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Agonistic sounds signal male quality in the Lusitanian toadsh
M. Clara P. Amorim
, Amparo Gonçalves
, Paulo J. Fonseca
MARE Marine and Environmental Sciences Centre, ISPA Instituto Universitário, Lisbon, Portugal
Centro de Ciências do Mar, Universidade do Algarve, Campus de Gambelas, 8000-810 Faro, Portugal
Division of Aquaculture and Upgrading, Portuguese Institute for the Sea and Atmosphere, I.P. (IPMA, I.P.), Lisbon, Portugal
Departamento de Biologia Animal and cE3c Centre for Ecology, Evolution and Environmental Changes, Faculdade de Ciências, Universidade de Lisboa, Lisbon, Portugal
Acoustic communication during agonis-
tic behaviour is widespread in shes.
Breeding Lusitanian toadsh males de-
fend nests from intruders with sounds.
Low fundamental frequency (high
mean pulse period) reected high an-
drogen levels.
The dominant harmonic frequency de-
creased with sonic muscle lipid content.
Spectral content of sh sounds signal
male traits that are key to ght out-
abstractarticle info
Article history:
Received 26 February 2015
Received in revised form 27 May 2015
Accepted 1 June 2015
Available online 3 June 2015
Acoustic communication
Temporal cues
Spectral cues
Steroid hormones
Territorial defence
Acoustic communication during agonistic behaviour is widespread in shes. Yet, compared to other taxa, little is
known on the information content of sh agonistic calls and their effect on territorial defence. Lusitanian toadsh
males (Halobatrachus didactylus) are highly territorial during the breeding season and use sounds (boatwhistles,
BW) to defend nests from intruders. BW present most energy in either the fundamental frequency, set by the
contraction rate of the sonic muscles attached to the swimbladder, or in the harmonics, which are multiples of
the fundamental frequency. Herewe investigated if temporal andspectral features of BW producedduring terri-
torial defence reect aspects of male quality that may be important in resolving disputes. We found that higher
mean pulse period (i.e. lower fundamental frequency) reected higher levels of 11-ketotestosterone (11KT), the
main teleost androgen which, in turn, was signicantly related with male condition (relative body mass andgly-
cogen content). BW dominant harmonic mean and variability decreased with sonic muscle lipid content. We
found no association between BW duration and malequality. Taken together, these results suggest that thespec-
tral content of sh agonistic sounds may signal male features that are key in ght outcome.
© 2015 Elsevier Inc. All rights reserved.
1. Introduction
Animals' interactions, such as conicts over limited resources, are
mediatedby the exchange of signals that provide information for adap-
tive decision making [1]. Territorial defence is often a key component of
an individual's tness and includes both signalling territorial ownership
Physiology& Behavior 149 (2015) 192198
Corresponding author.
E-mail addresses: (M.C.P. Amorim),
(C. Conti), (T. Modesto), (A. Gonçalves), (P.J. Fonseca).
0031-9384/© 2015 Elsevier Inc. All rights reserved.
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and chasing away intruders. In this context, signals should provide
species/sex-specic information to inform potential intruders of the
owner's presence on site, and also provide information on ghting abil-
ity and motivation for territorial confrontations [1]. Hence, information
content of territorial signals is expected to depend on whether a signal
serves only for territorial ownership advertisement, to reveal the
sender's quality and motivation, or both [1].
Multiplestudies show that vocalizations can inuence the resolution
of contests as they reect the sender's body size or condition (e.g.
[24]). For example, low frequency vocalizations may give a reliable
indication of a large body size and deter potential attackers [2] or, in the
case of humans, inuence the perception of leadership capacity [5].In
sh, the largest group of vertebrates exhibiting widespread evolution
of sound production [6], acoustic signals are often produced during con-
tests, including territorial defence [7]. In this taxon there is evidence for
salient information content in thetemporal pattern of sound pulses [6].
In addition, there is a strong selection pressure favouring complexity in
the spectro-temporal content of vocal signals [8]. Surprisingly, little is
known on whether temporal and spectral features of agonistic calls re-
ect the sender's quality and affect territorial defence (e.g. [7,9,10]). Im-
portantly, while it has been shown that in some shes larger individuals
produce longer, louder and lower frequency agonistic sounds [1114],
few studies have provided an integrative view on how agonistic sounds
reect multiple aspects of male quality such as physical and physiologic
Fishes from the Batrachoididae family have become a key
neuroethological model for studying acoustic communication in verte-
brates because mate attraction and territorial defence in theses shes
rely heavily on acoustic signalling [1517]. While mating advertisement
calls (boatwhistles and hums) produced by batrachoidid nest-holders
have been also implied in signalling territorial ownership and in spacing
out individuals [9,16,18], grunts are considered the main agonistic call [8,
15]. Within this family, Lusitanian toadsh (Halobatrachus didactylus)
males mainly defend their nests with agonistic boatwhistles (BW) that
are similar to mating advertisement BW except for presenting lower
dominant frequencies and weaker amplitude modulation [16].AsBW
have shown to be rather complex signals [8] they render the opportunity
to assess the salience of relevant sound features for territorial defence in
teleosts. Typically, BW are relatively long multiharmonic signals wherein
the fundamental frequency is determined by the ring rate of the vocal
acoustic neural network that drives the contraction rate of the paired
sonic muscles attached to the swimbladder [10].
Here we relate spectral and temporal features of the agonistic BW
with Lusitanian toadsh male quality, including biometric, condition,
and physiological features. We predictthat BW acoustic features convey
information on male quality including energetic reserves andhormonal
status which inuence the outcome of contest behaviour [19].
2. Methods
2.1. Subject males and maintenance
The Lusitanian toadsh, like other batrachoidids, has two xed male
reproductive phenotypes that differ ina suite of morphological and neu-
roendocrine traits [10,20]. Type I males nest under rocks or in crevices,
are territorial, vocalize to attract mates and provide parental care to the
eggs in the nest. In contrast, Type II males are not territorial and sneak
fertilizations [20].
We captured territorial males that spontaneously occupied articial
concrete shelters placed in the lower intertidal of the Tagus River es-
tuary (Military Air Force Base 6, Montijo, Portugal; 38° 42N; 8° 58W).
