Mitochondrial Cytochrome Oxidase I Sequence Polymorphisms Reveal Population Genetic Diversity of Wuchereria Bancrofti in Papua New Guinea
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MITOCHONDRIAL CYTOCHROME OXIDASE I SEQUENCE POLYMORPHISMS REVEAL POPULATION GENETIC DIVERSITY OF WUCHERERIA BANCROFTI IN PAPUA NEW GUINEA By AKSHAYA RAMESH Submitted in partial fulfillment of the requirements For the degree of Master of Science Thesis Advisor: Dr. Peter A Zimmerman Department of Biology CASE WESTERN RESERVE UNIVERSITY August, 2012 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Akshaya Ramesh candidate for the MS_ degree *. (signed) Dr. Roy Ritzmann (Chair of the committee) Dr. Peter Zimmerman Dr. Daniel Tisch Dr. Michael Benard (date) 5/22/2012 *We also certify that written approval has been obtained for any proprietary material contained therein. ii Dedicated to my beloved Father and Grandmother iii Table of Contents Chapter 1: Lymphatic Filariasis: Global burden and Epidemiology 1.0. Introduction 1 1.1. Global burden 1 1.1.2. Lymphatic filariasis in Papua New Guinea 1 1.2. Global Alliance to eliminate lymphatic filariasis (GAELF/GPELF) 2 1.3. Life cycle of the filarial nematode: Wuchereria bancrofti 4 1.4. Vectors of W. bancrofti in Papua New Guinea 5 1.5. Genetics of W. bancrofti populations 6 1.6. Objectives 7 Chapter 2: The complete mitochondrial genome sequence of the filarial nematode, W. bancrofti 2.0. Introduction 8 2.1. Methods 9 2.1.1. Genomic DNA extraction and amplification 9 2.1.2. W. bancrofti mitochondrial genome amplification strategy 10 2.1.3. Agarose gel electrophoresis and gel extraction 12 2.1.4. Sequence assembly and gene annotation 13 2.2. Results and Discussion 13 2.2.1. General features of the mitochondrial genome of W. bancrofti 13 2.2.2. Protein coding genes 15 2.2.3. Codon Usage and amino acid composition 17 2.2.4. Ribosomal RNA genes 17 2.2.5. Transfer RNA genes 18 2.2.6. Non coding regions 19 2.3. Conclusions and future directions 20 iv Chapter 3: Genetic diversity of Wuchereria bancrofti in Dreikikir district, East Sepik province, Papua New Guinea 3.0. Introduction 21 3.1. Methods 24 3.1.1. Study sites and sample selection 24 3.1.2. Genomic DNA extraction and amplification 25 3.1.3. PCR amplification of the Cytochrome oxidase1 (cox1) gene and visualization of the products 25 3.1.4. Sequencing of the cox1 gene 26 3.1.5. Sequence analysis: Alignment of sequences and Sequence editing 27 3.1.6. Genetic heterogeneity of W. bancrofti 29 3.1.7. Test for neutrality 29 3.1.8. Population structure of W. bancrofti 29 3.2. Results 30 3.2.1. Genetic heterogeneity of W. bancrofti 31 3.2.2. Test for compliance to the neutral model of evolution 33 3.2.3. Genetic structure of W. bancrofti populations 33 3.2.4. Isolation by distance 45 3.3. Discussion 47 3.3.1. Genetic heterogeneity of W. bancrofti populations 47 3.3.2. Population structure 49 3.3.3. What mechanisms are responsible for structuring populations of W. bancrofti in PNG? 50 3.4. Conclusions and future directions 52 References 54 v List of Tables Table 1: Fifteen primer sets and COX1 primers used in the present study to amplify the complete mt genome and study the genetic heterogeneity of W. bancrofti. 11 Table 2: Summary statistics for 14 individuals from 6 villages with the observed number of haplotypes, haplotype diversity and test for neutrality 32 Table 3: Matrix with the pairwise Fst values for the six villages 34 Table 4: Fst values for the individuals from five villages 35 Table 5: AMOVA statistic generated using Arlequin 3.0 and DnaSP 5.0 across the villages (n=6) 36 vi List of Figures Figure 1: Linear representation of the complete mitochondrial genome of W. bancrofti from a Papua New Guinean isolate 14 Figure 2: Map of the study sites, Adapted from Bockarie et al., 1998 23 Figure 3: Multidimensional Scaling plot for the six villages in the ESP using pairwise Fst 34 Figure 4: Haplotype network for W. bancrofti hosts (n=14) across six villages in the ESP 38 Figure 5: Haplotype network for W. bancrofti hosts Peneng 39 Figure 6: Haplotype network for W. bancrofti hosts Albulum1 40 Figure 7: Haplotype network for W. bancrofti hosts Albulum2 41 Figure 8: Haplotype network for W. bancrofti hosts Yautong1 42 Figure 9: Haplotype network for W. bancrofti hosts Yautong2 43 Figure 10: Haplotype network for W. bancrofti hosts Moihuak 44 Figure 11: Histogram of genetic distances among hosts in the 6 villages 45 Figure 12: Histogram of genetic distances among hosts in the 6 villages 45 Figure 13: Comparison of Fst between the Peneng and Moihuak with respect to the other study sites 46 Figure 14: IBD plot for the six villages in ESP, PNG 47 vii Acknowledgements It is a pleasure to thank the many people who made this thesis possible. I consider it a great privilege for the opportunity given to me to work at the Center for Global Health and Diseases at Case Western Reserve University. I would like to sincerely thank Dr. Peter Zimmerman, my advisor and mentor for his crucial guidance