Progress 12/14/04 to 12/13/09

Outputs
Progress Report Objectives (from AD-416) 1)Measure genetic variation in production traits in the USDA103 catfish line and characterize correlations between traits; 2)Develop genomic resources for integrating functional genomics into the catfish applied breeding program; 3)Increase biological efficiency through selective breeding of catfish to increase the reproductive efficiency in the production of blue-channel hybrid catfish for commercial production and develop rapid, molecular methods to distinguish channel catfish, blue catfish and blue-channel catfish spawns from each other; and evaluate the improved germplasm developed at 3 to 4 locations in commercial-sized ponds in addition to research ponds to ensure that the germplasm merits release to the U.S. catfish industry. Approach (from AD-416) To attain the first objective, we will measure growth and carcass yield in catfish after rearing in ponds; correlate levels of growth hormone, insulin-like growth factors I and II (IGF-I, IGF-II)and IGF binding proteins (IGFBPs) with growth efficiency in catfish families; quantify family variation in resistance to E. ictaluri in the 5th and 6th generations of the USDA103 line; and determine levels of gonadotropic hormones in spawning catfish and correlate with individual variation in reproductive efficiency. For the second objective, we will add interspecific conserved genes to the catfish genetic linkage map to facilitate comparative genomic analyses; identify chromosomal regions (quantitative trait loci) containing genes controlling carcass yield and resistance to ESC; develop molecular markers for disease resistance candidate genes based on comparative functionality with other species; and identify candidate genes and gene products controlling economically important traits using differential gene expression on oligonucleotide microarrays. For the third objective, we will develop and implement an index based on breeding values of traits and their economic values to select individuals with superior composite phenotypes; and quantify differences in performance for production traits between selected lines and the USDA103 base population. This is the final report of this project. Two generations of selection for survival to a bacterial pathogen within the USDA 103 catfish line led to a 10% improvement in survival. Matings of USDA 103 with USDA 102 line catfish led to offspring with improved fingerling production traits compared to the parent lines. During the term of this project, industry adoption of USDA103 germplasm did not meet expectations, so some resources devoted to continued development of this line were diverted to development of a channel catfish line with a more diverse genetic base. Thus, the Industry Pool line was developed from eggs obtained from eight commercial hatcheries. Initial tests showed improved survival of Industry Pool fish compared to USDA 103 in the fry to fingerling production phase. We used DNA markers to pedigree 190 pond-spawned Industry Pool families. Increased emphasis was placed on production of channel x blue catfish hybrids including laparoscopy to measure testis development in blue catfish males and artificial temperature cycling to accelerate catfish maturity. We cooperated with a commercial producer to develop catfish pituitary extract to induce ovulation for hybrid catfish production. We adapted hatching jar technology and the use of bromelain to improve catfish hybrid embryo survival. We developed a DNA test to identify hybrid catfish at all life stages to support commercial efforts to produce hybrids in ponds. We developed the first catfish physical map and 9,000 single nucleotide polymorphism markers useful for population management and selective breeding. We developed the first catfish oligonucleotide microarray to measure the expression of 19,000 genes, and showed expression of 64 genes significantly increased and 74 genes significantly decreased in spleen after stimulation of immune responses. Toll-like receptors and genes in the major histocompatability loci were characterized to determine their predictive utility in disease resistance. A quantitative assay was optimized for detection of channel catfish virus (CCV) in asymptomatic fish, and 10-50% of commercial and research fish carried CCV. The genes involved in the growth hormone-insulin like growth factor (IGF) pathway were characterized. Our research showed that plasma levels of IGF1 were correlated with weight gain. We were the first to show that levels of IGF2 gene expression were significantly elevated after administration of growth hormone and IGF2 gene expression levels in muscle were correlated with rate of growth, which differed from mammalian research. Therefore we learned that data from research in mammals may not be applicable to catfish physiology, and that IGF2 may serve as a biomarker for growth potential in catfish. Details of subprojects may be found in 6402-31000-008-01S �Hill Area Aquaculture�, and 6402-31000-008- 15S, �Acoustic Technologies for Evaluating Catfish Production." Refer to Project 6402-31000-009-00D for accomplishments (replacement for this project.) Significant Activities that Support Special Target Populations The USDA Census of Aquaculture conducted in 2000 classified 84% of catfish farms as small businesses, with annual sales of less than $500, 000. Of the 1,370 catfish farms in the United States, 38% (515) reported annual revenues of less than $25,000. This data is not available in the 2005 Census of Aquaculture. Increased foreign competition and higher feed and fuel prices have reduced the profit margins for these small businesses. Development of catfish lines with superior performance for commercially important traits, utilization of these lines in commercial culture, and utilization of production technology originating from the Catfish Genetics Research Unit will help solve production problems, increase efficiency and profitability for both small and large catfish farmers, and provide a quality product for consumers. Most catfish producers with limited acreage buy fingerlings from large breeders that are very likely to utilize improved brood stocks, and the development and use of improved catfish lines can quickly affect the profits of small producers. Because small farms do not enjoy the same economies of scale experienced by larger operations, breeding fish with improved production traits will be highly beneficial to small farmers. Average consumers also benefit from the increased availability of higher-quality, safer domestic products at a reduced price.

Impacts
(N/A)

Publications

Progress 10/01/08 to 09/30/09

Outputs
Progress Report Objectives (from AD-416) 1)Measure genetic variation in production traits in the USDA103 catfish line and characterize correlations between traits; 2)Develop genomic resources for integrating functional genomics into the catfish applied breeding program; 3)Increase biological efficiency through selective breeding of catfish to increase the reproductive efficiency in the production of blue-channel hybrid catfish for commercial production and develop rapid, molecular methods to distinguish channel catfish, blue catfish and blue-channel catfish spawns from each other; and evaluate the improved germplasm developed at 3 to 4 locations in commercial-sized ponds in addition to research ponds to ensure that the germplasm merits release to the U.S. catfish industry. Approach (from AD-416) To attain the first objective, we will measure growth and carcass yield in catfish after rearing in ponds; correlate levels of growth hormone, insulin-like growth factors I and II (IGF-I, IGF-II)and IGF binding proteins (IGFBPs) with growth efficiency in catfish families; quantify family variation in resistance to E. ictaluri in the 5th and 6th generations of the USDA103 line; and determine levels of gonadotropic hormones in spawning catfish and correlate with individual variation in reproductive efficiency. For the second objective, we will add interspecific conserved genes to the catfish genetic linkage map to facilitate comparative genomic analyses; identify chromosomal regions (quantitative trait loci) containing genes controlling carcass yield and resistance to ESC; develop molecular markers for disease resistance candidate genes based on comparative functionality with other species; and identify candidate genes and gene products controlling economically important traits using differential gene expression on oligonucleotide microarrays. For the third objective, we will develop and implement an index based on breeding values of traits and their economic values to select individuals with superior composite phenotypes; and quantify differences in performance for production traits between selected lines and the USDA103 base population. Significant Activities that Support Special Target Populations Research focused on genetic control of efficient growth, survival, and reproductive success, and also improving the efficiency of producing hybrid catfish. The USDA403 research line of catfish was compared with a new catfish line Industry Pool developed from commercial catfish. Growth was similar between the two lines but the Industry Pool line showed higher survival in pond culture. Selected matings based on resistance to E. ictaluri challenge in tanks showed that offspring of broodstock selected from high survival families were consistently showed higher survival than fish from non-select families. Catfish Genetics Research Unit (CGRU) research also focused on channel catfish X blue catfish F1 hybrids, and unit scientists interacted with commercial hybrid catfish producers to share research data and evaluate production techniques. Genomic tools were developed with cooperators to evaluate targeted and global gene expression changes in immune cells and tissues following exposure to pathogens. Evalution of the Industry Pool line in a single spawning year in showed evidence for genetic control of time of spawning in females and incidence of spawning in males. The enzyme bromelain was found useful to remove adhesiveness of catfish eggs to improve hatching success. Several experiments demonstrated the central role of the growth hormone/insulin-like growth factor pathway in catfish with the goal of identifying molecular genetic variation linked to efficient growth. Significant Activities that Support Special Target Populations Development of catfish lines with superior performance for commercially important traits, utilization of these lines in commercial culture, and utilization of production technology originating from the Catfish Genetics Research Unit will help solve production problems, increase efficiency and profitability for both small and large catfish farmers, and provide a quality product for consumers. Most catfish producers with limited acreage buy fingerlings from large breeders that are very likely to utilize improved brood stocks, and the development and use of improved catfish lines can quickly affect the profits of small producers. Because small farms do not enjoy the same economies of scale experienced by larger operations, breeding fish with improved production traits will be highly beneficial to small farmers. The USDA Census of Aquaculture conducted in 2000 classified 84% of catfish farms as small businesses, with annual sales of less than $500,000. Of the 1,370 catfish farms in the United States, 38% (515) reported annual revenues of less than $25, 000. This data is not available in the 2005 Census of Aquaculture. Technology Transfer Number of Web Sites managed: 2

Impacts
(N/A)

Publications

• Kobayashi, Y., Peterson, B.C., Waldbieser, G.C. 2008. Association of Cocaine- and Amphetamine-Regulated Transcript (CART) Messenger RNA Level, Food Intake, and Growth in Channel Catfish. Comparative Biochemistry and Physiology 151:219-225.
• Davis Jr, K.B., McEntire, M.E. 2009. Comparison of the cortisol and glucose stress response to acute confinement among white bass, Monrone chrysops, striped bass, Monrone saxatilis and sunshine bass, Monrone chrysops x Morone saxatilis. Journal of the World Aquaculture Society. 40(4):567-572.
• Davis Jr., K.B. 2009. Age at Puberty of Channel Catfish, Ictalurus punctatus, Controlled by Thermoperiod. Aquaculture. 292:244-247.
• Elibol-Flemming, B., Wolters, W.R., Waldbieser, G.C., Boyle, C.R., Hanson, L.A. 2009. Expression Analysis of Selected Immune Relevant Genes in Channel Catfish During Edwardsiella ictaluri Infection. Journal of Aquatic Animal Health 21:23-35.
• Lange, M.D., Waldbieser, G.C., Lobb, C.J. 2009. Patterns of Receptor Revision in Immunoglobulin H Chains of A Teleost Fish. Journal of Immunology 182:5605-5622.
• Majji, S., Thodima, V., Arnizaut, A., Deng, Y., May, W., Sittman, D., Waldbieser, G.C., Hanson, L., Cuchens, M.A., Bengten, E., Chinchar, V.G. 2008. Expression Profiles of Cloned Channel Catfish (Ictalurus punctatus) Lymphoid Cell Lines and Mixed Lymphocyte Cultures. Developmental and Comparative Immunology 33:224-234.
• Milev-Milovanovic, I., Majji, S., Thodima, J., Deng, Y., Hanson, L., Arnizaut, A., Waldbieser, G.C., Chinchar, V.G. 2009. Identification and Expression Analyses of Poly[I:C]-stimulated Genes in Channel Catfish (Ictalurus punctatus). Fish and Shellfish Immunology 26:811-820.
• Pearson, P.R., Small, B.C., Beecham, R.V., Sink, T.D., Labarre, S.B., Minchew, D.C. 2009. Effects of loading density on golden shiner survival during and after hauling. North American Journal of Aquaculture. 71:24-29.
• Peterson, B.C., Waldbieser, G.C. 2009. Effects of Fasting on IGF-I, IGF-II, and IGF-binding Protein MRNA Levels in Channel Catfish (Ictalurus punctatus). Domestic Animal Endocrinology 37:74-83.
• Small, B.C., Murdock, C.A., Bourgeois, A.L., Peterson, B.C., Waldbieser, G. C. 2008. Stability of Reference Genes for Real-Time PCR Analyses in Channel Catfish (Ictalurus punctatus) Tissues Under Varying Physiological Conditions. Comparative Biochemistry and Physiology 151:296-304.
• Liu, Z., Li, R.W., Waldbieser, G.C. 2008. Utilization of Microarray Technology for Functional Genomics in Ictalurid Catfish. North American Journal of Aquaculture.

