Alloparental behavior in the highly vocal Lusitanian toadfish

Abstract

In many fish species in which males guard nests with their eggs, parental care directed to genetically unrelated offspring may arise for example from nest takeovers or cuckoldry. Lusitanian toadfish (Halobatrachus didactylus) has exclusive male parental care and face intensive nest competition during the breeding season that may lead to care of foster eggs. Males of this species use visual displays and sounds when defending their nests frequently resulting in expulsion of the intruder without escalated confrontation. In this study we intended to investigate the existence of alloparental care in Lusitanian toadfish, a behavior whose adaptive significance is still poorly understood. Fish were randomly assigned to three different treatments: parental males in nests with their eggs, parental males with foster eggs and parental males without eggs. Nests with eggs with no nest holder or with females were used as controls. We performed three territorial intrusions over periods of 15 days and observed the acoustic and visual behaviors of residents and intruders. Egg survival was tallied from nests' photographs in all groups. Circulating steroid levels were measured in the three test groups and in another set of non-manipulated males. There were no differences in acoustic and visual territorial defense behaviors among treatments. Egg survival was similar between males (parental and alloparental) and significantly higher than in nests with no nest-tender. Females presented intermediate egg survival. All groups presented similar levels of testosterone and alloparental males showed higher 11-ketotestosterone levels but within the range of levels observed in non-manipulated males. Cortisol levels were similar in all male groups suggesting that experiments did not increase fish stress. The present results suggest the existence of alloparental care in this species.

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Alloparental behavior in the highly vocal Lusitanian toadfish
Andreia Ramos
a,
⁎, Paulo J. Fonseca
a
, Teresa Modesto
b
, Vítor C. Almada
c
, Maria Clara P. Amorim
c
a
Departamento de Biologia Animal, Centro de Biologia Ambiental, Faculdade de Ciências da Universidade de Lisboa, Bloco C2 Campo Grande, 1749‐016 Lisbon, Portugal
b
Centro de Ciências do Mar, Universidade do Algarve, Edifício 7, Campus de Gambelas 8005‐139 Faro, Portugal
c
Unidade de Investigação em Eco-Etologia, I.S.P.A. —Instituto Universitário, Rua Jardim do Tabaco 34, 1149‐041 Lisbon, Portugal
abstractarticle info
Article history:
Received 9 May 2012
Received in revised form 28 June 2012
Accepted 4 July 2012
Available online xxxx
Keywords:
Alloparental care
Batrachoididae
Egg survival
Hormone circulating levels
Sound production
Territorial defense
In many fish species in which males guard nests with their eggs, parental care directed to genetically unrelated
offspring may arise for example from nest takeovers or cuckoldry. Lusitanian toadfish (Halobatrachus
didactylus) has exclusive male parental care and face intensive nest competition during the breeding season
that may lead to care of foster eggs. Males of this species use visual displays and sounds when defending
their nests frequently resulting in expulsion of the intruder without escalated confrontation. In this study
we intended to investigate the existence of alloparental care in Lusitanian toadfish, a behavior whose adaptive
significance is still poorly understood. Fish were randomly assigned to three different treatments: parental
males in nests with their eggs, parental males with foster eggs and parental males without eggs. Nests with
eggs with no nest holder or with females were used as controls. We performed three territorial intrusions
over periods of 15 days and observed the acoustic and visual behaviors of residents and intruders. Egg survival
was tallied from nests' photographs in all groups. Circulating steroid levels were measured in the three test
groups and in another set of non-manipulated males. There were no differences in acoustic and visual territo-
rial defense behaviors among treatments. Egg survival was similar between males (parental and alloparental)
and significantly higher than in nests with no nest-tender. Females presented intermediate egg survival. All
groups presented similar levels of testosterone and alloparental males showed higher 11-ketotestosterone
levels but within the range of levels observed in non-manipulated males. Cortisol levels were similar in all
male groups suggesting that experiments did not increase fish stress. The present results suggest the existence
of alloparental care in this species.
