i SCHOOL OF PUBLIC HEALTH COLLEGE OF HEALTH SCIENCES UNIVERSITY OF GHANA, LEGON INFLUENCE OF TEMPERATURE ON THE GROWTH, DEVELOPMENT AND SUSCEPTIBILITY OF ANOPHELES GAMBIAE (S.L.) MOSQUITOES TO PYRETHROIDS BY THOMAS PEPRAH AGYEKUM (10637332) THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON, IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF DOCTOR OF PHILOSOPHY DEGREE IN PUBLIC HEALTH FEBRUARY, 2023 University of Ghana http://ugspace.ug.edu.gh i DECLARATION I, Thomas Peprah Agyekum, declare that, except for references to other people's work, which have been duly acknowledged, this thesis, submitted to the School of Public Health, University of Ghana, is the result of my original research, and that the thesis has not been presented for any degree elsewhere. This thesis write-up was done under the joint supervision of Professor Julius Fobil, Dr. John Arko-Mensah, Professor Jonathan Hogarh and Dr. Paul Kingsley Botwe. THOMAS PEPRAH AGYEKUM …………………. ………………… STUDENT (10637332) SIGNATURE DATE PROF. JULIUS FOBIL …………….…… ………………… (PRINCIPAL SUPERVISOR) SIGNATURE DATE DR. JOHN ARKO-MENSAH …………………. .……………….. (CO-SUPERVISOR) SIGNATURE DATE PROF. JONATHAN HOGARH …………………… ………………… (CO-SUPERVISOR) SIGNATURE DATE DR. PAUL KINGSLEY BOTWE …………………. ………………… (CO-SUPERVISOR) SIGNATURE DATE 24/02/2023 24/02/2023 24/02/2023 24/02/2023 24/02/2023 University of Ghana http://ugspace.ug.edu.gh ii DEDICATION This work is dedicated to God Almighty for His guidance and protection through this academic journey. I also dedicate this work to my parents, Mr. Samuel Agyei Agyekum and Madam Comfort Boakyewaa, and my siblings for all the support. To my lovely wife, Abigail and our son, Nana Yaw, I appreciate all the sacrifices and understanding. University of Ghana http://ugspace.ug.edu.gh iii ACKNOWLEDGEMENT I am most grateful to the Almighty God for giving me life, strength and enablement to complete this research. I would like to express my sincere thanks to my supervisors, Prof. Julius Fobil, Dr. John Arko-Mensah, Prof. Jonathan Hogarh and Dr. Paul K. Botwe, for guiding and directing me throughout my entire program. I am grateful to Prof. Thomas Robins for his support while I was at the University of Michigan during my experiential learning. I also acknowledge the support and help I received from all the GEOHealth Project mentors and team members. I am deeply thankful for your support. I appreciate Professors Kwasi Obiri-Danso (Former Vice Chancellor, KNUST), Philip Antwi- Agyei and Bernard Fei-Baffoe (Department of Environmental Science, KNUST), Dr. Duah Dwomoh (University of Ghana), and Prof. Sam Newton (School of Public Health, KNUST) for their encouragement and support. I am most grateful to my wife and son for their love, support and understanding. My profound gratitude goes to my parents (Mr. Samuel Agyei Agyekum and Madam Comfort Akua Boakyewaah), siblings (Georgina, Beatrice, Peter and Lydia) and in-laws for their prayers and support. A special mention to the late Prof Evans Afriyie-Gyawu for all the support and advice. I am grateful to Dr. Maxwell K. Billah and Mrs. Racheal Nkrumah (both at Department of Animal Biology and Conservation Science, University of Ghana), Dr. Samuel K. Dadzie and Dr. Kwadwo Frimpong (both at Department of Parasitology, Noguchi Memorial Institute for Medical Research (NMIMR), University of Ghana), Mr. Issah Ibrahim, Mr. Julius Adde, Staff of Vestergaard Vector Lab, NMIMR, Mr. Ahokpossi Eudon-Marcus, and Mr. Sylvester Coleman for their great help in carrying out this research work. Finally, my enormous thanks go to Mr. Kwadwo Owusu Akomea, Mrs. Comfort Awuah, Rev. and Mrs. Arkhurst (Central Presbytery Chairperson, PCG), Jessica Afi Anyonam Ahiakpah, University of Ghana http://ugspace.ug.edu.gh iv Jane Oppong, Emmanuel Agyare Sackey, Derrick Owusu-Ansah, Raymond Opatah and all those who in one way or the other contributed to the successful completion of this thesis. I appreciate your efforts, support, and motivation. God richly bless you all. ACKNOWLEDGEMENT OF FUNDING This study was financed by the ½ West Africa-Michigan CHARTER in GEOHealth with funding from the United States National Institutes of Health/Fogarty International Center (US NIH/FIC) (paired grant No 1U2RTW010110-01/5U01TW010101) and Canada's International Development Research Center (IDRC) (grant No. 108121-001). University of Ghana http://ugspace.ug.edu.gh v TABLE OF CONTENT CONTENTS PAGE DECLARATION......................................................................................................................i DEDICATION..........................................................................................................................ii ACKNOWLEDGEMENT..................................................................................................... iii TABLE OF CONTENT...........................................................................................................v LIST OF TABLES ................................................................................................................xiv LIST OF FIGURES ..............................................................................................................xvi LIST OF ABBREVIATIONS ........................................................................................... xviii OPERATIONAL DEFINITION OF TERMS...................................................................xxii ABSTRACT.........................................................................................................................xxiv PREFACE......................................................................................................................... xxviii CHAPTER ONE ...................................................................................................................... 1 INTRODUCTION.................................................................................................................... 1 1.1 Background ...................................................................................................................... 1 1.2 Problem Statement ........................................................................................................... 3 1.3 Conceptual Framework .................................................................................................... 5 1.4 Justification ...................................................................................................................... 7 1.5 General Objective ............................................................................................................. 7 1.5.1 Specific Objectives .................................................................................................... 7 1.6 Research Questions .......................................................................................................... 8 1.7 Research Hypotheses........................................................................................................ 8 University of Ghana http://ugspace.ug.edu.gh vi CHAPTER TWO ..................................................................................................................... 9 LITERATURE REVIEW ....................................................................................................... 9 2.1 Introduction ...................................................................................................................... 9 2.2 Biology of mosquitoes ..................................................................................................... 9 2.3 Life cycle of mosquitoes ................................................................................................ 11 2.3.1 Egg stage.................................................................................................................. 12 2.3.2 Larval stage .............................................................................................................. 13 2.3.3 Pupal stage ............................................................................................................... 14 2.3.4 Adult stage ............................................................................................................... 14 2.4 Common genera of mosquitoes ...................................................................................... 16 2.4.1 Anopheles mosquitoes ............................................................................................. 16 2.4.2 Aedes mosquitoes .................................................................................................... 19 2.4.3 Culex mosquitoes ..................................................................................................... 20 2.5 Anopheles and malaria transmission .............................................................................. 22 2.6 Control of mosquitoes .................................................................................................... 24 2.6.1 Use of mosquito bed nets......................................................................................... 24 2.6.2 Indoor Residual Spraying (IRS) .............................................................................. 25 2.6.3 Larval source management (LSM) .......................................................................... 26 2.7 Targets of stages of mosquito with insecticides ............................................................. 27 2.7.1 Larviciding............................................................................................................... 27 2.7.2 Adulticiding ............................................................................................................. 27 University of Ghana http://ugspace.ug.edu.gh vii 2.8 Class of insecticides and their targets ............................................................................ 28 2.8.1 Carbamate insecticides ............................................................................................ 29 2.8.2 Organochlorine (OC) insecticides ........................................................................... 29 2.8.3 Organophosphorus (OP) insecticides ...................................................................... 30 2.8.4 Pyrethroid insecticides ............................................................................................. 30 2.9 Mechanisms used by mosquitoes to develop an insecticide resistance phenotype ........ 31 2.9.1 Target site resistance ............................................................................................... 33 2.9.2 Cuticular resistance .................................................................................................. 34 2.9.3 Behavioral resistance ............................................................................................... 34 2.9.4 Metabolic resistance ................................................................................................ 35 2.10 Predominant insecticides in Ghana .............................................................................. 39 2.11 Effect of climate change and climate variability on insect vectors .............................. 40 2.12 Summary of reviewed literature on temperature, Anopheles mosquito growth, development and survival..................................................................................................... 42 2.12.1 Introduction ........................................................................................................... 42 2.12.2 Methods ................................................................................................................. 43 2.12.3 Synthesis of evidence and gaps in current literature on temperature, growth, development, and survival of Anopheles mosquito .......................................................... 45 2.12.4 Discussion .............................................................................................................. 