Fournier, J.P. et al. “If a Bird Flies in the Forest, Does an Insect Hear it?”
S1: Supplementary Methods
Flight sound recordings
Flight sounds during foraging were recorded from two bird species. The first was an Eastern
Phoebe (Sayornis phoebe), a small (16-21 g) tyrant flycatcher that is primarily an aerial insectivore.
During the breeding season phoebes hunt for flying or perched insects from dawn until dusk and
remain close (<20 m) to their nest. Audio and video recordings of Phoebes took place near the
buildings and forest surrounding Queen’s University Biological Station (44°34N, 76°19W) near
Chaffey’s Lock, Ontario, Canada, between May and August (2009-2010) (CCAC Permit # AUP B1015). Each morning, recording equipment was set up approximately 30 minutes in advance of placing
the first tethered moth to allow the birds to acclimate to the changes in their surroundings. Noctuidae
moths that had been collected the evening before at local ultra-violet lights were tethered between the
abdomen and thorax with fine cotton thread and suspended from a branch that was in clear view of
the hunting Phoebes and less than 10 m from the nest. Moths were placed between the recording
microphone and the birds’ hunting perch so that the birds would approach the microphone at more or
less the same angle during each attack. Since we did not have a portable field microphone that would
allow us to record across a broad frequency range, two microphones with different frequency
characteristics were used: an Earthworks (QTC40, Milford, NH, USA [4 Hz - 40 kHz ± 1 dB]) and a
custom-manufactured Avisoft (CM16, Berlin, Germany [5 – 200 kHz ± 6 dB]. Microphones were
inconspicuously clamped to the branch of the tree ~ 50 cm from the tethered moth. Sounds were
saved as .wav files to a Fostex-FR2 field recorder (Akishima, Tokyo, Japan) (16-bit, 192 kHz sampling
rate).
Sound files were analyzed using Raven Pro 1.3 software (Cornell Lab of Ornithology, Ithaca,
New York). The average wing-beat frequency (WBF) is defined as the number of wing-beat cycles (i.e.
up and down stroke) per second (Hz). WBF was measured from a 500 ms segment of the oscillogram
prior to the point of capture (in the Phoebe trials; 21 attacks from 8 birds, including means of 2-3
attacks per bird), or 500 ms to the point of landing (in the Chickadee trials; one ‘attack’ for each of 11
birds). Spectral and amplitude analysis was performed on 10 flight cycles prior to each attack trial for a
subset of 5 attacks (one from each of 5 birds). These attacks were selected for analysis based on 2
criteria: (1) the bird approached the microphone in a direct path toward the microphone (assessed
based on video recordings), and (2) that there was no interference by the bird hitting a branch or the
microphone itself, which would sometimes happen due to the leaves and branches surrounding the
equipment. Recordings made with the Earthworks microphone were hi-pass filtered to attenuate
frequencies below 300 Hz (Hingee and Magrath, 2009). The Avisoft microphone is not sensitive to
frequencies below ~ 5 kHz and therefore did not require filtering. Analysis was conducted with Raven
1.3 using a 512 pt dFFT with a Hanning Window; frequency resolution of 188 Hz. Phoebe flight sound
levels were estimated for a given distance by referencing the recorded sounds to a calibrated sound of
the same dominant frequency. To do this, a continuous pure tone centered at the mean dominant
frequency of bird flight (~1 kHz) was generated with a Tabor Electronics 50MS/s Waveform Generator
WW5061 (Tel Hanan, Israel) and broadcast through a generic Woofer. The sound was recorded using
the same equipment and settings as used in the field. The sound volume was adjusted until the output
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Fournier, J.P. et al. “If a Bird Flies in the Forest, Does an Insect Hear it?”
was equal to that of the bird flight recordings at the point of capture (which represented a distance of ~
50 cm from the microphone). The dB·peSPL values at the specified distance were measured with a
Brüel & Kjær (Nærum, Denmark) sound level meter (Type 2239) placed at the same location as the
microphone. Sound levels at higher frequencies were obtained from the spectrogram display of the
recorded sound.
Two conventional video cameras (Sony Steady Shot DCR-TRV19) were positioned within 5 10 meters from the tethered moth, at 90º angles from one another. A Sony ECM-MS907 (100 Hz – 15
kHz) microphone was connected to one video camera to later match video footage with audio
recordings (see below). These recordings were not used to characterize sounds. Using Raven Pro 1.3
software the audio recording from the video was aligned temporally with the audio recording from
recording microphone (Earthworks or Avisoft). This allowed for frame-by-frame analysis of the bird’s
foraging tactics and wing movements. Video footage was edited in iMovie 7.1.4 (Apple Computer, Inc.,
Cupertino, CA, USA).
The Black-capped Chickadee (Poecile atricapillus) is a small (10-14 g), passerine songbird
with an omnivorous diet that includes insects (Smith, 1997). The foraging technique we recorded in
chickadees differed from that of aerial insectivores, and involved flying up to a feeding station.
Although this method did not reflect a bird attacking an insect, it is very similar to the method used by
a Chickadee to glean insects off the forest floor, bark, or foliage (Robinson and Holmes, 1982;
Remsen and Robinson, 1990). Audio and video recordings of chickadees were performed at Mer
Bleue Bog (45°22'N 75°30'W) in Ottawa, Ontario, between January and May (2009-2010) (NCC
Permit # 10006). Chickadee flight sounds were recorded using a hand full of seed to entice them
towards the recording microphones (Earthworks and Avisoft, as noted above) that were positioned 15
cm away and directed toward the incoming bird. Chickadee flight sound levels (dB SPL) were
measured in the field using a Bruel & Kjaer Type 2239 sound level meter (AF weighting - RMS). As
well, sound levels were estimated from .wav files as for the Phoebe. All other details with respect to
equipment and analyses were as described above for the Phoebe.
