Courtship song components affect male and female Drosophila differently

dnim.Behav., 1995, 50, 827-839

Courtship song components affect male and female Drosophila differently STELLA

A. CROSSLEY*,

H. C. BENNET-CLARKt

& HELEN

T. EVERT*

*Department of Psychology, Monash University, Clayton, Australia TDepartment of Zoology, University of Oxford (Received 13 June 1994; initial acceptance 2 August 1994; final acceptance 13 January 1995; MS. number: 4678)

Abstract.The responses of male and female Drosophila melanogaster to male courtship songs were investigatedby videotaping single sexed groups of flies as they were exposed to simulated sounds: pulse, sine,pulse sine and rhythmic, and controls: white noise (males only), and silence (females only). Patternedsounds (1-min silence, 1-min sound) were delivered for 19 min: (1) in 6-dB steps from 60 to 108dB; and (2) at 84 dB. This method enabled sound intensity effects to be distinguished from the effectsof time of exposure. Pulse, pulse sine and rhythmic songs significantly increased male locomotory activity and male-male courtship interactions compared with initial measurements at no sound. Sine songhad similar effects but to a lesserdegree. White noise resulted in complete cessation of movement. Femalesbehaved differently from males: there was more movement at initial no sound, followed by decreased movement over time in all conditions. However, sine and rhythmic songs differed significantly fromthe silent control condition and maintained activity at a high level. An exposure of 1 min to pulse sinesong at 84 dB made females slow down showing that intensity level, and not length of exposure, wasthe critical stimulus. It is suggested that the different responsesof males and females to the various componentsof male courtship song are adaptive: female movement initially attracts males; while increasedmale movement plus decreased female movement enhances mating. 0 1995 The Association

for the Study of Animal

Behaviour

Thedemonstration that male Drosophila melanogaster make sounds during courtship (Shorey

‘pulse song’ and a sinusoidal hum ‘sine song’. Sine song precedes and/or follows some but not all 1962)has generated a large%ody of research on pulse bursts (Schilcher 1976a). The two sibling the genus Drosophila (Ewing 1983; Spieth & species differ in the inter-pulse interval of pulse Ringo 1983). Most of this has described the song which has a mean length of 34 ms for species-specific nature of song types within the D. melanogaster and of 48 ms for D. simulans. In genusand provided circumstantial evidence for addition, Kyriacou & Hall (1980) described a theirfunction in sexual isolation and sexual stimu- sinusoidally varying rhythm in mean inter-pulse lation (Crossley 1986). Songs of over a hundred interval for each of these two sibling speciesbut different species of Drosophila and the related with different periods of the oscillation (60 s Zaprionus have been described (Ewing 1983), but D. melanogaster, 30 s D. simulans). Pulse and sine experimentalconfirmation of the function of the song are produced by wing vibration during various song components is available for only a courtship (Crossley 1989). By using simulated fewspecies(Ikeda et al. 1981; Crossley & Bennet- songs to supplement the deficient courtship of Clark1993). The songs of D. melanogaster, and to wingless males, the role of song parameters in a lesserextent its sibling speciesD. simulans, have enhancing mating successin D. melanogaster has beenthose most extensively studied. These songs been confirmed for invariant pulse songs (Bennetconsistof trains of pulses arranged in bursts: Clark & Ewing 1967, 1969; Schilcher & Manning 1975; Schilcher 1976b) and for rhythmic pulse Correspondence: S. A. Crossley, Department of Psy- songs (Kyriacou & Hall 1982; Greenacre et al. chology, Monash University, Clayton, Victoria 3168, 1993). Mating is also improved in this speciesif Australia. H. C. Bennet-Clark is at the Department of Zoology, University of Oxford, South Parks Road, males and females are pre-stimulated with invariOxfordOX1 3PS, U.K. (email: henry.bennet-Clark@ ant pulse song before mixing (Bennet-Clark et al. 1973) and if females are primed with sine song zoology.oxford.ac.uk). 0003-3472/95/090827+ 13 $12.00/O

