Lusitanian toadfish song reflects male quality

Abstract

Lusitanian toadfish males that provide parental care rely on acoustic signals (the boatwhistle) to attract females to their nest. We test the hypothesis that male quality, namely male size and condition that are relevant for parental success, is reflected in vocal activity and boatwhistle characteristics and thus advertised to females. We recorded 22 males over a week during the peak of the breeding season. Calling rate and calling effort (percentage of time spent calling) strongly reflected male condition (lipid content of somatic muscles) and to a smaller extent sonic muscle hypertrophy and larger gonads. Males in better condition (increased body lipid and relative higher liver mass) also contracted the sonic muscles at faster rate as shown by the shorter boatwhistle pulse periods. Amplitude modulation reflected the degree of sonic muscle hypertrophy. None of the measured male quality parameters were good predictors of boatwhistle duration and dominant frequency. Altogether this study strongly suggests that Lusitanian toadfish males advertise their quality to females primarily with boatwhistle calling rate and calling effort, which mainly reflect male condition. Because pulse period had low variability, consistent with the existence of a vocal central pattern generator, we suggest that males that sustain sonic muscles contraction at a very fast rate close to their physiological limit may be honestly advertising their quality (condition). Similarly, males that produce boatwhistles with higher amplitude modulation, a feature that seems dependent on sonic muscle hypertrophy, could be more attractive to females.

Full text

2997
INTRODUCTION
Females are often the choosy sex and select mates based on direct
benefits such as food, parental care or a good territory, or on indirect
benefits such as genetic quality (Andersson, 1994). Males advertise
their attributes through courtship displays, which inform their mates
about their quality. Signals can evolve to be reliable or honest if
they bear a cost to the sender either because of production costs or
through increased vulnerability of attack by heterospecific or
conspecific receivers (Zahavi, 1975). Alternatively, index signals
are reliable because they cannot be faked due to physical or
physiological constraints that force the signal to reveal honest
information (Vehrencamp, 2000; Maynard Smith and Harper,
2003). Honest signals can also be relatively cost-free if signallers
and receivers share a common interest or when there is a threat of
retaliation by the receiver (Vehrencamp, 2000).
Acoustic signals are good examples of sexually selected traits
predominantly used by females of several taxa to identify, locate
and choose between potential mates (Andersson, 1994; Bradbury
and Vehrencamp, 1998). Different components of vocalisations
seem to convey honest messages that are relevant for mate choice
but may also be maladaptive for females if female preference has
evolved, for example, by male sensory exploitation (Ryan et al.,
1990). Examples of honest acoustic signal components are high
calling rates that are energetically costly to maintain or may attract
predators or parasites, and the fundamental frequency of
advertisement calls that may be dependent on the vocal apparatus
size and thus on the sender size (Maynard Smith and Harper, 2003).
Fish probably represent the largest group of sound-producing
vertebrates and often emit acoustic signals during courtship (Ladich,
2004). However, there are few studies that relate calling rate and
call characteristics with male quality and even fewer that show the
role of acoustic signals in mate choice. This is probably due to
technical limitations related to underwater recordings (in natural
contexts) and playbacks, rather than to the lack of a role of fish
calls on mate choice (Ladich, 2004). For example, female of the
bicolour damselfish Stegastus partitus (Pomacentridae) prefer
courtship chirps of lower frequency that indicate a larger male body
size (Myrberg et al., 1986). Females of the same species also favour
males with high courtship rates who have better body condition and
are less prone to cannibalise their eggs (Knapp and Kovach, 1991).
Although it has not been tested, the studies by Myrberg et al.
(Myrberg et al., 1986) and Knapp and Kovach (Knapp and Kovach,
1991) both suggest that S. partitus females could be also selecting
males with a high courtship chirping rate, which could be indicative
of a better male condition and parental ability. McKibben and Bass
have also shown that females of the midshipman Porichthys notatus
(Batrachoididae) prefer the more intense of two hums, the male
mating signals (McKibben and Bass, 1998). In addition, the
percentage of females responding to sound playback increased as
duration of calls increased and the pauses between calls decreased,
indicating that longer continuous calls resembling natural hums are
more attractive to females (McKibben and Bass, 1998).
Breeding males from the family Batrachoididae (toadfishes and
midshipmen) nest under rocks and produce advertisement calls
The Journal of Experimental Biology 213, 2997-3004
© 2010. Published by The Company of Biologists Ltd
doi:10.1242/jeb.044586
Lusitanian toadfish song reflects male quality
M. Clara P. Amorim1,*, J. Miguel Simões1, Nuno Mendonça2, Narcisa M. Bandarra2, Vitor C. Almada1and
Paulo J. Fonseca3
1Unidade de Investigação em Eco-Etologia, Instituto Superior de Psicologia Aplicada, Rua Jardim do Tabaco 34, 1149-041 Lisboa,
Portugal, 2Unidade de Valorização dos Produtos da Pesca e Aquicultura (U-VPPA), Instituto de Investigação das Pescas e do Mar
(IPIMAR), Instituto Nacional de Recursos Biológicos (INRB), Avenida de Brasília, 1449-006 Lisboa, Portugal and 3Departamento de
Biologia Animal e Centro de Biologia Ambiental, Faculdade de Ciências da Universidade de Lisboa. Bloco C2, Campo Grande,
1749-016 Lisboa, Portugal
*Author for correspondence (amorim@ispa.pt)
Accepted 18 May 2010
SUMMARY
Lusitanian toadfish males that provide parental care rely on acoustic signals (the boatwhistle) to attract females to their nest. We
test the hypothesis that male quality, namely male size and condition that are relevant for parental success, is reflected in vocal
activity and boatwhistle characteristics and thus advertised to females. We recorded 22 males over a week during the peak of the
breeding season. Calling rate and calling effort (percentage of time spent calling) strongly reflected male condition (lipid content
of somatic muscles) and to a smaller extent sonic muscle hypertrophy and larger gonads. Males in better condition (increased
body lipid and relative higher liver mass) also contracted the sonic muscles at faster rate as shown by the shorter boatwhistle
pulse periods. Amplitude modulation reflected the degree of sonic muscle hypertrophy. None of the measured male quality
parameters were good predictors of boatwhistle duration and dominant frequency. Altogether this study strongly suggests that
Lusitanian toadfish males advertise their quality to females primarily with boatwhistle calling rate and calling effort, which mainly
reflect male condition. Because pulse period had low variability, consistent with the existence of a vocal central pattern generator,
we suggest that males that sustain sonic muscles contraction at a very fast rate close to their physiological limit may be honestly
advertising their quality (condition). Similarly, males that produce boatwhistles with higher amplitude modulation, a feature that
seems dependent on sonic muscle hypertrophy, could be more attractive to females.
