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.     

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.     

Maternal provisioning of sequestered defensive steroids by the Asian snake <Emphasis Type="Italic">Rhabdophis tigrinus</Emphasis>

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.