MOLECULAR ANALYSIS OF MECHANISMS AND IDENTIFICATION OF FACTORS OF PYRETHROID RESISTANCE IN ANOPHELES GAMBIAE SENSU LATO IN SOUTHWESTERN NIGERIA AND SOUTHERN BENIN REPUBLIC Y By R A R IB Jean Rousseau DJOUAKA-FOLEFALCK B.Sc. (Cameroon), M.Sc. ZoologyY (Ib adan), S.I. 119677 SI T R A thesis in the Department of IZVoolo Egy submitted to the Faculty of Science in partial fulfilmeNnt of the requirements for the degree of U AN DOCTOR OF PHILOSOPHY AD of the IB UNIVERITY OF IBADAN 2011 ABSTRACT The development of resistance to insecticides by Anopheles mosquitoes continues to threaten the success of malaria control programmes in West Africa. Local data on mechanisms and factors causing resistance in the region are scanty. This study was designed to investigate the environmental factors and mechanisms implicated in resistance to pyrethroids by Anopheles gambiae in southwestern Nigeria and southern BYenin Republic. Larvae of Anopheles mosquito were collected in 2007 from 19 localitiesR in the six states of southwestern Nigeria and 18 localities in the seven divisions of sAouthern Benin and reared to adults. These were identified morphologically and wBith RPolymerase Chain Reactions (PCR). They were also bioassayed for susceptibility toI pyrethroids. Molecular characterisation of pyrethroid resistant phenotypes was caLrried out using PCR and microarray analyses of the expressed genes. DissolvYed Oxygen (DO) and pH were determined using a digital multipurpose meter wIhiTle physical appearances of breeding sites were assessed visually. Xenobiotic factoSrs such as Spilled Engine Oil (SEO) and agricultural pesticides that might contribuRte to the emergence of resistance in Anopheles populations were examined through bioassay. Associations between pyrethroid resistance with environmental factors and mIoVlecu Elar profiles of Anopheles were evaluated using Chi square. A. gambiae complexN genotyped in Nigeria comprised of 73.6 % A. arabiensis and 26.3 % A. gambiae sen sUu stricto; while those genotyped in Benin were 92.9 % A. gambiae s.s. and 7.0 % A. Nmelas. Pyrethroid resistance in Nigeria and Benin were recorded in 68.4 % and 94.4 %A of the localities examined respectively. Breeding sites contaminated with SEO (B-SDEO) or Pesticide Residues (B-PR) had low DO (B-SEO = 13.4 ± 1.5 mg/l, B-PR=1A2.2 ± 1.7 mg/l), the Non-contaminated Breeding sites (B-NC) had higher levels of IDBO (B-NC=33.1 ± 2.3) and mainly produced pyrethroid-susceptible Anopheles (p<0.05). Significant variations in pH were not recorded. Differences in habitation by resistant- Anopheles in breeding sites contaminated with SEO or pesticide residues were observed. A. gambiae found around the two agricultural sites (Houeyiho, Benin and Ajibode, Nigeria) exposed to synthetic pesticides showed significant levels of pyrethroid resistance with mortality rates of 70.0 % and 89.7% respectively. A. gambiae larvae survived at ii 2 SEO concentrations below 11.8x10-3 μL/cm . Ninety six percent of larval mortality resulted from direct cuticle contact with SEO whereas only four percentage mortality was from larval suffocation. A cross resistance phenomenon was recorded with SEO and pyrethroids. A. gambiae showed the presence of elevated frequencies of knock down resistance West (kdr-W) mutations in Benin samples (kdr-W ranged from 0.6 to 0.9) and absence of kdr-W in Nigeria samples. Two detoxification genes (CYP6P3 and CYP6M2) were up-regulated in resistant-Anopheles. Additional detoxification genes specifYic to agricultural and SEO sites were also over-expressed in the resistant populations. R There was an association between residual synthetic pesticides, spilAled engine oil and emergence of pyrethroid-resistance in A. gambiae in Nigeria anBd BeRnin Republic. The diversified profile of identified metabolic genes reflected the iInfluence of a range of xenobiotics on selection of resistance in mosquitoes. L Keywords: Anopheles, Pyrethroid resistance, XenobioticYs. Word count: 487 IT R SE IV U N AN BA D I iii DEDICATION This project is dedicated to GOD almighty, who has being my source of inspiration and sustenance. To my home institution, colleagues, family members and friends for their constant support in the course of this programme. Y AR R LIB ITY ER S V UN I AN D I B A iv CERTIFICATION This is to certify that this work was carried out by MR. Jean Rousseau DJOUAKA-FOLEFACK in Cell Biology and Genetics Unit of Department of Zoology, University of Ibadan, Ibadan, Nigeria. ………………………………………. Y (SUPERVISOR) R Dr. A. A. Bakare A B.Sc., M.Sc., Ph.D (Ibadan) R Cell Biology and Genetics Unit. IB Department of Zoology, L University of Ibadan, Ibadan, Nigeria. ITY S R VE UN I N A D A IB v ACKNOWLEDGEMENT I would like to take this opportunity to express deep gratitude to my supervisor Dr. A. Bakare and my co-supervisor, Dr. Samson Awolola for their constant encouragement, supervision and guidance during this PhD research. Sincerely, you have given me a lot during this long and very exciting research venture. My profound gratitude to Dr. Ousmane Coulibaly for his guidance and immense support throughout the duration oYf this programme. I appreciate all the academic and non-academic staffs of the Department of Zoology, University of Ibadan for the prompt and constant willingness to giveR a helping hand throughout this research study. A The financial support for this PhD research work was partiallRy provided by the LSTM (Prof. Janet Hemingway) and the UNICEF/UNDP/WoIrldB Bank/WHO Special Programme for Research and training in Tropical DiseaseLs (TDR). I appreciate my laboratory supervisors at the Liverpool School of TrYopic al Medicine (LSTM) in the United Kingdom for sharing their scientific knowTledge with me and providing the necessary training in microarray and other molecIular works. Your contribution was of paramount importance in this research. To ProSf. Janet Hemingway Director of LSTM, I am very thankful for your scientificE andR financial contributions and for covering all expenses during my trips to the LSTM and for providing all laboratory consumables needed for this research work. II rVemember the sleepless night asking myself if I will be able to complete this Ph.D.N program; Thanks Prof. Janet, you made this a reality and I sincerely appreciate it. U Huge thanNks g oes to my family for being incredibly supportive, understanding and patient not onAly throughout this study, but in everyday of my life. I will like to thank almighty DGod for giving me a good health and empowering me with more knowledge, the best wAeapon for survival. IB vi TABLE OF CONTENT Page Title page……………………………………………………………………………………i Abstract…………………………………………………………………………………….ii Dedication ………………………………………………………………………………. .iv Certification ……………………………………………………………………………….v Acknowledgement………………………………………………………………………Y…vi Table of contents …………………………………………………………………R………vii List of tables ……………………………………………………………………A……..…xiv List of figures …………………………………………………………B…R……………….xv List of appendix ……………………………………………………I…………………….xix CHAPTER ONE ………………………………………………L………………………..…1 1.0 INTRODUCTION ……………………………………Y…… …………………………..1 1.1 Aim of this study…………………………………IT…………………………………….4 1.2 Objectives of this study ……………………S……………………………………..…....5 CHAPTER TWO …………………………R…………………………………………..……6 2.0 LITERATURE REVIEW …………E……………………………………………...……6 2.1 Malaria disease and transmisIsioVn of malaria parasites………..………………………..6 2.2 The biology and the ecology of malaria vectors ...…………..………………….…….9 2.2.1 Adults …………………N………..…………………………………………………..11 2.2.2 Mating and bloo d-Ufeeding of adult Anopheles ……………………………………..11 2.2.3 The ecoAlogyN of breeding sites ………………………………………………………12 2.3 FactoDrs involved in malaria transmission ……………………………………….……12 2.3.1A Preferred sources for blood meals …………………………………………………13 2B.3.2 Life Span ………………………………………………………………………...…13 I2.3.3 Patterns of feeding and resting ……………………………………………….…….13 2.4 Major vectors of malaria in sub-Saharan Africa. …………………………………….13 2.4.1 The A. gambiae complex ...……………..………………………………………..…14 2.4.2 A. funestus group ...…………..………………………………………….………….14 2.5 Vector Identification ………...…………..………………………………………..…15 2.5.1 Application of molecular techniques in the identification of sibling species …...…16 vii 2.5.1.1 Restriction fragment length polymorphism ………………………………………18 2.6 Malaria vector control strategies ……………………………………………………..19 2.6.1 Insecticide-treated bed nets ………………………………………………………...19 2.6.1.1 Bednets ownership patterns in Nigeria …………………………………………...22 2.6.1.2 Bednets ownership patterns in Benin Republic …………………………………..23 2.6.2 Indoor residual spraying ……………………………………………………………24 2.6.3 Breeding sites reduction …………………………………………………………Y…25 2.6.4 Less implemented vector control strategies…………………………………R……...25 2.6.5 The development of genetically modified Anopheles (GMA) for malariAa control ..26 2.7 The use of insecticides in Public Health ………………………………R……………..26 2.7.1 Insecticides and mode of action ………………………………IB……………………27 2.7.1.1 Organochlorine ………………………..……………… L………………………….27 2.7.1.2 Organophosphate ...……………..…………………Y……………………………...30 2.7.1.3 Carbamates ……………………..……………T…………………………………...30 2.7.1.4 Pyrethroids ……………………..………S……I…………………………………...30 2.7.2 Protective mechanisms of Anopheles Rand development of resistance to insecticides... …………………………………………………………………..31 2.7.2.1 Resistance of Anopheles to inseEcticides ……………………..……………….…31 2.7.2.2 Behavioral mechanisms oIf VN resistance ……………………..…………………..…34 2.7.2.3 Metabolic mechanisms of resistance ……………………..………………………34 2.7.2.3.1 Cytochrome PU450s (monoxygenase or oxidase mechanisms) …………………34 2.7.2.3.2 Glutathione- S-transferase ……………………..………………………………..34 2.7.2.3.3 TargAet siNte modification (mutations) ……………………..…………………….34 2.8 DetecDtion of insecticide resistance in malaria vectors ……………………..………...36 2.8.1A Bio-assay for phenotyping insecticide resistance in vectors ……………………….36 I2B.8.2 The bottle tests with synergists ……………………..……………………………...38 2.8.3 Polymerase chain reaction (PCR) for target modification …………………………38 2.8.4 Microarray technique for detection of detoxification genes ……………………….38 2.8.5 Reported cases of resistance of A. gambiae to insecticides in Nigeria …………….39 2.8.6 Reported cases of resistance of A. gambiae to insecticides in Benin ………………40 viii 2.8.7 Reported cases of resistance of A. gambiae to insecticides in other African countries ………………………………………………………...…40 2.9 Factors favouring the emergence of resistance in mosquito populations ………….…41 2.9.1 The selection of insecticide resistance by ITNs and IRS ………………………..…41 2.9.2 Agricultural pesticide residues and other xenobiotics ……………………………...42 2.9.3 Types of mechanisms of resistance selected by ITNs, IRS, agricultural pesticideY residues and xenobiotics ……………………………………………………….…43 CHAPTER THREE ……………………………………………………………A…R………44 3.0 MATERIALS AND METHODS……………………………………………………..44 3.1 Susceptibility of Anopheles populations to pyrethroid in studied sitesR ………………44 3.1.1 Description of sampling sites …………………………………IB…………………....44 3.1.2 Collection of Anopheles larvae ………………………… …L……………………….44 3.1.3 Rearing of larvae to adult stage in the insectary ……Y……………………………...44 3.1.4 WHO susceptibility tests on adult Anopheles fIroTm surveyed localities ……………48 3.1.5 Mapping of permethrin susceptibility in Anopheles populations from studied localities …………………S…………………………………………48 3.2. Molecular characterization of AnophelRes populations from studied sites: PCR-species, PCR-forms, PCRE-kdr………………………………………………50 3.2.1 DNA extraction ………N……I V……………………………………………………….50 3.2.2 Polymerase chain reaction for A. gambiae speciation ……………………………...54 3.2.3 Polymerase chai n Ureaction for detection of the knock down (kdr) mutation in A. gambNiae populations ………………………………………………………..54 3.2.4 SpecificA primers used for PCR-species and PCR-kdr………………………………54 3.2.5 EleDctrophoresis of PCR products …………………………………………………..55 3.3 EAvaluation of potential contributions of agricultural pesticide residues and spilled IB petroleum products in the selection of pyrethroid resistance in Anopheles populations ……………………………………………………………………….55 3.3.1 Screening of pesticide residues in water and soil samples from vegetable sites …...55 3.3.1.1 Screening technique …………………………………………………………...…55 3.3.1.2 Collection of water and soil samples used for the bio-assay ……….…………….55 3.3.1.3 History of synthetic pesticides utilization by farmers in target agricultural sites...56 ix 3.3.2 Evaluation of the contribution of petroleum products in the selection for insecticide resistance in A. gambiae …………………………………………………………58 3.3.2.1 KAP studies on the empirical utilisation of petroleum products (PP) in rural communities ……………….………………………………………………….….58 3.3.2.2 Anopheles populations used for analysing the lethal activities of PP. ………...…58 3.3.2.3 Determination of lethal concentrations of 4 PP on larvae of A. gambiae ………..58 3.3.2.4 Identification of the mode of action of PP on Anopheles larvae………………Y.…58 3.3.3 Analysis of associations between the presence of petroleum products in breReding sites and the emergence of pyrethroid resistant populations of AnopheAles…….…61 3.3.3.1 Physico-chemical properties of breeding sites of resistant and susRceptible Anopheles ……………….…………………………………I…B…………………..61 3.3.3.2 Selection of oviposition spots by gravid females of AnoLpheles in localities where some breeding sites are contaminated with spilledY pet roleum products …………61 3.3.3.3 Monitoring of the development of Anopheles Tlarvae (resistant and susceptible strains) in breeding sites with petroleumS proIducts residues ………..………….…62 3.4 Screening of candidate metabolic genes overexpressed in pyrethroid resistant populations of A. gambiae from BeRnin and Nigeria ………………..………….…62 3.4.1 Selection of permethrin resistant EA. gambiae for micro-array analysis ……………62 3.4.1.1 Target preparation anNd mIic Vroarray hybridizations ……………….………………63 3.4.1.2 cDNA synthesis, labelling and hybridization ……………….……………………63 3.4.1.3 Array scanning aUnd visualization ……………….……………………………..…67 3.5. Data analysisN …… ………….………………………………….…………………….67 3.5.1 AnalysisA of data on the susceptibility level of Anopheles to permtehrin: ….………67 3.5.2 QueDstionaires, focuss group discussions and indepth-interviews with farmers and Apetroleum products users …………..…………..…………..……………………..67 I3B.5.3 Lethal activities of petroleum products on Anopheles larvae …………..……….…69 3.5.4 Cross analysis of the physico-chemical properties of breeding sites and the susceptibility status of emerging Anopheles populations …………………..….…69 3.5.5 Analysis of data on the oviposition preference of gravid Anopheles, eggs hatching rates and larval developments in simulated breeding sites …………..…………..69 x 3.5.6 Allelic frequencies of the kdr mutation in permethrin resistant and susceptible phenotypes of Anopheles mosquitoes analysed ………..……………………...…71 3.5.7 Analysis of micro-array spots for expressed metabolic genes identified in studied Anopheles populations …………………………………………………………....71 CHAPTER FOUR ………..…………………..………………………………………..…73 4.0 RESULTS ………..…………………..…………………………………………….…73 4.1 Screening of the susceptibility of Anopheles populations to pyrethroid in Y southwestern Nigeria and southern Benin ………..………………………R...…….73 4.1.1 Susceptibility to permethrin of A. gambiae populations in southwesternA Nigeria …73 4.1.2 Susceptibility to permethrin of A. gambiae collected in southern BRenin ………..…75 4.2 Molecular characterization of Anopheles populations from survIeByed sites in Nigeria and Benin (PCR-species, PCR-forms and PCR-kdr) ……L…..………………...…78 4.2.1 Molecular characterisation of mosquito populationsY from the southwestern Nigeria………..……………………………I…T………………………………...…79 4.2.2 Molecular characterisation of mosquito poSpulations from the southern Benin ……79 4.3 Evaluation of potential contributions ofR agricultural pesticides in the selection of pyrethroid resistance in Anopheles populations breeding around vegetable farms .…88 4.3.1 The use of synthetic pestici EIdeVs in the vegetable farm of Houeyiho in Benin ……...88 4.3.2 The use of synthetic pesticides in the vegetable farm of Ajibode in Nigeria ………88 4.3.3 Susceptibility to permeNthrin of A. gambiae collected around vegetable farms of HoueyihoU and Ajibode ………..……………………………………….…89 4.3.4 Assessment Nof th e presence of pesticides residues in breeding sites found around vegetable sites through monitoring of the hatching rates of Anopheles eggs ……90 4.3.5 AssDessmAent of the presence of pesticides residues in breeding sites found around Avegetable sites through monitoring of larval development rates……………….…93 I4B.4 Evaluation of the potential contributions of spilled petroleum products in the selection of pyrethroid resistance in A. gambiae populations ……………….….99 4.4.1 The empirical utilisation of petroleum products by rural communities in the southern Benin for mosquito control. ………..……………………………….….99 4.4.2 Analysis of lethal effect of petrol on permethrin resistant larvae of A. gambiae from Ladji and from Ojoo ………..…………………………….…100 xi 4.4.3 Analysis of lethal activity of kerosene on permethrin resistant larvae of A. gambiae from Ladji and from Ojoo ………………………………………………...……104 4.4.4 Analysis of lethal effect of new engine oil on permethrin resistant larvae of A. gambiae from Ladji and from Ojoo ……………………...……..…106 4.4.5 Analysis of lethal activity of used engine oil on permethrin resistant larvae of A. gambiae from Ladji and from Ojoo …………………….…………106 4.4.6 Identification of the mode of action of PP on A. gambiae larvae ……………….Y..107 4.4.7 Analysis of physico-chemical properties of breeding sites producing R pyrethroids resistant and susceptible populations of A. gambiae in A southwestern Nigeria and southern Benin…………………………R………….…110 4.4.8 Identification of the preferred types of breeding sites selectedI bBy pyrethroids susceptible and resistant strains of Anopheles for ovip osLitions ……..……….…115 4.4.9 Hatching rate of eggs laid by pyrethroids susceptiblYe and resistant strains in oily breeding sites from Nigeria and BeninT …………………………………118 4.4.10 Development of larvae of pyrethroid susSceptIible and resistant strains in oily breeding sites (rate of larvaeR getting to pupae stage) ………………...…118 4.5 Identification of detoxifying genes Eup-regulated in pyrethroids resistant Anopheles from sites of sIpVilled petroleum products and agricultural sites under pesticidesN utilisation ………………………………………………...120 4.5.1 Genotyping and bioassay of Anopheles populations prior to micro-array analysis ……… …U……………………………………………………………….120 4.5.2 IdentificatioNn of metabolic genes over transcribed on Anopheles samples from aAgricultural setting ……………………………………………………...…122 4.5.3 IdenDtification of metabolic genes over transcribed on Anopheles samples Afrom oil spillage site ………………………………………………………….…122 I4B.5.4 Comparative expression profiles of metabolic genes in permethrin resistant Anopheles from Akron in southern Benin and Ojoo in the southwestern Nigeria………………………………………………………….…122 CHAPTER FIVE ………………………………………………………………………..130 5.0 Discussion ………………………………………………………………………..…130 5.1 The susceptibility pattern of Anopheles populations to pyrethroid in xii southwestern Nigeria and southern Benin ………………………………..……..130 5.2 Genotyping of permethrin resistant phenotypes ………………………………….…131 5.3 The hatching rate of Anopheles strains in breeding sites simulated with soil and water samples from vegetable farms ……...………………………...…132 5.4 Water and soil samples from vegetable farms contain compounds that inhibit Anopheles larval development. …………………………………………………133 5.5 The implication of pesticides residues in the emergence of pyrethroid Y resistance in malaria vectors ………………………………………………R……134 5.6 The treatment of mosquito breeding sites with petroleum products and A the selection of pyrethroid resistance in malaria vectors ……B……R……………135 5.7 Identification of the mode of action of PP on A. gambiae larvaIe …………………..136 5.8 Inhibitory effects of petroleum on Anopheles oviposition anLd larval development ..136 5.9 Existence of a cross resistance between petroleum proYducts and permethrin in sampled Anopheles………………………I…T………………………………...137 5.10 Identification of detoxifying genes up-reguSlated in pyrethroids resistant Anopheles from sites of spilled petroleum products and from agricultural areas under pesticides utilisatioEn …R……………………………………………..138 5.11 Differential expression of mIetVabolic genes by A. gambiae populations collected around vegNetable farms and those from localities of spilled petroleum products ……………………………………………………………...140 CHAPTER SIX ……… U…………………………………………………………………142 6.0 CONCLUSIONN AND RECOMMENDATIONS …………………………………...142 References…A…………………………………………………………………………….145 AppendixD ………………………………………………………………………………..165 IB A xiii LIST OF TABLES Page 4.1 Susceptibility to permethrin of A. gambiae in southwestern Nigeria …………...…...74 4.2 Susceptibility of A. gambiae populations to permethrin in southern Benin…………..77 4.3 Distribution of members of A.gambiae complex in studied localities in Nigeria…….82 4.4 Allelic frequencies of the kdr mutation in Anopheles species from the studied localities in Nigeria…………………………………………………………R……Y.83 4.5 Distribution of members of A. gambiae complex in studied localities in Benin……..86 4.6 Distribution of the kdr alleles in Anopheles species from the studied locaAlities in Benin……………………………………………………………R……………..87 4.7 The use of pesticides in the vegetable farm of Houeyiho in BenIinB and Ajibode in Nigeria………………………………………L………………………...91 4.8 Comparison of the mean mortality rates to permethrinY of A. gambiae populations produced by oily and non-oily sites in NigerIiaT………………………………….112 4.9 Physico chemical parameters (pH and DO) Sof breeding sites producing susceptible and resistant populations of Anopheles in southwestern Nigeria………………..113 4.10 Comparison of the mean mortalityE ratRes to permethrin of A. gambiae populations produced by turbid and noInV-turbid sites in southern Benin……………………..114 4.11 Physico chemical parameters (pH and DO) of breeding sites producing susceptible and resistNant populations of Anopheles in southern Benin………….116 4.12 Molecular form, peUrcentage mortality and kdr frequency of A. gambiae from southNern Benin and southwestern Nigeria…………………………………123 4.13 CandidatAe metabolic genes from micro-array analysis of resistant and susceptible field samples from Akron and Orogun……………………………...125 4.14 ACanDdidate metabolic genes up-regulated in resistant populations of IB Anopheles from Ojoo …………………………………………………………...127 xiv LIST OF FIGURES Page 1.1 Trends of malaria in the world…………………………………………………………2 2.1 The life Cycle of malaria parasite……………………………………………………...8 2.2 The biological Cycle of Anopheles species…………………………….......................10 2.3 The exponential amplification of the gene in Polymerase Chain Reaction (PCR); (A) represents the denaturation phase, (B) the annealing or Y hybridization and (C) the amplification or extension phase.………….......R...........17 2.4 Chromosomal inversions in A. gambiae females; this mosquito is A heterokaryotypic for inversions 2Rb and 2Rc (A) as well as the R 2La inversion (B) ……………………………....................I...B................................20 2.5 The use of insecticide-treated bed nets (ITNs) for malaria vLector control …………...21 2.6 The different targets of insecticides in Anopheles mosYquit oes……………………….28 2.7 Chemical structure of some insecticides used in pTublic health……………………….29 2.8 Chemical equations of enzymatic activities deveIloped by mosquitoes to withstand insecticide lethal doses......S.................................................................32 2.9 Series of mechanisms of resistanceE to inRsecticides developed by Anopheles………….33 2.10 Mutation on the sodium chaInnVel in kdr resistant Anopheles from West and East AfricaN……………………………...................................................37 3.1 Map of study areas (Divisions and States) in Benin and Nigeria………………..........45 3.2 Identification of breUeding sites and field collection of Anopheles larvae……….........46 3.3 The horizontaNl pos ition of Anopheles larvae at the surface of water…………………47 3.4 WHO bioAassay steps for analysing the susceptibility status of Anopheles to inDsecticides……………………..……………………………………………........49 3.5 DANA extraction for PCR analysis of collected Anopheles samples……………..........51 I3B.6 DNA amplification (PCR reactions) of collected Anopheles samples………………..52 3.7 Agarose gel migration of PCR products of amplified Anopheles DNA……………...53 3.8 Map of sites selected for screening of pesticides residues in vegetable farm (Akpakpa-CREC, Houeyiho, Ajibode and UI- Ibadan)………………….............57 3.9 Quantification of the lethal activity of 4 petroleum products on Anopheles larvae introduced in plates containing different concentrations of PP……………60 xv 3.10 Map showing the 3 study sites selected for candidate metabolic gene search and their locations in relation to the control site (Orogun)………………............64 3.11 Steps for producing and labeling cDNA of Anopheles samples from target sites (Ojoo, Akron and Orogun) for micro-array hybridization…………………..........65 3.12 Gel Analysis of migrated total RNA prior to cDNA production……………………66 3.13 Steps for synthesis and printing of detoxifying gene probes of A. gambiae on the “Detox” chip array……………….…………………………………………Y…68 3.14 Visualisation and analysis of scanned microarray slide; the red spots correspRond to upregualtion of genes from resistant Anopheles on a given probe whiAle the green dots correspond to up regulation of genes from susceptible pBopulRations of Anopheles……………………………………………………I……………………72 4.1 Map of permethrin susceptibility status of Anopheles pop ulLations in the surveyed localities of southwestern Nigeria and southern BYenin…………………………...76 4.2a Members of the A. gambiae complex identifiedI iTn southwestern Nigeria…………..80 4.2b Samples from the locality of Challenge: the banding patern shows Anopheles gambiae ss (coded “g”) and A. arabRiensSis (coded “Ar”) living together in similar site………………………………………………………..…...81 4.3a Mechanisms of permethrin resistaEnce identified in southwestern Nigeria and southernN BenIi Vn……………………………………………………....84 4.4 Members of the A. gambiae complex identified in the southern Benin……………....85 4.3b The kdr Banding pUattern recorded with samples from Benin…………………….....89 4.5 Hatching rate Nof re sistant and susceptible strains of Anopheles in simulated water Aand soil samples from vegetable farms of Houeyiho ……………………...92 4.6 HatchDing rate of resistant and susceptible strains of Anopheles in simulated water and soil samples from vegetable farms of Ajibode…………………….......94 I4B.7 L Aarval development of resistant and susceptible strains of Anopheles in simulated water and soil samples from vegetable farms of Houeyiho…………....95 4.8 Larval development of resistant and susceptible strains of Anopheles in simulated water and soil samples from vegetable farms of Ajibode……………...97 4.9 The yield of rearing resistant and susceptible strains of Anopheles in simulated water and soil samples from vegetable farms of Houeyiho………..…..98 xvi 4.10 The yield of rearing resistant and susceptible strains of Anopheles in simulated water and soil samples from vegetable farms of Ajibode, Ibadan……………………………………………………………………..……..100 4.11 Utilisation of petroleum products for mosquito control in rural communities in southern Benin……………………….........................................102 4.12 Larvicidal effect of petroleum products on larvae of A. gambiae from Ladji; a similar mortality trend is recorded with most PP except petrol Y which shows a lower larvicidal effect .…….........................................A......R..........103 4.13 Larvicidal effect of petroleum products on larvae of A. gambiae from Ojoo.……..105 4.14 Comparative analysis of recorded HIC of petroleum products on larRvae of Anopheles gambiae from Ladji and from Ojoo larvae.……I..B..............................108 4.15 Comparative analysis of recorded LoC100 of petroleum p roLducts on larvae of A. gambiae from Ladji and Ojoo larvae.…….......Y................................................109 4.16 Mode of action of petroleum products on AnophTeles larvae (Mortality rates recorded with sieved anSd crIude or raw petroleum products from field contaminated breeding siRtes) .……......................................................111 4.17 Number of eggs laid by resistant (A. gambiae from Ojoo) and susceptible (A. gambiae from UEI) strains of Anopheles in oily and non-oily breeding sites frIomV southwestern Nigeria and southern Benin…..........117 4.18 Hatching rate of eggs laNid by resistant (A. gambiae from Ojoo) and susceptibl e U(A. gambiae from UI) strains of Anopheles in oily and non-oily bNreeding sites from Nigeria and Benin…..............................................119 4.19 Rate of lAarvae (larvae from hatched eggs) of A. gambiae from ODjoo and A. gambiae from UI developing to pupae stage in oily Aand non-oily breeding sites from southwestern Nigeria and southern Benin.......121 I4B.20 Candidate metabolic genes over transcribed on resistant Anopheles mosquitoes from the agricultural site of Akron when co-hybridized with the susceptible strain A. gambiae from Orogun…………….….……...........................................124 4.21 Candidate metabolic genes over transcribed on resistant Anopheles mosquitoes from the oil spillage locality of Ojoo when co-hybridized with the susceptible strain A. gambiae from Orogun.……....................................................................128 xvii 4.22 Cohort of expressed genes in resistant population of A. gambiae from the Agricultural site of Akron in Benin and the site of spilled oil of Ojoo in Nigeria.……...................................................................................129 Y RA R B L I ITY RS VE UN I AN D I B A xviii LIST OF APPENDIX Page 1. Indepth interview guide on knowledge, attitudes and practices (KAP) of communities on synthetic pesticides use in Agriculture …………………………165 2. Indepth interview guide on knowledge, attitudes and practices (KAP) of communities on the use of petroleum products (PP) for mosquito control ………Y166 3. WHO bioassay for insecticide susceptibility using adult mosquitoes …………R……167 4. Laboratory protocols and preparation of solutions …………………………A…….….169 5. Characteristics and toxicity of petroleum products on insects…………R……….