Crayfish Plague Agent Detected in Populations of the Invasive North American Crayfish Orconectes Immunis (Hagen, 1870) in the Rhine River, Germany

Aquatic Invasions (2013) Volume 8, Issue 1: 103–109 doi: http://dx.doi.org/10.3391/ai.2013.8.1.12 Open Access

Short Communication

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

Anne Schrimpf 1*, Christoph Chucholl 2, Thomas Schmidt 1 and Ralf Schulz 1 1 University Koblenz-Landau, Institute for Environmental Sciences, Fortstrasse 7, 76829 Landau, Germany 2 Fischereiforschungsstelle BW, Argenweg 50/1, 88085 Langenargen, Germany E-mail: [email protected] (AS), [email protected] (CC), [email protected] (TS), [email protected] (RS) *Corresponding author

Received: 18 October 2012 / Accepted: 14 December 2012 / Published online: 4 January 2013

Handling editor: Vadim Panov

Abstract

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 long- established 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. Key words: Crayfish plague, Aphanomyces astaci, calico crayfish, crustacean disease, Rhine River, invasive American crayfish species, real-time PCR, pathogen vector

Introduction Europe in the wild (Holdich et al. 2009; Chucholl and Pfeiffer 2010). Invasive non-indigenous species are one of the Although all North American crayfish species leading threats to freshwater biodiversity, are suspected to be carriers of A. astaci (OIE besides habitat deterioration (Sala et al. 2000; 2009), only three of the North American crayfish Gherardi 2007). All European freshwater cray- species present in the wild in Europe have been fish species (Crustacea, Decapoda, Astacidae) shown to be a carrier of the pathogen so far: the are highly threatened by Aphanomyces astaci signal crayfish [Pacifastacus leniusculus (Dana, (Schikora 1906), an invasive, crayfish-specific 1852)] (Unestam and Weiss 1970), the spiny- parasite causing crayfish plague, i.e. a fatal cheek crayfish [Orconectes limosus (Rafinesque, disease (Söderhäll and Cerenius 1999). 1817)] (Vey et al. 1983), and the red swamp Aphanomyces astaci originates from North crayfish [Procambarus clarkii (Girard, 1852)] America and was probably first introduced to (Diéguez-Uribeondo and Söderhäll 1993). These Europe in the late 1850s. Its natural hosts, North- three species belong to the “Old” non-indigenous American freshwater crayfish species, were not crayfish species in Europe, i.e. have been found during the first outbreaks of the disease in introduced into European waters before 1975 Europe but were repeatedly introduced later (summarized by Holdich et al. 2009). (Alderman 1996; Holdich et al. 2009). Out of a One “New” non-indigenous crayfish species in total of more than 460 crayfish species living in Europe is the North American calico crayfish North America (Crandall and Buhay 2011), [Orconectes immunis (Hagen, 1870)], which was today at least eight species are established in first recorded at two locations in the Upper

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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).

Rhine plain in the mid–1990s (Dussling and fishing bait seems more likely (see Gelmar et al. Hoffmann 1998; Dehus et al. 1999; Gelmar et al. 2006; Chucholl 2013). Since its discovery, the 2006). The pathway of introduction of calico crayfish has rapidly spread upstream and O. immunis is unclear; both an introduction from downstream in the Upper Rhine plain (Gelmar et the pet trade and as fishing bait by Canadian al. 2006; Chucholl 2012) and is currently soldiers had been suggested (Dehus et al. 1999; colonizing a stretch of more than 98 km, where it Gelmar et al. 2006). However, since the calico inhabits a wide spectrum of habitat types crayfish was not known in the German pet trade (summarized in Chucholl 2012; Figure 1). prior to its establishment in the Upper Rhine Despite its presence in Europe for almost two plain and because this species is popular as decades, its status as carrier of A. astaci is still fishing bait in North America, an introduction as unclear.

