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Non-destructive method for detecting Aphanomyces astaci, the causative agent of crayfish plague, on the individual level
Journal Pre-proofs Non-destructive method for detecting Aphanomyces astaci, the causative agent of crayfish plague, on the individual level Dora Pavić, Milan Čanković, Ines Sviličić Petrić, Jenny Makkonen, Sandra Hudina, Ivana Maguire, Tomislav Vladušić, Lidija Šver, Reno Hraš ćan, Karla Orlić, Paula Dragičević, Ana Bielen PII: DOI: Reference:
S0022-2011(19)30015-1 https://doi.org/10.1016/j.jip.2019.107274 YJIPA 107274
To appear in:
Journal of Invertebrate Pathology
Received Date: Revised Date: Accepted Date:
24 January 2019 27 October 2019 30 October 2019
Please cite this article as: Pavić, D., Čanković, M., Sviličić Petrić, I., Makkonen, J., Hudina, S., Maguire, I., Vladušić, T., Šver, L., Hraš ćan, R., Orlić, K., Dragičević, P., Bielen, A., Non-destructive method for detecting Aphanomyces astaci, the causative agent of crayfish plague, on the individual level, Journal of Invertebrate Pathology (2019), doi: https://doi.org/10.1016/j.jip.2019.107274
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Non-destructive method for detecting Aphanomyces astaci, the causative agent of crayfish plague, on the individual level Dora Pavić1, Milan Čanković2, Ines Sviličić Petrić2, Jenny Makkonen3, Sandra Hudina4, Ivana Maguire4, Tomislav Vladušić1, Lidija Šver1, Reno Hrašćan1, Karla Orlić4, Paula Dragičević4, Ana Bielen1,* 1
University of Zagreb, Faculty of Food Technology and Biotechnology, Department for Biochemical Engineering, Pierottijeva 6, 10000 Zagreb, Croatia 2
Ruđer Bošković Institute, Division for Marine and Environmental Research, Bijenička cesta 54, 10000 Zagreb, Croatia 3
University of Eastern Finland, Department of Environmental and Biological Sciences, P.O. Box 1627, FIN70211 Kuopio, Finland 4
University of Zagreb, Faculty of Science, Department of Biology, Rooseveltov trg 6, 10000 Zagreb, Croatia *
Corresponding author: [email protected]
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Abstract The pathogenic oomycete Aphanomyces astaci, transmitted mainly by invasive North American crayfish, causes the crayfish plague, a disease mostly lethal for native European crayfish. Due to its decimating effects on native crayfish populations in the last century, A. astaci has been listed among the 100 worst invasive species. Importantly, detecting the pathogen in endangered native crayfish populations before a disease outbreak would provide a starting point in the development of effective control measures. However, current A. astaci-detection protocols either rely on degradation-prone eDNA isolated from large volumes of water or, if focused on individual animals, include killing the crayfish. We developed a nondestructive method that detects A. astaci DNA in the microbial biofilm associated with the cuticle of individual crayfish, without the need for destructive sampling. Efficiency of the new method was confirmed by PCR and qPCR and the obtained results were congruent with the traditional destructive sampling method. Additionally, we demonstrated the applicability of the method for A. astaci monitoring in natural populations. We propose that the new method should be used in future monitoring of A. astaci presence in endangered European native crayfish individuals as an alternative to eDNA-based monitoring.
Keywords: Aphanomyces astaci detection, crayfish cuticle-associated biofilm, pathogenic oomycete, endangered native crayfish
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1. Introduction Pathogenic oomycete Aphanomyces astaci Schikora, 1906, is one of the major reasons for the decline of native European crayfish populations. Due to its detrimental effects and fast spread in European waterways, it was classified amongst the world’s 100 worst invasive alien species (Alderman, 1996; Lowe et al., 2000; OIE, 2018). It is transmitted primarily by North American invasive crayfish species that act as carriers in Europe and are mostly resistant to infection (Holdich et al., 2009). In contrast, native crayfish individuals most often develop highly contagious and lethal disease if in contact with A. astaci zoospores (Makkonen et al., 2013; Becking et al., 2015). Five known A. astaci genotypic groups differ in origin and virulence (Huang et al., 1994; Diéguez-Uribeondo et al., 1995; Kozubíková et al., 2011; Makkonen et al., 2012, 2013; Viljamaa-Dirks et al., 2013). Group A (As) is believed to be related to the first introduction of A. astaci into Europe in the 19th century and the primary carrier is unknown (Huang et al., 1994; ViljamaaDirks et al., 2013). The carriers of other A. astaci groups are North American invasive crayfish species: B (PsI) and C (PsII) originate from Pacifastacus leniusculus (Huang et al., 1994), D (Pc) from Procambarus clarkii (Diéguez-Uribeondo et al., 1995; Rezinciuc et al., 2014), and E (Or) from Faxonius limosus (Kozubíková et al., 2011). The genotypic groups vary in virulence, with genotypes B and E reported to be much more virulent than genotype A (Makkonen et al., 2012, 2013; Viljamaa-Dirks et al., 2013; Becking et al., 2015). Indeed, genotype A is mostly detected in native European crayfish populations (Astacus astacus and Astacus leptodactylus) that are chronically infected but vital, without mass mortalities (Jussila et al., 2011b; Viljamaa-Dirks et al., 2011; Kokko et al., 2012; Svoboda et al., 2012; Kušar et al., 2013). Continuous monitoring of the pathogen’s distribution is necessary in order to have a clear understanding about its spread through European waterways and to identify populations that are potential pathogen carriers (OIE, 2018). Morphological observations and microscopy of infected tissue can serve as a first indication of A. astaci presence, but cannot differentiate between A. astaci and related oomycetes (Cerenius et al., 1988; Viljamaa-Dirks and Heinikainen, 2006). Isolation and cultivation of A. astaci, followed by morphological and molecular identification, was required to confirm its presence in suspect animals (Huang et al., 1994; OIE, 2018), however, successful isolation of the pathogen was time consuming, frequently without satisfying results. Methods that rely on PCR-based detection of A. astaci DNA in the crayfish tissue were developed, making the diagnostic procedure faster and more sensitive. The most common assays use the internal transcribed region (ITS) as a marker region to identify A. astaci by standard (Oidtmann et al., 2006) or quantitative PCR (qPCR) (Vrålstad et al., 2009). These methods have been used extensively in crayfish plague monitoring (Cammà et al., 2010; Jussila et al., 2011b; Kokko et al., 2012; Svoboda et al., 2012; Filipová et al., 2013; Kušar et al., 2013; Maguire et al., 2016). Over the years, several other methods were developed, including qPCR-based detection of A. astaci chitinase genes (Hochwimmer et al., 2009), microsatellite markers useful to determine different A. astaci genotypes (Grandjean et al., 2014), mitochondrial DNA analysis that allows the detection and characterization of A. astaci haplotypes (Makkonen et al., 2018) and PCR-based A. astaci genotyping (Minardi et al., 2018). All these protocols use DNA samples as a starting point, isolated either directly from water (eDNA, Strand et al., 2011, 2012, 2014; Wittwer et al., 2018) or from a piece of crayfish cuticle after killing the animal (Oidtmann et al., 2004, 2006; Filipová et al., 2013).
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We aimed to eliminate the killing step (i.e. destructive sampling) and developed a non-destructive A. astaci detection method which is more suitable for crayfish plague monitoring in endangered native populations. After the initial chemotaxis of A. astaci zoospores toward the host cuticle (Cerenius and Söderhäll, 1984), zoospores encyst and subsequently germinate into hyphae that penetrate into the crayfish tissues through the cuticle (Unestam and Weiss, 1970; Cerenius et al., 2003; Cammà et al., 2010). Thus, we assumed that A. astaci DNA, originating from zoospores, cysts or hyphae, can be detected in the mixed epibiotic microbial communities scrubbed from the surface of live crayfish.
2. Material and methods 2.1. Sampling We collected adult freshwater crayfish (total length > 6 cm) in LiNi traps (Westman et al., 1978) or by hand at different locations in Croatia (Table 1). The disease status was previously tested for 3 of 15 locations. Some animals (Table 2) were collected in order to compare the results of A. astaci detection by newly developed swab-based method with standard A. astaci cuticle-based detection. In such cases, two types of samples were taken from each animal: (i) swab of the cuticle containing the epibiotic community (for non-destructive A. astaci detection, Fig. 1); and (ii) a sample of abdominal cuticle, collected after killing the animal (for standard destructive detection; Oidtmann et al., 2006). These two sample types were collected from animals brought directly from the field (natural population samples) and from animals infected with A. astaci zoospores in the laboratory (experimental samples) (Table 2). Additionally, swab samples of a larger number of animals were taken to demonstrate the applicability of the method for A. astaci monitoring in natural populations. Forty-six Austropotamobius torrentium individuals and 114 As. astacus individuals were sampled in the Plitvice Lakes National Park during 2017 and 2018, and 110 P. leniusculus individuals were sampled from the Korana River population during 2018.
2.2. Inoculation of animals with A. astaci zoospores Animals were held separately in 18 x 12 x 10 cm aquaria filled with approximately 1 L of water, and equipped with air pumps. The water temperature was ± 18 °C. Each crayfish was given one bottom feeding fish tablet twice a week and the water was changed the day after the feeding. Highly virulent Aphanomyces astaci strain B, PsI-genotype (UEF-T16B) isolated from P. leniusculus in Lake Tahoe, USA, was used. This strain was chosen because we inoculated (mostly) As. leptodactylus, a highly robust (although native) crayfish species. It has successfully spread from eastern to western Croatia over the past 50 years (Maguire and Gottstein-Matočec, 2004) and has sometimes been considered a cryptic invader due to its higher adaptive plasticity compared to other native species (Lucić et al., 2012). There is no literature data on A. astaci infection trials that used A. leptodactylus as a host, but we assumed that such advantageous life-history traits should make A. leptodactylus more resistant to A. astaci infection compared to other native species. Indeed, vital A. leptodactylus populations, including some percentage of B strain positive individuals, were found co-occurring the same locations as invasive B strain carrier P. leniusculus (Maguire et al., 2016). After a 1-month acclimatization period, animals were infected with 500 zoospores / mL of water, following the procedure described by Makkonen et al. (2012). We used a 4
moderate zoospore concentration of 500 zoospores / mL to infect the animals in order to mimic natural situation in the chronically infected population. Swabs and cuticle samples were taken immediately after death of the crayfish.