61 2.12.5 Conclusion ............................................................................................................. 70 CHAPTER THREE ............................................................................................................... 73 METHODOLOGY ................................................................................................................ 73 University of Ghana http://ugspace.ug.edu.gh viii 3.1 Introduction .................................................................................................................... 73 3.2 Study design, temperature selection and maintenance ................................................... 73 3.3 Maintenance of Anopheles gambiae (s.l.) mosquito colony .......................................... 74 3.4 Molecular identification of An. gambiae (s.l.) mosquitoes (Tiassalé strain) ................. 76 3.4.1 DNA extraction from adult mosquitoes ................................................................... 76 3.4.2 Identification of Anopheles gambiae (s.l.) members by polymerase chain reaction (PCR) ................................................................................................................................ 76 3.5 Temperature, growth and development of immature An. gambiae (s.l.) mosquitoes .... 78 3.5.1 Developmental time of immature mosquitoes, larval survival, and mortality ........ 78 3.5.2 Time to pupation, pupation success, and mortality ................................................. 79 3.5.3 Larval and pupal weight and size ............................................................................ 80 3.5.4 The number of adults produced (production capacity) and sex ratio ...................... 81 3.6 Temperature and the growth and development of adult An. gambiae (s.l.) mosquitoes 81 3.6.1 Adult longevity ........................................................................................................ 81 3.6.2 Estimation of length of gonotrophic cycle, biting rate and fecundity ..................... 82 3.6.3 Body weight, size, and proboscis length ................................................................. 83 3.7 Insecticide susceptibility and metabolic enzyme level .................................................. 84 3.7.1 Insecticide susceptibility .......................................................................................... 84 3.7.2 Methods for measuring mosquito metabolic enzyme level ..................................... 85 3.8 Quality Control ............................................................................................................... 87 3.9 Statistical Analysis ......................................................................................................... 89 University of Ghana http://ugspace.ug.edu.gh ix 3.9.1 Temperature and the growth and development of immature An. gambiae (s.l.) mosquitoes ........................................................................................................................ 89 3.9.2 Temperature and the growth and development of adult An. gambiae (s.l.) mosquitoes .......................................................................................................................................... 90 3.9.3 Insecticide susceptibility and expression of metabolic enzyme levels .................... 91 3.10 Ethical Approval .......................................................................................................... 92 CHAPTER FOUR .................................................................................................................. 93 RESULTS ............................................................................................................................... 93 4.1 Introduction .................................................................................................................... 93 4.2 Composition of An. gambiae (s.l.) (Tiassalé strain) mosquitoes ................................... 93 4.3 Effects of temperature on the growth and development of immature An. gambiae (s.l.) mosquitoes ............................................................................................................................ 94 4.3.1 Developmental time of the immature stages ........................................................... 94 4.3.2 Determination of larval mortality and survival under different temperature regimes .......................................................................................................................................... 95 4.3.3 Measurement of time to pupation, pupation success, and mortality ....................... 96 4.3.4 Measurement of larval and pupal weight and size .................................................. 98 4.3.5 Temperature, number of adults produced, and sex ratio of An. gambiae (s.l.) mosquitoes ...................................................................................................................... 101 4.4 Effects of temperature and the growth and development of adult An. gambiae (s.l.) mosquitoes .......................................................................................................................... 102 4.4.1 Adult longevity ...................................................................................................... 102 University of Ghana http://ugspace.ug.edu.gh x 4.4.2 Length of the gonotrophic cycle, biting rate and fecundity of An. gambiae (s.l.) mosquitoes ...................................................................................................................... 106 4.4.3 Measurement of body weight, size, and proboscis length of adult mosquitoes .... 107 4.5 Insecticide susceptibility and expression of metabolic enzyme levels ........................ 110 4.5.1 Mortality of An. gambiae (s.l.) mosquitoes after exposure to pyrethroid insecticides ........................................................................................................................................ 110 4.5.2 Knockdown resistance ratio (KRR) ....................................................................... 111 4.5.3 Influence of temperature and insecticide on the expression of metabolic enzyme113 CHAPTER FIVE ................................................................................................................. 119 DISCUSSION ....................................................................................................................... 119 5.1 Introduction .................................................................................................................. 119 5.2 Effects of temperature on the growth and development of immature An. gambiae (s.l.) mosquitoes .......................................................................................................................... 119 5.2.1 Hatching and development time of mosquitoes decreased with increasing temperature ..................................................................................................................... 119 5.2.2 Survival time of An. gambiae (s.l.) larvae decreased with increasing temperature ........................................................................................................................................ 120 5.2.3 Pupation success of mosquitoes decreased with increasing temperature .............. 121 5.2.4 Larval and pupal size decreased with increasing temperature .............................. 122 5.2.5 Number of adult mosquitoes produced decreased with increasing temperature ... 122 5.3 Effect of temperature on the growth and development of adult An. gambiae (s.l.) mosquitoes .......................................................................................................................... 123 University of Ghana http://ugspace.ug.edu.gh xi 5.3.1 Longevity of mosquitoes decreased with increasing temperature ......................... 123 5.3.2 Gonotrophic cycle length and biting rate of mosquitoes were unaffected by increasing temperature .................................................................................................... 124 5.3.3 Fecundity of mosquitoes decreased with increasing temperature ......................... 125 5.3.4 Body size and proboscis length of mosquitoes decreased with increasing temperature ........................................................................................................................................ 126 5.4 Effects of temperature on insecticide susceptibility and metabolic enzyme expression ............................................................................................................................................ 126 5.4.1 Susceptibility of mosquitoes to pyrethroids decreased with increasing temperature ........................................................................................................................................ 126 5.4.2 Expression of metabolic enzymes in mosquitoes increased with increasing temperature ..................................................................................................................... 127 CHAPTER SIX .................................................................................................................... 129 CONCLUSION AND RECOMMENDATIONS ............................................................... 129 6.1 Conclusion .................................................................................................................... 129 6.2 Recommendations ........................................................................................................ 130 6.3 Strength and limitations ............................................................................................... 131 6.3.1 Strength .................................................................................................................. 131 6.3.2 Limitations ............................................................................................................. 131 6.4 Future research direction .............................................................................................. 131 6.5 Contribution to knowledge ........................................................................................... 132 REFERENCES ..................................................................................................................... 133 University of Ghana http://ugspace.ug.edu.gh xii APPENDICES ...................................................................................................................... 193 Appendix I: Search terms and search results from databases ............................................ 193 Appendix II: List of studies excluded with reasons ........................................................... 194 Appendix III: Risk of bias in included studies using the SYRCLE tool ............................ 195 Appendix IV: Ambient and rearing water conditions for each temperature regime .......... 197 Appendix V: Gel photographs of the PCR performed ....................................................... 198 Appendix VI: Ethical approval for the study ..................................................................... 200 Appendix VII: Pairwise comparisons of the development of the immature stages of An. gambiae (s.l.) mosquitoes................................................................................................... 201 Appendix VIII: Two-group comparisons and the overall trend of the effect of temperature on An. gambiae (s.l.) larval survival .................................................................................. 202 Appendix IX: Two-group comparisons and an overall trend of the effect of temperature on adult An. gambiae (s.l.) longevity ...................................................................................... 202 Appendix X: Comparison by sex across temperature and an overall trend of the effect of sex on adult An. gambiae (s.l.) longevity ................................................................................. 