Name: a new approach that eliminates handling for studying aggression and the "loser" effect in drosophila melanogaster
Uploaded: 2015-12-30T15:00:00
Description: 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

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" s...

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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.

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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) 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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