Oviposition preference for and positional avoidance
of acetic acid provide a model for competing
behavioral drives in Drosophila
Ryan M. Josepha, Anita V. Devinenib, Ian F. G. Kingc, and Ulrike Heberleina,b,c,1
aProgram in Biological Sciences, and Departments ofbNeuroscience andcAnatomy, University of California, San Francisco, CA 94143-2822
Edited by Yuh Nung Jan, University of California School of Medicine, San Francisco, CA, and approved May 8, 2009 (received for review February 14, 2009)
Selection of appropriate oviposition sites is essential for progeny
survival and fitness in generalist insect species, such as Drosphila
melanogaster, yet little is known about the mechanisms regulating
how environmental conditions and innate adult preferences are
evaluated and balanced to yield the final substrate choice for egg-
deposition. Female D. melanogaster are attracted to food containing
acetic acid (AA) as an oviposition substrate. However, our observa-
tions reveal that this egg-laying preference is a complex process, as
avoidance for the same food. We show that 2 distinct sensory
modalities detect AA. Attraction to AA-containing food for the pur-
pose of egg-laying relies on the gustatory system, while positional
repulsion depends primarily on the olfactory system. Similarly, dis-
tinct central brain regions are involved in AA attraction and
repulsion. Given this unique situation, in which a single environ-
mental stimulus yields 2 opposing behavioral outputs, we propose
that the interaction of egg-laying attraction and positional aver-
balance competing behavioral drives and integrate signals in-
volved in choice-like processes.
choice behavior ? gustatory system ? olfactory system ?
mushroom body ? ellipsoid body
since a laid egg represents a marker for female position. Past
studies have used egg laying as a readout for conditions advan-
tageous to progeny development (1, 2), in which oviposition
preference effectively separates larvae of different sibling spe-
cies of Drosophila. Egg laying has also been used to detect
aversion toward compounds toxic to both larvae and adults (3,
4). Furthermore, numerous studies have used patterns of ovi-
position to distinguish subtle differences in host plant prefer-
ences, which have provided insights into resource requirements
and ecological behaviors of different Drosophila species (5, 6).
Despite numerous studies using oviposition-site selection as a
behavioral readout, direct study of the relevant sensory circuits
and the oviposition program itself have been initiated only
recently in D. melanogaster (7, 8). To investigate the genetic
mechanisms and neural circuits regulating this important be-
havioral choice in D. melanogaster, we developed a simple yet
robust 2-choice assay that utilizes acetic acid (AA), a naturally
occurring product of fruit fermentation, as an egg-laying attract-
ant (9, 10). However, in addition to verifying a strong egg-laying
preference for AA, we surprisingly observed D. melanogaster
show a strong positional aversion to the same AA-containing
food. We demonstrate that when sampling for oviposition sites,
females integrate input from distinct sensory modalities to
choose a particular behavioral output from 2 competing options:
ovipositional attraction for and positional repulsion to AA.
Egg-laying preference is primarily relayed through gustatory
neurons, while positional aversion is relayed through the olfac-
tory system. We also map central brain regions mediating these
competing behaviors. Taken together, the process by which
viposition provides a powerful yet simple means for mon-
itoring preference behavior in Drosophila melanogaster,
females integrate sensory information to execute these compet-
ing and interacting behaviors provides a tractable model for
studying choice-like behavior in D. melanogaster.
Egg-Laying Preference for and Positional Aversion to AA-Containing
Food. To investigate the mechanisms involved in egg-laying
preference, we devised a simple apparatus in which females are
allowed the choice to lay eggs on regular food or food containing
various concentrations of AA (Fig. 1A). Similar to previous
observations (9, 10), mated females laid approximately 91% of
their eggs on food containing 5% AA (Fig. 1 B and D; ?AA) as
compared to regular food (Fig. 1 B and D; ?AA), with an
oviposition index (OI) of ?0.82. It has been postulated that D.
