How animals use sensory information to weigh the risks vs. benefits of behavioral decisions remains poorly understood. Inter-male aggression is triggered when animals perceive both the presence of an appetitive resource, such as food or females, and of competing conspecific males. How such signals are detected and integrated to control the decision to fight is not clear. For instance, it is unclear whether food increases aggression directly, or as a secondary consequence of increased social interactions caused by attraction to food. Here we use the vinegar fly, Drosophila melanogaster, to investigate the manner by which food influences aggression. We show that food promotes aggression in flies, and that it does so independently of any effect on frequency of contact between males, increase in locomotor activity or general enhancement of social interactions. Importantly, the level of aggression depends on the absolute amount of food, rather than on its surface area or concentration. When food resources exceed a certain level, aggression is diminished, suggestive of reduced competition. Finally, we show that detection of sugar via Gr5a + gustatory receptor neurons (GRNs) is necessary for food-promoted aggression. These data demonstrate that food exerts a specific effect to promote aggression in male flies, and that this effect is mediated, at least in part, by sweet-sensing GRNs.

Funding: This study was supported by funding from the National Institutes of Health (NIH), Code: NIH-NRSA5T32GM07616, Grant/Project #: T32 GM007616, Grant/Project Title: PREDOCTORAL TRAINING IN BIOLOGY AND CHEMISTRY. This study was supported by the funding from the Howard Hughes Medical Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

A resolution of these issues would be facilitated by a quantitative analysis of aggressive behavior on variable food resources. Such analyses have been enabled by the development of machine vision-based automated aggressive behavior recognition software [26] , [43] – [46] . Here we report on the results of such an analysis, performed in the context of systematic and quantitative manipulations of food resource parameters and analyses of their effects on male-male social interactions. Our results set constraints, in a principled and rigorous manner, on models for how food promotes aggression. We also identify a key component of food and its chemoreceptor that are required for aggression.

Despite much progress, fundamental questions remain unanswered about how resources promote aggression. In particular, it is widely assumed that flies fight in the presence of food due to competition over a limiting resource or to claim territory for potential reproductive advantages [3] , [22] , [33] – [35] . However, other explanations have not been excluded. For example, increased aggression in the presence of food could simply be due to an increase in encounter frequency and/or duration between males attracted to the resource, or to an increase in aggressive drive or arousal. Food may also increase locomotor activity, promoting increased encounters and thereby indirectly enhancing aggression. In addition, most previous reports [3] , [10] , [22] , [24] , [27] – [29] , [31] , [32] , [36] – [40] measured male-male aggression in the presence of females, which added a potential confound, as presence of females can increase aggression on its own [23] , [41] , [42] . Finally, it is not clear whether food promotes aggression in a purely permissive or in an instructive manner.

As in many other species, Drosophila males exhibit a gender-specific repertoire of stereotyped aggressive behaviors [1] – [9] . Recent studies have identified some of the male-specific sensory signals and their physiological receivers relevant for aggression [1] , [2] , [4] , [10] – [20] . In particular, cuticular hydrocarbon pheromones, such as 11-cis-vaccenyl acetate (cVA) [1] , [3] , [9] , [21] – [27] and (z)-7-tricosene (7-T) [1] , [4] , [24] , [28] – [32] promote aggression through olfactory [1] , [2] , [4] , [5] , [10] – [20] and gustatory receptor neurons [10] , [12] – [18] , [21] . However, the detection of cues from conspecific males is a necessary but not sufficient condition for aggression: male flies will not fight unless a resource, such as food or females, is present [1] , [3] , [4] , [9] , [22] – [24] , [26] – [32] .

Metazoan organisms in nature constantly face behavioral choices. Depending on the actions selected, an animal may gain access to potential resources or risk starvation, predation or agonistic interactions. Aggression is an ideal system in which to study how the nervous system makes value-based decisions, as the decision to fight comes with apparent costs and benefits, and requires the assessment of a potential conflict: the detection of attractive resources and competitors who limit access to such resources.

