The widespread presence of North American alien crayfish in Europe is a major driver of native crayfish population declines, mainly because they are chronic carriers of the oomycete Aphanomyces astaci responsible for crayfish plague. Screening for the crayfish plague pathogen in host populations has become a common practice across Europe, but sampling usually covers spatial but not temporal variation. Our study focuses on the current situation in Czechia, where screening for A. astaci was first conducted in the mid-2000s. We provide data about the distribution and prevalence of this pathogen at almost 50 sites with three host crayfish: the spiny-cheek crayfish Faxonius limosus, signal crayfish Pacifastacus leniusculus, and marbled crayfish Procambarus virginalis. Among these sites were 20 localities that were resampled several years (usually more than a decade) after the original screening for A. astaci. We did not detect any A. astaci infection in two studied P. virginalis populations but documented several new hotspots of highly infected P. leniusculus in Czechia, and the first site with the coexistence of the latter with F. limosus. Our data suggest that despite some fluctuations, A. astaci prevalence in North American host populations generally does not tend to change significantly over time; we only observed two cases of a significant increase and one of a significant decrease. We no longer detected A. astaci in several originally weakly infected populations, but our data suggest it likely still persists in these areas and threatens native crayfish populations. At the single known site in the country where P. leniusculus and F. limosus coexist, we documented the presence of the same A. astaci genotype group in both crayfish species, likely due to interspecific transmission of the pathogen from the former host to the latter. However, genotyping of A. astaci in infected host individuals still supported the link between specific pathogen genotypes and crayfish hosts, suggesting that assessment of sources of mass mortalities from the pathogen genotyping is feasible in European regions where the mutual contact of different American crayfish species is uncommon.

Aphanomyces astaci, infection prevalence, interspecific pathogen transmission, invasive crayfish distribution, microsatellite genotyping, mitochondrial haplogroups, qPCR genotyping

Crayfish species native to Europe face numerous threats, such as habitat loss, deteriorating water quality, overfishing or predators, with various impacts in different regions of the continent (Souty-Grosset et al. 2006). However, the key reason for declines and local extinctions of European crayfish populations is the widespread presence of invasive non-native crayfish species of North American origin (Holdich et al. 2009; Richman et al. 2015). This is partly due to their superior competitive abilities (Lindqvist and Huner 1999; van Kuijk et al. 2021), but also because these crayfish are major chronic carriers of the oomycete Aphanomyces astaci, a pathogen causing crayfish plague. More information on various aspects of this disease is provided in several recent reviews (Jussila et al. 2015; Rezinciuc et al. 2015; Svoboda et al. 2017; Becking et al. 2022).

Three natural host species of A. astaci, the spiny-cheek crayfish Faxonius limosus, the signal crayfish Pacifastacus leniusculus and the red swamp crayfish Procambarus clarkii, have become particularly widespread throughout Europe, but several additional alien crayfish species of the North American genera Procambarus, Faxonius, Cambarellus and Australasian Cherax have been locally introduced as well (Holdich et al. 2009; Kouba et al. 2014; Weiperth et al. 2020). Procambarus clarkii and P. leniusculus have also been introduced to other continents (P. clarkii being the most widespread crayfish globally; Oficialdegui et al. 2020) and along with them the crayfish plague pathogen. The introduction and spread of A. astaci in new regions potentially threaten local crustacean populations, including native crayfish species (Peiró et al. 2016; Mrugała et al. 2017; Martín-Torrijos et al. 2018) and those introduced for aquaculture purposes (Hsieh et al. 2016; Putra et al. 2018).

Several studies have conducted surveys on the spatial distribution and/or prevalence of chronic A. astaci infections in North American crayfish populations (e.g., Sandstrӧm et al. 2014; Tilmans et al. 2014; James et al. 2017b). They have shown that the prevalence of A. astaci may substantially differ among species and regions as well as within regions (e.g., Tilmans et al. 2014; Maguire et al. 2016; Grandjean et al. 2017). Moreover, intensive screenings in localities of coexistence with the native noble crayfish Astacus astacus have suggested that not all North American crayfish populations host this pathogen (e.g., Schrimpf et al. 2013). Although host populations reaching 100% prevalence are no exception (e.g., Kozubíková et al. 2011a; Filipová et al. 2013), they usually exhibit much lower prevalence values, and individual infection loads tend to be low as well (e.g., Maguire et al. 2016; James et al. 2017b; Panteleit et al. 2019).

