The Prevalence of Aphanomyces Astaci in Invasive Signal Crayfish
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1 The prevalence of Aphanomyces astaci in invasive signal crayfish from the UK and implications for native crayfish conservation J. JAMES1*†, S. NUTBEAM-TUFFS2,3†, J. CABLE1,A.MRUGAŁA4, N. VIÑUELA-RODRIGUEZ4,A.PETRUSEK4 and B. OIDTMANN3 1 School of Biosciences, Cardiff University, Cardiff CF10 3AX, UK 2 The Roslin Institute, University of Edinburgh, Easter Bush, Midlothian EH25 9RG, UK 3 Centre for Environment, Fisheries and Aquaculture Science (Cefas), The Nothe, Weymouth, Dorset DT4 8UB, UK 4 Department of Ecology, Faculty of Science, Charles University, Viničná 7, CZ-12844 Prague 2, Czech Republic (Received 26 May 2016; revised 27 September 2016; accepted 19 October 2016) SUMMARY The crayfish plague agent, Aphanomyces astaci, has spread throughout Europe, causing a significant decline in native European crayfish. The introduction and dissemination of this pathogen is attributed to the spread of invasive North American crayfish, which can act as carriers for A. astaci. As native European crayfish often succumb to infection with A. astaci, determining the prevalence of this pathogen in non-native crayfish is vital to prioritize native crayfish popula- tions for managed translocation. In the current study, 23 populations of invasive signal crayfish (Pacifastacus leniusculus) from the UK were tested for A. astaci presence using quantitative PCR. Altogether, 13 out of 23 (56·5%) populations were found to be infected, and pathogen prevalence within infected sites varied from 3 to 80%. Microsatellite pathogen geno- typing revealed that at least one UK signal crayfish population was infected with the A. astaci genotype group B, known to include virulent strains. Based on recent crayfish distribution records and the average rate of signal crayfish population dispersal, we identified one native white-clawed crayfish (Austropotamobius pallipes) population predicted to come into contact with infected signal crayfish within 5 years. This population should be considered as a priority for translocation. Key words: Pacifastacus leniusculus, crayfish plague, white-clawed crayfish, Austropotamobius pallipes, Ark Sites. INTRODUCTION crayfish (Pacifastacus leniusculus), was first intro- duced into the UK from Sweden during the 1970s Crayfish plague, caused by the oomycete for aquaculture (e.g. Holdich and Reeve, 1991; Aphanomyces astaci, is arguably one of the most Alderman, 1996; Holdich et al. 1999, 2014). This deadly invasive parasites of freshwater ecosystems corresponded with mass declines in Britain’s histor- worldwide (Lowe et al. 2004; DAISIE, 2009). The ically abundant native white-clawed crayfish pathogen is thought to have been first introduced (Austropotamobius pallipes) (see Holdich and Reeve, into Europe (Italy) in 1859, and has subsequently 1991; Holdich and Sibley, 2009; Holdich et al. spread throughout most of the continent (reviewed 2009, 2014; James et al. 2014), to such an extent by Alderman, 1996; Holdich, 2003). In the second that since 2010 they have been categorized as endan- half of the 20th century the spread of A. astaci gered (IUCN, 2015). Whilst it was widely consid- throughout Europe was facilitated by the movement ered that reductions in native crayfish were, at least of non-native North American (henceforth referred partially, due to the transmission of A. astaci from to as American) crayfish (reviewed by Alderman, signal crayfish, screening and detection of this 1996; Holdich, 2003). Whilst American crayfish are pathogen in the UK did not occur until the early often asymptomatic carriers of the pathogen, in 1980s (Alderman, 1996). One of the first suspected native European crayfish infection is usually fatal outbreaks of plague in the UK was recorded from (Unestam and Weiss, 1970; Diéguez-Uribeondo the River Lee, Thames catchment, England in et al. 1997; Bohman et al. 2006; Kozubíková et al. 1981 (Alderman, 1996). The pathogen has since 2008; Oidtmann, 2012). Therefore, preventing the been reported in native crayfish from several other spread of this pathogen in regions with populations sites in England as well as Wales and Ireland of highly susceptible hosts is a conservation priority. (Alderman et al. 1984, 1990; Holdich and Reeve, One of the main American crayfish species respon- 1991; Alderman, 1996; Lilley et al. 1997; Holdich, sible for spreading A. astaci in Europe, the signal 2003). However, these reports have been based on pathogen morphology and disease symptoms in * Corresponding author. School of Biosciences, Cardiff fi University, Cardiff, CF10 3AX, UK. E-mail: jamesj12@ native European cray sh. Given that there are no cardiff.ac.uk morphological features that distinguish A. astaci † Both authors contributed equally to this study. from non-pathogenic Aphanomyces species (Royo Parasitology, Page 1 of 8. © Cambridge University Press 2017 doi:10.1017/S0031182016002419 Downloaded from https:/www.cambridge.org/core. University of Cardiff, on 16 Jan 2017 at 11:40:25, subject to the Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. https://doi.org/10.1017/S0031182016002419 J. James and others 2 et al. 2004;Oidtmann,2012), molecular confirmation −80 °C, before being stored individually in falcon is essential (Oidtmann et al. 2006; Vrålstad et al. tubes containing 95% molecular grade ethanol. 