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• W2885291121 abstract "•A bioluminescence-based system monitors neural activity in freely interacting flies•cVA-sensitive neurons respond more to male olfactory landmarks than to male bodies•Male, but not virgin-female, olfactory landmarks attract both sexes via Or67d neurons•Olfactory landmarks create a preferred site for interactions regardless of context To communicate with conspecifics, animals deploy various strategies to release pheromones, chemical signals modulating social and sexual behaviors [1Blomquist G.J. Bagnères A.G. Insect Hydrocarbons: Biology, Biochemistry, and Chemical Ecology. Cambridge University Press, 2010Crossref Scopus (424) Google Scholar, 2Tumlinson J.H. Silverstein R.M. Moser J.C. Brownlee R.G. Ruth J.M. Identification of the trail pheromone of a leaf-cutting ant, Atta texana.Nature. 1971; 234: 348-349Crossref PubMed Scopus (90) Google Scholar, 3Prokopy R.J. Hendrichs J. Mating behavior of Ceratitis capitata on a field-caged host tree.Ann. Entomol. Soc. Am. 1979; 72: 642-648Crossref Google Scholar, 4Karlson P. Lüscher M. ‘Pheromones’: a new term for a class of biologically active substances.Nature. 1959; 183: 55-56Crossref PubMed Scopus (837) Google Scholar, 5Wyatt T.D. Pheromones and Animal Behaviour. Cambridge University Press, 2003Crossref Google Scholar]. Importantly, a single pheromone induces different behaviors depending on the context and exposure dynamics [6Ejima A. Pleiotropic actions of the male pheromone cis-vaccenyl acetate in Drosophila melanogaster.J. Comp. Physiol. A Neuroethol. Sens. Neural Behav. Physiol. 2015; 201: 927-932Crossref PubMed Scopus (16) Google Scholar, 7Liu W. Liang X. Gong J. Yang Z. Zhang Y.-H. Zhang J.-X. Rao Y. Social regulation of aggression by pheromonal activation of Or65a olfactory neurons in Drosophila.Nat. Neurosci. 2011; 14: 896-902Crossref PubMed Scopus (111) Google Scholar, 8Sasaki T. Hölldobler B. Millar J.G. Pratt S.C. A context-dependent alarm signal in the ant Temnothorax rugatulus.J. Exp. Biol. 2014; 217: 3229-3236Crossref PubMed Scopus (31) Google Scholar]. Therefore, to comprehend the ethological role of pheromones, it is essential to characterize how neurons in the recipients respond to temporally and spatially fluctuating chemical signals emitted by donors during natural interactions. In Drosophila melanogaster, the male pheromone 11-cis-vaccenyl acetate (cVA) [9Butterworth F.M. Lipids of Drosophila: a newly detected lipid in the male.Science. 1969; 163: 1356-1357Crossref PubMed Scopus (121) Google Scholar] activates specific olfactory receptor neurons (ORNs) [10van der Goes van Naters W. Carlson J.R. Receptors and neurons for fly odors in Drosophila.Curr. Biol. 2007; 17: 606-612Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 11Kurtovic A. Widmer A. Dickson B.J. A single class of olfactory neurons mediates behavioural responses to a Drosophila sex pheromone.Nature. 2007; 446: 542-546Crossref PubMed Scopus (509) Google Scholar] to regulate diverse social and sexual behaviors in recipients [12Bartelt R.J. Schaner A.M. Jackson L.L. cis-vaccenyl acetate as an aggregation pheromone in Drosophila melanogaster.J. Chem. Ecol. 1985; 11: 1747-1756Crossref PubMed Scopus (244) Google Scholar, 13Ejima A. Smith B.P.C. Lucas C. van der Goes van Naters W. Miller C.J. Carlson J.R. Levine J.D. Griffith L.C. Generalization of courtship learning in Drosophila is mediated by cis-vaccenyl acetate.Curr. Biol. 2007; 17: 599-605Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 14Wang L. Anderson D.J. Identification of an aggression-promoting pheromone and its receptor neurons in Drosophila.Nature. 2010; 463: 227-231Crossref PubMed Scopus (208) Google Scholar, 15Xu P. Atkinson R. Jones D.N.M. Smith D.P. Drosophila OBP LUSH is required for activity of pheromone-sensitive neurons.Neuron. 2005; 45: 193-200Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar]. Physicochemical analyses have identified this chemical on an animal’s body [16Everaerts C. Farine J.-P. Cobb M. Ferveur J.