aggression in Drosophila.</a> Proc Natl Acad Sci U S A. 110, 6151-6156 (2013).</li><li>Dierick, H. A., Greenspan, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotonin+and+neuropeptide+F+have+opposite+modulatory+effects+on+fly+aggression.">Serotonin and neuropeptide F have opposite modulatory effects on fly aggression.</a> Nat Genet. 39, 678-682 (2007).</li><li>Williams, M. 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=Regulation+of+aggression+by+obesity-linked+genes+TfAP-2+and+Twz+through+octopamine+signaling+in+Drosophila.">Regulation of aggression by obesity-linked genes TfAP-2 and Twz through octopamine signaling in Drosophila.</a> Genetics. 196, 349-362 (2014).</li><li>Chase, I. D., Bartolomeo, C., Dugatkin, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aggressive+interactions+and+inter-contest+interval:+how+long+do+winners+keep+winning?.">Aggressive interactions and inter-contest interval: how long do winners keep winning?.</a> Anim. Behav. 48, 393-400 (1994).</li><li>Goessmann, C., Hemelrijk, C., Huber, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+formation+and+maintenance+of+crayfish+hierarchies:+behavioral+and+self-structuring+properties.">The formation and maintenance of crayfish hierarchies: behavioral and self-structuring properties.</a> Behav Ecol Sociobiol. 48, 418-428 (2000).</li><li>Sturtevant, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experiments+on+sex+recognition+and+the+problem+of+sexual+selection+in+Drosophila.">Experiments on sex recognition and the problem of sexual selection in Drosophila.</a> J Animal Behav. 5, 351-366 (1915).</li><li>Jacobs, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+light+on+mating+of+Drosophila+melanogaster.">Influence of light on mating of Drosophila melanogaster.</a> Ecology. 41, 182-188 (1960).</li><li>Dow, M. A., von Schilcher, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aggression+and+mating+success+in+Drosophila+melanogaster.">Aggression and mating success in Drosophila melanogaster.</a> Nature. 254, 511-512 (1975).</li><li>Hoffmann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+laboratory+study+of+male+territoriality+in+the+sibling+species+Drosophila+melanogaster+and+D.+simulans.">A laboratory study of male territoriality in the sibling species Drosophila melanogaster and D. simulans.</a> Anim. Behav. 35, 807-818 (1987).</li><li>Vrontou, E., Nilsen, S. P., Demir, E., Kravitz, E. A., Dickson, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=fruitless+regulates+aggression+and+dominance+in+Drosophila.">fruitless regulates aggression and dominance in Drosophila.</a> Nat Neurosci. 9, 1469-1471 (2006).</li><li>Nilsen, S. P., Chan, Y. B., Huber, R., Kravitz, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gender-selective+patterns+of+aggressive+behavior+in+Drosophila+melanogaster.">Gender-selective patterns of aggressive behavior in Drosophila melanogaster.</a> Proc Natl Acad Sci U S A. 101, 12342-12347 (2004).</li><li>Fernandez, M. P., Kravitz, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aggression+and+courtship+in+Drosophila:+pheromonal+communication+and+sex+recognition.">Aggression and courtship in Drosophila: pheromonal communication and sex recognition.</a> J Comp Physiol A Neuroethol Sens Neural Behav Physiol. 199, 1065-1076 (2013).</li><li>Alekseyenko, O. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+serotonergic+neurons+that+modulate+aggression+in+Drosophila.">Single serotonergic neurons that modulate aggression in Drosophila.</a> Current biology : CB. 24, 2700-2707 (2014).</li><li>Asahina, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tachykinin-expressing+neurons+control+male-specific+aggressive+arousal+in+Drosophila.">Tachykinin-expressing neurons control male-specific aggressive arousal in Drosophila.</a> Cell. 156, 221-235 (2014).</li><li>Andrews, J. 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=Octopamine+neuromodulation+regulates+Gr32a-linked+aggression+and+courtship+pathways+in+Drosophila+males.">Octopamine neuromodulation regulates Gr32a-linked aggression and courtship pathways in Drosophila males.</a> PLoS genetics. 10, e1004356 (2014).</li><li>Zwarts, 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=Complex+genetic+architecture+of+Drosophila+aggressive+behavior.">Complex genetic architecture of Drosophila aggressive behavior.</a> Proc Natl Acad Sci U S A. 108, 17070-17075 (2011).</li><li>Dierick, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+quantifying+aggression+in+male+Drosophila+melanogaster.">A method for quantifying aggression in male Drosophila melanogaster.</a> Nat Protoc. 2, 2712-2718 (2007).</li><li>Wang, 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=Hierarchical+chemosensory+regulation+of+male-male+social+interactions+in+Drosophila.">Hierarchical chemosensory regulation of male-male social interactions in Drosophila.</a> Nat Neurosci. 14, 757-762 (2011).</li><li>Davis, S. M., Thomas, A. L., Nomie, K. J., Huang, L., Dierick, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tailless+and+Atrophin+control+Drosophila+aggression+by+regulating+neuropeptide+signalling+in+the+pars+intercerebralis.">Tailless and Atrophin control Drosophila aggression by regulating neuropeptide signalling in the pars intercerebralis.</a> Nat Commun. 5, 3177 (2014).</li><li>Yuan, Q., Song, Y., Yang, C. H., 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=Female+contact+modulates+male+aggression+via+a+sexually+dimorphic+GABAergic+circuit+in+Drosophila.">Female contact modulates male aggression via a sexually dimorphic GABAergic circuit in Drosophila.</a> Nat Neurosci. 17, 81-88 (2014).</li><li>Perron, J. M., Huot, L., Corrivault, G. W., Chawla, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+carbon+dioxide+anaesthesia+on+Drosophila+melanogaster.">Effects of carbon dioxide anaesthesia on Drosophila melanogaster.</a> J Insect Physiol. 18, 1869-1874 (1972).</li><li>Joachim, D., Curtsinger, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotype+and+anesthetic+determine+mate+choice+in+Drosophila+melanogaster.">Genotype and anesthetic determine mate choice in Drosophila melanogaster.</a> Behav Genet. 20, 73-79 (1990).</li><li>Barron, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaesthetising+Drosophila+for+behavioural+studies.">Anaesthetising Drosophila for behavioural studies.</a> J Insect Physiol. 46, 439-442 (2000).</li><li>Greenspan, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fly Pushing: The theory and Practice of Drosophila Genetics. , (1997).</li><li>Cabral, L. G., Foley, B. R., Nuzhdin, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+sex+trade+with+violence+among+genotypes+in+Drosophila+melanogaster?.">Does sex trade with violence among genotypes in Drosophila melanogaster?.</a> PLoS One. 3 (4), e1986 (2008).</li></ol>