e 1995 The Association for the Study of Animal Behaviour

827

828

Animal

Behaviour,

(Schilcher 1976b; Kyriacou & Hall 1984) or with rhythmic pulse song (Kyriacou & Hall 1984) before introduction to wingless males. The conclusion is that pulse song functions as a species recognition signal, while sine song, and possibly also pulse song, has a priming effect on female receptivity. In the above experiments mating was used to assessthe function of song components. Mating, however, is the end point of complex interactions between the two sexes during courtship and measurements of mating successdo not provide information about behaviour prior to mating that may be critical to successfulmating (Connolly & Cook 1973; Crossley 1975). The female may summate courtship stimuli until she reachesa threshold for mating, and/or the song may influence behaviour in other ways so that mating is facilitated (Manning 1967a, b; Cook 1973). Pheromones also influence mating and must be controlled for in experiments by comparing flies at the same stage of maturity (Tompkins et al. 1982; Vaias et al. 1993). Measuring responses to song of males and females housed separately (Schilcher 1976a, b; Crossley & Bennet-Clark 1993) enables the sound stimuli to be separated from stimuli resulting from the presence of the other sex. Using this approach Schilcher (1976a) showed that D. melunogaster males respond to pulse song by moving and by courting other males. The threshold intensity of sound for the response was 88 dB (estimate). Males of the speciesD. parabipectinata subjected to simulated D. parabipectinata ‘short pulse song’ increase movement and courtship with an increase in the sound particle velocity level, but above 96 dB high locomotion impedes courtship and the number of courtship interactions decreases (Crossley & Bennet-Clark 1993). Schilcher (1976a) measured the effects of artificial songs on female D. melanogaster and reported that sine song and pulse song, delivered separately, decrease movement over a 2-min period of song exposure. In this paper we describe the behavioural responses of single sexed groups of males and of females of D. melanogaster to artificial songs. Our study was based on Schilcher’s (1976a) study but (1) more song types were tested, (2) subjects were exposed to sound over a longer test period, (3) males as well as females were subjected to increasing sound particle velocity levels, and (4) behavioural responseswere videotaped to facilitate their time/event recording.

50, 3

MATERIALS

AND METHODS

Flies We used a Glen Waverley (GW) strain of that had been derived from wildcaught females 5 years earlier. Stocks were maintained in a temperature and light controlled cabinet at 23”C, with a 12:12 h 1ight:dark cycle and with lights on at 0900 hours. The laboratory temperature was 23 f 1°C. The sexes were separated within 5 h of eclosion and wings were removed from males on the second day under light carbon dioxide anaesthesia. Flies were aged 34 days when tested between 1000 and 1500 hours. This procedure was used throughout the study. D. melanogaster

Apparatus Figure 1 shows the layout of the apparatus used for song simulation, sound delivery, behaviour observation and videorecording. Artljicial

song simulation

Separate function generators were used to produce sine song (Wavetek 134) and pulse song (Wavetek 156), and these were driven by an IBM computer program which specified song structure. White noise was provided by a white noise generator. Separate attenuators were used to adjust the levels of sine song and pulse song, as well as white noise, by known steps independently. A mixer-buffer allowed the outputs of up to four signal sources to be mixed and fed to a loud. speaker. Becausethis simulator uses triggered and gated oscillators, the inter-pulse interval could be specified independently of the carrier frequency within a pulse. This overcame a problem with the designs of Johnson & Cowling (1980) and Robinson & Ewing (1978) which used the ICL 8038 chip, which provided a continuously running sinusoidal output fed to a gate controlled by a pulse generator: the output from this type of design is a chopped coherent sine wave, so if carrier frequency is set to 333 Hz (period 3 ms) a train of identical pulses can be produced only if the inter-pulse interval is specified in exact multiples of the carrier frequency period (Johnson & Cowling 1980). The output from our simulator produced realistic models of the type of song that

IBM PC computer

Program song parameter specifications

song duration

I

1. Experimental

thermometer

Sine song frequency

Pulse and

Figure

I

form

set-up

for analysis

for delivering

Television monitor

Observation subsequent

White noiie generator

artificial

<

songs to Drosophila.

Video-recorder

O-60 dB step attenuator

Set sound levels

Time marker generator

Monitors sound signal

Videocamera

2 R ..

830

Animal

Behaviour,

produces, such as severalbursts of one song type followed by bursts of another song type followed by a repeat of the first song type followed by a period of silence.

