Heterospecific inter-pulse intervals of courtship song elicit female rejection in Drosophila biauraria

Anim.

Behav.,

1995, 50, 905-914

Heterospecific inter-pulse intervals of courtship song elicit female rejection in

Drosophila biauraria MASATOSHI

TOMARU, Institute

HIROSHI

of Biological

MATSUBAYASHI

Sciences,

University

& YUZURU

OGUMA

of Tsukuba, Japan

(Received 12 September 1994; initial acceptance 3 November final acceptance 13 January 1995; MS. number: 4728)

1994,

Abstract. The present study investigated the significance of song in sexual isolation in Drosophila Interspecific copulation was not observed with a heterospecific D. triauraria male that had intact wings, although it was often observed when the male’s wings were removed, indicating that heterospecific courtship song elicits female rejection, and prevents copulation. Artificially synthesized songswere played to a female with a wingless, antennaless male. The conspecific inter-pulse interval of artificial song increased mating frequency, whereas the heterospecific, longer or shorter, inter-pulse interval significantly decreased the frequency. The mating frequency with random noise and silence showed intermediate values. Female rejection behaviour was greater with heterospecific than with conspecificinter-pulse intervals of artificial song; with random song and silence female responses were intermediate. This indicates that the inter-pulse interval is used as a speciesdiscriminator in D. biauraria females.It is suggested that the female’s ability to discriminate inter-pulse intervals and her responses, rejection and acceptance, have evolved under the influence of reinforcement, reproductive character displacement, or both, which triggered sexual selection.

biauraria.

0 1995 The Association

for the Study of Animal

Behaviour

A female is a chooser; a male courts a female eagerly and less discriminatingly. These sexual differences suggest that female discrimination is more important than male discrimination in sexualinteraction (Trivers 1972). Female discrimination against heterospecific males may depend on species-specificmales’ signals. The sibling species of the Drosophila auraria complex (in the melunogaster species group) of easternAsia show strong sexual isolation towards each other (Kurokawa 1960; Kurokawa et al. 1982). They live in different microhabitats (Kurokawa 1967; Kimura 1987), but sympatric speciescan be caught together in bait traps, so it is likelythat in nature they will meet each other and could hybridize. Although some hybrid males are sterile,hybrid females are fertile (Kimura 1987) and can be back-crossed (Hara & Kurokawa 1984; Tomaru & Oguma 1994a). We therefore expectto see some form of pre-mating isolation, to prevent extensive introgression.

Courtship in the D. auraria complex is always initiated by a male (Spieth 1952; Oguma et al. 1987), but the female does not always play a passive role; she can accept or reject the male during attempted copulation. The courtship is frequently interrupted at a certain stage of the courtship sequences in interspecific crosses, for example, by a female fluttering her wings or kicking a male, and by a male ceasing to tap her body. The female may recognize a heterospecific male from his courtship song which is speciesspecific. These courtship songs consist of bursts of pulses (Tomaru & Oguma 1994b). In the sympatric species, the species-specificparameter of the song is the inter-pulse interval which in D. biauraria is 12.413.2 ms, in D. triauraria, 15.4-16.1 ms, in D. subauraria, 11.1-11.6 ms, and in D. auraria, 18.9-21.4 ms. The species-specific courtship song (Tomaru & Oguma 1994b) and the failure of wingless males to copulate in conspecific crosses(Grossfield 1968) suggest that the speciesspecific inter-pulse interval is the speciesdiscrimiCorrespondence: M. Tomaru, Institute of Biological nator. Although there are several reports that Sciences, University of Tsukuba, Tsukuba, Ibaraki 305, conspecificcourtship song makes females receptive Japan. H. Matsubayashi is now at the Department (Bennet-Clark & Ewing 1969; Ikeda et al. 1981; of Applied Biology, Kyoto Institute of Technology, Kyriacou & Hall 1982; Crossley & Bennet-Clark Matsugasaki, Kyoto 606, Japan. 0003-3472/95/100905

+ 10 $12.00/O

0 1995 The Association

905

for the Study

of Animal

Behaviour

Animal

906

Behaviour,

1993), there are no reports showing that the speciesspecific parameter of courtship song elicits female rejection. We investigated this possibility in the present study. We performed two kinds of experiments. In the first (crossability tests) we crossed males with or without wings in both conspecific and heterospecific crosses. In the second we played artificially synthesized songs to a female with a wingless, antennaless male. If the inter-pulse interval is the speciesdiscriminator, when a female is given heterospecific types of inter-pulse interval, she will reject a courting wingless male; and when a female is given a conspecific inter-pulse interval, she will accept a male.

