Physiological Entomology (2012) 37, 19–24
DOI: 10.1111/j.1365-3032.2011.00813.x
REVIEW ARTICLE
Volatile pheromone signalling in Drosophila DEAN P. SMITH University of Texas Southwestern Medical Center, Dallas, Texas, U.S.A.
Abstract. Once captured by the antenna, the male-specific pheromone 11-cis vaccenyl acetate (cVA) binds to an extracellular binding protein called LUSH that undergoes a conformational shift upon cVA binding. The stable LUSH–cVA complex is the activating ligand for pheromone receptors present on the dendrites of the aT1 neurones, comprising the only class of neurones that detect volatile cVA pheromone. This mechanism can explain the single molecule sensitivity of pheromone detection. The receptor that recognizes activated LUSH consists of a complex of several proteins, including Or67d, a member of the tuning odourant receptor family, Orco, a co-receptor ion channel, and SNMP, a CD36 homologue that may be an inhibitory subunit. In addition, genetic screens and reconstitution experiments reveal additional factors that are important for pheromone detection. Identification and functional dissection of these factors in Drosophila melanogaster Meigen should permit the identification of homologous factors in pathogenic insects and agricultural pests, which, in turn, may be viable candidates for novel classes of compounds to control populations of target insect species without impacting beneficial species. Key words. Behaviour, odorant binding protein, pheromone detection, signal transduction.
Introduction Insects use pheromones to trigger social behaviours in conspecifics. Pheromones are the dominant language for communication in insects, making the complex societies of social insects such as ants, bees and termites possible. However, pheromones are also used by most insect species to coordinate mating behaviour between appropriate individuals. Volatile pheromones act over a distance and are detected by olfactory neurones, whereas contact pheromones are detected by gustatory neurones upon direct contact with another individual. Volatile pheromone detection in many insects is exceptionally sensitive, approaching single molecule sensitivity (Kaissling & Priesner, 1970). In moths, sex pheromone sensitivity allows males to identify and locate females possibly over great distances, a feat that has intrigued naturalists for over 100 years (Fabre, 1916). Our research group at the University of Texas Southwestern Medical Center is interested in the initial steps of pheromone detection that account for this remarkable sensitivity: how the Correspondence: Dean P. Smith, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-9111, U.S.A. Tel.: +1 214 648 1650; e-mail: dean.smith@ utsouthwestern.edu © 2012 The Author Physiological Entomology © 2012 The Royal Entomological Society
presence of pheromone molecules are translated into electrical signals in primary olfactory neurones. The pheromone model system studied in the author’s laboratory is volatile cVA (11-cis vaccenyl acetate) pheromone signalling and induced behaviours in the genetically tractable insect Drosophila melanogaster Meigen. cVA is the major volatile pheromone in this species and is produced by males but is detected equally well by specialized olfactory neurones (T1 neurones) in both sexes. cVA induces sexually dimorphic behaviours, stimulating mating receptivity in females, but inhibiting courtship between males. The mechanisms underlying volatile pheromone detection are likely to be conserved among insects; therefore, the general principles and molecular components that are uncovered by studying Drosophila cVA detection are likely to be conserved in other insects.
Drosophila olfactory anatomy Volatile odourants (including pheromones) are detected by olfactory neurones located in the antenna (Fig. 1A). The dendrites of the olfactory neurones project into hair-like, fluid-filled structures called sensilla. The sensilla are classified into four groups based on morphology, although each 19
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Fig. 1. The anatomy of the Drosophila olfactory system. (A) Scanning electron micrograph of a Drosophila head showing the antennae (A) and maxillary palps (M). (B) High power view of the surface of the antenna showing the four major classes of sensillae. B, basiconic; C, coeloconic; I, intermediate; T, trichoid. Reproduced with permission from Landesbioscience, Fly 3, 290–297.
morphological group also appears to have functional specialization. The basiconic sensilla contain the dendrites of olfactory neurones that express odourant receptors tuned to food odourants (Hallem et al., 2004; Hallem & Carlson, 2006). The coeloconic sensilla neurones are tuned to acids and aldehydes, and are detected by variant glutamate receptors (Benton et al., 2009). Trichoid sensilla contain the dendrites of pheromonesensitive olfactory neurones (Clyne et al., 1997; Xu et al., 2005). Intermediate sensilla are intermediate in morphology between basiconic and trichoid sensilla, and one intermediate neurone is tuned to a fruit rind odourant but mediates oviposition behaviour (D. Ronderos & D. Smith, unpublished data). Thus, these four anatomic sensilla types contain olfactory neurones with specialized functions. Olfactory neurone activity is monitored using single sensillum electrophysiology (SSR). Sharp glass or tungsten electrodes are inserted into individual sensilla and the signals from the neurones within that sensillum are passed through a highimpedance amplifier, and action potentials are then recorded in response to odourant application. Single sensillum recordings have been instrumental in characterizing the organization and function of the Drosophila olfactory system. This technique, combined with the molecular genetic tools available in this system, allows the detailed characterization of most of the odourant receptors (Elmore & Smith, 2001; Hallem et al., 2004; Yao et al., 2005; Hallem & Carlson, 2006; Benton et al., 2009). Once the odourant response profile is obtained for the various basiconic neurone classes, it is possible to correlate receptors with the odourant sensitivity of each olfactory neurone class. This is accomplished by mis-expressing specific odourant receptors in a defined basiconic olfactory neurone lacking the endogenous odourant receptor and by measuring the odourant response profile of the mis-expressed receptor (i.e. the empty neurone approach) (Hallem et al., 2004). When the odourant spectrum for each basiconic receptor is characterized, these correlate well with the responses of the various basiconic neurone classes, allowing specific receptors to be correlated with functional classes of neurones (Hallem et al., 2004). However, a subset of receptors fail to function in basiconic neurones, and most of these are normally expressed in the trichoid sensillum zone of the antenna. This raises the
