Is it a bat or a male? A female moth (Syntomeida epilais, Lepidoptera: Erebidae: Arctiinae) adapts its acoustic signals for defense or courtship

Is it a bat or a male? A female moth (Syntomeida epilais, Lepidoptera: Erebidae: Arctiinae) adapts its acoustic signals for defense or courtship | 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 Research Article Is it a bat or a male? A female moth (Syntomeida epilais, Lepidoptera: Erebidae: Arctiinae) adapts its acoustic signals for defense or courtship Frank Coro This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5133806/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Mar, 2025 Read the published version in Journal of Comparative Physiology A → Version 1 posted 13 You are reading this latest preprint version Abstract Courtship behavior in the polka-dot wasp moth Syntomieda epilais is the most elaborate acoustic communication system known in the Erebidae. Both males and females must emit their acoustic signals for successful mating under natural conditions in the presence of insectivorous echolocating bats. Females of S. epilais were stimulated during their courtship period (between 02 ÷ 30 and 06 ÷ 30) with playback of conspecific male and female signals and of a sympatric bat attack sequence and their acoustic emissions were recorded. On the third night post-eclosion at the initiation of courtship behavior, females discriminate among these types of acoustic series, responding preferentially to conspecific male signals. In contrast, during the first two nights post-eclosion, they respond strongly to the bat attack sequence but not to conspecific male signals. It is demonstrated that post-mated female moths stop responding to conspecific male signals, while continuing to respond to the bat attack series. These and other novel observations suggest that these female moths can modulate their acoustic signals, according to the stimulating conditions for defense against bats or courtship, by varying their response thresholds and latencies. Acoustic behavior acoustic playbacks post-eclosion post-mating bat predation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Moth hearing and sound production in species with acoustic emission organs, have evolved under the evolutionary pressure of insectivorous echolocating bats (Conner 1999 ; Conner and Corcoran 2012 ; Nakano et al. 2015 ; ter Hofstede and Ratcliffe 2016 ; Barber et al. 2022 ). Most of the results published on moth acoustic emission organs have dealt with their function in bat-moth interactions. Blest et al. ( 1963 ) described the metathoracic tymbal organs in Erebidae, Arctiinae. Most of these moth species emit what is called a modulation cycle (MC). Each MC is composed of the active modulation half-cycle (MHC), the result of the inbuckling of the striated band on the surface of the tymbal organ, and the passive MHC due to the outbuckling of the tymbal organ (Blest et al. 1963 ; Fullard and Fenton 1977 ; Conner 1999 ). The first demonstration of Arctiinae moths using their acoustic emissions not only in their interactions with bats was reported by Conner ( 1987 ). This author recorded acoustic emissions from males of Cycnia tenera (Arctiinae) during the final moments before mating. Sanderford and Conner ( 1990 , 1995 ) reported sexually dimorphic acoustic signals used during courtship behavior in moths while studying the polka-dot wasp moth, Syntomeida epilas (Arctiinae, Euchromiina). In this species, males emit their MCs at a slower repetition rate (around 8.5 MC. s − 1 ) than females ( ca. 15 MC.s − 1 ). Females respond to their male signals in an antiphonal mode, and the female’s acoustic reply is essential for successful mating. Using artificially produced MCs presented at slow and high repetition rates, simulating male and female emissions, respectively, Sanderford ( 1992 , p. 54) reported that some S. epilais females were better than others at discriminating between male and female emissions. Sanderford ( 1992 , p. 36) also observed that S. epilais mating behavior is not seen during the first night post-eclosion (PE), very little courtship occurs during the second night, and normal mating behavior is usually fully developed by the third night PE. For mating, S. epilais females perch on the underside of the leaves of their most common host plant in Florida, Nerium oleander (Sanderford 1992 , p. 46). The acoustic signals emitted by both male and female S. epilais during courtship have been classified as used for long-distance communication (beyond 5 cm) in comparison to signals emitted by several other moth species (Nakano et al. 2009 , 2015 ). Several experiments stimulating different Arctiinae moth species with bat calls and conspecific male emissions to study their acoustic responses have been carried out in which the moths are held by their wings folded above the thorax to simulate flying, or while tethered (Corcoran et al. 2010 ; Nakano et al. 2013 ; Dowdy and Conner 2016 , 2019 ; Fernandez et al. 2020 ; Barber et al 2022 ). Tethered S. epilais moths have been used to study acoustic aposematism during their interactions with sympatric bats. The defensive use of acoustic signals in S. epilais is considered to be an example of Müllerian mimicry (Barber and Conner 2007 ; Conner and Corcoran 2012 ). Courtship behavior in S. epilais has been recognized as the most elaborate known acoustic communication system in the Erebidae, Arctiinae (Conner 1999 ; Sanderford 2009 ). In most other Noctuoidea and Pyraloidea moth species only males produce acoustic emissions during mating (Conner 1987 ; Simmons and Conner 1996 ; Nakano et al. 2010 , 2013 , 2015 ; Nakano 2023 ). My aim was to study a behavior involving the whole auditory system and the motor component generating these emissions in an intact moth under natural (outdoor) conditions. The questions addressed are: (1) do these females emit acoustic signals during their first two nights PE; (2) is mating affecting their acoustic behavior; and (3) Do their acoustic responses to acoustic stimuli differ depending on whether the females are perched or held by the wings above the thorax. In this study I demonstrate that virgin, perched S. epilais females with three or more nights PE discriminate among conspecific male and female signals, and a sympatric bat attack sequence, responding preferentially to their male emissions. I show that they emit acoustic signals during the first two nights PE responding to bat attack series and not to the male signals. This is the first report of post-mated female moths that stop responding acoustically to conspecific male signals and continue to respond to bat attack calls. For the first time it is shown here that a female moth adapts her acoustic response for defense against bats or for courtship by varying response threshold and latency. Materials and Methods Subjects S. epilais fourth- and fifth- instar larvae were collected in stands of Nerium oleander in Southeastern Florida, Miami Dade County. The larvae were fed with fresh oleander leaves under natural (outdoor) conditions in the terrace of my home (25 o 7’ 21.82” N – 80 o 4’ 24.80” W) open to my backyard. Prior to and after pupation, the insects were continuously exposed to temperatures, humidities, and photoperiods typical of Southeastern Florida between April and November. Once pupation occurred, they were checked daily for moth eclosion, upon which females and males were segregated in separate foam boxes that had screen wire sections allowing the moths to be exposed to the photoperiod of the environment. The moths were fed ad libitum with a watery honey solution in vials with cotton placed on top of the box. All the moths used during this study were later released to the natural environment where they had been collected as larvae. None of these moths were reared from eggs laid by females that mated under these conditions. 64/74 (86%) of the females stimulated with the playback of the three different acoustic series present in their environment during their courtship behavior hours (between 02 ÷ 30 and 06 ÷ 30) were virgin and were at least three nights PE. Nine females that were experimentally stimulated and responded acoustically to male acoustic series were placed in boxes with males where they mated. After mating, these moths were also acoustically stimulated and later returned to the sites where they had been collected as larvae. Acoustic recordings All the recordings were done using equipment from Avisoft Bioacoustics (Glienicke Germany): Condenser Microphone CM16/CMPA (10 kHz to 140 kHz ± 3 dB) attached to an UltraSoundGate 116H (frequency response from 20 Hz to 460 kHz) data acquisition device at 250 kHz sampling frequency via a laptop computer running Avisoft Recorder software. During the acoustic stimulation of females, the microphone was placed 5 cm behind a perched moth (Fig. 1 ), or one held by the wings above the thorax. Temporal variables from the female emissions in response to the acoustic stimuli were analyzed using BatSound (version 4.14) software (Pettersson Electronics and Acoustics AB, Sweden). All files were saved in .WAV format. Acoustic stimulation All acoustic stimulation series were delivered through an ultrasonic dynamic speaker with a flat (± 6 dB) frequency response from 5_ 70 kHz (Vifa, Avisoft Bioacoustics, Germany) placed 60 cm from the female (Fig. 1 ). The speaker received the electric signals from an UltraSoundGate Player 116 (frequency range: 1_125 kHz; Avisoft Bioacoustics, Germany) via a laptop computer running Avisoft Recorder software. A Calibrated 40 kHz (± 3 kHz) Reference Signal Generator (Avisoft Bioacoustics, Germany) was used. This device generates a 40 kHz sinusoidal signal at an amplitude of 75 dB SPL (± 3 dB), which was recorded at 250 kHz sampling frequency. This signal was used in all the amplitude calibration procedures in combination with Avisoft-SAS Lab Pro software. This reference signal was recorded at the gain setting of the UltraSoundGate 116H device used in the subsequent measurements of the three- stimulation series applied. Using this procedure ensures that all amplitude measurements done are referenced to this signal and expressed in dB SPL (0 dB = 20 µPa). For the calibration of the three acoustic stimuli applied, the microphone was placed directly in front and 60 cm from the loudspeaker, at the position where the moth perches (Fig. 1 ). All the acoustic series applied were digitized at a sampling frequency of 250 kHz (4 µs period) and played back at this same sampling frequency. All acoustic series presented to S. epilais females (Fig. 2 ) were playback of recordings made under natural (outdoor) conditions. No synthetic (artificial) acoustic stimuli were used. The male and female emissions were recorded by the author while the specimens were freely flying during their courtship behavior hours under outdoor conditions. The male series lasts 1.2 s and has 12 MCs with a mean (± SD) repetition rate of 9.3 ± 1.3 MC. s − 1 , calculated using the inverse of the interval between the MCs. The female emission series lasts 0.6 s and consists of 10 MCs with a mean (± SD) repetition rate of 15.9 ± 2.2 MC. s − 1 . The repetition rate of each of these S. epilais male and female emissions falls within the values described by Sanderford and Conner ( 1990 , 1995 ). The other stimulation series applied to S. epilais females was a playback of an attack sequence from a free-flying Mexican free-tailed bat, Tadarida brasiliensis , recorded in Florida (courtesy of Dr. Aaron Corcoran). This is the most abundant insectivorous bat in Florida that is sympatric with S. epilais during its courtship behavior hours. This bat attack series lasts 1.1 s and consists of 40 pulses varying in both duration and repetition rate. It consists of the following phases: search, early and late approach, and the buzz starting 1,005 ms from the beginning of the series (shown by the asterisk in Fig. 2 ). Each of these stimulation series was analyzed for the frequency components of its MCs or pulses (values as mean ± SD). The power spectrum of the click with highest amplitude in each of the 12 MCs in the male series (duration 545 ± 78 µs), in each of the 10 MCs in the female series (515 ± 50 µs) and in each of the first ten bat pulses (9.5 ± 4.5 ms, values between 14.5 _3.2 ms) was measured using Batsound software. The peak frequency of the male clicks was 34 ± 1 kHz, that of the female clicks 39 ± 0.5 kHz, and in the bat pulses analyzed 37 ± 2 kHz. The 20 dB range below the peak frequency in the male clicks is between 28 _ 40 kHz, in the female clicks between 34 _ 46 kHz, and in the first ten bat pulses it is between 35 _ 41 kHz. The three-stimulation series applied have the same frequency range. The three-stimulation series were also measured for the maximal amplitude of each of the male and female clicks, and for each of the 40 pulses in the bat attack sequence. The Avisoft SAS Lab Pro software with the 75 dB SPL calibration signal previously recorded together with the one-dimensional transformation function in RMS (logarithmic) was used for this purpose. As all these series were recorded at 250 kHz sampling frequency (4µs period), the averaging time used for these measurements was 100 µs. The median and interquartile range of the 12 male clicks is 94 dB SPL (range 91.5 _94.5 dB SPL), in the 10 female clicks the amplitude is 95 dB SPL, and in the 40 bat pulses it is 94 dB SPL (range 91.5 _ 97 dB SPL). These maximal amplitude values do not differ statistically (Kruskal-Wallis test KW = 3.6, p = 0.16). Thus, the maximal amplitude of each of the series applied was considered as 94 dB SPL (these series were presented with 3-dB increments). The acoustic responses of S. epilais female were analyzed using BatSound (version 4.14) software. These three-stimulation series (Fig. 2 ) were presented only once at each amplitude applied to each tested female in the following order: male- female- bat- female- male- bat- female- bat- male. There was a 5-s silence interval after each of these individual series. This silence interval is the same as that used by Dowdy and Conner ( 2019 ) when stimulating several Arctiinae moths with a playback of a sympatric bat attack sequence. The main feature of the female responses analyzed was the number of MCs during the applied series. The mean value from the