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Who is he and what is he to you? Recognition in Drosophila melanogaster
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Who is he and what is he to you? Recognition in Drosophila melanogaster Jean-Christophe Billeter1 and Joel D Levine2 The inability to discriminate friend from foe or the ‘one’ among many potential mates can have immediate life-threatening consequences or a long-term evolutionary impact. Successful social interactions depend on the ability to recognize and identify individuals within a social context. Once recognition occurs, a repertoire of behavioral responses becomes available and choices are made as interactions between individuals unfold. The vinegar fly, Drosophila melanogaster, displays a wide range of social activities and patterns of social interaction. If a male fly is unable to recognize other males or distinguish them from females, he may attempt to court both males and females alike, wasting energy and reducing his fitness. We review recent studies on the mechanisms of social recognition in this organism that pertain to both sides of an interaction: the generation of signals by one individual and the receiving and processing of these signals by others. Addresses 1 Center for Behavior and Neurosciences, University of Groningen, Groningen, The Netherlands 2 Department of Biology, University of Toronto at Mississauga, Mississauga, ON, Canada Corresponding author: Levine, Joel D ([email protected])
Current Opinion in Neurobiology 2013, 23:17–23 This review comes from a themed issue on Neurogenetics Edited by Ralph Greenspan and Christine Petit For a complete overview see the Issue and the Editorial Available online 23rd September 2012 0959-4388/$ – see front matter, # 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.conb.2012.08.009
A man we passed just tried to stare me down And when I looked at you You looked at the ground I don’t know who he is But I think that you do Dadgummit Who is he, and what is he to you? -Bill Withers ‘Who is he?’
Introduction Male vinegar flies fight one another and court females; they do not fight females and rarely court other males. Recognition of another’s sex determines an individual’s expectations about social interactions soon to follow. Wild-type males will fight females that have been www.sciencedirect.com
engineered to display male chemical signals [1]. When a female’s nervous system is masculinized she behaves like a male, and a male will court her but then attack her, showing that appropriate social interactions rely on honest identification signals [1]. For this review we focus on Drosophila melanogaster and ask: What does one fly know about another and how? We will discuss the generation of signals for recognition, and the receiving and processing of those signals. We highlight recent studies that examine sexual recognition and touch upon other features of recognition such as those observed when flies interact in groups.
Generating signals for social recognition Cuticular hydrocarbons
Flies produce several chemical signals used for social interactions. The most conspicuous of these are cuticular hydrocarbons (CH), which vary in their chemical formula, abundance, sex-specific expression and physical distribution [2–5] (Figure 1a). CH production requires abdominal cells called oenocytes, which can be genetically manipulated to alter the blend of hydrocarbons on the body surface [1,6], or ablated to eliminate the expression of CH [7] (Figure 1b). In D. melanogaster, females produce CH compounds called dienes (Figure 1A). These dienes are not found in closely related Drosophila species, suggesting that they act as species-specific and sex-specific signals [8]. Males from most sibling species do not normally court D. melanogaster females [9], but will court them vigorously if they lack CH [7,10]. Re-application of dienes on a D. melanogaster female devoid of her own CH completely blocks courtship by males from other species, and application of D. melanogaster dienes on females from sibling species blocks conspecific males from courting them [7]. The ability to produce dienes depends on an enzyme called DesaturaseF (DesatF) [11]. This enzyme is solely expressed in females, under the control of the sex-determination transcription factor Doublesex (Dsx) [12]. Species identity is encoded by differential expression of dienes, presumably caused by repeated loss of the Dsx binding site in the desatF promoter across species [12–14]. Therefore, dienes function as both a species and sex signal for the vinegar fly. The desat1 gene encodes a Desaturase, which is expressed in the oenocytes; it controls the synthesis of monoenes, a class of CH which includes 7-Tricosene Current Opinion in Neurobiology 2013, 23:17–23
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(a) Chromatograms representing cuticular hydrocarbons (CH) of single flies (either virgin female or virgin male) separated by retention time using gas chromatography. Each peak represents a specific hydrocarbon and the area under each peak (measured in millivolts: mV) is relative to the amount of the specific compound in a single fly. From 30–40 compounds can be analyzed using gas chromatography, but only the compounds discussed in this review are highlighted. cVA, cis-Vaccenyl Acetate (beige) is present only in males and not in virgin females. 7-T (blue), 7-Tricosene, is present in higher amounts in males than females. The dienes (pink), 7,11-Heptacosadiene (7,11-HD) and 7,1-Nonacosadiene (7,11-ND) are only found in females. The peak surmounted by an asterisk is a standard (hexacosane) spiked in each sample to control for equal loading. (b) Schematic representation of the distribution of different CH on male and female flies during courtship. cVA is made in the male internal reproductive tract and transferred to females via the ejaculate during mating. 7-T is made in the oenocytes (abdominal cells labeled green) and has limited volatility [5]. It may be sensed by females when coming in close contact or by wafting by the wing during courtship. Dienes, such as 7,11-HD, are made in the female oenocytes. The volatility of these compounds is unknown, but they appear to be sensed by contact through ppk receptors on the male forelegs and mouthparts (highlighted in pink) during the tapping and licking stage of courtship [54–57].
