List of Contributors xix
About the Editors xxiii
About the Book xxvii
Preface xxix
Acknowledgments xxxi
1 Introduction 1
Rainer Kurmayer, Kaarina Sivonen, and Nico Salmaso
1.1 A Brief Historical Overview 1
1.2 The Genetic Basis of Toxin Production 2
1.2.1 Microcystin and Nodularin 2
1.2.2 Cylindrospermopsin 5
1.2.3 Saxitoxin 6
1.2.4 Anatoxin 8
1.3 Application of Molecular Tools 8
1.4 Laboratory Safety Issues 13
1.5 References 14
2 Sampling and Metadata 19
Rainer Kurmayer, Guntram Christiansen, Konstantinos Kormas, Wim Vyverman, Elie Verleyen, Vitor Ramos, Vitor Vasconcelos, and Nico Salmaso
2.1 Introduction 19
2.2 Handling of Samples 20
2.3 Sample Contamination 21
2.4 Sampling 21
2.4.1 Quantitative Depth-Integrated and Discrete Sampling 21
2.4.2 Qualitative Plankton Net Sampling 22
2.4.3 Surface (Scum Material) Sampling 22
2.4.4 Benthic (Terrestrial) Cyanobacteria Sampling 22
2.4.5 Food Supplement Sampling 22
2.4.6 Isolation of Single Colonies/Filaments 22
2.5 Subsampling Food Supplement Samples 23
2.6 Sampling of Nucleic Acids 23
2.7 General Conclusions 24
2.8 References 24
SOP 2.1 Sampling and Filtration (DNA) 26
Rainer Kurmayer and Konstantinos Kormas
SOP 2.1.1 Introduction 26
SOP 2.1.2 Experimental 26
SOP 2.1.3 Procedure 27
SOP 2.1.4 Notes 28
SOP 2.1.5 References 29
SOP 2.2 Sampling of Benthic Cyanobacteria 29
Wim Vyverman and Elie Verleyen
SOP 2.2.1 Introduction 29
SOP 2.2.2 Experimental 30
SOP 2.2.3 Procedure 30
SOP 2.2.4 Notes 31
SOP 2.2.5 References 31
SOP 2.3 Isolation of Single Cyanobacteria Colonies/Filaments 32
Rainer Kurmayer
SOP 2.3.1 Introduction 32
SOP 2.3.2 Experimental 32
SOP 2.3.3 Procedure 33
SOP 2.3.4 Notes 33
SOP 2.3.5 References 33
SOP 2.4 Sampling Food Supplements 34
Vitor Ramos, Cristiana Moreira, and Vitor Vasconcelos
SOP 2.4.1 Introduction 34
SOP 2.4.2 Experimental 35
SOP 2.4.3 Procedure (Fig. 8.3) 35
SOP 2.4.4 Notes 36
SOP 2.4.5 References 36
SOP 2.5 Sampling and Filtration (RNA) 37
Rainer Kurmayer and Guntram Christiansen
SOP 2.5.1 Introduction 37
SOP 2.5.2 Experimental 37
SOP 2.5.3 Procedure 38
SOP 2.5.4 Notes 38
SOP 2.5.5 References 38
SOP 2.6 Sampling of Abiotic and Biotic Data and Recording Metadata 39
Elie Verleyen, Maxime Sweetlove, Dagmar Obbels, and Wim Vyverman
SOP 2.6.1 Introduction 39
SOP 2.6.2 Experimental 39
SOP 2.6.3 Type of Metadata and Additional Biotic and Abiotic Data 40
SOP 2.6.4 Notes 41
SOP 2.6.5 References 42
3 Isolation, Purification, and Cultivation of Toxigenic Cyanobacteria 43
Sigrid Haande, Iwona Jasser, Muriel Gugger, Camilla H.C. Hagman, Annick Wilmotte, and Andreas Ballot
3.1 Introduction 43
3.2 Methodical Principles for Cyanobacterial Isolation, Purification, and Cultivation 44
3.2.1 Sampling, Identification, and Treatments Prior to the Isolation of Cyanobacteria 44
3.2.2 Traditional Techniques for the Isolation and Purification of Cyanobacteria 45
