STRUCTURE, BEHAVIOR AND MECHANISMS UNDERLYING SENSATION OF CH503, a DROSOPHILA MELANOGASTER PHEROMONE

DOCTORAL DISSERTATION STRUCTURE, BEHAVIOR AND MECHANISMS UNDERLYING SENSATION OF CH503, A DROSOPHILA MELANOGASTER COURTSHIP PHEROMONE SHRUTI SHANKAR MSc Biochemical Technology A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY TEMASEK LIFE SCIENCES LABORATORY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2015 “DECLARATION I hereby declare that this thesis is my original work and I have written it in its entirety I have duly acknowledged all the sources of information which have been used in this thesis This thesis has also not been submitted for any degree to any university previously” ii ACKNOWLEDGEMENT I would like to thank my ‘guru’, Joanne, for accepting me as one of her first PhD students, giving me the opportunity to study in Singapore, and above all, for the discovery of CH503 and an enjoyable PhD experience Joanne has set a wonderful example for me, by being both a terrific scientific mentor and an extremely compassionate and warm person I am very grateful to Joanne, for encouraging me to think independently, giving me a lot of freedom to design experiments, and motivating me to put in my best effort at work I will always be thankful for all the time she has patiently invested in helping me edit numerous presentations, conference posters and papers I will never forget our long conversations over email, about science, my annoyances, good books and how to deal with life It made me happy to walk into lab each day, to work with Joanne and my lab mates I would like to thank Temasek Life Sciences Laboratory for providing me with excellent facilities and supporting my PhD studies A million thanks to my lab mates Kah Junn and Jia Yi for their valuable contributions-for generating the Gr68a mutant and other fly strains, meticulously carrying out immunostaining experiments and helping me with several screens I would also like to thank Ruifen for her help in generating the Gr68a mutant I am extremely grateful to Meredith for her help with establishing calcium imaging assays, for motivating me to keep trying harder and to think of alternate methods, during the times I was near to giving up, and helping me develop a keen interest in microscopy I would like to thank Wan Chin for her help with gustatory receptor screen and all my attachment students for their contributions and sharing my enthusiasm about this project I am also thankful to my lab mates Jacqueline, Yin Ning, Soon Hwee, and Emilie for being wonderful people to work with, and for their eternal willingness to help I have been extremely lucky to have Prof Kenji Mori as a collaborator I would like to thank Prof.Mori for introducing me to the fascinating concept of pheromone chirality, and giving me the opportunity to co-author many chemistry papers I thank my thesis committee members Dr.Adam-Claridge Chang, Dr.Tong-Wey Koh and Prof Daiqin, for their invaluable suggestions on my work I am deeply grateful to my parents and brother for supporting my decision to pursue a PhD, and for visiting me on several occasions to make sure I was doing well and for patiently listening to me talk about CH503 I am also grateful to have found friends like Ranjit and Ekta, who have given me great company and support over the years Lastly, I would like to thank my art teacher, ‘Lao Shi’, for making my weekends enjoyable iii TABLE OF CONTENTS TOPIC PAGENUMBER ACKNOWLEDGEMENT iii TABLE OF CONTENT iv LIST OF FIGURES vi ABBREVIATIONS ix SUMMARY xvii CHAPTER 1: INTRODUCTION 1.1 PHEROMONES MEDIATE INNATE BEHAVIORS IN ANIMALS 1.2 COURTSHIP BEHAVIOR OF DROSOPHILA MELANOGASTER 1.3 PHEROMONES OF DROSOPHILA MELANOGASTER 1.4 ORGANIZATION OF THE DROSOPHILA BRAIN 12 1.5 NEUROTRANSMITTERS OF DROSOPHILA MELANOGASTER 14 1.6 THE CHEMOSENSORY ORGANS OF DROSOPHILA MELANOGASTER 16 1.7 NEUROGENETIC CONTROL OF COURTSHIP BEHAVIOR 28 1.8 OLFACTORY PHEROMONE CIRCUIT 41 1.9 THE DISCOVERY OF CH503 AND RESEARCH HYPOTHESES 45 CHAPTER 2: MATERIALS AND METHODS 2.1 FLY STOCKS 49 2.2 COURTSHIP ASSAYS 49 2.3 IMMUNOHISTOCHEMISTRY 51 2.4 SPINNING DISK CONFOCAL MICROSCOPY 52 2.5 PROBOSCIS EXTENSION REFLEX (PER) ASSAY 55 2.6 DETERMINING THE VOLATILITY OF CH503 57 2.7 GENERATION OF TRANSGENIC FLIES 58 2.8 GENERATION OF ΔGr68a AND ΔGr68a-RESCUE (Gr68aRes) FLIES 60 2.9 CHEMICAL REAGENTS 60 iv CHAPTER 3: RESULTS PART 1: STRUCTURAL CHARACTERIZATION OF CH503 3.1 ELUCIDATION OF THE STEREOSTRUCTURE OF CH503 64 3.2 RESULTS AND DISCUSSION 65 3.3 BIOACTIVITY OF THE CH503 STEREOISOMERS 67 3.4 DISCUSSION 71 PART 2: THE CH503 NEURAL CIRCUITRY 3.5.1 MALE COURTSHIP BEHAVIOR IS INHIBITED IN A 74 DOSE DEPENDENT MANNER BY CH503 3.5.2 CH503 IS A LOW VOLATILITY CONTACT CUE AND IS 75 EFFECTIVE ONLY WHEN DETECTED ON FEMALE CUTICLES 3.5.3 CH503 IS DETECTED BY GUSTATION, NOT OLFACTION 77 3.5.4 MEASURING A TASTE RESPONSE TO CH503 USING THE 79 PROBOSCIS EXTENSION REFLEX (PER) ASSAY 3.5.5 Voila1/ TM3 GUSTATORY MUTANTS SHOW A REDUCED 82 RESPONSE TO CH503 3.5.6 (R,Z,Z)-CH503 IS DETECTED BY GR68A NEURONS ON THE MALE 85 FORELEG 3.5.7 EXPRESSION PATTERN OF GR68a 90 3.5.8 MEASURING PHYSIOLOGICAL RESPONSES TO CH503 92 FROM Gr68a NEURONS 3.5.9 THE ROLE OF ppk23 NEURONS IN CH503 DETECTION 97 3.5.10 IDENTIFICATION OF HIGHER ORDER NEURONS 103 INVOLVED IN CH503 DETECTION CHAPTER 4: DISCUSSION 117 CHAPTER 5: FUTURE DIRECTIONS 130 REFERENCES 133 v LIST OF FIGURES AND TABLES CHAPTER 1: INTRODUCTION Figure 1: Chemical structures of insect pheromones Figure 2: The hallmark courtship features of male Drosophila Figure 3: Pheromones of Drosophila Figure 4: General pathway for pheromone biosynthesis in Drosophila