Characteristics and possible function of wingbeat acoustic emissions of tortricid moths

Characteristics and possible function of wingbeat acoustic emissions of tortricid moths | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Characteristics and possible function of wingbeat acoustic emissions of tortricid moths Alejandro Martin-Gabarrella, Luis Elvira-Segura, Joaquin Baixeras, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7623152/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Most moths are nocturnal flying insects and some species have evolved ears to detect bats, or produce ultrasounds to dissuade or confuse them. Normally moths emit anti-bat ultrasounds when they hear bat echolocation calls, but recently it has been shown that deaf moths emit anti-bat ultrasounds continuously from specialized structures (aeroelastic tymbals) located on their wings. Structures resembling aeroelastic tymbals have been suggested from museum specimens in the family Tortricidae, and it has been proposed that members of this family can emit anti-bat ultrasounds. We recorded the wingbeat acoustic emission of three tortricid species that are predated by bats, and found that both sexes of all three species emit brief ultrasound pulses within the bat´s hearing frequency range at specific positions of the wingbeat cycle. However, the ultrasound pulses are very quiet (ca. 20 dB SPL at 10 cm), their emission is arbitrary (not all individuals and not every wing stroke produces them), and the wings do not bear specialized sound-producing structures. This evidence suggests that the ultrasound pulses emitted by tortricid moths during wingbeat would not be useful as an anti-bat defense mechanism. Tortricids are atympanic, so these faint wingbeat ultrasounds pulses are probably not useful in intraspecific communication either. Biological sciences/Ecology Earth and environmental sciences/Ecology Biological sciences/Zoology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Moths are mostly crepuscular or night active insects and undergo significant bat predation, so they have developed different strategies to avoid bats [ 1 ][ 2 ][ 3 ]. Several moth species have evolved tympanic organs to detect bat echolocation cries and escape predation [ 4 ][ 5 ]. In addition, toxic moths avoid bat predation by emitting ultrasounds that advertise their distastefulness, and some moths emit ultrasounds to interfere with bat echolocation [ 6 ][ 7 ]. Warning ultrasound signals are emitted by both sexes from specialized body structures that have evolved independently several times in moths [ 4 ]. Moths also employ ultrasound communication in courtship behavior, where usually the male is the signaller [ 5 ]. Anti-bat ultrasounds are normally emitted when moths hear bat echolocation signals, however it was shown recently that species in the family Yponomeutidae emit ultrasounds continuously when flying. These microlepidoptera (i.e., small moths) are toxic and advertise their toxicity with ultrasounds, but because they are atympanic and cannot hear bats, they emit the ultrasounds continuously as they fly, and so they are always protected [ 8 ]. Yponomeutid ultrasounds are produced from striated wing patches devoid of scales that act as buckling tymbals (so called aeroelastic tymbals) [ 8 ]. A follow up study based on museum specimens indicates that aeroelastic wing tymbals appear to be present in several moth lineages, including the tortricid subfamily Olethreutinae [ 9 ]. Tortricidae is a large family of atympanic microlepidoptera that undergoes significant bat predation [ 10 ][ 11 ][ 12 ], but they are not toxic [ 7 ], so they are not expected to emit warning acoustic signals. However, they could mimic anti-bat ultrasound signals of sympatric toxic species and gain protection by Batesian mimicry, as other moths do [ 13 ]. The goal of our study was to determine if tortricids produce ultrasound pulses during wingbeat that could be used in anti-bat defense. We focused on three Olethreutinae species which are main pests of apple, peach and grapevine worldwide [ 14 ]. Significant bat predation has been documented in the field for two of these species. In apple orchards the damage produced by Cydia pomonella L. increases by 30% when bats are excluded [ 15 ], and in vineyards Lobesia botrana (Denis & Schiffermüller). is heavily preyed upon by an assemblage of bat species, making up to 27% of the prey for some bats at peak moth density [ 16 ]. The third species, Grapholita molesta Busck, overlaps seasonally and geographically with the other two species, shares host plants with C. pomonella and is prayed by bats [ 15 ], but the impact of bat predation on this species has not been quantified. We recorded from tethered individuals inside an anechoic chamber and from free-flying individuals in a wind tunnel, and synchronized acoustic emission and wing position using a high-speed video camera. In addition, we examined wing morphology with scanning electron microscopy (SEM) to determine if the hyaline patches detected previously resemble the aeroelastic tymbals known in Yponomeutidae [ 8 ][ 9 ]. Results Tethered moths produce ultrasound pulses at specific moments of the wingbeat cycle Recordings of tethered individuals with the Dodotronics microphone showed that both sexes of all three species emit brief ultrasound pulses of low intensity that are visualized only after appropriate ultrasound filtering of the original signal (Fig. 1 , Figure S5a). The frequency of the ultrasound pulses ranged between 20 kHz and 50 kHz, with peaks at around 25 kHz and 40 kHz (Figure S5b). By superposing the plot of the ultrasound pulses on the plot of the wing-tip distance in the time domain, it was shown that the pulses are produced in specific wing positions of the wingbeat cycle (Fig. 1 e). One pulse is generally produced when the wings are near the top position in the upstroke stage (i.e., dorsal) and the other when the wing is near the bottom position during the downstroke (i.e., ventral), and independently of the direction in which the wings were moving (upwards or downwards). The production of the pulses was inconsistent, with individuals producing two pulses, one pulse, or no pulses in each wingbeat cycle (Table S1 ). Free flying moths produce ultrasound pulses of similar acoustic characteristics as those produced by tethered individuals Free-flying males were recorded and compared with tethered individuals to determine if tethering affected the acoustic emission. The ultrasound pulses of free-flying Lobesia botrana (LB) were too faint and could not be analysed. The ultrasound pulses registered from free-flying Cydia pomonella (CP) and Grapholita molesta (GM) were similar to the pulses of tethered individuals (see below), except that their amplitude was much lower, probably due to the increased distance between insect and microphone in free-flying males (Fig. 2 ). Characteristics of the acoustic emissions of tethered individuals In order to completely characterize the ultrasound and audible signals produced during wingbeat, we recorded tethered individuals of each sex and species with the B&K microphone inside the anechoic chamber. Not all individuals produced detectable ultrasound pulses. The percentage of tested individuals from which ultrasound pulses could be recorded ranged between 58% for LB males and females and 83% for GM females (Table S1 ). As with the Dodotronics microphone, the amplitudes recorded with the B&K microphone were also small, but the signal to noise ratio was relatively better with the B&K microphone. Spectral components were also similar to the ones obtained with the Dodotronics microphone, ranging between 20 kHz and 50 kHz, and peaking around 35–40 kHz, but with the B&K the section near 20 kHz was slightly damped (compare Fig. 3 b with Supplementary Figure S5b). Table 1 a shows the mean values for the ultrasound pulse parameters and Tables S2 and S3 show results from the statistical analyses. Peak frequency of the ultrasound pulses ranged between 31.7 kHz (GM females) and 38.9 kHz (LB males), and it was significantly higher in LB than in GM, and in males than in females. Ultrasound pulse intensity ranged between 16.7 dB at 10 cm (LB males) and 21 dB at 10 cm (LB females), and it was higher in males than in females. Pulse duration was similar among species and sexes (ranging between 0.9 and 1.20 ms). Inter-pulse frequency was negatively correlated with body size [ 17 ], as expected [ 18 ], being 53.4 Hz in CP, 63 Hz in LB and 73.5 Hz in GM, and lower in females (which are bigger) than in males (61.8 and 64.8 Hz, respectively). Table 1 Characteristics of the acoustic emissions recorded from tethered tortricid moths in an anechoic chamber using a Brüel & Kjaer microphone. a , high frequency pulses, indicating the main frequency, its intensity, the duration of the pulses and the number of pulses per second. b , low frequency aspect, indicating the main frequency and its intensity. a Species Sex N Main frequency, kHz (mean ± SEM) Intensity at 10 cm, dB SPL (mean ± SEM) Pulse duration, ms (mean ± SEM) Interpulse frequency, Hz (mean ± SEM) C. pomonella Female 8 33.13 ± 3.05 19.54 ± 1.60 1.13 ± 0.16 51.55 ± 1.48 C. pomonella Male 9 37.44 ± 0.83 18.08 ± 0.82 1.13 ± 0.13 55.31 ± 0.85 G. molesta Female 10 31.70 ± 1.64 19.09 ± 1.05 1.10 ± 0.13 72.40 ± 1.29 G. molesta Male 10 35.10 ± 1.20 16.97 ± 1.05 1.20 ± 0.14 74.52 ± 1.87 L. botrana Female 7 36.86 ± 1.88 21.06 ± 2.14 0.93 ± 0.12 61.25 ± 3.14 L. botrana Male 8 38.38 ± 1.48 16.75 ± 1.45 0.90 ± 0.06 64.80 ± 1.86 b Species Sex N Main frequency, Hz (mean ± SEM) Intensity at 10 cm, dB SPL (mean ± SEM) C. pomonella Female 10 52.40 ± 1.08 57.53 ± 1.00 C. pomonella Male 10 54.00 ± 1.26 56.19 ± 1.05 G. molesta Female 10 72.20 ± 1.49 53.28 ± 1.00 G. molesta Male 10 75.80 ± 1.90 50.98 ± 0.62 L. botrana Female 10 61.80 ± 1.23 53.01 ± 1.13 L. botrana Male 10 68.20 ± 1.35 51.16 ± 0.73 Table 1 b shows the mean values for the low frequency (i.e., audible) wingbeat acoustic parameters, and Tables S2 and S3 show results from the statistical analyses. All species produced a two-peaked low-frequency signal in each wingbeat cycle, with several frequency harmonics over this fundamental frequency (Fig. 4 ). In 80% of GM and CP individuals the strongest component was the fundamental frequency, irrespective of sex, however in LB the second harmonic doubled the intensity of the fundamental frequency (Fig. 4 ). CP was the loudest species (56.9 dB at 10 cm), while GM and LB were quieter (52.1 dB at 10-cm, and females were significantly louder than males (54.6 dB and 52.8 dB at 10 cm, respectively). GM had the highest fundamental frequency, followed by LB and CP, averaging 74 Hz, 65 Hz and 53 Hz, respectively (inversely proportional to the size of each species), and males had higher fundamental frequency than females, 66 Hz and 62 Hz, respectively. Wings do not have hyaline patches Results from microscopy are shown in Fig. 5 . Hyaline areas are shown under optical microscopy as areas that lack scales. The tortricid moth forewings examined did not reveal any area devoid of scales except for an oval area on the basal posterior margin on the ventral side. The hindwings revealed an unscaled area on the dorsal side coincident with the development of the axillary sclerites and basal veins. At stronger magnifications (SEM) the basal oval area of the forewings showed a dense field of acanthae, which are tilted obliquely towards the termen. The unscaled area, specifically on the basal dorsal side of the hindwings, appears as a smooth sclerotized plate, devoid of scales, in which the prominent main basal veins can be observed. On the hindwing of the Yponomeutid moths there is a basal hyaline patch between veins Cu2 and Cu1b equipped with a series of 10–15 transverse ridges. The area is distinguishable both from the dorsal side and the ventral side. Discussion Although tortricids are not toxic [ 7 ] they could mimic the ultrasound warning signals produced by sympatric toxic species to avoid bat predation. This type of Batesian mimicry has been described before in other moth species [ 13 ]. For anti-bat ultrasound signals to be effective, the signal must be loud enough to reach bats with enough time to affect their hunting behavior. Therefore, anti-bat ultrasound signals are relatively loud, in the order of 70–85 dB (at 10 cm) when produced by thoracic tymbals in tiger moths (Fam. Erebidae)[ 19 ], 64–68 dB (at 10cm) when produced by abdominal scales in hawkmoths (Sphingidae) [ 20 ], and 55-65dB (at 10 cm) when produced by wing aeroelastic tymbals by deaf and toxic microlepidoptera (i. e.: Yponomeutidae) [ 9 ]. Although tortricids produce ultrasounds during wingbeat, their intensity is extremely low, in fact it is one of the lowest acoustic ultrasound intensities recorded from moth wingbeat, as far as we know [ 21 ], with an amplitude near 20 dB at 10 cm. This is about 100 times lower sound pressure than the 60 dB (at 10 cm) warning signals that toxic deaf Yponomeutidae emit (note that dB is a relative logarithmic unit of measurement) [ 9 ]. The bat hearing threshold is close to the human threshold, at 2-20dB [ 22 ], so for a bat with a 10 dB hearing threshold the expected detection distance of a 60 dB (at 10 cm) moth ultrasound is about 30 m, giving the bat enough time to process the information and modify its behavior. However, this distance shortens to just 30 cm for the 20 dB (at 10 cm) tortricid ultrasound, so bats may not have enough time to process the information and will probably capture the moth. The ultrasound signal of tortricid moths would provide no protection against bats. Probably most Lepidoptera emit incidental low intensity ultrasounds as the wings rub against each other or against other parts of the body at some point during wingbeat [ 23 ]. Natural selection should keep these emissions below predator-detection threshold if they do not have any function, or modify them, possibly improving the mechanism of production, if they are to be used as signals. Moths produce anti-bat ultrasounds either from tymbals or by stridulation, with specialized body structures that have evolved independently and are present on different parts of the body, such as the thorax, wings and abdomen [ 4 ] [ 19 ] [ 5 ] [ 6 ]. Agassiz [ 24 ] describes the hindwing stridulatory organ of ermine moths, which has a hyaline area bearing a “stridularium” that is consistent with our own observation of Yponometutid specimens. However, although it was reported that museum specimens in the tortricid subfamily Olethreutinae may bear hyaline patches on their hindwings that could act as aeroelastic tymbals and emit ultrasounds for bat defense [ 9 ], detailed examination of the hindwings of our tortricid specimens did not reveal hyaline patches or aeroelastic tymbals. The absence of specialized sound-producing structures in tortricid wings agrees with the emission of very low intensity ultrasounds during wingbeat. In addition, ultrasound emission in tortricids was inconsistent, with some individuals producing it all of the time, other individuals not producing it (or emitting below the detection threshold), and some producing one pulse per wing beat and other two. If the ultrasound pulses had a signalling function, a specialized sound production organ and a more consistent emission should have been developed. The evolution of tympanic ears to detect bats has enabled moths to use acoustic communication in courtship behavior. Courtship acoustic signals of moths can be classified according to their intensity as either "loud" (ca. 60–100 dB at 10 cm) or "quiet" (ca. 20–30 dB at 10 cm) [ 21 ]. The intensity of the tortricid ultrasound pulses is within the range of the "quiet" courtship songs, however it is unlikely that they can hear them because detection of far-field airborne acoustic signals requires tympanic organs, which tortricids lack, whereas all the other moth species that use ultrasounds in courtship have tympanic organs [ 4 ][ 5 ]. The "quiet" courtship songs were first discovered, and are well described, in species of the genus Ostrinia , where courting males walk around females with their wings raised up vertically above their bodies and beat them at 76 Hz, about twice as fast as when flying (43 Hz) [ 25 ]. Friction of male-specific specialized scales on the underside of the forewings against specialized scales on the mesothorax produce the ultrasound signals of Ostrinia [ 25 ][ 26 ]. GM males wingfan around females in a similar fashion as Ostrinia , however in GM the function of wingfanning is to deliver a short-range male pheromone, emitted from extricable caudal structures ("hair pencils"), directly towards the antennae of the mostly stationary females [ 27 ]. It is possible that GM males emit ultrasounds while wingfanning to females (we did not record it), but even if they did, females probably could not hear them because they are atympanic. The low-frequency components of the wingbeat, however, could potentially be sensed with the mechanosensory Johnston organ located at the base of the antennae (pedicel), that responds to the particle-velocity component of sound waves [ 28 ]. So, if the carrying frequency of the audible components of the wingbeat, or its harmonics, make the antenna resonate, then GM could hear its own wingbeat. This is how mosquitoes, other diptera and honeybees hear each other [ 29 ]. One of the few examples of acoustic hearing in the audible range in moths involves the atympanic web clothings moth, Tineola bisselliella (Hum.) (Lepidoptera: Tineidae). Males of this species wingfan during courtship and the sonic components of the wingfanning attract unmated females and other males, but not mated females, which do not produce acoustic emissions during courtship [ 30 ]. In our study, tortricids emitted many harmonics during wingbeat, with a fundamental frequency at around 52–75 Hz, so it may be worth testing the resonant frequency of their antennae to explore the possibility that the antenna can detect intraspecific acoustic signals in the audible range in these moths. The overall conclusion of our study, the first of its kind in tortricids as far as we know, is that the ultrasound pulses that they emit are an unavoidable wingbeat byproduct with no apparent biological function in either predator defense or courtship. The physical attributes of the ultrasound pulses were similar between tethered and free-flying individuals, which shows that the restraining method did not alter the acoustic signal. The characteristics of acoustic signals emitted by wing-fanning male GM during courtship, and their possible effect on courtship, are worth exploring. The strategies employed by tortricids and other atympanic and non-toxic moths to scape bat predation deserve further attention [ 31 ][ 1 ], as it may contribute to the implementation of bats as biocontrol agents in agriculture and forestry [ 3 ]. Our results indicate that ultrasound emission would probably have a negligible effect on the application of ultrasounds to control tortricid pest [ 32 ], however the wingbeat frequency data presented could contribute towards the development of automated traps to identify captured species from their wingbeat patterns [ 33 ]. Methods Insects Cydia pomonella , Grapholita molesta , and Lobesia botrana (CP, GM, and LB, respectively) larvae were reared on a semi-artificial diet [ 34 ], and all the stages were kept under a 16h:8h day:night photoperiod at 25 ± 1°C. Pupae were separated by sex and adults were separated from pupae every other day and kept with unrestricted access to 10% sucrose in water. All insects tested were between 2 and 3 days old. Recording setups Wingbeat was recorded either from free flying insects in a flight tunnel, or from tethered insects, and these were recorded either in the laboratory room or inside an anechoic chamber. Two different microphones were employed, depending on availability and suitability. To record from free flying insects, we used only males because they are easily induced to fly to a pheromone source in a flight tunnel. The wind tunnel consisted of a 170 x 45 x 45 cm glass box with an air flow of 0.3 m s -1 and a temperature of 25 ± 1 ºC, as described in [ 35 ]. Between 4 and 8 pheromone-emitting females were placed in the upwind end of the tunnel inside a metal-wire cage at maximum calling time [ 36 ]. Males were released individually in the downwind side of the tunnel and flew upwind towards females. An ultrasound microphone pointing downwards was placed 10 cm downwind from the females, as close as possible to, but not interfering with, the flying path. Ten males of each species were tested. For the rest of the recordings the insects were tethered so they could beat their wings freely while staying in place (Fig. 1 ). They were briefly anesthetised with CO 2 , immobilized in a plastic pipette tip, the dorsal thoracic scales were removed with wipe paper, and a 0.71 mm x 40 mm tungsten entomological needle was attached to the clean thoracic area with a small drop of cyanoacrylate gel glue using a coarse micromanipulator (Supplementary Figure S1 ). The needle projected forward