Key words: fish, Batrachoididae,
Halobatrachus didactylus
, acoustic communication, mate choice, male condition, muscle lipid content.
THE JOURNAL OF EXPERIMENTAL BIOLOGY

2998
(boatwhistles or hums) from their nests to attract females by
contracting a pair of sonic muscles embedded in the sides of the
swimbladder (Fish, 1972; Brantley and Bass, 1994; dos Santos et
al., 2000). Batrachoidid males provide uniparental care until the
young are free-swimming and call to attract females until the nest’s
ceiling is fully covered with eggs and embryos (Brantley and Bass,
1994). Batrachoidids have become a model group in the study of
acoustic communication in teleosts (Bass and McKibben, 2003;
Sisneros, 2009) but little is known on the role of acoustic signals
in mate choice and reproductive success. The Lusitanian toadfish
Halobatrachus didactylus (Bloch and Schneider 1801) produces a
long (about 1s) tonal low-frequency advertisement sound, the
boatwhistle, which is highly stereotyped and shows considerable
inter-individual differences during short periods of time (<10min)
(Amorim and Vasconcelos, 2008). A recent study has shown that
the male’s sound-producing muscles are highly variable (c.v.40%)
and that its mass depends mostly on male length and condition,
suggesting that sounds could advertise male quality in this species
(Amorim et al., 2009). Because the boatwhistle seems to be the only
courtship signal in this species (M.C.P.A., P.J.F. and J.M.S.,
personal observation) [see also Brantley and Bass (Brantley and
Bass, 1994) for a detailed description of the mating behaviour of
P. notatus] we have hypothesised that it should contain information
relevant to the female for mate choice. In the present study, we test
the hypothesis that calling activity (calling rate and calling effort –
percentage of time calling) and boatwhistle characteristics (duration,
pulse period, dominant frequency and amplitude modulation) reflect
male quality. For that purpose we recorded 22 males, each over a
week during the peak of the breeding season, and measured their
body condition, body size, sonic muscle hypertrophy and
reproductive condition (gonad and accessory gland mass). Because
Lusitanian toadfish females seem to produce only one clutch of a
few large eggs per reproductive season (Modesto and Canário,
2003a; Costa, 2004) and have to probably rely on the parental ability
of one single male for their reproductive success, we predict that
boatwhistles should inform females of male size and condition,
which are relevant for nest defence and parental care.
MATERIALS AND METHODS
Study species
Breeding males of the Lusitanian toadfish, H. didactylus, form
conspicuous choruses from May to July (in Portugal) and defend
nests in estuarine shallow waters that can contain clutches from
different females (Amorim et al., 2006). Parental care is provided
until the young are free-swimming. Besides the nest-guarding male
(‘type I’) morphotype, there are sneaker males (‘type II’) that have
larger testis (sevenfold), smaller accessory glands (threefold; the
accessory glands are part of the male reproductive apparatus, secrete
mucosubstances and are connected to the spermatic duct) and lower
(sixfold) 11-ketotestosterone levels than nesting males (Modesto and
Canário, 2003a; Modesto and Canário, 2003b). Only type I males
produce boatwhistles during the breeding season. Sonic muscles of
type I males but not of type II males or of females suffer hypertrophy
during the breeding season (Modesto and Canário, 2003a),
concurrent with an increase in vocal activity (Amorim et al., 2006).
Sound recording
Sixty artificial hemicylinder-shaped concrete shelters capped at one
end were deployed 1.5m apart in an intertidal area of the Tagus
estuary (Portugal, Montijo, Air-Force Base 6; 38°42⬘N, 8°58⬘W)
that was only exposed to air during spring low tides. Water level
varied between 0m and 2.8m in the study area. The shelters were
large enough (internal dimensions: 50cm long⫻30cm wide⫻20cm
high) to house a large male and several females and were readily
used as nests during the breeding season (Amorim et al., 2010).
Three groups of 6–8 males (N22) that spontaneously occupied these
artificial concrete shelters were recorded over a period of eight days
in June and July in 2006 and 2007, during the peak of the
reproductive season (May to July in Portugal) (Modesto and
Canário, 2003a). Shelters (6–8) containing subject males were placed
1.5m apart in two rows and were at least 15m apart from the
remaining shelters. The entrance of the subject males’ shelters were
closed with a plastic mesh preventing fish from abandoning the nest
during recordings but allowing prey items to enter and possible
visual interactions with conspecifics.
One hydrophone (High Tech 94 SSQ hydrophone, High Tech
Inc., Gulfport, MS, USA; sensitivity –165dB re. 1V/Pa, frequency
response within ±1dB from 30Hz to 6KHz) was firmly attached
to an iron rod partially buried in the sand substrate, placed ~10cm
from each shelter entrance and from the substrate. Simultaneous
multi-channel recordings were made to a laptop connected to USB
audio capture devices (Edirol UA25, Roland, Osaka, Japan; 16bit,
6kHz acquisition rate per channel) controlled by Adobe Audition
2.0 (Adobe Systems Inc., Mountain View, CA, USA). Recorded
sounds could be attributed to each male due to the high acoustic
attenuation observed in shallow water (Fine and Lenhardt, 1983).
