Unlike other snakes, most species ofRhabdophispossess glands in their dorsal skin, sometimes limited to the neck, known as nucho-dorsal and nuchal glands, respectively. Those glands contain powerful cardiotonic steroids known as bufadienolides, which can be deployed as a defense against predators. Bufadienolides otherwise occur only in toads (Bufonidae) and some fireflies (Lampyrinae), which are known or believed to synthesize the toxins. The ancestral diet ofRhabdophisconsists of anuran amphibians, and we have shown previously that the bufadienolide toxins of frog-eating species are sequestered from toads consumed as prey. However, one derived clade, theRhabdophis nuchalisGroup, has shifted its primary diet from frogs to earthworms. Here we confirm that the worm-eating snakes possess bufadienolides in their nucho-dorsal glands, although the worms themselves lack such toxins. In addition, we show that the bufadienolides ofR. nuchalisGroup species are obtained primarily from fireflies. Although few snakes feed on insects, we document through feeding experiments, chemosensory preference tests, and gut contents that lampyrine firefly larvae are regularly consumed by these snakes. Furthermore, members of theR. nuchalisGroup contain compounds that resemble the distinctive bufadienolides of fireflies, but not those of toads, in stereochemistry, glycosylation, acetylation, and molecular weight. Thus, the evolutionary shift in primary prey among members of theR. nuchalisGroup has been accompanied by a dramatic shift in the source of the species’ sequestered defensive toxins.

Several Asian natricine snakes of the genus Rhabdophis feed on toads and sequester steroidal cardiac toxins known as bufadienolides (BDs) from them. A recent study revealed that species of the R. nuchalis Group ingest lampyrine fireflies to sequester BDs. Although several species of fireflies are distributed in the habitat of the R. nuchalis Group, only lampyrine fireflies, which have BDs, included in the diet of these snakes. Thus, we hypothesized that the R. nuchalis Group chemically distinguishes fireflies that have BDs from those that do not have BDs. We also predicted that the R. nuchalis Group detects BDs as the chemical cue of toxin source. To test these predictions, we conducted three behavioral experiments using R. chiwen, which belongs to the R. nuchalis Group. In the first experiment, R. chiwen showed a moderate tongue flicking response to cinobufagin, a compound of BDs. On the other hand, the snake showed a higher response to the chemical stimuli of lampyrine fireflies (BD fireflies) than those of lucioline fireflies (non-BD fireflies). In the second experiment, in which we provided live BD and non-BD fireflies, the snake voluntarily consumed only the former. In the third, a Y-maze experiment, the snake tended to select the chemical trail of BD fireflies more frequently than that of non-BD fireflies. These results demonstrated that R. chiwen discriminates BD fireflies from non-BD fireflies, but the prediction that BDs are involved in this discrimination was not fully supported. To identify the proximate mechanisms of the recognition of novel toxic prey in the R. nuchalis Group, further investigation is necessary.