Fish use these shelters as nests during the breeding season that spans
from May to July in Portugal (e.g. [17]). We used plastic round
swimming-pools (2.5 m diameter, 0.5 m water depth) as stock and ex-
perimental tanks. Tanks were placed on the sand just above the high
tide shoreline, near the collection area and under a shadow net cover
held 170 cm high to prevent excessive solar radiation and water
heating. Water temperature varied in all tanks from 18 to 26 °C
(mean = 21.4 °C), within the range of the estuary water temperature
variation during the same period. Tank water was renewed every
23 days, by pumping directly from the estuary. A natural light cycle
was maintained as tanks were outdoors.
2.2. Territorial experiments
We carried out territorial intrusion experiments following
Vasconcelos et al. [16]. Briey, 24 h before trials two subject males
were placed in an experimental tank provided with two roof tiles (in-
ternal dimensions 44 cm × 18 cm × 10 cm) placed 50 cm apart and
20 cm away from the tank's border. In each 1 h trial, two intruder
males were placed sequentially in the experimental tank, one at the
start of the trial and the second 30 min after, both remaining in the
tank until the end of the trial (Supplementary material, Video 1). This
experimental design resembles the natural chorusing aggregations,
where territorial males nest close together [21] and may sequentially
attract competitor males [17]. Visual and acoustic behaviours were
monitored during trials and noted in detail on paper. Fish were marked
with small cuts between the n rays to ensure their identity. Marking
did not cause any measurable change in the sh behaviour. Fish were
never used in more than one trial.
We placed one hydrophone (High Tech 94 SSQ, High Tech Inc.,
Gulfport, MS, USA; frequency response: 30 Hz to 6 kHz ± 1 dB; voltage
sensitivity: 165 dB re. 1 V/μPa) in front of each nest, at about 10 cm
from its entrance and from the tank bottom, attached to a wooden rod
kept over the tank. Simultaneous two channel recordings were made
with a USB audio capture device (Edirol UA-25, Roland, Osaka, Japan;
16 bit, 44.1 kHz acquisition rate per channel) connected to a laptop
and down-sampled to 6 kHz by Adobe Audition 3.0 (Adobe Systems,
San José, CA, USA). Recorded sounds could be attributed to a particular
territorial male (henceforth named nest-holder) due to the close prox-
imity of each hydrophone to one nest. Also, in the course of similar ex-
periments (e.g. [16]) we have observed that only nest-holders produce
sounds. In one exception (unpublished data), the intruder entered the
nest and also made BW but these could clearly be distinguished from
the nest-holder's sounds due to spectral differences.
At the end of the 19 trials, residents (n= 38) and intruders (n=
34) that engaged in social interactions were measured to the nearest
mm for total length (TL) and to the nearest g for total body mass (M).
Note that in four trials one of the intruders did not interact with nest-
holders. Residents measured mean ± sd (range) 42.0 ± 3.0 (37.549.5)
cm in TL and intruders 36.9 ± 2.7 (30.042.0) cm in TL. Residents pro-
duced on average 4.7 ± 5.9 (range: 022, mode = 1) BW during trials.
For this study we onlyconsidered resident males that made atleast 4
BW (n= 14; note that one male only had 2 BW suitable for analysis).
These nest-holders made an average of 10.4 ± 6.4 (422) BW during
a 1 h trial. All resident males not used for sound analysis and the in-
truders were returned to the estuary after trials. Territorial males that
made fewer than 4 BW did not differ in TL from more vocal males (t
test, pN0.05). They likely made fewer sounds because they experienced
fewer interactions with intruders than the males that were more vocal
(t= 3.34, d.f. = 36, pb0.01, mean no. of interactions = 4.9 vs. 2.8 for
more vocal vs. less vocal sh).
The 14 males used to relate sounds with male quality averaged
41.9 ± 1.8 (39.346.0) cmin TL and 1221 ± 199 (9701600) g in M. Im-
mediately after theend of trials, these males were sacriced with an ex-
cessive dosage of MS222 (tricaine methane sulphonate; Pharmaq,
Norway) buffered (1:1) with sodium bicarbonate. Blood samples were
collected from the caudal vein in heparinized syringes within 4 min
from rst handling of the sh. Plasma samples were subsequently sep-
arated by centrifugation (6000 rpm for 5 min) on site and stored on
ice until taken to the lab, where they were stored at 20 °C until steroid
quantication. Following blood sampling, males were immediately
193M.C.P. Amorim et al. / Physiology & Behavior 149 (2015) 192198
dissected and the mass of the gonads (M
), the liver (M
), and of the
sonic muscles (M
) was weighed to the nearest mg, and eviscerated
body mass to the nearest g (ME). A sample of body muscle (epaxial
muscle bres) was taken. Samples of body and sonic muscles were im-
mediately placed in ice and in dry ice until stored in the lab at 20 °C
and 80 °C for subsequent quantication of the lipid and glycogen con-
tent, respectively. Lipids and glycogen are important sources of energy
in sh and are often used as a direct measure of body condition [22].
They are also important metabolic substrates of sonic muscle activity,
i.e. sound production (e.g. [23]).
2.3. Lipid and glycogen quantication
Lipid content was measured following Olsson et al. [24].Muscle
samples were desiccated at 60 °C for 24 h and were weighed on a
scale (Sartorius RC210D, Göttingen, Germany) to the nearest 0.01 mg.
Lipids were extracted in 100 ml of petroleum ether (Sigma-Aldrich, St.
Louis, MO, USA) for 8 h. Relative body lipid content of each male was
measured as the difference in dry weight before and after lipid extrac-
tion expressed as g/100 g tissue dry weight.
Glycogen content was determined according to the method de-
scribed by Viles and Silverman [25]. Dry samples (25 mg) were
boiled with 15 ml of 33% potassium hydroxide for 15 min. After
cooling, 0.5 ml was taken and 50 μl of a saturated sodium sulphate solu-
tion and 2 ml of ethanol were added. Samples were placed in an ice bath
for 30 min for glycogen precipitation. Following centrifugation the pre-
cipitate was dissolved in 0.5 ml of distilled water again precipitated
with 1 ml of ethanol (for 30 min) and centrifuged. The precipitate was
re-dissolved in 0.4 ml of distilled water and 3 ml of anthrone-reagent
was added (prepared with concentrated sulphuric acid, water and
anthrone Merck 101468). This mixture was heated at 90 °C for
20 min. The absorbance was then measured at 620 nm. A calibration
curve was prepared using glycogen type II from oyster (Sigma G8751)
as a standard. The results were expressed as μg/mg tissue dry weight.
