Monitoring a Norwegian freshwater crayfish tragedy: eDNA snapshots of invasion, infection and extinction

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 status. This non‐invasive, animal welfare friendly method excludes the need for cage‐held susceptible crayfish in disease monitoring. Furthermore, eDNA monitoring is less likely to spread A. astaci than traditional methods. This study resulted in the implementation of eDNA monitoring for Norwegian crayfish plague and crayfish surveillance programmes, and we believe other countries could improve management strategies for freshwater crayfish using a similar approach.publishedVersio

A National Scale “BioBlitz” Using Citizen Science and eDNA Metabarcoding for Monitoring Coastal Marine Fish

Marine biodiversity is threatened by human activities. To understand the changes happening in aquatic ecosystems and to inform management, detailed, synoptic monitoring of biodiversity across large spatial extents is needed. Such monitoring is challenging due to the time, cost, and specialized skills that this typically requires. In an unprecedented study, we combined citizen science with eDNA metabarcoding to map coastal fish biodiversity at a national scale. We engaged 360 citizen scientists to collect filtered seawater samples from 100 sites across Denmark over two seasons (1 p.m. on September 29th 2019 and May 10th 2020), and by sampling at nearly the exact same time across all 100 sites, we obtained an overview of fish biodiversity largely unaffected by temporal variation. This would have been logistically impossible for the involved scientists without the help of volunteers. We obtained a high return rate of 94% of the samples, and a total richness of 52 fish species, representing approximately 80% of coastal Danish fish species and approximately 25% of all Danish marine fish species. We retrieved distribution patterns matching known occurrence for both invasive, endangered, and cryptic species, and detected seasonal variation in accordance with known phenology. Dissimilarity of eDNA community compositions increased with distance between sites. Importantly, comparing our eDNA data with National Fish Atlas data (the latter compiled from a century of observations) we found positive correlation between species richness values and a congruent pattern of community compositions. These findings support the use of eDNA-based citizen science to detect patterns in biodiversity, and our approach is readily scalable to other countries, or even regional and global scales. We argue that future large-scale biomonitoring will benefit from using citizen science combined with emerging eDNA technology, and that such an approach will be important for data-driven biodiversity management and conservation

Short-term temporal variation of coastal marine eDNA

Temporal variation in eDNA signals is increasingly explored for understanding community ecology in aquatic habitats. Seasonal changes have been addressed using eDNA sampling, but very little is known regarding short-term temporal variation that spans hours to days. To address this, we filtered marine water samples from a single coastal site in Denmark every hour for 32 h. We used metabarcoding to target both fish and broader eukaryote diversity and evaluated temporal changes in this marine community. Results revealed variation in fish species richness (15–27) and eukaryote class richness (35–64) across the 32 h of sampling, and we further evaluated sampling efforts needed to reach different levels of diversity saturation. Relative read frequency data for both fish and eukaryotes indicated a clear diel change in community composition, with different communities detected during daylight versus dark hours. The abundance signals in our data reflected biological variation rather than stochastic variation, since replicates taken at the same hour were more similar to each other than those taken at different hours. Our compositional results indicated a dynamic community, rather than a static pool of eDNA—even across a few hours. The fish data showed a daily pattern of relative species abundances, and the uncoupling of fish and broader eukaryote data suggest that variation in eDNA profiles across a single day can provide valuable information reflecting diel changes, at least for highly mobile organism groups. However, our results also point to several pitfalls in current eDNA experimental design, in which samples are taken over large areas without relative time-consistency or short-term replication. Our findings shed new light on short-term variation in coastal eDNA and have wide implications for experimental study design and for incorporating temporality into project conceptualization for future aquatic biodiversity monitoring.publishedVersio

Development of species-specific eDNA-based test systems for monitoring of freshwater crayfish

This MONIS 5 report focuses on freshwater crayfish and was funded by the Danish Environmental Protection Agency. The aim of the present study was to develop species-specific systems for tracking environmental DNA (eDNA) from nine species of freshwater crayfish in water samples. Among these nine species, only one (Astacus astacus) can be considered indigenous in Scandinavia, and among the other eight non-indigenous species; two (Astacus leptodactylus and Pacifastacus leniusculus) are frequently encountered in Scandinavian freshwater systems. The remaining seven non-indigenous species are rarely encountered in Scandinavia but have been recorded in the past from other places in Europe. It is our aim that the nine species-specific eDNA assays presented here will allow for continuous monitoring of both the one indigenous species, the two non-indigenous species more frequently encountered and help produce early warnings of the seven non-indigenous species that might disperse to Denmark

Udvikling af arts-specifikke eDNA-baserede testsystemer til overvågning af ferskvandskrebs

