Project ID:
Title: Genetic Predisposition of Smoltification in Rainbow Trout and Steelhead
Columbia River Inter-Tribal Fish Commission
729 NE Oregon, Suite 200
Portland, Oregon 97232
Total Funding Requested:
$89,978
Response prepared by:
Columbia River Inter-Tribal Fish Commission
Shawn Narum
Jeffrey Stephenson
Yakama Nation
Bill Sharp
Chris Frederiksen
Joe Zendt
FY 2007-09 Innovative Projects, Section 10 1

A. Abstract
This study focuses on the use of molecular methods to quantify the potential of resident rainbow trout to contribute to recovery of steelhead populations. Resident rainbow trout and anadromous steelhead life history types ( Oncorhynchus mykiss ) in the Mid-Columbia
River are listed as a threatened species under the Endangered Species Act (ESA). Since these life history types of O. mykiss interbreed, there is potential for rainbow trout to contribute to the recovery of decimated steelhead stocks in the Pacific Northwest. Both environmental and genetic factors determine if individual O. mykiss remain as resident rainbow trout, or undergo the necessary physiological changes (smoltification) to prepare for anadromy. While some of the associated environmental factors (i.e. water flow and temperature) have been evaluated, the genetic mechanisms that contribute to life history selection are unknown. This proposal seeks to identify quantitative genetic characteristics that predispose individual O. mykiss to smoltification. The study focuses on three tributaries to the Klickitat River with varying abundance of each life history type: 1) White Creek - near exclusive steelhead population, 2) upper Summit Creek - predominant resident population, and 3) Snyder Creek - mixed population with both life history types. The primary objective of this project is to identify genetic traits that could be used to recover threatened steelhead populations.
B. Technical and/or scientific background
In the Klickitat River sub-basin plan, recovery of steelhead populations is considered one of the highest priorities in this region. For example, steelhead in the upper Klickitat River represent a crucial component of genetic variation in this sub-basin and may potentially contribute to anadromous production and recovery of steelhead. However, little is known regarding smolt production or limitations of O. mykiss smoltification. These critical uncertainties are addressed by the objective of this proposal: quantifying the expression of smoltification in juvenile O. mykiss . The information gained from this study will be used to implement conservation management strategies to assist recovery of the upper
Klickitat River steelhead population and others in the sub-basin.
In order to demonstrate the utility of this approach, a pilot study was completed to evaluate the genetic expression of the Na-K-ATPase gene in hatchery reared smolts. The expression of this gene is expected to change relative to photoperiod (osmoregulation during smoltification), and we detected significant differential expression of one isoform
(α1a) of the gene over the course of development during rearing at Hagerman National
Fish Hatchery (Figure 1). We also observed a trend in expression of two isoforms relative to changing photoperiod (Figure 2). The results of this pilot study indicate that expression of genes known to play a major role in smoltification (i.e., Na-K-ATPase) may be evaluated to determine smolt quality and test the potential of resident fish to contribute to anadromy in natural populations.
FY 2007-09 Innovative Projects, Section 10 2

C. Rationale and significance to regional programs
Steelhead of the Columbia River basin have declined tremendously in the last century
(Busby et al. 1996) and are a critical biological component to maintaining the river ecosystem. Populations in the Klickitat River are of critical concern as human impacts have reduced steelhead abundance and are listed as threatened under the ESA. Further, steelhead are considered a keystone species as they contribute nutrients for a wide variety of not only aquatic species, but also terrestrial plants and animals. Despite the decreasing abundance of steelhead in the Columbia River basin, this species remains of great cultural value to tribal community. This proposal seeks to identify critical genetic information that will contribute to the recovery of this species of great biological and cultural significance in the Klickitat River.
In the Klickitat River subbasin plan, steelhead are listed as one of the highest priority species for research and recovery projects. Strategies in this sub-basin include restoring steelhead above previous barriers. As some of these passage barriers have been removed or improved (i.e., Castile Falls), native resident populations may retain smoltification abilities and can possibly contribute to anadromous production. This study may provide information to assist with anadromous production not only in the Klickitat River, but research findings are likely to be applicable to other subbasins. This includes rivers such as the White Salmon River that is scheduled to have anadromous fish passage at Condit
Dam. If possible, recovery of anadromous runs from native resident stocks is highly preferred over out-of-basin populations. This proposal offers a thorough evaluation of steelhead smolt production and quantification of genetic traits related to smoltification that may be widely applied.
D. Relationships to other projects
Evaluating smolt production and smoltification potential in the Klickitat River is complementary to other research programs in the subbasin to restore steelhead populations such as ongoing research by the YKFP (Yakima Klickitat Fisheries Project).
E. Project history (for ongoing projects)
Not applicable to this new project.
F. Proposal biological objectives, work elements, and methods
The biological objective of this proposal is to identify quantitative genetic characteristics that predispose O. mykiss to smoltification. This objective focuses on the use of quantitative genetic markers to evaluate the potential of resident rainbow trout to contribute to recovery of steelhead populations. Both environmental and genetic factors determine if individual O. mykiss remain as resident rainbow trout, or undergo the necessary physiological changes (smoltification) to prepare for anadromy. While some of the associated environmental factors (i.e., water flow and temperature) have been
FY 2007-09 Innovative Projects, Section 10 3

