2009 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 mid-West 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 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.
Progress for this project involved:.
1)experimental animal studies,.
2)work to clone & characterize growth-regulating genes in yellow perch and development of quantitative real-time polymerase chain reaction (PCR) quantification of genes that control growth & immunity,.
3)redirection & identification of new research objectives related to the new ARS/USDA viral hemorrhagic septicemia virus program initiative, &.
4)a material transfer agreement.
1) Completed animal studies: Two studies were undertaken to examine the control of genes involved with growth & immunity in yellow perch. Study 1: We injected juvenile yellow perch with bovine growth hormone (Posilac) at 5 doses (0, 10, 30, 90, and 270 micrograms/g body weight [BW]) every 3 weeks for 9 weeks. Animals in the 90- & 270-micrograms/g BW groups were significantly larger than controls at the end of the study. However, levels of intraperitoneal body fat were highest in these two groups which could contribute to the observed increases in body mass. Study 2: We previously reported an immune induction study using lipopolysaccharide (LPS) in perch. We found that key immune signaling genes were maximally induced in gill, liver, spleen, head kidney, & kidney at 6 hours post-treatment. To further explore the induction of these genes, we are acclimating animals to repeat the LPS induction, but animals will be sampled for tissues & whole blood at 6 hours post-injection.
2) Continue characterization of genes involved with growth & immunity in yellow perch: In other animals, it has been shown that the signaling of the growth-regulating genes (growth hormone [GH], prolactin [PRL], & insulin-like growth factor [IGF]) genes, and immunoregulatory genes (e.g., interleukin.
6)are controlled by the suppressor of cytokine signaling (SOCS) genes. Quantitative real-time PCR assays have been developed to quantify levels of SOCS 1 & 3 genes in yellow perch & growth hormones (GH, PRL, somatolactin [SL], & IGF-I), estrogen receptors (alpha & beta), & aromatase. We have found that estrogen stimulates expression of SOCS gene expression, and studies are being pursued to further clarify how GH, LPS, & estrogen signal via SOCS proteins/genes.
3) Program redirection/realignment: This program has recently been redirected to include a new (FY 2009) national initiative to address research needs relating to viral hemorrhagic septicemia virus in the Great Lakes aquaculture species. Efforts are underway to establish & implement program goals with line management & National Program Staff, renovate space, & purchase equipment. Since these efforts have required substantial time & effort, some activities have been delayed, such as preparation of our next 5-year project plan, until a new scientist is hired.
4) Material transfer agreement (MTA): A MTA is under development with Novartis Pharmaceuticals for the transfer of the pharmaceutical drug, fadrozole, which is an aromatase inhibitor that blocks the production of estrogen & is used to treat estrogen-dependent cancer (breast and uterine) in humans. This compound will be used in yellow perch as a tool to understand how estrogen promotes growth in yellow perch.
Investigate neural, and endocrine, mechanisms affecting growth and composition at animal and tissue levels: Animal production has benefitted from the use of monosex populations which exploits the higher growth exhibited by a particular sex. Yellow perch also exhibit sex-specific growth, but females grow faster than males, and growth can be further increased by estradiol. The way in which estradiol enhances growth in yellow perch is not understood. We have approached this problem by cloning and characterizing the genes for GH, prolactin, somatolactin, IGF-I, the estrogen receptors (alph- & beta-ERs), and aromatase in yellow perch. More recent work on wild (Lake Erie) yellow perch (sources for broodstock) showed sex-specific differences, wherein females had a higher body mass than males which was also accompanied by higher liver estrogen receptor-alpha mRNA levels. Wild males had higher liver IGF-I, higher liver estrogen receptor-beta, and liver aromatase mRNA levels. In both sexes, levels of pituitary GH and liver IGF-I mRNA were higher in spring than at other times of the year. This is the first study of its type in wild yellow perch and has established assays for measurement of these important genes. This work has been expanded by the addition of more than 21,000 yellow perch gene sequences to the National Center for Biotechnology Information (NCBI) GenBank. Using this information, we recently found that estradiol may stimulate growth via increased IGF-I gene expression, as well as changes in several important metabolic genes in the liver. Understanding the linkage between sex, growth, and estrogen will enable new approaches to manipulate gender for monosex culture to enhance growth and reduce size variation. The genes will be evaluated for use as selection markers to improve genetic gain in our broodstock program.
