Joseph et al. Oviposition preference for and positional avoidance of acetic acid: A model for competing behavioral drives in Drosophila melanogaster

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1 Oviposition preference for and positional avoidance of acetic acid: A model for competing behavioral drives in Drosophila melanogaster Ryan Joseph 2, Anita Devineni 3, Ian King 1, and Ulrike Heberlein 1,2,3,# Department of Anatomy 1, Programs in Biological Sciences 2 and Neuroscience 3, University of California at San Francisco, San Francisco, CA # Corresponding author: Ulrike Heberlein ulrike.heberlein@ucsf.edu Phone: FAX: Running title: Simple decision-making in Drosophila Key words: Choice behavior, oviposition, acetic acid, olfactory system, gustatory system, mushroom body, ellipsoid body. Classification: Biological Sciences, Neuroscience Manuscript Information: Number of Text Pages: 19 Number of Figures: 7 Number of Tables: 0 Abstract Word Count: 187 Total Number of Characters: ~47,000 (with spaces), ~40,000 (no spaces) Supplemental Information: Suppl. Figures: 8 Suppl. Tables: 2 Suppl. Figure legends and Methods: 1 1

2 Abstract 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 observations reveal that this egg laying preference is a complex process, as it directly opposes an otherwise strong, default behavior of positional avoidance for the same food. We show that two distinct sensory modalities detect AA. Attraction to AA containing food for the purpose of egg laying relies on the gustatory system, while positional repulsion depends primarily on the olfactory system. Similarly, distinct central brain regions are involved in AA attraction and repulsion. Given this unique situation, in which a single environmental stimulus yields two opposing behavioral outputs, we propose that the interaction of egg laying attraction and positional aversion for AA provides a powerful model for studying how organisms balance competing behavioral drives and integrate signals involved in choice like processes. 2

3 \body Introduction Oviposition provides a powerful yet simple means for monitoring preference behavior in D. melanogaster, since a laid egg represents a physical marker for female position. Past studies have used egg laying as a readout for conditions advantageous to progeny development (1, 2), in which oviposition preference effectively separates larva of different sibling species of Drosophila. Egg laying has also been used to detect aversion towards compounds toxic to both larvae and adults (3, 4). Furthermore, numerous studies have employed physical and temporal patterns of oviposition to distinguish subtle differences in host plant preferences, which have provided insights into resource requirements and ecological behaviors of different Drosophila species (5, 6). Despite the numerous studies utilizing oviposition site selection as a readout for behaviors such as feeding and toxin avoidance, direct investigation of the relevant sensory circuits and the actual oviposition program itself have been initiated only recently in D. melanogaster (7, 8). Thus, much of the genetic mechanisms and neural circuits that regulate this important behavioral choice remain to be characterized. To investigate the genetic and neural pathways involved in oviposition site selection, we developed a simple yet robust two choice assay that utilizes acetic acid (AA), a naturally occurring byproduct of fruit fermentation, as an egg laying attractant (9, 10). However, in addition to verifying a strong egg laying preference for AA, we observed that, surprisingly, D. melanogaster females show a strong positional aversion to the same AA containing food. We demonstrate that when sampling for oviposition sites, females integrate inputs from distinct sensory modalities to evaluate and choose a particular behavioral output from two competing options: ovipositional attraction for and positional repulsion to AA. Egg laying preference is primarily relayed through gustatory sensory neurons, while positional aversion is relayed through the olfactory system. We also map some of the central brain regions mediating these competing behaviors. Taken together, the process by which females integrate sensory information to execute these competing and interacting behavioral drives provides a tractable model for studying choice like behavior in D. melanogaster. Results Egg laying preference for and positional aversion to AA containing food To investigate the mechanisms involved in D. melanogaster 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 3

4 91% of their eggs on food containing 5% AA (Fig. 1B, D; +AA) as compared to regular food (Fig. 1B, D; - AA), with an oviposition index (OI) of (a positive index reflects attraction, a negative reflects repulsion). It has been postulated that D. melanogaster may utilize AA as an energy source (11), in which case oviposition would result from a preference for AA containing media as a feeding source. To test this hypothesis, we first observed the physical location of mated females 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 (Fig. 1B, E), meaning that on average 67% of females directly sampling the food selected the half of the dish lacking AA with regard to physical location. To test for feeding preferences, we employed a modified two choice assay in which different food dyes were mixed into the halves of the dish. After the sampling period, female gut contents were analyzed by thin layer chromatography to quantify the relative ingestion of the two dyes. Females 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 identified similar de coupling between adult taste and egg-laying preferences (7, 12). Interestingly, positional repulsion for AA containing food was stronger in virgin females and males (Fig. 1B). Given that virgin females lay fewer eggs than their mated counterparts (Fig. S1A), they likely search for oviposition substrates less frequently, and may therefore have less incentive to overcome their innate positional aversion for AA containing food. Males explore AA containing food even less frequently than virgin females. Thus, the positional aversion towards AA grows as the need to lay eggs is either diminished in virgin females or eliminated in males, 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 AA containing food (Fig. 1B); attraction to AA as an oviposition substrate is therefore an innate preference not affected by post mating behavioral modifications (13). Effect of acidity on egg laying and positional preferences To determine if oviposition preference and positional avoidance were specific to AA or elicited by the higher acidity of AA containing food, we analyzed these behaviors on foods containing acetic, hydrochloric (HCl), or sulfuric (H 2 SO 4 ) acids, titrated to equivalent ph values. At ph 3.5 (5% AA), females showed negligible oviposition preference for foods containing HCl (OI=+0.14) or H 2 SO 4 (OI=+0.04), while preference for AA containing food was high (OI=+0.88) (Fig. 2A). Similarly, the positional aversion observed with 5% AA (PI=-0.38) was essentially eliminated on food acidified with either HCl or H 2 SO 4 (Fig. 2A). Thus, egg 4

