BACTERIAL DIVERSITY in the INTESTINAL TRACTS of the FUNGUS-CULTIVATING TERMITES Macrotermes Michaelseni (Sjostedt) and Odontotermes Somaliensis (Sjostedt)
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BACTERIAL DIVERSITY IN THE INTESTINAL TRACTS OF THE FUNGUS-CULTIVATING TERMITES Macrotermes michaelseni (Sjostedt) AND Odontotermes somaliensis (Sjostedt) By: Lucy Mwende Mackenzie A thesis submitted in partial fulfillment for the degree of Master of Science in Botany (Microbiology) in the Jomo Kenyatta University of Agriculture and Technology .--....-..-..,. ......,__ -... , .•. .... .... - " I. C. I . P E. LIB!lARY ACC N c. Ot;.~.lj:';:...~ 2005 ····· ········· ··· . .... ~ -.\ ·\ ~: r ' =t·.1.'"" · LIAS.-, c' '>.. ......· -.~.. ...t ...... ..:;.... ..... '\'T'""FJn f\IA(~'.t lr ' ' 1 "" .t •.•. l .•..1 ..... •.. ..•.!' .... ..... , ,..,., •T"" .,.... i r ~ ' . ., £.1.L~~ . .1: ....... .' .• .' • • : .. ~ ~ ~ ••-. ..~ ..._ I.... .,.,, ... ~:-~ - ·-~-~......... ___.4'«___ _ Declaration The work described in this thesis was carried out at the International Center of Insect Physiology and Ecology during the period 2003-2004. The experimental work described is entirely my own. Information derived from published work is specifically acknowledged in the text and references appended. The contents of this thesis have not been submitted for any other award. t:-J~~-~l.ii.k:. _________ __ Date --------b/4-/a-----:...-.------' Lucy Mwende Mackenzie International Center of Insect Physiology and Ecology P.O Box 30772, Nairobi, 00100 Kenya. This thesis has been submitted for examination with our approval as supervisors. I ..--- -:y]..._: -/ 2 ( &IQ~~( Date _ _t_.j _ _.i-;- _____-.: __ _ ---------------~~r. Hamadi I. Boga Jomo Kenyatta University of Agriculture and Technology P.O. Box 62000, Nairobi, 00200 Kenya. V/ CS Date ---Jq ·--"'""------- Dr. Anne W. T. Muigai Jomo Kenyatta University of Agriculture and Technology P.O. Box 62000, Nairobi, 00200 Kenya. ~ ·~-;Al.\- o~ ----~~-=2- - -- - ---- - Date------------ -.... Dr. Gerardo Toledo Diversa Coiporation, 4955 Directors place San Diego, CA 92121 USA ii M1 _Jl!f£__ /ll..,.. ____________ _ Date -1-~~fg_~- Dr. Martin Keller Diversa Corporation, 4955 Directors place San Diego, CA 92121 USA Dr. Wilber Lwande International Center oflnsect Physiology and Ecology P.O Box 30772, Nairobi, 00100 Kenya ~!{ -~--- ---~----- Date L_ Dr. Ellie 0. Osir ---~Jf International Center of Insect Physiology and Ecology P.O Box 30772, Nairobi, 00100 Kenya. iii Dedication To my mother, Ruth and father, Charles. To my sisters Harriet, Tabitha, Christine, Gloria, and Catherine and my brother Nicholas. Thank you for all the support. IV Acknowledgements This work was financially supported by Diversa Corporation, San Diego, CA (USA). I am extremely indebted to Diversa for the research grant without which it would not have been possible to do this work I would like to thank the International Center oflnsect Physiology and Ecology, (ICIPE) Nairobi Kenya through the Director of Research and partnerships Prof. O.K Ole Moi Yoi for awarding me the Dissertation Research Internship Program (DRIP) fellowship and for availing the use of the institutions facilities. I W.Quld also like to thank the Botany Department at the Jomo Kenyatta University of Agriculture and Technology for the support provided through out my study period. I am grateful to all my supervisors Dr. Hamadi Boga, Dr. Anne Muigai, Dr. Ellie Osir, Dr. Wilber Lwande, Dr. Martin Keller and Dr. Gerardo Toledo for the guidance and encouragement that they provided through out the period of this study. I express my deepest gratitude to my parents, siblings and my fiance for the endless love and moral support accorded during this course. More thanks to Diversa Corporation for the training on molecular techniques and phylogenetic analysis. I thank all my colleagues in the Molecular Biology and Biotechnology Department (l.C.l.P.E) for the friendly working atmosphere provided in the laboratory. v TABLE OF CONTENTS Title ... ............................................................................................ i Declaration ... ................................................................................... ii Acknowledgements .......... ................. ................................................. v List ofFigures ................. .................................................................. xi List ofTables .................................................................................... x List of Plates ...................................................... ............................... xi Abstract ........................................................... ....... ................ ........ xii Abbreviations .............................................................. .... ................ xiii CHAPTER ONE ............................................................................... I I. 0 General Introduction ........................................................................ I I. I Justification ......................................................................... 4 I.2 Objectives .........................- .............................................. .... 7 CHAPTER TWO .............................................................................. 8 2.0 Literature Review ........................................................................... 8 2.1 Classification of termites ............................................................... .... 8 2.2 The Intestinal Tracts ofTermites ........................................... 9 2.2.1 The Intestinal Tracts of Lower Termites .......................... I2 2.2.2 The Intestinal Tracts of Higher Termites ......................... I2 2.3 Nutritional Biology ofTermites ................................................ 14 2.3.2 Wood feeding Termites ............................................. 15 2.3.3 Soil feeding Termites ................................................ I5 2.3.4 Fungus-cultivating Termites ....................................... 16 Vl 2.4 Microbial Diversity in Termite guts ........................................... 17 2.4.lSpirochetes .................... ..... .................. .................... 19 2.4.2 Lactic Acid Bacteria ............... ........................ .......... 19 2.4.3 Sulfate-reducing Bacteria ............ ............................... 20 2.4.4 Methanogenic Archaea ............................. ................. 20 2.4.5 Bacteroides .................................... ........................ 21 2.4.6 Actinomycetes ........................................................ 21 2.4. 7 Homoacetogenic Bacteria ........................................... 22 CHAPTER THREE ........................................................................... 24 3.0 Materials and Methods .......................................... ........................... 24 3.1 Termite Collection ...... ........................................................... 24 3.2 Gut Preparations for Dilutions ................................................... 25 3 .3 Growth Medium ................................................................... 25 3.4 Culture-dependent Methods ...................................................... 26 3.4.1 Plate Counts .......................................................... 26 3.4.2 MPN Determinations ................................................ 26 3.4.3 Molecular Characterization oflsolates........................... 27 3.4.3.1 DNA Extraction ............................................. 27 3.4.3.2 PCR Amplification ofBacterial 16S rDNA ............. 28 3.4.3.3 Phylogenetic Analysis ......... ............................. 29 3.5 Culture-independent Methods ................................................... 30 3.5.1 DNA Extraction........ .............................................. 30 3.5.2 PCR Amplification ofBacterial 16S rDNA .......................................... ........................ 31 vu 3.5.3 Cloning of PCR Product into Sequencing Vector ............... 32 3.5.4 Plasmid Extraction oflndividual Clones ...... .................... 33 3.5.5 Restriction Digest Analysis ................................ ......... 34 3.5.5.1 EcoRI Digestion ............................................. 34 3.5.5.2 RFLP .......................................................... 34 3.5.6 Phylogenetic Analysis ............................................... 34 CHAPTER FOUR ............................................................................. 36 4.0 Results ..................................................................... ................... 36 4.1 Enumeration ofMicroorganisms ................................................ 36 4.2 Characterization oflsolates .................... ........................... ........ 37 4.3 PCR Amplification of 16S rDNA Gene from Macrotermes michaelseni and Odontotermes somaliensis...... 41 4.4 Plasmid Purification........................ ............ ........................... 41 4.5 Eco RI Digestion.................................... ............................... 42 4.6 Restriction Length Polymorphism (RFLP) .................................... 43 4. 7 Diversity of Bacterial Community in Macrotermes michaelseni ........... 45 4.8 Diversity of Bacterial Community in Odontotermes somaliensis..... ..... 49 CHAPTER FIVE ............................................................ .................. 56 5.0 Discussion ................................................................................... 56 6.0 References ..................... ............ .. ....... ........................ ................ 67 Appendix ............................. ....... ............................................