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Tadpoles detect chemical signals released from predators and conspecifics, and those present in the environment, and adjust their behavioral responses. This study evaluated the swimming activity of Rhinella dorbignyi (Duméril and Bibron, 1941) tadpoles exposed to chemical signals, including cues from a predator fish Synbranchus marmoratus Bloch, 1975 and an injured conspecific; sublethal concentration of insecticide cypermethrin; and their combination. Swimming behavior (total distance moved, average speed, global activity, number of contacts between tadpoles) was evaluated in an individual (1) and groups of different size (3, 5, 7 and 10 tadpoles) using a video-tracking software tool. Predator exposure modified behavioral parameters, reducing encounters with predators and, therefore, mortality. Total distance moved and average speed increased in trials involving 1 tadpole and 3 interacting tadpoles exposed to injured conspecifics, whereas global activity increased in all group sizes, showing that gregarious tadpoles may be affected by alarm cues and their behavior may be disrupted. The insecticide treatments (alone and combined) increased parameters in all group sizes, causing hyperactivity due to its neurotoxic effect. The different responses observed after exposure to alarm cues and environmental signals in the different group sizes modified the normal behavior and the ecological dynamics of gregarious tadpoles.
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19
Signals from predators, injured conspecifics, and pesticide
modify the swimming behavior of the gregarious tadpole of
the Dorbigny
s Toad, Rhinella dorbignyi (Anura: Bufonidae)
L.M. Curi, A.P. Cuzziol Boccioni, P.M. Peltzer, A.M. Attademo, A. Bassó, E.J. León, and R.C. Lajmanovich
Abstract: Tadpoles detect chemical signals released from predators and conspecifics, as well as those present in the e nvi -
ronment, and adjust their behavioral responses. This study evaluated the swimming activity of Dorbigny
s Toad (Rhinella
dorbignyi (Duméril and Bibron, 1841)) tadpoles exposed to chemical signals, including cues from a predator fish, the marbled
swamp eel (Synbranchus marmoratus Bloch, 1795), and an injured conspecific; sublethal concentration of insecticide cypermethrin;
and their combination. Swimming behavior (total distance moved, mean speed, global activity, number of contacts between tad-
poles) was evaluated in an individual (1) and groups of different size (3, 5, 7, and 10 tadpoles) using a video-tracking software tool.
Predator exposure modified behavioral parameters, reducing encounters with predators and, therefore, mortality. Total distance
moved and mean speed increased in trials involving 1 tadpole and 3 i nteracting tadpoles exposed to injured conspecifics, whereas
global activity increased in all group sizes, showing that gregarious tadpoles may be affected by alarm cues and their behavior may
be disrupted. The insecticide treatments (alone and combined) increased parameters in all group sizes, causing hyperactivity due to
its neurotoxic effect. The different responses observed after exposure to alarm cues and environmental signals in the different
group sizes modified the normal behavior and the ecological dynamics of gregarious tadpoles.
Key words: alarm cues, behavior, chemical signals, burrowing toad, pesticides, Dorbigny
s Toad, Rhinella dorbignyi.
Résumé : Les têtards détectent des signaux chimiques émis par des prédateurs et des congénères et des signaux présents
dans le milieu et ajustent leurs actions comportementales. Létude se penche sur lactivité de nage de têtards du crapaud
de Dorbigny (Rhinella dorbignyi (Duméril et Bibron, 1841)) exposés à des signaux chimiques, dont des signaux dun poisson
prédateur, languille des mares marbrée (Synbranchus marmoratus Bloch, 1795), et dun conspécifique blessé, une concentra -
tion sublétale de linsecticide cyperméthrine, et leur combinaison. Le comportement de nage (distance totale de déplace-
ment, vitesse moyenne, activité globale, nombre de contacts entre têtards) a été évalué pour des individus (1) et des grou pes
de différentes tailles (3, 5, 7 et 10 têtards) à laide dun logiciel de suivi vidéo. Lexposition aux signaux de prédateur modifie
des paramètres comportementaux, réduisant le nombre de rencontres avec des prédateurs et, donc, la mortalité. La dis-
tance totale parcourue et la vitesse moyenne augmentent dans les essais impliquant 1 têtard et 3 têtards interagissant les
uns avec les autres exposés à des conspécifiques blessés, alors que l
activité globale augmente dans les groupes de toutes les
tailles, ce qui démontre que des signaux d
alarme peuvent avoir une incidence sur les têtards grégaires et perturber leur
comportement. L
insecticide (seul ou en combinaison) entraîne une augmentation des paramètres dans les groupes de
toutes les tailles, causant une hyperactivité en raison de son effet neurotoxique. Les différentes réactions observées après
l
exposition à des signaux d
alarme et des signaux environnementaux au sein des groupes de différentes tailles modifient le
comportement normal et la dynamique écologique des têtards grégaires. [Traduit par ladaction]
Mots-clés : signaux d
alarme, comportement, signaux chimiques, têtard fouisseur, pesticides, crapaud de Dorbigny, Rhinella
dorbignyi.