(7-T), the major male hydrocarbon (Figure 1a and b) [7,15–17,18]. Wild-type males court male and female desat1 mutants indiscriminately [19], suggesting that this gene controls the production of repellent signals for mate discrimination. Rhythmic expression of clock genes in the oenocytes controls desat1 expression, resulting in a differential expression of CH throughout the day [15,20]. Flies may deploy these signals to share timing information [21]. Clock gene expression in the brain and in the oenocytes also changes when a fly is in a group, concomitant with a change in CH [15,22]. The functional meaning of this social adjustment of clock gene expression is unclear, but it correlates with increased sexual activity in groups [15,23], as well as the ability of an individual to recognize other group members [22]. CH also signify the metabolic state and age of the fly, which affect attractiveness [24] through regulation of CH synthesis by the insulin pathway [25]. The CH are complemented by an additional pheromone called cis-Vaccenyl Acetate (cVA). cVA is made in the male reproductive tract and transferred to females during mating, reducing her attractiveness (Figure 1B) [2,7,26,27]. cVA regulates group size, acting both to increase aggregation by attracting other flies to a food Current Opinion in Neurobiology 2013, 23:17–23
source [28,29], and to increase dispersal by triggering male aggression [30]. However, cVA is not a recognition signal because its function depends on the social and physical environment [28,31,32]. For example, females counteract the negative effect of cVA on their attractiveness by producing dienes [7]. Courtship song
When a male courts a female he generates a ‘song’ by extending one wing and vibrating it (Figure 2). Song is thought to stimulate the female and playback of recorded song to females reduces the latency to mate [33]. The frequency pattern of the song consists of two recurrent components: pulse and sine song (see Figure 2). The time between bouts of pulse song is the InterPulse Interval (IPI). Both the IPI and the IPI cycle define a malespecific and species-specific signal [34–37]. The role of the song in intra-specific mate choice is less clear because there is little reported variation in song parameters between D. melanogaster males [38]. But females prefer males with longer pulse songs. Such songs are energy demanding and may act as an honest indicator of the male’s health [39]. The role of the sine song is more controversial. The sine song is a pure tone that lasts for approximately 1 s. It may not only function as an acoustic www.sciencedirect.com
Recognition in Drosophila Billeter and Levine 19
Figure 2 Pulse
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Current Opinion in Neurobiology
Production of sex-specific signals by males and reception by females. Males generate a courtship song by extending and vibrating one wing unilaterally [49]. The song is controlled by anatomically separated neuronal populations that may be joined in a circuit. This circuit includes a cluster of neurons called P1, located in the dorsal laeral protocerebrum, a region of the nervous system (highlighted in grey) located in the back of the head. P1 neurons (symbolized by orange dots) may command the activation of the song by sending a signal via pIP10 neurons (indicated by blue lines) to a neuronal cluster called vPR6 (green) in the ventral nerve chord (portion of the nervous system [grey] in the thorax) that may act as a central pattern generator that connects to vMS11 (red) motorneurons controlling wing muscles to generate the song [41]. The song consists of two components: sine song and pulse song, whose frequency and amplitude are schematized. The courtship song is detected by females through sound displacing the arista (blue), feather like structures on the second antennal segment. Females also appear to smell cVA produced by males via the Or67d odorant receptor located in the third antennal segment (pink) [66].
signal, but as a way to fan CH toward the female (discussed in [39]). Courtship song may serve both as a way to generate a species-specific acoustic signal and a way to transfer CH. A male-specific neuronal circuit controls the wing muscles and generates the song (Figure 2) [40,41]. The male-specific ability to generate this acoustic signal is controlled by the expression of sex-specific transcription factors called dsx and fruitless ( fru) [42]. Mutant analysis indicates that fru regulates production of the pulse song by shaping connectivity within the courtship song circuit [40,41,43]. The sine song also requires the function of dsx, which appears to controls the development of a male-specific set of neurons affecting the song circuitry [42,44]. Production of the species-specific IPI is affected by the period ( per) gene [34,45], but the mechanism underlying the contribution of per to song is not understood. It will be of great interest to understand how the fru-dependent and dsx-dependent genetic programs interact with per to control the generation of a speciesspecific signal. We note that the wing may play a role in the transmission of chemical, acoustic, and visual signals. Males use wings to generate the courtship song and potentially waft chemical signals, and females use wings to signal rejection [46]. Study of Drosophila signaling organs, such as the oenocytes and wings, generally show that these organs simultaneously generate multiple signals, offering a challenge to researchers trying to disentangle discrete information within these multimodal signals. www.sciencedirect.com
Detecting the signal Tasting pheromones
Flies touch one another when encountering each other [47] and when fighting or courting [46] (Figure 1b). While touching, taste and touch receptors, which are distributed throughout the fly [48], may come close enough to other flies to sense the CH they display [49,50]. Signaling by contact is efficient for sharing personal information because the signal is closely associated with the source. Taste signals are carried to the brain by sensory neurons in the legs and mouthparts. Dendrites from these neurons are housed in porous hair-like structures called sensilla, which are filled with lymph [48]. CH may enter the lymph through pores in the sensilla. There, they may bind to odorant binding proteins (OBP), which facilitate accessibility to their cognate receptors. CheB42, an OBP, is expressed in non-neuronal support cells of the leg sensilla [51,52]. Mutating the CheB42a gene alters both a male behavioral response to female-specific signals [51] and the physiological response of a sodium channel named pickpocket 25 ( ppk25) [52–54]. This suggests that CheB42 binds female CH and shuttles them to the ppk25 channel. Other members of the pickpocket ( ppk) family have been implicated as potential CH receptors [52–57]. Ppk23expressing neurons are functionally heterogeneous: one population responds to male CH (monoenes and cVA), and another population responds to female CH (dienes) [55]. Artificial activation of ppk23-expressing neurons triggers male–female courtship and inhibits male–male courtship [55,57], indicating that ppk23 neurons are Current Opinion in Neurobiology 2013, 23:17–23
