Safeguarding a Freshwater Sentinel: eDNA-Based Conservation of A. pallipes Across Borders

Autochthonous populations of crayfish have been declining in the past years due to water pollution, climate change, invasive species, and pathogens. In this context, project PALLIPES (Interreg Italia- Slovenija) aims to conserve and enhance the population and environment of Austropotamobius pallipes in the transboundary area between Italy and Slovenia. This species, listed as endangered in the IUCN Red List, is protected by the Habitat Directive and the Bern Convention. It is a biological indicator for freshwater ecosystems, hence the necessity to assess its population and state of the environment. An established tool for the monitoring of rare, cryptic, and invasive species is environmental DNA (eDNA). This method allows to trace species through their genetic material that can be detected in the environment. It is efficient at uncovering small local populations that might have been neglected by traditional methods (traps). Water samples of 1.5L were collected in the river Reka-Timavo at 120 different Slovenian sites between June and October 2024. Water was filtered using a peristaltic pump onto Sterivex 0.45μm filter units and then conserved until eDNA extraction. qPCR was performed on a species-specific assay based on hydrolysis probe, specific for A. pallipes. This step was performed also with probes specific to Procambarus clarkii (invasive crayfish) and Aphanomyces astaci (pathogen) to verify their presence/ absence in the sites where A. pallipes was not detected. Barcoding analysis revealed the presence of A. pallipes at 42 sites and P. clarkii at nine sites. No sites tested positive for A. astaci, suggesting that observed declines in A. pallipes populations are more likely attributable to environmental stressors such as water quality degradation and climate change, rather than to crayfish plague. Both P. clarkii and A. astaci are currently under further investigation as they have been detected in the past, and we cannot exclude their influence on the local populations yet. Moreover, P. clarkii eDNA could potentially originate from domestic wastewaters linked to human consumption, so presence evaluation is still underway. To conclude, A. pallipes is still present in the Slovenian territory, where conservation measures must be implemented to preserve the local population. The same methodology will be applied for the Italian territory for the creation of a shared strategy for conserving A. pallipes within the transboundary area.