The relative roles of vision and chemosensation in mate recognition of Drosophila melanogaster

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1 214. Pulished y The Company of Biologists Ltd (214) 217, doi:1.1242/je.1817 RESEARCH ARTICLE The relative roles of vision and hemosensation in mate reognition of Drosophila melanogaster Sweta Agrawal*, Steve Safarik and Mihael Dikinson ABSTRACT Animals rely on sensory ues to lassify ojets in their environment and respond appropriately. However, the spatial struture of those sensory ues an greatly impat when, where and how they are pereived. In this study, we examined the relative roles of visual and hemosensory ues in the mate reognition ehavior of fruit flies (Drosophila melanogaster) using a rooti fly dummy that was programmed to interat with individual males. By pairing male flies with dummies of various shapes, sizes and speeds, or oated with different pheromones, we determined that visual and hemial ues play speifi roles at different points in the ourtship sequene. Vision is essential for determining whether to approah a moving ojet and initiate ourtship, and males were more likely to egin hasing ojets with the same approximate dimensions as another fly. However, whereas males were less likely to egin hasing larger dummies, one started, they would ontinue hasing for a similar length of time regardless of the dummy s shape. The presene of female pheromones on the moving dummy did not affet the proaility that males would initiate a hase, ut did influene how long they would ontinue hasing. Male pheromone oth inhiits hase initiation and shortens hase duration. Colletively, these results suggest that male D. melanogaster use different sensory ues to progress through the ourtship sequene: visual ues are dominant when deiding whether to approah an ojet whereas hemosensory ues determine how long the male pursues its target. KEY WORDS: Courtship, Pheromones, Ojet reognition INTRODUCTION For many insets, visual ojet reognition is essential for a variety of ehaviors, inluding foraging, prey apture (Nordström, 213; Olerg et al., 2) and ourtship (Boeddeker et al., 23; Collett and Land, 197; Trishler et al., 21). However, little is known aout visual ojet lassifiation in the fruit fly Drosophila melanogaster Meigen. Whereas the overall iruitry of the motion vision pathway has een thoroughly studied (Borst, 29; Paulk et al., 213), previous researh on the role of vision in Drosophila has largely foused on vertial landmarks [i.e. stripe fixation (Götz, 1987), looming shapes (Card and Dikinson, 28) and large-field optomotor responses (Götz, 1968)]. Flying Drosophila exhiit innate turning reations that depend on ojet size (Maimon et al., 28) and an e trained to steer towards speifi visual senes (Dill et al., 1993; Tang et al., 24; Wolf and Heisenerg, 1991), suggesting that they possess some rude aility to reognize partiular shapes or patterns. University of Washington, Department of Biology, Kinaid 34, Seattle, WA , USA. *Author for orrespondene (sagrawal@uw.edu) Reeived 24 Marh 214; Aepted 14 May 214 One ehavior during whih visual ojet reognition might e partiularly important is ourtship. Courtship in Drosophila involves a sequene of highly stereotyped maneuvers made y the male in order to assess, attrat and opulate with a female (Greenspan and Ferveur, 2; Sturtevant, 191). Flies an reprodue in the dark (Spieth and Hsu, 19), demonstrating that vision is not required for suessful ourtship. However, males in the dark or with impaired vision take longer to initiate and omplete ourtship (Markow, 197) and lind males are out-ompeted y individuals with intat vision (Connolly et al., 1969). Furthermore, vision is required for ertain omponents of ourtship suh as orientation and hasing (Cook, 1979). In laoratory studies, the role of vision is likely further diminished eause ourtship experiments are often performed using small hamers (radius <1 m) in whih the female and male flies are the only ojets present (e.g. de la Paz Fernández et al., 21). Males in the wild fae a muh more diffiult task eause natural rots attrat many individuals of different speies (Sturtevant, 191). To find an appropriate mate, males must not only visually distinguish an ojet from the akground, they must also determine its gender, speies identity and reeptivity. Vision is ertainly not the only ue availale to males when assessing an ojet s identity. Flies also produe speies- and sexspeifi pheromones that are important in ourtship (Billeter and Levine, 213; Co and Jallon, 199; Ferveur, 2; Wiker- Thomas, 27). However, most of these ompounds are heavy, longhain hydroarons with limited volatility. Large groups of Drosophila in small spaes may olletively emit detetale levels of pheromones into the surrounding air or deposit them on the sustratum (Farine et al., 212), ut the soures of suh hemosensory ues would e diffiult for any individual to loalize. Males would undoutedly enefit from the aility to find females at a distane using vision or other ues. In this study, we examined the relative roles of visual and hemosensory ues in mate reognition using a remotely atuated fly dummy. By testing males with different dummies, we assessed the influene of size, shape, speed and pheromone ontent on ourtship. Males prefer hasing ojets that are similar in size to a female. However, whereas males were less likely to egin hasing larger dummies, one they started, they ontinued hasing for a similar length of time regardless of the target s shape or speed. Adding female pheromones to the dummy did not alter the proaility that a male would start hasing it, ut did inrease the duration of the hase. Colletively, these results suggest that different sensory ues play a dominant role at different stages of ourtship: visual ues determine whether males will approah a prospetive target, whereas hemosensory ues determine how long the male ontinues to hase. RESULTS For our experiments, we used a ehavioral apparatus alled Flyatar (Zaala et al., 212), onsisting of a remotely atuated fly dummy 2796

