Survey of Mesomycetozoans in Millipedes and Terrestrial Isopods of Southeastern New England By: Stephanie Langlois A Study Presented to the Faculty of Wheaton College in Partial Fulfillment of the Requirements for Graduation with Departmental Honors in Biology Norton, Massachusetts 5/20/17 2 Acknowledgements Thank you, First and foremost, to Professor Dyer for your support, encouragement, and enthusiasm throughout the duration of this project. This project was a huge learning curve for the both of us, and I am happy I had the privilege to work with you. Thank you for believing I could do this. To Zephorene Stickney-Helmreich for your advice and encouragement. You helped me complete this project in an organized and timely manner. Thank you for keeping me level-headed. To Professor Cato and Professor Celada for being on my thesis committee. Your advice and edits were greatly appreciated. I hope you enjoyed learning all about trichomycetes. To Professor Post, for your friendliness and always being super quick to get things done. To Jillian Amaral for her help finding such obscure sources. To Nathan Kelly, for digging through woodpiles with me in the dead of winter and rescuing me from the spiders. Someday, I may forgive you for falling asleep several times while reading my thesis introduction. To Laura Wajda, for being my one friend with a passion for science. It means the world to me that you think this stuff is cool. You are so smart, talented, and hardworking, and you inspire me every day. To all of my friends and family, who have spent way too much time listening to me talk about obscure gut fungi and protists. Thank you for listening. To the Wheaton biology department, for encouraging students to think outside the box and pursue their passions. With a government that does not believe in the value or validity of science, you have given Wheaton students the education they need to fight back. Your support for women in STEM makes all the difference. I do not think I could have gotten such a unique and empowering education anywhere else. 3 Table of Contents I. Introduction 1. Overview of Trichomcyetes a. Why are Mesomycetozoa Important? 2. History of Discoveries 3. The Phylogenetic Debate a. Harpellales b. Amoebidiales c. Asellariales d. Eccrinales 4. Trichomycetes Typically Found in Isopoda and Diplopoda Host Populations a. Genera Found in Millipedes (Diplopoda) b. Genera Found in Isopoda 5. The Importance of the White 1999 Study and this Subsequent New England Study II. Methods 1. Study Subjects 2. Collection of Isopods 3. Collection of Millipedes 4. Maintaining Millipedes and Isopods 5. Dissecting Isopods 6. Dissecting Millipedes 7. Slide Preparation of Isopoda 8. Slide Preparation of Millipedes 9. Analysis of Slides 10. Photographing Techniques III. Results 1. Prevalence of Mesomycetozoa in Isopods and Millipedes 2. Mesomycetozoa in Isopoda 3. Mesomycetozoa in Millipedes 4. Other Artifacts an Organisms Worth Noting a. Gregarines b. Isopod Eggs c. Unknown Arthropod d. Nematodes e. Filamentous Bacteria that Resembles Fungi f. Other Fungi Found on Slides that are not Trichomycetes 4 IV. Discussion 1. Overview of Findings 2. Mesomycetozoa in Isopoda 3. Mesomycetozoa in Millipedes a. Filamentous Bacteria on Enterobryus in Millipedes 4. Other Artifacts and Organisms found in the Isopod and Millipede Gut 5. Problems with Identification: An Update 6. A Comparison to the White 1999 Study 7. Re-evaluating the Evolutionary Placement of Eccrinales 8. Viewing Evolution as a Spectrum V. Fungal Dictionary VI. Works Cited 5 Abstract Trichomycetes are a group of symbiotic organisms found in the gut of arthropods. They were recently split into two fungal groups and two protistan groups. The protists have been recatagorized as Mesomycetozoa (also known as Ichthysporea). This group lies somewhere at the animal-fungal evolutionary divergence and can help pinpoint a more precise date for this divergence with further phylogenetic studies. However, with confusing phylogenies, obscure lifecycles, and not enough worldwide research, answers have been difficult to come by. This study surveyed the trichomycete populations in Julidae millipedes and terrestrial isopods in Southeast New England. We found that in SE New England, Porcellio and Oniscidea isopods likely carry Parataeniella and Eccrinoides and Julidae carry Enterobryus. Further research should focus on how hibernation and seasonality affects trichomycete prevalence in these host organisms. 