The crayfish plague pathogen Aphanomyces astaci

Publications of the University of Eastern Finland Dissertations in Forestry and Natural Sciences

Publications of the University of Eastern Finland

Dissertations in Forestry and Natural Sciences

isbn 978-952-61-1135-3

Jenny Makkonen

The crayfish plague pathogen Aphanomyces astaciGenetic diversity and adaptation to the host species

Crayfish plague, caused by the oomy-

cete Aphanomyces astaci, has caused a

dramatic decline of the native cray-

fish in Europe. The North-American

crayfish species introduced to Europe

are the main vectors of the disease,

which is endemic in North-America.

In this work, genetic diversity, physi-

ological properties and sporulation

dynamics of A. astaci were investi-

gated. The thesis provides new in-

formation about the diversity of the

crayfish plague strains from Finland,

and of the evolving host-parasite

relationship, between this lethal

parasite and its susceptible European

crayfish hosts.

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Jenny Makkonen

The crayfish plague pathogenAphanomyces astaciGenetic diversity and adaptation

to the host species

JENNY MAKKONEN

The crayfish plague pathogenAphanomyces astaci

Genetic diversity and adaptation to the host species

Publications of the University of Eastern FinlandDissertations in Forestry and Natural Sciences

No 105

Academic DissertationTo be presented by permission of the Faculty of Science and Forestry for public

examination in the Auditorium L22 in Snellmania Building at the University of EasternFinland, Kuopio, on June, 14, 2013, at 12 o’clock noon.

Department of Biology

Kopijyvä OyKuopio, 2013

Editors: Profs. Pertti Pasanen, Pekka Kilpeläinen and Matti Vornanen

Distribution:Eastern Finland University Library / Sales of publications

[email protected]/kirjasto

ISBN: 978-952-61-1135-3 (nid.)ISSNL: 1798-5668ISSN: 1798-5668

ISBN: 978-952-61-1136-0 (PDF)ISSN: 1798-5676 (PDF)

Author’s address: University of Eastern FinlandDepartment of BiologyP.O. Box 162770211 KUOPIOFINLANDemail: [email protected]

Supervisors: Professor Atte von Wright, Ph.D.University of Eastern FinlandInstitute of Public Health and Clinical NutritionP.O. Box 162770211 KUOPIOFINLANDemail: [email protected]

Docent Japo Jussila, Ph.D.University of Eastern FinlandDepartment of BiologyP.O. Box 162770211 KUOPIOFINLANDemail: [email protected]

University Lecturer Paula Henttonen, Ph.D.University of Eastern FinlandDepartment of BiologyP.O. Box 162770211 KUOPIOFINLANDemail: [email protected]

Researcher Harri Kokko, M.Sc.University of Eastern FinlandDepartment of BiologyP.O. Box 162770211 KUOPIOFINLANDemail: [email protected]

Reviewers: Professor Emerita Tellervo Valtonen, Ph.D.University of JyväskyläDepartment of Biological and Environmental ScienceP.O. Box 3540014 JYVÄSKYLÄFINLANDemail: [email protected]

Senior Lecturer Lage Cerenius, Ph.D.University of UppsalaDepartment of Organismal BiologyNorbyv. 18A75236 UPPSALASWEDENemail: [email protected]

Opponent: Assistant Professor Ivana Maguire, Ph.D.University of ZagrebDepartment of BiologyRooseveltov trg 610000 ZAGREBCROATIAemail: [email protected]

ABSTRACT

The crayfish plague disease is caused by the oomyceteAphanomyces astaci. The native European crayfish species arehighly susceptible to this disease and their population sizes aregenerally declining. Five genotypes of A. astaci are present inEurope; their original host species are from North America. InFinland, two genotypes, As and PsI, are responsible for thecrayfish plague epidemics. In recent years there have beenongoing discussions concerning reduced virulence of crayfishplague, especially of the As-genotype.

The objectives of this study were to 1) investigate thegenetic variation among the A. astaci strains, 2) to study thedifferences in the physiological properties of the A. astaci strains,especially the variation in their virulence, and 3) to determinethe sporulation of A. astaci during the acute infection of thecrayfish plague in noble crayfish and in chronically infectedsignal crayfish.

The genetic studies revealed that there was lowintraspecific variation in the internal transcribed spacer regionsand therefore, this region is a very suitable target for themolecular detection methods to be conducted at the specieslevel. In the chitinase genes, high genotype specific diversitywas observed and markers for the genotype identification werefound for As, Ps, and Pc-genotypes. The chitinase results alsorevealed that the As-genotype was closer to the Pc-genotype,while the two signal crayfish genotypes, which were identicalbased on the chitinase comparisons, were clearly distinguishablefrom either the As- or Pc-genotypes. The variation observedamong the strains of the As-genotype, also indicated that thepathogen has experienced high selection pressure during itsspread across the Europe.

In this PhD study, significant differences were observedin the virulence of the A. astaci strains. Based on a series ofinfection experiments, the tested A. astaci strains of PsI-genotype killed 100 % of the infected noble crayfish, generallywithin a week. The strains of the As-genotype were more

variable and in several experiments, some of the experimentalcrayfish survived. Minor differences were also observed in theresistance of different noble crayfish populations againstcrayfish plague.

Furthermore, the sporulation dynamics affecting thespread of A. astaci during acute infection and in chronicinfection were quantified. In the noble crayfish with an acutecrayfish plague infection, the maximum peak in the sporulationoccurred 1-3 days post mortem. In the chronically infected signalcrayfish, sporulation was at a constant level and it also occurredin cold water, although the highest spore amounts were releasedunder stress, moulting and death at the warm watertemperatures.

The results of this PhD study emphasize that the furtherspreading of signal crayfish should be prevented. The PsI-genotype of A. astaci is highly virulent and it most likely retainsits virulence, since its original host species has been importedinto Europe. It is also genetically different from the As-genotype. Furthermore, the carrier crayfish constantly sporulateand therefore pose a continuous risk to the surrounding noblecrayfish populations. Moreover, the carrier status analyses ofnoble crayfish should also be considered, when stockings arebeing planned. This would help to avoid the spread of lessvirulent forms of the crayfish plague, since some infected noblecrayfish can act as carriers of the As-genotype crayfish plague.

Universal Decimal Classification: 576.88, 582.244, 591.2, 591.557, 595.384.1

CAB Thesaurus: Aphanomyces astaci; genetic variation; genetic diversity;genotypes; chitinase; virulence; sporulation; spores; experimental infection;disease resistance; mortality; host parasite relationships; adaptation; crayfish;Astacus astacus; Pacifastacus leniusculus

Yleinen suomalainen asiasanasto: rapurutto; geneettinen muuntelu;genotyyppi; itiöt; infektiot; taudinkestävyys; kuolleisuus; sopeutuminen;ravut

Preface

This work was carried out in the Department of Biology,University of Eastern Finland (former Department ofBiosciences, University of Kuopio) during the years 2006-2013.The work was funded by the Finnish Cultural Foundation theNorth-Savo Regional Fund, the Ministry of Agriculture andForestry, EU’s Fisheries Guidance Fund, University of Kuopio,University of Eastern Finland, Kuopio Naturalists’ Society andOlvi Foundation.

I am very grateful to my supervisors Japo Jussila, PaulaHenttonen, Harri Kokko and Atte von Wright for their adviceand encouragement during this scientific learning process. Japoand Harri, your fascination towards the new experiments andthe new results pushed this work into its current level! Paula,your endless support and understanding greatly helped methrough this journey. I also wish to thank Raine Kortet andAnssi Vainikka for participating in the study planning and theirvaluable comments on several manuscripts and Ossi V.Lindqvist for his help with the funding applications andencouragement through all these years.

I sincerely thank the two preliminary examiners of thisthesis, Tellervo Valtonen and Lage Cerenius, for theirencouraging comments, and constructive criticism that theypresented. Thanks also to Ivana Maguire, for accepting the taskof being my opponent. To Ewen MacDonald and RoseannaAvento, I am thankful for their help with the spelling andgrammar of this thesis.

I also want to acknowledge Satu Viljamaa-Dirks fromEvira, for assigning some of the crayfish plague strains into ouruse. Without her help and without the strain collectionconducted by Evira, this work would not have reached itspresent scale. I would also like to thank Trude Vrålstad andDavid Strand for collaboration and co-authorship in thesporulation studies. I would also like to thank Javier Diéquez-

Uribeondo for allowing me to use the A. astaci life cycle picturein this PhD-summary. Thanks also to all the IAA-members, thefriendly atmosphere at the conferences offered a pleasant way tojoin the scientific community! FGFRI, thanks for collaboration.

Many thanks to all my co-workers at the Department ofBiology: Anna Karjalainen, Jouni Heikkinen, Salla Ruuska, AnnaToljamo, Tiina Korkea-aho, Tiina Pitkänen-Arsiola, LiisaNurminen, Jaakko Mononen, Leena Ahola, Marketta Lämsä andmany others, for countless hilarious moments and fruitfuldiscussions over these years!

Special thanks to Hobo Kukkonen, Helena Könönen,Raisa Malmivuori and Elina Reinikainen for their technicalassistance. Thanks also go to the Fish Research Unit staff MarkoKelo, Kauko Strengell and Mikko Ikäheimo for helping me withthe crayfish.

My warmest thanks goes to the students Hanna Kinnula,Laura Koistinen and Lars Granlund, who were all doing a verygood job in taking care of the experiments. Teemu Poutiainen:thanks for the lunch company and for keeping my freezerstocked with pikeperch.

I want to thank my parents Seija and Matti, and mysiblings Pirkko and Timo, and the whole Hankasalmi-Zoo fortheir support, but especially for releasing my thoughts fromwork on the weekends. At this point, I would also like tomention my dearest dog Aatu, whose endless snoring was themost relaxing sound, I could ever imagine during the writing ofthis dissertation, at home.

Finally, Tatu, you have been by me all these years (andso many more), always supporting me, in the end nothing elsereally matters!

Kuopio, May 2013 Jenny Makkonen

LIST OF ABBREVIATIONS

bp base pair

CHC communally housed crayfish

CHI chitinase

Ct threshold cycle

DNA deoxyribonucleic acid

Hd haplotype diversity

IHC individually housed crayfish

ITS internal transcribed spacer

ISSG Invasive Species Specialist Group of IUCN

IUCN International Union for the Conservation ofNature

MGB minor groove binder

NCBI National Center for Biotechnology Information

PCR polymerase chain reaction

PG1 peptone glucose agar

proPO prophenoloxidase

qPCR quantitative polymerase chain reaction

RAPD random amplification of polymorphic DNA

rDNA ribosomal DNA

SNP single nucleotide polymorphism

LIST OF ORIGINAL PUBLICATIONS

This thesis is based on data presented in the following articles,referred to by the Roman numerals I-VI.

I Makkonen J, Jussila J, Henttonen P and Kokko H. Geneticvariation in the ribosomal internal transcribed spacers ofAphanomyces astaci Schikora from Finland. Aquaculture 311: 48-53,2011.

II Makkonen J, Jussila J and Kokko H. The diversity of thepathogenic Oomycete (Aphanomyces astaci) chitinase genes withinthe genotypes indicate adaptation to its hosts. Fungal Genetics andBiology 49: 635-642, 2012.

III Makkonen J, Jussila J, Kortet R, Vainikka A and Kokko H.Differing virulence of Aphanomyces astaci isolates and elevatedresistance of noble crayfish Astacus astacus against crayfish plague.Diseases of Aquatic Organisms, 102: 129-136, 2012.

IV Makkonen J, Kokko H, Vainikka A, Kortet R and Jussila J. Dose-dependent mortality of the noble crayfish (Astacus astacus) todifferent strains of the crayfish plague (Aphanomyces astaci). Journalof Fish Diseases, Submitted manuscript, 2013.

V Makkonen J, Strand DA, Kokko H, Vrålstad T and Jussila J. Timingand quantifying the Aphanomyces astaci sporulation from the noblecrayfish suffering from the crayfish plague. Veterinary Microbiology,162: 750-755, 2013.

VI Strand DA, Jussila J, Viljamaa-Dirks S, Kokko H, Makkonen J,Holst-Jensen A, Viljugrein H and Vrålstad T. Monitoring the sporedynamics of Aphanomyces astaci in the ambient water of latentcarrier crayfish. Veterinary Microbiology, 160(1-2): 99-107, 2012.

