DISEASES OF AQUATIC ORGANISMS Vol. 35: 235-238,1999 Published February 26 Dis Aquat Org NOTE Crayfish plague epizootics in Germany-classification of two German isolates of the crayfish plague fungus Aphanomyces astaci by random amplification of polymorphic DNA Birgit Oidtmann1,*,Lage Cerenius2,Ines Schmidl, Rudolf ~offmann',Kenneth soderhal12 '~nstituteof Zoology, Fish Biology and Fish Diseases. University of Munich, Kaulbachstr. 37, D-80539 Munich, Germany '~ivisionof Physiological Mycology, University of Uppsala, Villavigen 6. S-752 36 Uppsala, Sweden ABSTRACT: Following 2 outbreaks of crayfish plague m cumb to infection. During the past century, the distrib- southern Germany, the causative agent, the oomycete fungus ution of non-native crayfish species has dramatically Aphanomyces astaci, was isolated from the diseased Astacus increased due to stocking activities, the natural spread astacus. The identity of the 2 strains was confirmed using established techniques, such as physiology, spore production of stocked populations, and the release of exotic and the fact that the isolated strains were hlghly virulent for crayfish by private aquarist or pond owners. In areas, A. astacus in infection experiments. The relationship between where American species have been introduced, mass these German strains and other A astaci strains was In- mortalities of European crayfish have frequently been vestigated using randomly amplified polymorphic DNA-poly- merase chain reaction (RAPD-PCR).The German strains were documented (Alderman 1996). found to be closely related to a strain that had been isolated Mass mortalities of Astacus astacus were observed in from PacLfastacus len~usculusfrom Lake Tahoe, USA. 1996 in 3 independent sites in southern Germany sep- arated by at least 80 km. In 2 of the outbreaks diseased . KEY WORDS Crayfish . Aphanomyces astaa RAPD-PCR crayfish were collected for further investigation. Previ- ous occurrences of mass mortality or disappearance of native crayfish stocks in southern Germany have also The crayfish plague fungus Aphanornyces astaci been observed (E. Bohl pers. comm.) and the course of Schikora (a member of the Saprolegniales),still consti- disease in these cases, i.e. the duration of mortality and tutes a threat to natural populations of European fresh- total losses, strongly suggests that crayfish plague water crayfish species. The cultivation of farmed Euro- was involved. The distribution of crayfish in Bavaria is pean crayfish species is at risk from disease unless a well documented as a result of stocking surveys (Bohl farm has its own safe water supply, e.g. spring water. 1989). Failing this, the stock is constantly threatened by up- Molecular techniques allow the determination of the stream contamination due to the illegal release of non- degree of relatedness of organisms belonging to one native disease-carrying crayfish or fishing equipment. species. Huang et al. (1994) and Dieguez-Uribeondo et The crayfish plague fungus was introduced into al. (1995) applied arbitrary primers and the polymerase Europe following the introduction of North American chain reaction (PCR) technique to study the genetic crayfish (Soderhall & Cerenius 1992). North American variation between different strains of Aphanomyces crayfish species have a low susceptibility for the dis- astaci. These studies allowed the assignment of 4 ease and may carry the fungus in their cuticle over groups: Group A comprises strains that were isolated long periods of time as a benign infection, but can die from Astacus astacus and also 1 strain from Astacus of plague if the individuals are stressed (Soderhall leptodactylus; these strains are proposed to have been & Cerenius 1992). In contrast, European species are in Europe for a long period of time. Group B includes highly vulnerable to the disease and regularly suc- isolates from both A. astacus in Sweden and Pacifasta- cus leniusculus from Lake Tahoe, USA. In this case, imported P. leniusculus probably served as a vector and infected native A. astacus. Group C consists of a O Inter-Research 1999 Resale of full article not permitted Dis Aquat Org 35: 235-238, 1999 sinale strain isolated from P. leniusculus Table I. Aphanomyces aslaci qenotype-- qroups- as identified from randomly originatingfrom Pitt Lake, canada, G~~~~ amplified polymorphic ~~~-~ol~merasechain reaction (RAPD-PCR)data by D also currently has only 1 member: a Huang et al. (1994),Vennerstrom et al. (1998),Diepez-Uribeondo et al. (1995), Dieguez-Uribeondo (pers. comm.) and this paper. Year: year of isolation strain isolated from Procambarus clarkii in Spain. The aim of the present study was Isolate Year Country Host to investigate whether the German strains could be assigned to 1 of these 4 groups. Astacus strain (Group A) Materials and methods. Fungi: German .I1 1962 Sweden Noble crayfish L1 1962 Sweden Noble crayfish strains M96/1 and M96/2: Two strains of U, 1970 Sweden Noble crayfish Aphanomyces astaci, designated