Environmental and genetic factors can modulate aggressiveness, but the biological mechanisms underlying their influence are largely unknown. Social experience with conspecifics suppresses aggressiveness in both vertebrate and invertebrate species, including Drosophila. We searched for genes whose expression levels correlate with the influence of social experience on aggressiveness in Drosophila by performing microarray analysis of head tissue from socially isolated (aggressive) vs. socially experienced (nonaggressive) male flies. Among {approx}200 differentially expressed genes, only one was also present in a gene set previously identified by profiling Drosophila strains subjected to genetic selection for differences in aggressiveness [Dierick HA, Greenspan RJ (2006) Nat Genet 38:1023–1031]. This gene, Cyp6a20, encodes a cytochrome P450. Social experience increased Cyp6a20 expression and decreased aggressiveness in a reversible manner. In Cyp6a20 mutants, aggressiveness was increased in group-housed but not socially isolated flies. These data identify a common genetic target for environmental and heritable influences on aggressiveness. Cyp6a20 is expressed in a subset of nonneuronal support cells associated with pheromone-sensing olfactory sensilla, suggesting that social experience may influence aggressiveness by regulating pheromone sensitivity

Baier

Ben-Shahar

Black

Carney

Champagne

Chen

D. J. Anderson

Dierick

Ganguly-Fitzgerald

H. Dankert

Kravitz

L. Wang

Levine

Loeber

Luciano

Manoli

Matsumoto

Meerts

Osborne

P. Perona

Robinow

Soma

Stevenson

Svetec

Tecott

Toda

van der Goes van Naters

Vosshall

Wang

White

Yeager

English

PubMed

Wang, Liming

Dankert, Heiko

Perona, Pietro

Anderson, David J.

