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United States Department of Agriculture

Agricultural Research Service

Research Project: IMPROVING GREAT LAKES AQUACULTURE PRODUCTION
2010 Annual Report


1a.Objectives (from AD-416)
1. Initiate a genomics program that will enable the future development of a genetically-defined/improved broodstock(s) of yellow. Specifically, genomic DNA from geographically-distinct populations of yellow perch will be collected and genetic diversity will be determined using microsatellite analyses. 2. Develop molecular and proteomic tools needed to advance the genetic improvement of yellow perch. Sub-objective 2.A. – Develop molecular tools needed for germplasm improvement. Sub-objective 2.B. – Develop proteomic tools needed for germplasm improvement. 3. Characterize physiological mechanisms that influence growth in yellow perch. 4. Improve culture technologies for year-round fingerling production and 1-year grow-out for yellow perch. Sub-objective 4.A. – Improving early life-history survival through dietary stimulation of the endocrine system. Sub-objective 4.B. – Improving nutrition for perch grow out. 5. Determine whether there is genetic variation for resistance to viral hemorrhagic septicemia (VHS) in yellow perch and identify pathways of host defense. 6. To develop and evaluate vaccines capable of protecting yellow perch and other Great Lakes region species of fish from VHS.


1b.Approach (from AD-416)
For objective 1, we will collect genomic DNA samples (fin clips) from reproductively active male and female from populations in the mid-Atlantic region to the Midwest region (as far west as North Dakota) of the U.S. Genomic DNA will be extracted from all fin clips and used directly for microsatellite polymerase chain reaction (PCR) using existing microsatellites. PCR products will be run on an ABI 3730 capillary Prism Analyzer and fragments binned into size categories for allele calling. We will analyze microsatellite alleles for both intra and inter-population characteristics such as levels of inbreeding, genetic diversity within a strain, and the genetic relatedness between different strains. For objective 2, we will generate expressed sequence tags (ESTs) and microsatellite markers (genetic markers) from cDNA libraries. These molecular genetic tools, and the attendant assays to quantify expression of genes of interest (e.g., real-time PCR), will enable us to distinguish broodstock strains, evaluate desired broodstock characteristics, and enhance future selection efforts via marker assisted selection. We will also develop proteomic tools that will enable us to measure and characterize the function of critical proteins (hormones) that underlie traits of economic interest. Hormones will be sequentially purified via low-pressure chromatography (size exclusion) and ultimately via reversed-phase high performance liquid chromatography. Hormone identity will be confirmed by electrophoresis and western blotting and mass spectrometry. We shall also produce antibodies to these hormones and, once produced, we will then develop assays to measure blood levels of these hormones in yellow perch. For objective 3, using a number of established bio-assays, we will use these purified hormones to determine how they regulate various physiological sectors in yellow perch. Once the hormone measurement assays are developed, we will then study how they are tied to important physiological traits in our broodstock. Collectively, this information can be used to make more informed decisions in a genetic selection program, as well as diversify our scientific approach to improve yellow perch germplasm. For objective 4, we will develop unique dietary approaches to improve early life-history survival. This is a proof-of-concept study to improve larval survival and swimbladder inflation through dietary stimulation of the endocrine system. For juvenile and adult perch, there is no perch-specific diet and many finfish diets are increasingly using protein sources from plants in dietary formulations. Such sources of protein also possess antinutrients (e.g., phytates) which can block absorption of essential nutrients needed for proper growth and development. To address this, we are formulating diets with various sources of plant protein as well as augmenting these diets with additional amounts of essential nutrients to ensure that the animals are not nutrient-deprived.


