The Curious Case of the Cane Toad (Rhinella marina): An Assessment of Exploratory
Behavior and Foraging Success of an Invasive Vertebrate in a Novel Environment
by
Amanda J. Arner, B.S.
A Thesis
In
BIOLOGY
Submitted to the Graduate Faculty of Texas Tech University in
Partial Fulfillment of the Requirements for
the Degree of
MASTER OF SCIENCES
Approved
Dr. Ximena E. Bernal, Ph.D. Chair of Committee
Dr. Rachel A. Page, Ph.D.
Dr. Richard E. Strauss, Ph.D.
Peggy Gordon Miller Dean of the Graduate School
August, 2012
Copyright 2012, Amanda J. Arner
ii
ACKNOWLEDGMENTS
I would like to take this opportunity to thank my friends, family, advisors and
mentors for helping me through the completion of my Masters of Science. I am truly
blessed to have such wonderful people in my life; without their motivation, support
and proverbial shoulders to cry on, I would not have made it to this point in my career.
Firstly I would like to thank my advisor, Dr. Ximena E. Bernal, for her support
through this process. Her guidance and trust in me and her other graduate students are
apparent and reflected in her advising practices. I would like to that my committee
members Dr. Rachel A. Page and Dr. Rich E. Strauss for their support as well; Dr.
Page for her generosity in allowing me to use her resources and lab space and
equipment for my field research, and Dr. Stauss for his continued advice on statistical
methods and interpretation of biological meaning.
Though my advisors guided me through the process of conducting an original
research project, I cannot claim success without acknowledging my graduate student
peers for their care and support. Graduate school is a time of intrinsic growth and
establishment of individual identity as a scientist and professional, and the following
people have helped shape who I have become over this journey; Lynne Beaty, Maria
Gaetani, Meghan Cromie, Elizabeth Waring, Janice Kelly, Jenny Strovas, and
Priyanka DeSilva. My undergraduate research assistants, Katelyn Jordan and Meagan
Phelps, have been a tremendous help in conducting my laboratory research.
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My research projects were monetarily supported in part by three entities, which
I would like to acknowledge as well. Dr. Ximena Bernal, thank you for allowing my
research to benefit from your start-up funding, and allowing me the flexibility to
expand the toad lab to suit my research needs. The Association of Biologists at Texas
Tech University provided me with grant funding to purchase research supplies while
conducting field work in Panama, and provided me with financial assistance to attend
several conferences to present my research. Lastly, I would like to thank the HHMI-
funded Center for the Integration of Science Education and Research for their funding
support through the Graduate Teaching Scholars program. This funding enabled me to
grow as a professional educator as well as a scientist, and has better prepared me for a
future in science education.
Last but certainly not least; I would like to thank my family for their support.
My mother, Jennifer Holcombe, my father and stepmother, Howard and Karen Arner,
and my sister, Megan Bryan – thank you for lending your ears and supporting me
through this difficult time in my life. Finally, I would like to thank my fiancé, Hil
Miller, for just about every single thing he does, especially for enduring the strain of a
long-distance relationship and accepting my fierce desire to remain in Lubbock and
finish my degree, regardless of the consequences.
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TABLE OF CONTENTS
Acknowledgments ......................................................................................................... ii
Abstract .......................................................................................................................... vi
List of Tables .............................................................................................................. viii
List of Figures ................................................................................................................ ix
I. Introduction ................................................................................................................. 1
Background ................................................................................................................. 1
Overview of Thesis Research ..................................................................................... 5
II. Exploration and foraging behavior of the Cane Toad (Rhinella marina) in its Native
Range .............................................................................................................................. 8
Introduction ................................................................................................................. 8
Methods .................................................................................................................... 11
Study Site .......................................................................................................................... 11
Preliminary Study ............................................................................................................. 11
Experimental Arena Design .............................................................................................. 12
Experimental Procedure .................................................................................................... 13
Experiment #1 – Changes in Exploration with Increased Experience ............................. 15
Experiment #2 – Changes in Foraging Behavior with Increased Experience .................. 15
Experiment #3 – The Role of Spatial and Visual Cues Involved in Learning ................. 17
Results ....................................................................................................................... 18
Exploratory Behavior and Experience .............................................................................. 18
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Foraging Behavior and Experience .................................................................................. 19
Use of Spatial and Visual Cues to Locate Bowls ............................................................. 20
Discussion ................................................................................................................. 21
III. Exploration and foraging behavior of the Cane Toad (Rhinella marina) from an
Invasive Population ...................................................................................................... 34
Introduction ............................................................................................................... 34
Methods .................................................................................................................... 36
Experimental Arena Design .............................................................................................. 36
Experimental Procedure .................................................................................................... 37
Experiment #1 – Changes in Exploration with Increased Experience ............................. 39
Experiment #2 – Changes in Foraging Behavior with Increased Experience .................. 40
Experiment #3 – The Role of Spatial and Visual Cues Involved in Learning ................. 41
Results ....................................................................................................................... 42
Exploratory Behavior and Experience .............................................................................. 43
Foraging Behavior and Experience .................................................................................. 43
Use of Spatial and Visual Cues to Locate Bowls ............................................................. 44
Discussion ................................................................................................................. 44
IV. Diet Flexibility and Foraging Behavior in a Novel Environment in the Leaf Litter
Toad (Rhinella alata) .................................................................................................... 54
V. Conclusion ............................................................................................................... 63
Bibliography ................................................................................................................. 65
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ABSTRACT
An individual’s ability to modify its behavior as a result of experience is a key
component of successful survival in a changing environment. This ability has been
studied in many taxa including vertebrates (e.g. mammals, birds and fish) and
invertebrates (e.g. insects and cephalopods), however little conclusive evidence exists
for learning in anuran species. Much of the research previously done in this area was
constrained by unsuccessful attempts to develop an experimental paradigm that
provided evidence in support of learning in these taxa. The research outlined in this
thesis synthesizes laboratory and field designs to provide a new approach to studying
learning abilities in anurans, pertaining to exploration and foraging behavior on an
individual scale. The cane toad, Rhinella marina, is an ideal study species to
determine the role of learning in anurans. Its well-known invasive capabilities and
colonization of new environments suggest that cane toads are experts at modifying
their behavior based on changes in the environment. By studying how exploratory
behavior in a novel environment is modulated by experience, we can make inferences
about the spatial learning abilities of this species. To examine the role of learning in
invasive potential, we conducted similar studies with cane toads from populations in
both the native and invasive ranges. To further provide evidence for learning in
anurans we asked the same questions about a congeneric species of toad from the
native range with differing life history strategies, the leaf litter toad (Rhinella alata).
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For cane toads in both the native and invasive ranges, individuals were
repeatedly tested in an exploratory arena in one of two treatments, with or without
food present. After initial training in the arena, toads were tested to determine if
movement and behavioral strategies changed over time, as experience with the arena
increased. Toads were given five trials each, with a sixth trial to tease apart the use of
associative learning or spatial cues for foraging behavior. The smaller leaf litter toads
were tested in an arena scaled to size based on their locomotor ability, and were tested
with food in the arena for five total trials.
Cane toads from both the native and invasive ranges showed a decrease in
movement and exploration over time, regardless of treatment group. Individuals in the
experimental treatment (food in bowls) ate more mealworms over time while still
decreasing overall movement. Leaf litter toads did not show any significant trends in
either foraging or exploratory behavior while in the arena, though a large proportion of
the individuals successfully learned to eat the novel food item used for feeding before
and during trials. Our results indicate that cane toad behavior is modulated by
experience with a novel environment and by the presence of food. This study
ultimately emphasizes the role of learning in foraging in cane toads, a characteristic
that may have facilitated their success as invaders.
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LIST OF TABLES
2.1 Observation Values for Behavior During First Experimental Trial ................. 25
2.2 Degree of Preference for Bowls Encountered .................................................. 26
3.1 Observation Values for Behavior during First Experimental Trial .................. 44
3.2 Degree of Preference for Bowls Encountered .................................................. 45
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LIST OF FIGURES
2.1 Experimental Arena Setup ................................................................................ 23
2.2 Bowl Locations for Experimental Trials .......................................................... 24
2.3 Total Path Length, Time Spent in Margin, and Number of Escape Attempts .. 27
2.4 Number of Escape Attempts Per Trial .............................................................. 29
2.5 Number of Total and Unique Bowl Encounters ............................................... 30
3.1 Experimental Arena Setup for Laboratory Experiment .................................... 43
3.2 Total Path Length and Time Spent in the Margin ............................................ 46
3.3 Latency to Leave Origin and Time to Find Food Bowl .................................... 47
3.4 Time to Eat Mealworm and Tortuosity of Path to Eat Mealworm ................... 48
3.5 Number of Total and Unique Bowl Encounters ............................................... 49
4.1 Diagram of Experimental Arena for leaf Litter Toads ..................................... 56
4.2 Latency to Leave Origin and Total Path Length per Trial ............................... 57
4.3 Number of Escape Attempts per Trial .............................................................. 58
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INTRODUCTION
Background
Animals interact with the environment around them in a variety of ways.
Individuals moving through an environment collect information such as the location of
beneficial resources and areas for future avoidance due to potential risk factors (Dall et
al. 2005). To provide the most benefit, this gathered information should be stored, and
used to influence future behavioral decisions. Learning, as defined by Shettleworth
(1998), is “a relatively permanent change in behavior as a result of experience”.