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involved in the detection of both female and male pheromones. Four members of the gustatory receptor (Gr) family have also been associated with detection of CH. Loss of both Gr68a [58] and Gr39a [59] reduces male-to-female courtship, implying a role in the detection of female dienes. Loss of Gr32a [31,60] and Gr33a [61] results in enhanced male–male courtship, indicating that they detect malespecific inhibitory signals such as 7-T or cVA. One complication is that gustatory sensilla contain several neurons expressing multiple sensory receptors [62]. For example, the receptor neuron that expresses Gr32a also expresses Gr33a, rendering association between a specific signal and its receptor difficult. Because of the well-established difficulty in generating electrophysiological data on gustatory sensilla, it remains unclear whether either Ppk or Gr directly respond to CH or if they are acting downstream of unknown CH receptors. Smelling pheromones
Four Odorant receptors (Or) respond to fly extracts: Or67d, Or47b, Or88a, and Or65a [63]. Or47b and Or88a respond equally to male and female extracts, but the specific compound(s) activating them remains unknown. Or67d and Or65a respond to cVA [64–67]. cVA is bound by an OBP, called Lush, that is present in the lymph bathing the trichoid sensilla [68]. Lush and cVA form a complex that activates the Or67d receptor, indicating that CH association with an OBP is a general mechanism for receptor activation [69]. Or67d mutant males show increased courtship toward males and mated females and reduced aggression toward males [30,31,66]. Therefore, this receptor may mediate both the courtshipsuppressing and aggression-enhancing effects of cVA [31]. Sexual recognition clearly requires olfactory signals, but knowledge of the mechanisms of olfactory pheromonal signaling are currently restricted to cVA. Hearing the song
Auditory sensory neurons are either sensitive to vibrational stimuli, such as courtship song, or to continuous stimuli such as gravity and wind [70–72]. Auditory receptor neurons in both D. melanogaster and D. simulans females show equal sensitivity to both species’ songs, indicating that song recognition is not connected to tuning of the sensory neurons to a conspecific song [73]. Female D. melanogaster neurons receiving input from auditory receptor neurons undergo sustained depolarization in response to conspecific IPI, but not to D. simulans IPI. Altered tuning of these neurons may explain the ability to differentiate conspecific song from heterospecific song, and suggests that early stage auditory interneurons already contain sufficient information for making distinctions between species. Current Opinion in Neurobiology 2013, 23:17–23
Extracting the information Individuals may respond differently to the same signal depending on their sex, species, and social experience. These differences could be linked to limited perception of signals due to receptors being expressed or tuned differently in different individuals. For instance, some chemoreceptors are expressed in a greater number of sensory neurons in males than in females [55–57,74], or are only expressed in one sex [58]. Another potential mechanism for connecting perception of signal with sex and species identity is ‘emission-reception coupling’ [18]. Desat1 controls signals indicating sexual identity through its metabolic action on CH synthesis in the oenocytes. This enzyme is expressed in other tissues, where it is required for males to discriminate between males and females [7,18]. Desat1 thus controls both production and reception of sex-specific signals through its activity in organs producing signals and organs receiving the signal [18]. Behavioral responses, such as courtship, depend on the simultaneous perception and integration of multiple sensory modalities. For instance, artificial activation of ppk23+-taste neurons in males only triggers courtship when combined with olfactory and visual inputs [55–57]. Studies of the fru gene are shedding light on how multiple cues may be integrated to regulate behavioral responses. fru is expressed in about 2000 neurons belonging to sensory neurons, interneurons, and motorneuron populations [74–77,78,79]. One cluster of 20 fru+ interneurons, named P1 (Figure 2), is sufficient to trigger elements of courtship behavior [41,50,80]. P1 neurons are stimulated when a male touches a virgin female with his forelegs but becomes attenuated if the female is perfumed with cVA [50]. Therefore, malespecific P1 neurons are not only activated, but also modulated by female-associated stimuli. Or67d sensory neurons, which detect cVA, synapse onto a defined set of projection neurons at a glomerulus called DA1 [66]. DA1 neurons project close to where P1 neurons arborize [78,79]. DA1 neurons may thus connect with P1 neurons, transferring information about the presence and quantity of cVA in the environment. P1 neurons are located in a part of the brain that receives projection from different sensory modalities [79] and could function to integrate information from different signals. For instance, male aggression is regulated by the perception of both cVA and 7-T sensed by Or67d (olfactory) and Gr32a (gustatory) [31]. Gr32a-expressing leg sensory neurons project close to a sexually dimorphic population of fru-expressing neurons, which themselves extend to where P1 dendrites ramify [49]. By virtue of their anatomic locations in the lateral protocerebrum, P1 neurons are candidate integrators of multimodal input from a distributed set of sensory input. Importantly, P1 neurons are only present in males and therefore only receive input from sensory signals in males [80]. This sexual www.sciencedirect.com
Recognition in Drosophila Billeter and Levine 21
dimorphism may explain how reception of cVA by Or67d sensory neurons in a male or a female fly can elicit different behaviors.