Figure 5: Structural components of the Drosophila brain Figure 6: Mode of action of neurotransmitters Table 1: Drosophila melanogaster neurotransmitters Figure 7: Schematic showing the frontal view of the fly head with the antennae and the maxillary palps Figure 8: The organization of the olfactory circuit in adult flies Figure 9: The gustatory organs of the fly Figure 10: The Drosophila sex-determination pathway Figure 11: Sex-specific splicing of the Drosophila sex-determination genes Figure 12: Morphological differences between male and female Drosophila Figure 13: The Organization of Dsx neuronal clusters in the CNS Figure 14: The Drosophila apoptosis pathway Figure 15: Sex-specific splicing of the fruitless gene Figure 16: Neuronal pathways mediating receptivity in female Drosophila Figure 17: Olfactory pheromone circuits Figure 18: The anatomical differences in the projection patterns of Or67d PNs in males and female flies Figure 19: The cVA circuit in the male brain comprising of neuronal clusters linked by synapses Figure 20: The bidirectional cVA circuit Figure 21 A]: Schematic of the UV-LDI-o-TOF MS method B] Picture of the anogenital region of a male fly Figure 22: The chemical structure of CH503 vi CHAPTER 2: MATERIALS AND METHODS Figure 1: Fly mounted on a coverslip for imaging studies Figure 2: Calcium imaging Figure 3: Proboscis Extension Reflex Assay Figure 4: Experimental setup to determine the volatility of CH503 Figure 5: The GAL4-UAS system CHAPTER 3-PART 1: STRUCTURAL CHARACTERIZATION OF CH503 Figure 1: The structural analogs of CH503 Figure 2: The stereoisomers of CH503 Figure 3: Bioactivity of CH503 stereoisomers Figure 4: Calculation of effect sizes for courtship data Figure 5: HPLC separation of CH503 stereoisomers Table 1: Bioactivity of CH503 analogs CHAPTER 3- PART 2: THE CH503 NEURAL CIRCUITRY Figure 1: (R,Z,Z)-CH503 is a courtship inhibitory pheromone Figure 2: CH503 has low volatility Figure 3: Flies can detect CH503 without the major olfactory organs Figure 4: CH503 inhibits the sucrose induced appetitive Proboscis extension response Figure 5: Study of Voila1/TM3 gustatory mutants Figure 6: Screen of foreleg specific Gustatory receptor neurons Figure 7: Knockdown of Gr68a or deletion of Gr68a reduces sensitivity to CH503 Figure 8: Deletion of Gr68a does not induce a suppression of the appetitive PER to CH503 Figure 9: Artificial activation of Gr68a neurons with TrpA1 Figure 10: Expression pattern of Gr68a GAL4 Figure 11: The response profile of male specifc Gr68a neurons to CH503 Figure 12: Physiological response of Gr68a to (R)-3-acetoxy-11-octacosen-1-ol Figure 13: Physiological response of Gr68a neurons to (S)-3-acetoxy-11-octacosen-1-ol Figure 14: Physiological response of Gr68a > Gr68a RNAi flies to (R,Z,Z)-CH503 Figure 15: Physiological response of ΔGr68a flies to (R,Z,Z)-CH503 vii Figure 16: Response of Gr68a neurons on the female foreleg to (S,Z,Z)-CH503 Figure 17: The response profile of ppk23 neurons to CH503 Figure 18: Color-coded time course images showing responses of Gr68a ans ppk23 neurons to CH503 Figure 19: RNAi mediated knockdown of ppk23 or ppk25 does not alter sensitivity to CH503 Figure 20: Screen to identify CH503 processing circuits Figure 21: Screen to identify CH503 processing circuits Figure 22: Courtship inhibition difference for central brain circuits Figure 23: Expression patterns of NPF GAL4 and c929 GAL4 neuronal circuits in the adult male fly brains Figure 24: Components of the NPF signalling pathway are not involved in CH503 detection Figure 25: Courtship inhibition difference for NPF signalling pathway Figure 26: Role of TK circuits in CH503 detection Figure 27: Co-localization of NPF and TK neuronal circuits Figure 28: Synaptic connectivity of Gr68a and TK circuits Figure 29: A model for gustatory pheromone perception Table 1: Gr68a cell counts in male and female forelegs Table 2: Sample sizes for calcium imaging experiments Table 3: Screen of GAL4 lines associated with higher order brain circuits for CH503 detection defects CHAPTER 4: DISCUSSION Figure 1: Line graph showing the tonic response pattern of a Gr68a neuron Figure 2: Line graph showing the phasic response pattern of a ppk23 neuron viii LIST OF ABBREVIATIONS 5HT serotonin 7,11-HD 7,11-Heptacosadiene 7,11-ND 7,11-Nonacosadiene 7-T 7-Tricosene AbdB Abdominal B AL Antennal Lobe AMMC Antennal Mechanosensory and Motor Centre CH503 3-acetoxy-11,19-octacosadien-1-ol cVA cis-vaccenyl acetate cVOH cis-vaccenol CX Central Complex Cyo curly Oster DA dopamine DCO Dorsal Cibarial Sense Organ DEG/ENaC Degenerin and epithelial Na+ channel Desat desaturase DLP Dorso-Lateral Protocerebrum dORKΔC Drosophila Open Rectifier Potassium Channel Dsx Doublesex DsxF Female specific Doublesex protein DsxM Male specific Doublesex protein DTi Diphtheria toxin EB Ellipsoid body Elav embryonic lethal abnormal visual system EPSP Excitatory Post Synaptic Potential ESI-MS Electrospray ionization- Mass Spectrometry FB Fan shaped body ix Fru Fruitless FruM Male specific Fruitless protein GABA Gamma-aminobutyric acid GAL 80 Galactose 80 GAL4 Galactose GCaMP green fluorescent protein (GFP), calmodulin, and M13 GC-MS Gas Chromatography-Mass Spectrometry GFP Green Fluorescent Protein GPCR G-Protein Coupled Receptor GR Gustatory Receptor GRASP GFP reconstitution across synaptic partners GRN Gustatory Receptor Neuron Hid Head involution defective Hox Homeobox HPLC High-performance liquid chromatography HPTLC High-performance thin layer liquid chromatography IR Ionotropic Receptor Kr-GFP Kruppel-Green Fluorescent Protein LH Lateral Horn LP Lateral Protocerebrum LSO Labral Sense Organ m/z