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RESEARCH ARTICLE
Amphibious hearing in a diving bird, the great cormorant
(Phalacrocorax carbo sinensis)
Ole Næsbye Larsen*, Magnus Wahlberg and Jakob Christensen-Dalsgaard
ABSTRACT
Diving birds can spend several minutes underwater during pursuit-dive
foraging. To find and capture prey, such as fish and squid, they probably
need several senses in addition to vision. Cormorants, very efficient
predators of fish, have unexpectedly low visual acuity underwater. So,
underwater hearing may be an important sense, as for other diving
animals. We measured auditory thresholds and eardrum vibrations in
air and underwater of the great cormorant (Phalacrocorax carbo
sinensis). Wild-caught cormorant fledglings were anaesthetized, and
their auditory brainstem response (ABR) and eardrum vibrations to
clicks and tone bursts were measured, first inan anechoic box in air and
then in a large water-filled tank, with their head and ears submerged
10 cm below thesurface. Both the ABR waveshape and latency, as well
as the ABR threshold, measured in units of sound pressure, were
similar in air and water. The best average sound pressure sensitivity
was found at 1 kHz, both in air (53 dB re. 20 µPa) and underwater
(58 dB re. 20 µPa). When thresholds were compared in units of
intensity, however, the sensitivity underwater was higher than in air.
Eardrum vibration amplitude in bothmedia reflected the ABR threshold
curves. These results suggest that cormorants have in-air hearing
abilities comparable to those of similar-sized diving birds, and that
their underwater hearing sensitivity is at least as good as their aerial
sensitivity. This, together with the morphology of the outer ear
(collapsible meatus) and middle ear (thickened eardrum), suggests
that cormorants may have anatomical and physiological adaptations
for amphibious hearing.
KEY WORDS: Auditory brainstem response, Audiogram,
Auditory adaptation, Auditory threshold curves, Bioacoustics,
Underwater hearing
INTRODUCTION
Amphibious living requires that the senses function well both in air
and underwater. The physical properties of air and water, however,
are radically different. Underwater sound environments differ from
terrestrial ones in terms of the speed and absorption of sound (e.g.
Larsen and Wahlberg, 2017), and underwater light environments
differ greatly from terrestrial ones regarding the spectral
composition of light, luminance and turbidity (Tremblay et al.,
2014). About 150 species of amphibious birds of seven orders are
so-called pursuit-dive foragers, i.e. they forage by diving from a
swimming position on the water surface to capture prey underwater
at depths ranging from a few meters to much more than 100 m
(White et al., 2007; Tremblay et al., 2014). At the same time, they
must breed and nest on land, fly between nesting and foraging sites,
and perform a range of other activities in air.
This amphibious lifestyle of diving birds requires adaptations
of their visual system to function optimally in both terrestrial and
aquatic environments. Terrestrial birds have excellent vision (Martin
and Osorio, 2008). Most day-active birds of prey, for instance,
primarily rely on vision to find their prey, whereas night-time
hunters, such as owls, have acute hearing and vision abilities adapted
for finding and catching prey in the dark (Payne, 1971; Kettler et al.,
2016; Beatini et al., 2018). When entering the water, however, the
eyes of terrestrially adapted birds lose the refractive power of their
cornea (Katzir and Howland, 2003). This results in the picture on
their retina becoming blurred and their visual field reduced, unless
compensated for by anatomical adaptations such as thickened and
flattened low-powered corneas, which serve to reduce the effects of
loss of corneal refraction underwater (Martin and Brooke, 1991;
Martin, 1999; Nelson, 2006). In addition, diving birds often have
unusually well-developed intraocular muscles, which may help
underwater accommodation by regulating the size of the pupil and
altering the shape of the lens (Nelson, 2006; White et al., 2007).
Much less is known about possible adaptations of the auditory
system of diving birds to the amphibious lifestyle. It is unknown
whether they use their sense of hearing underwater at all; for
instance, to avoid sound-emitting predators, to track down sound-
emitting prey, to orient relative to ambient underwater sound sources,
or perhaps even for underwater sound communication as suggested
in a recent study (Thiebault et al., 2019). In-air auditory threshold
curves of 8 species of diving birds have been determined using
auditory brainstem responses (ABRs) (Crowell et al., 2015) but the
underwater hearing ability of diving marine birds has so far been
studied in only two species: the long-tailed duck (Clangula
hyemalis; Therrien, 2014) performing only shallow dives and the
great cormorant (Phalacrocorax carbo; Johansen et al., 2016;
Hansen et al., 2017). The last two studies established that one diving
cormorant specimen could detect underwater sounds, but the derived
data were not sufficient to establish more objective psychophysical
hearing thresholds that could be compared between the two media.
This was probably due in large part to the methodology usedin these
studies, which required an enormous training effort and therefore
time, and thus provided only limited datafrom one to two individuals
of each species. However, these studies are very important as they
demonstrated that underwater sounds are perceived and can be used
behaviourally by the cormorant.
One way to obtain more data on diving birds’hearing abilities is
to use physiological techniques such as auditory evoked potentials
(AEPs). Such data collection has its own challenges, demanding the
experimental bird to be anaesthetized not only during testing in air
but also underwater, while breathing is maintained. Also, it is not
always trivial to compare physiological and psychophysical
Received 24 October 2019; Accepted 10 February 2020
Sound and Behaviour Group, Department of Biology, University of Southern
Denmark, Campusvej 55, DK-5230 Odense M, Denmark.
*Author for correspondence (onl@biology.sdu.dk)
O.N.L., 0000-0002-8325-0982; M.W., 0000-0002-8239-5485; J.C.-D., 0000-
0002-6075-3819
1
© 2020. Published by The Company of Biologists Ltd
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Journal of Experimental Biology (2020) 223, jeb217265. doi:10.1242/jeb.217265
Journal of Experimental Biology
thresholds for birds (Brittan-Powell et al., 2002, 2005).
Physiological techniques allow for several frequencies being
tested carefully during a limited amount of time both in air and
underwater, and therefore also a larger number of individual birds to
be tested than when using psychophysical techniques (Beatini et al.,
2018; Mooney et al., 2019a,b). However, the lower thresholds
obtained by psychophysical techniques mean that these remain the
‘gold-standard’for audiogram determination.
As a supplement to physiological data, anatomical investigations
of the middle and outer ears of diving birds may reveal adaptations to
underwater hearing. Aquatic mammals, turtles and frogs that are
secondarily adapted to hear in the aquatic environment have special
anatomical adaptations to cope with the high acoustic impedance of
water as compared with air (Møhl, 1967; Christensen-Dalsgaard and
Elepfandt, 1995; Christensen-Dalsgaard et al., 2012). Most notably,
the presence of air cavities, such as air-filled middle ear cavities,
has been shown to increase sensitivity of these ears (Christensen-
Dalsgaard and Elepfandt, 1995; Christensen-Dalsgaard et al., 2012),
and a recent modelling approach has shown that stiff eardrums, such
as the tympanic discs in aquatic frogs and turtles, can increase the
hearing efficiency of the underwater ear (Vedurmudi et al., 2018).
If marine birds can hear well underwater, it is likely that similar
anatomical features can be found in their outer and middle ears that
will be different from those found in terrestrially adapted bird ears.
Other anatomical adaptations, probably for protecting the eardrums
underwater by passive closure of the auditory meatus, have been
described in auks (Kartaschew and Iljitschow, 1964), and it is likely
that there will be adaptationsto protect the middle ear from the large
hydrostatic pressures incurred while diving. However, because of
the scarce hearing data available on these species, it is currently
unclear whether such protective adaptations should be considered as
being auditory or hydrostatic adaptations, or both.
