Variation in Courtship Ultrasounds of Three Ostrinia
Moths with Different Sex Pheromones
Takuma Takanashi1*., Ryo Nakano2., Annemarie Surlykke3, Haruki Tatsuta4, Jun Tabata5, Yukio
Ishikawa6, Niels Skals3,71 Forestry and Forest Products Research Institute, Ibaraki, Japan, 2 National Institute of Fruit Tree Science, Ibaraki, Japan, 3 Institute of Biology, University of Southern
Denmark, Odense, Denmark, 4 Graduate School of Agriculture, University of the Ryukyus, Okinawa, Japan, 5 Biodiversity Division, National Institute for AgroEnvironmental Sciences, Ibaraki, Japan, 6 Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan, 7 Nyborg, Denmark

Abstract
Moths use ultrasounds as well as pheromones for sexual communication. In closely related moth species, variations inultrasounds and pheromones are likely to profoundly affect mate recognition, reproductive isolation, and speciation. The
European corn borer, Ostrinia nubilalis, and its Asian congeners, Ostrinia furnacalis and Ostrinia scapulalis, exhibit withinspecies and between-species variation in their pheromone communication. Recently, we reported ultrasoundcommunication in O. furnacalis; however, variations in ultrasounds in the three congeners have not been addressed todate. Here we investigated features of ultrasound production and hearing in O. nubilalis and O. scapulalis, and comparedthem with those of O. furnacalis. As in O. furnacalis, males of O. nubilalis and O. scapulalis produced ultrasounds during
courtship by rubbing specialized scales on the wings against scales on the thorax. The covering of these scales with nailpolish muffled the sounds and significantly reduced mating success in O. nubilalis, showing the importance of ultrasoundsignaling in mating. The ultrasounds produced by O. nubilalis and O. scapulalis were similar, consisting of long trains of pairsof pulses with a main energy at 40 kHz, but distinctly different from the ultrasound produced by O. furnacalis, consisting ofgroups of pulses peaking at 50 kHz and with substantially more energy up to 80 kHz. Despite overall similarities, temporalfeatures and patterns of amplitude modulation differed significantly among the geographic populations of O. nubilalis andO. scapulalis, which differed in pheromone type. In contrast, no significant difference in hearing was found among the three
species with regard to the most sensitive frequencies and hearing threshold levels. The patterns of variations in the songs
and pheromones well reflected those of the phylogenetic relationships, implying that ultrasound and pheromone
communications have diverged concordantly. Our results suggest that concordant evolution in sexual signals such as
courtship ultrasounds and sex pheromones occurs in moths.
Citation: Takanashi T, Nakano R, Surlykke A, Tatsuta H, Tabata J, et al. (2010) Variation in Courtship Ultrasounds of Three Ostrinia Moths with Different Sex
Pheromones. PLoS ONE 5(10): e13144. doi:10.1371/journal.pone.0013144
Editor: Frederic Marion-Poll, INRA - Paris 6 - AgroParisTech, France
Received July 10, 2010; Accepted September 9, 2010; Published October 4, 2010
Copyright: ß 2010 Takanashi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported in part by research grants from the Japan Society for the Promotion of Science (T.T., R.N., Y.I.), the Forestry and Forest
Products Research Institute (FFPRI) (T.T., R.N., Y.I.), Encourage Model for Researchers with Family Responsibilities of FFPRI (TT), and the Danish Natural Science
Research Council (A.S., N.S.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: takanasi@affrc.go.jp. These authors contributed equally to this work.

ca. 46 dB SPL at 1 cm [6,7]. These songs increase the mating
success of the males by making the females motionless, whichcorresponds to the freezing response elicited by ultrasonic bat calls[7,8]. In addition to the sound pressure levels, temporal andspectral features of the sound should be under the control of
selection pressures imposed by conspecific female mates and/or
unwanted eavesdroppers. There is only one well-known example
in moths (i.e., Achroia grisella), where females show preference for
specific temporal and spectral features of ultrasonic calling songs ofconspecific males [3,9]. Despite the importance of the variation in
temporal and spectral features of the sound to mate recognition,
reproductive isolation, and speciation, no comparative study on
variations of ultrasonic courtship songs among closely related
moth species has been made to date.
Sexual communication using female sex pheromones iswidespread across various moth species [3,10]. Females of Ostrinia
species release specific sex pheromones in order to attract