melanogaster may use AA as an energy source (11), such that
oviposition preference would result from an attraction to AA-
containing media as a feeding source. To test this hypothesis, we
first observed the physical location of flies during the 3-h
oviposition assay. Surprisingly, females avoided food containing
5% AA (the concentration found naturally in vinegar), with a
position index (PI) of ?0.33 (Fig. 1 B and E). To test for feeding
(TLC) to quantify the relative ingestion of each dye. Flies
ingested essentially equal amounts of food containing or lacking
AA (Fig. 1C). Thus, oviposition-site selection does not reflect
innate positional or feeding preferences, and may be in direct
conflict with positional preference under ecologically relevant
conditions. Recent studies show similar decoupling between
adult taste and egg-laying preferences (7, 12).
stronger in virgin females and males (Fig. 1B). Since virgin
females lay fewer eggs than mated females (Fig. S1A), they likely
search for egg-laying substrates less frequently, and may there-
fore have less incentive to overcome their innate positional
aversion to AA-containing food. Males explore AA-containing
food even less frequently than virgin females. Thus, the posi-
tional aversion to AA grows as the need to lay eggs is diminished
or absent, implying that the attractive oviposition and repulsive
positional drives are in competition. However, mated and virgin
females showed equivalently high OI values in response to
Author contributions: R.M.J., A.V.D., and U.H. designed research; R.M.J. and A.V.D. per-
formed research; I.F.G.K. contributed new reagents/analytic tools; R.M.J. and A.V.D. ana-
lyzed data; and R.M.J. and U.H. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Freely available online through the PNAS open access option.
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
July 7, 2009 ?
vol. 106 ?
no. 27 www.pnas.org?cgi?doi?10.1073?pnas.0901419106
substrate is therefore an innate preference not affected by
post-mating behavioral modifications (13).
were specific to AA or elicited by the acidity of AA-containing
food, we analyzed these behaviors on foods containing acetic,
hydrochloric (HCl), or sulfuric (H2SO4) acids, titrated to equiv-
alent pH values. At pH 3.5 (5% AA), females showed negligible
oviposition preference for foods with HCl or H2SO4, while
preference for AA-containing food was high (Fig. 2A). Likewise,
the positional aversion observed with 5% AA was eliminated
when food was acidified with HCl or H2SO4(Fig. 2A); similar
positional responses were observed in males (Fig. S2). Addi-
tionally, HCl and H2SO4did not suppress egg-laying (Fig. 2B).
Thus, egg-laying preference for 5% AA cannot be solely ex-
plained by the food’s acidity at this pH.
However, when acidity was increased even further, foods with
HCl or H2SO4became attractive egg-laying substrates, while AA
became aversive (Figs. 2C and S3A). This aversion to lay eggs on
higher concentrations of AA was accompanied with increased
positional repulsion (Figs. 2D and S3B), suggesting that repul-
sion overrides attraction at higher AA concentrations. Positional
repulsion appeared to be specific for high AA concentrations,
rather than for the increased acidity of the food, as flies showed
no positional aversion to HCl or H2SO4at equivalently low pH
values (Fig. 2D). Thus, females show a specific attraction for AA
as an oviposition substrate that cannot be explained by increased
food acidity. Moreover, these data show that egg-laying and
positional preferences are in competition when tested with AA,
but not other acids, such that decreases in oviposition preference
are accompanied with increases in positional aversion.
To explore the idea that the choice of egg-laying substrate
reflects an active sampling and evaluation process, rather than
a simple reflex, we assayed flies in additional experimental
contexts. When tested in a ‘‘stripe assay’’ (Fig. S4A), in which
females sequentially encounter alternating segments of control
food and food with increasing concentrations of AA (see SI
Methods), they still showed high preference for 5% AA. Thus,
flies explored their environment before selecting a preferred
oviposition site. Further evidence for exploration of AA-
containing food is shown using single-fly locomotor traces
The Olfactory System Mediates Positional Aversion to AA. Although
the sensory inputs and genetic pathways involved in D. melano-
gaster oviposition preference are relatively uncharacterized, the
role of taste and olfaction in egg laying of other insects has been
investigated (1, 6, 14, 15). In addition, AA can be aversive to D.
melanogaster in certain olfactory assays (5, 16). We therefore
analyzed the behavior of flies with impaired or enhanced
olfactory organs, the third antennal segments (17, 18). Anten-
naectomized females, while normal for egg-laying preference
(Fig. 3A), lost their positional aversion to 5% AA (Fig. 3B).
