The old menace is back: Recent crayfish plague outbreaks in the Czech Republic

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Aquaculture 274 (2

The old menace is back: Recent crayfish plagueoutbreaks in the Czech Republic

E. Kozubíková a,⁎, A. Petrusek a,b, Z. Ďuriš c, M.P. Martín d, J. Diéguez-Uribeondo d, B. Oidtmann e

a Charles University in Prague, Faculty of Science, Department of Ecology, Viničná 7, Prague 2, CZ-12844, Czech Republicb University of South Bohemia in České Budějovice, Research Institute of Fish Culture and Hydrobiology, Zátiší 728/II, Vodňany, CZ-38925, Czech Republic

c University of Ostrava, Department of Biology, Chittussiho 10, Ostrava, CZ-71000, Czech Republicd Real Jardín Botánico CSIC, Departamento de Micología, Plaza Murillo 2, 28014 Madrid, Spain

e CEFAS Weymouth Laboratory, Barrack Road, The Nothe, Weymouth, Dorset DT4 8UB, United Kingdom

Received 5 September 2007; received in revised form 2 November 2007; accepted 12 November 2007

Abstract

The crayfish plague pathogen, Aphanomyces astaci (Saprolegniales, Oomycetes), is lethal for native European crayfish species and eliminateda number of their populations during the second half of the 19th and throughout the 20th centuries. The disease is spread by non-native NorthAmerican crayfish species, known to be carriers of the pathogen, which are currently thriving in many European countries. This represents one ofthe major threats to the recovery of populations of native crayfish (the noble crayfish Astacus astacus and the stone crayfish Austropotamobiustorrentium) in the Czech Republic. While only two suspected plague-caused crayfish mass mortalities were reported in the second half of the 20thcentury, five mass mortalities involving both native species were found to be caused by A. astaci between 2004 and 2007, and several additionalones, though unconfirmed, are likely. We discuss possible reasons for this apparent increase in the number of crayfish plague outbreaks, and wereport details of the four most recent mass mortalities in which the presence of A. astaci has been confirmed by molecular methods (PCR withspecies-specific primers and sequencing of the pathogen DNA) and microscopic analysis, and of additional suspicious cases of crayfishdisappearance. As these plague outbreaks have been found only accidentally, it is likely that their actual frequency is higher. The Czech Republicis probably not exceptional in this respect, and plague monitoring within the whole Central Europe deserves more attention.© 2007 Elsevier B.V. All rights reserved.

Keywords: Aphanomyces astaci; Oomycete; Orconectes limosus; Astacus astacus; Austropotamobius torrentium; Mass mortalities

1. Introduction

Crayfish plague, the most harmful disease of Europeanfreshwater crayfish, is caused by the parasitic saprolegniousoomycete Aphanomyces astaci Schikora, 1903 (Cerenius et al.,1988). This parasite is endemic to North America and is highlyspecialized to penetrate into and live in the crayfish cuticle. InEuropean and Australasian freshwater crayfish species, theinfection leads to the death of affected animals (Söderhäll andCerenius, 1999), and due to the high infectivity of the pathogen,

⁎ Corresponding author.E-mail address: [email protected] (E. Kozubíková).

0044-8486/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.aquaculture.2007.11.015

whole populations of susceptible crayfish can be lost in a shorttime.

North American crayfish species are usually able to limit anA. astaci infection (Unestam, 1969, 1972), as they can inhibit thegrowth of hyphae in the cuticle by fast and heavy melanization.Such an immune response is possible due to their ability tocontinuously produce prophenoloxidase, an enzyme involved inmelanin formation (Cerenius et al., 2003). In European species, theprocess of melanin formation in response to A. astaci infection isnot as efficient, leading to a higher susceptibility to the crayfishplague (Cerenius et al., 2003). In North American species, theparasite survives despite being inhibited in growth, and its infectiveunits – zoospores –may be released into the water (mostly duringmoulting of the crayfish host), so infected American crayfish serve

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209E. Kozubíková et al. / Aquaculture 274 (2008) 208–217

as vectors of the disease (e.g., Vey et al., 1983; Diéguez-Uribeondoand Söderhäll, 1993; Oidtmann et al., 2004).

Crayfish plague was spread into Europe at the end of the1850s, and during the end of the 19th and the beginning of the20th century it eradicated a huge number of populations ofnative European crayfish species across the whole continent(Alderman, 1996). The plague still threatens native crayfish inmany European countries, mainly due to the broad distributionof introduced American crayfish species (Souty-Grosset et al.,2006) whose populations act as a reservoir of the plague path-ogen. Crayfish mass mortalities caused by A. astaci have beenrecently recorded in Germany (Oidtmann et al., 1999), Spain(Diéguez-Uribeondo, 2006), and Sweden (Bohman et al.,2006).

