dissertations View metadata, citation and similar papers at core.ac.uk brought to you by CORE
provided by UEF Electronic Publications
| 105 | Jenny Makkonen | Makkonen | 105 | Jenny
Jenny Makkonen The crayfish plague pathogen Aphanomyces astaci Crayfish plague, caused by the oomy- cete Aphanomyces astaci, has caused a Genetic diversity and adaptation Jenny Makkonen dramatic decline of the native cray- to the host species fish in Europe. The North-American to the... adaptation and diversity Genetic – astaci Aphanomyces pathogen plague crayfish The crayfish species introduced to Europe The crayfish plague pathogen are the main vectors of the disease, which is endemic in North-America. Aphanomyces astaci In this work, genetic diversity, physi- ological properties and sporulation Genetic diversity and adaptation to the host species 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.
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 astaci
Genetic diversity and adaptation to the host species
Publications of the University of Eastern Finland Dissertations in Forestry and Natural Sciences No 105
Academic Dissertation To 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 Eastern Finland, Kuopio, on June, 14, 2013, at 12 o’clock noon.
Department of Biology Kopijyvä Oy Kuopio, 2013 Editors: Profs. Pertti Pasanen, Pekka Kilpeläinen and Matti Vornanen Distribution: Eastern Finland University Library / Sales of publications [email protected] www.uef.fi/kirjasto
ISBN: 978-952-61-1135-3 (nid.) ISSNL: 1798-5668 ISSN: 1798-5668 ISBN: 978-952-61-1136-0 (PDF) ISSN: 1798-5676 (PDF)
Author’s address: University of Eastern Finland Department of Biology P.O. Box 1627 70211 KUOPIO FINLAND email: [email protected]
Supervisors: Professor Atte von Wright, Ph.D. University of Eastern Finland Institute of Public Health and Clinical Nutrition P.O. Box 1627 70211 KUOPIO FINLAND email: [email protected]
Docent Japo Jussila, Ph.D. University of Eastern Finland Department of Biology P.O. Box 1627 70211 KUOPIO FINLAND email: [email protected]
University Lecturer Paula Henttonen, Ph.D. University of Eastern Finland Department of Biology P.O. Box 1627 70211 KUOPIO FINLAND email: [email protected]
Researcher Harri Kokko, M.Sc. University of Eastern Finland Department of Biology P.O. Box 1627 70211 KUOPIO FINLAND email: [email protected] Reviewers: Professor Emerita Tellervo Valtonen, Ph.D. University of Jyväskylä Department of Biological and Environmental Science P.O. Box 35 40014 JYVÄSKYLÄ FINLAND email: [email protected]
Senior Lecturer Lage Cerenius, Ph.D. University of Uppsala Department of Organismal Biology Norbyv. 18A 75236 UPPSALA SWEDEN email: [email protected]
Opponent: Assistant Professor Ivana Maguire, Ph.D. University of Zagreb Department of Biology Rooseveltov trg 6 10000 ZAGREB CROATIA email: [email protected]
ABSTRACT
The crayfish plague disease is caused by the oomycete Aphanomyces astaci. The native European crayfish species are highly susceptible to this disease and their population sizes are generally declining. Five genotypes of A. astaci are present in Europe; their original host species are from North America. In Finland, two genotypes, As and PsI, are responsible for the crayfish plague epidemics. In recent years there have been ongoing discussions concerning reduced virulence of crayfish plague, especially of the As-genotype. The objectives of this study were to 1) investigate the genetic variation among the A. astaci strains, 2) to study the differences in the physiological properties of the A. astaci strains, especially the variation in their virulence, and 3) to determine the sporulation of A. astaci during the acute infection of the crayfish plague in noble crayfish and in chronically infected signal crayfish. The genetic studies revealed that there was low intraspecific variation in the internal transcribed spacer regions and therefore, this region is a very suitable target for the molecular detection methods to be conducted at the species level. In the chitinase genes, high genotype specific diversity was observed and markers for the genotype identification were found for As, Ps, and Pc-genotypes. The chitinase results also revealed that the As-genotype was closer to the Pc-genotype, while the two signal crayfish genotypes, which were identical based on the chitinase comparisons, were clearly distinguishable from either the As- or Pc-genotypes. The variation observed among the strains of the As-genotype, also indicated that the pathogen has experienced high selection pressure during its spread across the Europe. In this PhD study, significant differences were observed in the virulence of the A. astaci strains. Based on a series of infection experiments, the tested A. astaci strains of PsI- genotype killed 100 % of the infected noble crayfish, generally within a week. The strains of the As-genotype were more variable and in several experiments, some of the experimental crayfish survived. Minor differences were also observed in the resistance of different noble crayfish populations against crayfish plague. Furthermore, the sporulation dynamics affecting the spread of A. astaci during acute infection and in chronic infection were quantified. In the noble crayfish with an acute crayfish plague infection, the maximum peak in the sporulation occurred 1-3 days post mortem. In the chronically infected signal crayfish, sporulation was at a constant level and it also occurred in cold water, although the highest spore amounts were released under stress, moulting and death at the warm water temperatures. The results of this PhD study emphasize that the further spreading of signal crayfish should be prevented. The PsI- genotype of A. astaci is highly virulent and it most likely retains its virulence, since its original host species has been imported into Europe. It is also genetically different from the As- genotype. Furthermore, the carrier crayfish constantly sporulate and therefore pose a continuous risk to the surrounding noble crayfish populations. Moreover, the carrier status analyses of noble crayfish should also be considered, when stockings are being planned. This would help to avoid the spread of less virulent forms of the crayfish plague, since some infected noble crayfish 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 of Biosciences, University of Kuopio) during the years 2006-2013. The work was funded by the Finnish Cultural Foundation the North-Savo Regional Fund, the Ministry of Agriculture and Forestry, EU’s Fisheries Guidance Fund, University of Kuopio, University of Eastern Finland, Kuopio Naturalists’ Society and Olvi Foundation. I am very grateful to my supervisors Japo Jussila, Paula Henttonen, Harri Kokko and Atte von Wright for their advice and encouragement during this scientific learning process. Japo and Harri, your fascination towards the new experiments and the new results pushed this work into its current level! Paula, your endless support and understanding greatly helped me through this journey. I also wish to thank Raine Kortet and Anssi Vainikka for participating in the study planning and their valuable comments on several manuscripts and Ossi V. Lindqvist for his help with the funding applications and encouragement through all these years. I sincerely thank the two preliminary examiners of this thesis, Tellervo Valtonen and Lage Cerenius, for their encouraging comments, and constructive criticism that they presented. Thanks also to Ivana Maguire, for accepting the task of being my opponent. To Ewen MacDonald and Roseanna Avento, I am thankful for their help with the spelling and grammar of this thesis. I also want to acknowledge Satu Viljamaa-Dirks from Evira, for assigning some of the crayfish plague strains into our use. Without her help and without the strain collection conducted by Evira, this work would not have reached its present scale. I would also like to thank Trude Vrålstad and David Strand for collaboration and co-authorship in the sporulation studies. I would also like to thank Javier Diéquez- Uribeondo for allowing me to use the A. astaci life cycle picture in this PhD-summary. Thanks also to all the IAA-members, the friendly atmosphere at the conferences offered a pleasant way to join the scientific community! FGFRI, thanks for collaboration. Many thanks to all my co-workers at the Department of Biology: Anna Karjalainen, Jouni Heikkinen, Salla Ruuska, Anna Toljamo, Tiina Korkea-aho, Tiina Pitkänen-Arsiola, Liisa Nurminen, Jaakko Mononen, Leena Ahola, Marketta Lämsä and many others, for countless hilarious moments and fruitful discussions over these years! Special thanks to Hobo Kukkonen, Helena Könönen, Raisa Malmivuori and Elina Reinikainen for their technical assistance. Thanks also go to the Fish Research Unit staff Marko Kelo, Kauko Strengell and Mikko Ikäheimo for helping me with the crayfish. My warmest thanks goes to the students Hanna Kinnula, Laura Koistinen and Lars Granlund, who were all doing a very good job in taking care of the experiments. Teemu Poutiainen: thanks for the lunch company and for keeping my freezer stocked with pikeperch. I want to thank my parents Seija and Matti, and my siblings Pirkko and Timo, and the whole Hankasalmi-Zoo for their support, but especially for releasing my thoughts from work on the weekends. At this point, I would also like to mention my dearest dog Aatu, whose endless snoring was the most relaxing sound, I could ever imagine during the writing of this dissertation, at home. Finally, Tatu, you have been by me all these years (and so many more), always supporting me, in the end nothing else really 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 of Nature 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. Genetic variation in the ribosomal internal transcribed spacers of Aphanomyces astaci Schikora from Finland. Aquaculture 311: 48-53, 2011.
II Makkonen J, Jussila J and Kokko H. The diversity of the pathogenic Oomycete (Aphanomyces astaci) chitinase genes within the genotypes indicate adaptation to its hosts. Fungal Genetics and Biology 49: 635-642, 2012.
III Makkonen J, Jussila J, Kortet R, Vainikka A and Kokko H. Differing virulence of Aphanomyces astaci isolates and elevated resistance 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) to different strains of the crayfish plague (Aphanomyces astaci). Journal of Fish Diseases, Submitted manuscript, 2013.
V Makkonen J, Strand DA, Kokko H, Vrålstad T and Jussila J.Timing and quantifying the Aphanomyces astaci sporulation from the noble crayfish 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 spore dynamics of Aphanomyces astaci in the ambient water of latent carrier 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 data analysis, and I wrote the first version of the manuscript. In papers III and IV, I performed the infection experiments, took part in the follow-up of the experiments, undertook the sample collection and analysis and wrote the first version of the manuscript. In papers V and VI, I participated in the study design which was made in collaboration with D. Strand and his supervisors. I also took part in the data collection and conducted the data analysis in the paper V. I also wrote some parts of manuscripts (V & VI). Contents
1 Introduction ...... 17
2 Literature review ...... 19 2.1 HOST SPECIES...... 19 2.1.1 European crayfish species ...... 19 2.1.2 North-American crayfish species ...... 20 2.1.3 Host responses to the infection ...... 21 2.2 CRAYFISH PLAGUE ...... 22 2.2.1 The history and the spread of the crayfish plague ...... 22 2.2.2 The A. astaci spreading routes in nature ...... 24 2.2.3 Genotypes ...... 25 2.2.4 Life cycle ...... 27 2.2.5 Other characteristics ...... 30 2.2.6 Infection mechanisms ...... 31 2.2.7 Molecular tools in research and diagnostics...... 32 2.2.8 Genetics ...... 33 2.3 AIMS OF THE STUDY ...... 34
3 Materials and methods ...... 35 3.1 APHANOMYCES ASTACI STRAINS ...... 35 3.2 THE CRAYFISH POPULATIONS ...... 38 3.3 STUDY DESIGNS...... 38 3.3.1 Genetic variation (I & II) ...... 38 3.3.2 Infection experiments (III & IV) ...... 39 3.3.3 Sporulation (V & VI) ...... 40
4 Results ...... 43 4.1 GENETIC VARIATION...... 43 4.1.1 Internal transcribed spacers (I) ...... 43 4.1.2 Chitinase gene (II) ...... 43 4.2 VARIATION IN THE DISEASE PROGRESS (III & IV) ...... 45 4.2.1 Virulence variation ...... 45 4.2.2 Resistance variation ...... 48 4.3 VARIATION IN SPORULATION ...... 49 4.3.1 Noble crayfish (V) ...... 49 4.3.2 Signal crayfish (VI) ...... 49
5 Discussion ...... 51 5.1 GENETIC VARIATION AMONG A. ASTACI...... 51 5.2 VARIATION IN THE DISEASE PROGRESS ...... 55 5.3 VARIATION IN SPORULATION ...... 58
6 Conclusions ...... 63
References ...... 67
1 Introduction
The crayfish plague, caused by Aphanomyces astaci (Schikora), is the most devastating crayfish disease known to date (Cerenius et al. 2009). Since the disease arrived in Europe in 1859 (Cornalia 1860), it has been responsible for dramatic collapses in populations in the native European crayfish stocks (Souty- Grosset et al. 2006). The European crayfish species are extremely susceptible to the disease, and are listed in the IUCN (International Union for Conservation of Nature) Red list as threatened, with a declining population trend (IUCN 2012). In contrast, A. astaci, is listed as one of the 100 worst invasive species in the world in Global Invasive Species Specialist Group of IUCN (Lowe et al. 2004), and it can be considered as a one of the main reasons for the reducing numbers of the native crayfish throughout Europe (Souty-Grosset et al. 2006). A. astaci is a member of the class of Oomycetes, the most famous representatives of which, with huge economical importance, are plant pathogens, like the potato late blight (Phytophthora infestans) (Birch & Whisson 2001) and other plant pathogenic species in the genus Phytophthora. Oomycetes not only include other destructive plant and animal pathogens, but also saprophytes that are beneficial to the environment (Margulis & Schwartz 2000). The plant pathogenic species have been most intensively studied with the recent research concentrating on the field of genetics which has attempted to clarify the evolution and virulence mechanisms of these pathogens (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 species of Oomycetes, A. astaci has been extensively examined (Cerenius et al. 2009), but details of its genetics are still largely unknown. A. astaci is a specific parasite of the crayfish, with no other hosts known to date (Unestam 1972; Oidtmann et al. 2002a; Diéguez-Uribeondo et al. 2009). A. astaci spreads clonally
17 via swimming zoospores and neither a sexual reproduction cycle 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 of Aphanomyces (Diéguez-Uribeondo et al. 2009). Five genotypes of A. astaci have been recognized to date (Huang et al. 1994; Diéguez-Uribeondo et al. 1995; Kozubíková et al. 2011a). The genotype links the pathogen lineage to its original host species introduced to Europe from North America (Kozubíková et al. 2011a). In Finland, observations about variations in the disease progress have been reported from the field from time to time. In some lakes, some of the population has survived the infection and eventually the noble crayfish has recovered by itself, even with no intensive restockings being made (Nylund & Westman 1992). Moreover, in some lakes, the disease progress has been very slow and infected individuals have been found for several years (Jussila et al. 2011a; Viljamaa-Dirks et al. 2011). In Turkey, the crayfish plague has also similarly been detected from the narrow-clawed crayfish (Astacus leptodactylus) populations and this has not been accompanied by any increased mortality or population collapses (Kokko et al. 2012; Svoboda et al. 2012). In this study, the genetic variation in A. astaci was investigated, especially among the isolates of Finnish origin, and among the two genotypes that are present throughout the country (Vennerström et al. 1998). One practical reason for this was that the genotype specific genetic markers have long been in the crayfish researchers’ top 10 -wish list. It is also important to link the genetic information to the differences observed in the physiological properties of the A. astaci strains, i.e. to the differences in virulence. In addition, the sporulation of A. astaci was studied during an acute infection of the crayfish plague in the noble crayfish (Astacus astacus) as well as in chronically infected signal crayfish (Pacifastacus leniusculus).
