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Substrate-borne vibratory communication during courtship in Drosophila melanogaster.
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PMID:  23103187     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Courtship in Drosophila melanogaster has become an iconic example of an innate and interactive series of behaviors. The female signals her acceptance of copulation by becoming immobile in response to a male's display of stereotyped actions. The male and female communicate via vision, air-borne sounds, and pheromones, but what triggers the female's immobility is undetermined. Here, we describe an overlooked and important component of Drosophila courtship. Video recordings and laser vibrometry show that the male abdomen shakes ("quivers"), generating substrate-borne vibrations at about six pulses per second. We present evidence that the female becomes receptive and stops walking because she senses these vibrations, rather than as a response to air-borne songs produced by the male fluttering the wings. We also present evidence that the neural circuits expressing the sex-determination genes fruitless and doublesex drive quivering behavior. These abdominal quivers and associated vibrations, as well as their effect on female receptivity, are conserved in other Drosophila species. Substrate-borne vibrations are an ancient form of communication that is widespread in animals. Our findings in Drosophila open a door to study the neuromuscular circuitry responsible for these signals and the sensory systems needed for their reception.
Authors:
Caroline C G Fabre; Berthold Hedwig; Graham Conduit; Peter A Lawrence; Stephen F Goodwin; José Casal
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2012-10-25
Journal Detail:
Title:  Current biology : CB     Volume:  22     ISSN:  1879-0445     ISO Abbreviation:  Curr. Biol.     Publication Date:  2012 Nov 
Date Detail:
Created Date:  2012-11-23     Completed Date:  2013-05-10     Revised Date:  2014-10-22    
Medline Journal Info:
Nlm Unique ID:  9107782     Medline TA:  Curr Biol     Country:  England    
Other Details:
Languages:  eng     Pagination:  2180-5     Citation Subset:  IM    
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Copyright © 2012 Elsevier Ltd. All rights reserved.
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MeSH Terms
Descriptor/Qualifier:
Animal Communication*
Animals
Drosophila melanogaster / physiology*
Female
Male
Sexual Behavior, Animal / physiology*
Vibration
Grant Support
ID/Acronym/Agency:
096645//Wellcome Trust; RG54425//Biotechnology and Biological Sciences Research Council; WT082987//Wellcome Trust; WT085521//Wellcome Trust; WT086986MA//Wellcome Trust
Comments/Corrections
Comment In:
Curr Biol. 2014 Sep 8;24(17):R812-4   [PMID:  25202876 ]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

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Journal Information
Journal ID (nlm-ta): Curr Biol
Journal ID (iso-abbrev): Curr. Biol
ISSN: 0960-9822
ISSN: 1879-0445
Publisher: Cell Press
Article Information
© 2012 ELL & Excerpta Medica.
License:
Received Day: 6 Month: 8 Year: 2012
Revision Received Day: 7 Month: 9 Year: 2012
Accepted Day: 25 Month: 9 Year: 2012
pmc-release publication date: Day: 20 Month: 11 Year: 2012
Print publication date: Day: 20 Month: 11 Year: 2012
Volume: 22 Issue: 22
First Page: 2180 Last Page: 2185
PubMed Id: 23103187
ID: 3502867
Publisher Id: CURBIO9891
DOI: 10.1016/j.cub.2012.09.042

Substrate-Borne Vibratory Communication during Courtship in Drosophila melanogaster
Caroline C.G. Fabre14∗ Email: c.c.g.fabre.03@cantab.net
Berthold Hedwig1
Graham Conduit2
Peter A. Lawrence1
Stephen F. Goodwin3
José Casal1
1Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 2EJ, UK
2King's College, 21 King's Parade, Cambridge CB2 1ST, UK
3Department of Physiology, Anatomy and Genetics, University of Oxford, Sherrington Building, Parks Road, Oxford OX1 3PT, UK
Corresponding author c.c.g.fabre.03@cantab.net
4Present address: Department of Physiology, Anatomy and Genetics, University of Oxford, Sherrington Building, Parks Road, Oxford OX1 3PT, UK

Results and Discussion
Characteristics of Male Quivering during Courtship

Pairs of flies were placed in a chamber and filmed at 30–150 frames per second. The behaviors of both the male and female were annotated and analyzed from initiation of courtship until copulation. In addition to well-known courtship behaviors, we observed frequent bouts of abdominal movements in the male that we refer to as “quivering” (Movie S1 available online). Quivering consists of up-and-down movements of the abdomen (Movie S2) with a frequency of 6.64 ± 0.78 beats per second (n = 12 bouts/12 flies). Quivering is a behavior specific to male courtship: we find that females and isolated males do not quiver, nor do males placed with other males (data not shown).

