Journal of Comparative Physiology A

, Volume 198, Issue 9, pp 683–693

Convergence of reference frequencies by multiple CF–FM bats (Rhinolophus ferrumequinum nippon) during paired flights evaluated with onboard microphones

Authors

  • Yuto Furusawa
    • Faculty of Life and Medical Sciences, Neurosensing Bionavigation Research CenterDoshisha University
  • Shizuko Hiryu
    • Faculty of Life and Medical Sciences, Neurosensing Bionavigation Research CenterDoshisha University
  • Kohta I. Kobayasi
    • Faculty of Life and Medical Sciences, Neurosensing Bionavigation Research CenterDoshisha University
    • Faculty of Life and Medical Sciences, Neurosensing Bionavigation Research CenterDoshisha University
Original Paper

DOI: 10.1007/s00359-012-0739-9

Cite this article as:
Furusawa, Y., Hiryu, S., Kobayasi, K.I. et al. J Comp Physiol A (2012) 198: 683. doi:10.1007/s00359-012-0739-9

Abstract

The constant frequency component of the second harmonic (CF2) of echolocation sounds in Rhinolophus ferrumequinum nippon were measured using onboard telemetry microphones while the bats exhibited Doppler-shift compensation during flights with conspecifics. (1) The CF2 frequency of pulses emitted by individual bats at rest (Frest) showed a long-term gradual decline by 0.22 kHz on average over a period of 3 months. The mean neighboring Frest (interindividual differences in Frest between neighboring bats when the bats were arranged in ascending order according to Frest) ranged from 0.08 to 0.11 kHz among 18 bats in a laboratory colony. (2) The standard deviation of observed echo CF2 (reference frequency) for bats during paired flights ranged from 50 to 90 Hz, which was not significantly different from that during single flights. This finding suggests that during paired flights, bats exhibit Doppler-shift compensation with the same accuracy as when they fly alone. (3) In 60 % (n = 29) of the cases, the difference in the reference frequency between two bats during paired flights significantly decreased compared to when the bats flew alone. However, only 15 % of the cases (n = 7) showed a significant increase during paired flights. The difference in frequency between two bats did not increase even when the reference frequencies of the individuals were not statistically different during single flights.

Keywords

Jamming avoidanceReference frequencyResting frequencyDoppler-shift compensation

Abbreviations

BF

Best frequency

CF

Constant frequency

CF2

Constant frequency of the second harmonic component

DSC

Doppler-shift compensation

DSCF

Doppler-shifted constant frequency

FM

Frequency modulated

Frest

Resting frequency

IC

Inferior colliculus

IPI

Interpulse interval

JAR

Jamming avoidance response

TF

Terminal frequency

Introduction

Bats exhibit flexibility in echolocation when catching insects and avoiding obstacles in complete darkness (Griffin 1958). In nature, echolocating bats live in roosts with many conspecifics, and bats flying in groups are often observed foraging in field (Surlykke and Moss 2000; Simmons 2005). Jamming sounds from neighboring bats and their own cluttered echoes from surrounding objects may cause acoustic interference. However, bats appear to have evolved adaptations to overcome such interference (i.e., the jamming avoidance response, JAR), allowing them to extract the necessary information from their own returning echoes that are close to or overlap with sounds from other bats in time or/and frequency range.

The echolocation sounds of bats in the families Rhinolophidae, Hipposiderids and mustached bats consist of a long constant frequency (CF) and a short frequency-modulated (FM) portion. The CF frequencies of second harmonic components (CF2) of pulses emitted by bats at rest (resting frequency Frest) differ among bat species [i.e., mustached bats, ∼61 kHz (Suga et al. 1987); Hipposideros terasensis, ∼70 kHz (Hiryu et al. 2005), Rhinolophus ferrumequinum, ∼85 kHz (Tian and Schnitzler 1997)]. Frest is constant within a narrow frequency range in each bat species, but differs slightly among individuals depending on physical constitution, sex, age, geography and morphometrics (Pye 1972; Suga et al. 1987; Jones et al. 1992; Jones and Ransome 1993; Jones 1999; Guillen et al. 2000; Hiryu et al. 2006). These differences may provide cues for bats to recognize their own individual echoes. On the other hand, FM bat species, such as Eptesicus fuscus, emit downward FM sounds with harmonics [i.e., fundamental component sweeping from 60 to 22 kHz when the bat approaches a target (Ghose and Moss 2003)]. The frequency structure of the emitted FM pulse is statistically different among individuals (Masters et al. 1991, 1995; Kazial et al. 2001). The inherent individuality in echolocation sounds may facilitate individual identification during echolocation in the presence of conspecifics for both CF–FM and FM bat species.

