Thesis committee Thesis supervisor Prof. dr. Marcel Dicke Professor of Entomology, Wageningen University Thesis co-supervisor Dr. Ir. Bart G.J. Knols Medical Entomologist, University of Amsterdam Other members Prof. dr. B.J. Zwaan, Wageningen University Prof. dr. P. Kager, University of Amsterdam Dr. Ir. P. Bijma, Wageningen University Dr. Ir. I.M.A. Heitkonig, Wageningen University This research was conducted under the auspices of the C. T. de Wit Graduate School for Production Ecology and Resource Conservation Behavioural, ecological and genetic determinants of mating and gene flow in African malaria mosquitoes Kija R.N. Ng’habi Thesis Submitted in fulfillment of the requirement for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. dr. M.J. Kropff, in the presence of the Thesis committee appointed by the Academic Board to be defended in public at on Monday 25 October 2010 at 11:00 a.m. in the Aula. Kija R.N. Ng’habi (2010) Behavioural, ecological and genetic determinants of mating and gene flow in African malaria mosquitoes PhD thesis, Wageningen University – with references – with summaries in Dutch and English ISBN – 978-90-8585-766-2 > Abstract Malaria is still a leading threat to the survival of young children and pregnant women, especially in the African region. The ongoing battle against malaria has been hampered by the emergence of drug and insecticide resistance amongst parasites and vectors, re- spectively. The Sterile Insect Technique (SIT) and genetically modified mosquitoes (GM) are new proposed vector control approaches. Successful implementation of these ap- proaches requires a better understanding of male mating biology of target mosquito species. This thesis explored the potential behavioural, ecological and genetic determi- nants of mating and gene flow in the major African malaria vectorAnopheles gambiae s. l. This thesis specifically investigated (i) the effect of larval density and nutrition on the mating competitiveness of adult male Anopheles gambiae s.s mosquitoes (ii) compared the physiological fitness of male Anopheles gambiae mosquitoes between laboratory and field populations, (iii) the potential for establishing a self-replicating Anopheles arabiensis population in an enclosed semi-natural environment and observ- ing its genetic variation over time, (iv) the development of a PCR-based method for assessing male mating success among inseminated female An. gambiae and (v) the population genetic structure of An. gambiae s. l. along the Kilombero valley (southern Tanzania). Results indicated that environmental factors experienced during larval devel- opment, affect adult male mating behaviour. These factors can be experimentally ma- nipulated during mass-rearing to enhance male mating success. Laboratory reared males are physiologically different from wild males. Field males have more lipids than laboratory-reared males. This thesis also reports the first ever establishment of an Anopheles population in an enclosed semi-natural environment. Conditions within the system were suitable for maintaining the genetic variation which is frequently lost un- der traditional laboratory cage conditions. The newly developed PCR-based method to assess insemination success will help to understand the mating biology and ecology of An. gambiae mosquitoes within the enclosed semi-field system. Results also show that the An. arabiensis populations of the Kilombero valley display a population substruc- ture whereas An. gambiae s. s. showed no population substructure. It is concluded that environmental factors, such as crowding and larval nutri- tion can be manipulated in the laboratory to enhance male mating success. Given the successful establishment of a semi-field mosquito population and the evidence that it does maintain more genetic variation than laboratory colonies, I suggest that stud- ies aimed at exploring male mating biology /ecology and feasibility of SIT/GM can be executed in an enclosed semi-field environment. Information obtained in the semi- field system may be more representative of field mosquitoes than that from caged colony mosquitoes. The results of this thesis, therefore, provide useful information to strengthen current and future vector control approaches. Chapter 1 General introduction p. 11 Chapter 2 The effect of larval density on the mating com- p. 41 petitiveness of adult Anopheles gambiae mos- quitoes Chapter 3 Effect of larval diet on the mating competitive- p. 59 ness of male Anopheles gambiae s.s Chapter 4 Physiological fitness of free-living and laborato- p. 77 ry-reared male Anopheles gambiae s.l. Chapter 5 Establishment of a self-propagating population p. 95 of the African malaria vector An. arabiensis un- der semi-field conditions Chapter 6 A new robust diagnostic polymerase chain reac- p. 119 tion (PCR)-based method for determining the mating status of female An. gambiae mosqui- toes Chapter 7 The impact of laboratory versus semi-natural p.127 colonization on genetic heterogeneity in the ma- laria mosquito An. arabiensis Patton Chapter 8 Genetic