203 Appendix XI: Pairwise comparisons of gonotrophic cycle length, biting rate and fecundity ............................................................................................................................................ 203 Appendix XII: Mortality of An. gambiae (s.l.) mosquitoes exposed to pyrethroids .......... 204 Appendix XIII: Median levels of metabolic enzyme levels in An. gambiae (s.l.) mosquitoes reared at different temperature regimes ............................................................................. 205 Appendix XIV: Pairwise comparisons of enzyme levels in An. gambiae (s.l.) mosquitoes reared at different temperature regimes ............................................................................. 206 University of Ghana http://ugspace.ug.edu.gh xiii Appendix XV: Mann-Whitney U Test between mosquitoes that were not exposed and those exposed to pyrethroids ....................................................................................................... 207 Appendix XVI: Measurements of An. gambiae (s.l.) mosquito body parts using Leica application Software ........................................................................................................... 208 Appendix XVII: Abstracts of publication related to this study .......................................... 209 University of Ghana http://ugspace.ug.edu.gh xiv LIST OF TABLES Table 1: Characteristics of included studies ............................................................................ 48 Table 2: Effects of temperature on immature stages of Anopheles mosquitoes ...................... 52 Table 3: Effects of temperature on the longevity of Anopheles mosquitoes ........................... 55 Table 4: Effects of temperature on the body size of Anopheles mosquitoes ........................... 56 Table 5: Effects of temperature on fecundity, length of the gonotrophic cycle, and biting rate of Anopheles mosquitoes ......................................................................................................... 58 Table 6: Effects of temperature on insecticide susceptibility, expression of enzymes and immune responses in Anopheles mosquitoes ........................................................................... 60 Table 7: List of primers used for molecular identification of Anopheles mosquitoes ............. 78 Table 8: Number of An. gambiae (s.l.) mosquitoes from each rearing temperature regime used for biochemical analysis of metabolic enzyme level ............................................................... 87 Table 9: Composition of An. gambiae (s.l.) mosquitoes ......................................................... 93 Table 10: Relationship between rearing temperature and development time of immature An. gambiae (s.l.) mosquitoes ........................................................................................................ 94 Table 11: Median survival times of An. gambiae (s.l.) larvae reared at different temperatures .................................................................................................................................................. 95 Table 12: Time to pupation, pupation success, and pupal mortality of An. gambiae (s.l.) mosquitoes reared at different temperature regimes ................................................................ 98 Table 13: An. gambiae (s.l.) larval and pupal weight and size at different temperature regimes .................................................................................................................................................. 99 Table 14: Relationship between temperature and An. gambiae (s.l.) larval and pupal measurements ......................................................................................................................... 100 Table 15: Number of adults produced, and sex ratios of An. gambiae (s.l.) mosquitoes reared at different temperature regimes ............................................................................................ 102 University of Ghana http://ugspace.ug.edu.gh xv Table 16: Median longevity of adult An. gambiae (s.l.) mosquitoes reared at different temperatures ........................................................................................................................... 103 Table 17: Mean gonotrophic cycle length, biting rate, and fecundity of An. gambiae (s.l.) mosquitoes reared at different temperature regimes .............................................................. 107 Table 18: An. gambiae (s.l.) mosquito weight, size and proboscis length at different temperature regimes ................................................................................................................................... 108 Table 19: Relationship between temperature and adult An. gambiae (s.l.) mosquito weight, body size and proboscis length .............................................................................................. 109 Table 20: Knockdown resistance ratio of An. gambiae (s.l.) mosquitoes (Tiassalé strain) at different rearing temperature regimes .................................................................................... 112 University of Ghana http://ugspace.ug.edu.gh xvi LIST OF FIGURES Figure 1: Conceptual framework showing the relationship between temperature and growth, development and susceptibility of mosquitoes to insecticides .................................................. 6 Figure 2: Life cycle of a mosquito ........................................................................................... 16 Figure 3: Adult Anopheles mosquito ....................................................................................... 18 Figure 4: Adult Aedes mosquito .............................................................................................. 20 Figure 5: Adult Culex mosquito ............................................................................................... 21 Figure 6: Summary of mechanisms used by mosquitoes to produce a resistance phenotype.. 33 Figure 7: PRISMA flow diagram of search phases with numbers of studies included/excluded at each stage ............................................................................................................................. 47 Figure 8: Climate control incubators (RTOP-1000D, Zhejiang, China) ................................. 74 Figure 9: Assessment of the developmental stages of the immature An. gambiae (s.l.) mosquitoes ............................................................................................................................... 79 Figure 10: Measurement of larval and pupal size using Leica EZ4 HD microscope ............. 80 Figure 11: Oviposition cups for fecundity assessment ............................................................ 83 Figure 12: Insecticide susceptibility test following WHO protocols (WHO, 2016a) .............. 85 Figure 13: Kaplan-Meier survival plots of An. gambiae (s.l.) larvae reared at different temperatures ............................................................................................................................. 96 Figure 14: Longevity of adult An. gambiae (s.l.) mosquitoes reared under different temperature regimes. .................................................................................................................................. 105 Figure 15: Insecticide susceptibility of Anopheles gambiae (s.l.) (Tiassalé strain) mosquitoes reared at different temperature regimes ................................................................................. 111 Figure 16: Median MFO level in An. gambiae (s.l.) mosquitoes reared at different temperature regimes. NB: No mosquito reared at 34 oC survived after being exposed to pyrethroids, hence, no enzyme level was measured .............................................................................................. 114 University of Ghana http://ugspace.ug.edu.gh xvii Figure 17: Median GST level in An. gambiae (s.l.) mosquitoes reared at different temperature regimes. NB: No mosquito reared at 34 oC survived after being exposed to pyrethroids, hence, no enzyme level was measured .............................................................................................. 115 Figure 18: Median α-esterase level in An. gambiae (s.l.) mosquitoes reared at different temperature regimes. NB: No mosquito reared at 34 oC survived after being exposed to pyrethroids, hence, no enzyme level was measured .............................................................. 117 Figure 19: Median β-esterase level in An. gambiae (s.l.) mosquitoes reared at different temperature regimes. NB: No mosquito reared at 34 oC survived after being exposed to pyrethroids, hence, no enzyme level was measured .............................................................. 118 University of Ghana http://ugspace.ug.edu.gh xviii LIST OF ABBREVIATIONS AAEP American Association of Equine Practitioners AChE Acetylcholinesterase ANOVA One-way analysis of variance ARPPIS African Regional Postgraduate Program in Insect Science CDC Centre for Disease Prevention and Control CDNB 1-chloro-2,4-dinitrobenzene CE Carboxylesterases CEC1 Cecropin CI Confidence interval CO2 Carbon Dioxide CTAB Cetyl Trimethyl Ammonium Bromide DDT Dichloro-diphenyl-trichloroethane DEF1 Defensin DFID Department for International Development DNA Deoxyribonucleic acid DTNB Dithio-bis2-nitrobenzoic acid ECDC European Centre for Disease Prevention and Control EFSA European Food Safety Authority EIP Extrinsic Incubation Period University of Ghana http://ugspace.ug.edu.gh xix EPA Environmental Protection Agency FAO Food and Agriculture Organization GABA Gamma-Amino Butyric Acid GSTs Glutathione-S-Transferases H2O2 Hydrogen Peroxide HD High Definition IGS Intergenic spacer IPCC Intergovernmental Panel on Climate Change IQR Interquartile Range IRAC Insecticide Resistance Action Committee IRS Indoor Residual Spraying ITNs Insecticide Treated Nets JEV Japanese Encephalitis Virus K3PO4 Potassium Phosphate KDR Knockdown Resistance KDT Knockdown time KRR Knockdown Resistance Ratio LED Light Emitting Diode LLINs Long-lasting insecticidal nets MFO Mixed-Function Oxidases University of Ghana http://ugspace.ug.edu.gh xx MGSTs microsomal Glutathione-S-Transferases NMIMR Noguchi Memorial Institute for Medical Research NOS Nitric Oxide Synthase NSE Non-Specific Esterases OC Organochlorine OD Optical Density OLS Ordinary least square OP Organophosphorus PBO Piperonyl butoxide PCR Polymerase Chain Reaction PMI President's Malaria Initiative POPs Persistent Organic Pollutants PRISMA Preferred Reporting Items for Systematic Reviews and Meta-Analyses RDL Resistance to Dieldrin rDNA ribosomal DNA RoB Risk of Bias ROS Reactive Oxygen Species SD Standard Deviation SDG Sustainable Development Goal SINV Sindbis Virus University of Ghana http://ugspace.ug.edu.gh xxi SYRCLE's Systematic Review Center for Laboratory Animal Experimentation's TMBZ Tetramethylbenzidine UG-IACUC University of Ghana Institutional Animal Care and Use Committee UNDP United Nations Development Program UNFCCC United Nations Framework Convention on Climate Change UNICEF United Nations International Children's Emergency Fund UV Ultra Violet VNVL Vestergaard Noguchi Memorial Institute for Medical Research Vector Labs WHO World Health Organization WMO World Meteorological Organization WNV West Nile Virus ZIKV Zika Virus University of Ghana http://ugspace.ug.edu.gh xxii OPERATIONAL DEFINITION OF TERMS Biting rate: The daily feeding rate of a vector on a host (Paaijmans et al., 2013a). Development time: It is the time (in days) from the hatching of mosquito eggs to adult emergence. Extrinsic incubation period: It describes the time it takes for parasites to develop in the mosquito from the point of ingestion via an infected blood meal through to the point at which sporozoites enter the salivary glands and the mosquito becomes infectious (Ohm et al., 2018). Fecundity: It is expressed as the number of eggs laid per female