Keeping track of the enemy Flight analyses of the host-seeking malaria mosquito Anopheles gambiae s.s. Jeroen Spitzen Propositions 1. The use of infrared light illuminates the behavioural ecology of nocturnal mosquitoes. (this thesis) 2. Human odour causes prolonged and highly convoluted flight tracks and the combination with heat is crucial to induce landings of host-seeking mosquitoes. (this thesis) 3. The taboo on decreasing the global human population size should be lifted and needs to be addressed in a multifaceted approach on political and societal agendas. 4. Introduction of the ‘polluter pays’ principle will result in more environmental benefits than stimulating a growing market of organic and sustainable products. 5. Practice what you teach augments science for impact. 6. With the increase in number of PhD candidates, it becomes extremely challenging for the reviewing committee to check the originality of propositions. Propositions belonging to the thesis, entitled ‘Keeping track of the enemy: Flight analyses of the host-seeking malaria mosquito Anopheles gambiae s.s.’ Jeroen Spitzen Wageningen, 23 May 2018 Keeping track of the enemy Flight analyses of the host-seeking malaria mosquito Anopheles gambiae s.s. Jeroen Spitzen Thesis committee Promotors Prof. Dr W. Takken Personal chair at the Laboratory of Entomology Wageningen University & Research Prof. Dr M. Dicke Professor of Entomology Wageningen University & Research Other members Prof. Dr M. Naguib, Wageningen University & Research Dr F.O. Okumu, Ifakara Health Institute, Tanzania Dr J.T. Bousema, Radboud University, Nijmegen, The Netherlands Dr M.A.H. Braks, National Institute for Public Health and the Environment, Bilthoven, The Netherlands This research was conducted under the auspices of the C.T. de Wit Graduate School for Production Ecology & Resource Conservation. Keeping track of the enemy Flight analyses of the host-seeking malaria mosquito Anopheles gambiae s.s. Jeroen Spitzen Thesis submitted in fulfilment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus, Prof. Dr A.P.J. Mol, in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Wednesday 23 May 2018 at 11 a.m. in the Aula. Jeroen Spitzen Keeping track of the enemy: Flight analyses of the host-seeking malaria mosquito Anopheles gambiae s.s., 188 pages. PhD thesis, Wageningen University, Wageningen, the Netherlands (2018) With references, with summaries in English and Dutch ISBN 978-94-6343-258-0 DOI 10.18174/441165 Abstract Female mosquitoes can transmit pathogens to their host during blood feeding and are an important vector of human diseases such as dengue, chikungunya, filariasis and malaria. After 15 years of decline in the number of fatal malaria cases, this decline came to a halt in 2016. Growing resistance against drugs and insecticides pose a serious threat for future human health. This thesis focuses on the behaviour of host-seeking malaria mosquitoes by analysing their flight paths during their approach to different host cues. Fundamental knowledge on the role of selected host cues was acquired. In addition, studies to support successful implementation of vector control interventions were performed in both wind- tunnel settings and the semi-field in Kenya. My research demonstrates that automated tracking systems can strengthen behavioural-ecological studies on disease vectors, in addition to conventional bio-assays such as olfactometers, by providing detailed information on the approach behaviour of mosquitoes to different targets. The attraction towards the host-sensory cue CO2 was investigated in an olfactometer bioassay. Trap catches of female Anopheles gambiae s.s. were enhanced by separation of the CO2 source from the source of human skin emanations. Close-range deterrent effects of CO2 were overcome by the simultaneous presence of skin emanations. Flight path analysis of mosquitoes in a wind tunnel, showed that exposure to human odour resulted in prolonged and highly convoluted flight tracks. The combination of odour with heat was crucial to induce landings of host-seeking mosquitoes. A semi- field study in Kenya revealed that house-entering mosquitoes approached the eave of a house in a wide angle to the house at eave level, where the proportion that entered uninterruptedly (23%) spent just a few seconds around the eave area. The presence of insecticide-treated nets inside a house did not repel mosquitoes as measured by the number of house entries. At close range, in a wind tunnel, free-flight exposure of mosquitoes to deltamethrin-treated nets in combination with human odour did not reveal any (excito-) repellent effect and resulted in lower mortality rates compared to standard bioassays where contact with the