50, 4

with an aspirator. The courtship behaviour was then observed for 30 min. Pairs showing courtship elements (orientation and attempted copulation) and copulation latency (time to copulate after introduction) were recorded. Observations were made within the first half of the light periods (0900-1300 hours) in a room with a constantly regulated temperature of 24.5 f 1XK We made four types of crosses with D. biauraria females: crosseswith winged D. biauraria males, with wingless D. biauraria males, with winged D. triauraria males and with wingless D. triauraria males. The same number of pairs was always observed simultaneously for each of the four cross types. Artificial Song Experiments

METHODS Drosophila

Stocks

The D. biauraria B16 (Tokyo) was used as the tester female strain. The male of this strain has an inter-pulse interval of 12.9 ms (Tomaru & Oguma 1994b). In crossability tests the D. triauraria T544 (Tsukuba) with an inter-pulse interval of 16.1 ms (Tomaru & Oguma 1994b) was used as a heterospecific male strain. The strains were maintained in glass vials (3 cm diameter x 10.5 cm high) containing standard Drosophila glucose-yeastcornmeal medium at 24.5 f l.O”C under a light:dark cycle of 14:lO h (lights on 0700-2100 hours). All flies were sexed without anaesthesia, within 12 h of their emergence, with an aspirator. The flies were kept in groups of 10 in a vial for 45 days and were transferred once to new vials before crossability tests or artificial song experiments.

Production

of artiJicia1 songs

We made mating chambers (15 mm inside diameter x 6.5 mm high) for the artificial song experiments with acoustically transparent nylon net floors and roofs (Fig. la). These chambers resemble those of Crossley & Bennet-Clark (1993), but are smaller. Recorded artificial songs were played back by a Sony cassette-tape recorder WM-R707 and transmitted to a cone loudspeaker (90 mm diameter) via a power amplifier. Six mating chambers were settled in a circle (Fig. lb) on a stainless steel mesh placed 25 mm above the loudspeaker (Fig. I b). Artificial songs were played back at a sound pressure level of 120 dB above 2 x 10p5Pa. Synthesized artljicial

songs

A burst of artificial song was synthesized as a train of single sine waves (Fig. 2a). The frequency Surgery of Flies of the sine wave was 110.5 Hz and the burst length was 400 ms; both parameters lay within the range In crossability tests the wings of male flies were removed with microscissorsunder carbon dioxide of the D. auraria complex (Tomaru & Oguma anaesthesiafor 3 min. About half the flies had their 1994b). Artificial songs consisted of regularly wings removed; the rest were used as winged male repeated ‘bursts with intervals of 50 ms (Fig. 2~). controls. All males experienced 3 min of anaes- Three types of pulse song were synthesized (Fig. thesia. In artificial song experiments, wings of all 2). The inter-pulse intervals of each pulse song D. biauraria males were removed and their anten- were 13, 16 and 11 ms, which correspond to and nae were also removed from the base with forceps mean values of D. biauraria, D. triauraria D. subauraria, respectively. The random noise under 3 min of carbon dioxide anaesthesia. song was synthesized as a burst of data points randomly distributed along the time axis (Fig. 2a). Crossability Tests Songs were synthesized on an MS-DOS machine, A pair of flies was introduced into a glass transmitted to a Sony cassette-tape recorder mating chamber (15 mm in diameter x 3 mm high), WM-R707 via a Canopus digital to analogue

Tomaru et al.: Songs elicit rejection (a) Nylon net Acrylic cylinder \

I

lbp view Plug in accesshole Sideview Cb)

