Vocal differentiation parallels development of auditory saccular sensitivity in a highly soniferous fish

Abstract

Vocal differentiation is widely documented in birds and mammals but has been poorly investigated in other vertebrates, including fish, which represent the oldest extant vertebrate group. Neural circuitry controlling vocal behaviour is thought to have evolved from conserved brain areas that originated in fish, making this taxon key to understanding the evolution and development of the vertebrate vocal-auditory systems. This study examines ontogenetic changes in the vocal repertoire and whether vocal differentiation parallels auditory development in the Lusitanian toadfish Halobatrachus didactylus (Batrachoididae). This species exhibits a complex acoustic repertoire and is vocally active during early development. Vocalisations were recorded during social interactions for four size groups (fry: <2 cm; small juveniles: 2-4 cm; large juveniles: 5-7 cm; adults >25 cm, standard length). Auditory sensitivity of juveniles and adults was determined based on evoked potentials recorded from the inner ear saccule in response to pure tones of 75-945 Hz. We show an ontogenetic increment in the vocal repertoire from simple broadband-pulsed 'grunts' that later differentiate into four distinct vocalisations, including low-frequency amplitude-modulated 'boatwhistles'. Whereas fry emitted mostly single grunts, large juveniles exhibited vocalisations similar to the adult vocal repertoire. Saccular sensitivity revealed a three-fold enhancement at most frequencies tested from small to large juveniles; however, large juveniles were similar in sensitivity to adults. We provide the first clear evidence of ontogenetic vocal differentiation in fish, as previously described for higher vertebrates. Our results suggest a parallel development between the vocal motor pathway and the peripheral auditory system for acoustic social communication in fish.

Full text

RESEARCH ARTICLE
Vocal differentiation parallels development of auditory saccular
sensitivity in a highly soniferous fish
Raquel O. Vasconcelos
1,2,
*
, Peter W. Alderks
3
, Andreia Ramos
1,2
, Paulo J. Fonseca
2
, M. Clara P. Amorim
4
and
Joseph A. Sisneros
3
ABSTRACT
Vocal differentiation is widely documented in birds and mammals but
has been poorly investigated in other vertebrates, including fish,
which represent the oldest extant vertebrate group. Neural circuitry
controlling vocal behaviour is thought to have evolved from conserved
brain areas that originated in fish, making this taxon key to
understanding the evolution and development of the vertebrate
vocal-auditory systems. This study examines ontogenetic changes in
the vocal repertoire and whether vocal differentiation parallels
auditory development in the Lusitanian toadfish Halobatrachus
didactylus (Batrachoididae). This species exhibits a complex
acoustic repertoire and is vocally active during early development.
Vocalisations were recorded during social interactions for four size
groups (fry: <2 cm; small juveniles: 2–4 cm; large juveniles: 5–7 cm;
adults >25 cm, standard length). Auditory sensitivity of juveniles and
adults was determined based on evoked potentials recorded from the
inner ear saccule in response to pure tones of 75–945 Hz. We show
an ontogenetic increment in the vocal repertoire from simple
broadband-pulsed ‘grunts’ that later differentiate into four distinct
vocalisations, including low-frequency amplitude-modulated
‘boatwhistles’. Whereas fry emitted mostly single grunts, large
juveniles exhibited vocalisations similar to the adult vocal repertoire.
Saccular sensitivity revealed a three-fold enhancement at most
frequencies tested from small to large juveniles; however, large
juveniles were similar in sensitivity to adults. We provide the first clear
evidence of ontogenetic vocal differentiation in fish, as previously
described for higher vertebrates. Our results suggest a parallel
development between the vocal motor pathway and the peripheral
auditory system for acoustic social communication in fish.
KEY WORDS: Hearing, Vocal differentiation, Acoustic communication,
Ontogeny, Batrachoididae
INTRODUCTION
A fundamental question for all vocal communication systems
concerns the relationship between the developmental processes of
vocal differentiation and auditory perception during ontogenetic
development. Vocal differentiation, or the progressive increment in
the number of call types with development, has been mostly
documented in songbirds and mammals (e.g. Moss et al., 1997;
Doupe and Kuhl, 1999; Hollén and Radford, 2009). An individual
may undergo this process via vocal learning from adults during
ontogeny (e.g. Brainard and Doupe, 2002). However, non-learner
species also exhibit considerable ontogenetic changes in their
vocalisations, which may result from the development of the vocal
motor system in both peripheral vocal apparatus and central neural
circuitry controlling vocal behaviour (e.g. Jürgens, 2002;
Derégnaucourt et al., 2009). Equally important to consider is that
acoustic signal perception originates through development of the
auditory periphery, inner ear and sensory receptors, and also central
neural pathways (e.g. Moore, 2002). Certain species are known to
undergo a refinement process during a sensitive phase when young
are first experiencing social acoustic signals in the auditory vocal
environment. However, such interaction between vocal and auditory
systems during development has only been clearly demonstrated in
higher vertebrates, namely songbirds and mammals (e.g. Moore,
2002; Miller-Sims and Bottjer, 2012).
Soniferous fish are good candidates to examine vocal
differentiation and auditory sensitivity during ontogeny because
they have relatively simple central and peripheral vocal
mechanisms. In addition, the neural circuitry that controls vocal
behaviour in vertebrates seems to have evolved from conserved
brain areas found in ancestral fish before they diverged into the
major clades (Bass et al., 2008). Thus, studies that investigate the
development of vocal-auditory systems in vocal fish are important
to gain a comprehensive understanding of the mechanisms
underlying social acoustic communication in all vertebrates.
Previous fish studies have reported ontogenetic changes in
acoustic signal characteristics, such as repetition rate, amplitude,
duration and dominant frequency. The refinement in some of these
signal characteristics with age is probably due to ontogenetic
changes in the size and/or resonance properties of the sound-
generating apparatus (e.g. Myrberg et al., 1993; Amorim and
Hawkins, 2005; Lechner et al., 2010). It remains unclear whether
fish that produce more complex acoustic signals exhibit vocal
differentiation, which could pot entially result from ontogenetic
modifications of the motor circuitry of the vocal pathways as in birds
or mammals (e.g. Aronov et al., 2008).
