Studying the Ancient Parasitic Cnidarian, Sphaerospora Elegans, Leads to Better Understanding of Oligochaete Diversity and Myxozoan Discovery

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

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]

©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 in their longest dimension

Species belonging to the Sphaerospora lineage vary in their pathogenicity to their hosts. S. renicola is one of the most studied Sphaerospora because of its known association with causing swim bladder inflammation (SBI), a pathogenic disease causing high mortality, in young carp (Eszterbauer and Székely 2004). Another pathogenic species, also in carp, is S. molnari, which causes high mortality due to gill infections (Eszterbauer et al., 2013). Significantly, it is not known what the infectious agent is to fish, although based on life cycle patterns from other myxozoan groups, it is presumed that Sphaerospora species require an invertebrate, alternate host, most likely an annelid. To better study and understand Sphaerospora, and broader host/evolution trends in Myxozoa, it is essential to

9 elucidate the life cycle of a Sphaerospora species, and thus develop a general model for life cycles within the Sphaerospora lineage. Sphaerospora elegans, the type species of this clade, is an ideal target for a life cycle discovery project. S. elegans is a known parasite within the local Willamette River system, based on previous work by Dr. Atkinson. S. elegans’ vertebrate host is the three- spined stickleback (Gasterosteus aculeatus L., 1758), in which the parasite is fairly benign and does not kill significant levels of sticklebacks among populations (Özer, 2003). Three- spined stickleback are a small (average length of 5 cm), non-anadromous, freshwater fish widespread across the temperate northern hemisphere, and found endemic in Oregon (Fuller, 2019). In previous observations by Dr. Atkinson, Sphaerospora myxospores have been identified morphologically within the renal tubules of sticklebacks collected from the Willamette River and its tributaries, and confirmed as S. elegans by DNA sequencing. Location of investigation and target species of S. elegans In the present study, we selected Dunawi Creek, a tributary of the Willamette River, to screen for S. elegans-infected stickleback and search for the unknown invertebrate host. This location (Fig. 2) was selected because sticklebacks with Sphaerospora infections have been found in this creek during previous work by Dr. Atkinson. Additionally, its close proximity to Oregon State University makes it a convenient location that is easily accessible for frequent sampling events. We were able to obtain the necessary state and local permits for collecting fish at a locality in Bruce Starker Arts Park, a public city park in Corvallis.

E F Figure 2. Site of investigation on Dunawi Creek. A/B) Sampling locations within Bruce Starker Arts Park. C- E) Images of the creek taken at the location of investigation, showing “re-wilded” area with trees killed by beaver, and the narrow vegetated creek, which is ideal for stickleback. F) Initial examination of sediment in dip net.

Oligochaete characterization and diversity In North America, there are some 170 freshwater oligochaete species belonging to five different families (Haplotaxidae, Opistocystidae, Naididae, Tubificidae, and Lumbriculidae) (Kathman & Brinkhurst, 1998). Visual observation of physical characteristics, such as the location of hair chaetae, the presence or absence of eyespots, gills, proboscis, and the color and size of the worm, allows the categorization of oligochaetes into families based on an established key (Kathman & Brinkhurst, 1998). This information, combined with cytochrome oxidase I (COI) sequence data, can definitively establish species identities. The COI is a universal gene found in almost all animal life on Earth (Yahalomi, Atkinson et al., 2020).

11 Methods Sample collection and site of investigation Field sampling occurred six times between June and December, 2019 (Table 1). Mud samples were collected using a hand net to scrape mud from the bottom of the creek, checked for bycatch of any vertebrate, before transferring sediment into 750-mL containers. Approximately 400 mL of mud was collected for each sample and topped off with creek water. Macrophyte (plant) samples were collected by hand or with a net, and placed in a lidded bucket with creek water to check for any bycatch of vertebrates. Mud and plant samples were stored in the lab at 15 ºC with aeration. Fish collections were completed using a hand net from the creek bank. The net was plunged out and downward to the bottom of the creek and pulled to the edge and out of the water and the content immediately transferred to a bucket with extra water. Any stickleback were separated, and immediately euthanized by an overdose of MS-222; any non-target species (typically Gambusia) were returned to the creek live. Two additional adult sticklebacks were obtained for use as control material, from Oregon Department of Fish and Wildlife field biologist from the McKenzie River, another tributary of the Willamette River.

