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Molecular analysis of flies selected for aggressive behavior


Aggressive behavior is pervasive throughout the animal kingdom, and yet very little is known about its molecular underpinnings. To address this problem, we have developed a population-based selection procedure to increase aggression in Drosophila melanogaster. We measured changes in aggressive behavior in the selected subpopulations with a new two-male arena assay. In only ten generations of selection, the aggressive lines became markedly more aggressive than the neutral lines. After 21 generations, the fighting index increased more than 30-fold. Using microarray analysis, we identified genes with differing expression levels in the aggressive and neutral lines as candidates for this strong behavioral selection response. We tested a small set of these genes through mutant analysis and found that one significantly increased fighting frequency. These results suggest that selection for increases in aggression can be used to molecularly dissect this behavior.

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Figure 1: Population cage and territorial assay.
Figure 2: Fighting parameters in arena assay at Gen11 (white bars) and 21 (gray bars).
Figure 3: Aggression quantification in population cage at Gen22 and Aggr-Neutr mixed populations and pairs.
Figure 4: Control behaviors tested in selected lines at Gen22 and 23.
Figure 5: Validation of array expression differences by qPCR.
Figure 6: Fighting frequency of mutants in arena assay and mutant validation.

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We would like to thank J. Sullivan for help with the behavioral analysis, D. Robinson for technical assistance, A. McCartney for help with the figures, J. Fleisher for help with the video files, B. van Swinderen for discussions and for comments on the manuscript and R. Andretic and S. Pangas for comments on the manuscript. This material is based on work supported by the US National Science Foundation under grant no. 0432063 (R.J.G. and H.A.D.) as well as by a grant from the Wacker Foundation (R.J.G.). R.J.G. is the Dorothy and Lewis B. Cullman Fellow at The Neurosciences Institute, which is supported by the Neurosciences Research Foundation.

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Authors and Affiliations



This study was designed by H.A.D. and R.J.G. H.A.D. performed the experiments, analyzed the data and wrote the manuscript.

Corresponding author

Correspondence to Herman A Dierick.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Figure 1

Arena assay and aspiration hole. (PDF 86 kb)

Supplementary Figure 2

Array expression profiles of gene in Table 2. (PDF 44 kb)

Supplementary Table 1

Biological categories of significant genes32 (P < 0.002). (PDF 123 kb)

Supplementary Video 1

Main male fighting elements in population cage. The video (three clips spliced together) was shot in a population cage (see Fig. 1a) focusing on one single territory at a time (a blue food cup 2 cm in diameter). The first clip shows wing threat behavior where males lift their wings up at a 45° angle in a threatening posture (which has also been referred to as “wings erect” behavior). The second clip shows the most common male fighting element, referred to as charging or lunging, where the territorial male charges at any intruder on the territory. Often this ends in a lunge where the attacking male lifts his front legs and drops down on the attacked male, who will typically be running away and receive the blow on his side or back. Occasionally, an intruder will not run away, resist the attacks and reciprocate the lunging attacks of the territory holder. This is shown in the third clip and often occurs in short repeated bouts. The two males will then lunge at each other and often box, wrestle and tussle in an attempt to gain control over the territory. We refer to this last behavior as escalated fighting. Such escalations are rare and occur in less than five percent of all aggressive encounters10,21. In the Aggr selected lines they can last for up to 10 minutes. (MOV 609 kb)

Supplementary Video 2

Main male fighting elements in scaled-down territorial assay (Fig 1c). The movie was shot in scaled-down territorial assay with two males, one mated female and small food territory (eppendorf cap filled with fly food). Three clips are spliced together, showing wing threat (slow motion), charging & lunging and escalation (slow motion). (MOV 651 kb)

Supplementary Video 3

Main male fighting elements in arena-assay. The movie shows one arena in the arena chamber. Three clips are spliced together. The first clip shows wing threat followed by a charge and holding, where the attacker grabs the wings of the other male. This often leads to a “roll over”, which is quantified as the intensity parameter in Fig 2d. The second clip shows a fighting sequence. The third clip shows both males in escalation mode, boxing and tussling. (MOV 762 kb)

Supplementary Video 4

Abdomen dipping. The movie was shot in the population cage focusing a single territory. Male streaks his genital area over the food surface while walking as if to mark the territory. Six separate clips are spliced together. (MOV 295 kb)

Supplementary Video 5

Aspiration of escalating males. The movie shows two males escalating on a blue food territory in the population cage. Aspirator comes in from the left and gently sucks up both males from the territory. (MOV 126 kb)

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Dierick, H., Greenspan, R. Molecular analysis of flies selected for aggressive behavior. Nat Genet 38, 1023–1031 (2006).

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