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Calico crayfish seem to replace the formerly 50 individuals of calico crayfish and 10 most abundant non-indigenous crayfish species individuals of spiny-cheek crayfish from the of the Rhine River, the spiny-cheek crayfish Germersheim site in 2011 and 32 calico crayfish (Gelmar et al. 2006; Chucholl et al. 2008), which individuals from the Bühl site in 2012. has been present in the Upper Rhine catchment Upon capture, crayfish were identified using for at least five decades (Holdich et al. 2009). In distinct features (Figure 2), and whole specimens laboratory experiments, calico crayfish were were transported to the laboratory and frozen at - shown to be superior to spiny-cheek crayfish in 20°C. To evaluate the A. astaci carrier status we direct aggressive interactions and competition used the TaqMan® minor groove binder (MGB) for shelter (Chucholl et al. 2008). Furthermore, real-time PCR (qPCR) according to Vrålstad et the calico crayfish exhibits a strongly r-selected al. (2009). This method is the most specific and life history: it is rather small (at most 50 mm in sensitive method to test for the presence of the carapace length), features a high fecundity (up to crayfish plague pathogen (Tuffs and Oidtmann 500 pleopodal eggs female-1), and has the fastest 2011). Before DNA-extraction, we visually recorded life cycle among the crayfish species checked all calico crayfish for mechanical present in Central Europe, combining a high damage and melanisation. DNA was extracted growth rate and rapid maturation (within the first from the soft abdominal cuticle, the inner joint summer) with short longevity (2.5 years) of two walking legs, a part of the uropods and (Chucholl 2012). melanised spots when present using a CTAB- Knowledge about the A. astaci carrier status method as described in Vrålstad et al. (2009). of alien crayfish populations is imperative for The qPCR reaction was performed on a native crayfish conservation, risk assessment and Mastercycler® ep realplex S (Eppendorf) using management strategies (Peay 2009). The aim of the TaqMan® Environmental Master Mix to the present study was to evaluate the status of avoid PCR inhibition (Strand et al. 2011). calico crayfish populations in the Rhine Differing from the published PCR program, the catchment as carrier of A. astaci. annealing temperature was increased to 62°C and the annealing time decreased to 15 seconds to further exclude possible false positive results (T. Methods Vrålstad, personal communication). A negative control consisting of 5 μl nuclease free water To assess the A. astaci carrier status of calico was included together with a standard series of crayfish in the Upper Rhine plain, we sampled genomic DNA from a pure A. astaci culture. two calico crayfish populations using traps and Data analysis was carried out using the software hand-held nets. One of the sampled populations Real Plex 2.2 (Eppendorf). was close to the currently known downstream The relative level of infection by the pathogen invasion front of O. immunis (Germersheim, was based on the strength of the qPCR signal. about 26 km north of Karlsruhe) and occurred in The number of observed PCR-forming units in sympatry with O. limosus, whereas the second the reaction was assigned to semi-quantitative sampled population was located near the site of agent levels (according to Vrålstad et al. 2009). original introduction (Bühl, about 38 km south of Individuals with agent levels A0 (no detection) Karlsruhe). The sampling site near the invasion and A are considered uninfected, individuals front was located in the Rhine River close to 1 with agent levels A2 and higher are considered Germersheim (approx. Rhine km 390, 49°15'N, infected by A. astaci (see Table 1 caption for 8°25'E, see Figure 1) and the second sampling details). 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 Results Bühl (8°04'N, 48°43'E). This site is in close proximity to the O. immunis occurrence reported Using A. astaci-specific real-time PCR from a by Dussling and Hoffmann (1998), i.e. one of the total of 92 crayfish a positive carrier status was two locations where calico crayfish were first found for about 60% of the tested crayfish (Table discovered in the mid-1990s. Calico crayfish is 1). At the sampling site Germersheim we found the exclusive crayfish species in this channel and 23 positive detections out of 50 calico crayfish previous to its establishment no crayfish had (46%) and six positive detections out of ten been known to occur there. In total, we collected spiny-cheek crayfish (60%). Furthermore,

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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.

Sampling Crayfish Positives Positives Agent levela Species

site (number) (number) (%) A0 A1 A2 A3 A4 A5 A6 A7 Germersheim O. limosus 10 6 60 3 1 3 2 1 Germersheim O. immunis 50 23 46 21 6 13 9 1 Bühl O. immunis 32 26 81 3 3 4 18 2 2 Total 92 55 60 27 10 17 30 5 2 1 a Agent levels refer to semi-quantitative categories based on the numbers of observed PFUs (PFUobs) from the A. astaci−specific real-time 3 3 PCR. Agent level A0: no detection; A1: below the limit of detection (PFUobs< 5); A2: 5 ≤ PFUobs < 50; A3: 50 ≤ PFUobs < 10 ; A4: 10 ≤ 4 4 5 5 6 6 PFUobs < 10 ; A5: 10 ≤ PFUobs < 10 ; A6: 10 ≤ PFUobs < 10 ; A7: 10 ≤ PFUobs. A0 and A1 are both considered negative.

