2006 Annual 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? Problems: Farmers need effective antimicrobial agents to control diseases and bacterial pests occurring in aquaculture. Blue-green algae (cyanobacteria) have a number of characteristics that make them undesirable in fishponds, but the most economically important problem related to their presence is that some species produce earthy or musty metabolites that give fish an undesirable “off-flavor.” Although the farmer closely manages most aspects of catfish farming, one important part of catfish farming that has eluded control by the farmer is the microbial community that develops in the production ponds. In particular, conditions in catfish aquaculture ponds are such that summertime phytoplankton communities are often dominated by species of noxious blue-green algae. The control of phytoplankton community structure in catfish ponds is therefore highly desirable and one approach is to discover safe compounds that selectively remove or prevent the growth of odor-producing blue-green algae but do not affect more desirable species of phytoplankton. Catfish diseases from pathogenic bacteria cost producers more each year than off-flavor related problems. There are currently only a limited number of compounds (drugs) approved for the management of bacterial disease outbreaks in catfish populations. The discovery of novel natural compounds that can be used to treat (therapeutants) and/or prevent the transmission of bacterial catfish pathogens from fish to fish would provide catfish farmers with additional management approaches for disease prevention and control. Solutions: The approach to solving both problems is to identify natural products from plants and other organisms that will control pathogenic bacteria and blue-green algae in catfish ponds. These products have new chemistries that have never before been synthesized by organic chemists and will result in totally novel, prototype antimicrobial agents representing new chemical classes with different toxicities, that operate by different modes of action than existing antimicrobial agents and, consequently, lack cross-resistance to chemicals currently used. How serious is the problem? Why does it matter? Human population growth and an emphasis on seafood as a healthy food item have increased the demand for fishery products. At the same time, the supply of seafood from traditional capture fisheries has stabilized because of over-exploitation or habitat destruction. One approach to meeting the shortfall between consumer supply and demand of fishery products is through aquaculture, the controlled cultivation of fish and shellfish. The largest and fastest growing segment of aquaculture in the United States is farm-raised channel catfish. Most of the 500 million pounds of catfish processed in 1997 were grown in the southeastern United States and Mississippi accounted for well over half of that production. Catfish that are off-flavor are unmarketable and farmers are forced to hold those fish in inventory until the make-up of the pond microbial community changes and the flavor leaves the fish. Holding market-sized fish in inventory imposes an economic burden on the farmer, and off-flavor is estimated to cost the industry well over $25 million a year. The discovery of new antimicrobial compounds is an urgent research objective due to the development of microbial resistance in medicine and agriculture and to the loss of current antimicrobial products. The public continues to have concerns about the use of antibiotics in agriculture and particularly from the use of medicated feeds that could lead to the development of antibiotic-resistant bacteria in the environment. The primary focus of this project is the discovery of bactericidal compounds that are not antibiotics. 2.List by year the currently approved milestones (indicators of research progress) FY 2005: Milestone: Bioassay to discover new natural compound-based leads for the prevention and treatment of the most problematic channel catfish diseases is developed. Anticipated accomplishment: Identify and receive compounds and extracts for evaluation (a continuing process). Complete bioassays to evaluate submitted samples. FY 2006: Milestone: Begin evaluating the most promising compounds to discover natural and natural-based compounds for use as selective algicides or bactericides in catfish disease management. Anticipated accomplishment: Each active compound is studied progressively (determine IC50, 6-well plate assay). Bioassay-guided fractionation of active extracts occurs. Active fractions undergo further testing to help isolate and identify active compounds. Determine water solubility and/or formulations to maintain compound in water column. FY 2007: Milestone: Toxicological profiles of active compounds are developed. Anticipated accomplishment: Determine water solubility and/or formulations to maintain compound in water column. Mode-of-action studies are initiated. Invention disclosure(s) filed. FY 2008: Milestone: Efficacy testing (limnocorrals) of promising algicidal compound(s) occurs. Anticipated accomplishment: Environmental persistence of test compound determined. Patent application(s) prepared if appropriate. Initiate contact with potential Cooperative Research and Development Agreement (CRADA) partner and develop partnership as appropriate. FY 2009: Milestone: Efficacy testing in small catfish ponds occurs. Anticipated accomplishment: Environmental fate of test compound determined. Determine effects on catfish and possible accumulation of test compound in catfish. Initiate contact with U.S. Environmental Protection Agency to discuss toxicity-test requirements for possible approval for use of test compound in catfish aquaculture. 