Factors involved in the evolution of firefly toxin utilization in natricine snakes genus Rhabdophis : approaches from chemical recognition, morphology, and feeding behavior

概要

Introduction
Toxin sequestration is widely known in invertebrates and vertebrates. 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. As amphibians are considered the plesiomorphic diet of Rhabdophis, it is presumed that a dietary transition of toxin source from toads to fireflies has occurred within the radiation of Rhabdophis. There are at least three possible factors that would facilitate the evolutionary acquisition of this novel prey: chemical similarity, habitat similarity, and morphological similarity. In the present study, I focused on the chemical and morphological similarity hypotheses to investigate how the firefly toxin utilization has evolved in the R. nuchalis Group.

Materials and Methods
I conducted four experimental studies including chemical recognition, feeding morphology, and prey-handling behavior to investigate how the toxin change has occurred. In the first study, I examined whether R. chiwen distinguishes lampyrine firefly larvae that have BDs from other species of firefly larvae. I conducted chemical preference tests, feeding tests, and Y-maze tests to examine the snakes’ preference toward these larvae. In the second study, I conducted chemical preference experiments using R. tigrinus to identify the chemical cues that the snake utilizes to recognize toads. In the third study, I examined morphological differences among four species of Rhabdophis (R. tigrinus, R. leonardi, R. nuchalis, and R. chiwen). I provided a detailed structural comparison of the cranial design and the feeding apparatus among the four species. In the fourth study, I described and compared prey handling behavior of R. chiwen feeding on earthworms and firefly larvae.

Results and Discussion
In the first and second studies, R. chiwen distinguished lampyrine fireflies from other fireflies that do not possess BDs. Both R. chiwen and R. tigrinus showed a high chemical preference to their respective toxin source, but neither species showed a high chemical preference toward a BD (cinobufagin). A subsequent experiment suggested that R. tigrinus recognizes toads by detecting the non-volatile and hydrophobic chemical components contained in the toad skin. These results suggest that the chemical cue that species of Rhabdophis utilize to recognize toxin source would be a combination of BDs or their precursors. In the third study, I found several differences in the cranial morphology among the four species of Rhadophis. For instance, frog-eating species possessed a larger head size compared to earthworm-eating species. These data on cranial morphology, along with the dietary information, suggest that, within the genus Rhabdophis, the reduction of head size has occurred. In the fourth study, R. chiwen took a longer time in handling firefly larvae than earthworms. A possible reason for this is that the exploitation of fireflies has occurred recently, and the behavioral specialization to firefly larvae has not yet evolved extensively in R. chiwen.

Conclusion
In the present study, I provided the basic dietary and morphological information of the species of Rhabdophis, especially the R. nuchalis Group. Although further investigation is necessary for confirmation, species of Rhabdophis would have an ability to recognize toxin source by detecting the chemical components related to BDs. Along with the dietary change, species of the R. nuchalis Group have reduced gape size and cranial morphology, suggesting that the adaptation to smaller prey has evolved.