2005 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? What does it matter? Although aquaculture production in the U.S. has shown impressive growth in the last decade, most of this has been realized from the increased production of warm-water fish species, primarily catfish. There are abundant cool and cold freshwater resources in the U.S. and an established and 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, development of genetically improved strains is needed. Our research aims to create production opportunities with genetically improved fish. Research is conducted on traditional selection and breeding approaches, chromosome set manipulation methods, and molecular physiological and genetic techniques to for producing superior strains of cool and cold water species. Molecular and physiological analyses are conducted to characterize traits of importance to aquaculture production, (e.g., growth and disease resistance), characterize the variation in these traits, and to identify the genes contributing to variation in the traits. These data will be used in conjunction with performance information from genetic groups to breed rainbow trout improved for aquaculture production. Techniques for genetic manipulations such as transgenesis and chromosome set manipulation will be developed and evaluated for improving fish growth and health. The results from these research approaches will provide the data, technology, resources, and direction from which superior strains of cool and cold water species will be developed for the aquaculture industry. Furthermore, superior germplasm will be identified and developed. The research in this project falls under National Program 106 - Aquaculture and addresses goals in genetic improvement, health mangement, reproduction and growth as described in the National Program Action Plan. Specifically these are: Selective breeding, specific breeding aids, characterization of genetic resources under genetic improvement; Immunology and disease resistance under health management; control of gender and fertility, control of reproduction, and gamete quality under reproduction; and regulation of feed intake, tissue growth, and interactions with reproduction under growth. 2.List the milestones (indicators of progress) from your Project Plan. Objective 1: Establish domesticated strains of rainbow trout. Under this objective strains of rainbow trout will be developed and improved, primarily through selective breeding, for dissemination to the trout industry. Objective 2: Functional definition of growth, stress, and reproductive traits. Each of these physiological processes is energetically demanding. By defining the hierarchy of energetic partitioning and the regulatory genes and proteins affecting partitioning, we will develop trout strains with superior productivity in various environments. Objective 3: Polyploidy induction to disrupt sexual development. Sexual development requires considerable energy resources, that must be diverted from growth. Furthermore, there is increasing interest in using sterile fish in culture situations. Development of tetraploid lines will enable the efficient production of all triploid (sterile) groups of trout. For Fiscal year 2005 our specific milestones were: Objective 1: a. Production of broodyear 2005, estimation of genetic parameters for growth, Flavobacterium resistance, genetic parameters for growth have been estimated and analysis of the genetic parameters for disease resistance are underway. The disease challenge work was completed in June. 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 For Fiscal year 2006 our specific milestones are: 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 (met) 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 BMPs 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 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 For Fiscal year 2007 our specific milestones are: 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 Conduct baseline genetic analyses (gene variability and karyotyping) on 4N parental fish used for crosses to make advanced generation 4N offspring For Fiscal year 2008 our specific milestones are: 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 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 ’06 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 ’06, 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 For Fiscal year 2009 our specific milestones are: 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 ’08 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.What was the single most significant accomplishment this past year? Crosses made with tetraploid rainbow trout–First generation tetraploid male and female rainbow matured this year and produced a number of tetraploid and triploid crosses for further evaluation. The tetraploid fish generated in this project offer the opportunity to produce 100% triploid offspring, which are desirable because they are sterile and potentially faster growing than typical diploid production animals. The offspring from these crosses provide (1) the initial generation of potential tetraploid lines for aquaculture and (2) the initial evaluation of the potential for the lines to produce superior triploid offspring for aquaculture production. Each tetraploid female was crossed with (1) a tetraploid male to produce a second generation with four sets of chromosomes and (2) a diploid male to produce triploid offspring. The tetraploid males were also crossed with diploid females to produce triploids from the reciprocal cross. All crosses yielded the expected results with respect to ploidy level (100% tetraploids for the first crosses and 100% triploids for the latter two). The triploids produced were analyzed in a growth study and initial results suggest they have a growth advantage from a very young age; this study is still ongoing. 