Drosophila

Sound delivery

A Sennheiser H.D. 400 headphone, attached to a stand, was mounted on a plenum chamber machined in a PVC block which communicated via a slot (32 mm wide x 7 mm high) to the sound duct. The sound duct consisted of three transparent Perspex channels (32 mm wide x 7 mm high) bolted together to form a continuous duct of overall length 62 mm. In sequence, from the headphone, there were: (1) a duct 12 mm long; (2) a duct or cell 25 mm long for observation; and (3) a terminating duct 25 mm long. Two holes in the wall of the observation cell permitted entry of flies and insertion of a temperature-monitoring thermistor probe of a digital thermometer (Bailey Model Bat-8c). Therefore, sound was delivered sideways across the width of the observation cell. The lid of the observation cell was interchangeable to allow either viewing or insertion of a microphone for calibration. The sound duct could be taken to pieces for washing. Calibration

An electret pressure microphone (Tandy Corporation 270-090) was modified to act as a particle velocity microphone (Bennet-Clark 1984). Calibrated against a Briiel & Kjaer Microphone type 4134 on a sound-level meter type 2218 in anechoic far-field conditions, its frequency response was flat to & 0.5 dB from 200 to 700 Hz and f 1.5 dB between 150 Hz and 1 kHz. The microphone was inserted into the observation cell to measure the particle velocity produced by the headphone. Other tests using a matched pair of probe microphones showed that the sound particle velocity was constant to within 1 dB throughout the observation cell. The frequency response of the headphone was measured in the observation cell. The particle velocity in the cell was constant to % 1 dB between 150 and 700 Hz. The apparatus allowed song patterns to be specified to a precision of 1 ms and sound particle velocity levels to be specified to * 1 dB over a dynamic range from 60 to 120 dB relative to 5 x 10 - ’ m per s (this is equivalent to a sound intensity of 10 ~ I2 W per m2).

50, 3

Artificial

Songs

Parameters for song simulations were basedon (GW) song, in particular, those sections in single pair courtships during which the male closely followed the female (Crossley 1988. 1989). Definitions of terms used in describing songs are given in Figure 2 in Crossley & Bennet. Clark (1993). The following songs were made. (1) Pulse. Regularly repeated bursts of pulse song. Each burst comprised 10 monocyclic pulses of carrier frequency 300 Hz. The inter-pulse inter. val was 34 ms, the burst period 1000 ms, and the burst duration was 309 ms. (2) Sine. Regularly repeated bursts of sine song. Each burst was one polycyclic pulse. The carrier frequency was 160 Hz and the burst period was 1000 ms. The burst duration was 500 ms. (3) Pulse sine. Alternating pulse song and sine song: 1 s of pulse song, followed by 1 s of sint song, followed by 1 s of pulse song, followed by 1s of sine song, etc.vhe carrier frequencies of pulse and sine song were 300 and 160 Hz, respectively. The inter-pulse interval of pulse song was 34 ms. (4) Rhythmic. Bursts of pulse song with 10 pulses per burst and an inter-pulse interval vary ing in a sinusoidal manner between 3 1 and 37 ms. The rate of change of a sine wave is greatest at the mean value and slowest at the maximum and minimum. This was modelled by allotting fewer burst periods at the 34-ms crossover and more burst periods at the 31-ms and 37-ms extremes. The total period of the rhythm was 60 s. The standard deviation of the mean of the inter-pulse interval was 2.14 ms; that of a sine wave of 3 ms amplitude is 2.12 ms. (5) White noise. Continuous sound from a white noise generator. Songs were recorded using a Revox (Type 77) tape-recorder, which was connected to the calibration microphone in situ in the cell. Audiotaped songs were digitized, stored on a computer, measured (Crossley 1988) and plotted (Fig. 2). In addition, specifications of artificial songs were checked on oscilloscope displays at the time of delivery.

D. melanogaster

Behaviour

Study

Treatment

1

Each trial used eight or nine flies of one sex. Males were wingless. Flies were tipped into the

Crossleyet al.: Song effects in Drosophila

831

males in response to white noise and stopped moving. The silent condition lasted as long as the song conditions and, like those, was videotaped throughout.

(a)

Treatment 2

I

I

100 ms Figure2. (a) Song of D. melanogaster GW strain recorded on videotape using a condenser microphone. (b) Artificial song, pulse sine recorded on a Revox 77 tape-recorder using a particle velocity microphone. Songsin (a) and (b) were digitized, stored on computer and plotted (Hewlett-Packard 7470A Plotter).

observationcell through a funnel and were videorecordedusing a National TV camera WV-1350 AEIA fitted with an achromatic close-up lens, 50mm in diameter and 20 cm in focal length, whichwas mounted above the cell, and a National Panasonic NV-V300 video-recorder. Videorecordinggave a clear in-focus view of the whole of the cell, superimposed time ito 1 s) and song condition as sound. Temperature within the cell wasmonitored and maintained at 23 f 1°C. The video-recordingbegan as soon as all the flies were in the cell. Flies were allowed to settle for 2 min, by which time the criterion for settling, of no more than two flies moving, was realized. The sound delivery system was switched on and a song condition, previously selected for the trial, was played for 1 min at 60 dB. The sound was switchedoff for 1 min of silence followed by 1 min of the same song at 66 dB, followed by 1 min of silence,song at 72 dB and thereafter in steps of 6 dB to a maximum sound level of 108 dB, 19 min after the test began. Males were exposed to all five song conditions. Femaleswere treated like males except that a silentcondition was substituted for white noise. The reason for omitting white noise was that silencehad been used as a control for females in previouswork (Schilcher 1976a). In addition, preliminary trials showed that females behaved