The authors have nothing to disclose.

The modern era of using Drosophila with its powerful genetic methods as a model organism for the study of aggression began about a dozen years ago with the introduction of novel experimental arenas in which reliable fighting behavior could be obtained with single pairs of flies 1,7,8,17,19. These arenas included desired resources (a food cup, potential mates) and a space for loser flies to retreat into. When results obtained with these arenas were added to previous knowledge about fruit fly fight behavior, Drosophila melanogaster was firmly established as a highly desirable model system for examining a complex behavior like aggression and is used in many laboratories. The summed results obtained revealed that: (a) flies exhibit aggression in competition for food resources, access to mates or for defense of territory 14,15; (b) fights are composed of a sequential series of stereotypical behavioral patterns 1; (c) male and female flies fight in same sex pairings (some patterns are male specific, some are female specific and some are shared by both sexes) 17; (d) only male fights go to high intensity levels and result in the formation of hierarchical relationships with clear &#171; winner &#187; and &#171; loser &#187; flies 17; (e) a single gene fruitless, of the sex determination hierarchy, determines whether flies fight like males or females 16 and (f) multiple hormonal substances including amines and peptides influence aggression in fruit flies 7,8,19,20.
Here, a novel approach is described for the study of fruit fly aggression in which handling of flies during experiments is eliminated. This offers a significant improvement in the experimental protocol as all behavioral measurements are improved 5,6, new information relating to the behavior has been gained and the learning and memory that accompanies male fly fights is greatly improved. To illustrate, only two previous studies have reported the formation of &#34;loser&#34; effects in this model system 3,4. In both of these, larger chambers than the ones described in this protocol were used, flies were transferred into and out of fight arenas by aspiration, and longer fight times and interval times between fights were necessary to generate clear &#34;loser&#34; effects. Our recent study demonstrated that eliminating handling from the experimental protocol, clear dominance relationships between pairs of D. melanogaster male flies are observed after only 20 min of interaction. This amount of time is sufficient to induce significant changes in fighting strategies in both flies during 1st fights and to reliably observe the formation of strong &#34;loser&#34; effects in 2nd fights. In addition, for the first time, clear short-term &#34;winner&#34; effects also were observed during 2nd fights 5,6.
For success using this protocol, some critical points need to be noted. To induce and observe robust aggressive behavioral patterns we recommend using 6-7 day-old male flies that have been held in social isolation since the late pupal stage. In general, the availability of resources that flies are willing to compete over (food, mates) is highly desirable in order to obtain strong fighting behavior and establishment of dominance relationships. During this protocol, flies are painted after anesthesia using CO2, and that may be of concern as our recently published study showing that anesthetizing flies seriously interferes with subsequent behavior 5. Possible ways to avoid anesthesia might be to not paint animals if it is not important to keep track of individuals. If painting is necessary, however, the anesthesia should be used 48 hr before the animals are used for experiments, or alternative approaches to identify individuals also might be used 31. To induce strong &#34;loser&#34; and &#34;winner&#34; effects, it is crucial that flies establish and maintain a dominance relationship during first fights. In the protocol presented here, using the wild-type CS strain at 25 &#176;C, 20 min of interaction is sufficient to generate clear &#34;winners&#34; and &#34;losers&#34; and to observe changes in the fight strategies of both individuals. In experiments performed at different temperatures than 25 &#176;C or with other genotypes than CS, however, the average time for establishing a robust dominance relationship might be different. Accordingly, the fighting period should be adjusted by performing sets of preliminary experiments. Finally, how pairs of flies are separated after their first fights might be important in preserving the memory of fight outcomes. It is probably important to reduce movement of the apparatus and to be gentle during the introduction of the plastic divider in order to minimize disturbance of the animals.
The design of the apparatus and the two-piece cover allowing separation of the behavioral chambers into two equal compartments, make it easy to separate the two flies after first fights and to introduce new opponents in 2nd fights. Also, as shown in Table 2, with small adjustments of the experimental protocol, the same apparatus can be used to study courtship, locomotion or indeed most fruit fly behaviors in what should be less stressful conditions for the experimental animals. Possible further improvements on the procedure might be to introduce some form of thermostatic control into the apparatus (piezoelectric element or a coil to circulate temperature-controlled fluid) to allow easy use of temperature sensitive reagents (shibirets1, dTRPA1 channel).
In summary, we have devised a novel apparatus that offers a new strategy for the study of aggression in fruit flies in which handling of flies prior to, during and after experiments was eliminated. All behavioral measures are improved with the new apparatus when compared to several other common ways of handling flies. Perhaps a more general comment to make is that essentially all behavioral experiments in laboratory environments involve experimenters picking up or in other ways, handling animals. Despite efforts to be gentle in this handling, this is likely to be stressful for the animals. It might be useful to devise methods to eliminate this handling whenever possible, so that animals might be observed under slightly more normal conditions.

