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J Comp Physiol A (2014) 200:811–821DOI 10.1007/s00359-014-0924-0

OrIgInAl PAPer

Convergent evolution of anti‑bat sounds

Aaron J. Corcoran · Nickolay I. Hristov

received: 11 March 2014 / revised: 10 June 2014 / Accepted: 13 June 2014 / Published online: 1 July 2014 © Springer-Verlag Berlin Heidelberg 2014

sympatric tiger moths. remaining differences may result from the small size of the E. unicolor tymbal. Four of the five sympatric clicking tiger moth species were unpalatable to bats (0–20 % eaten), whereas E. unicolor was palatable to bats (86 % eaten). Based on these results, we hypoth-esize that E. unicolor evolved tymbal organs that mimic the sounds produced by toxic tiger moths when attacked by echolocating bats.

Keywords Batesian mimicry · Bioacoustics · Predator defense · Sound production · Tymbal organ

Introduction

Because of their long evolutionary history together and their predominance in nocturnal aerial environments, insec-tivorous bats and their insect prey have complex morpho-logical and behavioral adaptations for out-competing each other in predator–prey encounters (Miller and Surlykke 2001; Conner and Corcoran 2012; Yager 2012). These interactions are heavily influenced by acoustic sensing—bats use ultrasonic echolocation to detect and track insects, and insects listen for bat echolocation to alert them of dan-ger. Convergent evolution of echolocation signal design is common in bats. For example, both the old world bat fam-ily rhinolophidae, and the new world bat Pteronotus par-nellii independently evolved constant-frequency (CF) echo-location, which relies on frequency differences between call and echo caused by Doppler shifts. Bats of a number of families have independently evolved specific forms of frequency-modulated (FM) echolocation, which relies on temporal separation of call and echo (Jones and Teeling 2006). In each case the detailed temporal and spectral char-acteristics of echolocation reflect optimization for biosonar

Abstract Bats and their insect prey rely on acoustic sens-ing in predator prey encounters—echolocation in bats, tympanic hearing in moths. Some insects also emit sounds for bat defense. Here, we describe a previously unknown sound-producing organ in geometrid moths—a protho-racic tymbal in the orange beggar moth (Eubaphe uni-color) that generates bursts of ultrasonic clicks in response to tactile stimulation and playback of a bat echolocation attack sequence. Using scanning electron microscopy and high-speed videography, we demonstrate that E. unicolor and phylogenetically distant tiger moths have evolved seri-ally homologous thoracic tymbal organs with fundamen-tally similar functional morphology, a striking example of convergent evolution. We compared E. unicolor clicks to that of five sympatric tiger moths and found that 9 of 13 E. unicolor clicking parameters were within the range of

Electronic supplementary material The online version of this article (doi:10.1007/s00359-014-0924-0) contains supplementary material, which is available to authorized users.

A. J. Corcoran Department of Biology, Biology-Psychology Building, University of Maryland, College Park, MD 20742, USA

A. J. Corcoran (*) Department of Biology, Wake Forest University, Winston-Salem, nC 27106, USAe-mail: aaron.j.corcoran@gmail.com

n. I. Hristov Center for Design Innovation, 301 n. Main St, Winston-Salem, nC 27101, USA

n. I. Hristov Department of life Sciences, Winston-Salem State University, 601 S. Martin luther King Jr. Drive, Winston Salem, nC 27110, USA

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as predicted by acoustics and radar/sonar theory (Jones and Holderied 2007).

Convergent evolution is also common in anti-bat insect hearing. Insects independently evolved hearing organs at least 19 times, and at least 14 of these ears are sensitive to the ultrasonic frequencies of bats (Yager 1999; Yack and Dawson 2008). In many cases the evolution of hearing is thought to be a direct response to the predation pressure of echolocating bats, while in others, bat detection was an added function to hearing organs used for intraspecific communication. With few exceptions these hearing organs are broadly similar in morphology and function; however, they occur nearly everywhere on the insect body, including the thorax, abdomen, legs, wings, and mouthparts (Yager 1999; Yack and Dawson 2008). This extraordinary conver-gence underscores the importance of ultrasonic hearing in nocturnal insects, and the relative ease with which insects can evolve hearing organs from previously existing propri-oceptive structures.

