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How effective are acoustic signals in territorial defence in the Lusitanian toadfish?

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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. Subject males were assigned to three groups: muted, sham and unmanipulated. Males were muted by making a cut and deflating the swimbladder (the sound producing apparatus) under anaesthesia. Sham males suffered the same surgical procedure except the swimbladder cut and deflation. Toadfish nest-holder males reacted to intruders mainly by emitting sounds (sham and unmanipulated) and less frequently with escalated fights. 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 remaining groups probably due to their inability to vocalise. Together, our results show that fish acoustic signals are effective deterrents in nest/territorial intrusions, similar to bird song.
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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 intruders 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 signalduring 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 individualfishs 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 senders
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 intruders 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 residents
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-outeffect of bird song (Krebs, 1976).
To experimentally test the keep-out signalhypothesis, 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: 07 approaches) and 84%
experienced partial or total intrusions (range: 09). Muted fish
experienced fewer approaches (KruskalWallis 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 intruders 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
(KruskalWallis 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 residents 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 KruskalWallis 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.
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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 reactionsin 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 intruders 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 reactionor
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
(KruskalWallis test, P<0.05). Post hoc tests indicated a marginally
non-significant difference (P=0.07) between muted and unmanipulated
males for no reaction.
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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 subjects 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°42N, 8°58W). 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 reactionor
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 residentintruder 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.
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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 23 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 malemale 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 residentintruder interactions with sound
production did not differ between vocal groups (MannWhitney 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 tanks 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
residents and the intruders 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 (32.448.0) cm, 43.9 (36.650.0) cm and 40.5
(26.847.0) cm, respectively. We used a total of 64 intruders with a mean
(range) TL of 39.5 (27.050.0) 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 mouthmouth fighting. The number of times
residents showed no apparent reaction (no reaction, i.e. no visible or
audible behaviour) upon and intruders approach or nest intrusion was also
measured. We also tallied the duration of residentintruder 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
KruskalWallis 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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898
RESEARCH ARTICLE The Journal of Experimental Biology (2015) 218, 893-898 doi:10.1242/jeb.116673
The Journal of Experimental Biology
... All of these attributes can contribute prominently in determining the outcome of aggressive conflicts (e.g. Davies and Halliday, 1978;Clutton-Brock and Albon, 1979;Bradbury and Vehrencamp, 2011;Amorim, 2015;Conti et al., 2015;Billings, 2018). ...
... Resources such as shelters and territories are continually defended by an owner to gain fitness advantages (Conti et al., 2015;Arnott and Elwood, 2007). Danionella dracula males swim closely around nest sites that contain spawning crevices. ...
... Future studies would benefit from examining whether and how males might utilize acoustic communication and other signaling modalities (e.g. vision) to structure possible dominance hierarchies and determine nest ownership in a community (Conti et al., 2015;Chase et al., 2002;Amorim and Almada, 2005;Arnott and Elwood, 2009;Barata et al., 2007;Myrberg and Riggio, 1985). While the acoustic parameters we describe here can be correlated with features of agonistic displays, it is essential to first study what components, if any, of this information are perceived by members of a contest and any observers before characterizing a behavior as a signal or a cue, or to determine what features may or may not mediate mutual or selfassessment (Bradbury and Vehrencamp, 2011;Arnott and Elwood, 2009). ...
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Acoustic behavior is widespread across vertebrates, including among fishes. We report robust acoustic displays during aggressive interactions for a laboratory colony of Danionella dracula, a miniature and transparent species of teleost fish closely related to zebrafish (Danio rerio), which are hypothesized to be sonic based on the presence of a hypertrophied muscle associated with the male swim bladder. Males produce bursts of pulsatile sounds and a distinct postural display–extension of a hypertrophied lower jaw, a morphological trait not present in other Danionella species—during aggressive, but not courtship interactions. Females show no evidence of sound production or jaw extension in such contexts. Novel pairs of size-matched or -mismatched males were combined in resident-intruder assays where sound production and jaw extension could be linked to individuals. In both dyad contexts, resident males produced significantly more sound pulses than intruders. During heightened sonic activity, the majority of highest sound producers also showed increased jaw extension. Residents extended their jaw more than intruders in size-matched, but not -mismatched contexts. Larger males in size-mismatched dyads produced more sounds and jaw extensions compared to their smaller counterparts, and sounds and jaw extensions increased with increasing absolute body size. These studies establish D. dracula as a sonic species that modulates putatively acoustic and postural displays during aggressive interactions based on residency and body size, providing a foundation for further investigating the role of multimodal displays in a new model clade for neurogenomic and neuroimaging studies of aggression, courtship, and other social interactions.