mosquito (Mamai et al., 2017). Gonotrophic cycle length: The average number of days that gravid mosquitoes took to oviposit after taking a blood meal (Mala et al., 2014). Immature mosquito: This consists of the egg, larval and pupal stages of mosquitoes. Larval source management: the management of aquatic habitats (water bodies) that are potential larval habitats for mosquitoes to prevent the completion of development of the immature stages (WHO, 2013a). Longevity: It is the number of days a mosquito lives after emergence. Metabolic enzymes: They are enzyme systems that insects have developed to help them naturally detoxify insecticides and other foreign compounds (FAO, 2012; Gatton et al., 2013). Number of adults produced: The number of adult mosquitoes emerged from the total number of larvae. Pupation success: It is the proportion of larvae that pupated from the larval stage. Resistant population: a population is considered resistant if its response to an insecticide in detection tests drops significantly below its normal response (Georghiou & Mellon, 1983). University of Ghana http://ugspace.ug.edu.gh xxiii Susceptible population: a population that has not been subjected to insecticidal pressure and in which resistant individuals are either absent or rare (WHO, 2016a). Time to pupation: It is estimated as the time from egg hatching to the onset of pupation. University of Ghana http://ugspace.ug.edu.gh xxiv ABSTRACT Background: Anopheles mosquitoes are responsible for transmitting malaria and lymphatic filariasis. They are among the notable vector species for their crucial role in transmitting malaria. The survival of the vector is of great interest as it affects its ability to transmit diseases. The biology and ecology of mosquitoes are strongly dependent on ambient temperature. The mosquito's life cycle includes four stages: egg, larva, pupa and adult. Indeed, the rearing temperature of the immature stages (egg, larva, and pupa) can significantly impact the completion of the life cycle, the overall fitness of the adult, and ability to transmit disease. In recent years, global warming and possible future warmer climate have prompted many studies to focus on the effects of elevated temperatures on both the morphology and the biology of various species, including vectors. Despite the importance of temperature variability on An. gambiae (s.l.) mosquito's development and survival, there is still the need to explore how and whether or not elevated temperatures associated with climate change is likely to reduce or increase the vector's population dynamics by modifying the life cycle, reduce the efficacy of insecticides, and increase the expression of metabolic enzymes in An. gambiae (s.l.) mosquitoes. Objective: This study aimed to investigate the influence of elevated temperatures on the growth and development of An. gambiae (s.l.) mosquitoes, and the effectiveness of pyrethroid insecticides in such higher temperatures. Methods: Anopheles gambiae (s.l.) eggs were obtained from colonies established in the laboratory and were incubated, hatched and reared under eight temperature regimes (25, 28, 30, 32, 34, 36, 38 and 40 °C) using climate-controlled incubators (RTOP-1000D, Zhejiang, China), with photoperiod of 12:12 (L:D) and 80 ± 10% relative humidity. Larvae were fed 10 mg of TetraFin goldfish flakes (Tetra Werke, Melle, Germany). All adults were fed with a 10% University of Ghana http://ugspace.ug.edu.gh xxv sugar solution soaked in cotton wool. In addition, female mosquitoes used to estimate fecundity and longevity were blood-fed using a guinea pig on day four (4) post-emergence. Larvae were monitored daily for development to the next stage. The time to pupation, pupation success, number of adults produced, and sex ratio of the newly emerged adult was recorded. Molecular identification of An. gambiae (s.l.) mosquitoes was done using polymerase chain reaction (PCR) to identify the composition of sibling species in the An. gambiae complex. Larval survival and adult longevity were monitored every 24 hours, and data were analyzed using Kaplan-Meier survival analysis. Furthermore, analysis of variance (ANOVA) was used to assess the relationship between temperature and development time, time to pupation, length of the gonotrophic cycle, biting rate and fecundity. Kruskal-Wallis test was also used to assess the relationship between temperature and pupation success, pupal mortality, the number of adults produced, and sex ratio. Digital images of larvae, pupae, adult wings and proboscis were captured using stereo microscope with inbuilt camera (Leica EZ4 HD, Leica Microsystems Limited, Switzerland) and body parts were measured using Leica Application Software, version 3.4.0 (Leica Microsystems Limited, Switzerland). Data on larval, and pupal weight and size, adult weight, size and proboscis length were log-transformed and analyzed using ordinary least square (OLS) regression with robust standard errors. In addition, three to five-day-old non-blood-fed An. gambiae (s.l.) mosquitoes were used for insecticide susceptibility test following the WHO bioassay protocol. Batches of 20 – 25 non-blood-fed female adult An. gambiae (s.l.) mosquitoes from each temperature regime (25 – 32 °C) were exposed to two pyrethroid insecticides (0.75% permethrin and 0.05% deltamethrin). The knockdown rate after 60 min and mortality at 24 h were recorded. The levels of four metabolic enzymes (MFO, GST, α-EST and β-EST) were examined in both mosquitoes that were not exposed and those exposed to pyrethroids. University of Ghana http://ugspace.ug.edu.gh xxvi Results: An. gambiae (s.l.) mosquitoes used in this study consisted of An. gambiae (s.s.) and An. coluzzii. Development time of immature mosquitoes significantly decreased (F(5, 24) = 133.55, P < 0.001) with increasing temperature. Log-rank test showed that larval survival (X2(6) = 5353.12, P < 0.001) decreased with increasing temperature. In addition, Kruskal- Wallis test showed that the number of adults produced (X2(5) = 28.16, P < 0.001) decreased with increasing temperature, with male mosquitoes disproportionately produced at higher temperatures than females. Larval (βlarval size = 0.11, 95% CI; 0.14, 0.09, P < 0.001) and pupal (βpupal size = 0.12, 95% CI; 0.14, 0.10, P < 0.001) size significantly decreased with increasing temperature. Furthermore, longevity of both blood-fed (log-rank test; X2(4) = 904.15, P < 0.001) and non-blood-fed (log-rank test; X2(4) = 1163.60, P < 0.001) mosquitoes decreased with increasing temperature. The results further showed that the fecundity of mosquitoes significantly (F(2,57) = 3.46, P = 0.038) reduced with increasing temperature. Body size and proboscis length also decreased with increasing temperature. The mortality of An. gambiae (s.l.) mosquitoes to pyrethroids decreased at temperatures above 28 oC. Mosquitoes reared at higher temperatures were more resistant to the insecticides tested (deltamethrin and permethrin) and had more elevated enzyme levels than those reared at low temperatures (P < 0.05). Conclusion: Mosquitoes could not breed beyond temperatures at 36 oC. Therefore, if the ambient environmental temperatures rise to 36 oC, possibly as a consequence of climate change, it is likely to reduce or inhibit malaria transmission and perhaps its eradication in a future warmer temperature. In conclusion, warmer temperature is potentially hostile to a considerable proportion of emerging mosquitoes and may inhibit their survival such that the numbers of potential vectors may decrease. This study contributes to the knowledge on the relationship between temperature and growth and development of An. gambiae (s.l.) University of Ghana http://ugspace.ug.edu.gh xxvii mosquitoes and provides helpful information for modelling vector population dynamics in a future warmer climate. University of Ghana http://ugspace.ug.edu.gh xxviii PREFACE Publications related to this work Agyekum, T. P., Botwe, P. K., Arko-Mensah, J., Issah, I., Acquah, A. A., Hogarh, J. N., Dwomoh D., Robins T. G., & Fobil, J. N. (2021). A systematic review of the effects of temperature on Anopheles mosquito development and survival: implications for malaria control in a future warmer climate. International Journal of Environmental Research and Public Health, 18(14),7255. https://doi.org/10.3390/ijerph18147255. Agyekum, T. P., Arko-Mensah, J., Botwe, P. K., Hogarh, J. N., Issah, I., Dwomoh, D., . . . Fobil, J. N. (2022). Effects of elevated temperatures on the development of immature stages of Anopheles gambiae (s.l.) mosquitoes. Tropical medicine and international health, 27, 338– 346. https://doi.org/10.1111/tmi.13732. Agyekum, T. P., Arko-Mensah, J., Botwe, P. K., Hogarh, J. N., Issah, I., Dwomoh, D., . . . Fobil, J. N. (2022). Effects of Elevated Temperatures on the Growth and Development of Adult Anopheles gambiae (s.l.) (Diptera: Culicidae) Mosquitoes. Journal of Medical Entomology, 59(4), 1413-1420. https://doi.org/10.1093/jme/tjac046. Agyekum, T. P., Arko-Mensah, J., Botwe, P. K., Hogarh, J. N., Issah, I., Dadzie, S. K., . . . Fobil, J. N. (2022). Relationship between temperature and Anopheles gambiae sensu lato mosquitoes' susceptibility to pyrethroids and expression of metabolic enzymes. Parasites & vectors, 15(1), 163. https://doi.org/10.1186/s13071-022-05273-z. University of Ghana http://ugspace.ug.edu.gh https://doi.org/10.3390/ijerph18147255 https://doi.org/10.1111/tmi.13732 https://doi.org/10.1093/jme/tjac046 https://doi.org/10.1186/s13071-022-05273-z 1 CHAPTER ONE INTRODUCTION 1.1 Background Anopheles mosquitoes represent a major public health threat because of the diseases they transmit (Benelli, 2015), which overall, forms a significant part of all morbidity and mortality records (Brito et al., 2013). They are responsible for transmitting pathogens such as malaria parasites, arboviruses and filarial worms (Gendrin & Christophides, 2013). In Ghana, malaria is an endemic disease, and the prevalence of malaria still accounts for 38.0% of all outpatient visits with children under 5 years being the most vulnerable groups (Ejigu & Wencheko, 2021). The control of diseases transmitted by Anopheles mosquito through infective bites rely strongly on chemical insecticides; use of impregnated treated nets (ITNs), outdoor spraying and indoor residual spraying (IRS) (Nnko et al., 2017). In an attempt to control the vector, four main classes of insecticides – pyrethroids, organochlorines, carbamates, and organophosphates have been used historically (Liu et al., 2011; Baffour-Awuah et al., 2016). Of all these insecticides, the pyrethroid class has been widely used to control Anopheles mosquitoes in recent years. However, there have been reports of increasing resistance of the vector to the insecticide (Hunt et al., 2011; Ranson et al., 2011; Dadzie et al., 2017; Mouhamadou et al., 2019). Anopheles mosquitoes are poikilotherms (i.e. have to survive and adapt to environmental stress), therefore their growth and development characteristics rely on ambient temperatures (Reinhold et al., 2018). These characteristics comprise the length of gonotrophic cycles, biting rate, fecundity, survival and development of the immature mosquitoes (Vantaux et al., 2016). Consequently, any factor capable of modifying any of these characteristics can influence the University of Ghana http://ugspace.ug.edu.gh 2 potential of mosquitoes to transmit diseases. Temperature is a typical example of the factors that could affect the ecology and biology of Anopheles mosquitoes and their potential to transmit diseases (Alto & Bettinardi, 2013; Ezeakacha & Yee, 2019). Over the past two decades, atmospheric temperature has been increasing (UNFCCC, 2007), and this is projected to affect the development times and the vectorial capacity of Anopheles mosquitoes (Mohammed & Chadee, 2011). Climate parameters such as temperature, humidity, and rainfall not only can substantially affect the growth and development of mosquitoes, but also the sporogonic development of malaria parasites, Plasmodium