Natural Products for Malaria Vector Control: Flora, Fish and Fungi
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Natural products for malaria vector control: flora, fish and fungi Annabel F. V. Howard Thesis committee Thesis supervisors Prof. dr. ir. W. Takken Personal chair at the Laboratory of Entomology, Wageningen University Prof. dr. M. Dicke Professor of Entomology, Wageningen University Thesis co-supervisor Dr. J.J. Githure Head of Human Health, International Centre of Insect Physiology and Ecology, Kenya Other members Prof. dr. ir. R. Rabbinge Wageningen University Dr. W.J. de Kogel Plant Research International, Wageningen University and Research Centre Prof. dr. E. van Donk Netherlands Institute of Ecology, Maarssen Dr. W.J. Ravensberg Koppert B. V., Berkel en Rodenrijs This research was conducted under the auspices of the C. T. de Wit Graduate School for Production Ecology and Resource Conservation Natural products for malaria vector control: flora, fish and fungi Annabel F. V. Howard Thesis submitted in fulfilment of the requirements 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 on Tuesday 5th October 2010 at 11 a.m. in the Aula Annabel F. V. Howard (2010) Natural products for malaria vector control: flora, fish and fungi PhD thesis, Wageningen University – with references – with summaries in Dutch and English ISBN 978-90-8585-720-4 Table of Contents Abbreviations 7 Abstract 9 Chapter 1 General Introduction 15 Chapter 2 Malaria vector control options available to rural African 51 communities in the context of an integrated vector management strategy: a review PART I EXAMINING THE EFFECT OF AZADIRACHTA INDICA (THE 87 NEEM TREE) ON A MAJOR MALARIA VECTOR Chapter 3 Laboratory evaluation of the aqueous extract of Azadirachta 89 indica (the neem tree) wood chippings on Anopheles gambiae s.s. mosquitoes Chapter 4 Effects of a botanical larvicide derived from Azadirachta indica 109 (the neem tree) on oviposition behaviour in Anopheles gambiae s.s. mosquitoes PART II THE USE OF LARVIVOROUS FISH FOR MOSQUITO 125 CONTROL IN WESTERN KENYA Chapter 5 Abandoning small-scale fish farming in western Kenya leads to 127 higher malaria vector abundance Chapter 6 Malaria mosquito control using edible fish in western Kenya: 143 preliminary findings of a controlled study PART III CAN ENTOMOPATHOGENIC FUNGI BE USED TO CONTROL 155 INSECTICIDE-RESISTANT MOSQUITOES? Chapter 7 Pyrethroid resistance in Anopheles gambiae s.s. leads to 157 increased susceptibility to the entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana Chapter 8 The entomopathogenic fungus Beauveria bassiana reduces 177 blood feeding in wild insecticide-resistant mosquitoes in Benin, West Africa Chapter 9 Summarizing discussion 209 References 225 Samenvatting 253 Acknowledgements 259 Curriculum vitae 261 List of publications 263 PE&RC Education Certificate 265 Abbreviations Abbreviations ACT Artemisinin-based combination therapy AZA Azadirachtin BC Beauveria bassiana window, control bednet BP Beauveria bassiana window, permethrin bednet Bti Bacillus thuringiensis var. israelensis CC Control window, control bednet CI Confidence interval COMBI Communication for Behavioural Impact CP Control window, permethrin bednet CREC Centre de Recherche Entomologique de Cotonou DDT Dichlorodiphenyltrichloroethane DT Dispersible tablet df Degrees of freedom EC Emulsion concentrate EIP Extrinsic incubation period EIR Entomological inoculation rate FD Fisheries Department GC Gonotrophic cycle GST Glutathione-S-transferase HBI Human blood indices HPLC High performance liquid chromatography HR Hazard Ratios icipe International Centre of Insect Physiology and Ecology IE50 The concentration that causes 50% inhibition of emergence IE90 The concentration that causes 90% inhibition of emergence IEC Information Education and Communication IGR Insect growth regulators IPM Integrated pest management IRS Indoor residual spraying ITN Insecticide-treated bednet ITU International toxic units IVM Integrated vector management 7 Abbreviations kdr Knockdown resistance KEMRI Kenya Medical Research Institute KNAW Royal Dutch Academy of Arts and Sciences L:D Light : Dark LI 1st instar mosquito larva LII 2nd instar mosquito larva LIII 3rd instar mosquito larva LIV 4th instar mosquito larva LSHTM London School of Hygiene and Tropical Medicine MC Metarhizium anisopliae window, control bednet MDG Millennium Development Goals MFO Mixed function oxidase MP Metarhizium anisopliae window, permethrin bednet N Number NCP Neem seed cake powder NWM Not well maintained OR Odds Ratios PE&RC Production Ecology and Resource Conservation RH Relative humidity SDA Sabouraud Dextrose Agar SE Standard error SKK Anopheles gambiae s.s. Suakoko strain s.l. Sensu lato SNK