2006 Annual Report 4d.Progress report. This report serves to document research conducted under a Specific Cooperative Agreement (No. 58-6228-5-009) between ARS and the Aquaculture/Fisheries Center, University of Arkansas at Pine Bluff. Additional details of research can be found in the report for the parent CRIS 6228-41630-003-00D. Research is on-going and addresses objectives of the ARS Project Plan "Improved Warm Water Aquaculture Products and Technologies." Producing Disease-Free Golden Shiner Broodstock: Development of a quantitative PCR assay for Ovipleistophora: Eggs were collected from a commercial farm. Eggs and fry were collected with and without treatments to disinfect egg surfaces. The experiment involved three independent trials with 11 treatments run in replicate (50-100 fish per replicate). The DNA from all of the samples has been purified. Validation of the TaqMan quantitative PCR for Ovipleistophora (using an LNA modified fluorescent probe) is almost complete. Sensitivity is 100 copies or less, and it does not cross react with other microsporeans. Several of the study samples have been tested and clearly show that there is egg-associated vertical transmission of Ovipleistophora. In order to eradicate Ovipleistophora, it is critical to know if the parasite is vertically transmitted in or on eggs. Our work has shown that it is transmitted in an egg associated way. Farmers and Extension specialists are using this information in the development of strategies to eradicate the parasite. Development and Application of New Molecular Diagnostics for Improving Biosecurity in Aquaculture:. 1)Epidemiology of GHNV: Our studies of goldfish herpesvirus (GHNV) have demonstrated that GHNV is an important and widespread pathogen of goldfish, that it most likely persists in fish in a latent form, and that it is extremely difficult to prevent vertical transmission of the virus even when eggs are disinfected and well water is used in the hatchery. Publication of our assay has resulted in adoption of the assay in laboratories in the US, Europe, the Middle East, and Australia.. 2)Discovery and epidemiology of AGCRV: Our discovery and characterization of this virus has helped us to explain a number of undiagnosed fish kills and has clarified epidemiology of FHMRV and CGRV. It has also led to new insights into the taxonomy of the aquareovirus family.. 3)Development of PCR for the new catfish herpesvirus will allow us to proceed with surveys for this pathogen in the US.. 4)Our work with SVCV PCR has identified assays useful for detecting the strains most problematic in North America.. 5)Our participation in an international ring trial of KHV primers (that includes one set developed by us) is leading to increased sensitivity in diagnostic assays run in our lab to assist with diagnosis and prevention of KHV in the US. Aeromonas salmonicida Infections in Cyprinid Fishes: Epidemiology of Ulcer Disease: PCR assays have been developed and used to show that almost all koi ulcer cases are the result of infection by Aeromonas salmonicida achromogenes. We have also shown that the bacteria are still present following a variety of traditional therapeutic approaches. This work has been performed on field samples donated from all over the US and has provided insights immediately useful in the control of this important disease. Reducing Catfish Losses from Processing Plant Dockages: The survey revealed that 95% of catfish loads delivered to processing plants between 1997 and 2002 were assessed dockages that resulted in average losses of 2.45% per load or $0.066/kg of catfish marketed over the study period. Out-of-size discounts constituted the greatest losses. Dockage losses can be reduced by shifting either to longer-term single-batch production or more intensive grading. Longer-term production results in fewer smaller fish that would incur dockage losses. However, cash flow constraints require more intensive early-season grading. The grader choice depended on the dockage tolerance level and rate, the frequency distribution of sizes of catfish in the population, the efficiency of the grading technology, and the cost of the grading method. Larger farms minimize losses with intensive active grading (UAPB grader). Arkansas Catfish Farmers and Marketing Contracts: A sample of 40 catfish farmers were surveyed to elicit basic information on factors such as farm size, participation in contractual arrangements, perceptions about contracting, and reasons for contracting. Results from the study provide insights and perspectives on alternative market arrangements for catfish farmers. The minimum water acreage indicated was 45 acres; maximum acreage was 2,500 acres, with an average of 393 acres. Twenty-one respondents had sales contracts. Out of this number, 17 had contracts that indicated quantities to supply. Twenty-two respondents sold an average of 92% of fish to independent processors