Location: Dairy Forage Research2010 Annual Report
1a. Objectives (from AD-416)
The Great Lakes region is home to approximately 32% of the U.S. population which consumes more than 1.5 billion pounds (valued at $23 billion, 2004 dollars) of seafood products annually. Additionally, the Great Lakes sports and commercial fisheries are estimated to generate $4.5-$5.0 billion and $1 billion per year, respectively. To maintain these fisheries, as well as provide access to fresh seafood (finfish) products in this region, stocking efforts rely heavily upon regional aquaculture producers, as well as producers who service the fresh seafood (finfish) markets. Yellow perch are a high-value food fish ($10.00-$15.00/lb retail) and current ARS (and ARS partner) efforts are aimed at developing improved yellow perch germplasm and rearing/production practices to advance aquaculture in this region. The recent emergence of viral hemorrhagic septicemia virus (VHSv) in the Great Lakes region of North America has led to dramatic multi-species epidemics (affecting 28 Great Lakes fish species). This disease poses a serious threat to yellow perch (aquaculture) production and profitability in this region and, thus, scientific efforts of this ARS program. Consequently, additional work is needed to ensure the cost-effective production of pathogen-free stocks of cultured yellow perch. To address these needs, we shall undertake the following objectives: 1) Evaluate methods to improve yellow perch fingerling quality; 2) Continue to use molecular genetic tools and genetic selection to identify sources of variation growth in our yellow perch broostocks; 3) Quantitatively determine breeding values and heritability estimates for growth in our broodstock strains; 4) Characterize the innate immune response and early pathogen recognition to viral mimetics and VHSv in vitro and in vivo; 5) Use a recently developed disease challenge model for VHSv infection to identify sources of variation in VHSv susceptibility in our yellow perch broodstock; and 6) Utilize the disease challenge model to optimize and validate and a rapid (cost-effective) method (StaRT-PCR) for detection of VHSv in yellow perch.
1b. Approach (from AD-416)
The aquaculture “tool box” for yellow perch early life stage production has been established; however, several parameters need additional research to advance the technology & make it more cost-effective. The aim of this work is to test commercially available micro-diets as substitutes for the food constituents presently used, which include Green Tank Water (GTW), Brine Shrimp Nauplii (BSN), & Beef Liver and Heart mixture (BLHM). The goal of the proposed research is to reduce the cost of the first-feeding technology & improve survival of yellow perch. Aim 1: Improve survival & quality of first feeding larvae by reducing dependence upon “Green Tank Water”. Introduce fish starter diets within the first-feeding window (1-2 days post hatch). The aim is to decrease dependence on the use of processed mammalian tissue diets, such as beef liver & heart. Diets will be progressively replaced with available commercial larval starter diets. Aim 2: Evaluate performance measures of genetically defined broodstock progeny (first-feeding, swim bladder inflation, development, survival & growth & condition) that are reared under either a control dietary regimen or substituted dietary regimens. Aim 3. Continue development of genetically defined perch broodstock: The top 25-35% performing fish/strains (from the performance trial performed in 2007) have been separated, pit-tagged, & genotyped with appropriate microsatellites for a particular strain. These fish have been held (2008-2009) under photoperiod & temperature cycling to mimic their geographic origin & have begun to spawn (February-March 2009). One strain has already spawned & parental crosses performed in February 2009. These new fish will undergo another 12-month performance trial & will be sampled for tissues for deep sequencing and microarray analyses to identify biochemical pathways associated with fast growth. Aim 4. Expressed sequence tag and microsatellite development: The new larval perch library has been developed & test-sequenced. This past year the techniques were worked out to produce primary cell culture of granulocytes (presumably macrophages) from the perch head kidney, & a cDNA library has been made from plated perch macrophages stimulated with LPS. EST clones from this library will be sequenced for immune gene discovery needed to characterize the innate immune system in yellow perch. We will continue to mine all of the ESTs from all libraries for microsatellites & for SNPs which will add genotyping capabilities. For microsatellites, we will test new ones to determine their appropriateness for use in pedigree tracking. Overall, greater than 25,000 ESTs clones have been sequenced & annotated, & this work will continue. Additional aims of this SCA include: Aim 5: Use genomic and bioinformatic techniques to identify & develop molecular markers (genes of interest) for immune response to viral hemorrhagic septicemia virus challenge in yellow perch. Materials for genomic studies will come from challenge work using GLWI/ARS genotyped yellow perch strains. Aim 6: Use of new real-time quantitative PCR technologies to develop a rapid method to detect VHSV in yellow perch.
3. Progress Report
This year, the cooperators expanded their scientific aims to include characterization of the disease process and susceptibility to viral hemorrhagic septicemia (VHS) virus in yellow perch broodstocks. Overall, project emphasis is on broodstock development to improve survival, growth, and disease resistance to VHS (and VHS detection) in yellow perch. For broodstock development, second-generation progeny (F2) have been genotyped and passive integrated transponder (PIT)-tagged, and are undergoing sexual maturation for 1 year (March 2010-March 2011), using photo-thermal manipulation, with the aim to produce the third-generation (F3) progeny in April 2011. To increase our ability to pedigree (genotype) desirable broodstock phenotypes (those with high growth and low VHS susceptibility), additional effort is underway to identify and utilize single nucleotide polymorphisms (SNPs) for genotyping yellow perch. SNPs will be identified from ongoing DNA sequencing projects from whole perch larvae and from high complexity tissue libraries. These efforts have initially resulted in the characterization of approximately 9,000 new unigenes from yellow perch immune cells. In collaboration with researchers at the U.S. Geological Survey, Western Fisheries Research Center, Seattle, WA and at the University of Toledo, a standardized VHS disease-challenge model has been developed to evaluate disease susceptibility and characterize the disease process in yellow perch broodstock exposed to the Midwest strain of VHS. Disease-challenged animals that survived exposure to VHS are being genotyped to identify parental crosses that contribute to improved survival. Tissues from animals exposed to the standardized VHS challenge assay are also being used to optimize and validate a rapid, cost-effective, high-sensitivity assay that detects this pathogen in yellow perch. Additional efforts have resulted in the development of husbandry methods that improve larval survival and reduce reliance upon live diet sources, resulting in substantial progress toward achieving high-quality yellow perch fingerlings. Activities to monitor the cooperators of this agreement included e-mail correspondence, on-site meetings, and quarterly reports. ARS personnel and the cooperators also met at the 2010 World Aquaculture Society Meeting in March 2010 and at the North Central Regional Aquaculture Center (USDA) annual meeting in February 2010.