mass to charge ratio MB Mushroom Body ME Medulla mRNA messenger Ribo Nucleic Acid NPF long Neuropeptide F NPFR1 Neuropeptide F Receptor Oe- oenocyteless Orco Odorant receptor co-receptor ORN Olfactory Receptor Neuron x CHAPTER 5: FUTURE DIRECTIONS This study on CH503, provides a framework to understand the neuronal and evolutionary basis of gustatory pheromone perception in Drosophila melanogaster In this thesis work, I investigated two main hypotheses First, I tested the hypothesis that (R,Z,Z)-CH503 was the most potent stereoisomer of the pheromone However, contrary to what was hypothesized, I found that this naturally occurring stereoisomer had strong courtship inhibitory effects at a dose of 166ng/fly and above, in comparison with the ‘S’ stereoisomer that is a potent courtship inhibitor at low doses These studies were further carried out on other Drosophila species and has led to the finding that CH503 evolved by a mechanism known as sensory exploitation (Ng et al., 2014) Second, I tested the hypothesis, that CH503, analogous to the volatile pheromone cis-vaccenyl acetate, would be detected as an olfactory modality via a single receptor However, as described in this thesis, the detection of CH503 involves the gustatory receptor Gr68a in the peripheral nervous system and the NPF and TK circuits in the central brain Neurons expressing the ion channel ppk23 were additionally, shown to physiologically respond to CH503 (Shankar et al., 2015) These findings raise further questions about the CH503 detection mechanisms both in Drosophila melanogaster and distantly related species Some of the unanswered questions include:  Does Gr68a mediate CH503 detection in other Drosophila species? Functional cloning and expression of the Gr68a gene from other species, in Drosophila melanogaster Gr68a mutants would help to address this question It might be interesting to determine if the observed differences in sensitivities to CH503 (Ng et al., 2014), are due to structural changes in the Gr68a receptor  Does the Gr68a receptor respond physiologically to all the synthetic stereoisomers of CH503? This work shows that Gr68a mediates the detection of (S,Z,Z)-CH503 and (R,Z,Z)-CH503 The role of Gr68a in detection of the remaining stereoisomers of 130 CH503 was not investigated The strongest anti-aphrodisiac properties were exhibited by the (S,E,E)-CH503 stereoisomer In order to test the role of Gr68a as the sole mediator of CH503 detection more rigorously, it will be important to determine if this receptor can detect and physiologically respond to the (S,E,E)-CH503 stereoisomer at low dosages  Ppk23 neurons respond physiologically to CH503, these neurons also contribute to courtship inhibitory behavioral response induced by the pheromone? Despite the large physiological response observed from many ppk23 neurons to CH503, knocking down the levels of ppk23 using RNAi, did not significantly alter the sensitivity of male flies to CH503 Furthermore, ppk23 deletion mutants displayed very low baseline levels of courtship To further understand the contribution of ppk23 neurons, it would be imperative to also test if transiently silencing the ppk23 circuit using a temperature – sensitive shibire transgene would alter sensitivity to CH503 It would also be interesting to find out, if over expression of ppk23 in a Gr68a mutants would restore the response to CH503  Given the possibility that Drosophila gustatory pheromones have more than one detection pathway (e.g 7-Tricosene), other classes of ppk neurons, GR’s or IR’s contribute to CH503 detection? Apart from ppk23, neurons expressing ppk25 (Liu et al., 2012b) and ppk29 (Thistle et al., 2012) and the ionotropic receptor IR20a (Koh et al., 2014) are also known to play a role in courtship behavior Furthermore, it was found that the CH503 induced courtship inhibition difference for Gr66a> Rpr, Gr22e>Rpr and Gr28b>Rpr was small To test if these chemosensory neurons are also involved in CH503 detection, it would be necessary to study ppk25, ppk29, Gr22e, Gr66a, Gr28b and IR20a gene deletion mutants 131  Do TK neurons connect with P1 command neurons in the central brain? In this study a potential overlap of the above neuronal pathways was not investigated In order to establish connectivity, approaches such as GFP reconstitution across synaptic partners (GRASP) could be used Powerful imaging techniques such as photo activation and two photon microscopy that was used in delineation of the Or67d circuit, could potentially be applied to thoroughly map the CH503 circuit 132 REFERENCES: Akerboom, J., Chen, T.W., Wardill, T.J., Tian, L., Marvin, J.S., Mutlu, S., Calderon, N.C., Esposti, F., Borghuis, B.G., Sun, X.R., et al (2012) Optimization of a GCaMP calcium indicator for neural activity imaging J Neurosci 32, 13819-13840 Alekseyenko, O.V., Chan, Y.B., Fernandez Mde, L., Bulow, T., Pankratz, M.J., and Kravitz, E.A (2014) Single Serotonergic Neurons that Modulate Aggression in Drosophila Current biology : CB 24, 2700-2707 Andrews, J.C., Fernandez, M.P., Yu, Q., Leary, G.P., Leung, A.K., Kavanaugh, M.P., Kravitz, E.A., and Certel, S.J (2014) Octopamine neuromodulation regulates Gr32a-linked aggression and courtship pathways in Drosophila males PLoS genetics 10, e1004356 Antony, C., Davis, T.L., Carlson, D.A., Pechine, J.M., and Jallon, J.M (1985) Compared behavioral responses of maleDrosophila melanogaster (Canton S) to natural and synthetic aphrodisiacs Journal of chemical ecology 11, 1617-1629 Argiolas, A., and Melis, M.R (2013) Neuropeptides and central control of sexual behaviour from the past to the present: a review Prog Neurobiol 108, 80-107 