Aggression in animal systems is strongly associated with the acquisition and holding of resources such as food, territory and mates. Given the important role this plays in the fitness of individuals, it is not surprising that aggression has evolved across the animal kingdom. As an adaptive trait that directly benefits individuals, a strong learning and memory component is associated with aggression. In competition for social rank, previous fighting experience influences the outcome of later contests. In general, prior losing experience increases, and winning experience decreases, the probability of losing later contests (called &#34;loser&#34; and &#34;winner&#34; effects). &#34;Loser&#34; effects have been observed in a wide range of species, and some reports suggest that these can last for several days while &#34;winner&#34; effects usually are of shorter duration 2,10,11.
The first report of aggressive behavior in fruit flies (D. ampelophila) was by Sturtevant in 1915 in a paper concerned with sex recognition and sex selection 12. Half a century later, more complete examinations of male fruit fly aggression were made, that described most of the behavioral patterns observed during fruit fly fights. These experiments were mostly carried out in small groups of male and female flies observed over hours 13-15. Recently, with the addition of powerful genetic tools, dyadic single sex fight setups and shorter times of observation, D. melanogaster has emerged as an important model system for the study of the biology of aggression 1,16. Analysis of fights between same sex pairs of male and female flies demonstrated that aggression involves stereotypical behavioral patterns that transition from one to the other in a statistically reliable manner 1,17. Some of the behavioral patterns observed are sex-specific while others are observed in fights in both sexes. Male fights go to higher intensity levels than female fights and result in the formation of dominance relationships with clear &#34;winners&#34; and &#34;losers&#34;. At present, many laboratories have started investigating the biochemical 18, neural 7,19-21, and genetic 22 underpinnings of aggression. Unfortunately, a meta-analysis of studies to gain insight into fight dynamics and the formation and maintenance of dominance relationships is problematic due to the use of a multiplicity of experimental procedures in different laboratories. Essentially all of the techniques described in the literature involve handling and manipulation of flies to introduce them into arenas 19,23,24 and during the behavioral experiment 3,4 to transfer flies out of arenas. Gentle aspiration is the most common way to manipulate flies 3,4,23-25, but cold or CO2 anesthesia are also used 9,26, even though previous studies already have reported that these procedures have deleterious effects on fly behavior 27,28. A period of at least 24 hr is recommended after the use of any anesthetics to minimize their effects on behavior 29,30.
Flies learn from previous fighting experience and modify their behavioral pattern usage in new situations, suggesting that learning and memory accompany and are consequences of agonistic encounters. In that sense, fly fights resemble to operant conditioning learning situations in which flies learn that a strategy has worked and then use it more and more often during subsequent encounters. Flies change their fighting strategies after dominance relationships have been established during fights, with winners lunging more and more and losers less and less. After a period of separation, previous losers show much more submissive behavior and are highly likely to lose 2nd fights when paired with na&#239;ve flies or previous winners 3,4. However, the absence of an experimental procedure that excludes handling of flies has made detailed studies of &#34;loser&#34; effects difficult. In a recent study, we compared two experimental procedures routinely used in laboratories (aspiration and cold-anesthesia) to introduce flies into behavioral chambers to the new procedure that eliminates handling. The results showed that cold-anesthesia had far greater negative effects on aggression than aspiration, but even aspiration reduced the level of aggressiveness by flies. Aspiration did, however, cause highly significant effects on the learning and memory that accompany aggression. Following identical protocol with two experimental procedures (aspiration and no handling), a robust &#34;loser&#34; effect was only observed when the handling of flies was eliminated from the experimental procedure 5,6.
Ideally, studies of Drosophila aggression in laboratories should include environmental situations that flies normally encounter in the wild (competition for resources, territory to defend and space to escape). In addition, experimental conditions should be optimized to reliably induce, observe and interpret the behavior under examination (try to minimize animal handling, limit the use of CO2 as anesthetic, and standardize the experimental procedures). In attempting to address most or all of these issues, we designed a new behavioral apparatus that eliminates handling of flies before, during and after introducing them to behavioral arenas. With these apparatus, flies use their innate negative geotaxis to be moved and transferred into and out of the fight chambers. By eliminating handling of flies, the protocol aims to: (a) reduce the behavioral variability among individuals; (b) decrease the time necessary for flies to interact and generate clear dominance relationships (males); and (c) reliably induce strong enough behavioral changes to allow formation of &#34;loser&#34; effects.
This protocol describes a new experimental apparatus and a step by step procedure to analyze aggression and to allow the formation of a strong &#34;loser&#34; effect in D. melanogaster. We expect that this behavioral apparatus can be easily adapted for the study of other social behaviors exhibited by fruit flies.

