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Summer 2006 Chemical Evidence for Dietary Sequestration in the Asian tigrinus Deborah A. Hutchinson Old Dominion University

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Recommended Citation Hutchinson, Deborah A.. "Chemical Evidence for Dietary Toxin Sequestration in the Asian Snake Rhabdophis tigrinus" (2006). Doctor of Philosophy (PhD), dissertation, Biological Sciences, Old Dominion University, DOI: 10.25777/d4ng-k993 https://digitalcommons.odu.edu/biology_etds/61

This Dissertation is brought to you for free and open access by the Biological Sciences at ODU Digital Commons. It has been accepted for inclusion in Biological Sciences Theses & Dissertations by an authorized administrator of ODU Digital Commons. For more information, please contact [email protected]. CHEMICAL EVIDENCE FOR DIETARY TOXIN SEQUESTRATION

IN THE ASIAN SNAKE RHABDOPHIS TIGRINUS

by

Deborah A. Hutchinson M.S. August 2001, Old Dominion University B.A. May 1999, University of San Diego

A Dissertation Submitted to the Faculty of Old Dominion University in Partial Fulfillment of the Requirement for the Degree of

DOCTOR OF PHILOSOPHY

ECOLOGICAL SCIENCES

OLD DOMINION UNIVERSITY August 2006

Approved by:

Alan H. Savjlzk/'fbirector)

Mark J. Buuer IV (Member)

Akira Mori (Member)

Rose (Member)

Frank C. Schroeder (Member)

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

CHEMICAL EVIDENCE FOR DIETARY TOXIN SEQUESTRATION IN THE

ASIAN SNAKE RHABDOPHIS TIGRINUS

Deborah A. Hutchinson Old Dominion University, 2006 Director: Dr. Alan H. Savitzky

Rhabdophis tigrinus (: ) is an oviparous, bufophagous

(toad-eating) snake from eastern Asia that possesses defensive integumentary glands

on the neck known as nuchal glands. These glands are used in antipredator displays and

typically contain . Whereas toads are known to synthesize

bufadienolide from cholesterol precursors, we found that chemically

undefended R. tigrinus must sequester from ingested toads in order to

exhibit these compounds in their nuchal glands. Chemically defended females are

capable of provisioning their embryos with these toxins so their unfed hatchlings

possess defensive bufadienolides prior to consuming toads themselves. All of the

hatchling R. tigrinus from an island with a dense population of toads were chemically

defended regardless of their diet in the laboratory. In contrast, none of the hatchlings

from an island lacking toads possessed bufadienolides in their nuchal glands until after

they consumed bufonid prey. Proton NMR and HPLC analyses demonstrated that

newly acquired bufadienolides from ingested toads can be transferred from the dam to

the embryos as late as 12 days prior to oviposition, suggesting that at least some

transport of toxins occurs within the oviduct. Although hatchlings are provisioned with

most of the bufadienolide compounds possessed by the dam, there may be chemical

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. selectivity to this process. In a feeding experiment with gravid R. tigrinus from various

localities, most clutches had been provisioned with moderate to large quantities of

bufadienolides. In the provisioned clutches, the most abundant bufadienolide in the

unfed hatchlings was almost always the same compound, although it generally was not

the most abundant bufadienolide in the dam. R. tigrinus is the first amniote

known to have evolved specialized defensive structures dependent upon sequestered

dietary compounds, either obtained directly from prey or provisioned by the dam.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. This dissertation is dedicated to my parents,

Emilie M. Hutchinson

and

Thomas W. Hutchinson,

both of whom taught me the importance of education.

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

ACKNOWLEDGMENTS

Many people were necessary for the successful completion of this dissertation.

First and foremost, I wish to thank my advisor Alan Savitzky who provided me with

years of positive influence, unique research opportunities, and emotional and financial

support during the completion of my Master’s and Doctoral Degrees. Additionally, this

project would not have been possible without our collaboration with Akira Mori of

Kyoto University, who first hypothesized that Rhabdophis tigrinus sequesters its

defensive bufadienolides from dietary toads. Dr. Mori was also directly involved in the

feeding experiments presented in this dissertation. I was honored to conduct research in

Japan with Dr. Mori and in Jerrold Meinwald’s laboratory at Cornell University. I am

grateful for all of the time invested by Frank Schroeder to train me on the use and

interpretation of NMR spectroscopy and HPLC at Cornell. Sven Possner also assisted

me in this regard and graciously shared his lab space and equipment with me. Dr.

Schroeder and Xiaogang Wu spent many hours determining the structures of

bufadienolides in our samples. I also thank my wonderful family (Trevor Adams,

Thomas and Emilie Hutchinson, Judy and Isabella Fercioni, and Maggie) for their

continuous support during this rewarding and often challenging journey.

I extend gratitude to the remaining members of my dissertation committee, as

well as other individuals for their support. Ian Bartol, Mark Butler IV, and Robert Rose

contributed valuable comments on the writing of this dissertation. Daniel Sonenshine

graciously donated his time, advice, and laboratory equipment to me in the early stages

of our HPLC work. Gordon Burghardt assisted in the design of initial feeding

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experiments. I benefited from discussions on herpetology and experimental design with

William Resetarits, Jr., Christopher Binckley, Barbara Savitzky, and Victor Townsend,

Jr. I also thank Jackie Grant for supplying us with a transcutaneous

stimulator and advising us on its use. John Daly and Thomas Spande kindly donated

the telocinobufagin for our dilution curves.

I am indebted to many individuals (mainly students) who helped me care for

research and provided intellectual stimulation and emotional support. I extend

special gratitude to the past and present students in the Herpetology Laboratory at Old

Dominion University. Specifically, I thank Julie Ray, Angie and Gabe Rivera, John

Allsteadt, Kathy Roberts, Chris Petersen, Scott Goetz, Kelsey Holzman, Shannon

Davis, Dalyn Kuklock, Monica McGarrity, Gabrielle Mueller, Erica Schmutzler, and

Kira White.

I am also grateful to numerous additional individuals in and the United

States who assisted with the collection and husbandry of animals as well as the

collection of samples and data entry. This list includes Trevor Adams, Jay Bolin,

Jennifer Bradford, Gordon Burghardt, Matthew Close, Takashi Haramura, Masami

Hasegawa, Azusa Hayano, Tatsuya Hishida, Isami Ikeuchi, Yohei Kadota, Akira

Katayama, Noriko Kidera, Toshiro Kuroki, Alan Lemmon, Taku Mizuta, Koji

Mochida, Yoshihisa Mori, Emily Moriarty Lemmon, Eiko Nagata, Aya Nakadai,

Sumio Okada, Taku Okamoto, Hidetoshi Ota, Julie Ray, Barbara Savitzky, Kunio

Sekiya, Tomohiko Shimada, Hirohiko Takeuchi, Koji Tanaka, Mamoru Toda, Victor

Townsend Jr., and Ken Yoda.

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Due to the collaborative nature of this project, I used the pronoun “we”

throughout my three main dissertation chapters. This was done to reflect the

contributions of all authors of these chapters, which will be converted to manuscripts

for publication. The authors of Chapter II are myself, Akira Mori, Alan Savitzky,

Gordon Burghardt, Xiaogang Wu, Jerrold Meinwald, and Frank Schroeder. The

authors of Chapter III are the same as those of Chapter II, with the exception of

Xiaogang Wu. For Chapter IV, the authors include those of Chapter II, with the

exception of Xiaogang Wu and Gordon Burghardt.

Funding for this project was provided by the National Science Foundation

(IBN-0429223 and IOB-0519458 to Alan Savitzky and Jerrold Meinwald); Old

Dominion University (Dissertation Fellowship and Dominion Graduate Scholarship to

Deborah Hutchinson); the Society for Integrative and Comparative Biology (Grant-in-

Aid of Research to Deborah Hutchinson); Sigma Xi, The Scientific Research Society

(Grant-in-Aid of Research to Deborah Hutchinson); Kyoto University (Grant for

Biodiversity Research of the 21st Century COE A14 to Akira Mori and Dept, of

Zoology); the Kyoto University Museum (Visiting Faculty Award to Alan Savitzky);

the Japan-US Cooperative Science Program (Japan Society for the Promotion of

Science to Akira Mori and NSF INT-9513100 to Gordon Burghardt); and the Fujiwara

Natural History Foundation (10th Annual Grant for Scientific Research to Akira Mori).

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

Page

LIST OF TABLES...... xi

LIST OF FIGURES...... xiii

Chapter

I. INTRODUCTION...... 1 Predators of Toxic Prey ...... 1 Dietary Toxin Sequestration ...... 4 Bufophagous '...... 5 Rhabdophis tigrinus...... 7 Nuchal Glands ...... 7

II. DIETARY TOXIN SEQUESTRATION IN RHABDOPHIS TIGRINUS....12 Introduction ...... 12 Methods ...... 15 Experimental Design and Sample Collection ...... 15 Chemical Analyses ...... 17 Results ...... 19 Discussion ...... 32 Acknowledgments ...... 36

III. GEOGRAPHIC VARIATION OF CHEMICAL DEFENSE IN RHABDOPHIS TIGRINUS...... 38 Introduction ...... 38 Methods ...... 42 Experimental Design and Sample Collection ...... 42 Chemical Analyses ...... 43 Results ...... 46 Kinkazan ...... 46 Ishima...... 51 Discussion ...... 54 Acknowledgments ...... 56

IV. MATERNAL PROVISIONING OF DEFENSIVE STEROIDS BY RHABDOPHIS TIGRINUS...... 57 Introduction ...... 57 Methods ...... 59 Experimental Design and Sample Collection ...... 59 Chemical Analyses ...... 63

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IV. MATERNAL PROVISIONING OF DEFENSIVE STEROIDS BY RHABDOPHIS TIGRINUS Results ...... 67 Prey Chemistry and Preference ...... 67 Dams Fed Nontoad Prey ...... 69 Dams Fed Toads ...... 77 Discussion ...... 88 Acknowledgments ...... 94

V. CONCLUSIONS...... 96

REFERENCES...... 99

APPENDIX A. NMR AND MS DATA ON BUFADIENOLIDES ...... 109

VITA...... 128

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Table Page

2.1. HPLC Retention Times of Bufadienolide Compounds in Nuchal Gland Fluid of Rhabdophis tigrinus and Parotoid Gland Secretion of Toads ...... 23

2.2. Bufadienolides in Nuchal Gland Fluid of Rhabdophis tigrinus Dams and Their Hatchlings, Fed Controlled D ie ts...... 33

3.1. Presence or Absence of Bufadienolides in Hatchling Rhabdophis tigrinus from Kinkazan, Fed Controlled Diets ...... 48

3.2. Bufadienolide Compounds in Hatchling Rhabdophis tigrinus from Kinkazan and Ishima, Fed Controlled Diets ...... 49

3.3. Presence of Bufadienolides in Hatchling Rhabdophis tigrinus from Ishima, Fed Controlled Diets ...... 52

4.1. Bufadienolides in Nontoad-Fed Rhabdophis tigrinus Dams and Their Hatchlings Fed Controlled Diets ...... 70

4.2. Bufadienolides in Toad-Fed Rhabdophis tigrinus Dams and Their Hatchlings Fed Controlled Diets ...... 79

A. 1. NMR and MS Data for Bufadienolide Compound 1 ...... I ll

A.2. NMR and MS Data for Bufadienolide Compound 2 ...... 112

A.3. NMR and MS Data for Bufadienolide Compound 3 ...... 113

A.4. NMR and MS Data for Bufadienolide Compound 4 ...... 114

A. 5. NMR and MS Data for Bufadienolide Compound 5 ...... 115

A.6. NMR and MS Data for Bufadienolide Compound 6 ...... 116

A.7. NMR and MS Data for Bufadienolide Compound .7 ...... 117

A. 8. NMR and MS Data for Bufadienolide Compound .8 ...... 118

A.9. NMR and MS Data for Bufadienolide Compound .9...... 119

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Table Page

A.10. NMR and MS Data for Bufadienolide Compound 10 ...... 120

A. 11. NMR and MS Data for Bufadienolide Compound 11 ...... 121

A.12. NMR and MS Data for Bufadienolide Compound 12 ...... 122

A. 13. NMR and MS Data for Bufadienolide Compound 13 ...... 123

A.M. NMR and MS Data for Bufadienolide Compound 14 ...... 124

A. 15. NMR and MS Data for Bufadienolide Compound 15 ...... 125

A. 16. NMR and MS Data for Bufadienolide Compound 16 ...... 126

A.17. NMR and MS Data for Bufadienolide Compound 17 ...... 127

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LIST OF FIGURES

Figure Page

2.1. Nuchal glands of Rhabdophis tigrinus...... 13

2.2. Proton NMR spectra of prey fed to hatchling Rhabdophis tigrinus...... 20

2.3. Bufadienolides and identified from parotoid gland secretions of fowleri and B. terrestris...... 22

2.4. Proton NMR spectra of nuchal gland fluid from adult and hatchling Rhabdophis tigrinus...... 25

2.5. Seventeen bufadienolides isolated from the nuchal gland fluid of Rhabdophis tigrinus...... 28

2.6. HPLC chromatograms of pooled nuchal gland fluid from toad-fed hatchling Rhabdophis tigrinus bom to chemically undefended dam s ...... 29

2.7. HPLC chromatograms of nuchal gland fluid from the chemically defended Rhabdophis tigrinus dam and her offspring, indicating presence of identified bufadienolides ...... 30

3.1. Hatchling Rhabdophis tigrinus on Ishima ...... 41

3.2. HPLC chromatograms of nuchal gland fluid from Rhabdophis tigrinus...... 45

3.3. UV absorbance spectrum of a bufadienolide ...... 47

3.4. Aromatic region of 'H-NMR spectra from the nuchal gland fluid of Rhabdophis tigrinus hatchlings from clutch #2 laid by a dam collected on Kinkazan Island, which lacks toads ...... 50

3.5. Aromatic region of 'H-NMR spectra from the nuchal gland fluid of Rhabdophis tigrinus hatchlings from clutch #12 laid by a dam collected on Ishima, which has a dense population of toads ...... 53

4.1. Arrangement of cages for hatchling Rhabdophis tigrinus in the feeding trials ...... 62

4.2. HPLC chromatograms of nuchal gland fluid from Rhabdophis...... 66

4.3. Aromatic region of 'H-NMR spectra of prey extracts...... 68

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Figure Page

4.4. Aromatic region of 'H-NMR spectra of the nuchal gland fluid of Rhabdophis tigrinus dam #1 and three of her hatchlings ...... 71

4.5. Aromatic region of 'H-NMR spectra from Rhabdophis tigrinus dam #2 and three of her hatchlings ...... 73

4.6. Aromatic region of 'H-NMR spectra from Rhabdophis tigrinus dam #4 and three of her hatchlings ...... 74

4.7. Aromatic region of 'H-NMR spectra from Rhabdophis tigrinus dam #6 and three of her hatchlings ...... 75

4.8. Aromatic region o f 1 H-NMR spectra from Rhabdophis tigrinus dam #7 and three of her hatchlings ...... 76

4.9. Aromatic region o f 1 H-NMR spectra from Rhabdophis tigrinus dam #9 and three of her hatchlings ...... 78

4.10. Aromatic region o f 1 H-NMR spectra from Rhabdophis tigrinus dam #3 and three of her hatchlings ...... 80

4.11. Aromatic region of 'H-NMR spectra from Rhabdophis tigrinus dam #5 and her two hatchlings ...... 82

4.12. Aromatic region o f 1 H-NMR spectra from Rhabdophis tigrinus dam #8 and three of her hatchlings ...... 83

4.13. Aromatic region o f 1 H-NMR spectra from Rhabdophis tigrinus dam #10 and three of her hatchlings ...... 85

4.14. Aromatic region of 'H-NMR spectra from Rhabdophis tigrinus dam #11 and three of her hatchlings ...... 86

4.15. Aromatic region o f 1 H-NMR spectra from Rhabdophis tigrinus dam #12, her two hatchlings, and one yolk sample ...... 87

A. 1. Bufadienolide compound 1 showing the numbering of carbons for which data are presented in Table A. 1 ...... 110

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CHAPTER I

INTRODUCTION

Predators of Toxic Prey

Predators that repeatedly consume toxic prey must be able to withstand the

negative effects of the toxins and, presumably, should also gain benefits from

consuming the prey item that outweigh the costs of its toxicity. If both toxic and

nontoxic prey are available in a predator’s habitat, one might expect that nontoxic prey

would be preferred by the predator. However, if the predator gains a specific benefit

from consuming toxic prey, such as becoming chemically defended by sequestering

ingested toxins, the toxic prey may be preferred. An additional benefit would result if

the predator is able to provision its offspring with such defensive toxins. Even if the

predator does not sequester the toxins from its prey, a benefit is still gained if the

predator can increase its prey base by exploiting a resource that is avoided by other

predators.

The ability to tolerate ingested toxins may impose physiological tradeoffs on

the predator, such as reduced growth rates or decreased locomotor ability. Monarch

butterflies (Danaus plexippus) consume the leaves of sandhill milkweed

(.Asclepias humistrata), which contain high concentrations of cardiac glycosides. The

butterflies gain the benefit of becoming chemically defended by consuming the toxic

, but the first-instar larvae suffer reduced mass, decreased growth rates, and lower

survival rates due to ingestion of the toxins (Zalucki et al., 2001). Among ,

some individual garter snakes (Thamnophis sirtalis) experience decreased locomotor

The model for this dissertation is the Journal of Chemical Ecology.

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speed due to the presence of toxin-resistant sodium channels, which are necessary to

survive ingestion of the newt granulosa (Brodie and Brodie, 1999a, 1999b;

Geffeney et al., 2002). These newts contain (TTX), a found in

other animals such as puffer fish ( Takifugu); Thamnophis sirtalis is the only

known that is able to survive the ingestion of Taricha (Nussbaum et al., 1983). The

snakes exhibit variability in their levels of resistance to tetrodotoxin, and those that

have the most TTX-resistant sodium channels in their skeletal muscle suffer the

greatest reduction in crawling speed (Geffeney et al., 2002). However, if the snake

becomes chemically defended from eating the toxic newts, other animals may avoid

consuming it, regardless of its inability to flee (Williams et al., 2004).

Most toads of the genus Bufo (Bufonidae) are not as toxic as Taricha, but they

contain numerous compounds that can be deadly to predators including mammals and

certain snakes (Hitt and Ettinger, 1986; Sakate and Oliveira, 2000). Their defensive

compounds are concentrated in granular glands of the skin and may also be present in

the blood, viscera, and eggs (Licht, 1968). Toads contain high concentrations of

defensive compounds in their large parotoid glands, which are aggregations of granular

glands behind the head (Hutchinson and Savitzky, 2004). Some compounds in the

parotoid secretion inhibit hydrolysis of ATP and ion transport by the sodium-potassium

pump (Lichtstein et al., 1992). The toxins may have hemotoxic, neurotoxic,

cardiotonic, cardiotoxic, vasoconstrictive, hypotensive, or other effects on predators

(Habermehl, 1981, 1992). Mammalian predators on toads often consume only the

internal organs, while discarding the parotoid glands or the entire skin to avoid

ingesting higher concentrations of toxins (Groves, 1980).

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Among the defensive compounds contained in the parotoid secretion of Bufo

are a family of cardiotonic steroids (Zelnik et al., 1964; Chen and Kovarikova, 1967;

Cannon and Hostetler, 1976). These steroids include bufadienolides and their

derivatives, which are synthesized by toads from cholesterol (Doull et al., 1951;

Siperstein et al., 1957; Porto and Gros, 1971; Porto et al., 1972). Bufadienolides, such

as , consist of a steroidal skeleton bound to an a-pyrone ring at Cl 7, whereas

bufotoxins are bufadienolides bound to suberylarginine at C3 (Meyer and Linde, 1971).

The unsaturated pyrone ring of these compounds is essential for their cardiotonic

effects (Habermehl, 1981). Bufadienolides act by inhibiting the sodium-potassium

pump in a -like fashion, resulting in greater intracellular calcium levels and

causing prolonged contraction of (Reuter et al., 2002; Blaustein et al.,

2003). In cats, bufadienolides have LD 50 values of 0.09-0.57 mg/kg depending on the

specific compound that is administered intravenously (Michl and Kaiser, 1963). The

levels of cardiotonic steroids in individual toads vary in response to the degree of salt

acclimation of the animals (Lichtstein et al., 1992) and to the presence or absence of

predators in the larval habitat (Benard and Fordyce, 2003).

Bufonid skin secretions also contain biogenic amines, which consist of

derivatives of phenylethylamine and tryptamine (Erspamer, 1954; Deulofeu and

Ruveda, 1971; Habermehl, 1981; Erspamer, 1994; Toledo and Jared, 1995). Those

derived from phenylalanine include the catecholamines epinephrine, norepinephrine,

epinine, and dopamine (Deulofeu and Ruveda, 1971). The indolalkylamines, derived

from tryptamine, include bufotenine, bufotenidine, bufothionine, bufoviridine, O-

methylbufotenine, and serotonin (Deulofeu and Ruveda, 1971).

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Dietary Toxin Sequestration

Some predators have evolved both tolerance to toxic prey and the ability to

recycle ingested toxins for use in their own defense against higher order predators.

There Eire numerous examples of dietary compounds being sequestered and used for

defense among invertebrates and anamniote vertebrates (Duffey, 1980; Nishida, 2002).

A familiar example among invertebrates is that of monarch butterflies (Danaus

plexippus), which modify and store ingested from milkweeds (Brower,

1984; Nelson, 1993). As another example, female fireflies in the genus Photuris

consume male fireflies of the related genus Photinus and sequester their lucibufagins,

which are structurally similar to the defensive bufadienolides of toads (Eisner et al.,

1997). Female Photuris convert and store ingested lucibufagins, which are toxic to

their predators (Gonzalez et al., 1999b; Knight et al., 1999). Nudibranch mollusks are

known to sequester metabolites from ingested corals (McPhail et al., 2001). A well-

known example among anamniote vertebrates is the sequestration of alkaloidal toxins

from ants, beetles, and millipedes by Neotropical poison (Dendrobatidae), their

Malagasy analogues (Mantellidae), and a few other anurans (Garraffo et al., 1993;

Daly, 1998; Spande et al., 1999; Jones et al., 1999; Daly et al., 2003). In some

dendrobatids, pumiliotoxin alkaloids are sequestered from formicine ants and are

stored unaltered in the skin of the frogs for defense (Daly et al., 1994; Saporito et al.,

2004a).

Despite being a well-known phenomenon among invertebrates and anamniote

vertebrates such as , there is a paucity of experimental evidence supporting

the sequestration of dietary toxins in amniotes (, , and mammals). As one

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of the few examples, New Guinean birds in the genera and Ifrita sequester

from ingested arthropods, most likely melyrid beetles in the genus

Choresine (Dumbacher et al., 1992; Dumbacher et al., 2000; Dumbacher et al., 2004).

The only reported sequestration of dietary toxins in snakes is the transitory retention of

tetrodotoxin (TTX) by Thamnophis sirtalis following the consumption of the TTX-

containing newt Taricha granulosa (Williams et al., 2004). TTX was detected in the

blood, liver, kidney, heart, and skeletal muscle of Thamnophis sirtalis as early as one

hour following the consumption of Taricha (Williams et al., 2004). In fact,

Thamnophis sirtalis retains enough TTX in its liver weeks after consuming one

Taricha to make the snake dangerous or deadly to its avian and mammalian predators

(Williams et al., 2004).

Bufophagous Snakes

Snakes that consume toads, either occasionally or preferentially, are referred to

as bufophagous. Unlike mammalian predators that can selectively consume the internal

organs of toads while avoiding the highly toxic skin, snakes must consume their prey

whole. Therefore, bufophagous snakes must tolerate the physiological effects of high

concentrations of toxic compounds in whole toads. Ingestion of toads or their eggs has

resulted in sickness or death of humans, mice, dogs, and nonbufophagous snakes such

as Elaphe, Opheodrys, and Salvadora (Licht, 1967, 1968; Hitt and Ettinger, 1986;

Sakate and Oliveira, 2000; Savitzky et al., unpublished data). However, there are many

of bufophagous snakes in the derived superfamily Colubroidea that consume

whole toads with no ill effects.

