Simultaneous detection of invasive signal crayfish, endangered white-clawed crayfish and the crayfish plague pathogen using environmental DNA
Introduction
Invasive non-native species have become important drivers of global environmental change (Vitousek et al., 1996), although the importance of their impacts on biodiversity remains controversial (Russell and Blackburn, 2017). Their spread has been favoured by human-mediated activities (Crowl et al., 2008) in addition to natural dispersal, and, as a consequence have also become common vehicles for the introduction of novel pathogens (Randolph and Rogers, 2010). Invasive non-native species extend the geographic range of the pathogens they carry and facilitate host-switching (Peeler et al., 2011). In turn, pathogens play an important role in the evolution of communities but can also threaten the survival of native populations (Altizer et al., 2003). Co-introductions of parasites with non-native hosts are common; invasive species may bring novel infectious diseases that can infect native competitors, but can also act as hosts and effective dispersers for native diseases (Strauss et al., 2012). Invasive pathogens can have devastating effects on vulnerable native hosts, as their virulence tends to be higher than in the non-native species (Lymbery et al., 2014). Such pathogens seem particularly frequent in freshwater species, potentially reflecting the high susceptibility of freshwater ecosystems to non-native invasions (Moorhouse and Macdonald, 2015). Thus, early detection of both non-native hosts and parasites is critical for the control and management of the impacts caused by introduced diseases.
Detection of non-native species often occurs when populations have already established, spread from original source and altered the local environment (Vander Zanden et al., 2010; Zaiko et al., 2014). This is particularly the case in aquatic environments, where juveniles or larvae at the initial stages of introduction often have a patchy distribution, are difficult to identify using morphological techniques, and are easily missed by monitoring programmes (Pochon et al., 2013). Early detection is needed to make management actions such as eradication and control of invasive species more efficient and/or effective (Lodge et al., 2016) and as such is becoming fundamental for the management and control of aquatic invasive species (AIS; Vander Zanden et al., 2010). Analysis of environmental DNA (eDNA), i.e. free DNA molecules released from sources such as faeces, skin, urine, blood or secretions of organisms, is proving increasingly useful for detecting species that are difficult to identify and locate by more traditional and time-consuming methods (Biggs et al., 2015), such as endangered species (Dejean et al., 2011) and AIS at the early stages of their introduction (Bohmann et al., 2014; Dejean et al., 2012). Although still a relatively new tool, eDNA is becoming widely used for conservation (Biggs et al., 2015; Laramie et al., 2015; Spear et al., 2015; Thomsen and Willerslev, 2015) and protocols are being refined to increase its accuracy and reliability (Goldberg et al., 2016; Wilson et al., 2016). Quantitative PCR (qPCR) is commonly used to target particular species in eDNA samples (e.g. Ficetola et al., 2008; Thomsen et al., 2012) and, coupled with in vitro controls and amplicon sequencing, has proved a reliable method for the detection of invasive and endangered aquatic species (Klymus et al., 2015; Spear et al., 2015). In addition, qPCR is widely used to detect infectious agents in environmental samples (Guy et al., 2003), and can be particularly useful for the early detection of aquatic pathogens which can be introduced simultaneously with non-native species (Ganoza et al., 2006; Strand et al., 2014). High Resolution Melting (HRM) analysis is a qPCR-based method which facilitates identification of small variations in nucleic acid sequences by differences in the melting temperature of double stranded DNA depending on fragment length and sequence composition (Ririe et al., 1997). Analysis of HRM curves has been widely used for SNP genotyping as a fast method to discriminate species (Yang et al., 2009), including natives and invasives (Ramón-Laca et al., 2014). HRM has the potential for being used in AIS identification, including aquatic invasive pathogens, but it has not yet been applied to their detection from eDNA samples. We have used this method to investigate the distribution of the invasive signal crayfish (Pacifastacus leniusculus), carrier of the crayfish plague agent (Aphanomyces astaci) which is highly infective for native species (e.g. Austropotamobius pallipes), and the potential coexistence between native and invasive crayfish in UK populations.
Invasive non-native crayfish have been globally introduced, mainly for human consumption, and are known to seriously impact native ecosystems through predation, competition, disease transmission and hybridisation (e.g. Lodge et al., 2012). In Europe, non-indigenous crayfish mostly of North American origin have outnumbered their native counterparts in much of their range and represent one of the main threats to their persistence (Holdich et al., 2009). The distribution and abundance of native European crayfish species has been strongly influenced by high mortality rates associated with contracting crayfish plague (Schrimpf et al., 2012) through the introduction of North American freshwater crayfish around 1850 (Alderman, 1996). P. leniusculus was one of the first non-native species introduced to Europe and in the UK is displacing the native crayfish (A. pallipes) which has been classified as endangered in the UK (IUCN, 2017). Its success has been attributed to preadaptation, niche plasticity, the aggressive nature of the species (Chapple et al., 2012; Pintor et al., 2008) and/or the competitive advantage provided by the crayfish plague (Bubb et al., 2005; Dunn et al., 2009; Edgerton et al., 2004; Griffiths et al., 2004).
By using a novel approach to simultaneously identify both AIS and their major associated pathogens, we analysed the distribution of the highly invasive signal crayfish (P. leniusculus), the native crayfish (A. pallipes) and the crayfish plague pathogen (A. astaci) in areas where the presence of the signal crayfish is severely impacting the native populations, to identify potential areas of coexistence and refugia for the native species. We expected to find coexisting populations of both species more likely in locations where the crayfish plague has been historically and continually absent.
Section snippets
Ex situ optimisation of eDNA methods
In order to optimise eDNA protocols an ex-situ pilot experiment was conducted by placing individual P. leniusculus in three isolated tanks, each with 2 L of water. After 24 h, they were removed and two 15 ml water samples were taken from each tank. The sampling was repeated 24 and 48 h after removal. Two ultrapure water blanks and four tank blanks (with no crayfish in) were also taken as controls during each sampling period. Immediately after collection, a standard method of preserving and
Ex-situ optimisation
Optimisation of eDNA protocols was carried out ex-situ by placing individual P. leniusculus in isolated tanks for 24 h and sampling water from those tanks 24 and 48 h after removal. Reference DNA from the ex-situ study was successfully extracted and amplified in triplicate from P. leniusculus and A. pallipes positive controls and species confirmed by Sanger Sequencing of the 83 bp fragment of the 16S mtDNA. DNA from signal crayfish was detected in all water samples taken at different time
Discussion
By using a novel multiplex approach we could simultaneously detect the presence of the endangered white clawed crayfish and the highly invasive North American signal crayfish within a catchment that was free of crayfish plague. In contrast, we did not detect any native crayfish or coexistence of both species in tributaries where the pathogen was identified. A common impact of invasive species on native populations is the transmission of pathogens. Many non-native species not only introduce
Author contributions & competing interests
SC & CVR designed the study; CVR & TUW performed the analyses; JC & JJ contributed samples and information; SC & CVR wrote the paper with contributions of all the authors. Authors declare that they have no competing interests.
Data accessibility
All data is currently included in the Data in brief associated article.
Acknowledgements
This research was funded by the Welsh Government and Higher Education Funding Council for Wales (HEFCW) through the Sêr Cymru National Research Network for Low Carbon Energy and Environment (AQUAWALES; NRN-LCEE) and by the Environment Agency UK (SC170011). We thank: Jennifer Nightingale, Oliver Brown and Adam Petrusek for crayfish/DNA samples, Stephen Marsh-Smith, Louis MacDonald-Ames & Hayden Probert, for logistics and information on crayfish trapping; Tony Rees and members of Merthyr Tydfil
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