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United States Department of Agriculture

Agricultural Research Service

Research Project: IMPROVING GREAT LAKES AQUACULTURE PRODUCTION
2008 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.


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.


3.Progress Report
Progress for this project involved: (1) setting up animal holding facilities once space became available in August 2007; (2) experimental animal studies, and (3) work to clone and characterize growth-regulating genes in yellow perch.

(1) Animal holding facilities: One experimental recirculating fish-holding system was designed and set up with 12 x 30-ft gallon tanks, a sump system, mechanical filters, chemical filters, UV sterilizer, degassing tower and a ½ hp chiller. Also, two separate flow-through systems were designed and set up. They consist of 8 x 15-ft gallon tanks, a header tank with a 1500W heater, degassing column, feeders, lights, aeration, and an activated carbon sump for studies utilizing steroids. Lastly, 4 x 6-ft diameter tanks were set up for holding animals under flow-through conditions. (2) Completed animal studies: Three studies were undertaken to examine the control of genes involved with growth of yellow perch. Study 1-- this study simply involved a fasting (5-week) versus feeding study in adult yellow perch. After 5 weeks, tissues (brain, pituitary, gill, liver, kidney, head kidney, stomach, skin, muscle and plasma) were sampled to examine the expression of genes involved with growth. Study 2 -- Immature animals were fed a diet with 15 mg/kg of estrogen or an untreated diet. Lengths and weights were collected every two weeks for 12 weeks, and then sampled for tissues as above. Dietary estrogen treatment significantly increased weight and length yellow perch at all time points. Study 3 -- this study involved the time-course effects of lipopolysaccharide (LPS: stimulates the immune response) in yellow perch. Our aim is to understand common molecular pathways involved with growth and immunity. Equal numbers of male and female yellow perch were injected with 10 micrograms/gram body weight LPS or saline (control), and sampled at 3, 6, 12, 24, 48, and 72 hours after injection and tissues collected as above. (3) Characterize genes involved with growth and immunity: In yellow perch, GH does not stimulate growth, which suggests a condition of GH insensitivity. We hypothesize that the estrogen-dependent growth seen in yellow perch may be a causal factor underlying GH insensitivity and that any genetic gain in growth may be offset by decreases in immunity. We have cloned genes which encode for a family of proteins that regulate the signaling of hormones that control growth and immunity. We have cloned other growth-regulating genes including prolactin, growth hormone, somatolactin, insulin-like growth factor-I, the estrogen receptors (alpha and beta), aromatase, and myostatin 1 & 2. We are isolating ribonucleic acid (RNA) from the tissues of animals from the described studies, and the expression patterns, and control, of these genes will be examined.

This work addresses the following components in the NP-106 action plan: Genetic Improvement parts C (Selective Breeding for Economically-Important Traits) and D (Development of Genomics Resources); Integrated Aquatic Animal Health part C (immunology and Disease Resistance); and Growth and Development, and Nutrition part b (Tissue Growth and Development).


4.Accomplishments
1. Increase biological efficiency through selective breeding in yellow perch. Yellow perch progeny from pair crosses of wild perch populations from the Perquimans River (NC), Choptank River (MD), and Lake Winnebago (WI) were used in a performance trial to examine head-to-head growth performance for a 12-month period. Perquimans River fish attained a final average weight of 138.12 g, Choptank River fish, 126.70 g; and Winnebago fish, 52.08 g. Final lengths, weights, and absolute growth rates were not significantly different between Choptank and Perquimans fish, but both of these groups were significantly larger than the Winnebago fish. Condition factors also differed between the strains and at the end of the performance, the Choptank fish had the highest condition factor followed by Perquimans River and then Lake Winnebago fish. At present, the top 25-35% performers for each strain are being pit-tagged, genotyped and then put under photoperiod and temperature regimes to cycle them for spawning next spring (2009) to produce the F2 generation. These efforts will result in improved yellow perch germplasm. (National Program 106, Action Plan Component: Genetic Improvement, part a -- Conserve, Characterize, and Utilize Genetic Resources; and part b -- Selective Breeding for Economically Important Traits).

2. Develop genomic resources for integrating functional genomics into existing aquaculture research programs (yellow perch). Individual cDNA libraries were constructed for livers & brains of yellow perch controls and treated with dietary estradiol. From each of the 4 libraries, 4,000 expressed sequence tags (ESTs) were obtained & analyzed, providing 16,000 ESTs in total. In the liver libraries, thirteen genes were down regulated and 11 upregulated in response to estrogen treatment. In the brain libraries, 9 genes down-regulated and 11 up-regulated due to estrogen treatment. Primers have been designed for the potentially regulated genes for qualitative polymerase chain reaction (QPCR) analysis. The data support the differential regulation determined by the bioinformatic analysis. ESTs (8,000) were also obtained from cDNA libraries of perch ovaries that had been stimulated with human chorionic gonadotropin & progestational steroids. A cDNA library was constructed from 2-day-old yellow perch larvae (before feeding), partially sequenced, and the identified ESTs were analyzed for microsatellites (type I markers). These efforts have yielded information on genetic structure among various U.S. perch populations, & have identified new genes & genetic sequences that have directed genetic selection to improve cultivar traits and markers for pedigree tracking to further improve/support future selective breeding efforts. The new technologies & resources that are developed from this program will continue to support innovation and improvement in perch aquaculture (National Program 106, Action Plan Component: Genetic Improvement, part a -- Conserve, Characterize, and Utilize Genetic Resources; part c -- Genomic Resources; and part e -- Bioinformatics and Statistical Analysis Tools).

