TACTILE DISCRIMINATION IN THREE

SPECIES OF GARTER SNAKE,

(THAMNOPHIS)

The members of the Committee approve the doctoral dissertation of Vicki Lynne Keathley

Roger L. Mellgren Supervising Professor

Veme C. Cox

Raymond L. Jackson

Martha A. Mann

Yuan B. Peng

Dean of the Graduate School

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TACTILE DISCRIMINATION IN THREE

SPECIES OF GARTER SNAKE,

(THAMNOPHIS)

by

VICKI LYNNE KEATHLEY

Presented to the Faculty of tiie Graduate School of

The University of Texas at Arlington in Partial Fulfillment

of the Requirements

for the Degree of

DOCTOR OF PHILOSOPHY

THE UNIVERSITY OF TEXAS AT ARLINGTON

MAY 2004

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Copyright ©by Vicki Lynne Keathley 2004

All RightsReserved

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGEMENTS

This research was conducted under the supervision of Roger L. Meligren. I

appreciate Ms instruction and guidance throughout tMs very long process. I also very

much appreciate the other members of my cominlttee, Veme C. Cox, Raymond L.

Jackson, Martha M. Mann and Yuan B, Peng for their time, patience and many

contributions to tMs project

I would also like to acknowledge the technical help and si^port provided by

Golden Strader. Thank you for your many rescues.

I could not have acMeved tMs goal without the constant and unwavering support

of my family and friends. Not once did they ever question my motivation or resolve, or

tempt me to do something else when I needed to work. appreciateI their understanding

and forgiveness for the times I was mentally and/or physically checked out Thank you

to my parents, Beverly and Darrell Keathley,my sister, Alane Mulleo, and my son.

Hunter M. Davis, who wasespecially understanding.

My dear husband, Rodney A. Carver, colleague, friend and life companion has

trulybeen a life saver over these last few intense years. On tough days, he was always

there to encourage and restore my confidence and on gooddays ready to celebrate my

accomplishments. Priceless.

Aprill2,2004

IV

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABSTRACT

TACTILE DISCRIMINATION IN THREE

SPECIES OF GARTER SNAKE,

{THAMNOPHIS)

Publication No.

Vicki Lynne Keathley, Ph.D.

The University o f Texas at Arlington, 2004

Supervising Professor; Roger L. Mellgren

Tactile perception and the utilization of tactile information has been little

studied in snakes. Three questions were addressed in this study. 1) When given the

choice, would subjects choose one type of tactily differentiated substrate over another?

2) If tactile sensations are a factor in the discrimination of a preferred substrate, would

compromising the cephalicmechanorecqjtors disrupt that discrimination? 3) Could

snakes demonstrate acquisition of a behavior by utilizing tactile information from the

environment? Three species of gartersnake served as subjects, JhammpMs: marcimms,

radix and sirtalis. In Phase 1 of Experiment 1, each half of a rectangular box was

covered with one o f three substrates: large, medium or small irregularly shaped black

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. rocks. Results revealed that all subjects preferred large over medium and medium over

small rocks. Phase 2 was identical to Phase 1, except the cephalic mechanoreceptora

were covered with plastic wrap. In each substrate combination, less time was spent on

the preferred side when the mechanoreceptors were covered (Phase 2)tiran when they

were not covered (Phase 1). Experiment 1 supports the hypothesis that compromising

the cephalic mechanoreceptors interferes with the ability to discriminate a preferred

side. In Experiment 2, subjects were trained in a Y-maze with tactily different substrates

(large vs. medium rocks) in each arm for a food reward. Medium size rocks were the

only cue to the correct arm (going against their preferred substrate as determined in

Experiment 1). After meeting a criterion of 70% correct in 20 trials, probe trials were

conducted in the dark. Five of eight subjects reached criterion: all witihin 65 trials or

less. Subjects as a group, performed significantly better in the last twenty trials. Three

of the four subjects on which probes were run met or exceeded the 70% criterion. Five

of the eight showed a reduction in running time across trials. There were no species

differences. Evidence iftom this study suggests tactile cues from the substrate may be

acquired and used by snakes to navigate the environment.

VI

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS

ACKNOWLEDGEMENTS ...... iv

ABSTRACT ...... v

LIST OF ILLUSTRATIONS ...... ix

LIST OF TABLES ...... xi

Ch^ter

1. INTRODUCTION AND BACKGROUND ...... 1

1.1 Mechanoreceptors ...... 12

2. EXPERIMENT 1—SUBSTRATE PREFERENCE ...... 27

2.1 Phases ofExjwriment 1 ...... 29

2.1.1 Phase 1 ..... 29

2.1.2 Phase 2 ..... 29

2.1.3 Phase 3 ...... 30

2.1.4 Phase 4 ...... 31

2.2 Method of lavestigatioe ...... 31

2.2.1 Subjects...... 31

■ 2.2.2 Appratus ...... 32

2.2.3 Procedure ...... 32

2.3 Results—^Experimeat 1...... 35

2.3.1 Time on preferred side...... 35

vii

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.3.2 Time on preferred side under cover, no-cover conditions...... 36

2.4 Discussion—Experiment 1 ...... 40

2.4.1 Substrate preference...... 40

2.4.2 Effect o f the cover variable .... 44

3. EXPERIMENT 2—TACTE.E DISCRIMINATION IN A Y-MAZE...... 46

3.1 Method of Investigation ...... 50

3.1.1 Subjects...... 50

3.1.2 Apparatus ...... 51

3.1.3 Procedure ...... 51

3.2 Results—^Experiment 2 ...... 55

3.2.1 drorce...... a...... 55

3.2.2 Running time...... 63

3.3 Discussion—^Experiment 2 ..... 67

3.3.1 ^^rm c l s o r c e 67

3.3.2 Running time...... 72

4. SUMMARY AND CONCLUSIONS...... 78

...... a...... 8^1

BIOGRAPHICAL INFORMATION., ...... 99

V lll

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF ILLUSTRATIONS

Figure Page

1.1 Papilla and sunrounding tissues of a mechanoreceptor ...... 14

1.2 Stippled areas depict the distribution of mechanoreceptors on the head o f a typical coluhrid snake ...... 15

2.1 Head scales of atypical garter snake ...... 30

2.2 Shadowed areas depict the qjproximate scale area covered by the plastic wrap ...... 34

2.3 Total mean time spent on large and medium rocks in the no-cover and cover conditions ...... 37

2.4 Total mean time spent on large and small rocks in the no-cover and cover conditions ...... 38

2.5 Total mean time spent on medium and small rocks in the no-cover and cover conditions ...... 38

2.6 Total mean time spent on preferred and non-preferred substrates (cover vs. co-cover) ...... 40

3.1 Y-maze as it would appear if left was the correct side (medium rocks)...... 47

3.2 Cumulative successes for subject M l...... 59

3.3 Cumulative successes for subject M 2 ...... 60

3.4 Cumulative successes for subject M 3 ...... 60

3.5 Cumulative successes for subject R 1 ...... 61

3.6 Cumulative successes for subject R 2 ...... 61

3.7 Cumulative successes for subject M 4 ...... 62

IX

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3.8 Cmmilative successes for subject MS ...... 62

3.9 Cumulative successes for subject SL...... 63

3.10 Correct and incorrect running times (RT) for those that made criterion and those that did not ...... 66

X

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF TABLES

Table Page

2.1 Substrate preference data for each species...... 36

2.2 Total time (sec.) and percent spent on preferred side for each of the four phases...... 36

3.1 Comparisons between first twenty and last twenty trials...... 56

3.2 Subject performance across all trials ...... 58

3.3 RT summary statistics for the two trial blocks...... 64

3.4 Post hoc RT comparisons...... 66

XI

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 1

INTRODUCTION AND BACKGROUND

Herpeton, root of the word, herpetology, meanta creeping thing to the early

Greeks. The creeping, serpentine body plan, “a head, a tail, and no legs”, not only

facilitates, but requires the skin of snakes to be in close physical contact with the

environment at all times. Taction, then, might be expected to play an important roll in

the interpretation of the environment for these animals. Unfortunately, studies on the

ftinctional and behavioral aspects o f ophidian taction are not abundantly represented in

the literature.

One m i^ t assume that limblessness would severely limit the lifestyle and

locomotive options available to makes. This does not seem to be die case, however. For

instance, backwrd slanting teetih. and expandable jaws are morphologicaladaptations

which facilitate swallowing by allowing the animal to, in a sense, walk over their prey

(Cundall, 1987). They are not handicapped by a lack of the appendages with which

other animals would bring food to the mouth. A brief survey o f serpentine lifestyles

fimher illustrates this point

In addition to the four standard modes o f terrestrial locomotion:rectilmear,

concertina, sidewinding, and lateral undulation (Edwards, 1985;Bellaire, 1970), a few

more exotic methods have emerged. Fossorialtypes are often tunnelers which possess

1

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. thickened, blunt heads and a spine tipped tail for push off (Caiamaria) or they may be

sand swimmers (Eryx) witib nostrils that opai and close as needed (Greene, 1997).

Many snakes are aquatic and thus swimmers and divers. Those that are exclusively

aquatic may have special adaptations such as die bilaterally flattened tails of seasnakes

(Dunson, 1975) or die dorsaily placed nostrils and eyes of anacondas(Emectes), file

snakes (Aerochordm) and others (Pove! & Van Der Koolj, 1997). Most arboreal groups

merely climb and possess prehensile tails to cmise amor^ the trees, but, one unusual

genus {Chrysopeied) utilizes a method of jumping and gliding (facilitated by mi

ad^tation of the ventral scales) to move about in the forest canopy (Heyer &

Pongsapipatana 1970; Bellairs, 1970). Each of these varied species possesstactile

sensation, yet little is known about the effects of taction on thenmwelt of this class of

vertebrates.

Snakes incorporate several foraging and predation tactics. Themajority of

species engage in active hunting, stalking, or ambushing prey. Representatives of these

feeding strategies can be found in all o f the afore mentioned habitats. In addition, there

are some specialists. Patch-nosed snakes (Salvadord) and hognosed snakes(Heterodon)

both possess rostral scales modified for diggingup food items; lizard eggs and toads,

respectively (Greene, 197; Platt, 1969). One unusual serpent from P o t(Bothriopsis

bilimata), lures prey with the end o f the tail, which is of contrasting color to the rest o f

the body. This caudal luring isfound in the adults o f several other species, as well the

neonates o f some. (Henderson, 1970; Murphy, Carpenter & Gillingham, 1978; Sazima

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. & Puorto, 1993). These many examples make it easy to safely conclude that snakes Just

as other vertebrates, occupy many ecological niches and exhibit many lifestyles.

In addition to the challenges of moving about tofind food and water in these

diverse habitats, suitable cover, thermoregulation sites, hibernation locales, escape

options, and mates must be located—someperhaps several times a day

(thermoregulation sites) and others only once a year (mates). Therefore,whether finding

food or escaping a predator, snakes must be able to recognize thecomponents of a

particular environment in order to orient precisely and direct appropriate behaviors

within that environment. This would include their macro as well as micro habitat For

example, in some species, locating hibemacula may require individuals to migrate many

miles fromtheir summer ranges, and then subsequently return (Gregory & Stuart,

1975). Amazingly, Gregory(1974) also reportsred-sided garter snakes{Thamnophis

sirtalis parietalis) return to the same den site year after year. Snakes are also enable o f

relocating feeding areas. Theyhave been reported to return to points within dieir home

range vdiere food was abundant before they were displaced 500 meters by

experimenters (Weatherhead & Robertson, 1990).

In recent years, the objective for many researchers has been to establish the

proximate mechanians by which these limbless and little studied vertebrates

accomplish such feats. Research has centered around establishing the environmental

cues to which these creatures respond, and the relative importance of these cues to the

performance o f various behaviors.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Although the majority of snakes possess sensory systems that allow for vision,

olfaction, chemoreception, thermoreception, and taction (which Includes vibration),

most o f the attention has been on vision, olfaction and chemoreception.Ford and

Burghardt (1993) consider vision, chemoreception and taction the most important

sensorymodalities.

Studies on seasonal migrations,aggregation, foraging and others serve to

illustrate the approachtaken in most investigations as well as the dominance of vision,

olfaction and chemoreception as factors in ophidian behavioral research. Taction, which

has received the least amount of attention and is the subject of the present study will be

discussed following an overview of the other three senses. As one will note, the roles of

vision, olfactionand chemoreception often overlap. Taction has not been controlled for,

and has not been considered a part of the orientation equation.

As mentioned, the fidelity shown by some snakes in returning to densites is

remarkable. Using radio-telemetry, Brown and Parker (1976) found that ninety percent

o f the monitored snakes {Coluber constrictor mormon) in their field study returned not

only toa den complex, but to specific areas within a den complex, many o fthem

traversing the same path each year. They suggestedthe snakes may have utilized solar

cues as tiiis had been shown in rattlesnakes (Landreth, 1973) for basic direction and

landmarks and/or olfaction for specific areas. More recently, Lawson and Secoy (1991)

have found evidence for use of solar cues in navigation by the plains garter snake

{Thamnophis radix). When displaced, subjects clearly oriented toward their home range

in the absence o f terrestrial landmarks. This lends some support for Landreth’s

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. contmtioiL la this situation vision was important, but only for the directional cues

provided by polarized light not by local landmark cues. It was not clear how diey found

specific sites.

LeMaster and colleagues (LeMaster, Moore, & Mason, 2001) hypothesized that

red-sided garter snakes utilized pheromone cues (via chemoreception)to locate

potential mates and winter hibemacula. In an experiment conducted with actively

migrating snakes, they tested the response of snakes to pheromonetrails (laid down by

the experimenters on the natural substrate) under two conditions: the spring mating

season and the autumn migration. They found evidence for pheromone trail-following

only in males exposed to female trails during the breeding season. Femalesdid not

respond to pheiomones fix>m malesor other females.No snakes responded to

idieromones during theautumn migrationperiod. This indicates the chemosensory sense

in this species had little influence on the ability to find distant hibemacula and further,

chemoreceptionmay only be utilized under very specific circumstances, such as during

the breeding season. This does not rule out the possibility of visual, chemical, ortactile

cues being exploited inthe pursuit of specific den sites (micro habitats) once the general

area has been located. To muddy the water fijilher, Aere is evidence that at least two

species o f lizards have visual acuitywithin the ultraviolet range o fthe light spectrum.