2.3. Non-destructive sampling protocol Non-destructive sampling included thorough scrubbing of crayfish surface. Each crayfish was placed in a Petri dish filled with 0.1 % NaCl, 0.15 M Tween 20 solution and thoroughly scrubbed with a sterile brush wetted with the same solution (Fig. 1a, b). To avoid cross contamination, sterile instruments/consumables were used for each individual. The obtained suspension was returned to a 50mL Falcon tube (Fig. 1c, d). During the initial development of the protocol, the whole volume of the suspension (30 – 50 mL per crayfish) was centrifuged at 4,000 x g for 15 min at 4 °C. However, this step was later omitted because we obtained the same results using the modified protocol. Namely, the suspension was left to settle for 30 min at 4 °C, and then approximately 2 mL of the suspension was removed from the bottom of the tube by sterile Pasteur pipette, transferred to a new tube and centrifuged at 10,000 x g for 15 min at 4 °C (centrifuge Centric 200R, Domel, Slovenia). The supernatant was removed and the pellet of epibiotic cells was frozen at -20 °C until DNA isolation (Fig. 1e).
2.4. DNA extraction For DNA extraction from swabs, we tested several extraction kits: NucleoSpin® Microbial DNA kit (Macherey Nagel, Germany), DNeasy PowerSoil Kit (QIAGEN, Germany), QIAamp DNA Investigator Kit (QIAGEN, Germany) and ZymoBIOMICS DNA Miniprep Kit (Zymo Research, USA) and found no significant differences in yield or purity of the DNA (data not shown). Finally, we chose NucleoSpin® Microbial DNA kit (Macherey Nagel, Germany), a kit specifically designed for DNA isolation from microbial communities, with mechanical disruption of cell wall structures of microbial cells as the first step. Prior to DNA isolation from swab and cuticle samples, we confirmed the efficiency of this extraction protocol with samples of pure A. astaci mycelium, using approx. 30 mg of hyphae wet weight previously grown in PG-1 liquid medium (Unestam, 1965) as a starting biomass (data not shown). For the protocol, the samples were lysed by agitation (medium strength, 20 min) on a Vortex Mixer (Corning, USA). We used Macherey Nagel Bead Tubes Type B (provided in the kit), although Bead Tubes Type C from the same kit were also efficient for lysis. DNA elution from the column was performed using the initial 100-μL eluate for a second elution in order to increase yield and concentration of the final sample. Quantity and quality of DNA samples were assessed by agarose gel electrophoresis and QuantiFluor ONE dsDNA Dye on a Quantus Fluorometer (Promega, Germany).
2.5. PCR and qPCR A. astaci DNA in the extracted total DNA was detected following protocols described previously (Oidtmann et al., 2006; Vrålstad et al., 2009; Strand et al., 2014). Primers 42 (5’ GCT TGT GCT GAG GAT GTT CT 3’) and 640 (5’ CTA TCC GAC TCC GCA TTC TG 3’) were used to specifically amplify parts of the ITS region of A. astaci by standard PCR (Oidtmann et al., 2006). It is noteworthy that, during development of the method, we sometimes obtained non-specific PCR-products, especially when using DNA isolated from 5
swab samples as a template. This can be explained by higher diversity in the swab samples (DNA from the total epibiotic community), than from the cuticle samples (mostly crayfish DNA). We therefore increased the annealing temperature from 59 °C (Oidtmann et al., 2006) to 61 °C to increase the specificity (data not shown). In addition, because a recent study showed that ITS‐based molecular assays for A. astaci can yield unspecific amplifications (Viljamaa-Dirks and Heinikainen, 2018), we sequenced all the amplicons (i.e. positives) that yielded a sufficiently strong PCR signal (Sanger sequencing, Microsynth, Austria). The resulting sequences were compared with NCBI database using blastn (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch) and their identity to A. astaci ITS region was confirmed. Detection and relative quantification of A. astaci DNA was done by qPCR using primers AphAstITS39F (5′-AAGGCTTGTGCTGGGATGTT-3′), AphAstITS-97R (5′-CTTCTTGCGAAACCTTCTGCTA-3′) and MGB probe AphAstITS-60T (5′-6-FAM-TTCGGGACGACCC-MGBNFQ-3′), as described earlier (Vrålstad et al., 2009). We used a modified annealing step to increase the assay specificity; instead of 58 °C / 60 s (Vrålstad et al., 2009), we used 62 °C / 30 s (Strand et al., 2014). Quantitative PCR results were further classified into semi-quantitative categories of pathogen load based on their PCR forming units (PFU) values, ranging from A0 (no traces of A. astaci DNA) to A5 (high amount of A. astaci DNA in the sample, estimated PFU 104 and 105) (Vrålstad et al., 2009).
2.6. Statistical analyses The data we obtained were tested to analyze whether they met the assumptions for parametric tests (i.e. normality/homoscedasticity; Zar, 1999. Since both raw and transformed data (logarithmic transformation of continuous data, square-root for discrete data) violated these assumptions, nonparametric analogues were used instead (Mann-Whitney U test, Spearman Rank; Zar, 1999). Furthermore, sensitivity testing of both methods (cuticle and swab samples) was performed according to procedures described in detail in Wittwer et al. (2018). The statistical significance was set at p = 0.05.
3. Results 3.1. Comparison of A. astaci detection by the newly developed swab-based method with standard cuticlebased detection We compared the results of detection of A. astaci from non-destructive swab samples with traditional detection from cuticle samples (Table 3). Results of the standard PCR allowed us to differentiate between A. astaci-positive (amplicon of the expected product size of 569 bp) and negative individuals (no amplification). We obtained congruent results using both swab (i.e. non-destructive method) and cuticle samples (i.e. traditional destructive method) for all tested animals (Table 3, Fig. 2). The amplicons were confirmed as A. astaci by sequencing (data not shown). Subsequent qPCR assays confirmed the results of the standard PCR. Only four samples, AA2 S, AA3 S, PL1 S and PL1 C, were negative as determined by conventional PCR, while qPCR detected low agent level (A1), probably a consequence of higher sensitivity of qPCR (Tuffs and Oidtmann, 2011; Kušar et al., 6
2013). Differences in average PFU per reaction between cuticle and swab samples were not statistically significant (Mann–Whitney U test, p > 0.05). Furthermore, swab and cuticle DNA samples originating from the same individual contained comparable A. astaci agent levels with no statistically significant differences (Mann–Whitney U test, p > 0.05). However, our results indicated that the cuticle method was slightly more sensitive in A. astaci detection than the newly developed swab method (Fig. 3). When analyzing individual sample pairs, the same agent level was measured for both sample types in 6/14 samples, whilst in a few cases the agent level was (slightly) higher for swab sample (4/14) or for cuticle sample (4/14) (Table 3). A discrepancy in qPCR results was obtained for one infected crayfish (AT3), where agent level A5 was detected in cuticle sample, while the swab sample detected the agent at level A1. This result could be a consequence of low DNA concentration in the swab sample (0.432 ng/µL) compared to the cuticle sample (117.5 ng/µL) for this pair. Although the average DNA concentration in swab samples was lower than in the cuticle samples (15.8 ng/µL vs. 74.8 ng/µL, respectively), the difference in the concentration was the highest for the individual AT3, leading to the observed discrepancy in qPCR results. 3.2. Applicability of the newly developed method for A. astaci monitoring in natural populations We demonstrated the applicability of our newly developed swab method for Aphanomyces astaci monitoring in natural populations. We collected a large number of swab samples (270 in total) from native crayfish (As. astacus, Au. torrentium) and invasive crayfish (P. leniusculus). We chose locations that were previously shown to contain A. astaci (Maguire et al., 2016) and with current populations that were viable and without signs of gross mortality. Next, we performed standard PCR assays (Oidtmann et al., 2006) for all collected swab DNA samples to detect A. astaci on individual animals. Of 114 samples taken from As. astacus population in the Plitvice Lakes National Park, 16 were A. astaci-positive (14 %, 2017-2018). In Au. torrentium population in Plitvice Lakes National Park 1 out of 46 tested individuals was A. astaci-positive (2 %, 2017-2018), and 6 % of P. leniusculus population in the Korana River was A. astaci-positive (7/110, 2018). When the PCR signal was strong enough for sequencing, the amplicons were confirmed as A. astaci, 2 out of 17 positives from the Plitvice Lakes National Park and 3 out of 7 positives from the Korana River.