spp. within their bodies (Guerra et al., 2010; Hay et al., 2010; Afrane et al., 2012). Regards to Anopheles mosquitoes, studies conducted on growth and development characteristics and temperature have focused mainly on the development of the immature (egg, larvae and pupae) stages (Christiansen-Jucht et al., 2014) with little attention paid to the adult. Temperature variations can stress the adult mosquito, and the stress can make them more susceptible to external stressors leading to death (Lafferty & Mordecai, 2016). This may seem to imply that insecticide exposure (stressor) could induce high mortality or may go a long way to increase insecticide resistance. On the contrary, temperature has been shown to affect the efficacy of insecticides against mosquito species such as An. arabiensis, An. funestus, An. gambiae (s.s.), and An. stephensi (Glunt et al., 2014; Oxborough et al., 2015; Glunt et al., 2018). It is unclear how rearing temperature may affect the efficacy of insecticides and the susceptibility of or resistance of Anopheles gambiae mosquitoes (the predominant malaria vector in Ghana) to insecticides. In addition, temperature affects the mosquito's immune system (Murdock et al., 2012a; Murdock et al., 2013), decreases molecular stability while increasing enzyme function and membrane permeability (Lafferty & Mordecai, 2016). Yet, it is still University of Ghana http://ugspace.ug.edu.gh 3 unclear how variations in rearing temperature affect the expression of metabolic enzymes in mosquitoes (Qin et al., 2014; Camara et al., 2018). A growing body of literature recognizes the importance of how temperature affects the development and survival of An. gambiae (s.l.) mosquito since the parasite's development and disease transmission depend on the survival of the vector (Rajatileka et al., 2011). In sub- Saharan Africa, An. gambiae (s.l.) mosquito is one of the most predominant and important malaria vectors (Baffour-Awuah et al., 2016; Riveron et al., 2016). Elevated temperatures associated with climate change are likely to influence population dynamics of mosquitoes, affect the efficacy of insecticides and expression of metabolic enzymes. 1.2 Problem Statement Temperature variations could directly affect the dynamics of vector‐borne diseases (Polgreen & Polgreen, 2017; Roberts et al., 2018) by modifying the risk of disease transmission, especially where the extrinsic incubation period (EIP) gets closer to the lifespan of the vector (Alto & Bettinardi, 2013). A projected increase in temperature in Africa (Sylla et al., 2016) is anticipated to affect malaria transmission by altering key growth and development characteristics of Anopheles mosquitoes (Davies et al., 2016). However, studies that have examined either the net effects of rearing or ambient temperature on the growth and development of both the immature stages and adult An. gambiae (s.l.) mosquitoes are rare (Christiansen-Jucht et al., 2014, 2015; Shapiro et al., 2017). Temperature affects the population growth rates of mosquitoes by altering traits that enable these adult insects to live successfully in their habitats (Lyons et al., 2013). As a result of the high thermal conductivity of rearing water coupled with the inability to escape adverse conditions, the immature stages of mosquitoes are mostly affected by temperature (Oliver & Brooke, 2017). This may affect the growth and development of the immature stages of An. University of Ghana http://ugspace.ug.edu.gh 4 gambiae (s.l.) mosquitoes (Afrane et al., 2012; Davies et al., 2016) and the overall fitness of the adult An. gambiae (s.l.) mosquito by altering key growth and development characteristics such as fecundity, body size, adult longevity, blood-feeding behavior, gonotrophic cycle length, and biting rate (Davies et al., 2016; Shapiro et al., 2017; Ezeakacha & Yee, 2019). These characteristics can affect mosquito survival and parasite development and influence disease transmission (Christiansen-Jucht et al., 2014). The control of mosquitoes have focused on mainly the adult mosquito; however, vector control measures have not achieved the expected results owing to challenges such as inadequate financing (Shretta et al., 2016; WHO, 2017), gaps in control management (Kokwaro, 2009), and vector resistance to insecticides (Badolo et al., 2012; Riveron et al., 2015; Yewhalaw & Kweka, 2016). The toxicity and bioaccumulation of insecticides are influenced by temperature (Lushchak et al., 2018) and an increase in temperature could worsen vector control measures by reducing the efficacy of the insecticides, further increasing resistance (Glunt et al., 2018). Furthermore, an increase in temperature could increase the expression of metabolic enzymes (Angilletta Jr et al., 2009). Increase in metabolic enzymes could increase the detoxification of insecticides in mosquitoes, reduce the effectiveness of the insecticide, and increase resistance of mosquitoes to insecticides (Nardini et al., 2012; Panini et al., 2016). This may present a significant threat to malaria control and affect the achievement of sustainable development goals, especially goal 3 (ensure healthy lives and promote wellbeing for all ages) (UNDP, 2015). Due to the sensitivity of mosquitoes to temperature, several studies have focused on the effects of temperature on mosquito and their ability to transmit diseases (Paaijmans et al., 2013a; Ciota et al., 2014). However, there are still data gaps regarding the effects of elevated temperatures on adult An. gambiae (s.l.) mosquitoes’ traits such as fecundity, gonotrophic cycle length and University of Ghana http://ugspace.ug.edu.gh 5 biting. For instance, few studies have considered the impact of temperature on gonotrophic cycle length and biting rate (Shapiro et al., 2017). Only a few studies; if any at all, have evaluated the effects of different rearing temperatures on the sex ratio of emerged adult An. gambiae (s.l.) mosquitoes. This provides significant information on the population dynamics of mosquitoes and could inform control interventions. Furthermore, there is little information on the effects of rearing temperatures on the susceptibility of adult An. gambiae (s.l.) mosquitoes to pyrethroid insecticides and the expression of metabolic enzyme levels (Kristan et al., 2018). 1.3 Conceptual Framework This study considered only the effects of temperature on An. gambiae (s.l.) mosquitoes. However, varying climate parameters such as temperature, rainfall and humidity could strongly affect the growth and development of mosquitoes (Abiodun et al., 2016). Temperature is considered one of the most significant factors that affects biological processes and physiological functions, including growth and reproduction in ectotherms such as mosquitoes (Ezeakacha & Yee, 2019). It also plays a vital role in the growth and development of mosquitoes (Oliver & Brooke, 2017) and could modify the completion of the entire life cycle. Temperature could affect the growth and development of both the immature (egg, larvae and pupae) and adult stages of mosquitoes. The conditions experienced at the immature stages could affect the overall fitness of the adult mosquito by affecting the longevity, fecundity, body size, length of the gonotrophic cycle and biting rate (Figure 1). Temperature could significantly affect the metabolism and survival rate of insects and the efficacy of insecticides (Jaleel et al., 2020). Expression of metabolic enzymes such as mixed- function oxidase (MFO), Glutathione-S-transferases (GSTs), acetylcholinesterase (AChE), and non-specific esterase (NSE) in mosquitoes could detoxify insecticides more rapidly and render University of Ghana http://ugspace.ug.edu.gh 6 the insecticides ineffective. This could lead to the resistance of mosquitoes to insecticides. Overall, the effects of temperature on the growth and development of both immature and adult mosquitoes, expression of metabolic enzymes and susceptibility of mosquitoes to insecticides could alter the vector's population dynamics and ultimately affect mosquito control efforts. Figure 1: Conceptual framework showing the relationship between temperature and growth, development and susceptibility of mosquitoes to insecticides Immature mosquito (egg, larvae and pupae) Adult mosquito Growth and development Growth and development Insecticide susceptibility Expression of metabolic enzymes (GST, MFO & NSE) Resistance to insecticides (phenotypic & biochemical) Temperature variability University of Ghana http://ugspace.ug.edu.gh 7 1.4 Justification Malaria is still a major public health concern in Ghana, and vector control has been a major intervention to reduce malaria burden. Thus, climate change or increasing temperature threatens the current control measures and studies that will increase our knowledge to be better prepared in developing policies for control is needed. It is not clear how temperature may affect the susceptibility of An. gambiae (s.l.) mosquitoes to insecticides or expression of metabolic enzymes. Therefore, understanding how temperature affects the growth and development, and the susceptibility of An. gambiae (s.l.) mosquitoes to insecticides in elevated temperatures is critical. This will help policymakers put measures to mitigate the effects of temperature on the mosquito population. In addition, findings could guide mosquito control interventions and safeguard public health. 1.5 General Objective The general objective of the study was to investigate the influence of elevated temperatures on the growth and development, and susceptibility of An. gambiae (s.l.) mosquitoes to pyrethroid insecticides. 1.5.1 Specific Objectives The specific objectives were to; • Examine the influence of temperature on the developmental stages of An. gambiae (s.l.) mosquitoes. • Assess the relationship between temperature and the growth and development of adult An. gambiae (s.l.) mosquitoes. • Evaluate the effects of varying temperatures on the susceptibility of An. gambiae (s.l.) mosquitoes to pyrethroids and expression of metabolic enzymes. University of Ghana http://ugspace.ug.edu.gh 8 1.6 Research Questions This research sought to answer the under listed questions; • Is there any relationship between temperature and the developmental stages of An. gambiae (s.l.) mosquitoes? • Is there any relationship between temperature and growth and development of adult An. gambiae (s.l.) mosquitoes? • What are the effects of temperature on An. gambiae (s.l.) mosquito's susceptibility to pyrethroids and expression of metabolic enzymes? 1.7 Research Hypotheses The underlisted hypotheses guided the study. • Increasing temperature will influence the developmental stages of An. gambiae (s.l.) mosquitoes. • Increasing temperature will influence the growth and development of adult An. gambiae (s.l.) mosquitoes. • Varying temperatures from 25 oC through to 40 oC will affect the susceptibility of An. gambiae (s.l.) mosquitoes to pyrethroids and expression of metabolic enzymes. University of Ghana http://ugspace.ug.edu.gh 9 CHAPTER TWO LITERATURE REVIEW 2.1 Introduction This chapter provides an overview of key terms and concepts relevant to this thesis. The chapter presents biology of mosquitoes, control measures, insecticides used to control mosquitoes and resistance mechanisms. The existing gaps and limitations in the literature are also highlighted by assembling and evaluating the available evidence of the relationship between temperature and the development as well as survival of Anopheles mosquitoes. Research articles published up to March 2021 were systematically retrieved from PubMed, Science Direct, Scopus, ProQuest, Web of Science, and Google Scholar databases. The search strategies used keywords such as Anopheles, mosquito, malaria, temperature, temp*, season*, survival, insecticide resistance/susceptibility, metabolic enzyme, and longevity. 