4a.List the single most significant research accomplishment during FY 2006. Several promising natural compounds were discovered using the bactericide bioassay to have significant antibacterial activity towards the two most common catfish bacterial pathogens. 4b.List other significant research accomplishment(s), if any. None. 4c.List significant activities that support special target populations. This research project has brought together university researchers (Mississippi State University and University of Mississippi), USDA-ARS scientists, and catfish producers to evaluate methods for reducing and managing off-flavor problems in catfish aquaculture. These studies provided extension personnel with a guide to work with producers in implementing management strategies. 4d.Progress report. Additional plant extracts have been screened to discover promising natural algicides. A rapid antibacterial bioassay has been used to evaluate natural compounds, crude extracts from plants, and extract fractions to discover antibacterial compounds to help manage the most common bacterial diseases in catfish aquaculture. Several promising natural compounds were discovered to have significant antibacterial activity towards the most common catfish disease-causing bacteria. Research under a Non-Funded Cooperative Agreement between ARS and Nature Diagnostics, Inc. of Blacksburg, VA, resulted in development of one novel method for synthesizing the patented algaecide. The lead ARS scientist of inhouse Project 6408-41000-005-00D evaluated a sample of this novel algaecide as synthesized by Nature Diagnostics, Inc. Results from the bioassay evaluation found toxicity towards the odor-producing species of blue-green algae to be about 20 times less toxic than the algaecide when produced by the method outlined in the patent (U.S. #6,949,250). Based upon chemical analysis of the sample provided by Nature Diagnostics, Inc., impurities are present in the sample and this may account for the reduced activity. Nature Diagnostics, Inc., plans to continue to refine their method to enhance recovery and purity of the end product. In addition, this commercial partner plans to develop other additional synthetic methods to develop a more cost-effective method for manufacturing the patented algaecide. 5.Describe the major accomplishments to date and their predicted or actual impact. In FY 2006, a Non-Funded Cooperative Agreement was initiated with a commercial partner for the development of a patented natural-based algicide that is derived from a natural compound found in annual ryegrass. During the past 5 years, this project has made significant discoveries in the area of natural products for use as selective algicides in catfish aquaculture. This project has established an algicide discovery “pipeline” by establishing a number of excellent collaborations with university and commercial/industry scientists that facilitated the movement of experimental compounds from the laboratory to the efficacy testing in catfish aquaculture ponds. An experimental method for testing natural and natural-based compounds as selective blue-green algicides in catfish production ponds was developed and has been used during the past six summers. One promising natural-based compound is undergoing preliminary development. 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? Once natural and natural-based compounds that have potential for control of diseases or blue-green algae have been identified, promising compounds will have to be further evaluated, developed, and marketed by the industry to the catfish farmer. One commercial partner has been found for development of the patented natural-based algicide. If a cost-effective method for manufacturing the natural-based algicide can be developed, this technology should be transferred within three years. 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). Schrader, K.K. Natural-based algicides with potential use to manage off-flavor in catfish aquaculture. Seventh Symposium on Off-flavours in the Aquatic Environment. Oct. 2-7, 2005, Cornwall, Canada. Schrader, K.K., and Harries, M.D. Bioassay-guided isolation of antibiotics and natural compounds against the catfish pathogens Edwardsiella ictaluri and Flavobacterium columnare. Aquaculture America. 2006. Schrader, K.K. 2005. Barley straw extracts are nontoxic toward musty blue-green algae. The Catfish Journal. 20(3):12. (Trade Journal) Hanson, T.R., and K.K. Schrader. 2006. Off-flavors in channel catfish managed with low-dose applications of copper sulfate. Global Aquaculture Advocate. 9(1):58-59. (International Technical Journal). Review Publications Schrader, K. 2005. Evaluation of several commercial algicides for control of odor-producing cyanobacteria. Journal of Aquatic Plant Management. 43: 100-102. Schrader, K., Dennis, M.E. 2005. Cyanobacteria and earthy/musty compounds found in commercial catfish (ictalurus punctatus) ponds in the mississippi delta and mississippi-alabama blackland prairie. Water Research. 39(13):2807-2814. Schrader, K., Harries, M.D. 2006. A rapid bioassay for bactericides against the catfish pathogens edwardsiella ictaluri and flavobacterium columnare. Aquaculture Research. 37(9):928-937.