2006 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? Although aquaculture production in the U.S. has shown impressive growth in the last decade, most of this has come from the increased production of warm-water fish species, primarily catfish. There are abundant cool and cold freshwater resources in the U.S. and a vital aquaculture industry that has the capability to contribute more substantially to overall aquaculture production. The genetic quality of currently available strains of aquaculture species is a major constraint to realizing increased output and greater production, and therefore, the development of strains genetically improved for commercial production is needed. Our research aims to create production opportunities with genetically improved fish. Research is conducted on integrating traditional selection and breeding approaches, chromosome set manipulation methods, and molecular physiological and genetic techniques for producing superior strains of cool and cold water species. The project has three specific goals:. 1)to produce strains of rainbow trout with superior growth and disease resistance performance,. 2)to define the architecture of energetic partitioning and the regulatory genes and proteins affecting partitioning of energy into growth, stress, and reproductive traits,. 3)to establish a tetraploid line of rainbow trout for the production of triploid, reproductively sterile offspring by crossing with diploids. The research to be undertaken is aligned with National Program 106-Aquaculture and addresses the goals of genetic improvement, reproduction and early development, and growth, development and nutrition. Specifically, we address: Genetic improvement: traditional animal breeding, broodstock development, germplasm preservation, molecular genetics to address improvement of growth rates, feed efficiency, survival, disease resistance, and product quality; conservation and utilization of important aquatic germplasm for use by producers. Reproduction and early development: improvement of systems to enhance reproductive efficiency. Growth, development and nutrition: elucidate factors affecting growth rates, feed conversion and feed formulations and feeding strategies to reduce dependence on marine fish-based protein in aquaculture diets. Aquaculture producers and the public will benefit from improved productivity of superior strains rainbow trout being developed, and the reduced environmental impact of fish with greater efficiency. Additionally, this research supports the integration of selective breeding with identification of the genes contributing to variation in the traits. Improved integration of traditional breeding practices with molecular genetic information will speed improvement resulting in a supply of healthy foods that are produced more efficiently. 2.List by year the currently approved milestones (indicators of research progress) Year 1 (FY2005): Objective 1: a. Production of broodyear 2005, estimation of genetic parameters for growth and Flavobacterium psychrophilum resistance. b. Screen 20 families for growth at our center, North Carolina State University, Hagerman, Idaho and the Conservation Fund Freshwater Institute c. Test antimicrobial peptide transgenic fish for disease resistance d. Cryopreserve milt from NCCCWA rainbow trout males Objective 2: a. Evaluate in vivo effects of growth hormone (GH) secretogogues on GH release in rainbow trout b. Assess the use of GH secretogogues to alter feeding in rainbow trout and develop methods to measure feed efficiency and genetic variation in feed efficiency c. Determine association between stress responsiveness (plasma cortisol following a crowding stress), and disease resistance to Yersinia ruckeri Objective 3: a. Define pressure treatment protocols for induction of tetraploidy (4N) in four lines of rainbow trout and produce several 4N families of rainbow trout by crossing 1st generation 4N parents b. Produce several 3N families by crossing 4N X 2N parents and compare to normal diploids for viability and growth Year 2 (FY2006): Objective 1. a. Production of broodyear 2006, estimate genetic and phenotypic correlations between growth and Flavobacterium resistance b. Estimate the proportion of total variation due to genotype x environmental effects and screen families for genotype x diet interaction c. Test transgenic fish for disease resistance, identify muscle specific promoter d. Cryopreserve milt from NCCCWA and transgenic fish Objective 2. a. Evaluate in vivo effects of growth hormone (GH) secretogogues on GH release in rainbow trout b. Contrast endocrine response to fasting and re-feeding to identify hormones and genes that regulate feeding and growth, and determine the relationship between feed efficiency of individuals and their full-sib families c. Determine association between stress response (plasma cortisol following a crowding stress) and response to ACTH d. Identify ovarian genes altered in response to follicle recruitment after unilateral ovariectomy, and Identify changes in expression of growth factors during ovarian development Objective 3. a. Produce four 4N families from five selected NCCCWA families and make ten 2nd generation 4N families of even-year lines with defined genetic backgrounds b. Evaluate production traits of (1) 3Ns from crosses, (2) 3Ns from pressure treatment and (3) 2Ns, all from even-year lines and of similar genetic background and compare stress response