Table 1: Dunawi Creek sample collection date, location and sample types collected.

Date Location GPS Coordinates Sample types for Sticklebacks oligochaete found & examinations. collected? 06/15/19 by Dunawi Creek 44°32'56.6"N Mud No S.A. 123°18'13.3"W 6/28/2019 Dunawi Creek 44°32'56.6"N Mud No 123°18'13.3"W 44°32'57.0"N 123°18'14.8"W 44°32'58.2"N 123°18'17.7"W 07/15/2019 Dunawi Creek 44°32'56.6"N Mud No 123°18'13.3"W Water plants 08/15/2019 Dunawi Creek 44°32'56.6"N Mud Yes 123°18'13.3"W 09/03/2019 Dunawi Creek 44°32'56.6"N Mud No 123°18'13.3"W Water plants 12/03/2019 Dunawi Creek 44°32'56.6"N Mud No 123°18'13.3"W

12 Three-spined Stickleback examination A visual inspection and routine necropsy were completed on all three-spined sticklebacks (Fig. 3). The kidney was removed, and a piece was retained and examined for parasite myxospores or developmental stages using a compound microscope, with attention paid to searching for Sphaerospora infections in the tubules. The gallbladder and posterior gut were also examined via microscopy for myxozoan infections, as there are literature records of myxozoans in these tissues from other localities. A portion of the kidney was frozen for DNA analysis (see below).

Figure 3: Three-spined stickleback prior to necropsy to examine via microscopy for Sphaerospora and other myxozoan infections within the renal tubules, the gallbladder, and the posterior gut.

Oligochaete sorting and examination Creek mud was transferred incrementally into sample cups and ‘fluffed up’ with extra dechlorinated tap water to suspend all components. it was then poured rapidly onto a 140 µm stainless steel screen, which retained all annelids. Samples were rinsed with dechlorinated tap water and transferred into petri dishes for examination under a dissecting microscope (Fig. 4). Oligochaetes were removed from plant samples for visual inspection by placing plant clippings from the sample bucket into small containers with dechlorinated water. Leaves and stems were forcefully shaken in the water to remove annelids and small debris. The water and debris was then poured through the 140 µm screen, and the collected solid residues were placed in a petri dish for examination.

Figure 4. Examining screened sediment to clean and remove oligochaetes. Individual oligochaetes were removed from the bulk samples, and placed onto glass slides with water and a coverslip for examination using a compound microscope. Based on physical features of the oligochaetes (including hair chaete, eyespots, proboscis, gills; Fig. 5), I established a system for binning worms into operational taxonomic units (OTUs). These OTUs were a convenient way to track how many worms of each type were examined, and to begin to understand the diversity of potential hosts in the creek.

Figure 5: Examples of morphological characteristics used to sort oligochaetes into operational taxonomic unit (OTU) types: A/B) different types of chaete; C) posterior gills; D) anterior proboscis.

Simultaneously, we examined each worm for myxozoan infections. Published work from myxozoan infections in annelids has shown that the parasites typically proliferate and develop actinospores in the intestinal tract or within the coelom, and have a characteristic cell-within-cell structure. There are typically eight spherical shapes clustered together forming a packet-like form called a pansporocyst (Fig. 6) (Rocha, Alves, Antunes, Azevedo, & Casal, 2019).

Figure 6: Micrograph of aurantiactinomyxon pansporocysts developing in the intestinal epithelium of Tubificoides pseudogaster, a marine oligochaete, depicting the typical appearance a myxozoan infection. I was looking for an infection with similar presentation (Rocha et al., 2019).

