Progress 07/15/07 to 07/14/08
Outputs
OUTPUTS: Tank trials were conducted to examine the mechanisms underlying compensatory growth and to examine the potential regulation of leptin during altered states of growth, feed consumption and energy balance. We cloned and sequenced the leptin gene in striped bass and its hybrid, and show that gene expression is localized to the liver. Hepatic leptin mRNA expression was suppressed during periods of feed deprivation. Leptin levels are subsequently increased during refeeding or the compensatory growth response. The increase in leptin was accompanied by elevations in feed intake and replenishment of energy reserves. We have identified over 80 candidate genes that are potentially involved in the transition of oocytes from primary to secondary growth, which includes deposition into the oocyte of neutral lipids via unknown mechanisms and phospholipids bound to the vitellogenin (Vtg) yolk precursor protein produced by the liver. Up- or down-regulation of the gene transcripts is being verified by an orthogonal method based on rtqPCR assays and will be further validated by gene microarray technology. In addition, mechanisms of oocyte growth and lipidation based on the hepatically synthesized yolk precursors (vitellogenins, Vtgs) were further investigated. In the present work, we discovered that the C-type Vtgs lack an ovarian or oocyte receptor (VtgR) and that the A- and B-type Vtgs preferentially bind to different receptor moieties, namely the dimer and monomer, respectively, of the classical VtgR, which we previously discovered in temperate basses (genus Morone). Research has been conducted on the effects of organic carbon loading on the performance of three state-of-the-art nitrifying biofilter designs in intensive recirculating aquaculture systems is being tested. Biofilters have been evaluated in baseline conditions and loaded with total organic carbon. The effect of salinity on biofilters will be conducted shortly. Results of these studies have been disseminated to producer groups via direct contact with North Carolina hybrid striped bass growers while conducting research on their farms (Westside Fisheries Carolina Fisheries, CastleHayne Fisheries, Austin Brothers Fisheries); through multiple presentations at national and international meetings that include the annual NC Aquaculture Development Conference sponsored the North Carolina Cooperative Extension Service, the World Aquaculture Society, International Symposium on Fish Endocrinology, International Symposium on the Reproductive Physiology of Fish, Biannual meeting of the Asian Pacific Chapter of the World Aquaculture Society and as various seminars and workshops (NC and Australia) at universities and aquaculture institutes. Knowledge gained in this project has has also been used in the design of a large-scale aquaculture facility in North Carolina and an aquaculture laboratory in Florida. PARTICIPANTS: Individuals: Russell Borski, Harry Daniels, Tom Losordo, Craig Sullivan (Principal Investigators, North Carolina State University), and Dennis DeLong (Extension Specialist, North Carolina State University managed the project. Naoshi Hiramatsu (Postdoctoral Fellow), Adam Luckenbach (Postdoctoral fellow), Christian Tipsmark (Postdoctoral Fellow), Ryan Murashige (Research Technician), Robin Sanderson (Research Technician), Richard Jones, (Research Technician), Kaori Hiramatsu (Research Specialist), Matthew Picha (Ph.D. graduate student), Eugene Won (M.S. graduate student), Christina Strom (M.S. graduate student), Andrew Baltzegar (Ph.D. graduate student) Miller Johnstone (Ph.D. graduate student), Marc Turano (Ph.D. graduate student), Benjamin Reading (Ph.D. graduate student), Sandra Page (Ph.D. graduate Student), Amanda Boury (M.S. graduate student), Todd Guerdat (graduate student), Sean Bailey (undergraduate student), Jennifer Schmidt (undergraduate student), Beth Sommerville (undergraduate student), Jordan Pung (undergraduate student), Valerie Williams (undergraduate student), and Cherie Gothreau (undergraduate student) conducted the research activities, analyses and other work for this and related research. Partner Organizations: Keith Hairr, Flounder Grower (Owner, Carolina Flounder, LLC) Lee H. Brothers, hybrid striped bass grower (Owner, Carolina Fisheries); William Watson, hybrid striped bass grower (Owner, Westside Fisheries); North Carolina Sea Grant; Golden LEAF Foundation; Pamlico Aquaculture Field Laboratory, North Carolina State University; Amy Riedel (Aquatic EcoSystems, Inc) coordinated workshop in Florida. Collaborators and contacts: Steffen Madsen (Professor, Department of Biology, University of Southern Denmark), John Godwin (Assoc. Professor, Department