CONTROL OF BLUE-GREEN ALGAE IN AQUACULTURE PONDS

1 SRAC Fifteenth Annual Progress Report, December, 2002 20 Reporting PeriodJanuary 1, 1999 - August 31, 2002 Funding LevelYear 1 .. $307,574 Year 2 .. 275,970 Year 3 .. 252,703 Total .. $836,247 ParticipantsUniversity of Tennessee (Lead Institution) ..J. Larry WilsonUniversity of Arkansas at Pine Bluff ..Nathan M. StoneAuburn University ..David Bayne, Thomas J. Popma, Claude BoydClemson University ..David A. Brune, John A. Collier, SchwedlerUniversity of Georgia ..Gary J. Burtle, George W. Lewis, Eloise L. StyerLouisiana State University ..Robert R. RomaireMississippi State University ..John A. Hargreaves, Susan K. Kingsbury,Edwin H. RobinsonUniversity of G. NagleNorth Carolina State V. Daniels, Ronald G. HodsonUSDA, ARS, K. ShraderUSDA, ARS, SRRC ..Paul V. Zimba, Casey C. GrimmAdministrativeDr. Greg Weidemann, Associate Director AdvisorArkansas Agricultural Experiment StationUniversity of ArkansasFayetteville, ArkansasCONTROL OF BLUE-GREEN ALGAEIN AQUACULTURE PONDSC ontrol of BLUE-GREEN ALGAE in AQUACULTURE chemical CONTROL methodologies to prevent the establishment of noxious blue -greenalgal novel selective BLUE-GREEN algicides identified through laboratory , identify, and test allelopathic chemicals produced by competing blue -greenalgae and other micro-organisms found in local aquatic OBJECTIVESSRAC Fifteenth Annual Progress Report, December.

2 2002 nutrient manipulation to promote desirable phytoplankton community nitrogen-to-phosphorus ratios in the the availability of phosphorus from pond bottom the availability of inorganic trace metal potassium levels in the salinity levels in the water circulation as a means of altering the environment to promote desirablephytoplankton community the use of plankton-feeding fish to alter the environment to promote desirablephytoplankton community the development of phytoplankton communities in the Partitioned BENEFITSC ontrol of BLUE-GREEN ALGAE in AQUACULTURE PondsThe overall goal of this project is to identifymethods of controlling or eliminating blue -greenalgae from AQUACULTURE PONDS . The ability tocontrol algal communities in PONDS could benefitfarmers in several ways. Excessive abundance ofblue- green ALGAE , especially when combined withtheir habit of growing in surface scums, can causelow dissolved oxygen concentrations and otherwater quality aberrations that affect fish growthand health.

3 Therefore, the ability to CONTROL thecomposition of blooms could result in better fishgrowth and lower costs for aeration and otherwater quality management largest and fastest growing segment of aqua-culture in the United States is farm-raised channelcatfish. Catfish that are off-flavor are unmar-ketable, and farmers are forced to hold thosefish in inventory until composition of the pondmicrobial community changes and flavor im-proves. Holding market-sized fish in inventoryimposes an economic burden on farmers, andoff-flavor is estimated to cost the industry wellover $20 million a mortalities associated with blue -greenalgae are common in the early use of high rates of granular fertilizersmay be a factor in these excessive ALGAE blooms,especially in baitfish PONDS that have been inproduction for years and have accumulatedsediments. Phosphorus in the groundwater inLonoke County, Arkansas, also contributes tonutrient loading.

4 In addition, golden shinersmay stimulate phytoplankton production byforaging on larger species of zooplankton thatwould otherwise feed on the may indirectly contribute toSRAC Fifteenth Annual Progress Report, December, 2002 22 CONTROL of BLUE-GREEN ALGAE in AQUACULTURE PondsObjective 1. Develop chemical CONTROL methodologiesto prevent the establishment of noxious BLUE-GREEN AND PRINCIPAL ACCOMPLISHMENTSU niversity of Mississippi. More than 4,000plant and ALGAE extracts have been prepared fromseveral thousand collections of plants,cyanobacteria ( BLUE-GREEN ALGAE ) and collections now include more than 300collections of tropical cyanobacteria and marinealgae, and 450 collections of aquatic and wetlandplants, cyanobacteria (collections and cultures), ALGAE , and thousands of collections of higherplants. These extracts were evaluated for selectiveblue- green algicidal activity in a rapid bioasayusing 96-well microtiter extract evaluations were conducted usingOscillatoria agardii as the cyanobacterial testorganism, and Selenastrum capricornutum as achlorophyte CONTROL for nonspecific algicidalactivity.

5 These organisms were grown in con-tinuous flow culture and provide uniform algalmaterial for biological evaluation. Lipid extractsof 33 tropical marine cyanobacteria and algaewere evaluated in these assays at an initial con-centration 100 ppm. Two related species ofmarine green ALGAE and one species of marinebrown ALGAE were found to contain substancesthat were selectively algicidal against O. production through phosphorusrelease due to their bottom feeding of methods with the potential toreduce excessive blooms (pond renovation,sodium nitrate application) will provide farmerswith information to make informed decisionswhen weighing possible benefits against the of the treatments and managementpractices considered in this project have beenpromoted for controlling BLUE-GREEN ALGAE , buttheir effectiveness has not been documented. Itis anticipated that this research will reveal which,if any, of these treatments are beneficial.