5 laying preference for AA cannot be solely explained by the food s acidity at this ph. Flies laid the same number of eggs on these substrates (Fig. 2B), thus HCl and H 2 SO 4 do not suppress egg laying. When food acidity was increased even further, however, foods acidified with HCl or H 2 SO 4 became attractive, while AA containing food became aversive (Fig. 2C). This aversion to lay eggs on higher concentrations of AA was accompanied with increased positional repulsion (Fig. 2D), suggesting that the latter may override the attraction to lay eggs on higher, non-ecologically relevant AA concentrations. Importantly, positional repulsion appeared to be specific to high concentrations of AA, rather than to the increased acidity of the food, as positional aversion for HCl and H 2 SO 4 containing foods was negligible (Fig. 2D). Males, which failed to show positional aversion to HCl or H 2 SO 4 at ph=3.5, showed strong aversion to AA containing food of the same ph (Fig. S2). These data indicate that females show a specific attraction for AA as an oviposition substrate that cannot be explained by increased food acidity. Moreover, these experiments revealed that oviposition and positional preferences are in competition when tested with AA, such that decreases in oviposition preference were always accompanied with increases in positional aversion. In contrast, these two behaviors could be completely uncoupled on foods acidified with other acids; higher concentrations of HCl and H 2 SO 4, while significantly enhancing OIs, produced essentially neutral PIs. Taken together, these data demonstrate that egg laying and positional preferences induced by AA are competitive and selective for AA. Oviposition and positional preferences for AA in different experimental paradigms To explore the notion that the choice of egg laying substrate reflects a sampling and evaluation process, we carried out experiments in which mated females encountered various concentrations of AA in two distinct experimental settings. When presented with the choice to lay eggs on food with or without AA in the two choice assay (Fig. 1A), flies showed gradually increasing OIs as AA concentrations rose from 0.1 to 5% AA; this was followed by a reduction in OIs at AA concentrations greater than 5% (Fig. 3A). Yet, flies laid comparable number of eggs on all AA concentrations (Fig. S3). When tested in a stripe assay (Fig. 3B), in which females encounter alternating segments of control food and food supplemented with various concentrations of AA (see Suppl. Methods), they essentially showed the same degree of preference for AA: low preference for 0.25 and 10%, but high preference for 5% AA. Thus, female flies explored their environment prior to selecting a preferred oviposition site, and did not simply laid their eggs on the first AA containing substrate encountered. Further evidence for exploration of AA containing food is provided by high resolution single fly traces (Fig. S4). 5

6 The olfactory system mediates positional aversion to AA Although the sensory inputs and genetic pathways involved in D. melanogaster oviposition preference are relatively uncharacterized, the role of taste and olfaction in oviposition behaviors 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). To identify the sensory modalities involved in egg laying and positional preferences, we analyzed the behaviors of flies with impaired or enhanced olfaction. To impair olfaction, we surgically removed the primary olfactory organs, the third antennal segments (17, 18). Antennaectomized females, while normal for egg-laying preference (Fig. 3A), exhibited a loss of aversion for food containing 5% AA (PI=+0.12; Fig. 3B). Thus, olfaction plays an essential role in positional aversion to AA, but is not involved in oviposition preference. Consistent with these data is our observation that synaptic silencing of antennal projection neurons also disrupted positional aversion (Table S2). Males lacking antennae also showed significantly diminished positional aversion to 5% AA when compared to unoperated males (Fig. S5). 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, encoding RhoGAP18B) show an elevated olfactory startle response to ethanol and other odorants (19) and are therefore suspected to possess an enhanced sense of smell. Consistent with this hypothesis, positional aversion to AA was increased in whir 1 females (PI=-0.87; Fig. 4B), an effect that was significantly diminished by antennal removal (PI=-0.14; Fig. 4B). Furthermore, the increased repulsion exhibited by whir 1 females was accompanied by egg laying aversion for AA containing food (OI=-0.34; Fig. 4A), an effect that was strongly ameliorated by antennaectomy (OI=+0.61; Fig. 4A). Similarly, removing the antenna of whir 1 males reduced their excessive positional repulsion to AA (Fig. S5A). We next tested the responses to high concentrations of AA, which we had found to eliminate oviposition preference and enhance positional aversion (Fig. 2C,D). When females were presented with the choice of regular food and food containing 10% AA, their normally strong oviposition preference for AA was essentially abolished (OI=+0.07; Fig. 4C), and associated with an elevated positional repulsion (PI=-0.84; Fig 4D). Removal of the third antennal segment restored egg laying preference to nearly normal levels (OI=+0.54; Fig. 4C) and also normalized positional aversion (PI=-0.29; Fig. 4D). Taken together, these data provide examples in which the egg laying preference for AA is overridden by a strong positional aversion caused by excessive olfactory input, further illustrating competition between the two behavioral choices. However, the egg laying preferences of both antenaectomized whir 1 females (Fig. 4A) and wild type females exposed to 10% AA (Fig. 4C) were still 6