We quantified 30 ethograms of completed courtships (Experimental Procedures) in wild-type Oregon-R flies (Figures 1 and S1) for courtship behaviors including wing fluttering alone (about one-third of total courtship time), abdominal quivering alone (one-seventh of total courtship time), and simultaneous wing fluttering and abdominal quivering (one-seventh of total courtship time). We also recorded whether females were moving or stationary (Figure 1); they were immobile for one-third of the total courtship time (Figure S1B).

The most interesting results come from when the two behaviors of the males are compared relative to the simultaneous behavior of the female (Figure 1). Male quivering occurs 69% of the time that females are stationary but only 10% of the time that females are moving (Figure 1). In contrast, males flutter their wings about as often, independently of whether females are stationary or moving (53% and 42%, respectively; Figure 1). Although males flutter their wings for approximately half of the time that the females are stationary (53%), we note that males are also quivering for two-thirds of this 53%. However, when males flutter during female movement (42%), the males quiver for only one-seventh of this 42% (Figure 1). Thus, the data show that female immobility can coincide with male fluttering but mainly occurs when he is also quivering. Statistical analysis of the data shows that male quivering behavior (quivering or not) and female behavior (stationary or moving) are strongly associated, whereas the comparable association between male fluttering and female behavior also exists but is weaker (Figure 1 and S1C). Similar results were found for male and female pairs of another wild-type strain, Canton S (Figures S1E–S1G).

Our results do not support the general perception that signals generated by male wing fluttering act alone to diminish movement of females [7]. They show instead that quivering of the abdomen coincided with female immobility much more than wing fluttering did (Figure 1 and Movie S1). Also, and consistent with this finding, bouts of quivering vary in duration and depend on whether the female is moving (average duration only 1 s; Figure S1B) or stationary (average duration about 3 s; Figure S1B). In contrast, bouts of fluttering were longer when the female was moving than when she was immobile (Figure S1B).

If the wings of the wild-type male were amputated, males quivered more frequently than the wild-type (t test, p = 0.011) and for longer periods (p = 0.029); females placed with these males stopped moving more than when paired with intact males (p = 0.017) (Figures S2A–S2C). We also used males carrying mutations in the sex-determination genes doublesex (dsx) and fruitless (fru) because neurons expressing these genes drive male-specific behavior [5, 13, 14]. These mutations had no clear effects on the percentage of the courtship time that males fluttered their wings (dsx p = 0.45 and fru p = 0.24), although the pattern of fluttering was different from the wild-type (data not shown). However, dsx (Figures S2D–S2F) and fru (Figures S2G–S2I) mutant males quivered less than normal males (dsx p = 9.27 × 10−5 and fru p = 2.17 × 10−9). Importantly, we observed that their wild-type female partners stopped less than when courted by wild-type males (dsx p = 0.026 and fru p = 0.006) (Figures S2D–S2I, compare with Figure 1). Thus, if the male quivering is increased or decreased by intervention, the wild-type females stop more or less often, respectively (Figures S2A–S2I). These findings argue (but do not prove) that female stopping is a response to quivering and not a cause of quivering.

A mild activation of dsx-expressing neurons or fru-expressing neurons by forcing expression of Drosophila TRPA1 (Movie S3) triggered quivering in solitary males—as well as a mélange of other courtship behaviors (Movie S3) [15]. When stronger conditions were used to activate the fru-expressing neurons, quivering was induced also in females, arguing that appropriate neurons and circuitry are present but latent in the female. It follows that some of these neurons direct the abdominal quivering of the male during courtship and that the neuronal circuitry differs between normal males and females.

How Might Females Sense Male Quivering?

Our observations suggest that quivering of the male abdomen is sensed by the female and causes her to stop walking. We therefore asked how the tremor of the male abdomen might be transmitted to the female. One possibility is that the female could see quivering—vision is known to be used during courtship [7, 9]. To investigate, we performed courtship assays in the “dark” using infrared light that flies cannot detect [16]. Males quivered normally and again there was a strong coincidence between quivering and female immobility, suggesting that vision is not an important component (Figures S2J–S2L).