Publications are reprinted with permissions from Elsevier(papers I, II, V and VI) and Inter-Research (paper III).

AUTHOR’S CONTRIBUTION

In papers I and II, I performed all the laboratory work and dataanalysis, and I wrote the first version of the manuscript. Inpapers III and IV, I performed the infection experiments, tookpart in the follow-up of the experiments, undertook the samplecollection and analysis and wrote the first version of themanuscript. In papers V and VI, I participated in the studydesign which was made in collaboration with D. Strand and hissupervisors. I also took part in the data collection and conductedthe data analysis in the paper V. I also wrote some parts ofmanuscripts (V & VI).

Contents

1 Introduction ............................................................................. 17

2 Literature review ..................................................................... 192.1 HOST SPECIES...................................................................... 192.1.1 European crayfish species ...................................................... 192.1.2 North-American crayfish species ........................................... 202.1.3 Host responses to the infection .............................................. 212.2 CRAYFISH PLAGUE ............................................................ 222.2.1 The history and the spread of the crayfish plague ................... 222.2.2 The A. astaci spreading routes in nature ............................... 242.2.3 Genotypes ............................................................................. 252.2.4 Life cycle ............................................................................... 272.2.5 Other characteristics ............................................................. 302.2.6 Infection mechanisms ............................................................ 312.2.7 Molecular tools in research and diagnostics ........................... 322.2.8 Genetics ................................................................................ 332.3 AIMS OF THE STUDY ......................................................... 34

3 Materials and methods ........................................................... 353.1 APHANOMYCES ASTACI STRAINS .................................. 353.2 THE CRAYFISH POPULATIONS ....................................... 383.3 STUDY DESIGNS.................................................................. 383.3.1 Genetic variation (I & II) ...................................................... 383.3.2 Infection experiments (III & IV) ............................................ 393.3.3 Sporulation (V & VI) ............................................................ 40

4 Results ...................................................................................... 434.1 GENETIC VARIATION........................................................ 434.1.1 Internal transcribed spacers (I) ............................................. 434.1.2 Chitinase gene (II) ................................................................ 434.2 VARIATION IN THE DISEASE PROGRESS (III & IV) ...... 454.2.1 Virulence variation ............................................................... 45

4.2.2 Resistance variation .............................................................. 484.3 VARIATION IN SPORULATION ....................................... 494.3.1 Noble crayfish (V) ................................................................. 494.3.2 Signal crayfish (VI) ............................................................... 49

5 Discussion ................................................................................ 515.1 GENETIC VARIATION AMONG A. ASTACI.................... 515.2 VARIATION IN THE DISEASE PROGRESS ...................... 555.3 VARIATION IN SPORULATION ....................................... 58

6 Conclusions ............................................................................. 63

References ..................................................................................... 67

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1 Introduction

The crayfish plague, caused by Aphanomyces astaci (Schikora), isthe most devastating crayfish disease known to date (Cereniuset al. 2009). Since the disease arrived in Europe in 1859(Cornalia 1860), it has been responsible for dramatic collapsesin populations in the native European crayfish stocks (Souty-Grosset et al. 2006). The European crayfish species areextremely susceptible to the disease, and are listed in the IUCN(International Union for Conservation of Nature) Red list asthreatened, with a declining population trend (IUCN 2012). Incontrast, A. astaci, is listed as one of the 100 worst invasivespecies in the world in Global Invasive Species Specialist Groupof IUCN (Lowe et al. 2004), and it can be considered as a one ofthe main reasons for the reducing numbers of the nativecrayfish throughout Europe (Souty-Grosset et al. 2006).

A. astaci is a member of the class of Oomycetes, themost famous representatives of which, with huge economicalimportance, are plant pathogens, like the potato late blight(Phytophthora infestans) (Birch & Whisson 2001) and other plantpathogenic species in the genus Phytophthora. Oomycetes notonly include other destructive plant and animal pathogens, butalso saprophytes that are beneficial to the environment(Margulis & Schwartz 2000). The plant pathogenic species havebeen most intensively studied with the recent researchconcentrating on the field of genetics which has attempted toclarify the evolution and virulence mechanisms of thesepathogens (Kamoun 2003; Lamour et al. 2007; Stukenbrock &McDonald 2009; Raffaele et al. 2010; Jiang & Tyler 2012; Raffaele& Kamoun 2012). With respect to the animal pathogenic speciesof Oomycetes, A. astaci has been extensively examined(Cerenius et al. 2009), but details of its genetics are still largelyunknown.

A. astaci is a specific parasite of the crayfish, with noother hosts known to date (Unestam 1972; Oidtmann et al.2002a; Diéguez-Uribeondo et al. 2009). A. astaci spreads clonally

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via swimming zoospores and neither a sexual reproductioncycle nor any resting stages have been unequivocally described(Söderhäll & Cerenius 1999), only the clonal life cycle is known,and it seems to resemble the other animal pathogenic species ofAphanomyces (Diéguez-Uribeondo et al. 2009). Five genotypes ofA. astaci have been recognized to date (Huang et al. 1994;Diéguez-Uribeondo et al. 1995; Kozubíková et al. 2011a). Thegenotype links the pathogen lineage to its original host speciesintroduced to Europe from North America (Kozubíková et al.2011a).

In Finland, observations about variations in the diseaseprogress have been reported from the field from time to time. Insome lakes, some of the population has survived the infectionand eventually the noble crayfish has recovered by itself, evenwith no intensive restockings being made (Nylund & Westman1992). Moreover, in some lakes, the disease progress has beenvery slow and infected individuals have been found for severalyears (Jussila et al. 2011a; Viljamaa-Dirks et al. 2011). In Turkey,the crayfish plague has also similarly been detected from thenarrow-clawed crayfish (Astacus leptodactylus) populations andthis has not been accompanied by any increased mortality orpopulation collapses (Kokko et al. 2012; Svoboda et al. 2012).

In this study, the genetic variation in A. astaci wasinvestigated, especially among the isolates of Finnish origin,and among the two genotypes that are present throughout thecountry (Vennerström et al. 1998). One practical reason for thiswas that the genotype specific genetic markers have long beenin the crayfish researchers’ top 10 -wish list. It is also importantto link the genetic information to the differences observed in thephysiological properties of the A. astaci strains, i.e. to thedifferences in virulence. In addition, the sporulation of A. astaciwas studied during an acute infection of the crayfish plague inthe noble crayfish (Astacus astacus) as well as in chronicallyinfected signal crayfish (Pacifastacus leniusculus).

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2 Literature review

2.1 HOST SPECIES

2.1.1 European crayfish speciesThere are five indigenous crayfish species living in Europe(Astacus astacus, Astacus leptodactylus, Astacus pachypus,Austropotamobius pallipes and Austropotamobius torrentium)(Souty-Grosset et al. 2006). The geographical range of thesespecies has drastically declined, mainly due crayfish plague,interspecific competition and destruction of the habitat(Holdich 2002), or because of the introduction of the non-indigenous crayfish species (Gherardi & Holdich 1999).

In Finland, noble crayfish (A. astacus) is the nativespecies. The original distribution area, determined for the firsttime in 1859, has covered the southern and central parts ofFinland, south from the Kristiinankaupunki-Savonlinna-Sortavala axis, (Järvi 1910), corresponding to approximately 61°parallel of northern latitude (Cukerzis 1988). However, thedistribution has been expanded to cover the whole country byhuman interventions (Cukerzis 1988; Skurdal & Taugbøl 2002;Kilpinen 2003). Genetic studies have shown that thepopulations have been partially mixed all around the country(Alaranta et al. 2006).

The noble crayfish, like all the European crayfishspecies, is highly susceptible to crayfish plague (Unestam1969a; Unestam & Weiss 1970) and the population sizes aregenerally on the decline (Souty-Grosset et al. 2006).Nevertheless, in Finland commercially catchable noble crayfishpopulations still exist in sufficient numbers and thus the aim ofmaintaining the current highly valuable populations still tendsto be viewed from a fisheries management perspective ratherthan from an environmental conservation angle (Pursiainen &Ruokonen 2006; Reynolds & Souty-Grosset 2012).

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2.1.2 North-American crayfish speciesThree North American crayfish species, the signal crayfish (P.leniusculus), the red swamp crayfish (Procambarus clarkii) andthe spiny-cheek crayfish (Orconectes limosus), are now widelydistributed throughout Europe. Furthermore, singlepopulations of the marble crayfish (Procambarus sp.) as well asseveral Orconectes spp. have been discovered (Souty-Grosset etal. 2006). All the North American crayfish are considered ascarriers of A. astaci, although the prevalence of the diseaseseems to vary depending on environmental factors, or otherunknown reasons (Kozubíková et al. 2009; Skov et al. 2011;Vrålstad et al. 2011).

The signal crayfish (P. leniusculus) stockings were startedduring the 1960’s in Sweden (Abrahamsson 1969) and Finland(Westman 1973) to generate new crayfish populations into thelakes, where previous noble crayfish introductions had failed.One of the main reasons for these introductions was that thesignal crayfish was believed to have exceptionally highresistance against crayfish plague (Unestam 1969a; 1972;Unestam & Weiss 1970). Later it was realized that signalcrayfish could act as a chronic carrier of the disease (Persson &Söderhäll 1983); in fact the signal crayfish has been the mainvector and source for the A. astaci infections after itsintroductions into the environment (Huang et al. 1994).

The signal crayfish distribution area (Jussila &Mannonen 2004; Kirjavainen & Sipponen 2004) in Finlandcovers the original distribution area of noble crayfish(Pursiainen & Ruokonen 2006), but illegal introductions haveoccurred also outside the official distribution area (Jussila &Mannonen 2004). The eastern and northern parts of the countryare all that remain for the noble crayfish. There are noblecrayfish populations also existing in the signal crayfish area(Hyytinen et al. 2000; Kirjavainen & Sipponen 2004), but theretheir status is extremely vulnerable (Oidtmann et al. 2002a;Kirjavainen & Sipponen 2004).

Mass mortalities of the signal crayfish attributable to thecrayfish plague are generally uncommon, although the signalcrayfish can also develop an acute and fatal infection of the

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crayfish plague during stressful conditions (Unestam 1972;Persson et al. 1987; Cerenius et al. 1988). In Finland, the firstreported acute mortality and population collapse caused bycrayfish plague was detected in Lake Puujärvi (Karjalohja)signal crayfish, in 1996 (Helsingin Sanomat, 23.8.1996; Tulonenet al. 1998), and since that date the population has not beencompletely recovered. In Sweden, large-scale mortalities havealso occurred in natural waters (Smith & Söderhäll 1986) andthe population densities in the several productive signalcrayfish lakes have been falling in recent years for unknownreasons (Pakkasmaa 2006; Lennart Edsman 2012, personalcommunication).

The signs of the chronic crayfish plague, i.e. melanizedspots and broken limbs (Unestam & Weiss 1970), are commonlyseen in the cuticle of the signal crayfish and if these are present,the marketplace value of the crayfish is reduced (Tulonen et al.1998; Henttonen 2012; Jussila et al. 2013).

2.1.3 Host responses to the infectionInvertebrates lack antibodies and their immune defense relieson the innate immune system (Söderhäll & Cerenius 1998).Moreover, their open circulatory system demands effectiveclotting of any wounds (Söderhäll & Cerenius 1999).

When A. astaci hyphae penetrates through the crayfishbody cavity, �-1,3-glucans of its cell wall are recognized by thecrayfish blood cells via the non-self recognition system. Thislaunches the prophenoloxidase system (proPO-system) which isthe main defense reaction that the crayfish possesses against A.astaci infection (Söderhäll & Cerenius 1998). In this reaction, thepathogen is first encapsulated with semigranular blood cells.Then, a layer of granular blood cells is aggregated around thecapsule and the proPO-system is activated duringdegranulation of the granular cells (Unestam & Nylund 1972).The final outcome of activation of the proPO-system is apathogen surrounded by sticky melanin. Although thepathogens growth and dispersal are restricted, it is still alive(Söderhäll & Cerenius 1999).

The reason for the increased resistance of the signalcrayfish against the crayfish plague is the constantly activated

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proPO-system, which therefore reacts rapidly to the infection(Cerenius et al. 2003). In the noble crayfish, proPO-system willbe only activated, as a response to the infection, but often thereaction is too little and too late to combat successfully againstthe disease (Cerenius et al. 2003). Secondary bacterial or fungalinfections may disturb the constantly ongoing defense reactionand subsequently, also a signal crayfish can die from a A. astaciinfection (Söderhäll & Cerenius 1992). Psorospermium haeckeliinfections may also weaken the signal crayfish immune system(Thörnqvist & Söderhäll 1993).

