Crayfish plague agent detected in populations of the invasive North American crayfish Orconectes immunis ( Hagen , 1870 ) in the Rhine River , Germany

Crayfish plague, caused by the parasitic oomycete Aphanomyces astaci, has driven indigenous European crayfish species to regional extinction in many parts of Europe and is among the leading threats to the remaining populations. A. astaci is known to be carried by longestablished invasive crayfish species of North American origin, which are also the main vectors of the plague pathogen. In this study, we examined whether a new invasive crayfish of North American origin, the calico crayfish (Orconectes immunis), also carries A. astaci. Orconectes immunis is a recent invader of the Upper Rhine plain, where it seems to displace its invasive predecessor Orconectes limosus, which is a known carrier of the agent of the crayfish plague. Using real-time PCR, we identified the calico crayfish as the fourth invasive crayfish species to be a carrier of the crayfish plague pathogen in Europe and we confirmed the infection with A. astaci in O. limosus. These findings support the concern that all North American crayfish species in European waters are carriers of the crayfish plague pathogen. Such knowledge should prove useful for conservation efforts, management, legislation, and public education about the spread of crayfish plague and non-indigenous crayfish species.


Introduction
Invasive non-indigenous species are one of the leading threats to freshwater biodiversity, besides habitat deterioration (Sala et al. 2000;Gherardi 2007).All European freshwater crayfish species (Crustacea, Decapoda, Astacidae) are highly threatened by Aphanomyces astaci (Schikora 1906), an invasive, crayfish-specific parasite causing crayfish plague, i.e. a fatal disease (Söderhäll and Cerenius 1999).Aphanomyces astaci originates from North America and was probably first introduced to Europe in the late 1850s.Its natural hosts, North-American freshwater crayfish species, were not found during the first outbreaks of the disease in Europe but were repeatedly introduced later (Alderman 1996;Holdich et al. 2009).Out of a total of more than 460 crayfish species living in North America (Crandall and Buhay 2011), today at least eight species are established in Europe in the wild (Holdich et al. 2009;Chucholl and Pfeiffer 2010).
Although all North American crayfish species are suspected to be carriers of A. astaci (OIE 2009), only three of the North American crayfish species present in the wild in Europe have been shown to be a carrier of the pathogen so far: the signal crayfish [Pacifastacus leniusculus (Dana, 1852)] (Unestam and Weiss 1970), the spinycheek crayfish [Orconectes limosus (Rafinesque, 1817)] (Vey et al. 1983), and the red swamp crayfish [Procambarus clarkii (Girard, 1852)] (Diéguez-Uribeondo and Söderhäll 1993).These three species belong to the "Old" non-indigenous crayfish species in Europe, i.e. have been introduced into European waters before 1975 (summarized by Holdich et al. 2009).
One "New" non-indigenous crayfish species in Europe is the North American calico crayfish [Orconectes immunis (Hagen, 1870)], which was first recorded at two locations in the Upper Rhine plain in the mid-1990s (Dussling and Hoffmann 1998;Dehus et al. 1999;Gelmar et al. 2006).The pathway of introduction of O. immunis is unclear; both an introduction from the pet trade and as fishing bait by Canadian soldiers had been suggested (Dehus et al. 1999;Gelmar et al. 2006).However, since the calico crayfish was not known in the German pet trade prior to its establishment in the Upper Rhine plain and because this species is popular as fishing bait in North America, an introduction as fishing bait seems more likely (see Gelmar et al. 2006;Chucholl 2013).Since its discovery, the calico crayfish has rapidly spread upstream and downstream in the Upper Rhine plain (Gelmar et al. 2006;Chucholl 2012) and is currently colonizing a stretch of more than 98 km, where it inhabits a wide spectrum of habitat types (summarized in Chucholl 2012; Figure 1).Despite its presence in Europe for almost two decades, its status as carrier of A. astaci is still unclear.
Calico crayfish seem to replace the formerly most abundant non-indigenous crayfish species of the Rhine River, the spiny-cheek crayfish (Gelmar et al. 2006;Chucholl et al. 2008), which has been present in the Upper Rhine catchment for at least five decades (Holdich et al. 2009).In laboratory experiments, calico crayfish were shown to be superior to spiny-cheek crayfish in direct aggressive interactions and competition for shelter (Chucholl et al. 2008).Furthermore, the calico crayfish exhibits a strongly r-selected life history: it is rather small (at most 50 mm in carapace length), features a high fecundity (up to 500 pleopodal eggs female -1 ), and has the fastest recorded life cycle among the crayfish species present in Central Europe, combining a high growth rate and rapid maturation (within the first summer) with short longevity (2.5 years) (Chucholl 2012).
Knowledge about the A. astaci carrier status of alien crayfish populations is imperative for native crayfish conservation, risk assessment and management strategies (Peay 2009).The aim of the present study was to evaluate the status of calico crayfish populations in the Rhine catchment as carrier of A. astaci.