Thus, olfaction is essential for positional aversion to AA, but is
not required for oviposition preference. Consistent with these
data are our observation that silencing antennal projection
neurons disrupted positional aversion (Table S1). Males lacking
antennae also showed diminished aversion to 5% AA (Fig. S5C).
To analyze the effect of enhanced olfactory input, we tested
1) mutant flies with an increased sense of smell and 2) wild-type
flies exposed to higher AA concentrations. Mutations in white
rabbit (whir) show an elevated olfactory startle response to
ethanol and other odorants (19) and are suspected to possess an
for assaying oviposition and positional preference. Mated females were pre-
sented a dish in which one-half contains food mixed with a compound of
choice, and the other contains food mixed with an equivalent volume of
(PI) were calculated during the 3-h sampling period (see Methods for OI and
males in response to 5% AA. As the egg-laying rate decreased for each
consecutive group (left-to-right, shaded triangle), the positional aversion
response increased (*, P ? 0.05;**, P ? 0.01; 1-way ANOVA, Bonferroni
post-test; n ? 17). No significant differences in OI values were observed
between female groups (Student’s unpaired t test). (C) Females showed no
preference for consuming food containing AA. Mated females were pre-
sented the following 2-choice food combinations: ?AA/?AA, Blue #1/Green
#3 (black bar); ?AA/?AA, Blue #1 ? 5% AA/ Green #3 (green bar); ?AA/?AA,
5% AA ? Blue #1/Green #3 (blue bar). After feeding, the amount of dye in fly
gut contents was quantified by TLC. (D) Females deposited the majority of
eggs on 5% AA substrate (?AA). (E) A single time point showing females
instead of regular food.
(A) Mated female egg laying and positional responses were assayed using
food titrated to pH ? 3.5 with AA, HCl, or H2SO4. Control H2O-supplemented
food has pH ? 4.5. Significant responses at pH ? 3.5 were only observed for
AA-containing substrate (**, P ? 0.01; 1-way ANOVA, Dunnett’s multiple
comparison post-test; n ? 9). (B) Total number of eggs laid were comparable
on food supplemented with AA, HCl, or H2SO4(n ? 9). (C) Egg-laying prefer-
ences and (D) positional preferences of mated females for foods titrated to
different pH values using AA, HCl, and H2SO4(see Table S2 for acid concen-
trations). There were significant differences between the dose-response
curves for AA when compared with HCl and H2SO4(linear regression;***, P ?
0.0001; n ? 4–8).
July 7, 2009 ?
vol. 106 ?
no. 27 ?
enhanced sense of smell. Consistent with this hypothesis, posi-
tional aversion to AA was increased in whir1females, an effect
that was significantly diminished by antennal removal (Fig. 3B).
Furthermore, the increased positional repulsion exhibited by
whir1females was accompanied by egg-laying aversion for AA-
containing food; this effect was also strongly ameliorated by
antennaectomy (Fig. 3A). Similarly, removing the antenna of
whir1males reduced their excessive positional repulsion to AA
(Fig. S5C). We next tested responses to a high concentration
(10% AA), which normally eliminates oviposition preference
and enhances positional aversion (Fig. S5 A and B). Removing
antennae restored egg-laying preference to nearly normal levels
and normalized positional aversion (Fig. S5 A and B). Positional
aversion was not completely eliminated in antenaectomized
whir1females and wild-type flies exposed to 10% AA (Fig. S5 B
and C), suggesting that either olfactory neurons on the maxillary
palps or other sensory modalities are engaged at high AA
concentrations. Despite this caveat, our data show that olfactory
neurons in the third antennal segment are the primary sensors
inducing positional aversion to AA.