Here, we report on the hearing abilities of the great cormorant in
air and underwater using physiological methods. Great cormorants
have been characterized as foot-propelled visually guided pursuit-
dive foragers (Ashmole, 1971; Tremblay et al., 2014). They are
globally distributed and have very flexible foraging habits, hunting
wherever fish are available in coastal waters and freshwater lakes
(Grémillet et al., 1998). Great cormorants hunt solitarily in waters
where turbidity is high and light levels are low, hunting even at night
and into the dark Arctic winter (Grémillet et al., 2006; Tremblay
et al., 2014). They may also hunt socially when turbidity is very
high, driving fish prey towards the surface (Van Eerden and
Voslamber, 1995). Most of their dives last for about 30 s and reach
depths of 6–10 m with 35 m as the extreme (Grémillet et al., 2006).
They have highly pliable eye lenses and powerful intraocular
muscles that may accommodate the loss of corneal refractive power
underwater (Glasser and Howland, 1996). Their diving activity
(frequency, duration and depth) is modulated by ambient light
levels such that in Greenland waters, the deepest and longest
duration dives are observed at midday, whereas duration and depth
are lower at dawn and lowest during the night (White et al., 2008).
Such a circadian entrainment is expected if their primary sense for
hunting is vision and if potential prey performs vertical light-
dependent migration. It is therefore highly surprising that great
cormorants’visual acuity underwater is poor at the light levels that
they are known to encounter during natural dives (White et al.,
2007). Their visual acuity is comparable to unaided human vision
underwater, which means that diving cormorants can detect
individual prey only at distances of less than 1 m. This is
surprising as they are known to be highly efficient hunters and are
supposed to be visually guided.
This seeming contradiction could possibly be explained by the
use of tactile cues (Voslamber et al., 1995) as in harbour seals,
which use their vibrissae for sensing water currents (Dehnhardt
et al., 2001). Close-quarter prey detection could then be combined
with special foraging techniques, e.g. brief short-distance pursuit
and/or rapid neck extension to capture prey at very short range
(White et al., 2008). However, no vibrissae-like organ has been
described in cormorants or other diving birds. Another possibility,
not considered in the literature, is that great cormorants (and other
diving birds) underwater may use supplementary information
obtained through their sense of hearing.
To get an impression of the cormorants’underwater hearing
ability compared with that in air, we measured AEPs, assumed to
represent ABRs, to determine hearing thresholds using identical
criteria, with the same specimens measured first in air and then
underwater. In this way, it was possible to directly compare their
hearing sensitivity in the two media, and judge to what extent
cormorants have special adaptations for underwater hearing. It is
unclear, however, how such specializations would be manifested in
their outer and middle ear, and therefore we made some additional
anatomical investigations to determine possible adaptations to the
aquatic environment in these structures, as the anatomy of
the cormorant ear has been described only in general terms (Saiff,
1978). Our observations were supplemented with recordings of
eardrum vibrations in air and underwater to compare with
observations in amphibious vertebrates (Christensen-Dalsgaard
et al., 2012).
MATERIALS AND METHODS
In mid-to-late June in each of the years 2012, 2013, 2014 and 2015,
five great cormorant (Phalacrocorax carbo sinensis Staunton 1796)
fledglings were collected from treetop nests in the cormorant
breeding colonies at Nørresø, (55°08′40″N; 10°22′25″E) and
Brændegård Sø (55°07′52″N; 10°23′03″E), Funen, Denmark. The
fledglings were subsequently transferred to a 6×3×2 m
3
outdoor
aviary at the Department of Biology, University of Southern
Denmark, where they were provided with water and fish (capelin
and sprat) ad libitum for the 4–16 days that they were kept until
experiments were carried out. The fledglings were judged to be
4–6 weeks old and very close to leaving the nest when captured,
based partly on frequent and regular observations of the colony
(Jacob Sterup, personal information) and partly on their mass at the
time of the experiment (see Table 1; Dunning, 2007). Collection of
Table 1. Overview of the cormorant fledglings used for auditory
brainstem response (ABR) recordings in 2013–2015
Cormorant
ID
Mass
(kg) Year
No. of
frequencies in air
No. of frequencies
underwater
2 2.2 2013 6 N/A
3 2.3 2013 4 N/A
4 2.1 2013 5 5
A 1.9 2014 6 N/A
B 1.8 2014 4 4
C 1.6 2014 6 5
D 2.3 2014 6 5
E 2.1 2014 6 N/A
F 1.8 2015 6 7
G 1.8 2015 8 3
H 2.0 2015 7 8
I 1.7 2015 5 9
Mean±s.d. 2.0±0.2 12 individuals 8 individuals
N/A indicates that no underwater ABR signals were recorded (either due to
unknown technical problems or because the bird died during recording).
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RESEARCH ARTICLE Journal of Experimental Biology (2020) 223, jeb217265. doi:10.1242/jeb.217265
Journal of Experimental Biology
birds and experiments were carried out with permission from the
Danish Nature Agency (J.nr. SNS-342-00056 and nst-342-00122 to
O.N.L. and M.W.) and the Danish Animal Experimentation
Inspectorate (J.nr. 2012-15-2934-00067 to O.N.L.) for using up to
five cormorant fledglings per year. The birds collected in 2012 were
used for pilot ABR measurements, for recording of eardrum
vibrations in air and underwater, and for anatomical examinations,
whereas the birds collected in 2013–2015 were used for the ABR
experiments reported here. As a supplement, we dissected the ear
region of the head of two adult cormorants: a 3 year old female
cormorant that had been kept in captivity at the University of
Southern Denmark’s Marine Research Centre and had died from
natural causes in 2017, and a wild cormorant found dead there in
2018 and frozen within a few hours of death.
Preparation for ABR experiments
For recording, each experimental bird was anaesthetized by
intrapectoral injections of a Ketamine–Rompun mixture. The initial
dose was 20 mg kg
−1
ketamine hydrochloride and 5 mg kg
−1
xylazine hydrochloride; supplementary doses of 10 mg kg
−1
ketamine hydrochloride and 2.5 mg kg
−1
xylazine hydrochloride
were administered as needed to keep the birds anaesthetized for
3–5 h. We had to keep the dose this low, as higher doses such as
40 mg kg
−1
ketamine hydrochloride plus 20 mg kg
−1
xylazine
hydrochloride normally used to anaesthetize small birds like
starlings (e.g. Klump and Larsen, 1992) and budgerigars (e.g.
Larsen et al., 2006) immediately overdosed the cormorants. At the
end of experiments, each bird was overdosed with injection of
pentobarbital i.p. beforewaking up, as required by the Danish Animal
Experimentation Inspectorate.
To maintain core temperature, the anaesthetized bird was placed
on a heating blanket (Heat Therapy pump model TP702, Gaymar
Instruments Inc., Orchard Park, NY, USA) in a customized metal
frame holder shaped such that the bird gently rested its head, kept in
place with masking tape, in an upright, close to normal resting
position. On each side of this metal frame holder, another metal rod
shaped like an inverted Vwas welded such that the whole metal
frame holder could be attached to an overhead holder like a gondola
under a balloon. This arrangement was also used in the underwater
setup (see Fig. 1). Then, three subdermal needle electrodes (16 mm,
27-gauge needles, isolated to the tip, 170 cm lead, Tucker-Davis
Technologies, Alachua, FL, USA) were gently pushed through the
skin parallel to the surface. Two differential electrodes were placed
right behind the left ear meatus (the active electrode) and on the
vertex of the head (the inverting electrode), respectively, with
reference to the third ground electrode placed in the nape of the
neck. Note that the placement of the active and inverting electrode
here was opposite to that of Brittan-Powell et al. (2002, 2005),
Crowell et al. (2015) and Beatini et al. (2018). Therefore, the ABR
signals recorded here are inverted relative to the ones in those
studies.
