Infection strategies of pathogenic Oomycetes in fish.

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E. B. Gareth Jones, Kevin D. Hyde, Ka-Lai Pang (Eds.)
Freshwater Fungi
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Freshwater Fungi
and Fungal-like Organisms
Edited by
E. B. Gareth Jones, Kevin D. Hyde and Ka-Lai Pang
DE GRUYTER
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Editors
E. B. Gareth Jones
King Saud University
Riyadh 11451
Kingdom of Saudi Arabia
E-Mail: torperadgj@gmail.com
Kevin D. Hyde
Mae Fah Luang University
Chiang Rai
Thailand
E-Mail: kdhyde1@gmail.com
Ka-Lai Pang
Institute of Marine Biology and Center of Excellence for the Oceans
National Taiwan Ocean University
2 Pei-Ning Road
Keelung 20224
Taiwan, R.O.C.
E-Mail: klpang@ntou.edu.tw
ISBN 978-3-11-033345-9
e-ISBN 978-3-11-033348-0
Library of Congress Cataloging-in-Publication data
A CIP catalog record for this book has been applied for at the Library of Congress.
Bibliographic information published by the Deutsche Nationalbibliothek
The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliograe; detailed
bibliographic data are available on the Internet at http://dnb.dnb.de.
© 2014 Walter de Gruyter GmbH, Berlin/Boston
Typesetting: Compuscript Limited, Shannon, Ireland
Printing and binding: CPI books GmbH, Leck
Cover image: The cover shows the helicoid conidium of Helicomyces roseus, a common freshwater
asexual fungus found on submerged cellulosic materials. Photo by Dr Nattawut Boonyuen.
∞ Printed on acid-free paper
Printed in Germany
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Preface
This book Freshwater fungi and Fungal-like Organisms is the outcome of recent research
of the leading world mycologists on selected topics on fungi in streams, rivers, lakes
and meltwater. The aim of the book is to bring together the present state of knowl-
edge concerning freshwater fungi, especially topics often neglected in other treatise,
to highlight their importance to science and the challenges facing freshwater myco-
logy, as well as to consider future research. The book brings together many subjects
not covered in other monographic volumes, such as freshwater yeasts, lichens, fresh-
water basidiomycetes, fungal-like organisms, their potential industrial application,
and their role in the decomposition of complex organic matter in rivers and lakes. We
particularly focus on their role as pathogens of commercially important freshwater
animals, such as fish, crustaceans and molluscs, the diseases of mammals, including
humans, the catastrophic devastation caused by chytrids parasitic on frogs and other
reptiles.
Texts in freshwater biology rarely include fungi and fungal-like organisms in their
volumes and thus ignore their important contributions and roles in freshwater eco-
systems, in particular, their role in the breakdown and sequestration of pollutants in
freshwater habitats. Fungi play a major role in the decomposition of complex organic
compounds yielding nutrients for other aquatic organisms in the web of life in fresh-
water ecosystems.
The opening section of the book documents the current knowledge on the phy-
logeny of obligate freshwater fungi: Ascomycota (Dothideomycetes, Sordariomyce-
tes, and other classes); Basidiomycota; asexual fungi; yeasts; Chytridiomycota and
Blastocladiomycota. The second section is devoted to the phylogeny of fungal-like
organisms: Microsporidia; Pythiales and Peronosporales. The biodiversity of fungi in
selected habitats is reviewed in the third section: economic importance of zoosporic
Mesomycetozoean pathogenic in fish; oomycetes and zoosporic organisms of amphi-
bians, fish and freshwater invertebrates; and pythiosis of mammals, including man.
The concluding section considers the ecology of lichens, aquatic trichomycetes, fungi
found on decaying fronds of palms in a peat swamp, the role of fungi in leaf decompo-
sition and breakdown of wood in tropical streams, yeasts in extreme aquatic environ-
ments and the role of fungi in polluted waters. The epilogue considers the importance
of fungi and fungal-like organisms, and the direction of future research.
It is expected that this book will be essential reading for mycologists, microbio-
logists, freshwater biologists and limnologists interested in freshwater fungi and will
serve as a useful reference work on their occurrence and role in freshwater ecosystems.
We thank all the authors for agreeing to write for this volume and for delivering
their chapters on time. We are much indebted to Frank Gleason for suggestions during
the preparation of this volume, especially the section on fungal-like organisms. Our
thanks also go to all the staff at De Gruyter for conceiving this volume and for their
support in its publication (Simone Witzel, Nicole Karbe, and Hannes Kaden).
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Contents
Preface v
List of contributing authors xvii
E. B. Gareth Jones, Kevin D. Hyde and Ka-Lai Pang
1 Introduction 1
1.1 Origin of freshwater fungi and fungal-like organisms 4
1.2 Classification of freshwater fungi 5
1.3 Estimated number of freshwater fungi 6
1.4 World distribution 8
1.5 Endophytic fungi 8
1.6 Predacious fungi 9
1.7 Bioactive compounds 10
1.8 Barcoding of freshwater fungi 12
1.9 One name one fungus ruling 13
1.10 Role of fungi in freshwater habitats 14
1.11 Objectives and outline of the volume 15
1.12 Phylogeny of true freshwater fungi 15
1.13 Phylogeny of fungus-like organisms 15
1.14 Biodiversity of freshwater fungi and fungus-like organisms 16
1.15 Ecology 16
Acknowledgments 16
References 17
Phylogeny of freshwater fungi 23
Carol A. Shearer, Ka-Lai Pang, Satinee Suetrong and Huzefa A. Raja
2 Phylogeny of the Dothideomycetes and other classes of freshwater
fissitunicate Ascomycota 25
2.1 Introduction 25
2.2 Geographical distribution patterns 26
2.3 Substrate distribution patterns 26
2.4 Morphological adaptations 26
2.5 Systematics 28
2.5.1 General introduction 28
2.5.2 Current phylogenetic placement based on molecular
systematics 32
2.5.2.1 Dothideomycetes-Pleosporomycetidae-Pleosporales 32
2.5.2.2 Pleosporales incertae sedis 36
2.5.3 Zopfiaceae, Dothideomycetes, family incertae sedis 38
2.5.4 Dothideomycetes incertae sedis 38
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viii Contents
2.5.4.1 Jahnulales 38
2.5.4.2 Natipusillales 39
2.5.4.3 Minutisphaera clade 39
2.5.4.4 Freshwater asexual morphs with affinities to Dothideomycetes 39
2.6 Conclusions 40
Acknowledgments 40
References 40
Lei Cai, Dian-Ming Hu, Fang Liu, Kevin D. Hyde and E. B. Gareth Jones
3 The molecular phylogeny of freshwater Sordariomycetes and
discomycetes 47
3.1 Introduction 47
3.2 Materials and methods 48
3.2.1 Taxon sampling 48
3.2.2 Phylogenetic analysis 48
3.3 Discussion 48
3.3.1 Sordariomycetidae 56
3.3.1.1 Annulatascaceae 56
3.3.1.2 Magnaporthales 60
3.3.1.3 Calosphaeriales 60
3.3.1.4 Coniochaetales 61
3.3.1.5 Diaporthales 61
3.3.1.6 Sordariales 61
3.3.2 Sordariomycetidae incertae sedis 62
3.3.3 Hypocreomycetidae 62
3.3.3.1 Savoryellales 62
3.3.3.2 Microascales 63
3.3.3.3 Hypocreales 63
3.3.4 Xylariomycetidae 64
3.3.4.1 Xylariales 64
3.3.4.2 Phyllachorales 64
3.3.4.3 Trichosphaeriales 64
3.3.5 Discomycetes 64
3.3.5.1 Helotiales 64
3.3.5.2 Pezizales 65
3.3.5.3 Rhytismatales 66
3.4 Concluding remarks 66
Acknowledgments 66
References 67
E. B. Gareth Jones, Darlene Southworth, Diego Libkind and Ludmila Marvanová
4 Freshwater Basidiomycota 73
4.1 Group 1 freshwater yeasts 82
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Contents ix
4.1.1 Agaricomycotina 83
4.1.1.1 Tremellomycetes 83
4.1.2 Pucciniomycotina 85
4.1.2.1 Cystobasidiomycetes 85
4.1.2.2 Microbotryomycetes 85
4.1.2.3 Microbotryomycetes Incertae sedis 86
4.1.3 Ustilaginomycotina 87
4.1.3.1 Ustilaginomycetes 87
4.2 Group 2 filamentous fungi 87
4.2.1 Agaricomycotina 87
4.2.1.1 Agaricomycetes 87
4.2.1.2 Exobasidiomycetes 92
4.2.1.3 Tremellomycetes 92
4.2.2 Pucciniomycotina 92
4.2.2.1 Atractiellomycetes 92
4.2.2.2 Classiculomycetes 93
4.2.2.3 Microbotryomycetes 94
4.2.3 Ustilaginomycotina 95
4.2.3.1 Ustilaginomycetes 95
Basidiomycota—incertae sedis 95
4.3 Group 3 endophytes 99
4.4 Adaptation to freshwater habitats 99
Acknowledgments 100
References 100
Dian-Ming Hu, Lei Cai, E. B. Gareth Jones, Huang Zhang, Nattawut Boonyuen
and Kevin D. Hyde
5 Taxonomy of filamentous asexual fungi from freshwater habitats,
links to sexual morphs and their phylogeny 109
5.1 Introduction 109
5.2 Morphological taxonomy 110
5.2.1 Hyphomycetes 110
5.2.2 Coelomycetes 112