-7

i 6.5 mm

Loudspeaker \

Topview

907

introduced with an aspirator into the mating chamber for artificial song experiments. Artificial songs were played to the pair and the courtship behaviour was observed for 30 min. The number of pairs showing orientation, the number of attempted copulations and female responses (accepting a male, wing fluttering and kicking) and copulation latency were recorded. Fluttering and kicking are kinds of female rejection behaviour. Observations were made during the light period (0900-1630 hours) in a room at 24.5 f 1~0°C.Since the equipment to produce artificial song can produce only one kind of song at a time, not only song conditions but also the time of experiment may affect the results. To avoid such effects, we conducted five types of song experiments on the same day and randomized the order of song types by three different 5 x 5 Latin squares. We identified four types of female responses during attempted copulation: copulation without fluttering or kicking, copulation with fluttering or kicking, failure to copulate without fluttering or kicking, and failure to copulate with fluttering or kicking. In copulation with fluttering or kicking a female first flutters her wings or kicks the male but eventually accepts him. We did not record any rejection behaviour other than fluttering or kicking.

‘40 mm

Loudspeaker Figure 1. Diagram of the equipment used for the artificial song experiments. (a) A mating chamber. (b) Experimental settings. Six mating chambers were arranged in a circle above a loudspeaker. converter Sound Master, and recorded on a Hitachi Maxell US II tape. They were played back

from a loudspeaker and received by a Sony eleccondenser microphone ECM-55B for analysis. Although there wera’some additional pulses with themain pulses (Fig. 2b), the inter-pulse intervals of artificial song were not altered in playback [TableI). Pulses of courtship song in D. biauraria, D. triauraria and D. subauraria consist of one or wo additional pulses (Tomaru & Oguma 1994b).

tret

Statistical

We used the chi-squared test for contingency tables to detect differences between several courtship elements under different song conditions (Zar 1984). Multiple comparisons followed as pair-wise comparisons of the Fisher exact test with the sequential Bonferroni technique (Rice 1989). After applying the Bartlett homoscedasticity test on logarithmically transformed copulation latenties, we used the analysis of variance (ANOVA) to detect differences under different song conditions (Zar 1984).

RESULTS Crossability

Observation with artificial

songs

A pair of flies, a D. biauraria female and a wingless, antennaless D. biauraria male, was

Procedures

Tests

Figure 3 shows the mating frequencies (the percentage of mating pairs compared to the number of pairs showing attempted copulation) of

908

Animal

Behaviour,

50, 4

Tomaru et al.: Songs elicit rejection Table

909

I. Parameters of artificial songs sampled from a burst for each song type Played back

Synthesized Song me

Number of pulses per burst

Inter-pulse interval Wt

Burst length W

Number of pulses per burst

Inter-pulse interval (m4 t

ms 16ms llms

32 26 37

12.99 f0.000 15.99 f 0~000

402.79 399.66 395.92

32 26 37

12.93 f OGl7 16.06 f 0.057 10.98 f 0.036

13

11.oo + 0.000

Burst length (ms) 400.86 401.47 395.37

Artificial song synthesized on a personal computer.

winged, conspecific wingless and heterospecific wingless males (Tables II and III). Since interspecific copulation was decreased when the male’s wings were intact, we can conclude that the heterospecific courtship song elicits female rejection that prevents copulation. There was no effect of cross type on mating speed (z* s~=499 f 63.1 s for conspecific winged males, 512 & 65.5 s for conspecific wingless males, and 498 & 62.5 s for heterospecific wingless males; Bartlett test: P>O.2; ANOVA: 1;,,,,=0.269, PBO.7). Artificial

Y 0

I

1

5

10

1

A

15 20 Time (min)

I

\

25

30

Figure 3. Cumulative mating frequency in the crossability tests. n : Conspecific winged; 0: conspecific wingless; 0: heterospecific wingless; A: heterospecific winged. D. biauraria females in different male cross types and Table II shows the numbers of pairs showing orientation, attempted copulation and copulation frequency of pairs with attempted copulation. No heterospecific (D. triauraria) winged males copulated with a D. biauraria female, whereas more than 60% of D. triauraria wingless males showing attempted copulation had successful matings. The mating frequency of heterospecific winged males was significantly lower than that of conspecific

