Archaeal diversity and community structure in the compartmented gut of higher termites Doctoral thesis submitted in partial fulfillment for the award of a doctoral degree “Doktorgrad der Naturwissenschaften (Dr. rer. nat.)” to the Faculty of Biology, Philipps-Universität Marburg by James Oluoch Nonoh from Siaya County, Kenya Marburg/Lahn 2013 II This thesis was part of the major project on “Environmental drivers of methanogenic community structure in termite guts” and was funded by the Deutsche Forschungsgemeinschaft (DFG) under the topic Sonderforschungsbereich (SFB 987). The theme of my study was developed by Prof. Dr. Andreas Brune, the Group leader and main supervisor. The work described in this thesis was carried out in the Laboratory of Prof. Dr. Andreas Brune, at the Department of Biogeochemistry, Max Planck Institute for Terrestrial Microbiology, Marburg, Germany, between October 2009 and March 2013, under the direct supervision of Prof. Dr. Andreas Brune. The thesis was submitted to the Dean, Faculty of Biology, Philipps-Universität, Marburg, on: 08.04.2013 First reviewer Prof. Dr. Andreas Brune Second reviewer Prof. Dr. Ralf Conrad Date of oral examination 21.05.2013 III Declaration I do hereby certify that my doctoral thesis titled: “Archaeal diversity and community structure in the compartmented gut of higher termites” was carried out with legally and professionally authorized scientific methods and devices. All samples were legally and procedurally obtained from Kenya by myself, with permission from the Kenya National Council of Science and Technology (NCST) and the Kenya Wildlife Service (KWS), and with due approval from all the other relevant government authorizing agencies in Kenya. Unless specifically indicated, the experimental work described in this thesis was executed by me. Information obtained from published work is appropriately acknowledged, both in the text and in references therein appended. This thesis is the culmination of my original work and the contents herein, either in full or in part, have not been previously submitted for examination to any university for any award. Marburg/Lahn, April 2013 James O. Nonoh IV Acknowledgements With sincerity, I would like to thank my supervisor Prof. Dr. Andreas Brune for his support during my doctoral work. I sincerely thank the German Academic Exchange Service (DAAD) for the PhD fellowship. Your support, including the many trainings and capacity building workshops organized for the DAAD scholarship holders ensured that I had a focused but smooth stay in Germany and strengthened my resolve to participate effectively in nation building upon completion of my studies. I am grateful to IMPRS for organizing numerous trainings and support in workshops that all complemented very well with my research work. I am grateful to the Kenya National Council for Science and Technology (NCST) for facilitating my PhD study by granting me research permit to get samples from Kenya. On the same strength, I acknowledge the support from the Kenya Wildlife Service (KWS) for giving me permission to collect samples from Kenya for my research study. Your policy in promoting capacity building in science through the numerous fees waivers on my student research permit is highly commendable. I acknowledge the support of Harrison Kibogo and David Wainaina (both of ICIPE) during sampling, without which this work would not have been possible. On numerous occasions, Harrison Kibogo accepted to use his personal car to take me to the field for sampling upon request, despite the difficulty in accessing some sampling sites due to bad weather. David Wainaina, of Molecular Biology Department (ICIPE), always devoted his time, way outside his official commitments, to prepare and package the samples for shipment to our laboratories. I would like to thank all my colleagues in the “Termite Group” for their immense support during my stay at MPI. Many thanks to all of you for lending me a helping hand whenever I was in need, either during actual experimental work, or through the numerous V discussions. Special thanks to Roey Angel, Kristof Brenzinger and Franziska Brandt (Department of Biogeochemistry) for their support in qPCR analysis. I am deeply indebted to my late loving parents for all the support they gave me and for laying in me a good foundation that has no doubt contributed immensely to my successful achievements not only in academics, but in all aspects of my life. Thank you Dad and Mum for inculcating in me strong values that continues to shape and motivate my opinions in life. You always reminded me to work hard, but objectively, to believe strongly in myself despite contrary views and to trust none but myself and above all, to always take good care of all that I have worked so hard to achieve, for in the end, it will benefit not only myself, but all in need. These are the ideals that I strongly hold to date. With pleasure, I would like to express my sincere gratitude to my loving wife Lilian, and my sons Joshua and Emmanuel for their prayers, endless love and overwhelming support throughout my PhD work. It was not an easy decision to stay away from the people I love so dearly in my life, for such a long period of time. Your strength and understanding, and your commitment to support me at whatever cost, always made me to be even stronger, and in the end, it ensured that the only reason I sacrificed everything to be away from you, is well accomplished. VI The following manuscripts were published, submitted or in preparation by the date of submission of this thesis Published Paul, K., Nonoh, J.O., Mikulski, L., Brune, A. (2012). “Methanoplasmatales,” Thermoplasmatales-related archaea in termite guts and other environments, are the seventh order of methanogens. Appl. Environ. Microbiol. 78: 8245–8253. In preparation Nonoh, J.O., Paul, K., Boga, H.I., Köhler, T., Meuser, K., Kamanda, D.K. and Brune, A. Diversity and community structure of archaeal populations in the gut of higher termites (Blattodea: Termitidae). Nonoh, J.O., Dietrich, C., and Brune, A. Distribution of archaeal populations and methanogenic potentials in the highly compartmented gut of soil-feeding termites (Cubitermes ugandensis and Ophiotermes sp.). Nonoh, J.O., Dietrich, C., and Brune, A. Microenvironment and community structure of Archaea and Bacteria in the highly compartmented gut of Amitermes sp. (Isoptera: Termitidae). VII Table of contents Declaration .............................................................................................................................. iii Acknowledgements ................................................................................................................ iv Table of contents ................................................................................................................... vii Chapter One ..............................................................................................................................1 General introduction .................................................................................................................1 Ecological importance of termites.......................................................................................1 Higher termite phylogeny and feeding behavior ................................................................1 Methanogenesis in higher termites ......................................................................................5 Substrate range of methanogenic archaea ...........................................................................6 Methanogenic substrates utilized by termite gut methanogens .........................................7 Methane emission rates in termites ...................................................................................12 Community composition of archaea in higher termites ...................................................16 Objectives of the study ...........................................................................................................20 References ...............................................................................................................................22 Chapter Two ...........................................................................................................................31 Diversity and community structure of archaeal populations in the gut of higher termites (Blattodea: Termitidae) ..........................................................................................................31 Abstract ...................................................................................................................................32 Introduction .............................................................................................................................33 Methods ...................................................................................................................................34 Termites ..............................................................................................................................34 DNA extraction and purification .......................................................................................35 Amplification of archaeal SSU 16S rRNA genes ............................................................36 Generation of clone libraries .............................................................................................37 Phylogenetic analysis of sequence data ............................................................................38