Develop genomic resources for integrating functional genomics into existing aquaculture research programs: Individual cDNA libraries were constructed for livers, brains, and ovaries of yellow perch. Approximately 7,265 liver, 7,394 brain, and 7,200 ovarian expressed sequence tags (ESTs, which are fragments of unique genes) have recently been released/published on the NCBI EST database (http://www.ncbi.nlm.nih.gov/dbEST/index.html) (accession #s FK818865-FK826129, GO569903-GO577297, GO653210-GO660517). In addition, over 200 unique microsatellite sequences were also submitted and are also available at NCBI (accession #s EU153815-EU153821, EU283965-EU284009, EU281734-EU281845, EU277783-EU277832). cDNA libraries from the livers of yellow perch treated with estradiol (and control) were analyzed to determine genes that were consistently up- and down-regulated with estradiol treatment. From the liver libraries, we found 13 genes to be higher in the control versus estrogen fish, and 11 higher in estrogen compared to controls. In the brain libraries, we found 9 genes elevated in controls above estrogen-treated fish, and 11 higher in estrogen versus controls. More recently, a cDNA library was constructed from 2-day-old yellow perch larvae (prior to feeding), and sequencing of that library has begun. The goal is to obtain 8,000 additional ESTs, resulting in more than 28,000 ESTs to date. All of the ESTs are also being mined for microsatellites (type I markers: greater than 214). The new microsatellite markers have enabled clarification of genetic structure among various U.S. perch populations. From EST sequences, we have identified new genes and genetic sequences that have been used:.
Increase biological efficiency through selective breeding in yellow perch: F1 fish from our three yellow perch broodstock strains (Perquimans River, NC, Choptank River, MD, and Lake Winnebago, WI), which demonstrated superior growth in a 2008 performance trial, have been identified and genotyped using new microsatellite markers. In spring of 2009, we produced an F2 generation from 40 pair-wise (F1) crosses per strain. These new (F2) progeny are being evaluated for growth and have also been disseminated to researchers (via cooperative agreements) to evaluate strain & family differences for growth on soy-based diets and for disease resistance to the Midwest strain of the viral hemorrhagic septicemia virus.
1)to direct genetic selection to improve cultivar traits, and.
2)for pedigree tracking to further improve/support future selective breeding efforts.
Relational databases containing genomic and physiological data and core genomic analyses capabilities: To support further improvement in our yellow perch broodstock program, GLWI participants have developed gene sequences from various yellow perch tissues. Attendant with the increase in genomic sequence information is the need to interface gene sequencing instrumentation with databases and to store and process this information. To meet these data acquisition and processing needs, GLWI participants have developed new sequence alignment and identification programs with complete (on-site) National Center for Biotechnology Information (NCBI) protein, nucleotide and EST databases, together with investigator-derived sequence databases. To operate and manage this information, a 4-node Apple Workgroup Cluster for Bioinformatics along with BioTeam's Inquiry software has been developed. Each node of the cluster contains 2 – 2.0 GHz G5 processors, a 500-gigabyte hard drive, and 8 gigabytes of RAM. An automatic data backup system, consisting of an Apple XServe RAID system with a capacity of 2 x 1.4 terabytes, has been developed to maintain all databases, files, and software. To facilitate seamless data acquisition from DNA sequencers to sequence analysis programs, a GLWI bioinformatics technician has developed a custom pipeline for the automated analysis of DNA sequences using existing analysis programs and proprietary programs. This pipeline has improved gene sequence acquisition and sequence quality, which has enabled the recent submission of greater than 21,000 yellow perch gene sequences (ESTs: expressed sequence tags) to the NCBI. This information has resulted in a recent publication describing gene pathways which mediate the growth promoting effects estrogen in yellow perch.