In addition to inherent interindividual differences, many researchers are interested in whether bats actively exhibit momentary JAR against conspecific sounds during echolocation, i.e., if they change the characteristics of their emitted signals to minimize interference from conspecific sounds. Some experiments that have characterized the JAR in FM echolocating bats were conducted on bats that were foraging with conspecifics (Miller and Degn 1981; Obrist 1995; Surlykke and Moss 2000; Ulanovsky et al. 2004; Bartonicka et al. 2007; Gillam et al. 2007; Chiu et al. 2008, 2009; Necknig and Zahn 2011). For example, when flying together with conspecifics in the field, Tadarida teniotis has been observed to shift the terminal frequency (TF) of its FM sweep pulse downward or upward to maintain frequency separation between individuals (Ulanovsky et al. 2004). In another study, when playback stimuli consisting of recorded echolocation sounds were presented to the bats (Tadarida brasiliensis) through an ultrasonic speaker while they were foraging on insects in the field, the bats shifted their call frequency away from the playback frequency (Gillam et al. 2007). In the laboratory recordings, when a pair of bats (Eptesicus fuscus) were flying together and capturing tethered targets in a room, differences in call parameters between the two bats increased significantly compared to recordings taken when the bats were flying alone (Chiu et al. 2009). Another study demonstrated that when CF jamming tones whose frequency corresponded to the TF of Eptesicus fuscus were presented to stationary bats for target detection tasks (a two-alternative forced-choice experiment), the animals shifted their TFs away from the frequency of the jamming stimulus (Bates et al. 2007). In all of these studies, FM bat species exhibited JAR, that is, they significantly changed the frequency of their echolocation pulses in the presence of conspecific sounds or sounds that partially overlapped with the frequency range of their own sounds.

CF–FM bats compensate for the Doppler shift of returning echoes by adjusting the CF2 frequency of their emitted pulse, and accordingly maintain constant echo frequencies within a narrow frequency range (reference frequency) to which their auditory system is highly sensitive (Doppler-shift compensation; Schnitzler 1968; Simmons 1974; Gaioni et al. 1990). The Doppler-shifted CF (DSCF) processing area is a physiologically specialized region that is located within the primary auditory cortex of mustached bats; it allows the bats to detect frequency differences as small as 50 Hz in an echo CF2 (Suga 1984; Riquimaroux et al. 1991). However, it remains unknown whether CF–FM bats flying in a group with overlapping or close reference frequency ranges shift their reference frequency ranges to avoid acoustic interference from conspecific sounds. Currently, few studies have investigated the echolocation behaviors of CF–FM bats flying with conspecifics. For instance, Jones et al. (1994) studied CF–FM bats (Hipposideros speoris) that show partial Doppler-shift compensation [the bats compensate for echo CF2 incompletely (Habersetzer et al. 1984). The frequency response characteristics of hearing in H. speoris shows sensitivity near the reference frequency, but is less pronounced than in Rhinolophus bats (Schuller 1980)]. When three H. speoris were flying together in a cage, a trend for small frequency shifts (<1 kHz) was observed, but it was not distinct. In Asellia tridens, a hipposiderid bat that exhibits Doppler-shift compensation as strongly as Rhinolophus bats (Gustafson and Schnitzler 1979), no evidence was found to support the hypothesis that the bats shift the frequency of their emitted pulse during group flight for jamming avoidance (Jones et al. 1993).

In other studies, Hipposideros terasensis (Hiryu et al. 2006) and mustached bats (Suga et al. 1987; Gaioni et al. 1990) continually showed a long-term gradual shift in Frest over several months in a laboratory colony. H. terasensis also showed short-term changes (daily changes) according to the presence of neighboring conspecific bats [i.e., an increase of 1.12 kHz was observed in a new bat within 1 day when it was placed in a colony with members that had higher Frest (Hiryu et al. 2006)]. Because the reference frequency is typically slightly higher than the bat’s Frest [e.g., 50–300 Hz for R. ferrumequinum at an Frest of 81–84 kHz (Schuller et al. 1974); 50–250 for Pteronotus parnellii parnellii at an Frest of ∼61 kHz (Gaioni et al. 1990)], it is likely that the reference frequencies also changed in the short- or long term according to shifts in Frest. Interestingly, a previous study that used Telemike recordings of echoes demonstrated that the reference frequency of H. terasensis during single flights shifted between recording days (Riquimaroux et al. 2002). These findings suggest that the reference frequency in CF–FM bats is not fixed at a particular frequency, but rather is flexible on at least a daily basis. In other words, CF–FM bats can momentarily change their own reference frequency as needed, i.e., for JAR related to neighboring conspecific sounds.