To further show that oviposition and positional preferences
are competing drives, we asked if reduced olfactory input would
increase egg-laying preference for AA. Because OIs approach
saturation at 5% AA (Fig. 3A), an increase would be concealed
by a ‘‘ceiling effect.’’ We therefore analyzed responses to 0.25%
AA, a concentration that yielded a moderately attractive egg-
laying response and no positional avoidance (OI ? ?0.34, PI ?
?0.03; Fig. S5D). Antennaectomized females exhibited in-
creased egg-laying preference and a small but significant shift to
a more positive positional preference (OI ? ?0.55, PI ? ?0.10;
Fig. S5D). Thus, even low AA concentrations are detected by the
olfactory system and perceived as slightly repulsive. These data
support our hypothesis that olfactory-based aversion competes
with egg-laying attraction for AA.
The Gustatory System Mediates Oviposition Attraction to AA. Our
data indicated that a sensory modality other than olfaction
mediates egg-laying preference; a likely candidate was the
gustatory system. Gustatory bristles are present on the primary
taste structures: the labellum, front legs, wing margins, and the
ovipositor (20). To test if gustatory neurons mediate egg-laying
preference, we assayed pox-neuro (poxn) mutants, in which taste
bristles are transformed into mechanosensory bristles lacking
central nervous system (23). Homozygous poxn?M22-B5females
showed reduced egg-laying preference (OI ? ?0.28; Fig. 4A)
when compared to wild-type, poxn?M22-B5heterozygous, and
poxn?M22-B5homozygous flies carrying the SuperA transgenic
construct (23) that rescues all poxn defects. These data implicate
taste receptors in the egg-laying attraction for AA. However,
positional aversion was also reduced in homozygous poxn?M22-B5
females (PI ? ?0.09; Fig. 4B), likely due to abnormalities in
olfactory processing centers in the mutant (23). To overcome
these issues, we tested transgenic strains in which poxn expres-
sion was restored in a tissue-specific manner. The full-1 and -152
transgenes restore normal brain morphology and chemosensory
bristles to poxn?M22-B5flies, except for taste organs found on the
labellum (23). poxn?M22-B5females carrying the full-1 or full-152
transgene showed diminished AA egg-laying preference (OI ?
?0.12, ?0.23, respectively; Fig. 4A), but still maintained a robust
positional aversion to 5% AA (PI ? ?0.47, ?0.42, respectively;
Fig. 4B). In fact, positional aversion to 5% AA was enhanced
when compared with control strains. To confirm that gustatory
and not olfactory pathways mediate egg-laying responses to AA,
we removed the third antennal segments from the poxn-rescue
lines. As expected, antennaectomized flies showed reduced
positional aversion to AA, while oviposition indices were un-
changed (Fig. 4). Overall, these data show that females use taste
neurons on the labellum to recognize AA as an egg-laying
attractant, and that reduced egg-laying preference leads to a
compensatory increase in positional repulsion.
Brain Centers Involved in Egg-Laying and Positional Preferences for
AA. Thus far, our data has identified peripheral sensory systems
that induce egg-laying and positional responses to AA, and
exhibited reduced OI to 5% AA when compared to wild-type (wt) females.
Restoration of normal OI values was seen in whir1lacking antenna (?ant)
when compared with unoperated (?ant) whir1females. (B) whir1exhibited
excessive repulsion to 5% AA when compared with wt females. Removal of
0.001; 1-way ANOVA, Bonferroni’s post-test for comparisons between geno-
Role of olfaction in oviposition and positional choices. (A) whir1
ANOVA revealed genotype as the primary source of variation (80.42%, F ?
F ? 0.71, P ? 0.61; n ? 7). OIs were significantly lower in poxn?M22-B5homozy-
gotes and in poxn?M22-B5?/?flies carrying the full-1 and full-152 transgenes,
when compared with wild type (Ctl), poxn?M22-B5?/?heterozygotes and poxn
carrying the complete rescue SuperA transgene. (a ?***, P ? 0.001 when
compared with Ctl, poxn?M22-B5?/?, and SuperA lines; 1-way ANOVA, Bonfer-
roni’s post-test for comparisons between genotypes of the same antennal
condition; n ? 12). Antennaectomy had no effect on OI values (yellow bars).