…… 171 LI B SI TY ER NI V U AN BA D I xix CHAPTER ONE INTRODUCTION Malaria is the most important parasitic disease in the world (Holt et al., 2002). It is thought to be responsible for 500 million cases of illness and up to 2.7 million deaths annually, more than 90% of which occur in sub-Saharan Africa (Holt et al., 2002). Malaria morbidity and mortality in tropical Africa remain disproportionately high (Figure 1.1), compare to other malaria-endemic areas of the World (Gallup et al., 2001R). TYhis is partly due to three efficient vectors of the subgenus Cellia: A. gambiae, A. arabiensis, and A. funestus. These species occur together geographically across sub-SaharAan Africa and can inhabit the same villages, shelter in the same houses, and feed oBn thRe same individuals (Sharakhov et al., 2002). I Till very recent, the focus of malaria control programLmes in Africa was on the management of sick children through early treatment Ywith effective antimalarial drugs (Sirima et al., 2003). However, this cannot be the finTal strategy considering the life cycle of the parasite and the various hosts this parasitSe paIsses through (Miller et al., 2002). With the fast spread of resistance of Plasmodium species to easily available drugs coupled with the increasing price of effective antiE-maRlaria treatments and the absence of vaccines, National malaria control progIraVms (NMCP) are becoming increasingly reliant on strategies targeting the mosqNuito vectors (Toure et al., 2001). The control of malaria vectors aim at reducing insect vector densities, their longevities or limitin gU their contacts with humans and therefore reducing diseases transmission, moNrbidity and mortality (Toure et al., 2001). This can be done through various methoAds including: chemical (using insecticides based vector control tools such as LLINs aDnd larviciding ), physical (destruction of breeding sites and environmental sanitaAtion), or with biological (using predators or other natural parasites of the vector) I(BLawler and Lanzaro, 2005) methods. Considering the distribution of malaria disease in sub-Saharan Africa (Snow et al., 1999) and taking into consideration the bio-ecology of vectors (Depinay et al., 2004), Anopheles control strategies mainly used are based on synthetic insecticides (adulticides and larvicides) with focus on indoor residual spraying (IRS), insecticide treated nets (ITNs) of long lasting impregnated nets (LLINs) type and breeding sites treatment. ITNs constitute effective protective barriers for controlling 1 Y RA R LIB ITY RS IV E U N N 1994 1966 DA 1946 Fig.1.1 Trends of malaria in the world A Source: Gallup et al. (2001) IB 2 malaria vectors and have been found to significantly reduce malaria transmission rate (Carnevale et al., 1988). IRS have shown effectiveness in localities under malaria endemic conditions; this strategy significantly reduces the densities of malaria vectors, rapidly stops the transmission of the disease by destroying adult female Anopheles perpetuating the transmission in endemic areas (Brooke et al., 2000). Breeding site treatments destroy mosquito at the larval stage and thereby, reduce the overall densities of Anopheles Yin the communities. Unfortunately the emergence of mosquito populations capable of withstanding insecticide exposure is threatening these control measures (HeAminRgway and Ranson, 2000). The emergence of new populations of Anopheles that are resisRtant to the type of insecticides used in public health is increasingly growing (AkogbIetBo et al., 1999; Brooke et al., 2001; Awolola et al., 2002). This resistance phenomenLon affects mainly the major vectors of malaria: A. gambiae s.l. and A. funestus s.l. YThe resistance of A. gambiae to pyrethroids is now well established in West, CentrTal, East and South Africa. In South Africa, resistance to pyrethroids has been detecSted Iin A. funestus (Coetzee et al., 1999). In West Africa, A. gambiae resistance to the Rfour major classes of insecticides available for public health has been reported (Elissa et al., 1993; Akogbeto et al., 1999; Chandre et al., 1999; Awolola et al., 2002; DiabIaVte et E al., 2002; Fanello et al., 2003). Pyrethroids are the only insecticide licensed forN impregnation of nets and house spraying, hence resistance to this class of insecticides is of concern, particularly as there has been a substantial increase (>60% coverage) in th e Unumber of people using bednets in Africa (WHO Report, 2005). InsecticideN resistance can occur via target site insensitivity and/or metabolic detoxificationA. Target site resistance to pyrethroids and DDT in A. gambiae is due to a substitutiDon at a single codon in the sodium channel gene, and is referred to as knock-downA resistance (kdr). Two kdr alleles occur in A. gambiae; a leucine to phenylalanine IsBubstitution, known as West kdr (Martinez-Torres et al., 1998) and a leucine to serine substitution known as East kdr (Ranson et al., 2000). Metabolic resistance is predominantly caused by elevated activities of one or more members of three large multigene enzyme families; cytochrome P450 monooxygenase (P450s), glutathione S- transferases (GSTs) and carboxylesterases (COEs). In A. gambiae these gene families have 111, 31 and 51 members respectively (Ranson et al., 2002). 3 The emergence of resistance to pyrethroids in a number of African mosquito populations and its potential implications on the efficacy of current vector control tools highlights the importance of research into the effect of resistance on malaria transmission including the underlying mechanisms causing the resistance (Hemingway and Ranson, 2000). Long lasting impregnated nets used in most countries for malaria vector control are treated with pyrethroid insecticides hence, the emergence of pyrethroid resistance in Anopheles species is likely to threaten the efficacy this major malaria preventive Ytool . Results of studies from various regions of Africa suggest the need for close monRitoring of resistance, the identification of factors selecting for resistance and thAe analysis of mechanisms of resistance using advanced and highly sensitive moleculaRr tools. Several human practices have been identified as facItoBrs implicated in the emergence of insecticide resistance in malaria vectors; misus eL and overuse of agricultural pesticides by farmers have probably led to widespread iYnsecticide resistance in mosquito vector populations (Akogbeto et al., 2006; ChouaTibou et al., 2008). Molecular tools currently available have been helpful in ideSntifIying few mechanisms and pathways responsible for resistance (Martinez TorreRs et al., 1998; Ranson et al., 2000). However, these tools need to be more sensitive fEor further investigations on the various mechanisms of insecticide resistance. The type of responses anIdVN the mechanisms of resistance developed by mosquito populations when subjected to selection pressures from human activities or from environmental change s Uhave always been highly complex and difficult to predict. These adaptive mechaniNsms coupled with series of responses to environmental stimulis need to be further anaAlysed using Anopheles samples from various sites subjected to a wide range of stimulDis. The nature of mosquito habitats and the presence of different xenobiotics in their Abreeding sites could have a profound influence on mosquito development and the IsBelection of different resistance mechanisms in malaria vectors at larval stage and adult stages. 1.1 Aim of this study. This study aims at identifying factors responsible for the emergence of insecticide resistance and to analyse the mechanisms of pyrethroid resistance developed by A. gambiae in the southwestern Nigeria and southern Benin Republic. 4 1.2 Objectives of this study 1- Identify factors contributing to the emergence of pyrethroid resistance in A. gambiae species from southwestern Nigeria and southern Benin Republic. 2- Analyse the implication of target site mutation (kdr) in the selection for resistance in A. gambiae sl. in the southwestern Nigeria and southern Benin Republic 3- Analyse the implication of metabolic genes (Detox-genes) in the selection for resistance in A. gambiae sl.in the southwestern Nigeria and southeRrn BYenin Republic. A R LI B SI TY R E NI V N U A D I B A 5 CHAPTER 2 LITERATURE REVIEW 2.1 Malaria disease and transmission of malaria parasites Malaria remains a major killer, particularly in sub-Saharan Africa, with more than 1 million deaths among children every year. The reported case of malaria around the world is put at 243 million, with an estimated death of 863,000 (WHO, 2009). Eighty-nine percent of these deaths occurred in Africa (World Malaria Report, 2009). As at 2008Y, half of the people around the world are reported to be at risk of malaria. Out oRf the 109 countries where malaria is reported to be endemic, 45 are within the WHO AAfrica region. Nigeria accounted for one fourth of all estimated malaria cases in tRhe WHO African region in 2006 (World Malaria Report, 2009). IB In Nigeria, malaria accounts for much of the disease buLrden with about 97% of the approximately 150 million people at risk. It accounYts fo r 25% of all infant related mortality, 30% of child related mortality and 11% ofT maternal mortality (WHO Statistics, 2007; World Malaria Report, 2009). A large percIentage of the population affected with this disease live in extreme poverty in ruraSl areas with few having access to good healthcare facilities (Otubanjo and MafEe, 2R002; Amexo et al., 2004; Obrist et al., 2007). The human malaria parasite in Nigeria includes: Plasmodium falciparum, Plasmodium ovale and PlasNmodIiu Vm malariae. However, P. falciparum is responsible for more than 95% of all malaria cases transmitted. Malaria parasites are usually transmitted through the bites of in fUected female mosquitoes of the genus Anopheles. This species is widely distributedN across the different ecological zones in Nigeria where suitable sub Saharan climaAtic conditions exist (Molineaux and Gramiccia, 1980; Kiszewski, 2004). StDudies on malaria transmission conducted in Northern Nigeria have identified eleveAn species of Anopheles mosquitoes: A. gambiae sensu stricto, A. arabiensis, A. IfBunestus, A. rufipes, A. pharoensis, A. wellcomei, A. squamosus, A. coustani, A. maculipalpis, A. nili and A. pretoriensis of which 2 species; A. gambiae and A. funestus were regarded as main vectors (Bruce – Chwatt, 1951; Hanney, 1960; Service 1965; Boreham et al., 1979; Molineaux and Grammicia 1980; Rishikesh et al., 1985). Most of the studies in Southern Nigeria have focused on the bionomics and the characterization of breeding sites of malaria vectors such as A. gambiae s.s., A. arabiensis, 6 A. melas, A. funestus, A. nili, A. pharoensis, A. coustani and A. moucheti (Barber and Olinger, 1931; Muirhead-Thomson, 1947; Mattingly, 1949; Service, 1961; Nwoke et al., 1993). Reviewed studies on malaria vectors characterisation in Nigeria relied mainly on the use of morphological keys of identification (Okorie, 1973; Mafiana et al.,1998; Aigbodion and Odiachi 2003). However, the advent of molecular and immuno diagnostic tools such as PCR, RT-PCR, micro-array and enzyme link imunosorbant assay (ELISA) have alleviated the difficulties associated with identifying morphological indistinguisYhable members belonging to species complexes and the incrimination of Anopheles spRecies that are involved in malaria transmission (Service, 1993). Despite the deAvelopment of molecular techniques, only few studies have utilized them in malaria Rvector research in Nigeria (Awolola et al., 2003; Onyabe and Conn, 2003; Okwa et aIl.B, 2007; Oyewole et al., 2007). L The life cycle of malaria parasites is complexY an d passes through two hosts: Human, and Anopheles species. In humans the cycle Tinvolves trophozoites and merozoites production followed by differentiation into gameItocytes; in the invertebrate hosts, the parasites are ingested by mosquitoes atR the Sgametocytes stage during blood feeding. These gametocytes ex-flagellate and mate to produce zygotes. From the zygotes develops the ookinetes, the oocystes and fIinVally E the sporozoite stage. At this last step, the parasites migrate massively to the salNivary gland of Anopheles where they are ready for inoculation during another blood feeding of the mosquito (Fig. 2.1) (Bruce et al., 1980). Malaria contr olU is based on prompt diagnosis, appropriate drug treatment, protection of higNh-risk groups, and control of the mosquito vector. Optimism that mosquito-borne diseases such as malaria, dengue, and filariasis can be effectively controlledD or Aeven eradicated with inexpensive drugs, vaccines, or insecticides has been sorelAy tested (WHO, 1961) . Today, the efficacy of drugs is becoming debatable, vaccine IdBevelopment is slow, and mosquitoes are becoming resistant to insecticides, including those used to treat bed nets (Scoot et al., 2002). Current malaria control initiatives encourage the use of insecticide treated nets/long lasting impregnated nets (ITN‟s/LLINs), indoor residual spraying (IRS) and larviciding in an integrated vector management (IVM) strategy (Beier et al., 2008). Pyrethroids treated bednets, offer personal protection from mosquito bites and have been 7 RY RA LI B ITYS ER NI V U DA N A I B Fig. 2.1 The life Cycle of malaria parasite Source: NIH /CDC (2004) 8 found to significantly reduce malaria transmission (Trigg and Wernsdorfer, 1999; Maxwell, 2002). Despite the encouraging results recorded from Insecticide Treated Nets (ITNs), resistance of the major vector to pyrethroid insecticides have emerged and is now widespread in West Africa (Chandre et al., 1999; Hemingway and Ranson, 2000; Nwane et al., 2009). Insecticide resistance is reported to threaten most malaria control intervention tools; for example in Benin, reduced efficacy of ITN and IRS has been associated with insecticide resistance (Nguessan et al., 2007). Y 2.2 The biology and the ecology of malaria vectors R Like all mosquitoes, Anophelines go through four stages in their liAfe cycle: egg, larva, pupa, and adult (Fig.2.2). The first three stages are aquatic anRd last 5-14 days, depending on the species and the ambient temperature. The adult IstaBge is when the female Anopheles mosquito acts as malaria vector. The adult femal esL can live up to a month (or more in captivity) but most probably do not `live moreY than 2-3 weeks in nature (Bruce Chwatt, 1993). IT 2.2.1 Eggs and larvae S Adult females lay 50-200 eggs per Roviposition. Eggs are laid singly and directly on water and are unique in having floats Eon either side. Eggs are not resistant to drying and hatch within 2-3 days (Fig.2.2) (BrVuce Chwatt, 1993). Mosquito larvae have a wIell-developed head with mouth spikes (brushes) used for feeding, a large thora N xU and a segmented abdomen. Anopheles larvae lack a respiratory siphon and for thiNs reason position themselves so that their body is parallel to the surface of the water. ALarvae breathe through spiracles located on the 8th abdominal segment and thereforeD must come to the surface frequently. The larvae spend most of their time feeding on alAgae, bacteria, and other microorganisms in the surface microlayer. They dive below tBhe surface only when disturbed. Larvae swim either by jerky movements of the entire Ibody or through propulsion with the mouth brushes. Larvae develop through 4 stages, or instars, after which they metamorphose into pupae. At the end of each instar, the larvae moult, shedding their exoskeleton, or skin, to allow for further growth. The larvae occur in a wide range of habitats but most species prefer clean, unpolluted water (Fig.2.2) (Bruce Chwatt, 1993). 9 AR Y BR Y LI SI T ERV UN I AN AD IB Fig. 2.2 The biological Cycle of Anopheles species Source: Modified after CDC pictures, NIH /CDC (2004) 10 2.2.2 Pupae and adults The pupa is comma shaped when viewed from the side. The head and thorax are merged into a cephalothorax with the abdomen curving around underneath. As with the larvae, pupae must come to the surface frequently to breathe, which they do through a pair of respiratory trumpets on the cephalothorax. After a few days as a pupa, the dorsal surface of the cephalothorax splits and the adult mosquito emerges (Fig.2.2) (Bruce Chwatt, 1993). Y Adult Anopheles have slender bodies with 3 sections: head, thorax and Rabdomen. The head is specialized for acquiring sensory information and for fReediAng. The head contains the eyes and a pair of long, many-segmented antenInBae. The antennae are important for detecting host odours as well as odours of breeding sites where females lay eggs. The head also has an elongate, forward-projecting pro bLoscis used for feeding, and two sensory palps. The thorax is specialized for locomotYion. Three pairs of legs and a pair of wings are attached to the thorax. The abdomen is sTpecialized for food digestion and egg development. This segmented body part expaSndsI considerably when a female takes a blood meal. The blood is digested overR time serving as a source of protein for the production of eggs, which gradually filEl the abdomen. Anopheles mosquitoes caInV be distinguished from other mosquitoes by the palps, which are as long as the prNoboscis, and by the presence of discrete blocks of black and white scales on the winUgs. Adult Anopheles can also be identified by their typical resting position: males and f emales rest with their abdomens sticking up in the air rather than parallel to the suNrface on which they are resting. The duration from egg to adult varies considerably Aamong species and is strongly influenced by ambient temperature. MosquitoDes can develop from egg to adult in as little as 5 days but usually take 10-14 days in troApical conditions (Fig.2.2) (Bruce Chwatt, 1993). I2B.2.3 Mating and blood-feeding of adult Anopheles Adult mosquitoes usually mate within a few days (2-3 days) after emerging from the pupal stage. In most species, the males form large swarms, usually around dusk, and the females fly into the swarms to mate. Males live for about a week, feeding on nectar and other sources of sugar. Females will also feed on sugar sources for energy but usually require a blood meal for the development of eggs. After obtaining a full blood meal, the 11 female will rest for a few days while the blood is digested and eggs are developed. This process depends on temperature but usually takes 2-3 days in tropical conditions (NIH /CDC, 2004). During blood digestion, female‟s abdomen undergo series of changes from unfed (tiny abdomen) to blood-fed (red abdomen) then semi-gravid (half red and half whitish) to gravid (whitish); then the female lays eggs and resumes host seeking. This marks the end of a gonotrophic cycle and the beginning of a new one. The cycle repeats itself until the female dies. Females can survive up to a month (or longer in captivityY) but most probably do not live longer than 1-2 weeks in nature. Their chances oRf survival depend on temperature and humidity, but also their ability to successfuRlly oAbtain a blood meal while avoiding host defences. 2.2.4 The ecology of breeding sites IB Gravid Anopheles lay their eggs in different types of bLreeding sites depending on the species (Savage, 1990). Most Anopheles species pYrefe r clean water and edges of streams, while others thrive in irrigation areas, ricIe Tfields, grassy ditches and reservoirs. Some species require extensive vegetative covSer, for oviposition while some will prefer water bodies with dark or light bottomed pools. Others will prefer swamps and other permanent water bodies laden with EdissRolved organic matter (Mc Crae, 1983; 1984; Huang et al., 2005). Many of these sites develop into zones of transmission due to the concomitant increase of humNan pIo Vpulations moving to these areas. Ecological disturbance as a direct result of human activity may also increase the number of breeding sites. Road building and maintena nUce projects often impede drainage of runoff from rainfall. Clogged drainage ditches aNlong roads left by logging and construction activities are ideal places for floodwater mosquitoes. Around the house, objects such as empty cans, discarded tyres, potted plaDnts,A and similar objects used as a result of human activities are often responsible for thAe collection of rainwater which allows mosquitoes to breed (Kitron, 1989, Tadei I1B998). 2.3 Factors involved in malaria transmission Knowledge of the biology and behaviour of Anopheles mosquitoes can help comprehend how malaria is transmitted and can aid in designing appropriate control strategies. Factors that affect a mosquito's ability to transmit malaria include its innate 12 susceptibility to Plasmodium, its host choice and its longevity (MacDonald, 1957; Bruce- Chwatt, 1993). 2.3.1 Preferred sources for blood meals One important behavioural factor is the degree to which an Anopheles species prefers to feed on humans (anthropophily) or animals such as cattle or pigs (zoophily). Anthrophilic Anopheles are more likely to transmit the malaria parasites from one pYerson to another. Most Anopheles mosquitoes are not exclusively anthropophilic or zoophilic. The primary malaria vectors in Africa, A. gambiae and A. funestus, areR strongly anthropophilic and, consequently, are two of the most efficient malaria Avectors in the world (MacDonald, 1957). R 2.3.2 Life Span IB Once ingested by a mosquito, malaria parasites must uLndergo development within the mosquito before they are infectious to humans. The Ytime required for development of the parasite in the mosquito ranges from 10-21 Tdays (extrinsic incubation period), depending on the parasite species and the temperIature. If a mosquito does not survive longer than the extrinsic incubation perioRd, tShen she will not be able to transmit any malaria parasites (MacDonald, 1957). 2.3.3 Patterns of feeding and resting E Anopheles mosquitoNes aIr Ve crepuscular (active at dusk or dawn) or nocturnal (active at night). Some Anopheles mosquitoes feed indoors (endophagic), while others feed outdoors (exophagiUc). After blood feeding, some Anopheles mosquitoes prefer to rest indoors (endophilNic) w hile others prefer to rest outdoors (exophilic). Biting by nocturnal, endophagic AAnopheles can be markedly reduced through the use of insecticide-treated bed nets (ITNDs) or through improved housing construction (e.g. window and door screens) wherAeas, exophagic vectors are best controlled through breeding sites destruction. IEBndophagic Anopheles have an increase contact with humans and consequently are likely to be able to transmit more cases of malaria (MacDonald, 1957). 2.4 Major vectors of malaria in sub-Saharan Africa There are several species of malaria vectors in Africa. Two of these species have been reported to be widely distributed and being able to efficiently transmit the malaria 13 parasite: A. gambiae sl. and the A. funestus sl. Both species belong to a complex comprising of morphologically indistinguishable species. 2.4.1 The A. gambiae complex A. gambiae is the principal vector of malaria in tropical Africa. It has the capacity to colonise sunlit, temporary small water bodies that are scattered, around human dwellings (Minakawa, 1999; Gimnig, 2001). The A. gambiae complex was initially considered to be of a single species until much later when it was confirmRed Yusing molecular tools to be made up of six named species: A. gambiae (senAsu stricto), A. arabiensis, A. merus, A. melas, A. bwambe, and A. quadriannulatus (Hunt et al., 1998). All members of A. gambiae complex are morphologically identicRal but have few molecular differences. A. gambiae ss. and A. arabiensis are the mIoBst widespread of these groups with A. arabiensis broadly distributed in arid regions (LCoetzee et al., 2004). Both species occur in sympatry and are often found breeding iYn temporary stagnant water often associated with human activities (Coetzee et al., 20I0T4). Another member of the group, A. quadriannulatus is known to have a restricted distribution which is limited to south-east Africa and Ethiopia (Fettene and Temu, R2003S), A. merus and A. melas are salt water species, and their breeding is confined Erespectively to coastal regions of Africa (Moreno et al., 2004). 2.4.2 A. funestus group IV Members of the A. fNunestus group are widespread throughout sub-Saharan Africa and Madagascar (Gill ieUs and De Meillion, 1968, Mouchet et al., 1998). Species of this group include A. Nfunestus ss., A. parensis, A. aruni, A. vaneedeni, and A. rivulorum. Of these speciesA, A. rivulorum has few morphological features which can be used for identificaDtion at adult stage (Gillies and Coetzee, 1987). The vectorial capacities within the gAroup members vary significantly; for example, A. funestus s.s has been reported to IhBave the highest vectorial capacity in the funestus group (Gillies and De Meillion, 1968). Other members of the funestus complex have very low vectorial capacities. An isolated conducted on malaria transmission in Tanzania reported the presence of P. falciparum sporozoite in the salivary glands of A. rivulorum (Wilkes et al., 1996). Working in the laboratory, an experimental transmission was obtained with Anopheles vaneedeni (De Meillon et al., 1977). Cohuet et al. (2003) described a new taxon closely related to A. 14 rivulorum, based on biological, morphological and genetic characters. The species, provisionally called „A. rivulorum-like’, is present in Burkina Faso (Hacket et al., 2000) and Cameroon, and is clearly different from the A. rivulorum of South Africa (Gillies and Coetzee, 1987). This new taxon does not seem to play any role in malaria transmission. While members of the A. gambiae complex are well characterised, the species composition of members of the A. funestus group however, is still being determined (Mouchet et. al., 1993). In Nigeria and Benin Republic, data on A. funestus grouYp are almost inexistent. Few unpublished reports exist on the description of this specRies and its implication on malaria transmission in both countries. The absence Aof extensive information on A. funestus s.l. probably results from the difficultBies Rfor colonising this species in the laboratory. I 2.5 Vector Identification L In the epidemiology of any vector borne diseasYes, i t is essential to identify and incriminate the responsible vector species. IdentificIaTtion of the vectors therefore becomes an important component of a vector borne diseSase control programme. Most arthropods of medical importance are readily identified through taxonomic keys with morphological characteristics that are species specificE (GilRlies and De Meillon, 1968; Gillies and Coetzee, 1987). However there is a limIitVation to the use of morphological characteristics in distinguishing related organNisms sharing similar morphological features. There are a number of biological species sharing morphologically identical taxon but reproductively isolated. These popul atUions are known as cryptic species, sibling species or isomorphic species such as mNembers of A. funestus complex or A. gambiae complex (Hunt, 1998). Vector identifAication has helped to quantify the role of several cryptic species belonging to major grDoup in disease transmission (Coetzee et al., 2004). The occurrence of species compAlexes is often accompanied by different genetic variations presenting different IbBehaviours and vectoral capacities. As a result, control managers lacking adequate techniques may spend scarce resources to control non-vector species (Coetzee, 2004). In addition, proper species identification allows appropriate decision making (Weeto, 2004). Several features were used for identifying Anophele species: (i) the horizontal position of larvae at the surface of water, (ii) the grey-darkish dots on wings and (iii) the triangular segmentation of the posterior part of the abodomin (Gillies and De Meillon, 1968). 15 2.5.1 Application of molecular techniques in the identification of sibling species Different molecular techniques are available for species identification. These techniques include: DNA and RNA probes Restriction Fragment Length Polymorphism (RFLPs) described by Fanello et al.(2002) and Garros et al. (2004), Random amplified polymorphic DNA (RAPD – PCR), Single strand conformational polymorphism (SSCP) by Koekemoer et al. (1999). These techniques have been extensively used to characterize the major vectors of malaria. Other techniques used are crossing experiments, mitotiYc and meiotic karyotypes, polythene chromosomes, electrophoretic banding paRttern and advanced molecular techniques such as the use of micro-array chips. A 2.5.1.1 The Polymerase Chain Reaction Assay (PCR) R The use of PCR has created a revolution in diagnostic resIeaBrch by providing new ways of studying parasites, vectors and their hosts (GreenwoLod, 2002). The technique involves repeated amplification of small fragments of DNYA present in a test sample. The detection of nucleotide changes involves the use Tof specific primers. This technique allows a rapid and efficient analysis of a large numIbers of samples collected during field studies. The purpose of a PCR is to make hSuge copies of a gene. PCR has a major advantage because it utilises DNA whEich iRs relatively robust and can be transported from field and stored in the laboratoryI Vfor long periods (up to 20 years) and with less storage requirements (Long et al., 19N95; Li et al., 1997). Using DNA and RNA based techniques; members of mosquito complexes can be distinguished from a weUll preserved fragment of the insect body. Several PCR protocols have been developed to distinguish members of the A. gambiae complex (Collins et. al., 1987; Scott et. al.N, 1993; Hill and Crompton, 1994), A. funestus group (Koekemoer et al., 2002), anDd tAhe sibling species or molecular forms of A. gambiae s.s (Favia et al., 1997).There are three major steps in a PCR which are repeated for 25 or 40 cycles IdBepen Ading on the protocols. These are: denaturation for opening of DNA strands, annealing for specific binding of primers and extension for amplification of the products (Fig. 2.3). They are carried out in an automated thermocycler, which can heat and cool the tubes with the reaction mixture within the programed timing. The denaturation temperature in cycling reaction for A. gambiae and A. funestus speciation is 94º C and 99ºC respectively; during this step, both strand of the DNA are 16 AR Y LIB R TY RS I VE NI N U DA A IBFig. 