responses to the three series at each amplitude was analyzed. A given stimulation series was considered to have female response if there was one or more female MCs in at least one of the three series applied at each amplitude. The threshold of a specimen for each of the three-stimulation series presented was considered as the lowest amplitude at which the female responded with one or more MCs to at least one of the three series of the same type presented. The threshold values mentioned in the text are expressed as median and its interquartile range. The latency of the response was measured as the interval between the onset of the stimulus and the first female MC in the response. The mean value from at least two latency measurements at a given amplitude was considered for further analysis. The latency values mentioned in the text are the median and its interquartile range. Figure 2 Discrimination ability. To test the ability of S. epilais females to discriminate among the three-stimulation series applied, 55 perched, virgin, three or more nights PE females were stimulated during their courtship hours. Each female was stimulated with the three series only once using a wide range of amplitudes with 3-dB increments. All these recordings were obtained in outdoor conditions (see Fig. 1 ). These results obtained on 26 different nights, are from nine females in October - November 2018, and from 46 females between April and October 2019. During 17 of these nights, between two and five different females were acoustically stimulated during their courtship period. Between 33 and 42 of each of the three-stimulation series were applied once to each of these females, for a total of 2,115 series presented (overall, 6,345 series analyzed). The starting amplitudes of the stimuli presented varied between 55- and 64-dB SPL, depending on the threshold of the specimen to the male emission series to which most of these females showed the lowest value. As shown in Fig. 2 , the duration of the playback of the male emission and the bat attack series are similar: 1.2 s in the male series and 1.1 s in the bat series. These two-stimulation series differ in the number of acoustic stimuli presented: 12 male MCs and 40 bat pulses. To compare the female response to each of these series at each of the amplitudes applied, the number of female MCs per second (MC.s − 1 ) was analyzed. The male series and the female series have a similar number of MCs (12 in the male series and 10 in the female series) but at a different repetition rate. To compare the female response to each of these two series, the number of female MCs was used expressed as percentage of applied MCs at each of the applied stimulus amplitude. Time after eclosion. To study the possible effects of the first three nights PE of the females on their acoustic emissions, ten females from the same batch were examined (previously collected as larvae on the same day, fed until they pupated, and separated individually on the day of their eclosion). Females from each batch were acoustically stimulated only once while perched on the first three consecutive nights PE with the three-acoustic series previously described. The number of acoustic series presented on each night depended on the threshold of the specimens to these stimuli. Virgin versus post-mated. To test the possible effects of mating on the acoustic emissions of S. epilais females, I stimulated another group of the same nine perched females both while virgin and after mating. Each of the acoustic stimuli was presented only once to each female while they were perched during the hours of their normal courtship behavior. The virgin moths were tested 4.6 ± 1.3 (mean ± SD) nights PE, and they were stimulated again after they mated 5.9 ± 1.3 nights PE. The post-mated females were stimulated 24_48 hours after mating. The nine virgin females were stimulated with a total 1,071 series (357 series of each type), while a total of 234 series (78 series of each type) were presented to the post-mated moths. Holding by the wings. To study the effect of holding the moth by its wings, I acoustically stimulated the same 17 virgin females with three or more nights PE both while perched and when held by their wings folded above the thorax. All acoustic stimuli were presented only once to each female while perched or held by the wings. While perched, these females were presented with a total of 1,980 acoustic series (660 series of each type); when held by the wings, a total of 522 series (174 of each type) were applied. Latency. To study the changes in latency to the bat attack series, I selected the same seventeen females stimulated while perched and when held by the wings and the same nine females stimulated while virgin and after mating. When not responding to the male series all these females show a threshold above 80 dB SPL to the bat series. Therefore, the data was pooled for the latency at amplitudes of 88-, 91-, and 94-dB SPL. Statistics. Before applying a statistical test to a set of data, it was tested for normality (Kolmogorov-Smirnov test). When applying a paired test, the effectiveness of the pairing was also tested. Comparison between two or three data sets was analyzed using either parametric or non-parametric tests, according to the characteristics of the data. The statistical significance for all tests was set at p- value < 0.05. Statistical tests were done using GraphPad InStat 3 and OriginLab 8.6. All graphs were done using OriginLab 8.6. Results Syntomeida epilais females discriminate among three acoustic series present in their environment during their courtship behavior hours Since both males and females of S. epilais use acoustic communication during courtship behavior that takes place in the presence of flying insectivorous bats emitting their echolocation calls, discrimination among these acoustic emissions present in their environment becomes critical for successful mating. The comparison of the percentage of the series with female response showed statistically significant differences indicating more male series with female response (at 96%), less female response to bat attack series (at 70%), and the least response to female emission series (around 44%) (Fig. 3 a). In the large female population (55 virgin, perched, and at least three nights PE moths) studied between April and November, the responses also differed considerably regarding their coefficient of variation: only 3.3% for the male series, 35% for bat series, and 62% with female series. These data suggest that under these conditions, the female acoustic response to the male signals has the highest behavioral significance, facilitating the courtship acoustic interactions needed for mating. Some females better than others discriminated the male signals from female signals and bat attack sequences. For example, one of the 55 females did not respond to any of the 36 female series nor to any of the 36 bat attack sequences presented, while it responded to 92% of the 36 male series applied. Four females did not respond to female series at any of the amplitudes presented, while responding between 15% and 47% of bat attack series, and between 92% and 100% of the male series. The comparison of the female threshold to each of the acoustic series also showed statistically significant differences with the lowest value (58 dB SPL, interquartile range 55_61 dB SPL) for the male series, an intermediate value for the bat attack series (61 dB SPL, range 58_64 dB SPL), and the highest threshold for the female series (70 dB SPL, range 64_76 dB SPL) (Fig. 3 b). The comparison of the female responses to the male series and the bat attack sequence at each of the stimulus amplitude applied (Fig. 3 c) showed that in the whole amplitude range analyzed (from 58 to 94 dB SPL) the females responded with significantly more MC.s − 1 to the male series than to the bat attack series during courtship. The comparison of the female responses at each of the stimulus amplitude analyzed showed that in the whole amplitude range they responded significantly more to the male series than to the female series (Fig. 3 d). When comparing the responses in the whole amplitude range applied in each of the 55 stimulated females, in 46 of them (84%) there are statistically significant more MC.s − 1 to the male series than to the bat attack sequence (N = 11_13, Wilcoxon or paired t test, p 0.05) in their acoustic response to these same two stimuli. The comparison of the responses in each of these 55 females to the male and female signals showed that in all of them there are statistically significant more responses to the male signals than to the female emissions (same paired tests, p < 10 − 3 ). Even five of these females (9%) did not respond to any of the female signals in the whole amplitude range applied while responding to all the male signals. These results suggest that these perched, virgin, three + nights PE females are better discriminating between the male and female signals than between their conspecific male emission and the bat attack sequence. The abovementioned nine females were tested at eight different nights during which one or more females (for a total of 16) were also stimulated. All the other 16 tested females discriminated between their conspecific male signals and the bat attack sequence. Even one of them did not respond to any of the bat attack signals in the whole amplitude range applied. These results strongly suggest that the ability to discriminate among these acoustic signals by this population of S. epilais females depends on some internal condition that seems to vary in each specimen, as for example under the effects of a juvenile hormone. Effects of the first three nights post-eclosion on females’ acoustic emissions Ten different virgin, perched females were stimulated during the first, second-, and third-nights PE with the three-stimulating series described in Fig. 2 . During their first- and second-nights PE, these females respond randomly to these acoustic stimuli. This means that the same female may respond to some of these acoustic series on the first night but to none of them during the second night PE. Also, a female may not respond to any of these three series on the first night, but to some of them during the second night PE. During the first two nights PE, the females only responded to the bat attack sequence and the female series, while on the third night PE they also responded to the male series. During the first- and second-nights PE, the ten females were stimulated with a total of 450 series (150 series of each type). They responded to less than 1% of the male series presented, while responding to more than 70% of the bat series and to more than 20% of the female series (Fig. 4 a). The ten females studied during their third night PE were stimulated with a total of 1,200 series (400 series of each type). It is during the third night PE that the females start responding to 97% of the applied male series. The female responses to the bat attack series during the first three nights PE did not differ significantly (Friedman test Fr = 0.24, p = 0.89). However, the responses to the female series were higher during the third night PE than during the first two nights (Kruskal-Wallis test KW = 8.6, p = 0.01; Dunn’s test, p < 0.05) (Fig. 4 a). Likewise, the females’ threshold to the stimulation series changes strongly during the first three nights PE (Fig. 4 b). During the first two nights, only one of the ten females responded to one of the male series applied at the highest amplitude (94 dB SPL). During their third night PE, their threshold to the male series decreased considerably to 55 dB SPL (range 55_58 dB SPL) (Fig. 4 b). When responding to the bat attack series during the first night the threshold was 88 dB SPL (range 82_91 dB SPL), on the second night it was 91 dB SPL (range 88_94 dB SPL), whereas it decreased significantly to 61 dB SPL (range 55_64 dB SPL) during their third night (Friedman test Fr. =16.6.7, p < 10 − 4 ; Dunn’s test, p < 0.01). The threshold to the female series showed a similar tendency: during the first night PE at 91 dB SPL (range 91_94 dB SPL), second night at 94 dB SPL (range 88_94 dB SPL) and decreased to 67 dB SPL (range 64_76 dB SPL) during the third night (Kruskal-Wallis test KW = 17.4, p = 0.0004; Dunn’s test, p < 0.01) (Fig. 4 b). Effects of mating on females’ acoustic emissions Another factor not studied in moths and possibly affecting the acoustic emissions in S. epilais females is mating. While virgin and perched, the nine tested females responded to 93% of the male series applied; after mating and perched, they only responded to 2% of these series (Fig. 5 a). The comparison of the responses to the bat attack and the female series expressed in percentage of series with female response in their virgin or post-mated condition showed no significant difference between them (Mann-Whitney test and unpaired t test (p > 0.05) (Fig. 5 a). The male series is almost completely ineffective in evoking a response from post-mated females, whereas the bat attack series and the female series still evoke a response from these moths (Fig. 5 b). The post-mated females also show a statistically significant increase of their threshold to these two stimuli. Regarding the bat series it is at 88 dB SPL (range 85_91 dB SPL) in the post-mated females as compared to 61 dB SPL (range 58_64 dB SPL) in the virgin females for an increase of 27 dB. Similarly, a significant increase from 73 dB SPL (range 69_73 dB SPL) to 94 dB SPL (range 91_94 dB SPL) was obtained when comparing the threshold to the female series in the virgin and post-mated females, respectively. Perched or Held by the Wings: does it affect the females’ acoustic responses? Syntomeida epilais virgin females respond differently when stimulated under these two conditions. When perched, 17 virgin females responded to 97% of the male series presented. When held by the wings, they responded to only 3% of the male series (Fig. 6 a). When perched, S. epilais females respond to around 60% of the bat series presented, while they statistically significantly increase their respond to around 80% when held by the wings. These 17 females do not show a statistically significant difference in their response to the female series under these two conditions (Fig. 6 