Introduction
Moths have tympanal ears sensitive to ultrasound. The tuning of
hearing to bat calls as well as the degeneration of hearing in batfree areas indicates that ears of moths have most likely evolved tocounteract predation by insectivorous bats [1,2]. The location andmorphology of ears vary across superfamilies of moths, suggesting
the independent evolution of ears in each taxon after thedivergence of superfamilies [2,3]. Subsequent to the developmentof ears, a relatively small number of moth species developed
sound-producing organs, and utilized the sound either for defense
against bats or rival males, or for attracting mates [3–5]. The
ultrasounds used in these contexts are characterized by high soundpressure levels (SPL) ranging from 76 to 125 dB SPL at a distanceof 1 cm [6].
Males of the Asian corn borer moth, Ostrinia furnacalis(Crambidae), produce low-intensity ultrasonic courtship songs of
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conspecific males from a distance [11]. The European corn borer,
O. nubilalis, and the Adzuki bean borer, O. scapulalis, have a similar
pheromone, a mixture of (Z)-11- and (E)-11-tetradecenyl acetates(E11- and Z11-14:OAc) [12,13], whereas O. furnacalis uses a
mixture of different positional isomers, (Z)-12- and (E)-12tetradecenyl acetates (Fig. 1) [14,15]. Interestingly, O. nubilalis
and O. scapulalis show similar polymorphisms in the blend ofpheromone components: E-type females produce a pheromone
with 97–99% E11-14:OAc and 1–3% Z11-14:OAc, whereas Ztype females produce a pheromone with the opposite blend[12,13,16–19]. This variation in the two species is geneticallycontrolled by a single autosomal locus with two alleles [17,19]. In
O. nubilalis, male behavioral responses to the female pheromone
types are controlled by sex-linked gene(s) [17]. There seems to be
Z and E ‘races’ of O. nubilalis that produce and respond to Z and E
type pheromones, respectively [16,17]. The Z race occurs in mostregions of Europe and North America, whereas the E race occurs
in limited regions [16,20–22]. At some localities in France where
the Z and E races of O. nubilalis co-occur, strong assortative mating
within the races was found to be occurring [22–24]. In Japan, theextent of assortative mating between the pheromone types of O.
scapulalis seems to vary among localities [19].
The present study was designed to explore variation of courtship
ultrasounds in three Ostrinia moths, O. nubilalis, O. scapulalis, and O.
furnacalis. Ostrinia moth species are ideal for such research purposes
because they show remarkable diversity in sex pheromone
communication [11]. First, sound-producing organs of O. nubilalis
and O. scapulalis and the function of ultrasound in the mating
behavior of O. nubilalis were examined. Second, we investigatedvariations in the courtship songs in the populations of O. nubilalis
and O. scapulalis (Table 1) in terms of temporal and spectralfeatures, sound levels, and amplitude modulations. Third, hearing

in Ostrinia species was analyzed with reference to reception of maleultrasounds.

Results
Ultrasound Production and Mating Behavior
During courtship, ultrasounds were emitted by the males of O.
nubilalis and O. scapulalis in association with quick vibrations of thewings raised upright. We found that males of these species possess
sex-specific scales on the mesonotum (dorsal plate of themesothorax) and on the proximal part of the forewings (Fig. 2A–
E). These scales were morphologically similar to those of O.
furnacalis; however, no scale-less membranous area lay adjacent tothem [7].
The covering of the male-specific wing scales and thoracic scaleswith nail polish substantially reduced the levels of ultrasounds in O.
nubilalis and O. scapulalis. Behavioral experiments with O. nubilalis
showed that a majority (74%) of the females readily acceptedsham-operated males, whereas 42% accepted muted males[Table 2; Likelihood ratio (LR) test in a generalized linear model(GLM), binomial error with logit link, x21,3 = 5.4, P = 0.021].
These results show that male-specific wing scales and thoracic
scales play an important role in ultrasound production, anddemonstrate the importance of sound communication forincreasing mating success.

Differences and Similarities in Ultrasounds of Three
Moths
Pulse structures. The ultrasound of O. nubilalis and O.
scapulalis (Fig. 3A and B, Audio S1 and S2) clearly differed fromthat of O. furnacalis (Fig. 3C, Audio S3) [25]. While the ultrasoundof O. furnacalis consisted of chirps (groups of pulses), the ultrasounds

Figure 1. Phylogenetic relationships of Ostrinia moths and sex pheromones. Minor components of sex pheromones are indicated by anasterisk. O. nubilalis and O. scapulalis exhibit polymorphism in sex pheromones (Z and E types). Z11-14:OAc, E11-14:OAc, Z12-14:OAc, and E12-14:OAc
denote (Z)-11-tetradecenyl acetate, (E)-11-tetradecenyl acetate, (Z)-12-tetradecenyl acetate, and (E)-12-tetradecenyl acetate, respectively. The
phylogenetic tree was constructed by the neighbor-joining method using mitochondrial COII gene sequences [29].
doi:10.1371/journal.pone.0013144.g001

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Table 1. Localities of Ostrinia populations collected in Europe and Japan for the analyses of ultrasound.

Species

Pheromone aCountry

Code

City

O. nubilalisZ

GermanyDa

Darmstadt (49.8uN, 8.6uE)’’’’

FranceTo

Toulouse (43.6uN, 1.4uE)

’’

E

’’

Wa

Warloy (50.0uN, 2.6uE)

’’

’’

’’

Pa

Paris (48.4uN, 2.0uE) & Lille (50.6uN, 3.0uE)

O. scapulalis

Z

Japan

Mo

Morioka (39.7uN, 141.1uE)

’’

’’

’’

Tz

Tsuchizawa (39.3uN, 141.2uE)

’’

E

’’

Fk

Furukawa (38.5uN, 140.9uE)

’’

’’

’’

