A multidisciplinary approach to tackle the problem of the zoonotic parasite Anisakis in fish Miguel Bao Domínguez BSc Marine Science – University of Vigo, Spain Doctorate itinerary of the Master in Aquaculture – University of Vigo, Spain A thesis presented for the degree of Doctor of Philosophy in Systems Biology at the University of Aberdeen April 2017 Declaration I declare that this thesis has been composed by me and has not been accepted in any previous application for a degree. All quotations have been distinguished by quotation marks and the sources of information specifically acknowledged. Chapter 2 has been published jointly as: Bao, M., Garci, M. E., Antonio, J. M., Pascual, S., 2013. First report of Anisakis simplex (Nematoda, Anisakidae) in the sea lamprey (Petromyzon marinus). Food control, 33(1), 81-86. doi: 10.1016/j.foodcont.2013.02.009. Contribution I have done all the work, with the exception of the following contributions: J. M. A. contributed to perform sea lamprey necropsies, and to the enzymatic peptic digestion of the viscera and muscle tissues. M. E. G. contributed during sea lamprey field sampling and photography. S. P. contributed to the molecular analysis which was carried out by staff member (Mariana Noelia Cueto Rivas) of the “Instituto de Investigaciones Marinas – Consejo Superior de Investigaciones Científicas” (IIM-CSIC) (Vigo, Spain), performed the statistic tests and supervised the final content of the work. Chapter 3 has been published jointly as: Bao, M., Mota, M., Nachón, D. J., Antunes, C., Cobo, F., Garci, M. E., Pierce, G. J., Pascual, S., 2015. Anisakis infection in allis shad, Alosa alosa (Linnaeus, 1758), and twaite shad, Alosa fallax (Lacépède, 1803), from Western Iberian Peninsula Rivers: zoonotic and ecological implications. Parasitology Research, 114(6), 2143-2154. doi: 10.1007/s00436-015-4403-5. Contribution I have done all the work, with the exception of the following contributions: M. M., D. J. N., C. A., F. C., and M. E. G contributed collecting the fish and parasite data. S. P. contributed to the molecular analysis which was carried out by staff member (Mariana Noelia Cueto Rivas) of the IIM-CSIC. G. P. designed the statistical analysis. All co- authors contributed to the final content of the work. Chapter 4 has been published jointly as: 2 Bao, M., Strachan, N. J. C., Hastie, L. C., MacKenzie, K., Seton, H. C., Pierce, G. J., 2017. Employing visual inspection and Magnetic Resonance Imaging to investigate Anisakis simplex s.l. infection in herring viscera. Food Control, 75, 40-47. doi: 10.1016/j.foodcont.2016.12.030. Contribution I designed the study and have done all the work, with the exception of the following contributions: H. S. developed the MRI protocol. L. H. contributed to Anisakis inspection in herring. K. M. performed the morphological identification of ascaridoid samples. H. S. contributed to the Magnetic Resonance Imaging (MRI) investigations, analysis of results and writing of the paper. N. S. and G. P. contributed to the ideas behind the study and reviewed the paper. All co-authors contributed to the final content of the work. Chapter 5 has been produced jointly and is currently under review as: Bao, M., Pierce, G. J., Strachan, N. J. C., Martínez, C., Fernández, R., Theodossiou, I. (under review). Consumers’ attitudes and willingness to pay for Anisakis-free fish. Fisheries Research. Contribution I have done all the work, with the exception of the following contributions: G. J. P., N. S. C. M., R. F. and I. T. contributed to questionnaire design and distribution. G. J. P., N. S. and I. T. helped analysing results and supervised the final content of the work. Chapter 6 has been published jointly as: Bao, M., Pierce, G. J., Pascual, S., González-Muñoz, M., Mattiucci, S., Mladineo, I., Cipriani, P., Bušelić, I., Strachan, N. J. C., 2017. Assessing the risk of an emerging zoonosis of worldwide concern: anisakiasis. Scientific reports, 7, 43699. doi: 10.1038/srep43699. Contribution I have done all the work, with the exception of the following contributions: N. S. contributed to developing the quantitative risk assessment model. I. M., I. B., S. M. and P. C. provided data of Anisakis spp. infection levels in anchovy from the Mediterranean Sea. S. P. provided data of Anisakis spp. infection levels in anchovy from North-East 3 Atlantic waters. N.S. and G.P. supervised the work. All co-authors contributed to the final content of the work. Miguel Bao Domínguez 4 Acknowledgements Non foi sen tempo!, which in my homeland (Galicia) could be translated as: about time! But, first, I would like to thank many people that helped me to successfully conclude this PhD project. The work presented in this thesis started five years ago (2012) at “Instituto de Investigaciones Marinas – Consejo Superior de Investigaciones Científicas (IIM-CSIC)”. I am sincerely thankful to my