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United States Department of Agriculture

Agricultural Research Service

Research Project: Combating Viral Hemorrhagic Septicemia and Improving Yellow Perch Aquaculture for the Great Lakes Region
2011 Annual Report

1a.Objectives (from AD-416)
The main problems in yellow perch culture are the lack of genetically defined broodstock with enhanced traits for year-round production, poor larval survival, slow growth, and disease susceptibility. These problems are being pursued via a long-term genetic selection program to produce superior germplasm and complementary studies to understand the physiological basis for performance traits of interest. This project aims to integrate genetic, molecular, physiological, and nutritional approaches to develop superior pathogen-free broodstocks and improved production methods for commercial industry. We will focus on the following objectives: Objective 1: Develop yellow perch broodstock, define growth and viral hemorrhagic septicemia (VHS)-resistant phenotypes, characterize genetic diversity, and evaluate genotype x environment interaction for growth. Objective 2: Characterize critical pathways involved in growth and VHS 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.

1b.Approach (from AD-416)
For objective 1, we will characterize important phenotypes and genotypes in yellow perch broodstocks. Third-generation progeny will undergo performance testing for improved growth and survival and decreased susceptibility to the viral hemorrhagic septicemia (VHS) virus. This will involve evaluation of genotype x environment interactions in laboratory and industrial settings. This information will provide estimates for the heritability of desired traits (growth and VHS resistance) and a better understanding of the sources of phenotypic variation for these traits. For objective 2, we will generate genomic resources to aid in development of molecular tools to genotype and quantify expression of genes involved with growth and immunity in yellow perch. We will also develop proteomic tools that will enable us to measure and characterize the function of critical proteins (hormones and immune markers) important to growth and immunity for this species. Genomic tools will come from next-generation sequencing efforts to characterize the transcriptomes (expressed genes) of key tissues involved with growth and immunity. Proteomic tools will be developed to characterize biochemical pathways that underlie growth and immunity in yellow perch. Lastly, in vitro methods will be used to characterize how viral proteins impact cellular antiviral recognition and response pathways that impact how yellow perch combat VHS infection, and how the virus might evade or suppress immune pathways in this species. For objective 3, we will evaluate and test use of larval specialty micro-diets (SMD) as substitutes for live-prey diets to improve larval survival and standardize and reduce overall costs of producing high-quality yellow perch fingerlings. We will evaluate performance measures (first-feeding, swim bladder inflation, development, survival and growth) of genetically defined larval perch broodstock progeny that are reared under either a control live-diet regimen (typical for industry) or a dietary regimen where live prey is progressively substituted with SMD. For objective 4, we will utilize a standardized VHS challenge model to characterize the disease process and susceptibility of perch broodstocks to VHS infection. We will also develop new diagnostic tools to detect VHS and use these detection tools to evaluate how vaccines and vaccination strategies increase protective immunity against VHS infection. For the challenge assay, genetically defined perch will be exposed to varying doses of VHS virus, and survivors will be re-infected with VHS to characterize resistance and protective immunity to this pathogen. To detect this pathogen, we will develop and validate a novel polymerase chain reaction (PCR) assay that reliably speeds up VHS detection in a cost-effective manner. For vaccination and vaccination strategies, we will characterize protective immunity in perch, evaluating the efficacy and duration of existing and new vaccines for VHS and how new and existing adjuvants extend the efficacy of these vaccines.