Sounds from a neighbouring male were ~27dB lower than of a
subject male. Water temperature was measured every 3h during
recording periods and averaged 23°C (range: 19.5–28°C). All
subject fish experienced similar water temperature variability during
recordings; hence, the effect of temperature on call parameters
should be similar for all fish. Each male was recorded for an average
of 35h (range: 11–56h).
Sound analysis
Boatwhistles have been described in detail in Amorim and
Vasconcelos (Amorim and Vasconcelos, 2008) and, as in other
batrachoidids (e.g. Thorson and Fine, 2002), are characterised by
an initial shorter pulsed part followed by a longer tonal segment
(dos Santos et al., 2000; Amorim and Vasconcelos, 2008). We
analysed boatwhistles for total sound duration (ms, measured from
the start of the first pulse to the end of the last pulse), pulse period
of the tonal segment (ms, average peak-to-peak interval of six
consecutive pulses in the middle of this segment), dominant
frequency of the tonal segment (Hz, the frequency with maximum
energy in this part of the sound), and amplitude modulation [the
ratio between the mean amplitude (root mean square, r.m.s.) of the
initial and of the tonal segments; r.m.s. amplitude is a measurement
native to Raven software, Cornell Laboratory of Ornithology,
Ithaca, NY, USA]. Temporal variables and amplitude modulation
were measured from oscillograms, and dominant frequency from
power spectra computed with a 2048 points FFT conditioned by a
Hamming window, with a time overlap of 50.0% and a 10Hz filter
bandwidth. These acoustic parameters are depicted in Fig.1. Calling
rate (number of boatwhistles emitted per hour) was tallied for each
fish. Calling effort (number of hours calling / number of hours
recorded ⫻100) was also calculated per fish. Sound analysis was
carried out with Adobe Audition 2.0 and Raven 1.2.1 for Windows.
Morphometric analysis
At the end of recordings subject males were killed with an excessive
dosage of MS 222 (tricaine methane sulphonate; Pharmaq, Skøyen,
Oslo, Norway). In the laboratory, each subject male was measured
to the nearest mm for total length (TL), and to the nearest g for
M. C. P. Amorim and others
THE JOURNAL OF EXPERIMENTAL BIOLOGY

2999Toadfish song reflects male quality
eviscerated body mass (ME). Males (N22) used in this study
averaged 42.9cm (range: 37.9–47.7cm) in total length and 1207g
(range: 857–1612g) in eviscerated mass. The gonads (MG), the
accessory glands (MAG) and the liver (ML) mass were tallied to the
nearest mg. Sonic muscles, which are embedded in the sides of the
swimbladder, were gently cut from the swimbladder wall with a
pair of fine dissection scissors and were also weighed to the nearest
mg (MSM). The mass of the accessory glands was included in the
measurements as they are part of the male reproductive apparatus
and increase in mass during the breeding season in nesting males
(Modesto and Canário, 2003a).
A sample of body muscles was taken (hypaxial muscle fibres)
and the lipid fraction of both somatic and sonic muscles was
quantified as an additional measurement of body and sonic muscle
condition. Lipids are an important source of energy in fish and are
often used as a direct measure of body condition (Chellapa et al.,
1995). Lipids are also one of the major metabolic substrates of sonic
muscles during prolonged aerobic activity (Fine et al., 1986).
All experimental procedures comply with Portuguese animal
welfare laws, guidelines and policies.
Lipid analysis
The fish somatic and sonic muscle homogenates (10g) were
extracted using 30ml of chloroform: methanol (1:2vol./vol.) with
a polytron system. In addition, a saturated sodium chloride solution
(4ml), chloroform containing 50p.p.m. (parts per million) of
butylhydroxytoluene (10ml) and water (10ml) were added to split
the system into an aqueous and an organic phase (Bligh and Dyer,
1959). The mixture was sonicated during 15min. Complete
separation of the two phases was obtained by adding isopropanol;
the total mixture was centrifuged and the chloroform phase
transferred to a weighed tube. The chloroform was evaporated under
nitrogen prior to gravimetrical determination of total lipids.
Statistical analysis
We examined eight potential predictors of call parameters. We
included total length (log10TL) as a metric of body size. We used
residuals of the simple linear regression of sonic muscle mass on
eviscerated body mass (RMSM) as a metric of sonic muscle
hypertrophy. This metric gives a measure of an observed sonic
muscle mass relative to a mean expected value (given by the
regression model) for a given body size. In other words, a male
with a high positive residual of RMSM will have heavier than average
sonic muscles for his size. Likewise we used the residuals of the
simple linear regressions of gonads, accessory glands and liver mass
on eviscerated body mass (RMG, RMAG, RML, respectively) as
metrics of these parameters controlled for the influence of body
size. In addition, we used the residuals of MEon TL (COND) as a
metric of body condition. We log10-transformed TL and mass data
to meet the assumptions of normality and to linearise allometric
relationships. We further considered the lipid content of somatic
and sonic muscles (LipidM and LipidSM, respectively) as possible
predictors of call parameters.
We first generated a correlation matrix of the six measured
call parameters (calling rate and effort, and boatwhistle
characteristics) and morphological traits to examine general
relationships among the variables across all individuals. We then
used multiple regression analysis to assess the statistical
significance of each physical parameter as a predictor of male
mating call parameters with a stepwise selection procedure
(P≤0.05 to add and P≥0.10 to remove).
Because three of our dependent variables, calling rate, calling
effort and pulse period, were highly correlated (Table1), we
included these variables in a factor analysis to generate a single
factor that would combine and explain most of the variance in this
group of variables. Pulse rate (the inverse of pulse period and
equivalent to sonic muscle contraction rate) was used instead of
pulse period because the former had a positive loading in the first
principle component, similar to the other two variables. The scores
of the first factor can be viewed as an index of ‘vocal performance’,
i.e. of length and rate of calling and of muscle contraction rate during
sound production. The first factor explained 75% of the total data
variance with call rate, effort and pulse rate presenting a factor-
loading score of 0.90, 0.89 and 0.81, respectively. We used this first
factor (hereafter called vocal performance) in a multiple regression
analysis with the same eight aforementioned predictors to further
analyse data.