One o f them, the desert iguana,Dipsosaurus dorsalis, appears to locate pheromone

trails, which absorb ultraviolet light waves, o f potential mates in this manner (Alberts,

1989; Fleishman, Loew, & Leal, 1993) rather than throughchemoreception.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Research on the significance of the chemorecejptive sense in snakes has received

the most attention, but it is not always consistent. In a laboratory study on the

aggregative behavior o f eastern garter snakes(Thamnophis sirtalis), Heller and Halpren

(1982) tested the ability o f snakes to return to a preferred shelter that retained

previouslydeposited chemical cues of the subjects. Snakes which had received

vomeronasal nerve lesions (no chemoreceptive ability) were unable to locate the site

when tested individually. However, when tested with a group of snakes, they were able

to find the shelter. It is suggested lesionedmakes were able to utilize sensory modalities

other than chemoreception, such as the sight or touch of the other snakes to findthe

preferred shelter. However, it appears that a functioningvomeronasal system was

essential if conspecificcues (visual or tactile) were not available. A second experiment

demonstrated it was not necessary that an aggregationevent be proceeded by cues

deposited fiom a priorevent. Snakes were able to congregate in a novel, clean

environment after their vision had been occluded or following olfactory nerve cuts; and

contrary to the first experiment, to some degree, after the vomeronasal ducts o f the

snakes had been sutured shut (which inhibited chemoreception). Later, avomic snakes

(made so by vomeronasal nerve cuts, which is a more efficient method than duct suture)

tested within the natural setting of their home range,were found to move as frequently

and returned to previously used sites as often as sham operated subjects.(Graves,

Halpem, & Gillingham, 1993). Home range use and aggregation behavior would both

be considered movements within a micro habitat Taken together,diese experiments

imply that chemoreception is not an obligatory component of activity within the micro

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. habitat o f some snakes and that visual and/or tajctile cues may suffice under some

conditions.

On the contrary, chemical signals do appear to be critical for certain other

reptilian behaviors. One example is the feeding and foraging behavior of rattlesnakes

(Chiszar, Lee, Smith, & Radcliff, 1992). Pitvipers and some boids are unique in that

they possess facial pits, which at short distances can detect infrared heat sources. This

special sense is important in the detection of warm blooded prey. Rattlesnakes sit and

wait—often alongrodent trails—to ambush prey that come within detection range,and

thus striking distance. Once struck, they release the envenomated rodent, which may

move several meters fromthe snake before it succumbs to the venom. The rattlesnake

then locates the prey by following the prey’s chemical trail via chemoreception. Prey

trails are only followed subsequent to a strike; this is referred to as strike induced

chemosensory searching (SICS). It is well establishedthat chemoreception plays a vital

role inM s poststrike phase of the predatory sequence. (For a complete review o f this

phenomenon see Chiszar & Scudder, 1980; Chiszar, Radcliff, Scudder & DuvaU,1983.)

Haverly and Kardong (1996) have since analyzed the degree to which visual,

chemosensory, and infrared cues areutilized in the prestiike and strike phase in flie

rattlesnake, Crotalus viridisoreganm. Visual and infrared cues were found to play

dominant and equal roles during the prestiike and strike phases. Performance did not

change significantly if one or the other was occluded. However,if bofri sense modalities

were occluded, performance was diminished, indicating that chemoreception could not

compensate fbr loss o f the other two senses during the prestrike and strike phases.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Hence, chemoreception has a very specific fijnction in the feeding behaviors of these

snakes, that is, it is critical only in the poststrike phase of the feeding sequence.

Interestingly, the banded rock rattlesnake (Crotalus lepidm Mauberi) is much less adept

at poststrike prey trailing than the many representatives of the viridis species. This may

be due to a difference in the prey normally taken by the two snakes. The banded rock

rattlesnake specializes on lizards, which are not released when envenomated so there is

little need for prey trailing. It is not known whether this distinction is a product o f

ontogeny or genetics. Regardless, the animal’s environment and the availability o f prey

could have shaped both the acuity and behavioral use of chemoreception. (Chiszar,

Radcliff, & Feiler, 1986).

Chemoreception is important to many other species for prey recognitionand

attack (Burghardt & Pruitt, 1975), prey location (Gillin^iam, Rowe & Weins, 1990)

and predator detection (Bogert, 1941; Miller&, Outzke, 1999). There is also ample

evidence that courting and reproductive behaviors are dependent on chemoreception as

well (Kubie, Vagvolgyi & Halpem, 1978; Garstka, Camazine & Crews, 1982; Andren,

1982).

Ol&ction is often closely associated with chemoreception, but the two systems

connect to the brain along separate pathways.Input fiom the olfactorymucosa (airborne

odorants) terminates in the main olfactorybulb, while input fiom the vomeronasal

organ (non-volatile chemicals fiom the substrate) terminates in the accessory olfactory

bulb. (For a complete review of chemical senses in reptiles see Halpem, 1992.) The

volatile nature of air borne odorants suggests that alarger environmental area might be

8

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. affected by these cues than by the vomeronasal cues which are restricted to the

substrate. As a result, olfactionmay be more important for general orientation and

attention. Althoughdirect evidence has not been established, Cowles and Phelan (1958)

put forth this very suggestionearly on. Odor chemicals transferred through water

(Teather, 1991) would be regarded the same as airbome odor chemicals (Halpem,

Halpem, Erichsen, & Borghjid, 1997). Unlike chemoreception, olfaction has not been

shown to play an essential role in specific behaviors. For example, it is not critical for

courtship in garter snakes, where,as mentioned, chemoreception is critical (Kubie et.

al., 1982). There is, however, some evidence for olfactory mediated responsesto

predator odor (Weldon, 1982) and conspecific alarm pheromones (Graves & Duvall,

1988). Since both of these stimuli would often occur vdien the anuml was in an

inattentive or resting state, thenotion of olfaction serving to alert ftte animal to specific

types of global environmental change is an inviting one.

Although snakes are not genially thought of as possessing keen eyesight, a

number o f studies and observations indicate vision is an importantseme modality ina

number o f species. Modem snakes beganappearing during the Cretaceous period,

which ended some 65 million years ago. Scholars believe burrowing ancestors, with

much reduced eyes, existed as far back as 90 million years (Bellairs, 1970). This is

important because the subsequent evolutionary process seems to have been directed by

Ae need for, (and to have produced), a respectable pair o f eyes. In terms o f anatomical

sophistication, then, one would expect reasonable visual ability to be the result. As

snakes evolved froma subterranean to a terrestrial existence, the abridged ophidian eye

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. was, according to Underwood, (1970) reinvented, A lthou^ this theory is not without

controversy, (see Coats & Ruta, 2000) snA:es do possess eyes which are unique among

the reptiles (Reperant, Rio, Ward, Hergueta, Miceli& Lemire, 1992). They do not

possess movable eyelids, for instance. Instead, die eyes are covered by a clear scale

which protectsthe eye fromevery possible environmental incursion. There are also

species specific dissimilarities. For example, the retinas of predominately nocturnal

species contain only rod-like photoreceptors in contrast to some diumal species which

possess only cones (Sillman, Govardovskii, Rohlich, Southard, & Loew, 1997).

Nocturnal species are also more likely to have elliptical, rather than round pupils (Pope,

1955). Additionally, lens accommodation is a specialty in the snakes. The lens is not

deformed as in othervertebrates, but instead, throu^ muscle contractions,is pushed

forward to achieve greater focus (Ford & Burghardt,1993). Some aquatic snakes also

have the ability to contract the pupil for enhanced acuity underwato (Schaeffel & de

Queiroz, 1990).

Behaviorally, vision has been shown to play a role in fo rcin gactivities in both

experienced and ludve snakes (Drummond, 1985). The garter snake, (Thamnophis

radix) when exposed to live sunfish swimming in a sealed Plexiglas container, exhibits

elevated rates of tongue flicking, a clear predatory response. Garcia and Drummond

(1995) demonstrated that Thamnophis melanogaster, an aquatic species, respond to

models ofprey which are consistent in size with natural prey. These studies indicate

that visual cues (even inanimate ones) are sufficient to initiate a predatory response in

the absence of chemosensorycues. Teather (1991) found discordant results when he

10

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. weighed olfaction against vision in a study designed to test the relative Importance of

each. Snakes foraged longer and more often when both cues were available compared to

either alone, and chemical cues were more powerful elicitors than visual cues.

However, vision may have been a less important cue only because of the iqwcific

situation. In a separate experiment, vision was more important for snakes foraging on

fish in a bowl with a contrasting background (white) than with a cryptic background.

Similarly, brown tree snakes, Boiga irregularis will ignore a clear container (not even

tongueflicking) if they can see that it holds no prey, even though the container is awash

in prey odor. Theybehave in quite the opposite manner if the containers are not

transparent. If theycannot visually assess the presence or absence o f prey they will

inspect and tongue flick an enclosurewhich contains chemical prey cues. The principal

point being the importance of the experimental situationwhen trying to determine

which cues are dominant. (For additional commentary on the brown tree snake problem

onGuam, see Chiszar, 1990).

It has been suggestedthat visual cues are a principal component o f snake

antipredator behavior. Neonatal snakes respond more defensively to larger stimuli, such

as human hands and predator models as compared to cottonswabs and finger models

(Herzog, Bowers, & Burghardt, 1989).Young snakes also reacted more defensively (in

order) to realistic artificial glass eyes, round black spots, blackbars and no eyespots

(Bern & Herzog, 1994). Adult snakes have been shown to exhibit stable, ascending

defensive behaviors in response to escalating threats, as well (Bowers, Bledsoe, &

Burghardt, 1993). To date, similar experiments using animals with occluded vision have

11

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ttot been reported. Thus, the ftmdamental importance of vision in predator detection has

not been adequately demonstrated (extraneous cues such as vibratioiis could have an

effect), nor has it been established for general survival. A case in point is a population

of island tiger snakes (Notechis scutatus) on Camac Island near Western Austrailia.

These snakes would not naturally occur there. They are descendents o f eighty snakes

released on the island 70 years earlier. A significant number of the animals captured in a

field study were either blind orhalf blind, apparentlyas the result of nest defense by

silver gulls. Despite their condition, there was no difference in body condition, grovrth

rate, survival rate or reproductive rate compared to snakes on the island with normal

vision (Bonnet, Bradshaw, Shine, & Pearson, 1999). Alfiiough this implies vision is not

necessary for survival it must be noted that there were no natural predators on the

island. All that can be concluded is that vision was not necessary for feeding and mating

to occur in this instance. In the balance, visual cues do appear tobe important for

survival in some species.

1.1 Mechanoreceptors

As mentioned, functional and behavioral studies on reptilian taction have not

received critical attention. This is unfortunate because the physiological aspect of

taction, that is, the fwrceptionsderived fiom the transduction of mechanical energy into

electeophysical energy v ia , mechanoreceptorsin the skin of snakes, suggest tactility

may play a significant role.

The structure of the snake epidermis has been clearlydescribed by Maderson

(1965). Although tibe scales appear to be separate from one another, in reality, just as

12

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. human skin is unintermpted, all layers of the dermis and epidermis (which includes the

outer surface of the scale) are contkuous from scale to scale. This phenomenon occurs

because there is a difference in proportion o f two of the outer layers in specific areas of

the skin. The inner most layer of the epidermis, the stratum germinativum, is a

proliferation ofcells which produce the new upper layers which are subsequently

exposed with the periodic sloughing o f the skin. The outer surface o f the scale (stratum

comeum) is composed o f two layers of homy, keratinized, material; the beta layer and

the alpha layer. The P-layer makes up the outer surface of the scale and is more

keratinized which makes it considerably thicker and harder than the d-layer. The d-layer

is thinner and more pliable. It predominates on the under surface ofthe scale and

between the scales, especially along the hingearea. Both of these layers (pardcuiariy

the p-layer)are reduced in areas which overlie tactile sensory structures. These tactile

sensory structures have been referred to as: touch corpuscles, tactile sense organs, skin

sense organs, touch papillae, sensory pustules, and tubercles (Jackson, 1977). Jackson’s

choice, touch corpuscles, will be applied here.

Touch corpuscles appear as tiny elevated structures on the scale surface which

are surrounded by a craterlike structure (Jackson & Doetsch, 1977a). Utilizing scanning

electron microscopy (SEM) Jackson and Sharawy (1980) found these structures on the

scales o f the Texas rat snake (Elcphe obsoleta lindheimeri) which were 16-25

micrometers in diameter. The central bump (or peg as they called it) was 4-5

micrometers high. In allinstances, the pegs extended just above the rim o f the crater

(Figure 1.1). Thisspecialized structure appears as a minute bump when viewed under a

13

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. dissecting microscofje. Some species possess a bristle like structure surrounded by a

crater (Povel & Van Der Kooij, 1997).

Central Peg ’Rim

Beta Keratin Alpha Keratin

Central C e l Basal Epiderm al CeU

Dermis Nerve Fibers

Figure 1.1 P^illa and surrounding tissues of a mechanoreceptor.

Numbering in die hundreds on thecephalic scales (Figure 1.2), each touch

corpuscle is formed by a dermal papilla rising anduplifting the keratinized layers.

Basically, these corpuscles are associated one to one with an underlying papilla. Nerve

fibers fromthe dermal layerenter at the base o f the papilla and terminate in the area just

beneath Ihe peg. The nerve ends are separated firomthe alpha kaatlii layer by a thin

crown of epidermal cells.

14

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. m

Figure 1.2 Stippled areas depict the distribution of mechanoreceptors on the head o f a typical coiubrid snake.

Electrophysiological studies performed by Jackson and Doetsch (1977a)

confirmed that these touch corpiiscles were specialized, rapidly ad^ting

mechanoreceptors. Ihey tested the sensitivity of 146 maxillary nerve fil^re dispersed

among thesupralabial scales of the Texas rat snake (Elaphe obsoletalindheimeri).

Cephalic scales contain the greatest number o f corpuscles, particularly in the rostral and

chin area, and the scales along the margin of the lips (see Figure 2.1 for illustration).

Individual corpuscles and the skin around the corpuscles were stimiilated with cotton

swabs, wooden dowels and stainless steel probes; a tuning fork was used to test

responses to a vibratory stimulus. Their significant findings are as follows. The majority

(86%) of fibers responded to mechanical stimulation o f the skin surrounding the

corpuscles. That is, most o f the nerve fibers were affiliated with receptors in the

epidermis surrounding the touch corpuscles. DOring & Miller (1979) subsequently

grouped this majority into receptors with and without Schwann cell specialization,

complex unencapsulated receptors with varying terminal endings (fiee, branched

lanceolate, or branched coiled) and, though rare,Merkel cells which are very similar to

15

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. those in manunals, fish and birds (lamellated receptors were also similar to those of

birds and mammals).