like

The method was as in treatment 1, except that the same sound level (84 dB) was used at each song delivery. With males two songs were used in treatment 2, sine and pulse sine; and with females one song: pulse sine. Treatment 2 enabled the effects of sound level to be distinguished from the effects of length of song exposure. Every song condition was tested on each day of experimentation and the order of testing was randomized. Flies were subjected to one song condition only. Behaviour

Categories

Choice of behaviour categories for study stemmed from preliminary study and descriptions of behaviour claimed to influence the course of courtship (Connolly & Cook 1973; Crossley 1975). We recorded movement and courtship. (1) Movement was walking and running independently of any other fly. Synchronized movement of one fly followed by another was classified as courtship and not included here. Courtship was observed only in males. Flight movements did not occur. (2) Courtship was recorded in males only. A male followed or chased another male. Standing and orienting to another male, as to a female during heterosexual courtship, was included when it occurred but was rare. Licking and attempted copulation were not seen. We recorded behaviour categories for each fly from the videotapes using a Microprocessor event recorder. Responses throughout the period of song exposure were measured by recording the duration of each behaviour category in the second minute of the initial silent period and in each of the succeeding 1-min periods of song exposure. This procedure was followed for male movement and courtship and for female movement for songs in treatments 1 and 2. We used 24 males for each set of seven song conditions (five treatment 1, two treatment 2), a total of 168 males. Forty females were exposed for each set of six song conditions (five treatment 1, one treatment 2) a total of 240 females. All males

Animal

832

Behaviour,

50, 3

Table I. ANOVA results summary showing behavioural response to song and sound level together with criticai values at PeO.05 for Fisher’s least significant difference test (lsd) F

Source Male Behaviour Movement Tl Sine Tl, T2 Pulse sine Tl, T2 Courtship Tl Sine Tl, T2 Pulse sine Tl, T2 Female Bebaviour Movement Tl Pulse sine Tl, T2 Silence, pulse sine T2

Song

Sound level

Song x sound level

lsd(a)

lsd(b)

49.55*** (4,1035) 1.40 (1,414) 66.77*** (1,414)

16444*** (9,1035) 23.82*** (9,414) 129.34*** (9,414)

21.23*** (36,1035) 2.33* (9,414) 50.71*** (9,414)

0,452 0,578 0.360

0,580 0,859 0403

51.33*** (4,1035) 3.45 (1,414) 0.09 (1,414)

70.82*** (9,1035) 6.12*** (9,414) 33.06*** (9,414)

9,76*** (36,1035) 264* (9,414) 6.93*** (9,414)

0.177 0.177 0.238

0,193 0.204 0,258

30.52*** (9,1755) 26.61*** (9,702) 27.20*** (9,702)

2.56*** (36,1755) 8.88*** (9,702) 4.69*** (9,702)

0.413 0.433 0.435

0,601 0,597 0646

I.17 8.98** 7.50**

(4,1755) (1,702) (1,702)

Tl is treatment 1 and T2 treatment 2. Degrees of freedom are given in parentheses. lsd(a) refers to difference! between sound levels within a sone. and lsd(b) refers to differences between songs. *p
were measured for both behaviour categories for all seven songs at all 10 sound levels including initial silence. Movement of all females was measured at all 10 sound levels for all six conditions tested. Statistical

Analysis

Data analysis was carried out on the total duration (s) of each behaviour category per fly in each minute measured in each condition. In males, movement and courtship were analysed using a two-way ANOVA with repeated measures on one factor. The song condition comprised the between factor while the sound level was the within factor. Female movement was analysed as described for the male behaviour categories. All variates were transformed to natural logarithms before analysis to satisfy the ANOVA assumptions of homogeneity of variances. Examination of residuals and estimates showed the transformations to be appropriate. Data are transformed to their natural logarithm in Table I and Fig. 3. Separate ANOVAs were used to compare responses to the same songs that were played in both treatments 1 and 2 (sine and pulse sine for males, pulse sine for females). The silent condition (treatment 1) was compared in an ANOVA with the pulse sine condition in treatment 2. The interest in this

comparison was to see behaviour change OWI the course of the experiment in response te unchanging sound intensity levels. The GeisserGreenhouse correction (Keppel 1982) was applied in each ANOVA and did not change the sign& cance of the effects. The Fisher’s least significant difference test (Carmer & Swanson 1973; Keppd 1982) was used to assesswithin- and between. group differences. Regression analysis was uset to compare female responses to increasing sount levels in experimental conditions with theil responses in the silent condition. RESULTS

