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Agonistic sounds signal male quality in the Lusitanian toadfish
M. Clara P. Amorim
a,
⁎,CarlottaConti
a
,TeresaModesto
b
, Amparo Gonçalves
c
, Paulo J. Fonseca
d
a
MARE —Marine and Environmental Sciences Centre, ISPA —Instituto Universitário, Lisbon, Portugal
b
Centro de Ciências do Mar, Universidade do Algarve, Campus de Gambelas, 8000-810 Faro, Portugal
c
Division of Aquaculture and Upgrading, Portuguese Institute for the Sea and Atmosphere, I.P. (IPMA, I.P.), Lisbon, Portugal
d
Departamento de Biologia Animal and cE3c —Centre for Ecology, Evolution and Environmental Changes, Faculdade de Ciências, Universidade de Lisboa, Lisbon, Portugal
HIGHLIGHTS
•Acoustic communication during agonis-
tic behaviour is widespread in fishes.
•Breeding Lusitanian toadfish males de-
fend nests from intruders with sounds.
•Low fundamental frequency (high
mean pulse period) reflected high an-
drogen levels.
•The dominant harmonic frequency de-
creased with sonic muscle lipid content.
•Spectral content of fish sounds signal
male traits that are key to fight out-
come.
GRAPHICAL ABSTRACT
abstractarticle info
Article history:
Received 26 February 2015
Received in revised form 27 May 2015
Accepted 1 June 2015
Available online 3 June 2015
Keywords:
Acoustic communication
Fish
Temporal cues
Spectral cues
Steroid hormones
Territorial defence
Acoustic communication during agonistic behaviour is widespread in fishes. Yet, compared to other taxa, little is
known on the information content of fish agonistic calls and their effect on territorial defence. Lusitanian toadfish
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 reflect aspects of male quality that may be important in resolving disputes. We found that higher
mean pulse period (i.e. lower fundamental frequency) reflected higher levels of 11-ketotestosterone (11KT), the
main teleost androgen which, in turn, was significantly 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 fish agonistic sounds may signal male features that are key in fight outcome.
© 2015 Elsevier Inc. All rights reserved.
1. Introduction
Animals' interactions, such as conflicts 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 fitness and includes both signalling territorial ownership
Physiology& Behavior 149 (2015) 192–198
⁎Corresponding author.
E-mail addresses: amorim@ispa.pt (M.C.P. Amorim), conticarlotta@hotmail.it
(C. Conti), tmodesto@ualg.pt (T. Modesto), amparo@ipma.pt (A. Gonçalves),
pjfonseca@fc.ul.pt (P.J. Fonseca).
http://dx.doi.org/10.1016/j.physbeh.2015.06.002
0031-9384/© 2015 Elsevier Inc. All rights reserved.
Contents lists available at ScienceDirect
Physiology & Behavior
journal homepage: www.elsevier.com/locate/phb
and chasing away intruders. In this context, signals should provide
species/sex-specific information to inform potential intruders of the
owner's presence on site, and also provide information on fighting 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 influence the resolution
of contests as they reflect the sender's body size or condition (e.g.
[2–4]). 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, influence the perception of leadership capacity [5].In
fish, 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-
flect the sender's quality and affect territorial defence (e.g. [7,9,10]). Im-
portantly, while it has been shown that in some fishes larger individuals
produce longer, louder and lower frequency agonistic sounds [11–14],
few studies have provided an integrative view on how agonistic sounds
reflect multiple aspects of male quality such as physical and physiologic
features.
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 fishes
rely heavily on acoustic signalling [15–17]. 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 toadfish (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 firing 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 toadfish male quality, including biometric, condition,
and physiological features. We predictthat BW acoustic features convey
information on male quality including energetic reserves andhormonal
status which influence the outcome of contest behaviour [19].
2. Methods
2.1. Subject males and maintenance
The Lusitanian toadfish, like other batrachoidids, has two fixed 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 artificial
concrete shelters placed in the lower intertidal of the Tagus River es-
tuary (Military Air Force Base 6, Montijo, Portugal; 38° 42′N; 8° 58′W).