4. Discussion We developed a new method for A. astaci detection that enables pathogen monitoring without destructive sampling of the crayfish hosts. To test the method, we collected samples consisting of infected crayfish as well as uninfected animals and isolated in parallel the total DNA from surface swabs and from cuticle samples. Our results demonstrated that the non-destructive method we developed is comparable with the standard destructive methods (Oidtmann et al., 2006; Vrålstad et al., 2009). During the A. astaci infection process, zoospores that contact the crayfish cuticle encyst and then germinate into hyphae that penetrate through the exoskeleton. The hyphae subsequently spread into deeper tissues and organs, leading to development of lethal disease (Söderhäll and Cerenius, 1999). Although it is generally presumed that the majority of A. astaci biomass in infected animals is located inside the body (Unestam and Weiss, 1970; Cerenius and Söderhäll, 1984), we showed that the pathogen is also present on the cuticle of infected or carrier crayfish and can be collected by scrubbing the surface 7
of the animal. We hypothesize that the A. astaci DNA we detected in the swab samples originates from zoospores, cysts or, in the terminal stages of the disease, possibly hyphae (Unestam and Weiss, 1970; Cerenius et al., 2003; Cammà et al., 2010), but additional studies are needed to unravel this issue. The presence of A. astaci DNA in the swab samples enabled us to develop a non-destructive method for A. astaci detection in crayfish individuals and to circumvent destructive sampling used previously (Oidtmann et al., 2004, 2006; Vrålstad et al., 2009; Strand et al., 2011; Grandjean et al., 2014; Maguire et al., 2016). Our results showed no significant overall difference between swab and cuticle sample pairs in the A. astaci load, although these two sample types were quite different, both in composition and in average DNA concentration. Swab samples contained DNA from mixed epibiotic microbial communities (e.g. bacteria, fungi, oomycetes) originating from the surface of the animal. On the other hand, cuticle samples were small and presumably contained mostly DNA of the crayfish, as well as A. astaci DNA if the animal is infected. The fact that these two sample types showed comparable A. astaci load can be explained by the following: the density of A. astaci biomass is much higher in the tissues of the infected animals (cuticle sample) than on the cuticle surface (swab sample) (Unestam and Weiss, 1970); however, the large scrubbing surface for the swab samples compared to the small segment of cuticle sample probably compensates for this difference. Although the agent level detected was the same for most sample pairs, we observed slightly higher, albeit statistically insignificant, overall sensitivity of the cuticle-based procedure (i.e. slightly higher pathogen levels based on qPCR results), probably due to the overall higher DNA yield obtained for cuticle samples. In spite of this, the absolute status of individual animals was the same, either A. astaci-positive or A. astaci-negative, and, importantly, with no false negatives in swab method. This confirms the applicability of the newly developed method for monitoring, where discriminating between presence and absence of the pathogen is of pivotal importance for any management activities. For some sample pairs, the agent level detected was slightly higher for either the swab or the cuticle sample. We hypothesize that carrier crayfish (perhaps with a small amount of hyphae on the surface) or recently infected crayfish (with attached zoospores) have a higher probability of detection of A. astaci DNA in the swab. In contrast, detection in cuticle samples is more probable in symptomatic animals that have densely packed A. astaci hyphae inside the body. However, testing this assumption was out of the scope of this study and in vivo infection trials coupled with microscopy are needed to confirm it. Our novel non-destructive method has two main applications: (i) crayfish plague monitoring focused on individual crayfish in native, symptom-free populations, and (ii) as initial step in in vivo infection trials (see below). Current non-destructive sampling methods are based on the detection and quantification of A. astaci in environmental DNA samples (eDNA) isolated from natural waters (Strand et al., 2011, 2014; Wittwer et al., 2018). In comparison, our method is focused on A. astaci-detection in individual animals and thus is best suited for A. astaci-monitoring in native European crayfish populations (As. astacus, As. leptodactylus, Au. torrentium, Au. pallipes) that could harbor the low pathogenic A. astaci A strain without massive mortality events (Makkonen et al., 2012, 2013). In these cases, the low population densities may also decrease the possibilities for eDNA detection. To demonstrate the applicability of the swab-based method in such settings, we detected a low percentage of A. astacipositive As. astacus and Au. torrentium individuals in the Plitvice Lakes National Park. Previously, the A strain of A. astaci was detected in this As. astacus population using standard cuticle-based methods 8
(10/14 in 2012 and 2013), while the tested Au. torrentium individuals collected in the Plitvice lakes were pathogen-free (0/3 in 2012) (Maguire et al., 2016). Similarly, apparently healthy crayfish populations have often been shown to contain a variable percentage of A. astaci-positive animals (see for instance: Jussila et al., 2011b; Kokko et al., 2012; Svoboda et al., 2012; Filipová et al., 2013; Kušar et al., 2013). Further studies are needed to compare our method with eDNA-monitoring in such situations. Presumably, the probability of A. astaci DNA occurrence would be higher on the crayfish surface than in a large body of water, but this remains to be tested. Also, eDNA must be isolated from large volumes of water and can often be degraded (Strickler et al., 2015; Wittwer et al., 2018). Our non-destructive sampling method potentially offers certain advantages over the standard cuticle-based monitoring because it allows screening for pathogen presence using a much larger sample of protected crayfish individuals and it can be coupled with standard field work in population ecology or conservation. Moreover, our method could also be beneficial during the implementation of conservation programs, for example, to choose an A. astaci-free donor population of native crayfish for translocation to ark sites in order to preserve endangered native crayfish species biodiversity (Peay, 2009). Another possible application of our method is for in vivo infection trials where it can be used to confirm the A. astaci-negative status of animals prior to setting up infection experiments. Until present, animals with no clinical signs of the disease were used in infection trials, even though their A. astacinegative status could not be tested (Jussila et al., 2011a, 2013; Makkonen et al., 2012; Aydin et al., 2014). Some populations previously used in infection trials were shown to contain a significant number of A. astaci-positive animals (Jussila et al., 2011b, 2013). Because it is known that infected animals or carriers can be asymptomatic (Vrålstad et al., 2009) the erroneous assumption that animals were A. astaci-free could negatively affect the results of these experiments. In the future, our method should be a starting point for development of fast A. astaci detection methods that could be carried out directly in the field. Fast and simple on-site detection methods that do not require specialized equipment or skilled personnel are currently being developed in the more economically important freshwater aquaculture sector, and are most commonly antibody-based (e.g. Sheng et al., 2018) or utilize inexpensive loop-mediated isothermal amplification (LAMP) (e.g. Chen et al., 2018). If such monitoring protocols were developed for A. astaci, we could exclude infected individuals while disinfecting and returning the healthy animals, providing a breakthrough in crayfish plague management.
5. Conclusions Aphanomyces astaci is designated as one of the major threats for many crayfish species, with the outcome of the infection varying depending on the crayfish species, A. astaci strain and other factors (Jussila et al., 2013; Makkonen et al., 2013; Becking et al., 2015; Svoboda et al., 2017). All European crayfish species are highly susceptible to this pathogen, and crayfish plague is one of the important reasons that three of five European freshwater species, As. astacus, Au. torrentium, Au. pallipes, are listed as endangered and included in the International Union for Conservation of Nature Red List, Bern Convention and EU Council Directive 92/43/EEC (Holdich et al., 2009; Edsman et al., 2010). The 9
conservation of these species is a priority both on national and EU levels, and all effective management plans should include A. astaci monitoring. We propose that A. astaci monitoring in endangered native crayfish species should, in the future, be carried out using non-destructive methods, namely those based on the detection of A. astaci eDNA in water (Strand et al., 2011, 2014; Wittwer et al., 2018) and the method we describe here, which is focused on A. astaci detection on the surface of individual animals. Our method offers many advantages, including monitoring of a large number of samples without killing endangered crayfish. Because the results of our method are comparable with the cuticle-based A. astaci detection, they can be compared with the results of previous monitoring studies.
Acknowledgments The authors are thankful to Lidija Buzuk, Lea Ljubej, Nives Marčina, Ivan Radosavljević, Dunja Šikić and Marija Vuk for technical assistance during the study. Also, authors are grateful to Satu Viljamaa-Dirks for advice on A. astaci cultivation and sporulation.
Funding information The research has been supported by grants from The Environmental Protection and Energy Efficiency Fund of Republic of Croatia, Plitvice Lakes National Park and Croatian Science Foundation (grant UIP-2017-051720).