3.Progress Report
This program was redirected to address viral hemorrhagic septicemia (VHS) virus in Great Lakes aquaculture species. A new project plan for FY10-FY15 entitled “Combating Viral Hemorrhagic Septicemia and Improving Yellow Perch Aquaculture for the Great Lakes Region” has been approved by the Office of Scientific Quality Review. The plan describes integrated research involving yellow perch broodstock development, nutrition, growth, & VHS susceptibility. To implement this initiative, we have developed a lease arrangement for research space, purchased equipment to increase research capacity, & set up a disease research laboratory. We are also recruiting for a new ARS scientist to address VHS-specific research aims. We aim to understand the hormonal basis for why female perch grow faster than male perch, so that we can improve how we select for better growth. In a 421-day study, we saw positive growth up to 25 g & 130 mm total length, but we did not see sexually dimorphic growth. However, we did see correlations between growth & expression of important growth-related genes. To reevaluate this, we built an enclosure to control the photoperiod & temperature. We populated this system with equal numbers of larger (80 ± 1 g body weight, both sexes) yellow perch. During this acclimation period, we experienced an equipment malfunction that resulted in premature termination of this study. Characterization of genes involved with growth and immunity. In other animals, it has been shown that signaling of the growth-regulating hormones (GH, prolactin [PRL] & insulin-like growth factor [IGF]) and immuno-regulatory cytokines (e.g., interleukin.
6)are controlled by suppressors of cytokine signaling (SOCS) proteins. Quantitative real-time polymerase chain reaction (PCR) assays have been developed to quantify levels of suppressor of cytokine signaling (SOCS 1, 2 and.
3)genes in yellow perch. We have found that immune challenge (LPS injection) rapidly induces levels of SOCS 1 & 3 and that estrogen modulates SOCS gene expression, suggesting possible interactions between growth, sex, & immunity. To evaluate these interactions, we will be studying the expression of these genes in fast-growing & immune-challenged male and female yellow perch. Additional effort is underway to sequence the transcriptomes (genes) from yellow perch brain, pituitary, liver, & muscle. A material transfer agreement (MTA) is being developed with Elanco Dairy (a division of Eli Lily) for the transfer of the sustained release growth hormone implant, Posilac. This material will be used in yellow perch to evaluate whether growth hormone has growth-promoting actions in this species.


4.Accomplishments
1. Increase biological efficiency through selective breeding in yellow perch. In Spring 2009, we produced the second generation of three yellow perch broodstocks (i.e., Perquimans & Choptank Rivers and Lake Winnebago). To assess heritability, we mated two females per male (40 pair-wise crosses), resulting in full-sib and half-sib progeny per cross. Equally mixed progeny from these crosses were evaluated for growth performance (2009-2010) over a 12-month period under fresh water flow-through conditions at 20 degrees C and 16 h light:8 h dark photoperiod. We found that the second-generation animals reached market size two months sooner than the first (their parents’) generation. Using growth data, second-generation animals were divided into the top and bottom 20%, and two intermediate classes, 30% above and below the mean. All these fish were genotyped to determine the parental cross contribution to each size class, and their growth was compared with the growth of the first generation (parents). This information has enabled us to determine which parental crosses contribute to the fastest and lowest growing groups within each broodstock strain. The top 30% performing second-generation fish/strain have been individually tagged, genotyped, and are being held under photoperiod and temperature cycling (1 year) to allow for normal gonadal maturation and spawning in Spring 2011 to produce the third generation. In 2011, the third-generation progeny will be evaluated for growth and survival in collaboration with commercial producers. Disease resistance to the Midwest strain of the viral hemorrhagic septicemia virus will be evaluated with other scientists.

2. Genomic resources for integrating functional genomics into existing aquaculture research programs. A primary cell culture system for head kidney granulocytic phagocytes of yellow perch was developed and used to study the effects of various pathogens on the perch immune system. Using this culture system, we have also characterized about 9,000 new gene sequences from the head kidney cells. This new gene sequence information has enabled us to characterize and identify:.
1)a suite of perch-specific immune genes,.
2)the impact and molecular pathways involved following infection by the viral hemorrhagic septicemia virus (VHS) in yellow perch, and.
3)new molecular markers (called single nucleotide polymorphisms) for genotyping. These new molecular markers will be used for pedigree tracking to further improve selective breeding efforts in our yellow perch broodstocks.

