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The Pennsylvania State University The Graduate School Department of Entomology FUNGAL PARASITES THAT MANIPULATE ANT BEHAVIOR: TOWARD A MECHANISTIC UNDERSTANDING OF AN EXTENDED PHENOTYPE A Thesis in Entomology by Maridel Anne Fredericksen 2016 Maridel Anne Fredericksen Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science August 2016 ii The thesis of Maridel Anne Fredericksen was reviewed and approved* by the following: David P. Hughes Assistant Professor of Entomology and Biology Thesis Advisor Christina M. Grozinger Distinguished Professor of Entomology Thomas C. Baker Distinguished Professor of Entomology István Mikó Research Associate Gary W. Felton Department Head of Entomology *Signatures are on file in the Graduate School iii ABSTRACT Parasite manipulation of host behavior represents an extended phenotype of a parasite’s genes through a host’s body. Studying the proximate mechanisms by which a parasite induces abnormal behavior in its host can help us understand how behavior in general is regulated and how such specialized symbioses evolve. Fungi from the species complex Ophiocordyceps unilateralis manipulate ants to die in elevated locations that are suitable for the fungus to transmit to new hosts. This association provides a promising system to discover mechanisms of manipulation using new molecular tools. However, current progress in this system is limited because we lack a cellular context with which to interpret –omics data, and research so far has focused on only a handful of the hundreds of species that exist in nature. In this thesis, I use two approaches to explore this fungal parasite’s exploitation strategies and their effects on ant behavior. In Chapter 2 I provide a context for molecular studies by characterizing the cell-level interactions between parasite and host during manipulated host biting behavior. Using three-dimensional microscopy and automated image-segmentation techniques, I demonstrate that the fungus invades host muscle fibers and joins together to form extensive fungal networks throughout the ant’s body. In Chapter 3 I expand our current knowledge on the diversity of the manipulation by comparing transmission strategies of several fungal species. I reveal that infected ants in the Brazilian Amazon display species-specific patterns in biting behavior, and I show that the fungi infecting these ants exhibit periodic patterns of spore release. These findings serve as parameters for future studies to discover the processes underlying these patterns. Overall, the results from this thesis advance the field toward a mechanistic understanding of behavioral manipulation by parasites. iv TABLE OF CONTENTS List of Figures ......................................................................................................................... vi List of Tables ........................................................................................................................... vii Acknowledgements .................................................................................................................. viii Chapter 1 Introduction ............................................................................................................. 1 Behavioral Manipulation: progress and limitations ......................................................... 2 Ophiocordyceps unilateralis s.l.: a promising new system .............................................. 4 Thesis objectives .............................................................................................................. 7 Chapter 2 Life inside a controlled ant: 3D visualization reveals fungal parasite networks in manipulated hosts ......................................................................................................... 10 ABSTRACT ..................................................................................................................... 10 SIGNIFICANCE .............................................................................................................. 11 INTRODUCTION ........................................................................................................... 11 RESULTS ........................................................................................................................ 15 O. unilateralis s.l. and B. bassiana are present throughout the host and cause gross changes in muscle fiber arrangement. ..................................................... 15 The fungus O. unilateralis s.l. invades muscle fibers and forms fusions between fungal cells ....................................................................................................... 16 Fungal fusions create extensive networks ................................................................ 18 DISCUSSION .................................................................................................................. 19 MATERIALS and METHODS ........................................................................................ 23 Infection and Sample Collection .............................................................................. 23 Histology .................................................................................................................. 25 Serial Block-Face Scanning Electron Microscopy ................................................... 25 Data collection.......................................................................................................... 26 Statistical Analysis ................................................................................................... 27 Deep Learning Model ............................................................................................... 27 ACKNOWLEDGEMENTS ............................................................................................. 28 Chapter 3 Zombie ant diversity in a Brazilian rainforest: relating extended phenotype to transmission ..................................................................................................................... 34 ABSTRACT ..................................................................................................................... 34 INTRODUCTION ........................................................................................................... 35 METHODS ...................................................................................................................... 38 Field Collections ...................................................................................................... 38 Spore Clocks ............................................................................................................ 39 Identification ............................................................................................................ 40 Biting substrates ....................................................................................................... 41 Data Analysis: Interaction Network ......................................................................... 42 v RESULTS ........................................................................................................................ 42 Host biting location .................................................................................................. 42 Timing of spore release ............................................................................................ 45 DISCUSSION .................................................................................................................. 47 Biting location: Infected ants show species-specific patterns .................................. 48 Timing of spore release: Fungi show consistent patterns ......................................... 52 ACKNOWLEDGEMENTS ............................................................................................. 54 Chapter 4 Conclusions and Future Directions ......................................................................... 64 Appendix A Supplementary Material for Chapter 2 ....................................................... 69 Appendix B Supplementary Material for Chapter 3 ....................................................... 76 Ant morphospecies descriptions ............................................................................... 76 REFERENCES................................................................................................................. 83 vi LIST OF FIGURES Figure 2-1. Distribution of O. unilateralis s.l. in the host body .......................................... 30 Figure 2-2. Fungal abundance in muscle tissue ................................................................... 31 Figure 2-3. Fungal behaviors observed in O. unilateralis s.l.-infected ant muscles .......... 32 Figure 2-4. 3D reconstruction of fungal networks surrounding muscle fibers................. 33 Figure 3-1. Ant/plant interaction network ........................................................................... 55 Figure 3-2. Ant/substrate interaction network .................................................................... 56 Figure 3-3. Diversity of ants found on spiny palms compared to non-spiny palms ......... 57 Figure 3-4. Biting locations of ants on spiny palms ............................................................ 58 Figure 3-5. Volume of spore release for all specimens combined ...................................... 59 Figure 3-6. Spore count over two days ................................................................................

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