UNIVERSITY OF GHANA COLLEGE OF BASIC AND APPLIED SCIENCES IN VITRO STUDIES OF THE EFFECT OF ANOPHELES GAMBIAE MIDGUT BACTERIA ON THE DEVELOPMENT OF PLASMODIUM FALCIPARUM BY AMETSI, WILLIAMS GODWIN (10393712) THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF MPHIL. IN MOLECULAR CELL BIOLOGY OF INFECTIOUS DISEASES DEGREE DEPARTMENT OF BIOCHEMISTRY, CELL AND MOLECULAR BIOLOGY DECEMBER, 2021 University of Ghana http://ugspace.ug.edu.gh i DECLARATION I, Godwin Williams Ametsi, do certify that this project aside other cited works was carried out by me under the supervision of Dr. Jewelna Efua Birago Akorli and Dr. Yaw Aniweh, and that reference made to the works of others have been duly acknowledged. I certify that no part of this thesis has been previously submitted for a degree or any other qualification ………………………………….. Date: …………….…………… AMETSI WILLIAMS GODWIN 10393712 (STUDENT) ………………………………….. Date: …………………………… DR. JEWELNA EFUA BIRAGO AKORLI (PRINCIPAL SUPERVISOR) …………… Date: ………………………… DR. YAW ANIWEH (CO-SUPERVISOR) 20/12/2021 20/12/2021 20/12/2021 University of Ghana http://ugspace.ug.edu.gh ii ABSTRACT During blood feeding, female Anopheles mosquitoes may ingest Plasmodium gametocytes which undergo transformation in the gut and develop into sporozoites that are infectious to humans. Bacteria inhabit the mosquito gut, and the number and diversity of these bacteria change following blood feeding. The presence of some bacteria species results in the reduced intensity of developing Plasmodium parasites. Little attention has been given to understanding this direct mechanism of bacteria on Plasmodium parasites, and the effects of bacteria on malaria parasite developmental genes are not completely understood. This limits the scope of how gut bacteria, for example Enterobacter and Serratia, which have been found with anti- Plasmodium effects can be further explored for alternative disease control strategies. Therefore, this study investigated the lethal effect of cell-free secreted bio-products of E. cloacae and S. marcescens on a key Plasmodium parasite developmental gene (Gamete release gene, GAMER) for its potential as a target for malaria transmission-blocking. Plasmodium falciparum 3D7 and Dd2 cultures at 1% parasitaemia were independently exposed to spent Luria-Bertani (LB) medium from varying concentrations of Enterobacter cloacae and Serratia marcescens. The parasite killing effect of the bacteria were assessed with SYBR green fluorescent assay after 48 hours of co-culture. Spent media with final bacteria concentration between 10e+10-10e+20 reduced parasitaemia (P<0.001) compared to parasite culture without bacteria treatment. Using real-time (quantitative) PCR, it was found that the expression of GAMER was down regulated by 2 folds after 1 hour of screening P. falciparum 3D7 with cell- free spent medium of E. cloacae cultured for 8 hours in LB broth (Ec-8). However, the expression of GAMER was unaffected after 6 and 12 hours of screening P. falciparum 3D7 with Ec-8. These data provide information for further studies on gene and protein targets for transmission blocking interventions. University of Ghana http://ugspace.ug.edu.gh iii DEDICATION This work is dedicated to my mother, Mrs. Beatrice Setor for her continuous encouragement over the years and her selfless contribution to my education. God bless you; mum and I love you so dearly. University of Ghana http://ugspace.ug.edu.gh iv ACKNOWLEDGEMENT I am eternally grateful to God Almighty for being with me throughout the period of this project work. I wish to express my profound gratitude to my able supervisors; Dr. Jewelna Efua Birago Akorli of the Department of Parasitology, Noguchi Memorial Institute for Medical Research (NMIMR), University of Ghana and Dr. Yaw Aniweh, of the West African Centre for Cell Biology of Infectious Pathogens (WACCBIP), University of Ghana for their invaluable support, advice and suggestions towards the successful completion of this thesis. I applaud you for all the kind gestures you extended to me in diverse ways. As mentors you have taught me so much more. My sincere thanks to Rev. Dr. W. S. K. Gbewonyo for his assistance and encouragement in my academic career. You motivated me when I lost hope; I certainly needed that push to get the work done. I would like to acknowledge Naa Shormey Nortey for her encouragement and support throughout my MPhil studies. The immense assistance of Mark Clenam Tsifoanya-Tetteh, Isabella Georgina Gjameh, Sandra Adelaide King, Millicent Opoku, Esinam Abla Akorli, Lydia Okyere, Seraphim Tetteh and Kate Sagoe of the Department of Parasitology, Noguchi Memorial Institute for Medical Research, University of Ghana is gratefully acknowledged. My gratitude to Mr. Jacob Donkoh, Jersley Chirawurah, Felix Ansah and all lecturers of the Department of Biochemistry, Cell and Molecular Biology, University of Ghana, Legon for the knowledge they have imparted to me throughout my years at the department. Thank you all and God richly bless you. I sincerely thank my colleagues especially Emmanuel Edem Adade, Kojo Otieku Oworae Samuel Asenso, Joshua Labadah and Ibrahim Nuru for their support. University of Ghana http://ugspace.ug.edu.gh v I am very grateful to my mum, siblings and friends for their support I acknowledge the West African Centre for Cell Biology of Infectious Pathogens (WACCBIP) for funding my first year and this research work through a WACCBIP-DELTAS Postdoctoral fellowship to Dr Jewelna E. B. Akorli (ACE02-WACCBIP: Awandare; DE-15-007: Awandare). University of Ghana http://ugspace.ug.edu.gh vi TABLE OF CONTENTS DECLARATION ........................................................................................................................ i ABSTRACT ............................................................................................................................... ii DEDICATION ......................................................................................................................... iii ACKNOWLEDGEMENT ........................................................................................................ iv TABLE OF CONTENTS .......................................................................................................... vi LIST OF FIGURES .................................................................................................................. xi LIST OF TABLES ................................................................................................................... xii LIST OF ABBREVIATIONS ................................................................................................ xiii CHAPTER ONE ........................................................................................................................ 1 1.0 GENERAL INTRODUCTION ........................................................................................ 1 1.1 Introduction .................................................................................................................. 1 1.2 Rationale ....................................................................................................................... 5 1.3 Hypothesis .................................................................................................................... 5 1.4 Objectives ..................................................................................................................... 6 1.4.1 Main objective ........................................................................................................... 6 1.4.2 Specific objectives ..................................................................................................... 6 CHAPTER TWO ....................................................................................................................... 7 2.0 LITERATURE REVIEW ................................................................................................. 7 2.1 Epidemiology of malaria .............................................................................................. 7 2.2 Transmission of malaria ............................................................................................... 8 University of Ghana http://ugspace.ug.edu.gh vii 2.3 The Plasmodium parasite ............................................................................................ 11 2.3.1 Plasmodium falciparum ........................................................................................... 11 2.3.2 Plasmodium vivax .................................................................................................... 12 2.3.3 Plasmodium malariae .............................................................................................. 12 2.3.4 Plasmodium ovale.................................................................................................... 13 2.3.5 Plasmodium knowlesi .............................................................................................. 13 2.3.6 Plasmodium cynomolgi............................................................................................ 14 2.4 The Anopheles vector ................................................................................................. 14 2.4.1 Mosquito life cycle .................................................................................................. 15 2.5 Control of malaria ....................................................................................................... 18 2.5.1 Diagnosis of malaria ................................................................................................ 18 2.5.2 Treatment of Malaria ............................................................................................... 21 2.5.2.1 Chemotherapy ....................................................................................................... 21 2.5.2.2 Vector Control ...................................................................................................... 22 2.6 Challenges with current control strategies .................................................................. 24 2.6.1 Antimalarial Drug Resistance .................................................................................. 24 2.6.2 Vaccines................................................................................................................... 25 2.6.3 Insecticide resistance ............................................................................................... 28 2.7 The Anopheles mosquito microbiota and parasite transmission ................................. 28 2.7.1 The Anopheles mosquito microbiota ....................................................................... 28 2.7.2 Role of mosquito microbiota in larval development ............................................... 30 University of Ghana http://ugspace.ug.edu.gh viii 2.7.3 Role of mosquito microbiota on the nutrition of Anopheles mosquitoes ................ 30 2.7.4 Role of mosquito microbiota in vector competence ................................................ 32 2.8 Paradigm shift to bacteria-mediated malaria control strategies.................................. 32 2.8.1 Paratransgenesis....................................................................................................... 34 2.8.1.1 Challenges with paratransgenic interventions ...................................................... 35 2.8.2 Transgenesis ............................................................................................................ 36 CHAPTER THREE ................................................................................................................. 38 3.0 MATERIALS AND METHODS ................................................................................... 38 3.1 Reviving of archived bacteria isolates ........................................................................ 38 3.2 Growth curve estimation Enterobacter cloacae and Serratia marcescens .................. 