Project manager Jesper Harbo AndersenThis MONIS 5 report focuses on freshwater crayfish and was funded by the Danish Environmental Protection Agency. The aim of the present study was to develop species-specific systems for tracking environmental DNA (eDNA) from nine species of freshwater crayfish in water samples. Among these nine species, only one (Astacus astacus) can be considered indigenous in Scandinavia, and among the other eight non-indigenous species; two (Astacus leptodactylus and Pacifastacus leniusculus) are frequently encountered in Scandinavian freshwater systems. The remaining seven non-indigenous species are rarely encountered in Scandinavia but have been recorded in the past from other places in Europe. It is our aim that the nine species-specific eDNA assays presented here will allow for continuous monitoring of both the one indigenous species, the two non-indigenous species more frequently encountered and help produce early warnings of the seven non-indigenous species that might disperse to Denmark.Danish Environmental Protection AgencypublishedVersio

Monitoring a Norwegian freshwater crayfish tragedy: eDNA snapshots of invasion, infection and extinction

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 status. This non‐invasive, animal welfare friendly method excludes the need for cage‐held susceptible crayfish in disease monitoring. Furthermore, eDNA monitoring is less likely to spread A. astaci than traditional methods. This study resulted in the implementation of eDNA monitoring for Norwegian crayfish plague and crayfish surveillance programmes, and we believe other countries could improve management strategies for freshwater crayfish using a similar approach.publishedVersio

Seasonal turnover in community composition of stream-associated macroinvertebrates inferred from freshwater environmental DNA metabarcoding

Macroinvertebrate communities are crucial for biodiversity monitoring and assessment of ecological status in stream ecosystems. However, traditional monitoring approaches require intensive sampling and rely on invasive morphological identifications that are time-consuming and dependent on taxonomic expertise. Importantly, sampling is often only carried out once in a year, namely during late winter–spring, where most indicator taxa have larval stages in the streams. Hence, species with divergent phenology might not be detected. Here, we use environmental DNA (eDNA) metabarcoding of filtered water samples collected in both spring and autumn from five streams in Denmark to address seasonal turnover in community composition of stream macroinvertebrates. We find that eDNA read data from the same stream sampling site clearly show different communities in spring and autumn, respectively. For three of the five streams, season even appears to be a more important factor than sampling site for explaining the variation in community composition. Finally, we compare eDNA data with a near-decadal dataset of taxon occurrences in the same five streams based on kick sampling conducted through a national monitoring program. This comparison reveals an overlap in species composition, but also that the two approaches provide complementary rather than identical insights into community composition. Our study demonstrates that aquatic eDNA metabarcoding is useful for species detection across highly diverse taxa and for identifying seasonal patterns in community composition of freshwater macroinvertebrates. Thus, our results have important implications for both fundamental research in aquatic ecology and for applied biomonitoring.publishedVersio

eDNA results 2015 - Statistical analysis

eDNA results from 2015 used in statistical analysi

eDNA results 2014-2017

eDNA results from 2014-2017 in Haldenvassdraget watercourse

Monitoring of environmental DNA from nonindigenous species of algae, dinoflagellates and animals in the North East Atlantic

Monitoring the distribution of marine nonindigenous species is a challenging task. To support this monitoring, we developed and validated the specificity of 12 primer-probe assays for detection of environmental DNA (eDNA) from marine species, all nonindigenous to Europe. The species include sturgeons, a Pacific red algae, oyster thief, a freshwater hydroid from the Black Sea, Chinese mitten crab, Pacific oyster, warty comb jelly, sand gaper, round goby, pink salmon, rainbow trout and North American mud crab. We tested all assays in the laboratory, on DNA extracted from both the target and non-target species to ensure that they only amplified DNA from the intended species. Subsequently, all assays were used to analyse water samples collected at 16 different harbours across two different seasons during 2017. We also included six previously published assays targeting eDNA from goldfish, European carp, two species of dinoflagellates of the genera Karenia and Prorocentrum, two species of the heterokont flagellate genus Pseudochattonella. Conventional monitoring was carried out alongside eDNA sampling but with only one sampling event over the one year. Because eDNA was relatively fast and easy to collect compared to conventional sampling, we sampled eDNA twice during 2017, which showed seasonal changes in the distribution of nonindigenous species. Comparing eDNA levels with salinity gradients did not show any correlation. A significant correlation was observed between number of species detected with conventional monitoring methods and number of species found using eDNA at each location. This supports the use of eDNA for surveillance of the distribution of marine nonindigenous species, where the speed and relative easy sampling in the field combined with fast molecular analysis may provide advantages compared to conventional monitoring methods. Prior validation of assays increases taxonomic precision, and laboratorial setup facilitates analysis of multiple samples simultaneously. The specific eDNA assays presented here can be implemented directly in monitoring programmes across Europe and potentially worldwide to infer a more precise picture of the dynamics in the distribution of marine nonindigenous species