-F. Drosophila cuticular hydrocarbons revisited: mating status alters cuticular profiles.PLoS ONE. 2010; 5: e9607Crossref PubMed Scopus (203) Google Scholar, 17Yew J.Y. Dreisewerd K. Luftmann H. Müthing J. Pohlentz G. Kravitz E.A. A new male sex pheromone and novel cuticular cues for chemical communication in Drosophila.Curr. Biol. 2009; 19: 1245-1254Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar] and in its local environment [18Farine J.-P. Ferveur J.-F. Everaerts C. Volatile Drosophila cuticular pheromones are affected by social but not sexual experience.PLoS ONE. 2012; 7: e40396Crossref PubMed Scopus (61) Google Scholar, 19Keesey I.W. Koerte S. Retzke T. Haverkamp A. Hansson B.S. Knaden M. Adult frass provides a pheromone signature for Drosophila feeding and aggregation.J. Chem. Ecol. 2016; 42: 739-747Crossref PubMed Scopus (34) Google Scholar]. However, because these methods are imprecise in capturing spatiotemporal dynamics, it is poorly understood how individual pheromone cues are released, detected, and interpreted by recipients. Here, we developed a system based on bioluminescence to monitor neural activity in freely interacting Drosophila, and investigated the active detection and perception of the naturally emitted cVA. Unexpectedly, neurons specifically tuned to cVA did not exhibit significant activity during physical interactions between males, and instead responded strongly to olfactory landmarks deposited by males. These landmarks mediated attraction through Or67d receptors and allured both sexes to the marked region. Importantly, the landmarks remained attractive even when a pair of flies was engaged in courtship behavior. In contrast, female deposits did not affect the exploration pattern of either sex. Thus, Drosophila use pheromone marking to remotely signal their sexual identity and to enhance social interactions. To communicate with conspecifics, animals deploy various strategies to release pheromones, chemical signals modulating social and sexual behaviors [1Blomquist G.J. Bagnères A.G. Insect Hydrocarbons: Biology, Biochemistry, and Chemical Ecology. Cambridge University Press, 2010Crossref Scopus (424) Google Scholar, 2Tumlinson J.H. Silverstein R.M. Moser J.C. Brownlee R.G. Ruth J.M. Identification of the trail pheromone of a leaf-cutting ant, Atta texana.Nature. 1971; 234: 348-349Crossref PubMed Scopus (90) Google Scholar, 3Prokopy R.J. Hendrichs J. Mating behavior of Ceratitis capitata on a field-caged host tree.Ann. Entomol. Soc. Am. 1979; 72: 642-648Crossref Google Scholar, 4Karlson P. Lüscher M. ‘Pheromones’: a new term for a class of biologically active substances.Nature. 1959; 183: 55-56Crossref PubMed Scopus (837) Google Scholar, 5Wyatt T.D. Pheromones and Animal Behaviour. Cambridge University Press, 2003Crossref Google Scholar]. Importantly, a single pheromone induces different behaviors depending on the context and exposure dynamics [6Ejima A. Pleiotropic actions of the male pheromone cis-vaccenyl acetate in Drosophila melanogaster.J. Comp. Physiol. A Neuroethol. Sens. Neural Behav. Physiol. 2015; 201: 927-932Crossref PubMed Scopus (16) Google Scholar, 7Liu W. Liang X. Gong J. Yang Z. Zhang Y.-H. Zhang J.-X. Rao Y. Social regulation of aggression by pheromonal activation of Or65a olfactory neurons in Drosophila.Nat. Neurosci. 2011; 14: 896-902Crossref PubMed Scopus (111) Google Scholar, 8Sasaki T. Hölldobler B. Millar J.G. Pratt S.C. A context-dependent alarm signal in the ant Temnothorax rugatulus.J. Exp. Biol. 2014; 217: 3229-3236Crossref PubMed Scopus (31) Google Scholar]. Therefore, to comprehend the ethological role of pheromones, it is essential to characterize how neurons in the recipients respond to temporally and spatially fluctuating chemical signals emitted by donors during natural interactions. In Drosophila melanogaster, the male pheromone 11-cis-vaccenyl acetate (cVA) [9Butterworth F.M. Lipids of Drosophila: a newly detected lipid in the male.Science. 1969; 163: 1356-1357Crossref PubMed Scopus (121) Google Scholar] activates specific olfactory receptor neurons (ORNs) [10van der Goes van Naters W. Carlson J.R. Receptors and neurons for fly odors in Drosophila.Curr. Biol. 2007; 17: 606-612Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 11Kurtovic A. Widmer A. Dickson B.J. A single class of olfactory neurons mediates behavioural responses to a Drosophila sex pheromone.Nature. 