... All of these attributes can contribute prominently in determining the outcome of aggressive conflicts (e.g. Davies and Halliday, 1978;Clutton-Brock and Albon, 1979;Bradbury and Vehrencamp, 2011;Amorim, 2015;Conti et al., 2015;Billings, 2018). ...
... Resources such as shelters and territories are continually defended by an owner to gain fitness advantages (Conti et al., 2015;Arnott and Elwood, 2007). Danionella dracula males swim closely around nest sites that contain spawning crevices. ...
... Future studies would benefit from examining whether and how males might utilize acoustic communication and other signaling modalities (e.g. vision) to structure possible dominance hierarchies and determine nest ownership in a community (Conti et al., 2015;Chase et al., 2002;Amorim and Almada, 2005;Arnott and Elwood, 2009;Barata et al., 2007;Myrberg and Riggio, 1985). While the acoustic parameters we describe here can be correlated with features of agonistic displays, it is essential to first study what components, if any, of this information are perceived by members of a contest and any observers before characterizing a behavior as a signal or a cue, or to determine what features may or may not mediate mutual or selfassessment (Bradbury and Vehrencamp, 2011;Arnott and Elwood, 2009). ...
Preprint
Individuals can reveal their relative competitive ability or mate quality through acoustic communication, varying signals in form and frequency to mediate adaptive interactions including competitive aggression. We report robust acoustic displays during aggressive interactions for a laboratory colony of Danionella dracula, a recently discovered miniature and transparent species of teleost fish closely related to zebrafish (Danio rerio). Males produce bursts of pulsatile, click-like sounds and a distinct postural display, extension of a hypertrophied lower jaw, during resident-intruder dyad interactions. Females lack a hypertrophied lower jaw and show no evidence of sound production or jaw extension under such conditions. Novel pairs of size-matched or mismatched males were combined in resident-intruder assays where sound production and jaw extension could be linked to individuals. Resident males produce significantly more sound pulses than intruders in both dyad contexts; larger males are consistently more sonic in size-mismatched pairs. For both conditions, males show a similar pattern of increased jaw extension that frequently coincided with acoustic displays during periods of heightened sonic activity. These studies firmly establish D. dracula as a sound-producing species that modulates both acoustic and postural displays during social interactions based on either residency or body size, thus providing a foundation for investigating the role of these displays in a new model clade for neurogenomic studies of aggression, courtship and other social interactions.
... The Lusitanian toadfish, H. didactylus, is a gregarious vocal species with an unusually rich repertoire for a fish (Amorim et al., 2008) that relies on acoustic communication for mate finding and attraction (Vasconcelos et al., 2012) and for the spacing out of territorial males (Vasconcelos et al., 2010;Conti et al., 2015). The more commonly produced soundthe boatwhistle (BW)is used both to attract females and repel possible intruders (Vasconcelos et al., 2010(Vasconcelos et al., , 2012Conti et al., 2015). ...
... The Lusitanian toadfish, H. didactylus, is a gregarious vocal species with an unusually rich repertoire for a fish (Amorim et al., 2008) that relies on acoustic communication for mate finding and attraction (Vasconcelos et al., 2012) and for the spacing out of territorial males (Vasconcelos et al., 2010;Conti et al., 2015). The more commonly produced soundthe boatwhistle (BW)is used both to attract females and repel possible intruders (Vasconcelos et al., 2010(Vasconcelos et al., , 2012Conti et al., 2015). A reduction in BW active space will likely affect mate detection distance and vocal interactions amongst neighbouring territorial males, with implications for fitness. ...
... The Lusitanian toadfish, H. didactylus, is a gregarious vocal species with an unusually rich repertoire for a fish (Amorim et al., 2008) that relies on acoustic communication for mate finding and attraction (Vasconcelos et al., 2012) and for the spacing out of territorial males (Vasconcelos et al., 2010;Conti et al., 2015). The more commonly produced soundthe boatwhistle (BW)is used both to attract females and repel possible intruders (Vasconcelos et al., 2010(Vasconcelos et al., , 2012Conti et al., 2015). ...
... The Lusitanian toadfish, H. didactylus, is a gregarious vocal species with an unusually rich repertoire for a fish (Amorim et al., 2008) that relies on acoustic communication for mate finding and attraction (Vasconcelos et al., 2012) and for the spacing out of territorial males (Vasconcelos et al., 2010;Conti et al., 2015). The more commonly produced soundthe boatwhistle (BW)is used both to attract females and repel possible intruders (Vasconcelos et al., 2010(Vasconcelos et al., , 2012Conti et al., 2015). A reduction in BW active space will likely affect mate detection distance and vocal interactions amongst neighbouring territorial males, with implications for fitness. ...