2.3 The exponential amplification of the gene in Polymerase Chain Reaction (PCR); (A) represents the denaturation phase, (B) the annealing or hybridization and (C) the amplification or extension phase. Source: Oceanexplorer (2011) 17 disrupted leading to two single stranded DNA. The annealing (hybridization) temperature in a cycling reaction for species specific PCR assay for A. gambiae and A. funestus is 50º C and 45ºC respectively. At this relatively low temperature, several reactions are possible. The primers through the Brownian motion jiggle around in the reaction mixture for identifying the matching DNA sequence in the single stranded DNA template. Hydrogen bonds are constantly formed and broken between the single stranded primer and the single stranded template until the matching sequence is identified. At the identification oYf the matching sequence, the primers fit properly and more stable bonds that last aR little bit longer are formed between the single stranded DNA template and the primAer. Once this initial hybridization is successful the polymerase enzyme can attach andR starts copying the template (extension phase). IB o During the extension phase, the temperature is slighLtly increased from 45 C to 72ºC. This is the ideal temperature for polymerase reaYctio ns. The primers having few bases built in, form stronger hydrogen bonds with temTplates. Primers that are not properly matched become loose (primer-dimers) and doS notI add to the extension of the fragment. The complementary bases (dNTPs) to thRe template are coupled to the primer by the polymerase enzyme which adds dNTPE‟s from 5‟ to 3‟, reading the template from 3‟ to 5‟ side. 2.5.1.2 Restriction fragment lenIgVth polymorphism Detection of nucleotNide changes by this method involves subsequent analysis of the PCR products b y Usequence-specific analytical methods such as digestion with restriction enzymNes or hybridization with probes directed at specific nucleotides. This method is parAticularly employed in the identification of the 2 molecular forms of the A. gambiae Ds.s. Here, a digesting enzyme (HhaI) is added to the PCR reactions following the protoAcol described by Scott et al. (1993). Since the restriction site for the HhaI enzyme I(BGCGC) lies within the A. gambiae fragment amplified in the assay, it is therefore possible to differentiate between the two molecular forms (Fanello et al., 2002). The technique here described by Fanello et al., (2002) has been well standardized and is gradually becoming a routine laboratory technique in several countries for characterising molecular forms of A. gambiae ss ( A. gambiae “M” form and A. gambiae “S”). 18 2.5.1.3 Cytogenetic techniques for A. gambiae speciation As A. gambiae has tracked humans across temporally and spatially diverse habitats it appears to have been forced to undergo extensive ecological adaptation, which in turn is driving population divergence (Bradley, 2010). The first evidence for ecological adaptation of A. gambiae came via the examination of chromosomal inversions, which occur when a segment of a chromosome breaks off, flips 180 degrees, and becomes reinserted into same position (Hoffmann and Rieseberg, 2008). This event causesY gene order within the inversion to be reversed relative to that of an ancestral orR standard chromosome. Due to this rearrangement, when homologous chromosomes aAttempt to pair in a heterokaryotype (an individual heterozygous for an invertRed and standard chromosome) a characteristic loop is created due to the inability oIfB the two chromosomes to linearly align. In the ovarian nurse cells of semi-gravid LA. gambiae females, giant polytene chromosomes are formed (Fig. 2.4). By vieYwin g the characteristic banding pattern and/or loops of these chromosomes under aI mTicroscope, one can determine which inversions are present (della Torre, 1997). ColSuzzi (1985) believed that genes within the inversions were responsible for speciation and, used inversion frequency data to name five non-Linnaean chromosomal forms – BEamaRko, Guinea-Bissua, Forest, Mopti, and Savanna, each of which he predicted to bIe Vin the early stages of speciation (Coluzzi et al., 1985; Toure et al., 1998; Powell et al., 1999). 2.6 Malaria vector contNrol strategies Vector control aUims to decrease contacts between humans and vectors of human diseases. Control Nof mosquitoes may prevent malaria as well as several other mosquito-borne diseaseAs. Socio-economic improvements of households combined with vector reductionD efforts and effective treatments have led to the elimination of malaria diseases in seAveral North American countries (Schofield and White, 1984). Vector control IsBtrategies for controlling malaria are mainly focussed on: insecticide-treated bed nets, indoor residual spraying and larviciding (larval reduction). 2.6.1 Insecticide-treated bed nets Insecticide-treated bed nets (ITNs) such as long lasting insecticide trateted nets (LLINs) are a form of personal protection that has repeatedly been shown to reduce severe disease and mortality due to malaria in endemic regions (Fig.2.5). ITNs have been shown 19 Y RA R LI B ITY ER S NI V U Fig. 2.4 Chromosomal inversions in A. gambiae females; this mosquito is heteroAkarNyotypic for inversions 2Rb and 2Rc (A) as well as the 2La inversion (B) D Source: Sharakhov and Sharakhova (2008). A IB 20 Y RA R LI B SI TY ER IV U N N DA BFiAg. 2.5 The use of insecticide-treated bed nets (ITNs) for malaria vector control I Source: WHO (2003). 21 to reduce malaria mortalities by about 20% and morbidity to 50% (UNICEF, 2003). Untreated bed nets form a protective barrier around persons using them. The application of a residual insecticide greatly enhances the protective efficacy of bed nets. Besides their lethal activities, some insecticides have repellent properties that reduce the number of mosquitoes that enters the house and attempt to feed. There are several types of nets available in the market; these nets may vary by size, material, and/or treatment. Most ITNs are made of polyester (permaNet®) but nets are also availabYle in cotton, or polyethylene (Olysetnet®). Currently, only pyrethroid insecticides areR approved for use on ITNs (WHO, 2006). These insecticides have very low mammaliaAn toxicity but are highly toxic to insects and have a rapid knock-down effect, even Rat very low doses. Pyrethroids have a relatively high residual effect: they do not rapIiBdly break down unless washed or exposed to sunlight. Previously, nets had to be reLtreated at intervals of 6-12 months and more frequently if the nets were washed. NeYts ar e retreated by simply dipping them in a mixture of water and insecticide and allowTing them to dry in a shady place. The need for frequent retreatment, the lack of underSstanIding of the importance of bednets, and the additional cost for insecticides resultedR in very low retreatment rates in most African countries and constituted the major bEarrier to full implementation of ITNs in endemic countries (Binka et al., 1998). TIhVis condition has led to the development of long lasting insecticides treated nets (LLINs) (WHO, 2006). More recently, several companies have developed long-lasting insecNticide-treated nets (LLINs) that retain lethal concentrations of insecticide for at least 3U years (WHO, 2006). Although NITNs have shown their effectiveness for malaria control, some key strategic quesAtions remain unanswered: should ITNs be sold or provided free of charge to the groupDs most at risk? Should ITNs be provided to the entire households or exclusively to chAildren and pregnant women? (Maxwell et al., 2006) I2B.6.2 Bednets ownership patterns in Nigeria In 2004, a survey conducted in 5 sentinel sites: Kano and Maiduguri in the North, Nsukka, in the East and Ibadan and Lagos in the southwestern Nigeria showed that the percentage of households owning a bednet in Nigerian was very low in 2000, but significantly increase from 0% in several localities in 2000 to more than 10% in 2004 (NetMark, 2004). 22 In 2004, Ibadan remained lowest at 10% and Maiduguri highest at 51%. Rural ownership has increased more quickly than urban ownership. In 2000, the percent of households that owned a net was 13% in urban areas and 11% in rural areas. In 2004, 23% of urban and 29% of rural households owned at least one net (NetMark, 2004). Ibadan had the lowest proportion of untreated nets (6%) and Nsukka the highest (20%). The data on baby nets were not available in 2000. In 2004 baby net ownership is relatively high: 40% of households owned a baby net with a built-in frame in the 5 sentinel sites (NetMYark, 2004). Baby net ownership ranged from 15% in Kano to 52% in the city of LagRos. There was a small rise in the percent of children under five sleeping under a haAnging net the prior night, from 8.8% in 2000 to 10.3% in 2004. The proportion rangedR from a low of 3% in Ibadan site to a high of 17% in Maiduguri site. The totalI Bnumber of women of reproductive age in all households sampled was 2,528; of Lthese, 703 were from net owning households. The total number of pregnant womeYn in the households sampled was 249 and, of these, 76 were from net-owning hoIuTseholds (NetMark, 2004). Recently, LLINs are massively being introduced in NigSeria through the Global Funds initiatives (USAID, 2010) 2.6.3 Bednets ownership patterns in BenRin Republic In Benin Republic, the naItiVonal E malaria control program (NMCP) places emphasis on the use of LLINs for the Nprevention of malaria among children under five and pregnant women. A survey conducted in 2006 by the Ministry of Health showed that more than half of all households (56% )U owned at least one mosquito net of any type (treated or untreated nets). The coveraNge rate in rural areas was 50% and 66% in urban settings. Twenty five percent of neAt owners reported owning at least one ITN and only 20% of children under five and Dpregnant women said that they had slept under an ITN the previous night. Based uponA these data, the NMCP is supporting a four-pronged approach to net distribution in IBBenin, which includes free distribution through health centers during ante natal care (ANC) and immunization clinic visits, distribution of highly-subsidized ITNs through community-based channels, free distribution through mass campaigns, and the sale of ITNs in the commercial market (USAID, 2010). At the health center level, ITNs are distributed through antenatal kits that include antimalarial drugs, and other drugs, at a cost of about $1 per kit. These kits are subsidized by USAID, UNICEF and the World Bank. In 23 the local market, prices of treated nets (LLINs) range from $7 to $12. At the hospital settings, these long lasting treated nets are subsidized by USAID at a cost price of $2 each (USAID, 2010). 2.6.4 Indoor residual spraying Many endophilic malaria vectors are particularly susceptible to control through indoor residual spraying (IRS). This strategy involves coating the walls and other surfaces of a house with a residual insecticide. For several months, the residual insecticide wiYll kill mosquitoes and other insects that come in contact with these surfaces (WHRO, 2006). Contrary to ITNs, IRS does not directly prevent people from being bitten bAy mosquitoes. Rather, it kills mosquitoes after they have fed, if they come to rest on thRe sprayed surface. IRS thus prevents transmission of infection to other persons. IRSI isB an efficient approach to the control of malaria transmission as chances of killingL an Anopheles mosquito is repeated every time the mosquito rests on the treated waYll an d before it reaches the age of transmitting mature sporozoites (Curtis et al., 200I0)T. Historically the best vector control results have been achieved by IRS, e.g. the reduction of the incidence of malaria in India from about 75 million cases per year inR the S1930s to about 110 000 in the 1960s (a reduction of 99.8%), the near eradEication of previously holoendemic malaria from Zanzibar, in the 1960s, and the mIaVnagement of malaria catastrophe in South Africa in the late 90s (Brooke et al., 2000). Results obtained so fNar with insecticide-treated nets (ITNs) have not matched these achievements. HoweverU, the use of ITNs requires less equipment and labour, and may be more feasible in mNany circumstances (Curtis et al., 1998; Kroeger et al., 2002). IRS with DDT and dielAdrin was the primary malaria control method used during the global malaria eradicatioDn campaign from 1955 to 1969 (MacDonald, 1957). The campaign did not achieAve its stated objective but it did eliminate malaria from several areas and sharply IrBeduced the burden of malaria disease in others (MacDonald, 1957). Resistance to DDT and dieldrin coupled with their negative impact on the environment resulted to the introduction of new insecticides which unfortunately were more expensive and could not sustainable in poor endemic countries. The negative publicity of DDT and dieldrin insecticides used in IRS campaigns accounted negatively for the up scaling of this malaria control strategy. However, the recent success of IRS in 24 reducing malaria cases in South Africa (more than 80% reduction of malaria prevalence) has revived interest in this malaria prevention tool and has also reignited the necessity of using DDT in some countries for malaria vector control (WHO, 2009). 2.6.5 Breeding sites reduction Breeding sites reduction is the method of choice for mosquito control when the mosquito species targeted are concentrated in a small number of discrete habitats. The larval habitats may be destroyed by filling depressions that collect water, by draYining swamps, or by ditching marshy areas to remove standing water. ContainerR-breeding mosquitoes are particularly susceptible to source reduction as people can bAe educated to remove or cover standing water in cans, cups, and rain barrels arouBnd hRouses. Mosquitoes that breed in irrigation water can be controlled through careful Iwater management. The use of larvicides in malaria control requires several prerequis itLes: the knowledge of laying behaviors of Anopheles in the locality, the survey, mapYping and monitoring of breeding sites and finally the composition and chemical IaTctivity of the larvicide to be used. Larvicides could be biological or synthetic. Biological larvicides include Bacillus thuriengiensis, Bacillus sphaericus, larvivoRurs sSpecies of fish etc. The control of A. culicifaciesE larvae using larvivorous fish is reported to be working well in Karnatakastate,I IVndia (Yapabandara et al., 2002). However, it does not seem to be applicable to thNose Anopheline mosquitoes which typically breed in small puddles that frequently alternate between dryness and being re-filled with rainwater, and in situations where th erUe are many such sites within mosquito-flight range of a village. This applies to maNny A. gambiae rural breeding sites (Curtis et al., 2003). Several trials to combat mosqAuito larvae with bacillus have been successfully conducted at low scale (HougardD et al., 1983; Becker et al., 1994; Hougard and Back, 2003). These bacteria releaAse toxins which are ingested by larvae and have a cytotoxic activity in the midgut IcBells of insect larvae. 2.6.6 Less implemented vector control strategies Other vector control strategies with less implementation in communities include: (i) fogging or outdoor spraying which is primarily reserved for emergency situations such as halting epidemics or rapidly reducing adult mosquito populations when they have become severe pests; (ii) the use of repellents such as DEET (Fradin and Day, 2002), 25 wearing light-coloured clothes, long pants and long-sleeved shirts, (NH-USA, 2009); (iii) the spray of petroleum products in mosquito breeding sites, (Burton, 1967; Thevagasayam et al., 1979); (iv) the genetic modification of malaria vectors aims to develop mosquitoes that are refractory to the parasite. This approach is still several years from applications in the field (Lorena, 2003). 2.6.6.1 The development of genetically modified Anopheles (GMA) for malaria control Y Curtis (1968) proposed the basic concept of genetic control of vecRtor borne diseases since 1968, but major advances in the molecular manipulation oAf Drosophila melanogaster during the 1980s encouraged re-evaluation of this ideRa. The Anopheles genome sequence provides an architectural scaffold for mappingI, Bidentifying, selecting, and exploiting desirable insect vector genes. It also promotesL understanding of mosquito biochemistry, physiology, and behavior as well as of Ymala ria epidemiology, and spurs development of new public health interventions (HemTingway et al., 2002). A strain of A. stephensis that is unable tSo traInsmit malaria in mice has already been engineered (Tu et al., 2001; Ito et al., 2R002). The next big challenges are: driving of refractory genotypes in wild strains (Tu et al., 2001), studying the bio-ecology of engineered mosquitoes (Scott IeVt al E., 2002) and getting information on stability of engineered genes (Tu et al., 2001), understanding of oxidative stress which appears to be important in refractory strainNs to resist parasite infections and to drive refractory gene into wild populations of A noUpheles (Hemingway et al., 2002), and getting communities fully involved in the pNrocess. The use of genetically modified insect vectors in the field will require carefuAl consideration of bio-safety, ecological, ethical, legal, and social issues to ensure puDblic acceptance (WHO, 1991). 2.7 TAhe use of insecticides in public health IB A great variety of insecticides are used in public health for malaria vectors control. These insecticides can be grouped under 6 main families: organochlorine, organophosphate, carbamates, pyrethroids, growth regulator and bacterial toxins. Currently, a total of 12 insecticides from these families are used in public health against mosquitoes at adult stage: 7 pyrethroids, 3 organophosphate compounds, 1 carbamate and the dichloro diphenyl trichloroethane (DDT) (WHOAfro, 2003). 26 2.7.1 Insecticides and mode of action Insecticides usually act on the nervous system of insects and more specifically on the transfer of nervous impulses. The insecticidal activity results in either tetanisation (organochlorine and pyrethroids) or paralysis (organophosphates and carbamates) leading to death of mosquitoes. The nervous impulse moves through sodium channels to synapses where it is transmitted to the next axon through neuro transmitters (acetylcholine). After the transfer of nervous impulses, the synthesized acetylcholine is degraded and its acYtivity stopped by acetylcholinesterase, an enzyme whose function is to degrade acetRylcholine. Insecticides are capable of disturbing this transfer of impulse by either maintAained opening of the sodium channel or acting on the chlorine channel or iBnhibRiting activities of acetylcholinesterase (Fig. 2.6) I 2.7.1.1 Organochlorine L This family is subdivided into 3 subgroups baseYd on their chemical structure and mode of action. The main members of the family areT: DDT and its analogues, lindane and cyclodiene. DDT was discovered in 1939 by PaSul MIuler in Switzerland and tested in 1942 as an anti-mosquito spray in army camps in the USA and UK. In 1944, DDT was tested for the first time as an IRS insecticide Ein ciRvilian areas at Voluntoro, Italy. This insecticide was highly successful (Singh eIt Val., 1962). In 1950, DDT water dispersible powder containing 50 to 75 % tecNhnical grade DDT was made available and its remarkable convenience in application prompted it to be an ultimate choice in anti-malaria campaigns. Organochlorine effica cyU in agriculture and public health generated a great interest of WHO and led to lNaunching of malaria eradication programme in the 50s (Mouchet, 1994). DDT has a coAmplex chemical structure (Fig. 2.7). Its activity is focused on peripheral and central nDervous system of insects (Hassal, 1990); it has a rapid knock down effect on mosqAuito populations (Fig. 2.6). Despite these high performances, its bioaccumulation in ItBhe environment and the appearance of cases of resistance in some regions prompted WHO to stop using and even to ban it in many countries. Lindane and cyclodiene are subgroups in the family of organochlorine. A known member of this subfamily is Dieldrine. Their activities are focused on the central nervous system, where they inhibit chlorine channels, the main receptors of gamma-aminobutyrique acid (GABA) (Fig. 2.6). This set of insecticides was also banned because of their bioaccumulation, toxicity and the 27 RY Cyclodiene (Dieldrine) BR A LI Y Sodium Channel Chlorine Channel (GABA) IT RS IV E AChE N Pyrethroi dsU + DDT Organophosphates AN Carbamates ADFig. 2.6 The different targets of insecticides in Anopheles mosquitoes IB Source: Modified after WHO picture, WHO (2006) 28 Y AR LIB R SI TY R VE UN I DA N IB A Fig. 2.7 Chemical structure of some insecticides used in public health Source: Krieger (2001) 29 emergence of cases of resistance. 2.7.1.2 Organophosphate These are derived from phosphoric acid, and replaced the organochlorine because of their relatively low toxicity. The members of this family of insecticides used in public health are: malathion and fenitrothion. When coupled with oxygen molecules, organophosphates are good inhibitors of acetylcholinesterase (Keith, 2005). This enzyme degrades activities of acetylcholine which neuromediates cholinergic synapses locaYted in the central nervous system of insects. The fixation of organophospRhates on acetylcholinesterase leads to accumulation of acetylcholine at the synaptic juAnction. When the level of acetylcholine becomes too high, the acetylcholine receBptorRs are blocked. It is this blockage that leads to paralysis and death of insects (Figs. 2.6I and 2.7) 2.7.1.3 Carbamates L These are synthetically derived from eserine. ThYey a ct like organophosphates by inhibiting activities of acetylcholinesterase (Keith,T 2005).The family are made up of carbamate and bendiocarb. These insecticides are Iderived from carbonic acids. They are less used because of their cost and their toxRicityS on mammals (Figs. 2.6 and 2.7) 2.7.1.2.4 Pyrethroids They are synthesized IfVrom Epyrethrines which are natural extracts from Chrysanthemum cinerariaefNolium flowers. First generations of pyrethroids were very volatile and therefore less persistent. With more studies conducted on these compounds, this instability was oveUrcome and more stable molecules were developed (Elliot et al., 1978). Pyrethroids ar e divided in to two groups based on their α radicals: group I: permethrin, groupN II: deltamethrin, lambda-cyhalothrin and cypermethrin. Pyrethroids act on sodiuDm chAannel by keeping it opened and therefore accelerate the speed of nervous impuAlses (Figs. 2.6 and 2.7). The insect ends up dying by tetanisation. IB Pyrethroids have a rapid knockdown effect coupled with high excito-repulsive action and are less toxic to mammals at operational doses (Darriet, 1984). These features explain why pyrethroids were quickly welcome and used for nets impregnation (WHO, 1984). However, the emergence of populations of insects capable to withstand lethal doses of this family of insecticides threatens the efficacy of current malaria control strategies. 30 2.7.2 Protective mechanisms of Anopheles and development of resistance to insecticides All living organisms have natural mechanisms of protection against harmful compounds including insecticides. In Anopheles, 3 types of enzymatic activities are normally observed when they get in contact with insecticides: Esterase, oxydase and glutathion-s-transferase (Figs. 2.8 and 2.9). With esterase activities, ester bonds found on the insecticide are destroyed and the insecticide is converted into carboxylicR acidY and alcoholic compounds which can easily be metabolized by the insect. The enzyme acting during this conversion is known as carboxylesterase. Insecticides can beA oxidized by oxidase enzymes to produce alcoholic compounds which can also be eRasily degraded by the mosquito. With glutathion S. transferase, insecticides activIitBy is conjugated by a tripeptide known as glutathione and this conjugation leads to Lsequestration of insecticide and reduced activity on the target site (Chandre et al., 1Y999 ). In area of high insecticides pressure and constant exposure of mosquitoes to inseTcticides, these protective phenomena are enhanced and, a proportion of insects in the pIopulation will gradually inherit a high capability of withstanding lethal doses of Sinsecticides: this condition is known as resistance. R 2.7.2.1 Resistance of Anopheles to insEecticides Resistance is defined as: the occuIrVrence in a population of a group of individual capable of tolerating doses of chemicalNs which under normal condition could kill the majority of the population” (Hamon aUnd Mouchet, 1961). Resistance in mosquito populations has a genetic backup anNd its spread is due to allelic selections from spontaneous mutations or migrations. TAoxicity of an insecticide results from interaction between the insecticide and the biologDical set up of the mosquito. Various steps are necessary for this to take place: the insecAticide must get in contact with the insect, enter the insect, be transformed into a ImBetabolite and carried to the target site for expression. All these steps are governed by either one or several genes of which any structural or functional modification could lead to resistance (Soderlund and Bloomquist, 1990). These modifications can lead to changes in the behaviour of the insects like the capacity of the insects to identify and escape from areas under insecticide treatments (this is known as behavioural resistance), the ability of the insects to over-express detoxification genes for metabolising the insecticides also 31 1- Esterases: RY R-COO-C H +H O R-COOH + C H -AOH 2 5 2 2 5 BR 2- Oxydases: LIY R-H+O +2H TR-OH+H O 2 2 RS I 3- GlutathioEn -S-transferases: Tripetide cNonIj Vugation and insecticides by glutathion N U Fig.2.8 CAhemical equations of enzymatic activities developed by mosquitoes to D withstand insecticide lethal doses I B A 32 RY BR A LI ITY RS IV E UN N Fig.2.9D SerAies of mechanisms of resistance to insecticides developed by Anopheles A Source: Modified after WHO picture, WHO (2006) I B 33 referred as metabolic resistance and finally the modification of the insecticide target sites in the insects known as target site resistance (Fig. 2.9). 2.7.2.2 Behavioral mechanisms of resistance Behavioral mechanisms are rarely observed in mosquito populations. The mechanism is based on the ability of insects to run away from areas which have been treated with insecticides, thereby avoiding contact with the insecticide. WithY the publishing of mosquito genome, investigations are currently focused on genes responsible for neurosensory perception, and chemical detection by the mosquito (RAansRon et al., 2002). 2.7.2.3 Metabolic mechanisms of resistance R Three families of proteins are largely responsible for metabIoBlizing insecticides: the cytochrome-P450s (oxidase), carboxylesterases (esterase)L and the glutathione-S- transferases (GST). A recent analysis of the A. gambiYae g enome identified 111 genes putatively encoding P450s, 51 genes encoding foIr Tcarboxylesterases and 31 genes for glutathione transferases (Ranson et al., 2002). 2.7.2.3.1 Cytochrome P450s (monoxygenRase oSr oxidase mechanisms) Cytochrome P450s exist in insEects in very diverse families. Certain subfamilies of P450s have been implicated in the metabolism of insecticides (Feyereisen, 1999). Elevated P450s activities hav VNe beIen widely implicated in resistance to pyrethroids in many species, but the lack of sensitivity of biochemical assays designed to detect increases in P450s in individual in seUcts and the paucity of knowledge on the role of individual P450 enzymes in inseNcticide metabolism have prevented an accurate assessment of this mechanism A(Ranson et al., 2003). Recently, elevated expression of a particular P450 gene hasD been associated with resistance to pyrethroids in A. gambiae from East Africa (Nikou et al., 2003), but this preliminary finding needs further verification. IEBster Aase The family of carboxylesterase proteins is extensive in insects. This includes enzymes like acetylcholinesterases which is found at the nervous synapse and responsible for degrading acetylcholine. Carboxylesterase proteins do not hydrolyse organophosphates but act by sequestration because of their high affinity with this family of insecticides (Cuany et al., 1993). Insensitive acetylcholinesterase (Ace-1) has been reported in malaria 34 vectors from Sri Lanka (Karunaratne et al., 1999). In West Africa, Djogbenou et al. (2008) identified and mapped the distribution of Ace-1 in A. gambiae samples from Benin and Burkina Faso. Elevated frequencies of Ace-1 mutation are associated with resistance to organophosphate and carbamate (Djogbenou et al., 2008). Depending on esterases involved, resistance can be specific to a particular insecticide or can confer broad- spectrum resistance to a number of different insecticides (Oakeshott et al., 1999). Y 2.7.2.3.2 Glutathione-S-transferase R This compound binds on insecticides and produces less toxic produActs. The most significant one is the DDT-ase which degrades DDT insecticide. GlutaRthione transferases have been implicated in resistance to DDT in several ABnopheles populations (Prapanthadara et al., 1993; 2000). Recently, a glutathione tranIsferase responsible for resistance to DDT in A. gambiae has been elucidated (Ra nLson et al., 2001). In other insects such as Drosophila, glutathione transferase has aYlso been implicated in resistance to pyrethroids (Vontas et al., 2001) and to organophIoTsphates (Huang, 1998). 2.7.2.3.3 Target site modification (knock-dowSn mutation) Targets sites for insecticides are eitRher receptors or enzymes of the nervous system like acetylcholinesterase, sodium chaEnnel and the gamma acetyl-buturic-acid (GABA) receptors. Structural modificationIsV of these targets either reduce binding affinity or change the synthesis of enzymes Nleading to resistance. Target modifications are powerful mechanisms of resista ncUe in the sense that they lead to cross resistance of all families of insecticides targetNing the same pathway. It is associated to point or multiple mutations on nucleotide seAquences. Mutations affecting sodium channels and GABA receptors have been idenDtified in various species of mosquitoes (Coustau and French-Constant, 1995; MartAinez-Torres et al., 1998). Once insecticide resistance is developed, the genes can pBersist in the insect population for 30 years or more, but at low levels. I The knock down resistance (kdr) is a target site modification generated by a mutation in the voltage-gated sodium channel of the insect‟s nervous system. This target is similar for both DDT and pyrethroid insecticides. This resistance mechanism has evolved at least twice in A. gambiae (Matinez-Torres et al., 1998; Ranson et al., 2000) and is now present at very high levels in some regions of Africa (Akogbeto et al., 1999; Chandre et al., 1999). With kdr bearing A. gambiae collected from West Africa, the point mutation on 35 the sodium channel leads to different amino acids synthesis: leucine is replaced by phenyl alanine (Leu-Phe). In East Africa, the same kdr mutation leads to the replacement of leucine by serine (Leu-Ser) (Fig. 2.10). The kdr has also been detected in A. sacharovi (Luleyap et al., 2002) and A. stephensi (Enayati et al., 2003). Once identified, the mutation can be detected using polymerase chain reaction (PCR) techniques (Matinez- Torres et al., 1998). The knock down resistance mechanism has evolved at least twice in A. gambiae (Matinez-Torres et al., 1998; Ranson et al., 2000) and is now present atY very high levels in some regions of Africa (Akogbeto et al., 1999; Chandre et al., 199R9). 