Methods
To assess the A. astaci carrier status of calico crayfish in the Upper Rhine plain, we sampled two calico crayfish populations using traps and hand-held nets.One of the sampled populations was close to the currently known downstream invasion front of O. immunis (Germersheim, about 26 km north of Karlsruhe) and occurred in sympatry with O. limosus, whereas the second sampled population was located near the site of original introduction (Bühl, about 38 km south of Karlsruhe).The sampling site near the invasion front was located in the Rhine River close to Germersheim (approx.Rhine km 390, 49°15'N, 8°25'E, see Figure 1) and the second sampling site was located with an aerial distance of about 63 km to the first sampling site at a small channel that is connected to the Rhine River near Bühl (8°04'N, 48°43'E).This site is in close proximity to the O. immunis occurrence reported by Dussling and Hoffmann (1998), i.e. one of the two locations where calico crayfish were first discovered in the mid-1990s.Calico crayfish is the exclusive crayfish species in this channel and previous to its establishment no crayfish had been known to occur there.In total, we collected 50 individuals of calico crayfish and 10 individuals of spiny-cheek crayfish from the Germersheim site in 2011 and 32 calico crayfish individuals from the Bühl site in 2012.
Upon capture, crayfish were identified using distinct features (Figure 2), and whole specimens were transported to the laboratory and frozen at -20°C.To evaluate the A. astaci carrier status we used the TaqMan ® minor groove binder (MGB) real-time PCR (qPCR) according to Vrålstad et al. (2009).This method is the most specific and sensitive method to test for the presence of the crayfish plague pathogen (Tuffs and Oidtmann 2011).Before DNA-extraction, we visually checked all calico crayfish for mechanical damage and melanisation.DNA was extracted from the soft abdominal cuticle, the inner joint of two walking legs, a part of the uropods and melanised spots when present using a CTABmethod as described in Vrålstad et al. (2009).The qPCR reaction was performed on a Mastercycler ® ep realplex S (Eppendorf) using the TaqMan ® Environmental Master Mix to avoid PCR inhibition (Strand et al. 2011).Differing from the published PCR program, the annealing temperature was increased to 62°C and the annealing time decreased to 15 seconds to further exclude possible false positive results (T.Vrålstad, personal communication).A negative control consisting of 5 μl nuclease free water was included together with a standard series of genomic DNA from a pure A. astaci culture.Data analysis was carried out using the software Real Plex 2.2 (Eppendorf).
The relative level of infection by the pathogen was based on the strength of the qPCR signal.The number of observed PCR-forming units in the reaction was assigned to semi-quantitative agent levels (according to Vrålstad et al. 2009).Individuals with agent levels A 0 (no detection) and A 1 are considered uninfected, individuals with agent levels A 2 and higher are considered infected by A. astaci (see Table 1
A. astaci was detected in 26 of the 32 tested calico crayfish from Bühl (81%).Most individuals from both species and both sampling sites contained very low to moderate levels of agent DNA (A 1 to A 4 ).However, two calico crayfish from the Bühl site were infected at a very high level (A 6 ) and one spiny-cheek crayfish with mechanical damage from the Germersheim site was exceptionally highly infected (A 7 ).Melanised areas were observed in eight calico crayfish from the Bühl site, in four calico crayfish from the Germersheim site, all of which were tested positive, and in one highly infected (A 7 ) spiny-cheek crayfish specimen.

