Behavior Genetics, Vol. 31, No. 3, May 2001 (©2001)
Compared Ontogenesis of Courtship Song Components
of Males from the Sibling Species, D. melanogaster
and D. simulans
Bruno Moulin,1,2 Fanny Rybak,1 Thierry Aubin,1 and Jean-Marc Jallon1
Received 7 July 2000—Final 2 Feb. 2001
The courtship song of Drosophila is known to be an important signal involved in sex and species
recognition. It consists of pulse song and sine song, which have been studied in a quantitative
way with different parameters. For the first time the setting of both components of the acoustic
signaling is described and compared for males belonging to the sibling species D. melanogaster
and D. simulans. At early ages, these two species share similar interpulse interval values but
maturation establishes the species specificity of this character. For D. melanogaster the variations of several acoustic parameters take place in two successive periods, whereas for D. simulans the majority of the acoustic features does not change much with age. In D. melanogaster,
copulation success seems to be linked to the maturation of the acoustic performance, which is
not the case for D. simulans.
KEY WORDS: Ontogeny; courtship song; Drosophila.
INTRODUCTION
In these species, a courting male typically orients
toward a female and then proceeds to tap her with his
foretarsi, to extend one wing and vibrate it, to lick her
genitalia with his proboscis, and, finally, to attempt to
copulate (Manning, 1959). The stimuli involved are
visual, chemical (either through contact or airborne
factors), and auditory, but the importance of each channel of information transmission might vary between
species. Such information may be produced and sensed
by either sex. The male is traditionally considered to
be the sex partner which initiates courtship after responding to female visual and chemical stimuli, while
females would make the final decision after analysing
the male acoustic signals (Ewing, 1983).
In this stereotyped courtship, the acoustic signals
are produced by the male’s wing vibrations. For six of
the eight species of the subgroup, the song consists
of two discrete elements known as pulse song and sine
song; however, two other species D. sechellia and
D. yakuba have not been shown to perform the sine
song (Cowling and Burnet, 1981; Cobb et al., 1989).
The pulse song is composed of tone pulse trains. The
Ethological isolation mechanisms are barriers to
mating which are due to incompatibility of behaviours,
especially courtship (Mayr, 1963). Drosophila is a
good model system for such studies as courtship behavior, first described in D. melanogaster by Bastock
and Manning (1955), was soon compared with that in
the sympatric species D. simulans (Manning, 1959).
Actually, D. melanogaster and D. simulans are members of the so-called melanogaster subgroup, consisting of nine closely related species which display similar qualitative traits in courtships, and have been the
objects of very detailed descriptions (Cobb et al., 1986;
Welbergen et al., 1987).
1
NAMC—CNRS UMR 8620, Université Paris-Sud, F-91405 Orsay
Cedex, France.
2
To whom correspondence should be addressed at Laboratoire de
Neurobiologie de l’Apprentissage, de la Mémoire, et de la Communication (NAMC), CNRS UMR 8620, Bâtiment 446, Université
Paris-Sud, F 91405 Orsay Cedex, France. E-mail: Bruno.Moulin@
ibaic.u-psud.fr
299
0001-8244/01/0500-0299$19.50/0 © 2001 Plenum Publishing Corporation
300
time between consecutive pulses is referred to as the interpulse interval (IPI). It is a species-specific parameter which might allow females to recognize conspecific
males and increase their sexual receptivity (BennetClark and Ewing, 1969; Kyriakou and Hall, 1982).
The sine song consists of a modified sine wave which
seems important for the female sexual stimulation (von
Schilcher 1976a, b).
Most courtship observations reported up to now
involved mature flies, in which all the components of
the courtship are well tuned. However, flies of either
sex are not ready to court and mate immediately after
imaginal hatching. Jallon and Hotta (1979) have reported the building-up of male courting ability during
days, following wing extension. Moreover, the age of
the flies appears to be an important factor for the crossability of the two species (Ashburner, 1989), in which
the acoustic signaling is to play a role.
Thus it seems important to describe the ontogenies of courting and mating abilities of both sibling
species D. melanogaster and D. simulans. In the present study, fine changes with age of both pulse and sine
songs will also be studied in details (a few new parameters are introduced to describe these love song
components better). Finally, we look for correlations
between the maturation of the male copulating ability
and that of parameters of the acoustic signaling.