When the acute stage of the disease is reached, paralysisof the abdomen is often the only visible symptom and thisoccurs 1-2 days prior to death (Unestam & Weiss 1970). It hasbeen postulated that the neurotoxic effects are the major causeof death of the crayfish (Nybelin 1934; Schäperclaus 1954).Other reported symptoms are uncoordinated movement,described as walking on stilts, combined with spasmodic limbtremor and tail movements (Schikora 1906). The duration ofpre-mortality behavior is dependent on the water temperatureand the given zoospore challenge dose (Alderman & Polglase1988).

2.2 CRAYFISH PLAGUE

2.2.1 The history and the spread of the crayfish plagueThe first outbreaks of the crayfish plague (A. astaci) weredetected in Lombardy, Northern Italy, in 1859, where hugenumbers of crayfish suddenly started to die (Cornalia 1860).During the next three decades, the disease swept acrosscontinental Europe (Fig. 1) (Alderman 1996) and reachedFinland via Russia in 1893, when the first mass mortalities weredetected in Lake Saimaa (Järvi 1910). From Finland, the diseasethen spread to Sweden in 1907 and finally to Norway as late as1971 (Alderman 1996).

The vector for this first wave of the crayfish plague isunknown (Huang et al. 1994; Alderman 1996; Kozubíková et al.2011a), although some sources have suggested that it couldhave been the North-American spiny-cheek crayfish (Orconectes

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limosus) which was introduced to Europe in 1890 (Souty-Grosset et al. 2006). However, this theory has been laterdiscounted, because O. limosus carries a different genotype of A.astaci in comparison to that responsible for the first invasion(Kozubíková et al. 2011a). Nonetheless, the original vector wasmost likely an infected crayfish of North-American origin(Unestam 1969a; Unestam 1972; Unestam 1975), either beingintroduced on purpose, or accidentally in the ballast water tankof an Atlantic crossing ship (Alderman 1996).

Figure 1. Spread of the crayfish plague (A. astaci) through Europe with maindistribution areas of the native European crayfish species. The colored regions refer tothe distribution areas of the native species: red refers to A. leptodactylus, blue to A.astacus, yellow to A. torrentium and green to A. pallipes. The map is modified andredrawn after Alderman (1996).

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Soon after its emergence in Finland, the crayfish plaguecaused a dramatic drop in the previously thriving crayfishbusiness and trade to the neighboring countries (Järvi 1910).After the crayfish plague epidemics, crayfish populations wereoften restocked into the lakes, but these often failed forunknown reasons, the infection remained in the lake in a“chronic” form and when the population density of the noblecrayfish (A. astacus) had increased to an exploitable level, thedisease struck again causing a new population collapse (Fürst1995; Erkamo et al. 2010). One proposal for this phenomenonwas the labyrinth structure of Finnish lakes, which offeredprotective areas for subpopulations and enabled a slowmigration of the disease (Westman & Nylund 1978; Nylund &Westman 1992).

As a solution to this problem, re-stockings with signalcrayfish (P. leniusculus) was started during the 1960’s (Westman1973).

2.2.2 The A. astaci spreading routes in natureA. astaci can exist in three forms: mycelium (in the crayfishcuticle), a cyst and a swimming zoospore (in the ambientwater). The infectious stage of A. astaci is the zoospore(Cerenius & Söderhäll 1984a; Oidtmann et al. 2002a). Thepostulated pathways of the transmission are 1) crayfish, 2)contaminated water and items that have been in contact withcontaminated water, and 3) animals that have been feeding onthe infected crayfish (Oidtmann et al. 2002a). During the firstwave of the crayfish plague, man made actions, like the crayfishtrade, were the most likely reason for the rapid dispersal of thedisease (Alderman 1996).

The second, still ongoing wave of the disease started inthe 1970’s and can be traced to introduction of North Americancrayfish species (mainly P. leniusculus, P. clarkii), which in mostcases, carry a chronic A. astaci infection (Persson & Söderhäll1983; Vogt 1999; Oidtmann et al. 2006; Kozubíková et al. 2011b).These introduced species spread the crayfish plague to newareas when new habitats become colonized. The dispersal ratesfor the invasive species seem to be rather high, as in P.

25

leniusculus, a downstream dispersal rate of up to 18-24.4 km yr-1

has been reported (Hudina et al. 2009). The susceptibleEuropean crayfish species can only act as a vector duringcrayfish plague outbreaks (Oidtmann et al. 2002a).

Several treatments and disinfectants have beenexamined in attempts to stop the spread of the crayfish plague.Under natural conditions, liming (Rennerfelt 1936; Svensson etal. 1976) and electrical barriers (Unestam et al. 1972) have beentested with some success, but they have not been sufficientlyeffective. The zoospore production from an infected crayfishcan be prevented with high concentrations of MgCl2, more than20 mM, (Rantamäki et al. 1992).

Powerful chemical treatments can be used in thedisinfection of contaminated equipment, e.g. hypochlorite(Alderman & Polglase 1985) and peracetic acid, which can bealso used to disinfect water in aquaculture or fish transport(Jussila et al. 2011b) as well as in crayfish farming to preventdisease spread among the crayfish individuals (Kouba et al.2012).

2.2.3 GenotypesThe distribution and prevalence of A. astaci in North-America isstill largely unknown, because the infections do not evoke anysymptoms or mortality in its indigenous host species. If newspecies are introduced to Europe in the future, most likely alsonew genotypes will arrive with those animals (Huang et al.1994).

Currently, five genotypes of A. astaci are recognized(Table 1) and have been identified with the random RAPD-PCR-technique (random amplification of polymorphic DNA –polymerase chain reaction), which amplifies a “DNAfingerprint” with arbitrary primers (Huang et al. 1994; Diéguez-Uribeondo et al. 1995; Kozubíková et al. 2011a). Group A (As-genotype) is related to the first invasion of A. astaci, before thestart of signal crayfish (P. leniusculus) introductions. Group B(PsI-genotype) and group C (PsII-genotype) are found from thesignal crayfish, and also from the noble crayfish (A. astacus)again after the signal crayfish introductions. The PsI-genotype

26

is linked to the signal crayfish found in Lake Tahoe and LakeHennessey in USA, which were the main sources for theanimals brought to Sweden (Abrahamsson 1969) and Finland(Westman 1973). A single isolate of the PsII-genotype has beenlinked to the introduction of the signal crayfish from Lake Pitt(Canada) to Sweden (Huang et al. 1994) but this genotype is notwidely present in Europe (Söderhäll & Cerenius 1999). Group D(Pc-genotype) is linked to the red swamp crayfish (Procambarusclarkii) introductions (Diéguez-Uribeondo & Söderhäll 1993;Diéguez-Uribeondo et al. 1995), which were initiated in Spain in1973 (Henttonen & Huner 1999). This genotype also has ahigher temperature optimum in comparison to the threepreviously described genotypes (Diéguez-Uribeondo et al.1995). The group E (Or-genotype) linked to the introductions ofthe spiny-cheek crayfish (O. limosus) was isolated recently byKozubíková et al. (2011a). Although the spiny-cheek crayfishhas not been widely stocked for fisheries management purposes,it has colonized vast areas of central Europe and is inexorablyspreading towards the Baltic and northern Europe (Filipová etal. 2011; Pârvulescu et al. 2012). O. limosus has also beendocumented to coexist with native A. leptodactylus populations(Hudina et al. 2009). The resistance (Schikora 1906; Unestam1969a) and carrier status (Vey et al. 1983) of O. limosus againstthe crayfish plague has been known for a long time, but theisolation process, which is essential in order to undertakegenotyping, is often complicated in the weakly infected NorthAmerican crayfish species (Oidtmann et al. 2006; Vrålstad et al.2009).

Table 1. Genotypes of A. astaci.Genotype Original host Reference

As unknown Huang et al. (1994)

PsI P. leniusculus (Lake Tahoe) Huang et al. (1994)

PsII P. leniusculus (Lake Pitt) Huang et al. (1994)

Pc P. clarkii Diéguez-Uribeondo et al. (1995)

Or O. limosus Kozubíková et al. (2011a)

27

In Finland, As- and PsI-genotypes are present(Vennerström et al. 1998) and both of them are responsible forcrayfish plague epidemics every year. The PsI-genotype hasbeen commonly isolated from the official signal crayfishdistribution area, whereas the As-genotype infections has beendiagnosed from the northern and eastern parts of the country,where viable noble crayfish stocks still exist (Hyytinen et al.2000; Pursiainen & Ruokonen 2006). The As-genotype is alsoknown to be present in Turkey (Huang et al. 1994), butthroughout continental Europe, it is the PsI-genotype formcausing the majority of infections (Royo et al. 2004). At present,the situation is similar in Spain (Diéguez-Uribeondo &Söderhäll 1999), Great Britain (Lilley et al. 1997), Germany(Oidtmann et al. 1999a) and Sweden (Huang et al. 1994).

2.2.4 Life cycleSexual and asexual life cycles are recognized in theAphanomyces spp. The sexual stage guarantees geneticdivergence and a resting phase, while the asexual life cycle isresponsible for the dispersal of the pathogen (Diéguez-Uribeondo et al. 2009). The sexual life cycle is commonly foundfrom the saprophytic and plant pathogenic species ofAphanomyces, but animal pathogenic species (i.e. A. astaci, A.invadans, A. repetans) generally lack this phase (Söderhäll &Cerenius 1999; Royo et al. 2004; Diéguez-Uribeondo et al. 2009).In fact, Rennerfelt (1936) described the sexual structures of A.astaci, but because other researchers have not been able toduplicate the experiments with success, it has been postulatedthat Rennerfelt was perhaps studying some other species ofAphanomyces, or a mixed culture (Unestam 1969b; Söderhäll &Cerenius 1999).

Parasitic species of Aphanomyces spp. can undergorepeated zoospore emergence (RZE) for at least threegenerations (Cerenius & Söderhäll 1984a), which has beenproposed to represent an adaptation to the parasitic life style,because the RZE will confer on the spore a new opportunity tofind a suitable host after a short resting period (Cerenius &Söderhäll 1985). Zoospores of saprophytic Aphanomyces species

28

lack this feature and they tend to germinate very readily(Cerenius & Söderhäll 1985; Söderhäll & Cerenius 1999; Royo etal. 2004). The zoospores orientate towards a variety ofnutritious surfaces, including the soft cuticle of the crayfish, viachemotaxis. The chemotactic effect is similar in manyAphanomyces species, but this does not explain the specificity ofA. astaci as a crayfish parasite (Cerenius & Söderhäll 1984b).

When the zoospores of A. astaci find a suitable host theyencyst and germinate. Encystment can be artificially induced byion or temperature treatment (Svensson & Unestam 1975), aswell as with organic compounds or a mechanical stimulus(Cerenius & Söderhäll 1984a; Cerenius & Söderhäll 1985). Theencysted zoospore germinates forming a penetration peg within1 h (Nyhlén & Unestam 1975). Germination is often successfulin wounds, body openings and in the soft cuticle (i.e. joints,ventral abdominal cuticle) of the crayfish (Unestam & Weiss1970). The hyphae grow inside the cuticle and when the acutestage of the disease is reached, the hyphae can be foundgrowing along the ventral nerve cord (Unestam & Weiss 1970).When the crayfish is dying, the hyphae burst out of the cuticleinto the ambient water and form sporangia. Primary spores areformed in sporangia and then released from the hyphal tip,forming clusters of primary cysts. Secondary swimmingzoospores are released from the primary cysts into the ambientwater (Rennerfelt 1936; Svensson & Unestam 1975; Svensson1978; Söderhäll & Cerenius 1987) (Fig. 2).

29

Figure 2. Life cycle of A. astaci, originally drawn and published by Diéguez-Uribeondo et al. (2006). 1) secondary zoospore (the infective unit), 2) encystingzoospore, 3) crayfish epicuticle, 4) germinating cyst, 5) cuticle penetration, 6a)melanized hyphae (chronic infection in the North-American crayfish), 6b)unmelanized hyphae (acute infection in the native European crayfish species or in theimmune stressed North-American crayfish), 6c) melanized spots in the crayfishcuticle as a macroscopic sign of an infection of A. astaci, 7) sporangium of A. astaci, 8)clusters of primary cysts, i.e. sporeballs, 9) secondary cyst. The zoospore responds tounspecific stimuli forming a secondary cyst that will not germinate and instead willform a new zoospore, i.e. RZE. This process can be repeated up to three timesdepending on the conditions. 10) Non-viable cyst.

