High duty cycle pulses suppress orientation flights of crambid moths

Journal of Insect Physiology 83 (2015) 15–21

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High duty cycle pulses suppress orientation flights of crambid moths Ryo Nakano a,b,⇑, Fumio Ihara a, Koji Mishiro a, Masatoshi Toyama c, Satoshi Toda a a

Breeding and Pest Management Division, NARO Institute of Fruit Tree Science, 2-1 Fujimoto, Tsukuba, Ibaraki 305-8605, Japan Department of Biological Sciences, University of Toronto Scarborough, Toronto, ON M1C 1A4, Canada c Grape and Persimmon Research Division, NARO Institute of Fruit Tree Science, 301-2 Mitsu, Akitsu, Higashi-hiroshima, Hiroshima 739-2494, Japan b

a r t i c l e

i n f o

Article history: Received 15 August 2015 Received in revised form 23 October 2015 Accepted 4 November 2015 Available online 5 November 2015 Keywords: Acoustic startle response Arms race Echolocation Evasive behavior Tympanal organ

a b s t r a c t Bat-and-moth is a good model system for understanding predator–prey interactions resulting from interspecific coevolution. Night-flying insects have been under predation pressure from echolocating bats for 65 Myr, pressuring vulnerable moths to evolve ultrasound detection and evasive maneuvers as counter tactics. Past studies of defensive behaviors against attacking bats have been biased toward noctuoid moth responses to short duration pulses of low-duty-cycle (LDC) bat calls. Depending on the region, however, moths have been exposed to predation pressure from high-duty-cycle (HDC) bats as well. Here, we reveal that long duration pulse of the sympatric HDC bat (e.g., greater horseshoe bat) is easily detected by the auditory nerve of Japanese crambid moths (yellow peach moth and Asian corn borer) and suppress both mate-finding flights of virgin males and host-finding flights of mated females. The hearing sensitivities for the duration of pulse stimuli significantly dropped non-linearly in both the two moth species as the pulse duration shortened. These hearing properties support the energy integrator model; however, the threshold reduction per doubling the duration has slightly larger than those of other moth species hitherto reported. And also, Asian corn borer showed a lower auditory sensitivity and a lower flight suppression to short duration pulse than yellow peach moth did. Therefore, flight disruption of moth might be more frequently achieved by the pulse structure of HDC calls. The combination of long pulses and inter-pulse intervals, which moths can readily continue detecting, will be useful for repelling moth pests. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction For flying nocturnal insects, the main predators are nightcruising bats that emit ultrasonic pulses for echolocation (Miller and Surlykke, 2001). Moths, as a countermeasure, have evolved ultrasound-sensitive ears composed of tympanal organs and also have evolved evasive maneuvers against bat echolocation sounds to avoid being preyed upon (Roeder, 1962; Minet and Surlykke, 2003; Conner, 2014; Nakano et al., 2015). Noctuid moths (Noctuoidea, Noctuidae) possess two auditory neurons (A1 and A2 cells) in each ear, giving them one of the simplest sound receivers in animals [note that notodontid moths (Noctuoidea, Notodontidae) have only one auditory neuron (Surlykke, 1984)]. Therefore, noctuid moths, a large taxonomic group of lepidoptera, have been rigorously studied as a model auditory system that evolved under strong predation pressure from echolocating bats (Roeder, 1962; Adams, 1971; Paul, 1973; Agee and Orona, 1988; Boyan et al., 1990; Windmill et al., 2006; ter Hofstede et al., 2013; Pfuhl et al., ⇑ Corresponding author at: Department of Biological Sciences, University of Toronto Scarborough, Toronto, ON M1C 1A4, Canada. E-mail address: [email protected] (R. Nakano). http://dx.doi.org/10.1016/j.jinsphys.2015.11.004 0022-1910/Ó 2015 Elsevier Ltd. All rights reserved.