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Conclusion Social recognition is based on the ability of an individual to classify and connect with others. Despite fast advances in this field, most features of recognition remain to be elucidated. What part does memory play in the recognition process? Studies on aggression in Drosophila show that flies recognize one another as individuals. Flies discriminate between winners and losers of previous fights, suggesting that memory plays a role when individuals identify one another based on past experience together [81]. How is the information from sexual signals extracted? Although signals for species and sex have been determined, the receptors that mediate these signals and the neural circuits that extract the information are not fully determined. How these cues are integrated to influence a behavioral response will be the subject of fascinating research. The identification of neural circuits that mediate recognition for fighting and mating will be key for understanding more complex group phenomena. For instance, some Drosophila females suppress the production of same-strain offspring after having mated with a same-strain male in the presence of a male from a different strain [23]. This indicates that females can recognize group composition when making reproductive decisions. We expect that it will soon be possible to identify inherited features of recognition, as well as neuronal circuits that facilitate patterns of interaction which emerge as flies co-operate and resolve conflicts in groups.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest
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51. Park SK, Mann KJ, Lin H, Starostina E, Kolski-Andreaco A, Pikielny CW: A Drosophila protein specific to pheromonesensing gustatory hairs delays males’ copulation attempts. Curr Biol 2006, 16:1154-1159. 52. Ben-Shahar Y, Lu B, Collier DM, Snyder PM, Schnizler M, Welsh MJ: The drosophila gene CheB42a is a novel modifier of Deg/ENaC channel function. PLoS ONE 2010, 5:e9395. 53. Lin H, Mann K, Kinser R, Starostina E, Pikielny C: A Drosophila DEG/ENaC channel subunit is required for male response to female pheromones. Proc Natl Acad Sci U S A 2005, 102:1283112836. 54. Starostina E, Liu T, Vijayan V, Zheng Z, Siwicki KK, Pikielny CW: A Drosophila DEG/ENaC subunit functions specifically in gustatory neurons required for male courtship behavior. J Neurosci 2012, 32:4665-4674. 55. Thistle R, Cameron P, Ghorayshi A, Dennison L, Scott K: Contact chemoreceptors mediate male–male repulsion and male– female attraction during Drosophila courtship. Cell 2012, 149:1140-1151. 56. Lu B, LaMora A, Sun Y, Welsh MJ, Ben-Shahar Y: ppk23dependent chemosensory functions contribute to courtship behavior in Drosophila melanogaster. PLoS Genet 2012, 8:e1002587. 57. Toda H, Zhao X, Dickson BJ: The Drosophila female aphrodisiac pheromone activates ppk23+ sensory neurons to elicit male courtship behavior. Cell Rep 2012 http://dx.doi.org/10.1016/ j.celrep.2012.05.007. 58. Bray S, Amrein H: A putative Drosophila pheromone receptor expressed in male-specific taste neurons is required for efficient courtship [Internet]. Neuron 2003, 39:1019-1029. www.sciencedirect.com
Recognition in Drosophila Billeter and Levine 23
59. Watanabe K, Toba G, Koganezawa M, Yamamoto D: Gr39a, a highly diversified gustatory receptor in Drosophila, has a role in sexual behavior. Behav Genet 2011, 41:746-753. 60. Miyamoto T, Amrein H: Suppression of male courtship by a Drosophila pheromone receptor. Nat Neurosci 2008, 11:874-876. 61. Jiao Y, Moon SJ, Montell C: A Drosophila gustatory receptor required for the responses to sucrose, glucose, and maltose identified by mRNA tagging. Proc Natl Acad Sci USA 2007, 104:14110-14115. 62. Montell C: A taste of the Drosophila gustatory receptors. Curr Opin Neurobiol 2009, 19:345-353. 63. der Goes van Naters van W, Carlson JR: Receptors and neurons for fly odors in Drosophila. Curr Biol 2007, 17:606-612. 64. Ha TS, Smith DP: A pheromone receptor mediates 11-cisvaccenyl acetate-induced responses in Drosophila. J Neurosci 2006, 26:8727-8733. 65. Ejima A, Smith BPC, Lucas C, der Goes van Naters van W, Miller CJ, Carlson JR, Levine JD, Griffith LC: Generalization of courtship learning in Drosophila is mediated by cis-vaccenyl acetate. Curr Biol 2007, 17:599-605. 66. Kurtovic A, Widmer A, Dickson BJ: A single class of olfactory neurons mediates behavioural responses to a Drosophila sex pheromone. Nature 2007, 446:542-546. 67. Ronderos DS, Smith DP: Activation of the T1 neuronal circuit is necessary and sufficient to induce sexually dimorphic mating behavior in Drosophila melanogaster. J Neurosci 2010, 30:2595-2599. 68. Xu P, Atkinson R, Jones DNM, Smith DP: Drosophila OBP LUSH is required for activity of pheromone-sensitive neurons. Neuron 2005, 45:193-200. 69. Laughlin JD, Ha TS, Jones DNM, Smith DP: Activation of pheromone-sensitive neurons is mediated by conformational activation of pheromone-binding protein. Cell 2008, 133:12551265. 70. Kamikouchi A, Shimada T, Ito K: Comprehensive classification of the auditory sensory projections in the brain of the fruit fly Drosophila melanogaster. J Comp Neurol 2006, 499:317-356. 71. Kamikouchi A, Inagaki HK, Effertz T, Hendrich O, Fiala A, Go¨pfert MC, Ito K: The neural basis of Drosophila gravitysensing and hearing. Nature 2009, 458:165-171. 72. Yorozu S, Wong A, Fischer BJ, Dankert H, Kernan MJ, Kamikouchi A, Ito K, Anderson DJ: Distinct sensory