2007; 446: 542-546Crossref PubMed Scopus (509) Google Scholar] to regulate diverse social and sexual behaviors in recipients [12Bartelt R.J. Schaner A.M. Jackson L.L. cis-vaccenyl acetate as an aggregation pheromone in Drosophila melanogaster.J. Chem. Ecol. 1985; 11: 1747-1756Crossref PubMed Scopus (244) Google Scholar, 13Ejima A. Smith B.P.C. Lucas C. van der Goes van Naters W. Miller C.J. Carlson J.R. Levine J.D. Griffith L.C. Generalization of courtship learning in Drosophila is mediated by cis-vaccenyl acetate.Curr. Biol. 2007; 17: 599-605Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 14Wang L. Anderson D.J. Identification of an aggression-promoting pheromone and its receptor neurons in Drosophila.Nature. 2010; 463: 227-231Crossref PubMed Scopus (208) Google Scholar, 15Xu P. Atkinson R. Jones D.N.M. Smith D.P. Drosophila OBP LUSH is required for activity of pheromone-sensitive neurons.Neuron. 2005; 45: 193-200Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar]. Physicochemical analyses have identified this chemical on an animal’s body [16Everaerts C. Farine J.-P. Cobb M. Ferveur J.-F. Drosophila cuticular hydrocarbons revisited: mating status alters cuticular profiles.PLoS ONE. 2010; 5: e9607Crossref PubMed Scopus (203) Google Scholar, 17Yew J.Y. Dreisewerd K. Luftmann H. Müthing J. Pohlentz G. Kravitz E.A. A new male sex pheromone and novel cuticular cues for chemical communication in Drosophila.Curr. Biol. 2009; 19: 1245-1254Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar] and in its local environment [18Farine J.-P. Ferveur J.-F. Everaerts C. Volatile Drosophila cuticular pheromones are affected by social but not sexual experience.PLoS ONE. 2012; 7: e40396Crossref PubMed Scopus (61) Google Scholar, 19Keesey I.W. Koerte S. Retzke T. Haverkamp A. Hansson B.S. Knaden M. Adult frass provides a pheromone signature for Drosophila feeding and aggregation.J. Chem. Ecol. 2016; 42: 739-747Crossref PubMed Scopus (34) Google Scholar]. However, because these methods are imprecise in capturing spatiotemporal dynamics, it is poorly understood how individual pheromone cues are released, detected, and interpreted by recipients. Here, we developed a system based on bioluminescence to monitor neural activity in freely interacting Drosophila, and investigated the active detection and perception of the naturally emitted cVA. Unexpectedly, neurons specifically tuned to cVA did not exhibit significant activity during physical interactions between males, and instead responded strongly to olfactory landmarks deposited by males. These landmarks mediated attraction through Or67d receptors and allured both sexes to the marked region. Importantly, the landmarks remained attractive even when a pair of flies was engaged in courtship behavior. In contrast, female deposits did not affect the exploration pattern of either sex. Thus, Drosophila use pheromone marking to remotely signal their sexual identity and to enhance social interactions. Conventional neural recording techniques such as electrophysiology or fluorescence-based imaging require the fly to be tethered during investigation. To record the activity of specific neurons in a fly freely navigating a three-dimensional space, we developed a method utilizing the strengths of the Gal4-UAS binary expression system and bioluminescence (Figure 1) [20Martin J.R. Rogers K.L. Chagneau C. Brûlet P. In vivo bioluminescence imaging of Ca2+ signalling in the brain of Drosophila.PLoS ONE. 2007; 2: e275Crossref PubMed Scopus (66) Google Scholar, 21Naumann E.A. Kampff A.R. Prober D.A. Schier A.F. Engert F. Monitoring neural activity with bioluminescence during natural behavior.Nat. Neurosci. 