38 3.3 Preparation of bacteria culture cell-free spent medium .............................................. 39 3.4 Malaria Parasite Culturing .......................................................................................... 40 3.4.1 Thawing of laboratory parasite isolates ................................................................... 40 3.4.2 Washing of human O+ erythrocytes (RBCs) ........................................................... 40 3.4.3 Culturing Plasmodium falciparum in vitro .............................................................. 41 3.5 Growth inhibition assay (GIA) ................................................................................... 41 3.6 Expression of GAMER gene following co-culture of P. falciparum gametocytes and cell-free bio-products of E. cloacae and S. marcescens ................................................... 42 3.6.1 Co-culture of P. falciparum parasites with cell-free bio-products of E. cloacae and S. marcescens.................................................................................................................... 42 3.6.2 Total RNA extraction from malaria parasites .......................................................... 43 3.6.3 Complementary DNA (cDNA) synthesis ................................................................ 44 University of Ghana http://ugspace.ug.edu.gh ix 3.6.4 Gene expression analysis using RT q-PCR ............................................................ 44 3.7 Data analyses .............................................................................................................. 45 CHAPTER FOUR .................................................................................................................... 47 4.0 RESULTS....................................................................................................................... 47 4.1 Growth curve of Enterobacter cloacae and Serratia marcescens .............................. 47 4.2 Standard curve for Enterobacter cloacae and Serratia marcescens) ......................... 48 4.2.1 Effect of bacteria cell-free spent medium on P. falciparum 3D7 and Dd2 strains . 49 4.2.2 Resistance indices of E. cloacae and S. marcescens cell-free spent medium on Plasmodium falciparum .................................................................................................... 54 4.2.3 Pattern of the anti-Plasmodial effects of bacteria cell-free spent media ................. 56 4.3 Expression of GAMER gene following exposure of Plasmodium to bacteria cell-free spent medium .................................................................................................................... 57 CHAPTER FIVE ..................................................................................................................... 61 5.0 DISCUSSION ................................................................................................................ 61 5.1 LIMITATIONS OF THE STUDY……………………………………………………..56 CHAPTER SIX ........................................................................................................................ 65 6.0 CONCLUSION AND RECOMMENDATION ............................................................. 65 6.1 Conclusion .................................................................................................................. 65 6.2 Recommendations ...................................................................................................... 65 REFERENCES ........................................................................................................................ 67 APPENDICES ....................................................................................................................... 112 University of Ghana http://ugspace.ug.edu.gh x University of Ghana http://ugspace.ug.edu.gh xi LIST OF FIGURES Figure 1: Global distribution of malaria.. ............................................................................. 8 Figure 2: The life cycle of Plasmodium falciparum ............................................................. 10 Figure 3:The life cycle of the Anopheles mosquito. ............................................................ 17 Figure 4:Adult male and female Anopheles mosquitoes. ................................................... 18 Figure 5: Plasmodium falciparum developmental changes as they travel through the mosquito gut.. ......................................................................................................................... 34 Figure 6: Plate map for the design of the growth inhibition assay of Plasmodium falciparum 3D7 and Dd2 strains. .......................................................................................... 43 Figure 7:Growth curve of Enterobacter cloacae and Serratia marcescens ........................ 47 Figure 8: Concentration of Serratia marcescens (log transformed) versus mean absorbance (OD600). ............................................................................................................. 48 Figure 9: Growth curves of P. falciparum 3D7 and Dd2 strains treated with cell-free spent medium produced by Enterobacter cloacae cultured for 24 hours. ................................... 50 Figure 10: Growth curves of P. falciparum 3D7 and Dd2 strains treated with cell-free spent medium produced by Serratia marcescens cultured for 24 hours. .......................... 51 Figure 11: P. falciparum 3D7 and Dd2 strains treated with cell-free metabolites produced by Enterobacter cloaacae cultured for 24 hours.. ................................................................ 56 Figure 12: P. falciparum 3D7 and Dd2 strains treated with cell-free metabolites produced by Serratia marcescens cultured for 24 hours...................................................................... 57 Figure 13: Standard curve of endogenous control. ............................................................. 58 Figure 14: Standard curve of target gene. ........................................................................... 59 University of Ghana http://ugspace.ug.edu.gh xii LIST OF TABLES Table 1: Average IC50 (mg/μL) following in vitro treatment of Plasmodium falciparum with spent media from Enterobacter cloacae and Serratia marcescens ............................. 52 Table 2: Resistance indices of Enterobacter cloacae cell-free spent medium on Plasmodium falciparum 3D7 and Dd2 strains ........................................................................................... 55 S1 Table. Measurement of Optical cell density (OD600) of Enterobacter cloacae ........ 112 S2 Table. Enterobacter cloacae colony counts .................................................................. 114 S3 Table. Measurement of Optical cell density (OD600) of Serratia marcescens........... 115 S4 Table. Serratia marcescens colony counts ..................................................................... 116 S5 Table. Cell-free spent medium of Enterobacter cloacae cultured at selected time points .................................................................................................................................................... 0 S6 Table. Cell-free spent medium of Serratia marcescens cultured at selected time points .................................................................................................................................................... 1 S7 Table. Primer sequences used in this study ...................................................................... 1 S8 Table. RT-qPCR reaction set up ....................................................................................... 2 S9 Table. RT-qPCR reaction conditions ................................................................................ 2 S10 Table. cDNA reaction set up ........................................................................................... 2 S11 Table 14. cDNA synthesis reaction conditions ............................................................... 3 University of Ghana http://ugspace.ug.edu.gh xiii LIST OF ABBREVIATIONS ACTs - Artemisinin based combination therapies AQ - Amodiaquine AS - Artesunate Bti - Bacillus thuringiensis subspecies Israelensis CQ - Chloroquine DHFR - Dihydro folate reductase DHPS - Dihydropterate synthase DNA - Deoxyribonucleic acid GAMER - Gamete release gene HADO - Haloacid dehalogenase domain ookinete protein gene HRP2 - Histidine-rich protein 2 IPT - Intermittent preventive treatment IPTc - Intermittent preventive treatment in children younger than 5 years IPTi - Intermittent preventive treatment in infants IPTp - Intermittent preventive treatment in pregnant women IRS - Indoor residual spraying ITNs - Insecticide-treated nets IVM - Integrated vector management LAMP - Loop-mediated isothermal amplification University of Ghana http://ugspace.ug.edu.gh xiv LLINs - Long-lasting insecticide-treated bed nets PCR - Polymerase chain reaction PE - Protective efficacy Pf - Plasmodium falciparum PfCRT - Plasmodium falciparum chloroquine resistant transporter, PfEMP1 - Plasmodium falciparum erythrocyte membrane protein 1 PfHRP2 - Plasmodium falciparum histidine-rich protein 2 PfHRP3 - Plasmodium falciparum histidine-rich protein 3 pLDH - Plasmodium lactate dehydrogenase PVM - Parasitophorous vacuolar membrane qPCR - Quantitative polymerase chain reaction qPCR - Quantitative real time polymerase chain reaction RDT - Rapid diagnostic test RNA - Ribonucleic acid RT-qPCR - Reverse transcriptase real time polymerase chain reaction s.l - Anopheles gambiae senso lacto s.s - Anopheles gambiae senso stricto SIT - Sterile insect technique SP - Sulfadoxine-Pyrimethamine TBV - Transmission blocking vaccine University of Ghana http://ugspace.ug.edu.gh xv TTM - Transfusion-transmitted malaria WHO - World Health Organization University of Ghana http://ugspace.ug.edu.gh 1 CHAPTER ONE 1.0 GENERAL INTRODUCTION 1.1 Introduction Malaria is an infectious disease caused by an obligate intracellular protozoan parasite of the genus Plasmodium and transmitted through the bites from infected female Anopheles mosquitoes. Although there are several species responsible for malaria, the six (6) species known to infect humans are Plasmodium falciparum, P. vivax, P. ovale, P. malariae, P. knowlesi and P. cynomolgi (World Health Organization, 2021). In 2020, the World Health Organization (WHO) reported 241 million malaria cases worldwide with 627, 000 deaths; 95% of these cases were recorded in Africa. Children (≤ 5 years) and pregnant women are the most susceptible (World Health Organization, 2021). The disease is mainly controlled by activities that effectively kill mosquitoes and the use of drugs that target the parasites within the human host. Vector control interventions remain important in fighting malaria (Benelli and Beier, 2017). These typically exploit the unique breeding, feeding and resting dynamics of the Anopheles vector. The consistent use of pyrethroids for indoor residual spraying (IRS) and in insecticide- treated nets (ITNs) represent key means to eliminate the vectors. Larvivorous fishes are also employed as biological agents for reducing mosquito population (Howard, Zhou and Omlin, 2007; Kondrashin et al., 2017). Other biological strategies include the use of niche competitors, entomopathogens, (Ramirez et al., 2018) and plasmodial symbiotic organisms (Geissbühler et al., 2009; Abdul-Ghani, Al-Mekhlafi and Alabsi, 2012). Personal protective interventions for the at-risk population and roll-out of small-scale environmental management strategies are also helpful in vector control (Kondrashin et al., 2017). The WHO initiated an Integrated Vector Management approach to achieve significant results in the control of mosquito-borne diseases University of Ghana http://ugspace.ug.edu.gh 2 as most strategies have not been successful when used as stand-alone projects (Ministry of Health-Republic of Ghana, 2009; WHO, 2015; Herdiana, Sari and Whittaker, 2018). Malaria treatment (Chemotherapy) involves the use of anti-malarial medicines to