(B) PIs of the flies shown in A. Most genotypes (unoperated, purple bars)
maintained positional repulsion to 5% AA, although poxn?M22-B5?/?flies
showed significantly reduced aversion when compared to Ctl flies.
poxn?M22-B5?/?carrying the full-1 and full-152 transgenes showed en-
hanced aversion, when compared with poxn?M22-B5?/?carrying the com-
plete-rescue SuperA transgene (b ?*, P ? 0.05, when compared with
SuperA; c ?*, P ? 0.05 when compared with Ctl; 1-way ANOVA, Bonfer-
roni’s post-test; n ? 12). All genotypes demonstrated significant reductions
in positional aversion upon removal of antennae (magenta bars), with the
exception of poxn?M22-B?/?(2-way ANOVA, Bonferroni’s post-test within
genotypes for ?ant vs. ?ant; n ? 12).
Role of gustatory system in oviposition and positional choices. (A) OI
shown that behavioral outputs of the 2 preference pathways are
in competition. To identify higher-order brain regions that may
mediate and integrate signals from these competing pathways,
we silenced specific neuronal populations by expressing a tem-
perature-sensitive Shibire transgene, UAS-Shits(24), under the
control of various GAL4 lines. 58 GAL4-expressing lines were
crossed to UAS-Shits, and their progeny were assayed for egg
laying and positional preferences at the permissive (23 °C) and
restrictive (30 °C) temperatures (Table S1).
Three GAL4 lines with highly selective expression in the
mushroom body (MB) lost egg-laying preference for 5% AA.
Two representative lines, GAL45-120and GAL45-98, showed
strongly reduced oviposition preference at 30 °C in the presence
of UAS-Shits(Figs. 5A and S6A). Meanwhile, positional aversion
5A and S6A), providing evidence for dissociation between the
competing behavioral choices toward AA. Expression of GAL4
in both the GAL45-120and GAL45-98lines, visualized with a
UAS-GFP transgene (25), was preferentially found in the MB,
some lateral neurons (LNs), and a few scattered cells in the brain
(Figs. 5B and S6B). Assays conducted with pdf-GAL4/UAS-Shits
flies, which express GAL4 specifically in LNs, did not affect egg
laying or positional responses (Fig. S7). Furthermore, we did not
detect GFP expression in olfactory and gustatory neurons of
GAL45-120and GAL45-98lines. Thus, the observed phenotypes
were not due to silencing of LNs or sensory systems.
We also identified 4 lines with highly specific expression in the
ellipsoid body (EB) ring neurons that exhibited disrupted posi-
tional aversion to 5% AA. Two representative lines, GAL44-67
and GAL42-72showed reductions in positional aversion to 5%
AA in the presence of UAS-Shitsat 30 °C (Figs. 5C and S6C)
when compared with the singly transgenic controls. Egg-laying
preference in the experimental flies was essentially unchanged
(Figs. 5C and S6C). GAL44-67and GAL42-72lines express GAL4
primarily in the EB ring neurons, (Figs. 5D and S6D); peripheral
sensory structures revealed no GFP expression. Of note, females
showed increased positional aversion to 5% AA at 30 °C (Figs.
5 and S6), likely due to enhanced olfactory input caused by
higher volatility of AA at 30 °C; this effect was consistent across
all genotypes and thus, did not confound data interpretation.
GAL44-67and GAL42-72also showed disrupted positional aver-
sion in the presence of UAS-Shitsat 23 °C (Figs. 5C and S6C), an
effect likely caused by residual function of the UAS-Shitstrans-
gene in neurons that are particularly sensitive to synaptic silenc-
ing (26). We were unable to determine if the disruption in
positional aversion seen upon silencing EB neurons was associ-
ated with an increase in egg-laying preference, as the latter was
nearly maximal at 5% AA. Attempts to carry out these tests at
lower AA concentrations were unsuccessful, as changes in
positional responses were too subtle for definitive conclusions.
