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Vocal behavior predicts reproductive success in a teleost fish

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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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Behavioral Ecology
doi:10.1093/beheco/arr199
Advance Access publication 12 December 2011
Original Article
Vocal behavior predicts reproductive success in
a teleost fish
Raquel O. Vasconcelos,
a
Rita Carricxo,
a
Andreia Ramos,
a
Teresa Modesto,
b
Paul J. Fonseca,
a
and
M. Clara. P. Amorim
c
a
Departamento de Biologia Animal, Centro de Biologia Ambiental, Faculdade de Cieˆncias da
Universidade de Lisboa, Bloco C2 Campo Grande, 1749-016 Lisbon, Portugal,
b
Centro de Cieˆncias do
Mar, Universidade do Algarve, Campus de Gambelas, 8000-810 Faro, Portugal, and
C
Unidade de
Investigacxa
˜o em Eco-Etologia, I.S.P.A. – Instituto Universita´rio, Rua Jardim do Tabaco 34, 1149-041
Lisbon, Portugal
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
Many studies on communication systems have centered on
the relationship between signals and reproductive suc-
cess. Determining the characteristics of signals that lead to en-
hanced mating success may help in understanding how
a communication system may have evolved and how sexual se-
lection may have shaped signaling (Andersson 1994;Bradbury
and Vehrencamp 1998).
Acoustic signals are well-known examples of sexually se-
lected traits typically used by females of several taxa to identify,
locate, and select between potential mates (Andersson 1994;
Bradbury and Vehrencamp 1998). The effect of acoustic sig-
naling on mate attraction has been broadly investigated pri-
marily in insects, anurans, and birds. These studies reported
that features of males’ calling, such as song complexity, rep-
ertoire size, amplitude, singing effort, and conspecific acous-
tic interactions, can be indicators of individual quality. This
can be related with males’ resources, health condition, learn-
ing ability, developmental resilience to stress or social skills
(e.g., Searcy and Andersson 1986;Kroodsma and Byers 1991;
Nordby et al. 1999;White et al. 2010).
Teleost fishes most likely represent the largest group of sound-
producing vertebrates that have evolved a variety of mechanisms
to produce vocalizations crucial to social interactions, including
mate attraction (Ladich & Myrberg 2006;Myrberg and Lugli
2006). However, a link between vocal behavior and reproductive
success has never been directly shown. Few studies have dem-
onstrated the role of certain acoustic signals in mate choice and
related sound features with male size. For example, females of
the bicolor damselfish (Stegastus partitus, Pomacentridae) prefer
not only males that produce courtship chirp sounds of lower
dominant frequency, which are positively correlated with body
size (Myrberg et al. 1986), but also males with higher rates of
courtship visual displays (Knapp and Kovach 1991). A male’s
courtship rate correlates positively with mating success and sub-
sequent egg survival, suggesting that chirp sound rate may also
relate to reproductive success; however, this has never been di-
rectly shown (Knapp and Kovach 1991). The existence of re-
dundant multimodal signals has been previously reported
(revised in Partan and Marler 2005). Male vocal features, such
as dominant frequency, seem to be a common indicator of body
size in vocal fish species (e.g., croaking gouramis, Ladich et al.
1992; mormyrids, Crawford 1997).
Representatives of the family Batrachoididae, which include
toadfishes and the plainfin midshipman fish (Porichthys notatus),
have emerged as one of the main study models for both behav-
ioral and neurobiological studies in fish acoustic communica-
tion (Bass and McKibben 2003). Winn (1972) reported female
phonotaxis toward a male whose mate advertising signals, called
boatwhistles, were emitted at higher calling rates (CRs) in
Address correspondence to Raquel Vasconcelos. E-mail: rfvasconcelos@
fc.ul.pt.
Received 19 May 2011; revised 23 October 2011; accepted 23
October 2011.
The Author 2011. Published by Oxford University Press on behalf of
the International Society for Behavioral Ecology. All rights reserved.
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Opsanus tau. Female midshipman fish also showed phonotaxis
toward hum-like (mate attraction) sounds, namely longer, high-
er amplitude and higher fundamental frequency tone stimuli
(McKibben and Bass 1988). Recently, Amorim et al. (2010)
reported that male vocal activity and fine acoustic features of
mating call (boatwhistle) reflect several aspects of male mor-
phological characteristics in another batrachoidid, the Lusita-
nian toadfish (Halobatrachus didactylus). Males that contracted
the sonic muscles at faster rates, as shown by the shorter boat-
whistle pulse periods, were in better condition (increased
body lipid and higher liver mass), and boatwhistle amplitude
modulation reflected the degree of sonic muscle hypertrophy.
Besides, this study also suggested that Lusitanian toadfish males
advertise their quality based on boatwhistle CR and calling ef-
fort (CE), which was correlated with measurements of physical
condition.
The major goal of this work was to verify whether vocal behav-
ior (CR, CE, and signal features) can predict reproductive suc-
cess, as measured by the number of eggs in a male’s nest, in
a vocal teleost. For this purpose, we tested the Lusitanian toad-
fish (H. didactylus), which strongly depends on acoustic com-
munication for mating; males are polygynous, defend nests,
and provide parental care. We also investigated the influence
of sex steroids (11-ketotestosterone [11-KT] and testosterone
[T]) that typically peak in the breeding season (Modesto and
Cana´rio 2003a), in the vocal performance and reproductive
success. Moreover, the morphometric traits of males were also
related with vocal parameters and number of eggs.
