Field Application of an Edna Assay for the Threatened White-Clawed Crayfish Austropotamobius Pallipes
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bioRxiv preprint doi: https://doi.org/10.1101/562710; this version posted February 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. eDNA assay for Austropotamobius pallipes 1 Field application of an eDNA assay for the threatened white-clawed crayfish 2 Austropotamobius pallipes 3 Siobhán Atkinson1,2*, Jeanette E.L. Carlsson2, Bernard Ball2, Mary Kelly-Quinn1, Jens Carlsson2 4 1. School of Biology and Environmental Science/Earth Institute, University College 5 Dublin, Dublin, Ireland 6 2. Area52 Research Group, School of Biology and Environmental Science/Earth Institute, 7 University College Dublin, Dublin, Ireland 8 *Corresponding author: Siobhán Atkinson, [email protected] 9 1 bioRxiv preprint doi: https://doi.org/10.1101/562710; this version posted February 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. eDNA assay for Austropotamobius pallipes 10 Abstract 11 The white-clawed crayfish Austropotamobius pallipes has undergone extensive declines 12 within its native range in the last century. Because of its threatened status, European 13 legislation requires the species to be regularly monitored and that Special Areas of 14 Conservation (SACs) be designated for it. Knowledge of the distribution of this species is vital 15 for addressing these needs. This study presents an environmental (e)DNA assay to detect A. 16 pallipes in water samples, based on the mitochondrial cytochrome oxidase I (COI) gene, 17 utilizing species-specific primers, a minor groove binding (MGB) probe and quantitative PCR. 18 The results of this study indicate that eDNA is an effective tool for detecting A. pallipes in a 19 lotic system, and could provide a valuable, non-invasive method for determining the 20 distribution of this species. 21 Keywords: eDNA, qPCR, conservation, non-invasive, detection, native species, river. 22 Introduction 23 The white-clawed crayfish Austropotamobius pallipes is a relatively large, long-lived (>10 24 years) crustacean that inhabits both rivers and lakes (Reynolds et al. 2010). It requires 25 alkaline conditions for survival and is commonly found in waterbodies overlying 26 carboniferous limestone bedrock (Lucey and McGarrigle 1987). A. pallipes is one of the five 27 indigenous crayfish species in Europe (Holdich et al. 2009). This once abundant species has, 28 however, become greatly reduced or locally extinct across large parts of its native range 29 during the last century (Grandjean et al. 1997). Pollution (Demers and Reynolds 2002, Lyons 30 and Kelly-Quinn 2003), habitat loss and disease (Matthews and Reynolds 1992) have 31 contribution to this decline. Of particular concern is the crayfish plague, caused by the 32 fungus Aphanomyces astaci (Holdich et al. 2009). A. pallipes possesses no resistance to this 2 bioRxiv preprint doi: https://doi.org/10.1101/562710; this version posted February 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. eDNA assay for Austropotamobius pallipes 33 fungus, so eradication of entire populations of A. pallipes is possible following an outbreak 34 (Reynolds et al. 2010). Despite this, Ireland is considered one of the few remaining 35 strongholds for A. pallipes within Europe. One of the reasons for this is that the invasive 36 signal crayfish Pacifastacus leniusculus, which can impose wide-ranging impacts on 37 ecosystems and act as a vector for the crayfish plague (Vaeßen and Hollert 2015), has not 38 been reported in Ireland to date. However, crayfish plague has reached Ireland’s rivers, and 39 large changes in the distribution of A. pallipes have been attributed, at least in part, to past 40 plague outbreaks (Demers et al. 2005). Additionally, there is at present an outbreak of 41 crayfish plague in Ireland (National Biodiversity Data Centre 2018), and hence there is a 42 pressing need for a non-invasive, rapid and cost-effective sampling method that can be used 43 to establish the distribution of the species. 