18 2 Literature review
2.1 HOST SPECIES
2.1.1 European crayfish species There 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 these species 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 native species. The original distribution area, determined for the first time in 1859, has covered the southern and central parts of Finland, south from the Kristiinankaupunki-Savonlinna- Sortavala axis, (Järvi 1910), corresponding to approximately 61° parallel of northern latitude (Cukerzis 1988). However, the distribution has been expanded to cover the whole country by human interventions (Cukerzis 1988; Skurdal & Taugbøl 2002; Kilpinen 2003). Genetic studies have shown that the populations have been partially mixed all around the country (Alaranta et al. 2006). The noble crayfish, like all the European crayfish species, is highly susceptible to crayfish plague (Unestam 1969a; Unestam & Weiss 1970) and the population sizes are generally on the decline (Souty-Grosset et al. 2006). Nevertheless, in Finland commercially catchable noble crayfish populations still exist in sufficient numbers and thus the aim of maintaining the current highly valuable populations still tends to be viewed from a fisheries management perspective rather than from an environmental conservation angle (Pursiainen & Ruokonen 2006; Reynolds & Souty-Grosset 2012).
19 2.1.2 North-American crayfish species Three North American crayfish species, the signal crayfish (P. leniusculus), the red swamp crayfish (Procambarus clarkii) and the spiny-cheek crayfish (Orconectes limosus), are now widely distributed throughout Europe. Furthermore, single populations of the marble crayfish (Procambarus sp.) as well as several Orconectes spp. have been discovered (Souty-Grosset et al. 2006). All the North American crayfish are considered as carriers of A. astaci, although the prevalence of the disease seems to vary depending on environmental factors, or other unknown reasons (Kozubíková et al. 2009; Skov et al. 2011; Vrålstad et al. 2011). The signal crayfish (P. leniusculus) stockings were started during the 1960’s in Sweden (Abrahamsson 1969) and Finland (Westman 1973) to generate new crayfish populations into the lakes, where previous noble crayfish introductions had failed. One of the main reasons for these introductions was that the signal crayfish was believed to have exceptionally high resistance against crayfish plague (Unestam 1969a; 1972; Unestam & Weiss 1970). Later it was realized that signal crayfish could act as a chronic carrier of the disease (Persson & Söderhäll 1983); in fact the signal crayfish has been the main vector and source for the A. astaci infections after its introductions into the environment (Huang et al. 1994). The signal crayfish distribution area (Jussila & Mannonen 2004; Kirjavainen & Sipponen 2004) in Finland covers the original distribution area of noble crayfish (Pursiainen & Ruokonen 2006), but illegal introductions have occurred also outside the official distribution area (Jussila & Mannonen 2004). The eastern and northern parts of the country are all that remain for the noble crayfish. There are noble crayfish populations also existing in the signal crayfish area (Hyytinen et al. 2000; Kirjavainen & Sipponen 2004), but there their status is extremely vulnerable (Oidtmann et al. 2002a; Kirjavainen & Sipponen 2004). Mass mortalities of the signal crayfish attributable to the crayfish plague are generally uncommon, although the signal crayfish can also develop an acute and fatal infection of the
20 crayfish plague during stressful conditions (Unestam 1972; Persson et al. 1987; Cerenius et al. 1988). In Finland, the first reported acute mortality and population collapse caused by crayfish plague was detected in Lake Puujärvi (Karjalohja) signal crayfish, in 1996 (Helsingin Sanomat, 23.8.1996; Tulonen et al. 1998), and since that date the population has not been completely recovered. In Sweden, large-scale mortalities have also occurred in natural waters (Smith & Söderhäll 1986) and the population densities in the several productive signal crayfish lakes have been falling in recent years for unknown reasons (Pakkasmaa 2006; Lennart Edsman 2012, personal communication). The signs of the chronic crayfish plague, i.e. melanized spots and broken limbs (Unestam & Weiss 1970), are commonly seen 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 infection Invertebrates lack antibodies and their immune defense relies on the innate immune system (Söderhäll & Cerenius 1998). Moreover, their open circulatory system demands effective clotting of any wounds (Söderhäll & Cerenius 1999). When A. astaci hyphae penetrates through the crayfish body cavity, -1,3-glucans of its cell wall are recognized by the crayfish blood cells via the non-self recognition system. This launches the prophenoloxidase system (proPO-system) which is the main defense reaction that the crayfish possesses against A. astaci infection (Söderhäll & Cerenius 1998). In this reaction, the pathogen is first encapsulated with semigranular blood cells. Then, a layer of granular blood cells is aggregated around the capsule and the proPO-system is activated during degranulation of the granular cells (Unestam & Nylund 1972). The final outcome of activation of the proPO-system is a pathogen surrounded by sticky melanin. Although the pathogens growth and dispersal are restricted, it is still alive (Söderhäll & Cerenius 1999). The reason for the increased resistance of the signal crayfish against the crayfish plague is the constantly activated
21 proPO-system, which therefore reacts rapidly to the infection (Cerenius et al. 2003). In the noble crayfish, proPO-system will be only activated, as a response to the infection, but often the reaction is too little and too late to combat successfully against the disease (Cerenius et al. 2003). Secondary bacterial or fungal infections may disturb the constantly ongoing defense reaction and subsequently, also a signal crayfish can die from a A. astaci infection (Söderhäll & Cerenius 1992). Psorospermium haeckeli infections may also weaken the signal crayfish immune system (Thörnqvist & Söderhäll 1993). When the acute stage of the disease is reached, paralysis of the abdomen is often the only visible symptom and this occurs 1-2 days prior to death (Unestam & Weiss 1970). It has been postulated that the neurotoxic effects are the major cause of death of the crayfish (Nybelin 1934; Schäperclaus 1954). Other reported symptoms are uncoordinated movement, described as walking on stilts, combined with spasmodic limb tremor and tail movements (Schikora 1906). The duration of pre-mortality behavior is dependent on the water temperature and the given zoospore challenge dose (Alderman & Polglase 1988).
2.2 CRAYFISH PLAGUE
2.2.1 The history and the spread of the crayfish plague The first outbreaks of the crayfish plague (A. astaci) were detected in Lombardy, Northern Italy, in 1859, where huge numbers of crayfish suddenly started to die (Cornalia 1860). During the next three decades, the disease swept across continental Europe (Fig. 1) (Alderman 1996) and reached Finland via Russia in 1893, when the first mass mortalities were detected in Lake Saimaa (Järvi 1910). From Finland, the disease then spread to Sweden in 1907 and finally to Norway as late as 1971 (Alderman 1996). The vector for this first wave of the crayfish plague is unknown (Huang et al. 1994; Alderman 1996; Kozubíková et al. 2011a), although some sources have suggested that it could have been the North-American spiny-cheek crayfish (Orconectes
22 limosus) which was introduced to Europe in 1890 (Souty- Grosset et al. 2006). However, this theory has been later discounted, 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 was most likely an infected crayfish of North-American origin (Unestam 1969a; Unestam 1972; Unestam 1975), either being introduced on purpose, or accidentally in the ballast water tank of an Atlantic crossing ship (Alderman 1996).
Figure 1. Spread of the crayfish plague (A. astaci) through Europe with main distribution areas of the native European crayfish species. The colored regions refer to the 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 and redrawn after Alderman (1996).
23 Soon after its emergence in Finland, the crayfish plague caused a dramatic drop in the previously thriving crayfish business and trade to the neighboring countries (Järvi 1910). After the crayfish plague epidemics, crayfish populations were often restocked into the lakes, but these often failed for unknown reasons, the infection remained in the lake in a “chronic” form and when the population density of the noble crayfish (A. astacus) had increased to an exploitable level, the disease struck again causing a new population collapse (Fürst 1995; Erkamo et al. 2010). One proposal for this phenomenon was the labyrinth structure of Finnish lakes, which offered protective areas for subpopulations and enabled a slow migration of the disease (Westman & Nylund 1978; Nylund & Westman 1992). As a solution to this problem, re-stockings with signal crayfish (P. leniusculus) was started during the 1960’s (Westman 1973).
2.2.2 The A. astaci spreading routes in nature A. astaci can exist in three forms: mycelium (in the crayfish cuticle), a cyst and a swimming zoospore (in the ambient water). The infectious stage of A. astaci is the zoospore (Cerenius & Söderhäll 1984a; Oidtmann et al. 2002a). The postulated pathways of the transmission are 1) crayfish, 2) contaminated water and items that have been in contact with contaminated water, and 3) animals that have been feeding on the infected crayfish (Oidtmann et al. 2002a). During the first wave of the crayfish plague, man made actions, like the crayfish trade, were the most likely reason for the rapid dispersal of the disease (Alderman 1996). The second, still ongoing wave of the disease started in the 1970’s and can be traced to introduction of North American crayfish species (mainly P. leniusculus, P. clarkii), which in most cases, carry a chronic A. astaci infection (Persson & Söderhäll 1983; Vogt 1999; Oidtmann et al. 2006; Kozubíková et al. 2011b). These introduced species spread the crayfish plague to new areas when new habitats become colonized. The dispersal rates for the invasive species seem to be rather high, as in P.
24 leniusculus, a downstream dispersal rate of up to 18-24.4 km yr-1 has been reported (Hudina et al. 2009). The susceptible European crayfish species can only act as a vector during crayfish plague outbreaks (Oidtmann et al. 2002a). Several treatments and disinfectants have been examined in attempts to stop the spread of the crayfish plague. Under natural conditions, liming (Rennerfelt 1936; Svensson et al. 1976) and electrical barriers (Unestam et al. 1972) have been tested with some success, but they have not been sufficiently effective. The zoospore production from an infected crayfish can be prevented with high concentrations of MgCl2, more than 20 mM, (Rantamäki et al. 1992). Powerful chemical treatments can be used in the disinfection of contaminated equipment, e.g. hypochlorite (Alderman & Polglase 1985) and peracetic acid, which can be also used to disinfect water in aquaculture or fish transport (Jussila et al. 2011b) as well as in crayfish farming to prevent disease spread among the crayfish individuals (Kouba et al. 2012).