Data on the development of hearing abilities in fishes is relativ ely
limited compared with what is kno wn for other taxa. The available
studies across various taxonomic groups rev eal common principles,
namely ontogenetic incr eases in auditory sensitivity (e.g. birds,
Dmitrieva and Gottlieb, 1994; anurans, Boatright-Hor o witz and
Simmons, 1995; mammals, Reimer, 1995; r eptiles, Werner et al.,
1998), an extension of the frequency hearing range (e.g. mammals,
Rübsamen, 1992) and a shift in the most sensitive frequencies (e.g.
anurans, Boa tright-Horo witz and Simmons, 1995). Ontogenetic
studies of fish hearing show diverse results, ranging from no
differences (Popper, 1971; Zeddies and Fa y , 2005), to expansion of
Received 8 April 2015; Accepted 7 July 2015
1
Institute of Science and Environment, University of Saint Joseph, Rua de Londres
16, Macau S.A.R., People’s Republic of China.
2
Departamento de Biologia Animal
and Centre for Ecology, Evolution and Environmental Changes (cE3c),
Universidade de Lisboa, Bloco C2 Campo Grande, Lisbon 1749-016, Portugal.
3
Departments of Psychology and Biology, University of Washington, Seattle, WA
98195, USA.
4
MARE – Marine and Environmental Sciences Centre, Departamento
de Biociências, ISPA – Instituto Universita
́
rio, Rua Jardim do Tabaco 34, Lisbon
1149-041, Portugal.
*Author for correspondence (raquel.vasconcelos@usj.edu.mo)
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© 2015. Published by The Company of Biologists Ltd
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Journal of Experimental Biology (2015) 218, 2864-2872 doi:10.1242/jeb.123059
Journal of Experimental Biology

the detectable frequency range (Higgs et al., 2002, 2003; Alderks and
Sisneros, 2011), up to increases in auditory sensitivity with size/age
(Kenyon, 1996; Wysocki and Ladich, 2001; Sisneros and Bass, 2005).
Although some effort has been made to understand how fish
vocal behaviour and auditory sensitivity develop during ontogeny,
the question of whether there is concurrent ontogenetic development
of the vocal motor and auditory systems for social communication
remains unresolved. A few existing studies on this topic show
that sound detection develops prior to the fish’s ability to generate
perceivable sounds (Wysocki and Ladich, 2001; Vasconcelos
and Ladich, 2008). However, acoustic communication might
be absent during early developmental stages because of poor
hearing sensitivity, as in the gourami (Trichopsis vittata) (Wysocki
and Ladich, 2001). Yet, at least in some species, it may occur
during a wide range of developmental stages, as in the catfish
(Synodontis schoutedeni) (Lechner et al., 2010). Nevertheless, these
studies only focused on simple-pulsed sounds produced in distress/
agonistic contexts and they do not report any evidence of
vocal differentiation that would facilitate acoustic communication
in these species.
In this study, we used the highly soniferous Lusitanian toadfish
Halobatrachus didactylus (Bloch and Schneider 1801)
(Batrachoididae, subfamily Halophryninae) to investigate the
relationship between vocal differentiation and auditory sensitivity.
This mari ne teleost relies on acoustic communication to mediate
social interactions early in development. The Lusitanian toadfish
exhibits an unusually large vocal repertoire that consists of about
five different vocalisations used in various social contexts, including
long tonal sounds (Amorim et al., 2008; Vasconcelos et al., 2010).
Vasconcelos and Ladich (2008) using the auditory evoked potential
(AEP) recording technique showed that the Lusitanian toadfish
exhibits an ontogenetic increase in auditory sensitivity at the lowest
(100 Hz) and highest tested frequencies (800–1000 Hz). These same
authors recorded distress grunt sounds from different developmental
stages and verified that the ability to communicate acoustically
seems to occur when juveniles are able to generate perceivable
grunts of higher amplitude and lower dominant frequency. However,
whether such ontogenetic auditory improvements occur at the
level of the auditory endorgans or more centrally in the auditory
pathway has never been investigated. Also, previous work only
analysed changes in the acoustic features of grunt calls produced in a
distress context and nothing is known about development of the
vocal repertoire. We propose that this model system offers an
unmatched opportunity to investigate how the vocal motor and
auditory systems develop during ontogeny enhancing social acoustic
communication.
The aim of this study was twofold: (1) to verify how the vocal
repertoire develops during ontogeny; and (2) to compare changes in
the vocal behaviour with the auditory sensitivity at different
developmental stages in the Lusitanian toadfish H. didactylus.
Vocalisations were recorded during social interactions in different
size groups, whereas auditory sensitivity was measured from
equivalent juvenile and adult size groups based on auditory evoked
responses from populations of hair cells in the saccule (main
auditory endorgan in most teleosts).
RESULTS
Development of the vocal repertoire
Behavioural observations revealed that both fry and juveniles
readily occupied available shelters, becoming territorial within
24 h. Individuals exhibited both visual and acoustic signalling
during competition for space and food. Territorial visual displays
included not only mouth opening and spreading of pectoral fins and
opercula, but also more aggressive behaviour such as attacks, with
occasional bites towards potential intruder s.
Acoustic signalling was detected in all size groups analysed,
including fry. Single grunts and grunt trains were produced during
agonistic interactions while competing for food or space. Long
grunt trains and double croaks were generally produced when
individuals were inside the shelters and were not accompanied by
any obvious social interaction. In contrast, the relatively rare
boatwhistles recorded among large juveniles were detected only
during active nest defence when the territorial individual was facing
a potential intruder.