Our final regression models complied with all assumptions of
multiple linear regression. All model residuals were normally
distributed. Further residual analysis was performed using
0.30.5 0.7 0.90.1
DF
0.5 1.0 1.5 2.00
Frequency (kHz)
PP 10 ms
C
0.30.5 0.7 0.90.1
A
D
B
Sound duration
DF
20
10
0
–10
–20
1.0
0.8
0.6
0.4
0.2
0
0
–10
–20
–30
–40
–50
–60
Time (s)
Frequency (kHz) Relative amplitude
Relative amplitude (dB)
Fig.1. Oscillogram (A), sonogram (B) and
power spectrum (D) of a boatwhistle. Sound
duration, the initial (fine continuous line) and
the tonal (fine dashed line) phases of the
boatwhistle are depicted in the oscillogram.
In the sonogram and in the power spectrum
the dominant frequency (DF) of the sound is
shown. (C)Detail of the boatwhistle tonal
phase waveform depicting the pulse period
(PP). Note that the pulse period is inversely
related to the fundamental frequency and not
to the dominant frequency of the boatwhistle.
THE JOURNAL OF EXPERIMENTAL BIOLOGY

3000
Durbin–Watson statistics, residual plots as well as multicollinearity
tests (variance inflation factors, VIF).
All statistical analyses were performed using SPSS for Windows
(16.0, SPSS Inc., Chicago, IL, USA).
RESULTS
Acoustic activity
Acoustic activity varied greatly among subject males. All males
produced boatwhistles (BW) during the study period but calling rate
varied markedly among males and within males. The average calling
rate varied from 0.1 to 361.7BWh–1 per male (overall mean calling
rate39.9BWh–1; Table2). Only seven out of the 22 recorded males
exhibited average calling rates higher than 10BWh–1 during the
study period, and the maximum calling rate observed for each male
varied between 2 and 1071BWh–1 (mean244.5BWh–1). Males
vocalised for a different number of days [mean (range)5 (2–8)]
and for a different number of hours (calling effort, Table2) but
remained in silence most of the time.
Boatwhistle characteristics were consistent with previous
descriptions (e.g. Vasconcelos et al., 2010) and showed a large
between-individual variation (Table2).
Predictors of male acoustic characteristics and activity
Correlation analysis showed that both calling rate and calling effort
were significantly positively related with lipid content of the
somatic muscles, relative gonad mass and relative sonic muscle mass
(Table3). Boatwhistle duration was positively correlated with body
condition (Table3). Pulse period was negatively correlated with lipid
content of the somatic muscles and relative sonic muscle mass
(Table3), indicating that males that exhibited an average faster sonic
muscle contraction rate (i.e. shorter pulse period) had larger sonic
muscles and higher lipid levels in the body.
The best regression models for each dependent variable showed
that calling rate, calling effort and pulse period strongly reflect male
condition measured by the lipid content in the somatic muscles
(Table4). Body lipid showed high partial correlations with calling
rate, calling effort and pulse period (r0.89, 0.94 and –0.61,
respectively, Table4) and accounted for most of the variation
explained by each regression model (Table4). Lipid content of the
somatic muscles explained 77% (out of 82% explained by the full
model, Table4), 88% (out of 94%) and 31% (out of 46%) of calling
rate, calling effort and pulse period variability, respectively. Males
with higher body lipid content showed a significantly higher calling
rate, called for more hours and contracted the sonic muscles faster
during sound production, exhibiting shorter pulse periods in a
boatwhistle (Fig.2). Calling rate was further predicted by relative
sonic muscle mass, which explained a further 5% of its variability.
Males with heavier-than-average sonic muscles called at a higher
rate (Fig.2). Gonad mass also explained 3% of calling effort
variability, and males with relatively larger gonads spent longer
periods calling (Fig.2). Liver mass accounted for an additional 15%
of pulse period variation, and males with heavier livers for a given
size tended to contract the sonic muscles faster during sound
production as observed by their shorter pulse periods (Fig.2).
Consistently, vocal performance, a new variable that combined the
former dependent variables was only predicted by the lipid content
of the somatic muscles, the strongest predictor for these three
acoustic parameters (Table4).
Additionally, relative sonic muscle mass showed a weaker but
significant positive effect on amplitude modulation explaining 22%
of its variation (Table4; Fig.2). Both boatwhistle duration and
dominant frequency were not predicted by any of the independent
variables as regression models were not significant (P>0.05).
DISCUSSION
The boatwhistle produced by Lusitanian toadfish nesting males is
the major mate attraction signal and therefore essential for male
reproductive success (dos Santos et al., 2000) [see Bass and
McKibben (Bass and McKibben, 2003) for other batrachoidids].
This call shows a large inter-individual variation (Amorim and
Vasconcelos, 2008) (present work) and could thus be used to
discriminate among males. But can females use acoustic cues to
choose a mate? Our work shows that male vocal activity and mating
call characteristics reflect several aspects of male quality.
Calling rate and calling effort
High calling rate and increased calling effort strongly reflected good
male body condition measured by the lipid content of the somatic
muscles. The lipid fraction of the body
muscles explained 77% and 88% of the
variability of calling rate and effort observed
over a week in our focal males. In animals
where males provide parental care,
indicators of male parental ability such as
body condition are expected to play a
substantial role in intersexual
communication and be under strong mate
selection by females (Andersson, 1994).
Fish unguarded eggs are quickly eaten by
M. C. P. Amorim and others
Table1. Correlations between the six acoustic variables showing the strong relations between calling rate, calling effort and pulse period
Calling rate Calling effort Duration Pulse period Dominant frequency Amplitude modulation
Calling rate – 0.76*** 0.00 –0.67*** 0.27 0.41
Calling effort – –0.06 –0.55* 0.20 0.33
Duration – –0.31 0.00 0.00
Pulse period – –0.12 –0.52*
Dominant frequency – –0.07
Amplitude modulation –
Values shown are Spearman rank correlation coefficients. Significant differences are indicated by asterisks, i.e. *
P
<0.05; ***
P
<0.001;
P
-values are uncorrected
for multiple tests.