Insects have independently evolved the ability to pro-duce ultrasound in response to echolocation of attacking bats at least four times. In each case the sound production mechanism is strikingly different. The large subfamily of tiger moths (erebidae, Arctiinae) are best known for this ability, which is conveyed by paired metathoracic tym-bal organs that generate sound through cuticular buckling (Blest et al. 1963; Fullard and Heller 1990). less studied are tiger beetles, which appear to make anti-bat sounds by beating their hindwings against their elytra (Yager and Spangler 1997), certain hawkmoths, which stridulate geni-tal valves against their last abdominal segment (gopfert and Wasserthal 1999; Barber and Kawahara 2013), and certain saturniid caterpillars, which produce chirps through mandibular “tooth strikes” (Bura et al. 2009). Peacock but-terflies (Inachis io) also produce ultrasound as part of a multimodal display when disturbed during torpor, however, this defense is not elicited by ultrasound (Mohl and Miller 1976).

Only tiger moth anti-bat sounds have been studied in detail regarding their effect on bats (reviewed by Conner and Corcoran 2012). Depending on the clicking rate and presence or absence of defensive chemicals, acoustic moths can be categorized as using one of three defenses: acous-tic aposematism [typified by low clicking rates and pres-ence of toxic chemicals (Dunning 1968; Hristov and Con-ner 2005a, b)], acoustic mimicry [low clicking rates and not chemically defended (Barber and Conner 2007)] and sonar jamming [high clicking rates and typically no chemi-cal defense (Fullard et al. 1979; Corcoran et al. 2009, 2011; Corcoran and Conner 2012)]. Some animals may also com-bine defensive strategies, for example using partial jam-ming to enhance the effect of an acoustic aposematic signal (ratcliffe and Fullard 2005). Moth clicks have also been

shown to startle bats, but this effect is ephemeral, indicat-ing that startle would only be effective when clicking moths are rare (Bates and Fenton 1990). We are not aware of any study demonstrating startle as the primary acoustic defense of moth species.

While conducting research on acoustic signaling in tiger moths, we discovered that the geometrid moth Eubaphe unicolor (robinson 1869) produces ultrasound in response to both tactile stimulation and ultrasonic playback of a recording of a bat attack. To our knowledge there are no prior published accounts of anti-bat ultrasound produc-tion in the family geometridae (reviewed by nakano et al. 2013). We used scanning electron microscopy and high-speed videography to examine the sound production mechanism of E. unicolor, and compared results with the mechanism of sound production in the dogbane tiger moth Cycnia tenera (Hubner 1818). We also compared the acous-tic emissions of E. unicolor to five sympatric tiger moths and conducted palatability trials using big brown bats (Ept-esicus fuscus, Beauvois 1796). Our results demonstrate a striking level of convergence between the sound-producing structure and acoustic emissions of E. unicolor and sympa-tric tiger moths. As E. unicolor was found palatable to bats and clicked at relatively low rates, we propose that this ani-mal mimics the clicks of sympatric toxic tiger moths.

Methods

Animals

Eubaphe unicolor and tiger moths were collected by attracting insects to white sheets using ultraviolet lights set on the grounds of the Southwestern research Station, which is operated by the American Museum of natural History, and located in the Chiricahua mountains near Por-tal, Arizona. Partly because of its tiger moth-like clicking response, E. unicolor was previously mis-identified as the tiger moth Virbia fragilis (Corcoran et al. 2010). Insects were held individually in 30 ml plastic cups until experi-ments were conducted on either the night of capture, or the following night.

Big brown bats were captured on the grounds of the Southwestern research station using mist nets and held in captivity for up to 2 weeks. Bats were fed mealworms and wild-caught moths and provided water ad libitum. They were allowed to fly for 1–2 h per night for exercise in an outdoor flight cage (3.5 m × 5 m × 3 m).

localizing the Eubaphe unicolor sound organ

To find the E. unicolor sound organ the moths were induced to click using tactile stimulation while an observer

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listened to the output of a “bat detector” (Pettersson elek-tronik D200, Uppsala, Sweden) and looked for movement of the cuticle on live moths under a dissecting microscope. This was aided by removing scales with tape, and putative sound organ structures were ablated using an insect pin. After finding what was believed to be paired sound organs, the acoustic structures of seven moths were ablated one at a time using an insect pin. The acoustic response of the moths to tactile stimulation was recorded after one tymbal was ablated and after both tymbals were ablated. A sham operation was conducted on seven other moths using the procedure described above, except the mesothoracic epis-terna were punctured instead of the prothoracic episterna.