2010; 13: 513-520Crossref PubMed Scopus (153) Google Scholar]. We chose Gal4 lines that each label a specific type of neuron to express a bioluminescent calcium indicator, tandem-dimer Tomato-aequorin (tdTA) [22Bakayan A. Vaquero C.F. Picazo F. Llopis J. Red fluorescent protein-aequorin fusions as improved bioluminescent Ca2+ reporters in single cells and mice.PLoS ONE. 2011; 6: e19520Crossref PubMed Scopus (43) Google Scholar] or GFP-aequorin (GFP-aeq) [23Pfeiffer B.D. Truman J.W. Rubin G.M. Using translational enhancers to increase transgene expression in Drosophila.Proc. Natl. Acad. Sci. USA. 2012; 109: 6626-6631Crossref PubMed Scopus (222) Google Scholar], which relies on a chromophore, coelenterazine (Cz), to emit light. This eliminates excitation light that causes cellular damage, strong cuticular autofluorescence burying the true signal, and, most importantly, the need to maintain the neurons of interest in the focal plane of an optical device, thereby freeing the animal from any physical constraint. Because the specificity of the Gal4 line ensures the source of signals, we can monitor neural activity by collecting all of the photons emitted from the fly with photo-multiplier tubes (PMTs) surrounding the behavioral arena (Figure 1A). The dimensions of the arena and the spatial arrangement of the PMTs were optimized using numerical simulation to detect 95% of the emitted photons. To minimize the noise, the animal’s movements were recorded without any external illumination using a thermal camera. The image contrast was improved by creating a cold background by placing a Peltier element behind the arena (Figure 1A). We first examined the potential of our method by simultaneously recording the fly’s behavior and the activity of gustatory receptor neurons (GRNs). A transgenic fly expressing tdTA only in sugar-sensing Gr5a GRNs [24Chyb S. Dahanukar A. Wickens A. Carlson J.R. Drosophila Gr5a encodes a taste receptor tuned to trehalose.Proc. Natl. Acad. Sci. USA. 2003; 100: 14526-14530Crossref PubMed Scopus (160) Google Scholar] was released in an arena containing droplets of sucrose solution (Figure 1B). When the fly was away from the sugar source, the number of detected photons was fluctuating around a basal level reflecting the sum of noise, spontaneous neuronal activity, and auto-oxidation of Cz. The photon count increased as soon as the fly extended its proboscis to taste the sugar and returned to baseline when the fly stopped feeding (Figure 1B; Video S1). To quantify the sensitivity and the dynamic range of the probe under controlled conditions, we tethered individual flies and monitored the responses of Gr5a GRNs to different concentrations of sucrose solution applied to the labellum (Figures S1A and S1B). Responses to sucrose became significantly larger than those to water at 250 mM and further increased at 500 mM (Figures 1C and S1B). This sensitivity is comparable to GCaMP recording [25Inagaki H.K. Ben-Tabou de-Leon S. Wong A.M. Jagadish S. Ishimoto H. Barnea G. Kitamoto T. Axel R. Anderson D.J. Visualizing neuromodulation in vivo: TANGO-mapping of dopamine signaling reveals appetite control of sugar sensing.Cell. 2012; 148: 583-595Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 26Marella S. Fischler W. Kong P. Asgarian S. Rueckert E. Scott K. Imaging taste responses in the fly brain reveals a functional map of taste category and behavior.Neuron. 2006; 49: 285-295Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar], although not as high as electrophysiological single-sensillum recording [27Dahanukar A. Foster K. van der Goes van Naters W.M. Carlson J.R. A Gr receptor is required for response to the sugar trehalose in taste neurons of Drosophila.Nat. Neurosci. 2001; 4: 1182-1186Crossref PubMed Scopus (207) Google Scholar, 28Hiroi M. Marion-Poll F. Tanimura T. Differentiated response to sugars among labellar chemosensilla in Drosophila.Zool. Sci. 2002; 19: 1009-1018Crossref PubMed Scopus (128) Google Scholar]. It was also comparable to the fly’s behavioral sensitivity to sucrose presented to the labellum assessed by proboscis extension reflex [29Harris D.T. Kallman B.R. Mullaney B.C. Scott K. Representations of taste modality in the Drosophila brain.Neuron. 