Extensive epistasis for olfactory behaviour, sleep and waking activity in Drosophila melanogaster.
ABSTRACT Epistasis is an important feature of the genetic architecture of quantitative traits, but the dynamics of epistatic interactions in natural populations and the relationship between epistasis and pleiotropy remain poorly understood. Here, we studied the effects of epistatic modifiers that segregate in a wild-derived Drosophila melanogaster population on the mutational effects of P-element insertions in Semaphorin-5C (Sema-5c) and Calreticulin (Crc), pleiotropic genes that affect olfactory behaviour and startle behaviour and, in the case of Crc, sleep phenotypes. We introduced Canton-S B (CSB) third chromosomes with or without a P-element insertion at the Crc or Sema-5c locus in multiple wild-derived inbred lines of the Drosophila melanogaster Genetic Reference Panel (DGRP) and assessed the effects of epistasis on the olfactory response to benzaldehyde and, for Crc, also on sleep. In each case, we found substantial epistasis and significant variation in the magnitude of epistasis. The predominant direction of epistatic effects was to suppress the mutant phenotype. These observations support a previous study on startle behaviour using the same D. melanogaster chromosome substitution lines, which concluded that suppressing epistasis may buffer the effects of new mutations. However, epistatic effects are not correlated among the different phenotypes. Thus, suppressing epistasis appears to be a pervasive general feature of natural populations to protect against the effects of new mutations, but different epistatic interactions modulate different phenotypes affected by mutations at the same pleiotropic gene.
[show abstract] [hide abstract]
ABSTRACT: In 2000, Drosophila melanogaster joined the ranks of vertebrates and invertebrates with a defined behavioral sleep state. The characterization of this sleep state revealed striking similarities to sleep in humans: sleep in flies has both circadian and homeostatic components, it is influenced by sex and age, and it is affected by pharmacological agents such as caffeine and antihistamines. As in mammals, arousal thresholds in flies increase with sleep deprivation. Furthermore, changes in brain electrical activity accompany the change from wake to sleep states. Not only do flies and vertebrates share these behavioral and physiological traits of sleep, but they are likely to share at least some genetic mechanisms underlying the regulation of sleep as well. This article reviews the methods currently used to identify and characterize the Drosophila sleep state. As these methods become more refined and our understanding of Drosophila sleep more detailed, the powerful techniques afforded by this organism are likely to unveil deep insights into the function(s) and regulatory mechanisms of sleep.Methods in enzymology 02/2005; 393:772-93. · 1.90 Impact Factor
Extensive epistasis for olfactory behaviour, sleep and waking
activity in Drosophila melanogaster
SHILPA SWARUP1, 2, SUSAN T. HARBISON1, 2, LAUREN E. HAHN1,
TATIANA V. MOROZOVA2, 3, AKIHIKO YAMAMOTO2, 3,
TRUDY F. C. MACKAY1, 2AND ROBERT R. H. ANHOLT1, 2, 3*
1Department of Genetics, North Carolina State University, Raleigh, NC 27695-7614, USA
2W. M. Keck Center for Behavioral Biology, North Carolina State University, Raleigh, NC 27695-7617, USA
3Department of Biology, North Carolina State University, Raleigh, NC 27695-7617, USA
(Received 3 November 2011; revised 21 December 2011; accepted 5 January 2012)
Epistasis is an important feature of the genetic architecture of quantitative traits, but the dynamics of epistatic
interactions in natural populations and the relationship between epistasis and pleiotropy remain poorly
understood. Here, we studied the effects of epistatic modifiers that segregate in a wild-derived Drosophila
melanogaster population on the mutational effects of P-element insertions in Semaphorin-5C (Sema-5c) and
Calreticulin (Crc), pleiotropic genes that affect olfactory behaviour and startle behaviour and, in the case of Crc,
sleep phenotypes. We introduced Canton-S B (CSB) third chromosomes with or without a P-element insertion at
the Crc or Sema-5c locus in multiple wild-derived inbred lines of the Drosophila melanogaster Genetic Reference
Panel (DGRP) and assessed the effects of epistasis on the olfactory response to benzaldehyde and, for Crc, also
on sleep. In each case, we found substantial epistasis and significant variation in the magnitude of epistasis. The
predominant direction of epistatic effects was to suppress the mutant phenotype. These observations support a
previous study on startle behaviour using the same D. melanogaster chromosome substitution lines, which
concluded that suppressing epistasis may buffer the effects of new mutations. However, epistatic effects are not
correlated among the different phenotypes. Thus, suppressing epistasis appears to be a pervasive general feature
of natural populations to protect against the effects of new mutations, but different epistatic interactions
modulate different phenotypes affected by mutations at the same pleiotropic gene.
Epistasis is an integral feature of the genetic
architecture of quantitative traits (Anholt & Mackay,
2004; Flint & Mackay, 2009; Mackay et al., 2009).
Epistasis occurs when the effect of variation at one
locus is suppressed or enhanced by the genotype at
another locus. Epistatic interactions can bias esti-
mates of the effects of quantitative trait loci (QTLs) in
mapping populations when present but not accounted
for (Carlborg et al., 2006); enable inferences of genetic
networks affecting complex traits (Phillips, 2008); and
affect predictions of long-term response to artificial
and natural selection (Carlborg et al., 2006; Phillips,
2008). Epistasis is difficult to detect in classical quan-
titative genetic analyses based on resemblance be-
tween relatives in outbred populations (Falconer &
Mackay, 1996), and epistatic interactions contribute
largely additive genetic variation in outbred popu-
lations when the contributing alleles are rare (Hill
et al., 2008). However, epistatic interactions are com-
mon in experiments designed to examine their effects
on trait means in QTL mapping populations. For
et al., 1995; Gurganus et al., 1999; Dilda & Mackay,
2002), wing morphology (Weber et al., 1999), lifespan
(Leips & Mackay, 2000, 2002) and startle-induced
locomotor behaviour (Jordan et al., 2006). In mice,
epistasis has been reported between QTLs affecting
growth, body weight and morphometry (Brockmann
et al., 2000; Cheverud et al., 2001; Workman et al.,
* Corresponding author: Robert R. H. Anholt, Department of
Biology, Box 7617, North Carolina State University, Raleigh,
NC 27695-7617, USA. Tel: (919) 515-1173. Fax: (919) 515-1801.