In addition, there have been a few attempts, using various methodological approaches, to evaluate whether the prevalence of A. astaci differs over time. Nylund and Westman (2000) and Jussila et al. (2017) estimated the pathogen prevalence in P. leniusculus populations from gross symptoms, i.e., the presence of melanised lesions; however, these symptoms or their absence do not always correspond to results of A. astaci molecular detection (Kozubíková et al. 2009). Matasová et al. (2011) examined temporal changes in A. astaci prevalence in three F. limosus populations over three to six years using molecular diagnostics. They did not observe significant temporal variation in one highly infected and one very lowly infected population. However, the prevalence of an intermediately infected population decreased below the detection level over six years (Matasová et al. 2011). Unfortunately, the extent of that study was rather limited (a single host species, few populations, and a moderate time scale), thus its results cannot be generalised.

In Central and Western Europe, the key crayfish plague reservoirs are invasive North American crayfish populations (Holdich et al. 2009), although chronic A. astaci infections have also been documented in some native European crayfish populations (reviewed in Svoboda et al. 2017) and in the Chinese mitten crab Eriocheir sinensis (Schrimpf et al. 2014; Svoboda et al. 2014). However, unlike in Turkey, Finland, or Eastern European countries (Svoboda et al. 2017), no cases of chronic infections in native crayfish species in the territory of Czechia have been documented, despite dedicated efforts (Mojžišová et al. 2020). Currently, two of the three main crayfish plague carriers in Europe (F. limosus, P. leniusculus) are widespread in this country (Kouba et al. 2014; Mojžišová et al. 2020), and asymptomatic infections by A. astaci in Czech populations of F. limosus, and to a lesser extent of P. leniusculus, have been well documented (Kozubíková et al. 2009, 2011a; Rusch et al. 2020). The third species, P. clarkii, has not yet been documented from the wild in Czechia. However, populations of the marbled crayfish Procambarus virginalis, another proven A. astaci carrier (Keller et al. 2014; Mrugała et al. 2015), have recently been documented in the country, presumably originating from ornamental aquaria (Patoka et al. 2016, and unpubl. data).

Although all three invasive crayfish documented from Czechia (F. limosus, P. leniusculus, P. virginalis) have been included in the list of invasive alien species of the European Union concern according to Regulation (EU) No 1143/2014, their spread in the country continues, either unaided (due to active dispersal along watercourses), or due to unauthorised human-mediated introductions. As a result, new populations of all three species are being discovered (see map in Mojžišová et al. 2020).

Given that North American crayfish species pose the greatest risk as vectors of crayfish plague, country-wide screenings for the presence of A. astaci in their populations have been performed in several countries. This study follows up the screening of Czech populations carried out more than a decade ago in pioneering studies that applied molecular diagnostics to study the distribution and prevalence of A. astaci in North American asymptomatic hosts (Kozubíková et al. 2006, 2009). In samples collected between 2004 and 2006, F. limosus populations showed great variability in A. astaci prevalence, reaching up to 100%, while P. leniusculus populations seemed to be infected less intensively, with prevalence not exceeding 37% (Kozubíková et al. 2009, 2011a). Since then, both species have expanded not only in the originally invaded areas, but also with numerous populations appearing in non-adjacent places, including areas bordering Austria, Germany and Poland (Štambergová et al. 2009; Mojžišová et al. 2020). Some of the newly reported borderland P. leniusculus populations, recently screened for A. astaci along with environmental DNA samples, have shown very high prevalence values (Rusch et al. 2020), in contrast to Czech populations examined previously. We presume that these highly infected populations could have been founded from sources other than the remaining Czech populations, possibly having their origin across the country border. Despite the limited distribution of P. leniusculus in Czechia and low A. astaci prevalences reported from most populations there, its importance as a local crayfish plague reservoir is also indicated by genotyping of A. astaci from crayfish plague outbreaks. Four mass mortalities of the native noble crayfish were caused by A. astaci genotypes assumed to originate from this host species (Kozubíková-Balcarová et al. 2014; M. Mojžišová, unpubl. data).