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representations of wind and near-field sound in the Drosophila brain. Nature 2009, 458:201-205. 73. Tootoonian S, Coen P, Kawai R, Murthy M: Neural representations of courtship song in the Drosophila brain. J Neurosci 2012, 32:787-798. This study of neural coding reports intracellular recordings from secondorder neurons receiving input from auditory sensory neurons stimulated with courtship songs from different Drosophila species. These experiments suggest how acoustic signals may be coded before the species information it carries can be extracted by post-synaptic neurons. 74. Stockinger P, Kvitsiani D, Rotkopf S, Tiria´n L, Dickson BJ: Neural circuitry that governs Drosophila male courtship behavior. Cell 2005, 121:795-807. 75. Billeter J-C, Goodwin SF: Characterization of Drosophila fruitless-gal4 transgenes reveals expression in male-specific fruitless neurons and innervation of male reproductive structures. J Comp Neurol 2004, 475:270-287. 76. Lee G, Foss M, Goodwin S, Carlo T, Taylor B, Hall J: Spatial, temporal, and sexually dimorphic expression patterns of the fruitless gene in the Drosophila central nervous system. J Neurobiol 2000, 43:404-426. 77. Manoli DS, Foss M, Villella A, Taylor BJ, Hall JC, Baker BS: Malespecific fruitless specifies the neural substrates of Drosophila courtship behaviour. Nature 2005, 436:395-400. 78. Cachero S, Ostrovsky AD, Yu JY, Dickson BJ, Jefferis GSXE: Sexual dimorphism in the fly brain. Curr Biol 2010, 20:1589-1601. 79. Yu JY, Kanai MI, Demir E, Jefferis GSXE, Dickson BJ: Cellular organization of the neural circuit that drives Drosophila courtship behavior. Curr Biol 2010, 20:1602-1614. These two papers [78,79] in the same issue of Current Biology highlight the importance of neuroanatomy as a prerequisite for the analysis of circuits underlying social behaviors. They show the extent of sexual dimorphism in the Drosophila brain and indicate that certain brain areas receive input from several sensory modalities at once. Although they did not establish synaptic connectivity, these studies establish a framework in which to begin the systematic dissection of the circuitry underlying courtship behavior. 80. Kimura K-I, Hachiya T, Koganezawa M, Tazawa T, Yamamoto D: Fruitless and doublesex coordinate to generate male-specific neurons that can initiate courtship. Neuron 2008, 59:759-769. 81. Yurkovic A, Wang O, Basu AC, Kravitz EA: Learning and memory associated with aggression in Drosophila melanogaster. Proc Natl Acad Sci U S A 2006, 103:17519-17524.
Current Opinion in Neurobiology 2013, 23:17–23
Who is he and what is he to you? Recognition in Drosophila melanogaster Jean-Christophe Billeter1 and Joel D Levine2 The inability to discriminate friend from foe or the ‘one’ among many potential mates can have immediate life-threatening consequences or a long-term evolutionary impact. Successful social interactions depend on the ability to recognize and identify individuals within a social context. Once recognition occurs, a repertoire of behavioral responses becomes available and choices are made as interactions between individuals unfold. The vinegar fly, Drosophila melanogaster, displays a wide range of social activities and patterns of social interaction. If a male fly is unable to recognize other males or distinguish them from females, he may attempt to court both males and females alike, wasting energy and reducing his fitness. We review recent studies on the mechanisms of social recognition in this organism that pertain to both sides of an interaction: the generation of signals by one individual and the receiving and processing of these signals by others. Addresses 1 Center for Behavior and Neurosciences, University of Groningen, Groningen, The Netherlands 2 Department of Biology, University of Toronto at Mississauga, Mississauga, ON, Canada Corresponding author: Levine, Joel D ([email protected])
Current Opinion in Neurobiology 2013, 23:17–23 This review comes from a themed issue on Neurogenetics Edited by Ralph Greenspan and Christine Petit For a complete overview see the Issue and the Editorial Available online 23rd September 2012 0959-4388/$ – see front matter, # 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.conb.2012.08.009
A man we passed just tried to stare me down And when I looked at you You looked at the ground I don’t know who he is But I think that you do Dadgummit Who is he, and what is he to you? -Bill Withers ‘Who is he?’
Introduction Male vinegar flies fight one another and court females; they do not fight females and rarely court other males. Recognition of another’s sex determines an individual’s expectations about social interactions soon to follow. Wild-type males will fight females that have been www.sciencedirect.com
engineered to display male chemical signals [1]. When a female’s nervous system is masculinized she behaves like a male, and a male will court her but then attack her, showing that appropriate social interactions rely on honest identification signals [1]. For this review we focus on Drosophila melanogaster and ask: What does one fly know about another and how? We will discuss the generation of signals for recognition, and the receiving and processing of those signals. We highlight recent studies that examine sexual recognition and touch upon other features of recognition such as those observed when flies interact in groups.