The online version of this article is published within an Open Access environment subject to the conditions of the Creative Commons
Attribution-NonCommercial-ShareAlike licence <http://creativecommons.org/licenses/by-nc-sa/2.5/>. The written permission of
Cambridge University Press must be obtained for commercial re-use.
Genet. Res., Camb. (2012), 94, pp. 9–20.
f Cambridge University Press 2012
2002; Klingenberg et al., 2004; Yi et al., 2006).
Epistasis is also a prominent feature of the genetic
architecture of growth rate in Arabidopsis (Kroymann
& Mitchell-Olds, 2005), chickens (Carlborg et al.,
2006) and yeast (Steinmetz et al., 2002; Sinha et al.,
Although epistatic interactions have been detected
in QTL mapping experiments, it is easier to study
epistasis in crosses among lines with reduced genetic
heterogeneity in largely homozygous genetic back-
grounds (Eshed & Zamir, 1996; Clark & Wang, 1997;
Sambandan et al., 2006). Drosophila melanogaster is
an excellent model system to study epistasis affecting
quantitative traits due to the ease of constructing
chromosome substitution and introgression lines, and
generating mutations in a common homozygous
genotype. Epistasis has been documented for ag-
gressive behaviour by constructing chromosome
substitution lines in which small segments of one
genotype were introgressed into a different genetic
background (Edwards & Mackay, 2009). Epistasis for
aggression was also evident from behavioural and
whole genome transcriptional analyses of an ensemble
of co-isogenic hyper-aggressive P-element insertion
lines (Zwarts et al., 2011). Epistasis for metabolic
activity was revealed by constructing all possible
two-locus genotypes for several pairs of P-element
insertion mutations (Clark & Wang, 1997). Diallel
cross analysis of co-isogenic P-element insertion
lines enabled identification of epistatic networks of
genes affecting negative geotaxis (Van Swinderen &
Greenspan, 2005), olfactory avoidance behaviour
(Fedorowicz et al., 1998; Sambandan et al., 2006),
aggression (Zwarts et al., 2011) and startle behaviour
in Drosophila (Yamamoto et al., 2009).
Previously, Yamamoto et al. (2009) created pairs
of chromosome substitution lines in which isogenic
Canton-S B (CSB) chromosomes with P-element in-
sertions in genes affecting startle behaviour and their
P-element free co-isogenic control chromosomes were
substituted into different homozygous wild-derived
D. melanogaster genotypes. This design enables the
quantification of the extent to which naturally segre-
gating variants modify the effects of single mutations,
as well as the magnitude of variation of epistasis
among the different lines. This study reported wide-
spread suppressing epistasis of naturally segregating
modifiers on startle behaviour. Since the magnitude of
suppressing epistasis was proportional to the magni-
tude of the mutational effect of the P-element inser-
tion on startle behaviour, it was concluded that
suppressing epistasis buffers the effects of new muta-
tions in natural populations.
P-element insertions at genes previously implicated
in startle behaviour, Semaphorin-5C (Sema-5c) and
Calreticulin (Crc) also affect olfactory behaviour
(Sambandan et al., 2006) and in the case of Crc, sleep
phenotypes (Harbison & Sehgal, 2008). The objective
of the present study was to ask whether suppressing
epistasis by naturally segregating modifiers on behav-
ioural traits is a general principle or unique to the
startle response, and, moreover, to assess whether the
effects of the same P-element insertion on different
phenotypes is modulated by the same or different
2. Materials and methods
(i) Drosophila stocks
P-element insertion lines for Crc and Sema-5c, which
were generated as part of the Berkeley Drosophila
Gene Disruption Project (Bellen et al., 2004), contain
single P[GT1] insertions generated in the isogenic
w1118, CSB background. Crc and Sema-5c have pleio-
tropic effects on olfactory avoidance of benzaldehyde
(Sambandan et al., 2006; Rollmann et al., 2007),
bristle number (Norga et al., 2003) and startle re-
sponse (Yamamoto et al., 2008, 2009), and Crc also
has pleiotropic effects on sleep traits (Harbison &
Sehgal, 2008). Both Crc and Sema-5c are located on
chromosome 3 (C3). The construction of chromo-
some substitution lines carrying either a CSB C3 or
the Crc and Sema-5c P[GT1] mutations on the same
CSB C3 in inbred lines of the Drosophila melanogaster
derived from a Raleigh (North Carolina) population
of wild D. melanogaster, has been reported previously
(Yamamoto et al., 2009). Thirteen chromosome sub-
stitution lines with P-element insertions at Sema-5c
and 14 chromosome substitution lines with P-element
insertions at Crc and the corresponding controls were
used in this study (Fig. 1). All flies were reared in large
mass cultures on cornmeal/molasses/agar medium at
25 xC and a 12 h light/12 h dark cycle (lights on at
6:00 am; lights off at 6:00 pm).
(ii) Behavioural assays
We measured olfactory behaviour for C3 substitution
lines with Crc and Sema-5c mutations, and the cor-
responding CSB C3 substitution lines contempo-
raneously using a modification of the ‘dipstick’ assay
(Anholt et al., 1996), as described previously (Swarup
et al., 2011). We measured olfactory behaviour
of single-sex groups of 50 flies/replicate and three
replicates/sex for each line. Assays were conducted
between 2:00 and 4:00 pm using 0.3% (v/v) benzal-
dehyde (Sigma-Aldrich, St. Louis, MO). Replicate
measurements on individual lines were collected over
multiple days to account for environmental variation.