5.2.3 Asexual-sexual connections 112
5.3 Phylogeny 113
5.3.1 Dothideomycetes 114
5.3.1.1 Capnodiales 114
5.3.1.2 Dothideales 114
5.3.1.3 Hysteriales 117
5.3.1.4 Jahnulales 117
5.3.1.5 Mytilinidiales 117
5.3.1.6 Pleosporales 117
5.3.1.7 Tubeufiales 118
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x Contents
5.3.2 Leotiomycetes 119
5.3.3 Orbiliomycetes 120
5.3.3.1 Orbiliales 120
5.3.4 Sordariomycetes 120
5.3.4.1 Glomerellales 123
5.3.4.2 Hypocreales 123
5.3.4.3 Sordariales 123
5.3.4.4 Savoryellales 124
5.4 Discussion 125
Acknowledgment 126
References 126
Martha J. Powell and Peter M. Letcher
6 Phylogeny and characterization of freshwater Chytridiomycota
(Chytridiomycetes and Monoblepharidomycetes) 133
6.1 Introduction 133
6.2 Chytridiomycetes 138
6.2.1 Order 1. Chytridiales (Chytridiaceae, Chytriomycetaceae) 138
6.2.2 Order 2. Spizellomycetales (Spizellomycetaceae,
Powellomycetaceae) 140
6.2.3 Order 3. Rhizophlyctidiales (Rhizophlyctidaceae, Sonoraphlyctidaceae,
Arizonaphlyctidaceae, Borealophlyctidaceae) 141
6.2.4 Order 4. Rhizophydiales (10 families described) 141
6.2.5 Order 5. Lobulomycetales (Lobulomycetaceae) 144
6.2.6 Order 6. Cladochytriales (Cladochytriaceae, Nowakowskiellaceae,
Septochytriaceae, Endochytriaceae) 144
6.2.7 Order 7. Polychytriales (no families described) 146
6.3 Incertae sedis 147
6.4 Monoblepharidomycetes (Harpochytriales, Monoblepharidales,
Hyaloraphidiales) 148
Acknowledgments 148
References 148
Phylogeny of fungus-like organisms 155
Ray Kearney and Frank H. Gleason
7 Microsporidia 157
7.1 Ecology 160
7.2 Classification 162
7.3 Evolutionary origins 165
7.4 Cell structure and spore significance 166
7.5 Metabolism 167
7.6 Genome structure 168
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Contents xi
7.7 Discussion and conclusion 168
7.8 Further research avenues 170
References 171
Agostina V. Marano, Ana L. Jesus, Carmen L. A. Pires-Zottarelli, Timothy Y. James,
Frank H. Gleason and Jose I. de Souza
8 Phylogenetic relationships of Pythiales and Peronosporales
(Oomycetes, Straminipila) within the “peronosporalean
galaxy” 177
8.1 Introduction 177
8.2 The monophyly of Chromalveolata and the relationships
between heterotrophic straminipile lineages 178
8.3 Major lineages within the Oomycetes: the
“galaxies” 179
8.4 The “peronosporalean galaxy”: a marine origin? 179
8.5 Ecological and economical significance 180
8.6 The phylogeny of Pythiales and Peronosporales 181
8.6.1 Clade 1: Albuginales 185
8.6.2 Clade 2: Pythiales 185
8.6.2.1 Pythiogeton 186
8.6.2.2 Pythium, Lagenidium and Phytopythium 186
8.6.3 Clade 3: Peronosporales 187
8.6.3.1 Downy mildews 188
8.6.3.2 Phytophthora and Peronophythora 188
8.6.3.3 Halophytophthora and Salisapilia 190
8.7 Conclusions and future perspectives 192
Acknowledgments 194
References 194
Biodiversity of freshwater fungi 201
Sally L. Glockling, Wyth L. Marshall, Rodolphe E. Gozlan, Agostina
V. Marano, Osu Lilje and Frank H. Gleason
9 The ecological and economic importance of zoosporic Mesomycetozoean
(Dermocystida) parasites of freshwater fish 203
9.1 Phylogeny 203
9.2 Life cycles 205
9.3 The zoospore 206
9.4 Symptoms of disease 207
9.5 Ecological and economic significance 209
9.6 Discussion and conclusion 211
Acknowledgment 212
References 212
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xii Contents
Mohammad N. Sarowar, Marcia Saraiva, Casey N. Jessop,
Osu Lilje, Frank H. Gleason and Pieter van West
10 Infection strategies of pathogenic oomycetes in fish 217
10.1 Introduction 217
10.2 Taxonomy of oomycetes pathogenic to fish 221
10.3 Physical adaptation and strategy for infection:
macroscopic infection, the face of infection on hosts 223
10.4 Oomycete zoospores, the first line of attack 224
10.5 Triggers for zoospore formation, waking up the beast 225
10.6 Encystment and germination, one step closer to infection 225
10.7 Repeated zoospore emergence, the back-up plan 227
10.8 Chemotactic response of zoospores, the specialization 228
10.9 Proteins and amino acids as substrates for growth 229
10.10 Sexual reproduction, seeing through the bad times 231
10.11 Molecular adaptation and strategy in setting infection:
microscopic infection 231
10.12 Host responses to oomycete infections 233
10.13 The animal trade is responsible for the spread of pathogens into novel
and wild ecosystems 234
10.14 Future perspectives 235
Acknowledgments 236
References 236
Frank H. Gleason, Jodi L. Rowley, Casey N. Jessop and Osu Lilje
11 Zoosporic parasites of amphibians 245
11.1 Chytridiomycota 245
11.2 Mesomycetozoea 247
11.3 Oomycota (oomycetes or water moulds) 250
11.4 Perkinsozoa 251
11.5 The Fisher concept of emerging infectious diseases (EIDs) 252
11.6 Host switching by parasites 252
11.7 Genetic variation in parasite populations 254
11.8 Proteases 255
11.9 International animal trade 255
11.10 Discussion and conclusion 256
Acknowledgments 257
References 257
Angkana Chaiprasert and Theerapong Krajaejun
12 Pythiosis 263
12.1 History 263
12.2 Biology 263
12.3 Molecular typing 265
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Contents xiii
12.4 Epidemiology 266
12.5 Pathogenesis 266
12.6 Clinical features 267
12.6.1 Human pythiosis 267
12.6.2 Animal pythiosis 269
12.7 Diagnosis 269
12.8 Management 271
12.9 Research direction 272
Acknowledgment 273
References 274
Frank H. Gleason, Sergey A. Karpov, Osu Lilje, Deborah J. Macarthur, Floris
F. van Otgen and Telesphore Sime-Ngando
13 Zoosporic parasites of phytoplankton 279
13.1 The main groups of zoosporic parasites and parasitoids of
phytoplankton 280
13.1.1 Aphelidea 280
13.1.2 Chytridiomycota 283
13.1.3 Blastocladiomycota 293
13.2 Ancient interactions 294
13.3 Novel food webs 295
13.3.1 Vorticella communities attached to cyanobacterial filaments 295
13.3.2 Communities involving other protists 295
13.4 Host parasite dynamics 296
13.5 Conclusion 298
Acknowlegments 299
References 300
Sally L. Glockling, Agostina V. Marano, Osu Lilje and Frank H. Gleason
14 Zoosporic parasites of freshwater invertebrates 305
14.1 Parasites in the Blastocladiomycota and Chytridiomycota 306
14.2 Parasites in the Oomycota 307
14.3 Parasites in the Mesomycetozoea 311
14.4 Parasites of crayfish 312
14.4.1 Crayfish plague 312
14.4.2 Psorospermium haekeli 313
14.5 Parasites of mosquitoes, blackflies and midges 313
14.5.1 Coelomomyces 314
14.5.2 Lagenidium giganteum 315
14.5.3 Pythium 316
14.5.4 Leptolegnia 316
14.5.5 Crypticola 316
14.5.6 Amoebidium and Paramoebidium 317
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xiv Contents
14.6 Parasites of Daphnia 317
14.7 Parasites of rotifers and nematodes 318
14.7.1 Sommerstorffia spinosa 320
14.7.2 Aquastella 321
14.8 Parasites of protozoans 321
14.9 Discussion 321
Acknowledgments 324
References 324
Ecology 331
Holger Thüs, André Aptroot and Mark R. D. Seaward
15 Freshwater lichens 333
15.1 Ecology 336
15.1.1 Habitats and diversity of freshwater lichens 336
15.1.2 Collecting and identifying freshwater lichens 338
15.2 Physiological challenges for freshwater lichens 339
15.2.1 Water saturation and diffusion resistance 339
15.3 Freshwater lichens as a food source for other organisms 343
15.4 Biogeography of freshwater lichens 344
15.5 Zonation 345
15.6 Lichen trimlines 348
15.7 Freshwater lichen communities 349
15.8 Freshwater lichens as bioindicators 350
15.9 Water quality 351
15.10 Conservation 352
Acknowledgments 353
References 353
Robert W. Lichtwardt
16 Aquatic Trichomycetes 359
16.1 Trichomycetes, an ecological group 359
16.2 Phylogenetic considerations 359
16.3 Distribution and success of Trichomycetes 364
16.4 Variations in symbiotic associations 365
16.5 Medical implications 367
Acknowledgments 368
References 368
Umpava Pinruan, Aom Pinnoi, Kevin D. Hyde and E. B. Gareth Jones
17 Tropical peat swamp fungi with special reference to palms 371
17.1 Material and methods 373
17.1.1 Sample collection 373
17.2 Results 373
17.2.1 Abundance of fungi on four palms (Eleiodoxa conferta,
Licuala longicalycata, Metroxylon sagu and Nenga pumila) 373
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Contents xv
17.2.1.1 Eleiodoxa conferta 379
17.2.1.2 Licuala longicalycata 380
17.2.1.3 Metroxylon sagu 380
17.2.1.4 Nenga pumila 380
17.2.2 Fungal diversity 381
17.2.3 Percentages overlap in fungal diversity between the four palms 382
17.3 Conclusion 383
Acknowledgments 385
References 386
Verónica Ferreira, Vladislav Gulis, Cláudia Pascoal and Manuel A. S. Graça
18 Stream pollution and fungi 389
18.1 The importance of aquatic hyphomycetes in woodland streams 389
