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RESEARCH ARTICLE
How effective are acoustic signals in territorial defence in the
Lusitanian toadfish?
Carlotta Conti
1
, Paulo J. Fonseca
2
, Marta Picciulin
3,
* and M. Clara P. Amorim
1,‡
ABSTRACT
The function of fish sounds in territorial defence, in particular its
influence on the intruder’s behaviour during territorial invasions, is
poorly known. Breeding Lusitanian toadfish males (Halobatrachus
didactylus) use sounds (boatwhistles) to defend nests from intruders.
Results from a previous study suggest that boatwhistles function as a
‘keep-out signal’during territorial defence. To test this hypothesis we
performed territorial intrusion experiments with muted Lusitanian
toadfish. Males were muted by making a cut and deflating the
swimbladder (the sound-producing apparatus) under anaesthesia.
Toadfish nest-holder males reacted to intruders mainly by emitting
sounds (sham-operated and control groups) and less frequently with
escalated bouts of fighting. When the nest-holder produced a
boatwhistle, the intruder fled more frequently than expected by
chance alone. Muted males experienced a higher number of
intrusions than the other groups, probably because of their inability
to vocalise. Together, our results show that fish acoustic signals are
effective deterrents in nest/territorial intrusions, similar to bird song.
KEY WORDS: Batrachoididae, Halobatrachus didactylus,‘Keep-out’
signal, Muting experiments, Sound production, Teleost fish,
Territorial behaviour
INTRODUCTION
An individualfish’s probabilityof surviving and reproducing depends
to a large extent on its social behaviour in which communication
takes a major role. In contests for the establishment of social
hierarchies and territories, differences in fighting ability between
contestants influence the outcome of disputes (Parker, 1974; Arnott
and Elwood, 2009). Fighting ability or resource-holding potential
(Parker, 1974) is often related to size, but also to other factors such as
development of weaponry, physiological state, sex and residency
status (Turner and Huntingford, 1986; Enquist and Leimar, 1987;
Arnott and Elwood, 2009). Hence, when a contest occurs, opponents
typicallystart a ritualised sequence of displays that facilitate opponent
assessment and when asymmetries between contestants are large the
contest should be settled without the need for costly combats (Enquist
and Leimar, 1983, 1987).
Empirical evidence shows that acoustic signals are often used in
mutual assessment during agonistic interactions in mammals
(Clutton-Brock and Albon, 1979), birds (Krebs, 1976; Krebs
et al., 1978; Searcy and Beecher, 2009), anurans (Davies and
Halliday, 1978; Cocroft and Ryan, 1995) and fishes (Ladich and
Myrberg, 2006), because acoustic features may signal the sender’s
quality. For example, lower-frequency calls usually reflect larger
body size and hence better competitive ability because larger vocal
organs and vocal tracts produce and radiate lower frequencies more
efficiently (Bradbury and Vehrencamp, 1998). Also, other features
such as calling rate or sound amplitude may be condition dependent
(Clutton-Brock and Albon, 1979; Prestwich, 1994; Wyman et al.,
2008; Amorim et al., 2010a).
In fish, different studies have shown that several properties of
acoustic signals are related to body size. Larger fish tend to produce
lower-frequency (e.g. Ladich, 1998; Myrberg et al., 1993; Lobel and
Mann, 1995; Connaughton et al., 2000), louder (Ladich, 1998;
Connaughton et al., 2000; Lindström and Lugli, 2000; Amorim et al.,
2013) and longer sounds (Wysocki and Ladich, 2001; Amorim and
Hawkins, 2005; Amorim and Neves, 2008) than smaller individuals.
Also, the levelof calling activity may reflect the amount of fat reserves
(Amorim et al., 2010a, 2013; Pedroso et al., 2013).