There is an assumption that distinct A. astaci genotype groups known from Europe are linked to their original North American crayfish carrier (for more details, see Ungureanu et al. 2020). Thus, various genotyping assays applicable on either axenic A. astaci cultures (Huang et al. 1994; Rezinciuc et al. 2014) or on mixed genome samples (e.g., Grandjean et al. 2014; Makkonen et al. 2018; Minardi et al. 2019; Di Domenico et al. 2021) should allow tracking the source of infection in crayfish plague outbreaks. Although a recent study using mtDNA sequencing has shown that A. astaci haplotypes are not host species-specific (Martín-Torrijos et al. 2021), these haplogroups likely include multiple strains that may differ from each other in variable nuclear markers targeted by other genotyping methods (e.g., RAPD: Huang et al. 1994; microsatellites: Grandjean et al. 2014) or in their physiological properties. Despite the increasing number of genotyping methods and studies applying them, there is still only limited data about A. astaci strains genotyped directly from North American crayfish hosts in Europe that would support the link between host species and pathogen strains co-introduced with them (reviewed in Ungureanu et al. 2020). On the contrary, some evidence for the interspecific transmission of A. astaci strains between North American hosts has been provided, both from captivity (Mrugała et al. 2015) and from the wild in the invaded range (James et al. 2017a).

Our study had thus three aims: (i) to update data about the A. astaci distribution and prevalence in Czechia including recently discovered alien crayfish populations; (ii) to investigate potential long-term temporal changes in A. astaci prevalence in populations of two alien crayfish species resampled after more than a decade; and (iii) to genotype A. astaci in representative host individuals from multiple populations to further test the assumption that distinct A. astaci genotypes causing crayfish plague outbreaks in Europe are specifically linked to their North American crayfish carriers.

Our data, extending the pilot study by Matasová et al. (2011), evaluated for the first time long-term changes in the prevalence of the crayfish plague pathogen in chronically infected invasive crayfish species on larger temporal and spatial scales. We statistically compared A. astaci prevalence in 14 sampling sites with P. leniusculus and F. limosus after more than ten years, and screened new sites with the documented presence of alien crayfish. Consistently with previous studies (Kozubíková et al. 2009, 2011a), the proportion of infected populations and prevalence values tended to be higher for F. limosus than for P. leniusculus. However, several hotspots of infected P. leniusculus were recently discovered in the country.

In our study, significant changes in A. astaci prevalence after a decade were observed only infrequently. Some fluctuations of A. astaci prevalence may reflect seasonality (Matasová et al. 2011), changes in host population density, or possibly the stress level to which the crayfish hosts are exposed. This might have caused the highly significant increases in A. astaci prevalence observed in two previously studied F. limosus populations. In several previously crayfish populations with low prevalences and infection levels (Kozubíková et al. 2011a), we no longer detected any trace of A. astaci DNA; however, the wide overlap of prevalence confidence intervals (see Table 2) indicates that the pathogen presence cannot be ruled out, and the decrease of prevalence was not significant in such cases.