2014; Zhemchuzhnikov et al., 2014). Relatively quiet ultrasounds seem to evoke a slight turning away from the sound source in flying moths (Roeder, 1962, 1964, 1966, 1967). On the other hand, the intense and frequent ultrasonic pulses (high pulse repetition rate = large number of pulses s
1) of an approaching bat cause high firing rates in both auditory A1 and A2 cells, eliciting steep turns, looping/zigzag flight, and/or diving to the ground (Roeder, 1998; Corcoran et al., 2013). However, the relationship between behavioral and auditory responses is not well understood, particularly from the viewpoint of the temporal aspects of sound stimuli (Roeder, 1964; Fullard et al., 2007; Ratcliffe et al., 2009). Insectivorous bats are divided into two types on the basis of duty cycle (ratio of pulse duration per pulse period between the onsets of one pulse and the next one) of echolocation pulses in the search phase; LDC [low (<25%) duty cycle] and HDC [high (P25%) duty cycle] bats (Fenton et al., 2012). Most of them (ca. 85%) are LDC bats that emit downward frequency-modulated (FM) pulses with relatively short duration (Schnitzler and Kalko, 2001; Jones and Teeling, 2006; Fenton et al., 2012). Fewer predatory HDC bats (15%) generate long pulses with a constantfrequency (CF) component, including horseshoe bats Rhinolophus spp. (Rhinolophoidea, Rhinolophidae) and Old World leaf-nosed

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bats Hipposideros spp. (Rhinolophoidea, Hipposideridae). Several studies have reported that these HDC bats can prey on many tympanate moths, especially noctuids (Vaughan, 1997; Bogdanowicz et al., 1999; Jones and Waters, 2000; Longru et al., 2005; Pavey et al., 2006; Schoeman and Jacobs, 2011). Probably owing to the species richness and large population sizes of LDC bats, the defensive behaviors of moths against LDC bat calls have been the main focus of studies into bat–moth arms races (but see Skals and Surlykke, 2000; Alem et al., 2011; Cordes et al., 2014; Nakano et al., 2013, 2014). Simulated echolocation calls of approaching LDC bats, e.g., 50-kHz pulses of 3-ms duration with a 30-ms inter-pulse interval (=9% DC and 30 pulses s
1) of >90 dB SPL (sound pressure level in decibel; 0 dB SPL = 20 lPa) at the moth position, interferes with the free flight of noctuoid moths (Roeder, 1964; Hartley, 1992; Ratcliffe et al., 2011; Corcoran et al., 2013). The moth ear decreases the detection threshold as duration of sound pulse increases (Surlykke et al., 1988; Tougaard, 1998). HDC long pulses, hence, are easily detected by moths (Jones and Waters, 2000) and may have more repulsive effect than LDC pulses, but little is known about the evasive reaction caused by HDC pulses. In eastern Asia, pyralids and crambids (Pyraloidea) unlike noctuids are rarely preyed upon by the greater horseshoe bat Rhinolophus ferrumequinum (Rhinolophoidea, Rhinolophidae) which is a representative HDC bat in Asia (Longru et al., 2005). Though data from light traps indicated that population density of pyralids/ crambids is lower than noctuids, the number of pyralids/crambids preyed by bats is much lower than expected from their density [Kitching et al., 2000; Longru et al., 2005 (crambids are regarded as pyralids in these literatures)]. Pyralids and crambids (e.g., the waxmoths and the corn borers) possess tympanal organs sensitive to a broad frequency range (Agee, 1969; Pérez and Zhantiev, 1976; Spangler and Takessian, 1983; Heller and Krahe, 1994; Skals and Surlykke, 2000; Takanashi et al., 2010; Moir et al., 2013). Besides the high-frequency sensitivity, we predict that the temporal structure of echolocation calls of attacking HDC bats is a key for the avoidance of HDC bats in pyralids and crambids. Here, on the basis of difference in pulse structure of the sympatric bats [LDC bats, the Japanese house bat Pipistrellus abramus and the eastern longfingered bat Myotis macrodactylus (Vespertilionoidea, Vespertilionidae); HDC bat, R. ferrumequinum], we examined the hearing threshold and orientation-flight suppression in two species of Japanese crambid moths.