2.8 Detection of insecticide resistance in malaria vectors A The detection of insecticide resistance in Anopheles popuRlations is highly important for health policies and decision making on the type of vIeBctor control strategy to implement in a given locality. This detection provides informaLtion on the susceptibility to insecticides of mosquito populations and the potenYtial mechanisms of insecticide resistance involved. Four tools are routinely used forT basic detection of resistance in field Anopheles populations: the “WHO bioassays” in tuIbes with adult mosquitoes, the “bottle test” with synergists, bio-chemical assays Rto deStermine elevated enzyme activities related to resistance, and polymerase chaiEn reaction (PCR) for detection of target sites modification in the mosquito. 2.8.1 Bio-assay for phenotyNpingI i Vnsecticide resistance in vectors In this bio-assay, the WHO test kits used for the purpose is composed of impregnated papers p reUpared using silicone oil (Dow Corning 556) with technical grade insecticides. FemNales of Anopheles are exposed to different impregnated papers for one hour and the Amortality recorded after 24hours monitoring in the insectary (WHO, 1986). This assaDy segregates resistant and susceptible phenotypes and allows the characterisation of AnAopheles populations as resistant when the mortality rate of exposed mosquitoes is IlBower than 95% or susceptible when higher than 95%. The validation of results from this bio-assay depends immensely on the total number of exposed mosquitoes. Ideally mosquitoes should all be of the same age and the average number of exposed mosquitoes higher than 100 (WHO, 1986). The main difficulty in this diagnostic technique is getting enough Anopheles (minimum of 100) from the same locality all aged between 2-5 days. 36 Y AR TVVIGNLVVLNL Sensitive R TVVIGNFVVLNL kMdors Wqueistot B TVVIGNSVVLNL kAdfrri cEaa st AfricYa LI I II I II I IT V Out-Side S Axon 1 2 3 4 1 2 E3 64 R 1 2 3 64 1 2 3 4 6 5 6 5 5 5 In-Side Axon IV COON H N 2 H UN Fig. 2.10D MuAtation on the sodium channel in kdr resistant Anopheles from West and A East Africa B Source: Chandre (2004) I 37 2.8.2 The bottle tests with synergists The bottle bioassay described by Allister and Brogdon (1999) can be used to assess the biochemical mechanisms of resistance developed by mosquito populations collected in the field. The technique is based on coating of bottles with the insecticide solution to be tested and introducing female mosquitoes into these bottles. Once resistance is detected, another set of coated bottle is prepared using 2 synergists: piperonyl butoxide (PBO) and S.S.S-tributlyphosphorotrithioate (DEF). PBO is used for detecting the presenYce of elevated oxidase activities in the mosquito whereas DEF is for esteraseA (AlRlister and Brogdon, 1999). 2.8.3 Polymerase chain reaction (PCR) for target modification R PCR analysis provides insight information on the sequIenBce arrangements, the presence or absence of specific nucleotides in the DNA of th eL field collected mosquitoes. This sequences arrangement profile is used for moleculaYr characterisation of mechanisms of resistance in sampled Anopheles populations.I TThe most common PCR for target modification is the PCR kdr used in knock doSwn resistance. The technique is based on detection of single nucleotide polymorpRhisms following DNA extractions and using appropriate primers (Martinez- TorresE et al., 1998). This PCR allows determination of various resistant alleles (RR, RISV, SS) and their respective frequencies in mosquito populations could be calculNated. The acetylcholinesterase target site mutation (Ace-1) known to confer carbamate and organophosphate resistance could also be screened in field population of A. gambiaUe using PCR protocols described by Weill et al. (2004). 2.8.4 MicroarrayN tech nique for detection of detoxification genes This TAechnique provides an accurate platform for analysing series of up and down regulatedD genes at a single trial. Contraty to classical PCR analysis which screens for a limiteAd number of genes at a time, the micro-array technology provides simultanous IiBnformation on several gene responses (hundreds to thousands of gene responses) following a given stimulation. The microarray “detox chip” for example which is used for screening detoxifying genes expressed in Anopheles mosquitoes, contains fragments of 230 A. gambiae genes from families associated with metabolic based insecticide resistance. These gene fragments included 103 P450s, 31 COEs, 35 GSTs, 41 Redox genes, 5 ATP binding-cassette transporters, tissue-specific genes and housekeeping genes 38 (David et al., 2005).Each gene represented on the microarray is either obtained by PCR amplification or artificially synthesized. To keep cross hybridization between closely related genes to a minimum, gene-specific segments are selected between 70 and 300 bp in length as probes which are spotted in duplicate onto gamma-amino-propyl-silane- coated glass slides (UltraGaps, Corning) by using a Biorobotics Micro- Grid II printer (BioRobotics, Cambridge, U.K.). 2.8.5 Reported cases of resistance of A. gambiae to insecticides in Nigeria Y A limited number of extensive studies have been conducted on vector resRistance to insecticides in Nigeria. Awolola (2003) studied the resistance of A.A gambiae to insecticides in Lagos, Nigeria. The study identified the presence Bof rResistance in some Anopheles populations and established the presence of M and S mIolecular forms existing as single or in sympatry in some localities of southwestern LNigeria. Mojca (2003) also reported on the low presence of kdr mutation on AnophYeles populations from Ogun state in the South West Nigeria. Awolola et al. (2005I)T investigated the distribution of the molecular M and S forms of A. gambiae and the knock down resistance (kdr) gene associated with pyrethroid and DDT resisRtanceS in A. gambiae s.s. at 13 localities across Nigeria. The report showed that the ovEerall collection was a mix of the molecular M and S forms across the mangrove (I63V:37%), forest (56:44%), and transitional (36:64%) ecotypes, but almost a pureN collection of the S form in the Guinea and Sudan-savanna. Results of insecticide susceptibility tests showed that mosquitoes sampled at seven localities were suscep tiUble to permethrin, deltamethrin, and DDT, but populations of A. gambiae resistantN to these insecticides were recorded at six other localities mainly in the transitional anAd Guinea-savanna ecotypes. ThDe kdr gene was found only in the molecular S forms, including areas where both formAs were both M and S forms are in sympatric. No hybrid M/S has been found during ItBhe analysis of samples from the field; this suggest as strong mating barrier between the two molecular forms of A. gambiae ss. The overall frequency of the kdr alleles in populations of Anopheles was low: <47% in forest, 37–48% in the transitional, and 45– 53% in Guinea-savanna. The data suggest that pyrethroid resistance in A. gambiae in Nigeria is not as widespread when compared to neighboring West African countries (Awolola et al., 2005). 39 2.8.6 Reported cases of resistance of A. gambiae to insecticides in Benin In Benin, the first report of Anopheles resistance was by Akogbeto and Yakoubo (1999). This resistance resulted from the exposure to DDT of populations of A. gambiae from meridian regions during IRS campaign. Resistance in Benin is related to two phenomena: (i) the massive use of DDT and dieldrin for house-spraying applications in southern villages of Benin from 1953 to 1960 during WHO programmes of malaria eradication (Joncour, 1960) and (ii) the massive use of organochlorine in agricuYltural settings during the 1950s (WHO, 1976). R Pyrethroids were introduced for agricultural use in Benin in the 1970As, and after 30 years of continuous use, cases of resistance may be found in someB popRulations of insects (Akogbeto et al., 1999). N‟Guessan (2007) analysed the operatioInal impact of pyrethroid resistance on the efficacy of ITNs and IRS. Results from tLhe study established a link between pyrethroid resistance caused by kdr and the Yfailure of ITNs in Benin. Using biochemical assays Corbel et al. (2007) impliIcaTted the detoxification enzymes in conferring resistance to pyrethroids, DDT, dieldrin and carbosulfan in A. gambiae populations from four localities in Benin. RDjogSbenou et al. (2008) identified and mapped the distribution of Acer-1 in A. gamEbiae samples from Benin. He reported elevated frequencies of Acer-1 mutatioIn Vassociated with resistance to organophosphate and carbamate in Benin (Djogbenou et al., 2008). 2.8.7 Reported cases of rNesistance of A gambiae to insecticides in other African countries U The resistaNnce of vectors to insecticides is a real handicap to the use of insecticide-treated materiAals and the implememtation of IRS (N‟Guessan et al., 2007). The first cases of resistaDnce were mentioned in the 1950s and 1960s with identified populations of A. gambAiae capable of withstanding lethal doses of organochlorine. In 1950-1960, this IrBesistance phenomenon was mostly limited to dieldrin and hexachlorocyclohexane (HCH) (Coz and Hamon 1963). In Africa, the first cases of dieldrin resistance in A. gambiae were recorded in Burkina Faso in 1960 (Elissa et al., 19993; Coz et al., 1999). Ten years later, the identification of dieldrin resistance and cases of DDT resistance were reported in Togo, Senegal, Nigeria, Cameroon and Guinea (WHO, 1976). With pyrethroids, cases of resistance were published relatively late in the 1990s. The first cases of pyrethroid 40 resistance were reported in West African countries such as in Côte d'Ivoire (Elissa et al., 1993), Benin (Akogbeto and Yakoubo, 1999), Burkina Faso (Diabate, 1999; Chandre et al., 1999), Côte d'Ivoire (Chandre et al., 1999) and Mali (Fanello et al., 2003). Other reports followed from Kenya in East Africa (Vulule et al., 1994), and Cameroon in Central Africa and in the Central African Republic (Etang et al., 2003). Two main mechanisms of resistance have so far been identified in A. gambiae from agricultural settings in the West Coast of Africa: the kdr mutation and enzyYmatic mechanisms of resistance. kdr mutations have been mostly recorded in settinRgs where agricultural practices are associated with the use of pesticides. This mechAanism is well spread in Burkina Faso (Diabate et al., 2001), and in Cote d‟Ivoire (ElisRsa et al., 1993). In the northern area of Cameroon, enzymatic mechanisms of resistaIncBe have been identified in various localities (Etang et al., 2003). L Studies conducted by Diabate et al. (2002) Yhigh lighted increased levels of resistance genes, kdr, in A. gambiae collected inI Tcotton-growing areas and constantly subjected to insecticide treatments, as compared to the low frequency of kdr recorded in rural areas where farmers are restricted Rto foSod crops for local consumption with no pesticides. In Côte d'Ivoire, the kdr mEutation identified in resistant strains of A. gambiae was probably selected as a resultI oVf the massive use of DDT and pyrethroids against pests in cotton fields (Chandre et aNl., 1999; Diabate et al., 2002). 2.9 Factors favouring the emergence of resistance in mosquito populations Several enviro nmUental and human practices have been implicated in the emergence of insecticide reNsistance in diseases vectors. These factors include the large scale implementation of ITNs (Vulule et al., 1999) and IRS (Joncour et al., 1960) in communiDties,A the agricultural pesticides (Chandre et al.,1999; Diabate et al.,2002) and the pAresence of pollutants (xenobiotics) in mosquito breeding habitats (Poupardin et al., I2B008; Muhammad et al., 2009). These factors either select for target sites mutations in mosquitoes or cross induce the expression of several detoxifying genes. 2.9.1 The selection of insecticide resistance by ITNs and IRS In the Western Kenya, the permethrin tolerance (PT) of a population of the mosquito A. gambiae increased following the introduction of permethrin-impregnated nets for malaria control in certain villages near Kisumu (Vulule et al., 1999). A similar 41 observation was made several years later by Aram (2004) who showed that after one year of ITN use, there was a reduction in permethrin susceptibility in A. gambiae from ITN villages but not from villages without ITNs. In Cote d‟Ivoire, Corbel et al. (2003) analysed Anopheles mosquitoes in experimental hut trial and found that insecticide resistance was selected by carbamate impregnated bednets. The study, also demonstrated that when carbamate were combined with pyrethroids on the same net, no such selection was recorded. Y 2.9.2 Agricultural pesticide residues and other xenobiotics R The implication of agriculture in the selection of insecticide resistanAce in malaria vectors has been well documented (Chandre et al., 1999; Diabate et alR., 1999; Akogbeto and Yacoubo, 1999). Akogbeto et al. (2003) later demonstrated IthBat various insecticides are used against pests in vegetable farms and in cotton plantatLions. These insecticides are mainly pyrethroids, organophosphate compounds and caYrbam ates. Insecticides are highly used in vegetable and cotton farming (AkogbetoT et al., 2003). Data on insecticide susceptibility tests always show elevated resistSanceI on mosquito populations collected in or around agricultural farms under pesticidRes use (Mouchet et al., 1988). This is because insecticide residues are left in water and soil after farms spraying. When these residues are in contact with mosquito larvaeI, Vthey E exercise a selection pressure on larvae and this pressure results to gradual dNevelopment of insecticide resistance in mosquito populations (Akogbeto et al 2004.). In the Republic of Benin, insecticides were introduced in agriculture in the 1970 sU. It is not impossible after 30 years of use to record some cases of resistance. SeveraNl authors mainly from West Africa and Cameroon incriminate pesticides used in agricuAlture as a factor selecting insecticide resistance in mosquitoes (Georghiou and LaguDnes 1991; Chandre et al., 1999; Martin et al., 2000; Diabate et al, 2002; N‟guAessan et al., 2003). IB Experiments conducted on larvae of Aedes aegypti showed that constant exposure to sub-lethal doses of xenobiotics such as the herbicide atrazine, the polycyclic aromatic hydrocarbon fluoranthene and the heavy metal copper increased their tolerance to insecticides (Poupardin et al., 2008). Results of the experiement revealed a moderate increase in larval tolerance to permethrin following exposure to fluoranthene and copper while larval tolerance to temephos increased moderately after exposure to atrazine, copper 42 and permethrin. This study revealed the potential of xenobiotics found in polluted mosquito breeding sites to affect their tolerance to insecticides, possibly through the cross- induction of particular detoxification genes. 2.9.3 Types of mechanisms of resistance selected by ITNs, IRS, agricultural pesticide residues and xenobiotics Surveys conducted in Kenya showed that the frequency of the kdr mutation prior to ITN introduction was between 3 and 4 % target communities. After ITN introductioYn, the kdr mutation increased in ITN and neighboring villages from 4% to 8% (ArRam et al., 2004). In addition to the kdr mutation, Vulule (1999) used biochemiAcal test with synergists to establish higher oxidase levels in mosquito populations Rfrom villages with ITNs than a comparison population from villages without impreIgBnated nets. He further speculated that the use of impregnated nets selected for higherL oxidase and esterase levels in A. gambiae to metabolize permethrin acquired from thYe ne ts. Both oxidase and esterase mechanisms could confer cross-resistance to other pTyrethroids. Evidence for selection for an insensitive acetylcholinesterase mechanismS byI carbamate impregnated bednets was later established by Corbel (2003). With Agricultural pesticides, twEo mRain mechanisms of resistance have so far been identified in A. gambiae populaItioVns collected in or around agricultural settings in West Africa: the kdr mutation anNd enzymatic mechanisms of resistance (Elissa et al., 1993; Diabate et al., 2001; Etang et al., 2003; Chouaibou et al., 2008). U N AD A I B 43 CHAPTER 3 MATERIALS AND METHODS 3.1 Susceptibility of Anopheles populations to pyrethroid in studied sites Populations of Anopheles were collected from various localities and analysed for their susceptibility to permethrin. 3.1.1 Description of sampling sites Mosquito samples were collected from six states in southwestern Nigeria naYmely Ogun, Oyo, Lagos, Osun, Ondo and Ekiti states (fig. 3.1). In the RepublRic Benin, mosquito larvae were collected from 6 administrative divisions in the soAuthern region manely the Zou, Plateau, Couffo, Atlantique, Mono and Littoral. StanRding water points found in each site from these administrative divisions and staIteBs were systematically scrutinized for mosquito larvae (Fig. 3.2). Sites with breeding Lwater containing Anopheles larvae were considered as sampling sites for this researchY. The names of the sites were obtained fIroTm the local communities and the geographical coordinates (latitudes and longituSdes) of each sampling site were determined using a geographical positioning system (RGPS) device (GPS Garmin® Model 60). Each geographical coordinate as well as the name of the locality were projected on the map of Benin and Nigeria using Arch view 3.1E software. 3.1.2 Collection of AnophelNes laIrv Vae Contrary to other families of mosquitoes (Culex, Aedes and Mansonia), Anopheles larvae lack caudal sip hUon and therefore are recognized by their morphology and their horizontal positioNn on water surface (Bruce-Chwatt, 1980) (Fig. 3.3). This regular presence of AAnopheles larvae at the surface of water allows proper breathing of individuaDls. Anopheles larvae were scooped into large vessels, were sieved and concentrated in small buckets, washed with clean water and taken to the insectary for IrBearin Ag to adult stage. 3.1.3 Rearing of larvae to adult stage in the insectary Anopheles larvae samples collected in the field were reared in three different insectaries depending on the sites of collection and the available facilities for keeping and conveying samples from the field to the laboratory. All samples collected in the southwestern part of Nigeria were reared in the insectary of the Department of Zoology, 44 AR Y BR Y LI T RS I E IV U N DA N IB A Fig. 3.1 Map of study areas (Divisions and States) in Benin and Nigeria 45 RY BR A LI ITY ER S V UN I N AD A Fig. 3.2 Identification of breeding sites and field collection of Anopheles larvae I B 46 RY RA IB L Y IT RS IV E N Fig. 3.3 The horizo nUtal position of Anopheles larvae at the surface of water (left) N Source: Bruce-Chwatt (1980) DA I B A 47 University of Ibadan, Nigeria. The insectaries of the Centre for Research in Entomology of Cotonou (CREC) based in Cotonou, Republic of Benin and that of the International Institute of Tropical Agriculture (IITA-Benin) for samples collected in the Republic of Benin. At the insectary, mosquito larvae were reconditioned into clean vessels of 50 cm diameter and 15 cm depth containing Well water and covered with mosquito nets. Larvae were fed through the pores of nets mesh with a powder of cat/dogs biscuits bought from local super market. At the adult stage, mosquitoes were transferred into cages and fedY with 10% sucrose solutions for 2-5 days. R 3.1.4 WHO susceptibility tests on adult Anopheles from surveyed localitiAes Two to five-day old Anopheles emerging from each identifieBd bRreeding sites, were bio-assayed using discriminating doses of insecticides. Bio-aIssays were carried out following WHO protocols (1986). Females of A. gambiae weLre exposed for one hour to papers impregnated with diagnostic dosages of permethYrin ( 0.75%). For each test, 5 test tubes were used: one for the control and four foIrT exposed mosquitoes. Control tubes contained filter papers impregnated with silicSon oil (insecticide carrier), while treated papers were impregnated with diagnostic doses of insecticide plus carrier. An average of 20 mosquitoes was introduced in each tuRbe, making a total of 100 females Anopheles exposed to each insecticide. After oEne hour, mosquitoes were transferred to holding containers and provided witNh coItt Von pads with 10% sucrose solution. An average of 50 females of Anopheles from each locality was exposed to permethrin. Mortality rates were recorded at 24 hours U(Fig. 3.4 and Appendix.3) and the susceptibility status of the population was gNraded as susceptible if the mortality rate is between 97% to 100%; as reduced susceAptibility if the mortality rate is between 95% to 95% and as resistant if the mortalityD rate is below 95% according to WHO protocol (1986). 3.1.5A Mapping of permethrin susceptibility in Anopheles populations from studied IlBocalities The susceptibility of malaria vectors to permethrin was mapped using data (latitude and longitude) on the geographical locations where Anopheles were collected. Mortality rates following exposure to permethrin of Anopheles populations from surveyed locality were projected onto the maps of Nigeria and Benin using Arc-view 3.1 software and then after, converted as PDF files. 48 Y AR LIB R SI TY ER NI V U AN FigA. 3.4D WHO bioassay steps for analysing the susceptibility status of Anopheles to B insecticides (WHO, 1996). I 49 3.2 Molecular characterization of Anopheles populations from studied sites: PCR- species, PCR-forms, PCR-kdr In each locality, an average of 30 Anopheles that had been exposed to permethrin was subjected to PCR Analysis. The 30 mosquitoes for PCR analysis were sorted from the group of Anopheles that had survived the exposure to permethrin and those that died after 24 hours post exposure to permethrin impregnated papers (making an average of 15 survivors and 15 dead for each locality). PCR-species (Koekemoer et al., 1999) Ywere conducted on Anopheles to identify the various members of A. gamAbiaeR complex encountered in each site. The next set of PCR focused on molecular foRrms (Favia et al., 1997) and involved only A. gambiae ss. from the previous screeningB. The PCR forms sub-grouped the A. gambiae ss. into 2 molecular forms: A. gambiae ssI. (M) and A. gambiae ss. (S). The last series of PCRs investigated the presence of the kdLr mutation which confers a pyrethroid resistant phenotype to Anopheles populationYs. E ach PCR process involved 3 main steps: DNA extraction, Polymerase chain reIacTtions and electrophoresis (Figs. 3.5, 3.6 and 3.7). 3.2 .1 DNA extraction S DNA extraction for each adultE femRale was carried out by adding 100 ml of lysis buffer (0.1 MNaCl, 0.2 M sucrIosVe, 0.1 M Tris-HCl, 0.05 M EDTA, pH 9.1 and 0.5% SDS) onto the mosquito samNple in an eppendoorf tube (1.5ml). A sterile laboratory pestle was used to grind andU homogenize mosquito samples in each eppendoorf tube. The homogenate was incub ated at 65º C for 1 h, and 15 µl of 8 M potassium acetate solution was then added aNnd centrifuged for 10 min at 14,000 rpm. The supernatant was pipetted without disturAbing the pellets and the deposit containing pellets was transferred into a new o eppendooDrf tube and filled up with 400µl of 100% ethanol (ice cold ethanol from -20 C freezAer). The ethanol suspension was well mixed and incubated at room temperature for 5 ImBinutes then centrifuged at 14,000 rpm for 15 minutes. Ethanol is removed and the pellets oare re-suspended in 200µl of 70% ethanol (ice cold ethanol from -20 C freezer), centrifuged at 14,000 rpm for 20 minutes and pipetted off without disturbing the DNA pellets. After this series of ethanol washing, the DNA pellets were finally air-dried and o suspended in 100µl of sterile distilled water, stored at -20 C before PCR analysis (Fig. 3.5 and Appendix.4). 50 AR Y R LI B ITY ER S V UN I AN FiDg. 3.5 DNA extraction for PCR analysis of collected Anopheles samples BAI 51 AR Y LIB R ITYS VE R UN I DA N Fig. 3.6 DNA amplification (PCR reactions) of collected Anopheles samples BAI 52 AR Y R LIB ITY RS VE UN I AN Fig. D3.7 garose gel migration of PCR products of amplified Anopheles DNA BAI 53 3.2.2 Polymerase chain reaction for A. gambiae speciation Each DNA extracts from mosquito samples was used as template for PCR synthesis. A thermo-cycler made GenePro model B-480 was set for amplifications of DNA fragments. For species identification (Koekemoer et al.,1999). A master mix solution containing 1.25µl of PCR buffer, 1.25µl of dNTPs, 1µl of each primer (Universal, A. gambiae ss, A. arabiensis, A. melas, A. quadrianulatus.), 0.5µl of MgCl2 and 4.9µl of sterile distilled water was prepared for each PCR tube and 2µl of extracted DNA saYmple was added to the master mix. The thermocycler was loaded with DNA sampRles to be o amplified and programmed at 94 C for 10 minutes (denaturation phase), theAn 30 cycles of o o o 94 C for 30 seconds, 50 C for 30 seconds and 72 C for 30 seconds R(hybridization and extension phase). After the amplification, the PCR product was mIBigrated on 1% agarose gel (Figs. 3.6 and 3.7). L 3.2.3 Polymerase chain reaction for detection of the kYnock down (kdr) mutation in A. gambiae populations T For the identification of kdr mutations Iin collected Anopheles samples, the protocol described by Martinez-Torres etR al. S(1998) was used. A master mix solution containing 2.5µl of PCR buffer, 0.5µl Eof dNTPs, 0.3µl of MgCl2, 1.25µl of Agd1 (primer), 1.25 µl of Agd2 (primer), 2.5µl of the Agd3 (primer), 2.5 µl of Agd4 (primer), 0.125 of Taq and 13µl of sterile distilled VNwateIr was prepared for each PCR tube and 1µl of extracted DNA sample was added to the master mix. The Agd1 and Agd2 primers correspond to the standard A. gambiae bUanding whereas the Agd1 and Agd3 correspond to the resistant genotype and theN Agd2 and Agd4 to the susceptible genotype. The thermocycler was o oloaded with DANA samples to be amplified and programmed at 95 C for 15 minutes, 94 C o o ofor 30 seDconds, 35 cycles of 48 C for 30 seconds, 72 C for 1 minute and 72 C for 10 minuAtes. After the amplification, the PCR product was migrated on 1% agarose gel I(BAppendix. 4). Specific primers used for PCR-species and PCR-kdr The members of A. gambiae complex were discriminated as gambiae ss, arabiensis, and melas. Using respectively the following primers sequences: CTGGTTTGGTCGGCACGTTT, AAGTGTCCTTCTCCATCCTA, GTGACCAACCCACTCCCTTGA described by Koekemoer et al. (1999). The kdr gene 54 mutation was localized on the DNA template using 4 primers described by Martinez- Torres et al. (1998): ATAGATTCCCCGACCATG, AGACAAGGATGATGAACC, AATTTGCATTACTTACGACA, and CTGTAGTGATAGGAAATTTA. 3.2.3 Electrophoresis of PCR products For the migration of PCR products, an agarose gel (1%) is prepared. 1% agarose powered is dissolved in TBE buffer, heated to boiling temperature for 10 minutes, cooled to the room temperature and 10µl of ethydium bromide added and gently swRirledY. The ethydium bromide is a radioactive compound which fixes the nucleotides mAaking them to fluoresce in the presence of UV light. The gel is caste in the electrophoresis tank and wells made on it with electrophoresis comb and allowed to solidify at rBoomR temperature. The size of the comb and therefore the number of wells was selected Ibased on the number of PCR products (samples) to migrate at a time. The voltage wa s Lset at 100 and the migration on the gel was fixed for approximately 1hour. MolecuYlar weight markers ranging from 1000 to 100 bp were simultaneously loaded onI tThe gel and used as the ladder for interpreting the size of each amplified DNA (Fig. 3.7). 3.3 Evaluation of potential contributionsR of aSgricultural pesticide residues and spilled petroleum products in the selection oEf pyrethroid resistance in Anopheles populations Soil and water samples IwVere collected from agricultural sites under synthetic pesticides application and froNm areas of spilled petroleum products. 3.3.1 Screening of pesticide residues in water and soil samples from vegetable sites The screening Uof pesticide residues was conducted on samples from the agricultural sites oNf Houeyiho in Cotonou and Ajibode in Ibadan. 3.3.1.1 ScreenAing technique AD bioassay focused on factors capable of inhibiting the normal growth of AnopAheles larvae in breeding sites was conducted (MIM-WHO, 2003). The protocol used IiBn this evaluation is mainly based on comparison of the growth of larvae in test breeding sites (water and soil samples from agricultural settings under pesticide pressure) and in control breeding sites (water and soil samples from similar areas with no pesticides). 3.3.1.2 Collection of water and soil samples used for the bio-assay Surface water and soil samples (1 cm of the upper layer) were collected from the vegetable farms of Houeyiho in Benin and Ajibode in Ibadan, Nigeria (Fig. 3.8). These 55 samples were considered as test samples. For control samples, soil and water were collected from the premises of the insectary of the Centre for Research in Entomology of Cotonou–Akpakpa (CREC-Akpakpa) and the premises of the insectary of the Department of Zoology, of the University of Ibadan (UI) (Fig. 3.8). Both control sites have no previous history of pesticide contamination. Soil and water samples collected in the field were taken to the insectary where they were used for reconstituting Anopheles breeding sites, 5 replicates of each type of breeding sites were reconstituted with samples fromY each target locality. Breeding sites reconstitution was based on (i) a mixture of 1R0g of top (surface) soil from vegetable farms and 1000 ml of water from the coRntroAl sites and (ii) 1000ml of water collected in the vegetable farm (water used for watering vegetables). Water samples from CREC-Akpakpa were designated as CREC-IwBater similarly; samples from the premises of the UI-insectary were recorded as UI-watLer. 3.3.1.3 Monitoring of larval development in simulatedY bre eding sites An average of 200 eggs of the susceptible TA. gambiae strain from kisumu was inoculated in each artificial breeding site (soSil anId water samples from Houeyiho and Ajibode, CREC-Akpakpa and UI-insectary). A similar inoculation was repeated with the resistant A. gambiae strain from ladji and fRor the different types of artificial breeding sites simulated. More than 4,000 eggs werEe inoculated and monitored during this biological evaluation. The variations in haItcVN hing rates of eggs, the larvae developmental rate and yields of rearing larvae of resistant A. gambiae from Ladji and susceptible A. gambiae from Kisumu to adu ltU mosquitoes in test and control artificial breeding site were determined compared and plotted. During this follow-up experiment, larvae in all artificial breeding sitesA weNre fed with similar quantities and types of food (a powder from cat biscuits mDixed with yeast). 