3.2.3 Culture Media Preparation 47
3.2.4 Cultivation Conditions 48
3.3 General Conclusions 49
3.4 References 49
SOP 3.1 Isolation, Purification, and Clonal Isolate Testing 51
Sigrid Haande, Camilla H.C. Hagman, and Andreas Ballot
SOP 3.1.1 Introduction 51
SOP 3.1.2 Experimental 51
SOP 3.1.3 Procedure 52
SOP 3.1.4 Notes 54
SOP 3.1.5 References 54
SOP 3.2 Isolation of Picocyanobacterial Cells by Flow Cytometer (FCM) Sorting 55
Ewa Koz³owska and Iwona Jasser
SOP 3.2.1 Introduction 55
SOP 3.2.2 Experimental 56
SOP 3.2.3 Procedure 56
SOP 3.2.4 Notes 58
SOP 3.2.5 References 59
SOP 3.3 Axenization 60
Muriel Gugger
SOP 3.3.1 Introduction 60
SOP 3.3.2 Experimental 60
SOP 3.3.3 Procedure 61
SOP 3.3.4 Notes 63
SOP 3.3.5 References 63
SOP 3.4 Culture Media (Solid and Liquid) 64
Sigrid Haande, Camilla H.C. Hagman, and Andreas Ballot
SOP 3.4.1 Introduction 64
SOP 3.4.2 Experimental 64
SOP 3.4.3 Procedure 65
SOP 3.4.4 Notes 68
SOP 3.4.5 References 68
SOP 3.5 Strain Maintenance (Living Cultures) 69
Sigrid Haande, Camilla H.C. Hagman, and Andreas Ballot
SOP 3.5.1 Introduction 69
SOP 3.5.2 Experimental 69
SOP 3.5.3 Procedure 70
SOP 3.5.4 Notes 72
SOP 3.5.5 References 73
SOP 3.6 Cryopreservation and Recovery 73
Muriel Gugger
SOP 3.6.1 Introduction 73
SOP 3.6.2 Experimental 74
SOP 3.6.3 Procedure 75
SOP 3.6.4 Notes 78
SOP 3.6.5 References 78
4 Taxonomic Identification of Cyanobacteria by a Polyphasic Approach 79
Annick Wilmotte, H. Dail Laughinghouse IV, Camilla Capelli, Rosmarie Rippka, and Nico Salmaso
4.1 Introduction 79
4.2 Nomenclature and Classification of Cyanobacteria 82
4.3 Microscopy 84
4.3.1 Light Microscopy 84
4.3.2 Autofluorescence Microscopy 86
4.4 Molecular Markers: Single Loci 87
4.5 Molecular Markers: Multiple Loci 94
4.5.1 Multilocus Sequence Typing (MLST) and Multilocus Sequence Analysis (MLSA) 94
4.5.2 Genome-Based Extension of MLST and MLSA 96
4.6 Molecular Typing Methods Based on Gel Electrophoresis 96
4.7 Denaturing Gradient Gel Electrophoresis (DGGE) 97
4.8 Taxonomic and Molecular Databases 97
4.9 The Polyphasic Approach 98
4.10 Final Considerations 105
4.11 References 106
SOP 4.1 Taxonomic Identification by Light Microscopy 120
Nico Salmaso, Rosmarie Rippka, and Annick Wilmotte
SOP 4.1.1 Introduction 120
SOP 4.1.2 Experimental 121
SOP 4.1.3 References 124
SOP 4.2 Polyphasic Approach on Cyanobacterial Strains 125
Nico Salmaso, Camilla Capelli, Rosmarie Rippka, and Annick Wilmotte
SOP 4.2.1 Introduction 125
SOP 4.2.2 Experimental 126
SOP 4.2.3 References 131
5 Nucleic Acid Extraction 135
Elke Dittmann, Anne Rantala-Ylinen, Vitor Ramos, Vitor Vasconcelos, Guntram Christiansen, and Rainer Kurmayer
5.1 Introduction 135
5.2 Specific Extraction Procedures and Storage 137
5.2.1 DNA Extraction from Laboratory Strains 137
5.2.2 DNA Extraction from Field Samples 137
5.2.3 DNA Extraction from Food Supplements 137