2. Materials and methods 2.1. Insects We used two species of Japanese crambid moths: the yellow peach moth Conogethes punctiferalis and the Asian corn borer moth Ostrinia furnacalis (Pyraloidea, Crambidae). They were derived from natural populations captured in June, 2013, at the NARO Institute of Fruit Tree Science (36.05°N, 140.10°E; Tsukuba, Ibaraki, Japan). C. punctiferalis, of which main host plants are fruits of peach, apple and chestnut trees, was collected as larvae from young peach fruits and reared on an artificial diet of Silk-mateTM 2M (Nosan Corp., Yokohama, Japan), oak sawdust, and water. Mated females of O. furnacalis, of which main hosts are stalks of corn and millet, were captured with an insect net in low vegetation, and larvae of their offspring were raised on a diet of Silk-mateTM 2M and water. They all were kept under a constant temperature of 24 °C and photoperiod of 16:8 h light:dark cycle. We sexed newly-emerged adult moths (0 day old) on the basis of genital morphology, and housed males and females individually in nylon mesh cages (30  30  30 cm) with a supply of water until used in the follow-

ing experiments. All insects were maintained without any disturbance, harmful manipulations and distress until used in experiments below. 2.2. Auditory frequency sensitivity We recorded auditory nerve responses to 5–100 kHz pure tone pulses in C. punctiferalis to obtain auditory sensitivity curve (audiogram) of the moth. A virgin 2 to 5-day-old moth anesthetized with CO2 was placed ventral side up on a block of dental wax after all wings and legs were removed at the base and trochanters, respectively; it has not been reported that these procedures interfere with auditory nerve activity (Agee, 1969; Skals and Surlykke, 2000; ter Hofstede et al., 2011). We fastened the connection between the prothorax and mesothorax with a staple and the abdominal tip with a couple of tiny pins to the block. To record extracellular action potentials from a nerve branch (1N1) containing four axons of auditory cells (A1–4 cells) in each tympanal organ on the ventral side of the first abdominal segment (Minet and Surlykke, 2003), we dissected the metathoracic right coxa and inserted a stainless steel hook recording electrode around the nerve branch. An indifferent reference electrode was inserted into the abdomen. Neural responses were amplified (ER-1, gain 1000, 10 Hz to 10 kHz band-pass filter; Cygnus Technology, Delaware Water Gap, PA, USA) and digitized (USB-1604HS, 16-bit, 192 kHz sampling; Measurement Computing, Norton, MA, USA) with the software (DASYLabÒ Basic ver. 12.0; Measurement Computing). We placed an electrostatic speaker [ES1 speaker (±11 dB from 4 to 110 kHz) connected to an ED1 speaker driver; Tucker-Davis Technologies, Alachua, FL, USA] at the same height as the moth, 10 cm from the moth’s right tympanic membrane, and at an angle of 90° to the moth’s body axis. Then, we broadcast sound stimuli through the USB-1604HS and the DASYLabÒ (192 kHz sampling) toward the right tympanic membrane. Sounds were recorded with the microphone [type 4939 1/400 microphone (grid-off) connected to a type 2670 preamplifier and type 2690 NexusTM conditioning amplifier (20 Hz to 140 kHz band-pass filter); Brüel and Kjær, Nærum, Denmark] placed at a distance of 1 mm from the position of moth’s left ear, and digitized simultaneously with nerve responses via USB-1604HS. In a single session we presented a sequence of 27 pulses, of which each pulse-duration was 20 ms (1 ms rise/fall time) and delivered at a constant rate of 2 pulses s
1 with increment of 1 dB. The carrier frequency of the pure tone pulse, ranging 5–100 kHz, was randomly chosen at a 5 kHz interval. Sound and neural data, originally saved as separate .asc files on a laptop PC, were converted to a single stereo .wav file and analyzed with BatSound 3.31 (Pettersson Elektronik, Uppsala, Sweden). Then the minimum intensities (dB SPL r.m.s.) evoking 1–2 action potentials of auditory cell (A1) in responses to a pulse stimulus presented were investigated. We statistically derived the average and its 95% confidence intervals of auditory sensitivity curve from a generalized additive mixed model (GAMM) with a random effect of individual ID (ten females and seven males) on the gamm4 package of R 3.1.0 (R Core Team, 2014). Data previously obtained from O. furnacalis [six females and a male; captured in Akiruno, Tokyo where the three bat species (P. abramus, M. macrodactylus and R. ferrumequinum) are commonly observed; Nakano et al., 2008] were re-analyzed by a GAMM. Species comparison of the audiograms between C. punctiferalis and O. furnacalis was performed by likelihood ratio test (LRT). 2.3. Auditory pulse-duration sensitivity By means of the same technique above, we examined the three moth species’ hearing thresholds to 50 kHz pure tones with varied