a). When perched, all these 17 females responded to male series with a median threshold value of 56 dB SPL (range 55_61 dB SPL). When held by the wings, only four of them responded, but at much higher amplitudes, between 85- and 94-dB SPL. The threshold to the bat series increases significantly, from 64 dB SPL (range 61_70 dB SPL) while perched, to 85 dB SPL (range 82_91 dB SPL) when held by the wings (Fig. 6 b). With the female series, the threshold increase is from 70 dB SPL (range 64_79 dB SPL) while perched to 91 dB SPL (range 85_91 dB SPL) when held by the wings. For both these stimuli, the increase of threshold is 21 dB when the moth is held by the wings. Do S. epilais females adapt their acoustic signals for defense or courtship? Results depicted in Figs. 4 , 5 , and 6 , strongly suggest that S. epilais females modulate the emission of their acoustic signals according to their physiological condition (during their first two nights PE vs. with three or more nights PE) and mating status (virgin or post-mated). While virgin, the females also responded to acoustic stimulations differently depending on being perched or held by the wings. In all these situations, S. epilais females show changes in their responsiveness: when females do not respond to conspecific male acoustic signals, they have a statistically significant higher threshold to the bat attack sequence (increase of more than 20 dB) than when they also respond to conspecific male signals. When the females do not respond to male emissions, they also show changes in their latency to the bat attack series. Figure 7 a shows the increase in the latency to the bat series in the same female specimen when stimulated at the same acoustic amplitude while perched (upper oscillogram) and when held by the wings (lower oscillogram). The response latencies to the bat stimulus of 17 females when perched and held by the wings, respectively, increased significantly (Fig. 7 b) when the females do not respond to the male signals. A similar comparison of nine females in their virgin or post-mated state also shows a significant increase (Fig. 7 c) of their response latencies when females do not respond to their male acoustic signals. Similar results to those shown in Fig. 7 b, c was obtained when comparing the latency to the bat attack sequence applied to the ten females stimulated during the first three nights PE. During the first two nights PE, when they were not responding to the male series (see Fig. 4 a), the latency to the bat series had a statistically significantly longer median value 1,020 ms ( range 970_1,060 ms, n = 31) than during the third night − 320 ms, (range 220_695 ms, n = 27), when they were already responding to the male series (Mann-Whitney test, U = 49, p < 10 − 4 ). Discussion Conspecific male signals preferred. The 55 perched, virgin, S. epilais females three or more nights PE tested between April and November not only responded preferentially to their male acoustic signals, but also with much less variability than to the other two acoustic series present in their environment during their courtship behavior hours. These females also respond with more MCs and a lower threshold to the male series than to the other two-stimulation series applied. These females are also able to discriminate among these three acoustic series present in their environment during their courtship hours in a wide and ecologically relevant amplitude range, between 58- and 94-dB SPL, responding with more MCs to the male series from the lowest to the highest amplitude applied (see Fig. 3 c, d). With this discrimination ability, S. epilais females ensure the intraspecific acoustic antiphonal communication within this wide amplitude range essential for successful mating. The intensity coding ability of S. epilais females in a wide ecologically relevant amplitude range, is reported for the acoustic responses in an intact moth for the first time. These results emphasize the difference in discrimination ability among Noctuoidea females. For example, Nakano et al. ( 2010 ) report that Spodoptera litura (Noctuidae) females do not distinguish between conspecific male songs and insectivorous bat calls. Delayed post-eclosion courtship. Sanderford ( 1992 , p. 36) observed that S. epilais mating behavior is not seen during the first night PE, very little courtship occurs during the second night and normal mating behavior is usually fully developed by the third night PE. The proposed explanation was that moths were not able to produce their acoustic emissions because their tymbal organs were not yet sclerotized and hard enough to generate the ultrasonic signals needed for courtship. However, according to the results shown in Fig. 4 the tymbal organs in S. epilais females are already fully functional during the first- and second-night PE since the females can acoustically respond to the bat attack and female series albeit at stimulus amplitudes above 80 dB SPL. These are the first results from stimulating virgin, perched Erebidae (Arctiinae) female moths with different acoustic series present in their environment during their first three nights PE. The fact that S. epilais females acoustically respond to the bat attack series before they emit their acoustic signals in response to conspecific male emissions, supports Conner’s ( 1999 ) statement that: “in both their sensory and motor aspects, the weapons of bat/moth warfare have frequently evolved into components of courtship systems”. So far, an increase of auditory sensitivity in any female moth occurring between their eclosion and the time at which they are participating in courtship behavior has been an unanswered question. The female responses to the male series on the third night PE showing the threshold at 55 dB SPL, as well as the threshold decrease by more than 20 dB when responding to the bat attack and female series, may be explained by the secretion of a hormone between the second- and third-night PE that increases their auditory sensitivity. This would allow them to acoustically interact with conspecific males during courtship behavior hours starting on the third night PE as previously described by Sanderford and Conner ( 1990 , 1995 ) and confirmed by my personal observations. The role of juvenile hormone? Stimulation of the nine post-mated females showed that they practically do not respond to the male series, while still responding to the bat series at higher amplitudes (above 80 dB SPL). As post-mated females fly for laying their fertilized eggs, they will continue interacting acoustically with insectivorous bats, trying to avoid them. These are the first results indicating strong effects of mating on the acoustic behavior of female moths. These results in S. epilais females could be explained considering that in female crickets it has been shown that mating abolishes or reduces their phonotactic behavior when they are presented with their male acoustic signals (Koudele at al. 1987; Loher et al. 1993 ; Lickman et al. 1998 ). Also, in female crickets Choi et al. ( 2012 ) demonstrated that Juvenile Hormone III makes them more selective for conspecific male calling songs. If a similar effect of JH III occurs in S. epilais females, this could help explain the variability of their acoustic responses among the specimens of the same population to different acoustic stimuli. An example of this variability was reported by Sanderford ( 1992 , p. 54) describing the results from 24 virgin, perched S. epilais females differently discriminating between male and female synthetic analog emissions. Another case is provided by my own experiments with playbacks of conspecific male and female series presented to 55 virgin, perched females. Results from these same 55 females, also show variability among them with respect to their discrimination ability between their male conspecific signal and the bat attack sequence. All these findings might be explained assuming that S. epilais females with higher concentration of the juvenile hormone will discriminate better in favor of their conspecific male signal, as reported for the phonotaxis in female crickets. With respect to the significant loss of auditory sensitivity to the bat attack sequence and the female series in post-mated S. epilais females (Fig. 5 b), for crickets Pollack ( 2016 ) states that “the mechanisms linking JH and poor sensitivity to ultrasound are completely unexplored”. Differences within the Erebidae. The only previous report that I have found of acoustically stimulating the same female moths when stationary (= perched) and while flying tethered, is by Nakano et al. ( 2013 ). These authors found that perched Manulea (formerly Eilema ) japonica females do not emit acoustic signals in response to conspecific male song, while they do produce them in response to constant frequency (CF) and frequency modulated (FM) bat calls. While flying tethered, these females still respond significantly less to conspecific male song than to CF and FM bat calls. These results clearly differ from those described here in S. epilais virgin females stimulated under these two similar conditions. The result of this comparison increases the diversity of acoustic behaviors for defense and courtship, previously described among Erebidae female moths. According to Sanderford ( 2009 ) in the majority of the arctiid (= arctiinae) mating systems studied, the sounds emitted during courtship do not appear to differ substantially from those used in bat defense. Recently, Fernandez et al. ( 2020 ) described that when held by the wings Bertholdia trigona (Erebidae: Arctiinae) females’ response to bats tends to continue throughout and after playback of the bat emissions, while this behavior was not seen in female responses to male acoustic signals. Also, B. trigona female responses to conspecific male emissions had a lower number of MCs than to the bat emission. The results obtained in S. epilais females differ from those reported in this other Arctiinae moth. Importance of behavioral context. To my knowledge, the results from S. epilais females are the first report of changes in the acoustic responses of female moths depending on the behavioral context: courtship or defense. Virgin, perched, and three or more nights PE females respond to more than 90% of the applied conspecific male signals showing a threshold at 58 dB SPL, thus increasing the probability of acoustically interacting with flying males emitting their signals. When perched during their first two nights PE and after mating, as well as when stimulated while virgin and held by the wings, they practically do not respond to conspecific male signals, showing significantly higher threshold and longer latency in their responses to the bat attack signals. Under the latter conditions, their first MC in response to the bat attack sequence preferentially falls during the buzz phase. The significant increase of the threshold to the bat attack sequence in S. epilais females when they are not responding to conspecific male acoustic signals is the first report of such change in moths. A possible explanation of the behavioral advantage of only responding to the bat attack sequences above 80 dB SPL may come from the behavior of some rainforest crickets (Romer and Holderied 2020; Romer 2021). The bat avoidance behavior of these crickets has thresholds around 80 dB SPL and their response is always a short cessation of flight. These authors suggest that this high behavioral threshold rejects the irrelevant background noise, including bat calls below 80 dB SPL from bats that are further away. A similar behavioral significance may explain this change in S. epilais females when they are held by the wings or while perched and not responding to conspecific male signals. Conclusion The results presented in this paper open new research questions in moth bioacoustics. For example, which are the sensory and neural bases of the discrimination among the three different acoustic series presented? What are the possible effects of Juvenile Hormone on the auditory system of moths? Which are the mechanisms involved in changing the acoustic emissions in the same virgin female when stimulated while perched or held by the wings? Also, we can ask a comparative question: are some or all the unique features described here for females of Syntomeida epilais present in females of other moth species? Declarations Competing interests: The author declares no competing interests. Conflict of interest: The author declares no conflict of interest. Funding: Miami Dade College bought all the hardware and software used in this study and funded the author to assist with the preparation of this manuscript. Author Contribution The author (Frank Coro) conceived and designed the study, performed the research, analyzed the data, originated all the figures, and wrote the paper. Acknowledgments I dedicate this paper to my late colleague Dr. Natacha Portilla, who in the early 1980’s encouraged me to study the acoustic emissions in moths with two-celled ears during their courtship behavior. Friedrich G. Barth, Peter Narins, William E. Conner, all Professor Emeritus, and Professor Manfred Kössl provided very helpful comments and suggestions on earlier versions. Through all these years, while the results were obtained, Mark Sanderford shared his knowledge on the courtship behavior of Syntomeida epilais and commented on earlier versions. I thank Aaron Corcoran for providing the digital recording of the attack sequence of Tadarida brasiliensis . My thanks to Miami Dade College for supplying the equipment and software used in this research. References Barber JR, Conner WE (2007) Acoustic mimicry in a predator prey interaction. PNAS 104:9331–9334. https://doi.org/10.1073/pnas.0703627104 Barber JR, Plotkin D, Rubin JJ, Homziak TN, Leavell BC, Peter R, Houlihan PR, Minera KA, Breinholt JW, Quirk-Royala B, Padrón PS, Nunez M, Kawahara AY (2022) Anti-bat ultrasound production in moths is globally and phylogenetically widespread. PNAS 119:e2117485119. https://doi.org/10.1073/pnas.2117485119 Blest AD, Collett TS, Pye JD (1963) The generation of ultrasonic signals by a New World Arctiid moth. Proc. R. Soc. Lond. B 158:196–207. https//doi.org/10.1098/rspb.1963.0042 Choi R, Atkins G, Stout J (2012) The effects of injecting Juvenile Hormone III into the prothoracic ganglion