Acoustic recording

Sound recordings were made from 10 male and 13 female E. unicolor. All recordings were made in an open room, 4 × 12 m wide and 3 m high. Sound-absorbent foam cov-ered all surfaces within 2 m of the recording setup, and sounds were broadcast along the long axis of the room with maximal distance to the far wall. Moths were suspended approximately 1 m above a sound-foam-covered table with their wings clamped together dorsally in a hemostat. Moths were induced to click both with tactile stimulation of the thorax and head with a blunt probe, and separately with ultrasound playback of a 2.1 s recording of a big brown bat capturing a tethered moth in an anechoic flight room. The amplitudes of calls in the sequence were modified to reflect the intensity profile of an attack sequence as heard by a moth being attacked by a bat in the field (Corcoran et al. 2013). Call amplitude increased from search phase through early approach phase, where it plateaued at 100 dB peak equivalent Sound Pressure level (peSPl, Stapells et al. 1982) and remained at that level through the end of the attack. Pulse interval (the duration between successive calls) decreased from 115 ms in search phase to 6 ms in ter-minal buzz phase. This recording was used previously for stimulating clicking in tiger moths at the same field loca-tion (Corcoran et al. 2010). Big brown bats are common at the site where moths were collected (personal observation, A.J.C.), and are known to include moths in their diet (Clare et al. 2014). Sounds were broadcast from a Binary Acous-tics Technology (Tucson, AZ) AT100 ultrasound speaker. The speaker was placed 10 cm posterior of the suspended moth’s thorax and sounds were broadcast with a maximum amplitude of 100 dB peSPl at 10 cm. Moth clicks were recorded using an Avisoft Bioacoustics (Berlin, germany) USgH digital recording unit with an Avisoft CM16/CMPA ultrasound microphone (±3 dB from 15 to 140 kHz) and digitized at a 300 kHz sample rate. The microphone was placed 10 cm from the moth’s thorax facing the moth’s left

side. reference sound recordings have been deposited at the Macaulay library archive, Cornell University.

Acoustic measurements

Eubaphe unicolor clicks had the same general acoustic structure as the well-documented tiger moth clicks: two bursts of clicks (the active and passive half modulation cycles) separated by a silent interval. Therefore, 13 acous-tic parameters measured previously from tiger moths clicks (Barber and Conner 2006; Corcoran et al. 2010) were used to characterize the sounds made by E. unicolor. Modu-lation cycle duration, mc, is the total duration of the two click bursts and silent interval; modulation half cycle, mhc, is the duration of the first burst of clicks; inter-cycle silent interval, isi, is the duration of the silent interval; clicks is the number of clicks in the active half modulation cycle; click duration, cdur, is the duration of individual clicks; dominant frequency, d, is the frequency with maximum energy as measured from a power spectrum of a modula-tion cycle; the maximum and minimum click frequencies, −15 and +15 dB kHz, are the frequencies below and above the dominant frequency where power spectral density is 15 dB below the maximum; click amplitude, dB peSPl, is the maximum peak equivalent sound pressure level of the clicks at 10 cm (dB peSPl re. 2 × 10 −5 µPa). Temporal measurements were made manually in BatSound Pro 3.3 from oscillograms. Click amplitude was determined by cal-ibrating the voltage readings from the ultrasound recording unit. To do so, a pure tone stimulus of known amplitude (as measured with a Bruel and Kjaer 2610 measuring amplifier with a ¼ in. B&K microphone with grid off) at the mean dominant frequency of the moth clicks (84 kHz) was broad-cast and recorded by the Avisoft recording unit. Acoustic parameters were measured and averaged from three modu-lation cycles per individual for both the recordings made from tactile stimulation and ultrasound stimulation. Four additional acoustic parameters were measured relating to the clicking response made during ultrasound playback: the time from the first click to the end of the recording, T-end; the duration of the pulse interval of the two calls preceding the first click, PI; the maximum clicking rate over a 100 ms period, clicks s−1; and the maximum duty cycle of the moth clicks, maxDC. Duty cycle is defined as the percentage of time occupied by sound. maxDC was measured by splitting the playback recording into 100 ms sections, counting the maximum number of clicks that occur in one section, mul-tiplying that number by the mean click duration and divid-ing the result by 100 ms (Barber and Conner 2006). results were compared to previously published click parameters of sympatric tiger moths (Corcoran et al. 2010). Some click-ing parameters (PI, T-end) were measured from the original

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tiger moth sound files from a previous study (Corcoran et al. 2010).