2 (214) doi:1.1242/je.1817 List of areviations CH utiular hydroaron VA is-vaenyl aetate GCMS gas hromatography mass spetrometry oe with genetially alated oenoytes (Fig. 1A). We first paired males with moving dummies of various shapes and sizes and measured the total time spent hasing. Overall, hases with the dummy appeared qualitatively similar to those with atual females (Fig. 1B; supplementary material Movie 1). We never saw males performing ourtship maneuvers not direted at the dummy nor in the dark (data not shown). Chase sequenes were identified in post-proessing using an automated ehavioral lassifier (Fig. 1C). As seen in Fig. 2A, the size and shape of the dummy learly influene males propensity for hasing. Males spent little time hasing when paired with dummies that were larger or very differently shaped than another fly. Males also spent little time hasing a larger, isometrially saled version of a small attrative dummy. These results suggest that males an indeed distinguish and ehave differently towards different ojets. Next, we systematially studied the influene of shape y pairing males with ylindrial dummies of onstant height (.8 mm) ut inreasing diameter (Fig. 2B), or uoidal dummies of onstant width ( mm) ut inreasing height (Fig. 2C). Males paired with dummies of smaller radius or dereased height spent the most time hasing (Fig. 2B and 2C, respetively). For a suset of ehavioral trials, we sored eah sequene for the presene of A B mm C mm Index swith Coated glass over C Atuated magnet Atuating magnet Servo motor Fig. 1. Flyatar is a viale system for understanding soial interations in Drosophila melanogaster. (A) Shemati representation of Flyatar, shown in vertial ross-setion, not drawn to sale. (B) Series of overlaid stills progressing left to right. Left panel depits an example hase etween a male and a female D. melanogaster. Right panel depits an example hase etween a male D. melanogaster and the dummy. (C) Three example hases (at 3 frames s 1 ) identified y our automated ehavior lassifier. Triangles indiate the positions and orientations of male flies, and the gray squares indiate the positions and orientations of the dummy. Red triangles represent fly position efore the hase, green during the hase, and lue after the end of the hase. Blak arrows indiate the diretion the dummy is initially moving. unilateral wing extension, a ourtship-speifi male ehavior. We found that males not only hased less when paired with taller dummies, they also demonstrated fewer wing extensions during those hases (Fig. 2D). We were not ale to reord song prodution, and do not know the extent to whih wing extension orrelates with singing. In order to determine whether size or shape of the dummy affets the distane at whih males first notie the dummy, we alulated the distane etween the fly and dummy when the fly first oriented towards and visually fixated the dummy (Fig. 3). This fixating maneuver is the earliest ourtship ehavior we an measure. Beause males do not always first fixate the dummy efore approahing and hasing it, we had to first identify those hases that egan with a fixating maneuver (Fig. 3A), whih we defined as a turn made y the male π/8 rad that rought the dummy into the frontal third of the male s field of view. The distane etween the male and dummy when this turn ours is plotted in Fig. 3. Overall, dummy size and shape do not appear to influene the distriution of distanes at whih the male will fixate the dummy. The total hasing time is a funtion of the numer of hases initiated and the durations of those hases. In experiments in whih we manipulated dummy height, males paired with the tallest dummies initiated fewer hases (Fig. 4A, ottom). One they had initiated a hase, however, males ontinued hasing for a similar amount of time regardless of the dummy s height (Fig. 4A, top). Even as we varied the dummy s speed over a large range from 1. to 13.4 mm s 1, the hase duration remained roughly onstant at 1 s (Fig. A). This result suggests that the temporal duration of the hase might e ontrolled y an internal lok that is not strongly influened y the speed of the target or proprioeptive feedak during walking. There were, however, onsistent differenes etween fast and slow hases. For example, males hasing a dummy moving at 13.4 mm s 1 tended to stay diretly ehind the dummy and made few lateral, irling motions (Fig. B,C; supplementary material Movie 2). In ontrast, males hasing a dummy moving at 1 mm s 1 spent more time moving laterally and irling to the sides of the dummy. Males initiated fewer hases when paired with wider dummies (Fig. 4B, ottom). Chase duration also generally dereased as dummy width inreased (Fig. 4B, top). Taken at fae value, this result suggests that the width of an ojet influenes male ehavior more than the height. However, the measurements of hase duration are statistially prolemati eause males rarely hased the larger dummies, resulting in few oservations. In addition, unlike inreasing height, inreasing the dummy s width inreases its ontat area with the arena floor, whih may have inreased the virations aused y the dummy s motion, inadvertently affeting the male s ourtship ehavior. Nevertheless, the results of Fig. 4A and Fig. A suggest that ojet shape is most important for determining initial attrativeness, ut one males egin hasing, they will ontinue hasing for a fixed amount of time if no other information is availale. We next examined whether hemial ues, suh as pheromones, ould influene hase initiation or duration. We oated dummies with pheromone mixtures extrated from male or virgin female flies. In eah experiment, we used gas hromatography mass spetrometry (GCMS) to quantify the amount of pheromone applied to the dummy. Although we tried to apply a onsistent and physiologially relevant amount of pheromone in eah trial, the amount of pheromone deposited on the dummy varied (Fig. 6). Additionally, eause of differenes in the surfae hemistry and morphology of the dummies ompared with real flies, we do not know how muh of the hexane- 2797