7 lower than that of wild type females exposed to 5% AA (Fig. 4A,C). Similarly, the positional aversion was not completely eliminated in antenaectomized whir 1 females and wild type females exposed to 10% AA (Fig. 4B,D). These data suggest that either olfactory neurons on the maxillary palps or other sensory modalities become engaged at high AA concentrations not normally found in the flies natural habitat. Despite this caveat, our data indicate that the olfactory neurons housed in the third antennal segment are the primary sensory input producing positional aversion to AA containing food. To further test the idea that oviposition and positional preferences are competing drives, we asked if reduced olfactory input would increase oviposition preference for AA. Since oviposition preference approaches saturation at 5% AA (OI=+0.82; Fig. 4A,C), an increase in OI 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. S5B). Antennaectomized females exhibited a significant increase in egg laying preference and a small but significant shift to a more positive positional preference (OI=+0.55, PI=+0.10; Fig S5B). These data show that even very low AA concentrations are still detected by the olfactory system and perceived as slightly repulsive, and further support the hypothesis that olfactory-based aversion competes with egg laying attraction for AA. The gustatory system mediates oviposition attraction to AA Our data indicated that at least one other sensory modality was involved in the behavioral responses, since preference for AA containing food as an oviposition substrate persisted and even increased in flies lacking their third antennal segment. A likely candidate was sensory input from 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 egg laying preference for AA was mediated by gustatory neurons, we analyzed pox neuro (poxn) mutant flies, in which taste bristles are transformed into mechanosensory bristles that do not express gustatory receptors (21, 22); null mutants also have structural defects in the central nervous system (23). Homozygous poxn M22-B5 females showed reduced egg laying preference (OI=+0.28; Fig. 5A) when compared to wild type, poxn M22-B5 heterozygous, and poxn M22-B5 homozygous females carrying the SuperA transgenic construct (23) that rescues all poxn defects (OI=+0.83, +0.96, +0.89, respectively; Fig. 5A). These data implicate taste receptors in the attraction towards AA for egg laying purposes. However, the positional aversion to AA was also reduced in homozygous poxn M22-B5 females (PI=-0.09; Fig. 5B), likely due to abnormalities in olfactory processing centers in the mutant (23). To further refine the gustatory inputs for taste based egg laying preference and bypass issues related to abnormal olfactory brain centers, we 7

8 tested several transgenic strains in which poxn expression was restored in a tissue specific manner. The full- 1 and full-152 transgenes restore normal adult brain morphology and chemosensory bristle development to poxn M22-B5 flies, with the exception of taste organs found on the labellum (23). poxn M22-B5 homozygous females carrying either the full-1 or full-152 transgene showed diminished AA egg laying preference (OI=+0.12, +0.23, respectively; Fig. 5A), but still maintained a robust positional aversion to 5% AA (PI=- 0.47, -0.42, respectively; Fig. 5B). In fact, the positional aversion to 5% AA was enhanced when compared to control strains. Taken together, these data show 1) that females use taste neurons on the labellum to recognize AA as an egg laying attractant, and 2) that the reduced egg laying preference observed in their absence leads to enhanced positional repulsion. To confirm that the responses to AA are largely mediated by gustatory and olfactory pathways, we removed the third antennal segments from poxn lines. As expected, antennaectomized poxn M22-B5 homozygotes or homozygotes carrying the full-1 or full-152 transgenes showed a loss or strong reduction in positional aversion to AA (PI=0.00, -0.10, respectively; Fig. 5B). Oviposition indices remained similar to their already reduced values (Fig. 5A). Thus, olfaction and taste are the primary sensory modalities that participate in the environmental assessment leading to the different behavioral choices in response to AA. Higher brain centers involved in egg laying preference and positional aversion to AA Thus far, our data have identified the peripheral sensory systems that induce egg laying and positional responses to AA and shown that the behavioral outputs of the two preference pathways are in competition, since reductions in egg laying preference are always associated with an increase in positional aversion, and vice versa. Two extreme models could explain this competition: 1) the two sensory signals are independently processed in the female brain and simply compete at the behavioral output level, since a fly can only be in one place at any given time, or 2) the information from taste and olfactory based drives converges within the female brain, such that signals from both pathways are evaluated and integrated before driving the behavioral choice. The latter model requires that neurons within the female brain, particularly in higher order structures involved in complex processing, be involved in both the egg laying and positional behaviors. To identify such potential brain regions, we synaptically silenced specific neuronal populations by expressing a temperature sensitive Shibire transgene, UAS-Shi ts (24), under the control of various GAL4 lines. 58 GAL4 expressing lines were crossed to UAS-Shi ts, and their female progeny was assayed for egg laying and positional preferences at the permissive (23 C) and restrictive (30 C) temperatures (Table S2). Three GAL4 lines with highly selective expression in the mushroom body (MB) exhibited diminished egg 8