30

2.2.5 Other characteristicsThe first successful isolations of A. astaci were made by Nybelin(1934) and Rennerfelt (1936) on crayfish blood agar.Subsequently, several typical characteristics in agar culture forA. astaci have been described. The hyphae are unseptated anduniform (Ø 8-10 μm). Septa are only present when thesporangia are formed. The hyphal tips are rounded and thehyphae grow inside the crayfish cuticle, not on its surface(Cerenius et al. 1988). When the sporangia are formed, thebranches become slightly wider (Ø 10-12 μm) (Alderman &Polglase 1986). Encysted primary spores (Ø 9-11 μm) arespherical and usually there are 15-30 primary spores in eachspore cluster (Alderman & Polglase 1986).

Sporulation of cultured A. astaci hyphae can be inducedunder laboratory conditions by replacing the culture media(Unestam 1965) with lake water (Unestam 1966a; Unestam1969b; Cerenius et al. 1988) or a diluted salt solution (Unestam1966a; Unestam 1969c; Cerenius & Söderhäll 1984a). Zoosporeformation and germination can be enhanced with CaCl2

treatment and inhibited with MgCl2 or KCl (Cerenius &Söderhäll 1984a).

Laboratory tests have shown that zoospores are infectiveif present at temperatures between 2 and 25 °C (Unestam1969b). The optimal temperatures for the sporangial formationand the release of the zoospores lie between 16 and 24 °C but itcan still occur at temperatures as low as 4 °C (Alderman &Polglase 1986). At temperatures between 16 and 24 °C, thezoospores may continue swimming for at least 48 h (Alderman& Polglase 1986). Unestam (1969a) reported that in aquariumconditions at 14 °C, zoospores stay infectious for one week, butat lower temperatures (0 - 10 °C) the survival period is moreprolonged, up to 14 days (Alderman 2000). Under favorableconditions, when the capacity of repeated zoospore emergenceis taken into account, the survival time may be even longer(Cerenius & Söderhäll 1985; Evans & Edgerton 2002). Also thehyphal growth rate (Alderman & Polglase 1986) and thecrayfish mortality rate (Alderman et al. 1987) are temperature-dependent: the optimal temperature for the hyphal growth on

31

RGY-agar is between 22 and 24 °C (Alderman & Polglase 1986).In laboratory infection trials, crayfish mortality occurs morerapidly at temperatures near 20 °C in comparison to lowertemperatures (Alderman et al. 1987). Boiling (100 °C, 1 min) andfreezing (-20 °C, 72 h) of the mycelia (Oidtmann et al. 2002a) orthe infected crayfish (Alderman 2000) kill the pathogen andwater temperatures constantly above 30 °C are also fatal(Oidtmann et al. 2002a).

2.2.6 Infection mechanismsThe chemotaxis observed in A. astaci focus on the joints and thetips of the walking legs of the crayfish (Cerenius & Söderhäll1984b), also the germination and penetration sites are usuallyfound in the soft cuticle and body openings (Unestam & Weiss1970). A germinating spore will secrete proteases when itpenetrates through the epicuticle (Nyhlén & Unestam 1975).The secretion of chitinase starts before the germ tube starts tobranch (18 h after encystment) and the chitinous layers of thecrayfish endocuticle will be reached during the next 1-2 days(Söderhäll et al. 1978). In contrast to the saprophytic species, A.astaci constitutively secretes chitinase during its vegetativegrowth (Unestam 1966b; Andersson & Cerenius 2002).However, chitinase is not expressed in the zoospores, which donot encounter chitin (Söderhäll et al. 1978). Therefore, theconstitutive chitinase production may be an adaptation to theparasitic life style and is a reflection that this specializedparasite is unable to survive without its crayfish host(Andersson 2001). No genotype or strain specific differences inchitinase production have been observed. Chitinase is alsospeculated to be a potential virulence factor, since it has such acritical role during the infection process (Andersson 2001;Andersson & Cerenius 2002).

Some other enzymes that may involve the pathogenesishave also been recognized, i.e. the trypsin proteinase (AaSP2),which is expressed in the mycelia growing in the crayfishplasma. AaSP2 may have a role in combating the crayfishdefense reactions (Bangyeekhun et al. 2001).

32

2.2.7 Molecular tools in research and diagnosticsUntil the 21st century, crayfish plague diagnostics relied on themicrobiological isolation of A. astaci from the diseased crayfishand a successful re-infection of healthy crayfish (Alderman &Polglase 1986; Cerenius et al. 1988). Some modifications to thedescribed method were developed (Oidtmann et al. 1999b;Viljamaa-Dirks & Heinikainen 2006) in attempts to improve thedetection level, which was often poor because of the manypotential contaminants that tended to overgrow the ratherslowly growing A. astaci (Oidtmann et al. 1999b; Edgerton et al.2004; Oidtmann et al. 2004; Oidtmann et al. 2006; Viljamaa-Dirks & Heinikainen 2006; Vrålstad et al. 2009). In Finland,infection experiments were replaced by RAPD-PCR (Huang etal. 1994), which enabled the A. astaci genotype identification, inaddition to the species detection from a pure culture. Eventoday, it is still necessary to conduct new isolations for researchpurposes and genotype identification.

The first PCR-methods for the detection of A. astaciDNA from the crayfish cuticle samples were developed byOidtmann et al. (2002b; 2004), but the methods were notsensitive enough for the detection of A. astaci from the NorthAmerican carrier crayfish. The method was then furtherdeveloped as a more sensitive nested-PCR (Oidtmann et al.2006), which enabled carrier status analyses from the NorthAmerican crayfish, although it lacked specificity, because thenew closely related Aphanomyces species isolated from thecrayfish, Aphanomyces frigidophilus (Ballesteros et al. 2006) andAphanomyces repetans (Royo et al. 2004), also gave a positivesignal in this test (Oidtmann et al. 2006; Ballesteros et al. 2007).

Recently, two specific and sensitive quantitative PCR(qPCR) applications for the A. astaci detection have beendeveloped (Hochwimmer et al. 2009; Vrålstad et al. 2009).TaqMan® Minor Groove Binder (MGB) qPCR targets theinternal transcribed spacer (ITS) regions of A. astaci (Vrålstad etal. 2009). This method is highly sensitive and specific; theclosely related Aphanomyces species do not cause any crossreactions, or false positives (Vrålstad et al. 2009; Tuffs &Oidtmann 2011). The method has also been applied to the A.

33

astaci spore detection directly from water filtrates (Strand et al.2011). Another recent qPCR application is based on thechitinase gene amplification (Hochwimmer et al. 2009). Basedon a comparative study, detection based on the ITS-regions(multicopy region) was reported to be 10-100 times moresensitive, than could be achieved with the chitinase gene(approximately three target copies per genome) based detection(Tuffs & Oidtmann 2011).

2.2.8 GeneticsWhen this study started in 2006, there were few A. astacisequences available in NCBI (National Center forBiotechnology Information) GenBank, and those were mainlysequences of ITS-regions, produced during the development ofthe specific PCR-method (Oidtmann et al. 2002b; 2004). Only asingle chitinase gene sequence was available (Andersson &Cerenius 2002). Since then, the amount of genetic informationhas increased: the raw data for the transcriptome of A. astaci hasbeen recently published in NCBI Genbank in 2013 and also thegenome sequencing project of A. astaci is underway.

Since the RAPD-PCR papers were published (Huang etal. 1994; Diéguez-Uribeondo et al. 1995), it has been obviousthat there is high genetic diversity existing between thedifferent genotypes of A. astaci (Huang et al. 1994; Diéguez-Uribeondo et al. 1995), but the location of these differences inthe genome have remained unknown. Some phylogeneticstudies have been carried out on the ITS-regions, revealing thatdifferent isolates and genotypes of A. astaci are very similar andthe amount of intraspecific variation is near to zero (Diéguez-Uribeondo et al. 2009). This has been postulated to be a result ofthe dispersal via clonal zoospores (Diéguez-Uribeondo et al.2009). The low variation rate on the ITS-regions is alsoadvantageous, when this region is used as a target in thespecies-specific PCR applications (Oidtmann et al. 2006;Vrålstad et al. 2009).

34

2.3 AIMS OF THE STUDY

The aims of the PhD study were to explore the genetic diversityexisting in the different strains of A. astaci, and if possible, tolink this information to the observed differences in thephysiological characteristics and differences in the diseaseprogress in the noble crayfish. Another important reason forstudying the genetic diversity was to find suitable geneticmarkers for the recognition of the different genotypes of A.astaci. In addition, it was desired to compare the sporulationkinetics of A. astaci during the acute and chronic infection.

The specific aims of this study were as follows (with referencesto the publications):

1. To explore the genetic diversity and to developgenotype specific markers for the A. astaci genotypes. (I,II)

2. To investigate under controlled laboratory conditionsthe differences in disease progress among the A. astacistrains isolated from Finland. (III, IV)

3. To study the differences among the Finnish noblecrayfish (A. astacus) populations in crayfish plagueresistance. (III, IV)

4. To compare the sporulation of A. astaci during an acuteand chronic infection. (V, VI)

35

3 Materials and methods

3.1 APHANOMYCES ASTACI STRAINS

Strains of UEF (Table 2) were isolated and maintained in PG1-agar (Unestam 1965) in the University of Eastern Finland;strains from Evira, National Veterinary Institute of Norway(NVI) and Uppsala University were obtained as DNA, with theexception of the isolates Evira6462/06 and Evira8372/09, whichwere obtained as live hyphae to be used in the infectionexperiments. RAPD-genotypes of the Evira strains weredetermined by Viljamaa-Dirks et al. (2013) and of UppsalaUniversity strains by Huang et al. (1994) and Diéguez-Uribeondo et al. (1995).

In the infection experiments (III & IV), four A. astacistrains were used (Table 2). For clarity reasons, they werenamed PsI-Puujärvi (UEF8866-2), As-Kemijoki (UEFT2B), As-Kivesjärvi (Evira6462/06) and As-Pajakkajoki (Evira8372/09).

Tabl

e 2.

A. a

stac

i iso

late

s, th

eir o

rigin

s, iso

latio

n ye

ars,

RAPD

gen

otyp

es, h

osts

and

aut

hors

. Iso

late

s are

of F

inni

sh o

rigin

, if n

ot st

ated

oth

erw

ise.

Hos

ts a

re a

bbre

viat

ed a

s fol

low

s: SC

= sig

nal c

rayf

ish,

NC

= no

ble c

rayf

ish

and

RSC=

red

swam

p cr

ayfis

h. A

utho

rs U

nive

rsity

of E

aste

rn F

inla

nd(U

EF),

Finn

ish F

ood

Safet

y A

utho

rity

Kuop

io R

esea

rch

Uni

t (Ev

ira),

Upp

sala

Uni

vers

ity (U

U) a

nd N

atio

nal V

eter

inar

y In

stitu

te of

Nor

way

(NVI

). In

pap

er I,

inte

rnal

tran

scrib

ed s

pace

r (IT

S) se

quen

ces a

nd in

the p

aper

II, c

hitin

ase g

ene s

eque

nces

are

com

pare

d.