2.2.3 Genotypes The distribution and prevalence of A. astaci in North-America is still largely unknown, because the infections do not evoke any symptoms or mortality in its indigenous host species. If new species are introduced to Europe in the future, most likely also new 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 “DNA fingerprint” 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 the start of signal crayfish (P. leniusculus) introductions. Group B (PsI-genotype) and group C (PsII-genotype) are found from the signal crayfish, and also from the noble crayfish (A. astacus) again after the signal crayfish introductions. The PsI-genotype
25 is linked to the signal crayfish found in Lake Tahoe and Lake Hennessey in USA, which were the main sources for the animals brought to Sweden (Abrahamsson 1969) and Finland (Westman 1973). A single isolate of the PsII-genotype has been linked to the introduction of the signal crayfish from Lake Pitt (Canada) to Sweden (Huang et al. 1994) but this genotype is not widely present in Europe (Söderhäll & Cerenius 1999). Group D (Pc-genotype) is linked to the red swamp crayfish (Procambarus clarkii) introductions (Diéguez-Uribeondo & Söderhäll 1993; Diéguez-Uribeondo et al. 1995), which were initiated in Spain in 1973 (Henttonen & Huner 1999). This genotype also has a higher temperature optimum in comparison to the three previously described genotypes (Diéguez-Uribeondo et al. 1995). The group E (Or-genotype) linked to the introductions of the spiny-cheek crayfish (O. limosus) was isolated recently by Kozubíková et al. (2011a). Although the spiny-cheek crayfish has not been widely stocked for fisheries management purposes, it has colonized vast areas of central Europe and is inexorably spreading towards the Baltic and northern Europe (Filipová et al. 2011; Pârvulescu et al. 2012). O. limosus has also been documented to coexist with native A. leptodactylus populations (Hudina et al. 2009). The resistance (Schikora 1906; Unestam 1969a) and carrier status (Vey et al. 1983) of O. limosus against the crayfish plague has been known for a long time, but the isolation process, which is essential in order to undertake genotyping, is often complicated in the weakly infected North American 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)
26 In Finland, As- and PsI-genotypes are present (Vennerström et al. 1998) and both of them are responsible for crayfish plague epidemics every year. The PsI-genotype has been commonly isolated from the official signal crayfish distribution area, whereas the As-genotype infections has been diagnosed 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 also known to be present in Turkey (Huang et al. 1994), but throughout continental Europe, it is the PsI-genotype form causing 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 cycle Sexual and asexual life cycles are recognized in the Aphanomyces spp. The sexual stage guarantees genetic divergence and a resting phase, while the asexual life cycle is responsible for the dispersal of the pathogen (Diéguez- Uribeondo et al. 2009). The sexual life cycle is commonly found from the saprophytic and plant pathogenic species of Aphanomyces, 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 to duplicate the experiments with success, it has been postulated that Rennerfelt was perhaps studying some other species of Aphanomyces, or a mixed culture (Unestam 1969b; Söderhäll & Cerenius 1999). Parasitic species of Aphanomyces spp. can undergo repeated zoospore emergence (RZE) for at least three generations (Cerenius & Söderhäll 1984a), which has been proposed to represent an adaptation to the parasitic life style, because the RZE will confer on the spore a new opportunity to find a suitable host after a short resting period (Cerenius & Söderhäll 1985). Zoospores of saprophytic Aphanomyces species
27 lack this feature and they tend to germinate very readily (Cerenius & Söderhäll 1985; Söderhäll & Cerenius 1999; Royo et al. 2004). The zoospores orientate towards a variety of nutritious surfaces, including the soft cuticle of the crayfish, via chemotaxis. The chemotactic effect is similar in many Aphanomyces species, but this does not explain the specificity of A. astaci as a crayfish parasite (Cerenius & Söderhäll 1984b). When the zoospores of A. astaci find a suitable host they encyst and germinate. Encystment can be artificially induced by ion or temperature treatment (Svensson & Unestam 1975), as well as with organic compounds or a mechanical stimulus (Cerenius & Söderhäll 1984a; Cerenius & Söderhäll 1985). The encysted zoospore germinates forming a penetration peg within 1 h (Nyhlén & Unestam 1975). Germination is often successful in wounds, body openings and in the soft cuticle (i.e. joints, ventral abdominal cuticle) of the crayfish (Unestam & Weiss 1970). The hyphae grow inside the cuticle and when the acute stage of the disease is reached, the hyphae can be found growing along the ventral nerve cord (Unestam & Weiss 1970). When the crayfish is dying, the hyphae burst out of the cuticle into the ambient water and form sporangia. Primary spores are formed in sporangia and then released from the hyphal tip, forming clusters of primary cysts. Secondary swimming zoospores are released from the primary cysts into the ambient water (Rennerfelt 1936; Svensson & Unestam 1975; Svensson 1978; Söderhäll & Cerenius 1987) (Fig. 2).
28 Figure 2. Life cycle of A. astaci, originally drawn and published by Diéguez- Uribeondo et al. (2006). 1) secondary zoospore (the infective unit), 2) encysting zoospore, 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 the immune stressed North-American crayfish), 6c) melanized spots in the crayfish cuticle 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 to unspecific stimuli forming a secondary cyst that will not germinate and instead will form a new zoospore, i.e. RZE. This process can be repeated up to three times depending on the conditions. 10) Non-viable cyst.
29 2.2.5 Other characteristics The 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 for A. astaci have been described. The hyphae are unseptated and uniform (Ø 8-10 μm). Septa are only present when the sporangia are formed. The hyphal tips are rounded and the hyphae grow inside the crayfish cuticle, not on its surface (Cerenius et al. 1988). When the sporangia are formed, the branches become slightly wider (Ø 10-12 μm) (Alderman & Polglase 1986). Encysted primary spores (Ø 9-11 μm) are spherical and usually there are 15-30 primary spores in each spore cluster (Alderman & Polglase 1986). Sporulation of cultured A. astaci hyphae can be induced under laboratory conditions by replacing the culture media (Unestam 1965) with lake water (Unestam 1966a; Unestam 1969b; Cerenius et al. 1988) or a diluted salt solution (Unestam 1966a; Unestam 1969c; Cerenius & Söderhäll 1984a). Zoospore formation and germination can be enhanced with CaCl2 treatment and inhibited with MgCl2 or KCl (Cerenius & Söderhäll 1984a). Laboratory tests have shown that zoospores are infective if present at temperatures between 2 and 25 °C (Unestam 1969b). The optimal temperatures for the sporangial formation and the release of the zoospores lie between 16 and 24 °C but it can still occur at temperatures as low as 4 °C (Alderman & Polglase 1986). At temperatures between 16 and 24 °C, the zoospores may continue swimming for at least 48 h (Alderman & Polglase 1986). Unestam (1969a) reported that in aquarium conditions at 14 °C, zoospores stay infectious for one week, but at lower temperatures (0 - 10 °C) the survival period is more prolonged, up to 14 days (Alderman 2000). Under favorable conditions, when the capacity of repeated zoospore emergence is taken into account, the survival time may be even longer (Cerenius & Söderhäll 1985; Evans & Edgerton 2002). Also the hyphal growth rate (Alderman & Polglase 1986) and the crayfish mortality rate (Alderman et al. 1987) are temperature- dependent: the optimal temperature for the hyphal growth on
30 RGY-agar is between 22 and 24 °C (Alderman & Polglase 1986). In laboratory infection trials, crayfish mortality occurs more rapidly at temperatures near 20 °C in comparison to lower temperatures (Alderman et al. 1987). Boiling (100 °C, 1 min) and freezing (-20 °C, 72 h) of the mycelia (Oidtmann et al. 2002a) or the infected crayfish (Alderman 2000) kill the pathogen and water temperatures constantly above 30 °C are also fatal (Oidtmann et al. 2002a).
2.2.6 Infection mechanisms The chemotaxis observed in A. astaci focus on the joints and the tips of the walking legs of the crayfish (Cerenius & Söderhäll 1984b), also the germination and penetration sites are usually found in the soft cuticle and body openings (Unestam & Weiss 1970). A germinating spore will secrete proteases when it penetrates through the epicuticle (Nyhlén & Unestam 1975). The secretion of chitinase starts before the germ tube starts to branch (18 h after encystment) and the chitinous layers of the crayfish 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 vegetative growth (Unestam 1966b; Andersson & Cerenius 2002). However, chitinase is not expressed in the zoospores, which do not encounter chitin (Söderhäll et al. 1978). Therefore, the constitutive chitinase production may be an adaptation to the parasitic life style and is a reflection that this specialized parasite is unable to survive without its crayfish host (Andersson 2001). No genotype or strain specific differences in chitinase production have been observed. Chitinase is also speculated to be a potential virulence factor, since it has such a critical role during the infection process (Andersson 2001; Andersson & Cerenius 2002). Some other enzymes that may involve the pathogenesis have also been recognized, i.e. the trypsin proteinase (AaSP2), which is expressed in the mycelia growing in the crayfish plasma. AaSP2 may have a role in combating the crayfish defense reactions (Bangyeekhun et al. 2001).
31 2.2.7 Molecular tools in research and diagnostics Until the 21st century, crayfish plague diagnostics relied on the microbiological isolation of A. astaci from the diseased crayfish and a successful re-infection of healthy crayfish (Alderman & Polglase 1986; Cerenius et al. 1988). Some modifications to the described method were developed (Oidtmann et al. 1999b; Viljamaa-Dirks & Heinikainen 2006) in attempts to improve the detection level, which was often poor because of the many potential contaminants that tended to overgrow the rather slowly 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 et al. 1994), which enabled the A. astaci genotype identification, in addition to the species detection from a pure culture. Even today, it is still necessary to conduct new isolations for research purposes and genotype identification. The first PCR-methods for the detection of A. astaci DNA from the crayfish cuticle samples were developed by Oidtmann et al. (2002b; 2004), but the methods were not sensitive enough for the detection of A. astaci from the North American carrier crayfish. The method was then further developed as a more sensitive nested-PCR (Oidtmann et al. 2006), which enabled carrier status analyses from the North American crayfish, although it lacked specificity, because the new closely related Aphanomyces species isolated from the crayfish, Aphanomyces frigidophilus (Ballesteros et al. 2006) and Aphanomyces repetans (Royo et al. 2004), also gave a positive signal 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 been developed (Hochwimmer et al. 2009; Vrålstad et al. 2009). TaqMan® Minor Groove Binder (MGB) qPCR targets the internal transcribed spacer (ITS) regions of A. astaci (Vrålstad et al. 2009). This method is highly sensitive and specific; the closely related Aphanomyces species do not cause any cross reactions, or false positives (Vrålstad et al. 2009; Tuffs & Oidtmann 2011). The method has also been applied to the A.
32 astaci spore detection directly from water filtrates (Strand et al. 2011). Another recent qPCR application is based on the chitinase gene amplification (Hochwimmer et al. 2009). Based on a comparative study, detection based on the ITS-regions (multicopy region) was reported to be 10-100 times more sensitive, than could be achieved with the chitinase gene (approximately three target copies per genome) based detection (Tuffs & Oidtmann 2011).
2.2.8 Genetics When this study started in 2006, there were few A. astaci sequences available in NCBI (National Center for Biotechnology Information) GenBank, and those were mainly sequences of ITS-regions, produced during the development of the specific PCR-method (Oidtmann et al. 2002b; 2004). Only a single chitinase gene sequence was available (Andersson & Cerenius 2002). Since then, the amount of genetic information has increased: the raw data for the transcriptome of A. astaci has been recently published in NCBI Genbank in 2013 and also the genome sequencing project of A. astaci is underway. Since the RAPD-PCR papers were published (Huang et al. 1994; Diéguez-Uribeondo et al. 1995), it has been obvious that there is high genetic diversity existing between the different genotypes of A. astaci (Huang et al. 1994; Diéguez- Uribeondo et al. 1995), but the location of these differences in the genome have remained unknown. Some phylogenetic studies have been carried out on the ITS-regions, revealing that different isolates and genotypes of A. astaci are very similar and the amount of intraspecific variation is near to zero (Diéguez- Uribeondo et al. 2009). This has been postulated to be a result of the dispersal via clonal zoospores (Diéguez-Uribeondo et al. 2009). The low variation rate on the ITS-regions is also advantageous, when this region is used as a target in the species-specific PCR applications (Oidtmann et al. 2006; Vrålstad et al. 2009).
33 2.3 AIMS OF THE STUDY
The aims of the PhD study were to explore the genetic diversity existing in the different strains of A. astaci, and if possible, to link this information to the observed differences in the physiological characteristics and differences in the disease progress in the noble crayfish. Another important reason for studying the genetic diversity was to find suitable genetic markers for the recognition of the different genotypes of A. astaci. In addition, it was desired to compare the sporulation kinetics of A. astaci during the acute and chronic infection.
The specific aims of this study were as follows (with references to the publications):
1. To explore the genetic diversity and to develop genotype specific markers for the A. astaci genotypes. (I, II)
2. To investigate under controlled laboratory conditions the differences in disease progress among the A. astaci strains isolated from Finland. (III, IV)
3. To study the differences among the Finnish noble crayfish (A. astacus) populations in crayfish plague resistance. (III, IV)
4. To compare the sporulation of A. astaci during an acute and chronic infection. (V, VI)
34 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 the exception of the isolates Evira6462/06 and Evira8372/09, which were obtained as live hyphae to be used in the infection experiments. RAPD-genotypes of the Evira strains were determined by Viljamaa-Dirks et al. (2013) and of Uppsala University strains by Huang et al. (1994) and Diéguez- Uribeondo et al. (1995). In the infection experiments (III & IV), four A. astaci strains were used (Table 2). For clarity reasons, they were named PsI-Puujärvi (UEF8866-2), As-Kemijoki (UEFT2B), As- Kivesjärvi (Evira6462/06) and As-Pajakkajoki (Evira8372/09).