3 (214) doi:1.1242/je.1817 A Total time spent hasing (s) B 3 a a d a a a a, Width (mm) C Height (mm) D Time hasing with wing extension (%) Height (mm) 3 mm Fig. 2. The shape and size of a dummy influene males likelihood of ourtship. Gray irles indiate the response of a single male during a 1 min ehavioral trial. Blak dashes plot the population mean. Groups with the same letter are not signifiantly different (P<., plus Bonferroni orretion). (A C) Total time males spent hasing when paired with a partiular dummy. Dummy shape is indiated along the horizontal axis. In A, dummies have the following dimensions, from left to right (in mm): (n=22), (n=33), (n=31), (n=2), (n=24). In B and C, dummy shape is modified along a single axis, as represented y the shape in the upper right orner of eah panel. In B, sample sizes from left to right are as follows: n=22, n=2, n=2, n=2, n=23, n=27. In C, sample sizes from left to right are as follows: n=33, n=42, n=36, n=36. Data from the first olumns of B and C are repliated in A. (D) Perent of time spent hasing that males also had a wing extended. Ten flies were randomly seleted and examined for eah dummy shape, ut data for only those that demonstrated hasing are shown. From left to right: n=8, n=1, n=9, n=7. extrated pheromone is atually detetale y males. Therefore, we also onduted omplementary experiments with immoilized, wildtype flies mounted on top of the dummy. Males paired with dummies perfumed with female utiular hydroarons (CHs) spent more time hasing ompared with males paired with lank dummies (Fig. 7A). Males were not any more likely to initiate hases towards a perfumed dummy (Fig. 7C); instead, they hased the perfumed dummies approximately twie as long as lank ones (Fig. 7B). We oserved similar results when males were presented with dummies on whih a female fly was mounted. The presene of the female roughly douled the hase duration ut did not influene the numer of hases initiated. Males paired with dummies perfumed with male pheromone or dummies with males mounted on them spent less time hasing (Fig. 7A). This result was due to oth shorter hase durations (Fig. 7B) and dereased numers of hases initiated (Fig. 7C). To examine whether this repression of hase initiation was due to the immediate influene of male-speifi pheromones or a hange in ehavior over time, we plotted a umulative distriution of when, over the duration of a ehavioral experiment, males initiated hases (Fig. 7D). The slopes of all traes are remarkaly onstant over the 1 min trial, showing that males were not gradually learning to avoid the male-perfumed and male-mounted dummies. This result implies that male flies an sense male pheromones over a great enough distane to prevent hase initiation. We also traked wing extension for a suset of these trials (Fig. 7E). Males extended their wings signifiantly more to female-perfumed dummies than to lank dummies, ut they did not extend their wings signifiantly less to A B C D 8 a a a a 8 a, a, a a, a 8 a a a a Fly dummy distane at hase start (mm) 3 mm Width (mm) Height (mm) Fig. 3. Males distane from the dummy when visually fixating and initiating hasing does not vary with dummy shape. (A) Example hase. Triangles indiate the positions and orientations of male flies, and the squares indiate the positions and orientations of the dummy. The lak arrow shows the initial diretion of dummy travel. Blue shapes indiate the first frame that is lassified as a hase y our lassifier (see Materials and methods). We then evaluated the prior frames to find the frame in whih the dummy first entered the frontal third of the fly s visual field assuming no head rotation. If, in order to ring the dummy into the frontal third of its visual field, the fly made a turning maneuver π/8 (green shapes), the fly was lassified as having made an orienting maneuver, whih is the earliest ourtship ehavior we an measure. The distane etween the fly and the dummy when this orienting maneuver was made is plotted in B D. Gray irles represent the responses of males per hase during a 1 min ehavioral trial. Blak dashes represent the mean of eah trial type. In B, dummies have the following dimensions, from left to right (in mm): (n=76), (n=24), (n=1), (n=), (n=21). Dummy shape is indiated along the horizontal axis. In C and D, dummy shape is modified along a single axis, as represented y the shape in the upper right orner of eah panel. In C, sample sizes from left to right are as follows: n=76, n=2, n=1, n=1, n=14, n=21. In D, sample sizes from left to right are as follows: n=24, n=317, n=186, n=

