Genetic diversity of Manayunkia speciosa in the Klamath River basin By: Dan Horner Mentors: Sascha Hallett Jerri Bartholomew Hello my name is Dan Horner, And this summer I was also fortunate enough to work in Jerri Bartholomew’s Lab. My research was focused on the genetic diversity of the polychaete worm seen here, Manayunkia speciosa, in the Klamath River Basin. The discovery a decade ago that this polychaete was involved in the lifecycle of the parasite Ceratomyxa shasta has led to the worm being the focus of increased research

Background Ceratomyxa shasta is a microscopic, spore-forming parasite Causes intestinal necrosis and mortality of Klamath River juvenile salmonids 60% of out migrating juvenile salmonids infected Severe economical and ecological impacts The parasite Ceratomyxa shasta is a myxosporean that is characterized by it’s complex life cycle involving an invertebrate and vertebrate host. The spores are approximately 10 micrometers across, making them visible only under a microscope So what we are seeing in these top two pictures are the…. [LEFT] the myxospore stage that infects the polychaete, M. speciosa, [RIGHT] The actinospore stage is then seen here on the right, this is the parasite stage that infects the fish. This bottom shows a severe infection from C. shasta that is characterized by the distended abdomen Juvenile salmonids, which refers to trout and salmon species, are particularly susceptible. According to a recent survey, approximately 60% of the out-migrating population of Chinook salmon was infected. The high prevalence of C. shasta infection in the Klamath has led to some formidable economical effects on the fishing community. 2

Economic Effects of C. shasta Low salmon numbers forced regulatory closures of 2006 salmon fishing Loss of $150 million to the region's economy 1. Along many parts of the Klamath communities are economically dependent upon the salmon population for both fishing and tourism. 2. Recently in 2006, low salmon population numbers forced regulatory closures to many locations along the Klamath River, resulting in approximately 150 millions dollars of lost profit to the local economy 3. These serious economics effects have to led to the necessity of over 2 million dollars spent annually on C. shasta related research. Over $2 million spent on C. shasta research annually

Salmonid Populations Salmonid species at risk: - Steelhead - Coastal cutthroat trout - Coho salmon* - Spring and fall-run chinook *Classified as an endangered species in 1997(ESA) and 2002 (CESA)

C. shasta Lifecycle Myxospore Actinospore Salmonid M. speciosa Every player in the C. shasta lifecycle can now be seen. Just to give perspective, M. speciosa is around the length and width of a human childs eyelash Additionally, C. shasta spores are microscopic, so research can become difficult when handing such small organisms. What is important to notice in this lifecycle is that the parasite has two distinct stages of life as seen in the earlier slide. The myxospore stage is shed from the infected salmonid and settles to the river bottom where is infects the invertebrate polychaete host, M. speciosa. M. speciosa then releases actinospores in the water infecting the vertebrate host, a salmonid spp., thus completing the lifecycle. Further, in any complex lifecycle, removing one of required hosts form the cycle will prevent the proliferation of the parasite In this study, I was seeing simply if any intraspecie genetic differences existed within the M. speciosa Klamath populations. Thus when looking at the C. shasta lifecycle, having a polychaete host that is genetically resistant to infection would protect salmonids from parasite infection. Intraspecies differences could affect the suitability of this polychaete as the definitive host

Hypothesis Objective: Manayunkia speciosa in the Pacific Northwest are the same species Objective: Determine if Klamath River polychaetes belong same species, and if so, determine if distinct strains of M. speciosa exist within the Klamath River Basin Thus my working hypothesis for this summer is…. Additionally then concerning the unusual distribution of the parasite…

Primary Locations of Research Klamath Lake Iron Gate Dam The primary locations for my research can be seen here labeled in red along the Klamath River The key thing to notice here is that 3 sites from above and below the Iron Gate Dam were chosen These sites were chosen primarily due to the extensive amount of data that existed from previous studies done by the Bartholomew lab. 7

Willamette River Willamette Klamath 1. Acted as a necessary outgroup for intraspecific phylogenetic comparisons Willamette 2. A known infectious zone for C. shasta Klamath 3. 300 miles NW of primary sample site, the Klamath. The Willamette River was chosen for several reason in addition to it’s close proximity to lab are Oregon State University. Having an outgroup is important when analyzing genetic diversity because it shows polarity and can show direction of evolution. It is also a known infectious zone for C. shasta It is geographically isolated from the Klamath draining into the Columbia Finally, it’s M. speciosa are genetically separated with both region sharing separate evolutions over time. 4. Genetically isolated river basin for comparison to the Klamath