4b.List other significant accomplishments, if any. Methods to control secretion of growth hormone (GH)- We have newly demonstrated the existence of a endocrine axis that controls growth hormone (GH) secretion in teleosts. Understanding this growth axis will allow us to better manipulate and optimize growth in production of rainbow trout. We have determined that novel GH-secretogogues (GHS) stimulate GH release (via the GHS-receptor) in tilapia, channel catfish and rainbow trout, thus providing the first evidence for the presence of this regulatory axis in teleosts. Ghrelin is a newly identified peptide that has been found to be the natural ligand for the GHS-R. We extended our work to rainbow trout and have found that ghrelin, and other GH secretogogues, do stimulate the growth axis (GH, IGF-I and IGF-binding proteins) and feed intake in this species. It is expected that this work will result in novel ways to promote growth in commercially-important teleosts. Identification of genetic variation for resistance to bacterial coldwater disease-We conducted a successful exposure of 75 full-sib families to a challenge with Flavobacterium psychrophilum, the causative agent of bacterial coldwater disease. This disease is of major concern to the trout producing industry and we aim to determine whether there is genetic variation for resistance to this bacteria. A standardized challenge was applied to 45 replicated and 30 unreplicated family groups. Mortality ranged from 28 to 99% and was similar in replicates. Genetic relationships among multiple families with low mortality indicate that there is a genetic component to resistance to this bacteria. Through selective breeding to enhance resistance we expect to deliver fast growing, healthier fish to the trout industry and ultimately to consumers. Stress response and resistance to enteric redmouth disease-We determined that stress response, measured as plasma cortisol levels, was not associated with resistance to enteric redmouth disease in either stressed or unstressed fish. Families of rainbow trout from our broodstock have a five-fold range in plasma cortisol levels in response to a crowding stress. Concern that increased stress responsiveness might result in fish with decreased disease resistance was addressed this year. Using Yersinia ruckeri, the causative agent of enteric redmouth disease in a challenge, we determined that cortisol response was not related to survival following exposure to the pathogen. Continued studies of associations between stress response indicators and performance traits will provide the information on which to base selection decisions to address the negative effects of stress in aquaculture production facilities. Identification of new genes involved with energy balance regulation- Pro-opiomelanocortin (POMC) is a pre-pro-hormone in which one gene codes for multiple peptide hormones. Some the resulting hormones, a-melanocyte stimulating hormone and b-endorphin, are involved in feed intake through neuropeptide signaling in the hypothalamus. We identified, sequenced and determined the tissue distribution of these new genes encoding POMC which suggests complex regulation of POMC peptides and feeding and energy balance. Tracking expression of these genes will allows us to better understand energy balance and efficiency in rainbow trout. Determining the role of these genes in feed intake and efficiency will help us to selectively breed for faster growing more efficient fish. 4c.List any significant activities that support special target populations. None 4d.Progress report. 1930-31000-007-01S: This report serves to document research conducted under a specific cooperative agreement between ARS and the University of Connecticut. This project has progressed to the second generation and lines with targeted transgenes appear to have enhanced disease resistance. Other groups of transgenic rainbow trout are being bred to genetic purity for testing in FY 2006. 1930-31000-007-02R: This report serves to document research conducted under a reimbursable agreement between ARS and CSREES NRI. Work under this agreement has begun, and tools for quantifying growth axis hormones and neuropeptides have been developed and validated. 1930-31000-007-03S: This report serves to document research conducted under a specific cooperative agreement between ARS and North Carolina State University. In 2005 fish from 20 genetic groups were sent to the NCSU extension facility in Fletcher, NC for rearing and growth monitoring. These same genetic groups were reared 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. 1930-31000-007-04S: This report documents research conducted under a specific cooperative agreement between ARS and University of Idaho. Fish from 20 genetic groups were sent to the University of Idaho, Hagerman facility for rearing. These same groups are reared at our Leetown facility. Each genetic group was divided into replicate tanks and fed either a control diet high in fishmeal content or a diet high in protein derived from plant sources (soybean meal). Performance on the fishmeal diet was better in every family, and there were differences between families; however, there were no families that performed disproportionately well or badly on soybean based diets. An additional 18 families will be investigated under the same protocol this year to verify and extend these results. 1930-31000-007-05S: This report serves to document research conducted under a Specific Cooperative agreement between ARS and the University of Connecticut. 1. Cecropin transgenic fish exhibit resistant to a viral pathogen IPNV: We have confirmed that 5 families (S9#746, A12#944, A13#831, Cec230#3255 and S9-A26) of F2 Rbt99 trsansgenic fish are resistant to the viral pathogen IHNV. 2. F1 transgenic fish with CF-17 transgene have been produced: We have successfully produced 10 families of F1 transgenic fish carrying CF-17 transgene. 3. Cryopreservation of trout sperm: We have successfully cryopreserved 21 familiesof Rbt09 transgneic fish. 4. Some bacterial pathogens may develop resistant to antimicrobial peptides: We have demonstrated conclusively that some bacterial, pathogens can gain resistant to antimicrobial peptide via physiological adaption, bur will return to non-resistant status if the selection pressure is removed. 