ABR recordings in air
For ABR recordings in air, the metal frame holder with the bird in
sitting position on the heating blanket was transferred to a custom-
built sound-attenuated and anechoic booth (inner dimensions
135×105×95 cm
3
; for specifications, see Jensen and Klokker,
2006). Here, the metal frame was placed on the floor such that the
bird’s head was located right below a ½ inch microphone (type
40AF, G.R.A.S. Sound and Vibration, Holte, Denmark) directed
towards the speaker and calibrated with a sound level calibrator
(type 4230, Brüel & Kjær Sound & Vibration Measurement a/s,
Nærum, Denmark). The microphone output used for system
calibration was amplified and high-pass filtered (0 dB
amplification; high-pass cut-off 20 Hz; power module, type
12AA, G.R.A.S. Sound and Vibration) and recorded on a laptop
computer. Sound stimuli (click sounds or single-frequency tone
bursts of 25 ms duration, including 2 ms long ramp-up and ramp-
down segments at the start and end to avoid spectral smearing) were
delivered at a repetition rate of 25 Hz by a battery powered
loudspeaker (Creative D100, Creative Technology, Crawley, UK)
located 90 deg to the left of the body–beak axis and 70 cm away
from the left ear of the experimental bird.
Sound stimulation, recording and data analysis were performed
using custom-made software (QuickABR) running on a digital
signal processor (type TDT RM2, Tucker-Davis Technologies) and
a PC as in previous studies from this laboratory (Christensen-
Dalsgaard et al., 2011, 2012). AEPs, which are assumed to reflect
the ABR, were passed from the subdermal needle electrodes via
cables shielded with grounded aluminium foil through a low-
impedance headstage and a preamplifier (type RA4LI and RA4PA,
Tucker-Davis Technologies), where they were amplified (74 dB),
digitized (sampling rate 25 kHz, 16 bits), acquired by the RM2
signal processor, and stored on a PC.
ABR recordings underwater
For subsequent ABR recordings underwater, the still deeply
anaesthetized bird was first tracheostomized in the neck about
2 cm posterior to the corner of the mouth and an 8 mm diameter
flexible plastic tube was inserted about 2 cm into the proximal part
of the severed trachea. The trachea was tightly attached to the plastic
tube and sealed with superglue; a piece of string was attached to the
end of the 14 cm long plastic tube. By this arrangement, the bird
breathed freely as before the operation. In addition, the bird’s body
was wrapped in bubble wrap to help maintain body temperature and
the ABR electrodes were reattached. The anaesthetized bird was
again placed in the customized metal frame holder, which was now
attached like a gondola to a photo tripod hanging upside-down from
the ceiling (Fig. 1) above the centre of a 100×90×60 cm
3
water tank
Water tank
Tracheostomy tube
ABR electrode Hydrophone
Electrode cable Speaker
Fig. 1. Setup for recording auditory brainstem responses (ABRs)
underwater. The cormorant’s body rested on a metal frame attached to an
upside-down photo tripod that could be lowered to place the bird’s head
underwater. The cormorant was lowered until the head was submerged to a
depth where the ears were located 10 cm below the water surface and next to
the hydrophone. The tracheostomy tube was 1–2 cm above the water surface.
For further explanation, see Materials and Methods.
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RESEARCH ARTICLE Journal of Experimental Biology (2020) 223, jeb217265. doi:10.1242/jeb.217265
Journal of Experimental Biology
used in a previous study when also the tank’s sound field was
investigated (for further information on the acoustics of the tank,
see Christensen-Dalsgaard et al., 2012). The photo tripod could be
turned and fixed such that the bird’s body axis on the gondola frame
holder was oriented about 40 deg relative to vertical with the bird’s
head facing downwards (see Fig. 1). Finally, the bird in the gondola
frame holder was lowered by means of the photo tripod, until the
ears of the experimental bird were positioned 10–11 cm below the
water surface, while the end of the tracheostomy tube was kept
above the water surface. In this position, the bird kept breathing
normally. We chose to place only the bird’s head below the water
surface, as placing the whole body underwater lowered the body
temperature unacceptably fast and released air bubbles, which raised
the ambient noise level and interfered with the ABR measurements.
ABR signals were recorded using the same stimuli and recording
setup as for in-air recordings.
For sound stimulation, we used an underwater speaker (type
UW30, Lubell Labs Inc., Columbus, OH, USA) hanging on the
inner wall of the water tank about 45 cm from the bird’s left ear
(Fig. 1). It was powered by a power amplifier (type DD-8, Xelex
AB, Stockholm, Sweden) that received signal input from the same
digital signal processor and laptop used for stimulation in air. Sound
pressure levels next to the bird’s head were recorded with a small
hydrophone (type TC4013, Reson, Slangerup, Denmark), amplified
by a hydrophone amplifier (type A1105, Etec ApS, Frederiksværk,
Denmark), and calibrated with a pistonphone calibrator (type 4223,
Brüel & Kjær).
Experimental procedure for ABR recordings
Initially, both in air and underwater, intense click stimuli (half-cycle
4 kHz pulse with a relatively flat spectrum to above 4 kHz)
presented at 25 Hz were used to produce clear ABRs that defined the
duration of the post-stimulus response window (usually from about
3 ms to about 8 ms after calculated sound stimulus arrival at the left
ear), within which to judge the threshold. Latencies from click-
stimulus arrival at the exposed ear to maximum top of wave II in
the ABR response (Fig. 2) were determined by correcting for
transmission time in the two media (0.7 m/344 m s
−1
=2.0 ms in air
and 0.45 m/1500 m s
−1
=0.3 ms underwater). To compare click
response latencies underwater and in air, 26 dB was added to in-air
dB values to transform the dB scale from a reference pressure of
20 µPa to 1 µPa (e.g. Larsen and Wahlberg, 2017). Click amplitudes
were stated in peak equivalent dB (Fig. 2); this is the root mean
square (RMS) dB value of a sinusoid with the same peak amplitude
as the click.
Subsequently, 400 single-frequency tone bursts were delivered at
a repetition rate of 25 Hz for each of the frequencies 250, 500, 1000,
2000, 2500, 3000, 4000 and 6000 Hz, and the responses were
averaged for each frequency. The phase was changed 180 deg for
each burst to reduce microphonics. For each frequency, the initial
sound pressure was set to produce clear ABR responses and then the
sound pressure was reduced in a series of 5 dB steps. Like Brittan-
Powell et al. (2010) and Beatini et al. (2018), we used the visual
detection method, defining the ABR threshold as the sound pressure
2.5 dB below the lowest stimulus level at which a response could
be visually detected on the trace between 4 and 8 ms (see also
Brittan-Powell et al., 2002, 2005). Thresholds were determined
independently by two of the authors (J.C.-D. and O.N.L.) as well
as by T. Bojesen Lauridsen, an MSc student who had experience in
judging ABR data from frog recordings.
Thresholds determined by this method critically depend on the
recorded noise level both between air and water and across
experimental animals. The noise level of the ABR recordings was
estimated by dividing all recordings at each stimulus level into two
batches by assigning every even numbered recording into batch 1
and every odd numbered recording into batch 2. Subsequently, each
of the two batches was averaged. The difference between the two
batch averages was taken as an estimate of the ABR noise, whereas
the average of the two batches was an estimate of signal+noise.