evaluated, the genetic mechanisms that contribute to life history selection are unknown.
This proposal seeks to identify quantitative genetic characteristics that predispose individual O. mykiss to smoltification. The study focuses on three tributaries to the
Klickitat River with varying abundance of each life history type: 1) White Creek - near exclusive steelhead population, 2) upper Summit Creek - predominant resident population, and 3) Snyder Creek - mixed population with both life history types. The study will utilize quantitative genetics technology such as real-time PCR and microarrays to identify genes that regulate expression of smoltification in O. mykiss . This objective will identify genetic traits that could be used to recover threatened steelhead populations.
Specific deliverables include:
1) Identification of genes that predispose individual O. mykiss to anadromy.
2) Modeling of genetic and environmental factors that may assist recovery of steelhead.
3) Management plans to maximize genetic traits for anadromy in the population.
Objective 1 . Identify quantitative genetic characteristics that predispose O. mykiss to smoltification.
Work Element 1.1 – Field sampling to collect tissues from resident and smolting O. mykiss
Sampling Methods:
Tissues (gill, brain, and liver) will be collected from juvenile O. mykiss at four time intervals (n = 10 each time) relative to known physiological development of smoltification. April and May are the peak of many hormonal cues (i.e. NaK-ATPase) that indicate smoltification (Boeuf 1993). The sampling time periods are spaced at three month intervals to account for gene expression prior to (February), during (May), and after (August and November) expected peak smoltification.
Three sampling sites were chosen to represent a predominant steelhead population (White
Creek), an exclusively resident rainbow trout population (Summit Creek), and a population with a mixture of resident rainbow trout and anadromous steelhead (Snyder
Creek). An additional site on the upper Klickitat R. above Castile Falls (a primarily resident but possibly mixed population) will be added if access and flow conditions are conducive to sampling.
Individual O. mykiss will be sampled with beach seines or electrofishing by field personnel and transported live to a facility for tissue dissection. Immediately prior to dissection, fish will be sacrificed with an overdose of MS222. Target organs (gill, brain, and liver) will be removed and stored in RNAlater to preserve RNA quality. Fin clips will also be taken and stored in ethanol to preserve DNA.
FY 2007-09 Innovative Projects, Section 10 4