N
21 except for the correlation between calling rate and calling endurance where
N
22.
Table 2. Descriptive statistics for the dependent acoustic variables
N
Mean s.d. Range c.v.
Calling rate 22 39.9 87.7 0.1–361.7 2.19
Calling effort 22 30.7 20.1 2.4–67.7 0.66
Boatwhistle duration (ms) 21 681.9 154.4 383.2–1049.7 0.23
Pulse period (ms) 21 19.2 1.1 17.4–22.4 0.06
Dominant frequency (Hz) 21 117.9 35.0 52.7–181.1 0.30
Amplitude modulation 21 0.7 0.2 0.5–1.3 0.26
Coefficient of variation (c.v.)s.d./mean.
THE JOURNAL OF EXPERIMENTAL BIOLOGY

3001Toadfish song reflects male quality
predators and females must rely on male brood protection for the
survival of their offspring (Sargent and Gross, 1993). Consequently,
females benefit from choosing good fathers, and more so if they
are single spawners such as batrachoidids (Brantley and Bass, 1994;
Modesto and Canário, 2003a). Parental care in the Lusitanian
toadfish is costly because type I males experience reduced feeding,
fan the eggs and defend their nest vigorously for at least 30 days
(till the fry becomes free swimming) (Modesto and Canário, 2003a;
Vasconcelos et al., 2010), consistent with the marked decrease in
the male’s condition (hepatossomatic index and the Fulton’s
condition factor, K) during the spawning season (Modesto and
Canário, 2003a). Our results suggest that Lusitanian toadfish females
should favour males that call at a higher rate and for prolonged
periods, as they would be in better condition and could provide better
parental care. Consistently, larger nesting males of the batrachoidid
P. notatus sampled at the end of the breeding season presented higher
body condition (K) and a larger number of viable late-stage embryos
in the nest (Sisneros et al., 2009), suggesting that body condition
is an honest indicator of parental ability in batrachoidids. Similarly,
in the sand goby (Pomatoschistus minutus), another teleost with
prolonged male parental care, only males with adequate body
condition initiate nest building and breeding (Lindström, 1998a),
and food supplemented males stay longer at the nest, mate sooner
and manage to get more eggs than non-fed males with lower
condition (Lindström, 1998b). Parental common goby
(Pomatoschistus microps) males with higher energy reserves are
also probably less likely of filial cannibalism than males with lower
condition, as observed in other fish (Kvarnemo et al., 1998). Future
work will need to address whether calling rate and effort reflect
male parental ability in the Lusitanian toadfish. In birds, call rate
and other song features may be reliable indicators of parental quality
(Dolby et al., 2005).
In our study species, the ability to call at higher rates and to sustain
calling for longer periods also reveal, although to a much smaller
Table 3. Relationship between male physical and acoustic characteristics (Spearman rank correlation)
Calling rate Calling effort Duration Pulse period Dominant frequency Amplitude modulation
LipidM
r
0.51* 0.61** 0.03 –0.60** –0.05 0.50*
N
20 20 20 20 20 20
LipidSM
r
–0.06 –0.09 –0.25 0.39 0.33 –0.37
N
22 22 21 21 21 21
log10
TL r
–0.27 –0.18 –0.02 0.30 –0.15 –0.21
N
22 22 21 21 21 21
RM
G
r
0.49* 0.63** 0.05 –0.31 0.07 0.14
N
22 22 21 21 21 21
RM
AG
r
0.36 0.42 0.28 –0.29 –0.08 0.41
N
22 22 21 21 21 21
RM
L
r
0.12 –0.09 0.15 –0.30 –0.19 0.09
N
22 22 21 21 21 21
RM
SM
r
0.58** 0.47* 0.21 –0.53* 0.13 0.41
N
22 22 21 21 21 21
COND
r
0.04 0.08 0.51* –0.35 –0.35 0.07
N
22 22 21 21 21 21
Significant differences are indicated by asterisks, i.e. *
P
<0.05; **
P
<0.01;
P
-values are uncorrected for multiple tests. LipidM – total lipid content of somatic
muscles; LipidSM – total lipid content of sonic muscles.
TL
– total length;
RM
G– residuals of gonad mass;
RM
AG – residuals of accessory gland mass;
RM
L– residuals of liver mass;
RM
SM – residuals of sonic muscle mass; COND – body condition.
Table4. Table for predictors of male call parameters (calling rate, calling effort and boatwhistle characteristics)
Included Model
Dependent variable predictor
B
s.e.m.
tPr F
significance
R
2DW VIF
Calling performance Intercept –1.50 0.36 –4.19 0.001
LipidM 3.65 0.78 4.68 <0.001 0.74
F
1,1821.90
P
<0.001 0.55 1.7 1.00
Calling rate LipidM 2.11 0.26 8.18 <0.001 0.89 1.04
RM
SM 0.27 0.12 2.38 0.03 0.49
F
2,1841.92
P
<0.001 0.82 1.8 1.04
Calling effort LipidM 73.69 6.11 12.06 <0.001 0.94 1.05
RM
G6.46 2.68 2.41 0.03 0.49
F
2,1885.86
P
<0.001 0.91 1.3 1.05
Pulse period Intercept 20.49 0.44 46.22 <0.001
LipidM –3.03 0.96 –3.14 0.006 –0.61 1.00
RM
L–0.44 0.21 –2.15 0.046 –0.46
F
2,177.13
P
0.006 0.46 2.0 1.00
Amplitude modulation Intercept 0.736 0.039 18.69 <0.001
RM
SM 0.086 0.038 2.27 0.036 0.47
F
1,185.16
P
0.04 0.22 1.9 1.00
Calling rate was log10 (
x
+1)-transformed to meet the linear regression model assumptions.
r
– partial correlation between the dependent variable and the
predictor, controlling for the effects of the other predictors in the model. LipidM – total lipid content in the somatic muscles.