The remaining fourteen percent of nerve fibers responded exclusively to

mechanical stimulation o f the corpuscle unit, specifically, lateral displacement of the

peg extending above the crater. These rapidly adapting units were most sensitive to

mechanical stimiili moving rapidly across the skin surface. A discharge was elicited

with the initial movement of the peg (“on” response) and again when the peg returned

to the normal position (“off’ response). Corpuscle units were also sensitive to 256 Hz

vibratory stimuli, firing approximately 26 times in a 0,1 second interval. No res{»nse

occurred when static, sustained pressure was placed on the peg or rim of the crater.

Although it was necessary to individually stimulate each corpuscle in order to

determine which neurons enervated which corpuscles, the greatest discharge was seen

when all of the corpuscles (ei^ t on average) associated with a nerve fiber were

simultaneously stimulated. A corpuscle group(those associated witha single neuron)

acted as a single unit withthe receptive field (RJF) o f the neuron confined to the

perimeter of the group. No RF’s were found to be largerthan 1 mm^. This, of course,

confines all RF’s to one scale. The study also revealed an input of about six neurons per

mm^. This is supported by Jackson and Sharawy, (1980) which foundan averse of

44.8 corpuscles per mm^ on the rostral scales (the most anterior scale o f die head) of

eight Texas rat snakes{Elaphe obsolete llindheimeri). Presumably, spatial summation

of the evoked discharges and detailed information from the very small RF’s would

provide additional tactile information(beyond that o f the non specialized units)

16

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. coaceming the nature and location of stimuli contactiog the skin. If many corpuscles

units were stimulated simultaneously, along the oral mucosa, for instance, summation

could potentially have a particularly significant impact on feeding activity. Or, as

Jackson and Sharawy (1980) have suggested, the entire cephalic region may function as

a probing apparatus (much like rat vibrissae) for directly transducing somatosensory

infoimation from the environment. The symmetric distribution of the corpuscles suggest

this possibility as well.

Although both types of receptors (corpuscle and non specialized units in the skin

surrounding the corpuscles) are sensitive to mechanical stimulation, the corpuscles are

morphologicallyunique (encapsulated), and spatiallyunique (occur only on the head)

and thus may serve a different frinction tiian the surrounding skin receptors. In a

separate report, Jackson and Doetsch (1977b) identified the proportions of the more

ubiquitous, non specialized nerve endings as: r^idly adapting (68%), slowly adapting

(26%), and intermediately adapting (6%) units. Similar to other vertebrates, rapidly

adapting (RA) units had the highest response thresholds and fired only during the active

phase of skin displacement (both vertical and parallel movement). In addition, most of

the tested fibers (72%) reliably fired at the on set o f each cycle of a 256-Hz vibratory

stimulus (about 16 action potentials per 0.1 seconds). Slowlyadapting (SA) units

responded to both dynamic and sustained deformation orpressure on the skin. At the

initial stimulation of a sustained stimulus, an eruption of AP spikes was observed

followed by responses that steadily decreased over a period of about 1 2 seconds

(although theresponses ranged from 3 to 300 seconds).In contrast to RA units, SA

17

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. units were not as sensitive to vibratory stiiiiuli (256 Hz). They consistentiy fired just

31% of the time. In short, RA units dischargeonly to dynamic skin distortion or contact

and respond reliably to high frequency vibratory stimuli. In contrast, action potentials

are elicited firom SA units during both dynamic and static contact and were responsive

to vibratory stimuli only one-third o f the time. The receptive fields (RF) o f each neuron

were much larger than those found in the encapsulated corpuscles, and were not

necessarily confined within the margin of a scale as in the encapsulated units.

Approximately 70% spanned more than one scale. RA units had the smallest RF’s

(mean =12 mm^) and were concentrated on the most anteriorsupralabial scales.

Conversely, the RF’s of SA units were larger (mean =18.3 mm^) and increased in

number on the posterior supralabials.

As an example, data from Jackson and Doertsch (1977b) show a first upper

labial scale (which is just posterior to the rostral scale) whichcontained theR Fof one

RA nerve fiber (the RF was small and confined to one scale) whereas theR Fof a SA

fiber spanned 4 scales. The authors suggest that there is little disparity between these

findings and the trigeminal receptive fields o f mammals. Proske (1969b) came to a

similar conclusion concerning thevibration sensitivity of reptiles and mammals. Thus,

tactile sensitivity between tibese two vertebrate classes may serve similar functions.

Gaither and Stein (1979) have shown that the visual and somatic receptive fields of the

green iguana{Iguana iguana)are quite similar to mammals and are processed and

represented in homologous brain structures. Furtihermore, the somatotopic

18

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. representations in the brain are exaggerated for the face and upper limbs and the

receptive fields in these areas are the smMlest of the body.

Jackson and Doetsch (1977b) point out the ratio of RA, lA, and SA units they

examined in the facial area of the rat snake were very similar to those in the dorsal and

ventral thorasic area of the Australian black snake, Pseudechis porthyriacus, (Proske,

1969a). This finding supports the previously mentioned notion of an extra sensory

function for the very different touch corpuscles of the facial region. As Proske (1969b)

fturther notes, the presence of vibrasensoiy nerve endings on the trunk and belly region

(and lack of encapsulated corpuscles) suggests a significant role in the detection of

environmental events, such as ground vibrations created by approaching predators. This

is also supported by the fact that snakes frequently cany the head raised above the

ground. It is tiberefore logical to assume tiiat vibration detection does not require the

more sophisticated enc^sulated corpuscle found on the rat snake head and that ground

vibrations may be predominately detected by nonencapsulated receptors. Cephalic

encapsulated corpuscles may serve a different fimction, whichin addition may be

species specific. In support o f this, is a report by Terashima & Liang (1994) inwhich

two types of nonencapsulated mechanoreceptors were identified in the orofacial region

of the pit viper Trimeresunisflavoviridis.; a slowly adapting touch receptor and a

rapidly adapting vibrotactile receptor which maybe analogous to the nonencapsulated

SA and RA receptors described by Jackson & Doetsch (1977b). The presence of

encrqjsulated receptors was not established. (Similar procedures and stimuli were

applied in both studies.) Species differences may account for the absence of

19

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. encapsulated corpuscles like tiiose found in the rat smke. Pit vipers are C8|>able of

radiant heat perceptton via infiared receptors located within the pit area of the head,

which is located just above the supralabial scales. This sensory ability may supersede

the need for any additional specialized nerve endings.

Mammals generally follow the same p^em s (adaptation rates, RF’s, stimulus

requirements) as those described for reptiles, however, wilts, such as the rapidly

adapting Pacinian corpuscles (which are vibration sensitive) are encapsulated

(Greenspan & Bolanowski, 1996). In reptiles, both RA and SA receptor types have been

identified in the pit viper, Trimeresurus flavoviridis (Terashima and Liang, 1994),

common boa constrictor.Boa constrictor constrictor (Bodnar, Poulos, & T ^p er, 1975),

green lizard,Lacerta viridis (Baily, 1969) and alligator.Alligator mississippiemis (

Sitninoff & Kruger, 1968). Liang, Terashima, & Zhu, (1995) have reported

tbermosensitive, thermo-mechanosensitive, and mechanical nociceptive neurons as

well.

Many animals, such as the star-nosed mole (Catania, 1999), the echidna ( Iggo,

Gregory, & Proske, 1996) and channel catfish (Lamb &Caprio, 1992; Hoagland &

Abe, 1933) extend their area o f environmental definition by utilizing mechanosensory

appendages. Arachnids rely on sensory hairs (Brownell & Farley, 1979; Krapf, 1986),

and many mammals, sea lions, (Zimmer, 2001) and rats (Quic-Robles, Valdivieso &

Guajando, 1989; Brecht,PreilowsM, & Merzenich, 1997) for instance, make use of the

vibrissae for this purpose. A few snakes possess what may be homologousstructures

(Bellairs, 1970), although the functions of these structures remain unclear. One

20

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. exception may be the water snake, Erpeton tmtaadatnm, which is festooned with an

elaborate pair of scale covered, innervated tentacles near the end of the upper snout

(usually about one-sixth the length of the head). Winokur (1977) has suggested these

tentacles may serve as vibration detectors which would augment Ae animals ambush

foraging strategy.Elongated tactile papillae have also been identified in the genera,

Rrinotyphlops, the monotypic genus,Xenotyphlops (Young & Wallach, 1998) andthe

sea snake Emydocephalus (Guinea, 1996). While the translucent and pliable structures

of Aese species are not as long as Aose ofErperon (110 um vs. 15 mm), Aeir existence

helps siqjport an argument for tactile differentiation of Ae environment, which, m boA

Aese cases, might be fiirAer enhanced wiA Aese structures.

Chin tobercles, along wiA various oAer knobs and protoberances of Ae ventral

surface, have been described in snakes as secondary sex characteristics wiA tactile

fimction. They appearto be essential m courtship and mating sequences m some species

(Blanchard, 1931; Harrison, 1933; Pisani, 1976). Noble (1937) concluded, afier

covering the chin tubercles wiA tape, that cMn ptessmg by Ae made on Ae female’s

body or head was an autostimiilating event which was a necessary componentto

successful mating. Unfortunately,Kubie et. a l., (1978) were not able to replicate Aese

findings. Gillingham (1987) postulates that chinrubbing stimulates the female, alAough

Aere is no evidence to support this work. Clearly, it is obligatory that tactile events

accompany snake mating, just as wiA mating m all vertebrateanimals. It is Aerefore

difficult to discOTi precisely vriien courtship begins, which tactile events are essential to

reproductive success, or to define clearly a proximate function foreach event.

21

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Nevertheless, before intromission aind coitus transpires, the formal sequence of

courtship events are described as the tactile-chase, mount, and tactile alignment During

these stages, several tactile acts such as chin pressing, cephalocaudal w ves, (rapid skin

contractions), caudalcephalic waves, biting, undulation, and several oAer stereotyped

behaviors may occur (Carpenter & Ferguson, 1977; Gillingham, 1974,1979,1987;

Akester, 1983). The primary role of Ae tactile sense in snakes has most often been

associated wiA Aese “tactile releasmg mechanisms” which occur during courtship

(Carpenter &, Ferguson, 1977). Evidence suggests Aese behaviors are as crucial to

successfiil mating as chemosensoiy cues. Unfortunately, taction has not been Aought of

as usefiil m any oAer situation save mating. Auxiliary fiinctions for tactility in snakes

have received inadequate consideration.

Several major pomtshave been emphasized Aus far. Snakes are Averse and

quite complex creatures. They share qualitetively Ae same sense modalities as all oAer

vertebrates, and Acti some. AlAough Ae eim tellm g for chemoreception in Aese

animals has been Ae most popular and esteblished force behind most snake research, it

is clear that Aey are capable of utilizing oAer sense modalities, especially when Ae

situation requires Aem to do so. Many oAer animals process environmental information

through tactile sensory structures. And finally, research tells us snakes teve Ae

morphological means to process tactile information as well.

The major aims of this stody were (!) to assess Ae possibility timt garter snakes

exhibit substrate preferences, (2) determine Ae extent to which tactie information can

be Asmpted by physically covaing Ae cephalic mechanoreceptors, and (3) determine

22

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. whether snakes could utilize trctile information (in the form of substrate cues). In a

foraging situation.

Phase 1 of the first experiment was performed in order to assess which types of

substrates were preferred by the subjects. Subjects were placed in a rectangular open

field where half of the field was covered with one substrate and the other half a different

substrate. Preference was indicated by the amount o f time spent on each side. In the

beginning, foursubstrates were tested, with a total of six combinations for each subject:

large black rocks, medium black rocks, small black rocks, and a black,flat surface. (The

flat, Plexiglas substrate was subsequently dropped because the subjects could not move

about on it efficiently.) If it was shown that a subject did prefer one side over the other

in each of the three combinations,(small"medium, small-large,medium-large). Phase 2

was conducted. Phase 2 was identical to Phase 1, except that the animal’s head was

wrapped with a thin piece of transparent plastic kitchen wrap. The wrap was applied In

order to compromise the mechanoreceptorso f the head, particularly those around the

mouth, the supposition being that, by placing a thin plastic barrier between the

mechanoreceptorsand the surface o f the rocks, any sensations perceived by the snake

would be muted. The animal could still see, breathe flmough thonostrils and utilize the

tongue. The hypothesis was that, if themechanoreceptors were Indeed compromised,

the animal would not be able to as easily identify the preferred side (as identified in

Phase 1) and would therefore spend significantly less time there as compared to Phase

1. Additionally, a third pjbase was run, which was a control with tiie plastic wrap

23

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. present, but not placed where it would prevent tactile reception, and a Phase 4, which

was a repeat of Phase 2.

In the second experiment, subjects were trained to choose the correct arm of a

Y-maze for a food reward. The correct arm, which was changed randomly from trial to

trial, was covered with one of the three possible sizes of black rocks—small, medium,

or large. The size of rock, in the correct arm, was always the same for each subject and

was not the preferred rock of the subject as identified in Phase. 1 of the iSrst experiment.

This was necessary in order to eliminate the possibility that the size of rock designating

the correct arm biased the choice of the subject because it was the size favored by the

subject. Thus, the incorrect arm was always covered withthe preferred rock and the

subject would have to overcome its preference for this substrate in ordbr to receive the

food reward. In order to assess whether the snakes could make die choice using taction

alone, after a subject reached criterion (70%), probe trials were ran randomly (coin flip)

in complete darkness for a total of ten trials, success was Judged by the same criterion.

Running times, percent of correct choices, number and length of runs, and the

cumulative percent of correct choices over trials were analyzed. The results of statistical

tests have, in most cases throu^out this p ^ r , been evaluated on the basis of the

traditional p < .05 criterion for statistical significance. However, because this long held

and widely revered criterion is nevertheless arbitrary, in cases where test results are in

the neighborhood of the criterion, the exact p-value is given sothat readers can evaluate

for themselves the meaningfulness and significance (or lackthereof) of the test

outcome.