Manayunkia genetic background No genetic data exists for M. speciosa However, Manayunkia spp. have been sequenced from Lake Baikal - M. sp.TP2002 - M. zenkewitschii - M. athalasia All Manayunkia were sequenced using universal primers targeting the Cytochrome Oxidase c subunit 1 gene Before beginning to analyze the Klamath’s M. speciosa population genetically, we had to research it’s pre-existing genetic history and build from there. However, no genetic data exists for M. speciosa on a species level. Thus we determined best approach in sequencing Klamath River M. speciosa DNA would be based on the method used in prior genus level efforts at genetic characterization. Three different Manayunkia spps have been sequenced, all from Lake Baikal, using existing universal primers targeting the Cytochrome Oxidase subunit 1 gene. Thus we entered into our project with various scientific elements that could be put together to build a platform from which to genetically characterize the Klamath M. speciosa populations. Lake Baikal

Methods 1. Extract DNA from collected samples 2. Specific CO1 gene amplified through PCR 3. PCR product sequenced and analyzed for novel genetic characteristics

Cytochrome Oxidase Subunit 1(CO1) CO1 gene: - Subunit 1 is a 710 bp mitochondrial gene - Codes for 13 proteins - Could possibly be used as a universal barcode for all species For Nematodes: The Cytochrome Oxidase subunit 1 gene was then chosen for genetic sequencing for differentiation between our Klamath and Willamette sites. So a little more about the actual gene…. CO1 is a electron carrier in the electron transport chain, thus it is present in every oxygen utilizing species This universal presence makes it an ideal gene for across the board species identification, or barcoding Further, this particular gene accumulates genetic variation quicker than it’s counterpart nuclear DNA. Making it ideal for intraspecies differentiation. The actual subunit 1 of Cytochrome Oxidase is only 710 bp long But it’s tendency to absorb genetic variations has caused many to believe it could act as an universal barcode for all species. - 0-6% variability: same species - 10-20% variability: different species Subunit 1

Interspecies variance values consistent with known species variability Species/Location Amplified # bp d-val sp2002 zenk athalasia Willamette M. athalasia 675 16% 22% - 19% M. sp2002 576 17% M. zenkewitschii 565 20% 520 Williamson 505 2.1% I5 Site 530 3.2% Interspecies variance values consistent with known species variability Known species variability This table then compares the values between the Lake Baikal species, the Klamath samples, and the outgroup Willamette samples. We established the known species variability values by comparing to the already existing Manyunkia CO1 data from Lake Baikal. As you can see, all these values fell within the stated nematode range of 10-20% for different species. Further, we compared our M. speciosa intraspecies values of the Willamette and one site above the Iron Gate Dam, the Williamson, and one site below the dam, referred to as the I5 site. These as well, seen in the bottom right, were also within the stated intraspecies nematode range of 0-6%. Intraspecies variance also within acceptable level

Current Genotypic Results Klamath Genotypes Willamette Genotypes

There are several mathematical approaches that can be used to analyse DNA sequence data to investigate relationships. You always analyse the data in multiple ways to determine which patterns have the most support – nodes on a tree. The two phylogenetic analyses we used were maximum parsimony and Bayesian.

Klamath and Willamette If we now look at the geographic distribution of M.s. genotypes within the Klamath Basin: we had expected that Iron Gate Dam may have influenced the distribution of genotypes, but its presence appears insignificant - perhaps too soon (40 years) for it to impact the CO1 gene? RATHER the defining geographic feature is Klamath Lake – and we see two main clusters of genotypes – those from the Klamath Lake and those from below the Klamath Lake.

Conclusion All the Manayunkia used in previous C. shasta research have been the species, Manayunkia speciosa. Distinct genotypes appear to exist, but further research is necessary

Ongoing Research Hudson River, NY Deschutes River, OR Cowlitz River, WA Fraser River, B.C. Lake Superior, WI 17

Acknowledgements Stephen Atkinson Gerri Buckles Charlene Hurst Sarah Bjork Jill Pridgeon Sue-Jie Koo Kevin Ahern Jerri Bartholomew Sascha Hallett HHMI