Progeny Marker for Salmonids
Statement of Work
October 1, 2004 to September 30, 2005
Bonneville Power Administration
Project 2002-030-00

PROJECT GOALS

Develop methodology and assess an elemental compound for marking the progeny of hatchery steelhead/rainbow trout-Onchorhynchus mykiss broodstock.

BACKGROUND

The Umatilla River Summer Steelhead Program artificially supplements Umatilla steelhead using wild endemic broodstock to prevent domestication.  Hatchery reared steelhead are the progeny of about 115 wild parents taken annually from a cross section of the run.  Historically, hatchery steelhead comprised from 10 to 60 percent of the adult returns (1986 to 1998; CTUIR et al., 2000).  Hatchery reared endemic summer steelhead are frequently observed digging redds and spawning naturally during spawning surveys (Contor et al. 1998).  The current management goal and assumption is that returning hatchery-reared steelhead reproduce successfully and enhance natural production.  However, because the supplementation program uses endemic steelhead for broodstock, there remains an inability to evaluate the reproductive success of hatchery fish using genetic markers (without very expensive and detailed DNA parent-progeny analysis involving thousands of fish per year for multiple years).  This project will evaluate how well strontium concentrations can be artificially elevated in the progeny of female trout that were injected prior to spawning.  
For the progeny mark to be successful, eggs must absorb the strontium before spawning and incorporate it into boney tissues during early life stages of growth.  Past studies have shown that erythromycin phosphate can pass from maturing females into the developing ova and resultant embryos (Haukenes and Moffitt, 1999).  Strontium is a likely candidate for marking otoliths because strontium can substitute for calcium in the calcium carbonate matrix of the otolith.  Strontium is ideal because its binding characteristics are similar to calcium (i.e. similar ionic radius and identical valence).  Normally, boney structures contain strontium at levels relative to background concentrations in the environment (Kalish, 1990).  
The elemental composition of fish bony structures, such as fin rays, scales and otoliths, provide a natural record of the chemical composition of environments experienced by individual fish (Gallahar and Kingsford, 1996; Wells et al., 2000).  The concentration of strontium is greater in seawater than most fresh water habitats.  Therefore, analysis of Sr: Ca ratios across the otolith of a fish can describe the anadromous history of that fish.  Further comparison of Sr: Ca ratios in the primordia (core region) and freshwater growth region can be used to determine maternal life histories (resident or anadromous).  Rieman (et al, 1994) analyzed otolith microchemistry to determine whether adult spawners were the progeny of migratory sockeye salmon or non-migratory kokanee.  Zimmerman and Reeves (2000 and 2002) repeated similar findings with steelhead and rainbow trout.
Researchers artificially increased chemicals such as strontium in otoliths, scales, vertebrae and/or dorsal spines of fish directly (Schroder et al. 1995, Behrens-Yamada and Mulligan 1982, Snyder et al., 1992, and Pollard et al., 1999).  We hypothesize that we can mark the otoliths of juvenile rainbow trout by administering the marking agent to their female parents.  We expect fish to mobilize stable strontium within the blood plasma by the yolk precursor vitellogenin, and into the developing ova.  Ultimately, progeny will incorporate strontium (at small but elevated rates) into the primordia of developing otoliths because fish substitute small amounts of strontium for calcium in bony structures (Kalish 1990).  Some ion exchange occurs between the female and her eggs in the period before spawning and such exchange may continue to influence the yolk content prior to spawning (Alderdice 1988).  Continuing ion exchange late in the development stage of eggs prior to spawning should provide elevated strontium concentrations in the otoliths of the progeny.
The primordia of salmonid otoliths are the first calcified structures to form in the developing embryo and are present at least several weeks before hatching (Rieman and Myers, 1994).  We do not expect to find elevated levels of strontium in scales or fin rays because those boney structures develop much later than otoliths.  However, because researchers have artificially increased strontium in bony structures of fish through direct methods (Behrens-Yamada and Mulligan 1982, Snyder et al.1992, Pollard et al., 1999), we will conduct some additional strontium testing on scales and fin rays of marked progeny to determine if non-lethal sampling alternatives would be practical.
Researchers have proven radioactive isotopes to be effective markers of fish.  However, marking fish with stable salts such as strontium chloride has many advantages over radioactive tagging.  Radioactive materials are comparatively expensive, environmentally unattractive, and a potential threat to human health.  The threat to human health is an important concern with radioactive isotopes because anglers may catch and consume the steelhead that would receive the proposed marker.
Guillou and de la Noue (1987) suggested using strontium to mark farm-reared brook trout destined for human consumption.  In fact, fish artificially marked with strontium in that particular study did not demonstrate any abnormal increase in strontium content of muscle or skin tissue.  We will be introducing strontium in the fish in a different manner (injected into the peritoneum or dorsal sinus); therefore, we will examine the concentrations of strontium in muscle and skin tissues to ensure there is no over-accumulation of strontium.
After developing and testing the marker, CTUIR will inject hatchery steelhead (identified by a fin clip) with the marker medium at the Three Mile Dam Trap on the Umatilla River.  The marked adults released at Three Mile Dam will spawn naturally and surviving progeny would carry the mark throughout their lives.  The chemical composition of otoliths primordia from a sample of naturally produced progeny would indicate the ratios of progeny from marked (hatchery reared) and unmarked (wild) females.  This would provide a means of evaluating the relative reproductive success of the endemic hatchery-raised females that spawn naturally.
LOCATION OF PROJECT
CTUIR will conduct this research project primarily at Oregon State University (OSU), Corvallis, Oregon and Three Mile Falls Dam adult holding facilities.  Otolith preparation as well as project oversight and administration will conducted at the Tribal Offices in Pendleton, Oregon.
OBJECTIVES AND TASKS