3. Laboratory challenge models to assess pathogenesis of disease. Over the past two years, we have collaborated with scientists from the U.S. Geological Survey (USGS), Western Fish Research Center, Fish Health Laboratory (Seattle, WA) to develop the yellow perch as the standard challenge model for the Midwest viral hemorrhagic septicemia (VHS) virus strain. We have provided perch from our broodstocks for this research. During this time, we worked with the USGS Challenge Facility to further develop the model and to collect relevant tissues in order to describe the immune response in yellow perch following VHS exposure. We now have a standardized exposure route, viral dose range, and exposure time for subsequent studies to evaluate differences in yellow perch broodstock VHS susceptibility. Additionally, samples from these studies are being used to optimize a newly developed standardized reverse transcription polymerase chain reaction technique (StaRT-PCR) that rapidly detects the VHS pathogen in yellow perch in a cost-effective manner.

4. Rapid and automated methods to detect infectious and non-infectious pathogens and toxins in aquatic animals. Inherent with the standardized reverse transcription polymerase chain reaction (StaRT-PCR) assay is the quality control necessary to detect the viral hemorrhagic septicemia (VHS) virus rapidly, reliably, accurately, and economically. Additionally, this test will have the ability to distinguish actively replicating virus (i.e., is transmissible) in yellow perch. The method has been developed and is undergoing further optimization to enable detection of this pathogen in other Great Lakes finfish species such as white perch (Morone americana), smallmouth bass (Micropterus dolomieui), round goby (Neogobius melanostomus), and muskellunge (Esox masquinongy). We have also expanded the use of this detection assay to determine how the VHS infection process works at the cellular level using fish-derived cell lines such as the epithelioma papulosum cyprini (EPC) cell line. This information should yield valuable clues with which to design ways to interfere with the viral disease/infection process in finfish.

5. Improve embryo and early life stage handling, rearing, and culture techniques to enhance survival and health. Feeding trials have reduced the dependence on parts of the live-diet regimen for the early life stage feeding of yellow perch. This new regimen consists of a variety of larval diets from natural (live) to nonliving commercial specialty microdiets (SMD) for larval fish. Compared with a semi-natural fresh process high-protein diet, yellow perch larvae fed the SMD exhibited a 45% survival at the fingerling stage, as compared to 5% survival for larval perch fed the semi-natural diets. These results were used to determine the best management practice for reducing diet components and maintain high fingerling survival and cost effectiveness. In a follow-up study, we modified our initial approach to include an extended presentation, from the on-set of first feeding from 7 to 13 days (live diets were present at this time), and then continuing the SMD regimen for the remaining period of the feeding study (out to 42 days). Fingerling survival increased to 20-25%. We will continue to evaluate different diets and the best management practice approach for yellow perch fingerling production. Utilizing the same diet regimen from the previous feeding trial, we found that young-of-the-year perch that were produced from 2- and 6-year-old broodstock exhibited different survival rates: 70% for those with 6-year-old parents and 17% for those with 2-year-old parents. It is uncertain if broodstock age will play a role in yellow perch fingerling production and diet development.

6. Neural and endocrine mechanisms affecting growth and composition at animal and tissue levels. Yellow perch exhibit sex-specific growth, wherein females grow faster than males, and growth can be further increased by dietary estradiol administration. The way in which estradiol enhances growth in yellow perch is not understood, nor do we understand how other hormones promote growth in this species. To evaluate the hormonal control of growth in male and female perch, we conducted a 421-day growth study under culture conditions in juvenile perch from 102-421 days post-hatch. We found that the growth pattern in both sexes matched seasonal temperatures, with growth being the highest during spring, summer, and fall, and minimal during winter. At the end of the study, size (weight or length) was not significantly different between the two sexes; however, we did find that growth was positively correlated with expression of key growth-regulating genes such as growth hormone (GH) and insulin-like growth factor-I (IGF-I). This correlation suggests that GH and IGF-I are involved with growth in perch, which is a new and important finding for this species. We also found that levels of the estrogen receptor-beta were also correlated with growth and GH and IGF-I, which is quite unique, but not well understood at this time. In separate studies wherein yellow perch were fed diets containing estrogen, we found that the growth-promoting effect of dietary estradiol involves increased IGF-I gene expression as well as changes in several important metabolic genes in the liver, thus, confirming our belief that IGF-I is involved with growth in yellow perch. Understanding the linkage between sex, growth, and estrogen will enable new approaches to manipulate gender for monosex culture or enhance growth to reduce size variation. Additionally, these genes will be evaluated for use as selection markers to improve genetic gain for growth in our broodstock improvement program.