possibility that additional signal transduction factors may be necessary in the pheromone-sensing neurones. Indeed, Or67d, the tuning odourant receptor expressed exclusively by the cVAsensitive T1 neurones (Ha & Smith, 2006; Kurtovic et al., 2007), does not respond to volatile cVA when expressed in the empty neurone system (Laughlin et al., 2008). Using genetic screens to identify cVA-insensitive mutants, a number of novel signalling factors required for pheromone detection by trichoid neurones have been uncovered that are not required for the detection of general odourants. To identify and characterize the components required for cVA signalling in Drosophila, a combination of genetic screens, mutagenesis, electrophysiology, structural studies and behavioural assays have been used. The first gene product uncovered that is required for cVA detection was the odourantbinding protein LUSH. lush was identified in an enhancer-trap genetic screen (Kim et al., 1998b). A transposable element carrying a gene for LacZ was mobilized randomly throughout the genome, and individual lines were established, each carrying a unique transposon inserted somewhere in the genome. LacZ expression is dependent on capturing local enhancers upon integration; thus, by screening the lines for LacZ expression restricted to the antennae, it was possible to identify genes expressed exclusively in the antennae. The gene lush was identified as being expressed exclusively in the trichoid sensillum zone of the antennae (Kim et al., 1998a). LUSH is a member of the Drosophila odourant-binding protein family, a large family of genes expressed in chemosensory organs (Galindo & Smith, 2001). Lepidopteran OBP family members include the pheromone-binding proteins known to be secreted into the extracellular lymph space bathing the olfactory neurone dendrites and binding directly to pheromones (Vogt & Riddiford, 1981). However, at that time, the role of these proteins in vivo was unknown. LacZ expression revealed that LUSH was expressed exclusively in the ventral–lateral surface of the antennae, where trichoid sensilla are localized, and anti-LUSH antiserum showed that this protein is secreted into the sensillum lymph of all trichoid sensilla by non-neuronal supporting cells (Kim et al., 1998b; Shanbhag et al., 2005). To study the potential effects that loss of the LUSH protein has on cVA detection, the transposon in the lush enhancer-trap stock was remobilized to generate a 2-kb genomic deletion that removed the entire coding sequence of the lush gene (Kim et al., 1998a). Indeed, when the pheromone responses of lush 1 mutants to cVA pheromone were assayed, it was found that these mutants are insensitive to cVA pheromone (Fig. 2) (Xu et al., 2005). A wild-type, transgenic copy of the lush gene restores cVA sensitivity to the mutants (Fig. 2) (Xu et al., 2005), confirming that the loss of LUSH protein accounts entirely for the cVA detection defect. The fact that LUSH is required for the activation of cVA-sensitive neurones eliminated the possibility that LUSH functions to remove cVA from the sensillum lymph because a loss of a pheromone remover would not be expected to block pheromone sensitivity. Furthermore, recombinant LUSH protein produced in bacteria and infused into the sensillum lymph through the recording pipette also restores cVA sensitivity to lush 1 mutants (Xu et al., 2005). This indicates that LUSH is directly required for cVA
© 2012 The Author Physiological Entomology © 2012 The Royal Entomological Society, Physiological Entomology, 37, 19–24
Volatile pheromone signalling
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Fig. 2. 11-cis Vaccenyl acetate (cVA) sensitivity in wild-type and lush 1 mutants. TOP, 1-s recording traces after a 300-ms pulse of air blown across paper impregnated with 1 μL of a 1 : 100 dilution of cVA. Wild-type shows a robust burst of action potentials after a latency that is largely a result of the time for the air pulse to reach the preparation. lush 1 mutants are insensitive to cVA, although a wild-type transgenic copy of lush (rescue) restores cVA sensitivity to the mutants. APO3, a pheromone-binding protein from the moth species Antherea polyphemus fails to confer cVA sensitivity to lush 1 mutants, demonstrating binding protein specificity. Bottom: dose–response curves for cVA in wild-type and lush 1 mutants. Reproduced with permission from Elsevier Inc, Neuron 45, 193–200.
signalling, and not for the development of the cVA detection mechanism. When analyzing the lush 1 mutants, a second, unexpected phenotype associated with loss of the extracellular LUSH protein was noted. The spontaneous activity of the pheromone-sensitive T1 neurones was reduced by more than 400-fold in the absence of pheromone. However, the loss of LUSH in other trichoid sensilla had no effect on the spontaneous activity of those neurones. This ruled out the possibility of a nonspecific osmotic change affecting T1 spontaneous activity upon the loss of abundant extracellular LUSH protein because this loss would be identical in other trichoid sensilla where LUSH is also normally expressed. Therefore, LUSH is not a simple pheromone carrier that transports hydrophobic cVA to the pheromone-sensitive T1 neurones through the sensillum lymph because the loss of a carrier protein would not be expected to reduce the spontaneous activity in these neurones in the absence of pheromone. Instead, LUSH alone appears
to have agonist activity, even when there is no pheromone present. This insight revealed that LUSH was more likely to function as a signalling factor than as a simple pheromone carrier. To account for the pheromone-independent agonist activity of LUSH, it was speculated that LUSH may undergo conformational activation upon binding pheromone, and that the active conformation may be the ligand for neuronal receptors. If a fraction of the LUSH molecules can spontaneously isomerize to the activated state at a low rate, this could account for the spontaneous activity in wild-type flies that is lacking in lush 1 mutants. If pheromone stabilizes or induces this activated LUSH state, this could account for the requirement of LUSH in cVA detection. This model makes two testable predictions. First, if LUSH undergoes a conformational activation upon cVA binding, it should be possible to observe differences in the X-ray crystal structure of LUSH with and without cVA pheromone bound.
© 2012 The Author Physiological Entomology © 2012 The Royal Entomological Society, Physiological Entomology, 37, 19–24
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Fig. 3. Ribbon diagram of the relevant portions of LUSH with and without 11-cis vaccenyl acetate (cVA) bound. Overlaid structures with cVA present (purple) or absent (green). cVA, in magenta binds to LUSH ejecting F121 from the binding pocket. This, in turn, disrupts the salt bridge between D118 and K87 that normally stabilizes the inactive conformation of LUSH. This results in flipping of the Cterminus of LUSH, which is most apparent in the orientation of Q120. Reproduced with permission from Elsevier Inc, Cell 133, 1255–1265.
