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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
2013 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:
A third generation (F3) of yellow perch broodstock were selected from a performance trial and cycled for reproduction this year. Using a newly developed method for external gender identification in yellow perch, we developed back-up F3 broodstocks using a bimodal selection regime that enabled us to select the best performing male and female animals of each stock, which should increase selection pressure for this trait. Progeny from pair-wise crosses of improved and unimproved domesticated strains were produced and distributed to commercial participants as part of on-going genotype x environment studies to characterize performance and enhance methods for sustainable yellow perch production. For one study, we are evaluating performance of fingerlings fed fishmeal-based diets versus fishmeal-free diets in a novel production system. The yellow perch for this project were produced, raised to fingerling size, and subsequently feed-trained on the test diets for approximately 50 days at which time the fingerlings were transferred to the cooperating farm for evaluation. In a second study, 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 larvae was compared to non-genetically improved larvae. Initial results from this study show positive trends for the genetically improved larvae in the areas of increased hatching success, larvae survival, feeding, and growth. We have also completed another nutritional study aimed at evaluating the mineral supplementation required to sustain efficient growth using diets formulated with plant-based proteins. Three isocaloric fishmeal-free diets (all plant-based protein sources) with similar macronutrient content (40% crude protein/10% fat) were developed, but differing in how much mineral mix was added to the diets. Growth performance and feed acceptance was evaluated over a 16-week period. Fish fed a commercial fishmeal-based reference diet showed the highest feed intake and growth rates. Within the plant-based diets tested, fish fed the intermediate level of mineral supplementation showed the highest feed intake and growth. However, the overall low feed intake in the plant-based diets suggests these diets were less palatable than the reference diet. The standardized reverse transcriptase polymerase chain reaction (StaRT-PCR), which is a newly developed test for detecting the viral hemorrhagic septicemia virus (VHSv) in yellow perch, has been adapted to a more rapid and cost-effective format using real-time PCR technology. Like the StaRT-PCR assay, this new testing format shows high specificity to the VHSv pathogen, also displays a high dynamic range in quantifying virus levels and has significantly lower false positive and false negative events than other detection methods.


4.Accomplishments
1. Mineral requirements are determined for yellow perch consuming plant protein-based feeds. Little is known of the nutritional requirements for growth of yellow perch in aquaculture systems; consequently, commercial feed formulations are based on nutritional requirements for rainbow trout, and these diets contain large quantities of fishmeal and oil that are too costly for other species. Plant proteins and oils are less expensive and more sustainable than fishmeal, but inclusion at high rates can reduce palatability, nutrient availability, disease resistance, and fish performance. ARS researchers at Milwaukee, Wisconsin conducted a nutritional study to determine the practical micronutrient (mineral) requirements for efficient growth in yellow perch consuming sustainable plant protein sources. Using a fishmeal-free diet provided by collaborators, three diets were formulated with similar macronutrient content (40% crude protein/10% fat), but differing in how much mineral mix was added to the diets. Fish fed the mid-level amount of mineral showed the highest feed intake and growth, suggesting that this mineral supplementation level is adequate for use with plant-based proteins. Researchers also compared fish fed the plant protein-based diet to fish fed a commercial fishmeal-based diet with a similar macronutrient profile. Although both groups of fish showed gains in body weight and body length, yellow perch fed the commercial fishmeal-based diet encountered fewer deaths and disease, ate more of the feed, and were able to convert more of the feed into body mass. This study confirms the potential of developing diets for yellow perch with practical mineral mixes, and the partial substitution of fishmeal for plant-based proteins. Use of properly formulated aquafeeds that utilize plant-based proteins will increase the sustainability and profitability of the aquaculture industry.

2. Rapid test for detecting the viral hemorrhagic septicemia virus (VHSv) in yellow perch has been completed and published. VHSv is one of the world’s most challenging finfish diseases. A new and especially virulent strain (IVb) emerged in the North American Great Lakes in 2003, threatening fisheries, baitfish, and aquaculture industries. Previous diagnostic tests for VHSv have been inaccurate, time-consuming and costly. Working with collaborators, ARS scientists at Milwaukee, Wisconsin have developed a standardized reverse transcriptase polymerase chain reaction (StaRT-PCR) test that is specific to the VHSv pathogen, is rapid, and displays a higher range of accuracy and detection than existing tests. In 2013, the StaRT-PCR test was converted to an easier-to-use real-time polymerase chain reaction platform using a two-color detection system. This new assay format will enable standard veterinary diagnostic labs to readily adapt this technology, and it will improve accuracy and reduce the time needed to reliably and cost-effectively detect the VHSv pathogen in economically and ecologically important finfish species. The assay is currently under regulatory review before it can be classified as an accepted method internationally.

3. Specific genes of the viral hemorrhagic septicemia virus (VHSv) differentially affect the fish immune system. VHSv is one of the world’s most challenging finfish diseases. A new and especially virulent strain (IVb) emerged in the North American Great Lakes in 2003, threatening fisheries, baitfish, and aquaculture industries. In an effort to understand how VHSv invades the host fish, collaborators previously found that two specific proteins of the VHSv pathogen negatively affect the immune response in fish, which enables the VHSv pathogen to infect and replicate within host fish cells. One of these proteins, the VHSv M, has been assessed in detail. Additional ARS-funded research has identified a potential mechanism for how this viral protein reduces the fish immune response following pathogen exposure. Additionally, researchers have identified a naturally occurring mutant that differs from the Great Lakes type IVb strain M protein by only one amino acid. This mutant significantly alters the ability of the host to inhibit viral replication, which allows the virus to escape the fish viral detection and response pathways. This knowledge will enable scientists to develop vaccines and therapeutics that disrupt the life-cycle of this pathogen and minimize its economic impact to the aquaculture industry.


Review Publications
Pierce, L.R., Willey, J.C., Crawford, E.L., Palsule, V.V., Leaman, D.A., Faisal, M., Kim, R.K., Shepherd, B.S., Stanoszek, L.M., Stepien, C.A. 2013. A new StaRT-PCR approach to detect and quantify fish viral hemorrhagic septicemia virus (VHSv): enhanced quality control with internal standards. Journal of Virological Methods. 189:129-142.

Shepherd, B.S., Rees, C.B., Sepulveda Villet, O.J., Palmquist, D.E., Binkowski, F.P. 2013. Identification of gender in yellow perch by external morphology: validation in four geographic strains and effects of estradiol. North American Journal of Aquaculture. 75: 361-372.

Last Modified: 9/21/2014
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