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Bufophagous snakes occur in three families — Viperidae, Elapidae, and

Colubridae — and many share common morphological and antipredatory attributes. An

apparent commonality among bufophagous snakes is the presence of enlarged adrenal

glands, although the significance of this morphological trait is unknown (Smith and

White, 1955; Spaur and Smith, 1971; Savitzky et al., unpublished data). Batesian

mimicry seems to be used by many bufophagous snakes as well. Some examples of

nonvenomous, bufophagous snakes that are patterned like venomous ones include

species of Heterodon, Lystrophis, Xenodon, and Waglerophis (Greene, 1997).

Elaborate antipredatory behaviors, such as letisimulation (death-feigning), are

also shared among many bufophagous snakes. Death-feigning has been reported in the

African elapid Hemachatus (Hulme, 1951; Branch, 1998), as well as many

bufophagous colubrids. The Asian Rhabdophis, all three species of the North American

Heterodon, the Eurasian Natrix, and the South American Lystrophis all exhibit death-

feigning as a defense (MacDonald, 1947; Edgren, 1955; Hamilton and Pollack, 1956;

Schmidt and Inger, 1957; Broadley, 1958; Myers and Arata, 1961; Heusser and

Schlumpf, 1962; Platt, 1969; Gehlbach, 1970; McDonald, 1974; Mutoh, 1983;

Burghardt and Greene, 1988; Mori et al., 1996). While death-feigning, Heterodon

platirhinos may experience bradycardia (MacDonald, 1947). Interestingly, the North

American Masticophis, which is not bufophagous but does prey on Heterodon, also has

been reported to feign death (Hamilton and Pollack, 1956; Tucker, 1989). The African

anuran specialist Causus, a viperid, does not death-feign, but it exhibits the same

elaborate inflation and striking behaviors as the New World Heterodon and Xenodon

(Greene, 1997).

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Rhabdophis tigrinus

The Yamakagashi,Rhabdophis tigrinus (Serpentes: Colubridae: Natricinae)

occurs in southeastern , , southeastern , Korea, the large Japanese

islands of Honshu, Shikoku, and Kyushu, and some of the smaller Japanese islands

(Nakamura and Ueno, 1963; Paik and Yang, 1986; Toriba and Sawai, 1990; Zhao and

Adler, 1993). R. tigrinus primarily consumes frogs, including bufonids such as Bufo

japonicus (Fukada, 1959; Moriguchi, 1982; Mori and Moriguchi, 1988). Unfed

hatchlings display higher tongue flicking scores for bufonid extracts than for other prey

extracts, demonstrating an innate preference for toads (Mori, 2004). However, R.

tigrinus occurs on some islands where toads are absent, reducing the prey base for

those populations. Even in regions where Rhabdophis and Bufo are sympatric, the

availability of toads as prey may be highly seasonal. By the time the eggs ofR. tigrinus

hatch around late August, the toads that metamorphosed the previous spring may be

too large for hatchling snakes to ingest (Mori, 2004). Thus, it is possible that a juvenile

R. tigrinus would not encounter a toad of ingestible size until the following spring,

when newly metamorphosed toads are available. Also at this time of year, juveniles of

R. tigrinus consume many Bufo tadpoles (Goris and Maeda, 2005).

Nuchal Glands

Rhabdophis tigrinus possesses defensive glands on its neck known as nuchal

glands, which were first described by Kenji Nakamura in 1935. These glands occur in

12 species in the Asian natricine genera Rhabdophis, Macropisthodon, and Balanophis

(Nakamura, 1935; Smith, 1938; Jiang and Zhao, 1983; Stuebing and Lian, 2002). R.

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tigrinus has a series of about 10-20 pairs of nuchal glands that begins a few millimeters

behind the head and extends caudally (Nakamura, 1935; Smith, 1938; Toriba and

Sawai, 1990). The nuchal glands develop in the dermis and are of mesodermal origin

(Fukada, 1992a). They are deeply embedded in the stratum compactum and are

surrounded by connective tissue that penetrates the glands, but they lack direct

muscular attachment to assist in expelling their contents (Nakamura, 1935; Roberts,

2000). The nuchal glands consist of vacuolated cells and blood capillaries, but lack

lumina and ducts (Nakamura, 1935; Smith, 1938; Roberts, 2000). When R. tigrinus is

threatened, it arches or flattens its neck, directing the two parasagittal rows of nuchal

glands toward its attacker (Smith, 1938; Mori and Burghardt, 2001). When pressure is

applied to the nuchal region, the contents of the glands escape through ruptures of the

thin skin between overlying scales, in a holocrine fashion (Nakamura, 1935; Roberts,

2000). The pressure required to expel the glands’ contents may arise internally from the

force of the snake’s axial muscles, or externally by manually squeezing the glands or

presumably from a predator’s bite.

The nuchal gland fluid of Rhabdophis tigrinus is an irritant that serves a

defensive function (Nakamura, 1935). The use of *H-NMR and 13C-NMR spectroscopy

revealed that the nuchal gland fluid contains tetra-hydroxylated bufadienolides and the

tri-hydroxylated bufadienolide gamabufotalin, which is also found in the parotoid

glands of toads (Akizawa et al., 1985a; Akizawa et al., 1985b). Other tissues of R.

tigrinus have been reported to lack bufadienolides, and the nuchal glands of

Rhabdophis are the only ophidian structures known to possess cardiotonic steroids

(Akizawa et al., 1985a). The nuchal gland fluid irritates mucous membranes and

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 9

reportedly causes corneal injury when splashed into the eyes of humans, rabbits, or

dogs (Nakamura, 1935; Kitazume, 1953; Kawashima, 1957,1959; Suzuki, 1960; Asahi

et al., 1985). Most reported natural predators on R. tigrinus are hawks, but fish,

salamanders, raccoon dogs, and other snakes have also been reported to prey on this

species (Tanaka and Mori, 2000). Field observations of encounters between

Rhabdophis and its predators are lacking, but it is hypothesized that contact between

nuchal gland fluid and the eyes or mouth of a or mammal serves as an effective

deterrent (Mori, 2004).

Despite containing some of the same toxins, the parotoid glands of toads and

the nuchal glands of Rhabdophis tigrinus exhibit very different morphologies and,

presumably, differ physiologically. The parotoid glands synthesize cardiotonic steroids

(bufadienolides and bufotoxins) from cholesterol and other precursor molecules within

secretory syncytia (Doull et al., 1951; Siperstein et al., 1957; Porto and Gros, 1971).

These precursor molecules presumably are delivered to the parotoid glands by

subcutaneous capillaries that lie basal to the myoepithelium (Hutchinson and Savitzky,

2004). Nuchal glands, however, are believed to be incapable of producing these

compounds because they lack a secretory epithelium and their cells lack the Golgi

apparatus and smooth endoplasmic reticulum (Roberts, 2000). This unusual

morphology suggests that the snakes do not synthesize bufadienolides, but rather

supports the hypothesis, first proposed by Akira Mori, that R. tigrinus sequesters

bufadienolides in the nuchal glands from toads that are consumed as prey.

Furthermore, the nuchal glands contain dense networks of capillaries that permeate the

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 10

glands, suggesting that prey compounds are delivered to the glands via the blood

(Savitzky and Mori, unpublished data).

In addition to death-feigning (see above), Rhabdophis tigrinus employs a suite

of antipredator behaviors that involve the nuchal glands, including neck flattening,

neck arching, neck butting, and directing the nuchal region toward the predator

(Fukada, 1961; Mori et al., 1996). Among natricines, behaviors such as these have only

been observed in snakes that possess nuchal glands (Mori, 2004), however neck

arching has also been observed in Masticophis Jlagellum (Colubrinae; Stitt, 2003),

which lacks nuchal glands. Defensive displays involving the nuchal glands are used

more often than flight among R. tigrinus that occur sympatrically with toads and when

snakes are exposed to low temperatures (Mori et al., 1996; Mori and Burghardt, 2000,

2001). Furthermore, hatchling R. tigrinus reared on toads use their nuchal glands in

defense more often than hatchlings fed nontoad diets, lending support to the

sequestration hypothesis and suggesting that the snakes can assess their chemically

defended status (Mori, 2004).

Thus, several lines of evidence suggest that Rhabdophis tigrinus does not

synthesize the bufadienolides found in its nuchal glands and may instead sequester

those compounds from ingested toads. The lack of a secretory epithelium and secretory

organelles in the nuchal glands (Roberts, 2000) lends morphological support to this

hypothesis, and behavioral evidence demonstrates that the use of nuchal glands in

defensive displays varies with the presence or absence of toads at a site (Mori et al.,

1996; Mori and Burghardt, 2000). The chapters that follow present chemical evidence

from three dietary experiments, which cumulatively confirm the sequestration of

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. dietary bufadienolides in nuchal glands by R. tigrinus. Chapter II provides the initial

evidence for the dependence of R. tigrinus on dietary toads for the accumulation of

bufadienolides in nuchal gland fluid. Chapter III demonstrates the lack of defensive

compounds in nuchal glands of R. tigrinus from an island lacking toads, as well as the

presence of bufadienolides in R. tigrinus from an island with a dense population of

toads. Finally, Chapter IV provides evidence for the maternal provisioning of defensive

bufadienolides by R. tigrinus.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 12

CHAPTER II

DIETARY TOXIN SEQUESTRATION IN RHABDOPHIS TIGRINUS

Introduction

Many invertebrates are known to sequester dietary toxins for defensive

purposes (Duffey, 1980; Nishida, 2002), including such classic cases as milkweed

insects (Brower, 1984) and sea slugs (McPhail et al., 2001). However, vertebrate

examples of dietary toxin sequestration are rare. The brilliantly colored Neotropical

poison frogs (Dendrobatidae), their Malagasy analogues (Mantellidae), and a few other

anurans sequester alkaloidal toxins from ants, beetles, and millipedes (Garraffo et al.,

1993; Daly, 1998; Spande et al., 1999; Jones et al., 1999; Daly et al., 2003; Saporito et

al., 2004b). Two genera of New Guinea birds, Pitohui and Ifrita, apparently also

sequester alkaloids from arthropod prey (Dumbacher et al., 1992; Dumbacher et al.,

2000; Dumbacher et al., 2004). Some populations of garter snakes ( Thamnophis

sirtalis) accumulate tetrodotoxin after consuming newts ( Taricha granulosa), rendering

the snake's tissues toxic for several weeks as the compound gradually clears from the

tissues (Williams et al., 2004). Here we report on a sophisticated system of chemical

sequestration in the Asian natricine snake Rhabdophis tigrinus.

Rhabdophis tigrinus possesses a series of paired structures known as nuchal

glands in the dorsal skin of the neck (Figure 2.1; Nakamura, 1935; Smith, 1938; Toriba

and Sawai, 1990; Fukada, 1992b; Roberts, 2000). The glands function in antipredator

defense and are associated with specific behaviors that direct the nuchal region toward

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 2.1. Nuchal glands of Rhabdophis tigrinus. (a) Snake in typical defensive posture

(“neck arch”), with head bent and dorsal skin of neck exposed to predator. Arrow

indicates ridge formed by the underlying nuchal glands, (b) Vascular cast of skin in

ventral view, showing the dense reticulum of capillaries in the paired nuchal glands.

Blood vessels have been filled with yellow latex and the surrounding tissues have been

cleared with methyl salicylate. Anterior is toward the left. Bar, 5 mm. (c) Transverse

section through a pair of nuchal glands, showing the absence of a secretory epithelium,

lumen, or duct. The blue tissue is dermal collagen, which forms a dense capsule around

each gland. The glands empty by rupturing through the thin skin between adjacent

scales. Trichrome stain. Bar, 1 mm.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 14

an attacking predator (Smith, 1938; Fukada, 1992b; Mori et al., 1996; Mori and

Burghardt, 2001). The contents of the nuchal glands have been shown to irritate

mucous membranes and cause comeal injuries (Nakamura, 1935; Kitazume, 1953;

Kawashima, 1957, 1959; Suzuki, 1960; Asahi et al., 1985), and the glands are known

to contain bufadienolides (Akizawa et al., 1985a; Akizawa et al., 1985b).

Bufadienolides are cardiotonic steroids that act by inhibiting the sodium-potassium

pump in a digitalis-like fashion (Melero et al., 2000). The steroidal toxins in nuchal

glands are similar to those found in the skin secretions of toads (Bufonidae; Erspamer,

1994), which are often consumed by R. tigrinus (Mori and Moriguchi, 1988).

Whereas toads are capable of producing bufadienolides from cholesterol

precursors (Doull et al., 1951; Siperstein et al., 1957; Meyer, 1966; Porto and Gros,

1971; Porto et al., 1972; Erspamer, 1994), several lines of evidence suggest that snakes

in the genus Rhabdophis do not synthesize their defensive bufadienolides. Compared to

individuals that occur sympatrically with toads, R. tigrinus from a toad-free island

(Kinkazan) engage less frequently in defensive displays that emphasize the nuchal

glands and more often flee when threatened (Mori and Burghardt, 2000). This suggests

that Kinkazan snakes lack toxins in their nuchal glands and have evolved to rely more

heavily on flight to escape predation. Histological and ultrastructural evidence reveals

that nuchal glands lack secretory epithelia and certain organelles typical of secretory

cells, such as the Golgi apparatus and endoplasmic reticulum (Roberts, 2000).

Furthermore, a dense network of capillaries in the nuchal glands suggests that

bufadienolides may be transported to the glands via the plasma (Figure 2.1).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 15

We tested the sequestration hypothesis by rearing Rhabdophis tigrinus

hatchlings from four different dams (mothers) on controlled diets either containing or

lacking toads. Our objectives were to assess whether unfed hatchlings contain

bufadienolides in their nuchal glands and to determine whether the hatchlings are

capable of sequestering these toxins from ingested toads. The results of this experiment

provide the first chemical evidence that the defensive system of R. tigrinus relies upon

the sequestration of bufadienolide toxins from bufonid prey. Further evidence is

presented suggesting that female snakes possessing high levels of bufadienolides can

provision their ingestively naive offspring with these defensive compounds.

Methods

Experimental Design and Sample Collection

Four gravid female Rhabdophis tigrinus were collected in Kyoto and Okayama

Prefectures, Japan. They were fed only nonbufonid frogs that lack bufadienolides

(Rhacophorus arboreus and/or Rana nigromaculata) before being shipped to the

United States along with their clutches in July 2004. Nuchal gland fluid was collected

from the chemically defended dam (#4) in the U.S. after being fed goldfish ( Carassius

auratus)', the other three dams (#1-3) appeared too emaciated after oviposition to be

subjected to sampling. Nuchal gland fluid was collected from these three dams post

mortem, at which time they had only been fed nonbufonid frogs and fish in captivity.

To collect nuchal gland fluid we placed a section of a laboratory tissue

(Kimwipe; Kimberly-Clark) over the dorsal surface of the neck and gently squeezed

until the glands ruptured through the skin. The tissue was then inserted into a vial of

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 16

HPLC-grade methanol with forceps, sealed with a teflon-lined cap, and stored at -20 °C

for later analysis. We changed gloves, rinsed forceps in deionized water, and dried the

forceps with a Kim wipe between each individual. At least one control vial (Kim wipe

without glandular fluid, placed into methanol with forceps) was prepared near the end

of the sampling sequence on each day that samples were collected.

The four clutches of eggs were incubated on moist vermiculite in a 30 °C

incubator until hatching. A small volume of nuchal gland fluid was collected from

three unfed hatchlings from each clutch. We were careful to express only a few glands,

to ensure that we could resample these individuals at later dates; nuchal glands do not

appear to regenerate after they have ruptured, at least initially (personal observation).

Sampled hatchlings were randomly assigned to different feeding groups. One group

was fed only fishes ( Pimephales promelas), another was fed nonbufonid frogs (juvenile

Scaphiopus holbrookii or metamorphic Spea multiplicata), and the third group was fed

toads ( Bufo fowleri, B. quercicus, and/or B. terrestris). We used more than one species

of Bufo as prey because the availability of toads was limited. All individuals in the

toad-fed group received metamorphic B. terrestris until our supply was exhausted. We

then fed this group juvenile B. fowleri, cut portions of previously frozen adult B.

terrestris, and/or juvenile B. quercicus. Hatchlings that were not sampled in the naive

state were fed fish, toads, or both. After the snakes had fed for several days, we

resampled their nuchal gland fluid. Some individuals were switched from one of the

nontoad-fed groups to the toad-fed group to detect the effect of a change in diet on the

composition of nuchal gland fluid. A few hatchlings survived long enough to allow

three samples of nuchal gland fluid to be collected, but most died after the second

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 17

sample was taken. Sampling of nuchal gland fluid was unlikely to have been a factor in

the deaths of these snakes, which are notoriously difficult to maintain in captivity for

extended periods, either as adults or young. Our animal use protocols were approved

by the Old Dominion University IACUC.

Samples of prey items were also collected for chemical analyses. For juvenile

and adult toads, we collected skin secretions either by squeezing the parotoid glands

directly onto a Kimwipe or by stimulating the skin with a transcutaneous amphibian

stimulating device (TAS; Grant and Land, 2002). We also prepared whole-body

extracts of the fish and whole-skin or whole-body extracts of the small frogs and toads.

Chemical Analyses

Methanol extracts of nuchal gland fluid and prey items were evaporated to

dryness, reconstituted in deuterated methanol (methyl-d3 alcohol-d), and analyzed with

proton nuclear magnetic resonance spectroscopy ('H-NMR) prior to fractionation to

determine the presence of bufadienolides and other compounds of interest (Taggi et al.,

2004). Initial analyses were used to determine the presence or absence of

bufadienolides by identifying NMR-spectroscopic signals corresponding to the

pyranone protons of bufadienolides. In samples of nuchal gland fluid where the identity

of NMR-spectroscopic signals could not unambiguously be determined on the basis of

one-dimensional proton NMR spectra alone (e.g., in cases of severe signal overlap in

the aromatic region), additional phase-sensitive dqf-COSY spectra were acquired

(Taggi et al., 2004). NMR analyses were performed with Unity INOVA 500 and 600

MHz spectrometers (Varian, Inc.) equipped with Oxford magnets (Oxford Instruments)

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 18

and HCN or DBG probes. Preliminary tests indicated that an additional

dichloromethane extraction did not elute additional compounds from samples of nuchal

gland fluid, so this step was eliminated from the protocol. Further corroboration for the

presence or absence of bufadienolides in nuchal gland fluid was obtained via HPLC

analyses with UV-detection, monitoring wavelengths between 200 and 400 nm.

Bufadienolides have characteristic UV-spectra with absorption maxima at

approximately 200 and 300 nm. For these analyses, an Agilent 1100 series HPLC

equipped with a quaternary pump, diode array detector, reversed phase 25 cm x 10 mm

Supelco Discovery® HS C l8 column, and an automated sample injector was used. A

solvent gradient system was employed, starting with a 20:80 methanol:water mixture,

the methanol content of which was increased linearly from 20% at three minutes to

100% at 25 minutes, using a constant flow rate of 3.4 ml/min. The injection volumes of

the samples ranged from 10-25 pL.

For isolation and identification of individual bufadienolides, samples of nuchal

gland fluid from Rhabdophis tigrinus and parotoid gland secretion from toads were

fractionated using the same HPLC system as above. Fractions of interest were collected

with a Foxy® 200 fraction collector. Individual fractions thus obtained were

characterized with two-dimensional NMR spectroscopy to elucidate chemical

structures of the bufadienolides. In addition, isolated bufadienolides were analyzed by

positive-ion electrospray mass spectrometry using a Micromass Quattro II

spectrometer.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 19

Results

Chemical analyses confirmed the presence of bufadienolides in the parotoid

secretions of Bufo terrestris and B. fowleri, as well as in whole-skin extracts of juvenile

B. quercicus, all of which were used as prey for toad-fed Rhabdophis tigrinus

hatchlings (Figure 2.2). Major components of the bufadienolide mixtures produced by

B. fowleri and B. terrestris, which had not previously been analyzed in detail, were

isolated via HPLC and subsequently identified by NMR and mass spectroscopy (Figure

2.3, Table 2.1). Surprisingly, bufadienolides were absent in the skin of metamorphic B.

terrestris, which were also fed to hatchling snakes in the toad-fed group. Later analyses

of whole-body extracts revealed that most metamorphic B. terrestris lack

bufadienolides altogether, although some have minute amounts (Figure 2.2). We also

confirmed that bufadienolides were absent from whole-body extracts of Pimephales

promelas and skin extracts of Spea multiplicata and Scaphiopus holbrookii, which

served as negative controls in the sequestration studies (Figure 2.2).

The nuchal gland fluid from Rhabdophis tigrinus varied in color and opacity

both among individuals and through time within an individual; this variation was most

easily observed in adults. Samples containing bufadienolides tended to be colorless to

light yellow and translucent, or white and opaque. Samples lacking bufadienolides

often were brilliant yellow and translucent. Using HPLC-MS and NMR, we identified

the yellow compounds as lumichrome (7,8-dimethylalloxazine), lumiflavin (7,8,10-

trimethylisoalloxazine), and their derivatives (Goto, 1975). Lumichrome was the major

component in the yellow nuchal gland fluid; lumiflavin was present in lesser quantity.

Both compounds are degradation products of riboflavin (vitamin B2).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 20

: t o o Adult Bufo terrestris m

500 £

400

300

•200 •; 100

10,0 8.0 6.0 4.0 2.0 0.0

Juvenile Bufo fowleri 20 10 ju ULiJu_L -*L 0 £

Juvenile Bufo quercicus

Metamorphic Bufo terrestris <-500

8.0 7.5 7.0 6.5 6.0 c Fish (Pimephales)

I , Yu I, itfi i .jlik. i <. ^ j .... m.—*. 1-5 5

Nonbufonid 2 (Spea) JO 0 is •5^ to Nonbufonid oc frog (Scaphiopus)

.UX o - ju j u - i M i------8.0 7.5 7.0 6.5 6.0 ppm

Fig. 2.2. Proton NMR spectra of prey fed to hatchling Rhabdophis tigrinus. Gray bars

highlight three areas diagnostic of bufadienolides.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 21

Fig. 2.2. Continued, (a) Full spectrum showing evidence of large quantities of

bufadienolides in the parotoid gland secretion from an adult toad (Bufo terrestris),

pieces of which were fed to some hatchling R. tigrinus. (b) Aromatic region of spectra

from the parotoid secretion of a juvenile B. fowleri, whole-skin extract of a juvenile B.

quercicus, and pooled whole-body extracts of three metamorphic B. terrestris.

Bufadienolides are clearly present in the former two samples, whereas the metamorphic

B. terrestris contain only trace quantities of these compounds. The horizontal black bar

indicates peaks that extend beyond the displayed scale, (c) Aromatic region of spectra

from extracts of nonbufonid prey fed to hatchling R. tigrinus. As expected, whole-body

extracts of the fish (Pimephales promelas) and skin extracts of the nonbufonid frogs

(Spea multiplicata and Scaphiopus holbrookii) show no evidence of bufadienolides.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 22

NH NH A OH hn ^ nh 2 O HN.

COOH COOH

T2: R,=OH, R2=CHO. R3= H T3: R,=H, R2=CH3. R3=OH IS: R,=OH, R2=CH3, R3=H

OH j [ HN N H , HO

{Chf2)n/V* HN% HO COOH

T4: n=1 T7 T8: R=OH T5; n=2 T9: R=H

F ig . 2 .3 . Bufadienolides and bufotoxins identified from parotoid gland secretions of

Bufo fowleri and B. terrestris. Major components from B. fowleri include compounds

T1-T6, whereas those in B. terrestris include T3 and T6-T9. Compounds Tl, T4, and

T6 are new natural products.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. T a b l e 2.1. HPLC R etention Times of Bufadienolide Compounds in N uchal G land Fluid of Rhabdophis tigrinus and Parotoid G land Secretion of Toads

Retention Compound time (min.) 1 14.41 2 15.31 3 17.14 4 17.35 5 17.86 6 18.76 7 18.76 8 19.64 9 21.10 10 22.16 11 24.04 12 24.56 13 25.63 14 26.12 15 27.39 16 28.96 17 30.31 T1 26.62 T2 27.52 T3 27.64 T4 28.41 T5 30.11 T6 30.78 T7 21.90 T8 22.90 T9 27.85

Bufadienolides are identified by compound number (see

Figures 2.3, 2.5); compounds from toads are prefixed by

the letter T.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 'H-NMR spectroscopic analyses revealed substantial differences in the quantity

of bufadienolides present in the nuchal gland fluid of different snakes. Of the four

dams that had not been fed toads in captivity, two had no bufadienolides even though

large volumes of fluid were obtained from their nuchal glands; the fluid consisted of

water, trace amounts of nonbufadienolide steroids (primarily cholesterol), lipids, and

traces of carbohydrates (Figure 2.4). Another dam exhibited only trace amounts of

bufadienolides. The absence of substantial quantities of bufadienolides in these dams

suggests that they had not consumed toads in their natural habitat recently or at any

time in their lives.