35 36 Table 2. A. astaci isolates, their origins, isolation years, RAPD genotypes, hosts and authors. Isolates are of Finnish origin, if not stated otherwise. Hosts are abbreviated as follows: SC= signal crayfish, NC= noble crayfish and RSC= red swamp crayfish. Authors University of Eastern Finland (UEF), Finnish Food Safety Authority Kuopio Research Unit (Evira), Uppsala University (UU) and National Veterinary Institute of Norway (NVI). In paper I, internal transcribed spacer (ITS) sequences and in the paper II, chitinase gene sequences are compared.
Water body and/or Isolation RAPD Studied in paper Isolate code Host Author a geographical origin yeargenotype I II III & IV UEF7203 Lake Kukkia, Luopioinen 2003 PsI SC UEF x x UEF7204 Lake Kukkia, Luopioinen 2003 PsI SC UEF x x UEF7208 Lake Kukkia, Luopioinen 2003 PsI SC UEF x x UEF8140-1 Lake Pyhäjärvi, Säkylä 2003 PsI SC UEF x x UEF8140-2 Lake Pyhäjärvi, Säkylä 2003 PsI SC UEF x x UEF8140-5 Lake Pyhäjärvi, Säkylä 2003 PsI SC UEF x x UEF8147 Lake Pyhäjärvi, Säkylä 2003 PsI SC UEF x x UEF8866-1 Lake Puujärvi, Karjalohja 2003 PsI SC UEF x x UEF8866-2 Lake Pyhäjärvi, Säkylä 2003 PsI SC UEF x x x UEFT2B River Kemijoki, Taivalkoski 2007 nab NC UEF x x UEFKJ4 River Kemijoki, Koskenkylä 2010 nab NC UEF x Evira6462/06 Lake Kivesjärvi, Paltamo 2006 As NC Evira x x Evira8372/09 River Pajakkajoki, Kuhmo 2009 As NC Evira x x Evira6672/05 Lake Taulajärvi, Tampere 2005 As NC Evira x Evira4426/03 Lake Kelvänjärvi, Lieksa 2003 As NC Evira x x Evira5596/04 River Pyhäjoki, Venetpalo 2004 As NC Evira x Evira5727/04 River Pyhäjoki, Joutenniva 2004 As NC Evira x Evira10278/05 Crayfish farm pond, Paltamo 2005 As NC Evira x EviraK105/99 Lake Päijänne, Jyväskylä 1999 As NC Evira x EviraK071/99 River Lieksanjoki, Lieksa 1999 As NC Evira x EviraK104/98 Lake Konaanjärvi, Hauho 1998 As NC Evira x Table 2. Continued.
Water body and/or Isolation RAPD Host Studied in Isolate code Author geographical origin yeargenotype species I II III & IV EviraK047/99 Lake Korpijärvi, Mäntyharju 1999 PsI NC Evira x Evira3697/03 Lake Iso-Kuivajärvi, Hartola 2003 PsI NC Evira x x Evira7862/03 River Pyhäjoki, Oulaistenkoski 2003 PsI NC Evira x x Evira5721/04 River Pyhäjoki, Mieluskoski 2003 PsI NC Evira x Evira6458/03 Lake Lievestuoreenjärvi, Lievestuore 2003 PsI SC Evira x Pl Lake Tahoe, USA 1970 PsI SC UU x Pc Spain 1992 Pc RSC UU x L1 Ämmern, Sweden 1962 As NC UU x x Kv Swedenc 1978 PsII SC UU x VI03629 Ostfold, Halden, Norway 2005 nab SC NVI x x aRAPD-genotypes published in Huang et al. (1994), Diéguez-Uribeondo et al. (1995) and Viljamaa-Dirks et al. (2013). b Not analyzed with RAPD. c Isolated in Sweden from signal crayfish which originated from Pitt Lake, Canada. 37 3.2 THE CRAYFISH POPULATIONS
The crayfish populations used in the infection experiments and sporulation studies are presented in Table 3.
Table 3. Crayfish species and populations, crayfish amounts and catchment years used in 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 2 surrounding the 5.8S rRNA gene of 18 A. astaci strains were sequenced and compared. In paper II, partial chitinase gene sequences were produced and studied from 28 A. astaci strains (Table 2). In both studies (I & II), the target fragment of A. astaci DNA was first amplified with the polymerase chain reaction (PCR) with suitable primers. For the ITS-regions, universal primers ITS1 and ITS4 (White et al. 1990) producing a 720 bp amplicon were used. For the sequencing of the chitinase gene, primers AaChiF and AaChiR, producing a 384 bp PCR amplicon containing a partial chitin binding site and partial catalytic domain, were designed (paper II). PCR reactions were carried out from DNA extracted from cultured mycelia of A. astaci. Detailed descriptions of the methods for the DNA extraction, PCR amplification, cloning and sequencing are given in papers I and II. Sequencings were made from PCR amplicons directly and after a molecular cloning of
38 the PCR amplicon into a plasmid vector. The initial aim was to obtain a general view of the studied genetic region of each strain and subsequently to conduct a molecular cloning process in order to obtain more detailed information of the internal variation inside a multicopy region or -gene. Sequence analysis and alignments of ITS regions were built with MultAlin (Corpet 1988) and ChromasPro 1.41 (Technelysium Ltd., Australia) computer programs. BioEdit 7.0.8 was used to calculate nucleotide differences (Hall 1998) and DnaSP (Librado & Rozas 2009) for the divergence parameters. Chitinase gene sequences were analyzed and aligned with GeneiousPro 5.3.6 (Drummond et al. 2011). Phylogenetic maximum likelihood trees were made with PhyML (Guindon & Gascuel 2003) and the possible recombination events were determined with DualBrothers (Suchard et al. 2003; Minin et al. 2005). The sequences produced in these studies are available in NCBI (National Center for Biotechnology Information) GenBank with 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 test different combinations of crayfish populations and A. astaci strains. The experiments were carried out on the largest possible scale, but it was not possible to implement a full cross- examination of the different combinations, because of the limited time, space and hands. Zoospore emergence from the cultured mycelia of A. astaci was induced by lake water washings as described by Cerenius 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 Lake Koivujärvi crayfish, where different zoospore densities (1, 10, 100 and 1000 spores ml-1) were tested (Table 4). The infection experiments were conducted in an air-conditioned laboratory and the temperature was adjusted to a constant + 18 °C.
39 Table 4. Crayfish populations, A. astaci strains and zoospore densities used in the infection 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, the behavior and any possible deaths of crayfish were monitored daily. Dead crayfish were removed immediately to avoid secondary infections by zoospores being released from moribund individuals. The dead crayfish were individually packed and stored in a freezer (-20 °C) for further DNA extractions and qPCR analyses (Vrålstad et al. 2009) of the infection status. Detailed descriptions of the methodological techniques are explained in the relevant articles (III & IV). The infection statuses of the crayfish were classified according to Vrålstad et al. (2009) as follows: agent levels A0 (no detection) and A1 (threshold cycle, Ct >39) were assessed as the negative results and amplification below the limit of detection, respectively. Agent level A2 corresponded to very low levels of A. astaci DNA and the detection was below the limit of quantification (Ct 39.0-34.7). Agent level A3 indicated the presence of low levels of A. astaci DNA (Ct 34.6-30.0) and A4 designated a moderate infection status (Ct 30.0-26.2). Kaplan-Meier survival test (Log Rank, Mantel-Cox) was used to evaluate differences in mortality among experimental groups (SPSS 17.0). A non-linear regression was used to study the effect of inoculation spore dose in the Expt 6.
3.3.3 Sporulation (V & VI) In the sporulation studies, three different experimental setups were used. First (V), the noble crayfish (n=9) were infected with A. astaci strain PsI-Puujärvi (UEF8866-2) in a separate spore bath (20 h) containing 1820 spores ml-1 in lake water. After incubation,
40 the crayfish were rinsed in fresh water for 1 h and then rinsed again in fresh clean water before their transfer into the individual monitoring tanks, which contained 8 l of fresh and spore-free lake water. The laboratory temperature was set at + 18 °C. The infected crayfish were monitored twice a day (9.00 am and 21.00 pm) and simultaneously, 1 ml water samples were collected in triplicate from each tank. Water samples were immediately frozen (-20 °C) for further spore quantity analyses with qPCR (Strand et al. 2011). When a moribund crayfish was observed, the water circulation was immediately stopped to avoid the spread of the spores to the adjacent monitoring tanks. The sample collection was continued for seven days post mortem. Quantitative PCR analyses were run from freeze-dried spore eluates in a method described in detail in paper V. Each sample was analyzed as non-diluted (1x) and diluted (10x) DNA, to monitor possible inhibition in the PCR reaction. The amount of A. astaci ITS copies in a sample was estimated as PCR forming units (PFU, the amount on target DNA copies in a genome), according to Vrålstad et al. (2009), which was then translated into spores by dividing by 177. The amount of PFU’s per spore (177) was estimated by a dilution series from freeze-dried spores similarly as described by Strand et al. (2011). In addition, there were two separate experiments conducted to determine A. astaci sporulation of the signal crayfish (P. leniusculus) from two locations (a crayfish farm in Orivesi and Lake Saimaa) with a known carrier status of A. astaci (VI). Two experiments were made with both populations: the IHC (individually housed crayfish) experiment consisted of ten replicate 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 of four replicate tanks at room temperature (17-23 °C). Each tank contained 20 signal crayfish in 100 l volume of lake water.
41 In both experiments, water samples were collected weekly: in the IHC-setup, a 1 l water sample was collected from each 2 l tank. In the CHC-setup, three 1 l water samples (two samples from 5 cm below the water surface and one sample by siphoning the bottom of the tank) were collected from each tank. After the sampling, the crayfish were fed and two days later, the tanks were cleaned and new water was added. Hence, each sample represented the spore amount released from the signal crayfish during a period of five days. The amount of A. astaci spores in the water samples were quantified with vacuum filtration (3 μm polycarbonate filters, Millipore IsoporeTM) and DNA extraction (Strand et al. 2011) was made from the filtrate. Quantitative PCR (Vrålstad et al. 2009) was applied as quadruplicate (duplicate undiluted and duplicate 10x diluted) for each sample. Detailed criteria and the methodology for the estimation of spore amounts in the samples are explained in the paper VI. Statistical analyses were made with R (2.13.2), where a linear mixed-effect model was applied to estimate the spore numbers (V & VI). The crayfish populations were compared with the logistic regression model (VI).
42 4 Results
4.1 GENETIC VARIATION
4.1.1 Internal transcribed spacers (I) In total, 32 sequences from 17 Finnish A. astaci isolates and one Swedish isolate (Table 2) were produced (I). The sequences that were generated directly from the PCR amplicons (n=13), were all identical, i.e. no genotype or strain specific mutations were observed. Among the cloned sequences (n=23), several single nucleotide polymorphisms (SNP) were found. Altogether, 31 SNPs were counted in the 720 bp region, which represented polymorphism level of 3.2 % among the sequence set cloned in this study. The highest levels of polymorphisms (5.2 %) were observed in the ITS1-region, but some polymorphisms (n=3, polymorphism level 1.9 %) were also detected in the conserved 5.8S rDNA gene. In the ITS2-region, polymorphism level was 4.2 % (I, table 3). Intragenomic variation (variation between the different copies of the multicopy region in the genome) was observed in the strains UEF8822-2 and UEF7208, from which various clones were produced. Haplotype divergence (Hd) inside a strain UEF8866-2 was actually slightly higher (Hd=0.833) than the intraspecific 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) in comparison 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 sequenced directly after the PCR amplification. Among the PCR amplicons, 14 SNPs classified the As- and PsI-genotypes and four SNP differences were observed
43 between the As- and Pc-genotypes (Fig. 3A). The two signal crayfish (P. leniusculus) related genotypes PsI and PsII were identical 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) analysis made with PhyML (Guindon & Gascuel 2003). Moreover, the CHI2-group was divided into three subgroups, named CHI2A, CHI2B and CHI2C (Fig. 3B). Group CHI1 (n=72) was the most commonly detected group, each strain studied had CHI1-sequence(s) present in its genome. CHI2 was the most heterogenic group, the subgroups described above were divided as follows: CHI2A was the most common group (n=38) and it was present in both genotypes As and 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 group and 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 as group CHI1B as well, since this group displayed the typical nucleotide changes observed in both groups CHI1 and CHI2. However, based on the results of the ML-analysis, the group was named as CHI2C (Fig. 3B). Possible recombination sites were also detected between the CHI2A and CHI3 groups, among the strains of Ps-genotype. Chitinase sequencing can be applied also in genotyping purposes as described in paper II, i.e. using PCR to amplify the DNA extracted from the crayfish cuticle samples. This method is applicable in cases where there is severe infection and high amounts of A. astaci DNA present in the crayfish cuticle. If the analysis is made after TaqMan® MGB qPCR (Vrålstad et al. 2009), which is the recommendation, this corresponds to amplification with less than 30 cycles (A3 agent level, > 1000 PCR forming units).
44 A)
B)
Figure 3. Differences observed in the chitinase sequences classify the different genotypes of A. astaci. A) Location of the polymorphic sites in the PCR-amplicons. B) Maximum likelihood tree (Guindon & Gascuel 2003) of cloned sequences showing the three main chitinases (CHI1, CHI2 and CHI3) and the division of CHI2 into three subgroups (A, B and C).Figure 3B printed with permission from Elsevier.
