Using polymerase chain reaction to characterize recent prey consumption by the slender running crab spider, Tibellus Simon (Araneae: Philodromidae) by Brandes Willem Struger-Kalkman A Thesis Presented to The University of Guelph In partial fulfilment of requirements for the degree of Master of Science in Environmental Sciences Guelph, Ontario, Canada © Brandes Struger-Kalkman, February, 2016 ABSTRACT USING POLYMERASE CHAIN REACTION TO CHARACTERIZE RECENT PREY CONSUMPTION BY THE SLENDER RUNNING CRAB SPIDER, TIBELLUS SIMON (ARANEAE: PHILODROMIDAE) Brandes Struger-Kalkman Advisor: University of Guelph, 2015 Professor J.M. Schmidt This thesis investigates the use of polymerase chain reaction (PCR) as a tool for characterizing the recent prey consumption of spiders. A methodology using PCR was developed to detect a 543-bp region of DNA from the cytochrome c oxidase subunit I gene of Drosophila suzukii (Matsumura). Feeding experiments involving slender running crab spiders, Tibellus Simon spp., revealed the robust detection of target DNA from the extracts of Tibellus for up to 60 h after one D. suzukii was consumed. Further experiments showed that consuming multiple D. suzukii extended detection to 96 h in Tibellus. A field study demonstrated that the method detected recent predation by 31% of Tibellus collected in the field. However, an evaluation of the specificity of the method determined that modification is necessary for precisely identifying the prey of field spiders. Avenues of future research on spider predation in both the laboratory and field are discussed. ACKNOWLEDGEMENTS I am forever grateful to everyone that has helped to make this project a success. First and foremost, I want to thank my advisor, Dr. Jonathan Schmidt, and my co-advisor, Dr. Marc Habash. Jonathan, I thank you first for developing my interest in spiders. As you remember, I gave them no credit whatsoever until you opened my eyes to their potential. I also want to acknowledge your support, guidance, and criticism throughout this process, all of which have helped me develop my own critical thinking and broaden my views (by „turning things on their head‟). I wish to also highlight your wisdom and generosity, but most of all your patience, which knows no limits. Marc, I found your knowledge of the molecular side of this research to be very insightful. I thank you for helping me develop experiments to explore the intricacies of my work and for your input in developing this thesis. I also want to thank Dr. Alex Smith and Dr. Paul Sibley for their reviews of my thesis and for their contributions during my defense. I want to thank Dr. Adam Chippindale and Dr. Justin Renkema for contributing Drosophila spp. with which we could rear spiders and conduct feeding experiments. I also give thanks to Gillian Ferguson (Ontario Ministry of Agriculture, Food, and Rural Affairs) for arranging sampling visits in Leamington greenhouses. This allowed me to gain some hands-on experience with spiders in crop systems. The spiders collected from these greenhouses will not go untested. I also need to thank Jonathan Gaiero, whose help was fundamental in developing my PCR assay. I want to thank Kiera Belley and Alicia Newman for their assistance both inside and outside of the laboratory. Both had a big hand in collecting and maintaining spiders as well as in rearing Drosophila. Without their efforts, research would have been far more challenging. I also want to thank you, Kiera, for our philosophical discussions and for your help with my molecular work. I am so grateful to Amanda Poole for her dedication. Amanda, you kept my spirits up when they were sinking, you kept me aligned with my goal, and you enured my “creative process”. I am also grateful to Michael Tomascik for the same reasons and more. Mike, you and I spent a lot of time collecting, feeding, identifying, observing, and discussing our spiders. I have benefited both as a person and as an academic from our experiences and accomplishments throughout this endeavour. I wish you nothing but the best in life, friend, and let us not end our adventures yet. iii Ever since I was young, I have had a curiosity about bugs and I have my family to thank for helping me to develop this interest. My fascination with insects probably began one summer well over a decade ago when my brother Arih and I collected whatever insects we could catch and pinned them inside egg cartons. I owe everything to Arih; he has always been by my side and has always helped me to be the best I can be. I am also thankful for my mother and