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Aphanomyces astaci, by random amplification of polymorphic DNA. Aquaculture 126, 1–9. Jussila, J., Kokko, H., Kortet, R., Makkonen, J., 2013. Aphanomyces astaci PsI-genotype isolates from different Finnish signal crayfish stocks show variation in their virulence but still kill fast. Knowl. Manag. Aquat. Ecosyst. 411, 10. Jussila, J., Makkonen, J., Kokko, H., 2011a. Peracetic acid (PAA) treatment is an effective disinfectant against crayfish plague (Aphanomyces astaci) spores in aquaculture. Aquaculture 320, 37–42. Jussila, J., Makkonen, J., Vainikka, A., Kortet, R., Kokko, H., 2011b. Latent crayfish plague (Aphanomyces astaci) infection in a robust wild noble crayfish (Astacus astacus) population. Aquaculture 321, 17– 20. Kokko, H., Koistinen, L., Harlioğlu, M.M., Makkonen, J., Aydın, H., Jussila, J., 2012. Recovering Turkish narrow clawed crayfish (Astacus leptodactylus) populations carry Aphanomyces astaci. Knowl. Manag. Aquat. Ecosyst. 12. Kozubíková, E., Viljamaa-Dirks, S., Heinikainen, S., Petrusek, A., 2011. Spiny-cheek crayfish Orconectes limosus carry a novel genotype of the crayfish plague pathogen Aphanomyces astaci. J. Invertebr. Pathol. 108, 214–216. Kušar, D., Vrezec, A., Ocepek, M., Jenčič, V., 2013. Aphanomyces astaci in wild crayfish populations in Slovenia: First report of persistent infection in a stone crayfish Austropotamobius torrentium population. Dis. Aquat. Organ. 103, 157–169. Lowe, S., Browne, M., Boudjelas, S., De Poorter, M., 2000. 100 of the world’s worst invasive alien species: a selection from the global invasive species database. Auckland. Lucić, A., Hudina, S., Faller, M., Cerjanec, D., 2012. A comparative study of the physiological condition of native and invasive crayfish in Croatian rivers. Biologia (Bratisl). 67, 172–179. Maguire, I., Gottstein-Matočec, S., 2004. The Distribution Pattern of Freshwater Crayfish in Croatia. Crustaceana 77, 25–47. Maguire, I., Jelić, M., Klobučar, G., Delpy, M., Delaunay, C., Grandjean, F., 2016. Prevalence of the pathogen Aphanomyces astaci in freshwater crayfish populations in Croatia. Dis. Aquat. Organ. 118, 45–53. Makkonen, J., Jussila, J., Kortet, R., Vainikka, A., Kokko, H., 2012. Differing virulence of Aphanomyces astaci isolates and elevated resistance of noble crayfish Astacus astacus against crayfish plague. Dis. Aquat. Organ. 102, 129–136. Makkonen, J., Jussila, J., Panteleit, J., Keller, N.S., Schrimpf, A., Theissinger, K., Kortet, R., Martín-Torrijos, L., Sandoval-Sierra, J.V., Diéguez-Uribeondo, J., Kokko, H., 2018. MtDNA allows the sensitive detection and haplotyping of the crayfish plague disease agent Aphanomyces astaci showing clues about its origin and migration. Parasitology 145, 1210–1218. Makkonen, J., Kokko, H., Vainikka, A., Kortet, R., Jussila, J., 2013. Dose-dependent mortality of the noble crayfish (Astacus astacus) to different strains of the crayfish plague (Aphanomyces astaci). J. Invertebr. Pathol. 115, 86–91. Minardi, D., Studholme, D.J., van der Giezen, M., Pretto, T., Oidtmann, B., 2018. New genotyping method for the causative agent of crayfish plague (Aphanomyces astaci) based on whole genome data. J. Invertebr. Pathol. 156, 6–13. Oidtmann, B., Geiger, S., Steinbauer, P., Culas, A., Hoffmann, R.W., 2006. Detection of Aphanomyces 12
astaci in North American crayfish by polymerase chain reaction. Dis. Aquat. Org. 72, 53–64. Oidtmann, B., Schaefers, N., Cerenius, L., Söderhäll, K., Hoffmann, R.W., 2004. Detection of genomic DNA of the crayfish plague fungus Aphanomyces astaci (Oomycete) in clinical samples by PCR. Vet. Microbiol. 100, 269–282. OIE, 2018. Crayfish Plague (Aphanomyces astaci). Manual of Diagnostic Tests for Aquatic Animals. Chapter 2.2.2., http://www.oie.int/en/international-standard-setting/aquatic-manual/accessonline/. Peay, S., 2009. Selection criteria for “ark sites” for white-clawed crayfish, in: Crayfish Conservation in the British Isles. Leeds, UK, Peak Ecology Ltd. Rezinciuc, S., Galindo, J., Montserrat, J., Diéguez-Uribeondo, J., 2014. AFLP-PCR and RAPD-PCR evidences of the transmission of the pathogen Aphanomyces astaci (Oomycetes) to wild populations of European crayfish from the invasive crayfish species, Procambarus clarkii. Fungal Biol. 118, 612– 620. Sheng, X., Tang, Q., Zhang, L., Tang, X., Xing, J., Zhan, W., 2018. Development and application of a rapid semiquantitative immunochromatographic test strip to detect white spot syndrome virus. Aquaculture 495, 773–779. Söderhäll, K., Cerenius, L., 1999. The crayfish plague fungus: history and recent advances. Freshw. Crayfish 12, 11–35. Strand, D.A., Holst-Jensen, A., Viljugrein, H., Edvardsen, B., Klaveness, D., Jussila, J., Vrålstad, T., 2011. Detection and quantification of the crayfish plague agent in natural waters: direct monitoring approach for aquatic environments. Dis. Aquat. Organ. 95, 9–17. Strand, D.A., Jussila, J., Johnsen, S.I., Viljamaa-Dirks, S., Edsman, L., Wiik-Nielsen, J., Viljugrein, H., Engdahl, F., Vrålstad, T., 2014. Detection of crayfish plague spores in large freshwater systems. J. Appl. Ecol. 51, 544–553. Strand, D.A., Jussila, J., Viljamaa-Dirks, S., Kokko, H., Makkonen, J., Holst-Jensen, A., Viljugrein, H., Vrålstad, T., 2012. Monitoring the spore dynamics of Aphanomyces astaci in the ambient water of latent carrier crayfish. Vet. Microbiol. 160, 99–107. Strickler, K.M., Fremier, A.K., Goldberg, C.S., 2015. Quantifying effects of UV-B, temperature, and pH on eDNA degradation in aquatic microcosms. Biol. Conserv. 183, 85–92. Svoboda, J., Kozubíková, E., Kozák, P., Kouba, A., Koca, S.B., Diler, Ö., Diler, I., Policar, T., Petrusek, A., 2012. PCR detection of the crayfish plague pathogen in narrow-clawed crayfish inhabiting Lake Eğirdir in Turkey 98, 255–259. Svoboda, J., Mrugała, A., Kozubíková-Balcarová, E., Petrusek, A., 2017. Hosts and transmission of the crayfish plague pathogen Aphanomyces astaci: a review. J. Fish Dis. 40, 127–140. Tuffs, S., Oidtmann, B., 2011. A comparative study of molecular diagnostic methods designed to detect the crayfish plague pathogen, Aphanomyces astaci. Vet. Microbiol. 153, 343–353. Unestam, T., 1965. Studies on the crayfish plague fungus Aphanomyces astaci I. Some factors affecting growth in vitro. Physiol. Plant. 18, 483–506. Unestam, T., Weiss, D.W., 1970. The host-parasite relationship between freshwater crayfish and the crayfish disease fungus Aphanomyces astaci: responses to infection by a susceptible and a resistant species. J. Gen. Microbiol. 60, 77–90. 13
Viljamaa-Dirks, S., Heinikainen, S., 2006. Improved detection of crayfish plague with a modified isolation method. Freshw. Crayfish 15, 376–382. Viljamaa-Dirks, S., Heinikainen, S., 2018. A tentative new species Aphanomyces fennicus sp. nov. interferes with molecular diagnostic methods for crayfish plague. J. Fish Dis. 42, 413–422. Viljamaa-Dirks, S., Heinikainen, S., Nieminen, M., Vennerström, P., Pelkonen, S., 2011. Persistent infection by crayfish plague Aphanomyces astaci in a noble crayfish population - A case report. Bull. Eur. Assoc. Fish Pathol. 31, 182–188. Viljamaa-Dirks, S., Heinikainen, S., Torssonen, H., Pursiainen, M., Mattila, J., Pelkonen, S., 2013. Distribution and epidemiology of genotypes of the crayfish plague agent Aphanomyces astaci from noble crayfish Astacus astacus in Finland. Dis. Aquat. Organ. 103, 199–208. Vrålstad, T., Knutsen, A.K., Tengs, T., Holst-Jensen, A., 2009. A quantitative TaqMan (R) MGB real-time polymerase chain reaction based assay for detection of the causative agent of crayfish plague Aphanomyces astaci. Vet. Microbiol. 137, 146–155. Westman, K., Pursiainen, M., Vilkman, R., 1978. A new folding trap model which prevents crayfish from escaping. Freshw. Crayfish 4, 235–242. Wittwer, C., Nowak, C., Strand, D.A., Vrålstad, T., Thines, M., Stoll, S., 2018. Comparison of two water sampling approaches for eDNA-based crayfish plague detection. Limnologica 70, 1–9. Zar, J.H., 1999. Biostatistical Analysis, 4th ed. Prentice Hall, London, UK.
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Figure captions
Figure 1. Sampling of crayfish epibiotic communities for A. astaci detection. (a) – (b) Scrubbing the crayfish with the sterile brush. (c) – (d) Pouring the obtained suspension of epibionts into the 50-mL Falcon tube. (e) Obtained pellet of epibiotic cells in 2-mL tube after centrifugation.
Figure 2. An example of standard PCR A. astaci detection results in pairs of swab and cuticle samples. S – DirectLoad™ 50 bp DNA StepLadder; + - positive control with pure A. astaci genomic DNA used as a template; - - negative control.
Figure 3. Comparison of detected pathogen levels for cuticle and swab sample pairs.
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Highlights
We have developed a novel non-destructive Aphanomyces astaci-detection protocol. The method detects A. astaci DNA in cuticle-associated biofilm of infected animals. The new method is applicable in monitoring of A. astaci in endangered crayfish species.
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Table 1. Sampling localities, coordinate system (HTRS 96/TM) and crayfish species present in each location. x
y
Crayfish species
Previous data on the disease status of the location (year)
459441
5082277
AT
n.t.
429177
4970612
AA
n.t.
Burgeti (National Park Plitvice)
430094
4971437
AA
Positive (2012, 2013)*
Rječica (National Park Plitvice)
430040
4970656
AT
n.t.
Prijeboj (National Park Plitvice)
433656
4965730
AT
n.t.
Prštavac (National Park Plitvice)
429195
4970868
AA
n.t.
Sartuk (National Park Plitvice)
424969
4977264
AT
n.t.
Belajske Poljice (River Korana)
426295
5034353
AL, PL
n.t.
Karlovac business zone (River Korana)
427002
5035063
AL, PL
n.t.
Ladvenjak (River Korana)
430444
5030260
AL, PL
AL positive (2012, 2013), PL negative (2012)*
Lučica (River Korana)
420953
5023112
PL
n.t.
Sučevići (River Korana)
423012
5020504
AL, PL
n.t.
Šćulac (River Korana)
423355
5026240
AL, PL
AL negative (2012)*, PL n.t.
Motičnjak (Varaždin)
491159
5129909
AA
n.t.
3rd Maksimir Lake (Zagreb)
462659
5076222
AL
n.t.
Sampling locality Bliznec (Nature Park Medvednica) Bakinovac (National Park Plitvice)
AL – Astacus (Pontastacus) leptodactylus; AA - Astacus astacus; AT - Austropotamobius torrentium; PL – Pacifastacus leniusculus; n.t. – not tested * Maguire et al., 2016
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Table 2. Sampled crayfish from each locality. Sampling Sex Sampling date locality *AL1 Maksimir m April 2017 *AL2 Maksimir m April 2017 *AL3 Maksimir m April 2017 *AL4 Maksimir m April 2017 *AL5 Maksimir m April 2017 *AL6 Maksimir m April 2017 *AT2 Bliznec m September 2017 *AT3 Bliznec f September 2017 AL7 Maksimir m April 2017 AT1 Prijeboj m March 2017 AA1 Motičnjak m May 2017 AA2 Motičnjak m May 2017 AA3 Burgeti m April 2017 PL1 Ladvenjak m September 2018 * Animals infected in the laboratory AL - Astacus (Pontastacus) leptodactylus; AA - Astacus astacus; AT - Austropotamobius torrentium; PL – Pacifastacus leniusculus Code
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Table 3. Comparison of non-destructive detection of A. astaci from swab samples (S) and traditional detection from the cuticle samples (C). Agent level A0 = 0 PFU; A1 = <5 PFU; A2 = between 5 and 50 PFU; A3 = between 50 and 103 PFU; A4 = between 103 and 104 PFU; A5 = between 104 and 105 PFU (Vrålstad et al., 2009).