Received 9 April 2021. Accepted 28 August 2021.
L.M. Curi. Consejo Nacional de Investigaciones Cientí ficas Técnicas (CON ICET), Buenos Aires, Argentina; Instituto de Ictiología del Nordeste (INICNE),
Facultad de Ciencias Veterinarias, Universidad Nacional del Nordeste (FCV, UNNE), Sargento Cabral 2139, CP 3400, Corrientes, Argentina.
A.P. Cuzziol Boccioni, P.M. Peltzer, A.M. Attademo, and R.C. Lajmanovich. Consejo Nacional de Investigaciones Científicas Técnicas (CONICET),
Buenos Aires, Argentina; Laboratorio de Ecotoxicología, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral (FBCB-UNL-
CONICET), Ciudad Universitaria, Paraje
El Pozo
, RN 168, Km 472, CP 3000, Santa Fe, Argentina.
A. Bassó. Laboratorio de Ecotoxicología, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral (FBCB-UNL-CONICET), Ciudad
Universitaria, Paraje El Pozo, RN 168, Km 472, CP 3000, Santa Fe, Argentina.
E.J. León. Consejo Nacional de Investigaciones Científicas Técnicas (CONICET), Buenos Aires, Argentina; Laboratorio de Ecotoxicología, Facultad de
Bioquí mica y Ciencias Biológicas, Universidad Nacional del Litoral (FBCB-UNL-C ONICET), Ciudad Universitaria, Paraje El Pozo, RN 168, Km 472,
CP 3000, Santa Fe, Argentina; Instituto Nacional de Limnología, Laboratorio de Biodiversidad y Conservación de tetrápodos (INALI-UNL- CONICET), Ciudad
Universitaria, Paraje El Pozo, RN 168, Km 472, CP 3000, Santa Fe, Argentina.
Corresponding author: Lucila M. Curi (email: lucilacuri@gmail.com).
© 2021 The Author(s). Permission for reuse (free in most cases) can be obtained from copyright.com.
Can. J. Zool. 100: 1927 (2022) dx.doi.org/10.1139/cjz-2021-0075 Published at www.cdnsciencepub.com/cjz on 27 October 2021.
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Introduction
In the last decade, studies on the behavior of amphibian tadpoles
exposed to multiple stressors suggested not only behavioral shifts
but also morphological and physiological modifications (Üveges
et al. 2019; Saura
Mas and Benejam 2019). Ecotoxicological studies
address several biological traits to determine the toxicity of single
or complex chemical mixtures on amphibians (Robles-Mendoza
et al. 2011; Cuzziol Boccioni et al. 2020). Moreover, behavioral end-
points proved to be effective biomarkers in contamination studies,
since they are influenced by both endogenous and exogenous
factors (Mann et al. 2009; Egea-Serrano et al. 2012). In amphibian
tadpoles, impairment of swimming capacity has been linked to
disturbances in neural, endocrine, and metabolic processes (Widder
and Bidwell 2008) and has been used as an indicator of sublethal
effects associated with exposure to several toxic chemicals (Denoël
et al. 2012; Junges et al. 2017). The analysis of locomotor behavior in
aquatic organisms (Juszczak et al. 2006; Henry et al. 2019) is useful
to demonstrate the sublethal effect of some toxic compounds, as
demonstrated in a few ecotoxicological studies (Peltzer et al. 2013;
Egea-Serrano and Tejedo 2014; Da Costa Araujo and Malafaia 2020).