Isola

te c

od

eW

ate

r b

od

y a

nd

/o

r

geog

rap

hic

al

ori

gin

Isola

tion

RA

PD

gen

oty

pe

aH

ost

Au

tho

rS

tud

ied

in

paper

year

III

III

& I

V

UEF7

203

Lake

Kukki

a,

Luopio

inen

2003

PsI

SC

UEF

xx

UEF7

204

Lake

Kukki

a,

Luopio

inen

2003

PsI

SC

UEF

xx

UEF7

208

Lake

Kukki

a,

Luopio

inen

2003

PsI

SC

UEF

xx

UEF8

140-1

Lake

Pyh

äjä

rvi, S

äky

lä2003

PsI

SC

UEF

xx

UEF8

140-2

Lake

Pyh

äjä

rvi, S

äky

lä2003

PsI

SC

UEF

xx

UEF8

140-5

Lake

Pyh

äjä

rvi, S

äky

lä2003

PsI

SC

UEF

xx

UEF8

147

Lake

Pyh

äjä

rvi, S

äky

lä2003

PsI

SC

UEF

xx

UEF8

866-1

Lake

Puujä

rvi, K

arj

alo

hja

2003

PsI

SC

UEF

xx

UEF8

866-2

Lake

Pyh

äjä

rvi, S

äky

lä2003

PsI

SC

UEF

xx

x

UEFT

2B

Riv

er K

emijoki

, Taiv

alk

osk

i2007

na

bN

CU

EF

xx

UEFK

J4R

iver

Kem

ijoki

, Kosk

enkyl

ä2010

na

bN

CU

EF

x

Evi

ra6462/0

6

Lake

Kiv

esjä

rvi, P

altam

o2006

As

NC

Evi

rax

x

Evi

ra8372/0

9

Riv

er P

aja

kkajo

ki,

Kuhm

o2009

As

NC

Evi

rax

x

Evi

ra6672/0

5

Lake

Taula

järv

i, T

am

per

e2005

As

NC

Evi

rax

Evi

ra4426/0

3

Lake

Kel

vänjä

rvi, L

ieksa

2003

As

NC

Evi

rax

x

Evi

ra5596/0

4

Riv

er P

yhäjo

ki,

Ven

etpalo

2004

As

NC

Evi

rax

Evi

ra5727/0

4

Riv

er P

yhäjo

ki,

Joute

nniv

a2004

As

NC

Evi

rax

Evi

ra10278/0

5

Cra

yfis

h f

arm

pond,

Paltam

o2005

As

NC

Evi

rax

Evi

raK105/9

9

Lake

Päijänne, Jy

väsk

ylä

1999

As

NC

Evi

rax

Evi

raK071/9

9

Riv

er L

ieks

anjo

ki,

Lieks

a1999

As

NC

Evi

rax

Evi

raK104/9

8

Lake

Konaanjä

rvi, H

auho

1998

As

NC

Evi

rax

36

Tabl

e2.

Cont

inue

d.

Isola

te c

od

eW

ate

r b

od

y a

nd

/o

r

geog

rap

hic

al

ori

gin

Isola

tion

RA

PD

gen

oty

pe

Host

speci

es

Au

tho

rS

tud

ied

in

year

III

III

& I

V

Evi

raK047/9

9

Lake

Korp

ijärv

i, M

änty

harj

u1999

PsI

NC

Evi

rax

Evi

ra3697/0

3

Lake

Iso

-Kuiv

ajä

rvi, H

art

ola

2003

PsI

NC

Evi

rax

x

Evi

ra7862/0

3

Riv

er P

yhäjo

ki,

Oula

iste

nko

ski

2003

PsI

NC

Evi

rax

x

Evi

ra5721/0

4

Riv

er P

yhäjo

ki,

Mie

lusk

osk

i2003

PsI

NC

Evi

rax

Evi

ra6458/0

3

Lake

Lie

ves

tuore

enjä

rvi, L

ieves

tuore

2003

PsI

SC

Evi

rax

Pl

Lake

Tahoe,

USA

1970

PsI

SC

UU

x

Pc

Spain

1992

Pc

RSC

UU

x

L1Äm

mer

n,

Sw

eden

1962

As

NC

UU

xx

Kv

Sw

eden

c1978

PsII

SC

UU

x

VI0

3629

Ost

fold

, H

ald

en,

Norw

ay

2005

na

bSC

NVI

xx

a RA

PD-g

enot

ypes

pub

lishe

d in

Hua

ng e

t al.

(199

4), D

iégu

ez-U

ribe

ondo

et a

l. (1

995)

and

Vilj

amaa

-Dir

ks e

t al.

(201

3).

bN

ot a

naly

zed

with

RA

PD.

cIs

olat

ed in

Sw

eden

from

sign

al c

rayf

ish

whi

ch o

rigi

nate

d fr

om P

itt L

ake,

Can

ada.

37

38

3.2 THE CRAYFISH POPULATIONS

The crayfish populations used in the infection experiments andsporulation studies are presented in Table 3.

Table 3. Crayfish species and populations, crayfish amounts and catchment years usedin the experiments.

Species Origin n Year Paper

Noble crayfish (A. astacus) ESeppä crayfish farm, Haapavesi 60 2010 III

Noble crayfish (A. astacus) ESeppä crayfish farm, Haapavesi 9 2010 V

Noble crayfish (A. astacus) Lake Rytky, Kuopio 94 2010 III

Noble crayfish (A. astacus) Lake Viitajärvi, Tervo 138 2010 III

Noble crayfish (A. astacus) Lake Viitajärvi, Tervo 120 2011 IV

Noble crayfish (A. astacus) Lake Mikitänjärvi, Hyrynsalmi 84 2011 IV

Noble crayfish (A. astacus) Lake Koivujärvi, Kiuruvesi 101 2011 IV

Signal crayfish (P. leniusculus) Lake Saimaa 100 2010 VI

Signal crayfish (P. leniusculus) Crayfish farm, Orivesi 100 2010 VI

3.3 STUDY DESIGNS

3.3.1 Genetic variation (I & II)In paper I, internal transcribed spacer (ITS) -regions 1 and 2surrounding the 5.8S rRNA gene of 18 A. astaci strains weresequenced and compared. In paper II, partial chitinase genesequences were produced and studied from 28 A. astaci strains(Table 2).

In both studies (I & II), the target fragment of A. astaciDNA was first amplified with the polymerase chain reaction(PCR) with suitable primers. For the ITS-regions, universalprimers ITS1 and ITS4 (White et al. 1990) producing a 720 bpamplicon were used. For the sequencing of the chitinase gene,primers AaChiF and AaChiR, producing a 384 bp PCR ampliconcontaining a partial chitin binding site and partial catalyticdomain, were designed (paper II).

PCR reactions were carried out from DNA extracted fromcultured mycelia of A. astaci. Detailed descriptions of themethods for the DNA extraction, PCR amplification, cloning andsequencing are given in papers I and II. Sequencings were madefrom PCR amplicons directly and after a molecular cloning of

39

the PCR amplicon into a plasmid vector. The initial aim was toobtain a general view of the studied genetic region of each strainand subsequently to conduct a molecular cloning process inorder to obtain more detailed information of the internalvariation inside a multicopy region or -gene.

Sequence analysis and alignments of ITS regions werebuilt with MultAlin (Corpet 1988) and ChromasPro 1.41(Technelysium Ltd., Australia) computer programs. BioEdit 7.0.8was used to calculate nucleotide differences (Hall 1998) andDnaSP (Librado & Rozas 2009) for the divergence parameters.Chitinase gene sequences were analyzed and aligned withGeneiousPro 5.3.6 (Drummond et al. 2011). Phylogeneticmaximum likelihood trees were made with PhyML (Guindon &Gascuel 2003) and the possible recombination events weredetermined with DualBrothers (Suchard et al. 2003; Minin et al.2005).

The sequences produced in these studies are available inNCBI (National Center for Biotechnology Information) GenBankwith access numbers GU320213-GU320248 (I) and JQ173169-JQ173371 (II).

3.3.2 Infection experiments (III & IV)A total of six infection experiments were conducted to testdifferent combinations of crayfish populations and A. astacistrains. The experiments were carried out on the largest possiblescale, but it was not possible to implement a full cross-examination of the different combinations, because of the limitedtime, space and hands.

Zoospore emergence from the cultured mycelia of A.astaci was induced by lake water washings as described byCerenius et al. (1988). In the six infection experiments (III & IV),zoospore densities varied between 260 and 2440 spores ml-1,except in the experiment that was conducted with LakeKoivujärvi crayfish, where different zoospore densities (1, 10,100 and 1000 spores ml-1) were tested (Table 4). The infectionexperiments were conducted in an air-conditioned laboratoryand the temperature was adjusted to a constant + 18 °C.

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Table 4. Crayfish populations, A. astaci strains and zoospore densities used in theinfection trials.

Expt Crayfish population A. astaci strain Spores ml-1

1 L. Viitajärvi & L. Rytky PsI-Puujärvi, As-Kemijoki 2 250

2 F. ESeppä & L. Viitajärvi As-Kivesjärvi, As-Pajakkajoki 2 440

3 L. Rytky & L. Viitajärvi As-Kivesjärvi, As-Kemijoki 810

4 L. Viitajärvi PsI-Puujärvi, As-Kivesjärvi, As-Kemijoki 670

5 L. Mikitänjärvi PsI-Puujärvi, As-Kivesjärvi, As-Kemijoki 260

6 L. Koivujärvi PsI-Puujärvi & As-Kivesjärvi 1, 10, 100, 1000

During these experiments, the water quality, thebehavior and any possible deaths of crayfish were monitoreddaily. Dead crayfish were removed immediately to avoidsecondary infections by zoospores being released frommoribund individuals. The dead crayfish were individuallypacked and stored in a freezer (-20 °C) for further DNAextractions and qPCR analyses (Vrålstad et al. 2009) of theinfection status. Detailed descriptions of the methodologicaltechniques are explained in the relevant articles (III & IV).

The infection statuses of the crayfish were classifiedaccording to Vrålstad et al. (2009) as follows: agent levels A0 (nodetection) and A1 (threshold cycle, Ct >39) were assessed as thenegative results and amplification below the limit of detection,respectively. Agent level A2 corresponded to very low levels ofA. astaci DNA and the detection was below the limit ofquantification (Ct 39.0-34.7). Agent level A3 indicated thepresence of low levels of A. astaci DNA (Ct 34.6-30.0) and A4designated a moderate infection status (Ct 30.0-26.2).

Kaplan-Meier survival test (Log Rank, Mantel-Cox) wasused to evaluate differences in mortality among experimentalgroups (SPSS 17.0). A non-linear regression was used to studythe effect of inoculation spore dose in the Expt 6.

3.3.3 Sporulation (V & VI)In the sporulation studies, three different experimental setupswere used. First (V), the noble crayfish (n=9) were infected withA. astaci strain PsI-Puujärvi (UEF8866-2) in a separate spore bath(20 h) containing 1820 spores ml-1 in lake water. After incubation,

41

the crayfish were rinsed in fresh water for 1 h and then rinsedagain in fresh clean water before their transfer into theindividual monitoring tanks, which contained 8 l of fresh andspore-free lake water. The laboratory temperature was set at +18 °C.

The infected crayfish were monitored twice a day (9.00am and 21.00 pm) and simultaneously, 1 ml water samples werecollected in triplicate from each tank. Water samples wereimmediately frozen (-20 °C) for further spore quantity analyseswith qPCR (Strand et al. 2011). When a moribund crayfish wasobserved, the water circulation was immediately stopped toavoid the spread of the spores to the adjacent monitoring tanks.The sample collection was continued for seven days postmortem.

Quantitative PCR analyses were run from freeze-driedspore eluates in a method described in detail in paper V. Eachsample was analyzed as non-diluted (1x) and diluted (10x) DNA,to monitor possible inhibition in the PCR reaction. The amountof A. astaci ITS copies in a sample was estimated as PCR formingunits (PFU, the amount on target DNA copies in a genome),according to Vrålstad et al. (2009), which was then translatedinto spores by dividing by 177. The amount of PFU’s per spore(177) was estimated by a dilution series from freeze-dried sporessimilarly as described by Strand et al. (2011).

In addition, there were two separate experimentsconducted to determine A. astaci sporulation of the signalcrayfish (P. leniusculus) from two locations (a crayfish farm inOrivesi and Lake Saimaa) with a known carrier status of A. astaci(VI).

Two experiments were made with both populations: theIHC (individually housed crayfish) experiment consisted of tenreplicate individual tanks, each containing 2 l of lake water at+18 °C temperature and a similar setup at +4 °C temperature.The CHC (communally housed crayfish) experiment consisted offour replicate tanks at room temperature (17-23 °C). Each tankcontained 20 signal crayfish in 100 l volume of lake water.

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In both experiments, water samples were collectedweekly: in the IHC-setup, a 1 l water sample was collected fromeach 2 l tank. In the CHC-setup, three 1 l water samples (twosamples from 5 cm below the water surface and one sample bysiphoning the bottom of the tank) were collected from each tank.After the sampling, the crayfish were fed and two days later, thetanks were cleaned and new water was added. Hence, eachsample represented the spore amount released from the signalcrayfish during a period of five days.