Tz

Tsuchizawa (39.3uN, 141.2uE)aGenetic polymorphism in female sex pheromone blend. Z-type females produce 97–99% (Z)-11-tetradecenyl acetate and 1–3% (E)-11-tetradecenyl acetate, whereas Etype females produce the opposite blend [12,13,19].
doi:10.1371/journal.pone.0013144.t001

of O. nubilalis and O. scapulalis consisted of long trains of pairs ofpulses, pulse 1 and pulse 2, which exhibit different temporalfeatures and amplitudes. In the pair, pulse 1 was defined as the one
with the shorter pulse interval (pulse duration plus inter-pulseinterval) (Fig. 3A).
Spectral features and sound levels. The pulses of O.
nubilalis and O. scapulalis were broadband with most energy andpeak sound levels (mean = 45 dB SPL) at high frequencies (<30–
60 kHz) (Fig. 4A–C). O. furnacalis had a broader frequency
distribution (<35–80 kHz) than the other species, but
approximately the same peak sound level (46 dB SPL) (Fig. 4C).
We compared features of the ultrasound between the populations of O. nubilalis and O. scapulalis, which differ in pheromone
types, i.e., three populations of O. nubilalis [Z types from Germany(Darmstadt) and France (Toulouse), and an E type from France(Warloy)] and two populations of O. scapulalis [Z type (Morioka)
and E type (Furukawa) from Japan]. Overall, we found that both
spectral features and sound levels differed between pulses 1 and 2of O. nubilalis and O. scapulalis and among the five populations of
the two species. In a population of Z-type O. nubilalis (Darmstadt),
the variation in individual spectra of pulses 1 and 2 was small(Fig. 4A). Variations between pulses 1 and 2 were also small in two
populations of O. nubilalis, Z type (Toulouse) and E type (Warloy)(Fig. 4B), and in the two populations of O. scapulalis, Z type(Morioka) and E type (Furukawa) (Fig. 4C). Peak sound levels of
pulse 1 and pulse 2 differed significantly in the five populations ofO. nubilalis and O. scapulalis (ANOVA using the LR test in a
generalized linear mixed model, Gaussian error with log link,
x21,7 = 27.0, P,0.0001), and so did peak frequencies of pulse 1 andpulse 2 (x21,7 = 84.8, P,0.0001) (Fig. 4A–C). Among the five
populations, peak sound levels including pulses 1 and 2 differedsignificantly (x21,7 = 3.9, P = 0.049), whereas peak frequencies did
not.
Amplitude modulations. Using autocorrelation functions,
we compared amplitude modulations of pulse 1 and pulse 2 among
the five populations of O. nubilalis and O. scapulalis (Fig. 5).
Differences in the amplitude modulations of waveforms in Z-type
O. nubilalis (Toulouse) and E-type O. nubilalis (Warloy) are wellcharacterized in the patterns of autocorrelation coefficients (Fig. 5A
and B). Autocorrelation coefficients of the pulses differedsignificantly among the five populations (MANOVA, F4,61 = 3.1,
P,0.0001), but there was no significant difference between pulse 1
and pulse 2 in the five populations. Subsequently, discriminantfunction analyses and canonical variate analyses (CVA) were
performed to examine how the autocorrelation coefficients forpulses 1 and 2 differed among the three populations of O. nubilalis
PLoS ONE | www.plosone.org[Z type (Darmstadt and Toulouse) and E type (Warloy)] and two
populations of O. scapulalis [Z type (Morioka) and E type(Furukawa)]. Plots of CVA scores showed that the confidence
ellipses were well separated from each other in terms of the
populations, while the difference between pulse 1 and pulse 2 wassmall throughout the populations (Fig. 5C).
Temporal features. Levels of variation in pulse durationand pulse interval were high among the populations of O. nubilalis,but low in O. scapulalis. To further analyze populational differencesin the ultrasounds, temporal features of pulse 1 were comparedamong four European populations of O. nubilalis [Z types(Darmstadt and Toulouse) and E types (Warloy and Paris)] andfour Japanese populations of O. scapulalis [Z types (Morioka andTsuchizawa) and E types (Tsuchizawa and Furukawa)] (Table 1,3). In O. nubilalis, there were significant populational differences inpulse duration (LR test in GLM, quasi error with log link,
F2,21 = 5.4, P = 0. 013) and pulse interval (F2,21 = 17.4, P,0.0001)
In contrast, a significant populational difference was found only inpulse intervals in O. scapulalis (F2,21 = 6.4, P = 0.006). Similarly,
temporal features of pulse 2 were compared among four
populations of O. nubilalis and O. scapulalis (Table 3). A
significant populational difference was found in pulse duration(F2,21 = 23.9, P,0.0001) in O. nubilalis, whereas no significant
difference in any features was detected in O. scapulalis. No
significant difference in temporal features was found between thetwo sympatric (Tsuchizawa) populations of O. scapulalis differing inpheromone type either.

