GENOMIC ANALYSES OF THE ANOPHELES PUNCTULATUS GROUP: INSIGHTS INTO MOSQUITO BIOLOGY AND IMPLICATIONS FOR VECTOR CONTROL AND DISEASE TRANSMISSION by KYLE JOSEPH LOGUE Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation Advisors: Dr. Peter A. Zimmerman, Ph.D. and Dr. David Serre, Ph.D. Department of Biology CASE WESTERN RESERVE UNIVERSITY May, 2016 SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Kyle Joseph Logue Candidate for the degree of Doctor of Philosophy*. Committee Chair Ryan Martin, PhD Committee Member Christopher Cullis, Ph.D Committee Member Michael Benard, Ph.D Committee Member Peter A. Zimmerman, Ph.D Committee Member David Serre, Ph.D Date of Defense January 25, 2016 *We also certify that written approval has been obtained for any proprietary material contained therein ii Table of Contents Table of Contents iii List of Tables ix List of Figures xii Acknowledgements xv List of Abbreviations xvii Glossary xix Abstract 1 I. Chapter 1. The biology of Anopheles punctulatus Species in Papua New Guinea 3 A. Introduction: Anopheles mosquitoes and disease 4 B. Systematics and distribution of Anopheles species in Papua New Guinea 7 1. Discovery of the Anopheles punctulatus group 7 2. Species determination 8 a. Morphology 8 b. Cross-mating studies 9 c. Molecular techniques 10 3. Geographic distribution 12 4. Species distribution and larval habitats 14 a. An. punctulatus s.s. 15 b. An. koliensis 16 c. An. farauti s.s. 16 d. An. hinesorum 17 e. An. farauti 4 17 f. Minor An. punctulatus species 18 5. Species relationships 19 C. Vector ecology of An. punctulatus species 22 1. Host feeding patterns 22 a. Methods used to identify the blood meals of hematophagous insects 22 b. Host feeding patterns of Anopheles in Papua New Guinea 26 2. Vectorial status 29 a. Malaria 29 b. Lymphatic filariasis 31 D. Vector control 33 iii 1. History of vector control 33 2. Vector control in Papua New Guinea 36 E. Genomic data available for Anopheles 38 F. Dissertation aims 40 II. Chapter 2. Genome Sequencing of the Anopheles punctulatus Sibling species and examination of divergence among species 44 A. Introduction 45 B. Methods 48 1. Mosquito collections and species identification 48 2. Whole genome sequencing and de novo assembly of mitochondrial genomes 49 a. Whole genome sequencing of five An. punctulatus mosquitoes 49 b. De novo assembly of mitochondrial genome 50 3. Targeted sequencing of mitochondrial genomes of An. punctulatus mosquitoes 52 a. Long range primer design for targeted sequencing of mitochondrial genomes 52 b. Long range amplification and multiplex sequencing 55 c. De novo assembly of mitochondrial genomes 55 4. De novo assembly of the nuclear genome of four An. punctulatus species 58 5. Genome assembly comparisons between Anopheles mosquitoes 59 a. An. punctulatus species 59 b. An. punctulatus species and An. gambiae complex 59 c. An. farauti s.s. genome assemblies 60 6. Phylogenetic analysis and molecular dating of Anopheles mosquitoes using protein-coding genes of the mitochondrial genomes 60 a. Reconstruction of evolutionary history 60 b. Molecular dating 62 7. Phylogenetic analysis of four AP species using orthologous regions of the nuclear genome 62 C. Results 63 1. Sequencing and assembly of mitochondrial and nuclear genomes 63 a. Mosquitoes sequenced 63 b. Assembly of mitochondrial genomes 64 c. Sequencing and de novo assembly of the nuclear genome of four wild-caught mosquitoes of the An. punctulatus group 65 2. Genome comparison of Anopheles mosquitoes 67 iv a. Comparison of the genome assemblies of An. punctulatus species 67 b. Comparison of An. farauti s.s. genome assembly to An. farauti s.s. genome assembled by the Broad 70 3. Phylogenetic analysis 70 a. Phylogeny of Anopheles mosquitoes using the mitochondrial genome 70 b. Molecular dating of Anopheles using mitochondrial genome 73 c. Phylogeny of four AP species using multiple nuclear loci 75 D. Discussion 78 1. Genome assembly and comparative analyses 79 2. Molecular dating of Anopheles species using mitochondrial genome 81 3. Evolutionary relationships of An. punctulatus species 83 4. Implications for vector control initiatives 84 III. Chapter 3. Using Genomic Data to Investigate if Introgression is Occurring Between An. punctulatus Sibling Species 86 A. Introduction 87 B. Methods 90 1. Identification of putative introgression candidates using sequence divergence between orthologous regions of the nuclear genome 90 2. Identification of Single Nucleotide Polymorphisms (SNPs) in orthologous regions of the An. punctulatus s.s. and An. farauti 4 genomes 91 3. Number of shared polymorphisms expected under different population histories 92 4. Characterization of the historical demography of An. farauti 4 and An. punctulatus s.s. 94 C. Results 96 1. Phylogenetic analysis does not provide any evidence of introgression 96 2. Analyses of shared polymorphisms between An. farauti 4 and An. punctulatus s.s. does not support recent gene flow between these species 98 3. Characterizing