We used the Lusitanian toadfish as the study species for var-
ious reasons. This species relies heavily on acoustic communi-
cation to find mates in the breeding season. Halobatrachus
didactylus exhibits a rich vocal repertoire rare among fishes,
which comprises at least 5 different vocalizations (Amorim
et al. 2008), including a complex amplitude-modulated adver-
tisement call (Amorim and Vasconcelos 2008). Phylogenetic
analysis indicated that Lusitanian toadfish represents a basal
lineage in the Batrachoididae, providing an excellent model
for understanding integrated mechanisms underlying the evo-
lution of acoustic communication in fishes (Rice and Bass
2009). Moreover, the Lusitanian toadfish is highly tolerant
to experimental manipulations, displays the full acoustic rep-
ertoire, and mates in seminatural situations (Amorim et al.
2010;Vasconcelos et al. 2010).
METHODS
Study species
The Lusitanian toadfish, H. didactylus (Batrachoididae), is a ben-
thic marine fish that inhabits estuaries and coastal zones of the
Eastern Atlantic and the Mediterranean (Roux 1986). During
the reproductive season, from May to July in Portugal, territo-
rial males (‘‘type I’’) build nests under rocks in aggregations in
shallow waters and attract females to spawn by emitting long
advertisement calls (boatwhistles), forming conspicuous cho-
ruses (Amorim et al. 2006;Amorim and Vasconcelos 2008).
Besides boatwhistles, Lusitanian toadfish also produces other
pulsed sounds, such as grunt trains, long grunt trains, croaks,
double croaks, and associations between some of these calls
(Amorim et al. 2008). Some of these sounds are known to be
used during agonistic interactions, such as territorial defense
(e.g., grunt train, Vasconcelos and Ladich 2008;Vasconcelos
et al. 2010), but the function of such vocal plasticity remains
unclear. Females deposit the eggs on the roof of the nest, which
are guarded by the male until the offspring are free swimming
(dos Santos et al. 2000). Like other batrachoidids, this species is
sexually dimorphic: sneaker or ‘‘type II’’ males are typically small-
er, with a higher gonadosomatic index and smaller sonic muscles
relativeto type I male (Modesto and Cana´rio 2003a,2003b), and
attempt opportunistic fertilizations by parasitizing the nests.
Test subjects
Prior to the onset of the breeding season, 60 concrete nests
were placed along an intertidal area of the Tagus River estuary
(Military Air Force Base, Montijo, Portugal) to create an aggre-
gation of artificial shelters that were easily accessible at low
tides during the whole breeding season, from May to July.
These hemicylinder-shaped nests (internal dimensions: 50
cm long, 30 cm wide, and 20 cm high) were placed along
the shore approximately 1.5 m apart in 2 rows.
We used a group of these nests (6 or 7) to confine type I
toadfish males that spontaneously occupied these shelters. In
total, we recorded 56 males (34–49.5 cm total length [TL];
627–2097 g eviscerated body mass [ME]). At the end of the
recording protocol, tested type I males did not differ in body
mass and body condition from the other territorial type I
males found in the nests along the shore in the study area
(One-way ANOVA: F
1,77
¼0.44–0.75; P.0.05). The male
morphotype was easily identified on the basis of body size
and secretion of their large accessory glands when gently
pressed near the vent (Modesto and Cana´rio 2003a). All
animals used in this study, including conspecifics attracted
to the experimental nests, were dissected after being sacri-
ficed with an excessive dosage of MS222 (tricaine methane
sulphonate; Pharmaq, Norway) at the end of the study.
Each subject was measured to the nearest millimeter for TL
and to the nearest gram for ME. The gonads (M
G
), the male
accessory glands (M
AG
), and the liver (M
L
) mass were tallied
to the nearest milligram. Sonic muscles, which are embedded
in the swimbladder walls, were gently cut and weighed to the
nearest milligram (M
SM
). All experimental procedures com-
plied with Portuguese animal welfare laws, guidelines, and
policies.
Figure 1
Oscillogram of a typical mate advertisement boatwhistle produced by
a type I male Lusitanian toadfish during the reproductive season (a).
Experimental setup (b), showing a vocal type I toadfish male (MI
N
)
confined inside the artificial nest, a female (F) inside the nest laying
eggs and a satellite type I toadfish male (MI
S
) that was often found
outside the nest close to the entrance. A hydrophone (H) was placed
in front of the nest entrance in order to record male’s vocal activity.
Illustration by Marta Bolgan.
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Experimental setup
The vocal activity of 9 different groups of type I males was
recorded over a period of 6 h per day (centered in the full
tide) for about 2 weeks. Each experimental group was com-
posed of 6 or 7 fish. These recordings were performed
throughout the peak of the toadfish breeding season (May–
July) in 2008 and 2010.
Nests were surrounded with a plastic net to prevent vocal type
I males fromescaping and to ensure individual identity through-
out the recordings (see Vasc oncelos et al. 20 10;Figure 1).
A small opening (10 cm wide, 5 cm high) was created at the
entrance of each of these nests to allow females and, eventually,
small type I or type II males to enter. These are typically smaller
(generally TL ,30 cm and body mass ,500 g) than the tested
type I males (Amorim et al. 2009). Plastic nets did not affect
propagation of acoustic signals and allowed possible visual in-
teractions with free-swimming conspecifics, as well as the en-
trance of prey items in the nest. All unoccupied nests within
15 m from a subject male were also wrapped in plastic nets to
prevent further occupations during the study.
One hydrophone (High Tech 94 SSQ, Gulfport, MS; fre-
quency range: 30 Hz–6 kHz, 61 dB; voltage sensitivity: 2165
dB re. 1 V/lPa) was placed at about 10 cm from the entrance
of each experimental nest and 10 cm up from the substrate.
The recording system also included audio capture devices
Edirol UA-25 (Roland, 16 bit, 6 kHz acquisition rate per chan-
nel) connected to a laptop to perform simultaneous multi-
channel recordings, which were controlled with Adobe
Audition 2.0 (Adobe Systems Inc., 2005). Sounds captured
from each hydrophone were stored in approximately 60-min
duration wave files and, therefore, about 6 recording sessions
were acquired per day for each fish.
