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www.vetinst.no eDNA monitoring of a crayfish plague outbreak in Norway – snapshots of invasion, infection and extinction David Strand 1,2 , Stein Ivar Johnsen 3 , Johannes Rusch 1 , Steen Wilhelm Knudsen 4 , Sune Agersnap 4 , William Brenner Larsen 4 , Peter Rask-Møller 4 , Trude Vrålstad 1 1 Norwegian Veterinary Institute, 2 Norwegian Institute for Water Research, 3 Norwegian Institute for Nature Research, 4 University of Copenhagen, Natural History Museum of Denmark 0 1 2 3 P S N 0 1 2 3 P S N 0 1 2 3 P S N 0 1 2 3 P S N 0 1 2 3 P S N 0 1 2 3 P S N Noble Crayfish (N) Signal Crayfish (S) Crayfish Plague (P) 0 1 2 3 P S N 0 1 2 3 P S N 0 1 2 3 P S N 0 1 2 3 P S N 0 1 2 3 P S N 0 1 2 3 P S N 0 1 2 3 P S N 0 1 2 3 P S N 0 1 2 3 P S N 0 1 2 3 P S N 0 1 2 3 P S N 0 1 2 3 P S N 0 1 2 3 P S N 0 1 2 3 P S N 0 1 2 3 P S N 0 1 2 3 P S N 0 1 2 3 P S N 0 1 2 3 P S N 0 1 2 3 P S N 0 1 2 3 P S N 0 1 2 3 P S N Water flow Study site and methods Two and five locations were surveyed in 2014 and 2015, respectively eDNA samples were taken from upstream and downwards, and with some distance to the nearest cage to avoid eDNA from caged crayfish 960 trap nights determined CPUE estimates of illegal signal crayfish eDNA sampling and analysis includes the following steps 3x 5L water samples were filtered on site at each location through glass fiber filters, using a peristaltic pump, tubing and a filter holder Filters were stored on ice and transported to the lab within 12 hours DNA was extracted from filters using a large volume CTAB protocol Each sample was screened for the presence of A.astaci, A. astacus and P. leniusculus using probe based qPCR assays 1,2 References 1. Strand et al, 2014. Detection of crayfish plague spores in large freshwater systems. Journal of Applied Ecology. 2014 4;51(2):544–553 2. Agersnap et al (in prep). Environmental DNA (eDNA) detection and quantification of noble, signal and narrow-clawed crayfish (Decapoda – Astacoidea) Background Crayfish plague, caused by the oomycete Aphanomyces astaci, is the main reason for the drastic decline of European noble crayfish (Astacus astacus). North American signal crayfish, often illegally spread in Europe, are carriers of this pathogen (Fig.1) Monitoring of noble crayfish using baited traps in the Norwegian Lake Rødnessjøen in 2014 revealed illegally introduced signal crayfish (Pacifastacus leniusuculus) Detection of A.astaci suggested an emerging crayfish plague outbreak in the lake. Classic crayfish plague monitoring uses caged noble crayfish as living bait to monitor disease spread, crayfish monitoring uses baited traps (crayfish/trapnight=CPUE) We hypothesize that environmental DNA (eDNA) monitoring offers a better alternative, using non-invasive methods without sacrificing or disturbing live crayfish. Here, we compare eDNA monitoring with cage monitoring during the emerging outbreak in the natural noble crayfish population in Lake Rødnessjøen. Figure 1 - Crayfish plague (A. astaci; a-c) is lethal to European noble crayfish (right). American signal crayfish (left) is a natural host and carrier of this pathogen. Results and conclusions In this study, we were able to: Follow the frontline of the upstream spreading crayfish plague outbreak that reached ~20 km in one year over two interconnected lakes (Rødnessjøen and Skullerudsjøen) Detect the increase, peak, decline and disappearance of eDNA from A. astaci and noble crayfish in the water during noble crayfish mortalities and subsequent local extinction Detect eDNA of illegally introduced signal crayfish at low abundances (0.12 CPUE) at the location where the outbreak originated (loc. 1, Fig.3) Establish that eDNA monitoring often revealed the pathogen in the water weeks before mortalities attributed to crayfish plague was observed in the caged noble crayfish. We conclude that environmental DNA (eDNA) monitoring is a rapid and powerful tool for separate or simultaneous surveillance of threatened noble crayfish, invasive signal crayfish and the crayfish plague pathogen A. astaci in natural waters. Figure 3 – Timeline with corresponding location maps showing the spread of crayfish plague (red) during one year based on classic cage monitoring and eDNA monitoring. eDNA monitoring of noble crayfish (green) and signal crayfish (yellow) provides supplementary information matching the outbreak situation. Sampling locations are numbered 1-5. Corresponding graphs show eDNA results of the 3 target species from each location: 0 = no detection; 1 = detection below quantification limit (LOQ); 2 = ~80-500 Crayfish eDNA copies/L water, or ~3-100 crayfish plague spores/L; 3 = >500 Crayfish eDNA copies/L water, or >100 Crayfish plague spores/L water. Detection of A. astaci eDNA in the water is also indicated directly on the map in red along with the condition of caged crayfish (living or dead with crayfish plague diagnosis). Figure 2 – eDNA copy estimates of noble crayfish and A. astaci spores (copies converted to spores) at location 3 (Fig. 3) from April-Sept. Estimates below limit of quantification (LOQ) are included to demonstrate increase/decrease of eDNA/spores 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 4 5 5 4 4 4 4 4 5 5 5 3 3 3 3 4 4 4 4 5 5 5 5 5 2014 Oct - Nov 2015 April May June July Aug - Sept Live caged crayfish Observed crayfish mortality in the cage Figure 4 – eDNA sample and analysis procedure, from on-site water filtering to qPCR. QR-code: See our eDNA sample/filtration procedure on YouTube The TARGET project (243907 – Targeted strategies for safeguarding noble crayfish against alien and emerging threats) is supported by Norwegian Research Council 2