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
Parental care evolves when the benefits of taking care of the off-
spring outweighs the costs (Farmer and Alonzo, 2008). Parental care
is often given by females (as in mammals), and contribute to a differ-
ential reproductive effort between the sexes (Alcock, 2009). However,
paternal care is the most common form of parental care in teleost fish-
es with external fertilization (Blumer, 1979) and has evolved due to
the lower cost of care for males than for females (Gross and Sargent,
1985). When females provide parental care, they not only lose mating
opportunities but grow less and take longer to produce new eggs due
to reduced feeding and extra energy spent in care. In contrast, costs of
paternal care are reduced in fish as males often need to defend a terri-
tory/nest to attract females to mate with, and can attract more mates
while caring for existing broods (Alcock, 2009). In fish, parental care
consists in preparing and defending nests or territories, to guard,
clean and aerate the eggs and embryos, as well as to provide nutrition-
al support for the brood to become independent (Gross and Sargent,
1985).
When the competition for nests or mates is intense, care for foster
offspring (i.e. alloparental care) may result from nest takeovers or
from increased sneaking fertilizations. Alloparental care is also ob-
served in group-living communal or cooperative breeders (Taborsky,
1985). While the adaptive advantages of parental care to their own off-
spring are obvious, the care for offspring of other males canbe consid-
ered maladaptive (Smith and Wootton, 1995; Trivers, 1972). Indeed,
alloparental care can be considered a mistake(lack of foster egg recog-
nition) or a by-product of lack of alternatives. However, alloparental
care may also bring direct benefits. For example, males with foster
eggs can be more attractive to females (eggs in the nest effect;
e.g. Rohwer, 1978)orbenefit from decreased egg predation due to
confusion or dilution effects (Wisenden, 1999). Despite the advantages
of alloparental care there are alsomany disadvantages such as increased
expenses of care, reduced space for new eggs, and higher risks of egg
infection (Constanz, 1985; Manica, 2002). Studying the existence of
alloparental care in different species will allow to better understand
its adaptive role and the underlying behavioral and physiological
mechanisms.
Batrachoididae males, toadfish and midshipmen, defend their
nests in the reproductive season. These species may present alterna-
tive reproductive tactics exhibiting two male morphotypes. In such
Journal of Experimental Marine Biology and Ecology 434-435 (2012) 58–62
⁎Corresponding author. Tel.: +351 217500000.
E-mail address: andreiamcramos@gmail.com (A. Ramos).
0022-0981/$ –see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jembe.2012.07.002
Contents lists available at SciVerse ScienceDirect
Journal of Experimental Marine Biology and Ecology
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Author's personal copy
cases only type I males or “singing males”use acoustic signals to attract
mates, providing parental care to multiple clutches of eggs (Brantley
and Bass, 1994; Gray and Winn, 1961; Modesto and Canário, 2003). In
contrast, type II males neither build nests nor acoustically advertise to
females, but instead satellite or sneak spawn to steal fertilizations
from type I males (Brantley and Bass, 1994). During the period that
type I males spend defending their nests and providing parental care,
they have fewer opportunities to feed, decreasing their physical condi-
tion (Modesto and Canário,2003; Sisneros et al.,2009). Because in these
fish allopaternal care could exist likely due to frequent nest takeovers
and parasitic fertilizations and since parental care is prolonged and
costly, they provide excellent models to study the existence and the
adaptive value of allopaternity.
Our aim was three-fold. First, we aimed to study the existence of
allopaternal care in Lusitanian toadfish, by comparing both nest de-
fense under territorial intrusions and egg survival between clutches
guarded by parental males and alloparental males that were given a
nest with 100% foster eggs. Second, we wanted to verify if the use of ag-
onistic behavior during nest defense, including sound production, dif-
fered between males with different genetic relation to offspring. Finally,
we checked for physiological differences (androgen and cortisol levels)
that likely underlie territorial defense behavior including sound emission
(Remage-Healey and Bass, 2005). We predicted that if males do not show
alloparental care, (1) parental males would defend their nests more ag-
gressively than alloparental males and would show higher levels of an-
drogens. We also predicted that (2) parental males would have higher
offspring survival.