2.4. Hormone quantication
Plasma levels of testosterone (T), 11 keto-testosterone (11KT) and
cortisol (CORT) were measured by radioimmunoassay (RIA). 11KT and
CORT are respectively the predominant androgen and corticosteroid in
teleost sh, including the Lusitanian toadsh [20]. Previously to the as-
says, 100 μl plasma samples were extracted for free and conjugated ste-
roids using the techniques described in Scott and Canário [26] and
Damasceno-Oliveira et al. [27]. RIA methodology and cross reactions
of antisera for T, 11KT and CORT were described in Vasconcelos et al.
[17]. The results of the different fractions (free and conjugated) were
summed. Intra-assay and inter-assay precision (coefcient of variation)
was 6.3% and 5.2% for T, 3.9% and 5.5% for 11KT, 7.8% and 10.3%for CORT,
2.5. Behavioural and sound analyses
We tallied the number of agonistic residentintruder interactions,
which started with the intruder's approach (when the intruder reached
within a body length from the nest) and partial or total intrusions in the
nest (hereafter named intrusions). For resident sh we tallied the fre-
quency of agonistic escalated behaviours (escalated ght, EF) such as
chasing, bite attempts, bites and mouthmouth ght. Non-escalated vi-
sual behaviour such as approach or mouth opening with the extension
of ns was not observed. We also considered the number of emitted
BW by the resident.
Acoustic analysis was performed using Adobe Audition 3.0 and
Raven 1.2 for Windows (Bioacoustics Research Program, Cornell Lab-
oratory of Ornithology, Ithaca, NY, USA). We measured BW duration
(Dur, ms), from the start of the rst pulse to the end of the last pulse,
and pulse period (PP) of the BW tonal phase [cf. 28] (ms, average
peak-to-peak interval of six consecutive pulses in the middle of this
segment Fig. 1a). We calculated the mean and coefcient of variation
(CV, CV = SD / mean) for the BW duration and pulse period per individ-
ual. Because the dominant frequency (frequency with the highest ener-
gy component in the BW tonal phase) can be in either the fundamental
frequency (H0) or one of its harmonics (H1 to H3) [28], we further cal-
culated the most common dominant harmonic for each sh (i.e. the
mode). We then averagedthe observed frequency values for the sounds
that presented that harmonic as the dominant frequency, hereafter
named dominant harmonic (DH, Hz; Fig. 1a). As a measureof its variabil-
ity we used the ratio between the number of sounds that presented the
DH by the total number of sounds. This ratio indicates if a male produces
BW with the dominant frequency always in one particular harmonic
(DH = 1) or in different harmonics (DH b1). Temporal patterns were
measured from the oscillogram and frequency measures from the
power spectrum (sampling frequency 8 kHz, Hamming window, lter
bandwidth 10 Hz). We analysed an average ± sd (range) of 7.3 ± 2.5
(410) BW per male.
2.6. Statistical analyses
We investigated whether the number of BW or the number of EF
made by nest-holders was correlated with the number of intruder inter-
actions (only intrusions in the case of EF) and with the nest-holder's
quality (see below) with Pearson correlation. We tested if BW acoustic
parameter (Dur mean, Dur CV, PP mean, PP CV, DH, DH variability)
reected nest-holder's features with linear regression analyses, exclud-
ing the male for which only 2 BW were analysed. Dependent variable,
i.e. acoustic parameters were not correlated (Pearson correlation,
pN0.05). We log
-transformed the dominant harmonic mean and var-
iability to meet the assumption of normality.
We considered the following descriptors for resident quality: steroid
levels (T, 11KT, CORT), lipid content of somatic and sonic muscles
(LipidM and LipidSM, respectively), glycogen content of somatic and
sonic muscles (GlycM and GlycSM, respectively), and total length
TL). We also used the residuals of the simple linear regression of
sonic muscle, gonads and liver mass on eviscerated body mass (RM
, respectively) as metrics of these parameters controlled for
the inuence of body size. For example a high positive residual of
indicates a heavier than average sonic mass for a given body
size. In addition, we used the residuals of ME on TL (COND) as a metric
of body condition. 11KT, GlycM and COND were positively correlated
with each other (Pearson correlation, r=0.750.77, pb0.01). LipidSM
was also related with RM
(r=0.57,pb0.05). The remaining male de-
scriptors were unrelated. To avoid multicollinearity and to reduce the
number of explanatory variables we excluded GlycM, COND and RM
from analyses. We further excluded T as it is physiologically related
with 11KT [20].
Our nal regression models complied with all assumptionsof multi-
ple linear regression. All model residuals were normally distributed.
Further residual analyses were performed using DurbinWatson statis-
tics, residual plots as well as multicollinearity tests (variance ination
factors, VIF). All statistical analyses were performed using SPSS for Win-
dows (20.0, SPSS Inc., Chicago, IL, USA).
3. Results
Nest-holders experienced one to nine interactions with intruders
(mean ± SD = 4.6 ± 2.2), from which 46.2% were approaches, 4.6%
were approaches followed by intrusions, and 49.2% were direct partial
or total intrusions. None experienced a nest takeover. Nest-holders
only responded to intruders' approaches with BW (Fig. 1b). During in-
trusions nest-holders reacted with BW or with BW followed by EF
(when the intruder would not ee upon a BW). In a few occasions, sub-
ject males did not apparently react, i.e. did not show a visible change in
visual or acoustic behaviour, or immediately escalated their behaviour
194 M.C.P. Amorim et al. / Physiology & Behavior 149 (2015) 192198
(Fig. 1b). The number of BW was correlated with the number of interac-
tions (r=0.61,pb0.05) but not with male features. The number of EF
was in turn only correlated with own body glycogen levels (r=0.56,
Agonistic BW Dur averaged 640 ms (range 437933 ms) and pre-
sented a relatively high coefcient of variation (mean = 0.3, range
0.10.5). PP averaged 23 ms (range 19.726.9 ms) and was more stereo-
typed (mean CV = 0.05, range 0.010.07). DH was most commonly
found on the rst harmonic (DH mean = 89 Hz, range 41146 Hz),
and on average males maintained a particular dominant frequency
band in 70% of BW, i.e. DH variability averaged 0.7 (range 0.51.0).
Only three out of 13 males produced BW always with the same domi-
nant frequency, i.e. with DH variability = 1.