The amount of A. astaci spores in the water samples werequantified with vacuum filtration (3 μm polycarbonate filters,Millipore IsoporeTM) and DNA extraction (Strand et al. 2011) wasmade from the filtrate. Quantitative PCR (Vrålstad et al. 2009)was applied as quadruplicate (duplicate undiluted and duplicate10x diluted) for each sample. Detailed criteria and themethodology for the estimation of spore amounts in the samplesare explained in the paper VI.

Statistical analyses were made with R (2.13.2), where alinear mixed-effect model was applied to estimate the sporenumbers (V & VI). The crayfish populations were comparedwith the logistic regression model (VI).

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4 Results

4.1 GENETIC VARIATION

4.1.1 Internal transcribed spacers (I)In total, 32 sequences from 17 Finnish A. astaci isolates and oneSwedish isolate (Table 2) were produced (I). The sequences thatwere generated directly from the PCR amplicons (n=13), were allidentical, i.e. no genotype or strain specific mutations wereobserved. Among the cloned sequences (n=23), several singlenucleotide polymorphisms (SNP) were found. Altogether, 31SNPs were counted in the 720 bp region, which representedpolymorphism level of 3.2 % among the sequence set cloned inthis study. The highest levels of polymorphisms (5.2 %) wereobserved in the ITS1-region, but some polymorphisms (n=3,polymorphism level 1.9 %) were also detected in the conserved5.8S rDNA gene. In the ITS2-region, polymorphism level was4.2 % (I, table 3).

Intragenomic variation (variation between the differentcopies of the multicopy region in the genome) was observed inthe strains UEF8822-2 and UEF7208, from which various cloneswere produced. Haplotype divergence (Hd) inside a strainUEF8866-2 was actually slightly higher (Hd=0.833) than theintraspecific variation (variation among the different isolates)among the isolates of PsI-genotype (Hd=0.800). In addition,divergence was higher in the As-genotype (Hd=1.000) incomparison to the PsI-genotype (Hd=0.800).

4.1.2 Chitinase gene (II)Chitinase gene sequences (n=203) were obtained from 28 A.astaci strains (Table 2). I.e., 176 were cloned and 27 sequenceddirectly after the PCR amplification.

Among the PCR amplicons, 14 SNPs classified the As-and PsI-genotypes and four SNP differences were observed

44

between the As- and Pc-genotypes (Fig. 3A). The two signalcrayfish (P. leniusculus) related genotypes PsI and PsII wereidentical and were therefore subsequently combined as Ps-genotypes.

The cloned sequences were divided into three groups(CHI1, CHI2 and CHI3) in a maximum likelihood (ML) analysismade with PhyML (Guindon & Gascuel 2003). Moreover, theCHI2-group was divided into three subgroups, named CHI2A,CHI2B and CHI2C (Fig. 3B).

Group CHI1 (n=72) was the most commonly detectedgroup, each strain studied had CHI1-sequence(s) present in itsgenome. CHI2 was the most heterogenic group, the subgroupsdescribed above were divided as follows: CHI2A was the mostcommon group (n=38) and it was present in both genotypes Asand Ps, whereas the subgroups CHI2B (n=11) and CHI2C (n=9)were only found from those strains belonging to the As-genotype. Group CHI3 (n=46) was the most homogenic groupand it was observed only in the Ps-genotypes.

Based on the typical nucleotide changes for each group,group CHI2C of the As-genotype could have been named asgroup CHI1B as well, since this group displayed the typicalnucleotide changes observed in both groups CHI1 and CHI2.However, based on the results of the ML-analysis, the group wasnamed as CHI2C (Fig. 3B). Possible recombination sites werealso detected between the CHI2A and CHI3 groups, among thestrains of Ps-genotype.

Chitinase sequencing can be applied also in genotypingpurposes as described in paper II, i.e. using PCR to amplify theDNA extracted from the crayfish cuticle samples. This method isapplicable in cases where there is severe infection and highamounts of A. astaci DNA present in the crayfish cuticle. If theanalysis is made after TaqMan® MGB qPCR (Vrålstad et al. 2009),which is the recommendation, this corresponds to amplificationwith less than 30 cycles (A3 agent level, > 1000 PCR formingunits).

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Figure 3. Differences observed in the chitinase sequences classify the differentgenotypes of A. astaci. A) Location of the polymorphic sites in the PCR-amplicons. B)Maximum likelihood tree (Guindon & Gascuel 2003) of cloned sequences showing thethree main chitinases (CHI1, CHI2 and CHI3) and the division of CHI2 into threesubgroups (A, B and C).Figure 3B printed with permission from Elsevier.

4.2 VARIATION IN THE DISEASE PROGRESS (III & IV)

4.2.1 Virulence variationExtensive variation in the virulence among the A. astaci strainswas observed in the six infection experiments (Table 5). PsI-Puujärvi, which was the only PsI-genotype strain used in theseexperiments, killed 100 % of the infected noble crayfish withinless than one week. Only one exception was observed, in a LakeMikitänjärvi crayfish population where deaths started 11 daysafter the inoculation and 100 % mortality was only achieved at

B)

A)

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day 37. The high virulence of PsI-Puujärvi was also present inExpt 6, with different spore dosages: 1 spore ml-1 density of PsI-Puujärvi spores killed 100 % of the Lake Koivujärvi crayfishmore rapidly than 1000 spores ml-1 of As-Kivesjärvi spores (Fig.4).

The As-Kivesjärvi strain was used in five infectionexperiments, of which in three experiments, 100 % mortality wasachieved during the follow-up period. In two experiments (Expt4 & 5) made with Lake Viitajärvi and Lake Mikitänjärvi crayfishand a slightly lower spore dosage (670 and 260 spores ml-1,respectively), in Lake Viitajärvi crayfish cumulative mortalityreached 50 % during the 49-day follow-up and in LakeMikitänjärvi crayfish, only 9 % mortality occurred during the 99-day follow-up. In contrast, there was 100 % mortality in LakeKoivujärvi crayfish infected with As-Kivesjärvi in the dose-effectexperiment, with all the different spore dosages (1-1000 sporesml-1) being tested (Fig. 4). Strain As-Pajakkajoki was used only ina single infection experiment, where it evoked 100 % mortality inViitajärvi and ESeppä crayfish, and there was no statisticallysignificant difference in comparison with the As-Kivesjärvistrain.

The As-Kemijoki strain was used in four of the sixinfection experiments. Increased mortality was observed only ina single experiment (Expt 1) and also in that case, only in theLake Rytky crayfish population. Lake Viitajärvi crayfish, testedsimultaneously under the same conditions and with the samespore batch, remained viable. In Expt 3, 17.0 % mortality wasobserved in Lake Viitajärvi crayfish, while in Lake Rytkycrayfish, the mortality was much less, only 4.5 % (one crayfish).

Based on the quantitative PCR (Vrålstad et al. 2009)analyses (III & IV), the crayfish infected with As-Kemijokiremained mainly negative, and thus were uninfected, oralternatively only very low agent levels (A1-A2, indicating A.astaci DNA amount of less than one spore) were detected.

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Table 5. Crayfish populations and numbers (n), A. astaci strains and spore densitiesused in the infection experiments. The mean day of death refers to the number of daysafter inoculation, when 50 % mortality was achieved. Mortality rate (%) describes thetotal mortality achieved during the follow-up period. Corrected day of death (DoD)refers to the estimated day of death, when the effect of the zoospore density is excluded.

Population n StrainSpores

mL-1

Day of Death (DoD)

Mean±SD

(days)

Mortality

rate (%)

Corrected

DoD2

Expt 1LakeViitajärvi

12 Control 43.01 0.0 n/d

12 As-Kemijoki 2 250 43.01 0.0 14.4

12 PsI-Puujärvi 2 250 5.2±0.1 100.0 3.3

LakeRytky

12 Control 42.3±0.7 8.0 n/d

12 As-Kemijoki 2 250 27.0±2.3 84.0 n/d

12 PsI-Puujärvi 2 250 5.8±0.1 100.0 3.3


12 Control 25.3±0.7 8.0 n/d

24 As-Kivesjärvi 2 440 10.2±0.4 100.0 14.0

24 As-Pajakkajoki 2 440 15.8±1.0 100.0 n/d

FarmedEseppä

12 Control 22.8±1.5 33.0 n/d

24 As-Kivesjärvi 2 440 13.2±0.8 100.0 14.0

24 As-Pajakkajoki 2 440 17.5±0.9 100.0 n/d

Expt 3LakeRytky

12 Control 50.3±0.7 8.5 n/d

22 As-Kemijoki 810 50.4±0.6 17.0 n/d

24 As-Kivesjärvi 810 15.6±0.5 100.0 19.4

LakeViitajärvi

6 Control 51.0±0.0 0.0 n/d

12 As-Kemijoki 810 44.5±4.2 4.5 n/d

24 As-Kivesjärvi 810 16.6±0.5 100.0 19.4


12 Control 47.01 0.0 n/d

18 As-Kemijoki 670 47.01 0.0 n/d

18 As-Kivesjärvi 670 15.1±1.8 50.0 20.3

18 PsI-Puujärvi 670 4.9±0.1 100.0 4.8

Expt 5LakeMikitänjärvi

12 Control 93.5±4.5 18.2 n/d

24 As-Kemijoki 260 97.7±1.3 4.6 n/d

24 As-Kivesjärvi 260 77.4±8.7 4.6 24.8

24 PsI-Puujärvi 260 20.4±1.8 100.0 5.9

1 No deaths occurred in the group, the mean refers to the length of the follow-up period.2 n/d, not determined.

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Figure 4. Mortality of Lake Koivujärvi noble crayfish (A. astacus) infected with PsI-Puujärvi and As-Kivesjärvi strains of the crayfish plague (A. astaci) at differentzoospore densities (1, 10, 100 and 1000 spores ml-1).

4.2.2 Resistance variationIncreased resistance towards the strains of the As-genotype wasobserved in several infection experiments, especially in thoseconducted with low spore dosages (less than 1000 spores ml-1 inthe experiments 3, 4 and 5). No gender- or size-relateddifferences in resistance were observed.

Between the experiments 4 (Lake Viitajärvi) and 5 (LakeMikitänjärvi), results were compared among each other using theestimated dose-response curve (IV). The day of death given sporedose corrections indicated that Lake Mikitänjärvi noble crayfishhad a significant time lag in the observed day of death, as thePsI-Puujärvi infected crayfish died approximately 3.5x later thanthe model predicted. Lake Mikitänjärvi crayfish infected with As-Kivesjärvi exhibited no mortality whereas the model predictedan average 24.8 day of death. The Lake Viitajärvi noble crayfish,on the other hand, died on the average slightly earlier during As-Kivesjärvi infection, in 25 % less time, than predicted given theequal inoculation dose, while the observed day of death duringPsI-genotype was similar to the estimate from the dose-responsecurve.

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4.3 VARIATION IN SPORULATION

4.3.1 Noble crayfish (V)Those noble crayfish suffering an acute and fatal infection of A.astaci (PsI-genotype) were producing a high amount of spores,the infective units of A. astaci, into the ambient water system.Some sporulation was observed already 48 h premortem and then,24 h post mortem, the sporulation started to increase and itreached its maximum at 48 h and it remained significantly higherin comparison to premortem levels until 96 h post mortem. In thisexperimental setup, the sporulation level declined below thepremortem levels at 120-156 h post mortem.

Based on this data, it was estimated (mean with 90th and10th percentile) that a noble crayfish having an acute infection ofA. astaci, could release 1.7 million spores (573 000; 2 829 000) intothe ambient water during the sporulation peak. The biggestnumber of spores released by a single crayfish individual wasestimated at approximately 3.2 million spores passing into theambient water during this sporulation peak, if one assumes ahomogenous distribution of the spores.

4.3.2 Signal crayfish (VI)The estimated number of spores (mean with 90th and 10th

percentile) released over five days were 30 698 (43, 75 101) for theindividually housed crayfish and 9 577 (161, 22 789) for thecommunally housed crayfish. As an overall mean estimate, asignal crayfish individual that was carrying A. astaci (without anacute infection) released 22 106 (67; 28 466) spores during a fiveday time period. Higher variation in the amount of sporulationwas observed among the individually housed crayfish than inthe communally housed crayfish, which most likely indicatedthat there was extensive variation in extent of sporulationbetween different signal crayfish individuals.