In aquatic environments, chemical signals are a major source
of information for animals, which can assess the state of the envi-
ronment and obtain information about the presence of predators
(Mitchell et al. 2017). In addition, chemoreception may be one of
the most effective sensory modalities for detecting environmental
stressors (Hagman et al. 2009). In natural environments, tadpoles
are exposed to a variety of natural and allochthonous chemical sub-
stances derived from anthropogenic activities (Hanlon and Relyea
2013). The effect of predation on a community is mainly driven by
the ability of predators and prey to detect each other (Thetmeyer
and Kils 1995). Chemical communication during predation events
consists of signals (Wilson and Lefcort 1993) that can originate
from the predators (known as kairomones; Vázquez et al. 2017),
or chemical cues released by conspecifics under predator attack
(known as alarm pheromones or alarm cues; Schoeppner and
Relyea 2005; Mitchell et al. 2017). Amphibian tadpoles can recog-
nize alarm cues, which alert them of the presence of predation
risk; thus, alarm cues confer an adaptive and survival advantage
(Kiesecker et al. 1996; Faulkner et al. 2017).
The changes in behavior and (or) morphology of tadpoles in
response to the presence of predators is known as predator-
induced phenotypic plasticity (Benard 2004) and have been fre-
quently studied using anuran tadpoles as a model organism
(McCollum and Leimberger 1997; Mogali et al. 2011; Maher et al.
2013). Tadpoles can avoid areas with presence of predators by
reducing their activity and changing several biological traits,
such as time to reach metamorphosis (Gazzola et al. 2015; Mogali
et al. 2016; Üveges et al. 2019), coloration, and morphology
(Relyea 2001; Van Buskirk et al. 2003). Both types of signals (pred-
ator presence and tadpole injury due to predator attack) produce
several ecological adjustments to reduce the risk of predation
(Crossland and Shine 2012; Faulkner et al. 2017; Crossland et al.
2019). Moreover, some amphibians, like toads of the Bufonidae
family, have epidermal secretory cells (giant cells) that release
their secretion only when the skin is injured, eliciting a
scape
reaction
in the tadpole school; therefore, these cells play an im-
portant role in chemical signaling defense mechanisms against
predators (Regueira et al. 2016).
The presence of stress factors can alter the recognition or
response to the alarm cues (Burraco et al. 2013). Different studies
investigated the interaction between presence of predators and
exposure to agrochemicals in anuran tadpoles (Burraco et al.
2013; Moore et al. 2015; Mikó et al. 2017). However, few studies
focused on the synergistic effect of the presence of predators, sig-
nals of predation (injuries) from a conspecific, and pesticides in
gregarious anuran tadpoles (Sontag et al. 2006; Wells 2007; Wei
et al. 2014; Balestrieri et al. 2019). The deleterious effect of
agrochemical contamination on anuran behavior was frequently
investigated (see Relyea and Mills 2001; Zala and Penn 2004;
Boone et al. 2007). The synergistic effect of different chemical
stressors (natural and allochthonous) is environmentally rele-
vant due to the massive amounts of pollutants reaching water
bodies and the collapse of aquatic ecosystems worldwide (Bashir
et al. 2020; Bassem 2020).
Therefore, this study aimed to evaluate the swimming activity
in gregarious tadpoles of Dorbigny
s Toad (Rhinella dorbignyi
(Duméril and Bibron, 1841)) (Anura: Bufonidae) exposed to differ-
ent signals: (i) chemical cues of a predator, the marbled swamp
eel (Synbranchus marmoratus Bloch, 1795) (Synbranchidae), (ii) alarm
cues of an injured conspecific, (iii) sublethal concentration of a
common pesticide that occurs in water bodies (cypermethrin), and
(iv) a combination of the chemical stressors at the individual level
and in groups of different sizes. We expected greater negative
effects of the combination of chemical stimuli on the swimming
behavior parameters of these gregarious anuran tadpoles than
of each signal individually; we also expect a modification of nor-
mal ecological performance of tadpoles due to these alterations.
Materials and methods
Test species
Rhinella dorbignyi tadpoles were used as test organisms. This
anuran species is widely distributed in the Neotropical region,
with occurrence in Argentina, Brazil, Paraguay, and Uruguay (Cei
1980). Adults occur in forests, grasslands, and agricultural ecosys-
tems such as rice plantations (Narvaes et al. 2004). The tadpoles
are commonly found in temporary and semi-permanent ponds
displaying schooling gregarious behavior during the reproduc-
tive period (November to March; Sánchez et al. 2009). In Argen-
tina, this toad species is categorized as
not threatened
(Vaira
et al. 2012).
Several fragments of gelatinous egg strings of R. dorbignyi were
randomly collected from temporary ponds in natural floodplains
of the ParaRiver (31°39034.700S, 60°35031.100W), Argentina, with
collection permission of the Ministry of Environment of the Prov-
ince of Santa Fe, Argentina (EXP. 02101-0018518-1). This site was
previously found to be unpolluted (Attademo et al. 2014, 2015).