on phonotaxis by female crickets Gryllus bimaculatus . Physiol Entomol 37:201–205. https://doi.org/10.1111/j.1365-3032.2011.00811.x Conner WE (1987) Ultrasound: its role in the courtship of the arctiid moth, Cycnia tenera . Experientia 43:1029–1031. https://doi.org/10.1007/BF01952230 Conner WE (1999) Un Chant D’Appel Amoureux’: Acoustic Communication in Moths. J Exp Biol 202:1711–1723. https://doi.org/10.1242/jeb.202.13.1711 Conner WE, Hristov NI, Barber JR (2009) Sound Strategies: Acoustic Aposematism, Startle, and Sonar Jamming. In: Conner WE (ed) Tiger Moths and Woolly Bears: Behavior, Ecology, and Evolution of the Arctiidae, Oxford University Press, pp 177–191 Conner WE, Corcoran AJ (2012) Sound Strategies: The 65-Million-Year-Old Battle Between Bats and Insects. Annu Rev Entomol 57:21–39. https://doi.org/10.1146/annurev-ento-121510-133537 Corcoran AJ, Conner WE, Barber JR (2010) Anti-bat tiger moth sounds: Form and function. Curr Zool 56:358–369. https://doi.org/10.1093/czoolo/56.3.358 Dowdy NJ, Conner WE (2016) Acoustic Aposematism and Evasive Action in Select Chemically Defended Arctiine (Lepidoptera: Erebidae) Species: Nonchalant or Not? PLoS ONE 11:e0152981. https://doi:10.1371/journal.pone.0152981 Dowdy NJ, Conner WE (2019) Characteristics of tiger moth (Erebidae: Arctiinae) anti-bat sounds can be predicted from tymbal morphology. Front Zool 16:45. https://doi.org/10.1186/s12983-019-0345-6 Fernandez Y, Dowdy NJ, Conner WE (2020) Extreme Duty Cycles in the Acoustic Signals of Tiger Moths: Sexual and Natural Selection Operating in Parallel. Integr Org Biol 2:obaa046. https://doi.org/10.1093/iob/obaa046 Fullard JH, Fenton MB (1977) Acoustic and behavioral analyses of the sounds produced by some species of some species of Nearctic Arctiidae (Lepidoptera). Can J Zool 55:1213–1224. https://doi.org/10.1139/z77-160 Koudele K, Stout J, Reichert D (1987) Factors which influence female crickets’ ( Acheta domesticus ) phonotactic and sexual responsiveness to males. Physiol Entomol 12:67–80. https://doi.org/10.1111/j.1365-3032.1987.tb00725.x Lickman K, Murray A-M, Cade WH (1998) Effect of mating on female phonotactic response in Gryllus integer (Orthoptera: Gryllidae). Can J Zool 76:1263–1268. https://doi.org/10.1139/z98-061 Loher W, Weber T, Huber F (1993) The effect of mating on phonotactic behaviour in Gryllus bimaculatus (De Geer). Physiol Entomol 18:57–66. https://doi.org/10.1111/j.1365-3032.1993.tb00449.x Nakano R (2023) Multiple functions of ultrasonic courtship song in moths. In Acoustic Communication in Animals . In:Y. Seki (ed) Springer Nature Singapore Pte Ltd., pp 47–61. https//doi.org/10.1007/978-981-99-0831-8_3 Nakano R, Takanashi T, Fujii T, Skals N, Surlykke A, Ishikawa Y (2009) Moths are not silent, but whisper ultrasonic courtship songs. J Exp Biol 212:4072–4078. https/doi.org/10.1242/jeb.034266 Nakano R, Takanashi T, Skals N, Surlykke A, Ishikawa Y (2010) To females of a noctuid moth, male courtship songs are nothing more than bat echolocation calls. Biol Lett 6:582–584. https://doi.org/10.1098/rsbl.2010.0058 Nakano R, Takanashi T, Surlykke A, Skals N, Ishikawa Y (2013) Evolution of deceptive and true courtship songs in moths. Sci. Rep. 3:2003. https://doi.org/10.1038/srep02003 Nakano R, Takanashi T, Surlykke A (2015) Moth hearing and sound communication. J Comp Physio A 201:111–121. https://doi.org/10.1007/s00359-014-0945-8 Pollack GS (2016) Hearing for Defense. In: Pollack GS (eds) Insect Hearing, Springer Handbook of Auditory Research, pp 81–98. https://doi.org/10.1007/978-3-319-28890-1_4 Römer H (2021) Neurophysiology goes wild: from exploring sensory coding in sound proof rooms to natural environments. J Comp Physiol A 207:303–319. https://doi.org/10.1007/s00359-021-01482-6 Römer H, Holderied M (2020) Decision making in the face of a deadly predator: high-amplitude behavioural thresholds can be adaptive for rainforest crickets under high background noise levels. Philos Trans R Soc B 375:20190471. https://doi.org/10.1098/rstb.2019.0471 Sanderford MV (1992) Acoustic courtship communication of the Polka-dot wasp moth, Syntomeida epilais Walker (Lepidoptera, Arctiidae, Ctenuchinae). Dissertation, Wake Forest University Sanderford MV (2009) Acoustic Courtship in the Arctiidae. In: Conner WE (ed) Tiger Moths and Woolly Bears: Behavior, Ecology, and Evolution of the Arctiidae, Oxford University Press, pp 193–206 Sanderford MV, Conner WE (1990) Courtship sounds of the polka-dot wasp moth Syntomeida epilais . Naturwissenschaften 77:345–347. https://doi.org/10.1007/BF01138395 Sanderford MV, Conner WE (1995) Acoustic courtship communication in Syntomeida epilais Wlk. (Lepidoptera: Arctiidae, Ctenuchinae). J Insect Behav 8:19–31. https://doi.org/10.1007/BF01990967 Simmons RB, Conner WE (1996) Ultrasonic signals in the defense and courtship of Euchaetes egle Drury and E. bolteri Stretch (Lepidoptera: Arctiidae). J Insect Behav 9:909–919. https://doi.org/10.1007/BF02208978 Ter Hofstede HM, Ratcliffe JM (2016) Evolutionary escalation: the bat-moth arms race. J Exp Biol 219:1589–1602. https://doi.org/10.1242/jeb.086686 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 27 Mar, 2025 Read the published version in Journal of Comparative Physiology A → Version 1 posted Editorial decision: Revision requested 27 Oct, 2024 Reviews received at journal 26 Oct, 2024 Reviews received at journal 20 Oct, 2024 Reviews received at journal 16 Oct, 2024 Reviews received at journal 14 Oct, 2024 Reviewers agreed at journal 26 Sep, 2024 Reviewers agreed at journal 26 Sep, 2024 Reviewers agreed at journal 25 Sep, 2024 Reviewers agreed at journal 25 Sep, 2024 Reviewers invited by journal 24 Sep, 2024 Editor assigned by journal 23 Sep, 2024 Submission checks completed at journal 23 Sep, 2024 First submitted to journal 22 Sep, 2024 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5133806","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":370887987,"identity":"d5af86cc-9e4e-416f-b412-756521dabdf1","order_by":0,"name":"Frank Coro","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAmElEQVRIiWNgGAWjYBAC9gYQWcHACKIliNLCcwBEniFZC2MbSVrYDx/78HHeNtn+BuaDt3mI0sKTljxz5rbbxjMOsCVbE6XFXoLHmJl32+3EDQw8ZtLE2QLWMgekhf8bKVoawLawEakF6BfGGceAfjnMZmw5hygt7IcPM3youS3b39788MYbYrQgADNpykfBKBgFo2AU4AMAffEsIsiRkCoAAAAASUVORK5CYII=","orcid":"","institution":"Miami Dade College","correspondingAuthor":true,"prefix":"","firstName":"Frank","middleName":"","lastName":"Coro","suffix":""}],"badges":[],"createdAt":"2024-09-22 19:44:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5133806/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5133806/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00359-025-01739-4","type":"published","date":"2025-03-27T15:57:43+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":71268484,"identity":"b5b64781-9e4a-4245-b9b4-aa892b8812df","added_by":"auto","created_at":"2024-12-12 18:23:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":221528,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDevices\u003c/strong\u003e used for ultrasonic stimulation of and recording from \u003cem\u003eS. epilais\u003c/em\u003e females at the back terrace of my home open to the backyard (outdoor conditions). Females were placed inside a foam cup surrounded by an acoustically transparent cloth to which they perch. The temperature, humidity, and level of illumination under which the acoustic stimulation and recordings were done are those typical of Miami Dade County (Southeastern Florida) from April to November. All the results presented were obtained between 02÷30 and 06÷30. A photo of a female \u003cem\u003eS. epilais \u003c/em\u003eis shown\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5133806/v1/dde171c67f9eed112025d7a9.png"},{"id":71268049,"identity":"9e79b611-fb91-4636-97a6-aae040c2e269","added_by":"auto","created_at":"2024-12-12 18:15:21","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":155072,"visible":true,"origin":"","legend":"\u003cp\u003eOscillograms and spectrograms of the \u003cstrong\u003eacoustic stimuli\u003c/strong\u003e used in this study, all of which are playbacks of recordings obtained under natural conditions. The \u003cem\u003eS. epilais \u003c/em\u003emale emission series lasts 1.2 s and has 12 MCs. The \u003cem\u003eT. brasiliensis \u003c/em\u003eattack sequence, recorded while this bat was freely flying in Florida, lasts 1.1 s and has 40 pulses of decreasing duration and at increasing repetition rate. The asterisk shows the buzz that starts at 1,005 ms after the beginning of this attack series. The \u003cem\u003eS. epilais \u003c/em\u003efemale emission series lasts 0.6 s and has 10 MCs. The spectrograms of these stimulation series show that their frequency range is between 20- and 45- kHz. The horizontal bar below each oscillogram represents 200 ms\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5133806/v1/42aaa4a48301d36c9782efd6.jpeg"},{"id":71268487,"identity":"737ec03b-429b-4843-8715-886bb00a4d30","added_by":"auto","created_at":"2024-12-12 18:23:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":26808,"visible":true,"origin":"","legend":"\u003cp\u003eResults from stimulating 55 perched, virgin \u003cem\u003eS. epilais \u003c/em\u003efemales three or more nights PE with the playback of \u003cstrong\u003ethree different acoustic series\u003c/strong\u003e present in their natural environment during their courtship hours. \u003cstrong\u003ea \u003c/strong\u003eComparison of the percentage of each stimulation series with female response. Letters show statistically significant differences among the results (Friedman test Fr = 95.9, p\u0026lt;10\u003csup\u003e-4\u003c/sup\u003e; Dunn’s post-test p\u0026lt;10\u003csup\u003e-3\u003c/sup\u003e) (values as mean + SD).\u0026nbsp; \u003cstrong\u003eb \u003c/strong\u003eComparison of the female threshold for each series. Letters show statistically significant differences among the results (Kruskal Wallis test KW=53, p\u0026lt;10\u003csup\u003e-4\u003c/sup\u003e; Dunn’s post-test p\u0026lt;0.01) (values as median + 75 percentile). \u003cstrong\u003ec, d \u003c/strong\u003eComparison of the responses to signal amplitudes between 58- and 94-dB SPL in 3-dB steps. All data shown are the mean ± SEM and the tests used for comparison were Mann-Whitney, Wilcoxon, or Paired \u003cem\u003et\u003c/em\u003e test, p\u0026lt;10\u003csup\u003e-3\u003c/sup\u003e\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5133806/v1/2768b7fcd9539f6e0375077a.png"},{"id":71268492,"identity":"60be308a-e70e-426e-bf3b-48e489ba7e68","added_by":"auto","created_at":"2024-12-12 18:23:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":25077,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of the \u003cstrong\u003efirst three nights post-eclosion\u003c/strong\u003e on the responses of ten virgin, perched females to the male, bat attack, and female series. \u003cstrong\u003ea \u003c/strong\u003ePercentage of series\u003cstrong\u003e \u003c/strong\u003ethat had female response at each of the tested nights PE (values as mean + SD). \u003cstrong\u003eb \u003c/strong\u003eThreshold in dB SPL of the female response to each of the series applied (values as median + 75 percentile). Letters show the results of Friedman test or Kruskal-Wallis test followed by Dunn’s test applied to the data from the first three nights PE. For more details see main text\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5133806/v1/abfe11687f96342c63d63ff0.png"},{"id":71268045,"identity":"4d3c2d97-a870-4af1-bf4b-f736f9398b60","added_by":"auto","created_at":"2024-12-12 18:15:20","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":14716,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of \u003cstrong\u003emating\u003c/strong\u003e on the responses of the same nine perched females to the male, bat attack, and female series while virgin and after mating. \u003cstrong\u003ea \u003c/strong\u003ePercentages of series with female response under these two physiological conditions (values as mean + SD). Post-mated females practically stop responding to their male conspecific signals. There is no statistical difference in the responses to the bat attack sequence (Mann-Whitney test, U = 31.5, p =0.45) nor to the female series (unpaired \u003cem\u003et \u003c/em\u003etest, \u003cem\u003et\u003c/em\u003e =1.4, p = 0.18) while virgin or post-mated. \u003cstrong\u003eb \u003c/strong\u003eFemale threshold to each of the three acoustic series applied when virgin and after mating (values as median + 75 percentile). Asterisks show statistically significant increase in the threshold to the bat series (Wilcoxon test W=45, p=0.004) and to the female series (Mann-Whitney test U=0.0, p=10\u003csup\u003e-3\u003c/sup\u003e) when post-mated\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5133806/v1/a0a439c15657815f37451e49.png"},{"id":71268050,"identity":"eb1aac9b-e841-45c1-af0e-488c4022f04b","added_by":"auto","created_at":"2024-12-12 18:15:21","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":23635,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of being \u003cstrong\u003eperched or held by the wings\u003c/strong\u003e above the thorax on the acoustic responses of the same seventeen virgin females, three or more nights PE. \u003cstrong\u003ea \u003c/strong\u003ePercentage of series with female response under these two conditions (values as mean + SD). They respond significantly more to the male series while perched than when held by the wings (Wilcoxon test W=153, p\u0026lt;10\u003csup\u003e-4\u003c/sup\u003e). When held by the wings, they respond significantly more to the bat attack sequence (Mann-Whitney test U=76, p=0.02), while there are no statistical differences in the responses to the female series (unpaired t test t = 0.9, p = 0.38). \u003cstrong\u003eb \u003c/strong\u003eFemale threshold when perched or held by the wings (values as median + 75 percentile). While held by the wings, the threshold to the bat series increases significantly (paired t test t=14.9, p\u0026lt;10\u003csup\u003e-4\u003c/sup\u003e) as also increases the threshold to the female series (Mann-Whitney test U=7.0 p\u0026lt;10\u003csup\u003e-4\u003c/sup\u003e). Asterisks show statistically significant differences in the variables analyzed in each of these two conditions\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-5133806/v1/1513b9352bee64d85504bc73.png"},{"id":71268044,"identity":"8679f0a0-96a5-4c3e-9ca6-992832925f17","added_by":"auto","created_at":"2024-12-12 18:15:20","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":41470,"visible":true,"origin":"","legend":"\u003cp\u003eIncrease in the \u003cstrong\u003elatency to the bat attack sequence\u003c/strong\u003e in the same 26 females when they are responding to conspecific male signals as compared to when they are not responding to the male series. \u003cstrong\u003ea\u003c/strong\u003e Responses from the same female specimen to the bat attack series presented at 94 dB SPL while perched (upper recording) and when held by the wings (lower recording). The arrows show the first female MC in each recording: in the upper oscillogram it appears during the search phase, while in the lower one it falls at the buzz phase of the bat attack series. The horizontal bar represents 200 ms. \u003cstrong\u003eb \u003c/strong\u003eComparison of the latency to the bat series (as the median value + 75 percentile) in the same seventeen females stimulated while perched (n = 41) and when held by the wings (n = 40). When held by the wings, the latency is statistically significantly higher (Mann-Whitney test U = 352, p\u0026lt;10\u003csup\u003e-4\u003c/sup\u003e). \u003cstrong\u003ec\u003c/strong\u003e Same as in \u003cstrong\u003eb\u003c/strong\u003e, for the same perched nine females that were stimulated when virgin (n = 23) and after mating (n = 20). The post-mated females show a significantly higher latency to the bat series (Mann-Whitney test U = 46.5,\u003c/p\u003e\n\u003cp\u003ep\u0026lt;10\u003csup\u003e-4\u003c/sup\u003e). Asterisks indicate a statistically significant difference\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-5133806/v1/ca365066f24b35da954039b6.png"},{"id":79604882,"identity":"52e42b14-51fe-49ab-aea8-4031548dd996","added_by":"auto","created_at":"2025-03-31 16:08:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1513755,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5133806/v1/05260eb8-9edf-423c-abdd-977914ee7727.