Code *AL1 S *AL1 C *AL2 S *AL2 C *AL3 S *AL3 C *AL4 S *AL4 C *AL5 S *AL5 C *AL6 S *AL6 C *AT2 S *AT2 C *AT3 S *AT3 C AL7 S AL7 C AT1 S AT1 C AA1 S AA1 C AA2 S AA2 C AA3 S AA3 C PL1 S PL1 C
Days till death 17 10 28 7 7 7 15 6 / / / / / / / / / / / /
Swab Cuticle Swab Cuticle Swab Cuticle Swab Cuticle Swab Cuticle Swab Cuticle Swab Cuticle Swab Cuticle Swab Cuticle Swab Cuticle Swab
Standard PCR detection (Oidtmann et al., 2006) positive positive positive positive positive positive positive positive positive positive positive positive positive positive positive positive negative negative negative negative negative
Cuticle
negative
n. d.
A0
Swab Cuticle Swab Cuticle Swab Cuticle
negative negative negative negative negative negative
0.39 n. d. 1.7 n. d. 0.54 1.3
A1 A0 A1 A0 A1 A1
Sample type
* Animals infected in the laboratory
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qPCR detection (Vrålstad et al., 2009) Average PFU per reaction
Agent level
115.6 83.0 54.4 11.0 33.9 90.5 4.3 12.5 88.9 38.6 2.8 762.0 202.0 191.1 4.8 40 044.3 n. d. n. d. n. d. n. d. n. d.
A3 A3 A3 A2 A2 A3 A1 A2 A3 A2 A1 A3 A3 A3 A1 A5 A0 A0 A0 A0 A0
AL - Astacus leptodactylus; AA - Astacus astacus; AT - Austropotamobius torrentium; PL – Pacifastacus leniusculus; n. d. – not detected; PFU – PCR-forming unit
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S0022-2011(19)30015-1 https://doi.org/10.1016/j.jip.2019.107274 YJIPA 107274
To appear in:
Journal of Invertebrate Pathology
Received Date: Revised Date: Accepted Date:
24 January 2019 27 October 2019 30 October 2019
Please cite this article as: Pavić, D., Čanković, M., Sviličić Petrić, I., Makkonen, J., Hudina, S., Maguire, I., Vladušić, T., Šver, L., Hraš ćan, R., Orlić, K., Dragičević, P., Bielen, A., Non-destructive method for detecting Aphanomyces astaci, the causative agent of crayfish plague, on the individual level, Journal of Invertebrate Pathology (2019), doi: https://doi.org/10.1016/j.jip.2019.107274
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Non-destructive method for detecting Aphanomyces astaci, the causative agent of crayfish plague, on the individual level Dora Pavić1, Milan Čanković2, Ines Sviličić Petrić2, Jenny Makkonen3, Sandra Hudina4, Ivana Maguire4, Tomislav Vladušić1, Lidija Šver1, Reno Hrašćan1, Karla Orlić4, Paula Dragičević4, Ana Bielen1,* 1
University of Zagreb, Faculty of Food Technology and Biotechnology, Department for Biochemical Engineering, Pierottijeva 6, 10000 Zagreb, Croatia 2
Ruđer Bošković Institute, Division for Marine and Environmental Research, Bijenička cesta 54, 10000 Zagreb, Croatia 3
University of Eastern Finland, Department of Environmental and Biological Sciences, P.O. Box 1627, FIN70211 Kuopio, Finland 4
University of Zagreb, Faculty of Science, Department of Biology, Rooseveltov trg 6, 10000 Zagreb, Croatia *
Corresponding author: [email protected]
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Abstract The pathogenic oomycete Aphanomyces astaci, transmitted mainly by invasive North American crayfish, causes the crayfish plague, a disease mostly lethal for native European crayfish. Due to its decimating effects on native crayfish populations in the last century, A. astaci has been listed among the 100 worst invasive species. Importantly, detecting the pathogen in endangered native crayfish populations before a disease outbreak would provide a starting point in the development of effective control measures. However, current A. astaci-detection protocols either rely on degradation-prone eDNA isolated from large volumes of water or, if focused on individual animals, include killing the crayfish. We developed a nondestructive method that detects A. astaci DNA in the microbial biofilm associated with the cuticle of individual crayfish, without the need for destructive sampling. Efficiency of the new method was confirmed by PCR and qPCR and the obtained results were congruent with the traditional destructive sampling method. Additionally, we demonstrated the applicability of the method for A. astaci monitoring in natural populations. We propose that the new method should be used in future monitoring of A. astaci presence in endangered European native crayfish individuals as an alternative to eDNA-based monitoring.
Keywords: Aphanomyces astaci detection, crayfish cuticle-associated biofilm, pathogenic oomycete, endangered native crayfish
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1. Introduction Pathogenic oomycete Aphanomyces astaci Schikora, 1906, is one of the major reasons for the decline of native European crayfish populations. Due to its detrimental effects and fast spread in European waterways, it was classified amongst the world’s 100 worst invasive alien species (Alderman, 1996; Lowe et al., 2000; OIE, 2018). It is transmitted primarily by North American invasive crayfish species that act as carriers in Europe and are mostly resistant to infection (Holdich et al., 2009). In contrast, native crayfish individuals most often develop highly contagious and lethal disease if in contact with A. astaci zoospores (Makkonen et al., 2013; Becking et al., 2015). Five known A. astaci genotypic groups differ in origin and virulence (Huang et al., 1994; Diéguez-Uribeondo et al., 1995; Kozubíková et al., 2011; Makkonen et al., 2012, 2013; Viljamaa-Dirks et al., 2013). Group A (As) is believed to be related to the first introduction of A. astaci into Europe in the 19th century and the primary carrier is unknown (Huang et al., 1994; ViljamaaDirks et al., 2013). The carriers of other A. astaci groups are North American invasive crayfish species: B (PsI) and C (PsII) originate from Pacifastacus leniusculus (Huang et al., 1994), D (Pc) from Procambarus clarkii (Diéguez-Uribeondo et al., 1995; Rezinciuc et al., 2014), and E (Or) from Faxonius limosus (Kozubíková et al., 2011). The genotypic groups vary in virulence, with genotypes B and E reported to be much more virulent than genotype A (Makkonen et al., 2012, 2013; Viljamaa-Dirks et al., 2013; Becking et al., 2015). Indeed, genotype A is mostly detected in native European crayfish populations (Astacus astacus and Astacus leptodactylus) that are chronically infected but vital, without mass mortalities (Jussila et al., 2011b; Viljamaa-Dirks et al., 2011; Kokko et al., 2012; Svoboda et al., 2012; Kušar et al., 2013). Continuous monitoring of the pathogen’s distribution is necessary in order to have a clear understanding about its spread through European waterways and to identify populations that are potential pathogen carriers (OIE, 2018). Morphological observations and microscopy of infected tissue can serve as a first indication of A. astaci presence, but cannot differentiate between A. astaci and related oomycetes (Cerenius et al., 1988; Viljamaa-Dirks and Heinikainen, 2006). Isolation and cultivation of A. astaci, followed by morphological and molecular identification, was required to confirm its presence in suspect animals (Huang et al., 1994; OIE, 2018), however, successful isolation of the pathogen was time consuming, frequently without satisfying results. Methods that rely on PCR-based detection of A. astaci DNA in the crayfish tissue were developed, making the diagnostic procedure faster and more sensitive. The most common assays use the internal transcribed region (ITS) as a marker region to identify A. astaci by standard (Oidtmann et al., 2006) or quantitative PCR (qPCR) (Vrålstad et al., 2009). These methods have been used extensively in crayfish plague monitoring (Cammà et al., 2010; Jussila et al., 2011b; Kokko et al., 2012; Svoboda et al., 2012; Filipová et al., 2013; Kušar et al., 2013; Maguire et al., 2016). Over the years, several other methods were developed, including qPCR-based detection of A. astaci chitinase genes (Hochwimmer et al., 2009), microsatellite markers useful to determine different A. astaci genotypes (Grandjean et al., 2014), mitochondrial DNA analysis that allows the detection and characterization of A. astaci haplotypes (Makkonen et al., 2018) and PCR-based A. astaci genotyping (Minardi et al., 2018). All these protocols use DNA samples as a starting point, isolated either directly from water (eDNA, Strand et al., 2011, 2012, 2014; Wittwer et al., 2018) or from a piece of crayfish cuticle after killing the animal (Oidtmann et al., 2004, 2006; Filipová et al., 2013).
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We aimed to eliminate the killing step (i.e. destructive sampling) and developed a non-destructive A. astaci detection method which is more suitable for crayfish plague monitoring in endangered native populations. After the initial chemotaxis of A. astaci zoospores toward the host cuticle (Cerenius and Söderhäll, 1984), zoospores encyst and subsequently germinate into hyphae that penetrate into the crayfish tissues through the cuticle (Unestam and Weiss, 1970; Cerenius et al., 2003; Cammà et al., 2010). Thus, we assumed that A. astaci DNA, originating from zoospores, cysts or hyphae, can be detected in the mixed epibiotic microbial communities scrubbed from the surface of live crayfish.