3.3.1.A4 History of synthetic pesticides utilization by farmers in target agricultural IsBites The level of synthetic pesticide use by communities in the vegetable farms where samples were collected was investigated through quantitative and qualitative surveys. After getting farmers consents on the study, they were subjected to quantitative and qualitative questionnaires to generate data on: the families of insecticide used in vegetable farming, the various doses applied for pests control, the frequency of treatments, the origin 56 Y RA R LI B SI TY R IV E UN N AD A IB Fig. 3.8 Map of sites selected for screening of pesticides residues in vegetable farms (Akpakpa-CREC, Houeyiho, Ajibode and UI- Ibadan). 57 of insecticides and the place of purchase (Appendix.1). Both the discussion guide (qualitative) and the questionnaire (quantitative) were tested and validated at the International Institute of Tropical Agriculture (IITA, Benin-Cotonou) Cotonou, Station. At Houeyiho, a total of 20 farmers approved the consent forms and underwent both the quantitative and the qualitative interviews, while at Ajibode, 10 farmers were interviewed and probed on their agricultural practices. This investigation also included direct observations of farm treatments by farmers. More emphasis was placed on the qualiYtative aspect of this survey. R 3.3.2 Evaluation of the contribution of petroleum products in Rthe Aselection for insecticide resistance in A. gambiae 3.3.2.1 KAP studies on the empirical utilisation of petroleuLm IpBroducts (PP) in rural communities Prior to assessing the larvicidal activities of YPP, questionnaires, focus group discussions (FGD), and in-depth questionnaires Tand interviews (Appendix.2) were conducted in 3 rural communities namely GbSodjoI, Ladji, and Ketonou in the Southern Benin were PP are still used for larval control. The interview focused on: i) the empirical use of PP, ii) the mode of application, iEii) tRhe period when PP are mostly used for breeding site treatments and iv) the frequeInVcy of utilization of PP (Appendix.2). A total of 65 key respondents were intervieweNd in the 3 communities. 3.3.2.2 Anopheles populations used for analysing the lethal activities of PP Two local resis taUnt strains of Anopheles were used for this experiment: A. gambiae from Ladji i and NA. gambiae from Ojoo. Both strains were used as reference resistant mosquitoes foAr assessing the lethal activities of PP in areas of pyrethroid resistance. A. gambiae Dpopulations from ladji were collected from the rural locality of Ladji, at 7 km fromA Cotonou, the economic capital of the Republic of Benin and A. gambiae populations IfBrom Ojoo were collected from the locality of Ojoo in Ibadan, Oyo state. Both strains were maintained in the insectary throughout the duration of this experiment. 3.3.2.3 Determination of lethal concentrations of 4 PP on larvae of A. gambiae Larvae of A. gambiae from Ladji and A. gambiae from Ojoo were exposed to four petroleum products: petrol, kerosene, engine oil and used engine oil from mechanic -3 -3 workshops. Known volumes of PP ranging from 0.12 x 10 µl to 9820 x 10 µl were 58 2 introduced in 255 cm plates half filled with well water ( approximately 100ml) and each containing 25 larvae (second to third stage larvae) of Anopheles gambiae from Ladji and A. gambiae from Ojoo (Fig. 3.9). Larvae were fed throughout the experiment with biscuit and yeast. Each breeding site treatment with PP in each plate was replicated four times for each population of Anopheles (A. gambiae from Ladji and A. gambiae from Ojoo), making a total of 100 larvae exposed to each tested concentration of PP. Similarly, coYntrol breeding sites with no traces of petroleum products were constituted and monitored in tandem. Mortality rates as well as the number of adults emerging from each breReding site were determined. The lowest concentration (LoC100) capable of iAnhibiting the development of larvae to the adult stage was determined; it correspoBndsR to 100% mortality of exposed larvae. I The highest concentration (HiC) not having any observLable effect on the growth of larvae also known as the NOEL (No effect level) was dYeter mined; it corresponds to 0% mortality of exposed larvae. In between both conTcentrations, the LC50 (concentration leading to a mortality of 50 % of exposed larvaSl poIpulation) was also determined for each PP. Data generated were pooled and used Rfor plotting the curve of activity of each PP on strains of A. gambiae from Ladji and A. gambiae from Ojoo. Thus, the HiC and the LoC100 corresponding to petrol, keVroseEI ne, engine oil and waste oil was determined by dose response analysis during this laboratory assessment. In between the HiC and the LoC100, the LC50 was determined forN each PP. 3.3.2.4 Identification oUf the mode of action of PP on Anopheles larvae Two potenNtial modes of action of petroleum products were analysed during this study: i) the kAilling of larvae by "suffocation" through the oil film produced by PP at the surface oDf breeding sites, and ii) the direct lethal activity through contact and ingestion of dissoAlved particles of PP in breeding sites, referred here as "contact toxicity". Water IsBamples with visible residues of petroleum products were collected from the locality of Ojoo, an area of spilled waste engine oils by mechanics and oil retailers. These samples from the field known as “crude samples” were used in the laboratory for the simulation of two types of breeding sites: i) The first set of breeding sites known as "unsieved or crude" was directly reconstituted by putting two litres of the water from the field into laboratory bowls. ii) The second set of breeding sites known as "sieved or clean" was reconstituted 59 Y Engine Oil Fuel Kerosene Waste Oil AR LIB R ITY ER S IV UN AN D I B A Fig. 3.9 Quantification of the lethal activity of 4 petroleum products on Anopheles larvae introduced in plates containing different concentrations of PP 60 by sieving to clean two litres of the crude sample from the field. The determination of the mode of action of petroleum products on mosquito larvae was based on statistical comparisons of larval mortalities recorded in the “unsieved or crude” simulations and the mortalities in the “sieved/clean” simulations. One hundred larvae of A. gambiae from Ojoo were introduced in each bowl containing sieved or unsieved petroleum and reared to adults. Four replicates were made for each type of simulation, making a total of 400 larvae monitored in "un-sieved/crude" and 400 larvae in "sieved/clean" breeding sites. CoYntrol bowls were constituted alongside using well water with no trace of petroleumR products and containing the same number of Anopheles larvae. For each typRe of Abreeding site (sieved and un-sieved), mortality rates were determined and statistical tests of comparison used to associate larval lethality with either sieved or un-sieved simIuBlations. 3.3.3 Analysis of associations between the presence oLf petroleum products in breeding sites and the emergence of pyrethroid resistaYnt p opulations of Anopheles The association between the nature (physicTo-chemical properties) of breeding sites and the susceptibility statut of emerging populIations of Anopheles was analysed. 3.3.3.1 Physico-chemical properties of breeSding sites of resistant and susceptible Anopheles R Fresh water samples weIreV co Ellected from each identified breeding site in the southwest Nigeria and the southern Benin and their physico-chemical properties determined. The physical thiNckness of the water and the presence of oil layer at the surface of the breeding sites w eUre visually appreciated and recorded. The dissolved oxygen (DO) and the pH of the Nbreeding site; 2 parameters which are likely to affect the development of Anopheles laArvae and their susceptibility status were determined by a digital portable multi-meDter (Handheld Digital-Multimeter® model M3640D). These physico-chemical paramAeters were either measured in the field or water samples were preserved in a cooler IaBnd later analysed in the laboratory. 3.3.3.2 Oviposition preferences of gravid females of Anopheles in localities where breeding sites are partially contaminated with spilled petroleum products 50 gravid females of A. gambiae ready to lay eggs were introduced into a cage provided with 2 different bowls of breeding sites for oviposition. The first bowl contained water with petroleum product residues collected at Ojoo and the second bowl had well 61 water with no trace of petroleum products and served as control sample. This experiment was carried out subsequently with A. gambiae from Ojoo and A. gambiae from UI strain introduced in cages containing the two simulated oviposition bowls.This oviposition experiment was repeated with other sets of oviposition bowls contaminated with PP residues collected at Akpakpa in Benin. Each oviposition cage was made in 4 replicates of 50 mosquitoes resulting in a total of 200 females for each strain monitored respectively on petroleum contaminated samples from Ojoo and Akpakpa. The number of eggs laYid by each strain of Anopheles in the breeding site with or without petroleum proRducts was counted and compared. Chi square (χ2) statistical test was used to compRare Athe number of eggs laid in each type of simulated site and to determine the preferred oviposition habitat of pyrethroid resistant and susceptible Anopheles. IB 3.3.3.3 Monitoring of the development of Anopheles larvaeL (resistant and susceptible strains) in breeding sites with petroleum products resYidue s Two hundred eggs of A. gambiae were intIroTduced into breeding sites containing two liters of water contaminated with petroleuSm residues from areas of spilled petroleum products in Ojoo and Akpakpa. The hatching rate and the number of larvae getting to pupae stage were determined for the reRsistant strain A. gambiae from Ojoo and the susceptible strain A. gambiae from UI.E Four replicates were analysed for each experiment. Control breeding sites madeN of cIle Van well water with no trace of petroleum products were also seeded with eggs of the same species and monitored in tandem with test breeding sites. The number of h atUched eggs and the number of larvae progressing to the pupae stage in each type of brNeeding sites (oily breeding sites and the controls) and for each strain of Anopheles (thAe resistant and the susceptible strain) were determined. 3.4 ScreDening of candidate metabolic genes overexpressed in pyrethroid resistant popuAlations of A. gambiae from Benin and Nigeria IB This experiment focused on the identification of differential expressions of metabolic genes in pyrethroid resistant Anopheles populations which are exposed to different types of xenobiotics such as spilled petroleum and agricultural pesticides. Anopheles larvae exposed to petroleum products were collected from Ojoo in Nigeria whereas larvae exposed to pesticide residues were collected from the vegetable farm of 62 Akron. The susceptible Anopheles populations from the locality of Orogun in Nigeria (Fig. 3.10) served as control samples in this experiment. 3.4.1 Selection of permethrin resistant A. gambiae for micro-array analysis Thousands of fourth instar larvae and pupae were collected from the breeding pools identified in the 3 target localities: Ojoo, Orogun and Akron (Fig. 3.10). Larvae and pupae were maintained in the laboratory and reared to adults (F0). Batches of 20 females of A. gambiae (one day old) which had emerged from larvae collected directly froYm the field were introduced into WHO susceptibility tubes (WHO, 1986) containiRng papers coated with 0.75% permethrin. After an exposure time of one hour, mosAquito samples from Akron and Ojoo were transferred into insecticide free tubes and Rmaintained for 24 hours with sugar solutions. At the end of the 24 hr, dead mosquiItoBes were discarded and alive or selected individuals (permethrin resistant populationL) were maintained in cages with sugar solutions for 3 days then after, a minimum oYf 30 permethrin resistant females (3 pools of 10 females also known as 3 biological reTplicates) from Ojoo and Akron were preserved in RNA-later solution for microarrSay aInalysis as described by David et al., (2005). Susceptible mosquitoes from OroRgun were subjected to WHO tubes containing control papers impregnated with silicEon oil (insecticide carrier) instead of permethrin. They were also maintained for 3I dVays with sugar solutions and a minimum of 60 females preserved in RNA-later solution. 3.4.2 Target preparation anNd microarray hybridizations Total RNA (tR NUA) was extracted in pools of 10 mosquitoes from the preserved samples (30 AnoNpheles from Ojoo; 30 Anopheles from Akron and 60 Anopheles from Orogun). TheA extraction was done with the PicoPure™ RNA isolation kit (Arcturus) accordingD to the manufacturer's instructions. After extraction, the quantity of extracted tRNAA was measured by a spectrophotometer (Nanodrop) (Fig. 3.11) and its quality was IbBased on electrophoresis migrations of 1µL of extracted tRNA on 1% agarose gel at a voltage of 75 for 60 minutes (Fig. 3.12) 3.4.3 cDNA synthesis, labelling and hybridization Extracted RNA was amplified using a RiboAmp™ RNA amplification kit (Arcturus). Amplified RNAs were checked for quantity by spectrophotometry, then reverse transcribed into labelled cDNA and competitively hybridized to the array. Cy3 and 63 Y RA R LI B ITY RS VE NI N U A BA D I Fig. 3.10 Map showing the 3 study sites selected for candidate metabolic gene search and their locations in relation to the control site (Orogun) 64 Extraction of tRNA from Anopheles Y R Reverse transcription for st A building the 1 DNA strand BR Ynd LI Building of the complementaTry 2 strand (cDNA ) SI R VETranscription of cDNA to I (aRNA ) N U DA N Reverse transcription and production of labelled DNA for hybridization A IB Fig. 3.11 Steps for producing and labeling cDNA of Anopheles samples from target sites (Ojoo, Akron and Orogun) for micro-array hybridization 65 RY BR A LI SI TY ER NI V N U A AD I B Fig. 3.12 Gel analysis of migrated total RNA prior to cDNA production Cy5 dyes were used for labelling of test and control samples. Labels were swapped between samples, making 2 technical replicates from each biological replicate and hence a 66 total of six labelled targets for each comparison (test and control samples) (Fig. 3.13). Labelled cDNA targets from the agricultural site of Akron and the oil spillage locality of Ojoo sites were competitively hybridised with the susceptible population of Anopheles samples from Orogun to the A. gambiae detox chip array which was printed with a physical rearrangement of the detoxifying probes (David et al., 2005; Muller et al., 2008). 3.4.4 Array scanning and visualization After visual inspection of each array, spot and background intensities Ywere calculated from the scanned array images using GenePix Pro 5.1 soAftwaRre (Axon Instruments). Raw intensities were then analysed with Limma softwarRe package version 2.4 (Lima 2.4) For the comparisons between the two groups of samples, selected (resistant) vs. unselected (susceptible), estimates for technical replIicBates (dye-swaps) were first averaged and then compared between the two groups asL described by Muller et al. (2008). The probes printed on the microarray include 103Y cyt ochrome P450s, 31 esterases, 35 glutathione S-transferases and 85 additional gIenTes such as peroxidases, reductases, superoxide dismutases, ATP-binding cassetteS transporters, tissue specific genes and housekeeping genes (Fig. 3.13). 3.5. Data analysis R Series of statistical analyIsVis w Eere conducted on the various laboratory and field data collected during this research study. 3.5.1 Analysis of data on thNe susceptibility level of Anopheles to permethrin Mortality rates oUf Anopheles post exposure to permethrin were computerized on Excel software and the corresponding susceptibility status of Anopheles populations were graded as resiAstanNt (R), susceptible (S) or reduced susceptibility (RS) (WHO, 1986). Data on the lDatitudes and longitudes of surveyed localities were analysed with Arc-view softwAare and projected on maps of Benin and Nigeria. I3B.5.2 Questionnaires and group discussions and in-depth interviews with farmers and petroleum products users Qualitative data from focus group discussions and in-depth interviews were tape reordered, transcribed and analysed with the software Text base beta. A Printing of Know genes (Probes) Cloning and isolation of DNA DfNraAgm freangtsm oef nAt. cglaomnbiniage 67 and isolation Amplification of each fragment (genes) by PCR Purification of each gene fragment Automated printing of the short fragment on the slide ******** **CHIP* Know series of genes also ******** called Probes ******** RY RA B Y LI T SI R VE I UN N Fig. 3.13 StepAs for synthesis and printing of detoxifying gene probes of A. gambiae on D the “Detox” chip array I B A Quantitative data were analysed with Epi Info sofware. Data were mainly descriptive and presented as percentages. 68 3.5.3 Lethal activities of petroleum products on Anopheles larvae Mortality rates corresponding to given concentrations of petroleum products were determined in the laboratory. Data generated from the laboratory assays were used to plot dose response curves of mortality rates and petroleum concentrations in breeding sites with Excel and SPSS software package. The HiC and LoC of each petroleum product were determined through laboratory bio-assays. The HiC and LoC of the 4 types of petroleum products tested were compared for assessing the relative efficacy ofY each petroleum products of Anopheles larvae. The mode of action of petroleum prRoducts on Anopheles larvae was identified by analysing the percentage mortalities of laArvae in sieved and un-sieved breeding sites. R 3.5.4 Cross analysis of the physico-chemical properties of bIrBeeding sites and the susceptibility status of emerging Anopheles populations L Data on the physico-chemical properties (DOY, pH , and the presence of oil particles) of breeding sites were cross-analysed withT data on the permethrin susceptibility of Anopheles. The mean DO and the meanS pHI of breeding sites producing resistant Anopheles was determined and compaRred with the mean DO and the mean pH of breeding sites producing permethrin susceptible Anopheles. The number of breeding sites with petroleum residues producing pEermethrin resistant Anopheles was also compared with the number of breeding IsVites with petroleum residues producing permethrin susceptible Anopheles. The Nstatistical analysis of variance was conducted on variables for associating the presen cUe of petroleum products in breeding sites and the emergence of pyrethroid resistaNnt Anopheles in studied localities. With data collected in vegetables farms, the meAan DO and the mean pH of breeding sites producing permethrin resistant AnopheleDs was also determined and compared with the mean DO and mean pH of breedAing sites producing susceptible populations of Anopheles. I3B.5.5 Analysis of data on the oviposition preference of gravid Anopheles, eggs hatching rates and larval developments in simulated breeding sites The mean number of eggs laid by resistant Anopheles (A. gambiae from Ojoo) in breeding sites with petroleum residues was determined and compared with the mean number of eggs laid in control breeding sites with no petroleum residues. Similar calculations were made with the mean number of eggs laid by the susceptible strain (A. 69 gambiae from UI). The comparison of means was based on the analysis of variance, for identifying whether or not permethrin resistant and susceptible strains of Anopheles prefer laying their eggs in breeding sites containing petroleum products. The number of eggs laid by A. gambiae from Ojoo and A. gambiae from UI in the various simulated breeding sites was plotted (histograms) with Excel 2000 Software. The hatching rate was calculated as the percentage of the number of eggs hatched out of the 200 eggs inoculated in each simulated breeding site. The percentage of haYtched eggs of resistant Anopheles (A. gambiae from Ojoo) in simulated breeding sites cRontaining petroleum residues was determined and compared with the percentage ofA hatched eggs inoculated in control breeding sites. Similar calculations were madBe wiRth the percentages of hatched eggs of susceptible Anopheles (A. gambiae frIom UI) in petroleum contaminated and in non-contaminated (controls) breeding siLtes. The recorded hatching rates of eggs of resistant and susceptible Anopheles inocYulate d in simulated breeding sites was plotted (histograms) with Excel 2000 Software. T With data recorded in vegetable farms, Ia similar calculation was made. The percentage of hatched eggs from resistant SAnopheles (A. gambiae from Ladji) and susceptible Anopheles (A. gambiae froEm KRisumu) in the various simulated breeding sites was determined and compared wIiVth the percentage of hatched eggs in control breeding sites. The larval development rate was recorded as the percentage number of pupae recorded out of the number oNf first instar larvae that hatched from the 200 eggs inoculated in each simulated bre edUing site. The number of pupae from resistant A. gambiae from Ojoo and suscepNtible A. gambiae from UI in simulated breeding sites containing 2petroleum resAidues was determined and compared (χ analysis) with the number of pupae recorded Din control breeding sites. Similar calculations and comparisons were made with data Afrom vegetable farms. The number of pupae of resistant A. gambiae from Ladji and of IsBusceptible A. gambiae from Kisumu recorded in the various simulated breeding sites was determined and compared with the percentage of pupae in control breeding sites. The recorded hatching rates of eggs of resistant and susceptible Anopheles inoculated in simulated breeding sites was plotted (histograms) with Excel 2000 Software. For all comparisons made, the level of significance for statistical tests conducted was fixed at a probability value of 5%. 70 3.5.6 Allelic frequencies of the kdr mutation in permethrin resistant and susceptible phenotypes of Anopheles mosquitoes analyzed In each surveyed locality, the allelic frequency of the kdr mutation (R) was calculated based on the formulae (Wigginton, et al., 2005) : 2RR+RS ƒ(R)= 2(RR+RS+SS) with RR being the homozygous kdr resistant mosquitoes, RS the heterozygous and SYS the susceptible mosquitoes. This allelic frequency was determined for the sub popuRlations of dead and lived females of Anopheles exposed to permethrin. In each stRudieAd locality, the statistical comparison of the allelic frequency in dead and live moBsquito was conducted and the probability value determined for associating (or not) the kIdr mutation to recorded resistant phenotypes. The map for the distribution of kdr mutaLtions in analysed mosquito populations was made using Arc view software. Y 3.5.7 Analysis of micro-array spots of expressed ImTetabolic genes identified in studied Anopheles populations The colour significance on micrRo arSray analysis program was based on fold changes (more than 2 folds). The GenEe Pix pro 5.1 software and the Limma 2.4 software packages were used for analysiIngV the color intensities of expressed genes in screened populations of Anopheles (Fig.3.14). The lists of metabolic genes up-regulated in permethrin resistant AnopheNles collected from the vegetable site of Akron and the spilled petroleum products sit e Uof Ojoo were determined. N DA I B A 71 RY RA LI B SI TY R IV E UN AN ADFig. 3.14 Visualisation and analysis of scanned microarray slide. IB CHAPTER 4 RESULTS 72 4.1 Screening of Anopheles populations for the susceptibility to pyrethroid in southwestern Nigeria and southern Benin Anopheles larvae were collected in the various sites and reared to the adult stage for bioassays. 4.1.1 Susceptibility to permethrin of A. gambiae populations in southwestern Nigeria Following the exposure of females of A. gambiae to permethrin impregnated papers, a good spread of resistance was recorded in the 6 surveyed states of Oyo, OYsun, Ondo, Ekiti, Ogun and Lagos in southwestern Nigeria. Out of the 19 populatRions of A. gambiae s.l. collected from these states, 14 populations were clearlyA resistant to permethrin, whereas 5 were within the range of susceptibility (Table 4.1R). In Oyo state, 8 sites with Anopheles breeding spots werIeB identified during this study: IITA, Ojoo, Ajibode, Basorun, UI, orogun, Challeng e Land Oja-tuntun. Permethrin resistance was clearly recorded in Anopheles populationYs from Basorun (mortality rate of Anopheles = 70%), Ojoo (mortality rate of AnopheleTs = 80%), Ajibode (mortality rate of Anopheles = 90%), Challenge (mortality ratSe oIf Anopheles = 81%) and Oja-tuntun (mortality rate of Anopheles = 81%). A. gaRmbiae populations collected from UI, IITA and Orogun were all susceptible: UI (mortality rate of Anopheles =100%), IITA (mortality rate of Anopheles =100% ) and OroguInV (mo Ertality rate of Anopheles =100% ). In Osun state, 2 sites with Anopheles breeding spots were identified during this study: Modakeke and LagNere. Permethrin susceptibility was recorded in Anopheles populations from Mo dUakeke (mortality rate of Anopheles = 97% ) while permethrin resistance was fouNnd in Anopheles populations from Lagere (mortality rate of = 94%). In OndAo state, 2 sites with Anopheles breeding spots were identified: Illesha garage (Akure) aDnd Owena. Anopheles populations from the two localities were all resistant to permAethrin: Ilesha garage (mortality rate of Anopheles = 89% ) and Owena (mortality rate IoBf Anopheles =75%). In Ekiti state, 2 sites with Anopheles breeding spots were identified: Ati-kankan 1 and Ati-kankan 2 both at Ado-Ekiti. Permethrin resistance was recorded with Anopheles TABLE 4.1 Susceptibility to permethrin of A. gambiae in southwestern Nigeria States Localities Latitude Longitude Anopheles Mortality Susceptibility (main city) tested rates Oyo IITA 7°22'12.0" 3°53'24.0" 95 100% Susceptible 73 (Ibadan) Ojoo 7°27'52.9" 3°54'58.7" 80 80% Resistant (Ibadan) Ajibode 7°19'48.0" 3°54'00.0" 85 90% Resistant (Ibadan) Bashorun 7°25'18.4" 3°56'08.3" 75 70% Resistant (Ibadan) UI 7°26'21.8" 3°53'21.8" 80 100% Susceptible (Ibadan) Challenge 7°26'17.9" 3°56'14.1" 80 81% ResiYstant (Ibadan) R Orogun 7°18'36.0" 3°54'36.0" 80 100% A Susceptible (Ibadan) Oja-tuntun 8°08'28.2" 4°14'3.5" 83 B81%R Resistant (Ogbomoso) Osun Lagere 7°28'37.1" 4°33'17.9" 78 I 94% Resistant (Ife) L Modakeke 7°28'37.1" 4°32'19.9" 8Y0 97% Reduced Sus. (Ife) Ondo Ilesha 7°16'13.3" 5°09'57.7I"T 77 89% Resistant garage (Akure) Owena 7°24'03.2" 5°00'45.0" 78 75% Resistant (Owena) S Ekiti Ati-kankan 1 7°37'06.0" 5R°13'14.4" 83 85% Resistant (Ado-ekiti) Ati-kankan 2 7°37 EI'0V6.5" 5°13'14.4" 80 88% Resistant (Ado-ekiti) Ogun Imowo N6°50'57.5" 3°55'51.4" 76 96% Reduced Sus. (Ijebu-ode) Mobafulo 6°47'59.1" 3°53'52.0" 75 80% Resistant ( Ijebu-ode) U Ogere 1 N 07°26'12.3" 4°37'16.2" 4 75% Resistant (LagAos-Ibadan rDoad) Ogere 2 07°26'12.3" 4°37'16.2" 40 88% Resistant A(Lagos-Ibadan B road) ILagos Badagry 6°24'36.0" 2°53'24.0" 50 98% Susceptible Susceptibility levels: From 97% to 100% mortalities (97% excluded) correspond to susceptible insect populations; 95% to 97% mortalities (95% excluded) correspond to reduced susceptibility in insect populations; below 95 % mortalities correspond to resistant insect populations (WHO/ANVR/MIM, 2003). populations from the two sites. 85% mortality rate of Anopheles was recorded at Ati- kankan 1 and 88% at Atikankan 2. 74 In Ogun state, 4 sites with Anopheles breeding spots were identified during this study: Imowo (Ijebu-Ode), Mobalufo (Ijebu-Ode), Ogere 1 (Lagos-Ibadan road), and Ogere 2 (Lagos Ibadan road). Permethrin resistance was recorded with Anopheles populations from Ogere 1 (mortality rate of Anopheles = 75%); Ogere 2 (mortality rate of Anopheles = 88%) and Mobalufon (mortality rate of Anopheles = 80%). A reduced susceptibility pattern was noticed with Anopheles populations from Imowo (mortality rate of Anopheles = 96%). Y In Lagos state, Anopheles breeding sites were found at Badagry where pRermethrin susceptibility was recorded with the Anopheles populations (mortality rate oAf Anopheles = 98%). Samples from Lagos city could not reach the insectary for BbioR-assays as they all died during the trip back to the laboratory. In most screened breIeding sites, cadavers of larvae were found on water surface. L The latitudes and the longitudes of the 19 suYrvey ed sites were used for the mapping of susceptibility status of Anopheles popIulTations to permethrin in southwestern Nigeria. Data generated showed that 13 bioassayed populations were found to be resistant to permethrin. (Fig. 4.1). S 4.1.2 Susceptibility to permethrin of EA. gRambiae collected in southern Benin Following the exposure oIfV females A. gambiae to permethrin impregnated papers and 24h monitoring of mortality rates of exposed females, resistance was recorded in the 6 surveyed divisions (Zou, PNlateau, Mono, Couffo, Oueme, Atlantique) of the Southern Benin. Out of the 18 pUopulations of A.gambiae sl. collected from these divisions, 17 populations wereN clearly resistant to permethrin, whereas only one population was susceptible (Table 4.2). InD theA Zou division, 2 sites with Anopheles breeding spots were identified: Seto and AAbomey. Permethrin resistance was clearly recorded in Anopheles populations from ISBeto (mortality rate of Anopheles = 80%) and Abomey (mortality rate of Anopheles = 83%). In Plateau division, 3 sites with Anopheles breeding spots were identified during the surveyed period: the site of Pobe, Sakete and Ifangni. Resistance was recorded in 75 RY RA LI B SI TY R IV E UN AN BA D I Fig. 4.1 Map of permethrin susceptibility status of Anopheles populations in the surveyed localities of southwestern Nigeria and southern Benin 76 TABLE 4.2 Susceptibility of A. gambiae populations to permethrin in southern Benin Divisions Localities Latitude Longitude Anopheles Mortality Susceptibility Tested Rates Zou Seto 2°59'39.4" 2°04'43.2" 50 80% Resistant Abomey 7°10'48.0" 1°58'48.0" 60 83% Resistant Plateau Pobe 6°57'36.0" 2°40'48.0" 50 80% RResistYant Sakete 6°42'36.0" 2°36'00.0" 55 91% A Resistant Ifangni 06°40'12.0" 2°43'48.0" 100 98R% Susceptible Mono Lokossa 6°36'36.0" 1°42'36.0" 80 IB93% Resistant Grand- Popo 6°16'12.0" 1°48'00.0" 60 L 90% Resistant Couffo Valehoue 8°01'34.6" 2°29'49.3" Y75 92% Resistant Oueme Gogbo 6°41'05.2" 2°28'37.1I" T 80 90% Resistant Akron 6°25'48.0" 2°38'S24.0" 103 23% Resistant Ketonou 6°24'00.0" 2R°32'24.0" 70 81% Resistant Atlantique Niaouli 6°49V'48.0E" 2°32'24.0" 75 67% Resistant Gbodjo 6°2I5'48.0" 2°18'00.0" 116 83% Resistant Pahou UN6°21'36.0" 2°10'48.0" 106 31% Resistant Akpakpa 6°21'00.0" 2°27'36.0" 110 69% Resistant A LadNji 6°23'40.1" 2°26'32.5" 100 65% Resistant DGbedjromede 6°21'36.0" 2°25'48.0" 112 36% Resistant A Houeyiho 6°21'36.0" 2°23'24.0" 76 70% Resistant ISusceptibility levels: From 97% to 100% mortalities (97% excluded) correspond to susceptible insect populations; 95% toB 97% mortalities (95% excluded) correspond to reduced susceptibility in insect populations; below 95 % mortalities correspond to resistant insect populations (WHO/ANVR/MIM, 2003). 