Asahina, K., Watanabe, K., Duistermars, B.J., Hoopfer, E., Gonzalez, C.R., Eyjolfsdottir, E.A., Perona, P., and Anderson, D.J (2014) Tachykinin-expressing neurons control malespecific aggressive arousal in Drosophila Cell 156, 221-235 Balakireva, M., Gendre, N., Stocker, R.F., and Ferveur, J.F (2000) The genetic variant Voila causes gustatory defects during Drosophila development The Journal of neuroscience : the official journal of the Society for Neuroscience 20, 3425-3433 Balakireva, M., Stocker, R.F., Gendre, N., and Ferveur, J.F (1998) Voila, a new Drosophila courtship variant that affects the nervous system: behavioral, neural, and genetic characterization The Journal of neuroscience : the official journal of the Society for Neuroscience 18, 4335-4343 Bargmann, C.I (2012) Beyond the connectome: how neuromodulators shape neural circuits BioEssays : news and reviews in molecular, cellular and developmental biology 34, 458-465 Bartelt, R.J., Schaner, A.M., and Jackson, L.L (1985) cis-Vaccenyl acetate as an aggregation pheromone inDrosophila melanogaster Journal of chemical ecology 11, 1747-1756 Benton, R., Sachse, S., Michnick, S.W., and Vosshall, L.B (2006) Atypical membrane topology and heteromeric function of Drosophila odorant receptors in vivo PLoS biology 4, e20 Benton, R., Vannice, K.S., Gomez-Diaz, C., and Vosshall, L.B (2009) Variant ionotropic glutamate receptors as chemosensory receptors in Drosophila Cell 136, 149-162 Berry, J.A., Cervantes-Sandoval, I., Nicholas, E.P., and Davis, R.L (2012) Dopamine is required for learning and forgetting in Drosophila Neuron 74, 530-542 133 Besson, J.M., and Chaouch, A (1987) Peripheral and spinal mechanisms of nociception Physiol Rev 67, 67-186 Billeter, J.C., Atallah, J., Krupp, J.J., Millar, J.G., and Levine, J.D (2009) Specialized cells tag sexual and species identity in Drosophila melanogaster Nature 461, 987-991 Billeter, J.C., Rideout, E.J., Dornan, A.J., and Goodwin, S.F (2006) Control of male sexual behavior in Drosophila by the sex determination pathway Current biology : CB 16, R766776 Bousquet, F., Nojima, T., Houot, B., Chauvel, I., Chaudy, S., Dupas, S., Yamamoto, D., and Ferveur, J.F (2012) Expression of a desaturase gene, desat1, in neural and nonneural tissues separately affects perception and emission of sex pheromones in Drosophila Proceedings of the National Academy of Sciences of the United States of America 109, 249-254 Bray, S., and Amrein, H (2003) A putative Drosophila pheromone receptor expressed in male-specific taste neurons is required for efficient courtship Neuron 39, 1019-1029 Brieger, G., and Butterworth, F.M (1970) Drosophila melanogaster: identity of male lipid in reproductive system Science 167, 1262 Burke, C.J., Huetteroth, W., Owald, D., Perisse, E., Krashes, M.J., Das, G., Gohl, D., Silies, M., Certel, S., and Waddell, S (2012) Layered reward signalling through octopamine and dopamine in Drosophila Nature 492, 433-437 Busch, K.E., Laurent, P., Soltesz, Z., Murphy, R.J., Faivre, O., Hedwig, B., Thomas, M., Smith, H.L., and de Bono, M (2012) Tonic signaling from O(2) sensors sets neural circuit activity and behavioral state Nat Neurosci 15, 581-591 Bussell, J.J., Yapici, N., Zhang, S.X., Dickson, B.J., and Vosshall, L.B (2014) Abdominal-B neurons control Drosophila virgin female receptivity Current biology : CB 24, 1584-1595 Butcher, R.A., Fujita, M., Schroeder, F.C., and Clardy, J (2007) Small-molecule pheromones that control dauer development in Caenorhabditis elegans Nature chemical biology 3, 420-422 Cameron, P., Hiroi, M., Ngai, J., and Scott, K (2010) The molecular basis for water taste in Drosophila Nature 465, 91-95 Chen, Y.W., Weng, R., and Cohen, S.M (2011) Protocols for use of homologous recombination gene targeting to produce microRNA mutants in Drosophila Methods Mol Biol 732, 99-120 Chiang, A.S., Lin, C.Y., Chuang, C.C., Chang, H.M., Hsieh, C.H., Yeh, C.W., Shih, C.T., Wu, J.J., Wang, G.T., Chen, Y.C., et al (2011) Three-dimensional reconstruction of brainwide wiring networks in Drosophila at single-cell resolution Curr Biol 21, 1-11 Clyne, P., Grant, A., O'Connell, R., and Carlson, J.R (1997) Odorant response of individual sensilla on the Drosophila antenna Invertebrate neuroscience : IN 3, 127-135 134 Coyne, J.A., Crittenden, A.P., and Mah, K (1994) Genetics of a pheromonal difference contributing to reproductive isolation in Drosophila Science 265, 1461-1464 Crickmore, M.A., and Vosshall, L.B (2013) Opposing dopaminergic and GABAergic neurons control the duration and persistence of copulation in Drosophila Cell 155, 881-893 Crocker, A., and Sehgal, A (2008) Octopamine regulates sleep in drosophila through protein kinase A-dependent mechanisms The Journal of neuroscience : the official journal of the Society for Neuroscience 28, 9377-9385 Datta, S.R., Vasconcelos, M.L., Ruta, V., Luo, S., Wong, A., Demir, E., Flores, J., Balonze, K., Dickson, B.J., and Axel, R (2008) The Drosophila pheromone cVA activates a sexually dimorphic neural circuit Nature 452, 473-477 de Bruyne, M., Foster, K., and Carlson, J.R (2001) Odor coding in the Drosophila antenna Neuron 30, 537-552 DeGennaro, M., McBride, C.S., Seeholzer, L., Nakagawa, T., Dennis, E.J., Goldman, C., Jasinskiene, N., James, A.A., and Vosshall, L.B (2013) orco mutant mosquitoes lose strong preference for humans and are not repelled by volatile DEET Nature 498, 487-491 Demir, E., and Dickson, B.J (2005) fruitless splicing specifies male courtship behavior in Drosophila Cell 121, 785-794 Dickson, B.J (2008) Wired for sex: the neurobiology of Drosophila mating decisions Science 322, 904-909 Dierick, H.A., and Greenspan, R.J (2007) Serotonin and