2. Material and methods 2.1. Sampling We collected adult freshwater crayfish (total length > 6 cm) in LiNi traps (Westman et al., 1978) or by hand at different locations in Croatia (Table 1). The disease status was previously tested for 3 of 15 locations. Some animals (Table 2) were collected in order to compare the results of A. astaci detection by newly developed swab-based method with standard A. astaci cuticle-based detection. In such cases, two types of samples were taken from each animal: (i) swab of the cuticle containing the epibiotic community (for non-destructive A. astaci detection, Fig. 1); and (ii) a sample of abdominal cuticle, collected after killing the animal (for standard destructive detection; Oidtmann et al., 2006). These two sample types were collected from animals brought directly from the field (natural population samples) and from animals infected with A. astaci zoospores in the laboratory (experimental samples) (Table 2). Additionally, swab samples of a larger number of animals were taken to demonstrate the applicability of the method for A. astaci monitoring in natural populations. Forty-six Austropotamobius torrentium individuals and 114 As. astacus individuals were sampled in the Plitvice Lakes National Park during 2017 and 2018, and 110 P. leniusculus individuals were sampled from the Korana River population during 2018.
2.2. Inoculation of animals with A. astaci zoospores Animals were held separately in 18 x 12 x 10 cm aquaria filled with approximately 1 L of water, and equipped with air pumps. The water temperature was ± 18 °C. Each crayfish was given one bottom feeding fish tablet twice a week and the water was changed the day after the feeding. Highly virulent Aphanomyces astaci strain B, PsI-genotype (UEF-T16B) isolated from P. leniusculus in Lake Tahoe, USA, was used. This strain was chosen because we inoculated (mostly) As. leptodactylus, a highly robust (although native) crayfish species. It has successfully spread from eastern to western Croatia over the past 50 years (Maguire and Gottstein-Matočec, 2004) and has sometimes been considered a cryptic invader due to its higher adaptive plasticity compared to other native species (Lucić et al., 2012). There is no literature data on A. astaci infection trials that used A. leptodactylus as a host, but we assumed that such advantageous life-history traits should make A. leptodactylus more resistant to A. astaci infection compared to other native species. Indeed, vital A. leptodactylus populations, including some percentage of B strain positive individuals, were found co-occurring the same locations as invasive B strain carrier P. leniusculus (Maguire et al., 2016). After a 1-month acclimatization period, animals were infected with 500 zoospores / mL of water, following the procedure described by Makkonen et al. (2012). We used a 4
moderate zoospore concentration of 500 zoospores / mL to infect the animals in order to mimic natural situation in the chronically infected population. Swabs and cuticle samples were taken immediately after death of the crayfish.
2.3. Non-destructive sampling protocol Non-destructive sampling included thorough scrubbing of crayfish surface. Each crayfish was placed in a Petri dish filled with 0.1 % NaCl, 0.15 M Tween 20 solution and thoroughly scrubbed with a sterile brush wetted with the same solution (Fig. 1a, b). To avoid cross contamination, sterile instruments/consumables were used for each individual. The obtained suspension was returned to a 50mL Falcon tube (Fig. 1c, d). During the initial development of the protocol, the whole volume of the suspension (30 – 50 mL per crayfish) was centrifuged at 4,000 x g for 15 min at 4 °C. However, this step was later omitted because we obtained the same results using the modified protocol. Namely, the suspension was left to settle for 30 min at 4 °C, and then approximately 2 mL of the suspension was removed from the bottom of the tube by sterile Pasteur pipette, transferred to a new tube and centrifuged at 10,000 x g for 15 min at 4 °C (centrifuge Centric 200R, Domel, Slovenia). The supernatant was removed and the pellet of epibiotic cells was frozen at -20 °C until DNA isolation (Fig. 1e).
2.4. DNA extraction For DNA extraction from swabs, we tested several extraction kits: NucleoSpin® Microbial DNA kit (Macherey Nagel, Germany), DNeasy PowerSoil Kit (QIAGEN, Germany), QIAamp DNA Investigator Kit (QIAGEN, Germany) and ZymoBIOMICS DNA Miniprep Kit (Zymo Research, USA) and found no significant differences in yield or purity of the DNA (data not shown). Finally, we chose NucleoSpin® Microbial DNA kit (Macherey Nagel, Germany), a kit specifically designed for DNA isolation from microbial communities, with mechanical disruption of cell wall structures of microbial cells as the first step. Prior to DNA isolation from swab and cuticle samples, we confirmed the efficiency of this extraction protocol with samples of pure A. astaci mycelium, using approx. 30 mg of hyphae wet weight previously grown in PG-1 liquid medium (Unestam, 1965) as a starting biomass (data not shown). For the protocol, the samples were lysed by agitation (medium strength, 20 min) on a Vortex Mixer (Corning, USA). We used Macherey Nagel Bead Tubes Type B (provided in the kit), although Bead Tubes Type C from the same kit were also efficient for lysis. DNA elution from the column was performed using the initial 100-μL eluate for a second elution in order to increase yield and concentration of the final sample. Quantity and quality of DNA samples were assessed by agarose gel electrophoresis and QuantiFluor ONE dsDNA Dye on a Quantus Fluorometer (Promega, Germany).
2.5. PCR and qPCR A. astaci DNA in the extracted total DNA was detected following protocols described previously (Oidtmann et al., 2006; Vrålstad et al., 2009; Strand et al., 2014). Primers 42 (5’ GCT TGT GCT GAG GAT GTT CT 3’) and 640 (5’ CTA TCC GAC TCC GCA TTC TG 3’) were used to specifically amplify parts of the ITS region of A. astaci by standard PCR (Oidtmann et al., 2006). It is noteworthy that, during development of the method, we sometimes obtained non-specific PCR-products, especially when using DNA isolated from 5
swab samples as a template. This can be explained by higher diversity in the swab samples (DNA from the total epibiotic community), than from the cuticle samples (mostly crayfish DNA). We therefore increased the annealing temperature from 59 °C (Oidtmann et al., 2006) to 61 °C to increase the specificity (data not shown). In addition, because a recent study showed that ITS‐based molecular assays for A. astaci can yield unspecific amplifications (Viljamaa-Dirks and Heinikainen, 2018), we sequenced all the amplicons (i.e. positives) that yielded a sufficiently strong PCR signal (Sanger sequencing, Microsynth, Austria). The resulting sequences were compared with NCBI database using blastn (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch) and their identity to A. astaci ITS region was confirmed. Detection and relative quantification of A. astaci DNA was done by qPCR using primers AphAstITS39F (5′-AAGGCTTGTGCTGGGATGTT-3′), AphAstITS-97R (5′-CTTCTTGCGAAACCTTCTGCTA-3′) and MGB probe AphAstITS-60T (5′-6-FAM-TTCGGGACGACCC-MGBNFQ-3′), as described earlier (Vrålstad et al., 2009). We used a modified annealing step to increase the assay specificity; instead of 58 °C / 60 s (Vrålstad et al., 2009), we used 62 °C / 30 s (Strand et al., 2014). Quantitative PCR results were further classified into semi-quantitative categories of pathogen load based on their PCR forming units (PFU) values, ranging from A0 (no traces of A. astaci DNA) to A5 (high amount of A. astaci DNA in the sample, estimated PFU 104 and 105) (Vrålstad et al., 2009).
2.6. Statistical analyses The data we obtained were tested to analyze whether they met the assumptions for parametric tests (i.e. normality/homoscedasticity; Zar, 1999. Since both raw and transformed data (logarithmic transformation of continuous data, square-root for discrete data) violated these assumptions, nonparametric analogues were used instead (Mann-Whitney U test, Spearman Rank; Zar, 1999). Furthermore, sensitivity testing of both methods (cuticle and swab samples) was performed according to procedures described in detail in Wittwer et al. (2018). The statistical significance was set at p = 0.05.
3. Results 3.1. Comparison of A. astaci detection by the newly developed swab-based method with standard cuticlebased detection We compared the results of detection of A. astaci from non-destructive swab samples with traditional detection from cuticle samples (Table 3). Results of the standard PCR allowed us to differentiate between A. astaci-positive (amplicon of the expected product size of 569 bp) and negative individuals (no amplification). We obtained congruent results using both swab (i.e. non-destructive method) and cuticle samples (i.e. traditional destructive method) for all tested animals (Table 3, Fig. 2). The amplicons were confirmed as A. astaci by sequencing (data not shown). Subsequent qPCR assays confirmed the results of the standard PCR. Only four samples, AA2 S, AA3 S, PL1 S and PL1 C, were negative as determined by conventional PCR, while qPCR detected low agent level (A1), probably a consequence of higher sensitivity of qPCR (Tuffs and Oidtmann, 2011; Kušar et al., 6
2013). Differences in average PFU per reaction between cuticle and swab samples were not statistically significant (Mann–Whitney U test, p > 0.05). Furthermore, swab and cuticle DNA samples originating from the same individual contained comparable A. astaci agent levels with no statistically significant differences (Mann–Whitney U test, p > 0.05). However, our results indicated that the cuticle method was slightly more sensitive in A. astaci detection than the newly developed swab method (Fig. 3). When analyzing individual sample pairs, the same agent level was measured for both sample types in 6/14 samples, whilst in a few cases the agent level was (slightly) higher for swab sample (4/14) or for cuticle sample (4/14) (Table 3). A discrepancy in qPCR results was obtained for one infected crayfish (AT3), where agent level A5 was detected in cuticle sample, while the swab sample detected the agent at level A1. This result could be a consequence of low DNA concentration in the swab sample (0.432 ng/µL) compared to the cuticle sample (117.5 ng/µL) for this pair. Although the average DNA concentration in swab samples was lower than in the cuticle samples (15.8 ng/µL vs. 74.8 ng/µL, respectively), the difference in the concentration was the highest for the individual AT3, leading to the observed discrepancy in qPCR results. 3.2. Applicability of the newly developed method for A. astaci monitoring in natural populations We demonstrated the applicability of our newly developed swab method for Aphanomyces astaci monitoring in natural populations. We collected a large number of swab samples (270 in total) from native crayfish (As. astacus, Au. torrentium) and invasive crayfish (P. leniusculus). We chose locations that were previously shown to contain A. astaci (Maguire et al., 2016) and with current populations that were viable and without signs of gross mortality. Next, we performed standard PCR assays (Oidtmann et al., 2006) for all collected swab DNA samples to detect A. astaci on individual animals. Of 114 samples taken from As. astacus population in the Plitvice Lakes National Park, 16 were A. astaci-positive (14 %, 2017-2018). In Au. torrentium population in Plitvice Lakes National Park 1 out of 46 tested individuals was A. astaci-positive (2 %, 2017-2018), and 6 % of P. leniusculus population in the Korana River was A. astaci-positive (7/110, 2018). When the PCR signal was strong enough for sequencing, the amplicons were confirmed as A. astaci, 2 out of 17 positives from the Plitvice Lakes National Park and 3 out of 7 positives from the Korana River.