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pulse duration. Duration of these pulses were 1, 3, 5, 10, 15, 20, 25, 30, 40 and 50 ms (0.1 ms rise/fall time), which cover the pulse duration of the approach phase echolocation calls emitted by the sympatric insectivorous LDC/HDC bats: P. abramus and M. macrodactylus, both of which emit short duration pulses, and R. ferrumequinum, which emits long duration pulses (Tian and Schnitzler, 1997; Funakoshi and Takeda, 1998; Koyanagi et al., 2003; Lee and Lee, 2005; Fujioka et al., 2011; Fenton et al., 2012; Luo et al., 2012; Mantani et al., 2012). Due to the fact that 1–5 ms duration pulses recorded at the sampling frequency of 192 kHz cannot be analyzed by fast Fourier transformation analysis with P1024 sampling points, sound intensity was measured as peak equivalent SPL in dB (dB peSPL) with reference to a known peak signal voltage from a sound calibrator (type 4231, 94 dB SPL, 1 kHz; Brüel and Kjær) (Stapelles et al., 1982). Varied duration pulses were randomly chosen and broadcast at preparations of ten females and ten males of C. punctiferalis and five females and four males of O. furnacalis. Species comparison of the duration-thresholds between C. punctiferalis and O. furnacalis was performed by LRT in GAMM. 2.4. Behavioral test We compared the repulsive effects of ultrasonic pulses on hostfinding flight in mated females of C. punctiferalis and on matefinding flight in virgin males of O. furnacalis. These orientation flights were induced by attractants (see below) placed upwind in an acrylic flight tunnel (11.5 cm internal-diameter  66 cm long; wind speed 0.25 cm s
1). Although the repulsive effect may be underestimated owing to the restricted evasive-behaviors in the small space of the tunnel, we have already confirmed that simulations of LDC and HDC bat calls suppressed mate-finding flight of male C. punctiferalis in the same settings of the tunnel (Nakano et al., 2014). In this study, host-finding flight in mated females of C. punctiferalis was examined. We tried to test mated females’ flight in the other two species as well, but we could not obtain the sufficient number of mated females of the same condition. Because the timing of flights for oviposition was unknown in female C. punctiferalis, we checked when mated females oriented toward a host plant in the following way. We used 4–5 days old virgin females and gravid females that had mated 1 or 2 d prior in the mesh cage. No host plants were available to these females before the experiment. As an attractant, a slice of young apple fruit, known as one of their oviposition substrates (Luo and Honda, 2015), was placed at the center of a square sticky board (10  10 cm; Sumitomo Chemical, Tokyo, Japan). We set this sticky trap at the most upwind part of the tunnel with screen steel meshes fitted at each end to delimit moth movement and introduced five females of the same mating status into the downwind end. The number of females captured on the sticky board was counted 30 min after release of the moths. Since C. punctiferalis is nocturnal, we ran experiments in the last hour of the photophase (L16), the entire scotophase (D1–D8), and the first hour of the photophase (L1) (5–9 replicates for each time of day and mating status). We changed the sticky board and attractant every trial. Under conditions that females were trapped most frequently we played sound stimuli (described later) during 15 min flighttunnel experiments, to examine the negative effects of LDC/HDC bat calls on the host-finding flight. For O. furnacalis, we released five virgin 2–4 day old males in the downwind end of the tunnel in the last 2 h of the scotophase (D7– D8), when the moths actively mate (Nakano et al., 2008). As an attractant, we used a commercial rubber septum impregnated with the synthetic female sex pheromones (E)- and (Z)-12-tetradcenyl acetate (E/Z12–14:OAc) (Sankei Chemical, Kagoshima, Japan). This attractant is originally used for a month in field surveys monitoring the emergence of wild male moths of O. furnacalis. Therefore, we