population structure of sympatric malar- p. 141 ia vectors An. arabiensis and An. gambiae sensu stricto in a malaria endemic region of Southern Tanzania Chapter 9 Clarification of anomalies in the application of a p. 161 2La molecular karyotyping method for the ma- laria vector An. gambiae Chapter 10 Summarising discussion p. 173 Summary p. 189 Curriculum vitae p. 195 List of publications p. 197 Acknowledgements p. 201 PE&RC PhD Education Certificate p. 205 > Contents Chapter 1 General introduction p. 11 Chapter 2 The effect of larval density on the mating com- p. 41 petitiveness of adult Anopheles gambiae mos- quitoes Chapter 3 Effect of larval diet on the mating competitive- p. 59 ness of male Anopheles gambiae s.s Chapter 4 Physiological fitness of free-living and laborato- p. 77 ry-reared male Anopheles gambiae s.l. Chapter 5 Establishment of a self-propagating population p. 95 of the African malaria vector An. arabiensis un- der semi-field conditions Chapter 6 A new robust diagnostic polymerase chain reac- p. 119 tion (PCR)-based method for determining the mating status of female An. gambiae mosqui- toes Chapter 7 The impact of laboratory versus semi-natural p.127 colonization on genetic heterogeneity in the ma- laria mosquito An. arabiensis Patton Chapter 8 Genetic population structure of sympatric malar- p. 141 ia vectors An. arabiensis and An. gambiae sensu stricto in a malaria endemic region of Southern Tanzania Chapter 9 Clarification of anomalies in the application of a p. 161 2La molecular karyotyping method for the ma- laria vector An. gambiae Chapter 10 Summarising discussion p. 173 Summary p. 189 Curriculum vitae p. 195 List of publications p. 197 Acknowledgements p. 201 PE&RC PhD Education Certificate p. 205 10 C������1 General introducti on Ng’habi KR 11 Chapter 1 The research reported in this thesis focused on the determinants of gene flow, and in particular the ecological, behavioural and genetic determinants of mating success, in African malaria mosquitoes. This introductory chapter aims to give an overview of the malaria burden, control efforts and the prospects for new vector control approaches. Then it gives an overview of ecological, behavioural and genetic determinants of mating success and gene flow in mosquito vectors. 12 General introduction Background 1 Malaria Burden espite intensified control efforts, malaria remains one of world’s most burdensome Ddiseases (Hay et al., 2005; Hay et al., 2004; Snow et al., 2005). Malaria accounts for an estimated 240 million cases and 280, 000 deaths worldwide, with over 80% occurring in African region (WHO, 2009); the majority of these are children of under five years of age (WHO, 2008). Pregnant women are another vulnerable group as malaria causes serious adverse effects including abortion, low birth weight and maternal anaemia (Adam et al., 2005; Newman et al., 2003; Rogerson et al., 2007). The economic toll of malaria has been high in Africa and has been linked with poverty (Sachs and Malaney, 2002). Malaria is caused by a protozoan belonging to the genus Plasmodium. There are five malaria parasite species of this genus that can infect humans. The first two includes the P. vivax, and P. malariae. For P. ovale recent evidence indicates that it may be consisting of two species (Duval et al., 2009) and P. falciparum is the fifth malaria parasite. P. falciparum is the most deadly species responsible for most of the deaths occurring in Africa (Breman et al., 2007). It has been considered that P. falciparum is a strictly human parasite, however recent studies have found that P. falciparum is not an exclusively human parasite and is a close relative of P. reichenowi, a chimpanzee parasite (Prugnolle et al., 2010). In addition, P. knowlesi recently been incriminated as the sixth human malaria parasite (Prugnolle et al., 2010; White, 2008a). Malaria vectors Only some seventy Anopheles species can vector malaria (Service, 1993). Of the 422 known Anopheles species, members of the An. gambiae s.l. Giles complex are responsible for malaria transmission in Africa (White, 1974). The An. gambiae complex consists of seven sibling species (Hunt et al., 1998). Two members of the complex, An. gambiae s.s. Giles and An. arabiensis Patton are considered to be the major vectors of malaria in Africa. Although they are mostly found in sympatry, the two species differ greatly in their behaviour and physiology. Anopheles arabiensis is more zoophilic and exophilic (Gillies, 1955), and able to tolerate higher temperatures and lower humidities thanAn. gambiae s.s. (Kirby and Lindsay, 2004) whereas An. gambiae s.s. is more anthropophilic and endophilic (Gillies, 1955). As a result An. arabiensis is widely distributed in arid areas (Coetzee et al., 2000) and more likely to change its behaviour in response to interventions such as residual insecticides than An. gambiae s.s. (Geissbühler et al., 2007). An. arabiensis is therefore believed to replace An. gambiae s.s. (Lindblade et al., 2006) in some areas and to be present as the sole vector of malaria in other places.