Flies between 4 and 7 days old were collected a day
prior to the assay and food deprived for 2 h in a 50 ml
conical tube containing a cotton wool swab tip
S. Swarup et al. 10
(referred to as ‘odour tube’). The measurement is
initiated by depositing 0.1 ml of odorant solution on
the cotton wool swab tip in the odour tube. The odour
tube is then connected to a collection tube and flies are
given 2 min to partition between the tubes. At the end
of the assay, a response index (RI) is calculated as
follows: RI=number of flies in the collection tube/
total number of flies. An RI of 1 indicates the highest
avoidance response to benzaldehyde, while 0 indicates
indifference (or attraction) to the odorant.
We measured sleep and waking activity of the Crc
chromosome substitution lines and their respective
controls by recording locomotion of virgin male
and female flies for seven continuous days using the
Drosophila Activity Monitoring (DAM) System
(Trikinetics, Waltham, MA). Each fly was housed
separately in an activity monitor tube. The DAM sys-
tem uses an infrared beam to detect movement in the
monitor tube; the movement is recorded as activity
counts in 1-min intervals. We eliminated flies that died
after 7 days of recording from the analysis. We used a
custom C++program to compute day and night sleep
duration in minutes, and waking activity as counts per
waking minute. Sleep is defined as inactivity lasting
5 min or longer (Hendricks et al., 2000; Shaw et al.,
2000; Huber et al., 2004, Ho & Sehgal, 2005).
(iii) Mutational effects and epistatic interactions
We estimated the effects (2a) of each mutation on ol-
factory behaviour and sleep phenotypes in the CSB
background as the deviation of the mean phenotypic
value of the homozygous mutant from that of the
CSB control (Falconer & Mackay, 1996). We used
Student’s t tests to assess the significance of the dif-
ference in phenotypic values between mutant and
We estimated the epistatic interaction for each
DGRP line as the difference between the expected and
observed phenotypic values. There are two chromo-
some substitution lines for each DGRP line, one with
a mutant C3 and the other with a wild-type C3. The
observed phenotypic value of each DGRP line is the
mean of the line with the mutant C3. The expected
phenotypic value of each line is the difference between
the mean of the line with the wild-type C3 and the
estimate of 2a for the appropriate mutation obtained
in the pure CSB background. We assessed the sig-
nificance of epistatic interactions in each DGRP line
background by performing three-way fixed effect
analyses of variance (ANOVAs) using the model: Y=
where Y is the observed value, m is the overall mean,
G is the effect of the presence or absence of the P-
element, S is the effect of sex, L is the random effect
of the DGRP line versus CSB genetic backgrounds,
GrL, GrS, LrS and GrLrS are the interaction
terms, and e is the environmental variance between
replicates. A significant interaction term (GrL and/
or GrLrS) indicates epistasis. To assess variation in
epistatic effects among DGRP lines, we performed
similar mixed model ANOVAs across all genotypes,
treating the DGRP genotypes and interactions with
them as random effects. Finally, to determine the
significance of epistatic interactions among different
wild-derived genetic backgrounds, we estimated indi-
vidual epistatic effects for each background and tested
for significance using ANOVA.
Fig. 1. Generation of co-isogenic CSB C3 substitution lines in inbred DGRP genetic backgrounds. The left side of
the diagram illustrates the three major D. melanogaster chromosomes in co-isogenic CSB lines, with arrows indicating
the locations of P-element insertions in Sema-5c and Crc. The right side of the diagram illustrates the introduction of
CSB C3 with or without P-element insertions into different DGRP lines, indicated with different colours (Yamamoto et al.,
Extensive epistasis for olfactory behaviour11
(i) Effects of Crc and Sema-5c mutants on olfactory
To assess the effects of naturally segregating epistatic
modifiers on P-element insertional mutations that
affect olfactory behaviour, we selected P-element in-
sertions in the Sema-5c and Crc genes that have large
effects on olfactory avoidance behaviour towards
benzaldehyde (Sambandan et al., 2006; Rollmann
on responsiveness to benzaldehyde using our modified
behavioural assay. To analyse the data, we used a
two-way ANOVA model, Y=m+L+S+LrS+E,
where m is the overall mean, L is the fixed effect of line,
S is the fixed effect of sex, LrS is the linersex in-
teraction term and E is the environmental variance.
As we observed a significant line effect (P<0.0001),
but no significant sex (P=0.99) or linersex effect
(P=0.90), measurements of sexes separately were
pooled for analyses. The RI at 0.3% (v/v) benzal-
dehyde for the CSB control was 0.98¡0.01 (n=3 re-
plicates/sex/genotype, 50 individuals per replicate),
5c and Crc mutants were 0.68¡0.03 and 0.56¡0.04,
respectively (n=3 replicates/sex/genotype, 50 in-
dividuals per replicate for each mutant), significantly
lower than the CSB control (P<0.0001; Fig. 2a).
(ii) Effects of Crc mutations on sleep
Like Sema-5c, the Crc locus is a hotspot for P-element
insertions (Spradling et al., 2011). Previously a P-
element insertion allele at Crc (CrcBG02566) was found
to affect sleep phenotypes (Harbison & Sehgal, 2008),
but this insertion was at a different site than the
P-element insertion allele (CrcBG01724) previously im-
plicated in startle behaviour (Yamamoto et al., 2009).
Although both insertions are in the first exon, it is
tinct phenotypic effects (Rollmann et al., 2006, 2008).
Therefore, we assessed the effects of the CrcBG01724al-
lele on day and night sleep and waking activity. There
were significant differences between CrcBG01724and the
co-isogenic control for night sleep in both sexes
(P<0.0001), for day sleep for males (P<0.0001), and
for wakingactivity in females(P=0.0024) (Fig. 2b–d).
There was a significant sex effect for day sleep and
waking activity (P<0.0001), and a significant sexr
line interactionfor day sleep (P<0.0001, the mutation
increases day sleep only in males).
