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/
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no. 27 www.pnas.org?cgi?doi?10.1073?pnas.0901419106
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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