SALMON RESTORATION PROGRAM       WASHINGTON STATE UNIVERSITY THE UNIVERSITY OF IDAHO & NATIONAL MARINE FISHERIES       WSU/UI and NMFS SALMON RESTORATION RESEARCH PROGRAM Executive Summary Objective ­ Establish a multi-investigator program in the area of Fish Reproduction and Salmon Biology to enhance research activities and promote interactions between the investigators. The benefit is an integrated program to address and apply basic aspects of fish reproduction and biology to the improvement of native salmon stocks in the Northwest. Organization- Utilizes the organization and existing structure for the Center for Reproductive Biology at the University of Idaho and Washington State University to help administer and integrate the research programs with the Northwest Fisheries Sciences Center, National Marine Fisheries Services. Summary - The collaborative fish reproduction research proposed will address concerns regarding the conservation and enhancement of native salmon populations. The products expected from these projects are (1) an increase in the number of salmonid populations represented and the inclusion of maternal genomes in a developing germplasm repository, (2) a more complete accounting of the genetic sex of the wild populations and a more complete understanding of the genes on the Y chromosome, (3) new methods to estimate genetic damage in salmonid populations, (4) increases in egg and sperm quality, (5) identification of the interactions between disease, immunology and reproduction and (6) identification of mechanisms of olfactory imprinting to develop hatchery practices that will minimize straying and negative interactions between hatchery and wild salmon. All of the projects are designed to identify possible insults that may compromise the reproductive performance of threatened and endangered native populations of fish. The salmon biology research directly addresses many of the questions and issues raised in the NWRSC Salmon Research Plan. Projects - The central theme of the proposed projects is an integrated approach to a basic understanding of the mechanisms controlling salmon biology and reproduction. The outcomes of these research activities are expected to provide (1) new information with which to make informed decisions and (2) new or improved procedures to increase the reproductive efficiency of captive broodstock programs. WSU/UI PROJECTS - Project 1 - Dr. Joseph Cloud, UI, Establishing a Germplasm Repository for ESA-listed Anadromous Salmonids in the Columbia Basin. Project 2 - Dr. James Nagler, UI; Gary Thorgaard, WSU; and Ruth Phillips, WSU, Genetic Sex of Wild Chinook Salmon Project 3 - Dr. Gary Thorgaard, WSU, Genetic analysis of domestication behavior in Oncorhynchus mykiss Project 4 - Dr. Rolf Ingermann, UI, Impact of Stress on Gamete Quality in Salmonids Project 5 - Dr. Douglas Call and Terry McElwain, WSU, Microarray detection of multiple pathogens in managed and wild salmon populations Project 6- Dr. Ken Cain, UI, Reduction of disease-related impacts on important salmonid stocks through broodstock immunization against key pathogens. Project 7 - Dr. Graham Young, UI, Regulation of steroid production in salmonids and impacts of environmental contaminants. Project 8 - Dr. Ruth Phillips, WSU-Vancouver, Mapping the Male Specific Genes on the Y Chromosome in Chinook Salmon. NMFS PROJECTS - Project 9 - Dr. Penny Swanson, REUTD, Northwest Fisheries Service Center and Dr. Briony Campbell, University of Washington, Environmental and endocrine regulation of salmon and marine fish reproduction. Project 10 - Lyndal Johnson, EC, Northwest Fisheries Service Center, Contaminant effects on fish reproduction. Project 11 -Dr. Andrew Dittman, REUTD, Northwest Fisheries Service Center, "Mechanism of olfactory imprinting and homing, and impacts of hatchery practices on straying in salmon" Project 12 - Dr. Nat Scholz, EC, Northwest Fisheries Service Center, Contaminant effects on fish neurobiology and development Project 13 - Dr. Mark Strom, REUTD, Northwest Fisheries Service Center, Fish migratory health and disease. Project 14 - Dr. Brian Beckman and Dr. Don Larsen, REUTD, Northwest Fisheries Service Center, Effects of genetics and environment on salmon life history pathways. Project 15 - Dr. Linda Park, CB, Northwest Fisheries Service Center, Mapping genes for development, age of maturity and Growth. NMFS SALMON RESEARCH PLAN RELEVANCE The Northwest Fisheries Science Center (NWFSC) of the National Marine Fisheries Service (NMFS) has developed a "Salmon Research Plan" That contains specific questions and issues regarding salmon recovery and science. The Cooperative Institute for Salmon Research and Science between the NWFSC of NMFS and the Center for Reproductive Biology of WSU and UI will provide direct basic research to address a number of these major questions. The research focus on Salmon Biology in this program directly applies biological research to these questions. Examples of these items are as follows: 1. How can we identify the specific attributes of a viable salmon ESU so that we can in turn provide quantitative goals for recovery? 2. To what extent do hydropower operations contribute to the declining population trends evident in many salmon populations, and how can we quantify the benefits of major alterations in hydropower operations? 3. To what extent do hatchery operations of any kind contribute to or mitigate the risk of extinction faced by small wild salmon populations? 4. Can we establish explicit links between salmon productivity and habitat attributes that can be protected or restored via management actions? 5. Is there a way of making the ideal of "ecosystem and multi-species management" operational for salmon? 6. Global climate change is upon us. Should our strategies for salmon recovery take this climate change (as it alters ocean conditions, the terrestrial environment, and fundamental physical processes) into explicit consideration? The proposed research program provides the biological components to these questions in regards to research, performance measures and identification of the mechanistic issues. Examples of research impacts and interactions are listed below. SELECTED RESEARCH INTERACTIONS The following are examples of possible research interactions between the University and NWFSC researchers. These research themes are to illustrate areas of scientific interaction. Areas of collaborative research are not limited to these themes. Collaborations in these and related areas will facilitate the development of specific biological performance measures that can be used in salmon recovery efforts. - The Genomic Core laboratory associated with the Center for Reproductive Biology, as well as University researchers can be accessed by NMFS scientists to provide advanced technology in micro-arrays and bio-informatics. This can include simple access to the Core laboratory or assistance in experimental design and technique development. The principle of access also applies to the other seven Core Laboratories of the Center for Reproductive Biology. - Apply advances in genetic mapping and genetic toxicology to assess the effects of genetic damage on for complex traits such as reproduction, smoltification, disease resistance and domestification behavior. - Evaluate the impact of sex reversal and YY male Chinook in the population are present for both groups. Research will address the molecular and cellular mechanisms of how this phenomena occurs and its impact on the population. - Molecular and genetic comparisons of wild and hatchery fish with emphasis on evaluating and improving supplementation efforts and broodstock operations for Pacific salmon. - Improved understanding of the etiology of diseases of Pacific salmon and methods to reduce the impact of diseas. Coordination would facilitate research on a variety of diseases (such as BKD, CWD, IHN, etc.) rather than narrowly focusing on only one issue. One example may be in detecting virulence factors associated with important salmonid pathogens, or in utilizing advanced diagnostic techniques (such as micro-arrays), to assess pathogen loads relative to salmonid populations, watersheds or habitats. - Determine factors affecting disease transmission during hatchery supplementation and barge transportation through the application of advanced genomics approach developed by University scientists. - Research on genetic archive procedures for storing male and female gamete from wild fish populations. - Determine those factors in freshwater life stages that limit salmon productivity in the Columbia River basin tributaries. Research will involve assessment of the fitness of juvenile salmon related to abiotic (e.g., habitat condition, water quality) and biotic (e.g., pathogen levels, stream productivity) factors. REPRESENTATIVE RESEARCH IMPACTS The reproductive consequences of female chinook salmon carrying male-specific genetic markers in a number of different populations that spawn in the Columbia River will be determined. The search for the environmental factor(s) within the spawning and early rearing habitat that is responsible for causing the appearance of male-specific genetic markers in female chinook salmon is being pursued. Additional diagnostic markers for the X and Y chromosomes of salmon are being sought to facilitate research on the sex reversal problem. Lines of fish are being established and characterized which will be useful in the assessment of levels of genetic damage present in steelhead and chinook salmon populations Better measures of sperm quality are being developed in order to improve the fertility of captive broodstock Salmon eggs can now be stored at refrigeration temperatures for extended periods of time; this result will allow broodstock hatchery managers greater flexibility in their genetic programs. Methods are being developed that will result in the inclusion of the female genetics in the Northwest salmonid sperm bank (i.e. cryopreservation of female gamete). Ongoing studies will determine if factors associated with unfavorable aquatic habitats induce apoptosis and poor gamete quality. Immunization strategies for ESA-listed broodstock in captive breeding programs are being developed to reduce disease outbreaks and limit transmission of pathogens to progeny. Strategies to reduce the infection rates among salmon as they return to their spawning grounds are being sought Methods to detect multiple pathogens simultaneously in the migrating salmon or in their habitat based on genomic DNA microarrays is being developed; these diagnostic tools are expected to provide answers faster and with less error Molecular tools needed to understand fundamental aspects of steroid hormone production have been developed. These will be used to analyze the mechanism of action of environmental contaminants on steroid-producing tissue Develop rearing and release strategies that will minimize straying of hatchery-reared salmon and thereby minimize the genetic and ecological impacts of hatchery fish on wild fish. PROJECT 1 SUMMARY Title: Cryopreservation of female salmonid germplasm Investigator: Joseph G. Cloud Objectives: The overall goal of this project is to develop new methodologies to store and recover germplasm of female salmonids. The specific objectives of the project are as follows: Develop the methodology to cryopreserve and transplant sexually immature salmonid ovaries into histocompatible recipients Determine if cyclosporin, the compound used to improve transplantation rates of human tissues, will inhibit T lymphocyte production in salmonids and improve salmonid ovarian tissue survival rates following transplantation Test whether the ovaries of spawned female salmon have functional oogonia that will develop into fertile eggs when transplanted to a suitable recipient. Summary: Pacific salmon in the Columbia Basin exist as a number of spawning aggregates. These subpopulations, and the genetic diversity contained within them, have been threatened with localized extirpation. As a result, a number of the steelhead and chinook salmon populations have been officially listed under the Endangered Species Act. Since the genetic diversity within existing spawning aggregates is not replaceable and should be conserved to protect present and future opportunities, including the evolutionary process in salmon, a regional salmonid germplasm repository has been established. At present, this germplasm repository is based on the storage of cryopreserved spermatozoa. The goal of this project is to expand the capability of this repository to include the cryopreservation and storage of cellular materials that can develop into fertile eggs. This goal will provide a means of conserving the mitochondrial genome of populations that are at risk and a more efficient means of reestablishing a preserved population should it become extinct in the wild. Specific Aims: A sperm bank has been established to conserve the genetic diversity of some of the fishes within the Columbia Basin. Since 1992, sperm from selected populations of chinook salmon and steelhead that spawn within the Snake River drainage have been systematically collected, frozen and stored in liquid nitrogen. This