of 2N and 3N animals Year 3 (FY2007): Objective 1. a. Produce BY 2007 germplasm through selective breeding and estimate genetic parameters for quality traits, and evaluate realized heritability for growth b. Estimate the proportion of total variation due to GxE effects for reproductive traits and estimate the proportion of total variation due to genotype x diet effects on growth c. Test transgenic fish for disease resistance, breed transgenic lines to homozygosity d. Produce >10 all female families with sex reversed males Objective 2. a. Determine effects of sex steroids on pituitary responsiveness to GH secretogogues b. Determine effect of GH secretogogue-supplemented feed on growth, feed intake and efficiency and longitudinal evaluation of feed efficiency over time c. Determine heritability of stress responsiveness (plasma cortisol following a crowding stress) Objective 3. a. Make ten 2nd generation 4N families of odd-year lines with defined genetic backgrounds b. Evaluate production traits of. 1)3Ns from crosses,. 2)3Ns from pressure treatment and. 3)2Ns, all from odd-year lines and of similar genetic background and compare disease resistance of 2N and 3N animals c. Conduct baseline genetic analyses (gene variability and karyotyping) on 4N parental fish used for crosses to make advanced generation 4N offspring Year 4 (FY 2008): Objective 1. a. Produce BY 2008 germplasm through selective breeding and estimate correlations with quality traits and evaluate realized heritability for Flavobacterium resistance b. Estimate the proportion of total variation due to genotype x diet effects on reproduction c. Test anti-microbial peptide (AMP) transgenic fish for disease resistance and amplify disease resistant transgenic lines to introgress 10 crosses into a Leetown line d. Produce >30 all female families with sex reversed males Objective 2. a. Evaluate variation in endocrine measures of growth and associations with growth performance in brood year 2006 fish b. Determine if fish selected for an improved stress response to crowding, also exhibit an improved stress response to a temperature or salinity stress c. Evaluate growth factors, identified from studies in 2006, for their ability to affect ovarian development Objective 3. a. Make ten 3rd generation 4N families of even-year lines with defined genetic backgrounds b. Determine the combination of 4N and 2N parental lines that yield superior 3N offspring for commercial production c. Analyze advanced generations of 4N fish for changes in gene content/frequency and chromosomal make-up Year 5 (FY 2009): Objective 1. a. Produce BY 2009 germplasm through selective breeding and estimate correlations with quality traits and evaluate realized heritability for Flavobacterium resistance b. Estimate the proportion of total variation due to genotype x diet effects on reproduction c. Test AMP transgenic fish for disease resistance and amplify disease resistant transgenic lines to introgress 10 crosses into a Leetown line d. Produce >70 all female families with sex reversed males Objective 2. a. Evaluate variation in endocrine measures of growth and associations with growth performance in brood year 2008 fish b. Identify QTL for stress responsiveness c. Identify genes and hormones in eggs that affect egg quality Objective 3. a. Make ten 3rd generation 4N families of odd-year lines with defined genetic backgrounds b. Identify 4N rainbow trout lines to be released and tested under commercial conditions c. Define the genetic stability/volatility of different 4N lines of fish and evaluate commercial applicability 4a.List the single most significant research accomplishment during FY 2006. Breeding for resistance to bacterial cold-water disease NP 106 Genetic improvement action plan item b. Selective breeding for commercially important traits. In FY 2006, it was discovered that the resistance of rainbow trout to Flavobacterium psychrophilum the causative agent of bacterial cold-water disease has a substantial genetic component and a wide range of breeding values. This suggests that selection for resistance to bacterial cold-water disease will improve resistance to this pathogen in our broodstock. The data from 75 families, generated in conjunction with the other projects at NCCCWA (1930-31000-008 and 1930-32000-003) was analyzed using the “Survival Kit” software. The selection of breeders for the 2007 broodstock will be accomplished based on breeding values for resistance to F. psychrophilum. Field trial of these fish along with control groups is planned, and is expected to offer growers a fish resistant to the most important bacterial disease affecting trout culture. 