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In endangered crayfish conservation projects, it is paramount to map the distribution of the causative agent of crayfish plague, Aphanomyces astaci, in native populations. Considering the inapplicability of the destructive cuticular sampling protocol for monitoring endangered populations, we explored the use of non-invasive sampling techniques to detect this pathogen with molecular assays. In the present study, we exploited environmental DNA (testing increasing water volumes combined with different filter porosities) and cuticular swabs to collect A. astaci DNA. In addition, we evaluated the impact of the storage method on DNA preservation during field activities. After the first evaluations performed on both highly infected Austropotamobius pallipes and carrier Procambarus clarkii specimens in laboratory conditions, these sampling techniques were applied to wild populations of white-clawed crayfish. Our findings highlight better results with the filtration of 5 L of water with filters of 2.7 µm porosity for eDNA analysis and demonstrate that cuticular swabbing is equally effective as the World Organisation of Animal Health’s protocol. Storage in absolute ethanol proved to be the best solution to preserve swabs and filter samples for up to a week at room temperature. In conclusion, we suggest an integration of both sampling methods when monitoring A. astaci for conservation purposes.

Cherax destructor is a crayfish endemic to south-eastern Australia and one of the last alien crayfish to be introduced in Italy. In the Laghi di Ninfa Natural Reserve (Latium region, Central Italy), the species was probably introduced in 1999, but only reported for the first time in 2008. Nearby this area, the most widespread alien crayfish is the red swamp crayfish Procambarus clarkii. In the Natural Reserve, between 2008 and 2013 and during spring and summer, crayfish sampling was carried out with baited traps to assess the distribution of C. destructor and its possible relationship with P. clarkii. Cherax destructor was first recorded in 2008; few P. clarkii were detected in the cultivation ponds where C. destructor was present in 2012 and 2013. Moreover, crayfish plague analyses evidenced a positive result in two out of the 12 sampled P. clarkii. Cherax destructor is now completely absent from the Natural Reserve, while P. clarkii has spread in the area and was probably responsible for this eradication since C. destructor is vulnerable to crayfish plague which was also detected in the area. An ecosystem restoration project in the area favoured the spread of. P. clarkii; the implications of this intervention are discussed.