A significant decrease to below the detection level in the F. limosus population from the Pšovka brook, already reported by Matasová et al. (2011), thus remains a notable exception. That study indicated a decrease in the prevalence of A. astaci to below the detection limit over six years (2004–10), and we did not detect A. astaci in the same brook stretch even a decade later (Table 2). Several kilometres upstream from that area, close to a zone where F. limosus was getting into contact with A. astacus as this latter species recolonised the stream, no A. astaci was detected in 2012 and 2013 (Kozubíková-Balcarová et al. 2014). However, we recently found A. astaci infections in three out of 15 tested F. limosus individuals there (Table 1; Rusch et al. 2020), indicating that the pathogen continues to persist within the host population and thus is an ongoing threat to native crayfish. In fact, a sudden disappearance of A. astacus from a several-km-long stretch of the brook upstream of the contact zone was observed by local conservation authorities in autumn 2021 (L. Beran, pers. comm.), presumably due to an unreported crayfish plague outbreak, as predicted by Kozubíková-Balcarová et al. (2014). Therefore, any presumed disappearance of the pathogen from a previously infected host population should be considered with caution.

To obtain more reliable data about the occurrence of A. astaci in populations where the pathogen prevalence may be low, very high numbers of individuals per population need to be examined (see Schrimpf et al. 2013). Negatively tested individuals in the low dozens per site, as in our present study, cannot prove the absence of the pathogen. One example of a likely underestimation of A. astaci occurrence may be the Vysočina region (sites no. 34 to 41 in Fig. 1, Table 1), the area of the first successful introduction of P. leniusculus to the Czech territory (Filipová et al. 2006). We did not reliably confirm the pathogen in recently collected samples from anywhere in this region, including populations with a previously reported A. astaci presence (Table 2; Kozubíková et al. 2011a). Although this may possibly represent a long-term regional decrease of A. astaci prevalence, disappearance of the pathogen from the entire region is highly unlikely. In this context, it should be noted that in one out of nine crayfish individuals from the newly screened population in the Dolní Tis fishpond (site no. 40), a trace amount of A. astaci DNA was consistently detected (Table 1).

An extreme case where the absence of A. astaci detection likely represents a false negative result at the whole-population level might be the Vysokopecký pond (site no. 6). There, we confirmed the infection in two F. limosus individuals in 2017, but three years later no trace of A. astaci DNA was detected either in 20 individuals from the pond or in 15 individuals from the adjacent Litavka brook (Table 1). The long-term presence of the pathogen in the brook may be nevertheless assumed, as a crayfish plague outbreak caused by A. astaci genotype group E was confirmed in the section just below the Vysokopecký pond in 2011 (Kozubíková-Balcarová et al. 2014)

A contrasting difference between older and more recent samples, but in the opposite direction, was also observed in the Bobrava river in the south-eastern part of the country (site no. 45). Consistently high A. astaci prevalence (≥70%) in P. leniusculus was detected there in 2018 and 2020 (Table 1), but the pathogen was not detected by qPCR in 10 individuals collected approx. 12 km upstream in 2010 (E. Kozubíková-Balcarová, unpubl. data). Genotyping of the pathogen in P. leniusculus from this river confirmed the genotype group B, which is generally associated with this host species. Corresponding strains have been repeatedly isolated and/or genotyped from P. leniusculus originating in the USA (Huang et al. 1994; Makkonen et al. 2019) as well from individuals collected across its invaded range in Europe (reviewed in Ungureanu et al. 2020). Considering that the nearest locality to the Bobrava river known to host crayfish infected by A. astaci is inhabited by F. limosus (site no. 23, located within the same river basin), we presume that the long-term persistence of A. astaci in its original P. leniusculus host is a likely explanation for its recent confirmation in the Bobrava.

Genotype group B was also confirmed in all other genotyped individuals of P. leniusculus from Czech localities (Fig. 1). Populations of that species with sufficient infection levels to allow genotyping were located only in the southern part of the country. Except for the Bobrava river mentioned above, all those localities are close to the state border with Austria (in two cases, in the immediate vicinity). According to the species occurrence database of the Nature Conservation Agency of the Czech Republic (accessed 12/2021), they were all discovered after 2010. Nevertheless, A. astaci had likely been spreading from P. leniusculus to native crayfish earlier: four mass mortalities of native A. astacus caused by genotype group B have been confirmed in various regions of Czechia (Kozubíková-Balcarová et al. 2014; M. Mojžišová, unpubl.). The first was reported in 2007 from the tributary of Pěnenský pond (site no. 31), in which we confirmed the presence of infected P. leniusculus and genotyped the pathogen only a decade later.