Next, we asked whether quivering might be associated with release of male-specific pheromones via the cuticle. In Drosophila, pheromones are low-volatility hydrocarbons and are produced by the abdominal oenocytes of both male and female [7]. By using RNA interference, we reduced the expression of the sex-determination gene transformer (tra), but only in the female nervous system [17]. The result was neuronally masculinized females that showed male-like behavior directed toward normal females; these masculinized females exhibited abdominal quivering, which wild-type females never do (Figures 2 and S3 and Movie S4). Their wild-type female partners tend to become immobile when the masculinized females exhibit abdominal quivering (for quantitation, see Figures 2 and S3). We have presented evidence that, in normal courtship and as a response to the male quivering, the females tend to stop. But these neuronally masculinized females have a female anatomy, and accordingly their oenocytes produce only female pheromones [18]. Yet, when they quiver, their wild-type female partners tend to stop moving; we deduce that male pheromones are not the relevant signal emitted during quivering.

Could abdominal quivering generate an acoustical signal? It seems that it does not; a variety of detectors, including insectavox microphones [19] (Experimental Procedures), which we used successfully to record the wing song, all failed to detect any air-borne sound associated with quivering (data not shown). To determine whether quivering generates substrate-borne vibrations, the courting pair was placed on a membrane; so that any possible vibrations caused by wing fluttering could be avoided, the male's wings were amputated. A laser vibrometer was used to measure any oscillations of the membrane (Figure 3A). The results show that bouts of quivering coincided precisely with rhythmic vibrations of the substrate (Figures 3B and 3C and Movie S5). Pulse-like vibrations occurred during quivering with a repetition rate of 6.44 ± 0.32 pulses per second (n = 225 pulses/10 flies; Figures 3B and 3C), with each pulse lasting for about 5 ms (Figure 3C). These fit well with the visual analysis (6.64 ± 0.78 beats per second), arguing that each beat corresponds with a single pulse. There is considerable regularity in the time intervals between pulses (Figure 3), giving a pulse interval of 165.61 ± 4.21 ms (n = 225 pulses/10 flies). There is some indication of variation in the amplitude of the abdominal beats during quivering (Movie S2), which may correspond with the observed variation in amplitude of the substrate vibration pulses. In some arthropods that employ substrate-borne vibratory communication during courtship, patterned variation in amplitude may assist species recognition [20], raising the possibility that Drosophila does the same. In Drosophila, as in other arthropods, tremulations may be transmitted via the legs of the male to the substrate. The substrate-borne vibrations that result may be perceived by the female either by chordotonal organs present on the proximal tibia or carried through the body to the Johnston's organ at the base of the antenna [20–22], or in both of these ways.

During courtship several behaviors may act synergistically to aid copulation. Our evidence argues that quivering is particularly important because it correlates strongly with the female ceasing movement, considered to be a sign of receptivity [7]. Furthermore, quivering may explain why males of, for example, D. heteroneura, D. melanogaster, and D. silvestris that have amputated wings or an otherwise impaired serenade can still elicit acceptance by the female [1, 23–25].

We found both abdominal quivering and associated substrate-borne vibrations are conserved in other Drosophila species (Figure 4). In D. sechellia and D. yakuba (Figures 4A, 4B, S4A, and S4B, and Movie S6), we observed vibrations in the substrate with a pulse repetition rate of 7.13 ± 0.96 (D. sechellia; n = 50 pulses/5 flies) and 6.80 ± 0.49 (D. yakuba; n = 19 pulses/5 flies) (Figure 4C). The pulse interval is 157.56 ± 11.13 ms (n = 50 pulses/5 flies) for D. yakuba and 173.37 ± 8.70 ms (n = 19 pulses/5 flies) for D. sechellia. The frequency and length of the quivering bouts varied (data not shown). In both species, males simultaneously quiver the abdomen and flutter their wings more frequently than D. melanogaster (Figures 4, S4A, and S4B; compare with Figures 1 and S1). Importantly, in both species, quivering was again strongly associated with female immobility (Figure 4 and Movie S6). Less-detailed, yet similar, observations were made on different Drosophila species: some from the same group as D. melanogaster (D. biarmipes, D. mauritiana, and D. simulans), and others from more distant groups (D. mojavensis and D. willistoni).