4 (214) doi:1.1242/je.1817 A Average hase duration (s) Numer of hases a a, a a, B Height (mm) Width (mm) Fig. 4. Dummy shape affets males likelihood of initiating a hase ut not hase duration. Gray dots indiate the response of a single male during a 1 min ehavioral trial. Blak dashes plot the population mean. Groups with the same letter are not signifiantly different (P<., plus Bonferroni orretion). Top panels plot the average hase duration during the 1 min ehavioral trials; ottom panels plot the total numer of hases males initiated towards the dummy. (A) Responses to hanges in height of uoid dummy with a 1.6 x1.6 mm ase. In the top and ottom panels, sample sizes from left to right are as follows: n=32, n=41, n=32, n=26. (B) Responses to hanges in width of ylindrial dummy with a.8 mm height. In the top and ottom panels, sample sizes from left to right are as follows: n=2, n=9, n=6, n=4, n=2, n=2. male-perfumed dummies (P<.), suggesting that female pheromones promote wing extension ut male pheromones do not inhiit it. In addition to wild-type flies, we also mounted onto dummies flies that had een manipulated to no longer produe CHs via geneti alation of their oenoytes (oe flies). oe flies onstitute the most fly-like visual stimuli we an provide that also lak the majority of hemosensory ues normally availale. As seen in Fig. 7A, males responses to dummies mounted with either oe males or oe females were idential, suggesting that males annot distinguish etween male and female flies when oenoytes are asent. Males did hase dummies mounted with oe flies more than they hased unperfumed dummies (Fig. 7A), suggesting that the presene of the fly ody does inrease their responsiveness. However, our data do not resolve whether this effet was due to a hange in hase initiation or hase duration. The mean hase duration of males paired with oe fly-mounted dummies was slightly greater than that of males paired with unperfumed dummies and slightly less than that of males paired with female-perfumed or female-mounted dummies (Fig. 7B). However, none of these effets were statistially signifiant at the P<. level. The numer of hases did inrease for males paired with dummies mounted with oe flies, ut these results were only signifiant at the P<. level a a a,, when ompared with lank dummies or the female-perfumed dummies. We also examined whether the presene of pheromone or a fly ody altered the distane at whih the male first fixated the dummy (Fig. 7F). Again, no onditions tested appeared to have an effet, with the exeption of the dummy mounted with an oe male. So far, all our experiments were performed using moving stimuli. We next tested whether males might e attrated to a stationary dummy or a stationary path of pheromones that was not assoiated with a visual ojet. To present a pheromone stimulus in the asene of a strong visual ue, we plaed a small irle of filter paper oated with 4 μl of either hexane or hexane with dissolved female CHs in the arena. As shown y the spatial distriutions plotted in Fig. 8, males spent little time in the viinity of either the pheromone path or the hexane solvent (filter paper with hexane:.92±.63 s, filter paper with hexane and female CHs:.8±.4 s). However, when we plaed a stationary dummy in the same loation as the filter paper, males spent signifiantly more time near the dummy ompared to the filter paper (P<.), suggesting that a stationary visual ojet is an attrative ue. Coating the stationary dummy with female pheromones inreased its attrativeness, as males spent signifiantly more time near a oated dummy than a lank dummy (219.34±173.3 s versus 71.63±3.39 s, P<.). By attrativeness, we refer simply to a male s tendeny to spend time near an ojet and do not distinguish etween long-distane attration and loal preferene. Pheromones are important for distinguishing not only the gender of a potential mate ut also its speies identity (Wyatt, 23). We hypothesized that males should hase dummies perfumed with pheromones from other speies less than those perfumed with onspeifi pheromones. We extrated and perfumed the dummies with pheromone from a losely related speies, Drosophila simulans. This speies o-ours with D. melanogaster in the wild and is visually idential (Sturtevant, 192). As demonstrated in Fig. 9, males spend less time hasing dummies perfumed with female D. simulans pheromones ompared with dummies perfumed with onspeifi female pheromones. Indeed, the response to dummies perfumed with female D. simulans pheromones was not statistially different from the response to non-perfumed dummies (P<.). This result suggests that D. simulans female pheromones do not have an inhiitory effet on D. melanogaster males, ut rather that female D. melanogaster pheromones promote ourtship. The response to dummies perfumed with male D. simulans pheromones was idential to the response of males presented with dummies perfumed with male D. melanogaster pheromones, suggesting that the inhiitory omponent of male pheromones may e shared etween the two speies. DISCUSSION By pairing male flies with dummies of various shapes, sizes, speeds and pheromone oatings, we determined that visual and hemial ues are important at different points in the ourtship sequene of D. melanogaster (Fig. 1). Males appear to use a simple visual filter to deide whether to approah a moving ojet, and then ontinue hasing for a fixed amount of time in the asene of additional ues (Figs 4, ). With more information, suh as the presene of utiular pheromones, males will hoose to either stop or ontinue hasing (Fig. 7). Female CHs do not affet whether the male will initiate a hase, they only influene how long the male ontinues hasing one he has already egun. Male pheromones, however, are likely deteted at a greater distane and an inhiit hase initiation (Fig. 7). Both the shape of the dummy and its pheromone oating influene the amount of wing extension exhiited during a ourtship out (Fig. 2D, Fig. 7E). Finally, D. simulans pheromone does not inrease 2799