18.2 Effects of nutrient enrichment on stream fungi 391
18.3 Effects of heavy metals and acidification on stream fungi 394
18.4 Ecological and toxicological effects of engineered nanoparticles on
stream fungi 395
18.5 Effects of organic xenobiotics on stream fungi 397
18.6 Effects of thermal pollution on stream fungi 398
18.7 Effects of the interaction among factors on stream fungi 403
18.8 Conclusions 404
Acknowledgments 404
References 405
Felix Bärlocher and Kandikere R. Sridhar
19 Association of animals and fungi in leaf decomposition 413
19.1 History 413
19.2 Effects of the leaf-fungus complex on invertebrate consumers 416
19.2.1 Nutritional value of mycelium vs. leaf substrate 416
19.2.2 Modifications of leaf substrate 417
19.2.3 Do invertebrates differ in their feeding strategies? 420
19.2.4 What factors ultimately determine food choice and feeding
selectivity? 421
19.2.5 Stoichiometric considerations 423
19.2.6 Stimulation of fungi by invertebrate feeding 424
19.2.7 Anthropogenic changes 424
19.2.8 Research outside temperate regions 426
19.3 Effects of invertebrate consumers on the leaf-fungus complex 428
19.3.1 Invertebrate ingestion of conidia 429
19.3.2 Invertebrate ingestion of the leaf-fungus complex 429
19.4 Conclusions 431
Acknowledgments 432
References 432
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xvi Contents
Diego Libkind, Gabriel Russo and María Rosa van Broock
20 Yeasts from extreme aquatic environments: hyperacidic
freshwaters 443
20.1 Introduction 443
20.2 The River Agrio-Lake Caviahue acidic aquatic system 444
20.2.1 Yeast occurrence 445
20.2.2 Yeast diversity 446
20.3 Comparative yeast diversity study between RAC and the acidic
environments of the Iberian Pyrite Belt (IPB) 451
20.4 Acidic rock drainage (ARD) yeasts ecoclade 454
20.5 Physiological aspects of acidophilic yeasts 456
20.6 Possible ecological roles of yeasts in acidic aquatic
environments 457
20.7 Final remarks 458
Acknowledgments 459
References 460
Nattawut Boonyuen, Somsak Sivichai and E. B. Gareth Jones
21 Decomposition of wood in tropical habitats 465
21.1 Review of fungal diversity on wood in freshwater streams 466
21.2 Colonization of 15 timbers exposed at two locations in Thailand 467
21.2.1 Materials and methods 467
21.2.2 Results 468
21.2.3 Rate of decay of selected timbers at two contrasting freshwater
ecosystems in Thailand 472
21.2.4 Discussion 473
21.2.4.1 Fungal community 473
21.2.4.2 Decay of wood in freshwater habitats 474
Acknowledgments 476
References 477
Ka-Lai Pang, Kevin D. Hyde and E. B. Gareth Jones
22 Epliogue 481
22.1 Introduction 481
22.2 Freshwater fungi 481
22.3 Freshwater fungus-like organisms 482
22.4 Knowledge gaps and future work in freshwater mycology 482
22.5 Conclusions 486
References 486
Index 489
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List of contributing authors
André Aptroot
G. van der Veenstraat 107
NL-3762 XK Soest
The Netherlands
E-Mail: andreaptroot@gmail.com
Felix Bärlocher
63B York Street
Dept. Biology
Mt. Allison University
Sackville, NB, E4L 197
Canada
E-Mail: fbaerlocher@mta.ca
Nattawut Boonyuen
National Center for Genetic Engineering
and Biotechnology (BIOTEC)
113 Thailand Science Park,
Phahonyothin Road
Khlong Nueng, Khlong Luang
Pathum Thani 12120
Thailand
E-Mail: nattawut@biotec.or.th
María Rosa van Broock
Laboratorio de Microbiología
Aplicada y Biotecnología (MABB)
Universidad Nacional del Comahue
Centro Regional Universitario Bariloche
(CRUB)-CONICET (Consejo Nacional de
Investigaciones Científicas y
Tecnológicas)
CCT Patagonia Norte. Quintral 1250
(8400) Bariloche
Río Negro
Argentina
E-Mail: queemiegvb@yahoo.com.ar
Lei Cai
State Key Laboratory of Mycology
Institute of Microbiology
Chinese Academy of Sciences
NO.1, Beichen West Road, Chaoyang
District
Beijing 100101
China
E-Mail: mrcailei@gmail.com
Angkana Chaiprasert
Department of Microbiology
Faculty of Medicine Siriraj Hospital
Mahidol University
Bangkok 10700
Thailand
E-Mail: angkana.cha@mahidol.ac.th
José I. de Souza
Núcleo de Pesquisa em Micologia
Instituto de Botânica
Av. Miguel Stéfano 3687
04301-012, São Paulo, SP
Brazil
E-Mail: jisouza@yahoo.com.br
Verónica Ferreira
IMAR-CMA
Department of Life Sciences
Faculty of Science and Technology
University of Coimbra
P.O. box 3046
3001-401 Coimbra
Portugal
E-Mail: veronica@ci.uc.pt
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xviii List of contributing authors
Frank H. Gleason
School of Biological Sciences
Level 5, Carslaw (F07)
The University of Sydney
NSW 2006
Australia
E-Mail: frankjanet@ozemail.com.au
Sally Glockling
135 Brodrick Road
Hampden Park
Eastbourne BN22 9RA
UK
E-Mail: sally@glockling.com
Rodolphe E. Gozlan
School of Conservation Sciences
Bournemouth University
Talbot Campus
Fern Barrow
Poole
Dorset BH12 5BB
UK
E-Mail: rgozlan@bournemouth.ac.uk;
rudy.gozlan@ird.fr
Manuel A. S. Graça
IMAR-CMA
Department of Life Sciences
Faculty of Science and Technology
University of Coimbra
P.O. box 3046
3001-401 Coimbra
Portugal
E-Mail: mgraca@ci.uc.pt
Vladislav Gulis
Department of Biology
Coastal Carolina University
P.O. Box 261954
Conway, SC 29528-6054
USA
E-Mail: vgulis@coastal.edu
Kevin D. Hyde
School of Science
Mae Fah Luang University
333 Moo 1, Tambon Tasud
Muang District, Chiang Rai 57100
Thailand
E-Mail: kdhyde1@gmail.com
Dian-Ming Hu
College of Biology and Bioengineering
Jiangxi Agricultural University
Nanchang
Jiangxi Province, 330045
China
E-Mail: hudianming1@gmail.com
Timothy Y. James
Department of Ecology and
Evolutionary Biology
University of Michigan
Kraus Natural Science Bldg., Rm. 1008
830 North University
Ann Arbor, MI 48109-1048
USA
E-Mail: tyjames@umich.edu
Casey N. Jessop
2 School of Biological Sciences
F07, University of Sydeny
Camperdown
NSW 2006
Australia
E-Mail: cjes1587@uni.sydney.edu.au
Ana L. de Jesus
Núcleo de Pesquisa em Micologia
Instituto de Botânica
Av. Miguel Stéfano 3687
04301-012, São Paulo, SP
Brazil
E-Mail: analuciajesus@hotmail.com
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List of contributing authors xix
E. B. Gareth Jones
Department of Botany and Microbiology
College of Science
King Saud University
Riyadh 11451
Kingdom of Saudi Arabia
E-Mail: torperadgj@gmail.com
Sergey Karpov
St. Petersburg State University
Universitetskaya emb 7/9
St. Petersburg, 199034
Russia
E-Mail: sakarpov4@gmail.com
Ray Kearney
The Department of Infectious Diseases
and Immunology
The University of Sydney
NSW 2006
Australia
E-mail: ray.kearney@sydney.edu.au
Theerapong Krajaejun
Department of Pathology
Faculty of Medicine Ramathibodi
Hospital
Mahidol University
Bangkok 10400
Thailand
E-Mail: theerapong.kra@mahidol.ac.th
Peter M. Letcher
The University of Alabama
Department of Biological Sciences
1332 SEC, Box 870344
Tuscaloosa, AL 35487
USA
E-Mail: letch006@bama.ua.edu
Diego Libkind
Laboratorio de Microbiología Aplicada
y Biotecnología (MABB)
Universidad Nacional del Comahue
Centro Regional Universitario Bariloche
(CRUB)-CONICET (Consejo Nacional de
Investigaciones Científicas y Tecnológi-
cas)
CCT Patagonia Norte. Quintral 1250
(8400) Bariloche
Río Negro
Argentina
E-Mail: libkindfd@comahue-conicet.gob.ar
Robert Lichwardt
Department of Ecology & Evolutionary
Biology
University of Kansas
Lawrence, KS 66049-7534
USA
E-Mail: licht@ku.edu
Osu Lilje
School Biological Sciences
F07, University Sydney
Camperdown
NSW 2006
Australia
E-Mail: osu.lilje@sydney.edu.au
Fang Liu
State Key Laboratory of Mycology
Institute of Microbiology
Chinese Academy of Sciences
NO.1, Beichen West Road, Chaoyang
District
Beijing 100101
China
E-Mail: orchid.lf@gmail.com
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xx List of contributing authors
Deborah J. Macarthur
P.O. Box W29
Watsons Bay
NSW 2030
Australia
E-Mail: deborah.macarthur@sydney.edu.au
Agostina V. Marano
Núcleo de Pesquisa em Micologia
Instituto de Botânica
Av. Miguel Stéfano 3687
04301-012, São Paulo, SP
Brazil
E-Mail: agosvm@hotmail.com
Wyth L. Marshall
BC Centre for Aquatic Health Services
871a Island Hwy
Campbell River
BC V9W 2C2
Canada
E-Mail: wythmarshall@yahoo.ca
Ludmila Marvanová
Masaryk University, Faculty of Science
Institute of Experimental Biology
Czech Collection of Microorganisms
University Campus Bohunice, Kamenice
753/5
625 00 Brno, Czech Republic.
E-Mail: ludmila@sci.muni.cz
Floris van Ogtrop
Faculty of Agriculture and Environment
C81, University of Sydney,
Sydney, NSW 2006,
Australia
E-mail: floris.vanogtrop@sydney.edu.au
Ka-Lai Pang
Institute of Marine Biology and Center
of Excellence for the Oceans
National Taiwan Ocean University
2 Pei-Ning Road, Keelung 20224
Taiwan (R.O.C.)
