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1a.Objectives (from AD-416):
Current ARS, and ARS partners’, efforts are directed at advancing aquaculture in the Great Lakes region. The Great Lakes region is home to approximately 32% of the U.S. population but produces less than 3% of the total U.S. aquaculture production. The Great Lakes region consumes 1.5 billion pounds of seafood annually (valued at $23 billion, 2004 dollars). Yellow perch are a high-value food fish ($12.00-$20.00/lb retail) and current ARS, and ARS partners’, efforts are aimed at developing improved yellow perch broodstocks and rearing/production practices to advance aquaculture in this region. The emergence of the viral hemorrhagic septicemia virus (VHSv) in the Great Lakes region of North America has led to multi-species epidemics in wild populations. This disease poses a serious threat to aquaculture production and profitability in this region, and thus is the impetus for the scientific efforts of the ARS program. To address the needs of the Great Lakes region we shall undertake the following objectives:.
1)Continue cooperative efforts (maintain and breed) to evaluate, improve, and disseminate genetically defined F3 & F4 yellow perch broodstock and progeny;.
2)Continue research on the use of specialty micro-diets and live-diets for production of high-quality perch fingerlings and initiate evaluation of diets for efficient grow out of perch fingerlings;.
3)Initiate StaRT-PCR technology transfer and IP discussions with USDA and University of Toledo;.
4)Refine and simplify procedures for use of SMIS standards in the StaRT-PCR assay; and.
5)Evaluate pathogenicity and mutagenicity of major VHSv proteins using fish cell lines.

1b.Approach (from AD-416):
1) Continue cooperative efforts to genetically improve yellow perch broodstocks and perform limited germplasm releases with stakeholders (UWM-SFS). We will initiate discussions with the Ohio State University regarding scientific avenues for collaboration to evaluate genetically improved yellow perch. As part of a limited germplasm release using excess F3 broodstock and F4 progeny, we will work with stakeholders to perform on-farm selection. We will also produce and evaluate F4 Choptank and Perquimans broodstocks. For selected stakeholders we will complete 20-40 pair-wise crosses per participant from the F3 broodstock strains and transfer the fertilized F4 egg ribbons for performance evaluation and on-farm selection at different commercial sites. Excess F3 broodstock will be transferred to a single commercial site (Bell Aquaculture). For limited germplasm releases UWM will work cooperatively and efficiently with ARS to develop required agreements (MTRA or CRADA) to document technology transfer. 2) Investigate the use of specialty micro-diets (SMD) and live-diets for the production of high-quality perch fingerlings and initiate evaluation of diets for cost-effective grow out of perch fingerlings (UWM-SFS). We will use Otohime & Reed Mariculture SMDs to improve yellow perch larval and fingerling survival to identify a substitution to live diets, which will support commercial fingerling production. Controlled, replicate studies will use progeny from F3 broodstock. The first-feeding progeny from these broodstocks will be presented with the specialty micro-diets, with development-based adjustments over a period of 42 days. Production costs will be estimated to develop an informative model that will be evaluated at a commercial site. 3) StaRT-PCR Technology Transfer: (UT): Actively engage USDA agencies APHIS & ARS in technology transfer. University of Toledo (UT) should provide USDA with the StarRT-PCR IP under a research-only license that enables USDA agencies APHIS & ARS to use the StaRT-PCR assay and derivatives for research and evaluation purposes without conflict or licensing fees. Subject to negotiation of the actual terms any improvements to the technology made by USDA should be transferred to UT but be available at no cost to USDA for any purpose. All improvements by UT resulting from further funding by USDA will similarly be made available under the license. 4) VHSv Assay Refinement (UT): Optimize and simplify the use of SMIS in quantitative VHSv pathogen assays. Characterize optimal SMIS ratio ranges to enable better transferability of the StaRT-PCR technology to other labs within a 10^2 to 10^6 molecule range that correspond to the early stages of VHS infection. 5) VHSv Mutagenicity and Pathogenicity Evaluation (UT): Characterize how VHSv proteins and newly identified mutations affect virulence and pathogenicity in fish cell lines. We will use EPC cell lines that have been transiently transfected with VHSv proteins and mutations to evaluate viral replication and the host viral detection and response pathways.

3.Progress Report:

This project is related to four objectives of the parent project. Objective 1: Develop yellow perch brood-stock, define growth and VHS resistant phenotypes, characterize genetic diversity, and evaluate genotype x environment interaction for growth. Objective 2: Characterize critical pathways involved in growth and VHSv resistance in yellow perch through gene expression and physiological studies. Objective 3: Improve early survival and methods for producing feed trained fingerlings. Objective 4: Develop and evaluate challenge assays, detection tools and vaccines for protecting yellow perch and other Great Lakes region species of fish from VHS. New avenues of research have been pursued with regard to urban aquaculture and aquaponics. An extensive cost analysis of commercial-scale yellow perch production utilizing recirculating aquaculture systems (RAS) and aquaponic systems is being conducted. All aspects of production, including labor, fish feed, and energy costs, are being collected and catalogued, along with cost information on the design, material selection, and construction of these systems. This economic information will be used to develop economic models for the setup and operation of RAS and aquaponic systems. To determine the optimum fish stocking density to vegetable plant ratio in aquaponic systems, five aquaponic systems are being operated with initial stocking densities between 1000 to 2500 fingerlings to measure the production as a function of density and water quality. Initial results have shown that yellow perch in aquaponic systems may be able to tolerate elevated levels of total ammonia nitrogen and nitrite from what was originally described. Monthly sampling efforts for growth, food conversion, and condition factors are being calculated to track the progress of this investigation. Under a joint cooperative agreement, on-the-farm research is being conducted to compare the performance of yellow perch fed fishmeal-based diets versus fishmeal-free diets. The yellow perch fingerlings for this project were obtained by using domesticated broodstocks where eggs were artificially fertilized, incubated, hatched and raised to fingerling size. The fingerlings were subsequently feed-trained on the test diets used in this study for approximately 50 days, at which time the fingerlings were transferred to the cooperating farm for evaluation. Under a joint cooperative agreement, 24 fertilized egg ribbons from genetically improved broodstock were provided to a cooperator to evaluate a new incubation system at their commercial facility. In addition to testing the new incubation system, performance between these genetically improved sac fry was compared to non-genetically improved sac fry. Initial results from this study show positive trends in the areas of increased hatching success, sac fry survival feeding and growth. Studies using fish cell lines that stably express major viral hemorrhagic septicemia virus (VHSv) type IVb proteins have shown that the M and P genes negatively influence fish cell function, allowing the VHSv pathogen to survive and replicate within the host cell. By contrast, expression of another VHSv gene (Nv) was found to stimulate the host (cell) innate immune response. Work on the M protein is wrapping up while Nv studies will be expanded in the coming year. The standardized reverse transcriptase polymerase chain reaction (StaRT-PCR) method for detecting the VHSv pathogen in aquaculture species was developed, perfected, and modified into a real-time PCR platform. The new assay was evaluated with challenged fish, including the yellow perch Choptank strain, juvenile muskellunge, and wild caught fishes.