The vocal repertoire increased in the number of call types with
increasing fish size, from simple broadband-pulsed sounds in fry
and small juveniles to four different vocalisations in large juveniles
and adults that included more complex amplitude modulated
harmonic calls. Fig. 1 depicts the four representative call types
produced by large juveniles, a grunt call produced by fry and a long
grunt train generated by an adult.
Group size had a significant effect on the calling rate of all sound
types: single grunts (H=26.63, d.f.=3, P<0.001), grunt trains
(H=9.60, d.f.=3, P=0.029), long grunt trains (H=22.69, d.f.=3,
P<0.001), double croaks (H=18.39, d.f.=3, P<0.001) and
boatwhistles (H=35.63, d.f.=3, P<0.001) (see Fig. 2A–E). Single
grunt production rate ranged from 1.65±0.63 calls h
−1
per fish
(mean number of sounds per session divided by the total number of
individuals present) at the fry stage to none for the adults. Grunt
trains also decreased in production rate with growth, ranging from
1.05±0.27 (fry) to 0.33±0.13 calls h
−1
per fish (adults). In contrast,
long grunt train production rate increased with increasing fish size,
from 0.01±0.01 (fry) to 0.65±0.30 calls h
−1
per fish (adults).
Double croaks were only produced by large juveniles (0.05±0.30
calls h
−1
per fish) and adults (0.23±0.06 calls h
−1
per fish).
Similarly, boatwhistles were only detected among large juveniles
(0.02±0.02 calls h
−1
per fish) and adults (10.38±3.10 calls h
−1
per
fish). Overall, the significant decrease in single grunt and grunt train
production rates throughout development was accompanied by a
significant increase in the calling rate of long grunt trains, double
croaks and boatwhistles (Fig. 2F). While fry and small juveniles
emitted exclusively various grunt-type sounds, large juveniles
already exhibited the full adult vocal repertoire composed of grunt
trains, long grunt trains, double croaks and boatwhistles.
Development of the saccular auditory sensitivity
In order to compare the dynamic range of the response magnitudes
or relative gain of the evoked saccular potentials between different
size groups, the iso-level response profiles obtained for each
individual under 130 dB re. 1 µPa sound stimulation were
normalised and expressed relative to a value of 0 dB at the best
frequency (BF, defined as the frequency that evoked the lowest
saccular potential response threshold). Each data point of the iso-
level profile (mean value in voltage) was converted into dB relative
to the value measured at the BF. The data was then averaged to
construct a relative gain plot for each group (Fig. 3A). Normalised
iso-level curves obtained from the three different size groups (small
and large juveniles and adults) were not significantly different
(repeated-measures ANOVA, F
2,36
=0.27, P>0.05). However, the
range of relative gain from 75 to 945 Hz of small juveniles (range
28 dB) was about 10 dB lower than that of large juveniles and adults
(38 and 37 dB, respectively).
The three size groups had best auditory saccular sensitivity at the
lowest frequencies tested, between 75 and 165 Hz, and showed a
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RESEARCH ARTICLE Journal of Experimental Biology (2015) 218, 2864-2872 doi:10.1242/jeb.123059
Journal of Experimental Biology

gradual decrease in sensitivity with frequency up to 945 Hz
(Fig. 3B). Mean auditory thresholds increased from 110 dB re.
1 µPa at 85 Hz (in adults) up to 151 dB at 785 Hz (small juveniles)
and 945 Hz (adults). The threshold at the BF varied significantly
with size: 100–133 dB (small juveniles), 97–136 (large juveniles)
and 88–124 dB (adults) (one-way ANOVA, F
2,47
=6.31, P<0.01).
Auditory threshold curves obtained from the three different
size groups revealed significant overall differences within the
frequency range 75–425 Hz, (repeated-measures ANOVA,
F
2,51
=5.80, P<0.01; Fig. 3B). In addition, results indicated a
significant interaction between size and frequency (F
34,867
=2.11,
P<0.001), meaning that the effect of size on auditory sensitivity is
frequency dependent. LSD post hoc tests showed significant group-
specific differences between small and large juveniles at all test
frequencies within 105 and 425 (except at 385 Hz). Auditory
thresholds of large juveniles did not differ from that of adults at any
frequency.
DISCUSSION
A major goal of this study was to investigate how the relatively large
and complex vocal repertoire previously identified in the Lusitanian
toadfish adults (Amorim et al., 2008) develops throughout
ontogeny. Additionally, the focus of this study was to determine
whether vocal differentiation parallels the development of the
auditory sensitivity in this species. We showed that the number of
stereotyped vocalisations increases in large juveniles above 5 cm
standard length (SL) and that such vocal differentiation is coincident
with an increase in saccular auditory sensitivity.
Ontogenetic vocal differentiation
The Lusitanian toadfish exhibited a gradual increase in the vocal
repertoire, or number of stereotyped vocalisations, with increasing
size throughout development. The vocal repertoire changed from
simple broadband-pulsed grunts, mostly produced in fry and small
juveniles, to four different call types, including amplitude-
modulated and harmonic sounds (e.g. double croaks and
boatwhistles) in large juveniles and adults. Interestingly, large
juveniles already exhibited the full adult vocal repertoire, although
differences in calling rates were found compared with the adults.
Previous studies in other fish species reported developmental
changes in acoustic features of vocalisations such as amplitude,
dominant frequency and temporal patterns (e.g. gurnards, Amorim
and Hawkins, 2005; catfishes, Lechner et al., 2010; gouramis,
Henglmüller and Ladich, 1999; Wysocki and Ladich, 2001).
However, these studies never reported developmental changes in the
vocal repertoire or vocal differentiation, probably because the vocal
repertoire was more restricted and the sounds produced were not as
elaborate as, for instance, those produced by batrachoidids (Amorim
et al., 2008).
Vasconcelos and Ladich (2008) reported developmental changes
in the acoustic features of grunt calls produced in a distress context
by Lusitanian toadfish ranging from 3 to 32 cm SL. Dominant
frequency, sound duration and number of pulses decreased, whereas
pulse period and sound level increased with increasing fish si ze.