Two native crayfish species can be found in the CzechRepublic — the noble crayfish Astacus astacus and the stonecrayfish Austropotamobius torrentium (Ďuriš and Kozák, 2000;Souty-Grosset et al., 2006). A. astacus is an important organismfor human consumption and aquaculture in some parts of Europe(Souty-Grosset et al., 2006); however, this crayfish is notexploited in Czechia, and is listed as an endangered species andprotected by local law. Three additional species occurring inopen waters of the area are of alien origin. The narrow-clawedcrayfish Astacus leptodactylus, also susceptible to the crayfishplague, was introduced from Eastern Europe. The remaining twospecies, the spiny-cheek crayfish Orconectes limosus and thesignal crayfish Pacifastacus leniusculus, are of North Americanorigin. The spiny-cheek crayfish is widespread in the watershedsof the Elbe and the Vltava rivers, and has also been released intoa number of isolated water bodies. Details about the historicaland present distribution of this species in the Czech Republichave been recently summarised in Petrusek et al. (2006) andFilipová et al. (2006a). Many Czech O. limosus populations aresources of the plague pathogen — chronic infections byA. astaci in several populations have been directly confirmedby DNA analysis (Kozubíková et al., 2006 and unpublisheddata). The signal crayfish P. leniusculus is known from a fewlocalities scattered across the Czech Republic (Filipová et al.,2006b), but so far it does not seem to have spread significantly.Its distribution in Central Europe is nevertheless still increasing:it has been recently confirmed in the river Morava in Slovakia(Petrusek and Petrusková, 2007), from where it may coloniseadditional areas of the eastern Czech Republic. In many otherEuropean countries, however, it is widely distributed and repre-sents a more serious threat to native crayfish than O. limosus(Pöckl and Pekny, 2002; Bohman et al., 2006; Diéguez-Uribeondo, 2006; Souty-Grosset et al., 2006).

The wave of crayfish plague infection that spread acrossEurope over 100 years ago also hit the Czech territory(Krupauer, 1968; Alderman, 1996). However, no confirmedreports of plague outbreaks exist for most of the 20th century,until the end of the 1990s. In 1998 and 1999, two massmortalities of the noble crayfish of suspected plague origin werereported from Central Bohemia (Kozák et al., 2000a,b). Thefirst unambiguously confirmed plague outbreak in the countryduring the past few decades took place in 2004 in Silesia, in thebrook Křivec in the watershed of the river Olše/Olza

(Kozubíková et al., 2006). In the present study, we describedetails of four additional crayfish plague outbreaks recorded inthe Czech Republic between 2005 and 2007, some of which werecently reported in a brief note (Kozubíková et al., 2007). Weadd further molecular evidence (sequences of the amplifiedDNA) of the presence of the crayfish plague pathogen in all ofthese crayfish plague outbreaks, and discuss potential sources ofinfection. We also describe other recent suspicious cases ofcrayfish disappearance which might have been possibly causedby disease, and discuss possible reasons for the apparentincrease in the prevalence of crayfish plague on the Czechterritory.

2. Material and methods

2.1. Localities and sample collection

Samples of dying or dead crayfish were collected from four accidentallyrecorded crayfish mass mortalities: 1) a tributary of the pond Pěnenský, 2) theBojovský brook, 3) the Úpořský brook, and 4) the river Olše/Olza (locations ofthe three latter outbreaks were reported in Kozubíková et al., 2007). Othersuspicious cases of crayfish population disappearances, from which sampleswere not available, were either recorded by chance or during regular crayfishpopulation surveys. The positions of all localities are marked in Fig. 1; fordetails of each case, see Results.

Additionally, we included twelve individuals of the American spiny-cheekcrayfish O. limosus in the analysis, from the brook Prudník in Silesia. This aliencrayfish population, as a potential reservoir of the crayfish plague pathogen, maybe linked to the apparent disappearance of the noble crayfish A. astacus from theadjacent river Osoblaha (Ďuriš and Horká, 2007).

The samples were analysed for the presence of A. astaci, either fresh orpreserved for later processing by deep-freezing (−80 °C) or in 96% ethanol.Circumstances of the mass mortalities, such as the behaviour of affectedcrayfish, the potential presence of other crayfish species or the impact on otheraquatic organisms, were recorded. Localities where the mass mortality occurredin 2005 were checked the following season (2006) for the presence of crayfishby an inspection of suitable shelters.

2.2. Detection of A. astaci

To detect the presence of A. astaci in the samples, we used a moleculartechnique combined with microscopic examination. Firstly, the soft abdominalcuticle of the tested crayfish specimens was dissected and examined using lightmicroscopy. We usually checked four to six individuals for the presence ofmycelium corresponding to A. astaci (Cerenius et al., 1988): branched, aseptate,round-tipped hyphae 8–10 μm in diameter growing inside the cuticle.Microphotographs of hyphae were taken using a Nikon DXM1200F digitalcamera mounted on the microscope.

