The Pennsylvania State University The Graduate School Department of Biology OF HORSENETTLE & HORNWORMS: THE DAMAGE MECHANISMS OF NON-GLANDULAR TRICHOMES AND THEIR EFFECTS ON HERBIVORES A Dissertation in Biology by M. Alexandra Serpi Ó 2019 M. Alexandra Serpi Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy August 2019 The dissertation of M. Alexandra Serpi was reviewed and approved* by the following: James H. Marden Associate Director of Operations, Huck Institutes of the Life Science Professor of Biology Chair of Committee Rudolf Schilder Assistant Professor of Entomology and Biology Tanya Renner Assistant Professor of Entomology Stephen W. Schaeffer Associate Department Head of Graduate Education Professor of Biology Tracy Langkilde Professor and Head of the Department of Biology Dissertation Advisor *Signatures are on file in the Graduate School iii ABSTRACT In their endless struggle to survive, organisms constantly suffer damage from their surrounding environment. Pathogens infect. Toxins poison. Predators bite. Based on their own struggle for survival, plants have evolved numerous mechanisms to defend themselves against herbivory. These mechanisms are divided into two, broad categories: chemical and physical defenses. Chemical defenses include distasteful and toxic compounds. Physical defenses include spines, leaf toughness, and trichomes that make it difficult for herbivores to consume plants. Trichomes are particularly diverse, grow in many forms, and may or may not contain a distal gland. While glandular trichomes have been shown to defend plants by trapping or poisoning small herbivores, non-glandular trichomes are less well-studied. This dissertation explored the short-term, long-term, and transgenerational effects experienced by a lepidopteran herbivore after consuming non-glandular trichomes. To examine the effects of trichomes alone, I removed the trichomes from Horsenettle (Solanum carolinense) and added them to an artificial diet that lacked other leaf chemicals or structures. To examine the effects of consuming trichomes in situ on leaves, I created two treatments, one with trichomes and one with trichomes removed, using the same species to maintain consistent leaf chemistry. I observed the effects on Tobacco Hornworms (Manduca sexta) after consuming these diets by measuring their growth, efficiency, and survival. I collected eggs of this first generation to explore potential, trichome-induce transgenerational effects on offspring growth, efficiency, and survival. Finally, I sought to understand which characteristics of non-glandular trichomes damaged larvae. Together, these studies revealed that non-glandular trichomes, consumed as part of a natural diet, chemically and physically damaged hornworms. The resulting damage to the midgut epithelium allowed the gut contents to leak into the surrounding hemocoel which lead to energetic iv resources being diverted from growth to other metabolic processes; likely tissue repair and immune response mechanisms. As a result, larvae ate less diet and gained less mass. Their offspring also diverted energy to other metabolic processes, were smaller, and more likely to survive. Ultimately, this collection of work not only provides a detailed example of the long-term and transgenerational effects of damage, but also identified the mechanism behind the damage. v TABLE OF CONTENTS LIST OF FIGURES ..................................................................................................................... vii ACKNOWLEDGEMENTS ......................................................................................................... xi CHAPTER 1 INTRODUCTION ................................................................................................. 1 STUDY SYSTEM ............................................................................................................... 8 Tobacco hornworms (Manduca sexta) ....................................................................... 8 Horsenettle (Solanum carolinense) .......................................................................... 11 REFERENCES ................................................................................................................. 16 CHAPTER 2 DAMAGE FROM NON-GLANDULAR TRICHOMES SUPPRESSES THE GROWTH AND DIET CONSUMPTION OF AN HERBIVORE .......................................... 28 ABSTRACT ..................................................................................................................... 28 INTRODUCTION ............................................................................................................. 29 METHODS ...................................................................................................................... 32 RESULTS ........................................................................................................................ 37 Diet Consumption & Efficiency following Acute and Chronic Exposure ................. 37 Size, Development Time, & Survival following Chronic Exposure .......................... 40 Reproduction following Chronic Exposure .............................................................. 43 DISCUSSION ................................................................................................................... 45 CHAPTER 3 NON-GLANDULAR TRICHOMES CHEMICALLY AND PHYSICALLY DAMAGE HERBIVORES .......................................................................................................... 65 ABSTRACT ..................................................................................................................... 65 INTRODUCTION ............................................................................................................. 66 vi METHODS ...................................................................................................................... 70 Modified Trichomes Experiment ............................................................................... 70 Fluorescent Diet Experiment .................................................................................... 73 RESULTS ........................................................................................................................ 75 Modified Trichomes Experiment ............................................................................... 75 Fluorescent Diet Experiment .................................................................................... 77 DISCUSSION ................................................................................................................... 79 REFERENCES ................................................................................................................. 89 CHAPTER 4 DAMAGE FROM NON-GLANDULAR TRICHOMES HAS TRANSGENERATIONAL CONSEQUENCES FOR AN HERBIVORE ............................. 94 ABSTRACT ..................................................................................................................... 94 INTRODUCTION ............................................................................................................. 95 METHODS ...................................................................................................................... 98 RESULTS ...................................................................................................................... 103 Offspring Color & Size ........................................................................................... 103 Offspring Diet Consumption & Efficiency .............................................................. 109 Offspring Development, Survival, & Reproduction ................................................ 114 DISCUSSION ................................................................................................................. 117 REFERENCES ............................................................................................................... 130 APPENDIX TRICHOME DENSITY CALCULATION ...................................................... 137 vii LIST OF FIGURES Figure 1-1: Timeline of Data collection. ................................................................................ 14 Figure 1-2: Tobacco Hornworm (Manduca sexta). ............................................................... 15 Figure 1-3: Horsenettle (Solanum carolinense). .................................................................... 15 Figure 2-1: Comparison of diet consumption and efficiency metrics for fifth instar hornworm larvae after 48 hours of feeding on treatment diets. Acute larvae were reared on Ordinary Diet from hatching to fifth instar before exposure to treatment diets (n = 110). Chronic larvae were reared on treatment diets from hatching to fifth instar (n = 550). Metrics are organized in columns from left to right: mean amount of diet consumed (g), mean mass gain (g), percent of diet excreted as frass (%), mean efficiency of conversion (ECI, %), and mean metabolic gap (%). Treatments are represented by bars on each graph from left to right: Ordinary Diet, Trichome Diet, Shaved Leaves, and Whole Leaves. Gray bars indicate diets without trichomes; black bars indicate diets with trichomes. Error bars indicate standard error. Different letters on bars indicate statistically significant differences between groups using a Tukey post hoc comparison. Effect sizes for Trichome presence and Natural Diet Base are listed under each bar graph and were calculated using Cohen’s f2 (Small effect: f2 > 0.02,