under contract without a predetermined price. Only two sold to independent processors under contract with a predetermined price, averaging 65% of all sales. Ten respondents sold an average of 77% of fish to independent processors without a contract. For sales to cooperatives/associations, one respondent sold 50% under contract with a predetermined price, four sold an average of 73% without a predetermined price and 5 sold an average of 80% without a contract. On a scale of 1-5, where 1 = strongly disagree, and 5 = strongly agree, the average score for certain contractual statements obtained were: A) A predetermined fish price with processor will, on the average, result in a higher price than selling in the cash market (3.1); B) My main fish marketing goal is to reduce marketing risks rather than increase my net sales price (2.7); C) Processors have too much control over the cash price of fish (4.4); D) A minimum price guarantee for fish producers should be provided through government payments (2.6); E) Assistance is needed to help forecast catfish prices six months ahead (3.5); F) I am willing to accept a lower fish price to reduce price risk (2.3); and, G) A revenue insurance program for fish farmers should be developed by the government (2.7). Biomass Scanning Technology: Preliminary work with the VAKI biomass scanner revealed two major problems. First, the swimming behavior of catfish appears to be a major obstacle. The infrared array created by the VAKI system depends on "straight swimming" fish in order to create a reasonable biomass estimation. Channel catfish tend to swim too close to the bottom of the frame and also have a tendency to flash or turn to the side as they pass through the array. This results in continuous error readings being generated. A second obstacle to pond use is the extraordinary high turbidity of these waters. Due to these seemingly insurmountable obstacles, the research focus has been re-directed to the use of sonar to generate biomass estimation. Current collaboration is underway with Dr. Jim Chambers with the National Center for Physical Acoustics (NCPA) at the University of Mississippi. A sonar-based system is currently being developed at NCPA and will be further field tested in conjunction with the SRAC inventory project scheduled for the fall of 2007. Preliminary Study of the Efficacy and Accuracy of the Catfish Trawl as an Inventory Assessment Tool: A catfish trawl has been used in various projects to capture random samples of fish from commercial catfish ponds. An experienced operator may setup the trawl and land up to a 1,000 lb of fish in less than an hour. The objective of this study is to determine if the trawl could be used to assess the size distribution of fish inventories in commercial catfish ponds. The trawl was pulled across 3 commercial catfish ponds. All captured fish were individually weighed to assess the size distribution of the population. The day following the trawl pull, the ponds were seined twice with a fingerling seine to capture all the fish in the pond. Seined fish were transferred to a sock, and a random sample of fish was captured from the sock with a hoop-net. Sampled fish were individually weighed to assess the size distribution of the population. The average fish weight obtained from the trawl and from the seine samples were compared for each pond with t-test statistics. For the three ponds tested, the average weight of fish captured with the trawl was significantly larger (P< 0.05) than the average weight of fish sampled from the seine. However, the range of fish sizes caught by each method appeared similar, suggesting that the trawl has the capacity of capturing fish of all sizes. The quantity of fish captured with the trawl did not appear to be related to the quantity of fish in the pond. Development and Testing of New Fish Farming Equipment: An in-pond floating food fish grader, which was developed at UAPB by modifying the Greenland and Gill (1972) horizontal grading platform to avoid the problems resulting from lifting fish out of the water, has been shown to significantly reduce the number and weight of sub-harvestable fish sent to the processing plant. Additional refinements to the grading system may enable it to function more efficiently and to be integrated more effectively into the harvesting process. This project is working on refinements to three components of the UAPB grading system: the crowder boat that is used in conjunction with the grader, a loading platform, and the "Seinitizer" (a seine-sanitizing reel). Additional design work was done on the crowder boat to move fish into the eductor box using a water jet system to move fish through the sock with minimal crowding and stress. Initial testing has been completed. A loading platform was developed for the in-pond grader that is integrated with the eductor box and facilitates loading fish for harvest and reduces stress on