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a new approach that eliminates handling for studying aggression and the "loser" effect in drosophila melanogaster

Aggressive behavior in Drosophila melanogaster is composed of the sequential expression of stereotypical behavioral patterns (for analysis see 1). This complex behavior is influenced by genetic, hormonal and environmental factors. As in many organisms, previous fighting experience influences the fighting strategy of flies and the outcome of later contests: losing a fight increases the probability of losing later contests, revealing &#34;loser&#34; effects that likely involve learning and memory 2-4. The learning and memory that accompanies expression of complex social behaviors like aggression, is sensitive to pre-test handling of animals 5,6. Many experimental procedures are used in different laboratories to study aggression 7-9, however, no routinely used protocol that excludes handling of flies is currently available. Here, we report a new behavioral apparatus that eliminates handling of flies, using instead their innate negative geotactic responses to move animals into or out of fighting chambers. In this protocol, small circular fight arenas containing a food cup are divided into two equal halves by a removable plastic slider prior to introduction of flies. Flies enter chambers from their home isolation vials via sliding chamber doors and geotaxis. Upon removal of plastic sliders, flies are free to interact. After specified time periods, flies are separated again by sliders for subsequent experimentation. All of this is done easily without handling of individual flies. This apparatus offers a novel approach to study aggression and the associated learning and memory, including the formation of &#34;loser&#34; effects in fly fights. In addition, this new general-purpose behavioral apparatus can be employed to study other social behaviors of flies and should, in general, be of interest for investigating experience-related changes in fundamental behavioral processes.

