Monitoring Chrysaora hysoscella (Cnidaria, Scyphozoa) in the Belgian part of the North Sea using eDNA Number of words: 34 752 Jamael Abato Stamnummer: 01501018 Promotor: Prof. dr. Colin Janssen Copromotor: Dr. Jana Asselman Master’s Dissertation submitted to Ghent University in partial fulfilment of the requirements for the degree of Master of Science in Environmental Sanitation Academiejaar: 2016 - 2017 Copyright The author, the promoters and the thesis tutor give the permission to use this dissertation for consultation and to copy parts of it for personal use. Every other use is subject to the copyright laws; mention explicitly the source when using results from this dissertation. Gent, June 2017 i Acknowledgements I would like to express my gratitude to Vlaamse Interuniversitaire Raad University Development Cooperation (VLIR-UOS) for supporting my masters study here in Ghent University. Indeed, this opportunity has exposed me to a lot of learnings, technologies and has given me the chance to explore Ghent and Europe which made me appreciate and understand European culture and way of life. My sincere appreciation to Sylvie Bauwens for her kind support from the start of my application in my masters programme and for VLIR-UOS scholarship. Special thanks to Prof. Dr. Colin Janssen for accepting me to work in his laboratory for my thesis and for his constructive criticisms which improved the paper. I would also like to express my sincere thanks to Dr. Jana Asselman and Tara Grosemans for being kind and for imparting their knowledge about this research topic. Thank you for guiding and helping me during my laboratory works and for the efforts in editing my manuscript to improve the whole paper. Lastly, thanks to all the staff of GhenTox Lab for attending my needs during my laboratory works. I would also like to give thanks to my friends and colleagues who in their own ways have motivated me to continue working on this study and who had extended their help. Lastly, I thank God for His blessings and for his guidance throughout the duration of my stay and study here in Ghent. ii Table of Contents Copyright .............................................................................................................................................. i Acknowledgements .............................................................................................................................. ii List of Figures ...................................................................................................................................... vi List of Tables ....................................................................................................................................... vii Abstract ............................................................................................................................................. viii Chapter 1: General Introduction ......................................................................................................... 1 1.1. Background of the study ..................................................................................................... 1 1.2. Statement of the problem .................................................................................................. 3 1.3. Significance of the study ..................................................................................................... 4 Chapter 2: Literature Review .............................................................................................................. 5 2.1. Jellyfish diversity ...................................................................................................................... 5 2.2. Study species: Chrysaora hysoscella (Linnaeus, 1767) ............................................................ 5 2.3. Ecological and societal roles of jellyfish ................................................................................... 7 2.3.1. Regulating services ............................................................................................................ 8 2.3.2. Provisioning services ......................................................................................................... 9 2.3.3. Supporting services ......................................................................................................... 10 2.3.4. Cultural services .............................................................................................................. 14 2.4. Anthropogenic causes of jellyfish bloom ............................................................................... 14 2.4.1. Climate change ................................................................................................................ 16 2.4.2. Cultural eutrophication ................................................................................................... 17 2.4.3. Fishing ............................................................................................................................. 19 2.4.4. Aquaculture ..................................................................................................................... 20 2.4.5. Construction .................................................................................................................... 20 2.4.6. Alien invasion .................................................................................................................. 21 2.5. Consequences of jellyfish bloom ........................................................................................... 24 2.5.1. Net-based fisheries ......................................................................................................... 24 2.5.2. Aquaculture ..................................................................................................................... 25 2.5.3. Tourism and human health ............................................................................................. 26 2.6. Environmental DNA (eDNA): a new research tool ................................................................. 27 Chapter 3: Methodology ................................................................................................................... 31 3.1. Decontamination of the materials ......................................................................................... 31 iii 3.2. Water sampling for Environmental DNA (eDNA) ................................................................... 31 3.3. eDNA extraction ..................................................................................................................... 33 3.4. Measuring the purity of extracted eDNA: NanoDrop ............................................................ 35 3.5. Testing primer specificity: PCR ............................................................................................... 35 3.6. Verification of primer specificity: gel-electrophoresis ........................................................... 36 3.7. Detection and quantification of C. hysoscella eDNA from the samples: qPCR ...................... 38 Chapter 4: Results ............................................................................................................................. 40 4.1. eDNA extraction ..................................................................................................................... 40 4.2. C. hysoscella primer specificity .............................................................................................. 40 4.3. qPCR analysis of eDNA samples ............................................................................................. 42 4.3.1. Presence or absence of C. hysoscella eDNA ................................................................... 42 4.3.2. Tracing the possible cause of false positive results ........................................................ 43 4.3.3. Cq means of the eDNA samples ...................................................................................... 45 4.3.4. Spatial and temporal variation in the eDNA distribution ............................................... 47 Chapter 5: Discussion ........................................................................................................................ 51 5.1. C. hysoscella (scyphozoa) and its eDNA in the Belgian coast ................................................ 51 5.2. eDNA approach as a tool in monitoring C. hysoscella in the BPNS ........................................ 60 Chapter 6: Conclusions and recommendations ................................................................................ 65 References......................................................................................................................................... 66 Appendices ........................................................................................................................................ 87 Appendix 1. The nine study locations established in the Belgian part of the North Sea (BPNS) with their corresponding coordinates and depths. ...................................................................... 87 Appendix 2. Protocol on CTAB-based extraction of high-molecular weight DNA from eDNA filters. (Responsible: Jana Asselman) ............................................................................................ 88 Appendix 3. Protocol on primer and probe design for qPCR. (Responsible: Tara Grosemans) .... 93 Appendix 4. Protocol on PCR for eDNA. (Responsible: Tara