Progress 10/01/07 to 09/30/08

Outputs
Progress Report Objectives (from AD-416) 1)Measure genetic variation in production traits in the USDA103 catfish line and characterize correlations between traits; 2)Develop genomic resources for integrating functional genomics into the catfish applied breeding program; 3)Increase biological efficiency through selective breeding of catfish to increase the reproductive efficiency in the production of blue-channel hybrid catfish for commercial production and develop rapid, molecular methods to distinguish channel catfish, blue catfish and blue-channel catfish spawns from each other; and evaluate the improved germplasm developed at 3 to 4 locations in commercial-sized ponds in addition to research ponds to ensure that the germplasm merits release to the U.S. catfish industry. Approach (from AD-416) To attain the first objective, we will measure growth and carcass yield in catfish after rearing in ponds; correlate levels of growth hormone, insulin-like growth factors I and II (IGF-I, IGF-II)and IGF binding proteins (IGFBPs) with growth efficiency in catfish families; quantify family variation in resistance to E. ictaluri in the 5th and 6th generations of the USDA103 line; and determine levels of gonadotropic hormones in spawning catfish and correlate with individual variation in reproductive efficiency. For the second objective, we will add interspecific conserved genes to the catfish genetic linkage map to facilitate comparative genomic analyses; identify chromosomal regions (quantitative trait loci) containing genes controlling carcass yield and resistance to ESC; develop molecular markers for disease resistance candidate genes based on comparative functionality with other species; and identify candidate genes and gene products controlling economically important traits using differential gene expression on oligonucleotide microarrays. For the third objective, we will develop and implement an index based on breeding values of traits and their economic values to select individuals with superior composite phenotypes; and quantify differences in performance for production traits between selected lines and the USDA103 base population. Significant Activities that Support Special Target Populations The ADODR collaborates with the cooperating principle investigators and has continuously monitored performance on this extramural agreement through personal meetings, telephone, and email correspondence to ensure that the project is progressing in accordance with the objectives of the agreement. Research to determine optimal management of pond dissolved oxygen concentrations was evaluated under different operating schemes (constant aeration; manual control; or automated control). Continuous use of the aerators significantly increased electricity costs, but this cost was offset by the increased cost of labor and gasoline for manual monitoring protocols, leading to no significant differences in the partial net returns between these two aeration schemes. The automatically controlled aeration devices that worked best in following programmed protocol in ponds showed an approximate 30% increase in partial net returns over those of the average constant and manually monitored schemes partial net returns. The evaluation of whether production results achieved in small experimental earthen ponds (0.05 ha) assigned to the fingerling to stocker (second) phase of the modular system for catfish could be verified under commercial scale conditions was completed. Verification of the modular system proved to be successful and preliminary economic analysis indicates a reduced cost of production as compared to the current multiple batch system for catfish farmers. This management scheme also offers greater efficiency, improved control of inventory, a reduced loss due to bird depredation, and a better opportunity to adjust to processor desire for particular sizes of harvested fish. Evaluation of organic fertilization/feeding 2 strategies as part of the low-input management practices for the earthen pond culture of the freshwater prawn Macrobrachium rosenbergii was completed. The results of the experiments using different fertilizers and different feeding strategies provide for a cost-dependent strategy of selection of a known organic fertilizer that has been demonstrated to be equally successful when compared to other, known to be successful fertilizers. Producers will be afforded more flexibility in managing variable costs of an operation because a variety of effective fertilizers is available for use, and stocking densities can be increased without sacrificing mean harvest weight due to density dependent growth factors. NP 106, Component: 1, 2, 3, 4, Problem Statement: 1b, 1c, 2c, 3a, 3c, 3e, 4b, 4e. Significant Activities that Support Special Target Populations Development of catfish lines with superior performance for commercially important traits, utilization of these lines in commercial culture, and utilization of production technology originating from the Catfish Genetics Research Unit will help solve production problems, increase efficiency and profitability for both small and large catfish farmers, and provide a quality product for consumers. Most catfish producers with limited acreage buy fingerlings from large breeders that are very likely to utilize improved brood stocks, and the development and use of improved catfish lines can quickly affect the profits of small producers. Because small farms do not enjoy the same economies of scale experienced by larger operations, breeding fish with improved production traits will be highly beneficial to small farmers. The USDA Census of Aquaculture conducted in 2000 classified 84% of catfish farms as small businesses, with annual sales of less than $500,000. Of the 1,370 catfish farms in the United States, 38% (515) reported annual revenues of less than $25, 000. This data is not available in the 2005 Census of Aquaculture. Technology Transfer Number of Web Sites managed: 4

Impacts
(N/A)

Publications

• Barrero, M., Small, B.C., Waldbieser, G.C., D'Abramo, L., Hanson, L., Kelly, A. 2007. Effect of Carp Pituitary Extract and Luteinizing Hormone Releasing Hormone on Reproductive Steroids, Cathepsins, and Incidence of Spawning in Young-Adult Channel Catfish. North American Journal of Aquaculture, accepted for publication 04/16/2007.
• Bengten, E., Quiniou, S.M., Hikima, J., Waldbieser, G.C., Warr, G.W., Miller, N.W., Wilson, M. 2006. Structure of the Catfish IGH Locus: Analysis of the Region Including the Single Functional IGHM Gene. Immunogenetics 58:831-844.
• Bilodeau, A.L., Bosworth, B.G., Peterson, B.C. 2008. Differences in mortality, growth, and immune response among genetic groups of catfish exposed to virulent Edwardsiella ictaluri.. Fish and Shellfish Immunology 24:82-89
• Clay, L.A., Wang, S.Y., Wolters, W.R., Peterson, B.C., Waldbieser, G.C. 2005. Molecular Characterization of the Insulin-Like Growth Factor-i (IGF- I) Gene in Channel Catfish (Ictalurus punctatus). Biochimica et Biophysica Acta - Gene Structure and Expression. 1731(3):139-148.
• Davis Jr, K.B., Goudie, C.A., Simco, B.A. 2007. Sex Genotype and Sex Phenotype Contribute to Growth Differences Between Male and Female Channel Catfish. North American Journal of Aquaculture 69:324-329.
• Edholm, E.S., Stafford, J.L., Quiniou, S.M., Waldbieser, G.C., Miller, N.W. , Bengten, E., Wilson, M. 2007. Channel catfish, Ictalurus punctatus, CD4- like molecules. Developmental and Comparative Immunology 32:172-187.
• Evenhuis, J., Bengten, E., Snell, C., Quiniou, S.M., Miller, N.W., Wilson, M. 2007. Characterization of Additional Novel Immune Type Receptors in Channel Catfish, Ictalurus punctatus. Immunogenetics. 59:661-671.
• Karsi, A., Wolters, W.R., Waldbieser, G.C. 2005. Assignment of immune- related genes to the channel catfish, ictalurus punctatus, genetic map. Animal Genetics 36:502-506.
• Li, R.W., Silverstein, P.S., Waldbieser, G.C. 2005. Genomic characterization and expression analysis of the baculoviral iap repeat containing 2 (birc2) gene in channel catfish, ictalurus punctatus. Animal Genetics 36:537-539.
• Li, R.W., Waldbieser, G.C. 2006. Genomic organization and expression of the natural killer cell enhancing factor (nkef) gene in channel catfish, ictalurus punctatus (rafinesque). Fish and Shellfish Immunology 20:72-82.
• Peterson, B.C., Small, B.C., Waldbieser, G.C., Bosworth, B.G. 2008. Endocrine responses of fast and slow growing families of channel catfish. North American Journal of Aquaculture 70:240-250.
• Silverstein, P.S., Li, R.W., Murdock, C.A., Waldbieser, G.C. 2007. Poly i:c inhibits the expression of channel catfish virus immediate-early gene orf 1 at early times after infection. Fish and Shellfish Immunology 23:479- 484.
• Small, B.C., Davis Jr, K.B., Peterson, B.C. 2008. Elucidating the Effects of Cortisol and Stress on Economically Important Traits in Channel Catfish. North American Journal of Aquaculture 70:223-235.
• Waldbieser, G.C., Wolters, W.R. 2007. Definition of the USDA103 Strain of Channel Catfish (Ictalurus punctatus). Animal Genetics. 38:180-183.
• Wang, S., Xu, P., Thorsen, J., Zhu, B., De Jong, P.J., Waldbieser, G.C., Kucuktas, H., Liu, Z. 2007. Characterization of a BAC Library from Channel Catfish Ictalurus punctatus: Indications of High Rates of Evolution Among Teleost Genomes. Marine Biotechnology 9:701-711.
• Williams, M., Waldbieser, G.C., Dyer, D.W., Gillaspy, A.F., Lawrence, M.L. 2008. Characterization of the RRN Operons in the Channel Catfish Pathogen Edwardsiella ictaluri. Journal of Applied Microbiology 104:1790-1796.
• Yang, F., Waldbieser, G.C., Lobb, C. 2006. The Nucleotide Targets of Somatic Mutation and the Role of Selection in Immunoglobulin Heavy Chains of a Teleost Fish. Journal of Immunology 176:1655-1667.
• Davis Jr., K.B. 2006. Management of physiological stress in finfish aquaculture. North American Journal of Aquaculture. 68:116-121.
• Li, M., Peterson, B.C., Janes, C.L., Robinson, E.H. 2006. Comparison of diets containing various fish meal levels on growth performance, body composition, and insulin-like growth factor-i of juvenile channel catfish ictalurus punctatus of different strains. Aquaculture 253:228-235.
• Stafford, J.L., Wilson, M., Nayak, D., Quiniou, S.M., Clem, L.W., Miller, N.W., Bengten, E. 2006. Identification and Characterization of a Fc Receptor Homolog in an Ectothermic Vertebrate, the Channel Catfish (Ictalurus punctatus). Journal of Immunology.
• Stafford, J.L., Bengten, E., Du Pasquier, L., Mcintosh, R.D., Quiniou, S.M. , Clem, L.W., Miller, N.W., Wilson, M. 2006. A novel family of diversified immunoregulatory receptors in teleosts is homologous to both mammalian fc receptors and molecules encoded within the leukocyte receptor complex. Immunogenetics 59:758-773.