5 (214) doi:1.1242/je.1817 A Average hase duration (s) C mm s 1 a a a a a a Speed (mm s 1 ) 2 mm s B 3.4 mm s 1 1 mm s 1 mm s 1. >1. 1 mm s mm s mm s 1 2 mm Fig.. Dummy speed does not affet hase duration, ut does affet males irling tendeny. (A) Average hase duration. Males were paired with uoid dummies ( mm 3 ) moving at the indiated speed. Gray irles indiate the single male responses during a 1 min ehavioral trial. Blak dashes plot the population mean. Groups with the same letter are not signifiantly different (P<., plus Bonferroni orretion). Sample sizes from left to right are: n=22, n=21, n=22, n=31, n=29, n=27. (B) Example traes of a male hasing a uoid dummy moving at 1 mm s 1 (left) or 13.4 mm s 1 (right), 2. frames s 1. Triangles indiate the positions and orientations of male flies, and the gray squares indiate the positions and orientations of the dummy. Red triangles represent fly position efore the hase, green during the hase, and lue after the end of the hase. Blak arrows indiate the diretion the dummy is initially moving. Drawn to different sales. (C) 2D histogram of the positions of male flies during hases in dummy-entered oordinates. The gray square indiates the dummy s position and size. Fly position is measured from its enter. Data from all hases from all males are pooled for eah panel and the speed of the dummy is indiated at the ottom of eah panel. Sample sizes are the same as in A. Color sale represents perent oupany. male hase duration (Fig. 9), implying that female D. simulans lak the CH omponents that prolong hases. Males responded similarly to dummies whether they were perfumed with male D. melanogaster or D. simulans pheromone, perhaps eause males of oth speies produe similar inhiitory ompounds. The aility to visually distinguish among dummies and seletively pursue only those that math speifi riteria has een extensively studied in other arthropods (Wehner, 1981) inluding dragonflies (Olerg et al., 2), lowflies (Boeddeker et al., 23) and horseshoe ras (Barlow et al., 1982). Our study suggests that D. Relative aundane (ng) * * * all CHs VA 7-T 7-P 7,11-HD 7,11-ND Fig. 6. Dummies were perfumed with a physiologially relevant amount of pheromone. Blue and red filled and unfilled irles indiate the amount of pheromone omponent found on an individual fly (filled) or dummy (unfilled). Color indiates gender, with lue indiating pheromone omponent extrated from male flies or male-perfumed dummies and red indiating the pheromone omponent extrated from females or female-perfumed dummies. Blak dashes represent the population mean, and asterisks indiate statistially signifiant differenes (*P<. plus Bonferroni orretion). Aundane was determined y omparison to an internal standard, otadeane. CH, utiular hydroaron; VA, is-vaenyl aetate; 7-T, 7-triosene; 7-P, 7- pentaosene; 7,11-HD, 7,11-heptaosadiene; 7,11-ND, 7,11-nonaosadiene. Sample sizes, from left to right: n=1, n=1, n=1, n=1. melanogaster males an also distinguish amongst ojets using a simple visual filter in the initial stages of ourtship. This hypothetial filter assesses the shape of the target and its size and influenes oth whether the male will initiate a hase and how muh time it spends extending a wing. Given the poor spatial resolution of Drosophila eyes (Buhner, 1984), it is not surprising that the visual filter that governs ourtship is quite oarse. In our experiments, males oasionally hased dummies that were very dissimilar in size and shape to a female (Fig. 2). Furthermore, males were unale to distinguish etween male- and female-mounted dummies in the asene of pheromones (Fig. 7), suggesting that they annot distinguish etween sexes using visual ues. Nevertheless, given the importane of ourtship for reprodutive suess, one might have expeted the males to exhiit a it more seletivity. One explanation is that our experimental proedure of isolating males soon after elosion yielded less seletive animals. Alternatively, males may simply not need to disriminate fine details in the early stages of ourtship, and perhaps eing less seletive even offers an advantage when ompeting with other males. Also, in situations in whih the male is presented with a hoie of many ojets with different shapes, the small ias in the male s shape preferene may result in his hasing the most femalelike shape. Nonetheless, our study shows that males ehave differently towards different visual ojets and thus exhiit some level of disrimination during the earliest stage of ourtship. By raising flies in omplete darkness, Spieth and Hsu (Spieth and Hsu, 19) proved that vision is not neessary for mating in D. melanogaster. However, later studies revealed that male ourtship ehavior differs markedly in the light and dark. Whereas males in oth situations exhiit many of the same ourtship maneuvers, males in the dark never orient towards and fixate a female, nor do they extensively hase (Cook, 198). Rather, males use a very different strategy in the dark to loate females and initialize ourtship: they extend oth wings and walk in a zig-zag pattern of motion until they ollide with another fly (Cook, 198; Crossley and Zuill, 197; Krsti et al., 29). Should the male thus enounter a fly in the dark, he would already e lose enough to sample its pheromones and hoose whether to ourt it. In ontrast, males in the 28