3.Progress Report
Under objective 1, we continued to improve genetic resources for yellow perch by generating the third generation (F3) of genetically-improved broodstocks. In addition to on-site performance trials to evaluate growth of F3 progeny, an additional 75,000 F3 progeny, from two broodstock strains, were shipped to two industry partners who are currently evaluating growth and performance under differing culture environments. Additionally, F3 progeny have been sent to cooperators to perform cross x strain tests for susceptibility to the viral hemorrhagic septicemia virus (VHSv). Results are expected to impact selection criteria to achieve further genetic gain in our yellow perch broodstocks. Under objective 2, we continued to characterize critical pathways that underlie growth and susceptibility to the VHS pathogen. Using current genomic techniques, we generated over 3 million unique sequences from tissues of yellow perch that were reared under varying growth conditions and from tissues exposed to pathogen mimetics. Analysis of this sequence information resulted in at least 20,000 redundant annotated gene sequences that will enhance ongoing research to characterize the physiological and genetic basis for traits of interest (growth and disease susceptibility) and improve methods for genotyping yellow perch broodstocks. Under objective 3, we continued to develop and test methods that improve early survival and feed training of yellow perch fingerlings. Improvements to early feeding and rearing (on-site) have resulted in higher egg survival from each egg strand, which means that fewer broodstock animals have to be used to produce more progeny, thus reproductive success has been improved. Under objective 4, a standardized disease challenge assay was developed to characterize the disease process of yellow perch exposed to the VHS virus. This assay was used to screen our broodstocks for phenotypes (families and strains) that show lower susceptibility to this pathogen. Tissues from these exposed fish are used to characterize genes involved with the immune response in yellow perch and complementary studies resulted in the establishment of experimental fish cell lines that are yielding insights into the viral recognition and response pathways in fish. Substantial progress was made in refining and validating the standardized reverse transcriptase polymerase chain reaction (StaRT-PCR) that is being developed as a rapid, cost-effective, method for detecting the VHS virus. The progress resulting from this research should impact efforts to select yellow perch broodstocks that display high growth and low susceptibility to the VHS pathogen, enable the design of therapies that interfere with the VHSv life-cycle, facilitate the development and testing of vaccines that minimize the effects of the VHS pathogen, and enable cost-effective surveillance and detection of the VHS pathogen in agricultural and fisheries production settings.

1. Reproductive efficiency increased in domestic yellow perch broodstock. As part of an ongoing research effort to increase yellow perch fingerling production cost-effectively, three early life stage husbandry trials have been conducted by ARS researchers at Milwaukee, Wisconsin, from July 2010 through March 2011. The primary research objective was to adjust the density variable relative to eggs, sac-fry, larvae and post-larvae in flow-through systems. The first baseline husbandry trial included starting with a high density of egg ribbons (15) for each tank of 2 replicate tanks. The diets for first-feeding sac-fry through the fingerling stage were standardized for all three trials. Fingerling production for each of the replicate trials was significantly low; approximately 4800 and 4200 fish per tank for the trial period of June 25, 2010 through August 23, 2010. The second trial was conducted with a reduced number of egg ribbons for each tank of two replicate tanks. Fingerling production increased significantly to approximately 30,000 fish per tank for the trial period of October 7, 2010 through December 3, 2010. The third trial was conducted using the same protocol using 9 egg ribbons for each tank of two replicate tanks. Fingerling production increased slightly to 35,000 fish per tank for the trial period of December 28, 2010 through March 4, 2011. Based on the results from these three trials, the goal of increasing yellow perch fingerling production as a direct function of density has been accomplished and reproductive efficiency has been improved in our broodstocks. These results will also be beneficial to the yellow perch aquaculture industry.

2. Third generation of genetically-improved yellow perch has been developed. In Spring 2010, select performers of the second generation for each of the 4 strains of perch (Perquimans, Choptank, Winnebago, Green Bay) were environmentally cycled for spawning in 2011. In Spring 2011, full and half-sib crosses were completed on the Perquimans, Choptank and Winnebago strains to produce third generation growth-selected progeny for each strain. To more fully evaluate the growth performance of these genetically-improved yellow perch strains, under varying environments, third generation fingerlings have been disseminated (in excess of 100,000 feed trained fingerlings) for on-site and off-site rearing trials. Selection for growth has decreased the time to market size (between the first and second generations) by 1.5 months and further gains are expected in our third generation. ARS researchers at Milwaukee, Wisconsin, have developed a standardized disease challenge assay that is being used to perform a cross strain comparison of survival to evaluate variation (strain x parental cross) for susceptibility of yellow perch broodstocks to the Midwest strain of the viral hemorrhagic septicemia virus (VHSv). This work has resulted in a better understanding of the disease process and immune pathways affected by VHS infection in genetically-defined yellow perch. Knowledge of the disease process, and affected immune pathways, is essential to identify ways to interfere with this pathogen via development of novel prevention and treatment strategies. These efforts continue to result in improvement of commercially-important (growth and disease susceptibility) traits that will increase productivity of yellow perch farming.