2. Materials and methods
2.1. Study species
The Lusitanian toadfish, Halobatrachus didactylus (Bloch & Schneider),
is a member of the Batrachoididae family, and is a benthic fish that in-
habits estuaries and coastal zones of the Eastern Atlantic and the Mediter-
ranean, usually found partly buried in soft sediment or concealed in rock
crevices (Roux, 1986).
Between May and July (reproductive season of Lusitanian toadfish
in Portugal), breeding males defend their nests under rocks in shal-
low waters and attract females to spawn with advertisement calls
(boatwhistles) until the roof of the nest is full of eggs and embryos
(dos Santos et al., 2000). The eggs are attached to the roof by an adhe-
sive disk, and guarded by a male until the brood is free-swimming
(Roux, 1986). During this period (c. 30 days), the competition for
shelters in shallow waters increases (Amorim et al., 2010) and shelter
owner substitutions occur, including in nests with eggs (Vasconcelos
et al., 2010). Nest-holding males defend their nests with the emission
of agonistic boatwhistles, frequently resulting in the expulsion of the
intruder, or with chases and bites when fights escalate (Vasconcelos
et al., 2010).
2.2. Test subjects and maintenance
We used an experimental procedure similar to that conducted by
Vasconcelos et al. (2010). Concrete nests (internal dimensions: 50 cm
long, 30 cm wide and 20 cm height) with a hemicylinder shape capped
at one end were placed in the Tagus River estuary (Military Air Force
Base 6, Montijo, Portugal; 38° 42′N; 8° 58′W). These nests were readily
occupied by toadfish in the breeding season and were only exposed to
air during spring low tides, when males could be collected. Experimental
males (type I) were maintained in round stock tanks (plastic swimming-
pools, 2 m diameter and water depth c. 0.5 m), near the intertidal toad-
fish nesting area. Stock tanks were provided with shelters (roof tiles) to
allow them to develop territorial behavior. Experimental tanks were sim-
ilar to the stock tanks but with 2.5 m diameter and had two nests 50 cm
apart from the border. All tanks were under a shadow net cover held circa
170 cm high to prevent excessive solar water heating. Water tempera-
ture variedfrom 19 to 26 °C, within the range of the estuary water tem-
perature variation during the same period. The renovation of water was
done every 2–3 days, by pumping water directly from the estuary. The
light cycle was natural as tanks were outdoors.
2.3. Territorial experiments
Two subjects were placed in each experimental tank and given a
period of acclimation of 48 h before the beginning of the observation.
Subject fishes were randomly assigned to three different treatments:
(1) alloparental males, i.e. parental males that were given nests with
eggs from another male (AP), (2) parental males in nests with their
own eggs (PWE), (3) parental males that were given nests with no
eggs (PNE). We also used two control groups in experimental tanks.
One consisted of nests with eggs but with no males (ENM), to control
for egg survival in nests in the same conditions as those of AP and PWE
groups but with no nest holder to provideparental care. The other con-
trol group comprised nests with eggs occupied by a female (FWE). The
latter group aimed to check the effect of the simple presence of a fish in
egg survival since females do not provide parental care.
We used 36 residents (n =10 PNE; n =14 PWE and n=12 AP) kept
in pairs (18 pairs) in the experimental tanks.Each pair of resident males
was submitted to 3 intrusions. We used two territorial males in each
tank to simulate a more realistic scenario since this species lives in ag-
gregations (Amorim et al., 2010). Resident males were always different
in each experiment and varied in total length (TL= 35–47 cm) and in
body mass (498–2010 g). Intruders were never used twice in the
same day and ranged 32–50 cm in total length (TL) and 477–1915 g
in body mass. Fish were marked with small cuts between the fin rays
to insure identity during trials. Marking did not cause any measurable
change in the fish behavior.
At the start of each trial we inserted a male intruder in the tank, and
registered all behaviors and sounds produced by the residents and the
intruder during 30 min. After this period we placed a new male (second
intruder) in the tank, and registered all behaviors of the four fish for an-
other period of 30 min.