target different pathways of the parasite. Currently, Artemisinin-based combination therapy (ACT) has been recommended by WHO as frontline antimalarial drugs for the treatment of malaria (World Health Organization, 2019). However, increased reports of parasite resistance to antimalarial drugs (World Health Organization, 2010) especially ACTs (Dondorp et al., 2009; Ariey et al., 2014) threaten the global efforts to fight the disease. It is in this regard that vaccines are being considered as alternatives for use as drugs in malaria treatment. Vaccines have shown sufficient efficacy in the control and prevention of diseases like smallpox (Rieckmann et al., 2017), poliomyelitis and measles. However, there is currently no effective anti-malaria vaccine as the most promising anti-malaria vaccine RTS,S/AS01 undergoing WHO-recommended pilot implementation in some parts of Africa, has been reported to have low prevention and efficacy indices (Moorthy and Okwo-Bele, 2015) and its efficacy declines over time (RTS, 2014; Olotu et al., 2016). The periodic emergence of anti-malarial drug resistance, lack of effective anti- malarial vaccines and failing vector-control methods may have contributed to the estimated 627, 000 malaria-related deaths reported globally in 2020, compared with the 416, 000 estimated deaths in 2019 as recorded in the WHO 2021 World Malaria Report. This alarming statistic calls for the need for a paradigm shift to identify novel approaches for the control of malaria. Mosquito midgut microbiota is found to be major players in the ecology and physiology of mosquitoes including impact on resistance to mosquito control agents (Berticat et al., 2002; Duron et al., 2006), fitness (Strand, 2018) and vector competence (Dennison, Jupatanakul and Dimopoulos, 2014). In recent times, there has been heightened interest in research to identify suitable commensal microbes (bacteria, fungi and/or viruses) in disease vectors such as blood- University of Ghana http://ugspace.ug.edu.gh 3 sucking bugs, tsetse flies and mosquitoes that can be explored for control of diseases like leishmaniasis (Karimian et al., 2019), trypanosomiasis and malaria (Azambuja, Garcia and Ratcliffe, 2005; Geiger et al., 2009). This knowledge has been important for the development of novel microbial-mediated (e.g Wolbachia) mosquito control strategies (Moreira et al., 2009; Iturbe‐Ormaetxe, Walker and O’ Neill, 2011; Kamtchum-Tatuene et al., 2017; Niang et al., 2018; O’Neill, 2018). The presence of some microbes, mainly bacteria species in the mosquito midgut, has significant effects on vector competence. Several bacteria families including Enterobacteriaceae, Acetobacteriaceae and Neisseriaceae have been associated with the regulation of Anopheline vector competence and anti-Plasmodial activities (Cirimotich et al., 2011; Boissière et al., 2012; Gendrin and Christophides, 2013). For example, the colonization of the midgut with Chromobacterium (Csp_P) reduces malaria and dengue infection in vector mosquitoes (Ramirez et al., 2014). Asaia sp. also activate the immune system of An. stephensi to produce anti-microbial peptides that reduce P. berghei infection in the vector (Capone et al., 2013). Enterobacter and Serratia are genera of bacteria belonging to the Enterobacteriaceae family and play key roles in parasite development via direct interactions with bacteria-produced anti- Plasmodium factors (Cirimotich et al., 2011), secretion of various biomolecules such as enzymes and toxins (Azambuja, Garcia and Ratcliffe, 2005), and by the formation of a physical barrier that obstructs the contact between Plasmodium ookinetes and the midgut epithelium (Bando et al., 2013; Bahia et al., 2014; Song et al., 2018). Enterobacter sp. and Serratia sp. also inhibit the sporogonic development of P. vivax in Anopheles albimanus (Gonzalez-Ceron et al., 2003). Serratia and Enterobacter, therefore, have the potential for use in the control of malaria parasites in various Anopheles vectors and can be further explored for a novel intervention to control the disease. University of Ghana http://ugspace.ug.edu.gh 4 The vector-control potential of an Enterobacter species in Aedes and Culex mosquitoes was investigated by incorporating mosquito-larvicidal genes, cry4B from Bacillus thuringiensis subsp. israelensis (Bti) and binary toxin genes from Bacillus sphaericus into the genome of E. amnigenus strain isolated from Anopheles (Tanapongpipat et al., 2003). It was established that the engineered E. amnigenus strain expressed the mosquito lethal genes which led to the death of the Aedes and Culex mosquito larvae (Tanapongpipat et al., 2003). The parasite inhibiting potential of E. agglomerans was demonstrated by engineering the E. agglomerans to secrete anti-Plasmodium molecules and this significantly suppressed the development of P. falciparum and P. berghei oocysts (Wang et al., 2011). Several studies have demonstrated that mosquito vector defence against malaria parasites is regulated by the activation of the vector’s immune system (Meister et al., 2009; Bahia et al., 2014). RNA transcription profiles of aseptic and septic mosquitoes identified several genes that are highly expressed by enteric bacteria, including several anti-Plasmodium factors (Dong, Manfredini and Dimopoulos, 2009). A comprehensive transcriptional profile of the genes involved in the development of Plasmodium berghei in Anopheles gambiae midgut revealed two genes, Gamete release gene (GAMER) and Haloacid dehalogenase (HAD) domain ookinete protein gene (HADO), that play critical roles in the development of parasites in the vector (Akinosoglou et al., 2015). Gamete release (GAMER) gene encodes a 10.7kD protein that is associated with male gamete release. Inhibition of this gene results in significantly low ookinete numbers. Haloacid dehalogenase (HAD) domain ookinete protein (HADO) gene encodes a 44.7 kD amino acid protein that has a putative magnesium phosphatase role. The protein is found on the concave periphery of ookinetes. Disruption of HADO compromises ookinete development leading to a significant decline in oocyst numbers (Akinosoglou et al., 2015). University of Ghana http://ugspace.ug.edu.gh 5 1.2 Rationale In a recent study, an Enterobacter sp. isolated from wild Anopheline populations in Zambia rendered mosquitoes resistant to infection with Plasmodium falciparum by blocking the development of ookinete, oocysts and sporozoites, and this has been shown to occur via a mosquito-independent mechanism with the parasite that involves the secretion of reactive oxygen species (Cirimotich et al., 2011). This suggests that the effects of bacteria on parasites in mosquito vectors is not exclusively through an immune-dependent mechanism and could be a direct interaction between bacteria and parasite. Little attention has been given to understanding this direct mechanism of bacteria on Plasmodium parasites, and the effects of bacteria on malaria parasite developmental genes are not completely understood. This limits the scope of how gut bacteria, for example Enterobacter and Serratia, which have been found with anti-Plasmodium effects can be further explored for alternative disease control strategies. Therefore, this study investigated the lethal effect of cell-free secreted bio-products of E. cloacae and S. marcescens on a key Plasmodium parasite developmental gene (GAMER) for its potential as a target for malaria transmission-blocking. 1.3 Hypothesis Anti-Plasmodial activities of E. cloacae and S. marcescens isolated from Anopheles mosquito midgut are achieved through the secretion of bio-products that affect the expression of Plasmodium developmental genes. University of Ghana http://ugspace.ug.edu.gh 6 1.4 Objectives 1.4.1 Main objective The main aim of this study is to determine the direct effects of E. cloacae and S. marcescens secreted bio-products (cell-free spent media) on P. falciparum in vitro. 1.4.2 Specific objectives The specific objectives of the study are: 1. Compare the concentration-dependent anti-plasmodial effect of cell-free spent media of Enterobacter cloacae and Serratia marcescens in vitro 2. Investigate the regulation of GAMER gene following exposure to Enterobacter cloacae cell-free spent media in vitro University of Ghana http://ugspace.ug.edu.gh 7 CHAPTER TWO 2.0 LITERATURE REVIEW 2.1 Epidemiology of malaria Malaria is an infectious parasitic disease caused by Plasmodium species and transmitted by female Anopheles mosquitoes. Malaria is generally associated with non-specific symptoms such as fever, chills and fatigue. However, anaemia, lactic acidosis, multiple-organ failure and coma have also been observed in severe cases of the disease (Evans et al., 2006). The clinical manifestations of malaria are caused by the asexual blood stages of the parasites (Maitland and Marsh, 2004; Arévalo-Herrera et al., 2015, 2016). The disease is characterized by high mortality and morbidity. Malaria is predominantly found in the tropical and subtropical regions of Africa, Asia and the South America (Figure 1). There were 241 million reported cases and 627, 000 deaths in 2020 with women and children (below 5 years) being the most vulnerable (World Health Organization, 2021). In sub-Saharan Africa, malaria is caused predominantly by Plasmodium falciparum (Breman, Alilio and Mills, 2004; Snow et al., 2005) whereas P. vivax is predominant in America and South-East Asia (Guerra et al., 2010). Both P. falciparum and P. vivax cause majority of the malaria cases across the word (Snow et al., 2005). Plasmodium ovale and P. malariae are responsible for a few cases globally (Gebru et al., 2017). Although P. knowlesi was initially considered a rare zoonotic species, it is now known to infect humans (Singh and Daneshvar, 2013; Vythilingam et al., 2014; World Health Organization, 2019). Another species, P. cynomolgi has also recently been found to cause malaria in human hosts (Ta et al., 2014; Hartmeyer et al., 2019; Imwong et al., 2019). University of Ghana http://ugspace.ug.edu.gh 8 Figure 1: Global distribution of malaria. Most malaria cases occur in Africa with a few cases in America and South-East Asia. (Source: World Malaria Report 2021). 2.2 Transmission of malaria The lifecycle of the Plasmodium parasite involves an invertebrate vector and a vertebrate host. Anopheles mosquitoes are the only efficient vectors of malaria and transmit the disease from person to person through their bites. Malaria infections may be acquired via blood transfusions or the use of contaminated needles. However, transfusion-transmitted malaria (TTM) is rare with the risk of infection greatly reduced by the screening of blood donors (Owusu-Ofori et al., 2013; Verra et al., 2018). The female Anopheles vector ingests male and female gametocytes from an infected human host during a blood meal. The chemical and physical environment in the mosquito midgut immediately triggers gametocyte transformation into gametes (Billker et al., 1997, 1998) which fuse to form zygotes that develop into motile ookinetes within the vector midgut within 24 hours post-ingestion (Yassine and Osta, 2010). These motile forms (ookinetes) cross the peritrophic matrix surrounding the blood meal (Weiss et al., 2014) and transverse the midgut epithelium to form oocysts at the basal lamina. After 7 to 10 days the oocysts eventually develop into the sporozoites (Yassine and Osta, 2010) that spread through University of Ghana http://ugspace.ug.edu.gh 9 the haemolymph to all parts of the vector including the salivary gland (Smith, Vega-Rodríguez and Jacobs-Lorena, 2014; Sierra et al., 2015). Infective sporozoites (about 20-200) (Kappe, Kaiser and Matuschewski, 2003) are injected into the vertebrate host dermis during the mosquito’s subsequent blood feeding process. The sporozoites stay within the dermis for at least 5 minutes (Matsuoka et al., 2002; Amino et al., 2006; Yamauchi Lucy M. et al., 2007) and they travel to the liver to invade the hepatocytes (Figure 2). An asexual exo-erythrocytic cycle is set up in the liver