of Zoology, North Carolina State University), Wade Watanabe (University of North Carolina Wilmington), Kevin Main (Mote Aquaculture, Saratota, Florida) Akihiko Hara (Dean, Faculty of Fisheries, Hokkaido University, Japan) and Takahiro Matsubara (Assistant Director, National Fisheries Research Institute, Kushiro, Japan) Training or professional development: The research outlined in the proposed studies provided the research training and experiences for several postdoctoral fellows, graduate students, and undergraduate students; involved work with a hybrid striped bass, tilapia, and flounder producers and workshops to educate the public and science community about aquaculture. TARGET AUDIENCES: This research targeted individuals, including students, in aquaculture sciences, the general aquaculture industry, aquaculture producers using recirculating systems, producers of hybrid striped bass nationwide and especially in eastern North Carolina. Results of this work have been disseminated to producers and scientists and students through various mechanisms that include scholarly publications, presentations at international, national and State symposia and conferences dealing with aquaculture issues, direct instruction of graduate and undergraduate students in courses taught at N.C. State University, hands on training of postdoctoral, graduate and undergraduate students during execution of the research, and direct contact with finfish growers during research conducted, in part, on their farms. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Partial compensatory growth was observed in HSB grown in tanks and ponds during the realimentation period when fish were fed daily following periods of feed deprivation. Studies show that compensatory growth is accompanied by higher specific growth rates, hyperphagia and improved feed conversion efficiency relative to those animals maintained on a continuous daily feeding regime. Cyclic feeding regiments also improved or reduced phosphorous levels in ponds. These findings have the potential to fundamentally improve production practices while simultaneously reducing the impact on pond water quality. Of the several nucleic acid primers that have been designed against known mammalian and fish leptin cDNA sequence we successfully cloned leptin in striped bass. Thus, our results are the first to sequence leptin, an important metabolic hormone, in Perciformes, which represent the most advanced, numerous, and diverse group of fishes. We show that leptin is upregulated with the deposition of energy reserves and during states of feeding, suggesting leptin may function to enhance feed intake in fish. Results of our research on oogenesis and egg quality revealed that a many genes are activated in association with the transition of oocytes from primary to secondary growth. In silico analyses for gene annotation is ongoing to Ddfinitive identify these genes and their potential involvement in oocyte lipidation. Results of our research on oogenesis and egg quality revealed that expression of many score of genes are specifically activated or deactivated in association with the transition of oocytes from primary growth to secondary growth (lipidation and vitellogenic growth). This work also has led to discovery of 1,000s of new ovarian genes in Morone species, changes in whose transcription can now be evaluated during oocyte growth and in association with good versus poor egg quality. These developments will allow us to build a realistic model of the changes in gene expression that underlie good (or poor) egg quality in farmed fishes. For the biofiltration work, baseline freshwater nitrification tests were completed earlier in 2007 for the three types of filters. During these evaluations, total organic carbon (TOC) and chemical oxygen demand (COD) measurements were taken. Nitrification of all biofilters was significantly affected by an increase in TOC concentration of 25%. Further increases in TOC reduced nitrification rates by 50 - 70%. In fact, studies on the fluidized bed filters were ceased when the fluidized sand bed began to vary in expansion from complete collapse to uncontrollable expansion. The impact of the information generated in this study to date clearly indicates thresholds of organic carbon loading beyond which the nitrification rates in all biofilters tested are not stable.
Publications
- Amano, H., Fujita, T., Hiramatsu, N., Kagawa, H., Sawaguchi, S., Matsubara, T., Sullivan, C.V., and A. Hara. 2008. Molecular alteration of three forms of vitellogenins and their derivative yolk proteins during oocyte growth and maturation in grey mullet (Mugil cephalus). Cybium, the International Journal of Ichthyology. 32(2) suppl.: 156-158.