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
2–3 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]. Briefly, 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 fin rays to ensure their identity. Marking
did not cause any measurable change in the fish 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 (37.5–49.5)
cm in TL and intruders 36.9 ± 2.7 (30.0–42.0) cm in TL. Residents pro-
duced on average 4.7 ± 5.9 (range: 0–22, 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 (4–22) 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 fish).
The 14 males used to relate sounds with male quality averaged
41.9 ± 1.8 (39.3–46.0) cmin TL and 1221 ± 199 (970–1600) g in M. Im-
mediately after theend of trials, these males were sacrificed 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 first handling of the fish. 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
quantification. Following blood sampling, males were immediately
193M.C.P. Amorim et al. / Physiology & Behavior 149 (2015) 192–198
dissected and the mass of the gonads (M
G
), the liver (M
L
), and of the
sonic muscles (M
SM
) was weighed to the nearest mg, and eviscerated
body mass to the nearest g (ME). A sample of body muscle (epaxial
muscle fibres) 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 quantification of the lipid and glycogen con-
tent, respectively. Lipids and glycogen are important sources of energy
in fish 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 quantification
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 quantification
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 fish, including the Lusitanian toadfish [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 (coefficient of variation)
was 6.3% and 5.2% for T, 3.9% and 5.5% for 11KT, 7.8% and 10.3%for CORT,
respectively.
2.5. Behavioural and sound analyses
We tallied the number of agonistic resident–intruder 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 fish we tallied the fre-
quency of agonistic escalated behaviours (escalated fight, EF) such as
chasing, bite attempts, bites and mouth–mouth fight. Non-escalated vi-
sual behaviour such as approach or mouth opening with the extension
of fins 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 first 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 coefficient 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 fish (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, filter
bandwidth 10 Hz). We analysed an average ± sd (range) of 7.3 ± 2.5
(4–10) 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)
reflected 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
10
-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
(log
10
TL). We also used the residuals of the simple linear regression of
sonic muscle, gonads and liver mass on eviscerated body mass (RM
SM
,
RM
G
,RM
L
, respectively) as metrics of these parameters controlled for
the influence of body size. For example a high positive residual of
RM
SM
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=0.75–0.77, pb0.01). LipidSM
was also related with RM
L
(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
L
from analyses. We further excluded T as it is physiologically related
with 11KT [20].
Our final regression models complied with all assumptionsof multi-
ple linear regression. All model residuals were normally distributed.
Further residual analyses were performed using Durbin–Watson statis-
tics, residual plots as well as multicollinearity tests (variance inflation
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 flee 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) 192–198
(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,
pb0.05).
Agonistic BW Dur averaged 640 ms (range 437–933 ms) and pre-
sented a relatively high coefficient of variation (mean = 0.3, range
0.1–0.5). PP averaged 23 ms (range 19.7–26.9 ms) and was more stereo-
typed (mean CV = 0.05, range 0.01–0.07). DH was most commonly
found on the first harmonic (DH mean = 89 Hz, range 41–146 Hz),
and on average males maintained a particular dominant frequency
band in 70% of BW, i.e. DH variability averaged 0.7 (range 0.5–1.0).
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 reflect male quality.
Both Dur mean and Dur CV were not significantly related with any
male quality variable (regression analyses, pN0.05). However, several
male quality variables were significantly associated with pulse period
and spectral characteristics of BW. PP mean increased with 11KT
(R
2
= 0.63) and decreased with relative gonad mass (RM
G
:R
2
=0.22,
Table 1,Fig. 2a,b). PP CV was negatively affected by CORT (R
2
= 0.37;
Fig. 2c). DH mean was negatively associated with LipidSM (R
2
=0.35,
Table 1,Fig. 3a). DH variability decreased with LipidSM (R
2
= 0.52)
Rel. amplitude (dB)
0.4 0.60.20
Frequency (kHz)
-40
-20
0
DH
Sound duration
6 PP
100 ms
(a)
72.7
0
0
27.3
Approach
45.7
42.9
5.7 5.7
Intrusion
BW
BW+EF
EF
NR
(b)
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).BW—emission of boatwhistles; BW + EF —boatwhistles followed by escalated fight; EF —escalated fight; 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
significant (r—partial 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 inflation factor.)