Estuary water temperature during the recording period
ranged between 19.5 and 24 C, and the water level varied
from air exposure in the lower spring tides up to 2.8 m at
high tide.
Every 2 weeks, when the tide was low enough to access the
nesting experimental area (spring tides), any fish found
outside close to the subject males’ nests (i.e., partially un-
der or on the side near the entrance) were identified (sex
and male type), euthanized, and dissected in the laboratory.
This procedure was only possible in the first breeding cycle
(2008) when there were more specimens spawning in the
study area.
Recorded type I males were also removed, anesthetized in an
MS222 bath, and blood samples were collected within 5 min of
handling the specimen. After finishing the recordings and re-
moving the type I males from the respective nest, a photograph
was taken whenever there were eggs attached to the roof of the
nest. The number of eggs that seemed intact and viable (e.g.,
remains of egg membranes without yolk were not considered)
was determined using the software Image J (Wayne Rasband,
National Institute of Health). This software allowed analyzing
the pictures with detail and marking each egg. All eggs were
recognized and counted individually.
Sound analysis
All test fish produced several types of vocalizations that in-
cluded agonistic grunts, croaks, and mate-advertising boat-
whistles. We only quantified and analyzed the boatwhistles
(Figure 1a) because these are the signals used for nest owner-
ship advertisement and to attract females during the repro-
ductive season in Batrachoididae (McKibben and Bass 1998;
Amorim and Vasconcelos 2008). Acoustic analysis was per-
formed using Raven 1.2 for Windows (Bioacoustics Research
Program, Cornell Laboratory of Ornithology, Ithaca, NY).
Recorded sounds could be attributed to particular nest hold-
ers due to proximity of the hydrophones to the subject males
and because of the high sound attenuation along short dis-
tances with low water depth (21 dB between nests).
The following parameters were calculated for each individ-
ual: mean CR (CR
mean
), as the averaged number of sounds
emitted per hour (i.e., per recording session); maximum CR
(CR
max
), as the maximum CR per hour; active CR (CR
active
),
as averaged number of sounds emitted per hour excluding
sessions without subject’s vocal activity; and CE, percentage
of time spent calling, that is, number of 15 min intervals with
calling activity divided by the total number of recorded 15 min
intervals multiplied by 100. CRs were tallied on a minute basis.
Moreover, in order to relate sound features with the males’
vocal performance and reproductive success, we analyzed 15
boatwhistles per male (with high signal-to-noise ratio) selected
randomly from all 56 fish. For the acoustic analysis, we adop-
ted the classification used by Amorim and Vasconcelos (2008)
that considers 3 distinct phases in the boatwhistle (beginning
[P1], middle [P2 or tonal phase], and end [P3]), based on
differences in pulse period and dominant frequency. The
acoustic parameters measured were total duration (ms), from
the start of the first pulse to the end of the last pulse; ampli-
tude modulation, by dividing the mean (root mean square)
amplitude measured in P1 by the one measured in P2; dom-
inant frequency (of P2), as the highest energy component
within the sound power spectrum (sampling frequency
8 kHz, Hamming window, filter bandwidth 10 Hz); fundamen-
tal frequency (of P2), calculated as the inverse of the average
pulse period measured in the tonal phase.
Hormone assays
In order to determine whether sex steroid levels were related to
the vocal performance and reproductive success of recorded
toadfish, blood samples were collected from the caudal vein
in heparinized syringes. This procedure was performed after
finishing the recording protocol for each test group recorded
in 2010.
Plasma was separated by centrifugation (5000 rpm for 5 min)
andstoredat24C. Plasma samples (50 ll) were diluted in
phosphate buffer (450 ll) containing 0.5 g/L of gelatin
(pH 7.6) and denatured at 80 C for 60 min. After samples
cooled, steroids, T (17b-hydroxyandrost-4-ene-3-one), 11-KT
(17b-hydroxyandrost-4-ene-3,11-dione), and cortisol (11b,17,21-
trihydroxy-pregn-4-ene-3,20-dione) were measured by radioim-
munoassay (RIA). Details of the RIAs methodology have been
published elsewhere (Scott et al. 1984). RIAs were performed
using duplicate amounts (100 ll) of denatured samples. Cross
reactions of antisera used in RIAs for T, 11-KT, and cortisol were
described previously in Kime and Manning (1982),Scott et al.
(1984),andRotllant et al. (2005), respectively. For each hor-
mone, circulating plasma levels from all animals were measured
within the same assay. Average intraassay and interassay coeffi-
cient of variations for RIAs were 1.0% and 5.2% for T, 1.3% and
5.5% for 11-KT, 6.4% and 10.3% for cortisol, respectively.
Statistical analysis
Following Amorim et al (2010), we used residuals of the sim-
ple linear regression of sonic muscle mass on ME (RM
SM
)as
a metric of sonic muscle hypertrophy. Likewise, we used the
residuals of the simple linear regressions of gonads, accessory
glands, and liver mass on ME (RM
G
,RM
AG
, and RM
L
, respec-
tively) to control for the influence of body size. This metric
gives a measure of an observed organ mass relative to a mean
expected value (given by the regression model) for a given
body size. Moreover, we used the residuals of ME on TL
O. Vasconcelos et al. Prediction of reproductive success in a vocal fish 377
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(COND) as a metric of body condition. We only used the total
body mass to calculate COND when comparing recorded
males and free-swimming territorial type I males, as the latter
were not sacrificed and ME was not determined. We log
10
-
transformed TL and ME to meet the assumptions of normality
and to linearize allometric relationships.