All of these previous studies have generated important results. However, the sound recordings for bats flying in groups in all of these studies were conducted using a stationary microphone located on the ground except for the Telemike study (Riquimaroux et al. 2002). Therefore, Doppler error in the recorded sounds due to the relative flight velocity between the bat and the microphone always occurs and makes it difficult to estimate small frequency changes. In addition, the assignment of recorded sounds to individual bats is difficult for bats flying in groups when stationary microphones are used. The most important issue to address is the echo frequencies that the flying bats listen to, rather than the frequencies that they broadcast. For CF–FM bat species, the reference frequency is important in terms of JAR.

Therefore, in the present study, the echolocation sounds of the bats (Rhinolophus ferrumequinum nippon) flying in pairs were measured using onboard telemetry microphones (Telemike; Riquimaroux and Watanabe 2000) in a flight chamber. The Telemike recordings allowed us to resolve the problems mentioned above, because both the emitted pulses and the returning echoes were recorded individually by each of the microphones that were mounted on the backs of two bats. We hypothesized that each bat would shift its reference frequency to avoid acoustic interference during paired flight when their reference frequency ranges were similar or overlapped. The reference frequency of each bat was investigated by measuring the CF2 frequency of its echoes while exhibiting Doppler-shift compensation.

Materials and methods

Subjects

Japanese horseshoe bats (Rhinolophus ferrumequinum nippon, body mass, 20–30 g) were used in this study. The bats were captured in a natural cave in Hyogo Prefecture, Japan under license and in compliance with current Japanese laws. The animals were housed in a temperature- and humidity-controlled colony room (3 × 4 × 2 m) at Doshisha University in Kyoto, Japan. The bats were allowed to fly freely and were provided access to food (mealworms) and water. The day and night cycle of the room was set to 12-h dark:12-h light.

R. ferrumequinum nippon uses compound echolocation signals, each consisting of a CF component with a second harmonic around 68–70 kHz being strongest, plus an accompanying initial short upward FM sweep (2–8 kHz, ending at 68–70 kHz) and a terminal short downward FM sweep (beginning at 68–70 kHz and extending 8–12 kHz lower) (Hiryu et al. 2008).

Measurements of echolocation behaviors during flight

Eight bats were used in single and paired flights. The flight experiments were conducted in a flight chamber that was 8 (L) × 3 (W) × 2 m (H) under long wavelength lighting with red filters (>650 nm) to avoid any visual effects on the bats. The chamber was constructed of steel plates to minimize interference from external electromagnetic noise and waves used by FM radio stations. In the flight chamber, three plastic chains were suspended vertically from the ceiling of the chamber as obstacles to provide the bats with motivation for echolocation during flight. Each chain was 2-m long with oval links that were 6.5-cm long and 3.5-cm wide, made of plastic rings. The placements of these three chains were changed randomly before every recording. A landing mesh [1 (W) × 1 m (H)] was attached to the frontal wall (referred to as the target wall) at a height of 1.5 m above the floor. Bats were released from one end of the flight chamber by the experimenter and were allowed to fly longitudinally in the flight chamber. After release, the bats continued to make U-turns before landing or flying directly toward and landing on the mesh.

The experimental procedure was as follows. First, each bat of the pair flew alone in the flight chamber (single flight before paired flight; S1). Then, two experimenters released each of two bats simultaneously, and the bats flew together in the chamber for a few minutes (paired flight). After that, each bat flew alone again (single flight after paired flight; S2). The combined experimental sessions were completed within 1 h. When the bats demonstrated fatigue, we discontinued the experiment. We assessed whether the bats shifted their own reference frequency ranges during paired flight by comparing the recordings to those during single flights. The recording and analysis procedures are described below.

Sound recordings by Telemike

Echolocation pulses emitted by bats and returning echoes were recorded by the Telemike mounted on the back of each bat during flight. The recording procedure used for the Telemike was the same as in a previous study (Hiryu et al. 2008). The Telemike consisted of a 1/8-inch omni-directional condenser microphone (Knowles, Model FG-3329, Itasca, IL, USA), a miniature custom-designed FM transmitter unit, a 1.5-V hearing aid battery (Sony, Type SR621SW, Tokyo, Japan) and a transmitting antenna. The Telemike was attached to the back of the bat with a piece of double-sided adhesive tape, with the microphone pointed forward and positioned approximately 1 cm above the noseleaf, at the center of the right and left pinnae of the bat. Because the Telemike weighed 0.6 g (including the battery), it was small and light enough to be carried by the bats. Accordingly, the bats did not exhibit any fatigue during the experiments. The transmitter of the Telemike produced FM radio signals with a carrier frequency between 100 and 105 MHz or 90 and 95 MHz, which was received by an FM radio antenna (RadioShack Corporation, Model15-1859, TX, USA) that was hung from the ceiling in the flight chamber. Then the signals were demodulated using a custom-made FM receiver. The signals from the receiver were band-pass filtered between 20 and 150 kHz (NF Corporation, Model 3625, Yokohama, Japan), digitized by a DAT recorder (SONY, Model SIR-1000W, Tokyo, Japan, 16-bit, 384 kHz), and stored as files on the hard disk of a personal computer. The total frequency response of the Telemike system was flat within ±4 dB, between 20 and 100 kHz.