Progress 10/01/06 to 09/30/07

Outputs
Progress Report Objectives (from AD-416) 1)Measure genetic variation in production traits in the USDA103 catfish line and characterize correlations between traits; 2)Develop genomic resources for integrating functional genomics into the catfish applied breeding program; 3)Increase biological efficiency through selective breeding of catfish to increase the reproductive efficiency in the production of blue-channel hybrid catfish for commercial production and develop rapid, molecular methods to distinguish channel catfish, blue catfish and blue-channel catfish spawns from each other; and evaluate the improved germplasm developed at 3 to 4 locations in commercial-sized ponds in addition to research ponds to ensure that the germplasm merits release to the U.S. catfish industry. Approach (from AD-416) To attain the first objective, we will measure growth and carcass yield in catfish after rearing in ponds; correlate levels of growth hormone, insulin-like growth factors I and II (IGF-I, IGF-II)and IGF binding proteins (IGFBPs) with growth efficiency in catfish families; quantify family variation in resistance to E. ictaluri in the 5th and 6th generations of the USDA103 line; and determine levels of gonadotropic hormones in spawning catfish and correlate with individual variation in reproductive efficiency. For the second objective, we will add interspecific conserved genes to the catfish genetic linkage map to facilitate comparative genomic analyses; identify chromosomal regions (quantitative trait loci) containing genes controlling carcass yield and resistance to ESC; develop molecular markers for disease resistance candidate genes based on comparative functionality with other species; and identify candidate genes and gene products controlling economically important traits using differential gene expression on oligonucleotide microarrays. For the third objective, we will develop and implement an index based on breeding values of traits and their economic values to select individuals with superior composite phenotypes; and quantify differences in performance for production traits between selected lines and the USDA103 base population. Significant Activities that Support Special Target Populations Evaluation of a fingerling to stocker phase (2nd phase) of a 3-phase modular system for the culture of channel catfish continued. A verification study was conducted in 4-acre ponds to determine whether results achieved previously in small experimental ponds were applicable to commercial scale conditions. Fingerlings were stocked (123,550/ha) into ponds in early April. After 142 days, individual mean weight for all ponds ranged from 150 to 186 g. Fish were then fed until November and harvested in January. At final harvest, mean individual weight ranged from 109 to 159 g, a good size for stocking the final growout (phase-3) ponds. Survival averaged 47%, approximately 20% less than that obtained in small ponds. Mortality was primarily attributed to disease. Fish harvested from the phase-2 ponds were stocked into phase-3 ponds at a density of either 5,000 or 6,500 per acre. Partial harvest of these ponds is scheduled for late July to determine what percentage of the harvested population has achieved market size. An economic analysis of the second and third phases of culture is planned to determine whether the 3-phase system offers an effective alternative management strategy for commercial catfish production. Three different organic fertilization strategies were evaluated for freshwater prawn production. The organic fertilizer, consisting of a 50:50 combination of corn gluten pellets and range cubes, was applied daily at rates of 23 to 54 lb/A. At harvest, no significant differences in mean harvest weight, survival, and mean production were found among the different treatments. Survival ranged from 68 to 77% and harvest weight ranged from 41 to 44 g. The equivalent production achieved with the lowest rate of fertilization translates into a 39% reduction in fertilizer costs per kilogram of freshwater prawn produced. Combined with previous results that evaluated qualitative sources of fertilizer, a 56% reduction in fertilizer costs per weight of prawn produced has been realized, and total operational costs attributed to the cost of feed have been reduced by 22%. The economic feasibility of domestic production of freshwater prawn in the US has been substantially increased, and the harvest size fills a size and quality niche that is absent in foreign imports. The production costs and benefits of different aeration protocols for the management of oxygen in production systems are currently under investigation. These investigations are designed to reduce cost of production while maintaining sufficient water quality. Accomplishments Use of catfish pituitary extract for inducing ovulation in channel catfish females. Channel x blue catfish hybrids exhibit excellent performance for growth, meat yield, and disease resistance and there is considerable interest in commercial production of hybrids. However, production of hybrid fry is dependent on hormome induced ovulation of female catfish which is expensive and inconsistent. Use of catfish pituitary extract for inducing ovulation in catfish, as an alternative to existing products (Luteinizing Hormone Releasing Hormone (LHRH) and carp pituitary extract), could lower costs and spur development of high value product from readily available, low-value processing waste (catfish heads) . Catfish heads were collected from 2 processing plants; and pituitaries were removed, extracted in ethanol and acetone, and dried. Catfish pituitary extract was compared to carp pituitary extract purchased from a commercial vendor for effectiveness in inducing ovulation in catfish. Catfish pituitary was similar to carp pituitary for percentage of females ovulating, number of eggs produced per kg of female body weight, and fertility of embryos at 48 hours post-fertilization. This project is being conducted in conjunction with a commercial catfish farmer and has resulted in development of an investigative new animal drug request by the U.S. Fish and Wildlife Service (USFWS) for use of catfish pituitary as a spawning aid for catfish. Based on the successful results from the initial trial, plans are being made to expand production of catfish pituitary for larger trials in spring 2008. This research supports NP106 Action Plan Component 1b (GENETIC IMPROVEMENT: Selective breeding for economically important traits). Estimation of phenotypic and genetic (co)variances for economically important traits. -- Estimation of phenotypic and genetic (co)variances for economically important traits are required for development of a breeding program to produce superior catfish germplasm for release to catfish producers. Data were collected for growth, resistance to enteric septicemia of catfish, and processing yield for families from the third generation of selection of the NWAC103 channel catfish line and the best performing animals were selected as future broodstock based on an index for these traits. Selected broodstock were mated to produce another generation of families which are currently being evaluated for growth, resistance to ESC, and processing yield (fourth generation of selection of the NWAC103 channel catfish line = G5 population). This information is being used in a continued selection program to develop superior germplasm for release which will benefit producers, processors, and consumers. This research supports NP106 Action Plan Component 1b (GENETIC IMPROVEMENT: Selective breeding for economically important traits). Identification of molecular genetic markers linked to economically important traits in channel catfish x blue catfish backcross hybrids. -- Eight full-sib backcross hybrid families (channel catfish female x channel-blue F1 hybrid male) were produced in spring 2007 and backcross fish will be grown and individuals will be measured for a series of economically important traits: growth, body composition, body shape, meat yield and composition, and ESC resistance. These fish will also be genotyped at a series of highly variable microsatellite markers that cover the entire genome. Data will be analyzed to identify markers that are associated with performance for traits listed above. Originally the project plan indicated marker-trait association would be examined in F2 families produced from channel catfish lines divergently selected for carcass yield. However, because the channel x blue hybrid is divergent for a series of traits including meat yield, growth, body shape and composition, and disease resistance it was determined that searching for linkage between markers and trait performance would be more efficient in the backcross population. This research supports NP106 Action Plan Component 1b (GENETIC IMPROVEMENT: Selective breeding for economically important traits). Pilot testing of experimental catfish lines on commercial farms. -- The mission of the Catfish Genetics Research Unit (CGRU) is to produce improved catfish germplasm for release to commercial catfish farmers. It is important to determine how germplasm being evaluated for release will perform in a commercial production environment, which may differ from the original research environment. A project was initiated to establish and test a protocol for evaluation of experimental catfish lines on commercial farms. Juvenile catfish from a line currently being developed at the CGRU are being compared to juvenile catfish from a control line (developed by mixing fish from 10 commercial farms). All fish were hatched and reared to the juvenile stage at the CGRU under similar conditions (age, stocking rates, diets, and feeding rates). Juveniles from each line were stocked on 6 commercial farms and 2 research stations with at least 1 replicate pond per line per location. Fish will be raised until harvestable weight and cooperating farmers will provide records on the weight of feed delivered, weight of fish harvested, and other significant observations (significant disease problems etc.). This research supports NP106 Action Plan Component 1b (GENETIC IMPROVEMENT: Selective breeding for economically important traits). Reproductive efficiency among catfish strains. -- Sustained production of channel catfish depends on reproductively efficient fish, and although a variety of breeding programs to improve channel catfish traits for commercial aquaculture have existed over the years, few comparisons have been made between selectively bred strains for reproductive parameters related to spawning. In order to evaluate potential strain differences for reproductive traits, four selectively bred commercial strains of channel catfish were evaluated in a 12-month study, during which critical reproductive indices were measured, including various sex steroids, proteins, and enzymes. Among the four strains no differences were observed for any of the traits measured, including spawning frequency. These data are being used to assess what controls reproductive success in channel catfish in order to develop tools to rapidly identify strains or individuals with greater reproductive potential. This research supports NP106 Action Plan Component 1b (GENETIC IMPROVEMENT: Selective breeding for economically important traits). Effects of winter feeding on growth, meat yield, and visceral composition in channel, blue, and channel x blue hybrid catfish. -- Catfish farmers typically dramatically reduce or cease feeding because catfish have lower feeding activity at cold temperatures. Additionally, wet weather patterns common during the winter in much of the southeastern U.S. make it difficult to get feeding equipment to ponds. Little is known about differential response of channel, blue, and channel x blue catfish to winter feeding. A study was conducted to evaluate growth, meat yield, and visceral composition in channel, blue, and channel x blue hybrid catfish that were either not fed (control) from November to February or fed a sinking pellet 2 times per week. Channel and hybrid catfish that were fed gained more weight than blue catfish that were fed. Channel and hybrid catfish that were not fed did not lose weight, but blue catfish that were not fed lost weight. Fed fish from all 3 groups were fatter and had higher fillet yield than unfed fish from the same groups. Fillet yield was highest for hybrid catfish, than for channel catfish or blue catfish regardless of feeding regime. This research supports NP106 Action Plan Component 1b (GENETIC IMPROVEMENT: Selective breeding for economically important traits). Effects of diet and feeding strategies on production traits and processing yield of catfish. -- Cooperative efforts to determine effects of diet composition and feeding strategies on catfish growth and processing yield have continued with Mississippi State University nutritionists. USDA Catfish Genetics Unit employees assist in this cooperative effort by providing facilities, labor, and expertise for measurement of processing yield (fillet, carcass and nugget yield). This research supports NP106 Action Plan Components 1b (GENETIC IMPROVEMENT: Selective breeding for economically important traits) and Component 4 (Growth and Development, and Nutrition). Production of a physical map of the catfish genome. -- A large amount of molecular genetic have been collected from the catfish genome, but that data have not yet been organized in the context of the 29 catfish chromosomes. Over 46,000 bacterial artificial chromosome (BAC) clones, each containing a fragment of the catfish genome, were organized into a physical map of re-assembled catfish genomic fragments. This physical map is being anchored to the genetic map, and 200 genetic markers are shared between the two maps to date. The clones were assembled into 1, 782 clusters which will facilitate location of all catfish DNA sequences discovered to date. The map will improve the efficiency of finding genes controlling important production traits and finding genetic markers useful for selection of superior catfish broodstock. This research supports NP 106 Action Plan component 1c (GENETIC IMPROVEMENT; Genomic Resources). Locating conserved genes in the catfish genome. -- The catfish genetic map contains DNA markers located throughout the genome, but the catfish- specific markers are not useful for comparisons of genome structure between species. Therefore, we produced DNA markers for genes that are conserved between species, specifically expressed sequences tags (ESTs) that contain variable DNA sequences termed microsatellites. Out of 309 EST markers developed, 137 (44%) could be placed on the catfish genetic map because there was no microsatellite variation in the mapping population. These genes provide anchoring points for comparison of the catfish genome with other species, which will assist further development of the catfish genetic map and its use for marker assisted selective breeding. Production of completely inbred catfish. -- Inbreeding can be used to concentrate positive genetic alleles from a population, remove deleterious recessive alleles, and provide genetically identical animals to serve as a baseline for measurement of trait variation. Because there are no inbred catfish lines, we produced completely homozygous catfish via pressure treatment of catfish eggs activated by sperm that had been treated with ultraviolet light. Testing of resulting offspring with molecular genetic markers revealed that each genetic marker that was heterozygous (containing two variants) in the mother was homozygous (containing only one of the variants) in the offspring. Each marker also showed a complete lack of paternal contribution to the offspring genomes. Because the homozygous founders provide a source of genomic DNA for which all loci are homozygous, this will help researchers identify members of multiple copy gene families and also serve as a superior source of genomic DNA for the sequencing of the catfish genome. Development of homozygous catfish lines from these founders will also permit the measurement of environmental variation between tanks and ponds. This research supports NP 106 Action Plan component 1c (GENETIC IMPROVEMENT; Genomic Resources). Sex-specific growth differences in catfish. -- Male channel catfish grow faster than female catfish, however the relative contribution of the genotype and phenotype to the difference in growth should be known before an all male strain of catfish is developed for use in aquaculture. All male catfish with an XY genotype were produced by mating a YY male with a normal, XX female. A portion of fish from each family were hormonally feminized and the growth of the two groups was measured. The results demonstrated that sex genotype and phenotype both contributed to the male growth advantage in channel catfish and these data provide further evidence that monosex male culture would produce an economic gain of about 5% for the industry. This research supports NP106 Action Plan Component 1d (GENETIC IMPROVEMENT; Specific Breeding Aids). Accelerated maturation of catfish broodstock. -- A major difficulty in genetic selection of channel catfish is that most fish do not reproduce until they are three years old. If sexual maturation could be accelerated, then fish could be spawned earlier and the process of selection for desirable characteristics would be more rapid. Groups of channel catfish were held in outside ponds, in indoor tanks maintained at a constant 26 C, or under a cycled temperature regime of 4 months at 26 C and 2 months at 12 C and each group were fed when they would eat. The fish held indoors were the largest, however, fish from the 4-2 month cycled group had over 50% spawning success compared to only 2 spawns in the pond raised fish. These data suggest it is possible to decrease the time to sexual maturation by exposing fish to shortened annual cycles and that annual cycles are more important in determining puberty than the size of the fish. This research supports NP106 Action Plan Component 1d (GENETIC IMPROVEMENT; Specific Breeding Aids). Identification of Toll-like receptors in catfish. -- Losses to disease are a major economic burden to catfish producers, and understanding the molecular pathways in the catfish immune system can help us identify catfish with superior innate immunity. Toll-like receptors (TLR) play a key role in the recognition of microbes by vertebrates. We have identified and obtained the DNA sequence for ten TLR genes in channel catfish in addition to the 3 previously published. Expression studies are now underway to examine TLR gene activity in catfish after bacterial and viral infection and to determine if DNA sequence variation in TLR genes is predictive of enhanced disease resistance. This research supports NP 106 Action Plan component 2c (INTEGRATED AQUATIC ANIMAL HEALTH MANAGEMENT; Immunology and Disease resistance). Jar hatching of catfish eggs.-- Channel x blue catfish hybrids exhibit excellent performance for growth, meat yield, and disease resistance and there is considerable interest in commercial production of hybrids. However, production of hybrid fry requires hormone-induced ovulation and strip spawning of female catfish which typically results in poor fertility and hatch thus, it constrains widespread production of hybrids. Hybrid egg masses typically have low fertility, and the unfertilized eggs serve as a substrate for fungal and bacterial growth which spreads to viable embryos and greatly reduces hatch. Egg masses can be separated by chemical treatment and incubated in hatching jars which results in the more buoyant unfertilized eggs separating from good eggs. Although catfish eggs can be hatched in standard hatching jars, the capacity of these jars is too small to be useful given the volume of eggs hatched at a commercial catfish farm. A �large-volume hatching jar� was developed from a commercially available rotifer hatching tube and successfully used to hatch channel catfish eggs. Results with channel catfish eggs suggest each jar could be used to hatch 20 kg of eggs (~ 30 spawns) with hatch rates similar or better than traditional hatching of egg masses in troughs. The large-volume jars should yield greater improvements in hatching with hybrid eggs because initial fertility will be lower than it was for the channel catfish spawns used in our initial trials. Trials with large-volume hatching jars will be conducted at two commercial farms producing hybrids during the spring of 2008. This research supports NP106 Action Plan Component 3c (REPRODUCTION AND EARLY DEVELOPMENT: Gamete and Zygote Quality). IGF-II as an indicator of growth in channel catfish. -- Emerging evidence points to important roles for insulin like growth factor-I and �II (IGF-I and IGF-II) in fish growth. Abundance of IGF-I and IGF-II mRNA was investigated in slow and fast growing USDA303-strain families of catfish. While liver and muscle IGF-I mRNA levels were similar between families, muscle IGF-II mRNA levels were higher in fast growing families of fish compared to the slower growing families of fish. We also found that cortisol levels were negatively correlated to weight gain among the fast and slow growing families of catfish. Differences in levels of IGF- II mRNA in fast growing fish support IGF-II�s role in growth regulation of channel catfish. IGF-II could potentially be a candidate gene whose expression may reflect differences in muscle growth. The relationship between stress responsiveness and weight gain warrants further investigation into its utility in our selective breeding program. This research supports NP 106 Action Plan component 4b, GROWTH AND DEVELOPMENT, AND NUTRITION; Tissue Growth and Development). Significant Activities that Support Special Target Populations Development of catfish lines with superior performance for commercially important traits, utilization of these lines in commercial culture, and utilization of production technology originating from the Catfish Genetics Research Unit will help solve production problems, increase efficiency and profitability for both small and large catfish farmers, and provide a quality product for consumers. Most catfish producers with limited acreage buy fingerlings from large breeders that are very likely to utilize improved brood stocks, and the development and use of improved catfish lines can quickly affect the profits of small producers. Because small farms do not enjoy the same economies of scale experienced by larger operations, breeding fish with improved production traits will be highly beneficial to small farmers. The USDA Census of Aquaculture conducted in 2000 classified 84% of catfish farms as small businesses, with annual sales of less than $500,000. Of the 1,370 catfish farms in the United States, 38% (515) reported annual revenues of less than $25, 000. These data are not available in the 2005 Census of Aquaculture. Technology Transfer Number of Web Sites managed: 1 Number of Non-Peer Reviewed Presentations and Proceedings: 27 Number of Newspaper Articles,Presentations for NonScience Audiences: 4

Impacts
(N/A)

Publications

• Barrero, M., Small, B.C., D'Abramo, L., Hanson, L., Kelly, A. 2007. Comparison of Estradiol, Testosterone, Vitellogenin and Cathepsin Profiles Among Two-year-old Channel Catfish (Ictalurus punctatus) Females from Four Selectively Bred Strains. Aquaculture 264:390-397.
• Venn Beecham, R., Small, B.C., Minchew, C.D. 2006. Using portable lactate and glucose meters for catfish research: acceptable alternatives to established laboratory methods?. North American Journal of Aquaculture 68:291-295.
• Bosworth, B.G., Wolters, W.R., Silverstein, J., Li, M.H., Robinson, E.H. 2006. Family, strain, gender, and dietary protein effects on production and processing traits of norris and NWAC103 strains of channel catfish, Ictalurus punctatus. North American Journal of Aquaculture 69:106-115.
• Bosworth, B.G., Small, B.C., Gregory, D.J., Kim, J.M., Black, S., Jerrett, A.R. 2006. Effects of rested-harvest using the anesthetic aqui-s on channel catfish, ictalurus punctatus, physiology and fillet quality. Aquaculture 262:302-318.
• Li, M.H., Robinson, E.H., Bosworth, B.G. 2007. Effects of Dietary Protein Concentration and L-carnitine on Growth, Processing Yield, and Body Composition of Channel X Blue Catfish Hybrids. North American Journal of Aquaculture 69:229-234.
• Li, M.H., Robinson, E.H., Oberle, D.F., Bosworth, B.G. 2006. Effects of Dietary Protein Concentration and Feeding Regimen on Channel Catfish Ictalurus punctatus Production. Journal of the World Aquaculture Society 37:370-377.
• Manning, B.B., Li, M.H., Robinson, E.H., Peterson, B.C. 2006. Enrichment of channel catfish (ictalurus punctatus) fillets with conjugated linoleic acid and omega-3 fatty acids by dietary manipulation. Aquaculture 261:337- 342.
• Peterson, B.C., Small, B.C. 2007. Effects of GH on Immune and Endocrine Responses of Channel Catfish Challenged with Edwardsiella ictaluri. Comparative Biochemistry and Physiology A 146:47-53.
• Quiniou, S.M., Waldbieser, G.C., Duke, M.V. A first generation bac-based physical map of the channel catfish genome. Biomed Central (BMC) Genomics.
• Robinson, E.H., Li, M.H., Oberle, D.F., Bosworth, B.G. 2006. Efficacy of high-protein "finishing" diets on growth, fattiness, and processing yield of channel catfish, ictalurus punctatus. Journal of Applied Aquaculture 18:37-52.
• Small, B.C., Murdock, C.A., Waldbieser, G.C., Peterson, B.C. 2006. Reduction in channel catfish hepatic growth hormone receptor expression in response to food deprivation and exogenous cortisol. Domestic Animal Endocrinology (online). . Domestic Animal Endocrinology.

