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Downloaded from the NCBI Nucleotide Database (Accession Numbers Are Shown in Supplementary Table 4.3.8 a and B) Analysis of Population Structure of Simulium damnosum sensu lato In the Ecological Transition Zone of Central Ghana Submitted by Ernest Tawiah Gyan BSc. Environmental Science, 2006 University of Cape Coast MPhil Zoology, 2013 University of Ghana A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy School of Life Sciences College of Health Sciences and Engineering La Trobe University Victoria, Australia October 2019 Table of Contents Analysis of Population Structure of Simulium damnosum sensu lato In the Ecological Transition Zone of Central Ghana ........................................................................................................................... Table of Contents ................................................................................................................................... I Abstract ............................................................................................................................................... VIII Statement of Authorship ..................................................................................................................... IX Dedication .............................................................................................................................................. X Acknowledgement ................................................................................................................................ XI List of Abbreviations ......................................................................................................................... XIII List of Figures, Tables and Supplementary Sheets .......................................................................... XV List of Figures .................................................................................................................................. XV List of figures of chapter 1 ......................................................................................................... XV List of figures of chapter 2 ......................................................................................................... XV List of figures of chapter 3 ........................................................................................................ XVI List of figures of chapter 4 ........................................................................................................ XVI List of figures of chapter 5 ....................................................................................................... XVII List of Supplementary Figures...................................................................................................... XIX Supplementary Figures A .......................................................................................................... XIX Supplementary Figures B .......................................................................................................... XIX Supplementary Figures C ........................................................................................................... XX List of Tables .................................................................................................................................. XXI List of tables of chapter 2 .......................................................................................................... XXI List of tables of chapter 3 .......................................................................................................... XXI List of tables of chapter 4 ......................................................................................................... XXII List of tables of chapter 5 ......................................................................................................... XXII List of Supplementary Table ....................................................................................................... XXIII List of Supplementary Sheets ...................................................................................................... XXV Chapter 1: General Introduction ......................................................................................................... 1 1.1 Onchocerciasis: a general background ..................................................................................... 1 1.2 Onchocerciasis: Life cycle of Onchocerca volvulus .................................................................. 3 1.3 Onchocerciasis: organization, control and elimination ........................................................... 5 1.3.1 Onchocerciasis Control Program in West Africa (OCP) ................................................. 5 1.3.2 African Program for Onchocerciasis Control (APOC) .................................................... 9 1.3.3 Expanded Special Project for Elimination of Neglected Tropical Diseases (ESPEN) . 11 1.3.4 Onchocerciasis Elimination Programme for the Americas ............................................ 12 1.3.5 Treatment ........................................................................................................................... 14 I 1.3.6 Ivermectin ........................................................................................................................... 15 1.3.7 Global geographical distribution of onchocerciasis ........................................................ 17 1.4 Life cycle and biology of Simulium blackflies ......................................................................... 19 1.5 Distribution of Simulium blackflies ......................................................................................... 22 1.6 Simulium vector complexes and Onchocerca-simulium complexes ....................................... 27 1.7 Blackfly diversity and evolution: a need to combine morphology, cytology and molecular genetics ............................................................................................................................................. 29 1.8 Transmission Zone .................................................................................................................... 36 1.9 Aims, scope and justification of the study ............................................................................... 38 1.9.1 Justification (Rationale) of the study................................................................................ 38 1.9.2 Justification (Rationale) of sampling area ....................................................................... 39 1.9.3 Scope of the study ............................................................................................................... 40 1.9.4 Aims of the study ................................................................................................................ 40 Chapter 2: Amplicon Sequencing ...................................................................................................... 41 2.1 Introduction ............................................................................................................................... 41 2.1.1 Genetic diversity and its utility in population structure determination........................ 41 2.1.2 Standard for estimating degree of genetic diversity ....................................................... 44 2.1.3 Purpose of study, specific objectives and mode of assessment ....................................... 45 2.1.4 Hypotheses .......................................................................................................................... 46 2.2 Materials and Methods ............................................................................................................. 48 2.2.1 Sample collection ................................................................................................................ 48 2.2.2 Sample processing .............................................................................................................. 49 2.2.3 DNA extraction with Qiagen’s DNeasy Blood & Tissue Kit .......................................... 49 2.2.4 DNA concentration determination with Qubit® Fluorometer ...................................... 49 2.2.5 Amplification of short amplicons by polymerase chain reaction (PCR) ....................... 50 2.2.6 Gel electrophoresis ............................................................................................................. 50 2.2.7 Molecular phylogenetics analysis ..................................................................................... 51 2.2.8 Meta-analysis ...................................................................................................................... 52 2.2.9 Ethical clearance ................................................................................................................ 54 2.3 Results ........................................................................................................................................ 55 2.3.1 Meta-analysis assessment of diversity and divergence in different species .................. 55 2.3.2 Sample and sequence characteristics ............................................................................... 67 2.3.3 Polymorphism and diversity assessment of the mitochondrial and nuclear amplicon sequences ...................................................................................................................................... 72 2.3.4 Polymorphism and divergence between populations using the mitochondrial
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