When paired fight experiments were conducted, each bat was mounted with its own Telemike, each with a different carrier frequency (100–105 and 90–95 MHz), which allowed us to record the emitted pulses and returning echoes for each bat individually.

Sound analysis

The acoustic characteristics of the pulses and turning echoes during flight were analyzed from a spectrogram of Telemike data using custom Matlab routines on a personal computer. Each pulse and echo was extracted from the sound recordings and their second harmonic components were analyzed. The CF2 frequency was determined from the frequency at the peak energy location in the spectrogram of each sound with a frequency resolution of 46 Hz using a fast Fourier transform over 8,192 sample points.

Because each Telemike recorded pulses and echoes of both bats during paired flight, the following procedure was used to analyze individual-specific pulses and echoes. Rhinolophus ferrumequinum nippon exhibit Doppler-shift compensation for returning echoes during stereotyped landing flight (Hiryu et al. 2008). Therefore, the reference frequency of a bat can be measured based on the CF2 frequency in echoes with relation to some target. Hence, we measured echo delays from all of the observed calls based on the spectrograms of the Telemike recordings. As the bats approached the target wall for landing, echo delays from the target wall decreased continually with flight time. By examining the consecutive decreases in the echo delay with respect to flight time, the bat’s own echoes from the target wall were identified. The CF2 frequencies of these selected echoes were measured for individual bats. During single flights, we analyzed an individual’s echoes from the target wall using the above procedure to measure CF2 frequencies before and after paired flights (S1 and S2).

The reference frequency was determined from the mean of the CF2 frequency for 20 personal echoes for each bat. During each paired flight, three landing approaches were selected from one flight session for each bat at intervals of approximately 30–60 s, so that the reference frequencies were obtained three times for each bat per one flight session (see Fig. 4; P1, P2 and P3).

Measurements of daily changes in Frest

CF2 frequencies of echolocation pulses emitted by bats at rest (Frest) were recorded for individual bats in our laboratory colony (18 bats) on each experimental day. Sound recordings were taken for individual bats after they were placed in a cage [25 (L) × 25 (W) × 40 cm (H)] to isolate them from conspecifics. A condenser microphone (Brüel and Kajer, 4939, Naerum, Denmark) was placed approximately 5 cm below the noseleaf of the bat hanging from the ceiling of the cage. Echolocation pulses recorded by the microphone were amplified (Brüel and Kajer, 5935L, Naerum, Denmark), high-pass filtered (20 kHz; NF Corporation, model 3625, Yokohama, Japan) and digitally recorded with a 16-bit conversion at a sampling rate of 384 kHz with a SONY SIR-1000W DAT recorder (Tokyo, Japan). The data were stored as files on the hard disk of a personal computer. In addition to the Frest recordings that were taken on experimental days, Frest for all of the bats were measured weekly to investigate long-term changes in Frest in the colony members.

The Frest of bats were analyzed using spectrogram of recorded sound sequences using custom Matlab routines on a personal computer. Twenty pulses were randomly selected from each recording for all bats, and the CF2 frequency of the pulse was determined with a frequency resolution of 23.4 kHz. The mean of the 20 pulses was calculated for each bat on each day and is referred to as Frest.

For statistical comparisons, either Student’s t test, one-way factorial analysis of variance (ANOVA) or the F-test was used, when appropriate, to test for significant differences in call parameters between data sets.

Results

Long-term changes in Frest for 18 bats

Figure 1 shows intraindividual changes and interindividual differences in Frest for the 18 bats in the colony over 3 months (for 87 days from 1 September to 24 November 2008). The Frest of Rhinolophus ferrumequinum nippon generally ranged from 67.5 to 69 kHz (bat #908 was eliminated due to an unnatural decrease after 60 days). The extent of the change in Frest, which was defined as the difference between the maximum and minimum Frest of an individual bat over the recording period, ranged from 2.01 to 0.12 kHz, and the mean change in Frest was 0.5 kHz (Table 1). Frequency differences in the mean Frest between 1 September and 24 November were significant for all bats (Student’s t test; P < 0.001 for all bats except P < 0.01 in #692, Table 2). Throughout the 3-month recording period, the bats showed long-term gradual decreases in their Frest by 0.22 kHz on average.
https://static-content.springer.com/image/art%3A10.1007%2Fs00359-012-0739-9/MediaObjects/359_2012_739_Fig1_HTML.gif
Fig. 1

Intraindividual changes in Frest for 18 bats in a laboratory colony over a 3-month observation period (from 1 September to 24 November 2008). Recordings were conducted every week. Bat #909 died on 22 October 2008. In addition, #908 showed an unnatural decrease in Frest, which appeared to be due to failing health