above the insect head, between the eyes and antenna, so that it did not interfere with wing or antennal movement. After < 10 minutes the insect was removed from the pipette tip by gently pulling out from the needle. Most individuals did not beat their wings spontaneously, although their bodies were hanging in the air, so a gentle stream of air was blown over them to stimulate several seconds of wingbeat during which the recording took place. Tethering setup 1 was used to synchronize sound production with wing position during the wingbeat cycle. This setup was placed directly on the laboratory bench, and the needle holding the inset was attached to a pin holder connected to a coarse micromanipulator which allowed insect rotation in 3 axes (Supplementary Figure S2). The microphone pointed horizontally towards the left side of the insect, as close to the insect as possible, but without touching its wings during wingbeat (for acoustic intensity calculations we estimated 0.5-cm distance between insect and microphone). A high-speed IR video camera filmed the insect from behind at 50 cm-distance. To synchronize sound production with wing position, five 10-ms-long pulses were sent simultaneously to a red LED light (660 nm, 160–650 mcd, LedTech, UK) placed 10-15cm cm in front of the insect in the camera field of view, and to a piezo buzzer (6 kHz pure tone, RND 430, RND, Switzerland) placed 15–20 cm away from the insect. LED pulses were captured with the camera and sound pulses were recorded with the microphone. From the video recordings we manually measured the distance between wingtips and plotted it as a function of time. Ultrasound pulses (see below) were superposed to this plot to reveal at which point of the wing cycle the ultrasound pulses were produced. Recordings with ultrasound pulses of 10 individuals of each species and sex were obtained. Tethering setup 2 was used to characterize the acoustic emissions, and it was placed inside of an anechoic chamber (Supplementary Figures S1 and S2). An insect pin was fixed to the insects as indicated above, and the pin was attached to a plastic holder fitted with a ball joint in an articulated arm using washers and magnets, which allowed free orientation adjustment in all three axes (Supplementary Figure S1 ). Microphone position was set as in setup 1 and there was no camera, piezo buzzer or LED (Supplementary Figure S2). The number of GM, LB and CP recorded ranged between 12 and 16, and the number that produced ulktrasound pulses ranged bwteen 7 and 10 (Supplementary Table S1 ). Microphones In order to determine the part of the wing-beat cycle where ultrasound pulses were produced (tethering setup 1), and to record the wing beat of free-flying individuals (wind tunnel), we used an affordable ultrasound microphone (Ultramic 250k, Dodotronic, Italy). The microphone was calibrated against an already-calibrated measurement ultrasound microphone (Supplementary Figure S3). The Dodotronic microphone digitized the signal internally at 250 kHz sampling frequency. We set the recording sampling frequency at 250 kHz and conditioned the signal using a high-pass filter at 18 kHz with 24 dB decay per octave in Audacity 3.1.1 software [ 37 ]. Amplitude in dB SPL from these pulses was not calculated with this microphone because we were just interested in the occurrence of ultrasound pulses or on the difference between tethered and free-flying moths. In order to completely characterize the ultrasound emissions produced by wing-beating tethered individuals in the anechoic chamber (tethering setup 2) we used a (relatively) flat-response ultrasound microphone (Brüel & Kjaer -B&K- Type 4939-A-001, ¼-inch, calibration curve shown in Supplementary Figure S3). The microphone was fitted with pre-amplifier (Type 2670, B&K) and signal-conditioner (Type 1708, B&K), with a x10 pre-amplification and a linear filter. After analogue filtering, the signal was digitized and sampled with a digital oscilloscope (Picoscope 5242D with Picoscope 6.14.69 software, Pico Technology, United Kingdom), at 595 kHz sampling frequency and 16-bits resolution. The voltage scale was set to 20 mV range with AC coupling. Unit conversion of pulse amplitude from Volts to sound pressure level (SPL) in dB (relative to 20 µ Pa) for the B&K microphone is described in the Supplementary material. Distance attenuation was also calculated to be able to compare our data to other references (Supplementary material). Acoustic sampling and processing When using the Dodotronic microphone, each insect was recorded for a few seconds while wingbeating until we observed clear pulses (after filtering in Audacity). Next, we recorded a few seconds of background noise with the insect not beating its wings. From each of these recordings we selected a single 12,500 sample-points section, which corresponds to 50 ms at 250 kHz sampling rate, about 25 to 35 complete wingbeat cycles, depending on sex and species. Fast Fourier Transform (FFT) algorithm and no windowing was used to obtain the spectra in Matlab [ 38 ]. The background noise spectrum was subtracted from the wingbeat spectrum in each individual, and the resulting spectrum was corrected by applying the calibration curve indicated above. With the B&K microphone we obtained ca. 20 different 500-ms-long recordings from each individual. Recordings were checked with the spectrogram feature of Picoscope to find the one with the best signal-to-noise ratio. The selected recording was filtered [ 38 ] using an elliptic 10 kHz to 100 kHz bandpass filter, with stop band limits at 2 kHz and 150 kHz using a value of passband ripple of 0.1 dB and 60 dB of attenuation at the stop band (Supplementary Figure S3). After filtering, ultrasound pulses that exceeded an intensity threshold, which was set manually in each individual recording, were detected, and a Tukey window of 300 points was applied around them. The same filter was applied to the inter-pulse sections, which were used as noise relative to the ultrasound pulses. Then, a 2 14 -point FFT was applied to all the windows, pulses and inter-pulses were averaged separately, and the noise spectrum was subtracted from the signal spectrum. The resulting spectrum was multiplied by the calibration curve to obtain the final result. In order to ensure that all frequencies higher than 10 kHz are part of the signal, we also applied an elliptic filter from 500 Hz to 40 kHz with a stopband from 200 Hz to 80 kHz and a passband parameter of 0.1dB and 30 dB attenuation at the stop band (Supplementary Figure S3). The signal was also windowed using a 10000 points Tukey window to allow the lower frequencies. For the analysis of low-frequency (i.e., audible) wingbeat signals, we used a first-order low-pass filter with a 30 dB attenuation per octave from 10kHz and then performed a 2 14 points FFT without windowing to the entire 0.5-sec-long signal. Microscopic examination of the wings Wings were dry-spread perpendicular to the body axis in order to allow adequate extension of the wing membranes. In this position, the upper side of each wing will be considered dorsal, while the lower side will be considered ventral. The presence of hyaline areas was determined using a stereomicroscope (MZ9.5, Leica microsystems), and the images were obtained with a Leica Z16 stereomicroscope equipped with a DFC500 camera. Software LAS 4.0 was used for image capture. Automontage was used to increase the depth of field. For scanning electron microscopy (SEM), wings were mounted on electron microscope stubs under the stereo microscope at 40–60 x, the wing surface to be glued was gently cleaned from scales with a fine brush (number 000) to ensure better adherence, and the loose scales were removed with the help of a syringe connected to an air pump. The wings were removed from the thorax at the base with the help of point-tip tweezers and were arranged on Hitachi-type electron microscope stubs (15 or 25 mm, depending on wing size) with double-side carbon tabs (Ted Pella© 16084-20), with the cleaned side down. Stubs were then coated with AuPd using a Sputter Coater Polaron SC7640 for 400 seconds (at ca. 3 Å/s). Stubs were examined and photographed using a SCIOS 2 Electron Microscope at 4 kV. Voucher specimens and SEM stubs of examined wings are deposited in the collection of the Natural History Museum at the University of Valencia (Spain). The Yponomeutid specimen was taken from this collection. All images were edited using Photoshop CS3 (Adobe®). Edition was restricted to background cleaning and photo merge. Several individuals were processed until the desired results were obtained. Consequently, different images of the same sex and species may not belong to the same specimen. Statistical analyses In order to analyse differences in acoustic intensity, dominant frequency (both low and high frequencies), ultrasound pulse duration, and inter-pulse frequency (i.e., time between the pulse from one wingbeat to the next one, if there were two pulses per wingbeat we focused only on one of them), we used Generalized Linear Models (GLM) with a gaussian family link, as a function of sex and species \(\:\sim{(species+sex)}^{n}\) , being n the order of the best fitting model. Using ANOVA and the Akaike Information Criterion (AIC) we compared hierarchically different order models from the simplest (n = 0, no dependency) to the most complex (n = 2, 2nd order interaction) and took the most complex model that was significantly different from the previous one and with lower AIC value. Statistical analyses were run in R 4.1.2. software [ 39 ]. For significant variables, pairwise comparisons between levels of a parameter were made using emmeans() package in R [ 40 ]. The data are included in the Supplementary file. Declarations Funding C.G. was funded by a grant from the Ministerio de Ciencia e Innovación (MICINN, Grant No. PID2019-107030RB-C22), and A. M.-G. was funded by a Ph.D. fellowship from Agència de Gestió d'Ajuts Universitaris i de Recerca (AGAUR, 2021-FISDU-00093) Author Contribution A.M.-G. performed the acoustic experiments. A.M.-G. and L.E.-S analysed the acoustic recordings. X.B. performed the microscopic examination of the wings. All the authors contributed equally to the manuscript. Acknowledgement Jesús Uriol (Acustics Ambient S.L., Lleida, Spain) helped with the design of the anechoic chamber. Data Availability The acoustic measurements are included in the Supplementary file. References Jacobs, D. S. & Bastian, A. Non-auditory defenses of prey against bat predation. In Predator–Prey Interactions: Co-Evolution Between Bats and Their Prey 43–71Springer, (2016). Kawahara, A. Y. et al. 