This section presents the design of the behavioral chamber and the analysis of a typical set of behavioral experiments following the protocol described above measuring aggression and the formation of a &#34;loser&#34; effect. Also illustrated are samples of other behaviors that can be measured using this apparatus.
Figure 6 shows a schematic representation of the new apparatus. The apparatus is composed of three cylindrical wells (dimension: 22.86 mm diameter, 17 mm height) that are the fighting chambers. The two-piece cover closes the fighting chambers on the top and allows insertion of a thin opaque plastic divider that splits the chambers into two equal sections. On each side of the apparatus, sliding walls with three holes (15.88 mm diameter) allow loading of flies into the fighting chambers by negative geotaxis directly from their isolation vials. After flies enter the fighting chambers, the sliding walls are used to close the sides of the all chambers. At this point, the two flies are within the fight arenas but cannot interact due to the dividers. By removing these, the flies are free to interact and the subsequent behavioral patterns can be observed and recorded.
Aggression experiments: 38 fights between pairs of wild type CS males were performed. Different behavioral parameters were scored while flies were on food cups and the dynamics of fights were analyzed (Table 1). An average of 24 encounters (brief meetings between flies), 54 lunges (an indicator of higher intensity fighting) were observed during a 20 min time period, with the 1st lunge usually delivered during the 2nd encounter. On average, the 1st encounter, the 1st lunge and the time to establish dominance were seen 2, 4 and 8 min after the start of recording. In most cases, the last 12 min of fights consist of repetitive sequences of high intensity encounters with subordinate animals approaching, being attacked and retreating, reinforcing the dominance or submissive status of each animal.
&#34;Loser&#34; effect experiments: In order to search for the formation of a &#34;loser&#34; effect, 21 1st fights of 20 min duration were carried out between pairs of wild type CS males. The chamber sliding divider then was inserted to separate the flies. After 10 min, 2nd 20 min fights were performed between (a) previous losers and familiar winners or (b) previous losers and unfamiliar winners and these were analyzed as above (Figure 7). In both conditions, 20 previous losers lost their 2nd fights (95.5%), demonstrating a robust &#34;loser&#34; effect. The fight outcomes were compared to the expected value of 50-50 with a two-tailed Chi-square analysis and a highly significant &#34;loser&#34; effect was found. Other behavioral changes were observed in losing individuals between the 1st and 2nd fights accompanying the formation of the &#34;loser&#34; effect 5.
Other behaviors than aggression can be studied in the apparatus: to illustrate, courtship and locomotion experiments were performed (Table 2). For courtship, male and female flies were loaded by negative geotaxis into the behavioral chambers, dividers were removed and behavioral courtship patterns were scored. 34 experiments were performed and the latencies to court (time between the 1st encounter and the 1st courtship behavior = 7 sec) and to copulate (time between the 1st encounter and copulation = 4 min) were analyzed. Only 2 males did not succeed in copulating during the 15 min time of observation. The Courtship Vigor Index (the fraction of time that males spent courting females during a 15 min period after the 1st courtship behavioral pattern) was scored at 75% (Table 2). For locomotion either single flies or group of 10 flies were introduced into chambers and the total number of midline crossings during 5 min were counted. Single flies cross the midline of the chamber on average 73 times during 5 min, while individual flies in groups cross the midline on average 45 times/5 min (Table 2). This decrease observed when flies are in groups can be explained by the reduced space available per fly and by the time they spend interacting with each other instead of exploring the territory.
<img alt="Figure 6" src="/files/ftp_upload/53395/53395fig6.jpg" /> 
