Studying the ancient parasitic cnidarian, Sphaerospora elegans, leads to better understanding of oligochaete diversity and myxozoan discovery. by David Lehrburger A THESIS submitted to Oregon State University Honors College in partial fulfillment of the requirements for the degree of Honors Baccalaureate of Science in Integrative Biology (Honors Associate) Presented March 12, 2020 Commencement June 2020 2 AN ABSTRACT OF THE THESIS OF David Lehrburger for the degree of Honors Baccalaureate of Science in Integrative Biology presented on March 12, 2020. Title: Studying the ancient parasitic cnidarian, Sphaerospora elegans, leads to better understanding of oligochaete diversity and myxozoan discovery. Abstract approved:_____________________________________________________ Stephen Atkinson Myxozoans are microscopic parasites, related to corals and jellyfish. Most Myxozoa probably have two-host lifecycles that require a vertebrate (typically fish) and invertebrate (annelid or bryozoan). However no life cycle is known from the Sphaerospora lineage. I hypothesized that the life cycle of Sphaerospora elegans, a myxozoan parasite of three-spined stickleback (Gasterosteus aculeatus) requires an alternate annelid host. I sampled fish and annelids from a creek known to have the parasite. PCR and DNA sequencing demonstrated that the fish currently are infected. I quantified annelid diversity in the creek by establishing 18 unique morphological groups and sorting oligochaetes into these groups, then sequencing representatives from each group to test morphological categorization versus DNA identity. DNA resolved the 18 morpho-groups into 16 nominal species, from 10 genera and two families. Using visual and molecular screening, I discovered two (of 674) oligochaetes had myxozoan infections: neither infection was S. elegans. Neither myxozoan could be identified by sequence matches (>98%) to known species: the most similar myxozoans were Myxidium truttae (96%) and Myxoboulus arrabonensis (92%). This method validates the direct examination of annelids to discover myxozoan infections, but could not preclude involvement of a non-annelid hosts in the life cycle of S. elegans. Key Words: Myxozoan, sphaerospora, parasite, annelid, worm Corresponding e-mail address: [email protected] 3 ©Copyright by David Lehrburger March 12, 2020 4 Studying the ancient parasitic cnidarian, Sphaerospora elegans, leads to better understanding of oligochaete diversity and myxozoan discovery. by David Lehrburger A THESIS submitted to Oregon State University Honors College in partial fulfillment of the requirements for the degree of Honors Baccalaureate of Science in Integrative Biology (Honors Associate) Presented March 12, 2020 Commencement June 2020 5 Honors Baccalaureate of Science in Integrative Biology project of David Lehrburger presented on March 12, 2020. APPROVED: _____________________________________________________________________ Stephen Atkinson, Mentor, representing Microbiology _____________________________________________________________________ Jerri Bartholomew, Committee Member, representing Microbiology _____________________________________________________________________ Michael Blouin, Committee Member, representing Integrative Biology _____________________________________________________________________ Toni Doolen, Dean, Oregon State University Honors College I understand that my project will become part of the permanent collection of Oregon State University, Honors College. My signature below authorizes release of my project to any reader upon request. _____________________________________________________________________ David Lehrburger, Author 6 Outline: • Myxozoa are a large group of microscopic, parasitic Cnidaria. • Myxozoans have two-host life cycles that alternate between vertebrate (usually fish) and invertebrate hosts (usually annelids or bryozoans). • Myxozoa are sub-divided into two classes: Malacosporea (in bryozoans; with fewer than 20 species) and Myxosporea (in annelids >2000 species). Myxosporea is further divided into three main lineages: “marine”, “freshwater” and the earliest diverging lineage, Sphaerospora sensu stricto. • Several species of Sphaerospora cause diseases in aquaculture • No life-cycle is known for Sphaerospora (it is unknown exactly how fish become infected) • My goal was to identify the invertebrate host of the type species, Sphaerospora elegans, a common kidney parasite of three-spined sticklebacks. Abstract Myxozoans are microscopic parasites, related to corals and jellyfish. Most Myxozoa probably have two-host lifecycles that require a vertebrate (typically fish) and invertebrate (annelid or bryozoan). However no life cycle is known from the Sphaerospora lineage. I hypothesized that the