Table 1

Intraindividual difference between the maximum and minimum Frest for 18 bats in a laboratory colony during a 3-month recording period from 1 September to 24 November 2008

ID

Maximum Frest (kHz)

Minimum Frest (kHz)

Frequency change (kHz)

675

68.26

67.22

1.04

676

68.59

68.28

0.30

677

68.16

67.66

0.50

682

68.32

67.89

0.43

689

68.56

67.76

0.80

690

68.40

68.06

0.34

692

68.71

68.48

0.23

693

67.70

67.53

0.17

901

67.78

67.53

0.25

902

67.62

67.34

0.28

903

69.31

68.84

0.47

904

67.98

67.50

0.48

905

68.95

68.45

0.50

906

68.88

68.57

0.31

907

68.83

68.53

0.29

908

67.98

65.98

2.01

909

67.72

67.60

0.12

910

68.43

68.01

0.42

Mean ± SD

68.34 ± 0.49

67.85 ± 0.67

0.50 ± 0.44

Table 2

Comparison of Frest between 1 September and 24 November 2008

ID

Frest [kHz] (mean ± SD)

Frequency difference (kHz)

1 September 2008

24 November 2008

675

68.01 ± 0.09

68.24 ± 0.03

0.23***

676

68.55 ± 0.04

68.28 ± 0.05

−0.26***

677

68.06 ± 0.04

67.92 ± 0.10

−0.14***

682

67.89 ± 0.09

68.14 ± 0.03

0.24***

689

68.44 ± 0.02

67.76 ± 0.09

−0.68***

690

68.40 ± 0.03

68.19 ± 0.03

−0.21***

692

68.56 ± 0.04

68.49 ± 0.07

−0.07**

693

67.63 ± 0.04

67.53 ± 0.05

−0.11***

901

67.78 ± 0.04

67.53 ± 0.05

−0.25***

902

67.54 ± 0.05

67.39 ± 0.05

−0.15***

903

69.31 ± 0.05

68.88 ± 0.03

−0.43***

904

67.98 ± 0.03

67.50 ± 0.04

−0.48***

905

68.86 ± 0.05

68.39 ± 0.08

−0.47***

906

68.80 ± 0.03

68.66 ± 0.02

−0.14***

907

68.83 ± 0.03

68.65 ± 0.02

−0.18***

908

67.95 ± 0.05

65.98 ± 0.07

−1.97***

909

67.72 ± 0.02

910

68.43 ± 0.02

68.01 ± 0.05

−0.42***

Mean

68.32 ± 0.04

68.10 ± 0.05

−0.22***

Bat #909 died on 21 October 2008 and bat #908 was eliminated from the average due to an unnatural decrease in Frest. A Student’s t test was used to detect significant changes. ** P < 0.01; *** P < 0.001

Frequency differences in Frest between individuals in the colony (interindividual differences) were investigated on each recording day. For example, Table 3 shows Frest on 1 September 2008 for the 18 bats in ascending order, which were distributed between 67.54 (#902) and 69.31 kHz (#903). The standard deviation (SD) of individual Frest ranged from 0.02 to 0.09 kHz, and the mean of the SD was 0.03 kHz. Interindividual differences in Frest between neighboring bats (neighboring Frest) ranged from 0.00 to 0.44 kHz, with a mean of 0.10 kHz. Except for four pairs of neighboring bats, 97 % of all combinations of the 18 bats in the colony (149/153; number of combinations was 18C2 = 153) showed significant interindividual differences in Frest on a given day.
Table 3

Interindividual differences in Frest between neighboring bats

ID

Frest [kHz] (mean ± SD)

Neighboring Frest (kHz)

902

67.54 ± 0.05

 

693

67.63 ± 0.04

0.09***

909

67.72 ± 0.02

0.08***

901

67.78 ± 0.04

0.06***

682

67.89 ± 0.09

0.12***

908

67.95 ± 0.05

0.06*

904

67.98 ± 0.03

0.02 NS

675

67.98 ± 0.09

0.00 NS

677

68.06 ± 0.04

0.08*

690

68.40 ± 0.03

0.34***

910

68.43 ± 0.03

0.03***

689

68.44 ± 0.02

0.00 NS

676

68.55 ± 0.04

0.11***

692

68.56 ± 0.04

0.01 NS

906

68.80 ± 0.03

0.24***

907

68.83 ± 0.03

0.03***

905

68.86 ± 0.05

0.04*

903

69.31 ± 0.05

0.44***

Mean ± SD

0.03 ± 0.03

0.10 ± 0.12

Data were taken from 1 September 2008. Neighboring Frest represents interindividual differences in Frest between neighboring bats when the bats were arranged in ascending order according to Frest. A Student’s t test was used. NS not significant (probability P > 0.05); * P < 0.05; ** P < 0.01; *** P < 0.001