project, a collaborative effort of the Nez Perce Tribe, University of Idaho and Washington State University, has resulted in the acquisition of a genome resource bank for these targeted populations. A sperm bank provides an efficient and inexpensive means to conserve the nuclear genetics of a population. But the inclusion of eggs in this repository would provide for the conservation of the mitochondrial or cytoplasmic genome of a population, and it would provide a more efficient means of reestablishing a population from the stored, cryopreserved materials. However, since the cryopreservation and storage of fish eggs and embryos is not technically feasible because of their large stores of yolk, an alternative approach is to cryopreserve the female germ cells before yolk deposition and to transplant them to a proper recipient to complete vitellogenesis post-thaw. This approach is feasible because sexually immature ovaries can be transplanted and because sexually immature salmonid testes that have been cryopreserved, stored in liquid nitrogen and transplanted post-thaw to histocompatible recipients have produced fertile sperm (Cloud et al., unpublished data). The aims of this project are (1) to develop the methodology to cryopreserve and store sexually immature salmonid ovaries in liquid nitrogen, (2) to reduce tissue rejection following organ transplantation in salmonids using cyclosporin, and (3) to determine if spawned ovaries of female salmon have the capability of producing fertile eggs. 1. The development of the methodology to cryopreserve sexually immature ovaries will examine two variables: the cooling rate during cryopreservation and the percent of cryoprotectant in the freezing media. The experiment will be designed as a factorial so that all combinations can be compared. Upon thawing, the ovaries will be incubated in vitro for three days, fixed and sectioned. The resultant histology of the tissues post-thaw will be used to identify the three best treatments. Ovaries from these three treatments will be transplanted to histocompatible recipients to determine if the ovaries, post-thaw, can produce fertile eggs. 2. Cyclosporin (Novartis Pharma AG, Basel, Switzerland; administered s. c. dissolved in olive oil) will be administered at varying concentrations (1, 3, 9, 27 mg/kg) at varying timed sequences (daily for one or two weeks; followed by every second day for one or two weeks; followed by once a week for one or two months) to rainbow trout. The endpoints will be the numbers of T lymphocytes and the rate of success following ovarian transplantation among unrelated females. 3. The ovaries of spawned salmon may contain oogonia and may produce fertile eggs if transplanted to a proper recipient. If the ovarian tissue of spawned, wild salmon can be "recycled", it would provide an alternative source of female germplasm (these tissues could be collected after spawning; collection of the tissue would not compromise the reproduction of the female because it would be post-spawn). Conclusion: The unique combination of genes that constitute a stock of salmonids is protected by federal mandate because of the unique phenotype that is expressed. As the numbers of individuals in each of the protected populations decrease, there is a greater probability of losing the diversity of the genetic composition and the stock itself. The maintenance of fish populations is a difficult problem given all of the possible factors that can combine to cause a population decline. Correcting the problems that are contributing to the decline of the population can have an extended time line; in fact, the reversal of the global climate changes may require very long time periods. The establishment of genome resource banks (GRB) is a strategy that is being utilized world wide to conserve the genetic makeup of threatened and endangered populations of all types of plants and animals. While a GRB will not provide corrective measures to restore a population, a GRB will provide the time that is sometimes required for change, and it will provide a measure of genetic insurance during the times in which corrective measures are being instituted. Two parts of this project will require a collaboration with other laboratories. Our studies with the use of cyclosporin to increase the survival of the transplanted tissues will be done in collaboration with Dr. Ken Cain and his laboratory. Additionally, testing the potential of spawned salmon ovaries as a source of female germplasm will be done in collaboration with Dr. Penny Swanson's group. Salmon Recovery Impact The number of populations in which male and female germplasm has been cryoconserved can be clearly monitored. The numbers of embryos that can be derived from the frozen materials is a measure of the quality of the germplasm stored in liquid nitrogen and an estimate of the potential of the repository.   PROJECT 2 SUMMARY Title: Genetic sex of wild chinook salmon Investigators: James J. Nagler, Department of Biological Sciences, University of Idaho, Gary H. Thorgaard and Ruth Phillips, School of Biological Sciences, Washington State University Objectives: determine the temporal and spatial incidence of male-specific DNA markers in wild female chinook salmon in the Columbia River Basin develop additional, novel genetic markers for the X and Y chromosomes in chinook salmon test for the presence of an abnormal YY genotype in wild male chinook salmon from the Columbia River Basin study environmental factors (e.g., temperature, environmental estrogens) that could cause sex reversal or gonadal impairment in chinook salmon examine the early viability of offspring from females that carry male-specific genetic markers Summary: In 1999, we investigated whether wild male and female fall chinook salmon (Oncorhynchus tshawytscha) spawning naturally in the Columbia River were faithfully expressing their genetic sex. A molecular test using the polymerase chain reaction (PCR) was available that is based on a DNA marker (OtY1) specific for the Y-chromosome in this species. We tested if chinook salmon from the Hanford Reach of the Columbia River with a male phenotype possessed this marker, and conversely, if phenotypic females did not. Our results showed that a high proportion (84%) of phenotypic female salmon were positive for the male-specific DNA marker. This finding was significant because this observation had not been observed in other populations of female chinook salmon sampled from British Columbia and Alaska. This unusual situation bears investigation to determine how widespread this phenomenon is within the chinook salmon of the Columbia River basin, what (if anything) is causing this, and whether it could be affecting the ability of these fish to maintain their populations. Additional tests for genetic sexing of chinook salmon are needed in order to increase the confidence level in making conclusions regarding the source of apparent females with male-specific genetic markers which are being found in some populations. The Thorgaard lab at Washington State University (WSU) is involved in developing such tests using the amplified fragment length polymorphism (AFLP) method. Haploid embryos carrying either X or Y-chromosomes have been produced by androgenesis and DNA from those embryos is being tested to identify markers specific for the X and Y chromosome. To date, one convincing new male-specific marker has been identified and characterized. One explanation for our observations was that some of the female salmon sampled from the Hanford Reach began life as males. That is, genetically they are XY males, as opposed to XX females. These fish would have been "sex-reversed", a phenomenon well known from laboratory studies on fishes treated with high doses of estrogens. Male salmonids can be sex-reversed to fully functional females by treatment with estrogenic steroid hormones during embryonic development. The opposite is possible when females are similarly treated with androgenic steroid hormones. Because all the chinook salmon sampled in this study with a male phenotype (wild and hatchery) tested positive with our test (i.e., all carry at least one Y-sex chromosome) we suspect a feminizing environmental factor(s). A number of current environmental contaminants from industry, agriculture and domestic sewage effluent are estrogenic in fish. It remains to be determined whether Columbia River water contains sufficient concentrations of these compounds to cause the effects reported. Another environmental factor to consider is temperature. Temperature shifts during embryonic development have caused significantly skewed sex ratios in a limited number of fish species (one report exists in sockeye salmon that showed a positive effect). Wild embryonic chinook salmon in the Hanford Reach do experience daily temperature fluctuations due to upstream hydroelectric dam operations. The effect of elevated temperature on early gonad development needs to be examined in chinook salmon. Finally, the reproductive consequences of females bearing male-specific markers is unknown. It is speculated that their reproductive performance (e.g. egg/embryo viability) could be compromised. Experiments are planned to investigate this question and establish a significant consequence (or not) for this phenomenon. Specific Aims: Specific Aim 1: We continue to collect adult chinook salmon tissue samples from three different field study sites (Hanford Reach/Priest Rapids Hatchery, Yakima River, and Ives Island complex, below Bonneville dam). In Fall 2002, samples from a total of 50 female and 50 male chinook salmon were collected from the Hanford Reach, 50 female and 50 male from the Priest Rapids Hatchery, 50 female and 25 male from the Yakima River, and 36 female and 20 male from Ives Island. Gonad tissue samples were fixed in 95% ethanol; total length and phenotypic sex data for each fish was recorded. Sampling will continue in Fall 2003. Specific Aim 2: A genetic marker showing Y chromosome specific linkage in chinook, chum and coho salmon has been recovered by AFLP screening of haploid chinook salmon embryos produced by androgenesis. Nested PCR primers designed from this sequence show sex-specific amplification. This region includes an apparent pseudo-gene of an open reading frame found autosomally in all Pacific salmonids tested. The test is not successful for rainbow trout or pink salmon but shows promise for use with sockeye salmon. Our approach should be applicable to isolating sex-specific sequences in other fish species. This marker (OtY2-WSU) should have applications for investigating possible sex reversal events in different Pacific salmon species. Specific Aim 3: Two approaches are being investigated to detect male chinook salmon with a YY genotype. The first is to individually breed male chinook salmon from the Columbia River with a known XX female using in vitro fertilization (i.e. single pair matings), and examine the phenotypic sex of the offspring. If all-male offspring (i.e. XY) are produced in any of the matings then the male parent must have a YY genotype. Fifty single pair matings (~300 eggs per family), from fish sampled at the Priest Rapids Hatchery, were conducted in the Fall 2002 and the offspring are being raised to a size at which they can be examined. The second is the development of a test based on quantitative real-time PCR to detect male chinook salmon that have a YY genotype as compared to a XY genotype. A PCR primer set for the male-specific GHp in chinook salmon has been investigated for this purpose. The real-time method is based on a 96-well plate format and has the attributes of being rapid with a high sample throughput and there is no post-PCR gel electrophoresis required. The method is currently operational to identify genotype (i.e. XY from XX), however it has proven not to have the resolution to distinguish a YY from a XY genotype. Work in this aim will continue using the new male-specific marker described in Aim 2 above. Specific Aim 4: The early embryo viability of each of the 50 single-pair mated families, described above in Specific Aim 3, have been followed from fertilization until they hatched (e.g., cleavage stage, embryonic keel, eyed, hatch). It remains to determine the sexual genotype of the mother and correlate embryo viability with females that were positive or negative for the Y-linked markers. Specific Aim 5: Temperature is a significant environmental factor that fluctuates both seasonally and daily in the Columbia River, due to annual and hydroelectric dam activities respectively. We propose to investigate temperature alterations on the developing salmon gonad, while juvenile are in freshwater residence before leaving for the ocean. Conclusions: Information on the incidence of male-specific DNA markers in females in consecutive years is needed to predict population effects. We have collected samples for four consecutive years (1999-2002) from the Hanford Reach and propose to follow this population for another year, to provide an overall 5-year picture. Our reference population at the Priest Rapids Hatchery has been similarly sampled and we propose to continue to sample there in Fall 2003. To ascertain whether