neuropeptide F have opposite modulatory effects on fly aggression Nat Genet 39, 678-682 Ejima, A., and Griffith, L.C (2008) Courtship initiation is stimulated by acoustic signals in Drosophila melanogaster PloS one 3, e3246 Ejima, A., Smith, B.P., Lucas, C., van der Goes van Naters, W., Miller, C.J., Carlson, J.R., Levine, J.D., and Griffith, L.C (2007) Generalization of courtship learning in Drosophila is mediated by cis-vaccenyl acetate Current biology : CB 17, 599-605 Fabre, C.C., Hedwig, B., Conduit, G., Lawrence, P.A., Goodwin, S.F., and Casal, J (2012) Substrate-borne vibratory communication during courtship in Drosophila melanogaster Current biology : CB 22, 2180-2185 Fan, P., Manoli, D.S., Ahmed, O.M., Chen, Y., Agarwal, N., Kwong, S., Cai, A.G., Neitz, J., Renslo, A., Baker, B.S., et al (2013) Genetic and neural mechanisms that inhibit Drosophila from mating with other species Cell 154, 89-102 Feinberg, E.H., Vanhoven, M.K., Bendesky, A., Wang, G., Fetter, R.D., Shen, K., and Bargmann, C.I (2008) GFP Reconstitution Across Synaptic Partners (GRASP) defines cell contacts and synapses in living nervous systems Neuron 57, 353-363 135 Feng, K., Palfreyman, M.T., Hasemeyer, M., Talsma, A., and Dickson, B.J (2014) Ascending SAG neurons control sexual receptivity of Drosophila females Neuron 83, 135148 Ferrero, D.M., Moeller, L.M., Osakada, T., Horio, N., Li, Q., Roy, D.S., Cichy, A., Spehr, M., Touhara, K., and Liberles, S.D (2013) A juvenile mouse pheromone inhibits sexual behaviour through the vomeronasal system Nature 502, 368-371 Gendre, N., Luer, K., Friche, S., Grillenzoni, N., Ramaekers, A., Technau, G.M., and Stocker, R.F (2004) Integration of complex larval chemosensory organs into the adult nervous system of Drosophila Development 131, 83-92 Gendron, C.M., Kuo, T.H., Harvanek, Z.M., Chung, B.Y., Yew, J.Y., Dierick, H.A., and Pletcher, S.D (2014) Drosophila life span and physiology are modulated by sexual perception and reward Science 343, 544-548 Goralski, T.J., Edstrom, J.E., and Baker, B.S (1989) The sex determination locus transformer-2 of Drosophila encodes a polypeptide with similarity to RNA binding proteins Cell 56, 1011-1018 Gordon, M.D., and Scott, K (2009) Motor control in a Drosophila taste circuit Neuron 61, 373-384 Grosjean, Y., Grillet, M., Augustin, H., Ferveur, J.F., and Featherstone, D.E (2008) A glial amino-acid transporter controls synapse strength and courtship in Drosophila Nature neuroscience 11, 54-61 Grosjean, Y., Rytz, R., Farine, J.P., Abuin, L., Cortot, J., Jefferis, G.S., and Benton, R (2011) An olfactory receptor for food-derived odours promotes male courtship in Drosophila Nature 478, 236-240 Hall, J.C (1994) The mating of a fly Science 264, 1702-1714 Hardy, R.W., Tokuyasu, K.T., and Lindsley, D.L (1981) Analysis of spermatogenesis in Drosophila melanogaster bearing deletions for Y-chromosome fertility genes Chromosoma 83, 593-617 He, C., Cong, X., Zhang, R., Wu, D., An, C., and Zhao, Z (2013) Regulation of circadian locomotor rhythm by neuropeptide Y-like system in Drosophila melanogaster Insect molecular biology 22, 376-388 Heilig, M (2004) The NPY system in stress, anxiety and depression Neuropeptides 38, 213224 Heinrichs, V., Ryner, L.C., and Baker, B.S (1998) Regulation of sex-specific selection of fruitless 5' splice sites by transformer and transformer-2 Molecular and cellular biology 18, 450-458 Hewes, R.S., Park, D., Gauthier, S.A., Schaefer, A.M., and Taghert, P.H (2003) The bHLH protein Dimmed controls neuroendocrine cell differentiation in Drosophila Development 130, 1771-1781 136 Hiroi, M., Meunier, N., Marion-Poll, F., and Tanimura, T (2004) Two antagonistic gustatory receptor neurons responding to sweet-salty and bitter taste in Drosophila Journal of neurobiology 61, 333-342 Hoyer, S.C., Eckart, A., Herrel, A., Zars, T., Fischer, S.A., Hardie, S.L., and Heisenberg, M (2008) Octopamine in male aggression of Drosophila Current biology : CB 18, 159-167 Ignell, R., Root, C.M., Birse, R.T., Wang, J.W., Nassel, D.R., and Winther, A.M (2009) Presynaptic peptidergic modulation of olfactory receptor neurons in Drosophila Proc Natl Acad Sci U S A 106, 13070-13075 Ito, M., Masuda, N., Shinomiya, K., Endo, K., and Ito, K (2013) Systematic analysis of neural projections reveals clonal composition of the Drosophila brain Current biology : CB 23, 644-655 Jallon, J.M (1984) A few chemical words exchanged by Drosophila during courtship and mating Behavior genetics 14, 441-478 Jefferis, G.S., Potter, C.J., Chan, A.M., Marin, E.C., Rohlfing, T., Maurer, C.R., Jr., and Luo, L (2007) Comprehensive maps of Drosophila higher olfactory centers: spatially segregated fruit and pheromone representation Cell 128, 1187-1203 Jenett, A., Rubin, G.M., Ngo, T.T., Shepherd, D., Murphy, C., Dionne, H., Pfeiffer, B.D., Cavallaro, A., Hall, D., Jeter, J., et al (2012) A GAL4-driver line resource for Drosophila neurobiology Cell reports 2, 991-1001 Kaplan, J.M., and Horvitz, H.R (1993) A dual mechanosensory and chemosensory neuron in Caenorhabditis elegans Proc Natl Acad Sci U S A 90, 2227-2231 Katayama, N., Abbott, J.K., Kjaerandsen, J., Takahashi, Y., and Svensson, E.I (2014) Sexual selection on wing interference patterns in Drosophila melanogaster Proceedings of the National Academy of Sciences of the United States of America 111, 15144-15148 Keleman, K., Vrontou, E., Kruttner, S., Yu, J.Y., Kurtovic-Kozaric, A., and Dickson, B.J (2012) Dopamine neurons modulate pheromone responses in Drosophila courtship learning Nature 489, 145-149 Kent, C., Azanchi, R., Smith, B., Formosa, A., and Levine, J.D (2008) Social context influences chemical communication