Figure 6.&#160;Schematic representation of the behavioral chamber. (A) Side view of the apparatus. The new apparatus is composed of three separate parts: (a) a plastic block containing three individuals fighting chambers; (b) two sliding walls with three holes (15.88 mm diameter) in each; and (c) the two-piece cover on the top. (B) Top view of the apparatus. The dimensions of the plastic block (a) are 36.83 mm by 123.19 mm. Each individual fighting chamber has a diameter of 22.86 mm and a height of 17 mm. Holes on the side that allow the loading of the flies have a diameter of 15.88 mm. <a href="https://www.jove.com/files/ftp_upload/53395/53395fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/53395/53395fig7.jpg" /> 
Figure 7.&#160;The formation of a robust &#34;loser&#34; effect. A significant &#34;loser&#34; effect (percentage of losers that lose 2nd fights is observed when previous losers were paired (a) with familiar winners (n = 21, *** p &#60;0.0001) or (b) with unfamiliar winners for their 2nd fights (n = 21, *** p &#60;0.0001) (Lose: 95.5 %, Win: 4.5%, No draws). <a href="https://www.jove.com/files/ftp_upload/53395/53395fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table fo:keep-together.within-page="1">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Behavioral parameters</td>
			<td style="width:88px;">Unit</td>
			<td style="width:88px;">Time (sec)</td>
			<td style="width:88px;">n</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Encounters</td>
			<td>24 &#177; 2</td>
			<td></td>
			<td align="right">38</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lunges</td>
			<td>54 &#177; 6</td>
			<td></td>
			<td align="right">38</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Number of&#160; encounters before 1st lunge</td>
			<td>2 &#177; 0.3</td>
			<td></td>
			<td align="right">38</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Time of the 1st encounter</td>
			<td></td>
			<td>145 &#177; 20</td>
			<td align="right">38</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Time of the 1st lunge</td>
			<td></td>
			<td>229 &#177; 26</td>
			<td align="right">38</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Time of dominance</td>
			<td></td>
			<td>475 &#177; 56</td>
			<td align="right">34</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Latency to lunge</td>
			<td></td>
			<td>87 &#177; 20</td>
			<td align="right">38</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Latency to dominance</td>
			<td></td>
			<td>342 &#177; 51</td>
			<td align="right">34</td>
		</tr>
	</tbody>
</table>
Table 1: Dynamics of agonistic interactions between pairs of male flies. Mean values &#177; SEM are calculated for each parameter scored as indicators of aggressiveness. The total number of encounters and lunges, and the number of encounters before the 1st lunge were scored. Times of the 1st encounter, 1st lunge and dominance, and the latencies to lunge and to dominance are presented in sec &#177; SEM (n &#62;34).
<table fo:keep-together.within-page="1">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Behavioral parameters</td>
			<td style="width:88px;">Time (sec)</td>
			<td style="width:88px;">Unit</td>
			<td style="width:88px;">(%)</td>
			<td style="width:88px;">n</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Courtship behavior</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Latency to court</td>
			<td>7 &#177; 3</td>
			<td></td>
			<td></td>
			<td align="right">34</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Latency to copulate</td>
			<td>273 &#177; 62</td>
			<td></td>
			<td></td>
			<td align="right">32</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Courtship Vigor Index</td>
			<td></td>
			<td></td>
			<td>75 &#177; 0.05</td>
			<td align="right">34</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Locomotion</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Total midline crosses (single fly)</td>
			<td></td>
			<td>73 &#177; 4</td>
			<td></td>
			<td align="right">48</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Total midline crosses (group of flies)</td>
			<td></td>
			<td>45 &#177; 3</td>
			<td></td>
			<td align="right">20</td>
		</tr>
	</tbody>
</table>
Table 2: Analysis of courtship behavior and locomotion. Mean values &#177; SEM are calculated for the latencies to court and copulate (in sec). A courtship vigor index (in % of time spent courting after 1st courtship display) was scored after males and females were loaded into behavioral chambers by negative geotaxis (n &#62;32). Locomotion, when single flies or groups of 10 flies were loaded into chambers, was scored by counting the total number of midline crossings during 5 min (n = 48 and 20).