our observations of male-specific DNA markers in Hanford Reach females were somehow unique, we obtained samples from other rivers in the watershed. Samples of fall chinook salmon (males and females) from two other locations, the Yakima River and the Ives Island complex, will be obtained in Fall 2003. This will provide a 4-year consecutive sample similar to that for the Hanford Reach/ Priest Rapids Hatchery. Potential primer combinations (totaling 1024) will be used to screen for sex-linked markers in androgenetic DNA samples. This will allow us to screen close to 20,000 different PCR products for association with the Y or X chromosomes. Based on the success of isolating a new Y-linked marker, we believe that there is a good probability of isolating additional male- and possibly female-linked markers. Any promising markers identified will be characterized and tested across a panel of populations and species. A possible outcome of XY females in Columbia River chinook salmon populations, that would be producing ~50% of their eggs carrying a Y-chromosome, is the generation of YY "supermale" fish. All the offspring of these fish would be male that ultimately could cause skewed population sex ratios. Laboratory studies with chinook salmon (and other salmonids) demonstrate that YY males are sexually viable; the extent to which they are prevalent in the wild is unknown. We propose three complimentary approaches to determine whether YY chinook salmon males exist. First, we will continue development of a real-time quantitative PCR method using the new male-linked marker sequence discovered by the Thorgaard lab and apply it to chinook salmon DNA samples we have inventoried. Following from the development of the GHp real-time PCR assay we have considerable experience and the method development should proceed rapidly. Secondly, we will complete the analysis of the offspring from each the 50 families produced from the single-pair matings done in Fall 2002. This will involve the determination of phenotypic sex in a 40 fish sub-sample from each family to arrive at the sex ratio for each family. A family composed of all males would indicate a YY father. We propose to repeat this experiment in the Fall 2003. Finally, in collaboration with Dr. Ruth Phillips (WSU-Vancouver) we propose to use a fluorescence in situ hybridization (FISH) method that Dr. Phillips developed, using the OtY1 marker on white blood cell nuclei, to detect YY individuals (2 Y-chromosomes painted) from XY (1 Y-chromosome painted) or XX individuals (no chromosomes painted). An important question to address is whether the reproductive fitness of females carrying Y-chromosome linked genetic markers is compromised. Do these females produce eggs that are of the same quality as females negative for the genetic markers? We propose to complete the genetic analysis of females used to provide eggs for the 50 families generated in November 2002 and correlate this information with embryo survival in each case. Some offspring will be retained and grown out to determine sex ratio and assess proper gonad development. This experiment will be repeated in the Fall 2003. Salmon Recovery Impacts: This proposal will investigate how prevalent the phenotypic alteration of wild chinook salmon is in the Columbia River basin and what the long-term reproductive implications are for these populations. The development of methods to rapidly test large numbers of fish DNA samples to determine sexual genotype and detect male chinook salmon with a YY genotype are expected outcomes. With the evidence that we have isolated a new male-specific marker for Pacific salmon, this marker can now be used for addressing the issue of possible sex reversal in Pacific salmon as well as for evolutionary studies of sex chromosome evolution and sequence differences among populations. These studies will demonstrate whether male fish are being sex reversed to a female phenotype. Finally, habitat alterations such as inappropriate temperature fluctuations due to dam operations could affect the proper gonad development of juvenile salmon. This proposal will investigate ways to quantify this impact. PROJECT 3 SUMMARY Title: Genetic analysis of domestication behavior in Oncorhynchus mykiss Investigator: Gary H. Thorgaard Staff: Paul Wheeler (Research Technologist III) Objectives: Apply standardized behavioral assays to compare behaviors related to domestication in clonal lines of rainbow trout and steelhead (Oncorhynchus mykiss) Attempt to genetically map these behaviors using QTL (quantitative trait locus) analysis Test the generality of any QTLs identified in crosses involving outbred strains of Oncorhynchus mykiss and chinook salmon, Oncorhynchus tshawytscha. Summary: The role of hatcheries in salmon and steelhead restoration has become increasingly controversial. In some instances it has been shown that modifying the hatchery environment to one that is more similar to that in nature can improve return rates of released smolts. Evidence has also emerged that fish stocks which have been reared in hatcheries for a number of generations may develop behavioral differences related to their rearing conditions. This domestication process may be similar to those that have occurred in other animals when they are brought into captivity in a human-controlled environment. Understanding the nature of the genetic changes that occur during domestication could help in monitoring the process and in evaluating the status of existing stocks. This goal seemed unattainable a few years ago but today increasingly sophisticated methods of genetic analysis are making it realistic. A set of methods known as QTL (quantitative trait locus) analysis are being applied that merge genetic mapping using DNA markers with phenotypic measurements of traits in genetic crosses to allow the number and location of chromosome regions influencing a trait to be identified. Ultimately, these methods can lead to an understanding of the specific genes associated with variations in a trait. We would like to apply these approaches to studying domestication-related behavioral differences among strains of Oncorhynchus mykiss. Our efforts will complement those of other program participants, including Ruth Phillips of WSU, who is seeking to map the sex chromosome in chinook salmon, Andrew Dittman of NMFS, who will seek to map olfactory receptor genes in salmonids, Brian Beckman of NMFS, who is interested in strain differences for complex traits in salmonids, and Linda Park of NMFS, who is interested in genetic mapping in chinook salmon. An important tool in our efforts are the clonal lines of Oncorhynchus mykiss which we have developed in our laboratory using the chromosome set manipulation methods of androgenesis and gynogenesis. We will make these lines available to other program participants. All individuals within each line are essentially genetically identical to each other and as such represent a valuable resource for repeatable experimentation. We are currently propagating seven clonal lines which have variations for traits such as development rate, disease resistance, immune response, numbers of meristic elements and tendency to undergo smoltification. We have successfully used these lines in genetic mapping and in QTL analysis of other complex traits such as development rate and immune response. Genetic mapping using these lines is efficient and cost-effective because of the types of markers and crosses we use in our studies. Through the efforts of Megan Lucas and Rob Drew, graduate students in our lab, we have recently confirmed substantial differences among the lines for behaviors related to domestication, such as position in the water column and startle response. We now seek to build on these preliminary results in an effort to better understand the genetic changes with domestication and adaptation to the hatchery environment in salmonids. Initial work with the clonal lines can help us to identify mechanisms which can then be tested in other strains of Oncorhynchus mykiss, as well as in strains of chinook salmon. Specific Aims: Specific Aim 1: Apply standardized behavioral assays to compare behaviors related to domestication in clonal lines of rainbow trout and steelhead (Oncorhynchus mykiss). The behavioral tests which we have applied to four of the clonal lines will be extended to three additional lines which include a wider range of domestication histories. Additional tests which can be efficiently and quantitatively applied to larger numbers of individuals (needed for objective 2) will also be developed. Specific Aim 2: Attempt to genetically map these behaviors using QTL (quantitative trait locus) analysis. Lines which are most divergent for the behaviors studied under objective 1 will be crossed and progeny will produced from the hybrids between the lines by androgenesis (induced all-paternal inheritance). The resulting doubled haploid progeny are segregating for DNA markers and traits differing between the parent lines. Measurement of the markers and traits and statistical analysis of their association patterns allows chromosome regions associated with variations in the traits to be identified. Specific Aim 3: Test the generality of any QTLs identified in crosses involving outbred strains of Oncorhynchus mykiss and chinook salmon, Oncorhynchus tshawytscha. Although the use of clonal lines makes the identification of QTLs far more efficient, it also provides results which are specific to the lines being studied. We will test the hypothesis that QTLs identified using the clonal line crosses represent more general mechanistic changes associated with domestication. This will involve conducting crosses using outbred strains of steelhead and chinook salmon and examining whether markers associated with QTLs in the clonal line crosses also segregate with domestication-related behavioral differences in these crosses. Large-scale DNA marker studies are much more expensive and difficult to conduct in such crosses than in the clonal crosses so these studies will be focused on the candidate regions identified in the clonal crosses.   Conclusion: Domestication may be an impediment to the utilization of hatcheries in recovery programs for salmon and steelhead. By utilizing clonal line crosses of Oncorhynchus mykiss , we believe that we efficiently address this issue and can begin to develop an understanding of the genetic basis for domestication process in salmonids. This could in the long term provide a method to monitor domestication-related changes in stocks being used in recovery programs. Our project would interact with several other projects in the program, including those of Phillips from WSU and Beckman, Dittman and Park from NMFS which involve genetic mapping and evaluation of strain differences. Performance measures developed: Develop improved genetic map for salmonids. Increased understanding of genetic basis for domestication-related behavioral differences among strains that are involved in adaptation to the hatchery environment.   Salmon recovery impact: The changes associated with domestication may be impacting the success of recovery efforts for salmon and steelhead that involve hatchery strains. We propose to evaluate the genetic basis of such changes using clonal lines of Oncorhynchus mykiss and then extend these studies to other strains. Our studies will help to evaluate the degree to which hatchery programs can contribute to recovery programs for endangered populations and how hatchery programs might be more effectively used in such efforts. PROJECT 4 SUMMARY Title: Impact of physiological stress on gamete quality in salmonids Investigator: Rolf L. Ingermann Objectives: Establish to what extent salmonid gametes are sensitive to oxidative stress in vitro. Establish how variables such as environmental hypercapnia, hypoxia, and hyperoxia compromise gamete function and quality. Establish how the intact adult maintains the integrity of its gametes. Summary: Chemical aspects of the habitat such as dissolved O2 and CO2 tensions and physical aspects such as temperature, impediments to upward migration, and handling procedures during artificial propagation, may physiologically stress the adult salmonid. Since physiological stressors may influence gamete production and quality, and ultimately affect the likelihood of generating sufficient numbers of viable embryos, developing a thorough understanding of their roles is of fundamental importance in salmonid biology. We have found that low PO2, low pH, and high PCO2 all reversibly reduce sperm motility and fertility in vitro in chinook salmon and steelhead trout. We have established that low PO2 compromises sperm function by interfering with the production of ATP; ATP is the fuel used by the molecular motors of the flagellum (dynein ATPase) to propel the sperm. Low extracellular pH, as well as elevated PCO2, appears to interfere with sperm motility by altering intracellular pH. We have found by phosphorus NMR spectroscopy that intracellular pH is maintained at about 0.5 pH units below that of the extracellular fluid and at least some of the pH sensitivity of sperm motility is attributable to the pH sensitivity of dynein