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Entomol. 57 , 21–39 (2012). van der Geest, L. P. S. & Evenhuis, H. H. Tortricid Pests: Their Biology, Natural Enemies and Control. World Crop Pests Vol. 5Elsevier,. (1991). Ancillotto, L. et al. A bat a day keeps the pest away: Bats provide valuable protection from pests in organic apple orchards. J. Nat. Conserv. 78 , 126558 (2024). Baroja, U. et al. Bats actively track and prey on grape pest populations. Ecol. Indic. 126 , 107718 (2021). Navarro-Roldán, M. A., Avilla, J., Bosch, D., Valls, J. & Gemeno, C. Comparative effect of three neurotoxic insecticides with different modes of action on adult males and females of three tortricid moth pests. J. Econ. Entomol. 110 , 1740–1749 (2017). Darveau, C. A. Insect flight energetics and the evolution of size, form, and function. Integr. Comp. Biol. 64 , 586–597 (2024). Corcoran, A. J. & Hristov, N. I. Convergent evolution of anti-bat sounds. J. Comp. Physiol. A . 200 , 811–821 (2014). Barber, J. R. et al. Anti-bat ultrasound production in moths is globally and phylogenetically widespread. Proc. Natl. Acad. Sci. USA 119, e2117485119 (2022). Nakano, R. & Nagamine, K. Loudness-duration tradeoff in ultrasonic courtship songs of moths. Front. Ecol. Evol. 7 , 244 (2019). ter Hofstede, H. M. & Ratcliffe, J. M. Evolutionary escalation: the bat–moth arms race. J. Exp. Biol. 219 , 1589–1602 (2016). Clark, C. J. Ways that animal wings produce sound. Integr. Comp. Biol. 61 , 696–709 (2021). Agassiz, D. J. L. Do small ermine moths sing? Possible stridulatory sound production in Yponomeutidae (Lepidoptera). J. Nat. Hist. 52 , 1229–1236 (2017). Nakano, R. et al. Moths produce extremely quiet ultrasonic courtship songs by rubbing specialized scales. Proc. Natl. Acad. Sci. USA 105, 11812–11817 (2008). Takanashi, T. et al. Variation in courtship ultrasounds of three Ostrinia moths with different sex pheromones. PLoS ONE . 5 , e13144 (2010). Baker, T. C. & Cardé, R. T. Courtship behavior of the oriental fruit moth ( Grapholitha molesta ): experimental analysis and consideration of the role of sexual selection in the evolution of courtship pheromones in the Lepidoptera. Ann. Entomol. Soc. Am. 72 , 173–188 (1979). Windmill, J. F. C. & Jackson, J. C. Mechanical specialization in insect ears. In Insect Hearing (eds Pollack, G. S. et al.) 143–164 (Springer, (2016). Göpfert, M. C. & Hennig, R. M. Hearing in insects. Annu. Rev. Entomol. 61 , 257–276 (2016). Takács, S., Mistal, C. & Gries, G. Communication ecology of webbing clothes moth: attractiveness and characterization of male-produced sonic aggregation signals. J. Appl. Entomol. 127 , 127–133 (2003). Lloyd, N., Wilson, J. M. & Barclay, R. M. Behaviors of western spruce budworm moths ( Choristoneura occidentalis ) as defences against bat predation. J. Insect Behav. 19 , 533–544 (2006). Nakano, R., Ito, A. & Tokumaru, S. Sustainable pest control inspired by prey–predator ultrasound interactions. Proc. Natl. Acad. Sci. USA 119, e2211007119 (2022). van Klink, R. et al. Emerging technologies revolutionise insect ecology and monitoring. Trends Ecol. Evol. 37 , 872–885 (2022). Ivaldi-Sender, C. Techniques simples pour un élevage permanent de la tordeuse orientale, Grapholita molesta (Lepidoptera: Tortricidae) sur milieu artificiel. Ann. Zool. Ecol. Anim. 6 , 337–343 (1974). Navarro-Roldán, M. A., Amat, C., Bau, J. & Gemeno, C. Extremely low neonicotinoid doses alter navigation of pest insects along pheromone plumes. Sci. Rep. 9 , 8150 (2019). Navarro-Roldán, M. A. & Gemeno, C. Sublethal effects of neonicotinoid insecticide on calling behavior and pheromone production of tortricid moths. J. Chem. Ecol. 43 , 881–890 (2017). Audacity Team. Audacity®: Free Audio Editor and Recorder. Version 3.1. (2021). The MathWorks Inc. MATLAB version: 23.2.0.2515942 (R2023b), Natick, Massachusetts. (2023). https://www.mathworks.com R Core Team. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. (2024). https://www.R-project.org/ Lenth, R. V. & emmeans Estimated Marginal Means, aka Least-Squares Means. R package version 1.10.5. (2022). https://CRAN.R-project.org/package=emmeans Additional Declarations No competing interests reported. Supplementary Files MartinGabarrellasupplementary.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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11:20:28","extension":"html","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":114509,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7623152/v1/3ba15211f7121ec8ea4e0cee.html"},{"id":92406407,"identity":"e8a837fe-fbff-47ff-917f-5a05ea367f8a","added_by":"auto","created_at":"2025-09-29 11:20:28","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":411704,"visible":true,"origin":"","legend":"\u003cp\u003eWing position and ultrasound pulse emission. Insects were tethered and recorded with microphone and video camera (\u003cstrong\u003ea\u003c/strong\u003e) in order to obtain the ultrasound pulses (\u003cstrong\u003ed\u003c/strong\u003e) and the wing position (\u003cstrong\u003eb\u003c/strong\u003e), and from these the point of the wingbeat at which the ultrasound pulses where produced (\u003cstrong\u003ee\u003c/strong\u003e, semi-circles, not drawn to scale, dorsal (red), ventral (blue), endpoint of the wing trajectory (grey), lines and shaded areas represent mean ± SEM, respectively, N=10)\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7623152/v1/f65163763bb3049592df9233.png"},{"id":92406394,"identity":"658b7bd9-08bf-486d-96f4-0da0072efe92","added_by":"auto","created_at":"2025-09-29 11:20:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":332273,"visible":true,"origin":"","legend":"\u003cp\u003eUltrasound pulses recorded with an Dodotronic microphone from free-flying and from tethered and wingbeating male \u003cem\u003eC. pomonella\u003c/em\u003e and \u003cem\u003eG. molesta\u003c/em\u003e moths. Free flying \u003cem\u003eL. botrana\u003c/em\u003e did not produce detectable ultrasound pulses. \u003cstrong\u003ea\u003c/strong\u003e, representative waveform showing the ultrasound pulses. \u003cstrong\u003eb\u003c/strong\u003e, normalized spectral components of the ultrasound pulses (N=10).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7623152/v1/da4201f6578a369daf7d2261.png"},{"id":92407452,"identity":"3d38d374-5c6e-4951-bdca-28165d460d94","added_by":"auto","created_at":"2025-09-29 11:28:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":404013,"visible":true,"origin":"","legend":"\u003cp\u003eProperties of the ultrasound pulses produced by the wingbeat of tethered tortricid moths recorded with a B\u0026amp;K microphone in an anechoic chamber. \u003cstrong\u003ea\u003c/strong\u003e, representative traces of ultrasound pulses. \u003cstrong\u003eb\u003c/strong\u003e, frequency spectra, solid lines and shaded areas represent mean ± SEM, respectively. N = 7-10.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7623152/v1/54bf580529ca15099ed4696d.png"},{"id":92406398,"identity":"74712512-ca1c-4427-8b04-948f7769b25f","added_by":"auto","created_at":"2025-09-29 11:20:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":408367,"visible":true,"origin":"","legend":"\u003cp\u003eProperties of the low frequency aspects of the acoustic emission produced by the wingbeat of tethered tortricid moths recorded with a B\u0026amp;K microphone in an anechoic chamber. \u003cstrong\u003ea\u003c/strong\u003e, representative waveforms. \u003cstrong\u003eb\u003c/strong\u003e, frequency spectra, solid lines and shaded areas represent mean ± SEM, respectively. N = 10.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7623152/v1/a96b6821af46dbd1b61a165d.png"},{"id":92407453,"identity":"b58ebd5f-6417-4c11-aeb1-ead4869bd37d","added_by":"auto","created_at":"2025-09-29 11:28:27","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":928785,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the wings in the three species of tortricids relative to \u003cem\u003eYponomeuta sp\u003c/em\u003e. \u003cstrong\u003ea\u003c/strong\u003e, \u003cstrong\u003ee\u003c/strong\u003e, \u003cstrong\u003ei\u003c/strong\u003e, \u003cem\u003eCydia pomonella\u003c/em\u003e. \u003cstrong\u003eb\u003c/strong\u003e, \u003cstrong\u003ef\u003c/strong\u003e, \u003cstrong\u003ej\u003c/strong\u003e, \u003cem\u003eGrapholita molesta\u003c/em\u003e. \u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003eg\u003c/strong\u003e, \u003cstrong\u003ek\u003c/strong\u003e, \u003cem\u003eLobesia botrana\u003c/em\u003e. \u003cstrong\u003ed\u003c/strong\u003e, \u003cstrong\u003eh\u003c/strong\u003e, \u003cstrong\u003el\u003c/strong\u003e, \u003cem\u003eYponomeuta sp\u003c/em\u003e. \u003cstrong\u003ea\u003c/strong\u003e-\u003cstrong\u003ed\u003c/strong\u003e, general aspect of the wings in dorsal view with stereomicroscope. \u003cstrong\u003ee\u003c/strong\u003e-\u003cstrong\u003eg\u003c/strong\u003e, SEM image of the forewing and hindwing top side. Boxes magnified in \u003cstrong\u003ei\u003c/strong\u003e-\u003cstrong\u003ek\u003c/strong\u003e. \u003cstrong\u003eh\u003c/strong\u003e, show detail of the top side of the hindwing of \u003cem\u003eYponomeuta sp\u003c/em\u003e. showing the hyaline zone. \u003cstrong\u003ei\u003c/strong\u003e-\u003cstrong\u003el\u003c/strong\u003e, SEM image of the proximal part of the hind wing, top side. Scale bars, \u003cstrong\u003ea\u003c/strong\u003e, \u003cstrong\u003ed\u003c/strong\u003e-\u003cstrong\u003eg\u003c/strong\u003e = 2 mm, \u003cstrong\u003eb\u003c/strong\u003e-\u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003eh\u003c/strong\u003e-\u003cstrong\u003el\u003c/strong\u003e = 1 mm.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7623152/v1/16b847d62bbc9c5800c2d06b.jpeg"},{"id":93367262,"identity":"8031b053-5ca1-4f2f-9f68-2320bb1f8709","added_by":"auto","created_at":"2025-10-13 05:32:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3113609,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7623152/v1/3586711c-84e8-483e-9218-f2295601b3f8.pdf"},{"id":92406419,"identity":"b0dc3720-c583-4ba8-85e4-14f4b53768df","added_by":"auto","created_at":"2025-09-29 11:20:28","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":3328613,"visible":true,"origin":"","legend":"","description":"","filename":"MartinGabarrellasupplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-7623152/v1/8a5229ad9165e42a4a1240d3.