Sound organ functional morphology

Scanning electron microscopy was used to image the sound organ in E. unicolor. In addition, high-speed video of the sound organ was taken through an objective microscope at 500 frames-per-second using a Vision research (Wayne, nJ, USA) Phantom v310 camera. Video was coupled with synchronized ultrasound recording using the Avisoft recording system. For comparison, high-speed video and audio were also made of the tymbal organ of C. tenera, a model tiger moth species (Fullard and Heller 1990).

Palatability trials

We examined five tiger moth species with geographic ranges that overlap with E. unicolor in the southern United States. All but C. tenera were found to commonly co-occur with E. unicolor at our field location, while several other species were found in insufficient numbers to provide reli-able measurements of acoustic properties and palatability to bats.

To determine the palatability of E. unicolor and com-mon sympatric tiger moth species, moths were presented as potential food to big brown bats. After being brought into captivity bats were fed a combination of mealworms and wild-caught noctuid, geometrid, and notodontid moths until the bats would readily eat moths when prompted. Bats were held in a gloved hand and presented food items from forceps. low-intensity red light was used for illumination to minimize visual cues associated with food items. After 2–3 nights of feeding, bats readily ate a diet exclusively of moths, at which time palatability trials began.

For palatability trials bats were fed eight palatable con-trol moths and eight experimental moths—including E. unicolor and various tiger moth species depending on avail-ability—in a random order. The sound-producing structures of acoustic moths were ablated prior to being presented as food. Moths were held by the abdomen in the forceps and allowed to beat their wings approximately 1 cm in front of the bat’s mouth. The bat would then either lunge at the food item with its open mouth and consume the moth, or turn its head away with disinterest. In some cases the bat appeared to inspect the moth with its nose or start to bite into the food item before rejecting it. If the bat rejected a food item or appeared disinterested it was fed half of a mealworm and presented the moth a second time shortly after eating the mealworm. This ensured the bat was rejecting the moth as a food item and not simply ignoring it. If the moth was eaten on either the first or second attempt it was consid-ered palatable. If it was rejected on both attempts it was

considered unpalatable. If part of the moth was eaten it was considered partly palatable with the head counting 1/6, the thorax 1/3 and the abdomen 1/2 of the moth (Hristov and Conner 2005b). Palatability for a moth species was taken as the averaged percentage of the moths consumed by bats. This method provides results that are similar to releasing silenced moths and observing whether free-flying, wild bats consume or drop moths after capture (Corcoran and Conner 2012; n. Dowdy, personal communication).

Results

Eubaphe unicolor has paired tymbal organs on the protho-racic episternum (Fig. 1a). Thirteen of 14 ablations of the tymbal resulted in loss of sound production ability, com-pared with 0 of 14 for sham operations, a highly signifi-cant difference (Fisher’s exact test; P < 0.0001). The sin-gle ablation that did not remove sound production caused a reduction in the amount of clicking, and a second ablation of the same organ completely removed sound production.

All 23 moths tested produced ultrasound in response to tactile simulation and 18 of 23 moths (8 of 10 males; 10 of 13 females) produced ultrasonic clicks in response to playbacks of a bat echolocation attack sequence (Fig. 1b, c). Acoustic properties of clicks produced by males and females were not significantly different (data not shown; Student’s t test; P > 0.1).

Eubaphe unicolor produce clicks through the sequential buckling and relaxation of a tymbal organ that is highly similar in functional morphology to the independently evolved tiger moth tymbal (Fig. 2). The E. unicolor tym-bal is a convex piece of thinned cuticle covering an air sac. A row of minute ridges run along the tymbal’s dorsal–pos-terior edge; this structure appears to function in the same way as the “striated band” (or microtymbals) often found in tiger moth tymbals (Blest et al. 1963; Fullard and Heller 1990). During sound production the posterior edge of the tymbal moves anteriorly (for video, see Online resource 1). A wave of buckles propagates along the tymbal from the posterior–ventral edge in the anterior and dorsal directions. The microtymbals allow the buckling to occur as a series of discrete deformations that each generates a broadband ultrasonic click. After a short silent interval, a second burst of clicks is emitted as the tymbal sequentially returns to its resting state. This sound-producing process is functionally the same in E. unicolor and in tiger moths having a striated band, such as C. tenera (Fig. 2; Online resource 2).