Generating signals for social recognition Cuticular hydrocarbons
Flies produce several chemical signals used for social interactions. The most conspicuous of these are cuticular hydrocarbons (CH), which vary in their chemical formula, abundance, sex-specific expression and physical distribution [2–5] (Figure 1a). CH production requires abdominal cells called oenocytes, which can be genetically manipulated to alter the blend of hydrocarbons on the body surface [1,6], or ablated to eliminate the expression of CH [7] (Figure 1b). In D. melanogaster, females produce CH compounds called dienes (Figure 1A). These dienes are not found in closely related Drosophila species, suggesting that they act as species-specific and sex-specific signals [8]. Males from most sibling species do not normally court D. melanogaster females [9], but will court them vigorously if they lack CH [7,10]. Re-application of dienes on a D. melanogaster female devoid of her own CH completely blocks courtship by males from other species, and application of D. melanogaster dienes on females from sibling species blocks conspecific males from courting them [7]. The ability to produce dienes depends on an enzyme called DesaturaseF (DesatF) [11]. This enzyme is solely expressed in females, under the control of the sex-determination transcription factor Doublesex (Dsx) [12]. Species identity is encoded by differential expression of dienes, presumably caused by repeated loss of the Dsx binding site in the desatF promoter across species [12–14]. Therefore, dienes function as both a species and sex signal for the vinegar fly. The desat1 gene encodes a Desaturase, which is expressed in the oenocytes; it controls the synthesis of monoenes, a class of CH which includes 7-Tricosene Current Opinion in Neurobiology 2013, 23:17–23
18 Neurogenetics
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(a) Chromatograms representing cuticular hydrocarbons (CH) of single flies (either virgin female or virgin male) separated by retention time using gas chromatography. Each peak represents a specific hydrocarbon and the area under each peak (measured in millivolts: mV) is relative to the amount of the specific compound in a single fly. From 30–40 compounds can be analyzed using gas chromatography, but only the compounds discussed in this review are highlighted. cVA, cis-Vaccenyl Acetate (beige) is present only in males and not in virgin females. 7-T (blue), 7-Tricosene, is present in higher amounts in males than females. The dienes (pink), 7,11-Heptacosadiene (7,11-HD) and 7,1-Nonacosadiene (7,11-ND) are only found in females. The peak surmounted by an asterisk is a standard (hexacosane) spiked in each sample to control for equal loading. (b) Schematic representation of the distribution of different CH on male and female flies during courtship. cVA is made in the male internal reproductive tract and transferred to females via the ejaculate during mating. 7-T is made in the oenocytes (abdominal cells labeled green) and has limited volatility [5]. It may be sensed by females when coming in close contact or by wafting by the wing during courtship. Dienes, such as 7,11-HD, are made in the female oenocytes. The volatility of these compounds is unknown, but they appear to be sensed by contact through ppk receptors on the male forelegs and mouthparts (highlighted in pink) during the tapping and licking stage of courtship [54–57].
(7-T), the major male hydrocarbon (Figure 1a and b) [7,15–17,18]. Wild-type males court male and female desat1 mutants indiscriminately [19], suggesting that this gene controls the production of repellent signals for mate discrimination. Rhythmic expression of clock genes in the oenocytes controls desat1 expression, resulting in a differential expression of CH throughout the day [15,20]. Flies may deploy these signals to share timing information [21]. Clock gene expression in the brain and in the oenocytes also changes when a fly is in a group, concomitant with a change in CH [15,22]. The functional meaning of this social adjustment of clock gene expression is unclear, but it correlates with increased sexual activity in groups [15,23], as well as the ability of an individual to recognize other group members [22]. CH also signify the metabolic state and age of the fly, which affect attractiveness [24] through regulation of CH synthesis by the insulin pathway [25]. The CH are complemented by an additional pheromone called cis-Vaccenyl Acetate (cVA). cVA is made in the male reproductive tract and transferred to females during mating, reducing her attractiveness (Figure 1B) [2,7,26,27]. cVA regulates group size, acting both to increase aggregation by attracting other flies to a food Current Opinion in Neurobiology 2013, 23:17–23
source [28,29], and to increase dispersal by triggering male aggression [30]. However, cVA is not a recognition signal because its function depends on the social and physical environment [28,31,32]. For example, females counteract the negative effect of cVA on their attractiveness by producing dienes [7]. Courtship song
When a male courts a female he generates a ‘song’ by extending one wing and vibrating it (Figure 2). Song is thought to stimulate the female and playback of recorded song to females reduces the latency to mate [33]. The frequency pattern of the song consists of two recurrent components: pulse and sine song (see Figure 2). The time between bouts of pulse song is the InterPulse Interval (IPI). Both the IPI and the IPI cycle define a malespecific and species-specific signal [34–37]. The role of the song in intra-specific mate choice is less clear because there is little reported variation in song parameters between D. melanogaster males [38]. But females prefer males with longer pulse songs. Such songs are energy demanding and may act as an honest indicator of the male’s health [39]. The role of the sine song is more controversial. The sine song is a pure tone that lasts for approximately 1 s. It may not only function as an acoustic www.sciencedirect.com
Recognition in Drosophila Billeter and Levine 19
Figure 2 Pulse
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Current Opinion in Neurobiology
Production of sex-specific signals by males and reception by females. Males generate a courtship song by extending and vibrating one wing unilaterally [49]. The song is controlled by anatomically separated neuronal populations that may be joined in a circuit. This circuit includes a cluster of neurons called P1, located in the dorsal laeral protocerebrum, a region of the nervous system (highlighted in grey) located in the back of the head. P1 neurons (symbolized by orange dots) may command the activation of the song by sending a signal via pIP10 neurons (indicated by blue lines) to a neuronal cluster called vPR6 (green) in the ventral nerve chord (portion of the nervous system [grey] in the thorax) that may act as a central pattern generator that connects to vMS11 (red) motorneurons controlling wing muscles to generate the song [41]. The song consists of two components: sine song and pulse song, whose frequency and amplitude are schematized. The courtship song is detected by females through sound displacing the arista (blue), feather like structures on the second antennal segment. Females also appear to smell cVA produced by males via the Or67d odorant receptor located in the third antennal segment (pink) [66].