4.2 VARIATION IN THE DISEASE PROGRESS (III & IV)
4.2.1 Virulence variation Extensive variation in the virulence among the A. astaci strains was observed in the six infection experiments (Table 5). PsI- Puujärvi, which was the only PsI-genotype strain used in these experiments, killed 100 % of the infected noble crayfish within less than one week. Only one exception was observed, in a Lake Mikitänjärvi crayfish population where deaths started 11 days after the inoculation and 100 % mortality was only achieved at
45 day 37. The high virulence of PsI-Puujärvi was also present in Expt 6, with different spore dosages: 1 spore ml-1 density of PsI- Puujärvi spores killed 100 % of the Lake Koivujärvi crayfish more rapidly than 1000 spores ml-1 of As-Kivesjärvi spores (Fig. 4). The As-Kivesjärvi strain was used in five infection experiments, of which in three experiments, 100 % mortality was achieved during the follow-up period. In two experiments (Expt 4 & 5) made with Lake Viitajärvi and Lake Mikitänjärvi crayfish and a slightly lower spore dosage (670 and 260 spores ml-1, respectively), in Lake Viitajärvi crayfish cumulative mortality reached 50 % during the 49-day follow-up and in Lake Mikitänjärvi crayfish, only 9 % mortality occurred during the 99- day follow-up. In contrast, there was 100 % mortality in Lake Koivujärvi crayfish infected with As-Kivesjärvi in the dose-effect experiment, with all the different spore dosages (1-1000 spores ml-1) being tested (Fig. 4). Strain As-Pajakkajoki was used only in a single infection experiment, where it evoked 100 % mortality in Viitajärvi and ESeppä crayfish, and there was no statistically significant difference in comparison with the As-Kivesjärvi strain. The As-Kemijoki strain was used in four of the six infection experiments. Increased mortality was observed only in a single experiment (Expt 1) and also in that case, only in the Lake Rytky crayfish population. Lake Viitajärvi crayfish, tested simultaneously under the same conditions and with the same spore batch, remained viable. In Expt 3, 17.0 % mortality was observed in Lake Viitajärvi crayfish, while in Lake Rytky crayfish, 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-Kemijoki remained mainly negative, and thus were uninfected, or alternatively only very low agent levels (A1-A2, indicating A. astaci DNA amount of less than one spore) were detected.
46 Table 5. Crayfish populations and numbers (n), A. astaci strains and spore densities used in the infection experiments. The mean day of death refers to the number of days after inoculation, when 50 % mortality was achieved. Mortality rate (%) describes the total 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.
Day of Death (DoD) Spores Population n Strain Mean±SD Mortality Corrected mL-1 (days) rate (%) DoD2 Expt 1 12 Control 43.01 0.0 n/d Lake 12 As-Kemijoki 2 250 43.01 0.0 14.4 Viitajärvi 12 PsI-Puujärvi 2 250 5.2±0.1 100.0 3.3
Lake 12 Control 42.3±0.7 8.0 n/d Rytky 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
Expt 2 12 Control 25.3±0.7 8.0 n/d Lake 24 As-Kivesjärvi 2 440 10.2±0.4 100.0 14.0 Viitajärvi 24 As-Pajakkajoki 2 440 15.8±1.0 100.0 n/d
Farmed 12 Control 22.8±1.5 33.0 n/d Eseppä 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 3 12 Control 50.3±0.7 8.5 n/d Lake 22 As-Kemijoki 810 50.4±0.6 17.0 n/d Rytky 24 As-Kivesjärvi 810 15.6±0.5 100.0 19.4
Lake 6 Control 51.0±0.0 0.0 n/d Viitajärvi 12 As-Kemijoki 810 44.5±4.2 4.5 n/d 24 As-Kivesjärvi 810 16.6±0.5 100.0 19.4
Expt 4 12 Control 47.01 0.0 n/d Lake 18 As-Kemijoki 670 47.01 0.0 n/d Viitajärvi 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 5 12 Control 93.5±4.5 18.2 n/d Lake 24 As-Kemijoki 260 97.7±1.3 4.6 n/d Mikitänjärvi 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.
47 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 different zoospore densities (1, 10, 100 and 1000 spores ml-1).
4.2.2 Resistance variation Increased resistance towards the strains of the As-genotype was observed in several infection experiments, especially in those conducted with low spore dosages (less than 1000 spores ml-1 in the experiments 3, 4 and 5). No gender- or size-related differences in resistance were observed. Between the experiments 4 (Lake Viitajärvi) and 5 (Lake Mikitänjärvi), results were compared among each other using the estimated dose-response curve (IV). The day of death given spore dose corrections indicated that Lake Mikitänjärvi noble crayfish had a significant time lag in the observed day of death, as the PsI-Puujärvi infected crayfish died approximately 3.5x later than the model predicted. Lake Mikitänjärvi crayfish infected with As- Kivesjärvi exhibited no mortality whereas the model predicted an 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 the equal inoculation dose, while the observed day of death during PsI-genotype was similar to the estimate from the dose-response curve.
48 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 it reached its maximum at 48 h and it remained significantly higher in comparison to premortem levels until 96 h post mortem. In this experimental setup, the sporulation level declined below the premortem levels at 120-156 h post mortem. Based on this data, it was estimated (mean with 90th and 10th percentile) that a noble crayfish having an acute infection of A. astaci, could release 1.7 million spores (573 000; 2 829 000) into the ambient water during the sporulation peak. The biggest number of spores released by a single crayfish individual was estimated at approximately 3.2 million spores passing into the ambient water during this sporulation peak, if one assumes a homogenous 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 the individually housed crayfish and 9 577 (161, 22 789) for the communally housed crayfish. As an overall mean estimate, a signal crayfish individual that was carrying A. astaci (without an acute infection) released 22 106 (67; 28 466) spores during a five day time period. Higher variation in the amount of sporulation was observed among the individually housed crayfish than in the communally housed crayfish, which most likely indicated that there was extensive variation in extent of sporulation between different signal crayfish individuals. Those signal crayfish that survived until the end of the experiment released 2 763 (26; 5 490) spores per week in the absence of deaths and moultings. A significant increase in the sporulation was observed one week prior to their death. The
49 trend was detected in 14 of the 18 crayfish, but it was absent in four crayfish. Increased sporulation was observed also during the moulting events. The substitute crayfish (n=7), that were added to the tanks during the experiment to replace the dead individuals, released high spore levels during the first 1-2 weeks when they were introduced into the experimental system.
50 5 Discussion
5.1 GENETIC VARIATION AMONG A. ASTACI
The ITS-regions of the A. astaci strains were highly similar (I). In fact, the sequences of PCR-amplicons (produced without molecular cloning) were 100 % identical. In the cloned sequences, 96.8 % similarity was observed confirming the results of previous studies (Oidtmann et al. 2004; Diéguez-Uribeondo et al. 2009; Vrålstad et al. 2009). The difference in the similarity percentages obtained with two different approaches is a consequence of the methodology: the PCR amplicon sequence presents a combined result of the most common sequence in the multicopy region, while the sequence obtained after molecular cloning process represents a single, randomly selected ITS-copy (Ganley & Kobayashi 2007). Nonetheless, minor differences among the different copies, so called intragenomic variation, do exist (Gandolfi et al. 2001; Ganley & Kobayashi 2007) and it has been 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 intraspecific variations in the ITS-regions have been reported (Diéguez- Uribeondo et al. 2009). It has been postulated that the main mechanism for the homogenization of ITS copies is recombination 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 been claimed that the laboratory cultivation and the lack of sexual recombination may diminish the variation and the number of rDNA copies affect the polymorphism levels (Ganley & Kobayashi 2007). Based on the study of Diéguez-Uribeondo et al. (2009), the saprophytic and plant parasitic species of Aphanomyces with a known sexual life cycle display a higher intraspecific variation level in comparison to the clonally
51 spreading animal pathogenic lineage of Aphanomyces. These results are at odds with the current theory of concerted evolution. It has also being claimed that concerted evolution may not be as efficient as has been suggested, since in some species of fungi, surprisingly high intragenomic variation has been observed (Simon & Weiß 2008). Moreover, the low level of intraspecific variation in A. astaci is a highly advantageous feature, because most of the current molecular techniques used in the detection of A. astaci target the ITS-regions. If there were high intragenomic- and intraspecific variation, this would lead to false negative diagnoses and to unreliability of these detection methods (Oidtmann et al. 2006; Vrålstad et al. 2009; Tuffs & Oidtmann 2011). In the chitinase genes of A. astaci, several genotype specific marker sites were detected (II). Based on the previous studies, there seem to be at least 3-4 chitinase gene copies present in the genome of A. astaci (Andersson & Cerenius 2002; Hochwimmer et al. 2009) and the observed variation can be due to either differences between the gene copies, or to allelic variation (heterozygosis), also known as the single nucleotide polymorphisms (SNPs). In the PCR amplicons, 14 polymorphic sites distinguished 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 greater influence on the genotype than the geographical origin of the crayfish population in North America. One interesting finding was the similarity between the As- and Pc-genotypes, which were distinguished only by four sites. Based on the chitinase gene sequences, these genotypes were similar, although in RAPD-PCR they were clearly distinguishable (Huang et al. 1994; Diéguez-Uribeondo et al. 1995), therefore the differences must be present in other genetic locations. Transcriptome comparisons are likely needed to arrive at definite conclusions. Nevertheless, it would be tempting to speculate that the original host of the As-genotype may have
52 been closer to Procambarus spp. than Pacifastacus spp. This is also supported by the theory of accidental transport of the vector species in the ballast water tanks of the ships (Alderman 1996), since the European-bound ships left from the east coast of North America, where the procambarids are the native species (Hobbs 1988). The comparison of the chitinase sequences produced with a molecular cloning process indicated the presence of three groups of chitinases, possibly being derived from three different genes. Some of the chitinase forms were specific for either the As-genotype (CHI2B, CHI2C) or the Ps-genotypes (CHI3). The observed differences most likely indicate that the genotypes have co-evolved with their original host species for a rather long time. Altogether, the As-genotype seemed to be more variable in comparison to the Ps-genotypes. This result is logical, since the As-genotype of A. astaci is constantly undergoing a higher selection pressure in comparison to the Ps-genotypes. This may lead to lowered virulence and a better adaptation to the new host 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 in Oomycetes (Dodds 2010; Raffaele et al. 2010), since the pathogens need to adapt to their hosts by altering their virulence (Ebert 1994). Another possible explanation for the observed variation could be the multiple introductions of the As-genotype into Europe, thus having a wider origin in comparison to Ps- genotypes. Furthermore, the group CHI2C of the As-genotype included a SNP which was introduced a translation stop codon into the beginning of the catalytic domain of the gene; therefore the As-genotype seems to have a nonfunctional chitinase copy in its genome. Chitinase is believed to be a potential virulence factor of A. astaci, because it has an essential role in the penetration into crayfish cuticle (Andersson & Cerenius 2002). Some chitinases may also have a role in the cell wall modulating processes. The observed genetic differences may be one possible
53 reason for the differences in the virulence of these genotypes. Based on the studies conducted on the plant pathogenic Oomycetes, adaptation to the new host species is most likely to cause mutations and copy-number changes in hundreds of effector genes, located in the variable repeat-rich partitions of the genome, which are linked to the pathogenicity and the host range (Jiang & Tyler 2012). The observed differences in the chitinase gene sequences can also be used for genotyping purposes. The genotyping with chitinase PCR can be made directly from the crayfish cuticle samples. This method is not as sensitive as qPCR, but it can be utilized in the cases of acute infections or clear visible symptoms of a crayfish plague, when high amounts of A. astaci hyphae are present in the crayfish cuticle. For example, it was possible to detect the As-genotype infection (unpubl. results) from the Turkish narrow-clawed crayfish (A. leptodactylus) displaying symptoms 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 also linked to the A. astaci diagnostics. Homogenic ITS-regions proved suitable for the detection at the species level, while the chitinase gene sequencing, albeit with more limited sensitivity (Tuffs & Oidtmann 2011), could be used in the detection of the known genotypes. Unfortunately, the recently isolated Or- genotype (Kozubíková et al. 2011a) was not available when this study was conducted and thus the information of its chitinase gene variability is not available. Furthermore, a recent study by our research group has shown that the genetic diversity detected in the chitinase gene of A. astaci represents only a tiny proportion of the true genetic variation existing between the As- and Ps-genotypes. A large amount of new polymorphic sites have been identified all over the genome, when the complete transcriptomes of A. astaci genotypes are compared (unpubl. results).