These developmental changes were most likely associated with the
growth of the sound-producing apparatus, i.e. swimbladder and
intrinsic sonic muscles, which increase in si ze both ontogenetically
and seasonally (Modesto and Canário, 2003a). Nevertheless, the
development of the vocal motor pathways in the hindbrain may also
have contri buted for the vocal changes observed (Fine et al., 1984;
Fine, 1989; Knapp et al., 1999).
In the present study, we investigated how the full vocal repertoire
produced within a social context changes with growth in the
Lusitanian toadfish, from fry to the adult stage. The vocal
differentiation observed in this fish species most likely results
from developmental changes in the premotor and motor vocal
pathways located in the hindbrain. Knapp et al. (1999) reported that
the vocal motor system, including sexual differentiation features,
develops early in the larvae stage, well before any evidence of
sexual maturation and vocal activity in Porichthys notatus
Frequency (Hz)
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BC
Large juvenile grunt trainLarge juvenile single grunt
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200 ms
Frequency (Hz)
Frequency (Hz)
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Frequency (Hz)
A
Fry single grunt
Fig. 1. Spectrograms and oscillograms of representative vocalisations
produced by Lusitanian toadfish during social interactions. (A) Single
grunt call produced by the earliest developmental stage (fry) considered in this
study. Sound has been filtered <100 Hz to increase signal-to-noise ratio
(SNR). (B–E) Sounds emitted by large juveniles, which already exhibited the
full vocal repertoire. (F) Long grunt train produced by an adult ( juvenile
recordings of this sound type displayed poor SNR). Sampling frequency 8 kHz;
hamming window, 30 Hz filter bandwidth.
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RESEARCH ARTICLE Journal of Experimental Biology (2015) 218, 2864-2872 doi:10.1242/jeb.123059
Journal of Experimental Biology

(Batrachoididae, subfamily Porichthinae). However, these authors
also reported that motoneurons, which directly establish the firing
rate of sonic muscles and vocal features, establish their connections
with the sonic muscle prior to establishing connections with the
premotor neurons in the hindbrain. More recently, Chagna ud and
Bass (2014) showed that the central pattern generator (CPG)
anatomy of two batrachoidids (P. notatus and Opsanus tau;
subfamilies Batrachoidinae and Porichthinae, respectively) differs
at the levels of both the pacemaker-motoneuron circuit and the
afferent pre-pacemaker neurons and this may contribute to the
species-specific patterning of frequency and amplitude-modulated
vocalisations. Together, this suggests that the maturation of CPG
and patterns of connectivity between hindbrain vocal nuclei may
underline the vocal differentiation observed in the Lusitanian
toadfish. Future studies should investigate this hypothesis and also
whether steroid levels play a role in vocal patterning and plasticity,
as reported for adult P. notatus (Bass, 2008). The ability to extend
the vocal repertoire to include more elaborated calls through
maturation of central vocal nuclei has been predominantly described
in birds (Aronov et al., 2008) and mammals (Jürgens, 2002).
Behavioural observations of fry and juveniles suggested that the
different vocalisations are produced during specific social contexts.
Single grunts and grunt trains were typically emitted during
agonistic interactions while competing for either food or territory,
as observed by Vasconcelos and Ladich (2008) and Vasconcelos
et al. (2010). Long grunt trains and double-croaks, which had so far
been recorded only in adults in semi-natural conditions (Amorim
et al., 2008), were also produced by juveniles inside their shelters
but without any obvious social interaction or visual display,
possibly to signal shelter occupancy. Finally, one of the most
surprising results was that large juveniles of Lusitanian toadfish
were capable of producing long harmonic boatwhistles. This signal
was produced while the resident was facing an intruder, similar to
agonistic boatwhistles previously recorded in territorial male
adults (Vasconcelos et al., 2010). Such findings strongly suggest
that similar to P. notatus (Knapp et al., 1999), the CPG must be fully
developed at this developmental stage long before sexual maturity,
which occurs around 30–35 cm total length for all morphotypes in
this species (Pereira et al., 2011). Boatwhistles have been commonly
described as mate advertisement calls among batrachoidids, used by
nesting males to attract females for spawning (e.g. Vasconcelos
et al., 2012). However, Vasconcelos et al. (2010) showed that
Lusitanian toadfish also uses this signal in agonistic contexts during
active nest defence. Although the sex of immature individuals used
in the present study was not determined, it is likely that both males
and females were present in the observation tanks. Our current
findings further support the agonistic role of boatwhistles in this
species, but future work should investigate potential sex-specific
differences and the social role of the vocal repertoire in this species.
Ontogenetic development of periphe ral audito ry sensitivity
Comparisons of auditory sensitivity measured from populations of
hair cells within the saccule across different size Lusitanian toadfish
revealed a threefold ontogenetic increase in sensitivity at most test
Calling rate (calls h
–1
per fish)
7
6
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10
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b
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d
a
a,b
a,b
b
A
C
E
D
B
F
a,b
b
*
***
***
***
3.5
Fig. 2. Variation in the calling rates across
different size groups for each sound type
produced by Lusitanian toadfish. (A) Single
grunt, (B) grunt train, (C) long grunt train,
(D) double croak and (E) boatwhistle. Overall
differences are based on Kruskal–Wallis tests,
followed by pairwise comparison post hoc
tests to verify group-specific differences.
*P<0.05; *** P<0.001. Data are medians
±10th, 25th, 75th and 90th percentiles.
(F) Overall variation in the mean calling rate
(±s.d.) of each sound type across different size
groups. Groups: fry, <2.0 cm SL (N=12–20
fish, 10 sessions); small juveniles, 2.4–4.9 cm
SL (N=17, 10); large juveniles, 5.0–8.69 cm
SL (N=12, 10); adults, 25–35 cm (N=6, 10).