Hearing of Ultrasounds
In a population of Z-type O. nubilalis (Darmstadt), the individual
hearing threshold curves showed a steep increase in sensitivity over
30 kHz, and gradual decrease beyond 60 kHz (Fig. 6A). The
individual variation was low in both sexes. The hearing thresholdcurves of E-type O. nubilalis (Paris) as well as Z-type and E-type O.
scapulalis (Matsudo) showed similar patterns (Fig. 6B). No
significant difference between the sexes was found in the mostsensitive frequencies (LR test in GLM, quasi error with log link,
F1,19 = 3.9, P = 0.050) or in the thresholds at these frequencies(F1,19 = 2.5, P = 0.12). No significant difference was found in themost sensitive frequencies and their threshold levels among thespecies or pheromone types (frequency: F2,22 = 0.2, P = 0.75; mean
6 SD = 45.769.5 kHz, threshold: F2,22 = 0.9, P = 0.40;
42.663.5 dB SPL).
Hearing in Ostrinia moths was tuned to the features of courtship
ultrasound. In populations of O. nubilalis and O. scapulalis, the3

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Figure 2. Ultrasound-producing scales on the wings and thoraxes of the males of O. nubilalis and O. scapulalis. A) Photograph showing
areas bearing the male-specific scales (indicated by dotted boxes) in O. nubilalis (Z type, Darmstadt). Left and right arrowheads indicate the malespecific scales on the right of the notum (dorsal plate of mesothorax) and basal part of the right forewing, respectively. The right tegula was removed
to show the male-specific scales. Scale bar: 500 mm. B, C) Scanning electron micrographs showing male-specific scales (indicated by a black dotted
ellipse) on the right mesothorax (B) and on the right forewing (white dotted ellipse) in O. nubilalis (Z type, Darmstadt) (C). D, E) Male-specific scales(black dotted ellipse) on the right mesothorax (D) and those (white dotted ellipse) on the right forewing in O. scapulalis (E type, Matsudo) (E). Notethat some scales have naturally fallen out. Scale bars: B–E, 200 mm. See Table 1 for collection sites of the populations examined.
doi:10.1371/journal.pone.0013144.g002

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Mechanisms of Ultrasound Production

Table 2. Influence of male sound production on mating
success in Z-type Ostrinia nubilalis.

When producing courtship ultrasounds, O. nubilalis and O.
scapulalis rub specialized scales on the wings against those on thethorax, as in O. furnacalis. The two species have sound-producing
scales similar to those of O. furnacalis, but lack a scale-less area on
the forewing (Fig. 2), which probably functions as a resonator of
40–50 kHz sound (Fig. 4) [7]. The lack of a sound resonator may
partly explain the difference between the sound of O. nubilalis (O.
scapulalis) and that of O. furnacalis.
Temporal features, peak frequencies, and peak sound levels
differed between pulses 1 and 2 in O. nubilalis and O. scapulalis, butpatterns of amplitude modulations did not (Table 3, Fig. 3–5).
These results suggest that the two pulses are generated by two
different actions: up- and downstrokes of the wings. Because
shorter pulse intervals are associated with upstrokes and longer
pulse intervals with downstrokes in O. furnacalis [7], O. nubilalis andO. scapulalis are likely to produce pulse 1, which has shorter
intervals, via upstrokes and pulse 2, which has longer intervals, via
downstrokes. Considering the proposed mechanism of productionof pulses 1 and 2 in O. nubilalis and O. scapulalis, variations in pulse
amplitudes are likely to arise from the shift in pulse production
between the right and left sides, e.g., the synchronicity of up- anddownstrokes of the two wings and/or symmetry of soundproducing organs on the two sides.
The wing beat rate during the production of ultrasound canbe estimated from the pulse repetition rate [7]. The wing beatrate in O. nubilalis and O. scapulalis estimated from the repetition
rates of pulses 1 and 2 (mean pulse interval = 9.4 ms) was 53
cycles/s. This estimate is lower than the rate in O. furnacalis
during ultrasound production (74 cycles/s) but close to that inO. furnacalis during free flight (42 cycles/s) [7]. Thus, thedifference in wing beat rates among Ostrinia species is a plausible
cause for the differences in the temporal features of theultrasound.

Female responseMaletreatment aN

AcceptanceRejection% mated

Sham

27

20

7

74

Muted

26

11

15

42

a

Sham, ordinary scales on both mesothorax and forewings were covered with
nail polish; Muted, sound scales on mesothorax and forewings were covered
with nail polish (see Fig. 1). A significant difference in female response is foundbetween the male treatment groups (Likelihood ratio test in a generalized
linear model, binomial error with logit link, x21,3 = 5.35, P = 0.021).
doi:10.1371/journal.pone.0013144.t002

frequency range of male ultrasounds (38.3 and 36.1 kHz for mean
peak frequencies of pulses 1 and 2, respectively) was well within themost sensitive frequency range of hearing (<30–60 kHz) (Fig. 4,6). A distance of 1–2 cm corresponds to the natural distance
between a female and a courting male. At this distance, the peaksound levels of pulse 1 and pulse 2 (mean 6 SD) were
45.763.3 dB SPL, and 44.364.8 dB SPL, respectively. Thus,
the hearing threshold levels at the most sensitive frequencies(42.663.5 dB SPL) were significantly lower than the levels of pulse
1 (LR test adjusted with a false discovery rate [26] in GLM, quasi
error with log link, F1,58 = 12.20, P = 0.0018) and marginally lower
than those of pulse 2 (F1,58 = 2.2, P = 0.13). These results showedthat peak sound levels are above the hearing thresholds in
populations of O. nubilalis and O. scapulalis.