father, Klari Kalkman and Stephen Struger, who, both rooted in biology, provided a supportive environment in which I could continue to collect and study insects. I also want to acknowledge my grandparents, Wim and Janet Kalkman and John and Theresa Struger, for always encouraging my pursuit of entomology, whether it was by sending cards decorated with pictures of insects or by supplying jars in which I could keep specimens. I cannot mention everybody that has helped to support me, but I also want to acknowledge the Ellis family, Rebecca “Russ” David, the Monticello “family”, and Savannah Vince for their encouragement and support. A special thank you goes to Dr. Gard Otis, whose expert advice made this entire thesis possible. iv TABLE OF CONTENTS ACKNOWLEDGEMENTS iii TABLE OF CONTENTS v LIST OF TABLE viii LIST OF FIGURES ix ABBREVIATIONS x CHAPTER 1. INTRODUCTION 1.1 Overview of the importance of spiders to agriculture 1 1.2 Choice of method to detect prey consumption by spiders 1.2.1 Evolving beyond observational methods 5 1.2.2 Discussion of detection methods 7 1.3 Molecular analysis of spider prey using PCR 1.3.1 Review of laboratory feeding assays 14 1.3.2 Summary of field studies with comparison to lab studies 27 1.3.3 Research gaps considered 33 1.4 Objectives and model organism choices 1.4.1 Research goals and objectives 35 1.4.2 Choice of detection method 36 1.4.3 Choice of model spider and prey 37 CHAPTER 2. MATERIALS AND METHODS 2.1 General protocols 2.1.1 Collection and maintenance of spiders 41 2.1.2 Collection and rearing of Drosophila 42 2.1.3 Feeding protocol 43 2.1.4 Molecular protocols 45 2.1.5 Statistical analysis 49 2.2 Suitability of selected primers for detecting prey DNA 2.2.1 Validation of DNA detection using selected primers 50 2.2.2 Specificity of the 543-bp PCR assay 52 2.2.3 Sensitivity of the 543-bp PCR assay 55 v 2.2.4 The detection of D. suzukii DNA in spiders 56 2.3 Optimizing the post-consumption detection of D. suzukii DNA in Tibellus using PCR 2.3.1 Effect of Tibellus sample storage on the detection of consumed D. suzukii DNA 60 2.3.2 Effect of thawing Tibellus on the detection of consumed D. suzukii DNA 61 2.3.3 Efficiency of extracting Tibellus DNA 61 2.3.4 Replicability of the PCR result 62 2.4 Application of the optimized PCR detection method to feeding and field experiments 2.4.1 The decay of a 543-bp region of D. suzukii DNA detected in Tibellus 63 2.4.2 Effect of multiple prey on the detection of D. suzukii DNA in Tibellus 67 2.4.3 Effect of scavenging on the detection of D. suzukii DNA in Tibellus 69 2.4.4 Screening field-collected Tibellus for prey DNA using the 543 bp PCR assay 70 CHAPTER 3. RESULTS AND DISCUSSION 3.1 Suitability of selected primers for detecting prey DNA 3.1.1 Validation of DNA detection using selected primers 71 3.1.2 Specificity of the 543-bp PCR assay 73 3.1.3 Sensitivity of the 543-bp PCR assay 80 3.1.4 The detection of D. suzukii DNA in spiders 83 3.2 Optimizing the post-consumption detection of D. suzukii DNA in Tibellus using PCR 3.2.1 Effect of Tibellus sample storage on the detection of consumed D. suzukii DNA 85 3.2.2 Effect of thawing Tibellus on the detection of consumed D. suzukii DNA 87 3.2.3 Efficiency of extracting Tibellus DNA 88 3.2.4 Replicability of the PCR result 90 3.3 Application of the optimized PCR detection method to feeding and field experiments 3.3.1 The decay of a 543-bp region of D. suzukii DNA detected in Tibellus 94 3.3.2 Effect of multiple prey on the detection of D. suzukii DNA in Tibellus 103 3.3.3 Effect of scavenging on detection of D. suzukii DNA in Tibellus 106 3.3.4 Screening field-collected Tibellus for prey DNA using the 543 bp PCR assay 109 CHAPTER 4. CONCLUSIONS 112 REFERENCES 117 APPENDIX I – LITERATURE SOURCES FOR PREY DETECTION METHODS 124 APPENDIX II – SPIDERS STUDIED USING POLYMERASE CHAIN REACTION 125 vi APPENDIX III – SPIDERS COLLECTED IN CANADIAN AGROECOSYSTEMS 129 APPENDIX IV – OCCURRENCES OF TIBELLUS SPECIES 130 APPENDIX V – SITES FOR THE COLLECTION OF SPIDERS 132 APPENDIX VI – PRELIMINARY EXPERIMENT TO DETERMINE OPTIMAL PRIMER CONCENTRATION FOR THE 543-BP PCR ASSAY 133 APPENDIX VII – RESULTS FOR IN SILICO AND IN VITRO SPECIFICITY TESTS 135 APPENDIX VIII – PRELIMINARY EXPERIMENT TO PROBE SENSITIVITY OF THE 543-BP PCR ASSAY 139 APPENDIX IX – PRELIMINARY EXPERIMENT TO EFFECT VARIATION IN BRIGHTNESS OF PCR RESULTS AMONG FED SPIDERS 140 APPENDIX X – RESULTS OF MULTIPLE-PREY FEEDING EXPERIMENTS 142 vii LIST OF TABLES Table 1.1 Molecular half-lives of prey DNA detected in two spider species using PCR.