cells where merozoites are subsequently released after 2-16 days (depending on the parasite species) (Cox, 2001; Sturm et al., 2006; Tarun et al., 2006) into host circulation following the rapture of infected hepatocytes (Cowman, Berry and Baum, 2012; Cowman et al., 2016). The released merozoites attach to and invade erythrocytes. Within the red blood cell, the merozoite is transformed into a ring or early trophozoite form, which in turn develops into a mature trophozoite that undergoes asexual multiplication to form a schizont containing numerous merozoites. The erythrocytic schizont ruptures, releasing merozoites that re-invade uninfected host red blood cells, thereby completing the erythrocytic cycle (Cowman, Berry and Baum, 2012). After several rounds of asexual replication in the host, lasting between 24 and 72 hours, (Venugopal et al., 2020), ring-stage parasites commits to gametocyte production by expressing gametocyte exported proteins on the surface of the infected red blood cells (Tibúrcio et al., 2015). The early stages of the gametocyte (Stages I-IV) take about 7-10 days to mature (Gardiner and Trenholme, 2015) and they remodel the surface structure of infected red blood cells to sequester in internal organs such as the heart, brain, spleen, gut and bone marrow (Aguilar et al., 2014; Joice et al., 2014) but the mature forms are found in the bloodstream after 12 days (Tibúrcio et al., 2015; Neveu and Lavazec, 2019). University of Ghana http://ugspace.ug.edu.gh 10 Figure 2: The life cycle of Plasmodium falciparum (Source: (Cowman, Berry and Baum, 2012)). Plasmodium sporozoites injected into the host’s bloodstream travel to the liver to invade host hepatocytes and develop into merozoites that are released into host circulation following the rapture of the infected hepatocytes. Released merozoites invade uninfected host red blood cells, develop into trophozoites and finally maturing into schizonts. Raptured schizonts release merozoites that re-invade uninfected host red blood cells setting up an asexual blood stage cycle. Gametocytes, developing from the asexual blood-stage are picked by female Anopheles mosquitoes and transformed into gametes in the mosquito midgut yielding zygotes. Emerging zygotes develop into ookinetes, oocysts and finally sporozoites that migrate to the salivary gland. University of Ghana http://ugspace.ug.edu.gh 11 2.3 The Plasmodium parasite Plasmodium sp. are obligate intracellular apicomplexan parasites that infect primates and other mammals such as birds, and reptiles. There are currently over 200 species identified (Watson, 1967) but only six of these; P. falciparum, P. vivax, P. malariae, P. ovale, P. knowlesi and P. cynomolgi) infect humans (Cowman et al., 2016). Plasmodium parasites have a diverse morphology and a very complex life cycle. They grow and develop in both the invertebrate vector and the vertebrate host (Venugopal et al., 2020), the sexual life stage occurs in the invertebrate host. In extreme cases, the parasites can be found in several organs such as bone marrow, heart and spleen (Aguilar et al., 2014; Joice et al., 2014). The blood-stage forms of Plasmodium spp. are responsible for the non-flu like symptoms of malaria (Maitland and Marsh, 2004; Arévalo-Herrera et al., 2015, 2016; Escalante et al., 2015) 2.3.1 Plasmodium falciparum Globally, this parasite is responsible for the majority of malaria cases and is the deadliest of all the Plasmodium species (Snow et al., 2005). P. falciparum invades red blood cells of all ages (Simpson et al., 1999) and the asexual blood stages exhibit a characteristic 48-hour cycle or tertian pattern (Ziegler, 1980; Barnes, 1986). It takes approximately 9 to 14 days after Plasmodium falciparum infection for the symptoms to manifest. Characteristic to P. falciparum infections, infected red blood cells adhere to the endothelial linings of blood vessels and sequestration in the cerebral microvasculature causes cerebral malaria. Cerebral malaria is associated with impaired consciousness and in severe cases coma (Idro et al., 2010; Rénia et al., 2012). The parasite is the most studied and several strains of P. falciparum have been adapted for in vitro blood cultures (Trager and Jensen, 1976). University of Ghana http://ugspace.ug.edu.gh 12 2.3.2 Plasmodium vivax The parasite is transmitted globally in the tropics except for West Africa. This is because the West African human population lacks the Duffy binding receptor which P. vivax requires to establish infection (Guerra, Snow and Hay, 2006; Culleton et al., 2008). Recent reports of P. vivax infections have, however, been made on Duffy negative individuals (Michon et al., 2007; Guerra et al., 2010). The parasite infects reticulocytes (young red blood cells) (Kitchen, 1938; Vryonis, 1939; Simpson et al., 1999; Tjitra et al., 2008; Poespoprodjo et al., 2009). The symptoms of P. vivax infection has a characteristic tertian pattern or 48-hour cycle (Ziegler, 1980; Barnes, 1986; McKenzie, Jeffery and Collins, 2002) and incubation period of 12 to 18 days (Powell, 1986; Yeom and Park, 2008; Brasil et al., 2011; Kim et al., 2013). In endemic areas, the parasites undergo relapse every three weeks (Hill and Amatuzio, 1949; Coatney, Cooper and Young, 1950; Anstey et al., 2012). 2.3.3 Plasmodium malariae Plasmodium malariae is globally distributed and coincides with P. falciparum infection. They are widespread throughout sub-Saharan Africa, Southeast Asia, South America and Europe (Collins and Jeffery, 2007). The parasite infects old red blood cells (normocytes) in primate hosts (Simpson et al., 1999; Ashley and White, 2014). Plasmodium malariae has a 72-hour cycle (quartan) (Powell, 1986) and it takes 18 to 40 days after infection for the symptoms of the disease to manifest (Ziegler, 1980; Barnes, 1986). The sporozoites of P. malariae takes about 1 hour to get to the liver and they mature in about 15 days. There are no quiescent liver stage forms (hypnozoites) or recrudescence as is the case in human P. vivax and P. ovale infections (Collins and Jeffery, 2007). University of Ghana http://ugspace.ug.edu.gh 13 2.3.4 Plasmodium ovale This parasite is found predominantly in tropical regions of Africa and Asia (Collins and Jeffery, 2005; Mueller, Zimmerman and Reeder, 2007; Smith et al., 2008). The global burden of P. vivax infection is often underestimated due to the difficulty in the detection of the parasite using microscopy especially at low parasitaemia (Rousset, Couzineau and Baufine-Ducrocq, 1969; Faye et al., 1998, 2002; Mueller, Zimmerman and Reeder, 2007). There are two distinct species of P. ovale parasites; Plasmodium ovale curtisi (classic type) and Plasmodium ovale wallikeri (variant type) and they both infect reticulocytes with a characteristic tertian or 24- hour cycle (Powell, 1986; Sutherland et al., 2010; Ngotho et al., 2019). P. ovale has an incubation period of 12 to 18 days and has the potential to form hypnozoites extending for not less than 10 months (Collins and Jeffery, 2005; Richter et al., 2010). 2.3.5 Plasmodium knowlesi This parasite is naturally known to infect long-tailed and short-tailed macaques (Butcher, Cohen and Garnham, 1970). The first case of zoonosis was discovered in Kapit Division of Sarawak, Malaysian Borneo (Singh et al., 2004). The parasite has since then successfully infected people in the Asian territories (Cox-Singh and Singh, 2008) and currently considered the fifth Plasmodium parasite to naturally infect man (White, 2008). The life cycle of the parasite is similar to that described in P. falciparum (Fig. 1) and it is transmitted by Anopheles leucosphyrus mosquitoes (Peyton, 1989; Sallum, Peyton and Wilkerson, 2005). No clinical symptoms are associated with parasite development in the liver. However, the development of the parasite in the erythrocytes comes with unique symptoms that follow a 24-hour (quotidian) cycle (Watson, 1967). The parasite is morphologically similar to P. malariae (Cox-Singh and Singh, 2008) but distinguishable using molecular techniques. The incubation period of the University of Ghana http://ugspace.ug.edu.gh 14 parasite is between 11 to 12 days (Watson, 1967) and P. knowlesi infections are readily treatable when diagnosed early. 2.3.6 Plasmodium cynomolgi Macaques are the natural host of this Plasmodium parasite (Butcher, Cohen and Garnham, 1970). However, P. cynomolgi has been found to also infect humans (Imwong et al., 2019) where it invades the reticulocytes (Russell and Cooke, 2017; Chua et al., 2019). The parasite has remarkable morphological and biological similarities to P. vivax which is traceable to their genetic make-up (Tachibana et al., 2012; Pasini et al., 2017). The life cycle of the parasite is similar to other Plasmodium species (Fig. 2). Plasmodium cynomolgi also has dormant forms (hypnozoites) that may be activated to cause relapse infections weeks to months after the primary infection (Krotoski et al., 1982; White, 2011; Joyner, Barnwell and Galinski, 2015; Joyner et al., 2016). With P. cynomolgi infection, clinical symptoms are often mild but may result in severe complications which can quickly lead to death (Rahimi et al., 2014). The parasite plays a vital role in scientific research where they are used as models for studying Plasmodium (Pacheco et al., 2011; Fonseca et al., 2017; Voorberg-van der Wel et al., 2017; Chua et al., 2019). 2.4 The Anopheles vector Mosquitoes are vectors life-threatening infectious diseases such malaria, lymphatic filariasis, dengue and chikungunya virus (Gendrin and Christophides, 2013). The prevalence of these infectious diseases relies on the distribution, abundance and vector competence of mosquitoes (Kibuthu et al., 2016). Approximately 40 Anopheles species are competent vectors of malaria. Anopheles mosquitoes belong to the Order Diptera, Family Culicidae and Subfamily Anophelinae. They are found all over the world except in the Antarctica (Ebenezer et al., 2014). University of Ghana http://ugspace.ug.edu.gh 15 The predominant mosquito species in Africa are Anopheles arabiensis, An. funestus, An. gambiae, An. melas, An. merus, An. moucheti and An. nili (Sinka et al., 2010). In Ghana, Anopheles gambiae s.l are the predominant mosquito species. Anopheles funestus, and An melas are also present and their distribution is ecological (Appawu et al., 2004; de Souza et al., 2010; Tuno et al., 2010). 