Discussion
Variation in Ultrasounds
Considerable variation in courtship ultrasound was observed
among the three Ostrinia species. The songs of O. nubilalis and O.
scapulalis were distinctly different from the song of O. furnacalis inspectral and temporal features, while the sound levels of the songsof the three species were similar (44–46 dB SPL at 1 cm). The
songs of O. nubilalis and O. scapulalis were composed of pairs ofpulses, clearly contrasting to the chirp structure of the song of O.
furnacalis (Fig. 3). The bandwidth of the songs of O. nubilalis and O.
scapulalis (main energies in 30–60 kHz) was narrower than that ofO. furnacalis (35–80 kHz) (Fig. 4). Interestingly, the temporalfeatures (pulse duration and pulse interval) and the patterns ofamplitude modulation differed among the populations of O.
nubilalis and O. scapulalis with different pheromone types (Table 3,
Fig. 3, 5). O. nubilalis showed extensive variation in temporalfeatures among the populations, whereas O. scapulalis showed
relatively small variation (Table 3).
The patterns of variation in the songs of the three species (Fig. 3,4) were consistent with the phylogenetic relationships: O. nubilalis
and O. scapulalis are very closely related to each other, but relatively
distantly related to O. furnacalis (Fig. 1) [27–29]. The presence/
absence of discrete within-species variation in pheromones, i.e.,
pheromone type, is also consistent with the phylogenetic relationships of these species (Fig. 1). Thus, the patterns of variation in songs
and pheromones suggest concordant divergence. Ecological factors
such as temporal isolation and host-plant use, and geographic
factors [22,23,30] may explain the levels of variation of the songs in
the two species. Further study of the genetic basis to the variation ofsongs in Ostrinia moths would shed light on the evolution ofultrasound and pheromone communication.
PLoS ONE | www.plosone.org

Sexual Communications via Ultrasound and Pheromones
Electrophysiological recordings showed that the hearing of O.
nubilalis and O. scapulalis was matched to the frequencies and soundlevels of the songs (Fig. 4, 6). Thus, both males and females arecapable of hearing the song during courtship. Furthermore,hearing was very similar across populations of O. nubilalis and O.
scapulalis (Fig. 6). The similarity extended even to O. furnacalis [7].
Given these findings, the spectral discrimination of male courtshipsongs is not likely to occur in the three species. These similarities
suggest that the physiological properties of sound perception havebeen conserved as a result of predatory pressures imposed by
insectivorous bats. Subsequent to the evolution of ultrasonichearing, the males of three Ostrinia species may have acquired theability to produce quiet courtship ultrasounds that could be heard
only by females in close proximity. Quiet ultrasound is adaptive inpreventing eavesdropping by conspecific rivals and/or natural
enemies including bats [6–8,31].
Males of O. nubilalis produce ultrasonic songs for copulation(Table 2). In O. furnacalis, the ultrasonic songs cause females tofreeze, facilitating copulation [7,8]. Ultrasounds simulating bat
echolocation calls elicited freezing behavior, i.e., cessation of
movement and pheromone release in O. nubilalis and other moths[3,31–34]. Likewise, the songs of O. nubilalis and O. scapulalis
would elicit female freezing behavior, presumably without any
discrimination between conspecific songs and bat calls. A lack of
discrimination was also found in Spodoptera litura, which belongs
to the superfamily Noctuoidea [31]. However, A. grisella of the
Pyraloidea to which Ostrinia also belongs, discriminates between
its own songs and bat calls [3,34]. Behavioral observations
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Figure 3. Courtship ultrasounds of O. nubilalis, O. scapulalis, and O. furnacalis. A) Ultrasounds of O. nubilalis (Z type, Darmstadt). Upper:
oscillogram showing the entire song train. Middle: expanded oscillogram showing pulse pairs including pulse 1 (P1) and pulse 2 (P2), which exhibit
different temporal features and amplitudes. Lower: spectrogram of the three pulse pairs. P1 is defined as the pulse in a pulse pair with the shorter
pulse interval. PD and PI denote pulse duration and pulse interval, respectively. B) Pulse pairs of O. scapulalis (Z type, Morioka). C) A pulse group of O.
furnacalis. Audio files of Z-type O. nubilalis, Z-type O. scapulalis and O. furnacalis are available online in the supplementary materials (Audio S1, S2, andS3). See Table 1 for collection sites of the populations examined.
doi:10.1371/journal.pone.0013144.g003

indicate that Hecatesia thyridion (Noctuoidea) also can discriminate
between bats and conspecifics [35]. Therefore, features ofultrasonic communication are well diverged possibly due todifferent selection pressures imposed by conspecific mates andinsectivorous bats even in the same superfamilies of moths.
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Female pheromone communication for mate attraction prevails
across various taxonomic groups of moths, that is, more than 16 of
25 superfamilies [3,10]. Such communication is considered tohave originated from common ancestors of moths and their closely
related order, Trichoptera [3,36]. In contrast, some species
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Figure 4. The frequency–sound pressure level distribution of pulses in O. nubilalis, O. scapulalis, and O. furnacalis. Black lines are spectraof pulse 1 (P1), and red lines are those of pulse 2 (P2). A) Thin lines are individual spectra of P1 and P2, and thick lines are the mean spectra of P1 andP2 in Z-type O. nubilalis (Darmstadt, n = 5). One spectrum with low peak level of P2 differed from all others, and might be a noise. B) Solid lines are themean spectra of P1 and P2 in Z-type O. nubilalis (Toulouse, n = 9), and dotted lines are the mean spectra of P1 and P2 in E-type O. nubilalis (Warloy,n = 5). C) Solid lines are the mean spectra of P1 and P2 in Z-type O. scapulalis (Morioka, n = 7), and dotted lines are the mean spectra of P1 and P2 in Etype O. scapulalis (Furukawa, n = 7). The blue line is the mean spectrum of O. furnacalis (n = 5) obtained from Nakano et al. (2008) [7]. See Table 1 forcollection sites of the populations examined.
doi:10.1371/journal.pone.0013144.g004