Second, if LUSH is the true ligand for the receptors expressed on pheromone-sensitive T1 neurones, it should be possible to generate dominant LUSH mutants that activate T1 neurones in the absence of cVA pheromone. To obtain insight into the possible structural effects of cVA binding on LUSH, we have collaborated with John Laughlin and David Jones of the University of Colorado Health Sciences Center in Denver (Laughlin et al., 2008). Recombinant LUSH protein lacking the signal sequence was expressed in bacteria and refolded slowly under conditions to allow proper disulfide bond formation and then purified. Protein crystals were grown in the presence or absence of cVA pheromone and analyzed. The first major finding from solving the crystal structure of LUSH with and without cVA bound was that cVA is completely enveloped by LUSH. This rules out the possibility that a unique pheromone/LUSH surface is recognized by neuronal receptors. Second, cVA-bound LUSH has a different conformation than apoLUSH. cVA binding to LUSH ejects the C-terminus of LUSH from the cVA binding pocket. Phenylalanine 121 appears to be the key residue contacting cVA in the binding pocket, leading to disruption of the salt bridge between aspartate 118 and lysine 87, with subsequent flipping of the orientation of the C-terminal loop of LUSH (Fig. 3). If this conformational shift in LUSH is important for the activation of T1 neuronal receptors, mutating phenylalanine 121 to alanine should impair the ability of cVA–LUSH complexes to activate T1 neurones because cVA will be less able to induce the conformational shift. Indeed, cVA binding is not impacted by the F121A mutation, although the F121A mutant
C
Fig. 4. LUSHD118A activates T1 neurones in the absence of 11-cis vaccenyl acetate (cVA). (A) Trace of T1 neuronal activation upon infusing recombinant LUSHD118A into the T1 sensillum lymph of lush 1 mutants through the recording pipette. (B) Comparison of infusing wild-type (wt) LUSH and LUSHD118A into the T1 sensillum lymph of lush 1 mutants. (C) Overlaid structures of LUSH with and without cVA bound together with the structure of apoLUSHD118A . apoLUSHD118A and cVA bound LUSH have almost identical conformational shifts (arrow). Reproduced with permission from Elsevier Inc, Cell 133, 1255–1265.
Fig. 5. Current model for 11-cis vaccenyl acetate (cVA) detection in Drosophila. Left, in the absence of cVA, LUSH is in the inactive state, and SNMP is bound to the Or67d/Orco complex, inhibiting ion fluxes. Right, in the presence of cVA, LUSH undergoes a conformational activation that allows it to interact with SNMP, which releases the Or67d/Orco complex, allowing ions to flow.
version of LUSH, when infused into the sensillum lymph of lush 1 mutants through the recording pipette, requires 50fold more cVA to achieve the same activation compared with infusing wild-type LUSH protein at the same concentration (Laughlin et al., 2008). By contrast, mutating phenyalanine 121 to the bulkier amino-acid tryptophan results in a LUSH protein that activates T1 neurones at cVA concentrations fivefold lower than wild-type LUSH. This finding comprises good
© 2012 The Author Physiological Entomology © 2012 The Royal Entomological Society, Physiological Entomology, 37, 19–24
Volatile pheromone signalling evidence that the LUSH conformational shift induced by cVA is important for activating T1 neurones. Finally, disrupting the D118/K87 salt bridge present in apoLUSH by mutating D118 to alanine (D118A) produced a dominant allele of LUSH. When LUSHD118A protein is infused into the sensillum lymph of lush 1 mutants, the T1 neurones activate without any cVA (Fig. 4A, B). When John Laughlin and David Jones solved the X-ray crystal structure of apoLUSHD118A , it was gratifying to see that the C-terminal loop showed the identical conformation shift as cVA-bound LUSH (Fig. 4C) (Laughlin et al., 2008). These data indicate that conformational changes in LUSH are sufficient to mediate activation of T1 neurones, and the role of cVA is to induce the activated conformation in the LUSH protein.
The neuronal LUSH receptor To identify the molecular components of the receptor expressed by T1 neurones that mediate cVA sensitivity, a genetic screen was undertaken for cVA-insensitive mutants. Three thousand homozygous flies with mutagenized third chromosomes were screened for defective cVA responses using SSR (Jin et al., 2008). Sixteen mutants were recovered that could be divided into several phenotypic classes based on the T1 SSRs. The class that turned out to be very interesting was insensitive to any concentration of cVA, although it still showed spontaneous activity in the T1 neurones. The presence of spontaneous activity in the T1 neurones ruled out nonspecific mutations affecting neuronal viability or the ability to fire action potentials. This class of mutants was named the vains mutants (cVA insensitive with spontaneous activity). Complementation analysis revealed four different genes mutated to produce the vains phenotype. Whole genome sequencing combined with deletion mapping experiments revealed the vains mutants corresponded to lesions in Orco, Or67d, snmp and a transcription factor gene that is not be discussed further here. Orco (Or co-receptor, formerly Or83b) (Larsson et al., 2004) is an ion channel that associates with ‘tuning’ odourant receptor subunits to produce odourant-gated ion channels (Sato et al., 2008; Wicher et al., 2008). Orco mutants were expected in the mutant screen because all Or proteins require this ion channel to function. Two mutant alleles of Orco were identified in the screen, both comprising strong mutations affecting splicing signals and resulting in frameshift mutations (Jin et al., 2008). Mutants in Or67d were also expected and recovered, given that this receptor had previously been identified as a T1-specific receptor conferring cVA sensitivity to other trichiod neurones that are normally insensitive to cVA (Ha & Smith, 2006). Or67d mutants were subsequently generated showing a loss of cVA sensitivity in T1 neurones (Kurtovic et al., 2007). Both Orco and Or67d mutants have infrequent spontaneous action potentials, similar to that observed in lush 1 mutants. The third vains gene, snmp, was unexpected, and encodes a homologue of human CD36, which is a scavanger receptor expressed on macrophages that is required to take up lipids and oxidized cholesterol in lipoprotein complexes leading to
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atherosclerosis (Coburn et al., 2000; Bonen et al., 2007; Guest et al., 2007; Thorne et al., 2007). Of note, snmp mutants have a T1 neurone spontaneous activity rate that is 20-fold higher than wild-type (Benton et al., 2007; Jin et al., 2008). Antibodies to SNMP infused into the sensillum lymph through the recording pipette produce a similar phenotype, indicating that SNMP is present on the surface of the dendrites (Jin et al., 2008). Interestingly, dominant LUSHD118A fails to activate T1 neurones from flies mutant for Or67d, Orco or snmp (Laughlin et al., 2008). Taken together, these data suggest that SNMP acts as an inhibitory subunit in the receptor complex, and all three of these components act downstream of LUSH. Benton et al. (2007) studied the role of SNMP in cVA sensitivity independently from our studies and suggested that SNMP may function to transfer cVA from LUSH to the neuronal receptor. It is worth noting, however, that dominant LUSH activates T1 neurones in the absence of cVA, although this is not the case if SNMP is missing (Laughlin et al., 2008). Therefore, SNMP must have a role in T1 neurone activation that is independent of cVA transfer. Figure 5 shows a current working model for cVA signal transduction. Finally, there are additional gene products important for cVA detection that remain to be described. There are several mutants recovered from a third chromosome screen that affect cVA sensitivity. We have just started screening the second and X chromosomes for genes important for cVA sensitivity located on other chromosomes. When all relevant cVA sensitivity factors have been identified, it is expected that the misexpression of all these factors in the empty basiconic neurone system will reconstitute T1 neuronal levels of cVA sensitivity (Laughlin et al., 2008).
