EFFECTS OF EXTREME TEMPERATURES ON THE SURVIVAL OF THE QUARANTINE STORED-PRODUCT PEST, TROGODERMA GRANARIUM (KHAPRA BEETLE) AND ON ITS ASSOCIATED BACTERIA DIANA MARIA WILCHES CORREAL Bachelor of Science in Biology, Los Andes University, 2011 Bachelor of Science in Microbiology, Los Andes University, 2012 A Thesis Submitted to the School of Graduate Studies of the University of Lethbridge in Partial Fulfilment of the Requirements for the Degree MASTER OF SCIENCE Department of Biological Sciences University of Lethbridge LETHBRIDGE, ALBERTA, CANADA ©Diana Maria Wilches Correal, 2016 EFFECTS OF EXTREME TEMPERATURES ON THE SURVIVAL OF THE QUARANTINE STORED-PRODUCT PEST, TROGODERMA GRANARIUM (KHAPRA BEETLE) AND ON ITS ASSOCIATED BACTERIA DIANA MARIA WILCHES CORREAL Date of Defence: November 25/2016 Dr. Robert Laird Associate Professor Ph.D. Co-supervisor Dr. Kevin Floate Research Scientist Ph.D. Co-supervisor Agriculture and Agri-Food Canada Lethbridge, Alberta Dr. Paul Fields Research Scientist Ph.D. Thesis Examination Committee Member Agriculture and Agri-Food Canada Winnipeg, Manitoba Dr. Theresa Burg Associate Professor Ph.D. Thesis Examination Committee Member Dr. Mukti Ghimire Plant Health Safeguarding Ph.D. U.S. Department of Agriculture Specialist Seattle, Washington Dr. Tony Russell Assistant Professor Ph.D. Chair ABSTRACT Trogoderma granarium is a pest of stored-grain products in Asia and Africa, and a quarantine pest for much of the rest of the world. To evaluate extreme temperatures as a control strategy for this pest, I investigated the effect of high and low temperatures on the survival (Chapters 2 and 3) and on the microbiome (Chapter 4) of T. granarium. The most cold- and heat-tolerant life stages were diapausing-acclimated larvae and diapausing larvae, respectively. Trogoderma granarium can be controlled (Probit 9) with an exposure of 70 d to −15°C or 1.2 h to 60°C. High temperatures affected the microbiome; an effect of low temperatures was not detected. While the microbiome changed with life stage, it was dominated by Spiroplasma bacteria. Further research is necessary to understand the Spiroplasma-T. granarium relationship. Future research should also investigate combinations of extreme temperatures with other techniques to shorten the time required for mortality. iii ACKNOWLEDGEMENTS A project such as my thesis would not have been possible without the support and encouragement of a team of amazing people. I would like to start by thanking my supervisors Dr. Kevin Floate, Dr. Paul Fields, and Dr. Robert Laird, who provided the opportunity for me to take on this project and continued their support and encouragement at every step in its development. Dr. Hailey Catton and Dr. Chad Laing eased the burden of my statistical and bioinformatic analyses. If it were not for the insect material provided by Dr. Muhammad Sagheer, my project would not have even made it out of the starting gate. Or perhaps the same could be said for the research funding provided by Agriculture and Agri-Food Canada. For all the emotional support and inspiration, I would like to thank my lab mates, fellow graduate students, Canadian and Colombian friends, and, of course, my family. Lastly, thanks to my lab mentor Paul Coghlin, whose barista skills and cinnamon buns made the mornings that much more pleasant. This team came together for me and gave me the learning experience of a lifetime. Thank you everyone. iv TABLE OF CONTENTS Abstract ........................................................................................................................................ iii Acknowledgements ...................................................................................................................... iv Table of Contents ..........................................................................................................................v List of Tables ............................................................................................................................. viii List of Figures ............................................................................................................................... ix List of Abbreviations ....................................................................................................................x CHAPTER 1: GENERAL INTRODUCTION AND LITERATURE REVIEW ....................1 Objectives .....................................................................................................................................1 Literature review ..........................................................................................................................1 1. Study organism .....................................................................................................................2 2. Control of Trogoderma granarium.......................................................................................4 3. Extreme temperatures ...........................................................................................................5 3.1 Mechanisms of temperature tolerance ...........................................................................6 3.1.1 Mechanisms of cold tolerance ........................................................................7 3.1.2 Mechanisms of heat tolerance .........................................................................7 3.2 Factors affecting thermal tolerance ................................................................................8 3.2.1 Life history of the species and symbiosis with microorganisms ....................8 3.2.2 Stage of development ......................................................................................9 3.2.3 Diapause ........................................................................................................10 3.2.4 Relative humidity ..........................................................................................14 3.2.5 Acclimation ...................................................................................................14 3.2.6 Temperature and duration of exposure .........................................................15 4. Control of dermestids with extreme temperatures ..............................................................15 4.1 Low temperature ..........................................................................................................16 4.2 High temperature .........................................................................................................17 4.3 Models..........................................................................................................................19 5. The insect microbiome and its effects on the host..............................................................22 5.1 Effect of temperature on microbiome-insect relationship ...........................................27 5.2 Effect of microbiome on insect survival at extreme temperatures ..............................29 5.3 The microbiome of Trogoderma granarium................................................................29 5.4 Tools to assess microbiome effects on insect hosts .....................................................33 Structure of the thesis and goals of each chapter ......................................................................37 CHAPTER 2: CONTROL OF Trogoderma granarium WITH LOW TEMPERATURES .56 Abstract ......................................................................................................................................56 Introduction ................................................................................................................................57 Methods ......................................................................................................................................62 Study species and life stages tested ........................................................................................62 Experiment 1: Effect of density on diapause ..........................................................................63 Experiment 2: Supercooling points ........................................................................................63 Experiment 3: Determination of the most cold-tolerant life stage .........................................64 v Experiment 4: Cold-tolerance of diapausing-acclimated larvae at different low temperatures ................................................................................................................................................65 Data analyses ..........................................................................................................................65 Results ........................................................................................................................................67 Experiment 1: Effect of density on diapause ..........................................................................67 Experiment 2: Supercooling point (SCP) ...............................................................................67 Experiment 3: Determination of the most cold-tolerant life stage .........................................68 Experiment 4: Cold-tolerance of diapausing-acclimated larvae at different low temperatures ................................................................................................................................................69 Discussion ..................................................................................................................................70