The effect of pond dyes on mosquitoes and other freshwater invertebrates A thesis submitted for the degree of Doctor of Philosophy School of Biological Sciences Natali Ortiz Perea January 2018 Declaration I confirm that this is my own work and the use of all material from other sources has been properly and fully acknowledged. Natali Ortiz Perea ii ABSTRACT Freshwater habitats are important because they represent two percent of Earth’s water resources, are highly diverse in aquatic organisms and are the most productive and threatened ecosystem worldwide. Pollution, urbanization and climatic changes are responsible for drastic changes in these ecosystems. The creation of new ponds offers an opportunity to increase biodiversity, landscape connectivity and provide new habitat for organisms. However, new ponds might be a good habitat for mosquitoes to lay eggs. Mosquitoes have worldwide distribution and are responsible for most of the vector-borne diseases, affecting thousands of people and causing millions of deaths. British mosquitoes currently do not carry human diseases, but they are a biting nuisance. Their distribution, abundance, species composition and potential for mosquito disease transmission are intimately linked to the physical environment. Culex pipiens is commonly found in UK gardens and is a potential vector of viruses including the West Nile Virus. However, any environmental factors that significantly change the distribution and population of Cx. pipiens could impact future risks of disease transmission. Pond dyes are a cosmetic product for garden ponds and lakes; they inhibit algal growth and improve the overall appearance of the water body reflecting surrounding planting. The dyes block red light from entering the water, interrupting the process of photosynthesis and therefore inhibiting the growth of certain aquatic plants such as algae. Although these dyes are non-toxic to fish and invertebrates, their use in urban gardens raises questions linked to mosquito oviposition, since coloured water can be an attractant. iii This research focussed on the impact of pond dyes on mosquitoes and freshwater macroinvertebrates. Gravid female Culex mosquitoes preferred to lay eggs in black dyed water under laboratory conditions and in a semi-field choice test but there was no evidence that these results translated into a difference in a fully natural ecosystem. Mosquito survivorship in black dyed water was significantly reduced; Dyes of other colours had no impact on oviposition but did impact survival in both field and laboratory conditions. The impact of black pond dye on small artificial ponds found effects depending on the experimental method. In ponds that were cleared of all animals but they reseeded with known species of macroinvertebrates dyes impacted negatively on the biodiversity, evenness and abundance of species. By contrast, in the second pond study, where dye was applied to the existing macroinvertebrate communities, no differences were detected between treated and untreated ponds in respect to biodiversity or abundance. A final study on the impact of black pond dye on mosquito larval predation by Chaoborus flavicans and Gammarus pulex found no difference in predation. Considering the concerns over potential future spread of disease in urban environments and the properties of these dyes, more studies are needed to understand how aquatic animals and interactions between species respond in presence of colour dyes. iv ACKNOWLEDGEMENTS I would like to express the deepest appreciation to my supervisor Dr Amanda Callaghan for giving me the guiding, comments, support and the opportunity to work with her and for her help, patience and encouragement throughout this work. I would like to extend a special thanks to the committee members Professor Mark Fellowes and Dr Graham Holloway for giving me feedback during the process of my thesis, helpful guidance of statistically advice and collection of Gammarus pulex. I would like to extend a special thanks to Ross Cuthbert for his collaboration, help and patience to analyse the functional response data. I thank the project students I have worked; Rebecca Gander, Oliver Abbey, Tania Smith, Sophie Connor and Alice Hurley for their help and company in the field. I would like to thank all my friends both from Room 8 and outside University for their support, company and good moments. I am extremely grateful to my parents and sister for their entire support, encouragement, patience, love and advice during this process. I also thank the sponsor Colciencias who funded my studies with a PhD scholarship and Colfuturo who helped me with a grant. v Table of Contents ABSTRACT ...................................................................................................................................... iii ACKNOWLEDGEMENTS ............................................................................................................... v LIST OF ABREVIATIONS .............................................................................................................. ix INDEX OF FIGURES ........................................................................................................................ x INTRODUCTION ............................................................................................................................ 16 CHAPTER 1. LITERATURE REVIEW.......................................................................................... 18 1.1. Freshwater environments ................................................................................................. 18 1.1.1. Ponds ........................................................................................................................ 20 1.1.2. Other standing water in urban habitats ..................................................................... 27 1.2. Introduction to Mosquitoes .............................................................................................. 29 1.2.1. Mosquitoes as vectors of disease .............................................................................. 34 1.3. Infectious diseases in Europe ........................................................................................... 37 1.4. Mosquitoes in Great Britain. ............................................................................................ 40 1.5. Culex pipiens .................................................................................................................... 45 1.6. Mosquito predators in Great Britain ................................................................................. 47 1.7. Aims and objectives of the study ..................................................................................... 51 CHAPTER 2. Pond dyes are Culex mosquito oviposition attractants ............................................. 55 2.1 Introduction ...................................................................................................................... 55 2.2 Materials and Methods ..................................................................................................... 58 2.2.1 Trapping wild gravid female mosquitoes ................................................................. 58 2.2.2 Oviposition preferences of wild mosquitoes ............................................................ 58 2.2.3 Emergence study ...................................................................................................... 60 2.2.4 Wild population numbers in dye treated and untreated artificial containers ............ 60 2.2.5 Statistics ................................................................................................................... 62 2.3 Results .............................................................................................................................. 64 2.1 Mosquito oviposition selection in laboratory conditions and oviposition selection in the tent 64 2.2 Wild population numbers in dye treated and untreated artificial containers ................ 64 2.3 Emergence study: field conditions ............................................................................... 66 2.4 Discussion ........................................................................................................................ 68 CHAPTER 3. The effect of pond dyes on oviposition and survival in wild UK Culex mosquitoes 73 3.1 Introduction ...................................................................................................................... 73 vi 3.2 Materials and Methods ..................................................................................................... 77 3.2.1 Trapping wild gravid females................................................................................... 77 3.2.2 Pond dyes ................................................................................................................. 79 3.2.3 Oviposition preferences of wild mosquitoes ............................................................ 79 3.2.4 Emergence study ...................................................................................................... 80 3.2.5 Natural colonisation of containers with or without dyes. ......................................... 82 3.2.6 Statistical analysis .................................................................................................... 82 3.3 Results .............................................................................................................................