Those signal crayfish that survived until the end of theexperiment released 2 763 (26; 5 490) spores per week in theabsence of deaths and moultings. A significant increase in thesporulation was observed one week prior to their death. The

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trend was detected in 14 of the 18 crayfish, but it was absent infour crayfish. Increased sporulation was observed also duringthe moulting events. The substitute crayfish (n=7), that wereadded to the tanks during the experiment to replace the deadindividuals, released high spore levels during the first 1-2 weekswhen they were introduced into the experimental system.

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5 Discussion

5.1 GENETIC VARIATION AMONG A. ASTACI

The ITS-regions of the A. astaci strains were highly similar (I). Infact, the sequences of PCR-amplicons (produced withoutmolecular cloning) were 100 % identical. In the cloned sequences,96.8 % similarity was observed confirming the results ofprevious studies (Oidtmann et al. 2004; Diéguez-Uribeondo et al.2009; Vrålstad et al. 2009). The difference in the similaritypercentages obtained with two different approaches is aconsequence of the methodology: the PCR amplicon sequencepresents a combined result of the most common sequence in themulticopy region, while the sequence obtained after molecularcloning process represents a single, randomly selected ITS-copy(Ganley & Kobayashi 2007). Nonetheless, minor differencesamong the different copies, so called intragenomic variation, doexist (Gandolfi et al. 2001; Ganley & Kobayashi 2007) and it hasbeen estimated that between 138 and 177 (Strand et al. 2011) ITS-copies are present in the genome of A. astaci.

In some Aphanomyces species, also intraspecificvariations in the ITS-regions have been reported (Diéguez-Uribeondo et al. 2009). It has been postulated that the mainmechanism for the homogenization of ITS copies isrecombination via concerted evolution (Elder & Turner 1995;1996) with the diversity evolving during the asexual generations(Gandolfi et al. 2001). On the other hand, it has also beenclaimed that the laboratory cultivation and the lack of sexualrecombination may diminish the variation and the number ofrDNA copies affect the polymorphism levels (Ganley &Kobayashi 2007). Based on the study of Diéguez-Uribeondo et al.(2009), the saprophytic and plant parasitic species ofAphanomyces with a known sexual life cycle display a higherintraspecific variation level in comparison to the clonally

52

spreading animal pathogenic lineage of Aphanomyces. Theseresults are at odds with the current theory of concertedevolution. It has also being claimed that concerted evolutionmay not be as efficient as has been suggested, since in somespecies of fungi, surprisingly high intragenomic variation hasbeen observed (Simon & Weiß 2008).

Moreover, the low level of intraspecific variation in A.astaci is a highly advantageous feature, because most of thecurrent molecular techniques used in the detection of A. astacitarget the ITS-regions. If there were high intragenomic- andintraspecific variation, this would lead to false negativediagnoses and to unreliability of these detection methods(Oidtmann et al. 2006; Vrålstad et al. 2009; Tuffs & Oidtmann2011).

In the chitinase genes of A. astaci, several genotypespecific marker sites were detected (II). Based on the previousstudies, there seem to be at least 3-4 chitinase gene copiespresent in the genome of A. astaci (Andersson & Cerenius 2002;Hochwimmer et al. 2009) and the observed variation can be dueto either differences between the gene copies, or to allelicvariation (heterozygosis), also known as the single nucleotidepolymorphisms (SNPs).

In the PCR amplicons, 14 polymorphic sitesdistinguished the As- and Ps-genotypes. The two signal crayfish(P. leniusculus) related genotypes (PsI and PsII) were identical,which might indicate that the host species has a greaterinfluence on the genotype than the geographical origin of thecrayfish population in North America.

One interesting finding was the similarity between theAs- and Pc-genotypes, which were distinguished only by foursites. Based on the chitinase gene sequences, these genotypeswere similar, although in RAPD-PCR they were clearlydistinguishable (Huang et al. 1994; Diéguez-Uribeondo et al.1995), therefore the differences must be present in other geneticlocations. Transcriptome comparisons are likely needed to arriveat definite conclusions. Nevertheless, it would be tempting tospeculate that the original host of the As-genotype may have

53

been closer to Procambarus spp. than Pacifastacus spp. This is alsosupported by the theory of accidental transport of the vectorspecies in the ballast water tanks of the ships (Alderman 1996),since the European-bound ships left from the east coast of NorthAmerica, where the procambarids are the native species (Hobbs1988).

The comparison of the chitinase sequences producedwith a molecular cloning process indicated the presence of threegroups of chitinases, possibly being derived from three differentgenes. Some of the chitinase forms were specific for either theAs-genotype (CHI2B, CHI2C) or the Ps-genotypes (CHI3). Theobserved differences most likely indicate that the genotypeshave co-evolved with their original host species for a rather longtime. Altogether, the As-genotype seemed to be more variable incomparison to the Ps-genotypes. This result is logical, since theAs-genotype of A. astaci is constantly undergoing a higherselection pressure in comparison to the Ps-genotypes. This maylead to lowered virulence and a better adaptation to the newhost species (Ebert 1994), because based on current knowledge,its original host species was not widely introduced in Europe(Souty-Grosset et al. 2006; Kozubíková et al. 2011a). In general,host jumps are known to accelerate the evolution speed inOomycetes (Dodds 2010; Raffaele et al. 2010), since thepathogens need to adapt to their hosts by altering their virulence(Ebert 1994). Another possible explanation for the observedvariation could be the multiple introductions of the As-genotypeinto Europe, thus having a wider origin in comparison to Ps-genotypes.

Furthermore, the group CHI2C of the As-genotypeincluded a SNP which was introduced a translation stop codoninto the beginning of the catalytic domain of the gene; thereforethe As-genotype seems to have a nonfunctional chitinase copy inits genome. Chitinase is believed to be a potential virulencefactor of A. astaci, because it has an essential role in thepenetration into crayfish cuticle (Andersson & Cerenius 2002).Some chitinases may also have a role in the cell wall modulatingprocesses. The observed genetic differences may be one possible

54

reason for the differences in the virulence of these genotypes.Based on the studies conducted on the plant pathogenicOomycetes, adaptation to the new host species is most likely tocause mutations and copy-number changes in hundreds ofeffector genes, located in the variable repeat-rich partitions ofthe genome, which are linked to the pathogenicity and the hostrange (Jiang & Tyler 2012).

The observed differences in the chitinase gene sequencescan also be used for genotyping purposes. The genotyping withchitinase PCR can be made directly from the crayfish cuticlesamples. This method is not as sensitive as qPCR, but it can beutilized in the cases of acute infections or clear visible symptomsof a crayfish plague, when high amounts of A. astaci hyphae arepresent in the crayfish cuticle. For example, it was possible todetect the As-genotype infection (unpubl. results) from theTurkish narrow-clawed crayfish (A. leptodactylus) displayingsymptoms of the crayfish plague, such as intense melanisation,but no acute mortality (Kokko et al. 2012).

Both of the genetic regions studied here have been alsolinked to the A. astaci diagnostics. Homogenic ITS-regionsproved suitable for the detection at the species level, while thechitinase gene sequencing, albeit with more limited sensitivity(Tuffs & Oidtmann 2011), could be used in the detection of theknown genotypes. Unfortunately, the recently isolated Or-genotype (Kozubíková et al. 2011a) was not available when thisstudy was conducted and thus the information of its chitinasegene variability is not available.

Furthermore, a recent study by our research group hasshown that the genetic diversity detected in the chitinase gene ofA. astaci represents only a tiny proportion of the true geneticvariation existing between the As- and Ps-genotypes. A largeamount of new polymorphic sites have been identified all overthe genome, when the complete transcriptomes of A. astacigenotypes are compared (unpubl. results).

55

5.2 VARIATION IN THE DISEASE PROGRESS

Based on the experimental series of six A. astaci infections (III &IV), a significant virulence variation between the As- and thePsI-genotypes could be demonstrated. The PsI-Puujärvi strainwas an aggressive killer and 100 % acute mortality with thetested zoospore densities was generally achieved within a week,although in the previous experiments, also occasional survivorshave been observed (Jussila et al. 2011b). Based on the latestexperiment (unpubl. data), a rapid 100 % mortality, similarly asdescribed in the experiments made here with PsI-Puujärvi, hasalso been detected with several other PsI-genotype strains of A.astaci.

The tested As-genotype strains were more variable intheir virulence features, the symptomatic stage started later andlasted longer and the acute mortality did not appear as rapidlyas observed with the PsI-Puujärvi. As-Kivesjärvi was the mostvirulent of the tested As-genotype strains, while the As-Pajakkajoki provoked fatalities somewhat later, although thedifference was not statistically significant (III). Although themortality rate of As-Kivesjärvi and As-Pajakkajoki strains wasslower in comparison to the PsI-Puujärvi infections, both strainscaused 100 % mortality in two experiments (III, Expts 2 & 3). Inthe next experiments (IV, Expts 4 & 5), As-Kivesjärvi infectionswere not as efficient, since 50 % mortality occurred in LakeViitajärvi crayfish (Expt 4) and no mortalities were observedamong Lake Mikitänjärvi crayfish (Expt 5). The strain As-Kemijoki caused mortality only in a single experiment (Expt 1)where severe mortality (84 %) was observed only in the LakeRytky crayfish population, whereas Lake Viitajärvi crayfishinfected under the same conditions with the same spore batchremained alive and asymptomatic (III). In three other infectionexperiments (Expts 3, 4 & 5), the As-Kemijoki caused noincreased mortality in comparison to the control group (III, IV).

As a side observation, that the crayfish infected with thePsI-Puujärvi became clearly symptomatic one day prior to theirdeaths. The symptomatic crayfish had an agitated behavior with

56

intensive scratching of their cuticle; this scratching wasespecially focused on the eyes and the joints of the walking legs.Among the As-Kivesjärvi infected crayfish, the symptomaticstage of the disease lasted longer, 2 days on the average, and insome individual cases for as long as 3-4 days. With this strain,the main symptom was the loss of the balance and a partialparalysis: the crayfish tended to lie on their backs, but whendisturbed physically, an escape reaction occurred promptly.With both tested strains, the appearance of symptoms wasinvariably followed by death. The surviving crayfish exhibitedno symptoms of the disease (III, IV).

The As-Kemijoki strain seemed to be almost avirulent,although it has been responsible for crayfish mortalities in RiverKemijoki (Lapland, Finland). The Kemijoki epidemics started in2006 and are still ongoing (Petri Muje, personal communication).The As-Kemijoki isolate was from the year 2007 and it has beenreported that years of laboratory cultivation have convertedsome A. astaci isolates to being avirulent: this has happened dueto the loss of the zoospore motility (Unestam & Weiss 1970;Unestam & Svensson 1971). However, the zoospores of As-Kemijoki were motile when the experimental infections wereprepared and therefore, different mechanisms of avirulence musthave occurred to explain this phenomenon. Based on the qPCRresults from the surviving crayfish, it appears as if the sporescould not have attached to the crayfish cuticle, since most of thetested crayfish remained negative (uninfected) in the qPCR (III,IV). The others showed very low agent levels, corresponding to aDNA amount of less than a spore being present in their tissue.

One possible explanation for the observed avirulencecould also be temperature adaptation. The strain was causingepidemics in rather cold environmental conditions in RiverKemijoki and it needs to be evaluated, whether its temperatureoptimum is lower than expected. Earlier, strains adapted towarmer environments have been reported from the NorthAmerican red-swamp crayfish (P. clarkii) in Spain (Diéguez-Uribeondo et al. 1995).

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The variables reported to be responsible for thedifferences in the mortality rate during the crayfish plagueinfection are the zoospore dosage and ambient watertemperature (Unestam & Weiss 1970; Alderman et al. 1987;Alderman 2000). Here, the effect of the water temperature wasremoved by performing this experimental series in an air-conditioned laboratory space, where the temperature wasadjusted to be a constant +18 °C. With respect to the zoosporedosage effect, an experiment was conducted to examine theeffect of the spore dosage on the mortality rate (Expt 6, IV). LakeKoivujärvi crayfish were infected with PsI-Puujärvi and As-Kivesjärvi at different dosages of A. astaci spores (1, 10, 100 &1000 spores ml-1). A logarithmic response to the spore dosagewas observed. All the tested spore densities caused 100 %mortality, although at the lower densities, the mortality rate wasslower (IV). This experiment also emphasized the high virulenceof the PsI-Puujärvi: 1 spore ml-1 dosage of PsI-Puujärvi provokedacute mortality faster than 1000 spores ml-1 of As-Kivesjärvi (IV).