We asked whether these acoustic parameters reect male quality.
Both Dur mean and Dur CV were not signicantly related with any
male quality variable (regression analyses, pN0.05). However, several
male quality variables were signicantly associated with pulse period
and spectral characteristics of BW. PP mean increased with 11KT
= 0.63) and decreased with relative gonad mass (RM
Table 1,Fig. 2a,b). PP CV was negatively affected by CORT (R
= 0.37;
Fig. 2c). DH mean was negatively associated with LipidSM (R
Table 1,Fig. 3a). DH variability decreased with LipidSM (R
= 0.52)
Rel. amplitude (dB)
0.4 0.60.20
Frequency (kHz)
Sound duration
6 PP
100 ms
5.7 5.7
Fig. 1. Nest-holder territorial defence behaviour. (a) Oscillogram (left) and power spectrum (right) of an agonistic boatwhistle, the main response to intruders. Sound duration (thick
dashedline), six pulse periods (6PP,solid line) and dominant harmonic (DH)are depicted. (b)Proportion (%) ofthe nest-holder'sreactions to intruder approac hes (n= 33) and intrusions
in the nest (n=35).BWemission of boatwhistles; BW + EF boatwhistles followed by escalated ght; EF escalated ght; NR no reaction.
Table 1
Results of linear regression analyses assessing the association of male quality with boatwhistle features. Re gression mo dels for boa twhistle duration (mean and CV) were not
signicant (rpartial correlation between the dependent variable and the predictor, controlling for the effects of the other predictors in the model. DW Durbin Watson sta-
tistics, VIF variance ination factor.)
Dependent variable Included predictor BSEM tprF Model signicance R
PP mean Intercept 19.74 0.50 39.27 b0.001
11KT 1.87 0.30 6.21 b0.001 0.91 1.02
1.15 0.33 3.45 0.009 0.77 F
= 22.88 pb0.001 0.85 2.14 1.02
PP CV Intercept 0.07 0.01 5.53 b0.001
CORT 0.007 0.003 2.27 0.049 0.60 F
= 5.17 p= 0.049 0.37 2.80 1.0
DH mean
Intercept 2.15 0.10 21.19 b0.001
LipidSM 0.037 0.02 2.45 0.03 0.59 F
= 5.98 p= 0.03 0.35 1.73 1.0
DH Intercept 0.32 0.01 23.61 b0.001
LipidSM 0.013 0.002 6.46 b0.001 0.93 1.13
0.015 0.01 2.84 0.03 0.73
0.017 0.01 2.63 0.03 0.71 F
= 16.02 p= 0.002 0.87 2.21 1.14
Data were log
195M.C.P. Amorim et al. / Physiology & Behavior 149 (2015) 192198
and with RM
= 0.18), but increased withRM
=0.13,Table 1,
Fig. 3bd).
4. Discussion
The Lusitanian toadsh used BW as the primary response to in-
truders, both during approaches and while facing actual intrusions in
the nest (Fig. 1). Nest-holders typically only resorted to escalated ght
when opponents did not ee after rst receiving BW, and only after
the intruder entered the nest. These results are consistent with BW sig-
nalling territorial ownership and serving as a warning of a possible at-
tack. Congruently, BW have been suggested as deterrent signals in
territorial intrusions [16]. In addition, BWmay also function in resource
holding potential assessment, if acoustic features relate to ghting abil-
ity [19].
We found that circulating steroid hormones were associated with
pulse period mean and CV. Males with elevated levels of 11KT pro-
duced BW with higher mean pulse period, i.e. with lower fundamen-
tal frequency [4]. In turn, 11KT was positively correlated with male
condition (relative body mass and glycogen content). Males with
higher CORT levels produced BW with less variable fundamental fre-
quencies (lower PP CV). Our results are consistent with the well-
known role of sex and stress steroids as regulators of acoustic signals
used during social behaviour [10,29]. Steroids not only shape vocal
control circuits in sh but also modulate vocal motor patterning
through rapid non-genomic effects on the activity of the hindbrain
vocal central pattern generator [30]. Such effects have been exten-
sively shown in other batrachoidids. In the plainn midshipman
sh, Porichthys notatus, intramuscular injections of 11KT and CORT
rapidly increase the durations of ctive calls but do not affect funda-
mental frequency [31]. 11KT and CORT administered exogenously
via implanted food items, also increase calling rate of mating calls
in territorial Gulf toadsh, Opsanus beta [32]. While our study points
to different effects of 11KT and CORT on BW features they are consis-
tent with steroids modulating the activity of the central mechanisms
of sound production. Indeed, the vocal central pattern generator of
batrachoidids [30,33] expresses abundant androgen mRNA receptors
[29] and plays an essential role in setting the duration and pulse rep-
etition rate of calls [34].
Despite the trade-off with, e.g. the immune system, elevated levels
of steroids are also socially adaptive since they regulate ght or ight re-
sponses and the allocation of energy resources [35,36].11KTlevelsare
usually elevated in territorial batrachoidid males in the pre-nesting pe-
riod and in the rst half of the breeding season,encompassing territorial
establishment, courtship and spawning (e.g. [20,37]) In these periods,
social challenges modulate the releaseof sex or stress steroid hormones,
while the release of these hormones in turn facilitates adequate re-
sponses to changes in social environment. This reciprocal interaction
between steroids and behaviour is known as the challenge hypothesis
[38]. Consequently, having uctuations of circulating steroid levels
encoded in the fundamental frequency of the toadsh agonistic
boatwhistle is likely relevant in opponent assessment, as the intruders
may obtain valuable information on the nest-holder's agonistic motiva-
tion, energetic availability and reproductive status.
Our present results further suggest that dominant harmonic
mean and variability signal sonic muscle condition (relative mass
and mainly lipid content). We found that males with higher lipid
levels and relative mass made BW with lower dominant harmonics.
In addition, males with greater lipid content in their sonic muscles
presented a lower variability in the BW dominant harmonic. That
is, they tended to present the maximum spectral energy in the
same harmonic. Sonic muscle features are androgen dependent.