Three worms of each visual OTU were saved for DNA extraction and COI sequencing to identify the species/genotype. Any annelids with suspected myxozoan parasite infections were photographed, and saved for DNA extraction and molecular

15 sequencing. Mud and water samples were discarded after removal of all annelids. This process was repeated until the entirety of each sample was thoroughly examined. Tissue digestion, PCR, purification, sequencing and identification DNA was extracted from fish and worm samples using Qiagen™ DNeasy™ blood and tissue kit, then stored at -20 C until PCR. The extracted DNA was amplified using PCR with Thermo Scientific™ Dream Taq™

Hot Start DNA polymerase, Thermo Scientific ™ Dream Taq ™ 10X Buffer with MgCl2, BSA, and dNTPs with specific primers chosen for each assay type (Sphaerospora, Myxozoa or oligochaete ID): For detection of S. elegans, I used universal eukaryote SSU primer ERIB1 (Barta et al., 1997) and S. elegans-specific reverse primer Se1000R (Atkinson, unpublished). PCR program: an initial denaturing at 94 ºC for 3:00, followed by 32 cycles of denaturing at 94 ºC for 0:20, annealing at 64 ºC for 0:30, and extending at 72 ºC for 1:30. The final extension period was 7:00 at 72 ºC. All annelids that tested negative for S. elegans were subjected to a second PCR to detect presence of other myxozoans. I used universal forward primer ERIB1 (Barta et al., 1997), with a general myxozoan reverse primer Act1R (Hallett & Diamant, 2001) or myxozoan forward primer MYXGEN4f (Diamant et al., 2004) and universal reverse primer ERIB10 (Barta et al., 1997). The PCR program began with an initial denaturing at 94 ºC for 3:00, followed by 34 cycles of 94 ºC for 0:25, 55 ºC for 0:30, and 72 ºC for 0:45. There was then a final extension of 72 ºC for 7:00. Annelids from each OTU, plus any that gave myxozoan PCR positive results, were amplified for identification by sequencing. I amplified mitochondrial COI using “Barcode of Life” primers LCO-1490 and HCO-2198 (Folmer, Black, Hoeh, Lutz, & Vrijenhoek, 1994). The PCR program had a 3:00 initial denature step followed by 4 cycles of denaturing at 94 ºC for 0:20, annealing at 45 ºC for 0:40, and extending for 0:40 at 72 ºC. This was followed by 34 cycles of denaturing at 94 ºC, annealing at 51 ºC and extending at 72 ºC for the same time as the previous cycles. The final extension was 7:00 at 72 ºC. All PCR products were visualized in 2% agarose gel with electrophoresis at 160 V for 40 minutes. Quality products were purified using the Qiagen™ DNA purification kit prior to submission for Sanger sequencing by the Center for Genome Research and Biocomputing (CGRB) at Oregon State University, using one of the PCR primers. DNA sequences were used as search queries on NCBI GenBank® to determine the closest known annelid species. Sequences with more than a 97% nucleotide identity were considered similar enough to be the same species.

16 Alignment and phylogenetic analysis An alignment was made of all oligochaete COI sequences generated in this project, plus selected reference taxa based on the BLAST matches from the GenBank searches, plus a marine non-clitellate annelid as an outgroup taxon. Sequences were aligned within MEGA Software using MUSCLE. I trimmed the alignment to remove low-quality regions at each end. Several low quality sequences were removed from the final alignment. A Maximum likelihood phylogenetic analysis was conducted using RaxML with the GTR+I+G model of nucleotide substitution (as identified as the most appropriate model in previous annelid analyses; Atkinson pers. comm.), with a bootstrap of 100 replicates. The consensus tree was annotated with taxon and clade information using Adobe Photoshop. Results Investigation of sticklebacks Results of screening three-spined sticklebacks are shown in Table 2. Two adult sticklebacks were collected from the McKenzie River by Oregon Department of Fisheries and Wildlife (ODFW), both of these fish had mature Sphaerospora myxospores in their kidney tubules (Fig. 1). From Dunawi Creek, I saw very few fish, and fewer sticklebacks specifically: I only caught stickleback on one collection date, and these fish were juveniles. Necropsies revealed only myxozoan-like developmental stages in the renal tubules. No myxozoan infections were seen in other tissues examined. PCR testing of all sticklebacks revealed that all fish with either mature Sphaerospora spores or developmental stages, were positive. In addition, two Dunawi Creek fish that were visually negative tested positive by PCR for Sphaerospora. DNA sequencing confirmed the species present was S. elegans in both McKenzie River and Dunawi Creek fish (Table 3).

17 Table 2: The prevalence of infection of S. elegans in three-spined stickleback screened visually and by molecular methods.