Objective 1. Determine blood clearance rate of strontium chloride hexahydrate (SrCl2-6H20) from adult steelhead/rainbow trout-Onchorhynchus mykiss given intraperitoneal cavity injections.

Objective 2. Determine strontium calcium (Sr/Ca) ratios of otoliths sampled from juvenile steelhead/trout from anadromous maternal parents from various locations within the Pacific Northwest.

Objective 3. Verify sample quality for all otoliths samples (2003-2005) and the precision of the wavelength dispersive microprobes.  

Objective 4. Analyze data gathered from the experimental trials and report results to BPA and the scientific community.


LITERATURE CITED

Alderdice, D.F.  1988.  Osmotic and ionic regulation in teleost eggs and larvae, p. 163-251. In  Rieman, B.E., D.L. Myers, and R.L. Nielsen.  1994.  The use of otolith microchemistry to discriminate O. nerka of resident and anadromous origin.  Can. J. Fish. Aquat. Sci. 51:68-77.

Behrens-Yamada, S., and T.J. Mulligan.  1982.  Strontium marking of hatchery-reared coho salmon, Oncorhynchus kisutch Walbaum, identification of adults.  J. Fish Biol.  20:5-9.

Contor, C. R.; Hoverson, E. and Kissner, P.  1998.  Umatilla Basin Natural Production Monitoring and Evaluation, Annual Progress Report 1996-1997. Portland: Confederated Tribes of the Umatilla Indian Reservation, Department of Natural Resources.

CTUIR et al.  Prepared for the Northwest Power Planning Council (NPPC).  2001.  Umatilla Subbasin/Willow Creek Subbasin Summary (Draft).

Gallahar, N.K. and M.J. Kingsford.  1996.  Factors influencing Sr/Ca ratios in Girella elevata.  J. Fish Biol.  48:174-186.

Guillou, A., and J. de la Noue.  1987.  Use of strontium as a nutritional marker for farm-reared brook trout.  Progressive Fish-Culturist.  49:34-39.

Haukenes, Alf H. and Christine M. Moffitt.  1999.  Concentrations of erythromycin in maturing chinook salmon after intraperitoneal injection of one of two drug formulations.  Journal of Aquatic Animal Health.  11:61-67.