Work Element 1.2 – Genetics Laboratory Analysis
Laboratory Methods:
RNA and DNA will be extracted from tissue samples for analyses using a Qiagen 3000 robot and DNeasy/RNeasy protocols from Qiagen. DNA will be PCR amplified with microsatellite loci that map to a quantitative trait locus (QTL) for smoltification (Nichols,
K. pers. comm.). These fragments will be scored on an automated genotyping instrument and allele frequencies will be calculated.
RNA extracted from tissues will be hybridized to microarrys (GRASP; Rise et al. 2003) to test the genome for differential expression. We will use a “reference design” where all experimental samples are compared to a common reference sample. RNA from five to ten individuals will be pooled from each site. We will label the RNA with Cy3 and Cy5 using RT-PCR (reverse transcription PCR) and then hybridize the products to GRASP microarrays containing 16,000 expressed sequence tags. Pairwise tests of each treatment will be performed with a “dye flip” (alternating the dye label to control for fluorescence strength). Samples from each site will be pooled, and a total of 12 microarrays will be necessary (4 time periods x 3 sites). Genes with at least two fold differential expression will be targeted and validated with Applied Biosystem TaqMan assays.
Applied Biosystem TaqMan assays will be used to quantitate expression of known NaK-
ATPase or thyroid genes in RNA from tissue samples. This information will be used to validate differential expression from microarrays.
Work Element 1.3 – Statistical Analysis
Statistics Methods:
Differences in QTL allele frequencies will be tested for significance among the three sample sites. Genes with at least two fold differential expression (average of three replicates) will be searched in GenBank to determine if the genes have been annotated.
Annotated genes of interest will be validated with gene specific TaqMan assays and the level of differential expression will be quantified among treatment groups.
Work Element 1.4 – Interpretation/Reporting
This experiment will provide information to determine the level of genetic predisposition of O. mykiss for smoltification. Differentially expressed genes will be correlated to treatment groups with variables that include water temperature and flow, thermal units, and food resources. This analysis will determine the combination of genetic traits and environmental factors that will be used in recovery of steelhead in the Klickitat River sub-basin.
All results will be reported in a report to the Bonneville Power Administration, and a publication submitted to a journal.
FY 2007-09 Innovative Projects, Section 10 5

Proposed time line
Calendar Year 2008
July-August – Optimize QTL and microarray analysis
August – collect tissue samples (Time 1)
November – collect tissue samples (Time 2)
Calendar Year 2009
February – collect tissue samples (Time 3)
May – collect tissue samples (Time 4)
Jan.-June. – QTL and microarray analysis
July-Aug. – Statistical Analysis of data / Write report
September – Submit report to BPA
G. Facilities and equipment
YKFP has the existing infrastructure to support sampling programs in the Klickitat River subbasin. This includes staff, field gear, and office space. Genetic analysis will be completed at the Hagerman Fish Culture Experiment Station operated by University of
Idaho and Columbia River Intertribal Fish Commission staff. The Hagerman site houses multiple laboratories (including genetics, nutrition, and culture) with staff and office space. The genetics laboratory is fully equipped with all major hardware to complete the objectives of this project. Funding from this project will provide money for salaries, consumable laboratory supplies, minor laboratory equipment, and equipment maintenance.
H. References
Banks, M. A., M. S. Blouin, B. A. Baldwin, and V. K. Rashbrook. 1999. Isolation and inheritance of novel microsatellites in chinook salmon ( Oncorhynchus tshawytscha ). Journal of Heredity 90:281-288. See erratum May/June 1999 for primer sequence corrections.
Boeuf, G. 1993. Salmonid smolting: a pre-adaptation to the oceanic environment. In
(J.C. Rankin and F.B. Jensen, Eds.) Fish Ecophysiology, pp. 105-125. Chapman and Hall, London.
Buchholz, W., S. J. Miller, and W. J. Spearman. 1999. Summary of PCR primers for salmonid genetic studies. U.S. Fish and Wildlife Service, Alaska Fisheries
Progress Report 99-1.
Busby, P. J., T. C. Wainwright, G. J. Bryant, L. J. Lierheimer, R. S. Waples, F. W.
Waknitz, and I. V. Lagomarsino. 1996. Status review of West Coast steelhead from Washington, Idaho, Oregon, and California. NOAA Technical
Memorandum NMFS NWFSC-27.
Cairney, M., J. B. Taggart, and B. Hoyheim. 2000. Characterization of microsatellite
FY 2007-09 Innovative Projects, Section 10 6