In all but one case, we identified A. astaci genotypes that were expected to be found in European populations of their respective North American crayfish carriers (Fig. 1, Table 1). This indicates that original sources of the pathogen in crayfish mass mortalities can be reasonably assumed from genotyping results in regions where the coexistence of different North American invasive hosts is uncommon or absent. However, in a single locality in Czechia where the syntopic presence of P. leniusculus and F. limosus was discovered in 2020 (Malý Klikovský pond; site no. 25), we unambiguously identified A. astaci genotype group B in both host species. This indicates a likely interspecific transmission of A. astaci from P. leniusculus to F. limosus, as in a previously reported case from the UK where the recipient taxon was a member of the virile crayfish species complex, Faxonius cf. virilis (James et al. 2017a). Regions where multiple invasive A. astaci carriers coexist or may come into contact, such as the Netherlands (Tilmans et al. 2014) and Hungary (Weiperth et al. 2020), may thus yield hardly predictable host taxon – pathogen genotype combinations, making it difficult to track the origin of possible crayfish plague outbreaks.

Our experience with the inconsistent success of the applied genotyping methods confirms that characterising A. astaci genotypes chronically infecting their original carriers is challenging, and various methodological approaches may complement each other. In the relatively rare cases when a heavy infection of an American host is observed, all genotyping methods are likely to succeed. In already preserved material, increasing the pathogen DNA concentration in the isolate, such as with the use of the GlycoBlue Coprecipitant in our study, may increase the chance for successful genotyping. Alternatively, when live crayfish are available, the growth of the pathogen may be enhanced by their exposure to stress (as in Kozubíková et al. 2011b) or by analysis of host moults, which seems particularly promising (Martín-Torrijos et al. 2021).

Our data suggest that long-term significant changes in A. astaci prevalence in its North American hosts were not common within the studied populations. In several originally weakly infected populations (in particular of P. leniusculus) we no longer detected the pathogen, but it is likely that it persists in the area. The re-appearance of infected F. limosus individuals in the Pšovka brook (moreover, associated with the recent disappearance of susceptible A. astacus from an adjacent section of the brook) confirms that A. astaci prevalence at low levels (<5%) still poses a threat to local native crayfish. The preventive rescue transfer of A. astacus from the Pškovka to another local watershed without alien crayfish (Kozubíková-Balcarová et al. 2014) was apparently a timely measure that contributed to the species conservation in the region. However, highly infected populations of invasive crayfish (and nearby populations of the susceptible native species) should be prioritised in any management strategies. In this context, the apparent cross-border invasion by strongly infected P. leniusculus, likely of different origin from the long-established weakly infected populations of that species in Czechia, is of particular concern.

Despite evidence of the apparent interspecific transmission of A. astaci from P. leniusculus to F. limosus at one site, our results generally support the link between specific pathogen genotypes and particular North American crayfish hosts invading European waters. This suggests that A. astaci genotyping is a relevant approach to tracking of sources of the pathogen in crayfish plague outbreaks in Central and Western European countries. Overall, our study highlights the importance of routine country-wide screening for relevant aquatic wildlife pathogens as an integral part of relevant conservation strategies. In the case of A. astaci, the screening accuracy might be improved by combining the analyses of host tissues and environmental DNA (e.g., Rusch et al. 2020; Troth et al. 2020; Sieber et al. 2022).

We thank all colleagues who helped providing the samples, in particular Jiří Hladovec and Jiří Patoka, and the Nature Conservation Agency of the Czech Republic for access to the species occurrence database. This study was funded by the Technology Agency of the Czech Republic [project no. TH02030687] and Charles University [project SVV 260436]. JS, EK-B and AP conceived the project. JS, EK-B, EŠ, AP, MB, AK and MM collected material (with additional assistance), MM, EK-B and RS performed laboratory analyses. MM and AP wrote the draft, which was then commented by all other co-authors.