Species that have the ability to learn about the environment around them potentially
increase access to resources, prolonging survival and ultimately increasing fitness. The
evolution of such a behavior can be defined in terms of environmental predictability.
Stephens’ learning model (1993) predicts that individual learning should evolve when
the environment stays constant throughout an individual’s lifetime but changes across
generations. These changes can be either predictable (i.e. seasonal) or unpredictable
(i.e. habitat destruction), and negate the benefits of an innate behavioral regime
without the modification that learning provides.
The ability to learn has been identified in a variety of taxa (e.g. primates: Drea
2006; Hayes et al. 1953; Poirier & Hussey 1982; marine mammals: Deecke 2006
rodents: Mead 1957; Poucet et al. 1986; birds: Sol 2002; fish: Warburton 1990; and
insects: Durier and Rivault 2001). Studies of learning in anurans have produced
negative or conflicting results (Suboski 1992 and references therein). Though there is
CHAPTER I
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overwhelming evidence of learning ability in larval anurans (e.g. Ferrari & Chivers
2008; Gonzalo et al. 2007; Sonta et al. 2006), this ability is not as apparent in adult
anurans. Greding (1971) summarized the dichotomous findings of this field best when
he stated, “Abott (1894) concluded that frogs and toads are quite stupid, but Schaffer
(1911) found them capable of learning.” Turn of the century research programs
investigating learning ability in frogs and toads primarily focused on attempts to
condition individuals using traditional learning paradigms successful in other taxa
(Suboski 1992). During the cognitive revolution of the 1960s and 70s, learning in
anurans was revisited but studies at this time also failed to provide clear evidence for
the presence or absence of learning in this group. Common problems cited include
lack of response to shock stimuli (Thompson & Boice 1975) and methodological
constraints due to the diverse locomotion of anurans (Brattstrom 1990).
In 1992 Milton Suboski conducted a comprehensive review of learning
experiments in reptiles and amphibians and made two important comments concerning
the direction of the field. First, he described an issue he called the releaser-induced
inhibition model (Suboski 1992), which states that the typical behaviors researchers
associate with learning may not be applicable for herpetofaunal species given the
nature of their locomotor behavior and differences in life history strategies. Second,
Suboski revealed particular behaviors likely to be influenced by learning such as
spatial navigation and orientation, but had not yet been addressed due to the
significant complexity of these behaviors and lack of appropriate testing paradigm.
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Recent studies addressing spatial navigation (Bilbo et al. 2000), landmark
learning (Crane & Mathis 2011), foraging success (Gibbons et al. 2005), and
reinforcement learning strategies (Muzio et al. 1992) suggest that learning does occur
in amphibians despite weak and conflicting evidence from previous studies. Further
studies that examine learning in anurans are necessary to improve our understanding
of the ecological factors that mediate the evolution of learning in amphibians and other
vertebrate taxa. Studies that allow us to examine whether learning is widespread in
this group, and how its evolution (or lack of) could be modulated by different natural
history strategies would provide valuable insights. The research experiments outlined
in this thesis aim to further investigate the role of spatial learning in exploratory and
foraging behavior in anurans, and provide baseline information about these behaviors
in a novel environment and how they are modulated by experience.
Spatial learning can be defined as an individual’s ability to gather information
about the spatial orientation of resources in its current environment (Paulissen 2008)
including information such as location of foraging patches, presence of predators, and
potential mating or nesting locations. Spatial learning could also benefit animals that
encounter novel environments, or areas they have not previously visited. For species
introduced into non-native areas, learning about the spatial location of resources and
potential risks could provide useful information in such a novel environment (Amiel et
al. 2011; Russell et al. 2010; Sol 2002). Larger brain size has been linked to both
cognitive abilities and invasion success (Amiel et al. 2011; Sol et al. 2005), yet spatial
learning has not been specifically addressed as a potential factor aiding invasion
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success and should be investigated further as its presence is likely a factor in the
success of invasive animals.
The cane toad (Rhinella marina) is a large, terrestrial anuran species
possessing several life history traits that make it an ideal candidate to investigate
spatial learning. The native range of these toads extends from southern Texas/northern
Mexico through Central America and northern South America. Cane toads are also
found in well-established invasive populations in Australia, South Florida, Hawaii,
and several Caribbean islands (Somma 2012 and references therein). This species
exhibits several traits proposed to facilitate range expansion, including a semi-
terrestrial niche, presence of parotid glands, and a large body size (Alexander 1964;
Van Bocxlaer et al. 2010). The natural history and behavior of these toads is well
documented (e.g. Krakauer 1968; Zug & Zug 1979), and descriptions in several
sources report behaviors that indicate the capacity for learning (Alexander 1965;
Hagman & Shine 2008) . For example, observations suggest individuals have the
ability to quickly learn novel food resources and locations (Alexander 1965), which
could potentially lead to a fitness advantage over other species of anurans in the same
habitat. Cane toads are opportunistic feeders and often seen in urban areas near
artificial lights and other prey attractants (personal observation). Because of their
broad, flexible diet and habitat use requirements, and historical invasion success, cane
toads represent an ideal species for investigating learning abilities in the anuran clade.
As a generalist species and successful colonizer, cane toads represent an
anuran species with a wide range of suitable habitats and niche requirements. Not all
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anurans, however, are as flexible in their niche specifications. The leaf litter toad,
Rhinella alata, is a sympatric bufonid in the cane toad’s native range, yet possesses a
very different life history strategy. Contrary to the cane toad, leaf litter toads are
highly specific in their habitat use and diet requirements (Toft 1981; Parmelee 1999).
A comparative study addressing similar questions about exploratory ability and
foraging success would reveal whether learning behavior in anurans could potentially
be modulated by differences in life history strategies.
Overview of Thesis Research
While learning can be easily defined in terms of animal behavior, it is not so
easily measured across taxa. Spatial learning, for the purposes of this thesis, will be
defined and measured by a suite of variables that describe movement and exploration
and how such behaviors change with experience in a novel environment.
In the following chapters, three studies are outlined that examine how
exploratory and foraging behavior change as experience with a novel environment
increases. The first chapter details the main study, testing this ability in cane toads
from a population in Gamboa, Panama, within the species’ native range. Documenting
and describing the exploratory behaviors of cane toads from a typical native
population is essential for establishing a baseline for comparison to populations in the
invasive range or other species. The second chapter outlines a study addressing the
same question as the first chapter, except conducted in a laboratory setting with cane
toads from the invasive range in South Florida. This study was designed to shed light
on any possible differences in behavior between cane toads from the native and
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invasive range to determine if spatial learning and exploratory behavior are
comparable between the two populations. The final chapter describes a similar study
conducted on a smaller scale with the leaf litter toad, Rhinella alata, a sympatric
species in parts of its native range. This study provides a behavioral comparison point
for a congeneric species of toad with a different life history strategy.
If the species tested here are able to learn about their environment and modify
their behavior accordingly, we expect to see that exploratory behavior decreases as
experience with the novel environment increases. When there is no new information
available (i.e. when all possible locations in the arena have been visited), we expect to
see individuals to greatly reduce behaviors associated with exploration. If this
information is gained for later use, then such learning should be reflected in how the
toads use the space in subsequent experimental trials.
Understanding how an anuran species uses the environment on an individual
scale will add valuable information to the field of anuran behavior. Little is known
about anuran space use, other than in the context of mating behavior and habitat
distribution. This study is the first of its kind, in that it was designed specifically to
determine behavioral strategies of individual anurans as experience in an environment
increases. Shifting from the historical stimulus-response learning experiments to a
more open-ended experiment provides evidence to determine not just if cane toads are
learning about a novel environment, but how their movement and foraging behavior is
modulated as experience increases and information is acquired. Knowing how a
successful invasive anuran such as the cane toad gathers and uses information about
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novel environments will further elucidate possible connections between learning and
invasive potential, particularly related to exploratory behavior and foraging success.
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EXPLORATION AND FORAGING BEHAVIOR OF THE CANE TOAD
(RHINELLA MARINA) IN ITS NATIVE RANGE
Introduction
Gathering and using information about the environment is a key component of
successful survival (Dall et al. 2005). The most effective way to gather such
information is by systematic exploration, or movement around an area to acquire
information about potential resources and risk factors (Russell et al. 2009).
Exploration is most beneficial when encountering an environment or situation for the
first time, and individuals are likely to explore an unknown area because of the
potential benefits within, possibly greater than those that are already known (Krebs et
al. 2009). Exploration provides the function for gathering information that can later be
used to modify behaviors associated with space use, resulting in learning about one’s
environment.
Learned information can be used to create cognitive maps or to memorize the
spatial location of potential resources. Spatial learning allows an animal to consider
orientation, distance, and complexity of an environment, and traverse the most optimal
path between its current position and its target, usually a resource (i.e. food or water).
In areas of high predation risk, knowing the fastest route to a resource or refugia may
mean the difference between life and death (Paulissen 2008). Spatial learning has been
studied in a wide range of taxa (e.g. primates: Drea 2006, Hayes et al. 1953, Poirier &
Hussey 1982; rodents: Mead 1957, Poucet et al. 1986; marine mammals: Deecke
CHAPTER II
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2006; birds: Sol 2002; fish: Warburton 1990; reptiles: Paulissen 2008; insects: Durier
and Rivault 2001), concluding that species across a wide range exhibit some form of
spatial learning. Although spatial learning in anuran amphibians has not been studied
to the same extent as in other taxa, evidence from the literature suggests that frogs and
toads have the ability to spatially orient in their environment (Landler & Gollmann
2011; Sinsch 2006; Landreth & Ferguson 1966).