Subsequently, a cuticle sample from each crayfish was frozen in liquidnitrogen and powdered in a sterile mortar, and the total genomic DNA wasextracted using the QIAamp DNA Mini Kit (Quiagen, USA) according to themanufacturer's instructions. Details of the cuticle dissection and DNA extractionare given in Oidtmann et al. (2006). The identical extraction protocol was usedon fresh samples and those preserved by freezing or in ethanol.

To check for the presence of A. astaci DNA in the isolates, a fragment of theITS (internal transcribed spacer in the rDNA region) was amplified by PCRusing the species-specific primers “42” and “640”, which amplify anapproximately 550 bp-long ITS fragment (Oidtmann et al., 2006). DNA isolatefrom the A. astaci laboratory culture M96/1 (genetic group B from Oidtmannet al., 1999) was used as a positive control. Amplified DNAwas visualised on anagarose gel, and the presence of a PCR product corresponding to the positivecontrol was considered as a positive detection of A. astaci in the tested crayfish.Negative controls (lacking DNA template) were run in each PCR reaction to ruleout any reagent contamination.

Fig. 1. Approximate locations of the crayfish mass mortalities and crayfish disappearance in 2004–2007. 1: Bojovský brook, May 2005, A. astacus; 2: Úpořský brook,June 2005, A. torrentium; 3: Klíčava brook, April 2005, A. astacus; 4: Hýskovský brook, summer 2006, A. torrentium; 5: River Olše/Olza, July–August 2006,A. astacus; 6: River Osoblaha, September 2006, A. astacus; 7: brook near Horní Pěna, June–July 2007, A. astacus and Koštěnický brook, 2004–2005, A. astacus.Black diamond close to 5 indicates a plague outbreak in the Křivec brook, recorded in 2004 (described in Kozubíková et al., 2006).

210 E. Kozubíková et al. / Aquaculture 274 (2008) 208–217

2.3. Sequencing and sequence analysis

One or two samples were chosen from each mass mortality where crayfishwere available for testing, and amplified DNA was sequenced to confirm thedetection of A. astaci. A nested PCR amplification was employed to gain enoughhigh-quality product for sequencing. The general primer pair ITS5 and ITS4 wasused as outer primers for amplification of the rRNA gene cluster (White et al.,1990). Subsequently, the A. astaci-specific primers “42” and “640”were used onthe resulting amplicon. Amplification reactions using Ready-To-Go PCR Beads(Amersham Biosciences, UK) followed the protocol and cycling parametersdescribed in Diéguez-Uribeondo et al. (2007). Prior to sequencing, the ampli-fication products were purified from an agarose gel using the QIAquick Gel PCRpurification kit (Qiagen, CA, USA). Both strands were separately cycle-sequenced using primers “42” and “640” on an ABI PRISM 3700 capillarysequencer. Sequencher (Gene Codes Corporation, USA) was used to identify theconsensus sequence from the two strands of the ITS nrDNA of each isolate.

Resulting 548 bp-long sequences were aligned using ClustalW (Thompsonet al., 1994) with the following reference sequences from GenBank: A. astaci ofall four known genetic groups (accession nos. AY683895, AY310499,AY683894, AY683896) and, according to a BLAST search, the two othermost similar taxa, Aphanomyces invadans (accession nos. AY283642 andAY082907) and Aphanomyces frigidophilus (accession no. AY647192). Thealignment was checked manually, trimmed to an unambiguously alignablefragment available for all sequences (518 bp) and further analysed in thesoftware package MEGA version 3.1 (Kumar et al., 2004). We calculated thepairwise sequence divergence (using the Kimura 2-parameter model) and

constructed a neighbour-joining tree; branch support was estimated with 500bootstrap replicates; positions with gaps were excluded from the analysis.

3. Results

3.1. Details of the crayfish mass mortalities

Localities of the described losses of native crayfish populationswere recorded in three regions of the Czech Republic (Fig. 1, inset).Two of the confirmed plague outbreaks and two of the plague-suspected cases of crayfish disappearance were recorded in CentralBohemia (watersheds of the Berounka and Vltava rivers; Fig. 1 —large map), one confirmed and one suspected case in the fishpondregion of South Bohemia, and additional confirmed and suspectedcases in the Odra (Oder) watershed close to the border with Poland inSilesia (north-eastern part of the Czech Republic; Fig. 1 — nos. 5+6,Fig. 2). Stone crayfish populations were affected in two cases inCentral Bohemia; the remaining involved noble crayfish.