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Characteristics and possible function of wingbeat acoustic emissions of tortricid moths","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMoths are mostly crepuscular or night active insects and undergo significant bat predation, so they have developed different strategies to avoid bats [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e][\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e][\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Several moth species have evolved tympanic organs to detect bat echolocation cries and escape predation [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e][\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In addition, toxic moths avoid bat predation by emitting ultrasounds that advertise their distastefulness, and some moths emit ultrasounds to interfere with bat echolocation [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e][\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Warning ultrasound signals are emitted by both sexes from specialized body structures that have evolved independently several times in moths [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Moths also employ ultrasound communication in courtship behavior, where usually the male is the signaller [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAnti-bat ultrasounds are normally emitted when moths hear bat echolocation signals, however it was shown recently that species in the family Yponomeutidae emit ultrasounds continuously when flying. These microlepidoptera (i.e., small moths) are toxic and advertise their toxicity with ultrasounds, but because they are atympanic and cannot hear bats, they emit the ultrasounds continuously as they fly, and so they are always protected [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Yponomeutid ultrasounds are produced from striated wing patches devoid of scales that act as buckling tymbals (so called aeroelastic tymbals) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. A follow up study based on museum specimens indicates that aeroelastic wing tymbals appear to be present in several moth lineages, including the tortricid subfamily Olethreutinae [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Tortricidae is a large family of atympanic microlepidoptera that undergoes significant bat predation [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e][\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e][\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], but they are not toxic [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], so they are not expected to emit warning acoustic signals. However, they could mimic anti-bat ultrasound signals of sympatric toxic species and gain protection by Batesian mimicry, as other moths do [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe goal of our study was to determine if tortricids produce ultrasound pulses during wingbeat that could be used in anti-bat defense. We focused on three Olethreutinae species which are main pests of apple, peach and grapevine worldwide [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Significant bat predation has been documented in the field for two of these species. In apple orchards the damage produced by \u003cem\u003eCydia pomonella\u003c/em\u003e L. increases by 30% when bats are excluded [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], and in vineyards \u003cem\u003eLobesia botrana\u003c/em\u003e (Denis \u0026amp; Schifferm\u0026uuml;ller). is heavily preyed upon by an assemblage of bat species, making up to 27% of the prey for some bats at peak moth density [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The third species, \u003cem\u003eGrapholita molesta\u003c/em\u003e Busck, overlaps seasonally and geographically with the other two species, shares host plants with \u003cem\u003eC. pomonella\u003c/em\u003e and is prayed by bats [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], but the impact of bat predation on this species has not been quantified.\u003c/p\u003e\u003cp\u003eWe recorded from tethered individuals inside an anechoic chamber and from free-flying individuals in a wind tunnel, and synchronized acoustic emission and wing position using a high-speed video camera. In addition, we examined wing morphology with scanning electron microscopy (SEM) to determine if the hyaline patches detected previously resemble the aeroelastic tymbals known in Yponomeutidae [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e][\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eTethered moths produce ultrasound pulses at specific moments of the wingbeat cycle\u003c/h2\u003e\u003cp\u003eRecordings of tethered individuals with the Dodotronics microphone showed that both sexes of all three species emit brief ultrasound pulses of low intensity that are visualized only after appropriate ultrasound filtering of the original signal (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Figure S5a). The frequency of the ultrasound pulses ranged between 20 kHz and 50 kHz, with peaks at around 25 kHz and 40 kHz (Figure S5b). By superposing the plot of the ultrasound pulses on the plot of the wing-tip distance in the time domain, it was shown that the pulses are produced in specific wing positions of the wingbeat cycle (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). One pulse is generally produced when the wings are near the top position in the upstroke stage (i.e., dorsal) and the other when the wing is near the bottom position during the downstroke (i.e., ventral), and independently of the direction in which the wings were moving (upwards or downwards). The production of the pulses was inconsistent, with individuals producing two pulses, one pulse, or no pulses in each wingbeat cycle (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eFree flying moths produce ultrasound pulses of similar acoustic characteristics as those produced by tethered individuals\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFree-flying males were recorded and compared with tethered individuals to determine if tethering affected the acoustic emission. The ultrasound pulses of free-flying \u003cem\u003eLobesia botrana\u003c/em\u003e (LB) were too faint and could not be analysed. The ultrasound pulses registered from free-flying \u003cem\u003eCydia pomonella\u003c/em\u003e (CP) and \u003cem\u003eGrapholita molesta\u003c/em\u003e (GM) were similar to the pulses of tethered individuals (see below), except that their amplitude was much lower, probably due to the increased distance between insect and microphone in free-flying males (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCharacteristics of the acoustic emissions of tethered individuals\u003c/h3\u003e\n\u003cp\u003eIn order to completely characterize the ultrasound and audible signals produced during wingbeat, we recorded tethered individuals of each sex and species with the B\u0026amp;K microphone inside the anechoic chamber. Not all individuals produced detectable ultrasound pulses. The percentage of tested individuals from which ultrasound pulses could be recorded ranged between 58% for LB males and females and 83% for GM females (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). As with the Dodotronics microphone, the amplitudes recorded with the B\u0026amp;K microphone were also small, but the signal to noise ratio was relatively better with the B\u0026amp;K microphone. Spectral components were also similar to the ones obtained with the Dodotronics microphone, ranging between 20 kHz and 50 kHz, and peaking around 35\u0026ndash;40 kHz, but with the B\u0026amp;K the section near 20 kHz was slightly damped (compare Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb with Supplementary Figure S5b).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea shows the mean values for the ultrasound pulse parameters and Tables S2 and S3 show results from the statistical analyses. Peak frequency of the ultrasound pulses ranged between 31.7 kHz (GM females) and 38.9 kHz (LB males), and it was significantly higher in LB than in GM, and in males than in females. Ultrasound pulse intensity ranged between 16.7 dB at 10 cm (LB males) and 21 dB at 10 cm (LB females), and it was higher in males than in females. Pulse duration was similar among species and sexes (ranging between 0.9 and 1.20 ms). Inter-pulse frequency was negatively correlated with body size [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], as expected [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], being 53.4 Hz in CP, 63 Hz in LB and 73.5 Hz in GM, and lower in females (which are bigger) than in males (61.8 and 64.8 Hz, respectively).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eCharacteristics of the acoustic emissions recorded from tethered tortricid moths in an anechoic chamber using a Br\u0026uuml;el \u0026amp; Kjaer microphone. \u003cb\u003ea\u003c/b\u003e, high frequency pulses, indicating the main frequency, its intensity, the duration of the pulses and the number of pulses per second. \u003cb\u003eb\u003c/b\u003e, low frequency aspect, indicating the main frequency and its intensity.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ea\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSpecies\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSex\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eN\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMain frequency, kHz (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eIntensity at 10 cm, dB SPL (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ePulse duration, ms (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eInterpulse frequency, Hz (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eC. pomonella\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFemale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e33.13\u0026thinsp;\u0026plusmn;\u0026thinsp;3.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e19.54\u0026thinsp;\u0026plusmn;\u0026thinsp;1.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e51.55\u0026thinsp;\u0026plusmn;\u0026thinsp;1.48\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eC. pomonella\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e37.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e18.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e55.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.85\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eG. molesta\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFemale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e31.70\u0026thinsp;\u0026plusmn;\u0026thinsp;1.64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e19.09\u0026thinsp;\u0026plusmn;\u0026thinsp;1.