9 laying preference for 5% AA. Two representative lines, GAL and GAL showed strongly reduced oviposition preference at 30 C in the presence of UAS-Shi ts (OI=-0.18, Fig. 6A; OI=+0.12, Fig. S6A). Meanwhile, positional aversion to 5% AA was unaffected in the experimental and control females (Figs. 6A and S6A). Expression of GAL4 in both the GAL and GAL lines, visualized with a UAS-GFP transgene (25), was preferentially found in the MB, some lateral neurons (LNs), and a few additional neurons scattered throughout the brain (Figs. 6B and S6B). Assays conducted with pdf-gal4/uas-shi ts flies, which express GAL4 specifically in PDF positive LNs, did not affect egg laying or positional preferences (Fig. S7). Furthermore, we did not detect GFP expression in olfactory and gustatory neurons of GAL /UAS- GFP and GAL /UAS-GFP females (data not shown), excluding the possibility that the observed phenotypes were due to silencing of sensory systems. These data suggest that while the MB is essential for egg laying preference, it is not required for the aversive positional response, and provide the first experimental evidence for dissociation between the competing behavioral choices towards AA. We also identified fourlines with highly specific expression in the ellipsoid body (EB) ring neurons that selectively disrupted positional aversion to 5% AA. Two representative lines, GAL and GAL showed significant reductions in positional aversion to 5% AA in the presence of UAS-Shi ts at 30 C (PI=- 0.03, Fig. 6C; PI =-0.28, Fig. S6C) when compared to GAL /+, GAL /+, and UAS-Shi ts /+ controls. Egg laying preference in these flies was essentially unchanged (Figs. 6C and S6C). The GAL and GAL lines express GAL4 primarily in the cell bodies of the EB ring neurons, likely in the R2, R3 and R4 subclasses (Figs. 6D and S6D). Peripheral sensory structures revealed no GFP expression (data not shown). Of note, females generally showed higher positional aversion to 5% AA at 30ºC than 23 C (Figs. 6 and S6, 7), likely due enhanced olfactory input caused by increased volatility of AA at 30 C; this effect was consistent across all genotypes and did therefore not confound data interpretation. Lines GAL and GAL also showed disrupted positional aversion in the presence of UAS-Shi ts at 23ºC (Figs. 6C and S6C). This effect is likely due to residual function of the strong UAS-Shi ts transgene in neurons that are particularly sensitive to synaptic silencing, as has been reported (26). We are unable to determine if the disruption in positional aversion seen upon silencing EB neurons was associated with an increase in egg laying preference, as the latter is nearly maximal at 5% AA; attempts to carry out these experiments at lower concentrations of AA were unsuccessful, as positional aversion was too low in all genotypes. To further investigate whether the MB and EB function in separate or interconnected neural circuits to regulate the behavioral responses to AA, we tested flies in which both brain regions were silenced simultaneously. The presence of crosstalk between the two circuits could manifest itself as non additive 9