We used Mann–Whitney (M-W) Utests to compare morpho-
logical traits (log
10
TL, COND, RM
G
, and RM
SM
) between re-
corded type I males and nest-satellite conspecifics. We also
used M-W Utests to compare vocal parameters (CR
mean
,
CR
max
,CR
active
, and CE) and the morphological traits
(log
10
TL, COND, RM
G
, and RM
SM
) between males with eggs
and males without eggs. These tests were also adopted to
compare boatwhistle features between males with and without
eggs, as well as to compare hormonal levels between tested
males and free-swimming males from the same study area.
We examined general relationships among the variables
across all individuals, including morphological traits, calling
parameters (including boatwhistle features), and steroid levels,
by performing Spearman correlations. As a large number of
statistical tests were made, the overall level of significance
needed to be adjusted for multiple tests (to control the type I
error rate). Strict application of the Bonferroni method can
strongly reduce the power of statistical tests. Such power loss
can be avoided by choosing an experiment wise error rate
higher than the usually accepted 5%. We used 10% as sug-
gested by Wright (1992) and Chandler (1995), which has been
adopted in similar studies (e.g., Møller and Petrie 2002). So, we
adopted a significance criterion of P,0.01. We then consid-
ered 9 potential predictors of reproductive success (number of
eggs), including morphological traits and calling parameters
(log
10
TL, COND, RM
G
,RM
SM
,RM
L
,CR
mean
,CR
max
,CR
active
,
and CE). We used multiple regression analysis to assess the
statistical significance of each variable as a predictor of number
of eggs with a stepwise selection procedure (P0.05 to add
and P0.10 to remove). Our final regression modes complied
with all assumptions of multiple linear regression. All model
residuals were normally distributed. Further residual analysis
was performed using Durbin–Watson statistics, residual plots
as well as multicollinearity tests (variance inflation factors, VIF).
Parametric tests were only performed when data were nor-
mally distributed and variances were homogeneous. Statistical
analyses were performed using SPSS for Windows (16.0, SPSS
Inc., Chicago, IL).
RESULTS
Vocal behavior of nesting males and attraction of
conspecifics
Most of the nesting toadfish males started to vocalize, pre-
dominantly with boatwhistles, within 24 to 48 h of confinement
and interacted acoustically in a chorus, similarly to free-swim-
ming toadfish. From the total of 56 toadfish males confined in
the artificial nests, 51 toadfish (91.1%) showed vocal activity
and 16 vocally active fish (28.6%) presented egg clutches in
their nests, indicating that they successfully attracted mates.
From the 28 males recorded in the first breeding season, 11
vocally active specimens (39.3%) attracted other conspecific
males, which were outside the nests, close to the nest’s entrance,
and partially buried in the substrate (Figure 1b). These fish,
which were in a position typically occupied by sneakers (type
Figure 2
Comparison of total length, TL (a), body condition, COND (b), residuals of gonads mass, RM
G
(c), and residuals of sonic muscle mass, RM
SM
(d) between nest-holder type I toadfish males and nest-satellite type I toadfish males found outside the nests. Plots show medians, 10th, 25th,
75th, and 90th percentiles as boxes and whiskers. *P,0.05, **P,0.01, ***P,0.001, Mann–Whitney Utests.
378 Behavioral Ecology
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II males), were mostly small type I males (N¼10, 91%) with
significantly lower TL (M-W Utest: U¼13.5, N
nest-holder fish
¼11,
N
nest-satellite fish
¼7, P¼0.023) and COND (Utest: U¼2, N
nest-
holder fish
¼11, N
nest-satellite fish
¼7, P,0.001, Figure 2a) relative
to the vocalizing nesting type I males. Moreover, these type I
males found outside the nest also had significantly lower M
SM
(Utest: U¼0–12, N
nest-holder fish
¼11, N
nest-satellite fish
¼7, P,
0.001, Figure 2b), M
AG
(Utest: U¼0–12, N
nest-holder fish
¼11,
N
nest-satellite fish
¼7, P¼0.016) and M
G
(Utest: U¼16, N
nest-holder
fish
¼11, N
nest-satellite fish
¼7, P¼0.042). Onlyone type II male was
found outsidethe nest, among the 11 collected males. Within the
same study period, also 3 gravid females were found inside the
nests during low tides (see Figure 1b).
The mean and maximum CRs varied considerably among
individuals, namely between 0 and 2.81 bw min
21
(0.34 6
0.64 bw min
21
,mean6standard deviation [SD]) and 0–20.46
bw min
21
(3.60 65.55 bw min
21
), respectively. The CE also
differed greatly between males, that is, 0–46% (15% 613%,
mean 6SD).
Predictors of reproductive success
The mean, active, and maximum CRs were significantly higher in
nesting toadfish males with eggs than in males without any
clutches (M-W Utest, U(CR
mean
)¼70; U(CR
active
)¼103; U
(CR
max
)¼66.5; N
eggs
¼14, N
no eggs
¼40, P,0.001, Figure 3a).
The CE also differed significantly between these 2 fish groups (U
test: U¼102, N
eggs
¼14, N
no eggs
¼40, P,0.001, Figure 3b).
No differences were found in terms of TL, COND, RM
G
,and
RM
MS
(Utests: U(TL) ¼230.5; U(COND) ¼241; U(RM
G
)¼
277; U(M
MS
)¼255; N
eggs
¼14, N
no eggs
¼40, P.0.05).
Correlation analysis showed that most morphometric fea-
tures and vocal parameters were not significantly correlated
(see Table 1). Calling rates (CR
mean
,CR
active
, and CR
max
)
and the CE were highly positively correlated with the number
of eggs (Table 1).