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Journal of Experimental Biology

frequencies. Saccular sensitivity increased by 10 dB between the
smaller (2.4–4.9 cm SL) and the larger juveniles (5.0–8.7 cm),
which already exhibited similar auditory thresholds to adults.
Vasconcelos and Ladich (2008), using the AEP recording
technique, also reported reduced auditory sensitivity in small
juveniles (2.8–3.8 cm SL, equivalent to the smallest size class used
in this study) compared with large juveniles (5.4–6.6 cm SL) and
adults. However, significant differences were only found at 100 Hz
(∼7 dB less) and at higher frequencies of 800 and 1000 Hz (∼15
and 11 dB, respectively). Auditory thresholds recorded using the
AEP technique were considerably lower, name ly 22– 40 dB less
(within about 100–500 Hz), compared with those reported in this
study using the auditory saccular potentials. The AEP recording
technique measures the overall synchronous neural electrical
activity induced by acoustic stimulation and includes the evoked
responses from potentially one or more endorgans (i.e. saccule,
lagena and utricle), the primary afferents of the eighth nerve and
central auditory nuclei (Kenyon et al., 1998). Therefore, the
summed AEP neural response is expected to be more complex and
of different amplitude compared with the receptor-specific
responses reported in this study. Saccular potential recordings
reflect only the receptor potentials recorded from a limited
population of hair cells within the saccular macula close to the
recording electrode.
Our findings are in contrast to those reported from a study
conducted in the batrachoidid P. notatus, which showed via saccular
potential recordings that auditory threshold curves were similar
throughout development (Alderks and Sisneros, 2011) (Fig. 4). The
smallest juvenile midshipman fish tested (2–3 cm SL) had auditory
sensitivity similar to that of large juveniles (7–8 cm SL) and
adults (>9 cm SL), with auditory thresholds varying from ∼115 dB
re. 1 µPa (at 75–145 Hz) to 150 dB (at 945 Hz). Such species-
specific differences within Batrachoididae may be due to their
divergent life histories. An important behavioural difference
between these two species is that P. notatus juveniles do not
reveal aggressive territorial behaviour: they engage in far fewer
social interactions than H. didactylus. The vocal activity of
P. notatus seems to start much later in development, probably
associated with sexual maturity and reproductive behaviours (our
unpublished observations). Moreover, Vasconcelos et al. (2011)
showed that there is a notable difference in seasonal and gender
saccular sensitivity variation in these two species. While in
P. notatus saccular sensitivity is increased during the breeding
season when adult males start vocalising to attract females (Sisneros,
2009), in H. didactylus the sensitivity does not change seasonally and
vocal activity is detected all year round (Vasconcelos et al., 2011).
Studies on the ontogeny of auditory sensitivity in fish have shown
varying results. For instance, Popper (1971) and Zeddies and Fay
(2005), using acoustically evoked behavioural responses, found no
differences among different size goldfish Carassius auratus and
zebrafish Danio rerio, respectively. Although in the latter, acoustic
startle response thresholds were adjusted as the fish develop, in
order to maintain appropriate reactions to relevant stimuli switching
150
140
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110
75 100 200 300 500 800 1000
Small juveniles (25, 27)
Large juveniles (8, 13)
Adults (32, 40)
Small juveniles (17, 18)
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Adults (6, 11)
Frequency (Hz)
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160
100
90
B
A
0
–10
–20
–30
–40
–50
–60
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Fig. 3. Comparison of the mean relative gain and auditory threshold
curves of the evoked potentials recorded from the saccule in Lusitanian
toadfish of different size groups. (A) Comparison of the mean (±s.d.) relative
gain curves based on responses to iso-level pure tones at 130 dB re. 1 μPa.
The iso-level response data were normalised, i.e. a relative value of 0 dB was
assigned to the peak response for each recording and the remaining data for
other frequencies were expressed in relative dB (relative to best frequency). (B)
Comparison of the mean (±s.d.) auditory threshold curves between different
size groups. Number of animals and number of recordings per group,
respectively, are indicated in parentheses. Group sizes: small juveniles, 2.4–
4.9 cm; large juveniles, 5.0–8.69 cm; adults, 25–35 cm, standard length. SPL,
sound pressure level.
150
140
130
120
110
75 100 200 300 500 800 1000
H.d. small juveniles (25, 27)
H.d. large juveniles (8, 13)
H.d. adults (32, 40)
P.n. small juveniles (42, 42)
P.n. large juveniles (20, 20)
P.n. adults (43, 43)
Frequency (Hz)
SPL (dB re. 1 µPa)
Fig. 4. Comparison of mean auditory threshold curves across different
size groups in the Lusitanian toadfish and the midshipman fish. Two
Batrachoidae species are compared: the Lusitanian toadfish (Halobatrachus
didactylus; H.d.) and the midshipman fish (Porichthys notatus; P.n.). Note that
within Lusitanian toadfish, small juveniles differed significantly from large
juveniles and adults, which revealed higher auditory sensitivity and produced
the full vocal repertoire. In contrast, in the midshipman fish, the auditory
sensitivity of different size groups is similar and vocal behaviour has only been
detected in adults (based on Alderks and Sisneros, 2011). Number of animals
and number of recordings per group are indicated in parentheses. Illustrations
represent a typical small juvenile of each species. SPL, sound pressure level.
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Journal of Experimental Biology

from particle motion, at larvae stage, to sound pressure sensitivity,
in juveniles and adults. Yet, Higgs et al. (2003), using AEP
recordings and sound pressure stimulation, reported an expansion of
the maximum detectable frequency from 200 Hz to 4000 Hz with
increasing zebrafish size, which coincided with the development of
the Weberian ossicles and sensitivity to sound pressure.