To further investigate whether the MB and EB function in
separate or interconnected pathways, we simultaneously si-
lenced both regions in ‘‘double-GAL4’’ flies carrying GAL45-120,
GAL44-67and UAS-Shits. Cross-talk between the 2 circuits could
manifest as nonadditive (synergistic or epistatic) effects on the
behavioral choices. Compared with the respective single GAL4/
UAS-Shitslines, double-GAL4 females showed disruptions of
oviposition and positional preference that were essentially the
sum of those seen with the individual GAL4 lines (Fig. S8),
which suggests the MB and EB function in largely separate
pathways to affect egg-laying attraction and positional repulsion
to 5% AA, respectively.
Our data provide a neurobehavioral model in which AA, a single
ecologically relevant input, is detected by separate sensory
systems to generate 2 distinct behavioral outputs: gustatory-
based egg-laying attraction and olfactory-based positional re-
pulsion (Fig. 6). We postulate D. melanogaster has an innate
positional repulsion to the smell of AA. However, when needing
to lay eggs, the attraction for AA overrides this positional
repulsion, thereby allowing females to deposit their eggs on
females at permissive (23 °C) and nonpermissive (30 °C) temperatures. (A)
GAL45-120/UAS-Shitsexhibited reduced egg-laying preference for 5% AA at
30 °C, while maintaining normal positional aversion. (B) Brain GAL4 expres-
sion of GAL45-120, visualized by crossing to UAS-CD8.GFP, revealed strong
expression in the mushroom body (MB) and a few lateral neurons (LNs). (C)
GAL44-67/UAS-Shitsexhibited strong reduction in positional aversion at 23 °C
strong expression in neurons that project to the ellipsoid body ring (EB). The
locations of cell bodies (cb), dendrites (d), and axonal terminals (t) are indi-
cated. (A and C:*, P ? 0.05;**, P ? 0.01;***, P ? 0.001 by 1-way ANOVA with
Bonferroni’s post-test for comparisons between columns within the 23 °C
or 30 °C groups; n ? 8). (B and D: green ? UAS-CD8.GFP, red ? neuropil
Effect of silencing specific neuronal subsets with Shibirets. OI and PI
tional repulsion to AA. The gustatory (GS) and olfactory (OS) systems simul-
taneously detect input from a single compound, AA. Both sensory systems
relay the signals to higher order centers of their respective circuits for pro-
cessing and subsequent execution of motor programs (MS ? motor systems)
leading to oviposition preference (OP) or positional avoidance (PA). Compe-
tition between behavioral drives could occur: (1) in the female brain where
neurons of the 2 pathways interact to simultaneously evaluate competing
signals, such that either oviposition preference (OP) or positional avoidance
(3) a combination of both central integration and behavioral output compe-
tition; (4) and (5) as directional inhibitory interactions between either the
outputs. Red intersection lines represent negative interactions.
Models for the interaction between egg-laying attraction and posi-
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vol. 106 ?
no. 27 ?
AA-containing food. Other studies have revealed opposing
behavioral responses to a single compound; when detected as
carbonation by the gustatory system, CO2is attractive (27), but
when detected as an odorant, it is aversive (28). However, our
experimental setup is unique in that opposing behavioral re-
sponses to a single stimulus (AA) are concurrently induced and
the 2 behavioral drives.
these competing drives. In 1 extreme model, information gath-
ered by the olfactory and gustatory systems would be processed
by a set of common neurons, where concurrent evaluation of
sensory input from both pathways would result in the selection
of either repulsion or attraction before a final motor program for
these neurons simultaneously integrate sensory inputs to drive
either egg-laying or positional behaviors. In the alternative
extreme model, gustatory and olfactory signals would be inde-
pendently processed by parallel neural circuits, such that attrac-
tion and repulsion only compete at the behavioral output level,
after motor-program selection, since a female fly can only be in
1 place at a given time (Fig. 6, no. 2). Combinatorial models
invoking both central integration and competition of behavioral
outputs are also possible (Fig. 6, no. 3–5).