Progress 10/01/05 to 09/30/06

Outputs
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? The specific problem area is to improve economically important traits of channel catfish through an applied breeding program that incorporates new biotechnologies and addresses all areas of quantitative and qualitative genetics, reproduction, and molecular and cellular genetics. Genetically improved germplasm will be developed, evaluated, and released to commercial producers. Research areas have been identified through research planning sessions with industry, state and federal research, and cooperative extension representatives. All research focuses on problems of critical importance to the catfish industry that can be solved through genetic improvement. The applied breeding program for catfish genetic improvement focuses on identifying economically important traits, determining the amount and sources of phenotypic and genetic variation for those traits, determining the best approaches to improve the traits through genetic selection, and incorporating molecular genetic and specific breeding aid technologies into the applied breeding program. The project's overall objective is to develop a breeding program to improve catfish production. Specific objectives are: 1)characterize variation for important traits; 2)develop molecular markers and a genetic map, and incorporate molecular markers into the breeding program; 3) conduct research to develop management protocols for optimizing growth, production and fish health of genetically improved catfish lines released to commercial industry; and 4)release genetically improved germplasm to commercial producers. The planned research falls under National Program 106, Aquaculture. It addresses the Genetic Improvement, Integrated Aquatic Animal Health Management, and Growth and Development, and Nutrition components of the Action Plan. There has been limited genetic improvement of aquaculture stocks so there are major opportunities for improvement through traditional animal breeding, broodstock development, germplasm preservation, molecular genetics, and allied technologies. Commercial catfish farmers, like all aquaculture producers, essentially utilize fish stocks that are not many generations removed from wild, unselected stocks. Research will address improvement of growth rates, feed efficiency, survival, disease resistance, fecundity, fillet yield and product quality; environmental tolerances; genetic characterization and gene mapping; and conservation and utilization of important aquatic germplasm. Development of catfish lines or germplasm with superior performance for commercially important traits and utilization of these lines in commercial culture will help solve production problems, increase efficiency and profitability, and provide a quality product for consumers. 2. List by year the currently approved milestones (indicators of research progress) FY 2005 (Year 1) (1) Measure genetic variation in production traits in the USDA103 line and characterize correlations between traits. (2) Begin ranking of Teneration 4 families for ESC resistance: Replicated disease challenges of Generation 4 families are currently underway and will be completed by 2005. (3) Validate GTH-II immunoassay. (4) Develop genomic resources for integrating functional genomics into the catfish applied breeding program. (5) Produce F1 carcass yield families. (6) Obtain real-time PCR results for TLR3, TLR5, lysozyme C, lysozyme G. (7) Produce oligonucleotide microarray. (8) Increase biological efficiency through selective breeding of catfish and transfer improved catfish germplasm to the U.S. catfish industry. FY 2006 (Year 2) (1) Determine growth and carcass yield breeding values for Generation 4 (G4) fish. (2) Complete development of fluoroimmunoassay for IGF2 and real-time PCR assay for IGF binding proteins. (3) Complete ranking of Generation 4 families for ESC resistance. (4) Develop genomic resources for integrating functional genomics into the catfish applied breeding program. (5) Begin placement of additional conserved genes on linkage map. (6) Develop web-based database of molecular markers and linkage map. (7) Produce F1 ESC resistance families. (8) Begin identification of additional Toll-like receptors. (9) Increase biological efficiency through selective breeding of catfish and transfer improved catfish germplasm to the U.S. catfish industry. (10) Begin to develop individual selection index for Generation 4 broodfish. FY 2007 (Year 3) (1) Measure genetic variation in production traits in the USDA 103 line and characterize correlations between traits. (2) Begin calculation of correlations for growth efficiency and IGF molecular data. (3) Begin ranking of Generation 5 families for ESC resistance. (4) Begin correlation of GTH-II response and reproductive success. (5) Continue placement of additional conserved genes on linkage map. (6) Produce F2 carcass yield families. (7) Complete identification of additional Toll-like receptors. (8) Begin identification of candidate genes for ESC resistance/susceptibility. (9) Complete development of individual selection index for Generatoin 4 broodfish. (10) Begin collection of data from the comparison of Generation 5 fish with Generation 2 fish (NWAC103). FY 2008 (Year 4) (1) Determine growth and carcass yield breeding values for G5. (2) Continue calculation of correlations for growthefficiency and IGF molecular data. (3) Rank Generationi 5 families for ESC resistance. (4) Continue correlation of GTH-II response and reproductive success. (5) Continue placement of additional conserved genes on linkage map. (6) Produce F2 disease resistance families. (7) Begin correlation of molecular data with ESC resistance. (8) Continue identification of candidate genes for ESC resistance/susceptibility. (9) Begin identification of candidate genes for incidence of spawning. (10) Begin development of individual selection index for G5 broodfish. (11) Complete collection of data from comparison of Generation 5 with Generation 2 (NWAC103). FY 2009 (Year 5) (1) Complete calculation of correlations for growth efficiency and IGF molecular data. (2) Complete correlation of GRH-II response and reproductive success. (3) Continue placement of additional conserved genes on linkage map. (4) Identify QTLs for carcass yield and ESC resistance. (5) Calculate correlation of molecular data with ESC resistance. (6) Continue identification of candidate genes for ESC resistance/susceptibility. (7) Continue identification of candidate genes for incidence of spawning. (8) Complete development of individual selection index for G5 broodfish. (9) Produce selected fingerlings for release to industry. 4a List the single most significant research accomplishment during FY 2006. Toll-like receptor (TLR) gene expression was tested in families of channel catfish that showed strong differentiation in response to ESC challenge (high and low susceptibility). When exposed fish were compared to controls, expression of the TLR5 gene in stomach and liver was elevated 200 fold in fish with low susceptibility to ESC compared to only a 2-fold increase in fish with high susceptibility. Thus the TLR5 gene will serve as a candidate gene for further testing, and potentially a good candidate for marker-assisted selection of catfish with improved survivability. 4b List other significant research accomplishment(s), if any. Feed intake is highly correlated to growth in channel catfish and the efficiency of growth affects the profitability of catfish production. In terrestrial livestock, ghrelin is a potent regulator of feeding and the catfish ghrelin gene was sequenced in our laboratory last year. More recently, research was conducted to identify the regulatory mechanisms of ghrelin by sequencing and characterizing the gene for ghrelin-receptor. Utilizing this information, a bio-assay has been developed to determine the effects of ghrelin administered directly into the brain on its receptor and ultimately on feed regulation and efficient growth. The new insight provided may aid in the identification of catfish that are more efficient at converting feed into weight gain. Selective breeding for such fish would aid commercial catfish producers by reducing the amount feed and associated cost required to get their fish to a marketable size. This research contributes to Program Component GROWTH AND DEVELOPMENT, AND NUTRITION; a. Regulating Feed Intake. Reproductive efficiency is important for the sustained production of channel catfish; however, the hormonal and genetic regulation of catfish reproduction is poorly understood. Steroidogenic factor-1 (SF-1) is a transcription factor with the potential to control the production of estradiol, an important steroid for successful reproduction. Research was conducted that characterized the seasonal regulation of SF-1 and correlated its expression to estradiol production. This information will be utilized to identify broodfish with greater reproductive potential and may aid in the development of new tools for inducing spawning. This research contributes to Program Component REPRODUCTION AND EARLY DEVELOPMENT; a. Control of Reproduction. Insulin like growth factor-I (IGF-I) plays an important role in growth of mammals, but its function in catfish is poorly understood. Research in cooperation with Mississippi State University examined the role of IGF-I in three different strains of catfish. Results showed that plasma IGF-I concentrations were positively correlated to weight gain. IGF-I may be an indicator of fast growth and identifying polymorphisms in IGF genes may have potential for use in DNA marker-assisted selection programs. This work addresses Objective 1: Measure genetic variation in production traits in the USDA103 line and characterize correlations between traits. This research contributes to Program Component GROWTH AND DEVELOPMENT, AND NUTRITION. A coordinated regulation of fish growth and immune response to disease is essential to maintain homeostasis. Although the mechanisms through which the endocrine and immune system communicate is not known in catfish, a better understanding of this system may provide insight into how to select for growth and disease resistance. Research examined the effects of virulent Edwardsiella ictaluri (E. ictaluri) on growth hormone (GH)- injected channel catfish. Results showed that disease downregulated insulin like growth factor-I and growth hormone receptor, components involved in the regulation of growth, while GH treatment stimulated levels of lysozyme, a component of the nonspecific immune system. Defining the mechanisms through which E. ictaluri affects the immune system will be crucial in developing more effective methods for enhancing the immune system through selective breeding. This work addresses Sub- objective 2.3: Develop molecular markers for disease resistance candidate genes based on comparative functionality with other species. This research contributes to Program Component INTEGRATED AQUATIC ANIMAL HEALTH MANAGEMENT; c. Immunology and Disease Resistance. Selective breeding of channel catfish for resistance to a bacterial pathogen has shown some success in the laboratory. A multitrait selection index was utilized to select for growth, carcass yield, feed efficiency, and resistance to enteric septicemia of catfish (ESC). After two generations of selection, 62 families of USDA303 catfish were screened for ESC susceptibility and were compared to 29 families of the parent strain USDA103 in two replicate experimental challenges. The USDA303 catfish population showed a 10% improvement in resistance to ESC than NWAC103 catfish during replicate challenges. This suggests that resistance to ESC has a genetic component and selective breeding could be useful in providing some increase in ESC survivorship in the pond environment. This research contributes to Program Component INTEGRATED AQUATIC ANIMAL HEALTH MANAGEMENT; c. Immunology and Disease Resistance. Development of ESC resistant line is continuing. Survivors from the 10 most resistant families were pit-tagged and released into ponds. These fish were used to produce F1 resistant families. The spawning rate was 50%. Fish from these families are being evaluated for growth and susceptibility to ESC. These fish have the potential to become a selected line that will eventually be crossed with an improved growth strain in order to improve susceptibility to ESC with minimum sacrifice to growth characteristics. This research contributes to Program Component INTEGRATED AQUATIC ANIMAL HEALTH MANAGEMENT; c. Immunology and Disease Resistance. Survivorship to ESC (Enteric Septicemia of Catfish) varies among and within strains of commercially raised catfish, however the immunological basis for differences in susceptibility are not well-understood. Mortality, pathogen levels (bacterial DNA), responses of immune receptor expression (TLR3 and TLR5), and lysozyme activity were measured during experimental challenge with virulent Edwardsiella ictaluri in channel catfish (Ictalurus punctatus) strains: USDA103, Red River (RR), USDA103 x Red River, and Blue catfish (I. furcatus), as well as USDA103 x Blue. Fish were sampled throughout a 21 day challenge. Mortality and lysozyme activity were positively correlated. There was a slight negative correlation between expression of TLR3 in kidney tissue and mortality late in challenge. Understanding these differences will be crucial for enhancing the immune system through selective breeding and in developing disease management protocols. This research contributes to Program Component INTEGRATED AQUATIC ANIMAL HEALTH MANAGEMENT; c. Immunology and Disease Resistance. Estimation of phenotypic and genetic (co)variances for economically important traits are required for development of a breeding program to produce superior catfish germplasm for release to catfish producers. Data from feeding trials and challenges with pathogenic bacteria that cause Enteric Septicemia of Catfish (ESC) have been collected from full and half-sib families and are currently being analyzed to determine heritabilities and genetic correlations for these traits. Heritabilities and genetic correlations for growth and carcass yield will be estimated when data for these traits are collected from the same families in the fall of 2006. This information is being used to develop superior germplasm for release to which will benefit producers, processors, and consumers. This research contributes to Program Component GENETIC IMPROVEMENT; b. Selective breeding for economically important traits. Information on the relative economic value of various production traits is needed to determine the amount of emphasis that should be applied to selection for improvement in different traits. Research is currently underway to estimate the relative economic value of traits important in farmed catfish (growth, feed conversion, meat yield, disease resistance). Data collection and analysis should be complete for most traits by the end of 2006. This research contributes to Program Component GENETIC IMPROVEMENT; b. Selective breeding for economically important traits. Economic losses associated with diseases are an increasing problem for farm-raised catfish producers. A study to compare production traits of USDA 103 strain channel catfish, USDA 102 strain channel catfish, USDA 103 x USDA 102 crossbred, and USDA 103 strain channel catfish x blue catfish hybrids is underway. Hybrids had the best fingerling performance of all groups. Of the channel catfish groups, the USDA 103 x USDA 102 cross was superior to the USDA 103 and USDA 102 for fingerling production traits. The same crosses are currently being evaluated for foodfish production and processing traits. Initial results indicate crossbreeding and hybridization could enhance catfish production. This research contributes to Program Component GENETIC IMPROVEMENT; b. Selective breeding for economically important traits. It is important to understand the effects of dietary protein and feeding frequency on important traits such as meat yield of catfish (the percentage of whole fish weight that is saleable meat) do determine the most profitable combination of dietary protein and feeding frequency. Effects of dietary protein level and frequency of feeding on meat yield of channel catfish were determined. Meat yield increased as dietary protein level and feeding frequency increased. Catfish farmers need to consider the impact of dietary protein level and feeding frequency on fish growth and meat yield in determining the optimal protein level and feeding frequency. However, the optimal combination of dietary protein level and feeding frequency changes with the price of feed and the price of fish. This research contributes to Program Component QUALITY, SAFETY AND VARIETY OF AQUACULTURE PRODUCTS FOR CONSUMERS; b. Interaction of Genetics and Nutrition. Channel catfish x blue catfish hybrids are superior to channel catfish for several production traits, however, commercial use of hybrids is limited by inability to produce large numbers of hybrid fry. Blue male catfish must be sacrificed and their testes removed to provide sperm for hybrid production but testes development of blue male catfish varies considerably among individuals and undeveloped males are frequently killed which leads to increased costs for hybrid production. During the spring of 2006 we assessed the use of laparoscopic examination to assess development of blue catfish testes. We determined laparoscopic examination is a fairly quick, accurate, non-lethal method for assessing testes development in blue catfish. This research contributes to Program Component GENETIC IMPROVEMENT; b. Selective breeding for economically important traits. Channel catfish x blue catfish hybrids are superior to channel catfish for several production traits, but poor fertility and hatch are factors which impede large-scale production of hybrids. Catfish eggs are adhesive and are typically hatched as a large egg mass, but poor fertility typical of hybrid egg masses leads to increased bacterial and fungal infection and reduced hatch. We compared jar-hatching of hybrid eggs after chemical elimination of the adhesiveness to traditional hatching of egg masses and found use of jars increased hatch to 6.8 hybrid fry per gram of eggs compared to 4.8 fry per gram with traditional hatching methods. This research contributes to Program Component REPRODUCTION AND EARLY DEVELOPMENT; e. Early Life Stage Development and Survival. Channel catfish x blue catfish hybrids are superior to channel catfish for several production traits, but little is know about the effect of using different parental strains on performance of hybrids. A study was initiated to compare ESC resistance, growth, and processing traits of hybrids produced from 3 channel catfish strains and 2 blue catfish strains. This information will be useful in determining which channel and blue catfish strains produce the best hybrid offspring for commercial production. This research contributes to Program Component GENETIC IMPROVEMENT; b. Selective breeding for economically important traits. Growth trials conducted at the Catfish Genetics Research Unit (CGRU) have shown that blue catfish may have potential as an alternative to channel catfish for commercial production. However, little is known about machine settings required for commercial processing of blue catfish. We worked with a commercial processor and found that minor adjustments to fillet