2.2 Biology of mosquitoes Mosquitoes are regarded as the most important groups of arthropods, primarily because of their role in disease transmission (Do Nascimento et al., 2018). They belong to the family Culicidae and form the core of global entomological research because of their role as vectors in transmitting a wide array of debilitating parasitic and viral diseases that affect both humans and animals (Becker et al., 2010). Mosquitoes are slender insects with long legs, and are usually identified with scales on their bodies and their long proboscis (Harbach, 2007). About forty- one (41) genera of mosquitoes have been reported, and about 3500 species have already been reported from different parts of the world (Do Nascimento et al., 2018). These mosquitoes are grouped into two subfamilies of Anophelinae and Culicinae (Wilkerson et al., 2015; Foster et University of Ghana http://ugspace.ug.edu.gh 10 al., 2017). There are three best-known genera of mosquitoes: Anopheles, Aedes, and Culex (Wilkerson et al., 2015). Globally, mosquitoes are the most important insect pests that affect humans and animals (Gouge et al., 2016). They serve as vectors for several diseases, including malaria, dengue, lymphatic filariasis, and many other arboviral diseases, including Lassa and yellow fevers, which are accountable for hundreds of millions of morbidity and millions of deaths annually (WHO, 2019b). Projections by the World Health Organization (WHO) show that mosquito- borne diseases are the leading causes of morbidity and mortality in developing countries (WHO, 2005). However, not all mosquito species are of public health importance. From a medical perspective, the most important species belong to the following genera; Aedes, Anopheles, and Culex (ECDC, 2014). In addition to their impact on human health, mosquitoes play a crucial role in natural ecosystems. They serve as important pollinators and food sources for some animals such as amphibians, reptiles, birds, and mammals (Wong & Jim, 2016). Mosquitoes are very successful insects because they can acclimatize to a wide range of habitats (Becker et al., 2010). Aside the deserts and perpetually frozen areas, mosquitoes are ubiquitous, and found in humid tropics, subtropics, warm moist climates, and the temperate and cool regions (Do Nascimento et al., 2018). The activities of mosquitoes are, to some extent, species- specific. For instance, while some species are active at night or sunset, others are active during the daytime (Harbach, 2007). The flight habits of mosquitoes are also species dependent. Most local species linger around their breeding places whereas other species travel very far from their breeding habitats. Usually, the female mosquitoes cover a longer flight distance compared to the males. It has been reported that some mosquitoes move as far as 75 miles (about 121 km) from the breeding sites, though on the average they are mostly within a mile or 2 miles from their breeding habitat (AAEP, 2016). University of Ghana http://ugspace.ug.edu.gh 11 Generally, mosquitoes are highly attracted to humans and are well adapted to breeding places created by human activities. The use of water storage containers in animal husbandry and other farming activities such as fish ponds and irrigation systems offer suitable breeding conditions for anthropophilic mosquitoes (Egbuche et al., 2016). With regards to feeding, both the male and female mosquitoes depend on plant nectar and fruit sap for energy. However, the female counterparts require blood meal as an additional dietary requirement and source of protein for the development of their eggs (Silver, 2008). When it comes to blood meal sources, different species prefer different host animals. Whereas some feed on humans and birds, others prefer other animal hosts (Gouge et al., 2016). The geography of the mosquito reveals a wide global diversity, and in Ghana, the following species; Culex, Aedes, Anopheles, and Mansonia mosquitoes, have been reported across the country (Ughasi et al., 2012; Kudom, 2015; Kudom et al., 2015a; Owusu-Asenso, 2018). 2.3 Life cycle of mosquitoes The life cycle of mosquito (Figure 2) is a complete metamorphosis (Becker et al., 2010) which comprises four different stages: egg, larvae, pupae and adult, and the whole cycle requires almost two weeks (Tokachil et al., 2017). The adult stage is free-flying, while the first three stages are aquatic (Jackman & Olson, 2002). In addition, the first three stages are called the aquatic or immature stages. Only the adults are involved in disease transmission, although the dynamics of the immature stages (larvae and pupae) play a significant role in determining the fitness of the adult mosquito for disease transmission (Li, 2009). University of Ghana http://ugspace.ug.edu.gh 12 2.3.1 Egg stage The eggs of mosquitoes differ significantly among the major groups of species and individual species (Eldridge, 2008). In order to find potential oviposition sites, gravid mosquitoes depend on olfactory and visual signals. As these mosquitoes come closer to a site, they use olfactory, visual, and tactile alerts to appraise the quality of the site for oviposition (Day, 2016). Mosquitoes oviposit one at a time, and they float on the surface of the water. The eggs are laid either singly (e.g., Anopheles and Aedes species) or stuck together in floating rafts (e.g. Culex species). Some species (e.g. Aedes) oviposit just above the water line or on wet mud (these eggs hatch only when inundated with water) while Culex and Anopheles species lay their eggs on water (Osman, 2010). Other species like the Mansonia lay eggs as submerged clusters attached to roots, stems, and leaves of aquatic vegetation (Day, 2016). In addition, adult females oviposit in numerous different ways depending on the species. Females lay between 100 to 300 eggs (Genoud et al., 2019) after a blood meal. The eggs are vulnerable to desiccation and hatch within the second or third day; however, hatching may take up to two to three weeks in colder climates (Coleman, 2009). Many mosquito species usually oviposit during the dawn and twilight periods (Day, 2016). Generally, when the mosquitoes oviposit, the eggs are white but grow dark within few hours (Farnesi et al., 2017). In general, mosquitoes can be grouped into two categories per their egg-laying behavior as well as whether or not their embryos undergo diapause (innately dogged resting period) or dormancy period (externally triggered resting period) (Becker et al., 2010). In the first category, called rapid hatch (Day, 2016), the embryos do not undergo diapause or dormancy and hatch after the embryonic development is completed. However, in the second category (delayed hatch), the eggs do not hatch right after oviposition (Becker et al., 2010) but later when conditions are favorable for the eggs to hatch. Under this category, the eggs are usually drought-resistant, stay alive for long periods outside the water, and hatch shortly after being re-flooded (Day, 2016). University of Ghana http://ugspace.ug.edu.gh 13 2.3.2 Larval stage Mosquito larvae inhabit different water bodies, including temporary or permanent, extremely polluted or clean, stagnant or flowing, small or large (Becker et al., 2010). The larva has a well- developed head and a mouth with brushes to feed, a large thorax, and a fragmented abdomen (Pwalia, 2014). The larval stage is the longest of the three immature stages (Beck-Johnson et al., 2013) and the only stage where feeding occurs, making it vital for nutritional reserves accumulation to develop the adult in the pupal stage. The larval stage responses to fluctuating conditions are crucial in population size regulation and mosquito control (Owusu et al., 2017). Larvae progress into four instar stages before reaching the pupal stage (Madzlan et al., 2016). At each molt, the head capsule is amplified to the full-size features of the subsequent instar, though the body continues to grow. Thus, one can use how big the head capsule is as a correct morphometric pointer for the larval instar (Becker et al., 2010). The larvae feed on dissolved foods in the breeding sites during the first two instars. During the third and fourth instar, they mainly survive on bacteria, algae, and other microorganisms to hoard sufficient energy for the transformation and further developments that take place during the pupal phase (Barfi, 2015). In terms of appearance and morphology, there is a significant difference between the larvae and the adults. The larval stage survives in water, and their feeding behavior and breathing structures clearly show this. In general, it is easy to classify mosquitoes to species at the larval stage than using the adults (Eldridge, 2008). Except for Anopheles larvae that lay parallel to the water surface because of lack of respiratory siphon, most mosquito larvae have a respiratory siphon, which dangles from the water surface (Ponlawat & Harrington, 2009; Osman, 2010). The larvae (e.g., Ae. vexans) occasionally come together in particular places at the breeding sites to reduce the chance of predation of any single larva (Becker et al., 2010). University of Ghana http://ugspace.ug.edu.gh 14 2.3.3 Pupal stage The pupal stage comes after the larval stage and it is the third stage of the mosquito's life cycle and also the final stage of the aquatic or immature stages (Kauffman et al., 2017). The mosquito pupae are also known as tumblers and present fewer characters beneficial for identification (Eldridge, 2008). The pupa looks like a comma shape with the head and thorax merged into a cephalothorax and the abdomen located below it. At this stage, key transformations occur, resulting in the metamorphosis of larval tissues into adult tissues (Coleman, 2009). Naturally, the pupal head and thorax are joined into a protuberant cephalothorax which possesses anterolaterally two respiratory trumpets. These are linked to the mesothoracic spiracles of the emerging adults to supply oxygen (Becker et al., 2010). In addition, the pupae have two big structures known as paddles that project from the tip of the abdomen (Eldridge, 2008). The pupal phase, a resting and non-feeding stage, is when the mosquito turns into an adult and usually takes about two days (Osman, 2010). 2.3.4 Adult stage This is the final stage of the mosquito's life cycle and the only stage which is not aquatic. This stage is finalized when gas is pushed through the pupal and the pharate adult cuticle and into its midgut. The newly emerged adult mosquito moves carefully to avoid dropping onto the water surface as its appendages linger partially in the exuvia (Becker et al., 2010). Upon its emergence, the adult mosquito seeks a safe hideout in the neighboring vegetation to enable its wings to develop fully (Agyekum, 2017). Like other insects, the adult mosquito has three different body regions: head, thorax, and abdomen (Eldridge, 2008). At emergence, the male mosquitoes are not sexually developed, as they have to spin their hypopygium via 180o before they are ready to mate with the female mosquitoes (usually takes about a day) (Becker et al., 2010). Because of this, the male mosquitoes emerge earlier (usually University of Ghana http://ugspace.ug.edu.gh 15 1 – 2 days) than the females to reach sexual maturity at the same time as the incipient females (Genoud et al., 2019). The male mosquitoes mate with the females as soon as they emerge (Diabate & Tripet, 2015). After mating, the females hoard more sperm in their spermathecae (a receptacle in which sperm is stored after mating) to inseminate several egg batches without copulation (Becker et al., 2010). In terms of morphology, there are differences between male and female mosquitoes. The female mosquitoes possess short palpi and an extended stiletto-like proboscis that in most species has structures called stylets adapted for piercing the skin of their host for a blood meal. However, the male mosquitoes have long hairy palpi in addition to a long fleshy proboscis adapted for sucking plant nectar and other fluids from fruits and flowers (Eldridge, 2008). Mosquitoes feed on plant nectar and other fluids from fruits and flowers, but the female, in addition to this, requires a blood meal to develop its eggs (Nyasembe & Torto, 2014). In most mosquito species, the female needs a blood meal to complete oogenesis (production or development of an ovum). This has caused mosquitoes to develop an intricate host-seeking behavior to identify and feed on a potential host. Generally, this host-seeking distribution is dependent on the species, host accessibility, and season (Verdonschot & Besse-Lototskaya, 2014). Most species are non-autogenous, implying that after copulation, the female mosquitoes require a blood meal to complete the egg development (Becker et al., 2010). After the adult mosquitoes have emerged, they are ready to commence their life cycle all over again by feeding, mating, and laying eggs. University of Ghana http://ugspace.ug.edu.gh 16 Figure 2: Life cycle of a mosquito 2.4 Common genera of mosquitoes The well-known and medically essential genera of mosquitoes include Anopheles, Aedes, and Culex mosquitoes (Lebl et al., 2015). 