Figure 2 shows the weekly changes in mean neighboring Frest among the 18 bats over the 3-month recording period (#908 was eliminated from the last 3 weeks of analysis because of an unnatural decrease in Frest). The mean neighboring Frest ranged from 0.08 to 0.11 kHz (0.10 ± 0.07 kHz) and did not change significantly during the 3-month period (ANOVA, F12,190 = 1.80,P = 0.999). Although each bat showed a slight decrease in Frest over the 3-month period, there was no change in the interindividual difference between Frest.
https://static-content.springer.com/image/art%3A10.1007%2Fs00359-012-0739-9/MediaObjects/359_2012_739_Fig2_HTML.gif
Fig. 2

Weekly changes in neighboring Frest among 18 bats (interindividual differences in Frest between two bats with the closest Frest; neighboring bats) over a 3-month recording period. Bat #908 was eliminated from the analyses for the last 3 weeks because of an unnatural decrease in Frest

Doppler-shift compensation during paired flight

Figure 3a shows the spectrograms of the second harmonic components of pulse–echo pairs for two bats that were flying together. During paired flight, each bat maintained a constant echo CF2 frequency by changing the CF2 frequency of its emitted pulses. Accordingly, the spectrograms suggested that the bats may have conducted adequate Doppler-shift compensation for their own echoes, even during paired flight. For further analysis, returning echoes from each bat during paired flights were separated (see “Materials and Methods”). Figure 3b shows an example of changes in CF2 frequencies in pulse–echo pairs for two bats flying together. Data were taken from the last few seconds that were recorded before Bat A landed on the landing mesh. The CF2 frequencies of the echoes from the target wall were kept constant within narrow frequency ranges for each bat (mean ± SD; Bat A: 68.13 ± 0.07 kHz, Bat B: 68.14 ± 0.08 kHz). The mean values were defined as the reference frequencies of the individual bats.
https://static-content.springer.com/image/art%3A10.1007%2Fs00359-012-0739-9/MediaObjects/359_2012_739_Fig3_HTML.gif
Fig. 3

a Spectrograms of the second harmonic components of pulse–echo pairs in each bat during paired flight (top panel Bat A, bottom panel Bat B). The sounds reaching each bat were individually recorded using a Telemike. b Representative changes in the CF2 frequencies of the pulse (solid triangles) and the echo (open circles) from the target wall during paired flight for Bat A (blue) and Bat B (red) before landing (the two bats flew in the same direction toward the target wall). Data were taken from the last few seconds that were recorded before Bat A landed on the landing mesh. c The distributions of standard deviations (SDs) for the reference frequencies (the mean of CF2 frequencies of returning echoes from the target wall that were frequency compensated by the bats) during paired and single flights. Twenty echoes were analyzed for SD from each flight case. For each bat pair, three paired (P1–P3) and two single flight (S1 and S2) cases were conducted, respectively, and a total of 16 combinations among 8 bats were analyzed (n = 96 for paired flights, n = 64 for single flights). The bottom and top boundaries of the box indicate 25 and 75 % of the distribution of the data, respectively. The whiskers indicate the 10th and 90th percentiles. The solid lines within the box represent the mean values (paired flight; 67.0 Hz, single flight 63.0 Hz)

Figure 3c shows the distributions of SD for reference frequencies during single and paired flights. Data were taken from 16 pairs of 8 bats. SDs during paired flights ranged from 50 to 90 Hz (67 ± 17 Hz, n = 96), which was not significantly different from single flights (63 ± 18 Hz, n = 64, P > 0.12). The bats compensated for the Doppler shift in CF2 frequencies in echoes during paired flights with the same accuracy as when they were flying alone.

Changes in echo CF2 frequencies

Figure 4a shows representative changes in reference frequencies for the pairing of bats #913 and #916. The interindividual differences in Frest between these two bats was 0.10 kHz on the experimental day (significant difference, P < 0.001), which corresponded to the mean neighboring Frest in our laboratory colony (0.10 ± 0.07 kHz; Fig. 2). On the other hand, the pairing of #910 and #682 showed the smallest interindividual difference in Frest among all of the bats on that experimental day (0.03 Hz, P < 0.01, Fig. 4b). In the single flights before the paired flights (S1), the difference in reference frequencies between the two bats (∆F) was significant for both pairs (P < 0.001; ∆F = 0.08 kHz between #913 and #916 in Fig. 4a; ∆F = 0.06 kHz between #682 and #910 in Fig. 4b). However, ∆F was not significant during the paired flights (P1–P3 in Fig. 4a, b). In both pairs, neither bat changed its reference frequency range to expand ∆F during paired flight. Rather, ∆F during paired flights decreased compared to during S1. As shown in Fig. 4b, the two bats changed their reference frequencies in an identical direction from P1 to P3, resulting in ∆F remaining small throughout paired flights (∆F = 0.00 kHz for P1 and 0.02 kHz for P2 and P3). When the two bats of each pair flew alone after the paired flights (S2), ∆F between #913 and #916 became significantly different (P < 0.01), but that between #910 and #682 still remained similar (P = 0.11).
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Fig. 4