54 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 the PsI-genotypes could be demonstrated. The PsI-Puujärvi strain was an aggressive killer and 100 % acute mortality with the tested zoospore densities was generally achieved within a week, although in the previous experiments, also occasional survivors have been observed (Jussila et al. 2011b). Based on the latest experiment (unpubl. data), a rapid 100 % mortality, similarly as described in the experiments made here with PsI-Puujärvi, has also been detected with several other PsI-genotype strains of A. astaci. The tested As-genotype strains were more variable in their virulence features, the symptomatic stage started later and lasted longer and the acute mortality did not appear as rapidly as observed with the PsI-Puujärvi. As-Kivesjärvi was the most virulent of the tested As-genotype strains, while the As- Pajakkajoki provoked fatalities somewhat later, although the difference was not statistically significant (III). Although the mortality rate of As-Kivesjärvi and As-Pajakkajoki strains was slower in comparison to the PsI-Puujärvi infections, both strains caused 100 % mortality in two experiments (III, Expts 2 & 3). In the next experiments (IV, Expts 4 & 5), As-Kivesjärvi infections were not as efficient, since 50 % mortality occurred in Lake Viitajärvi crayfish (Expt 4) and no mortalities were observed among 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 Lake Rytky crayfish population, whereas Lake Viitajärvi crayfish infected under the same conditions with the same spore batch remained alive and asymptomatic (III). In three other infection experiments (Expts 3, 4 & 5), the As-Kemijoki caused no increased mortality in comparison to the control group (III, IV). As a side observation, that the crayfish infected with the PsI-Puujärvi became clearly symptomatic one day prior to their deaths. The symptomatic crayfish had an agitated behavior with
55 intensive scratching of their cuticle; this scratching was especially focused on the eyes and the joints of the walking legs. Among the As-Kivesjärvi infected crayfish, the symptomatic stage of the disease lasted longer, 2 days on the average, and in some individual cases for as long as 3-4 days. With this strain, the main symptom was the loss of the balance and a partial paralysis: the crayfish tended to lie on their backs, but when disturbed physically, an escape reaction occurred promptly. With both tested strains, the appearance of symptoms was invariably followed by death. The surviving crayfish exhibited no symptoms of the disease (III, IV). The As-Kemijoki strain seemed to be almost avirulent, although it has been responsible for crayfish mortalities in River Kemijoki (Lapland, Finland). The Kemijoki epidemics started in 2006 and are still ongoing (Petri Muje, personal communication). The As-Kemijoki isolate was from the year 2007 and it has been reported that years of laboratory cultivation have converted some A. astaci isolates to being avirulent: this has happened due to the loss of the zoospore motility (Unestam & Weiss 1970; Unestam & Svensson 1971). However, the zoospores of As- Kemijoki were motile when the experimental infections were prepared and therefore, different mechanisms of avirulence must have occurred to explain this phenomenon. Based on the qPCR results from the surviving crayfish, it appears as if the spores could not have attached to the crayfish cuticle, since most of the tested crayfish remained negative (uninfected) in the qPCR (III, IV). The others showed very low agent levels, corresponding to a DNA amount of less than a spore being present in their tissue. One possible explanation for the observed avirulence could also be temperature adaptation. The strain was causing epidemics in rather cold environmental conditions in River Kemijoki and it needs to be evaluated, whether its temperature optimum is lower than expected. Earlier, strains adapted to warmer environments have been reported from the North American red-swamp crayfish (P. clarkii) in Spain (Diéguez- Uribeondo et al. 1995).
56 The variables reported to be responsible for the differences in the mortality rate during the crayfish plague infection are the zoospore dosage and ambient water temperature (Unestam & Weiss 1970; Alderman et al. 1987; Alderman 2000). Here, the effect of the water temperature was removed by performing this experimental series in an air- conditioned laboratory space, where the temperature was adjusted to be a constant +18 °C. With respect to the zoospore dosage effect, an experiment was conducted to examine the effect of the spore dosage on the mortality rate (Expt 6, IV). Lake Koivujärvi 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 dosage was observed. All the tested spore densities caused 100 % mortality, although at the lower densities, the mortality rate was slower (IV). This experiment also emphasized the high virulence of the PsI-Puujärvi: 1 spore ml-1 dosage of PsI-Puujärvi provoked acute mortality faster than 1000 spores ml-1 of As-Kivesjärvi (IV). Previously, the zoospore dosage effects have been tested with the narrow-clawed crayfish (A. leptodactylus) and the results have been comparable to the present findings (Alderman et al. 1987). Unestam & Weiss (1970) reported that a zoospore density of 25 spores ml-1 inoculated into aquarium water caused 50 % mortality (LD50) in the noble crayfish (A. astacus) with a further challenge of 250 spores ml-1 causing 100 % mortality during the 36 days follow-up. Cerenius et al. (1988) have also claimed that 10 000 spores ml-1 will always evoke 100 % mortality of crayfish, but in the present experimental conditions with large water volumes (Jussila et al. 2011b), it was not possible to achieve such high infection doses. It was much more difficult to demonstrate the variation in the resistance than the variation in the virulence, because the direct comparison of different experiments was not possible. The physiological status (i.e. moulting cycle, reproduction cycle, and infections other than A. astaci) could have had some impact on crayfish survival, at least in P. leniusculus (Nylund & Westman
57 1983), and therefore only the experiments conducted simultaneously were directly comparable. We also found out that there is a logarithmic response in the mortality initiation and A. astaci spore amount. With higher spore amounts used in the infection, crayfish started to die earlier (Fig. 4). Based on the day of the dead estimates, it was possible to compare to some extent the Lake Viitajärvi (Expt 4) and Lake Mikitänjärvi (Expt 5) populations. Lake Mikitänjärvi crayfish have been reported to carry A. astaci infection (Jussila et al. 2011a), and this could have caused increased resistance against the new secondary infections of A. astaci. When the effect of the zoospore dosage was taken into account, the mortality rate of Lake Mikitänjärvi crayfish after PsI-Puujärvi infection was approximately 3.5 times more slow than encountered in the Lake Viitajärvi population. However, despite the delayed mortality, 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, as only a few individuals died after the As-genotype infections (IV).
5.3 VARIATION IN SPORULATION
The sporulation from the noble crayfish (A. astacus) having an acute infection of the crayfish plague was measured from the PsI-Puujärvi infected crayfish, at 12 h intervals (V). The effect of the inoculation zoospore doses was excluded by performing the infections in separate infection tanks and rinsing the crayfish in clean water before their transfer into the monitoring tanks. Therefore, one can assume that the spores detected in the water of the monitoring tank were spores which had been released from the moribund crayfish individuals. Sporulation was also measured from the As-Kemijoki infected noble crayfish, but since no deaths occurred during the experiment, also the spore measurements with qPCR remained negative (unpubl. results). Moderate amounts of spores were detected already shortly after the infections and premortem of the crayfish individuals. The main peak in the sporulation occurred 24-96 h
58 postmortem. Thus during the sporulation peak, as many as 3.2 million spores could be being released from a single crayfish, presuming that the spores were distributed homogenously. Sporulation had significantly declined by the sixth day postmortem, 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 motile period of the zoospores. Therefore the actual time period for zoospore 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 allows the zoospore to become encysted and start another motile period after a short resting period (Cerenius & Söderhäll 1984a; Cerenius & Söderhäll 1985). There was an extensive variation in the spore release between the experimental crayfish and between the different measurement points. Part of the variation may be explained by the sampling, since the spores more likely orientated towards the solid particles in the water column (Svensson 1978) and for that reason they might not have been equally distributed within the water column. The variation between the individuals was also high. An individual crayfish, that died one day after the infections, was sporulating considerably less than those individuals that died later. One could speculate that this individual was weakened and died for some other unknown reasons 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 the individual crayfish. The water sampling for the spore quantification was made at a level 15 cm above the tank bottom, mainly to avoid measuring any encysted spores which had accumulated on the tank bottom. If the measurements had been made directly from the bottom, larger spore estimates would have been obtained, similarly as in paper VI.
59 In the signal crayfish (P. leniusculus), the sporulation pattern during the chronic infection was rather different (VI) compared to that of noble crayfish (V). A signal crayfish carrying A. astaci continuously released a moderate number of spores (~ 2 700 spores per week), provided it was not moulting or succumbing to the infection. Spores were also produced in cold water (+4 °C). Moreover, moultings and deaths increased the sporulation levels, as predicted (Oidtmann et al. 2002a). The amount of sporulation increased one week prior to death, which indicates that weakened or stressed individuals can develop an acute crayfish plague infection, similar findings have been reported earlier (Unestam 1972; Persson et al. 1987; Cerenius et al. 1988). In the case of individually housed crayfish, stress induced sporulation was also observed, as the substitute crayfish released higher numbers of spores during the first 1-2 weeks after being placed into the experimental system. Probably due to the accumulation of the encysted spores onto the tank bottom and the taxis of the spores towards the nutrients (Cerenius & Söderhäll 1984b), the spore numbers in the communally housed crayfish were considerably higher on the tank 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 bottom where they quickly become attached to the crayfish. Therefore, the spore estimates based on the water samples taken from the water column are likely to underestimate the actual numbers of spores that the infected individuals were releasing. The signal crayfish that were used in this study (VI) were severely infected with the A. astaci overall prevalence being 95 %. In the carrier crayfish populations, the prevalence and infection status is highly variable (Kozubíková et al. 2009; Skov et al. 2011; Vrålstad et al. 2011) and thus in the populations with a lower infection 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 ambient water, not only during their moulting phase or death. Similar
60 indirect evidence has been gained earlier when the red swamp crayfish (Procambarus clarkii), i.e. when susceptible species have been placed in the same tanks with their red swamp crayfish counterparts, the susceptible crayfish became infected with the crayfish plague (Diéguez-Uribeondo & Söderhäll 1993). Thus, it is apparent that these carrier crayfish species pose a constant threat to susceptible species, and this risk is not dependent on the phase of the crayfish life cycle.
61 62 6 Conclusions
The agents of most emerging diseases often originate from other geographical areas or from another host species. The crayfish plague is an excellent example of this phenomenon. After its arrival in Europe in the form of the As-genotype, the pathogen has been subjected to high selection pressure as it spreads into its new environment infecting the highly susceptible European crayfish host species. After the first wave of the disease, new crayfish plague genotypes were brought to Europe with the North American crayfish species introduced here to fill the lakes emptied of native crayfish. These genotypes are most likely experiencing less selection pressure, since their original host species are present in Europe, although the environment is different. This thesis provides new information about the genetic variability of A. astaci. With respect to the two studied genetic regions, the intraspecific variation in the ITS-regions was low (I) and based on these results, the ITS-regions can be viewed as highly suitable target region for developing species-specific molecular detection methods. Genotype specific diversity was observed in the chitinase genes of A. astaci (II). Based on the chitinase sequence comparisons, the two in this sense identical signal crayfish genotypes, PsI and PsII, were clearly distinguishable from the As- and Pc-genotypes. In contrast, the As- and Pc-genotypes were rather similar to each other and this similarity may also reflect their original host species in North America. The variation observed among the strains of the As- genotype also indicate, that this clonally spreading crayfish pathogen has been subjected to high selection pressure during its 150 year history in Europe. The genetic differences observed in the chitinase sequences also permit the genotype detection with PCR and sequencing, directly from the crayfish cuticle. Until recently, the genotyping has relied on the RAPD-PCR method, which means
63 that genotyping was possible only if a pure culture of A. astaci could be successfully obtained. As a result of this study (II), the specific markers for the genotype detection are now available for researchers and based on these results, the genotyping can also be further developed towards applications with higher sensitivities. Moreover, significant differences in the physiological properties, i.e. in the differing virulence of the A. astaci strains, were observed (III, IV). Based on the series of the infection experiments made with different A. astaci strains and crayfish populations, it can be stated that the PsI-genotype, which is linked to the signal crayfish introductions, is a highly virulent killer. A. astaci strains of this genotype killed 100 % of the infected noble crayfish (A. astacus) within a few days. It is also less likely that the virulence of the PsI-genotype will become reduced during the next decades, because the signal crayfish is now widely present throughout Europe and there is a host- pathogen relationship of A. astaci PsI-genotype and the signal crayfish in existence between these two species. The tested strains of As-genotype were more variable in their virulence and in several experiments, part of the noble crayfish populations survived, or even evaded the infection under laboratory conditions, with limited water volumes and no protective areas provided for the experimental crayfish. Based on these results, one can recommend that the carrier status analyses of noble crayfish should be considered, when the stockings are planned. This would help to avoid the spread of the less virulent forms of the crayfish plague, since it seems that also some infected noble crayfish can act as latent carriers of crayfish plague, without displaying any visible symptoms of the disease. During our experiments (III, IV), there were also indications that small differences do occur in the resistance features of different noble crayfish populations. Some populations with a known history of crayfish plague had a slower mortality rate in comparison to the populations without previous epidemics. Although the mortality rate was slower, all of the crayfish still died from the A. astaci infection made with the PsI-Puujärvi. However, future studies will examine this topic in
64 more detail, and more definitive remarks can be expected to be gained in the future. Furthermore, in this thesis, the sporulation dynamics affecting the spread of A. astaci during the acute infection and in the chronic infection were quantified with modern qPCR techniques (V, VI). In the noble crayfish, moderate amounts of A. astaci spores were released premortem and the maximum peak in the sporulation was achieved 24-96 h postmortem. In the signal crayfish, the rate of sporulation was constant and it also occurred in the cold water, although the highest spore amounts were released under stress, moulting and death in the water temperatures corresponding to the summer period. All the results obtained in this thesis underline the fact that the further spreading of the signal crayfish should be prevented more efficiently. The crayfish plague in the signal crayfish is genetically different from As-genotype and furthermore, it is highly virulent. In addition, infected signal crayfish are constantly sporulating and therefore posing a continuous risk to the surrounding waters, when not only crayfish, but also fish, or any contaminated equipments are being transported.