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did not replace a lure for four weeks. We set a pheromone lure in the center of the sticky board and placed this trap at the upwind end of the tunnel. The repulsive effects of sound stimuli were assayed by the number of males trapped in a 15-min trial with noise or simulated bat calls. We presented three sound stimuli: (i) continuous background room noise after band-pass filter of 50 ± 1 kHz, (ii) 50 kHz LDC pulse train with 5 ms pulse duration (PD; 1 ms rise/fall time) and 11 ms inter-pulse interval (IPI), and (iii) 50 kHz HDC pulse train with 30 ms PD (1 ms rise/fall time) and 30 ms IPI. We simulated these pulses based on the temporal structures of the approach phase calls of P. abramus and M. macrodactylus (LDC bats; Fujioka et al., 2011; Luo et al., 2012) and R. ferrumequinum (HDC bat; Tian and Schnitzler, 1997; Mantani et al., 2012), respectively. Noise and bat-like sounds were broadcast at 22 dB peSPL and 100 dB peSPL, respectively, at a distance of 10 cm from the ES1 speaker membrane to reproduce the natural sound intensities at the position of a moth being attacked by a bat (Corcoran et al., 2013). We placed the speaker at the upwind end of the tunnel and directed it downwind, which means that the distance between the speaker and the screen mesh of the tunnel was 0 cm. Thus, subject moths were exposed to room noise of 22 dB peSPL or bat call simulations of roughly 85–100 dB peSPL on the basis of the moth position in the tunnel. In each experiment, the stimulus was looped for 15 min. Effect of sound stimuli (explanatory variable) on capture rate (response variable) was analyzed by LRT in generalized linear models (GLM) with a binomial error distribution and logit link function in R 3.1.0. In multiple comparisons, the P-value was adjusted by controlling the false discovery rate (Benjamini and Hochberg, 1995). 3. Results 3.1. Frequency threshold The audiograms, hearing threshold curves for various sound frequencies, of C. punctiferalis and O. furnacalis showed similar trends and no significant species difference (LRT in GAMM, v21 = 0.68, P = 0.41) (Fig. 1A). Estimated best frequencies and 95% confidence intervals (CI) were 49.8 (CI: 38.3–62.2) kHz at 35.1 (33.2–36.9) dB SPL r.m.s. and 47.9 (21.2–69.9) kHz at 37.8 (35.5–40.1) dB SPL r.m. s., respectively. These hearing thresholds to 70 kHz (the call frequency of a sympatric HDC bat R. ferrumequinum; Mantani et al., 2012) were 4.3 dB and 4.0 dB higher in C. punctiferalis and O. furnacalis (Fig. 1A) than thresholds to 50 kHz (the call frequencies of LDC bats P. abramus and M. macrodactylus) (Fukui et al., 2004; Fujioka et al., 2011; Luo et al., 2012). 3.2. Duration threshold Pulse duration significantly affected the hearing thresholds in both the two crambid species (C. punctiferalis, LRT in GAMM, v22 = 49.64, P < 0.0001, N = 20; O. furnacalis, v22 = 16.21, P = 0.00030, N = 10) (Fig. 1B). As duration of pulse stimuli shortened, the thresholds rose non-linearly. The negative slopes fitted with
3.5 dB per doubling of duration in the range of <8.0 ms in C. punctiferalis (R2 = 0.97) and
3.1 dB per doubling of duration in the range of <9.2 ms in O. furnacalis (R2 = 0.95). Species difference was found in the threshold curve, and C. punctiferalis had significantly lower thresholds than O. furnacalis (LRT in GAMM, v21 = 7.72, P = 0.0055). Compared with the thresholds for 30 ms pulse (C. punctiferalis, 41.8 dB peSPL; O. furnacalis, 45.0 dB peSPL) which corresponds to the pulse duration of approaching R. ferrumequinum (Mantani et al., 2012), the thresholds for 5 ms pulse (C. punctiferalis, 48.9 dB