10 (synergistic or epistatic) effects on the behavioral choices. We therefore tested double GAL4 females, carrying GAL , GAL and UAS-Shi ts, for egg laying and positional responses to 5% AA. Compared to the respective single GAL4/UAS-Shi ts lines, the double GAL4 females showed disruptions of oviposition and positional preferences that were essentially the sum of those seen with the individual GAL4 lines (Fig. S8). Thus, our data suggests the MB and EB function in largely separate pathways to affect egg laying attraction and positional repulsion to 5% AA, respectively. These results do not, however, eliminate the possibility of an interaction between the two circuits occurring upstream of the parallel segments involving the MB and EB (see Discussion). Discussion Our results demonstrate that a single environmental stimulus, AA, at ecologically relevant concentrations, generates two competing behavioral responses, egg laying attraction and positional repulsion, when sensed by the gustatory and olfactory systems, respectively. We postulate that females exhibit an innate positional repulsion to the smell of AA, which is also seen in males. However, when needing to lay eggs, a taste based attraction for AA overrides this positional repulsion, thereby allowing females to deposit their eggs on AA containing food. These data allow us to construct a neurobehavioral model in which one environmental input is sensed by two separate sensory systems to generate distinct behavioral outputs (Fig. 7). Other studies have shown that flies can display opposing behavioral responses to a single compound; when detected as carbonation by the gustatory system, CO 2 is attractive (27), but when detected as an odorant, it is aversive (28). However, our experimental setup is distinct in that opposing behavioral responses to a single stimulus (AA) are concurrently induced and assayed, which affords direct observation of the competition between the two behavioral drives. Environmental and genetic factors influence the sensory inputs that balance the two competing behavioral drives. When females encounter AA concentrations higher than those typically found in nature, or if their sense of smell is genetically enhanced, as in whir 1 mutants, positional repulsion dominates and females avoid AA containing food as an egg laying substrate. Conversely, when their sense of smell is disrupted, attraction towards AA containing food increases, even at very low concentrations of AA. Similarly, when females are unable taste AA with their labellum, as in poxn null mutants carrying full-1 or full-152 transgenes, the reduction in egg laying preference for AA containing food is associated with an increase in positional aversion for the same food. Thus, in addition to demonstrating that egg laying attraction and positional aversion to AA are mediated by taste and olfactory systems, respectively, our data 10

11 provide evidence for competition between the two behaviors, since changes in preference for one cause compensatory shifts in preference in the other. Several models can be invoked to explain these data. In one extreme model, the information gathered by the olfactory and gustatory systems would be integrated and processed in higher brain centers, where concurrent evaluation of sensory input from both pathways would result in the selection of either repulsion or attraction before a final motor program is executed (Fig. 7, #1). In the alternative extreme model, the gustatory and olfactory signals would be processed by parallel neural circuits such that attraction and repulsion only compete at the behavioral output level after motor program selection, since a female fly can only be in one place at any given time (Fig. 7, #2). Combinatorial models invoking both central integration and competition of behavioral outputs are also possible (Fig. 7, #3-5). Models that involve competition through central integration imply that signals converge on common neurons in higher brain centers, and therefore synaptically silencing such neurons would be expected to disrupt both egg laying attraction and positional repulsion. In our limited Shi ts screen of 58 GAL4 lines, we did not identify such a brain region. However, we did identify higher order brain structures involved in regulating each of the individual preference pathways. The MB appears to be involved in taste mediated attraction to AA for egg laying purposes. Since the MB has been primarily studied for its role in olfactory learning and memory (29, 30), it was surprising that in our paradigm it appears to regulate the taste based behavior. However, a neural connection between the suboesophageal ganglion, which receives gustatory inputs, and the MB has been described recently in honeybees (31), providing a potential neuroanatomical link between gustatory inputs and the MB. On the other hand, the EB (likely the R1 and R4 ring neurons) appears to play a role in the olfactory based positional repulsion to AA. Our data is consistent with recent studies showing that the EB plays a role in spatial memory (32), as well as olfactory-related responses (30, 31). Exactly how and where the MB and EB function in the neural circuits that regulate AA attraction and repulsion remains to be determined. However, the results obtained with MB and/or EB silencing, allow us to draw some important conclusions regarding the models presented in Figure 7. Experiments with poxn flies showed that abrogating gustatory inputs upstream of potential central integration 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 responses and 11

12 therefore some cross-talk between the olfactory and gustatory inputs must occur centrally, and 2) strongly suggests that the MB functions after of such cross talk, 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. S7), in which the MB and EB were silenced simultaneously, revealed an additive effect, leading us to hypothesize that the EB functions in parallel to the MB to control motor program involved in positional aversion. However, more complex scenarios can also be envisioned. Our data clearly show that disrupting peripheral sensory input causes compensatory shifts in egglaying attraction and positional aversion (Fig. 2C-D, 4, 5, S5B). Thus, despite evidence for central integration, competition between behavioral outputs also contributes to the overall response of female flies when choosing a substrate for oviposition. Such competition arises from a logistical issue; females lay equal numbers of eggs on regular food or food 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 two 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. 7, #3-5). We suggests that our paradigm could be employed as a simple model for choice like behavior in D. melanogaster females. Supporting this possibility, a recent study by Yang et al. (8) employs a different paradigm as a readout for simple decision making, in which females use their gustatory system to evaluate bitter and sweet egg laying substrates. Our setup differs in that it uses a single compound to stimulate competing drives via two distinct sensory modalities. Both systems provide powerful new paradigms to study the molecular and neural bases of simple decision making in D. melanogaster. Methods Fly stocks: Behavioral analysis and white rabbit (whir 1 ) experiments were performed in w 1118 Berlin genetic background. The poxn M22-B5 lines used were in a mixed w Berlin background, in which flies contained the original poxn M22-B5 second chromosome, but all other chromosomes were from the w 1118 Berlin strain. UAS- Shibire ts transgenic flies contained two insertions of the transgene in a w 1118 Canton S background. Unless otherwise noted, flies were reared in constant light, 25 C, 70% humidity on cornmeal/molasses/yeast food. Two-choice oviposition and positional assays: The two 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- 12