in D melanogaster males Current biology : CB 18, 1384-1389 Kim, W.J., Jan, L.Y., and Jan, Y.N (2013) A PDF/NPF neuropeptide signaling circuitry of male Drosophila melanogaster controls rival-induced prolonged mating Neuron 80, 11901205 Koganezawa, M., Haba, D., Matsuo, T., and Yamamoto, D (2010) The shaping of male courtship posture by lateralized gustatory inputs to male-specific interneurons Current biology : CB 20, 1-8 137 Koh, T.W., He, Z., Gorur-Shandilya, S., Menuz, K., Larter, N.K., Stewart, S., and Carlson, J.R (2014) The Drosophila IR20a Clade of Ionotropic Receptors Are Candidate Taste and Pheromone Receptors Neuron 83, 850-865 Kohatsu, S., Koganezawa, M., and Yamamoto, D (2011) Female contact activates malespecific interneurons that trigger stereotypic courtship behavior in Drosophila Neuron 69, 498-508 Kohl, J., Ostrovsky, A.D., Frechter, S., and Jefferis, G.S (2013) A bidirectional circuit switch reroutes pheromone signals in male and female brains Cell 155, 1610-1623 Krashes, M.J., DasGupta, S., Vreede, A., White, B., Armstrong, J.D., and Waddell, S (2009) A neural circuit mechanism integrating motivational state with memory expression in Drosophila Cell 139, 416-427 Kurtovic, A., Widmer, A., and Dickson, B.J (2007) A single class of olfactory neurons mediates behavioural responses to a Drosophila sex pheromone Nature 446, 542-546 Kwon, J.Y., Dahanukar, A., Weiss, L.A., and Carlson, J.R (2014) A map of taste neuron projections in the Drosophila CNS Journal of biosciences 39, 565-574 Lacaille, F., Hiroi, M., Twele, R., Inoshita, T., Umemoto, D., Maniere, G., Marion-Poll, F., Ozaki, M., Francke, W., Cobb, M., et al (2007) An inhibitory sex pheromone tastes bitter for Drosophila males PloS one 2, e661 Laissue, P.P., and Vosshall, L.B (2008) The olfactory sensory map in Drosophila Adv Exp Med Biol 628, 102-114 Larsson, M.C., Domingos, A.I., Jones, W.D., Chiappe, M.E., Amrein, H., and Vosshall, L.B (2004) Or83b encodes a broadly expressed odorant receptor essential for Drosophila olfaction Neuron 43, 703-714 Lebestky, T., Chang, J.S., Dankert, H., Zelnik, L., Kim, Y.C., Han, K.A., Wolf, F.W., Perona, P., and Anderson, D.J (2009) Two different forms of arousal in Drosophila are oppositely regulated by the dopamine D1 receptor ortholog DopR via distinct neural circuits Neuron 64, 522-536 Lee, Y., Kim, S.H., and Montell, C (2010) Avoiding DEET through insect gustatory receptors Neuron 67, 555-561 Levine, J.D., and Millar, J.G (2009) Chemical signalling: laser on the fly reveals a new male-specific pheromone Current biology : CB 19, R653-655 Li, Y., Zhou, Z., Zhang, X., Tong, H., Li, P., Zhang, Z.C., Jia, Z., Xie, W., and Han, J (2013) Drosophila neuroligin regulates sleep through modulating GABA transmission The Journal of neuroscience : the official journal of the Society for Neuroscience 33, 1554515554 138 Ling, F., Dahanukar, A., Weiss, L.A., Kwon, J.Y., and Carlson, J.R (2014) The molecular and cellular basis of taste coding in the legs of Drosophila The Journal of neuroscience : the official journal of the Society for Neuroscience 34, 7148-7164 Liu, H., and Kubli, E (2003) Sex-peptide is the molecular basis of the sperm effect in Drosophila melanogaster Proceedings of the National Academy of Sciences of the United States of America 100, 9929-9933 Liu, L., Leonard, A.S., Motto, D.G., Feller, M.A., Price, M.P., Johnson, W.A., and Welsh, M.J (2003) Contribution of Drosophila DEG/ENaC genes to salt taste Neuron 39, 133-146 Liu, N.Y., He, P., and Dong, S.L (2012a) Binding properties of pheromone-binding protein from the common cutworm Spodoptera litura Comparative biochemistry and physiology Part B, Biochemistry & molecular biology 161, 295-302 Liu, T., Starostina, E., Vijayan, V., and Pikielny, C.W (2012b) Two Drosophila DEG/ENaC channel subunits have distinct functions in gustatory neurons that activate male courtship The Journal of neuroscience : the official journal of the Society for Neuroscience 32, 1187911889 Liu, W.W., and Wilson, R.I (2013) Glutamate is an inhibitory neurotransmitter in the Drosophila olfactory system Proceedings of the National Academy of Sciences of the United States of America 110, 10294-10299 Liu, Y., and Yang, C.H (2014) Unveiling the secrets to her heart Neuron 83, 3-5 Mane, S.D., Tepper, C.S., and Richmond, R.C (1983) Studies of esterase in Drosophila melanogaster XIII Purification and characterization of the two major isozymes Biochemical genetics 21, 1019-1040 Maniatis, T., and Tasic, B (2002) Alternative pre-mRNA splicing and proteome expansion in metazoans Nature 418, 236-243 Manning, A (1966) Corpus allatum and sexual receptivity in female Drosophila melanogaster Nature 211, 1321-1322 Marcillac, F., Grosjean, Y., and Ferveur, J.F (2005) A single mutation alters production and discrimination of Drosophila sex pheromones Proceedings Biological sciences / The Royal Society 272, 303-309 Marella, S., Mann, K., and Scott, K (2012) Dopaminergic modulation of sucrose acceptance behavior in Drosophila Neuron 73, 941-950 Masek, P., and Scott, K (2010) Limited taste discrimination in Drosophila Proceedings of the National Academy of Sciences of the United States of America 107, 14833-14838 Meunier, N., Marion-Poll, F., Rospars, J.P., and Tanimura, T (2003) Peripheral coding of bitter taste in Drosophila Journal of neurobiology 56, 139-152 139 Miyamoto, T., and Amrein, H (2008) Suppression of male courtship by a Drosophila pheromone receptor Nature neuroscience 11, 874-876 Miyamoto, T., Chen, Y., Slone, J., and Amrein, H (2013) Identification of a Drosophila glucose receptor using Ca2+ imaging of single chemosensory neurons PloS one 8, e56304 