machines set for channel catfish were required for processing of blue catfish. This information was made available by a presentation to the industry at the Catfish Processors Conference held at Mississippi State University, Starkville, MS, in May, 2006. This research contributes to Program Component QUALITY, SAFETY AND VARIETY OF AQUACULTURE PRODUCTS FOR CONSUMERS; g. Processing. Genetic improvement of economically significant traits in channel catfish is needed for the long term viability of the industry. This research is to identify and characterize candidate genes involved in growth and lipid metabolism. Two candidate genes involved in growth have been identified and characterized including, insulin-like growth factor-I and -II (IGF-I and -II). The further development of molecular tools involved in growth and lipid metabolism and their characterization is the foundation for understanding the functions of the candidate genes for these traits in channel catfish and could lead to the genetic improvement of these economically significant traits. This research contributes to Program Component GROWTH AND DEVELOPMENT, AND NUTRITION; b. Tissue Growth and Development. Feed costs have a major influence on total production costs in the catfish industry and, selection for feed efficiency has improved total production efficiency in several animal systems. Since accurate measurement of feed efficiency is extremely difficult and expensive to accomplish in individuals, an indirect selection criterion, such as lipid content would be very beneficial. We are conducting studies to determine if lipid content correlates with feed efficiency. Preliminary results indicate a significant relationship between feed efficiency and lipid content in analyses with whole body channel catfish. These studies will provide useful information to farmers and could greatly reduce production costs. This research contributes to Program Component GROWTH AND DEVELOPMENT, AND NUTRITION; b. Tissue Growth and Development. Genetic control of resistance to pathogens may be manifest as changes in gene expression during or after pathogen exposure, but the problem is in measuring many genes at once. We utilized genomic technology to produce the first catfish DNA microarray. This array contained DNA probes for 19, 000 catfish genes and an initial experiment showed it was capable of detecting differences in gene expression due to pathogen exposure. The catfish microarray will be useful for efficiently measuring levels of expression of many genes at once and helping researchers identify candidate genes that influence production traits. This research contributes to Program Component GENETIC IMPROVEMENT; c. Genomic Resources. The molecular mechanisms involved in catfish responses to bacterial pathogens are not well understood. We used the newly developed catfish microarray to measure levels of expression of 19,000 genes in the spleen after injection of bacterial cell wall components that elicit immune reponses. The results showed expression of 64 genes was significantly increased and expression of 74 genes was significantly decreased after injection. These experiments provided researchers with candidate genes for further analysis to determine whether differences in responses correlate with improved survival after pathogen challenge. This research contributes to Program Component INTEGRATED AQUATIC ANIMAL HEALTH MANAGEMENT; c. Immunology and Disease Resistance. 4c List significant activities that support special target populations. Development of catfish lines with superior performance for commercially important traits and utilization of these lines in commercial culture will help solve production problems, increase efficiency and profitability for both small and large catfish farmers, and provide a quality product for consumers. Most catfish producers with limited acreage buy fingerlings from large breeders that are very likely to utilize improved brood stocks, and the development and use of improved catfish lines can quickly affect the profits of small producers. Because small farms do not enjoy the same economies of scale experienced by larger operations, breeding fish with improved production traits will be highly beneficial to small farmers. The USDA Census of Aquaculture conducted in 2000 classified 84% of catfish farms as small businesses, with annual sales of less than $500,000. Of the 1,370 catfish farms in the United States, 38% (515) reported annual revenues of less than $25, 000. 4d Progress report. This is the last year of research conducted under Specific Cooperative Agreement, 6402-31000-008-02S, "Candidate Genes for Catfish Disease Resistance" with Mississippi State University. Problem: Enteric Septicemia of catfish (ESC, caused by the gram negative, facultative intracellular bacterium Edwadsiella ictaluri) is the most economically important infectious disease to channel catfish aquaculture. The basis for pathogen resistance in mammals is often due to genes whose products function in the early stages of infection. Yet, little is known about the early response of channel catfish to E. ictaluri infection. Approach: Our objective was to observe the expression profile of select genes during the first 96 hours of infection by E. ictaluri. This involved the development of real-time rtPCR assays for transcripts of genes encoding an acute phase proteins (transferrin, serum amyloid P), macrophage responsive protein (Nramp), cell stress responsive protein (heat shock 70-HSP70), neutrophil proteins (beta integrin and E1B) and 18s ribosomal RNA. Then the expression of these genes were evaluated in two trials in which Channel catfish that were injected intraperitoneally with E. ictaluri or medium (controls) and then sampled. In the first trial the fish were sampled (n=7) at 6, 12, 24, 48, and 96 h after injection. The spleen, liver, anterior kidney (AK), and gut were sampled for RNA extraction. The posterior kidneys (PK) were collected for bacterial counts. In the second trial whole blood, liver and spleen were sampled for RNA expression analysis and tumor necrosis factor alpha (TNF alpha) was added to the gene expression profile. Also blood smears were taken for whole blood counts and samples of the liver and spleen were embedded in OCH medium and quick frozen for cryostat sectioning. Results: The first trial demonstrated that most transcripts were found universally but, SAP and transferrin were liver-specific. Expression of EB1 (AK, spleen and liver), Hsp70 (AK and spleen), Beta1-integrin (liver) and Nramp (spleen and gut) significantly increased by 48 hpi. Transferrin was strongly up-regulated and SAP was down-regulated by 72 hpi, suggestive of positive and negative acute phase reactants. The second ESC challenge experiment, EB1, Hsp70, Beta1-integrin, TNF alpha and Nramp expression were demonstrated in the peripheral blood. SAP and transferrin expression were not detected. TNF alpha was upregulated at 24h in response to infection. The spleen also demonstrated a 24h TNF alpha induction and this was followed by the EB1, Hsp70, Beta1-integrin, and Nramp induction at 48h and 72h. Cryostat sections were used for histochemical analysis of leukocyte population and for laser capture micro dissection (LCM) to evaluate tissue specific gene expression within the spleen. Analysis of the 72 hr samples demonstrate a marked increase in neutrophils throughout the spleen in infected fish. Neutrophils were focused around the periarterial lymphoid sheaths (PALS) in non-infected fish. LCM sampling of both tissue types from infected and non-infected fish and real-time quantitative PCR on the RNA extracts supports the data indicating differential distribution of neutrophils. EB-1 was non- detectable in parenchymal tissue, and expressed at a relatively low level in the PALS of non-infected tissue compare to high levels of expression throughout both tissues types in infected fish. As a comparison, HSP70 was expressed in both tissues in both groups but expression was highest in the infected parenchymal tissue. Impact: By evaluating expression of genes involved in the inducible portion of the innate defenses during the early stages of ESC we were be able to identify the critical time points that determine ESC outcome. The initial response occurs by 24 hours and secondary responses were apparent by 48 hours post-infection. This will allow researchers to focus on this critical window to identify the genetic basis of ESC resistance and factors that enhance or suppress the protective response. This is the last report of research conducted under Specific Cooperative Agreement, 6402-31000-008-03S, "Channel Catfish Molecular Markers" with Auburn University. The collaborative project has had several objectives. The very first was to identify DNA markers within genes of known functions, particularly those already mapped on the human and zebrafish linkage maps, are valuable for mapping the catfish genome because they provide landmarks for comparative genome mapping. The second was to characterize certain innate immune-related genes, in particular the CXCL8 gene that appear to have multiple versions of cDNAs that may be exploited as a marker for mapping of this gene. Third, after initial development of genetic linkage maps using various resource families, it is important to integrate the maps using the same resource DNA. For the first objective, we took the approach of "intron tagging". By amplification and sequence analysis of introns of known genes, polymorphic markers can be identified because intron sequences are highly variable and often contain microsatellites. On the basis of progress made in previous years, in the last year, we have completed the objective. We designed primers for 50 genes to amplify their introns. Of the 50 pairs of primers designed, 42 produced products. These PCR segments were sequenced. Sequence analysis of the 42 introns indicated that 8 (19%) included microsatellite sequences in them (Table 1). The sequences have been deposited to GenBank available for public use and their accession numbers are included in the publication. The information was transferred to the laboratory whose personnel conducted mapping analysis of the polymorphic markers. This work was published in Animal Genetics (Serapion et al., 2004). Interleukin-8 is a CXC type chemokine produced in response to stimulation by pro-inflammatory cytokines or bacterial lipopolysaccharides. We cloned and characterized interleukin-8 cDNAs and its genomic segments containing all the exons and introns from channel catfish (Ictalurus punctatus). Multiple interleukin-8 cDNA clones were identified during analysis of expressed sequence tags (ESTs). Sequence analysis indicated presence of four types of alternatively spliced and two types of alternatively polyadenylated transcripts. Analysis of the genomic DNA of the locus confirmed that all four types of transcripts were likely derived from partial splicing. Channel catfish interleukin-8 gene has four exons and three introns with highly conserved splice sites as compared to interleukin-8 genes of other organisms. In spite of the structural conservation through evolution, the piscine interleukin-8 genes showed a much greater sequence divergence than their counterparts among mammals. RT-PCR indicated that two of the four splicing forms were expressed at high levels whereas the other two aberrant splicing forms were not detectable. Expression of interleukin-8 gene was up-regulated in channel catfish and blue catfish after infection with pathogenic bacteria Edwardsiella ictaluri. This work was published in Developmental and Comparative Immunology (Chen et al., 2005). After consulting with CGRU scientist, an expanded objective was initiated: Integration of various linkage maps by using the same resource markers. In the last several months, experiments have been initiated to genotype microsatellite markers that were mapped using the channel catfish resource families. In this case, genotyping was conducted using PCR with channel catfish x blue catfish interspecific resource DNA. Several points are important for this objective: 1)integration of existing genetic maps will enhance the resolution of the linkage maps; and 2)use of the same set of microsatellite primers in different resource families will allow comparison of the mapping populations and their impact on genetic distances, particularly between the intraspecific and interspecific families. To date, almost 50 microsatellite primer pairs have been tested and genotyped with 64 fish from an interspecific family. 5. Describe the major accomplishments to date and their predicted or actual impact. Major accomplishments expected over the life of the project are: 1) continued improvement in performance of catfish line USDA103; 2) development and publication of a second-generation catfish genetic linkage map containing conserved genes useful for comparative mapping; and 3)continued development of a multi-trait selection program utilizing the USDA103 and other catfish germplasm to develop additional improved lines for future release to commercial producers. Action Plan components: Genetic Improvement sections b and c. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? To date, the main transfer of science and technology has been scientific publication in peer-reviewed journals, popular press, discussions with catfish producers, and a joint ARS-MAFES extension workshop to educate catfish producers in the methods for producing hybrid catfish. The most significant transfer of technology under the previous project from the Catfish Genetics Research Unit was the development of the USDA103 catfish line, which demonstrated superior growth performance. Broodfish from this line, named NWAC103, were made available to commercial producers through a joint release by ARS and MAFES beginning February 1, 2001 and completed in February 2002. Another germplasm release is planned for the end of this project, but this will depend on acceptance and approval by partners and stakeholders. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Avery, J, J. Steeby, B.G. Bosworth, B.C. Small. 2005. Producing hybrid catfish fry: Workshop Manual. Mississippi State University, Delta Branch Experiment Station, Stoneville, MS. http://www.msstate. edu/dept/tcnwac/HybridManual05.pdf. p. 115. Beecham, R.V., B.C. Small, and C.D. Minchew. 2006. Using portable lactate and glucose meters for catfish research: an Inexpensive alternative to established laboratory methods? Proc. Aquaculture America 2006, Las Vegas, NV. p. 22. Bilodeau, L, Peterson, BC. Immune response of catfish strains exposed to virulent Edwardsiella ictaluri. Abstract, VIIth International Congress on the Biology of Fish in St. Johns, Canada, July 18-23, 2006, p. 61. Bosworth, B., B. Small, J. Steeby, and J. Avery. 2005. Producing Hybrid Catfish Fry: Workshop Manual. Mississippi State University Thad Cochran National Warmwater Aquaculture Center, Stoneville, MS. (workshop manual). Bosworth, B.G. and E.L. Torrans. 2006. Production and processing traits of blue, catfish, blue catfish x channel catfish hybrids, and three strains of channel catfish. Aquaculture America, Las Vegas, NV. Abstract No. 29. Bosworth, B.G. and E.L. Torrans. 2006. Production and processing traits of blue, catfish, blue catfish x channel catfish hybrids, and two strains of channel catfish. Catfish Farmers of America, San Antonio, TX. Abstract No. 4. Li, M., B.C. Peterson, C.L. Janes, and E.H. Robinson. Comparison of diets containing various fish meal levels on growth performance, body composition, and insulin like growth factor-I of juvenile channel catfish of different origin. Abstract, Catfish Farmers of America Meeting. San Antonio, TX, Feb. 23-25, 2006, p. 36. Li, M.H., Robinson, E.H., Mischke, C.C., Torrans, E.L., Bosworth, B.G. 2006. Effects of organic fertilization and "organic" diets on production of channel catfish in earthen ponds. Aquaculture America, Las Vegas, NV. Abstract No. 162. Murdock, CA, Small, BC, Bilodeau, AL, Peterson, BC, Waldbieser, GC. Evaluation of reference genes for quantitative PCR analyses in channel catfish tissues under varying physiological conditions. Abstract, VIIth International Congress on the Biology of Fish in St. Johns, Canada, July 18-23, 2006, p. 168. Peterson B.C. and B.C. Small. 2005. Effects of restricted feed intake on circulating levels of IGF-I and abundance of hepatic IGF-I and GH receptor mRNA in channel catfish. J. Anim. Sci. 83(s1):283. Peterson, B.C., Small, B.C., Bilodeau, A.L. 2006. Effects of Posilac on immune and endocrine responses of channel catfish challenged with Edwardsiella ictaluri. Abstract, 2006 ADSA-ASAS Joint Annual Meeting. Minneapolis, MN, July 9-13, 2006, p. 352. Peterson, BC, Small, BC. Feeding frequency, growth, ghrelin, and NYP mRNA in Norris and NWAC103 channel catfish (Ictaluris punctatus). Abstract, VIIth International Congress on the Biology of Fish in St. Johns, Canada, July 18-23, 2006, p. 183. Peterson, B.C., A.L. Bilodeau, B.C. Small. 2006. Overview of the GH-IGF- I network in regulating growth in channel catfish. Proc. Aquaculture America 2006, Las Vegas, NV. p. 231. Quiniou, S.M.A., G.C. Waldbieser. 2006. Toward production of a BAC- based physical map of the channel catfish genome. Plant and Animal Genomes XIV Conference, San Diego, CA. January 2006. P636. Small, B.C. 2006. Improvements in channel catfish growth after two generations of selection and comparison of performance traits to blue catfish and hybrid catfish fingerlings in an aquarium rack system. Proc. Aquaculture America 2006, Las Vegas, NV. p. 292. Small, B.C. 2005. Growth Improvements in the USDA103 line: Two generations later. The Catfish Journal. 9(12):11. (popular publication). Small, B. and B. Bosworth. 2005. Overview of AQUI-S research to assess effects on catfish physiology and fillet quality. 11th Annual Drug Approval Coordination Workshop. United States Fish and Wildlife Service Aquatic Animal Drug Approval Partnership Program, Bozman, MT. pp.1-4. (workshop publication). Small, B.C. 2006. Managing Hatch Rates and Diseases in Catfish Eggs. SRAC Publication No. 1804, Southern Regional Aquaculture Center. Small, B.C. and B.G. Bosworth. 2005. Overview of AQUI-S research to assess effects on catfish physiology and fillet quality. Proceedings of the 11th Annual Aquaculture Drug Approval Coordination Workshop. Bozeman, MT. pp. 1-4. Small, B.C., B.C. Peterson, and C.A. Murdock. 2006. Food deprivation and the catfish endocrine system. Proc. Aquaculture America 2006, Las Vegas, NV. p. 293. p. 17. N.G. Chatakondi, D.R. Yant, A.M. Kelly, B.C. Small, and R.A. Dunham. 2006. Past, present and future of hybrid catfish in us aquaculture. Proc. Aquaculture America 2006, Las Vegas, NV. p. 53. Small, B.C.. 2006. Improving therapeutic treatments of channel and hybrid catfish egg diseases. 2006 Catfish Culture Research Symposium- Catfish Farmers of America Annual Meeting, San Antonio, TX. Tucker, C., P.S. Silverstein, A. Camus, A.L. Bilodeau, D. Wise, and G.C. Waldbieser. 2005. Channel catfish virus disease and NWAC103 catfish. The Catfish Journal. 19(5):8. Waldbieser, G.C. 2005. Invited review of "Aquaculture and Fisheries Biotechnology: Genetic Approaches" (by Rex A. Dunham). The Quarterly Review of Biology. 80:254-255. Waldbieser, G.C. "Development of a Genomics Laboratory to Support Agricultural Research". Marine Eco-Genomics Regional Research Partnership Assessment Workshop. Medical University of South Carolina, Charleston, SC. Feb. 2006.