Dependent variable Included predictor BSEM tprF Model significance R
2
DW VIF
PP mean Intercept 19.74 0.50 39.27 b0.001
11KT 1.87 0.30 6.21 b0.001 0.91 1.02
RM
G
−1.15 0.33 −3.45 0.009 −0.77 F
2,8
= 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
1,9
= 5.17 p= 0.049 0.37 2.80 1.0
DH mean
a
Intercept 2.15 0.10 21.19 b0.001
LipidSM −0.037 0.02 −2.45 0.03 −0.59 F
1,11
= 5.98 p= 0.03 0.35 1.73 1.0
DH Intercept 0.32 0.01 23.61 b0.001
Variability
a
LipidSM −0.013 0.002 −6.46 b0.001 −0.93 1.13
RM
SM
−0.015 0.01 −2.84 0.03 −0.73
RM
G
0.017 0.01 2.63 0.03 0.71 F
3,7
= 16.02 p= 0.002 0.87 2.21 1.14
1.02
a
Data were log
10
-transformed.
195M.C.P. Amorim et al. / Physiology & Behavior 149 (2015) 192–198
and with RM
SM
(R
2
= 0.18), but increased withRM
G
(R
2
=0.13,Table 1,
Fig. 3b–d).
4. Discussion
The Lusitanian toadfish 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 fight
when opponents did not flee after first 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 fighting 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 fish 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 plainfin midshipman
fish, Porichthys notatus, intramuscular injections of 11KT and CORT
rapidly increase the durations of fictive 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 toadfish, 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 fight or flight 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 first 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 fluctuations of circulating steroid levels
encoded in the fundamental frequency of the toadfish 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)
18
20
22
24
26
28
Pulse period mean (ms)
-3 -2 -1 0 1 2
RM
G
20
22
24
26
28
Pulse period mean (ms)
01234567
Cortisol (ng/ml)
0.00
0.02
0.04
0.06
0.08
Pulse period CV
(a) (b)
(c)
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
G
). Relationbetween pulse periodvariability (coefficient of variation,
CV) with circulating cortisol (c). Lines of univariate regressions and 95% confidence interval bands are shown.
196 M.C.P. Amorim et al. / Physiology & Behavior 149 (2015) 192–198
Sonic muscle mass, morphological and physiological traits adapted
for rapid contraction are driven seasonally by androgens in
batrachoidids and other vocal fish, paralleling the increased vocal ac-
tivity in the breeding season (e.g. [39–42]). For example, androgens
drive an increase of mitochondria-filled 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 plainfin midshipman diverge between calling and
non-calling type I males [43]. Lipids, likely reflecting 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 fibres
could provide a ‘sink’for 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 efficacy [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 toadfish, the oyster toadfish 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 fine variability in spectral parameters of
fish calls can be perceived by fish.
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.physbeh.2015.06.002.
Acknowledgements
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 field work. Fish drawings
02468101214
LipidSM (g)
1.6
1.8
2.0
2.2
Log10-DH (Hz)
0 2 4 6 8 101214
LipidSM (g)
0.15
0.20
0.25
0.30
0.35
Log
10
-DH variability
-3 -2 -1 0 1 2
RMSM
0.15
0.20
0.25
0.30
0.35
Log
10
-DH variability
-3 -2 -1 0 1 2
RM
G
0.15
0.20
0.25
0.30
0.35
Log
10
-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
SM
) and (d) relative gonad mass (RM
G
). Lines of univariate regressions and 95% confidence interval bands are shown.
197M.C.P. Amorim et al. / Physiology & Behavior 149 (2015) 192–198
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).
References
[1] J.W. Bradbury, S.L. Vehrencamp, Principles of Animal Communication, Sinauer Asso-
ciates, Sunderland, MA, 1998.
[2] N.B. Davies, T.R. Halliday, Deep croaks and fighting assessment in toads Bufo bufo,
Nature 274 (1978) 683–685, http://dx.doi.org/10.1038/274683a0.
[3] W.D. Brown,A.T. Smith, B. Moskalik, J. Gabriel, Aggressive contests in house crickets:
size, motivation and the information content of aggressive songs, Anim. Behav. 72
(2006) 225–233, http://dx.doi.org/10.1016/j.anbehav.2006.01.012.
[4] M.C.P. Amorim, J.M. Simões, N. Mendonça, N.M. Bandarra, V.C. Almada, P.J. Fonseca,
Lusitanian toadfish song reflects male quality, J. Exp. Biol. 213 (2010) 2297–3004,
http://dx.doi.org/10.1242/jeb.044586.