ATPase. Although this gives an indication of how depressed intracellular pH affects the sperm, the specific mechanisms which underlie pH regulation by sperm are unknown. Aspects of the environment that promote struggling and/or metabolic stress within the adult male are likely to be associated with elevated internal CO2 levels, reduced systemic pH, and possibly with reduced O2 delivery to the sperm, and thus compromise sperm quality and function. These findings also have implications in artificial reproduction of captive broodstocks as CO2 is used as a fish anesthetic for gamete collection. Less clear, but no less significant, is whether environmental hypercapnia and/or hypoxia affect these variables within the adult and indirectly impact gamete quality. Whether and how such environmental factors influence egg quality is largely unknown. Recent data suggest that the chemical composition of the activating solution within which eggs are fertilized has a profound effect on sperm motility. For example, dilution of semen with deionized water results in very poor sperm motility while dilution with 50% ovarian fluid results in excellent motility. Current activities are focused on determining which ions/elements of the activating solution are particularly important in permitting full semen motility and hence, maximal fertility. It is likely that the sperm duct epithelium controls the capacity for sperm to become motile by altering the pH of the reproductive tract. Future studies will focus on the mechanism by which the epithelium exerts this influence and by what mechanisms the epithelium is regulated. A potential physiological stressor of salmonids is hyperoxia. Mammalian sperm, including human sperm, show oxidative damage associated with exposure to elevated O2 tensions which includes DNA fragmentation. Since salmonids are exposed to elevated dissolved O2 below dam spillways, for example, and since their sperm are routinely stored unfrozen during artificial reproduction under 100% O2, it appears likely that salmonid sperm are exposed to oxidative stress. Since such stressors may have significant deleterious effects on the quality of salmonid gametes, their effects warrant careful study. Specific Aims: The overall aim of this study is to establish and characterize the direct effects of potential stressors on salmonid gametes as well as their indirect effects on gametes via actions on the adult. Only with a thorough understanding of the effects of internal and external stressors on salmonid gamete quality can the effects of those stressors be eliminated or reduced. Specific aims include: Establish the consequences of gamete oxidative stress on fertilization. Determine the mechanism by which sperm regulate intracellular pH. Establish whether the initiation, or some stage thereafter, is responsible for the pH sensitivity of motility. Determine whether and the extent to which the physiology of the intact male can overcome deleterious effects of acidosis or hypercapnia on sperm function. Characterize the ionic composition of egg fertilizing solutions which permit maximal sperm motility and fertility. Examine the extent to which the sperm duct epithelium regulates sperm function. Conclusion: Stressors within and outside the adult salmonid may have direct and/or indirect effects on gametes that compromise their ability to generate viable offspring. We have recently found that factors such as low O2, high CO2 and low pH all reduce sperm motility and fertilizing ability in vitro. This study seeks to establish the basic mechanisms that underlie these observations. It also seeks to determine whether exposure of the adult to such environmental variables also negatively impacts sperm function or whether the intact adult possesses physiological compensatory mechanisms. Further, based on available information in the scientific literature, we suspect that potential stressors such as hyperoxia have significant deleterious effects on gametes that reduce their ability to generate viable young. With such information it may be possible to eliminate these stressors or mitigate their impacts. For example, it may give the rationale to replace CO2 as an anesthetic where practical or allow CO2 to volatilize out of semen samples prior to use. Furthermore, since even modest levels of CO2 (~1 kPa) have demonstrable, deleterious effects on sperm function, it is not inconceivable that increases in CO2 associated with global climate change (and especially with schemes to eliminate CO2 by maintenance in aqueous deposits) may have subtle, sublethal effects on fish reproduction. Ultimately, only with a thorough knowledge of the effects of potential stressors on gamete quality can their effects be minimized and maximal reproductive success of precious salmonids be ensured. Performance Measures Developed: Test for: sperm motility and fertilizing ability egg fertility and embryonic development reproductive tract function DNA fragmentation as an indicator of oxidative stress Salmon Recovery Impact: This proposal will analyze potential internal and external stressors that may impact salmonid gamete quality. Our long-term goal is to use this information in evaluating procedures to lessen exposures to identified stressors as well as help to establish ways to mitigate the deleterious effects of stressors.   PROJECT 5 SUMMARY Title: Microarray detection of multiple pathogens in managed and wild salmonid populations Investigators: Douglas R. Call Objective: To develop and apply a DNA microarray detector for simultaneous detection of multiple salmonid pathogens. Summary: Virtually every stage of salmonid life-history is impacted by disease. Despite the potential importance of disease, however, there is very little information available on the distribution and ecology of infectious disease agents relative to fish populations, watersheds, or habitats. The lack of data on disease ecology reflects the fact that fish pathogen assays, typically culture-based, are too cumbersome to apply to large numbers of samples. For example, an assay for the causative agent of bacterial kidney disease (BKD; Renibacterium salmoninarum) can take 3-19 weeks. A second consequence of this situation is that disease surveillance is piecemeal if it occurs at all. Thus, important but unexpected foreign pathogens (e.g., Piscirickettsia salmonis) might not even be detected until significant expansion has occurred in U.S. territory. One solution to difficult diagnostics is to use molecular techniques such as polymerase chain reaction (PCR). PCR is used to generate many copies of a specific gene sequence so that if present, the gene sequence can be detected using a method such as agarose electrophoresis. In this case, we are using a DNA microarray to interrogate the PCR products and identify pathogen specific sequences. Microarrays are composed of a lattice pattern of spots. Each spot is the location for a sequence specific "probe." PCR products ("targets") are labeled and hybridized to the array whereupon targets anneal to complementary probe sequences and are detected using a fluorescent imaging system. The arrays can be designed to accommodate multiple pathogen specific sequences for a PCR product that has highly conserved primer sequences. Alternatively, the arrays can be designed to detect multiple products from a multiplex PCR reaction. Both strategies will be employed for our project. Microarray detectors can be applied to diagnostics and surveillance, and they can be used for detecting planktonic pathogens when coupled to appropriate filtration technologies. Arrays developed from this project can be incorporated into the nation's first aquatic animal health inspection service based at the Washington Animal Disease Diagnostic Laboratory (WADDL, Pullman, WA). Using these assays, WADDL will provide the expertise to evaluate the health status of fish relative to changes in their native habitat, and to detect foreign and domestic infectious diseases that may threaten salmonid recovery efforts. The array technology can be used to monitor disease status of fish raised in resource augmentation hatcheries and can be used in conjunction with other salmon reproduction projects to better define factors limiting salmonid populations. Specific aims 1. Array validation and sensitivity testing We have coupled PCR amplification of the 16S rDNA gene with an oligonucleotide-based microarray detector suitable for distinguishing between 18 bacterial targets. We have had to redesign the assay to generate a smaller fragment of the 16S gene (179 bp vs. original 530 bp product) because secondary structure interfered with probe annealing for some targets. We have also modified our array production process to adapt to a new spotter system and to reduce between batch variance in signal quality. Now that these parameters have been defined, we are in the process of sensitivity testing. Sensitivity is a function of the array detector, baseline PCR reaction and interference from complicating bacterial and eukaryotic DNA in the sample. Preliminary data indicate that our assay is very sensitive under idealized conditions (<10 genomic copies), but that PCR template bias can complicate analyses from mixed communities. This objective will completely define sensitivity as a function of each phase of the detection process. 2. Transfer the current array probes to a bead-based format. While the 16S pathogen detection system is suitable for simultaneous detection of bacterial pathogens, the current format represents a bottleneck in sample processing. We have already improved our assay over conventional formats by printing arrays on Teflon masked slides that afford the ability to process up to 12 samples on a single slide. Furthermore, to generate quantitative data for analysis requires a time consuming image processing step. For this objective we will transfer our current 16S detection system to a microbead-based assay format. In this latter format, each 16S probe is affixed to a uniquely colored bead (5 _m in diameter). During the assay, PCR products are mixed with the beads in a manner that capitalizes on solution phase kinetics as opposed to reliance on passive diffusion as required with the planar arrays. This should enhance sensitivity while reducing time required to complete the assay. Finally, the samples are then quantified using a flow-cytometer. This produces digital data that requires no image analysis and can be processed in a 96-well format. Consequently, the entire assay can be converted to a high throughput system both in terms of detection, interpretation and sample handling. We have recently acquired the requisite flow-cytometer that will be used for this objective. Conclusion There is sufficient DNA polymorphism in the 16S gene to clearly distinguish between 18 species of bacteria and thus the microarray detector has excellent potential as a diagnostic or surveillance tool both with tissue and environmental samples. Sensitivity analyses currently underway will help define the limits of the assay and indicate where further improvements can be made including transferring the assay to a more efficient, bead-based format. Research Impact: The long-term goals of this research include developing and implementing a DNA microarray based assay that will be used to determine the prevalence and distribution of disease agents relative to salmonid populations, watersheds and habitats. This system will also assist with automatic detection of emerging pathogens should they enter U.S. territories and WADDL will be able to adapt the system to the aquatic animal health program. Rapid diagnosis of endemic or foreign animal diseases can facilitate management decisions and significantly mitigate the consequences of disease outbreaks. The simple array design is also universally portable so that once published, any research group or diagnostic lab can adopt the system for pathogen detection. Future collaborations: Discussions are underway regarding potential collaborations between NMFS personnel (Dr. M. Strom and others), WSU (Dr. D. Call) and U. of Idaho (Dr. K. Cain). Activities include research focused on studying the genetic diversity of Renibacterium salmoninarum including assessment of geographic variation and differences between virulent and avirulent strains. Additional ideas include phylogenetic analysis and marker development for Aeromonas species, and generating preliminary data needed to develop a whole genome sequencing project for Flavobacterium psychrophilum (causative agent of cold water disease). PROJECT 6 SUMMARY: Title: Reduction of disease-related impacts on important salmonid stocks through broodstock immunization against key pathogens. Investigator: Kenneth D. Cain Objectives: The long-term goal of this project is to develop strategies that reduce the risk of vertical transmission and disease occurrence in captively-reared and/or hatchery managed salmonids. Therefore, the objectives are: To develop effective vaccine preparations for immunization of salmonid broodstocks to reduce/inhibit vertical transmission of infectious agents. To increase our understanding of the role of maternal transfer of immunity in fish through detection