Song Experiments

The mating frequency was highest with the conspecific (13 ms) song condition, intermediate with silence and random noise and lowest with the heterospecific (11 and 16 ms) songs (Fig. 4, Tables IV and V), showing that heterospecific types of inter-pulse interval elicited female rejection of males. Fluttering or kicking was more frequent with 16 ms (heterospecific) song than with 13 ms (conspecific) song but not more than with 11 ms (heterospecific) song (Tables IV and V). For attempted copulations, fluttering and kicking were most frequent with the heterospecific (16 and 11 ms) song and least frequent with the conspecific (13 ms) song (Fig. 5, Table V), suggesting that heterospecific songs elicited fluttering or kicking and conspecific song inhibited such behaviour. Copulation frequency per attempted copulation was intermediate with random song or silence and highest with conspecific (13 ms) song (Fig. 5, Table V). The heterospecific inter-pulse intervals (16 and 11 ms) inhibited copulation and the conspecific inter-pulse interval (13 ms) accelerated it. More pairs attempted copulation with the silence than with the other song types (Tables IV

910

Animal

Behaviour,

50, 4

Table II. Number of pairs showing orientation and attempted frequency in the crossability tests of D. biuurariu females heterospecific D. friuururiu males

Male Conspecific Conspecific Heterospecific Heterospecific

type

Attempted copulation

Orientation

winged wingless winged wingless

copulation, and copulation with conspecific males or

55 55 50 47

Copulation

49 47 33 36

87.8” 76.6” Ob 63.9” 71.81*

xv N (observed pairs)=57. Values with the same superscript (pair-wise comparison of Fisher exact test with the a=0.05 for six tests (k=6), see Table III). tChi-squared value comparing numbers of pairs with (but showing attempted copulation). *Pco+Ol. Table III. crossability

Male

Pair-wise comparisons tests with D. biuuroriu

of Fisher females

exact

Conspecific Conspecific Heterospecific

winged wingless winged

*Significantly (k=6).

different

0.1220

with

the sequential

and V) suggesting artificial song alters male behaviour. There was no effect of song type on mating speed (x&s~=635 * 79.3 s for 13 ms song, 743 f 164.6 s for 16 ms song, 742 f. 117.9 s for 11 ms song, 492 f 99.8 s for random song and 829 f 85.9 s for silence; Bartlett test: P>O.9; ANOVA: F4,,99= 1.345, P>O.2). DISCUSSION Heterospecific courtship song elicited fluttering and kicking and inhibited copulation in D. biauraria females, although courting males were accepted when there was no song or random song (Tables II and IV, Figs 3,4 and 5). This is the first report clearly demonstrating female rejection in the presence of heterospecific courtship song. No D. melanogaster females have been reported to show such rejection; both mating frequency and mating speed decrease in the presence of hetero-

letter are not significantly different sequential Bonferroni technique at copulation

tests for

Conspecific wingless

type

Bonferroni

(%)

and without

copulation

copulation

frequencies

in the

Heterospecific winged

Heterospecific wingless

o~oooo* o~oooo*

0.0096 0.1536 o~oooo*

technique

at (x=0.05

for

six tests

specific songs but not as much as if there is no song (Bennet-Clark & Ewing 1969) or if random song is played (Kyriacou & Hall 1982). Crickets and katydids do not reject or avoid a male in the presence of heterospecific sounds either (Gwynne & Morris 1986). The artificial song experiments also demonstrate that a conspecific inter-pulse interval inhibits female fluttering and kicking and increases the mating frequency (Table IV, Fig. 5). Depending on the inter-pulse interval, therefore, a female given an artificially synthesized courtship song either rejects or accepts a courting male. Clearly it is the female responsesthat play an important role in sexual isolation, and the inter-pulse interval is a speciesdiscriminator. With random noise and silence, the frequencies of fluttering and/or kicking and of copulation were intermediate to those with heterospecific and conspecific songs (Fig. 5). Assuming that female responses observed with random song and silence represent the initial female responses without any

Tomaru et al.: Songs elicit rejection

0

5

10

15

20

25

30

Time (min) Figure 4. Cumulative mating frequency in the artificial songexperiments; 13 ( n ) ms song is conspecific, 11 (a) and 16 (A) ms songs are heterospecific. 0: Silence; x : random.

soundstimuli, a heterospecific inter-pulse interval elicitsfluttering and kicking and inhibits female acceptance,and a conspecific inter-pulse interval