and minisatellite loci in Atlantic salmon ( Salmo salar L.) and cross-species amplification in other salmonids. Molecular Ecology 9:2175-2178.
Condrey, M. J., and P. Bentzen. 1998. Characterization of coastal cutthroat trout
(Oncorhynchus clarki clarki ) microsatellites and their conservation in other salmonids. Molecular Ecology 7:787-789.
Evans, A.F. and R.E. Beaty 2001. Identification and Enumeration of Steelhead
( Oncorhynchus mykiss ) Kelts in the Juvenile Collection Systems of Lower
Granite and Little Goose dams, 2000. Annual Report to US Army Corps of
Engineers, Walla Walla District.
Kalinowski, ST. 2003. Genetic Mixture Analysis 1.0. Department of Ecology, Montana
State University, Bozeman MT 59717. Available for download from http://www.montana.edu/kalinowski.
McConnell, S., L. Hamilton, D. Morris, D. Cook, D. Paquet, P. Bentzen, and J. Wright.
1995. Isolation of salmonid microsatellite loci and their application to the population genetics of Canadian east coast stocks of Atlantic salmon.
Aquaculture 137:19-30.
Moran, P. 2003. Genetic structure of Oncorhynchus mykiss populations in the Grande
Ronde River, Imnaha River, and adjacent regions of the Snake River Basin. Final
Report submitted to the U.S. Fish & Wildlife Service. Available from paul.moran@noaa.gov.
Morris, D. B., K. R. Richard, and J. M. Wright. 1996. Microsatellites from rainbow trout ( Oncorhynchus mykiss ) and their use for genetic study of salmonids.
Canadian Journal of Fisheries and Aquatic Sciences 53:120-126.
Narum, S. R., S. Boe, P. Moran, and M. Powell. In press. Small scale genetic structure and variation in steelhead trout of the Grande Ronde River, Oregon, U.S.A.
Transactions of the American Fisheries Society, in press.
Nelson, R. J., and T. D. Beacham. 1999. Isolation and cross species amplification of microsatellite loci useful for study of Pacific salmon. Animal Genetics
30:228-229.
Olsen, J. B., P. Bentzen, and J. E. Seeb. 1998. Characterization of seven microsatellite loci derived from pink salmon. Molecular Ecology 7:1087-1089.
Olsen, J. B., S. L. Wilson, E. J. Kretschmer, K. C. Jones, J. E. Seeb. 2000.
Characterization of 14 tetranucleotide microsatellite loci derived from sockeye salmon. Molecular Ecology, 9:2185–2187.
Rannala, B., and J. L. Mountain. 1997. Detecting immigration by using multilocus genotypes. Proceedings of the National Academy of Sciences (U.S.) 94:9197-
9201.
Rise, M.L., and 23 coauthors. 2003. Development and application of a salmonid EST database and cDNA microarray: data mining and interspecific hybridization characteristics. Genome Research 14:478-490.
FY 2007-09 Innovative Projects, Section 10 7

I. Key personnel

Shawn Narum, Lead Geneticist, 3 months (FTE) per year

Bill Sharp, 0.5 months (FTE) per year

Chris Frederiksen, Lead Modeling Scientist, 2 months (FTE) per year

Joe Zendt, 0.5 months (FTE) per year
FY 2007-09 Innovative Projects, Section 10 8

SHAWN R. NARUM
Columbia River Inter-Tribal Fish Commission
Hagerman Fish Culture Experiment Station
3059-F National Fish Hatchery Rd.
Hagerman, ID 83332
208-837-4071 nars@critfc.org
Education
Ph.D., Natural Resources, University of Idaho, 2006
M.S., Marine Science, University of San Diego, 2000
B.S., Fishery Biology, Colorado State University, 1996
Appointment
2002-present Lead Geneticist, Columbia River Inter-Tribal Fish Commission
Experience
My research interests include population structure and genetic diversity of natural populations of salmonids to assist with conservation efforts. Much of my research has focused on variation in life history types, interaction of hatchery and wild stocks, mixed stock analyses, and genetic control of smoltification.
Selected Publications
Narum , S. R., and M. R. Campbell. In press . Genetic variation and structure of Chinook salmon life history types in the Snake River. Transactions of the American Fisheries Society.
Narum , S. R., W. D. Arnsberg, A. J. Talbot, M. S. Powell. 2007. Reproductive isolation following reintroduction of Chinook salmon with alternative life histories.
Conservation Genetics, DOI 10.1007/s10592-006-9268-9.
Seeb, L. W, A. Antonovich, M.A. Banks, T.D. Beacham, M.R. Bellinger, S. M. Blankenship, M.
Campbell, N.A. Decovich, J.C. Garza, C.M. Guthrie III, T. A. Lundrigan, P. Moran, S.R.
Narum , J.J. Stephenson, K.J. Supernault, D.J. Teel, W.D. Templin, J.K.Wenburg, S.F.
Young, C.T. Smith. In press . Development of a Standardized DNA Database for
Chinook Salmon. Fisheries, in press.
Narum , S. R., S. Boe, P. Moran, M. Powell. 2006. Small-scale genetic structure and variation in steelhead of the Grande Ronde River, Oregon, USA. Transactions of the
American Fisheries Society 135:979-986.
Narum , S. R. 2006. Beyond Bonferroni: Less conservative analyses for conservation genetics. Conservation Genetics 7:783-787.
Narum , S.R, M. Powell, and A. Talbot. 2004. A distinctive microsatellite locus that differentiates ocean from stream type Chinook salmon ( Oncorhynchus tshawytscha ) in the Columbia River Basin. Transactions of the American Fisheries Society 133:1051-
1055.
FY 2007-09 Innovative Projects, Section 10 9