of antigen-specific immunoglobulin (Ig) in eggs and newly hatched fry. To determine mechanisms of pathogen entry and persistence within the egg. Summary: Disease-associated mortality represents a significant risk to restoring and rebuilding depressed anadromous salmonid populations. With the listing of a number of salmonid stocks under the Endangered Species Act (ESA) and the establishment of captive broodstock programs to preserve genetic integrity of select stocks, the threat of infectious disease and the impact it could have on these programs is a concern. Recent loss of high numbers of endangered Redfish Lake sockeye salmon due to an IHN outbreak illustrates this. A number of fish pathogens are difficult to control and can be transmitted vertically (from parent to progeny) either on or within the eggs, or cause disease in valuable broodstock directly. Two bacterial pathogens that are transmitted within eggs and cause severe losses in salmon and steelhead are Renibacterium salmoninarum (causative agent of bacterial kidney disease) and Flavobacterium psychrophilum (causing bacterial coldwater disease). If immunity could be enhanced in adult fish, the risk of disease in progeny would be reduced. This project will focus on developing vaccination strategies to reduce the risk of vertical transmission. The potential role of maternal transfer of Ig to eggs and fry in relation to disease protection will be established. Initial focus will be primarily on coldwater disease (CWD) but collaborations with NMFS scientists to address vaccine strategies for bacterial kidney disease (BKD) are being established. This project will be aimed at reducing disease occurrence by devising broodstock immunization strategies that would both decrease pathogen numbers in fish prior to spawning, and potentially transfer antibody to the egg to enhance protection through early life stages. Specific Aims: Standard iodophore treatment of eggs during fertilization is usually effective at eliminating pathogens on the surface of eggs, but pathogens capable of remaining within eggs are difficult to control and can cause high mortalities in young fish. One possible means of reducing pathogen loads within the egg and conferring some level of immunity to the young is to immunize broodstock prior to spawning. This project will investigate the feasibility of such an approach by immunizing broodstock against F. psychrophilum prior to and during egg development. This bacterial pathogen causes high mortalities in salmon and steelhead, and can be transmitted vertically to offspring on and within eggs. Another important component of this project will be to determine the mechanisms and development stage in which a pathogen enters the egg. We will sample eggs from infected adults during different stages and identify bacterial presence using immunohistochemistry, PCR, and culture techniques. The role infected males play in pathogen transmission to eggs during fertilization will also be determined. By identifying stages and mechanisms of bacterial entry into the eggs, it may be possible devise additional control strategies. If immunity could be enhanced in adult fish prior to egg collection and fertilization, then pathogen numbers should decrease and maternal transfer of immunoglobulin to the egg and fry may enhance early protection from disease. This accompanied by an efficacious vaccination program during early rearing may reduce disease-related mortalities during smolt migration, a time when many of these pathogens are encountered. Practical methodology to implement this into captive broodstock and other existing programs will be developed. Conclusion: Vertical transmission of disease presents a dilemma when attempting to control and manage for disease outbreaks. Adult fish can be tested for the presence of pathogens and the relative risk of disease to their offspring speculated, but progeny must be constantly monitored and if infection levels are high then offspring may be culled. Even if disease occurrence is low, the risk of a carrier state developing or transfer of pathogens to other fish during times of stress may be high. Current disease management practices tend to ignore a preventative approach to disease control. Immunization would focus on prevention. Studies have shown that adult fish can mount a strong immune response and maternal transfer of antibody to the eggs does occur. If vaccine preparations can be developed to take advantage of this then infection levels should be reduced. Salmon Recovery Impact: Currently, captive broodstock and Kelt reconditioning programs have been established in the Northwest to preserve the genetic integrity of specific anadromous fish stocks. Since disease outbreaks in these captive populations could result in complete genetic loss of a stock, vaccination strategies such as those outlined here would be very appealing. The development of a broodstock immunization program followed by proper immunization of juvenile fish has the potential to increase survival of anadromous fish during early life stages and post-release. This may prove beneficial for captive broodstock programs as well as hatchery based mitigation/recovery programs. Many reports indicate that survival of smolts infected with bacterial or viral pathogens is reduced in response to stressors encountered during migration. If preventative measures that enhance the fish's ability to fight infection can be implemented then long-term survival may be enhanced. With increased concern over rebuilding anadromous stocks in this region, there is a strong need to develop improved strategies that limit disease-related mortality. PROJECT 7 SUMMARY Title: Regulation of steroid production in salmonids and impacts of environmental factors Investigator: Graham Young Objectives: To determine the mechanisms through which steroid hormone synthesis is regulated by endogenous factors, and to assess the impacts of environmental factors on these mechanisms Summary: Steroid hormones regulate pivotal processes in the body including many aspects of reproduction (sex steroids), homeostasis (corticosteroid effects on hydromineral balance, energy metabolism, immune system function) and behavior (sex steroids and corticosteroids). Steroid hormones are the terminal products of hormonal signals originating in hypothalamus and transduced through the secretion of tropic hormones from the pituitary. These tropic hormones (gonadotropins - sex steroids; adrenocorticotropin ­ corticosteroids) stimulate steroid hormone synthesis by the gonads or interrenal cells. The synthesis of steroids involves an array of steroidogenic proteins and requires the intracellular trafficking of cholesterol to the mitochondria, performed by steroidogenic acute regulatory protein (StAR), the cleavage of the side chain of cholesterol, and the subsequent conversion of pregnenolone to various intermediary metabolites and bioactive steroids, involving a number of enzymes which are probably transcriptionally regulated. Thus, there are multiple sites for the regulation of steroid hormone synthesis by endogenous factors, including direct effects on the steroid-producing cells, and indirect effects via actions higher up on these axes. Furthermore, multiple sites therefore exist where environmental factors may potentially have impacts on the timely production of steroid hormones. Environmental stressors, ranging from environmental contaminants to inappropriate temperatures in wild fish, and stressors associated with captive rearing are known to impact reproductive and homeostatic processes, and some of these effects are correlated with altered steroid hormone production. Understanding how environmental factors impact on both reproduction and on homeostatic processes requires a good understanding of the basic endogenous mechanisms that regulate these pathways. We therefore need to understand how these steroidogenic proteins are regulated both at the transcriptional and translational levels. Current understanding of how steroid synthesis in fish is regulated is largely limited to studies on the effects of tropic hormones on steroid production by steroidogenic tissue maintained in vitro. In order to increase knowledge of the molecular and cellular aspects of steroid hormone production, we have recently cloned cDNAs encoding StAR from rainbow trout and coho salmon, and have also cloned cDNAs encoding several key steroidogenic enzymes. cDNAs for most of the other steroidogenic enzymes have been donated to us. Our collaboration with Dr Penny Swanson's group also allows us to look at higher levels of the axes controlling steroid production, since her laboratory has assays for salmon gonadotropins, and methods for analysing expression of gonadotropin receptors, insulin-like growth factor I, etc. In addition, some of the methods developed in this program will be of utility to the research of Dr. Swanson's group Efforts toward a fundamental understanding of the endogenous regulation of steroidogenesis will generate a number of tools that will be used to examine the potential disruption of steroidogenesis by environmental factors. We will focus on two additional areas of direct relevance to salmon recovery. The first will analyze the impact of environmental contaminants, termed endocrine disrupting chemicals (EDCs), on reproductive and homeostatic processes in salmonids and their mode of action. EDCs have deleterious, sub-lethal effects on vertebrate animals. Development, reproduction and growth are often impaired, and in many cases, this has been associated with the disruption of the endocrine systems controlling these processes, and linked to the steroid hormone-like actions of EDCs, which are often estrogenic. We already have evidence that specific sites the steroid biosynthetic pathway are quite sensitive to estrogenic effects: expression of StAR in gonads is severely downregulated in response to low doses of estradiol, and expression of cholesterol side-chain cleavage enzyme in interrenal is similarly inhibited in response to estradiol. In addition to documenting impacts and mechanisms of EDCs, we also aim to identify additional, sensitive and specific biomarkers for EDCs: plasma vitellogenin induction has been classically used as a marker for estrogenic exposure but recent work, for example, indicates that inhibition of pituitary follicle-stimulating hormone is a more (10-fold) sensitive biomarker. This area of research is highly complementary to Lindahl Johnson's project and we anticipate working closely with her group. The second area of relevance to salmon recovery will focus on non-chemical environmental stressors. It has been well documented that a number of stressors inhibit reproductive processes through mechanisms that are not well understood but which may partly be mediated by the stress hormone, cortisol. Salmonids migrating through river systems are subject to a number of environmental stressors that include dam passageways, and areas of elevated temperature in dam-impounded water. Salmonids reared in captivity are also exposed to a suite of stressors. Elevated temperatures, for example, are known to inhibit processes associated with ovulation and spermiation, and this may partially explain the frequent asynchrony in development between male and female captive broodstock, and between captive broodstock and wild spawning salmon. Understanding these deleterious effects of temperature, and especially the periods of development that are critically sensitive, is of direct relevance to improving the effectiveness of captive broodstock in salmon recovery programs. In addition, understanding these processes is of direct utility over a longer time scale in predicting the impact of global warming on salmon recovery programs. This area of research has strong collaborative linkages with the research of Dr. Swanson's team. Specific aims: The specific aims of the project are: 1. Develop real time PCR assays to quantify mRNA levels for StAR and steroidogenic enzymes. 2. Use these tools and others to assess the effects of tropic hormones (gonadotropins, adrenocorticotropin), growth factors implicated in regulating steroid production, and endogenous steroids at physiological doses on steroidogenic processes in gonads and interrenal at different developmental stages using both in vivo and in vitro exposure. 