3.1.1. Confirmed cases of crayfish plague

3.1.1.1. Bojovský brook, Central Bohemia, May 2005. Dying anddead noble crayfish, as well as a few apparently healthy-lookingindividuals but with diurnal activity (altogether around 10 individuals)

Fig. 2. Extent of the mass mortality of Astacus astacus in the Olše watershed in 2006.



were found in the Bojovský brook (left-side tributary of the riverVltava) approximately 1 km north-east of Bojov (49°53′ N, 14°21′ E;Fig. 1 — number 1) on May 14, 2005. No crayfish were found furtherdownstream, although in the previous season they had been present andespecially abundant in the stretch of brook examined. No negativeeffects on fish or macrozoobenthos (including isopods and amphipods)were observed. No other crayfish species were found in the brook (norhave they been reported from the adjacent part of the river, the Vranéwater reservoir), and no noble crayfish or any signs of the presence ofcrayfish were recorded in subsequent checks in May 2006 and 2007.

Two moribund crayfish individuals were used for PCR detection ofA. astaci — one deep-frozen and one preserved in ethanol. Hyphaewith morphological characteristics corresponding to A. astaci mycelia(Fig. 3) were observed in the cuticle of four specimens, and PCRdetection was positive for both analysed individuals.

3.1.1.2. Klíčava and Úpořský brooks, Central Bohemia, April–June2005. Two crayfish mortalities, probably interrelated, were recordedat about the same time as the previous crayfish plague outbreak in theKřivoklátsko Protected Landscape Area, approximately 40 km north-west of the above-described outbreak. Several dead (and no living)noble crayfish were observed by fishermen in the Klíčava brook (left-side tributary of the Berounka), south of Ruda and upstream from theKlíčava reservoir (50°06′ N, 13°52′ E; Fig. 1 — number 3) on April30, 2005. This observation of dead crayfish was ominous, as the brookhad been densely populated by crayfish in the previous seasons(Agency for Nature Conservation and Landscape Protection, unpub-lished data). Populations of fish (e.g., bullhead Cottus gobio, browntrout Salmo trutta or perch Perca fluviatilis) and lampreys (Lampetraplaneri) appeared to be unaffected. No other crayfish species areknown to have been present at this locality and no noble crayfish wererecorded in the brook in the following season (2006).

Unfortunately, no crayfish samples were available from the Klíčavabrook; therefore the potential presence of the crayfish plague pathogencould not be confirmed. However, indirect evidence exists that thislocality might have served as a source of infection for the crayfishplague outbreak described below (see Discussion).

Less than two months later (on June 26, 2006), a mass mortality ofthe stone crayfish was recorded in the Úpořský brook (right-sidetributary of the Berounka) in the Týřov nature reserve, east of Skryje(49°58′ N, 13°48′ E; Fig. 1 — number 2). At least several hundredindividuals were affected. Decaying crayfish carcasses, moribund

Fig. 3. Photomicrograph of hyphae in the soft abdominal cuticle of Astacusastacus from the mass mortality in the Bojovský brook (sample preserved in96% ethanol).

crayfish, as well as apparently unaffected individuals (but often withdiurnal activity) were observed in the stream at the same time. Duringthe following weeks the crayfish mortality spread upstream, and wasstopped only by an approximately 2 m high pond dam with a verticalwater overflow, which apparently acted as an efficient migration barrierto crayfish. The stone crayfish population upstream from the pond hadnot been affected, and survived into the following season. No crayfishcould be found downstream from the pond in 2006. No other crayfishspecies were found at this locality.

Five freshly dead or dying stone crayfish were analysed for thepresence of A. astaci. The specimens were processed immediately aftertransport to the laboratory, without previous preservation. Although nohyphae were observed in the crayfish abdominal cuticles, all fiveindividuals tested positive for the DNA of A. astaci by PCR.

3.1.1.3. River Olše and its tributaries, Silesia, July–August 2006.Next confirmed crayfish plague outbreak occurred in a large stretch ofthe River Olše (Polish toponym: Olza) and its tributaries in Silesia,around the town of Jablunkov (Fig. 1 — number 5; Fig. 2). The massmortality stretched at least from Bukovec close to the border withPoland (49°34′ N, 18°49′ E) to Bystřice (49°38′ N, 18°43′ E), whichrepresents over 15 km of river. Dead, dying, as well as living noblecrayfish were first recorded in the River Olše on July 10, 2006.Subsequently, a mass mortality of crayfish was observed at variousplaces in the river in July and August 2006, apparently spreading bothupstream and downstream. Over 600 dead crayfish were removed byfishermen from the stretch of river around Jablunkov alone.

Several specimens of the noble crayfish were sampled on July 12,2006 at two sites ca 14 km apart (Písek u Jablunkova, 49°33′ N, 18°48′E, and Bystřice, 43°37′ N, 18°43′ E). Two specimens from each sitewere analysed for the presence of A. astaci by PCR, and all four testedpositive. Hyphae of the same diameter and type of growth as A. astaciwere observed in the cuticle of only one of the crayfish analysed.

