CELLULOLYTIC AND XYLANOLYTIC GUT ENZYME ACTIVITY PATTERNS IN MAJOR SUBTERRANEAN TERMITE PESTS By JOSEPH ANTHONY SMITH A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007 1 © 2007 Joseph Anthony Smith 2 To my family, friends and teachers, without whom this degree would not have been possible 3 ACKNOWLEDGMENTS I would like to thank the following people and institutions for their support. I thank Procter and Gamble for the funding that allowed me to conduct this research. I thank my committee, Dr. Phil Koehler, Dr. Mike Scharf, Dr. Lonnie Ingram, and Dr. Phil Brode, for their input and feedback on my research. I thank Bruce Ryser for supplying Formosan subterranean termites for my research. I thank my fellow students, and Cynthia Tucker in particular, for their intelligent input and collaboration. I thank my family and friends for their moral support. Finally, I would like to especially thank Dr. Phil Koehler for his efforts as my advisor and committee chair. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES...........................................................................................................................8 LIST OF FIGURES .......................................................................................................................10 ABSTRACT...................................................................................................................................11 INTRODUCTION .........................................................................................................................13 LITERATURE REVIEW ..............................................................................................................15 Wood Molecular Structure .....................................................................................................15 Termite Taxonomy .................................................................................................................16 Termite Digestive System Anatomy.......................................................................................17 Symbiont Taxonomy and Anatomy........................................................................................18 Symbiont Assemblages in Termite Hindguts .........................................................................19 Termite Carbohydrolase Types and Locations.......................................................................20 Termite Carbohydrolase Structural and Functional Characteristics.......................................21 BUFFER AND PH OPTIMIZATION FOR TERMITE CARBOHYDROLASE ASSAYS.........28 Introduction.............................................................................................................................28 Materials and Methods ...........................................................................................................29 Termite Collection, Dissection, and Enzyme Extraction ................................................29 Buffer Optimization Assays ............................................................................................29 Gut Region Assays Across PH........................................................................................31 Data Analysis...................................................................................................................32 Results.....................................................................................................................................33 Buffer Optimization Assays ............................................................................................33 Gut Region pH Optimization Assays ..............................................................................34 Discussion...............................................................................................................................34 Activity Magnitudes ........................................................................................................34 Activity Changes with pH ...............................................................................................35 Differences in Gut Regions .............................................................................................35 Conclusions .....................................................................................................................36 CARBOHYDROLASE ACTIVITY PATTERNS IN RETICULITERMES FLAVIPES AND COPTOTERMES FORMOSANUS WORKERS AND SOLDIERS.......................................43 Introduction.............................................................................................................................43 Materials and Methods ...........................................................................................................44 Termite Collection, Dissection, and Enzyme Extraction ................................................44 Termite Photography.......................................................................................................45 5 Soluble Protein Assays....................................................................................................45 Endoglucanase Assays.....................................................................................................45 Exoglucanase Assays.......................................................................................................46 Beta-Glucosidase Assays.................................................................................................46 Xylanase Assays..............................................................................................................47 Beta-Xylosidase Assays ..................................................................................................48 Data Analysis...................................................................................................................48 Results.....................................................................................................................................49 Termite Gut and Frontal Gland Comparisons .................................................................49 Worker Cellulolytic Activities ........................................................................................49 Worker Xylanolytic Activities ........................................................................................50 Soldier Cellulolytic Activities .........................................................................................50 Soldier Xylanolytic Activities .........................................................................................50 Gut Soluble Protein Levels..............................................................................................51 Discussion...............................................................................................................................51 CHANGES IN RETICULITERMES FLAVIPES GUT CELLULOLYTIC ACTIVITIES IN RESPONSE TO DIET............................................................................................................63 Introduction.............................................................................................................................63 Materials and Methods ...........................................................................................................64 Termite Collection...........................................................................................................64 Termite Diets and Feeding ..............................................................................................65 Termite Dissection and Enzyme Extraction....................................................................65 Endoglucanase Assays.....................................................................................................66 Exoglucanase and Beta-glucosidase Assays ...................................................................67 Data Analysis...................................................................................................................67 Results.....................................................................................................................................68 Termite Gut Observations ...............................................................................................68 Endoglucanase Activities ................................................................................................69 Exoglucanase Activities ..................................................................................................69 Beta-glucosidase Activities .............................................................................................70 Discussion...............................................................................................................................70 CHANGES IN RETICULITERMES FLAVIPES GUT XYLANOLYTIC ACTIVITIES IN RESPONSE TO DIET............................................................................................................77 Introduction.............................................................................................................................77 Materials and Methods ...........................................................................................................78