Ecology of Mosquito Vectors in Relation to Avian Malaria in Zoological Gardens in the United Kingdom Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor in Philosophy by Arturo Hernández Colina September 2019 This thesis is the product of my work and the assistance and advice from others is fully acknowledged. The present research was done in the Department of Epidemiology and Population Health of the Institute of Infection and Global Health at the University of Liverpool in collaboration with Chester Zoo and Flamingo Land. Arturo Hernández Colina ii Acknowledgements I am profoundly thankful for the support and advice of my supervisor, Professor Matthew Baylis, who encouraged and motivated me throughout my PhD. Without the inspiration that he provoked in me I would not be able to finish this journey with a big smile. I extend this acknowledgement to my supervisors, Dr Marcus Blagrove and Professor Rob Christley, for their contributions, advice, teachings and friendship. This project would not have been possible without the collaboration and great efforts of the zoo teams. From Chester Zoo: many thanks to Javier Lopez, Lindsay Eckley, Victoria Davis and Mark Vercoe, who took part since the initial planning of the project to the analysis of the final outputs. Likewise, to Andrew Woolham and the penguins and parrots’ team, and Wayne McLeod and the birds’ team who gave us access to the animal exhibits to operate the traps and helped to collect nets. From Flamingo Land: Ross Snip and especially Dawn Ward who committed to the endeavour of operating the traps over there. Thank you very much for welcoming us and for giving your dedication to this project. Thanks to Jenny Hesson for her mosquito mentoring and friendship, taking the time to teach me the laboratory techniques and answer my constant questions. Also, to Julian Chantrey for his endless enthusiasm and contributions to the project and for being part of my progress assessment panel providing advice along the way as well as Jan van Dijk. The technicians’ team in Leahurst deserve a big thanks for doing the invisible work that makes all other works possible: David Jones, Jenny Llewelyn, Karen Ryan, and very specially, Kenneth Sherlock, who contributed in many aspects of this research and shared his entomologic expertise with me. I am extremely thankful for the hard work and commitment from colleagues and students who supported our research and gave their precious time and effort. In particular, Emily Lomax, Freya Townsend and Amber Maddox who were brave enough to identify mosquitoes and assist the lab work for endless hours to get the analysis of blood-fed mosquitoes done. Their efforts were rewarded with First-class honours for their projects or graduation with distinction. I am very thankful for the assistance of, again, Lindsay Eckley, Javier López, Julian Chantrey, Mark Vercoe, Dawn Ward, Andrew Owen and Andrew Moss, who reviewed the questionnaire and helped improving its content and design. Javier, Julian, Mark and especially Lindsay also assisted the challenging task of contacting and inviting the participants. Thanks to all the participating institutions and their dedicated staff who spent the time answering the survey. I am also thankful for the approval and support from BIAZA. I appreciate immensely the keen assistance of Marie McIntyre and Hannah Vineer for their advice on stats models, and of Cyril Caminade for his recommendations on weather analysis and getting hold of the weather data. iii The final version of this thesis was refined with the expert advice of my examiners, Professor Andy Fenton and Dr Francesco Baldini. I am very thankful for their contributions, their time and interest, which certainly improved the quality of this work. I want to thank CONACYT (Science and Technology National Council, Mexico) for funding my doctorate and making all this possible, to the SEP (Public Education Ministry, Mexico) for the complementary grant that I was awarded to support my doctorate studies, to the Houghton Trust for funding our research and conference attendance, and to Chester Zoo for also contributing to cover the expenses of the project. I want to thank the marvellous community of Leahurst campus, fellow PhD colleagues, academics and staff, who bring life to this site and whom I admire. A special mention for Elsa who offered me her friendship and took me through a very helpful induction to life in the UK, Leahurst and the University, making me feel integrated. I am in debt to my friends who shared with me numerous great moments during these years: Sören, Andrea, Steph, Maya, Veysel, Marcus, Cyril, Marisol, Maria, Jordana, Jack. Also, to the friends across the pond: Lalo, Alex, Alicia, Eri, Horacio, Neida, Ari, Fran, Luiyo, Gladys, José, Mr. Daniel and Gina. For sharing great moments and for the adventures that await. I also include in the list “Los Delgados” family, with whom I share fantastic memories from all life and have been always there offering support and love. I extend this