13 substrates were made by mixing the appropriate volume of experimental compound or H 2 O into molten 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 two samples of food-substrate into each half (see Supplemental Methods for detailed description). For each test, recently-eclosed females were collected and mated for 2-3 days. Flies were gently knocked into the assay bottle without anesthesia to eliminate C0 2 -based artifacts. Flies were allowed to sample for 3 h. To determine oviposition preference, the amount of eggs on each half of the two choice dish was counted, and an oviposition index (OI): [OI = (# eggs laid on experimental food - # eggs laid on control food) / # 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. Number of flies was totaled, averaged, and a position index (PI) was calculated: [PI = (flies on experimental food - flies on control food) / (flies on experimental food + flies on control food). Variants of this two-choice setup, including the stripe assay and single fly tracking (Fig. 3B, C), are described in Suppl. Methods. Two-choice feeding assays: 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% H 2 0) 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 Suppl. Methods for detailed protocol). Generation of food of different acidity: We empirically measured the concentrations of AA, HCl, and H 2 SO 4 that yielded food-substrate mixtures with equivalent ph values between 2.0 and 4.5 using ph indicator strips. To verify these measurements, hardened food was re-heated, diluted 1:10 in distilled water, and the ph of the resulting solution was measured using a ph meter. Acid concentrations that yield equivalent ph values are listed in Table S1. Surgeries: Females were anesthetized with C0 2, and the third antennal segment was removed with a set of sharp forceps. Flies were allowed to recover for 2 days before testing. Brain regions involved in egg-laying and positional preference: We selected 58 lines with GAL4 expression in restricted neuronal subsets of the adult fly brain (Table S2). GAL4 lines were crossed to flies carrying UAS-Shi ts transgenes. Mated GAL4/UAS-Shi ts, GAL4/+, and UAS-Shi ts /+ females were placed at room temperature (23 C) or in an incubator (30ºC) 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 switched to dark conditions for the remainder of the assay for optimal egg-laying. 13

14 Immunohistochemistry: GAL4/UAS-CD8.GFP fly brains were immunostained with an antibody against GFP and nc82, and imaged using a Leica confocal microscope (see Suppl. Methods for details). Statistics: All statistical analyses were performed using GraphPad Prism, Version 4.0 (GraphPad Softwate, Inc., San Diego CA). Statistics was performed independently on oviposition preference data and position preference data. Error bars in figures represent mean ± standard error of the mean (S.E.M). Acknowledgements We thank W. Boll and M. Noll for pox-neuro flies and extremely useful insights, L. Luo and E. Marin for GAL , GAL4 4-67, and GAL brain images, F. Wolf for single-fly traces, A. Rothenfluh for advice and use of whir alleles, and members of the Heberlein lab for exiting and heated discussions. Funding provided by an NSF predoctoral fellowship (A.D.), an NRSA/NIDA (I.K.) and grants from NIH/NIAAA (U. H.). References 1. Richmond RC, Gerking JL (1979) Oviposition site preference in Drosophila. Behav Genet 9: Chess KF, Ringo JM (1985) Oviposition site selection by Drosophila melanogaster and Drosophila simulans. Evolution 39: Possidente B, Mustafa M, Collins L (1999) Quantitative genetic variation for oviposition preference with respect to phenylthiocarbamide in Drosophila melanogaster. Behav Genet 29: Moreteau B, R'Kha S, David JR (1994) Genetics of a nonoptimal behavior: oviposition preference of Drosophila mauritiana for a toxic resource. Behav Genet 24: Amlou M, Moreteau B, David JR (1998) Genetic analysis of Drosophila sechellia specialization: oviposition behavior toward the major aliphatic acids of its host plant. Behav Genet 28: Barker JS, Starmer WT, Fogleman JC (1994) Genotype-specific habitat selection for oviposition sites in the cactophilic species Drosophila buzzatii. Heredity 72(4): Matsuo T, Sugaya S, Yasukawa J, Aigaki T, Fuyama Y (2007) Odorant-binding proteins OBP57d and OBP57e affect taste perception and host-plant preference in Drosophila sechellia. PLoS Biol 5: e Yang CH, Belawat P, Hafen, E, Jan L-Y, Jan Y-N (2008) Drosophila egg-laying site selection as a system to study simple decision-making processes. Science 319: Laudien H, Iken HH (1977) Ecological imprinting and protein biosynthesis. Experiments with Drosophila melanogaster Meigen Z Tierpsychol 44:

15 10. Eisses KT (1997) The influence of 2-propanol and acetone on oviposition rate and oviposition site preference for acetic acid and ethanol of Drosophila melanogaster. Behav Genet 27: Parsons PA (1982) Acetic acid vapour as a resource and stress in Drosophila. Australian Journal of Zoology 30: Harada, E., Haba, D., Aigaki, T., Matsuo, T. (2008). Behavioral analyses of mutants for two odorantbinding protein genes, Obp57d and Obp57e, in Drosophila melanogaster. Genes Genet. Syst. 83, Kubli E (2003) Sex-peptides: seminal peptides of the Drosophila male. Cell Mol Life Sci 60, Higa I, Fuyama Y (1993) Genetics of food preference in Drosophila sechellia. I. Responses to food attractants. Genetica. 88: Mitchell BK, Itagaki H, Rivet MP (1999) Peripheral and central structures involved in insect gustation. Microsc Res Tech 47: Fuyama Y (1976) Behavior genetics of olfactory responses in Drosophila. I. Olfactometry and strain differences in Drosophila melanogaster. Behav Genet 6: de Bruyne M, Foster K, Carlson JR (2001) Odor coding in the Drosophila antenna. Neuron 30: Keller A, Vosshall LB (2007) Influence of odorant receptor repertoire on odor perception in humans and fruit flies. Proc Natl Acad Sci U S A 104: Rothenfluh A, Threlkeld RJ, Bainton RJ, Tsai L-T, Lasek AW, Heberlein U (2006) Distinct behavioral responses to ethanol are regulated by alternate RhoGAP18B isoforms. Cell 127: Stocker RF (1994) The organization of the chemosensory system in Drosophila melanogaster: a review. Cell Tissue Res 275: Awasaki T, Kimura K (1997) pox neuro is required for development of chemosensory bristles in Drosophila. J Neurobiol 32: Clyne PJ, Warr CG, Carlson JR (2000) Candidate taste receptors in Drosophila. Science 287: Boll W, Noll M. (2002) The Drosophila Pox neuro gene: control of male courtship behavior and fertility as revealed by a complete dissection of all enhancers. Development 129: Kitamoto T (2001) Conditional modification of behavior in Drosophila by targeted expression of a temperature-sensitive shibire allele in defined neurons. J Neurobiol 47: Marin EC, Jefferis GS, Komiyama T, Zhu, H, Luo L (2002) Representation of the glomerular olfactory map in the Drosophila brain. Cell 109:

16 26. Kimura K, Hachiya T, Koganezawa M, Tazawa T, Yamamoto D (2008) Fruitless and doublesex coordinate to generate male-specific neurons that can initiate courtship. Neuron 59: Fischler W, Kong P, Marella S, Scott K (2007) The detection of carbonation by the Drosophila gustatory system. Nature 448: Suh GS, Wong AM, Hergarden AC, Wang JW, Simon AF, Benzer S, Axel R, Anderson DJ (2004) A single population of olfactory sensory neurons mediates an innate avoidance behaviour in Drosophila. Nature 431: Zars T (2000) Behavioral functions of the insect mushroom bodies. Curr Opin Neurobiol 10: Heisenberg M (2003) Mushroom body memoir: from maps to models. Nat Rev Neurosci 4: Schroter U, Menzel R (2003) A new ascending sensory tract to the calyces of the honeybee mushroom body, the subesophageal-calycal tract. J Comp Neurol 465: Neuser K, Triphan T, Mronz M, Poeck B, Strauss R (2008) Analysis of spatial orientation memory in Drosophila. Nature 453: Figure Legends Fig. 1. Egg-laying and positional responses to acetic acid (AA). (A) Apparatus for assaying oviposition and positional preference. Mated females were presented 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 water. An oviposition preference index (OI) and a positional preference index (PI) were calculated for the females during the 3 h sampling period (see Methods for OI and PI formulas). (B) Comparison of OI and PI values of mated females, 5-6 day old virgins, 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, one 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 presented the following two choice food combinations: a) -AA/-AA: Blue #1/Green #3 (black bar); b) +AA/-AA: Blue #1 + 5% AA/ Green #3 (green bar); c) - 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 spending the majority of time on media lacking AA. Photos were taken after 3 h (D) and at the 1 h (E) time point. To facilitate photography in D and E, 0.5% agarose was utilized instead of regular food. 16