Most studies conducted on fish, however, have revealed
improvements in hearing abilities throughout development,
similar to our data on the Lusitania toadfish and to other taxa
(e.g. amphibians, Boatright-Horowitz and Si mmons, 1995; reptiles,
Werner et al., 1998; birds, Dmitrieva and Gottlieb, 1994; mammals,
Reimer, 1995). For instance, Kenyon (1996), using behavioural
conditioning techniques, reported ontogenetic increases in auditory
sensitivity in the damselfish (Stegastes partitus)of45dBre.1µPa
at their most sensitive frequency (300 Hz). Wysocki and Ladich
(2001), using the AEP recording technique in different size
croaking gourami (Trichopsis vittata), found an increase in
sensitivity of about 14 dB re. 1 µPa at 0.8–3.0 kHz and a shift in
the best frequency from 2.5 kHz to 1.5 kHz. Likewise, Lechner et al.
(2010, 2011) reported a considerable increase in auditory sensitivity
and shift in the best frequency with development in two catfish
species. Lechner et al. (2010) reported an ontogenetic increase in
Synodontis schoutedeni auditory sensitivi ty of 26 dB re. 1 µPa and
a change in the best frequency range from 2–3 kHz down to
0.3–1 kHz. According to Lechner et al. (2011), aud itory sensitivity
in Lophiobagrus cyclurues increased up to 40 dB re. 1 µPa during
ontogeny and the small juveniles were unable to detect frequencies
higher than 2–3 kHz, whereas large juveniles showed best
sensitivity to higher frequencies of 4–6 kHz. Recently, Caiger
et al. (2013) described an ontogenetic enhancement of auditory
abilities in the hapuka (Polyprion oxygeneios) based on AEP
measurements, i.e. a 22× increase in auditory sensitivity and an
expansion of the auditory bandwidth (from the maximum 800 Hz to
1000 Hz) from larvae to the juvenile stage.
Although relatively few studies have examined the structure–
function relationships in the developing fish ear, increases in
auditory sensitivi ty during ontogeny in fish have been typically
explained as a result of general inner ear development with
continued addition of hair cells (Popper and Hoxter, 1984;
Lombarte and Popper, 1994; Lu and DeSmidt, 2013), number of
auditory nerve ganglion cells, innervation patterns of the eighth
nerve (Corwin, 1983; Popper and Hoxter, 1984) and/or increase in
swimbladder size and development of the morphological
connections (Weberian ossicles ) to the ear (Lechner et al., 2011).
Another possibility could be the otolith enlargement with fish
growth, providing increased inertial mass for the accelerometer-like
auditory endorgan (Gauldie, 1988).
Future work should identify potential morphological differences
in the inner ear throughout ontogeny in the Lusitanian toadfish, with
particular focus on early developmental stages (e.g. hair cell
addition and density; otolith size and density) that may contribute to
the changes observed in the peripheral auditory sensitivity. This
species does not present accessory morphological hearing structures
and the swimbladder shape may actually shield the nearby ears
during sound production (Barimo and Fine, 1998). Nevertheless,
the potential effect of the swimbladder on auditory sensitivity
especially at lower frequencies has yet to be investigated.
Parallel development o f vocal–auditory systems
In this study we show that the developmental stage, when large
juveniles start producing the full adult vocal repertoire, is coincident
with a significan t enhancement in auditory saccular sensitivity. Our
data provide clear evidence that the development of the vocal motor
control system parallels the development of the peripheral auditory
system in a vocal fish species. We sugg est that this parallel
development may serve an important and conserved function in the
development of social acoustic communication among vertebrates.
Juveniles of vocal vertebrate species often gradually transform
primitive unstructured vocalisations into complex, stereotyped calls
that constitute the adult repertoire (e.g. birds, Tchernichovski et al.,
2001; Derégnaucourt et al., 2009). Among birds and mammals,
previous research showed that auditory feedback is crucial during
the vocal differentiation phase and that the acoustic signals
perceived from the auditory–vocal environment play an important
role in the refinement of the neural connections within the vocal
circuitry (e.g. Miller-Sims and Bottjer, 2012). The onset of coupling
between the vocal motor and auditory system throughout the
evolution of vertebrates remains unknown. Future studies should
investigate the potential coupling of auditory–vocal systems in fish
by testing whether early acoustic experience can shape vocal
behaviour. Such studies have the potential to ultimately shed light
onto the mechanisms and evolution of vocal behaviour common to
all vertebrates (Bass et al., 2008).
MATERIALS AND METHODS
Animals
Toadfish were collected in Portugal, mostly from the Tagus River estuary
but also from the Mira River estuary (only the small and large juveniles used
for auditory measurements were collected from this location). Collections
were carried either by trawling or directly from an intertidal nesting area
from February to June. Although we are aware of size differences among
populations from Tagus and Mira (Costa, 2004), this study mostly focuses
on comparisons of either auditory sensitivity or sound production among
animals from the same population.
Based on previous behavioural observations and auditory information
(Vasconcelos and Ladich, 2008), fish were classified into four different size
groups: fry, 1.7–2.00 cm standard length (SL), 0.19–0.30 g body mass;
small juveniles, 2.4–4.9 cm SL, 0.60–3.10 g; large juveniles, 5.0–10.60 cm
SL, 3.17–15.02 g; and adults, 25–35 cm SL, 279–651 g. Fish within these
size ranges, with the exception of the smallest group (fry: N=12–20) that
was only studied in terms of vocal behaviour, were either used for saccular
sensitivity measurements (small juveniles: N=25; large juveniles: N=8;
adults: N=32, including 27 type I or nest-guarding males and 5 females) or
vocal recordings (small juveniles: N=17, large juveniles: N=12, adults:
N=6). Note that type I males nest under rocks in the breeding season from
where they vocalize in choruses to attract gravid females and care for the
offspring, in contrast to the smaller and less vocal type II or sneaking males,
which attempt opportunistic fertilisation (Modesto and Canário, 2003b).
The juvenile groups used in this study represent early development stages
and were probably less than one year old (Pereira et al., 2011). The
Lusitanian toadfish reaches sexual maturity at 30–35 cm total length (around
4–6 years old) in females and both male morphotypes (Pereira et al., 2011).
