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Method Article

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

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DOI:

10.3791/53395

December 30th, 2015

In This Article

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....

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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.

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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.......

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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.......

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Disclosures

The authors have nothing to disclose.

Acknowledgements

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: http://mikesmachine.com).

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 ....

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Drosophila melanogaster pupaBloomington Stock Center
Standard fly foodFabricated in-house. 
Behavioral chambersMike machineFabricated in-house. Directly  contact the compagny for more informations.
Borosilicate glass vialsVWR International 47729-57616 x 10 mm
CottonFisherbrand22-456-881Any brand can be used
Pasteur pipettesVWR International 53300-567
Paintbrush Blick Art Material06157-7030Round , Size 3/0
ToothpickN/AN/AAny brand can be used
Acrylic paint (blue/white)Blick Art Material01637-5172/01637-1022Any brand can be used
Dry active yeastSigmaYSC2-500G
Srew cap tubeVWR International 10011-394 15 mm diameter, 10 mm height
EppendorfVWR International 22363212
TapeN/AN/AAny brand can be used
Plastic slicesElectron Microscopy Sciences70329-4022 x 40 x 0.25 mm Thickness
Light source (bulb)VWR International 500003-418Any brand can be used
TimerVWR International 62344-641Any brand can be used
IncubatorPercivalDirectly contact the constructor for more informations.
Carbon Dioxide Dry (CO2)Medical-Technical Gases, Inc14H31
BinocularNikonSMZ-745Any brand can be used
Camera (SONY Handycam  HDR-CX330)B&HSOHDRCX330BAny brand can be used
ComputerWith a minimum of 1.4 Ghz Processor, running Microsoft Windows or Machintosh HD
ClipWrapDownload onlineAny importing software can be used
QuickTime PlayerDownload onlineAny reading software can be used
GraphPad SoftwareOnlineAny statistical software can be used

References

  1. Chen, S., Lee, A. Y., Bowens, N. M., Huber, R., Kravitz, E. A. Fighting fruit flies: a model system for the study of aggression. Proc Natl Acad Sci U S A. 99, 5664-5668 (2002).
  2. Hsu, Y., Earley, R. L., Wolf, L. L. Modulation of aggressi....

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Tags

Drosophila AggressionBehavioral ChamberNegative GeotaxisFly Handling EliminationIsolation Vial PreparationPlastic Divider UseVideo Recording AnalysisDominance Relationship FormationSocial Behavior Study