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? This project is part of NP106 Aquaculture, and contributes to the action plan through the development of brood stock/genetic improvement programs for carnivorous fish (trout). With the world population steadily increasing, agricultural research is playing a progressively more critical role in meeting the rising demand for food and at the same time attempting to improve the nutritional value of that food. As the world's need for renewable sources of protein becomes more important, with declining space and reduced wild animal populations, it will be necessary to raise agricultural livestock in limited space, on readily renewable and relatively inexpensive food sources, and in an ecologically sound manner. If commercial aquaculture producers fail to develop alternative food sources they will be unable to meet future consumer demands and their products will most likely be relegated to a secondary food source and hence, unfeasible as a protein resource for most of the world's economically underprivileged population. This lack of commercial development could also lead to the loss of billions of dollars in lost revenue, employment opportunities, and exportation products. In addition to presenting the world with a renewable and healthy food source, the development of fish feeds containing cereal grains will provide a sustainable market for cereal grain farmers. Barley and oats represent important commodities for feed, food and malt use in the United States. The continued viability of U.S. industries that depend on these crops will depend on continuing improvements in the efficiency with which they can be grown and used. This goal of this project is to change the type of ingredients used in aquaculture diets from marine source based to sustainable plant sources (particularly small grains). Requirements for plant-derived aquatic feed ingredients are high protein, favorable amino acid levels, decreased phytate levels, increased energy from oils and carbohydrates, presence of omega-3 fatty acids and the improvement of palatability by lowering fiber and antinutritional components. Grain and grain by-products have been components of prepared feeds for fish for over 70 years. Wheat middlings were one of the first feed ingredients used to extend meat mixtures used to rear trout, and remains a key component today. Corn is the main grain component in catfish feeds, primarily because of its geographical proximity to areas where catfish farming is located. Other small grains have been considered as components of aquaculture feeds, but concerns about crude fiber and phytate levels in these grains have limited their use. Recently, genetically enhanced cultivars of barley containing lower levels of phytic acid have been evaluated as components of fish feeds, and found to be superior to normal cultivars in nutrient bioavailability. Development of hulless cultivars of barley and oats further open the prospect of higher utilization of these grains in fish feeds. Historically cultivars of barley and oats were chosen for cultivation on the basis of their agronomic performance and value for livestock feed or as components of human foods. However, there are other germplasm sources available that possess characteristics of interest with respect to fish production. In order to capitalize on the benefits of these genetically diverse cultivars they must first be evaluated for their use in fish feeds. Researchers at the National Small Grains Germplasm Research Facility are currently screening their vast repository of genetically diverse small grain cultivars for strains that possess traits that might accentuate their performance in formulated fish diets. Not only is it necessary to change the diets of commercially produced fish, but it is also necessary to change the fish such that they can efficiently utilize these new plant-based feeds. During the history of aquaculture there has been limited genetic improvement in aquaculture stock. In order for aquaculture to expand in the manner necessary to meet future consumer demands we must follow what has been done historically with other domesticated agricultural livestock. Two of the main elements necessary to increase availability of animal products are the improvement in animal health and nutrition and the means to produce it in a cost efficient manner. In the advancement of all domesticated stock, genetics has played a major role in meeting the two prior mentioned demands. Genetically enhanced stocks are more resistant to disease, reach market size more rapidly, and demonstrate an improved feed conversion ratio. For aquaculture to continue its growth, genetically enhanced domesticated stocks of fish will need to be generated that are specific for the consumption of the most cost efficient and readily available diet. A successful conclusion to this research will enable the total replacement of fishmeal in trout feeds with sustainable plant-derived ingredients, while maintaining optimal production efficiencies 2.List the milestones (indicators of progress) from your Project Plan. Milestone 1 (16 months) - Genetic analysis on the different strains of fish. Milestone 2 (30 months)- Molecular markers for traits linked to growth on cereal grain diets. Formulated cereal grain diets. Milestone 3 (60 months)- Testing of progeny generated from marker selected broodstock. 