Less known is how acoustic communication affects agonistic
interactions in fish, but in at least a few species sounds seem to be
used in mutual assessment and influence fight outcome (reviewed in
Ladich and Myrberg, 2006; Raffinger and Ladich, 2009). However,
studies on the function of sounds in territorial defence are scarce, in
particular in its influence on the intruder’s behaviour during
territorial invasions by conspecifics. For example, playing back
click sounds to skunk loaches Yasuhikotakia morleti during
territorial intrusions made residents increase the number of lateral
displays performed at intruders (Valinski and Rigley, 1981) whereas
the playback of rachet sounds to brown bullhead catfish Ameiurus
nebulosus decreased the number of attacks residents made at
intruders (Rigley and Muir, 1979). These experiments clearly show
that sounds can have a major role in modulating the resident’s
territorial behaviour. However, the deterrent function of sounds on
territorial intrusion has seldom been demonstrated. Playbacks of
conspecific sounds in the absence of a resident male have been
shown to have a deterrent effect in territorial intrusion in the bicolor
damselfish Stegastes partitus (Myrberg, 1997) and in the painted
goby Pomatoschistus pictus (Pereira et al., 2014), equivalent to the
‘keep-out’effect of bird song (Krebs, 1976).
To experimentally test the ‘keep-out signal’hypothesis, we used
the vocal Lusitanian toadfish Halobatrachus didactylus Bloch and
Schneider 1801. In the reproductive season (May to July in
Portugal) males occupy rock crevices or excavate under rocks in
shallow water and attract females with long tonal sounds (∼800 ms)
named boatwhistles (dos Santos et al., 2000; Modesto and Canário,
2003; Amorim et al., 2006). Females deposit their eggs under the
roof of the nest and males guard the eggs of multiple females until
the offspring is able to swim away (Ramos et al., 2012; Roux, 1986).
During this period competition for nests is high (Amorim et al.,
2010b) and males actively defend the nest from intruders with visual
Received 12 November 2014; Accepted 12 January 2015
1
MARE –Marine and Environmental Sciences Centre, ISPA Instituto Universitário,
1149-041 Lisboa, Portugal.
2
Departamento de Biologia Animal, cE3c - Centre for
Ecology, Evolution and Environmental Changes, Faculdade de Ciências,
Universidade de Lisboa, 1749-016 Lisboa, Portugal.
3
Facoltàdi Scienze
Matematiche, Fisiche e Naturali, Dipartimento di Biologia –CSEE, University of
Trieste, 34127 Trieste, Italy.
*Present address: Independent scholar, Italy
‡
Author for correspondence (amorim@ispa.pt)
893
© 2015. Published by The Company of Biologists Ltd
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The Journal of Experimental Biology (2015) 218, 893-898 doi:10.1242/jeb.116673
The Journal of Experimental Biology
and acoustic behaviour (Vasconcelos et al., 2010; Ramos et al.,
2012). Recently, Vasconcelos and colleagues (Vasconcelos et al.,
2010) have proposed that the boatwhistle functions as a ‘keep-out’
signal and suggested that vocalising may be an effective means to
avoid territorial intrusions and escalated levels of fighting in the
Lusitanian toadfish. However, the study of Vasconcelos et al.
(2010) cannot exclude the possibility that chemical or other cues
could also be at play. In the Lusitanian toadfish, vocalisations are
generated by vibration of the swimbladder caused by the contraction
of intrinsic sonic muscles (dos Santos et al., 2000); muting can
therefore be easily achieved by making a cut and deflating the
swimbladder under anaesthesia. Males can still contract the sonic
muscles but sounds become inaudible while fish behaviour appears
unaltered. Here, we used muting experiments to verify whether
acoustic signals (i.e. boatwhistles) are effective deterrents of
territorial intrusions in this species. We compared the dynamics of
territorial defence and the number of intrusions among muted and
control males (sham-operated and unmanipulated residents). We
further tested whether intruders fled more frequently than expected
by chance alone when the nest-holder made a boatwhistle.
RESULTS
Interaction dynamics
Intruding males readily swam towards the shelters and often
approached and tried to enter them. 44% of the resident males
(N=57) experienced approaches (range: 0–7 approaches) and 84%
experienced partial or total intrusions (range: 0–9). Muted fish
experienced fewer approaches (Kruskal–Wallis test: N=57, H=6.78,
P<0.05) but a greater number of intrusions (H=9.65, P<0.01) than
other groups (Fig. 1). However, the total number of interactions
(approach+intrusion) did not differ among groups (H=4.99, P>0.05;
Fig. 1).
The resident males responded to an intruder’s approach either by
producing sounds (mainly boatwhistles) or exhibiting escalated
levels of fighting (mostly bites and mouth wrestling). During
intrusions, the nest-holder response was similar but the proportion
of escalated fights was higher and the number of vocalisations lower
than during approaches (Table 1). Also, in contrast to approaches,
the production of boatwhistles could proceed to a fight if the
intrusion persisted. On many occasions there was no apparent
reaction from the resident (‘no reaction’).