2015; 86: 1449-1460Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar]. eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiJhNDRmZjY1ZTNkMDk2NmE3NDZhZTMxZDY1YjIyMDIzZCIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc4ODAyMjg1fQ.JEUKJcd_7fvvInqJnwJc5EYMNSDoYvzXWTFtZEqQdGYcZD-zSBUJPbcbIOfinRJHZeIHtnoAkgqdUmTM2RsuIIHroJ77Gt0XUWzCUdLOcQF7z9cI2bVbhsnO5d-_9Qz3RmI20B59NkrSKlhVhDPxxVcUF1n78I-aoCJSOPcGUT3qMwE3vqDgGiOfrJT-CdnIN-H8XNUou7cG_CjV2ll5BdpsNouaaqr-vwq26ZmblO0Iup-4Y-0EmUHbPnFoCo4TLjR_osd6Q_IGr5HvAlYO2SJfZHuWuCAR0fgHm3no0th3RjqjE9lP9IEie-kIKpT1G7VbKynraxvaEhn2WQH2Ag Download .mp4 (0.54 MB) Help with .mp4 files Video S1. Activity of Sugar-Sensitive Gr5a GRNs in a Freely Behaving Fly, Related to Figure 1Activity of sugar sensitive Gr5a GRNs in a freely behaving fly (Gr5a-Gal4>tdTA). The blue trace represents the number of emitted photons acquired at 1 Hz. The inset shows the ongoing behavior in an arena recorded with a thermal camera. A white bar corresponds to the sucrose containing region. Images are processed by subtracting the background from the original images and enhancing the contrast. The video is sped up 3 times. The data corresponds to that shown in Figure 1B. We next asked whether our method is applicable to second-order gustatory neurons in the central brain. We chose to focus on NP1562-Gal4-positive gustatory projection neurons downstream of Gr5a GRNs because (1) they also respond to sucrose, (2) there are fewer than 20 of them, allowing us to test a challenging case, and (3) NP1562-Gal4 does not label any taste sensory neurons [30Kain P. Dahanukar A. Secondary taste neurons that convey sweet taste and starvation in the Drosophila brain.Neuron. 2015; 85: 819-832Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar], thus ensuring that bioluminescence signals originate in central neurons. For this recording, a small patch of cuticle and perineural sheath on the dorsal part of the brain was removed to enhance the permeation of Cz. As a result, we were able to detect transient signals each time the freely behaving fly extended its proboscis to taste sucrose (Figures 1D and 1E; Video S2). The tasting behavior-triggered average response was larger than the control, average activity at random time points during the experiment as well as average activity following the extension of a proboscis away from the sucrose (Figure 1F). Thus, the method is sensitive enough to detect the feeding-dependent activity of gustatory central neurons. eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiIyY2E0N2Y0ZGEwMDMwNDljMDBmYmZjOGYwOGQwYmIzYiIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc4ODAyMjg1fQ.FoAVUMJdnnuY0_ABEaCDnzG3_Ur-s6y7uucIyc7ZSt7gSLaBqixoT7NKISMqEburMTrfwaaqWMDQRExwayJ8IYedxhg1s7G5KWxra5YP1OTaHyIz0jpZf5LJKhAFdvx0r7GsCFIgG5xX3R32kup-a4ft50uyabb_2bL-f-XhhxSpdXFy3EBd0zM2h_Zz2DjkvlTdQ2tOOnwBmWZgEyVcWhJegLZp36q9aStgrrX9KgivXCtESsLZgha5mfT8PCv-bDcyMjN6xAlCVV4UfmCCQfRa807rZC57iROmObdq9505fyVFlOJSqYtrTxZReeqRMnR6gAeU3qQKVJV6EauBgQ Download .mp4 (0.88 MB) Help with .mp4 files Video S2. Activity of Second-Order Gustatory Neurons in a Freely Behaving Fly, Related to Figure 1Activity of second-order gustatory neurons in a freely behaving fly (NP1562-Gal4>GFP-aeq). The blue trace represents the number of emitted photons acquired at 5 Hz. The inset shows the ongoing behavior in an arena recorded with a thermal camera. A block of 1 M sucrose agar is visible on the left. Images are processed by enhancing the contrast. The video is sped down by 60%. The data corresponds to that shown in Figure 1D. We finally expressed the bioluminescent probe in Or67d ORNs, which are exclusively tuned to cVA [10van der Goes van Naters W. Carlson J.R. Receptors and neurons for fly odors in Drosophila.Curr. Biol. 2007; 17: 606-612Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar]. We collected the photons emitted from a tethered fly placed above various odors (Figure S1). The bioluminescent flies showed no sign of neural activity in response to our panel of odors, except for cVA at 10−4 dilution or higher concentration (Figures S1D and S1E). These results confirm the specificity of Or67d ORN activity (see also STAR Methods). To test with more naturalistic stimuli, we exposed flies to the walls of arenas that have housed either males or virgin females. As expected, Or67d ORNs responded only to the wall of the arena treated by males, the unique producers of cVA (Figure S1F) [9Butterworth F.M. Lipids of Drosophila: a newly detected lipid in the male.Science. 