3. Using techniques and knowledge from the above, we will assess the effects of EDCs with known steroid-mimicking actions on steroidogenic processes in gonads and interrenal at different developmental stages, using both in vivo and in vitro exposure and environmentally realistic concentrations. Assessing whether the sensitivity of steroidogenic tissue to these factors changes with developmental stage is important, since embryonic/larval stages are highly sensitive to EDCs. 4. Analyze the impact of elevated temperatures and physical stressors (e.g., confinement stress) on steroidogenesis and gametogenesis at several stages of sexual maturation to identify critical sensitive periods. Conclusions: Environmental stressors, whether chemical or physical, may impact on a number of regulatory processes essential for development, homeostasis and reproduction, and at a number of different levels. This is critical for both sexually mature fish that are undergoing normal reproductive cycles, but also earlier stages of development. As in mammals, it is likely that these larval and juvenile phases are most sensitive to the impact of natural and anthropogenic stressors. Disruption of normal developmental programs may severely affect the development and reproductive success of these fish. Since steroid hormones are key mediators both in reproduction and in the regulation of immune function, homeostasis and metabolism, any disruption of steroid biosynthesis is therefore likely to have severe consequences. The proposed research will increase understanding of the endogenous regulation of steroid hormone synthesis and the impact of environmental stressors. Salmon Recovery Impact: In order to support salmon recovery programs, a fundamental understanding is needed on the molecular and biochemical processes involved in development and reproduction and the specific mechanisms by which these processes can be disrupted through effects on the steroidogenic machinery. These tools will also be of utility is assessing the mechanisms underlying the numerous reproductive problems of captively-reared salmonid broodstock, such as precocious or delayed puberty, failure to undergo sexual maturation, poor egg quality, and asynchrony between sexes in the timing of maturation This research program has strong collaborative linkages with the research programs of the groups of Dr. Penny Swanson and Lindahl Johnson at the Northwest Fisheries Science Center. It also has direct relevance to several of the NMFS Salmon Research Plan topics, including the impact of hydropower operations on salmon (thermal and physical stressors, Topic 2), the links between salmon productivity and habitat (EDCs and thermal stressors, Topic 4), and the impact of global climate change (thermal stressors, Topic 6).         PROJECT 8 SUMMARY Title: Comparative Genome Mapping in Pacific Salmon Investigator: Ruth B. Phillips Other Investigators: Linda Park, Gary H. Thorgaard Summary: Major genome projects are underway on several salmonid species including rainbow trout and Atlantic salmon. The purpose of these projects is to identify specific genes underlying traits of importance to aquaculture including growth, temperature tolerance and disease resistance. A long-term goal of the Conservation Biology Division of National Marine Fisheries Service is to identify the genetic loci associated with local adaptation in natural populations of salmon. There is also special interest in the genetic basis of disease resistance since infectious disease is a significant problem for endangered species being maintained in conservation hatcheries. The comparative genome approach to the identification of disease resistance genes should be especially informative for coho and chinook salmon, since there are significant species differences in the resistance to different pathogens in these two species. In order to identify genes underlying specific traits, draft genome maps are needed for each species and these maps need to be correlated with the more detailed map for rainbow trout. In the proposed project we will develop draft genome maps for chinook and coho salmon and relate these maps to the rainbow trout map. We will use a combination of genetic and physical mapping and work closely with Linda Park (salmon maps) and Gary Thorgaard (rainbow map).   Proposed Research: In the proposed research we will prepare genome maps for coho and chinook salmon and correlate them with the rainbow trout map. This will involve both physical and genetic mapping. We are currently preparing a set of BACs that contain genes or microsatellite markers that are linked to each of the rainbow trout chromosome pairs. Because of the large number of chromosome rearrangements that have occurred between the karyotypes of trout and salmon, we will need to extend this set to include one for each of the fifty rainbow trout chromosome arms. We will also need to use rainbow trout markers that can be scored in chinook and coho crosses, so the genetic maps can be tied to the physical maps. Although preliminary results suggest that many of the rainbow trout BACs containing protein-coding genes will hybridize to salmon chromosomes, it may be necessary and desirable to isolate BACs directly from the chinook BAC library which is currently in preparation (R. Devlin, Dept. of Fisheries and Oceans, Canada, pers. com.). This library should be available commercially later in the year. Specific Aims: 1-Test rainbow trout genetic markers (protein-coding loci and microsatellite loci) from each linkage group to identify a set which can be amplified and mapped in chinook salmon. Include as many genes as possible that might be candidates for disease resistance and factors involved in reproductive success and alternative life histories. 2-Map at least two markers from each rainbow trout chromosome arm in chinook crosses (in collaboration with Linda Park of NMFS and Kerry Naish of University of Washington). 3-Screen either the rainbow trout BAC library or the chinook BAC library for clones that contain genetic markers that can be scored in chinook salmon from each rainbow trout linkage group and if possible each rainbow trout chromosome arm. 4-Prepare labeled probes from DNA of BAC clones and use them in hybridization experiments with chinook chromosomes in order to prepare a framework physical map for chinook and relate it to the rainbow trout map. 5-Repeat steps 1-4 for coho salmon. 6-Compare the maps of chinook and coho salmon with each other and with rainbow trout. Conclusions: Comparative salmonid genome mapping provides essential information needed for identification of genes underlying important phenotypic traits. Many of these traits are being studied by other investigators in the WSU/UI and NMFS Salmon Restoration Research Program . These traits include disease resistance, which is especially important in hatchery rearing of endangered species (Ken Cain), temperature tolerance that may be important in adaptation of salmon to changes in the environment, smoltification and age at maturation that are involved in alternative life histories (Linda Park, Brian Beckman, Penny Swanson), and other traits that are altered in domestication (Gary Thorgaard). In my lab we have a special interest in genes involved in sex determination and sexual differentiation and genes involved in disease resistance. Several of the other investigators are using a genetic approach for investigation of these traits and this project should give them the tools needed for identification of the genes involved in these important phenotypes. Research Impact: This project will map a large number of genetic markers and protein-coding genes in chinook salmon and coho salmon. These will include candidate genes involved in a number of processes important to the salmon recovery program. These include disease resistance that could improve survival of endangered stocks being reared in conservation hatcheries, temperature tolerance which may be important in adaptation of salmon to global warming, variation in time at sexual maturity that may enable salmon to adapt to seasonal environmental change, life history variation that confers the ability of salmon to adapt to different local environments in the Pacific Northwest, and genes involved in male and female sexual differentiation which could be important in assessing impacts of environmental pollutants on sex reversal. PROJECT 9 SUMMARY Title: Environmental and endocrine regulation of salmon reproduction Investigator: Penny Swanson, Resource Enhancement Utilization Technologies Division, NWFSC and Dr. Briony Campbell, University of Washington Objectives: Summary: In Pacific salmon, both genetic and environmental factors influence reproductive success: however, estimates of heritabilities for some reproductive fitness characters can be quite low emphasizing the importance of phenotypic plasticity in response to changes in environmental conditions. Clearly, maintaining genetic traits that confer the ability of salmon to adapt to environmental change is crucial for long-term population survival. However, understanding the short and long term effects environmental factors on the salmon reproductive system will provide important information that is needed to improve present salmon management practices, and to evaluate the impacts of habitat restoration efforts, global warming and environmental contaminants. In seasonally breeding animals, such as Pacific salmon, photoperiod and temperature directly influence the seasonal timing of reproduction and gamete/embryo viability, and indirectly influence age of maturity, adult body size, fecundity, and egg size by affecting food availability, growth, development rate and energy status. Environmental factors, such as water temperature and chemical contaminants, can also have detrimental effects on salmon reproduction by inhibiting fundamental physiological process needed for gamete production. It is well established that external (environmental) and internal (e.g. energy status) information is perceived and processed by the brain, which in turn, regulates reproduction through the endocrine system. A better understanding of the underlying endocrine mechanisms involved in regulating reproduction and how environmental factors influence the reproductive system are needed to improve methods to control reproduction of fish in a captive environment and monitor the impacts of environmental change on wild fish. Members of the Physiology Team at the NWFSC are conducting both applied and basic research on the endocrine regulation of reproduction in Pacific salmon. The applied research is directed primarily toward solving specific problems with reproduction of salmon that have been encountered in captive broodstock programs that were established to prevent extinction of Redfish Lake sockeye salmon and 8 stocks of Snake River spring chinook salmon. These problems include: highly variable survival of embryos to the eyed stage, early age of maturation of male fish, asynchronous timing of spawning of males and females, seasonally delayed maturation of captively-reared adults compared to wild parent stocks, and poor reproductive success of captively reared adults when released into their native habitat. Our research focuses on evaluating the roles of water temperature and growth in determining the age of maturation, fecundity, egg size, seasonal timing of spawning and gamete quality. In conjunction with these applied studies we are conducting basic research on the physiology and regulation of gonadotropins, and the regulation of puberty onset in chinook and coho salmon. The goals of this research are to characterize early cellular and endocrine changes during the onset of puberty in salmon, determine critical periods in the life cycle when growth/nutritional status influences the onset and completion of puberty, and ultimately, determine how rearing environment in the wild or captivity influences age of maturity in salmon. Both immunoassays and quantitative real time PCR assays have been developed to monitor several points of the growth and reproductive endocrine axis. Using these assays, we propose to examine the mechanism whereby environmental factors influence the salmon reproductive system. Collaborative research with other NWFSC, UI and WSU scientists in several areas is envisioned: 1) identification of candidate genes involved in regulating age of maturity, 2) determining potential effects of habitat conditions (water temperature) and endocrine disrupting chemicals on the growth and reproductive endocrine systems, 3) determining the mechanism whereby growth influences early stages of spermatogenesis and oogenesis.         