6 (214) doi:1.1242/je.1817 A B C * * * * * ** * * * * Total time spent hasing ( 1 2 s) D Chases initiated Mounted Mounted Mounted Mounted oe mounted oe mounted Average hase duration (s) Time (min) Mounted E Time hasing with wing extension (%) Mounted oe mounted oe mounted Numer of hases a a * * * * * Mounted Mounted oe mounted oe - mounted Mounted Mounted oe mounted oe mounted Fig. 7. The presene of pheromones affets hase duration ut not the numer of hasing outs. (A C,E,F) Colored and gray filled and unfilled irles represent the responses of single males during a 1 min ehavioral trial. In all ases, the dummy used was uoid ( mm 3 ), whih was then further modified as indiated on the horizontal axis. oe mounted refers to dummies mounted with a fly with genetially alated oenoytes (see Materials and methods). Blak dashes represent the population mean, and asterisks indiate statistially signifiant differenes (*P<. plus Bonferroni orretion). The doule asterisk indiates that the population was signifiantly different from all other onditions in that panel (**P<. plus Bonferroni orretion). (A) Total time males spent hasing when paired with the indiated dummy. Sample sizes from left to right: n=33, n=22, n=12, n=29, n=1, n=12, n=1. (B) Average hase duration as performed y the male towards the dummy. (C) Numer of hases initiated y the male towards the dummy. (D) Cumulative distriution of when, over the 1 min ehavioral trial, males initiated a hase. Data for all males were averaged (dark line), with surrounding fill indiating s.e.m. (E) Perent of time spent hasing that males also had a wing extended. Groups with the same letter are not signifiantly different (P<. plus Bonferroni orretion). Ten flies were randomly seleted and examined for eah ondition, ut only those that demonstrated hasing are shown. From left to right: n=8, n=1, n=8. (F) As desried in Fig. 3, we identified hases that egan with the male making an orienting maneuver to fixate the dummy prior to approahing and hasing the dummy. The distane etween the fly and the dummy when this orienting maneuver was made is plotted. From left to right: n=24, n=1, n=41, n=181, n=72, n=11, n=14. F Fly dummy distane at hase start (mm) * * light typially egin ourtship y visually fixating the potential mate at a distane and then approahing and hasing her (Greenspan and Ferveur, 2). Thus, males appear to have two different modes for loating females and initiating ourtship. In the light, visual ues dominate the male s searh, whereas in the dark, tatile and hemosensory ues take preedene. The fat that oth visual and the omination of hemosensory and tatile ues an initiate ourtship helps reonile the results of our experiments with other reent studies examining ourtship initiation (Kohatsu et al., 211; Pan et al., 212). In oth of these prior studies, vision alone was insuffiient to evoke male ourtship. For example, Kohatsu and o-workers (Kohatsu et al., 211) presented tethered males with a female adomen ut oserved no hasing unless males first touhed the adomen. One possiility for the disrepany with our results is that tethering may raise the arousal threshold neessary to eliit hasing. In the other study, Pan and o-workers (Pan et al., 212) reported that males would not hase simple ruer and dummies until they onurrently ativated P1 neurons. However, the visual stimulus used in this prior study was essentially two-dimensional, eause the ruer and dummies were loated underneath a lear arena floor on whih the flies walked. Our experiments demonstrate that height is an important fator in ourtship initiation and the stimulus used y Pan and oworkers may not have een the right shape (or ould not e viewed on the orret region of the retina) to initiate ourtship without onurrent neuronal ativation. One a male has initiated ourtship, our experiments demonstrate that pheromones then influene hase duration. How and at what distane are the males deteting the pheromones? The majority of Drosophila pheromones are heavy hydroarons of low volatility. 281

7 (214) doi:1.1242/je.1817 Filter paper Stationary dummy Hexane Hexane + CHs 1 mm. >1. Fig. 8. Males are not attrated to a stationary soure of pheromone without a visual ue. 2D histogram of the positions of male flies in an arena with either a irle of filter paper (top panels) or a stationary dummy measuring mm 3 (ottom panels). The filter paper and dummy were oated either in hexane (left panels) or hexane with dissolved female CHs (right panels). Color sale represents perent oupany. The lue irle surrounds an area 1 mm in diameter around the filter paper or dummy and total oupany (s) was measured inside the irle. Average oupany values with 9% onfidene intervals are as follows, from left to right: top panels,.92±.63 s and.8±.4 s; ottom panels, 71.63±3.9 s, 219±173.3 s. From left to right, sample sizes for top panels: n=11, n=1 and for ottom panels: n=17, n=17. Farine and o-workers (Farine et al., 212) found that approximately.2% of a fly s CHs are volatile enough to e reovered using solid phase miroextration fier under natural onditions, ut olleting detetale amounts required plaing 1 2 flies in small 8 ml vials for 2 h. Furthermore, several studies that have plaed males in a ehavioral hamer in the presene of air that had een piped over virgin females failed to detet an inrease in male ourtship (Antony and Jallon, 1982; Tompkins et al., 198). A study y Tompkins and Hall (Tompkins and Hall, 1981) did show that female extrat ould inrease male ourtship ehavior at a distane, ut at 8 mm the effet was no longer signifiant. In our experiments, we found that males ould fixate and approah dummies at distanes greater than 8 mm (Fig. 3). This result, omined with our oservation that female pheromones did not inrease the numer of hases initiated, suggests that vision, when availale, is the predominant ue used y males to initiate hases. However, in our experiments, male pheromone did lower males propensity to initiate ourtship, suggesting that males an sense male pheromones at a far enough distane so as to inhiit ourtship