BEHAVIORS MEDIATE APHID INTERACTIONS WITH NATURAL ENEMIES AND THE ENVIRONMENT A Dissertation Submitted to the Graduate Faculty of the North Dakota State University of Agriculture and Applied Science By James Kopco In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY Major Department: Entomology November 2016 Fargo, North Dakota North Dakota State University Graduate School Title Behaviors mediate aphid interactions with natural enemies and the environment By James Kopco The Supervisory Committee certifies that this disquisition complies with North Dakota State University’s regulations and meets the accepted standards for the degree of DOCTOR OF PHILOSOPHY SUPERVISORY COMMITTEE: Jason Harmon Chair Marion Harris Erin Gillam Ned Dochtermann Approved: November 2, 2017 Frank Casey Date Department Chair ABSTRACT Behavior is a crucial component of ecology that mediates how animals interact with one another and with the environment. Behaviors can allow animals to avoid the harmful effects of things like competition, predation, and extreme abiotic conditions. However, animals often have constraints that limit the potential benefits of their behaviors, so we addressed what factors contribute to these constraints in plant-aphid-wasp systems. Parasitoids of aphids are tiny wasps that lay their eggs in aphids, where the larva feeds and develops. Each aphid can only sustain a single parasitoid, so parasitoids mark aphids when they lay an egg to discourage others from laying additional eggs. Not all parasitoids mark aphids the same way, and whether species with different marks can recognize one another’s mark was unclear. We found that parasitoids with different marks fail to respond to one another’s marks. Because these parasitoids laid eggs in aphids that were already parasitized, competition between them will occur through mortal combat between the larvae rather than egg-laying restraint in the adults. Parasitoid attack induces aphid behaviors such as fighting, running away, or dropping off the host plant. These behaviors can deter or evade the attacker, but they cost the aphid an opportunity to feed. This feeding interruption decreases the number of offspring the aphids produce. When aphids run from a threat, they usually resettle on the newest leaves of a plant, where their reproduction is enhanced. However, when they drop from a plant to escape, they resettle randomly and do not benefit from the better location, indicating that more vigorous responses can lose benefits. We also showed that aphids exhibit similar movement and resettling behaviors when disturbed by heat shocks. However, this behavior did not affect aphid reproduction or survival when induced by a heat shock. While movement to new leaves appears to be a general response to several disturbances, its benefits to aphids only occur in specific situations. We have shown that behaviors can be constrained by unfamiliar stimuli and the general context in which they are invoked. These constraints can dictate how insects interact with each other and the environment. iii ACKNOWLEDGEMENTS I would like to express gratitude to the many people who helped me survive graduate school and finish this dissertation. My adviser, Dr. Jason Harmon, offered tremendous help, both with the nuts-and- bolts science and with staying sane as I worked. Dr. Deirdre Prischman-Voldseth was also an invaluable mentor, offering advice, encouragement, and professional development opportunities every step of the way. I also benefitted tremendously from the camaraderie and collaboration of the lab team, especially Aleix Valls, Jackie Nelson, Erin McClain, Tyler Follman, Joe Alfonso, and Jamin Dreyer, who all contributed insights and encouragement. Of course, I had support from outside the entomological community as well that helped push me through to complete this dissertation. The unwavering support of my family, especially during the most stressful parts of graduate school, kept me sane and optimistic. Nick, Mom, and Dad, your non-stop support was fantastic. Liz really pushed me in the final stretches of writing this dissertation, and her support made all the difference in the world. The colorful cards from Aunt Kathy, smothered in stickers and full of corny jokes were always a welcome pick-me-up. Aunt Andi and Uncle Ray plus long-term friends like Ken and Alex were always encouraging and game to commiserate. And of course, the huge Kopco family gatherings every Christmas offered a valuable mid-winter boost of encouragement, even if I had to do some Facebook creeping to make sure I know everyone’s kids’ names. iv TABLE OF CONTENTS ABSTRACT……...……………………………………………………………………………………………………iii ACKNOWLEDGMENTS……...……………………………………………………………………………………..iv LIST OF TABLES …..……..…………………………..