The egg strings were immediately (1 h) transported in plastic
flasks containing pond water and maintained in laboratory con-
ditions until the eggs hatched (Gosner stage 19; Gosner 1960).
Embryos and then tadpoles were maintained in glass aquaria
with dechlorinated tap water until they reached Gosner stage 35
(Gosner 1960). Tadpoles were fed boiled lettuce ad libitum and
water in the aquaria was changed every 48 h. At the start of the
experiment, mean (6SD) total length of tadpoles (9.66 6 0.24 mm)
was measured with a digital caliper (0.001 mm accuracy) and
mean (6SD) mass (0.09 6 0.02 g) was measured with a digital bal-
ance (0.001 g accuracy).
A total of 1040 tadpoles at Gosner stage 36 were taken from the
stock for the study. Tadpoles were treated according to the crite-
ria of the American Society of Ichthyologists and Herpetologists
(2004) and with the approval from the Animal Ethics Committee
of the Faculty of Biochemistry and Biological Sciences of the
National University of the Littoral (FBCB 388/06).
Experimental design
Chemical signals
Three chemical signals were selected and tested alone and in
combination as follows: (i) cues from the predator fish S. marmoratus (PC),
(ii) cues from injured conspecifics (IC), (iii) sublethal concentration
of the pesticide cypermethrin (10
l
g/L) (CY), and (iv) a combination
of signals; illustrated in Fig. 1. In total, eight treatments were per-
formed, including individual and combined treatments, which
were applied to different group sizes (see Fig. 1).
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21
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Fig. 1. Experimental conditions used to evaluate Dorbigny
s Toad (Rhinella dorbignyi) tadpoles, showing the compounds and the number of
individuals assayed in each group. Color version online.
(i)
Treatment involving chemical cues from predator
Synbranchus marmoratus is widely distributed across Central and
South America (from Mexico to northern Argentina; Kullander
2003). This eel species prefers shallow rivers with calm water,
streams, lagoons, and estuaries, where it lives in caves (Maggese
et al. 1993). Its conservation status is
not threatened
(Daniels
and Maíz-Tome 2019). It was selected because it is a potential
native predator of anuran tadpoles (Maffei et al. 2011; Junges
et al. 2012). Fish juveniles used in predator chemical treatment
experiments were collected from an unpolluted pond in the flood-
plain of Paraná River (Santa Fe province, Argentina; 31°42
0
34.0
00
S,
60°34
0
60
00
W). During 1 week, before the start of different trials,
similar-sized eels (mean (
6
SD) length = 24
6
1.3 cm; mean (
6
SD)
mass = 12.8
6
1.45 g) were acclimated to bioassay conditions and
fed non-experimental tadpoles daily (between 2 and 3 tadpoles).
The day before experimentation, one eel was randomly selected
and housed in a flask containing 1 L of clean water to obtain
water for each treatment involving the chemical cues from a
predator (PC treatment). Then, 200 mL of experimental water
prepared for each PC treatment was randomly used for the dif-
ferent trials. The procedure was repeated for the four treat-
ments containing PC and for each replicate (n = 20).
(ii)
Treatment involving conspeci
c alarm cues
The alarm cues for each IC treatment were obtained by par-
tially injuring the caudal fin of one tadpole (technique modified
from Mitchell and McCormick 2013). This partial lesion was
repeated for each treatment involving IC treatment at individ-
ual (1) and group (3, 5, 7, and 10 tadpoles) sizes and the replicates.
This procedure did not compromise tadpole survival. After being
injured, the tadpoles were maintained in a Petri dish containing
200 mL dechlorinated water for a maximum of 1 h to ensure the
persistence of the alarm cue in the water (Ferrari et al. 2010).
Then, this water with chemical cues from injured tadpoles was
used for the present treatment and in combination in the differ-
ent group sizes. When experiments finished, injured tadpoles
were maintained together until they completed metamorphosis.
(iii)
Sublethal insecticide concentration treatment
Cypermethrin was selected due to its extensive use in soybean
and maize crop farming in Argentina and its deleterious effects
on toad tadpoles at different biological levels (Izaguirre et al.
2001; Casco et al. 2006). Cypermethrin (CAS No. 52315-07-8; water
solubility 4
10
l
g/L; Biga and Blaustein 2013) is a pyrethroid with
a broad spectrum of insecticidal activity based on neurotoxicity.