Impacts
(N/A)

Publications

• Small, B.C., Chatakondi, N. 2006. Efficacy of formalin as an egg disinfectant for improving hybrid catfish (ictalurus punctatus x i. furcatus) hatching success. North American Journal of Aquaculture 68:9-13.
• Small, B.C. 2006. Improvements in channel catfish growth after two generations of selection and comparison of performance traits to blue catfish and hybrid catfish fingerlings in an aquarium rack system. Aquaculture America Conference 2006, Las Vegas, NV. p. 292.
• Kaiya, H., Small, B.C., Bilodeau, A.L., Shepherd, B.S., Kojima, M., Hosoda, H., Kangawa, K. 2005. Purification, cdna cloning and characterization of ghrelin in channel catfish, ictalurus punctatus. General and Comparative Endocrinology 143:201-210.

Progress 10/01/04 to 09/30/05

Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? The specific problem area is to improve economically important traits of channel catfish through an applied breeding program that incorporates new biotechnologies and addresses all areas of quantitative and qualitative genetics, reproduction, and molecular and cellular genetics. Genetically improved germplasm will be developed, evaluated, and released to commercial producers. Research areas have been identified through research planning sessions with industry, state and federal research, and cooperative extension representatives. All research focuses on problems of critical importance to the catfish industry that can be solved through genetic improvement. The applied breeding program for catfish genetic improvement focuses on identifying economically important traits, determining the amount and sources of phenotypic and genetic variation for those traits, determining the best approaches to improve the traits through genetic selection, and incorporating molecular genetic and specific breeding aid technologies into the applied breeding program. The projects overall objective is to develop a breeding program to improve catfish production. Specific objectives are: 1) characterize variation for important traits; 2) develop molecular markers and a genetic map, and incorporate molecular markers into the breeding program; 3) conduct research to develop management protocols for optimizing growth, production and fish health of genetically improved catfish lines released to commercial industry; and 4) release genetically improved germplasm to commercial producers. The planned research falls under National Program 106, Aquaculture. It addresses the Genetic Improvement, Integrated Aquatic Animal Health Management, and Growth and Development, and Nutrition components of the Action Plan. There has been limited genetic improvement of aquaculture stocks so there are major opportunities for improvement through traditional animal breeding, broodstock development, germplasm preservation, molecular genetics, and allied technologies. Commercial catfish farmers, like all aquaculture producers, essentially utilize fish stocks that are not many generations removed from wild, unselected stocks. Research will address improvement of growth rates, feed efficiency, survival, disease resistance, fecundity, fillet yield and product quality; environmental tolerances; genetic characterization and gene mapping; and conservation and utilization of important aquatic germplasm. Development of catfish lines or germplasm with superior performance for commercially important traits and utilization of these lines in commercial culture will help solve production problems, increase efficiency and profitability, and provide a quality product for consumers. 2. List the milestones (indicators of progress) from your Project Plan. Objective 1: Measure genetic variation in production traits in the USDA103 line and characterize correlations between traits. Sub-objective 1.1: Measure growth and carcass yield in catfish after rearing in ponds. -Determine growth and carcass yield breeding values for Generation 4 (G4) fish. -Determine growth and carcass yield breeding values for Generation 5 (G5) fish. Sub-objective 1.2: Correlate levels of growth hormone, insulin-like growth factors I and II (IGF-I, IGF-II) and IGF binding proteins (IGFBPs) with growth efficiency in catfish families. -Develop a fluoroimmunoassay for IGF2 and real-time PCR assay for IGF binding proteins. -Calculate correlations for growth efficiency and IGF molecular data. Sub-objective 1.3: Quantify family variation in resistance to E. ictaluri in the 4th and 5th generations of the USDA103 line. -Rank Generation 4 families for ESC resistance. -Rank Generation 5 families for ESC resistance. Sub-objective 1.4: Determine levels of gonadotropic hormones in spawning catfish and correlate with individual variation in reproductive efficiency. -Validate GTH-II immunoassay. -Correlate GTH-II response and reproductive success. Objective 2. Develop genomic resources for integrating functional genomics into the catfish applied breeding program. Sub-objective 2.1: Add interspecific conserved genes to the catfish genetic linkage map to facilitate comparative genomic analyses. -Add two hundred expressed sequences to linkage map. -Place additional conserved genes on linkage map. -Develop web-based access to database of molecular markers and linkage map. Sub objective 2.2: Identify chromosomal regions (quantitative trait loci) containing genes controlling carcass yield and resistance to ESC. -Produce F1 carcass yield families. -Produce F1 ESC resistance families. -Produce F2 carcass yield families. -Produce F2 disease resistance families. -Identify QTLs for carcass yield and ESC resistance. Sub-objective 2.3: Develop molecular markers for disease resistance candidate genes based on comparative functionality with other species. -Obtain real-time PCR results for TLR3, TLR5, lysozyme C, lysozyme G -Identify additional Toll-like receptors. -Correlate molecular data obtained above with ESC resistance. Sub-objective 2.4: Identify candidate genes and gene products controlling economically important traits using differential gene expression on oligonucleotide microarrays. -Produce oligonucleotide microarray. -Identify candidate genes for ESC resistance/susceptibility. -Identify candidate genes for incidence of spawning. Objective 3: Increase biological efficiency through selective breeding of catfish and transfer improved catfish germplasm to the U.S. catfish industry. Sub-objective 3.1: Develop and implement an index based on breeding values of traits and their economic values to select individuals with superior composite phenotypes. -Determine economic values for traits. -Develop individual selection index for G4 broodfish. -Develop individual selection index for G5 broodfish. Sub-objective 3.2: Quantify differences in performance for production traits between selected lines and the USDA103 base population. -Obtain data from comparison of G5 with G2 (NWAC103). -Produce fingerlings for release to industry. 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Measure genetic variation in production traits in the USDA103 line and characterize correlations between traits. Milestone Substantially Met 2. Begin ranking of Generation 4 families for ESC resistance: Replicated disease challenges of Generation 4 families are currently underway and will be completed by October 2005. Milestone Substantially Met 3. Validate GTH-II immunoassay. Milestone Substantially Met 4. Develop genomic resources for integrating functional genomics into the catfish applied breeding program. Milestone Substantially Met 5. Produce F1 carcass yield families. Milestone Fully Met 6. Obtain real-time PCR results for TLR3, TLR5, lysozyme C, lysozyme G. Milestone Substantially Met 7. Produce oligonucleotide microarray. Milestone Fully Met 8. Increase biological efficiency through selective breeding of catfish and transfer improved catfish germplasm to the U.S. catfish industry. Milestone Substantially Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? FY 2006 Objective 1: Measure genetic variation in production traits in the USDA103 line and characterize correlations between traits. Sub-obj. 1.1 Determine growth and carcass yield breeding values for Generation 4 fish. Sub-obj. 1.2 Complete development of fluoroimmunoassay for IGF2 and real- time PCR assay for IGF binding proteins. Sub-obj. 1.3 Complete ranking of Generation 4 families for ESC resistance. Objective 2. Develop genomic resources for integrating functional genomics into the catfish applied breeding program. Sub-obj. 2.1 Complete addition of 200 expressed sequences to linkage map. Begin placement of additional conserved genes on linkage map. Develop web-based database of molecular markers and linkage map. Sub-obj. 2.2 Produce F1 ESC resistance families. Sub-obj. 2.3 Begin identification of additional Toll-like receptors. Objective 3: Increase biological efficiency through selective breeding of catfish and transfer improved catfish germplasm to the U.S. catfish industry. Sub-obj. 3.1 Complete determination of economic values for traits. Begin to develop individual selection index for Generation 4 broodfish. FY 2007 Objective 1: Measure genetic variation in production traits in the USDA103 line and characterize correlations between traits. Sub-obj. 1.2 Begin calculation of correlations for growth efficiency and IGF molecular data. Sub-obj. 1.3 Begin ranking of Generation 5 families for ESC resistance. Sub-obj. 1.4 Begin correlation of GTH-II response and reproductive success. Objective 2. Develop genomic resources for integrating functional genomics into the catfish applied breeding program. Sub-obj. 2.1 Continue placement of additional conserved genes on linkage map. Sub-obj. 2.2 Produce F2 carcass yield families. Sub-obj. 2.3 Complete identification of additional Toll-like receptors. Sub-obj. 2.4 Begin identification of candidate genes for ESC resistance/susceptibility. Objective 3: Increase biological efficiency through selective breeding of catfish and transfer improved catfish germplasm to the U.S. catfish industry. Sub-obj. 3.1 Complete development of individual selection index for Generation 4 broodfish. Sub-obj. 3.2 Begin collection of data from the comparison of Generation 5 fish with Generation 2 fish (NWAC103). FY 2008 Objective 1: Measure genetic variation in production traits in the USDA103 line and characterize correlations between traits. Sub-obj. 1.1 Determine growth and carcass yield breeding values for G5. Sub-obj. 1.2 Continue calculation of correlations for growth efficiency and IGF molecular data. Sub-obj. 1.3 Complete ranking of Generation 5 families for ESC resistance. Sub-obj. 1.4 Continue correlation of GTH-II response and reproductive success. Objective 2. Develop genomic resources for integrating functional genomics into the catfish applied breeding program. Sub-obj. 2.1 Continue placement of additional conserved genes on linkage map. Sub-obj. 2.2 Produce F2 disease resistance families. Sub-obj. 2.3 Begin correlation of molecular data with ESC resistance. Sub-obj. 2.4 Continue identification of candidate genes for ESC resistance/susceptibility. Sub-obj. 2.4 Begin identification of candidate genes for incidence of spawning. Objective 3: Increase biological efficiency through selective breeding of catfish and transfer improved catfish germplasm to the U.S. catfish industry. Sub-obj. 3.1 Begin development of individual selection index for G5 broodfish. Sub-obj. 3.2 Complete collection of data from comparison of Generation 5 with Generation 2 (NWAC103). 4a What was the single most significant accomplishment this past year? The USDA103 line of channel catfish was developed and evaluated at the Thad Cochran National Warmwater Aquaculture Center and released under the name NWAC103 to commercial producers in 2001. After two generations of selectively breeding USDA103 channel catfish for rapid growth, a new experimental channel catfish line (USDA303) has been developed. Research was conducted to assess growth improvements in the USDA303 line of channel catfish, and showed a marked improvement in USDA303 channel catfish growth. Two generations of selection for increased growth resulted in a 21% increase in USDA303 channel catfish body weight compared to USDA103 catfish at the end of the study. Continued improvements in growth through selective breeding will lead to more efficient production for U.S. catfish farmers. 4b List other significant accomplishments, if any. The growth hormone-insulin like growth factor (GH-IGF) system plays an important role in growth of mammals, but its function in catfish is poorly understood. Assays were developed that allowed the measurement of GH, GH receptor (GHR), IGF-I, and IGF-II mRNA as well as an assay to measure protein levels of IGF-I. Research that examined regulatory mechanisms of the GH-IGF system in catfish found that IGF-I and IGF-II may play important roles in growth and that GH and GHR may play important roles in regulating the action of IGF-I. Identifying polymorphisms in GH or IGF genes may have potential for use in DNA marker-assisted selection programs. Insulin-like growth factors-I and II (IGF-I and IGF-II) are potential regulators of catfish growth and may also play roles in immune function. We measured levels of gene expression for IGF-I, IGF-II, and Toll-like receptors 3 and 5 in developing catfish as well as the role of IGF-I in catfish challenged with virulent Edwardsiella ictaluri. Results showed that growth (IGF-I and IGF-II) and immune (TLR3 and TLR5) associated genes could be functional and play important roles during embryogenesis and early development of catfish and that IGF-I decreases as Edwardsiella ictaluri in the blood increases. Understand mechanisms regulating catfish growth and immune function may lead to the ability to genetically select groups of individuals with improved growth and disease resistant phenotypes. Glucocorticoids are known to impede somatic growth in a number of vertebrate species. In order to better understand the mechanisms through which they may act in channel catfish, we examined the effects of feeding cortisol on the growth hormone (GH)/insulin-like growth factor-I (IGF-I) /IGF-binding protein (IGFBP) system. Similar studies were conducted in collaboration with the Harry K. Dupree Stuttgart National Aquaculture Research Center in palmetto and sunshine bass. The catfish results showed that cortisol administration increased a 20-kDa IGFBP in circulation and decreased plasma IGF-I levels. This research identified a 20 kDa IGFBP as a possible important regulator of growth. One mechanism through which cortisol may impede growth of catfish is through an increase in a low molecular weight IGFBP which may lead to inhibitory effects on the action of IGF-I. Lower molecular IGFBPs may be used as a marker of growth or stress. Molecular genetic markers have the potential to improve the selection for desired traits, such as growth and feed efficiency, in catfish by serving as predictors of genetic merit. Two genes known to regulate growth and feeding in other animals are growth hormone receptor and ghrelin. Research was conducted to identify these genes in catfish, determine their roles in catfish growth, and identify differences in sequence and gene expression patterns. Both genes were identified in catfish, found to be potent regulators of growth hormone, and to play central roles in the regulation of catfish growth. This new insight into the regulation of catfish growth together with observed differences in sequence and expression patterns of the two genes may prove to be useful tools for identifying superior families of catfish. Reproductive efficiency is important for the sustained production of channel catfish; however, the hormonal and genetic regulation of catfish reproduction is not understood. Ongoing research has identified several genes with the potential to regulate catfish reproductive efficiency. One gene in particular, steroidogenic factor-1 (SF-1), was identified in reproductive tissues of mature channel catfish and has the potential to control the production of steroids important for successful reproduction. Identification of factors regulating reproductive development will allow for the genetic selection of superior broodfish and provide information toward the development of tools for spawning induction. Development of ESC resistant line. ESC is the most prevalent disease affecting commercial catfish farms. 100 families (full-sib groups) were screened and ranked for susceptibility to ESC infection (with virulent Edwardsiella ictaluri) in three separate challenge experiments. Survivors from the 10 most resistant families were pit-tagged and released into ponds. In 2 years, these fish will be used for breeding and heritability of resistance to ESC will be measured. This project addresses component 1. 3 of the project plan and meets the milestone for ranking G4 families for ESC resistance. This project also addresses component 2.2 and its milestone of completion in yr2 in that the fish that will be used to produce the F1 resistant families have been selected, pit-tagged, and released into the ponds. TLR expression in resistant catfish. Blue x channel catfish hybrids show increased resistance to ESC infection. Lysozyme activity and expression of two toll-like receptor (TLR) genes, which show association with immune response to ESC, were measured in back-cross hybrid (channel x blue/channel) fish. Both lysozyme activity and expression of both TLR genes increased after exposure to virulent Edwardsiella ictaluri, however the patterns of expression differed from what was previously found for channel catfish. This research will improve efforts towards selectively breeding catfish for disease resistance. Detection of CCV carriers with genetic detection assay. Channel catfish virus (CCV) may be transmitted both vertically and horizontally. A quantitative genetic assay was optimized for detection of CCV in asymptomatic fish. Both broodfish and fingerlings from both commercial farms and research populations were determined to be carriers of CCV. The incidence of CCV carriers was on average 1050% of fish sampled. This information will be used for screening and assessment of CCV susceptibility in future projects. Channel catfish virus (CCV) accounts for approximately 5% of the annual losses to disease on commercial farms. However, localized CCV outbreaks can produce large losses of fingerlings on individual farms. In cooperation with investigators from Mississippi State University, an extensive series of viral challenges was performed to determine the relative susceptibility of various catfish lines to CCV, especially the NWAC103 line that was developed at the Catfish Genetics Research Unit. NWAC103 performance was average for the catfish strains tested, with no significant increase or decrease in susceptibility to CCV compared with other strains. These experiments provided initial data that will be used to examine the genetic control of resistance to CCV in channel catfish. Stressors in production systems reduce the efficiency of catfish production. Research was designed to develop a tool for characterizing levels of physiological stress in catfish. The proopiomelanocortin (POMC) gene was cloned which provided DNA sequence useful for studying factors which regulate POMC gene activity. This molecular tool will allow us to measure variation in stress responses between fish, and correlate the responses of catfish with resistance to diseases and efficient growth. Genes encoding the Major Histocompatability Complex (MHC) class I and II molecules have been identified in a number of fish species, including the channel catfish, but there is still a dearth of knowledge concerning their functional roles in fish immune responses. Animals from two families were identified that were matched or mismatched for MHCI and MHC II types, and genetic linkage and cell culture studies were performed. These studies demonstrated that MHC class I and II genes do not exist on the same chromosome in catfish as they do in mammals. Preliminary functional studies also indicated spontaneous non-specific cytotoxic responses between cells of different fish are likely mediated by differences in the MHC class I, but not class II, region molecules. This research provides insights into the control of catfish immune responses. In mammals, CD45 plays an important role in T and B lymphocyte cell receptor and cytokine signaling, however very little is known about its role in fish. We cloned the catfish CD45 gene and determined the DNA sequence. The catfish genome contains one functional CD45 