Two representative changes in reference frequencies for two bats between single and paired flights. a Bat #913 versus #916; these two bats showed an average difference in the Frest (0.10 kHz, P < 0.001). b Bat #910 versus #682; these two bats showed the smallest interindividual difference in Frest (0.03 kHz, P < 0.01) on the experimental day. Single flight sessions were conducted before (S1) and after (S2) the paired flights. Data for paired flights were taken from three approach flights during one recording session (P1–P3). A Student’s t test was applied for the differences in reference frequencies between the two bats (∆Fs); **P < 0.01; ***P < 0.001

Figure 5a shows the relationship between ∆Fs during single flights and those of paired flights for all 16 pairs of 8 bats. An enlarged view of the plots with ∆Fs < 0.3 kHz is shown in Fig. 5b. Three plots (S1 vs. P1, S1 vs. P2, S1 vs. P3) were created for each pair (all plots for each pair have the same color; n = 48). We found that in 60 % (n = 29) of the cases, ∆Fs during paired flights were smaller than during single flights. However, only 15 % of the cases (n = 7) showed a significant increase in ∆Fs during paired flights (P < 0.05). This suggests that when two bats were flying together, they did not increase their ∆Fs, but rather tended to shift their own reference frequencies to become much closer.
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Fig. 5

Relationship between ∆Fs of paired bats between single and paired flights. a All recorded data. Three data points (S1 vs. P1, S1 vs. P2, S1 vs. P3) were plotted for each pair of bats. A total of 16 pairs (n = 48) were analyzed. b Enlarged view including the pairs that showed less than 0.3 kHz in ∆F (13 pairs, n = 39). A dashed line indicates no change in ∆F between single and paired flights

Discussion

Intraindividual changes in Frest

Based on the long-term recordings of Frest, the mean intraindividual change between the beginning and the end of the 3-month recording period was −0.22 kHz, indicating that all of the colony members showed a gradual decrease in Frest (Table 2). This finding is consistent with previous studies that demonstrated flexibility of Frest over long term for H. terasensis (Hiryu et al. 2006) and mustached bats (Suga et al. 1987; Gaioni et al. 1990).

The frequency representation in the DSCF area in the auditory cortex of the mustached bat differs among individuals depending on their Frest (Suga et al. 1987). On the other hand, Frest changes instantaneously with an increase in body temperature (Huffman and Henson 1993a) or due to flight activities (Henson et al. 1990). In addition, such short-term changes in Frest are accompanied by frequency shifts in cochlear resonance as well as tonotopic shifts in the frequency response of the auditory neurons (Huffman and Henson 1993b). We confirmed that the reference frequency of each individual changed over a short interval between single and paired flight sessions (i.e., the interval between S1 and P1 was <10 min; three landing approaches during the paired flight were selected every 30–60 s). Furthermore, the distributions of SD for reference frequencies were not significantly different between single and paired flights, indicating that during paired flight, bats exhibited Doppler-shift compensation with the same accuracy as during single flight. We suggest that CF–FM bats have the flexibility to momentarily change their reference frequency. The auditory system is likely capable of quickly adapting to short-term intraindividual changes in reference frequencies.

Paradoxical shifts in reference frequency ranges during paired flight

JAR has been well studied in electric fish, such as Eigenmannia, which exhibit electrolocation by producing electric organ discharges to sense electric field interference (Bullock et al. 1972a, b). Watanabe and Takeda (1963) first demonstrated that these fish shift their own discharge frequency in a direction to increase the difference in frequency between themselves and the jamming stimulus. Shifts in the signal frequencies emitted by electric fish are easy-to-understand behavioral adaptations by animals to avoid interference from jamming source, such as conspecifics, which has been frequently referred to in bat research in the context of JAR.

In the present study, bats did not shift their own reference frequency ranges to increase the difference in frequency between themselves and paired bats even when the reference frequencies of the individuals were not statistically different during single flights. This result appears to be inconsistent with previous JAR studies that have suggested that bats change the characteristics of their emitted signals to minimize interference from conspecific sounds (Habersetzer 1981; Miller and Degn 1981; Obrist 1995; Surlykke and Moss 2000; Ibáńez et al. 2004; Ulanovsky et al. 2004; Bartonicka et al. 2007; Gillam et al. 2007; Chiu et al. 2008, 2009; Necknig and Zahn 2011); the same has been found in electric fish (Watanabe and Takeda 1963; Bullock et al. 1972a, b). One of the reasons for this inconsistency is that these previous bat studies investigated the frequencies of emitted pulses rather than the echo frequencies that the bats actually listen to during flight. Furthermore, because recorded sounds included the Doppler shift caused by the bat’s own flight, small frequency shift (<1 kHz) were difficult to detect with adequate accuracy. Thus, it appears to be too early to conclude that the bats during flight with conspecifics shift their own signal frequency away from that of conspecifics, similar to the JAR of electric fish.