Kalish, J.M.  1990. Use of otolith microchemistry to distinguish the progeny of sympatric anadromous and non-anadromous salmonids.  Fish. Bull.  88:657-666. In  Rieman, B.E., D.L. Myers, and R.L. Nielsen.  1994.  The use of otolith microchemistry to discriminate O. nerka of resident and anadromous origin.  Can. J. Fish. Aquat. Sci. 51:68-77.

Pollard, Morgan J., Michael J. Kingsford, and Stephen C. Battaglene. 1999.  Chemical marking of juvenile snapper, Pagrus auratus Sparidae), by incorporation of strontium into dorsal spines.  Fish. Bull. 97:118-131.

Reed, S.J.B. 1996. Electron Microprobe Analysis and Scanning Electron Miroscopy in Geology. Cambridge University press. Great Britain, pp. 158-159.

Rieman, B.E., D.L. Myers, and R.L. Nielsen.  1994.  The use of otolith microchemistry to discriminate O. nerka of resident and anadromous origin.  Can. J. Fish. Aquat. Sci. 51:68-77.

Schroder, S.L., C.M. Knudsen, and E.C. Volk.  1995.  Marking salmon fry with strontium chloride solutions.  Can. J. Fish Aquatcult.  0(suppl. 2):79-83.

Snyder, R.J., B.A. McKeown, K. Colbow, and R. Brown.  1992.  Use of dissolved strontium in scale marking of juvenile salmonids: effects of concentration and exposure time.  Can. J. Fish. Aquat. Sci.  49:780-782.

Wells, Brian K., Gretchen E. Bath, Simon R. Thorrold, and Cynthia M. Jones.  2000.  Incorporation of strontium, cadmium, and barium in juvenile spot (Leiostomus xanthurus) scales reflects water chemistry.  Can. J. Fish. Aquat. Sci.  57:2122-2129.

Zimmerman, Christian E. and Gordon H. Reeves.  2000. Population structure of sympatric anadromous and nonanadromous Oncorhynchus mykiss: evidence from spawning surveys and otolith microchemistry. Can. J. Fish. Aquat. Sci. 57:2152-2162.

Zimmerman, Christian E. and Gordon H. Reeves.  2002. Identification of steelhead and resident trout progeny in the Deschutes River, Oregon, revealed with otolith microchemistry.  Transaction of the American Fisheries Society 131;986-883.

[From Excel File:]
The relative reproductive success of hatchery reared endemic steelhead and their naturally reared cohorts is a critical uncertainty associated with strategies for the restoration, mitigation and enhancement of salmon and steelhead the Umatilla River Basin and abroad.    
  
Determining the reproductive success of hatchery fish spawning in the wild has been listed as important research need identified by ISRP, ISBA, subbasin plans, and RPAs in the draft NMFS Biological Opinion as a critical uncertainty (Subbasin Plan; section 1.4.3, page 1-29; Appendix H, Umatilla Basin RM&E Plan, Management Objective 3, Assumption 3e, 3g, 5a, M&E Objective 3e, 3g and 5a, pages H-2 and H-3).

The development of the progeny mark will provide a useful and cost effective tool to determine the reproductive success of hatchery and naturally produced steelhead spawning in the wild without the need for expensive and detailed genetic pedigree analysis.
Pedigree analysis is a valuable tool but requires the handling of every single adult fish at the interception point.  In the case of the Umatilla River, that would mean the physical handling of thousands and thousands of adult salmon and steelhead including the non-target species given the logistics at the adult trap and thus can only be deployed on the smaller tributary scale.

The progeny mark would allow sub-sampling of the adult returns and related progeny which will reduce costs and associated handling stress on target and non-target species and will need to be tested along side a pedigree type analysis (Subbasin Plan, Appendix, pages H-10, H-21 and H-77).

Determining the reproductive success of hatchery fish in the wild and reducing stress to returning adult salmon and steelhead are goal consistent with the current subbasin plan and will aid in the adaptive management of the Umatilla Basin Fish Restoration Project (Subbasin Plan, Section 2.5, page 2-7 and Section 5.6 page 5-88, evaluating management strategies).