2009). The only reports in the scientific literature of Samples collected between September 2009 and A. astaci detection in the UK using molecular July 2010 from 17 sites were processed at Cefas methods are from another introduced crayfish (n =8–30 animals per site), whereas those harvested species, Orconectes cf. virilis (see Tilmans et al. during May–September 2014 were analysed at 2014), which is restricted to a single catchment CUNI (n =20–30 animals per site, Table 1). (James et al. 2016). From each crayfish, a section of tail fan and soft Gaining a comprehensive understanding of abdominal cuticle were harvested for A. astaci A. astaci distribution in the UK is essential for screening. For animals processed in CUNI soft native crayfish conservation. It is generally consid- cuticle from two limb joints and any sections of mel- ered that the only way of ensuring the sustainability anized cuticle were also collected and pooled of white-clawed crayfish in the UK is through the (Svoboda et al. 2014). At Cefas tissue samples establishment of isolated ‘Ark Sites’ free from non- from the tail fan and soft abdominal cuticle were native crayfish and at low risk of their invasion analysed separately (mean: 60 and 78 mg of tissue (Peay, 2009). Resources for implementing such con- per host sample for the tail fan and soft abdominal servation measures are, however, limited and so the cuticle, respectively). For these samples, tissue dis- selection of native crayfish populations for transloca- ruption was conducted in fast prep tubes containing tion needs to be a well-informed process. Native lysis matrix A (MP Biomedicals, Cambridge, UK) crayfish populations in close vicinity to A. astaci- and DNA subsequently extracted using the Qiamp infected invasive crayfish populations are at higher DNeasy Biorobot investigator kit (Qiagen, Hilden, risk of extirpation than those neighbouring unin- Germany), according to the manufacturer’s guide- fected ones (Söderbäck, 1994; Westman et al. 2002; lines. At CUNI, for each animal, all collected Schulz et al. 2006; Dunn et al. 2009; Schrimpf tissue samples were amassed (40–50 mg per host et al. 2013). Co-existence of native crayfish with sample) and ground together in liquid nitrogen. invasive crayfish for several years has been observed DNA was then extracted using DNeasy tissue kit in the absence of A. astaci (see Söderbäck, 1994; (Qiagen, Hilden, Germany) in accordance with Westman et al. 2002; Schulz et al. 2006; Dunn manufacturer’s instructions. et al. 2009; Schrimpf et al. 2013). Therefore, it is All samples were tested for A. astaci presence with of greater urgency to translocate native crayfish the TaqMan MGB qPCR as described in Vrålstad populations at high risk of A. astaci transmission, et al. (2009) with the following slight alterations at than those in close proximity to uninfected invasive Cefas and CUNI, respectively: an elongation of the crayfish. decontamination step from 2 to 5 min (Tuffs and Here, we used quantitative PCR (qPCR) to assess Oidtmann, 2011), an increase in the annealing tem- the prevalence and intensity of infection with A. perature from 58 to 60 °C and decreased synthesis astaci in 23 populations of invasive signal crayfish time from 60 to 30 s (Svoboda et al. 2014). At in England and Wales. Using these data in combin- Cefas and CUNI qPCRs were run on a Step one ation with long-term white-clawed crayfish distribu- Plus real-time cycler (Applied Biosystems) and an tion records (James et al. 2014) we identified native iQ5 BioRad thermal cycler, respectively. Negative crayfish populations at high risk of infection with controls were used in every step of the procedure; A. astaci (determined by their proximity to an these remained negative in all cases. The amount A. astaci-infected signal crayfish population). of A. astaci DNA in each sample was estimated Given that A. astaci genotypes differ in virulence based on the calibration curve of a set of standards. (Makkonen et al. 2012; Becking et al. 2015), when At Cefas the quantity of pathogen DNA in these possible, we also genotyped the strain of A. astaci. standards ranged from 1 ng to 10 fg, in a 10-fold dilution series. At CUNI four standards were included containing 3 × 410,3×48,3×44 and 3 × METHODS 42 PCR forming units (PFU) of pathogen DNA. For this study, invasive signal crayfish (P. leniuscu- At Cefas each sample was run in triplicate and an lus) from the UK were screened for the presence of average taken when calculating pathogen DNA con- A. astaci using similar molecular methods in two centrations. At CUNI each isolate was run twice, separate laboratories: the Centre for Environment, undiluted and a 10-fold diluted replicate to test for Fisheries and Aquaculture Science, Weymouth, inhibition that may affect the efficacy of pathogen UK (Cefas); and Charles University, Prague, detection (Vrålstad et al.