Androgen levels (T and 11-KT) were not correlated with any
of the several vocal activity parameters nor with the number of
eggs (Table 1). Androgen and cortisol levels were compared
for tested animals versus free-swimming fish collected in the
same study area and showed that tested animals presented
significantly higher cortisol levels but T and 11-KT levels were
not significantly different (Table 2). In addition, males with
clutches obtained similar number of eggs compared with non-
restrained control males (Table 2).
Most boatwhistle acoustic features were neither correlated with
the vocal performance nor with the number of eggs (Spearman
correlations: R¼20.49 to 0.24, N¼24, P.0.01). Only pulse
period was negatively correlated with CR
max
(R¼20.55, N¼24,
P¼0.006). Nevertheless, toadfish males with eggs produced
generally boatwhistles with lower dominant frequencies when
compared with males without eggs (M-W Utest, U¼31, N
eggs
¼13, N
no eggs
¼11, P¼0.018) but similar duration (Utest, U¼
63, P.0.05), amplitude modulation (Utest, U¼47, P.0.05)
and pulse period (Utests: U¼65, P.0.05).
The best predictors for the number of eggs were the maxi-
mum CR and the CE—Table 3,Figure 4a,b.CR
max
was the
variable that most explained the variability of the number of
eggs, namely 52% (out of 58% explained by the full model).
Secondly, CE explained 6% out of 58% by the model.
Although morphometric features did not account for varia-
tion of reproductive success, they explained some of the variabil-
ity found in the vocal parameters. TL, COND and RM
SM
explained 29%, 6%, and 7% (out of 42% explained by the full
model) of the CR
max
variability, respectively. In addition, TL was
the only variable that explained CE variability (56%, Ta bl e 3).
DISCUSSION
The relation between characteristics of acoustic signaling that
lead to enhanced reproductive success can provide important
means to understand how vocal communication systems have
evolved. This approach has been focused in several taxa such
as insects, anurans, and birds. Although teleost fishes may
represent the largest group of sound-producing vertebrates
and use acoustic signals during various social interactions in-
cluding courtship, the relationship between vocal behavior
and reproductive success has never been clearly shown. This
study is the first to experimentally demonstrate that sound
production influences reproductive success in a teleost fish.
Inter- and intrasexual attraction
Almost all Lusitanian toadfish tested in this study showed vocal
activity and several presented egg clutches in their nests, indi-
cating that they successfully attracted gravid females. Besides
mates, vocal males also attracted other conspecific ‘‘satellite’
males, which remained outside the nests close to the en-
trance. In Batrachoididae, nest-parasitizing males have been
described as type II males that attempt opportunistic fertiliza-
tions, which are characterized by smaller body size, accessory
glands, and sonic muscle mass, but larger gonads than type I
males (or nest holders) (Brantley and Bass 1994;Modesto and
Cana´ rio 2003a). Our data, however, showed that the nest-sat-
ellite males were mostly type I but with significantly lower TL,
condition, sonic muscle, accessory gland mass and gonad
Figure 3
Comparison of maximum calling rate, CR (a) and calling effort, CE
(b) between type I toadfish males with and without clutches in the
nests. Plots show medians, 10th, 25th, 75th, and 90th percentiles as
boxes and whiskers. *P,0.05, **P,0.01, ***P,0.001, Mann–
Whitney Utests.
O. Vasconcelos et al. Prediction of reproductive success in a vocal fish 379
by guest on May 29, 2013http://beheco.oxfordjournals.org/Downloaded from
mass than the nest-holder vocal males. Such finding suggests
that smaller type I males with inferior quality (body size and
condition) may adopt a sneaking behavior due to space com-
petition. The data presented were collected during a summer
season with particularly high occupancy rates of the artificial
nests placed along the intertidal study area. Alternative mat-
ing tactics are not necessarily fixed throughout life and may
change depending on the social environment. For instance, in
the gobiid fish Bathygobius fuscus, larger males are always nest
holders but males of smaller size employ both tactics, nest
holder or sneaker (Taru et al. 2002). With the decrement of
larger males, sneaking males change their mating tactic to
nest holding. Also, in cichlid fish, Oreochromis mossambicus,
small males often adopt alternative female-like mating tactics
(Oliveira and Almada 1999). Our results provide evidence of
a similar mating behavioral plasticity in type I Lusitanian toad-
fish males, which probably depends on the social contexts
and/or environmental constraints (nest availability). Similar
nest-parasitizing behavior has been observed for type I males
of the batrachoidid P. notatus (Lee and Bass 2006), even
though type II males of both batrachoidid species show fixed
alternative mating tactics (Brantley and Bass 1994).
Species that breed in aggregations typically exhibit higher
levels of competition among males that can ultimately result
in higher number of males unable to defend a territory, and
also in higher sexually motivated territorial males that
lose the ability to discriminate between sexes (Oliveira and
Almada 1999). Future studies should investigate whether
nest-satellite type I males are truly sneakers and attempt op-
portunistic fertilizations, whether this tactic is maintained
throughout the whole breeding season, and which social
and environmental features are responsible for shaping such
mating behavior.
Vocal behavior and reproductive success
Our data indicated that the CR (mean, active, and maximum
CRs),aswellastheCE,weresignicantlyhigherinnesting
males with eggs than in males without clutches. The physical
features of nesting males, such as TL, body condition, and
gonads and sonic muscle masses, were not correlated with
the number of eggs, which indicates that the reproductive suc-
cess in H. didactylus primarily relies on the vocal performance.
Both higher CRs and calling in a regular fashion during long
periods of time (increased CE) result in a more conspicuous
vocal output or male advertisement, which probably facilitates
detection, localization, and selection by females. Higher adver-
tising CRs might be important to indicate spawning readiness
or motivation of males and in synchronizing gamete release
(Amorim et al. 2003). Previous studies with other batrachoidids
revealed that gravid females show phonotaxis when advertising
calls are played back at relatively high rates (e.g., O. tau,Winn
1972;P. notatus,McKibben and Bass 1998).