MATERIALS AND METHODS
Flies
Flies of the D. melanogaster Canton S strain (an
old laboratory strain) and D. simulans strain Seychelles
(collected in the Seychelles archipelago in 1981) were
grown on a standard corn/agar medium at 25 ⫾ 0.5°C
under a 12:12-h light:dark cycle. Sexes were separated
within 1 h of emergence under light carbon dioxide
anesthesia. Flies were then transferred to autoclaved
food vials. Virgin males, with wings of similar size,
were aged individually, while virgin females were aged
in groups of five flies.
During recording sessions, the temperature (measured with an electronic thermometer) was kept constant at 25 ⫾ 1°C. It was regulated by a heating/cooling
system. Females used for the courtship tests all had the
same age, 4 days for D. melanogaster, and 6 days for
D. simulans. For males of each species six age classes
were selected: 12 h, 17 h, 22 h, 27 h, 33 h, and 3 days
for D. melanogaster and 1, 2, 3, 4, 5, and 6 days for
D. simulans.
Moulin, Rybak, Aubin, and Jallon
Copulation Kinetics
In copulation kinetics we followed the variations
of the cumulative number of pairs of flies mating versus time. A couple of flies, involving a mature female
and a male of a different age, were introduced in a
cylindrical chamber 10 mm in internal diameter and
5 mm in depth. This chamber was built from Teflon.
Its roof was made of transparent Perspex to allow behavioral observations. A small aperture in the wall
allowed the introduction of flies into the chamber. The
number of matings was counted each minute over
30 min. For each species and each age class, 50 couples were observed.
Recording Method
We have used the method described by Aubin
et al. (2000), which is based on the simultaneous
recording of the acoustic signal to be studied with ambient noise and the ambient noise alone. A simple subtraction between these two signals allows the isolation
of the signal of interest with a good signal-to-noise
ratio. Recordings were made in a room without any particular acoustic insulation. To record the signals emitted by the flies, the same chamber as in the copulation
kinetics experiments was used. As its size corresponded
to the diameter of the microphone grid, the floor of
the chamber was the microphone itself, protected by a
fine nylon mesh. We used two 4176 Bruel & Kjaer
1/2 prepolarized condenser microphones, which are pressure sensitive, coupled to a preamplifier. The distance
between both microphones was 6 cm. Both signals were
recorded simultaneously from either microphone on
the two channels of a Sony TCD3 DAT recorder (frequency response curve flat between 20 Hz and 20 kHz)
at a sampling frequency of 48 kHz. The signal-to-noise
ratio and dynamic range were both more than 90 dB. All
recordings were made between 0900 and 1200 AM.
Analysis Method
The signal recorded on the two channels of the
DAT recorder corresponded to 16 bits of digital data.
These data were transferred to a PC computer by means
of an Audiomedia III PCI acquisition card interface and
then stored as files on the hard disk of the computer for
all subsequent processing and spectral analysis. There
was no time delay between the data in both files. Subtraction between files was carried out using a software
written in C programming language.
Ontogeny of Drosophila Males’ Courtship Song
301
Signals were examined in the amplitude versus
time domain, using the Syntana package software built
in our laboratory (Aubin, 1994).
As described by Burnet et al. (1977), and IPI was
measured as the time in milliseconds between the major
positive peaks of two successive pulses. Each IPI was
measured peak to peak directly on the computer screen
with the help of a cursor with a reference time base.
Time values measured were automatically stored in an
ASCII file for subsequent analysis. Time resolution as
measured on the screen was of 125 ⫻ 10⫺6 s. Following the procedure described by Wheeler et al. (1988),
the maximum IPI value was defined as 100 ms. Thus,
any “silence” interval between two pulses longer than
this value was considered to be an interburst interval.
For measurements of brief sound impulses in the frequency domain, a FFT analysis is not satisfactory (Randall and Tech, 1987) and so a frequency analysis was
performed using the zero-crossing method (F ⫽ 1/T;
F, frequency in Hz; T, period of the pulse in s).