E-Mail: klpang@ntou.edu.tw
Cláudia Pascoal
CBMA – Centre of Molecular and
Environmental Biology
Department of Biology
University of Minho
Campus de Gualtar
4710-057 Braga
Portugal
E-Mail: cpascoal@bio.uminho.pt
Aom Pinnoi
National Center for Genetic Engineering
and Biotechnology (BIOTEC)
113 Thailand Science Park,
Phahonyothin Road
Khlong Nueng, Khlong Luang
Pathum Thani 12120
Thailand
E-Mail: aom5736@yahoo.com
Umpava Pinruan
National Center for Genetic Engineering
and Biotechnology (BIOTEC)
113 Thailand Science Park,
Phahonyothin Road
Khlong Nueng, Khlong Luang
Pathum Thani 12120
Thailand
E-Mail: umpava328@gmail.com
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List of contributing authors xxi
Carmen L. A. Pires-Zottarelli
Núcleo de Pesquisa em Micologia
Instituto de Botânica
Av. Miguel Stéfano 3687
04301-012, São Paulo, SP
Brazil
E-Mail: zottarelli@uol.com.br
Martha J. Powell
The University of Alabama
Department of Biological Sciences
1332 SEC, Box 870344
Tuscaloosa, AL, 35487
USA
E-Mail: mpowell@biology.as.us.edu
Huzefa Raja
Department of Chemistry and Biochemistry
The University of North Carolina at
Greensboro
435 Sullivan Science Building
301 McIver Street
Greensboro, NC 27412
USA
E-Mail: mpowell@biology.as.us.edu
Jodi Rowley
Australian Museum Research Institute
Australian Museum
6 College Street
Sydney NSW 2010
Australia
E-Mail: Jodi.Rowley@austmus.gov.au
Gabriel Russo
Laboratorio de Microbiología Aplicada
y Biotecnología (MABB)
Universidad Nacional del Comahue
Centro Regional Universitario Bariloche
(CRUB)-CONICET (Consejo Nacional de
Investigaciones Científicas y Tecnológicas)
CCT Patagonia Norte. Quintral 1250
(8400) Bariloche
Río Negro
Argentina
E-Mail: gavilano@gmail.com
Telesphore Sime-Ngando
Laboratoire Microorganismes: Génome
et Environnement
UMR CNRS 6023
Bât. Biologie A, 24 avenue des Landais,
BP 80026
63171 Aubière Cedex
France
E-Mail: Telesphore.SIME-NGANDO@
univ-bpclermont.fr
Holger Thüs
Life Sciences Department
Division of Genomic and Microbial
Diversity
The Natural History Museum
Cromwell Road
London SW7 5BD
UK
E-Mail: h.thues@nhm.ac.uk
Marcia Saraiva
Aberdeen Oomycete Laboratory
School of Medical Sciences
University of Aberdeen
Foresterhill AB25 2ZD
UK
E-Mail: m.saraiva@abdn.ac.uk
Mohammad N. Sarowar
Aberdeen Oomycete Laboratory
School of Medical Sciences
University of Aberdeen
Foresterhill AB25 2ZD
UK
E-Mail: n.sarowar@bau.edu.bd
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xxii List of contributing authors
Mark R. D. Seaward
Dept. of Achaeological, Environmental &
Geographical Sciences (AGES)
University of Bradford
Bradford BD7 1DP
UK
E-Mail: m.r.d.seaward@bradford.ac.uk
Carol A. Shearer
Department of Plant Biology
University of Illinois
Rm. 265 Morrill Hall
505 S. Goodwin Ave.
Urbana, IL 61801
USA
E-Mail: carolshe@illinois.edu
Somsak Sivichai
Darlene Southworth
Department of Biology
Southern Oregon University
1250 Siskiyou Blvd.
Ashland, OR 97520
USA
E-Mail southworth@sou.edu
Kandikere R. Sridhar
Department of Biosciences
Mangalore University
Mangalagangotri 574 199
Mangalore, Karnataka
India
E-Mail: kandikere@gmail.com
Satinee Suetrong
National Center for Genetic Engineering
and Biotechnology (BIOTEC)
113 Thailand Science Park,
Phahonyothin Road
Khlong Nueng, Khlong Luang
Pathum Thani 12120
Thailand
E-Mail: satinee.na@gmail.com
Pieter van West
Aberdeen Oomycete Laboratory
School of Medical Sciences
University of Aberdeen
Foresterhill AB25 2ZD
UK
E-Mail: p.vanwest@abdn.ac.uk
Huang Zhang
Faculty of Environmental Science and
Engineering
Kunming University of Science and
Technology
Kunming, Yunnan Province, 650500
China
E-Mail: zhanghuang2002113@gmail.com
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Mohammad N. Sarowar, Marcia Saraiva, Casey N. Jessop,
Osu Lilje, Frank H. Gleason and Pieter van West
10 Infection strategies of pathogenic
oomycetes in fish
10.1 Introduction
Studies on the evolutionary lineage of oomycetes show that these pathogens are capable
of infecting different hosts, ranging from algae, plants, protists, fungi and arthropods
to vertebrate animals (van West 2006; Phillips et al. 2008; Beakes et al. 2012). Animal
pathogenic oomycetes are often neglected in spite of significant economic importance
due to lack of study and scarcity of data (Diéguez-Uribeondo et al. 2009). Outbreaks of
mycotic diseases, including saprolegniosis (causative agent Saprolegnia spp.) and epi-
zootic ulcerative syndrome (EUS) (causative agent Aphanomyces invadans) can result
in enormous economic losses in aquaculture threatening both production targets and
even the long-term viability of the industry as well as natural ecosystems (van West
2006; Phillips et al. 2008; Sarowar et al. 2013; van den Berg et al. 2013). For example,
saprolegniosis has resulted in heavy losses in salmonid hatcheries and “winter kill” in
catfish aquaculture (Bangyeekhun et al. 2001; Torto-Alalibo et al. 2005). It has been esti-
mated that 10% of all hatched salmon in the aquaculture industry succumb to Saproleg-
nia infections (van West 2006). The best-studied fish pathogenic oomycetes belong to
the order Saprolegniales, which include the genera Saprolegnia, Achlya and Aphanomy-
ces. Several other genera of Saprolegniales also have a significant impact on freshwater
ecosystems by infecting different host organisms and their eggs (Tab. 10.1) (Kiesecker
et al. 2001; Fernández-Benéitez et al. 2008; Ruthig 2009).
Shortly after 1905, and extending to about 1970, reports of saprolegniosis in the
wild became more frequent and reflected increasing recognition while the cause of
the disease was still in some dispute. Numerous reports support the presumption that
all freshwater fish are susceptible to some oomycete infection under the right con-
ditions (Srivastava 1980), since certain water moulds are consistently isolated from
fish lesions. Most notable are Saprolegnia, Achlya, and Aphanomyces species from the
order Saprolegniales and Pythium undulatum from the order Peronosporales (Noga
1993; El-Sharouny and Badran 1995). Most of our basic and applied knowledge of
oomycete infections in freshwater fish is based on studies of salmonids due to their
economic and ecological relevancy.
Oomycetes pathogenic to animals are zoosporic eukaryotic microorganisms –
often referred to as water moulds (Coker 1923; Seymour 1970). They are fungal-like cos-
mopolitan organisms distributed in a variety of aquatic environments and ecological
niches. Together with the brown algae and diatoms, they are classified as Stramenopi-
les (Heterokonts). The oomycetes however have lost their plastids in the evolutionary
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218 10 Infection strategies of pathogenic oomycetes in fish
Tab. 10.1: Oomycetes species reported from tissues of bony fishes (Osteichthyes) or their eggs
(Adapted from Czeczuga et al. 2002; Boys et al. 2012; Czeczuga et al. 2013).
Oomycete family Isolates Known as
pathogen (Y/N)
Observed on dead (D)
or live (L) fish
Saprolegniaceae Achlya ambisexualis* Y D
A. americana* Y D
A. caroliniana* Y D & L
A. crenulata N D
A. debaryana N D
A. diffusa* Y D
A. dubia* Y D
A. flagellata – L
A. intricate* Y D
A. klebsiana* Y D
A. oblongata N D & L
A. oligacantha N D
A. orion* Y D
A. polyandra N D & L
A. prolifera* Y D & L
A. proliferoides* Y D
A. radioasa* Y D
A. rodriqueziana N D
A. treleaseana N D
Saprolegnia ansiospora – L
S. diclina* Y D & L
S. eccentrica N D & L
S. ferax* Y D & L
S. hypogyna N D
S. litoralis N D & L
S. mixta* Y D & L
S. monoica* Y D
S. parasitica* Y D & L
S. pseudocrustosa N D
S. shikotsuensis* Y D & L
S. torulosa* Y D
S. uliginosa N D
Dictyuchus monosporus Y D
D. pisci – L
Isoachlya monilifera Y D & L
Thraustotheca clavata N D
Leptolegniaceae Aphanomyces frigidophilus – L
A. Invadans* Y L
A. irregularis N D
A. laevis – L
A. stellatus N D
Leptolegnia caudata Y D & L
(continued)
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10.1Introduction  219
process (Baldauf 2003; Robideau et al. 2011). Water moulds are phylogenetically very
distant from the Kingdom Eumycota, the true Fungi, unlike their initial appearance
would suggest. For example, there are several clearly defined developmental stages in
the life cycle that distinguish them from true fungi. Indeed the Stramenopiles are cha-
racterized by forming free-swimming spores, called zoospores that have two flagella
of which one flagellum has tinsel hairs (Walker and van West 2007).
Common disease symptoms include visible white and gray cotton wool like
patches of filamentous mycelium growing on the skin and gills of fish (Stueland
et al. 2005). The rapid growing infections cause large wounds on the skin and head
of the fish (Fig. 10.1), resulting in impaired osmoregulation and hemo-dilution,
whereas extensive gill infections cause respiratory failure, which leads to morta-
lity. Unlike Saprolegnia infection, A. invadans is capable of invading through fish
muscle and other tissues, and induce a granulomatous host response characteristic
of EUS lesions (Lilley and Roberts 1997). Fish are more prone to saprolegniosis and
EUS in winter, when presumably lower water temperatures cause the fish to become
immuno-compromised (Bly and Clem 1992; Bly et al. 1992; Oidtmann 2012).
Pathogenic oomycetes in the order Saprolegniales, such as Saprolegnia and Apha-
nomyces species, are responsible for infections on fish in aquaculture, wild population
and even recreational fish tanks (Willoughby and Pickering 1977; Neish and Hughes
Oomycete family Isolates Known as
pathogen (Y/N)
Observed on dead (D)
or live (L) fish
Leptomitaceae Leptomitis lacteus* Y D & L
Pythiaceae Pythium afertile N D
P. aquatile N D
P. arrhenomanes N D
P. butleri N D
P. diclinum – L
P. dissotocum N D
P. hemmianum N D
P. intermedium N D
P. myriotylum N D
P. ostracodes N D
P. periplocum N D
P. proliferum – L
P. tenue N D
Zoophagus insidians N D
* Species known as pathogens; sh specimens include eggs of African catsh, Clarias gariepinus,
muscle sections of monkey goby, Neogobius fluviatilis; racer goby, Neogobius gymnotrachelus;
Chinese sleeper, Perccottus glenii; and stone moroko, Pseudorasbora parva and found on live sh
bony herring, Nematalosa erebi; Golden Perch, Macquaria ambigua; Murray cod, Maccullochella
peelii; and spangled perch, Leiopotherapon.
Tab. 10.1: (continued)
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220 10 Infection strategies of pathogenic oomycetes in fish
Fig. 10.1: Salmonid fish and eggs infected with Saprolegniosis. (A) Farmed salmon (Salmo salar),
(B) farmed rainbow trout (Onchorhynchus mykiss). The white/gray patches on the skin, head
and tail of the fish are signs of severe saprolegniosis. (C) Salmon eggs completely destroyed by
saprolegniosis. The pictures were taken by Herbert van den Berg and Rodrigo Belmonte.