Results

The level of aggression depends on the absolute amount of food If food specifically enhances aggression, how do flies measure it? The answer to this question sets constraints on the sensory systems that are involved, and ultimately how the brain uses this information to guide the decision to fight. We first examined the effect of changing the area over which food (at a fixed concentration) is distributed, using a modifiable arena (Figure S4c). Consistent with previous reports [22], [27], we observed a dose-dependent relationship between the size of the food patch and the level of aggression (Figure 2a). Next we investigated whether this dose-dependent increase was due to an effect on either proximity, arousal, or general social interactions. Although, we observed a slight increase in locomotion as the size of the food patch increased (Figure 2b), this enhanced aggression was seen even when normalized by locomotion (Figure 2c). Furthermore, the inter-fly distance distribution was not changed by any of the differently sized food arenas that were tested (Figure S5b). Unlike aggression, male-male courtship showed no change in response to the change in the amount of food (Figure 2d), suggesting that the dose-dependent effect of food does not reflect a general increase in social interactions. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 2. Flies measure the level of total nutrients to increase the level of aggression, rather than the area of food. (a) Aggression increases as the size of food patch increases. See Figure S4 for schematic diagrams of the arena used. n = 41, 39, and 52 male-male pairs for 0, 79, 707 mm2, respectively. Same pairs are further analyzed for Figures 2b-d. (b) Locomotion also increases in some cases (0 vs. 707 mm2) as the size of food increases. (c) Aggression normalized by locomotion is significantly increased in the presence of food. (d) Male-male courtship normalized by locomotion is not changed by the presence of food. (e) Left: Increasing the concentration of food while keeping the size of food constant (707 mm2) increases aggression. Right: Increasing the size of food while keeping the concentration constant also increases aggression. The concentration-dependent increase in aggression is quantitatively similar to the size-dependent increase in aggression. The absolute nutritional content remains the same between the left and the right (1∶235 = 3 mm2, 1∶54 = 13 mm2, etc). Some of the data in E are the same as those used in A and are replotted here for comparison purposes. n = 41, 22, 16, 29, 28, 31, 36, 37, 39, 27, and 52 male-male pairs from left to right. https://doi.org/10.1371/journal.pone.0105626.g002 Previous studies did not distinguish whether the increase in aggression caused by increasing the size of food patch was due to an increase in area, total food amount or both [22], [27]. We therefore investigated whether changing the concentration of food while keeping the arena area constant would yield a similar result. Indeed aggression in a fixed-size arena increased as the concentration of food increased (Figure 2e left). In fact, when we compared the level of aggression in the cases where the areas of food were different (Figure 2e right) but the caloric content was matched, the level of aggression was indistinguishable (see Figure S5a for side-by-side comparisons). These data are incompatible with the notion that flies assess the quality of food in the context of aggression by using a physical dimension of food territory, such as area or perimeter circumference. Instead, these results suggest that the level of aggression depends upon the absolute amount of food in the substrate.

Flies display territorial behavior Territorial behavior refers to overt or implied defense of an area by one or a group of animals at the exclusion of others [66]. Although the term territoriality is frequently used when referring to aggression in Drosophila [3], [22], previous studies have not distinguished between the defense of a territory (territoriality) from the defense of a resource per se [6], [35]. To investigate this issue, we observed in more detail the spatial distribution of a pair of flies with respect to food resources of different areas. As mentioned earlier, flies preferentially occupy the area where food is present (Figure 1b and 4a). In addition, we observed that as the area of the food patch was increased, the position heat map showed an apparent circular “donut” shape (Figure 4a), suggesting an increased preference of flies to remain near the periphery of the food patch. This observation suggested that flies may defend the perimeter of the food, rather than the entire food resource, when the size of the patch is large. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 4. Flies display territorial behavior. (a) Top row: Schematic diagrams show the arenas with different size of food being used. Bottom row: Position heat-map of a pair of flies presented with different sizes of food. The heat-maps display two features: 1) flies spend a lot of time on top of food and 2) they spend a lot of time near the border of the food area. n = 41, 29, 86 and 41 male-male pairs from left to right. (b) Position heat map compares the distribution of flies on 30 mm and 45 mm diameter food when there is only 1 fly in the arena (left) and when there are two flies (right). 2-fly data from one experiment are individually averaged. n = 30 and 52 for 30 mm diameter food, single and pairs of flies, respectively. n = 25 and 41 for 45 mm diameter food, single and pairs of flies, respectively. The pairs are further analyzed in Figures 4c – 4f. (c and d) These histograms show the amount of time flies spend at different distances from the border of 30 mm (c) food and 45 mm (d) patch. The schematic diagrams of the behavioral setups are overlaid for visualization. Briefly, the x-axis is aligned so that 0 denotes the border of food patch while negative values indicate the distance inward from food border (inside the food patch) and positive values indicate the distance outward from the food border (outside of food patch). The blue line denotes when there is a single fly in the arena while the orange line denotes when there is a pair of flies. Lines indicate the median while shaded area denotes the interquartile range. (e) Presence of another fly increases the amount of time flies spend in Zone B (“interaction zone”) for both 30 mm and 45 mm food patches. (f) Presence of another fly does not change the amount of time flies spend on the food patch (Zone A). https://doi.org/10.1371/journal.pone.0105626.g004 To distinguish whether this phenomenon was related to aggression, or simply reflected an innate preference of flies to occupy the boundary of a food patch, we compared the distribution of single flies and fly pairs for two different sizes of food patches (Figure 4b). In order to quantify these distributions with respect to the food patch area, we measured the amount of time flies spent as a function of the distance from the food patch border patches, and aligned the histograms to the border defined as 0 mm (Figures 4c and 4d). In both 30 mm and 45 mm diameter patches, we observed two peaks defining three zones in the histograms, which we refer to as Zones A, B, and C (Figures 4c and d, lower). Zone A comprised the food patch itself and exhibited a peak in the fly distribution at the border. Zone B comprised the area between the food border peak and a second peak, located approximately 15–20 mm from the outside edge of the arena. Zone C comprised the perimeter area of the arena. Since Zone A was the area occupied by the food patch, fly occupation of this area simply reflected their natural attraction to food. Zone C could, in part, reflect thigmotactic tendencies of flies [67], [68], since in the absence of food, a similar peak around 15–20 mm from the edge of the arena was also observed (Figure S7a). To investigate whether these experimental peaks were different from a random distribution, which would be expected if flies behaved as if they were randomly moving particles, we calculated a random distribution from the area in the bins at each indicated distance from the food border and compared it to the experimental distribution (Figure S7b). These comparisons revealed that in the absence of a food patch (blue line), flies behaved similarly to randomly moving particles (teal colored line). In contrast, in the presence of a 30 mm diameter food patch, fly positions (orange) were not randomly distributed. In both single and paired fly experiments, there were two peaks dividing these three zones in both 30 mm diameter (Figure 4c, blue for single fly and orange for paired fly experiments) and 45 mm diameter food patches (Figure 4d). Nevertheless, we observed a noticeable difference in the distribution of flies within Zone B. Pairs of flies appeared to spend more time in this zone than did single flies. To quantify these differences, we calculated the area under the curves in Zone A and Zone B for single vs. paired flies. Single male flies spent significantly less time than did flies in pairs in Zone B for both 707 mm2 and 1590 mm2 food patches (Figure 4e). In contrast, when we calculated the amount of time flies spent in the food area (Zone A), we found that the presence of an opponent male made no difference (Figure 4f). These data indicate that the presence of an opponent does not enhance attraction to food; instead it only increases the amount of time flies spend in the area just outside the food border, suggesting that fighting flies adopt a “perimeter defense” strategy. These data are consistent with the notion that when the size of the food patch is large (Figure 4a, 177 mm2 vs. 1590 mm2), Drosophila males fight over access to a food-containing territory, rather than just over the food resource itself.