In his review on learning in reptiles and amphibians, Suboski (1992) suggested
that spatial navigation and orientation are likely candidates influenced by learning but
have not been studied extensively due to the aforementioned problems associated with
developing a consistently reliable research paradigm. Evidence suggests that studies
using arena designs or reward systems similar to those found in the natural habitat of
the study species have had more luck with obtaining consistent and interpretable
results than those that follow standard laboratory protocols. Incorporating water into
the experimental design, Bilbo et al. (2000) used a variation on the classic Morris
water maze to identify spatial and cue-based learning in leopard frogs (Rana pipiens).
Water has also been used as a reward for the aquatic Bombina orientalis (Brattstrom
1990), as slightly desiccated individuals learned the shortest path to water at the end of
a maze. A more recent study using water as the reward mechanism indicates that toads
have the capacity for detailed spatial learning using cues available in the environment
(Daneri et al. 2011). Experimental designs incorporating structures that show a high
degree of similarity to those found in the species’ natural environment have also
revealed the presence spatial learning in anurans. Using an artificial cave-like
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vivarium, Lüddecke (2003) found that the dendrobatid frog Colostethus palmatus
preferentially retreated to larger, wetter caves, and did so at a faster rate over time.
Arguably, studies which test spatial learning and navigation in an organism’s true
environment will provide the most reliable information about these abilities
(Timberlake 1984). Such studies in anurans are minimal to date, but indirect kin
recognition via spatial learning has been shown in the strawberry poison dart frog,
Oophaga pumilio (Stynoski 2009).
Though previous studies provide evidence for navigation via spatial learning in
anurans, no study has looked at the effects of exploration and movement on foraging
success, in either a laboratory or field setting. The research presented in this study
investigates the exploratory behavior of the cane toad (Rhinella marina = Bufo
marinus), and how exploration contributes to foraging success in a novel environment
through the use of spatial learning.
Drawing inspiration from traditional open-field maze designs used to test
exploration (Hall 1934) we tested wild-caught cane toads from their native range in
the novel environment of an experimental arena. We asked the questions: 1) How does
exploratory behavior change as experience with a novel environment increases? 2)
How does foraging behavior change as experience with a novel environment
increases? and 3) How do cane toads use visual cues in their environment to locate
food? The first two questions will elucidate connections between experience and
movement in a novel environment, while the third will provide evidence as to whether
or not spatial learning exists in this species.
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Methods
Study Site
Individual cane toads were collected within and around the town of Gamboa,
Panama (9°07.0'N, 79°41.9'W), at a facility of the Smithsonian Tropical Research
Institute. All toads were collected and tested between July -August 2010, and June -
August 2011. Sex of the toads was determined upon capture. Male toads were
primarily used for this study, with female toads used when we were unable to locate
males in a timely manner. Females used were within the size range of the males,
thereby having the same locomotor abilities as the males.
Preliminary Study
During the summer of 2010 we conducted a preliminary study to determine the
locomotor abilities and general movement strategies of cane toads in their native
range. Five male toads were captured during July and tested in a variety of arena
designs and trial time lengths, using canned dog food as a food reward. Toads were
tested in a circular arena (240cm diameter by 60 cm high) made from cinderblocks in
an outdoor enclosure every other day for 10 days. Cinderblocks and bricks were used
in the arena to create complexity in the environment, and the amount and arrangement
of these items were changed throughout the experiment to determine if block
placement affects toad movement. Regardless of the block arrangement three food
bowls were placed in the arena during all trials, containing 50 g (2 oz) of canned dog
food. During the preliminary study we measured the time it took a toad to leave the
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origin, find a food bowl, and eat the dog food within. General trends in movement and
space use were also observed and recorded.
The results of the preliminary study suggested that cane toads required a
moderate amount of complexity to stimulate movement; we found that in situations of
little complexity (eg. Arenas with 1-3 block formations made with less than 5 blocks
each) and high complexity (eg. Arenas with block formations made with more than 5
blocks each), toads did not move as much. Cane toads required at least 60 minutes to
explore the arena, identify and consume food resources. Four of the five toads in this
experiment did not leave the origin for 6-10 minutes during the first trial, and three of
the five toads required more than 20 minutes to find and eat food from one bowl.
Based on these results we designed a final arena and experimental paradigm to
maximize the amount of information gathered about exploratory behavior and
foraging success in a novel environment.
Experimental Arena Design
The exploratory arena used in the 2011 experiments was developed based on
concepts derived from traditional maze designs such as the radial arm maze (Olton et
al. 1977), open field maze (Hall 1934; Walsh & Cummins 1976) and the Morris water
maze (Bilbo et al. 2000). The arena consisted of a circular plastic wading pool 244 cm
diameter x 46 cm tall, extended to 76 cm in height. The arena floor was covered with a
mixture of 3-5cm cleaned and dried leaf litter and soil. Three types of pre-configured
block formations were randomly placed at equal spacing along the margin of the arena
to promote exploration by creating a spatially complex environment (Figure 1). Six
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plastic bowls (6cm tall by 15cm in opening diameter) were placed in the arena in a
randomized block design. Each bowl contained one mealworm (2.5-4 cm long) for the
experimental group and remained empty for the control group. Infrared lights with a
30-ft range (Clover electronics) and a high resolution outdoor security camera
(Supercircuits model # PC88WR) were placed 167 cm above the arena for video
recording of trials. Individuals began each trial from a haphazardly preselected point
along the edge of the arena, held constant throughout the experiment.
Experimental Procedure
Toads were assigned to a treatment group (experimental = food in bowl,
control = no food in bowl) and moved into the arena in their individual hide, which
serves as a familiar origin point for entry into the arena. Each toad was tested for 60
minutes between the hours of 20:00 and 02:00 the following morning, during noted
foraging times (Zug & Zug 1979). Trials were video-recorded for further analysis. The
number of mealworms and bowls eaten from were recorded for each trial. Although
toads are not known to use chemical cues when foraging (Martof 1962), the arena was
sprayed liberally with aged water between trials to account for any chemosensory cues
left behind by the previous trial.
Toads were first introduced to the arena in a series of training trials, in which
each toad had 60 minutes to explore the arena. During this time, toads in the
experimental group were given the opportunity to find the bowls offering a food
reward. Toads in the control group also encountered bowls; however no food reward
was presented. The individual encountering a food bowl and successfully eating a
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mealworm marked the end of the training period. If a toad did not eat by the fifth trial
it was not tested further. Since toads in the control group were not offered a food
reward, the training period for these individuals ended when they encountered an
empty food bowl. Control group toads were fed one mealworm in their housing bin
every other day, after their trial was completed.
Toads were tested for five trials after initially finding and eating a mealworm
during training. After the fifth trial, toads were tested for a sixth and final trial, in
which the bowls were moved to new locations. We scored whether the toad first
visited a location that previously held a bowl, or the new location of the bowl for this
trial. This trial allowed us to determine if the toads were using the relative location of
the food within the environment or were associating the food bowls themselves with
mealworms despite their location in the arena.
The behavioral software package Ethovision XT (version 8.0) was used to
analyze each video-recorded trial at the rate of 1 fps (frame per second). Unless
otherwise stated, all variables were calculated using the program’s built in analysis
functions. Videos were recorded to an external DVR box (Supercircuits model
#DMR80U) and imported into Ethovision for analysis. All statistical analyses were
conducted in SPSS (version 19) with alpha = 0.05 unless otherwise stated.
To determine if specific bowls were preferentially visited during the main
experimental trials, the frequency of visits to each bowl was calculated and compared
to the null hypothesis that if toads were encountering bowls randomly, then each bowl
should be eaten out of an equal number of times. The likelihood of eating out of a
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specific bowl first during each trial was also calculated, based on bowls that had been
eaten out of first during previous trials.
Experiment #1 – Changes in Exploration with Increased Experience
To determine how cane toads modify their movement in the arena as
experience increases the following variables were measured: time spent moving (TM),
total path length (TPL), time spent in the margin, or outer 25% of the arena (TMAR),
latency to leave the origin (LO), time to encounter a bowl (TB), and number of escape
attempts (ESC). Latency to leave origin and number of escape attempts were recorded
directly from videos by an observer. If toads are changing their behavior as experience
in the arena increases, then we expect a reduction of time allocated to behaviors that
are considered ‘exploratory’ (i.e. total time moving), and an increase of behaviors that
indicate familiarity with the environment (i.e. decreased thigmotaxis).
To examine the changes in exploratory behavior across trials, the variables for
each group were analyzed separately using Friedman tests. Highly correlated variables
(rho ≥ 0.90) were selectively removed from analysis based on biological relevance, to
prevent overlapping interpretations. The number of escape attempts was scored as a
count variable, and was square root-transformed before analysis.
Experiment #2 – Changes in Foraging Behavior with Increased Experience
To determine if foraging behaviors changed across trials in the experimental
group, serving as a proxy for learning about the location of food resources in the
environment, we measured bowl encounters during each trial. A bowl encounter is
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16
defined as a toad physically touching a bowl or coming within a three-centimeter
radius of the bowl for greater than two seconds. The total number of bowl encounters
(BE), as well as the number of unique bowl encounters (how many of the six bowls
available in the arena were encountered by the toad), were scored for each trial.
