Summary1. Indigenous European freshwater crayfish (ICS) are threatened due to invasive North American freshwater crayfish that are natural carriers of Aphanomyces astaci which causes crayfish plague. Infectious A. astaci zoospores are released from carrier crayfish, but little is known about the spore abundance in water systems that either host non-indigenous crayfish species (NICS) or experience crayfish plague outbreaks. We tested two large-scale filtering approaches to generate new insight about the abundance and dynamics of A. astaci spores in natural freshwater systems. 2. Depth filtration (DF) and dead-end ultrafiltration (DEUF) followed by A. astaci-specific quantitative real-time PCR was used to monitor A. astaci spores in large Nordic lakes hosting A. astaci-positive Pacifastacus leniusculus, the dominating NICS in Northern Europe. Crayfish and water were sampled together to compare the A. astaci pathogen load in tissues, A. astaci prevalence in the population and the corresponding spore density in water. Samples were also obtained from a river where indigenous noble crayfish suffered from acute crayfish plague. The sensitivity of the filtering techniques was evaluated using simulation of random events. 3. We detected A. astaci spores in lakes hosting NICS with both filtering methods but predominantly at concentrations below c. 1 spore L À1 . We found a significant positive association between A. astaci spore density in water, the A. astaci prevalence in the corresponding NICS population and the tissue pathogen load. Water from the river with the ongoing crayfish plague outbreak contained overall c. 43 times more spores L À1 than water hosting NICS. Both filtering techniques proved suitable and equally sensitive, but simulations suggest that an optimization of the spore recovery could yield a 10-fold increase in the DEUF-method sensitivity. 4. Synthesis and application. Our study demonstrates a low amount of pathogen spores are present in aquatic environments with non-indigenous crayfish species, emphasizing the need for large-volume filtering techniques for successful detection. The approach can be used for risk assessments and to improve conservation and management strategies of crayfish in Europe. Applications of this method include targeted disease surveillance, habitat evaluation prior to crayfish re-stockings and water monitoring that can minimize disease transmission and spread, for example in crayfish farms and prior to fish movements for stocking purposes.

1. The European noble crayfish Astacus astacus is threatened by crayfish plague caused by the oomycete Aphanomyces astaci, which is spread by the invasive North American crayfish (e.g. signal crayfish Pacifastacus leniusculus). Surveillance of crayfish plague status in Norway has traditionally relied on the monitoring survival of cage-held noble crayfish, a method of ethical concern. Additionally, trapping is used in crayfish population surveillance. Here, we test whether environmental DNA (eDNA) monitoring could provide a suitable alternative to the cage method, and a supplement to trapping.2. We took advantage of an emerging crayfish plague outbreak in a Norwegian watercourse following illegal introduction of disease-carrying signal crayfish, and initiated simultaneous eDNA monitoring and cage-based surveillance, supplemented with trapping. A total of 304 water samples were filtered from several sampling stations over a 4-year period. eDNA data (species-specific quantitative real-time PCR [qPCR]) for the presence of A. astaci, noble and signal crayfish within the water samples were compared to cage mortality and trapping. 3. This is the first study comparing eDNA monitoring and cage surveillance during a natural crayfish plague outbreak. We show that eDNA monitoring corresponds well with the biological status measured in terms of crayfish mortality and trapping results. eDNA analysis also reveals the presence of A. astaci in the water up to 2.5 weeks in advance of the cage method. Estimates of A. astaci and noble crayfish eDNA concentrations increased markedly during mortality and vanished quickly thereafter. eDNA provides a snapshot of the presence, absence or disappearance of crayfish regardless of season, and constitutes a valuable supplement to the trapping method that relies on season and legislation. 4. Synthesis and applications. Simultaneous eDNA monitoring of Aphanomyces astaci (crayfish plague) and relevant native and invasive freshwater crayfish species is well-suited for early warning of invasion or infection, risk assessments, habitat evaluation and surveillance regarding pathogen and invasive/native crayfish 1662 | Journal of Applied Ecology STRAND eT Al.

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Noble crayfish Astacus astacus is threatened in Europe due to invasive crayfish carrying the crayfish plague agent Aphanomyces astaci. Norway is among the last countries in which the introduction of non-indigenous crayfish has been limited through strict legislation practices. However, North American signal crayfish Pacifastacus leniusculus were recently discovered in a watercourse that has been repeatedly hit by the plague. We mapped the distribution and relative density (catch per unit effort) of signal crayfish within this lake, and performed agent-specific real-time PCR to estimate the prevalence of A. astaci in the population. The resulting length frequencies and relative density estimates clearly demonstrate a well-established signal crayfish population, in which 86.4% of the analysed individuals were confirmed carriers. The success of detection was significantly higher (84.1%) in the crayfish tailfan (i.e. uropods) than in the soft abdominal cuticle (38.4%), which is commonly used in prevalence studies. We therefore propose tailfan (uropods and telson) as the preferred tissue for studying A. astaci prevalence in signal crayfish populations. The likelihood of detecting an A. astaci-positive signal crayfish increased significantly with increasing crayfish length. Further, large female crayfish expressed significantly higher PCR-forming units values than large males. In surveys primarily exploring the presence of A. astaci-positive individuals in a population, large females should be selected for molecular analyses. Our study demonstrates that a potent crayfish plague infection reservoir, evidently originating from the illegal human introduction of signal crayfish, has permanently been established in Norway.

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