- Davis, L., Hiramatsu, N., Sullivan, C.V., Hirano, T., and E.G. Grau. 2008. Estrogen regulation of multiple vitellogenin and estrogen receptor genes and of the growth hormone-insulin like growth factor axis in tilapia. Cybium, the International Journal of Ichthyology 32(2) suppl.: 242-243.
- Uchida, K., Moriyama, S., Breves, J.P., Fox, B.K., Pierce, A.L., Borski, R.J., Hirano, T., and Grau, E.G. (2008) cDNA cloning and isolation of somatolactin in Mozambique tilapia and effects of seawater acclimation, confinement stress, and fasting on its pituitary expression. General and Comparative Endocrinology. In press.
- Picha, M.E., Turano, M.J., Tipsmark, C.K. Borski, R.J. (2008) Regulation of Endocrine and Paracrine Sources of Insulin-like Growth Factors and Growth Hormone Receptor During Compensatory Growth in a Teleost Fish. Journal of Endocrinology. In press
- Turano, M.J., Borski, R.J., and Daniels H.V. (2008) Effects of cyclic feeding on compensatory growth of hybrid striped bass (Morone chrysops x M. saxitilis) foodfish and water quality in production ponds. Aquaculture Research. 39:1514-1523.
- Reading, B.J., Hiramatsu, N., Sawaguchi, S., Matsubara, T., Hara, A., Lively, M.O., and Sullivan, C.V. 2008. Conserved and variant molecular and functional features of multiple vitellogenins in white perch (Morone americana) and other teleosts. Marine Biotechnology (in press).
- Davis, L.K., Pierce, A.L., Hiramatsu, N., Sullivan, C.V., Hirano, T., and Grau, E.G. 2008. Sex-specific expression of multiple vitellogenins, estrogen receptors and growth hormone receptors, and effects of 17B-estradiol in the male tilapia (Oreochromis mossambicus). Gen. Comp. Endocrinol. 156 (2008) 544-551.
- Hiramatsu, N., Mihoka, I., Ideuchi, H., Fujita, T., Amano, H., Matsubara, T., Sullivan, C.V., and A. Hara. 2008. Differential production and uptake of dual vitellogenins in Japanese medaka (Oryzias latipes). Cybium, the International Journal of Ichthyology 32(2) suppl.: 260.
- Reading, B.J., Hiramatsu, N., Matsubara, T., Hara, A., and C.V. Sullivan. 2008. Deduced primary structures of three vitellogenins and specific binding to multiple ovarian receptors in white perch (Morone americana). Cybium, the International Journal of Ichthyology 32(2) suppl.: 159-161.
- Sawaguchi, S., Ohkubo, N., Amano, H., Hiramatsu, N., Hara, A., Sullivan, C.V., and T. Matsubara. 2008. Controlled accumulation of multiple vitellogenins into oocytes during vitellogenesis in the barfin flounder, Varasper moseri. Cybium, the International Journal of Ichthyology 32(2) suppl.: 262.
- Davis, L.K., Hiramatsu, N., Hiramatsu, K., Reading, B.J., Matsubara, T., Hara, A., Sullivan, C.V., Pierce, A.L., Hirano, T., and E. Gordon Grau. 2007. Induction of three vitellogenins by 17B-estradiol with concurrent inhibition of the growth hormone-insulin-like growth factor 1 axis in a euryhaline teleost, the tilapia (Oreochromis mossambicus). Biology of Reproduction 77(4): 614-625.
- Tipsmark, C.K., Strom, C.N., Bailey, S.T. Borski, R.J. (2008) Leptin stimulates pituitary prolactin release through an extracellular signal-regulated protein kinase (ERK) dependent pathway. Journal of Endocrinology.196, 275-281.