2.4.1 Mosquito life cycle The Anopheles mosquito has four developmental stages namely; egg, larva, pupa and adult (Rozendaal, 1997). The early developmental forms (eggs, larvae, pupae) are aquatic and are often found in sunlit pools (Rozendaal, 1997), often man-made habitats such as burrow-pits, drains, brick-pits, hoof-prints, and permanent habitats like water holes or rainwater collecting in natural depressions (Gimnig et al., 2001; Imbahale et al., 2011; Mattah et al., 2017). Anophelines survive and thrive in habitats with optimum environmental conditions like temperature and humidity (Ndoen et al., 2010; Beck-Johnson et al., 2013). Their abundance and distribution are seasonal (Amaechi et al., 2018; Sanei-Dehkordi et al., 2019) and are most prevalent with high rainfall periods (Mahgoub, Kweka and Himeidan, 2017). The increase in mosquito population during rainy seasons could explain the high number of malaria cases recorded in the rainy seasons across sub-Saharan Africa (McMichael, 2013; M’Bra et al., 2018). Anopheles mosquitoes lay eggs in singles on the surfaces of water (Fig. 3). The eggs hatch in 1 to 2 days and it takes about 10 days for the larvae to progress across all four developmental phases (first instar to fourth-instar) at optimum temperatures between 26-28 ºC (Impoinvil et al., 2007). Fourth (4th) instar larvae take 2-3 days to emerge into pupae at an optimum temperature between 25-28 ºC and the pupal-to-adult transition takes 1-2 days depending on environmental cues such as temperature and average humidity (Bayoh and Lindsay, 2003; University of Ghana http://ugspace.ug.edu.gh 16 Kirby and Lindsay, 2009). Besides the morphologically distinguishable features between male and female adults (Fig 4), these also differ in terms of nutrition and dispersion. Generally, both male and female adult mosquitoes feed on plant nectar 24-36 hours post-emergence for all their energy requirements (Foster and Takken, 2004; Manda et al., 2007; Barredo and DeGennaro, 2020). The adult male mosquitoes continue to feed on the sugar meals to maintain their reproductive abilities (Gouagna et al., 2014). After 5 days of feeding on nectar, gravid adult female mosquitoes feed on blood for the development of their fertilised eggs (Foster and Takken, 2004; Hansen et al., 2014). Adult female mosquitoes rely on cues such as carbon dioxide (Gillies, 1980; Erdelyan et al., 2012), exhaled chemicals (lactic acid) (Acree et al., 1968) and a range of odourants (acetates, alcohols, and ketones) to locate their primate hosts (Hallem et al., 2004; Potter, 2019). The digestion of the blood meal is temperature-dependent and takes about 2-3 days in the tropics and 5-8 days in temperate environments (Paskewitz, 1995). Generally, Anopheles mosquitoes feed only once per oviposition or gonotrophic cycle, however, newly emerged mosquitoes may feed two or more times during their first oviposition cycle (Venkat Rao, 1943; Thomson, 1948; Edman, Webber and Kale, 1972; Ezemuoka et al., 2020). University of Ghana http://ugspace.ug.edu.gh 17 Figure 3:The life cycle of the Anopheles mosquito. The female adult Anopheles vector lays eggs on water surfaces. The eggs hatch into larvae, develop to pupae and emerge as adults. Eggs, larvae and pupae are found in water. Adult mosquitoes are terrestrial. (Photo source: (Williams and Pinto, 2012). University of Ghana http://ugspace.ug.edu.gh 18 Figure 4:Adult male and female Anopheles mosquitoes. Distinctive features of adult male and female Anopheles mosquitoes. Adult male mosquitoes have feathery antennae that help them sense their potential mates' wingbeats. However, female mosquitoes have uniquely plain antennae with mouthparts adapted for blood feeding. Their proboscis is long, slender relatively smooth compared to the males (Photo source: (Penn Vet | Nikon SMZ 1000) accessed at https://www.vet.upenn.edu/research/core-resources-facilities/imaging-core/instruments- applications/nikon-smz-1000) 2.5 Control of malaria 2.5.1 Diagnosis of malaria The early and accurate diagnosis of malaria is a committed step in the efforts deployed to control the disease. The clinical or presumptive diagnosis of malaria makes use of the signs and symptoms of the disease. However, the accuracy and specificity of this method have been extremely low. This means that exclusive dependence on clinical features or symptoms for malaria diagnosis results in abuse of anti-malarial drugs, under treatment and/or overtreatment (Pillay et al., 2019). Because of this, the World Health Organization (WHO) recommends that parasitological tests should be carried out for all suspected malaria cases using rapid diagnostic University of Ghana http://ugspace.ug.edu.gh https://www.vet.upenn.edu/research/core-resources-facilities/imaging-core/instruments-applications/nikon-smz-1000 https://www.vet.upenn.edu/research/core-resources-facilities/imaging-core/instruments-applications/nikon-smz-1000 19 tools (RDT) and/or microscopy before commencing treatment (World Health Organization, 2019). 2.5.1.1 Microscopy Microscopy is the gold standard for the detection of the malaria parasite in peripheral blood smears (Kilian et al., 2000; Njama-Meya, Kamya and Dorsey, 2004; Sousa-Figueiredo et al., 2012). However, this technique is time-consuming and requires technical expertise. The specificity and sensitivity of microscopy are also subjective (Pillay et al., 2019). The detection of malaria parasites at a very low parasitaemia using microscopy is often underestimated and even gets complicated in cases of mixed infections (Kilian et al., 2000; McKenzie et al., 2003; Singh et al., 2004). Despite these challenges, microscopy is inexpensive and allows for the identification and quantification of Plasmodium species where the expertise is available (Kilian et al., 2000). Molecular diagnostic methods including PCR and isothermal assays, and rapid diagnostic test kits (RDTs) (Lucchi et al., 2013) are highly sensitive and specific to parasite detection techniques compared to microscopy (Okell et al., 2009; Berzosa et al., 2018; Pillay et al., 2019). 2.5.1.2 Rapid Diagnostic Tests (RDTS) RDTs are immunochromatographic tests that detect specific parasite antigens such as histidine- rich protein 2 (HRP2), Plasmodium lactate dehydrogenase (pLDH) or aldolase, which are often produced during the erythrocytic cycle (Ugah et al., 2017). This diagnostic technique gives reliable results (Bharti et al., 2008) but the sensitivity wanes with parasitaemia below 300– 500 parasites/μL (Kim et al., 2008). Moreover, some parasites have undergone mutations by deleting their histidine-rich protein 2 and 3 (PfHRP2 and PfHRP3) making them evade University of Ghana http://ugspace.ug.edu.gh 20 detection by the RDTs (Mariette et al., 2008; Koita et al., 2012; Wurtz et al., 2013; Amoah, Abankwa and Oppong, 2016). 2.5.1.3 Polymerase chain reaction (PCR) Polymerase chain reaction detects parasite DNA and can identify infections below the detection limits of microscopy and RDTs (Rogawski et al., 2012). The gene targets used in PCR include the 18S ribosomal RNA gene and cytochrome b gene which give reliable results (Steenkeste et al., 2009). However, conventional PCR is not applicable as point-of-care diagnostic tools in many malaria-endemic areas because they are expensive, and require a high level of skills (Steenkeste et al., 2009; Rogawski et al., 2012). The loop-mediated isothermal amplification (LAMP) is also a type of PCR which gives accurate, specific, and sensitive results (Cuadros et al., 2017; Piera et al., 2017; Vásquez et al., 2018). LAMP is a simple molecular diagnostic tool hinged on the principle of isothermal amplification, which requires little or no special equipment or laboratories and provides results in about 1 hour (Hopkins et al., 2013). LAMP requires a small amount of blood sample and it is also tolerant to inhibitory substances present in blood samples (such as haemoglobin and immunoglobulin) making it convenient for use in low- resourced settings (Port et al., 2014; Yongkiettrakul et al., 2014). 2.5.1.4 Future of malaria diagnostics There are attempts to automate the detection methods to allow for a quicker, simpler and cost- effective diagnosis of the disease (Laroche et al., 2017; Poostchi et al., 2018). The presence of an automated system will provide adequate information on infected red blood cell count of patients to enable the monitoring of parasite post-treatment and provide an early marker of drug resistance. It will also provide an opportunity to identify asymptomatic cases, and potentially University of Ghana http://ugspace.ug.edu.gh 21 preventing the spread of blood transfusion-related malaria (Purwar et al., 2011; Abbas et al., 2018; Pillay et al., 2019). 2.5.2 Treatment of malaria 2.5.2.1 Chemotherapy Malaria treatment is a core component of the interventions championed by the WHO to facilitate the control and eradication of malaria. The major objective of malaria treatment is to alleviate the symptoms, prevent relapses and block the spread or transmission of the disease. Therefore, antimalarial drugs target asexual erythrocytic stages, liver or tissue forms and gametocytes (Fig. 2) with much attention given to the drugs that target the blood-stage forms (Delves et al., 2012; Bosson-Vanga et al., 2018). Generally, the kind of treatment regimen deployed depends on the degree of severity of the disease. For example, case management for uncomplicated and severe malaria involves the use of ACTs to facilitate parasite clearance. For fatal cases of uncomplicated malaria, patients are immediately placed on fluids for the resuscitation of patient and preventing their possible death as a result of dehydration and followed with supportive care (Maitland and Marsh, 2004; Hodgson and Angus, 2016). In Southeast Asia, multidrug-resistant P. falciparum is treated with combinations of artemisinin derivatives and mefloquine or atovaquone plus proguanil (Kain, 1996). However, Chloroquine is used to mainly treat infections with P. vivax, and in some cases P. malariae and P. ovale (Kain, 1996; Olliaro and Mussano, 2009). Currently, WHO strongly recommends the use of ACTs for the treatment of uncomplicated P. falciparum malaria in children and adults (except pregnant women in their first trimester) (White, 2004; Dondorp et al., 2009; World Health Organization, 2022). In P. falciparum endemic areas, pregnant women (in their first trimester) are put on quinine and clindamycin (or Sulfadoxine-Pyrimethamine (SP) in areas where SP is still effective) University of Ghana http://ugspace.ug.edu.gh 22 medications during antenatal visits to prevent from malaria-related maternal anaemia, deaths and low-birth weight infants (Shulman, 1999; Nzila, Okombo and Molloy, 2014; World Health Organization, 2022). This therapy, generally called intermittent preventive treatment (IPT), is also applicable to infants who form the majority of the at-risk individuals (Schellenberg et al., 2001; Greenwood, 2006). IPT schemes have been extensively studied in infants (IPTi), children younger than 5 years (IPTc) and pregnant women (IPTp) and have been demonstrated to be protective against malaria and its related adverse outcomes (Schultz et al., 1994; Shulman, 1999; Greenwood, 2006; McGready, 2009; Bojang et al., 2010; Konaté et al., 2011; Matangila et al., 2015). It has also been demonstrated that Artemisinin combination therapies (ACTs) (SP, DP, SP + AS, AQ + AS) when used in IPT, had acceptable protective efficacy (PE) against clinical malaria (Matangila et al., 2015; Al Khaja and Sequeira, 2021). In early and late pregnancy, intravenous artesunate is the drug of choice for treating complicated malaria during early and late pregnancy, respectively (Al Khaja and Sequeira, 2021). People traveling to and returning from malaria-endemic areas are advised to take chemoprophylactics (Wickremasinghe et al., 2017) and this approach is targeted at reducing cases of imported malaria. Chemoprophylaxis treatment regimens administer drugs such as chloroquine, doxycycline and atovaquone-proquanil. 