Michael Oliewo Aluma

In recent years, European countries have intensified efforts to control or limit the spread of invasive freshwater crayfish and the crayfish plague pathogen Aphanomyces astaci , while working to conserve native species such as the noble crayfish ( Astacus astacus ). Although crayfish shed relatively low amounts of DNA into their environment, environmental DNA (eDNA) approaches have proven effective for detecting their presence. A range of protocols and equipment is currently used in eDNA‐based monitoring of freshwater crayfish. To evaluate how methodological variation influences detection accuracy, we conducted the first European interlaboratory ring test using eDNA to detect A. astacus , the invasive signal crayfish Pacifastacus leniusculus , a chronic carrier of A. astaci , and the pathogen itself. The aim is to harmonize monitoring methods for crayfish and disease surveillance across laboratories. Eleven teams from thirteen European countries participated, each using its own equipment and protocols to collect and filter water from indoor tanks and outdoor ponds where the presence of A. astacus and P. leniusculus had been experimentally manipulated, as well as from a natural lake containing a P. leniusculus population. The resulting samples were analyzed in each team's laboratory. Despite methodological differences, all teams successfully detected DNA from both crayfish species in indoor tanks (3–10 crayfish/m ³ ). However, detection accuracy declined in outdoor ponds where crayfish density was an order of magnitude lower (0.32 crayfish/m ³ ). Detection was most variable for A. astaci , likely due to its very low prevalence in the host stock. Our study demonstrates the challenges of achieving consistent eDNA results across laboratories and highlights the importance of interlaboratory comparisons. It also underscores the need to identify sources of variability and error, an essential step toward developing robust and standardized protocols. This multinational intercalibration and exchange of knowledge improved methodology and enhanced reliability in crayfish detection.

In Estonia, three invasive North American crayfish species—Pacifastacus leniusculus, Faxonius limosus, and Procambarus virginalis—have been detected through the annual monitoring program. To protect Astacus astacus, the only native freshwater crayfish species in Estonia, rapid and effective conservation‐based management actions are necessary. Recently, the environmental DNA (eDNA) approach has been increasingly used in Europe to detect crayfish species and the crayfish plague pathogen Aphanomyces astaci. Our study explored the potential of integrating the eDNA approach into ongoing annual monitoring programs for invasive crayfish species and A. astaci. We also evaluated the relationship between eDNA concentration and signal crayfish population density at a single location. We filtered 139 eDNA samples from 16 water bodies and screened them for A. astacus, P. leniusculus, and A. astaci using singleplex qPCR assays. A subset of the samples was also screened for P. virginalis and F. limosus. Crayfish eDNA was detected in nine out of 14 water bodies where presence was confirmed by trapping, resulting in a 64% detection efficiency. Detection of P. virginalis was only observed in samples with amplifications below the limit of detection, and A. astaci eDNA was found in only one water body hosting invasive crayfish species. Although we could not establish a convincing quantitative correlation between the estimated P. leniusculus eDNA concentration and crayfish population density, we conclude that the eDNA approach is promising and, with further optimization, it can be integrated into routine monitoring of crayfish and crayfish plague pathogen as a supplement to traditional trapping methods.

Invasive non-indigenous crayfish species (NICS) are a major threat to the existence of native crayfish populations in European freshwater ecosystems. The discovery of signal crayfish Pacifastacus leniusculus, marbled crayfish Procambarus virginalis, and spiny-cheek crayfish Faxonius limosus in Estonia has increased the risk of extinction of Estonia’s only native crayfish species, the noble crayfish Astacus astacus. The aim of this study was to give an overview of the status, distribution, and impacts of P. leniusculus, F. limosus, and Procambarus virginalis on A. astacus populations and assess the effect of trapping on NICS abundance. Annual monitoring of crayfish has been carried out since 2008 as part of A. astacus conservation and the NICS eradication plan. In this study, we present data from nine sampling locations monitored from 2010 to 2022. The spread of NICS continues to increase beyond their distribution areas, and in two sampling locations P. leniusculus and A. astacus live in sympatry. Our results suggest that trapping has a limited effect on population abundance, as NICS have already caused the extinction of two A. astacus populations. However, intensive trapping should continue simultaneously with sensitive molecular techniques to monitor the spread of NICS.