This insecticide has been detected at levels ranging from 3.5 to
194 lg/L in Argentine surface water (Marino and Ronco 2005). The
concentration of 10
l
g/L used for CY treatments was selected
according to a previous study (Cabagna et al. 2006) and toxic values
for other aquatic organisms (World Health Organization (WHO)
1989). Cypermethrin was obtained from Argengric, Argentina.
The commercial product consisted of 2.5% of the active ingredi-
ent formulated in aqueous xylene.
Experimental conditions and behavioral endpoints
Individual (1 tadpole) and group (3, 5, 7, and 10 tadpoles) behav-
ioral responses were recorded for 5 min in a Petri dish (15 cm di-
ameter and 2 cm height) containing 200 mL in each chemical
treatment. Eight treatments (with five replicates each) were per-
formed in the laboratory, as shown in Fig. 1. Dechlorinated water
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Can. J. Zool. Vol. 100, 2022
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Fig. 2. Swimming behavior of Dorbigny
s Toad (Rhinella dorbignyi) exposed to treatments with different chemical cues (control, cue from
injured conspecifics (IC), sublethal concentration of cypermethrin (CY), cue from predator fish (PC)), based on total distance moved and
mean speed (A), global activity (B), and number of contacts (C).
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Curi et al.
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was used as control treatments. The laboratory conditions con-
sisted of 24
6
2 °C and the digital recordings were made between
the hours of 1400 and 1800. Different individuals were used in
Table 1. Summary of treatments showing their effects (increase
:
or
decrease
;
with respect to controls) on different endpoints of the
swimming behavior of Dorbignys Toad (Rhinella dorbignyi) tadpoles.
each trial, treatment, and replicate to ensure a mean of response
that can vary at intraspecific and interindividual levels.
Treatment
Tadpole
groups
Distance
moved
Mean
speed
Global
activity
No. of
contacts
Total distance moved (cm), mean speed (cm/s), and global activ-
ity (cm2) were evaluated individually (n = 1) and in interactions of
an increasing number of tadpoles (n = 3, 5, 7, and 10). Likewise,
the number of interactions (contacts between pairs) was also ana -
lyzed. These parameters were quantified and analyzed using the
Smart video-tracking software (version 3.0.02; PANLAB HARVARD
APP
A
R
A
TUS
V
R
).
Statistical analysis
MANOVA (Wilksl multivariate test statistic) was used to deter-
mine overall significant differences among treatments involving
chemical cues in the response vectors (four behavior parameters:
total distance moved, global activity, mean speed, and number of
contacts). If MANOVA was significant, then an ANOVA (F test) fol-
lowed by a Tukey
s or a Dunnett
s post hoc test was used to test for
treatment significance. Shapiro
Wilk
s test and Levene
s median
test were used to assess normality and homogeneity of variance,
respectively, of the data (Zar 1999). Statistical analyses were per-
fo
rm
ed
u
s
i
n
g
t
h
e
I
B
M
V
R
SPSS
V
R
so
ft
wa
re
v
e
r
s
io
n
1
8
(
p
<
0.
0
5
)
.
Results
The four variables measuring tadpole swimming behavior
were significantly altered after exposure to the different chemi-
cal signals in all group sizes: 1 tadpole (
l
= 0.49, F
[28,48]
= 2.26,
p < 0.006), 3 tadpoles (l = 0.51, F[42,280] = 5.86, p < 0.0001), 5 tad-
poles (l = 0.037, F[42,505] = 12.2, p < 0.0001), 7 tadpoles (l = 0.004,
F[42,730] = 38.8, p < 0.0001), and 10 tadpoles (l = 0.016, F[18,314] =
58.3, p
<
0.0001). The individual analysis of each MANOVA and
the ANOVA for each behavior variable are shown in Supplemen-
tary Table S1.1
Total distance moved and mean speed
Total distance moved and mean speed were the most affected
parameters in all the treatments and group sizes (Fig. 2A, Table 1).
Both parameters were higher than the control in all treatments;
this difference was significantly higher in the CY and CY + PC treat-
ments with 3 and 5 interacting tadpoles than in the control. Total
distance moved and mean speed were also significantly lower in PC
and IC + CY + PC treatments with 7 interacting tadpoles than in the
control (Dunnett
s post hoc test, p
<
0.05; Fig. 2A, Table 1). In the
group with 10 interacting tadpoles, these parameters were signifi-
cantly higher in IC and CY than in the control (Dunnett
s post hoc
test, p < 0.05; Fig. 2A, Table 1).