77 Pobe (mortality rate of Anopheles = 80%) and Sakete (mortality rate of Anopheles = 91%). A. gambiae populations from the site of Ifangni were susceptible (mortality rate of Anopheles = 98%). In Mono division, two sites with Anopheles breeding spots were identified: Lokossa and Grand-Popo. Permethrin resistance was recorded in Anopheles populations from Lokossa (mortality rate of Anopheles = 93%) and Grand-Popo (mortality raYte of Anopheles = 90%). In Couffo division, only one site, Valehoue, with Anopheles breeding Rspots was identified. Permethrin resistance was recorded in Anopheles populations wiAth a mortality rate of 92 %. R In Oueme division, 3 sites with Anopheles breeding spots IwBere identified: Gogbo, Akron and Ketonou. Permethrin resistance was recorded in ALnopheles populations from Gobo (mortality rate of Anopheles = 90%), Akron (mortaYlity rate of Anopheles =23%) and Ketonou (mortality rate of Anopheles = 81%). T In Atlantique division, 7 sites with AnopIheles breeding spots were identified during this study: the sites of Niaouli, GRbodSjo, Pahou, Akpakpa, Ladji, Gbedjromede, Houeyiho. Permethrin resistance wasE clearly recorded in Anopheles populations from Niaouli (mortality rate of Anopheles = 67%), Gbodjo (mortality rate of Anopheles =83%), Pahou (mortality rate of AnoNphelIes V = 31%), Akpakpa (mortality rate of Anopheles = 69%), Ladji (mortality rate of Anopheles = 65%), Gbedjromede (mortality rate of Anopheles = 36%) and Houeyiho (m oUrtality rate of Anopheles = 70%) (Table 4.2). The latituNdes and the longitudes of the 18 surveyed sites were used for the mapping of pAermethrin susceptibility in the southern Benin (Fig. 4.1). Data generated revealed Dwide presence of resistance in most surveyed sites (Fig. 4.1). 4.2. AMolecular characterization of Anopheles populations from surveyed sites in INBigeria and Benin (PCR-species, PCR-forms and PCR-kdr) PCR analyses were conducted on a total of 288 female Anopheles. Out of the total of 288 Anopheles analysed, 141 of these individuals survived lethal doses of permethrin while 147 individuals died after exposure to permethrin. 78 4.2.1 Molecular characterisation of Anopheles from the southwestern Nigeria The polymerase chain reaction for species differentiation identified 76 A. gambiae ss. (26%) and 212 A. arabiensis (74%) (Fig.4.2a). At Ojoo and Ajibode all samples analysed were A. gambiae ss. whereas at the locality of Challenge both A. gambiae ss and A. arabiensis were found living in sympatry with 73% and 27% for A. gambiae ss. and A. arabiensis respectively. Except at Challenge where, Anopheles species collected were a mix of A. gambiae ss and A. arabiensis (Fig.4.2b), populations of Anopheles fouYnd in other surveyed localities of this region were mainly A. arabiensis (Table 4.R3). When identified samples of A. gambiae ss. were subjected to PCR-form diRffereAntiation, they were all identified as “M” forms. Molecularly characterized sBamples of Anopheles gambiae ss and arabiensis from the surveyed localities were suIbjected to PCR-kdr for investigations on the sodium channel structure. Only 1 mos quLito found in the locality of Challenge in Ibadan had a modified sodium channel at hYeterozygous form (RS). Analysis conducted on A. arabiensis samples did not show IanTy trace of kdr mutations (Table 4.4). The map of molecular mechanisms of permethrin resistance developed by Anopheles populations in this region of Nigeria showRed aS high presence of non-kdr mutants among females that had survived permethrin eExposure (Fig. 4.3) 4.2.2 Molecular characterisatioInV of mosquito populations from the southern Benin PCR analyses were conducted on a total of 438 female Anopheles. 196 of these individuals survived lethal dNoses of permethrin and 242 individuals died after exposure to permethrin (Table 4 .5U). The polymerase chain reaction for species differentiation identified 407 AN. gambiae ss. (93%) and 31 A. melas (7%). No A. arabiensis was identified in this region of Benin (Fig. 4.4). Two members of the A. gambiae complex namely AD. meAlas and A. gambiae ss. were found in sympatry in the sites of Houeyiho, KetoAnou, Pahou and Grand popo. The proportions of A. melas at Houeyiho, Ketonou, IPBahou and Grand- popo were 1%, 2%, 5% and 3% respectively whereas those of A. gambiae ss were respectively 99%, 98%, 95%, and 97 % (Table 4.5). When samples of A. gambiae ss. were subjected to PCR-form differentiation, they were all identified as “M” forms. Elevated frequencies of kdr alleles ranging from 0.9 to 0.5 were found in the 196 females Anopheles that had survived permethrin exposure (Table 4.6). The map of molecular mechanisms of permethrin resistance developed by Anopheles populations 79 AR Y R LIB ITY RS IV E U N Fig. 4.2a MemAbeNrs of the A. gambiae complex identified in southwestern Nigeria A DB I 80 RY RA LI B ITYS VE R UN I N Fig. 4.2b ThAe banding pattern of samples from Challenge, Ibadan showing A. gambiae Dss (coded “g”) and A. arabiensis (coded “Ar”) living together in similar site. I B A 81 TABLE 4.3 Distribution of members of A. gambiae complex in studied localities in Nigeria PCR-Species PCR- Forms Divisions Localities No. % % % % % Tested gambiae ss arabiensis melas “S” “M” Oyo IITA 20 100% - - - 100% Ojoo 30 100% - - - Y100% Ajibode 24 100% - - - R 100% Bashorun 20 100% - - A- 100% UI 18 100% - - R - 100% Challenge 30 73% 27% IB- - 100% Orogun 26 100% - L - - 100% Oja-tuntun 20 - 10Y0% - - - Osun Lagere 10 - IT100% - - - Modakeke 6 - S 100% - - - Ondo Ilesha garage 10 - R 100% - - - Owena 20 VE- 100% - - - Ekiti Ati-kankan 1 10 I - 100% - - - Ati-kankan 2 1N0 - 100% - - - Ogun Imowo U8 - 100% - - - MobafulNo 10 - 100% - - - OgerAe1 2 - 100% - - - DOgere2 8 - 100% - - - LagBos ABadagry 6 - 100% - - - I 82 TABLE 4.4 Allelic frequencies of the kdr mutation in Anopheles species from the studied localities in Nigeria Survivors Dead Divisions Localities Anopheles RR RS SS f-kdr Anopheles RR RS SS f-kdr tested tested Oyo IITA 0 - - - - 0 - - - - Ojoo 15 0 0 0 0 15 0 0 0 0 Ajibode 8 0 0 0 0 8 0 0 R0Y 0 Bashorun 15 0 0 0 0 15 0 A0 0 0 UI 0 - - - - 0 R- - - - Challenge 15 0 1 14 0.03 15 IB 0 0 0 0 Orogun 0 - - - - L0 - - - - Oja- 15 0 0 0 0 15 0 0 0 0 tuntun Y Osun Lagere 4 0 0 0 IT0 4 0 0 0 0 Modakeke 2 0 0 0S 0 2 0 0 0 0 Ondo Ilesha 8 0 0R 0 0 8 0 0 0 0 garage Owena 15 IV0 E0 0 0 15 0 0 0 0 Ekiti Ati- 12 0 0 0 0 12 0 0 0 0 kankan 1 Ati- N U9 0 0 0 0 9 0 0 0 0 kankan 2 Ogun Inowo N 3 0 0 0 0 3 0 0 0 0 MobAafulo 5 0 0 0 0 5 0 0 0 0 ODgere 1 1 0 0 0 0 1 0 0 0 0 AOgere 2 4 0 0 0 0 4 0 0 0 0 LaIgBos Badagry 1 0 0 0 0 1 0 0 0 0 83 RY RA LI B TY RS I E IV U N DA N A IB Fig. 4.3a Mechanisms of permethrin resistance identified in southwestern Nigeria and southern Benin 84 AR Y R LI B ITYS VE R UN I N Fig. 4.4 MemAbers of the A. gambiae complex identified in the southern Benin D A IB 85 TABLE 4.5 Distribution of members of A. gambiae complex in studied localities in Benin PCR- Species PCR-Forms Divisions Localities Anopheles % % % % % tested gambiae ss arabiensis melas S YM Zou Seto 20 100% - - R- 100% Abomey 24 100% - - A- 100% Plateau Pobe 15 100% - IB R- - 100% Sakete 16 100% - L - - 100% Ifangni 10 100% - - - 100% Mono Lokossa 18 100% ITY- - - 100% Grand- Popo 21 97% - 3% - 100% Couffo Valehoue 21 1R00%S - - - 100% Oueme Gogbo 23 E100% - - - 100% Akron 30 IV 100% - - - 100% Ketonou N30 98% - 2% - 100% Atlantique Niaouli U 30 100% - - - 100% Gbodjo N 30 100% - - - 100% PahAou 30 97% - 5% - 100% DAkpakpa 30 100% - - - 100% A Ladji 30 100% - - - 100% IB Gbedjromede 30 100% - - - 100% Houeyiho 30 99% - 1% - 100% 86 TABLE 4.6 Distribution of the kdr alleles in Anopheles species from the studied localities in Benin Survivors Dead Divisions Localities Anopheles RR RS SS f-kdr Anopheles RR RS YSS f-kdr tested tested Zou Seto 10 5 2 3 0.6 10 0 R2 8 0.1 Abomey 12 9 2 1 0.83 12 0A 1 11 0.04 Plateau Pobe 10 5 3 2 0.65 10B R0 2 8 0.1 Sakete 6 3 2 1 0.66 LI15 0 3 12 0.25 Ifangni 0 0 0 0 0Y 15 0 0 15 0 Mono Lokossa 3 1 2 0 T0.66 15 0 2 13 0.33 Grand- Popo 6 4 1 S1I 0.75 15 0 1 14 0.08 Couffo Valehoue 6 2 R3 1 0.58 15 0 4 11 0.33 Oueme Gogbo 8 E5 2 1 0.75 15 0 1 14 0.06 Akron 15 IV 5 8 2 0.6 15 0 6 9 0.2 Ketonou N15 8 5 2 0.7 15 1 3 11 0.16 Atlantique Niaouli U 15 5 8 2 0.6 15 0 2 13 0.06 Gbodjo 15 10 3 2 0.76 15 0 3 12 0.1 PahoAu N 15 12 2 1 0.86 15 0 1 14 0.03 DAkpakpa 15 8 4 3 0.66 15 0 5 10 0.16 A Ladji 15 13 2 0 0.93 15 0 6 9 0.2 IB Gbedjromede 15 10 5 0 0.83 15 1 4 10 0.2 Houeyiho 15 5 9 1 0.63 15 1 3 11 0.16 87 in this southern region of Benin showed a high presence of kdr mutants among females that had survived permethrin exposure (Figs. 4.3a and 4.3b) 4.3 Evaluation of potential contributions of agricultural pesticides to selection of pyrethroid resistance in Anopheles populations breeding around vegetable farms Several analyses of soil and water samples were conducted in the laboratory to assess the presence of pesticide residues in water bodies surrounding farms under synthetic pesticides treatments. Y 4.3.1 The use of synthetic pesticides in the vegetable farm of Houeyiho in BeRnin In the vegetable farm of Houeyiho, all the 20 farmers interviewed agAreed that it is impossible to grow vegetables without using pesticides. Some vegetabRles like cucumber, lettuce and green cabbages are very sensitive to pests and have toI bBe treated very often to avoid pests attack. Farmers also agreed that without pesticide Ltreatments, the yield is very low and most vegetables are destroyed before getting toY ma turity. Among the pesticides used, 2 were the most mentioned: the Decis® (deltamTethrin EC 10.75 g/l), and Kinikini® (mixture of cyfluthrin and malathion) (Table 4S.7). IOther local products made from neem (Azadirachta indica) seeds and papaya leRaves were also mentioned by farmers as being effective in controlling cabbage nematodes. The supply chain for pesticides was also probed but very few farmers could prEovide information on where and how they got the pesticides; more than 50% of inteIrVviewed farmers got the pesticides from informal or non- registered suppliers. In moNst cases, vegetable farmers received a basic training from extension workers of thUe ministry of agriculture in Benin. The training is focussed on pest management stratNegie s such as the recognition of pests, periods of treatment, the type of pesticides to use and, the basic health safety measures. More than 30% of interviewed farmers Ddo nAot respect manufacturer‟s instructions on the use of pesticides. In the vegetAable farm at Houeyiho, cabbages were treated 2 times each week and during the 65 IdBays for the maturity of cabbage; this corresponds to an average of 18 treatments for each complete round of vegetables development. Investigations in vegetable farms also revealed that 72 treatments of pesticides are done annually at Houeyiho. 4.3.2 The use of synthetic pesticides in the vegetable farm of Ajibode in Nigeria At Ajibode, farmers also agreed that it was impossible to grow vegetables without using pesticides. The pesticides mainly used by farmers are lambda cyhalothrin 88 Y RA R LI B SI TY VE R NI N U Fig. 4D.3b TAhe kdr Banding pattern recorded with mosquito samples from Benin A I B 89 commercially known as Karate® and cypermethrin known as Cyper-Di-force®. No traditional mixture of plant leaves and seeds is used for vegetable pests control at Ajibode (Table 4.7). Empty containers of pesticides were found at the Ajibode farm. Trainings on good practices for pests management in vegetable farms are not properly organized at Ajibode. Farmers use pesticides irrespectively of the indicated doses. The number of pest treatments in vegetable farming increases with the intensity of pests attack recorded Yin the farms. 4.3.3 Susceptibility to permethrin of A. gambiae collected around vegetableR farms of Houeyiho and Ajibode A 76 and 85 females Anopheles collected around vegetable fBarmsR of Houeyiho and Ajibode were exposed to permethrin impregnated papers. MortalitIy rates of 70% and 90% were recorded at Houeyiho and Ajibode respectively. Ano pLheles populations collected around the vegetable farms were recorded to be resistant Yto permethrin (Table 4.2) 4.3.3.1 Assessment of the presence of pesticide IreTsidues in Anopheles breeding sites found in surveyed vegetable farms through mSonitoring of eggs hatching rates The control breeding sites used in this experiment (the control breeding site made of well water collected at the CREC and rEeferRred here as CREC-water) offered favourable conditions for the hatching of egIgsV of both A. gambiae from Kisumu and Ladji (more than 70% hatching rates for bothN strains). When 10 g of surface soil from agricultural areas under insecticide treatments were added into 1000 ml of water of the control breeding, a decrease in hatching ratUes was observed with both strains but more significantly with the susceptible strain A. gambiae from Kisumu. The hatching rates of A. gambiae from Kisumu and AA. gaNmbiae from Ladji dropped from 75% and 86% in control breeding sites to 7% anDd 37% respectively in the test breeding sites (Fig. 4.5). The statistical comparison of boAth results revealed a relatively high toxic impact of the simulations on A. gambiae IfBrom Kisumu eggs (P=0.000). Data from this experiment revealed the inhibition of the hatching rate of Anopheles eggs in breeding sites reconstituted with soil samples collected in vegetable farms under synthetic pesticide treatments. With breeding sites reconstituted with soil from irrigation pool mixed with the irrigation water, a relatively low hatching inhibition was recorded. Both strains A. gambiae from Kisumu and A. gambiae from Ladji gave hatching rates of 58% 90 TABLE 4.7 The use of pesticides in the vegetable farm of Houeyiho in Benin and Ajibode in Nigeria Y Locations R Vegetable farming practices Houeyiho (Benin) Ajibode (NAigeria) Main synthetic insecticide Decis® (Deltamethrin Karate®R (lambda used EC 10,75 g/l) cyhalothrin) Kinikini® (cyfluthrin + LCIypBer-Di force® malathion) (Cypermethrin) Locally made insecticides Neem extracts+ pTapaYya No extract leaves and grainIs identified Treatment doses for pests 40 g of (Neem + About 50ml of the control in vegetable farms papaya mSixture) is product (Karate®) diluEted Rin 15L of water is diluted in 15 L of water Frequency of vegetable farms IVThe number of The number of treatment N treatment is high, and treatment is high, with synthetic and local depends on the level of and depends on the pesticides U pest attacks level of pest attacks Types of vegetaNbles grown by Brassica oleracea, Brassica oleracea, farmers A Amaranthus blitoides, Amaranthus Daucus carota, blitoides, D Cucumis sativus, A Lactuca sativa IBOrigin of pesticides used by Formal and informal Pesticides used by farmers chain supply system farmers are obtained commercially 91 Y RA R LIB ITY S ER NI V U N Fig. 4.5 HatcAhing rate of resistant and susceptible strains of Anopheles in simulated water anDd soil samples from vegetable farms of Houeyiho BAI 92 and 72% respectively (Fig. 4.5). In the last simulation made with irrigation water alone, the recorded hatching rate for eggs of A. gambiae from Kisumu and A. gambiae from Ladji was 38% and 60% respectively (Fig. 4.5). For samples from the vegetable site at Ajibode, Nigeria, the control breeding sites used was UI-water collected from a spring identified in the Zoological garden. This control offered favourable conditions for the hatching of eggs of both A. gambiae from Kisumu and A. gambiae from Ladji (70% and 72% hatching rates respectively). WhYen 10 g of the surface soil from the agricultural area of Ajibode was added into 1000 mRl of water from the control site (UI-water), a remarkable inhibition of the hatchinAg rates of A. gambiae from Kisumu and A. gambiae from Ladji was recorded. TRhe hatching rates dropped from 70% and 72% as earlier recorded in control breediIngB sites to 5% and 20% respectively in the test breeding sites (Fig. 4.6). L In the second set of simulated breeding sites Yreco nstituted with soil from the irrigation pool mixed with the irrigation water, a IreTlatively low hatching inhibition was recorded. Hatching rates of 55% and 65% were recorded with eggs of A. gambiae from Kisumu and A. gambiae from Ladji respectivelyS (Fig. 4.6). In the third simulation made excluRsively with irrigation water from the Ajibode farm, the hatching rate for eggs IoVf A. Egambiae from Kisumu and A. gambiae from Ladji was 30% and 55% respectively. 4.3.3.2 Assessment of the pNresence of pesticides residues in Anopheles breeding sites found in surveyed ve geUtable farms through monitoring of larval development rates The monitNoring of larval development rates in the control samples made of well water from thAe CREC (CREC-water), showed that 98% and 92% of larvae of A. gambiae from LadDji and A. gambiae from Kisumu respectively got to the pupae stage (Fig. 4.7). In the teAst breeding sites constituted of samples from the agricultural settings of Houeyiho, a IdBecrease in the development rate was recorded in the two simulations made in the laboratory. In the first test simulation made of soil from the vegetable farm diluted with CREC-water, 42% and 61% of A. gambiae from Kisumu and A. gambiae from Ladji respectively got to the pupae stage. In the second test simulation made of wet soil and irrigation water from the vegetable farm, 85% and 96% of A. gambiae from Kisumu and A. gambiae from Ladji respectively got to the pupae stage (Fig. 4.7). 93 AR Y R LIB ITY S ER NI V N U Fig. 4.6 HatcAhing rate of resistant and susceptible strains of Anopheles in simulated water anDd soil samples from vegetable farms of Ajibode BAI 94 RY BR A LI ITYS ER NI V Fig. 4.7 Larval deve lUopment of resistant and susceptible strains of Anopheles in simulated water Nand soil samples from vegetable farms of Houeyiho AD A IB 95 In the third set of test breeding sites made of irrigation water alone, 63% and 70% larval development rates were recorded for A. gambiae from Kisumu and A. gambiae from Ladji respectively (Fig. 4.7). In the vegetable site of Ajibode, the control breeding sites made with 1000 ml water collected from a spring flowing in the Zoological garden (UI-water) gave a mean percentage of 90% and 89% larvae of A. gambiae from Ladji and A. gambiae from Kisumu respectively getting to the pupae stage (Fig. 4.8). When 10 g of the surfacYe soil from the agricultural area of Ajibode was added to 1000 ml of water of thRe control breeding site, the development of larvae showed a relatively low rate ofR firstA instars larvae reaching the pupae stage: 40% and 50% for larvae of A. gambiae from Kisumu and A. gambiae from Ladji respectively (Fig. 4.8). IB In breeding sites reconstituted with soil from irrigaLtion pool mixed with the irrigation water from Ajibode, 75% and 80% of larvae ofY A. gambiae from Kisumu and A. gambiae from Ladji respectively reached the pupae stTage (Fig. 4.8). In the last set of breeding sites made SexclIusively with the irrigation water from Ajibode farm, 55% and 60% of larvae ofR A. gambiae from Ladji and A. gambiae from Kisumu respectively got to pupae stageE (Fig. 4.8). 4.3.3.3 Assessment of the preseInVce of pesticides residues in Anopheles breeding sites found in surveyed vegetablNe farms through monitoring mosquito productivity The proportions of A. gambiae eggs reaching adult stage were low in simulated breeding sites. In the coUntrol breeding site made with CREC-water, 46% and 48% of the 200 eggs of A. Ngambiae from Kisumu and the 200 eggs of A. gambiae from Ladji respectively rAeached the adult stage. In breeding sites containing 100 g of surface soil from the Dvegetable site of Houeyiho, the yield of adult production dropped significantly to 2% aAnd 21% for A. gambiae from Kisumu and A. gambiae from Ladji respectively IcBompared to the yield recorded in the control breeding sites (46% and 48% for A. gambiae from Kisumu and A. gambiae from Ladji respectively) (Fig. 4.9). When eggs were inoculated into the artificial breeding sites made with soil and watering water from the agricultural site of Houeyiho, an adult production of 49% and 68% was recorded for A. gambiae from Kisumu and A. gambiae from Ladji respectively. In the last set of breeding sites reconstituted with water of irrigation from the vegetable farm of Houeyiho, 20% of 96 RY RA LI B ITY ER S V NI N U A Fig. 4.8 DLarval development of resistant and susceptible strains of Anopheles in simuAlated water and soil samples from vegetable farms of Ajibode I B 97 RY RA LI B ITY RSE NI V U AN Fig. AD4.9 The yield of rearing resistant and susceptible strains of Anopheles in IsBimulated water and soil samples from vegetable farms of Houeyiho 98 adults of A. gambiae from Kisumu and 39% of A. gambiae from Ladji were produced from the 200 eggs inoculated. The growth of the resistant strain A. gambiae from Ladji was less affected by simulations than that of the susceptible strain A. gambiae from Kisumu. The yield of resistant strains was always high compared to susceptible strains: 2%, 49% and 20% for A. gambiae from Kisumu versus 21%, 68% and 39% for A. gambiae from Ladji in the various simulated breeding sites (Fig. 4.9). With samples from Ajibode, the proportions of adults Anopheles recorded Yin the control breeding site (UI-water) were 50% and 53% of the 200 eggs of A. AgamRbiae from Kisumu and the 200 eggs of A. gambiae from Ladji respectively. RIn breeding sites containing 100 grams of surface soil from the vegetable site at AjibBode, the yield of adult production was very low with both strains: 5% and 10% for A. gaImbiae from Kisumu and A. gambiae from Ladji respectively A. gambiae from Ladji (FLig. 4.10). When eggs were inoculated into breeding sites reconstituted with soiYl an d watering water from the agricultural site of Ajibode, an adult production oIfT 35% and 60% was recorded for A. gambiae from Kisumu and A. gambiae from SLadji respectively (Fig. 4.10). In breeding sites made of irrigation water from the vegetable farm of Ajibode, 15% of adults of A. gambiae from Kisumu and 30% of AE. gamRbiae from Ladji were produced from the 200 eggs inoculated (Fig. 4.10). 4.4 Evaluation of the potentiaIl Vcontributions of spilled petroleum products in the selection of pyrethroid resiNstance in A. gambiae populations Several experiemUents were conducted in the field and the laboratory to assess the lethal effect of peNtrole um products on mosquito larvae, their modes of action on mosquito larvae and their contributions in the selection of pyrethroid resistance in malaria vectors. 4.4.1 ThDe emApirical utilisation of petroleum products for mosquito control in rural commAunities of the southern Benin I B The use of petroleum products for mosquito control in rural communities of the southern Benin was confirmed through interviews conducted in 4 communities Gbodjo, Ladji, and Ketonou. Petroleum products are used against several insects of great nuisance like: mosquito larvae, flies and cockroaches. The petroleum products mainly used in these communities are kerosene and waste engine oil from mechanics. Others, such as petrol and engine oil, are said by the interviwed individuals to be used occasionally. 99 AR Y BR Y LI ITS ER NI V U N Fig. 4.10 ThAe yield of rearing resistant and susceptible strains of Anopheles in simulateDd water and soil samples from vegetable farms of Ajibode, Ibadan BAI 100 These products are either sprayed on the ground, on table surfaces, on standing water points and in latrines. Out of a total number of 65 key respondents interviewed in the three communities, 73% spray kerosene in standing water points and latrines to control mosquito growth and nuisance, whereas 9% use waste engine oil, 5% engine oil and 2% petrol (Fig. 4.11). Information extracted from the interviewed populations also showed that 11% do not know much about using petroleum products for pests control.Y This investigation also revealed that this traditional technique of controlling vectors using petroleum products is transferred in the community from parents to offspring wRithout any scientific explanation to support the efficacy of this strategy. RA 4.4.2 Analysis of the lethal effect of petrol on permethrin IrBesistant larvae of A. gambiae from Ladji and A. gambiae from Ojoo Experiments conducted in the laboratory on the larvicidYal ac ti Lvities of petrol on larvae of A. gambiae from Ladji showed 100% mortality when breeding sites contained in -3 2 laboratory bowls were treated with petrol at concIenTtrations of 7.856 × 10 μl/cm . The lowest concentration (LoC100) of petrol capableS of producing 100 % mortality was 7. 856 -3 2 × 10 μl for each cm of treated breedinRg sites (Fig. 4.12.). The highest concentration (HiC) of petrol yielding no larvicidal aEctivity on the resistant strain A. gambiae from Ladji -3 2 was 11.8 × 10 μl for each cm I oVf sprayed surface. The LC50 of petrol on larvae of A. -3 2gambiae from Ladji was N2.946 × 10 μl/cm . Treatments of breeding sites with -3 2concentrations of pet roUl below 7.856 × 10 μl/cm did not kill 100% of larvae of A. gambiae from LaNdji but rather selects some survivals (Fig. 4.12.). When this experiment was conducteAd with the permethrin resistant strain A. gambiae from Ojoo, the larvicidal activitiesD of petrol on this strain showed 100% mortality when breeding sites were sprayed -3 2with Apetrol at concentration of 10.120 × 10 μl/cm . The lowest concentration (LoC100) of -3 2pBetrol capable of producing 100% mortality was 10.120 × 10 μl for each cm of treated Ibreeding site. The highest concentration (HiC) of petrol yielding no larvicidal activity on the -3 resistant strain A. gambiae from Ojoo was recorded at concentration of 393 × 10 μl for 2 each cm of sprayed surface. The LC50 of petrol on larvae of A. gambiae from Ojoo was -3 2 7856 × 10 μl/cm . Treatments of breeding sites with concentrations of petrol below -3 2 10.120 × 100 μl/cm kill less than 100% of larvae of A. gambiae from Ojoo and therefore 101 Y None Petrol R Waste Oil 7 2 A 6 11% 3% 9% BRI Eng. Oil 3 L 4% SI TY ER Kerosene IV 4873% UN N Fig. 4.11 UAtilisation of petroleum products for mosquito control in rural communDities in southern Benin BAI 102 RY RA LI B ITY RSE NI V U Fig. 4.12 DLarvA N icidal effect of petroleum products on larvae of A. gambiae from Ladji. A I B 103 select some survivals (Fig. 4.13). 4.4.3 Analysis of the lethal activity of kerosene on permethrin resistant larvae of A. gambiae from Ladji and A. gambiae from Ojoo The treatment of breeding sites with kerosene was very effective on larvae of A. -3 gambiae from Ladji at concentrations ranging from 11.8 × 10-3μl to 3.930 × 10 μl per 2 cm . At these concentrations, larval mortalities increased from 0% to 100% (Fig. Y4.12). Larvicidal activities of kerosene on larvae of A. gambiae from Ladji showed 100% mortality when breeding sites contained in laboratory bowls were mixed withR petrol at -3 2 concentration of 3.930 × 10 μl/cm . The lowest concentration (LoRC100A) of kerosene -3capable of producing 100% mortality was, therefore, recorded at 3B.930 × 10 μl for each 2cm of treated breeding site (Fig. 4.12). The highest concentraItion (HiC) of kerosene yielding no larvicidal activity on the resistant strain A. gambiLae from Ladji was recorded -3 2 at concentrations of 11.8 × 10 μl for each cm of sprayedY sur face. -3 2 The LC50 of kerosene on larvae of A. gambIiaTe from Ladji was 1.964 × 10 μl/cm . - 3 2Treatments of breeding sites with concentratioSns of kerosene below 3.930 × 10 μl/cm (the LoC100) did not kill 100% of larvae of RA. gambiae from Ladji but rather selected some survivals (Fig. 4.12). When this experiment waIsV repe Eated on the permethrin resistant strain A. gambiae from Ojoo, the larvicidal actNivity of kerosene on this strain showed 100% mortality when -3 2breeding sites were mixed with kerosene at concentrations of 10.020 × 10 μl/cm . The lowest concentration (LUoC100) of kerosene capable of producing 100% mortality was -3 210.020 × 10 μl for each cm of treated breeding sites (Fig. 4.13). The highest concentrationA (HiNC) of kerosene yielding no larvicidal activity on the resistant strain A. -3 2gambiae Dfrom Ojoo was 393 × 10 μl for each cm of treated surface. The LC50 of -3 2kerosAene on larvae of A. gambiae from Ojoo was 5892 × 10 μl/cm . Treatments of B -3 2Ibreeding sites with concentrations of kerosene below 10.020 × 100 μl/cm killed less than 100% of larvae A. gambiae from Ojoo and resulted in the selection of few survivals (Fig. 4.13). 104 AR Y R LI B ITYS VE R I U N Fig. 4.13 LarAvicidNal effect of petroleum products on larvae of A. gambiae from Ojoo AD I B 105 4.4.4 Analysis of the lethal effect of new engine oil on permethrin resistant larvae of A. gambiae from Ladji and A. gambiae from Ojoo Engine oil was effective on the resistant strain A. gambiae from Ladji after -3 2 treatments of simulated breeding sites with concentrations between 11.8 × 10 μl/cm and -3 2 3.930 × 10 μl/cm . At these concentrations, larval mortalities increased from 0% to 100% (Fig. 4.12). Larvicidal activities of engine oil on larvae of A. gambiae from Ladji showed 100% mortality when breeding sites contained in laboratory bowls were treatedY with -3 2 engine oil at concentration of 3.930 × 10 μl/cm . The lowest concentratioAn (LRoC100) of -3 2engine oil capable of producing 100% mortality was 3.930 × 10 μl for eRach cm of treated breeding site (Fig. 4.12). The highest concentration (HiC) of engine oil yielding no -3 larvicidal activity on the resistant strain A. gambiae from Ladji wIasB 11.8 × 10 μl for each 2 cm of treated surface. The LC50 of engine oil on larvae of AL. gambiae from Ladji was -3 21.964 × 10 μl/cm . Treatments of breeding sites with coYncentrations of engine oil below -3 2 3.930 × 10 μl/cm (the LoC100) killed less thanI 1T00% of larvae of A. gambiae from Ladji and resulted in the selection of few individuals (Fig. 4.12). When this experiment was replicRatedS with the permethrin resistant strain A. gambiae from Ojoo, the larvicidal aEctivity of engine oil on this strain showed 100% -mortality when breeding sites weIreV treated with engine oil at concentration of 10.020 × 103 2μl/cm . The lowest concenNtration (LoC100) of engine oil capable of producing 100% -3 2mortality was 10.020 × 10 μl for each cm of treated breeding sites (Fig. 4.13). The highest concentration (HUiC) of engine oil yielding no larvicidal activity on the resistant -3 2strain A. gambiaeN from Ojoo was recorded at concentration of 393 × 10 μl for each cm of sprayed surfaAce (Fig. 4.13). The LC50 of engine oil on larvae of A. gambiae from Ojoo -3 2was 5.89D2 × 10 μl/cm . Treatments of breeding sites with concentrations of engine oil belowA -3 2 10.020 × 110 μl/cm killed less than 100% of larvae of A. gambiae from Ojoo and IrBesulted in the selection of few individuals survivals (Fig. 4.13). 