Statistical Analysis
When values were not normally distributed, nonparametric tests were used: Kruskal–Wallis test for
K independent samples and Mann–Whitney U test for
two independent samples. Significance levels were corrected using the Bonferonni Dunn–Sidak method (Ury,
1976). The Spearman’s rank test was used for correlation coefficient estimations. An ANOVA test was used
for D. melanogaster and D. simulans intrapulse frequencies and for D. simulans intrasine frequencies, as
these data were normally distributed.
For each distribution of IPI, the values of the kurtosis coefficient (k) were calculated according to Sokal
and Rohlf (1981).
RESULTS
Copulation Kinetics Analysis
Figure 1 compares copulation kinetics of pairs involving a mature female and a male of a different age,
both belonging to either D. melanogaster (Fig. 1a) or
D. simulans (Fig. 1b).
For D. melanogaster, all females were 4 days old.
While no courtship behavior of males less than 12 h
old could be observed, the copulation kinetics of 33h-old conspecific males were similar to those of 3 days
old. They reached a close saturation level (艐90%) in
about 15 min and thus were considered mature for
Fig. 1. Copulation kinetics for males of different ages in
D. melanogaster (a) and D. simulans (b) together with a mature —
4-day-old (me)/6-day-old (sim)—female.
1a: 䊉 12 h; * 17 h; x 22 h; 䉱 27 h; 䊏 33 h; 䉬 3 days.
1b: 䊉 1 day; * 2 days; x 3 days; 䉱 4 days; 䊏 5 days; 䉬 6 days;
ⵧ 7 days.
their courtship performing ability. Comparing the kinetic curves with males of different ages with a
Kruskal–Wallis test yielded the value H ⫽ 50.67, with
p ⬍ .0001 (df ⫽ 4; n ⫽ 80). With males of 22 and 27 h
the levels of copulation in 30 min were lower, respectively, 56 and 76%. For younger males (12–17 h), the
proportions of copulations were very low, reached 8
and 16%, respectively, in 30 min. A comparison with
a Mann–Withney U test showed that each series of
values was significantly different from each other
( p ⬍ .005) except for 22 and 27 h (Zc ⫽ 1.55266). So,
whichever the tested age, at least a few D. melanogaster males displayed courtship and succeeded
in copulating with mature females, although their
percentages were very different.
In contrast for D. simulans, with 6-day-old females,
conspecific males less than 1 day never displayed
302
Moulin, Rybak, Aubin, and Jallon
courtship during 30 min, and only a few did so with
2 days of age. Copulation kinetics presented in Fig. 1b
were compared for males with different ages (1–7 days)
with a Kruskal–Wallis test: H ⫽ 54,65 with p ⬍ .0001
(df ⫽ 5; n ⫽ 95). From the age of 3 days, a larger number mated, which increased up to 6 days. At that age 72%
of D. simulans males mated in 30 min, a lower number
than that obtained with mature D. melanogaster males.
The 1- to 2-day groups were different from the 3-, 4-, or
5-day groups ( p ⬍ .01) and the 6- to 7-days groups were
different from the 3-, 4-, and 5-days groups ( p ⬍ .05).
Percentages of Courtship Durations Spent in
Singing
During courtship, D. melanogaster and D. simulans males produce songs consisting, in both species,
of bursts of pulse song (PS) and bouts of sine song (SS)
(Fig. 2). For each species and age class, the signals produced during 10 independent courtships involving different males were recorded and analyzed. The PS and
SS total durations were measured in each courtship sequence, and then the proportions of each component
were calculated.
For D. melanogaster males (Fig. 3a), the fractions
of courtship duration spent in pulse song (%PS) and in
sine song (%SS) both increased with age up to 33 h in
a linear way, although the correlation coefficient was
higher with the former parameter (r ⫽ .98 and r ⫽ .87,
respectively). Thus the %SS evolution parallels that of
the %PS. There was also a linear increase in the maximum percentage of copulation with the two parameters, age and %PS (Fig. 3b). The correlation coefficient
value obtained with a Spearman test was r ⫽ 1.
Changes in both percentages with age were very
different for D. simulans males. Actually there was
nearly no variation of the fraction of the courtship time
spent in pulse song (%PS) during male maturation. In
this species, however, significant differences for the
parameter %SS were observed between 2, 3, and 4 days
versus 6 days ( p ⬍ .01) (data not shown).