Models that involve competition through central integration
imply that signals converge on common neurons in higher brain
centers, and therefore, silencing these neurons would be ex-
pected to disrupt both egg-laying attraction and positional
repulsion. In our limited Shitsscreen, we did not identify such a
region. However, we did find higher-order structures that reg-
ulate each individual preference pathway. The MB appears to
mediate taste-based attraction to AA for egg-laying purposes.
Given the role of the MB in olfactory learning and memory (29,
30), it was surprising that it regulates taste-based behavior in our
paradigm. However, a neural connection between the suboe-
sophageal ganglion, which receives gustatory input, and the MB
has been described recently in honey bees (31), providing a
possible neuroanatomical link. Meanwhile, the EB (likely the R1
and R4 ring neurons) plays a role in the olfactory-based posi-
tional repulsion to AA. Our data are consistent with studies
showing the EB plays a role in olfactory-related tasks (30, 31)
and spatial memory (32).
Exactly how and where the MB and EB function in the neural
circuits that regulate AA responses remains to be determined.
However, the results obtained with MB and/or EB silencing
allow us to draw important conclusions regarding the models
presented in Fig. 6. Experiments with poxn flies showed that
abrogating gustatory input upstream of potential central inte-
gration in the brain not only impaired egg-laying preference for
AA, but also caused a concomitant enhancement of positional
aversion. In contrast, synaptic silencing of the MB, while also
causing a robust decrease of egg-laying preference, did not result
in a compensatory increase in positional aversion. These data 1)
argue against competition of behavioral outputs as the sole
mechanism responsible for selection between behavioral re-
sponses, and therefore, some cross-talk between olfactory and
gustatory inputs must occur centrally, and 2) strongly suggests
that the MB functions downstream of such cross-talk, after a
positional response has been chosen, as its silencing affects only
the motor program involved in egg-laying preference without
altering positional aversion. With regards to the EB, our double-
GAL4 experiments (Fig. S8) revealed an additive effect, leading
us to hypothesize that the EB functions in parallel to the MB to
control the motor program involved in positional aversion. Thus,
potential cross-talk between the 2 circuits could also occur
upstream from the EB.
Our data clearly show that disrupting peripheral sensory input
causes compensatory shifts in egg-laying attraction and posi-
tional aversion (Figs. 3, 4, and S5). Thus, despite evidence for
central integration, competition between behavioral outputs
contributes to the overall response of flies when choosing a
substrate for oviposition. Such competition arises from a logis-
supplemented with 5% AA when not given a choice, but when
provided with the choice of both oviposition substrates, they lay
approximately 90% of their eggs on AA-containing food. Since
laying an egg takes time (8), and females cannot be in 2 places
at the same time, the OI and PI values must be at least partially
correlated. Thus, our data supports a model where both central
integration and competition of behavioral outputs mediate the
choice-like behavior elicited when females encounter different
oviposition substrate options (Fig. 6, no. 3).
We suggest that our paradigm can be used as a simple model
for choice-like behavior in D. melanogaster. Supporting this
possibility, a recent study by Yang et al. (8) employs a different
paradigm for simple decision-making, in which females use their
gustatory system to evaluate bitter and sweet egg-laying sub-
strates. Our model differs in that it uses a single compound to
stimulate competing drives via 2 distinct sensory modalities.
Both systems provide powerful new paradigms to study the
molecular and neural bases of simple decision-making in D.