There are several small differences between the tymbals of E. unicolor and tiger moths. The E. unicolor tymbal is a modified prothoracic episternum (Fig. 1a), while the tiger moth tymbal is a modified metathoracic episternum. The striated band of E. unicolor is on the posterior edge of the

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tymbal and buckling begins at the tymbal’s ventral side; in tiger moths the striated band is on the anterior edge of the tymbal and buckling is initiated at the dorsal side (Fig. 3). In addition, the buckling of C. tenera propagates only over the striated band, while in E. unicolor buckling propagates over the striated band and anteriorly into a non-striated sec-tion of the tymbal.

Four of five sympatric tiger moth species were found unpalatable to bats (0–20 % consumed; Table 1). The only palatable tiger moth species was the sonar-jamming Bertholdia trigona. The acoustic properties of the 4 toxic tiger moths were analyzed separately from B. trigona because of B. trigona’s extraordinary sound-producing abilities (Corcoran et al. 2009). The acoustic clicks from toxic tiger moths that are sympatric with E. unicolor were

highly variable, with temporal properties covering a range of approximately 200–500 % of the minimum values, and spectral values having a range of 200–300 % of the mini-mum (Table 1). Maximum clicking rates varied by over 600 % of the minimum (not including B. trigona). Moths typically clicked in response to pulse intervals produced in late-approach or early-terminal phase (8.7–31.6 ms), sev-eral 100 ms before capture would occur.

Eubaphe unicolor produced clicks with acoustic prop-erties within the range of sympatric toxic tiger moths for most temporal properties, including modulation cycle dura-tion, modulation half-cycle duration, inter-cycle silent inter-val, click number per active half modulation cycle, maxi-mum duty cycle, and the start of clicking in the playback (Table 1). The temporal property click duration was below

Fig. 1 Sound production by the moth Eubaphe unicolor. a Image of the head and thorax of Eubaphe unicolor. The tymbal organ, tb, is a modified prothoracic episternum. The striated band, sb, is on the posterior–dorsal edge of the tymbal and is more visible in Fig. 3. cx1, prothracic coxa; cx2, mesothoracic coxa; ep2 mesothoracic epister-num; ep3, metathoracic episternum. Image courtesy of nick Dowdy. b example spectrogram of Eubaphe unicolor phonoresponding to

playback of a big brown bat (Eptesicus fuscus) echolocation attack sequence. Shown are the last 1 s of a 2.1 s playback b and a close-up c showing the acoustic structure of the bat calls and moth clicks. The bat increases the rate of echolocation emissions throughout the portion of the recording that is shown. note the moth’s acoustic response, which begins in the bat’s approach phase of echolocation

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the range of sympatric tiger moths, and clicking rate was slightly above the upper range of tiger moths, but similar to C. tenera. Spectral parameter values of E. unicolor were either near the higher range-limit or above that of sympatric tiger moths. E. unicolor clicks had an amplitude that was on average 1 dB less than the lowest-amplitude tiger moth. E. unicolor were consumed by bats on 10 of 11 feeding trials.

Discussion

We report the first known sound-producing structure in a geometrid moth—a prothoracic tymbal organ that produces bursts of ultrasonic clicks in response to tactile stimulation and playback of the ultrasound of an attacking bat (Fig. 1). Sound production for use in courtship is known for a single species of geometrid moth, although its sound-production

organ has not been documented (nakano et al. 2009). Our results suggest that E. unicolor clicks are used for bat defense. Further experiments pitting moths against free-flying bats are required to confirm this finding and mating experiments are needed to test whether clicks are also used in courtship. This is the fifth known sound production mech-anism used in response to bat echolocation, with the others being the tymbal organ of tiger moths, the beating of hind-wings against elytra in tiger beetles, genital stridulation in certain hawkmoths, and mandibular tooth strikes of certain saturniid caterpillars (Blest et al. 1963; Yager and Spangler 1997; Bura et al. 2009; Barber and Kawahara 2013).