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Is it a bat or a male? A female moth (Syntomeida epilais, Lepidoptera: Erebidae: Arctiinae) adapts its acoustic signals for defense or courtship","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMoth hearing and sound production in species with acoustic emission organs, have evolved under the evolutionary pressure of insectivorous echolocating bats (Conner \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Conner and Corcoran \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Nakano et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; ter Hofstede and Ratcliffe \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Barber et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Most of the results published on moth acoustic emission organs have dealt with their function in bat-moth interactions. Blest et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1963\u003c/span\u003e) described the metathoracic tymbal organs in Erebidae, Arctiinae. Most of these moth species emit what is called a modulation cycle (MC). Each MC is composed of the active modulation half-cycle (MHC), the result of the inbuckling of the striated band on the surface of the tymbal organ, and the passive MHC due to the outbuckling of the tymbal organ (Blest et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1963\u003c/span\u003e; Fullard and Fenton \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1977\u003c/span\u003e; Conner \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). The first demonstration of Arctiinae moths using their acoustic emissions not only in their interactions with bats was reported by Conner (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1987\u003c/span\u003e). This author recorded acoustic emissions from males of \u003cem\u003eCycnia tenera\u003c/em\u003e (Arctiinae) during the final moments before mating.\u003c/p\u003e \u003cp\u003eSanderford and Conner (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1990\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1995\u003c/span\u003e) reported sexually dimorphic acoustic signals used during courtship behavior in moths while studying the polka-dot wasp moth, \u003cem\u003eSyntomeida epilas\u003c/em\u003e (Arctiinae, Euchromiina). In this species, males emit their MCs at a slower repetition rate (around 8.5 MC. s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) than females (\u003cem\u003eca.\u003c/em\u003e 15 MC.s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Females respond to their male signals in an antiphonal mode, and the female\u0026rsquo;s acoustic reply is essential for successful mating. Using artificially produced MCs presented at slow and high repetition rates, simulating male and female emissions, respectively, Sanderford (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1992\u003c/span\u003e, p. 54) reported that some \u003cem\u003eS. epilais\u003c/em\u003e females were better than others at discriminating between male and female emissions. Sanderford (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1992\u003c/span\u003e, p. 36) also observed that \u003cem\u003eS. epilais\u003c/em\u003e mating behavior is not seen during the first night post-eclosion (PE), very little courtship occurs during the second night, and normal mating behavior is usually fully developed by the third night PE. For mating, \u003cem\u003eS. epilais\u003c/em\u003e females perch on the underside of the leaves of their most common host plant in Florida, \u003cem\u003eNerium oleander\u003c/em\u003e (Sanderford \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1992\u003c/span\u003e, p. 46). The acoustic signals emitted by both male and female \u003cem\u003eS. epilais\u003c/em\u003e during courtship have been classified as used for long-distance communication (beyond 5 cm) in comparison to signals emitted by several other moth species (Nakano et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2009\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSeveral experiments stimulating different Arctiinae moth species with bat calls and conspecific male emissions to study their acoustic responses have been carried out in which the moths are held by their wings folded above the thorax to simulate flying, or while tethered (Corcoran et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Nakano et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Dowdy and Conner \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Fernandez et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Barber et al \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Tethered \u003cem\u003eS. epilais\u003c/em\u003e moths have been used to study acoustic aposematism during their interactions with sympatric bats. The defensive use of acoustic signals in \u003cem\u003eS. epilais\u003c/em\u003e is considered to be an example of M\u0026uuml;llerian mimicry (Barber and Conner \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Conner and Corcoran \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCourtship behavior in \u003cem\u003eS. epilais\u003c/em\u003e has been recognized as the most elaborate known acoustic communication system in the Erebidae, Arctiinae (Conner \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Sanderford \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). In most other Noctuoidea and Pyraloidea moth species only males produce acoustic emissions during mating (Conner \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; Simmons and Conner \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Nakano et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2010\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Nakano \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). \u003cb\u003eMy aim\u003c/b\u003e was to study a behavior involving the whole auditory system and the motor component generating these emissions in an intact moth under natural (outdoor) conditions. The questions addressed are: (1) do these females emit acoustic signals during their first two nights PE; (2) is mating affecting their acoustic behavior; and (3) Do their acoustic responses to acoustic stimuli differ depending on whether the females are perched or held by the wings above the thorax.\u003c/p\u003e \u003cp\u003eIn this study I demonstrate that virgin, perched \u003cem\u003eS. epilais\u003c/em\u003e females with three or more nights PE discriminate among conspecific male and female signals, and a sympatric bat attack sequence, responding preferentially to their male emissions. I show that they emit acoustic signals during the first two nights PE responding to bat attack series and not to the male signals. This is the first report of post-mated female moths that stop responding acoustically to conspecific male signals and continue to respond to bat attack calls. For the first time it is shown here that a female moth adapts her acoustic response for defense against bats or for courtship by varying response threshold and latency.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSubjects\u003c/h2\u003e \u003cp\u003e \u003cem\u003eS. epilais\u003c/em\u003e fourth- and fifth- instar larvae were collected in stands of \u003cem\u003eNerium oleander\u003c/em\u003e in Southeastern Florida, Miami Dade County. The larvae were fed with fresh oleander leaves under natural (outdoor) conditions in the terrace of my home (25\u003csup\u003eo\u003c/sup\u003e 7\u0026rsquo; 21.82\u0026rdquo; N \u0026ndash; 80\u003csup\u003eo\u003c/sup\u003e 4\u0026rsquo; 24.80\u0026rdquo; W) open to my backyard. Prior to and after pupation, the insects were continuously exposed to temperatures, humidities, and photoperiods typical of Southeastern Florida between April and November. Once pupation occurred, they were checked daily for moth eclosion, upon which females and males were segregated in separate foam boxes that had screen wire sections allowing the moths to be exposed to the photoperiod of the environment. The moths were fed \u003cem\u003ead libitum\u003c/em\u003e with a watery honey solution in vials with cotton placed on top of the box. All the moths used during this study were later released to the natural environment where they had been collected as larvae. None of these moths were reared from eggs laid by females that mated under these conditions.\u003c/p\u003e \u003cp\u003e64/74 (86%) of the females stimulated with the playback of the three different acoustic series present in their environment during their courtship behavior hours (between 02\u0026thinsp;\u0026divide;\u0026thinsp;30 and 06\u0026thinsp;\u0026divide;\u0026thinsp;30) were virgin and were at least three nights PE. Nine females that were experimentally stimulated and responded acoustically to male acoustic series were placed in boxes with males where they mated. After mating, these moths were also acoustically stimulated and later returned to the sites where they had been collected as larvae.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAcoustic recordings\u003c/h3\u003e\n\u003cp\u003eAll the recordings were done using equipment from Avisoft Bioacoustics (Glienicke Germany): Condenser Microphone CM16/CMPA (10 kHz to 140 kHz\u0026thinsp;\u0026plusmn;\u0026thinsp;3 dB) attached to an UltraSoundGate 116H (frequency response from 20 Hz to 460 kHz) data acquisition device at 250 kHz sampling frequency via a laptop computer running Avisoft Recorder software. During the acoustic stimulation of females, the microphone was placed 5 cm behind a perched moth (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), or one held by the wings above the thorax. Temporal variables from the female emissions in response to the acoustic stimuli were analyzed using BatSound (version 4.14) software (Pettersson Electronics and Acoustics AB, Sweden). All files were saved in .WAV format.\u003c/p\u003e\n\u003ch3\u003eAcoustic stimulation\u003c/h3\u003e\n\u003cp\u003eAll acoustic stimulation series were delivered through an ultrasonic dynamic speaker with a flat (\u0026plusmn;\u0026thinsp;6 dB) frequency response from 5_ 70 kHz (Vifa, Avisoft Bioacoustics, Germany) placed 60 cm from the female (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The speaker received the electric signals from an UltraSoundGate Player 116 (frequency range: 1_125 kHz; Avisoft Bioacoustics, Germany) via a laptop computer running Avisoft Recorder software. A Calibrated 40 kHz (\u0026plusmn;\u0026thinsp;3 kHz) Reference Signal Generator (Avisoft Bioacoustics, Germany) was used. This device generates a 40 kHz sinusoidal signal at an amplitude of 75 dB SPL (\u0026plusmn;\u0026thinsp;3 dB), which was recorded at 250 kHz sampling frequency. This signal was used in all the amplitude calibration procedures in combination with Avisoft-SAS Lab Pro software. This reference signal was recorded at the gain setting of the UltraSoundGate 116H device used in the subsequent measurements of the three- stimulation series applied. Using this procedure ensures that all amplitude measurements done are referenced to this signal and expressed in dB SPL (0 dB\u0026thinsp;=\u0026thinsp;20 \u0026micro;Pa). For the calibration of the three acoustic stimuli applied, the microphone was placed directly in front and 60 cm from the loudspeaker, at the position where the moth perches (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). All the acoustic series applied were digitized at a sampling frequency of 250 kHz (4 \u0026micro;s period) and played back at this same sampling frequency.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAll acoustic series presented to \u003cem\u003eS. epilais\u003c/em\u003e females (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) were playback of recordings made under natural (outdoor) conditions. No synthetic (artificial) acoustic stimuli were used. The male and female emissions were recorded by the author while the specimens were freely flying during their courtship behavior hours under outdoor conditions. The male series lasts 1.2 s and has 12 MCs with a mean (\u0026plusmn;\u0026thinsp;SD) repetition rate of 9.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3 MC. s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, calculated using the inverse of the interval between the MCs. The female emission series lasts 0.6 s and consists of 10 MCs with a mean (\u0026plusmn;\u0026thinsp;SD) repetition rate of 15.