Caltech Authors - Main

Supporting Information
Wang et al. 10.1073/pnas.0801327105
SI Text
Cumulative Probability Distribution (CDP) Analysis for Fig. S2. The
most sensitive criterion for fighting is that a given pair of flies
exhibits at least one lunge during the 20-min observation period.
However, arbitrarily less sensitive (more stringent) criteria can
also be used. We wished to examine systematically how criteria
of different stringencies affected the comparison between hous-
ing conditions and genotypes. To do this, we first measured the
total number of lunges exhibited by each pair of flies, during the
20-min observation period. We then plotted the data as a
cumulative probability distribution curve, for each genotype and
housing condition (heterozygous/single-housed; homozygous/
single-housed; heterozygous/group-housed; homozygous/group-
housed). Due to different genetic backgrounds, separate com-
parisons were made for P-element heterozygotes vs.
homozygotes (Cyp6a20/ vs. Cyp6a20/, A), and Df heterozy-
gotes vs. Df/P-element homozygotes (Df(2R)BSC11/ vs.
Cyp6a20/Df, B).
In each case, group-housed heterozygous controls (A,
Cyp6a20/ and B, Df(2R)BSC11/) were chosen as the ‘‘ref-
erence’’ datasets for comparison to other conditions/genotypes.
For each stringency criterion (lunge number), a Kruskal–Wallis
ANOVA (significance level  0.05) was performed on each
dataset from the four curves. If there was significant difference
between the four datasets, then a Mann–Whitney U test (sig-
nificance level  0.05) was performed between the ‘‘reference’’
dataset and the three other ‘‘test’’ datasets. This analysis per-
mitted a comparison of the aggressiveness of different genotypes
under different housing conditions, at progressively higher strin-
gencies.
At the origin of each curve, when X  0, the group-housed
heterozygous controls (A, Cyp6a20/ Group housing, and B,
Df(2R)BSC11/ Group housing) are significantly different from
all other groups. As the lunge threshold criterion (X) increases,
the difference between the four datasets becomes gradually
indistinguishable. This is to be expected, because if the lunge
threshold were set arbitrarily high (e.g., at least 1,000 lunges in
20 min), then none of the pairs in any condition or genotype
would be scored as having a fight (fighting frequency  0), and
therefore all of the curves would be statistically indistinguish-
able. The question is, how do the different curves behave as they
approach this point of statistical equivalence? The asterisks
above the reference curve represent points that are statistically
different from ‘‘test’’ datasets in some or all of the other three
curves. ‘‘N.S.’’ above the remaining curves represents the point
after which these curves become indistinguishable from the
reference curve.
Four major conclusions can be drawn from this analysis:
1. The behavior of the three ‘‘test’’ curves with respect to each
other is clearly different in A and B. In general, f lies con-
taining the Df(2R)BSC11 deficiency chromosome (B) are
more aggressive than those containing the P-element inser-
tion chromosome (A). This difference could reflect a differ-
ence in the genetic backgrounds of CS and Df(2R)BSC11/Cyo
f lies, or haplo-insufficiency of genes other than Cyp6a20 that
are encompassed by the deficiency. Therefore, the most
conservative conclusions are those that can be drawn from the
analysis of the P-element insertion data (A).
2. The three ‘‘test’’ datasets in A and B are indistinguishable
from each other by Kruskal-Wallis ANOVA, in the range X 
0–19. In addition, all ‘‘test’’ datasets are significantly different
from the ‘‘reference’’ (heterozygous/group-housed) dataset
for threshold criteria X  0 (A) or X  0–4 (B), at each of the
individual lunge threshold values within these intervals. This
indicates that the Cyp6a20 homozygous mutation renders
f lies equally aggressive under group-housed and single-
housed conditions, and that under both housing conditions,
these mutant flies are more aggressive than group-housed
heterozygous controls. This supports the conclusion that the
suppressive effect of enriched social experience on aggres-
siveness is mediated, at least in part, by Cyp6a20.
3. The curves for homozygous P-element mutant flies, in either
the single- or group-housing conditions, become statistically
indistinguishable from the reference curve at the same lunge
criterion (X  1, Cyp6a20/ group housing, and Cyp6a20/
single housing, A). This indicates that single-housed homozy-
gous flies are no more aggressive than group-housed homozy-
gous flies, compared with group-housed heterozygous flies.
This would suggest that most or all of the difference in
aggressiveness between single and group-housed heterozy-
gous flies is due to Cyp6a20, and therefore that this gene is the
primary, if not the exclusive, mediator of the effect of social
experience on aggressiveness. However, in experiments using
the deficiency-containing chromosome (B), the two homozy-
gous curves reach statistical equivalence with the ‘‘reference’’
curve at different points (X  5 for Cyp6a20/Df, group
housing, X  11 for Cyp6a20/Df, single housing, B). This
indicates that under single housing conditions, the homozy-
gous mutants are still somewhat more aggressive than they are
under group housing conditions; subject to the caveats in 1),
this would imply that additional genes besides Cyp6a20 me-
diate the effect of social experience on aggressiveness.
4. The curves for single-housed flies (homozygous or heterozy-
gous) become indistinguishable from the reference curve at
the same point in A (X  1, Cyp6a20/, single housing and
Cyp6a20/, single housing). This implies that single-housed
flies are no more aggressive in the presence or absence of
Cyp6a20, compared with group-housed heterozygous flies.
This would suggest that Cyp6a20 exclusively functions in
mediating the effect of group housing to suppress aggressive-
ness. However, in B, the two single-housing curves reach
statistical equivalence with the reference curve at different
points (X  8 for Df(2R)BSC11/, single housing, X  11 for
Cyp6a20/Df, single housing). This suggests a difference of
aggressiveness between single-housed heterozygous (Df/)
and homozygous (Df/P-element) Cyp6a20 mutants. If so, then
subject to the caveats in 1), Cyp6a20 may function not only in
mediating the effect of group housing on aggressiveness, but
also in controlling the level of aggressiveness under single
housing conditions.
Wang et al. www.pnas.org/cgi/content/short/0801327105 1 of 4
Fig. S1. Aggression arena. The apparatus was modified from an original design by Hoyer SC et al. [Hoyer SC, et al. (2008) Octopamine in male aggression of
Drosophila Curr Biol 18:156–167]. The dimensions of each arena were 5.0 cm (D)  11.4 cm (H). The center of the arena contains an inner square well (1.2 cm 
1.2 cm) filled with sucrose-apple juice-agar medium, and an outer square well (2.2 cm  2.2 cm) filled with 1% agar medium. Fluon was painted on the inner
surface of the wall of each arena, to prevent flies from climbing out. Arenas were covered by transparent plastic lids to prevent flies from escaping and to facilitate
video capture.
Wang et al. www.pnas.org/cgi/content/short/0801327105 2 of 4
Fig. S2. Cumulative probability distributions of lunging thresholds for Cyp6a20 mutant analysis. For each lunge threshold criterion (x axis), the cumulative
probability (y axis) represents the cumulative percentage of pairs that can be accounted for by X lunges or less (in 20 min). (A) Cumulative probability distributions
for Cyp6a20/ and Cyp6a20/ (n  6, same datasets used in Fig. 3, error bars represent SEM). (B) Cumulative probability distributions for Cyp6a20Df/ and
Cyp6a20Df/- (n  6, same data used in Fig. 3, error bars represent SEM). Curves that approach 100% more rapidly indicate a lower level of aggressiveness. Squares
represent homozygous mutants (Cyp6a20/ in A and Cyp6a20/Df in B), triangles represent heterozygous controls (Cyp6a20/ in A and Df(2R)BSC11/ in B).
Open and solid symbols indicate single- and group-housing conditions, respectively (refer to SI Text).
Wang et al. www.pnas.org/cgi/content/short/0801327105 3 of 4
Other Supporting Information Files
Dataset S1
Fig. S3. Larval Cyp6a20 expression is enriched in salivary gland (SG). For B, C-S third instar larvae were dissected in PBS and both SG and SG-removed larval
tissue were collected. Equal amount of RNA extracted from both tissues were reverse-transcribed and simultaneously amplified using RpL32 and Cyp6a20 primers,
producing 423 bp and 962 bp, respectively. RpL32 (CG7939) was used as an internal control of gene expression. PCR products were loaded on 1.2% agarose gel
and visualized by ethidium bromide. (A) GFP expression was seen in SG of P[GawB]NP2593/UAS-mCD8GFP (Left, arrow head), but not in SG of P[GawB]NP2593/
(Right) larva. (Scale bar: 1 mm.) (B) RT-PCR confirmed Cyp6a20 transcripts were enriched in SG.
Wang et al. www.pnas.org/cgi/content/short/0801327105 4 of 4


A common genetic target for environmental and heritable influences on aggressiveness in Drosophila

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A common genetic target for environmental and heritable influences on aggressiveness in Drosophila

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