911

inhibits rejection. Therefore although these two behaviour patterns are qualitatively different, the inhibition between rejection and copulation is reciprocal. Whether the reciprocal inhibition observed at a behavioural level comes from the inhibition in the central nervous system remains unresolved. In nature females can compare and choose males on a food substrate. So they can use both absolute and relative criteria in their mate choice. In the present study each female heard only one kind of song, so we could not detect whether females use relative mate choice criteria. Since the heterospecific inter-pulse interval elicited female rejection, however, it is clear that a female uses absolute criteria when she rejects a courting male. It is nevertheless likely that female discrimination for courting males using both absolute and relative criteria is more precise than if only an absolute criterion is used (Hoikkala & Aspi 1993). Males of D. triuuraria frequently attempted to copulate with D. biuuraria females (Table II) and the two species are sympatric (Kurokawa 1967; Kimura 1987) so it is plausible that sexual isolation between them depends on the female’s discrimination ability for the sounds elicited by a courting male in nature and that both relative and absolute criteria play a role in sexual isolation. Although songs from males with intact wings inhibited interspecific copulation completely within 30 min (Fig. 3), in more than 20% of pairs, copulations were observed with synthesized

Table IV. Number of pairs showing orientation and attempted copulation, and frequencies of copulation and rejection under five song conditions in the artificial song experiments in D. biauraria

Song type

Orientation

13ms 16ms 11 ms Random Silence x’t

86

7.5 83 83 88

Attempted copulation 54b 4tP 53"b 42b 72" 18.72**

Copulation W) 66.7" 31.3b 30.2b 47.6"b 5 1.4ab 20.61**

Copulation with fluttering or kicking (%) 22.2b 66.7-56.3ab 30.0ab 37.8ab 11.85*

N (observed pairs)=90. Values with the same superscript letter are not significantly different (pair-wise comparison of Fisher exact test with the sequenti: Bonferroni technique at ~0.05 for 10 tests (k= lo), see Table V); 13 ms song is conspec fit, 16 and 11 ms songs are heterospecific. tChi-squared value. *f-0.05;

**I-0.001.

Animal

912

Behaviour,

50, 4

Table V. Pair-wise comparisons of Fisher exact tests for behaviour in the artificial song experiments with D. biuurariu

Song type 13ms

16ms

16ms

11 ms

Random

1.oooo

1.oooo

04004* oaO40* ooooo* omoo*

0.0002* 0.0196 oaooo* o~oooo*

0.0743 0.0475 1~0000 0.0080 0.053 1

0.0040* 0.1022 0.2030 o~oooo*

1~0000

0.1082

1 .oooo 0.7160 1 .oooo 1 .oooo

0.0849 0.0345 0.0271 0.0214

0.0083 0.0386 0.0566 0.1584 0.4020

0.0879 0.0635 0.1061 0.0298 0.0121

O+IO65 0.0275 0.1737 0.1756 0.3241

llms

Random

Silence

o~oooo*

o~oooo* 0.7034 0.7718 1 .oooo 1 .oooo

Rows: 1, attempted copulation; 2, copulation; 3, between copulation with fluttering or kicking and copulation without; 4, fluttering and kicking for each attempted copulation; 5, copulations for each attempted copulations; 13 ms song is conspecific, 16 and 11 ms are heterospecific. *Significantly different with the sequential Bonferroni technique at a=O.OS for 10 tests (k= 10)

heterospecific inter-pulse intervals (Fig. 4). There are two reasons why the artificial heterospecific songs could not inhibit mating completely. First, the artificial songs failed perfectly to mimic real songs; some song parameters may have been lacking (Bennet-Clark & Ewing 1969; von Schilcher 1976; Ikeda et al. 1981; Kyriacou & Hall 1982; Crossley & Bennet-Clark 1993). For example, the artificial song may fail to reach the female at the appropriate time. Although artificial songs were constantly repeated in bursts of pulse song (Fig. 2c), songs produced by males are less frequently repeated during courtship. Therefore, different timings allowed wingless males to copulate even with the heterospecific inter-pulse interval. Since synthesized artificial songs were fixed for burst length (400 ms) and interburst interval (50 ms), the number of pulses given to females differed: 11 ms songs included 40% more pulses than 16 ms songs did (Table I). A female may discriminate a courting male on the number of pulses, but this cannot explain why