We hypothesized that toads in the experimental group will learn that food is available
in the bowls in the arena, and will continuously seek out food during trials. Thus, we
predicted that toads in this group will increase bowl encounters and unique bowl
encounters over time. Four additional variables related with food consumption were
recorded in the experimental group: Time from leaving the origin to eating first
mealworm (TE), path length from leaving the origin to eating first mealworm (PLE),
tortuosity, or curvature, of the path to eat first mealworm (T), and total number of
mealworms eaten per trial (MLS). PLE was calculated using point coordinate data
exported from Ethovision, and TE and MLS were directly measured from the videos
by an observer. Tortuosity was calculated by dividing the length of the curve (total
path length) by the Euclidean distance between its ends (Benhamou 2004). We
predicted that if toads are modifying foraging behavior in the arena due to availability
of food resources, they should take less time to find food, cover a shorter distance to
reach food bowls, and eat a greater number of mealworms as experience with the
arena increases (i.e. as information about the location of food resources is learned).
As in the analysis for the first experiment, a series of Friedman tests were used
to analyze the four foraging-related variables. BE, UBE and MLS were measured as
count variables, and thus were square root-transformed. Highly correlated variables
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were excluded a priori from the analysis, keeping the variable that provided greater
biological insights given the questions posited in this study.
Experiment #3 – The Role of Spatial and Visual Cues Involved in Learning
The purpose of the final experiment was to determine what type of spatial cues
cane toads are using to located food in the environment. During the final trial we
moved the bowls to new locations that did not previously contain bowls (Figure 2). If
cane toads in the experimental group are using spatial cues in the arena to locate
bowls, then toads should first visit locations that previously contained bowls. If the
toads, however, are associating the visual cue of the bowl with the presence of food in
a more specific context, then we expect the toads to move to the new bowl locations
first before visiting the old bowl locations, if at all.
In this experiment we recorded whether each toad first visited an old bowl
location or a new bowl location after leaving the origin. A visit to a bowl location was
scored if the toad entered the zone where the bowl was previously located and
remained static for two seconds or more, the same criterion used to determine bowl
encounters during all other trials. We predict that toads in the control group, having
learned no association between bowls and food items, will visit old and new locations
with equal probabilities, while toads in the experimental treatment will preferentially
visit bowl locations depending on the specific cues used. These observations were
statistically analyzed using binomial probabilities.
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Results
We tested a total of 20 adult toads, ten assigned to the experimental group and
ten to the control (SVL =114.83 + 11.3mm, mass= 145.00 + 45.65g). In the
experimental group, the majority of the toads ate during the first trial (7 out of 10). Of
the remaining toads one ate on the second and fifth trial each. One toad failed to eat
during the training trials and was excluded from further analysis, resulting in a total
sample size of N=19. None of the variables differed significantly between the
treatments groups for the first trial (Table 1), indicating that both groups had
equivalent behaviors at the beginning of the experiment when the arena was
considered a novel environment.
Both groups showed a degree of preference for encountering certain bowls in
the arena (Table 2), with bowls five and six being encountered much more frequently
than the others. For toads in the experimental group, bowl five was preferred for bowl
encounters that resulted in a successful meal (38% of all food visits) and comprised
the first food meal during the trial in more than half of the bowl visits that resulted in a
meal.
Exploratory Behavior and Experience
In experiment #1, time spent moving and total path length were highly
correlated (rho = 0.94, p < 0.001). The total path length of each toad during trials is
more representative of movement in the context of exploration and total area covered.
Measuring the amount of time spent moving for anurans does not make biological
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sense for our research questions, given that toads move in a start-stop fashion with
long bouts of immobility. Thus, the total amount of time moving could be small, but a
much larger area in the arena could be covered overall.
There was a significant difference across trials for total path length (control: χ2
= 29.14, p < 0.001; experimental: χ2 = 23.89, p < 0.001), time spent in the margin
(control: χ2 = 21.03, p < 0.001; experimental: χ2 = 19.27, p < 0.01), and number of
escape attempts (control: χ2 = 20.29, p = 0.001; experimental: χ2 = 22.95, p < 0.001),
in both the control and experimental group (Figure 3). There was also a significant
difference in the time the toads spent at the margins of the arena between groups in
trials 3-6, but not for trial 2 (t-test bootstrapped 1000 times: trial 2: t = -.325, p =
0.749; trial 3: t = -2.55, p = 0.025; trial 4: t = -2.90, p = 0.012; trial 5: t = -3.24, p =
0.01; trial 6: t = -2.19, p < 0.043). There were no significant differences in either the
latency to leave the origin or the time to find a bowl across trials for either group
(Figure 4).
Foraging Behavior and Experience
For the four variables measured in the experimental group, a significant
correlation exists for the time to eat (TE) and path length to eat (PLE) variables,
similar to the correlation between TM and TPL. The path length to eat variable was
excluded from further analysis to account for the redundancy of variables given that
tortuosity incorporates this measurement and describes the qualities of the toad’s
movement more robustly than simply looking at path length.
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There was a significant difference in the total number of mealworms eaten per
trial (χ2 = 15.81, p < 0.01), however there was no significant difference in the time the
toads took to eat or the directionally of their path to the food (TE: χ2 = 8.30, p = 0.14;
T: χ2 = 9.81, p = 0.08). Given our sample size, however, the low estimate of critical
probability suggests a trend of tortuosity changing across trials from more to less
curved paths to food.
The total number of bowls encountered and the unique number of bowls
encountered changed significantly across trials for the control group (BE: χ2 = 20.12, p
= 0.001; UBE: χ2 = 11.08, p = 0.05), but did not change significantly for the
experimental group (BE: χ2 = 4.79, p = 0.44; UBE: χ2 = 4.00, p = 0.55; Figure 5). The
difference between the mean values for UBE for each group, however, increased as
time progressed, with significant differences between treatment groups in trials five
and six (trial 5: t = 2.33, p < 0.05; trial 6: t = 2.20, p < 0.05).
Use of Spatial and Visual Cues to Locate Bowls
In the final trial of the experiment (experiment #3), we examined if toads
visited old or new bowl locations first. Toads in the control group visited old and new
bowl locations at near equal numbers (old locations = 6, new locations = 4). There was
a significant difference, however, in bowl location visits for the experimental group
(binomial probability, p < 0.05). Toads that received food in the bowls visited
significantly more old bowl locations first than new bowl locations (old locations = 7,
new locations = 2).
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Discussion
The results of this experiment indicate that cane toads are able to change their
exploratory and foraging behavior as a result of experience in a novel environment.
Individuals in both groups decreased exploration as experience with the environment
increase, as seen by decreases in total path length and time spent near the walls of the
arena. Toads in both groups also gradually ceased to try and escape from the arena
over time. We found that differences in behavior were likely due to the presence (or
absence) of food in the environment. Contradictory to our predictions based on results
from the preliminary study measures of latency did not change as experience
increased, indicating that time may not be as important a factor in exploration and
foraging success for anurans as for other species.
Regardless of the presence of food in the arena, toads in this study decreased
their total path length as experience with the arena increased. Decreases in exploratory
behavior have been shown to occur in rats that colonize novel environment (Russell et
al. 2010). This decrease in thigmotaxic behavior, or movements in contact with walls,
as experience increases suggests an increased in bolder behavior associated with
foraging in a safe environment (Simon et al. 1993). This observation is consistent with
anecdotal reports of cane toad foraging in urban environments at pet bowls and light
sources (Alexander 1965; Krakauer 1968). In all individuals, attempts to escape
diminished over trials until they stopped completely, presumably as information is
acquired and remembered about previously unsuccessful escape routes.
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Toads in the experimental group modified their behaviors to increase foraging
success. Individuals in this group spent a greater amount of time away from walls
than those in the control group, likely due to their motivation to find food in the
environment. Toads in this group had a lower average number of escape attempts per
trial, and extinguished escape attempts more quickly than in the control group. This
may indicate that when a food reward is present, this reward is preferentially
considered over the possibility of escape. Interestingly, the mean number of bowl
encounters decreased for this group, yet the number of unique bowls encountered in
the arena increased as time progressed. These changes in bowl visitation behavior
suggests that the toads are learning that bowls do not regenerate food rewards within
the same trial, yet other food rewards are present at different bowl sites. This
ultimately led the toads to increase their foraging efficiency by eating, on average,
more mealworms during trials as experience increased. Though speculative, this is
consistent with the literature regarding foraging at nonrenewable patches (e.g.
Devenport et al. 1998).
Though the number of mealworms eaten increased as experience in the arena
increased, the time it took for toads to eat the first meal did not change significantly
over trials. This finding provides evidence that time may not be a motivating factor
during exploration for this species. This is contradictory our predictions, and to
findings of exploration and learning in other species (e.g. Durier & Rivault 2001;
Mettke-Hofmann et al. 2002; Russell et al. 2010). This lack of time-modulated
behavior may be a reason why previous studies report inconclusive results regarding
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the presence of learning in anurans (e.g. Grubb 1976; McGill 1960; Suboski 1992).
Movement, rather than time, seems to be the determining factor of exploratory
behavior in this species. For ectotherms a tradeoff may exist between errors in
movement the speed at which an individual moves.
