(Farlow Ex Gomont) Comb. Nov

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1997, p. 3104–3110 Vol. 63, No. 8 0099-2240/97/$04.0010 Copyright © 1997, American Society for Microbiology Evidence for Paralytic Shellfish Poisons in the Freshwater Cyanobacterium Lyngbya wollei (Farlow ex Gomont) comb. nov. 1 1 1 2 2 W. W. CARMICHAEL, W. R. EVANS, Q. Q. YIN, P. BELL, AND E. MOCZYDLOWSKI Department of Biological Sciences, Wright State University, Dayton, Ohio 45435,1 and Departments of Pharmacology and Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 065102 Received 30 January 1997/Accepted 6 June 1997 Lyngbya wollei (Farlow ex Gomont) comb. nov., a perennial mat-forming filamentous cyanobacterium prev- alent in lakes and reservoirs of the southeastern United States, was found to produce a potent, acutely lethal neurotoxin when tested in the mouse bioassay. Signs of poisoning were similar to those of paralytic shellfish poisoning. As part of the Tennessee Valley Authority master plan for Guntersville Reservoir, the mat-forming filamentous cyanobacterium L. wollei, a species that had recently invaded from other areas of the southern United States, was studied to determine if it could produce any of the known cyanotoxins. Of the 91 field samples collected at 10 locations at Guntersville Reservoir, Ala., on the Tennessee River, over a 3-year period, 72.5% were toxic. The minimum 100% lethal doses of the toxic samples ranged from 150 to 1,500 mg kg of lyophilized L. wollei cells21, with the majority of samples being toxic at 500 mg kg21. Samples bioassayed for paralytic shellfish toxins by the Association of Official Analytical Chemists method exhibited saxitoxin equiv- alents ranging from 0 to 58 mg g (dry weight)21. Characteristics of the neurotoxic compound(s), such as the lack of adsorption by C18 solid-phase extraction columns, the short retention times on C18 high-performance liquid chromatography (HPLC) columns, the interaction of the neurotoxins with saxiphilin (a soluble saxitoxin-binding protein), and external blockage of voltage-sensitive sodium channels, led to our discovery that this neurotoxin(s) is related to the saxitoxins, the compounds responsible for paralytic shellfish poisonings. The major saxitoxin compounds thus far identified by comparison of HPLC fluorescence retention times are decarbamoyl gonyautoxins 2 and 3. There was no evidence of paralytic shellfish poison C toxins being produced by L. wollei. Fifty field samples were placed in unialgal culture and grown under defined culture conditions. Toxicity and signs of poisoning for these laboratory-grown strains of L. wollei were similar to those of the field collection samples. The presence in reservoirs and lakes of the southeastern a potent postsynaptic, cholinergic nicotinic agonist that causes a United States of Lyngbya sp., a cyanobacterium in the family depolarizing neuromuscular blockade and has been associated Oscillatoreaceae, has been documented for at least 100 years with Anabaena flos-aquae, Anabaena spiroides, Anabaena circina- but has become more common during the past 2 decades. lis, and Oscillatoria spp. Anatoxin A(s) [antx-A(s)], which is Lyngbya wollei (Farlow ex Gomont) comb. nov. was recently produced by strains of Anabaena (21), is a potent inhibitor of identified as the species responsible for these occurrences (28). acetylcholinesterase. Saxitoxin (STX) and neosaxitoxin Benthic mats of this cyanobacterium can reach densities of 6.6 (neoSTX) were shown to be the major neurotoxins present in kg (fresh weight) m22 (28) to 13.8 kg (fresh weight) m22 (5). Aphanizomenon flos-aquae (20). In addition, Anabaena circina- Visible growth of L. wollei occurs as dense floating mats, while lis water blooms from the Murray River system in Australia the majority of the L. wollei biomass remains subsurface (28). have recently been shown to produce paralytic shellfish poisons In the southeastern United States, L. wollei overwinters in the (PSPs) (4, 15). form of benthic mats and rises to form surface mats during the When establishing whether compounds that cause acute or warmer months. acute lethal toxicity exist within a potentially biotoxic cya- Although there have been no reports of toxic compounds nobacterium, the general procedure for initial screening is the being produced by freshwater Lyngbya spp., the production of mouse bioassay. Although there are limitations to the use of toxic compounds by marine Lyngbya spp. is well-known. Lyng- this bioassay, it does a good job of allowing one to distinguish bya toxin A and derivatives thereof have been identified as the between the known neurotoxins and hepatotoxins of cyanobac- causative agents for the dermatitis produced by Lyngbya ma- teria through observation of signs of poisoning and survival juscula (7). Lyngbya toxin A has also been shown to be a potent times (8). Once toxicity has been established with