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
11-ketotestosterone (ng/ml)
Pulse period mean (ms)
-3 -2 -1 0 1 2
Pulse period mean (ms)
Cortisol (ng/ml)
Pulse period CV
(a) (b)
R2 = 0.63 R2 = 0.22
R2 = 0.37
Fig. 2. Relation between pulse period mean with(a) circulating11-ketotestosterone and (b)relative gonad mass(RM
). Relationbetween pulse periodvariability (coefcient of variation,
CV) with circulating cortisol (c). Lines of univariate regressions and 95% condence interval bands are shown.
196 M.C.P. Amorim et al. / Physiology & Behavior 149 (2015) 192198
Sonic muscle mass, morphological and physiological traits adapted
for rapid contraction are driven seasonally by androgens in
batrachoidids and other vocal sh, paralleling the increased vocal ac-
tivity in the breeding season (e.g. [3942]). For example, androgens
drive an increase of mitochondria-lled sarcoplasm in sonic muscles
necessary for sustained aerobic activity of muscle contraction [39]
that is key to reproductive success [17]. The effect of steroids on
sonic muscle function also depends on the expression of hormone
receptors and enzymes involved in steroid signalling pathways,
which in the plainn midshipman diverge between calling and
non-calling type I males [43]. Lipids, likely reecting previous forag-
ing success and health status, are one of the major metabolic sub-
strates of sonic muscles during prolonged aerobic contraction and
can noticeably decrease during the period of maximal vocal activity
[40]. In this context, low and stable BW dominant harmonics are in-
formative of androgen driven traits, such as sonic muscle mass, and
likely of the individuals foraging competitive ability. Other hypothe-
ses should also be taken into consideration. For example, harmonic
variability could be associated with the lipid levels in sonic muscles.
As steroids are lipophilic hormones, high lipid levels in muscle bres
could provide a sinkfor 11KT [44] and enhance the aforementioned
morphological and physiological traits for sustained rapid contrac-
tion. Alternatively, harmonic variability could be an epiphenomenon
related to intrinsic muscle contraction variability unrelated with
male quality.
In summary, agonistic BW not only function to fend off potential
nest intruders but also potentially convey relevant information for
male assessment. Agonistic BW with low and stable fundamental
and dominant frequencies signal elevated steroid levels that mediate
social interactions and trigger high sonic muscle condition required
for sustained sound production. Low sound frequency typically sig-
nals male quality even when not related with body size [5].Aninter-
esting result in the present study is the information associated with
BW frequency stability. Signal stability may increase redundancy in
information and therefore communication efcacy [1]. The present
work also suggests high physiological condition associated with
low frequency stereotyped signals as t hese were made by individuals
with better body and sonic muscle condition and high steroid levels.
Recent studies have pointed to the existence of non-linear acous-
tic components in batrachoidid BW [8,45]. For example, similar to
the Lusitanian toadsh, the oyster toadsh BW start with a broad-
frequency non-harmonic grunt-like component [28,45,46].Oneof
the proposed mechanisms underlying this chaotic initial grunt
phase of the BW is the arrhythmic (but in-phase) recruitment of
the bilateral sonic muscles encoded by the vocal central neural
network [45]. This raises the hypothesis that the stability of the fun-
damental and dominant frequencies of BW is also advertising preci-
sion in the neural circuitry underlying the vocal pattern generator,
which may be dependent on development issues. Future studies
should address the role of vocal stability vs. plasticity in vocal inter-
actions and whether such ne variability in spectral parameters of
sh calls can be perceived by sh.
Supplementary data to this article can be found online at http://dx.
We thank the Air Force Base No. 6 of Montijo (Portugal) for allowing
this study in their military establishment. We are grateful to André
Alves and Daniel Alves for the help with the eld work. Fish drawings
LipidSM (g)
Log10-DH (Hz)
0 2 4 6 8 101214
LipidSM (g)
-DH variability
-3 -2 -1 0 1 2
-DH variability
-3 -2 -1 0 1 2
-DH variability
(a) (b)
(c) (d)
R2= 0.35 R2= 0.52
R2= 0.18 R2= 0.13
Fig. 3. Relation between the dominant frequency mean (DH) with sonic muscle lipid content (LipidSM). (a) Relation between the dominant frequency variability with (b) sonic muscle
lipid content (LipidSM), (c) relative sonic muscle mass (RM
) and (d) relative gonad mass (RM
). Lines of univariate regressions and 95% condence interval bands are shown.
197M.C.P. Amorim et al. / Physiology & Behavior 149 (2015) 192198
in Fig. 1 were made by Manuel Vieira. This study was funded by Science
and Technology Foundation, Portugal (project PTDC/MAR/118767/
2010, pluriannual programme UI&D 331/94 and UI&D 329, and grant
SFRH/BPD/41489/2007 to M.C.P.A).
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198 M.C.P. Amorim et al. / Physiology & Behavior 149 (2015) 192198
... In teleosts, acoustic signals are used during competitive feeding (aMoriM & hawkinS 2005), distress or alarm situations (knight & laDich 2014), territorial interactions (longrie et al. 2013;Picciulin et al. 2006), conspecific identification, as well as during courtship and agonistic displays (colleye & ParMentier 2012;ParMentier et al. 2010a), mate choice (aMoriM et al. 2015), mate quality assessment (aMoriM et al. 2015) or coordination of gamete release (loBel 1992). Descriptions of these sounds are based on various acoustic characteristics, including different temporal features (e.g., number of pulses, period of pulses), dominant frequencies and amplitude. ...
... In teleosts, acoustic signals are used during competitive feeding (aMoriM & hawkinS 2005), distress or alarm situations (knight & laDich 2014), territorial interactions (longrie et al. 2013;Picciulin et al. 2006), conspecific identification, as well as during courtship and agonistic displays (colleye & ParMentier 2012;ParMentier et al. 2010a), mate choice (aMoriM et al. 2015), mate quality assessment (aMoriM et al. 2015) or coordination of gamete release (loBel 1992). Descriptions of these sounds are based on various acoustic characteristics, including different temporal features (e.g., number of pulses, period of pulses), dominant frequencies and amplitude. ...