3. Investigate neural, and endocrine, mechanisms affecting growth and composition at animal and tissue levels. The combination of a high demand (and high cost) for yellow perch, and reduced supply of wild sources, is driving the need for perch aquaculture. Despite this need, there are a two bottle-necks in yellow perch aquaculture that we are addressing,.
1)slow growth to market size (~14-16 months) and small size at harvest. Physiologically, yellow perch display an estrogen-dependent, sexually-dimorphic growth rate where females grow faster, and larger, than males which pose concerns for a selective breeding program. Additionally, growth hormone (GH) does not appear to stimulate growth which suggests a condition of GH insensitivity. We hypothesize that the estrogen-dependent growth seen in yellow perch may be a causal factor underlying GH insensitivity and that any genetic gain in growth (due to selection) may be offset by decreases in immunity. Consequently, we have characterized and published sequences for the growth-regulating hormones in yellow perch. These hormones include pituitary hormones, growth hormone (GH), prolactin (PRL) and somatolactin (SL), their associated growth factors (insulin-like growth factors: IGF-I, IGF-II), and steroid receptors (estrogen receptors) and a steroidogenic enzyme (aromatase). An understanding of the hormonal pathways responsible for estrogen-dependent growth in yellow perch will provide unprecedented insights into how female sex steroids promote growth in this species as well as developing ways to manipulate and select for more uniform growth among the sexes. Such an understanding will be essential to selecting for improved growth in this species while, potentially, minimizing any deleterious effects on other important phenotypic traits such as sexual maturation and immunity. (National Program 106, Action Plan Component: Growth and Development, and Nutrition, part b -- Tissue Growth and Development).

4. Develop methods to alter timing of reproductive development (yellow perch). Photothermal regimes were used to develop out-of-cycle spawning perch broodstocks. Gametes were collected & artificially fertilized from wild broodstock in 3/2007. These eggs produced a F1 generation that was manipulated photothermally to become a July spawning broodstock & subsequently transferred to private industry. Gametes were collected & artificially fertilized from wild broodstock of the Sassafras River (MD) in 3/2003. These eggs produced an F1 generation that was manipulated photothermally to become a January spawning broodstock. F1 broodstock egg production was: normal spawners, 7,525,000 (2005), 6,320,000 (2006),16,435,000 (2007) &14,400,000 (2008); Out-of-cycle spawners: 5,805,000 (2005), 9,985,000 (2006), 23,665,000 (2007) & 22,800,000 (2008). Jan 2007 eggs resulted in F2 fingerlings that were photothermally conditioned to become January spawning broodstock (2009-2010). Fingerlings produced from the Jan spawning of the Sassafras River strain in 2006 were raised for 14 months in a recirculating aquaculture system (RAS) at constant temperature between 20-22 degrees C. Adult fish were selected to become additional broodstock, & manipulated photothermally to spawn in Oct 2007. These fish produced 11 million eggs to make a F3 generation. The history of this F3 generation is: great-grandparents were wild March spawners, grandparents were captive January spawners, & parents were captive Oct 2007 spawners. These F3 generation perch were evaluated for the timing of first-feeding, general behavior, early mortality syndrome, & transition to a commercial diet. These techniques will be used to induce maturity of the F1 generation (National Program 106, Action Plan Component: Genetic Improvement, part d -- Specific Breeding Aids; Plan Component: Reproduction and Early Development, part a -- Control of Reproduction).


5.Significant Activities that Support Special Target Populations
“Improving Growth in Finfish: Approaches and Mechanisms”. U.S. Trout Farmers Association meeting, September 18-20, Hilton Milwaukee Center, Milwaukee, WI. Oral presentation.

USDA-ARS, NP-106 (Aquaculture) National Stakeholder Workshop, Kansas City, MO, April 15-17, 2008. ARS/USDA Lead SY recorded and organized the “Percid” and “Physiology” break-out sessions. SCA participants were also in attendance.

Early Life Stage Culture Workshop held in conjunction with the USDA/North Central Regional Aquaculture Center, November 3, 2007, in southwestern Wisconsin. Used a variety of species as examples.

20th Annual Native American Fish and Wildlife Society Conference, Great Lakes Region “Northern Wisconsin Aquaculture Demonstration Facility Overview”. Presented by Northern Aquaculture Demonstration Facility (NADF) Manager, Lac du Flambeau, WI, September 13, 2007. Wisconsin Aquaculture Industry Advisory Committee and the Wisconsin Aquaculture Association Meeting, “Rearing Advanced Growth Walleye in Northern Wisconsin”. Presented by NADF Manager, Bayfield, WI, September 7, 2007.

“Viral Hemorrhagic Septicemia Virus Symposium”. Organized by NADF and the Wisconsin Veterinary Diagnostic Laboratory, Madison, WI, August 9, 2007.

Workshop on the “Introduction to Aquaculture” and Advanced Aquaculture” presented at the Wisconsin Association of Agricultural Educators (WAAE) Summer Conference on June 25-28, 2007, Madison, Wisconsin. Presented by Great Lakes Water Institute SCA cooperators.

NADF Field Day and VHS Biosecurity Workshop on “How to Protect Your Farm from VHS and Other Diseases”. Organized by the Northern Aquaculture Demonstration Facility, Bayfield, WI, June 14, 2007.


6.Technology Transfer

Number of Non-Peer Reviewed Presentations and Proceedings4

Review Publications
Gahr, S.A., Vallejo, R.L., Weber, G.M., Shepherd, B.S., Silverstein, J., Rexroad III, C.E. 2008. Effects of short term growth hormone treatment on the rainbow trout (Oncorhynchus mykiss) liver and muscle transcriptomes. Physiological Genomics. 32:380-9.

Last Modified: 8/22/2014
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