24

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. This study could shed liglrt on one aspect of habitat selection in snakes. This

phenomenon—the non-random distributioE of a species within a larger available

environment—has been established in snakes (Burger & Zappalorti, 1988; Weatherfiead

& Charland, 1985). It is the hows and whys that beg further research. Suitable habitat

for a given species is usually delineated by specific physical factors such as degree of

canopy cover, soil type, flora variety, percent and nature of ground cover, ahitude,

temperature, etc. Presumably, type o f substrate would fall into this category o f physical

features. The term microhabitat describes more specific preferences, which could be

related to food, such as ambush site selection (Shine & Li-Xin, 2002) or the soil

moisture contento f terrestrial retreat sites (Elick & Sealander, 1972) for example. Some

selections are quite definite. The arboreal broad-headed snakes o fsouthern Australia

choose a specific species of tree for retreat sites, and the tree must also be dead, large

not small, and have many branches and hollows (Webb & Shine, 1997). Of course

habitat selection in snakes is a complicated subject (for reviews see Reinat, 1993;

Heatwole, 1977). Seasonal and reproductive changes, ecdysis, interspecific competition,

prey density and predation risk are all probably highly correlated with habitat choice.

The apparent preference for specific structural features, including substrate

characteristics, could be related to any of the above and could, o f course, be an artifect

of any of the above, or other relevant, variables. Basically, structural features can be

used reliably to describe and locate species specific habitats (Reinert & Zappalorti,

1988), however, as Reinert (1993) points out, whether snakes utilize structural features

as cues for locating preferredhabitate is still an open question. Thus, because the snakes

25

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. in the Y-maze study bad only substrate cues to discrimiiiate between the two arms of

the ntiuze, the quiestioii of snakes using stmctural cues to find specific places (whether

through vision or taction) is relevant to this study.

Three species ofThammphis were used in this study, each from distinct and

geographically separated areas. Very little is known about habitat use in the

Thamnophines, and virtually nothing is known about microhabitat substrate

preferences. Typical habitat descriptions are very broad, such as “degree of forestation”

As so little is known, differences in preferred substrate among the three species, was

also analyzed.

While it is often mentioned as a potential factor, with the exception of the

previously cited reproductive investigations, the ability of snakes to utilize tactile cues

has not been investigated, nor has die notion ofsubstrate preference in snakes.

Snakes, like most reptiles, are difficult subjects, but flie remarkable abilities of

these animals invites a thorough investigation of their perceptual processes and

resources. Considering the sensitivity o f the skin and given the variation in usage and

complexity of the other sense modalities, taction should be evaluated as well. The

prospect o f tactile sensations serving as cues for snakesseems quite plausible and is

therefore a perceptual resource that deserves further attention.

26

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 2

EXPERIMENT 1—SUBSTRATE PREFERENCE

The subjects were three species of young garter snakes: Thammphis marcianm,

Thammphis radix, and Thammphis sirialis. All three species are active foragers, and

therefore they can be relied upon to move about and investigate the apparatus.

Additionally, members of the genusThammphis were chosen because they have been

utilized successfiillyin several previous experiments. The three species are indigenous

to three different geographical areas of the UnitedStates. The radix occupy the central

plains area, the sirtalis, the entire eastern half ofthe country,west coast and southern

Canada, and the marcianm the western half o f Texas (ifromabout Dallas west) through

the southern halfof Arizona, down into Mexico and reaching updtrou^ die western

half of Oklahoma into southern Kansas. Although there are some areas of overlap,

basically, the sirtalis occupy the wetter zones, the radix the h i^ , drier plains, and the

marcianm the arid southern regions.

This experiment had tiiree objectives. The first was to establish die substrate

preference of each subject. The second was to provide evidence that physically covering

the mechanoreceptorson the cephalic scales could disrupt transduction of tactile cues

and the third objective was to determine whether these three distinct populations of

snakes would show differential preferences to the three types of substrates. The very

27

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. different habitats occupied by the three species suggest these species may exhibit

different preferences for substrate type. Although there was no specific theoretical

premise to this last goal, there is some literature which suggests individual species

might choosemicrohabitats based onthe push-point density o f a particular substrate

(Kelley, Arnold & Gladstone, 1997). Push points are various irregularities in the

substrate against which the snake pushes laterally to move forward. (A lack of push-

pomts is the most probable explanation forthe difficulty the snakes exhibited in trying

to move about on the flat, barren Plexiglas the fourth substrate originally planned for

this experiment.)

The experiment was conducted in fourphases. Each of the four were run in

identical rectangular Plexiglas boxes (77 cm long x51 cm wide x 12 cm high). The

sides of the clear, open box were covered with white peqper; the bottom of the box was

constructed of black Plexiglas. Subjectswere observed via an overhead web camera

linked to an adjacent PC. For all trials, each half of the field (bisected on the short axis)

was covered with one o f the three types of substrate; large, black,irregularly shaped

rocks (mean width, 31 mm.), medium, black, irregularly shaped rocks (mean width, 16

mm.), and small, black, irregularly shaped rocks (mean width, 7 mm.), Thus, there

were three possible combinations: small-medium, snail-large, medium-large. Each

subject was run once in each substrate combination for Phases 1-4. The amounto f time

the subject spent on each side during the trial was recorded as well asthe number of

times the subject crossed from side to side, the later being recorded primarily to

document that a subject clearly sampled both sides.

28

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. IWIti.iWilU

2.1.1 Phase 1

Each subject was run in each substrate condition, in the manner just described.

The purpose here was to establish substrate preferences. Preference was determined by

comparing the time each subject spent on each substrate in each o f the three substrate

conditions.

2.1.2 Phase 2

Each subject was run in each substrate condition with the head wrapped with a

piece of clear, plastic wrap fromjust behind the nostrils to just behind the eye (see

Figure 2.1). This procedure covered approximately the third through the sixth upper and

lower labial scales. The purpose here was to determine if covering a significant portion

of the snakes mechanoreceptors would result in a reduced edacity for tactile

perception. Reduced capacity was determined by comparingtime spent on preferred

substrates (established in Phase 1) while in the “no-cover” state as opposed to the

“cover” state o f Phase 2. In theory, subjects should spend less time on the preferred

substrate in the “cover” state if the transduction o f tactile information has been

diminished, there by preventing them from discriminating the preferred side.

29

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Frontal Parietal

Parletals Frontal

Eostral

Figure 2.1 Head scales of a ^ ic a i garter snake {Thammphis)

2.1.3 Phase 3

Each subject was run in each condition with a piece of cleaTj plastic wrap (same

size as Phase 2) wrapped around the snake’s head just behind the parietal scales. This

procedure did not cover a significant number of cephalic mechanoreceptors. This phase

was to act as a control for the wrapping procedure in Phase 2. Efifectiveness of the

control was determined by comparing time spent on the preferred substrates between

Phase 1 and Phase 3. There should be no significant difference between the two.

30

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2J. 4 Phase 4

This Phase was Identical to Phase 2 and was conducted to replicate the results o f

that phase.

2.2 Method o f Investigation

2.2.1 Subjects

The subjects were five 12 month old checkered garter snakes (Thamnophis

marcianm), two 9 month old plains garter snakes{Thamnophis radix), and two 12

month old eastem garter snakes{Thamnophis sirtalis). A third sirtalis completed Phase

1 but died before Phase 2 was begun. Data firom this subject were not included in the

analyses. All subjects in each species were firom a single clutch, and all were males.

They were purchased firoma private dealer.

The subjects were housed individually in commercial plastic shoe boxes, 20 cm

W X 30 cm L X 10 cm D. with air holes drilled in the sides and tops for ventilation. This

type of enclosure is standard and widely used by many commercial snake breeders. The

box substrate was covered with institutional paper toweling.Animals were normally fed

in their enclosure thrice weekly, but were not fed during testing. They were maintained

on a diet of feeder fish with a calcium/phosphorus/B i supplement added every two

weeks. Water was available ad libitum. The housing room vrasmonitored daily and kept

at an ambient temperature of 28 C. Overhead florescent ceiling fixtures were litfirom

9:00 A.M . to 4:00 P.M ., daily.

31

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.2.2 Apparatus

The testing apparatus was a 77 cm. x 51 cm. clear, Plexiglas, rectangular box,

with a black Plexiglas bottom. The walls were 31 cm. high. During testing, the box

rested on a standard black lab table (151 cm. x 78 cm.) and the outside walls were

covered with white cardboard. In addition to the florescent lighting, two incandescent

fixtures were mounted 150 cm. directly over the box. A Veo Connect web camera was

mounted, lens down, between the two light fixtures at a distance of 160 mm above the

box. The camera was connected to a PC which rested on an adjacent lab table. Subjects

were observed on tibe PC monitor via the overhead camera and camera software.

The small rocks (mean widfli: 7 mm.), medium rocks {mem width: 16 mm,),

and largerocks (mean width: 31 mm.) were all impermeable and irregular in shape.

2.2.3 Procedure

The marcianus were tested over a period o f four months (March—June of

2003). T. radix and sirtalis were tested during July and August o f2003. Trials were

conducted between 1 0 : 0 0 a.m. and 2 : 0 0 p.m., once a day for six consecutive days

(which constituted one phase).

The random order for trial substrate combinations, for each subject, was

determined (counterbalance) before tibe trials began. Random subject order was

assigned each day. Placement of substrate type (whether on the left or right side o f the

box fromthe investigators perspective) was arranged so that the subject never had the

same substrate typeon the same side on two consecutive days. For example, if Ae

subject’s assigned combination on day 1 was L-M (large-medium),the huge rocks

32

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. would be placed on the left side. If on day 2 the assigned combination was L-S (large-

small) the large rocks would be placed on the right side. Thus, side was counterbalanced

within substrate combination in each o f the four phases.

Each trial (including within-subject trials) started with a clean box and clean,

dry rocks. Bach of the two types o f rock for a given substrate combination covered one

half of tire rectangular field o f the box to a depth of approximately 3 cm. The individual

subjects were brought into the test room (kept at the same temperature as the housing

room) for the day’s trial after the rocks had been placed in the box and the camera

checked. The subjects were picked up and cupped in the left hand with the right hand

over the snake. Each was gently placed onto the very center of the box, straddling both

substrates (sides), while the investigator continued to keep the right hand positioned

over, but not touching the snake. Once the snake was on thesurface, it invariably froze

and remained so for a time. Once the investigator was out o f sight,the snake usually

began to move within about a minute. Trial time ( 20 minutes) started with the first

movement. The recording o f time spent on the left or right dso started with the first

movement. The side on which the snake’s head and upper third o f body laid determined

whether it was on the left or right side. Number of crosses from one side tothe other

were also recorded. At the conclusion o f a trial, the subject was removed to its home

box and returned to the housing room.

Test boxes were washed after each trial with warm tap water and a liquid hand

sanitizer. Rocks were washed in hot water and allowed to air dry overnight on large lab

trays.

33

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The procedure for the cover phases (Phase 2 and Phase 4) were sb follows.

Plastic wrap was cut into small 1 cm x 5 cm strips. The subject was held at the neck

with the thumb and index finger of the left hand. The plastic strip was then wrapped

around the snake’s head with the forward part of the strip starting just below the

subject’s left eyeand ending just below the subjects left eye. Thus, the subject’s eyes

and head scales were covered with two layers of wrap fk)m just behind the nostrils

(anterior side of the prefrontal scales)to just behind the eyes (posterior side of the

parietal scales, see Figure 2.2). In Phase 3 (control) the same size plasticstrip was

wrapped around the head in the same fashion except the edge o f thewrap began at the

posterior side o f the parietal scales. Therefore, the only mechanoreceptors likely to be

compromised were those few that would be found on the posterior (7® or8 *) upper

labial scales.

UiODeriUbiiiils Parietal

RoM

ILnwirJUbUt

Figure 2.2 Shadowed areas depict the approximate scale areacovered by the plastic wr^.

34

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. After the cover was placed around the snake’s head, it was put back into its

home cage for a period of two minutes, allowingthe snake to acclimate to the cover.

This also gave time to insured the cover was securely wrapped, before the trial was

started. From this point, the procedure was the same as in Phase 1. After the trial, the

cover was removed with surgical scissors and the animal was retumed to its home cage.

2.3 Results—Experiment 1

2.3.1 Time on preferred side

No differences for preferred substrate were found between species. Each subject

preferred the same substrate in each of the substrate pairs during Phase 1.Large rocks

were preferred over both of the other substrates, and the medium rocks were preferred

over the small (preference was large > medium > small). See Table 2.1.

Collapsed across species, subjects spent proportionately more time on the large

rocks than on the medium rocks, .70 vs. .30, respectively (z = 1.69,p< .05). They spent

more time on the large rocks than die small in that pair, .69 vs. .31, respectively, (z =

1.61,p < .05). And, although not statistically significant, they spent more time on the

medium rocks than the small in that pair, .61 vs. .39, respectively, (z = .93, p= .18). In

light of these findings, the rest of the analysis will presume that large rocks are

preferred over medium, and medium are preferred over small (Table 2.2).

35

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 2.1 Sabstrate preference data for each species.

S ubject Large-Medium Large- Small Medium - Small

Preferred Time* on ' Preferred Time* on Preferred Time* on Side Preferred I Side Preferred Side Preferred Ml L 870 L 742 M 825 M2 L 1067 L 957 M 614 M3 L 734 1 L 990 M 825 M4 L 771 L 720 M 771 MS L 815 L . 756 M 923 Mean 851.40 833.00 791.60 P 0.71 0.69 0.66 S1 L 637 L 790 M 724 S2 L 942 L 930 M 610 Mean 789.50 860.00 667.00 P 0.66 0.72 0.56 R1 L 779 L 895 M 638 I R2 L 923 L 720 M 624 Mean 851.00 807.50 6 3 1 J0 P 0.71 0 J 7 0.63 * numbers in seconds

Table 2.2 Total time (sec.) and percent spent on preferred side for each of the four

L*-S M*-S Total Time Percent TotalTime Percent TotalTime Percent Phase 1 7538 70% 7500 69% 6554 61% No-cover Phase 2 6026 56% 6041 56% 5665 52% Cover Phases 7138 66% 6616 61% 5956 55% No-cover Phase 4 4894 46% 6649 62% 5842 54% Cover * Indicates overall preference, numbers in seconds

2.3.2 Time on preferred side under the cover, no- cover conditions.

No significant differences in time spent on the preferred side were found

between Phases 1 (no-cover) & 3 (control/no-cover) in the large-medium pair, {M DIFF

- 44.44 sec., t% = .55, p > .05), the large-small pair(MDIFF = 98.22 sec., t» = .1.65, p

>.05) or the medium-small pair {MDIFF = 66.44 sec., % = .83, jo > .05) so the data were

36

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. collapsed across these phases, as were the data forPhases 2 & 4 (these two conditions

were identical), for the following analyses. Separate 2 x 2 ANOVAs on each o f the

substrate pairs (large vs. medium, large vs. small, and medium vs. small) and the cover

vs. no-cover condition, revealed a significant or nearly significant interaction effect in

all three conditions (F1 4 7 = 25.63, p < .05; F\,n = 3.49,/? = .07. and F1 4 7 = 3.78,/?