Ponsard). O. nubilalis collected from maize and mugwort in France
are known to use Z- and E-type pheromone, respectively [21,23].
In addition, a culture of Z-type pheromone established from larvae
collected from maize in Darmstadt (49.8uN, 8.6uE), Germany [38](provided by Peter Witzgall and Gabor Szöcs), and that of E-type
pheromone established from larvae collected from mugwort inParis (48.4uN, 2.0uE) and Lille (50.6uN, 3.0uE) (collectively
referred to as ‘‘Paris’’), northern France [24] were used (provided
by Laurent Pélozuelo). Mugwort populations of O. nubilalis in
France with E-type pheromone were recently proposed to beconsidered as O. scapulalis [39], but in the present study, we refer tothe two populations from Warloy and Paris as O. nubilalis,
according to the established practice [e.g., 24,28]. We believe that
our interpretation of the results would essentially be unaffected by
the proposed change in the species name of the populations from
Warloy and Paris. In O. scapulalis, adult females collected at
Morioka (39.7uN, 141.1uE) [19] and Tsuchizawa (39.3uN,
141.2uE), northern Japan and Matsudo (35.7uN, 139.9uE), central
Japan were used to establish three different cultures of Z-type
pheromone. To establish three different cultures of E-type
pheromone, adults collected at Furukawa (38.5uN, 140.9uE)[19], northern Japan, Tsuchizawa, and Matsudo were used. The
sex pheromone of several individuals per O. scapulalis culture waschecked by gas chromatography as described by Tabata et al.
(2003) [18] and Takanashi et al. (2005) [19]. All cultures of Ostrinia
were maintained in the laboratory as described by Takanashi et al.
(2005) [19] and Nakano et al. (2006) [25].

derived from only four superfamilies (Pyraloidea, Noctuoidea,
Geometroidea and Uranioidea) have further acquired ultrasoundproduction for mate attraction, courtship, territoriality and bat
defense [2,5,37]. There are eleven other superfamilies of eared
moths, of which eight have not yet been examined for courtship
ultrasound [3]. We predict that a growing number of moths will befound to use ultrasonic courtship songs for sexual communication.
Interestingly, ultrasounds, predominantly produced by males, arecomponents of multi-modal sexual communication in the
behavioral sequence of mating [2,5], in concert with female
and/or male pheromones. Our findings also suggest concordant
evolution in ultrasounds and pheromones occurs across mothsuperfamilies.

Materials and Methods
Animals
Cultures of O. nubilalis and O. scapulalis monomorphic in the
production of Z-type pheromone [97–100% of (Z)-11-tetradecenyl
acetate and 0–3% of (E)-11-tetradecenyl acetate] or E-type
pheromone [0–1% of (Z)-11-tetradecenyl acetate and 99–100%of (E)-11-tetradecenyl acetate] were established from insects
collected at different localities in Europe and Japan (Table 1), as
described by Takanashi et al. (2005) [19] and Pélozuelo et al.
(2007) [24]. For O. nubilalis, larvae were collected from maize at
Toulouse (43.6uN, 1.4uE), southern France, and from mugwort at
Warloy (50.0uN, 2.6uE), northern France (provided by Sergine

Figure 5. Autocorrelation coefficients of pulse amplitudes in O. nubilalis and O. scapulalis. A) Examples of waveforms of pulse 1 in Z-type O.
nubilalis (Toulouse) and in E-type O. nubilalis (Warloy). Two figures are drawn to the same scale. B) The autocorrelation coefficients of the amplitudes
were calculated up to 30 lags corresponding to 0.1 ms in Z-type O. nubilalis (Toulouse) (left) and E-type O. nubilalis (Warloy) (right). C) Plots of the firsttwo canonical variate scores for autocorrelation coefficients of pulse envelops in O. nubilalis and O. scapulalis (n = 33). Ninety-five percent confidence
ellipses of the centroid of the first two canonical variate scores are shown. The first and second canonical variates (CV1 and CV2) explained 73.9% of
the total variance (46.8% attributed to CV1, and 27.1% to CV2), successfully summarizing overall differences. nub Z, Z-type O. nubilalis (Darmstadt andToulouse); nub E, E-type O. nubilalis (Warloy); sca Z, Z-type O. scapulalis (Morioka); sca E, E-type O. scapulalis (Furukawa). P1 and P2 denote pulse 1 andpulse 2, respectively. See Table 1 for collection sites of the populations examined.
doi:10.1371/journal.pone.0013144.g005

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Variation in Courtship Songs

Table 3. Temporal features of pulse 1 and pulse 2 in geographical populations of Ostrinia nubilalis and Ostrinia scapulalis.