Discussion
Using the currently most reliable molecular detection method for the agent of crayfish plague (Vrålstad et al. 2009;Tuffs and Oidtmann 2011), we have shown for the first time an A. astaci infection in calico crayfish.We found infections in this species at two sampling sites in the Upper Rhine plain with an aerial distance of about 63 km.Moreover, we confirmed the infection in spiny-cheek crayfish co-existing with calico crayfish.The relatively low agent levels (≤A 4 ) of most infected crayfish are typical for North American crayfish species, which show an evolved defense reaction against A. astaci that normally prevents further spread of A. astaci hyphae within their body (Cerenius et al. 2003) and results in a latent infection.Thus, North American crayfish do usually not suffer from the disease but continuously release spores into the water with elevated spore levels prior to and during molting and mortalities (Strand et al. 2012).In the present study we found one spinycheek crayfish with a physical injury.This injury may have stressed its immune system and thus explains the high infection status (A7) of this individual.The immune response can be visually observed in infected crayfish as melanised spots.However, melanised spots alone are not a good indicator for the infection status with A. astaci because they also appear as a reaction to physical injury.In the present study, melanisation was found in only 13 of the 29 positive tested crayfish.Therefore, the absence of melanised spots does not conclusively indicate the absence of an infection with A. astaci.
The positive verification of calico crayfish as carrier of the crayfish plague agent is worrying.Particularly, given the fast and successful spread of this "New" non-indigenous crayfish species in the Upper Rhine plain.Moreover, the species replaces the "Old" non-indigenous crayfish, spiny-cheek crayfish, from preferred habitats (Gelmar et al. 2006;Chucholl et al. 2008;Chucholl 2012).Preliminary field observations suggest that calico crayfish inhabit a wider spectrum of habitats than spiny-cheek crayfish (Chucholl 2012).Specifically, calico crayfish were found in shallow temporary backwaters adjacent to the Rhine River and brooks draining from the Schwarzwald, which are habitats from which spiny-cheek crayfish are typically absent.Calico crayfish might therefore spread A. astaci into habitats that were previously not colonized by spiny-cheek crayfish.However, to date, calico crayfish have not come into contact with indigenous European crayfish species (Chucholl 2012).
We assume that the calico crayfish will continue its fast invasion of the Rhine River and connected waterways.Artificial channels that connect the Rhine River to other large river catchment areas, such as the Danube, the Rhône, the Odra, and the Elbe, promote a fast spread of invasive aquatic species throughout Europe (Bij de Vaate et al. 2002) and will most likely also facilitate the further active spread of calico crayfish.In addition to active range expansion, secondary introductions, i.e. translocations of calico crayfish by humans, are also a potential mechanism of spread and have already occurred in Germany and possibly in France (Chucholl and Dehus 2011;Collas et al. 2011).Public and stakeholder information is therefore imperative to mitigate the risk of further secondary introductions, which are a major threat to otherwise isolated populations of indigenous European crayfish.
Finally, it is important to note that there is accumulating evidence that different North American crayfish species are carriers of different strains of A. astaci (Huang et al. 1994, Kozubíková et al. 2011) and that these strains vary in their virulence (Jussila et al. 2011;Viljamaa-Dirks et al. 2011).An important question is therefore whether calico crayfish carry a different and possibly new A. astaci strain.This question is closely linked to the question of whether a) calico crayfish were already carriers of A. astaci when they were introduced to the Rhine catchment or whether b) calico crayfish were initially uninfected and got infected later, when they came into contact with A. astaci-carrying spiny-cheek crayfish.The positive verification of A. astaci in the single species population close to Bühl that has existed for about 14 years is a strong indication that calico crayfish were already carriers of the agent of crayfish plague when they were introduced to the Rhine catchment.In this case, calico crayfish may carry a new and possibly more virulent strain.However, we cannot rule out the possibility of recent human influence (e.g. by fishing gear that was contaminated with A. astaci spores) or an infection via animals (e.g.predatory fish feeding on infected crayfish; Oidtmann et al. 2002).Furthermore, A. astaci originating from spiny-cheek crayfish in the Rhine River could have reached the population by gradually infecting calico crayfish that are presumably distributed in the whole channel that connects the Rhine River with the sampling site in a stepping-stone manner.In the case of the Germersheim population A. astaci can be transmitted from calico crayfish to spiny-cheek crayfish and vice versa.We hope to resolve the question of the origin of A. astaci in European calico crayfish populations in the future, when methods become available that facilitate the assignment of A. astaci strains and the detection of new strains.
The positive verification of calico crayfish as a carrier of A. astaci adds another species to the list of highly dangerous non-indigenous species.In particular, it adds another species to the list of A. astaci-carrying crayfish species.The result of this study has to be implemented in native crayfish conservation strategies.A site where calico crayfish is present has to be considered as a reservoir for A. astaci.

Figure 1 .
Figure 1.The known distribution of Orconectes immunis in Europe as summarized in Chucholl (2012) with data from Chucholl and Dehus (2011; triangles), Collas et al. (2011; diamonds) and Gelmar et al. (2006; circles), completed with unpublished data from Chucholl (squares).Black stars indicate the sampling sites of this study.The international River Basin District Rhine (data: European Environment Agency, 2011) is colored light grey.Data of waters and national borders: GADM (2012).

Figure 2 .
Figure 2. Orconectes immunis ♀ (top) and O. limosus ♂ (bottom) from the Rhine River.Arrows denote key characters to distinguish the two species (modified from Gelmar et al. 2006 and Chucholl et al. 2008): dn -distinct tooth followed by a notch on the dactylus of the chelipeds (only present in O. immunis); ht -hair tufts on the ventral side of the chelae joints of the 1st and 2nd pereiopod (only present in O. immunis); db -distinct dark bandage adjacent to the orange cheliped tips (only present in O. limosus); hp -hepatic spines (only present in O. limosus).

Table 1 .
Real-time PCR detection of Aphanomyces astaci in investigated specimens of Orconectes immunis and Orconectes limosus from the two sampling sites from the Upper Rhine plain in Germany and percentage of specimens detected positive.For each sampling site, number of analyzed crayfish, number and proportion of infected individuals, and relative level of infection according to Vrålstad et al. (2009) are given.