=.06, respectively). In the large-medium condition the effect o f rock size was

significant = 27.18, p < .05), large were preferred over medium (M = 710.97 sec.

vs. M = 491.89). There was no main effect for the cover vs. no-cover factor, (Fi,n =

.009,/?> .05). See Figure 2.3.

Large m Mledlum 900 815.33 800 ® 700 600 aOS=64- 600.28 500 383.50 400 300 no cover co\er M®«fcinn ■ -Large Figure 2.3 Total meantime spent on large and medium rocks in the no-cover and cover conditions.

In the large-small condition, the large rock substrate wasprrferred over the

small (M = 746 sec. vs. M = 459,0 sec.), Fixi - .48.34,/? < .05. Again, there was no

main effect for the cover vs. no-cover factor, F^n - .02, p > .05. See Figure 2.4.

37

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. L a n p i ¥S. S m a ll 900 . 787.22 800 ' M JQQ _ S QQQ _ Jl 5QQ _ 42 a0 0 _ _ ------^ 495.00 400 - 300 - I ' 1 no cover cover ------Sub# ------Large Figure 2.4 Totd mean time spent on large and small rocks in the no-cover and cover conditions.

In the medium vs. small combination, the medium substrate was preferred over

the small (M = 653.43 sec. vs. M- 541.28 sec.), F i,n = 9.42, je? < .05. Once again, there

was no main effect for the cover vs. no-cover fector,F\^n = .1118,/» >.05. See Figure

2.5.

Medium vs. Small

611.78 ! 511.83 570.72

nocxjiar — -Smeii! Figure 2.5 Total mean timespent on medium and small rocks in the no-cover and cover conditions.

38

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Because these results indicate clearly which rocks were preferred in each of the

substrate conditions, it was determined that the preference scores could be collapsed

across substrate conditions. Thus, a preferred/non-preferred vs. cover/no-cover

ANOVA was constructed and carried out. Preferred scores are defined as the amount of

time spent by each subject on the preferred side in each of the three substrate

conditions, non-preferred scores are simply the total minus the time spent on the

preferred side. For example, 1200 (total trial time) minus 870 (time on preferred side)

equal 330 (non-preferred score). This analysis on collapsed data corroborated the

previous one. In the no-cover condition, the difference in time allocation between

preferred and non-preferred sides was large(Af = 764.92 see. and M = 439.44 sec.,

respectively, = 25.56, p .05).< On average, 64% of time was allocated to the

preferred side. When trials were conducted in the cover condition, this difference was

dramatically reduced {M- 641.13 sec. and M = 555.33 sec., respectively) to tiie point

that just slightlymore than half the time (54%) was spent on the preferred side(Figure

2.6). Overall however, there was more time allocated to the preferred substrate (Fi,s3 =

75.29,p < .05).

39

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Preferred y s . Nonipreferred Substrate 900 764.74 800 •S 700 © 600 641.13 5.33 I 500 .,.438.44 400 300 no cover cover Preferred -Ntm-preferred Figure 2.6 Total mean time spent on preferred and non-preferred substrates (cover vs. no-cover).

Using the same collapsed data, mean times on the prefored side for the cover

condition (collapsed over Phase 2 and Plm e 4) and for the no-cover condition (Phase 1

and Phase 3 combined) were compared. The mean times on the preferred side in the

cover and no-cover conditions were significantly different from one another (MDIFF =

123.61 sec., 33.14), = 3.73,/r < .05.

An analysis of the number of crosses during cover and no-cover trials revealed

no difference between the two conditions. Mean number of crosses was 12.74 in the no­

cover condition and 12.41 in the cover(SE = 1.42, fe = .23,p = .82).

2.4 Discussion—Experiment 1

2.4.1 Substrate preference

Experiment 1 was predicated on the assumption that the subjects would establish

and maintain habitat preferences. In nature, preferences would range from the more

general,macro habitat to specific site locations which would vary according to the

animals current activity (foraging, resting, thermoregulating, etc.). It was further

40

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. assumed that, given a choice of three habitats (i.e., three substrates), one, orpossibly

two, would be preferred over the others, and that preference would be made evident by

the allocation of time spent by the subjects on those substrates. The results of this

experiment demonstrate clearly the affinitypossessed by the subjects for the large rocks

in this particular situation. The notion that time allocation wasa expression of

“preference” implies therewas cause and effect relationship between the two, i.e.

preference leads to more time spent on the substrate. However, it is possible that

substrate preference grew out of the time allocated to the substrate at the beginning of

the experiment. For instance, if the subject bolted to one side initially and stayed for a

while it might have developed a preference forthat side. The fact that all o f the subjects

exhibited the same preference argues againstthis possibility. This systematic

component lessons the possibility that preference grew out of time spent on a substrate

due to initial random choice. The crossing data also clearly show that each subject

sampled both sides and there was no difference between the subjects on this factor.

No predictions were made concerning substrate preference. These data, alone,

simply could not uphold hypotheses such as: large rocks will bepreferred because there

are potentially more hiding places. Phase l(and Phase 3) were designed primarily to

establish preference which is an essential component to the rest of Experiment 1 and

Experiment 2. Secondary to this objective was establishment of a base line from which

future studies, designed to address more direct questions based on the ecology and

natural history of particular species, could be launched. That said, speculations will be

offered, none the less.

41

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The idea that there is some structural or physical quality of the large rocks which

was attractive to the snakes is a very plausible one. Reinert (1993) and Reinert &

Zappalorti (1988) have generated considerable researcharound this possibility with

studies on habitat preference in Timber Rattlesnakes (Crotalus horridus). In one study,

150 random samples o f the habitat o f two different populations of this snake were

collected (mountain and coastal plain). Analysis revealed the habitats were different.

The mountain habitat had denser canopy cover, more fallen logs and less ground

vegetation than did the coastal plain habitat. Principal component analysis on ten

identical structural variables in the two habitats revealed that the first and second

components o f the mountain population were canopy structure and fallen logs. In the

coastal plain analysis, ground vegetation structure andfallen logs (even though there

were less of them) were the first and second cmnponents. Plainly, the two habitats

shffled two of the top three structural components, differing only inthe total amount of

each. He also noted that the structures ofthe two canopies were different in the two

habitats, but the amounts of canopy closure did not differ. It is also interesting that the

amount of ground vegetation was more important tothe coastal plain population where

there were fewer fallen logs. It looks as if some type of ground cover is a highpriority

for this ^)ecies. It would be interesting to know whether preference simply developed

for that cover which is most abundant or whether, given a choice, they would choose

one over the other (e.g., logsover vegetation). The principal point is that structure is

important to Timber Rattlesnakes, and it appears that it is also important to the snakes in

this study. The larger rocks created many more crevices and spaces for the snakes to

42

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. explore than did the medium rocks. These openings in the substrate could have been

perceived as outlets for escape or hiding places. For the rattlesnakes, thelogs,-may have

provided shelter, or perhaps rodents hiding in the logs provide a reliable food source.

The garter snakes may also have been looking for food. They are active foragers, hence,

their natural behavior is to poke and prod among leaf litter, along stream banks, etc.

searching for prey. The rattlesnakes may prefer the logs for ambush sites. Tsairi &

Bouskila (2004) found (under experimental conditions) that the desert viper, Echis

coloratus, chose ambush sites solely on the basis o f cover, to the point of ignoring sites

with prey odor but no-cover. Although this is probably less relevant for garter snakes,

when they are in situations where multiple prey are immediately present, such as frogs

or fish stranded in a puddle, moving to a strikingspot with cover would probably be

advantageous. In the case of the rattlers, the canopy,might also have provided escape

from the sun, or possibly from birds of prey. For the smallish garter snakes in this study,

die larger rocks may have provided a sense of overhead cover. They did, on occasion,

loop their bodies around and between the lowest part o f the rocks as though they were

trying to avoid overhead threats.

Another important aspect of the Reinert (1993) study centers on the innate

nature o f the preferences of the two populations o f snakes. The three species in this

study may share similar innate preferences, which would be one explanation forwhy

they all preferred the same substrate even though they have dissimilar natural habitats.

Reinert makes the point that the mountain and coastal habitats lookedvery different

froma human perspective. This observation leads one to wonder whether the natural

43

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. habitats of the three species in this study are really that diiBfeirent fiom one another from

a snake’s point of view. As for their view of the substrates in this study, the more

complex (less uniform) structure of the large rock substrate may possibly have been

perceived as providing more cover. Hence, an innate rale of thumb for garter snakes

may be to move toward the more structurally complex environment.

2.4.2 Effect of the cover variable

The evidence from Experiment 1 upholds the hypothesis that compromising the

cephalic mechanoreceptors interferes with the snakes’ ability to discriminate the

preferred substrate. Without the cover, the subjects always spent more time on the

preferred side of the pair of substrates ( large for the large-mediumpair, large for the

large-small pair and medium for the medium-smali pair.) But, when the cover was on,

the difference in amount of time spent on the two substrates was significantly smaller.

This effect was most dramatic in the large-medium pair. Without the cover, subjects

spent about twice as much time on the large side, but withthe cover an equal amount of

time was spent on each side.

If they had been constantly on the move, going around the perimeter the entire

length o f the trial, time scores for the preferred and non-preferred sides would have

been close to equal. It could be argued then, that the small difference in time spent on

the two substrates in the cover vs. no-cover conditions, was not due to the cover but

hyperactivity. The fact there were no differences in the number of crosses between the

two conditions, rules this out, as does the control condition (ifcover, se, caused

hyperactivity a difference between Phase 1 and Phase 3, would have been evident.) Had

44

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. they been trying to rub offthe cover, they would likely have stayed on the substrate that

was most likely to facilitate that endeavor, a scenario that was not played out in the

control condition (Phase 3).

It is possible the subjects were using vision as well as taction to discriminatethe

two substrates, and that the plastic wrap, though clear, interfered with their visual

perception. However, a discrimination based on something as gross as brightness, which

is probably the only discrimination the snakes could make, should not have been

affected by a clear cover. The degree of visual acuity in snakes is not known, but, even

amongthose considered to have the best acuity (diumal, arboreal, ambush predators),

which visually can detect minute marks such as the breathing movements o f prey, the

prey is lost to the bacl^round if it stops moving (Lillywhite & Henderson, 1993).

Recall that the snakes still had use of their chemosensory sense. Therefore, if

they had been usingthe tongue to discriminate thesurfeces, they should have been able

to do so in the cover condition.

It follows, then, that the mechanoreceptors along the margin o f the lips and chin,

are likely the primary source of information concerning the substrates. It seems entirely

plausible that the plastic wrap could have prevented lateral movement o f the corpuscle

pegs of these receptors, and have also created sustained static pressure on the peg and

rim of the crater, which would result in loss o fresponse, and thus, little or no

information being garneredfrom the receptor.

45

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTERS

EXPERIMENT 2—TACTILE DISCRIMINATION IN A Y-MAZE

The goalof Experiment 2 was to provide evidence that snakes can use tactile

cues, alone, to successfully traverse a Y-maze for a food reward. To this end, it was

first necessary to train them in the maze with two tactily different substrates ^ c h

they could possibly differentiate visually, and then test them later under circumstances

which eliminated vision and left only taction as a resource for interpreting the

environment. In the first stage o f this cjqperiment, subjects were trained to choose the

correct arm o f a Y-maze where the only cue indicating the correct arm was the size o f

rock covering the floor o f the runway and arms o f the maze (Figure3.1). There have

been only a handful o f Y-maze studies done with snakes, and no discriminations of

this type have been reported. Indeed, o f the six Y or T-maze studies reported by

Burghm'dt (1977), three reported dismal results for place learning (with no cues). In

two others, the results were clouded by control issues. The lone study with solid

results was one by Halpem (1988) in ’sriiich prominent visual stimuli marked the

entrances to the two arms (a bold, stripe vs. a dot pattern.). There were no prominent

visual cues in this study. The arms o f the maze were presumably more difficult to

visually discern because the substrates were the same color (black) and shiny. In fact.

46

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. the entire inside of the maze was black and shiny, so there was very little visual

contrast between the arms. Nevertheless, it was possible that differences in the

Figure 3 .1 Y-maze as it would appear if left was the correct side (medium rocks).

47

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. reflective densities o f the two rock types, as well as the stnictural differences, were

discemable visually by the snakes. However, as mentioned previously, regardless of the

cue used by the snakes, any acquisition o f the behavior would support the notion of

snakes’ ability to utilize natural, structural cues as guides to specific microhabitats and

would be evidence gladly received. But plainly, in concert with the assertion of this

study, it was presumed the two rock types were also tactily distinguishable and thus

were exploitable as cues to the environment; the result being, evidence to support this

supposition.

Data fiom Experiment 1, indicated that the entire subject pool preferred the

largerocks overall m d the medium over the small. As a result, largeand medium were

the sizes chosen as the two rock types used in the Y-maze. The reward (a fish bit) was

always located in the arm with medium (less peferred) rocks; the incorrect arm always

contained the large(preferred) rocks. Recall, this arrangement was used in order to

ensure that the snakes were not initially drawn to an arm because (for whatever

ophidian reason) they were partial to that particular rock. Arm choice (AC) and running

time (RT) were recorded. A small “well” was dug into the rocks three centimeters fiom,

and in the center of, the end of the correct arm. The fish bit was placed into the well,

which was below the level o f the rock substrate, so that it was not visible until the snake

was right above it A choice was considered made when the snakes head came within

eight centimeters o f the end o f an arm. If the wrong arm was initially chosen, the

subject was allowed to make the correction. Time did not end until the subject reached

the food goal.

48

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The correct arm—^left or right—was quasi randomly switched with each trial.

Trials for all subjects were run in the following repeating pattern for a total o f no less

than 60 trials: LRLR RLRL LRRL RLLR. This method was chosen in order to

eliminate the possibilities of long runs of one side or the other which would have been

possible had the arms been truly randomly chosen. An early long run, for an individual

subject might have created an advantage for that individual which would make it

difficult to compare to the other subjects. Had sixty coin tosses been applied, long runs

(especially in the first or last trials) could have an unpredictable effect, even if all

subjects were run identically.