fish by reducing the need to crowd fish. Initial testing of the loading platform for channel catfish production has been completed. A commercial scale "Seinitizer" (seine-sanitizing system) has been incorporated into a traditional seine reel design and was tested during commercial catfish harvesting operations. The initial testing of the crowder boat design indicated that it could be operated successfully under commercial conditions. The initial testing of this equipment under commercial conditions indicated the need for some additional minor design modifications. In the coming year, the spool height will be increased to accommodate larger socks, and the angle of the guideposts will need to be changed to facilitate loading operations. Field trials of the loading platform indicated that catfish can be loaded successfully into a loading basket using the eduction loading system. These trials indicated the need for an improved method of controlling the flow of fish from the eductor box to the platform. A gate valve control mechanism has been developed and will be incorporated into the loading platform design in the next year. Initial testing of the "Seinitizer" has indicated that seines can be disinfected with this equipment and that normal harvesting operations are not impeded. Several minor design modifications will be incorporated in this next year to better distribute sanitizing solution across the net and improve ease of use. Effect of Periodic Precision Grading During the Production Season: This is a multiple-year study. Twelve 0.1-ha earthen ponds were each stocked with 2,273 kg/ha channel catfish (0.45 kg) and 16,250 fingerlings/ha 12.7-cm fish in March 2005. In 2005, a total of 3,532 + 1,309 kg/ha were harvested from the live-car graded treatments, as compared with 4,273 + 286 kg/ha from the in-pond grader treatments. These yields were not significantly different (P >0.05). On February 28, an additional 15,000 12.7-cm fingerlings/ha were understocked and additional partial harvests conducted on March 13-14, 2006, and July 13-14, 2006. To date, the total weight of fish harvested in the respective treatments are 6,395 + 1,586 kg/ha with live car grading and 7,118 + 445 kg/ha with the UAPB grader. The total weights harvested, to date, are not significantly different (P>0.05). However, there appears to be a trend of higher and less variable yields in the ponds graded with the UAPB grader. Also, observations from the sampling data appear to show more clear distinction among year classes of fish in ponds graded with the UAPB grader than in ponds graded with the traditional live car. This study is still underway. More in-depth analysis of data will be done to determine if apparent trends are sufficient to warrant extending this study into a 3rd year or if it will be terminated and harvested completely in Fall 2006. The Effect of Stocking Density on the Growth and Production of Tank-Hatched Golden Shiners: Variability trial. - This 2006 study is currently in progress. Methods being used for this trial are identical to the 2004 low density trial with the following changes. Water was first added to the ponds on May 2. Ponds were fertilized with cottonseed meal only. Hatchery fry were stocked May 5 at a rate of 30,000 or 1,500,000/acre (six replicates per density). Crumbled pellets were fed for a total of 4 wk prior to the switch to floating pellets. Fish are being sampled every 2 wk. Fish are scheduled to be harvested July 18-19 (11 wk). Determination of Effects of Fish Density and Temperature on Growth of Commercial Freshwater Baitfish Species: A 10-wk trial was conducted in a flow-through aquaria system to determine the growth response of juvenile golden shiners to increasing density. Fifteen golden shiners (average wt of 1.14 g per fish) were stocked into each of 20, 110-L aquaria. A 1-mm 2-mesh tank divider confined the fish to 100%, 50%, or 25% of total tank water volume (96-L), creating initial loading rates of 1.8, 3.6, and 7.1 g/1,000 cm3 (5 replicates per density). Each tank received a constant inflow of water at a rate of approximately 100 ml/min, equivalent to one exchange per 18 hr. Water temperature was maintained at 28 +1.5 deg C with heaters (250 watt Jager TS Automatic Heater). An external filter unit (Penguin Aquarium Power Filter 200B) provided circulation, aeration, and filtration of water within each tank. Each tank was also aerated with 2.5-cm air stone supplied by a low pressure blower. Fish were confined to the portion of the tank closest to the water inflow. Tanks were covered with acrylic lids to prevent fish from jumping out. An automatic timer controlled the room lights, with a 12-hr-light, 12-hr-dark cycle, and changes in lighting were instantaneous. Fish were fed twice daily to satiation with a 32% crude protein commercial minnow feed in the form of extruded pellets that had been crumbled and sieved, resulting in particle sizes