Detailed Ontogeny of Acoustic Parameters
To characterize thoroughly the changes with age
of pulse song, the following parameters were measured
at different ages: pulse intrasignal frequency (IPF), median interpulse interval (IPI), mean number of pulses
per burst (NPB), and mean number of bursts per minute
(NBM). For the sine song we measured the variations,
with male age, of three parameters: sine intrasignal
frequency (SSF), mean sine bout duration (SbD), and
mean number of sine bouts per minute (SbM).
Fig. 2. Comparison of the love song components of mature males of either D. melanogaster (a) or D. simulans (b).
Ontogeny of Drosophila Males’ Courtship Song
303
Table I. Variations with Male Age of Intrasignal Pulse
Frequency (IPF), Sine Song Intrasignal Frequency (SSF),
and Mean Sine Bout Duration (SbD) for D. melanogaster
(a) and D. simulans (b) (Mean ⫾ SD)
Age
IPF (Hz)
SSF (Hz)

SbD (ms)
a
12
17
22
27
33
h
h
h
h
h
179.3
185.7
187.5
193.9
197.5
⫾
⫾
⫾
⫾
⫾
18.0
24.1
25.2
20.7
14.1
152.3
147.8
151.8
151.9
157.1
⫾
⫾
⫾
⫾
⫾
5.6
6.3
5.6
5.1
4.4
⫾
⫾
⫾
⫾
⫾
⫾
26.0
28.8
27.4
20.0
26.9
25.8
373.1
503.8
523.7
754.6
521.9
⫾
⫾
⫾
⫾
⫾
31.3
73.5
65.4
79.8
45.0
b
1
2
3
4
5
6
Fig. 3. (a) Variations in the percentages of pulse song (%PS) and
sine song (%SS) durations with age during total courtship for
D. melanogaster males. Left scale: %PS (䊊). Right scale: %SS (ⵧ).
The variations were linear, following the equations %PS ⫽ 2.19x ⫹
220.18 (— —) and %SS ⫽ 9.04x ⫺ 909.33 (. . . .). (b) Linear increase in the maximum percentage of copulation with the two parameters; age and %PS.
Drosophila melanogaster Male Songs
For D. melanogaster males, 16,709 pulses for five
age groups were studied. The pulse intrasignal frequency values (IPF) showed significant differences
with age (ANOVA F test, p ⬍ .001) (Table I). The contrast method revealed that the difference was more
marked between the two groups of either younger males
(12–17 h) and older males (27–33 h), but between these
two age classes, the IPF value increased by only 7%.
IPI values have been calculated for different individuals of different ages (Table II). Figure 4 compared
the IPI values distributions at different ages. At 12 h
their distribution was multimodal but became clearly
unimodal at 22 h. Then remaining unimodal, its width
day
days
days
days
days
days
484.7
482.0
480.5
487.5
483.6
477.0
⫾
⫾
⫾
⫾
⫾
⫾
36.1
46.4
37.5
40.6
49.3
51.3
189.9
192.1
193.7
190.1
193.9
192.7
593.5 ⫾ 93.5
524.4 ⫾ 65.3
440.8 ⫾ 68.3
463.5 ⫾ 51.1
520.4 ⫾ 83.4
1022.9 ⫾ 166.5
became narrower with a marked increase in the kurtosis coefficient value. The age variations of median IPI
values shown in Table II and Fig. 5a were clearly biphasic: it markedly decreased between 12 and 17 h (⫺32%
in 5 h) and then varied only slowly (⫺16% in 2 days).
The total variation is almost ⫺50%.
