Genetic diversity of Manayunkia speciosa

in the Klamath River basin

By: Dan HornerMentors: Sascha Hallett

Jerri Bartholomew

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

Economic Effects of C. shasta

• Low salmon numbers forced regulatory closures of 2006 salmon fishing

• Loss of $150 million to the region's economy

• Over $2 million spent on C. shasta research annually

Salmonid Populations Salmonid species at risk:

- Spring and fall-run chinook

- Steelhead

- Coho salmon*- Coastal cutthroat trout

*Classified as an endangered species in 1997(ESA) and 2002 (CESA)

C. shasta Lifecycle

MyxosporeActinospore

Salmonid

M. speciosa

Intraspecies differences could affect the suitability of this polychaete as the definitive host

Hypothesis

• Manayunkia speciosa in the Pacific Northwest are the same species

• Determine if Klamath River polychaetes belong same species, and if so, determine if distinct strains of M. speciosa exist within the Klamath River Basin

Objective:

Primary Locations of Research

Iron Gate Dam

Klamath Lake

Willamette River

1. Acted as a necessary outgroup for intraspecific phylogenetic comparisons

2. A known infectious zone for C. shasta

3. 300 miles NW of primary sample site, the Klamath.

4. Genetically isolated river basin for comparison to the Klamath

Klamath

Willamette

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

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)

Subunit 1

• CO1 gene:

- Could possibly be used as a universal barcode for all species

- Subunit 1 is a 710 bp mitochondrial gene

- Codes for 13 proteins

• For Nematodes:

- 10-20% variability: different species

- 0-6% variability: same species

Species/Location Amplified # bp

d-valsp2002

d-valzenk

d-valathalasia

d-valWillamette

M. athalasia675 16% 22% - 19%

M. sp2002576

-19% 17% -

M. zenkewitschii565 20% - - -

Willamette520 - - 19% -

Williamson505 - - 19% 2.1%

I5 Site530 - - 19% 3.2%

Interspecies variance values consistent with known

species variability

Intraspecies variance also within acceptable level

Known species variability

Current Genotypic Results

Klamath Genotypes

Willamette Genotypes

Klamath and Willamette

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

Fraser River, B.C.

Lake Superior, WI

Deschutes River, OR

Cowlitz River, WA

Hudson River, NY

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