The behavioural effects of T1 neuronal activation Several groups have studied the behavioural effects of cVA detection on fruit flies (Ejima et al., 2007; Kurtovic et al., 2007; Ronderos & Smith, 2010; Liu et al., 2011; Wang et al., 2011). There is general agreement that cVA mediates the recognition of sex between individuals in this species (Ejima et al., 2007; Kurtovic et al., 2007; Ronderos & Smith, 2010). cVA detection induces sexually dimorphic behaviours, inducing enhanced receptivity to courtship in females, and reducing courtship and social avoidance in males. This is consistent with cVA being present on the male cuticle, thus reducing inappropriate mating attempts between males and enhancing courtship in females. In a study using tetanus toxin expressed in subsets of olfactory neurones, it was suggested that cVA is detected both by Or67d-expressing T1 neurones and Or65a-expressing T2 neurones and that mating behaviour is mediated through the Or65a circuit (Ejima et al., 2007; Liu et al., 2011). However, the Or65a driver used to express tetanus toxin or other transgenes also expresses these genes in other neurones in the central nervous system (Ejima et al., 2007). This raises concerns about the effects on other circuits in the brain that might impact courtship behaviour. Evidence has been obtained showing that cVAmediated mating behaviour requires the Or67d-expressing T1
© 2012 The Author Physiological Entomology © 2012 The Royal Entomological Society, Physiological Entomology, 37, 19–24
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neurones (Kurtovic et al., 2007; Ronderos & Smith, 2010). Both groups report that Or67d mutants have reduced female receptivity and increased male–male courtship. Our own group has further evidence that cVA detection occurs exclusively though the T1 neurones (Ronderos & Smith, 2010). Transgenic flies expressing dominant LUSHD118A constitutively and specifically activate the T1 neurone circuit (Ronderos & Smith, 2010). The behaviour of these transgenic flies was exactly what would be predicted with male courtship inhibited toward females and female receptivity to courtship diminished (Ronderos & Smith, 2010). Both of these effects of LUSHD118A are eliminated when the downstream receptor Or67d is missing (Ronderos & Smith, 2010). Dominant LUSHD118A has no effect on Or65a neurones, nor any other class of trichoid or basiconic olfactory neurones, except T1 neurones (Laughlin et al., 2008). Therefore, T1 neurones appear to be necessary and sufficient for mediating all cVA mating behaviours. Elucidation of the gene products and mechanisms mediating volatile pheromone detection in Drosophila should help in the provision of approaches manipulating the behaviour of other insects with agricultural and pathological significance. The discovery of novel gene products required for pheromone detection in Drosophila using genetics screens is anticipated, as well as an understanding of their roles in pheromone signal transduction. Homologues in other species might prove to be useful targets for disrupting pheromone-mediated behaviours.
References Benton, R., Vannice, K., Michnick, S. & Vosshall, L. (2007) An essential role for a CD36-related receptor in pheromone detection in Drosophila. Nature, 450, 289–293. Benton, R., Vannice, K.S., Gomez-Diaz, C. & Vosshall, L.B. (2009) Variant ionotropic glutamate receptors as chemosensory receptors in Drosophila. Cell, 136, 149–162. Bonen, A., Han, X.X., Habets, D.D. et al. (2007) A null mutation in skeletal muscle FAT/CD36 reveals its essential role in insulinand AICAR-stimulated fatty acid metabolism. American Journal of Physiological Endocrinology and Metabolism, 292, E1740–E1749. Clyne, P., Grant, A., O‘Connell, R. & Carlson, J.R. (1997) Odorant response of individual sensilla on the Drosophila antenna. Invertebrate Neuroscience, 3, 127–135. Coburn, C.T., Knapp, F.F. Jr, Febbraio, M. et al. (2000) Defective uptake and utilization of long chain fatty acids in muscle and adipose tissues of CD36 knockout mice. Journal of Biological Chemistry, 275, 32523–32529. Ejima, A., Smith, B.P., Lucas, C. et al. (2007) Generalization of courtship learning in Drosophila is mediated by cis-vaccenyl acetate. Current Biology, 17, 599–605. Elmore, T. & Smith, D.P. (2001) Putative Drosophila odor receptor OR43b localizes to dendrites of olfactory neurons. Insect Biochemistry and Molecular Biology, 31, 791–798. Fabre, J.H. (1916) The Life of the Caterpillar. Dodd, Mead and Company, Inc., New York, New York. Galindo, K. & Smith, D.P. (2001) A large family of divergent Drosophila odorant-binding proteins expressed in gustatory and olfactory sensilla. Genetics, 159, 1059–1072. Guest, C.B., Hartman, M.E., O‘Connor, J.C. et al. (2007) Phagocytosis of cholesteryl ester is amplified in diabetic mouse macrophages and is largely mediated by CD36 and SR-A. PLoS ONE, 2, e511.