initiation. Several male-speifi pheromones, suh as isvaenyl aetate (VA), are light ompared with other CHs and volatile to some degree (Farine et al., 212; Jallon et al., 1981), and ould funtion as a long-distane ue that inhiits ourtship initiation. Whether the same male pheromones are responsile for oth inhiiting male hase initiation and trunating hases is urrently unknown. Another male-speifi pheromone, 7-triosene, whih is heavier than VA, has also een shown to derease male male ourtship (Billeter et al., 29; Wang et al., 211) and might e involved in dereasing hase duration. Although fly pheromones may not at as the primary ue to initiate ourtship, one a male approahes the perfumed dummy, he might detet the CHs y either olfation or gustation. Both olfatory (van der Goes van Naters and Carlson, 27) and gustatory (Bray and Amrein, 23; Miyamoto and Amrein, 28; Watanae et al., 211) neurons respond to fly CHs. Unfortunately, our experiments do not provide the visual resolution neessary to determine whether males atually ontated the dummies with their legs, wings or mouthparts during hases. However, given the distriution of fly dummy distanes we measured during a typial hase (Fig. C), males were ertainly lose enough to do so. The pheromones responsile for inreasing the duration of hasing outs are not known, ut several female-speifi pheromones have een shown to stimulate male wing extension and inhiit A B C 1 * * 9 n.s. n.s. 4 n.s. Total time spent hasing ( 1 2 s) n.s. D. sim perf D. sim perf Average hase duration (s) D. sim perf D. sim perf Numer of hases * D. sim perf D. sim perf Fig. 9. Allospeifi pheromone does not eliit an inrease in hasing out length. Colored and gray filled and unfilled irles represent the responses of individual males during a 1 min ehavioral trial. In all ases, the dummy used was uoid ( mm 3 ), whih was then further modified as indiated on the horizontal axis. D. sim perf, Drosophila simulans perfumed. Blak dashes represent the population mean, and asterisks indiate statistially signifiant differenes (*P<. plus Bonferroni orretion; n.s., not signifiant). (A) Total time males spent hasing. Sample sizes, from left to right: n=33, n=22, n=12, n=18, n=11. (B) Average hase duration, as initiated y males towards the dummy. (C) Total numer of hases initiated y the male towards the dummy. 282

8 (214) doi:1.1242/je.1817 A Pheromone B Initial visual filter C Chemosensory ues D E Non-female CHs, no additional info, inhiitory signals Dereasing female speed, tatile ues, ovipositor extrusion, other stimulatory ues F Fig. 1. Shemati representation of ourtship and the range of sensory ues enountered. Dark green outline represents the likely spatial sale of gustatory ues, light green the range of olfatory ues and light gray the range of visual ues, not drawn to sale. Courtship egins (A) when the male fly first orients towards the likely female. Using a oarse initial visual filter, the male deides whether to approah and hase the female (B). This approah rings the male into range of the female s volatilized pheromones. Based on the gathering of olfatory and gustatory information y tapping (C) or liking (D) the female, males will ontinue hasing for a given amount of time. The lak of further sensory information or the presene of inhiitory signals will ause the male to aort ourtship (E). Alternatively, the presene of other enouraging or exitatory signals will lead to eventual opulation (F). interspeies ourtship, inluding the 7,11-dienes nonaosadiene and 7-11-heptaosadiene (Antony and Jallon, 1982; Marilla et al., 2). Sensory ues have a spatial struture that will greatly impat how and when animals pereive them. As a result, when determining whih sensory ues are relevant for a given ehavior, it is ruially important to study suh ehaviors on a spatial sale that is ethologially plausile. By using a larger arena in whih males were free to approah or disengage with their targets, we were ale to disset at whih stages of ourtship different sensory ues eome relevant, and thus how they are used y males to progress through the ourtship sequene. This approah is essential if we are to etter understand the neural mehanisms underlying the early stages of male ourtship ehavior. MATERIALS AND METHODS Animals Unless otherwise noted, all flies were reared on standard medium in a 16 h:8 h light:dark yle at 2 C. Behavioral experiments were performed on 2- to 4-day-old male fruit flies, D. melanogaster of the Canton-S strain. Males were olleted under light anesthesia (CO 2 ) within a few hours of elosion and housed individually in food vials. A single male was aspirated into the ehavioral hamer at the start of eah ehavioral trial. We alated adult oenoytes y rossing +: PromE(8)-Gal4, tup- Gal8 ts ;+ with +: UAS-StingerII, UAS-hid/CyO;+. Progeny were kept at 18 C until elosion. Adult progeny were olleted under CO 2 anesthesia and kept at 2 C for at least 24 h. Adults were then sujeted to three overnight heat treatments at 3 C (on days 2, 3, and 4) and returned to 2 C etween treatments. We verified alations y examining flies for green fluoresent protein fluoresene on day. For experiments in whih a fly was mounted on the dummy, we first anesthetized flies with old, removed their legs and wings, and then glued them on top of the dummy using UV-ured glue (Newall XUVG-1, Lotite 314). The dummy was plaed into the ehavioral hamer and rotated to move head first. Hydroaron extration and analysis Cutiular hydroarons (CHs) were extrated from 2- to 7-day-old male and virgin female D. melanogaster and male and female D. simulans. Flies were olleted under CO 2 anesthesia and housed in food vials in same-sex groups. To extrat CHs