Field validation of an eDNA assay for the endangered white-clawed crayfishAustropotamobius pallipes

Abstract

The white-clawed crayfish Austropotamobius pallipes has experienced extensive 18 population declines in its native range in the last century and is now a threatened species. 19 European legislation therefore requires A. pallipes populations to be monitored regularly and that 20 Special Areas of Conservation (SACs) be designated in part of their range. These needs cannot 21 be met without knowledge about the distribution of this species. However, common crayfish 22 sampling approaches can be time consuming, expensive, or yield false negatives at low 23 population densities. We therefore present an environmental DNA (eDNA) assay to detect A. 24 pallipes in water samples with species-specific primers, a minor groove binding (MGB) probe, 25 and quantitative PCR, based on the mitochondrial cytochrome oxidase I (COI) gene. The results 26 reflected the expected distribution of A. pallipes, based on the known locations of this species in 27 the catchment areas sampled. The results of this study indicate that eDNA is an effective tool for 28 detecting A. pallipes in a lotic system, and could therefore provide a valuable, non-invasive 29 method for determining its distribution. 30Austropotamobius pallipes, Ireland, river 32 33 3 The white-clawed crayfish Austropotamobius pallipes is a relatively large, long-lived (>10 y) 34 crustacean that inhabits both rivers and lakes (Reynolds et al. 2010). This species requires 35 alkaline conditions for survival and is commonly found in waterbodies overlying carboniferous 36 limestone bedrock (Lucey and McGarrigle 1987). Austropotamobius pallipes is 1 of the 5 37 indigenous crayfish species in Europe (Holdich et al. 2009) and was once abundant throughout 38 Europe. However, over the last century, populations of this species have been greatly reduced or 39 gone locally extinct across large parts of its native range (Grandjean et al. 1997). Pollution 40 (Demers and Reynolds 2002, Lyons and Kelly-Quinn 2003), habitat loss, and disease (Matthews 41 and Reynolds 1992) have all contributed to this decline. The crayfish plague, caused by the 42 fungus Aphanomyces astaci (Holdich et al. 2009) is of particular concern because A. pallipes 43 possesses no resistance to this fungus. Thus, entire populations of A. pallipes can be eradicated 44 following an outbreak (Reynolds et al. 2010). 45In spite of the crayfish plague, Ireland is considered one of the few remaining strongholds 46 for A. pallipes within Europe. One reason for this may be that the invasive signal crayfish 47 49 Ireland. However, crayfish plague has reached Irish rivers, and large changes in the distribution 50 of A. pallipes have been partially attributed to past plague outbreaks (Demers et al. 2005). 51Additionally, there is currently an outbreak of crayfish plague in Ireland (National Biodiversity 52 Data Center 2018). Thus, there is a pressing need for a non-invasive, rapid, and cost-effective 53 sampling method that can be used to establish the distribution of this species. 54 Austropotamobius pallipes is protected ...

Development and application of eDNA-based tools for the conservation of white-clawed crayfish

“…Currently, many species-specific eDNA assays only cover in-silico, in-vitro and sometimes basic in-situ validation steps (Dickie et al, 2018;Lacoursière-Roussel et al, 2016). Already published white-clawed crayfish eDNA assays have shown some promising first results but yet need to go through the required thorough level of in-situ evaluation (Atkinson et al, 2019;Robinson et al, 2018). Here we illustrate that sampling methods can differ strongly in performance and recommend rigorous testing of eDNA assays to optimise sampling strategies.…”

Influence of distance from source population and seasonality in eDNA detection of white‐clawed crayfish, through qPCR and ddPCR assays

“…Several primers and probe sets specific to the white-clawed crayfish have been developed and optimized within the literature (Table 1), targeting amplicon length ranging from 96 bp to more than 250 bp, and targeting a region of COI (Atkinson et al, 2019;Chucholl et al, 2021;Troth et al, 2020) or 16S genes (Manfrin et al, 2022).…”

“…To choose the most suitable PCR set (primers and probe) for specimens found in France (our study area), we carried out an in-silico alignment using Geneious Pro R10 software (https://www.genei ous.com; Kearse et al, 2012), consisting in aligning these experimental primers and probe sets given in the literature (Table 1) with the genetic data of A. pallipes from French territory (from GenBank) (Appendix C). The experimental set giving the most satisfactory results (amplicon size < 120 bp and no mismatch) was the one developed by Atkinson et al (2019). The others gave greater degrees of nucleotide incompatibility of the probe (Chucholl et al, 2021), or one of the two primers (Troth et al, 2020), some with excess mismatches (Manfrin et al, 2022).…”

“…These standards were generated using DNA extracted from A. pallipes tissue (40 ng/μL, quantified using a NanoDrop® 1000 Spectrophotometer) following the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) Guidelines (Bustin et al, 2009), and concentrations ranged from 4 × 10 −1 to 4 × 10 −5 ng/μ L. Positive signals were considered when a Ct value <42 (i.e., considered "false positive" if above), by qPCR and one positive droplet (setting the FAM amplitude level = 2000), by ddPCR. A site was noted as "harbouring A. pallipes" if at least one replicate of the six technical replicates (per station and per PCR method) was positive (Atkinson et al, 2019;Bedwell & Goldberg, 2020).…”

“…In freshwater ecosystems, the first species-specific eDNAbased approach was developed by Ficetola et al (2008), proving its effectiveness to detect an invasive frog (Rana catesbeiana) in France. Since a large number of studies and methodological development were conducted to improve eDNA-based applications (Schenekar, 2022), in particular as an aquatic conservation tool to monitor taxa of interest (e.g., invasive, rare, threatened taxa), using eDNA collected from river water samples (e.g., Atkinson et al, 2019;Dubreuil et al, 2021;Harper et al, 2018;Piggott, 2016). However, detection and interpretation of eDNA signals from water samples in rivers can be challenging due to natural dilution, degradation (environmental factors, biological processes), and transport conditions (Barnes & Turner, 2015).…”