this bioassay, tumor promoter and an activator of protein kinase C. Com- there are several methods available for cleanup, quantitative pounds cytotoxic to a murine leukemia cell line (P-388) and an analysis, and identification of the toxin(s) (8, 12). The purpose influenza virus (PR8) were found in extracts of a cultured of this study was to determine if the L. wollei isolates from Lyngbya sp. Immunosuppressive peptides have also been iso- Gunterville Reservoir produce any of the biotoxins associated lated from L. majuscula (16). with cyanobacterial blooms and to characterize any biotoxins Neurotoxic alkaloids have been isolated from species and present. strains of cyanobacteria in the genera Anabaena, Aphanizom- enon, Oscillatoria, and Trichodesmium (8). Anatoxin A (antx-A) is MATERIALS AND METHODS Collection of samples. L. wollei samples were collected at Guntersville Res- ervoir, located on the Tennessee River near Guntersville, Ala. Collection of field * Corresponding author. Phone: (937) 775-3173. Fax: (937) 775- samples was initiated in November 1991, continued through June 1994, and took 3320. place on 10 different days. Sampling locations included Siebold, Ossawintha, 3104 VOL. 63, 1997 PSP IN LYNGBYA WOLLEI 3105 Waterfront, Boshart, and County Park. A complete listing and location of sam- TABLE 1. Frequency distribution of toxicity in field samples of pling locations plus dates can be found in a Tennessee Valley Authority report L. wollei collected from Guntersville Reservoir (9). The samples were stored in an ice chest for transport to the laboratory, cleaned of debris, rinsed with distilled water, lyophilized, and stored at 220°C. % of total samples MLD ,mgkg21a No. of samples Extraction of samples for chemical and biological assay. The lyophilized L. 100 (n 5 91) wollei samples were extracted with 25% methanol in water (adjusted to pH 3.5 with acetic acid) or 25% methanol in 50 mM acetic acid at 50 ml per g of 150 2 2 material. The mixture was stirred for 1 to3hatroom temperature and allowed 250 13 14 to steep for 12 h at 4°C. The majority of the cellular debris was removed either 500 25 27.5 by filtration through a Buchner funnel with no filter paper or by squeezing the 1,000 18 20 material in the barrel of a disposable syringe. The methanol was removed by 1,500 8 9 evaporation at 25 to 30°C. Depending on the size of the sample, the filtrate was b clarified by centrifugation at either 20,000 3 g for 30 min at 5°C or 16,000 3 g NT 25 27.5 for 2 min at room temperature. a Based on the weight of lyophilized samples. Bioassay. Estimations of the minimum 100% lethal dose (MLD ) were 100 b NT, nontoxic at 1,500 mg kg21. carried out with duplicate male mice (ICR-Swiss, 16 to 25 g) injected intraperi- toneally (i.p.). Symptoms of poisoning and survival times were noted and com- pared with previous mouse bioassay results from known cyanobacterial toxins. On the basis of survival times (a few minutes to 1 h), it was possible to determine pended in sucrose-free MOPS-NaN3-EDTA buffer and centrifuged for 15 min at whether neurotoxins or hepatotoxins were present. On the basis of signs of 7,700 3 g. The supernatant was centrifuged for 45 min at 100,000 3 g, and the poisoning, it was possible to determine whether antx-A(s) (which results in final pellet was resuspended in 0.3 M sucrose buffer at a concentration of ;5mg anticholinesterase signs of poisoning) or antx-A (which results in depolarizing of protein ml21 and stored in aliquots at 275°C. neuromuscular blocking agent signs of poisoning) were present. If these signs The standard assay mixture for a [3H]STX binding competition assay with were not present, then PSP-type toxins were suspected. When PSP-type toxicity unlabeled STX (Calbiochem-Novabiochem) or L. wollei extracts contained 4.2 was observed, assays were done according to the Association of Official Analyt- nM [3H]STX (Amersham), 20 mM MOPS-NaOH (pH 7.4), 0.1 mM EDTA, and ical Chemists (AOAC) mouse lethality bioassay for PSPs (3). All mouse bioas- either 0.2 M choline chloride (for garter snake or bullfrog serum) or 0.2 M says were carried out according to the guidelines and protocols of the Wright sodium chloride (for rat brain microsomes) in a final volume of 0.35 ml. The State University laboratory animal use guidelines. The bioassays were done with binding reaction was initiated by the addition of plasma or microsomes and extracts of L. wollei since adding the lyophilized cells to water resulted in a equilibrated for 1 to2hat0°Cbefore removal of the free toxin at 4°C by passing suspension that could not be drawn into a syringe needle. triplicate 100-ml aliquots of the reaction mixture, followed by a wash solution of Culture of L. wollei. The cleaned, rinsed samples were blotted with filter paper, 0.5 ml of 100 mM Tris-HCl, pH 7.2, through small cation-exchange columns.

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