7. Alternative sources of ingredients to replace or reduce fish meal and fish oils in aquaculture diets. We sought to determine if differences in responses of two strains of yellow perch broodstock to dietary soybean meal were genetically based. At the start of the study, Strain A was initially larger than Strain B (58.6 g and 55.4 g, respectively). Fish were fed diets that only varied by the percentage of fishmeal (25, 35, 45, or 55%) that was replaced with soybean meal (SBM). Control groups of animals from each strain were also fed a fishmeal-based diet. All animals were fed their respective diets at 1.5% body/day weight for eight weeks. When SBM was included at rates greater than 35%, percent weight gain, feed efficiency (FE), and specific growth rate (SGR) significantly decreased in both strains. FE was highest in fish fed diets containing no SBM and lowest when fed diets containing 55% SBM. SGR was highest in fish fed diets containing no SBM and decreased substantially when fed diets containing more than 45% SBM. SBM inclusion affected liver size and the ratio of peritoneal fat relative to body weight. Liver size decreased and peritoneal fat increased with SBM inclusion. Tissue samples were collected for genetic, histological, and proximate composition. Overall, there were no differences in growth or FE between strains of yellow perch; both strains performed well with nearly 35% of the fishmeal replaced with SBM. High variation in growth may have limited statistical differences between strains; however, this variation may indicate that substantial genetic variation exists (within families) for selecting perch that perform well on higher SBM diets.


5.Significant Activities that Support Special Target Populations
“Viral Hemorrhagic Septicemia (VHS) Immersion Challenge in Juvenile Muskellunge Using StaRT-PCR: A Quantification Study.” The International Association for Great Lakes Research (IAGLR) Annual Conference, May 17-21, 2010, Toronto, Ontario, Canada. Oral presentation given by University of Toronto collaborators. Target population: stakeholders, fisheries managers and researchers. "Characterization and Regulation of Suppressor of Cytokine Signaling (SOCS) Genes in Yellow Perch (Perca flavescens)". The 2010 Aquaculture America Meeting, World Aquaculture Society, March 1-5, 2010, San Diego, California. Oral presentation given by ARS personnel. Target population: producers, consumers and researchers. "Development of Genetically-Defined Yellow Perch Broodstocks: Results of Performance Trial of F1 Generation”. The 2010 Aquaculture America Meeting, World Aquaculture Society, March 1-5, 2010, San Diego, California. Oral presentation given by ARS personnel. Target population: producers, consumers and researchers. "New Insights into the Recognition of Gram-Negative Lipopolysaccharides by Trout Macrophages”. The 2010 Aquaculture America Meeting, World Aquaculture Society, March 1-5, 2010, San Diego, California. Oral presentation given by University of Wisconsin-Milwaukee cooperators. Target population: producers, consumers and researchers. North Central Region Aquaculture Strategic Planning Meeting, February 19-21, 2010, East Lansing, Michigan. Presentations were given on progress of the ARS/Great Lakes Water Institute (University of Wisconsin-Milwaukee) [UWM/GLWI] program. ARS and GLWI personnel participated in work groups to aid in identifying relevant research issues to the North Central Region. Target population: stakeholders. “Improving Great Lakes Aquaculture Production”. The Annual Meeting of Seafood Science and Technology Society of America.” October 26-29, 2009, New Orleans, Louisiana. Oral presentation by UWM/GLWI cooperator: Target population: producers, consumers and researchers.


Review Publications
Grzybowski, M., Sepulveda-Villet, O.J., Stepien, C.A., Rosauer, D., Binkowski, F., Klaper, R., Shepherd, B.S., Goetz, F. 2010. Genetic Variation of 17 Wild Yellow Perch Populations from the Midwest and East Coast Analyzed Via Microsatellites. Transactions of the American Fisheries Society. 139:270-287.

Last Modified: 10/31/2014
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