Correspondingly, none of the sampled offspring of these three dams exhibited

bufadienolides in their nuchal glands upon hatching (Figure 2.4). Furthermore, no

bufadienolides were detected in subsequent samples of nuchal gland fluid from these

offspring when they were fed fish or nonbufonid anurans (Figure 2.4). No other

potentially defensive compounds, such as biogenic amines, were found in the nuchal

gland fluid of Rhabdophis tigrinus, although these compounds are known from

pelobatid and bufonid frogs (Deulofeu and Ruveda, 1971; Erspamer, 1994), which

were used as prey for some hatchlings. However, snakes from all three clutches that

were fed toads rapidly and consistently accumulated bufadienolides in their nuchal

glands (Figure 2.4). For example, one hatchling that did not possess bufadienolides

after feeding on fish ( PimephalesprOrnelas') for 8.5 weeks exhibited these compounds

in its nuchal gland fluid three days after feeding on juvenile Bufo quercicus for two

consecutive days. One hatchling that was sampled three times over 64 days exhibited

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 25

a Dam #3. fed 20 nonbufonid prey Dam #1 10 h too 0 & Dc Hatchling, unfed U 2 - 50 Hatchling, fed fish !0 t . Hatchling, fed toads ; $ -0 ~c -2 T 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 8.0 7.5 7.0 6,5 6.0

Dam #4. fed Day 0 nonbufonid prey 150

100 100 50 0 50 _ .ikA. 0 § Day 15 (8 20 Hatchling, unfed 40 *In 0 ■20 1 JLu .. i...... 0 Hz'

F ig . 2.4. Proton NMR spectra of nuchal gland fluid from adult and hatchling

Rhabdophis tigrinus. (a) Full spectrum of dam #1 after being fed nonbufonid prey,

showing the absence of bufadienolides (note the absence of peaks in the three regions

diagnostic of bufadienolides, highlighted in gray), (b) Aromatic region of spectra from

dam #3 and her hatchlings. The dam lacked bufadienolides as did her hatchlings, which

only accumulated the toxins when fed toads.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2 6

Fig. 2.4. Continued, (c) Aromatic region of spectra from a hatchling bom to dam #3.

This individual lacked bufadienolides at hatching (day 0), but accumulated increasing

quantities as toads were consumed during the following two months, (d) Aromatic

region of spectra of a chemically defended dam (#4) and her offspring. Top spectrum:

Dam, showing presence of bufadienolides, despite having not been fed toads in

captivity. Middle spectrum: Unfed hatchling, which was provisioned with

bufadienolides by its chemically defended dam. Bottom spectrum: Hatchling fed North

American toads. This individual possessed bufadienolides provisioned from its mother

and sequestered additional types of bufadienolides from its bufonid prey.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2 7

continual accumulation of bufadienolides as additional toads were consumed (Figure

2.4). The results from these three clutches provide the first chemical evidence that

dietary toxins are sequestered by R. tigrinus from bufonid prey.

The fourth dam had very high concentrations of bufadienolides in her nuchal

glands, including one major bufadienolide and at least seven minor ones (Figure 2.4).

Interestingly, her offspring possessed bufadienolides in their nuchal glands

immediately upon hatching (Figure 2.4). The hatchlings from this clutch retained

bufadienolides in their nuchal glands for at least 8.5 weeks regardless of their diet in

the laboratory. However, toad-fed individuals accumulated additional types of

bufadienolides that they did not possess upon hatching (Figure 2.4).

The structures of bufadienolides in the nuchal glands of hatchlings and dams

were elucidated via HPLC fractionation of pooled nuchal gland fluid samples obtained

from toad-fed hatchlings bom to dam #4. Pure samples of individual bufadienolides

were isolated and subsequently characterized by MS and two-dimensional NMR-

spectroscopy. Seventeen bufadienolides were identified, six of which are new natural

products (Figure 2.5, Tables 2.1, A.1-A.17). Eight of these bufadienolides were

present only after the snakes had consumed North American toads (Compounds 1-4, 9,

12,13, 17; Figure 2.6). At least eight bufadienolides were naturally present in the

defended dam, six of which were provisioned to her offspring in amounts large enough

to be identified, although trace quantities of the other two bufadienolides may also

have been present (Figure 2.7). All of the bufadienolides identified in Rhabdophis

tigrinus lacked fatty acid or amino acid side chains. Notably, the suberylarginine side

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 28

R2 R, R4 *5 7 R,=H, R2=OH 1 R ^H , R2=OH, R3=H, R4=OH, Rs=H 11: R^OH, R2=H 2 Rt=H, R2=OH, R3=H, R4=H, Rs=OH 3 R^OH, R2=H, R3=OH, R4=H, R 5 R^OH, R2-H, R3=H, R4=OH, R5=OH 6 R^OH, R2=H, R3=H, R4=H, R5=OH 8 R^OH, R2=H, R3=H, R4=OH, R5=H 10 R^OH, R2=H, R3=H, R4=H, R5=H 12 R ^H , R2=OH, R3=H, R4=H, R5=H

H OH

M iR ^ O H ;!/: R,= H

F ig . 2 .5 . Seventeen bufadienolides isolated from the nuchal gland fluid of Rhabdophis

tigrinus. Compounds 2-6 and 9 are new natural products.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 2.6. HPLC chromatograms of pooled nuchal gland fluid from toad-fed hatchling

Rhabdophis tigrinus bom to chemically undefended dams. Retention time in minutes

(min) and UV-absorbance in milli-absorbance units (mAU) are displayed.

Bufadienolides are identified by number (see Figure 2.5). (a) Clutch #1. (b) Clutch #2

(c) Clutch #3.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 30

6/7 1400

1200

1000

800

600

400 15 200 T 8 i 11 1 0 JU-JL.t 5 i 16 b

z> < 6/7 E 20

c 250

200

150

100 6.7

10 15 20 25 30 35 . m m

Fig. 2.7. HPLC chromatograms of nuchal gland fluid from the chemically defended

Rhabdophis tigrinus dam and her offspring, indicating presence of identified

bufadienolides (see Figure 2.5).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 2.7. Continued. Retention time in minutes (min) and UV-absorbance in milli-

absorbance units (mAU) are displayed, (a) Dam #4 fed a nonbufonid diet in captivity,

(b) Pooled samples of unfed hatchlings bom to dam #4. (c) Pooled samples of toad-fed

hatchlings bom to dam #4.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. chain characteristic of bufotoxins was absent from the components of the nuchal gland

fluid. Bufotoxins are C3-acylated bufadienolides that occur as major components of the

skin secretions of both American and Japanese toads. Interestingly, the bufadienolides

identified from R. tigrinus are generally more highly hydroxylated than the

bufadienolides found in their prey (Figure 2.3). A summary of the bufadienolides in the

four dams and their offspring fed controlled diets is displayed in Table 2.2.

Discussion

Our studies provide the first direct chemical support for the hypothesis that

Rhabdophis tigrinus sequesters defensive bufadienolides from a dietary source. We

found that when R. tigrinus lack bufadienolides upon hatching, they must acquire those

toxins from ingested toads. In contrast, the presence of bufadienolides in the unfed

offspring of the chemically defended dam is best explained as maternal provisioning of

toxins sequestered by the dam from toads she ingested. Apparently, female R. tigrinus

with high concentrations of bufadienolides can provision their hatchlings with these

defensive compounds, presumably via the yolk. Subsequently, hatchlings from the

chemically defended clutch increased the diversity of bufadienolides in their arsenal

after they were fed North American toads, which differ from Japanese toads in some

components of their skin secretions. Interestingly, all of the undefended dams were

collected in Tomi-son (Okayama Prefecture), whereas the defended dam was from

Miyama-cho (Kyoto Prefecture). Toads (Bufo japonicus) are common in Miyama-cho,

but their local abundance in Tomi-son is not known, although that region is within the

species' range.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 33

Table 2.2. Bufadienolides in N uchal G land Fluid of R h a b d o p h i s t i g r i n u s Dams and Their H atchlings, Fed C ontrolled Diets

Clutch #1 Clutch #2 Clutch #3 Clutch #4 (N=l 1) (N=4) (N=8) (N=10)

Dam, None (n = l) 6£Z (n-1) None (n = l) fed fish, frogs

Hatchlings, None (n=3) None (n=4) None (n=3) unfed

Hatchlings, fed None (n=8) None (n=l) None (n=6) fish

Hatchlings, fed None (n=l) None (n=l) None (n=2) frogs

Hatchlings, fed toads

Bufadienolides are identified by compound number; see Figure 2.5. The most

abundant bufadienolide in each category is underlined. Compounds 6 and 7 co-elute

from the HPLC column, so they could not always be distinguished. N, number of

hatchlings per clutch; n, number of samples analyzed by NMR; most hatchlings were

sampled more than once to detect the effect of diet on nuchal gland composition.

Comparable samples were pooled to obtain adequate quantities of bufadienolides for

identification with HPLC. White background indicates absence of bufadienolides;

darkening shades of gray indicate increasing quantities of bufadienolides in individual

NMR samples, relative to each other.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 34

It was not possible to quantify precisely the amount of bufadienolides in

sequential samples of nuchal gland fluid because the fluid cannot be uniformly

expressed from the glands. However, any sampling bias would tend to favor larger

quantities of bufadienolides in the initial collection because individual glands

apparently do not regenerate after they rupture, at least not initially (personal

observation). This results in fewer glands being available for extraction in subsequent

samples. We attempted to standardize the volume of nuchal gland fluid collected at

each sampling period, but the third sample obtained from several hatchlings usually

resulted in noticeably smaller volumes of fluid. Therefore, the gradual accumulation of

bufadienolides observed in one toad-fed hatchling over three sampling periods almost

certainly represents a real phenomenon and not a sampling artifact.

The absence of C3-acylated bufadienolides (bufotoxins) in the nuchal gland

fluid suggests that Rhabdophis tigrinus selectively sequesters non-acylated

bufadienolides from toads or cleaves the suberylarginine side chains from ingested

bufotoxins prior to storage. Free genins, such as non-acylated bufadienolides, have

greater bioavailability and are more easily metabolized than cardiotonic steroids with

side chains, such as glycosides (Melero et al., 2000). Therefore, it is likely that non-

acylated bufadienolides are more easily sequestered by R. tigrinus than bufotoxins.

Chemical modification of sequestered bufadienolides may take place in the liver of R.

tigrinus after their uptake in the digestive tract.

The HPLC-MS and NMR data indicate that bufadienolides in the nuchal gland

fluid of Rhabdophis tigrinus are generally more polar than the steroidal toxins of toads

and have undergone additional hydroxylation after uptake by the snakes. Modification

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 35

of sequestered dietary toxins by hydroxylation has been reported in predaceous fireflies

(Gonzalez et al., 1999b), and dendrobatid frogs (Daly et al., 2003). In dendrobatids,

hydroxylation dramatically increases the toxicity of some sequestered alkaloids (Daly

et al., 2003). Removal of the hydroxyl group on C14 or its conversion to a C14-C15

epoxide group (as in compounds T8 and T9) has been shown to reduce the cardiotonic

activity of bufadienolides (Habermehl, 1981; Melero et al., 2000). However, the effect

of additional hydroxyl groups on the potency of cardiotonic steroids is dependent on

their specific positions and orientations (Melero et al., 2000). Thus, although it is

possible that the additional hydroxylation of bufadienolides by Rhabdophis increases

their toxicity, this remains to be tested.

Nothing is yet known of the processes of uptake and storage of bufadienolides

in Rhabdophis. However, the presence of dense capillary networks within the nuchal

glands suggests that the toxins are transported to those structures via the circulatory

system. This hypothesis is supported by a preliminary analysis of the plasma from one

female R. tigrinus, which was found to contain at least two bufadienolides (Hutchinson

et al., unpublished data).

The correlation between the yellow color of nuchal gland fluid and the absence

of bufadienolides may result from structural similarities between the yellow

compounds (lumichrome and lumiflavin) and the pyrone ring of bufadienolides. There

may be a protein in Rhabdophis plasma that transports bufadienolides from the

intestines or liver to the nuchal glands, similar to the globulins that bind certain cardiac

glycosides in mammalian plasma (Schoner, 2002). If bufadienolides out-compete

lumichrome and lumiflavin for binding and transport to the nuchal glands,

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 36

accumulation of the two riboflavin derivatives may be greater when dietary

bufadienolides are absent, resulting in the bright yellow glandular fluid of chemically

undefended R. tigrinus.

We do not yet know whether the bufadienolides persist indefinitely within the

nuchal glands or whether the toxins must be replenished by periodically ingesting

toads. In any event, our results raise important questions regarding the interplay among

diet, antipredator defense, and fitness in Rhabdophis tigrinus. Among amniote

vertebrates, the nuchal glands of R. tigrinus are the first defensive structures known to

be dependent upon sequestered dietary compounds.

Acknowledgments

We thank Trevor Adams, Jay F. Bolin, Jennifer Bradford, Matthew Close, Alan

Lemmon, Emily Moriarty Lemmon, Yohei Kadota, Koji Mochida, Aya Nakadai, Julie

Ray, Barbara A. Savitzky, Mamoru Toda, and Victor R. Townsend, Jr., for collecting

animals in the field; Shannon J. Davis, Dalyn Kuklock, Gabrielle T. Mueller, Julie M.

Ray, Erica M. Schmutzler, Koji Tanaka, and Kira White for animal care; Jacqueline

Grant for construction and advice on the TAS; and John Daly and Thomas Spande for

offering methodological advice. This work was supported by the National Science

Foundation (IBN-0429223 and IOB-0519458 to AHS and JM); Old Dominion

University (Dissertation Fellowship to DAH); the Society for Integrative and

Comparative Biology (Grant-in-Aid of Research to DAH); Sigma Xi, The Scientific

Research Society (Grant-in-Aid of Research to DAH); Kyoto University (Grant for

Biodiversity Research of the 21st Century COE A14 to AM and Dept, of Zoology); the

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 37

Kyoto University Museum (Visiting Faculty Award to AHS); and the Japan-US

Cooperative Science Program (Japan Society for the Promotion of Science to AM and

NSF INT-9513100 to GMB).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 38

CHAPTER III

GEOGRAPHIC VARIATION OF CHEMICAL DEFENSE

IN RHABDOPHIS TIGRINUS

Introduction

Chemically defended animals may synthesize their defensive toxins from

nontoxic precursors or sequester intact toxins from environmental sources, notably the

diet (Duffey, 1980; Brower, 1984; Daly et al., 1994; Gonzalez et al., 1999a; Nishida,

2002; Dumbacher et al., 2004; Saporito et al., 2004a; Williams et al., 2004). Animals

capable of synthesizing their own toxins would not be expected to vary in levels of

chemical defense over a geographic range unless there was genetic variation in the

ability to synthesize toxins or geographic variation in their suite of predators

(Thompson, 2005). Greater geographic variation may exist among animals dependent

on prey for sequestered chemical defenses, especially if the prey species is distributed

unevenly or has geographically variable toxicity itself (Hanifin et al., 1999; Thompson,

2005).

Rhabdophis tigrinus is an oviparous Asian snake (Colubridae: Natricinae) that

possesses unusual defensive glands on its neck (Nakamura, 1935; Smith, 1938). The

contents of the nuchal glands have been shown to irritate mucous membranes and

cause corneal injuries (Nakamura, 1935; Kitazume, 1953; Kawashima, 1957, 1959;

Suzuki, 1960; Asahi et al., 1985), and the glands are known to contain bufadienolide

steroids (Akizawa et al., 1985a; Akizawa et al., 1985b). Bufadienolides are cardiotonic

steroids that act by inhibiting the sodium-potassium pump in a digitalis-like fashion

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 39

(Melero et al., 2000). The steroidal toxins in nuchal glands are similar to those found in

the skin secretions of toads (Bufonidae; Erspamer, 1994), which are often consumed by

R. tigrinus (Mori and Moriguchi, 1988). Whereas toads are capable of synthesizing

their defensive bufadienolides from cholesterol (Doull et al., 1951; Siperstein et al.,

1957; Porto and Gros, 1971; Porto et al., 1972), R. tigrinus is dependent on dietary

toads from which it can sequester these compounds (Chapter II).

The majority of reported natural predators on Rhabdophis tigrinus are hawks,

but fish, salamanders, raccoon dogs ( Nyctereutes), and other snakes have also been

reported to prey on this species (Tanaka and Mori, 2000). Field observations of

encounters between R. tigrinus and its predators are lacking, but presumably contact

between the irritating nuchal gland fluid and the eyes or mouth of a bird or mammal

would serve as an effective deterrent (Asahi et al., 1985; Mori, 2004).

Rhabdophis tigrinus occurs in southeastern China, Taiwan, southeastern Russia,

Korea, the large Japanese islands of Honshu, Shikoku, and Kyushu, and some of the

smaller Japanese islands (Nakamura and Ueno, 1963; Paik and Yang, 1986; Toriba and

Sawai, 1990; Zhao and Adler, 1993). R. tigrinus primarily consumes frogs, including

the Japanese toad Bufo japonicus (Fukada, 1959; Moriguchi, 1982; Mori and

Moriguchi, 1988). Unfed hatchlings display higher tongue flick scores for bufonid

extracts than for other prey species, demonstrating a preference for toads in this species

(Mori, 2004). However, some islands where R. tigrinus occurs lack toads, limiting the

prey base for those populations.

In this study, we analyzed the chemistry of nuchal gland fluid of hatchling

Rhabdophis tigrinus from two small Japanese islands: Ishima (Tokushima Prefecture)

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4 0

where toads are abundant, and Kinkazan (Miyagi Prefecture) where toads are absent.

Snakes on Ishima include Achalinus spinalis, Amphiesma vibakari, Dinodon orientale,

Elaphe climacophora, Elaphe quadrivirgata, Euprepiophis conspicillatus (= Elaphe

conspicillata\ Utiger et al., 2002), Gloydius blomhoffii, and Rhabdophis tigrinus

(personal observation; Akira Mori, personal communication). Ishima harbors two

species of lizards ( Eumeces latiscutatus and Takydromus tachydromoides) and one

species of turtle ( Chinemys reevesii; personal observation; Akira Mori, personal

communication). Ishima also supports dense populations of anurans, including Bufo

japonicus, Hyla japonica, and Rana rugosa (personal observation; Akira Mori,

personal communication). In contrast, there are no toads on Kinkazan; the only anuran

present is Rana tagoi (Nagata and Mori, 2003). Snakes on Kinkazan include Elaphe

climacophora, Elaphe quadrivirgata, Euprepiophis conspicillatus, Gloydius

blomhoffii, and Rhabdophis tigrinus (Nagata and Mori, 2003), and there are no

mammalian predators on Kinkazan (Akira Mori, personal communication).

On a visit to Ishima in 2005, we noted that many hatchling Rhabdophis tigrinus

on that island possessed unusually large nuchal ridges, presumably formed by enlarged

nuchal glands (personal observation; Figure 3.1). This morphological feature

apparently correlates with a large quantity of bufadienolides sequestered from the

abundant toad population on Ishima.

In this study, we sought to determine any differences in chemical composition

in the nuchal gland fluid of hatchling Rhabdophis tigrinus from Kinkazan and Ishima,

which we reared on controlled diets. Our findings provide further support for the

hypothesis that R. tigrinus must sequester the defensive bufadienolides in its nuchal

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 41

F ig . 3.1. Hatchling Rhabdophis tigrinus on Ishima. Note the large nuchal ridge on the

dorsal surface of the neck, formed by underlying nuchal glands.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 42

glands from dietary toads. We also provide evidence for the maternal provisioning of

these compounds on Ishima, which harbors a dense population of toads.

Methods

Experimental Design and Sample Collection

The hatchling Rhabdophis tigrinus used in this study were the subjects of a

previous experiment on the effects of diet on antipredatory behavior. For that study, 12

gravid females were collected in 2002 on Kinkazan and Ishima (six females from each

island). They were transported to Kyoto University where they were fed only fish and

nonbufonid frogs that lack bufadienolides (Rana nigromaculata). Following

oviposition, the eggs were incubated under conditions approximating natural thermal

and light cycles until hatching. The temperature of the enclosure housing the eggs and

snakes was maintained between 25 and 30°C. The hatchlings were reared on controlled

diets of either exclusively nontoad prey that lacked bufadienolides or a combination of

nontoad prey and toads. The nontoad prey items used were fish ( Oryzias latipes),

nonbufonid frogs (Hyla japonica), and salamanders (Hynobius tokyoensis and

Hynobius lichenatus). The toads used as prey were metamorphic Bufo japonicus. Any

snakes that died prior to the start of the feeding experiment were frozen for future

chemical analyses. At the end of the experiment in spring of 2003, all remaining snakes

were frozen individually in plastic bags. Several metamorphic toads from the batch

used as prey were frozen as well.

In 2005, nuchal gland fluid was collected from the hatchlings after allowing

them to thaw. From each snake, we expressed all nuchal glands onto a portion of

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 43

Kimwipe (Kimwipes wipers S-200) while wearing nitrile, polyethylene, or latex

gloves; the Kimwipe was then placed into a vial of methanol with forceps and covered

with a teflon-lined cap. The forceps were rinsed in methanol from a glass pipette and

dried with a Kimwipe between each individual; gloves were also changed between

individuals. At least one control vial consisting of a Kimwipe in methanol was

prepared at the end of each sampling session. Hatchlings from all 12 clutches were

sampled (six clutches from Ishima and six from Kinkazan), but one clutch is

represented by only one sample. Two frozen metamorphic toads (0.12 g each) were cut

into pieces and extracted individually in methanol for chemical analyses. All samples

were stored at -20° C.

Chemical Analyses

Methanol extracts of nuchal gland fluid and prey items were shipped on dry ice

to Old Dominion University, stored at -20° C, and transported to Cornell University for

chemical analyses. All samples were evaporated to dryness, reconstituted in deuterated

methanol (methyl-d3 alcohol-d), and analyzed to determine the presence or absence of

bufadienolides. The unfractionated samples were analyzed initially with 'H-NMR on

Unity INOVA 400, 500, or 600 MHz spectrometers (Varian, Inc.) equipped with

Oxford magnets (Oxford Instruments) and HCN or DBG probes.

A subset of samples (seven from Kinkazan and 13 from Ishima) was analyzed

further with an Agilent 1100 series HPLC equipped with a quaternary pump, diode

array detector, and automated sampler. Select samples of nuchal gland fluid from unfed

or fish-fed hatchling Rhabdophis tigrinus were reconstituted in a known volume of

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. methanol to obtain quantitative data. A glass pipette attached to a hypodermic needle

was used to transfer methanol into each vial. Samples from unfed R. tigrinus hatchlings

from Kinkazan were reconstituted in 300 pL methanol. Most of the samples from

unfed R. tigrinus hatchlings from Ishima were reconstituted in 1500 pL because of

their high concentrations of bufadienolides. The samples were fractionated through a

reversed-phase 25 cm x 10 mm Supelco Discovery® HS C l8 column. The solvent

gradient used was 20:80 methanol:water, which was increased linearly to 100%

methanol at 3.4 ml/min. The injection volumes of the samples were 20-40 pL each (25

pL for the standardized samples), and each run lasted 53 minutes.

To identify bufadienolides in the samples analyzed by HPLC, we compared

them with the 17 previously identified compounds from a similar feeding experiment

(See Chapter II, Figure 2.5). The previously fractionated compounds were reanalyzed

with HPLC to determine any changes in retention time before comparing the two sets

of data (Figure 3.2). Of the 17 bufadienolides identified in Rhabdophis tigrinus,

compounds 6 and 7 co-elute, so they could not be distinguished from one another.