Visual Visual Visual Date Source Other Molecular Fish Length Sex gallbladder intestines kidney examined location infections results exam exam exam

1 7/18/19 McKenzie - - + w+

2 7/18/19 McKenzie - - + +

3 8/15/19 Dunawi 29 mm F - - - Eye cyst +

4 8/15/19 Dunawi 36 mm M - - w+ +

5 8/15/19 Dunawi 34 mm M - - - +

6 8/15/19 Dunawi 21 mm - - - -

7 8/15/19 Dunawi 30 mm - - w+ w+

Table 3: Sequencing results from three of the three-spined sticklebacks that were molecularly positive for S. elegans from both the McKenzie River and Dunawi Creek demonstrating the current presence of S. elegans within the Willamette River system.

Fish Source Location Sequencing Percent match GenBank Results Accession 1 McKenzie S. elegans 99.2% JX286618 2 Dunawi S. elegans 99.8% JX286618 3 Dunawi S. elegans 99.9% JX286618

Investigation of the oligochaetes I developed morphological descriptions of 18 OTUs, and binned oligochaetes into these. Data of the characteristics observed and the types of oligochaetes and their matched molecular identity is provided in Table 4.

- -

N/A

pairs per per pairs

Small

unknown

segment.

HC: seg II; seg HC:

Light brown brown Light

and orange. and

simple pairs. simple

- -

tail.

thin

and and N/A

White

6 pair 6

unknown

segment.

textured

HC: seg. II;seg. HC:

Wrinkley bundles per per bundles

Long and simple pairs; simple

N/A

unknown

segment.

bundles per per bundles

HC: pairs of HC: pairs

simple HC; 2 simple

- -

15 97

HC.

N/A

Small

unknown

Gray and Gray and

medium white; dark dark white;

movement.

long simple simple long

purple/black purple/black

Twitchy/jerky Twitchy/jerky

Proboscis

coelomocytes

HC: seg II; seg HC: very

g II;g

- -

110

large

99.6%

thicker Naididae

Small Small / mouth.

segmens.

intestines.

HC on last last on HC vaga Dero

medium HC: se HC:

pairs; 4 pair pair 4 pairs; per bundles

segment. No No segment.

head with anterior: bifid anterior:

and simple in in simple and

Brown / black / black Brown

Narrow tail,

aracteristics and the corresponding species match species corresponding match the and aracteristics

gray

- -

limnaei

Small

Large

99.6%

Naididae

mouth.

white

Chaetogaster Chaetogaster

- -

12 16

Nais Nais

Slow Slow

pairs

White

moving

Small elinguis

bifid HC HC bifid

98.0%

Naididae

HC: seg II; seg HC: and simple

11 53

Dero Dero

pairs

Gills

digitata

Large

bifid HC HC bifid

Dark red Dark

Sporadic Sporadic Naididae

swimming

100.0%

simple and simple

HC: Seg II; Seg HC:

- -

sp.

pairs

head

bifid HC HC bifid

83.4% Pink and and Pink

Medium HC: seg II, seg HC:

Bulbous Bulbous simple and simple Tubificidae

Aeolosoma Aeolosoma

yellow spots yellow

213

pairs.

Gills

spots.

bifid HC HC bifid

Sporadic Sporadic

Naididae

intestines intestines

with black black with

spots; red red spots;

swimming

100.0%

Medium

simple and simple yellow with

HC: seg VI, seg HC:

Light orange orange Light obtusa Dero

- -

Small 99.7%

segment

Naididae

coelom

10 pairs per pairs per 10

medium HC: seg II; seg HC: and simple

Squiggly Squiggly brown; pink brown; vasculature

diastrophus

Orange, red, red, Orange,

Chaetogaster Chaetogaster

bifid HC pairs;HC bifid

he NCBI GenBank database. database. NCBI he GenBank

7 3

Nais Nais

dark White

No HC No

Large

99.4%

Sporadic Sporadic variabilis

Naididae

swimming

- -

large

Yellow

88.3%

orange gut orange Tubificidae

Limnodrilus Limnodrilus

Medium hoffmeisteri

5 3

Pink

Pristina Pristina

Large

jenkinae

93.4%

Naididae

- - chaetae chaetae

Large

98.6% Dark red red Dark touched.

on ventral. on

Tubificidae

No HC, but but HC, No

Limnodrilus Limnodrilus

Curls up when when up Curls

coelomocytes.

robust robust claparedeanus

intestines; many many intestines;

Small

purple

99.6% bifid HC; HC; bifid

Sporadic Sporadic

Naididae

HC pairs. HC

swimming

posterior = posterior

HC: seg VI, seg HC: = 3 Anterior

coelomocytes.