thanks to Anne Hignell, who has been also there and supported me, especially during the first weeks after my arrival. I owe a great thanks to my family, my parents, and brothers, who gave me the most exceptional support always and have encouraged me in my odyssey. Regardless of the distance, they have been with me, and I can always feel the warmth of their hearts. A unique thanks to Merit Gonzalez Olvera who decided to cross the ocean to join me and share this adventure with me thoroughly and unconditionally. She contributed immensely to the project joining me in the field samplings and doing the parasite testing. Her constant support and love kept me optimistic and motivated but more importantly, she has given great joy to my life and countless extraordinary moments. iv v Abstract Avian malaria is one of the most serious diseases in penguins under human care and could become a severe threat to the conservation of vulnerable wild populations. It is caused by the Haemosporidia parasites of the genus Plasmodium and needs a mosquito vector for its transmission. We captured mosquitoes during two years in Chester Zoo (Cheshire) and one year in Flamingo Land (Yorkshire); both zoos house Humboldt penguins (Spheniscus humboldti). The mosquito temporal and spatial abundance across the seasons and sites were analysed. It was found that Culex pipiens, the principal avian malaria vector in Europe, was the most abundant species. There was a peak in the mosquito abundance during the summer as expected, but it was at different months between sites and years. The abundance of mosquitoes also varied among sampling areas; one area in Chester Zoo captured a greater proportion of mosquitoes than the others in both years, and in Flamingo Land, we also found an area with consistent high catches. Blood-fed mosquitoes were captured and analysed to identify the host on which they had fed. Different proportions of blood-fed mosquitoes were captured by areas and months; more were collected during the summer and in certain areas that not in all cases were related to a high abundance of un- fed mosquitoes. Most of these mosquitoes were Culex pipiens and Culiseta annulata; it was confirmed that the first one prefers to feed on birds and the second one on non-human mammals. However, many Culex pipiens fed on humans, which alert us about the possible nuisance for visitors and the potential transmission risk of zoonotic diseases. A partially identified Culicinae mosquito, likely to be Culex pipiens, and an Anopheles maculipennis s. l. fed on penguins; so, they could be involved in avian malaria transmission. It was found that mosquitoes travel variable distance after feeding and therefore, the control measures against mosquitoes should cover more than the areas of immediate concern. The environmental variables were analysed to understand the drivers of the diverse mosquito captures. The temperature was the most important variable related to mosquito abundance, and the dense vegetation, proximity to mosquito oviposition sites and closeness to animal exhibits were also significant. Therefore, the temperature could guide actions for mosquito control and avian malaria prevention and avoiding those surrounding features near the penguin exhibits could prevent high densities of mosquitoes. Many aspects of avian malaria epidemiology are uncertain so, through an online survey, the knowledge of the staff in zoos and wildlife parks about the disease was gathered. It was found that avian malaria had affected penguins in more than half of the answering institutions, involving mainly Humboldt and African penguins (Spheniscus demersus) with high lethality rates; therefore, efforts on preventive actions are encouraged. Avian malaria parasites were found in Culex pipiens mosquitoes and their saliva, wild birds and penguins, suggesting that the transmission process happens locally. Mosquito populations are dynamic, and the biosurveillance of their populations is needed to better understand their role as disease vectors and to implement effective control measures at the right time, assisting in this way the prevention of avian malaria in captive penguins. vi Table of Contents Acknowledgements iii Abstract vi Table of contents vii List of figures xii List of tables xiv Chapter One. General Introduction 1 1.1 Introduction 2 1.2 Diseases of Wildlife 2 1.2.1 Wildlife diseases and public health 4 1.2.2 Wildlife diseases and domestic animals 4 1.2.3 Wildlife diseases and conservation 5 1.2.3.1 Conservation approaches 7 1.2.3.2 Disease ecology in zoos 9 1.3 Diseases Affecting Wild Birds 9 1.3.1 Disease ecology of wild birds 9 1.3.2 Vector-borne diseases of wild birds 10 1.3.3 Environmental effects on wild bird diseases 11 1.4 Haemosporidians of Wild Birds 12 1.4.1 Life cycle of haemosporidians 13 1.4.2 Avian malaria in wild birds 15 1.4.3 Ecology of avian malaria vectors 17 1.4.3.1 Culex pipiens 18 1.4.4 Avian malaria in penguins 21 1.4.5 Avian malaria prevention and control 22 1.5 Objectives 23 Chapter Two.