Environmental DNA sampling as a surveillance tool for cane toad Rhinella marina introductions on offshore islands

Containing the spread of established invasive species is critical for minimizing their ecological impact. Effective containment requires sensitive sampling methods capable of detecting new introductions when invaders are at low density. Here we explore whether environmental DNA (eDNA) sampling could be used as a surveillance tool to detect new incursions of aquatic invasive species on offshore islands. We develop an eDNA molecular assay for invasive cane toads ( Rhinella marina ) in Australia, validate our assay on the mainland, and apply it to an offshore island (Moreton Island) that is a target of ongoing cane toad surveillance. Our eDNA assay correctly identified four mainland sites at which cane toads were observed, as well as a fifth site within 1 km of known populations. Five additional sites outside the cane toad’s current distribution tested negative for cane toad eDNA. Site occupancy detection models indicated that two water samples and three qPCR replicates were sufficient to achieve a cumulate detection probability > 0.95. Applying our eDNA assay to samples from 19 sites on an offshore island over a 2-year period revealed the absence of cane toad eDNA, in line with our current understanding of cane toad distribution. Our results suggest that eDNA sampling could be strategically applied to meet the Australian Commonwealth’s objective of maintaining cane toad-free offshore islands.