Toads in the control group did not receive any external motivation to explore
the arena other than the novel environment itself. Individuals in this group still
explored the arena, presumably due to unknown benefits hidden within (Krebs et al.
2009). Escape from the enclosed environment seemed to be the motivating factor for
these toads, as they consistently attempted to escape from the arena, as many as 27
times per trial. By the final trial, however, toads in this group had completely ceased
escape attempts, similar to toads exposed to a food reward. Although not surprising,
this behavior reveals cognitive reasoning ability, in that cane toads are making
appropriate decisions that incorporate recent experience by learning that they could
not successfully escape from the arena.
When examining the specific cues used by cane toads to learn the location of
food, our results indicate that individuals remember the relative location of the food in
the arena rather than specific features associated with the food (e.g. bowl). Upon
leaving the origin, individuals were seen hopping to sites that previously contained a
bowl. Here they stayed for several seconds to several minutes, raising up and
extending their forelimbs, changing the orientation of their bodies, and generally
moving about the area where the bowl was previously located, as if confused. After
this period of investigation, however, individuals continued to move about the
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environment until they found a food bowl in a new location, and in most instances
successfully ate during the trial. Though reversal learning has yet to be investigated in
anurans, evidence for this ability exists in lizards (Leal & Powell 2011) and turtles
(López 2003). Now that how toads find food resources in an environment has been
established, reversal learning could be investigated through this spatial context.
Studies that incorporate stimuli or arena designs similar to those found in the
natural setting of the study species often have more interpretable results and greater
real-world applicability of findings (Timberlake 1984). Previous studies that found
evidence of learning in anurans have used rewards such as water (Brattstrom 1990;
Daneri et al. 2011) and arena designs mimicking a natural cave system (Lüddecke
2003), and necessary offspring rearing locations (Stynoski 2009). This study is the
first to use an an ecologically-relevant design to determine exploratory behavior of
adult anurans in a novel environment. The use of food rewards located in the novel
arena to test spatial navigation and learning in adult anurans promotes more realistic
behavioral responses and gives us a better idea of what these individuals might do
when encountering a novel environment in the wild.
Cane toads are an excellent candidate species for investigating foraging and
learning in a novel environment because of their history as generalist, invasive species
that survive in a range of environments. Now that baseline behaviors for native-range
cane toads in a novel environment has been established, extending this design to other
anuran species would provide a framework to directly investigate the role that this
learning ability could have played in their success at invading new habitats. Finally,
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this study highlights the use of relative spatial location in how cane toads learn about
where to find food. Studies that further investigate the cues used by anurans in spatial
learning will provide valuable insights about their foraging ecology and flexibility of
life history strategies.
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Figure 2.1 Experimental Arena Setup
The arena used for experiments contained 2-3 cm of dirt and leaf litter substrate, six
bowls which served as the location for food resources, and block features to promote
movement and exploration in the environment. Each toad started from the same point
(origin) for all trials. Bowls were numbered to keep track of which bowls toads were
visiting and eating from during each trial.
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Figure 2.2 Bowl Locations for Experimental Trials
To test whether toads were using cues from the environment to determine the location
of food bowls, bowls were moved from their original locations (A) to new locations
that did not previously contain bowls (B) for the final experimental trial.
A B
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Table 2.1 Observation Values for Behavior During First Experimental Trial
Control Experimental
1st trial Mean SE Mean SE p TM 1255.86 132.62 1296.96 126.67 0.99 TPL 8113.76 735.10 8940.98 975.41 0.31 TMAR 2906.01 269.50 3226.12 53.12 0.44 LO 96.29 11.54 57.72 16.46 0.20 TB 217.22 83.48 231.34 80.21 0.22 BE 31.6 3.91 31.11 4.18 0.51 UBE 3.5 0.40 3.44 0.41 0.84 ESC 7.9 2.14 10.89 2.66 0.18 *p values found using a t-‐test, bootstrapped 1000 times at α=0.05
Observations for the exploration variables did not differ significantly between the
control and experimental groups for the first trial, indicating that the toads had similar
behaviors in an environment where they had no previous experience.
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Table 2.2 Degree of Preference for Bowls Encountered
Bowl # Total Visits* (C) Total Visits* (E) Food VisitsϮ 1st Eaten in TrialӔ 1 131 0.10 128 0.10 4 0.05 1 0.02 2 212 0.17 216 0.17 16 0.18 7 0.15 3 46 0.04 84 0.07 13 0.15 6 0.13 4 165 0.13 183 0.14 7 0.08 3 0.06 5 365 0.29 333 0.26 33 0.38 25 0.52 6 340 0.27 324 0.26 14 0.16 6 0.13
* Number of times bowl was encountered during all trials Ϯ Number of times bowl was eaten from during all trials Ӕ Number of times bowl was the first eaten from during all trials
Total bowl encounters summed across all trials for both the control (C) and
experimental (E) groups. The number in italics represents the proportion of the total
that each bowl represents Bowls 5 and 6 were encountered more often than other
bowls in both the experimental (E) and control (c) groups.
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Figure 2.3 Total Path Length, Time Spent in Margin, and Number of Escape Attempts
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Three variables were significant across trials for both treatment and control groups;
TPL (top), TMAR (middle), and ESC (bottom). Both total path length and number of
escape attempts decreased steadily over time. Time spent in the margin initially
decreased steadily for the experimental group in trials 1-4 before reaching a level
threshold held relatively constant for trials 5 and 6. The control group saw a more
gradual decrease in TMAR over time.
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Figure 2.4 Latency to Leave Origin and Time to Encounter Bowl
Neither of the variables associated with time changed significantly for either
treatment group as experience with the arena environment increased. LO (above, top)
showed almost no change over time, while the time to encounter a food bowl (bottom)
highly varied per individual tested, independent of treatment group.
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Figure 2.5 Number of Total and Unique Bowl Encounters
The number of total bowl encounters (top) substantially decreased for both treatment
groups between trials 1 and 6, however there were not substantial decreases between
intermediate trials, or between groups. The change in number of unique bowl
encounters (bottom) was not significant in either treatment group, however there is a
decreasing trend in the control group and an increasing trend in the experimental
group.
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EXPLORATION AND FORAGING BEHAVIOR OF THE CANE TOAD
(RHINELLA MARINA) FROM AN INVASIVE POPULATION
Introduction
When a species is introduced to a non-native area, individuals either establish a
small, sustained population or die out within a few generations. Occasionally, an
introduced species flourishes in its new environment and the population grows larger
each generation, successfully expanding from the introduction site (Lockwood et al.
2005). Many factors have been suggested to contribute to a species invasive potential
and ultimate success, however introduction effort, or propagule pressure, is the only
factor that is well-studied and consistent across invasive species (Lockwood et al.
2005). Behavioral characteristics may also influence the invasion success of
vertebrates (Holway & Suarez 1999), and behavioral flexibility has been identified as
a likely characteristic of invasive species (Sol 2002; Adamo & Lozada 2009).
Learning ability has been highlighted as a factor that promotes invasion success
(Amiel et al. 2011; Roudez et al. 2007; Sol 2002; Sol et al. 2008). If the ability to learn
is present in an invasive species to a greater extent than in native species that occupy
similar trophic levels, the invasive species could potentially outcompete natives and
receive a fitness advantage. This idea has been tested in a native and invasive species
of crab (Roudez et al. 2007), and similar results were found in native and invasive
crayfish when tested for association between a novel odor and predation risk (Hazlett
et al. 2002). If learning is a potential contributor to invasive success, then this ability
CHAPTER III
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should be present in the native populations prior to introduction. It is unlikely that
learning evolves in the introduced populations separate from the native populations,
due to the short time span over which introduced species often become invasive.
Learning is expected to be favored when resources in an environment are
predictable within the individual’s lifetime, but are not predictable between
generations (Stephens 1993). In a novel environment, such as those encountered by
the original individuals of an introduced or invasive species, resources are unknown
and therefore unpredictable. Because information about resources and environmental
factors is unknown, learning about the environment through exploration is a key
component of successful colonization (Russell et al. 2010). Therefore, we would
predict that there is a strong selective pressure on initial individuals of an introduced
species to learn about the environment, and those who do so will have higher survival
and reproductive success. The capacity to learn has been shown to have a genetic
component (Mery & Kawecki 2002), meaning this ability will be passed on to the
offspring of those individuals who are successful. Learning comes at a cost to
individuals who no longer need the ability (Mery & Kawecki 2004), so there is a
potential tradeoff between learned and innate behaviors depending on predictability of
the environment, consistent with Stephen’s learning model.
To elucidate possible differences in learning ability between cane toads in
native and invasive populations, we investigated exploratory and foraging behavior in
individuals from the invasive range in South Florida. The findings from chapter 2
suggest that cane toads from the native range change their foraging behavior with
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increased experience in a novel environment, and modulate their movements and
space use to more efficiently find food. If selection for the ability to learn is higher in
the invasive range, then cane toads from the invasive population should demonstrate
higher learning capabilities than the native population in Gamboa, Panama.