Gray color with with color Gray

Nais communis Nais

simple and bifid simple

- -

Red

tubifex

pairs of pairs Tubifex

Large

bifid HC. bifid

100.0%

simple and simple Tubificidae

HC: seg II, seg HC: 3

- -

1 5

sp.

Brown Brown

Large

99.1% and pink and

simple and simple Tubificinea

Tubificidae

HC: seg VI; seg HC: vasculature

bifid HC pair HC bifid

clusters along along clusters

intestines; red red intestines;

: Operational taxonomic units (OTUs) created to describe oligochaetes from Dunawi Creek, with morphological ch behavioral and morphological with Creek, Dunawi from describe oligochaetes (OTUs) to units created taxonomic :Operational

Size

OTU:

Body Body Gills?

Family 19

Species

match to

Behavior Additional

reference

Reference

Eyespots?

color/spots

Configuratio

Percent DNA PercentDNA

Hair Chaete?Hair

Characteristics

No. observed

Table4 determined by similarity reference COI gene, with mitochondrial of similarity by species determined t from Sequences for Aulodrilus acutus and Ilyodrilus templetoni were observed in the results from Sanger sequencing. These species were not included in Table 4 because their genetic data did not match the other species of oligochaetes belonging to the same OTU type. A. acutus was believed to belong to the T. tubifex OTU while I. templetoni was categorized with species of the Chaetogaster diastrophus OTU. Eight different OTU groups resulted in a unanimous, singular, molecular identification, while six of the OTU groups were established by combining multiple OTU characterizations into a single group based on their shared molecular data. Additionally, there were four different OTU groups that were not strongly matched with a molecular identification on GenBank. This indicates that there were misidentifications that occurred during the matching of oligochaetes to the OTUs categorized, and that categorization of oligochaetes by these OTUs alone is not definitive enough to determine species identity, and should be paired with molecular data for verification.

Type 17 Type 18 Type 1 Type 2 Type 4 1% 0% 1% 3% 3%

Type 16 Type 5 0% 0%

Type 3 Type 6 4% 2% Type 15 Type 7 14% 0% Type 8 11% Type 14 16%

Type 13 0% Type 11 Type 8% Type 9 12 32% 2% Type 10 0%

Figure 7. Species diversity of oligochaetes observed in Dunawi Creek based on numbers of individual worms scanned belonging to each OTU type.

20 In addition, the family diversity of observed species was calculated. Families Tubificidae and Naididae accounted for all the species isolated from Dunawi Creek. 11.3% of the observed oligochaetes belonged to Tubificidae while the observed familial diversity of Nadidae was 88.7%. The oligochaete COI gene sequences were used to create a phylogenetic tree of the collected oligochaetes from Dunawi Creek (Fig. 8). Sequences from GenBank were used in the alignment with 68 oligochaete sequences from Dunawi Creek, to provide established reference points and inform phylogenetic relationships.

Figure 8: Consensus tree of novel COI sequences from Dunawi Creek oligochaetes, with reference sequences from GenBank. Annotations show inferred family/genus/species groupings within the tree.

22 Parasites found Figure 9 contains examples of the non-myxozoan oligochaete parasites observed during this investigation. In addition, I identified two worms with morphologically different myxozoan infections, in different sites of development within the annelid host. Figures 10 and 11 show the morphology of the myxozoans, with packets of developing spores either in the lumen of the intestine, or in the coelomic cavity of the host. The parasites were each visible as numerous clusters of cells, with larger and more developed spores towards the posterior end of the worm. No mature actinospores were visible. PCR tests using Sphaerospora primers were negative. PCR tests with general myxozoan primers were positive, and sequencing showed both parasites to be different species: one had a 92% sequence identity with Myxobolus arrabonensis (GenBank accession number MH375073), and the other a 96% identity with Myxidium truttae (GenBank accession number AF201374).

A B C D

Figure 9. Micrographs of non-myxozoan parasites observed during the examination of oligochaetes: A/B) endoparasitic ciliate species; C) nematomorpha larvae; D) ectoparasitic ciliates.