Miyamoto, T., Slone, J., Song, X., and Amrein, H (2012) A fructose receptor functions as a nutrient sensor in the Drosophila brain Cell 151, 1113-1125 Monastirioti, M., Linn, C.E., Jr., and White, K (1996) Characterization of Drosophila tyramine beta-hydroxylase gene and isolation of mutant flies lacking octopamine The Journal of neuroscience : the official journal of the Society for Neuroscience 16, 3900-3911 Moon, S.J., Lee, Y., Jiao, Y., and Montell, C (2009) A Drosophila gustatory receptor essential for aversive taste and inhibiting male-to-male courtship Current biology : CB 19, 1623-1627 Mori, K (2007) Significance of chirality in pheromone science Bioorganic & medicinal chemistry 15, 7505-7523 Mori, K., Shikichi, Y., Shankar, S., and Yew, J.Y (2010) Pheromone synthesis Part 244: Synthesis of the racemate and enantiomers of (11Z,19Z)-CH503 (3-acetoxy-11,19octacosadien-1-ol), a new sex pheromone of male Drosophila melanogaster to show its (S)isomer and racemate as bioactive Tetrahedron 66, 7161-7168 Mucignat-Caretta, C., Caretta, A., and Cavaggioni, A (1995) Acceleration of puberty onset in female mice by male urinary proteins The Journal of physiology 486 ( Pt 2), 517-522 Ng, S.H., Shankar, S., Shikichi, Y., Akasaka, K., Mori, K., and Yew, J.Y (2014) Pheromone evolution and sexual behavior in Drosophila are shaped by male sensory exploitation of other males Proceedings of the National Academy of Sciences of the United States of America 111, 3056-3061 Ni, L., Bronk, P., Chang, E.C., Lowell, A.M., Flam, J.O., Panzano, V.C., Theobald, D.L., Griffith, L.C., and Garrity, P.A (2013) A gustatory receptor paralogue controls rapid warmth avoidance in Drosophila Nature 500, 580-584 Rajashekhar, K.P., and Singh, R.N (1994) Neuroarchitecture of the tritocerebrum of Drosophila melanogaster The Journal of comparative neurology 349, 633-645 Rideout, E.J., Dornan, A.J., Neville, M.C., Eadie, S., and Goodwin, S.F (2010) Control of sexual differentiation and behavior by the doublesex gene in Drosophila melanogaster Nature neuroscience 13, 458-466 Robertson, H.M., Warr, C.G., and Carlson, J.R (2003) Molecular evolution of the insect chemoreceptor gene superfamily in Drosophila melanogaster Proceedings of the National Academy of Sciences of the United States of America 100 Suppl 2, 14537-14542 Rodrigues, V., and Siddiqi, O (1981) A gustatory mutant of Drosophila defective in pyranose receptors Molecular & general genetics : MGG 181, 406-408 140 Root, C.M., Denny, C.A., Hen, R., and Axel, R (2014) The participation of cortical amygdala in innate, odour-driven behaviour Nature 515, 269-273 Root, C.M., Ko, K.I., Jafari, A., and Wang, J.W (2011) Presynaptic facilitation by neuropeptide signaling mediates odor-driven food search Cell 145, 133-144 Ruta, V., Datta, S.R., Vasconcelos, M.L., Freeland, J., Looger, L.L., and Axel, R (2010) A dimorphic pheromone circuit in Drosophila from sensory input to descending output Nature 468, 686-690 Scott, D (1986) Sexual mimicry regulates the attractiveness of mated Drosophila melanogaster females Proceedings of the National Academy of Sciences of the United States of America 83, 8429-8433 Shanbhag, S.R., Park, S.K., Pikielny, C.W., and Steinbrecht, R.A (2001) Gustatory organs of Drosophila melanogaster: fine structure and expression of the putative odorant-binding protein PBPRP2 Cell and tissue research 304, 423-437 Shang, Y., Donelson, N.C., Vecsey, C.G., Guo, F., Rosbash, M., and Griffith, L.C (2013) Short neuropeptide F is a sleep-promoting inhibitory modulator Neuron 80, 171-183 Shankar, S., Chua, J.Y., Tan, K.J., Calvert, M.E., Weng, R., Ng, W.C., Mori, K., and Yew, J.Y (2015) The neuropeptide tachykinin is essential for pheromone detection in a gustatory neural circuit Elife Shikichi, Y., Shankar, S., Yew, J.Y., and Mori, K (2013a) Synthesis and Bioassay of the Eight Analogues of Male Pheromone CH503 (3-Acetoxy-11,19-octacosadien-1-ol) of the Fruit Fly Drosophila melanogaster Bioscience, Biotechnology, and Biochemistry 77, 1931 1938 Shikichi, Y., Shankar, S., Yew, J.Y., and Mori, K (2013b) Synthesis and bioassay of the eight analogues of the CH503 male pheromone (3-acetoxy-11,19-octacosadien-1-ol) of the Drosophila melanogaster fruit fly Bioscience, biotechnology, and biochemistry 77, 19311938 Shiraiwa, T., and Carlson, J.R (2007) Proboscis extension response (PER) assay in Drosophila Journal of visualized experiments : JoVE, 193 Shohat-Ophir, G., Kaun, K.R., Azanchi, R., Mohammed, H., and Heberlein, U (2012) Sexual deprivation increases ethanol intake in Drosophila Science 335, 1351-1355 Siegel, R.W., and Hall, J.C (1979) Conditioned responses in courtship behavior of normal and mutant Drosophila Proceedings of the National Academy of Sciences of the United States of America 76, 3430-3434 Sitaraman, D., Zars, M., Laferriere, H., Chen, Y.C., Sable-Smith, A., Kitamoto, T., Rottinghaus, G.E., and Zars, T (2008) Serotonin is necessary for place memory in Drosophila Proceedings of the National Academy of Sciences of the United States of America 105, 5579-5584 141 Siwicki, K.K., Riccio, P., Ladewski, L., Marcillac, F., Dartevelle, L., Cross, S.A., and Ferveur, J.F (2005) The role of cuticular pheromones in courtship conditioning of Drosophila males Learning & memory 12, 636-645 Slone, J., Daniels, J., and Amrein, H (2007) Sugar receptors in Drosophila Current biology : CB 17, 1809-1816 Starostina, E., Liu, T., Vijayan, V., Zheng, Z., Siwicki, K.K., and Pikielny, C.W (2012) A Drosophila DEG/ENaC subunit functions specifically in gustatory neurons required for male courtship behavior The Journal of neuroscience : the official journal of the Society for