Watch this Scientific Journal Video about A New Approach that Eliminates Handling for Studying Aggression and the "Loser" Effect in Drosophila melanogaster at JoVE.com

A New Approach that Eliminates Handling for Studying Aggression and the "Loser" Effect in Drosophila melanogaster

During fruit fly fights, the behavioral patterns observed, fight dynamics, and associated learning and memory are influenced by experimental conditions. The protocol presented here describes a novel procedure that entirely eliminates handling of flies during experiments. This improves fight dynamics and allows formation of strong &#34;loser&#34; effects.

A New Approach that Eliminates Handling for Studying Aggression and the "Loser" Effect in Drosophila melanogaster

Aggressive behavior in Drosophila melanogaster is composed of the sequential expression of stereotypical behavioral patterns (for analysis see 1). This ...

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Nanyang Technological University

Research

JoVE Journal

Neuroscience

9.5K Views.  Harvard Medical School. During fruit fly fights, the behavioral patterns observed, fight dynamics, and associated learning and memory are influenced by experimental conditions. The protocol presented here describes a novel procedure that entirely eliminates handling of flies during experiments. This improves fight dynamics and allows formation of strong "loser" effects.

Video: A New Approach that Eliminates Handling for Studying Aggression and the "Loser" Effect in Drosophila melanogaster

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed in sensory hairs called sensilla, which cover the surface of olfactory organs. The surface of each sensillum is covered with tiny pores, through which odorants pass and dissolve in a fluid called sensillum lymph, which bathes the sensory dendrites of the OSNs housed in a given sensillum. The OSN dendrites express odorant receptor (OR) proteins, which in insects function as odor-gated ion channels4, 5. The interaction of odorants with ORs either increases or decreases the basal firing rate of the OSN. This neuronal activity in the form of action potentials embodies the first representation of the quality, intensity, and temporal characteristics of the odorant6, 7.
Given the easy access to these sensory hairs, it is possible to perform extracellular recordings from single OSNs by introducing a recording electrode into the sensillum lymph, while the reference electrode is placed in the lymph of the eye or body of the insect. In Drosophila, sensilla house between one and four OSNs, but each OSN typically displays a characteristic spike amplitude. Spike sorting techniques make it possible to assign spiking responses to individual OSNs. This single sensillum recording (SSR) technique monitors the difference in potential between the sensillum lymph and the reference electrode as electrical spikes that are generated by the receptor activity on OSNs1, 2, 8. Changes in the number of spikes in response to the odorant represent the cellular basis of odor coding in insects. Here, we describe the preparation method currently used in our lab to perform SSR on Drosophila melanogaster and Anopheles gambiae, and show representative traces induced by the odorants in a sensillum-specific manner.

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

Electrophysiological responses of olfactory sensory neurons to odorants can be measured in insects using single sensillum recordings. In this video article we will demonstrate how to perform single sensillum recordings in the antennae of the vinegar fly (Drosophila melanogaster) and the maxillary palps of the malaria mosquito (Anopheles gambiae).