Full-text available
The ability to produce sounds for acoustic communication is well known in different grunt species (Haemulidae). However, most of the sounds have not been described and the sound-producing mechanism of very few grunt species has been deeply studied. Additional data is needed to search for synapomorphy in the sonic mechanism. This study describes acoustic features and branchial anatomy in Haemulon aurolineatum. Correlations were found between some acoustic features and standard length, showing the largest specimens produced shorter, lower-pitched grunts of higher intensity. Examinations of acoustic features and branchial anatomy show that H. aurolineatum uses the same stridulatory mechanism described previously in H. flavolineatum. The unusual feature of Haemulon species concerns the fourth ceratobranchials. These appear to be part of the lower pharyngeal jaws since they possess firmly attached teeth that face the upper pharyngeal jaws. The stridulation results from the rubbing of both pharyngeal and fourth ceratobranchial teeth. This mechanism is probably common to the 23 Haemulon species, but additional information is needed regarding the mechanism of other Haemulinae species to produce stridulatory sounds. Fourth ceratobranchials could constitute a key element of Haemulinae ability to produce sounds providing an eventual synapomorphic aspect of the mechanism in the family.
... Many fishes use sounds for communication purposes in a wide range of behavioral contexts related to aggression (e.g., competitive feeding, intra-and interspecific chase, and territory defence), distress or alarm situations, conspecific identification, and reproduction (e.g., courtship interactions, mate choice, mate quality assessment, and coordination of gamete release ; Lobel et al., 2010;Amorim et al., 2015). The increasing number of studies concerning fish vocal abilities indicates that acoustic communication is an important aspect of teleost biology in freshwater and at sea and should be integrated as such in the study of this group. ...
Many fishes use sounds to communicate in a wide range of behavioral contexts. In monitoring studies, these sounds can be used to detect and identify species. However, being able to confidently link a sound to the correct emitting species requires precise acoustical characterization of the signals in controlled conditions. For practical reasons, this characterization is often performed in small sized aquaria, which, however, may cause sound distortion, and prevents an accurate description of sound characteristics that will ultimately impede sound-based species identification in open-water environments. This study compared the sounds features of five specimens of the silverspot squirrelfish Sargocentron caudimaculatum recorded at sea and in aquaria of different sizes and materials. Our results point out that it is preferable to record fish sounds in an open-water environment rather than in small aquaria because acoustical features are affected (sound duration and dominant frequency) when sounds are recorded in closed environments as a result of reverberation and resonance. If not possible, it is recommended that (1) sound recordings be made in plastic or plexiglass aquaria with respect to glass aquaria and (2) aquaria with the largest dimensions and volumes be chosen.
... Today, fish acoustic communication is considered an important aspect of teleost social behavior across a wider taxonomic spectrum since fish sounds have been reported in many different unrelated taxa (Parmentier et al. 2021;Lobel et al. 2010). Acoustic signals mediate fish social interactions in a wide range of activities such as distress or alarm situations, conspecific identification, courtship and agonistic interactions, mate choice, mate quality assessment, and coordination of gamete release (Amorim et al. 2015). ...
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All anemonefish species can produce two types of sounds. The first class concerns agonistic sounds that are produced during territory defence and probably to establish social hierarchy between individuals. The second class relates to submissive sounds that are emitted in reaction to aggressive acts by dominant individuals. In both types of sounds, irrespective of the sexual status, frequency is highly related to fish size: smaller individuals produce pulses of higher frequency and shorter duration than larger individuals. Consequently, these sonic features within a group may convey information on the social rank of the emitter within the group. This relationship between fish size and both dominant frequency and pulse duration could concern all the Amphiprionini tribe. It highlights the use of a highly conservative vocalization mechanism. Aggressive sounds are initiated by buccal jaw teeth snapping caused by rapid mouth closure attributed to a sonic ligament. We hypothesize that the slam provokes bone vibrations. As the close association of the rib cage and the swimbladder wall would be analogous to a membrane loudspeaker, vibrations would cause shaking of this membrane and cause the second part of the sound. The sound-producing mechanism related to submissive sounds is still not known.
... Underwater playbacks further show that toadfishes, including Gulf toadfish, distinguish call types and PRRs (Fish, 1972;Winn, 1972;McKibben and Bass, 1998;McKibben and Bass, 2001;Remage-Healey and Bass, 2005). Individual differences in boatwhistle PRR are further linked to male quality and aggressive state in the Lusitanian toadfish, Halobatrachus didactylus (Vasconcelos et al., 2012;Amorim et al., 2015b). More broadly, behavioral evidence supports a role for PRR in individual and species recognition in other soniferous teleosts (Gerald, 1971;Myrberg and Spires, 1972;Myrberg and Riggio, 1985;Maruska et al., 2007;Amorim et al., 2015a). ...
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Precise neuronal firing is especially important for behaviors highly dependent on the correct sequencing and timing of muscle activity patterns, such as acoustic signaling. Acoustic signaling is an important communication modality for vertebrates, including many teleost fishes. Toadfishes are well known to exhibit high temporal fidelity in synchronous motoneuron firing within a hindbrain network directly determining the temporal structure of natural calls. Here, we investigated how these motoneurons maintain synchronous activation. We show that pronounced temporal precision in population-level motoneuronal firing depends on gap junction-mediated, glycinergic inhibition that generates a period of reduced probability of motoneuron activation. Super-resolution microscopy confirms glycinergic release sites formed by a subset of adjacent premotoneurons contacting motoneuron somata and dendrites. In aggregate, the evidence supports the hypothesis that gap junction-mediated, glycinergic inhibition provides a timing mechanism for achieving synchrony and temporal precision in the millisecond range for rapid modulation of acoustic waveforms.
... For many species of fish, including Epinephelidae, sound plays a critical role in reproduction and therefore the survival and success of the species (Mann and Lobel, 1995;Bass and Mckibben, 2003;Luczkovich et al., 2008;Walters et al., 2009;Mann et al., 2010;Montie et al., 2016Montie et al., , 2017. Effective communication requires both species and mate recognition for reproduction (Myrberg and Lugli, 2006;Amorim et al., 2015). In known soundproducing groupers, acoustic signals are used by different taxa for recognition, attracting mates, defending territories, agonism and as an alarm system against predators (Mann et al., 2010;Schärer et al., 2012aSchärer et al., ,b, 2013Schärer et al., , 2014Rowell et al., 2018). ...