Measured in this way, the median ABR noise (RMS) was 0.19 µV in
air and 0.23 µV in water (i.e. 1.7 dB higher in water than in air).
Across birds, the ABR noise varied between 0.83 and 1.59 µV, or up
to 5.6 dB. We therefore concluded that the noise levels in air
and water were sufficiently similar to make threshold comparisons
meaningful.
Statistics
To test whether the relationship between peak amplitude and latency
differed in air and underwater, we conducted a random coefficient
mixed model (RCMM) with peak amplitude, medium (air and
water) and the interaction between them. The model had a common
intercept, whereas the common slope for peak amplitude was
omitted to enable model convergence. Individual was treated as
subject. We used a Box–Cox transformation of latency (λ=−2)
(Sokal and Rohlf, 1981) to enable residuals not to deviate from
assumptions regarding normality and homoscedasticity.
Sound level (dB re. 20 µPa)
02468101214 0246810121416
66
76
86
96
106
116
97
102
107
112
122
117
127
Sound level (dB re. 1 µPa)
B
A
–0.5
0.0
0.5
1.0
1.5
–1.0
0.0
1.0
2.0
3.0
Time (ms)
Fig. 2. ABR to clicks. (A) Typical in-air ABR to a click
stimulus (individual F). With the active electrode at the
exposed ear, three deflections, marked I–III, were always
observed at the highest stimulus levels. The interpolated heat
diagram below the click response showsthe change in delay
and peak amplitude for successive 5 dB reductions of
stimulus sound level. The peak indicated by II in the response
is clearly detected over a 30 dB range, whereas the trough
indicated by I and the smaller peak indicated by III are
detectable only over a range of up to about 20 dB. Colour
coding −0.5 to +1.5 corresponds to amplitude peak–peak in
µV. (B) Underwater ABRs from individual F. Colour coding
−1.0 to +3.0 corresponds to amplitude peak–peak in µV.
4
RESEARCH ARTICLE Journal of Experimental Biology (2020) 223, jeb217265. doi:10.1242/jeb.217265
Journal of Experimental Biology
To test whether the threshold curves in air differed from those
underwater, we first related the in-air pressure–dB thresholds to the
same reference value of 1 µPa as in water byadding 26 dB to each of
the in-air data points. As thresholds may also be expressed in
intensity units, we related the in-air to the underwater intensity
thresholds by recalculating in-air and underwater thresholds to dB
re. 1 pW m
−2
(see Larsen and Wahlberg, 2017, for details). Note
that the intensity is the far-field intensity calculated from the
pressure measurements. As thresholds for individuals were
measured at multiple frequencies in both air and water, we used a
repeated measures ANOVAwith individual as the repeated measure.
Frequency and medium (air and water) were used as fixed effect
discrete parameters in the model. The repeated measures analysis
applied a first-order autoregressive covariance matrix. We tested the
model first for differences between underwater and in-air pressure
thresholds, and second for differences between underwater and in-
air intensity thresholds. The residuals for both models abided with
assumptions regarding normality and homoscedasticity. All
analyses were made in SAS v. 9.4 (SAS Institute, Cary, NC,
USA) using ‘Proc mixed’.
Outer and middle ear anatomy
Upon termination of ABR experiments, the overdosed birds, and
later the two deceased adult cormorants, were decapitated and the
heads kept frozen at −80°C for later examination. Thawed heads
were examined under a stereo microscope (Leica M60 with camera
IC80 HD, Leica Microsystems GmbH, Wetzlar, Germany). The ear
region was located, and dissection proceeded by removing feathers,
skin and bone to reveal the overall anatomy of the ear region
including eardrum and columella, to be photographed and measured
using callipers (Mitutoyo, Japan) with a vernier scale.
Laser Doppler vibrometer recordings of eardrum vibrations
in air and underwater
We investigated eardrum vibrations of three cormorants both in air
and underwater. For the experiments in air, we placed the deeply
anaesthetized birds in an anechoic room after surgically removing
feathers, skin and muscle tissue posterior to the meatus to be able to
direct a laser beam to the centre of the eardrum. We recorded
eardrum vibrations with a laser Doppler vibrometer (OFV-505
sensor and OFV-5000 vibrometer, Polytec, Waldbronn, Germany)
with the laser head placed approximately 1 m from the bird’s ear.
The bird was stimulated by sound from 12 loudspeakers (JBL 1G,
Lansing, James B., Los Angeles, CA, USA) placed at 30 deg
intervals on a circle 1 m from the bird’s ear. The sound stimulus was
a 100 ms frequency sweep from 200 to 8000 Hz repeated 10 times,
and the loudspeakers were calibrated and centred acoustically by a
½ inch pressure microphone (type 4192, Brüel & Kjær) placed over
the head of the animal. Stimulation, calibration and data acquisition
were controlled by Tucker-Davis System 2 hardware (Tucker-Davis
Technologies) and customized software (‘DragonQuest’;
Christensen-Dalsgaard and Manley, 2005). Sound at the ear
opening was measured by a probe microphone (type 4082, Brüel
& Kjær), allowing calculation of the transfer function between
sound stimulation and eardrum vibration velocity.
In water, we used the same water tank as for the underwater ABR
measurements, and the same method as reported earlier for
underwater measurements in turtles (Christensen-Dalsgaard et al.,
2012). An important modification, however, was that we measured
eardrum vibrations in the severed heads of three freshly killed and
decapitated birds, to have a cleaner sound field in the tank than the
presence of the intact bird would allow, by avoiding, for instance,
re-radiated sound from the lungs. Briefly, the eardrum was
surgically exposed and one small piece of reflecting film (3M,
3M Center, St Paul, MN, USA) was glued on the eardrum centre and
another on the head for control measurements. The head was then
tied to a PVC platform and suspended 13 cm below the water
surface, approximately 45 cm above an underwater loudspeaker
(type UW30, Lubell Labs Inc.). Sound was measured by a
hydrophone (type 8103, Brüel & Kjær) located approximately
1 cm from the eardrum and conditioned by a conditioning amplifier
(type Nexus 2, Brüel & Kjær). The laser Doppler vibrometer (model
as above) was focused on the reflecting foil through a transparent
window in the tank. The particle velocity in the direction of
tympanum movement was measured from the pressure gradient
between two closely spaced hydrophones (type 8103, Brüel &
Kjær), as described previously (Christensen-Dalsgaard et al., 2012).
Eardrum and particle velocity measurements were controlled by
a digital signal processor (type TDT RM2, Tucker-Davis
Technologies) and customized software. The speed of sound in
the tank was 1370 m s
−1
(Christensen-Dalsgaard et al., 2012). We
compared the underwater laser measurements with a model of fish
swim bladder vibration in an underwater sound field using
coefficients for damping and stiffness taken from fish tissue
(Alexander, 1966; see also Christensen-Dalsgaard et al., 2012).
In SI units, the model states that:
v¼p, 100 ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ð3V=4pÞ
3
p
v2ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ð500 ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ð3V=4pÞ
3
p2v21Þ2þ200 ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ð3V=4pÞ
3
p2v2
q!;
ð1Þ
where vis vibration velocity, pis pressure, Vis cavity volume and ω
is angular frequency.
RESULTS
ABR and latency
The waveshape of the ABR, especially to click sounds, was similar
in air and in water (Fig. 2). It typically had three phases, one
negative and two positive ones, here denoted I, II and III. Phase II
had the largest amplitude and was most prominent and consistent,
and therefore it could reliably be detected over much larger stimulus
ranges than phase I and III.