The subjects fix»m Experiment 1 were used in this study. Therefore, all subjects

had equal experience with the two substrates. They were given three trials per day for at

least twenty consecutive days. Criterion was set at 70% correct in twenty consecutive

trials. This was expected to be reached at around 60 total trials. Subjects were given up

to ten extra trials in order to meet criterion. The animals were tested in the same

windowless room as inExperiment 1 and observed with the same equipment as used in

Experiment 1.

If a subject reached criterion, probe trails were begun. This meant the second or

third trial of the day (randomly chosen) was run in total darkness. There was no light

coming into the room or lightfrom any source within the room, i.e. the computer and

surge protector strip were both shut off; there were no windows, and the single door was

sealed. Prior to the probe trial, a line approximately 1 cm long and 3 mm wide of

phosphorescent paint was applied to the top, center o f thesubjects head, just posterior to

49

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. the parietal scales, and allowed to dry. Tiny dots of the paint had also been ^plied to

the top perimeter edge (not visible to the subject) of the apparatus in symmetrically

strategic places so the experimenter could track the snakes orientation and position

within the maze during the trial. The snakewas observed from above with the

experimenter standing at the base o f the runway (behind the start box) and until the

snake turned into an arm. The experimenter was then able to walk around the left or the

right of the maze to observe the behavior of the snake within the arm.

A total often probe trials were run, with criterion for AC success being 70%.

Running times, numbers of runs, and lengths of runs, were also recorded. RT across

trials and cumulative successes across trials were analyzed.

3.1 Method of Investigation

2). 1.1 Subjects

The subjects were the same as those utilized in Experiment 1, except for die

second sirtalis, which did not respond to the reward in 2 0 consecutive trials, so was

eliminated. They were maintained (housing and husbandry) in exactly the same

manner as in Experiment 1, with a singleexception. During testing, the only food

they received was that consumed during their three daily trials. The amount was

equivalent toa 1 cm long feeder fish. Young snakes can easily feed on adaily basis,

especially whenthe amount is less than they would naturally consume at a single

repast.

Although most snakes ate the rewardat each trial, they did not always do so—

and this was not froma lack o f finding the reward, they also occasionally regurgitated

50

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. the food. TMs meant, unfortunately, that it was not possible to maintain all of the

subjects at equivalent motivational levels, either individually or as a group. A record of

reward consumption was kept, and Is discussed later as a possible factor in individual

performance.

3.1.2 Apparatus

The Y-mazB was constructed of clear Plexiglas whichwas spray painted

black on the outside. The main runway was 50 cm long; theaims were each 40 cm

long.The walls were 30 cm tall, and the width of each aom, and of the runway, was

15 cm. The angle of the arms of the Y was 90°, relative to each other. The individual

pieces o f the maze construction were joined together (with a heavy duty liquid

adhesive) in a manner such that Ihe end product resembled a large cookie cutter.That

is, there was no bottom. A lab table top (black and shiny), made of a composite

material, served as the bottom. An S cm wide clear Plexiglasstrut was glued across

the Y from the outer comer of the left arm to the outer comer of the right arm for

stability.

Rocks, lightingand camera set up were identical to those o f Experiment 1.

3.1.3 Procedure

Trials witih T. marcianus and T sirtalis were conducted in September and

October of2003. The T. radix trials were run in November, 2003. Trials were

administered between 10:00 am. and 2:00 p.m. Subject order was chosen randomly

each day.

51

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Prior to beginning regular trials, subjects were given two 20 minute pretrials in

the Y-maze, without rocks, but with a fish bit located on the floor of the maze Just

beyond the entrance to each of the arms. This was done to habituate the snakes to the

apparatus and start box and to alow tibteoi to become familiar with finding food and

feeding in the Y-maze. It had been previously established (in this lab) that snakes of this

size could not detect air borne odors at a distance equivalent to the length of die arms of

the maze.

The left (L) and right (R) order of correct arm presentation in the maze was

worked out prior to the experiment Trials for aU subjects were run in this repeating

pattern: LRLR RLRL LRRL RLLR, for a total of at least 60 trials. Subjects were given

three trials per day, which meant there were six possible combinations of correct-side

order for the three daily trials. At the end of sixty trials, each subject had received three

each of RRL, RLL, LRR, AND LLR, and four each of RLR and LRL.

Prior to the experiment, masking tape was laid down on a black lab table in a

manner which formed an outline of the maze 5 mm wider than the maze itself. To set up

the Y-maze, two rowso f large rocks were placed on the table so that they made a

straight line down the center of the runway outline, the length of the runway. Next, the

maze was placed on the table, within the outline, over the rocks, and a white, mesh

plastic screen was inserted at the base o f the runway, to formthe start box. The medium

rocks were then poured (with a small scoop) into the maze, entirely covering the floor

on the appropriate side o f the rock line toa depth of approximately2 cm. fromthe:

box to the end of the correct arm. The large rocks previously laid down normally did

52

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. not move, but if they did, corrections were made to keqp the line delineating the two

sides straight and centered. The large rocks were then added to the same depth, covering

the floor of the other side of the maze.

A well approximately 15 mm. in diameter was formed in the rocks of each arm,

3 cm. from the end, in the center of the arm. The bottom of the well was actually the

table top. The fish bit was then placed in the well.

The subject was brought into the testing room in the home cage, and

subsequently transferred to the start box where it remained for 1 minute. Time started as

soon asthe screen was removed. The time elapsed between the start time and the first

movement of the snake was not recorded. This time varied ctmsiderably between

subjects and fiom trial to trial within subjects. On some occasionsthe animal simply

froze in the start box, presumably startled by the removal of the screen. Or, the subject

bolted out onlyto freeze a few centimeters outside die start box. Sometimes, a subject

spent a good deal of time examining the start box and/or the rocks tiieat edge o f the

start box before moving forward.All behavior subsequent to removing the screen was

considered part of the trail.

A choice was considered made when the subject’s head came within eight

centimeters of the back wsdl of an arm. Correction was allowed if the subject initially

made the wrong choice. Time ended when the subject actually found the reward. The

snake was allowed to consume the reword before it was removed to tie home cage and

the housing room.

53

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Ajfter the trial, the rocks, inside o f maze, and lab table were washed with a hand

sanitizer solution and water. The rocks were then spread on lab trays to dry for the next

day’s trials. The table top and maze were dried with paper toweling and the maze was

set up again with new rocks. The intertrial interval (ITI) was 20 minutes.

Probe trials were begun after a subject reached a criterion o f 70% correctin 20

trials. If this had not bear met by 60 trials, but was possible within another 10 trials,

then the subject was given 10 more trials. Probe trials were run on the second or third

trial o f the day for a total of 10 trials. A 70% criterion was applied to the probe trials.

Subjects were given two 20 minute pre- probe trials, in the Y-maze in the

darkened room. As was the case in regular trials,there were no rocks in the maze, but

one fish bit was placed in the maze at the choice point of the arm openings.

Twenty minutes prior to the probe trial, a 1 cm long and3 mm wide stripe of

phosphorescent acrylic paint was applied to the back o f the snake’s head just behind the

parietal scales. The snake was returned to the home cage while the paint dried. (It was

found fliat the snake’s water bowl had to be removed while the paint dried because they

would invariably get in the dish and the paint would come off. They would also do this

at fire conclusion o f the trial when the paint was dry.)

The Y-maze was set up as before, but for the probe trials, the computer, surge

strip, and lightswere turned off. With the md o f a flash light, the subject was brought

into the test room in the home cage, then placed in the start box for 1 minute. The flash

light was set on the lab table turned away firom theapparatus, so there was a small

amount of ambient light in the roomwhile the animal was in the start box. After 1

54

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. minirte in the start box the trial started with these actions executed quickly in the

followii^ order: flash light oflf, screen lifted and timer started. The experimenter,

standing at the base of the Y, tracked the subjects actions from above, moving to the left

or right side of the runway as needed, in order to observe the subject when it entered an

arm. The experimenter was no less than 30 centimeters from the maze at any given

time. Small dots of phosphorescent paint on the top edge of the maze allowed the

experimenter to gauge the subjects position and easily identifywhen the snake had

come to within 8 centimeters of the back of the arm, which indicated an arm choice. As

in regular trials, the subject was allowed to make the adjustment to the correct arm if

initiallywrong and in due course consume the reward. The trial ended when the reward

was reached.

Inter trial activities (washing apparatus, etc.) and elapsed time (20 minutes) were

the same as for regular trials

3.2 Results—Experiment 2

3.2.1 Arm choice

Proportiono f correct choices forthe first and last twenty trials, as well as the

binomial probabilities associated with thosechoices, are shown in Table 3.1 for each

subject. Number o f runs and length of longest run,whether correct (+) or incorrect (-)

for each of these two blocks are alsoshown. A run is defined as a sequence of like

events, where a single event, different from the one before and after, is also counted as a

run. For example, in the following sequence there are four runs:TTFTTTF. A

particularly large or small number of runs (given a certainN) would indicate non-

55

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. randomness. Ten or more runs would be expected in a Hock of twenty trials, hence,

nine or less would be considered non-random. A run of five or more like events out of

twenty would also be non-random. These tests were computed according to the method

of Bruning & Kintz (1987).

Table 3.1 Comparison between first twenty and last twenty trials First Twenty Trials Last Twenty Trials Prop. Binominal #of Longest Prop. Binominal #of Longest Subject Correct Probability Runs Run Correct Probability Runs Run M1 .65 .1315 13 4 (+) .90 .0000* 6* 7*(+) M2 .55 .4119 13 3(+/-) .85 .0012* 6* 11* w

M3 .55 .4119 12 . 1 .75 .0206* 11 6*(+)

Rl .70 .0576 11 5*(+) .70 .0576 8* ...... 7*W ...... R2 .65 .1315 7* 9*(+) .75 .0206* 9* 7*(+) M4 .30 .9793 5* 8*(-) .50 .5880 14 4(+) MS .35 .9423 7* 5*(-) .55 .4119 13 3{+) 81 .65 .1315 8* ...± t L . .55 .4119 11 ...... * Significmt at j? < .05 leveL

All subjects performed at chance levels {p> .05) on proportion of correct

choices made during the first twenty trials.

In the first block o f 20 trials, four o f the eight subjects (R2, M4, M5, & SI)

exhibited fewer runs than would be expected by chance (p < .05). Again, four subjects

(R l, R2, M4, & M5) showed runs o f greater than expected lengths. It should be noted

that the longest runs for two ofthe four subjects (R l, R2) were correct runs over the last

5 and 9 trials of the block, respectively. The other two (M4, M5) both had runs of

incorrect choices.

56

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. In contrast to behavior in the first twenty trials, four of the eight subjects, (M l,

M2, M3, R2) exhibited significant proportions of correct choices in thelast twenty trials

ip < .05), a fifth subject was very close with a result of .70 ip ~ .0576). A related-

samples t-test on the total proportion o f correct choices made by all of the subjects

during each o fthe two trial blocks confirmed a significant difference between the two

blocks (r7 = -3.01,jp< .05). Subjects made more correct choices during the last twenty

trials, (M= .69, SD - .1488) than during the first twenty trials (M= .55,SD - .1473).

Four subjects (M l, M2, R l, R2) showed fewer runs in the last 20 trials than

would be expected by chance ip < .05), and five (M l, M2, M3, R l, R2) demonstrated

above chance performance in the length of longestrun (j? < .05).

Five subjects (M l, M2, M3, R l, R2) reached significant levels o f success over

all trials by the time they attained criterion. The number of trials required to reach

criterion ranged firom 50 to 65. Probe trials were run on four of the five vriiich met the

criterion o f 70% correct in 20 trials. Attempts to run probe trials on subject M3 were

thwarted and eventually abandoned because the subject would not leave the start box

during the trial.) Of the fourwhich met criterion, three (M l, M2, & R l) chose the

correct arm during probe trials a significant number of timesip < .05). See Table 3.2.

57

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 3.2 Subject performance across all trials. # Of Trials Percent Subject Made Proportion Probes Proportion To Ate Criterion Correct Run Correct Criterion Reward M1 yes 55 .73* .93 yes .90* M2 yes 50 .62* .93 yes .90* M3 yes 65 .66* .75 no n/d

Rl yes . 61 .66* .97 yes .70* R2 yes 60 .62* .98 yes .60 Percent Made # Total Proportion Probes Proportion Subject Ate Criterion Trials Correct Run Correct Reward M4 no 60 .42 .45 no na M5 no 66 .50 .48 no na S1 no 64 .56 .73 no na

Figures 3.2 through 3.9 depict cumulative successes across total trials for each

subject. Since the dimensions of the graphs are Cumulative Successes (vertical) and

Cumulative Trials (horizontal), slope on these graphs is, by definition, the ratio of

cumulative successes to cumulative trials. Thereforethe slope of the line from any

given point on the graphback through the origin represents the cumulativeproportion

o f successes up to that point. At the same time, the local slope of the line throu^the

first and last points of a given set of points on the graphrepresents the proportion of

successes for that set of points. For example, a run o f allcorrect trials will parallel

perfectly the 100% Success line, and therefore have a slope o f 1. By the same token, a

run of all incorrect trials will have a slope o f zero, and thus be parallel the x-axis.

A good example of the utility of these facts is seen in thegraph o f subject M2

(Figure 3.3). Thisgraph can be seen as having two distinct parts with a cusp at trial 38

(represented by the bottompoint ofthe first long run of successes). The cumulative

successes to that point are 20 out of 38 trials, or a proportion o f .53 (the same as the 58

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. slope of the line through that point and the origin, and closely parallel to the random

performance line with a slope of .50). If we look only at the trials after number 38, the

slope of the line through the last trial (number 73) back through the point at trial 38, we

see that the line nearly parallels the perfect performance line. The actual slope o f that

portion o f the line, and thus the proportion correct in that group of trials is 31 of 35, or

.89, just slightly less than a slope o f 1.

Subject M1 Y-maze Performance

E 5

10 2 3 3 0 4 0 S ) e 0 70 80 90 C u m T r ia te

- Actual S u c c e s s e s ------100% S u c c e s s e s -Random Outcom e

Figure 3.2 Cumulative successes for subject M l.

59

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Subject M2 Y-niaze Perfonmance m

80 70

60 50

40 E 3 30 u 20 10 0 0 10 20 30 40 50 70 80 90 Cum TrUris

-Mual Successes -100% Successes -Random Outcome

Figure 3.3 Cumulative successes for subject M2.

Subject M3 YHmaze Perfbrmanee

I

E s

0 10 20 30 40 50 60 70 80 90 Cum T rlA -Astual Successes 100% Success® -Random Outcome

Figure 3.4 Cumulative successes for subject M3.