In the weeks following the first report of the mass mortality, dyingcrayfish were also recorded in the lower reaches of some of the Olšetributaries (Fig. 2), such as theKostkov inNávsí (49°35′N, 18°46′E), theŽabinec in Bystřice (left-side tributary of the Hluchová, approximately600 m upstream from the Olše; 49°38′ N, 18°43′ E), and the Ošetnickýbrook in Ošetnice (right-side tributary of the Lomná, approximately1.5 km upstream from the Olše; 49°34′ N, 18°45′ E). No signs of thepresence of crayfish were found in the affected watercourses in 2007.

3.1.1.4. Brooks near Jindřichův Hradec, South Bohemia, June–July2007. Tens of dead, dying and still-living noble crayfish wererecorded in the lower part of a small brook discharging into thePěnenský pond near Horní Pěna, South Bohemia (49°06′N, 15°03′E) atthe end of June 2007. During the following weeks, the mortality spreadupstream into an approximately 600 m long stretch of the brook belowthe Sígl pond, as well as into a small side tributary of the brook, andcrayfish completely disappeared from the lowest section where the massmortality had been originally observed. As in other cases, no adverseeffects were observed in fish or aquatic macroinvertebrates. Threecrayfish individuals were used for PCR detection of A. astaci, all testingpositive. Hyphae corresponding to A. astaci mycelia were observed inthe soft abdominal cuticle of three out of six examined specimens.

A dense population of noble crayfish also completely disappearedfrom an upper part of the nearby Koštěnický brook (upstream of theKačležský pond, 5–7 km eastwards from the above mortality) in 2004or 2005. Although no direct information is available, crayfish plaguemay be suspected in this case as well, as no fish mortalities werereported from the brook or fishponds located on it, and there were noobvious sources of pollution in its largely forested watershed.

Fig. 4. Neighbour-joining tree showing divergence between analysed Aphanomyces sequences from crayfish mass mortalities and reference data from GenBank.(Kimura 2-parameter distance, complete deletion of gaps). The first five sequences originate from Czech crayfish plague outbreaks (one sample per locality is shown);codes at reference A. astaci sequences indicate strains and respective genetic groups. GenBank accession numbers are shown in parentheses; scale bar indicates 1%divergence.


3.1.2. Additional suspicious cases of the disappearance of nativecrayfish populations

3.1.2.1. Hýskovský brook, Central Bohemia, Summer 2006. Apopulation of the stone crayfish A. torrentium apparently disappearedin 2006 from the Hýskovský brook (left-side tributary of the Berounkanear Hýskov, 50°00′ N 14°03′ E, approximately 18 km from both theKlíčava and Úpořský brooks, Fig. 1— number 4). The presence of thespecies at this locality was confirmed in 2005. A healthy male wasobserved on June 26 (Agency for Nature Conservation and LandscapeProtection of the Czech Republic, unpublished data), and a number ofsuitable shelters in the lower reach of the stream were occupied bycrayfish in summer (David Fischer, pers. comm.). Due to the relativelyscarce supply of shelters, the population probably could never havebeen very dense.

In 2006, no living crayfish were found in the stream. However, deadindividuals were repeatedly observed during the season. On August 3, asingle dead individual was found in the lower reach of the stream; onOctober 19, two dead and partly decayed individuals were found in theupper reach of the stream, above any likely source of pollution (DavidFischer, pers. comm.). As in the previous cases, no other crayfishspecies are known to occur in the same locality.

3.1.2.2. River Osoblaha, Silesia, September 2006. Indirect evidenceof a recent noble crayfish disappearance exists from the River Osoblahanear the Czech–Polish border in Silesia (50°18′ N 17°45′ E; nearestsettlement: Studnice, Fig. 1 — number 6). In its lower part, in closevicinity of the confluence with the Prudník brook, traces of recentcrayfish activity (dense crayfish burrows in the river bank, certainlyless than a year old) were observed in October 2006, but no crayfishspecimens were found despite intensive searches at the beginning andrepeatedly at the end of October 2006 (Ďuriš and Horká, 2007). Thenearest confirmed presence of noble crayfish in the Osoblaha was onlyapproximately 8.3 km upstream from the confluence with the Prudník,above the village Bohušov, and further upstream up to Arnultovice(Ďuriš and Horká, 2007).