6 Introduction Overview of Trichomycetes Figure 1: Pictures of the four main groups of Trichomycetes. Source: Powerpoint by Emma R. Wilson. Boise State University. “Overview of Trichomycetes” Trichomycetes, meaning “hair-like fungi”, are an endocommensal polyphyletic*1 group comprised of both fungi and protists. Trichomycetes are currently classified into four main taxa. There are two fungal families, Asellariales and Harpellales, and two protistan families now placed within the Mesomycetozoans, Eccrinales and Amoebidiales (White 2011). Trichomycetes inhabit the gut of many aquatic insects, isopods, millipedes, and some species of crab all over the world. Their hosts are detrivores or scavengers, and many hosts of Trichomycetes are found in freshwater environments. Only a few species of Harpellales have shown more parasitic tendencies, while the rest of the trichomycetes seem benign. Historically, Trichomycetes phylogeny has been fraught with reclassifications and reorganizations of taxonomical categories. In fact, nowadays the term Trichomycetes is 1 *denotes the first use of a word defined in supplementary dictionary found on pg. 78 7 solely a convenient term to describe the organisms in the four orders. Researchers now refer to Trichomycetes with a lower case “t” to symbolize this association derived from convenience (Lichtwardt 2001). This is how I will refer to these organisms for the remainder of the thesis. However, many of the host isopods and millipedes studied in this project have protistan Mesomycetozoans, not the classically defined fungal trichomycetes. This reclassification has important implications towards our understanding of the animal-fungal divergence. This polyphyly and the benign nature of trichomycetes incite intrigue surrounding these organisms. There is a evolutionary tendency for ecological relationships to move on a spectrum from parasitic and harmful to obligate and benign. As research develops, an evolutionary narrative may develop about the evolution of benign trichomycete hosts with their current symbionts. This account may also inform research on terrestrialization. It could also support research concerning the basal divergence of Animalia and Fungi. However, further research is crucial because trichomycete studies have been spasmodic and primarily centralized in the Midwestern United States and France. 8 Why Are the Mesomycetozoa Important? Figure 2: A phylogenetic tree of all Eukaryotes. Note the divergence of fungi, mesomycetozoa, and animals in the Opisthokonts. Although Mesomycetozoans seem to branch from the animalia lineage, this has not been proven definitively. They remain in an undefined location branching around the divergence of animals and fungi. Also note the extreme degree of separation between the fungi, where Asellariales and Harpellales are classified, and the Mesomycetozoa, where the Eccrinales and the Amoebidiales are classified. Source: https://clas-pages.uncc.edu/matthew-parrow/ While studying fish parasites in 1998, researchers came across an unexpected finding: there is an ancient but extant phylum at the animal-fungal divergence (Ragan 1998). They named it the DRIP clade (an acronym for its original members: Dermocystidium, rosette agent, Ichthyophonus, and Psorospermium), which was later renamed Mesomycetozoea. Since then, organisms from protist, animal, and fungal groups have been reclassified into this diverse, yet monophyletic protistan group. Many of them are parasitic or infectious (very different from the symbiotic trichomycetes also placed in this group), which substantiates them as a subject of interest for infectious disease researchers and ecologists (Glocking et al. 2013). 9 There has been no conclusive evidence indicating the exact position of the Mesomycetozoans on a phylogenetic tree. The lack of chitin* in the walls of Eccrinales and Amoebidiales would indicate that they were closer to the Animalia lineage. However, Ichthyophonus hoferi, a close relative of Amoebidiales and Eccrinales, does have chitinous cell walls, which is usually a fungal trait (Glocking et al. 2013). As the orders and genera of the Mesomycetozoa undergo phylogenetic analysis, they may help pinpoint a more precise date for the divergence of animals and fungi. History of Discoveries Although a historically fungal grouping categorized by location in the host gut, the two orders of trichomycete groups discovered first, the Eccrinales and the Amoebidiales, are actually Mesomycetozoan protists. In 1848 American naturalist, Joseph Leidy, was the first to publish about trichomycetes (although “trichomycete” was not yet a term). He had discovered a type of Eccrinales, called Enterobryus, in the hindgut of a millipede, but classified it as a species of algae (Lichtwardt 2001). After this initial discovery, trichomycete research was conducted almost entirely in France. In 1853, Charles Robin wrote about another species of Enterobryus, and he was the first to speculate about a fungal origin. Until 1929, only species of Enterobryus and Amoebidium had been discovered out