All fish, with the exception of the adults recorded in the field, were
transported to the laboratory at the University of Lisbon (Lisbon, Portugal)
where they were kept in stock aquaria ( juveniles in 50–60 litre tanks; adults
in 80 litre tanks) and maintained at 21±2°C under a 12 h light:12 h dark
cycle. Vocal–acoustic recordings of fry and juveniles were carried out in the
same laboratory, while those of adults were performed in semi-natural
conditions in the intertidal area. For saccular potential recordings, both small
and large juveniles and adults were shipped to the University of Washington
(Seattle, WA, USA), where they were maintained in 40 litre and 80–250 litre
tanks, respectively, under similar light and temperature conditions.
After auditory recordings fish were killed by immersion in a 0.025% ethyl
p-aminobenzoate saltwater bath. For the adults, both sex and male types
were confirmed (Modesto and Canário, 2003b).
Electrophysiological experiments followed National Institutes of Health
guidelines for the care and use of animals and were approved by the
University of Washington Institutional Animal Care and Use Committee.
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All behavioural experimental procedures comply with Portuguese animal
welfare laws, guidelines and policies. All animals used in this study,
including those shipped from Portugal to the University of Washington
(USA), adapted rapidly to captivity and behaved normally in the stock tanks,
which suggests that these animals were not exposed to overly stressful
conditions.
Sound recordings
In order to investigate vocal differentiation among fry and juveniles, a group of
each size class was placed in 50–60 litre recording tanks (fry: N=12–20, small
juveniles: N=17, large juveniles: N=12 fish) provided with several shelters
(halved ceramic flower pots), sand substrate, and maintained at 22±2°C.
Two hydrophones (High Tech 94 SSQ, Gulfport, MS, USA; frequency range:
30 Hz–6 kHz ±1 dB; voltage sensitivity: −165 dB re. 1 V μPa
−1
) were placed
in the middle of each recording tank at about 10–20 cm apart and ∼1–2cm
from the bottom. Hydrophones were high pass filtered (>10 Hz) for
decoupling the DC component, and connected to an audiocapture device
Edirol UA-25 (Roland, Osaka, Japan; 16 bit, 44.1 kHz acquisition rate per
channel) and then to a laptop, to perform double-channel recordings down-
sampled to 8 kHz by Adobe Audition 3.0 (Adobe Systems Inc., San José, CA,
USA). Sound recordings were performed for 60 min. Fish behaviour was
monitored throughout the recording session in order to verify the behavioural
context of sound production. Food was provided at ∼30 min from the start
of recording to stimulate social interactions and sound production. A total of
10 consecutive sessions (1−2 per day) were carried out for each group.
The vocal repertoire of toadfish adults was recorded under semi-natural
conditions following the method described by Vasconcelos et al. (2010,
2012). Briefly, during the toadfish breeding season (May–June), males that
spontaneously occupied six concrete shelters placed close to the lower limit
of an intertidal area of the Tagus estuary were recorded with a similar audio
chain as above: a High Tech 94 SSQ hydrophone placed next to each nest,
connected to an audiocapture device (M-Audio Fast Track Ultra 8R,
Cumberland, RI, USA) and then to a laptop controlled by Adobe Audition
3.0. Nest entrances were closed with a plastic net to prevent fish from
escaping and to ensure male identity throughout the recordings. Plastic nets
did not affect acoustic signals and allowed both the entrance of small
prey and possible interactions with free-swimming conspecifics. We
continuously recorded six type I adult males for 10 days and randomly
selected a 1 h session from each day for sound analysis.
Sound recordings were analysed using Raven 1.2 for Windows
(Bioacoustics Research Program, Cornell Laboratory of Ornithology,
Ithaca, NY, USA). Toadfish vocalisations were identified based on
previous works (Amorim et al., 2008; Vasconcelos et al., 2010). Sounds
were classified into: single grunts, grunt trains, long grunt trains, double
croaks and boatwhistles. In this study, croak-like sounds were classified as
single grunts because in juveniles dominant frequencies are higher and single
grunts are typically longer (Vasconcelos and Ladich, 2008). This makes it
difficult to distinguish single grunts, commonly produced among juveniles,
from croaks previously reported in adults (Amorim et al., 2008).
The calling rate for each sound type was determined per hour/session and
then aver aged per gr oup for all size classes. In the recordings of fry and
juveniles conducted in tanks, it was sometimes not clear which individual was
vocalising. Therefore, calling rates were calculated based on the total number
of sounds divided by the number of individuals present in the tank.
Although sound production was pooled from single groups for each size
class, all animals used in this study were active and displayed social
behaviour towards conspecifics, including the adults that attracted females
to their nests. Therefore, we are confident that our sound recordings reflect
size-specific socially relevant vocal activity. Also, data collected on sound
production were not intended to thoroughly describe the association
between specific vocalisations and the behavioural context, as this would
have required a larger sample size. Our intent was instead to detect broad
size-related differences in the vocal repertoire that gradually occur with fish
development.
Measuring peripheral auditory sensitivity
We used the evoked saccular potential recording method to measure
auditory thresholds of populations of hair cells within the saccule based on
previous studies (Sisneros, 2007; Vasconcelos et al., 2011). Briefly, fish
were first anaesthetised in a 0.025% ethyl p-aminobenzoate saltwater bath
and then immobilised by an intramuscular injection of pancuronium
bromide: juveniles, ∼0.5 mg kg
−1
; adults, 2–4mgkg
−1
. The saccule was
then exposed by dorsal craniotomy and a barrier of denture cream was built
up around the cranial cavity to allow the fish to be lowered below the water
surface. A teleost ringer solution was used to prevent the cranial cavity from
drying out and to dilute any bleeding.
Test subjects were placed in a round saltwater-filled tank (30 cm
diameter, 24 cm high) and positioned 10 cm above an underwater speaker
(UW-30, Telex Communications, Burnsville, MN, USA) that was
embedded in gravel in the centre of the tank. During experiments, fresh
seawater (at 21±1°C) was perfused through the fish’s mouth and over the
gills. The recording tank was placed on a vibration-isolated table housed
inside an acoustic attenuation chamber (Industrial Acoustics, New York,
NY, USA) with all the recording and stimulus generation equipment located
outside the chamber.
