1. 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

2. 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

3. 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

4. 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)

5. 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

6. 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…

7. 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

8. Willamette River Willamette Klamath
1. Acted as a necessary outgroup for intraspecific phylogenetic comparisons
Willamette
2. A known infectious zone for
C. shasta
Klamath
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

9. 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

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

11. 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
% variability: different species
Subunit 1

12. 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

13. Current Genotypic Results
Klamath Genotypes
Willamette Genotypes

14. 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.

15. 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.

16. 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

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

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