1980; Lilley and Roberts 1997; Bruno and Wood 1999; Hussein and Hatai 2002). One of
the most destructive pathogens on fishes is Saprolegnia parasitica, which is endemic
to all freshwater habitats around the world and is to some extent responsible for the
decline of natural populations of salmonids and other freshwater fishes (Neitzel et al.
2004; van West 2006). The devastating infections are a major problem in the salmon
aquaculture which includes hatcheries and farms in Scotland, Scandinavia, Chile,
Japan, Canada, and the USA. Saprolegniosis costs the aquaculture industry tens
of millions of pounds annually (Lilley and Roberts 1997; Hussein and Hatai 2002;
Phillips et al. 2008). In the USA, “winter kill” in farmed catfish caused by
S. parasitica results in financial losses of up to 50%, which represents an economic
loss of about $40 million (Bruno and Wood 1999).
Epizootic ulcerative syndrome (EUS) is another devastating mycotic disease
caused by the pathogen A. invadans affecting both wild and farmed fishes. It was first
characterized and described in Japan in 1971 (Egusa and Masuda 1971). Since then
A. invadans has been responsible for major epidemics in freshwater and estuarine
fishes in Asia, Australia, North America and Africa. The rapid spread of the disease
has substantially affected livelihoods of fish farmers and fishermen especially in Asia
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10.2Taxonomy of oomycetes pathogenic to fish  221
and Africa (Lilley et al. 2001, 2003). The infective life stage of A. invadans is the free-
swimming zoospore that attaches to a fish host, encysts and germinates to develop
vegetative hyphae invading and ramifying through host tissues (Lilley and Roberts
1997; Kiryu et al. 2003). The hyphae will invade the fish skin, muscular tissue and
may reach the internal organs eventually killing the fish. The likely pathway of trans-
mission of S. parasitica and A. invadans from infected to native fish is through the
formation of a zoosporangium developing from the mycelium in fish tissue at the fish
surface, followed by the formation of primary zoospores that are released from the
zoosporangium.
This review focuses on the different aspects of the infection strategy of oomy-
cetes that are pathogenic to fish. We review the physical strategy for infection with
different life stages of the pathogens, which shaped their pathogenicity towards fish
and molecular strategy in setting infection, which illustrates recent molecular work
to support the notion that pathogenic oomycetes are capable of manipulating host
immune responses in fish.
10.2 Taxonomy of oomycetes pathogenic to fish
Most oomycetes that are pathogenic to animals belong to the subclass Saprolegniomy-
cetidae, which has two orders: Saprolegniales and Leptomitales (Phillips et al. 2008).
Kützing (1843) formally erected the Saprolegniales but considered members of the
order to be nonchlorophyllous algae. The first account (English) monograph of the
family was published by Humphrey (1893) and revised by Coker (1923). Subsequently
Moreau and Moreau (1935, 1938), Coker (1935), Dick (1969, 1972, 1978, 2001), Miller and
Ristanovic (1975), Powell and Blackwell (1998) and Padgett et al. (2000) have made
observations about systematic concepts in water moulds, most of which emphasize
the significant morphological plasticity especially within the family Saprolegniaceae.
The genera were distinguished by the structure and shape of asexual sporangia and
mode of zoospore release (Seymour 1970; Daugherty et al. 1998). The species diffe-
rentiation was based on morphological characteristics of their sexual structures, i.e.
oogonia, oospores, and antheridia (Seymour 1970; Johnson et al. 2005). The traditi-
onal taxonomy often led to complicacy in identifying species, many species exhibit
very similar or overlapping characters. For example, isolates of Saprolegnia often fail
to produce sexual structures at all in vitro especially isolates of fish pathogenic S.
parasitica and A. invadans. This has resulted in taxonomic confusion regarding isola-
tes pathogenic to fish.
Advances in molecular biology have made it possible to perform complemen-
tary speciation at the taxon level by DNA sequencing. These developments not only
provide more confidence but are also time-saving and are valuable in avoiding ques-
tionable or synonymous speciation (Robideau et al. 2011). The increasing availability
of molecular sequence data of the members of Saprolegniales has guided studies into
redrawing the phylogenetic tree. Analysis of different regions of the ribosomal RNA
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222 10 Infection strategies of pathogenic oomycetes in fish
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10.3Physical adaptation and strategy for infection 223
(rRNA) gene, i.e. small ribosomal subunit (18S), internal transcribed spacer (inclu-
ding ITS1, 5.8S and ITS2) and large ribosomal subunit of rRNA gene (28S) determined
phylogenetic data of the family Saprolegniaceae to be clustered in 10 robust clades
(Hulvey et al. 2007). Interestingly most Saprolegnia isolates that are obtained from
salmonid lesions fall into the same cluster based on ITS sequences of the isolates (Fig.
10.2) (Diéguez-Uribeondo et al. 2007). DNA sequence databases have been used as the
main resource for identifying species with similar morphological features (Tautz et al.
2003; Hulvey et al. 2007). However, the major limitation of the databases is that there
is a lack of biodiversity studies on the Saprolegniaceae. To confound this there are a
number of incorrectly identified DNA sequences in the available public databases.
Inevitably, this situation will result in incorrect identification of species (Bridge et al.
2003) and makes it difficult to understand the true extent of all possible oomycete
species that are pathogenic to fish (Hulvey et al. 2007). Several studies have attemp-
ted to clarify the existing morphology based on traditional taxonomy using the full
ITS sequences (Diéguez-Uribeondo et al. 2007; Nilsson et al. 2009).
10.3 Physical adaptation and strategy for infection:
macroscopic infection, the face of infection on hosts
It has often been assumed that oomycetes pathogenic to fish set infection at the epi-
dermis level, and probably, where the epithelial cells are ruptured (Rucker 1944).
However, it has also been suggested that Saprolegnia species can gain entrance into
susceptible animals by penetrating directly into the intact epidermis, or entering into
natural body openings or wounds. The invading organism also may enter at points
where the epidermis is not scaly (Willoughby 1971) or is thin. Saprolegnia species are
known to infect gills, skin and fins of mature fish and also eggs from aquatic animals
such as fish and amphibians (Hussein et al. 2001; van West 2006; Petrisko et al. 2008;
Ghiasi et al. 2010; Thoen et al. 2011; Cao et al. 2012). Lesions associated with hyphae
belonging to the Saprolegniaceae develop at the site of infection and are usually
accompanied by necrosis, ulceration (Bootsma 1973) and inflammation (Vishniac and
Nigrelli 1957). The hyphae of some water moulds can penetrate into the dermal areas
(connective tissue) in fish and can also adhere closely to the stratified epithelial cells
of the epidermis. According to Bootsma (1973), necrosis may be accompanied by cell
proliferation.
Fig. 10.2: Phylogeny of different orders of oomycetes. (A) Phylogenetic relationships of different
orders of oomycetes. The maximum likelihood tree was constructed retrieving the internal
transcribed spacer (ITS) regions sequence of the rRNA gene of the isolates from the GenBank.
The animal pathogenic oomycetes are highlighted (*) and they are from a range of genera within
the oomycetes. (B) The order Saprolegniales is extended into the maximum likelihood tree using
the isolates that are most frequent from fish. Percentile bootstrap values for both trees based on
1,000 replications are indicated at the nodes (Adapted from Phillips et al. 2008).
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224 10 Infection strategies of pathogenic oomycetes in fish
In the late 1970s Neish (1977) observed the course and progression of saproleg-
niosis in a few Pacific salmons. Hyphae at first destroyed the multicellular stratified
squamous epithelium followed by penetration into the basement membrane. Sub-
sequently, as the infection proceeded, the hyphae grew into the stratum spongiosum
of the dermis and then into the collagen bundles of the stratum compactum. From
the dermis, hyphae can penetrate into the hypodermis, and then into the subtending
musculature. The histopathology of the invasion of salmonid fry was investigated by
Miyazaki et al. (1977). They found hyphal embolisms in the blood of infected fish, and
mycelium throughout the abdominal cavity. Hyphae of Saprolegnia sp. was found to
have penetrated into the stomach lumen at the pyloric region, and grew from sites in
the stomach wall into the pancreas, pyloric caeca, spleen, abdominal musculature,
and visceral blood vessels.
10.4 Oomycete zoospores, the first line of attack
One of the key characteristics that separate oomycetes from true fungi is the forma-
tion of free-swimming zoospores. The epidemic spread of the oomycete diseases is
based on the very rapid dispersal by zoospores from host to host that are released
from zoosporangia. The released zoospores can actively swim which probably incre-
ase the chance of finding new hosts. The number of zoospores released per zoospo-
rangium varies from species to species. The zoospores of Saprolegnia species have a
single nucleus and lack cell walls and can swim with the help of two flagella each of
differing lengths; one tinsel (or anterior) flagellum, and one whiplash (or posterior)
flagellum. The anterior flagellum is probably responsible for pulling the zoospore
through the water whereas the posterior flagellum acts as a rudder for steering the
cell (Judelson and Blanco 2005). Generally, zoospores are “bean” shaped, due to the
ventral groove into which the two flagella are inserted, both in a lateral orientation
(Dick 1973, 1990, 2000).
Zoospores are also essential for targeting the site of infection, which is often
referred to as the homing response (van West et al. 2003). Oomycete zoospores have
evolved intriguing homing responses. For example, they are able to make use of
electro tactic as well as chemotactic clues (Carlile 1983; Donaldson and Deacon 1993;
Morris and Gow 1993; van West et al. 2002, 2003 ). Once a suitable site, or a host,
has been found, the zoospore undergoes encystment and becomes immobilized. This
involves the rapid production of a cell wall surrounding the zoospore. The flagella are
either discarded or retracted into the cell which depends on the species (Dick 2001).
Subsequently the cyst attaches to the host.
Many pathogenic isolates of Saprolegnia and Aphanomyces species have two
different types of motile zoospores. Initially a primary zoospore is released from the
zoosporangium. It then encysts, and later a secondary zoospore can emerge from the
cyst. Both primary and secondary zoospores have two flagella, an anterior and a pos-
terior flagellum, although the lengths of the flagella in the secondary zoospores may
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10.5Triggers for zoospore formation, waking up the beast 225
be twice as long (Walker and van West 2007). The secondary zoospores are considered
the better swimming spore and are more comparable to zoospores from the Peronos-
porales, such as, Phytophthora and Pythium (Beakes 1987). Secondary zoospores have
a longer motile period and are considered crucial for successful infection.
The production of two generations of zoospores (diplanetism), often morphologi-
cally different, is one of the defining characteristics of the “saprolegnian line” or “sap-
rolegnian galaxy” of oomycetes (Sparrow 1976; Barr and Désaulniers 1987; Dick 2001).