We found an effect of treatment on the number of ‘no reactions’
(Kruskal–Wallis test: approach, N=25, H=7.04, P<0.05; intrusion,
N=8, H=10.56, P<0.01) but not on escalated levels of fighting
(approach, H=2.36, P>0.05; intrusion, H=1.76, P>0.05) during
approaches and intrusions. Muted fish showed the highest
occurrences of ‘no reaction’(Figs 2 and 3).
The duration of interactions (one-way ANOVA, F
2,144
=1.22,
P>0.05) and of interaction sequences (F
2,88
=0.91, P>0.05) did not
differ among groups (Fig. 4). The production of boatwhistles (BWs)
did not affect interaction duration in any interaction type: approach,
intrusion or approach followed by intrusion (two-way ANOVA,
BW: F
1,138
=0.12, P>0.05; interaction type: F
2,138
=19.53, P<0.001;
BW×interaction type F
2,138
=0.05, P>0.05).
There were marginally non-significant differences in takeovers
of muted and vocal fish nests (χ
2
=3.25, d.f.=1, P=0.07). Overall,
nest takeovers occurred infrequently. From the 48 residents that
experienced intrusions, 14 got replaced. A total of 23% (3 in 13),
18% (3 in 17) and 44% (8 in 18) of unmanipulated, sham-operated
and muted males got replaced by intruders, respectively. We
found no differences in time until nest takeover (i.e. sequence of
interaction duration until nest takeover) among treatments
(F
2,12
=0.42, P>0.05; Fig. 4). In nest takeovers, intruders and
residents were of similar sizes, the difference in total lengths
averaging 0.9%.
Intruder response to resident’s behaviour
Intruders usually fled when they heard a boatwhistle either while
approaching (85%, N=33) or intruding a nest (76%, N=25). The
probability of fleeing upon hearing a boatwhistle was significantly
higher thanexpected by chanceboth during approaches (binomial test,
N=33, P<0.001) or intrusions (binomial test, N=25, P<0.05). When
intruders received escalated agonistic behaviour, the chances of
fleeing were also higher than chance (binomial test, N=46, P<0.01)
and they fled 74% of the time. When intrusions were successful, the
intruder either stayed in the shelter with the resident or replaced him.
DISCUSSION
Experimental approaches to investigate the functional significance of
agonistic sounds in fish and other animals include sound exposure
0
2
4
6
8
Number of approaches
a
b
a,b
0
2
4
6
8
10
Number of intrusions
a
b
a,b
Muted Sham Unmanipulated
Treatment
0
5
10
15
Number of interactions
Fig. 1. Number of approaches, intrusions and interactions experienced
by resident male toadfish. Dots indicate medians whereas boxes and error
bars depict quartiles and range. Different letters indicate pairwise differences
given by post hoc Kruskal–Wallis tests. In the case of approaches, differences
are marginally non-significant (P=0.06) and for intrusions differences are
significant at the level of P<0.01.
894
RESEARCH ARTICLE The Journal of Experimental Biology (2015) 218, 893-898 doi:10.1242/jeb.116673
The Journal of Experimental Biology
through playback, exclusion of fish sounds by keeping opponents in
separate tanks or by muting individuals, the use of mirrors to level
visual interactions while testing the function of sound and correlative
analyses (Ladich and Myrberg, 2006). Although muting procedures
are more invasivethan the widely usedplayback approach (McGregor,
1992), they avoid the concurrent presentation of acoustical and visual
stimuli in playback tests, usually needed to elicit behavioural
responses in fish (Ladich and Myrberg, 2006). Muting experiments
have only been carried out twice (Valinski and Rigley, 1981; Ladich
et al., 1992) probably because many vocal fish species have unknown
sound-producing mechanisms (Ladich and Fine, 2006). Also, when
the mechanism is known, its deactivation typically results in alteration
or impairment of behaviour (Ladich and Myrberg, 2006). However, in
fishes that use swimbladder mechanisms, such as the Lusitanian
toadfish, swimbladder deflation does not impair sonic muscle
contraction but results in a marked decrease of sound amplitude
(Skoglund, 1961), causing the sounds to become inaudible while
behaviour remains apparently unaltered. Such fish species are ideal to
investigate the function of acoustical signalling in social contexts
because the outcome of social interactions of mute fish can be
compared with those of vocal animals. Unlike most fish species
(Ladich and Myrberg, 2006), the Lusitanian toadfish has the
advantage that a great component of agonistic interactions relies on
acoustic signalling performed with no accompanying visual displays
(Vasconcelos et al., 2010), thus avoiding the confounding effects of
the interplay of different sensory channels. Here, we experimentally
investigated whether sounds (boatwhistles) made by the Lusitanian
toadfish have an active role in preventing territorial intrusion by
comparing territorial defence between muted fish and two control
groups, sham-operated and unmanipulated males.