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(A)

Ostrinia furnacalis

Conogethes punctiferalis 1 cm

(B)

Fig. 1. Hearing threshold curves in two crambid moths. (A) Sensitivity for 20 ms duration pulses of 5–100 kHz. Left: C. punctiferalis (N = 10$ + 7#). Right: O. furnacalis (N = 6$ + 1#), adapted from Nakano et al. (2008). (B) Sensitivity for 50 kHz pulses of 1–50 ms duration. Left: C. punctiferalis (N = 10$ + 10#). Right: O. furnacalis (N = 5$ + 4#). Semitransparent gray circles and black lines with semi-transparent gray bands denote individual data points and averages with their 95% CI estimated by GAMMs (see Section 2 for details). Top images in (A) are adult males of each species.

peSPL; O. furnacalis, 56.6 dB peSPL), the pulse duration of approaching P. abramus and M. macrodactylus (Fujioka et al., 2011; Luo et al., 2012), heightened by 7.0 dB in C. punctiferalis and 11.6 dB in O. furnacalis (Fig. 1B). 3.3. Host-finding flight In the first hour of scotophase (D1 on x-axis of Fig. 2A), females of C. punctiferalis were more often captured by the sticky trap with an oviposition host plant (LRT in GLM, v29 = 44.40, P < 0.0001) (Fig. 2A). Although females that had mated the day prior to the experiment oriented toward the host plant in D1 more frequently than virgin females (average ± SE of capture rate, 0.46 ± 0.14 vs. 0.12 ± 0.08), the highest capture rate was found in females mated 2 d before the experiment (0.79 ± 0.09, v22 = 12.84, P = 0.0016). Therefore, subsequent trials to examine the repulsive effects of sound stimuli on host-finding flight in C. punctiferalis were performed only in D1 with females that had mated 2 d before. 3.4. Flight disruption Compared to background noise, both LDC and HDC bat-like pulses of 50-kHz pure tone significantly depressed the capture rate of mated females of C. punctiferalis (average ± SE, noise, 0.64 ± 0.10, N = 5$  5 replicates; LDC pulse, 0.28 ± 0.05, N = 5$  5 replicates; HDC pulse, 0.10 ± 0.07, N = 5$  6 replicates) (LRT in GLM, noise vs. LDC pulse, v21 = 6.68, adjusted P = 0.014; noise vs. HDC pulse, v21 = 18.73, adjusted P < 0.0001) (Fig. 2B, left). Capture rates were not statistically different between the LDC and HDC pulses presented (v21 = 3.00, adjusted P = 0.083).

For O. furnacalis, the number of males captured by the trap was slightly lower when the LDC short pulse was presented vs. noise playback, but the difference was not significant (noise, 0.68 ± 0. 80, N = 5$  5 replicates; LDC pulse, 0.52 ± 0.05, N = 5$  5 replicates; v21 = 1.34, adjusted P = 0.25) (Fig. 2B, right). The capture rate with the HDC long pulse (0.16 ± 0.12, N = 5$  5 replicates) was lowest among the treatments (noise vs. HDC pulse, v21 = 14.70, adjusted P = 0.00038; LDC pulse vs. HDC pulse, v21 = 7.50, adjusted P = 0.0092). 4. Discussion In this study, we corroborate that long ultrasonic pulses similar to the echolocation calls of HDC bats such as horseshoe bats Rhinolophus spp. suppressed both mate-finding and host-finding flights of tympanate crambid moths on the basis of their auditory nerve responses. Although short pulses of LDC bats, e.g., mouseeared bats Myotis spp., also depressed orientation flights of mated females (and virgin males; Nakano et al., 2014) of yellow peach moth C. punctiferalis, the repulsive effect of LDC pulses was not apparent in males of Asian corn borer moth O. furnacalis. Generally, the HDC bats emit long pulses composed of a high (P70 kHz) constant-frequency component which a lot of moth species (e.g., noctuids) are not the most sensitive to; their auditory nerves (A1 cells) can respond to 70 kHz sounds 15 dB louder than the best frequency (Jones, 1999; Surlykke et al., 1999; Miller and Surlykke, 2001; ter Hofstede et al., 2013; Nakano et al., 2015). On the other hand, in our crambid moths, 4 dB more energy than 50 kHz sounds of the best frequency (the call frequency of sympatric LDC bats) is needed for detecting 70 kHz sounds, meaning relatively high sensitivities to even 70 kHz sounds among tympa-