signal, but as a way to fan CH toward the female (discussed in [39]). Courtship song may serve both as a way to generate a species-specific acoustic signal and a way to transfer CH. A male-specific neuronal circuit controls the wing muscles and generates the song (Figure 2) [40,41]. The male-specific ability to generate this acoustic signal is controlled by the expression of sex-specific transcription factors called dsx and fruitless ( fru) [42]. Mutant analysis indicates that fru regulates production of the pulse song by shaping connectivity within the courtship song circuit [40,41,43]. The sine song also requires the function of dsx, which appears to controls the development of a male-specific set of neurons affecting the song circuitry [42,44]. Production of the species-specific IPI is affected by the period ( per) gene [34,45], but the mechanism underlying the contribution of per to song is not understood. It will be of great interest to understand how the fru-dependent and dsx-dependent genetic programs interact with per to control the generation of a speciesspecific signal. We note that the wing may play a role in the transmission of chemical, acoustic, and visual signals. Males use wings to generate the courtship song and potentially waft chemical signals, and females use wings to signal rejection [46]. Study of Drosophila signaling organs, such as the oenocytes and wings, generally show that these organs simultaneously generate multiple signals, offering a challenge to researchers trying to disentangle discrete information within these multimodal signals. www.sciencedirect.com
Detecting the signal Tasting pheromones
Flies touch one another when encountering each other [47] and when fighting or courting [46] (Figure 1b). While touching, taste and touch receptors, which are distributed throughout the fly [48], may come close enough to other flies to sense the CH they display [49,50]. Signaling by contact is efficient for sharing personal information because the signal is closely associated with the source. Taste signals are carried to the brain by sensory neurons in the legs and mouthparts. Dendrites from these neurons are housed in porous hair-like structures called sensilla, which are filled with lymph [48]. CH may enter the lymph through pores in the sensilla. There, they may bind to odorant binding proteins (OBP), which facilitate accessibility to their cognate receptors. CheB42, an OBP, is expressed in non-neuronal support cells of the leg sensilla [51,52]. Mutating the CheB42a gene alters both a male behavioral response to female-specific signals [51] and the physiological response of a sodium channel named pickpocket 25 ( ppk25) [52–54]. This suggests that CheB42 binds female CH and shuttles them to the ppk25 channel. Other members of the pickpocket ( ppk) family have been implicated as potential CH receptors [52–57]. Ppk23expressing neurons are functionally heterogeneous: one population responds to male CH (monoenes and cVA), and another population responds to female CH (dienes) [55]. Artificial activation of ppk23-expressing neurons triggers male–female courtship and inhibits male–male courtship [55,57], indicating that ppk23 neurons are Current Opinion in Neurobiology 2013, 23:17–23
20 Neurogenetics
involved in the detection of both female and male pheromones. Four members of the gustatory receptor (Gr) family have also been associated with detection of CH. Loss of both Gr68a [58] and Gr39a [59] reduces male-to-female courtship, implying a role in the detection of female dienes. Loss of Gr32a [31,60] and Gr33a [61] results in enhanced male–male courtship, indicating that they detect malespecific inhibitory signals such as 7-T or cVA. One complication is that gustatory sensilla contain several neurons expressing multiple sensory receptors [62]. For example, the receptor neuron that expresses Gr32a also expresses Gr33a, rendering association between a specific signal and its receptor difficult. Because of the well-established difficulty in generating electrophysiological data on gustatory sensilla, it remains unclear whether either Ppk or Gr directly respond to CH or if they are acting downstream of unknown CH receptors. Smelling pheromones
Four Odorant receptors (Or) respond to fly extracts: Or67d, Or47b, Or88a, and Or65a [63]. Or47b and Or88a respond equally to male and female extracts, but the specific compound(s) activating them remains unknown. Or67d and Or65a respond to cVA [64–67]. cVA is bound by an OBP, called Lush, that is present in the lymph bathing the trichoid sensilla [68]. Lush and cVA form a complex that activates the Or67d receptor, indicating that CH association with an OBP is a general mechanism for receptor activation [69]. Or67d mutant males show increased courtship toward males and mated females and reduced aggression toward males [30,31,66]. Therefore, this receptor may mediate both the courtshipsuppressing and aggression-enhancing effects of cVA [31]. Sexual recognition clearly requires olfactory signals, but knowledge of the mechanisms of olfactory pheromonal signaling are currently restricted to cVA. Hearing the song
Auditory sensory neurons are either sensitive to vibrational stimuli, such as courtship song, or to continuous stimuli such as gravity and wind [70–72]. Auditory receptor neurons in both D. melanogaster and D. simulans females show equal sensitivity to both species’ songs, indicating that song recognition is not connected to tuning of the sensory neurons to a conspecific song [73]. Female D. melanogaster neurons receiving input from auditory receptor neurons undergo sustained depolarization in response to conspecific IPI, but not to D. simulans IPI. Altered tuning of these neurons may explain the ability to differentiate conspecific song from heterospecific song, and suggests that early stage auditory interneurons already contain sufficient information for making distinctions between species. Current Opinion in Neurobiology 2013, 23:17–23
