Title MOLECULAR ECOLOGY OF CHIGGER MITES (ACARI: TROMBICULIDAE) AND ASSOCIATED BACTERIA IN THAILAND Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor in Philosophy by Kittipong Chaisiri November 2016 AUTHOR’S DECLARATION Apart from the help and advice acknowledged, this thesis represents the unaided work of the author ..................................................... Kittipong Chaisiri November 2016 This research was carried out in the Department of Infection Biology, Institute of Infection and Global Health, University of Liverpool ii ABSTRACT Molecular ecology of chigger mites (Acari: Trombiculidae) and associated bacteria in Thailand Kittipong Chaisiri Chiggers are the tiny six-legged larval stage of mites in the family Trombiculidae. These mites, particularly the genus Leptotrombidium, act as important vectors of Orientia tsutsugamushi, the causative agent of scrub typhus disease in the Asia-Pacific region (including Thailand). Although the medical impact of these mites has been recognized in the country due to the increasing incidence of the disease in humans, knowledge of the ecology and epidemiological role of these mites is still very limited to date. A systematic review of mite-associated bacteria was conducted from 193 publications (1964 - January 2015) providing a reference database of bacteria found in mites of agricultural, veterinary and medical importance. Approximately 150 bacterial species were reported from 143 mite species with Cardinium, Wolbachia and Orientia as the dominant genera. Nationwide field sampling of small mammals from 13 locations in Thailand revealed a high diversity of chigger mites. From approximately 16,000 mites isolated from 18 host species examined (1,574 individual animals), 38 chigger species were found including three species new to science (i.e., Trombiculindus kosapani n. sp., Helenicula naresuani n. sp. and Walchia chavali n. sp.) and 10 new records for the first time in the country. Brief taxonomic information for the morphological identification of chiggers is provided. A combination of autofluorescent and brightfield microscopy was demonstrated to be a novel approach to study both the morphology and DNA profile of the same individual chigger. Most chigger species showed low host specificity. The diversity of chiggers on hosts was influenced by host intrinsic (i.e., host phylogeny and maturity) and extrinsic factors (i.e., habitat and geographical location). Chigger species richness and host-chigger network connectance were found to be interrelated variables explaining human scrub typhus incidence in Thailand. Chigger-associated bacteria were investigated for the first time using an Illumina MiSeq 16S rRNA amplicon sequencing approach. DNA of O. tsutsugamushi was detected in the chigger population as expected. In addition to O. tsutsugamushi, Borrelia and Mycobacterium were identified aspotential pathogens of human and animals. Potential symbiotic bacteria of arthropods; e.g., Candidatus Cardinium, Pseudonocardia, Rickettsiella and Wolbachia were also discovered for the first time in chiggers. An important technical limitation was that chigger DNA starting quantity (individual specimens versus pooled mites) was found to have a significant impact on the apparent microbiome profile. These outcomes from the studies of chigger taxonomy and the ecology of host-chigger interactions, as well as the composition of the microbiome in chiggers, are of key importance to the chigger research field, providing essential information for disease epidemiology with vector control implications. Keywords: Chiggers, Trombiculidae, molecular ecology, microbiome, bacteria, 16S rRNA amplicon sequencing, Illumina MiSeq, Thailand iii ACKNOWLEDGEMENTS This PhD thesis would be impossible to finish without the help and support from several people and organizations. First, I would like to express my gratitude to my primary supervisor, Dr Ben Makepeace for his patience in supervising me along the 4 years of this PhD programme. Although we faced a number of difficulties working with the challenging issues of specimens and laboratory procedures, he remained behind me until the PhD thesis was finally completed. I would like to equally thank also my two co-supervisors, Dr John McGarry and Prof. Steve Paterson for their great help and support. I am indebted to Prof. Serge Morand (Centre d’Infectiologie Christophe Mérieux of Laos), my formal MSc supervisor who has taught me and offered great scientific/academic opportunities since 2008, when his research projects, CERoPath (Community Ecology of Rodents and Their Pathogens in Southeast-Asia, granted from French National Research Agency: ANR 07BDIV 012) and BioDivHealthSEA (Local Impacts and Perceptions of Global Changes: Health, biodiversity and zoonoses in Southeast Asia, granted from French National Research Agency: ANR 11CPEL 002) commenced in Thailand. I would like to take this opportunity to acknowledge the two projects providing chigger specimens and allowing me to participate in extensive field surveys across the region. In addition to Prof. Serge Morand, I credit the following people from the CERoPath dream team for their contribution in the field surveys: Dr Vincent Herbreteau, Dr Alexis Ribas, Yannick Chaval, Khomson Satchasataporn, Noppawan Thaprathom, Praphaiphat Siribat, iv Anamika Karnchanabanthoeng, Sompote Temchuen, as well as others who are not mentioned here. I acknowledge provincial and local public health offices as well as primary health care units (Ministry of Public Health Thailand) and their staff for great support during my fieldwork. I also acknowledge the Bureau of Epidemiology Thailand, and would like to particularly thank to Dr Soawapak Hinjoy who provided data of human scrub typhus incidence in Thailand for epidemiological analysis in this thesis. I owe a big thank you to Dr Alexandr Stekolnikov (Zoological Institute of Russian Academy of Sciences, Saint-Petersburg) for the critical help identifying chigger materials and describing the three new species. Although we have never met in person, his contribution was very important for the whole thesis work. Also, I would like to show my appreciation to Dr Ratree Takhampunya and Sommai Promsathaporn (Armed Forces Research Institute of Medical Sciences, Bangkok) for allowing me to be trained on the basics of chigger preparation and identification at their Institute. I also owe a lot of thanks to Prof. Daniel Paris (Mahidol-Oxford Research Unit, Bangkok) for a great collaboration and his innovative idea of UV- and fluorescent-microscopy applications for chigger imaging. Heartfelt thanks should also be expressed to the others who supported me in data analysis and laboratory work while I was based in the University of Liverpool: Christina Gill (Institute of Infection & Global Health) and Dr Alistair Darby v (Institute of Integrative Biology) for their bioinformatics advice in the microbiome analysis; Dr Alison Beckett (Institute of Translational Medicine) for chigger specimen processing for electron microscopy (both SEM and TEM); Dr Luca Lenzi (Institute of Integrative Biology) for analysing whole genome data of Leptotrombidium deliense; Dr Catherine Hartley (Institute of Infection & Global Health) for specimen importation and a lot of help in the laboratory; as well as people in the Veterinary Parasitology group and other colleagues in the Department of Infection Biology for their help and encouraging words. Importantly, I would like to acknowledge Mahidol-Liverpool Chamlong Harinasuta PhD Scholarships 2013 (University of Liverpool, Liverpool, UK and Mahidol University, Bangkok, Thailand) for financial support and an invaluable opportunity to do my PhD at the University of Liverpool, as well as the Faculty of Tropical Medicine, Mahidol University (my affiliation), that allowed me to take educational leave for the whole period of the PhD programme. Last but not least, I would like to express appreciation to my family: parents, brother, precious wife and children, as my PhD would never have been completed without their love and patience. vi DEDICATION This work is dedicated to my parents, wife and children for their love and patience vii TABLE OF CONTENTS Title .............................................................................................................................................. i AUTHOR’S DECLARATION ............................................................................................................. ii ABSTRACT ................................................................................................................................... iii ACKNOWLEDGEMENTS ................................................................................................................ iv DEDICATION .............................................................................................................................. vii TABLE OF CONTENTS ................................................................................................................. viii LIST OF TABLES ........................................................................................................................... xii LIST OF FIGURES ........................................................................................................................ xiv LIST OF ABBREVIATIONS .......................................................................................................... xviii CHAPTER 1: GENERAL INTRODUCTION: CHIGGER