Institute of Infection, Veterinary and Ecological Sciences Torix Rickettsia: aspects of diversity, host range and symbiont-host interaction. Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor in Philosophy Panupong Thongprem December 2020 DECLARATION I hereby declare that the work in this thesis is based on research carried out at the Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool. The work contained in this thesis is my own, unless otherwise acknowledged and cited. The data that have already been published in scientific journals are specifically declared in each chapter with authors contribution described. This thesis is original and has not been previously submitted for any other degree. Panupong Thongprem December 2020 i ABSTRACT Rickettsia bacteria have traditionally been considered as the aetiologic agent of deadly arthropod-borne diseases in humans and livestock. However, more recent studies have discovered Rickettsia as non-vertebrate pathogens that are actually important to invertebrate evolution as symbionts. Recently, Rickettsia in the ‘torix’ clade were described from glossiphoniid leeches. This clade has since been observed to infect a wide range of invertebrate species and is thought to be most common in host species associated with freshwater habitats. This leads to a general hypothesis that torix Rickettsia are a common endosymbiont of freshwater taxa. However, this hypothesis is yet to be formally tested. To assess this hypothesis, I firstly investigated in-depth a freshwater-associated insect order, the Odonata (dragonflies and damselflies), in which torix Rickettsia had not been previously recorded. This study revealed the first incidence of torix Rickettsia in odonates, present in roughly 10% of the screened species. Maternal transmission of this endosymbiont was observed in a damselfly (Coenagrion puella), and this strain has likely driven mtDNA introgression between the insect and its sister species (C. pulchellum). Then, I expanded the screen to test for torix Rickettsia in other invertebrate taxa and compared the infection frequency between freshwater and terrestrial communities. Fisher’s exact test indicated that the proportions of infected species from freshwater community is significantly higher than the terrestrial group in three representative insect orders. In addition to this broad screen, torix Rickettsia in a few blood-feeding insects are recorded for the first time, including mosquitos (Anopheles plumbeus), black flies (Simulium aureum) and the common bed bug (Cimex lectularius). Bed bugs were then established as a model system to study biological impacts of torix Rickettsia carriage. Symbionts in the bed bug were transmitted via matrilines only. There were no signs of reproductive parasitism, sex ratio distortion or cytoplasmic incompatibility phenotypes. Torix Rickettsia only express mild parasitic impacts on C. lectularius biology by slowing development time and reducing fecundity. Finally, this thesis raises three questions for onward study; i) why torix Rickettsia are abundant in freshwater biomes, ii) how do torix strains transition into terrestrial species and iii) how torix Rickettsia are associated with broad spectrum of eukaryotic hosts. Possible scenarios for these three questions are discussed for future study. ii ACKNOWLEDGEMENTS I would like to express my great appreciation to ‘Development and Promotion of Science and Technology Talents Project (DPST)’ for the financial support and opportunity to study in University of Liverpool, UK, along with the ‘Office of Educational Affairs, Royal Thai Embassy’ for being responsible for my academic life. However, the completion of my PhD degree would not have been possible without the support of these following people. First and foremost, I would like to thank my supervisor, Prof Greg Hurst, for his kind hospitality, consistent support and useful guidance during these four years of my research and personal development. Not only was he a major reason of my PhD success, but he also was a major supplier of chocolates and foods, and especially ice-creams on a hot summer day. Secondly, I would like to thank Dr Lucy Weinert for her kind response to be the external examiner in my viva. This thesis would have not been accomplished without the useful guidance of my assessors, Prof David Atkinson and Dr Kate Baker, temporary assessors, Dr Meriel Jones and Dr Seth Barribeau, and my secondary supervisor, Dr Alistair Darby. I was lucky to receive help from two people with expertise in the common bed bug, Dr Oliver Otti and Dr Sophie Evison. They guided me in all the works in bed bug experiments. Grateful thanks to Otti for a major blood resource of bed bug productions and his kind hospitality when I was visiting the lab in University of Bayreuth, Germany. I had great pleasure of working with Prof David Thompson and Dr M. Olalla Lorenzo- Carballa in my first publication, and thanks for their odonate samples. I also would like to acknowledge other contributors who contributed specimens and DNA samples for this research, i.e., Craig Maccadam, Dr Anthony Cook, Dr Steffon Roth and Dr Klaus Reinhardt. Special thanks to Dr Jack Pilgrim who firstly taught me PCR and FISH techniques. He always proof-reads and comments on my works since the first-year report, including my first publication and until this thesis in the end. Thanks to people in the Hurst group who always support and share all happy memories together. In particular, I’m very grateful to Dr Stefanos Siozios and Dr Ewa Chrostek for their guidance and assistance in teaching me all these years. Thanks to Dr Louise Reynolds for her comments on every aspect of my oral presentation practices, Dr Emily Hornett for her comments in my manuscripts. Thanks to Helen Davison for helping me in all field and the works until we finished the publication from Chapter 2. Thanks also to Andrew Williams for his help in BEV-like symbiont screening, Jordan Jones and Sam Edwards for their blood donation to supply my bed bugs when they were hungry. Thanks go to all academic staff and PhD colleagues in Biosciences building for their help and support, my Thai friends in Liverpool for the company in all situations and Chaba Chaba Thai restaurant in Allerton for being my shelter over these years. Finally, my deep and sincere gratitude go to my family, my mom, dad and sister, for their encouragement, support and love. P.S. “My deepest condolences go to all invertebrates that were sacrificed, especially numbers of bed bugs that were left to die for nothing during the pandemic of COVID-19” iii Table of Contents DECLARATION ........................................................................................................ i ABSTRACT ............................................................................................................. ii ACKNOWLEDGEMENTS ........................................................................................ iii Table of Contents .................................................................................................. iv CHAPTER 1 Introduction ........................................................................................ 1 1.1 Heritable endosymbionts ........................................................................................ 2 1.1.1 Primary and secondary endosymbionts ............................................................................... 2 1.1.2 Symbionts that manipulate host reproduction .................................................................... 4 1.1.3 Cytoplasmic incompatibility (CI) .......................................................................................... 8 1.1.4 Fitness impacts of symbiont association ............................................................................ 10 1.2 Rickettsia .............................................................................................................. 13 1.2.1 Rickettsia in the aspects of arthropod borne disease ........................................................ 13 1.2.2 Arthropod-associated Rickettsia ........................................................................................ 15 1.2.3 Transmission patterns and impact on host biology ........................................................... 15 1.2.4 Host range .......................................................................................................................... 16 1.2.5 Diversity of Rickettsia ......................................................................................................... 17 1.3 Torix group Rickettsia ............................................................................................ 20 1.3.1 Incidence of torix group Rickettsia ..................................................................................... 20 1.3.2 Torix Rickettsia hosts ......................................................................................................... 20 1.4 General objectives, study system and directions for this thesis ............................. 21 1.4.1 Chapter 2: Diversity of Rickettsia in order Odonata .......................................................... 21 1.4.2 Chapter 3: Screening of freshwater and terrestrial arthropod taxa .................................. 21 1.4.3 Chapter 4: Incidence of torix Rickettsia in the common bed bug ...................................... 22 1.4.4 Chapter 5: Torix Rickettsia-Bed bug interaction ...............................................................