1969; 163: 1356-1357Crossref PubMed Scopus (121) Google Scholar, 31Guiraudie-Capraz G. Pho D.B. Jallon J.-M. Role of the ejaculatory bulb in biosynthesis of the male pheromone cis-vaccenyl acetate in Drosophila melanogaster.Integr. Zool. 2007; 2: 89-99Crossref PubMed Google Scholar]. Although gas chromatography-mass spectrometry (GC-MS) studies have shown that males and females release several dozens of other compounds, none of them is exclusively produced in males [18Farine J.-P. Ferveur J.-F. Everaerts C. Volatile Drosophila cuticular pheromones are affected by social but not sexual experience.PLoS ONE. 2012; 7: e40396Crossref PubMed Scopus (61) Google Scholar, 19Keesey I.W. Koerte S. Retzke T. Haverkamp A. Hansson B.S. Knaden M. Adult frass provides a pheromone signature for Drosophila feeding and aggregation.J. Chem. Ecol. 2016; 42: 739-747Crossref PubMed Scopus (34) Google Scholar]. Therefore, our result suggests that the major, if not the sole, substance detected by Or67d ORNs in the flies’ social environment is cVA. These results demonstrate that our system can detect neural activity from just one type of sensory and central neuron in response to active sampling of a stimulus by the unrestrained fly. We then observed the activity of Or67d ORNs in response to natural cVA signals exchanged by interacting flies. We introduced a pair of males in a recording arena. One of the flies, referred to as lum, received a Cz injection rendering the calcium probe functional, whereas the partner fly (control) did not. The filtered and normalized photon count showed many peaks corresponding to ORN responses to natural cues (Figures 2A and S2). Because cVA has been spotted on male genitalia and cuticle [16Everaerts C. Farine J.-P. Cobb M. Ferveur J.-F. Drosophila cuticular hydrocarbons revisited: mating status alters cuticular profiles.PLoS ONE. 2010; 5: e9607Crossref PubMed Scopus (203) Google Scholar, 17Yew J.Y. Dreisewerd K. Luftmann H. Müthing J. Pohlentz G. Kravitz E.A. A new male sex pheromone and novel cuticular cues for chemical communication in Drosophila.Curr. Biol. 2009; 19: 1245-1254Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 32Scott D. Richmond R.C. Evidence against an antiaphrodisiac role for cis-vaccenyl acetate in Drosophila melanogaster.J. Insect Physiol. 1987; 33: 363-369Crossref Scopus (28) Google Scholar], we expected these neural responses to coincide with the physical interaction between flies. However, although flies encountered each other throughout the arena, most of the peaks were triggered in a particular region (Figure 2B). At the time of ORN excitation, the average distance between lum and the center of this region was shorter than the typical fly body length (L, 2.5 mm), whereas the distance from lum to control was significantly longer (Figure 2C). These results are not in line with the hypothesis that cVA is detectable on the fly body, and rather suggest that the major source of cVA is placed at a specific region in the environment. During the first minutes of the example recording described above, Or67d ORNs responded mostly in the right side of the arena (Figure 2D). At a certain point (T = 540 s), one fly seemed to release a drop that we refer to as a landmark from the tip of its abdomen, which was confirmed by a post hoc visual observation of the arena (Figure 2D). In some other experiments, we were even able to observe the landmark appear on the video (Figure 2E, T = 95 s; Video S3). ORNs started to respond only after this marking behavior, again in a restricted region around the landmark (Figures 2E, S3A, and S3B). In fact, a multivariate analysis showed that the number of detected photons is dependent on the distance between lum and the landmark, but not on the distance between flies (Figure 2F). Neural signals were not detected while the flies were physically interacting away from the marked region (Figure 2G). eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiI5MzNlZGZkZjczNWE0ODQ5MTQ3ZjJhYzFlOTcyNTcyZCIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc4ODAyMjg1fQ.bVhL8Gwd0NPEigH2gdT36j1jwj6Fc0eqeMp5Fr2tFT3MmbjjAgu_fQi7SJyQRxbLtIgEEMmcy1B6wF2HDAT0N-AZpAkzJVSAchaWv8U2uWvwSkQZ8r7av8F0B5ykbBBpMoNdxTUjNbB7_LC_tKBxDkciHsxLbPPj9Ak1qQweEFKiOWCvQG-tvG68KIlDDC7oyhFdIqWeskjudbE7ZNoTEe9itwyt28DCymaipQ1hdBzO3JPxjkPAkS7LvFoKJ-9s2qfjw6foJrWXr09kHJ4Tn9XQeD3GLz5p5w0_slP1-be93Z512o3kYVn4eLU8X-aMvbkkuXD_bhniDcpXa6BdhA