Development of environmental DNA detection assays for snakes in paddy fields in Japan

Paddy ecosystems are among the most biodiversity-rich ecosystems, but they can be degraded by field consolidation or the abandonment of rice cultivation. Snakes are the top predators in paddy ecosystems; therefore, understanding the distribution of snakes is important for the conservation of paddy biodiversity. Traditionally, surveys of snakes have been primarily conducted by sighting and trapping, but such methods require encountering snakes during surveys and require expertise in species identification. In this study, we developed environmental DNA (eDNA) detection assays for snakes as a method for surveying a wide area relatively easily. We designed species-specific assays for three common snake species in Japanese paddy ecosystems: Elaphe quadrivirgata, Rhabdophis tigrinus, and Gloydius blomhoffii. After confirming the detection of target species DNA from the rearing environment, the assays were applied to field samples. Eighty-seven and 51 samples were collected from paddy fields, reservoirs, and ditches in summer and autumn, respectively. We collected eDNA samples from eight traditional paddy areas that had not been consolidated, eight consolidated paddy areas, and two abandoned paddy areas in southeastern Hyogo Prefecture, Japan. We attempted to detect the DNA of the three target species in these samples using our eDNA assays and succeeded in identifying the DNA of R. tigrinus and G. blomhoffii in three areas and one area, respectively. Statistical analysis suggested that land-use type affected the detection of R. tigrinus eDNA. This study is the first attempt to survey snakes in paddy ecosystems using eDNA analysis and will contribute to the development of paddy ecosystem monitoring.