The latency from click stimulus arrival at the ear to time of highest
amplitude of phase II in the ABR was determined as a function of
stimulus amplitude after compensating for transmission time from the
loudspeaker and underwater speaker to the ear (Fig. 3). Both in-air
and underwater latencies seemed to follow the same general trend of
decreasing latency with increasing peak equivalent sound pressure, as
indicated by the non-significant interaction between peak amplitude
and medium (RCMM F
1,53
=0.27, P=0.61). The latencies ranged
from about 6.0 ms to about 3.5 ms at the highest stimulus amplitudes.
In this comparison, in-air stimuli had been presented at higher
amplitudes than the underwater stimuli. Peak amplitude showed a
significant negative relationship to latency, i.e. higher peak amplitude
resulted in shorter latency (RCMM F
1,53
=85.94, P<0.0001,
slope=−0.00033). Medium (air versus water) did not affect latency
time (RCMM F
1,53
=0.01, P=0.90).
The similarity between in-air and underwater latencies may be
interpreted to mean that the same physiological brain processes
occur in the experimental bird in both media. However, three
individuals (UW 4, UW C and UW G) displayed longer latencies at
5
RESEARCH ARTICLE Journal of Experimental Biology (2020) 223, jeb217265. doi:10.1242/jeb.217265
Journal of Experimental Biology
very high stimulus amplitudes and were therefore measured at only
2–3 click sound amplitudes underwater. This might indicate that the
general state of these experimental birds had deteriorated somewhat
when they were transferred from the in-air to the underwater setup.
ABR thresholds in air
In-air hearing thresholds to pure tone bursts were obtained for 12
birds. Thresholds varied considerably (range 15–30 dB) from bird
to bird and from frequency to frequency (Fig. 4A). Slightly different
positions of the measuring electrode relative to the ear opening may
account for some of the variation between individuals in the
recorded ABR.
The average in-air ABR threshold curve was U-shaped and had
best frequency (BF) at 1 kHz and a tuning Q
10dB
of 2. The average
threshold at BF was 53 dB re. 20 µPa. The 10 dB bandwidth
(i.e. 10 dB above threshold at BF) of the average threshold curve
was 2.0 kHz. The slope of the average threshold curve from BF
towards lower frequencies was about 15 dB per octave.
ABR thresholds underwater
Underwater hearing thresholds were obtained from 8 of the 12 birds
tested in air (Fig. 4B). Just as in air, there was considerable threshold
variation from bird to bird, especially at frequencies below 2.5 kHz
(range 10–40 dB). Just as in air, BF of the average threshold curve
was at 1 kHz and Q
10dB
was 2. The average of all underwater
thresholds at BF was 84 dB re. 1 µPa. The 10 dB bandwidth of the
average threshold curve was 2.0 kHz. The slope of the average
threshold curve from BF towards lower frequencies was 8 dB per
octave. The three individuals with longer latencies underwater also
had higher underwater thresholds, again suggesting deteriorated
conditions for these three birds. Therefore, a second average
was calculated based on the data from the remaining five subjects.
These two average curves were generally similar with only a few
dB deviation.
Comparison of threshold curves in the two media
To directly compare the shape and sensitivity of ABR threshold
curves in air and underwater, the sound pressure threshold values
were all related to 1 µPa. This is equivalent to adding 26 dB to the in-
air thresholds, which had been measured relative to 20 µPa (Fig. 4A).
A repeated measures ANOVA found that the sound pressure
thresholds underwater and in air did not differ significantly
(Fig. 5A; F
1,7
=1.33, P=0.29). However, the threshold differed
significantly between frequencies (F
7,22
=18.93, P<0.0001). Least
square means indicated that thresholds were significantly lower for
frequencies between 500 and 2500 Hz compared with those of higher
and lower frequencies tested (Fig. 5A). There was a non-significant
frequency–medium interaction effect (F
7.22
=0.66, P=0.71).
Hearing thresholds may also be expressed in intensity terms
by relating the dB values to 1 pW m
−2
(for details, see Larsen and
Wahlberg, 2017). The underwater intensity thresholds were
significantly lower than those in air (Fig. 5B; F
1,7
=68.96,
P<0.0001). Frequencies also differed significantly, with those
between 500 and 2500 Hz having lower thresholds than lower and
higher frequencies (F
7,22
=18.30, P<0.0001). Finally, the frequency–
medium interaction was non-significant (F
7.22
=0.80, P=0.59).
Anatomy of the outer and middle ear
The ear opening was often difficult to localize, even under a surgical
microscope. In the two adult individuals, it was found about 40 mm
from the centre of the ipsilateral eyeball and about 29 mm from the
ipsilateral corner of the mouth (Fig. 6A). Here, the opening is
covered with a dense layer of 15–16 mm long feathers
indistinguishable from the other feathers covering the head. Thus,
these feathers lacked the open structure of the ear coverts of, for
instance, songbirds.
100
2
3
4
A
B
C
D
E
F
G
H
I
4
B
C
D
F
G
H
I
90
80
70
60
50
40
100 1000 10,000
140
130
120
110
100
90
80
70
60
100 1000
Frequency (Hz)
Threshold (dB re. 1 µPa) Threshold (dB re. 20 µPa)
10,000
A
B
Fig. 4. Scatter plots of ABR audiograms from cormorant fledglings
collected over 3 years. (A) In-air threshold curves of 12 cormorant fledglings.
(B) Underwater threshold curves of 8 of the 12 fledglings. Solid line indicates
average values. Bird ID is indicated in the key (see Table 1).
3.0
80 90 100 110
Peak equivalent (dB re. 1 µPa)
120 130 140 150
3.5
4.0
4.5
5.0
Latency (ms)
5.5
6.0
6.5
7.0
UW 4
UW B
UW C
UW D
UW F
UW G
UW H
UW I
Air 4
Air B
Air C
Air D
Air F
Air G
Air H
Air I
Fig. 3. Latency. Click response latency of the phase II deflection for the 8 birds
whose ABRs were measured both in air and underwater (UW). Bird ID is
indicated in the key (see Table 1).
6
RESEARCH ARTICLE Journal of Experimental Biology (2020) 223, jeb217265. doi:10.1242/jeb.217265
Journal of Experimental Biology
In the adult cormorants, the external ear formed a lens-shaped
naked recess (like a ‘satellite dish’, typically about 2.5 mm deep,
7.5 mm long and 4.0 mm wide), the long axis of which was almost
parallel to the beak (Fig. 6B,C). The actual bean-shaped ear opening
(about 2 mm by 1 mm; Fig. 6C) was located at the centre of the
naked recess. The bottom of the naked recess was very resilient and
applying a very slight pressure with two needles easily closed the ear
opening along its long axis (Fig. 6D,E). The meatus was spoon-
shaped at the bottom of the naked recess and led to a short passage
posterior to the quadratum bone (Fig. 6C) that bent down into a
pocket-like chamber, 7–8 mm high and 6–7 mm in diameter, i.e.
with a volume of about 0.1 ml. The axis of the meatus chamber from
surface to bottom was not perpendicular to the head surface but
directed forward about 45–50 deg relative to the beak axis (Fig. 6C).
Fledgling ears only differed from adult ears by the lens-shaped outer
ear being partly covered with skin at its two ends.