4. Discussion We developed a new method for A. astaci detection that enables pathogen monitoring without destructive sampling of the crayfish hosts. To test the method, we collected samples consisting of infected crayfish as well as uninfected animals and isolated in parallel the total DNA from surface swabs and from cuticle samples. Our results demonstrated that the non-destructive method we developed is comparable with the standard destructive methods (Oidtmann et al., 2006; Vrålstad et al., 2009). During the A. astaci infection process, zoospores that contact the crayfish cuticle encyst and then germinate into hyphae that penetrate through the exoskeleton. The hyphae subsequently spread into deeper tissues and organs, leading to development of lethal disease (Söderhäll and Cerenius, 1999). Although it is generally presumed that the majority of A. astaci biomass in infected animals is located inside the body (Unestam and Weiss, 1970; Cerenius and Söderhäll, 1984), we showed that the pathogen is also present on the cuticle of infected or carrier crayfish and can be collected by scrubbing the surface 7
of the animal. We hypothesize that the A. astaci DNA we detected in the swab samples originates from zoospores, cysts or, in the terminal stages of the disease, possibly hyphae (Unestam and Weiss, 1970; Cerenius et al., 2003; Cammà et al., 2010), but additional studies are needed to unravel this issue. The presence of A. astaci DNA in the swab samples enabled us to develop a non-destructive method for A. astaci detection in crayfish individuals and to circumvent destructive sampling used previously (Oidtmann et al., 2004, 2006; Vrålstad et al., 2009; Strand et al., 2011; Grandjean et al., 2014; Maguire et al., 2016). Our results showed no significant overall difference between swab and cuticle sample pairs in the A. astaci load, although these two sample types were quite different, both in composition and in average DNA concentration. Swab samples contained DNA from mixed epibiotic microbial communities (e.g. bacteria, fungi, oomycetes) originating from the surface of the animal. On the other hand, cuticle samples were small and presumably contained mostly DNA of the crayfish, as well as A. astaci DNA if the animal is infected. The fact that these two sample types showed comparable A. astaci load can be explained by the following: the density of A. astaci biomass is much higher in the tissues of the infected animals (cuticle sample) than on the cuticle surface (swab sample) (Unestam and Weiss, 1970); however, the large scrubbing surface for the swab samples compared to the small segment of cuticle sample probably compensates for this difference. Although the agent level detected was the same for most sample pairs, we observed slightly higher, albeit statistically insignificant, overall sensitivity of the cuticle-based procedure (i.e. slightly higher pathogen levels based on qPCR results), probably due to the overall higher DNA yield obtained for cuticle samples. In spite of this, the absolute status of individual animals was the same, either A. astaci-positive or A. astaci-negative, and, importantly, with no false negatives in swab method. This confirms the applicability of the newly developed method for monitoring, where discriminating between presence and absence of the pathogen is of pivotal importance for any management activities. For some sample pairs, the agent level detected was slightly higher for either the swab or the cuticle sample. We hypothesize that carrier crayfish (perhaps with a small amount of hyphae on the surface) or recently infected crayfish (with attached zoospores) have a higher probability of detection of A. astaci DNA in the swab. In contrast, detection in cuticle samples is more probable in symptomatic animals that have densely packed A. astaci hyphae inside the body. However, testing this assumption was out of the scope of this study and in vivo infection trials coupled with microscopy are needed to confirm it. Our novel non-destructive method has two main applications: (i) crayfish plague monitoring focused on individual crayfish in native, symptom-free populations, and (ii) as initial step in in vivo infection trials (see below). Current non-destructive sampling methods are based on the detection and quantification of A. astaci in environmental DNA samples (eDNA) isolated from natural waters (Strand et al., 2011, 2014; Wittwer et al., 2018). In comparison, our method is focused on A. astaci-detection in individual animals and thus is best suited for A. astaci-monitoring in native European crayfish populations (As. astacus, As. leptodactylus, Au. torrentium, Au. pallipes) that could harbor the low pathogenic A. astaci A strain without massive mortality events (Makkonen et al., 2012, 2013). In these cases, the low population densities may also decrease the possibilities for eDNA detection. To demonstrate the applicability of the swab-based method in such settings, we detected a low percentage of A. astacipositive As. astacus and Au. torrentium individuals in the Plitvice Lakes National Park. Previously, the A strain of A. astaci was detected in this As. astacus population using standard cuticle-based methods 8
(10/14 in 2012 and 2013), while the tested Au. torrentium individuals collected in the Plitvice lakes were pathogen-free (0/3 in 2012) (Maguire et al., 2016). Similarly, apparently healthy crayfish populations have often been shown to contain a variable percentage of A. astaci-positive animals (see for instance: Jussila et al., 2011b; Kokko et al., 2012; Svoboda et al., 2012; Filipová et al., 2013; Kušar et al., 2013). Further studies are needed to compare our method with eDNA-monitoring in such situations. Presumably, the probability of A. astaci DNA occurrence would be higher on the crayfish surface than in a large body of water, but this remains to be tested. Also, eDNA must be isolated from large volumes of water and can often be degraded (Strickler et al., 2015; Wittwer et al., 2018). Our non-destructive sampling method potentially offers certain advantages over the standard cuticle-based monitoring because it allows screening for pathogen presence using a much larger sample of protected crayfish individuals and it can be coupled with standard field work in population ecology or conservation. Moreover, our method could also be beneficial during the implementation of conservation programs, for example, to choose an A. astaci-free donor population of native crayfish for translocation to ark sites in order to preserve endangered native crayfish species biodiversity (Peay, 2009). Another possible application of our method is for in vivo infection trials where it can be used to confirm the A. astaci-negative status of animals prior to setting up infection experiments. Until present, animals with no clinical signs of the disease were used in infection trials, even though their A. astacinegative status could not be tested (Jussila et al., 2011a, 2013; Makkonen et al., 2012; Aydin et al., 2014). Some populations previously used in infection trials were shown to contain a significant number of A. astaci-positive animals (Jussila et al., 2011b, 2013). Because it is known that infected animals or carriers can be asymptomatic (Vrålstad et al., 2009) the erroneous assumption that animals were A. astaci-free could negatively affect the results of these experiments. In the future, our method should be a starting point for development of fast A. astaci detection methods that could be carried out directly in the field. Fast and simple on-site detection methods that do not require specialized equipment or skilled personnel are currently being developed in the more economically important freshwater aquaculture sector, and are most commonly antibody-based (e.g. Sheng et al., 2018) or utilize inexpensive loop-mediated isothermal amplification (LAMP) (e.g. Chen et al., 2018). If such monitoring protocols were developed for A. astaci, we could exclude infected individuals while disinfecting and returning the healthy animals, providing a breakthrough in crayfish plague management.
5. Conclusions Aphanomyces astaci is designated as one of the major threats for many crayfish species, with the outcome of the infection varying depending on the crayfish species, A. astaci strain and other factors (Jussila et al., 2013; Makkonen et al., 2013; Becking et al., 2015; Svoboda et al., 2017). All European crayfish species are highly susceptible to this pathogen, and crayfish plague is one of the important reasons that three of five European freshwater species, As. astacus, Au. torrentium, Au. pallipes, are listed as endangered and included in the International Union for Conservation of Nature Red List, Bern Convention and EU Council Directive 92/43/EEC (Holdich et al., 2009; Edsman et al., 2010). The 9
conservation of these species is a priority both on national and EU levels, and all effective management plans should include A. astaci monitoring. We propose that A. astaci monitoring in endangered native crayfish species should, in the future, be carried out using non-destructive methods, namely those based on the detection of A. astaci eDNA in water (Strand et al., 2011, 2014; Wittwer et al., 2018) and the method we describe here, which is focused on A. astaci detection on the surface of individual animals. Our method offers many advantages, including monitoring of a large number of samples without killing endangered crayfish. Because the results of our method are comparable with the cuticle-based A. astaci detection, they can be compared with the results of previous monitoring studies.
Acknowledgments The authors are thankful to Lidija Buzuk, Lea Ljubej, Nives Marčina, Ivan Radosavljević, Dunja Šikić and Marija Vuk for technical assistance during the study. Also, authors are grateful to Satu Viljamaa-Dirks for advice on A. astaci cultivation and sporulation.
Funding information The research has been supported by grants from The Environmental Protection and Energy Efficiency Fund of Republic of Croatia, Plitvice Lakes National Park and Croatian Science Foundation (grant UIP-2017-051720).