Spanish supervisor (Dr. Santiago Pascual) and to many work colleagues at IIM-CSIC who helped me, directly or indirectly, while I was studying the epidemiology of Anisakis in sea lamprey and shads of Western Iberian Peninsula rivers. Santi, I will always thank you for giving me the opportunity to face this challenge, the facilities and resources of the ECOBIOMAR Group to develop my research, and for the enlightening supervision of my work. José, Garci, Mariana and María Teresa thank you very much for your invaluable help during the field work, sampling and lab work, and for the splendid time I had with you all. Many thanks to all my PhD colleagues, Álvaro, Jorge, Marcos, María Gregori, María Llarena, Lorena, and to Helena for your help, encouragement and for the nice chats/time talking about science and common live. You are great scientists and inspiring persons. Thanks a lot to Prof. Ángel Guerra and Dr. Ángel F. González for your warm welcome to ECOBIOMAR, for your help, for the very helpful conversations/advices, and for the good times at the office and during the coffee time. I also want to extend my thanks to ALL the colleagues of the IIM-CSIC for your help any time I needed it. It was superb being part of your family during those years. I would like to acknowledge the University of Aberdeen for not only funding me, but also providing me with an excellent environment of work/education and continuous opportunities of training. I also acknowledge the EU FP7 PARASITE project for the funding, and for the opportunity to meet and learn from outstanding experts in the field of Anisakis and associated diseases. My profound gratitude to my Aberdeen-based supervisors Prof. Norval Strachan, Prof. Graham Pierce and Prof. Ioannis Theodossiou. Firstly, Graham, I am eternally thankful for the opportunity you gave me to come to Aberdeen. Norval and Graham my deepest gratitude for your time, patience, encouragement, guidance, teaching, constant support and inspiring supervision of my thesis. Ioannis thank you very much for the teaching and supervision of the economic part (chapter five) of the thesis. I sincerely thank Dr. Lee 5 Hastie for his help during fish sampling at the Oceanlab, for your help/company at the office, and for many other collaborations we carried out together and really nice moments (for instance, many tasks within the EU FP7 PARASITE project, “sea lice” and “freshwater pearl mussel” projects, chapter four, etc.). Thanks a lot to Dr. Ken MacKenzie for reviewing chapter one, for your collaboration in chapter four, for providing me the “anisakid” bibliography I wasn’t able to find anywhere else, for your collaboration/help in many other projects (“macroparasites of shads” paper, etc.), and for the really nice moments (for instance, good times at the EU FP7 PARASITE project meeting at Hamburg and in the 9th International Symposium on Fish Parasites in Valencia, etc.). I wish you all the best. I also wish to re-express my sincere gratitude to all the co-authors and collaborators of the chapters that compose this thesis, which contribution was much appreciated and has been properly detailed above. M. E. Garci, J. M. Antonio, M. Mota, D. J. Nachón, C. Antunes, F. Cobo, L. Hastie, K. MacKenzie, H. Seton, C. Martínez, R. Fernández, M. González-Muñoz, S. Mattiucci, I. Mladineo, P. Cipriani and I. Bušelić, a million thanks to you all! Thanks a lot to the lunch/badminton club friends (Eakapon, Ahmed, Douglas, Ottavia, Yeh-Fang, Ayham, Abdo and Manu) for the good times and exciting conversations we had together. You made my life in the grey Aberdeen much more enjoyable. My best wishes for you all in your future in which I hope we will meet again. Also, many thanks to my office colleagues (Niall, Ewan, Alan, Josh) for your help with English grammar and nice conversations at work. Thanks to my lifetime friends from Vigo (some still living there and other living abroad). Thank you for being there every time I come back home and for the daily funny chats we have thanks to the social media. This thesis is dedicated to my family, and so I wish to express my endless gratitude to them. Especially dedicated to Mum and Dad, thanks for your never-ending love, understanding, support and confidence in me. Thanks to my little brother for your understanding when I left home, for your support, and for being at home with Mum and Dad. Also, thanks to the rest of my family and to my family members in law for your understanding, support and permanent caring. Last, but certainly not least, no acknowledgement would be complete without giving thanks to my future wife, Belén. 6 For moving with me since the first day to Aberdeen and for being brave throughout these years, for listening to me in the hardest times, for your understanding, constant encouragement and endless trust, for your love and support, and for keeping me attached to the real world.