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? Rainbow trout is an important aquaculture fish species both for its commercial value as a food fish As well as for its recreational value to fishermen in the northern United States and Canada. A major handicap of large-scale culturing of this fish species is the disease outbreak resulting from infection by bacterial, fungal, parasitic and viral pathogens. Development of strains of rainbow trout resistant to pathogen infection via genetic manipulation will be the solution to this problem. To resolve this problem we have initiated work to produce new rainbow trout strains with increased resistance to infection by bacteria, viruses, fungi and parasites by genetic engineering. Furthermore, we have also conducted studies to characterize genes that are responsive to infection by pathogens in rainbow trout in order to develop alternative strategies of disease protection in rainbow trout. 2.List the milestones (indicators of progress) from your Project Plan. The overall goals for the entire project are: (1) Develop rainbow trout brood stocks with disease resistant genetic trait for aquaculture. (2) Identify and characterize the genes responsive to induction by cecropin B in rainbow trout macrophage cells. (3) Develop rainbow trout brood stock with enhanced muscle growth for aquaculture by down-regulating MSTN gene. Milestones for FY2005: I. Conduct challenge studies of F2 Rbt99 fish with a viral pathogen, IHNV. II. Characterize F2 transgenic fish with respect to transgene inheritance and transgene expression. III. Continue to breed homozygous fish from F2 Rbt-99 transgenic fish to homozygous. IV. Continue screening genes in trout macrophage cells that are regulated by antimicrobial peptides. V. Produce RNAi gene constructs and test the feasibility of these constructs in down regulating MSTN-1 gene in a rainbow trout cell line in vitro. Milestones for FY2006: I. Conduct challenge studies of F3 Rbt99 and F2 Rbt-02 fish against A. salmonicida and IHNV. II. Initiate breeding of Rbt-99 transgenic fish to homozygous by androgenesis. III. Microarray screening of genes that are regulated by antimicrobial peptide. IV. Complete characterization of cecropin resistant bacterial pathogens for infectivity to hosts. V. Cryopreserve sperm of F2 Rbr02 fish. VI. Identify strong muscle-specific promoters from rainbow trout. VII. Investigate whether antimicrobial peptide can serve as an adjuvant in vaccination. Milestones for FY2007: I. F4 Rbt99 challenge studies. II. Breed Rbt02 to homozygous. III. Cryopreserve F3 Rbt99 sperm and F2Rbt-02 sperm. IV. Genotyping homozygous Rbt99 fish. V. Constructing and testing RNAi for MSTN VI. Breeding homozygous Rbt02 fish VII. Confirm microarray analysis results by real-time PCR VIII. Publish results of research with Rbt99 transgenic fish. Milestones for FY2008 I. Conduct scale-up challenge studies with homozygous Rbt99 fish. II. Conduct F3 Rbt02 challenge studies. III. Cryopreservation of F3 Rbt02 sperm. IV. Genotyping homozygous Rbt02 fish. V. Produce P1 transgenic fish carrying RNAi for MSTN. Milestones for FY2009: I. Genotyping P1 transgenic fish carrying RNAi for MSTN. II. Conduct scale-up challenge studies with homozygous Rbt02 fish III. Cryopreserve sperm samples of homozygous Rbt99 and Rbt02 fish IV. Comparing the expression patterns of immune responsive genes in transgenic fish and non-transgenic fish. Milestones for FY2010: I. Establish F1 family of transgenic fish carrying RNAi of MSTN. II. Re-confirm the results of scale-up challenge studies with newly established animals by crossing homozayours transgenic fish (i.e., Rbt99 and Rbt02) with non-transgenic fish. III. Prepare manuscripts reporting results of challenge studies with Rbt02 fish; and the expression patterns of immune responsive genes in transgenic fish and non-transgenic fish. 4a.What was the single most significant accomplishment this past year? We demonstrated conclusively that cecropin B or CF-17 peptide can induce inflammatory responses in macrophages. The induction of the inflammatory response does not further block the effect of LPS induction of inflammatory response in macrophage cells. These results suggest that cecropin or CF-17 could serve as an adjuvant in fish vaccination. 4b.List other significant accomplishments, if any. 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 transgenic 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 sperm from 21 families of Rbt09 transgenic 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. 4c.List any 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 the University of Connecticut. Additional details of research can be found in the report for the parent CRIS 1930-31000-007-00D Utilizing Genetics for Enhancing Cool and Cold Water Aquaculture Production. 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. (1) We have discovered that transgenic model fish (medaka) carrying cecropin transgene are resistant to infection by bacterial pathogens. (2) Cecropin and the synthetic analog (CF-17) can inhibit the propergation of IHNV and other important fish virus, suggesting that CF-17 may be an excellent candidate for controlling viral diseases in fish. (3) We have obtained evidence to show that F2 transgenic fish carrying cecropin B transgene exhibited resistant to bacterial and viral pathogens. (4) Although prolonged exposure of Gram-negative fish pathogens to cecropin may result in selection of resistant cells via physiological adaptation, yet these cells possess no infectivity to fish hosts. These results suggest that cecropin is far safer than any commercially available antibiotics. (5) We have provided conclusive evidence that cecropin B or CF-17 possessed pro-inflammatory effect to macrophage, suggesting that cecropin or CF-17 has direct effect to the immune system of the fish. Furthermore, it also suggests that these peptides can serve as an adjuvant in vaccination. 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? None 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). None