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2 MC. s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The repetition rate of each of these \u003cem\u003eS. epilais\u003c/em\u003e male and female emissions falls within the values described by Sanderford and Conner (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1990\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). The other stimulation series applied to \u003cem\u003eS. epilais\u003c/em\u003e females was a playback of an attack sequence from a free-flying Mexican free-tailed bat, \u003cem\u003eTadarida brasiliensis\u003c/em\u003e, recorded in Florida (courtesy of Dr. Aaron Corcoran). This is the most abundant insectivorous bat in Florida that is sympatric with \u003cem\u003eS. epilais\u003c/em\u003e during its courtship behavior hours. This bat attack series lasts 1.1 s and consists of 40 pulses varying in both duration and repetition rate. It consists of the following phases: search, early and late approach, and the buzz starting 1,005 ms from the beginning of the series (shown by the asterisk in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eEach of these stimulation series was analyzed for the frequency components of its MCs or pulses (values as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD). The power spectrum of the click with highest amplitude in each of the 12 MCs in the male series (duration 545\u0026thinsp;\u0026plusmn;\u0026thinsp;78 \u0026micro;s), in each of the 10 MCs in the female series (515\u0026thinsp;\u0026plusmn;\u0026thinsp;50 \u0026micro;s) and in each of the first ten bat pulses (9.5\u0026thinsp;\u0026plusmn;\u0026thinsp;4.5 ms, values between 14.5 _3.2 ms) was measured using Batsound software. The peak frequency of the male clicks was 34\u0026thinsp;\u0026plusmn;\u0026thinsp;1 kHz, that of the female clicks 39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 kHz, and in the bat pulses analyzed 37\u0026thinsp;\u0026plusmn;\u0026thinsp;2 kHz. The 20 dB range below the peak frequency in the male clicks is between 28 _ 40 kHz, in the female clicks between 34 _ 46 kHz, and in the first ten bat pulses it is between 35 _ 41 kHz. The three-stimulation series applied have the same frequency range.\u003c/p\u003e \u003cp\u003eThe three-stimulation series were also measured for the maximal amplitude of each of the male and female clicks, and for each of the 40 pulses in the bat attack sequence. The Avisoft SAS Lab Pro software with the 75 dB SPL calibration signal previously recorded together with the one-dimensional transformation function in RMS (logarithmic) was used for this purpose. As all these series were recorded at 250 kHz sampling frequency (4\u0026micro;s period), the averaging time used for these measurements was 100 \u0026micro;s. The median and interquartile range of the 12 male clicks is 94 dB SPL (range 91.5 _94.5 dB SPL), in the 10 female clicks the amplitude is 95 dB SPL, and in the 40 bat pulses it is 94 dB SPL (range 91.5 _ 97 dB SPL). These maximal amplitude values do not differ statistically (Kruskal-Wallis test KW\u0026thinsp;=\u0026thinsp;3.6, p\u0026thinsp;=\u0026thinsp;0.16). Thus, the maximal amplitude of each of the series applied was considered as 94 dB SPL (these series were presented with 3-dB increments).\u003c/p\u003e \u003cp\u003eThe acoustic responses of \u003cem\u003eS. epilais\u003c/em\u003e female were analyzed using BatSound (version 4.14) software. These three-stimulation series (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) were presented only once at each amplitude applied to each tested female in the following order: male- female- bat- female- male- bat- female- bat- male. There was a 5-s silence interval after each of these individual series. This silence interval is the same as that used by Dowdy and Conner (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) when stimulating several Arctiinae moths with a playback of a sympatric bat attack sequence.\u003c/p\u003e \u003cp\u003eThe main feature of the female responses analyzed was the number of MCs during the applied series. The mean value from the responses to the three series at each amplitude was analyzed. A given stimulation series was considered to have female response if there was one or more female MCs in at least one of the three series applied at each amplitude. The threshold of a specimen for each of the three-stimulation series presented was considered as the lowest amplitude at which the female responded with one or more MCs to at least one of the three series of the same type presented. The threshold values mentioned in the text are expressed as median and its interquartile range. The latency of the response was measured as the interval between the onset of the stimulus and the first female MC in the response. The mean value from at least two latency measurements at a given amplitude was considered for further analysis. The latency values mentioned in the text are the median and its interquartile range.\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eDiscrimination ability.\u003c/b\u003e To test the ability of \u003cem\u003eS. epilais\u003c/em\u003e females to discriminate among the three-stimulation series applied, 55 perched, virgin, three or more nights PE females were stimulated during their courtship hours. Each female was stimulated with the three series only once using a wide range of amplitudes with 3-dB increments. All these recordings were obtained in outdoor conditions (see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These results obtained on 26 different nights, are from nine females in October - November 2018, and from 46 females between April and October 2019. During 17 of these nights, between two and five different females were acoustically stimulated during their courtship period. Between 33 and 42 of each of the three-stimulation series were applied once to each of these females, for a total of 2,115 series presented (overall, 6,345 series analyzed). The starting amplitudes of the stimuli presented varied between 55- and 64-dB SPL, depending on the threshold of the specimen to the male emission series to which most of these females showed the lowest value.\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the duration of the playback of the male emission and the bat attack series are similar: 1.2 s in the male series and 1.1 s in the bat series. These two-stimulation series differ in the number of acoustic stimuli presented: 12 male MCs and 40 bat pulses. To compare the female response to each of these series at each of the amplitudes applied, the number of female MCs per second (MC.s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was analyzed. The male series and the female series have a similar number of MCs (12 in the male series and 10 in the female series) but at a different repetition rate. To compare the female response to each of these two series, the number of female MCs was used expressed as percentage of applied MCs at each of the applied stimulus amplitude.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTime after eclosion.\u003c/b\u003e To study the possible effects of the first three nights PE of the females on their acoustic emissions, ten females from the same batch were examined (previously collected as larvae on the same day, fed until they pupated, and separated individually on the day of their eclosion). Females from each batch were acoustically stimulated only once while perched on the first three consecutive nights PE with the three-acoustic series previously described. The number of acoustic series presented on each night depended on the threshold of the specimens to these stimuli.\u003c/p\u003e \u003cp\u003e \u003cb\u003eVirgin versus post-mated.\u003c/b\u003e To test the possible effects of mating on the acoustic emissions of \u003cem\u003eS. epilais\u003c/em\u003e females, I stimulated another group of the same nine perched females both while virgin and after mating. Each of the acoustic stimuli was presented only once to each female while they were perched during the hours of their normal courtship behavior. The virgin moths were tested 4.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3 (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD) nights PE, and they were stimulated again after they mated 5.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3 nights PE. The post-mated females were stimulated 24_48 hours after mating. The nine virgin females were stimulated with a total 1,071 series (357 series of each type), while a total of 234 series (78 series of each type) were presented to the post-mated moths.\u003c/p\u003e \u003cp\u003e \u003cb\u003eHolding by the wings.\u003c/b\u003e To study the effect of holding the moth by its wings, I acoustically stimulated the same 17 virgin females with three or more nights PE both while perched and when held by their wings folded above the thorax. All acoustic stimuli were presented only once to each female while perched or held by the wings. While perched, these females were presented with a total of 1,980 acoustic series (660 series of each type); when held by the wings, a total of 522 series (174 of each type) were applied.\u003c/p\u003e \u003cp\u003e \u003cb\u003eLatency.\u003c/b\u003e To study the changes in latency to the bat attack series, I selected the same seventeen females stimulated while perched and when held by the wings and the same nine females stimulated while virgin and after mating. When not responding to the male series all these females show a threshold above 80 dB SPL to the bat series. Therefore, the data was pooled for the latency at amplitudes of 88-, 91-, and 94-dB SPL.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistics.\u003c/b\u003e Before applying a statistical test to a set of data, it was tested for normality (Kolmogorov-Smirnov test). When applying a paired test, the effectiveness of the pairing was also tested. Comparison between two or three data sets was analyzed using either parametric or non-parametric tests, according to the characteristics of the data. The statistical significance for all tests was set at \u003cem\u003ep-\u003c/em\u003evalue\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Statistical tests were done using GraphPad InStat 3 and OriginLab 8.6. All graphs were done using OriginLab 8.6.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eSyntomeida epilais\u003c/b\u003e \u003cb\u003efemales discriminate among three acoustic series present in their environment during their courtship behavior hours\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSince both males and females of \u003cem\u003eS. epilais\u003c/em\u003e use acoustic communication during courtship behavior that takes place in the presence of flying insectivorous bats emitting their echolocation calls, discrimination among these acoustic emissions present in their environment becomes critical for successful mating. The comparison of the percentage of the series with female response showed statistically significant differences indicating more male series with female response (at 96%), less female response to bat attack series (at 70%), and the least response to female emission series (around 44%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). In the large female population (55 virgin, perched, and at least three nights PE moths) studied between April and November, the responses also differed considerably regarding their coefficient of variation: only 3.3% for the male series, 35% for bat series, and 62% with female series. These data suggest that under these conditions, the female acoustic response to the male signals has the highest behavioral significance, facilitating the courtship acoustic interactions needed for mating.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSome females better than others discriminated the male signals from female signals and bat attack sequences. For example, one of the 55 females did not respond to any of the 36 female series nor to any of the 36 bat attack sequences presented, while it responded to 92% of the 36 male series applied. Four females did not respond to female series at any of the amplitudes presented, while responding between 15% and 47% of bat attack series, and between 92% and 100% of the male series.\u003c/p\u003e \u003cp\u003eThe comparison of the female threshold to each of the acoustic series also showed statistically significant differences with the lowest value (58 dB SPL, interquartile range 55_61 dB SPL) for the male series, an intermediate value for the bat attack series (61 dB SPL, range 58_64 dB SPL), and the highest threshold for the female series (70 dB SPL, range 64_76 dB SPL) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eThe comparison of the female responses to the male series and the bat attack sequence at each of the stimulus amplitude applied (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec) showed that in the whole amplitude range analyzed (from 58 to 94 dB SPL) the females responded with significantly more MC.s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to the male series than to the bat attack series during courtship. The comparison of the female responses at each of the stimulus amplitude analyzed showed that in the whole amplitude range they responded significantly more to the male series than to the female series (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003eWhen comparing the responses in the whole amplitude range applied in each of the 55 stimulated females, in 46 of them (84%) there are statistically significant more MC.s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to the male series than to the bat attack sequence (N\u0026thinsp;=\u0026thinsp;11_13, Wilcoxon or paired t test, p\u0026thinsp;\u0026lt;\u0026thinsp;0.03). The other nine females did not show a statistically significant difference (same paired tests, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) in their acoustic response to these same two stimuli. The comparison of the responses in each of these 55 females to the male and female signals showed that in all of them there are statistically significant more responses to the male signals than to the female emissions (same paired tests, p\u0026thinsp;\u0026lt;\u0026thinsp;10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e). Even five of these females (9%) did not respond to any of the female signals in the whole amplitude range applied while responding to all the male signals. These results suggest that these perched, virgin, three\u0026thinsp;+\u0026thinsp;nights PE females are better discriminating between the male and female signals than between their conspecific male emission and the bat attack sequence.