Meanwhile there was a gradual increase in the
mean number of bursts per minute (NBM) with age up
to 33 h, a parameter which then leveled off (Fig. 5a). A
Kruskal–Wallis test revealed a significant difference
Table II. Variations with Age of the Median IPI (⫾ Median Error),
Kurtosis Coefficient (k), and Mean Number of Pulses per Burst

(NPB ) for D. melanogaster (a) and D. simulans (b) Males (⫾SE)
Age
IPI (ms)
k

NPB
a
12
17
22
27
33
h
h
h
h
h
65.5
44.6
43.3
40.5
37.8
⫾
⫾
⫾
⫾
⫾
4.7
2.4
1.6
1.2
0.7
⫺0.75
⫺0.07
1.23
3.64
5.88
4.1
4.3
4.6
5.8
6.2
⫾
⫾
⫾
⫾
⫾
0.5
0.5
0.5
0.6
0.5
⫺0.40
0.34
⫺0.01
0.04
⫺0.10
0.10
5.6
6.5
5.4
5.0
5.4
5.5
⫾
⫾
⫾
⫾
⫾
⫾
1.1
1.0
1.1
1.0
1.0
1.0
b
1
2
3
4
5
6
day
days
days
days
days
days
57.0
55.7
50.9
55.3
49.7
50.6
⫾
⫾
⫾
⫾
⫾
⫾
2.1
1.2
1.8
2.1
1.0
2.0
304
Moulin, Rybak, Aubin, and Jallon
Fig. 4. Distributions of D. melanogaster male IPI values for five age groups.
Ontogeny of Drosophila Males’ Courtship Song
305
Drosophila simulans Male Songs
With Drosophila simulans males 16,079 pulses
were studied and the same parameters calculated as for
D. melanogaster males, but far fewer differences with
age were detected. For the parameter IPF (Table Ib),
an ANOVA test showed no significant difference between different ages (F ⫽ .3373, p ⫽ .8901). The distributions of IPI values looked unimodal and broad
whatever the age (see Fig. 6 and the kurtosis coefficient values with small variations in Table II). There
was a small decrease in the median IPI value with age
but differences were significant only between the extreme age groups (1–2 and 5–6 days) (⫺11%). The
parameter ( NBM) increased in a significant way between 1 and 2 days ( p ⬍ .05) but then did not change
much (Fig. 5b). The marked increase observed at
5 days is not continuous with the other age groups
[opposite to the variation between the groups of 1– 4
days ( p ⬍ .01) and 3–6 days ( p ⬍ .05)]. Moreover,
the(NPB) values showed no significant differences between groups of different ages including the 5-day
group (p ⫽ .2919).
Two thousand four hundred eighteen sine song
bouts were analyzed for the same age groups of D. simulans males. No significant differences were seen for
SSF values at different ages (F ⫽ .1445, p ⫽ .9814)
(Table Ib). Paired comparisons of (SbD) and (SbM)
values also did not show a significant difference between all age classes except for the 6-day group.
Fig. 5. Age-dependent variations of median InterPulse interval (IPI
〫) and mean Number of Bursts per Minute ( NBM 䊏) values for
D. melanogaster (a) and D. simulans (b).
between all age groups ( p ⬍ .001). There was also an
increasing tendency with age of the mean number of
pulses per burst (NPB) but differences appeared significant only between the two age groups (12–17–22 h)
and (27–33 h) ( p ⬍ .05) (Table II).
The ontogeny study of sine song involved the
analysis of 4880 sine bouts. Concerning the sine intrasignal frequency (SSF) parameter, only the 33-h group
was significantly different from all the others ( p ⬍ .01),
but the difference was small (⫹4%) (Table I). For the
mean duration of sine bouts (SbD), the performance of
the youngest males (12 h) was much shorter (⫺40%)
than that of the others; however, the 27-h group value
seemed abnormally high (Table I). Finally, there was
also a marked increase in the number of sine bouts per
minute (SbM) up to 27 h and then a plateau (data not
shown).
DISCUSSION
At maturation, the acoustic parameters of males of
both species, D. melanogaster and D. simulans, described here are similar to those described by other authors (Wheeler et al., 1988; Ritchie et al., 1994). The
small differences observed might be related to differences in the experimental conditions, especially the
temperature (Peixoto and Hall 1998), and differences
in strains, genetic backgrounds, and breeding conditions. During male ontogenesis, both components of the
mating song have been studied in detail and in both
species. However, the IPI rhythm described by Kyriakou and Hall (1989) has not been measured. Some of
the parameters studied are described for the first time,
especially the sine song bout duration.
