Toxin Production by Gambierdiscus toxicus Isolated from the Florida Keys JOHN A. BABINCHAK, DAVID J. JOLLOW, MICHAEL S. VOEGTLlNE, and THOMAS B. HIGERD Introduction al., 1984) were extracted and partitioned compared with a highly toxic clonal G. between solvents ofdifferent polarities. toxicus isolated in Hawaii (Sawyer et al., Ciguatera is a tropical fish-borne The biological and chemical nature, 1984). disease in which the lipid-soluble neuro­ however, ofthe limited quantity of lipid­ toxin, ciguatoxin, is believed to be trans­ soluble dinoflagellate toxin obtained by Materials and Methods ferred through the food chain and bio­ these nonstringent extraction, partition­ Benthic dinoflagellate samples were concentrated primarily in carnivorous ing, and purification techniques can collected from the Florida Keys at a site reef fish to levels toxic to humans. Since only be conjecture. Cultured G. toxicus previously involved in ecological studies ecological research in the South Pacific readily produce a toxin distinguished on benthic dinoflagellates associated implicated Gambierdiscus toxicus as the from ciguatoxin on the basis of higher with ciguatera (Bomber, 1985). Samples probable cause of ciguatera (Yasumoto molecular weight and lower polarity. were collected in December 1983, and et al., IfJ77), this dinoflagellate had been This toxin has been tentatively identified in February, May, July, and December collected from the wild, grown uni­ as maitotoxin (Yasumoto et aI., lfJ79) , 1984, from Knight Key, which con­ algaliy, and extracted for toxins that have a toxin first isolated from the gut of sistently had a high G. toxicus popula­ been analyzed principally by mouse surgeonfish, Acanthurus sp. (Yasumoto tion. This site, station 3 of Bomber bioassay. et aI., 1fJ76). (1985), is located in Florida Bay, 0.8 kIn Evidence that G. toxicus produces the Unialgal cultures, initiated with up to east of seven-mile bridge (lat. 42°44' fish-extracted ciguatoxin is based on 30 cells, are used in most studies on tox­ 20"N, long.·81°07'I6"W). The area con­ reports in which either wild cells (Yasu­ in production by cultured G. toxicus. sists ofan algal reef depauperate in coral moto et aI., lfJ77, lfJ79; Bagnis et aI., The objectives of this study were to with strong tidal currents and up to 95 1980; Shimizu et aI., 1982) or cultured isolate and culture clonal G. toxicus col­ percent cover by Halimeda spp. cells (Yasumoto et aI., lfJ79; Tindall et lected concurrently from a single site in (Bomber, 1985). Pieces of macroalgae the Florida Keys, and compare the were collected in plastic jars with sea­ quantity and nature of the toxins pro­ water, shaken, and the seawater filtered duced. Toxicity, as measured in mice through a 250 ~m Nitex 1 sieve. The (LD ), was determined using whole filtrate was then refiltered through a 25 ABSTRACT-The toxicities of six clonal so Gambierdiscus toxicus cultures collected cells or nonfractionated extracted tox­ ~m Nitex sieve and the retentate ana­ concurrently from Knight Key, Fla., were ins, and the relative polarity of the ex­ lyzed microscopically for G. toxicus. compared with the toxicity ofthe Hawaiian tracted toxins was analyzed by high Samples with high populations of G. G. toxicus strain, 739. W 50 values obtained performance liquid chromatography toxicus were suspended in 1 liter poly­ using mouse bioassay demonstrated a hundredfold range in whole-cell toxicity. The (HPLC) (Higerd et al., 1986). The toxic carbonate bottles containing 700 rnl Hawaiian and two Floridian strains had characteristics ofthe Florida isolates are filtered (20 ~m) seawater for shipment comparable mouse toxicity (LD5oJ ofabout to the laboratory. 2.5 x 10 4 cells/kg. Two additional groups The medium used for isolation and ofFloridian strains had toxicities ofabout John A. Babinchak and Thomas B. Higerd are with growth of G. toxicus was Provasoli's ES 2 X 105 and >1 million cells/kg, respective­ the Charleston Laboratory, Southeast Fisheries ly. Fractionation of methanol extracts by Center, National Marine Fisheries Service, enriched seawater medium as modified high-performance liquid chromatography NOAA, P.O. Box 12607, Charleston, SC 29412. by 1. West (McLacWan, lfJ73). Seawater suggests that toxins produced by different David 1. Jollow is with the Department of Phar­ macology, Medical University of South Carolina, was collected either at the Florida Keys clones of G. toxicus are indistinguishable Charleston, SC 29425. And Michael S. Voegtline from each other but are more polar than fish and Thomas B. Higerd are with the Department toxin. Isolates ofOstreopsis heptagona, also of Basic and Clinical Immunology and Micro­ 'Mention of trade names or commercial firms does isolatedfrom Knight Key, had relatively low biology, Medical University of South Carolina, not imply endorsement by the National Marine 6 toxicities (W50 > 5 X 10 cells/kg). Charleston, SC 29425. Fisheries Service, NOAA. 48(4), 1986 53 sampling site, or at two sites from 7 to 20 /-1m Nitex filter and resuspending the Table 1.