[5] C.A. Klofstad, R.C. Anderson, S. Peters, Sounds like a winner: voice pitch influences
perception of lea dership capacity in both men and wome n, Proc. R. Soc. B 279
(2012) 2698–2704, http://dx.doi.org/10.1098/rspb.2012.0311.
[6] F. Ladich, Sound production and acoustic communication, in: G. von der Erude, J.
Mogdans, B.G. Kapoor (Eds.), The Senses of Fish Adaptations for the Reception of
Natural Stimuli, Kluwer, Dordrecht and Narosa, New Delhi 2004, pp. 210–230.
[7] F. Ladich, A.A. Myrberg Jr., Agonistic behavior and acoustic communication, in: F.
Ladich, S.P. Collin,P. Moller, B.G. Kapoor(Eds.), Communication in Fishes, vol. 1, Sci-
ence Publishers, Enfield, NH 2006, pp. 121–148.
[8] A.N. Rice, B.R. Land, A.H. Bass, Nonlinear acoustic complexity in a fish ‘two-voice’
system, Proc. Biol. Sci. B 278 (2011) 3762–3768, http://dx.doi.org/10.1098/rspb.
2011.0656.
[9] L. Remage-Healey, A.H. Bass, Rapid elevations in both steroid hormones and vocal
signaling during playback challenge: a field experiment in Gulf toadfish, Horm.
Behav.47(2005)297–305, http://dx.doi.org/10.1016/j.yhbeh.2004.11.017.
[10] K.P. Maruska, J.A. Sisneros, Sex steroid-dependent modulation of acoustic com-
municatio n systems in fishes, i n: F. Ladic h (Ed.), Sound Communication in Fishes,
Springer-Verlag, Wien 2015, pp. 207–233.
[11] A.A. Myrberg Jr., S.J. Ha, H. Shamblott, The sounds of bicolor damselfish
(Pomacentrus partitus): predictors of body size and a spectral basis for individual
recognition and assessment, J. Acoust. Soc. Am. 94 (1993) 3067–3070, http://dx.
doi.org/10.1121/1.407267.
[12] F. Ladich, Sound characteristics and outcome of contests in male croaking gouramis
(Teleostei), Ethology 104 (1998) 517–529, http://dx.doi.org/10.1111/j.1439-0310.
1998.tb00087.x.
[13] L.E. Wysocki, F. Ladich, The ontogenetic development of auditory sensitivity, vocal-
ization and acoustic communication in the labyrinth fish Trichopsis vittata,J.Comp.
Physiol. A. 187 (2001) 177–187.
[14] M.C.P. Amorim, S.S. Pedroso, M. Bolgan, J.M. Jordão, M. Caiano, P.J. Fonseca, Painted
gobies sing their quality out loud: acoustic rather than visual signals advertise male
quality and contribute to mating success, Funct. Ecol. 27 (2013)289–298, http://dx.
doi.org/10.1111/1365-2435.12032.
[15] A.H. Bass, J.R. McKibben, Neural mechanisms and behaviors for acoustic communi-
cation in teleost fish, Prog. Neurobiol. 69 (2003) 1–26, http://dx.doi.org/10.1016/
S0301-0082(03)00004-2.
[16] R.O. Vasconcelos, J.M. Simões, V.C. Almada, P.J. Fonseca, M.C.P. Amorim, Vocal be-
haviour during territorial intrusions in the Lusitanian toadfish: boatwhistles also
function as territorial ‘keep-out’signals, Ethology 116 (2010) 155–165, http://dx.
doi.org/10.1111/j.1439-0310.2009.01722.x.
[17] R.O. Vasconcelos,R. Carriço, A. Ramos, T. Modesto,P.J. Fonseca, M.C.P. Amorim,Vocal
behavior predicts reproductive success in a teleost fish, Behav. Ecol. 23 (2012)
375–383, http://dx.doi.org/10.1093/beheco/arr199.
[18] H.E. Winn, Vocal facilitation and the biological significance of toadfish sounds, in:
W.N. Tavolga (Ed.), Marine Bio-acoustics, vol. 2, Pergamon Press, Oxford, UK 1967,
pp. 283–304.