To determine the quantities of bufadienolides present in the standardized

samples, we obtained a dilution curve using known concentrations of telocinobufagin

(3-beta,5,14-trihydroxy-5-beta-bufa-20,22-dienolide). The equation obtained from the

linear trend line fitted to the telocinobufagin dilution curve was used to convert the

cumulative area (mAU) of bufadienolide peaks at 280 nm into a total quantity of

bufadienolides in each sample. The 280 nm wavelength was selected because

bufadienolides have absorbance maxima at approximately 200 and 300 nm, and the

latter exhibits a more stable baseline and experiences less interference from the UV

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. F ig . 3 .2 . HPLC chromatograms of nuchal gland fluid from Rhabdophis tigrinus.

Retention time in minutes (min) and UV-absorbance in milli-absorbance units (mAU)

are displayed, (a) Unfractionated reference bufadienolides from a previous experiment

(Chapter II). (b) An unfed hatchling R. tigrinus from Ishima from this study.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4 6

absorbance of methanol. Peaks produced by bufadienolides (both structurally known

and unknown) were identified by their UV absorbance spectra (Figure 3.3), and only

those peaks with areas greater than 15.0 mAU (milli-absorbance units) were used to

calculate the total quantity of bufadienolides in each standardized sample.

Results

Kinkazan

Samples of nuchal gland fluid from hatchling Rhabdophis tigrinus from six

clutches laid by dams collected on Kinkazan were analyzed for the presence or absence

of bufadienolides. Unfed hatchlings were available from only one clutch, but

hatchlings fed exclusively nonbufonid prey should exhibit similar chemistry in their

nuchal gland fluid. This is because, among the species used as prey in this experiment,

bufadienolides are only known from the toads (Bufo japonicus). A summary of the

presence or absence of bufadienolides is presented in Table 3.1. Specific bufadienolide

compounds found in hatchlings from both islands are presented in Table 3.2.

Figure 3.4 displays 'H-NMR spectra from clutch #2, which are representative

of the hatchlings from Kinkazan. The unfed and nontoad-fed hatchlings from Kinkazan

had no bufadienolides in their nuchal gland fluid. However, hatchlings fed a

combination of metamorphic toads (Bufo japonicus) and fish (Oryzias latipes)

possessed large quantities of bufadienolides (Figure 3.4, Table 3.1). Toad-fed

hatchlings from three clutches sequestered at least eight bufadienolides from their

bufonid prey, with compound 11 being the most abundant bufadienolide in each

sample (Table 3.2).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4 7

600

300 400 500 n m

F ig . 3.3. UV absorbance spectrum of a bufadienolide.

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Table 3.1. Presence or Absence of Bufadienolides in H atchling R h a b d o p h i s TIGRINUS FROM KlNKAZAN, FED CONTROLLED DIETS

Clutch 1 Clutch 2 Clutch 3 Clutch 4 Clutch 5 Clutch 6 (N=12) (N=17) (N=14) (N=18) (N=13) (N=15)

Hatchlings, n=2 unfed i l l i l l H 111illllll Hatchlings, fed nontoad n=3 n=3 n=3 n=4 n=3 n=3 prey

Hatchlings, fed toads

White represents a lack of bufadienolides; black, presence of bufadienolides; cross-

hatching, no samples available. N represents the number of hatchlings per clutch; n,

number of hatchlings analyzed by ]H-NMR.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 3.2. Bufadienolide Compounds in H atchling R h a b d o p h i s t ig r in u s from Kinkazan and Ishima, Fed C ontrolled Diets

Kinkazan Ishima

Hatchlings, None (n=2) unfed

Hatchlings, None (n=2) fed nontoad prey

Hatchlings, fed toads

Combined data for hatchlings pooled by locality and

feeding treatment; n, number of hatchlings analyzed by

HPLC. White represents a lack of bufadienolides; black,

presence of bufadienolides. Bufadienolides are identified

by compound numbers (see Figure 3.2). The compounds

listed include only those shared by all hatchlings in a

given category. The most abundant bufadienolide is

underlined; that in the hatchlings from Ishima was not

always consistent.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 50

Hatchling, unfed 100

)||i^|illW)iiiiiOpii>Mpii»wii»Mii miliWii w «l»l 0

-2 |T Hatchling, fed frogs, fish -c 13 & -5 ® .5 ■£,

K400 I 0) Hatchling, fed toads, fish 300 “

200

100

0

8.0 7.5 7.0 6,5 6,0 ppm

Fig. 3.4. Aromatic region of ’H-NMR spectra from the nuchal gland fluid of

Rhabdophis tigrinus hatchlings from clutch #2 laid by a dam collected on Kinkazan

Island, which lacks toads. Gray bars highlight peaks in the three regions diagnostic of

bufadienolides, which are evident only in the hatchling fed toads.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 51

Ishima

A strikingly different pattern emerged from the analysis of hatchling

Rhabdophis tigrinus from Ishima, which harbors a dense population of toads (Bufo

japonicus). Table 3.3 summarizes the presence of bufadienolides in six clutches from

Ishima. Figure 3.5 displays 'H-NMR spectra from hatchlings in clutch #12, which are

representative of the hatchlings from Ishima.

The unfed hatchlings typically possessed very large quantities of bufadienolides

(Figure 3.5, Table 3.3). Specifically, three unfed hatchlings from clutch #12 each

possessed between 1.4-2.0 mg of bufadienolides in their nuchal gland fluid. Similarly,

one unfed hatchling from clutch #11 exhibited 1.6 mg of bufadienolides, although a

different unfed hatchling from the same clutch exhibited only 0.1 mg.

The hatchlings that were fed exclusively on nontoad prey, as well as those fed

bufonid prey, possessed large quantities of bufadienolides in their nuchal gland fluid,

although not as much as in the majority of unfed hatchlings. For example, a

quantitative analysis of one fish-fed hatchling revealed 1.3 mg of bufadienolides in the

nuchal gland fluid. The unfed hatchlings from Ishima possessed at least six

bufadienolide compounds in their nuchal gland fluid, all but one of which (compound

17) were present in the hatchlings fed nonbufonid prey (Table 3.2). Hatchlings fed

toads exhibited six new bufadienolides in addition to the six or seven that they

presumably possessed prior to feeding (Table 3.2).

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Table 3.3. Presence of Bufadienolides in H a t c h l i n g Rhabdophis tigrinus f r o m Is h i m a , F e d C o n t r o l l e d D ie t s

Clutch 7 Clutch 8 Clutch 9 Clutch 10 Clutch 11 Clutch 12 (N=21) (N=7) (N=14) (N=14) (N=18) (N=43)

Hatchlings, unfed

Hatchlings, fed nontoad prey

Hatchlings, fed toads

Black represents the presence of bufadienolides; cross-hatching, no samples available.

N represents number of hatchlings per clutch; n, number of hatchlings analyzed by

1 H-NMR.

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Hatchling, unfed 1500

1000

500 1 0 » » % * * * « i I Hatchling, fed fish 300

200 -8

100

______k ______L,...... J 1 . 8.0 7.5 7.0 6.5 6.0 ppm

Fig. 3.5. Aromatic region of 'H-NMR spectra from the nuchal gland fluid of

Rhabdophis tigrinus hatchlings from clutch #12 laid by a dam collected on Ishima,

which has a dense population of toads. A very large quantity of bufadienolides is

evident in the unfed hatchling (note scale on y-axis), providing evidence for maternal

provisioning of these compounds. Large quantities of bufadienolides were also present

in hatchlings fed nontoad prey as well as in those fed bufonid prey; however, the

quantities were not as large as in the unfed hatchlings.

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Discussion

Our results clearly demonstrate that the chemical defense of hatchling

Rhabdophis tigrinus varies geographically. Hatchlings from Kinkazan are chemically

undefended at hatching and after feeding on nonbufonid frogs, fish, and/or

salamanders. However, hatchlings from Kinkazan are capable of sequestering large

quantities of defensive bufadienolides when artificially fed toads in the laboratory.

Previous analyses of three adult R. tigrinus from Kinkazan demonstrated that the adults

from this island also lack bufadienolides in their nuchal glands (Hutchinson et al.,

unpublished data), which provides further evidence that this species is incapable of

synthesizing defensive bufadienolides even in the adult stage. Therefore, R. tigrinus on

Kinkazan are chemically undefended against their predators, which is likely the reason

that these snakes disproportionately tend to flee rather than use their nuchal glands in

defensive displays (Mori and Burghardt, 2000). However, because these snakes possess

the ability to sequester toxins from bufonid prey, they could regain their chemical

defense in the event of an introduction of Bufo to Kinkazan. Despite the lack of

mammalian predators on this island, the presence of bufadienolides in the nuchal gland

fluid presumably would be an effective deterrent against birds of prey (Mori, 2004).

In contrast, most of the hatchling Rhabdophis tigrinus from Ishima, where toads

are abundant, possessed very large quantities of bufadienolides in their nuchal gland

fluid at the time of hatching. These compounds presumably were provisioned to the

offspring by the dams, although the latter were not available for chemical sampling. In

support of this hypothesis, a dam from Ishima collected in a different year showed very

large quantities of bufadienolides in her nuchal glands, suggesting that she could

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 55

transfer some of these toxins to her offspring during pregnancy (see Chapters II, IV).

Most of the compounds evident in unfed hatchlings in this study were retained in

hatchlings fed nonbufonid prey, although in lower quantities. The toad-fed hatchlings

doubled the number of bufadienolides in their arsenal by sequestering additional

compounds, and in some cases possessed larger quantities of bufadienolides than

nontoad-fed hatchlings.

The majority of unfed hatchling Rhabdophis tigrinus from Ishima possessed

larger quantities of bufadienolides than nontoad-fed hatchlings from that island. It is

likely that this result is due, at least partially, to the fact that unfed hatchlings died and

were frozen prior to the start of the experiment, whereas most individuals used in the

feeding trials were sacrificed 7-9 months later. This observation suggests that

bufadienolides provisioned by dams persist only temporarily in the nuchal glands of

hatchlings. It is not known how long these compounds persist, but it is likely that the

offspring must ingest toads at some point during their lives, and perhaps continuously,

in order to replenish their supply of these defensive compounds.

The difference in the presence of bufadienolides in the nuchal gland fluid of

hatchling Rhabdophis tigrinus from Ishima and Kinkazan may have a major influence

on the fitness of hatchlings on these two islands. The large quantities of bufadienolides

found in the nuchal gland fluid of hatchlings from Ishima possibly result in enlarged

nuchal ridges and more effective defensive displays involving the glands. In the

absence of chemical defense, hatchlings from Kinkazan have evolved to rely more

heavily on flight to escape predation (Mori and Burghardt, 2000).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 56

Acknowledgments

We thank the following individuals for their contributions to this study: Masami

Hasegawa, Isami Ikeuchi, Taku Mizuta, Eiko Nagata, Tomohiko Shimada, and Ken

Yoda for collecting Rhabdophis; Takashi Haramura, Azusa Hayano, Yoshihisa Mori,

Eiko Nagata, and Koji Tanaka for husbandry; Noriko Kidera for assistance with the

sampling of nuchal gland fluid and data entry; Thomas Spande and John Daly for

supplying the telocinobufagin standard; and Xiaogang Wu for assistance with HPLC.

This work was supported by the National Science Foundation (IBN-0429223 and IOB-

0519458 to AHS and JM); Old Dominion University (Dissertation Fellowship to

DAH); the Society for Integrative and Comparative Biology (Grant-in-Aid of Research

to DAH); Sigma Xi, The Scientific Research Society (Grant-in-Aid of Research to

DAH); Kyoto University (Grant for Biodiversity Research of the 21st Century COE

A14 to AM and Dept, of Zoology); the Kyoto University Museum (Visiting Faculty

Award to AHS); the Japan-US Cooperative Science Program (Japan Society for the

Promotion of Science to AM and NSF INT-9513100 to GMB); and the Fujiwara

Natural History Foundation (10th Annual Grant for Scientific Research to AM).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 57

CHAPTER IV

MATERNAL PROVISIONING OF DEFENSIVE STEROIDS

BY RHABDOPHIS TIGRINUS

Introduction

Chemically defended animals may synthesize their defensive toxins from

nontoxic precursors or sequester intact toxins from environmental sources, notably the

diet (Duffey, 1980; Brower, 1984; Daly et al., 1994; Gonzalez et al., 1999a; Nishida,

2002; Dumbacher et al., 2004; Saporito et al., 2004a; Williams et al., 2004). Regardless

of how the adults become toxic, their offspring would enjoy a selective advantage if

they were defended by maternal provisioning of these chemicals. When toxins are

deposited in the eggs of oviparous species, those toxins may serve as deterrents against

egg predation, but may not be incorporated into the embryos’ tissues. However, if

provisioning takes place via the yolk, for example, the embryos may absorb toxins

along with nutrients and thus become chemically defended themselves. A mechanism

of toxin delivery such as this would ensure that the young are at least temporarily

defended chemically from the time of hatching, thus eliminating the need for the young

to expend energy immediately to synthesize toxins or to search for toxic prey from

which to sequester defensive chemicals.

There are many examples of defensive toxins in the eggs of chemically

defended species. Eggs of rough-skinned newts (Taricha granulosa) and a Costa Rican

harlequin frog (Atelopus chiriquiensis) are provisioned with tetrodotoxin, a potent

neurotoxin (Pavelka et al., 1977; Hanifm et al., 2003). Toxic eggs are also known from

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 58

toads of the genus Bufo (Licht, 1968) and moths in the genus Utethesia (Eisner and

Meinwald, 1995; Eisner et al, 2000). Eggs of fireflies (Photuris) are endowed with

betaine synthesized by the female as well as sequestered lucibufagins when available

(Gonzalez et al., 1999a). Maternal provisioning of toxins in eggs may deter oophagy in

these taxa, but it does not ensure that the offspring will be chemically defended after

hatching unless the toxins are incorporated into the developing embryos.

Rhabdophis tigrinus is an oviparous Asian snake (Colubridae: Natricinae) that

possesses unusual defensive glands on its neck (Nakamura, 1935; Smith, 1938). The

contents of the nuchal glands have been shown to irritate mucous membranes and

cause comeal injuries (Nakamura, 1935; Kitazume, 1953; Kawashima, 1957, 1959;

Suzuki, 1960; Asahi et al., 1985), and the glands are known to contain bufadienolides

(Akizawa et al., 1985a; Akizawa et al., 1985b). Bufadienolides are cardiotonic steroids

that act by inhibiting the sodium-potassium pump in a digitalis-like fashion (Melero et

al., 2000). The steroidal toxins in nuchal glands are similar to those found in the skin

secretions of toads (Bufonidae; Erspamer, 1994), which are often consumed by R.

tigrinus (Mori and Moriguchi, 1988). Whereas toads are capable of synthesizing their

defensive bufadienolides from cholesterol (Doull et al., 1951; Siperstein et al., 1957;

Porto and Gros, 1971; Porto et al., 1972), R. tigrinus is dependent on dietary toads

from which it can sequester these compounds (Chapter II).

The offspring of Rhabdophis tigrinus would enjoy a selective advantage if the

dams (mothers) were capable of provisioning their progeny with sequestered defensive

compounds. In that case, the unfed offspring would possess defensive toxins upon

hatching and would not need to find and consume a toad to become chemically

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 59

defended, at least initially. In this study we explored the mechanism of maternal

provisioning of defensive bufadienolides in R. tigrinus. We analyzed nuchal gland fluid

samples from 12 gravid females and their offspring, fed on diets either containing or

lacking toads. We present evidence for the maternal provisioning of toxins in R.

tigrinus both late in pregnancy as well as by deposition in yolk.

Methods

Experimental Design and Sample Collection

Twelve gravid Rhabdophis tigrinus were collected between 27 May and 12

June 2005 from Okayama Prefecture: Tomi-son (n = 1), Kagamino-cho (n = 1),

Mikamo-son (n = 2), and Sinjo-son (n = 1); Hiroshima Prefecture: Shohbara City (n =

1); Kumamoto Prefecture: Yamato-cho (n = 1); Kyoto Prefecture: Miyama-cho (n = 3);

and Tokushima Prefecture: Naka-gun (n = 1) and Ishima Island (n = 1). The gravid

females were fed frogs (primarily Rana nigromaculata, but also Hyla japonica and

Rhacophorus arboreus) and fishes, specifically Ayu ( Plecoglossus altivelis), Iwashi

(Sardinops melanosticutus), Nijimasu ( Oncorhynchus mykiss), and Wakasagi

(Hypomesus nipponensis). Additionally, some of the dams were fed a single adult toad

{Bufo japonicus) either prior to or shortly following oviposition.

Samples of nuchal gland fluid were collected from the Rhabdophis tigrinus

dams while they were gravid and unfed (in captivity), and again after oviposition (at

which point they had been fed in the laboratory). The nuchal gland fluid was collected

by expressing a few glands onto a portion of Kimwipe (Kimwipes wipers S-200) while

wearing powder-free NBR nitrile gloves (AS ONE brand); the Kimwipe was then

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 6 0

placed in a vial of methanol with forceps and covered with a teflon-lined cap. We were

careful to express only a few glands to ensure that we could resample individuals at

later dates; nuchal glands do not appear to regenerate after they have ruptured, at least

initially. The forceps were rinsed in methanol from a glass pipette and dried with a

Kimwipe between individuals; gloves were also changed between individuals. A

control vial consisting of a Kimwipe in methanol was prepared at the end of each

sampling session. All samples were stored at -20°C.

Oviposition took place at Kyoto University between 29 June and 16 July 2005.

The eggs were incubated under conditions approximating natural thermal and light

cycles until hatching, which occurred between 10 and 19 August 2005. The

temperature of the enclosure housing the eggs and snakes was maintained between 25

and 30°C. Two eggs from clutch #12 were dissected a few days after oviposition to

collect yolk for chemical analyses.

Some hatchlings from each clutch had a few of their nuchal glands expressed

for chemical analyses prior to the onset of feeding. Two clutches had only two

hatchlings each (clutches #5 and 12), and all of these unfed offspring were sampled. At

least three unfed hatchlings were sampled from each of the other 10 clutches. Unfed

offspring from most clutches were sampled on 17 August 2005. However, clutches 2

and 11 hatched after this date, so those offspring were sampled on 11 September 2005,

prior to being fed.

We used a balanced, randomized design to assign feeding treatments to the

hatchlings. We first randomly assigned all of the hatchlings in a clutch to a feeding

treatment (toads or fish) using a random number table (Rohlf and Sokal, 1995).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 61

Numbers CM designated the fish-fed group and 5-9 designated the toad-fed group. If

the distribution of feeding treatments was not as close to 50/50 as possible among the

hatchlings within a clutch, we randomly selected individuals to re-assign to feeding

treatments in order to obtain a balanced design. However, for the clutches with only

two hatchlings each (Clutches 5 and 12), we assigned all offspring to the fish-fed group

to determine how long they were able to maintain any bufadienolides provisioned by

the dam. In the other 10 clutches, we ensured that all of the previously sampled

hatchlings were not in the same feeding group. Hatchlings were housed individually in

screen-topped plastic containers with paper towel as a substrate; water was available ad

libitum. The hatchlings were grouped by clutch on three shelves in the same ambient-

temperature facility at Kyoto University where the eggs had been incubated (Figure

4.1). Some juveniles that hatched early were fed fish ( Oryzias latipes) to sustain them

until the beginning of the feeding trials.

Samples of nuchal gland fluid were collected from almost all hatchlings on 11

September 2005, the day before the start of the feeding trial. One hatchling from

Clutch #5 and both hatchlings from Clutch #12 were excluded from this sampling

session to ensure that they could be resampled later. One hatchling from Clutch #9 had

escaped by this date.

During the feeding trial, all Rhabdophis tigrinus hatchlings were offered food

on the same days. Hatchlings in the fish-fed group were given one live fish (medaka,

Oryzias latipes), which typically weighed between 0.07 and 0.14 g. Hatchlings in the

toad-fed group were offered three thawed metamorphic toads ( Bufo japonicus), which

weighed a total of 0.10-0.16 g; the toads had been frozen since spring 2005. The fish

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 4.1. Arrangement of cages for hatchlingRhabdophis tigrinus in the feeding trials.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 63

were placed in the snakes’ water bowls, whereas the toads were clumped together on a

plastic dish placed in the cage. The snakes were left with their food items overnight,

and any snakes that had not eaten by the following day were force-fed. Food was

offered to the hatchlings on three dates: 12, 14, and 16 September, with any necessary

force-feedings taking place on 13, 15, and 17 September. Nuchal gland fluid was

resampled from all of the hatchlings on 21 September 2005. Following the feeding

trial, all hatchlings that were exhausted of nuchal gland fluid were euthanized by

freezing. The remaining 37 hatchlings were fed two Oryzias every three days until their

natural death, at which time they were frozen. Our animal use protocols conformed to

institutional policies and practices.

Samples of prey items fed to hatchling and adult Rhabdophis tigrinus were

prepared for chemical analyses. Bufo japonicus metamorphs and Oryzias latipes, both

of which were used as prey for hatchling Rhabdophis, were prepared as whole-body

extracts in methanol. Skin secretions from Rana nigromaculata, which were fed to

adult Rhabdophis, were collected using a transcutaneous amphibian stimulator (Grant

and Land, 2002). Parotoid gland secretions from adult B. japonicus were collected by

manually squeezing the glands. Skin secretions from anurans were collected on

sections of Kim wipes and stored in methanol at -20°C.

Chemical Analyses

Samples of nuchal gland fluid and prey extracts were shipped on dry ice to Old

Dominion University, maintained at -20°C, and transported to Cornell University for

chemical analyses. Samples of nuchal gland fluid required no preparation prior to

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 64

evaporation, but the whole-body samples of prey in methanol (fish and metamorphic

toads) were homogenized with a glass tissue grinder and filtered prior to chemical

analysis. Yolk samples were placed on dry ice and lyophilized prior to extraction in

methanol. Subsequently, all samples were evaporated to dryness, reconstituted in

deuterated methanol (methyl-d3 alcohol-d), and analyzed with 'H-NMR on a Unity

INOVA 600 MHz spectrometer (Varian, Inc.) equipped with an Oxford magnet

(Oxford Instruments) and an HCN probe.

Following analysis with 1 H-NMR, we re-evaporated all samples to dryness and

reconstituted them in a standardized volume of methanol for HPLC. A glass pipette

attached to a hypodermic needle was used to transfer methanol into each vial. Most

samples were placed in 300 pL methanol, but this amount occasionally resulted in

samples that were too concentrated or inadequately dissolved. To correct for this, we

used 900 pL of methanol for yolk samples and 1500 pL for parotoid gland secretions

of toads and samples of adult Rhabdophis tigrinus nuchal gland fluid that contained

large quantities of bufadienolides. Samples were analyzed with an Agilent 1100 series

HPLC equipped with a quaternary pump, a diode array detector, and an automated

sampler. The samples were fractionated through a reversed phase 25 cm x 10 mm

Supelco Discovery® HS Cl 8 column. The solvent gradient was 20:80 methanol:water,

which was increased linearly to 100% methanol at 3.4 ml/min. The injection volumes

of the samples were 25 pL each, and each run lasted 53 minutes.

To identify bufadienolides, we compared the samples with the 17 previously

identified compounds from a similar feeding experiment in 2004 (see Figure 2.5). The

previously fractionated compounds were reanalyzed with HPLC to detect any changes

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. in retention time before comparing the two sets of data (Figure 4.2). The reference

compounds eluted approximately 14-18 seconds earlier than in 2004, which was

consistent with the elution times of compounds in this experiment. More than 17

individual bufadienolides were present in many of the samples from 2005 (e.g., one

dam exhibited 26 bufadienolides). Of the 17 identified bufadienolides from

Rhabdophis, compounds 6 and 7 co-elute, so they could not be distinguished from one

another. Compound 11 co-eluted with lumichrome (a degradation product of

riboflavin), when both compounds were present.

To summarize the data for hatchlings with the same feeding histories, only the

compounds that were present in each individual are displayed in the tables. However,

samples that contained very little nuchal gland fluid, due to exhaustion of the glands,

were excluded from the analysis if their bufadienolide content was markedly different

from other individuals in the same clutch and feeding treatment. Identified

bufadienolides were only counted as present when the area of their HPLC peak

measured greater than 15.0 mAU (milli-absorbance units).