Ultimately, CR and CE may provide information about the
singing males, and it is possible that these vocal features are
used as honest signals for mate choice by females. Our data
indicated that TL, condition, and sonic muscle mass com-
bined partially explained the variability found in the calling
performance among individuals. Moreover, besides the likely
higher physiological and metabolic costs (Mitchell et al. 2008;
but see Amorim et al. 2002), the production of boatwhistles at
high rates and in a regular fashion may also impose ecological
costs, such as the attraction of predators and the time spent
calling and not in other activities (Ryan 1988;Gannon et al.
2005). Females strongly benefit from choosing good males,
especially when they are single spawners as batrachoidids
(Brantley and Bass, 1994;Modesto and Cana´rio, 2003a).
Fish-unguarded eggs are quickly eaten by predators, and
Table 1
Correlations between morphometric features, vocal performance, and number of eggs in the Lusitanian toadfish males
TL COND RM
G
RM
SM
CR
mean
CR
max
CR
active
CE T 11-KT
TL — 20.02 (54) 0.27 (54) 20.05 (54) 20.22 (55) 20.25 (55) 20.34 (55) 20.06 (55) –0.42 (24) 20.17 (24)
COND 0.00 (54) 0.19 (54) 0.02 (54) 20.01 (54) 20.02 (54) 0.22 (54) 0.00 (23) 0.01 (23)
RM
G
0.36* (54) 0.05 (54) 0.03 (54) 20.05 (54) 0.11 (54) 0.01 (23) 0.03 (23)
RM
SM
0.23 (54) 0.22 (54) 0.20 (54) 0.21 (54) 0.05 (23) 0.38 (23)
CR
mean
0.97** (55) 0.92** (55) 0.86** (55) 0.11 (23) 0.04 (23)
CR
max
0.94** (55) 0.77** (55) 0.17 (23) 0.12 (22)
CR
active
0.68** (55) 0.08 (23) 0.11 (23)
CE 0.26 (23) 0.16 (23)
T 0.37 (23)
Neggs 20.12 (54) 20.08 (54) 0.01 (54) 0.07 (54) 0.58** (55) 0.59** (55) 0.49** (55) 0.50** (55) 0.45 (23) 20.05 (23)
Values shown are Spearman rank correlation coefficients. Nis indicated below respective correlation coefficients. COND, body condition; CR
active
,
active calling rate.
Significant differences are indicated by asterisks: *P,0.01, **P,0.001.
Table 2
Comparison of steroid levels (T, testosterone and 11-KT, 11-ketotestosterone) between tested confined fish and free-swimming fish with and
without eggs present in the nest
T (ng) 11-KT (ng) Cortisol (ng) Number of eggs
With eggs Test fish 0.53 60.36 (7) U¼19,
P.0.05
0.43 60.40 (7) U¼13,
P.0.05
16.66 613.32 (5) U¼3,
P¼0.013
994 6518 (14) U¼78,
P.0.05Free-swimming fish 1.00 61.05 (8) 2.75 63.53 (8) 3.99 62.47 (8) 810 6692 (14)
No eggs Test fish 0.47 60.24 (17) 0.63 60.59 (17) 21.82 610.37 (18)
Free-swimming fish 0.29 (2) 1.54 (2) 1.18 (2)
Mann–Whitney Utests performed between groups are indicated. Values are means 6SD; Nis indicated in parentheses.
380 Behavioral Ecology
by guest on May 29, 2013http://beheco.oxfordjournals.org/Downloaded from
females must rely on males’ brood protection to ensure the
survival of the offspring (Sargent and Gross 1993). Also, the
presence of a nesting male is critical for eggs’ survival as they
need to be aerated for proper development and to prevent
fungal infections (Ramos and Amorim, unpublished data).
Therefore, in species where males provide parental care, in-
dicators of male parental quality are expected to be important
in intersexual communication and be under strong mate se-
lection by females (Andersson 1994).
Amorim et al. (2010) also reported that increased boatwhis-
tle CR and CE strongly reflected good male condition given
by the lipid content of the somatic muscles in the Lusitanian
toadfish. Accordingly, we further demonstrate that these vocal
parameters affect the mating success in this species and seem
to inform receivers, that is, females and other competing
males, about the size and also quality of males. Lusitanian
toadfish males that are in good condition advertise their
spawning motivation through higher CRs and increased CE,
which affects their success in attraction of gravid females for
reproduction.
Moreover, our data showed that the toadfish males with eggs
that exhibited higher CRs also produced boatwhistles with
significantly lower dominant frequencies. This suggests a pos-
sible trade-off between CR and muscle contraction rate, as
calling more requires slower muscle contraction to avoid mus-
cle fatigue. Changes in muscle contraction rate have been
described to lower levels of muscle fatigue (e.g., Brantley
and Bass 1994). Moreover, dominant frequency may indicate
size of vocal male. Dominant frequency is negatively corre-
lated with body size in several fish species (e.g., Ladich et al.
1992;Myrberg et al. 1993).
Sisneros et al. (2009) reported that, in the batrachoidid
P. notatus at the end of the breeding season, larger nesting
males presented higher body condition (K) and larger num-
ber of viable late-stage embryos in the nest, suggesting that
body condition is an honest indicator of parental ability in
batrachoidids. In our study, we did not find a correlation be-
tween condition (comparable to K, but based on residuals—
see MATERIALS AND METHODS) and number of eggs. The
data obtained in terms of number of eggs was collected after 2
weeks of confinement of reproductive males in the experi-
mental nests. At this point, we were only evaluating the ability
to attract mates and to provide early parental care and not the
capacity of providing good parental care through later stages
of embryo development.