life cycle of Sphaerospora elegans, a myxozoan parasite of three-spined stickleback (Gasterosteus aculeatus) requires an alternate annelid host. I sampled fish and annelids from a creek known to have the parasite. PCR and DNA sequencing demonstrated that the fish currently are infected. I quantified annelid diversity in the creek by establishing 18 unique morphological groups and sorting oligochaetes into these groups, then sequencing representatives from each group to test morphological categorization versus DNA identity. DNA resolved the 18 morpho-groups into 16 nominal species, from 10 genera and two families. Using visual and molecular screening, I discovered two (of 674) oligochaetes had myxozoan infections: neither infection was S. elegans. Neither myxozoan could be identified by sequence matches (>98%) to known species: the most similar myxozoans were Myxidium truttae (96%) and Myxoboulus arrabonensis (92%). This method validates the direct examination of annelids to discover myxozoan infections, but could not preclude involvement of a non-annelid hosts in the life cycle of S. elegans. 7 Introduction Myxozoa is an ancient, obligately parasitic clade of cnidarians. Globally, myxozoans cause several severe diseases with economic impacts both in wild and aquaculture fisheries, and in at least one report, environmental effects (Okamura et al., 2015). They account for some of the most devastating diseases of salmonids, including whirling disease, proliferative kidney disease and enteronecrosis (gut rot) (Okamura et al., 2015). Myxozoans were first described in the eighteenth century. Since then, more than 2200 species have been identified globally, in both marine and freshwater environments (Patra, 2017). Based on species diversity in fish hosts, and the extensive distribution of Myxozoa across the Earth, it is estimated that there could be as many as 40,000 different species (Atkinson et al., 2018). Despite this large number of species, very few complete life cycles of myxozoans are known. As of 2017, from 2200 myxozoan species only 52 life cycles have been described, with 38 of these inferred from molecular data only (Patra, 2017). These life cycles all involve alternate hosts, most commonly a vertebrate (fish) and an annelid (oligochaete or “polychaete” worms), or in a few cases a bryozoan. This lack of knowledge makes life cycle discovery a continued area of interest within myxozoan research. For almost all myxozoans, infection cannot spread from vertebrate to vertebrate host, but instead must go through the annelid. The lifecycle includes two different spore stages: myxospores which are released from the vertebrate host, and the actinospores which are released from the invertebrate host. Upon release from either host, the spores must enter and infect the next host in the cycle, where replication and development of the new spore type occurs. These life cycles can last months or years, with long gaps of time in between hosts. Myxozoans belong to the phylum Cnidaria, which includes corals, jellyfish, and sea anemones (Patra, 2017). There are two myxozoan Classes: Malacosporea (in fish & bryozoans) and Myxosporea (in vertebrates & annelids) (Okamura et al., 2015). Myxosporea are further divided into three lineages, the Sphaerospora, “marine” myxosporea, and “freshwater” myxosporea, based on their host types, predominant environment, and ribosomal DNA sequence relationships (Fiala and Bartoŝová, 2010). Phylogenetic analyses suggests Sphaerospora emerged after Malacosporea, but before the freshwater and marine lineages (Holzer et al., 2018). Within each lineage, subclades of myxozoan species can be defined by commonalities of myxospore shape, DNA sequence features, and tissue and host specificity (Wolf and Markiw, 1984). 8 “Sphaerospora” aptly describes parasites in that lineage because the myxospores, which form within the vertebrate host, have a spherical morphology (Fig. 1) (Lom & Dyková 2006). Species within Sphaerospora most often infect the tubules within the kidney of their freshwater and marine hosts (Okamura et al., 2015). Sphaerospora species share the unusual genetic characteristic of having extraordinarily long small subunit ribosomal RNA (SSU): >3000 base pairs. This feature sets Sphaerospora apart because it is significantly longer than that of other myxozoan species and one of the longest among all eukaryotes (Okamura et al., 2015; Bartošová et al., 2013). This extremely long SSU makes laboratory amplification of Sphaerospora DNA difficult and, and is one reason why only 19 Sphaerospora species have SSU sequences deposited to GenBank, despite there being 103 different Sphaerospora species described (Patra, 2017). Figure 1. Myxospores of Sphaerospora elegans. The spores are approximately 15 µm