4b.List other significant research accomplishment(s), if any. Genes regulating egg development NP 106 Reproduction and early development action plan item c. Gamete and Zygote Quality. We have identified reproductive cycle changes in expression of members of the TGFbeta superfamily of growth factor peptides and some of their inhibitors. The most dramatic changes in gene expression of these factors were just before spawning, during the resumption of meiosis. In vitro studies of ovarian fragments near this stage of the reproductive cycle revealed that gene expression of these peptides was affected by a progestin steroid that is known to induce the resumption of meiosis. Most interesting is a 21-fold increase in the expression of the growth factor inhibitor BAMBI. We confirmed that injections of the progestin also elevated BAMBI expression in the ovary. Our data suggest the inhibition of TGFbeta peptides may be an important part of processes regulating egg development as the rainbow trout prepares for spawning and therefore be important for egg quality. Genotype x diet interactions in rainbow trout families NP106 Genetic improvement action plan item b. Selective breeding for commercially important traits. Over two years more than 30 families of NCCCWA rainbow trout were raised at the University of Idaho, Hagerman Fish Culture Experiment Station on two diets, one high in soybean meal and the other without soybean meal, to test the genotype x diet interaction. There were significant genetic and diet effects on feed intake, growth rate and feed efficiency, but the genotype x diet interaction effect was not important. This suggests that the fish selected for growth on today’s typical diets will be the best performers on diets of tomorrow if they incorporate higher levels of plant protein sources. This will encourage improvement of today’s lines of fish for better performance in the future 4c.List significant activities that support special target populations. None 4d.Progress report. This report serves to document research conducted under a specific cooperative agreement between ARS and North Carolina State University (1930-31000-007-03S, Evaluation of Genotype by Environment Interactions in Rainbow Trout). In 2006 a student was hired at NCSU to analyze the data from the 20 genetic groups grown at both the NCSU extension facility in Fletcher, NC in our Leetown, WV facility for performance comparison over multiple environments. The best performing groups in North Carolina were also the top performing groups in WV. Our analyses will provide estimates of the proportion of variation among environments due to genotype, environment and genotype x environment interaction. Additionally, this report documents research conducted under a specific cooperative agreement between ARS and University of Idaho (1930-31000-007-04S, Evaluation of Selected Rainbow Trout Lines Fed Grain Based Diets Under Farm Scale Conditions). Fish from 33 genetic groups were evaluated at the University of Idaho, Hagerman facility. Each genetic group was divided into 4 replicate tanks and fed either a control diet high in fishmeal content or a diet high in protein from soybeal meal. Family and diet effects were large, however the effects of family x diet were mostly small. The most important family x diet interaction effect was for feed efficiency. A reimbursable agreement was established for ARS scientist Brian Shepherd’s funding through the CSREES NRI program grant (1930-31000-007-02R Innovative approaches to growth enhancement in teleosts). Work under this agreement has was begun, and tools for quantifying growth axis hormones and neuropeptides were developed and validated. When Brian Shepherd moved to Wisconsin, the agreement was terminated (on Dec. 31, 2005) and moved with him. 5.Describe the major accomplishments to date and their predicted or actual impact. • NP 106 Genetic Improvement b.Selective breeding for economically important traits. Aquaculturists growing rainbow trout either for food or recreational purposes need genetically improved strains to improve production. The establishment of strains of rainbow trout with a broad genetic base is a major accomplishment that lays the groundwork for genetic improvement. The first generations of selection, have been for growth in our even year line (2004 and 2006), and following evaluation for resistance to bacterial coldwater disease in our 2005 broodfish we will select for resistance in 2007. This is a central part of objective 1. We have met with the US Trout Farmers’ Association to discuss germplasm release goals (2005 and 2006) and are developing a strategic plan to release germplasm. Our lines will be preliminarily tested on two farms in FY 2007. • NP 106 Genetic Improvement d.Specific breeding aids. A tetraploid line simplifies production of 100% sterile fish and provide intellectual property protection for breeders. Techniques for polyploidy induction have been optimized, and tetraploid offspring (F1) were produced from tetraploid parents meeting a CRIS milestone and the Action Plan goal of developing specific breeding aids. We have had contact with several commercial producers and breeders regarding development of tetraploid lines indicating the potential impact of this development. • NP 106 Genetic Improvement b. Selective breeding for economically important traits. Stress tolerance and disease resistance are two the high priority traits for genetic improvement identified with industry input. Genetic groups with high and low responsiveness to stress have been identified and a positive correlation between stress response and growth was identified. Additionally, no association was found between stress response and resistance to enteric redmouth disease. These lines formed the base for an ongoing study to identify QTL for stress responsiveness. • NP 106 Genetic Improvement a. Conserve, characterize, and utilize genetic resources. The lines of rainbow trout developed here are meant to serve the needs of producers throughout the country. The goal of evaluating our populations across several relevant environments relies on developing the necessary relationships and methods for moving fish over great distances. We have cooperated to evaluate