[19] Y.Y. Hs u, R.L. Earley, L.L. Wolf, Modulation of aggressive behaviou r by fighting
experience: mechanisms and contest outcomes, Biol. Rev. 81 (2006) 33–74, http://
dx.doi.org/10.1017/S146479310500686X.
[20] T. Modesto, A.V.M. Canário, Morphometric changes and sex steroid levels dur-
ing the annual reproductive cycle of the Lusitanian toadfish, Halobatrachus
didactylus, Gen. Comp. Endocrinol. 131 (2003) 220–231, http://dx.doi.org/
10.1016/S0016-6480(03)00027-3.
[21] M.C.P. Amorim, J.M. Simões, P.J. Fonseca, V.C. Almada, Patterns of shelter usage and
social aggregation by the vocal Lusitanian toadfish, Mar. Biol. 157 (2010) 495–503,
http://dx.doi.org/10.1007/s00227-009-1335-6.
[22] S. Chellappa, F.A. Huntingford, R.H.C. Strang, R.Y. Thomson, Condition factor and
hepatosomatic index as estimates of energy status in male three-spine stickleback,
J. Fish Biol. 47 (1995) 775–787, http://dx.doi.org/10.1111/j.1095-8649.1995.
tb06002.x.
[23] S. Mitchell, J. Poland, M.L. Fine, Does muscle fatigue limit advertisement calling in
the oyster toadfish Opsanus tau? Anim. Behav. 76 (2008) 1011–1016, http://dx.
doi.org/10.1016/j.anbehav.2008.03.024.
[24] K.H. Olsson, C. Kvarnemo, O. Svensson, Relative costs of courtship behaviours in
nest-building sand gobies, Anim. Behav. 77 (2009) 541–546, http://dx.doi.org/10.
1016/j.anbehav.2008.10.021.
[25] P. Viles, J. Silverman, Determination of starch and cellulose with anthrone, Anal.
Chem. 21 (1949) 950–953, http://dx.doi.org/10.1021/ac60032a019.
[26] A.P. Scott, A.V. Canário, 17α,20β-Dihydroxi-4-pregnen-3-one 20-sulphate: a major
new metabolite of the teleost oocyte maturation-inducing steroid, Gen. Comp.
Endocrinol. 85 (1992) 91–100, http://dx.doi.org/10.1016/0016-6480(92)90176-K.
[27] A. Damasceno-Oliveira, B. Fernandez-Duran, J. Goncalves, E. Couto, A.V.M. Canario, J.
Coimbra,Plasma steroid hormonelevels in female flounderPlatichthys flesusand the
influence of fluctuating hydrostatic pressure, Comp. Biochem. Phys. A 163 (2012)
272–277, http://dx.doi.org/10.1016/j.cbpa.2012.08.001.
[28] M.C.P. Amorim, R.O. Vasconcelos, Variability in the mating calls of the Lusitanian
toadfish Halobatrachus didactylus: potential cues for individual recognition, J. Fish
Biol. 72 (2008) 1355–1368, http://dx.doi.org/10.1111/j.1095-8649.2008.01974.x.
[29] P.M. Forlano, M. Marchaterre, D.L. Deitcher, A.H. Bass, Distribution of androgen re-
ceptor mRNA expression in vocal, auditory and neuroendocrine circuits in a teleost
fish, J. Comp. Neurol. 518 (2010) 493–512, http://dx.doi.org/10.1002/cne.22233.
[30] A.H. Bass, L. Remage-Healey, Central pattern generators for social vocalization:
androgen-dependent neurophysiological mechanisms, Horm. Behav. 53 (2008)
659–672, http://dx.doi.org/10.1016/j.yhbeh.2007.12.010.
[31] L. Remage-Healey, A.H. Bass, Rapid, hierarchical modulation of vocal patterning by
steroid hormones, J. Neurosci. 24 (2004) 5892–5900, http://dx.doi.org/10.1523/
JNEUROSCI.1220-04.2004.
[32] L. Remage-Healey, A.H. Bass, A rapid neuromodulatory role for steroid hormonesin
the control of reproductive behavior, Brain Res. 1126 (2006) 27–35, http://dx.doi.
org/10.1016/j.brainres.2006.06.049.
