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Summer 2006 Chemical Evidence for Dietary Toxin Sequestration in the Asian Snake Rhabdophis 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 (Colubridae: Natricinae) 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 bufadienolide toxins. Whereas toads are known to synthesize
bufadienolide steroids from cholesterol precursors, we found that chemically
undefended R. tigrinus must sequester bufadienolides 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 vertebrate
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
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. vii
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 amphibian
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 animals 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 Japan 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 Snakes '...... 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 bufotoxins identified from parotoid gland secretions of Bufo 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 plants
(.Asclepias humistrata), which contain high concentrations of cardiac glycosides. The
butterflies gain the benefit of becoming chemically defended by consuming the toxic
plant, 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 vertebrates,
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 Taricha granulosa (Brodie and Brodie, 1999a, 1999b;
Geffeney et al., 2002). These newts contain tetrodotoxin (TTX), a neurotoxin found in
other animals such as puffer fish ( Takifugu); Thamnophis sirtalis is the only animal
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 bufotoxin
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 bufotalin, 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 digitalis-like fashion, resulting in greater intracellular calcium levels and
causing prolonged contraction of cardiac muscle (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 cardenolides 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 frogs (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 amphibians, there is a paucity of experimental evidence supporting
the sequestration of dietary toxins in amniotes (reptiles, birds, and mammals). As one
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of the few examples, New Guinean birds in the genera Pitohui and Ifrita sequester
batrachotoxins 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
species 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 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 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 bird 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 frog (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. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 48 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). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 52 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. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 53 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. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 54 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 type, 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 ouabain 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. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 98 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. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 99 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 venom 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? 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Herpetology of China. Society for the Study of Amphibians and Reptiles, Oxford, OH. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 109 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. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 110 o 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.