of less than 0.85 mm. Fish were fed small amounts of feed at a time by hand until the fish did not respond to additional feed. Fish were harvested after 10 wk. Each fish was individually weighed and corresponding total length recorded. Two-way and one-way ANOVA were used to analyze data, and differences among means were determined using Tukeys HSD test. Results were considered significant at P < 0.05. Fish density had no effect on fish growth or final condition (Fulton's K). On average, fish gained 1.41 g over the 10-wk period. Survival in 2 tanks was below 80%, and these tanks were excluded from the study. Loading rates at harvest averaged 4.0, 7.1, and 15.9 g/1,000 cm3 in the 100%, 50%, or 25% of total tank volume treatments, respectively. Fish in the aquaria were more densely loaded than in culture ponds. For comparison, typical golden shiners yields in ponds are 500 to 1,000 lb/acre, or 0.06 to 0.12 g/liter (assuming an average depth of 3 ft). Water quality was acceptable for golden shiners and did not differ significantly among treatments. The feed conversion ratio did not differ among treatments, but was relatively high (4.1 - 5.4), implying that fish were fed to excess or that fine particles in the feed were not utilized by fish. Development of Practical Diets for Largemouth Bass, with Emphasis on Non-Fish Protein and Lipid Sources: Since the 5-year plan was initiated, most of the research objectives for the remaining years of the project have been addressed by another University. To avoid duplication of effort, it was necessary to change the focus of the remainder of the project. We are pursuing nutrition research with LMB in the following two areas:. 1)Carbohydrates and. 2)Vitamins. To date, Dr. Sink (Post-doctoral research associate in Dr. Lochmann's lab) has conducted some basic research with LMB, including the validation of insulin ELISA assays, conduct of glucose stimulation-insulin/blood glucose response tests, and development of insulin and glucose time response curves for LMB fed commercial pellets and pure glucose. Results from these tests will enable us to develop appropriate practical diets for feeding trials to study carbohydrate utilization further in LMB. 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). Wiese, N. and C. Engle. 2006. Cutting losses - model proposes use of in-pond bar graders to reduce dockages at catfish plants. Global Aquaculture Advocate. 31-33. Goodwin. Diseases of koi and goldfish. National Ornamental Goldfish Growers Association. World Aquaculture Society Annual Meeting, Las Vegas, Nevada, Feb 15, 2006. Goodwin. Prevention and Control of ulcer disease. AKCA Regional Koi Health Symposium. Tulsa, OK July 15, 2006. Subhadra, B., and R. Lochmann. 2006. Prostaglandin E2 Production in Largemouth Bass Fed Diets With Poultry Meal and Different Lipids. Aqua2006, Florence, Italy, May 9-13. Scientific Publications Goodwin, A. E., Khoo, L., LaPatra, S. E., Bonar, C., Key, D. W., Garner, M., Hanson, L. 2006. Goldfish Hematopoietic Necrosis Herpesvirus (Cyprinid herpesvirus. 2)In the USA: Molecular Confirmation of Isolates from Diseased Fish. Journal of Aquatic Animal Health. 18:11-18. Goodwin, D. K. Nayak, and R. S. Bakal. 2006. Natural infections of wild creek chubs (Semotilus atromaculatus ) and cultured fathead minnows (Pimephales promelas) by Chinese grass carp reovirus (Golden Shiner Virus). Journal of Aquatic Animal Health. 18:35-38. Goodwin, G.E. Merry, J. Sadler. 2006. Detection of the herpesviral hematopoietic necrosis disease agent (Cyprinid herpesvirus. 2)in moribund and healthy goldfish: Validation of a quantitative PCR diagnostic method. DAO 69:137-143. Kachowski, A., and N. Stone. Single season production of jumbo golden shiners. Abstracts, Student/Faculty Research Forum, University of Arkansas at Pine Bluff, March 22-23, 2006. p. 13. Wiese, N., C. Engle, J. Trimpey, K. Quagrainie and B. Green. 2006. Reducing catfish farm losses due to dockages assessed by processing plants. Journal of the World Aquaculture Society. 37(1):60-73. Wiese, N., C.Engle, J. Trimpey, K. Quagrainie, and B. Green. 2006. Reducing catfish farm losses due to dockages assessed by processing plants. Published abstract. 2006 Catfish Farmers of America Research Symposium, San Antonio, Texas. Engle, C.R., N. Wiese, J. Trimpey, K. Quagrainie, and B. Green. 2006. Reducing catfish farm losses due to dockages assessed by processing plants. Published Abstract. Annual Meeting of the U.S. Aquaculture Society, a Chapter of the World Aquaculture Society, Las Vegas, Nevada. Quagrainie, K. and C. Engle. 2006. Arkansas catfish farmers and marketing contracts: a survey. Published abstract. Aquaculture America 2006, Las Vegas, Nevada. Subhadra, B., R. Lochmann, S. Rawles, and R. Chen. 2006. Effect of dietary lipid source on the growth, tissue composition and hematological parameters of largemouth bass (Micropterus salmoides). Aquaculture. 255:210-222.