4.4.5 Analysis of the lethal activity of used engine oil on permethrin resistant larvae of A. gambiae from Ladji and A. gambiae from Ojoo Treatments with waste engine oil exhibited mortalities of 100% at concentrations - 3 2 equal or higher than 3.930 × 10 μl/cm . This concentration represents the lowest concentration of used engine oil (LoC100) producing 100% mortalities on larvae of A. 106 gambiae from Ladji (Fig. 4.12). The highest concentration (HiC) of used engine oil - yielding no larvicidal activity on the resistant strain A. gambiae from Ladji was 11.8 × 10 3 2 μl for each cm of treated surface. The LC50 of used engine oil on larvae of A. gambiae -3 2 from Ladji was 1.964 × 10 μl/cm . Treatments of breeding sites with concentrations of -3 2 used engine oil below 3.930 × 10 μl/cm (the LoC100) killed less than 100% of larvae of A. gambiae from Ladji and resulted in the selection of few individuals (survivals post treatments) (Fig. 4.12). Y When this experiment was replicated with the permethrin resistant Rstrain A. gambiae from Ojoo, the larvicidal activity on this strain showed 100% mAortality when -3 2 breeding sites were treated with used engine oil at concentration oBf 10R.020 × 10 μl/cm . The lowest concentration (LoC100) of waste engine oil capabIle of producing 100% -3 2 mortality was recorded at 10.020 × 10 μl for each cm o f Ltreated breeding sites (Fig. 4.13). The highest concentration (HiC) of used engine oiYl yielding no larvicidal activity on -3 2 the resistant strain A. gambiae from Ojoo was 393 I× T10 μl for each cm of treated surface (Fig. 4.13). The LC50 of used engine oil on larvae of A. gambiae from Ojoo was 5892 × -3 2 10 μl/cm . Treatments of breeding sites wRith cSoncentrations of engine oil below 10.020 × -3 2100 μl/cm killed less than 100% of Elarvae of A. gambiae from Ojoo and resulted in the selection of few individuals (Fig. 4.13). The comparison of activIitVies of the 4 petroleum products on permethrin resistant larvae of A. gambiae from LNadji and A. gambiae from Ojoo showed a relatively low lethal activity of petrol on UAnopheles larvae (Figs. 4.12 and 4.13) as recorded with the determined HiC aNnd L oC values (Figs. 4.14 and 4.15). 4.4.6 IdentifiAcation of the mode of action of PP on A. gambiae larvae A. gambiDae from Ladji was chosen in this experiment because of its relatively consistent susceAptibility to petroleum products compare to A. gambiae from Ojoo. The analysis of ItBhe mode of action of petroleum products on larvae of A. gambiae from Ladji showed that 100% (n = 400 exposed larvae) of larvae were killed in laboratory bowls containing water with mixtures of petroleum products collected in the field with oil pellicle (oil film) covering the surface of the water. However, when this pellicle was completely removed through series of sieving, the mortality decreased to 96% therefore, associating 4% of larval mortalities to suffocations from oil pellicle developed as a result of PP present in 107 AR Y LIB R TY RS I VE NI N U Fig. 4.14 CoAmparative analysis of recorded HIC of petroleum products on A. gambiae Dfrom Ladji and A. gambiae from Ojoo larvae A I B 108 RY BR A LI ITY RSE NI V U AN D Fig. A4.15 Comparative analysis of recorded LoCB 100 of petroleum products on A. Igambiae from Ladji and A. gambiae from Ojoo larvae 109 water surface (Fig. 4.16). 4.4.7 Analysis of physico-chemical properties of breeding sites producing pyrethroids resistant and susceptible populations of A. gambiae in southwestern Nigeria and southern Benin The nature of breeding sites was analysed in target localities. Two types of breeding sites were identified in the 19 localities surveyed in southwestern Nigeria. The first contained petroleum products particles and were nominated as “oily breedRing sYites”, while the second had clean water with no petroleum residues and were nominated as “non- oily breeding sites”. Out of the 19 sites surveyed in Nigeria, 13 were “oily bAreeding sites” and 6 were “non-oily breeding sites”. The mortality rates to permetRhrin of Anopheles populations emerging from oily breeding sites and non-oily wIeBre 82.1% and 97.2% respectively. Breeding sites identified in the field with petroleLum particles produced more permethrin resistant populations of Anopheles than breeYdin g sites not showing traces of petroleum products (P=0.001) (Table 4.8). T The mean pH of breeding sites producSing Ipermethrin resistant populations of A. gambiae was 7.9 whereas that of breeding sites producing permethrin susceptible populations of A. gambiae was 7.5. ThEe mRean DO of breeding sites producing permethrin resistant Anopheles was 14.34mIgV/l, while that of breeding sites producing permethrin susceptible Anopheles was 3N4.5mg/l (Table 4.9). In the surveyed sites of southern Benin, two types of breeding sites were identified in the 18 localities surveyed: 9 turbid breeding sites containing food d eUtritus and vegetation (nominated as “turbid breeding sites”) and 9 clean breeding siNtes with neither food particles nor vegetation; these were recorded as “non-turbid” bAreeding sites. TDhe mortality rates to permethrin of Anopheles populations emerging from turbid breedAing sites and non-turbid breeding sites were 65.5% and 75.62% respectively. IABlthough both types of breeding sites produced permethrin resistant Anopheles, it was found that the turbid breeding sites identified in the southern Benin could produce more mosquitoes with permethrin resistant status than the clean “non-turbid” breeding sites (Table 4.10). The mean pH of turbid breeding sites was found at 7.8 compared to the mean pH of clean non-turbid breeding sites which was 7.3. The mean DO recorded with turbid breeding sites was 12.2mg/l compared to the mean DO recorded with clean 110 RYA LIB R ITY RSE IV U N Fig. 4.16 MoAde oNf action of petroleum products on Anopheles larvae (Mortality rates recorded with sieved and crude or raw petroleum products from field contaminated breedAingD sites) I B 111 Y TABLE 4.8 Comparison of the mean mortality rates to permethrin oAf AR. gambiae populations produced by oily and non-oily sites in Nigeria Oily breeding sites IBN Ron Oily breeding sites Number of breeding sites 13 L 6 surveyed during the study Y Number of tested females of A. I8T49 546 gambiae from identified breeding sites S Mortality rates (mean) recorded with R 82.1% 97.2% Anopheles from each type of breeding E sites following exposure to permIetVhrin Variance (mortality rates in N 4.33 47.7 surveyed localities) Pv. Comparing the me anU mortalities Pv=0.00151 (following exposure to permethrin) recorded with moNsquito emerging from oilyD andA non-oily breeding sites IB A 112 TABLE 4.9 Physico-chemical parameters (pH and DO) of breeding sites producing susceptible and resistant populations of Anopheles in southwestern Nigeria Localities in Breeding Oxygen level in pH Anopheles Mortality Nigeria site type the breeding level tested rates (%) site (mg/l) Y IITA No oil 40 7.2 95 1R00 Ajibode No oil 25 9 85 A90 Ojoo Oily 12 7.9 80 R 80 Challenge Oily 12 75 80I B 81 Lagere Oily 20 7.7 L94 94 Ati-kankan 1 Oily 15 7.6 Y 83 85 Ati-kankan 2 Oily 15 I7T.7 80 88 Mobafulo Oily 13 S 7.9 75 80 Ogere 1 Oily 10 R 7.8 4 75 UI No oil V52E.5 7.9 80 100 Orogun No oil NI 30 7.1 80 100 Modakeke No oUil 35 7 80 97 Inowo No oil 19.5 9 76 96 Badagry ANNo oil 30 7 50 98 BashoDrun Oily 12 8 75 70 OAja-tuntun Oily 13 7.8 83 81 IBIlesha garage Oily 16.5 7.6 77 89 Owena Oily 10 9 78 75 Ogere 2 Oily 13 7.6 40 88 113 Y R A TABLE 4.10 Comparison of the mean mortality rates to permethrRin of A. gambiae populations produced by turbid and non-turbid sites in souLtheIrnB Benin Surveyed breeding sites Turbid breeding sites Clean breeding sites (with few traces Yof o il) (no oil traces) Number of breeding sites 9 T 9 Mean dissolved oxygen S12I.2 27.4 Mean pH level of breeding sites R 7.8 7.3 Mean mortality rates recorded with E 65.7% 75.6% permethrin following exposure of V emerging Anopheles I Permethrin resistance leUvel oNf High Less high emerging Anopheles AN D I B A 114 breeding sites 27.4mg/l (Table. 4.11). 4.4.8 Identification of the preferred types of breeding sites selected by pyrethroids susceptible and resistant strains of Anopheles for ovipositions When gravid females of the permethrin susceptible strain A. gambiae from UI were released in cages containing several breeding sites reconstituted with water samples collected in the locality of spilled petroleum products of Ojoo in Nigeria, out of a mean number of 1,900 eggs laid in each cage, 4.5% (86 eggs) of these eggs were oviposiYted in oily breeding sites and 95.5% (1814 eggs) in the clean breeding site (control sRite). With gravid females of the permethrin resistant strains A. gambiae from OjoAo released in similar cages, out of a mean number of 2,425 eggs laid in cages, 77% (1R875 eggs) of these eggs were oviposited in breeding sites with clean water (control IsiBte) whereas 23% (550 eggs) of these eggs were laid in breeding site with petroleum dLebris (Fig.4.17). Neither the resistant strain A. gambiae from Ojoo nor the suscepYtible strain A. gambiae from UI preferred laying eggs in oily breeding sites. HowevTer, it was observed that the resistant strain A. gambiae from Ojoo could lay a relatiSvelyI high number of eggs in oily breeding sites compared to the susceptible strain A. gambiae from UI (23% of eggs laidE by RA. gambiae from Ojoo versus 4.5% of eggs laid by A. gambiae from UI). When this experiment was replicated using water samples collected in the locality of sNpilleId Vpetroleum products of Akpakpa in southern Benin, the permethrin resistant strain A. gambiae from Ojoo laid 28% (725 out of 2525) of eggs in the oily breeding sites aUnd 72% (1800 out of 2525) in clean water (Fig. 4.17). With the sNusceptible strain A. gambiae from UI released in similar cages, 5% (105 out of 1880) Aof oviposited eggs were deposited in oily breeding sites and 75% (1775 out of 1880) Dwere laid in clean breeding sites (Fig. 4.17). With samples from Benin, neither the reAsistant strain A. gambiae from Ojoo nor the susceptible strain A. gambiae from UI IpBreferred laying eggs in oily breeding sites. However, it was observed that the resistant strain A. gambiae from Ojoo could lay a relatively high number of eggs in oily breeding sites compared to the susceptible strain from UI (28% of eggs laid by A. gambiae from Ojoo versus 5 % of eggs laid by A. gambiae from UI). 115 TABLE 4.11 Physico- chemical parameters (pH and DO) of breeding sites producing susceptible and resistant populations of Anopheles in southern Benin Localities Breeding O2 level pH level individuals Mortality site type in mg/l in % Y Seto Turbid 14 7 50 R80 Grand- Popo Turbid 12 7 60 RA 90 Valehoue Turbid 13 8 75 92 Akron Turbid 10 8.5 LI10B3 23 Niaouli Turbid 12 8.1 Y 75 67 Akpakpa Turbid 12 9 110 69 Ladji Turbid 12 IT7 100 65 Gbedjromede Turbid 12R S 7 112 36 Houeyiho Turbid E13 9 76 70 Abomey Clean V 24 7.1 60 83 Pobe CleanN I 20 7.6 50 80 Sakete CUlean 25 7.2 55 90 Ifangni N Clean 30 7 100 98 Lokossa A Clean 25 8.5 80 93 GogboD Clean 35 7 80 90 KeAtonou Clean 33 7.5 70 81 IBGbodjo Clean 30 7 116 83 Pahou Clean 25 7.2 106 31 116 AR Y LIB R SI TY R VE UN I N Fig. 4.17 NumAber of eggs laid by resistant (A. gambiae from Ojoo) and susceptible (A. gambAiae Dfrom UI) strains of Anopheles in oily and non-oily breeding sites from sBouthwestern Nigeria and southern Benin I 117 4.4.9 Hatching rate of eggs laid by pyrethroid susceptible and resistant strains in oily breeding sites The control breeding sites used in this experiment: The “clean breeding site from Ojoo in Nigeria” offered favourable conditions for the hatching of eggs of both A.gambiae from Ojoo and A. gambiae from UI. 84% (168 hatched eggs out of 200 eggs laid) and 87% (174 eggs out of 200 eggs laid) hatching rates were recorded in the control breeding sites with eggs of A. gambiae from Ojoo and A. gambiae from UI respectively (Fig. Y4.18). When eggs of the resistant strain A. gambiae from Ojoo and of the suscepAtibleR strain A. gambiae from UI were laid in oily breeding sites simulated with samples from Ojoo, a decrease in hatching rates was observed with both strains. TheB hatRching rates of A. gambiae from Ojoo and A. gambiae from UI dropped from 84I% and 87% in control breeding sites to 63% and 61% in the test breeding sites (Fig. 4L.18). In the site of Akpakpa, the locality of spilled Ypetroleum of Benin, the control breeding site (the clean breeding site from AkpTakpa in Benin) offered favourable conditions for the hatching of eggs of both A. gamIbiae from Ojoo and A. gambiae from UI. 80% (160 hatched eggs out of 200 eggs laSid) and 75% (150 hatched eggs out of 200 eggs laid) hatching rates were recordEed Rin the control breeding sites with eggs of A. gambiae from Ojoo and A. gamIbiVae from UI respectively (Fig. 4.18). When eggs of the resistant strains A. gambiae Nfrom Ojoo and of the susceptible strains A. gambiae from UI were laid in oily breeding sites reconstituted with samples from Akpakpa, a decrease in hatching rates was ob sUerved with both strains. The hatching rates of A. gambiae from Ojoo and A. gamNbiae from UI dropped from 80% and 75% in control breeding sites to 70% and 63%A in test breeding sites (Fig. 4.18). 4.D4.10 Development of larvae of pyrethroid susceptible and resistant strains in oily bAreeding sites (rate of larvae getting to pupae stage) IB The rates of first instar larvae getting to the pupae stage were low in all breeding sites containing traces of petroleum products. Very few larvae (1%) of the permethrin susceptible strain A. gambiae from UI could reach the pupae stage in breeding sites containing petroleum products residues from south western Nigeria (only 1 pupae was recorded out of the 122 L1 larvae that had emerged from hatched eggs) compared to the control breeding sites which yielded 64% of first instar larvae getting to pupae 118 Y RA R LIB ITY RS IV E U N AN Fig. 4.18 Hatching rate of eggs laid by resistant (A. gambiae from Ojoo) and susceAptibDle (A. gambiae from UI) strains of Anopheles in oily and non-oily breeding IsBites from Nigeria and Benin 119 stage (Fig. 4.19).With the permethrin resistant strains A. gambiae from Ojoo, 27% (34 out of 126 hatched individuals) of larvae were able to reach the pupae stage when reared in water with oily residues from Ojoo compared to the development rate recorded with samples in the control breeding sites where 66% of first instar larvae got to the pupae stage (Fig. 4.19). In the site of Akpakpa, the locality of spilled petroleum of Benin, the simulated control breeding sites (clean breeding Benin) offered favourable conditions foYr the development to pupae stage of first instar larvae of both A. gambiae from OjRoo and A. gambiae from UI: 63% and 60% respectively. In breeding sites with petrolAeum residues, the rates of first instar larvae getting to the pupae stage were low. VBery Rfew larvae (5%) of the permethrin susceptible strain A. gambiae from UI could reIach the pupae stage in breeding sites containing mixed petroleum products residuLes collected in petroleum polluted localities from southern Benin compared toY the development rate of 60% recorded in the control (Fig.4.19). T With the permethrin resistant strain A.S gamIbiae from Ojoo, 35% of larvae were able to reach the pupae stage when reareRd in water with oil residues from Akpakpa in Benin compared to the development Erate of 63% recorded in the control breeding sites (Fig. 4.19). 4.5 Identification of detNoxifIyi Vng genes up-regulated in pyrethroids resistant Anopheles from sites under synthetic agricultural pesticides utilisation and sites of spilled petroleum pro dUucts A competNitive hybridization was conducted on a microarray detox chip using permethrin reAsistant A. gambiae populations from the agricultural site of Akron and the site of spilled petroleum products of Ojoo and, the permethrin susceptible A. gambiae populatioDn from Oregun. I4B.5.1 A Genotyping and bioassay of Anopheles populations prior to micro-array analysis The genotypic analysis of Anopheles populations from the 3 localities (Akron, Ojoo and Orogun) selected for competitive hybridisation on micro-array chips showed that all mosquitoes samples were A. gambiae ss. When subjected to molecular forms analysis, results revealed that all sanples selected were A. gambiae ss of the “M” 120 RYA LIB R ITY ER S NI V U N Fig. 4.19 RatAe of larvae (larvae from hatched eggs) of A. gambiae from Ojoo and A. gambAiae Dfrom UI developing to pupae stage in oily and non-oily breeding sites from sBouthwestern Nigeria and southern Benin I 121 molecular form (Table 4.12). No individual was found with the East-kdr mutation. The West-kdr was found at high frequencies in A. gambiae from Akron populations, and neither mutation occurred in either A. gambiae from Orogun or A. gambiae from Ojoo. 4.5.2 Identification of metabolic genes over transcribed on Anopheles samples from agricultural setting The competitive hybridization of resistant A. gambiae samples from Akron and the susceptible Anopheles from Orogun on a “detox-chip” array exhibited different Ygene expression profiles (Fig. 4.20). The agricultural site of Akron revealed Aa reRmarkable expression of 5 genes from 2 groups of detoxification genes: the CYRP group and the CPLC group (not well documented yet). During samples analysis, oBver-expressions of the CYP6P3, CYP325D2 and CYP6M2 genes all belonging to the ICYP sub class and the CPLC8 and CPLC3 belonging to the group of genes involved iLn the cuticle synthesis were recorded. The expression levels obtained with theseY genes (CYP6P3, CYP325D2, CYP6M2, CPLC8 and CPLC3) in the resistant moIsqTuitoes from Akron were respectively 12.37; 5.05; 2.48; 8.03 and 5.24 fold changeSs as compared with the susceptible field population (A. gambaie from Orogun). The most striking result recorded with samples from the agriculture site of Akron waEs thRe 12.4-fold over expression of the cytochrome P450, CYP6P3 (Table 4.13 and Fig. 4.20). 4.5.3 Identification of metaNbolIic V genes over transcribed on Anopheles samples from oil spillage site A total of 9 d etUoxification genes were significantly expressed on samples from Ojoo, the localityN under oil spillages. 5 of the 9 genes (CYP6P3, CYP6N1, CYP6AG2, CYP6M2 anAd CYP6AK1) belong to the CYP sub-class whereas 2 (GSTD1-6 and GSTD11D) were from the GST sub-class and the last 2 (CPLC8 and TPX2) are involved in the cuAticle synthesis. The expression levels obtained with these genes (CYP6P3, CYP6N1, I4B.5.4 Comparative expression profiles of metabolic genes in permethrin resistant A. gambiae from Akron in southern Benin and Ojoo in the southwestern Nigeria In both resistant populations of mosquitoes (A. gambiae from Akron and A. gambiae from Ojoo) studied, the expression profiles of transcripts revealed the elevated expression of CYP6AG2, CYP6M2, CYP6AK1, GSTD1-6, GSTD11, CPLC8 and TPX2 in the resistant A. gambiae strain from Ojoo with respectively 7.37; 4.84; 2.71; 2.58; 2.19; 122 Y AR TABLE 4.12 Molecular form, percentage mortality and kdr freBquenRcy of A. gambiae from southern Benin and southwestern Nigeria I Collection site Cytotype % Mortality with W esLt- kdr East-kdr (N=30) Permethrin Frequency Frequency (N=80) Y (N=30) (N=30) Orogun M 100 IT 0 0 (control site) S Akron M 23R 0.86 0 (Agricultural) E Ojoo (Oil M V 80 0 0 contamination) NI N U DA I B A 123 Y RA R B Y LI T RS I E NI V U Fig. 4.20 CAandNidate metabolic genes over transcribed on resistant Anopheles mosquitoDes from the agricultural site of Akron when co-hybridized with the susceAptible strain A. gambiae from Orogun I B 124 Y R BR A I TABLE 4.13 Candidate metabolic genes from micro-arr ayL analysis of resistant and susceptible field samples from Akron and Orogun Y Site Identified Expressions Pvalues Genes SIle Tvel (log) (fold changes) CYP6P3 12.37 5.42 R CYP325D2 5.05 3.58 Akron E (Agricultural site) CNYP6IM V2 2.48 4.01 UCPLC8 8.03 4.01 N CPLC 5.24 3.49 DA I B A 125 4.07; 2.54; 2.62; and 5.02 fold changes as compared with the susceptible field population (Orogun) (Table 4.14 and Fig. 4.21). Two P450s namely CYP6P3 and CYP6M2 earlier identified on resistant populations of A. gambiae from Akron were also overexpressed on pyrethroid resistant samples from Ojoo. elevated expression of the CYP6P3 gene: more than 12 folds expression with sample from Akron and 7.37 folds with samples from Ojoo (Fig. 4.22). The CPLC8 belonging to the family of cuticular genes implicated in the synthesis and the thRicknYess of the cuticle in mosquitoes was also over transcribed in both samples. The expression levels recorded with CPLC8 was more than 8 folds with Anopheles populatiAons from the agricultural site of Akron and 2.62 folds on samples from the oil spBillagRe locality of Ojoo (Fig. 4.22). I The last similarity recorded among the 2 resistant Lpopulations of A. gambiae analysed was the up-regulation of the CYP6M2 gene. TYhe CYP6M2 gene which belongs to the subclass of CYP was expressed more thanI 2T times higher in pyrethroid resistant populations of A. gambiae from Benin and NiSgeria. Fold changes of 2.48 and 2.58 were recorded for A. gambiae from Akron anRd A. gambiae from Ojoo respectively when competitively hybridized with A. gambEiae from Orogun (Fig. 4.22). NI V U AN BA D I 126 Y R TABLE 4.14 Candidate metabolic genes up-regulated in resistant pAopulations of Anopheles from Ojoo R Site Identified Expressions IB P-values Genes level (fold L (log) chanYges) CYP6P3 7.37 9.27 CYP6N1 IT4.84 12.94 Ojoo CYP6AG2 S 2.71 9.25 (Oil contamination CYP6M2 R 2.58 8.39 site) CYP6AKE1 2.19 9.58 GSTIDV1-6 4.07 8.49 NGSTD11 2.54 6.11 U CPLC8 2.62 8.46 TPX2 5.02 11.06 DA N I B A 127 RY BR A LI ITY RS IV E U N DA N Fig. 4.21 Candidate metabolic genes over transcribed on resistant Anopheles mosqAuitoes from the oil spillage locality of Ojoo when co-hybridized with the IsBusceptible strain A. gambiae from Orogun 128 RY RA LI B ITY ER S NI V CFomigp. a4r.a2t2iv eC goehnoer te xopf ree sxUspiorensss iend A ggerniceus ltiunr er easnidst oainl ts ppiollpaugela stiitoens : of A. gambiae from the Agricultural siteN of Akron in Benin and the site of spilled oil of Ojoo in Nigeria A BA D I Chapter 5 Discussion 129 5.1 The susceptibility pattern of Anopheles populations to pyrethroid in southwestern Nigeria and southern Benin Series of insecticide bio-assays conducted in this study revealed a wide spread of insecticide resistance in southwestern Nigeria. The presence of Anopheles populations capable of withstanding diagnostic doses of permethrin was initially reported in southwestern Nigeria by Awolola et al. (2003) and Mojca et al. (2003). Although these initial studies were confined to Lagos and Ogun states respectively, the spreaYd of Anopheles resistance seems to go beyond those 2 states. Anopheles populatAionsR collected from 13 localities out of the 19 surveyed in the 6 states covered during this study were resistant to permethrin. In West African countries such as Benin, BurkRina Faso and Cote d‟Ivoire, several studies have shown the presence of permethrinI rBesistant populations of Anopheles (Akogbeto et al., 1999; Chandre et al., 1999; Diab aLte et al., 2003; N‟guessan et al., 2003). The Republic of Benin shares a highly popYulated and very business active geographic border with the southwestern region oTf Nigeria therefore, the permethrin resistance observed in southwestern Nigeria couldI originate from migrations of resistant strains of Anopheles from Benin RepublicS where high levels of resistance were documented as early as 1999 (Akogbeto Ret al., 1999) or, could be locally selected by specific environmental factors and humEan activities. The use of agricultNural Ip Vesticides was less recorded in surveyed localities of southwestern Nigeria. Agricultural pesticides utilisation was recorded only in the locality of Ajibode where veg eUtable farming is practiced at low scale. A. gambiae populations from Ajibode weNre found resistant to permethrin (mortality rate of 90%). Many reviews have implicaAted synthetic pesticide utilisation in agriculture as a potential source of insecticidDe resistance selection in malaria vectors (Yadouleton et al., 2009). The limited utilisAation of agricultural pesticides in surveyed sites, suggests that other environmental IfBactors or human practices account for the selection of pyrethroid resistance in malaria vectors in southwestern Nigeria. However, the relatively low rates of permethrin resistance recorded in all surveyed sites probably explain the progressive flow of pyrethroid resistant genes in Anopheles populations of the southwestern Nigeria. In the southern Benin, pyrethroid susceptible populations of Anopheles were almost inexistent. Out of the 18 populations of mosquitoes analysed, only one population 130 exhibited considerable level of susceptibility (mosquito populations from the locality of Ifangni). Ifangni is a rural area located in the south eastern Benin. Here, communities have low incomes and agriculture is mainly for household consumption and not market oriented. The local agriculture practiced at Ifangni is less demanding and farmers use little or no pesticides; this probably explains the absence of pyrethroid resistance in this locality. High levels of resistance were recorded in the southern Benin, with morYtality rates to permethrin being as low as 23%. Very low mortalities were recorded in Anopheles populations collected in or around the vegetable farms of Akron (Mortality of R23%) and Pahou (31%) which are constantly under pesticide treatments. This resultA confirms the contribution of agricultural pesticides utilisation in the selection for resRistance in malaria vectors (Diabate et al., 2002; N‟guessan et al., 2003; ChouaibouI eBt al., 2009). Vegetable cropping is consistently becoming an important source for incoLmes to many households in urban and periurban settings (Yadouleton et al., 2009). CYons umers demand for vegetables is periodical and significantly increases during fesTtivity periods. Vegetable farming is highly pesticide dependent with peaks of pesticSide Itreatments corresponding to periods of high market demands or high pest attacks (Yadouleton et al., 2009). Agricultural pesticides used by farmRs have similar active ingredients as those used in public health for mosquito control tEherefore; these pesticides could easily contribute to the selection for resistance in mIalVN aria vectors. The intensity of pyrethroid resistance was relatively low in southwestern Nigeria compare to southern Benin. This difference probably results from tUhe nature of factors selecting for resistance in both countries. In Benin the selection is mainly from agricultural pesticides utilisation whereas in Nigeria, most breedinAg sNites producing resistant Anopheles populations contained traces of petroleumD products, suggesting a cross resistance phenomenon between spilled petroleum products and pyrethroids. I5B.2 G Aenotyping of permethrin resistant phenotypes The southwestern region of Nigeria shares a border with the southern Benin where resistance has been documented as being mostly related to kdr mutations (Akogbeto et al., 1999). The proximity of the two countries could lead to suspicion of the presence of kdr alleles in Anopheles mosquitoes from Nigeria. Contrary to this suspicion, results obtained from PCR-kdr analysis did not show an association between permethrin resistant 131 phenotypes and the presence of kdr alleles in analysed mosquito populations (one kdr mutant Anopheles out of 514 analysed). These results stand in favor of a low gene flow among Anopheles populations from Benin and Nigeria. This reproductive barrier could be justified by a remarkable presence of 2 different members of A. gambiae complex in both countries: A. arabiensis in the south western Nigeria and A. gambiae ss. in Benin. In a recent study conducted by Noutcha and Anumudu (2009), at Igbora a rural community in Ibadan, both members of A. gambiae complex (A. gambiae ss. and A. arabiensis) wYhere found to be the main malaria vectors in the community. The low frequency oRf the kdr mutation recorded in the 19 surveyed localities of the south western NigAeria confirms earlier findings of Awolola et al. (2003), on Anopheles populations fromR Lagos and Mojca et al. (2003) who reported low presence of kdr mutations in AInBopheles samples from Ogun state in southwestern Nigeria. L In the southern Benin, 2 members of A. gambYiae complex were identified: A. gambiae ss and A. melas. The distribution of IbToth species in Benin was initially documented by Akogbeto et al. (1990). This study confirms the main occupancy of the southern Benin by A. gambiae ss. whichR conSstitutes 93% of the total A. gambiae sl. populations analysed. When samples oEf A. gambiae ss were further genotyped, they were all characterized as “M” moleculIarV forms as earlier reported by Akogbeto et al. (2003). Classical molecular Nanalysis and micro-array assays conducted on samples from the southwestern Nigeria and the southern Benin revealed that several detoxifying genes are up-regulated in p eUrmethrin resistant individuals. These findings demonstrate that permethrin resistaNnce does not relate exclusively to changes occurring on the sodium channel (kdr Amutation) but also to several other genes such as Ace-1 (Weill et al. 2004) and serieDs of detoxifying genes (David et al., 2005) which could be externally expressed as peArmethrin resistant phenotypes. I5B.3 The hatching rate of Anopheles strains in breeding sites simulated with soil and water samples from vegetable farms The biological screening of pesticide residues from water and soil samples collected in agricultural sites in Benin and Nigeria suggest that samples from agricultural settings under pesticide pressure contain inhibitory factors responsible for the reduced growth rate of susceptible larvae of A. gambiae from Kisumu, with a lesser inhibitory 132 effect on the development of the resistant Ladji strain. Hatching of Anopheles eggs when introduced in simulated breeding sites made with soil and water from agricultural sites of Houeyiho and Ajibode is a rapid phenomenon and does not give room for expression of inhibitory factors on embryos. The recorded hatching rates should be considered as signal of toxicity for samples collected within from agricultural settings under synthetic pesticide treatments. 