3. The number of genomic sequences for yellow perch has been increased. Very little is known of the genes and proteins (markers) that control growth in yellow perch. The lack of genetic and phenotypic markers is limiting the pace of genetic improvement for this species. To address this, ARS researchers at Milwaukee, Wisconsin, isolated ribonucleic acid (RNA) from the brain, pituitary, liver and muscle of yellow perch that were stimulated to grow faster using a novel growth promoter (estrogen). Normalized complementary deoxyribonucleic acid (cDNA) libraries were made from the RNA from each tissue, and then sequenced using a leading edge technology. Over 700,000 sequences were generated from each library (over 2.8 million), resulting in more than 20,000 assembled (unannotated) sequences per library. To identify (annotate) perch genes, these new sequences were systematically compared with known sequences of other species in public domain databases. This comparison identified yellow perch genes that are known to control feed intake, growth, metabolism, reproduction and immunity in other fish species. This new sequence information has increased the overall representation of genomic sequences from functionally important tissues of yellow perch and is being used to develop new markers that will increase the pace of genetic improvement in yellow perch. Genetically-improved yellow perch broodstocks, that display commercially-important traits, will increase profitability for domestic aquaculture producers.

4. Antiviral pathway identified in fish cells. Various fish cell lines have been developed to characterize viral recognition and response pathways in fish. ARS collaborators have individually expressed the major proteins of the viral hemorrhagic septicemia virus (VHSv) in fish cells to investigate infectivity and virulence of these proteins at a cellular level. Two VHSv proteins were found to inhibit antiviral responses in fish cells, decreasing production of the antiviral molecule, interferon. Furthermore the research indicated that interferon already present in the cell can block VHSv from multiplying. Collectively, this work suggests that the VHS virus adversely affects the cellular response to interferon to allow virus replication. Knowledge of how particular VHSv proteins affect the viral recognition and response pathway in fish will enable development of a more targeted and effective vaccine for treatment of this pathogen in important aquaculture species.

5. Development of a rapid test for detecting the viral hemorrhagic septicemia virus (VHSv) in aquaculture species. Collaborators and ARS researchers at Milwaukee, Wisconsin, are utilizing and optimizing the standardized reverse transcriptase polymerase chain reaction (StaRT-PCR) test to quantify live, replicating VHSv. The StaRT-PCR test was checked against human viruses, other fish viruses and various strains of the VHSv. Experiments have verified the StaRT-PCR test to be specific to VHSv and very sensitive. The StaRT-PCR assay will accelerate the ability to analyze susceptibility of perch strains to VHSv, facilitate development and evaluation of vaccines for this pathogen and reduce VHSv surveillance costs for aquaculture producers.

Review Publications
Shepherd, B.S., Aluru, N., Vijayan, M. 2011. Acute handling disturbance modulates plasma insulin-like growth factor binding proteins in rainbow trout (Oncorhynchus mykiss). Domestic Animal Endocrinology. 40:129-138.

Lynn, S.G., Wallat, G.K., Malison, J.A., Shepherd, B.S. 2011. Developmental expression and estrogen responses of endocrine genes in juvenile yellow perch (Perca flavescens). General and Comparative Endocrinology. 171:151-159.

Last Modified: 4/23/2014
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