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>

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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 beha...

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.

<table><tbody><tr><td>Drosophila melanogaster pupa</td><td>Bloomington Stock Center</td><td></td><td></td></tr><tr><td>Standard fly food</td><td></td><td></td><td>Fabricated in-house.&amp;nbsp;</td></tr><tr><td>Behavioral chambers</td><td>Mike machine</td><td></td><td>Fabricated in-house. Directly&amp;nbsp; contact the compagny for more informations.</td></tr><tr><td>Borosilicate glass vials</td><td>VWR International&amp;nbsp;</td><td>47729-576</td><td>16 x 10 mm</td></tr><tr><td>Cotton</td><td>Fisherbrand</td><td>22-456-881</td><td>Any brand can be used</td></tr><tr><td>Pasteur pipettes</td><td>VWR International&amp;nbsp;</td><td>53300-567</td><td></td></tr><tr><td>Paintbrush&amp;nbsp;</td><td>Blick Art Material</td><td>06157-7030</td><td>Round , Size 3/0</td></tr><tr><td>Toothpick</td><td>N/A</td><td>N/A</td><td>Any brand can be used</td></tr><tr><td>Acrylic paint (blue/white)</td><td>Blick Art Material</td><td>01637-5172/01637-1022</td><td>Any brand can be used</td></tr><tr><td>Dry active yeast</td><td>Sigma</td><td>YSC2-500G</td><td></td></tr><tr><td>Srew cap tube</td><td>VWR International&amp;nbsp;</td><td>10011-394&amp;nbsp;</td><td>15 mm diameter, 10 mm height</td></tr><tr><td>Eppendorf</td><td>VWR International&amp;nbsp;</td><td>22363212</td><td></td></tr><tr><td>Tape</td><td>N/A</td><td>N/A</td><td>Any brand can be used</td></tr><tr><td>Plastic slices</td><td>Electron Microscopy Sciences</td><td>70329-40</td><td>22 x 40 x 0.25 mm Thickness</td></tr><tr><td>Light source (bulb)</td><td>VWR International&amp;nbsp;</td><td>500003-418</td><td>Any brand can be used</td></tr><tr><td>Timer</td><td>VWR International&amp;nbsp;</td><td>62344-641</td><td>Any brand can be used</td></tr><tr><td>Incubator</td><td>Percival</td><td></td><td>Directly contact the constructor for more informations.</td></tr><tr><td>Carbon Dioxide Dry (CO&lt;sub&gt;2&lt;/sub&gt;)</td><td>Medical-Technical Gases, Inc</td><td>14H31</td><td></td></tr><tr><td>Binocular</td><td>Nikon</td><td>SMZ-745</td><td>Any brand can be used</td></tr><tr><td>Camera (SONY Handycam&amp;nbsp; HDR-CX330)</td><td>B&amp;amp;H</td><td>SOHDRCX330B</td><td>Any brand can be used</td></tr><tr><td>Computer</td><td></td><td></td><td>With a minimum of 1.4 Ghz Processor, running Microsoft Windows or Machintosh HD</td></tr><tr><td>ClipWrap</td><td>Download online</td><td></td><td>Any importing software can be used</td></tr><tr><td>QuickTime Player</td><td>Download online</td><td></td><td>Any reading software can be used</td></tr><tr><td>GraphPad Software</td><td>Online</td><td></td><td>Any statistical software can be used</td></tr></tbody></table>

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) th...

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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Research

JoVE Journal

Neuroscience

9.6K Views.  Harvard Medical School.  Tijdens fruitvlieg gevechten, de gedragspatronen waargenomen, vechten dynamiek, en de bijbehorende leren en geheugen worden beïnvloed door de experimentele omstandigheden. De hier gepresenteerde protocol beschrijft een nieuwe werkwijze die volledig elimineert hantering vliegen bij experimenten. Dit verbetert gevecht dynamiek en maakt de vorming van een sterke "verliezer" effecten. 

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

Studying Aggression in Drosophila (fruit flies)

Aggression is an innate behavior that evolved in the framework of defending or obtaining resources. This complex social behavior is influenced by genetic, hormonal and environmental factors. In many organisms, aggression is critical to survival but controlling and suppressing aggression in distinct contexts also has become increasingly important. In recent years, invertebrates have become increasingly useful as model systems for investigating the genetic and systems biological basis of complex social behavior. This is in part due to the diverse repertoire of behaviors exhibited by these organisms. In the accompanying video, we outline a method for analyzing aggression in Drosophila whose design encompasses important eco-ethological constraints. Details include steps for: making a fighting chamber; isolating and painting flies; adding flies to the fight chamber; and video taping fights. This approach is currently being used to identify candidate genes important in aggression and in elaborating the neuronal circuitry that underlies the output of aggression and other social behaviors.

Studying Aggression in Drosophila fruit flies

Aggression is an innate behavior that evolved in the framework of defending or obtaining resources.  This complex social behavior is influenced by ...