Suppression of the “primary” (or first-formed) generation of zoospore occurs widely
in the order Saprolegniales. However, non-motile aplanospores may be released from
the sporangium, forming balls of cysts, as in the genera Aphanomyces and Achlya, or
may be retained in the sporangium as in Dictyuchus and Thraustotheca (Beakes 1987;
Dick 2001). Zoospores of oomycetes contain an array of peripheral vesicles, which
upon encystment are discharged to form either a ventral “adhesive pad” or an outer
cyst coat layer (Beakes 1987; Gubler and Hardham 1988; Lehnen and Powell 1989;
Burr and Beakes 1994; Hardham 2005) which reflects evolutionary modification for
better attachment to hosts.
10.5 Triggers for zoospore formation, waking up the beast
The asexual zoosporangial phase of fish pathogenic isolates of oomycetes is mainly
responsible for dispersing free-swimming infectious propagules in order to find new
hosts. The zoosporangia are formed at the end of the hyphae and are separated from
the hyphae by basal septa. Specific triggers are known to set off zoospore release, e.g.
the lack of nutrients (Fuller and Jaworski 1987) or a sudden drop in temperature. The
presence and number of hosts might also bring about a queue in fish pathogenic oomy-
cetes to initiate sporulation. In most cases, the release of zoospores is finely orchestra-
ted with the host’s physiology. The number of zoospores being produced and released
can significantly increase with sudden drop of temperature (Bly et al. 1992). The latter
also leaves a cold-blooded animal like fish compromised, which increases the chance
of infection by pathogens (Bly et al. 1992). In case of A. invadans, infection in fish
occurs mostly at water temperatures of 18–22°C or less (Bondad-Reantaso et al. 1992).
These conditions also favor the sporulation of A. invadans (Lumanlan-Mayo et al. 1997)
and temperatures of 17–19°C have been shown to delay the inflammatory response of
some fish species to oomycete infection (Chinabut et al. 1995; Catap and Munday 1998).
10.6 Encystment and germination, one step closer to infection
Encystment and germination is considered to be vital for establishing a successful
infection. Zoospores undergo the rapid process of encystment, which involves shed-
ding the two flagella and the formation of a non-motile walled cyst (cytospore). This
could help zoospores to thrive a bit longer in the absence of a suitable host. Contact
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226 10 Infection strategies of pathogenic oomycetes in fish
of the zoospore with the host triggers encystment, which in turn initiates germination
and eventually causes infection.
The triggers that lead to encystment under laboratory conditions include agitation
by mechanical force and the presence of nutrients such as peptone (Dieguez-Uribeondo
et al. 1994), dissolved fish mucus or nutrient solutions such as bovine serum albumin,
haemoglobin and hempseeds (Willoughby 1986; Dieguez-Uribeondo et al. 1994). The
environmental factors that lead to encystment and germination in zoospores may vary
between species (Willoughby et al. 1983), but a single regulatory system is proposed to
control both encystment and germination cycles (Andersson and Cerenius 2002). The
encystment of zoospores has been found to be regulated by Ca2+ in Aphanomyces astaci,
Aphanomyces euteiches and even in Phytophthora parasitica (Cerenius and Söderhäll
1984a, b, 1985; Warburton and Deacon 1998). It is thought that this may also be the case
for other pathogenic oomycete species (Donaldson and Deacon 1992).
Cytological studies have identified key peripheral organelles associated with
encystment of zoospores and cysts. One of these is the kinetosome-associated orga-
nelle (or K-body) (Holloway and Heath 1977; Beakes 1983; Lehnen and Powell 1989,
1991). The K-body is the first organelle to be discharged by fusing with the plasma
membrane and forming a diffuse fibrillar pad (Lehnen and Powell 1989; Burr and
Beakes 1994). Secondary encystment vesicles (SEVs) have been described in S. diclina
and S. parasitica in association with encystment (Burr and Beakes 1994). Fully dif-
ferentiated SEVs in S. parasitica contain boathook spines whereas in S. diclina the
SEVs contain capsules shaped bodies (Beakes 1983). During encystment these vesic-
les break-up into smaller vesicles that appear to form bubbles on the surface of the
secondary zoospores before discharging and releasing an electron dense surface in
which, in the case of S. parasitica, the boathook spine bundles are embedded (Beakes
1983; Burr and Beakes 1994). The SEV discharge reacts with the lectin Concanavalin A
(Con A), which indicates the presence of mannose and glucose sugars. The discharge
becomes patchy in germlings and localised with the bundles of spines (Burr and
Beakes 1994). Burr and Beakes (1994) infer that the entrapment of this discharge
material in amongst the boathook spines may act as an adhesive complex that facili-
tates binding to fish. Con A induces strong encystment of S. diclina and S. parasitica
secondary zoospores (Burr and Beakes 1994). The lectin wheat germ agglutinin (WGA)
is known to react to N-acetyl glucosamine and binds to sites that correspond closely
with K-body loci on primary and secondary zoospores. WGA induces the encystment
of secondary zoospores of S. diclina, but not S. parasitica, which may correspond to
its relatively poor expression on S. parasitica (Burr and Beakes 1994). The expression
of N-acetyl glucosamine has also been observed on the adhesive pads of K2-bodies
of S. ferax spores attaching to a substrate (Lehnen and Powell 1989), hyphal adhe-
rence on plastic surfaces (Willoughby 1986) and S. parasitica germ tubes (Burr and
Beakes 1994). Fibrillar type vesicles release fibrillar material but there is no evidence
of spines (Burr and Beakes 1994). The lack of lectin reactive compounds in these
vesicles, suggests the absence of glycoconjugates with accessible residues of sugars.
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10.7Repeated zoospore emergence, the back-up plan 227
Evidence from Phytophthora cinnamomi suggests these vesicles are repositories for
storage proteins (Gubler and Hardham 1988, 1990).
In several Saprolegnia species, a set of hairs or boathooks or spines is produced
upon encystment on the surface. Although there is considerable variation both in
the length and number of the long-hooked hairs associated with the secondary cysts
(Pickering et al. 1979; Hatai et al. 1990; Fregeneda-Grandes et al. 2000), the size and
structure of the hairs seem to be species specific, although more evidence is required
for this characteristic to be used for species identification ( Hatai et al. 1990; Diéguez-
Uribeondo et al. 2007).
There is debate amongst scientists as to the function of hairs on the cysts. Some
suggest that they may be required for attachment to a host. Others imply that it slows
sedimentation when free flowing in a water column. These advantages would be con-
sistent with increasing the ability of cysts to attach themselves to passing fish, as has
been reported on several occasions (Willoughby and Pickering 1977; Willoughby et al.
1983; Burr and Beakes 1994; Quiniou et al. 1998). Long-hooked hairs of the cysts seem
to favor contact and attachment to the surface of the host or substratum (Pickering and
Duston 1983; Hallett and Dick 1986), and presence of bundles of long hairs appears
to be often related to pathogenicity (Pickering and Willoughby 1982; Fregeneda-Gran-
des et al. 2000). Reports suggest that adaptation to parasitism in Saprolegnia might
have occurred at cyst level by the development of long-hooked hairs to facilitate host
attachment and selection of a retracting germination (Diéguez-Uribeondo et al. 2007).
Diéguez-Uribeondo et al. (2007) found that most Saprolegnia isolates obtained from
salmonid lesions contained cysts with bundles of long-hooked hairs and suggested to
put them into the same cluster. Curiously, Saprolegnia isolates with the longest hairs
(Fregeneda-Grandes et al. 2000, 2001) (Type I) appeared to be less aggressive patho-
gens or even non-pathogenic, at least towards salmonid fish, than those with shorter
spines (Fregeneda-Grandes et al. 2000, 2001) (Type II) and these observations appear
to be supported by other studies (Hatai et al. 1990; Hussein and Hatai 1999; Stueland
et al. 2005). This could mean that the size and number of hairs might not be directly
related to the pathogenicity of the species.
10.7 Repeated zoospore emergence, the back-up plan
The release of primary motile zoospores from sporangia serves a minimal level of
dispersal from the parent colony. The released zoospores undergo encystment if a
suitable host is found. In several fish pathogenic species, such as, S. parasitica and
A. invadans the resulting cyst is capable of releasing a new zoospore. In several Sap-
rolegnia species the secondary zoospores can undergo repeated cycles of encystment
and zoospore release, which is known as “repeated zoospore emergence” or “poly-
planetism” (Cerenius and Söderhäll 1984b; Hatai et al. 1990; Diéguez-Uribeondo et
al. 1994, 2007). Repeated zoospore emergence may represent an adaptation to the
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228 10 Infection strategies of pathogenic oomycetes in fish
parasitic mode of life in the Saprolegniales as it enables zoospores to make several
attempts to find and colonise a suitable host (Cerenius and Söderhäll 1985; Diéguez-
Uribeondo et al. 2007, 2009). Aphanomyces invadans isolates have been shown to
produce one additional generation of motile zoospores from artificially encysted
secondary zoospores, but no further generations could be induced. This is in contrast
to A. astaci, where up to three successive generations can be induced (Cerenius and
Söderhäll 1984b, 1985).
10.8 Chemotactic response of zoospores, the specialization
Some aspects of the physiological and ecological properties of zoospores in general
have been reviewed by Hickman and Ho (1966), Chet and Mitchell (1976), Fuller (1977)
and Gleason and Lilje (2009). Motile zoospores can be released from zoosporangia
of many parasitic species in large numbers. The chemotactic properties of zoospo-
res of a few species of various groups of fungi and fungus-like organisms have been
studied in the laboratory. Some of the zoospores which infect plant and animal hosts
are known to be chemotactic, sense exudates, navigate in chemical gradients and can
swim for several hours. For example, Bimpong and Clerk (1970) found that zoospores
of the plant parasite Phytophthora palmivora remain motile in distilled water for 84 h
at 17˚C, for 24 h at 27˚C and for 1 h at 33˚C. Zentmyer (1961) reported that zoospores of
Phytophthora cinnamomi sense the presence of exudates from avocado roots, settle
on the surface of the roots and begin to encyst and germinate within 30 to 60 min in
the laboratory.
Much of the early research on chemotaxis in zoospores of oomycetes focused
on parasites of vascular plants, particularly species of Phytophthora. For example,
Khew and Zentmyer (1973) tested the responses of zoospores of P. cactorum, P. capsici,
P. cinnamomi, P. citrophthora and P. palmivora to 12 amino acids (alanine, arginine,
aspartate, asparagine, glutamate, glutamine, histidine, leucine, methionine, phe-
nylalanine, proline and serine). Zoospores of all five species were attracted to all
amino acids, but there was considerable variation in the magnitude of the respon-
ses to amino acids by different species. Halsall (1975) tested the responses of zoo-
spores of P. drechsleri, P. cryptogea, P. cinnamomi, P. nicotianae var. parasitica and
P. citricola to four amino acids (asparagine, glutamine, aspartate and glutamate) and to
sucrose and ethanol. Zoospores of all five species were attracted to all six compounds.