Christopher R. Frederiksen
Department of Natural Resources
Fisheries Program
Yakama Nation
(509) 966-5156
Education:
Montana State University, Bozeman, Montana
Bachelor of Science in Geo-Hydrology with Stream Ecology emphasis, 2001.
Experience:
Yakama Nation Lead Modeling Scientist (May 2004 – present)

Constructed EDT model outputs and interpretation used for Salmon Recovery
Planning process for Yakima and Klickitat Subbasins.

Developed variable stock recruitment curves characterizing natural production for Klickitat Spring Chinook

Modeled artificial production potential for an integrated spring chinook hatchery program based on habitat characteristics, Klickitat Subbasin.

Black Rock Feasibility Study- Provide modeling expertise to BOR for attribute refinements and adjustments.

Modeled population performance of resident rainbow and steelhead existing in sympatry

Participate in development of species-specific YKFP master plans

Supervise and train 1 FTE biologist in the use of the EDT model

Supervise and train 2 field technicians in data collection for EDT attributes.
Yakama Nation Modeling Scientist (Dec 2002 – April 2004)

Constructed Klickitat EDT model

Work with YKFP and FRM biologist to perform EDT analysis for specific habitat and supplementation projects

Collect variety of field data for both physical and biological EDT attributes

Supervised and trained 2 PTE field technicians
Yakama Nation Field Biologist (July 2002 – Nov 2002)

Conducted Spring Chinook redd surveys and carcass recovery

Assisted with screw trap installation and operations

Assisted with screw trap data analysis for smolt production estimates
FY 2007-09 Innovative Projects, Section 10 10

Joseph S. Zendt
Fisheries Biologist
Yakama Nation Fisheries Resource Management Program
P.O. Box 215
Klickitat, Washington 98628
Office Phone: 509-369-3184
Email: jzendt@ykfp.org
Education: M.S. in Fishery Biology, July 1999 - Colorado State University, Fort Collins, Colorado
B.S. in Biology, May 1994 - Bucknell University, Lewisburg, Pennsylvania
Employment: Fisheries Biologist, Yakama Nation Fisheries Resource Management Program,
Toppenish and Klickitat, Washington - Jan. 2000-present

Planned and conducted projects to monitor and evaluate fish populations and habitat the Klickitat River subbasin and in the forested area of the Yakama Reservation
(includes stream fish sampling, spawner surveys, habitat surveys)

Evaluated proposals for timber sales and other forest management activities, recommending modifications to minimize impacts to fisheries habitat

Member/Current chair of Klickitat Technical Advisory Group (part of Washington
Salmon Recovery Funding Board planning process)

Data management and analysis, development of project proposals and budgets, preparation of written and oral reports