Moth Neurophysiology
Moths (Trichoplusia ni) were obtained from the Insect Production Unit of the Canadian Forest
Service (Sault Ste. Marie, Ontario, Canada). The auditory nerve (IIIN1b) was exposed using the
standard dorsal dissection technique (e.g. Yack, 1992). Extracellular action potentials were recorded
using a stainless steel hook electrode referenced to a second stainless steel electrode inserted into
the moth’s abdomen, amplified (Grass Instruments P-55 preamplifier (West Warwick, RI, USA)), and
monitored on an oscilloscope and audio monitor. The neural response and sound stimuli (see below)
were recorded as .wav files to a Fostex FR-2 data recorder and later analyzed using Raven Pro 1.3
software. All neurophysiological recordings were performed inside a Faraday cage lined with sound
attenuating foam.
Auditory threshold curves were constructed to determine the overlap between the hearing of
Trichoplusia ni and bird flight sounds. Synthetic acoustic stimuli between 10 – 80 kHz were broadcast
as trapezoidal sound pulses of 30 ms duration (5ms rise/fall, linear ramp). Broadcast sounds were
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Fournier, J.P. et al. “If a Bird Flies in the Forest, Does an Insect Hear it?”
shaped using PC Tucker Davis software (RPvdsEX, v. 5.4; Alachua, FL, USA) and synthesized by a
Tucker Davis Technologies (TDT) digital signal processor (RX6 multifunction processor). Sound
pulses were attenuated using a TDT PA5 programmable attenuator and broadcast from a calibrated
Pioneer Ribbon Tweeter speaker (model ART-54F; Kanagawa, Japan). The speaker was placed 30
cm away from the preparation ipsilateral to the recording electrode. The speaker was calibrated by
broadcasting equal amplitude continual tones to a 0.25’’ Brüel and Kjaer Type 4939 microphone
(Naerum, Denmark) and Brüel and Kjaer Nexus conditioning amplifier, connected to a Tektronix
TDS2002 oscilloscope, and recorded as mV peak-to-peak for conversion to dB SPL (r.m.s. re 20
µPa.). Each frequency was broadcast in random order, at 1-5 kHz intervals. The auditory threshold
was defined as the lowest sound level at which neural spikes could be clearly heard (audiospeaker)
and seen (oscilloscope) in synchrony with the sound stimulus by two independent observers.
Flight sounds recorded from foraging wild phoebes were played to moth neural preparations
to assess the moth’s response to bird flight. Two types of playbacks were conducted. A full approach
was played back to the moth (n=16 moths) to determine how firing patterns are sustained throughout
a full approach sequence. These sounds were recorded from a phoebe capturing a moth in the field
(see previous section on bird flight recordings). To be consistent with the playback stimulus between
preparations, a single recording of a full approach was played back to different moths. In addition,
playbacks of 4 consecutive flight cycles (~200 ms in duration) (3 repetitions each) were used to
measure the mean number of A1 and A2 cell spikes elicited by one flight cycle and the mean A1 cell
inter-spike interval (ms) for both down-stroke and up-stroke. These were performed in a subset of
recordings (n=5 moths) that showed good signal to noise ratio where the auditory cells (A1, A2) and
the non-auditory B cell could be easily distinguished from one another. Statistical analysis was
performed using SPSS software V13.0 (IBM, New York, NY, USA). A Pearson correlation analysis
was used to correlate phoebe playback sound levels with the number of A1 cell spikes, and A1 cell
inter-spike intervals. All playbacks were broadcast using an Avisoft ScanSpeak speaker (1–120 kHz)
(at 30 cm from the moth neural preparation). Avisoft Recorder software, in combination with the
Avisoft USG Player116, was used to loop and control intensity of playbacks of bird flight signals to
moth neural preparations. Sound volume was controlled using a volume dial on USG Player116 and
broadcast at set intervals. Sounds were calibrated using methods described above.
Butterfly Neurophysiology
Audiograms were conducted on Morpho peleides butterflies to confirm their sensitivity to
sound frequencies between 1- 20 kHz (Lane et al. 2008, Lucas et al. 2009). As well, full flight
playbacks were conducted to determine how the compound action potential (CAP) responded to
playbacks of an approaching Phoebe at different sound levels. Butterflies were ordered from London
Pupae Supplies (Oxfordshire, UK: Permit number: P-2011-04393). Procedures for both rearing and
conducting audiograms were performed according to previously described methods (Lane et al. 2008,
Lucas et al. 2009). All recordings were performed on the 2N1cNIII nerve branch. Only full flight
approaches of Phoebes recorded with the Earthworks microphone were played back to the butterfly.
Equipment used for playback and calibration were otherwise the same for the moth described above.
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Fournier, J.P. et al. “If a Bird Flies in the Forest, Does an Insect Hear it?”
Detection Distances
Distances at which moths and butterflies would detect an approaching bird were estimated
using the inverse square law for sound attenuation over distance (Greenfield, 2002). This law states
for every doubling of distance from the sound source, there is a 6 dB reduction in sound level. By
using the sound level (dB SPL) of bird flight measured at a given distance in combination with the
insect’s response threshold (dB SPL), the detection distance was estimated.
Access to Data
Raw data files of representative flight sounds and neural responses are available at http://httpserver.carleton.ca/~jyack/. Due to the large size of original video and audio files, these are available
upon request.
References
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