Student-Newman-Keuls Spp. Species (plural) s.s. Sensu stricto UN United Nations UV Ultraviolet light VKPER Anopheles gambiae s.s. Valley de Kou pyrethroid-resistant strain WHA World Health Assembly WHO World Health Organisation WHOPES World Health Organisation Pesticide Evaluation Scheme WM Well maintained 8 Abstract Abstract Introduction Despite international organisations providing much focus over the past 10 years, malaria is still killing vast numbers of Africans, especially children. It is agreed that malaria can only be successfully controlled by using different control tools simultaneously in the spirit of integrated vector management (IVM), and that African communities will need to become more directly involved in mosquito control (Chapter 2). Using mosquito control tools in a way that requires almost no technical equipment or knowledge will open them up to the rural communities that are best placed to deploy them. In addition, widespread insecticide resistance is reducing the ability of insecticide-based tools to control mosquitoes. For these reasons, biological control and other natural mosquito control methods are being researched by many institutions. Several potential natural control tools are readily available in sub-Saharan Africa. If these tools prove effective and become operational, then it is possible that they will be sustainable because communities can intentionally produce the biological agents themselves, bringing a source of money to rural communities. This would be especially important in areas where infrastructure is poorly developed, and repeat applications of chemical control tools are not easily made. This thesis was designed to test the feasibility and effectiveness of a variety of natural products against both larval and adult malaria vector mosquitoes using low-tech methods in laboratory and field trials. Part I: Flora Azadirachta indica A. Juss (Meliaceae) (the neem tree) was chosen due to the already proved mosquitocidal properties, and its ready availability in Africa. We wanted to use neem in a way that could easily be deployed in resource-poor rural 9 Abstract areas. Laboratory studies were conducted to examine the larvicidal and pupicidal properties of a crude aqueous extract of neem wood against the principle African malaria vector, Anopheles gambiae Giles s.s. (Diptera: Culicidae) (Chapter 3). The results indicate that even a relatively low dose of 0.15 grams of dried neem wood in 1 litre of water was able to inhibit the emergence of 90% of mosquito adults when larvae were exposed during their first three larval instars. Even for the fourth (last) larval instar, just 0.6 g/l was required to prevent 90% emergence. Furthermore, neem-exposed larvae exhibited significantly increased development times when compared to the controls. Pupae were also killed by the aqueous neem extracts, and were subject to neem-induced emergence abnormalities, but the concentrations required to kill pupae were much higher than for larvae and not likely to be used operationally. High performance liquid chromatography (HPLC) analysis identified several polar constituents in the aqueous neem extracts including nimbin and salannin. However, azadirachtin was not present in significant amounts. The effect of this extract on the oviposition behaviour of adult female An. gambiae s.s. mosquitoes was then monitored (Chapter 4). The oviposition results show that when using 0.1 g/l of the crude aqueous neem extract, significantly more mosquitoes laid their eggs when compared to mosquitoes exposed to the control treatment. For the doses 10x and 100x higher, the same proportion of mosquitoes laid their eggs as in the control, indicating that even at much higher doses than required for successful larval control, female oviposition will not be detrimentally affected. Part II: Fish Larvivorous fish have previously been shown to effectively control mosquito numbers. Therefore, a census was carried out to examine the current status of fish farming in western Kenya (Chapter 5). Working with the Kenyan Fisheries Department we found that while fish farming is a favoured activity, 30% of the 261 10 Abstract ponds found did not contain fish. These “abandoned” ponds had significantly more An. gambiae s.l., Anopheles funestus Giles and culicine mosquitoes when compared to the ponds that still contained fish. Furthermore, An. gambiae s.l. was proportionally more abundant in the abandoned ponds when compared to the other mosquito types. Surprisingly, vegetation did not significantly affect mosquito distribution. Following our study, demand for fish to restock abandoned ponds increased by 67% when compared to the previous year. The overwhelming majority of fish being farmed in our census area were fish of