O. nubilalis

Pulse 1

Pulse 2N

O. scapulalis

Z type, Da

Z type, To

E type, Wa

E type, PaZ type, Mo

Z type, Tz

E type, Fk

E type, Tz

Duration

7.260.4

6.361.0

5.761.1

7.460.4*

7.360.7

7.661.0

6.660.7

6.960.7

Interval

8.960.4

7.260.9

6.960.9

9.060.2***

8.460.6

9.361.1

7.660.9

8.361.0***

Duration

8.860.8

6.960.8

7.260.3

9.260.6***

7.261.6

7.661.3

8.661.0

8.762.5

Interval

11.761.5

9.361.8

10.762.2

11.061.2

10.860.8

10.461.3

10.761.2

12.661.6

5

9

5

5

7

9

7

4

Values are the mean 6 SD.
*P,0.05, **P,0.01,***P,0.001 (Likelihood ratio test in a generalized linear mixed model). See Table 1 for the code of the geographical populations examined.
doi:10.1371/journal.pone.0013144.t003

Temporal features of sounds recorded by the three systems wereanalyzed using BatSound software. We measured two parameters
for the sound pulses: pulse duration and pulse interval (pulse
duration plus inter-pulse interval) (see Fig. 2). These parameters
were measured from four to nine males using three sequential
pulse pairs with two different pulse intervals and averaged as
representative individual values with respect to each pulse in thepair for the subsequent statistical analyses.
In order to extract structural patterns of pulse amplitudes
among different populations of moths, we used autocorrelation
function analyses [40]. We first obtained relative amplitude valuesof pulse pairs (three replicates of two different pulses within thepair) sampled at 300 kHz with BatSound software from 33
individuals of O. nubilalis [Z type (Darmstadt), Z type (Toulouse)
and E type (Warloy)] and O. scapulalis [Z type (Morioka) and E
type (Furukawa)]. We then calculated autocorrelation coefficientsof these values for up to 30 lags corresponding to 0.1 ms, and usedthem as explanatory variables in the following analyses. We
examined the differences in the autocorrelation coefficients among
the pulse pairs from the five populations of the two species using anested multivariate analysis of variance (MANOVA). Linear
discriminant function analyses were subsequently used to examine
how many individuals were correctly classified into the original
groups of the species, of the populations, and of the pulse pairs. In
order to compare the pattern of pulse amplitude modulation, acanonical variate analysis (CVA) [41] was performed for individual
means of autocorrelation coefficients. CVA is particularly usefulfor finding a quantitative classification rule (i.e., linear discriminantfunctions) using the values of explanatory variables to predict
membership of an object in one of the prespecified classes. In
CVA, coefficients of explanatory variables are calculated in orderto obtain the largest among-group variance to within-groupvariance ratio. All these analyses were performed with the R
statistical package [42].

Sound Recording and Analysis
Sounds were recorded at room temperature during the
scotophase using three different systems in laboratories at the
University of Tokyo and University of Southern Denmark. At the
University of Tokyo, we examined two populations of O. nubilalis[Z type (Toulouse) and E type (Warloy)] as described in Nakano
et al. (2008) [7]. A single male was confined with 5–10 virgin
females in a cubic mesh cage (18618618 cm), which was placedin a one-side opened soundproof box (40640670 cm). The
courtship ultrasounds were recorded with a 1/4-inch condenser
microphone (type 4939; Brüel & Kjær) connected to pre- andconditioning-amplifiers (types 2670 and 2690 with a 0.02–
100 kHz band-pass filter; Brüel & Kjær). We examined one
population of Z-type O. nubilalis (Darmstadt) and two populationsof O. scapulalis [Z type (Morioka) and E type (Furukawa)] using
another condenser microphone as described in Nakano et al.
(2006) [25]. A single male and 5–10 virgin females were placed ina small cylindrical mesh cage (diameter, 5.5 cm; height, 5 cm).
Ultrasounds from the male were recorded using a 1/4-inch
microphone (type 40BF, G.R.A.S.) connected to pre- andmeasuring amplifiers (types 2670 and 2608, Brüel & Kjær). The
position of the microphone was adjusted by hand so that themembrane was always at a distance of 1–2 cm from the courting
male on the inner surface of the cage, which corresponds to the
distance between the sound-producing male and female during
courtship. The acoustic signals obtained from the microphonesystems were digitized by an analog/digital converter (Wavebook
512A, IOtech) at a sampling rate of 300 kHz (12 bits), and highpass-filtered (.10 kHz) using BatSound 3.31 software (Pettersson
Elektronik AB). Power spectra were computed by using a Hann’s
window with an FFT size of 512 points. Relative sound amplitudes
were converted to sound pressure in dB peSPL (peak equivalentsound pressure level in decibels relative to 20 mPa rms) withreference to a sound level calibrator (type 4231 and 4230, Brüel &Kjær; 94 dB SPL at 1 kHz).
In addition to the above systems, sounds of E-type O. nubilalis(Paris) and of O. scapulalis [Z type (Tsuchizawa) and E type
(Tsuchizawa)] were recorded using a bat detector with amicrophone (D240x, Pettersson Elektronik AB) for the analysisof temporal features alone. Recordings on the detector with a 3.4s
digital memory (sampling at 307 kHz) were saved at a 10-fold
reduced clock rate onto a Sony MZ-B10 MD recorder, anddigitized and saved on a computer with a sampling rate of
44.1 kHz. The effective sampling rate for later analysis was
441 kHz because the sounds were recorded with the MD recorderat a 10-fold reduced speed.
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Scanning Electron Microscopy
The scales on the wings and thoraxes of Z-type O. nubilalis(Darmstadt) and E-type O. scapulalis (Matsudo) were observed as
described in Nakano et al. (2008) [7]. Briefly, a newly emerged
moth was killed and mounted on a coverslip. After drying in air,the sample was coated with platinum, and observed with ascanning electron microscope (S-2000, Hitachi, Ltd.) at 10 kV.