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astaci in North American crayfish by polymerase chain reaction. Dis. Aquat. Org. 72, 53–64. Oidtmann, B., Schaefers, N., Cerenius, L., Söderhäll, K., Hoffmann, R.W., 2004. Detection of genomic DNA of the crayfish plague fungus Aphanomyces astaci (Oomycete) in clinical samples by PCR. Vet. Microbiol. 100, 269–282. OIE, 2018. Crayfish Plague (Aphanomyces astaci). Manual of Diagnostic Tests for Aquatic Animals. Chapter 2.2.2., http://www.oie.int/en/international-standard-setting/aquatic-manual/accessonline/. Peay, S., 2009. Selection criteria for “ark sites” for white-clawed crayfish, in: Crayfish Conservation in the British Isles. Leeds, UK, Peak Ecology Ltd. Rezinciuc, S., Galindo, J., Montserrat, J., Diéguez-Uribeondo, J., 2014. AFLP-PCR and RAPD-PCR evidences of the transmission of the pathogen Aphanomyces astaci (Oomycetes) to wild populations of European crayfish from the invasive crayfish species, Procambarus clarkii. Fungal Biol. 118, 612– 620. Sheng, X., Tang, Q., Zhang, L., Tang, X., Xing, J., Zhan, W., 2018. Development and application of a rapid semiquantitative immunochromatographic test strip to detect white spot syndrome virus. Aquaculture 495, 773–779. Söderhäll, K., Cerenius, L., 1999. The crayfish plague fungus: history and recent advances. Freshw. Crayfish 12, 11–35. Strand, D.A., Holst-Jensen, A., Viljugrein, H., Edvardsen, B., Klaveness, D., Jussila, J., Vrålstad, T., 2011. Detection and quantification of the crayfish plague agent in natural waters: direct monitoring approach for aquatic environments. Dis. Aquat. Organ. 95, 9–17. Strand, D.A., Jussila, J., Johnsen, S.I., Viljamaa-Dirks, S., Edsman, L., Wiik-Nielsen, J., Viljugrein, H., Engdahl, F., Vrålstad, T., 2014. Detection of crayfish plague spores in large freshwater systems. J. Appl. Ecol. 51, 544–553. Strand, D.A., Jussila, J., Viljamaa-Dirks, S., Kokko, H., Makkonen, J., Holst-Jensen, A., Viljugrein, H., Vrålstad, T., 2012. Monitoring the spore dynamics of Aphanomyces astaci in the ambient water of latent carrier crayfish. Vet. Microbiol. 160, 99–107. Strickler, K.M., Fremier, A.K., Goldberg, C.S., 2015. Quantifying effects of UV-B, temperature, and pH on eDNA degradation in aquatic microcosms. Biol. Conserv. 183, 85–92. Svoboda, J., Kozubíková, E., Kozák, P., Kouba, A., Koca, S.B., Diler, Ö., Diler, I., Policar, T., Petrusek, A., 2012. PCR detection of the crayfish plague pathogen in narrow-clawed crayfish inhabiting Lake Eğirdir in Turkey 98, 255–259. Svoboda, J., Mrugała, A., Kozubíková-Balcarová, E., Petrusek, A., 2017. Hosts and transmission of the crayfish plague pathogen Aphanomyces astaci: a review. J. Fish Dis. 40, 127–140. Tuffs, S., Oidtmann, B., 2011. A comparative study of molecular diagnostic methods designed to detect the crayfish plague pathogen, Aphanomyces astaci. Vet. Microbiol. 153, 343–353. Unestam, T., 1965. Studies on the crayfish plague fungus Aphanomyces astaci I. Some factors affecting growth in vitro. Physiol. Plant. 18, 483–506. Unestam, T., Weiss, D.W., 1970. The host-parasite relationship between freshwater crayfish and the crayfish disease fungus Aphanomyces astaci: responses to infection by a susceptible and a resistant species. J. Gen. Microbiol. 60, 77–90. 13
Viljamaa-Dirks, S., Heinikainen, S., 2006. Improved detection of crayfish plague with a modified isolation method. Freshw. Crayfish 15, 376–382. Viljamaa-Dirks, S., Heinikainen, S., 2018. A tentative new species Aphanomyces fennicus sp. nov. interferes with molecular diagnostic methods for crayfish plague. J. Fish Dis. 42, 413–422. Viljamaa-Dirks, S., Heinikainen, S., Nieminen, M., Vennerström, P., Pelkonen, S., 2011. Persistent infection by crayfish plague Aphanomyces astaci in a noble crayfish population - A case report. Bull. Eur. Assoc. Fish Pathol. 31, 182–188. Viljamaa-Dirks, S., Heinikainen, S., Torssonen, H., Pursiainen, M., Mattila, J., Pelkonen, S., 2013. Distribution and epidemiology of genotypes of the crayfish plague agent Aphanomyces astaci from noble crayfish Astacus astacus in Finland. Dis. Aquat. Organ. 103, 199–208. Vrålstad, T., Knutsen, A.K., Tengs, T., Holst-Jensen, A., 2009. A quantitative TaqMan (R) MGB real-time polymerase chain reaction based assay for detection of the causative agent of crayfish plague Aphanomyces astaci. Vet. Microbiol. 137, 146–155. Westman, K., Pursiainen, M., Vilkman, R., 1978. A new folding trap model which prevents crayfish from escaping. Freshw. Crayfish 4, 235–242. Wittwer, C., Nowak, C., Strand, D.A., Vrålstad, T., Thines, M., Stoll, S., 2018. Comparison of two water sampling approaches for eDNA-based crayfish plague detection. Limnologica 70, 1–9. Zar, J.H., 1999. Biostatistical Analysis, 4th ed. Prentice Hall, London, UK.
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Figure captions
Figure 1. Sampling of crayfish epibiotic communities for A. astaci detection. (a) – (b) Scrubbing the crayfish with the sterile brush. (c) – (d) Pouring the obtained suspension of epibionts into the 50-mL Falcon tube. (e) Obtained pellet of epibiotic cells in 2-mL tube after centrifugation.
Figure 2. An example of standard PCR A. astaci detection results in pairs of swab and cuticle samples. S – DirectLoad™ 50 bp DNA StepLadder; + - positive control with pure A. astaci genomic DNA used as a template; - - negative control.
Figure 3. Comparison of detected pathogen levels for cuticle and swab sample pairs.
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Highlights
We have developed a novel non-destructive Aphanomyces astaci-detection protocol. The method detects A. astaci DNA in cuticle-associated biofilm of infected animals. The new method is applicable in monitoring of A. astaci in endangered crayfish species.
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Table 1. Sampling localities, coordinate system (HTRS 96/TM) and crayfish species present in each location. x
y
Crayfish species
Previous data on the disease status of the location (year)
459441
5082277
AT
n.t.
429177
4970612
AA
n.t.
Burgeti (National Park Plitvice)
430094
4971437
AA
Positive (2012, 2013)*
Rječica (National Park Plitvice)
430040
4970656
AT
n.t.
Prijeboj (National Park Plitvice)
433656
4965730
AT
n.t.
Prštavac (National Park Plitvice)
429195
4970868
AA
n.t.
Sartuk (National Park Plitvice)
424969
4977264
AT
n.t.
Belajske Poljice (River Korana)
426295
5034353
AL, PL
n.t.
Karlovac business zone (River Korana)
427002
5035063
AL, PL
n.t.
Ladvenjak (River Korana)
430444
5030260
AL, PL
AL positive (2012, 2013), PL negative (2012)*
Lučica (River Korana)
420953
5023112
PL
n.t.
Sučevići (River Korana)
423012
5020504
AL, PL
n.t.
Šćulac (River Korana)
423355
5026240
AL, PL
AL negative (2012)*, PL n.t.
Motičnjak (Varaždin)
491159
5129909
AA
n.t.
3rd Maksimir Lake (Zagreb)
462659
5076222
AL
n.t.
Sampling locality Bliznec (Nature Park Medvednica) Bakinovac (National Park Plitvice)
AL – Astacus (Pontastacus) leptodactylus; AA - Astacus astacus; AT - Austropotamobius torrentium; PL – Pacifastacus leniusculus; n.t. – not tested * Maguire et al., 2016
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Table 2. Sampled crayfish from each locality. Sampling Sex Sampling date locality *AL1 Maksimir m April 2017 *AL2 Maksimir m April 2017 *AL3 Maksimir m April 2017 *AL4 Maksimir m April 2017 *AL5 Maksimir m April 2017 *AL6 Maksimir m April 2017 *AT2 Bliznec m September 2017 *AT3 Bliznec f September 2017 AL7 Maksimir m April 2017 AT1 Prijeboj m March 2017 AA1 Motičnjak m May 2017 AA2 Motičnjak m May 2017 AA3 Burgeti m April 2017 PL1 Ladvenjak m September 2018 * Animals infected in the laboratory AL - Astacus (Pontastacus) leptodactylus; AA - Astacus astacus; AT - Austropotamobius torrentium; PL – Pacifastacus leniusculus Code
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Table 3. Comparison of non-destructive detection of A. astaci from swab samples (S) and traditional detection from the cuticle samples (C). Agent level A0 = 0 PFU; A1 = <5 PFU; A2 = between 5 and 50 PFU; A3 = between 50 and 103 PFU; A4 = between 103 and 104 PFU; A5 = between 104 and 105 PFU (Vrålstad et al., 2009).
Code *AL1 S *AL1 C *AL2 S *AL2 C *AL3 S *AL3 C *AL4 S *AL4 C *AL5 S *AL5 C *AL6 S *AL6 C *AT2 S *AT2 C *AT3 S *AT3 C AL7 S AL7 C AT1 S AT1 C AA1 S AA1 C AA2 S AA2 C AA3 S AA3 C PL1 S PL1 C
Days till death 17 10 28 7 7 7 15 6 / / / / / / / / / / / /
Swab Cuticle Swab Cuticle Swab Cuticle Swab Cuticle Swab Cuticle Swab Cuticle Swab Cuticle Swab Cuticle Swab Cuticle Swab Cuticle Swab
Standard PCR detection (Oidtmann et al., 2006) positive positive positive positive positive positive positive positive positive positive positive positive positive positive positive positive negative negative negative negative negative
Cuticle
negative
n. d.
A0
Swab Cuticle Swab Cuticle Swab Cuticle
negative negative negative negative negative negative
0.39 n. d. 1.7 n. d. 0.54 1.3
A1 A0 A1 A0 A1 A1
Sample type
* Animals infected in the laboratory
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qPCR detection (Vrålstad et al., 2009) Average PFU per reaction
Agent level
115.6 83.0 54.4 11.0 33.9 90.5 4.3 12.5 88.9 38.6 2.8 762.0 202.0 191.1 4.8 40 044.3 n. d. n. d. n. d. n. d. n. d.
A3 A3 A3 A2 A2 A3 A1 A2 A3 A2 A1 A3 A3 A3 A1 A5 A0 A0 A0 A0 A0
AL - Astacus leptodactylus; AA - Astacus astacus; AT - Austropotamobius torrentium; PL – Pacifastacus leniusculus; n. d. – not detected; PFU – PCR-forming unit
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