Table I shows the results of the ANOVAs. In, treatment 1 there were significant differences in response to sound level for males and females and to song conditions for males. However, significanr’ song by sound-level interactions for both sexes showed that females as well as males were differ. entially influenced by the different songs used in treatment 1. Responsesin treatments 1 and 2 were significantly different. Figure 3 illustrates the behavioural responses measured in treatment 1, and Table I gives the critical values for the least significant difference test (lsd) between means within and between

Crossley et al.: Song effects in Drosophila

A White noise 0 Sine l Pulse sine

Silence60

groups. Groups are the different conditions and these are designated by the song played after the initial silent period in each condition. The first minute of videotape measured was the second minute of the initial silent period. Male courtship and female movement did not differ between groups but there were differences in male movement: the white noise control elicited more movement than rhythmic (mean difference (md) 0.814>lsd 0.580, P1sd 0.580, PcO.05). These differences occurred because the ‘criterion of settling’ used did not give equivalent movement across all conditions at the start of the experiments. In the next minute, however, when sounds at 60 dB were played, there were no differences in movement across conditions (Fig. 3a) showing that a baseline level of movement was achieved. Had this not been the case it may have been difficult to unravel the effects of sound on male movement.

(a)

‘72 78

66

84

90

96 102 108

Particle velocity (dB) III

1

3

5

I

I

I

I

I

I

I

7

9

11

13

15

17

19

Time (min) A 0 . A

White Sine Pulse Pulse

noise

l

Rhythmic

833

(b)

sine

Male Behaviour

lence60

66

72

78

84

90

96 102108

Particle velocity (dB) I

III

1

3

III

5

7

9

II

11

13

15

I

17

19

Time (min) 51

4

I

1

Silence60

66

72

78

84

90

96 102 108

Particle velocity (dB) I

I

I

I

I

I

I

I

I

I

1

3

5

7

9

11

13

15

17

19

Time

(mid

Males responded to the white noise by becoming inactive. They stopped, spread their legs, and pressed their tarsi against the substrate. There were no courtship interactions elicited by white noise. In contrast, the experimental conditions pulse, pulse sine, rhythmic and sine elicited both movement and courtship chases. The threshold intensity of sound for each type of behaviour was determined by comparing means obtained for the initial no sound minute in each condition with means calculated for exposure at increasing sound intensity levels. For movement, the lowest sound threshold intensity was 66 dB for white noise (md 0.785>lsd 0.452, PcO.05) and 72 dB for the remaining conditions (pulse md 1.264; pulse sine md 1.560; rhythmic md 1.771; sine md 0.771; >lsd O-452, PcO.05). The lowest sound threshold intensity for male to male courtship was 72 dB measured for pulse (md 0.380>1sd 0.177, Plsd 0.177, PxO.05) and rhythmic (md

Figure 3. (a) Male movement, (b) male courtship and (c) female movement in response to sound at increasing intensity levels. In the silent control condition (c) the sound delivery apparatus was switched off for the duration of the experiment.

834

Animal Behaviour, 50, 3

0.264>lsd 0.177, PcO.05). With sine song, 96 dB was the lowest sound level that produced significantly increased courtship (md 0.201>lsd 0.177, PcO.05). There was no evidence that the highest sound intensity threshold for male behaviour was reached in these experiments since responseswere still obtained at the highest sound intensity used: 108 dB. Differences in levels of responses to song conditions at the same sound intensity indicated that males discriminated between songs. White noise at every sound intensity tested at and above 66 dB elicited significantly less movement from males than any other condition while songs containing pulse (pulsed songs): pulse, pulse sine and rhythmic produced more movement than white noise and did not differ in the amount of movement elicited. Sine song also increased male movement but more slowly and to a lesser extent than the three pulsed songs (Fig. 3a). For example, mean movement produced by sine song at 78 dB was significantly less than mean movement found in the pulse, pulse sine and rhythmic conditions at 78 dB (sine cf: pulse md 2.208, pulse sine md 1.890, rhythmic md 2.240>lsd 0.580, Plsd 0.193, PO.05). Female Movement