5.2.4 RNA Extraction from Laboratory Strains 138
5.2.5 RNA Extraction from Field Samples 138
5.2.6 Single Colony and Filament Analysis 138
5.2.7 Whole Genome Amplification 139
5.2.8 Nucleic Acid Storage 139
5.3 References 139
SOP 5.1 Standard DNA Isolation Technique for Cyanobacteria 140
Elke Dittmann
SOP 5.1.1 Introduction 140
SOP 5.1.2 Experimental 140
SOP 5.1.3 Procedure 141
SOP 5.1.4 Notes 141
SOP 5.1.5 References 142
SOP 5.2 DNA Isolation Protocol for Cyanobacteria with Extensive Mucilage 143
Guntram Christiansen, Elisabeth Entfellner, and Rainer Kurmayer
SOP 5.2.1 Introduction 143
SOP 5.2.2 Experimental 143
SOP 5.2.3 Procedure 144
SOP 5.2.4 Notes 145
SOP 5.2.5 References 145
SOP 5.3 Quantitative DNA Isolation from Filters 145
Rainer Kurmayer
SOP 5.3.1 Introduction 146
SOP 5.3.2 Experimental 146
SOP 5.3.3 Procedure 147
SOP 5.3.4 Notes 148
SOP 5.3.5 References 148
SOP 5.4 Genomic DNA Extraction from Single Filaments/Colonies for Multiple PCR Analyses 149
Guntram Christiansen, Chen Qin, and Rainer Kurmayer
SOP 5.4.1 Introduction 149
SOP 5.4.2 Experimental 149
SOP 5.4.3 Procedure 150
SOP 5.4.4 Notes 151
SOP 5.4.5 References 151
SOP 5.5 Whole Genome Amplification Using Bacteriophage Phi29 DNA Polymerase 151
Guntram Christiansen and Rainer Kurmayer
SOP 5.5.1 Introduction 151
SOP 5.5.2 Experimental 152
SOP 5.5.3 Procedure 152
SOP 5.5.4 Notes 152
SOP 5.5.5 Reference 153
SOP 5.6 DNA Extraction from Food Supplements 153
Vitor Ramos, Cristiana Moreira, and Vitor Vasconcelos
SOP 5.6.1 Introduction 153
SOP 5.6.2 Experimental 153
SOP 5.6.3 Procedure 154
SOP 5.6.4 Notes 155
SOP 5.6.5 References 156
SOP 5.7 RNA Extraction from Cyanobacteria 156
Guntram Christiansen and Rainer Kurmayer
SOP 5.7.1 Introduction 156
SOP 5.7.2 Experimental 156
SOP 5.7.3 Procedure 158
SOP 5.7.4 Notes 158
SOP 5.7.5 References 159
SOP 5.8 cDNA Synthesis 159
Guntram Christiansen and Rainer Kurmayer
SOP 5.8.1 Introduction 159
SOP 5.8.2 Experimental 159
SOP 5.8.3 Procedure 160
SOP 5.8.4 Notes 161
SOP 5.8.5 References 161
6 Conventional PCR 163
Elke Dittmann, Anne Rantala-Ylinen, Kaarina Sivonen, Ilona Ga²ga³a, Joanna Mankiewicz-Boczek, Samuel Cirés, Andreas Ballot, Guntram Christiansen, Rainer Kurmayer, Vitor Ramos, Vitor Vasconcelos, and Martin Saker
6.1 Introduction 163
6.2 Principle of PCR and Available Enzymes 164
6.2.1 Primer Development 165
6.2.2 Setup of PCR Conditions for DNA and Single Colony Analysis 168
6.2.3 Gel Electrophoresis and Documentation 168
6.2.4 Troubleshooting of PCR Results 168
6.2.5 PCR Product Downstream Processing (RFLP, Cloning, Sequencing) 169
6.3 Special Notes 170
6.4 References 170
SOP 6.1 PCR Detection of Microcystin Biosynthesis Genes Combined with RFLP Differentiation of the Producing Genus 172
Elke Dittmann
SOP 6.1.1 Introduction 172
SOP 6.1.2 Experimental 172
SOP 6.1.3 Procedure 173