genetic groups in Hagerman, Idaho, Fletcher, North Carolina, and Shepherdstown, West Virginia enabling us to characterize our germplasm in multiple environment. We have found no important genetic differences in growth performance across these environments. • NP 106 Genetic Improvement a. Conserve, characterize, and utilize genetic resources and b. selective breeding for economically important traits. The increasing demand for aquaculture products and the limited availability of fish meal as a protein source necessitates the development of plant protein based diets. It has been questioned whether fish that perform best on fish meal based diets will perform best on plant protein based diets.We have determined through 2 independent studies that genotype x protein source interactions are not significant in diets where the protein source has been either fish meal or plant proteins. This work has been conducted in conjunction with rainbow trout breeders, and scientists in the related project ‘Integration of Nutritional, Genetic and Physiological Approaches to Improve Production Efficiency of Rainbow Trout’ (CRIS # 5366-21310-003-00). This work demonstrates that selecting breeders based on performance on currently available diets will not hinder future progress on alternative protein source diets. 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? We have had contact from several industry groups regarding the production of tetraploid fish. These contacts may develop into cooperative agreements to transfer improved germplasm and to improve chromosome set manipulation technologies for salmonids. We are working with the US Trout Farmer's Association to initiate on-farm trials with our selectively bred germplasm. This will be the next step toward release of selectively bred improved germplasm to the US trout growers. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). 1) Hershberger, W.K., M.A. Hostuttler. 2006. Development of tetraploid rainbow trout may yield improved triploid production. Global Aquaculture Advocate Vol. 9(2): 80-82. log# 194224 2) Silverstein J.T. 2006. One fish, two fish, red fish and three blue fish. The Fish Line-Colorado Aquaculture Newsletter 18(2):5-6. log# 194251 3) Silverstein J.T., Palti Y, Vallejo R, Welch T, and Wiens G, 2006. Cracking the code: Selecting for resistance against bacterial cold-water disease. US Trout Farmers' Association Newsletter, Trout Talk: Fall 2006 log# 198916 Review Publications Leder, E.H., Silverstein, J. 2006. The Proopiomelanocortin Genes in Rainbow Trout (Oncorhynchus Mykiss): Duplications, Splice Variants, and Differential Expression. Journal of Endocrinology. 188:355-363. Gahr, S.A., Weber, G.M., Shepherd, B.S., Silverstein, J., Vallejo, R.L., Coulibaly, I., Rexroad III, C.E. 2005. Effects of Recombinant Bovine Somatotropin (rbst) on the Liver and Muscle Transcriptomes of High Growth Rate Rainbow Trout (Oncorhynchus Mykiss). Plant and Animal Genome Conference, Jan 2006. Abstract ID P534, pg 260. Silverstein, J. Cryopreservation activities and needs at the National Center for Cool and Cold Water Aquaculture. Aquaculture America Conference. Aquaculture America Book of Abstracts 2006, pg 287. Silverstein, J., Hardy, R., Casten, M., Barrows, F. 2006. Genotype diet interactions in rainbow trout families fed diets with and without high levels of soybean meal inclusion. International Symposium on Genetics in Aquaculture. p. 97. Silverstein, J. 2006. Producing all female rainbow trout: do neomales affect the growth of their sibilings?. Aquaculture America Conference February 13, 2006, Las Vegas, NV. Silverstein, J. 2006. Relationships among feed intake, feed efficiency and growth in juvenile rainbow trout (Oncorhynchus mykiss). North American Journal of Aquaculture.68:168-175. Silverstein, J., Brazil, B.L., Salem, M.S., Cox, M.K., Blemings, K.P., Yao, J. 2006. Ammonia excretion as an indicator of growth efficiency. Aquaculture America Conference. Aquaculture America Book of Abstracts. Pg. 285. Silverstein, J., Weber, G.M., Rexroad Iii, C.E., Vallejo, R.L. 2006. Genetics and genomics-intregration of breeding and molecular genetics programs. Meeting Abstract. Presented at BARD sponsored workshop on Aquaculture Genetics in Eilat, Israel, Feb 20-23, 2006. Gahr, S.A., Weber, G.M., Rexroad III, C.E. 2006. Fasting and refeeding effects the expression of the inhibitor of dna binding (id)genes in rainbow trout (Oncorhynchus mykiss) muscle. Comparative Biochemistry and Physiology. 144:472-477. Higgins, A., Silverstein, J., Engles, J., Wilson, M., Rexroad III, C.E., Blemmings, K. 2006. Starvation induced alterations in hepatic lysine metabolism in different families of rainbow trout (Oncorhynchus mykiss). Fish Physiology and Biochemistry Journal. 31(1):33-44. Lankford, S.E., Weber, G.M. 2006. Associations between plasma growth hormone, insulin-like growth factor-1 and cortisol, with stress responsiveness and growth performance in a selective breeding program for rainbow trout (oncorhychus mykiss). North American Journal of Aquaculture 68, 151-159. Palti, Y., Silverstein, J., Wieman, H., Phillips, J.G., Barrows, F.T., Parsons, J. 2005. Evaluation of family growth response to fish meal and gluten-based diets in rainbow trout (oncorhynchus mykiss). Aquaculture. 255 (1-4):548-556. Lankford, S.E., Weber, G.M. 2006. Potential roles of intraovarian growth factors during follicle maturation in rainbow trout (Oncorhynchus mykiss). Meeting Abstract.