\u003c/p\u003e \u003cp\u003eThe abovementioned nine females were tested at eight different nights during which one or more females (for a total of 16) were also stimulated. All the other 16 tested females discriminated between their conspecific male signals and the bat attack sequence. Even one of them did not respond to any of the bat attack signals in the whole amplitude range applied. These results strongly suggest that the ability to discriminate among these acoustic signals by this population of \u003cem\u003eS. epilais\u003c/em\u003e females depends on some internal condition that seems to vary in each specimen, as for example under the effects of a juvenile hormone.\u003c/p\u003e\n\u003ch3\u003eEffects of the first three nights post-eclosion on females’ acoustic emissions\u003c/h3\u003e\n\u003cp\u003eTen different virgin, perched females were stimulated during the first, second-, and third-nights PE with the three-stimulating series described in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. During their first- and second-nights PE, these females respond randomly to these acoustic stimuli. This means that the same female may respond to some of these acoustic series on the first night but to none of them during the second night PE. Also, a female may not respond to any of these three series on the first night, but to some of them during the second night PE.\u003c/p\u003e \u003cp\u003eDuring the first two nights PE, the females only responded to the bat attack sequence and the female series, while on the third night PE they also responded to the male series. During the first- and second-nights PE, the ten females were stimulated with a total of 450 series (150 series of each type). They responded to less than 1% of the male series presented, while responding to more than 70% of the bat series and to more than 20% of the female series (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). The ten females studied during their third night PE were stimulated with a total of 1,200 series (400 series of each type). It is during the third night PE that the females start responding to 97% of the applied male series. The female responses to the bat attack series during the first three nights PE did not differ significantly (Friedman test Fr\u0026thinsp;=\u0026thinsp;0.24, p\u0026thinsp;=\u0026thinsp;0.89). However, the responses to the female series were higher during the third night PE than during the first two nights (Kruskal-Wallis test KW\u0026thinsp;=\u0026thinsp;8.6, p\u0026thinsp;=\u0026thinsp;0.01; Dunn\u0026rsquo;s test, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eLikewise, the females\u0026rsquo; threshold to the stimulation series changes strongly during the first three nights PE (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). During the first two nights, only one of the ten females responded to one of the male series applied at the highest amplitude (94 dB SPL). During their third night PE, their threshold to the male series decreased considerably to 55 dB SPL (range 55_58 dB SPL) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). When responding to the bat attack series during the first night the threshold was 88 dB SPL (range 82_91 dB SPL), on the second night it was 91 dB SPL (range 88_94 dB SPL), whereas it decreased significantly to 61 dB SPL (range 55_64 dB SPL) during their third night (Friedman test Fr. =16.6.7, p\u0026thinsp;\u0026lt;\u0026thinsp;10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e; Dunn\u0026rsquo;s test, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The threshold to the female series showed a similar tendency: during the first night PE at 91 dB SPL (range 91_94 dB SPL), second night at 94 dB SPL (range 88_94 dB SPL) and decreased to 67 dB SPL (range 64_76 dB SPL) during the third night (Kruskal-Wallis test KW\u0026thinsp;=\u0026thinsp;17.4, p\u0026thinsp;=\u0026thinsp;0.0004; Dunn\u0026rsquo;s test, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEffects of mating on females\u0026rsquo; acoustic emissions\u003c/h2\u003e \u003cp\u003eAnother factor not studied in moths and possibly affecting the acoustic emissions in \u003cem\u003eS. epilais\u003c/em\u003e females is mating. While virgin and perched, the nine tested females responded to 93% of the male series applied; after mating and perched, they only responded to 2% of these series (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). The comparison of the responses to the bat attack and the female series expressed in percentage of series with female response in their virgin or post-mated condition showed no significant difference between them (Mann-Whitney test and unpaired t test (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eThe male series is almost completely ineffective in evoking a response from post-mated females, whereas the bat attack series and the female series still evoke a response from these moths (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). The post-mated females also show a statistically significant increase of their threshold to these two stimuli. Regarding the bat series it is at 88 dB SPL (range 85_91 dB SPL) in the post-mated females as compared to 61 dB SPL (range 58_64 dB SPL) in the virgin females for an increase of 27 dB. Similarly, a significant increase from 73 dB SPL (range 69_73 dB SPL) to 94 dB SPL (range 91_94 dB SPL) was obtained when comparing the threshold to the female series in the virgin and post-mated females, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePerched or Held by the Wings: does it affect the females’ acoustic responses?\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003eSyntomeida epilais\u003c/em\u003e virgin females respond differently when stimulated under these two conditions. When perched, 17 virgin females responded to 97% of the male series presented. When held by the wings, they responded to only 3% of the male series (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). When perched, \u003cem\u003eS. epilais\u003c/em\u003e females respond to around 60% of the bat series presented, while they statistically significantly increase their respond to around 80% when held by the wings. These 17 females do not show a statistically significant difference in their response to the female series under these two conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eWhen perched, all these 17 females responded to male series with a median threshold value of 56 dB SPL (range 55_61 dB SPL). When held by the wings, only four of them responded, but at much higher amplitudes, between 85- and 94-dB SPL. The threshold to the bat series increases significantly, from 64 dB SPL (range 61_70 dB SPL) while perched, to 85 dB SPL (range 82_91 dB SPL) when held by the wings (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). With the female series, the threshold increase is from 70 dB SPL (range 64_79 dB SPL) while perched to 91 dB SPL (range 85_91 dB SPL) when held by the wings. For both these stimuli, the increase of threshold is 21 dB when the moth is held by the wings.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDo\u003c/b\u003e \u003cb\u003eS. epilais\u003c/b\u003e \u003cb\u003efemales adapt their acoustic signals for defense or courtship?\u003c/b\u003e\u003c/p\u003e \u003cp\u003eResults depicted in Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, strongly suggest that \u003cem\u003eS. epilais\u003c/em\u003e females modulate the emission of their acoustic signals according to their physiological condition (during their first two nights PE \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003evs.\u003c/span\u003e with three or more nights PE) and mating status (virgin or post-mated). While virgin, the females also responded to acoustic stimulations differently depending on being perched or held by the wings. In all these situations, \u003cem\u003eS. epilais\u003c/em\u003e females show changes in their responsiveness: when females do not respond to conspecific male acoustic signals, they have a statistically significant higher threshold to the bat attack sequence (increase of more than 20 dB) than when they also respond to conspecific male signals. When the females do not respond to male emissions, they also show changes in their latency to the bat attack series.\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea shows the increase in the latency to the bat series in the same female specimen when stimulated at the same acoustic amplitude while perched (upper oscillogram) and when held by the wings (lower oscillogram). The response latencies to the bat stimulus of 17 females when perched and held by the wings, respectively, increased significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb) when the females do not respond to the male signals. A similar comparison of nine females in their virgin or post-mated state also shows a significant increase (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec) of their response latencies when females do not respond to their male acoustic signals.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSimilar results to those shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb, c was obtained when comparing the latency to the bat attack sequence applied to the ten females stimulated during the first three nights PE. During the first two nights PE, when they were not responding to the male series (see Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), the latency to the bat series had a statistically significantly longer median value 1,020 ms ( range 970_1,060 ms, n\u0026thinsp;=\u0026thinsp;31) than during the third night \u0026minus;\u0026thinsp;320 ms, (range 220_695 ms, n\u0026thinsp;=\u0026thinsp;27), when they were already responding to the male series (Mann-Whitney test, U\u0026thinsp;=\u0026thinsp;49, p\u0026thinsp;\u0026lt;\u0026thinsp;10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cb\u003eConspecific male signals preferred.\u003c/b\u003e The 55 perched, virgin, \u003cem\u003eS. epilais\u003c/em\u003e females three or more nights PE tested between April and November not only responded preferentially to their male acoustic signals, but also with much less variability than to the other two acoustic series present in their environment during their courtship behavior hours. These females also respond with more MCs and a lower threshold to the male series than to the other two-stimulation series applied. These females are also able to discriminate among these three acoustic series present in their environment during their courtship hours in a wide and ecologically relevant amplitude range, between 58- and 94-dB SPL, responding with more MCs to the male series from the lowest to the highest amplitude applied (see Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, d). With this discrimination ability, \u003cem\u003eS. epilais\u003c/em\u003e females ensure the intraspecific acoustic antiphonal communication within this wide amplitude range essential for successful mating.\u003c/p\u003e \u003cp\u003eThe intensity coding ability of \u003cem\u003eS. epilais\u003c/em\u003e females in a wide ecologically relevant amplitude range, is reported for the acoustic responses in an intact moth for the first time. These results emphasize the difference in discrimination ability among Noctuoidea females. For example, Nakano et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) report that \u003cem\u003eSpodoptera litura\u003c/em\u003e (Noctuidae) females do not distinguish between conspecific male songs and insectivorous bat calls.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDelayed post-eclosion courtship.\u003c/b\u003e Sanderford (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1992\u003c/span\u003e, p. 36) observed that \u003cem\u003eS. epilais\u003c/em\u003e mating behavior is not seen during the first night PE, very little courtship occurs during the second night and normal mating behavior is usually fully developed by the third night PE. The proposed explanation was that moths were not able to produce their acoustic emissions because their tymbal organs were not yet sclerotized and hard enough to generate the ultrasonic signals needed for courtship. However, according to the results shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e the tymbal organs in \u003cem\u003eS. epilais\u003c/em\u003e females are already fully functional during the first- and second-night PE since the females can acoustically respond to the bat attack and female series albeit at stimulus amplitudes above 80 dB SPL.\u003c/p\u003e \u003cp\u003eThese are the first results from stimulating virgin, perched Erebidae (Arctiinae) female moths with different acoustic series present in their environment during their first three nights PE. The fact that \u003cem\u003eS. epilais\u003c/em\u003e females acoustically respond to the bat attack series before they emit their acoustic signals in response to conspecific male emissions, supports Conner\u0026rsquo;s (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1999\u003c/span\u003e) statement that: \u0026ldquo;in both their sensory and motor aspects, the weapons of bat/moth warfare have frequently evolved into components of courtship systems\u0026rdquo;.\u003c/p\u003e \u003cp\u003eSo far, an increase of auditory sensitivity in any female moth occurring between their eclosion and the time at which they are participating in courtship behavior has been an unanswered question. The female responses to the male series on the third night PE showing the threshold at 55 dB SPL, as well as the threshold decrease by more than 20 dB when responding to the bat attack and female series, may be explained by the secretion of a hormone between the second- and third-night PE that increases their auditory sensitivity. This would allow them to acoustically interact with conspecific males during courtship behavior hours starting on the third night PE as previously described by Sanderford and Conner (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1990\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1995\u003c/span\u003e) and confirmed by my personal observations.