gene, which can be differentially expressed depending of the cell type and the level of activation of the cell. The catfish gene makes use of alternative exon splicing to produce variants of the molecule. This research provided basic knowledge of CD45 structure for future experiments that will discern the role of this molecule in the response of the catfish immune system to pathogens. Little is known about the role of repetitive elements in the channel catfish genome. A previously identified class of repetitive DNA sequences, termed XbaI elements, were visualized on channel catfish chromosomes using fluorescence in situ hybridization. The XbaI elements were located primarily at the centromere of every catfish chromosome. This information will assist researchers in the assembly of catfish genetic maps, the integration of genetic and physical genome maps, and the identification of chromosomal regions that control important production traits in catfish. The role of the natural killer cell is not well understood in the catfish immune system. The catfish gene encoding the Natural Killer Cell Enhancing Factor (NKEF) was identified and sequenced. The gene was found to be active in all major tissues, and NKEF gene expression slightly increased after exposure of fish to bacterial cell components. NKEF gene variation was used to place it on the catfish genetic map. This research provided tools to allow scientists to understand the function of natural killer cells and to explore whether DNA sequence variants in the NKEF gene are linked to disease resistance. Estimation of phenotypic and genetic (co)variances for economically important traits are required for development of a breeding program to produce superior catfish germplasm for release to catfish producers. Data is being collected for full and half-sib USDA 103 strain channel catfish families for growth, meat yield, and disease resistance to allow estimation of phenotypic and genetic (co)variances. This information is being used to develop superior germplasm for release to catfish producers which will benefit producers, processors, and consumers. Information on the relative economic value of various production traits is needed to determine the amount of emphasis that should be applied to selection for improvement in different traits. Research is currently underway to estimate the relative economic value of traits important in farmed catfish (growth, feed conversion, meat yield, disease resistance). This information will be used in combination with information on heritability of traits to more efficiently produce catfish germplasm. Meat yield, an economically important trait for farm-raised catfish, is affected by catfish strain/species and season of the year. A project comparing effect of season on meat yield of blue catfish, channel catfish and channel x blue catfish hybrids was completed. Hybrid catfish had higher whole carcass and fillet yield than blue or channel catfish during both the spring and fall. Blue catfish had higher carcass yield than channel catfish during both the spring and fall. Channel catfish had higher fillet yield than blue catfish in the fall, but blue catfish had higher fillet yield than channel catfish in the spring. Blue catfish had higher nugget yield (lower valued rib-meat) than channel or hybrid catfish. Catfish processors could increase processing yields by processing different genetic groups of catfish at certain times of the year. Economic losses associated with diseases are an increasing problem for farm-raised catfish producers. A study to compare fingerling growth and survival of 4 genetic groups of catfish was initiated, groups included: USDA 103 strain channel catfish, USDA 102 strain channel catfish, USDA 103 x USDA 102 crossbred, and USDA 103 strain channel catfish x blue catfish hybrids. Survival, growth, and disease resistance are being evaluated in these groups. The study should provide insight into disease resistance and growth that will be useful to farmers looking for groups of fish with improved disease resistance and to researchers attempting to understand genetics of disease resistance in catfish. It is important to understand the effects of dietary protein and feeding frequency on important traits such as meat yield of catfish (the percentage of whole fish weight that is saleable meat) do determine the most profitable combination of dietary protein and feeding frequency. Effects of dietary protein level and frequency of feeding on meat yield of channel catfish were determined. Meat yield increased as dietary protein level and feeding frequency increased. Catfish farmers need to consider the impact of dietary protein level and feeding frequency on fish growth and meat yield in determining the optimal protein level and feeding frequency. However, the optimal combination of dietary protein level and feeding frequency changes with the price of feed and the price of fish. 4c List any significant activities that support special target populations. Development of catfish lines with superior performance for commercially important traits and utilization of these lines in commercial culture will help solve production problems, increase efficiency and profitability for both small and large catfish farmers, and provide a quality product for consumers. Most catfish producers with limited acreage buy fingerlings from large breeders that are very likely to utilize improved brood stocks, and the development and use of improved catfish lines can quickly affect the profits of small producers. Because small farms do not enjoy the same economies of scale experienced by larger operations, breeding fish with improved production traits will be highly beneficial to small farmers. The USDA Census of Aquaculture conducted in 2000 classified 84% of catfish farms as small businesses, with annual sales of less than $500,000. Of the 1,370 catfish farms in the United States, 38% (515) reported annual revenues of less than $25, 000. 4d Progress report. Under a Specific Cooperative Agreement 6402-31000-008-02S, Candidate Genes for Catfish Disease Resistance, with Mississippi State University. The problem of Enteric Septicemia of catfish (ESC), caused by the gram negative, facultative intracellular bacterium Edwadsiella ictaluri is the most economically important infectious disease to channel catfish aquaculture. We addressed the basis for pathogen resistance in mammals are often due to genes whose products function in the early stages of infection. Yet, little is known about the early response of channel catfish to E. ictaluri infection. Our objective was to observe the expression profile of select genes during the first 96 hours of infection by E. ictaluri. This involved the development of real-time rtPCR assays for transcripts of genes encoding an acute phase proteins (transferrin, serum amyloid P), macrophage responsive protein (Nramp), cell stress responsive protein (heat shock 70-HSP70), neutrophil proteins (beta integrin and E1B)and 18s ribosomal RNA. Then the expression of these genes were evaluated in two trial in which Channel catfish that were injected intraperitoneally with E. ictaluri or medium (controls) and then sampled. In the first trial the fish were sampled (n=7) at 6, 12, 24, 48, and 96 h after injection. The spleen, liver, anterior kidney (AK), and gut were sampled for RNA extraction. The posterior kidneys (PK) were collected for bacterial counts. In the second trial whole blood, liver and spleen were sampled for RNA expression analysis and tumor necrosis factor (TNF) was added to the gene expression profile. Also blood smears were taken for whole blood counts and samples of the liver and spleen were embedded in OCH medium and quick frozen for cryostat sectioning. The first trial demonstrated that most transcripts were found universally but, Serum Alkaline Phosphatase (SAP) and transferrin were liver-specific. Expression of EB1 (AK, spleen and liver), Hsp70 (AK and spleen), 1- integrin (liver) and Nramp (spleen and gut) significantly increased by 48 hpi. Transferrin was strongly up-regulated and SAP was down-regulated by 72 hpi, suggestive of positive and negative acute phase reactants. The second ESC challenge experiment, EB1, Hsp70, 1-integrin, TNF and Nramp expression were demonstrated in the peripheral blood. SAP and transferrin expression were not detected. TNF was upregulated at 24h in response to infection. The spleen also demonstrated a 24h TNF induction and this was followed by the EB1, Hsp70, 1-integrin, and Nramp induction at 48h and 72h. Cryostat sections were used for histochemical analysis of leukocyte population and for laser capture micro dissection (LCM) to evaluate tissue specific gene expression within the spleen. Analysis of the 72 hr samples demonstrate a marked increase in neutrophils throughout the spleen in infected fish. Neutrophils were focused around the periarterial lymphoid sheaths (PALS) in non-infected fish. LCM sampling of both tissue types from infected and non-infected fish and real-time quantitative PCR on the RNA extracts supports the data indicating differential distribution of neutrophils. EB-1 was non-detectable in parenchymal tissue, and expressed at a relatively low level in the PALS of non-infected tissue compare to high levels of expression throughout both tissues types in infected fish. As a comparison, HSP70 was expressed in both tissues in both groups but expression was highest in the infected parenchymal tissue. By evaluating expression of genes involved in the inducible portion of the innate defenses during the early stages of ESC we were be able to identify the critical time points that determine ESC outcome. The initial response occurs by 24 hours and secondary responses were apparent by 48 hours post-infection. This will allow researchers to focus on this critical window to identify the genetic basis of ESC resistance and factors that enhance or suppress the protective response. This report documents research conducted under Specific Cooperative Agreement 6402-31000-008-03S, Channel Catfish Molecular Markers, with Auburn University. The very first objective was to identify DNA markers within genes of known functions, particularly those already mapped on the human and zebrafish linkage maps, are valuable for mapping the catfish genome because they provide landmarks for comparative genome mapping. The second was to characterize certain innate immune-related genes, in particular the CXCL8 gene that appear to have multiple versions of cDNAs that may be exploited as a marker for mapping of this gene. Third, after initial development of genetic linkage maps using various resource families, it is important to integrate the maps using the same resource DNA. For the first objective, we took the approach of intron tagging. By amplification and sequence analysis of introns of known genes, polymorphic markers can be identified because intron sequences are highly variable and often contain microsatellites. On the basis of progress made in previous years, in the last year, we have completed the objective. We designed primers for 50 genes to amplify their introns. Of the 50 pairs of primers designed, 42 produced products. These PCR segments were sequenced. Sequence analysis of the 42 introns indicated that 8 (19%) included microsatellite sequences in them (Table 1). The sequences have been deposited to GenBank available for public use and their accession numbers are included in the publication. The information was transferred to the Catfish Genetic Research Unit whose personnel conducted mapping analysis of the polymorphic markers. This work was published in Animal Genetics (Serapion et al., 2004). Interleukin-8 is a CXC type chemokine produced in response to stimulation by pro-inflammatory cytokines or bacterial lipopolysaccharides. We cloned and characterized interleukin-8 cDNAs and its genomic segments containing all the exons and introns from channel catfish (Ictalurus punctatus). Multiple interleukin-8 cDNA clones were identified during analysis of expressed sequence tags (ESTs). Sequence analysis indicated presence of four types of alternatively spliced and two types of alternatively polyadenylated transcripts. Analysis of the genomic DNA of the locus confirmed that all four types of transcripts were likely derived from partial splicing. Channel catfish interleukin-8 gene has four exons and three introns with highly conserved splice sites as compared to interleukin-8 genes of other organisms. In spite of the structural conservation through evolution, the piscine interleukin-8 genes showed a much greater sequence divergence than their counterparts among mammals. RT-PCR indicated that two of the four splicing forms were expressed at high levels whereas the other two aberrant splicing forms were not detectable. Expression of interleukin-8 gene was up-regulated in channel catfish and blue catfish after infection with pathogenic bacteria Edwardsiella ictaluri. This work was published in Developmental and Comparative Immunology (Chen et al., 2005). We initiated an expanded objective: Integration of various linkage maps by using the same resource markers. In the last several months, experiments have been initiated to genotype microsatellite markers that were mapped using the channel catfish resource families. In this case, genotyping was conducted using PCR with channel catfish x blue catfish interspecific resource DNA. Several points are important for this objective: 1) integration of existing genetic maps will enhance the resolution of the linkage maps; and 2) use of the same set of microsatellite primers in different resource families will allow comparison of the mapping populations and their impact on genetic distances, particularly between the intraspecific and interspecific families. To date, almost 50 microsatellite primer pairs have been tested and genotyped with 64 fish from an interspecific family. Under a Specific Cooperative Agreement 6402-31000-008-04S, Channel Catfish Breeding for Disease Resistance, with the University of Arkansas, Pine Bluff. Research was conducted to improve selective breeding of channel catfish resistant to ESC. Channel catfish families were selected based on ESC resistance and challenged with Edwardsiella ictaluri at the Catfish Genetics Research Unit and blood samples collected before and after challenge were tested for humoral immune function in the laboratory of Aquaculture/Fisheries Center, University of Arkansas at Pine Bluff. Serum complement activity is constitutively higher in ESC resistant fish, it remains high after E. ictaluri challenge and there is no clear relationship between serum lysozyme activity and ESC resistance. Selection of fish based on humoral immune function could produce catfish resistant to ESC and it may be adventitious to select for catfish with high serum complement activity. This research was terminated in FY 2004 when the graduate student supported by ARS funding left the university. Under a Specific Cooperative Agreement 6402-31000-008-05R, Sequence Characterization of the Channel Catfish Immunoglobin Heavy Chain Locus, with University of Mississippi. Research was conducted to identify DNA clones containing the catfish immunoglobin heavy chain locus, sequence these clones, and establish the gene structure in this locus. Two large DNA clones were identified and fragmented to make DNA sub-libraries. The sub-libraries were sequenced using the high-throughput capacity of the USDA, ARS, Mid South Area Genomics Laboratory. Two additional DNA clones have been identified that overlap the ends of the initial clones and sequencing is ongoing. Final completion of the sequencing is scheduled for FY 2005. This information will be useful in determining the structure of this important immunity-related molecule from a primitive vertebrate to help determine how these molecules have developed in more advanced species. It will also assist researchers in understanding the functionality of the catfish immune system. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. This project was initiated on 12/01/2004. Major accomplishments expected over the life of the project are: 1) Continued improvement in performance of catfish line USDA103; 2) development and publication of a second-generation catfish genetic linkage map containing conserved genes useful for comparative mapping; and 3) continued development of a multi- trait selection program utilizing the USDA103 and other catfish germplasm to develop additional improved lines for future release to commercial producers. Action Plan components: Genetic Improvement sections b and c. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? This current research project was initiated in November 2004. To date, the main transfer of science and technology has been scientific publication in peer-reviewed journals, popular press, discussions with catfish producers, and a joint ARS-MAFES extension workshop to educate catfish producers in the methods for producing hybrid catfish. The most significant transfer of technology under the previous project from the Catfish Genetics Research Unit was the development of the USDA103 catfish line, which demonstrated superior growth performance. Broodfish from this line, named NWAC103, were made available to commercial producers through a joint release by ARS and MAFES beginning February 1, 2001 and completed in February 2002. Another germplasm release is planned for the end of this project, but this will depend on acceptance and approval by partners and stakeholders. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Tucker C., Silverstein P, Camus A, Bilodeau L, Wise D, Waldbieser G. 2005. Channel catfish virus disease and NWAC103 channel catfish. The Catfish Journal. January 2005. p.8. Bilodeau, A.L., Small, B.C., Wolters,W.R., Wise, D.J. 2005. Early Host Response Improves Disease Resistance In Channel Catfish. Global Aquaculture Advocate. 2005. 8(3):84-85. Peterson, B.C., Small, B.C. 2005. The use of recombinant bovine growth hormone (rbGH; Posilac) to characterized the GH-IGF axis in channel catfish. [Abstract] Aquaculture 2005 Conference. New Orleans, LA. Jan 17- 20, 2005. p. 323. Peterson, B.C., Bilodeau, A.L. 2005. Development of real time PCR assays to measure levels of somatostatin-14 and somatostatin-22 mRNA in channel catfish. Abstract, 15th International Congress of Comparative Endocrinology Meeting. Boston, MA. May 22-27, 2005. p. 110. Small, B. C, Bilodeau, A.L. 2004. Cortisol, stress, and pathogen susceptibility in channel catfish (Ictalurus punctatus). Proceedings of the 5th International Symposium on Fish Endocrinology. p. O57. Small, B.C., Peterson, B.C. 2005. Establishment of a time-resolved fluoroimmunoassay for measuring plasma insulin-like growth factor I (IGF- I) in fish: effect of fasting on plasma concentrations and tissue mRNA expression of IGF-I and growth hormone (GH) in channel catfish. Proceedings of Aquaculture America 2005. p. 424. Small, B.C., Chatakondi, N. 2005. AQUI-STM reduces routine-handling stress in mature channel catfish. Proceedings of Aquaculture America 2005. p. 424. Small, B.C., Chatakondi, N. Optimizing chemotherapeutic treatment of hybrid catfish eggs for maximal hatching success. Proceedings of Aquaculture America 2005. p. 425. Peterson B.C., Small, B.C. 2005. Effects of recombinant bovine growth hormone on growth and the GH/IGF axis in channel catfish. Proceedings of Aquaculture America 2005. p. 323. Barrero, M., Small, B.C., DAbramo, L.R., Kelly, A.M., Hanson, L.A. 2005. Plasma steroid, cathpsin activity and egg size and protein content during in vivo oocyte maturation in four strains of channel catfish broodstock. Proceedings of Aquaculture America 2005. p. 23. Welker, T.L., Klesius, P.H., Arias, C.R., Small, B.C. 2005. Effect of hypoxia on stress and heat shock protein expression in channel catfish (Ictalurus punctatus Rafinesque). Proceedings of Aquaculture America 2005. p. 487. Small, B.C., Waldbieser, G.C., Murdock, C.A., Peterson, B.C. 2005. Molecular cloning, genomic organization and functional characterization of channel catfish growth hormone receptor: Changes in hepatic GHR mRNA expression following fasting and feeding exogenous cortisol. Proceedings of the XV International Conference of Comparative Endocrinology. Boston, MA. p.120. Murdock, C.A., Small, B.C., Waldbieser, G.C. 2005. Molecular cloning of channel catfish steroidogenic factor-1 and the effects of exogenous LHRH treatment on expression. Proceedings of the XV International Conference of Comparative Endocrinology. Boston, MA. p.101.

