A simple behavioral assay to measure social space
To study the regulation of local enhancement, we measured the distance between individual flies and their closest neighbor: their ‘social space’ (Mogilner et al. 2003). As described by others, we found that flies form groups in several different types of chambers (Fig. 1a, also see Bolduc et al. 2010; Lefranc et al. 2001; Navarro & del Solar 1975). However, locomotion in horizontally oriented chambers is dominated by exploration and dispersal rather than local enhancement, making it difficult to obtain stable measurements of social space (Fig. 1c, also see Lefranc et al. 2001; Simon & Dickinson 2010; Tinette et al. 2004). In our hands, horizontal group of flies do not assume a static position even after spending 1 h in the chamber. In contrast, in vertically oriented chambers, we find that the flies stop moving and assume a stable position within ∼20 min. The shape of the container also affected the flies' position and behavior, and we found that a triangular shape was most useful. Using the vertically oriented, triangular chamber shown in Fig. 1b, flies show a consistent sequence of behaviors amenable to acquisition of data on social space. For the first few minutes after placing the flies in the chamber, they display an escape response, manifested as negative geotaxis. Negative geotactic behavior ends as the flies crowd into the upper tip of the triangular assay chamber. They then move away from each other and locomote for several minutes (∼5–10 min for males, and up to 20 min for females) in an apparent attempt to reduce local crowding and to explore their new environment. At the end of the exploration phase, the flies remain essentially in one location and engage in sporadic grooming behavior for up to 45–55 min, and their social distance is stable throughout that time (Fig. 1f, cf. 15 vs. 45 min for males, 25 vs. 55 min for females). The relative stability of the distance between flies at this stage allows the easy acquisition of digital images for subsequent quantitation of the flies' location. Although it may also be possible to analyze social space using moving animals, the analysis would be complicated by variations in locomotor speed, and generally more technically demanding than the analysis of still pictures. To confine the image analysis of the final position to only two dimensions, the depth of the chamber was restricted to 0.47 cm (3/16′′). This arrangement prevented two flies from occupying the same x, y coordinate (Materials and methods).
Quantitation of the flies' position in the test chamber showed a surprisingly consistent distribution of distances between each fly and its closest neighbor. We find that 56% ± 3 (mean ± SEM) of the flies lie within 0–0.5 cm from their nearest neighbor (∼two body lengths in magnitude and comparable to social distances observed in other species –Mogilner et al. 2003), and 18% ± 2 are within 0.5–1 cm. The remaining 20% of the population are further apart as shown in Fig. 1d. The pattern of the fly's social space is the same in both genders, consistent with previous observations (Navarro & del Solar 1975 and Fig. 1d–f).
To confirm that the distance between the flies in our assay was governed by social interactions rather than the result of a random distribution, we used a computer simulation to map the location of 40 randomly placed dots (Fig. 2a). In addition, to account for the effects of centrophobism and geotaxis in individual flies lacking all social interactions, we assayed single flies 40 times and merged the data into a single combined image (Fig. 2b). We compared these distributions with that of 40 flies assayed together as described above (Fig. 2c). The flies assayed individually were not only attracted to the sides of the chamber (a well-described behavior –Simon & Dickinson 2010; Valente & Mitra 2007) but also moved away from the top. In contrast, we observed that the flies assayed in-group displayed less attraction to the sides and form a more robust aggregate at the top of the chamber. The flies that do not aggregate at the top and migrate to the sides of the chamber also form closely situated pairs (Fig. 2c).
For each condition, the data representing the distance between each fly (or random dot) was binned and used to generate a histogram, in which we represent the percentage of flies (y-axis) for every 0.5 cm increment (bins on the x− axis –Fig. 2d). The patterns of the three histograms differed significantly (Kolmogorov–Smirnov test, P < 0.00001) showing that interactions between the individual flies influence their distribution in the test chamber.