(iii) Epistasis between Crc and Sema-5c mutations
and wild-derived DGRP backgrounds
We used inbred DGRP lines in which C3 has been
replaced by a P-element free CSB wild-type C3 or a
Night sleep (min)
Day sleep (min)
Waking activity (counts/min)
P < 0·0001
P < 0·0001
P < 0·0001
P = 0·002
CSB Crc Sema-5c
Mean response index
P < 0·0001
Fig. 2. Effects of Crc and Sema-5c mutations on olfactory
behaviour and sleep phenotypes compared with the
co-isogenic CSB control. (a) Olfactory behaviour. Bars
represent mean response indices for pooled sexes; error
bars are standard errors of the mean. (b) Night sleep.
(c) Day sleep. (d) Waking activity. Bars represent mean
day and night sleep and waking activity for males and
females, separately, for the CSB control (open bars) or the
Crc mutant (black bars); error bars are standard errors of
S. Swarup et al. 12
co-isogenic C3 carrying a P-element insertion in Crc
or Sema-5c (Fig. 1; Yamamoto et al., 2009) to assess
the effects of naturally segregating epistatic modifiers
of the mutations on olfactory behaviour and sleep
phenotypes. We measured olfactory behaviour for 13
DGRP lines in which C3 was replaced by either an
isogenic CSB C3 or a co-isogenic CSB chromosome
with a Sema-5c mutation. We also assessed olfactory
behaviour and sleep phenotypes for 14 pairs of DGRP
there were no significant effects of sex or sexrline
interaction in the analyses of olfactory behaviour
among the chromosome substitution lines, whereas
these terms were significant for night sleep, day sleep
and waking activity, we report the results for olfac-
tory behaviour pooled across sexes, and the sleep and
activity data for males and females separately.
In the absence of epistasis, the expected phenotype
of a DGRP line bearing a Sema-5c or Crc mutation is
the difference between the effect (2a) of the mutation
type of the DGRP line with the CSB C3. Epistasis is
implicated by a significant difference between this ex-
pected value and the observed mean phenotype of the
DGRP line with a mutant C3. The significance of the
estimate of the epistatic effect is given by the P-value
of the genotype by line interaction in an ANOVA
comparing the effect of the mutation in CSB and the
DGRP line. Epistatic interactions that amplify the
effect of the mutation are considered enhancer effects,
whereas those that counteract the effect of the mu-
tation are defined as suppresser effects.
We found significant epistasis for olfactory behav-
iour in all but one instance (Sema-5c in RAL_437)
(Fig. 3, Table 1). In all cases where significant
epistatic interactions were observed for olfactory
behaviour, the epistatic effects were negative; i.e. the
observed responses of the substitution lines to
RAL_303 RAL_335 RAL_358 RAL_360 RAL_362 RAL_365 RAL_375
RAL_391 RAL_437 RAL_732 RAL_786 RAL_852
RAL_208 RAL_303 RAL_335 RAL_357
(a) Sema -5c
Fig. 3. Observed (closed bars) and expected (open bars) mean response indices for olfactory behaviour of (a) 13 DGRP C3
substitution lines with a P-element insertion at Sema-5c and (b) 14 DGRP C3 substitution lines with a P-element insertion
at Crc. The error bars indicate standard errors of the mean for pooled sexes. ns, not significant, *P<0.05, **P<0.01,
Extensive epistasis for olfactory behaviour13
benzaldehyde were greater than predicted based on
the estimate of 2a in the CSB background (Table 1,
Fig. 3). Since the effect of the mutations is to reduce
the response to benzaldehyde in the CSB background,
the negative difference between observed and expec-
ted olfactory behaviour in the DGRP lines indicates
suppression of the mutant effect in wild-type back-
grounds. We also found substantial and sex-specific
epistasis between DGRP lines and the Crc mutation
for night sleep, day sleep and waking activity (Fig. 4,
Table 2). For night sleep, epistatic interactions were
mostly suppressing, as for olfactory behaviour, with
few exceptions (e.g. RAL_358 and RAL_852 for
females and RAL_365 for males; Fig. 4a and b). The
Crc mutation increases day sleep in males (Fig. 2b);
thus, suppressing epistasis would counteract the Crc
mutation by reducing day sleep duration. Interest-
ingly, three genetic backgrounds showed epistatic
interactions for day sleep for females, two of which
were enhancer effects (Fig. 4c), indicating that muta-
tions with no effects on a phenotype in one genetic
background can have significant effects in other
backgrounds (i.e. the effect of the mutation was sup-
pressed in the CSB background). There were exten-
sive epistatic effects for male day sleep (Fig. 4d).
These effects were exclusively suppresser effects; that
is, epistasis caused day sleep duration to be dimin-
ished in mutants that gave rise to prolonged day sleep.
Few epistatic effects were observed for waking ac-
tivity, with suppresser effects for both sexes in the
RAL_365 background and enhancer effects for
females in RAS_391 and males in RAL_517 (Fig. 4e
We assessed whether there was significant variation
in epistasis among the wild-type and mutant C3 sub-
stitution lines for each trait, as indicated by a signifi-
cant genotype (wild-type versus mutant) by line
(DGRP line) interaction in the ANOVA. This term
was significant for all traits (Tables 3 and 4). Thus,
there is variation in the extent to which natural var-
iants modify mutational effects.
(iv) Pleiotropic epistatic effects
In addition to their effects on olfactory behaviour
(Sambandan et al., 2006; Rollmann et al., 2007), the
Sema-5c and Crc mutations also show reduced startle
behaviour (Yamamoto et al., 2008) and a mutation at
Crc has been associated with reduced night and day
sleep, and increased waking activity (Harbison &
Sehgal, 2008). To assess whether the same epistatic
modifiers affect the effects of the Sema-5c and Crc
mutations on multiple traits, we first asked whether
there was a correlation between the estimates of epi-
static effects for olfactory behaviour and those of
startle-induced locomotion, measured previously on
the same lines (Yamamoto et al., 2009). We did not
observe a significant correlation for either Sema-5c
(Fig. 5a) or Crc (Fig. 5b). Similarly, epistasis of
olfactory behaviour was not significantly correlated
with epistasis for day sleep and night sleep for Crc,
and the correlation with waking activity in males was
only nominally significant (P=0.04; Fig. 6). Epistasis
of day time sleep, night time sleep or waking activity
was also not correlated with epistasis of startle
behaviour (Supplementary Fig. S1 available at http://
journals.cambridge.org/grh). These results show that
different naturally segregating epistatic modifiers
modulate different phenotypes affected by mutations
at the same pleiotropic gene.
