Bufonid toads generally possess cardiotoxic steroids called bufadienolides as defensive chemicals. Although knowledge of the life stages at which the toad species possess the poison is important for our understanding of diversity of toxicity among bufonid toads, this knowledge is limited. In the present study, we revealed that the Japanese common toad, Bufo japonicus formosus, possesses toxins at the unfertilized egg stage by conducting a bioassay experiment. Recent studies documented that hatchlings of B. j. formosus have lethal toxic effects on native frog tadpoles (Rana pirica) in the invasive area of the toad (Hokkaido). In our bioassay experiment using R. pirica tadpole as a predator, no tadpoles died when they did not consume any prey item during two-days experimental period. However, approximately 90% of R. pirica tadpoles immediately died when they consumed an unfertilized egg of B. j. formosus. These results suggest that the toxin at the early life stages of B. j. formosus is, at least partly, provided from female parent.

Biological invasions adversely affect the survival of many native species, but long-term consequences of the novel pressures imposed by invaders on natives are less clear. If natives can adapt to the presence of the invader, the severity of impact will decline with time. On Hokkaido, Japan, alien toads (Bufo japonicus formosus) are highly toxic to native frog tadpoles (Rana pirica) that attempt to eat their hatchlings. Therefore, the arrival of toads potentially imposes selection pressure on native frogs in the context of behavioral response (feeding aversion) and physiological resistance to toad toxins. Here, we compared the consumption tendency and toxin resistance of R. pirica from several sites with different histories of toad invasion (0 to >100 years exposure). We found no evidence of adaptive response to invasive toads. The vast majority of R. pirica tadpoles from all sites readily consumed toad hatchlings and died, regardless of time since invasion or whether hatchlings were from sympatric versus allopatric toad populations. In contrast, tadpoles of a closely related species (Rana ornativentris), which co-occurs naturally with B. j. formosus on Honshu, readily consumed toad hatchlings without ill effect. Our results indicate that the lethal toxic effect of the alien toads on native frogs on Hokkaido is maintained over long time periods without mitigation through adaptation.

Understanding the distribution of invasive species and their reproductive area is crucial for their managements after invasion. While catch and observation surveys are still embraced, environmental DNA (eDNA) has been increasingly utilized as an efficient tool for identifying these species in the wild. In this study, we developed a Bufo-specific eDNA assay for detecting an invasive, toxic, and terrestrial toad species Bufo japonicus formosus in Hokkaido, Japan, and applied it to their reproductive area at watershed scale. The eDNA assay was field-validated in ponds where B. japonicus were observed, as well as in rivers downstream of the reproductive ponds. Thus, the assay provided us an opportunity to screen watersheds that include their reproductive area by collecting downstream water samples. Applying it to the Ishikari river basin, the largest river basin in Hokkaido (c.a., 14,330 km2), we detected toad eDNA at 32 out of 73 sampling sites. They are composed of eleven sites with species observation records nearby (all the sites with observation records within a 500 m radius) and 21 sites without such records. And those eDNA detections were from twelve out of 31 river systems in the entire river basin. A Bayesian, multiscale occupancy model supported high eDNA detectability among those sites. These results suggest that the eDNA assay can efficiently estimate the presence of reproductive area of the terrestrial toad even from a distant downstream of the watershed, and that it provides a powerful means of detecting new reproductive area and monitoring further spread of invasive species.