Global activity
The global activity was higher in groups with 3, 5, and 7 inter-
acting tadpoles exposed to IC, CY, IC + CY, IC + PC, CY + PC, and
IC + CY + PC than in the control (Dunnett
s post hoc test, p
<
0.05,
Figs. 2B and 3; Table 1). Moreover, the global activity was lower in
the group with 10 interacting tadpoles exposed to PC than in the
control (Dunnett
s post hoc test, p
<
0.05, Fig. 3, Table 1).
Intraspecific interaction
The number of contacts was higher in the groups with 3 and 5
interacting tadpoles exposed to CY and CY + PC, respectively
(Dunnett
s post hoc test, p
<
0.05, Fig. 2C, Table 1) than in the con-
trol. In contrast, in the group with 5 interacting tadpoles, the
number of contacts was lower in IC + CY + PC than in the control.
In the group with 7 interacting tadpoles, the number of contacts
was lower in all chemical signal treatments than in the control.
The number of contacts increased in the group with 10 interacting
Note: Combinations of signal mixtures are listed according to the acronym
of each involved treatment
treatment involving cues from injured conspecifics (IC),
cypermethrin (pesticid e) treatment (CY), and treatment involving cues from
predator fis h (PC). Arrows in boldface type ind icate changes (an incr ease or a
decreas e) that are significantly differ ent from the control.
tadpoles exposed to CY, but it declined in PC with respect to the
control (Dunnetts post hoc test, p < 0.05; Fig. 2C, Table 1).
Discussion
Predator chemical cues
Avoidance behavior is a more common response to alarm sig-
nals in Bufonidae than in other anuran families and it is useful to
prevent predation (Rödin Mörch et al. 2011). Our results showed a
decrease in total distance moved, mean speed, global activity,
and number of contacts in tadpoles exposed to fish predator
chemical cues, principally with a high number of interacting
tadpoles. Reduced activity decreases the probability of a random
encounter with predators and thus increases survivorship of tad-
poles (Chuang et al. 2019; Ramamonjisoa et al. 2019). Under
1
Supplementary table is available with the article at https://doi.org/10.1139/cjz-2021-0075.
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IC
1
:
:
:
3
:
:
;
5
;
;
;
7
;
;
;
10
:
CY
1
:
:
3
5
:
7
;
10
PC
1
:
:
3
:
:
:
:
5
;
;
:
;
7
;
;
;
;
10
;
;
;
IC + CY
1
:
:
3
;
5
:
:
=
7
;
10
:
:
:
:
IC + PC
1
:
:
:
3
=
5
:
:
;
7
:
:
;
10
:
:
:
:
CY + PC
1
:
:
:
3
:
5
7
:
:
;
10
:
:
:
;
IC + CY + PC
1
:
:
:
3
:
:
;
:
5
;
;
;
7
;
;
;
10
:
:
:
;
24
Can. J. Zool. Vol. 100, 2022
Published by Canadian Science Publishing
Fig. 3. Global activity of Dorbigny
s Toad (Rhinella dorbignyi) tadpoles exposed to treatments with different chemical cues (control (CO),
cue from injured conspecifics (IC), sublethal concentration of cypermethrin (CY), cue from predator fish (PC)). For each treatment, an
individual (1) and a group of different sizes (3, 5, 7, and 10 individuals), in order of occurrence in the figure from bottom to top, are
represented by their respective video track.
perceived predation risk, tadpoles usually react by changing the
level of activity according to the ratio between the amount of
predatory cue and conspecific density (Van Buskirk et al. 2011;
Gazzola et al. 2018). Our results revealed higher activity in treat-
ments involving 1, 3, and 5 interacting tadpoles, as indicated by
the low total distance moved, mean speed, and global activity
with respect to the control. In groups with a higher number of
interacting tadpoles (7 and 10), the anti-predatory strategy observed
in this gregarious tadpole consists of reducing their activity instead
of escaping. In several other studies involving other species, dif-
ferent species-specific behaviors were described such as freezing,
escape behavior, shelter seeking, avoidance of the predator
s vi-
cinity, and colony defense (Wisenden 2000; Mogali et al. 2019,
2020). Moreover, different defense responses to specific alarm sig-
nals were described for different predators (Schoeppner and Relyea
2009). Some authors reported that the presence of predators
not only reduced the activity of tadpoles, but also decreased
their growth rate (Van Buskirk 2002; McCoy 2007), modifying
their development and phenotypes (Benard 2004; Vonesh and
Warkentin 2006), and altering their morphology (McCollum and
Leimberger 1997; Ferrari et al. 2010; Gomez-Mestre and Díaz-Paniagua
2011).