The most obvious difference between the histograms was the relative size of the first and second bins. For flies tested together under ‘social conditions' (i.e. with other flies), the first bin was consistently larger than the second (Fig. 2d). In other words, there was a higher percentage of flies within two body lengths of each other as compared with those that were >2–4 body lengths from the nearest fly. In contrast, for flies tested separately, the first and second bins were roughly the same size. We therefore generated a simple ‘social space index’ (SSI) by subtracting the percentage of flies in the second bin from the percentage of flies in the first bin (Fig. 2e). Using this metric, we obtain a value of 0 or less when flies behave similarly to those tested individually (with the second bin containing a similar or higher percentage of flies than the first one), and therefore assign 0 as the baseline SSI representing social space in the absence of social interactions (such as in Fig. 2b). We obtain an SSI of 100 when all of the flies are in bin 1 (or 100% of the flies 0–0.5 cm from each other, two body lengths apart or less), which we define as a state of maximal social interaction. For 40 Cs flies of 3–4 days old, on average, we obtain an SSI of ∼40 (38.4 ± 4.6, n = 21 –Fig. 2e).
As explained above, we chose to use a vertical triangular chamber for most tests. However, we also confirmed that non-random aggregation patterns were also seen in horizontal circular chambers, using similar analysis (Fig. 2f–i). The percentage of flies that are two body lengths apart is more variable, seen as the larger bars representing the SEM in Fig. 2h as compared with Fig. 2d, which flies were visualized in the triangular chamber. However, the pattern of distribution is similar to that seen in the vertical triangular chamber and different from what would be seen in a random simulation (Fig. 2h– Kolmogorov-Smirnov test, P < 0.00001). Similarly, the SSI of a random simulation is close to 0, even in this chamber of a completely different size, shape and orientation (Fig. 2i).
For all of the experiments described below, we analyzed both the overall pattern of the histograms and the SSI. We show the SSIs here and the histograms in Fig. S1. To control for behavioral effects of environment seen in many other behavioral paradigms (e.g. associative learning Connolly & Tully 1998), each experiment was performed with a matched, internal control and in a temperature controlled environment. All attempts were made to keep the testing room at constant humidity; it was not feasible to humidify the test apparatus used here without disrupting the fly's behavior.
The most extensive previous studies of group formation show that flies are gregarious and cooperative, and that population size, presence of odorant, pheromones and genes affect this behavior (Lefranc et al. 2001; Tinette et al. 2004). Using the conditions we developed, we first tested whether the number of flies in the chamber would influence the SSI. Although 10 flies showed a lower SSI (Fig. 3a) and a statistically different distribution pattern as seen in the histograms (Fig S1a, supporting information), the distribution was constant using 20, 30 or 40 flies, (Fig. 3a and supporting information Fig. S1a) showing that above a certain threshold, social space did not vary with small changes in group size. For consistency, all further experiments were performed using 40 flies.
Figure 3. Social space is relatively independent of group size and correlated to social interactions. (a) Impact of group size. Graph represents the comparison of SSI ± SEM at densities of 10–40 flies per test chamber. The SSI was not statistically different across densities (one-way anova), but showed a trend toward a lower mean and higher variance at a density of 10 flies per chamber (n = 8 trials, number of male flies indicated). (b and c) Social space is affected by social experience. (b) Virgin flies show less social interaction. Graph represents the comparison of SSI ± SEM between 3–4 days old virgins (aged with the same gender), or mated (housed gender mixed) flies; males housed with males (virgin), n = 10 trials of ∼40 flies, males housed with females (gender mixed), n = 21 trials of 40 flies; females, virgin, n = 11 trials of ∼40, house gender mixed, n = 14 trials of 40 flies, t-tests indicate a significant difference of **P < 0.01. (c) Isolated flies show less social interaction. Graph represents the comparison of SSI ± SEM of flies ∼10 days old, collected from bottles at ∼3 days old and aged for 7 days either alone or socially enriched in groups of 40 flies of same gender. Males, n = 5–6 trials of ∼40 flies, females, n = 5 trials of ∼40, t-tests indicate a significant difference respectively of **P < 0.01 and *P < 0.05.
