Improving detection capabilities of a critically endangeredfreshwater invertebrate with environmental DNA using digital droplet PCR

Isogenus nubecula is a critically endangered Plecoptera species. Considered extinct in the UK, the species was recently rediscovered in one location of the river Dee in Wales after 22 years of absence. As many species belonging to the Perlodidae, this species can be a bio-indicator, utilised for assessing water quality and health status of a given freshwater system. However, conventional monitoring of invertebrates via kick-sampling for example, is an invasive and expensive (time consuming). Further, such methods require a high level of taxonomic expertise. Here, we compared the traditional kick-sampling method with the use of eDNA detection using qPCR and ddPCR-analyses. In spring 2018, we sampled eDNA from twelve locations on the river Dee. I. nubecula was detected using kick-sampling in five of these locations, three locations using both eDNA detection and kick-sampling and one location using eDNA detection alone - resulting in a total of six known and distinct populations of this critically endangered species. Interestingly, despite the eDNA assay being validated in vitro and in silico, and results indicating high sensitivity, qPCR analysis of the eDNA samples proved to be ineffective. In contrast, ddPCR analyses resulted in a clear detection of I. nubecula at four locations suggesting that inhibition most likely explains the big discrepancy between the obtained qPCR and ddPCR results. It is therefore important to explore inhibition effects on any new eDNA assay. We also highlight that ddPCR may well be the best option for the detection of aquatic organisms which are either rare or likely to shed low levels of eDNA into their environment.