5. Antimicrobial peptide may serve as an adjuvant: We have demonstrated conclusively that cecropin B or CF-17 peptide can induce inflammatory response in macrophage. The induction of the inflammatory response does not further block the effect of LPS induction of inflammatory response in macrophage. These results suggest that cecropin or CF-17 could serve as an adjuvant in fish vaccination. 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. This project started in the second quarter of FY 2005. Our major stakeholders are cool and cold water fish culturists growing fish either for food production or for live-hauling. We work closely with commercial breeders, commercial farmer/processors and commercial fish stocking operations. In addition we have close ties in the research community supplying research tools to scientists throughout the United States to advance efficient culture of cool and cold water fish. • 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 generation of selection, primarily for growth, occurred in 2004, and this year, 2005, we evaluated our population for resistance to bacterial coldwater disease. This is a central part of objective 1. We will meet with the US Trout Farmers Association this year (September 2005) to discuss germplasm release goals. This accomplishment is consistent with the NP 106 priority of genetic improvement. • 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. A tetraploid line would simplify production of 100% sterile fish and provide intellectual property protection for breeders. • 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. Identification of the relationship between traits of importance is a goal of our CRIS and meets the Aquaculture (NP 106) Action Plan’s second element regarding selective breeding. • The goal of evaluating our rainbow trout 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 genetic differences in growth performance across these environments. This work will establish whether there is a need for multiple selection programs or not. This is part of the 1st objective in our CRIS and is in accord with the NP 106 goal of characterizing germplasm. 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 transferred fish from families with known genetic background to other scientists to assist their studies and quantify genetic variation for particular traits (e.g. viral resistance). Through participation in producer group meetings such as US Trout Farmer’s and World Aquaculture Society meetings we are developing conduits to transfer our improved lines of rainbow trout to breeders and producers. 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). Silverstein, JT. 2005. Genetic Improvement in Aquaculture. Global Aquaculture Advocate. Feb.2005:50-53. Presentations Danley M, Mazik PM and Silverstein JT. 2005. Effects of feed frequency on the metabolic responses of hypercapnic rainbow trout. Aquaculture America, 2005, New Orleans Danley M, Mazik PM and Silverstein JT. 2004. Effects of feed frequency on the metabolic responses of hypercapnic rainbow trout. American Fisheries Society Conference, Madison WI. Hershberger, W.K., and M.A. Hostuttler. 2005. Development of tetraploid rainbow trout strains for production of improved triploids. Aquaculture America Conference, New Orleans, LA. Lankford, S.E. and Weber, G.M. 2005. Intraovarian regulators of folliculogenesis in rainbow trout: mRNA expression of transforming growth factor-¿ superfamily members. 15th International Congress of Comparative Endocrinology, Boston, MA. Abs 6.4. Leder EH and Silverstein JT. 2005. The pro-opiomelanocortin genes in the rainbow trout (Oncorhynchus mykiss). Society for Integrative and Comparative Biology Annual Meeting, San Diego, CA. Leder, EH and JT Silverstein. 2005. Expression of neuropeptides involved in energy homeostasis in developing rainbow trout. 15th International Congress of Comparative Endocrinology, Boston, Massachusetts. Leder, EH and JT Silverstein. 2005. The proopiomelanocortin genes in rainbow trout (Oncorhynchus mykiss): Multiple genes and differential expression. 15th International Congress of Comparative Endocrinology, Boston, Massachusetts. S.G. Lynn, J.A. Malison, B.S. Shepherd and W.J. Birdge. “Cloning, tissue-specific expression and responses to 17¿-estradiol of key endocrine genes in yellow perch, Perca flavescens. An oral presentation to be given at the 26th Annual Meeting of the Society of Environmental Toxicology and Chemistry (SETAC), Baltimore, MD, 13-17 November, 2005. B.S. Shepherd et al. “Effects of gradual salinity acclimation on the somatotropic axis in rainbow trout (Oncorhynchus mykiss)”. Given at the Finfish Physiology session at the World Aquaculture Society, Aquaculture America, New Orleans, January 17-20, 2005. Brian S. Shepherd (2004) “Approaches to Growth Enhancement in Salmonids: Impacts and Prospects”. An invited Departmental seminar given at the Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT, December 16th, 2004. Silverstein JT, Weber GM, Leder E and Blemings KP. 2005. Energy balance and physiological response to fasting and refeeding in rainbow trout fingerlings. Aquaculture America, 2005, New Orleans. Silverstein JT, Parsons J, Rexroad CE III, Palti Y. 2005 Heterosis and genetic distance in strain crosses of rainbow trout. Aquaculture America, 2005, New Orleans. Weber, G.M., Silverstein, J.T., Lankford, S.E., Gahr, S.A., and Blemings, K.P. 2005. Growth axis and target tissue response to fasting and refeeding in rainbow trout, Oncorhynchus mykiss, fingerlings. World Aquaculture Society Symposium, Aquaculture America 05, New Orleans, LA. Abs 481. Silverstein JT, Hostuttler M and Blemings KP. 2005. Strain differences in feed efficiency measured as residual feed intake in individually reared rainbow trout, Oncorhynchus mykiss (Walbaum). Aquaculture Research, 36: 704-711. The following is a Non-ARS Scientific Publication that was value to the project. Davis, KB, Simco, B, and Silverstein, JT. 2005. Relationship of gonadal development to body size and plasma sex hormone concentrations in female channel catfish. North American Journal of Aquaculture 67: 259-264.