……………………………………………………………viii LIST OF FIGURES ….……………………………………………………………………………………………….ix INTRODUCTION ……………………………………………………………………………………………………..1 References ………………………………………………………………………………………………………3 TO STING OR NOT TO STING: THE ROLE OF HOST MARKING MECHANISMS IN INTER-SPECIFIC MARK RECOGNITION BY PARASITOIDS ……………………………………………………………………….4 Abstract ………………………………………………………………………………………………………….4 Introduction ………………………………………………………………………………………………………5 Methods …………………………………………………………………………………………………………..8 Study System ……………………………………………………………………………………………….8 Rearing of Study Insects …………………………………………………………………………………..9 Sequential Exposure of Aphid Groups to Parasitoids ……..………………………………………….11 Parasitoid Response to Parasitized or Non-parasitized Aphids…………………………………12 Aphid Defensive Behaviors ………………………………………………………………..………..14 Exposure of Stung or Bystander Aphids to L. testaceipes ……………………………………………16 Results ……………………………………………………………………………………………………...…..18 Sequential Exposure of Aphid Groups to Parasitoids ……………...…………………………………18 Parasitoid Response to Parasitized or Non-parasitized Aphids ……...…………………………18 Aphid Defensive Behaviors………………………………………………………………………….20 Exposure of Stung or Bystander Aphids to L. testaceipes …………………………………………...20 Discussion ………………………………………………………………………………………………………22 Acknowledgments ……………………………………………………………………………………………..31 References ……………………………………………………………………………………………………..31 DISTURBANCES ALTER WITHIN-PLANT DISTRIBUTION TO THE POTENTIAL BENEFIT OF HERBIVOROUS INSECTS………...………………………………………………………………………………37 v Abstract …………………………………………………………………………………………………………37 Introduction ……………………………………………………………………………………………………..37 Methods …………………………………………………………………………………………………………41 Study System ……………………………………………………………………………………………..41 Aphids …………………………………………………………………………………………………41 Plants ………………………………………………………………………………………………….41 Experiments ……………………………………………………………………………………………….42 Time to Resettlement After Disturbance ………………………….…………………………….…42 Fitness of Aphids on Actively Growing Leaves vs. Old Leaves …………………………………44 Disturbance Frequency Experiments ..............................……………………..…………………45 Disturbance Frequency Experiment Analysis ………………………………………………….....46 Results….……………………………………………………………………………………………………….48 Time to Resettlement After Disturbance ...…………………………………….…………………..48 Fitness of Aphids on Actively Growing Leaves vs. Older Leaves ………………………………48 Disturbance Frequency ………………………………………………………..…………………….49 Discussion ………………………………………………………………………………………………………51 Acknowledgements ……………………………………………………………………………………………55 References ……………………………………………………………………………………………………..55 APHID BEHAVIORAL RESPONSES TO HEAT SHOCKS DO NOT AFFECT SHORT-TERM APHID FITNESS …………………………………………………………………………………………………………….61 Abstract …………………………………………………………………………………………………………61 Introduction ……………………………………………………………………………………………………..61 Methods …………………………………………………………………………………………………………64 Study System …………..………………………………………………………………………………….64 Behavioral Responses to Heat Shocks ……………..………………………………………………….66 Analysis of Behavioral Responses to Heat Shocks .............………………………………………….68 Fitness Effects………………………………………..……………………………………………………70 Analysis of Fitness Effects ……………………….………………………………………………………71 vi Results …………………………………….……………………………………………………………………72 Behavioral Responses to Heat Shocks ………………………………………………………………...72 Fitness Effects …………………………………………………………………………………………….74 Discussion ………………………………………………………………………………………………………77 Acknowledgements ……………………………………………………………………………………………83 References ……………………………………………………………………………………………………..83 CONCLUSIONS AND FUTURE DIRECTIONS …………………………………………………………………88 References ……………………………………………………………………………………………………..91 vii LIST OF TABLES Table Page 1. Treatments in the sequential exposure of whole aphid colonies experiment……...….....….…………….12 2. Variables analyzed in the sequential exposure of whole aphid colonies experiment ……...…...………..14 3. Specific timeline of disturbances …...……….…………………..………………………………..……………47 4. Coefficient estimates for effects of disturbance on actively growing leaves colonization ….…..…......…51 5. Temperatures at each height in behavioral responses to heat shocks experiment …....………..……….68 6. Treatments and sample sizes from the fitness effects experiment …....…………………………..……….71 7. Results of MANOVA analysis of the change in aphids in each fourth of the plants…..………..……..….74 8. Results of the ANOVAs testing changes in the number of aphids in each quarter of the plant .……..…75 viii LIST OF FIGURES Figure Page 1. Comparisons between characteristics of foraging behaviors (± SEM) for L. testaceipes (A, C, E, and G, open bars) and B. communis (B, D, F, H, shaded bars) …..……………………...…….19 2. Aphid defensive response to wasps …………...……………………………………………………………...21 3. The proportion of defensive or non-defensive