Variations of all acoustic parameters did not happen during the same age periods. It is possible to distinguish at least two phases for D. melanogaster. During the early phase, which takes place the first day after
306
Moulin, Rybak, Aubin, and Jallon
Fig. 6. Distributions of D. simulans male IPI values for six age groups.
Ontogeny of Drosophila Males’ Courtship Song
hatching, marked modifications of IPI and (SbD) occur,
which might fix the temporal pattern of the signal. Especially the D. melanogaster IPI values show their maximum variability at that time; their distribution is then
multimodal, with one mode close to that characteristic
for mature D. simulans males and one mode close to that
measured in mature D. melanogaster males; later the distribution becomes unimodal and species specific. This
period might be of behavioral plasticity, an hypothesis
which will have to be checked in the future.
During the second phase, several parameters
change gradually, particularly those involving the repetition of the acoustic performance and, consequently,
the quantity of signals produced. This is especially
the case for NPB and NBM and also the case for the
proportion of time spent in either pulse singing or sine
singing, which are multiplied by a factor of 4 or 5 in
D. melanogaster. These parameters are also well correlated with the success of copulation. They might reflect increasing amounts of wing display partially
linked to increasing motor activity.
For D. simulans, almost no copulations were observed during the early period, which lasts for about
2 days in that species. During that time D. simulans
slows its IPI down to about 50 ms, a value apparently
less precisely defined than the D. melanogaster speciesspecific value as suggested by Figs. 4–6 and experiments reported by Kawanishi and Watanabe (1980).
Later changes in any acoustic parameter are neither
large nor continuous. However, the number of copulations strongly increases between 2 and 6 days. No correlation is then observed—as expected—between
changes in copulation success and any acoustic parameter as in D. melanogaster.
But during that ontogeny period, other sensory
messages change, which might influence mature female
choice and lead to less or more copulation (Ford et al.,
1989). In the early phase of the study, 17–22 h, young
males of D. melanogaster and D. simulans bear similar long-chain hydrocarbons (Pechine et al., 1988).
These gender-appeal rich substances have disappeared
by about 20 h after hatching in the D. melanogaster
Canton S strain (Antony and Jallon, 1981), while
7-tricosene is building up in males of the D. melanogaster species and males and females of D. simulans.
7-Tricosene reaches its maximal abundance earlier in D. melanogaster males (about 3 days) than in
D. simulans males (about 6 days) (Jallon, 1984; Arienti et al., personal communication). Actually the increase with age of D. simulans Seychelles (Ferveur and
Jallon, 1993) seems to follow the increase in copula-
307
tion success with age reported in this study for males
D. simulans. 7-Tricosene, which stimulates D. simulans males’ precopulatory behavior (Jallon, 1984),
might also play a role in modulating female receptivity in D. simulans as already suggested for D. melanogaster females (Jallon, 1984; Scott, 1994). But as its
volatility is low, its diffusion might have to be helped
by wing movement as in Cullicaides mellus (Linley and
Carlson, 1983). In this case, the maturation of the wing
movement might be involved in developing two stimulating factors which may modulate females’ receptivity in a synergistic way. This interaction of chemical
message and wing movement might play a more important role in D. simulans than in D. melanogaster.
Although the present study was concerned with
only one strain of either species, it evokes some evolutionary comment between D. melanogaster and
D. simulans, two species which are closely related in
the melanogaster subgroup (Lemeunier et al., 1986).
At earlier ages, acoustic patterns are closer and close
to that of mature D. simulans. Moreover, at maturity,
the variability of D. simulans IPI values is greater than
that of D. melanogaster, as already noted by Watanabe
and Kawanishi while studying different strains (1981).
This suggests that the D. simulans acoustic system is
less precise. At maturity, the proportion of IPI displayed by D. simulans males with D. melanogaster
specificity was near 20%. And actually D. simulans
males are accepted more easily by D. melanogaster females than D. melanogaster males are accepted by
D. simulans females (Lee and Watanabe, 1987). Including the non-sexually dimorphic chemical signaling, the mate recognition system of D. simulans seems
more primitive than that of D. melanogaster.
ACKNOWLEDGMENTS
We are grateful to Ginette Laugé and Michael
Ritchie for comments.
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Edited by Lee Ehrman and Yong-Kyu Kin