We carried out three intrusion trials (as above) with the same res-
idents at the first, the fifth and the tenth day of the experiment to in-
vestigate any possible changes in the territorial defense that could be
caused by physiological changes due to the treatment effect. We hy-
pothesized that assigning a nest with 100% foster eggs or with no eggs
to parental males (with recent broods) could cause physiological and
behavioral changes that could possibly not be expressed immediately.
2.4. Behavior recording
Fish visual behavior, i.e. lateral displays (full extension of dorsal
and caudal fins in antiparallel position) and frontal displays (full ex-
tension of pectoral fins and opercula), was assessed through direct
observation and registered on paper. For residents we registered the
frequency of non-escalated behaviors such as mouth opening with
the extension of pectoral fins and opercula and escalated behaviors
including bite attempts and mouth–mouth fight (Vasconcelos et al.,
2010). For the intruders we tallied the number of approaches and at-
tempts to enter the nest. We defined approaches when the intruder
was at least within a body length from the nest and we considered at-
tempts to enter the nest when the intruders managed to get part of
the body inside the nest.
To record the acoustic signals we placed one hydrophone (High Tech
94 SSQ, Gulfport, MS, USA; frequency range: 30 Hz–6k
Hz,±1dB;volt-
age sensitivity: −165 dB re. 1 V/μPa) in front of each nest at about
10 cm both from its entrance and from the tank bottom, attached to an
wooden rod positioned over the tank. The two hydrophones were simul-
taneously recorded to a laptop connected to a 2 channel USB audio cap-
ture device (Edirol UA-25, Roland, Japan; 16 bit, 6 kHz acquisition rate
59A. Ramos et al. / Journal of Experimental Marine Biology and Ecology 434-435 (2012) 58–62

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per channel), controlled by Adobe Audition 2.0 (Adobe Systems Inc., San
Jose, CA, USA). Acoustic behavior during trials was also monitored with
headphones and registered on paper.
The acoustic signals were analyzed and quantified using also Adobe
Audition 2.0. We tallied the number of boatwhistles produced by resi-
dents as well as, grunts and other sounds that occurred less frequently.
Recordings of sonic muscles contraction activity during some experi-
ments showed that typically only residents produce boatwhistles dur-
ing territorial intrusions (J. Jordão, personal communication).
2.5. Hormones assays
To analyze the levels of testosterone (T), 11 ketotestosterone (11-KT)
and cortisol (Cort), we used 4 different groups: AP, PWE, PNE and
non-manipulated (NM) males that occupied spontaneously the artificial
nests placed in the estuary and that were not used in the experiments.
The NM group presented a size similar to the other experimental groups
(TL ranged 37–48 cm).
Immediately after the last observation (3rd intrusion), blood sam-
ples were collected. As soon as the nest was overturned, the male was
taken off the nest and immediately anesthetized in a box with sea
water and MS-222. Blood samples were collected from the caudal vein
using heparinized syringes, usually within 4 min from overturning the
nest. Plasma was immediately separated by centrifugation (5 min at
5000 RPM) and stored at −20 °C until steroid quantification by RIAs
(radioimmunoassays). The procedure to analyze T, 11-KT and Cort
levels in plasma was similar to that conducted by Vasconcelos et al.
(2012). After blood sampling, fish were allowed to recover from anes-
thesia and were returned to the estuary.
2.6. Eggs survival
We photographed and counted the eggs in each nest at the begin-
ning and end of each experiment to calculate the percentage of egg
survival for AP and PWE males, for nests with no males (ENM) and
for nests with females (FWE).
2.7. Statistical analysis
Statistical analyses were performed with STATISTICA for Windows
(v. 9.0, StatSoft, Inc. 2009). Parametric tests were only performed when
data were normally distributed and variances were homogeneous.
We compared the number of acoustic and visual behaviors among
treatment groups (independent factor with 3 levels, AP, PWE and
PNE) and among intrusions trials (repeated measures) using repeated-
measures ANOVA. We used resident male total length as a covariate
since it might influence contest behavior. However, since the effect of
male length was not significant the covariate was removed in further
analyses. We checked that the different treatment groups received a
similar amount of interactions by intruders (approaches and nest intru-
sions) alsowith repeated-measures ANOVA. Thenumber oftimes the in-
truders interacted with the resident males did not differ among treatment
groups (repeated-measures ANOVA: F
(2,33)
= 0.14, P> 0.05) or among the
three intrusions (repeated-measures ANOVA: F
(2,66)
=0.17, P > 0.05) en-
suring that any possible behavioral differences among groups were due to
treatment and not to intruder behavior.