2.5.2.2 Vector control Mosquito vector control, a means of disrupting malaria transmission, is the second pillar in the global efforts by the WHO to achieve total elimination and eradication of malaria. The World Health Organization recommends integrated vector management (IVM) techniques to control the vector through chemical, biological and genetic strategies. For these interventions to be effective, they must be tailored to meet the local contexts of the countries in which they are being implemented. Adult vector-control interventions involve the deployment of synthetic University of Ghana http://ugspace.ug.edu.gh 23 products to kill the adult mosquitoes by direct contact sprays or indirectly as preventive methods in indoor residual spraying (IRS) and long-lasting insecticide-treated bed nets (LLINs) Kokwaro (2009). In larval control, the breeding sites of the mosquito are targeted and can include clearing of aquatic weeds and filling of man-made and permanent habitats of the mosquitoes. Larvicides are also applied to water bodies to kill the immature stages of the mosquitoes (larvae or pupae) and they could act as mosquito stomach toxins, contact larvicides, surface agents, biological larvicides and insect growth regulators (Lacey, 2007). Other biological interventions make use of enthomopathogenic fungi, sterile insect technique (SIT), and engineered mosquitoes and new larval control tools currently being evaluated include lethal ovitraps and acoustic larvicide systems (Kokwaro, 2009; Abdul-Ghani, Al-Mekhlafi and Alabsi, 2012). Transgenesis and paratransgenesis are promising genetic vector-control strategies that hold promise for the effective control of malaria. Transgenesis involves genetic techniques that modify mosquito vectors to release anti-parasite effector molecules, making them refractory to parasites (Kokwaro, 2009; Abdul-Ghani, Al-Mekhlafi and Alabsi, 2012). Pararatransgenesis, on the other hand, involves the modification of mosquito microbiome to confer parasite resistance to the vector (Wang and Jacobs-Lorena, 2013). The prospect of paratransgenesis is hinged on the fact that the enteric microbes of mosquitoes are symbiotic and obtained mainly from the environment (Romoli and Gendrin, 2018). These approaches have been successful in the laboratory (Wang and Jacobs-Lorena, 2017) and yet to be rolled out on a global scale because of the varied and dynamic environmental conditions in the wild. University of Ghana http://ugspace.ug.edu.gh 24 2.6 Challenges with current control strategies 2.6.1 Antimalarial drug resistance The WHO and its partners intend to reduce malaria case incidence and malaria mortality by 90% before 2030 (WHO, 2015). Therefore, the reduced sensitivity of Plasmodium parasites to antimalarial drugs is a phenomenon that is frustrating global efforts to achieve this. Antimalarial drug resistance occurs when there is widespread exposure of parasites to antimalarial drugs often via poor compliance or inadequate treatment (Payne, 1988; White, 2004; Kokwaro, 2009; Maude et al., 2009). Monotherapy has also contributed to the increase in antimalarial drug resistance thereby justifying the need for combination therapy (Rodrigues Coura, 1987; Dondorp et al., 2009; Da Silva and Benchimol, 2014). Antimalarial drug resistance is confirmed via standard testing methods such as in vitro studies of resistance, detection of molecular markers of resistance and therapeutic drug efficacy studies; the latter remains the gold standard for determining drug resistance, decreased drug efficacy and for guiding drug policy (Talisuna, Bloland and D’Alessandro, 2004; Achan et al., 2011; WHO, 2017). Resistance to Chloroquine (CQ) was reported a few years after it was rolled-out in early 1950 which led to the resurgence of malaria in the 1980s (Antony and Parija, 2016; Blasco, Leroy and Fidock, 2017; Wotodjo et al., 2018). It is believed that P. falciparum resistance to chloroquine is due to mutations at position 76 of the Plasmodium falciparum chloroquine resistant transporter (PfCRT) gene. Plasmodium sp. with this point mutation pump out chloroquine at much higher rates than the wildtype (Chen et al., 2003; Valderramos and Fidock, 2006; Sá et al., 2009). Another point mutation in a related gene, Plasmodium falciparum multi- drug resistant transporter gene, pfMDR1, has also been implicated in chloroquine resistance (Foley and Tilley, 1997; Djimdé et al., 2001; Dorsey et al., 2001; White, 2004). Later, University of Ghana http://ugspace.ug.edu.gh 25 Sulphadoxine-Pyrimethamine (SP) replaced CQ for the treatment of CQ resistant parasites (Roper et al., 2004; Abiodun et al., 2011). Plasmodium parasites that are resistant to antifolates have point mutations in the Dihydro folate reductase (DHFR) and Dihydropterate synthase (DHPS) genes which reduce the affinity of the DHFR and DHPS enzyme complex to antifolates (Imwong et al., 2003; Amaratunga et al., 2016). More recently, evidence was reported of artemisinin-resistant P. falciparum in the Greater Mekong Subregion and Africa (Dondorp et al., 2009; Ashley et al., 2014; Leang et al., 2015; Antony and Parija, 2016; Phyo et al., 2016; Lu et al., 2017; Thanh et al., 2017; Bopp et al., 2018; Balikagala et al., 2021). Resistance to artemisinin and its derivatives have been proposed to be due to mutation in the kelch 13 gene (Bonnington et al., 2017; Zaw, Lin and Emran, 2019; Siddiqui et al., 2020; Uwimana et al., 2020, 2021; Asua et al., 2021; Balikagala et al., 2021). Currently, there is the need for improved surveillance for drug resistance across Africa, and the use of different ACTs or triple ACTs to halt the emergence and/or spread of resistance to both artemisinin and key partner drugs (van der Pluijm et al., 2020). This further supports the call for other control interventions to hasten parasite clearance and reduce the disease burden. 2.6.2 Vaccines Vaccines have been very effective and efficient in the control and prevention of diseases like smallpox, poliomyelitis and measles (Rieckmann et al., 2017). It is in this regard that several attempts have been made at developing potent vaccines for the control of malaria. Several vaccines targeting the different stages of the life cycle of the Plasmodium parasite are at various stages of clinical trials (Delves et al., 2012). It has been extremely difficult obtaining an effective anti-malaria vaccine for global use because the biology of the malaria parasite has not been completely understood (Gardner et al., 2002). There are a lot of parasite protein-coding University of Ghana http://ugspace.ug.edu.gh 26 genes that have not been functionally annotated although the entire genome of the malaria parasite has been completely sequenced and this impedes the discovery of effective vaccine targets (Gardner et al., 2002). The very few targets that have been found in the parasites are either polymorphic, redundant or do not elicit strong immune responses (Douglas et al., 2019). For example, the WHO-recommended pilot implementation of the anti-malaria vaccine RTS,S/AS01 in some parts of Africa showed the vaccine has low prevention and efficacy indices (Moorthy and Okwo-Bele, 2015). It has also been reported that the efficacy of the RTS,S/AS01 vaccine declines over time (RTS, 2014; Olotu et al., 2016). The host immune factors that offer protection against malaria is still under study (Taylor, Parobek and Fairhurst, 2012). However, the RTS, S/AS01 malaria vaccine research has made a lot of progress and is currently the vaccine of choice for children living in P. falciparum endemic (moderate to high transmission) areas (World Health Organization, 2022). It is highly likely that another vaccine with greater protective efficacy than the recommended RTS, S/AS01 may be approved (Datoo et al., 2021). Several attempts have been made to use the knowledge of the different stages of the Plasmodium parasite’s life cycle to produce blood (erythrocytic) stage vaccines, pre- erythrocytic stage vaccines and transmission blocking vaccines (TBV) (Arama and Troye- Blomberg, 2014). Pre-erythrocytic vaccine strategy targets the liver stages of the malaria parasite and aims at preventing malaria infection to the host. Scientists have attempted the use of attenuated whole sporozoites vaccines for malaria control (WSV) (Nussenzweig et al., 1967; Clyde et al., 1973; Rieckmann et al., 1974; Draper et al., 2018). Despite the high level of protection recorded among volunteers treated with the attenuated form of the whole sporozoites, it was impossible to roll out this intervention on a larger scale (Draper et al., 2018). Currently, whole sporozoite vaccines are being produced in large proportions. However, the University of Ghana http://ugspace.ug.edu.gh 27 efficacy of these vaccines in recent studies in malaria endemic areas is low compared to has previous reports (Mwakingwe-Omari et al., 2021). The circumsporozoite surface protein (CSP) antigen on the surface of the sporozoite was targeted as a replacement for the attenuated form of the whole sporozoite (Draper et al., 2018). A high level of protection was seen in mice immunized with CSP and this resulted in the development of sub-unit malaria vaccine, RTS, S (Stoute et al., 1997; Birkett, 2010; Crompton, Pierce and Miller, 2010). However, subunit vaccines have recorded low efficacy, and this has revived the interest in whole sporozoite vaccine (WSV) strategy. The blood stage vaccine approach aims to decrease the number of parasites in the blood in order to reduce the severity of the disease. This is premised on the fact that people who have constant exposure to malaria develop natural immunity to the disease over time (Marsh and Kinyanjui, 2006). It has been observed that vaccine containing proteins from the merozoite surface is feasible and can prevent malaria (Cohen, McGregor and Carrington, 1961; Sabchareon et al., 1991). The transmission blocking vaccine (TBV) strategies target the sexual stage of the malaria parasite and aims to prevent mosquitoes carrying malaria parasites from spreading them. TBV exploits antibodies to antigens such as Pfs25, Pfs48/45, Pfs28 and Pfs230 (Kaushal et al., 1983; Nikolaeva, Draper and Biswas, 2015) to block the sexual stages and prevent transmission of malaria (Huff, Marchbank and Shiroishi, 1958; Carter and Chen, 1976). Recently, an evidence of a multistage malaria vaccine has been successfully produced to a chimeric recombinant antigen composed of fragments of Pfs48/45, and the blood stage antigen glutamine rich protein (GLURP)(Theisen et al., 2014). Another study has produced human monoclonal antibodies directed against a portion of the CSP and tested it in healthy human participants (Gaudinski et al., 2021) This progress in relation to the expression of University of Ghana http://ugspace.ug.edu.gh 28 immunogenic transmission blocking antigens presents a lot of promise towards the possibility of the development of a transmission blocking malaria vaccine. 2.6.3 Insecticide resistance The fight to eliminate and eradicate malaria in most parts of the world especially, sub-Saharan Africa requires a combination of efforts chemotherapy (active antimalarial drugs), and effective vector-control methods. The use of chemical-based approaches for vector control is challenged by reduced efficacy of the currently recommended chemicals due to vector resistance to these synthetic compounds (Liu, 2015) which has occurred through constant exposure to agrochemicals (Nkya et al., 2013, 2014) and modification of key detoxification enzymes (Chandor-Proust et al., 2013). The periodic emergence of anti-malarial drug resistance, lack of effective anti-malarial vaccines and failing vector-control methods continues to militate against the successful elimination and eradication of malaria (World Health Organization, 2019). This calls for the need for a paradigm shift to identify novel approaches for the control of malaria. 2.7 The Anopheles mosquito microbiota and parasite transmission 2.7.1 The Anopheles mosquito microbiota In mosquitoes, symbiotic micro-organisms are found inhabiting organs such as midguts, salivary glands, and gonads (Pidiyar et al., 2004; Rani et al., 2009; Gusmão et al., 2010; Noden et al., 2011; Oliveira et al., 2011; Zouache et al., 2011; Strand, 2018). The diverse communities of microbes (bacteria, fungi and viruses) depends on ecological factors , diet and stage of insect development (Wang et al., 2011; Akorli et al., 2016; Benjamino et al., 2018; Jiménez-Cortés et al., 2018). Bacteria are the predominant community of the midgut microbial flora (Strand, University of Ghana http://ugspace.ug.edu.gh 29 2018) and thus, the most commonly studied (Guégan et al., 2018). Larvae have more diverse microbial communities than adults , as they feed directly on the micro-organisms in their aquatic environment (Wang et al., 2011; Gimonneau et al., 2014). Although, some bacteria are maintained through development from larvae to adults many are lost during metamorphosis (Lindh, Borg-Karlson and Faye, 2008). In An. gambiae, the number of operational taxonomic units (OTU) of bacteria are 3 times more in larval and pupal stages compared to the adult stages. Enterobacteriaceae and Flavobacteriaceae were present in all stages but the midgut of adult mosquitoes was mainly made up of Proteobacteria and Bacteriodetes (Moncayo et al., 2005). A profile of the microbiome of An. gambiae larvae and pupae revealed a 40% composition with cyanobacteria (Wang et al., 2011). The structural make-up of mosquitoes usually changes during metamorphosis. One key anatomical change that comes with moulting is the appearance of meconial peritrophic membrane (MPM1) at the pupal and adult emergence stages (Moncayo et al., 2005). MPM1 is believed to sterilize the midgut of adult mosquitoes (Moncayo et al., 2005). The microbes ingested by the mosquito larvae are sequestered by the MPM1 and eventually eliminated after the membrane is shed off when adult mosquitoes emerge (Moncayo et al., 2005). This could account for the different proportions of bacterial communities between the early developmental forms and the adult stages. The common bacteria genera encountered in the different mosquito species include Enterobacter, Serratia, Cedacea, Escherichia, Klebsiella, Pseudomonas, and Staphylococcus (detailed review in (Gendrin and Christophides, 2013). Bacteria found in mosquitoes influence various physiological functions including, nutrition, reproduction metabolism, and immunity (Jupatanakul, Sim and Dimopoulos, 2014). Microbes resident in mosquito habitats may influence the host-seeking behaviours of these disease vectors (Day, 2005; Verhulst et al., 2009, 2010, 2011). For instance, the level of attractiveness of humans to mosquitoes is dependent on the composition of skin microbiota of the host and University of Ghana http://ugspace.ug.edu.gh 30 the chemicals they secrete, and volatile compounds like lactic acid produced by Corynebacterium minutissimum attract An. gambiae (Verhulst et al., 2010). 