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe role of juvenile hormone?\u003c/b\u003e Stimulation of the nine post-mated females showed that they practically do not respond to the male series, while still responding to the bat series at higher amplitudes (above 80 dB SPL). As post-mated females fly for laying their fertilized eggs, they will continue interacting acoustically with insectivorous bats, trying to avoid them. These are the first results indicating strong effects of mating on the acoustic behavior of female moths. These results in \u003cem\u003eS. epilais\u003c/em\u003e females could be explained considering that in female crickets it has been shown that mating abolishes or reduces their phonotactic behavior when they are presented with their male acoustic signals (Koudele at al. 1987; Loher et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Lickman et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). Also, in female crickets Choi et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) demonstrated that Juvenile Hormone III makes them more selective for conspecific male calling songs. If a similar effect of JH III occurs in \u003cem\u003eS. epilais\u003c/em\u003e females, this could help explain the variability of their acoustic responses among the specimens of the same population to different acoustic stimuli. An example of this variability was reported by Sanderford (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1992\u003c/span\u003e, p. 54) describing the results from 24 virgin, perched \u003cem\u003eS. epilais\u003c/em\u003e females differently discriminating between male and female synthetic analog emissions. Another case is provided by my own experiments with playbacks of conspecific male and female series presented to 55 virgin, perched females. Results from these same 55 females, also show variability among them with respect to their discrimination ability between their male conspecific signal and the bat attack sequence. All these findings might be explained assuming that \u003cem\u003eS. epilais\u003c/em\u003e females with higher concentration of the juvenile hormone will discriminate better in favor of their conspecific male signal, as reported for the phonotaxis in female crickets.\u003c/p\u003e \u003cp\u003eWith respect to the significant loss of auditory sensitivity to the bat attack sequence and the female series in post-mated \u003cem\u003eS. epilais\u003c/em\u003e females (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb), for crickets Pollack (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) states that \u0026ldquo;the mechanisms linking JH and poor sensitivity to ultrasound are completely unexplored\u0026rdquo;.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDifferences within the Erebidae.\u003c/b\u003e The only previous report that I have found of acoustically stimulating the same female moths when stationary (=\u0026thinsp;perched) and while flying tethered, is by Nakano et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). These authors found that perched \u003cem\u003eManulea\u003c/em\u003e (formerly \u003cem\u003eEilema\u003c/em\u003e) \u003cem\u003ejaponica\u003c/em\u003e females do not emit acoustic signals in response to conspecific male song, while they do produce them in response to constant frequency (CF) and frequency modulated (FM) bat calls. While flying tethered, these females still respond significantly less to conspecific male song than to CF and FM bat calls. These results clearly differ from those described here in \u003cem\u003eS. epilais\u003c/em\u003e virgin females stimulated under these two similar conditions. The result of this comparison increases the diversity of acoustic behaviors for defense and courtship, previously described among Erebidae female moths.\u003c/p\u003e \u003cp\u003eAccording to Sanderford (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) in the majority of the arctiid (=\u0026thinsp;arctiinae) mating systems studied, the sounds emitted during courtship do not appear to differ substantially from those used in bat defense. Recently, Fernandez et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) described that when held by the wings \u003cem\u003eBertholdia trigona\u003c/em\u003e (Erebidae: Arctiinae) females\u0026rsquo; response to bats tends to continue throughout and after playback of the bat emissions, while this behavior was not seen in female responses to male acoustic signals. Also, \u003cem\u003eB. trigona\u003c/em\u003e female responses to conspecific male emissions had a lower number of MCs than to the bat emission. The results obtained in \u003cem\u003eS. epilais\u003c/em\u003e females differ from those reported in this other Arctiinae moth.\u003c/p\u003e \u003cp\u003e \u003cb\u003eImportance of behavioral context.\u003c/b\u003e To my knowledge, the results from \u003cem\u003eS. epilais\u003c/em\u003e females are the first report of changes in the acoustic responses of female moths depending on the behavioral context: courtship or defense. Virgin, perched, and three or more nights PE females respond to more than 90% of the applied conspecific male signals showing a threshold at 58 dB SPL, thus increasing the probability of acoustically interacting with flying males emitting their signals. When perched during their first two nights PE and after mating, as well as when stimulated while virgin and held by the wings, they practically do not respond to conspecific male signals, showing significantly higher threshold and longer latency in their responses to the bat attack signals. Under the latter conditions, their first MC in response to the bat attack sequence preferentially falls during the buzz phase.\u003c/p\u003e \u003cp\u003eThe significant increase of the threshold to the bat attack sequence in \u003cem\u003eS. epilais\u003c/em\u003e females when they are not responding to conspecific male acoustic signals is the first report of such change in moths. A possible explanation of the behavioral advantage of only responding to the bat attack sequences above 80 dB SPL may come from the behavior of some rainforest crickets (Romer and Holderied 2020; Romer 2021). The bat avoidance behavior of these crickets has thresholds around 80 dB SPL and their response is always a short cessation of flight. These authors suggest that this high behavioral threshold rejects the irrelevant background noise, including bat calls below 80 dB SPL from bats that are further away. A similar behavioral significance may explain this change in \u003cem\u003eS. epilais\u003c/em\u003e females when they are held by the wings or while perched and not responding to conspecific male signals.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe results presented in this paper open new research questions in moth bioacoustics. For example, which are the sensory and neural bases of the discrimination among the three different acoustic series presented? What are the possible effects of Juvenile Hormone on the auditory system of moths? Which are the mechanisms involved in changing the acoustic emissions in the same virgin female when stimulated while perched or held by the wings? Also, we can ask a comparative question: are some or all the unique features described here for females of \u003cem\u003eSyntomeida epilais\u003c/em\u003e present in females of other moth species?\u003c/p\u003e "},{"header":"Declarations","content":"\u003ch2\u003eCompeting interests:\u003c/h2\u003e\n\u003cp\u003eThe author declares no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author declares no conflict of interest.\u003c/p\u003e\n\u003ch2\u003eFunding:\u003c/h2\u003e\n\u003cp\u003eMiami Dade College bought all the hardware and software used in this study and funded the author to assist with the preparation of this manuscript.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eThe author (Frank Coro) conceived and designed the study, performed the research, analyzed the data, originated all the figures, and wrote the paper.\u003c/p\u003e\n\u003ch2\u003eAcknowledgments\u003c/h2\u003e\n\u003cp\u003eI dedicate this paper to my late colleague Dr. Natacha Portilla, who in the early 1980\u0026rsquo;s encouraged me to study the acoustic emissions in moths with two-celled ears during their courtship behavior. Friedrich G. Barth, Peter Narins, William E. Conner, all Professor Emeritus, and Professor Manfred K\u0026ouml;ssl provided very helpful comments and suggestions on earlier versions. Through all these years, while the results were obtained, Mark Sanderford shared his knowledge on the courtship behavior of \u003cem\u003eSyntomeida epilais\u003c/em\u003e and commented on earlier versions. I thank Aaron Corcoran for providing the digital recording of the attack sequence of \u003cem\u003eTadarida brasiliensis\u003c/em\u003e. My thanks to Miami Dade College for supplying the equipment and software used in this research.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBarber JR, Conner WE (2007) Acoustic mimicry in a predator prey interaction. 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J Exp Biol 219:1589\u0026ndash;1602. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1242/jeb.086686\u003c/span\u003e\u003cspan address=\"10.1242/jeb.086686\" targettype=\"DOI\" 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":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-comparative-physiology-a","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jcpa","sideBox":"Learn more about [Journal of Comparative Physiology A](http://link.springer.com/journal/359)","snPcode":"359","submissionUrl":"https://submission.nature.com/new-submission/359/3","title":"Journal of Comparative Physiology A","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Acoustic behavior, acoustic playbacks, post-eclosion, post-mating, bat predation","lastPublishedDoi":"10.21203/rs.3.rs-5133806/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5133806/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCourtship behavior in the polka-dot wasp moth \u003cem\u003eSyntomieda epilais\u003c/em\u003e is the most elaborate acoustic communication system known in the Erebidae. Both males and females must emit their acoustic signals for successful mating under natural conditions in the presence of insectivorous echolocating bats. Females of \u003cem\u003eS. epilais\u003c/em\u003e were stimulated during their courtship period (between 02\u0026thinsp;\u0026divide;\u0026thinsp;30 and 06\u0026thinsp;\u0026divide;\u0026thinsp;30) with playback of conspecific male and female signals and of a sympatric bat attack sequence and their acoustic emissions were recorded. On the third night post-eclosion at the initiation of courtship behavior, females discriminate among these types of acoustic series, responding preferentially to conspecific male signals. In contrast, during the first two nights post-eclosion, they respond strongly to the bat attack sequence but not to conspecific male signals. It is demonstrated that post-mated female moths stop responding to conspecific male signals, while continuing to respond to the bat attack series. These and other novel observations suggest that these female moths can modulate their acoustic signals, according to the stimulating conditions for defense against bats or courtship, by varying their response thresholds and latencies.\u003c/p\u003e","manuscriptTitle":"Is it a bat or a male? A female moth (Syntomeida epilais, Lepidoptera: Erebidae: Arctiinae) adapts its acoustic signals for defense or courtship","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-12 18:15:15","doi":"10.21203/rs.3.rs-5133806/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-10-27T15:39:30+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-27T00:20:50+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-20T18:19:37+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-16T20:17:21+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-14T18:37:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"253660596040581847977548579299460403976","date":"2024-09-26T15:15:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"180322898942840231330532395014672433148","date":"2024-09-26T12:36:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"96635714040928916718286411031403216050","date":"2024-09-25T21:39:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"328748967080617167952167521507866107397","date":"2024-09-25T06:02:35+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-09-24T14:11:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-09-23T14:35:04+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-09-23T14:34:23+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Comparative Physiology A","date":"2024-09-22T19:42:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-comparative-physiology-a","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jcpa","sideBox":"Learn more about [Journal of Comparative Physiology A](http://link.springer.com/journal/359)","snPcode":"359","submissionUrl":"https://submission.nature.com/new-submission/359/3","title":"Journal of Comparative Physiology A","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4efb5d48-6ebd-48de-b3ba-249aea8db713","owner":[],"postedDate":"December 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-03-31T16:02:15+00:00","versionOfRecord":{"articleIdentity":"rs-5133806","link":"https://doi.org/10.1007/s00359-025-01739-4","journal":{"identity":"journal-of-comparative-physiology-a","isVorOnly":false,"title":"Journal of Comparative Physiology A"},"publishedOn":"2025-03-27 15:57:43","publishedOnDateReadable":"March 27th, 2025"},"versionCreatedAt":"2024-12-12 18:15:15","video":"","vorDoi":"10.1007/s00359-025-01739-4","vorDoiUrl":"https://doi.org/10.1007/s00359-025-01739-4","workflowStages":[]},"version":"v1","identity":"rs-5133806","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5133806","identity":"rs-5133806","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}