2.4.1 Anopheles mosquitoes Anopheles mosquitoes belong to the order Diptera, sub-order Nematocera, family Culicidae and sub-family Anophelinae (Agyepong et al., 2012). Anopheles mosquitoes usually breed in transparent, sunny, temporal water bodies like swampy areas, mining sites, foot and hoof print, roadside puddles, drainage trenches, and edges of boreholes (Baffour-Awuah, 2012). In addition, Anopheles mosquitoes are not limited only to these habitats; they look out for different habitats for breeding. According to Mattah et al. (2017), these mosquitoes breed within the environs of deteriorating infrastructure like poorly maintained drains, culverts, broken water pipes, car tire imprints on unpaved roads, market gardens/urban agricultural sites, open tins/cans, pools at construction sites, low lying areas that are prone to flooding, among others. However, some Anopheles species breed even in polluted or contaminated water bodies. For University of Ghana http://ugspace.ug.edu.gh 17 instance, An. gambiae sensu lato (s.l.), also referred to as An. gambiae complex, has been found in swamp extremely polluted with organic matter (Sattler et al., 2005). This contradicts the conservative view that Anopheles mosquitoes breed only in clear or clean water habitats (Baffour-Awuah, 2012). Anopheles mosquitoes are the primary vectors responsible for the transmission of malaria in the world (Sokhna et al., 2013; Chabi et al., 2016; Huestis et al., 2017), and the vectors are continuously evolving (Sokhna et al., 2013). About 500 species of Anopheles has been described; however, only sixty (60) are reported to cause malaria (Sokhna et al., 2013). In addition to malaria-endemic areas, Anopheles mosquitoes that transmit malaria can also be found even in areas where malaria has been eradicated – these areas are always at risk of resurgence of the disease (Obacha, 2016). Many Anopheles species have been reported in the literature, and examples include but not limited to the An. gambiae complex, An. funestus group, An. nili group (Ossè et al., 2019), An. moucheti, An. vinckei (Paupy et al., 2013), An. minimus complex, An. dirus complex, and An. subpictus complex (Morgan et al., 2013). Most important malaria vectors in Africa belong to a species complex, and these species are sometimes difficult to differentiate morphologically. Previously, due to the complexities regarding this, sibling species in the complex have often been considered as a single unit, notwithstanding the significant differences among these sibling species (Wiebe et al., 2017). In sub-Saharan Africa, the An. gambiae complex and An. funestus groups are the main vectors responsible for causing malaria (Nnko et al., 2017; Gouignard et al., 2019). The An. gambiae complex, which is the focus of this study, was documented in the 1960s, and it has been reported to include the most important malaria vectors in sub-Saharan Africa, chiefly of the dangerous malaria parasite – Plasmodium falciparum (Bashir et al., 2018). The complex consists of nine (9) morphologically indistinguishable sibling species. They are An. gambiae University of Ghana http://ugspace.ug.edu.gh 18 sensu stricto (s.s.), An. arabiensis, An. quadriannulatus, An. melas, An. merus, An. bwambae, An. amharicus (Bass et al., 2007; Coetzee et al., 2013), An. coluzzii (Coetzee et al., 2013; Barron et al., 2018; Camara et al., 2018) and An. fontenillei (Barrón et al., 2019). In Ghana, some of the Anopheles species reported include An. gambiae (s.s.), An. funestus, An. coluzzii, An. pharaoensis, An. rufipes, An. melas and many more. The Anopheles species reported in the country are distributed based on ecological settings (Baffour-Awuah, 2012). For instance, An. gambiae (s.s.) and An. funestus – the most common species of Anopheles in the country, and An. coluzzii are found throughout the country (Baffour-Awuah et al., 2016). In most locations, An. coluzzii and An. gambiae (s.s.) sympatrically co-exist (Kudom, 2015). On the other hand, An. arabiensis predominates in the coastal savannah and northern regions. Anopheles rufipes and An. melas are also limited to the northern and coastal areas of the country, respectively (Baffoe-Wilmot et al., 2001; Yawson et al., 2004; De Souza et al., 2010). Figure 3: Adult Anopheles mosquito University of Ghana http://ugspace.ug.edu.gh 19 2.4.2 Aedes mosquitoes Aedes is the most prominent tribe of mosquitoes with about 1256 species categorized into ten (10) genera (in descending order); Aedes sensu, Verrallina, Armigeres, Psorophora, Eretmapodites, Heizmannia, Haemagogus, Zeugnomyia, Udaya, and Opifex (Wilkerson et al., 2015). Many species of Aedes exist, and they include but not limited to A. albopictus, A. aegypti, A. polynesiensis, A. scutellaris complex (WHO, 2009a), A. taeniorhynchus, A. vexans, A. sollicitans, A. togoi, A. atropalpus, A. triseriatus, and A. hendersoni (Day, 2016). However, A. aegypti, and A. albopictus are of significant public health importance because of their role in transmitting diseases (Kraemer et al., 2015). Some notable diseases they transmit include dengue, yellow fever, chikungunya and Zika viruses (Kweka et al., 2018). Aedes species can be found in natural and artificial receptacles capable of holding clear and clean water (Dom et al., 2013a; Dom et al., 2013b; Madzlan et al., 2016). Among the preferred breeding sites are earthen jars, ant traps, flower pots, drums, coconut shells, concrete tanks, and discarded tires (Simard et al., 2005; García-Rivera & Rigau-Pérez, 2006; Paupy et al., 2009). In addition, larvae can be found in natural sites like tree holes and bromeliads (García- Rivera & Rigau-Pérez, 2006). According to Dom et al. (2013b), Aedes aegypti usually prefers indoor artificial containers, and A. albopictus are more used to natural water receptacles found outdoors. The feeding behavior of Aedes differs among species. For instance, while A. albopictus is opportunistic and has zoophilic feeding behavior, A. aegypti is (except in African populations) highly anthropophilic (Paupy et al., 2009). In addition, A. albopictus is primarily a daytime and exophagic (feeds outdoors) mosquito and prefers to bite in the early morning and late afternoon. However, several exemptions have been documented based on the region, human habitat, season, and host availability (Paupy et al., 2009). Though the A. aegypti mosquito is also a University of Ghana http://ugspace.ug.edu.gh 20 daytime feeder and bites mainly in the morning or late afternoon, they generally rest in dark, indoor places like under beds, closets (García-Rivera & Rigau-Pérez, 2006) and behind curtains. In Ghana, studies have reported Aedes species in all the ecological zones (Appawu et al., 2006; Ughasi et al., 2012; Owusu-Asenso, 2018). Some of them include A. aegypti, A. africanus, and A. luteocephalus. Figure 4: Adult Aedes mosquito 2.4.3 Culex mosquitoes Culex mosquitoes consist of more than a thousand species distributed globally (Mullen & Durden, 2009). They are also the most prevalent mosquito species across the African continent (Nchoutpouen et al., 2019), and are vectors of many important disease causing viruses, including; West Nile virus (WNV), Sindbis virus (SINV), Japanese encephalitis virus (JEV), and a range of nematodes (Mullen & Durden, 2009; Weaver & Lecuit, 2015; Gould et al., 2017). Culex species are highly opportunistic and feed on both humans and animals. This behavior enhances their ability to transmit zoonotic diseases, making them a significant threat to public University of Ghana http://ugspace.ug.edu.gh 21 health (Weissenböck et al., 2010). Unlike Anopheles, Culex mosquitoes usually breed in turbid water (Mahgoub et al., 2017); however, there are instances where Anopheles and Culex larvae have been found together in breeding sites even though their breeding ecology differs (Emidi et al., 2017). There are so many species of Culex, but Culex pipiens complex is the most important species. It comprises six members: Cx. pipiens Linneaus, Cx. australicus Dobrotworsky and Drummond, Cx. quinquefasciatus Say, Cx. globocoxitus Dobrotworsky, Cx. pallens Coquillet, and Cx. molestus Forskll (Nchoutpouen et al., 2019). In Ghana, Culex species such as Cx. quinquefasciatus Say, Cx. thallassius, Cx. decens, Cx. fuscocephala, and Cx. perexiguus have been identified in different parts of the country (Opoku & Ansa-Asare, 2007; Kudom et al., 2015a, 2015b). Figure 5: Adult Culex mosquito University of Ghana http://ugspace.ug.edu.gh 22 2.5 Anopheles and malaria transmission Malaria remains a disease of public health concern in terms of morbidity and mortality caused by four species of the genus Plasmodium (P. falciparum, P. vivax, P. ovale and P. malariae). Malaria is a protozoan infection that attacks the red blood cells in the human body through the bite of an infected female anopheline mosquito (Wang et al., 2013). The incidence of malaria is approximately 300 – 500 million clinical cases, resulting in 1 million deaths each year, where children under 5 are the most affected (Traoré et al., 2020). In 2017, approximately 219 million cases of malaria were recorded, with 435 thousand deaths worldwide (WHO, 2019b). Vector control such as long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) remain key elements in reducing the transmission and burden of malaria in Africa (Otten et al., 2009). There have been increased global efforts to control and eliminate malaria over the past two decades and this has prevented about 1.5 billion cases and 7.6 million deaths (WHO, 2021). Notwithstanding, malaria remains a major public health issue in Ghana. The country is one of the eleven high-burden countries accounting for over 70% of the global malaria cases and deaths (Ghosh & Rahi, 2019), and part of the two highest-burden countries in Africa reporting the highest absolute increase in malaria cases in 2018 (WHO, 2019a). Over the years, Ghana has made some progress in the prevention and control of malaria as there has been a significant decline (28 % in 2011 to 14 % in 2019) of nationwide parasite prevalence among children under five years (based on microscopy) (President’s Malaria Initiative, 2022). In Ghana, the dominant species responsible for the transmission of the disease are An. gambiae (s.s), An. coluzzii, An. funestus, and An. arabiensis (Chabi et al., 2016). The key feature that makes these species efficient malaria vectors are their resting behavior and blood source preference. These behavioral differences affect the vectorial capacity, suitability, and University of Ghana http://ugspace.ug.edu.gh 23 effectiveness of vector control interventions (Akuamoah-Boateng et al., 2021). Additionally, environmental factors (temperature, relative humidity, vegetation, rainfall, etc) influence the survival of mosquitoes (Kristan et al., 2018; Agyemang-Badu et al., 2023). For instance, the larval stages of all species of Anopheles require water, thus the presence of a water source is related to increased distribution and density of larvae and an increased incidence of adult mosquitoes and consequently malaria (Ma