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e72.40\u0026thinsp;\u0026plusmn;\u0026thinsp;1.29\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eG. molesta\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e35.10\u0026thinsp;\u0026plusmn;\u0026thinsp;1.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e16.97\u0026thinsp;\u0026plusmn;\u0026thinsp;1.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e74.52\u0026thinsp;\u0026plusmn;\u0026thinsp;1.87\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eL. botrana\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFemale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e36.86\u0026thinsp;\u0026plusmn;\u0026thinsp;1.88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e21.06\u0026thinsp;\u0026plusmn;\u0026thinsp;2.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e61.25\u0026thinsp;\u0026plusmn;\u0026thinsp;3.14\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eL. botrana\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e38.38\u0026thinsp;\u0026plusmn;\u0026thinsp;1.48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e16.75\u0026thinsp;\u0026plusmn;\u0026thinsp;1.45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e64.80\u0026thinsp;\u0026plusmn;\u0026thinsp;1.86\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eb\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSpecies\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSex\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eN\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMain frequency, Hz (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eIntensity at 10 cm, dB SPL (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eC. pomonella\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFemale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e52.40\u0026thinsp;\u0026plusmn;\u0026thinsp;1.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e57.53\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eC. pomonella\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e54.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e56.19\u0026thinsp;\u0026plusmn;\u0026thinsp;1.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eG. molesta\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFemale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e72.20\u0026thinsp;\u0026plusmn;\u0026thinsp;1.49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e53.28\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eG. molesta\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e75.80\u0026thinsp;\u0026plusmn;\u0026thinsp;1.90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e50.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eL. botrana\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFemale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e61.80\u0026thinsp;\u0026plusmn;\u0026thinsp;1.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e53.01\u0026thinsp;\u0026plusmn;\u0026thinsp;1.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eL. botrana\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e68.20\u0026thinsp;\u0026plusmn;\u0026thinsp;1.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e51.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb shows the mean values for the low frequency (i.e., audible) wingbeat acoustic parameters, and Tables S2 and S3 show results from the statistical analyses. All species produced a two-peaked low-frequency signal in each wingbeat cycle, with several frequency harmonics over this fundamental frequency (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In 80% of GM and CP individuals the strongest component was the fundamental frequency, irrespective of sex, however in LB the second harmonic doubled the intensity of the fundamental frequency (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). CP was the loudest species (56.9 dB at 10 cm), while GM and LB were quieter (52.1 dB at 10-cm, and females were significantly louder than males (54.6 dB and 52.8 dB at 10 cm, respectively). GM had the highest fundamental frequency, followed by LB and CP, averaging 74 Hz, 65 Hz and 53 Hz, respectively (inversely proportional to the size of each species), and males had higher fundamental frequency than females, 66 Hz and 62 Hz, respectively.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eWings do not have hyaline patches\u003c/h3\u003e\n\u003cp\u003eResults from microscopy are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. Hyaline areas are shown under optical microscopy as areas that lack scales. The tortricid moth forewings examined did not reveal any area devoid of scales except for an oval area on the basal posterior margin on the ventral side. The hindwings revealed an unscaled area on the dorsal side coincident with the development of the axillary sclerites and basal veins. At stronger magnifications (SEM) the basal oval area of the forewings showed a dense field of acanthae, which are tilted obliquely towards the termen. The unscaled area, specifically on the basal dorsal side of the hindwings, appears as a smooth sclerotized plate, devoid of scales, in which the prominent main basal veins can be observed. On the hindwing of the Yponomeutid moths there is a basal hyaline patch between veins Cu2 and Cu1b equipped with a series of 10\u0026ndash;15 transverse ridges. The area is distinguishable both from the dorsal side and the ventral side.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAlthough tortricids are not toxic [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] they could mimic the ultrasound warning signals produced by sympatric toxic species to avoid bat predation. This type of Batesian mimicry has been described before in other moth species [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. For anti-bat ultrasound signals to be effective, the signal must be loud enough to reach bats with enough time to affect their hunting behavior. Therefore, anti-bat ultrasound signals are relatively loud, in the order of 70\u0026ndash;85 dB (at 10 cm) when produced by thoracic tymbals in tiger moths (Fam. Erebidae)[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], 64\u0026ndash;68 dB (at 10cm) when produced by abdominal scales in hawkmoths (Sphingidae) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], and 55-65dB (at 10 cm) when produced by wing aeroelastic tymbals by deaf and toxic microlepidoptera (i. e.: Yponomeutidae) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Although tortricids produce ultrasounds during wingbeat, their intensity is extremely low, in fact it is one of the lowest acoustic ultrasound intensities recorded from moth wingbeat, as far as we know [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], with an amplitude near 20 dB at 10 cm. This is about 100 times lower sound pressure than the 60 dB (at 10 cm) warning signals that toxic deaf Yponomeutidae emit (note that dB is a relative logarithmic unit of measurement) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The bat hearing threshold is close to the human threshold, at 2-20dB [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], so for a bat with a 10 dB hearing threshold the expected detection distance of a 60 dB (at 10 cm) moth ultrasound is about 30 m, giving the bat enough time to process the information and modify its behavior. However, this distance shortens to just 30 cm for the 20 dB (at 10 cm) tortricid ultrasound, so bats may not have enough time to process the information and will probably capture the moth. The ultrasound signal of tortricid moths would provide no protection against bats.\u003c/p\u003e\u003cp\u003eProbably most Lepidoptera emit incidental low intensity ultrasounds as the wings rub against each other or against other parts of the body at some point during wingbeat [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Natural selection should keep these emissions below predator-detection threshold if they do not have any function, or modify them, possibly improving the mechanism of production, if they are to be used as signals. Moths produce anti-bat ultrasounds either from tymbals or by stridulation, with specialized body structures that have evolved independently and are present on different parts of the body, such as the thorax, wings and abdomen [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Agassiz [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] describes the hindwing stridulatory organ of ermine moths, which has a hyaline area bearing a \u0026ldquo;stridularium\u0026rdquo; that is consistent with our own observation of Yponometutid specimens. However, although it was reported that museum specimens in the tortricid subfamily Olethreutinae may bear hyaline patches on their hindwings that could act as aeroelastic tymbals and emit ultrasounds for bat defense [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], detailed examination of the hindwings of our tortricid specimens did not reveal hyaline patches or aeroelastic tymbals. The absence of specialized sound-producing structures in tortricid wings agrees with the emission of very low intensity ultrasounds during wingbeat. In addition, ultrasound emission in tortricids was inconsistent, with some individuals producing it all of the time, other individuals not producing it (or emitting below the detection threshold), and some producing one pulse per wing beat and other two. If the ultrasound pulses had a signalling function, a specialized sound production organ and a more consistent emission should have been developed.