To determine the quantities of bufadienolides present in each sample, we

obtained a dilution curve using known concentrations of telocinobufagin (3-beta,5,14-

trihydroxy-5-beta-bufa-20,22-dienolide). The equation obtained from the linear trend

line fitted to the telocinobufagin dilution curve was used to convert the cumulative area

(mAU) of bufadienolide peaks at 280 nm into total quantity of bufadienolides in each

sample. The 280 nm wavelength was selected because bufadienolides have absorbance

maxima at approximately 200 and 300 nm, and the latter exhibits a more stable

baseline and experiences less interference from the UV absorbance of methanol. Peaks

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 66

250 10

200

150 3 < E 11 100 6.7

50 13 15 17 |14 12 16 JfsJW li J*a* , a A b

100

80

60 3 < 40 15

20 1 2 16 0 j v A,

10 15 20 25 30 35 min

Fig. 4.2.HPLC chromatograms of nuchal gland fluid from Rhabdophis.

(a) Unfractionated reference bufadienolides from a previous experiment (Chapter II).

(b) Dam #10 from this study, after being fed a toad in the laboratory.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 67

produced by bufadienolides (both structurally known and unknown) were identified by

their UV absorbance spectra (see Figure 3.3), and only those peaks with areas greater

than 15.0 mAU were used to calculate the total quantity of bufadienolides in each

sample. In any clutches where at least some samples of nuchal gland fluid contained a

peak produced by the co-elution of lumichrome and compound 11, the area of that peak

was excluded from calculations of bufadienolide quantity for all members of that

clutch. The total quantity of bufadienolides per sample was assigned to one of five

categories; if samples from hatchlings belonging to the same clutch and feeding

treatment fell into different categories, the smaller quantity was displayed as a

conservative measure.

Results

Prey Chemistry and Preference

The presence or absence of bufadienolides in certain species commonly used as

prey for Rhabdophis tigrinus was confirmed by 1 H-NMR and HPLC. Bufadienolides

were absent in the fishes ( Oryzias latipes) that were fed to hatchling Rhabdophis and in

the most common nonbufonid frog ( Rana nigromaculata) fed to adult Rhabdophis

(Figure 4.3). Bufadienolides were present in small quantities in the metamorphic toads

(Bufo japonicus) fed to hatchling Rhabdophis-, these compounds were abundant in the

parotoid secretion from adult B. japonicus, which were fed to some adult Rhabdophis

(Figure 4.3). One of the bufadienolides known from B. japonicus is gamabufotalin

(Melero et al., 2000), which was previously identified in the nuchal gland fluid of

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 68

Fish 200

MOO

1 XL «%««a>L

Frog 100 0 £ Dc

-200 2 ■ElX Toad metamorphs (8 100 £ 05

Adult toad 1500

1000

500

0

ppm 8.0 7.5 7.0 6.5 6.0

Fig. 4.3. Aromatic region of H-NMR spectra of prey extracts. Gray bars highlight

peaks in the three regions diagnostic of bufadienolides. Bufadienolides were absent in

both the whole-body extracts of fish ( Oryzias latipes) fed to hatchling Rhabdophis

tigrinus and in the skin secretion of the most common nonbufonid frog ( Rana

nigromaculata) fed to adult Rhabdophis. Small quantities of bufadienolides were

evident in the pooled whole-body extracts of Bufo japonicus metamorphs; large

quantities of these compounds were present in the parotoid gland secretion of an adult

B. japonicus.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Rhabdophis tigrinus (compound 10 in Figure 2.5; Akizawa et al., 1985a; Akizawa et

al„ 1985b).

Most hatchling Rhabdophis tigrinus in the toad-fed group readily consumed the

toads, but many hatchlings in the fish-fed group rejected the fish and had to be force-

fed. For example, on 13 September 2005, only two of 42 snakes (4.8%) had not eaten

all of their toads (in one of those cages the toads had become lodged under the water

dish and were inaccessible). In contrast, 29 of 44 snakes (66%) refused fish that day.

Similarly, on 15 September, only two snakes (4.8%) had not eaten all of their toads,

whereas 30 snakes (68%) had refused fish. On the last day of force-feeding (17

September), three snakes (7.1%) had to be force-fed toads, whereas 33 snakes (75%)

were force-fed fish. Overall, toads were rejected only 5.6% of the time, whereas fish

were rejected in 70% of cases.

Dams Fed Nontoad Prey

The results for female Rhabdophis tigrinus that were not fed toads in the

laboratory, and for their clutches, are provided in Table 4.1. R. tigrinus dams 1 and 2

both possessed bufadienolides in their nuchal gland fluid despite being fed only fish

and nonbufonid frogs in captivity. Dam #1 (Tomi-son, Okayama Prefecture) had a

small quantity of bufadienolides, and her offspring were only provisioned with a small

quantity of compound 10 (Figure 4.4, Table 4.1). The hatchlings from clutch #1 did not

accumulate any new bufadienolides when fed fish, but four new bufadienolides were

sequestered from ingested toads (Figure 4.4, Table 4.1). In contrast, dam #2

(Kagamino-cho, Okayama Prefecture) had a large quantity of bufadienolides in her

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 70

Table 4.1. Bufadienolides in Nontoad-FedR h a b d o p h i s t i g r i n u s Dams and Their H a t c h l in g s F e d C o n t r o l l e d Die t s

Clutch 1 Clutch 2 Clutch 4 Clutch 6 Clutch 7 Clutch 9 (N = 6) (N = 3) (N = 8) (N = 9) (N = 7) (N = 11)

Dam, 6/7. 8 ,9 , 10, 13 J 4 (n = l) 6/7 (n = l) unfed (n = l)

Dam, fed 6/7 (n = l) fish, frogs H

Hatchlings, None None 10 (n=3) unfed (n=3) (n=3)

Hatchlings, None None 1 0 (n=3) fed fish (n=2) (n=3)

6/7, 8. 10, Hatchlings, 8,10,11. 11, 13 11, 13 13 (n=3) (n=3) fed toads (n -3)

Total quantities of bufadienolides in the majority of samples per category are

represented by shades of gray: White, lack of bufadienolides; 25% gray, 1-100 pg;

50% gray, 100-500 pg; 75% gray, 500 pg-1 mg; 90% gray, 1-9 mg; cross-hatching,

no samples available. N represents the number of hatchlings per clutch; n, number of

samples analyzed (some hatchlings were sampled more than once, especially in the

small clutches). Bufadienolides are identified by number (see Figure 2.5), and the most

abundant bufadienolide per category is underlined.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 71

Dam #1. unfed 200 100

I l l ,1 -0 i i J ■ t . - 1 1 1 1 1 . . . , . V , . , 1

Hatchling, unfed 100 c 0 3 b k_(d ' * I : f: 1 1 A ! Lq (dL_ Hatchling, fed fish 100 0 ' 1 1. A 1 ' ‘ 1 ' 1 1 1 1 * « ' ' 1 ’ V ’ 1 1 '

Hatching, fed toads, fish

|

8.0 7.5 7.0 6.5 6.0 ppm

Fig. 4.4. Aromatic region of 'H-NMR spectra of the nuchal gland fluid of Rhabdophis

tigrinus dam #1 and three of her hatchlings. Gray bars highlight peaks in the three

regions diagnostic of bufadienolides. The small quantities of bufadienolides in the

unfed and fish-fed hatchlings provide evidence for maternal provisioning of dietary

toxins. Note the new peaks in the hatchling fed toads, representing additional

bufadienolides sequestered from prey.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 72

nuchal gland fluid, and she provisioned most of those compounds to her offspring in

moderate quantities (Table 4.1, Figure 4.5). Compound 17 was evident in the unfed

offspring as a minor component but was absent in the fish-fed group. However,

hatchlings from clutch #2 that were fed toads accumulated compound 17, so their

bufadienolide profiles appeared the same as those of the unfed offspring (Table 4.1).

Dam #4 (Mikamo-son, Okayama Prefecture) possessed a moderate amount of

bufadienolides before oviposition and after having been fed nonbufonid frogs and fish

in the laboratory (Figure 4.6, Table 4.1). About half of her compounds were

provisioned to her offspring in small quantities, and the fish-fed offspring had the same

bufadienolide profiles as the unfed ones (Table 4.1). Although the quantity of

bufadienolides in the toad-fed offspring did not change, they sequestered three new

bufadienolides from their bufonid prey (Table 4.1, Figure 4.6).

Dams 6 and 7 both possessed bufadienolides in their nuchal gland fluid. Dam

#6 (Shohbara city, Hiroshima Prefecture) had moderate to large quantities of

bufadienolides before oviposition and after being fed nonbufonid frogs and fish (Figure

4.7, Table 4.1). All of her bufadienolides were provisioned to her offspring in moderate

quantities, and surprisingly compound 1 was present in the fish-fed hatchlings,

although it was absent in the unfed hatchlings (Figure 4.7, Table 4.1). There was some

evidence for this compound in the unfed dam, although it was evident in too low a

quantity to be scored as present. The toad-fed hatchlings did not sequester any

additional bufadienolides (Figure 4.7, Table 4.1). Dam #7 (Yamato-cho, Kumamoto

Prefecture) possessed a low quantity of only one bufadienolide (compound 14), which

was not provisioned to her unfed offspring (Figure 4.8, Table 4.1). Her fish-fed

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 73

Dam #2, unfed

Hatchling, unfed

Hatchling, fed fish

500 to

Hatchling, fed toads

8.0 7.5 7.0 6.5 6.0 ppm

F i g . 4.5. Aromatic region of 'H-NMR spectra from Rhabdophis tigrinus dam #2 and

three of her hatchlings. Maternal provisioning of bufadienolides is evident from the

large peaks in the dam and her hatchlings.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 74

Dam #4, unfed 500

-1000 Dam, fed frogs and fish

-5 0 0

3c 2 Id 03 500 £ Hatchling, unfed CO c & ...... i ...... - HO JE 1 1 1 1 ' • i

500 Hatchling, fed fish

______H______

Hatchling, fed toads, fish 500

Htfl j til in t .Liiii» II ■ ■m u^

8.0 7.5 7.0 6.5 6.0 ppm

Fig. 4.6. Aromatic region of 'H-NMR spectra fromRhabdophis tigrinus dam #4 and

three of her hatchlings. The dam was fed only frogs and fish in the laboratory and did

not accumulate any new bufadienolides from these nonbufonid prey. The hatchlings

were provisioned with small quantities of two or three bufadienolides, and those that

were fed toads accumulated three new bufadienolides, evident from the new peaks in

the gray bars.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 75

:4000 Dam #6, unfed 3000

: 2000

: 1000

i r0

Dam, fed frogs, fish 2000

r1000

:0 42 c :3000 3 Hatchling, unfed 2 :2000 15k- ro. : 1000 .5 tn c *3 c

2000 Hatchling, fed fish

1000 | : 1 r " ...... *...... ** ...... “TT ...... 0

1-2000 Hatchling, fed toads, fish

1000

8iT 7.5 7.0 6.5 6.0 ppm

Fig. 4.7. Aromatic region of 'H-NMR spectra from Rhabdophis tigrinus dam #6 and

three of her hatchlings. The dam did not accumulate any new bufadienolides from the

nonbufonid prey, and all of her bufadienolides were provisioned to the hatchlings.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 76

500 Dam #7, unfed

■Mon m •fflM

Dc Hatchling, unfed <0 [ 100 2k. 0 03

to 03c Hatchling, fed fish 100 0 i—1—1—'—1—i—1—<—'—1—i—1—1—1—1—i—r-

Hatchling, fed toads, fish I- 100 0 mUNMM i 8.0 7.5 7.0 6.5 6.0 ppm

Fig. 4.8. Aromatic region of 'H-NMR spectra from Rhabdophis tigrinus dam #7 and

three of her hatchlings. The dam possessed a small quantity of only one bufadienolide

(compound 14), which was not provisioned to her offspring. Only the toad-fed

hatchlings sequestered bufadienolides from their prey.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 77

offspring had no bufadienolides, but her toad-fed offspring sequestered small quantities

of four bufadienolides from their bufonid prey (Figure 4.8, Table 4.1).

Dam #9 (Miyama-cho, Kyoto Prefecture) had a small quantity of one or two

bufadienolides (compound 6/7) prior to oviposition and after feeding on nonbufonid

frogs and fish in the laboratory (Figure 4.9, Table 4.1). Her unfed and fish-fed

hatchlings were all chemically undefended, but the hatchlings fed toads sequestered a

small quantity of two bufadienolides from their bufonid prey (Figure 4.9, Table 4.1).

Dams Fed Toads

Table 4.2 provides data for dams fed an adult toad (Bufo japonicus) in the

laboratory, and for their clutches. Dam #3 (Mikamo-son, Okayama Prefecture) was fed

a toad 19 days prior to oviposition. Analysis of her nuchal gland fluid revealed that she

possessed a moderate quantity of bufadienolides prior to eating the toad, and she

accumulated a large quantity of a new bufadienolide (compound 14) from this bufonid

meal, but lost compound 13 (Figure 4.10, Table 4.2). Her unfed offspring possessed a

moderate quantity of all of the bufadienolides that were present in the dam’s nuchal

gland fluid before she was fed a toad, as well as the newly accumulated compound 14

(Figure 4.10, Table 4.2). The quantity of bufadienolides in the fish-fed group was

smaller than that in the unfed hatchlings, and compound 13 was no longer evident

(Table 4.2). However, the quantity of bufadienolides in the toad-fed hatchlings was

generally higher than that in the fish-fed hatchlings and compound 13 was also

apparent in the toad-fed group (Table 4.2).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 78

Dam #9, unfed 200

500 Dam, fed frogs, fish

DC 03 0 £ -Qu. L^||^ ^ujEBy 4M

in c Hatchling, unfed 100 2c

mum

Hatchling, fed fish 100 ■o

Hatchling, fed toads 100 ■0 -i— .— i— i— |— —i— —I— 8.0 7.5 7.0 6.5 6.0 ppm

Fig. 4.9. Aromatic region of 'H-NMR spectra from Rhabdophis tigrinus dam #9 and

three of her hatchlings. The dam possessed a small quantity of one or two

bufadienolides prior to oviposition, none of which she provisioned to her offspring.

The toad-fed hatchlings sequestered two bufadienolides from their bufonid prey.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 79

Table 4.2. Bufadienolides in Toad-Fed R h a b d o p h i s t ig r i n u s Dams and Their H atchlings Fed C ontrolled Diets

Clutch 3 Clutch 5 Clutch 8 Clutch 10 Clutch 11 Clutch 12 (N = 13) (N = 2) (N = 10) (N = 11) (N = 5) (N = 2)

Dam, unfed

Dam, fed toad

Hatchlings, unfed

Hatchlings, fed fish

Hatchlings, fed toads

Total quantities of bufadienolides in the majority of samples per category are

represented by shades of gray: 25% gray, 1-100 pg; 50% gray, 100-500 pg; 75% gray,

500 pg-1 mg; 90% gray, 1-9 mg. N represents the number of hatchlings per clutch; n,

number of samples analyzed (some hatchlings were sampled more than once,

especially in the small clutches); cross-hatching, no samples available. Bufadienolides

are identified by number (see Figure 2.5), and the most abundant bufadienolide per

category is underlined (this factor was not consistent in hatchlings from clutch 12). The

only dams fed toads prior to oviposition were dams 3,5,10, and 11.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 80

Dam #3, unfed 2000

1000

J t i l l ..

Dam. fed toad - 2000

r 1000

-W_ c 3 & 2 Hatchling, unfed 1000 '■to e 500

.Ill ...... -.....- . 11...... 0

Hatchling, fed fish 500

. 1 i ...... t t ,

Hatchling, fed toads 500

u X 1 0 i , , “T—"r....i....'.... 1.... 1.... 1....1.....1....1....1 1 i 1 j * 8.0 7.5 7.0 6.5 6.0 ppm

Fig. 4.10. Aromatic region of *H-NMR spectra from Rhabdophis tigrinus dam #3 and

three of her hatchlings. Nineteen days prior to oviposition, the dam was fed a toad from

which she accumulated new bufadienolides, one of which she provisioned to her

offspring along with those that she possessed prior to this toad meal. Note the

appearance of new peaks in the gray bars after the dam was fed a toad and their

presence in all of her hatchlings.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 81

Dam #5 (Sinjo-son, Okayama Prefecture) was fed a toad 16 days prior to

oviposition, but she escaped before her nuchal gland fluid could be resampled. Before

being fed a toad in the laboratory, she possessed a moderate quantity of bufadienolides,

most of which were provisioned to her two offspring (Figure 4.11, Table 4.2). Her

unfed offspring possessed three bufadienolides that were not present in the nuchal

gland fluid of the unfed mother (compounds 3, 9, and 14); it is presumed that these

bufadienolides were sequestered by the dam from the toad she consumed in captivity

and were subsequently provisioned to her offspring (Figure 4.11, Table 4.2). The one

fish-fed hatchling had the same bufadienolide compliment as the unfed hatchlings, with

the exception of compound 17, which was absent in the fish-fed hatchling. Neither of

the two hatchlings from this clutch was fed toads.

Dam #8 (Miyama-cho, Kyoto Prefecture) possessed a moderate quantity of

bufadienolides prior to oviposition, most of which were provisioned to her offspring

(Figure 4.12, Table 4.2). The fish-fed hatchlings had lost compound 11, but otherwise

had the same bufadienolide profile as their unfed siblings (Table 4.2). The toad-fed

hatchlings did not increase the quantity of bufadienolides in their nuchal gland fluid,

but they sequestered two new bufadienolides (Figure 4.12, Table 4.2). Following

oviposition, the dam was fed a toad, from which she increased both the quantity and

number of bufadienolides in her nuchal gland fluid. After the toad meal, she had a large

quantity of bufadienolides, seven of which were new (Figure 4.12, Table 4.2).

Dam #10 (Miyama-cho, Kyoto Prefecture) was fed a toad nine days prior to

oviposition. She had the largest quantity of bufadienolides prior to oviposition of all

unfed dams (8.3 mg) and lower but still large quantities after consuming a toad in the

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 82

2000 Dam #5, unfed

1000

0 _ i 48 =Jc 2000 ^ (0 Hatchling, unfed XII— cC 1000 to 0}c JL r

Hatchling, fed fish 1000

. 1. ...J ...... J ...H...... r...... -0 ~T~ 8.0 7.5 7.0 6.5 6.0 ppm

F ig. 4.11. Aromatic region of 'H-NMR spectra from Rhabdophis tigrinus dam #5 and

her two hatchlings. The dam was fed a toad 16 days prior to oviposition, but she

escaped before having her nuchal gland fluid resampled. She presumably sequestered

new bufadienolides (compounds 3, 9, and 14) from her toad meal and provisioned them

to her offspring, evident as new peaks in the gray bars of the hatchlings’ spectra.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 83

Dam #8, unfed 1000

-5 0 0

_ k J li -0

Hatchling, unfed 200 0 49 'c 3

Hatchling, fed fish ■S 200 3U. «d 1 X______£ h0 5w 0)c Hatchling, fed toads, fish _c 200 X A -0

b 1500 Dam, fed toad

1000

500

0

8.0 7.5 7.0 6.5 6.0 ppm

Fig. 4.12. Aromatic region of 'H-NMR spectra fromRhabdophis tigrinus dam #8 and

three of her hatchlings. The dam possessed a moderate quantity of bufadienolides prior

to oviposition, most of which she provisioned to her offspring. The toad-fed hatchlings

accumulated two new bufadienolides from their prey. The dam sequestered seven new

bufadienolides from the toad she consumed in the laboratory after oviposition (see

Table 4.2).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 84

laboratory, when re-tested following oviposition (1.5 mg; Figure 4.13, Table 4.2). Her

unfed offspring possessed the largest quantities of bufadienolides of all 12 clutches

examined (more than 1 mg of bufadienolides in the majority of samples), and were

provisioned with most of the bufadienolides found in the dam (Figure 4.13, Table 4.2).

The quantity and number of bufadienolides in the fed offspring declined regardless of

prey , and no new bufadienolides were sequestered from the metamorphic toads

(Table 4.2). Dam #10 did not sequester any new bufadienolides from her toad meal, so

it was not possible to determine whether newly accumulated bufadienolides were

provisioned to her offspring in the nine days prior to oviposition (Table 4.2).

Dam #11 (Naka-gun, Tokushima Prefecture) was fed a toad 12 days prior to

oviposition. Samples of her nuchal gland fluid contained 2.4 mg of bufadienolides

before oviposition and 0.6 mg following toad consumption in the laboratory and

oviposition (Figure 4.14, Table 4.2). Her offspring were provisioned with moderate

quantities of bufadienolides, including compounds that were present in the unfed dam,

as well as a new bufadienolide (compound 14) sequestered from her bufonid meal in

the laboratory (Figure 4.14, Table 4.2). Fish-fed and toad-fed hatchlings possessed the

same quantities and types of bufadienolides as the unfed hatchlings, with the exception

of compound 9, which was lost in hatchlings fed both diets (Table 4.2).

A sample of nuchal gland fluid from dam #12 (Ishima Island, Tokushima

Prefecture) prior to oviposition contained 7.9 mg of bufadienolides (Figure 4.15). Yolk

samples from two of her oviposited eggs contained a large total quantity (more than 1

mg per egg) of all of the bufadienolides found in the dam’s nuchal gland fluid (Figure

4.15). Most of the bufadienolides found in the yolks were detected in the nuchal gland

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 85

Dam #10, unfed 3000

2000

1000

J k j | . J *.

Dam, fed toad 2000

1000

2000 c Hatchling, unfed 3 « -1000 3, U) C sc

1000 Hatchling, fed fish

500

II i il.. .. J ...... J L : 0

1000 Hatchling, fed toad

500

,JIl _ j j c------j------i ------0

8.0 7.5 7.0 6.5 6.0 ppm

Fig. 4.13. Aromatic region of 'H-NMR spectra fromRhabdophis tigrinus dam #10 and

three of her hatchlings. The dam possessed a large quantity of bufadienolides, most of

which she provisioned to her offspring in very large quantities. The dam consumed a

toad nine days prior to oviposition but did not accumulate any new (different)

bufadienolides from that meal.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 86

Dam #11, unfed : 3000

2000

hi 000 —I 0

2000

Dam. fed toad 1000

0 3c 1000 Hatchling, unfed (0 -500 -e 10

CO c

Hatchling, fed fish 1000

500

Hatchling, fed toads 1000

500

8.0 7.5 7.0 6.5 6.0 ppm

Fig. 4.14. Aromatic region of 'H-NMR spectra fromRhabdophis tigrinus dam #11 and

three of her hatchlings. The dam possessed a large quantity of bufadienolides, most of

which she provisioned to her offspring in moderate quantities. The dam consumed a

toad 12 days prior to oviposition, from which she sequestered compound 14 and

provisioned it to her offspring in addition to the compounds she possessed previously.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 87

Dam #12, unfed 5000

-i-JUL -0 T 500 Yolk

uiuJU

-5000 £ c Hatchling, unfed S' I la .sl h0 '<2 _c$ 2000 Hatchling, fed fish Mooo

5000 Dam, fed toad

-J l ~l-- I — i— — i— 8.0 7.5 7.0 6.5 6.0 ppm

Fig. 4.15. Aromatic region of 'H-NMR spectra fromRhabdophis tigrinus dam #12, her

two hatchlings, and one yolk sample. The dam possessed a large quantity of

bufadienolides, all of which were found in large quantities in the yolks of her

oviposited eggs. Most of these bufadienolides were evident in moderate quantities in

the nuchal gland fluid of the hatchlings. After oviposition, the dam accumulated an

additional bufadienolide (compound 14) from an ingested toad.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 88

fluid of hatchlings in moderate quantities. The hatchlings retained all but one of these

compounds after feeding on fish, although the quantity of bufadienolides decreased

somewhat after the hatchlings were fed (Figure 4.15, Table 4.2). Neither of the two

hatchlings from this clutch was fed toads. The dam accumulated one new compound

(14) from her bufonid meal, which was consumed after oviposition (Figure 4.15, Table

4.2).

The most abundant bufadienolide in the dams was more variable than that in the

hatchlings, and the bufadienolides sequestered from adult and metamorphic toads

differed. The major bufadienolide in most unfed dams was compound 6 or 7 (n = 6),

and the second most common major bufadienolide was compound 10 (n = 3). In

contrast, the most abundant bufadienolide in hatchlings that possessed them, regardless

of feeding treatment, was almost always compound 10 (gamabufotalin; 72 of 83

samples). Among the dams, compound 14 was the most frequently sequestered new

compound following consumption of an adult toad in captivity (n = 3). The most

common bufadienolides sequestered by hatchlings from metamorphic toads were

compounds 8, 11, and 13.