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(A)

(B)

Conogethes punctiferalis

Ostrinia furnacalis

**

***

NS

* * NS

Back- LDC HDC ground bat call bat call noise

Back- LDC HDC ground bat call bat call noise

Fig. 2. (A) Capture rates of female C. punctiferalis at different times. Females that had mated 2 d before the experiment (N = 300) were trapped most frequently in the first hour of scotophase (D1 on the x-axis). White and gray horizontal bars beneath the x-axis denote photophase and scotophase, respectively. (B) Repulsive effects of sound stimuli on moth orientation flights (mean ± SE). Left: host-finding flight in females of C. punctiferalis that had mated 2 d before the experiment. Right: mate-finding flight toward a synthetic sex pheromone in virgin males of O. furnacalis. The capture rates of C. punctiferalis significantly decreased in response to both bat calls compared to noise, whereas that of O. furnacalis to LDC bat call did not. NSP > 0.05; *P < 0.05; **P < 0.001; ***P < 0.0001 (LRT in GLM).

nate moths (Fig. 1A). Thus, the small disadvantage in perceiving 70 kHz sounds emitted from R. ferrumequinum is most likely to be offset by the high hearing sensitivities to the long duration pulses (Fig. 1). And inversely, the slight low-sensitivity to the short duration pulses of LDC bats in C. punctiferalis is probably offset by easy detection of the call frequency, 50 kHz. In O. furnacalis, however, the sensitivity to short pulse is notably low in comparison to C. punctiferalis, and therefore LDC short pulses were not effective in suppressing their flights (Figs. 1B and 2B). To link the results from neurophysiological and behavioral experiments where we used sound stimuli with different inter-pulse intervals might lead to an anecdotal interpretation. Nonetheless, it suggests that the high disruption of moth flight by simulated HDC pulses is relevant to the high hearing sensitivity to long duration pulses (Figs. 1 and 2B). The moth ear possesses the physiological characteristics of an energy detector which integrates energy of sounds vibrating a tympanic membrane (Fig. 3). Hence, detection threshold heightens as the duration of a sound stimulus falls below a certain length (Surlykke et al., 1988; Tougaard, 1998; Jones and Waters, 2000; Skals and Surlykke, 2000). In a pyralid (Galleria mellonella) and noctuid moths (Agrotis segetum, Noctua pronuba and Spodoptera lit-

toralis), the detection thresholds decrease by 1.7–2.5 dB per doubling of pulse duration (Waters and Jones, 1996; Tougaard, 1998; Skals and Surlykke, 2000). On the other hand, our crambid moths had slightly larger reductions than them (C. punctiferalis,
3.5 dB; O. furnacalis,
3.1 dB; Fig. 1B), implying taxonomic/species differences and/or geographic differences in the detection threshold. Why both hearing and behavioral responses to short pulses in O. furnacalis are insensitive remains unclear in this study, but ecological factors may be involved. Of the sympatric three insectivorous bat species (Yasui and Saito, 2010), the smallest LDC bat P. abramus aerially hunts tiny flies, mosquitoes and planthoppers, and seldom attacks moths (Hirai and Kimura, 2004). The aerial-hawking LDC bat M. macrodactylus, however, gives strong predation pressure on moths flying high like C. punctiferalis which uses peach and chestnut fruits as their larval host plants (Funakoshi and Takeda, 1998; Murayama et al., 2009). By contrast, the host plants of O. furnacalis are low-grasses and they usually fly near the ground. This behavior probably reduces the predation pressure from aerialhawking bats, as mentioned in Hawaiian noctuid moths (Fullard, 2001). HDC bat R. ferrumequinum is basically aerial-hawking predator, but also consumes fluttering moths on the ground