Previously, olfactory behaviour in D. melanogaster
has been used as a model trait to dissect the genetic
architecture of behaviour (Anholt, 2010) and dynamic
epistatic networks of pleiotropic genes have been im-
plicated as a major feature of the genetic ensembles
that underlie the manifestation of this behavioural
phenotype (Fedorowicz et al., 1998; Sambandan
et al., 2006). D. melanogaster can also serve as a gen-
etic model to study sleep (Hendricks et al., 2000;
Shaw et al., 2000). While epistasis can be hypothe-
sized from co-regulated gene expression networks
(Harbison et al., 2009), no previous study has quan-
tified the impact of epistasis on sleep in flies. In ad-
dition to modulation of behavioural phenotypes,
suppressing epistasismayexplain theparadox
Table 1. Epistatic interactions for olfactory behaviour
in DGRP chromosome substitution lines with Sema-5c
or Crc mutations
DGRP line Sema-5cCrc
The values indicate estimated epistatic effects for olfactory
RI of individual chromosome substitution lines with Sema-
5c or Crc mutations. ***P<0.0001; **0.0001<P<0.01;
*0.01<P<0.05; ns, P>0.05; nd, not determined.
S. Swarup et al.14
between developmental robustness in the face of gen-
etic variation, as illustrated by the effects of genetic
background modifiers on mutations in Sevenless and
Drosophila Epidermal Growth Factor Receptor that
affect development of photoreceptors (Polaczyk et al.,
The recent generation of a panel of diverse homo-
zygous wild-derived chromosome substitution lines
that carry the same homozygous CSB C3 with or
without a P-element insertion (Yamamoto et al.,
2009) enables analyses of the effects of naturally seg-
regating epistatic modifiers. We used chromosome
substitution lines with Sema-5c and Crc mutations
to analyse epistatic modulation of mutations that
affect olfactory behaviour, sleep and waking activity
in Drosophila in wild-derived genetic backgrounds.
Sema-5c has been implicated in early development
(Khare et al., 2000) and Crc, a calcium-binding
Night sleep (min)
Day sleep (min)
Night sleep (min)
Day sleep (min)
Waking activity (counts/min)
Waking activity (counts/min)
RAL_208 RAL_303RAL_357RAL_358RAL_362 RAL_365RAL_391 RAL_399RAL_517 RAL_714RAL_732RAL_786RAL_852
RAL_208RAL_303RAL_357RAL_358 RAL_362RAL_365RAL_391RAL_399 RAL_517RAL_714RAL_732 RAL_786RAL_852RAL_208 RAL_303RAL_357RAL_358RAL_362RAL_365RAL_391 RAL_399RAL_517 RAL_714RAL_732RAL_786 RAL_852
RAL_208RAL_303RAL_357 RAL_358RAL_362RAL_365RAL_391RAL_399RAL_517 RAL_714RAL_732 RAL_786RAL_852 RAL_208RAL_303RAL_357RAL_358 RAL_362RAL_365RAL_391 RAL_399RAL_517RAL_714RAL_732 RAL_786RAL_852
Fig. 4. Observed (closed bars) and expected (open bars) sleep phenotypes in DGRP C3 substitution lines with a P-element
insertion at Crc. (a) Night sleep in females. (b) Night sleep in males. (c) Day sleep in females. (d) Day sleep in males.
(e) Waking activity in females. (f ) Waking activity in males. The error bars indicate standard errors of the mean for sexes
separately. ns, not significant, *P<0.05, **P<0.01, ***P<0.001.
Extensive epistasis for olfactory behaviour15
chaperone, is involved in intracellular protein trans-
port, exocytosis and development of the nervous
system in Drosophila (Prokopenko et al., 2000).
Mutations in Sema-5c reduce olfactory avoidance
behaviour (Sambandan et al., 2006; Rollmann et al.,
2007) and startle behaviour (Yamamoto et al.,
2008). Mutations in Crc result not only in aberrant
chemosensory responses (Stoltzfus et al., 2003;
Table 2. Epistatic interactions for sleep phenotypes in DGRP chromosome
substitution lines with a Crc mutation
DGRP lineSexNight sleep (min)Day sleep (min)
The values indicate estimated epistatic effects of individual chromosome substitution
lines with a Crc mutation. m, males; f, females; *** P<0.0001; **0.0001<P<0.01;
*0.01<P<0.05; ns, P>0.05.
Table 3. ANOVAs of olfactory behaviour among DGRP lines with CSB and Sema-5c or Crc mutant third
Mutation Source of variationdf SS MSFP
Sema-5c Genotype (G)
df, degrees of freedom; SS: sums of squares (type III); MS, mean squares; ***P<0.0001; *0.01<P<0.05; ns, not significant.
S. Swarup et al.16
Sambandan et al., 2006) and reduced startle behav-
iour (Yamamoto et al., 2008), but also reduce day and
night sleep duration and increase waking activity
(Harbison & Sehgal, 2008). We confirmed the effects
of these P-element insertions in the CSB background
on olfactory behaviour using a recently developed
modified high throughput olfactory behavioural assay
(Swarup et al., 2011; Fig. 2a) and confirmed the ef-
fects on sleep, using the same P-element insertion line
in Crc previously implicated in startle behaviour
(Yamamoto et al., 2009) and olfaction (Sambandan
et al., 2006) (Fig. 2b).