in this project, constant support and encouragement. I would also like to thank Dr. Daniel Tisch for his help with the analysis of my data and motivation throughout my thesis writing period. I would like to take this opportunity to thank Dr. Michael Benard for introducing me to population genetics and lending me his expert views and precious time. I am extremely grateful to Dr. Scott Small, a post-doctoral fellow at the Zimmerman lab for constantly guiding me through the data; his untiring help, constant advice and support has substantially shaped the findings of my thesis. I unreservedly acknowledge with gratitude all the help and support provided by all the members of the Zimmerman lab. I deeply value the association with Dr. Rajeev Mehlotra, Krufinta Bun, Tenisha Phipps, Cara Halldin, Kyle Logue, Chad Schaber, Bangan John, Barnie Willie and Melinda Zikursh who have provided great help, caring and support besides practical advice in completion of this project. I would also like to thank Zachary Kloos, a great friend and fellow researcher for introducing me to Wuchereria bancrofti and helping me through my project. I wish to thank my best friend from high school (Soundarya Rangaraj), best friend as an undergraduate (Lakshmi Priya and Amrutha Pattamatta) and best friend in graduate school (Kirsten Eichelman) for their emotional support, entertainment and help through difficult times. viii I owe my gratitude to the Department of Biology at Case Western Reserve University and other members of the Center for Global Health and Diseases for extending their continuous support and guidance throughout my project. Last, and most importantly, I wish to thank my parents Sri Vidya Ramesh and Ramesh Veeraraghavan, my grandmother, Chandra Renganathan, my Aunt and Uncle for their never- ending love, care and support. To them I dedicate this thesis. ix List of Abbreviations ATP Annual Transmission Potential atp6 ATP synthase subunit 6 cob cytochrome b cox1-3 cytochrome c oxidase subunits 1-3 DALY disability-adjusted life year DEC Diethlycarbamazine ESP East Sepik province GAELF/GPELF Global Alliance to eliminate lymphatic filariasis gDNA Genomic DNA LD Linkage Disequilibrium LDR-FMA Ligase detection reaction-fluorescent microsphere assay LF Lymphatic filariasis MDA Mass drug administration MFI Median Fluorescence Intensity Mt Mitochondria nad1-6 NADH dehydrogenase subunits 1-6 nad4L NADH dehydrogenase subunit 4L nt nucleotide PacELF Pacific Program to Eliminate Lymphatic Filariasis PNG Papua New Guinea rrn ribosomal RNA trn transfer RNA WHO World Health Organization x Mitochondrial cytochrome oxidase I (COXI) sequence polymorphisms reveal population genetic diversity of Wuchereria bancrofti in Papua New Guinea Abstract by AKSHAYA RAMESH Wuchereria bancrofti is the primary causative agent of lymphatic filariasis, estimated to affect 120 million people in 80 countries. Several chemotherapeutic programs to eliminate this parasite have been introduced, which are likely to result in changes of the genetic structure in W. bancrofti populations. Despite constituting a major public health burden, this parasite remains poorly understood with respect to its mitochondrial sequence and population biology. To address this knowledge gap, the complete mitochondrial genome of W. bancrofti was sequenced following which a portion of the cytochrome oxidase 1 gene was amplified from individuals in the East Sepik Province of Papua New Guinea. The present study suggests that W. bancrofti populations are highly heterogeneous with a moderate genetic structure across the East Sepik Province. This study has facilitated exploration into W. bancrofti diversity and provides insights into patterns of transmission, an essential component of public health interventions aimed at eliminating lymphatic filariasis. xi Chapter 1 Lymphatic Filariasis: Global burden and Epidemiology 1.0. Introduction 1.1. Global burden Lymphatic filariasis (LF) is a neglected tropical disease, primarily of the poor, affecting around 120 million people worldwide and endemic in 80 countries. LF is endemic in Africa, South America, Indian subcontinent, South East Asia, the Pacific islands and the eastern Mediterranean. Around 118 million people are estimated to have clinical symptoms of the disease with 74 million being microfilaraemic, which includes hidden renal and lymphatic pathology; another 27 million have hydrocoele. Additionally, 16 million people are reported to have elephantiasis, a chronic form of the disease. The disability-adjusted life year (DALY) burden, a measure of overall disease burden expressed as the number of years lost due to ill-health, due to LF is 5.5 million (Global programme to eliminate lymphatic filariasis: Annual Report on Lymphatic Filariasis, 2002; Molyneux, Bradley, Hoerauf, Kyelem, & Taylor, 2003). 1.1.2. Lymphatic filariasis in Papua New Guinea Among the endemic islands, Papua New Guinea (PNG) has the highest estimated population at risk with almost 50% of the entire population at risk of infection.