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed ...

The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended upon this work to develop a simple quantitative measure of forepaw dexterity that is sensitive to lateralized impairments and age-dependent changes. Rodents learn skillful forepaw and digit movements to manage thin pasta pieces, which they eagerly consume. We have previously described methods for quantifying vermicelli handling in rats and showed that the measures are very sensitive to forelimb impairments resulting from unilateral ischemic lesions, middle cerebral artery occlusions and unilateral striatal dopamine depletion [Allred, R.P., Adkins, D.L., Woodlee, M.T., Husbands, L.C., Maldonado M.A., Kane, J.R., Schallert, T. &amp; Jones, T.A. The Vermicelli Handling Test: a simple quantitative measure of dexterous forepaw function in rats. J. Neurosci. Methods 170, 229-244 (2008)]. Here we present a more detailed protocol for this test in rats and compare it with a newly developed version for mice, the Capellini Handling Test. Rats and mice are videotaped while handling short lengths of uncooked vermicelli or capellini pasta, respectively, with a camera positioned to optimize the view of paw movements. Slow motion video playback allows for the identification of forepaw adjustments, defined as any distinct removal and replacement of the paw, or of any number of digits, on the pasta piece after eating commences. Forepaw adjustments per piece are averaged over trials per each testing session. Repeated testing permits sensitive quantitative analysis of changes in forepaw dexterity over time. Protocols for pre-testing habituation and handling practice, as well as procedures for characterizing atypical handling patterns, are described. Because rats and mice perform the pasta handling tests slightly differently, species-specific differences in administration and scoring of these tests are highlighted. All animal use was in accordance with protocols approved by the University of Texas at Austin Animal Care and Use Committee.

The Vermicelli and Capellini Handling Tests of forepaw dexterity take advantage of the natural inclination of rodents to manipulate food items using skillful forepaw and digit movements. Animals are videotaped while handling short strands of uncooked dry pasta. Slow motion video playback allows for the quantification of forepaw adjustments.

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended ...

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We developed a novel nanoinjection/electrophysiology assay that allows the detection of bioactive modulatory effects of compounds on the function of a neuronal circuit that mediates the escape response in Drosophila melanogaster3,4. Our in vivo assay, which uses the Drosophila Giant Fiber System (GFS, Figure 1) allows screening of different types of compounds, such as small molecules or peptides, and requires only minimal quantities to elicit an effect. In addition, the Drosophila GFS offers a large variety of potential molecular targets on neurons or muscles. The Giant Fibers (GFs) synapse electrically (Gap Junctions) as well as chemically (cholinergic) onto a Peripheral Synapsing Interneuron (PSI) and the Tergo Trochanteral Muscle neuron (TTMn)5. The PSI to DLMn (Dorsal Longitudinal Muscle neuron) connection is dependent on D&alpha;7 nicotinic acetylcholine receptors (nAChRs)6. Finally, the neuromuscular junctions (NMJ) of the TTMn and the DLMn with the jump (TTM) and flight muscles (DLM) are glutamatergic7-12. Here, we demonstrate how to inject nanoliter quantities of a compound, while obtaining electrophysiological intracellular recordings from the Giant Fiber System13 and how to monitor the effects of the compound on the function of this circuit. We show specificity of the assay with methyllycaconitine citrate (MLA), a nAChR antagonist, which disrupts the PSI to DLMn connection but not the GF to TTMn connection or the function of the NMJ at the jump or flight muscles.
Before beginning this video it is critical that you carefully watch and become familiar with the JoVE video titled "Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster " from Augustin et al7, as the video presented here is intended as an expansion to this existing technique. Here we use the electrophysiological recordings method and focus in detail only on the addition of the paired nanoinjections and monitoring technique.

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

A rapid in vivo assay to test for neuromodulatory compounds using the Giant Fiber System (GFS) of Drosophila melanogaster is described. Nanoinjections in the head of the animal along with electrophysiological recordings of the GFS can reveal bioactivity of compounds on neurons or muscles.

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

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 ...

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.

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.

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

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 ...

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 ...