More recently the properties of zoospores have been studied in parasites of
animals. For example, El-Feki et al. (2003) tested the responses of zoospores of
S. parasitica to metabolites (aspartate, alanine, leucine, tyrosine, histidine, arginine,
glucose and sucrose). Zoospores were attracted to all eight chemical compounds.
It is clear from these studies that zoospores of facultative parasites of both plant
and animal species are attracted to a large variety of exudates of metabolites from
tissues of their hosts. The chemotactic responses of zoospores, particularly attrac-
tion to amino acids, expedite quick arrival at uninfected and susceptible animal hosts
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10.9Proteins and amino acids as substrates for growth 229
which are rich in proteins. However, studies providing data on positive chemotaxis
of zoospores and metabolism capacities by parasites of amino acids and sugars also
suggest that it is also possible that some facultative parasites might be able to utilize
protein and carbohydrate compounds in both plant and animal substrates.
10.9 Proteins and amino acids as substrates for growth
Proteins from many sources, e.g. casein and keratin, are known to be good subst-
rates for the isolation of many species of oomycetes, particularly facultative para-
sites of animals, into pure culture and for their subsequent growth in liquid media
(Sparrow 1960). Living animal hosts and parts of dead animals, in particular, are
protein rich environments. Proteins must be digested outside the cell and the amino
acids produced must be transported into the cell prior to their catabolism. Smith
et al. (1994) demonstrated proteolytic activity of S. diclina, S. ferax and S. parasitica
by observing the clearing of casein in solid media. Jiang et al. (2013) documented the
presence of genes for serine, metallo- and cysteine proteases and genes for amino
acid transporters in the complete sequence of the genome of S. parasitica.
Once the protein substrates have been cleaved into their component amino acids
by proteases, the amino acid molecules must be catabolized and the energy in their
C-C bonds converted into high energy compounds, such as ATP. Some amino acid
molecules can be used directly for the synthesis of proteins or converted into nuc-
leotides for the synthesis of DNA in intermediary metabolism. Some saprotrophic
isolates of Saprolegnia, Achlya, Dictyuchus, Leptolegnia, Aphanomyces, Apodach-
lya, Leptomitus and Pythium grow rapidly on many but not all amino acids as sole
sources of carbon and nitrogen in liquid media (Tab. 10.2) (Gleason 1968; Gleason
et al. 1970a, b). Both saprotrophic and facultatively parasitic isolates of Saproleg-
nia can grow rapidly on mixtures of amino acids, and these isolates are capable of
removing the molecules of the amino acids from a mixture in the growth medium
(Gleason 1973; Nolan 1976). These data indicate that many oomycetes have the capa-
city for the digestion of proteins, the subsequent uptake of amino acids from a mixture
and their subsequent catabolism, and that carbohydrates do not need to be present
in the growth medium although many oomycetes can use carbohydrates as well as
proteins as carbon sources. Therefore, we would expect many species of oomycetes to
grow in protein rich environments such as dead or living animal hosts as saprotrophs
and facultative parasites. For example, Czechuga et al. (2002) isolated many species
of oomycetes from pieces of fish muscle tissue placed in lakes in Poland.
Sarowar et al. (2013) conducted challenge experiments to see whether Sapro-
legnia spp. isolates from insects and amphipods were able to infect and kill stonefly
nymphs, salmon eggs and frog embyos and found that S. diclina and S. ferax were
pathogenic to all three hosts whereas S. parasitica was less aggressive. They hypothe-
sised that S. parasitica may have evolved more specific affinity to live fish compared
to S. diclina and S. ferax which seem to be more saprotrophic and facultative parasites
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Tab. 10.2: The growth of ten species of oomycetes on single amino acid compounds as sole sources of carbon and nitrogen. The oomycetes were grown in
50 ml of growth media with only amino acid containing fixed Carbon amount of 4 g/l (pH 6.6 continuously) in a 125 ml Erlenmeyer flasks placed on a rotary
shaker. Dry weight indicates the amount of growth after various times of incubation (days). The conditions for growth and the composition of the growth media
are described in detail by Gleason (1968) and Gleason et al. (1970a, b). The blanks indicate that the amino acid was not tested.
Compound Dry weight (mg/flask) and days (in parenthesis)
Saprolegnia
sp.
Achlya
ambisexualis
Leptolegnia
eccentrica
Dictyuchus
sterile
Leptomitus
lacteus*
Apodachlya
punctata
Apodachlya
brachynema
Apodachlya
minima
Aphanomyces
laevis
Pythium
ultimum
Alanine  ()  ()  ()  ()  ()  ()  ()  ()  ()  ()
Glutamate  ()  ()  ()  ()  ()  ()  ()  ()  ()  ()
Aspartate  ()  ()  ()  ()  ()  ()  ()
Proline  ()  ()  ()  ()  ()  ()  ()  ()  ()  ()
Leucine  ()  ()  ()  ()  ()  ()  ()  ()  ()  ()
Hydroxyproline  ()  ()  ()
Arginine  ()  ()  ()  ()  ()  ()  ()  ()  ()
Phenylalanine  ()  ()  ()  ()  ()  ()  ()
Serine  ()  ()  ()  ()
Lysine  ()  ()
Glucose  ()  ()  ()  ()  ()  ()  ()  ()  ()  ()
* The growth of Leptomitus lacteus was measured after incubation for  days on a mixture of amino acids (asparagine, arginine, phenylalanine, glycine, serine,
threonine, isoleucine and valine). The dry weight was  mg. No growth was reported after  days with each of these amino acids as single sources of carbon.
230 10 Infection strategies of pathogenic oomycetes in fish
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10.10Sexual reproduction, seeing through the bad times 231
that adapt to seasonal variations in the presence of potential hosts (Sarowar et al.
2013). Whether the generalist nature of the latter species correlates with wider distri-
bution in the environment needs to be determined.
10.10 Sexual reproduction, seeing through the bad times
Sexual reproduction in oomycetes is oogamous and fertilization is achieved by the
direct transfer of a non-motile gametic nucleus from an accompanying antheridium
into the egg in the oogonium (Sparrow 1960, 1976; Karling 1981; Dick 2001). Ooga-
mous sexual reproduction is clearly one of the major evolutionary developments that
define more advanced oomycetes (Beakes et al. 2012). The sexual phase is thought
to enable survival under harsh conditions such as dryness and extreme temperature
until conditions become more favorable for germination. Sexual reproduction in Sap-
rolegnia begins with the formation of an oogonium and an antheridium. Fertiliza-
tion occurs when the antheridium grows towards and its nucleus fuses with the egg
nucleus in the oogonium. Oospores are released as enclosed eggs characterized by
a thick wall that enables survival under harsh conditions and germinate when con-
ditions improve (Beakes and Bartinicki-Garcia 1989; Diéguez-Uribeondo et al. 1996).
Sexually produced oospores differ in number, size and shape from species to species
allowing this feature to be one of the key tools for taxonomic speciation (Coker 1923;
Beakes et al. 2012). Under laboratory conditions, sexual reproduction of several Sap-
rolegnia species can be initiated by growth on nutrient-poor substrates or by reducing
the temperature. As sexual reproduction appears to be difficult to initiate in vivo, and
is sometimes impossible in vitro, it is not always possible to identify species based on
morphological characteristics of their sexual structures, especially the fish pathoge-
nic isolates (Beakes et al. 1994; Diéguez-Uribeondo et al. 2007, 2009). This severely
hampers the identification of Saprolegnia species, especially the isolates from salmo-
nid lesions. Sexual reproductive characters are fundamental to the identification of
the Peronosporomycetes (Dick 2001), but such structures have not been observed in
cultures of A. invadans.
10.11 Molecular adaptation and strategy in setting infection:
microscopic infection
In order to successfully establish an infection, oomycetes secrete effector proteins.
Effector molecules act as “ligands” that can regulate enzyme activity, gene expres-
sion, or cell signaling. Once inside or within the host cell surroundings the effector
proteins can alter the host defences by modulating host immune responses or by
inhibiting host cell functions, such as phagocytosis, proinflammatory responses,
apoptosis, and intracellular trafficking allowing the infection to progress (Dou 2008;
Wawra et al. 2012a, b). Depending on where the effectors fulfil their function, they are
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232 10 Infection strategies of pathogenic oomycetes in fish
classified as extracellular or apoplastic effectors when it acts on the host cell sur-
roundings, or intracellular effectors if they are able to translocate into host cells
where they can interfere with host defence mechanism (enzymatic machinery and/or
immune system) (Stassen and Van den Ackerveken 2011; Wawra et al. 2012a). Usually
effector molecules that mediate invasion of host cells are also secreted from mycelia.
Like plant cells, oomycetes secrete a number of hydrolytic proteins into the extracel-
lular space. For example, glycosyl hydrolases have been found in S. parasitica (Torto-
Alalibo et al. 2005), P. infestans (McLeod et al. 2003) and A. euteiches (Gaulin et al.
2008) and will aid in breaking down cell wall components and thus allow entry into
host tissues.
With the publication of the first complete genome sequence of S. parasitica,
several genes encoding serine proteases, metalloproteases and cysteine proteases,
were found to be largely extended in numbers (76, 69 and 85 genes, respectively)
when compared to other oomycete sequences, like P. sojae or P. infestans (Jiang
et al. 2013). Interestingly, an extended group of cysteine proteases (48 out of 85
genes) was found of which the papain-like peptidase C1 proteases were most pro-
minent. Cysteine proteins are mostly found in fruits. They seem to play several roles
in plant physiology and development. In addition, they are involved in signalling
pathways and in the response to biotic and abiotic stresses. In humans cysteine pro-
teases are responsible for MHC class II immune responses, apoptosis, hormone pro-
cessing, and extracellular matrix remodeling being important for bone development
(Grudkowska and Zagdanska 2004). In humans and livestock, fruits like papaya or pine-
apple that are rich in cysteine proteases are used against intestinal worm infections in
homeopathic medicine (Behnke et al. 2008). In the particular case of papain-like
peptidase C1 proteases it is known that it plays a role in defence against herbivorous
insects (van der Hoorn and Jones 2004). Nevertheless nothing is known about the
functions of cysteine proteases in S. parasitica. Notably, S. parasitica possesses one of
the largest repertoires of proteases (270 in total) among eukaryotes that are deployed
in waves at different points during infection as determined from RNA-Seq data (Jiang
et al. 2013).