Discussion

Our results provide further evidence for the sequestration and maternal

provisioning of defensive bufadienolides in Rhabdophis tigrinus. The chemical

analyses of nuchal gland fluid from dams and their hatchlings demonstrate that well-

defended dams provision larger quantities of bufadienolides to their offspring than

dams with small quantities of these compounds. Furthermore, there may be selectivity

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 89

in the provisioning of bufadienolides; the nuchal glands of most hatchlings exhibit the

same major bufadienolide, although typically it is not the major bufadienolide in the

defensive glands of the dams. Importantly, new bufadienolides were sequestered by

some dams fed toads late in pregnancy, and those compounds also were provisioned to

the offspring, suggesting that intraoviducal transfer of toxins can occur in addition to

provisioning via the yolk.

Although all 12 dams in this experiment possessed some bufadienolides prior to

being fed toads in the laboratory, the levels of chemical defense varied greatly among

individuals. Furthermore, there clearly was a relationship between the quantities of

bufadienolides in the nuchal gland fluid of the dams and those of their offspring. If the

total quantity of bufadienolides in a sample of nuchal gland fluid from a dam was

below 100 pg, her unfed offspring were not provisioned with these compounds or were

endowed with only minute quantities, which are unlikely to be biologically relevant. In

contrast, the offspring of dams with larger quantities of bufadienolides consistently

possessed larger quantities of bufadienolides in their own nuchal glands. Note that the

absolute quantities of bufadienolides reported here represent only a sample of the total

amount of bufadienolides in a snake at any given time; the entire contents of the

complete series of nuchal glands were not tested, to permit repeated sampling.

The most abundant bufadienolide in the nuchal gland fluid of the dams was

typically not the most abundant compound in the offspring. In 87% of the cases,

gamabufotalin (compound 10) was the most abundant bufadienolide in the hatchlings,

whereas this compound was typically not the most abundant in the nuchal gland fluid

of the dams. There are several possible explanations for this pattern. The maternal

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 90

provisioning of bufadienolides may be a selective process, whereby gamabufotalin has

greater bioavailability for embryos than other bufadienolides. If bioavailability of

various bufadienolides is the same for dams and embryos, the reason that

gamabufotalin is generally not the most abundant bufadienolide in most dams may be

due to a difference in the persistence of individual compounds. However, we found

gamabufotalin to be relatively stable, at least when stored at -20°C. Compound 11,

which was the most abundant bufadienolide in one nuchal gland fluid sample from a

dam, was also quite stable. In contrast, compounds 6 and 15, which were the most

prominent bufadienolides in some samples from dams, had decomposed in 8-11

months at -20°C. Therefore, persistence does not seem to provide a good explanation

for this pattern.

Another possible reason for the difference in prominent bufadienolides between

dams and their hatchlings is that the most abundant bufadienolide in the hatchlings

represents the most abundant compound in the dam at the time of vitellogenesis due to

her diet at that time. However, it seems unlikely that almost all of the dams would have

had gamabufotalin as their most abundant bufadienolide during vitellogenesis given the

variation present in nuchal gland fluid of dams at the time of oviposition. Therefore, it

appears likely that gamabufotalin is provisioned selectively to hatchlings regardless of

its relative concentration in the dams. Other processes cannot be ruled out, however,

such as modification of provisioned bufadienolides by embryos. Regardless of the

reason for the pattern, it may be beneficial to Rhabdophis tigrinus hatchlings to have

gamabufotalin as their most abundant bufadienolide. This compound is approximately

ten times more effective at inhibiting the sodium-potassium pump than

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 91

(Azuma et al., 1986), and thus, may be more effective as an antipredator compound

than other bufadienolides.

Obtaining evidence of sequestration of bufadienolides in hatchling Rhabdophis

tigrinus was complicated by the fact that so many clutches were provisioned with

bufadienolides by the dams. However, the two unprovisioned clutches (clutches 7 and

9) and the three clutches provisioned with small quantities of bufadienolides (clutches

1, 4, and 8) provide evidence of the sequestration of bufadienolides from metamorphic

toads. Specifically, compounds 6/7, 8, 10, 11, and 13 were shown to be sequestered

from metamorphic Bufo japonicus in cases where they were not already provided by

the dam.

It is not known how long provisioned bufadienolides persist in the offspring

without being replenished by consumption of toads. This study provides evidence that

some of the minor compounds present in the unfed offspring of Rhabdophis tigrinus

diminish within a matter of weeks. Furthermore, the quantity of bufadienolides in the

nuchal gland fluid either remained the same or decreased after the offspring were fed

fish. In the provisioned clutches, the toad-fed hatchlings typically possessed the same

quantity of bufadienolides as the fish-fed hatchlings, although the number of

bufadienolide compounds often was higher in the toad-fed group.

By the time Rhabdophis tigrinus hatch in the fall, the toads that had

metamorphosed the previous spring are juveniles, which possess parotoid glands and

presumably higher levels of bufadienolides than the metamorphic toads used as prey in

this experiment. These juvenile toads may be too large for hatchling R. tigrinus to

consume, although R. tigrinus is able to consume relatively larger prey items than some

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 92

other snakes (Mutoh, 1981). Recent field observations suggest that hatchlings can, in

some cases, consume juvenile toads in their first months of life (personal observation).

However, if juvenile toads generally cannot be consumed, the hatchling snakes must

rely on compounds provisioned by the dam for chemical defense until newly

metamorphosed toads are available as prey the following spring. It is clear from our

results that hatchling R. tigrinus are capable of sequestering bufadienolides from

metamorphic toads, even though the quantities of those compounds in this prey type

are small. The quantity of bufadienolides in the toad-fed hatchlings presumably would

have been greater if juvenile toads, rather than metamorphs, had been used as prey.

Furthermore, a preference for bufonid prey was clearly exhibited among the hatchlings,

which is consistent with previous findings for R. tigrinus (Mori and Moriguchi, 1988;

Mori, 2004).

There was significant variation in the quantity of bufadienolides in the nuchal

gland fluid of unfed dams, even within a locality. Three dams collected in Miyama-cho

(Kyoto Prefecture) had small, moderate, and large quantities of bufadienolides,

although toads (Bufo japonicus and B. torrenticola) are common in this area. These

results suggest that the level of chemical defense of an individual snake is dependent

on the number of toads it is able to catch and consume. It is not known how long these

sequestered compounds persist in the adults. Perhaps toads must be consumed on a

regular basis to maintain a large quantity of bufadienolides in the nuchal glands.

It is evident from our results that toads consumed during pregnancy can supply

bufadienolides to a dam’s late-stage embryos as well as to her nuchal gland fluid. In

two out of three dams that were fed a toad during pregnancy, the quantity of

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. bufadienolides in the dams’ nuchal gland fluid either stayed the same or increased after

eating the toad. In two cases, new bufadienolides were sequestered from the ingested

toad and also were provisioned to the offspring 2-3 weeks prior to oviposition. At that

stage of gestation, the eggs presumably are in the oviduct of the female, so any transfer

of toxins at that time would have to take place by diffusion. However, the presence of

bufadienolides in the yolk of fertile eggs of dam #12 indicates that bufadienolides are

also provisioned during vitellogenesis.

No clear patterns emerge from the comparison of bufadienolide quantities in the

nuchal gland fluid of the dams before and after consuming a toad in the laboratory. Of

the dams fed a toad during pregnancy, the quantity of bufadienolides in the nuchal

gland fluid of dams #10 and 11 decreased following oviposition. This may have

resulted from the transfer of bufadienolides to embryos late in pregnancy. It is unlikely

that this pattern was produced by the expression of more glands in the samples taken

from unfed, gravid females; this probably would not produce the large differences in

the quantity of bufadienolides between the two sets of samples. In the most extreme

case, the sample from dam #10 prior to being fed a toad contained 5.5 times more

bufadienolides than in the sample collected from her after consuming a toad and laying

eggs. However, this pattern was not observed in all dams that were fed toads during

pregnancy; the quantity of bufadienolides in the nuchal gland fluid of dam #3 more

than doubled following oviposition. Similarly, snakes fed toads following oviposition

did not exhibit consistent changes in the quantity of bufadienolides in their nuchal

gland fluid. The quantity of bufadienolides in dam #8 increased nearly six-fold after

being fed a toad, whereas that of dam #12 decreased by almost one-half.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 94

Maternal provisioning of sequestered bufadienolides appears to be a common,

but not universal, phenomenon in Rhabdophis tigrinus. Dams can endow their

offspring with bufadienolides from prey consumed both early and late in pregnancy.

These findings are unusual because the maternally provisioned bufadienolides by R.

tigrinus become incorporated into embryonic tissues, so that the hatchlings as well as

the eggs are chemically defended. Furthermore, some provisioning takes place

presumably long after vitellogenesis has occurred, suggesting that some toxins are

provisioned across the oviduct. It is also clear that hatchlings are capable of

sequestering additional bufadienolides from ingested toads, if those prey are available

to them. However, if hatchling R. tigrinus are unable to consume toads until the spring

following hatching, the persistence of maternally provisioned bufadienolides will be an

especially important factor in the ability of hatchling R. tigrinus to deter predators.

Given the vulnerability of hatchling snakes to predation (Burger, 1998), the presence of

defensive toxins presumably is an important contributor to offspring survival, and thus

maternal provisioning is expected to be subject to strong selective pressure.

Acknowledgments

We thank Yohei Kadota, Akira Katayama, Noriko Kidera, Toshiro Kuroki,

Masami Hasegawa, Koji Mochida, Aya Nakadai, Sumio Okada and Hirohiko Takeuchi

for their assistance collecting animals in the field; Yohei Kadota, Noriko Kidera and

Takashi Haramura for their assistance with the sampling of nuchal gland fluid; Tatsuya

Hishida and Koji Tanaka for assistance with husbandry; and Thomas Spande and John

Daly for supplying the telocinobufagin standard. This work was supported by the

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 95

National Science Foundation (IBN-0429223 and IOB-0519458 to AHS and JM); Old

Dominion University (Dissertation Fellowship to DAH); the Society for Integrative

and Comparative Biology (Grant-in-Aid of Research to DAH); Sigma Xi, The

Scientific Research Society (Grant-in-Aid of Research to DAH); Kyoto University

(Grant for Biodiversity Research of the 21st Century COE A14 to AM and Dept, of

Zoology); and the Japan-US Cooperative Science Program (Japan Society for the

Promotion of Science to AM and NSF INT-9513100 to GMB).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 96

CHAPTERV

CONCLUSIONS

The preceding three chapters provide chemical evidence for the dependence of

Rhabdophis tigrinus on a diet of toads for the sequestration of defensive

bufadienolides. R. tigrinus that are bom to chemically undefended dams lack

bufadienolides in their nuchal gland fluid at hatching and after feeding on nonbufonid

prey. These hatchlings only exhibit bufadienolides in their nuchal gland fluid after they

have eaten toads. However, chemically defended dams can provision their offspring

with bufadienolides that had been sequestered previously from toads ingested by the

dam. The offspring of these dams are chemically defended at hatching and do not need

to ingest toads to possess bufadienolides, at least initially. However, when provisioned

hatchlings consume toads, they typically sequester additional types of bufadienolide

compounds in their nuchal gland fluid.

The quantity of bufadienolides in the nuchal gland fluid of provisioned

hatchlings is correlated with that in the dams, resulting in well-defended dams having

offspring with large quantities of bufadienolides. Bufadienolides are provisioned to

embryos at least partially via the yolk. Additionally, dams are capable of sequestering

and provisioning bufadienolides from toads ingested as late as 2-3 weeks prior to

oviposition, suggesting that transfer of sequestered toxins also can occur by diffusion

across the oviduct.

Toxin sequestration in Rhabdophis tigrinus may involve both selectivity and

modification. Toads contain bufadienolides and bufotoxins, but the nuchal gland fluid

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 97

of R. tigrinus contains only the former. Therefore, R. tigrinus either selectively

sequesters bufadienolides from ingested toads or cleaves the suberylarginine side chain

from bufotoxins, thus converting them to bufadienolides. Furthermore, R. tigrinus

hydroxylates the sequestered bufadienolides, making them more polar and potentially

more toxic than the bufadienolides in toads. If bufadienolides are transported from the

digestive system to the nuchal glands via the plasma, as we suspect, modification of the

toxins may occur in the liver. However, at least one compound, gamabufotalin, is

found in both the parotoid gland secretion of toads and the nuchal gland fluid of R.

tigrinus, so this bufadienolide appears to be sequestered unaltered. Maternal

provisioning of bufadienolides may also involve selectivity; the major compound in the

dams typically is not the most abundant bufadienolide in the offspring.

The dependence of Rhabdophis tigrinus on toads for sequestered toxins results

in geographic variation in chemical defense of R. tigrinus due to the incomplete

sympatry of Rhabdophis and Bufo. On the island of Kinkazan, where toads are absent,

hatchling R. tigrinus lack bufadienolides in their nuchal gland fluid and do not

accumulate them when fed nonbufonid prey. Therefore, natural selection has resulted

in their reliance on flight to escape predation (Mori and Burghardt, 2000). In contrast,

R. tigrinus hatchlings on Ishima, an island that harbors a dense population of toads, are

provisioned with large quantities of bufadienolides from their dams, rendering them

well-defended at the time of hatching. Subsequently, ingested toads provide additional

types of bufadienolides and probably are necessary to maintain large quantities of

bufadienolides in the nuchal gland fluid over time.

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The discovery of dietary toxin sequestration by Rhabdophis tigrinus provides a

rare example of sequestered chemical defense among amniote vertebrates. The only

other examples of this phenomenon in amniotes are the sequestration of dietary

batrachotoxins in the skin and feathers of Pitohui and Ifrita (Dumbacher et al., 1992;

Dumbacher et al., 2000; Dumbacher et al., 2004) and the transitory storage of

tetrodotoxin from ingested newts in unspecialized tissues of Thamnophis sirtalis

(Williams et al., 2004). The involvement of the unusual nuchal glands, endowment of

dietary toxins to embryonic tissue, and intraoviducal provisioning of bufadienolides

late in pregnancy render the sequestration system of R. tigrinus unique. It is likely that

other snakes with nuchal glands also sequester bufadienolides from dietary toads, but

this remains to be explored. The dependence of R. tigrinus on toads for defensive

bufadienolides has important implications for the ecology and conservation of both

predator and prey.

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REFERENCES

AKIZAWA, T., YASUHARA, T., AZUMA, H., and NAKAJIMA, T. 1985a. Chemical structures and biological activities of bufodienolides in the nucho-dorsal glands of Japanese snake, Rhabdophis tigrinus. J. Pharmacobio-Dyn. 8:s-60.

AKIZAWA, T., YASUHARA, T., KANO, R., and NAKAJIMA, T. 1985b. Novel polyhydroxylated cardiac steroids in the nuchal glands of the snake, Rhabdophis tigrinus. Biomed. Res. 6:437-441.

ASAHI, H., KOHTARI, Y., CHIBA, K., and MISHIMA, A. 1985. Effect of the nucho- dorsal gland of the yamakagashi snake on the eye. Folia Ophthalmol. Japon. 36:379-383.

AZUMA, H., SEKIZAKI, S., AKIZAWA, T., YASUHARA, T., and NAKAJIMA, T. 1986. Activities of novel polyhydroxylated cardiotonic steroids purified from nuchal glands of the snake, Rhabdophis tigrinus. J. Pharm. Pharmacol. 38:388- 390.

BENARD, M. F. and FORDYCE, J. A. 2003. Are induced defenses costly? Consequences of predator-induced defenses in western toads, Bufo boreas. Ecology 84:68-78.

BLAUSTEIN, M. P., ROBINSON, S. W., GOTTLIEB, S. S., BALKE, C. W., and HAMLYN, J. M. 2003. Sex, digitalis, and the sodium pump. Molecular Interventions 3:68-72.

BRANCH, W. R. 1998. Field Guide to Snakes and Other Reptiles of Southern Africa. Ralph Curtis Books, Sanibel Island, FL.

BROADLEY, D. G. 1958. Some ecological notes on the British reptiles, with particular reference to their feeding habits. Br. J. Herpetol. 2:126-129.

BRODIE, E. D., Ill and BRODIE, E. D., JR. 1999a. Costs of exploiting poisonous prey: Evolutionary trade-offs in a predator-prey arms race. Evolution 53:626- 631.

BRODIE, E. D., Ill and BRODIE, E. D., JR. 1999b. Predator-prey arms races. BioScience 49:557-568.

BROWER, L. P. 1984. Chemical defence in butterflies, pp. 109-134, in: Richard I. Vane-Wright, P. R. Ackery (eds.). The Biology of Butterflies. Academic Press, London.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 100

BURGER, J. 1998. Antipredator behaviour of hatchling snakes: Effects of incubation temperature and simulated predators. Animal Behav. 56:547-553.

BURGHARDT, G. M. and GREENE, H. W. 1988. Predator simulation and duration of death feigning in neonate hognose snakes. Animal Behav. 36:1842-1844.

CANNON, M. S. and HOSTETLER, J. R. 1976. The anatomy of the parotoid gland in Bufonidae with some histochemical findings, II. Bufo alvarius. J. Morphol. 148:137-159.

CHEN, K. K. and KOVARIKOVA, A. 1967. Pharmacology and toxicology of toad venom. J. Pharmac. Sci. 56:1535-1541.

DALY, J. W. 1998. Thirty years of discovering arthropod alkaloids in amphibian skin. J. Nat. Prod. 61:162-172.

DALY, J. W., GARAFFO, H. M., SPANDE, T. F., JARAMILLO, C., and RAND, A. S. 1994. Dietary source for skin alkaloids of poison frogs (Dendrobatidae)? J. Chem. Ecol. 20:943-955.

DALY, J. W„ GARRAFFO, H. M., SPANDE, T. F., CLARK, V. C., MA, J. M., ZIFFER, H., and COVER, J. F., JR. 2003. Evidence for an enantioselective pumiliotoxin 7-hydroxylase in dendrobatid poison frogs of the genus Dendrobates. Proc. Natl. Acad. Sci. USA 100:11092-11097.

DEULOFEU, V. and RUVEDA, E. A. 1971. The basic constituents of toad , pp. 475-495, in: W. Bucherl, E. E. Buckley (eds.). Venomous Animals and Their Venoms. Academic Press, New York.

DOULL, J., DUBOIS, K. P., and GEILING, E. M. K. 1951. The biosynthesis of radioactive . Arch. Int. Pharmacodyn. Ther. 86:454-464.

DUFFEY, S. S. 1980. Sequestration of plant natural products by insects. Annu. Rev. Entomol. 25:447-477.

DUMBACHER, J. P., BEEHLER, B. M., SPANDE, T. F., GARRAFFO, H. M., and DALY, J. W. 1992. Homobatrachotoxin in the genus Pitohui: chemical defense in birds? Science 258:799-801.

DUMBACHER, J. P., SPANDE, T. F., and DALY, J. W. 2000. alkaloids from birds: A second toxic bird genus ( Ifrita kowaldi) from New Guinea. Proc. Natl. Acad. Sci. USA 97:12970-12975.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 101

DUMBACHER, J. P., WAKO, A., DERRICKSON, S. R., SAMUELSON, A., SPANDE, T. F., and DALY, J. W. 2004. Melyrid beetles ( Choresine): A putative source for the batrachotoxin alkaloids found in poison-dart frogs and toxic passerine birds. Proc. Natl. Acad. Sci. USA 101:15857-15860.

EDGREN, R. A. 1955. The natural history of the hog-nosed snakes, genus Heterodon: A review. Herpetologica 11:105-117.

EISNER, T„ EISNER, M„ ROSSINI, C„ IYENGAR, V. K„ ROACH, B. L„ BENEDIKT, E., and MEINWALD, J. 2000. Chemical defense against predation in an insect egg. Proc. Natl. Acad. Sci. USA 97:1634-1639.

EISNER, T., GOETZ, M. A., HILL, D. E., SMEDLEY, S. R , and MEINWALD, J. 1997. Firefly "femmes fatales" acquire defensive steroids (lucibufagins) from their firefly prey. Proc. Natl. Acad. Sci. USA 94:9723-9728.

EISNER, T. and MEINWALD, J. 1995. The chemistry of sexual selection. Proc. Natl. Acad. Sci. USA 92:50-55.

ERSPAMER, V. 1954. Pharmacology and indolealkylamines. Pharmacol. Rev. 6:425- 487.

ERSPAMER, Y. 1994. Bioactive secretions of the amphibian integument, pp. 178-350, in: G.T. Barthalmus H. Heatwole, and A.Y. Heatwole (eds.). Amphibian Biology. Surrey Beatty and Sons, Chipping Norton.

FUKADA, H. 1959. Biological studies on the snakes, V Food habits in the fields. Bull. Kyoto Gakugei Univ., B. 14:22-28.

FUKADA, H. 1961. Peculiar habits of the Japanese snake, Natrix t. tigrina (Boie). Bull. Kyoto Gakugei Univ., 18:13-16.B.

FUKADA, H. 1992a. Ch 8-Miscellany, pp. 130-138, in: H. Fukada (eds.). Snake Life History in Kyoto. Impact Shuppankai Co., Tokyo.

FUKADA, H. 1992b. Snake Life History in Kyoto. Impact Shuppankai Co., Tokyo.

GARRAFFO, H. M„ SPANDE, T. F„ DALY, J. W„ BALDESSARI, A., and GROS, E. G. 1993. Alkaloids from bufonid toads ( Melanophryniscus): decahydroquinolines, pumiliotoxins and homopumiliotoxins, indolizidines, pyrrolizidines, and quinolizidines. J. Nat. Prod. 56:357-373.

GEFFENEY, S., BRODIE, E. D., JR., RUBEN, P. C., and BRODIE, E. D., III. 2002. Mechanisms of adaptation in a predator-prey arms race: TTX-resistant sodium channels. Science 297:1336-1339.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 102

GEHLBACH, F. R. 1970. Death-feigning and erratic behavior in leptotyphlopid, colubrid, and elapid snakes. Herpetologica 26:24-34.

GONZALEZ, A., HARE, J. F„ and EISNER, T. 1999a. Chemical egg defense in Photuris firefly "femmes fatales". Chemoecology 9:177-185.

g o n z A l e z , a ., s c h r o e d e r , f . c ., a t t y g a l l e , a . b „ s v a t o s , a ., MEINWALD, J., and EISNER, T. 1999b. Metabolic transformations of acquired lucibufagins by firefly "femmes fatales". Chemoecology 9:105-112.

GORIS, R. C. and MAEDA, N. 2005. A Guide to the Amphibians and Reptiles of Japan. Krieger Publishing Company, Malabar, FL.

GOTO, T. 1975. Ch. 11. Non-alkaloidal nitrogen compounds, pp. 423-522, in: Koji Nakanishi, Toshio Goto, Sho Ito, Shinsaku Natori, Shigeo Nozoe (eds.). Natural Products Chemistry. Academic Press, Inc., New York, NY.

GRANT, J. B. and LAND, B. 2002. Transcutaneous amphibian stimulator (TAS): A device for collection of amphibian skin secretions. Herpetol. Rev. 33:38-41.

GREENE, H. W. 1997. Snakes: The Evolution of Mystery in Nature. University of California Press, Berkeley, CA.

GROVES, J. D. 1980. Mass predation on a population of the American toad, Bufo americanus. Am. Midi. Nat. 103:202-203.

HABERMEHL, G. G. 1981. Ch. 6. Amphibia (Amphibians), pp. 112-129. Venomous Animals and Their Toxins. Springer-Verlag, New York.

HABERMEHL, G. G. 1992. Chemistry and pharmacology of amphibian venoms, pp. 268-271, in: P. Gopalakrishnakone and C.K. Tan (eds.). Recent Advances Toxinology Research. National University of Singapore, Singapore.

HAMILTON, W. J., JR. and POLLACK, J. A. 1956. The food of some colubrid snakes from Fort Benning, Georgia. Ecology 37:519-526.

HANIFIN, C. T., BRODIE, E. D., Ill, and BRODIE, E. D., JR. 2003. Tetrodotoxin levels in eggs of the rough-skin newt, Taricha granulosa, are correlated with female toxicity. J. Chem. Ecol. 29:1729 - 1739.