PROJECT 10 SUMMARY Title: Exposure to and effects of endocrine-disrupting contaminants in Pacific salmon reproduction Investigator: Lyndal Johnson Objectives: To characterize exposure of threatened and endangered salmonids to endocrine-disrupting compounds in the environment and in the hatchery environment for broodstock: to assess the impacts of these contaminants on growth and reproductive function at environmentally realistic levels; and to use this information to develop sediment and water quality standards protective of salmon and provide guidance for habitat restoration efforts and broodstock rearing. Summary: There is a growing environmental concern about the adverse effects of endocrine disrupting contaminants, especially environmental estrogens, on aquatic organisms. Notable effects on fish health due to exposure to environmental estrogens are abnormalities in reproduction, development, behavior, and smoltification, all of which can reverberate at the population level. Numerous anthropogenic contaminants have been shown to possess estrogenic activity, including natural and synthetic hormones, alkylphenolic chemicals (surfactants), phthalates (plasticizers), and certain polychlorinated biphenyls and organochlorine pesticides. Other well-known classes of industrial contaminants, including dioxin-like PCBs and PAHs, may have anti-estrogenic activity. There are substantial data showing uptake of contaminants such as PCBs and DDTs by juvenile Pacific salmon. Recent surveys of Pacific Northwest estuaries show elevated body burdens of both classes of contaminants in juvenile salmon from the Hylebos and Duwamish Waterways in Puget Sound, and the Lower Columbia estuary. PCBs and DDTs have also been detected in hatchery fish and Chinook broodstock. In some cases, concentrations approach threshold levels for adverse biological effects, based on existing literature. Exposure to PCBs has been linked to reduced growth and reduced disease resistance in juvenile Chinook salmon, but effects of early exposure on reproductive development have not been investigated. More recent studies suggest that exposure to xenoestrogens may also be a concern. Joint studies by the Washington Department of Fish and Wildlife and the Northwest Fisheries Science Center's Ecotoxicology Program have demonstrated that Puget Sound flatfish are exhibiting signs of estrogenic exposure. Male English sole from contaminated areas in the Duwamish River, Elliott Bay, and Commencement Bay were found to contain significant levels of female-specific, estrogen-inducible yolk protein, vitellogenin, in plasma. Vitellogenin induction was particularly noticeable at sampling sites in the proximity of combined sewage outfalls, which are historically associated with release of estrogenic compounds. Juvenile salmon outmigrate through, and in some cases rear, in the same areas in Puget Sound, so it is likely that they are also being exposed. Additionally, salmonid habitats in upstream freshwater areas that receive agricultural runoff are at risk for the presence of estrogenic contaminants. However, on information on the extent of xenoestrogen exposure in Pacific salmon is very limited. The proposed project will apply a suite of biochemical and molecular biomarkers of estrogenic exposure and effects in chinook and/or coho salmon, thus providing important analytical tools that will aid in gauging the extent of such anthropogenic disturbances. This suite will include the biochemical measurement of plasma vitellogenin induction, which is a benchmark biomarker that has been used extensively for various fish species around the world. The suite will also include molecular biomarkers -- the upregulation of expression of key genes that are involved in the response to estrogenic exposure, such as the genes for the estrogen receptors, vitellogenin, and zona radiata (egg-shell) proteins. In addition to biomarkers of exposure, this effort would also incorporate biomarkers of effects, such as changes in gene expression of pituitary hormones (gonadotropins, prolactin, and growth hormone) and growth factors (IGFs) that are important components of hormonal signaling pathways. These biochemical and molecular biomarkers of exposure and effects would be monitored in laboratory exposure of juvenile salmon, using environmentally relevant compounds, concentrations, exposure durations, and routes of exposure (waterborne or dietary). Our assessment of endocrine and biochemical changes associated with exposure to endocrine-disrupting compounds will be combined with an assessment of several whole animal health measurements, such as the success of smoltification and subsequent outgrowth, early gonadal development, and sex determination. Laboratory exposures would be coupled with environmental monitoring of estrogenic compounds in sediment and water in the Puget Sound area, as well as assessment of estrogen exposure and effects in wild fish. In carrying out this project, we plan to work closely with other NWFSC, UI and WSU scientists in several areas: 1) measurement of estrogen-responsive hormones (e.g., FSH, LH, GH, insulin-like growth factors) and assessment of EDC effects on early gonadal development, in collaboration with Drs. Penny Swanson and Briony Campbell (NWFSC), 2) determining effects of endocrine-disruptors on steroid metabolism and StaR protein activity, with Dr. Graham Young (UI), and 3) effects on phenotypic sex and gamete quality, with Dr. James Nagler (UI). Specific Aims: 1) Determine the extent of environmental exposure to estrogenic substances in juvenile salmonids at sites where exposure has been demonstrated in English sole: Vitellogenin induction and exposure to estrogenic compounds has been observed in English sole from estuarine sites in Commencement Bay and Elliott Bay that are known to be used by out-migrant juvenile salmon. We propose to screen salmon from these sites for vitellogenin induction. Fish are already being collected for ongoing studies to monitor salmonid habitat use. 2) Develop analytical capabilities for measurement of selected estrogenic contaminants (e.g., alkylphenols) in sediments, prey, and fish tissues: Although methods have been developed for measurement of estrogenic compounds such as alkylphenols in sediments, water, and fish tissues, these analyses are not routinely performed in our laboratory. As one of the aims of this project, the staff from the Environmental Chemistry group at NWFSC would set up and validate these analyses, so concentrations of nonylphenol and other environmental estrogens could be measured in environmental samples and in laboratory-exposed fish. Analyses for other endocrine disrupting compounds that may be of interest, such as PCBs, DDTs, PAHs, and chlorinated pesticides, are performed routinely and could be incorporated into studies as necessary. 3) Determine effects of selected endocrine-disrupting compounds on the reproductive-endocrine axis of chinook or coho salmon during laboratory exposures. Vitellogenin induction is commonly measured as an indicator of xenoestrogen exposure, but a number of other proteins and hormones are highly estrogen-responsive, and in some cases are more directly linked to reproductive function. For examples, Harris et al. 2001 (Environ. Sci. Technol. 35:2909-2916) have demonstrated that the gonadotropic hormones, FSH and LH, may be affected by exposure to the estrogenic surfactant, nonylphenol, at concentrations well below those associated with vitellogenin induction. Estrogenic compounds may also have effects on hormones associated with the growth axis, such as growth hormone and insulin-like growth factors, as well as enzymes and protein involved in the process of steroidogenesis. Initially, we propose to measure these parameters in juvenile Chinook or coho salmon exposed to environmentally realistic doses of nonylphenol in the diet. We chose nonylphenol for our initial studies because it appears to be one of the more common and persistent estrogenic chemicals in the estuarine environment; future work will focus on additional contaminants of concern. Hormone analysis will be conducted in collaboration with Drs. Penny Swanson and Briony Campbell (NWFSC), while effects of endocrine-disruptors on processes of steroidogenesis will be examined in collaboration with Dr. Graham Young (UI). 4) Determine effects of selected endocrine-disrupting compounds on various aspects of reproductive performance, including early gonadal development: At Mukilteo field station, we have facilities for rearing salmon from the egg stage through smoltification, and holding them in saltwater. In conjunction with measuring concentrations of vitellogenin and hormone levels in exposed fish, we will monitor early gonadal gonadal development histologically to determine onset of puberty, for any abnormalities on egg and sperm development, and for discrepancies between genetic and phenotypic sex. If possible, fish will be held until adulthood for assessment of longer-term effects on reproductive performance. We anticipate collaboration with Dr. James Nagler (UI) and Drs. Penny Swanson and Briony Campbell (NWFSC) on this aspect of the project. 5) Determine dose-response relationships and threshold effect levels for impacts of estrogenic and anti-estrogenic compounds on reproductive function in salmon. Salmon will be exposed to endocrine-disrupting compounds at a range of environmentally relevant concentrations to determine threshold concentrations for biological effects. This information will be used to help develop sediment and water quality guidelines for the protection of listed salmon. Conclusions: Degradation in habitat quality as a result of chemical contamination is a potential contributor to the decline of native salmon stocks in the Pacific Northwest. Environmental estrogens and other endocrine-disrupting compounds are of particular concern because many are poorly regulated in spite of their potential impact on critical life processes such as growth and reproduction. Through this collaborative project, we will be able to use some of the most advanced techniques in fish reproductive endocrinology to examine the sublethal effects of environmental estrogens and other endocrine-disrupting compounds on salmon. Salmon Recovery Impact: Restoration of physical habitat has been heavily emphasized in salmon recovery efforts. However, habitat restoration can never be fully successful if fish health is compromised by poor water and sediment quality. Over the past few years, sediment and water quality issues have become more and more prominent in regards to listed salmon species. The NMFS Northwest Region has been involved in several Biological Opinions concerning issues of water quality and contaminant impacts on salmon, including reviews of USEPA and the State of Idaho's water quality standards, sediment quality standards for management of dredged material in the Lower Columbia, the development of water quality standards for current use pesticides, and impacts of bleached kraft mill effluent on salmon health. There is clearly a need for more accurate information on the sublethal effects of chemical contaminants on listed salmonid species, both for the development of adequate regulatory standards and for guidance in habitat restoration efforts. The need is particularly great in the case of endocrine-disrupting compounds, because although they may affect the regulation of critical life processes such as growth and reproduction, comprehensive studies of their complex effects on salmon species are rare. This effort will provide biologically meaningful information on the sublethal effects of endocrine-disrupting compounds on salmon growth and reproduction, and the extent of exposure to these substances in both wild and hatchery-reared fish. These data can be used for assessing potential impacts of endocrine-disrupting compounds, including environmental estrogens, on the productivity of listed ESUs, for the improvement of hatchery practices, and for the development of protective Water and Sediment Quality Criteria.       PROJECT 11 SUMMARY Title: Mechanism of olfactory imprinting and homing: impacts of hatchery practices on straying in salmon Investigator: ­ Dr. Andrew Dittman, Resource Enhancement Utilization Technology Division, NWFSC Objectives: The overall objective of this project is to develop rearing and release strategies that will minimize straying of hatchery-reared salmon and thereby minimize the genetic and ecological impacts of hatchery fish on wild fish. Specifically we will 1) develop and validate cost-effective and reliable cellular and molecular assays for olfactory imprinting in hatchery salmon and 2) use these assays to determine the developmental period(s) and environmental conditions that are critical for olfactory imprinting in hatchery-reared fish. Summary: A major uncertainty associated with hatchery reform and the conservation of naturally spawning salmon populations is the causes and consequences of straying (i.e. gene flow) between populations (both hatchery and wild). While artificial propagation may be a necessary tool for salmon recovery, removing salmon from their natural environment can have profound effects on the development, physiology, behavior and ecological interactions of fish when they are released back into their native environment. To appropriately manage salmon populations it is important to understand how hatchery and management practices (e.g. habitat alterations, transport, hatchery rearing and release procedures) will affect olfactory imprinting and ultimately homing and straying. In some cases inappropriate hatchery practices can result in extremely high straying rates and currently, there is no effective way to anticipate how different management practices will affect homing because there is no assay for whether salmon have imprinted to a particular site (beyond monitoring adult return patterns). The tendency to home to the natal stream to spawn is fundamental to the unique biology and management of Pacific salmon. Homing results in genetic isolation of populations of salmon uniquely adapted for conditions in their natal streams. The final freshwater stages of these homing migrations are governed by the olfactory discrimination of home-stream water. Prior to their seaward migration, juvenile salmon learn (imprint on) site-specific odors associated with their home stream and later use these retained odor memories to guide the final phases of their homing migration. This imprinting process is critical for the successful completion of the spawning