The 2-min silent period, which initiated all trials, was continued for the whole trial in the silent control condition and measurements were made for the same 1-min periods during which other conditions received sound stimulation. Figure 3c shows changes in female movement in response to songs at increasing sound intensities and to silence. There was no difference between

female groups in the initial minute prior to sound onset. However, females moved more than males, so that although all groups reached the settling criterion of no more than two flies moving before the onset of sound, they moved more than males in the first minute measured. The lowest sound intensity threshold for movement was established for females as for males by comparing the initial silent minute with successive levels of sound stimulation within each song condition. In both the pulse and pulse sine conditions, the 84-dB sound level and above produced sig nificantly smaller mean movement than the silent minute preceding these conditions (84 dB no sound cf: pulse md 0.847, pulse sine md 0.744>lsd 0.413, PcO.05). The rhythmic condition showed no significant cessation of movement over the initial silent minute until sound delivery at 102 dB and above (102 dB no sound cf: rhythmic md 0.511 >lsd 0.413, PcO.05). Female movement in the sine song conditionsdid not change significantly at any sound intensity level. Changes in movement within the silent control condition over time showed the extent to which female flies stopped moving in the absence of sound stimulation. Female movement measured early in the silent control condition, at 1, 3 and 5 min into the experiment, did not differ but there was significantly less movement shown at 7 min and in all later periods measured than at the start of this condition (min 1 cf: min 7 md 0.583>lsd 0.413, f-0.05). Female discrimination between songs and silencewas shown by comparing movement across conditions at the same sound intensity and experimental minute. There were no significant differencesin movement between conditions in the first 11 min of the experiment, but after this time there was a steady decline in movement in the pulse sine and silent control conditions with the result that at 108 dB level, i.e. the 19th experimental minute, the means for pulse sine and silence did not differ (md 0,169<1sd 0.601, PO.05) and those for both silence and pulse sine differed from rhythmic significantly (e.g. silence cf: rhythmic md 0.684>lsd 0.601, Plsd 0.601, PcO.05). Table II gives the results of the regression analysis. All conditions gave negative regression coefficients and t-tests showed that the slopes of

Crossley et al.: Song effects in Drosophila

II.

of regressionanalyses

835

ments were detected later (time 9 min, movement md 1.393>lsd 0.859, PcO.05; courtship md b t* P 0.354>lsd 0.204, Plsd 0.204, PcO.05). the regression lines were significant with movement decreasing with time. Comparison of the slopeof the regression line for the silent control DISCUSSION condition with the slopes of lines in each of the experimentalconditions showed significant differ- The pattern of significant results described above enceswith pulse, rhythmic and sine, but no sig- was consistent in substantiating the main connificant difference with the pulse sine condition. clusion that males without wings and females Therefore,only pulse sine caused females to slow respond differently to courtship song. down to the extent that they do so when left in The validity of the data was high for two silence. reasons. First, behaviour was event recorded off videotape to give a complete record of each fly’s behaviour, in contrast to previous work (Schilcher Comparison of Treatments 1 and 2 1976a; Crossley 8z Bennet-Clark 1993) where a In treatment 2 selected songs were delivered at a group of flies, as a whole, was measured using constant sound level of 84 dB instead of at the instantaneous sampling techniques. Our method increasingsound intensity levels used in treatment ensured the more accurate measurement with high 1. There was no difference in the level of male intra- and inter-observer reliability coefficients. movement,male courtship and female movement Second, each group served as its own control for measured in the preliminary no sound minute within-song comparisons. This is important in treatments 1 and 2 but males and females because Drosophila behaviour is influenced by respondedquickly to pulse sine at 84 dB resulting environmental factors which differ between samin significant differences between treatments 1 and ples (Connolly 1966). Therefore within-group dif2 in the first few minutes of sound delivery (male ferences in the present study are not due to movement,time 3 min, treatments 1 versus 2, md environmental fluctuations. 3,447>1sd0.403, PcO.05; female movement, time Males responded to pulse, pulse sine and Smin, treatments 1 versus 2, md 1.277, PcO.05; rhythmic songs by increasing locomotion and to Fig. 4). Pulse sine, at 84 dB, elicited more male white noise by decreasing it. A constant 84-dB courtshipthan that produced at the same time by pulse sine elicited as much movement in the first treatment 1 (time 3 min, treatments 1 versus 2, md minute of exposure as reached more gradually 0,462>1sd0.258, WO.05). by pulse, pulse sine and rhythmic songs with Sine song at 84 dB, like pulse sine, produced increasing sound intensity levels from 60 to effectson males earlier in treatment 2 than in 84 dB. This showed that the sound intensity treatment 1 but the response was more gradual so level and not the length of exposure was the that significant differences between the two treat- important stimulus.