5.4 Water and soil samples from vegetable farms contain compounds that inYhibit Anopheles larval development. R A. gambiae from Ladji is a permethrin resistant strain selected froAm a relatively polluted locality, the locality of Ladji in peripheral region of CotonoRu. This strain had probably developed over time capacities to withstand low lIevBels of toxicity. This assumption could explain the low inhibitory impact of breedi nLg sites on the hatching of A. gambiae from Ladji eggs as recorded in this study. YSimilar to hatching rates, larval development also varied with respect to strains (KisIuTmu or Ladji) of Anopheles inoculated and the types of artificial breeding sites simulated. In both vegetable farms (Houeyiho Rand SAjibode), breeding sites simulated with top soil collected around vegetables seemed to inhibit larval growth more than simulations with watering water and soil frIomV w Eatering pools. In these two simulations, inhibitory effects were less spectacularN. In breeding sites generated with water mixed with soil from watering pools, 85% of Anopheles Kisumu eggs were able to reach the pupal stage, whereas, in simulation s Uwith water mixed with soil collected around vegetables, only 42% of larvae were abNle to reach the pupal stage. A similar trend was recorded with samples from AjibodAe in Nigeria. This consistent difference in results (85% and 42% developmDental rate) suggests an unequal distribution of pesticide residues after treatment in thAe vegetable farm of Houeyiho. Soil directly under vegetable plants is more IcBontaminated with pesticide residues; it is subjected to several treatments and therefore receives a good amount of pesticide particles during treatments. During rain falls, these pesticide residues are washed and sediments in mosquito breeding sites were they exercise a selection pressure on mosquito larvae. 5.5 The implication of pesticide residues in the emergence of pyrethroid resistance in malaria vectors 133 Several chemicals (agricultural inputs) are used in vegetable farming at Houeyiho and Ajibode for pests control. These chemicals are mainly pyrethroids for Ajibode and, a combination of pyrethroids, organophosphates and carbamates for Houeyiho. These compounds are used as single formulations or as combinations of two to three insecticides of different families for generating a synergistic effect of insecticides and a better pest management. After pesticide treatments in agricultural settings, residues of insecticides get into mosquito breeding sites. These residues have lethal effects on larvae of Ysome populations of mosquito whereas they exert a selective pressure on other poRpulations, leading to a gradual tolerance of insecticide concentrations and the Aemergence of resistance. Insecticides used in public health against disease vectoBrs aRre similar to those used for years in agriculture. In Benin Republic, pyrethroiIds were introduced in agriculture in the 1970s and, after 30 years of continuous uLse, cases of resistance are certain to be found in some populations of insects. MoYst a uthors incriminate pesticides used in agricultural farms as the main source of seleIcTtion of resistance in mosquito species (Georghiou et al., 1991; Chandre et al., 1999, NS'guessan et al., 2003; Diabate et al., 2002). The intensification of exodus of young people from villages to towns, as a result of unemployment, has led to the deveElopmRent of agricultural spaces within urban and periurban areas for vegetable farImVing. These farms found in many West African countries are active throughout the yeaNr because of constant and high demands of urban populations. To keep the productivity high and avoid shortages of vegetables for urban consumers, farms are treated at relaUtively high frequencies. At Houeyiho an average of 72 pesticide treatments is condNucte d annually by farmers. This constant treatment of farms keeps high the level of inAsecticide residues in the soil and may explain the elevated toxicity observed with top soil. Data from this study indicate that factors inhibiting the hatching of A. gambAiae Deggs and the development of their larvae are insecticide residues resulting from IaBgricultural treatments with pesticides. The indirect biological assay developed by WHO (MIM-WHO, 2003) and used in this study revealed the presence of pesticides residues in mosquito breeding sites found in and around vegetable farms. Results obtained established the implication of synthetic agricultural pesticides in the selection for pyrethroid resistance in mosquito populations breeding in and around vegetable farms. 134 5.6 The treatment of mosquito breeding sites with petroleum products and the selection of pyrethroid resistance in malaria vectors Although abandoned long ago by National programs of malaria control in most African countries, petroleum products are still used in several rural communities considered in this study. This is probably due to the availability of these products, which are openly sold in many streets of West African countries such as Nigeria, Benin, Togo, and Niger. In Nigeria, several selling spots of engine oil were identified along the IbYadan Lagos express way. In Benin in addition to engine oil, petrol and gasoil are sold Rbeside the roads by retailers mainly women and their children. The low income of coAmmunities in rural settings also accounts immensely in the slow adoption of synBthetiRc insecticides and, therefore, solidifies their attachment to petroleum products as tIhe main malaria vector control tool. The use of petroleum products for vector control iLn studied rural communities is well known to the communities, is transferred from geYneration to generation and seems to have become a cultural practice. The availabiliItyT of these products and their relative cost-effectiveness suggests this method of mosquito control may have some benefit to the communities in rural areas. S A relatively low efficacy of petErol oRn Anopheles larvae was recorded in this study; this could be explained by its hIigVh volatility compared to kerosene, engine oil and used engine oil. The high volatility of petrol does not allow its persistence in the breeding sites. This low persistency resultsN in a low residual effect of this product in treated breeding sites. The relatively hi gUh efficacy of kerosene, engine oil and waste oil is likely to be due to their elevated Npersistency in breeding sites post treatments. The HiC and the LoC100 values determined for each petroleum product are key operational values: the LoC100 indicates Dthe Alowest effective quantity of petroleum products that kills 100% of A. gambiae larvae during breeding site treatments. The LoC100 is a cost effective IcBonce Antration for larviciding. On the other hand, the HiC also known as the NOEL corresponds to the quantity of petroleum products "wasted" in the environment during treatments of breeding sites. This value defines the threshold at which resistant populations of Anopheles could be gradually selected. The HiC values recorded in this study reflects the high probability of petroleum residues found in mosquito breeding sites to contribute to the emergence of pyrethroid resistance in analysed Anopheles populations. 135 Poupardin et al., (2008) demonstrated that several xenobiotics found in mosquito habitats contribute in the selection of insecticide resistance. It is possible that these products might have contributed through cross-resistance to the numerous cases of pyrethroid resistance recorded in Nigeria and Republic of Benin. The empirical use of PP by communities and the absence of knowledge on ideal concentrations to use during the treatment of breeding sites have over the years contributed to the spread in the environment of none active quantities of PP whiYch in return would have contributed to the selection of resistant populations of mosquRitoes. It is possible that the emergence of pyrethroid resistance in studied localitiesA has partially resulted from the presence in Anopheles breeding sites of petroRleum residues at concentrations below the LoC100 recorded in this study. TheI Bconstant exposure of Anopheles larvae to “friendly” concentrations of petroleum (LHiC) has probably induced over the years a cross resistance to pyrethroids. Y 5.7 Identification of the mode of action of PP on AT. gambiae larvae When A. gambiae larvae were reared iIn laboratory bowls containing water samples mixed with petroleum and oil peRllicleSs at the surface, a percentage mortality of 100 was recorded within 24 hrs. HoEwever, when the surface pellicle (oil film) was removed through series of sievinIg Vthe mortality rate of larvae remained as high as 96%. It is possible that some solublNe active compounds of petroleum products such as benzene, toluene, and xylene (McAuliffe, 1987) dissolve, diffuse into the water and are ingested by Anopheles larvae afte r Utreatment of the breeding sites. These compounds keep elevated larval mortalities despite the absence of oil film. It is likely that the mode of action of petroleum proAducNts in mosquito larvae is mostly through "contact toxicity" followed by the ingesDtion of dissolved petroleum compounds rather than the “suffocation” from the oil film A(Appendix.5). I5B.8 The inhibitory effects of petroleum on Anopheles oviposition and larvae development A low tolerance to oily breeding sites was recorded with pyrethroid susceptible Anopheles compare to resistant Anopheles during oviposition. This low tolerance translates the high selection pressure exercised on Anopheles species in localities where most breeding sites are covered with petroleum products. Similar to inhibiting oviposition, 136 petroleum products had a negative impact on the development of larvae of susceptible and resistant populations of A. gambiae. When a differential analysis of mortalities was conducted on the susceptible and the resistant populations, the destructive impact of petroleum appeared more prominent on the susceptible A. gambiae from UI compare to the resistant A. gambiae from Ojoo. This result suggests a high selection pressure of spilled petroleum products on Anopheles at larval stage. The inhibition of oviposition and larvae development by petroleum products in the laboratory justifies low presenYce of susceptible populations of Anopheles in oily breeding sites screened in theR surveyed localities. In locality of spilled petroleum products, clean breeding sites wAhich are free from petroleum particles may contain both resistant and susceptible sRtrain of Anopheles whereas, it was observed that petroleum contaminated breedinIgB sites mainly produce permethrin resistant strains of Anopheles. This segregational oLccupation of oily and non- oily breeding sites by resistant and susceptible popYulati ons of Anopheles probably explains the active selection played by spilled petrIolTeum products on mosquito species in some localities of southwestern Nigeria and southern Benin. 5.9 Existence of a cross resistance betwReenS petroleum products and permethrin in sampled Anopheles To establish the links bVetweEI en oil spillage and the selection of pyrethroid resistance several research sNteps were followed: At the initial step, the lethal activity of petroleum products on Anopheles larvae was established and the dose response curves of lethal activities of pe trUoleum products on Anopheles larvae revealed the existence of “Larvae-friendly”N concentrations of petroleum on mosquitoes. At the second setp of this research, the mAode of action of petroleum residues on mosquito larvae was established and this reveaDled that, when petroleum products are spilled in Anopheles breeding sites, the majoArity of larvae are killed by contact toxicity rather than suffocation.The thirth step of ItBhis analysis was mainly based on laboratory simulations and results obtained showed that petroleum products in Anopheles breeding sites exercise a severe selection pressure by killing most susceptible strains of A. gambiae and allowing the emergence of individuals found to be resistant to permethrin. Ojoo and Orogun are two localities of the southwestern Nigeria at less than 2 km. At Ojoo, A. gambiae populations are pyrethroid resistant whereas at Orogun, populations of A. gambiae are susceptible to permethrin. 137 These differences of susceptibility in both localities are probably justified by the nature of breeding sites which are mostly oily at Ojoo and relatively clean at Orogun. The reduced oxygen concentration identified in most breeding sites containing petroleum particles suggests that mosquitoes living in such breeding sites have developed elevated oxidative activities as survival strategies. This was confirmed through microarray analysis. The absence of the kdr mutations on samples emerging from oily breeding sites further confirms that petroleum products select for metabolic mechanisms of resisYtance and not target site mutations (kdr mutation). A. gambiae populations breedingR in water contaminated with petroleum products develop elevated detoxifyRing Aactivities for surviving petroleum residues found in their habitats. This study has established the existence of a corss resistance between petroleum products and pyIreBthroids. Nigeria is the first oil producer in Africa with over 2,45L5.260 barrels per day (EIA- US, 2010). Oil dumping is an old and generalizedY pr actice in this country. The management of used petroleum products is a critical Tand very sensitive issue in this part of the world and leads to wide-spread environmSenItal pollution and the development of various health hazards. Petroleum productsR are spilled on a wide-scale in the environment by mechanics, retailers, trailers and oEld cars. These products are washed by rains and accumulate in low-lying areas wIheVre, mixed with water, they constitute breeding spots for mosquitoes. Anopheles mayN lay their eggs in these oily breeding sites and selection pressures may be exercised during series of larval development cycles. 5.10 Identification oUf detoxifying genes up-regulated in pyrethroids resistant Anopheles from Nsites of spilled petroleum products and from agricultural areas under pesticiAdes utilisation TDhe impact of pyrethroid resistance in A. gambiae from the Southern Benin, belieAved to be mainly driven by kdr, has been implicated in the failure of LLIN's in the IrBegion (Nguessan et al., 2007). N'Guessan et al. (2007) used biochemical analysis of the mosquitoes to identify metabolic resistance and ruled out the involvement of P450s, GSTs and COEs. Analysing resistant populations of A. gambiae and Culex quinquefasciatus from the same locality, Corbel et al. (2007) demonstrated the presence of P450s, GSTs and COEs. This study confirmed the involvement of metabolic genes and moved a stage 138 further from biochemical analysis, by identifying series of metabolic genes up regulated in pyrethroid resistant strains. The gene showing greatest levels of over-expression in resistant populations of Anopheles analysed is CYP6P3. CYP6P3 is the ortholog of CYP6P9 from A. funestus, a mosquito in which target site resistance has not been reported. This gene has been genetically linked to pyrethroid resistance in A. funestus (Wondji et al., 2007) and is highly over expressed in a pyrethroid resistant colony (FUMOZ-R) from MozamYbique compared with the susceptible line FANG from Angola (>38 fold) (Amenya et aRl., 2008). Similarly in this study the CYP6P3 fold change between resistant anAd susceptible populations were the highest recorded by the detox chip for any gBeneR to date (7.4-fold, Ojoo and 12.4-fold, Akron). Interestingly, Muller (2008) emplIoyed the detox chip to investigate the effects on gene expression in A. arabiensis be foLre and after cotton spraying campaign and reported CYP6P3 to be down regulatedY after pesticides treatments. It is possible that CYP6P3 is switched-on in Anopheles IpoTpulations collected around vegetable farms during pesticides treatments and, after treatments this gene is switched off. The second metabolic gene identifRied Sin this study is CYP6M2 which was over-expressed in the two pyrethroid resEistant populations of Anopheles collected around vegetable farms and from areaIs Vof spilled petroleum products. Studies conducted by Muller (2008) on A. gambiaNe from Ghana also showed that this gene is up-regulated in pyrethroid resistant strains of Anopheles. These combined results from different authors working in different siUtes strongly support a role for both CYP6M2 and CYP6P3 in conferring pyrethrNoid resistance in A. gambiae populations analysed during this research. GSTDA1-6 and GSTD11 belong to the delta class of GSTs with the former being one of foDur alternatively spliced variants of GSTD1 (Ranson et al., 1998). Whilst the delta classA GSTs has been associated with resistance in other insects (Wang et al., 1991; Tang et IaBl., 1994; Vontas et al., 2001), this is the first study to implicate delta GSTs as conferring resistance in mosquitoes. GSTs and the peroxiredoxins are not thought to be able to metabolise pyrethroids directly. However, both classes of enzymes can protect against oxidative stress and may counteract the pyrethroid induced oxidative stress encountered by the mosquitoes (Rhee et al., 2005; Tang et al., 1994). GSTs may also play a passive 139 role in sequestering pyrethroids, thereby reducing the circulating levels of active insecticide (Kostaropoulos et al., 2005). The over expression of two cuticular precursor genes in both Akron and Ojoo resistant populations lends support to the hypothesis that mosquitoes may also protect themselves from insecticides by cuticular thickening, which leads to reduced penetration of insecticides. Compared with target-site and metabolic resistance, cuticular resistance is a less well understood mechanism and few studies have investigated the link betweeYn the insect cuticle and resistance (Stone et al., 1969; Apperson et al., 1975; NoppRun et al., 1989; Lin et al., 1993). However, over expression of CPLC8 has very Arecently been demonstrated in pyrethroid resistant A. gambiae from Nigeria (AwololRa et al., 2008) and A. stephensi (Vontas et al., 2007). The A. gambiae detox chip onIlBy contains three of the estimated 295 putative cuticular proteins in this species (HeNL et al., 2007), and all the three belong to the CPLC family about which very littlYe is known. The role of cuticular changes in resistance clearly warrants further investigTation, as it may prove to be just as an important defence mechanism as metabolic andS targIet-site resistance. 5.11 Differential expression of metaboliRc genes by A. gambiae populations collected around vegetable farms and those frEom localities of spilled petroleum products Microarray analysis shIowVed that the gene expression profiles of studied populations of mosquitoes cNorrelate with the nature of breeding sites hosting Anopheles larvae. A relatively high number of metabolic genes were over-expressed on samples from oil spillage area (Ojo oU) than those from Agricultural settings (Akron). 9 genes were overexpressed in Nthe oily site compare to 5 genes in the agricultural site. The kdr target site mutation Awas identified exclusively in the agricultural site of Akron. Although this mutation Dseems not to interfere with the expression of metabolic resistance, it remains possiAble that, in the absence of the kdr mutation as a protective mechanism, mosquitoes ItBend to compensate for this absence by developing many other defensive mechanisms such as the up regulation of several metabolic genes from different subclasses. This probably explains the relatively high number of metabolic genes recorded on samples from Ojoo compare to Akron. However, it is difficult at this stage to determine the relative contributions of kdr and metabolic resistance in mosquitoes with both mechanisms. It is nevertheless important to note that the highest level of permethrin resistance was observed 140 in the Akron population, which has both kdr and metabolic resistance and the lowest resistance observed in the Ojoo population which displays only metabolic resistance. AR Y R B L I SI TY R E NI V U AN D I B A 141 CHAPTER 6 CONCLUSION AND RECOMMENDATIONS This study investigated the possible mechanisms of resistance and factors contributing to the emergence of vector resistance in southwestern Nigeria and southern Benin. It was observed that massive utilization of agricultural pesticides and spillage of petroleum products are the two main factors which account for the emergence of pyrethroid resistance in A. gambiae populations collected in the Southern BeniRn anYd the South western Nigeria. The empirical use of petroleum products for treatment of mosquito Abreeding sites was confirmed by rural communities in Benin. Corroborative results Rfrom focus group discussions organized within communities and the laboratory analIysBis of samples revealed the lethal activity of petroleum products on Anopheles larvLae. While highlighting the mode of action of petroleum on Anopheles larvae whiYch is contact toxicity rather than suffocation, data from this study have also identifiedT the existence of some non-lethal or "larval-friendly" concentrations to which AnophSelesI larvae adapt to and gradually develop a cross resistance mechanism to pyrethroids. This study highlighted the EpoteRntial impact of oily breeding sites in the development of both susceptible and resistant strains of A. gambiae and, the contribution of spilled petroleum producNts inI t Vhe development and spread of pyrethroid resistance in malaria vectors in southwestern Nigeria. Similar to the implication of agricultural pesticides in the eme rgUence of pyrethroid resistance in mosquito populations, spilled petroleum producNts are washed by rains and accumulate in low-lying areas where, mixed with water, thAey constitute breeding spots for mosquitoes. Anopheles will lay their eggs in these oilyD breeding sites and larvae emerging from hatched eggs will undergo series of selecAtion pressure which ends with the emergence from these oily breeding spots of adult IpBopulations of A. gambiae which are capable to withstand lethal doses of pyrethroids. The molecular analysis of A. gambiae samples from both Nigeria and Benin revealed that permethrin resistance does not relate exclusively to changes occurring on the sodium channel; other mutations such as Ace-1 and series of detox genes are also externally expressed as permethrin resistance. The gene expression profiles of resistant and susceptible populations of A. gambiae directly from the field revealed the evidence of 142 metabolic resistance in mosquito samples from the Republic of Benin and Nigeria irrespective of the presence or absence of kdr. Furthermore two P450 genes, CYP6P3 and CYP6M2 which are strongly associated with permethrin resistance were identified in the resistant Anopheles following micro-array analysis. In addition, preliminary evidence for a role for cuticular resistance was provided from this study but this is an area that needs further investigation. Evidences that insecticides used outside malaria control activities (agricuYltural pesticides) and the contamination of breeding pools by spilled petroleum might bRe causing resistance to permethrin in A. gambiae has important implications Afor resistance management and the control of malaria vectors in Benin and Nigeria. R This research is multi-sectorial and encompasses agricuIltBure, environment and health sectors. Specific recommendations arising from this studLy are: (i) There is need to establish an information exchange Yplat form between agriculture and health policy makers on the negative impact of syInTthetic pesticide utilization on human health. (ii) There is need to develop cost effectiveR alteSrnatives (botanicals, biological control) for agriculture pests control and disseminaEte the developed alternatives to farmers through the proposed platform. (iii) Farmers should be sensiNtizedI Von the links between synthetic pesticides utilization and the emergence of populations of malaria vectors resistant to public health insecticides. (iv) Policies on environUmental protection should be reinforced and more emphasis should be placed on the mNana gement of waste petroleum products at community level. v) In rural communities where petroleum products are used for mosquito control, effective lethal dosDes shAould be made known to individuals through community sensitizations. (vi) RAesearchers should work towards the development of new families of insecticides IaBnd new vector control strategies (biological control strategies) which are cost effective, less toxic to humans and the environment. (vii) Research should be conducted on agro-ecosystems restoration as this could lead to the return of mosquito natural enemies/predators and the natural destruction of resistant malaria vectors. 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Indepth interview guide on knowledge, attitudes and practices (KAP) of communities on synthetic pesticides use in Agriculture 1- Quarter/Locality 2- Status of the resource person interviewed 3- Sex and age 4- The use of pesticides in the household Y 5- The types of pesticides used R 6- The purpose for pesticides use A 7- If Agricultural use of pesticides in mentioned, discuss the typRes of insecticides used in accordance with the cropping system IB 8- Frequency of pesticide treatments in vegetable farms L9- Doses of pesticides applied for pests control in veYgetable farming, 10- Origin of insecticides and the place of purchasTe 11- Safety precautions observed for reducing heIalth hazard when using pesticide 12- Trainings received on the use of pesRticidSes in agriculture. IV E UN AN D I B A 165 Appendix 2. Indepth interview guide on knowledge, attitudes and practices (KAP) of communities on the use of petroleum products (PP) for mosquito control 1- Quarter/Locality 2- Status of the resource person interviewed 3- Sex and age 4- The use of PP in the household 5- The types of PP used Y 6- The purpose for PP use R 7- If the use of PP for mosquito control in mentioned, discuss the tRypesA of PP used in accordance with the types of mosquito 8- Frequency of PP treatments in mosquito control IB 9- Doses of PP applied for mosquito control L 10- Origin of PP and the place of purchase Y 11- Safety precautions observed for reducing heIalTth hazard when using PP. S R IV E UN N DA I B A 166 Appendix3. WHO bio-assay for insecticide susceptibility using adult mosquitoes RYA BR Y LI ITS VE R WHO bioassay stepNs fIor analyzing the susceptibility status of Anopheles to insecticides.: (A-B-C- DU) is the transfer of female Anopheles into test tubes coated with impregnated papeNrs, (E) mosquito are kept for 1h in the exposure tube, ( F) mosquitoes are taken back to Aholding tube for 24h observation of mortality rates. TDhe WHO tube test kit consists of two plastic tubes (125 mm in length, 44 mm in diamAeter), with each tube fitted at one end with a 16-mesh screen. One tube (exposure tBube) is marked with a red dot, the other (holding tube) with a green dot. The holding tube Iis screwed to a slide unit with a 20 mm hole into which an aspirator will fit for introducing mosquitoes into the holding tube. The exposure tube is then screwed to the other side of the slide unit. Sliding the partition in this unit opens an aperture between the tubes so that the mosquitoes can be gently blown into the exposure tube to start the treatment and then blown back to the holding tube after the timed exposure (generally one hour). The filter- papers are held in position against the walls of the tubes by four spring wire clips: two 167 steel clips for attaching the plain paper to the walls of the holding tube and two copper clips for attaching the insecticidal paper inside the exposure tube. AR Y LIB R ITY S VE R I U N N A AD I B 168 Appendix4. Laboratory protocols and preparation of solutions DNA extraction (Livak, 1984). Livak grind buffer 1.6 ml 5M Nacl 5.48 g glucose 1.57 g Tris 10.16 ml 0.5 M EDTA Y 25 ml 20%SDS R Bring volume to 100 ml (with dH2O), filter sterilize, store 5ml aliquots aRt -20AoC. After thawing aliquot keep at 4oC for no more than 2 weeks. DNA extraction IB 1- L Heat LIVAK buffer to 65oC in heat block for 15 minutes and mix before useY to r e-dissolve precipitate 2- IT in a 1.5 ml eppendoorf grind 1 mosquito in 100 microL preheated LIVAK grind buffer. To optimize yield grind in 50microL first then rinse pestleR witSh further 50microL and transfer immediately to 65oC 3- IV E Incubate for 30 minutes at 65oC spin genNtly to collect condensations 4- add 14 microL of 8M K-acetate and mix 5- N . U incubate on ice for 30 minutes. 6- DA Centrifuge at 13000 rpAm for 20 minutes at 4oC. Transfer supernatant to a new 1.5 ml eppendoorf , be IBcareful not to transfer any debris . If desired, re-spin 20 minutes and transfer supernatant to new tube. 7- Add 200 microL EtOH, mix gently, and spin at 13,000 rpm for 15 minutes at 4oC. 8- Remove and discard supernatant, carefully rinse pellet in 100 microL ice cold 70% EtOH. 169 9- Dry pellet by leaving tubes on the bench for approximately 1 hour. 10- Re-suspend pellet in 10 microL TAE buffer. Other DNA extraction solution 8M KAc Grinding buffer Y 0.08M NaCl R 0.16M Sucrose A 0.06M EDTA R 0.5% SDS IB 0.1M Tris-HCl L• TA (Tris EDTA) Buffer Y 100ml 1M Tris (pH) T 20ml 0.5M EDTA I Make up volume to 1L. S • TAE (Tris Acetic EDTA) Buffer 50X (pH R8) 242g Tris E 37.2 g Na2 EDTA.2H2O IV 57.1M glycial acetic acid N Make up to 1 L U • Agarose gel For a 2.5% agAarosNe gel, dissolve 10g agarose in 400ml 1X TAE • Ethidium bromide DissoAlve D10mg EtBr crystals in 1ml distilled H2O I B 170 Y R Appendix5. Characteristics and toxicity of petroleum products on insecAts Chemical control of mosquito larvae with petroleum products (WHO, 19R70) Thickness of the product on water surface (Cm): IB Diesel: 0.04 L Gasoline: 0.30 TY Kerosene: 0.30 SI Boiling point: R o o Gasoline: 70 to 150 C E o o Kerosene: 150 -300 C IV o o Gas oil, fuel oils and diesel oNil: 250 -350 C Effect of petroleum on inUsects larvae 1- The activitAy of Npetroleum oil as insect larvicide or pupicides varies with their aromatic and parafDfin content and with their boiling range. Viscosity appears to be a function of boilinAg point. I2B- The covering capacity relates to the viscosity of the oil and has a suffocating activity on insects larvae 3- Aromatic vapors are toxic and paraffin vapors are generally inert o o 4- The more effective larvicides are found in the fraction produced in the 232 -316 C boiling range with a 1:1 mixture of aromatic and paraffin oils. 171 5- The more volatile fractions have greater toxicity than the less volatile 6- Film production at water surface is long with paraffin oils (saturated fractions), intermediate with intermediate fractions and short with aromatics 7- Paraffins spread well and films persist until destroyed by evaporation; aromatics form less lenses after application Y 8- Loss of activity seems to be more closely related to the reduction of the area covered by the oil film than with the evaporation of the more volatile fractions. R RA LIB TY RS I VE UN I DA N IB A 172