Impacts
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Publications

• Bilodeau, A.L., Peterson, B.C., Bosworth, B.G. 2005. Response of toll-like receptors, lysozyme, and igf-i in back-cross hybrid (f1 male (blue x channel) x female channel) catfish challenged with virulent edwardsiella ictaluri. Fish and Shellfish Immunology 20:29-39.
• Bilodeau, A.L., Waldbieser, G.C. 2005. Activation of tlr3 and tlr5 in channel catfish exposed to virulent edwardsiella ictaluri. Developmental and Comparative Immunology 29:713-721.
• Karsi, A., Waldbieser, G.C., Small, B.C., Wolters, W.R. 2005. Genomic structure of the proopiomelanocortin gene and expression during temporal stress in channel catfish, Ictalurus punctatus. General and Comparative Endocrinology.
• Peterson, B.C., Small, B.C. 2005. Effects of exogenous cortisol on IGFBPs and mRNA expression levels of IGF-I and GH in channel catfish. Domestic Animal Endocrinology 28:391-404.
• Peterson, B.C., Bosworth, B.G., Bilodeau, A.L. 2005. Differential expression of IGF-I, IGF-II, and toll-like receptors 3 and 5 mRNA during embryogenesis in hybrid (channel x blue) and channel catfish. Comparative Biochemistry and Physiology 141:42-47.
• Peterson, B.C., Waldbieser, G.C., Bilodeau, A.L. 2004. IGF-I and IGF-II mRNA expression in slow and fast growing families of USDA103 channel catfish (Ictalurus punctatus). Comparative Biochemistry and Physiology 139:317-323.
• Peterson, B.C., Waldbieser, G.C., Bilodeau, A.L. 2005. Effects of recombinant bovine somatotropin on growth and abundance of mRNA for IGF-I and IGF-II in channel catfish (Ictalurus punctatus). Journal of Animal Science 83:816-824.
• Quiniou, S.M., Wilson, M., Bengten, E., Waldbieser, G.C., Clem, L.W., Miller, N.W. 2004. Mhc rflp analyses in channel catfish full-sibling families: identification of the role of mhc molecules in spontaneous allogeneic cytotoxic responses. Developmental and Comparative Immunology.
• Small, B.C., Chatakondi, N. 2005. Routine measures of stress are reduced in mature channel catfish during and following Aqui-S anesthesia and recovery. North American Journal of Aquaculture 67:72-78.
• Small, B.C., Bilodeau, A.L. 2005. Effects of cortisol and stress on channel catfish (Ictalurus punctatus) pathogen susceptibility and lysozyme activity following exposure to Edwardsiella ictaluri. General and Comparative Endocrinology 142:255-261.
• Weber, T.E., Small, B.C., Bosworth, B.G. 2005. Lipopolysaccharide regulates myostatin and myod independently of an increase in plasma cortisol in channel catfish (Ictalurus punctatus). Domestic Animal Endocrinology 28(1):64-73.
• Weber, T.E., Bosworth, B.G. 2005. Effects of 28 day exposure to cold temperature or feed restriction on growth, body composition, and expression of genes related to muscle growth and metabolism in channel catfish. Aquaculture 246:438-492.