females were most receptive with the intermediate inter-pulse interval (13 ms). The number of pulses per burst is also detectable by females, but since it is not a species-specific parameter (Tomaru & Oguma 1994b), it cannot act as a species discriminator. Second, the sound pressure level of 120 dB may have been too high to inhibit copulation completely. The estimated sound pressure of real courtship song is less than 119 dB in D. melanogaster (Bennet-Clark & Ewing 1969). In D. parabipectinata, male movements and courtship interactions between males have been observed at sound levels between 66 and 120 dB (Crossley & Bennet-Clark 1993). Successful copulations of wingless males have been observed at sound levels of 84 and 100 dB in D. melanogaster, D. simulans and D. parabipectinata (Bennet-Clark & Ewing 1969; Kyriacou & Hall 1982; Crossley & Bennet-Clark 1993). Although artificial song experiments with altered sound level may be needed to examine the effective level in

Tomaru et al.: Songs elicit rejection

B 3 B 8

913 Number of

Rejection

Copulation

b

a

62

16ms-

a

b

69

11 ms

a

b

78

ab

ab

49

a

b

135

42.22*

30.66*

I

I

0

20

I

I

40 60 Response (%)

I

I

80

loo

x2

attempted

copulations

&rue 5. Female responses during attempted copulations; 13 ms song is conspecific, 11 and 16 ms songs are heterospecific. The chi-squared value in fluttering and kicking (Rejection) and in copulation frequency per attempted copulation (Copulation) between five song types are shown. Responses that have the same letters are not significantly different by pair-wise comparison with the sequential Bonferroni technique (Table V). *P
it is likely that the 120 dB of artificial song was sufficient to test the effectiveness of inter-pulse interval. More pairs attempted copulation with no song than with the artificial songs except 11 ms song (TableIV), suggesting that the flies’ sexual activity wasaffected by the artificial songs. All the males in all five song conditions had their antennae removed and it is unlikely that their consequent inability to receive pheromones differentially affected their sexual activity. It is also unlikely that a male altered his behaviour when he heard the artificial song. It is plausible that the female’s behaviour in the presence of artificial songs includessignals to enhance or to inhibit the male’s copulation attempts. Female behaviour influences male behaviour in several ways (Ewing 1983). Female movement, for example, is one of the important stimuli eliciting male courtship in the D. auraria complex (Y. Oguma, J.-M. Jallon, M. Tomaru & H. Matsubayashi, unpublished data). Although we did not record female behaviour quantitatively, the number (and possibly also quality) of female movements may have changed with the artificial songs. In most sympatric Drosophila species, premating isolation evolved more rapidly than post-matingisolation whereas pre-mating and postmating isolation evolved at comparable rates in

D. biauraria,

allopatric species(Coyne & Orr 1989). Coyne & Orr concluded that this observation could be explained by ‘reinforcement’. This phenomenon was also observed between the speciesof the D. auraria complex. Allopatric D. qua&aria females accept foreign males but females of D. biuuraria, D. triaurariu and D. auraria do not accept males of these species (Kurokawa et al. 1982). The inter-pulse intervals of courtship song differ between sympatric speciesbut not always between allopatric ones (Tomaru & Ogurna 1994b). The present study revealed that a female’s choice of male depends on the interpulse interval of his courtship song; heterospecific inter-pulse intervals elicit female rejection and conspecific inter-pulse intervals make a female receptive. This evidence implies that during speciation of the D. am-aria complex, reinforcement, reproductive character displacement, or both has occurred and that the selected male characteristic is the inter-pulse interval, as Sved (1981a, b) modelled mathematically. It is also possible that the evolutionary scenario of sexual selection was initiated by either reinforcement, reproductive character displacement, or both. If so, the direction of sexual selection might be restricted by the other sympatric species. This scenario suggests that species recognition plays an important role in sexual selection. Artificial song experiments with

ur, 50, 4 various whether variation

inter-pulse ltervals shollld clarify sexual selecti In is now operating on in the inter-pi Ise interval.

ACKNOWI

EDGMENTS

We thank Dr S. A. Crctssley for her kind advice for using specific mating chambers in the artificial song experiments. We also thank Dr S. A. Crossley and Dr D. M:lcer for critically reading the manuscript.

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