4a.What was the single most significant accomplishment this past year? The differential expression of several genes relating to trout growth, health and metabolism were assessed and determined for strains of rainbow trout reared on formulated cereal grain and commercial feeds. This research is necessary to understand the genetic components in trout involved in diet utilization and therefore allow the US aquaculture industry to remain competitive with global competition in the face of higher feed costs due to limited natural and protein resources and stricter environmental standards. To offset the economical burden of sustainable aquaculture, improved stocks of fish need to be generated that are more efficient in growing on alternative feed sources. Rainbow trout of distinct lineage were separated and reared on either a commercial diet or a diet containing a high level of barley (32%). After one year on the diet, tissue samples were taken and the changes in gene expression were evaluated in these fish through analysis using quantitative real-time PCR for several genes and microarray hybridization. The findings from this work will now allow researchers more efficient means to evaluate selected broodstock for performance improvement and aid in the evaluation of experimental formulated diets. 4b.List other significant accomplishments, if any. A patent was issued for the detection of Bacterial Kidney Disease. This test will be of valuable service in allowing researchers to evaluate the health of salmonids upon administration of a known dosage of a pathogenic bacterium. For this research a quantitative real-time PCR assay was devised to detect the presence of the bacterial genome and quantify its absolute copy number in infected tissue. With this test there now exists a method to assay dietary effect on fish health status by administration and monitoring of precise pathogen levels. A real-time PCR assay was developed by Dr. Ken Overturf to detect the presence of the bacterial genome responsible for Bacterial Kidney Disease and quantify its absolute copy number in infected tissue. This assay will allow researchers and clinicians to determine the effect of diet on fish health status by administration and monitoring of precise pathogen levels. A patent has been issued for this assay and licensees are being sought. 4c.List any significant activities that support special target populations. None. 4d.Progress report. None 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. This project was started as a joint collaboration between the Small Grains and Potato Germplasm Facility in Aberdeen, Idaho and the Hagerman Fish Culture Experiment Station in Hagerman, Idaho. With this being a new and unique area of research within the Agricultural Research Service, there has been rapid progress in this novel area of looking to enhance cereal grains for use in formulated fish diets and in improving trout strains for growth and the utilization of these new diets. During the span of this project we plan to develop cereal grains with accentuated positive traits for incorporation into fish feeds. Traits such as reduced phytic acid will aid in reducing phosphorus excretion into the environment, while increasing oil and protein levels and inserting them into cultivars possessing strong agronomic traits will aid in the production and marketing of a finished product. We also plan to have delineated a number of molecular markers that are closely linked to genes that allow rainbow trout to grow well and efficiently utilize a fish diet containing cereal grains in place of a large portion of the typically used fish meal and fish oil. Development of enhanced stocks of cereal grains for utilization in fish foods, formulated cereal grain fish diets, and improved rainbow trout stocks should have a major effect in the future production of food finfish in regards to volume and price of fish produced and its impact on the environment. 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? 5366-21310-003-00D replacing 5366-21310-002-00D per OSQR review. A patent was issued for the Detection of Bacterial Kidney Disease for the detection and quantification of this pathogen in salmonids using probes and primers designed by the projects scientist. Another test devised by this scientist is also being submitted for a patent. No known constraints. The Idaho Research Council is now looking for interested parties to market this project. 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). Overturf, K.E., Hardy, R.W. (2003) Trout selection on alternative protein diets. International Genetics for Applied Aquaculture. Puerto Varas, Chile Overturf, K. (2003) Genetic selection and molecular analysis of rainbow trout for enhanced growth and utilization of alternative diet sources. International Association for Genetics in Aquaculture. Puerto Varas, Chile Overturf, K., Casten, M. and Hardy, R. (2004) Two generations of rainbow trout selected for growth on diets utilizing an alternative protein source. World Aquaculture Society, Honolulu, Hawaii. Stone, D., Overturf, K. and Hardy, R. (2004) The use of exogenous dietary carbohydrase enzymes to improve digestibility and availability of nutrients in low phytate barley in rainbow trout. World Aquaculture Society, Honolulu, Hawaii. Johansen, K., and K. Overturf. Re-feeding after 30 day starvation affects the expression level of the muscle-specific genes myogenin, MEF2C and MEF2A in rainbow trout. World Aquaculture Society, Honolulu, Hawaii. Overturf, K., Bullock, D., LaPatra, S. and Hardy, R. (2004) Genetic selection and molecular analysis of domesticated rainbow trout for enhanced growth on alternative diet sources. Book Chapter, Genetics of Subpolar Fish and Invertebrates, Kluwer Academic Publishers, Norwell, MA. Review Publications Oveturf, K. and Johansen, K. (2005) Sequence, conservation, and cloning of rainbow trout Myf5. (2005) Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology 140: 533-541.