THE PENNSYLVANIA STATE UNIVERSITY SCHREYER HONORS COLLEGE DEPARTMENT OF VETERINARY AND BIOMEDICAL SCIENCES UNDERSTANDING OPHIOCORDYCEPS, THE ZOMBIE ANT FUNGUS: A CASE STUDY IN HOST BEHAVIORAL MANIPULATION BENJAMIN FOWLER SPRING 2016 A thesis submitted in partial fulfillment of the requirements for baccalaureate degrees in Immunology and Infectious Disease & Toxicology with honors in Immunology and Infectious Disease Reviewed and approved* by the following: David P. Hughes Assistant Professor of Entomology and Biology Thesis Supervisor Pamela A. Hankey Giblin Professor of Immunology Honors Adviser * Signatures are on file in the Schreyer Honors College. i ABSTRACT Millions of years of evolution have led many pathogens to develop unique strategies to maximize their transmission. Behavioral manipulation is one of these strategies and has captured wide audiences with the idea of zombie-like mind control. Ophiocordyceps is a genus of fungal parasites that infect insects, leading to the zombie-ant phenomenon, in which an infected ant leaves its home colony, climbs onto foliage above the forest floor, and subsequently dies. The species complex O. unilateralis even causes infected ants to bite into the plant’s flesh prior to death. Previous work has shown that this manipulation is essential for successful maturation of the fungus, but studies have not evaluated the role this manipulation may play in transmission of the fungus to susceptible host ants. Chapter 2 of this thesis introduces a novel strategy to evaluate the transmission potential of a particular species of the zombie-ant fungus, O. camponoti-atricipis. Experiments using spore-clocks and gravitational grids indicated that the fungus tightly concentrates infectious ascospore release during the early morning hours with a clear peak between 0500 and 0600 hours and that the release of spores is focused downwards by gravitropic growth. Chapter 3 of this thesis covers efforts to use phylogenetic analyses to assess the host specificity of O. unilateralis sensu lato. Together, these studies of transmission and host selectivity provide insights into the evolution of both behavioral manipulation and host specificity that help understand and model potential zoonotic diseases of humans. ii TABLE OF CONTENTS LIST OF FIGURES ..................................................................................................... iii ACKNOWLEDGEMENTS ......................................................................................... v Chapter 1 Introduction ................................................................................................. 1 References ........................................................................................................................ 6 Chapter 2 Transmission Potential ................................................................................ 8 Background ...................................................................................................................... 8 Methods ............................................................................................................................ 12 Sample Collection .................................................................................................... 12 Physical Characterization ......................................................................................... 13 Temporal Release Monitoring .................................................................................. 13 Results .............................................................................................................................. 14 Fecundity and morphological characteristics of spore producing body ................... 14 Timing of spore release ............................................................................................ 17 Discussion ........................................................................................................................ 18 Conclusions ...................................................................................................................... 22 References ........................................................................................................................ 23 Chapter 3 Host Specificity and Fungal Diversity ........................................................ 27 Background ...................................................................................................................... 27 Methods ............................................................................................................................ 30 Sample Collection .................................................................................................... 30 DNA Extraction, Cleaning, PCR, Sequencing, and Phylogenetic Analysis............. 30 Discussion ........................................................................................................................ 31 References ........................................................................................................................ 33 Chapter 4 Conclusions ................................................................................................. 37 Appendix A Attachment to Leaves ............................................................................. 40 Ophiocordyceps lloydii var. binata .................................................................................. 42 Ophiocordyceps unilateralis ............................................................................................ 50 References ........................................................................................................................ 53 Academic Vita ............................................................................................................. 54 iii LIST OF FIGURES Figure 1: A dead ant attached to a palm leaf in the Amazon with the fungus Ophiocordyceps camponoti-atricipis growing from its head ...................................................................... 4 Figure 2: Morphology of O. camponoti-atricipis. A) O. camponoti-atricipis specimen, the red arrow indicates the ascoma and the black brace indicates measured length to ascoma. B) Composite image of the internal and external morphology of the ascoma. An ostiole is indicated with a black arrow and a perithecium is indicated with a red arrow. C) A perithecium removed from an ascoma on a 25G needle (bar = 100 microns). D) Cluster of asci as they appear in a smashed perithecium, 100x. E) Ascus consisting of eight ascospores, 100x. F) Individual ascospore, 100x. ............................................................................... 11 Figure 3: Morphological and reproductive quantification of O. camponoti-atricipis. A) The distance of the fungal stalk (stroma) from the ant cadaver to the fruiting body (ascoma). The mean length to ascoma is 3.97 mm (n=40, s.d. 2.52 mm, range 1.03-11.5 mm). B) Inner circle (red): Growth of the fungal stalk. Stromal growth clustered around directly downwards. The mean distance from vertical is 25.8° (n=19, s.d. 21.1°, range 1.5-76.6°). Outer circle (blue): Direction of ant. The direction the ant faced in the same plane as the fungus was random and had no association with the angle of stromal growth. C) The number of ostioles present on a single ascoma. The mean ostiole count is 185 (n=10, s.d. 109, range 66-413). D) The mean count of the ascospores contained in a single perithecium is 2,212 (n=34, s.d. 761, range 719-4282). .................................................................................... 16 Figure 4: Hourly quantification of ascospores released by O. camponoti-atricipis. After either 12 hours (BD15_5, BD15_6, & BD15_7) or 24 hours (BD15_1, BD15_23, BD15_19, BD15_10, BD15_28, the number of ascospores released each hour was counted. Estimated values for specimens with indistinct hourly patches or such dense spore release that individual ascospores were indistinguishable are designated with dotted lines. The mean count of released ascospores is noted with a dashed line. ................................................ 17 Figure 5: Ophiocordyceps lloydii var. binata. Note the light tan mycelial growth covering the ant's body and attaching it to the leaf (red circle). ........................................................... 41 Figure 6: Higher magnification O. lloydii var. binata. Note that the ant's mandibles (red arrow) are not biting leaf tissue and that fungal growth is extensive around the base of the legs (red circle). .............................................................................................................................. 41 Figure 7: Ophiocordyceps lloydii var. binata mandibles. Note the clustering of fungal growth around the mouthparts (red circle). .................................................................................. 43 Figure 8: O. lloydii var. binata mycelium growing from the base of the legs viewed from the basal surface after removal of the leaf. Note how the growth radiates outwards. ............ 44 Figure 9: Higher magnification of the basal side of O. lloydii var. binata. Note the tubular mesh with a membrane (red arrow) between the strands. ......................................................... 45 iv Figure 10: Even higher magnification of the basal side of O. lloydii var. binata. Note the mycelium on the right and the tear in the membrane (red arrow) which reveals empty space below the membrane. ......................................................................................................