17 Fig. 2. Egg laying and positional responses for different acids and ph values. (A) Mated female egg laying and positional responses were characterized using food titrated to ph=3.5 with AA, HCl, or H 2 S0 4. Control H 2 O supplemented food has ph=4.5. Significant responses at ph=3.5 were only observed for AA containing substrate (**p<0.01, one 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 H 2 S0 4 (p>0.05, n 9). (C, D) Egg laying preferences (C) and positional preferences (D) of mated females for foods titrated to different ph values using AA, HCl, and H 2 S0 4 (see Table S1 for acid concentrations that yield equivalent ph values). There were significant differences between the dose response curves for AA when compared to HCl and H 2 S0 4 (linear regression; ***p<0.0001, n=4-8). Fig. 3. Characterization of egg laying substrate sampling. (A) Egg laying preferences for different concentrations of AA in individual two choice assays. Females showed attraction for AA as an egg laying substrate even at very low concentrations (0.25% AA), with a maximal preference for 3-5% AA. Preference diminished as the concentration increased. (B) Multiple choice stripe assays in which flies must traverse increasing concentrations of AA (***p<0.001; one way ANOVA, Bonferroni s post test revealed only 5% AA yielded significantly different preference compared to 0.25% AA, 10% AA, and all H 2 0 controls; n 6). Fig. 4. Role of olfaction in oviposition and positional choices. (A) Significant OI reduction was seen in whir 1 compared to wild type (wt) females. Restoration of normal OI values was seen in whir 1 lacking antenna ( ant) when compared to unoperated (+ant) whir 1 females. (B) whir 1 females exhibited excessive repulsion to 5% AA when compared with wt females. Removal of antenna in wt and whir 1 females caused a loss of positional aversion to 5% AA. (C) OI and (D) PI values for wt females +/- antennae at 5% and 10% AA. Removal of antenna reduced the highly negative PI seen at 10% AA, and increased attractive OI. Although reductions in positional aversion were observed in ant females at 10% AA (D), females still demonstrated some aversion to 10% AA. (A-D: ***p<0.001; one way ANOVA, Bonferroni s post test for comparisons between genotypes or AA% tests within either the +ant or -ant group. ***p<0.001; two way ANOVA, Bonferroni s post test for comparisons between +ant and ant columns of individual genotypes or AA% tests; n=8-12). 17

18 Fig. 5. Role of gustatory system in oviposition and positional choices. (A) OI values for poxn mutants and transgenic rescue lines at 5% AA (red bars); two way ANOVA revealed genotype as the primary source of variation (80.42%, F=153.3, p<0.0001), with negligible interaction with antennal condition (0.37%, F=0.71, p=0.61; n 7). OIs were significantly lower in poxn M22-B5-/- homozygotes and in poxn M22-B5-/- carrying full-1 and full-152 transgenes, when compared to wild type (Ctl), poxn M22-B5+/- heterozygotes, and poxn M22-B5-/- carrying the complete rescue SuperA transgene. (a = ***p<0.001 when compared to Ctl, poxn M22-B5+/-, and SuperA lines; one way ANOVA, Bonferroni s post test used 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 enhanced aversion, when compared to poxn M22-B5-/- carrying the complete rescue SuperA transgene (b = *p<0.05, when compared to SuperA; c = *p<0.05 when compared to Ctl; one way ANOVA, Bonferroni 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-B5-/- (two way ANOVA, Bonferroni s post test within genotypes for +ant vs. ant ; n 12). Fig. 6. Effect of silencing specific neuronal subsets with Shibire ts. OI and PI values for UAS-Shi ts /+ and GAL4/+ controls, and GAL4/UAS-Shi ts experimental females at permissive (23 C) and non permissive (30 C) temperatures. (A) GAL /UAS-Shi ts exhibited reduced egg laying preference for 5% AA at 30 C, while maintaining normal positional aversion. (B) Brain GAL4 expression of GAL , visualized by crossing to UAS-CD8.GFP, revealed strong expression in the mushroom body (MB) and a few lateral neurons (LNs). (C) GAL /UAS-Shi ts exhibited strong reduction in positional aversion at 23 C and 30 C, while maintaining normal egg laying attraction. (D) GAL drives 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 indicated. Control and experimental lines showed stronger positional aversion at 30 C, which is likely due to increased volatility of AA at 30ºC (A, C: *p<0.05, **p<0.01, ***p<0.001 by one way ANOVA with Bonferroni s post test for comparisons between columns within the 23ºC or 30ºC groups; n 8). (B, D: Green=UAS-CD8.GFP, red=neuropil marker nc82) Fig. 7. Models for the interaction between egg laying attraction and positional repulsion to AA. The gustatory (GS) and olfactory (OS) systems simultaneously detect input from a single compound, AA. Both 18

19 sensory systems relay the signals to higher order centers of their respective circuits for processing and subsequent execution of motor programs (MS=motor systems). Competition between behavioral drives could occur: 1) in the female brain where neurons of the two pathways interact to simultaneously evaluate competing signals, such that either oviposition preference (OP) or positional avoidance (PA) is selected before motor program execution; 2) at the behavioral output level after motor program selection where only the mutually exclusive nature of the behavioral outputs results in competitive shifts of behavioral responses; 3) a combination of both central integration and behavioral output competition; 4) and 5) as directional inhibitory interactions between either the gustatory (4) or olfactory (5) processing circuits and corresponding behavioral outputs. Red intersection lines represent negative interactions. 19

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