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Fish spawning aggregations (FSAs) consist of the temporary gathering of a large number of fishes at a specific location to spawn. Monitoring of FSA is typically conducted by divers, but surveys are often restricted to a limited area and dependent upon sea conditions, thus our knowledge of FSA dynamics is extremely limited. Fisheries independent research strives for new technology that can help remotely and unobtrusively quantify fish biomass and abundance. Since some fish species, such as groupers, produce sounds during reproductive behaviors, Eulerian passive acoustic monitoring provides information when divers cannot access the FSA site. Fish sounds provide an innovative approach to assess fish presence and potentially their numbers during reproductive events. However, this technology is limited by the sound propagation range, hence the distance between the fish emitting sounds and the hydrophone location. As such, this Eulerian monitoring approach implicitly creates a knowledge gap about what happens beyond the range of the recorders. Furthermore, the large datasets make the detection process by human ears and eyes very tedious and inconsistent. This paper reports on two innovative approaches to overcome these limitations. To facilitate fish call detections, we have developed an algorithm based on machine learning and voice recognition methods to identify and classify the sounds known to be produced by certain species during FSA. This algorithm currently operates on a SV3 Liquid Robotics wave glider, an autonomous surface vehicle which has been fitted to accommodate a passive acoustic listening device and can cover large areas under a wide range of sea conditions. Fish sounds detections, classification results, and locations along with environmental data are transmitted in real-time enabling verification of the sites with high detections by divers or other in situ methods. Recent surveys in the US Virgin Islands with the SV3 Wave Glider are revealing for the first time the spatial and temporal distribution of fish calls surrounding known FSA sites. These findings are critical to understanding the dynamics of fish populations because calling fish were detected several kilometers away from the known FSAs. These courtship associated sounds from surrounding areas suggest that other FSAs may exist in the region.
Animal weaponry has long captured the imagination of researchers and these weapons are frequently exaggerated in size. Large weapons are particularly common in species in which males defend females from potential rivals and sexual selection is generally credited with driving this pattern of exaggeration. Male New Zealand sheet-web spiders, Cambridgea foliata (Araneae: Desidae), possess chelicerae (jaws) that are substantially larger than those of female conspecifics. To investigate whether chelicerae exaggeration is selected for in the context of male–male competition, we staged contests between males and analysed how different components of resource-holding potential influenced the outcomes and durations of contests. We found that while males with large chelicerae were more likely to win contests, body condition and body size were better predictors of contest outcome. While contest durations were highly variable, there is some evidence that males make decisions about when to retreat from contests using self-assessment. As a result, only very large males are likely to reach the most escalated phase of fighting in which they lock chelicerae with their opponent. In this way, regardless of whether extra-long chelicerae impart any advantage over similarly sized opponents, exaggerated chelicerae are only used by especially large males and are therefore of little use to small males.
Fish sound production correlates with aggressive, reproductive and predator defense behaviors. Studies provide supporting evidence for communication associated with fighting, mate attraction, territoriality, neighbor and species recognition and female preference for male advertisement calls. Simple sounds can function as signals while call complexity is greatest during reproduction. Cue interception is being investigated among conspecifics and between prey and predators. Environmental conditions, predators and behavioral contexts influence call type, calling time or place and may affect amplitude, temporal and frequency parameters of sound signal design. Acoustic fishes encompass a diversity of different body plans, diel activity patterns, habitats and reproductive strategies.
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Acoustic communication is widespread among fishes, the largest extant group of vertebrates, and in many vocal teleost species it is essential for their reproductive and social behaviors. Recent evidence suggests that a fish’s internal hormonal state can profoundly influence its ability to produce and perceive social acoustic signals. Here, we review the current knowledge of how sex steroids can influence the auditory capabilities and vocal motor production of acoustic social signals in two incipient model teleosts, the plainfin midshipman fish Porichthys notatus and the African cichlid Astatotilapia burtoni. Studies of these model systems show that steroid-mediated improvements in vocal-acoustic processing are typically associated with reproductive readiness. This especially holds true for species that rely heavily on acoustic signaling during seasonal breeding such as the midshipman fish, as well as non-seasonally breeding species like cichlids that appear to use sound production as one component of a more complex multimodal courtship repertoire. Evidence for reproductive-state dependent plasticity in midshipman and cichlids is supported by changes in gonadal state, circulating sex-steroids (testosterone, 11-ketotestosterone, and estradiol), and steroid receptor expression in peripheral and central auditory structures that are concurrent with changes in auditory sensitivity and vocal motor production. This form of steroid-dependent vocal-acoustic plasticity is proposed to be an evolutionary labile solution for enhancing the detection and production of social acoustic signals. The abundance and diversity of vocal fish present unique future opportunities to explore how this solution may have shaped sexual selection and the evolution of acoustic communication systems in fishes.
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The relation between acoustic signaling and reproductive success is important to understand the evolution of vocal communi-cation systems and has been well studied in several taxa but never clearly shown in fish. This study aims to investigate whether vocal behavior affects the reproductive success in the Lusitanian toadfish (Halobatrachus didactylus) that relies on acoustic communication to attract mates. We recorded 56 nest-holding (type I) males during the breeding season and analyzed the calling performance and acoustic features of the mate advertising sounds (boatwhistles) exhibited over circa 2 weeks. Hormonal levels of the subjects and the number of eggs (reproductive success) present in the respective nests were quantified. Nesting males attracted both females and other males, namely smaller type I males with significantly lower total length (TL), body condition, sonic muscle mass, gonad mass, and accessory glands mass. Calling rate (CR), calling effort (CE) (% time spent calling), and sound dominant frequency were significantly higher in nesting males with clutches than in those without clutches. Sex steroids (11-ketotestosterone and testosterone) were not correlated with vocal parameters or number of eggs. Maximum CR and CE were the best predictors of the number of eggs. In addition, these vocal variables were best explained by male's TL, condition, and sonic muscle mass. We provide first evidence that vocal behavior significantly determines reproductive success in a vocal fish and show that acoustic signaling at higher and constant rates can operate as an indicator of the male's size and body condition and probably of elevated motivation for reproduction. Key words: acoustic communication, Batrachoididae, mate attraction, reproductive success, toadfish. [Behav Ecol 23:375–383 (2012)] INTRODUCTION
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A combination of field and laboratory investigations has revealed that the temporal patterns of testosterone (T) levels in blood can vary markedly among populations and individuals, and even within individuals from one year to the next. Although T is known to regulate reproductive behavior (both sexual and aggressive) and thus could be expected to correlate with mating systems, it is clear that the absolute levels of T in blood are not always indicative of reproductive state. Rather, the pattern and amplitude of change in T levels are far more useful in making predictions about the hormonal basis of mating systems and breeding strategies. In these contexts we present a model that compares the amplitude of change in T level with the degree of parental care shown by individual males. On the basis of data collected from male birds breeding in natural or captive conditions, polygynous males appear less responsive to social environmental cues than are monogamous males. This model indicates that there may be widely different hormonal responses to male-male and male-female interactions and presumably equally plastic neural mechanisms for the transduction of these signals into endocrine secretions. Furthermore, evidence from other vertebrate taxa suggests strongly that the model is applicable to other classes
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Sound communication is fundamental to many social interactions and essential to courtship and agonistic behaviours in many vertebrates. The swimbladder and associated muscles in batrachoidid fishes (midshipman and toadfish) is a unique vertebrate sound production system, wherein fundamental frequencies are determined directly by the firing rate of a vocal-acoustic neural network that drives the contraction frequency of superfast swimbladder muscles. The oyster toadfish boatwhistle call starts with an irregular sound waveform that could be an emergent property of the peripheral nonlinear sound-producing system or reflect complex encoding in the CNS. Here, we demonstrate that the start of the boatwhistle is indicative of a chaotic strange attractor and tested whether its origin lies in the peripheral sound-producing system or in the vocal motor network. We recorded sound and swimbladder muscle activity in awake, freely-behaving toadfish during motor nerve stimulation, and recorded sound, motor nerve and muscle activity during spontaneous grunts. The results show that rhythmic motor volleys do not cause complex sound signals. However arrhythmic recruitment of swimbladder muscle during spontaneous grunts correlates with complex sounds. This supports the hypothesis that the irregular start of the boatwhistle is encoded in the vocal pre-motor neural network, and not caused by peripheral interactions with the sound-producing system. We suggest that sound production system demands across vocal tetrapods have selected for muscles and motorneurons adapted for speed, which can execute complex neural instructions into equivalently complex vocalizations.