Convergence of sound production

The mechanism of sound production in E. unicolor is similar to that of tiger moths and demonstrates a notable

Time (ms)

Fre

quen

cy (

kHz)

0 2 4 6 8 10 12 140

25

50

75

100

125

Fre

quen

cy (

kHz)

a

b

f

g i

Time (ms)0 4 8 12 16

0

25

50

75

100

125d

c

−40 0

PSD (dB)

−40

−20

−20 0

PSD (dB)

e

h

j

Fig. 2 Comparison of independently evolved tymbal organs and acoustic emissions of the orange beggar moth Eubaphe unicolor (a–e) and the dogba orange ne tiger moth Cycia tenera (f–j). Moth images (a, f), tymbal organs (b, g), oscillograms (c, h), spectro-grams (d, i), and power spectral density (PSD) plots (e, j) are shown for each moth. Tymbal images are shown with anterior to the right and dorsal to the top. Arrows indicate the microtymbals (i.e., striated band) on the anterior margin of the C. tenera tymbal and the poste-

rior–dorsal margin of the E. unicolor tymbal. Spectrograms of C. ten-era and E. unicolor clicks both show one complete modulation cycle, which includes one burst of clicks from the active contraction and one burst of clicks from the passive relaxation of the tymbal organ. Some scales were removed from tymbal images for clarity. Scale bars show 0.5 mm. Cycnia tenera moth and tymbal images are reprinted with permission (Barber and Conner 2007; Hristov and Conner 2005a, b)

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example of convergent evolution (Figs. 2, 3). Both animals generate ultrasound through the contraction and relaxation of thoracic tymbal organs. Striae, or microtymbals, allow a single contraction or relaxation of the structure to generate several clicks. The result is a characteristic acoustic pattern of two bursts of clicks separated by a short silent interval (Fig. 2). It should be noted that this pattern is not unique to sounds used in anti-bat defense. For example, the males of some moths in the families nolidae, Crambidae, and Pyralidae have striated tymbals that produce a similar pat-tern of clicks that are used in intraspecific communication

(Spangler 1988; Skals and Surlykke 1999; nakano et al. 2012). Tymbal organs used for signaling mates have evolved at least six times in five moth families—Pyralidae, Crambidae, noctuidae, erebidae, and nolidae (Spangler 1986, 1988; Heller and Achmann 1993; Conner 1999; Skals and Surlykke 1999; nakano et al. 2010, 2012). It is noteworthy that tymbals have evolved on the pleura of all three thoracic segments—the metathorax of tiger moths, the mesothorax of some Crambid moths, and the prothorax of E. unicolor (Fig. 1a; Fullard and Heller 1990; nakano et al. 2012). This suggests that particular characteristics of

0 ms 2 ms 4 ms 6 ms

8 ms 10 ms 12 ms 14 ms

0 ms 2 ms 4 ms 6 ms 8 ms

10 ms 12 ms 14 ms 16ms 18 ms

a

b

Fig. 3 Still images from high-speed videos of one modulation cycle of the Cycnia tenera tymbal a and the Eubaphe unicolor tymbal b. The sounds produced from these tymbal activations are shown in

Fig. 1. The buckled tymbal region in each frame is encircled with a yellow line. Images are shown with anterior to the right and dorsal to the top. For movies, see Online resources 1 and 2

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Tabl

e 1

Aco

ustic

par

amet

ers

and

pala

tabi

lity

of A

rctii

ne a

nd g

eom

etri

d m

oths

Dat

a ar

e m

ean

(bol

d, to

p ro

ws)

and

S.D

. (bo

ttom

row

s)