et al., 2016). Malaria is transmitted through the bite of an infectious female Anopheles mosquitoes (WHO, 2019b). When a female Anopheles mosquito feeds on a Plasmodium-infected individual, gametocytes are taken up with the blood meal into the midgut. Gametocytes produce gametes that, once fertilized, rapidly develop into motile ookinetes, which cross the mosquito midgut epithelium and settle on its basal side (within 16 – 30 h post-blood-feeding) (Kirchner & Waters, 2019). Ookinetes then transform into sessile oocysts, producing thousands of individual sporozoites within 10 – 14 days. After oocyst rupture, sporozoites migrate to the salivary glands where they reside, ready to start a new infection when the mosquito feeds on a host again (Kirchner & Waters, 2019). Despite the efforts made to eliminate malaria, the transmission of malaria continues to occur and remains a critical public health issue, especially in Africa (Degefa et al., 2021). Factors such as increased insecticide resistance due to target site mutations, enhanced metabolic detoxification (Nwankwo, 2021), and behavioral resistance due to the preference of malaria vectors to bite outdoors and in the early evenings when people are indoors but unprotected (Degefa et al., 2021) contributes to the continual transmission of malaria globally. University of Ghana http://ugspace.ug.edu.gh 24 2.6 Control of mosquitoes Malaria vector control is the primary intervention for the global reduction and eradication of malaria (Kgoroebutswe et al., 2020). Vector control is measures of any kind against malaria- transmitting mosquitoes planned to limit the ability of mosquitoes to transmit disease (WHO, 2016b). The most common vector control measures include the use of insecticide-treated nets (ITN or LLIN) and indoor residual spraying (IRS) (WHO, 2017; Williams et al., 2018), with larval source management (LSM) as an additional control measure (Kgoroebutswe et al., 2020; McCann et al., 2021). 2.6.1 Use of mosquito bed nets One of the important tools of the Roll Back Malaria (RBM) strategy is the use of mosquito bed nets (insecticide-treated net (ITN) or long-lasting insecticidal nets (LLINs)) (Toé et al., 2009; Zöllner et al., 2015). An insecticide-treated net (ITN) is a bed net intended to provide a physical barrier against mosquitoes and also processed with residual insecticide to repel or kill mosquito vectors (Birget & Koella, 2015; Lindblade et al., 2015). On the other hand, long-lasting insecticidal nets (LLINs) - an effective alternative to insecticide-treated nets (ITNs), last longer than insecticide-treated bed nets (ITNs) and maintain their biological efficacy for about three (3) years (Sriwichai et al., 2016; Yang et al., 2018). The LLINs have insecticides coated around or incorporated into their fibers (Yang et al., 2018). The use of treated mosquito nets has had a great impact on reducing mosquito bites and in reducing malaria transmission (Mohammed, 2013; Castellanos et al., 2021). Policies aimed to promote universal access to bed nets (ITNs or LLINs) are developed in many malaria-endemic countries; however, the percentage of the people who slept under bed nets in 2015 in sub- Saharan Africa was estimated around 55 %, with most of them (about 68%) being children under-5 years old (WHO, 2015a). The usage of ITNs reduced malaria mortality rates in University of Ghana http://ugspace.ug.edu.gh 25 children under-5 years old by 55 % (Eisele et al., 2010; Admasie et al., 2018). Unfortunately, many households who received bed nets for free or subsidized prices do not use them (Baume & Franca-Koh, 2011). The low usage of bed nets has been attributed to fixing the bed net above the mat, house design, the feeling of suffocation and discomfort because of the relatively high temperatures in rooms (Toé et al., 2009). These reasons could grind down the achievements made in ITN use and reduce the effectiveness of malaria control programs. 2.6.2 Indoor Residual Spraying (IRS) Indoor residual spraying (IRS) of insecticides is a key method of reducing malaria vector transmission and has contributed to the decline in malaria prevalence globally (Tangena et al., 2020; Coleman et al., 2021). Indoor residual spraying (IRS) consists of applying a long-term, residual insecticide to potential mosquito hidden surfaces such as ceilings and interior walls of houses where mosquitoes might come into contact with the insecticide (WHO, 2013b). Indoor Residual Spraying (IRS) programs remain the most extensively used technique for controlling mosquitoes (Choi et al., 2019b) and are highly effective and can also significantly reduce malaria incidence and mortality (Gogue et al., 2020) on the condition that mosquito hide-outs in targeted communities are identified and sprayed (Agyekum, 2017). However, in recent years, IRS programs face challenges due to the increasing vector resistance to insecticides and the overall cost implications of the program implementation (WHO, 2020). The increased cost of IRS products has been linked to a reduction in IRS coverage throughout sub-Saharan Africa (Chaccour et al., 2021). This vector control method was introduced during the late 1940s when Dichloro-diphenyl- trichloroethane (DDT) was available and used to control mosquito vectors of malaria that entered houses (Van Den Berg et al., 2012). The success of house spraying for controlling University of Ghana http://ugspace.ug.edu.gh 26 malaria depends on applying an adequate and uniform dosage of insecticide on all potential resting places of the adult female mosquito (WHO, 2015b). Many sub-Saharan African countries have included IRS in their extensive malaria control plan in agreement with the Global Malaria Action Plan (GMAP) introduced by the WHO and Roll Back Malaria (RBM) Partnership. Globally, 185 million people (6 % of the global population at risk) were protected from malaria through the use of IRS in 2010 (WHO, 2011). However, in 2014, only 116 million (3.4 %) people were protected by IRS. There has been a reduction in the proportion of vulnerable population IRS protected (Cibulskis et al., 2016). 2.6.3 Larval source management (LSM) Larval source management (LSM) refers to managing mosquito breeding habitats to prevent the development of immature stages (eggs, larvae and pupae) or reduce the number of immature mosquitoes (WHO, 2013a; McCann et al., 2017). The goal of LSM is to reduce the number of adult mosquitoes that bite to prevent malaria transmission (Choi et al., 2019a). Four types of LSM exist, they include habitat modification (a permanent modification to the environment such as surface water drainage), habitat manipulation (a recurrent activity such as flushing of streams and water-level manipulation); larviciding (regular application of biological or chemical insecticides to water bodies); and biological control (introduction of natural predators into water bodies) (Fillinger & Lindsay, 2011; WHO, 2013a). It must be emphasized that larval source management (LSM) should not be considered a stand- alone intervention or replace core vector control interventions (using bed nets and IRS) but as an additional vector control measure (Fillinger & Lindsay, 2011; WHO, 2013a). Larval source management (LSM) provides the double benefits of reducing the numbers of house-entering mosquitoes and, importantly, those that bite outdoors (Fillinger & Lindsay, 2011). University of Ghana http://ugspace.ug.edu.gh 27 2.7 Targets of stages of mosquito with insecticides 2.7.1 Larviciding Larviciding denotes the process of killing the larvae of an insect (Baffour-Awuah, 2012). Many compounds comprising surface and oil films, bacteria larvicides, insect growth regulators, synthetic organic chemicals, and spinosyns can be used as larvicides (WHO, 2013a). Microbial larvicides such as Bacillus sphaericus or Bacillus thuringiensis or a combination of the two have proven to be effective in the control of mosquito larvae in different areas (Fillinger et al., 2009; Geissbühler et al., 2009; Fillinger & Lindsay, 2011; Baffour-Awuah, 2012; Maheu- Giroux & Castro, 2013; Afrane et al., 2016). These larvicides present numerous modes of actions against mosquito larvae. For instance, insect growth regulators thwart the development of the larvae, monolayers cause suffocation of mosquito larvae, and botanical or synthetic toxins directly interfere with the metabolic activities of insects (Fillinger & Lindsay, 2011). Larval control targets the aquatic stage of the mosquito by reducing mosquito larval sites, thus killing both indoor and outdoor biting mosquitoes (Fillinger & Lindsay, 2011). The suppression of mosquito larvae using larvicides serves as an excellent add-on to current methods such as the use of long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) (Antonio- Nkondjio et al., 2018). 2.7.2 Adulticiding Mosquito adulticide is a kind of insecticide used to eradicate adult mosquitoes (CDC, 2016). Adulticiding is just a word used to describe mosquito management activities targeted at adults. The control of the adult mosquito is the most common type of mosquito control (WHO, 2009b) and depend primarily on the use of long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) (Steketee & Campbell, 2010; Alemayehu et al., 2017; Camara et al., 2018). These are the most powerful and primarily used interventions (WHO, 2009b). In addition, in University of Ghana http://ugspace.ug.edu.gh 28 sub-Saharan Africa and other developing countries, some households use mosquito repellents such as mosquito coils to control adult mosquitoes (Agyekum, 2017). 2.8 Class of insecticides and their targets One way of controlling malaria is to prevent the infective mosquito from biting individuals, and this is done using insecticides (bed nets and indoor residual spraying) (Agyekum, 2017). Efforts from international agencies such as the US President's Malaria Initiative (PMI), World Bank, United Nations International Children's Emergency Fund (UNICEF), and Department for International Development (DFID), among others, as well as home governments to reduce malaria burden in sub-Saharan Africa have resulted in scaling up vector control measures, and this has resulted in a decline in malaria cases (WHO, 2011). The current control programs are primarily dependent on pyrethroid-based insecticides, which are the only recommended insecticides by the WHO for insecticide-treated nets (ITNs) (WHO, 2006). All the interventions aimed at the adult mosquito mainly depend on chemical insecticides. For instance, chemical insecticides such as pyrethroids are used for making long- lasting insecticidal nets (LLINs) (Essandoh et al., 2013), indoor residual spraying (IRS) (Da Cruz et al., 2019), and most mosquito repellents (Vences-Mejía et al., 2012; Hogarh et al., 2018). Pyrethroid insecticides are the only insecticide endorsed by the WHO to be used in most indoor residual sprays and treated bed nets (WHO, 2017). Other insecticides such as carbamates, organochlorines and organophosphates have been used to control mosquitoes (Cuervo-Parra et al., 2016). The efficacy of these insecticides has been challenged as mosquitoes have developed resistance to some classes of insecticides (Annan et al., 2014; Baffour-Awuah et al., 2016; Antonio-Nkondjio et al., 2017). Any modifications in mosquitoes could worsen the effectiveness of insecticides in the control of mosquitoes. University of Ghana http://ugspace.ug.edu.gh 29 2.8.1 Carbamate insecticides Carbamates are a class of insecticides structurally and mechanistically similar to organophosphate (OP) insecticides. Carbamates are N-methyl Carbamates derived from a carbamic acid and cause carboxylation of acetylcholinesterase at neuronal synapses and neuromuscular junctions (Silberman & Taylor, 2020). They are an important class of pesticides used worldwide in public health, among rural and urban settings. They include carboxyl (Sevin), aldicarb (Temik), and Propoxur (Baygon) (Adam & Lawson, 2010). Carbamate insecticides are synaptic poisons and bind to an enzyme found in the synapse