The eardrum was located in the chamber’s antero-medial wall and
was not visible from the outside because of the curved meatus. In
contrast to, for example, songbirds, the eardrum was not clearly
protruding into the chamber but was rather flat and it was difficult to
visually determine its exact rim because the eardrum proper was
covered by a loosely fitting external epithelium continuous with the
skin of the ear canal (named the ‘outer eardrum’by Schwartzkopff,
1949). When the outer eardrum was peeled off, the extent and slight
protrusion of the transparent ‘inner eardrum’into the meatus was
clearly visible. The intact two-layered eardrum in situ appeared very
thick and piston-like, especially around the three-pronged, about
2.0 mm by 1.3 mm, extra-columella’s eccentric attachment (area
about 4 mm
2
; Fig. 6F,G) to the almost circular ‘inner eardrum’(long
diameter about 6.5 mm and short diameter about 5.5 mm; area about
28 mm
2
); i.e. the extra-columella covered about 14% of the eardrum
area. From the extra-columella, the slightly curved, about 4.5 mm
long columella (Fig. 6F) led to the inner ear, to which it attached via
the oval–triangular 1 mm diameter footplate (area about 0.8 mm
2
;
Fig. 6H). The ratio of eardrum area to footplate area therefore was
about 35.
Eardrum vibrations in air
We collected eardrum responses to free-field air-borne sound in
three anaesthetized cormorants. The transfer functions (i.e. the
vibration spectrum divided by the sound spectrum recorded at the
eardrum; see example in Fig. 7A) showed a maximum vibration
amplitude around 1 kHz of about −21 dB re. 1 mm s
−1
Pa
−1
(0.1 mm s
−1
Pa
−1
) with ipsilateral stimulation (90 deg). Also, the
eardrum transfer functions showed a systematic change in vibration
amplitude with direction around 1 kHz, with a maximum difference
of about 6 dB between ipsilateral and contralateral stimulus
directions in the three birds.
Eardrum vibrations underwater
Underwater laser vibrometry measurements on three cormorant
heads showed that the eardrum vibrated with a maximum amplitude
of approximately −10 dB re. 1 mm s
−1
Pa
−1
(0.3 mm s
−1
Pa
−1
)at
1 kHz (Fig. 7B). The velocity amplitude was 20–30 dB higher than
that of the surrounding tissue, and approximately 30 dB higher than
underwater particle velocity amplitudes, which would be the source
of bone conduction stimulation of the ear. The observed peak
frequency corresponded to the peak frequency of a simple model
(Fig. 7B) based on sound-induced vibration of fish swim bladders
(Alexander, 1966; see Materials and Methods), assuming a cavity
volume of 0.2 ml (see also Christensen-Dalsgaard et al., 2012, for
details). This model, however, predicted lower vibration amplitudes
than observed, approximately 15 dB lower at 1 kHz. These post-
mortem measurements were performed approximately 10 min after
the birds were killed and therefore were probably not affected by
post-mortem changes in stiffness of the middle ear. Also, the
responses were comparable to, or even larger than, the in vivo
responses of the eardrum in air.
DISCUSSION
We have presented the first physiological measurements of
underwater sound sensitivity in a diving bird. Evidence, derived
from ABRs in air and underwater, from anatomy, and from laser
vibrometry suggest that the auditory threshold curves of cormorant
fledglings in both media are similar in shape and sensitivity to
sound pressure. However, the sensitivity underwater is higher than
in air in terms of intensity. Eardrum vibration amplitudes both in air
and underwater are maximal at the most sensitive frequencies,
suggesting that the underwater sensitivity is mediated by anatomical
specialization of the middle ear for underwater hearing. These
anatomical specializations will be detailed below.
Underwater and in air-sensitivity measured using the same
ABR methodology
We used the same ABR methodology to derive both in-air and
underwater hearing thresholds of the great cormorant. Compared
with previous studies, the relatively large sample size of specimens
70
100 1000 10,000
100 1000
Frequency (Hz)
10,000
80
90
100
Threshold (dB re. 1 µPa)Threshold (dB re. 1 pW m–2)
110
120
130 A
B
Air pressure
UW pressure
Air intensity
UW intensity
0
20
40
60
80
100
Fig. 5. Comparison of average ABR threshold in air and underwater
predicted from a repeated measures ANOVA model (least means
squares). (A) The model predicted average sound pressure curves (±s.e.) in
air and underwater (dB re. 1 µPa) that were not significant ly different. (B) When
measured as average intensity thresholds (dB re. 1 pW m
−2
), the model
predicted statistically different curves (average±s.e.) in air and underwater.
7
RESEARCH ARTICLE Journal of Experimental Biology (2020) 223, jeb217265. doi:10.1242/jeb.217265
Journal of Experimental Biology
tested as well as the consistency in methodologies of the
measurements in air and underwater on the same individuals
made it possible to more carefully analyse the differences in hearing
threshold between the two media than was possible in previous
studies on this species (Johansen et al., 2016; Maxwell et al., 2017;
Hansen et al., 2017) as well as on other species of marine birds
(Therrien, 2014; Crowell, 2016).
The cormorant in-air ABR threshold curve is shaped like in-air
audiograms of other birds of similar size (see Maxwell et al., 2017)
and shows similar sensitivity and shape to those of other diving
birds in air (Crowell et al., 2015). This gives confidence to the ABR
method for determining the general shape of the threshold curves,
even though the hearing thresholds derived with this method are
20–40 dB higher than those derived by psychophysics (Fig. 8A). A
large discrepancy between ABR thresholds and psychophysical
thresholds is also found in other species of birds (Brittan-Powell
et al., 2002) as well as in marine mammals (Wolski et al., 2003;
Houser and Finneran, 2006). The difference is usually explained by
the large methodological differences in the two approaches: ABR
measurements essentially tap into the nerve signals funnelled
through the animal’s brainstem from synchronous activity in many
auditory nerve fibres, whereas the psychophysical thresholds reflect
activity in only the most sensitive fibres and make use of the
animal’s entire decision process.
In addition, we used 25 ms duration pulse tones in the ABR
experiments. The peripheral auditory system is often modelled as a
leaky energy detector with a certain integration time, T
i
(Plomb and
Bouman, 1959). In this model, thresholds will remain constant for
stimulus tone durations longer than T
i
but increase when stimulus
duration is progressively shorter than T
i
. Therefore, with 25 ms
tones, much higher stimulus sound amplitude is required to deliver
sound energy than the 500 ms duration tones used in the
psychoacoustic experiments (Dooling and Searcy, 1985; Klump
and Maier, 1990). With an expected integration time for birds of
about 200 ms, this accounts for 10–15 dB of the difference between
ABR thresholds and psychoacoustic thresholds (Pohl et al., 2009,
2013). Therefore, even though the shape of average ABR threshold
curve mimics the psychophysically derived audiogram, it is
expected that the curves will not be comparable in terms of
sensitivity. However, ABR-derived threshold curves do allow
comparison between hearing sensitivity in the two media.
Finally, it might be argued that the relatively high thresholds in the
present study could be a consequence of using fledglings and not
adult birds, as avian auditory sensitivity increases during ontogeny.
However, this is not supported by data from other species of birds:
the increase in budgerigar (Melopsittacus undulatus) audiogram
sensitivity was found to be most marked during the first 3 weeks of
development, becoming indistinguishable from adult values at the
age of fledging (see fig. 3 in Brittan-Powell and Dooling, 2004).
As the cormorant chicks were judged to be 4–6 weeks old and very
close to fledging, we expect their threshold curves to be close to those
of adult birds. Any differencein threshold between juvenile and adult
birds is therefore expected to be smaller than the measurement
accuracy of this study, which is estimated to be about 6 dB.