Supervision of field technician crews
Research Technician, Colorado Cooperative Fish and Wildlife Research Unit, Colorado
State University, Fort Collins, Colorado - July-Dec. 1999
Fisheries Technician, Colorado Division of Wildlife, Denver, Colorado - April-June 1999
Graduate Research Assistant, Colorado Cooperative Fish and Wildlife Research Unit,
Colorado State University, Fort Collins, Colorado - July 1996-April 1999
Fisheries Technician, Virginia Department of Game and Inland Fisheries, Verona,
Virginia - May-June 1996 and March-December 1995
Commercial Fisheries Observer/Biologist, Saltwater, Inc., Anchorage, Alaska -
December 1995-April 1996
Publications:
Zendt, J. 2006. Yakima/Klickitat Fisheries Project - Klickitat Monitoring and Evaluation, 2005-2006
Annual Report. Project No. 199506335, BPA Report DOE/BP-00022506-1.
Zendt, J. and B. Sharp. 2006. Influences of Stocking Salmon Carcass Analogs on Salmonids in Klickitat
River Tributaries. Project No. 200105500, BPA Report DOE/BP-00007534-1.
Zendt, J. and R. Evenson. 2005. Yakima/Klickitat Fisheries Project - Klickitat Monitoring and Evaluation,
2004-2005 Annual Report. Project No. 199506335, BPA Report DOE/BP-00017757-1.
Zendt, J.S. and E.P. Bergersen. 2000. Distribution and abundance of the aquatic oligochaete host Tubifex tubifex for the salmonid whirling disease parasite Myxobolus cerebralis in the upper Colorado River basin.
North American Journal of Fisheries Management 20:502-512.
FY 2007-09 Innovative Projects, Section 10 11

WILLIAM R. SHARP
Department of Natural Resources
Fisheries Program
Yakama Nation
(509) 865-5121
EDUCATION:
B.S. Natural Resource Management with minor in Watershed Science. Colorado State
University. 1987
Instream Flow Incremental Methodology (IFIM) certification. Colorado State University
1988.
EXPERIENCE:
Yakama Nation Research Scientist IV (2005- present).

Project Manger directly responsible for managing and implementing all activities related to policy formulation and planning within the Klickitat Subbasin under the
Yakima Klickitat Fisheries Project.

Responsibilities include research design and development, data analysis and synthesis, report writing, budgeting, and subcontract development.

Administer NOAA-Fisheries Pacific Coastal Salmon Recovery and Washington
State Salmon Recovery habitat restoration grants awarded to the Yakama Nation.
 Function as team leader for the Northwest Power Planning Council’s development of Klickitat Fisheries Master Plan.

Manage biologists, data manager and watershed planner.
Fisheries Biologist I-III, Yakama Nation Fisheries Program, 1989- 2005

Functioned as lead biologist on the Klickitat Monitoring & Evaluation Project and conducted all aspects of project design, budgeting, salmonid population monitoring at all life stages and stream habitat inventory.

Managed all aspects of field crew supervision (crews of 2-15 technicians). This includes research design and development, daily field operation, data analysis and synthesis, report writing, and subcontract development.

Functioned as technical representative to the Washington State Salmon Recovery/
Watershed Planning effort and NOAA Salmon Recovery Plan development in the
Klickitat Subbasin.

Developed and implemented habitat restoration project in both the Yakima and
Klicitat Basins. Completed project to enhance in-channel and off-channel rearing habitat and create alcoves and velocity refugia to benefit depressed spring chinook and steelhead stocks.

Conducted adult salmon radio telemetry studies in the Yakima Basin.

Managed juvenile collection facilities in rivers and small tributaries, from large scale fixed collection facilities at dams to mobile and weir traps on tributaries.
Fisheries Biologist I, US Fish and Wildlife Service, 1988-1989

Conducted IFIM studies on rivers in Oregon and Washington
FY 2007-09 Innovative Projects, Section 10 12

1.40
Figure 1 . Mean gene expression of four Na-K-ATPase isoforms (relative to control gene
ARP) in steelhead smolts reared at Hagerman National Fish Hatchery. Significant differences in the α1a isoform are indicated by columns that do not share the same letter.
Error bars are ± s.e.m of 30 samples tested at each time period. a
1.20
a,b
1.00
0.80
0.60
b
α1a
α1b
α1c
α3
0.40
0.20
0.00
Nov Feb April
FY 2007-09 Innovative Projects, Section 10 13

Figure 2 . Gene expression of four isoforms of Na-K-ATPase (relative to control gene ARP) over changing photoperiod. The dark line indicates the number of daylight hours for each sample period at the latitude and longitude coordinates of the rearing site (Hagerman National
Fish Hatchery).
1.4
14
1.2
1.0
0.8
12
10
8
0.6
0.4
0.2
0.0
6
4
2
0
Nov Dec Jan Feb Mar April
α1a
α1b
α1c
α3
Daylight
FY 2007-09 Innovative Projects, Section 10 14