Previously, the zoospore dosage effects have been testedwith the narrow-clawed crayfish (A. leptodactylus) and the resultshave been comparable to the present findings (Alderman et al.1987). Unestam & Weiss (1970) reported that a zoospore densityof 25 spores ml-1 inoculated into aquarium water caused 50 %mortality (LD50) in the noble crayfish (A. astacus) with a furtherchallenge of 250 spores ml-1 causing 100 % mortality during the36 days follow-up. Cerenius et al. (1988) have also claimed that10 000 spores ml-1 will always evoke 100 % mortality of crayfish,but in the present experimental conditions with large watervolumes (Jussila et al. 2011b), it was not possible to achieve suchhigh infection doses.

It was much more difficult to demonstrate the variationin the resistance than the variation in the virulence, because thedirect comparison of different experiments was not possible. Thephysiological status (i.e. moulting cycle, reproduction cycle, andinfections other than A. astaci) could have had some impact oncrayfish survival, at least in P. leniusculus (Nylund & Westman

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1983), and therefore only the experiments conductedsimultaneously were directly comparable.

We also found out that there is a logarithmic response inthe mortality initiation and A. astaci spore amount. With higherspore amounts used in the infection, crayfish started to dieearlier (Fig. 4). Based on the day of the dead estimates, it waspossible to compare to some extent the Lake Viitajärvi (Expt 4)and Lake Mikitänjärvi (Expt 5) populations. Lake Mikitänjärvicrayfish have been reported to carry A. astaci infection (Jussila etal. 2011a), and this could have caused increased resistanceagainst the new secondary infections of A. astaci. When the effectof the zoospore dosage was taken into account, the mortalityrate of Lake Mikitänjärvi crayfish after PsI-Puujärvi infectionwas approximately 3.5 times more slow than encountered in theLake Viitajärvi population. However, despite the delayedmortality, all of the Lake Mikitänjärvi crayfish died from the PsI-Puujärvi infection, but a clear resistance towards both As-genotype strains was observed in Lake Mikitänjärvi crayfish, asonly a few individuals died after the As-genotype infections (IV).

5.3 VARIATION IN SPORULATION

The sporulation from the noble crayfish (A. astacus) having anacute infection of the crayfish plague was measured from thePsI-Puujärvi infected crayfish, at 12 h intervals (V). The effect ofthe inoculation zoospore doses was excluded by performing theinfections in separate infection tanks and rinsing the crayfish inclean water before their transfer into the monitoring tanks.Therefore, one can assume that the spores detected in the waterof the monitoring tank were spores which had been releasedfrom the moribund crayfish individuals. Sporulation was alsomeasured from the As-Kemijoki infected noble crayfish, butsince no deaths occurred during the experiment, also the sporemeasurements with qPCR remained negative (unpubl. results).

Moderate amounts of spores were detected alreadyshortly after the infections and premortem of the crayfishindividuals. The main peak in the sporulation occurred 24-96 h

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postmortem. Thus during the sporulation peak, as many as 3.2million spores could be being released from a single crayfish,presuming that the spores were distributed homogenously.Sporulation had significantly declined by the sixth daypostmortem, in comparison to its early stage.

The motile period of the zoospores lasts 48 h in +16-20 °C(Unestam 1966a; Alderman & Polglase 1986) and for that reason,the maximum sporulation peak partially reflects the motileperiod of the zoospores. Therefore the actual time period forzoospore release from a dead individual crayfish may be shorter.In addition, part of the observed length in the sporulation peak,as well as part of the variation in the observed zoospore levels,may be due to the repeated zoospore emergence, which allowsthe zoospore to become encysted and start another motile periodafter a short resting period (Cerenius & Söderhäll 1984a;Cerenius & Söderhäll 1985).

There was an extensive variation in the spore releasebetween the experimental crayfish and between the differentmeasurement points. Part of the variation may be explained bythe sampling, since the spores more likely orientated towardsthe solid particles in the water column (Svensson 1978) and forthat reason they might not have been equally distributed withinthe water column. The variation between the individuals wasalso high. An individual crayfish, that died one day after theinfections, was sporulating considerably less than thoseindividuals that died later. One could speculate that thisindividual was weakened and died for some other unknownreasons and because of the shorter time period after the infection,it had been less extensively colonized by the A. astaci hyphae.However, the overall sporulation pattern was similar in all of theindividual crayfish.

The water sampling for the spore quantification wasmade at a level 15 cm above the tank bottom, mainly to avoidmeasuring any encysted spores which had accumulated on thetank bottom. If the measurements had been made directly fromthe bottom, larger spore estimates would have been obtained,similarly as in paper VI.

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In the signal crayfish (P. leniusculus), the sporulationpattern during the chronic infection was rather different (VI)compared to that of noble crayfish (V). A signal crayfish carryingA. astaci continuously released a moderate number of spores (~2 700 spores per week), provided it was not moulting orsuccumbing to the infection. Spores were also produced in coldwater (+4 °C). Moreover, moultings and deaths increased thesporulation levels, as predicted (Oidtmann et al. 2002a). Theamount of sporulation increased one week prior to death, whichindicates that weakened or stressed individuals can develop anacute crayfish plague infection, similar findings have beenreported earlier (Unestam 1972; Persson et al. 1987; Cerenius et al.1988). In the case of individually housed crayfish, stress inducedsporulation was also observed, as the substitute crayfish releasedhigher numbers of spores during the first 1-2 weeks after beingplaced into the experimental system.

Probably due to the accumulation of the encysted sporesonto the tank bottom and the taxis of the spores towards thenutrients (Cerenius & Söderhäll 1984b), the spore numbers in thecommunally housed crayfish were considerably higher on thetank bottom in comparison to the numbers in the water column.Based on the current knowledge (Söderhäll & Cerenius 1999),most of the spores are likely to be found near to the bottomwhere they quickly become attached to the crayfish. Therefore,the spore estimates based on the water samples taken from thewater column are likely to underestimate the actual numbers ofspores that the infected individuals were releasing.

The signal crayfish that were used in this study (VI) wereseverely infected with the A. astaci overall prevalence being 95 %.In the carrier crayfish populations, the prevalence and infectionstatus is highly variable (Kozubíková et al. 2009; Skov et al. 2011;Vrålstad et al. 2011) and thus in the populations with a lowerinfection status the sporulation levels may be considerably lower.

In contrast to the previous beliefs (Oidtmann et al. 2002a),it was demonstrated (VI) that the signal crayfish could release A.astaci zoospores, the infective units, constantly into the ambientwater, not only during their moulting phase or death. Similar

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indirect evidence has been gained earlier when the red swampcrayfish (Procambarus clarkii), i.e. when susceptible species havebeen placed in the same tanks with their red swamp crayfishcounterparts, the susceptible crayfish became infected with thecrayfish plague (Diéguez-Uribeondo & Söderhäll 1993). Thus, itis apparent that these carrier crayfish species pose a constantthreat to susceptible species, and this risk is not dependent onthe phase of the crayfish life cycle.

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6 Conclusions

The agents of most emerging diseases often originate from othergeographical areas or from another host species. The crayfishplague is an excellent example of this phenomenon. After itsarrival in Europe in the form of the As-genotype, the pathogenhas been subjected to high selection pressure as it spreads into itsnew environment infecting the highly susceptible Europeancrayfish host species. After the first wave of the disease, newcrayfish plague genotypes were brought to Europe with theNorth American crayfish species introduced here to fill the lakesemptied of native crayfish. These genotypes are most likelyexperiencing less selection pressure, since their original hostspecies are present in Europe, although the environment isdifferent.

This thesis provides new information about the geneticvariability of A. astaci. With respect to the two studied geneticregions, the intraspecific variation in the ITS-regions was low (I)and based on these results, the ITS-regions can be viewed ashighly suitable target region for developing species-specificmolecular detection methods. Genotype specific diversity wasobserved in the chitinase genes of A. astaci (II). Based on thechitinase sequence comparisons, the two in this sense identicalsignal crayfish genotypes, PsI and PsII, were clearlydistinguishable from the As- and Pc-genotypes. In contrast, theAs- and Pc-genotypes were rather similar to each other and thissimilarity may also reflect their original host species in NorthAmerica. The variation observed among the strains of the As-genotype also indicate, that this clonally spreading crayfishpathogen has been subjected to high selection pressure during its150 year history in Europe.

The genetic differences observed in the chitinasesequences also permit the genotype detection with PCR andsequencing, directly from the crayfish cuticle. Until recently, thegenotyping has relied on the RAPD-PCR method, which means

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that genotyping was possible only if a pure culture of A. astacicould be successfully obtained. As a result of this study (II), thespecific markers for the genotype detection are now available forresearchers and based on these results, the genotyping can alsobe further developed towards applications with highersensitivities.

Moreover, significant differences in the physiologicalproperties, i.e. in the differing virulence of the A. astaci strains,were observed (III, IV). Based on the series of the infectionexperiments made with different A. astaci strains and crayfishpopulations, it can be stated that the PsI-genotype, which islinked to the signal crayfish introductions, is a highly virulentkiller. A. astaci strains of this genotype killed 100 % of theinfected noble crayfish (A. astacus) within a few days. It is alsoless likely that the virulence of the PsI-genotype will becomereduced during the next decades, because the signal crayfish isnow widely present throughout Europe and there is a host-pathogen relationship of A. astaci PsI-genotype and the signalcrayfish in existence between these two species.

The tested strains of As-genotype were more variable intheir virulence and in several experiments, part of the noblecrayfish populations survived, or even evaded the infectionunder laboratory conditions, with limited water volumes and noprotective areas provided for the experimental crayfish. Based onthese results, one can recommend that the carrier status analysesof noble crayfish should be considered, when the stockings areplanned. This would help to avoid the spread of the less virulentforms of the crayfish plague, since it seems that also someinfected noble crayfish can act as latent carriers of crayfishplague, without displaying any visible symptoms of the disease.

During our experiments (III, IV), there were alsoindications that small differences do occur in the resistancefeatures of different noble crayfish populations. Somepopulations with a known history of crayfish plague had aslower mortality rate in comparison to the populations withoutprevious epidemics. Although the mortality rate was slower, allof the crayfish still died from the A. astaci infection made with thePsI-Puujärvi. However, future studies will examine this topic in

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more detail, and more definitive remarks can be expected to begained in the future.

Furthermore, in this thesis, the sporulation dynamicsaffecting the spread of A. astaci during the acute infection and inthe chronic infection were quantified with modern qPCRtechniques (V, VI). In the noble crayfish, moderate amounts of A.astaci spores were released premortem and the maximum peak inthe sporulation was achieved 24-96 h postmortem. In the signalcrayfish, the rate of sporulation was constant and it also occurredin the cold water, although the highest spore amounts werereleased under stress, moulting and death in the watertemperatures corresponding to the summer period.

All the results obtained in this thesis underline the factthat the further spreading of the signal crayfish should beprevented more efficiently. The crayfish plague in the signalcrayfish is genetically different from As-genotype andfurthermore, it is highly virulent. In addition, infected signalcrayfish are constantly sporulating and therefore posing acontinuous risk to the surrounding waters, when not onlycrayfish, but also fish, or any contaminated equipments are beingtransported.

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Publications of the University of Eastern Finland Dissertations in Forestry and Natural Sciences

Publications of the University of Eastern Finland

Dissertations in Forestry and Natural Sciences

isbn 978-952-61-1135-3

Jenny Makkonen

The crayfish plague pathogen Aphanomyces astaciGenetic diversity and adaptation to the host species

Crayfish plague, caused by the oomy-

cete Aphanomyces astaci, has caused a

dramatic decline of the native cray-

fish in Europe. The North-American

crayfish species introduced to Europe

are the main vectors of the disease,

which is endemic in North-America.

In this work, genetic diversity, physi-

ological properties and sporulation

dynamics of A. astaci were investi-

gated. The thesis provides new in-

formation about the diversity of the

crayfish plague strains from Finland,

and of the evolving host-parasite

relationship, between this lethal

parasite and its susceptible European

crayfish hosts.

dissertatio

ns | 105 | Jen

ny M

ak

ko

nen

| Th

e crayfish plagu

e path

ogen Ap

han

omyces astaci – G

enetic diversity an

d adaptation to th

e...

Jenny Makkonen

The crayfish plague pathogenAphanomyces astaciGenetic diversity and adaptation

to the host species

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