RM
SM – residuals of sonic
muscle mass.
RM
G– residuals of gonad mass.
RM
L– residuals of liver mass. Results are from multiple regression analysis (stepwise procedure).
Regression models for boatwhistle duration and dominant frequency were not significant.
THE JOURNAL OF EXPERIMENTAL BIOLOGY

3002
extent, a higher degree of sonic muscle hypertrophy and relative
larger gonad mass. Muscle hypertrophy has been generally
associated with a higher capacity for calling (e.g. Connaughton et
al., 1997). The sonic muscle hypertrophy in H. didactylus males
observed in association to the reproductive season is accompanied
by an increase of total myofibril and sarcoplasm area and breeding
males show a larger sarcoplasm/myofibril area ratio than females
(Modesto and Canário, 2003b). A higher sarcoplasm/myofibril area
ratio has been interpreted as an adaptation to the increased speed
and fatigue resistance needed for boatwhistle production in the oyster
toadfish, Opsanus tau (Fine et al., 1990) and an increased
myofibrillar area should allow for more forceful contractions
resulting in higher amplitude calls (Connaughton et al., 1997).
Hence, Lusitanian toadfish males with larger sonic muscles should
M. C. P. Amorim and others
3
2
1
00 0.2 0.4 0.6 0.8 1.0
0 0.2 0.4 0.6 0.8 1.0
0 0.2 0.4 0.6 0.8 1.0
80
60
40
20
0
23
22
21
20
19
18
17
3
2
1
0
80
60
40
20
0
23
22
21
20
19
18
17
–3 –2 –1 0 1 2 3
RMSM
–3 –2 –1 0 1 2 3
RMG
–3 –2 –1 0 1 2 3
RML
–3 –2 –1 0 1 2 3
RMSM
1.5
1.0
0.5
Calling rate (BW h–1)
Calling effort
Pulse period (ms)
Amplitude modulation
LipidM
LipidM
LipidM
Fig.2. Relationship between
predictor variables of male
quality and call parameters.
Lines of univariate regressions
and 95% confidence interval
bands are shown. LipidM – lipid
content of body muscles;
RM
SM
– residuals of sonic muscle
mass;
RM
G– residuals of gonad
mass;
RM
L– residuals of liver
mass.
THE JOURNAL OF EXPERIMENTAL BIOLOGY

3003Toadfish song reflects male quality
sustain high calling rates and call at higher amplitudes, resulting in
a more conspicuous vocal output. The significant effect of relative
gonad mass suggests that calling rate and effort signal male mating
motivation as shown for fish and other vertebrates [e.g. fish (Fish,
1972); anurans (Burmeister and Wilczynski, 2001); mammals
(Vannoni and McElligott, 2009)]. Also, a higher than average calling
rate may signal other male traits such as a better immune system
[e.g. insects (Jacot et al., 2004)] or a higher fertilisation success
[e.g. anurans (Pfennig, 2000)].
Interestingly sonic muscle lipid content did not seem to predict
differences in the ability to sustain high calling rates over a week
but perhaps differences in lipid concentration would be visible in
the vocal performance of males in a longer time scale. For example,
Connaughton et al. (Connaughton et al., 1997) observed a
pronounced decrease in sonic muscle lipid in the weakfish
(Cynoscion regalis) during the peak of acoustic activity, which is
a month past the start of a high calling activity in this species.
Boatwhistle characteristics
Males in better condition (body lipid and higher liver mass) had shorter
pulse periods and hence contracted the sonic muscles faster during
sound production. However, we found a low between-male variability
for this parameter (c.v.6%), consistent with the existence of a vocal
central pattern generator in the hindbrain of batrachoidids that
establishes the patterned activity of the sonic muscles and hence the
pulse period of their calls (Bass and Baker, 1990). Consistently, O.
tau males also present a similar variability of fundamental frequency
of boatwhistles (c.v.6%) (Barimo and Fine, 1998), which is the
inverse of the pulse period. Lusitanian toadfish males that contract
the sonic muscles at a very fast rate could reliably be indicating to
females their better quality (condition) with the ability to sustain sonic
muscle contraction close to their physiological limit. Consistent with
this suggestion, males of the non-passerine bird brown skuas that
produce long difficult calls close to their performance limit are
honestly advertising their quality because they have a higher breeding
success and fledge more chicks (Janicke et al., 2008).
Relative sonic muscle mass showed a significant positive effect
on boatwhistles amplitude modulation. Amplitude modulation is an
important characteristic to distinguish boatwhistle emitted by nesting
males in different motivational contexts. Vasconcelos et al.
(Vasconcelos et al., 2010) have shown that the Lusitanian toadfish
also emits boatwhistles during territorial intrusions by other males
but these lack amplitude modulation, which seems characteristic of
a mating context. Consequently, it is possible that males can also
advertise their quality and motivation by increasing the amplitude
modulation of the mating boatwhistle, although this suggestion needs
to be tested.
Both boatwhistle duration and dominant frequency showed high
variability and were not predicted by any of the considered
independent variables. Boatwhistle duration was very variable
among males (see also Barimo and Fine, 1998; Amorim and
Vasconcelos, 2008). This parameter seems motivation dependent
in batrachoidids (M.C.P.A., P.J.F. and J.M.S., unpublished data)
(Thorson and Fine, 2002; Remage-Healey and Bass, 2005) and
probably translates the male’s physiological state (Remage-Healey
and Bass, 2005). Although pulse period (and hence the fundamental
frequency) showed little variability, the same was not true for the
dominant frequency because it may be represented by the
fundamental or by the first or the second harmonic (Amorim and
Vasconcelos, 2008).