Download .mp4 (9.6 MB) Help with .mp4 files Video S3. Activity of Or67d ORNs in Freely Interacting Flies, Related to Figure 2Activity of Or67d ORNs in a luminescent fly (lum) freely interacting with a non-luminescent control fly. The blue trace represents the number of emitted photons acquired at 5 Hz. Magenta squares indicate the peaks of neural activity. The inset shows the ongoing interactions between lum (magenta circle) and the control fly (green circle). The position of lum at the moment of each peak of neural activity is reported as a static magenta circle. The orange circle indicates the landmark-containing region. The video has been down sampled by selecting one every five frames and sped up 4 times. The data corresponds to that shown in Figure 2E. To quantify the temporal dynamics of bioluminescence signals, we calculated the average time course of significant signals (Figure S3C, top). The 20%–80% rise time was ∼500 ms, indicating that Or67d ORNs do not need to integrate more than several hundred milliseconds to detect the landmarks. The signal-triggered average of a fly position showed that the level of bioluminescence closely tracked the fly’s proximity to the landmark (Figure S3C, bottom), further confirming that landmarks are the cues that evoke these signals. To examine whether the difference in neuronal responses to landmarks and other males is merely due to the difference in time spent close to the two objects per encounter, we selected the time periods during which the fly approached either object at a particular speed and to a particular level of proximity and calculated the average bioluminescence signals triggered by these behaviors (Figure S3D; see STAR Methods). We found that significant signals were observed only when the fly approached the landmark even after normalizing for the behavior. Taken together, our results demonstrate that landmarks presented by males are the most salient Or67d-activating cues in this social environment and act at a short range. To examine the behavioral effects of these Or67d-activating landmarks, which are fecal deposits (Figure S4A), we monitored a male in a circular, flat arena whose geometry facilitates the visualization of landmark appearance (Figure 3A). Under dim light, the fly typically turned in a circle along the perimeter of the arena and placed landmarks accordingly (Figures S4B and S4C). However, just after marking, it changed the exploration pattern and spent significantly more time in the marked region (Figure 3B; Video S4). Based on the finding that each landmark activates Or67d ORNs within a short radius of ∼L (Figure 2), we quantified its attractiveness by computing the percentage of time the fly spent in this effective region pre and post marking. We found that the attractiveness increased after the landmark appearance and remained elevated for some period (>16 min) with a trend to decrease over time (Figure 3C). This was due to flies’ more frequent visit to the marked region as well as their longer stay within the region per each visit (Figures 3D and 3E). eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiI5M2NiMGNlNWZkNDA2NmM0MmE3YmRmZTUwZjZkOTc0MCIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc4ODAyMjg1fQ.qruQaBJW_1vi0oWINOsZKxCn-GLQBm9ACbNL77B3a6GL6TlFoyyFAziDUgtiHOFKzRHPD2xGvizKpZdv8xoyWuo0PCcUZjRc1i6GYiyGaZABBfIe5hMVNB1OyfEiefSx8lRCBOaGh8Tp8BVRB2cJkPSDJnU5g6QqAAw9mgilEKZtxzJ82cH4xLueSkzM6_b3SLcJSioZkakOGMKg3hszAAHNehNy-lD4IjAz_B20xrr_c912gB2h3x__j0iNF61PJoR1qg8nR4B-6b-CIChH1tTD9IulDwYtVAoqe8McJ5wdn-bhzlUP_6WNb-NvDy7Qa0Vgc6xs8FvJqUINhke95w Download .mp4 (8.82 MB) Help with .mp4 files Video S4. Flies’ Behavior before and after Landmark Deposition, Related to Figures 3 and 4Part 1 - Behavior of a male before and after the deposition of a landmark. A" @default.
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• W2885291121 title "Olfactory Landmark-Based Communication in Interacting Drosophila" @default.
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