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Progress 07/15/06 to 07/14/07
Outputs
OUTPUTS: Compensatory growth is a period of supra-accelerated growth that occurs when animals are alleviated of growth stunting conditions, i.e. environments with limited feed availability. Both tank and pond studies of compensatory growth have been completed to evaluate the effect cyclic feeding regimes (feed deprivation followed by normal ad libitum feeding) on production and water quality parameters of hybrid striped bass (HSB) food fish. Tank trials were conducted to examine the mechanisms underlying compensatory growth, including the appetite and growth regulatory hormones that might control rapid growth rates characteristic of compensatory growth. We also conducted experiments to evaluate how leptin, an appetite inhibitory hormone might regulate pituitary prolactin secretion. Additional studies examined the molecular determinants of egg quality in striped bass. GeneFishingTM DEG Screening (Seegene), which is based on annealing control primers and is a new type of PCR-based
differential display (DD), was used to discover potentially novel genes associated with substandard egg quality. We have identified over 80 candidate genes that are potentially involved in the transition of oocytes from primary to secondary growth, which includes deposition into the oocyte of neutral lipids via unknown mechanisms and phospholipids bound to the vitellogenin (Vtg) yolk precursor protein produced by the liver. We completed the cloning and detailed molecular and functional characterization of 3 types of Vtgs from the congeneric white perch antecedent to our cloning these genes from striped bass and developed a new model for vitellogenic oocyte growth based on our findings. In an additional line of work aimed at developing systems technologies for marine aquaculture that maximizes water reuse, the effects of organic carbon loading and salinity on the performance of three state-of-the-art nitrifying biofilter designs in intensive recirculating aquaculture systems is being
tested. The proposed biofiltration study is approximately 75% complete. Biofilters have been evaluated in baseline conditions and loaded with total organic carbon. Results of these studies have been disseminated to producer groups via direct contact with North Carolina hybrid striped bass growers while conducting research on their farms (Westside Fisheries Carolina Fisheries, CastleHayne Fisheries, Austin Brothers Fisheries); through multiple presentations at national and international meetings that include the annual NC Aquaculture Development Conference sponsored the North Carolina Cooperative Extension Service, the World Aquaculture Society, International Symposium on the Reproductive Physiology of Fish and as various seminars and workshops at universities and aquaculture institutes. Knowledge gained in this project has has also been used in the design of a large scale aquaculture facility in North Carolina and an aquaculture laboratory in Florida.
PARTICIPANTS: Individuals: Russell Borski, Harry Daniels, Tom Losordo, Craig Sullivan (Principal Investigators, North Carolina State University), and Dennis DeLong (Extension Specialist, North Carolina State University managed the project. Naoshi Hiramatsu (Postdoctoral Fellow), Adam Luckenbach (Postdoctoral fellow), Christian Tipsmark (Postdoctoral Fellow), Ryan Murashige (Research Technician), Robin Sanderson (Research Technician), Richard Jones, (Research Technician), Kaori Hiramatsu (Research Specialist), Matthew Picha (Ph.D. graduate student), Eugene Won (M.S. graduate student), Christina Strom (M.S. graduate student), Andrew Baltzegar (Ph.D. graduate student) Miller Johnstone (Ph.D. graduate student), Marc Turano (Ph.D. graduate student), Benjamin Reading (Ph.D. graduate student), Sandra Page (Ph.D. graduate Student), Amanda Boury (M.S. graduate student), Todd Guerdat (graduate student), Sean Bailey (undergraduate student), Jennifer Schmidt (undergraduate student), Beth
Sommerville (undergraduate student), Jordan Pung (undergraduate student), Valerie Williams (undergraduate student), and Cherie Gothreau (undergraduate student) conducted the research activities, analyses and other work for this and related research. Partner Organizations: Keith Hairr, Flounder Grower (Owner, Carolina Flounder, LLC) Lee H. Brothers, hybrid striped bass grower (Owner, Carolina Fisheries); William Watson, hybrid striped bass grower (Owner, Westside Fisheries); North Carolina Sea Grant; Golden LEAF Foundation; Pamlico Aquaculture Field Laboratory, North Carolina State University; Amy Riedel (Aquatic EcoSystems, Inc) coordinated workshop in Florida. Collaborators and contacts: Steffen Madsen (Professor, Department of Biology, University of Southern Denmark), John Godwin (Assoc. Professor, Department of Zoology, North Carolina State University), Wade Watanabe (University of North Carolina Wilmington), Kevin Main (Mote Aquaculture, Saratota, Florida) Akihiko Hara (Dean,
Faculty of Fisheries, Hokkaido University, Japan) and Takahiro Matsubara (Assistant Director, National Fisheries Research Institute, Kushiro, Japan) Training or professional development: The research outlined in the proposed studies provided the research training and experiences for several postdoctoral fellows, graduate students, and undergraduate students; involved work with a hybrid striped bass and flounder producers and workshops to educate the public and science community about aquaculture.