Biology

A Method to Test the Effect of Environmental Cues on Mating Behavior in Drosophila melanogaster

An individual's sexual drive is influenced by genotype, experience and environmental conditions. How these factors interact to modulate sexual behaviors remains poorly understood. In Drosophila melanogaster, environmental cues, such as food availability, affect mating activity offering a tractable system to investigate the mechanisms modulating sexual behavior. In D. melanogaster, environmental cues are often sensed via the chemosensory gustatory and olfactory systems. Here, we present a method to test the effect of environmental chemical cues on mating behavior. The assay consists of a small mating arena containing food medium and a mating couple. The mating frequency for each couple is continuously monitored for 24 h. Here we present the applicability of this assay to test environmental compounds from an external source through a pressurized air system as well as manipulation of the environmental components directly in the mating arena. The use of a pressurized air system is especially useful to test the effect of very volatile compounds, while manipulating components directly in the mating arena can be of value to ascertain a compound's presence. This assay can be adapted to answer questions about the influence of genetic and environmental cues on mating behavior and fecundity as well as other male and female reproductive behaviors.

A Method to Test the Effect of Environmental Cues on Mating Behavior in Drosophila melanogaster

We demonstrate an assay to analyze the environmental and genetic cues that influence mating behavior in the fruit fly Drosophila melanogaster.

An individual's sexual drive is influenced by genotype, experience and environmental conditions. How these factors interact to modulate sexual behaviors ...

Behavior

Appetitive Associative Olfactory Learning in Drosophila Larvae

In the following we describe the methodological details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with genetic interference, provides a handle to analyze the neuronal and molecular fundamentals of specifically associative learning in a simple larval brain.
Organisms can use past experience to adjust present behavior. Such acquisition of behavioral potential can be defined as learning, and the physical bases of these potentials as memory traces1-4. Neuroscientists try to understand how these processes are organized in terms of molecular and neuronal changes in the brain by using a variety of methods in model organisms ranging from insects to vertebrates5,6. For such endeavors it is helpful to use model systems that are simple and experimentally accessible. The Drosophila larva has turned out to satisfy these demands based on the availability of robust behavioral assays, the existence of a variety of transgenic techniques and the elementary organization of the nervous system comprising only about 10,000 neurons (albeit with some concessions: cognitive limitations, few behavioral options, and richness of experience questionable)7-10.
Drosophila larvae can form associations between odors and appetitive gustatory reinforcement like sugar11-14. In a standard assay, established in the lab of B. Gerber, animals receive a two-odor reciprocal training: A first group of larvae is exposed to an odor A together with a gustatory reinforcer (sugar reward) and is subsequently exposed to an odor B without reinforcement 9. Meanwhile a second group of larvae receives reciprocal training while experiencing odor A without reinforcement and subsequently being exposed to odor B with reinforcement (sugar reward). In the following both groups are tested for their preference between the two odors. Relatively higher preferences for the rewarded odor reflect associative learning - presented as a performance index (PI). The conclusion regarding the associative nature of the performance index is compelling, because apart from the contingency between odors and tastants, other parameters, such as odor and reward exposure, passage of time and handling do not differ between the two groups9.

Appetitive Associative Olfactory Learning in Drosophila Larvae

Drosophila larvae are able to associate odor stimuli with gustatory reward. Here we describe a simple behavioral paradigm that allows the analysis of appetitive associative olfactory learning.

In the following we describe the methodological  details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with ...

Drosophila Passive Avoidance Behavior as a New Paradigm to Study Associative Aversive Learning

This protocol describes a new paradigm for analyzing aversive associative learning in adult flies (Drosophila melanogaster). The paradigm is analogous to passive avoidance behavior in laboratory rodents in which animals learn to avoid a compartment where they have previously received an electric shock. The assay takes advantage of negative geotaxis in flies, which manifests as an urge to climb up when they are placed on a vertical surface. The setup consists of vertically oriented upper and lower compartments. On the first trial, a fly is placed into a lower compartment from where it usually exits&#160;within 3-15 s, and steps into the upper compartment where it receives an electric shock. During the second trial, 24 h later, the latency is significantly increased. At the same time, the number of shocks is decreased compared to the first trial, indicating that flies formed long-term memory about the upper compartment. The recordings of latencies and number of shocks could be performed with a tally counter and a stopwatch or with an Arduino-based simple device. To illustrate how the assay can be used, the passive avoidance behavior of D. melanogaster and D. simulans male and female were characterized here. Comparison of latencies and number of shocks revealed that both D. melanogaster and D. simulans flies efficiently learned the passive avoidance behavior. No statistical differences were observed between male and female flies. However, males were a little faster while entering the upper compartment on the first trial, while females received a slightly higher number of shocks in every retention trial. The Western diet (WD) significantly impaired learning and memory in male flies while flight exercise counterbalanced this effect. Taken together, the passive avoidance behavior in flies offers a simple and reproducible assay that could be used for studying basic mechanisms of learning and memory.