Acoustic stimuli were generated via the reference output signal of a lock-
in amplifier (SR830, Stanford Research Systems, Sunnyvale, CA, USA) that
passed the stimulus signal through an audio amplifier to the underwater loud
speaker. Prior to each experiment, we tested the speaker’s frequency
response by placing a mini-hydrophone (8103, Bruel and Kjaer; Naerum,
Denmark) in the position later occupied by the fish’s head, and then
measured the peak-to-peak voltage on an oscilloscope. This peak-to-peak
voltage was used in conjunction with an automated compensation script
written for Matlab (MathWorks, Natick, MA, USA) to calibrate the speaker
so that pressure level at all test frequencies was of equal amplitude within
±2 dB re. 1 µPa. This calibration procedure compensates for any resonant
frequencies of the tank to ensure that all test frequencies used were of equal
amplitude within 2 dB (i.e. the amplitude response of the speaker within the
tank was flat across all test frequencies). Sound pressure measurements of
the stimulus frequencies were measured using a spectrum analyser (Stanford
Research Systems SR780). Auditory stimuli consisted of eight repetitions of
single 500 ms tones from 75 Hz to 945 Hz (in 10–80 Hz increments)
presented randomly at a rate of one every 1.5 s.
Saccular potentials were recorded with glass electrodes filled with
3 mol l
−1
KCl (0.5–6MΩ). Electrodes were visually guided and placed in
the middle region of the saccular macula in either the left or right saccule.
Analog saccular potentials were preamplified (5A microelectrode amplifier,
Getting Instruments, San Diego, CA, USA), input into a lock-in amplifier
(SR830, Stanford Research Systems) and then stored on a computer running
a custom data acquisition Matlab script. The lock-in amplifier yields a DC
voltage output that is proportional to the component of the signal whose
frequency is locked to the reference frequency. The reference frequency was
set to twice the stimulation frequency, the amplifier sensitivity set to 50 mV
and the time constant to 100 ms. We used the 2nd harmonic of the stimulus
(twice the stimulus frequency) as the reference frequency because the
greatest evoked potential from the saccule in teleost fishes typically occurs at
twice the stimulus frequency due to the nonlinear response and opposite
orientation of hair cell populations within the saccule (Cohen and Winn,
1967). To estimate auditory thresholds, the saccular potentials were
recorded in response to single tone stimuli that were reduced in 3 dB
steps until the saccular response (mean voltage of eight evoked saccular
potential measurements) was no longer above background noise (mean
voltage measured without acoustic stimulation) ±2 s.d.
Background noise measurements were performed prior to the recording of
each threshold-tuning curve. Noise measurements followed the same
procedure of saccular potentials recordings except with the loud speaker
turned off. The background noise levels were consistently measured from
the recording electrode between 2–5µV.
Although toadfishes possess no hearing specialisations and thus are
primarily sensitive to particle motion (Fay and Edds-Walton, 1997), we
report in this study auditory thresholds based on sound pressure levels
(SPLs) for both technical reasons and comparison purposes with previous
studies using batrachoidid fishes (e.g. Sisneros, 2007; Alderks and Sisneros,
2011; Vasconcelos et al., 2011). Our aim was to compare the saccular
sensitivity between different size groups under identical experimental
conditions. Therefore, the auditory thresholds presented should not be
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RESEARCH ARTICLE Journal of Experimental Biology (2015) 218, 2864-2872 doi:10.1242/jeb.123059
Journal of Experimental Biology

considered as absolute values but instead as reliable data to perform
quantifiable comparisons between the different size/age groups.
Statistical analysis
Saccular sensitivity threshold curves and normalised iso-level curves from
different size groups were compared by repeated-measures ANOVA,
analysing auditory thresholds or iso-level responses to several frequencies
(75–425 Hz) in each fish (within subject factor) of different size groups
(between-subject factor). LSD post hoc tests were used to verify pairwise
group-specific differences. The threshold values at the best frequency (BF,
defined as the frequency that evoked the lowest saccular potential threshold)
were compared across groups with one-way ANOVA.
To analyse the development of the acoustic repertoire, calling rates for
each sound type from different size groups were compared with Kruskal–
Wallis, followed by pairwise comparison post hoc tests to verify group
specific differences. Parametric tests were used only when data were
normally distributed and variances were homogeneous. The statistical
analysis was performed using IBM SPSS v22 (IBM, Edicott, NY, USA) and
Statistica 7.1 (StatSoft, Tulsa, OK, USA).
Acknowledgements
We would like to thank the Air Force Base No. 6 of Montijo (Portugal) for allowing us
to conduct sound recordings of toadfish adults in their military establishment. We are
grateful to Daniel Alves and Patrı
́
cia Chaves for helping with fish sampling and sound
recordings, respectively.
Competing interests
The authors declare no competing or financial interests.
Author contributions
R.O.V. was responsible for conception of the study and experimental design,
performed most of data collection and analysis, and drafted the article. P.W.A.
contributed to auditory data collection and scientific drawings. A.R. contributed to
vocal data collection. P.J.F., M.C.P.A. and J.A.S. were involved in the conception of
the study and supervision of the experimental work. All authors revised the
manuscript prior to submission.
Funding
This study was supported by Fundo para o Desenvolvimento das Ciências e da
Tecnologia, Macau S.A.R. [ project 019/2012/A1]; Fundaça
̃
o para a Ciência e a
Tecnologia, Portugal [grants SFRH/BD/30491/2006 to R.O.V. and SFRH/BPD/
41489/2007 to M.C.P.A.; pluriannual programmes UI&D 331/94/FCT and UI&D
329/FCT]; and Royalty Research Fund, USA (grant from the University of
Washington to J.A.S.).
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