Methods
Cane toads were purchased from Carolina Biological Supply, who collected
them from areas in the South Florida invasive populations. Toads were housed at an
ACUC-approved animal facility at Texas Tech University (Lubbock, Texas), in
groups of 3-5 individuals in 50-gallon cattle tanks. Toads were kept under conditions
equivalent to those found in their native range of the tropics (80-85ºF, RH 85 %,
12L:12D cycle). Before the experiment, toads were removed from group housing and
housed individually to control their food intake. Individual housing bins were similar
to those used in the Gamboa experiment (Chapter 2); 24-liter plastic bins with 1-3cm
of peat moss substrate, a water container and a hide (ceramic flower pot). Toads were
randomly assigned to one of two treatment groups, control (no food in bowls) and
experimental (food in bowls).
Experimental Arena Design
The exploratory arena was developed based on concepts derived from
traditional maze designs such as the radial arm maze (Olton et al. 1977), open field
maze (Walsh & Cummins 1976) and the Morris water maze (Bilbo et al. 2000). The
arena consisted of a circular plastic wading pool 244 cm diameter x 46 cm tall,
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extended to 76 cm in height. The arena floor was covered with a mixture of 3-5cm
peat moss. Three types of pre-configured block formations were randomly placed at
equal spacing along the margin of the arena to promote exploration by creating a
spatially complex environment (Figure 1). Six plastic bowls (6cm tall x 15cm in
opening diameter) were placed in the arena in a randomized block design. Each bowl
contained one mealworm (2.5-4 cm long) for the experimental group and remained
empty for the control group. Infrared lights with a 30-ft range (Clover electronics) and
a high-resolution outdoor security camera (Supercircuits model # PC88WR) were
mounted on the ceiling approximately 160cm above the arena floor, and were used to
record all trials. Because cane toads are known to be visual foragers (Robins & Rogers
2004), LED lights were placed on the ceiling to give off an ambient light within the
range of natural moonlight (0.30 + 0.06 lux). Individuals began the trial from a
preselected point along the edge of the arena, which was held constant across all trials,
treatments, and individuals.
Experimental Procedure
Toads were moved into the arena in their individual hide structure from their
cage, which serves as a familiar origin point for trials. Each toad was tested for 60
minutes between the hours of 1430 and 1900, during their dark cycle. Trials were
video-recorded for further analysis. The number of mealworms eaten and location of
bowls eaten from was recorded for each trial. Although toad are not known to use
chemical cues when foraging (Martof 1962) the arena was sprayed liberally with aged
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water between trials to account for any chemosensory cues left behind by the previous
trial.
Toads were first introduced to the arena in a series of training trials, in which
each toad had 60 minutes to explore the arena. During this time, toads in the
experimental group were given the opportunity to find the bowls in the arena that
offered a food reward. Toads in the control group also encountered bowls; however no
food reward was presented. The end of the training period was marked by the
individual encountering a food bowl and successfully eating a mealworm. If a toad
did not eat by the fifth he was not tested in further trials. Since toads in the control
group were not offered a food reward, the training period for these individuals ended
when they encountered an empty food bowl. Control group toads were fed one
mealworm in their housing bin every other day, after their trial was completed.
Toads were tested for five trials after initially finding and eating a mealworm
during training. After the fifth trial, toads were tested for a sixth trial in which the
bowls were moved to new locations. We scored whether the toad first visited a
location that previously held a bowl, or the new location of the bowl for this trial. This
trial allowed us to determine if the toads were using the relative location of the food
within the environment or were associating the food bowls themselves with
mealworms despite their location in the arena.
The behavioral software package Ethovision XT (version 8.0) was used to
analyze each video-recorded trial at the rate of 1 fps (frame per second). Unless
otherwise stated, all variables were calculated using the program’s built in analysis
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functions. Videos were recorded to an external DVR box (Supercircuits model
#DMR80U) and imported into Ethovision for analysis. All statistical analyses were
conducted in SPSS (version 19) and critical p values are reported at a 95% confidence
level unless otherwise stated.
To determine if specific bowls were preferentially visited during the main
experimental trials, the frequency of visits to each bowl was calculated and compared
to the null hypothesis that if toads were visiting bowls randomly, then each bowl
should be eaten out of an equal number of times. The likelihood of eating out of a
specific bowl first during each trial was also calculated, based on bowls that had been
eaten out of first during previous trials.
Experiment #1 – Changes in Exploration with Increased Experience
To determine how cane toads modify their movement in the arena as
experience increases, the following variables were measured: time spent moving
(TM), total path length (TPL), time spent in the margin, or outer 25% of the arena
(TMAR), latency to leave the origin (LO), time to encounter a bowl (TB), and number
of escape attempts (ESC). Latency to leave origin and number of escape attempts were
recorded directly from videos by an observer. If toads are changing their behavior as
experience in the arena increases, then we expect a reduction of time allocated to
behaviors that are considered ‘exploratory’ (i.e. total time moving), and an increase of
behaviors that indicate familiarity with the environment (i.e. decreased thigmotaxis).
Due to sample size restrictions, data collected from this experiment were not
analyzed for statistical differences across trials or between groups. Correlations among
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variables were taken into account when describing the overall trends in behavior, and
highly correlated variables (rho ≥ 0.90) were selectively removed from analysis.
Experiment #2 – Changes in Foraging Behavior with Increased Experience
To determine if foraging behaviors changed across trials in the experimental
group, serving as a proxy for learning about the location of food resources in the
environment, we measured bowl encounters during each trial. A bowl encounter is
defined as a toad physically touching a bowl or coming within a three-centimeter
radius of the bowl for greater than two seconds. The total number of bowl encounters
(BE), as well as the number of unique bowl encounters (how many of the six bowls
available in the arena were encountered by the toad), were scored for each trial.
We hypothesized that toads in the experimental group will learn that food is available
in the bowls in the arena, and will continuously seek out food during trials. Thus, we
predicted that toads in this group will increase bowl encounters and unique bowl
encounters over time. Four additional variables related with food consumption were
recorded in the experimental group: Time from leaving the origin to eating first
mealworm (TE), path length from leaving the origin to eating first mealworm (PLE),
tortuosity, or curvature, of the path to eat first mealworm (T), and total number of
mealworms eaten per trial (MLS). PLE was calculated using point coordinate data
exported from Ethovision, and TE and MLS were directly measured by an observer
from the videos. Tortuosity was calculated by dividing the length of the curve (total
path length) by the Euclidean distance between its ends (Benhamou 2004). We
predicted that if toads are modifying foraging behavior in the arena due to availability
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41
of food resources, they should take less time to find food, cover a shorter distance to
reach food bowls, and eat a greater number of mealworms as experience with the
arena increases (i.e. as information about the location of food resources is learned).
As in the analysis for the first experiment, we did not have a sample size large
enough to support statistical analyses across trials or between groups. Correlations
were accounted for a priori, and variables that were highly correlated were excluded
from analysis based on biological meaning to the system.
Experiment #3 – The Role of Spatial and Visual Cues Involved in Learning
The purpose of the final experiment was to determine if cane toads in the
experimental group were using the relative spatial location of the food, or simply
associating the presence of food with bowls despite their location in the arena. During
the final testing trial we moved the bowls to new locations that did not previously
contain bowls (Figure 2). We predict that if toads are using spatial cues from the
environment to located food, then they should visit locations that previously held food
bowls before successfully locating a bowl in its new location. If the toads, however,
are associating the visual cue of the bowl with the presence of food, then we expect
the toads to move to the new bowl locations first before visiting the old bowl
locations, if at all.
In this experiment we recorded whether each toad first visited an old bowl
location or a new bowl location after leaving the origin. A visit to a bowl location was
scored if the toad entered the zone where the bowl was previously located and
remained static for two seconds or more, the same criterion used to determine bowl
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encounters during all other trials. We predict that toads in the control group, having
learned no association between bowls and food items, will visit old and new locations
with equal probabilities, while toads in the experimental treatment will preferentially
visit bowl locations depending on the specific cues used.
Results
We tested a total of 19 toads between September 5th and November 20th 2011
(Control = 8, Experimental = 11), with SVL 98.24 + 21.42mm and mass 104.31 +
6.33g. Due to methodological issues, three of the toads in the Experimental group
were unable to be used for analysis. Each toad in the control group completed 7 trials,
while six of the eight toads in the experimental group ate during the first trial. One
toad only ate once during the third trial, and one toad did not eat at all in the arena,
resulting in a final sample size of 14 (Control = 8, Experimental = 6). Due to the small
sample size and high variance associated with behavioral research, general trends seen
during trials are described here but no statistically analyze was performed. Toads in
both the control and experimental treatments behaved similarly during the training
trial when the arena was novel (Table 1), thus allowing us to draw conclusions about
their behavior based on experience gained in the arena.
Toads showed a preference for visiting certain bowls in the arena, as seen in
the previous study. The visitation preferences, however, differed slightly between the
two populations (Table 2). Bowl five was still a preferred bowl, with 26% of the total
bowl visits, but bowl two was visited most often, making up 29% of the total bowl
visits. Of bowl visits resulting in a successful meal, bowl five comprised 47% of all
Texas Tech University, Amanda Arner, August 2012
43
food visits and was the first bowl to be eaten from in a trial 58% of the time. The
second most-visited bowl for meals and first meals in a trial was bowl two, with 24%
of successful food visits and 23% of first meal visits.