Manipulation of yellow perch growth in a recirculating aquaculture system: Yellow perch (Perca flavescens), averaging 161 mm/51 g, were reared in 288-L circular tanks for 94 days under diel alternating (15/21ºC) and constant high (21ºC) or low (15ºC) water temperatures to test whether they would grow under fluctuating thermal regimes. Survival was higher in the variable temperature treatment versus high temperature treatment. Survival was 100% for the 15ºC group, 98% for the 15/21ºC group, and 85% for the 21ºC group. Feed conversion was significantly better in the 15/21ºC variable temperature tanks compared to the other treatments with values of 1.15 (SE +0.01), 1.30 (SE+0.15), and 1.41 (SE+0.03) for the 15/21ºC, 15ºC, and 21ºC treatment groups, respectively. Average growth rate in length per day was 0.29 mm, 0.34 mm, and 0.35 mm for treatment temperature 15ºC, 21ºC and 15/21ºC, respectively. Average growth in weight per day was 0.40 g, 0.33 g, and 0.41 g for treatment temperature 15ºC, 21ºC, and 15/21ºC, respectively. Final growth in length was only statistically greater between the variable temperature treatment group and all other temperature groups at the end of the study. Final growth in weight was only statistically different between the high (21ºC) temperature group and the variable (15/21ºC) temperature group at the end of the study, although the variable temperature (15/21ºC) tanks did have slightly higher average weights (0.41 g/day) throughout the study period. Overall, there were benefits to the fish in survival, feed conversion, and growth in the 15/21ºC variable temperature regime. This information may be useful to the commercial yellow perch grower as a way to reduce overall cost of heating water while also maximizing survival, growth, and feed conversion in their rearing systems.
5.Significant Activities that Support Special Target Populations
“Evaluation of Yellow Perch Grow-out in a 19C Water Recirculating System.” The International Recirculation Aquaculture Systems Conference, July 26, 2008, Roanoke, VA. Oral presentation.
“Improving Growth in Finfish: Approaches and Mechanisms.” U.S. Trout Farmers Association meeting, September 18-20, 2008, Hilton Milwaukee Center, Milwaukee, WI. Oral presentation.
USDA/ARS, GLWI and NADF personnel participated in the “North Central Region Aquaculture Strategic Planning Meeting.” February 26-27, 2009, Kansas City Airport Hilton, Kansas City, MO. A work group session was chaired by a Michigan State University aquaculture extension specialist.
USDA-ARS, NP-106 (Aquaculture). A regional yellow perch producers workshop, September 24-25, 2009, Olympia Resort and Conference Center, Oconomowoc, WI. This workshop was organized by the U.S. Dairy Forage Research Center, Madison, WI, and the Aquaculture National Program Staff, Beltsville, MD. Key stakeholders in the Midwest region were invited to participate.
|Number of Other Technology Transfer||3|
Lynn, S.G., Powell, K.A., Westneat, D.D., Shepherd, B.S. 2009. Seasonal and Sex-Specific mRNA Levels of Key Endocrine Genes in Adult Yellow Perch (Perca flavescens) from Lake Erie. Marine Biotechnology. 11:210-222.
Goetz, F.W., Rise, M.L., Rise, M., Goetz, G.W., Binkowski, F.P., Shepherd, B.S. 2009. Stimulation of Growth and Changes in the Hepatic Transcriptome by Estradiol-17-Beta in the Yellow Perch (Perca flavescens). Physiological Genomics. Available: http://physiolgenomics.physiology.org/cgi/content/abstract/00069.2009v1.