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 B10-15). 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). Spectraland 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 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 fromthat 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 niand 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 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 consecutiveflight 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 volumewas 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.
Detection Distances
Distancesat 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 Due to the large size of original video and audio files, these are available upon request.
References
Greenfield, M.D. 2002 Signalers and receivers: mechanisms and evolution of arthropod communication. New York, NY: Oxford University Press.
Hingee, M. Magrath, R.D. 2009 Flights of fear: a mechanical wing whistle sounds the alarm in a flocking bird. Proc. R. Soc. B.10, 1098-1110.
Lane, K.A., Lucas, K.M. & Yack, J.E. 2008 Hearing in a diurnal, mute butterfly, Morpho peleides (Papilionoidea, Nymphalidae). J. Comp. Neurol. 508, 677-686.
Lucas, K.M., Windmill, J.F.C., Robert, D. Yack, J.E. 2009 Auditory mechanics and sensitivity in the tropical butterfly Morpho peleides (Papilionoidea, Nymphalidae). J. Exp. Biol. 212, 3533-3541.
Remsen, J.V. Robinson, S.K. 1990 A classification scheme for foraging behaviour of birds in terrestrial habitats. Stud. Avian. Biol. 13, 144-160.
Robinson, S.K. Holmes, R.T. 1982 Foraging behaviour of forest birds: The relationship among search tactics, diet, and habitat Structure. Ecology. 63, 1918-1931.
Smith, W.J.1997 Displays of Sayornis Phoebe (Aves, Tyrannidae). Behaviour. 33, 283-322.
Yack, J.E. 1992 A multiterminal stretch receptor, chordotonal organ, and hair plate at the wing-hinge of Manduca sexta: Unravelling the mystery of the noctuid moth ear B cell. J. Comp. Neurol. 324:500-508.
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