Deborah A. Hutchinson

Dietary Sequestration of Defensive Steroids in Nuchal Glands of the Asian Snake Rhabdophis Tigrinus

The Asian snake Rhabdophis tigrinus possesses specialized defensive glands on its neck that contain steroidal toxins known as bufadienolides. We hypothesized that R. tigrinus does not synthesize these defensive steroids but instead sequesters the toxins from toads it consumes as prey. To test this hypothesis, we conducted chemical analyses on the glandular fluid from snakes collected in toad-free and toad-present localities. We also performed feeding experiments in which hatchling R. tigrinus were reared on controlled diets that either included or lacked toads. We demonstrate that the cardiotonic steroids in the nuchal glands of R. tigrinus are obtained from dietary toads. We further show that mothers containing high levels of bufadienolides can provision their offspring with toxins. Hatchlings had bufadienolides in their nuchal glands only if they were fed toads or were born to a dam with high concentrations of these compounds. Because geographic patterns in the availability of toxic prey are reflected in the chemical composition of the glandular fluid, snakes in toad-free regions are left undefended by steroidal toxins. Our findings confirm that the sequestration of dietary toxins underlies geographic variation in antipredatory behavior in this species and provide a unique example of sequestered defensive compounds in a specialized vertebrate structure.

Sequestered defensive toxins in tetrapod vertebrates: principles, patterns, and prospects for future studies

Chemical defenses are widespread among animals, and the compounds involved may be either synthesized from nontoxic precursors or sequestered from an environmental source. Defensive sequestration has been studied extensively among invertebrates, but relatively few examples have been documented among vertebrates. Nonetheless, the number of described cases of defensive sequestration in tetrapod vertebrates has increased recently and includes diverse lineages of amphibians and reptiles (including birds). The best-known examples involve poison frogs, but other examples include natricine snakes that sequester toxins from amphibians and two genera of insectivorous birds. Commonalities among these diverse taxa include the combination of consuming toxic prey and exhibiting some form of passive defense, such as aposematism, mimicry, or presumptive death-feigning. Some species exhibit passive sequestration, in which dietary toxins simply require an extended period of time to clear from the tissues, whereas other taxa exhibit morphological or physiological specializations that enhance the uptake, storage, and/or delivery of exogenous toxins. It remains uncertain whether any sequestered toxins of tetrapods bioaccumulate across multiple trophic levels, but multitrophic accumulation seems especially likely in cases involving consumption of phytophagous or mycophagous invertebrates and perhaps consumption of poison frogs by snakes. We predict that additional examples of defensive toxin sequestration in amphibians and reptiles will be revealed by collaborations between field biologists and natural product chemists. Candidates for future investigation include specialized predators on mites, social insects, slugs, and toxic amphibians. Comprehensive studies of the ecological, evolutionary, behavioral, and regulatory aspects of sequestration will require teams of ecologists, systematists, ethologists, physiologists, molecular biologists, and chemists. The widespread occurrence of sequestered defenses has important implications for the ecology, evolution, and conservation of amphibians and reptiles.

Maternal provisioning of sequestered defensive steroids by the Asian snake Rhabdophis tigrinus

Summary.Rhabdophis tigrinus obtains defensive steroids (bufadienolides) from its diet and sequesters those compounds in specialized structures on its neck known as nuchal glands. Hatchling snakes lacking these steroids must acquire them from toads consumed as prey. Here we show that females provision bufadienolides to their offspring in amounts correlated to the quantity in their own nuchal glands; thus, chemically protected mothers produce defended offspring. Bufadienolides can be provisioned to embryos via deposition in yolk and by transfer across the egg membranes within the oviducts. Maternally provisioned bufadienolides persist in the nuchal glands of juvenile snakes from the time of hatching in late summer until the following spring, when toads of ingestible size become abundant. Therefore, maternal provisioning may provide chemical protection from predators for young R. tigrinus in the absence of dietary sources of bufadienolides.

Nuchal glands: a novel defensive system in snakes

Of the various chemical defensive adaptations of vertebrates, nuchal glands are among the most unusual. First described in a Japanese natricine snake, Rhabdophis tigrinus, in 1935, these organs are embedded under the skin of the neck region as a series of paired glands that have neither lumina nor ducts. The major chemical components of the glandular fluid are bufadienolides, which are cardiotonic steroids also found in the skin secretion of toads. Here we review early studies of nuchal glands and briefly introduce our recent findings on the sequestration of bufadienolides from consumed toads and the maternal provisioning of those sequestered compounds. We summarize behavioral studies associated with the antipredator function of the nuchal glands, which have been conducted during our more than decade-long collaboration. Results of preliminary analyses on the possible costs of toad-eating and on the ultrastructure of the nuchal glands are also presented. Finally, we discuss the evolutionary origin of the nuchal glands and suggest future directions designed to understand the biological importance of these novel vertebrate organs, which have evolved in a limited number of snake species.

Chemical investigations of defensive steroid sequestration by the Asian snake Rhabdophis tigrinus

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.

Chemical defense of an Asian snake reflects local availability of toxic prey and hatchling diet.

Species that sequester toxins from prey for their own defense against predators may exhibit population-level variation in their chemical arsenal that reflects the availability of chemically defended prey in their habitat. Rhabdophis tigrinus is an Asian snake that possesses defensive glands in the skin of its neck (‘nuchal glands’), which typically contain toxic bufadienolide steroids that the snakes sequester from consumed toads. In this study, we compared the chemistry of the nuchal gland fluid of R. tigrinus from toad-rich and toad-free islands in Japan and determined the effect of diet on the nuchal gland constituents. Our findings demonstrate that captive-hatched juveniles from toad-rich Ishima Island that had not been fed toads possess defensive bufadienolides in their nuchal glands, presumably due to maternal provisioning of these sequestered compounds. Wild-caught juveniles from Ishima possess large quantities of bufadienolides, which could result from a combination of maternal provisioning and sequestration of these defensive compounds from consumed toads. Interestingly, juvenile females from Ishima possess larger quantities of bufadienolides than do juvenile males, whereas a small sample of field-collected snakes suggests that adult males contain larger quantities of bufadienolides than do adult females. Captive-born hatchlings from Kinkasan Island lack bufadienolides in their nuchal glands, reflecting the absence of toads on that island, but they can sequester bufadienolides by feeding on toads (Bufo japonicus) in captivity. The presence of large quantities of bufadienolides in the nuchal glands of R. tigrinus from Ishima may reduce the risk of predation by providing an effective chemical defense, whereas snakes on Kinkasan may experience increased predation due to the lack of defensive compounds in their nuchal glands.