Chemical investigations of defensive steroid sequestration by the Asian snake Rhabdophis tigrinus
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Rhabdophis tigrinus is an Asian natricine snake that possesses unusual defensive glands on the dorsal surface of its neck. These nuchal glands typically contain cardiotonic steroidal toxins known as bufadienolides, which are also abundant in the skin of toads. Feeding experiments demonstrated that toads consumed as prey are the ultimate sources of the bufadienolides in nuchal glands of R. tigrinus. Indeed, snakes on a toad-free Japanese island (Kinkasan, Miyagi Prefecture) lack these compounds in their nuchal glands, confirming that these snakes are unable to synthesize defensive bufadienolides. However, when snakes from Kinkasan are fed toads in the laboratory, they accumulate bufadienolides in their nuchal glands, indicating that they have not lost the ability to sequester defensive compounds from prey. In contrast, R. tigrinus from a toad-rich island (Ishima, Tokushima Prefecture) possess large quantities of bufadienolides, reflecting the abundance of toads from which these compounds can be sequestered. Feeding experiments involving gravid R. tigrinus demonstrated that bufadienolides can be provisioned to offspring so that hatchlings are chemically defended before their first toad meal. Maternal provisioning of bufadienolides can take place through two routes: by deposition in yolk and by diffusion in utero, even late in gestation. We applied bufadienolides to the surface of eggs from Kinkasan and found that the embryos are able to take up these compounds into their nuchal glands, demonstrating the feasibility of uptake across the eggshell. Female R. tigrinus provision bufadienolides to their offspring in direct proportion to their own level of chemical defense. By feeding toad-derived bufotoxins to R. tigrinus hatchlings, we determined that the sequestration of these compounds involves at least three types of modification: hydrolytic cleavage of suberylarginine side chains, hydroxylation, and epimerization.
KeywordsDietary toxins Toad Bufadienolides Nuchal glands Antipredator defense
We thank the following individuals for their contributions to the experiment involving topical application of bufadienolides to eggs: Hirohiko Takeuchi, Noriko Kidera, Ralph Saporito, and Seth Barribeau for assistance collecting R. tigrinus; Patrick Hatcher and Junyan Zhong for use of the 400 MHz NMR at ODU for initial analyses; and Georg Jander and Minsang Lee for use of an HPLC at Cornell. This work was supported by the National Science Foundation (IBN-0429223 and IOB-0519458 to A.H.S. and J.M., and INT-9513100 to G.M.B.); the Japan–US Cooperative Science Program (Japan Society for the Promotion of Science to A.M.); Kyoto University Grants for the Twenty-first Century COE A14 and the Global COE Program A06 (to Kyoto University); DuPont Crop Protection (Young Investigator Award to F.C.S.); the Society for Integrative and Comparative Biology and Sigma Xi, The Scientific Research Society (Grants-in-Aid of Research to D.A.H.); the Honor Society of Phi Kappa Phi (Love of Learning Grant to D.A.H.); Coastal Carolina University (Research Enhancement Grant to D.A.H.); and the American Philosophical Society (Franklin Research Grant to D.A.H.).
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