eDNA Detection of Native and Invasive Crayfish Species Allows for Year-Round Monitoring and Large-Scale Screening of Lotic Systems

Abstract

Effective management of both endangered native and invasive alien crayfishes requires knowledge about distribution, monitoring of existing and early detection of newly established populations. Complementary to traditional survey methods, eDNA sampling has recently emerged as a highly sensitive non-invasive detection method to monitor crayfish populations. To advance the use of eDNA as detection tool for crayfish we used a twofold approach: 1) we designed a novel set of specific eDNA-assays for all native (Austropotamobius torrentium, Austropotamobius pallipes, Astacus astacus) and the most relevant invasive crayfish species (Pacifastacus leniusculus, Faxonius limosus, Faxonius immunis) in Central Europe. To ensure specificity each primer pair was tested in silico, in vitro, and in situ; 2) we assessed the influence of spatio-temporal variables (distance to upstream population, season, stream size) on eDNA detection in seven streams using two different detection methods (qualitative endpoint PCR and quantitative droplet digital PCR, ddPCR). The newly developed eDNA assays successfully detected all crayfish species across different lotic and lentic habitats. eDNA detection rate (endpoint PCR) and eDNA-concentration (ddPCR) were significantly influenced by distance and season. eDNA detection was successful up to 7 km downstream of the source population and across all seasons, although detectability was lowest in winter. eDNA detection rate further decreased with increasing stream size. Finally, eDNA-concentration correlated positively with estimated upstream population size. Overall, we provide near operational eDNA assays for six crayfish species, enabling year-round detection, which represents a clear benefit over conventional methods. Due to its high sensitivity, eDNA detection is also suitable for the targeted search of as-yet unrecorded or newly emerging populations. Using quantitative ddPCR might further allow for a rough estimation of population size, provided that the identified spatio-temporal factors are accounted for. We therefore recommend implementing eDNA-detection as a complementary survey tool, particularly for a large-scale screening of data-deficient catchments or a year-round monitoring.

The amount of environmental DNA increases with freshwater crayfish density and over time

“…With the presented experiment we confirm that not only in fish but also freshwater crayfish population densities can be assessed with eDNA concentration. is that so only has been seen with clarity in one recent field study investigating streams (Chucholl et al, 2021). However, contrary what has been seen in fish, total biomass and crayfish size explained the measured amounts of eDNA equally Likely this is because the exoskeleton of the crayfish means that DNA is not excreted from the whole body surface as in fish.…”

“…Especially in lentic waterbodies, eDNA signals diffuse, whereas eDNA in lotic waterbodies will be more mixed and homogenously distributed; at least further downstream of the population and where the water is well‐mixed (Fremier et al, 2019; Stoeckle et al, 2021; Thalinger et al, 2021). For example, Chucholl et al (2021) recently described a correlation between the upstream population size of Austropotamobius spp. and the number of eDNA molecules contained in water (quantified via droplet digital PCR).…”

“…In fact, so far only a laboratory experiment by Dunn et al (2017) found a weak correlation of eDNA concentration and crayfish densities, but only in egg‐bearing females. From field investigations, there has been little to no correlation between the amount of crayfish eDNA and local population densities in lakes or river systems (Chucholl et al, 2021; Dougherty et al, 2016; Johnsen et al, 2020), where an overall downstream increase of eDNA has so far been observed (Rice et al, 2018).…”

Environmental DNA reveals invasive crayfish microbial associates and ecosystem-wide biodiversity before and after eradication

“…Molecular detection approaches also have variable levels of success (Chucholl et al, 2021;Harper et al, 2018;Troth et al, 2020). For example, the invasive Orconectes rusticus (Dougherty et al, 2016) and the endangered Cambaroides japonicus (Ikeda et al, 2016), can be reliably detected using amplification-based or universal barcoding assays, but for the globally invasive red swamp crayfish Procambarus clarkii, targeted detection assays have not been replicable to reach consensus as a stable monitoring approach (Cai et al, 2017;Mauvisseau et al, 2018;Thalinger et al, 2021).…”

The ‘Pritchard Trap’: A novel quantitative survey method for crayfish

“…artificial refuge traps (ARTs); Green et al, 2018), manual handsearches (Bradley et al 2015;Hilber et al, 2020), electrofishing (e.g. Alonso, 2001) and environmental DNA (Chucholl et al, 2021). However, these methods each have inherent limitations and biases, such as low spatial resolution (eDNA; Harper et al, 2018), or selecting for specific crayfish life stages, sexes or species (Price & Welch, 2009;Rabeni et al, 1997).…”