60

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Subject R1 Y^naze Peffoimnance

80

a>

E3 o

0 10 20 40 50 60 70 m 90 Cum Trials

•Actual Successes ■ 100% Successes -Random Outcome

Figure 3.5 Cumulative successes for subject Rl.

Subject R2 Y-maze Perfonnance 90

0 10 20 30 40 90 60 70 80 90 Ctnn Trfats

-Actual S u ccesses 100% Successes -Random Outcome

Figure 3.6 Cumulative successes for subject R2.

61

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Subject M4 Y°maze PerfDrmanee 90 80 70 m

50

40 E 5 30

10 0 0 10 20 40 5030 60 70 80 90 €^ m TrM s

-Astual Successes .-100% Successes -Random Outcome

Figure 3.7 Cumulative successes for subject M4.

Subject MS Y-maze Perfonnance 90

70

E 5

0 10 20 30 40so 60 70 80 90 Cum TrW s

-Artual Successes 100% Successes -Random Outcome

Figure 3.8 Cumulative successes for subject M5.

62

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Sutsjjwt SI Y-maze Perfonnance 9 0 T

3E O

0 10 20 30 4 0 SO 6 0 7 0 00 9 0 C u m T M s

-Astuai Successes ■ - 1 0 0 % Successes -Random Outcome

Figure 3.9 Cumulative successes for subject SI.

In addition to the planned data collection, observations were made on the

proportion of times, across trials, on which the subject ate the reward. Regression

analysis revealed that percent^e o f reward consumptionwas a reliable indicator of

overall success in the Y-maze. There was a significant positive correlation between

these two variables. As percent of reward consumption rose so did success, r(8 ) - .72,

^ < .0 5 ).

3.2.2 Runmng time

As a ^ u p , the subjects did not ©chibit a si^ fic a n t difference in RT between

the first block of twenty trials and the last block of twenty trials.

However, the five that made criterion ^ 1 , M2, M3, R l, R2) did, as a group,

show a significant decrease in RT fi-om thefirst twenty trials (M = 226.66 sec., SD =

63

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 161 sec.) to the last twenty trials (M = 168.82 sec., SD = 115 sec.); % - 2.99,p < .05.

Three of the mdividiials in this group also showed a decrease in RT from the first block

to the last block as well (see Table 3.3). Mean RT for subject M2 dropped from 247.05

sec. {SD = 130.89 sec.) to 185.7 sec. {SD = 21.9 sec.), t\g -2.0$,p < .05; subject Rl

dropped fix>m 255,55 sec. (SD = 218.7 sec.) to 136.5 sec. (SD = 83.49 sec.), tig = 2.02,p

< ,05; and subject R2 dropped from 149.9 sec. (SD = 119.87 sec.) to 90.7 sec. (SD =

69.54 sec.), tig - 2.01, p .05.<

Table 3.3 RT summary statistics for the two trial blocks. First Twenty Trials Last Twenty Trials Subject Mean Median Range Sum Mean Median Range Sum M1 219 170 528 4388 172 210 545 4941 M2* 247 210 545 4941 186 147 361 3714 M3 261 191 481 5228 260 228 662 5205 R1 * 266 201 930 5111 136 112 286 2726 R2* 150 109 445 2998 91 76 323 1814 M4 358 329 588 7164 397 235 1159 7937 MS 298 141 1194 5967 428 374 835 8564 81 415 384 1042 8304 331 205 1008 6617 * last 20 mean RT lowerthan first 2Q, p < MS, all nu m tes in seconds

Over all trials and subjects, there was a modest, negative relationsMp between

proportion of correct choices and RT. As proportion of correct choice went up, RT

came down, or, conversely, as errors went up, RT went up (r =.56, p = .15). The same

relationship was more clearly evident for the last twenty trials, across all subjects (r =

.$3,p < .02).

64

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. There exists an implicit a priori assumption that making mistakes in this type of

task is costlyin terms o f time to reach the goal. Therefore, the group of subjects which

made criterion (M l, M2, M3, R l, R2) would be expected to have the shortest RT’s by

virtue o f the fact they made fewer incorrect choices than the subjects that failed to meet

criterion (M4, M5, SI). An alternative hypothesis is: those that made criterion were just

physiologically speedier. To test this, group comparisons of RT’s for both those that

made criterion (MC), and those that failed to make criterion (FC) were broken down

into “correct choice” and “incorrect choice” RT categories. To even the field, only the

correct trial RT’s of the MC and FC groups were compared (across all correct

trials).The PC’s had significantly longer RT’s (M = 197.72 sec., SD = 126) than the

MC’s (M = 137.25 sec., SD = 78). (/1 3 1 = 4.29,/? = .0012.) This significant result

justified additional post hoc comparisons to further evaluate this finding. The MC’s

could still have been just naturally faster. Both groups were tested against themselves

(1** vs. last 20 trials) for correct and incorrect RT’s, and against each otherfor correct

and incorrect RT’s during the first and last twenty trials. Because the numbers of

observations were not equal, and in some cases the greatest variances were seen in the

smaller of the two group’s observations, t-tests using unpooled variance,where the

variances are not assumed equal, were run (Weiss, 1999). Results of these comparisons

are shown in Table 3.4 and Figure 3.10.

65

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 3.4. Post hoc RT comparisons. GROUPS CHOICE TRIALS MDIFF SD FCvs. MC incorrect 1st 20 109.71 282, 184 FC vs. MC incorrect last 20 283.97 * 331,154 FC vs. MC correct 1st 20 41.15 190.9,149.8 FC vs. MC correct last 20 75.72 ‘ 122.7. 76.8 FC incorrect 1st vs. last 20 85.78 282.1, 331.1 MC incorrect 1st vs. last 20 88.49 * 184, 154.1 FC correct 1st vs. last 20 22.48 144.1,122.66 MC correct 1st vs. last 20 12.09 77.51,76.82 * Btperiment-wWe p < .05; algnlfioanos crterion for each tesl was Ml * -3.23,p “ .0024, unpoofed f4es!s.

Mean- RT Correctvs. Incorrect Choice 600 500 400 300 200 100 0 L Correct Incorrect Correct Incorrect Failed Criterion Made Criterion ■ First 20 ■ Last 20 Figure 3.10 Correct and incorrect running tunes (RT) for those that made criterion and those that did not.

There were three comparisons where results were significantly different. In the

last twentytrials, those that made criterion had significantly lower RT’s than those that

did not on those trials where they initiallymade the wrong choice(1 3 9 = 4.09,/? = .0002,

see Figure 3,10). The MC’s also had shorter RT’s thandid the PC’s duringthe last

twenty trials on trials where they made the correct choice {U\ - -3.23, p = .0024).

Finally, MC’s were faster on the last twenty trials than their first twenty trials when they

66

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. made an incorrect choice {tsg = 2.10s j? = .04). Some of the comparisons that werenot

significmt are nonetheless important and will be explored in the discussion.

Comparison of the last 20 incorrect trials of PC’s vs. MC’s foimd a significant

difference in RT's (M- 541.43 sec., vs, M= 257.46 sec.), 139 = 4.09, j? < .05

3.3 Discussion - Experiment 2

3.3.1 Arm Choice

One of the goals of this study was to demonstrate the ability of snakes to utilize

substrate cues as discriminative stimuli allowing the successfijl navigation of a Y-maze.

The results indicate that these snakes have the ability to master the task. This ability is

reflected in the improved combined performance of the subjects during the last trial

blocks as compared to the first. This result is, in itself, noteworthy becauseno previous

studies have established the ability o f snakes to utilize natural, structural cues as guides

in their environment This will be addressed further in the general discussion. These

findingsare also important additions to the literature because there are so few well-

controlled learning studies in snakes (and none withThamnopMs marcianus.).

Additionally, the evidence fromthis study reinforces the notion that snakes can be

successful in appetitively motivated learning situations. Designs utilizing escape (to a

preferred place) as the reward have been conducted (Crawford & Holmes, 1966;

Fuenzalida & Ulrich, 1975; Holtzman, Harris, Aranguren, Bostocks, 1999), but in some

situations, (foraging experiments, for example) accessto food or water or even home

cage are moreappropriate rewards. 67

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The difficulty of working around the metabolism and physiological constraints

of reptiles is well known. In this experiment, young snakes worked very well because

they are growing rapidly, and will usually eat everyday. (The snakes in this study which

ate regularly, were consuming the equivalent of a one-inch fish, each day.) The

experiment would have been more difficult to do with adult subjects because their

hunger motivation would be more difficult to specify.

Several interesting thingswere found when the individual subject data were

examined. There were fairly clear and consistent differences between the group of five

subjects that made criterion and the group of threethat did not. Consideringthe first

block o f twenty trials, if the animals were behaving in a random manner, as expected,

they would 1 ) not show significant proportions o f correct choices,2 ) they would have a

high number o f runs (> 9), and 3) numbers o f observations in a run would be low (< 4).

The first point was indeed bome out; they did not show proportions ofcorrect choices

significantly above chance. On the second point, however, four of the eight subjects had

fewer and longer, runs than would be expected, but moreimportantly, threeof those

four (M4, M5, SI) were in the groiq) that did not make criterion. This may be a

reflection of these individual’s strongpropensity to stay on the preferred large rocks

rather flian venture onto the less preferred rocks during the early stages of the

experiment. This idea is also supported by the fact that two of the PC’s had longer runs

than expected, not on the correct side but onthe incorrect side (large rocks). Subject R2

(MC) also had a small number o f runs but this was due in part to a longer than expected

positive run of nine, which were the last nine trials of the first block. It could be argued

6 8

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. that the subject was begimsmg to make the association, but this subject went on to have

a similar long ran of six incorrect choices during the next fifteen trials; R2’s ran of

correct choice behavior was probably random. A more likely explanation for the other

subjects, (especially those that did not make criterion), at this stage o f the experiment,

was a tendency to cling, as it were, to the preferred larger rocks (which may have

provided more structural shelter), and thereafter to perseverate in the choice of those

familiar circumstances. Relevant to this interpretation, are two important points. First,

there are studies which have established individual variation in certain behaviors of

snakes, (differences in particular behavioral assays between individuals of the same

species, population or litter.) More importantly, these individual differences have been

shown to be consistent across time and across some situations. Brodie, (1993) has

demonstrated the consistencyof individual anti-predator behaviors in neonates through

their second year and across different thermal mvironmeots (Brodie & Russell, 1999).

Consistent individual variation has also been shown for mqrloratory behaviors (Chiszar

& Carter, 1975; Marmie, Kuhn, &, Chiszar, 1990), escape responses (Holtzman,et al

1999), and extremes in food preferences (Drummond & Burghardt, 1983). These

findings suggestAat once a behavior is established, it would likely persist.

Second, the bright and open environment o f both the maze and the preference

study box are not optimum snake habitat, primarily because there are no hiding places.

In fact, as previously mentioned, escape from an open situation to a “hiding” place has

been used as behavioral motivation in several studies (Crawford & Holmes, 1966;

Fuenzalida & Ulrich, 1975; Holtzman et ah 1999) precisely because it is within the

69

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. realm of natural snake behavior. Also, as previously mentioned, it is well established

that stroctoral features play an important part in the habitat selection of snakes (Reinert,

1993). It follows then, that the larger rocks provided more stroctural cover, and because

of this, were consistently sought out by some of the subjects. This postulate is most

evident in the cumulative successes graph for subject M4 (Figure 3.7). This subject had

an initial negative eight-run event, followed very closelyby another negative run of

five. Statistically, there is a low probability that this behavior was random. The

subject’s performance subsequently paralleled the random-performance line on the

graph o f cumulative successes for the duration of the experiment. In contrast, the gr^h

of subject M l makes obvious a pattem o f slow and steady progress as evidenced by a

slope which is steeper than the random-performance line, while subject M2 appears to

have had an “epiphany” of sorts with sudden improvement around the fortieth trial.

In general, those that made criterion (Ml, M2, M3, R l, R2) and those that did

not make criterion (M4, M5, SI) where in opposition on their patterns of runs, lengh of

runs and proportion of correct choices. Specifically, those that made criterion (MC)

went fiom expected random behavior in the first twenty trials to non-random

performance in the last twenty trials, indicating an acquisition of the behavior. On the

other hand, those that failed criterion (FC) went j&omnon-random to random behavior.

Ihe subjects’ propensityfor staying on the preferred substrate is the most likely

explanation for this pattem. The interesting thing about these subjects is thefact that

even when they became secure with both substrates, their behavior never departed

significantly fiomrandom, as evidenced by the fact that the slopes of their cumulative

70

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. correct choices gr^hs parallel, in each case, the random-behavior line for the duration

of the trials.

Another clue concerning the poor perfonnance of the PC’s is the fact that they

did not reliably consume the reward. In the Halpera (1988) studies, snakes chose the

correct arm of a Y-maze simply for the reward of tongueflicking prey odors on a

surface, so theoretically, not consuming the reward should not have prevented

acquisition of the behavior. In the present study subjects always found the reward and

presumably had a chemosensory perception of the reward. Consequently, even if they

were not motivated to eat on a particular trial, information about the

environment/reward should have been acquired and subsequently applied when the

incentive to eat was greater. This may have been the case, but a lack o f gustatory

motivation likely was not to blame for the lack of performance which might have bome

such out. To illustrate, M2 continued to eat and perform while in theprocess of ecdysis,

which is highly uncharacteristic, while M4 did not eat for fourteen straight days.

(Recall, the only food available was that obtained during the three daily trials.) A more

likely interpretation is that the possibility o f finding food was not the primary

motivation for the FC subjects’ movements in the maze, but rather, it was the

aforementioned drive to find a place to hide. In other words, the drive to e s c ^ the

situation was a higher order motivatioiL Indeed, these subjects were oftenobserved

coming into contact with and often crawling over the fish bit. Clearly tiieir lack of

consumption was not due to lack o f encountering tire reward. Two possibilities for this

71

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. fear response are perceived predation risk and handling stress (Moore, Thompson, &

Marler, 1991; Moore, LeMaster & Mason, 2000).

Three o f the four subjects that were given probe trials without visual cues chose

correctly a significant number of times. It is unfortunate more subjects were not

represented. However, if we assume their visual perception was virtually eliminated,

then these results are encouraging. It might argued that the effectiveness o f running the

animals in total darkness as a method of eliminating visual perception is not certain. To

some extent, such criticism is valid, however, the otiier methods considered, such as

placing paper or tape over the eye shields would have the same problems and were

considered more intrusive. The only absolute test would be to ablate the eyes or visual

nerves. In future studies, the possibility of surgically blinding the animals first, and then

testing whether or not they could learn the discrimination should be considered.

