Testing multiple environmental DNA substrates for detection of the cryptic and critically endangered burrowing freshwater crayfish engaewa pseudoreducta

Effective conservation of endangered species depends on knowledge of their distributions, but species detection can often be challenging. An example of this is provided by the Critically Endangered Margaret River burrowing crayfish (Engaewa pseudoreducta), which is highly cryptic. Due to the burrowing habit of this crayfish, detection of this species currently requires a great deal of effort, the results are often non-conclusive, and, as it involves manual excavation of their burrows, the habitat of this and other species is destroyed in the detection process. In response to these challenges, this study developed and optimized a species-specific probe-based qPCR assay targeting the 16S gene region to detect the target species from environmental samples. Three test substrates—chimney pellets (soil expelled by a crayfish as it digs its burrow), burrow scrapes (soil lining the inside of a burrow), and burrow water (water that is filling the burrow space)—were tested from 11 crayfish burrows thought to have been constructed by the target species to see if eDNA could be detected. DNA from the target species was successfully amplified from both chimney pellets (6/11 samples) and burrow scrapes (3/11 samples); however, E. pseudoreducta was not detected in any water samples. As previously stated, sampling techniques to confirm the presence of this species have relied on burrow excavation (resulting in habitat destruction) and were often not definitive; therefore, replacing the traditional survey method with a noninvasive eDNA-based technique could be of enormous benefit to the management and conservation of this (and similar) species.