Here, we have shown that calling rate, calling effort, pulse period
and amplitude modulation may honestly signal male quality, namely
male condition, spawning readiness and sonic muscle hypertrophy
in a batrachoidid. A recent study with P. notatus has shown that
females reveal best hearing sensitivity matching the higher harmonic
components of male advertisement calls during the breeding season
(Sisneros, 2009). This suggests the action of selective pressures for
these females to better detect and probably choose among different
males in dense breeding aggregations that are typical of
batrachoidids and other sound-producing teleosts. Although not
tested in fish, calling rate and time spent calling may influence
female choice in other species such as in insects and anurans
(reviewed in Gerhardt and Huber, 2002), in birds (e.g. Gentner and
Hulse, 2000) and in mammals (e.g. McComb, 1991). Calling
activity is also likely to be an important parameter for mate choice
in fish because sound duty cycle (the proportion of sound in a
stimulus) influences female preference (McKibben and Bass, 1998).
Whether females benefit from better brood care, better territories,
good genes or just ease of male location, remain still to be addressed
in fish.
LIST OF SYMBOLS AND ABBREVIATIONS
BW boatwhistle
COND residuals of the simple linear regressions of eviscerated body
mass on total length
LipidM lipid content of somatic muscles
LipidSM lipid content of sonic muscles
MAG accessory glands’ mass
MEeviscerated body mass
MGgonads’ mass
MLliver mass
MSM sonic muscle mass
RMAG residuals of the simple linear regressions of accessory glands
on eviscerated body mass
RMGresiduals of the simple linear regressions of gonad on
eviscerated body mass
RMLresiduals of the simple linear regressions of liver on
eviscerated body mass
RMSM residuals of the simple linear regressions of sonic muscles on
eviscerated body mass
TL total length
ACKNOWLEDGEMENTS
We would like to thank the Air Force Base No. 6 of Montijo (Portugal) for allowing
this study in their military establishment. This research was funded by the Science
and Technology Foundation, Portugal (project PDCT/MAR/58071/2004,
pluriannual program UI&D 331/94 and UI&D 329, grants SFRH/BPD/14570/2003
and SFRH/BPD/41489/2007).
REFERENCES
Amorim, M. C. P. and Vasconcelos, R. O. (2008). Variability in the mating calls of
the Lusitanian toadfish
Halobatrachus didactylus
: potential cues for individual
recognition.
J. Fish Biol.
72, 1355-1368.
Amorim, M. C. P., Vasconcelos, R. O., Marques, J. F. and Almada, F. (2006).
Seasonal variation of sound production in the Lusitanian toadfish,
Halobatrachus
didactylus
.
J. Fish Biol.
69, 1892-1899.
Amorim, M. C. P., Vasconcelos, R. O. and Parreira, B. (2009). Variability in the
sonic muscles of the Lusitanian toadfish (
Halobatrachus didactylus
): acoustic signals
may reflect individual quality.
Can. J. Zool.
87, 718-725.
Amorim, M. C. P., Simões, J. M., Fonseca, P. J. and Almada, V. C. (2010). Patterns
of shelter usage and social aggregation by the vocal Lusitanian toadfish.
Mar. Biol
.
157, 495-503.
Andersson, M. (1994).
Sexual Selection
. Princeton: Princeton University Press.
Bass, A. H. and Baker, R. (1990). Sexual dimorphisms in the vocal control system of
a teleost fish: morphology of physiologically identified neurons.
J. Neurobiol.
21,
1155-1168.
Bass, A. H. and McKibben, J. R. (2003). Neural mechanisms and behaviors for
acoustic communication in teleost fish.
Prog. Neurobiol.
69, 1-26.
Barimo, J. F. and Fine, M. L. (1998). Relationship of swim-bladder shape to the
directionality pattern of underwater sound in the oyster toadfish.
Can. J. Zool.
76,
134-143.
Bligh, E. and Dyer, W. (1959). A rapid method of total lipid extraction and purification.
Can. J. Biochem. Physiol.
37, 911-917.
Bradbury, J. W. and Vehrencamp, S. L. (1998).
Principles of Animal Communication
.
Sunderland: Sinauer Associates.
THE JOURNAL OF EXPERIMENTAL BIOLOGY

3004
Brantley, R. K. and Bass, A. H. (1994). Alternative male spawning tactics and
acoustic signals in the plainfin midshipman fish,
Porichthys notatus
(Teleostei,
Batrachoididae).
Ethology
96, 213-232.
Burmeister, S. S. and Wilczynski, W. (2001). Social context influences androgenic
effects on calling in the green treefrog (
Hyla cinerea
).
Horm. Behav.
40, 550-558.
Chellappa, S., Huntingford, F. A., Strang, R. H. C. and Thomson, R. Y. (1995).
Condition factor and hepatosomatic index as estimates of energy status in male
three-spined stickleback.
J. Fish Biol.
47, 775-787.
Connaughton, M. A., Fine, M. L. and Taylor, M. H. (1997). The effects of seasonal
hypertrophy and atrophy on fiber morphology, metabolic substrate concentration and
sound characteristics of the weakfish sonic muscle.
J. Exp. Biol.
200, 2449-2457.
Costa, J. L. (2004). The biology of the Lusitanian toadfish,
Halobatrachus didactylus
(Bloch and Schneider 1801), and its role in the structuring and functioning of the
biological communities; special reference to the Mira estuary population. PhD
Dissertation, University of Lisbon, Portugal.
Dolby, A. S., Clarkson, C. E., Haas, E. T., Miller, J. K., Havens, L. E. and Cox, B.
K. (2005). Do song-phrase production rate and song versatility honestly
communicate male parental quality in the Gray Catbird?
J. Field Ornithol.
76, 287-
292.
dos Santos, M., Modesto, T., Matos, R. J., Grober, M. S., Oliveira, R. F. and
Canário, A. (2000). Sound production by the Lusitanian toadfish,
Halobatrachus
didactylus
.
Bioacoustics
10, 309-321.
Fine, M. L. and Lenhardt, M. L. (1983). Shallow-water propagation of the toadfish
mating call.
Comp. Biochem. Physiol. A
76, 225-231.
Fine, M. L., Pennypacker, K. R., Drummond, K. A. and Blem, C. R. (1986).
Concentration and location of metabolic substrates in fast toadfish sonic muscle.