We found that mutational effects were generally
reduced in the chromosome substitution lines com-
pared with the original effect observed in the CSB
background. The presence of variation in epistatic
effects for each phenotype for each P-element inser-
tion indicates that different wild-derived genetic
backgrounds harbour different segregating epistatic
modifiers that alter the effect of the P-element
mutation. Although phenotypic measurements of a
larger number of chromosome substitution lines
might reveal correlations in epistatic measures among
olfactory behaviour, startle behaviour and sleep, the
lack of correlation of epistatic effects across these
phenotypes among the 27 lines that were available for
our study (Figs 5 and 6) suggests that different
Table 4. ANOVAs of sleep phenotypes and waking activity among DGRP lines with CSB and Crc mutant third
TraitSource of variation dfSSMSFP
Night sleepGenotype (G)
df, degrees of freedom; SS, sums of squares (type III); MS, mean squares; ***P<0.0001; **0.0001<P<0.01;
*0.01<P<0.05; ns, not significant.
0·20·4 0·6 0·8
Startle behaviour (Istartle)
0 0·20·4 0·60·8
Startle behaviour (Istartle)
Olfactory behaviour (Iolf)
Olfactory behaviour (Iolf)
Fig. 5. Relationship between the estimates of epistatic
interactions for olfactory behaviour (Iolf) and startle
induced locomotion (Istartle) in DGRP C3 substitution
lines. (a) Sema-5c: r2=0, P>0.05. (b) Crc: r2=0.186,
Extensive epistasis for olfactory behaviour 17
epistatic modifiers are likely to interact with the same
pleiotropic gene to modulate different phenotypes
(Fig. 7). This complex genetic architecture is in line
with previous conclusions that the manifestation of
complex behavioural phenotypes can be altered by
ensembles of epistatic genes (Sambandan et al., 2006;
Anholt, 2010; Zwarts et al., 2011). Independent
segregation of components of these ensembles in a
natural population will result in variation in epistatic
effects and these effects may express themselves
differently for different pleiotropic phenotypes as-
sociated with the same causal variant.
In conclusion, we have shown that epistasis appears
to be a pervasive general feature of natural popu-
lations and our results suggest that epistatic inter-
actions may protect against adverse effects of new
mutations. Furthermore, different epistatic inter-
actions modulate different phenotypes affected by
0·2 0·3 0·4 0·50·60·7
Night sleep (Isleep)
Olfactory behaviour (Iolf)
0·10·2 0·30·40·50·6 0·7
Olfactory behaviour (Iolf)
Olfactory behaviour (Iolf)Olfactory behaviour (Iolf)
Olfactory behaviour (Iolf)Olfactory behaviour (Iolf)
0·1 0·2 0·30·40·5 0·60·7
Day sleep (Isleep)
0·1 0·2 0·30·40·5 0·60·7
Waking activity (Isleep)
Night sleep (Isleep)
Day sleep (Isleep)
Waking activity (Isleep)
0·10·20·30·4 0·50·6 0·7
Fig. 6. Relationship between the estimates of epistatic interactions for olfactory behaviour (Iolf) and sleep phenotypes
(Isleep) in DGRP C3 substitution lines with a P-element insertion at Crc (a) Night sleep. Females: r2=0.001, P>0.05.
Males: r2=0.137, P>0.05. (b) Day sleep. Females: r2=0, P>0.05. Males: r2=0.008, P>0.05. (c) Waking activity.
Females: r2=0.086, P>0.05. Males: r2=0.319, P=0.044.
Fig. 7. Epistasis and pleiotropy. The diagram illustrates a
focal P-element-tagged gene (red circle) that forms part of
three genetic networks affecting different phenotypes,
indicated by green, blue and orange colours, respectively.
Gene ensembles that generate phenotype-specific epistatic
interactions with the focal gene, indicated by the dotted
arrows, are shown in corresponding muted colours.
S. Swarup et al.18
prevalence of epistasis in the genetic architecture of
complex traits is relevant to the design and interpret-
ation of genetic studies in human populations.
Widespread suppressing epistasis may account for the
‘missing heritability’ for human traits, such as height
(Manolio et al., 2009). Our study underscores the
importance of D. melanogaster as a model system for
the analysis of quantitative traits, as a similar detailed
analysis of epistasis under conditions in which we can
introduce a mutation in a range of tightly controlled
genetic backgrounds would not be possible in human
populations. Substitution of chromosomes with P-
element insertions in DGRP backgrounds will enable
future mapping of epistatic modifiers and, ultimately,
genome-wide characterization of epistatic interactions
between defined alleles and transposon-tagged sites
that affect organismal phenotypes.
at thesamepleiotropicgene. The
This work was supported by grants from the National
Institutes of Health (GM45146, GM59469) to TFCM and
Anholt, R. R. H. (2010). Making scents of behavioural
genetics: lessons from Drosophila. Genetics Research
(Cambridge) 92, 349–359.
Anholt, R. R. H., Lyman, R. F. & Mackay, T. F. C.
(1996). Effects of single P-element insertions on olfactory
behavior in Drosophila melanogaster. Genetics 143,
Anholt, R. R. H. & Mackay, T. F. C. (2004). Quantitative
genetic analyses of complex behaviours in Drosophila.
Nature Reviews Genetics 5, 838–849.
Bellen, H. J., Levis, R. W., Liao, G., He, Y., Carlson, J. W.,
Tsang, G., Evans-Holm, M., Hiesinger, P. R., Schulze,
K. L., Rubin, G. M., Hoskins, R. A. & Spradling, A. C.
(2004). The BDGP gene disruption project: single trans-
poson insertions associated with 40% of Drosophila
genes. Genetics 167, 761–781.
Brockmann, G. A., Kratzsch, J., Haley, C. S., Renne, U.,
Schwerin, M. & Karle, S. (2000). Single QTL effects,
epistasis, and pleiotropy account for two-thirds of the
phenotypic F(2). Variance of growth and obesity in
DU6irDBA/2 mice. Genome Research 10, 1941–1957.
