Epidemiology and Genomics of European Foulbrood (Melissococcus plutonius) of Honey Bees Edward George Haynes PhD The University of York Biology September 2013 Abstract European Foulbrood (EFB) is an important disease of honey bee larvae that has increased in prevalence in recent years, in both the UK and other countries. EFB is caused by the gram- positive bacterium Melissococcus plutonius. To date, most molecular epidemiology studies on M. plutonius have concentrated on developing detection methods, and using these to identify the bacteria in honey bees and honey bee hive products, though recently two genomes of M. plutonius have been published. In this thesis a genome sequence for the Type Strain is generated, and used to draw inferences about the accuracy of the published sequences. Genome sequence for other, field-collected isolates were generated and used to identify mobile genetic elements and to elucidate the evolutionary history of M. plutonius. The genome sequences were also used to design the first strain typing scheme for this pathogen, despite this pathogen being previously described as genetically homogenous. Previously undetectable diversity of M. plutonius is explored at a landscape level, showing geographical structuring of populations of the bacterium both within and among countries. The drivers of the observed structure are investigated, with both anthropogenic movements by beekeepers and natural transmission by bees implicated in the maintenance of M. plutonius population structure. This thesis demonstrates the role of the beekeeper in spreading the bacterium through the sale of live bees and through contaminated equipment. Asymptomatic larvae are shown to be carriers of the bacterium (and to go on to develop disease) and a potential role for social wasps as a vector of the pathogen was discovered. 2 List of Contents Abstract ............................................................................................................................................... 2 List of Contents ................................................................................................................................... 3 List of Figures ...................................................................................................................................... 7 List of Tables ..................................................................................................................................... 10 Acknowledgements ........................................................................................................................... 12 Author’s Declaration ......................................................................................................................... 13 1. Introduction .............................................................................................................................. 15 1.1. Honey Bees ............................................................................................................................ 15 1.1.1. Species and Distribution ................................................................................................. 15 1.1.2. The Keeping of Honey Bees ............................................................................................ 16 1.1.3. The Importance of Bees .................................................................................................. 18 1.1.4. Threats to the Honey Bee ............................................................................................... 20 1.1.5. Honey Bee Pathogens ..................................................................................................... 20 1.2. European Foulbrood .............................................................................................................. 21 1.3. Molecular Epidemiology ........................................................................................................ 24 1.3.1. Pathogen Detection ........................................................................................................ 24 1.3.2. Strain Typing ................................................................................................................... 25 1.4. Bacterial Genomics ................................................................................................................ 27 1.4.1. Next Generation Sequencing .......................................................................................... 27 1.4.2. Horizontal Gene Transfer ................................................................................................ 30 1.4.3. Inferring Gene Function .................................................................................................. 31 1.5. This Thesis .............................................................................................................................. 32 2. The Genome Sequence of Melissococcus plutonius, and the Comparative Analysis of Multiple Genomes ........................................................................................................................................... 33 2.1. Introduction ........................................................................................................................... 33 2.1.1. The Genome .................................................................................................................... 33 2.1.2. Genomics ........................................................................................................................ 34 2.2. Methods ................................................................................................................................. 35 2.2.1. Isolates Sequenced ......................................................................................................... 35 2.2.2. Sequencing ...................................................................................................................... 35 2.2.3. Generating the Type Strain Sequence ............................................................................ 37 2.2.3.1. Alignment, Visualisation and Rearrangement of Sequences ............................ 37 2.2.3.2. Gap-closing PCR ................................................................................................ 37 2.2.3.3. Long Range PCR ................................................................................................. 39 3 2.2.3.4. Annotation of Genome Sequence ..................................................................... 41 2.2.4. Phylogenetic Network ..................................................................................................... 41 2.2.4.1. Mapping Reads to the Reference Genome ....................................................... 41 2.2.4.2. Chromosome SNP Phylogeny ............................................................................ 41 2.2.5. Identification of Putative Mobile Genetic Elements ....................................................... 42 2.3. Results .................................................................................................................................... 42 2.3.1. First Rearrangement of the M. plutonius Genome ......................................................... 42 2.3.2. Second Rearrangement of the M. plutonius Genome .................................................... 42 2.3.3. Third rearrangement of the M. plutonius Genome ........................................................ 44 2.3.4. Long Range PCR ............................................................................................................... 49 2.3.5. Read Mapping and SNP Phylogeny ................................................................................. 49 2.3.6. Identification of Mobile Genetic Elements ..................................................................... 52 2.4. Discussion ............................................................................................................................... 57 2.4.1. Genome Orientation ....................................................................................................... 57 2.4.2. Genomic Comparisons .................................................................................................... 58 3. A typing scheme for the honey bee pathogen Melissococcus plutonius allows detection of disease transmission events and a study of the distribution of variants ......................................... 62 3.1. Introduction ........................................................................................................................... 62 3.2. Methods ................................................................................................................................. 63 3.2.1. Bacterial growth and DNA extraction ............................................................................. 63 3.2.2. Identification of loci ........................................................................................................ 63 3.2.3. Testing of locus variability............................................................................................... 64 3.2.4. International Isolates ...................................................................................................... 64 3.2.5. Case Studies of Suspected Honey bee Movements ........................................................ 66 3.2.6. Data Analysis ..................................................................................................................