Copeia
1986, 910-915.
Fine, M. L., Burns, N. M. and Harris, T. M. (1990). Ontogeny and sexual dimorphism
of sonic muscle in the oyster toadfish.
Can. J. Zool
. 68, 1374-1381.
Fish, J. F. (1972). The effect of sound playback on the toadfish. In
Behavior of Marine
Animals,
Vol. 2 (ed. H. E. Winn and B. Olla), pp. 386-434. New York: Plenum Press.
Gentner, T. Q. and Hulse, S. H. (2000). Female European starling preference and
choice for variation in conspecific male song.
Anim. Behav
. 59, 443-458.
Gerhardt, H. C. and Huber, F. (2002).
Acoustic Communication in Insects and
Anurans: Common Problems and Diverse Solutions.
Chicago and London: The
University of Chicago Press.
Jacot, A., Scheuber, H. and Brinkhof, M. W. G. (2004). Costs of an induced immune
response on sexual display and longevity in field crickets.
Evolution
58, 2280-2286.
Janicke, T., Hahn, S., Ritz, M. S. and Peter, H.-U. (2008). Vocal performance reflects
individual quality in a nonpasserine.
Anim. Behav
. 75, 91-98.
Knapp, R. A. and Kovach, J. T. (1991). Courtship as an honest indicator of male
parental quality in the bicolor damselfish,
Stegastes partitus
.
Behav. Ecol.
2, 295-
300.
Kvarnemo, C., Svensson, O. and Forsgren, E. (1998). Parental behaviour in relation
to food availability in the common goby.
Anim. Behav
. 56, 1285-1290.
Ladich, F. (2004). Sound production and acoustic communication. In
The Senses of
Fish Adaptations for the Reception of Natural Stimuli
(ed. G. von der Erude, J.
Mogdans and B. G. Kapoor), pp. 210-230. New Delhi: Kluwer, Dordrecht and
Narosa.
Lindström, K. (1998a). Effects of costs and benefits of brood care on filial cannibalism
in the sand goby.
Behav. Ecol. Sociobiol
. 42, 101-106.
Lindström, K. (1998b). Energetic constraints on mating performance in the sand goby.
Behav. Ecol.
9, 297-300.
Maynard Smith, J. and Harper, D. (2003).
Animal Signals
. Oxford: Oxford University
Press.
McComb, K. E. (1991). Female choice for high roaring rates in red deer,
Cervus
elaphus. Anim. Behav.
41, 79-88.
McKibben, J. R. and Bass, A. H. (1998). Behavioral assessment of acoustic
parameters relevant to signal recognition and preference in a vocal fish.
J. Acoust.
Soc. Am.
104, 3520-3533.
Modesto, T. and Canário, A. V. M. (2003a). Morphometric changes and sex steroid
levels during the annual reproductive cycle of the Lusitanian toadfish,
Halobatrachus
didactylus
.
Gen. Comp. Endocrinol.
131, 220-231.
Modesto, T. and Canário, A. V. M. (2003b). Hormonal control of the swimbladder
sonic muscles dimorphism in the Lusitanian toadfish
Halobatrachus didactylus
.
J.
Exp. Biol.
206, 3467-3477.
Myrberg, A. A., Jr, Mohler, M. and Catala, J. (1986). Sound production by males of a
coral reef fish (
Pomacentrus partitus
): its significance to females.
Anim. Behav
. 24,
923-933.
Pfennig, K. S. (2000). Female spadefoot toads compromise on mate quality to ensure
conspecific matings.
Behav. Ecol
. 11, 220-227.
Remage-Healey, L. and Bass, A. H. (2005). Simultaneous, rapid, elevations in steroid
hormones and vocal signalling during playback challenge: a field experiment in Gulf
toadfish.
Horm. Behav
. 47, 297-305.
Ryan, M. J., Fox, J. H., Wilczynski, W. and Rand, A. S. (1990). Sexual selection for
sensory exploitation in the frog
Physalaemus pustulosus
.
Nature
343, 66-67.
Sargent, R. C. and Gross, M. R. (1993). William’s principle: an explanation of parental
care in teleost fishes. In
Behaviour of teleost fishes
(ed. T. J. Pitcher), pp. 333-361.
London: Chapman and Hall.
Sisneros, J. A. (2009). Seasonal plasticity of auditory saccular sensitivity in the vocal
plainfin midshipman fish,
Porichthys notatus
.
J. Neurophysiol.
102, 1121-1131.
Sisneros, J. A., Alderks, P. W., Leon, K. and Sniffen, B. (2009). Morphometric
changes associated with the reproductive cycle and behaviour of the interidal-
nesting, male plainfin midshipman fish,
Porichthys notatus
.
J. Fish Biol.
74,18-36.
Thorson, R. F., Fine, M. L. (2002). Crepuscular changes in emission rate and
parameters of the boatwhistle advertisement call of the gulf toadfish,
Opsanus beta
.
Environ. Biol. Fish
. 63, 321-331.
Vannoni, E. and McElligott, A. G. (2009). Fallow bucks get hoarse: vocal fatigue as a
possible signal to conspecifics.
Anim. Behav.
78, 3-10.
Vasconcelos, R. O., Simões, J. M., Almada, V. C., Fonseca, P. J. and Amorim, M.
C. P. (2010). Vocal behaviour during territorial intrusions in the Lusitanian toadfish:
boatwhistles also function as territorial ‘keep-out’ signals.
Ethology
116, 155-165.
Vehrencamp, S. L. (2000). Handicap, index, and conventional signal elements of bird
song. In
Animal Signals: Signaling and Signal Design in Animal Communication
(ed.
Y. Espmark, T. Amundsen and G. Rosenqvist), pp. 277-300. Trondheim, Norway:
Tapir Academic Press.
Zahavi, A. (1975). Mate selection: a selection for a handicap.
J. Theor. Biol
. 53, 205-
214.
M. C. P. Amorim and others
THE JOURNAL OF EXPERIMENTAL BIOLOGY