The large variation in threshold obtained from individual birds of
the present study (Fig. 4) is readily explained by the ABR
methodology, which in humans has been shown to exhibit
inherent amplitude variation of 10–15 dB (e.g. Picton et al.,
2003). In addition, it is well known that small shifts in electrode
position can produce different signal amplitudes, which will have a
tremendous influence on the derived thresholds. The fact that in the
present study the birds needed to be anaesthetized, and in the case of
the underwater trials were subject to radical surgery, may also have
caused variation in the birds’responses depending on their general
A
B
C
D
40 mm
E
FG
H
Fig. 6. Anatomy of the right ear of a
2 year old female cormorant. (A) Right
side of the head. The red spot indicates
the location of ear opening under the
dense feather cover. (B) Close-up with
cover feathers brushed aside. (C) The
lens-shaped naked recess surrounding
the bean-shaped ear opening. The
black spot in the ear opening is the canal
leading to the eardrum chamber below
at a 45–50 deg angle relative to the
head surface with the beak direction to
the right. (D,E) Very light pressure
applied with two needles closes the ear
opening along its long axis. (F) The
excised columella with the extra-
columella at the top and the footplate at
the bottom. Part of the excised eardrum
is indicated with a red arrow.
(G) Eardrum with eccentric extra-
columella attachment seen from below
(from the middle ear). (H) Footplate to
the oval window seen from the inner ear.
8
RESEARCH ARTICLE Journal of Experimental Biology (2020) 223, jeb217265. doi:10.1242/jeb.217265
Journal of Experimental Biology
physiological state and well-being, which may be mirrored in the
different latency dependence on peak pressure (Fig. 3).
The fact that in-air ABR thresholds and psychophysical
audiograms are so similar in shape (Fig. 8A) gives us confidence
that the same would also be the case for the underwater hearing data.
However, comparison of the average underwater ABR threshold
curve with previously derived psychophysical data (Hansen et al.,
2017) showed a large difference in the shape of the two threshold
curves (Fig. 8B). The ABR threshold curve has its BF at 1 kHz and
tapers off at lower frequencies at a rate similar to that found in the
in-air audiogram. At higher frequencies, the underwater thresholds
are also monotonically increasing, but at a lower slope than for the
in-air audiogram (Fig. 4A). This is in great contrast to the
underwater psychophysical data (Hansen et al., 2017), where the
hearing thresholds varied by only 5 dB in the tested range of 1–
4 kHz, whereas there was a 31 dB difference in the average ABR
threshold in the same frequency interval (Fig. 8B). It should be
remembered, however, that the psychophysical thresholds were
derived from a single individual and with a limited dataset because
of methodological difficulties (Hansen et al., 2017). It is therefore
conceivable that more psychophysical data on several individuals
would result in underwater hearing thresholds more like the ones
derived here by the ABR method.
Comparing threshold curves in air and underwater
Despite these discrepancies, it seems safe to compare the in-air and
underwater ABR threshold curves in terms of both their shape and
their sensitivity, as they were derived from a relatively large dataset
using several individuals (8 of which produced both in-air and
underwater data) and very similar methodologies. The cormorant
average underwater ABR threshold curve seems slightly displaced
towards lower frequencies relative to the average in-air ABR
threshold curve (Fig. 4) but the statistical model predicts curves that
are not significantly different for the two media (Fig. 5A). True
seals, which are considered to be fully adapted for hearing in both
media, have their underwater BF above the in-air BF, and the high-
frequency slope is steeper in the underwater than in the in-air
audiogram (Reichmuth et al., 2013; Sills et al., 2014, 2015). In fact,
the weaker slopes of the average underwater threshold curve
and its slightly lower frequency emphasis are more like those of
another not aquatically adapted vertebrate, namely humans (Parvin
and Nedwell, 1995). So, judging from the shape of the average
–40
0.5 1 2
Eardrum vibration transfer function (dB re. 1 mm s–1 Pa–1)
Frequency (kHz)
3
100 1000 10,000
45
–35
–30
–25
Eardrum
Head
Particle velocity
Model
–90
–60
–30
0
+30
+60
+90
–20
–15 A
B
–50
–60
–40
–30
–20
–10
0
Fig. 7. Cormorant eardrum vibrations in air and underwater measured
with laser Doppler vibrometry. (A) In-air eardrum vibration velocity transfer
function as a function of sound source direction in the frontal hemi-field: 0 deg
corresponds to the beak direction, whereas +90 deg corresponds to the
ipsilateral source location. (B) Underwater vibration velocity transfer functions
of the eardrum and the surrounding skin on the head compared with the
measured particle velocity of the medium, close to the head, and the vibration
output of a simple model.
0
100 1000 10,000
100 1000
Frequency (Hz)
Threshold (dB re. 20 µPa)
Threshold (dB re. 1 µPa)
10,000
20
ABR air
Psychophysical air
40
60
80
100 A
B
60
80
70
100
90
110
120
130
ABR UW
Psychophysical UW
Fig. 8. Comparison between ABR thresholds and thresholds obtained
with behavioural methods. (A) Average ABR threshold curve (±s.e.) in air
from 12 cormorant fledglings compared with the psychophysical threshold
curve of a single adult cormorant in air (Maxwell et al., 2017). (B) Average ABR
threshold curve (±s.e.) underwater from 8 of the 12 cormorant fledglings
compared with the psychophysical threshold curve of a single cormorant
underwater (Hansen et al., 2017).
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RESEARCH ARTICLE Journal of Experimental Biology (2020) 223, jeb217265. doi:10.1242/jeb.217265
Journal of Experimental Biology
underwater threshold curve, cormorants may not have developed
fully aquatic hearing abilities and are more adapted to hearing in air.
Another way to judge whether cormorant hearing is adapted or
not for in-air and underwater hearing is to compare the magnitude of
the hearing thresholds obtained in the two media. Previous
comparisons between in-air and underwater thresholds have
focused on true seals and sea lions (Kastak and Schusterman,
1998, 1999; Møhl, 1968; Lombard et al., 1981; Mulsow and
Reichmuth, 2010; Reichmuth et al., 2013). Depending on whether
the acoustic signals were assumed to be detected in units of sound
pressure (in pascals) or intensity (in watts m
−2
), the authors reached
quite different conclusions as to whether the animals’hearing
abilities were in-air or water adapted, or both. This counter-intuitive
result can be elucidated with an example. Consider an animal with a
hearing threshold at a frequency of 1 mPa in air (or 34 dB re.
20 µPa) and 1 mPa (or 60 dB re. 1 µPa) underwater, with decibels
defined as:
dB ¼20log10
p
p0
;ð2Þ
where p
0
is the reference pressure of 20 µPa and 1 µPa in air and
water, respectively. It is evident that the in-air and underwater
decibels cannot be readily compared because of the different
reference units (this would result in us erroneously reporting a
higher threshold in water than in air, for identical sound pressures).
By comparing decibels in pressure units, 26 dB must be added to the
in-air decibels, as was done in Fig. 5A. This is a direct result of the
definition of air decibels:
dB ¼20log10
p
20 106
¼20log10
p
106
20log10ð20Þ
¼20log10
p
106
26:ð3Þ
To complicate matters further, it is often assumed that animals
detect acoustic energy or intensity, rather than acoustic pressure. In
the free acoustic field, the acoustic intensity (I) is defined as:
I¼p2
Z0
or
dB ¼10 log10
I
I0
;ð4Þ
where Z
0
is the characteristic acoustic impedance of the medium
(density multiplied by speed of sound). The reference acoustic
intensity is often chosen as I
0
=1 pW m
−2
, mainly taken from
audiology, where it is close to the human hearing threshold in air at
1 kHz.