[33] M.L. Fine, F.A. Chen, D.A. Keefer, Autoradiographic localization of dihydrotestoster-
one and testosterone concentrating neurons in the brain of the oyster toadfish,
Brain Res. 709 (1996) 65–80, http://dx.doi.org/10.1016/0006-8993(95)01275-3.
[34] B.P. Chagnaud, R. Baker, A.H. Bass, Vocalization frequency and duration are coded in
separate hindbrain nuclei, Nat. Commun. 2 (346) (2011) 1–11, http://dx.doi.org/10.
1038/ncomms1349.
[35] J.C. Wingfield, S. Lynn, K.K. Soma, Avoiding the ‘costs’of testosterone: ecological
bases of hormone–behavior interactions, Brain Behav. Evol. 57 (2001) 239–251,
http://dx.doi.org/10.1159/000047243.
[36] A.F.H. Ros, K. Becker, A.V.M. Canário, R.F. Oliveira, Androgen levels and energy me-
tabolism in Oreochromis mossambicus, J. Fish Biol. 65 (2004) 895–905, http://dx.
doi.org/10.1111/j.1095-8649.2004.00484.x.
[37] M.L. Fine, M.S. Johnson, D.W. Matt, Seasonal variation in androgen levelsin the oys-
ter toadfish, Copeia 2004 (2004) 235–244, http://dx.doi.org/10.1643/CP-03-073R1.
[38] J.C. Wingfield, R.E. Hegner, A.M. Dufty Jr., G.F. Ball, The “challenge hypothesis”: the-
oretical implications for patterns of testosterone secretion, mating systems and
breeding strategies, Am. Nat. 136 (1990) 829–846 (Stable URL: http://www.jstor.
org/stable/2462170).
[39] R.K. Brantley, J.C. Wingfield, A.H. Bass, Sex steroid levels in Porichthys notatus,afish
with alternative reproductive tactics, and a review of the hormonal bases for male
dimorphism among teleost fishes, Horm. Behav. 27 (1993) 332–347, http://dx.doi.
org/10.1006/hbeh.1993.1025.
[40] M.A. Connaughton, M.L. Fine, M.H. Taylor, The effects of seasonal hypertrophy and
atrophy on fiber morphology, metabolic substrate concentration and sound charac-
teristics of the weakfish sonic muscle, J. Exp. Biol. 200 (1997) 2449–2457.
[41] T. Modesto, A.V.M. Canário, Hormonal control of swimbladder sonic muscle dimor-
phism in the Lusitanian toadfish Halobatrachus didactylus, J. Exp. Biol. 206 (2003)
3467–3477, http://dx.doi.org/10.1242/jeb.00581.
[42] M.L. Fine, K.R. Pennypacker, Hormonal basis for sexual dimorphism of the sound-
producing apparatus of the oyster toadfish, Exp. Neurol. 92 (1986) 289–298,
http://dx.doi.org/10.1016/0014-4886(86)90081-6.
[43] R.M. Genova, M.A. Marchaterre, R. Knapp, D. Fergus, A.H. Bass, Glucocorticoid and
androgen signaling pathways diverge between advertisement calling and non-
calling fish, Horm. Behav. 62 (2012) 426–432, http://dx.doi.org/10.1016/j.yhbeh.
2012.07.010.
[44] G.T. Crossin, M. Poisbleau, L. Demongin, O. Chastel, T.D. Williams, M. Eens, P.
Quillfeldt, Migratory constraints on yolk precursors limit yolk androgen deposition
and underlie a brood reduction strategy in rockhopper penguins, Biol. Lett. 8 (2012)
1055–1058, http://dx.doi.org/10.1098/rsbl.2012.0476.
[45] C.P.H. Elemans, A.F. Mensinger, L.C. Rome, Vocal production complexity correlates
with neural instructions in the oyster toadfish (Opsanus tau), J. Exp. Biol. 217
(2014) 1887–1893, http://dx.doi.org/10.1242/jeb.097444.
[46] M.L. Fine, R.F. Thorson, Use of passiveacoustics for assessing behavioral interactions
in individual toadfish, T. Am. Fish. Soc. 137 (2008) 627–637, http://dx.doi.org/10.
1577/T04-134.1.
198 M.C.P. Amorim et al. / Physiology & Behavior 149 (2015) 192–198