Exploratory Behavior and Experience
Similar to the findings of the previous experiment, strong trends were seen in
both the total path length and time spent in the margin (Figure 2). Total path length
shows a strong decreasing trend for both experimental and control groups, with a
plateau beginning at trial 3. Conversely, time spent in the margin shows an increasing
trend for both groups across all trials. Toads in neither the control nor experimental
group tried to escape during trials, which may provide valuable insights for
interpreting their behavior in the arena. There were no changes in the latency to leave
the origin for either group as experience with the arena increased (Figure 3), which is
consistent with the findings of the first experiment.
Foraging Behavior and Experience
For both the time to eat the first mealworm and the tortuosity of the path to
reach such mealworm there is a strong decreasing trend during trials 1-5. Trial 6,
however, exhibits higher variation without fitting the trend from previous trials. There
were no apparent trends in the number of mealworms eaten per trial with increased
experience in the arena. For the experimental group, the total number of bowls
encountered and the number of unique bowls encountered seemed to decrease slightly
over time, however this trend may represent random variation in the small sample size.
Texas Tech University, Amanda Arner, August 2012
44
The control group showed a sharp decrease in both total and unique bowl encounters
between trials 1 and 2, but then a more shallow or non-existent decrease for the
remainder of the experiment.
Use of Spatial and Visual Cues to Locate Bowls
During the final trial of the experiment we moved the bowls to different
locations to determine if toads were using the relative location of the food in the arena
or associating the bowls with the presence of food. Of the six toads in the
experimental group, four visited old bowl locations first, one visited a new bowl
location first, and one did not encounter any bowls or bowl locations during the last
trial. In contrast, in the control group, one toad went to an old bowl location first, three
toads went to new bowl locations first, and four toads did not encounter any bowls or
bowl locations during this trial.
Discussion
The toads in the laboratory experiment acted both similarly and differently
from the toads in the field experiment. Toads in both experiments showed a marked
decrease in exploratory behavior, as defined by movement, as experience with the
arena increased. Interestingly, toads in the laboratory showed an increase in time spent
in the margin over trials probably due to their marked decrease in movement. In
general, toads in the control group would leave the origin, hop across the arena, and sit
on a cinder block or brick for the remainder of the trial. This marked decrease in
overall movement suggests a general lack of motivation to explore. Toads in the
Texas Tech University, Amanda Arner, August 2012
45
experimental group exhibited similar behaviors, except that they would visit food
bowls during the beginning of the trial then move to a block and remain stationary
until the trial was over.
The most interesting point to note, I think, is the fact that none of the toads
ever tried to escape from the exploratory arena. This raises the question of whether the
differences in behavior between the two populations are due to their native vs.
invasive origins or to the effects of captivity. By being kept under laboratory
conditions the toads’ decision-making processes could be affected when it comes to
spatial use. The difference in escape behavior highlights an unexpected confounding
effect of laboratory housing, which we cannot tease apart in this experiment.
Conducting this experiment with wild-caught toads in Florida would help disentangle
the effects of captivity from the population of origin for looking at differences in
learning abilities.
Overall, this experiment uncovers signs of potential differences in behavior
between the native and invasive populations. For instance, the toads in the laboratory
decreased their movement much more rapidly than the toads from the field experiment
and did not attempt to escape. One toad in the control group failed to even leave the
origin point during the sixth trial, and many toads simply did not move around as
much as the toads in the field experiment. These findings are contradictory to our
prediction that toads from the invasive range would be faster learners and show a more
rapid decrease in exploration and increase in foraging success than toads from the
Texas Tech University, Amanda Arner, August 2012
46
native range. Again, this could be an artifact of being in captivity, which would need
further testing to be conclusive.
Texas Tech University, Amanda Arner, August 2012
47
Figure 3.1 Experimental Arena Setup for Laboratory Experiment
The experimental arena in the laboratory at Texas Tech University was designed to be
an exact replica of the arena used during the field experiment in Gamboa, Panama. For
substrate, peat moss was used instead of leaf litter and other organic materials
collected from the field.
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48
Table 3.1 Observation Values for Behavior During First Experimental Trial
Control Experimental
1st trial Mean SE Mean SE p TM 600.98 190.81 693.69 440.89 0.95 TPL 4372.91 1602.39 5148.15 2595.33 0.50 TMAR 2005.65 438.82 1676.68 836.93 0.36 LO 213.21 65.26 149.32 78.54 0.06 TB 142.02 139.78 472.14 508.26 0.11 BE 16.50 6.19 19.83 12.98 0.70 UBE 3.00 0.93 4.50 1.38 0.04 ESC 0.00 0.00 0.00 0.00 - *p values found using a t-test or Mann-Whitney U test, where appropriate Observations for the exploration variables did not differ significantly between the
control and experimental groups for the first trial, indicating that the toads had similar
behaviors in an environment where they had no previous experience.
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49
Table 3.2 Degree of Preference for Bowls Encountered
Bowl # Total Visits Food Visits 1st Eaten in Trial 1 64 0.14 5 0.10 2 0.08 2 137 0.29 12 0.24 6 0.23 3 81 0.17 4 0.08 1 0.04 4 40 0.08 3 0.06 1 0.04 5 125 0.26 23 0.47 15 0.58 6 26 0.05 2 0.04 1 0.04
Similar to the results reported in chapter 1, the South Florida toads showed
preferential bowl visitation. Bowls five and two were visited 26% and 29% of the
time, respectively. Bowl five accounted for 47% of visits that ended in a successful
meal, and was the first bowl choice for a meal 58% of the time.
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50
Figure 3.2 Total Path Length and Time Spent in the Margin
Total path length (top) decreases over time, however time spent in the margin
(bottom) increases in both groups as experience increases. This finding is different
from that of the experiment with the native toads, in that both TPL and TMAR
decreased in the toads tested in Gamboa.
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51
Figure 3.3 Latency to Leave Origin and Time to Find Food Bowl
The latency to leave origin for both groups did not change as experience with the
arena increased, indicating that time is not an important factor in learning in either the
native or invasive populations. Time to find food bowl, however, showed differing
trends depending on group.
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52
Figure 3.4 Time to Eat Mealworm and Tortuosity of Path to Eat Mealworm
Both time to eat mealworm and tortuosity of the path to eat mealworm decreased over
time, but then increased during the final trial. This may be an artifact of small sample
size and high variance.
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53
Figure 3.5 Number of Total and Unique Bowl Encounters
Total bowl encounters and unique bowl encounters decreased over time for both
control and experimental groups. The control group’s decrease was initially much
more rapid than the experimental group, and remained lower across all trials,
presumably because toads in this group learned that there was no reason to encounter
these empty bowls.
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54
DIET FLEXIBILITY AND FORAGING BEHAVIOR IN A NOVEL
ENVIRONMENT IN THE LEAF LITTER TOAD (RHINELLA ALATA)
Interspecific competition for critical resources often promotes niche
specialization (MacArthur 1958). Morphological and behavioral traits associated with
reduction of competition among sympatric species abound (e.g. Sargeant 2007; Toft
1995). Similarly, diet differentiation among species in a community is thought to
result from interspecific competition avoidance (Vitt & Caldwell 1994). Foraging and
diet specialization persists due to those selective pressures imposed by other species in
the community that could potentially consume the same resource (Bolnick et al. 2002
and references therein). Complex assemblages of leaf litter arthropodivores are
common in Neotropical forested areas, potentially leading to increased competition for
shared, limited food resources (Caldwell & Vitt 1999). Consistently, in diverse leaf
litter anuran communities in Amazonian Peru and lowland Panama, species such as
poison arrow frogs (Dendrobatidae) and some toads (Bufonidae & Rhinellidae) are
specialized on eating ants (Toft 1980, 1981). Within Dendrobatids, such diet
specialization has been studied in detail. Ant specialization in this family, for instance,
has been associated with the evolution of multiple traits such as bright coloration, high
toxicity and greater aerobic capacity ( Santos & Cannatella 2011; J Santos et al. 2003).
In contrast, specialization in ant consumption in bufonids has yet to be explored.
Myrmecophagy is considered common in bufonids and species that primarily
inhabit leaf litter assemblages, such as the leaf litter toad, Rhinella alata (formerly
CHAPTER IV
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55
Bufo typhonius). This species is considered an ant specialist, consuming these insects
in greater proportion than are found in the environment (Toft 1980, 1981). In lowland
Panama ants comprise about 84% of this species’ diet, contrasting heavily with the
second preferred prey items (coleopterans) totaling only 14% of the diet (Toft 1980).
In populations in South American, however, leaf litter toads supplement their diet with
adult coleopterans that make up between 25% (Vitt & Caldwell 1994) to 60% of their
diet (Parmelee 1999). This species seems thus to be restricted to eating hard-bodied,
slow-moving arthropods, similar to other ant-specialist species in Neotropical leaf
litter communities (Parmelee 1999; Toft 1980). The diet flexibility of this species or
other ant specialist anurans has not yet been investigated. The purpose of this study is
to examine diet flexibility in R. alata and evaluate their exploratory and foraging
behavior in a novel environment. By presenting leaf litter toads with unfamiliar, soft-
bodied insect larvae we tested their ability and willingness to consume such novel
prey. To further examine the range of foraging behaviors utilized by this species, we
tested individuals who successfully learned to consume novel prey items in an
exploratory arena, to determine if they could further locate these novel prey items in
an unfamiliar environment.