In other taxa, higher CRs may signal male quality such as
a better immune system (e.g., insects, Jacot et al. 2004), pa-
rental quality (e.g., birds, Dolby et al. 2005) or higher fertil-
ization success (e.g., anurans, Pfennig 2000 and birds, White
et al. 2010).
Steroid plasma levels, vocal behavior, and reproductive
success
Our data showed that circulating androgen levels were not sig-
nificantly related with reproductive success or vocal behavior
in the studied animals. However, androgen levels in experi-
mental animals were generally lower (but not significantly
different) than those obtained from free-swimming toadfish
collected in the same study area during low tides (see Table
Table 3
Predictors of reproductive success (number of eggs), CR
max
, and CE of the Lusitanian toadfish
Predictor BSEM tPrF Model significance R
2
DW VIF
Neggs CR
max
1.20 0.33 3.67 0.001 0.762 F
1,53
¼35.32 ,0.001 0.581 2.20 2.03
CE 0.28 0.10 2.70 0.009 2.03
CR
maxa
log TL 2.10 0.42 0.536 ,0.001 0.651 F
3,53
¼12.28 ,0.001 0.424 1.92 1.92
COND 21.91 0.73 20.284 0.012 1.03
RM
SM
1.78 0.70 0.275 0.015 1.03
CE
a
log TL 9.18 1.13 8.104 ,0.001 0.747 F
1,53
¼65.68 ,0.001 0.558 1.33 1.00
COND, body condition.
a
Only morphometric parameters were considered for regression analysis.
Figure 4
Relationship between the best predictors, maximum calling rate, CR
(a) and calling effort, CE (b), and the reproductive success (number of
eggs) in the Lusitanian toadfish. The number of eggs was square-root
transformed and the maximum CR was log-transformed. Regression
equations: (a) SQRT (Neggs 10.5) ¼9.4 (log CR
max
)123.3; (b)
SQRT (Neggs 10.5) ¼13.6 (CE) 111.7.
O. Vasconcelos et al. Prediction of reproductive success in a vocal fish 381
by guest on May 29, 2013http://beheco.oxfordjournals.org/Downloaded from
2). In fact, androgen profiles of these free-swimming animals
were similar to those found for the same species in wild ani-
mals captured by beam trawler during the reproductive sea-
son (Modesto and Cana´ rio 2003a).
In contrast, cortisol circulating levels of confined males were
significantly higher than those obtained from free-swimming
toadfish at the same study site. This suggests that confinement
in the experimental nests was probably responsible for the
higher cortisol levels, although animals exhibited vocal activity,
successfully attracted mates, and had similar body condition
compared with the free-swimming fish. In addition, subject
males with clutches obtained similar number of eggs as free-
swimming males from the same site (Table 2), suggesting that
spawning success of the studied males was not altered by any
reaction to confinement. Therefore, we consider the hypoth-
esis that in confined animals increased cortisol levels could
potentially result in decreased androgen levels concealing any
possible relation with the male’s reproductive success. In com-
mon carp, chronically elevated cortisol levels affected all parts
of the brain–pituitary–gonad axis resulting in a strong
decrease of testicular production of androgens, including
11-KT (Goos and Consten 2002). Moreover, in vitro, physio-
logical levels of cortisol can inhibit the pathways that lead to
the production of 11-KT (Consten et al. 2002). In this context,
measured androgen levels probably did not reflect accurate
hormonal profiles of the specimens throughout the study pe-
riod and cannot provide precise information about normal
relationships between steroid plasma levels, vocal behavior,
and reproductive success. Future studies should investigate
whether androgens affect calling and spawning success in
nonconfined animals.
This study is the first to experimentally demonstrate that
sound production influences reproductive success in a teleost
fish. We suggest that signaling at higher rates and in a regular
fashion can operate as an honest signal of male quality and an
indicator of elevated motivation for reproduction in Batrachoi-
didae, and perhaps in other fish. Future studies should address
whether vocal behavior also signals parental quality, as in
other taxa.
FUNDING
Science and Technology Foundation, Portugal (project
PDCT/MAR/58071/2004, pluriannual program UI&D 331/
94 and UI&D, grants SFRH/BD/30491/2006 to R.O.V. and
SFRH/BPD/41489/2007 to M.C.P.A).
We thank the Air Force Base No. 6 of Montijo (Portugal) for allowing this
study in their military establishment. We are grateful to Marta Bolgan for
the illustration of the toadfish experimental nest. We also thank Silvia
Pedroso and Joana Jorda
˜o for their help with the fieldwork.
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The current study investigated the structure and function of the olfactory system of the Lusitanian toadfish, Halobatrachus didactylus, using histology and electrophysiology (electro‐olfactogram [EOG]), respectively. The olfactory system consists of a digitated anterior peduncle, of unknown function, containing the inhalant nostril. This then leads to a U‐shaped olfactory chamber with the olfactory epithelium—identified by Gαolf‐immunoreactivity—on the ventral surface. A large lacrimal sac is connected to this tube and is likely involved in generating water movement through the olfactory chamber (this species is largely sedentary). The exhalent nostril lies by the eye and is preceded by a bicuspid valve to ensure one‐way flow of water. As do other teleosts, H. didactylus had olfactory sensitivity to amino acids and bile acids. Large‐amplitude EOG responses were evoked by fluid from the anterior and posterior testicular accessory glands, and bile and intestinal fluids. Anterior gland and intestinal fluids from reproductive males were significantly more potent than those from non‐reproductive males. Male urine and skin mucus proved to be the least potent body fluids tested. These results suggest that chemical communication—as well as acoustic communication—may be important in the reproduction of this species and that this may be mediated by the accessory glands and intestinal fluid.
