the curious case of the cane toad (rhinella marina): an

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

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.

Texas Tech University, Amanda Arner, August 2012

iii

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.


iv

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


v

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


vi

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).


vii

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.


viii

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


ix

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


1

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


2

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.


3

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


4

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


5

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


6

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


7

novel environments will further elucidate possible connections between learning and

invasive potential, particularly related to exploratory behavior and foraging success.


8

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


9

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


10

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.


11

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


12

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


13

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


14

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


15

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


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


17

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.


18

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


19

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.


20

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).


21

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.


22

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


23

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


24

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,


25

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.


26

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.


27

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


28

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.


29

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.


30

Figure 2.3 Total Path Length, Time Spent in Margin, and Number of Escape Attempts


31

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.


32

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.


33

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.


34

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


35

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


36

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,


37

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


38

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


39

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


40

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


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


42

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


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.


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


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


46

native range. Again, this could be an artifact of being in captivity, which would need

further testing to be conclusive.


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.


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.


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.


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.


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.


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.


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.


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


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


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


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.


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


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.


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


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


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.


62

Escape Attempts (ESC)

Trial1 2 3 4 5

# of

esc

ape

atte

mpt

s

0

10

20

30

40

50


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.


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


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.


65

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