treated material is enforced. The knowledge obtained on the behavioural responses of mosquitoes to host cues has indirectly affected vector control tool implementations in the field. For example, in the development of an odour-baited trap, a CO2 release pipe was included that is separated from the attractive odour plume. The role of heat was exploited in the development of a repellent bioassay and a heat source was added to another trap model. Insights in house-entry behaviour and mosquito responses to bed nets support the successful implementation of push-pull systems, installation of eave tubes or implementing house improvement operations to reduce malaria transmission. An integrated vector management approach is required to further develop existing control tools by adding and improving alternative intervention techniques. 6 Table of Contents Abstract 6 Chapter 1 9 General Introduction Chapter 2 17 Keeping track of mosquitoes: A review of tools to track, record and analyse mosquito flight Chapter 3 35 Effect of human odours and positioning of CO2 release point on trap catches of the malaria mosquito Anopheles gambiae sensu stricto in an olfactometer Chapter 4 49 A 3D analysis of flight behaviour of Anopheles gambiae sensu stricto malaria mosquitoes in response to human odour and heat Chapter 5 81 Visualization of house-entry behaviour of malaria mosquitoes Chapter 6 97 Effect of insecticide-treated bed nets on house-entry by malaria mosquitoes: The flight response recorded in a semi-field study in Kenya Chapter 7 111 Absence of close-range excitorepellent effects in malaria mosquitoes exposed to deltamethrin-treated bed nets Chapter 8 129 General Discussion References 141 Summary 165 Samenvatting 171 Acknowledgements 177 Curriculum Vitae 182 List of publications 183 Education statement 186 Chapter 1 General Introduction Chapter 1 Introduction Blood-sucking arthropods cause nuisance because their bites can hurt and cause itching. The sound of an approaching mosquito in flight induces agitation of the host target. Many of them transmit pathogens that cause serious health problems across the world. It is this indirect effect why mosquitoes are considered the deadliest animals of planet earth. Among the mosquitoes that act as vectors of infectious diseases such as dengue, yellow fever, Zika or West-Nile virus, the genus Anopheles, with 465 species and over 50 unnamed members of species complexes, compiles 41 species considered dominant in transmitting the Plasmodium parasite to humans (Sinka et al., 2012 and references therein). This group caused an estimated number of 216 million human malaria cases in 2016, with approximately 445,000 deaths worldwide (World Health Organization, 2017a). Vector-control tools such as long- lasting-insecticide-treated nets (LLINs) and indoor residual spraying (IRS) currently lose their efficacy due to behavioural and physiological resistance (N’Guessan et al., 2007; Ranson et al., 2011). Alternative control methods are required. Many of these methods aim to interrupt mosquito-host interactions, e.g. by the use of odour-baited traps, push-pull systems, or house improvements (Homan et al., 2016; Menger et al., 2016; Tusting et al., 2016). For the innovation and successful implementation of alternative, sustainable tools it is necessary to understand mosquito behaviour in detail so that interventions can be made more precisely and effectively. This thesis focuses on the flight behaviour of one of the main vectors of malaria parasites, Anopheles gambiae Giles sensu stricto, while exposed to host cues. Automated tracking techniques were developed and used to unravel flight tracks of nocturnal mosquitoes. Fundamental knowledge on the role of specific host cues was acquired and applied studies to support successful implementation of vector control interventions were performed in both wind-tunnel settings or the semi-field in Kenya. Malaria Malaria is caused by protozoa of the genus Plasmodium. The parasite is transmitted to humans through bites of infected female mosquitoes belonging to the genus Anopheles (Diptera: Culicidae). Plasmodium falciparum is the main species causing (fatal) malaria and is dominant in Sub-Saharan Africa, while P. vivax is the dominant species in Asia and South America, although with fewer cases than P. falciparum (Vogel, 2013). The other three species that can cause human malaria are P. ovale, P. knowlesi and P. malariae (White et al., 2014). The parasite needs both humans and mosquitoes for its development. As a result of a mosquito bite, sporozoites travel via the saliva of an infected mosquito to the liver 10 General Introduction of their host. Here, merozoites are produced which can infect red blood cells. Within