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Odor and pheromone perception mutants
To test whether odor or pheromone perception might be involved in regulating social space between flies, we measured the SSI of mutants defective in olfaction. We tested paraslb1, a mutant in Cs background with broad olfactory defects: paralytic, which encodes a sodium channel (Lilly et al. 1994). We also tested two null alleles of Or83b (Or83b1 and Or83b2–Larsson et al. 2004), an odorant receptor, required for the perception of most odors and of the volatile pheromone cis-vaccenyl acetate (cVA), after outcrossing six times in our laboratory Cs background. We did not detect any differences between the SSI of these mutants and genetically matched controls (Fig. 4a,b and supporting information Fig. S1d,e).
Figure 4. Social space may depend on vision but not classical odor or cVA perception. (a–c) SSI is not modified in odor perception mutants; (a) an allele of parasbl1 compared with genetic background Cs, males, n = 6 trials of ∼40 flies. (b) Or83b1 and Or83b2 were outcrossed six times and compared with genetic background Cs, n = 6 trials of ∼40 flies (SSI ± SEM). (c) Flies show less social aggregation in darkness, under a red light. Graph represents the comparison of SSI in light and dark conditions (males, n = 15 trials of ∼40 flies, t-test indicates a significant difference of **P < 0.005). (d–f) Outcrossed mutants white disrupting the eye pigments localization show less social aggregation, normal geotaxis and reduced phototaxis. w1118Cs10 were outcrossed 10 times, indicated as w, compared with their genetic control Cs. (d) Reduction in performance in the fast phototaxis assay. Flies were given five times 15 seconds to go toward the light, in a counter-current apparatus (n = 8 trials of ∼40 male flies; SSI ± SEM –t-test indicates that Cs is different from w ***P < 0.0006). (e) No differences in negative geotaxis. In a climbing assay in the counter-current apparatus, the flies were giving 15 seconds to reach the upper vial (n = 5 trials of ∼40 male flies (SSI ± SEM) –t-test indicates no differences). (f) Decreased SSI in males and females white. In males, n = 21 trials for Cs and n = 18 trials for w (SSI ± SEM); t-test indicates that w is different from Cs (*P < 0.05 and ***P < 0.001). In females, n = 16 trials for Cs and n = 12 trials for w; t-test indicates that w is different from Cs (***P < 0.001).
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Possible effects of vision
To test the possible effects of decreased visual cues on social space, we measured the SSI of flies in red light placed in a dark room. In this condition, flies still interact socially, but on average are farther from their nearest neighbor than controls, and thus show a decreased SSI (Fig. 4c and supporting information Fig. S1f). These data suggested that social space might depend at least partially on visual cues.
To determine whether more subtle changes in vision might affect social space, we used a mutant of the white gene (w1118Cs10) in which eye pigmentation is essentially absent (Green 2010). Although white mutants are not blind, they show defects in visual acuity because of the diffusion of light across adjacent photoreceptor arrays (Stark & Wasserman 1974), and outcrossed w1118Cs10 flies display modest but consistent defects in fast phototaxis as compared with genetically matched Cs (Fig. 4d). To control for possible effects on motor behavior in the phototaxis test, we used a standard climbing assay. Cs and w1118Cs10 do not show differences in climbing consistent with the notion that fast phototaxis is decreased because of decreased visual abilities in w1118Cs10 (Fig. 4e).
In social space assays, we found that the w1118Cs10 males and females come to rest farther apart than their genetically matched controls and thus, showed a lower SSI (Fig. 4f and supporting information Fig. S1g), similar to flies assayed in darkness (Fig. 4c). These data suggest that genetic defects in visual behavior may affect social space and more generally show the ability of our assay to detect changes in social behavior in a genetic mutant.