M96/1 Sweden Noble cra;fish and M96/2, were isolated following nat- Sweden Noble crayfish Da Sweden Noble crayfish ural outbreaks of crayfish plague on R, Sweden Noble crayfish Astacus astacus in southern Germanv in Vb Sweden Noble crayfish spring 1996. A1 1988 Turkey Narrow-clawed crayfish K136 1995 Finland Noble crayfish The first isolate, M96/1, originsted from from a pond hatchery fed ~cirastacusstrain (Group Astacus astacus 1970 USA Signal crayfish by a tributary of the river Ammer. No si 1970 Sweden Noble crayfish exotic crayfish had been transferred to the ' Ti i970 Sweden Noble crayfish farm for the previous 10 yr. Seventy dis- SA 1971 Sweden Signal crayfish Yx 1973 Sweden Noble crayfish eased A. astacus 7.5 to 10.5 cm in length, 1974 Sweden Noble crayfish weighing 10 to 20 g, were collected from K121 1995 Finland Noble crayfish this site. FDL457 1990 England White-clawed crayfish FDL458 1990 England White-clawed crayfish The second isolate, M96/2, originated M96,1 1996 Germany Noble crayfish from Astacus astacus from a private pond ~96/2 1996 Germany Noble crayfish hatchery close to the river Kammel. The 1993 Spain White-clawed crayfish hatchery has a spring water supply. Only 3 Pacirastacus strain I1 (Group C) specimens (8 to l1 cm long, weighing 9 to 1978 Sweden Signal crayfish (from 30 g) were recovered following an out- Pitt Lake in Canada) break with associated mass mortality. Procambarus strain (Group D) Pc 1992 Spain Red swamp crayfish Both isolation and identification (incl.ud- ing zoospore production) of the German strains as Aphanomyces astaci were undertaken using fish were transferred into 10 l aquaria, containing aer- the method of Cerenius et al. (1988). Morphological ated tap water and brick tubes that allowed each cray- assignation of the fungi was performed as described by fish to hide. Only intermolt crayfish were used. During DiCguez-Uribeondo et al. (1995). the infection trial, the crayfish were not fed. Individu- Aphanomyces astaci strains for comparative study: als that died during the experiments were examined The strains chosen for comparison with the German for the presence of Aphanomyces astaci hyphae in the isolates were strain L1, isolated in 1962 from an out- cuticle. Detailed experimental conditions are shown in break of crayfish plague in Astacus astacus in Sweden, Table 2. and strain P1, isolated from Pacjfastacus leniusculus DNA preparation: Mycelia were produced by culti- from Lake Tahoe, USA, in 1970. Strain L1 represents vating the fungus at 20°C in 200 m1 PG1 medium, as an Aphanomyces astaci strain which has probably been present in Sweden for a Table 2. Infection trial parameters long time, whereas strain P1 may be typical of Aphanomyces astaci strains that have Parameter been introduced into Europe only recently (see also Table 1). Species Astacus astacus Challenge experiments: Challenge ex- No of ind. in experiment 10 periments were performed with German Water 10 1 Temperature 17°C isolates M96/1 and M96/2 using the Size of ind. 2.0-5.0 cm method of Rantamaki et al. (1992): Clean Strain M96/1 Strain M96/2 Control Astacus astacus were kept in 50 1 aquaria 1 1 in aerated tap water at 17°C for at least Spores ml-' at start of experiment 5150 l700 8 wk before the experiment. One week Motility. of spores- added 30 % 0% before the infection experiment the cray- I Oidtmann et al.: Crayfish plague epizootics in Germany 237 Table 3. Sequence of primers used the German strains can be assigned to Group B, as de- scribed by Dieguez-Uribeondo et al. (1995).This group Primer Sequence contains isolates from Pacifastacus leniusculus origi- nating from California, as well as isolates from Astacus 5'-GTTTCGCTCC astacus which have been infected as a result of proxim- B03 5'-CATCCCCCTG ity to P. leniusculus. This supports the hypothesis that the German outbreaks were caused by a recent intro- duction of Aphanomyces astaci. The most likely route described by Soderhall & Cerenius (1987). DNA was for A. astaci to Germany from the U.S. in recent years extracted using a Nucleon I1 kit following the manu- was probably via Sweden, vectored by P. leniusculus. facturer's (Scotlab) instructions for preparation of DNA The hatchery where strain M96/1 was isolated had from mycelial fungi. not had related disease problems for a period of at least Randomly amplified polymorphic DNA (RAPD)- 16 yr and thus may be regarded as free of crayfish PCR: The RAPD-PCR was performed as described by plague during that time. Wild stocks of Astacus astacus Huang et al. (1994) with slight modifications. The and Austropotarnobius torrentium were observed in RAPD reactions were carried out in a 50 p1 volume con- the upstream drain system prior to the epizootic and taining 1.6 ng fungal DNA, 1.5 mM MgCl,, 5 p1 10-X are currently still present. During the period 1984 to buffer (Arnersham), 200 pM of each dNTP (Pharma- 1989, crayfish stock surveys revealed no occurrence of cia), and 2.5 units of Ampli Taq-polymerase (USB).