6 Anypractice demonstrated to be effective incontrolling BLUE-GREEN ALGAE has considerablepotential for improving aquaculturemanagement and enhancing these results are promising, thecyanobacterium O. agardii is not known toproduce odorous compounds that cause off-flavor problems in AQUACULTURE . We have sinceestablished cultures of the O. perornata, afilamentous BLUE-GREEN alga that produces 2-methylisoborneol (MIB), the major taintingsubstance in catfish grown in northwestMississippi. These cultures are suitable for high-throughput extract the first phase of the project, 356 extractsfrom collections of aquatic and marinecyanobacteria, plants and ALGAE havesubsequently been evaluated in replicatebioassays using O. perornata. Forty-threeextracts (12%) were found to be strongly activeand cyanobacterial-selective at the concentrationof 100 ppm, used for initial screening.

7 Doseresponse data was obtained for these extracts athalf-log concentrations. Twelve extracts werefound to be effective at 30 ppm or lower. Thetwo most potent extracts were confirmed to betwo of the related marine green ALGAE species firstidentified in the initial bioassays that usedO. agardii rather than O. Fifteenth Annual Progress Report, December, 2002 23 CONTROL of BLUE-GREEN ALGAE in AQUACULTURE PondsBioassay-guided fractionation of several extractshas resulted in the purification of unusualcyanobacterial-selective natural products that areeffective against both Oscillatoria strains atconcentrations in the parts-per-billion chemical structures of several of thesemetabolites have been solved and are undergoingadditional toxicological and second phase of the program focused on theevaluation of plants found in tropical rainforestsand temperate regions throughout the world forcyanobacterial-selective algicidal activity.

8 Initialevaluations of plant extracts at 100 ppm picked upsome activity associated with less selectiveantimicrobial substances such as tannins. In orderto reduce the incidence of false-positive hits andto select for only those plants that contain potentand potentially more selective compounds, theprimary phase of the high-throughput screeningprogram evaluated plant extracts at 20 ppm. Extractplates were evaluated in duplicate. Plant extractsthat showed cyanobacterial-selective activity at 20ppm were reconfirmed by secondary evaluationsat 20, 10 and ppm. A bio-genetically diverserepository of plant extracts from over 170 plantfamilies collected was examined. These plants wereobtained from Peru, New Guinea, and the UnitedStates. This repository contained chemicallydistinct crude lipophilic extracts of separate plantparts (roots, leaves and stems, flowers, etc.) of eachspecies collected.

9 Over 2,300 crude extracts of morethan 1,050 species of higher plant extracts wereevaluated in this high-throughput screening 70 plant species showed some level ofselective activity against O. perornata. Since themajor goal of this project was to identifycyanobacteria-specific agents, a process of deselection was undertaken to excludecompounds with broad activity against othermicroorganisms ( BLUE-GREEN ALGAE are gram-negative bacteria). Thus, the active extracts thatshowed broad antimicrobial activity against apanel of biomedically important microorganisms(bacteria and fungi) were used to deselect extractsfrom further study. The microorganisms usedto dereplicate antibiotic-containing extracts wereStaphylococcus aureus, methicillin-resistantStaphylococcus aureus, Pseudomonas aeruginosa,and Trichophyton mentarophytes. In addition,all active extracts that showed mammaliantoxicity in the monkey kidney Vero cellcytotoxicity assay were also deselected for most potent cyanobacteria-selective extracts(that were not significantly antibacterial and werenon-toxic) were those obtained from the rootsand stems of a Peruvian collection of Dulaciacandida (family = Olacaceae).

10 Dulacia candidais a widely distributed tropical Amazonianshrub. The crude D. candida extracts showedextremely potent anti-cyanobacterial activity,approaching the parts per billion range. Thecomponents of the extract were separated andthe bioactivity was confined to several moder-ately non-polar chromatographic , the substance (or substances) respon-sible for the potent anti-cyanobacterial have notyet been addition, more than one hundred pure bio-logically active compounds from marineinvertebrates and ALGAE have been examined foranti-cyanobacterial activity. Some of these sub-stances inhibited ALGAE growth, but were eithercytotoxic or not selectively toxic to nuisanceblue- green Proprietary analogs of a promisingnatural product found in certain plants weretested in PONDS for selective activity against BLUE-GREEN ALGAE . Efficacy testing yielded promisingresults, and one of the best proprietarycompounds (as determined by laboratoryscreening studies) significantly reduced the MIB-producing cyanobacterium Oscillatoriaperornata and the cyanobacterium RaphidiopsisSRAC Fifteenth Annual Progress Report, December, 2002 24 Objective 2.