A simple assay to study social behavior in Drosophila: measurement of social space within a group - PubMed (original) (raw)

A simple assay to study social behavior in Drosophila: measurement of social space within a group

A F Simon et al. Genes Brain Behav. 2012 Mar.

Abstract

We have established a new simple behavioral paradigm in Drosophila melanogaster to determine how genes and the environment influence the behavior of flies within a social group. Specifically, we measure social space as the distance between two flies. The majority of Canton-s flies, regardless of their gender, are within two body lengths from each other. Their social experience affects this behavior, with social isolation reducing and mating enhancing social space respectively, in both males and females. Unlike several other social behaviors in the fly, including the formation of social groups themselves (a well-described behavior), social space does not require the perception of the previously identified aggregation pheromone cis-vaccenyl acetate. Conversely, performance of the assay in darkness or mutations in the eye pigmentation gene white increased social space. Our results establish a new assay for the genetic dissection of a fundamental mode of social interaction.

© 2011 The Authors. Genes, Brain and Behavior © 2011 Blackwell Publishing Ltd and International Behavioural and Neural Genetics Society.

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Figures

Figure 1

Figure 1. Social space in aggregates of wild type flies (Canton-s)

A) Group formation in different chambers. From left to right, clockwise: 250 ml Erlenmeyer flask, horizontal circular chamber (9 cm in diameter), horizontal rectangular chamber (18 × 24.5cm), 0.8 cm diameter tube (10 ml pipette), vertical triangle (height: 20 cm, base: 22 cm), inverted vertical triangle with same dimensions as vertical triangle (note that panels are not to scale). B) Vertical triangle assay chamber containing 40 flies (height: 15.3 cm, base: 15.3 cm, see methods). C – F) Quantitation of social space after 15 min. in the chamber. The graph represents the percentage of flies (±SEM) at the indicated distance from their closest neighbor, using bins of 0.5 cm. C) Quantitation of social space in a vertical triangular chamber and in a horizontal circular chamber, see methods for details (n=3 trials of males). D-E) Similar social space in a vertical triangle chamber for D) Males, n=21 trials of ~40 flies, and E) Females, n=16. Males and female behavior does not differ (Kolmogorov-Smirnov Test). F) Time spent in the chamber does not affect social space. Quantitation after different times in the chamber, males (n=12) after 15 min. and 45 min., females (n=6) after 25 min. and 55 min. Social space does not differ (Kolmogorov-Smirnov Test). For C to E, each trial consists of ~40 flies (3-5 days old).

Figure 2

Figure 2. A Social Space Index to determine social interactions

A-E) Vertical triangle chamber. A-C) Aggregation patterns of A) a random simulation of 40 dots; B) a merged image of 40 single flies; C) 40 flies assayed together. To increase visibility in the figure, the area of the marks representing each fly in (A) to (C), was increased (x 6) relative to their original size. Quantitation of social space was performed using the more precisely localized original mark. Comparison of A to B shows the effects of centrophobism and negative geotaxis; Comparison of B to C shows the added effects of interactions between individual flies. D) Quantification of social space between flies. The graphs represent the percentage of flies (±SEM) at the different distances from their closest neighbor, using bins of 0.5 cm. The 3 histograms differ significantly (Kolmogorov-Smirnov Test, P<00001). E) A simplified representation of the data is shown as a Social Space Index (SSI, defined as the value of the first bin minus the value of the second bin). An SSI score of =<0 suggests little or no social interaction (see text). F-I) Quantitation of behavior in a horizontal circular chamber. F) Aggregation patterns of a random simulation of 40 dots to compare to 40 flies G) (as in 2A-C the size of marks representing each fly was increased by a factor of 6 to enhance their visibility in the figure), H) Quantification of social space between flies in a circular chamber comparing the random simulation and the distribution of three trials of 40 flies tested together. The 2 histograms in (H) differ significantly (Kolmogorov-Smirnov Comparison indicates that the data-sets are different p<0.00001). I) Social Space Index comparing the simulation and 40 flies tested together in a horizontal chamber.

Figure 3

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 Social Space Index (SSI ±SEM) at densities of 10 to 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-C) Social space is affected by social experience. B) Virgin flies show less social interaction. Graph represents the comparison of Social Space Index (SSI±SEM) between 3-4 day 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 Social Space Index (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, *.

Figure 4

Figure 4. Social space may depend on vision but not classical odor or cVA perception

(A-C) Social Space Index is not modified in odor perception mutants; A) An allele of para (sbl1) compared to genetic background Canton-s, Males, n=6 trials of ~40 flies. B) Or83b1 and Or83b2 were outcrossed 6 times and compared to genetic background Canton-s, 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 Social Space Index 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 to their genetic control Canton-s (Cs). D) Reduction in performance in the fast phototaxis assay. Flies were given 5 times 15 sec. to go towards 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 sec. 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 Cs and w are different from w (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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