SOP 6.1.4 Notes 174
SOP 6.1.5 Reference 174
SOP 6.2 PCR Detection of Microcystin and Nodularin Biosynthesis Genes in the Cyanobacterial Orders Oscillatoriales, Chroococcales, Stigonematales, and Nostocales 175
Elke Dittmann, Joanna Mankiewicz-Boczek, and Ilona Ga²ga³a
SOP 6.2.1 Introduction 175
SOP 6.2.2 Experimental 175
SOP 6.2.3 Procedure 177
SOP 6.2.4 Notes 177
SOP 6.2.5 References 178
SOP 6.3 Genus-Specific PCR Detection of Microcystin Biosynthesis Genes in Anabaena/Nodularia and Microcystis and Planktothrix, Respectively 179
Anne Rantala-Ylinen and Kaarina Sivonen
SOP 6.3.1 Introduction 179
SOP 6.3.2 Experimental 179
SOP 6.3.3 Procedure 181
SOP 6.3.4 Notes 181
SOP 6.3.5 References 181
SOP 6.4 PCR Detection of Anatoxin Biosynthesis Genes Combined with RFLP Differentiation of the Producing Genus 182
Anne Rantala-Ylinen and Kaarina Sivonen
SOP 6.4.1 Introduction 182
SOP 6.4.2 Experimental 182
SOP 6.4.3 Procedure 183
SOP 6.4.4 Notes 184
SOP 6.4.5 Reference 184
SOP 6.5 PCR Detection of the Saxitoxin Biosynthesis Genes, sxtA, sxtX, sxtH, sxtG, and sxtI 185
Andreas Ballot and Samuel Cirés
SOP 6.5.1 Introduction 185
SOP 6.5.2 Experimental 187
SOP 6.5.3 Procedure 187
SOP 6.5.4 Notes 188
SOP 6.5.5 References 189
SOP 6.6 PCR Detection of the Cylindrospermopsin Biosynthesis Gene cyrJ 189
Samuel Cirés and Andreas Ballot
SOP 6.6.1 Introduction 189
SOP 6.6.2 Experimental 190
SOP 6.6.3 Procedure 191
SOP 6.6.4 Notes 191
SOP 6.6.5 References 192
SOP 6.7 PCR from Single Filament of Toxigenic Planktothrix 193
Qin Chen, Guntram Christiansen, and Rainer Kurmayer
SOP 6.7.1 Introduction 193
SOP 6.7.2 Experimental 193
SOP 6.7.3 Procedure 194
SOP 6.7.4 Notes 195
SOP 6.7.5 References 195
SOP 6.8 Analysis of Microcystin Biosynthesis Gene Subpopulation Variability in Planktothrix 196
Rainer Kurmayer
SOP 6.8.1 Introduction 196
SOP 6.8.2 Experimental 196
SOP 6.8.3 Procedure 197
SOP 6.8.4 Notes 197
SOP 6.8.5 References 198
SOP 6.9 PCR Detection of Microcystin Biosynthesis Genes from Food
Supplements 199
Vitor Ramos, Cristiana Moreira, and Vitor Vasconcelos
SOP 6.9.1 Introduction 199
SOP 6.9.2 Experimental 199
SOP 6.9.3 Procedure 201
SOP 6.9.4 Notes 202
SOP 6.9.5 References 203
7 Quantitative PCR 205
Anne Rantala-Ylinen, Henna Savela, Kaarina Sivonen, and Rainer Kurmayer
7.1 Introduction 205
7.2 Primer/Probe Design 206
7.3 Optimization 208
7.4 Absolute Quantification 208
7.5 Relative Quantification 209
7.6 Calibration of qPCR Results 209
7.7 General Conclusions 210
7.8 References 210
SOP 7.1 Optimization of qPCR Assays 211
Rainer Kurmayer
SOP 7.1.1 Introduction 211
SOP 7.1.2 Experimental 211
SOP 7.1.3 Procedure 212
SOP 7.1.4 Notes 213
SOP 7.1.5 References 213
SOP 7.2 Calibration of qPCR Results 214
Rainer Kurmayer
SOP 7.2.1 Introduction 214
SOP 7.2.2 Experimental 214
SOP 7.2.3 Procedure 215
SOP 7.2.4 Notes 217
SOP 7.2.5 References 217