To compare hormones levels (T, 11-KT and Cort) among treatment
groups (AP, PWE, PNE and NM) we used non-parametric Kruskall–Wallis
tests. Differential egg survival for AP, PWE, ENM and FEW groups was
tested with a one-way ANOVA.
3. Results
We observed that 71% of intruders (n= 108 in total) approached
the nests in less than 3 min. Intruding behavior (totalling 546 events)
consisted mostly of approaches to a nest (74%). Attempts to enter a
nest were less frequent (26%). From these, intruders succeeded in en-
tering the nest only 16 times (11%): 6 in nests with AP males, 6 in nests
with PWE males and 4 in nests with PNE males. In the 6 attempts to
enter PWE nests, 4 resulted in nest takeovers. In the other groups (AP
and PNE) no nest takeovers were observed.
Resident males typically responded to intruding events (79%). In the
case of an intruder's approach the most common reaction was sound pro-
duction (91.5%), and less frequently visual behavior (8.5%). Similarly,
when an intruder attempted to enter the nest the resident's main re-
sponse was sound production (70%). Thus, acoustic signaling, was the
most common reaction to intrusion behavior. Moreover, boatwhistles
were the most frequent sound emission, corresponding to 91% of the reg-
istered vocalizations as previously observed in this species (Vasconcelos
et al., 2010). All subject males emitted boatwhistles in at least one of
the 3 intrusion trials. There were no differences in both visual behavior
and sound production among the different male groups (Table 1). Like-
wise, there were no differences in the visual and acoustic behavior
along the three intrusion trials. Taking together, the analyses of our re-
sults point to the interaction between trial (repeated variable) and the
treatment variables was not significant (Table 1). Taken together the
data suggests similar motivation among AP, PWE and PNE males to de-
fend the nest and no changes in territorial defense during the course of
the experiment.
3.1. Eggs survival
Experimental nests taken from the estuary presented a variable
amount of dead eggs (mean= 12.0%, range: 0.2–72.2%), putting into
evidence a considerable level of egg mortality in nature. At the end
of the experiment egg survival was significantly higher in nests
with males (AP, PWE) than in control nests without a nest holder
(CRTL). Egg survival in nests with females was intermediate and did
not differ from the other treatments (one-way ANOVA F
(2,36)
=2.31;
Pb0.05, Fig. 1).
3.2. Hormone levels
T and Cort levels did not exhibit significant differences among male
groups (AP, PWE, PNE and NM) (Kruskall–Wallis test: T–N=35,
H=4.47, P>0.05; Cort–N= 35; H= 0.08, P > 0.05; Fig. 2AandC).
However, 11KT showed significant differences among groups (Kruskall–
Wallis test: N=35, H=16.89, Pb0.001; Fig. 2B) with the alloparental
and non-manipulated males presenting higher circulating levels than pa-
rental groups (with and without eggs). 11KT levels of AP males and of
other experimental male groups fell within the variability observed for
the non-manipulated males sampled in nature.
4. Discussion
Most research about parental care has focused in understanding
the fitness benefits of increased offspring survival and of the fitness
costs to the parent through decreased future reproductive opportuni-
ties (Gross and Sargent, 1985). However the existence of alloparental
care has received less attention (Constanz, 1985).
Table 1
Effects of group treatment (factor) and intrusion trials (repeated measures variable) on
non-escalated (exhibitions) and escalated agonistic visual behavior and on sounds
made in response to intruders. Data concerning sound production and non-escalated
visual behavior were log-transformed to meet ANOVA assumptions.
