Food is necessary for normal levels of male-male aggression but not male-male courtship and its effects are independent from locomotion or encounter duration.

<p>(a) Flies performed more lunges during the observation period in the presence of a 22×22 mm food patch. n = 171, 92 male-male pairs tested for apple juice food patch and agarose patch, respectively. (b) Flies performed more lunges in the presence of arena, which was entirely covered with food. n = 113 and 44, for uniform food and uniform agarose, respectively. (c) Top: Schematic diagram of the aggression assay arenas used. Left side shows the food patch configuration and right side shows the uniform food configuration. A pair of male flies is illustrated at scale for comparison. Bottom: Position heat map shows the average amount of time flies spend in a particular position in the arena. The data shown are averages of multiple pairs of flies (same sample numbers as Figures 1a and 1b). It uses a red-blue color map from MATLAB where deep red is high frequency (60 frames, which is roughly 2 seconds, are the deepest-red) and blue is 0. Every subsequent position heat map is presented in the same manner. On the left, flies are attracted to the patch of food while on the right the uniform food does not lead to attraction to a specific spot in the arena. (d) Uniform food does not change the amount of time flies spend at various distances from each other. The inter-fly distance histogram shows amount of time flies spend (y-axis) at a given distance from each other (x-axis). The distribution is not affected by the presence of food (1-way ANOVA). There is a very prominent peak around 3–4 mm, which ranges from 2 mm (less than 1 body length of flies) to 10 mm (3–4 body lengths), and accounts for around 50% of the 20 minutes assay. The area under the curve from 0 to 10 mm is hereafter referred to as “encounter duration”. The trace is the median trace from 72 and 44 male-male pairs for food and agarose, respectively. (e) Uniform food does not increase encounter duration. Assay is 20 minutes long (1200 seconds). Same number of samples as Figure 1d. (f) Locomotion (distance traveled) in a pair of flies is increased in the presence of food. Same number of samples as Figure 1d. (g) Normalization of aggression by locomotion by dividing the number of lunges by travel distance shows that food significantly increases aggression. Same number of samples as Figure 1d. (h) Number of one-wing extensions is not changed by the presence of uniform food. Manually scored data consisting of n = 17 and 18 pairs for food and agarose conditions, respectively. (i) Normalization of courtship (number of circling bouts) by locomotion shows that food decreases male-male courtship. Same number of samples as Figure 1d. (j) In the first three minutes, food progressively increases aggression (blue circle). In contrast, one-wing extension decreases (red circle). In the absence of food, lunges do not increase or decrease (blue box); courtship decreases (red box). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105626#pone.0105626.s009" target="_blank">Table S1</a> for statistics. Manually scored data of lunges and 1-wing extensions. n = 33 and 33 for food and agarose conditions for lunges. n = 34 and 31 for food and agarose conditions for one-wing extensions.