Sucrose is sufficient to promote aggression Foregoing data suggested that flies may use their chemosensory systems to measure the absolute nutritional content of the food to tune the level of aggression. Apple juice and fly culture food are complex mixtures containing a variety of odorants and tastants [69]–[71]. One obvious indicator of nutritional content in natural food resources is the concentration of sugar. Therefore, we tested whether pure sucrose, present in fly culture medium and food mix used in our experiments, would be sufficient to increase aggression in the absence of any other food component. Surprisingly we found that a small patch of 100 mM sucrose (see Figure S4e), comparable to concentrations found in fruits [33] and in laboratory fly food medium [70], was sufficient to promote aggression to a level comparable to that observed using the food substrate (Figure 5a and Figure S8d). Similar to uniform food, the ability of sucrose to increase aggression was not due to a difference in the encounter duration, because the presence of a patch of sucrose neither changed the overall distribution of the flies (Figure 5b), nor changed the encounter duration (Figure 5c and 5d). The presence of sucrose increased locomotion (Figure 5e), but the increase in aggression caused by sucrose remained significant following normalization to distance traveled (Figure 5f). In contrast, male-male courtship was not increased (Figure 5g). Thus pure sucrose can mimic the effect of food to increase aggression. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 5. Flies use sweet-sensing Gr5a+ GRNs to detect the concentration of sucrose in the food and tune the level of aggression accordingly. (a) 100 mM sucrose is sufficient to increases aggression. (b) Sucrose does not cause attraction, as it does not lead to an apparent change in the position heat map. n = 100 and 60 for 100 mM sucrose and agarose, respectively. Pairs are further analyzed from Figures 5b-5g. (c) Presence of sucrose does not change the amount of time flies spend near each other. (d) Encounter duration does not change in the presence of sucrose. (e) Sucrose increases locomotion. (f) Sucrose increases the number of lunges per meters traveled, which implies that the increase in aggression is not merely due to increased locomotion. (g) Sucrose does not change the number of circling per meters traveled. (h) Changing sucrose concentration increases and decreases aggression. The level of aggression is increased from 0 to 200 mM but becomes indistinguishable from no food condition at 800 mM. (*): 100 to 200 mM difference is significant when individually compared (P<0.05) but not after corrections for multiple comparisons. n = 32, 23, 10 and 26 from left to right. (i) Inhibiting the sugar-sensing Gr5a+ GRNs by expressing TNT decreases sucrose sensitivity (n = 3 and 3 for both genotypes. Each replicate has 10 male flies to calculate fraction of responders). (j) Inhibiting the sugar-sensing Gr5a+ GRNs by expressing TNT decreases food-promoted aggression compared to genetic controls. n = 36, 41, and 32 from left to right. https://doi.org/10.1371/journal.pone.0105626.g005 To examine the dose-dependency of aggression on sucrose, we compared the number of lunges in 100, 200 and 800 mM sucrose (Figure 5h, see Figure S4e). Similar to the results obtained with food (Figure S8d), we first saw an increase in aggression when we increased the concentration of sucrose from 100 to 200 mM. Moreover when we further increased the level of sucrose to 800 mM, the level of aggression was no different from the control condition (Figure 5h). Taken together, these data suggest that sucrose exhibits a bi-modal influence on aggression that is qualitatively similar to that seen with food.