SOP 7.3 Quantification of Potentially Microcystin/Nodularin-Producing Anabaena, Microcystis, Planktothrix, and Nodularia 218
Anne Rantala-Ylinen, Kaarina Sivonen, and Rainer Kurmayer
SOP 7.3.1 Introduction 218
SOP 7.3.2 Experimental 219
SOP 7.3.3 Procedure 219
SOP 7.3.4 Notes 221
SOP 7.3.5 References 221
SOP 7.4 Relative Quantification of Microcystis or Planktothrix mcy Genotypes Using qPCR 222
Rainer Kurmayer
SOP 7.4.1 Introduction 222
SOP 7.4.2 Experimental 222
SOP 7.4.3 Procedure 224
SOP 7.4.4 Notes 225
SOP 7.4.5 References 225
SOP 7.5 Quantification of Transcript Amounts of mcy Genes in Planktothrix 226
Guntram Christiansen and Rainer Kurmayer
SOP 7.5.1 Introduction 226
SOP 7.5.2 Experimental 226
SOP 7.5.3 Procedure 227
SOP 7.5.4 Notes 228
SOP 7.5.5 References 228
SOP 7.6 Quantification of Potentially Cylindrospermopsin-Producing Chrysosporum ovalisporum 229
Rehab El-Shehawy and Antonio Quesada
SOP 7.6.1 Introduction 229
SOP 7.6.2 Experimental 229
SOP 7.6.3 Procedure 230
SOP 7.6.4 Notes 231
SOP 7.6.5 References 231
SOP 7.7 qPCR Detection of the Paralytic Shellfish Toxin Biosynthesis Gene sxtB 231
Henna Savela
SOP 7.7.1 Introduction 231
SOP 7.7.2 Experimental 232
SOP 7.7.3 Procedure 233
SOP 7.7.4 Notes 234
SOP 7.7.5 References 234
SOP 7.8 Application of the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) Guidelines to Quantitative Analysis of Toxic Cyanobacteria 234
Henna Savela
SOP 7.8.1 Introduction 234
SOP 7.8.2 Sampling 235
SOP 7.8.3 Sample Preparation and DNA Extraction 235
SOP 7.8.4 Target Information and Oligonucleotide Design 235
SOP 7.8.5 qPCR Protocol 238
SOP 7.8.6 qPCR Validation 239
SOP 7.8.7 Data Analysis 239
SOP 7.8.8 Reference 239
8 DNA (Diagnostic) and cDNA Microarray 241
Anne Rantala-Ylinen, Kaarina Sivonen, and Annick Wilmotte
8.1 DNA (Diagnostic) Microarray 241
8.1.1 Introduction 241
8.1.2 Methodological Principles 242
8.1.3 General Conclusions 243
8.1.4 References 243
8.2 cDNA Microarray for Cyanobacteria 244
Hans C.P. Matthijs and J. Merijn Schuurmans
8.2.1 Introduction 244
8.2.2 Principles of Microarray Use 244
8.2.3 Considerations for Experimental Design 245
8.2.4 Microarray: Practical Approach 246
8.2.5 Microarray: Data Analysis 246
8.2.6 References 246
SOP 8.1 DNA-Chip Detection of Potential Microcystin and Nodularin Producing Cyanobacteria in Environmental Water Samples 248
Anne Rantala-Ylinen and Kaarina Sivonen
SOP 8.1.1 Introduction 248
SOP 8.1.2 Experimental 249
SOP 8.1.3 Procedure 250
SOP 8.1.4 Notes 253
SOP 8.1.5 References 253
SOP 8.2 cDNA Microarrays for Cyanobacteria 254
J. Merijn Schuurmans and Hans C.P. Matthijs
SOP 8.2.1 Introduction 254
SOP 8.2.2 Experimental 254
SOP 8.2.3 Procedure 256
SOP 8.2.4 Notes 259
SOP 8.2.5 Reference 261
9 Analysis of Toxigenic Cyanobacterial Communities through Denaturing Gradient Gel Electrophoresis 263
