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

Summary

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.

Abstract

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 "loser" 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 "loser" 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.

Introduction

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 "loser" and "winner" effects). "Loser" effects have been observed in a wide range of species, and some reports suggest that these can last for several days while "winner" 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 "winners" and "losers". 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 2^nd fights when paired with naïve flies or previous winners ^3,4. However, the absence of an experimental procedure that excludes handling of flies has made detailed studies of "loser" 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 "loser" 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 "loser" 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 "loser" 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.

Protocol

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 "loser" effect. Using this protocol, a short-term "winner" effect also has been recently reported ^5,6. Figure 1 shows the timeline of the behavioral experiments.

Figure 1. 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. Please click here to view a larger version of this figure.

1. Day 1: Social Isolation of Pupae

1. Raise and maintain fly stocks in an environmentally controlled 12 hr light/12 hr dark cycle incubator at 25 °C and 50% relative humidity.
2. 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.
3. 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).
4. Place isolation vials containing pupae into the 12 hr light/12 hr dark incubator at 25 °C and 50% relative humidity.

Figure 2. 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 °C and 50% relative humidity. Please click here to view a larger version of this figure.

2. Day 5: Painting the Flies for Individual Identification Purposes

1. 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.
2. Anesthetize fly with CO₂.
3. 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).
4. Let paint dry for approximately 10 sec, and gently transfer flies back to their original isolation vials.
5. Return isolation vials to the 12 hr light/12 hr dark incubator at 25 °C and 50% relative humidity.

Figure 3. 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. Please click here to view a larger version of this figure.

3. Day 7: Preparing the Apparatus

1. Prepare Food Cups:
   1. Heat fresh fly food until it melts.
   2. 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.
   3. 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).

Figure 4. 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. Please click here to view a larger version of this figure.

2. Prepare Individual Fighting Chambers:
   1. Thoroughly wash the apparatus and individual fighting chambers with water before use each time. Dry with paper towel before use.
   2. Place a food cup in the center of each fighting chamber and fix in place with a drop of putty.
   3. Cover fighting chambers with the apparatus lid (Figure 5).
   4. Place the apparatus into a high-humidity environment (60%) at 25 °C.
   5. Place a light source above the apparatus to illuminate the fighting chambers and set a video camera for recording from above.

Figure 5. 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. Please click here to view a larger version of this figure.

4. Day 7: Behavioral Experiment

1. Perform the behavioral experiments with 6-7 day-old male flies.
2. Insert plastic dividers into each fighting chamber.
3. Remove the cotton plug from the isolation tube and position the tube below an open hole on the side of the apparatus.
4. Let the fly enter into the fighting chamber by negative geotaxis, usually within a few seconds.
5. Close the sliding wall after the fly has left the tube and entered the chamber.
6. Repeat the procedure to introduce the second fly into the fight chamber on the other side of the apparatus.
7. Place the apparatus back in the illuminated videotaping position.
8. Begin video recording and remove the plastic divider to allow flies to interact.
   1. For Aggression Experiments:
      1. In these experiments, record fights for at least 20 min to ensure the formation of strong dominance relationships.
      2. After the desired time, stop video recording.
      3. Remove flies from the fighting chambers by reversing the procedure described above.
      4. Thoroughly wash the behavioral apparatus with mild detergent and water after each use and rinse.
   2. For "Loser" 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 "loser" effect.
      1. Follow the procedure described above to perform a first fight.
      2. CRITICAL STEP: To induce the formation of a "loser" mentality, ensure that first fights are at least 20 min long in order to establish strong dominance relationships.  After 20 min, re-insert the plastic divider into fighting chambers to separate the flies.
      3. After a 10 min period of rest, gently remove the plastic divider to allow flies to interact for second fights.
      4. At the end of the second 20 min fight, stop the video recording.
      5. Remove flies from behavioral chambers as above.
      6. Once again, thoroughly clean the behavioral apparatus.

5. Behavioral Analysis

1. Transferring Movies
   1. Connect cameras to the computer.
   2. Convert flies .MTS to .mov flies and join movies clips together, using video converting software and the option "join selected clips".
   3. Observe and analyze each movie with video reader software.
2. 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 "loser" effect.
   1. 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).
   2. 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.
   3. 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.
3. Calculating a "Loser" Effect
   1. Divide the number of fights in which previous losers lose 2^nd fights (Losers that lose: LL) by the total number of fights, and multiply by 100. The percentage obtained represents the "loser" effect:
      [(# flies LL) / (# total fight)] x 100
   2. Use a Chi-square test to compare whether the "loser" effect is statistically different from the expected value of 50%.

Results

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 "loser" 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 t...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Drosophila melanogaster pupa	Bloomington Stock Center
Standard fly food			Fabricated in-house.
Behavioral chambers	Mike machine		Fabricated in-house. Directly  contact the compagny for more informations.
Borosilicate glass vials	VWR International	47729-576	16 x 10 mm
Cotton	Fisherbrand	22-456-881	Any brand can be used
Pasteur pipettes	VWR International	53300-567
Paintbrush	Blick Art Material	06157-7030	Round , Size 3/0
Toothpick	N/A	N/A	Any brand can be used
Acrylic paint (blue/white)	Blick Art Material	01637-5172/01637-1022	Any brand can be used
Dry active yeast	Sigma	YSC2-500G
Srew cap tube	VWR International	10011-394	15 mm diameter, 10 mm height
Eppendorf	VWR International	22363212
Tape	N/A	N/A	Any brand can be used
Plastic slices	Electron Microscopy Sciences	70329-40	22 x 40 x 0.25 mm Thickness
Light source (bulb)	VWR International	500003-418	Any brand can be used
Timer	VWR International	62344-641	Any brand can be used
Incubator	Percival		Directly contact the constructor for more informations.
Carbon Dioxide Dry (CO2)	Medical-Technical Gases, Inc	14H31
Binocular	Nikon	SMZ-745	Any brand can be used
Camera (SONY Handycam  HDR-CX330)	B&H	SOHDRCX330B	Any brand can be used
Computer			With a minimum of 1.4 Ghz Processor, running Microsoft Windows or Machintosh HD
ClipWrap	Download online		Any importing software can be used
QuickTime Player	Download online		Any reading software can be used
GraphPad Software	Online		Any statistical software can be used