Together with the absence of any crayfish from the lower RiverOsoblaha, the presence of the spiny-cheek crayfish O. limosus wasconfirmed at two different places in an approximately 4.6 km longstretch of the Prudník located on the territory of the Czech Republic—one very close to the confluence with the Osoblaha, another furtherupstream, close to Slezské Pavlovice. This is the first record of thespiny-cheek crayfish in the eastern part of the Czech Republic. It is

thought to have migrated from Polish territory either downstreamthrough the Prudník, or possibly upstream through the Osoblaha (fordetails, see Ďuriš and Horká, 2007). This particular spiny-cheekcrayfish population seems to have been heavily infected by A. astaci,all twelve collected specimens tested positive for the presence of thepathogen by PCR. Although O. limosus has recently been reported tomake burrows in a muddy lake in the United Kingdom (Holdich andBlack, 2007), the burrows observed in the Osoblaha were unlikely tohave been made by this species, as such extensive burrowing has neverbeen observed in Central European localities. Given the proximity of anoble crayfish population, we presume that the burrows in theOsoblaha indicate the very recent disappearance of the latter species inthe lower stretch of the river adjacent to the confluence with thePrudník.

3.2. Sequencing of the PCR products

All sequences obtained from the PCR products from crayfishcollected during suspected crayfish plague outbreaks were confirmedto be of A. astaci. Their sequences were identical to all four referenceA. astaci sequences, while the divergence (Kimura 2-parameterdistance) from the two outgroups, A. invadans and A. frigidophilus,were 4.2% and 4.8%, respectively (Fig. 4).

4. Discussion

4.1. Confirmed plague outbreaks

Molecular analyses of DNA extractions, including sequen-cing from crayfish individuals sampled from the massmortalities, confirmed the presence of A. astaci DNA; thecrayfish plague was therefore the most likely cause of mortality.Other evidence, such as microscopic examination or thedevelopment of the outbreaks, also supports this diagnosis. Inall four localities where affected crayfish were available formolecular analysis, individuals were found simultaneously invarious conditions — dead (both recently and already decay-ing), dying, as well as alive and apparently intact but withunusual diurnal activity. In all localities where dead crayfish ortheir remains were noticed, fish and other aquatic invertebratesdid not seem to be affected; this rules out water pollution or


oxygen depletion as the cause of mortality. Mass mortalitieslimited to crayfish point to disease as the most likely cause,which is also supported by the spread of mortality in theupstream direction.

A. astaci is a parasite highly specific for freshwater crayfish(Unestam, 1972), therefore crayfish plague is a prime suspect inthe cases of mass mortalities of susceptible crayfish species.Nevertheless, it is not the only disease to cause crayfish mor-talities (Edgerton et al., 2004). Unfortunately, the crayfishplague pathogen cannot be distinguished from closely relatedOomycetes by morphological characters (Cerenius et al., 1988),so even the presence of hyphae growing inside the cuticle ofaffected animals cannot be regarded as definitive proof of thisdisease. The combination of all the above evidence corrobo-rated by the molecular detection and sequencing of the pathogenDNA, however, identifies the crayfish plague unambiguously.

The mass mortality of A. torrentium in the Úpořský brook,diagnosed as crayfish plague by PCR and confirmed by se-quencing, is particularly interesting because no hyphae wereobserved in the cuticles of any tested crayfish specimens (in theremaining confirmed plague outbreaks in the Czech Republic,we found hyphae in the cuticles of at least some animals ex-amined). Several hypotheses may explain the absence of ob-servable hyphae in samples examined from the Úpořský brook.Firstly, it is possible that the mycelium of the pathogen wasdeveloped in different parts of the crayfish body, such as limbjoints or eye stalks, and we just accidentally missed it in thesections of abdominal cuticle used for diagnosis. Secondly, thelimited sample size (five individuals), or the selection ofexamined crayfish, could have also affected the results of themicroscopic examination: the crayfish were collected while stillalive (though moribund) or very soon after death, so it ispossible that hyphae of A. astaci would have later developed insufficient density in the cuticle. The PCR-based detection ishighly sensitive to the presence of the pathogen DNA(Oidtmann et al., 2006) and can detect even early stages suchas attached and germinating zoospores or early stages of hyphaldevelopment, which would have been difficult to find by lightmicroscopy in fresh preparations of cuticle.

4.2. Suspected outbreaks, sources of infection and potential forrecovery of crayfish populations

Unfortunately, it was often impossible to obtain samples ofaffected crayfish from localities with suspected crayfish plagueoutbreaks. Until recently, information about the potential plaguethreat had not spread among stakeholders who are most likely torecord crayfish mortalities, such as fishermen, ichthyologists oramateur naturalists. We therefore often learned about mortalitiesafter a delay, and samples were no longer available. The neces-sity for suitable preservation of the moribund or dead animals(deep-freezing, ethanol suitable for DNA analyses) or immedi-ate transport to the laboratory may also limit the availability ofsuitable samples. The additional cases where we suspect poten-tial outbreaks of crayfish plague based on indirect evidencecannot certainly be considered as proven. However, some ofthem are worth discussing in more detail.