Coming back to our example, the acoustic intensity of 1 mPa is
2200 pW m
−2
(or +34 dB re. 1 pW m
−2
) in air and 0.67 pW m
−2
(or
−2dBre.1pWm
−2
) in water. In other words, for the same acoustic
pressure, the signal will carry 36 dB more intensity in air than
underwater. This also means that the same acoustic intensity will
result in acoustic pressures that are 36 dB higher in water than in air
(see Larsen and Wahlberg, 2017, for a thorough discussion of this
topic). Thus, our imagined animal would have identical hearing
thresholds in air and underwater when measured in units of pressure,
and we would probably conclude that the hearing system is adapted
to function equally well in both media. However, when measured in
units of intensity (or energy), the hearing thresholds would be much
higher in air than in water, and we would conclude that the ear
would be more adapted for underwater than for aerial hearing.
A similar analysis can be done on the results from the present
study. Assuming the cormorant detects acoustic pressure (Fig. 5A),
the hearing thresholds can be directly compared and there is no
difference between thresholds in the two media. Assuming the
cormorant detects acoustic intensity or energy (Fig. 5B), however,
the cormorant hearing thresholds are 20–40 dB lower underwater
than in air. Thus, without a better understanding of whether the
adequate stimulus is pressure or intensity, it is difficult to use
hearing thresholds to conclude whether the cormorant hearing
system is adapted for underwater hearing.
There is only one experiment known to us that has attempted to
determine what the adequate stimulus unit actually is: a study by
Finneran et al. (2002) on dolphins (Tursiops truncatus). Finneran
et al. (2002) determined that pressure (or rather, pressure-squared) is
the relevant unit for dolphins in water at low frequencies. Further
experimentation in other species and at other frequencies (Finneran
et al., 2002, only investigated the relatively low 100–300 Hz
frequency range) would be crucial for understanding not only what
the relevant stimulus unit is but also whether or not the hearing
thresholds of an animal are lower or higher in water than in air.
Until the dilemma of unit confusions is resolved, we cannot resolve
whether the cormorant’s hearing thresholds were lower in water than
in air. We can conclude, however, that the underwater hearing
thresholds are at least as low as, and possibly lower than, the in-air
ones. In addition, the results of Johansen et al. (2016) and of Hansen
et al. (2017), although somewhat preliminary and based on just one
animal, suggest that at the tested frequencies, the cormorant’shearing
thresholds in water were similar to what is found in animals that are
considered to have fully water-adapted hearing, such as seals and
porpoises at low frequencies. All this makes it plausible that
cormorants do have some adaptations for hearing underwater as have
many other secondarily adapted aquatic vertebrates (summarized by
Christensen-Dalsgaard and Manley, 2013).
The non-significant frequency–medium interaction suggests
that the thresholds did not differ between air and water. However,
this does not necessarily mean that the frequency response in the
two media was equal. It should be remembered that for practical
reasons the measurements were made only 10 cm below the water
surface. It is conceivable that any in-water adaptation of the hearing
system is not fully exploited that close to the water surface, and that
the magnitude of such effects could vary depending on depth. One
may also conceive more complicated interactions with the hearing
system and a more complicated acoustic field close to a water
surface that can only be resolved by further measurements.
Potential anatomical adaptations for underwater hearing
The sensitivity to underwater sound may be caused by special
anatomical features of the middle ear. The eardrum vibration
measurements (Fig. 7B) suggest that resonance of the air in the
middle ear cavity is important in driving tympanic vibrations. One
obvious difference between the cormorant middle ear and the
middle ear of, for instance, songbirds is the thick plate-shaped extra-
columella that covers about 14% of the double-layered tympanic
area (Fig. 6F). This is reminiscent of the tympanic disc structure
found in two aquatic-adapted middle ears, the ears of the clawed
frog Xenopus laevis (Christensen-Dalsgaard and Elepfandt, 1995;
Vedurmudi et al., 2018) and the turtle Trachemys elegans
(Christensen-Dalsgaard et al., 2012). The cormorants’eardrum
10
RESEARCH ARTICLE Journal of Experimental Biology (2020) 223, jeb217265. doi:10.1242/jeb.217265
Journal of Experimental Biology
vibration sensitivity to underwater sound measured by the transfer
function ( peak sensitivity 0.3 mm s
−1
Pa
−1
) is similar to the peak
sensitivity of turtles (0.3 mm s
−1
Pa
−1
) and clawed frogs (0.05–
0.25 mm s
−1
Pa
−1
). Possibly, some of these adaptations entail
modifications of the middle ear, such as stiffening of the tympanic
membrane, which in turn may explain the reduced sensitivity to air-
borne sound shown by both audiograms and eardrum vibrations. An
analytical model of the Xenopus ear suggests that a plate-like
tympanum is more efficient than a protruding thin membrane in
coupling sound energy in water to the inner ear (Vedurmudi et al.,
2018). Alternatively, a more solid eardrum could have evolved as a
protection of the ear against the increased pressures incurred when
the bird is diving.
Other characteristics of the outer and middle ear anatomy
described here are much like those for cormorants reported by Saiff
(1978) and for auks reported by Kartaschew and Iljitschow (1964).
The dense feather cover, the easily compressed ear opening and the
bent meatus may all protect the ear during the relatively shallow
cormorant dives rather than increase its sensitivity to underwater
sound. The fine structure of the eardrum–columella complex may
be an adaptation to underwater hearing but awaits comparative
studies using modern high-resolution techniques such as micro-CT
scanning (see e.g. Muyshondt et al., 2018).
Acknowledgements
We are greatly indebted to the estate management of Brahetrolleborg Skov og
Landbrug, represented by Søren Nielsen, for permission to catch cormorant
fledglings on their land. We are grateful to Jakob Sterup for observing the nesting
birds and (together with Thomas Bregnballe) for helping with fledgling retrieval from
the nests. The QuickABR software was developed by Christian Brandt. We thank
Tina Marie Huulvej and Simon Kongshøj Callesen for assisting with data collection
and Tanya Bojesen Lauridsen for independently determining ABR thresholds. We
are greatly indebted to Thorsten J. S. Balsby for helping with the statistical analysis.
Finally, we are grateful to Jakob Tougaard and two anonymous reviewers for very
useful comments to improve the manuscript.
Competing interests
The authors declare no competing or financial interests.
Author contributions
Conceptualization: O.N.L., M.W., J.C.-D.; Methodology: O.N.L., M.W., J.C.-D.;
Software: M.W., J.C.-D.; Validation: O.N.L., M.W., J.C.-D.; Formal analysis: O.N.L.,
M.W., J.C.-D.; Investigation: O.N.L., J.C.-D.; Resources: O.N.L., J.C.-D.; Data
curation: O.N.L., J.C.-D.; Writing - original draft: O.N.L.; Writing - review & editing:
O.N.L., M.W., J.C.-D.; Visualization: O.N.L., J.C.-D.; Supervision: O.N.L., M.W.,
J.C.-D.; Project administration: O.N.L.; Funding acquisition: O.N.L., M.W., J.C.-D.
Funding
This study was funded by the Carlsbergfondet (grants 2009-01-0292 to M.W. and
O.N.L., 2012-01-0662 to J.C.-D., M.W. and O.N.L., and 2013-01-0917 to M.W. and
O.N.L.), and by the Natur og Univers, Det Frie Forskningsråd (grant DFF-4002-
00536 to M.W.).
Data availability
The data are stored at the University of Southern Denmark and are available on
request to the authors.
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