Ha, T. & Smith, D. (2006) A pheromone receptor mediates 11cis vaccenyl acetate-induced responses in Drosophila. Journal of Neuroscience, 26, 8727–8733. Hallem, E.A. & Carlson, J.R. (2006) Coding of odors by a receptor repertoire. Cell, 125, 143–160. Hallem, E.A., Ho, M.G. & Carlson, J.R. (2004) The molecular basis of odor coding in the Drosophila antenna. Cell, 117, 965–979. Jin, X., Ha, T. & Smith, D. (2008) SNMP is a signaling component required for pheromone sensitivity in Drosophila. Proceedings of the National Academy of Sciences of the United States of America, 105, 10995–11000. Kaissling, K.-E. & Priesner, E. (1970) Die riechschwelle des seidenspinners. Naturwissenschaften, 57, 23–28. Kim, M.-S., Repp, A. & Smith, D.P. (1998a) LUSH odorant binding protein mediates chemosensory responses to alcohols in Drosophila melanogaster. Genetics, 150, 711–721. Kim, M.S., Repp, A. & Smith, D.P. (1998b) LUSH odorant-binding protein mediates chemosensory responses to alcohols in Drosophila melanogaster. Genetics, 150, 711–721. Kurtovic, A., Widmer, A. & Dickson, B.J. (2007) A single class of olfactory neurons mediates behavioural responses to a Drosophila sex pheromone. Nature, 446, 542–546. Larsson, M.C., Domingos, A.I., Jones, W.D. et al. (2004) Or83b encodes a broadly expressed odorant receptor essential for Drosophila olfaction. Neuron, 43, 703–714. Laughlin, J., Ha, T., Jones, D. & Smith, D. (2008) Activation of pheromone sensitive neurons is mediated by conformational activation of pheromone binding protein. Cell, 133, 1255–1265. Liu, W., Liang, X., Gong, J. et al. (2011) Social regulation of aggression by pheromonal activation of Or65a olfactory neurons in Drosophila. Nature Neuroscience, 14, 896–902. Ronderos, D. & Smith, D. (2010) Activation of the T1 neuronal circuit is necessary and sufficient to induce sexually dimorphic mating behavior in Drosophila melanogaster. Journal of Neuroscience, 30, 2595–2599. Sato, K., Pellegrino, M., Nakagawa, T. et al. (2008) Insect olfactory receptors are heteromeric ligand-gated ion channels. Nature, 452, 1002–1006. Shanbhag, S.R., Smith, D.P. & Steinbrecht, R.A. (2005) Three odorantbinding proteins are co-expressed in the sensilla trichodea of Drosophila melanogaster. Arthropod Structure and Development, 34, 153–165. Thorne, R.F., Mhaidat, N.M., Ralston, K.J. & Burns, G.F. (2007) CD36 is a receptor for oxidized high density lipoprotein: implications for the development of atherosclerosis. FEBS Letters, 581, 1227–1232. Vogt, R.G. & Riddiford, L.M. (1981) Pheromone binding and inactivation by moth antennae. Nature, 293, 161–163. Wang, L., Han, X., Mehren, J. et al. (2011) Hierarchical chemosensory regulation of male-male social interactions in Drosophila. Nature Neuroscience, 14, 757–762. Wicher, D., Schafer, R., Bauernfeind, R. et al. (2008) Drosophila odorant receptors are both ligand-gated and cyclic-nucleotideactivated cation channels. Nature, 452, 1007–1011. Xu, P., Atkinson, R., Jones, D.N. & Smith, D.P. (2005) Drosophila OBP LUSH is required for activity of pheromone-sensitive neurons. Neuron, 45, 193–200. Yao, C.A., Ignell, R. & Carlson, J.R. (2005) Chemosensory coding by neurons in the coeloconic sensilla of the Drosophila antenna. Journal of Neuroscience, 25, 8359–8367. Accepted 22 October 2011
© 2012 The Author Physiological Entomology © 2012 The Royal Entomological Society, Physiological Entomology, 37, 19–24
DOI: 10.1111/j.1365-3032.2011.00813.x
REVIEW ARTICLE
Volatile pheromone signalling in Drosophila DEAN P. SMITH University of Texas Southwestern Medical Center, Dallas, Texas, U.S.A.
Abstract. Once captured by the antenna, the male-specific pheromone 11-cis vaccenyl acetate (cVA) binds to an extracellular binding protein called LUSH that undergoes a conformational shift upon cVA binding. The stable LUSH–cVA complex is the activating ligand for pheromone receptors present on the dendrites of the aT1 neurones, comprising the only class of neurones that detect volatile cVA pheromone. This mechanism can explain the single molecule sensitivity of pheromone detection. The receptor that recognizes activated LUSH consists of a complex of several proteins, including Or67d, a member of the tuning odourant receptor family, Orco, a co-receptor ion channel, and SNMP, a CD36 homologue that may be an inhibitory subunit. In addition, genetic screens and reconstitution experiments reveal additional factors that are important for pheromone detection. Identification and functional dissection of these factors in Drosophila melanogaster Meigen should permit the identification of homologous factors in pathogenic insects and agricultural pests, which, in turn, may be viable candidates for novel classes of compounds to control populations of target insect species without impacting beneficial species. Key words. Behaviour, odorant binding protein, pheromone detection, signal transduction.
Introduction Insects use pheromones to trigger social behaviours in conspecifics. Pheromones are the dominant language for communication in insects, making the complex societies of social insects such as ants, bees and termites possible. However, pheromones are also used by most insect species to coordinate mating behaviour between appropriate individuals. Volatile pheromones act over a distance and are detected by olfactory neurones, whereas contact pheromones are detected by gustatory neurones upon direct contact with another individual. Volatile pheromone detection in many insects is exceptionally sensitive, approaching single molecule sensitivity (Kaissling & Priesner, 1970). In moths, sex pheromone sensitivity allows males to identify and locate females possibly over great distances, a feat that has intrigued naturalists for over 100 years (Fabre, 1916). Our research group at the University of Texas Southwestern Medical Center is interested in the initial steps of pheromone detection that account for this remarkable sensitivity: how the Correspondence: Dean P. Smith, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-9111, U.S.A. Tel.: +1 214 648 1650; e-mail: dean.smith@ utsouthwestern.edu © 2012 The Author Physiological Entomology © 2012 The Royal Entomological Society
presence of pheromone molecules are translated into electrical signals in primary olfactory neurones. The pheromone model system studied in the author’s laboratory is volatile cVA (11-cis vaccenyl acetate) pheromone signalling and induced behaviours in the genetically tractable insect Drosophila melanogaster Meigen. cVA is the major volatile pheromone in this species and is produced by males but is detected equally well by specialized olfactory neurones (T1 neurones) in both sexes. cVA induces sexually dimorphic behaviours, stimulating mating receptivity in females, but inhibiting courtship between males. The mechanisms underlying volatile pheromone detection are likely to be conserved among insects; therefore, the general principles and molecular components that are uncovered by studying Drosophila cVA detection are likely to be conserved in other insects.