Extracting the information Individuals may respond differently to the same signal depending on their sex, species, and social experience. These differences could be linked to limited perception of signals due to receptors being expressed or tuned differently in different individuals. For instance, some chemoreceptors are expressed in a greater number of sensory neurons in males than in females [55–57,74], or are only expressed in one sex [58]. Another potential mechanism for connecting perception of signal with sex and species identity is ‘emission-reception coupling’ [18]. Desat1 controls signals indicating sexual identity through its metabolic action on CH synthesis in the oenocytes. This enzyme is expressed in other tissues, where it is required for males to discriminate between males and females [7,18]. Desat1 thus controls both production and reception of sex-specific signals through its activity in organs producing signals and organs receiving the signal [18]. Behavioral responses, such as courtship, depend on the simultaneous perception and integration of multiple sensory modalities. For instance, artificial activation of ppk23+-taste neurons in males only triggers courtship when combined with olfactory and visual inputs [55–57]. Studies of the fru gene are shedding light on how multiple cues may be integrated to regulate behavioral responses. fru is expressed in about 2000 neurons belonging to sensory neurons, interneurons, and motorneuron populations [74–77,78,79]. One cluster of 20 fru+ interneurons, named P1 (Figure 2), is sufficient to trigger elements of courtship behavior [41,50,80]. P1 neurons are stimulated when a male touches a virgin female with his forelegs but becomes attenuated if the female is perfumed with cVA [50]. Therefore, malespecific P1 neurons are not only activated, but also modulated by female-associated stimuli. Or67d sensory neurons, which detect cVA, synapse onto a defined set of projection neurons at a glomerulus called DA1 [66]. DA1 neurons project close to where P1 neurons arborize [78,79]. DA1 neurons may thus connect with P1 neurons, transferring information about the presence and quantity of cVA in the environment. P1 neurons are located in a part of the brain that receives projection from different sensory modalities [79] and could function to integrate information from different signals. For instance, male aggression is regulated by the perception of both cVA and 7-T sensed by Or67d (olfactory) and Gr32a (gustatory) [31]. Gr32a-expressing leg sensory neurons project close to a sexually dimorphic population of fru-expressing neurons, which themselves extend to where P1 dendrites ramify [49]. By virtue of their anatomic locations in the lateral protocerebrum, P1 neurons are candidate integrators of multimodal input from a distributed set of sensory input. Importantly, P1 neurons are only present in males and therefore only receive input from sensory signals in males [80]. This sexual www.sciencedirect.com
Recognition in Drosophila Billeter and Levine 21
dimorphism may explain how reception of cVA by Or67d sensory neurons in a male or a female fly can elicit different behaviors.
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Conclusion Social recognition is based on the ability of an individual to classify and connect with others. Despite fast advances in this field, most features of recognition remain to be elucidated. What part does memory play in the recognition process? Studies on aggression in Drosophila show that flies recognize one another as individuals. Flies discriminate between winners and losers of previous fights, suggesting that memory plays a role when individuals identify one another based on past experience together [81]. How is the information from sexual signals extracted? Although signals for species and sex have been determined, the receptors that mediate these signals and the neural circuits that extract the information are not fully determined. How these cues are integrated to influence a behavioral response will be the subject of fascinating research. The identification of neural circuits that mediate recognition for fighting and mating will be key for understanding more complex group phenomena. For instance, some Drosophila females suppress the production of same-strain offspring after having mated with a same-strain male in the presence of a male from a different strain [23]. This indicates that females can recognize group composition when making reproductive decisions. We expect that it will soon be possible to identify inherited features of recognition, as well as neuronal circuits that facilitate patterns of interaction which emerge as flies co-operate and resolve conflicts in groups.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest
Ferna´ndez M, de LP, Chan Y-B, Yew JY, Billeter J-C, Dreisewerd K, Levine JD, Kravitz EA: Pheromonal and behavioral cues trigger male-to-female aggression in Drosophila. PLoS Biol 2010, 8:e1000541. This study shows that courtship and aggression are closely related male behaviors. Whether one or the other is performed depends on the recognition of both pheromonal and behavioral cues displayed by social partners. When those cues present conflicting information, such as when an individual has male pheromones but female behaviors, males have trouble resolving these paradoxical inputs. We note with humor that this is reminiscent of reaction formation in the Freudian sense.
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