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... The calling rate and calling effort (percentage of time spent calling) of Batrachoididae has been found to indicate male condition (Vasconcelos et al., 2012) because these parameters reflect sonic muscle hypertrophy and larger gonads (Amorim et al., 2010). Sound dominant frequency, amplitude and fatigue resistance may also indicate body size (Bose et al., 2018), with larger fish tending to produce lower frequency, louder and longer sounds than smaller individuals (Conti et al., 2015). Additionally, boatwhistles are involved in male competition, as closely located individuals will produce "jamming" signals. ...
... Despite the large number of experimental studies on toadfish vocalizations, surprisingly little is known about the occurrence and parameters of natural calls (Conti et al., 2015) and even less on the proximity of individual males. Previous studies have used invasive methods, such as locating and recording boatwhistles with SCUBA divers (Barimo and Fine, 1998) or restricting toadfish movements by placing individuals within artificial shelters (Zeddies et al., 2012). ...
... The calling rate and calling effort (percentage of time spent calling) of Batrachoididae has been found to indicate male condition (Vasconcelos et al., 2012) because these parameters reflect sonic muscle hypertrophy and larger gonads (Amorim et al., 2010). Sound dominant frequency, amplitude and fatigue resistance may also indicate body size (Bose et al., 2018), with larger fish tending to produce lower frequency, louder and longer sounds than smaller individuals (Conti et al., 2015). Additionally, boatwhistles are involved in male competition, as closely located individuals will produce "jamming" signals. ...
... Despite the large number of experimental studies on toadfish vocalizations, surprisingly little is known about the occurrence and parameters of natural calls (Conti et al., 2015) and even less on the proximity of individual males. Previous studies have used invasive methods, such as locating and recording boatwhistles with SCUBA divers (Barimo and Fine, 1998) or restricting toadfish movements by placing individuals within artificial shelters (Zeddies et al., 2012). ...
... A coustic signals are widely used among animals for multiple communication and behavioral purposes, including in the aquatic environment 1,2 . Bony fishes have evolved diverse sound generating mechanisms that are well-studied [3][4][5] and use them for intraspecific communication, often for agonistic and mating behaviors [6][7][8][9] . However, how acoustic communication can evolve via adaptation within species with respect to needs in their specific environments is unknown. ...
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Acoustic communication allows the exchange of information within specific contexts and during specific behaviors. The blind, cave-adapted and the sighted, river-dwelling morphs of the species Astyanax mexicanus have evolved in markedly different environments. During their evolution in darkness, cavefish underwent a series of morphological, physiological and behavioral changes, allowing the study of adaptation to drastic environmental change. Here we discover that Astyanax is a sonic species, in the laboratory and in the wild, with sound production depending on the social contexts and the type of morph. We characterize one sound, the "Sharp Click", as a visually-triggered sound produced by dominant surface fish during agonistic behaviors and as a chemosensory-, food odor-triggered sound produced by cavefish during foraging. Sharp Clicks also elicit different reactions in the two morphs in play-back experiments. Our results demonstrate that acoustic communication does exist and has evolved in cavefish, accompanying the evolution of its behaviors.
... While birdsong is one of the most well studied phenomena in animal communication, fewer studies have attempted to experimentally demonstrate the territorial function of vocalizations in other taxa. Speaker occupation experiments have been used in bicolor damselfish (Pomacentrus partitus; Myrberg, 1997) and painted gobies (Pomatoschistus pictus; Pereira et al., 2013), and muting experiments more recently in Lusitanian toadfish (Halobatrachus didactylus; Conti et al., 2015) to show that vocalizations serve as a "keepout" signal to other conspecifics. Due to the limitations of finding species amenable to such experimental designs, our understanding of the territorial function of vocalizations in mammals has been limited to observational field studies or playback experiments that induce an aggressive response in the territory holder (Smith, 1978;Grinnell et al., 1995;Barlow & Jones, 1997;Reby et al., 1999;Grinnell & McComb, 2001;Sharpe & Goldingay, 2009). ...
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Full-text available
In many species, territory defense is thought to be one of the primary functions of acoustic communication. North American red squirrels are a territorial species in which 'rattles' have long been thought to be the principal signal communicating territory ownership. These vocalizations have been assumed to deter intruders, thus reducing energy costs and the risk of injury associated with direct aggressive interactions. However, this hypothesis has not been directly tested. Here we used a speaker occupation experiment to test whether red squirrel rattles function to deter conspecific rivals. We studied 29 male squirrels and removed each individual from his territory twice in a paired design. During the experimental treatment we simulated the owner's presence after its removal by broadcasting the owner's rattle from a loudspeaker at the center of the territory once every seven minutes. During the control treatment the territory was left in silence after the temporary removal of the owner. We found that the presence of a speaker replacement reduced the probability of intrusion by 34% and increased the latency to first intrusion by 7%, providing support for the hypothesis that rattles play an active role in reducing intrusion risk. However, intrusions were not completely averted by the speaker replacement, indicating that vocalizations alone are not sufficient without other cues of the territory owner.