Conspecific alarm cues
The ability to recognize conspecific alarm cues confers an adapt-
ive advantage by alerting individuals about a risk (such as preda-
tor presence) and then eliciting a behavioral response (Faulkner
et al. 2017). Alarm cues can provide temporal and spatial informa-
tion about predation risk, allowing prey to regulate the expres-
sion of inducible defense in several aquatic species (Chivers and
Smith 1998). In our results, alarm cues from injured conspecific
tadpoles increased total distance moved and mean speed in all the
groups of interacting tadpoles, being reinforced with the increase
of global activity. As reported by Ferrari et al. (2010), intrinsic and
extrinsic factors affect the response to conspecific alarm cues.
Similarly, an increased swimming activity was observed when
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For personal use only.
Curi et al.
25
Published by Canadian Science Publishing
tadpoles were exposed to both alarm cues of predator presence
and injured conspecific. Specifically, as mentioned by Schoeppner
and Relyea (2005), the additional information provided by the si-
multaneous exposure to both kairomones (predator presence)
and pheromones (from attacked prey) provides complete and
effective anti-predator defenses in gregarious species. In addition,
Rödin Mörch et al. (2011) concluded that in solitary species (such
as the Cascades Frog, Rana cascadae Slater, 1939; the Northern Red-
legged Frog, Rana aurora Baird and Girard, 1852; the Yellow-bellied
Toad, Bombina variegata (Linnaeus, 1758)), the behavior of tadpoles
is typically unaffected by alarm cues, but in gregarious taxa,
avoidance is the most frequent response to alarm cues.
Furthermore, global activity was higher in R. dorbignyi tadpoles
exposed to injured conspecific cue in all of the evaluated group
sizes. In the presence of the injured conspecific alarm cues, tad-
poles displayed escape and disaggregation behavior; this behav-
ior was indicated by a lower number of contacts and increased
swimming activity, which probably allows tadpoles to reach a safer
location, as may occur in natural environments. However, in Rhinella
arenarum (Hensel, 1867) tadpoles exposed to injured conspecifics,
a decrease in individual activity was observed (Jungblut 2012).
These authors also found that tadpoles perceive different levels
of stimulus intensity or predation risk.
Synergisms between agrochemicals and natural signals
The swimming activity of R. dorbignyi was also affected by expo-
sure to cypermethrin alone or combined with other chemical sig-
nals. This insecticide is lipophilic, which facilitates its fast access
to various tissues, and has a high affinity to the central nervous
system due to its neurotoxic effect (Anadón et al. 1996), as demon-
strated in R. arenarum tadpoles (Izaguirre et al. 2000, 2001; Casco
et al. 2006). Cypermethrin effects incremented the behavioral pa-
rameters (total distance moved, mean speed, and global activity)
in the different group sizes. This effect can be associated with cyper-
methrin neurotoxicity, with short-term use of this pesticide causing
excitation. Likewise, several toxic compounds can inhibit cholines-
terase activity on non-target organisms, such as fish or amphibians,
and produce excitatory locomotion (Peltzer et al. 2013; Sandrini et al.
2013). Cypermethrin prolongs the normally transient increase in
sodium permeability of the nerve membrane during excitation,
leading to long-lasting trains of repetitive impulses in sense organs
and a frequency-dependent depression of the nerve impulse in
nerve fibers (WHO 1989). In our study, cypermethrin caused hyper-
activity, even when predator cues were also present, indicating
that individuals were not able to carry out their normal anti-
predatory activity. In biological terms, changes in tadpole loco-
motion are a threat to their survival, as even small changes can
limit the movements of animals in their habitats and, conse-
quently, reduce their ability to escape from predators (Araújo
and Malafaia 2020). Likewise, Relyea and Mills (2001) demon-
strated that carbaryl became even more lethal for Gray Treefrog
(Dryophytes versicolor (LeConte, 1825)) tadpoles when combined
with predatory stress. The tadpoles of Wood Frogs (Lithobates
sylvaticus (LeConte, 1825)) exposed to sublethal concentrations of
G
l
y
p
h
o
s
a
te
R
o
u
n
dup
V
R
f
aile
d
t
o
e
xh
ib
it
an
t
i
-
p
r
eda
t
o
r
be
h
a
v
io
r
(Moore et al. 2015). That work also provides considerable evi-
dence that injured conspecific cues are deactivated by Roundup,
and the tadpoles failed to reduce activity when we mixed the
alarm cue solution with this herbicide. The responses can vary
according to the differential sensitivity to the compounds. How-
ever, other authors did not find a synergistic effect between pred-
ator and chemical contamination (Reeves et al. 2011; Burraco et al.