Carlborg, O., Jacobsson, L., Ahgren, P., Siegel, P. &
Andersson, L. (2006). Epistasis and the release of genetic
variation during long-term selection. Nature Genetics 38,
Cheverud, J. M., Vaughn, T. T., Pletscher, L. S., Peripato,
A. C., Adams, E. S., Erikson, C. F. & King-Ellison, K. J.
(2001). Genetic architecture of adiposity in the cross of
LG/J and SM/J inbred mice. Mammalian Genome 12,
Clark, A. G. & Wang, L. (1997). Epistasis in measured
genotypes: Drosophila P-element insertions. Genetics 147,
Dilda, C. L. & Mackay, T. F. C. (2002). The genetic archi-
tecture of Drosophila sensory bristle number. Genetics
Edwards, A. C. & Mackay, T. F. C. (2009). Quantitative
trait loci for aggressive behavior in Drosophila melano-
gaster. Genetics 182, 889–897.
Eshed, Y. & Zamir, D. (1996). Less-than-additive epistatic
interactions of quantitative trait loci in tomato. Genetics
Falconer, D. S. & Mackay, T. F. C. (1996). Introduction
to Quantitative Genetics, 4/e. Reading, MA: Addison
Fedorowicz, G. M., Fry, J. D., Anholt, R. R. H. & Mackay,
T. F. C. (1998). Epistatic interactions between smell-
impaired loci in Drosophila melanogaster. Genetics 148,
Flint, J. & Mackay, T. F. C. (2009). Genetic architecture
of quantitative traits in mice, flies, and humans. Genome
Research 19, 723–733.
Gurganus, M. C., Nuzhdin, S. V., Leips, J. W. & Mackay,
T. F. C. (1999). High-resolution mapping of quantitative
trait loci for sternopleural bristle number in Drosophila
melanogaster. Genetics 152, 1585–1604.
Harbison, S. T., Carbone, M. A., Ayroles, J. A., Stone,
E. A.,Lyman,R. F.&
Co-regulated transcriptional networks contribute to
natural genetic variation in Drosophila sleep. Nature
Genetics 41, 371–375.
Harbison, S. T. & Sehgal, A. (2008). Quantitative genetic
analysis of sleep in Drosophila melanogaster. Genetics 178,
Hendricks, J. C., Finn, S. M., Panckeri, K. A., Chavkin, J.,
Williams, J. A., Sehgal, A. & Pack, A. I. (2000). Rest in
Drosophila is a sleep-like state. Neuron 25, 129–138.
Hill, W. G., Goddard, M. E. & Visscher, P. M. (2008). Data
and theory point to mainly additive genetic variance for
complex traits. PLoS Genetics 4, e1000008.
Ho, K. S. & Sehgal, A. (2005). Drosophila melanogaster:
an insect model for fundamental studies of sleep. Methods
in Enzymology 393, 772–793.
Huber, R., Hill, S. L., Holladay, C., Biesiadecki, M.,
Tononi, G. & Cirelli, C. (2004). Sleep homeostasis in
Drosophila melanogaster. Sleep 27, 628–639.
Jordan, K. W.,Morgan,
(2006). Quantitative trait loci for locomotor behavior in
Drosophila melanogaster. Genetics 174, 271–284.
Khare, N., Fascetti, N., DaRocha, S., Chiquet-Ehrismann,
R. & Baumgartner, S. (2000). Expression patterns of two
new members of the Semaphorin family in Drosophila
suggest early functions during embryogenesis. Mech-
anisms of Development 91, 393–397.
Klingenberg, C. P., Leamy, L. J. & Cheverud, J. M. (2004).
Integration and modularity of quantitative trait locus ef-
fects on geometric shape in the mouse mandible. Genetics
Kroymann, J. & Mitchell-Olds, T. (2005). Epistasis
and balanced polymorphism influencing complex trait
variation. Nature 435, 95–98.
Leips, J. & Mackay, T. F. C. (2000). Quantitative trait loci
for life span in Drosophila melanogaster: interactions with
genetic background and larval density. Genetics 155,
Leips, J. & Mackay, T. F. C. (2002). The complex genetic
architecture of Drosophila life span. Experimental Aging
Research 28, 361–390.
Long, A. D., Mullaney, S. L., Reid, L. A., Fry, J. D.,
Langley, C. H. & Mackay, T. F. C. (1995). High resol-
ution mapping of genetic factors affecting abdominal
bristle number in Drosophila melanogaster. Genetics 139,
Mackay, T. F. C., Richards, S., Stone, E. A., Barbadilla, A.,
Ayroles, J. F., Zhu, D., Casillas, S., Magwire, M. M.,
Cridland, J. M., Richardson, M. F., Anholt, R. R. H.,
Barro ´ n,M.,Bess,
Mackay,T. F. C. (2009).
T. J.& Mackay,T. F. C.
C., Blankenburg,K. P.,
Extensive epistasis for olfactory behaviour 19
Carbone, M. A., Castellano, D., Chaboub, L., Duncan,
L., Han, Y., Harris, Z., Javaid, M., Jayaseelan, J. C.,
Jhangiani, S. N., Jordan, K. W., Lara, F., Lawrence, F.,
Lee, S. L., Librado, P., Linheiro, R. S., Lyman, R. F.,
Mackey, A. J., Munidasa, M., Muzny, D. M., Nazareth,
L., Newsham, I., Perales, L., Pu, L.-L., Qu, C., Ra ` mia,
M., Reid, J. G., Rollmann, S. M., Rozas, J., Turlapati,
L., Worley, K. C., Wu, Y.-Q., Yamamoto, A., Zhu, Y.,
Bergman, C. M., Thornton, K., Mittleman, D. & Gibbs,
R. A. (2012). The Drosophila melanogaster Genetic
Reference Panel. Nature 482, 173–178.
Mackay, T. F. C., Stone, E. A. & Ayroles, J. F. (2009). The
genetics of quantitative traits: challenges and prospects.
Nature Reviews Genetics 10, 565–577.