We found a treatment effect on the number of approaches and
intrusions experienced by nest-holders. Muted fish had more
intrusions and fewer approaches than the remaining groups, but
experienced a similar number of interactions (approach+intrusion).
These results suggest that intruders initiated interactions equally with
all groupsbut were more likely to proceed to intrusions in the nests of
muted males, probably because these males were not able to make
audible sounds. This is consistent with the observed high numbers of
‘no reactions’in muted males. Muted fish probably attempted to
defend their shelters by making sounds, but as this species typically
emits soundswith no accompanying visualdisplays, attempts at sound
production could not be detected. Similarly, in the grasshopper
Chorthippus biguttulus, males muted by removing the forewings,
fictively stridulated with the same frequency and movement pattern as
intact animals (Kriegbaum and von Helversen, 1992).
Nest-holder Lusitanian toadfish mainly reacted to approaches and
intrusions with sounds and, less often, with higher levels of fighting.
There was no significant difference in the levels of escalated
fighting among the three treatment groups, either as a reaction to
approaches or to intrusions, suggesting that fish did not compensate
the lack of ability to produce sounds with increased levels of
aggressiveness. In contrast, muted skunk loach nest-holders
increase the number of visual displays, but lowered attacks, in
comparison to control fish in an attempt to prevent nest intrusion
(Valinski and Rigley, 1981).
Importantly, when nest-holders made boatwhistles, intruders
tended to flee. In this context, unmanipulated and sham groups had a
higher probability of preventing territorial intrusion than muted fish.
Escalated fights also had a higher than expected chance to expel the
intruder but are more costly because they can incur physical injuries
and are energetically demanding. Consistent with the keep-out
signal hypothesis, an average of 44% of intrusions resulted in nest
takeovers in muted males, compared with 20% for vocal males. The
difference in the proportion of nest takeovers seems to be caused by
the ability to vocalise and not by the intruder’s size. The difference
in total length between expelled nest-holders and successful
intruders was ∼1% for the three treatment groups, although size
differences in our experiments were generally higher with a mean
difference of 9%. Altogether, the present data strongly suggest that
boatwhistles are effective keep-out signals, lowering the probability
of territorial intrusions and therefore nest takeovers.
Table 1. Reactions to intruder approaches and nest intrusions
Treatment NR (%) BW (%) EF (%) BW+EF (%) N
Approach Muted 81.25 –18.25 –4
Sham 39.6 56.6 3.8 0 13
Unmanipulated 4.2 95.8 0 0 8
Intrusion Muted 64.2 –35.8 –18
Sham 32.2 37.7 20.7 9.4 17
Unmanipulated 43.1 23.3 25.9 7.7 13
Percentages were calculated per fish and then averaged for each treatment group. N, Number of fish that experienced an approach or an intrusion.
NR, no reaction; BW, boatwhistle sound; EF, escalated fighting; BW+EF, sound followed by escalated fighting.
0
1
2
No reaction
Muted Sham Unmanipulated
Treatment
0
1
2
3
4
Escalated fight
Fig. 2. Number of times resident male toadfish showed ‘no reaction’or
engaged in escalated levels of fighting when approached by intruders.
Dots indicate medians and boxes and error bars depict quartiles and range.
Treatment had a significant effect only on the number of ‘no reactions’
(Kruskal–Wallis test, P<0.05). Post hoc tests indicated a marginally
non-significant difference (P=0.07) between muted and unmanipulated
males for ‘no reaction’.