mc

mod

ulat

ion

cycl

e, m

hc m

odul

atio

n ha

lf c

ycle

, isi

int

er-c

ycle

sile

nt i

nter

val,

clic

ks n

umbe

r of

clic

ks i

n ac

tive

half

mod

ulat

ion

cycl

e, c

dur

clic

k du

ratio

n; p

eSP

L c

lick

ampl

itude

at

10 c

m, d

do

min

ant

freq

uenc

y, −

15 d

B F

min

imum

fre

quen

cy; +

15 d

B F

max

imum

fre

quen

cy, c

lick

rat

e cl

icks

per

sec

ond,

max

DC

max

imum

dut

y cy

cle,

PI

puls

e in

terv

al p

rece

ding

firs

t cl

icks

, T-e

nd

time

to e

nd o

f pl

ayba

ck,

%ea

ten

perc

ent o

f m

oths

eat

en b

y ba

ts, N

1 m

oth

sam

ple

size

for

aco

ustic

par

amet

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N2

mot

h sa

mpl

e si

ze f

or a

cous

tic p

layb

ack

para

met

ers,

N3

mot

h sa

mpl

e si

ze f

or

pala

tabi

lity,

N b

ats

bat

sam

ple

size

for

pal

atab

ility

. Se

e M

etho

ds f

or e

xpla

natio

n of

clic

k pa

ram

eter

s. T

iger

mot

h (B

. tr

igon

a, C

i. te

nuif

asch

ia,

Cy.

ten

era,

and

Euc

. an

tica

) ac

oust

ic d

ata

take

n fr

om C

orco

ran

et a

l. (2

010)

. Cyc

nia

tene

ra p

alat

abili

ty d

ata

take

n fr

om H

rist

ov a

nd C

onne

r (2

005b

). B

erth

oldi

a tr

igon

a pa

lata

bilit

y da

ta ta

ken

from

Cor

cora

n et

al.

(200

9)

Spec

ies

n1

mc

(m

s)m

hc

(ms)

Isi

(ms)

Clic

kscd

ur

(µs)

peSP

l

(dB

)d

(kH

z)−

15 d

B

F (k

Hz)

+15

dB

F

(kH

z)n

2C

lick

rate

max

DC

(%

)PI

(m

s)T-

end

(ms)

n3

n b

ats

% e

aten

Ber

thol

dia

trig

ona

629

.512

.25.

321

.528

080

.748

.721

.983

.16

1,56

443

.865

.61,

067

384

71

5.9

3.2

15.

470

2.1

8.7

10.9

5.6

121

3.4

42.3

655

Cis

then

e te

nui-

fasc

hia

614

.33.

96.

34.

833

072

58.4

35.2

68.8

635

211

.610

.821

85

320

0.8

1.5

2.3

2.2

506.

45.

513

.87

160

0.8

2.0

51

Cte

nuch

a ve

nosa

59.

81.

96

1.6

550

77.2

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819J Comp Physiol A (2014) 200:811–821

1 3

the thoracic pleura of moths may be preadapted for becom-ing tymbal organs, just as chordotonal proprioceptors com-mon to the insect body plan allowed the repeated evolution of tympanal hearing organs in numerous insects from seri-ally homologous structures (Yager 1999). Further mor-phological studies comparing out-groups lacking tymbal organs to tymbaled moths may reveal what characteristics have allowed the repeated evolution of lepidopteran tymbal organs.

not only are the sound-production mechanism of E. unicolor and tiger moth clicks similar, but also the spe-cific temporal and spectral acoustic features of clicking and the timing of the clicking response to the bat echolocation attack sequence (Table 1). The main differences of E. uni-color clicks compared to those of tiger moths are the higher frequency and lower amplitude, both of which might be explained by the small size of the E. unicolor tymbal organ. Eubaphe unicolor clicks are particularly broadband—they cover an 80-kHz range of frequencies. It has been sug-gested that broadband clicks are well-suited for signaling to bats, as they are directed at many bat species that hear best at different frequencies. In contrast, narrowband moth clicks are often used for intraspecific communication, and the acoustic energy can be concentrated in the frequency band where conspecifics are most sensitive (Skals and Sur-lykke 1999). Some tiger moths click for both purposes, and it is unclear whether their clicks are optimized for predator or conspecific receivers, or some compromise between the two (Conner 1999).

Anti-bat defensive function

What is the defensive function of clicking in E. unicolor? Since these moths were eaten by bats in all but one trial, their clicks cannot serve as honest signals of distaste-fulness, as do the clicks of many species of tiger moths (Dunning 1968; Hristov and Conner 2005a). Bats general-ize the aposematic message of tiger moth clicks between toxic species with differing clicking parameters (Muller-ian mimicry) and from toxic species to palatable species [Batesian mimicry (Barber and Conner 2007)]. The close similarity between the acoustic properties of E. unicolor clicks and that of sympatric toxic tiger moths suggests that E. unicolor may be an acoustic Batesian mimic of toxic tiger moths.