The presumed disappearance of noble crayfish from thelower stretch of the Osoblaha is a special case, as only the tracesof previous crayfish activity (burrows) were observed. In thiscase, however, we presume the likely involvement of thecrayfish plague because a heavily infected American crayfishspecies was present in the close vicinity (hundreds of metres).

As A. astaci cannot withstand long periods without itscrayfish host, and susceptible crayfish die out after contact withthis pathogen, North American crayfish populations representthe only currently known reservoir of infection. However, thesecrayfish carriers were found to occur in the same locality in onlytwo of the suspected plague outbreaks in the Czech Republic(the one mentioned above, and additional one observed in the1990s), and in none of the confirmed ones.

Nevertheless, both American species occurred in SouthBohemia, relatively close to the region where the plague out-break was observed in 2007. A dense spiny-cheek crayfishpopulation is present in the Zlatá stoka channel approximately22 km westwards (Petrusek et al., 2006), and some crayfishfrom this locality are certainly infected by A. astaci (E.Kozubíková, unpublished data). Signal crayfish used to bepresent even closer, in fishponds near Lomy u Kunžaku(Filipová et al., 2006b). These are located about 9 km eastwardsfrom the plague outbreak at Horní Pěna, and only 2 km from theKoštěnický brook where noble crayfish have recently disap-peared. However, the presence of signal crayfish in this regionwas confirmed for the last time in 2002; this species has nolonger been found in its original localities despite intensiveefforts between 2004 and 2006.

In the case of the Central Bohemian outbreaks, spiny-cheekcrayfish are present in the same watersheds, but their nearestconfirmed localities are in large rivers tens of kilometresdownstream (Petrusek et al., 2006); hence, the primary sourcesof infection remain unclear. However, transmission of thedisease among localities is possible not only directly withinfected crayfish, but also secondarily with water (e.g., byhuman activities), although such a transmission is probablymuch less likely. Zoospores are able to survive as long as a fewdays in wet conditions (Oidtmann et al., 2002), so an infectioncould be spread when water contaminated with live zoospores istransferred from a locality with a crayfish plague outbreak orwith the presence of infected American crayfish (Reynolds,1988).

The crayfish plague outbreak in the Úpořský brook may havebeen such a case, possibly caused by a transmission of thepathogen from the Klíčava brook, where crayfish had diedapproximately two months before. The same equipment wasused during monitoring of fish populations in both affectedwatercourses, and the Úpořský brook was visited within 24 hafter dead noble crayfish in the Klíčava were observed. Al-though the rubber boots, fishing nets and containers for holdingfish had been left to dry overnight, no special measures hadbeen taken to decontaminate them from potential water-bornepathogens. Therefore, A. astaci zoospores could possibly havebeen transferred between the two localities on insufficientlydried equipment. No obvious source of infection for the Klíčavapopulation is known.


The crayfish plague outbreak in the Olše River in 2006 is notthe first recent one in its watershed. Noble crayfish were eradi-cated by crayfish plague in the Křivec brook (left-side tributaryof the Olše in the town Třinec) in 2004 (Kozubíková et al.,2006). The source of infection in both of these cases isunknown. Although both outbreaks could possibly have beencompletely independent, with the pathogen of different origins,it is more likely that both had the same source of infection. Wepresume that A. astaci was repeatedly transferred from this as-yet unknown source. Alternatively, the pathogen might havepersisted in the watershed in a chronic infection. This has beendescribed from some Scandinavian lakes, where A. astaciapparently survives in sparse crayfish populations and massmortalities occur when the host population reaches a certaincritical density (Fürst, 1995). Whatever the cause of the latestoutbreaks, it is apparent that the remaining crayfish populationsin the Olše watershed are potentially threatened by the disease.Until the source of the pathogen is identified and eliminated,any crayfish reintroduction attempts there should be ruled out.

Investigation of the affected localities in the next season didnot reveal any presence of surviving crayfish. The same is truefor the Křivec brook, where the plague eradicated the noblecrayfish population in 2004 (Z. Ďuriš, unpublished data). It isalmost certain that whole populations downstream of the plagueoutbreaks were eliminated at these localities. The spread of thedisease upstream was stopped by the migration barrier in theÚpořský brook. This potentially might allow the remainingstone crayfish population to recolonise the lower stretch of thebrook, once the crayfish pass downstream through the fishpond.In the remaining streams, the potential for natural recolonisationis lower, though the survival of a small part of the population inthe highest reaches of the respective watercourses cannot beexcluded. In these cases, environmental conditions remainsuitable for crayfish, so unless infected crayfish (e.g., Americanspecies) remain undetected at the localities, the long-termsurvival of the pathogen is not possible and reintroduction fromthe nearest strong crayfish populations may be considered.However, before any reintroduction activities are carried out,the absence of A. astaci should be tested by long-term exposureof caged susceptible crayfish in the watercourses (Taugbol et al.,1993; Diéguez-Uribeondo et al., 1997), although this cannotcompletely rule out the unnoticed presence of A. astaci carriers.