-Whole-eell toxicity of cultured G. tox/cus 10 miles off Charleston, S.c., and fil­ retentate in 30 percent aqueous meth­ clones. tered onsite through a 20 /-1m Nitex sieve anol (v/v). This suspension was evapor­ Cell dia. LDso Clone Source (jAm)' (cells/kg) into 10 to 20 liter polycarbonate carboys. ated to dryness under a stream of nitro­ T39 Tern Island, HI 76 2.5 X 10' (4)' Upon arrival at the laboratory, the water gen. Cd20 Knight Key, FL 79 3.5 x 10' (4) was refiltered through 0.45 /-Iffi cellulose Toxicities of whole cells (LDso) were Cd4 Knight Key, FL 81 4.5 x 10' (3) Cd8 Knight Key, FL 94 2.5 x 10' (2) nitrate membranes in a Nuclepore radial determined by suspending the dried Cd10 Knight Key, FL 81 3.0 x 10' (2) flow cell into sterile polycarbonate car­ sample in phosphate buffered saline Cd9 Knight Key, FL 86 >2.3 x 10' (2) Cd13 Knight Key, FL 77 boys and refrigerated until used to pre­ containing 5 percent Tween 80 and in­ >2.5 x 10' (2) pare media. The vitamin mixture (stored jecting 0.2 ml of appropriate cell con­ 'Cellular measurements were obtained by measuring live individuals at 400 x with bright-field microscopy (n =30). frozen) and enrichments were prepared centrations intraperitoneally into female 'Bioassay analyses (n). in concentrated stocks, filter-sterilized ICR mice weighing approximately 20 g and added aseptically to the seawater. (Kelly et al., 1986). In addition to deter­ The prepared medium was filter-steril­ mining whole cell toxicities, the seven ized through 0.2 /-1m cellulose nitrate G. toxicus clones were grown and har­ membranes and used immediately or vested to obtain total cell densities refrigerated. greater than 1 x 106 cells. These sam­ and toxin production. This precipitate Clonal cultures of G. toxicus and Os­ ples were extracted in 80 percent aque­ was prevented by eliminating the Tris treopsis sp. were established from single ous methanol (v/v) for 48 hours at room buffer from ES medium. cells isolated with a micropipet, washed temperature and bioassayed or frac­ Five 0. heptagona clones isolated three or four times in sterile seawater, tionated by HPLC using a 50-100 per­ from Knight Key, prepared and bio­ and inoculated into 16 x 125 rom culture cent methanol linear gradient. Each assayed using the same procedure de­ tubes containing 5 mI ES medium. Os­ fraction was later assayed for toxicity by scribed for G. toxicus, had relatively treopsis spp. occur with G. toxicus in the mouse bioassay (Higerd et al., low toxicity (LDso > 5 x 106 cells/kg. the Florida sampling site (Bomber, 1986). Mouse mortalities were observed at in­ 1985) and have been shown to be toxic jections of 5 x 106 cells/kg, but higher by Nakajima et a1. (1981). Growth was Results and Discussion dosages were not assayed so a definitive monitored with an inverted microscope, Six G. toxicus clones were isolated LDso could not be calculated. Al­ and ifapparent within 30 days, the cul­ concurrently from a dinoflagellate sam­ though 0. siamensis and 0. ovata were tures were transferred to 25 mI mini­ ple collected at Knight Key, 20 Decem­ shown to be toxic by Nakajima et al. Fernbach flasks or 20 x 150 rom ber 1983 (Bomber, 1985, station 3). The (1981), their toxicities were also> 5 X culture tubes containing 15 mI ES Hawaiian strain, 139, was hand-carried 106 cells/kg. Besada et a1. (1982) re­ medium. After successful growth for from Hawaii to the SEFC Charleston ported no toxicity in Ostreopsis cultures 15-30 days, the cultures were placed in Laboratory. Five Ostreopsis clones isolated from the Caribbean Sea. 250 mI polycarbonate Erlenmeyer flasks represent a new species, 0. heptagona, The LDso values using whole cells of containing 100 mI ES medium. Stock with cellular lengths >100 /-1m (Norris G. toxicus are shown in Table 1. The cultures were maintained in 250 mI et al., 1985). G. toxicus clones were Hawaiian clone, 139, was similar in tox­ flasks and transferred to new medium identified microscopically by their icity to Cd20 and Cd4 from the Florida every 30 days. These cultures were cellular shape and the characteristic Keys and to 139 cultures grown in maintained at 27°C under an illumina­ "fishhook" apical pore slot (Adachi and Hawaii. Florida Key clones Cd8 and tion of 30-50 /-IE'M-2'S-1 and a 16:8 Fukuyo, 1979; Taylor, 1979). Further Cd10 were 10 times less toxic, while the hours light:dark cycle without aeration. taxonomic studies are in progress using toxicities of Florida Key clones Cd9 and For toxin bioassay, 100 mI cultures a chloral hydrate-hydriodic acid stain­ Cd13 were just evident with more than were inoculated into 2.8 liter Fernbach ing method (Schmidt et a1., 1978) and 2 x 106 cells/kg.