In previous studies, some individual red bats (Lasiurus borealis), but not big brown bats discriminated Batesian mimics from their models, allowing them to consume the palatable prey (Barber et al. 2009). This demonstrates the selective pressure on mimics to match the acoustic prop-erties of models. It might be predicted that similar selec-tive pressures would act on groups of toxic tiger moth species that share the burden of educating bats regarding

the relationship between moth clicks and toxic prey. Toxic tiger moths of a given region might, therefore, converge on a particular acoustic signal. However, we did not find evidence supporting this, as sympatric toxic tiger moths produced clicks with widely different temporal and spec-tral properties (Table 1). The variation between species and the large variation of clicking within species of toxic tiger moths (Table 1) should complicate the bat’s task of discriminating palatable and toxic prey. This increase the likelihood bats would mistake clicks of palatable E. uni-color for those of toxic tiger moths. It has been suggested that E. unicolor is a visual mimic of lycid beetles (linsley et al. 1961), raising the possibility of multimodal Batesian mimicry for defending against both visual and acoustic predators, a finding that might be predicted based on the presence of multimodal aposematic signaling (ratcliffe and nydam 2008).

Could E. unicolor clicks jam bat echolocation? High clicking rate, or high duty cycle—which equals clicking rate multiplied by click duration—have been suggested as the primary acoustic features required for an effective jam-ming signal (Miller 1991; Tougaard et al. 1998; Barber and Conner 2006; Corcoran et al. 2009, 2010). While it is agreed that clicking rate is an important measure for jam-ming, it is not clear whether click duration is important for jamming, as there have been no studies disambiguating the two factors. Therefore, we will address the likelihood of E. unicolor clicks jamming bats separately based on its duty cycle and clicking rate.

Only two clicking moth species have been pitted against bats to test the jamming hypothesis with proper controls: Euchaetes egle were unsuccessful at jamming and pro-duce 182 clicks s−1 with a 3.1 % maximum duty cycle, and Bertholdia trigona were highly successful at jamming and produce 1,564 clicks s−1 (maximum 4,500 clicks s−1) with a 43.8 maximum duty cycle (Hristov and Conner 2005a; Corcoran et al. 2010, 2011). Based on duty cycle, E. unicolor, at 3.8 %, would be predicted incapable of jam-ming bats. However, based on E. unicolor’s clicking rate (393 clicks s−1) the prediction is less clear, as this rate is between the two moths that have been tested. E. unicolor’s clicking rate is similar to that of C. tenera, whose clicks may partially jam bats to enhance their aposematic function (ratcliffe and Fullard 2005). It is possible that E. unicolor clicks partially jam bats, enhancing the effect of Batesian mimicry through distortion of the bat’s acoustic perception of prey. Behavioral experiments are required to test whether inter-family mimicry between E. unicolor and tiger moths occurs and whether jamming enhances Batesian mimicry.

In summary, E. unicolor has independently evolved a tymbal organ that is highly convergent with the tymbal of tiger moths, and appears to be used in the same behavio-ral context—anti-bat defense. We argue that the sounds of

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these palatable moths mimic the clicks of toxic sympatric tiger moths, and this effect might be enhanced by partial jamming of bat echolocation. This independent evolution of mimicry may have been facilitated by physical con-straints on sounds produced by tymbal organs. Our find-ings demonstrate a fifth independent evolution of anti-bat sound-producing organs in insects. Further morphological studies of tymbaled moths and out-groups lacking tymbals are required to understand the evolution of tymbal organs from the thoracic episternum. Comparative morphologi-cal and behavioral studies are also needed to determine the prevalence of clicking within the geometridae, and in other insects. The repeated evolution of sound-producing struc-tures for bat defense is further evidence of their survival value in the acoustically dominated aerial battles of bats and insects.

Acknowledgments We thank the staff of the Southwestern research Station for coordination of field research, and nick Dowdy for field assistance. William Conner and gerald Carter reviewed a for-mer version of this manuscript. Funding was provided by the national Science Foundation (grant number IOS-0951160), the American Museum of natural History (Theodore roosevelt grant) and by an institutional training grant (UMD T32 DC-00046) from the national Institute of Deafness and Communicative Disorders of the national Institutes of Health. All research on vertebrates was approved by the Wake Forest University Animal Care and Use Committee (IACUC #A09-094).

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