HANIFIN, C. T„ YOTSU-YAMASHITA, M„ YASUMOTO, T„ BRODIE, E. D., Ill, and BRODIE, E. D., JR. 1999. Toxicity of dangerous prey: Variation of tetrodotoxin levels within and among populations of the newt Taricha granulosa. J. Chem. Ecol. 25:2161 - 2175.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 103

HEUSSER, J. and SCHLUMPF, H. W. 1962. Totstellen bei der Barren-Ringelnatter - Natrix natrix Helvetica. Aquar. Terr. 2:214-218.

HITT, M. and ETTINGER, D. D. 1986. Toad toxicity. N. Engl. J. Med. 314:1517.

HULME, J. H. 1951. The "playing possum" habits of the ringhals cobra. Herpetologica 7:132.

HUTCHINSON, D. A. and SAVITZKY, A. H. 2004. Vasculature of the parotoid glands of four species of toads (Bufonidae: Bufo). J. Morphol. 260:247-254.

JIANG, Y. F. and ZHAO, E. M. 1983. Studies on amphibians and reptiles of Mt. Gongga region, , China. 3. Study of species-group nuchalis, genus Rhabdophis. Acta Herpetologica Sinica 2:59-62. (in Chinese with English abstract)

JONES, T. H., GORMAN, J. S. T., SNELLING, R. R., DELAB IE, J. H. C., BLUM, M. S., GARRAFFO, H. M„ JAIN, P., DALY, J. W„ and SPANDE, T. F. 1999. Further alkaloids common to ants and frogs: Decahydroquinolines and a quinolizidine. J. Chem. Ecol. 25:1179 - 1193.

KAWASHIMA, J. 1957. Disturbance of the eye by {Natrix tigrina). Jpn. Rev. Clin. Ophthalmol. 50:837-839. (in Japanese)

KAWASHIMA, J. 1959. Disturbance of the eye by snake venom {Natrix tigrina) II. Jpn. Rev. Clin. Ophthalmol. 53:834-837. (in Japanese)

KITAZUME, Y. 1953. On the increasing action of nuchal gland inclusions of the snake, Natrix tigrina, upon the blood pressure of the rabbit. Zool. Mag. 62:225- 227.

KNIGHT, M„ GLOR, R., SMEDLEY, S. R., GONZALEZ, A., ADLER, K., and EISNER, T. 1999. Firefly toxicosis in lizards. J. Chem. Ecol. 25:1981-1986.

LICHT, L. E. 1967. Death following possible ingestion of toad eggs. Toxicon 5:141- 142.

LICHT, L. E. 1968. Unpalatability and toxicity of toad eggs. Herpetologica 24:93-98.

LICHTSTEIN, D„ GATI, I., HAVER, E„ and KATZ, U. 1992. Digitalis-like compounds in the toad Bufo viridis: tissue and plasma levels and significance in osmotic stress. Life Sci. 51:119-128.

MACDONALD, A. S. J. 1947. "Shamming death" - snakes. J. Bombay Nat. Hist. Soc. 47:173.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 104

MCDONALD, H. S. 1974. Bradycardia during death-feigning of Heterodon platyrhinos Latreille (Serpentes). J. Herpetol. 8:157-164.

MCPHAIL, K. L„ DAVIES-COLEMAN, M. T., and STARMER, J. 2001. Sequestered chemistry of the arminacean nudibranch Leminda millecra in Algoa Bay, South Africa. J. Nat. Prod. 64:1183-1190.

MELERO, C. P., MEDARDE, M., and SAN FELICIANO, A. 2000. A short review on cardiotonic steroids and their aminoguanidine analogues. Molecules 2000:51- 81.

MEYER, K. 1966. Cardiotoxic steroids from toads. Mem. Inst. Butantan 33:433-440.

MEYER, K. and LINDE, H. 1971. Collection of toad venoms and chemistry of the toad venom steroids, pp. 521-556, in: W. Bucherl and E.E. Buckley (eds.). Venomous Animals and Their Venoms. Academic Press, Boston.

MICHL, H. and KAISER, E. 1963. Chemie and Biochemie der Amphibiengifte. Toxicon 1:175-228.

MORI, A. 2004. Another venom gland in snakes: Function, ultrastructure, and physiology of the nuchal glands of Rhabdophis tigrinus. Bull. Herpetol. Soc. Japan 2004:29-33.

MORI, A. and BURGHARDT, G. M. 2000. Does prey matter? Geographic variation in antipredator responses of hatchlings of a Japanese natricine snake {Rhabdophis tigrinus). J. Comp. Psychol. 114:408-413.

MORI, A. and BURGHARDT, G. M. 2001. Temperature effects on anti-predator behaviour in Rhabdophis tigrinus, a snake with toxic nuchal glands. Ethology 107:795-811.

MORI, A., LAYNE, D., and BURGHARDT, G. M. 1996. Description and preliminary analysis of antipredator behavior of Rhabdophis tigrinus tigrinus, a colubrid snake with nuchal glands. Japan. J. Herpetol. 16:94-107.

MORI, A. and MORIGUCHI, H. 1988. Food habits of snakes in Japan: A critical review. Snake 20:98-113.

MORIGUCHI, H. 1982. Appearance, movements and food habits of snakes at Minasegawa, Kanagawa, Japan. Snake 14:44-49.

MUTOH, A. 1981. Relationship between toad feeding and palatine bone elements in Rhabdophis tigrinus. Yasei (Wildlife) 6:27-30. (in Japanese)

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 105

MUTOH, A. 1983. Death-feigning behavior of the Japanese colubrid snake Rhabdophis tigrinus. Herpetologica 39:78-80.

MYERS, C. W. and ARATA, A. A. 1961. Remarks on "defensive" behavior in the hognose snake Heterodon simus (Linnaeus). Q. J. Florida Acad. Sci. 24:108- 110.

NAGATA, E. and MORI, A. 2003. A record of Elaphe conspicillata from Kinkazan Island, Miyagi Prefecture. Bull. Herpetol. Soc. Japan 2003:74-75. (in Japanese)

NAKAMURA, K. 1935. On a new integumental poison gland found in the nuchal region of a snake, Natrix tigrina. Mem. Coll. Sci. Kyoto Imp. Univ. 10B:229- 240.

NAKAMURA, K. and UENO, S. I. 1963. Japanese Reptiles and Amphibians in Colour. Hoikusha, Osaka, Japan.

NELSON, C. J. 1993. A model for glycoside storage by the monarch butterfly, pp. 83-90, in: S.B. Malcolm and M.P. Zalucki (eds.). Biology and Conservation of the Monarch Butterfly. Natural History Museum of Los Angeles County, Los Angeles, CA.

NISHIDA, R. 2002. Sequestration of defensive substances from plants by Lepidoptera. Annu. Rev. Entomol. 47:57-92.

NUSSBAUM, R. A., BRODIE, E. D„ JR., and STORM, R. M. 1983. Amphibians and Reptiles of the Pacific Northwest. University Press of Idaho, Moscow, ID.

PAIK, N. K. and YANG, S. Y. 1986. Taxonomic study on two subspecies of European grass snake (Rhabdophis tigrina) in Korea. Korean J. Syst. Zool. 2:79-92.

PAVELKA, L. A., KIM, Y. H., and MOSHER, H. S. 1977. Tetrodotoxin and tetrodotoxin-like compounds from the eggs of the Costa Rica frog, Atelopus chiriquiensis. Toxicon 15:135-139.

PLATT, D. R. 1969. Natural history of the hognose snakes Heterodon platyrhinos and Heterodon nasicus. Univ. Kansas Mus. Nat. Hist. Publ. 18:253-420.

PORTO, A. M., BARALLE, F. E„ and GROS, E. G. 1972. Biosynthesis of bufadienolides in toads III - Experiments with [2-14C]mevalonic acid, [20- 14C]3/?-hydroxy-5-pregnen-20-one and [20-14C]cholesterol. J. Biochem. 3:11-17.

PORTO, A. M. and GROS, E. G. 1971. Biosynthesis of the bufadienolide marinobufagin in toads Bufo paracnemis from cholesterol-20-14C. Experientia 27:506.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 106

REUTER, H., HENDERSON, S. A., HAN, T., ROSS, R. S., GOLDHABER, J. I., and PHILIPSON, K. D. 2002. The Na+-Ca2+ exchanger is essential for the action of cardiac glycosides. Circ. Res. 90:305-308.

ROBERTS, K. A. 2000. An ultrastructural survey of the nuchal glands of Rhabdophis tigrinus (Serpentes: Colubridae). MS thesis. Old Dominion University, Norfolk, Virginia.

ROHLF, F. J. and SOKAL, R. R. 1995. Statistical Tables. W. H. Freeman and Co., New York, NY.

SAKATE, M. and OLIVEIRA, P. C. L. D. 2000. Toad envenoming in dogs: effects and treatment. J. Venom. Anim. Toxins 6:52-62.

SAPORITO, R. A., GARRAFFO, H. M., DONNELLY, M. A., EDWARDS, A. L., LONGINO, J. T., and DALY, J. W. 2004a. Formicine ants: An arthropod source for the pumiliotoxin alkaloids of dendrobatid poison frogs. Proc. Natl. Acad. Sci. USA 101:8045-8050.

SAPORITO, R. A., GARRAFFO, H. M., DONNELLY, M. A., EDWARDS, A. L., LONGINO, J. T., and DALY, J. W. 2004b. Formicine ants: An arthropod source for the pumiliotoxin alkaloids of dendrobatid poison frogs. Proceedings o f the National Academy o f Sciences 101:8045-8050.

SCHMIDT, K. P. and INGER, R. F. 1957. Living Reptiles of the World. Doubleday and Co., Garden City, NY.

SCHONER, W. 2002. Endogenous cardiac glycosides, a new class of steroid hormones. Eur. J. Biochem. 269:2440-2448.

SIPERSTEIN, M. D., MURRAY, A. W., and TITUS, E. 1957. Biosynthesis of cardiotonic sterols from cholesterol in the toad, Bufo marinus. Arch. Biochem. Biophys. 67:154-160.

SMITH, H. M. and WHITE, F. N. 1955. Adrenal enlargement and its significance in the hognose snakes ( Heterodon). Herpetologica 11:137-144.

SMITH, M. A. 1938. The nucho-dorsal glands of snakes. Proc. Zool. Soc. London B 107:575-583, pi I.

SPANDE, T. F„ JAIN, P., GARRAFFO, H. M„ PANNELL, L. K„ YEH, H. J. C„ and DALY, J. W. 1999. Occurrence and significance of decahydroquinolines from dendrobatid poison frogs and a myrmicine ant: Use of *H and 1 C NMR in their conformational analysis. J. Nat. Prod. 62:5 -21.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 107

SPAUR, R. C. and SMITH, H. M. 1971. Adrenal enlargement in the hognosed snake Heterodonplatyrhinos. J. Herpetol. 5:197-199.

STITT, E. W. 2003. Natural history note: Masticophis flagellum cingulum (Sonoran Coachwhip) - Defensive behavior. Herpetol. Rev. 34:150-151.

STUEBING, R. B. and LIAN, T. F. 2002. Notes on the fire-lipped keelback Rhabdophis murudensis (Smith, 1925) (Ophidia: Colubridae: Natricinae) from northern Borneo. Raffles Bull. Zool. 50:227-230.

SUZUKI, R. 1960. Disturbance of the eye by snake venoms ( Natrix tigrina (Boie)). J. Clin. Ophthal. 14:1384-1387.

TAGGI, A. E., MEINWALD, J., and SCHROEDER, F. C. 2004. A new approach to natural products discovery exemplified by the identification of sulfated nucleosides in spider venom. J. Am. Chem. Soc. 126:10364-10369.

TANAKA, K. and MORI, A. 2000. Literature survey on predators of snakes in Japan. Curr. Herpetol. 19:97-111.

THOMPSON, J. N. 2005. The Geographic Mosaic of Coevolution. The Univ. of Chicago Press, Chicago.

TOLEDO, R. C. and JARED, C. 1995. Cutaneous granular glands and amphibian venoms. Comp. Biochem. Physiol. lllA:l-29.

TORIBA, M. and SAWAI, Y. 1990. Venomous snakes of medical importance in Japan, pp. 323-347, in: P. Gopalakrishnakone and L.M. Chou (eds.). Snakes of Medical Importance (Asia-Pacific Region). Venom and Toxin Research Group, National University of Singapore, Singapore.

TUCKER, M. 1989. Life history note: Masticophis flagellum (Coachwhip) - Behavior. Herpetol. Rev. 20:72.

UTIGER, U„ HELFENBERGER, N., SCHATTI, B„ SCHMIDT, C„ RUF, M., and ZISWILER, V. 2002. Molecular systematics and phylogeny of Old and New World ratsnakes, Elaphe auct., and related genera (Reptilia, , Colubridae). Russian J. Herpetol. 9:105-124.

WILLIAMS, B. L., BRODIE, E. D., JR., and BRODIE, E. D., III. 2004. A resistant predator and its toxic prey: Persistence of newt toxin leads to poisonous (not venomous) snakes. J. Chem. Ecol. 30:1901-1919.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 108

ZALUCKI, M. P., BROWER, L. P., and ALFONSO, A. M. 2001. Detrimental effects of latex and cardiac glycosides on survival and growth of first instar monarch butterfly larvae Danaus plexippus feeding on the sandhill milkweed Asclepias humistrata. Ecolog. Entomol. 26:212-224.

ZELNIK, R., ZITI, L. M., and GUIMARAES, C. V. 1964. A chromatographic study of the bufadienolides isolated from the venom of the parotid glands of Bufo paracnemis Lutz 1925. J. Chromatogr. 15:9-14.

ZHAO, E. M. and ADLER, K. 1993. Herpetology of China. Society for the Study of Amphibians and Reptiles, Oxford, OH.

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APPENDIX A

NMR AND MS DATA ON BUFADIENOLIDES

The following tables present results obtained from nuclear magnetic resonance

spectroscopy (NMR) and mass spectroscopy (MS). These data were used to assign

structures to bufadienolide compounds in the nuchal gland fluid of Rhabdophis

tigrinus. The NMR data presented include the chemical shift values (8) in parts per

million (ppm) for carbon and hydrogen atoms, which are identified by number (see

Figure A.l); proton-proton coupling constants ( J) in Hertz (Hz); Nuclear Overhauser

Effect Spectroscopy (NOESY) correlations between hydrogens through space; and

Heteronuclear Multiple Bond Correlations (HMBC) between hydrogens and carbons.

Coupling constants are only included for new natural products where this information

was essential for structural assignments. The MS data using positive ion electrospray

ionization (ESI+) are shown below each table as mass to charge ratios (m/z) for ionized

bufadienolides.

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HO,

OH HO" OH

Fig. A.I. Bufadienolide compound 1 showing the numbering of carbons for which data

are presented in Tables A.1-A.17.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. T a b l e A. 1. NMR a n d MS Da t a f o r B ufadienolide C o m p o u n d 1

Carbon NOESY HMBC 5 (ppm) Proton No. 5 (ppm) No. Correlations Correlations C-l 32.2 1-Ha 1.58 19 1-Hp 2.41 19 3,5 C-2 31.5 2-Ha 1.63 2-Hp 1.63 C-3 6 8 .6 3-H 3.96 2p, 4p C-4 42.7 4-Ha 1.95 9 4-Hp 1.53 6 a 2 ,3 ,5 , 10 C-5 76.0 C-6 37.2 6 -Ha 1.44 6 -Hp 1.74 19 C -l 24.8 7-Ha 1.32 15p 7-Hp 1.96 6 a 5 6 p, 8 C-8 41.6 8 -H 1.65 18, 19 C-9 45.7 9-H 1.86 10, 11, 19 C-10 41.7 C-l 1 69.1 11-H 3.72 18, 19 C-12 51.5 12-Ha 1.56 9 9, 11, 13, 17, 18 12-Hp 1.66 18 9, 11, 13, 14, 18 C-13 49.8 C-14 85.1 C-15 33.2 15-Ha 2.15 9, 12a 15-Hp 1.74 C-16 29.3 16-Ha 2.19 16-Hp 1.76 C-17 51.7 17-H 2.63 12a, 12p, 16a, 12, 13, 14, 16, 18(w) 2 0 , 2 1 ,2 2 C-l 8 17.9 18-CH3 0.74 12, 13, 14, 17 C-19 16.7 I9-CH3 1.00 1 ,5 ,9 , 10 C-20 124.3 C-21 150.4 21-H 7.46 16p(w), 17, 18 17, 20, 22, 24 C-22 148.9 22-H 7.94 16p, 17(w), 18 21,24 C-23 115.3 23-H 6.28 22 20, 24 C-24 164.7

w = weak NOE correlation.

MS (ESI+) m/z: (M+H)+ 419.2, (M+Na)+ 441.2.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 112

T a b l e A.2. NMR a n d MS Da t a f o r B ufadienolide C o m p o u n d 2

Carbon NOESYHMBC 8 (ppm) Proton No. 8 (ppm) J(H z) No. Correlations Correlations C -l 39.2 1-Ha 2.65 Jia,ip= 13.5, 2«, 2p, 19 J la,2p = 4 1-Hp 1.00 19 C-2 31.4 2-Ha 1.53 3la,2a — 3 2-Hp 1.63 J4P,3 = 4 lp(w) C-3 72.0 3-H 3.56 J4a,3= 11-2 lp, 2p, 4p, 5 C-4 37.5 4-Ha 1.38 3 ,5 4-Hp 1.58 5 C-5 50.2 5-H 1.49 34a,5 = 13.8, lp, 19 J4P.5 = 5 C-6 73.7 6-H 3.76 4«(w), 4p, 5, 10 7a> 7p C -l 30.3 7-Ha 1.98 7-Hp 1.56 J7P,8= 3.2 C-8 8-H 2.03 7a, 18, 19 C-9 43.7 9-H 1.81 4„, 12„ 10, 11, 19 C-10 36.7 C -l 1 68.7 11-H 3.73 8, 12p, 18, 19 C-12 51.4 12-Ha 1.56 9, 11, 13, 17, 18 12-Hp 1.69 18 9, 11, 13, 14, 18 C-13 49.8 C-14 84.6 C-15 33.1 15-Ha 2.18 12„ 15-Hp 1.74 C-16 29.3 16-Ha 2.22 16-Hp 1.77 C -l 7 51.9 17-H 2.61 12a, 12p, 16a, 12, 13, 14, 16, 18(w) 2 0 ,2 1 ,2 2 C-18 0.77 18-CH3 17.9 12, 13, 14, 17 C-19 26.1 19-CH-, 1.21 1 ,5 ,9 , 10 C-20 124.4 C-21 150.4 21-H 7.45 321,22 = 2.6 16p(w), 17, 18 17, 20, 22, 24 C-22 148.9 22-H 7.98 322,23 = 9.8 16p, 17(w), 18 2 1 ,2 4 C-23 115.3 23-H 6.28 22 20,24 C-24 164.5

w = weak NOE correlation.

MS (ESI+) m/z: (M+H)+ 419.2, (M+Na)+ 441.2.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 113

T a b l e A .3 . N M R a n d M S D a t a f o r B ufadienolide C o m p o u n d 3

Carbon NOESY HMBC 5 (ppm) Proton No. 5 (ppm) J ( Hz) No. Correlations Correlations

C-l 32.3 1-Ha 2.38 f la,ip = 14.5, 19, 2a, 2p 3la,2p 5 1-Hp 1.43 19 C-2 27.6 2-Ha 1.88 3 la,2a = 3.9 1.58 2-Hp Ip C-3 70.9 3-H 3.95 J4>3 = 3.9 4, 2n, 2p C-4 69.0 4-H 3.80 J4.5 = 11-8 2a, 7a, 9 C-5 45.6 5-H 1.54 J6a,5 = 3 lp , 19 C-6 21.5 6-Ha 1.98 J6a,7p= 3.9 5, 7a, 7p 6-Hp 1.61 3 6a,6p = 1 4 .5 19 C -l 22.0 7-Ha 1.35 J7a,7p= 13.8, 37a,8 = 11-8 7-Hp 1.84 C-8 41.9 8-H 1.70 18, 19 C-9 43.8 9-H 1.70 Jg 11 = 10.5 C-10 39.0 C-l 1 68.9 11-H 3.69 J l 1,12a = 10.5, l a(w), 8, 12p, j 11.12P = 5 18, 19 C-12 51.3 12-Ha 1.52 12-Hp 1.66 18 C-13 49.8 C-14 85.0 C-15 33.1 15-Ha 2.13 9, 12a 15-Hp 1.74 C-16 29.4 16-Ha 2.20 16-Hp 1.76 C-l 7 51.8 17-H 2.60 12a, 12p, 16a, 12, 13, 14, 16, 18(w) 2 0 ,2 1 ,2 2 C-l 8 18.0 18-CH3 0.74 12, 13, 14, 17 C-19 24.1 I9 -CH3 1.10 1, 5 ,9 , 10 C-20 124.2 C-21 150.4 21-H 7.45 321,22 = 2 .6 16p(w), 17, 18 17, 20, 22, 24 C-22 149.0 22-H 7.96 322,23 = 9 .8 16p, 17(w), 18 1 7 ,2 1 ,2 4 C-23 115.3 23-H 6.28 22 20, 24 C-24 164.5

w = weak NOE correlation.

MS (ESI+) m/z: (M+H)+ 419.2, (M+Na)+ 441.2.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 114

T a b l e A.4. NMR a n d MS Da t a f o r B ufadienolide C o m p o u n d 4

Carbon NOESY HMBC 8 (ppm) Proton No. 8 (ppm) J( Hz) No. Correlations Correlations C-l 33.8 1-Ha 2.69 2p, 19 2, 10, 5 1-Hp 1.95 J2a,ip= 13.2 19 C-2 38.8 2-Ha 2.74 •Jla,2a — 5.2, lp 1,3 Ea,2p = 15.7 2-Hp 2.06 C-3 215.8

C-4 50.0 4-Ha 3.08 •14a,4(5 = 14.5 7a, 9 3 ,5 4-Hp 2 . 0 0 6 a C-5 78.9 C- 6 36.4 6 -H„ 1.43 J6a,6p =13.2 6 -Hp 1.77 J6p,7a= 5, 19 J6p,7p= 3 C -l 25.2 7-Ha 1.30 J7a,8=13.2 7-Hp 2.06 Ep,8 = 5 6 a, 6 p C- 8 41.2 8 -H 1.78 J8>9= 13.2 18, 19 C-9 45.9 9-H 1.93 J9 1 1 10.5 C-10 42.1

C-l 1 68.7 11-H 3.82 Jll,12a = 10.5, 12p, 18, 19 Jl 1.12P = 5 C-12 51.3 12-Ha 1.60 12-Hp 1.72 18 C-13 49.8 C-14 84.9 C-15 33.1 15-H 2.15 15-H 1.74 C-16 29.4 16-Ha 2 .2 2 9, 1 2 a 16-Hp 1.76 C -l 7 51.8 17-H 2.63 1 2 a, 1 2 p, 12, 13, 14, 16a, 18(w) 16, 2 0 , 2 1 , 2 2 C-18 17.9 18-CH3 0.77 12, 13, 14, 17 C-19 16.5 I9 -CH3 1.10 1 ,5 ,9 , 10 C-20 124.2

C-21 150.5 21-H 7.46 El,22 ~ 2.6 16p (w), 17, 17, 20, 22, 18 24 C-22 149.0 22-H 7.96 322,23 = 9.8 16p, 17(w), 18 17,21,24 C-23 115.3 23-H 6.29 22 20, 24 C-24 164.5

w = weak NOE correlation.