migration and salmon that do not experience their natal water during appropriate juvenile stages are more likely to stray to non-natal sites. Determining the mechanisms and timing of olfactory imprinting in different salmon species is important for developing appropriate rearing and release strategies to reduce straying in hatchery or captively-reared fish. Defining these sensitive periods for imprinting will also allow ESA recovery teams to identify juvenile habitat or seasonal windows that are critical for imprinting and develop management strategies to minimize straying. Efforts to identify these sensitive periods have been hampered by the difficulty of experimentally assessing successful imprinting. Most studies have relied on large-scale field experiments involving the release of hundreds of thousands of tagged juvenile salmon to ensure that enough adults return to allow for appropriate statistical analysis. While such studies can provide critical information on the process of imprinting, they are often biased by recovery effort and require large numbers of juveniles, precluding the use of target populations that are threatened or endangered. Several alternative behavioral, physiological and biochemical techniques for assessing imprinting have been tested but all are technically difficult and/or expensive and are impractical for routine assessment of imprinting. The overall goal of this proposal is to develop simple, cost-effective molecular markers for olfactory imprinting. These markers should provide tools for determining the critical developmental periods and environmental conditions necessary for imprinting in hatchery-reared fish and ultimately for assessing straying in wild populations. Specific Aims: By identifying developmental periods that are important for olfactory imprinting, rearing and release strategies for each salmon species can be developed to lower stray rates in both production and recovery hatcheries. To determine the critical period(s) for imprinting, juvenile salmon are exposed to known odorants at key developmental stages that are associated with migrations or habitat shifts in wild fish and during periods that juveniles are typically released from hatcheries. Fish are subsequently tested for development of long-term memories of these odorants using molecular, electrophysiological, and behavioral assays. We have previously demonstrated that olfactory receptor neurons are sensitized to home-stream odors during the process of imprinting and the goal of this project is to exploit this sensitization to develop and validate new molecular tools for assessing imprinting. We are using cDNA arrays, real-time PCR and in situ hybridization to determine whether sensory neuronal populations in salmon change predictably during the process of imprinting. Ultimately these tools will used to identify the critical developmental periods and environmental conditions required for olfactory imprinting in Pacific salmon. Salmon Recovery impact: Understanding the causes and consequences of straying in both hatchery and wild fish is critical for understanding the requirements for viability in a salmonid ESU and subsequent quantitative goals for recovery. Salmon conservation and recovery efforts are confounded by the inability to define natural and human-influenced rates of migration between (meta)populations of salmon. Results from this research will help determine what are "natural" levels of movement (straying) between populations and what environmental conditions and human practices influence these stray rates. Determining the timing of olfactory imprinting in different salmon species is also critical for developing appropriate rearing and release strategies to reduce straying in hatchery or captively-reared fish. Many of the genetic and ecological concerns about the interactions of hatchery and wild fish are a result of straying. By developing new molecular tools for assessing imprinting we may soon be able to directly identify the critical developmental periods and environmental conditions required for olfactory imprinting in Pacific salmon and thereby develop hatchery and management practices that minimize straying. PROJECT 12 SUMMARY Title: Contaminant effects on fish neurobiology, behavior, and development Investigator: ­ Dr. Nat Scholz, Environmental Conservation Division, NWFSC Objectives: Our overall goal is to evaluate the effects of environmental pollutants on fish health, with an emphasis on threatened or endangered species of Pacific salmon. This work has the following objectives: Develop rapid, high-throughput phenotypic screens to evaluate developmental toxicity in fish exposed to common classes of environmental contaminants. Identify specific mechanisms or pathways of developmental toxicity in fish embryos and larvae. Determine the effects of pesticides and other neurotoxic chemicals on nervous system function in salmon. Evaluate the sublethal effects of environmental contaminants on behaviors that are essential for the survival, reproductive success, or migratory success of salmon. Summary: Salmon recovery planners are increasingly faced with the following question: how should habitat restoration activities be prioritized for river systems that have mixed chemical and physical degradation? This is a key question for natural resource managers who must confront the complex impacts of urbanization, agricultural land uses, and industrial activities on salmon habitats in the Columbia River Basin. Obviously, where pollution occurs, habitat-based recovery models for salmon should address the potential significance of chemical habitat deterioration. Unfortunately, specific determinants of chemical habitat quality (i.e., water and sediment contamination) are often excluded from habitat models. This is because (1) chemical habitat quality can be very complex and expensive to measure, and (2) there is a general absence of relevant toxicological data for most of the chemicals that salmon are exposed to. In the absence of empirical data for pollution, habitat recovery plans have generally placed a higher priority on the restoration of physical processes. Critically, this practice may undervalue the importance of chemical habitat quality and lead to predictive errors in recovery planning. To address these uncertainties, the Fish Neurobiology and Development Team is investigating the effects of ecologically realistic contaminant exposures on salmon health. The results of this research will guide the implementation of conservation measures for threatened and endangered species of salmon. Specific Aims: Development- A major goal of our ecotoxicological research is to generate new data that can be used to manage the recovery of salmon populations and at-risk species of marine fish in the Pacific Northwest. Unfortunately, it is not technically or logistically possible to conduct rapid and sensitive toxicological screens in salmon. There are two reasons for this. The first is the large number of environmental contaminants (alone and in mixtures) that need to be evaluated. The second is the fact that only a few, labor-intensive sublethal toxicological endpoints have been established for native fish species. For example, fish are particularly vulnerable to the harmful effects of pesticides and other environmental contaminants during early stages of development (embryos and larvae). Rapid and sensitive developmental screens in salmonids are not practical because (1) salmon embryos are only seasonally available, (2) the duration of embryonic and larval development is relatively protracted, (3) basic aspects of salmon developmental biology have not been described, (4) salmon embryos are opaque, which precludes conventional in vivo optical imaging techniques, and (5) ontogenetic and molecular markers for many critical developmental processes have not been worked out for salmon. Accordingly, we are proposing a novel use of the zebrafish (Danio rerio) as a surrogate experimental system for screening pesticides for harmful effects in fish. Zebrafish are a relatively new experimental model in the fields of vertebrate developmental biology, genetics, and toxicogenomics. Research in this system, however, is increasingly advancing our understanding of the mechanisms that control the differentiation and specification of the vertebrate embryo. Recently, the molecular and genomic tools available for zebrafish research have expanded considerably (i.e., the zebrafish genome project). Zebrafish are widely recognized as a useful model for studying human disease, and they have become a major National Institute of Health (NIH)-supported experimental model for studying fundamental mechanisms of development and developmental toxicity in humans and other vertebrates. For these reasons, we believe zebrafish will also be a good surrogate for salmon and other fish species. Neurobehavior - Toxic pesticides and metals are an emerging concern for the environmental health of anadromous salmonids throughout the Pacific Northwest. Pesticides are used extensively in many agricultural and urban watersheds, resulting in the widespread contamination of salmon habitat. In addition, copper and other metals are major constituents of urban stormwater runoff, irrigation return flows, and agricultural runoff. Copper and many pesticides (particularly the common organophosphates) are toxic to the salmon nervous system, and several studies on salmon have shown that these chemicals can interfere with critical behaviors at different life history stages. There is considerable concern within NOAA Fisheries that these environmental contaminants may impair the essential biological requirements (or behavioral patterns) of threatened or endangered species. Recent federal court decisions and regional and national Section 7 consultations under ESA have highlighted the need for targeted new research to more clearly understand the relationships between sublethal contaminant exposures and the physiology and behavior of salmon. To address existing data gaps, we will compare thresholds for neurophysiological and behavioral toxicity in juvenile Pacific salmon using a combination of digital video and in vivo electrophysiology. We will focus on olfactory function and predator avoidance behaviors that are specifically triggered by olfactory cues. Contaminant exposures will be ecologically realistic - that is, based on levels of pesticides and metals that have been measured in freshwater salmon habitats. We will use regression analyses and benchmark dose statistics to compare behavioral and neurophysiological toxicity thresholds. From these experiments, we should be able to establish whether physiological data can be extrapolated to behaviors that are essential for the survival of ESA-listed species.   Conclusions: Agriculture and urban land use activities are major sources of pesticides and other environmental contaminants, and these are eventually transported to rivers and streams that provide habitat for several species of Pacific salmon in the Columbia River Basin. Existing evidence indicates that many contaminants, including pesticides, hydrocarbons, and metals, are potentially toxic to fish at early life history stages. Still others can impair the salmon nervous system and interfere with behaviors that are critical for the survival of individual animals. Consequently, environmental contaminants may limit the viability and/or recovery of natural salmon populations that spawn and rear in river systems that have degraded water quality. We will use the zebrafish experimental model and techniques adapted from research biomedicine to better understand the impacts of water pollution on fish health. In addition, we will use neurophysiological and behavioral techniques to assess the effects of neurotoxic contaminants on the salmon nervous system. Salmon Recovery Impact: 1The aim of this work is to establish explicit links between habitat attributes (i.e., pollution), salmon health, and the productivity of wild populations, several of which are now listed as threatened or endangered under the Endangered Species Act. New data can be used to evaluate existing habitat conditions and identify contaminants that may limit salmon recovery. This work will also help natural resource managers identify restoration priorities in salmon habitats that have mixed physical and chemical degradation.   PROJECT 13 SUMMARY Title: Fish migratory health and disease Investigator: Dr. Mark S. Strom, Resource Enhancement and Utilization Technologies Division, NWFSC/NMFS Other Investigators: Dr. Linda D. Rhodes, REUTD, NWFSC/NMFS Objectives: The long term goals of this project are to: develop practical strategies to treat and prevent Bacterial Kidney Disease (BKD) of salmon, a prevalent disease caused by the bacterium Renibacterium salmoninarum develop methods to genetically distinguish strains of R. salmoninarum in order to determine the rate of BKD transmission from hatchery to wild stocks (and vice versa) determine the role of infectious disease in delayed mortality in juvenile Chinook salmon after multiple passage through hydroelectric dam bypass systems Summary: The Fish Health/Microbiology Team of the Integrat