R. alata individuals were collected at Gamboa, Panama (9°07.0'N, 79°41.9'W),
and its surrounding areas during June and July 2011. We collected 11 individuals
spanning a broad range of body sizes (SVL = 32.01 - 41.89cm, median = 36.77cm;
mass = 2.19 - 4.88g, median = 3.77g) to account for potential effects of body size on
diet as described in other bufonids (Duré, Kehr, & Schaefer 2009). Toads were
Texas Tech University, Amanda Arner, August 2012
56
brought back to the research station where they were housed individually in 11.5-L
plastic bins with mesh tops, with 2-3cm of leaf litter substrate, and two bowls for food
and water.
To determine if R. alata can successfully recognize novel insect larvae as a
consumable prey item, we offered toads the larva of the Darking Beetle, Tenebrio
molitor, originally from Eurasia (hereafter referred to as mealworms). Starting on the
day after capture, toads were offered two mealworms (< 6.35mm) in their food bowl
during their normal diurnal foraging period. Mealworms were left in the bowls until
the following day when the number of mealworms consumed was recorded for each
toad. Toads were offered mealworms every day, and were considered to have
successfully learned to consume the novel prey item if both mealworms were eaten for
at least two days in a row.
Eight of the eleven toads successfully learned to eat mealworms. While one
individual ate the mealworms the first time they were presented, the majority of toads
took 4 - 7 days (4.4 + 1.9 days) to successfully consume mealworms on a regular
basis. Neither body size nor mass were significantly correlated with the amount of
time it took individuals to successfully eat mealworms (Spearman rank test; body size:
rho= -0.225, p > 0.05; mass: rho= -0.01, p>0.05), suggesting that the size of the toads
does not determine whether or not R. alata individuals will eat a novel non-ant prey
item. Hunger levels when captured likely influenced willingness to consume non-
preferred or novel prey items in this species. In a similar study, however, with a
congeneric generalist species (cane toads = Rhinella marina) all individuals ate
Texas Tech University, Amanda Arner, August 2012
57
mealworms within 48 hours of being presented the novel food (Chapter II). Although
the leaf litter toads are not as quick to consume a novel food item as the cane toads,
the results here show that individuals of this ant-specialist species readily consume
non-ant prey items revealing the hidden diet flexibility of foraging decisions in this
species.
Once the toads were eating mealworms, we examined if R. alata can learn to
locate and successfully eat this now-familiar prey item in an unfamiliar landscape.
This experiment will help determine if R. alata are able to use spatial cues acquired
from a previously novel environment to find and remember the location of food
resources, and will demonstrate further foraging flexibility in this species. If
individuals can successfully recall the location of mealworms in an exploratory arena,
then we expect them to be able to find food in less time or distance travelled as
experience with the arena increases.
We used an exploratory arena following the design from Chapter 2. The arena
consisted of a circular enclosure made from ½” PVC pipe and 3 ml plastic sheeting,
measuring 100cm diameter and 60cm high, with 2-3cm of leaf litter and dirt substrate
(Figure 1). Seven toads were tested in 60 minutes trials between 10:00 and 16:00 in
the arena every other day for 10 days, totaling five trials for each toad. For each trial,
six bowls were placed in the arena in a randomized block design (3 on blocks, 3 on
substrate), each containing one mealworm (Figure 1). Trials were video recorded for
further analysis and characterization of toad behaviors.
Texas Tech University, Amanda Arner, August 2012
58
ImageJ (Rasband 2011) was used to calculate total path length (cm) and path
length to first eat food (cm) for each trial. Latency to leave the origin point and the
total time to reach a food bowl (in seconds) was measured directly via observation. To
further characterize behavior in the arena, the total number of bowl encounters and the
times those were visits to not previously visited bowls were recorded. Finally, the
number of times toads actively jumped against an outer wall of the arena (i.e. escape
attempts) was recorded.
Of the seven toads tested in the arena, three successfully found and consumed
a mealworm during the experiment (Trials eaten = 2.33 + 1.55, number of mealworms
eaten per trial = 1.1 + 0.17). Of the three individuals who ate in the arena, toads A and
C ate during more than one trial. Toad A ate during trials 2, 4 and 5, and toad C during
trials 3, 4 and 5. All three toads ate one mealworm per trial, with one exception being
toad C during trial 5. There were no apparent trends in either the time or distance
variables measured during trials (Figure 2). The overall number of escape attempts
decrease between trials 1 and 5 for all three toads, however the intermittent trials were
variable and showed no general trends for this variable (Figure 3). In comparison, cane
toads in a similar experiment showed significant reduction in both path length to find
the food in the arena and number of escape attempts as experience with the arena
increase, as seen in chapter two.
The results of this study provide evidence that the leaf litter toad, Rhinella
alata, can and will eat non-ant, unfamiliar prey items, suggesting this species has the
potential to exploit alternative and unfamiliar sources of food. Diet flexibility could
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59
aid in foraging success as the insect regime in an area changes temporally, especially
if there are marked difference in seasonality or where microhabitat use of several
species overlaps (Duré et al. 2009). Insect assemblages in the Neotropics change with
seasonality (Wolda 1988), however these differences do not consistently affect diet
content (Toft 1980). Once R. alata individuals have eaten this prey item, however,
they are unlikely to find and consume this food in the novel environment of an
experimental arena, and do not show any apparent signs of using spatial cues in the
environment to find food during subsequent trials. These findings do not match those
of the study conducted with the congeneric R. marina, which can readily find and
consume mealworms in an exploratory arena as experience increases. The differences
in learning abilities in these species may potentially be attributed to their differing life
history strategies; R. marina is an omnivorous generalist with a wide breadth of
habitable environments (Somma 2012), while R. alata are highly specific in their
habitat and diet requirements (Toft 1980). This differential use of niche space seems to
contribute to the differences in diet flexibility and foraging behaviors seen in these
species. This study provides a baseline for understand diet flexibility and exploratory
behavior in a novel environment in the leaf-litter ant specialist, Rhinella alata.
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60
Figure 4.1 Diagram of Experimental Arena for Leaf Litter Toads
Diagram of the experimental arena; 2-3 cm of leaf litter and dirt substrate covered the
bottom of the arena, while block formations promoted exploration and movement by
providing heterogeneous environmental conditions. All individuals started from the
origin for each trial, the location of which was held constant across individuals.
Food bowl
Blocks
Substrate
Origin
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61
Total Path Length (TPL)
Trial1 2 3 4 5
Dis
tanc
e (c
m)
0
1000
2000
3000
4000Toad AToad BToad C
Latency to Leave Origin (LO)
Trial
1 2 3 4 5
Tim
e (s
ec)
0
10
20
30
40
50
60Toad AToad BToad C
Figure 4.2 Latency to Leave Origin and Total Path Length per Trial
Variables measuring both time and distance traveled had highly variable observations,
indicating no apparent trend in exploratory movement.
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62
Escape Attempts (ESC)
Trial1 2 3 4 5
# of
esc
ape
atte
mpt
s
0
10
20
30
40
50
60Toad AToad BToad C
Figure 4.3 Number of Escape Attempts per Trial
The number of escape attempts showed a decreasing trend from the first to second
trials, and then sharply increased to the third trial. Trials 3-5 also show decreasing
trends, with the final trial showing the least variance among the three toads tested.
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63
CONCLUSION
In the most comprehensive review on reptile and amphibian learning to-date,
Milton Suboski recognized that we, as researchers, may be expecting a response from
these taxa that is not within the range of their normal behaviors (Suboski 1992). This
however doesn’t mean that reptiles and amphibians cannot learn. Since the review
there have been numerous studies on the learning abilities of lizards, snakes and turtles
(e.g. Aubret 2006; López Vargas, Gómez & Salas 2003; Paulissen 2008), and even a
few studies pertaining to learning in salamanders (e.g. Crane & Mathis 2011; Gibbons,
Ferguson, & Lee 2005). Recent studies of learning in anurans are scarce (e.g. Bilbo et
al. 2000; Brattstrom 1990; Greding 1971), but do provide evidence that anurans have
the capacity to learn. More research in this area is needed to understand learning in
this taxa, specifically related to spatial learning and navigation. The experiments
conducted in this thesis indicate that spatial learning ability exists in the cane toad,
Rhinella marina, and how this ability is associated with exploration and foraging
success. These findings are consistent with those of other studies using conditions
similar to those found in nature (e.g. Crane & Mathis 2011; Daneri et al. 2011;
Lüddecke 2003).
Generalist species such as the cane toad are able to occupy different niches
across a broad range of habitats, facilitating their ability to survive and reproduce
given heterogeneous conditions. This flexible life history strategy, combined with the
ability to learn about the environment around them, may be the reason that cane toads
are such successful colonizers of new habitats and make such prolific invasive species.
CHAPTER V
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64
Future studies on learning ability and exploratory behavior should be conducted on
other populations of cane toads, both in other areas of their native range and parts of
their invasive range, such as in Australia, Guam and Hawaii. Once the baseline
behavior for this species has been established in a native population, this methodology
could also be used to examine learning abilities in other terrestrial anurans. Simple
modifications could be made to the arena to allow for testing of both aquatic and
arboreal anurans, opening a host of possibilities for future studies on anuran learning
behavior.
The studies outlined in this thesis were designed to determine the baseline
movement and behavior of a well-known invasive anuran, and serve as a template for
future studies in this area. By asking the same questions about two different
populations of cane toads and a similar congeneric, sympatric toad, we were able to
accomplish this goal and describe how exploratory and foraging behaviors can change
as a result of experience in an environment.
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65
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