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(A)

(B)

Fig. 3. Electrophysiological traces of auditory cells in C. punctiferalis. Upper and lower oscillograms represent sound stimuli and neural responses, respectively. (A) Responses to 30, 50 and 70 dB SPL r.m.s. stimuli with 50 kHz and 20 ms duration pulses. (B) Responses to 1, 5 and 10 ms duration pulses with 50 kHz and 50 dB peSPL. Longer duration pulses elicit a greater number of spikes.

(Longru et al., 2005). Our results suggest that O. furnacalis is not exposed to the strong predation threat by LDC bats, but by HDC bats. In terms of practical use, i.e., behavioral regulation of moth pests by means of artificial ultrasound, we expect that a combination of sound frequencies of 20–60 kHz, corresponding to sympatric LDC bat calls, and long duration pulses of around 20– 30 ms, similar to HDC bat calls, is most likely to repel moths. The inter-pulse interval, which determines the pulse repetition rate (and duty cycle) and affects sensory adaptation of auditory processing in moths, must be studied in the future to efficiently repel moths approaching food-related materials. Acknowledgments We thank Andrew J. Masson (University of Toronto Scarborough, Toronto, Canada) for assisting with manuscript editing, and Shizuko Hiryu (Doshisha University, Kyotanabe, Japan) for advice on parameters of echolocation calls of Japanese bats. This work was supported by a Grant-in-Aid for Young Scientists (B) from the Japan Society for the Promotion of Science (JSPS) (grant number: 23780053, R.N.), the Ono Acoustic Research Fund (R.N.), a Kurata Grant (grant number: 1172, R.N.), and a Cross-Ministerial Strategic Innovation Promotion Program from the Council for Science, Technology and Innovation (CSTI) (‘‘Technologies for creating next-generation agriculture, forestry and fisheries”, R.N.). We dedicate this paper to Annemarie Surlykke, 1955–2015 (University of Southern Denmark), who provided new insights of bat echolocation and bat–moth interaction. References Adams, W.B., 1971. Intensity characteristics of the noctuid acoustic receptor. J. Gen. Physiol. 58, 562–579. http://dx.doi.org/10.1085/jgp.58.5.562. Agee, H.R., 1969. Acoustic sensitivity of the European corn borer moth, Ostrinia nubilalis. Ann. Entomol. Soc. Am. 62, 1364–1367. http://dx.doi.org/10.1093/ aesa/62.6.1364. Agee, H.R., Orona, E., 1988. Studies of the neural basis of evasive flight behavior in response to acoustic stimulation in Heliothis zea (Lepidoptera: Noctuidae): organization of the tympanic nerves. Ann. Entomol. Soc. Am. 81, 977–985. http://dx.doi.org/10.1093/aesa/81.6.977. Alem, S., Koselj, K., Siemers, B.M., Greenfield, M.D., 2011. Bat predation and the evolution of leks in acoustic moths. Behav. Ecol. Sociobiol. 65, 2105–2116. http://dx.doi.org/10.1007/s00265-011-1219-x. Benjamini, Y., Hochberg, Y., 1995. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Stat. Soc. Ser. B. 57, 289–300. Bogdanowicz, W., Fenton, M.B., Daleszczyk, K., 1999. The relationships between echolocation calls, morphology and diet in insectivorous bats. J. Zool. 247, 381– 393. http://dx.doi.org/10.1111/j.1469-7998.1999.tb01001.x.

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