Figure 10: Oligochaete with intestinal infection of a myxozoan that had a 92% SSU sequence identity to Myxobolus arrabonensis (GenBank accession number MH375073).

Figure 11: Oligochaete infected with a coelomic myxozoan species, which had a 96% SSU sequence identity with of Myxidium truttae (GenBank accession number AF201374).

24 DNA sequencing of COI from both host oligochaetes was successful, and sequences were 100% similar to each other, indicating that the hosts were the same species. Additionally, according to the visual examinations of these worms, they were both categorized as members of the same OTU group 8. The oligochaete COI gene sequences did not have high similarity with any reference oligochaete on GenBank; The closest resemblance for both oligochaetes was Limnodrilus hoffmeisteri with an identification < 82%. Discussion Sphaerospora and the three-spined sticklebacks The molecular data from developmental stages observed in the juvenile fish from Dunawi Creek confirmed that the myxozoan of Sphaerospora elegans was present currently at that locality. This positive result demonstrated that the exploration for S. elegans infections within the oligochaete populations was rational. I was surprised that stickleback were not abundant in Dunawi Creek in 2019, despite Dr. Atkinson’s data showing many fish visible in previous years. All of the sticklebacks from Dunawi Creek that were captured during this study were caught during a single visit to the field site. Attempts to catch fish on the other five visits were unsuccessful. These visits varied in time of day, which may explain the absence of sticklebacks in some sampling days. The sticklebacks collected on 8/15/2019 were all juveniles, which may indicate that they were spawning late in the year, and could help explain why sticklebacks were not found during previous visits. However, there were no sticklebacks caught during sampling days after 8/15/2019, indicating that other environmental factors may have been influencing the fish’s presence in Dunawi Creek in 2019. A standardization of collection dates/times could allow for a better understanding of the stickleback population in Dunawi Creek, and would limit the external environmental effects that may have affected this study (e.g. rain events, heat waves). Kidney tubules is the typical location for Sphaerospora infections to occur, however, my molecular data show that fish could be infected without having visible developmental stages. With juvenile fish, this observation is to be expected as the infections have reduced time to develop. The smallest fish tested negative for Sphaerospora while the next smallest tested positive. This may suggest the presence of a specific limit in stickleback development, before which Sphaerospora is unable to infiltrate and begin proliferation. Myxozoans found Two myxozoan species were observed during this study (Fig. 10), but I did not observe mature actinospores for either species. One was developing in the coelom of the

25 oligochaete host, and one in the intestine. I used multiple primer combinations to generate overlapping SSU sequences for each myxozoan, and assembled contigs for almost the entire SSU of each (~1950 bp and ~1900 bp respectively). Molecular data did not establish a definitive identity with any species already sequenced and added to GenBank (96% with Myxidium truttae and 92% similarity with Myxobolus arrabonensis). These could be alternate stages of novel myxozoans, or could be stages from known species that have yet to be sequenced. These discoveries are a good starting point for another undergraduate project. Oligochaete data The myxozoans were isolated from two different oligochaete hosts. Molecular analysis of the oligochaete’s COI gene did not produce a worm identity with high confidence (< 82% match with L. hoffmeisteri for each). However, upon completing an alignment of the two oligochaetes, their sequences were 100% identical. The phylogenetic tree (Fig. 8) places these two worms (identification codes L00206-14 and L00206-01) next to each other, and close to but not within the Limnodrilus clade. Additionally, the two oligochaetes were also categorized within the same OTU group based on their morphological characteristics. Like with the myxozoan species, the oligochaete molecular data demonstrated high quality traces, indicating that the primers worked well with the COI sequences, and that the problem is that a matching species does not yet exist in the GenBank database. Just like the myxozoans, I was unable to determine if the oligochaetes are novel species, or are known species that have yet to be sequenced. Oligochaete diversity and observations The morphological characteristics used to assign OTUs to oligochaetes were found to be more difficult to determine than initially expected. Related species can have very similar characteristics, making sorting difficult and potential misidentifications possible, lowering the confidence of the descriptions of species diversity and population proportions. Figure 11 summarizes the OTUs used to visually categorize the oligochaetes correlated with their closest COI genetic identity based on sequence similarity with known annelids from GenBank. It was apparent that multiple OTU classifications led to the same species identification, demonstrating some of the challenges that were associated with this morphological categorization component of this project (morphological traits appear to create a falsely high sense of species diversity).