Table

Results

Animal

Behaviour,

The effects of different songs on male to male

r

0 l

t

courtship

(a)

Sine Pulse sine

--

III1 Silence84

OL

84

84

I 84

I 84

I 84

I 84

I 84

I 84

Particle velocity (dB) I

II

1

3

5

I

III

7

9

III

11

13

15

17

19

Time (min) (b) Sine Pulse sine

0 l

m

Silence

84

84

84

84

84

84

84

84

84

Particle velocity (dB)

I

II

1

3

II

5

7

II

9

11

III

13

15

17

19

Time (min)

2 ‘i/t/ 1

1

01

50, 3

’

Silence

I

84

I

84

’

84

I

84

’

84

I

84

’

84

1 ’

84

I

tracking

mirrored

the effects on loco-

motion, except that at higher sound intensities there was a tendency for courtship interaction to decrease. A similar finding occurred when D. parabipectinata was stimulated by simulated song at high sound intensity (Crossley & BennetClark 1993): males moved too fast for courtship tracking to be sustained. Schilcher (1976a) also reported that pulse song elicited male movement and courtship. In his work, as with D. parabipectinata (Crossley & Bennet-Clark 1993), courtship comprised tracking, orientation, licking and attempted copulation. In contrast, in our work, licking and attempted copulations were absent. The difference may relate to the different sizes of container. Our cell was two to three times larger by volume than those used above and it is known that D. melanogaster display more sexual activity in small containers than in larger ones (Zawistowski &Richmond 1987). An interesting finding of this study, and one not previously reported, was that females moved more than males after introduction to the cell even after the settling criterion was reached. The difference was not that more females moved, it was that females, when they moved, continued moving for longer than males. Since female movement elicits courtship by males and is neccessaryfor tracking in courtship (Cook 1979; Tompkins et al. 1982),it is adaptive for females to be more spontaneously active than males. In females there was a decrease in movement in all groups tested across the 19-min experimental period. The two groups showing least movement in treatment 1, and which did not differ significantly in rate of change over time, were the silent control and the pulse sine conditions. The reason for the change was different in the two groups. In silence it was a case of settling following the disturbance caused by entry to the container. In contrast, with pulse sine stimulation, females responded once the sound-level threshold was reached. Evidence for this conclusion is given by the results of treatment 2 in which females were exposed to pulse sine at 84-dB sound intensity from the start of the experiment. The sharp

84

Particle velocity (dB) II

1

3

I

IllI

5

7

II

9

11

13

Time (min)

15

I

17

19

Figure 4. (a) Male movement, (b) male courtship and (c) female movement in response to sound delivery at 84 dB at every minute of experimental sound exposure.

Crossley et al.: Song efects in Drosophila

decreasein movement that resulted here, within I min, was evidence that females were responding to pulse sine song and not to the length of the soundexposure or the time elapsed since the start of the experiment. The rate of decrease in movementby females to sine and rhythmic song differedsignificantly from the silent control condition. Comparison of each group’s response to soundwith its initial response to silence as control wasimportant here for showing the effects of soundson female movement. Thus, although linear regression coefficients showed a slight decrease in movement in these groups, comparisonof movement in the first minute with movement in successive sound exposure minutes showedno differences for sine song and differencesonly with the last two exposure minutes for rhythmicsong. Therefore the levels of movement remainedhigh in the sine and rhythmic conditions in contrast to the pulse sine condition. This was evidencethat females discriminate between these songs.Female response to pulse song was unclear. Therewas a decrease in movement over time but therate of decreasediffered from that in the silent control condition. However, pulse song elicited decreasedmovement over initial silence, at the startof the experiments from the 1lth to the 19th minute of measurement, except for the 17th minute.If the result at 17 min was due to chance fluctuation,pulse as well as pulse sine song caused femalesto stand still. More work is required here. What is clear is that females in the presence of certaincomponents of courtship sounds behave as theydo in courtship and slow down (Cook 1973; Markow & Hanson 1981; Markow 1987). The results agree with those of previous workerswho found that pulse song reduces female activity(Bennet-Clark et al. 1973; Schilcher 1976a) butdo not support Schilcher’sfinding (1976a) that sinesong has similar effects. Different experimental procedures may explain the difference. Schilcherallowed a settling period of 1 min, a periodwhich in our work was not long enough to attain complete settling in females. He followed thiswith a 2- min silent period which acted as a within-group control for the following 2-min sound exposure period. We used successive I-minperiods of sound exposure interrupted with I min of silencefor 19 min and measured the total duration of locomotion per fly. In contrast, Schilcherestimated the amount of movement by notingthe number of flies moving per group per