895
RESEARCH ARTICLE The Journal of Experimental Biology (2015) 218, 893-898 doi:10.1242/jeb.116673
The Journal of Experimental Biology
Other studies support the importance of acoustic signals in
winning contests and in deterring territorial intrusion. In the
croaking gourami Trichopsis vittata, territorial males matched in
size with the opponent, had a significan tly higher chance of winning
the dispute when they were vocal than when muted. However, when
size differences increased, larger fish tended to win the fight
irrespective of the ability to vocalise (Ladich et al., 1992). Muted
skunk loaches also experienced more intrusions than control fish,
but differences in sizes between contestants were not mentioned
(Valinski and Rigley, 1981). The deterrent effect of sounds on
territorial intruders has been shown for the bicolor damselfish
(Myrberg, 1997) and for the painted goby (Pereira et al., 2014)
because intruders took longer to enter unoccupied territories/nests
associated with conspecific sound playback than silent ones. The
deterrent effect of agonistic acoustic signals on territorial intrusions
has traditionally been described for birds. Muting adversely affects
the ability to acquire and defend territories (e.g. McDonald, 1989)
and song playback from territories after removal of owners delays
occupation by intruders (e.g. Krebs et al., 1978).
Interestingly, the duration of interactions, including time to nest
takeover, did not differ between muted and vocal fish. This suggests
that the dynamics of mutual assessment, which involves reiteration
of behaviour between opponents (Enquist and Leimar, 1983, 1987),
was not altered by differences in vocal activity.
Our muting experiments did not cause alteration of the behaviour
in muted fish because all groups showed similar levels of escalated
fighting. Muting experiments in different taxa include examples
where the subject’s behaviour remains unaltered after being
silenced. For example, croaking gourami males prevented from
making sounds by cutting the two enhanced pectoral fin tendons
involved in sound production exhibited normal swimming
movements and agonistic behaviour (Ladich et al., 1992). Also, in
the study of Davies and Halliday (1978), silencing toad (Bufo bufo)
males did not seem to alter reproductive or agonistic behaviour.
Together, the results of this study provide experimental evidence
of the deterrent function of agonistic sounds in territorial defence in
fish. We show that acoustic signals play an active role in territorial
defence, decreasing the probability of escalated fighting and
intrusions, and thus probably reducing nest takeovers.
MATERIALS AND METHODS
Test males and maintenance
Prior to the beginning of the breeding season, 60 artificial hemicylinder
concrete shelters (50 cm long, 30 cm wide and 20 cm high) were placed
approximately 1.5 m apart in threerows, alongan intertidal areaof Tagus River
estuary (Military Air Force Base, Montijo, Portugal; 38°42′N, 8°58′W). Fish
spontaneously occupied these shelters and we were able to access the animals
at low spring tides during Mayto July 2011. We also used somefish caught by
local fisherman. Only territorial males were used and they were identified by
gently pressing their abdomen near the urogenital opening where they have
accessory glands that release a dark-brown seminal fluid, unlike females and
0
2
4
6
8
No reaction
a
b
a,b
Muted Sham Unmanipulated
Treatment
0
2
4
6
8
Escalated fight
Fig. 3. Number of times toadfish male residents showed ‘no reaction’or
engaged in escalated levels of fighting upon intrusions. Dots indicate
medians whereas boxes and error bars depict quartiles and range. Different
letters denote pairwise significant differences at P<0.01.
0
200
400
600
Interaction duration (s)
0
200
400
600
800
1000
Sequence duration (s)
Muted Sham Unmanipulated
Treatment
0
500
1000
1500
Time to nest takeover (s)
Fig. 4. Mean duration of resident–intruder interactions, sequence of
interactions and sequence of interactions that lead to nest takeover.
Temporal patterns of the dynamics of territorial defence did not differ among
groups (one way ANOVA, P>0.05). See the Materials and methods for details
of duration measurements.
896
RESEARCH ARTICLE The Journal of Experimental Biology (2015) 218, 893-898 doi:10.1242/jeb.116673
The Journal of Experimental Biology
sneaker males (Modesto and Canário, 2003). We maintained experimental
males in round stock tanks ( plastic swimmingpools 2 m in diameter and water
depth of 0.5 m) near the intertidal toadfish nesting area where males were
collected. Stock and experimental tanks (similar to the stock tanks but with
2.5 m diameter) were placed on the sand just above the high tide shoreline
under a shadow net coverheld 170 cm high to prevent excessive solarradiation
and water heating. Water temperature varied from 18 to 26°C (mean 21.4°C),
within the range of the estuary water temperature variation during the same
period. The water was changed every 2–3 days, by pumping water directly
from the estuary. A natural light cycle was maintained because the stock tanks
were outdoors.