Visual —exhibitions Visual —escalated Sound production
Source DF F P DF F P DF F P
Group 2,33 2.42 0.10 2,33 2.97 0.07 2,33 0.94 0.40
Intrusion 2,66 0.19 0.83 2,66 2.35 0.10 2,66 0.35 0.71
G× I 4,66 1.31 0.28 4,66 1.01 0.41 4,66 0.39 0.81
60 A. Ramos et al. / Journal of Experimental Marine Biology and Ecology 434-435 (2012) 58–62

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The results obtained in this study show no differences in territorial
defense among parental and alloparental males with eggs and males
without eggs, as observed by the lack of differences in both visual and
acoustic behaviors. This suggests that males defend their nests/shelters
in a similar fashion, independently of having their own eggs, foster eggs
or no eggs. Curiously, in a recent study parental Lusitanian toadfish
males with eggs were found to be more aggressive than males without
eggs (Vasconcelos et al., 2010). However, in contrast with the present
work, in (Vasconcelos et al., 2010) some males were already guarding
embryos, and these showed higher levels of aggressiveness during ter-
ritorial defense, which may explain the discrepancy with our study.
Parental care in fish mainly consists in nest defense and egg tending,
including aeration and cleaning (Gross and Sargent, 1985). Eggs in nests
occupied by males presented similar levels of survival and these levels
were significantly higher than when eggs were in nests without fish,
suggesting similar levels of care between alloparental and parental
males. However, we cannot exclude the possibility that egg survival
may have been influenced by the simple presence of a fish, i.e. by ‘pas-
sive care’. Movements inside the nest may have increased egg aeration
and fish may possess compounds in the epidermal mucus that might
have prevented fungus infection which could have increased egg sur-
vival (Knouft et al., 2003). The intermediate levels of egg survival
found for nests with females, which do not exhibit parental care, support
the argument that the simple presence of a fish inside the nest increases
egg survival, although these levels were not significantly different from
the ones found for unoccupied nests. This suggests that the significant dif-
ferences in egg survival between nests with males and nests with no fish
are possibly due to active care, pointing to the existence of alloparental
behavior in this fish. Nevertheless, we cannot rule out the ‘passive care’
hypothesis since subject males had larger body sizes than experimental
females likely causing larger water movement inside the nest and higher
mucus excretion.
Despite the fact that nests with males showed significantly higher
egg survival than unoccupied nests, the observed values were still low
(c. 30%). This low survival rate might be explained by the observed de-
tachment of some eggs from the nest, by egg cannibalism or simply by
egg death. During the long period of parental care males have decreased
opportunities to feed and experience a reduction in physical condition
throughout the breeding season (Modesto and Canário, 2003). Thus it
would be advantageous to ingest eggs that are nutritionally rich. Consis-
tent with this suggestion, eggs and embryos have been found in the
stomach of male midshipmen suggesting they could cannibalize part
of the clutch (Sisneros et al., 2009). However, cannibalism represents
an advantage to the male when they are able to distinguish their own
eggs from foster eggs, selectively consuming the eggs that they have
not sired. Cases like Pimephales promelas and Gasterosteus aculeatus are
some examples of cannibalism and, while the first can distinguish their
eggs (Sargent, 1989), G. aculeatus seems not to be able to do that
(FitzGerald and van Havre, 1987). According with (Wisenden, 1999),
many fish parents do not actively discriminate against non-descendant
eggs either because the cost of keeping them is very low or the cost of
eliminating them is too high. Alloparental care may be enhanced in spe-
cies in which females prefer to spawn with males that are already de-
fending nests with eggs (Rohwer, 1978), possibly causing some males
to adopt eggs as a tactic to attract females (Sargent, 1989; Unger and
Sargent, 1988). Whether Lusitanian toadfish males adopt foster eggs
after nest takeovers to enhance mate attraction still needs to be tested.
Our experimental design did not take into account the predation pres-
sure by heterospecifics. Crabs, for example, are typical egg predators that
Fig. 1. Variation in egg survival in the different groups. Dots and bars are means and
standard deviation. Different letters indicate pairwise significant differences given by
ANOVA post-hoc Tukey tests, Pb0.05. AP —alloparental; PWE —parental with eggs;
FWE —females with eggs; CTRL —control nests without a male or female.