Drosophila Passive Avoidance Behavior as a New Paradigm to Study Associative Aversive Learning

This work describes a simple behavioral paradigm that allows the analysis of aversive associative learning in adult fruit flies. The method is based on suppressing the innate negative geotaxis behavior due to the association formed between a specific environmental context and an electric shock.

This protocol describes a new paradigm for analyzing aversive associative learning in adult flies (Drosophila melanogaster). The paradigm is analogous to ...

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 ...

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. ...

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

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

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

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

Measuring and Altering Mating Drive in Male Drosophila melanogaster

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

Measuring and Altering Mating Drive in Male Drosophila melanogaster

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

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

Drosophila Courtship Conditioning As a Measure of Learning and Memory

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model organisms such as the fruit fly, Drosophila melanogaster. Drosophila is useful for understanding the basic neurobiology underlying cognitive deficits resulting from mutations in genes associated with human cognitive disorders, such as intellectual disability (ID) and autism. This work describes a methodology for testing learning and memory using a classic paradigm in Drosophila known as courtship conditioning. Male flies court females using a distinct pattern of easily recognizable behaviors. Premated females are not receptive to mating and will reject the male&#39;s copulation attempts. In response to this rejection, male flies reduce their courtship behavior. This learned reduction in courtship behavior is measured over time, serving as an indicator of learning and memory. The basic numerical output of this assay is the courtship index (CI), which is defined as the percentage of time that a male spends courting during a 10 min interval. The learning index (LI) is the relative reduction of CI in flies that have been exposed to a premated female compared to na&#239;ve flies with no previous social encounters. For the statistical comparison of LIs between genotypes, a randomization test with bootstrapping is used. To illustrate how the assay can be used to address the role of a gene relating to learning and memory, the pan-neuronal knockdown of Dihydroxyacetone phosphate acyltransferase (Dhap-at) was characterized here. The human ortholog of Dhap-at, glyceronephosphate O-acyltransferase (GNPT), is involved in rhizomelic chondrodysplasia punctata type 2, an autosomal-recessive syndrome characterized by severe ID. Using the courtship conditioning assay, it was determined that Dhap-at is required for long-term memory, but not for short-term memory. This result serves as a basis for further investigation of the underlying molecular mechanisms.

Drosophila Courtship Conditioning As a Measure of Learning and Memory

This protocol describes a Drosophila learning and memory assay called courtship conditioning. This classic assay is based on a reduction of male courtship behavior after sexual rejection by a non-receptive premated female. This natural form of behavioral plasticity can be used to test learning, short-term memory, and long-term memory.

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model ...

Drosophila Models for Genetic and Behavioral Traits of Autism Spectrum Disorder

Paradigms for Behavioral Assessment in Drosophila Model of Autism Spectrum Disorder

Autism Spectrum Disorder (ASD) encompasses a heterogeneous group of neurodevelopmental disorders with common behavioral symptoms including deficits in social interaction and ability for communication, enhanced restricted or repetitive behaviors, and also, in some cases, learning disability and motor deficit. Drosophila has served as an unparalleled model organism for modeling a great number of human diseases. As many genes have been implicated in ASD, fruit flies have emerged as a powerful and efficient way to test the genes putatively involved with the disorder. As hundreds of genes, with varied functional roles, are implicated in ASD, a single genetic fly model of ASD is unfeasible; instead, individual genetic mutants, gene knockdowns, or overexpression-based studies of the fly homologs of ASD-associated genes are the common means for gaining insight regarding molecular pathways underlying these gene products. A host of behavioral techniques are available in Drosophila which provide easy readout of deficits in specific behavioral components. Social space assay and aggression and courtship assays in flies have been shown to be useful in assessing defects in social interaction or communication. Grooming behavior in flies is an excellent readout of repetitive behavior. Habituation assay is used in flies to estimate the ability for habituation learning, which is found to be affected in some ASD patients. A combination of these behavioral paradigms can be utilized to make a thorough assessment of the human ASD-like disease state in flies. Using Fmr1 mutant flies, recapitulating Fragile-X syndrome in humans, and POGZ-homolog row knockdown in fly neurons, we have shown quantifiable deficits in social spacing, aggression, courtship behavior, grooming behavior, and habituation. These behavioral paradigms are demonstrated here in their simplest and straightforward forms with an assumption that it would facilitate their widespread use for research on ASD and other neurodevelopmental disorders in fly models.

Author Spotlight: Exploring Autism Spectrum Disorder Symptoms in Fruit Flies &#8212; Genetic Models and Behavioral Tests

Autism Spectrum Disorder (ASD) is associated with impaired social and communicative behavior and the emergence of repetitive behavior. For studying the interrelation between ASD genes and behavioral deficits in the Drosophila model, five behavioral paradigms are described in this paper for assaying social spacing, aggression, courtship, grooming, and habituation behavior.

Autism Spectrum Disorder (ASD) encompasses a heterogeneous group of neurodevelopmental disorders with common behavioral symptoms including deficits in ...

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

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