2.7.2 Role of mosquito microbiota in larval development Bacterial microbiota plays an important role in mosquito larval development (Lindh, Terenius and Faye, 2005). Bacteria are a major food source for mosquito larvae (Merritt, Dadd and Walker, 1992). This was demonstrated when larvae of Ae aegypti survived for several weeks on fish meal whereas growth was halted in larvae that were reared in antibiotics-treated water (Merritt, Dadd and Walker, 1992). The growth of Cx. quinquefasiatus larvae is stimulated in the presence of Pseudomonas aeruginosa cultured in a phosphorus-rich medium. However, the development of the larvae of Cx. tarsalis was hindered in the same medium (Peck and Walton, 2006). The larvae-to-pupae transition of An. gambiae were significantly hindered after gentamycin and penicillin-streptomycin were introduced in the larval rearing water (Touré et al., 2000). The introduction of Gentamycin into An. gambiae and An. quadriannulatus larval rearing water also tremendously affected the size of the larvae. Larvae in Gentamycin-treated larval rearing water were smaller in size compared to those reared in the absence of Gentamycin (Wotton et al., 1997). 2.7.3 Role of mosquito microbiota on the nutrition of Anopheles mosquitoes The composition of bacteria microbiota in adult mosquitoes is significantly affected by diet (Foster, 1995). Plant sap and nectar (sugar sources) are the main sources of food for adult mosquitoes. Nectar is primarily composed of carbohydrates and some traces of free amino acids (González-Teuber and Heil, 2009). Carbohydrates are used by mosquitoes to generate University of Ghana http://ugspace.ug.edu.gh 31 energy for flight (Sacktor and Wormser-Shavit, 1966) and free amino acids (proline) are required to fuel flight (Scaraffia and Wells, 2003). Sugar meals are abundant in carbohydrates with little protein. Hydrolytic enzymes are secreted into the mosquito midgut to help digest the ingested sugar. This leads to selective pressure for bacteria resident in the mosquito midgut. Female mosquitoes require blood meal for the nourishment of eggs (Foster, 1995). Bacteria resident in female mosquitoes aid the digestion of blood (Gusmão et al., 2010; Gaio et al., 2011). Immune and oxidative stress responses are down-regulated due to a temperature burst that ensues post blood-feeding which triggers increased bacteria proliferation (Oliveira et al., 2011) and influences bacteria diversity in the midgut (Wang et al., 2011). Blood meal reduces the diversity of bacteria in the mosquito midgut. Bacteria proliferation is skewed in favour of Enterobacteriaceace following a blood meal (Wang et al., 2011). This is because Enterobacteriaceae endure nitrosative and oxidative stresses that come with blood digestion. Thus, Enterobacteriaceae help maintain redox homeostasis (Wang et al., 2011). The midgut microbiome also enhances digestion via inhibition of enzymes necessary for nutrient absorption (Minard, Mavingui and Moro, 2013). Enterobacter and Serratia secrete hydrolytic enzymes which facilitate the digestion of blood in mosquitoes (Minard, Mavingui and Moro, 2013). Aseptic mosquitoes have reduced degradation of host red blood cells and low proteins (Gaio et al., 2011). In Ae. albopictus, Acinetobacter johnsonii and Acinetobacter baumanii have been linked with both nectar assimilation and blood digestion (Minard, Mavingui and Moro, 2013). Asaia bogorensis from An. stephensi was found to replenish the invertebrate host with vitamin B proving that gut microbiota of blood sucking dipterans could supply their host with essential nutrients (Douglas and Smith, 1989; Damiani et al., 2010). University of Ghana http://ugspace.ug.edu.gh 32 2.7.4 Role of mosquito microbiota in vector competence Vector competence is the ability of a vector to carry and spread pathogens (Lambrechts and Scott, 2009). Therefore, interventions that militate against pathogen transmission help reduce vector competence. The immune system of mosquitoes is a major player of vector competence. Mosquito midgut microbiota significantly influences the immunity of insect vectors (Gonzalez- Ceron et al., 2003). Mosquito midgut microbiota may secrete chemicals that have a direct or indirect impact on disease-causing agents (Habib et al., 2001; Chaudhary et al., 2013). Some gut resident bacteria like Enterobacter and Serratia have a direct impact on mosquito vector competence by inhibiting the maturation of Plasmodium and other pathogens such as fungi and viruses (Gonzalez-Ceron et al., 2003; Cirimotich et al., 2010; Cirimotich, Ramirez and Dimopoulos, 2011; Weiss et al., 2019). Enterobacter (Esp_Z) isolated from wild Anopheles populations in Zambia has been found to suppress Plasmodium parasite from developing before they are able to invade the midgut epithelium by exerting oxidative pressure through the secretion of reactive oxygen species (ROS) (Kumar et al., 2003; Molina-Cruz et al., 2008; Cirimotich et al., 2010; Dennison et al., 2016; Coon et al., 2017). An increase in the number of bacteria particularly after blood meal boosts the innate immune responses of the mosquito to control the bacteria load (Cirimotich et al., 2010). Generally, mosquitoes lack adaptive immunity (Garver et al., 2012). 2.8 Paradigm shift to bacteria-mediated malaria control strategies Several studies are currently being carried out to aid our understanding of the contribution of mosquito midgut microbiome to the disruption of pathogen transmission. In insects, the impact of Wolbachia on vector competence has been studied extensively over the years. Wolbachia are maternally acquired, obligate endosymbionts of many arthropods (Kittayapong et al., 2000; University of Ghana http://ugspace.ug.edu.gh 33 Moreira et al., 2009; Iturbe‐Ormaetxe, Walker and O’ Neill, 2011; Walker and Moreira, 2011; Herren et al., 2013). Wolbachia can influence insect reproduction via parthenogenesis, feminization and cytoplasmic incompatibility (CI) which enhance reproduction in infected insects. Cytoplasmic incompatibility ensures that the progeny requires Wolbachia (Dobson, Fox and Jiggins, 2002). Wolbachia-mediated cytoplasmic incompatibility resulted in the development of efficient vector control strategies such as incompatible insect technique (IIT) (Werren, Baldo and Clark, 2008; Atyame et al., 2011). Wolbachia also inhibits the multiplication and transmission of pathogens in various mosquito species. The pathogen intensity and lifespan of Ae. aegypti and Anopheles were greatly reduced in mosquitoes that did not have naturally resident Wolbachia species (Walker and Moreira, 2011). Wolbachia can also stimulate oxidative stress that suppresses the multiplication of dengue virus in Ae. aegypti (Pan et al., 2012). A major bottleneck of the Plasmodium life cycle, gametocyte-to ookinete-to oocyst transition (Fig 5), occurs 24-26 hours after blood-meal (Siciliano et al., 2020). Midgut bacteria species such as Asaia produce anti-Plasmodium effector proteins that confer refractoriness to mosquitoes (Damiani et al., 2010). Trans-infection of mosquitoes with bacteria such as Pantoea agglomerans also effectively inhibited the maturation of Plasmodium falciparum and P. berghei by 98% (Wang et al., 2012). The technique used P. agglomerans as a vehicle to convey antimalarial gene products to the lumen of the midgut of the mosquito vectors. (Wang et al., 2012). An Enterobacter species (Esp_Z) found in field-caught mosquitoes from Zambia fully or partly inhibited the establishment of ookinete, oocyst and sporozoite (Cirimotich, Ramirez and Dimopoulos, 2011). The Enterobacter sp. suppressed the development of the parasite before invading the midgut lumen of the mosquitoes. Co-infection of mosquitoes with Esp_Z and Plasmodium gametocytes resulted in reduced infection levels, but aseptic mosquitoes were less University of Ghana http://ugspace.ug.edu.gh 34 resistant. Similarly, the co-infection of An. albimanus with Serratia marcescens and P. vivax resulted in 1% oocysts infection, compared to 71% infection observed in aseptic mosquitoes (Gonzalez-Ceron et al., 2003). The significant association seen between the high level of Enterobacteriaceae and infection with Plasmodium parasites suggests that bacteria-parasite associations can be explored to identify novel strategies to combat malaria and other mosquito- borne diseases (Boissière et al., 2012). Figure 5: Plasmodium falciparum developmental changes as they travel through the mosquito gut. Plasmodium gametocytes develop into ookinetes that traverse the midgut epithelium to transform into oocyst and eventually sporozoites. (Source: Smith et al. 2014). 2.8.1 Paratransgenesis Synthetic insecticides are often used for the control of mosquitoes. The resistance of mosquitoes to insecticides, accompanied by environmental pollution and the potential harm to non-target organisms have triggered concerns about the continuous use of chemical based- University of Ghana http://ugspace.ug.edu.gh 35 approaches for mosquito control (David et al., 2010). Bacteria-mediated mosquito approaches have become a promising intervention (Lindh, 2007). Paratransgenesis is a technique that employs symbiotic organisms to transfer anti-pathogenic gene molecules to vector populations thereby, reducing the ability of the vectors to transmit pathogens (Beard, Cordon-Rosales and Durvasula, 2002). Successful paratransgenic interventions require that the bacteria be closely associated with the vector and the pathogen, microbiota must be cultivable and must be genetically transformable, the modified bacteria must be fit, modified bacteria must have an appropriate technique for reintroduction, and the modified bacteria species must be easy to disseminate in the vector population (Durvasula et al., 1997; Beard, Cordon-Rosales and Durvasula, 2002; Riehle and Jacobs-Lorena, 2005). Paratransgenesis has been beneficial in the control of American trypanosomiasis. The endosymbiont of Rhodnius prolixus (Tsetse vector) was modified to express a toxic peptide, cecropin A, which significantly inhibited Trypanaosma cruzi (pathogen) development (Durvasula et al., 1997). Asaia and Pantoea have also been successfully exploited in paratransgenic interventions (Dinparast Djadid et al., 2011). Escherichia coli was used as a vehicle to express two-anti-Plasmodium gene products (SM) and (PLA2) which suppressed the maturation of P. berghei confirming that paratransgenesis can be explored to curb malaria (Riehle et al., 2007). 