... Several species vocalisations are used in mutual assessment and influence the confrontation outcome [9,15]. For example, the male Lusitanian toadfish (Halobatrachus didactylus), may use boatwhistle vocalisations as a warning signal against other males [16]. Also, playback experiments of conspecific sounds in the absence of a resident male have been shown to play a territory guarding role against intrusions from potential competitors in the bicolour damselfish, Stegastes paritus [17], and in the painted goby, Pomatoschistus pictus [18]. ...
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Full-text available
Studies on the behavioural function of sounds are very rare within heterospecific interactions. John Dory (Zeus faber) is a solitary, predatory fish that produces sound when captured, but has not been documented to vocalize under natural conditions (i.e. in the wild). The present study provides the first in-situ recordings of John Dory vocalisations and correlates them to behavioural response of snapper (Pagrus auratus) a common species found through New Zealand. Vocalisations or ‘barks’, ranged between 200–600 Hz, with a peak frequency of 312 ± 10 Hz and averaged 139 ± 4 milliseconds in length. Baited underwater video (BUV) equipped with hydrophones determined that under natural conditions a John Dory vocalization induced an escape response in snapper present, causing them to exit the area opposite to the position of the John Dory. We speculate that the John Dory vocalisation may be used for territorial display towards both conspecifics and heterospecifics, asserting dominance in the area or heightening predatory status.
... In general, sound types in this family consist of growls and hums (Bass and McKibben 2003), croaks (dos Santos et al. 2000), short, broadband grunts (Thorson and Fine 2002a;Fine and Waybright 2015), and the harmonic Bboatwhistleĉ all used primarily for mate attraction (Bass and McKibben 2003). Calls have also been observed to be produced in the defense of the nest by males of Halobatrachus didactylus (Vasconcelos et al. 2010;Conti et al. 2015). Grunts are produced right before calls (Talvoga 1958) or as stand-alone utterances, singly or in combination, in agonistic and distress situations by both males and females (Gray and Winn 1961;dos Santos et al. 2000;Amorim et al. 2006;Maruska and Mensinger 2009;Fine and Waybright 2015). ...
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The soundscapes of many coastal habitats include vocalizations produced by species of the family Batrachoididae (toadfish and midshipman). We describe the calling and grunting behavior of male Amphichthys cryptocentrus, a tropical toadfish, and predict how these vocalizations are influenced by conspecifics. We recorded individual males, which produced broadband grunts and multi-note, harmonic “boatwhistle” calls. Grunts were either in combination with calls or stand-alone. We used a null model to test if these latter grunts were produced at random or in response to calls from conspecifics. The model supports the hypothesis that grunts were in response to calls from neighboring males, suggesting acoustic competition. Using the most conservative estimate of hearing abilities we predict that males responded to the second harmonic of neighbor’s calls (230 Hz) at amplitudes of approximately 100–125 dB re 1μPa2/Hz. We also observed that call and grunt rates increased when males were exposed to higher rates of acoustic activity from neighboring fish. Fish used grunts to respond to background calls that occurred at different amplitudes, suggesting they responded to the calls of multiple neighboring fish and not just the highest amplitude neighbor. This communication with multiple fish within hearing range suggests a communication network in which the spatial distribution of individual toadfish relative to one another will impact their vocal behavior. Thus, the density and distribution, and not just abundance, of these toadfish at a given site will influence the characteristics of the chorus and the role of this species in the local soundscape.
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Book
Playback is the technique of rebroadcasting natural or synthetic signals to animals and observing their response. The ability to present a putative signal in isolation, without the potential confounding effects of other activities of the signaller, is the main reason for the depth and range of our knowledge of communication systems. To date, playback of sound signals has predominated, but playback of electric signals and even video playback of visual signals suggests that playback will become just as prevalent in studies of communication in other sensory modalities. This book is one of the outcomes of a workshop on playback held at Thombridge Hall in the Peak District National Park, England during August 1991. There were two reasons for organising the workshop. First, the considerable and lively debate in the literature about the design and analysis of playback experiments -the pseudoreplication debate -was in danger of generating more heat than light. A workshop forum seemed the obvious place to clarify and, if possible, resolve the debate. Second, with the number of new playback and analysis techniques increasing rapidly, it seemed an opportune moment to discuss these techniques and to review some rapidly developing areas of interest in sound communication.