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Several batrachoidids have been known to produce sounds associated with courtship and agonistic interactions, and their repertoires have been studied acoustically and behaviourally. In contrast, sound production of the Lusitanian toadfish Halobatrachus didactylus, although often noted, has not been acoustically studied.This sedentary predator of Northeastern Atlantic coastal waters is usually found in sandy and muddy substrates, under rocks or crevices. Sound recordings were made in Ria Formosa, a lagoon complex in southern Portugal. The sound producing apparatus was studied in adult individuals of both sexes captured by local fishermen.It is shown that this species produces acoustic emissions similar to other batrachoidids. It produces a long, rhythmical, tonal sound, often in choruses, which is comparable to the boatwhistle or hum signals of Opsanus and Porichthys, and a complex of signals that were classified as grunts, croaks, double croaks and mixed calls (‘grunt-croak’). As in other toadfishes, H. didactylus presents sonic muscles connected to a bi-lobed swimbladder. Asynchronous contractions of the sonic muscles were detected when massaging the ventral surface of the fish.
Conference Paper
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Chapter
Parental care may be defined as an association between parent and offspring after fertilisation that enhances offspring survivorship. This phenomenon has attracted the attention of evolutionary biologists since Darwin; however, it was not until the recent insurgence of behavioural ecology and sociobiology (e.g. Williams 1966a; Trivers 1972; Alexander 1974; Wilson 1975) that the variety of parental care patterns in animals has attracted such rigorous study. Perhaps because we ourselves are mammals, we tend to think of parental care as being the principal occupation of females, possibly with some help from males. A survey of the vertebrates, however, reveals that mammals are merely at one end of the spectrum, with predominantly female care, and fishes are at the other end, with predominantly male care (Table 11.1; see also Chapter 10 by Turner, this volume). Within teleost fishes with external fertilisation (about 85 per cent of all teleost families), one finds that the four states of parental care, ranked in descending order of their frequencies, are: no care, male care, biparental care, and female care. This seemingly peculiar trend has attracted considerable attention from evolutionary biologists, who have proposed several hypotheses about the origins of parental care in fishes (see reviews by Maynard Smith 1977; Blumer 1979; Perrone and Zaret 1979; Baylis 1981; Gross and Shine 1981; Gross and Sargent 1985).
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The plainfin midshipman Porichthys notatus has two male reproductive morphs, ‘Type I’ and ‘Type II’, which are distinguishable by their physical traits alone. Type I males are eight times larger in body mass than Type II males and have a six-fold larger relative sonic (vocal) muscle mass than Type II males. In contrast, the testicles of Type II males are seven times larger than those of Type I males. This study demonstrates morph-specific patterns of reproduction, including acoustic signals, for Type I and II males. Field censuses of nests showed that only Type 1 males maintained nests. Type II males and females transiently appeared in these nests in association with each other. Infra-red video and hydrophone recordings in aquaria showed that Type I males maintained nests and readily vocalized. Long-duration ‘hums’ and sequences of short-duration ‘grunts’ were produced during advertisement and agonistic contexts, respectively. Humming Type I males attracted females to their nests, pair-spawned, and then guarded egg clutches alone. By contrast, Type II males neither acoustically courted females nor maintained available nest sites, but rather ‘sneak-’ or ‘satellite-spawned’ at the nests of Type I males. Type II males infrequently produced low amplitude, short duration grunts that were similar in spectral, temporal and amplitude characteristics to the grunts of females. Type II males appear to be obligate sexual parasites of the nest-building, mate-calling, and egg-guarding Type I males. The dimorphic body and vocal muscle traits of the two male morphs in the plainfin midshipman are thus paralleled by a divergence in their reproductive tactics and the properties of their acoustic signals.
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. The reproductive ecology of the gobiid fish Bathygobius fuscus was studied at Nobeoka, Miyazaki, Japan. Males of this species maintain small rock holes as a nest and females spawn an egg mass on the wall of the nest. The males employed two forms of mating tactic: nest holding and sneaking. A nest holder stayed in the nest and waited for a female to visit, whereas a sneaker intruded into a nest while a pair was engaged in reproduction. Males larger than 55 mm standard length were always nest holders; those of smaller size employed both tactics. As the larger males excluded the smaller males, the latter did not occupy a nest hole. With a decrease in the number of larger males, smaller males changed their mating tactic from sneaking to nest holding. The results suggest that male Bathygobius fuscus adopt a conditional strategy whereby they change their tactic depending on their social status.
Book
Why have males in many species evolved more conspicuous ornaments and signals such as bright colours, enlarged fins, and feather plumes, as well as larger horns and other weapons than females? Darwin's explanation for such secondary sex traits, the theory of sexual selection, became his scientifically perhaps most controversial idea. It suggests that the traits are favoured by competition over mates. After a long period of relative quiescence, theoretical and empirical research on sexual selection has erupted during the last decades. This book describes the theory and its recent development, reviews models, methods, and empirical tests, and identifies many remaining open problems. Among the topics discussed are the selection and evolution of mating preferences; relations between sexual selection, species recognition, and speciation; constraints on sexual selection; the selection of secondary sex differences in body size, weapons, and in visual, acoustic, and chemical signals. The rapidly growing study of sexual selection in plants is also reviewed. Other chapters deal with alternative mating tactics, and with the relationships among sexual selection, parental roles, and mating systems. The present review of this very active research field will be of interest to students, teachers, and research workers in behavioural and evolutionary ecology, animal behaviour, plant reproductive ecology, and other areas of evolutionary biology where sexual selection is a potential selection factor. In spite of much exciting progress, some of the main questions in the theory of sexual selection yet remain to be answered.