837

10 s. Therefore both the length of the experiment and the different parameters recorded may have contributed to the different results as well as other causessuch as genetic differences in strains. The song conditions delivered at increasing sound levelsshowed the threshold intensity for the responses of males and females to sound. White noise caused males to stand still at 66 dB and above. In contrast, all the other sounds tested on males elicited movement at and above 72 dB. This result is comparable to earlier findings: D. parabipectinata males 66 dB and D. melanogaster males 88 dB (estimated not directly measured; Schilcher 1976a; Crossley & BennetClark 1993). There was no evidence that a high sound intensity threshold was reached in our experiments because male and female Drosophila responded to sound at the highest sound level used: 108 dB. Schilcher (1976a) found failure to respond at about 114 dB (estimated). Female responsivenessto varying sound intensities is reported for the first time here. The response was a reduction in movement over initial no sound and the lowest threshold intensity measured was 84 dB for pulse song, and pulse sine song. Song in D. melanogaster is made up of pulse song, followed or preceded by sine song. Silent periods separate pulse and pulse plus sine bursts (Ewing 1983). Pulse song bursts occur without sine song but sine song seldom occurs in isolation. In many courtships, including those of l-day-old females (Crossley 1990) and ebony mutants (Crossley & Taylor 1983) sine song is lacking or rare. Song also varies in the number of pulses in a burst of song, variation in inter-pulse interval within a burst (Crossley 1988, 1989, 1990) and a rhythmic component in inter-pulse interval in the total song repertoire (Kyriacou & Hall 1989; Kyriacou et al. 1990). No song tested in this work approached the complexity of naturally occurring song but the closest to that of a mature 3-day-old male was pulse sine in treatment 2 and it is interesting that it was this song that produced the greatest decrease in locomotion in females. Since females characteristically come to a halt just prior to copulation and males sing sine and pulse song at about 84 dB at this time (Bennet-Clark 1975; Crossley 1990), this result mimics the natural situation. All songs containing pulse increased locomotion and courtship tracking by males and results did not differ for rhythmic which has been

Animal

838

Behaviour,

reported to have a sexually stimulating role superior to that of invariant pulse song (Kyriacou & Hall 1982) and so might be expected to influence behaviout in a manner to enhance mating. As groups were homosexual, mating behaviour was not the focus of this study except in so far as behaviour leading to mating was likely to be influenced by song. In this context the response to rhythmic by females was interesting. Female movement attracts courtship (Cook 1973) so that in failing to slow down in response to rhythmic song females may contribute to the enhanced mating successmeasured by several workers in response to simulated rhythmic songs (Kyriacou & Hall 1982, 1986; Greenacre et al. 1993). This explanation is unlikely, however, since matings occurred in the first few minutes in the latter experiments. Since females stand prior to copulation (Cook 1979; Markow 1987), feniales must have slowed down early on in the experiments of Greenacre et al. (1993) rather than continuing to move as they do in response to rhythmic. In view of the controversy concerning rhythms in song (Crossley 1988, 1989; Ewing 1988, 1989; Logan & Rosenberg 1989; Kyriacou et al. 1990), this response to rhythmic song is interesting. Further study is required to determine if the critical component is the rhythm or the variable inter-pulse interval, both of which were missing from the other songs tested. Many courtships between mature pairs comprise more sine song than pulse song (Crossley 1990) so it is surprising that sine song does not influence movement in females and, in males, that the effect of sine song is less than that of pulse songs. In addition, there is no evidence that sine song influences the sexes in a way that increases the likelihood of mating as much as the other components of songs do. Therefore Schilcher’s conclusion that sine song is sexually stimulating to females is not supported here. However, sine song does have some influence because the responsesof females to pulse sine were stronger than to pulse song, and males subjected to sine song moved more than they did in the initial silent period. ACKNOWLEDGMENTS We thank MS Cheryl Roberts and MS Sally Hunt for technical support, and MS Jacqui Tomkins and Dr Phillip McCloud, Head, Monash Univer-

50, 3

sity Statistical Consulting Service for statistical assistanceand advice. S.A.C. is especially grateful to Professor J. M. Cullen for his interest, and helpful discussions at all stages of the work H.C.B.-C. thanks Oxford University for sabbatical leave and Monash University for hospitality. The research

was supported

by an Australian

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