\u003c/p\u003e\u003cp\u003eThe evolution of tympanic ears to detect bats has enabled moths to use acoustic communication in courtship behavior. Courtship acoustic signals of moths can be classified according to their intensity as either \"loud\" (ca. 60\u0026ndash;100 dB at 10 cm) or \"quiet\" (ca. 20\u0026ndash;30 dB at 10 cm) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The intensity of the tortricid ultrasound pulses is within the range of the \"quiet\" courtship songs, however it is unlikely that they can hear them because detection of far-field airborne acoustic signals requires tympanic organs, which tortricids lack, whereas all the other moth species that use ultrasounds in courtship have tympanic organs [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e][\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The \"quiet\" courtship songs were first discovered, and are well described, in species of the genus \u003cem\u003eOstrinia\u003c/em\u003e, where courting males walk around females with their wings raised up vertically above their bodies and beat them at 76 Hz, about twice as fast as when flying (43 Hz) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Friction of male-specific specialized scales on the underside of the forewings against specialized scales on the mesothorax produce the ultrasound signals of \u003cem\u003eOstrinia\u003c/em\u003e [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e][\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. GM males wingfan around females in a similar fashion as \u003cem\u003eOstrinia\u003c/em\u003e, however in GM the function of wingfanning is to deliver a short-range male pheromone, emitted from extricable caudal structures (\"hair pencils\"), directly towards the antennae of the mostly stationary females [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. It is possible that GM males emit ultrasounds while wingfanning to females (we did not record it), but even if they did, females probably could not hear them because they are atympanic.\u003c/p\u003e\u003cp\u003eThe low-frequency components of the wingbeat, however, could potentially be sensed with the mechanosensory Johnston organ located at the base of the antennae (pedicel), that responds to the particle-velocity component of sound waves [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. So, if the carrying frequency of the audible components of the wingbeat, or its harmonics, make the antenna resonate, then GM could hear its own wingbeat. This is how mosquitoes, other diptera and honeybees hear each other [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. One of the few examples of acoustic hearing in the audible range in moths involves the atympanic web clothings moth, \u003cem\u003eTineola bisselliella\u003c/em\u003e (Hum.) (Lepidoptera: Tineidae). Males of this species wingfan during courtship and the sonic components of the wingfanning attract unmated females and other males, but not mated females, which do not produce acoustic emissions during courtship [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In our study, tortricids emitted many harmonics during wingbeat, with a fundamental frequency at around 52\u0026ndash;75 Hz, so it may be worth testing the resonant frequency of their antennae to explore the possibility that the antenna can detect intraspecific acoustic signals in the audible range in these moths.\u003c/p\u003e\u003cp\u003eThe overall conclusion of our study, the first of its kind in tortricids as far as we know, is that the ultrasound pulses that they emit are an unavoidable wingbeat byproduct with no apparent biological function in either predator defense or courtship. The physical attributes of the ultrasound pulses were similar between tethered and free-flying individuals, which shows that the restraining method did not alter the acoustic signal. The characteristics of acoustic signals emitted by wing-fanning male GM during courtship, and their possible effect on courtship, are worth exploring. The strategies employed by tortricids and other atympanic and non-toxic moths to scape bat predation deserve further attention [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e][\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], as it may contribute to the implementation of bats as biocontrol agents in agriculture and forestry [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Our results indicate that ultrasound emission would probably have a negligible effect on the application of ultrasounds to control tortricid pest [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], however the wingbeat frequency data presented could contribute towards the development of automated traps to identify captured species from their wingbeat patterns [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eInsects\u003c/h2\u003e\u003cp\u003e\u003cem\u003eCydia pomonella\u003c/em\u003e, \u003cem\u003eGrapholita molesta\u003c/em\u003e, and \u003cem\u003eLobesia botrana\u003c/em\u003e (CP, GM, and LB, respectively) larvae were reared on a semi-artificial diet [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], and all the stages were kept under a 16h:8h day:night photoperiod at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C. Pupae were separated by sex and adults were separated from pupae every other day and kept with unrestricted access to 10% sucrose in water. All insects tested were between 2 and 3 days old.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eRecording setups\u003c/h3\u003e\n\u003cp\u003eWingbeat was recorded either from free flying insects in a flight tunnel, or from tethered insects, and these were recorded either in the laboratory room or inside an anechoic chamber. Two different microphones were employed, depending on availability and suitability.\u003c/p\u003e\u003cp\u003eTo record from free flying insects, we used only males because they are easily induced to fly to a pheromone source in a flight tunnel. The wind tunnel consisted of a 170 x 45 x 45 cm glass box with an air flow of 0.3 m s\u003csup\u003e-1\u003c/sup\u003e and a temperature of 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1 \u0026ordm;C, as described in [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Between 4 and 8 pheromone-emitting females were placed in the upwind end of the tunnel inside a metal-wire cage at maximum calling time [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Males were released individually in the downwind side of the tunnel and flew upwind towards females. An ultrasound microphone pointing downwards was placed 10 cm downwind from the females, as close as possible to, but not interfering with, the flying path. Ten males of each species were tested.\u003c/p\u003e\u003cp\u003eFor the rest of the recordings the insects were tethered so they could beat their wings freely while staying in place (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). They were briefly anesthetised with CO\u003csub\u003e2\u003c/sub\u003e, immobilized in a plastic pipette tip, the dorsal thoracic scales were removed with wipe paper, and a 0.71 mm x 40 mm tungsten entomological needle was attached to the clean thoracic area with a small drop of cyanoacrylate gel glue using a coarse micromanipulator (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The needle projected forward above the insect head, between the eyes and antenna, so that it did not interfere with wing or antennal movement. After \u0026lt;\u0026thinsp;10 minutes the insect was removed from the pipette tip by gently pulling out from the needle. Most individuals did not beat their wings spontaneously, although their bodies were hanging in the air, so a gentle stream of air was blown over them to stimulate several seconds of wingbeat during which the recording took place.\u003c/p\u003e\u003cp\u003eTethering setup 1 was used to synchronize sound production with wing position during the wingbeat cycle. This setup was placed directly on the laboratory bench, and the needle holding the inset was attached to a pin holder connected to a coarse micromanipulator which allowed insect rotation in 3 axes (Supplementary Figure S2). The microphone pointed horizontally towards the left side of the insect, as close to the insect as possible, but without touching its wings during wingbeat (for acoustic intensity calculations we estimated 0.5-cm distance between insect and microphone). A high-speed IR video camera filmed the insect from behind at 50 cm-distance. To synchronize sound production with wing position, five 10-ms-long pulses were sent simultaneously to a red LED light (660 nm, 160\u0026ndash;650 mcd, LedTech, UK) placed 10-15cm cm in front of the insect in the camera field of view, and to a piezo buzzer (6 kHz pure tone, RND 430, RND, Switzerland) placed 15\u0026ndash;20 cm away from the insect. LED pulses were captured with the camera and sound pulses were recorded with the microphone. From the video recordings we manually measured the distance between wingtips and plotted it as a function of time. Ultrasound pulses (see below) were superposed to this plot to reveal at which point of the wing cycle the ultrasound pulses were produced. Recordings with ultrasound pulses of 10 individuals of each species and sex were obtained.\u003c/p\u003e\u003cp\u003eTethering setup 2 was used to characterize the acoustic emissions, and it was placed inside of an anechoic chamber (Supplementary Figures \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and S2). An insect pin was fixed to the insects as indicated above, and the pin was attached to a plastic holder fitted with a ball joint in an articulated arm using washers and magnets, which allowed free orientation adjustment in all three axes (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Microphone position was set as in setup 1 and there was no camera, piezo buzzer or LED (Supplementary Figure S2). The number of GM, LB and CP recorded ranged between 12 and 16, and the number that produced ulktrasound pulses ranged bwteen 7 and 10 (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eMicrophones\u003c/h3\u003e\n\u003cp\u003eIn order to determine the part of the wing-beat cycle where ultrasound pulses were produced (tethering setup 1), and to record the wing beat of free-flying individuals (wind tunnel), we used an affordable ultrasound microphone (Ultramic 250k, Dodotronic, Italy). The microphone was calibrated against an already-calibrated measurement ultrasound microphone (Supplementary Figure S3). The Dodotronic microphone digitized the signal internally at 250 kHz sampling frequency. We set the recording sampling frequency at 250 kHz and conditioned the signal using a high-pass filter at 18 kHz with 24 dB decay per octave in Audacity 3.1.1 software [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Amplitude in dB SPL from these pulses was not calculated with this microphone because we were just interested in the occurrence of ultrasound pulses or on the difference between tethered and free-flying moths.