44 Because of its vital role in freshwater ecosystem functioning and its threatened status 45 (Matthews and Reynolds 1992), A. pallipes is afforded protection under the EU Habitats 46 Directive and is listed under Annexes II and V. This means Ireland is required to regularly 47 monitor A. pallipes, and to designate Special Areas of Conservation (SACs) for the species 48 under Natura 2000 (Reynolds et al. 2010). Knowledge of the geographic distribution of A. 49 pallipes is essential for implementing these conservation measures. Traditional survey 50 methods include night viewing with a strong torch, modified quadrat samplers (DiStefano et 51 al. 2003), kick-sampling, baited traps and enclosures (Byrne and Lynch 1999), snorkeling 52 surveys (Reynolds et al. 2010) and SCUBA diving (Matthews and Reynolds 1992). While many 53 of these methods are effective, they are time-consuming, potentially costly, and may not be 54 effective when A. pallipes occur in low abundance. Furthermore, a license is required for 55 most traditional A. pallipes surveying methods. This is vital for ensuring the protection of A. 56 pallipes, however, a non-invasive methodology that does not require a license for assessing 3 bioRxiv preprint doi: https://doi.org/10.1101/562710; this version posted February 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. eDNA assay for Austropotamobius pallipes 57 its distribution would be beneficial. A promising new tool that has been shown to be both 58 effective at detecting species and to save significant time in the field (Sigsgaard et al. 2015) is 59 environmental DNA (eDNA) analysis. Environmental DNA is the collective term for DNA 60 present freely in the environment that has been shed by organisms for example in the form 61 of mucus, excrement, gametes, or blood (Taberlet et al. 2012, Thomsen and Willerslev 62 2015). The analysis has been successful in detecting species that occur in low abundance 63 such as rare or endangered species (Mächler et al. 2014, Laramie et al. 2015, Sigsgaard et al. 64 2015, Boothroyd et al. 2016, Carlsson et al. 2017), or invasive alien species (Jerde et al. 2011, 65 Goldberg et al. 2013, Fujiwara et al. 2016). Numerous studies have also shown eDNA to be 66 more sensitive than traditional methods at detecting species at low abundance (Dejean et al. 67 2012, Smart et al. 2015, Dougherty et al. 2016). Environmental DNA assays have been 68 successfully deployed for other crayfish species including Astacus astacus, Astacus 69 leptodactylus, P. leniusculus (Agersnap et al. 2017, Larson et al. 2017), Orconectes rusticus 70 (Dougherty et al. 2016, Larson et al. 2017), Procambarus clarkii (Cai et al. 2017) and Faxonius 71 eupunctus (Rice et al. 2018). 72 The aim of this study was to develop an MGB based qPCR assay to detect the presence of 73 the white-clawed crayfish, A. pallipes in water samples, and to test the reliability of the assay 74 by comparing the results with field observation data. 75 Methods 76 Study sites and crayfish distribution data 77 Eight sampling locations within seven different rivers were selected for field validation of the 78 assay (Table 1). The research presented here was carried out as part of a larger study 79 assessing the impact of river obstacles on freshwater fauna (e.g. Atlantic salmon, Salmo 4 bioRxiv preprint doi: https://doi.org/10.1101/562710; this version posted February 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. eDNA assay for Austropotamobius pallipes 80 salar, Atkinson et al. 2018). As part of this study, eDNA samples were collected above and 81 below a number of river obstacles (weir, ford crossing or bridge apron) in order to determine 82 whether they imposed migratory barriers on S. salar. The same samples collected for this S. 83 salar study were used for the present study. Out of the eight sites sampled, four were in 84 rivers with SAC status in which A. pallipes featured as a species of interest. Study rivers 85 within the River Barrow and River Nore SAC included the Delour and Dinin rivers and study 86 rivers within the Lower River Suir SAC included the Multeen and Duag rivers. Although the 87 Burren River is a tributary of the River Barrow, it does not have SAC status. 88 The current distribution of A. pallipes in these rivers was extrapolated using data provided 89 by the Irish Environmental Protection Agency (EPA). These data are point data based on 90 casual field observations made of A. pallipes by EPA staff whilst undertaking routine 91 biological monitoring between the years 2007 and 2016 (W. Trodd, Irish Environmental 92 Protection Agency, personal communication). In addition, it was possible at some sites to 93 confirm the presence of crayfish by field observations made while electrofishing for S. salar. 94 For the purposes of this study, A. pallipes observations were extrapolated to the wider 95 subcatchment level.