Iwona Jasser, Aleksandra Bukowska, Jean-Francois Humbert, Kaisa Haukka, and David P. Fewer
9.1 Introduction 263
9.2 Main Applications of the Method 264
9.3 Possible Applications 264
9.4 DGGE Procedure 265
9.5 General Conclusions Including Pros and Cons of the Method 267
9.6 Optimization of the Method and Troubleshooting 267
9.7 References 268
SOP 9.1 DGGE-mcyA Conditions 270
Aleksandra Bukowska and Iwona Jasser
SOP 9.1.1 Introduction 270
SOP 9.1.2 Experimental 270
SOP 9.1.3 Procedure 272
SOP 9.1.4 Notes 275
SOP 9.1.5 References 275
10 Monitoring of Toxigenic Cyanobacteria Using Next-Generation Sequencing Techniques 277
Li Deng, Maxime Sweetlove, Stephan Blank, Dagmar Obbels, Elie Verleyen, Wim Vyverman, and Rainer Kurmayer
10.1 Introduction 277
10.2 Specific Procedures 279
10.2.1 16S rRNA Gene Amplicon Library Preparation 279
10.2.2 Amplicon Purification, Quantification and Pooling 280
10.2.3 Sequencing 280
10.2.4 Bioinformatic Exploration of Sequencing Results 281
10.2.5 General Conclusions Including Pros and Cons of the Method 281
10.2.6 References 281
10.3 Bioinformatic Processing of Amplicon Sequencing Datasets 283
Maxime Sweetlove, Dagmar Obbels, Elie Verleyen, Igor S. Pessi, Annick Wilmotte, and Wim Vyverman
10.3.1 Introduction 283
10.3.2 Sequencing Platforms 283
10.3.3 Data Formats 284
10.3.4 Error Associated with NGS Data 285
10.3.5 OTU Delineation: Choosing a Similarity Threshold 286
10.3.6 Conclusions 286
10.4 References 286
SOP 10.1 Standard Technique to Generating 16S rRNA PCR Amplicons for NGS 288
Li Deng, Stephan Blank, Guntram Christiansen, and Rainer Kurmayer
SOP 10.1.1 Introduction 288
SOP 10.1.2 Experimental 288
SOP 10.1.3 Procedure 289
SOP 10.1.4 Notes 290
SOP 10.1.5 References 290
SOP 10.2 Bioinformatics Analysis for NGS Amplicon Sequencing 291
Maxime Sweetlove, Dagmar Obbels, Elie Verleyen, Igor S. Pessi, Annick Wilmotte, and Wim Vyverman
SOP 10.2.1 Introduction 291
SOP 10.2.2 Experimental 291
SOP 10.2.3 Practical Tips and Alternatives for Quality Filtering 298
SOP 10.2.4 References 298
11 Application of Molecular Tools in Monitoring Cyanobacteria and Their Potential Toxin Production 301
Vitor Ramos, Cristiana Moreira, Joanna Mankiewicz-Boczek, and Vitor Vasconcelos
11.1 Introduction 301
11.2 Possible Applications 303
11.3 Checklist of Publications, Applications and Lessons from Practice 315
11.3.1 Molecular-Based Studies on (Toxic) Cyanobacteria: Overview of Methods Being Used, and Generic Findings and Concerns 315
11.3.2 The Need for Complementary Approaches 316
11.3.3 Interpreting Results 316
11.3.4 Choice of Molecular Tools for Toxigenicity Assessment 317
11.3.5 Common and Possible Applications of Molecular Tools 318
11.4 General Conclusions 321
11.5 Acknowledgments 324
11.6 References 324
Appendix: Supplementary Tables 335
Cyanobacterial Species Cited in the Book 376
Glossary 379
Index 393