It is strange that substrate-borne signals have so far been overlooked in D. melanogaster, particularly as substrate-borne vibrations are well known in small invertebrates [26–28]. Tremulatory signals were detected during courtship of other arthropods, for example pentatomid bugs [20] and salticid spiders [22, 29, 30]. Such signals may be generated by up-and-down movements of the abdomen, similar to the quivering we observe, or by shaking of appendages [28, 29, 31–35]. Substrate-borne vibrations were thought to be unusual in Diptera; exceptions were the male and female reed fly, Lipara, which exchange signals as vibrations transmitted within the reed stems. The male signal appears to originate from tremulations of the abdomen [36]. Even two decades ago, abdominal movements were observed in D. silvestris, but this was then reported as a behavior unique to these flies of the Hawaiian islands [37]. Later, a repeated movement of the male abdomen that tapped the substrate was noted as part of a broad description of courtship behavior in D. melanogaster but not associated with any particular female behavior [38]. We have now characterized a male behavior, which we call quivering, that does not appear to include contact with the substrate and that generates substrate-borne vibrations. We do not know exactly how quivering produces these vibrations, but notice that the pulses themselves are short, suggesting some instantaneous element within the quiver beat.

The characteristics of substrate-borne signals depend on the material in which they are transmitted; they are robust and can propagate with little attenuation [21]. The frequency, amplitude, and modulation of these vibrations may carry information to the receiver about the sender [27, 39–41]. Substrate-borne signals may not be detectable by predators, as the latter may not possess suitable receptors [42]. It has not escaped our notice that vertebrates also use substrate-borne vibratory signaling [43].


Experimental Procedures
Fly Mutant and Wild-Type Stocks

Flies were raised on standard cornmeal medium under a 12:12 hr light:dark cycle and kept and tested at 25°C with 65% humidity. For the analysis of wild-type behavior, we used Oregon R (OrR) and Canton S (Cs). fru.Gal4 (fruGal4.P1.D) and elav.Gal4 (elavc155) flies were obtained from the Bloomington Stock Center. UAS.dTRPA1 flies were kindly provided by Stefan Pulver. UAS.traIR flies were obtained from the VDRC Stock Center. The dsx.Gal4 (dsxGal4.KI) line used was that described in [44]. For the analysis of the effect of mutations in the sex-determination genes, two allelic combinations were used: dsx1/Df(3R)dsx15 and fruΔC/Df(3R)4-40. For details of mutant alleles, see FlyBase [45]. Drosophila simulans, D. yakuba, D. mauritiana, D. sechellia, D. biarmipes, and D. willistoni flies were obtained from the University of California Drosophila Species Stock Center. D. mojavensis flies were kindly provided by Darren Parker. Adult flies were collected upon eclosion with light CO2 anesthesia. Before mating, individual males or small groups of five to ten virgin females were kept isolated in vials with fresh food. For some experiments, courting pairs were kept under infrared light [16] or the wings of collected males were cut off with microscissors and under anesthesia.

Behavioral Recording

Pairs of flies were tested in a single trial when they were 4–6 days old. Their behavior was recorded with a 10× macro lens and a Firewire Stingray F-033B camera (Allied Vision Technologies; Stadtroda, Germany) and acquired with “Astro IIDC” (Aupperle Services and Contracting; Calgary, Canada) into a laptop computer. For analysis of the wild-type, 30 courting pairs were recorded and analyzed. For other studies, a minimum of four pairs of flies was tested. Transparent plexiglass courtship chambers (10 mm diameter and 6 mm height) were assembled from two half chambers each of 3 mm height. Each fly was collected with a mouth aspirator and introduced into one half chamber. After a recovery period of 5 min, both halves were fused, and filming of the pair was commenced. Recording was started at the initiation of courtship and for approximately 600 s, or until copulation occurred. Each pair was tested only once. Before each test, chambers were washed with ethanol and dried.

Heat-Activation Experiments

Ectopic expression of the heat-activatable cation channel TRPA1 (dTRPA1) was obtained with the fru.Gal4 and dsx.Gal4 drivers in both males and females. The courtship chamber was inserted into a metal heating block set to produce a temperature of 26°C–27°C inside the chamber; at this temperature, we observed an effect on male but not female behavior with both drivers (Movie S3). We noted that at 29.5°C and only using the fru.Gal4 driver, the females began to quiver; however, the male's behavior became even more frenetic [15].

Behavior Annotations and Analysis

Movies were annotated with the “Annotation” software version 1.3, registering all standard male courting behaviors (such as orientating toward the female, following the female, proboscis extension, licking, tapping), in particular when males showed wing fluttering (this behavior comprises wing extension/vibration and scissoring) and/or abdominal quivering, and also whether the female was moving or immobile. The data for each movie were imported into Excel files. For statistical analysis and generation of diagrams, we used the R programming language and software environment [46]. All intervals shown in the paper are for 95% confidence level. We tested for associations between the three behavioral variables: female mobility, male quivering, and male fluttering; using the number of bouts, N, we fitted a series of generalized linear models with a Poisson error structure, N ∼female mobility + (or) male quivering + (or) male fluttering [47] (see legends to Figures S1C and S1D).