Behavioral Experiment
Mating success was compared between mute-operated andsham-operated males of Z-type O. nubilalis (Darmstadt). For
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Variation in Courtship Songs

Figure 6. Hearing threshold curves in O. nubilalis and O. scapulalis. A) Black lines are hearing threshold curves obtained from four females of Ztype O. nubilalis (Darmstadt), and black dotted lines are those from three males. The red line is a curve from both sexes (n = 7). B) The red dotted line
is a curve of E-type O. nubilalis (Paris, n = 5 for both sexes). The black line is a curve of Z-type O. scapulalis (Matsudo, n = 6) and black dotted line, Etype O. scapulalis (Matsudo, n = 6). The blue line is a curve from pooled populations of the two species. All curves were drawn using tensor productsmoothers implemented by generalized additive models [44]. See Table 1 for collection sites of the populations examined.
doi:10.1371/journal.pone.0013144.g006

muting, sound scales on the mesothorax and forewings werecovered with nail polish. For the sham control, ordinary scales,
instead of the sound scales, on the mesothorax and forewings werecovered with nail polish. All treatments were performed on CO2anesthetized virgin males under a stereomicroscope one day before
the experiments. Seven to ten pairs of operated males and intact
virgin females (2 or 3 days old) were introduced into a cubic meshcage (18618618 cm) during the late scotophase when the moths
showed high mating activity [25]. We counted the number ofsuccessful matings for 10 min after the introduction of the pairs.
The experiment was replicated three times using different insects.
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Electrophysiological Recording
In Ostrinia, a pair of tympanal nerves runs through a branch of
the first abdominal ganglion and the abdominal connective to athoracic ganglion [7,43]. The ventral portion of the thorax was
removed to expose the abdominal connective as described [43]with slight modifications. The connective was hooked onto a
recording tungsten electrode, and an indifferent silver electrodewas positioned in the flight muscle of the thorax. The connective
was covered with a 1:1 mixture of VaselineH and paraffin oil
during the recording to prevent desiccation. A Technics tweeter(EAS10TH400B) was placed 30 cm from the moth facing the10

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Variation in Courtship Songs

insect’s ear. Sound stimuli were 10?ms long pulses with a rise/falltime of 0.5 ms repeated at 1 Hz. Tympanal nerve activity was
band-pass filtered, amplified by a custom-built amplifier, anddisplayed on an oscilloscope through an audio monitor. The soundpressure threshold was defined as the pressure level necessary toelicit 1–2 spikes in at least eight out of 10 stimulations. Hearing
sensitivity was tested at 5–10 kHz steps in between 5 and 120 kHz.
The frequencies were tested in random order. Most sensitivefrequencies and threshold sound pressures at the most sensitivefrequencies were obtained from individual hearing curves, andthen the values were compared among both sexes of four different
populations of O. nubilalis [Z type (Darmstadt) and E type (Paris)]and O. scapulalis [Z type (Matsudo) and E type (Matsudo)].
The acoustic pulses were generated with an oscillator (Wavetek
model 186) controlled by a custom-built pulse generator that gave
shaped pulses with linear rise and fall times. The stimulus wasamplified (Xelex) and broadcast through a Technics tweeter. The
loudspeaker was calibrated before and after the experiments using
a 1/4 inch microphone (type 40BF, G.R.A.S.) according to themethod described in Sound recording and analysis.

Found at: doi:10.1371/journal.pone.0013144.s001 (0.05 MB
WAV)
Audio S2 Courtship ultrasound of Z-type Ostrinia scapulalis(Morioka). The ultrasound was slowed down 10 times. The
oscillogram of the ultrasound is shown in Fig. 2B.
Found at: doi:10.1371/journal.pone.0013144.s002 (0.05 MB
WAV)
Audio S3 Courtship ultrasound of Ostrinia furnacalis. The

ultrasound was slowed down 10 times. The oscillogram of theultrasound is shown in Fig. 2C.
Found at: doi:10.1371/journal.pone.0013144.s003 (0.05 MB
WAV)

Acknowledgments
We thank S. Ponsard, L. Pélozuelo, P. Witzgall, G. Szöcs and M.
Fukuzawa for providing insects, and N. Miura for providing phylogenetic
information. Thanks are also due to S. Ponsard, D. Bourguet and two
anonymous reviewers for helpful comments on the manuscript.

Author Contributions

Supporting Information

Conceived and designed the experiments: TT RN AS YI NS. Performed
the experiments: TT RN AS NS. Analyzed the data: TT RN HT YI NS.
Contributed reagents/materials/analysis tools: TT RN HT AS JT YI NS.
Wrote the paper: TT RN HT AS YI NS.

Audio S1 Courtship ultrasound of Z-type Ostrinia nubilalis(Darmstadt). The ultrasound was slowed down 10 times by
down-sampling of the recorded sound to make it audible to thehuman ear. The oscillogram of the ultrasound is shown in Fig. 2A.

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