2.8.1.1 Challenges with paratransgenic interventions The route for the re-introduction of the modified bacteria into wild mosquitoes is a major problem in paratransgenesis. Habitats of mosquito larvae are often used to transport transformed symbionts into adult mosquitoes but transtadial bacteria transfer is not efficient. Only about 0.7% of total bacteria is transferred from the fourth instar (larvae) to adult mosquitoes (Moll et al., 2001). The huge loss of larval microbiota in adult mosquitoes is due to the shedding of MPM1 and the intake of moulting fluid which contains antibiotics (Moll et al., 2001). Oviposition sites can also be treated with transformed endosymbionts (Riehle and University of Ghana http://ugspace.ug.edu.gh 36 Jacobs-Lorena, 2005). However, creating oviposition sites in and around houses may lead to an increase in the number of mosquitoes in domestic areas. The artificial egg-laying sites may not be used by adult female mosquitoes especially during the rainy seasons due to the multiplicity of oviposition sites (Riehle and Jacobs-Lorena, 2005; Riehle et al., 2007). Adult mosquitoes feed on nectar (sugar sources) after they emerge from the pupae (Impoinvil et al., 2004). Treatment of sugar sources with transformed endosymbionts may serve as a way to re- introduce bacteria into mosquitoes. Attractants can be employed to enhance the efficacy of the bait (Foster and Takken, 2004). 2.8.2 Transgenesis Transgenesis describes interventions that kill adult mosquitoes by transforming the mosquito vectors. It involves the use of mosquito lethal molecules. Some microbes such as Clostridium bifermentans serovar Malaysia and Bacillus substilis subspecies substilis can secrete insecticidal compounds (Yiallouros et al., 1994). Bacillus thuringiensis serovar israelis produces toxins which are encoded by the mosquito lethal genes, Cry and Cyt (Gonzalez, Brown and Carlton, 1982). It has been demonstrated that the Cry toxins are harmful to large insects belonging to orders Coleoptera, Lepidoptera, Hymenoptera and Diptera. However, only dipteran insects are susceptible to Cyt toxins (Schnepf et al., 1998). Death of mosquito larvae occurs due to breakdown of the midgut membrane of the larvae after the toxins have been activated by high pH in the midgut of the larvae (Bravo, Gill and Soberón, 2007). Similarly, Mts and Bin toxins secreted by Lysinibacillus sphericus are also extremely lethal to mosquito larvae (Berry, 2012). These toxins offer a lot of hope in our quest to fight malaria. They have been successfully experimented in the wild to kill An. gambiae mosquitoes in The Gambia and Ghana (Majambere et al., 2007; Nartey et al., 2013). University of Ghana http://ugspace.ug.edu.gh 37 University of Ghana http://ugspace.ug.edu.gh 38 CHAPTER THREE 3.0 MATERIALS AND METHODS 3.1 Reviving of archived bacteria isolates Preserved Enterobacter cloacae and Serratia marcescens previously isolated from the midgut of wild female Anopheles gambiae mosquitoes and characterized by MALDI-TOF and sequencing of bacterial 16S small subunit ribosomal ribonucleic acid (SSU rRNA) (Ezemuoka et al., 2020) were used for this study. Bacterial isolates were removed from cold storage and thawed to room temperature on a BIO LAB NS-8A clean bench (Bio Laboratories, France). The thawed bacteria samples (100µL) were spread on fresh LB agar plates and incubated overnight at 37oC to resuscitate the dormant bacteria cells. Subsequently, a colony was streaked on an LB agar plate and re-streaking was repeated to obtain pure/single bacteria colonies. Single colonies of Enterobacter cloacae and Serratia marcescens were aseptically transferred into 50mL sterile LB broth and incubated overnight at 37oC with shaking at 120rpm to obtain the stock bacterial cultures. 3.2 Growth curve estimation of Enterobacter cloacae and Serratia marcescens One millilitre (1mL) of Enterobacter cloacae (OD600= 0.64) and Serratia marcescens stock cultures (OD600= 0.59) were each transferred into 49mL sterile LB broth under aseptic conditions and incubated at 37oC with shaking at 120 rpm to obtain overnight cultures. Approximately 1mL of bacteria overnight culture i.e Enterobacter: OD600 = 0.55 or Serratia: OD600 = 0.50, was aliquoted into each of 15 Falcon tubes containing 50mL sterile LB broth and incubated as previously described. After 30 minutes, the first tube was removed from the incubator, placed on ice and optical cell density (OD600) was measured in triplicate. 1mL portion of the content of the Falcon tube (that has been cultured for 30-minutes) was serially University of Ghana http://ugspace.ug.edu.gh 39 diluted (10-fold dilution) in 1X PBS to 10-10-10-18 and 100μL of each final dilution was spread on LB agar plates in triplicates. The plates were placed in a 37oC incubator for overnight culturing. The procedure described above was repeated at 30-minutes intervals until the remaining 14 tubes had been assayed. The counting of single bacteria colonies was done manually using a counting chamber. The counting was done thrice per plate and an average was taken. OD600 was plotted against time using Graphpad prism v8 to obtain the growth curve for each bacterium. The number of colonies was also plotted against OD600 to estimate the concentration of bacteria cells. 3.3 Preparation of bacteria culture cell-free spent medium Five hundred microliters (500μL) each of Serratia marcescens (OD600= 0.68) and Enterobacter cloacae (OD600= 0.84) were grown in 50mL sterile LB broth for 0.5, 2.5, 4, 8, and 12 hours at 37oC with shaking at 120 rpm and optical cell density measured in triplicate. Cultures at each time point (OD600 for Serratia and Enterobacter were adjusted so that the starting concentration were similar at each time-point) were centrifuged at 14,000 rpm to separate the bacteria cells from the spent medium. The spent medium was further filtered using a 0.22-micron filter (Sigma Aldrich, USA) to remove excess cells. The optical cell density of the filtered spent medium was measured to confirm the exclusion of cells. About 500mL of each bacteria filtrate/extract (cell-free spent medium at different incubated time points) was freeze-dried and stored at 4oC until used to treat Plasmodium falciparum. University of Ghana http://ugspace.ug.edu.gh 40 3.4 Malaria parasite culturing 3.4.1 Thawing of laboratory parasite isolates Cryopreserved parasites (3D7 and Dd2) were retrieved, thawed at 37ºC for 2 minutes and transferred into 15mL tubes. The parasites were pelleted by centrifuging for 10 minutes at 1500 rpm and the supernatant containing 4.2% sorbitol (Sigma Aldrich, USA) and 0.9% NaCl (Sigma-Aldrich, USA) was discarded. 1mL of complete parasite medium (CPM) was added, mixed well and centrifuged at 1500 rpm for 10 minutes. The supernatant was discarded and washing with CPM was repeated. CPM was again added to the pelleted parasites and mixed. The content was transferred into a T-25 culture flask (Thermo Fisher, USA) containing 5mL complete parasite medium and 200μL packed red blood cells. The culture flask was flushed gently with mixed gas for about 45 seconds, tightly closed and carefully placed in the incubator at 37oC. 3.4.2 Washing of human O+ erythrocytes (RBCs) Human O+ and sickle cell negative blood was collected into sterile vacutainer tubes containing citrate phosphate dextrose and stored at 2-8oC for 48 hours. Plasma was collected and the blood was transferred to a sterile 15mL tube using a sterile pipette and centrifuged at 2000 rpm for 10 minutes. The serum and buffy coat which contains white blood cells was removed and 10mL of parasite washing media (PWM) was added to the tube, well mixed and centrifuged for further 10 minutes at 2000 rpm. The supernatant was aspirated, and the washing process was repeated twice with parasite washing media and stored at 2-8oC. University of Ghana http://ugspace.ug.edu.gh 41 3.4.3 Culturing Plasmodium falciparum in vitro Plasmodium falciparum Chloroquine-sensitive strains (3D7) and Chloroquine resistant laboratory strains (Dd2) were cultured in RPMI 1640 (Sigma Aldrich, USA) at 4% haematocrit and synchronized with 5% sorbitol (Sigma Aldrich, USA) to obtain rings, according to the malaria parasite culture SOP (Department of Parasitology, NMIMR) adapted from Trager and Jensen (1976). The gametocyte-stage of the parasites was purified with Percoll (Sigma Aldrich, USA). The parasite cultures were monitored every 48 hours by preparing thin smears, fixing in 100% methanol, staining with 10% Giemsa and viewing under the Leica microscope at 100X. The parasite culture medium was also changed and gassed with malarial gas (2% O2, 5.5% CO2, and 92.5% N2). 3.5 Growth inhibition assay (GIA) About 0.5g of the freeze-dried spent media was dissolved in 1mL of distilled water and centrifuged at 14000 rpm for 30 minutes. The supernatant was filtered with 0.22μ filter into a 1.5mL tube to obtain the stock extract solution. Two-fold serial dilution of the stock extracts were prepared using RPMI 1640 (Sigma Aldrich, USA) to get the working concentration of the extract. Ten microliters (10μL) of each serial dilution were aliquoted in triplicates into a 96-well culture plate containing 90μL of parasite (Dd2 or 3D7) culture at 1% parasitaemia and 2% haematocrit. To account for parasite growth, the negative control wells contained 100μL of only uninfected RBCs in complete parasite medium at 2% haematocrit and positive control wells contained 100μL of parasite mixture with no extract. The extract control wells had 100μL lyophilized LB medium added to 90μL of parasite (Dd2 or 3D7) culture at 1% parasitaemia and 2% haematocrit. The culture plates were subsequently gassed and incubated at 37oC for 72 hours in a modular incubator chamber (Billups-Rothenberg, Inc.). After 72 hours of incubation, University of Ghana http://ugspace.ug.edu.gh 42 the culture plates were removed from the incubation chamber and 100μL of SYBR Green-1 fluorescent (MSF) lysis buffer was added to each well and mixed by pipetting up and down a few times. The plates were then wrapped with aluminium foil and incubated in the dark at room temperature for an hour. The SYBR Green fluorescence intensities were measured with a Varioskan LUX multimode microplate reader (Thermo Fisher Scientific, USA) at excitation and emission wavelengths of 485nm and 530nm, respectively. Negative control wells of the parasites were used to blank the data generated from microplate reader by subtracting the average values of the control wells from the average values from the other wells. A graph of percentage parasitaemia versus logarithmic concentration of the extract was generated and IC50 values extrapolated on Graphpad Prism version 8. 3.6 Expression of GAMER gene following co-culture of P. falciparum gametocytes and cell-free bio-products of E. cloacae and S. marcescens 3.6.1 Co-culture of P. falciparum parasites with cell-free bio-products of E. cloacae and S. marcescens To investigate the expression of GAMER gene following treatment of gametocytes with bacteria extracts, IC50 concentration of each extract was prepared and used to perform GIA on asynchronous parasite culture with gametocyte parasitaemia of 0.09%. Briefly, 10μL of each IC50 concentration of extracts was aliquoted into 96-well culture plates containing 90μL/well Dd2 and 3D7 cultures at 1% parasitaemia and 2% hematocrit. Negative control wells contained 100μL of only uninfected RBCs in complete parasite medium at 2% hematocrit to normalize the effect of the experiment on red blood cells) and positive control wells contained 100μL of RBCs and parasites (infected red blood cells) in complete parasite medium at 2% hematocrit with no extract or cell-free spent medium to cater for parasite growth in the absence of the University of Ghana http://ugspace.ug.edu.gh 43 effect of bacteria cell-free spent medium). The extract control wells contained 100μL lyophilized LB medium added to 90μL of parasite (Dd2 or 3D7) culture at 1% parasitaemia and 2% haematocrit (to normalize the effect of cell-free LB medium on parasite growth). One hundred microliters (100μL) of the content of each well were taken at 0, 1, 6, 8, and 12 hours after co-culture into 1.5mL microcentrifuge tubes containing 1mL RNA later (Fisher, USA). The samples were stored at -80oC until R