26 Physical Characteristics of worms Worm Types based on DNA Sequencing (Visual Operational Taxonomic Units) Type # HC on II; short; red color 1

HC on VI; needle-like and HC in Tubificinea sp. 2 pair; brown clusters along intestines; red and pink vasculature; large size.

HC on II; long; red color 3 Tubifex tubifex

Eye spots; HC on VII; sporadic 4 swimming HC on VI; long and needle-like Nais communis 5 HC; purple eyespots; red intestines; small size

No HC; long; dark red 6 Limnodrilus claparedeanus

Long; HC; pink Color 7 Pristina jenkinae

No HC; yellow-orange gut; 8 medium-large size Limnodrilus hoffmeisteri

Eye spots; long, white color, 9 sporadic swimming Nais variabilis

HC on II; HC in groups of 2-5 10 pairs, one short and one long per pair; red color; medium Chaetogaster diastrophus size

27 HC on VI; HC in pairs, one short, one long; red intestines; 11 fast when swimming but slow movement; medium size HC on VI; long needle-like and 12 short pair HC; gills; yellow spots; medium size; sporadic swimming Dero obtusa HC on II; long needle-like and 13 short in pairs; red intestines

Eyespots; HC on IV; long, 14 needle-like HC; light orange color; slow movement; medium-large size HC on II; HC in groups of 3 Chaetogaster diastrophus pairs, each pair has one 15 needle-like and one short HC; orange-red-brown color; small size; pink vasculature on posterior end. HC on II; long and needle-like HC; pink & yellow spots on 16 Aeolosoma litorale body; bulbous head, medium size H HC C o on n IIV;I; lo longng a andnd n needleeedle-l-ilikeke HC;HC; gillsgills present;present; darkdark red red color; 17 Dero digitata cofastlor; andfast sporadic and spo rswimming;adic

swimming; largelarge size size

HC on II; long an d needle-like 18 HC; white/no color; slow Nais elinguis moving; small size No HC; white-gray color; small 19 size Chaetogaster limnaei

HC on II; very long and needle- like HC; white-no color; No sequence data 20 twitchy movement; small size; proboscis

No HC; pairs of simple chaete 21 No sequence data

Figure 11: Summary of the morphological characteristics used to differentiate the oligochaete worms and their associated species identities determined by COI sequences.

Another characteristic that may have affected the proportion of species types observed derives from the time taken to sort and examine mud samples. Some samples contained many annelids and required many hours to sort, and in some cases, the complete sample was not examined until several weeks after the sample was originally

28 collected. This time lapse between sample collection and sorting likely had an effect on the sample oligochaete populations and species diversities Summary of principal findings I determined through microscopic observation and molecular confirmation that Sphaerospora elegans lives within the Willamette River system, and that it actively infects the three-spined stickleback that inhabit the river. Through microscopy of the oligochaetes that live sympatricly with infected stickleback within Dunawi Creek, I established a morphological guide to species identification, and paralleled this with COI sequencing of the oligochaetes, which can be used to match morphological characteristics of oligochaetes to a tentative species identification. Oligochaetes were microscopically screened for visual myxozoan infections to search for the definitive host of S. elegans, or other myxozoan species. I found two oligochaetes that were infected by two different myxozoan species. Sequencing of these myxozoans showed that neither was S. elegans, and their sequences did not match any myxozoan already available on GenBank; indicating that these species have not yet been sequenced, or that they are novel myxozoans. Both infected oligochaetes had sequences that were identical, and are thus assumed to be the same species, but this host sequence also did not match the sequences available on GenBank, indicating similarly to the myxozoans, that the oligochaete species may not be sequenced, or is novel

Suggestions for future undergraduate projects: - Searching OSU sequence records from fish myxozoans to see if either of the myxozoans that I discovered in oligochaetes is already known (but unpublished) from a Willamette River fish host (Dr. Atkinson’s data), then describe this complete life cycle. - Additional annelid sampling in Dunawi Creek, targeting earlier times in the season, when juvenile fish may get infected. - Searching this river system for the vertebrate hosts of the two myxozoans that I did find in the oligochaetes, therefore establishing a life cycle discovery.

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