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Male Oyster Toadfish Opsanus tau produce an advertisement call, the boatwhistle, using sexually dimorphic sonic muscles attached to the swimbladder. The fundamental frequency and duration of the boatwhistle change seasonally suggesting hormonal modulation of the output of pattern generators in the brain. The toadfish has an unusual protracted reproductive cycle in which testes contain mature sperm throughout the year, and females develop large eggs during late summer and fall for spawning the following spring although some may mate in the fall. This study quantified gonad development and plasma androgens in males and females throughout a seasonal cycle to relate them to the prolonged reproductive cycle and to quantitative changes in boatwhistle parameters. Median levels of testosterone (T) and 11- ketotestosterone (11KT) in males peak in May during the early part of the spawning season (461 pg/mL for T and 3746 for 11KT) and decline to 153 and 43 pg/mL, respectively, in June although spawning continues into July. A minor increase in gonosomatic index (GSI) and levels of both androgens (180 and 94 pg/mL, respectively) occurs in October. Median levels of T (328 pg/mL) and GSI in females also peak in May. In June, T levels drop in spawned females but remain elevated in those still gravid. Ovaries start to develop in late summer, and T levels increase above levels of individuals spawned in June. A spring peak in T in unspawned females and increasing levels in the fall correlate with estradiol (E) levels. Androgen levels do not correlate with the seasonal cycle in boatwhistle parameters suggesting that some other factor is responsible.
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Most passive acoustics studies focus on daily and seasonal timing and location of choruses of calling fish, particularly sciaenids. Because male toadfish Opsanus spp. are stationary for extended periods, it is possible to extract detailed information about their calls and interactions, making them a powerful model for passive acoustics studies on commercially important species. Toadfishes of both sexes produce a short, pulsatile agonistic grunt, and males produce a “boatwhistle” advertisement call for male-male competition and to attract females. We identify unseen vocal individuals (oyster toadfish O. tau and Gulf toadfish O. beta) near a stationary hydrophone and describe call variability and changes over short- and long-term periods, source levels, call propagation, and directionality. Calls exhibit a directional pattern related to the heart-shaped swim bladder morphology, generating a maximal level behind the fish; grunt frequency spectra allow differentiation of individual callers over multiweek periods. Boatwhistle parameters of oyster toadfish calls change geographically, seasonally, and with temperature, and males call day and night. The Gulf toadfish call rate increases during twilight, when individuals produce shorter and simpler calls. Finally, nearby calling males compete acoustically by increasing their calling rates or producing a grunt (an acoustic tag) during another male's boatwhistle. Toadfishes have been successful models for addressing numerous questions in unseen fish by means of passive acoustics.
Fishes have evolved a diversity of sound-generating organs. These include vibrating the swimbladder and pectoral girdle by rapidly contracting muscles or rubbing bony elements against each other (stridulation) and plucking enhanced tendons. While the former mechanisms produce low-frequency, often harmonic signals (< 500 Hz), the latter usually generate broad-band pulsed sounds with frequencies up to a few kHz. The restriction of fish sounds to lower frequencies limits the distances over which sounds can propagate, especially in shallow waters where sound transmission is negligible below a certain frequency (cutoff frequency). Sounds are uttered in a variety of behavioral contexts, especially during agonistic interactions, courtship, spawning and in distress situations such as when they are disturbed or caught. The functional significance of sounds has seldom been investigated despite a wealth of behavioral studies. Acoustic signals may serve in reducing aggression, in assessing of the fighting ability of opponents, in species recognition, in attraction of mates and in mate choice. Is acoustic communication a driving force in the evolution of hearing sensitivities? In addition to numerous sound-producing organs and sound types, several fish taxa have evolved accessory hearing structures which result in a diversity of hearing abilities. However, the functional significance of this diversity remains unclear. Comparative studies revealed that sound characteristics do not always match hearing sensitivities. The conclusion is therefore that the selective pressures involved in the evolution of this diversity were other than those serving to optimize acoustic communication.
Evidence is provided that the ''chirp,'' a sound commonly produced by males of the bicolor damselfish (family: Pomacentridae) possesses an anatomical constraint: The peak frequency within its power spectrum reflects a clear inverse relationship to body size. For every 1-mm change in the standard length of a male (range: 50-69 mm), the peak frequency of its sounds shifts by approximately 20 Hz. The ultimate constraint appears to be the volume of an individual's gas bladder. This provides an individualistic feature to the sounds of different sized colony members, all of whose sounds possess an otherwise extremely stereotyped temporal pattern of their included pulses. This finding may aid in clarifying the mechanism that provides the clue for the already established acoustical recognition of individuals within colonies of the species.