MS (ESI+) m/z: (M+H)+ 417.2, (M+Na)+ 439.2.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 115

T a b l e A.5. NMR a n d MS Da t a f o r B ufadienolide C o m p o u n d 5

Carbon NOESY HMBC 8 (ppm) Proton No. 5 (ppm) J(H z) No. Correlations Correlations C-l 28.4 1-H 2 . 2 0 19 1-H 1.82 2p, 19 C-2 29.1 2-Ha 1.87 9 2-Hp 1.53 C-3 6 8 .6 3-H 4.09 2a) 2p, 4a, 4p C-4 37.9 4-Ha 1.96 9 4-Hp 1.52 C-5 76.4 C- 6 76.6 6 -H 3.57 7a> 7p C-7 31.3 7-H 2.07 7-H 1.53 C- 8 36.2 8 -H 2.04 18, 19 C-9 45.9 9-H 1.69 C-10 42.8 C -l 1 68.9 11-H 3.79 8 , 12p, 18, 19 C-12 51.5 12-Ha 1.55 11, 13, 17, 18 12-Hp 1 .6 8 18 9, 11, 13, 14, 17, 18 C-13 49.8 C-14 84.7

C-15 33.2 15-Ha 2.14 1 2 a 15-Hp 1.72 C-16 29.3 16-Ha 1.76 16-Hp 2 .2 1 C-l 7 51.8 17-H 2.62 1 2 a, 1 2 p, 16a, 12, 13, 14, 16, 18(w) 2 0 , 2 1 , 2 2 C-18 17.9 18-CH3 0.76 12, 13, 14, 17 C-19 19.6 I9 -CH3 1.19 1, 5,9, 10 C-20 124.3

C-21 150.5 21-H 7.46 hi,22= 2 .6 16p(w), 17, 18 17, 20, 22, 24

C-22 149.0 22-H 7.98 ^ 2 2 ,2 3 = 9.8 16p, 17(w), 18 2 1 ,2 4 C-23 115.3 23-H 6.29 2 2 20, 24 C-24 164.5

w = weak NOE correlation.

MS (ESI+) m/z: (M+H)+ 435.2, (M+Na)+ 457.2.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 116

T a b l e A .6 . N M R a n d M S D ata for Bufadienolide Compound 6

Carbon NOESYHMBC 8 (ppm) Proton No. 8 (ppm) J(H z) No. Correlations Correlations C-l 33.7 1-Ha 2.21 Jla,ip= 13-8 2„, 19 1-Hp 1.52 19 C-2 29.0 2-H 1.44 2-H 1.78 C-3 66.9 3-H 4.01 2a) 2p, 4a, 4p C-4 34.6 4-Ha 1.65 ■l4a,5 =1 3 .2 4-Hp 1.42 J4P.5 = 5.2 C-5 45.1 5-H 1.80 Ip, 19

C-6 73.5 6-H 3.69 *f 6 ,7 p = 3 4a(w), 4p, 5, 8, 10 7a, 7p C -l 30.7 7-Ha 1.56 J7a.8=13.2 7-Hp 1.94 Jtp.8=3.9 C-8 36.8 8-H 2.01 J8,9= 1 1.2 7p, 18, 19 7, 9, 10, 14, 15 C-9 43.7 9-H 1.69 J9,„ = 9.9 C-10 26.0 1.23 C-ll 68.6 11-H 3.75 J1 l,12ct= 1 1-8, 8, 12p, 18, 19 9, 10, 12 J1 i,i2p= 4.6 C-12 51.3 12-Ha 1.53 9, 11, 13, 17, 18 12-Hp 1.67 18 9 ,1 1 ,1 3 , 14, 17, 18 C-13 49.9 C-14 84.8 C-15 33.1 15-Ha 1.72 15-Hp 2.15 12a C-16 29.4 16-Ha 1.76 16-Hp 2.21 C-17 51.9 17-H 2.59 12a, 12p, 16a, 12, 13, 14, 16, 18(w) 2 0 ,2 1 ,2 2 C-18 17.9 18-CH3 0.76 12, 13, 14, 17 C-19 26.0 I 9 -CH3 1.23 1 ,5 ,9 , 10 C-20 124.2 II NJ 'o\ C-21 150.5 21-H 7.45 <— 1 16p(w), 17, 18 17, 20, 22, 24 to 'to C-22 149.1 22-H 7.98 •122,23 - 9.8 16p, 17(w), 18 17,21,24 C-23 115.2 23-H 6.28 22 20, 24 C-24 164.5

w = weak NOE correlation.

MS (ESI+) m/z: (M+H)+419.2, (M+Na)+ 441.2.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. T a b l e A.7. NMR a n d MS Da t a f o r B ufadienolide C o m p o u n d 7

Carbon NOESY HMBC 8 (ppm) Proton No. 8 (ppm) No. Correlations Correlations C-l 32.1 1-Ha 1.60 19 1-Hp 2.44 19 C-2 42.6 2-Ha 1.56 2-Hp 1 .8 6 C-3 68.5 3-H 3.95 2 p, 4p C-4 31.4 4-Ha 1.64 4-Hp 1.48 C-5 76.1 C-6 36.9 6 -Ha 1.48 6 -Hp 1.83 19 C -l 24.8 7-H 2.07 7-H 1.36 C-8 40.1 8 -H 2 . 1 0 18, 19 C-9 44.2 9-H 1.91 C-10 42.2 C -l 1 75.06 11-H 4.41 8 , 18, 19 C-12 214.6 C-13 63.4 C-14 85.6 C-15 45.1 15-H 1.56 15-H 1.69 C-16 28.9 16-Ha 1.74 16-Hp 2 .0 2 C-17 42.0 17-H 4.13 16a, 18(w) 12, 13, 16, 20, 2 1 , 2 2 C-18 17.7 18-CH3 0.91 12, 13, 14, 17 C-19 16.3 19-CH3 1 .1 1 1 ,5 ,9 , 10 C-20 122.9 C-21 151.4 21-H 7.52 16p(w), 17, 18 20, 22, 24 C-22 148.9 22-H 7.92 16p, 17(w), 18 2 1 ,2 4 C-23 115.6 23-H 6.31 2 2 2 0 ,2 4 C-24 164.2

w = weak NOE correlation.

MS (ESI+) m/z: (M+H)+ 433.2, (M+Na)+ 455.2.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. T a b l e A . 8. N M R a n d M S D ata for Bufadienolide Compound 8

Carbon NOESY HMBC 5 (ppm) Proton No. 6 (ppm) No. Correlations Correlations

C -l 27.4 1-Ha 2.28 2 „, 2 p, 19 1-Hp 1.80 2p, 19 C-2 29.2 2-Ha 1.92 9 2-Hp 1.55 C-3 69.1 3-H 4.13 2a> 2p, 4a, 4p C-4 37.8 4-Ha 2.17 7a, 9 4-H3 1.49 6 « C-5 76.2 C- 6 36.1 6 -Ha 1.36 7a 6 -Hp 1.69 19 C -l 24.5 7-Ha 1.26 7-Hp 1.96 6 a» 6 p C-8 41.2 8 -H 1 .6 8 18, 19 C-9 45.7 9-H 1.70 C-10 42.7 C -ll 69.0 11-H 3.74 8 , 18, 19 C-12 51.5 12-Ha 1.54 11, 13, 17, 18 12-Hp 1.67 9, 11, 13, 14, 18 C-13 49.7 C-14 85.0 C-15 33.1 15-Ha 2 . 1 2 1 2 a 15-Hp 1.72 7a C-16 29.3 16-Ha 2.19 16-Hp 1.75 C-17 51.6 17-H 2.62 18(w),12a, 12, 13, 14, 16, 1 2 p, 16a 2 0 , 2 1 , 2 2 C -l 8 17.9 18-CH3 0.74 12, 13, 14, 17 C-19 17.2 I9 -CH3 1.05 1 ,5 ,9 , 10 C-20 124.1 C-21 150.3 21-H 7.43 16p (w), 17, 18 20, 22, 24 C-22 148.8 22-H 7.94 16p, 17(w), 18 2 1 ,2 4 C-23 115.2 23-H 6.26 2 2 20, 24 C-24 164.7

w = weak NOE correlation.

MS (ESI+) m/z: (M+H)+419.2, (M+Na)+ 441.2.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 119

T a b l e A.9. NMR a n d MS Da t a f o r B ufadienolide C o m p o u n d 9

Carbon NOESY HMBC 5 (ppm) Proton No. 5 (ppm) J(Hz) No. Correlations Correlations C -l 75.6 1-H 4.56 f 1,2a ~ 2.6 , 2a, 2p, 9, 19 Ji,2p= 3.2 C-2 33.8 2-Ha 2.02 9 2-Hp 1.82 C-3 69.1 3-H 4.11 *143,3= 4 2a, 2p, 4a, 4p C-4 34.8 4-Ha 2.01 J4a,3 = 4 7« 4-Hp 1.44 6a C-5 32.7 5-H 2.10 4p, 6a, 6p, 19 C-6 27.3 6-Ha 1.38 6-Hp 1.81 19

C -l 22.5 7-Ha 1.38 J7 a ,8 = 13 7-Hp 1.88 *173,8 = 3 8 C-8 41.6 8-H 1.72 J8,9= 12 18, 19 C-9 45.7 9-H 1.62 J 9.11 = 10.5 7a, 12a C-10 42.5

C -l 1 68.3 11-H 3.71 *111,12a = 1 1*8 8, 12p, 18, 19 , J11.12P = 3 C-12 51.2 12-Ha 1.50 11, 13, 17, 18 12-Hp 1.67 18 11, 13, 14, 18 C-13 49.7 C-14 85.1 C -l 5 33.1 15-Ha 2.13 9, 12a 15-Hp 1.75 C-16 29.4 16-Ha 2.21 12a(w) 16-Hp 1.75 C-17 51.8 17-H 2.58 •Il7,16a= 9.2 12a, 12p, 16a, 12, 13, 14, 16, •ll7,163= 6.6 18(w) 2 0 ,2 1 ,2 2 C-18 17.9 18-CH3 0.74 12, 13, 14, 17 C-19 18.6 I9 -CH3 1.18 1 ,5 ,9 , 10 C-20 124.3 C-21 150.4 21-H 7.44 •121,22 = 2.6 16p (w), 17, 18 17, 20, 22, 24 C-22 148.9 22-H 7.96 •122,23 = 9.8 16p, 17(w), 18 21,24 C-23 115.3 23-H 6.28 2 2 20, 24 C-24 164.4

w = weak NOE correlation.

MS (ESI+) m/z: (M+H)+ 419.2, (M+Na)+ 441.2.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. T a b l e A. 10. NMR a n d MS Da t a f o r B ufadienolide C o m p o u n d 10

Carbon NOESY HMBC 5 (ppm) Proton No. 5 (ppm) No. Correlations Correlations C-l 33.1 1-Ha 2.36 2(1.84), 19 i -h 3 1.45 19 C-2 29.1 2-H 1.47 2-H 1.84 C-3 67.7 3-H 4.06 2a> 2p, 4a, 4p C-4 34.5 4-Ha 1.96 9 4-Hp 1.33 6„ C-5 39.0 5-H 1.71 Ip, 4p, 6a, 19 C-6 28.0 6-Ha 1.25 6-Hp 1.89 5, 8, 19 C -l 22.4 7-Ha 1.31 7-Hp 1.83 6a, 8 C-8 41.9 8-H 1.67 18, 19 C-9 42.6 9-H 1.78 12a 10, 11, 19 C-10 37.6 C -ll 69.0 11-H 3.68 8 or 12p, 18, 19 C-12 51.5 12-Ha 1.53 11, 13, 17, 18 12-Hp 1.66 18 9, 11, 13, 14, 18 C -l 3 50.0 C-14 85.2 C-15 33.1 15-Ha 2.16 12a 15-Hp 1.74 C-16 29.4 16-Ha 2.20 12a 16-Hp 1.76 C-17 51.8 17-H 2.61 12a, 12p, 16a, 12, 13, 14, 16, 18(w) 2 0 ,2 1 ,2 2 C-18 17.8 18-CH3 0.74 12, 13, 14, 17 C-19 24.3 19-CH3 1.08 1, 5,9, 10 C-20 124.4 C-21 150.3 21-H 7.44 16p(w), 17, 18 17, 20, 22, 24 C-22 149.0 22-H 7.96 16e, 17(w), 18 2 1 ,2 4 C-23 115.2 23-H 6.28 22 20, 24 C-24 164.7

w = weak NOE correlation.

MS (ESI+) m/z: (M+H)+ 403.2, (M+Na)+ 425.2.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. T a b l e A . 11. NMR a n d MS Da t a f o r B ufadienolide C o m p o u n d 11

Carbon NOESY HMBC 8 (ppm) Proton No. 8 (ppm) No. Correlations Correlations C -l 27.2 1-H 2.31 19, 2(1.54) 1-H 1.82 19 C-2 29.0 2-H 1.82 2-H 1.54 C-3 69.0 3-H 4.11 2

w = weak NOE correlation.

MS (ESI+) m/z: (M+H)+433.2, (M+Na)+ 455.2..

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. T a b l e A . 12. NMR a n d MS Da t a f o r B ufadienolide C o m p o u n d 12

Carbon NOESY HMBC 5 (ppm) Proton No. 5 (ppm) No. Correlations Correlations C -l 38.6 1-Ha 2.73 19, 2 „ 2p 1-Hp 0.97 19 C-2 31.8 2-Ha 1.59 2-Hp 1.65 lp C-3 72.5 3-H 3.59 lp, 2p, 4p, 5 C-4 37.3 4-Ha 1.74 4-Hp 1.48 5 C-5 44.7 5-H 1.33 Ip, 19 C-6 28.4 6-Ha 1.35 6-Hp 1.86 8, 19 C-7 22.6 7-Ha 1.85 4„ 7-Hp 1.37 C-8 42.1 8-H 1.65 18, 19 C-9 43.3 9-H 1.85 12« C-10 38.6 C -l 1 69.2 11-H 3.68 8, 18, 19 C-12 51.4 12-Ha 1.54 12-Hp 1.67 18 C-13 49.6 C-14 85.0 C -l 5 38.7 15-Ha 2.19 9, 12a 15-Hp 1.76 C-16 29.4 16-Ha 2.21 16-Hp 1.76 C -l 7 51.8 17-H 2.60 12a, 12p, 16a, 12, 13, 14, 16, 18(w) 2 0 ,2 1 ,2 2 C-18 18.0 18-CH3 0.74 12, 13, 14, 17 C-19 23.7 19-CHj 1.05 1 ,5 ,9 , 10 C-20 124.3 C-21 150.4 21-H 7.45 16p, 17, 18 20, 22, 24 C-22 149.0 22-H 7.96 16p, 17(w), 18 2 1 ,2 4 C-23 115.2 23-H 6.28 22 20, 24 C-24 164.5

w = weak NOE correlation.

MS (ESI+) m/z: (M+H)+ 403.2, (M+Na)+ 425.2.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. T a b l e A. 13. N M R a n d M S D a t a f o r B ufadienolide C o m p o u n d 13

Carbon NOESY HMBC 5 (ppm) Proton No. 6 (ppm) No. Correlations Correlations C -l 18.4 1-H„ 1.70 Ha 1-Hp 2.13 C-2 27.3 2-H 1.72 2-H 1.67 C-3 67.7 3-H 4.14 2a> 2p, 4a, 4p C-4 38.4 4-Ha 1.50 4-Hp 2.20 C-5 90.1 C-6 37.2 6-Ha 1.67 7a> 4p 6-Hp 2.10 C -l 25.0 7-Ha 1.38 7-Hp 2.15 C-8 42.7 8-H 1.95 18 C-9 40.3 9-H 1.71 4„ C-10 55.9 C -l 1 23.4 11-Ha 1.56 11 -Hp 1.19 18 C-12 41.3 12-Ha 1.43 12-Hp 1.52 18 C-13 49.1 C-14 85.3 C-l 5 32.1 15-Ha 2.07 12a 15-Hp 1.68 C-16 29.4 16-Ha 2.19 16-Hp 1.75 C-17 51.7 17-H 2.54 12, 13, 14, 20, 21,22 C-18 17.0 18-CH3 12, 13, 14, 17 C-19 209.1 19-H 10.06 6p, 8 1, 5, 10 C-20 124.5 C-21 150.3 21-H 7.43 17, 18 20, 22, 24 C-22 149.1 22-H 7.98 16p, 18, 23 21,24 C-23 115.2 23-H 6.28 20, 24 C-24 164.3

MS (ESI+) m/z: (M+H)+ 417.2, (M+Na)+ 439.2.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 124

T a b l e A . 14. NMR a n d MS D a t a f o r B ufadienolide C o m p o u n d 14

Carbon NOESY HMBC 5 (ppm) Proton No. 6 (ppm) No. Correlations Correlations C-l 26.0 1-H 1.80 2(1.57), 19 1-H 1.39 19 C-2 28.2 2-H 1.70 2-H 1.57

C-3 68.7 3-H 4.13 2 a > 2p, 4a, 4p 1,5

C-4 37.4 4-Ha 2.21 7' a> 97 4-Hp 1.50 C-5 76.1 C-6 35.7 6-Ha 1.38 6-Hp 1.72 19 C -l 24.5 7-Ha 1.24 7-Hp 1.99 6a, 6p, 8 C-8 41.6 8-H 1.62 18, 19 C-9 39.8 9-H 1.58 C-10 41.7 C -ll 22.4 11-Ha 1.48 11-Hp 1.27 18, 19 C-12 41.9 12-Ha 1.37 12-Hp 1.58 18 C-13 50.3 C-14 85.7 C-15 42.8 15-Ha 2.51 7a(w), 9, 12a 12, 16, 17 15-Hp 1.77 13, 14, 16, 17 C-16 73.0 16-H 4.49 12a, 15a, 15p, 13, 14 17

C-17 59.1 17-H 2.77 1 2 « , 1 2 p , 12, 13, 14, 15, 18(w) 16, 2 0 ,2 1 ,2 2 C-18 17.1 18-CH3 0.79 12, 13, 14, 17 C-19 17.1 I9 -CH3 0.93 1,5,9, 10 C-20 120.2 C-21 151.5 2 1-H 7.42 17, 18 17, 20, 22, 24 C-22 152.6 22-H 8.11 17(w), 18, 23 17,21,24 C-23 112.6 23-H 6.17 2 0 ,2 4 C-24 164.9

w = weak NOE correlation.

MS (ESI+) m/z: (M+H)+ 419.2, (M+Na)+ 441.2.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. T a b l e A . 15. NMR a n d MS Da t a f o r B ufadienolide C o m p o u n d 15

Carbon NOESY HMBC 5 (ppm) Proton No. 6 (ppm) No. Correlations Correlations C-l 25.9 1-H 1.41 19 1-H 1.82 2, 19 C-2 28.1 2-H 1.69 1 2-H 1.59 C-3 68.8 3-H 4.13 2a? 2p, 4a, 4p C-4 37.4 4-Ha 2.23 7«,9 4-Hp 1.49 6 a C-5 75.9 C-6 34.9 6 -Ha 1.32 7a 6 -Hp 1.69 19 4 ,5 ,7 C -l 23.7 7-Ha 0.98 7-Hp 1.62 6 a C-8 33.4 8 -H 2.04 6 3 , 7p, lip , 18, 7, 9, 14, 15 19 C-9 43.5 9-H 1.67 C-10 41.8 C-l 1 2 2 .2 11-Ha 1.59 1 2 a 11 -Hp 1.40 18, 19 C-12 40.5 12-Ha 1.50 12-Hp 1.77 Ha, Up, 18 C-13 45.9 C-14 73.4 C-15 63.0 15-H 3.60 7a, 7p(w) 16, 17 C-16 72.7 16-H 4.72 12„, 15, 17 15,20 C -l 7 52.8 17-H 2.70 12„, 1 2 p, 18(w) 12, 13, 14, 15, 16, 2 0 , 2 1 , 2 2 C-18 17.4 18-CHj 0.79 12, 13, 14, 17 C-19 17.0 19-CH3 0.96 1 ,5 ,9 , 10 C-20 119.5 C-21 152.4 2 1-H 7.40 17, 18 20, 22, 24 C-22 151.7 22-H 8.10 17(w), 18, 23 24 C-23 113.3 23-H 6.21 20, 24 C-24 164.5

w = weak NOE correlation.

MS (ESI+) m/z: (M+H)+417.2 , (M+Na)+ 439.2.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. T a b l e A . 16. N M R a n d M S D ata for Bufadienolide Compound 16

Carbon NOESY HMBC 5 (ppm) Proton No. 5 (ppm) No. Correlations Correlations C-l 25.9 1-H 1.43 19 1-H 1.84 19 C-2 28.2 2-H 1.58 2-H 1.62 C-3 68.7 3-H 4.10 4a, 4p C-4 37.5 4-Ha 2.16 9 4-Hp 1.50 C-5 75.9 C-6 35.5 6-H 1.86 6-H 1.40 C -l 27.0 7-H 1.48 7-H 1.89 C-8 35.3 8-H 2.29 7p, 18, 19 C-9 44.8 9-H 1.35 C-10 41.7 C -l 1 22.5 11 -Ha 1.63 11-Hp 1.42 18, 19 C-12 41.1 12-Ha 1.22 12-Hp 1.83 18 C -l 3 49.0 C-14 160.7 C-15 121.3 15-H 5.50 7a» 7p, 16 C-16 76.9 16-H 4.58 17 C-17 57.6 17-H 2.56 12a 12, 13, 16, 18, 2 0 ,2 1 ,2 2 C-18 22.6 18-CH3 1.04 12, 13, 14, 17 C-19 16.8 I9 -CH3 0.99 1 ,5 ,9 , 10 C-20 119.0 C-21 151.9 21-H 7.67 17, 18 20, 22, 24 C-22 150.3 22-H 7.84 17(w), 18, 23 2 1 ,2 4 C-23 114.4 23-H 6.28 20, 24 C-24 164.3

w = weak NOE correlation.

MS (ESI+) m/z: (M+H)+ 401.2, (M+Na)+ 423.2.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. T a b l e A . 17. NMR a n d MS Da t a f o r B ufadienolide C o m p o u n d 17

Carbon NOESY HMBC 8 (ppm) Proton No. 8 (ppm) No. Correlations Correlations C-l 26.0 1-Ha 1.38 19 1-Hp 1.80 19, 2p 2, 10, 19 C-2 28.3 2-Ha 1.69 2-Hp 1.58 C-3 68.8 3-H 4.13 2a, 2p, 4a, 4p C-4 37.5 4-Ha 2.21 7„,9 4-Hp 1.49 C-5 76.0 C-6 35.8 6-Ha 1.38 7a 6-Hp 1.73 19 C -l 24.6 7-Ha 1.26 7-Hp 1.95 6a, 6p, 8 C-8 41.8 8-H 1.65 18, 19 C-9 40.1 9-H 1.62 C-10 41.7 C -l 1 22.8 11-Ha 1.49 la 11-Hp 1.28 18, 19 C-12 41.8 12-Ha 1.45 12-Hp 1.53 Up, 18 C-13 49.5 C-14 85.9 C-15 32.9 15-Ha 2.07 9, 12a 15-Hp 1.70 C-16 29.5 16-Ha 2.19 13, 14, 15 16-Hp 1.73 C-17 51.9 17-H 2.56 12a, 12p, 16a, 12, 13, 14, 16, 18(w) 2 0 ,2 1 ,2 2 C-18 17.0 18-CH3 0.71 12, 13, 14, 17 C-19 17.1 I9 -CH3 0.93 1 ,5 ,9 , 10 C-20 124.8 C-21 150.2 2 1-H 7.43 16p, 17, 18 17, 20, 22, 24 C-22 149.1 22-H 7.99 16p, 17(w), 18 2 1 ,2 4 C-23 115.2 23-H 6.28 22 20, 24 C-24 164.7

w = weak NOE correlation.

MS (ESI+) m/z: (M+H)+ 403.2, (M+Na)+ 425.2.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 128

VITA

DEBORAH A. HUTCHINSON

Old Dominion University Department of Biological Sciences Norfolk, Virginia 23529-0266

Education

Doctor of Philosophy, Ecological Sciences, 2006 Old Dominion University, Norfolk, Virginia

Master of Science, Biology, 2001 Old Dominion University, Norfolk, Virginia

Bachelor of Arts, Biology, 1999 University of San Diego, San Diego, California

Scholarships and Fellowships

Dissertation Fellowship, Old Dominion University, 2005

Dominion Graduate Scholarship, Old Dominion University, 2001-2005

Alumni Association Outstanding Scholar Fellowship, Old Dominion University, 2004

Meredith Construction Company Scholarship, Old Dominion University, 2003

Virginia S. Bagley Endowed Scholarships, Old Dominion University, 1999 and 2003

Grants

Grant-in-Aid of Research, Sigma Xi, The Scientific Research Society, 2005 “Maternal Provisioning of Sequestered Dietary Toxins by Rhabdophis tigrinus (Serpentes: Colubridae)”

Grant-in-Aid of Research, Society for Integrative and Comparative Biology, 2005 “Dietary Toxin Sequestration in Two Populations of a Toad-Eating Snake, Rhabdophis tigrinus”

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