4.3. Crayfish plague as a future threat?

We are aware of only two documented cases of potentialcrayfish plague outbreaks from the Czech territory in the secondhalf of the 20th century (Kozubíková et al., 2006). On thecontrary, five confirmed and additional potential outbreaks havebeen recorded during just the last four years, between 2004 and2007. This apparent increase in the number of plague outbreakscan have several reasons. Firstly, it may be only an artefactcaused by previous underestimations of the danger posed by thecrayfish plague, and a tendency to ascribe any crayfish massmortality to water pollution; an increased focus on plaguedetection in the last few years and the availability of new toolsfor the diagnosis of A. astaci may therefore have biased the

data. On the other hand, American crayfish as potential vectorsof the plague pathogen have spread to many new localities in theCzech Republic in recent decades (Filipová et al., 2006a,b;Petrusek et al., 2006), which means an increase in the number ofchronic sources of infection from which the pathogen can besubsequently spread through various human activities.

Of the two potential plague vectors present in the country,O. limosus currently represents the more serious threat to thepopulations of native crayfish species, as it is widespreadespecially in the western half of the Czech territory (Petruseket al., 2006) and already proven to carry the plague pathogen inthe region (Kozubíková et al., 2006). The Czech populations ofA. torrentium, which are all distributed in the western part of thecountry (Kozák et al., 2002; Chobot, 2006), may be regarded asmore endangered by pathogen transmission from localities withO. limosus than A. astacus, which is distributed throughoutmost of the country (Chobot, 2006).

The recorded crayfish plague outbreaks most likely representonly the “tip of the iceberg”, a minor part of the real number ofaffected populations. Crayfish carcasses decay quickly, so wepresume that a number of plague-caused mass mortalitiesescape detection completely or remain unreported. Any suddendisappearance of crayfish from a water body for no apparentreason may be potentially caused by the crayfish plague.Additionally, most of the recent crayfish plague outbreaks havebeen located in two areas, Central Bohemia and Silesia. Thesebelong among centres of crayfish research in the CzechRepublic, which increases the likelihood of the timely detectionof outbreaks.

Although infected American crayfish populations areconsidered as the primary sources of A. astaci infections, directpathogen transmission can be linked to the syntopic occurrenceof O. limosus and susceptible crayfish species in only two casesof all the confirmed or suspected crayfish plague outbreakssince the 1990s. The pathways of zoospore transmission for theother plague outbreaks remain unknown. We may speculate thatO. limosus remains undetected downstream or upstream of theaffected watercourses. However, indirect mechanisms oftransmission may be considered as well — e.g., transfer ofinfected water with fish or on fishing gear from more distantlocalities with crayfish plague vectors or undetected plagueoutbreaks, natural long-distance dispersal of zoospores byanimal vectors, or releases of infected aquarium-kept Americancrayfish or even water from such aquaria.

Our results suggest that the occurrence of crayfish plague inthe Czech Republic is much more common than previouslythought. This disease seems to be a significant threat to thesuccessful recovery of native crayfish populations, even in areaswhere the presence of non-native American crayfish species hasnot been documented. The Czech Republic is probably notexceptional in this respect — it is likely that the situation inother Central European countries is similar, so plaguemonitoring deserves more attention. To prevent the undesirablespread of the pathogen, it is necessary to inform the generalpublic and especially stakeholders, such as fishermen or scubadivers, about the existence of the crayfish plague pathogen, thethreat posed by non-native crayfish species to the native fauna,


and measures needed to decontaminate potentially infectedequipment.

Acknowledgements

This work has been funded by the Grant Agency of theAcademy of Sciences of the Czech Republic (projectIAA601870701), the Grant Agency of the Charles University(GAUK 141/2005 B Bio), the Czech Ministry of Education(MSM0021620828 and MSM6007665809) and Ministerio deEducación y Ciencia, Spain (grant CGL2006-12732-C02-01).Eva Kozubíková was supported by the European CommunityProgramme “Structuring the European Research Area”, underSYNTHESYS at Real Jardín Botánico (CSIC). We thank thepersonnel of the Agency for Nature Conservation and Land-scape Protection of the Czech Republic, local authorities, andthe fishermen from Jablunkov, Třinec and Bystřice foradditional information and assistance. David Fischer providedinformation on the disappearance of crayfish from theHýskovký brook, Zdeněk Kučera kindly prepared the maps,and David Hardekopf and one anonymous referee corrected thelanguage of this paper.

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The old menace is back: Recent crayfish plague outbreaks in the Czech Republic

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