2013).
When tadpoles were exposed to the combination of the three
chemical signals, an increase in global activity in all the group
sizes and, therefore, a decrease in the number of contacts between
tadpoles, were observed. In this context, it can be assumed that
the tadpoles were overstimulated by synergisms of natural and
environmental signals. The increase in global activity and the
decrease in other swimming parameters could be due to signs of
cypermethrin toxicity, which acts synergistically with predator sig-
nals. Salibián (1992) described spontaneous arrhythmic flitter
movements in the early response of R. arenarum to deltamethrin.
Similarly, in our trials, such behavior could mean hyperactivity
but non-displacement, limited by the predator signals. The over-
excitation and continuous spasms of individuals are due to cypermeth-
rin exposure and could limit the normal
escape and disaggregation
and
anti-predatory
behaviors caused by conspecific and predator
signals, respectively. The alteration of swimming behavior and
social interaction represents an indirect impact of the pollutant
on population dynamics.
These results contribute to the understanding of how environ-
mental contamination influences the ecological dynamics of tad-
poles, principally on the normal anti-predatory and gregarious
behaviors of tadpoles. Due to the increased use of pesticides in
agricultural ecosystems, the effects of a particular chemical sub-
stance and its synergistic effects with other natural or anthropo-
genic factors are of ecological concern.
This study demonstrated that swimming behavior parameters
of amphibian tadpoles are suitable biomarkers to evaluate the
influence of different chemical signals in aquatic environments.
Swimming activity as a measurable response of the interaction
between exposure to toxicants and chemical signals provides ec-
ological insight into how contaminants modify intra- and inter-
specific interactions that ultimately affect the performance and
survival of organisms in the environment. These signals also altered
the gregariousness of tadpoles, which may have severe consequen-
ces on their survival in natural environments (Araújo and Malafaia
2020). Further studies are needed to elucidate the interaction mech-
anism of chemical signals from different sources and how those
signals modify the behavioral responses of tadpoles. Overall, the
study of those behavior endpoints may contribute to a better
understanding of susceptibility features throughout development
and natural history of gregarious species of anurans in response to
the allochthonous chemicals.
Competing interest statement
The authors declare that there are no competing or financial
interests.
Contributors
statement
R.C.L. and P.M.P. designed the experiments. L.M.C. and A.M.A.
performed the experiment. A.P.C., P.M.P., and L.M.C. analyzed
the data. L.M.C., P.M.P., and A.P.C. wrote the manuscript. E.J.L.,
A.B., and A.M.A. revised the manuscript.
Funding statement
This study was supported in part by National Agency for Pro-
motion of Science and Technology, Argentine (ANPCyT FONCyT
PICT, N° 1069), and Course of Action for Research and Science
Promotion, Argentine (CAI D-UNL, PIC 100004LI).
Data availability statement
The datasets used and (or) analyzed during the current study are
available from the corresponding author on reasonable request.
Acknowledgements
We thank C. Junges for laboratory assistance and J. Brasca for
English editing and comments.
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... As expected, the three species analyzed showed high sensitivity to GLU. swimming. [39][40][41] Noticeably, similar behavioral changes were described for fish species such as Poecilia reticulata, presenting loss of general activity and equilibrium; [42] in Oncohrhynchus mykiss [43] presented loss of coordination thus, showing that CYP similarly affects the behavior of phylogenetically distant organisms from communities in aquatic ecosystems. Thus, in this study, R. arenarum presented alterations in TD, showing irregular swimming and immobility when exposed to CP (Table 1), as it was observed in leopard frog tadpoles (Rana blairi) by Ruiz de Arcaute. ...
... Consequently, GLY sublethal effects include accelerated [54] or delayed development, [55] reduced size at metamorphosis, [54,55] developmental malformations, [56] and symptoms of oxidative stress. [57] Remarkably, the most active species R. arenarum, [58] seemed more sensitive to behavioral biomarkers than R. dorbignyi [41] and O. americanus, [59] considering that these species showed behavioral effects by CYP, GLY, and CP (Table 1). ...
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