Manolio, T. A., Collins, F. S., Cox, N. J., Goldstein, D. B.,
Hindorff, L. A., Hunter, D. J., McCarthy, M. I., Ramos,
E. M., Cardon, L. R., Chakravarti, A., Cho, J. H.,
Guttmacher, A. E., Kong, A., Kruglyak, L., Mardis, E.,
Rotimi, C. N., Slatkin, M., Valle, D., Whittemore, A. S.,
Boehnke, M., Clark, A. G., Eichler, E. E., Gibson, G.,
Haines, J. L., Mackay, T. F. C., McCarroll, S. A. &
Visscher, P. M. (2009). Finding the missing heritability of
complex diseases. Nature 461, 747–753.
Norga, K. K., Gurganus, M. C., Dilda, C. L., Yamamoto,
A.,Lyman,R. F., Patel,
Hoskins, R. A., Mackay, T. F. C. & Bellen, H. J. (2003).
Quantitative analysis of bristle number in Drosophila
mutants identifies genes involved in neural development.
Current Biology 13, 1388–1396.
Phillips, P. C. (2008). Epistasis – the essential role of gene
interactions in the structure and evolution of genetic sys-
tems. Nature Reviews Genetics 9, 855–867.
Polaczyk, P. J., Gasperini, R. & Gibson, G. (1998).
Naturally occurring genetic variation affects Drosophila
photoreceptor determination. Development Genes and
Evolution 207, 462–470.
Prokopenko, S. N., He, Y., Lu, Y. & Bellen, H. J. (2000).
Mutations affecting the development of the peripheral
nervous system in Drosophila: a molecular screen for
novel proteins. Genetics 156, 1691–1715.
Zwarts, L., Callaerts, P., Norga, K., Mackay, T. F. C. &
Anholt, R. R. H. (2008). Pleiotropic effects of Drosophila
neuralized on complex behaviors and brain structure.
Genetics 179, 1327–1336.
O¨zsoy, E. D., Yamamoto, A., Mackay, T. F. C. &
Anholt, R. R. H. (2006). Pleiotropic fitness effects of the
Tre1/Gr5a region in Drosophila. Nature Genetics 38,
Rollmann, S. M., Yamamoto, A., Goossens, T., Zwarts, L.,
Mackay, T. F. C. & Anholt, R. R. H. (2007). The early
developmental gene Semaphorin 5c contributes to olfac-
tory behavior in adult Drosophila. Genetics 176, 947–956.
Sambandan, D., Yamamoto, A., Fanara, J. J., Mackay,
T. F. C. & Anholt, R. R. H. (2006). Dynamic genetic
P. H.,Rubin, G. M.,
A. C.,Yamamoto, A.,
M. M.,Morgan,T. J.,
interactions determine odor-guided behavior in Droso-
phila melanogaster. Genetics 174, 1349–1363.
Shaw, P. J., Cirelli, C., Greenspan, R. J., & Tononi, G.
(2000). Correlates of sleep and waking in Drosophila
melanogaster. Science 287, 1834–1837.
Sinha, H., David, L., Pascon, R. C., Clauder-Munster, S.,
Krishnakumar, S., Nguyen, M., Shi, G., Dean, J., Davis,
R. W., Oefner, P. J., McCusker, J. H. & Steinmetz, L. M.
(2008). Sequential elimination of major-effect con-
tributors identifies additional quantitative trait loci con-
ditioning high-temperature growth in yeast. Genetics 180,
Spradling, A. C., Bellen, H. J. & Hoskins, R. A. (2011).
Drosophila P elements preferentially transpose to repli-
cation origins. Proceedings of the Natural Academy of
Sciences USA 108, 15948–15953.
Steinmetz, L. M., Sinha, H., Richards, D. R., Spiegelman,
J. I., Oefner, P. J., McCusker, J. H. & Davis, R. W.
(2002). Dissecting the architecture of a quantitative trait
locus in yeast. Nature 416, 326–330.
Stoltzfus, J. R., Horton, W. J. & Grotewiel, M. S. (2003).
Odor-guided behavior in Drosophila requires calreticulin.
Journal of Comparative Physiology A. Neuroethology
Sensory, Neural and Behavioral Physiology 189, 471–483.
Swarup, S., Williams, T. I. & Anholt, R. R. H. (2011).
Functional dissection of Odorant binding protein genes in
Drosophila melanogaster. Genes Brain and Behavior 10,
van Swinderen, B. & Greenspan, R. J. (2005). Flexibility in
a gene network affecting a simple behavior in Drosophila
melanogaster. Genetics 169, 2151–2163.
Weber, K., Eisman, R., Morey, L., Patty, A., Sparks, J.,
Tausek, M. & Zeng, Z. B. (1999). An analysis of poly-
genes affecting wing shape on chromosome 3 in
Drosophila melanogaster. Genetics 153, 773–786.
Workman, M. S.,Leamy,
Cheverud, J. M. (2002). Analysis of quantitative trait lo-
cus effects on the size and shape of mandibular molars in
mice. Genetics 160, 1573–1586.
Yamamoto, A., Anholt, R. R. H. & Mackay, T. F. C.
(2009). Epistatic interactions attenuate mutations affect-
ing startle behaviour in Drosophila melanogaster. Genetics
Research (Cambridge) 91, 373–382.
Yamamoto, A., Zwarts, L., Callaerts, P., Norga, K.,
Mackay, T. F. C. & Anholt, R. R. H. (2008). Neuro-
genetic networks for startle-induced locomotion in
Drosophila melanogaster. Proceedings of the Natural
Academy of Sciences USA 105, 12393–12398.
Yi, N., Zinniel, D. K., Kim, K., Eisen, E. J., Bartolucci, A.,
Allison, D. B. & Pomp, D. (2006). Bayesian analyses
of multiple epistatic QTL models for body weight and
body composition in mice. Genetical Research 87, 45–60.
Zwarts, L., Magwire, M. M., Carbone, M. A., Versteven,
M., Herteleer, L., Anholt, R. R. H., Callaerts, P. &
Mackay, T. F. C. (2011). Complex genetics architecture
of Drosophila aggressive behavior. Proceedings of the
Natural Academy of Sciences USA 108, 17070–17075.
L. J., Routman,E. J.&
S. Swarup et al.20