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A. astaci was detected in 26 of the 32 tested the Upper Rhine plain. Moreover, the species calico crayfish from Bühl (81%). Most replaces the “Old” non-indigenous crayfish, individuals from both species and both sampling spiny-cheek crayfish, from preferred habitats sites contained very low to moderate levels of (Gelmar et al. 2006; Chucholl et al. 2008; agent DNA (A1 to A4). However, two calico Chucholl 2012). Preliminary field observations crayfish from the Bühl site were infected at a suggest that calico crayfish inhabit a wider very high level (A6) and one spiny-cheek spectrum of habitats than spiny-cheek crayfish crayfish with mechanical damage from the (Chucholl 2012). Specifically, calico crayfish Germersheim site was exceptionally highly were found in shallow temporary backwaters infected (A7). Melanised areas were observed in adjacent to the Rhine River and brooks draining eight calico crayfish from the Bühl site, in four from the Schwarzwald, which are habitats from calico crayfish from the Germersheim site, all of which spiny-cheek crayfish are typically absent. which were tested positive, and in one highly Calico crayfish might therefore spread A. astaci infected (A7) spiny-cheek crayfish specimen. into habitats that were previously not colonized by spiny-cheek crayfish. However, to date, Discussion calico crayfish have not come into contact with indigenous European crayfish species (Chucholl Using the currently most reliable molecular 2012). detection method for the agent of crayfish plague We assume that the calico crayfish will (Vrålstad et al. 2009; Tuffs and Oidtmann 2011), continue its fast invasion of the Rhine River and we have shown for the first time an A. astaci connected waterways. Artificial channels that infection in calico crayfish. We found infections connect the Rhine River to other large river in this species at two sampling sites in the Upper catchment areas, such as the Danube, the Rhône, Rhine plain with an aerial distance of about 63 the Odra, and the Elbe, promote a fast spread of km. Moreover, we confirmed the infection in invasive aquatic species throughout Europe (Bij spiny-cheek crayfish co-existing with calico de Vaate et al. 2002) and will most likely also crayfish. The relatively low agent levels (≤A4) of facilitate the further active spread of calico most infected crayfish are typical for North crayfish. In addition to active range expansion, American crayfish species, which show an secondary introductions, i.e. translocations of evolved defense reaction against A. astaci that calico crayfish by humans, are also a potential normally prevents further spread of A. astaci mechanism of spread and have already occurred hyphae within their body (Cerenius et al. 2003) in Germany and possibly in France (Chucholl and results in a latent infection. Thus, North and Dehus 2011; Collas et al. 2011). Public and American crayfish do usually not suffer from the stakeholder information is therefore imperative disease but continuously release spores into the to mitigate the risk of further secondary water with elevated spore levels prior to and introductions, which are a major threat to during molting and mortalities (Strand et al. otherwise isolated populations of indigenous 2012). In the present study we found one spiny- European crayfish. cheek crayfish with a physical injury. This injury Finally, it is important to note that there is may have stressed its immune system and thus accumulating evidence that different North explains the high infection status (A7) of this American crayfish species are carriers of individual. The immune response can be visually different strains of A. astaci (Huang et al. 1994, observed in infected crayfish as melanised spots. Kozubíková et al. 2011) and that these strains However, melanised spots alone are not a good vary in their virulence (Jussila et al. 2011; indicator for the infection status with A. astaci Viljamaa-Dirks et al. 2011). An important because they also appear as a reaction to question is therefore whether calico crayfish physical injury. In the present study, carry a different and possibly new A. astaci melanisation was found in only 13 of the 29 strain. This question is closely linked to the positive tested crayfish. Therefore, the absence question of whether a) calico crayfish were of melanised spots does not conclusively indicate already carriers of A. astaci when they were the absence of an infection with A. astaci. introduced to the Rhine catchment or whether b) The positive verification of calico crayfish as calico crayfish were initially uninfected and got carrier of the crayfish plague agent is worrying. infected later, when they came into contact with Particularly, given the fast and successful spread A. astaci-carrying spiny-cheek crayfish. The of this “New” non-indigenous crayfish species in positive verification of A. astaci in the single

107 A. Schrimpf et al. species population close to Bühl that has existed Chucholl C (2012) Understanding invasion success: life-history for about 14 years is a strong indication that traits and feeding habits of the alien crayfish Orconectes immunis (Decapoda, Astacida, Cambaridae). Knowledge and calico crayfish were already carriers of the agent Management of Aquatic Ecosystems 404: 04 of crayfish plague when they were introduced to Chucholl C (2013) Invaders for sale: trade and determinants of the Rhine catchment. In this case, calico crayfish introduction of ornamental freshwater crayfish. Biological may carry a new and possibly more virulent Invasions 15: 125–141, http://dx.doi.org/10.1007/s10530-012- 0273-2 strain. However, we cannot rule out the Chucholl C, Dehus P (2011) Flusskrebse in Baden-Württemberg, possibility of recent human influence (e.g. by Fischereiforschungsstelle Baden-Württemberg (FFS), fishing gear that was contaminated with A. astaci Langenargen, Germany, 92 pp Chucholl C, Pfeiffer M (2010) First evidence for an established spores) or an infection via animals (e.g. predato- Marmorkrebs (Decapoda, Astacida, Cambaridae) population ry fish feeding on infected crayfish; Oidtmann et in Southwestern Germany, in syntopic occurrence with al. 2002). Furthermore, A. astaci originating Orconectes limosus (Rafinesque, 1817). 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We hope to resolve the question of de la réserve de Biosphère transfrontalière Vosges du Nord - Pfälzerwald 16: 18–36 the origin of A. astaci in European calico Crandall KA, Buhay JE (2011) Global diversity of crayfish crayfish populations in the future, when methods (Astacidae, Cambaridae, and Parastacidae––Decapoda) in become available that facilitate the assignment freshwater. Hydrobiologia 595: 295–301, http://dx.doi.org/10. 1007/s10750-007-9120-3 of A. astaci strains and the detection of new Dehus P, Dussling U, Hoffmann C (1999) Notes on the strains. occurrence of the calico crayfish (Orconectes immunis) in The positive verification of calico crayfish as Germany. Freshwater Crayfish 12: 786–790 a carrier of A. astaci adds another species to the Diéguez-Uribeondo J, Söderhäll K (1993) Procambarus clarkii Girard as a vector for the crayfish plague fungus, list of highly dangerous non-indigenous species. Aphanomyces astaci Schikora. 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