Fly Stocks. Behavioral analysis and white rabbit (whir1) experiments were
performed in w1118Berlin genetic background. The poxn?M22-B5lines used
were a mixed w Berlin background, in which flies contained the original
w1118Berlin strain. UAS-Shitstransgenic flies contained 2 insertions of the
transgene in a w1118Canton S background. Unless otherwise noted, flies
were reared in constant light, 25 °C, 70% humidity on cornmeal/molasses/
Two-Choice Oviposition and Positional Assays. The 2-choice apparatus was
assembled using plastic 6-oz round-bottom bottles with the base cut off and
replaced with a transparent 60-mm Petri dish lid. Food substrate was made by
mixing the appropriate volume of experimental compound or H2O into mol-
ten fly food at temperatures below the boiling point. Two-choice dishes were
made by dividing a 35-mm Petri dish lid with a razor blade, and pouring 2
samples of food-substrate into each half (see SI Methods for detailed descrip-
tion). For each test, 15–20 recently-eclosed females were collected and mated
for 2–3 days. Flies were gently knocked into the assay bottle without anes-
thesia to eliminate CO2-based artifacts, and allowed to sample for 3 h. To
determine oviposition preference, the amount of eggs on each half of the
on experimental food ? no. of eggs laid on control food) / no. of total eggs
laid]. For positional preference, the number of flies on each half of the dish
was counted at 15-min intervals for 3 h. The number of flies was totaled,
food ? flies on control food) / (flies on experimental food ? flies on control
tracking are described in SI Methods.
Feeding Assay. To assay feeding preferences, the food mixing protocol was
modified such that either Erioglaucine (FD&C Blue #1) or Fast Green FCF dye
(Green #3) was mixed into the experimental (5% AA) or control (5% H2O)
food. Females were allowed to feed for 4-h, after which they were frozen,
homogenized, and extracts centrifuged to remove insoluble material. Dyes in
the supernatant were separated by thin layer chromotography (see SI Meth-
ods for detailed protocol).
Generation of Food of Different Acidity. We empirically measured the concen-
trations of AA, HCl, and H2SO4that yielded food-substrate mixtures with
equivalent pH values between 2.0 and 4.5 by using pH indicator strips. To
verify these measurements, hardened food was reheated, diluted 1:10 in
distilled water, and the pH of the resulting solution was measured using a pH
Surgeries. Females were anesthetized with CO2, and the third antennal seg-
ment was removed with a set of sharp forceps. Flies recovered for 2 days
Brain Regions Involved in Egg-Laying and Positional Preference. We selected 58
(Table S1). GAL4 lines were crossed to flies carrying UAS-Shitstransgenes.
GAL4/UAS-Shits, GAL4/?, and UAS Shits/? females were placed at room tem-
perature (23 °C) or in an incubator (30 °C) and allowed to equilibrate for 30
min, after which the number of flies on each half of the dish was counted at
10-min intervals. After 8 time points (t ? 70 min), both the 23 °C and 30 °C
experiments were moved to the dark for the remainder of the assay for
optimal egg laying.
Immunohistochemistry. GAL4/UAS-CD8.GFP fly brains were immunostained
with an antibody against GFP and nc82 and imaged by using a Leica confocal
microscope (see SI Methods for details).
Statistics. All statistical analyses were performed using GraphPad Prism, Ver-
sion 4.0 (GraphPad Softwate, Inc.). Statistics were performed independently
on oviposition preference data and position preference data. Error bars in
figures, mean ? standard error of the mean (S.E.M).
ACKNOWLEDGMENTS. We thank W. Boll (Institute of Molecular Biology,
University of Zu ¨rich, Zu ¨rich, Switzerland) and M. Noll (Institute of Molecular
Biology, University of Zu ¨rich, Zu ¨rich, Switzerland) for pox-neuro flies and
extremely useful insights, L. Luo (Department of Biological Sciences, Stanford
University, Stanford, CA) and E. Marin (Department of Biological Sciences,
Stanford University, Stanford, CA) for GAL45-120, GAL44-67, and GAL42-72brain
images, F. Wolf (Ernest Gallo Clinic and Research Center, Emeryville, CA) for
single-fly traces, A. Rothenfluh (Department of Psychiatry, University of Texas
Southwestern Medical Center, Dallas, TX) for advice and use of whir alleles,
and D. Anderson and members of the Heberlein lab for exciting discussions.
Funding was provided by a National Science Foundation predoctoral fellow-
ship (to A.D.), a National Research Service Awards/National Institute on Drug
Abuse (to I.K.), and grants from National Institutes of Health/National Insti-
tute on Alcohol Abuse and Alcoholism (to U.H.).
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July 7, 2009 ?
vol. 106 ?
no. 27 ?