Subjects did appear to make more lateral head and upper body movements during the

probe trials, a behavior, which might be regarded by tiie tolerant among us, as searching

or “feeling” for the right substrate. It was a pattem reminiscent of the side to side

swimming motion o f sharks.

3.3.2 Runmng time

Snakes have a well deserved reputation for having a refractory nature when it

comes to experimental manipulations. But this study, and others of its nature, have

produced and will continue to produce, valuable information in the progression toward

an understanding o f the various mechanisms and processes which govem the lives of

these animals. Plainly, these are not rodents. A review of the literature suggests,

72

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. charitably, that ruxming time could be considered a tricky dependent variable when it

comes to snakes. (This is largely due to a deficiency in the number o f learning studies

involving snakes, particularly those involving reward and stimulus variables which

could have an impact on running time.) One o f the most valuable contributions o f this

study is a body of data which may contribute to a better understanding o f the

experimental conditions which warrant running time as a dependant variable, as well as

what kind o f meaning can be taken fix)m the results.

With rats we would be looking for a decreased latency to reach tiie goal across

successive trials as a clear sign they had learned the maze. In this experiment, only three

of the eight subjects showed a decrease in latency to reach the reward; a finding that

would initially suggest that the majority of the subjects did not acquire the behavior.

However, these three were in the group o f five which made criterion, and as a group,

they (the MC’s) were faster on the last block of trials than the PC’s. We know that

among individual snakes large differences can be found in burst rates and speed.

Chiszar & Carter (1975) noted large and consistent individual differences in eight

snakes traversing and open field. The fastest had a traversing rate of 11.1/squares per

minute, the slowest was .50/squares per minute and, they were very consistent in their

performance over a nine day period. One might infer firom this that the MC’s, as a

group, were physiologically faster. But, this was not the case. There was no difference

between the two groups (MC vs. FC) on either trial type (correct or incorrect) during the

first block o f trials. Clearly, the faster RT’s for the MC’s over all the trials must be

attributed to something else. Regression analysis on proportion correct and RT on the

73

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. last twenty trials suggestedttiere was a strong inveree relationsMp between the two

(Mgher proportions being associated with lower RT’s). It follows, then, that the MC’s

would show faster RT’s in the last vs. the jSrst block of correct trials. This did not

happen either. The overall superiority o f the MC’s running times can be attributed to the

responses they made to initially incorrect choices in the last trial block (see Figure

3.10). The relationship between the MC group’s RT and cumulative success apparently

had more to do with correcting mistakes than choosing correctly.Or possibly, as they

acquired the association they may have engaged in more directed responses. MC

subjects often crossed to the correct side precisely at the mouth o f the Y or made an

immediate U-tiim after entering theincorrect arm and then continued in a deliberate

feshion toward the goal. Sometimes subjects would stay on the correct sidefrom the

beginning. Conversely, on a number o f trials subjects wandered about in a circuitous

fashion, seemingly afflicted with some class of competingresponse such as exploring

the maze or lookingfor a way out o f the maze. Occasionally a subject would go to the

end of the wrong arm and subsequently return to the start box and thenrepeat the same

behavior seven or eighttimes. One subject in particular attempted to climb out of the

maze on almost every trial. Unfortunately these types o f observations are qualitative in

nature, but let the anecdotal record show, the subject just mentioned was not one o f the

ones that made criterion.

The mean RT’s o f the FC group were actually longer on incorrect trials during

their last twenty trials, as compared to their first twenty, which may be due to the

aforementioned idea that they had a tendency to engage in less directed behavims.

74

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Again, Figure 3.10 depicts the significant disparity between the responses of the MC’s

and the PC’s when at first choosing the wrong arm during the last twenty trials.

As the data indicate, actual physical speed had little to do with the successful

outcome o f the MC’s. The rat model interpretation simplydoes not apply in this

situation. One need only look at Figure3.10 torecognize that tihese five subjects did

acquire the behavior, a fact that just was not discemable by looking solely at the overall

RT results.

On correct trials the MC’s did not improve their RT from the early trials to the

latCT trials. Changes inRTin other snake studies present amixed set o f results. Kubie

and Halpem (1975) reported a decrease in RT for snakes in a situation where they were

foUowit^ a prey exteact trail in a Y-maze for a food reward. It was not clear whether die

reduction in RT was due to an actual increase in movement ^5eed, or less time engaged

in examining the trml onlater trials. In a morecomparable study, Crawford and Bartlett

(1966) did not find any change in RT for subjects traversing a strait runway for a food

and water reward. Conversely, Peretti and Carberry (1974) did find a decrease for

subjects seeking a water reward in a maze. Snakes tested In an escape

typically also exhibit reduced latencies and RT’s ( Kellogg &Pomeroy, 1936; Crawford

& Bartlett, 1966; Fuenzalida & Urlrich, 1975; Holtzman et al, 1999).

It seems RT may be an appropriate dependent variable for escape conditioning.

In only one of the aforementioned studies did subjects show any kind o f reduction in

errors (Kellogg& Pomeroy, 1936). Interestingly, these authors attributed the success of

their subjects not to available visual cues, but to tactile discrimination o f the apparatus.

75

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The Holtzman, et al (1999) exfseriinent might have kid better results (in error reduction)

had the subjects only cue, a large ^ t e card on the Mack background of an arena, been

more salient.

Cues aside, it seems very posslMe that the relevance of latencies and RT’s as

experimental dependent variaMes is related to the type of reward. For instance, the

natural approach of snakes to food is to slow down, not speed up. If they are in an active

predatory mode and detect or suspect a prey item, they slow their forward motion and

begin stalking the prey— behavior which would have obvious effects on experimental

runmng times. They do this even vhen fed the same food itan on a regular basis in then-

home cage. They slowly advance on the food item as though it might “get away”

(personal observation). Evidence supporting this proposition comes fiom a semi natural

field study in involving desert vipers (Tsairi & Bouskila, 2004). The primary focus of

that study was to determine if the snakes would choose ambush areas, (in a 7 m circular

enclosure divided into quarters), which contained rodent odor. During the study, they

observed that the snakes approached die rodent odor areas in a rectilinear fashion, the

slowest straight-line mode o f transport available to snakes. O tha areas were traversed

in either a sidewinding or serpentine motion, which are fester and involve a much less

subtle motion that might easily be detected by prey. (Incidentally, they found the snakes

chose ambush sites that were either elevated or contained structural components, prey

odor was not a factor.)

The approach which a water deprived snake makes to water, may be quite

different. A simple way to investigate this possibility would be to compare the RT’s of

76

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. two groups run in identical experimental sitiiattons, except for type of reward.

EstablisMng equivalent motivational states might be difficult, but predicting the

directionality of the outcome of the two groups over a baseline might be a start.

Analyzing tongue flickrate on the ^proachof subjects to food, water or esceq)e

routes would also be useful. For instance, one would expect higher tongue flickrates on

an approachto food than an approachto a means of escape (assuming the escape route

was not imbued with any chemical property.)

77

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 4

SUMMARY AND CONCLUSIONS

The intention of this study was to evaluate the perception and use of tactile

sensations in snakes. As sometimes happens in science, a wordor opinion gets bandied

about until it is generallyaccepted without anydemonstration of validity, or in this case,

non-validity. In the herpetological realm, generalimpressions of snake taction seem to

fall into three categories. First, snakes obviously feel things;they reqjondto touch,

temperature, or pain in an reflexive manner. Next, males intentionally touch females in

a unique way when mating, therefore, itis assumed that those perceptionsmust be

meaningful to the snakes involved, and must occur for successful m atii^. Taction is

also assumed to be involved in other types of social or physical contact between

conspecifics such as combat and aggregation (Heller & Halpem,1982; Gillingham,

Carpenter & Murphy, 1983). And third, the tactile sense in snakes is not hypotihesized

to have further functions except for the occasional reference as a possible, butnever

tested, cue. The present research has illuminated the role o f taction beyond the narrow

confines o f reflexive responses and social functions and therefore,has implications for a

much broader range ofbehaviors.

Whether it’s moving fromone lillypad to another or one continentto another,

animals use a myriad o f cues and mechanisms toget from one place to the nwct. Ample

78

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. research has shown that many animals utilize multiple cues (magnetic, solar, celestial,

polarized light, UV light, landmarks, etc.). The cue of choiceoften depends on the

strength and availability o f that particular cue. For instance, honeybees normally

navigate by using the sun as a compass, but on overcast days they utilize patterns of

polarized light as a guide (Able, 1991). It seems a reasonable guess that snakes could

also switch to the most salient available cue in any particular situation. This may have

been the situation during the Y-maze probe trials. Evidence from this study indicates

that, even if they had been utilizing visual or both visual and tactile cues during the

regular trials, in the dark, they may have switched to taction only. Of course, tactile

stimulation fromthe rocks could have been the primary cue. Conducting an identical y-

maze experiment, but training the subjects in thedark, or as mentioned previously,

surgicallyablating visual capability would be useful fiiture investigations.

Spo:iesspecific preferences for the three substrates offered in this study were

not found. This leaves the impression that habitat preferences between species may be

very subtle, and that predicting them will require a thorough understanding of the

ecologyo f the species. Generallyspeaking, this type of information is not available or is

exceedingly v^ue, due in no small part to the low profile (both literal and

metaphorical) nature o f these animals. The most promising new research involves the

tracking of habitat use via radio transmitters implanted in tifcie snakes. Using this

method, Fitzgerald, Shine & Lemckert (2002) found that differentqpedes of arboreal

snakes had preferences for different kinds of tree attributes. In the same field location,

carpet pythons {Morelia spilotamcdoweiii) |»eferred trees that offered dense vine

79

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cover; they did not utilize .tree hollows (Shine & Fitzgerald, 1996), whereas Stephen’s

banded snakes {HoplocephalusstephemO) preferred trees with hollows(which they

used as retreat sites). The authors attributed the behavior of the banded snakes to their

close phylogenetic relationship with terrestrial species. Hence, they shared the same

propensity for hiding places as their ground-dwelling kin. With this method, the

researchers were also able to document the reuse of particular trees by individuals. This

kind of specific information opens the door to predictions regarding habitat use as well

as to identifying the proximate cues involved in locating (and in some cases, relocating)

suitable habitat Although taction probably does not play a principal role in large-scale

movements, it may be a component of smaller-scale movements such as finding the

right tree or following the route to the hollow, oncethe tree has bem identified. For the

experimenter, to know which trees are preferred is a valuable piece of information.

When the snake leaves or is removed, tactile cues associated with the tree could be

altered. If th«e are disruptions inbehavior, some measure o f tactile dependence might

be implied. Field studies such as this will, of course, be necessary to determine how

well this, and fiiture, studies generalize to the real snake world.

It is very plam that more information is needed concerning the natural history of

the snakes being studied. We know, for instance, that most garter snakes are primarily

diurnal crefstures, and we recall that the retinas of some do not possess rods (Sillman et

al, 1997). This is important because a lack of rods would make nocturnal visual

discrimination difficult. However, some species are also known to forage at night An

examination of nocturnal foraging first in this genus (perhaps utilizing night vision

80

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. goggles) would help establish what types of cues they employ. A ininimum amount of

tongue flicking, for example, could point to cues other than chemical ones.

Unfortunately, due to the camera set up in this experimeiit, it was not possible to see

any tongue extrusion even during the lighted portion of the experiment, a shortcoming

that should be addressed in further studies.

Taction may be more directly involved with actual feeding than foraging. As a

general rule snakesswallow their prey head first (unless the prey is small, compared to

the head of the snake). To do otherwise is difficult and time consuming and

occasionally fatal. Research suggest they determine the “head” end by, direction of the

fur, and taper o f the body, cues which can best be discerned by tactile means

(Diefenback & Emslie, 1971). Greene’s (1974) study of ophiophagous snakes showed

convincingly that scale owcAap was the controlling cue in these snakes(which eat other

snakes), and, detecting it wasnot likely accomplished chemically or visually. He also

notes that snakes often“snout push” the prey before attemptingto swallow it, a

phenomenon diis author has also observed. Given the fact their labial scales are covered

with menchanoreceptors, this behavior couldplausibly be viewed as probii^ for tactile

information. Testing this idea in a feeding situation would be difficult, however.

Perhaps surgically severing the trigemental nerves on one side of the head might alter

the feeding behavior as compared to normal animals. A more practicalapproach might

be to test two groups o f animals in a water maze escape paradigm with tactile cues,

where one group hasthe mechanoreceptor nerves severed or compromised, perhaps

with a topical anesthetic.

81

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The only published study involving taction in snakes, was one carried out to

specifically address a captive management issue (Chiszar, Radcliff, Boyer & Behler,

1987). In it, the authors examined whether or not clear hide boxes would satisfy the

“cover seeking needs” ofcaptive cobras {Naja mossambica pallida). The boxes were

small enoughthe snake touched the sides o f the box when in it. They found that when

clear boxes wo’e the only choice, the snakes did Indeed use them. They surmised the

tactile stimulus of the walls of the box w «e satisfactory. But, when they were given the

choice of same size dark or clear boxes, they chose the dark box, leading to the

conclusion that darioiess is also important. When the two variables where again tested,

the clear box was small and in an open arena. The other side of the arena was covered

and dark, with no hide box. They again chose thedark side, but the use of the comers

mayhave provided enough tactile stimulationthat it was preferred. The authors wanted

to answer M s question because it is much safer to deal with venomous snakes when one

knows exactlywhere they are, but the truly interesting elementis that some measure of

what a snake is seeking,when on a quest for cover, is tactile stimulation. A simple

experiment to test how much stimulation is preferred would be to offer hide boxes of

varying size. That snakes seek this kind o f stimulation and show an ability for tactile

discrimination, as found in the present study, have practical spplications fijrall research

involving snakes. In short, tactile cues (or stimulation) is a variable for which one must

account

82

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Taken together, thefindings of this study present a solid appeal for additional

work in the area, both in and out of the lab. The greatest challenge will be deciding

what to do first.

83

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. BICXJRAPHICAL INFORMATION

Vicki L, Keathley bom in Dallas, Texas, received a Bachelor of Science in

Education from Texas Tech University in 1975. A Master of Science in Psychology was

granted in May, 2001. A Doctor of Philosophy in Experimental Psychology was

awarded in May, 2004.

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