\u003c/p\u003e\u003cp\u003eIn order to completely characterize the ultrasound emissions produced by wing-beating tethered individuals in the anechoic chamber (tethering setup 2) we used a (relatively) flat-response ultrasound microphone (Br\u0026uuml;el \u0026amp; Kjaer -B\u0026amp;K- Type 4939-A-001, \u0026frac14;-inch, calibration curve shown in Supplementary Figure S3). The microphone was fitted with pre-amplifier (Type 2670, B\u0026amp;K) and signal-conditioner (Type 1708, B\u0026amp;K), with a x10 pre-amplification and a linear filter. After analogue filtering, the signal was digitized and sampled with a digital oscilloscope (Picoscope 5242D with Picoscope 6.14.69 software, Pico Technology, United Kingdom), at 595 kHz sampling frequency and 16-bits resolution. The voltage scale was set to 20 mV range with AC coupling. Unit conversion of pulse amplitude from Volts to sound pressure level (SPL) in dB (relative to 20 \u003cem\u003e\u0026micro;\u003c/em\u003ePa) for the B\u0026amp;K microphone is described in the Supplementary material. Distance attenuation was also calculated to be able to compare our data to other references (Supplementary material).\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eAcoustic sampling and processing\u003c/h2\u003e\u003cp\u003eWhen using the Dodotronic microphone, each insect was recorded for a few seconds while wingbeating until we observed clear pulses (after filtering in Audacity). Next, we recorded a few seconds of background noise with the insect not beating its wings. From each of these recordings we selected a single 12,500 sample-points section, which corresponds to 50 ms at 250 kHz sampling rate, about 25 to 35 complete wingbeat cycles, depending on sex and species. Fast Fourier Transform (FFT) algorithm and no windowing was used to obtain the spectra in Matlab [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The background noise spectrum was subtracted from the wingbeat spectrum in each individual, and the resulting spectrum was corrected by applying the calibration curve indicated above.\u003c/p\u003e\u003cp\u003eWith the B\u0026amp;K microphone we obtained ca. 20 different 500-ms-long recordings from each individual. Recordings were checked with the spectrogram feature of Picoscope to find the one with the best signal-to-noise ratio. The selected recording was filtered [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] using an elliptic 10 kHz to 100 kHz bandpass filter, with stop band limits at 2 kHz and 150 kHz using a value of passband ripple of 0.1 dB and 60 dB of attenuation at the stop band (Supplementary Figure S3). After filtering, ultrasound pulses that exceeded an intensity threshold, which was set manually in each individual recording, were detected, and a Tukey window of 300 points was applied around them. The same filter was applied to the inter-pulse sections, which were used as noise relative to the ultrasound pulses. Then, a 2\u003csup\u003e14\u003c/sup\u003e-point FFT was applied to all the windows, pulses and inter-pulses were averaged separately, and the noise spectrum was subtracted from the signal spectrum. The resulting spectrum was multiplied by the calibration curve to obtain the final result. In order to ensure that all frequencies higher than 10 kHz are part of the signal, we also applied an elliptic filter from 500 Hz to 40 kHz with a stopband from 200 Hz to 80 kHz and a passband parameter of 0.1dB and 30 dB attenuation at the stop band (Supplementary Figure S3). The signal was also windowed using a 10000 points Tukey window to allow the lower frequencies.\u003c/p\u003e\u003cp\u003eFor the analysis of low-frequency (i.e., audible) wingbeat signals, we used a first-order low-pass filter with a 30 dB attenuation per octave from 10kHz and then performed a 2\u003csup\u003e14\u003c/sup\u003e points FFT without windowing to the entire 0.5-sec-long signal.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eMicroscopic examination of the wings\u003c/h2\u003e\u003cp\u003eWings were dry-spread perpendicular to the body axis in order to allow adequate extension of the wing membranes. In this position, the upper side of each wing will be considered dorsal, while the lower side will be considered ventral. The presence of hyaline areas was determined using a stereomicroscope (MZ9.5, Leica microsystems), and the images were obtained with a Leica Z16 stereomicroscope equipped with a DFC500 camera. Software LAS 4.0 was used for image capture. Automontage was used to increase the depth of field. For scanning electron microscopy (SEM), wings were mounted on electron microscope stubs under the stereo microscope at 40\u0026ndash;60 x, the wing surface to be glued was gently cleaned from scales with a fine brush (number 000) to ensure better adherence, and the loose scales were removed with the help of a syringe connected to an air pump. The wings were removed from the thorax at the base with the help of point-tip tweezers and were arranged on Hitachi-type electron microscope stubs (15 or 25 mm, depending on wing size) with double-side carbon tabs (Ted Pella\u0026copy; 16084-20), with the cleaned side down. Stubs were then coated with AuPd using a Sputter Coater Polaron SC7640 for 400 seconds (at ca. 3 \u0026Aring;/s). Stubs were examined and photographed using a SCIOS 2 Electron Microscope at 4 kV. Voucher specimens and SEM stubs of examined wings are deposited in the collection of the Natural History Museum at the University of Valencia (Spain). The Yponomeutid specimen was taken from this collection. All images were edited using Photoshop CS3 (Adobe\u0026reg;). Edition was restricted to background cleaning and photo merge. Several individuals were processed until the desired results were obtained. Consequently, different images of the same sex and species may not belong to the same specimen.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analyses\u003c/h2\u003e\u003cp\u003eIn order to analyse differences in acoustic intensity, dominant frequency (both low and high frequencies), ultrasound pulse duration, and inter-pulse frequency (i.e., time between the pulse from one wingbeat to the next one, if there were two pulses per wingbeat we focused only on one of them), we used Generalized Linear Models (GLM) with a gaussian family link, as a function of sex and species \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\sim{(species+sex)}^{n}\\)\u003c/span\u003e\u003c/span\u003e, being n the order of the best fitting model. Using ANOVA and the Akaike Information Criterion (AIC) we compared hierarchically different order models from the simplest (n\u0026thinsp;=\u0026thinsp;0, no dependency) to the most complex (n\u0026thinsp;=\u0026thinsp;2, 2nd order interaction) and took the most complex model that was significantly different from the previous one and with lower AIC value. Statistical analyses were run in R 4.1.2. software [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. For significant variables, pairwise comparisons between levels of a parameter were made using emmeans() package in R [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The data are included in the Supplementary file.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eC.G. was funded by a grant from the Ministerio de Ciencia e Innovaci\u0026oacute;n (MICINN, Grant No. PID2019-107030RB-C22), and A. M.-G. was funded by a Ph.D. fellowship from Ag\u0026egrave;ncia de Gesti\u0026oacute; d'Ajuts Universitaris i de Recerca (AGAUR, 2021-FISDU-00093)\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eA.M.-G. performed the acoustic experiments. A.M.-G. and L.E.-S analysed the acoustic recordings. X.B. performed the microscopic examination of the wings. All the authors contributed equally to the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eJes\u0026uacute;s Uriol (Acustics Ambient S.L., Lleida, Spain) helped with the design of the anechoic chamber.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe acoustic measurements are included in the Supplementary file.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eJacobs, D. S. \u0026amp; Bastian, A. Non-auditory defenses of prey against bat predation. 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(2024). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.R-project.org/\u003c/span\u003e\u003cspan address=\"https://www.R-project.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLenth, R. V. \u0026amp; emmeans Estimated Marginal Means, aka Least-Squares Means. R package version 1.10.5. (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://CRAN.R-project.org/package=emmeans\u003c/span\u003e\u003cspan address=\"https://CRAN.R-project.org/package=emmeans\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7623152/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7623152/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMost moths are nocturnal flying insects and some species have evolved ears to detect bats, or produce ultrasounds to dissuade or confuse them. Normally moths emit anti-bat ultrasounds when they hear bat echolocation calls, but recently it has been shown that deaf moths emit anti-bat ultrasounds continuously from specialized structures (aeroelastic tymbals) located on their wings. Structures resembling aeroelastic tymbals have been suggested from museum specimens in the family Tortricidae, and it has been proposed that members of this family can emit anti-bat ultrasounds. We recorded the wingbeat acoustic emission of three tortricid species that are predated by bats, and found that both sexes of all three species emit brief ultrasound pulses within the bat\u0026acute;s hearing frequency range at specific positions of the wingbeat cycle. However, the ultrasound pulses are very quiet (ca. 20 dB SPL at 10 cm), their emission is arbitrary (not all individuals and not every wing stroke produces them), and the wings do not bear specialized sound-producing structures. This evidence suggests that the ultrasound pulses emitted by tortricid moths during wingbeat would not be useful as an anti-bat defense mechanism. Tortricids are atympanic, so these faint wingbeat ultrasounds pulses are probably not useful in intraspecific communication either.\u003c/p\u003e","manuscriptTitle":"Characteristics and possible function of wingbeat acoustic emissions of tortricid moths","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-29 11:20:23","doi":"10.21203/rs.3.rs-7623152/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d0647c44-9452-4255-9060-fb08f8408ef7","owner":[],"postedDate":"September 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":55097012,"name":"Biological sciences/Ecology"},{"id":55097013,"name":"Earth and environmental sciences/Ecology"},{"id":55097014,"name":"Biological sciences/Zoology"}],"tags":[],"updatedAt":"2025-10-13T05:23:52+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-29 11:20:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7623152","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7623152","identity":"rs-7623152","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}