Drosophila olfactory anatomy Volatile odourants (including pheromones) are detected by olfactory neurones located in the antenna (Fig. 1A). The dendrites of the olfactory neurones project into hair-like, fluid-filled structures called sensilla. The sensilla are classified into four groups based on morphology, although each 19
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D. P. Smith B
Fig. 1. The anatomy of the Drosophila olfactory system. (A) Scanning electron micrograph of a Drosophila head showing the antennae (A) and maxillary palps (M). (B) High power view of the surface of the antenna showing the four major classes of sensillae. B, basiconic; C, coeloconic; I, intermediate; T, trichoid. Reproduced with permission from Landesbioscience, Fly 3, 290–297.
morphological group also appears to have functional specialization. The basiconic sensilla contain the dendrites of olfactory neurones that express odourant receptors tuned to food odourants (Hallem et al., 2004; Hallem & Carlson, 2006). The coeloconic sensilla neurones are tuned to acids and aldehydes, and are detected by variant glutamate receptors (Benton et al., 2009). Trichoid sensilla contain the dendrites of pheromonesensitive olfactory neurones (Clyne et al., 1997; Xu et al., 2005). Intermediate sensilla are intermediate in morphology between basiconic and trichoid sensilla, and one intermediate neurone is tuned to a fruit rind odourant but mediates oviposition behaviour (D. Ronderos & D. Smith, unpublished data). Thus, these four anatomic sensilla types contain olfactory neurones with specialized functions. Olfactory neurone activity is monitored using single sensillum electrophysiology (SSR). Sharp glass or tungsten electrodes are inserted into individual sensilla and the signals from the neurones within that sensillum are passed through a highimpedance amplifier, and action potentials are then recorded in response to odourant application. Single sensillum recordings have been instrumental in characterizing the organization and function of the Drosophila olfactory system. This technique, combined with the molecular genetic tools available in this system, allows the detailed characterization of most of the odourant receptors (Elmore & Smith, 2001; Hallem et al., 2004; Yao et al., 2005; Hallem & Carlson, 2006; Benton et al., 2009). Once the odourant response profile is obtained for the various basiconic neurone classes, it is possible to correlate receptors with the odourant sensitivity of each olfactory neurone class. This is accomplished by mis-expressing specific odourant receptors in a defined basiconic olfactory neurone lacking the endogenous odourant receptor and by measuring the odourant response profile of the mis-expressed receptor (i.e. the empty neurone approach) (Hallem et al., 2004). When the odourant spectrum for each basiconic receptor is characterized, these correlate well with the responses of the various basiconic neurone classes, allowing specific receptors to be correlated with functional classes of neurones (Hallem et al., 2004). However, a subset of receptors fail to function in basiconic neurones, and most of these are normally expressed in the trichoid sensillum zone of the antenna. This raises the
possibility that additional signal transduction factors may be necessary in the pheromone-sensing neurones. Indeed, Or67d, the tuning odourant receptor expressed exclusively by the cVAsensitive T1 neurones (Ha & Smith, 2006; Kurtovic et al., 2007), does not respond to volatile cVA when expressed in the empty neurone system (Laughlin et al., 2008). Using genetic screens to identify cVA-insensitive mutants, a number of novel signalling factors required for pheromone detection by trichoid neurones have been uncovered that are not required for the detection of general odourants. To identify and characterize the components required for cVA signalling in Drosophila, a combination of genetic screens, mutagenesis, electrophysiology, structural studies and behavioural assays have been used. The first gene product uncovered that is required for cVA detection was the odourantbinding protein LUSH. lush was identified in an enhancer-trap genetic screen (Kim et al., 1998b). A transposable element carrying a gene for LacZ was mobilized randomly throughout the genome, and individual lines were established, each carrying a unique transposon inserted somewhere in the genome. LacZ expression is dependent on capturing local enhancers upon integration; thus, by screening the lines for LacZ expression restricted to the antennae, it was possible to identify genes expressed exclusively in the antennae. The gene lush was identified as being expressed exclusively in the trichoid sensillum zone of the antennae (Kim et al., 1998a). LUSH is a member of the Drosophila odourant-binding protein family, a large family of genes expressed in chemosensory organs (Galindo & Smith, 2001). Lepidopteran OBP family members include the pheromone-binding proteins known to be secreted into the extracellular lymph space bathing the olfactory neurone dendrites and binding directly to pheromones (Vogt & Riddiford, 1981). However, at that time, the role of these proteins in vivo was unknown. LacZ expression revealed that LUSH was expressed exclusively in the ventral–lateral surface of the antennae, where trichoid sensilla are localized, and anti-LUSH antiserum showed that this protein is secreted into the sensillum lymph of all trichoid sensilla by non-neuronal supporting cells (Kim et al., 1998b; Shanbhag et al., 2005). To study the potential effects that loss of the LUSH protein has on cVA detection, the transposon in the lush enhancer-trap stock was remobilized to generate a 2-kb genomic deletion that removed the entire coding sequence of the lush gene (Kim et al., 1998a). Indeed, when the pheromone responses of lush 1 mutants to cVA pheromone were assayed, it was found that these mutants are insensitive to cVA pheromone (Fig. 2) (Xu et al., 2005). A wild-type, transgenic copy of the lush gene restores cVA sensitivity to the mutants (Fig. 2) (Xu et al., 2005), confirming that the loss of LUSH protein accounts entirely for the cVA detection defect. The fact that LUSH is required for the activation of cVA-sensitive neurones eliminated the possibility that LUSH functions to remove cVA from the sensillum lymph because a loss of a pheromone remover would not be expected to block pheromone sensitivity. Furthermore, recombinant LUSH protein produced in bacteria and infused into the sensillum lymph through the recording pipette also restores cVA sensitivity to lush 1 mutants (Xu et al., 2005). This indicates that LUSH is directly required for cVA
© 2012 The Author Physiological Entomology © 2012 The Royal Entomological Society, Physiological Entomology, 37, 19–24
Volatile pheromone signalling
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Fig. 2. 11-cis Vaccenyl acetate (cVA) sensitivity in wild-type and lush 1 mutants. TOP, 1-s recording traces after a 300-ms pulse of air blown across paper impregnated with 1 μL of a 1 : 100 dilution of cVA. Wild-type shows a robust burst of action potentials after a latency that is largely a result of the time for the air pulse to reach the preparation. lush 1 mutants are insensitive to cVA, although a wild-type transgenic copy of lush (rescue) restores cVA sensitivity to the mutants. APO3, a pheromone-binding protein from the moth species Antherea polyphemus fails to confer cVA sensitivity to lush 1 mutants, demonstrating binding protein specificity. Bottom: dose–response curves for cVA in wild-type and lush 1 mutants. Reproduced with permission from Elsevier Inc, Neuron 45, 193–200.