Fig. 2. Variation of (A) testosterone, (B) 11-ketotestosterone and (C)cortisol levels among
male groups. Plots show the medians, 10th, 25th 75th, and 90th percentiles as vertical
boxes with errors bars. Different letters indicate pairwise significant differences given by
Kruskall–Wallis post-hoc tests, Pb0.05. AP —alloparental males; PWE —parental males
with eggs; PNE —parental males with no eggs; NM —non-manipulated males.
61A. Ramos et al. / Journal of Experimental Marine Biology and Ecology 434-435 (2012) 58–62

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rapidly remove all eggs when males abandon their nests in nature
(unpublished data). Egg death in ENM was likely caused by fungus
(eggs became dark) but would have been much faster in the presence
of egg predators. Moreover, we cannot exclude the possibility of differ-
ential motivation between parental males (PWE) and alloparental
males in excluding predators, such as crabs from the nest, that might
have influenced the results of our experiments have they been con-
ducted in the natural habitat.
The lack of differences that weobserved in territorial defense behav-
ior among male groups is consistent with the general lack of differences
in androgen circulating levels. In fact, our different treatment groups
did not differ in T levels, although AP males showed higher levels of
11KT than the remaining groups. However, they showed a very high
variability in 11KT levels that fell within the range of the circulating
levels observed in nature, both in breeding males (non-manipulated
male group in this study) and in males in the post-breeding period
(Modesto and Canário, 2003). Thus, the presence of early-stage off-
spring (i.e. own or foster eggs) does not seem to alter androgen levels
that would mediate aggression towards intruders.
Although there is a general agreement on the mediation of agonistic
behavior and territorial defense displays by T and especially 11 KT
(Oliveira and Gonçalves, 2008), including calling behavior in teleosts
(Remage-Healey and Bass, 2005), the function of these hormones during
parental care in teleosts is more controversial. In a number of teleosts, an-
drogens have been shown to facilitate courtship and territorial behaviors,
but to suppress paternal care (Oliveira et al., 2002; Ros et al., 2004), al-
though exceptions where androgen levels remain elevated through pa-
rental care exist (e.g. Knapp et al., 1999; Rodgers et al., 2006). The few
studies that have focused on androgen mediation of nest defense during
parental care have shown that aggressive behavior of smallmouth bass
(Micropterus dolomieu) males towards a predator is not correlated to cir-
culating testosterone (or cortisol) (Hanson et al., 2009). However, in
other species (e.g. Neolamprologus pulcher), androgens may facilitate
nest defense (Desjardins et al., 2008). In the midshipman androgen levels
may reflect a compromise between investment in parental behavior ver-
sus continued courtship and/or territoriality (Knapp et al., 1999). This is
particularly important in males that can spawn with multiple females
over the course of the breeding season such as the Lusitanian toadfish.
Cortisol is a major stress hormone that can increase in plasma due
to long-term or acute stress (e.g. Clearwater and Pankhurs, 1997), or
it may be quickly released into the plasma in response to agonistic
challenges such as territorial intrusions (Remage-Healey and Bass,
2005). Similar plasma levels of this steroid in both experimental animals
and non-manipulated males suggests that experimental procedures did
not cause increased fish stress and that the lack of differences in territorial
defense behavior among treatments was not caused by pool confinement
and experimental procedures. Moreover, it suggests that territorial intru-
sions elicited a similar stress response among the different subject groups.
Cortisol data in our study suggest that the presence of eggs in the nest
(own or foster) did not affect the stress response induced by the agonistic
interaction between the intruders and nesting males.
5. Conclusion
This work suggests that Lusitanian toadfish provides alloparental
care to foster eggs. During the breeding season males arrive to mat-
ing areas in large densities increasing competition for nesting places
(Amorim et al., 2010) and nest takeovers are likely to occur upon nest
intrusions (Vasconcelos et al., 2010). In this context alloparental care
could have evolved if it conferred advantagesto the new nestoccupant.
Further work is needed to prove that the existence of eggs in the nest
represents an advantage in mate attraction. It is also still necessary to
test to which extent fish movements and the action of skin mucus re-
sults in ‘passive care’, such as observed in the case of nests occupied
by females in the present study. [RH]
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