Recording Vibrational Signals with Laser Vibrometry

Video and laser vibrometer recordings were conducted on a vibration-damped table in a soundproof room. Flies were placed into cylindrical chambers of approximately 10 mm in diameter and 6 mm in height, made of plastic. The top of this cylinder was a transparent film through which the flies were recorded using the Stingray F-33B camera with an attached blue filter (cutoff wavelengths at 395 and 480 nm). The bottom of the cylinder consisted of a piece of thermal foil, a membrane made of silver metallized polyester material, with an albedo of approximately 0.8 (Sub Zero Technology; Leicester, UK). The beam of a PSV-400 laser vibrometer (Polytec. Waldbronn, Germany) was directed perpendicular to the surface of this membrane (Figure 3). Signals were digitized with 12 bit amplitude resolution with a PCI MIO-16-E4 card (Analog Devices; Norwood, MA) and digitized with LabView (National Instruments; Austin, TX) on a PC. Signals were transformed into .wav data with the Neurolab software [48]. Video and laser vibrometer recordings were synchronized at the start by brief interruption of the laser path; this produces both a momentary peak in the oscillogram and a black frame in the video. Oscillograms were analyzed with the Raven software [49]. Neither an electret microphone (frequency response, 50 Hz to 13k Hz; sensitivity, 60 ± 3 dB) nor a piezoelectric transducer (resonant frequency, 2.8 ± 0.5 kHz) registered any air-propagated sound emitted during abdominal quivering. We do not know whether wing fluttering of Drosophila produces vibrational signals in the substrate.


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Supplemental Information

Movie S1. A Video Clip of a Pair of Courting Drosophila melanogaster, Related to Figure 1

First, the male chases the female and he flutters. Then he quivers, and she stops and cleans her legs. He moves a little closer to her and continues to quiver and flutter. She stops all activity, he quivers and later flutters. Then he moves toward her with abdomen curved and his wings fluttering, and she moves away. Played in real time.


Click here for additional data file (mmc1.mov)

Movie S2. A Video Clip Showing a Male Quivering during Courtship, Related to Figure 1

Quivering consists of vertical abdominal oscillations of low amplitude. The clip covers about 2 s of real time and is played at half speed.


Click here for additional data file (mmc2.jpg)

Movie S3. Video Clips of a Solitary dsx.Gal4, UAS. dTRPA1 Male, Left, and a Solitary fru.Gal4, UAS.dTRPA1 Male, Right, Related to the Results and Discussion and the Experimental Procedures

At the restrictive temperature, both types of males display several courtship behaviors, including quivering, even though they are solitary.


Click here for additional data file (mmc3.jpg)

Movie S4. Video Clip of Interaction between a Behaviorally Masculinized elav.Gal4, UAS.traIR Female and an Oregon-R Female, Related to Figure 2

The masculinized female (on the left) courts the wild type female and flutters, and she also displays bouts of quivering. The wild-type female is immobile.


Click here for additional data file (mmc4.jpg)

Movie S5. Video Clip Showing Simultaneous Quivering and Substrate-Borne Vibrations, Related to Figure 4

The male quivers and generates characteristic vibrations (see also Figure 3). To reduce interference with the oscillogram, the wings of the male have been amputated. The sound you hear is a transformation of the oscillogram record to audible frequencies; it is only there to help you link the oscillogram record with the video.


Click here for additional data file (mmc5.jpg)

Movie S6. Video Clips of Abdominal Quivering Displayed by Males in Courting Pairs of D. yakuba and D. sechellia, Related to the Results and Discussion
Click here for additional data file (mmc6.mov)

Document S1. Figures S1–S4


Acknowledgments

We thank L. Arnoult and P. Conduit for crucial discussions and the reviewers for constructive criticism; W. Amos for help with statistics; M. Bate, J. Berni, A. Bluon, S. Flynn, N. Gompel, H. ter Hofstede, B. Hudry, C. O'Kane, M. Landgraf, M. Neville, L. de Nerestang, H. Pavlou, B. Prud'homme, S. Pulver, C. Rezaval, C. Skjöldebrand, and B. Tiepman for advice and support; EPSRC for loan of the laser vibrometer to B.H., Wellcome Trust grants WT082987 and WT085521 to S.F.G., BBSRC grant RG54425 to B.H.; and Wellcome Trust grant WT086986MA to P.A.L. C.C.G.F. was supported by an EMBO long-term fellowship during part of this work.


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