signalling, and not for the development of the cVA detection mechanism. When analyzing the lush 1 mutants, a second, unexpected phenotype associated with loss of the extracellular LUSH protein was noted. The spontaneous activity of the pheromone-sensitive T1 neurones was reduced by more than 400-fold in the absence of pheromone. However, the loss of LUSH in other trichoid sensilla had no effect on the spontaneous activity of those neurones. This ruled out the possibility of a nonspecific osmotic change affecting T1 spontaneous activity upon the loss of abundant extracellular LUSH protein because this loss would be identical in other trichoid sensilla where LUSH is also normally expressed. Therefore, LUSH is not a simple pheromone carrier that transports hydrophobic cVA to the pheromone-sensitive T1 neurones through the sensillum lymph because the loss of a carrier protein would not be expected to reduce the spontaneous activity in these neurones in the absence of pheromone. Instead, LUSH alone appears
to have agonist activity, even when there is no pheromone present. This insight revealed that LUSH was more likely to function as a signalling factor than as a simple pheromone carrier. To account for the pheromone-independent agonist activity of LUSH, it was speculated that LUSH may undergo conformational activation upon binding pheromone, and that the active conformation may be the ligand for neuronal receptors. If a fraction of the LUSH molecules can spontaneously isomerize to the activated state at a low rate, this could account for the spontaneous activity in wild-type flies that is lacking in lush 1 mutants. If pheromone stabilizes or induces this activated LUSH state, this could account for the requirement of LUSH in cVA detection. This model makes two testable predictions. First, if LUSH undergoes a conformational activation upon cVA binding, it should be possible to observe differences in the X-ray crystal structure of LUSH with and without cVA pheromone bound.
© 2012 The Author Physiological Entomology © 2012 The Royal Entomological Society, Physiological Entomology, 37, 19–24
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D. P. Smith A
B
Fig. 3. Ribbon diagram of the relevant portions of LUSH with and without 11-cis vaccenyl acetate (cVA) bound. Overlaid structures with cVA present (purple) or absent (green). cVA, in magenta binds to LUSH ejecting F121 from the binding pocket. This, in turn, disrupts the salt bridge between D118 and K87 that normally stabilizes the inactive conformation of LUSH. This results in flipping of the Cterminus of LUSH, which is most apparent in the orientation of Q120. Reproduced with permission from Elsevier Inc, Cell 133, 1255–1265.
Second, if LUSH is the true ligand for the receptors expressed on pheromone-sensitive T1 neurones, it should be possible to generate dominant LUSH mutants that activate T1 neurones in the absence of cVA pheromone. To obtain insight into the possible structural effects of cVA binding on LUSH, we have collaborated with John Laughlin and David Jones of the University of Colorado Health Sciences Center in Denver (Laughlin et al., 2008). Recombinant LUSH protein lacking the signal sequence was expressed in bacteria and refolded slowly under conditions to allow proper disulfide bond formation and then purified. Protein crystals were grown in the presence or absence of cVA pheromone and analyzed. The first major finding from solving the crystal structure of LUSH with and without cVA bound was that cVA is completely enveloped by LUSH. This rules out the possibility that a unique pheromone/LUSH surface is recognized by neuronal receptors. Second, cVA-bound LUSH has a different conformation than apoLUSH. cVA binding to LUSH ejects the C-terminus of LUSH from the cVA binding pocket. Phenylalanine 121 appears to be the key residue contacting cVA in the binding pocket, leading to disruption of the salt bridge between aspartate 118 and lysine 87, with subsequent flipping of the orientation of the C-terminal loop of LUSH (Fig. 3). If this conformational shift in LUSH is important for the activation of T1 neuronal receptors, mutating phenylalanine 121 to alanine should impair the ability of cVA–LUSH complexes to activate T1 neurones because cVA will be less able to induce the conformational shift. Indeed, cVA binding is not impacted by the F121A mutation, although the F121A mutant
C
Fig. 4. LUSHD118A activates T1 neurones in the absence of 11-cis vaccenyl acetate (cVA). (A) Trace of T1 neuronal activation upon infusing recombinant LUSHD118A into the T1 sensillum lymph of lush 1 mutants through the recording pipette. (B) Comparison of infusing wild-type (wt) LUSH and LUSHD118A into the T1 sensillum lymph of lush 1 mutants. (C) Overlaid structures of LUSH with and without cVA bound together with the structure of apoLUSHD118A . apoLUSHD118A and cVA bound LUSH have almost identical conformational shifts (arrow). Reproduced with permission from Elsevier Inc, Cell 133, 1255–1265.
Fig. 5. Current model for 11-cis vaccenyl acetate (cVA) detection in Drosophila. Left, in the absence of cVA, LUSH is in the inactive state, and SNMP is bound to the Or67d/Orco complex, inhibiting ion fluxes. Right, in the presence of cVA, LUSH undergoes a conformational activation that allows it to interact with SNMP, which releases the Or67d/Orco complex, allowing ions to flow.
version of LUSH, when infused into the sensillum lymph of lush 1 mutants through the recording pipette, requires 50fold more cVA to achieve the same activation compared with infusing wild-type LUSH protein at the same concentration (Laughlin et al., 2008). By contrast, mutating phenyalanine 121 to the bulkier amino-acid tryptophan results in a LUSH protein that activates T1 neurones at cVA concentrations fivefold lower than wild-type LUSH. This finding comprises good
© 2012 The Author Physiological Entomology © 2012 The Royal Entomological Society, Physiological Entomology, 37, 19–24
Volatile pheromone signalling evidence that the LUSH conformational shift induced by cVA is important for activating T1 neurones. Finally, disrupting the D118/K87 salt bridge present in apoLUSH by mutating D118 to alanine (D118A) produced a dominant allele of LUSH. When LUSHD118A protein is infused into the sensillum lymph of lush 1 mutants, the T1 neurones activate without any cVA (Fig. 4A, B). When John Laughlin and David Jones solved the X-ray crystal structure of apoLUSHD118A , it was gratifying to see that the C-terminal loop showed the identical conformation shift as cVA-bound LUSH (Fig. 4C) (Laughlin et al., 2008). These data indicate that conformational changes in LUSH are sufficient to mediate activation of T1 neurones, and the role of cVA is to induce the activated conformation in the LUSH protein.