65 66 References
Abrahamsson S (1969) Signalkräftan — erfarenheter från USA och aspekter på dess inplantering i Sverige. Fauna och Flora 64:109- 116, in Swedish Alaranta A, Henttonen P, Jussila J, Kokko H, Prestegaard T, Edsman L, Halmekyto M (2006) Genetic differences among noble crayfish (Astacus astacus) stocks in Finland, Sweden and Estonia based on the ITS1 region. Bull Fr Pêche Piscic 380-381:965-976 Alderman DJ (1996) Geographical spread of bacterial and fungal diseases of crustaceans. Rev - Off Int Epizoot 15:603-632 Alderman DJ (2000) Summary final report: Effects of exposure to high and low temperatures on the survival of the crayfish plague fungus A. astaci in vitro and in vivo. Australian quarantine and inspection service, Canberra Alderman DJ, Polglase JL (1985) Disinfection for crayfish plague. Aquacult Res 16:203-205 Alderman DJ, Polglase JL (1986) Aphanomyces astaci: isolation and culture. J Fish Dis 9:367-379 Alderman DJ, Polglase JL (1988) Pathogens, parasites and commensals. In: Holdich DM, Lowery RS (eds) Freshwater crayfish: Biology, management, and exploitation. Croom Helm, London, pp 167-212 Alderman DJ, Polglase JL, Frayling M (1987) Aphanomyces astaci pathogenicity under laboratory and field conditions. J Fish Dis 10:385-393 Andersson G (2001) Differentation and pathogenicity within the Saprolegniaceae: Studies on the physiology and gene expression pattern in Saprolegnia parasitica and Aphanomyces astaci. Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 680, 41 p Andersson G, Cerenius L (2002) Analysis of chitinase expression in the crayfish plague fungus Aphanomyces astaci. Dis Aquat Organ 51:139-147 Ballesteros I, Martín MP, Diéguez-Uribeondo J (2006) First isolation of Aphanomyces frigidophilus (Saprolegniales) in Europe. Mycotaxon 95:335-340
67 Ballesteros I, Martín MP, Cerenius L, Söderhäll K, Tellería MT, Diéguez-Uribeondo J (2007) Lack of specificity of the molecular diagnostic method for identification of Aphanomyces astaci. Bull Fr Pêche Piscic 385:17-24 Bangyeekhun E, Cerenius L, Söderhäll K (2001) Molecular cloning and characterization of two serine proteinase genes from the crayfish plague fungus, Aphanomyces astaci. J Invertebr Pathol 77:206-216 Birch PRJ, Whisson SC (2001) Phytophthora infestans enters the genomics era. Mol Plant Pathol 2:257-263 Cerenius L, Söderhäll K (1984a) Repeated zoospore emergence from isolated spore cysts of Aphanomyces astaci. Exp Mycol 8:370-377 Cerenius L, Söderhäll K (1984b) Chemotaxis in Aphanomyces astaci, an arthropod-parasitic fungus. J Invert Pathol 43:278-281 Cerenius L, Söderhäll K (1985) Repeated zoospore emergence as a possible adaptation to parasitism in Aphanomyces. Exp Mycol 9:9- 63 Cerenius L, Söderhäll K, Persson M, Ajaxon R (1988) The crayfish plague fungus, Aphanomyces astaci: diagnosis, isolation and pathobiology. Freshw Crayfish 7:131-144 Cerenius L, Bangyeekhun E, Keyser P, Söderhäll I, Söderhäll K (2003) Host prophenoloxidase expression in freshwater crayfish is linked to increased resistance to the crayfish plague fungus, Aphanomyces astaci. Cell Microbiol 5:353-357 Cerenius L, Andersson MG, Söderhäll K (2009) Aphanomyces astaci and Crustaceans. In: Lamour K, Kamoun S (eds) Oomycete genetics and genomics: diversity, interactions, and research tools. John Wiley & Sons, Inc., pp 425-433 Cukerzis JM (1988) Astacus astacus in Europe. In: Holdich DM, Lowery RS (eds) Freshwater crayfish: biology, management, and exploitation. Croom Helm, London, pp 309-340 Cornalia E (1860) Sulla malattia dei gamberi. Atti Soc Ital Sci Nat 2:334-336, in Italian Corpet F (1988) Multiple sequence alignment with hierarchical clustering. Nucl Acids Res 16:10881-10890 Diéguez-Uribeondo J, Söderhäll K (1993) Procambarus clarkii Girard as a vector for the crayfish plague fungus, Aphanomyces astaci Schikora. Aquacult Fish Manage 24:761-765 Diéguez-Uribeondo J, Huang T, Cerenius L (1995) Physiological adaptation of an Aphanomyces astaci strain isolated from the freshwater crayfish Procambarus clarkii. Mycol Res 99:574-578
68 Diéguez-Uribeondo J, Söderhäll K (1999) RAPD evidence for the origin of an outbreak of crayfish plague in Spain. Freshw Crayfish 12:313-318 Diéguez-Uribeondo J, Cerenius L, Dyková I, Gelder SR, Henttonen P, Jiravanichpaisal P, Lom J, Söderhäll K (2006) Pathogens, parasites and ectocommensals. In: Souty-Grosset C, Holdich DM, Noël PY, Reynolds J, Haffner P (eds) Atlas of crayfish in Europe. Muséum National d’Histoire Naturelle, Paris, pp. 133- 149 Diéguez-Uribeondo J, Garcia MA, Cerenius L, Kozubíková E, Ballesteros I, Windels C, Weiland J, Kator H, Söderhäll K, Martin MP (2009) Phylogenetic relationships among plant and animal parasites, and saprotrophs in Aphanomyces (Oomycetes). Fungal Genet Biol 46:365-376 Dodds PN (2010) Genome evolution in plant pathogens. Science 330:1486-1487 Drummond AJ, Ashton B, Buxton S, Cheung M, Cooper A, Duran C, Field M, Heled J, Kearse M, Markowitz S, Moir R, Stones-Havas S, Sturrock S, Thierer T, Wilson A (2011) Geneious v5.4, Available from http://www.geneious.com/. Ebert D (1994) Virulence and local adaptation of a horizontally transmitted parasite. Science 265:1084-1086 Edgerton BF, Henttonen P, Jussila J, Mannonen A, Paasonen P, Taugbøl T, Edsman L, Souty-Grosset C (2004) Understanding the causes of disease in European freshwater crayfish. Conserv Biol 18(1):1466-1474 Elder JF, Turner BJ (1995) Concerted evolution of repetitive DNA sequences in eukaryotes. Q Rev Biol 70:297-320 Elder JF, Turner BJ (1996) Addendum to concerted evolution of repetitive DNA sequences in Eukaryotes. Q Rev Biol 71:79 Erkamo E, Ruokonen T, Alapassi T, Ruokolainen J, Järvenpää T, Tulonen J, Kirjavainen J (2010) Evaluation of crayfish stocking success in Finland. Freshw Crayfish 17:77-83 Evans LH, Edgerton BF (2002) Pathogens, parasites and commensals. In: Holdich DM (ed) Biology of Freshwater Crayfish. Blackwell Science, London, pp 377-423 Filipová L, Lieb DA, Grandjean F, Petrusek A (2011) Haplotype variation in the spiny-cheek crayfish Orconectes limosus: colonization of Europe and genetic diversity of native stocks. J N Am Benthol Soc 30:871-881
69 Fürst M (1995) On the recovery of Astacus astacus L. populations after an epizootic of the crayfish plague (Aphanomyces astaci Shikora). Freshw Crayfish 8:565-576 Gandolfi A, Bonilauri P, Rossi V, Menozzi P (2001) Intraindividual and intraspecies variability of ITS1 sequences in the ancient asexual Darwinula stevensoni (Crustacea: Ostracoda). Heredity 87:449-455 Ganley A, Kobayashi T (2007) Highly efficient concerted evolution in the ribosomal DNA repeats: Total rDNA repeat variation revealed by whole-genome shotgun sequence data. Genome Res 17:184-191 Gherardi F, Holdich DM (eds) (1999) Crayfish in Europe as alien species. How to make the best of a bad situation? A.A. Balkema, Rotterdam Guindon S, Gascuel O (2003) A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52:696-704 Hall TA (1998) BioEdit: A user friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser 41:95-98 Henttonen P (2012) Ravun taudinaiheuttajat, loiset ja päällysvieraat. In: Valtonen T, Hakalahti-Sirén T, Karvonen A, Pulkkinen K (eds) Suomen kalojen loiset. Gaudeamus Helsinki University Press, Tampere, pp 223-247, in Finnish Henttonen P, Huner JV (1999) The introduction of alien species in Europe: a historical introduction. In: Gherardi F, Holdich DM (eds) Crayfish in Europe as alien species. How to make the best of a bad situation? A.A. Balkema, Rotterdam, pp. 13-22 Hobbs HH (1988) Crayfish distribution, adaptive radiation and evolution. In: Holdich DM, Lowery RS (eds) Freshwater crayfish: biology, management, and exploitation. Croom Helm, London, pp. 52-82 Hochwimmer G, Tober R, Bibars-Reiter R, Licek E, Steinborn R (2009) Identification of two GH18 chitinase family genes and their use as targets for detection of the crayfish-plague Oomycete Aphanomyces astaci. BMC Microbiol 9:184, DOI:10.1186/1471- 2180-9-184 Holdich DM (2002) Present distribution of crayfish in Europe and some adjoining countries. Bull Fr Pêche Piscic 367(4):611-650 Huang T, Cerenius L, Söderhäll K (1994) Analysis of genetic diversity in the crayfish plague fungus, Aphanomyces astaci, by random amplification of polymorphic DNA. Aquaculture 126:1-9
70 Hudina S, Faller M, Luci A, Klobu ar G, Maguire I (2009) Distribution and dispersal of two invasive crayfish species in the Drava River basin, Croatia. Knowl Managt Aquatic Ecosyst 394- 395:09p1-09p11 Hyytinen L, Kirjavainen J, Lahti E, Muhonen J, Munne P, Niemi A, Sipponen M, Mannonen A, Halonen T (2000) Kalataloushallinnon rapustrategia. Maa- ja metsätalousministeriö, Hämeenlinna, in Finnish IUCN (2012) The IUCN red list of threatened species. Version 2012.2.