Territorial intrusion protocol
We carried out territorial intrusion experiments with resident and intruder
fish to simulate a context of male–male competition during territorial
defence. Resident males were randomly assigned to three treatments: muted,
sham-operated and unmanipulated males. Males were muted with a small
surgical procedure after they were anaesthetised with a benzocaine solution
(0.1 g l
−1
) for few minutes. A small incision in the abdominal area was made
and the swimbladder was deflated through a small cut to prevent sound
production. The abdominal opening was then closed with two stitches. To
control for possible effects of the surgery on toadfish territorial behaviour
(apart from the ability to vocalise) a sham-operated treatment was also used.
Sham-operated fish were given the same procedure as the muted group,
except for the actual swimbladder cutting and deflation, and they were still
able to vocalise normally. Fish were allowed to recover from anaesthesia
before being placed in the experimental tanks. Resident test males from the
unmanipulated group did not experience any surgical intervention and
controlled for possible effects of anaesthesia and surgery procedures. The
muting procedure was effective because muted males did not make sounds
during trials and the number of resident–intruder interactions with sound
production did not differ between vocal groups (Mann–Whitney test,
N
Sham
=20, N
unmanip
=19, U=154.5, P>0.05; Table 1).
Two males from the same experimental group were placed in an
experimental tank at least 24 h before the experiments, allowing them to
become territorial and recover from possible short-term surgery effects.
Each experimental tank was provided with two roof tiles as shelters (internal
dimensions 44×18×10 cm) placed approximately 50 cm apart and 20 cm
away from the tank’s border. All subject males readily occupied the empty
shelters and spent most of the time inside them, a normal territorial fish
behaviour (Vasconcelos et al., 2010). We placed one hydrophone (High
Tech 94 SSQ, High Tech Inc., Gulfport, MS, USA; frequency response:
30 Hz to 6 kHz ±1 dB; voltage sensitivity: −165 dB re. 1 V µPa
−1
) in front
of each nest, at about 10 cm from its entrance and from the tank bottom,
attached to a wooden rod kept over the tank. Simultaneous two-channel
recordings were made with a USB audio capture device (Edirol UA-25,
Roland, Osaka, Japan; 16 bit, 44.1 kHz acquisition rate per channel)
connected to a laptop and down-sampled to 6 kHz by Adobe Audition 3.0
(Adobe Systems, San José, CA, USA). Recorded sounds could be attributed
to a particular territorial male because of the proximity of each hydrophone
to one nest. Usually, only territorial males produce sounds (Vasconcelos
et al., 2010). In one exceptional case (M.C.P.A., unpublished data)<CQ2>,
we observed one intruder producing boatwhistles during intrusions but the
resident’s and the intruder’s sounds could clearly be distinguished due to
spectral differences.
In each trial, two intruder males (unmanipulated) were placed
sequentially in the experimental tank with an interval of 30 min between
intrusions and remained in the tank until the end of the trial (following
Vasconcelos et al., 2010). Our experimental design resembles the natural
chorusing aggregations, where territorial males nest very close together
(Amorim et al., 2010b) and may attract several competitor males
(Vasconcelos et al., 2012). It also aimed to increase the motivation of
subject males to become territorial and the number of territorial defence
interactions during trials, thus decreasing the need for a larger number of
operated males. The first intruder was not removed when the second was
introduced in the tank to avoid disturbing resident males. Intruders were
chosen randomly from stock tanks, but in most cases, residents and intruders
were matched in total length (TL) (mean total length difference resident
TL/intruder TL×100=7%; median=1%; range: −20% to 67%) with only
9 out of 57 residents experiencing size asymmetries larger than 20%. Fish
were labelled with marks in the fins (i.e. a small cut between the fin rays) to
identify them during trials. Marking did not cause any measurable change in
behaviour. Behavioural interactions and sound produced were registered for
60 min beginning with the placement of the first intruder male. After each
trial all specimens were measured for total length (TL) to the nearest mm and
weighed to the nearest gramme. We used a total of 18, 20 and 19 resident
males for the muted, sham-operated and unmanipulated treatments, with a
mean (range) TL of 41.3 (32.4–48.0) cm, 43.9 (36.6–50.0) cm and 40.5
(26.8–47.0) cm, respectively. We used a total of 64 intruders with a mean
(range) TL of 39.5 (27.0–50.0) cm.