A signal that might direct some secreted proteins for uptake by the host cell has
been found in plant pathogenic oomycetes. A short, conserved amino acid sequence
signature, named “RxLR” (arginine – any amino acid – leucine – arginine sequence),
occurs within 30 residues of the N-terminal signal peptide and is common to the
known oomycete avirulence proteins as well as to other secreted proteins of unknown
function (Ellis and Dodds 2011; Wawra et al. 2012b). In 2010, a host target protein,
SpHtp1, a putative effector from S. parasitica, was found to be delivered in vitro by
the pathogen into cells of a rainbow trout cell line (RTG-2). Although SpHtp1 con-
tains a RHLR sequence within the characteristic position for an RxLR motif, it is not
considered as a true RxLR effector due to the lack of a W-Y-sequence element and the
gene is not under diversifying selection as is usually the case for Phytophthora RxLR
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10.12Host responses to oomycete infections  233
effectors (Wawra et al. 2012a, b). SpHtp1 is translocated specifically into fish cells
independently of the pathogen.
Since 2005, more genome sequences of several oomycete pathogens have become
available. Computational analysis showed an enrichment of the RxLR-motif within
the secreted proteins of species only from the order Peronosporales with up to several
hundred putative RxLR-effectors (Tyler et al. 2006; Whisson et al. 2007; Haas et al.
2009; Baxter et al. 2010) whereas other oomycetes, e.g. S. parasitica show no enrich-
ment or could have different conserved motifs (Lévesque et al. 2010; Schornack et al.
2010; Kemen et al. 2011; Links et al. 2011; Jiang et al. 2013). Nevertheless, several genes
encoding host-targeting proteins (including SpHtp1) were identified in the genome of
S. parasitica. Most of these candidates have no homology to known proteins, and their
functions are currently unknown (Jiang et al. 2013).
Oomycetes evolved the ability to infect plants and animals independently of other
eukaryotic microbes, due to the development of unique mechanisms of pathogeni-
city. With the availability of a respectable molecular toolbox and a multitude of gene
sequences, significant progress has been made in understanding the molecular basis
of infection by oomycetes. However, most of our knowledge remains limited to eco-
nomically important species in the Phytophthora genus, and little is known about
infection by other pathogenic oomycetes (Kamoun 2003).
10.12 Host responses to oomycete infections
There is a lack of knowledge about animal responses to the presence of oomycete
pathogens. In the case of A. astaci, germination frequently takes place on the joints
or between the abdominal segments of the crayfish exoskeleton. The infection can be
seen as black spots due to heavy melanisation. This phenomenon occurs around the
hyphae of A. astaci within the cuticle of the crayfish cells. The melanization process
occurs due to the active form of the enzyme prophenoloxidase (proPO). Cerenius
et al. (2003) revealed that crayfish species that are resistant to A. astaci produce proPO
transcripts constitutively, whereas in susceptible crayfish the expression of this gene
is significantly lower. The authors suggest an adaptation of the resistant (North
American) crayfish to the presence of A. astaci by producing proPo continually,
enhancing their defence mechanism against A. astaci. A similar mechanism can be
found in insect larvae exposed to the pathogen Lagenidium giganteum (Golkar et al.
1993). In this case, the larvae of Anopheles gambiae induce an intense melanization
which encapsulates the germ tubes of the pathogen.
It has been assumed, but never proven, that small wounds on the epidermal layer
of the fish are possibly the point of entry for Saprolegnia. This would thus suggest that
the intact epidermis plays a key role in fish protection against infection by Saprolegnia.
The mucosal layer present on the epidermis acts as a protective shield and contains
several enzymes, such as proteases, that seem to help its protective role (Pickering
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234 10 Infection strategies of pathogenic oomycetes in fish
1994). Furthermore, Chivers et al. (2007) demonstrated that the epidermis of challenged
fish with cysts of either S. ferax or S. parasitica had an enhanced number of club cells
when compared to the epidermis of uninfected (control) fish. Club cells have an inte-
resting role in protection against pathogens. These cells are present in the surface
layers of the epidermis and when attacked by a pathogen it releases chemical alarm
cues. Interestingly, fish that had been immune-suppressed after exposure to the heavy-
metal cadmium (Cd) were not able to increase production of club cells in response
to Saprolegnia challenges (Chivers et al. 2007). This is very important once immune-
compromised fish have a higher incidence of saprolegniosis (Bly et al. 1994). The infec-
tion outcome can vary greatly not only due to factors that predispose fish to disease,
but also to difference in virulence of individual strains of Saprolegnia (Stueland et al.
2005). Saprolegniosis can progress rapidly and often inflammatory responses in the
fish do not occur (López-Dóriga and Martínez 1998).
10.13 The animal trade is responsible for the spread of pathogens
into novel and wild ecosystems
The oomycetes impact a wide array of taxa including; fish, insects, zooplankton,
nematodes, crayfish and amphibians (Phillips et al. 2008; Beakes et al. 2012). However,
they are most commonly known for their detrimental impact on aquatic taxa (finfish,
crustaceans and molluscs) (Arthur and Bondad-Reantaso 2002), as several outbreaks
involving the oomycetes have caused substantial economic damage and mass mortali-
ties in both novel ecosystems and in the wild (van West 2006). Saprolegnia parasitica
has caused global losses worth tens of millions of dollars in salmon and catfish farms
impacting Scotland, Scandinavia, Chile, Japan, Canada and the USA (van West 2006).
Additionally, S. parasitica could possibly partly explain the global declines in wild sal-
monids, having been reported to cause 22% mortality in the USA (Neitzel et al. 2004).
One major contributor that can be directly linked to oomycete outbreaks is the
global transport of aquatic taxa (Arthur and Bondad-Reantaso 2002). Live aquatic
taxa are transported for a number of reasons; 1) intentional introduction or transfer
into aquaculture facilities or natural waters, 2) intentional introduction of orna-
mental fish, and 3) unintentional introduction via ballast water of ships. In addi-
tion to the transport and introduction of non-native aquatic taxa, oomycetes can
be transported and introduced along with resistant non-native hosts, accidentally
introducing diseases into new local ecosystems (Phillips et al. 2008). While there
are many benefits to the global trade of aquatic taxa including the increase in aqua-
culture production, recreational fishing and biological control (Stewart 1991; Arthur
and Bondad-Reantaso 2002; Fisher and Garner 2007), oomcyetes can be highly
lethal to native hosts and can cause 100% mortality (Fisher et al. 2012). A. astaci,
commonly referred to as the crayfish plague, is responsible for eradicating cray-
fish Astacus astacus, A. leptodactylus and Austropotamobius pallipes populations
in the UK. Native species in the UK were exposed to this Oomycetes when resistant
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10.14Future perspectives 235
North American species Procambarus clarkii, Orconectes limosus and Pacifastacus
leniusculus were introduced (Phillips 2008). Aphanomyces invadans which causes
epizootic ulcerative syndrome (EUS) also commonly known as red spot in over a
hundred freshwater and estuarine fish species worldwide has been isolated in Asia,
Australia and the USA (Callinan et al. 1995; Lilley and Roberts 1997; Chinabut 1998;
Boys et al. 2012; Huchzermeyer and van der Waal 2012). In 2006, the pathogen was
also found in the Zambezi River System (ZRS) in Africa and by 2011 had spread
further along the ZRS, invading several new ecosystems (Huchzermeyer and van der
Waal 2012; Nsonga et al. 2013). The low host specificity of Aphanomcyes spp. incre-
ases its prevalence amongst a range of species, increasing the chance of disease
outbreaks (Huchzermeyer and van der Waal, 2012). This raises concerns about the
potential release of Aphanomyces spp. into new habitats as a result of global trade
of aquatic animals. Especially since global trade has increased from 25% to 38%
(57 million tonnes) in 1976 and 2010, respectively (The State of Food and Agriculture
1998; The 2012 State of World Fisheries and Aquaculture). This reflects how significant
the impact of the transport and introduction of oomycete infected individuals could be
for novel and wild ecosystems at both the local and the pan-continental scale.
10.14 Future perspectives
Oomycetes that are pathogens of animals are currently receiving substantially more
attention than in the past, which is primarily due to their roles in causing serious
economic damage in aquaculture. However, besides causing problems in fish farms,
these water moulds are also causing major problems in the environment. Remarkably
more than 150 years after their discovery, we still know very little about the infection
process both microscopically and at the molecular level. In fact we even don’t have
an answer to one of the most basic questions: “do oomycetes only infect wounded or
immune compromised animals, or can they also infect healthy animals?”
Recent developments regarding the basic molecular processes of Saprolegnia
species pathogenic to fish, the nature of the interactions with their hosts, and the
identification of genes and proteins involved in these processes and host immune
responses have increased our understanding of some aspects of the infection process
of the pathogen. The high level of genome plasticity and ever expanding reservoirs of
effectors proteins disclosed from the genome sequence of S. parasitica, will hopefully
help us to locate the most essential targets for genetic resistance breeding or chemical
controls. The unique metabolic features of oomycetes and common effector transloca-
tion pathways as reviewed in this chapter may offer key treatment targets.
Efforts to elucidate the molecular mechanisms by which Saprolegnia infect its
host and studying the host’s immune responses, have mainly focused on S. parasi-
tica infections on salmon and trout. New genomic approaches to gain insight into
the transcriptome of S. parasitica and the functional characterization of the virulent
genes will undoubtedly aid in our understanding of how the pathogen is able to cause
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236 10 Infection strategies of pathogenic oomycetes in fish
disease and henceforth leading to a novel control strategies to increase fish health,
reduce losses due to mycotic disease and increase production.
Acknowledgments
The authors would like to acknowledge the Biotechnology and Biological Scien-
ces Research Council (BBSRC, BB/G012075/1, PvW); Natural Environment Research
Council (NERC, NE/F012578/1, PvW); the University of Aberdeen (PvW); The Total
Foundation (PvW); and Commonwealth Scholarship Commission (BDCS-2010-42,
MNS), a Marie Curie Initial Training Networks with the SAPRO (Sustainable approa-
ches to reduce Oomycete (Saprolegnia) infections in aquacultures) grant PITN-GA-
2009-238550 (MS and PvW) for supporting our works. The authors thank Elayna
Truszewski, Department of Biological Sciences, Macquarie University for her editorial
assistance with preparation of the original manuscript.
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