On the other hand, a behavioral detection experiment revealed that the mustached bats can detect differences as small as 50 Hz in the frequencies of echo CF2 components, which corresponds to 0.08 % of Frest in this bat species (Riquimaroux et al. 1991, 1992). Ostwald (1984) demonstrated that there is overrepresentation of the CF2 frequency in the physiologically specialized area located in the primary auditory cortex of horseshoe bats. Although there are no behavioral data for Rhinolophus bats, if we assume that R. ferrumequinum nippon has at least 0.1 % of the Frest for frequency discrimination (∼70 Hz), the mean of neighboring Frest for colony members (0.1 kHz, see Fig. 2) may be large enough for the bats to discriminate between each other.

In the present study, the ∆Fs of bats during paired flights ranged widely from 0.0 to 1.2 kHz (Fig. 5). A total of 63 % of all paired flights (30/48) had ∆Fs that were smaller than the above assumption of frequency discrimination of ∼70 Hz, which suggests that the frequency differences might be large enough for bats to discriminate between neighboring bat calls. For CF–FM bat species that have highly sensitive auditory systems around the reference frequency for Doppler-shift compensation, it is not difficult to understand that the inherent interindividual variations in reference frequency may be large enough for the bats to discriminate between each other; this may have resulted in the absence of a significant increase in ∆Fs during paired flight in this study. However, it is puzzling that 11 of 16 bat pairs showed decrease in ∆Fs compared to when they were flying alone. This observation is the opposite to what has been shown in other animals known to exhibit JAR, including other bat species and electric fish.

When multiple bats are flying in the same area, variation in pulse design (i.e., CF2 frequencies in CF–FM bats) among conspecifics may facilitate the recognition of a bat’s own echoes from those of conspecifics (Jones et al. 1992). Suga et al. (1987) examined the variation in Frest among a large number (116) of mustached bats whose Frest ranged from 59.69 to 63.34 kHz, a spread of only 3.64 kHz with an average individual SD of 0.091 kHz. The authors suggested that the individual frequencies of the bats overlap with those of conspecifics. However, it remains unclear whether bats negotiate their Frest to avoid overlap in their frequency range while they are living together in a colony (e.g., the 116 bats used in that study were not living together in a colony).

In the laboratory colony of R. ferrumequinum nippon used in the present study, the Frest of the 18 bats ranged from 67.54 to 69.31 kHz, a spread of 1.77 kHz with an average individual SD of 0.03 kHz. The mean of the interindividual difference between neighboring Frest was 0.1 kHz, and 97 % of all combinations of the 18 bats in the colony showed significant interindividual differences in Frest on a given day excluding four pairs of neighboring bats (Table 3). These findings suggest that the bats living together in a colony may maintain minimum interindividual differences between neighboring bats, forcing the Frest into a narrow frequency band. This “scooting over” allows the CF–FM bats to allocate Frest for as many bats as possible in the narrow frequency band for which the species is highly sensitive.

In our previous study, Hipposideros terasensis changed their Frest by several kHz according to the presence of neighboring conspecific bats; i.e., they showed a long-term shift in the same direction among the colony members and converged their Frest with that of other bats in a short period of time (Hiryu et al. 2006). We suggest that rather than R. ferrumequinum nippon increasing their ∆Fs when they fly in groups, they may shift their reference frequencies in an identical direction, resulting in the differences among frequencies remaining low (see Fig. 5b). Social interactions may significantly affect Frest. Some previous studies have reported that bats can modify the frequency characteristics of their calls through their auditory experiences (vocal learning; Jones and Ransome 1993; Esser 1994; Boughman 1998; Knörnschild et al. 2010). Further investigation is required regarding the proximity of the reference frequency in the context of both echolocation and social interactions.

Acknowledgments

These experiments complied with the Principles of Animal Care, publication no. 86-23, revised in 1985, of the National Institutes of Health, and with current Japanese laws. All experiments were approved by the Animal Experiment Committee of Doshisha University. This work was partly supported by a Grant-in-Aid for Scientific Research on Innovative Areas (Grant No. 20200055), a Grant-in-Aid for Young Scientists (B) (Grant No. 21760318) of JSPS and an ONR grant (Grant No. 00014-07-1-0858).

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