the demographic history of An. punctulatus s.s. and An. farauti 4 103 D. Discussion 105 1. No evidence of contemporary gene flow among An. punctulatus sibling species 105 v 2. Discordant demographic history of An. farauti 4 and An. punctulatus s.s. species 108 3. Implications for disease transmission and implementation of vector control strategies in Papua New Guinea 109 IV. Chapter 4. Unbiased characterization of host feeding patterns of Anopheles punctulatus species by targeted high-throughput sequencing of the mammalian mitochondrial 16S rRNA 111 A. Introduction: Current and historical techniques used to evaluate host blood meals 112 B. Methods 115 1. Ethics 115 2. Sample collections 115 3. DNA isolation and molecular species identification 116 4. In silico assessment of mammalian mt 16S rRNA 116 a. Amplification range of mt 16S rRNA primers 116 b. Number of mammalian species amplified 117 c. Ability of primers to differentiate among mammalian species 117 5. Targeted high-throughput sequencing of mosquito host blood meals 118 a. Primer design of mammalian mt 16S ribosomal RNA genes and human mt genome hypervariable 118 b. Primer design for genotyping polymorphisms within the nuclear genome of An. punctulatus s.s. 118 c. Targeted high-throughput sequencing 120 6. Bioinformatic assessment of blood meal composition and population structure from individual mosquitoes 122 a. Filtering sequencing data for analysis 122 b. Identification of host blood meals from Anopheles mosquitoes 122 c. Identification of human individuals fed on by mosquitoes 123 d. Examination of population structure within An. punctulatus s.s. mosquitoes 124 C. Results 126 1. In silico assessment of mammalian mt 16S rRNA 126 a. Amplification range of universal mammalian 16S rRNA primers 126 b. Specificity of the universal mammalian 16S rRNA gene primer pairs 128 2. Application of assay to field-caught female Anopheles mosquitoes 128 a. Mosquitoes collected and sequencing reads 128 vi b. Mammalian blood hosts identified from blood fed Anopheles mosquitoes 134 c. Blood meal composition of individual An. punctulatus species 136 3. Examination of population structure within An. punctulatus s.s. 141 4. Evidence of mosquito blood meals from multiple human hosts 142 D. Discussion 145 1. Strength and utility of targeted sequencing approach to identify mammalian hosts blood meals from Anopheles mosquitoes 146 2. DNA profiling of human maternal lineages from field collected mosquitoes 148 3. Host feeding patterns of An. punctulatus mosquitoes in Papua New Guinea 150 V. Chapter 5. Project Summary 153 A. Summary 154 1. Generation of genomic data for four wild-caught An. punctulatus species 155 2. An. punctulatus species are deeply diverged and reproductively isolated 156 3. Development of an unbiased method to identify mammalian hosts fed on by An. punctulatus 160 B. Contribution of results to vector control initiatives and disease transmission 162 1. Vector control 163 2. Disease transmission and vector competence 165 C. Utility of high-throughput sequencing for understudied species 166 D. Future directions 169 VI. Appendix A. Accession numbers and location of deposited sequencing data 173 VII. Appendix B. Distribution of the sequence coverage per contig for the initial assemblies 175 VIII. Appendix C. Origin and dispersal of Anopheles mosquitoes based on analysis of mitochondrial genome sequences 178 IX. Appendix D. Putative genes that are introgressing in An. punctulatus mosquitoes 181 vii X. Appendix E. Primers used for SNP amplification and barcoding amplicons for high-throughput sequencing 190 XI. Appendix F. Composition of AP sibling species blood meals by village and AP species 199 XII. References 205 viii List of Tables Chapter 1 Table 1 Number of An. punctulatus sibling species screened by ELISA for the presence of P. falciparum, P. vivax and P. malariae 31 Table 2 Anopheles genomes assembled 39 Chapter 2 Table 3 Sample sequencing information 50 Table 4 Primers used to amplify mitochondrial genomes by long range PCR 53 Table 5 List of the samples used with their collection site or colony ID and corresponding NCBI accession numbers 57 Table 6 Summary of initial de novo genome assemblies before filtering out redundant contigs. 66 Table 7 Summary of the Anopheles punctulatus species genome Assemblies 67 Table 8 Summary of the An. farauti 4 assembly after sub-sampling sequencing reads to the coverage of An. punctulatus s.s. 68 Table 9 Percentage of DNA sequencing reads that align among species in the An. punctulatus group and An. gambiae complex 69 Table 10 Mean divergence times and 95% credibility intervals for selected Nodes 74 Table 11 Divergence times using the insect mitochondrial DNA mutation rate 75 ix Table 12 Number of pairwise nucleotide differences (lower diagonal) and percent divergence (upper diagonal) 78 Chapter 3 Table 13 Summary of the genetic diversity in Anopheles farauti 4 and Anopheles punctulatus s.s. 100 Table 14 Summary of the genetic diversity in An.