for ehavioral experiments, groups of several hundred samesex flies were anesthetized (CO 2 ) and plaed into 2 ml sintillation vials with 2 μl hexane per fly. The fly hexane mixture was agitated for 2 min. The hexane with dissolved CHs was pipetted into glass mirovials (Miroliter Analytial Supplies, Suwanee, GA, USA) and the flies were disarded. To extrat CHs for quantifiation purposes, individual flies or dummies perfumed with pheromone were plaed in glass mirovials ontaining μl hexane spiked with 1 ng ml 1 of otadeane (Sigma- Aldrih, St Louis, MO, USA) as an internal standard, agitated for 2 min, and then the fly or dummy was removed. The extrat was then analyzed using a GCMS onsisting of an HP 789A GC, a 97C Network Mass Detetor (Agilent Tehnologies, Palo Alto, CA, USA) and a DB GC olumn (J&W Sientifi, Folsom, CA, USA; 3 m,.2 mm,.2 μm) with helium as arrier gas. The olumn temperature profile egan at C (held for 4 min), ramped at 42. C min 1 to 13 C, followed y a ramp of 2 C min 1 to 23 C and a ramp of 3 C min 1 to 28 C, where it was held for 1 min. Chromatogram peaks were tentatively identified using the NIST mass spetral lirary (a. 12, spetra) and verified y hromatography with authenti standards and pulished Kovats indies. Peak areas for eah ompound were integrated using ChemStation software (Agilent Tehnologies). Hydroaron appliation Prior to appliation of CH extrats, dummies were washed with hexane. We then applied 4 μl of fly pheromone dissolved in hexane onto a dummy in a glass mirovial. The hexane was evaporated under nitrogen, leaving the CHs as a residue lightly oating the dummy. To apply pheromone to filter paper, we first used nitrogen to evaporate 4 μl of CH extrat or hexane to a redued volume etween and 3 μl. This volume was then pipetted onto the filter paper in 8 μl inrements. The hexane was then allowed to evaporate until the filter paper was visily dry. Behavioral assays The dummies used in all experiments were nikel-oated neodynium magnets (Armstrong Magnetis, In., Bellingham, WA, USA, and K&J Magnetis, In., Pipersville, PA, USA). Dummy shapes were limited as magnet manufaturers ould not make smaller dummies. Also, as the magnets get shorter or narrower, their field strength diminishes suh that they are no longer ompatile with our atuating system. All dummies were washed in hexane prior to the start of the experiment unless perfumed with pheromone. The ehavioral hamer [1 mm wide 3. mm deep (styled after Simon et al., 21)] was leaned with aetone and 7% ethanol every three trials when performing experiments with non-perfumed or fly-mounted dummies, and etween every trial when using perfumed dummies. After leaning, a dummy was plaed in the ehavioral hamer. A fresh dummy was used every three trials when unperfumed or mounted, and every trial when perfumed. Males were then aspirated into the ehavioral hamer. The experiment initiated one the 283

9 (214) doi:1.1242/je.1817 male egan walking. If the male did not walk for several minutes, or if its wings were damaged, the male was replaed. Experiments ran for 1 min. Room lights were turned off to ensure that light levels stayed onstant etween experiments. A amera (aa64-1gm; Basler) aove the ehavioral hamer automatially traked and reorded the movements of the fly and the dummy using ustom software (downloadale at ased on the Root Operating System (Willow Garage, Menlo Park, CA, USA). The dummy was programmed to move in a irle around the arena with a radius of 31 mm with a onstant speed of mm s 1 unless otherwise stated. Data and statistial analysis Data were analyzed using ustom ode written in MATLAB and Python. We developed a ehavioral lassifier to automatially identify hases. This lassifier was ased on three riteria: the dummy must e within the front third of the fly s field of view ([ π /3, π /3]), the fly to dummy distane had to e 7 mm, and oth onditions must e satisfied for at least 2 s. All identified hases were examined and verified, and ovious false positives were manually removed. We further onfirmed the auray of our lassifier using a tehnique similar to that used y Shneider et al. (Shneider et al., 212) y developing a test set of data (taken from trials involving a mm 3 dummy moving at mm s 1 ) with the dummy trajetories shifted ahead y 2 s (. times the length of one irling of the arena). Over this test set, our lassifier falsely identified only one hase, ompared with the 293 hases identified from the original data. Exept for Fig. 3, we defined the start of the hase as not when the male first fixated the dummy, ut rather when he approahed within 7 mm. Wing extension was sored manually. Aording to a two-sample F-test, most of our data had non-homogeneous varianes, and, as determined y the Kolmogorov Smirnov test, were not normally distriuted (P<.). As suh, we developed a nonparametri resampling method (Fisher s exat test) wherey we ould make pair-wise omparisons using the differene of means as our test statisti. In all omparisons we set a signifiane level of %, with Bonferroni orretions for numer of omparisons made. In eah figure, signifiane is denoted in two ways: either letter ode when every pair is ompared or rakets when only speifi omparisons were made. Signifiane for Fig. 8 was determined using a two-sample t-test. Aknowledgements We thank J. Levine and J. Atallah for advie and protools relating to pheromone perfuming, extration, and analysis, and for providing the oe fly lines. We also thank J. Riffell for his helpful disussions and help with pheromone analysis and the use of his GCMS. Competing interests The authors delare no ompeting finanial interests. 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