Behavioural analysis
Behaviour of residents and intruders was assessed by direct observation,
noted on paper and later tallied following Vasconcelos et al. (2010). Sound
production was simultaneously monitored with headphones that were
connected to the recording laptop. For residents, we registered the number
of non-escalated behaviours including mouth opening with the extension
of pectoral fins and opercula and escalated behaviours including chasing,
bite attempts, bites and mouth–mouth fighting. The number of times
residents showed no apparent reaction (‘no reaction’, i.e. no visible or
audible behaviour) upon and intruder’s approach or nest intrusion was also
measured. We also tallied the duration of resident–intruder interactions
and the sequence of interactions because duration of fighting is an
important measurement of mutual assessment (Enquist and Leimar,
1983). An interaction was considered as a set of consecutive behaviours
involving one resident and one intruder that started with the latter
approaching or intruding the nest and stopped when he fled to the border
of the tank or took over the nest. A sequence of interactions were a set of
consecutive interactions involving the same resident and intruder that were
not interrupted by an interaction with another male (usually the other
intruder) and that finished with either the intruder fleeing and not further
resuming the interaction or with a nest takeover. We tallied the number of
sounds emitted by the resident including agonistic boatwhistles or other
sound types (grunts, long grunt trains, croaks and double croaks; see
Amorim et al., 2008 for a description). For the intruders, we tallied the
number of approaches, intrusions in the nest (the intruder entering
partially or completely) and fleeing. We defined approaches when the
intruder was at least within a body length from the nest and an intrusion
when the intruders managed to get at least part of the body inside the nest.
Fleeing consisted of swimming away from the nest. These categories are
mutually exclusive but may be performed sequentially.
Statistical analysis
Statistical tests were performed with Statistica 12.0 for Windows (StatSoft,
Inc., Tulsa, OK, USA) and all data were transformed when necessary to
meet assumptions of the used parametric tests. When there was no normality
of the transformed data, non-parametric tests were used.
We compared the number of approaches, intrusions and total interactions
(approach+intrusion) experienced by the different treatment groups with
Kruskal–Wallis tests. Similarly, the responses of the residents (‘no reaction’
and escalated fights) were compared among treatment groups with Kruskal–
Wallis tests. Post hoc tests available in Statistica and described in Siegel and
Castellan (1988) were used for multiple comparisons between treatments.
The effect of treatment on interaction and sequence of interaction
durations was tested with one-way ANOVA. We tested whether the
production of boatwhistles altered interaction duration with a two-way
ANOVA that included the factor interaction type (with three levels:
approach, intrusion and approach+intrusion) and the factor boatwhistle
production (two levels: vocal and silent). We finally compared the duration
of the sequence of interactions until nest takeover among treatment groups.
Interaction and sequence of interaction durations were log-transformed to
meet the ANOVA assumptions.
A chi-square test of independence was performed to test whether when
there was an intrusion the variable nest takeover (nest takeover versus no
takeover) was independent of vocalising (vocal versus muted). The
probability of the intruder fleeing after receiving a boatwhistle or an
897
RESEARCH ARTICLE The Journal of Experimental Biology (2015) 218, 893-898 doi:10.1242/jeb.116673
The Journal of Experimental Biology
escalated attack by the resident, when approaching or intruding its nest, was
compared with what was expected to happen randomly with binomial tests.
Acknowledgements
We thank the Air ForceBase No. 6 of Montijo (Portugal ) forallowing this study in their
military establishment. We are grateful to Andreia Ramos for help with field work. We
thank Bruno Novais for analysing components of the behavioural data. We are also
grateful to the referees who helped to improve this paper. All experimental
procedures comply with Portuguese animal welfare laws, guidelines and policies.
Competing interests
The authors declare no competing or financial interests.
Author contributions
M.C.P.A. and P.J.F. were involved in conception of the study and experimental
design. C.C. conducted the study. M.C.P.A. carried out statisticalanalyses. M.C.P.A.
and C.C. drafted the article. All authors revised the article.
Funding
This study was funded by Science and Technology Foundation, Portugal ( project
PTDC/MAR/118767/2010, pluriannual program UI&D 331/94 and UI&D 329, grant
SFRH/BPD/41489/2007 to M.C.P.A.).
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