Neuroscience 32, 4665-4674 Stocker, R.F (1994) The organization of the chemosensory system in Drosophila melanogaster: a review Cell and tissue research 275, 3-26 Stocker, R.F., and Gendre, N (1989) Courtship behavior of Drosophila genetically or surgically deprived of basiconic sensilla Behavior genetics 19, 371-385 Taylor, B.J., Villella, A., Ryner, L.C., Baker, B.S., and Hall, J.C (1994) Behavioral and neurobiological implications of sex-determining factors in Drosophila Dev Genet 15, 275296 Thistle, R., Cameron, P., Ghorayshi, A., Dennison, L., and Scott, K (2012) Contact chemoreceptors mediate male-male repulsion and male-female attraction during Drosophila courtship Cell 149, 1140-1151 Toda, H., Zhao, X., and Dickson, B.J (2012) The Drosophila female aphrodisiac pheromone activates ppk23(+) sensory neurons to elicit male courtship behavior Cell reports 1, 599-607 van der Goes van Naters, W., and Carlson, J.R (2007) Receptors and neurons for fly odors in Drosophila Current biology : CB 17, 606-612 Vijayan, V., Thistle, R., Liu, T., Starostina, E., and Pikielny, C.W (2014) Drosophila pheromone-sensing neurons expressing the ppk25 ion channel subunit stimulate male courtship and female receptivity PLoS genetics 10, e1004238 von Philipsborn, A.C., Jorchel, S., Tirian, L., Demir, E., Morita, T., Stern, D.L., and Dickson, B.J (2014) Cellular and behavioral functions of fruitless isoforms in Drosophila courtship Current biology : CB 24, 242-251 Vosshall, L.B., Amrein, H., Morozov, P.S., Rzhetsky, A., and Axel, R (1999) A spatial map of olfactory receptor expression in the Drosophila antenna Cell 96, 725-736 Vosshall, L.B., and Stocker, R.F (2007) Molecular architecture of smell and taste in Drosophila Annual review of neuroscience 30, 505-533 Wang, L., and Anderson, D.J (2010) Identification of an aggression-promoting pheromone and its receptor neurons in Drosophila Nature 463, 227-231 142 Wang, L., Han, X., Mehren, J., Hiroi, M., Billeter, J.C., Miyamoto, T., Amrein, H., Levine, J.D., and Anderson, D.J (2011) Hierarchical chemosensory regulation of male-male social interactions in Drosophila Nature neuroscience 14, 757-762 Wang, Y., Pu, Y., and Shen, P (2013) Neuropeptide-gated perception of appetitive olfactory inputs in Drosophila larvae Cell reports 3, 820-830 Wang, Z., Singhvi, A., Kong, P., and Scott, K (2004) Taste representations in the Drosophila brain Cell 117, 981-991 Watanabe, K., Toba, G., Koganezawa, M., and Yamamoto, D (2011) Gr39a, a highly diversified gustatory receptor in Drosophila, has a role in sexual behavior Behav Genet 41, 746-753 Weiss, L.A., Dahanukar, A., Kwon, J.Y., Banerjee, D., and Carlson, J.R (2011) The molecular and cellular basis of bitter taste in Drosophila Neuron 69, 258-272 Wen, T., Parrish, C.A., Xu, D., Wu, Q., and Shen, P (2005) Drosophila neuropeptide F and its receptor, NPFR1, define a signaling pathway that acutely modulates alcohol sensitivity Proceedings of the National Academy of Sciences of the United States of America 102, 21412146 Weng, R., Chen, Y.-W., Bushati, N., Cliffe, A., and Cohen, S.M (2009) RecombinaseMediated Cassette Exchange Provides a Versatile Platform for Gene Targeting: Knockout of miR-31b Genetics 183, 399-402 Winther, A.M., Acebes, A., and Ferrus, A (2006) Tachykinin-related peptides modulate odor perception and locomotor activity in Drosophila Molecular and cellular neurosciences 31, 399-406 Woolf, C.J., and Walters, E.T (1991) Common patterns of plasticity contributing to nociceptive sensitization in mammals and Aplysia Trends Neurosci 14, 74-78 Wu, Q., Wen, T., Lee, G., Park, J.H., Cai, H.N., and Shen, P (2003) Developmental control of foraging and social behavior by the Drosophila neuropeptide Y-like system Neuron 39, 147-161 Wu, Q., Zhao, Z., and Shen, P (2005) Regulation of aversion to noxious food by Drosophila neuropeptide Y- and insulin-like systems Nat Neurosci 8, 1350-1355 Wu, Y., Ren, Q., Li, H., and Guo, A (2012) The GABAergic anterior paired lateral neurons facilitate olfactory reversal learning in Drosophila Learning & memory 19, 478-486 Yao, C.A., Ignell, R., and Carlson, J.R (2005) Chemosensory coding by neurons in the coeloconic sensilla of the Drosophila antenna The Journal of neuroscience : the official journal of the Society for Neuroscience 25, 8359-8367 Yew, J.Y., Dreisewerd, K., Luftmann, H., Muthing, J., Pohlentz, G., and Kravitz, E.A (2009) A new male sex pheromone and novel cuticular cues for chemical communication in Drosophila Current biology : CB 19, 1245-1254 143 Yu, J.Y., Kanai, M.I., Demir, E., Jefferis, G.S., and Dickson, B.J (2010) Cellular organization of the neural circuit that drives Drosophila courtship behavior Current biology : CB 20, 1602-1614 Yuan, Q., Joiner, W.J., and Sehgal, A (2006) A sleep-promoting role for the Drosophila serotonin receptor 1A Current biology : CB 16, 1051-1062 Zhou, C., Pan, Y., Robinett, C.C., Meissner, G.W., and Baker, B.S (2014) Central brain neurons expressing doublesex regulate female receptivity in Drosophila Neuron 83, 149-163 144 ... IN ANIMALS 1.2 COURTSHIP BEHAVIOR OF DROSOPHILA MELANOGASTER 1.3 PHEROMONES OF DROSOPHILA MELANOGASTER 1.4 ORGANIZATION OF THE DROSOPHILA BRAIN 12 1.5 NEUROTRANSMITTERS OF DROSOPHILA MELANOGASTER. .. COURTSHIP BEHAVIOR OF DROSOPHILA MELANOGASTER Male courtship behavior of Drosophila melanogaster has been extensively characterized and comprises of innate and learned aspects Courtship steps are executed... courtship behavior has been used as a paradigm to study how pheromones influence innate behaviors and to understand the neuronal basis of pheromone perception The anatomical, cellular and molecular basis