The neuronal LUSH receptor To identify the molecular components of the receptor expressed by T1 neurones that mediate cVA sensitivity, a genetic screen was undertaken for cVA-insensitive mutants. Three thousand homozygous flies with mutagenized third chromosomes were screened for defective cVA responses using SSR (Jin et al., 2008). Sixteen mutants were recovered that could be divided into several phenotypic classes based on the T1 SSRs. The class that turned out to be very interesting was insensitive to any concentration of cVA, although it still showed spontaneous activity in the T1 neurones. The presence of spontaneous activity in the T1 neurones ruled out nonspecific mutations affecting neuronal viability or the ability to fire action potentials. This class of mutants was named the vains mutants (cVA insensitive with spontaneous activity). Complementation analysis revealed four different genes mutated to produce the vains phenotype. Whole genome sequencing combined with deletion mapping experiments revealed the vains mutants corresponded to lesions in Orco, Or67d, snmp and a transcription factor gene that is not be discussed further here. Orco (Or co-receptor, formerly Or83b) (Larsson et al., 2004) is an ion channel that associates with ‘tuning’ odourant receptor subunits to produce odourant-gated ion channels (Sato et al., 2008; Wicher et al., 2008). Orco mutants were expected in the mutant screen because all Or proteins require this ion channel to function. Two mutant alleles of Orco were identified in the screen, both comprising strong mutations affecting splicing signals and resulting in frameshift mutations (Jin et al., 2008). Mutants in Or67d were also expected and recovered, given that this receptor had previously been identified as a T1-specific receptor conferring cVA sensitivity to other trichiod neurones that are normally insensitive to cVA (Ha & Smith, 2006). Or67d mutants were subsequently generated showing a loss of cVA sensitivity in T1 neurones (Kurtovic et al., 2007). Both Orco and Or67d mutants have infrequent spontaneous action potentials, similar to that observed in lush 1 mutants. The third vains gene, snmp, was unexpected, and encodes a homologue of human CD36, which is a scavanger receptor expressed on macrophages that is required to take up lipids and oxidized cholesterol in lipoprotein complexes leading to
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atherosclerosis (Coburn et al., 2000; Bonen et al., 2007; Guest et al., 2007; Thorne et al., 2007). Of note, snmp mutants have a T1 neurone spontaneous activity rate that is 20-fold higher than wild-type (Benton et al., 2007; Jin et al., 2008). Antibodies to SNMP infused into the sensillum lymph through the recording pipette produce a similar phenotype, indicating that SNMP is present on the surface of the dendrites (Jin et al., 2008). Interestingly, dominant LUSHD118A fails to activate T1 neurones from flies mutant for Or67d, Orco or snmp (Laughlin et al., 2008). Taken together, these data suggest that SNMP acts as an inhibitory subunit in the receptor complex, and all three of these components act downstream of LUSH. Benton et al. (2007) studied the role of SNMP in cVA sensitivity independently from our studies and suggested that SNMP may function to transfer cVA from LUSH to the neuronal receptor. It is worth noting, however, that dominant LUSH activates T1 neurones in the absence of cVA, although this is not the case if SNMP is missing (Laughlin et al., 2008). Therefore, SNMP must have a role in T1 neurone activation that is independent of cVA transfer. Figure 5 shows a current working model for cVA signal transduction. Finally, there are additional gene products important for cVA detection that remain to be described. There are several mutants recovered from a third chromosome screen that affect cVA sensitivity. We have just started screening the second and X chromosomes for genes important for cVA sensitivity located on other chromosomes. When all relevant cVA sensitivity factors have been identified, it is expected that the misexpression of all these factors in the empty basiconic neurone system will reconstitute T1 neuronal levels of cVA sensitivity (Laughlin et al., 2008).
The behavioural effects of T1 neuronal activation Several groups have studied the behavioural effects of cVA detection on fruit flies (Ejima et al., 2007; Kurtovic et al., 2007; Ronderos & Smith, 2010; Liu et al., 2011; Wang et al., 2011). There is general agreement that cVA mediates the recognition of sex between individuals in this species (Ejima et al., 2007; Kurtovic et al., 2007; Ronderos & Smith, 2010). cVA detection induces sexually dimorphic behaviours, inducing enhanced receptivity to courtship in females, and reducing courtship and social avoidance in males. This is consistent with cVA being present on the male cuticle, thus reducing inappropriate mating attempts between males and enhancing courtship in females. In a study using tetanus toxin expressed in subsets of olfactory neurones, it was suggested that cVA is detected both by Or67d-expressing T1 neurones and Or65a-expressing T2 neurones and that mating behaviour is mediated through the Or65a circuit (Ejima et al., 2007; Liu et al., 2011). However, the Or65a driver used to express tetanus toxin or other transgenes also expresses these genes in other neurones in the central nervous system (Ejima et al., 2007). This raises concerns about the effects on other circuits in the brain that might impact courtship behaviour. Evidence has been obtained showing that cVAmediated mating behaviour requires the Or67d-expressing T1
© 2012 The Author Physiological Entomology © 2012 The Royal Entomological Society, Physiological Entomology, 37, 19–24
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neurones (Kurtovic et al., 2007; Ronderos & Smith, 2010). Both groups report that Or67d mutants have reduced female receptivity and increased male–male courtship. Our own group has further evidence that cVA detection occurs exclusively though the T1 neurones (Ronderos & Smith, 2010). Transgenic flies expressing dominant LUSHD118A constitutively and specifically activate the T1 neurone circuit (Ronderos & Smith, 2010). The behaviour of these transgenic flies was exactly what would be predicted with male courtship inhibited toward females and female receptivity to courtship diminished (Ronderos & Smith, 2010). Both of these effects of LUSHD118A are eliminated when the downstream receptor Or67d is missing (Ronderos & Smith, 2010). Dominant LUSHD118A has no effect on Or65a neurones, nor any other class of trichoid or basiconic olfactory neurones, except T1 neurones (Laughlin et al., 2008). Therefore, T1 neurones appear to be necessary and sufficient for mediating all cVA mating behaviours. Elucidation of the gene products and mechanisms mediating volatile pheromone detection in Drosophila should help in the provision of approaches manipulating the behaviour of other insects with agricultural and pathological significance. The discovery of novel gene products required for pheromone detection in Drosophila using genetics screens is anticipated, as well as an understanding of their roles in pheromone signal transduction. Homologues in other species might prove to be useful targets for disrupting pheromone-mediated behaviours.
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