71 Kozubíková E, Filipová L, Kozák P, uriš Z, Martín MP, Diéguez- Uribeondo J, Oidtmann B, Petrusek A (2009) Prevalence of the crayfish plague pathogen Aphanomyces astaci in invasive American crayfishes in the Czech republic. Conserv Biol 23:1204- 1213 Kozubíková E, Viljamaa-Dirks S, Heinikainen S, Petrusek A (2011a) Spiny-cheek crayfish Orconectes limosus carry a novel genotype of the crayfish plague pathogen Aphanomyces astaci. J Invertebr Pathol 108:214-216 Kozubíková E, Vrålstad T, Filipová L, Petrusek A (2011b) Re- examination of the prevalence of Aphanomyces astaci in North American crayfish populations in Central Europe by TaqMan MGB real-time PCR. Dis Aquat Organ 97:113-125 Lamour KH, Win J, Kamoun S (2007) Oomycete genomics: new insights and future directions. FEMS Microbiol Lett 274:1-8 Librado P, Rozas J (2009) DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25:1451- 1452 Lilley JH, Cerenius L, Söderhäll K (1997) RAPD evidence for the origin of crayfish plague outbreaks in Britain. Aquaculture 157:181-185 Lowe S, Browne M, Boudjelas S, De Poorter M (2004) 100 of the world’s worst invasive alien species a selection from the global invasive species database. The invasive species specialist group (ISSG) of the world conservation union (IUCN), Auckland Margulis L, Schwartz KV (2000) Five kingdoms: An illustrated guide to the phyla of life on earth. WH Freeman & Co, New York, 941 p Minin VN, Dorman KS, Fang F, Suchard MA (2005) Dual multiple change-point model leads to more accurate recombination detection. Bioinformatics 21:3034-3042 Nybelin O (1934) Nya undersökningar över kräftpestens orsak Ny Svensk Fiskeritidskrift:110-114, in Swedish Nyhlén L, Unestam T (1975) Ultrastructure of the penetration of the crayfish integument by the fungal parasite, Aphanomyces astaci, Oomycetes. J Invert Pathol 26:353-366 Nylund V, Westman K (1983) Frequency of visible symptoms of the crayfish plague fungus Aphanomyces astaci on the American crayfish (Pacifastacus leniusculus) in the natural populations in Finland. Freshw Crayfish 5:277-283 Nylund V, Westman K (1992) Crayfish diseases and their control in Finland. Finn Fish Res 14:107-118
72 Oidtmann B, Cerenius L, Schmid I, Hoffmann R, Söderhäll K (1999a) Crayfish plague epizootics in Germany - classification of two German isolates of the crayfish plague fungus Aphanomyces astaci by random amplification of polymorphic DNA. Dis Aquat Organ 35:235-238 Oidtmann B, Schmid I, Rogers DW, Hoffmann RW (1999b) An improved isolation method for the cultivation of the crayfish plague fungus, Aphanomyces astaci. Freshw Crayfish 12:303-312 Oidtmann B, Heitz E, Rogers D, Hoffmann RW (2002a) Transmission of crayfish plague. Dis Aquat Organ 52:159-167 Oidtmann B, Bausewein S, Holzle L, Hoffmann R, Wittenbrink M (2002b) Identification of the crayfish plague fungus Aphanomyces astaci by polymerase chain reaction and restriction enzyme analysis. Vet Microbiol 85:183-194 Oidtmann B, Schaefers N, Cerenius L, Söderhäll K, Hoffmann RW (2004) Detection of genomic DNA of the crayfish plague fungus Aphanomyces astaci (Oomycete) in clinical samples by PCR. Vet Microbiol 100:269-282 Oidtmann B, Geiger S, Steinbauer P, Culas A, Hoffmann RW (2006) Detection of Aphanomyces astaci in North American crayfish by polymerase chain reaction. Dis Aquat Organ 72:53-64 Pakkasmaa S (2006) Ruotsin rapukansa koolla Jönköpingissä. Suomen Kalastuslehti 5:22-23, in Finnish Pârvulescu L, Schrimpf A, Kozubíková E, Cabanillas-Resino S, Vrålstad T, Petrusek A, Schulz R (2012) Invasive crayfish and crayfish plague on the move: first detection of the plague agent Aphanomyces astaci in the Romanian Danube. Dis Aquat Organ 98:85-94 Persson M, Söderhäll K (1983) Pacifastacus leniusculus Dana and its resistance to the parasitic fungus Aphanomyces astaci Schikora. Freshw Crayfish 5:292-298 Persson M, Cerenius L, Söderhäll K (1987) The influence of haemocyte number on the resistance of the freshwater crayfish, Pacifastacus leniusculus Dana, to the parasitic fungus Aphanomyces astaci. J Fish Dis 10:471-477 Pursiainen M, Ruokonen T (2006) Raputalouskatsaus 2006. Riista- ja Kalatalouden tutkimuslaitos, Helsinki, in Finnish Raffaele S, Kamoun S (2012) Genome evolution in filamentous plant pathogens: why bigger can be better? Nat Rev Microbiol 10:417- 430 Raffaele S, Farrer RA, Cano LM, Studholme DJ, MacLean D, Thines M, Jiang RHY, Zody MC, Kunjeti SG, Donofrio NM, Meyers BC,
73 Nusbaum C, Kamoun S (2010) Genome evolution following host jumps in the Irish potato famine pathogen lineage. Science 330:1540-1543 Rantamäki J, Cerenius L, Söderhäll K (1992) Prevention of transmission of the crayfish plague fungus (Aphanomyces astaci) to the freshwater crayfish Astacus astacus by treatment with MgCl2. Aquaculture 104:11-18 Rennerfelt E (1936) Untersuchungen über die Entwicklung und Biologie des Krebspestpilzes Aphanomyces astaci Schikora. Rep Inst Freshw Res Drottningholm 10:1-21, in German Reynolds J, Souty-Grosset C (2012) Management of freshwater biodiversity: crayfish as bioindicators. University Press, Cambridge, 374 p Royo F, Andersson G, Bangyeekhun E, Muzquiz JL, Söderhäll K, Cerenius L (2004) Physiological and genetic characterisation of some new Aphanomyces strains isolated from freshwater crayfish. Vet Microbiol 104:103-112 Schikora F (1906) Die Krebspest. Fischerei-Zeitung 9:529-532, in German Schäperclaus W (1954) Fischkrankheiten, 3. Auflage. Akademie Verlag, Berlin, 707 p, in German Simon UK, Weiß M (2008) Intragenomic variation of fungal ribosomal genes is higher than previously thought. Mol Biol Evol 25:2251- 2254 Skurdal J, Taugbøl T (2002) Astacus. In: Holdich DM (ed) Biology of freshwater crayfish. Blackwell Science, Oxford, pp 467-510 Skov C, Aarestrup K, Sivebæk F, Pedersen S, Vrålstad T, Berg S (2011) Non-indigenous signal crayfish Pacifastacus leniusculus are now common in Danish streams: Preliminary status for national distribution and protective actions. Biol Invasions 13:1269-1274 Smith V, Söderhäll K (1986) Crayfish pathology: an overview. Freshw Crayfish 6:199-211 Souty-Grosset C, Holdich DM, Noël PY, Reynolds J, Haffner P (2006) Atlas of crayfish in Europe. Muséum National d’Histoire Naturelle, Paris, 187 p Strand DA, Holst-Jensen A, Viljugrein H, Edvardsen B, Klaveness D, Jussila J, Vrålstad T (2011) Detection and quantification of the crayfish plague agent in natural waters: direct monitoring approach for aquatic environments. Dis Aquat Organ 95:9-17 Stukenbrock E, McDonald B (2009) Population genetics of fungal and Oomycete effectors involved in gene-for-gene interactions. Mol Plant Microbe Interact 22:371-380
74 Suchard MA, Weiss RE, Dorman KS, Sinsheimer JS (2003) Inferring spatial phylogenetic variation along nucleotide sequences: a multiple changepoint model. J Am Stat Assoc 98:427-437 Svensson E (1978) Interactions between a parasitic fungus, Aphanomyces astaci, Oomycetes, and its crayfish host. I. Motility, encystment, attachment, and germination of the zoospore. Acta Universitatis Upsaliensis 457, 18 p Svensson E, Unestam T (1975) Differectial induction of zoospore encystment and germination in Aphanomyces astaci, Oomycetes. Physiol Plantarum 35:210-216 Svensson E, Söderhäll K, Unestam T, Andersson B (1976) Liming: An overestimated method for preventing the spread of the crayfish plague. Rep Inst Freshw Res Drottningholm 55:132-135 Svoboda J, Kozubíková E, Kozák P, Kouba A, Bahadir S, Diler Ö, Diler I, Policar T, Petrusek A (2012) PCR detection of the crayfish plague pathogen in narrow-clawed crayfish inhabiting Lake Egirdir in Turkey. Dis Aquat Organ 98:255-259 Söderhäll K, Cerenius L (1987) Controlled growth and development in filamentous Oomycetes with empahis on Aphanomyces spp. In: Fuller MS, Jaworski A (eds) Zoosporic fungi in teaching and research. Southeastern Publising Corporation, Athens, Georgia, pp 264-267 Söderhäll K, Cerenius L (1992) Crustacean immunity. Annu Rew Fish Dis 2:3-23 Söderhäll K, Cerenius L (1998) Role of the prophenoloxidase- activating system in invertebrate immunity. Curr Opin Immunol 10:23-28 Söderhäll K, Cerenius L (1999) The crayfish plague fungus: history and recent advances. Freshw Crayfish 12:11-35 Söderhäll K, Svensson E, Unestam T (1978) Chitinase and protease activities in germinating zoospore cysts of a parasitic fungus, Aphanomyces astaci, Oomycetes. Mycopathologia 64:9-11 Thörnqvist PO, Söderhäll K (1993) Psorospermium haeckeli and its interaction with the crayfish defence system. Aquaculture 117:205-213 Tuffs S, Oidtmann B (2011) A comparative study of molecular diagnostic methods designed to detect the crayfish plague pathogen, Aphanomyces astaci. Vet Microbiol 153:343-353 Tulonen J, Järvenpää T, Erkamo E, Savolainen R, Westman K, Mannonen A (1998) Rapuvedet tuottaviksi. Riista- ja kalatalouden tutkimuslaitos, Helsinki, in Finnish
75 Unestam T (1965) Studies on the crayfish plague fungus Aphanomyces astaci I. Some factors affecting growth in vitro. Physiol Plantarum 18:483-505 Unestam T (1966a) Studies on the crayfish plague fungus Aphanomyces astaci II. Factors affecting zoospores and zoospore production. Physiol Plantarum 19:1110-1119 Unestam T (1966b) Chitinolytic, cellulolytic, and pectinolytic activity in vitro of some parasitic and saprophytic Oomycetes. Physiol Plantarum 19:15-30 Unestam T (1969a) Resistance to the crayfish plague in some American, Japanese and European crayfishes. Rep Inst Freshw Res Drottningholm 49:202-209 Unestam T (1969b) On the physiology of zoospore production in Aphanomyces astaci. Physiol Plantarum 22:236-246 Unestam T (1969c) On the adaptation of Aphanomyces astaci as a parasite. Physiol Plantarum 22:221-235 Unestam T (1972) On the host range and origin of the crayfish plague fungus. Rep Inst Freshw Res Drottningholm 52:192-198 Unestam T (1975) Defence reactions in and susceptibility of Australian and New Guinean freshwater crayfish to European- crayfish-plague fungus. Aust J Exp Biol Med Sci 53:349-359 Unestam T, Weiss DW (1970) The host-parasite relationship between freshwater crayfish and the crayfish disease fungus Aphanomyces astaci: responses to infection by a susceptible and a resistant species. J Gen Microbiol 60:77-90 Unestam T, Svensson E (1971) Physiological variation in strains of Aphanomyces astaci. Physiol Plantarum 25:414-416 Unestam T, Nylund JE (1972) Blood reactions in vitro in crayfish against a fungal parasite, Aphanomyces astaci. J Invert Pathol 19:94-106 Unestam T, Nestell CG, Abrahamsson S (1972) An electrical barrier for preventing migration of freshwater crayfish in running water: a method to stop the spread of the crayfish plague. Rep Inst Freshw Res Drottningholm 53:199-203 Vennerström P, Söderhäll K, Cerenius L (1998) The origin of two crayfish plague (Aphanomyces astaci) epizootics in Finland on noble crayfish, Astacus astacus. Ann Zool Fennici 35:43-46 Vey A, Söderhäll K, Ajaxon R (1983) Susceptibility of Orconectes limosus Raff. to the crayfish plague, Aphanomyces astaci Schikora. Freshw Crayfish 5:284-291
76 Viljamaa-Dirks S, Heinikainen S (2006) Improved detection of crayfish plague with a modified isolation method. Freshw Crayfish 15:376-382 Viljamaa-Dirks S, Heinikainen S, Nieminen M, Vennerström P, Pelkonen S (2011) Persistent infection by crayfish plague Aphanomyces astaci in a noble crayfish population - A case report. Bull Eur Ass Fish Pathol 31:182-188 Viljamaa-Dirks S, Heinikainen S, Torssonen H, Pursiainen M, Mattila J, Pelkonen S (2013) Distribution and epidemiology of the crayfish plague agent Aphanomyces astaci from noble crayfish Astacus astacus in Finland. Dis Aquat Organ 103: 199-208 Vogt G (1999) Diseases of European freshwater crayfish, with particular emphasis on interspecific transmission of pathogens. In: Gherardi F, Holdich D (eds) Crayfish in Europe as alien species. How to make the best of a bad situation? A.A. Balkema, Rotterdam, pp 87-102 Vrålstad T, Knutsen AK, Tengs T, Holst-Jensen A (2009) A quantitative TaqMan(R) MGB real-time polymerase chain reaction based assay for detection of the causative agent of crayfish plague Aphanomyces astaci. Vet Microbiol 137:146-155 Vrålstad T, Johnsen SI, Fristad RF, Edsman L, Strand D (2011) Potent infection reservoir of crayfish plague now permanently established in Norway. Dis Aquat Organ 97:75-83 Westman K (1973) The population of the crayfish, Astacus astacus L. in Finland and the introduction of the American crayfish Pacifastacus leniusculus Dana. Freshw Crayfish 1:41-55 Westman K, Nylund V (1978) Crayfish plague, Aphanomyces astaci, observed in the European crayfish, Astacus astacus, in Pihlajavesi waterway in Finland. A case study on the spread of the plague fungus. Freshw Crayfish 4:419-426 White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols: a guide to methods and applications. Academic Press, San Diego, pp 315-322
77 dissertations
| 105 | Jenny Makkonen | Makkonen | 105 | Jenny
Jenny Makkonen The crayfish plague pathogen Aphanomyces astaci Crayfish plague, caused by the oomy- cete Aphanomyces astaci, has caused a Genetic diversity and adaptation Jenny Makkonen dramatic decline of the native cray- to the host species fish in Europe. The North-American to the... adaptation and diversity Genetic – astaci Aphanomyces pathogen plague crayfish The crayfish species introduced to Europe The crayfish plague pathogen are the main vectors of the disease, which is endemic in North-America. Aphanomyces astaci In this work, genetic diversity, physi- ological properties and sporulation Genetic diversity and adaptation to the host species 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.
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