TARGET AUDIENCES: This research targeted individuals, including students, in aquaculture sciences, the general aquaculture industry, aquaculture producers using recirculating systems, producers of hybrid striped bass nationwide and especially in eastern North Carolina. Results of this work have been disseminated to producers and scientists and students through various mechanisms that include scholarly publications, presentations at international, national and State symposia and conferences dealing with aquaculture issues, direct instruction of graduate and undergraduate students in courses taught at N.C. State University, hands on training of postdoctoral, graduate and undergraduate students during execution of the research, and direct contact with finfish growers during research conducted, in part, on their farms.
PROJECT MODIFICATIONS: None to date.
Impacts
Partial compensatory growth was observed in HSB food fish grown in ponds and tanks during the realimentation period when fish were fed daily following periods of feed deprivation. It appears that three weeks of feed deprivation may be excessive and prevented complete growth compensation. Studies in both tanks and ponds show that compensatory growth is accompanied by higher specific growth rates, hyperphagia and improved feed conversion efficiency relative to those animals maintained on a continuous daily feeding regime. Pond total phosphorus (TP) was 32 % lower in the compensatory growth treatments than control ponds. Soluble reactive phosphorus was 41 % and 24 % lower in ponds offered the 3/3 and 3/6 week feed deprivation/refeeding cyclic regimes respectively. Validation of these findings has the potential to fundamentally improve production practices while simultaneously reducing the impact on pond water quality. Of the several nucleic acid primers that have been
designed against known mammalian and fish leptin cDNA sequence none have been successful in cloning hybrid striped bass leptin thus far. Nevertheless, we show that leptin is a potent stimulator of prolactin secretion in fish, suggesting both leptin and prolactin may act in concert in promoting fat deposition or in regulating energy metabolism in fish. Results of our research on oogenesis and egg quality revealed that a many genes are activated in association with the transition of oocytes from primary to secondary growth. In silico analyses for gene annotation is ongoing to Ddfinitive identify these genes and their potential involvement in oocyte lipidation. Research on Morone vitellogenins (Vtgs), transporters of phospholipids into the oocytes, led us to discover three different Vtgs (VgA, VgB, and VgC) in these species as well as multiple oocyte receptors for the three forms of Vtg. These findings led to our development of a new model for vitellogenesis in temperate basses and other
teleosts upon which future progress in discovering the molecular determinants of egg quality will be made. For the biofiltration work, baseline freshwater nitrification tests were completed earlier in 2007 for the three types of filters. During these evaluations, total organic carbon (TOC) and chemical oxygen demand (COD) measurements were taken. A dosing pump system was installed and a solution of sugar was pumped into the system to simulate an increased organic carbon load. Thus far, the nitrification of all biofilters has been significantly affected by an increase in TOC concentration of 25%. The TOC concentration was being increased further to determine the effect of very high TOC loads. The impact of the information generated in this study to date clearly indicates thresholds of organic carbon loading beyond which the nitrification rates in all biofilters tested are not stable. Results from the biofilter studies directly impacted how a large-scale fish farm in North Carolina was
designed. Similarly, results from this study also impacted the design of biofilters at a large research facility in Florida.
Publications
- Tipsmark, C.K., Strom, C.N., Bailey, S.T. Borski, R.J. (2008) Leptin Stimulates Pituitary Prolactin Release through an Extracellular Signal-Regulated Protein Kinase (ERK) Dependent Pathway. Journal of Endocrinology. In press
- Reading, B.J., Hiramatsu, N., Matsubara, T., Hara, A., and C.V. Sullivan. 2007. Deduced primary structures of three vitellogenins and specific binding to multiple ovarian receptors in white perch (Morone americana). Cybium, the International Journal of Ichthyology (in press).
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