Novel exotoxic principle(s) produced by the toxic dinoflagellate Alexandrium minutum : effects on brine shrimp (Artemia salina) and larval fish (Rhombosolea taparina)

A number of species of the dinoflagellate genus Alexandrium have been implicated in the production of Paralytic Shellfish Poisoning (PSP) toxins but occasionally have also been associated with the mortality of both cage-reared and wild raffish in such places as the Faroe Islands (A. tamarense) (Mortensen 1985), Taiwan (A. minutum) (Su et al. 1993) and Egypt (A. minutum) (Halim 1996). In the present work exocellular toxicity was investigated in two species of Australian dinoflagellates, A. minutum and Gymnodinium catenation. Both are known producers of neurotoxic PSP toxins, but these toxins are only known to exist endocellularly. Investigations took the form of animal based biological assays using the brine shrimp, Artemia sauna, and juvenile greenback flounder, Rhombosolea taparina, which were exposed to whole cell and cell-free cultures of the algae. Bioassays determined the time to mortality for both species, while pathological changes in major tissues and changes in physiochemical characteristics of the blood were characterised only in the flounder. Even though Artemia did not feed on toxic dinoflagellate cells of G. catenatum and A. minutum, cultures of these two species were found to kill Artemia within 24 hours. However, only A. minutum culture-filtrate was toxic to Artemia whereas G. catenatum culture-filtrate was not. Likewise, culture-filtrate of unialgal G. catenation cultures was found to be non-toxic to flounder with no pathological changes in gills or other tissues observed. This disproves the suspected involvement of G. catenation as the cause of clubbing necrosis gill syndrome (Clark et al. 1997) documented in farmed Atlantic salmon in Tasmania. In contrast, the culture-filtrate of unialgal A. minutum cultures was found to be highly toxic to flounder. Artemia and flounder mortality occurred within 24h of exposure to full strength A. minutum culture-filtrate. Flounder exhibited mild to severe histopathological gill changes including: swelling, degeneration and sloughing of the respiratory epithelium. The primary lamellar epithelial cells were so swollen in some areas that they displaced the respiratory epithelial cells into the interlaminar region. Also apparent were varying degrees of swelling, vacuolation, degeneration and cytoplasmic shrinkage of the gill chloride cells and hypertrophic or discharged mucocytes. Also common were antemortem blood clots in the heart ventricular tissue. Changes in blood physiochemical characteristics of flounder were also found with increases in blood potassium inducing a state of hyperkalemia. Hyperkalemia affects the contractile functioning of the heart, causing bradycardia and atrial standstill and can lead to cardiac arrest. Increases in gill-associated succinic dehydrogenase activity were observed but only when flounder were exposed to 60% A. minutum culture-filtrate mixed with 40% sterile seawater. No similar effect was observed in Artemia or flounder from exposure to seawater containing purified gonyautoxins (GTX) 1-4 in concentrations equivalent to the normal endocellular levels. Activity from the exocellular medium of A. minutum similar to that exhibited by Neurotoxic Shellfish Poisoning (NSP) toxins was revealed by neuroblastoma tissue culture assays (Manger et al. 1993), but neuroreceptor binding assays (Van Dolah et al. 1994) on the same material failed to detect brevetoxins. Lipid soluble A. minutum extracts also failed to show conclusive evidence for cytotcodcity to erythrocytes or gill tissues or toxicity to Artemia. This would discount the involvement of the fast acting spirolides (reported from A. ostenfeldii), which are lipophilic (Cembella et al. 1999). Endo/exotoxicity of A. minutum was followed over the growth cycle of the dinoflagellate in batch culture. Total Sodium Channel Blocking (SCB) toxin concentrations (STX equivalents) and profiles of individual GTX 1-4 components (endocellular only), as well as toxicity of the culture-filtrate to Artemia were examined. SCB activity was monitored by neuroreceptor binding assay. Peaks in toxicity of the exocellular medium appeared concurrent with peaks in the endocellular toxicity of GTXs (both occurring in early to late lag phase and declining as time progressed). The SCB toxicity of the medium was found to be three orders of magnitude higher (3.7-10.2 pg STX equivalents cell\\(^{-1}\\) ) than the endocellular SCB toxicity (169-610 fg STX equiv. cell\\(^{-1}\\)). This is one of the first reports conclusively demonstrating SCB toxicity in the exocellular medium of a dinoflagellate. Endocellularly, the gonyautoxins GTX\\(_{2}\\) and GTX\\(_{3}\\) were dominant early in culture growth (36- 64 mole%, epimeric total on day 48) but as time progressed GTX\\(_{1}\\)and GTX\\(_{4}\\) became increasingly important (58-93 mole %, epimeric total on day 182). Antibiotic treatment of dinoflagellate cultures reduced bacterial levels by up to 93% but this did not appear to affect SCB toxicity, although it did decrease toxicity of the exocellular medium to Artemia, which was highest later in culture growth. Toxicity of the A. minutum culture-filtrate towards Artemia did not appear to be correlated with exocellular or endocellular total SCB toxicity nor was it correlated with any individual toxic GTX fraction. It is concluded that, in addition to a Sodium Channel Blocking agent, A. minutum is producing a cytotoxic ichthyotoxin that is not a PSP toxin. This exotoxic principle is heat labile (reduced toxicity to Artemia above 80°C), but is not a gonyautoxin or brevetoxin. In fish A. minutum culture medium produces severe pathological gill lesions which resemble some of those caused by the raphidophyte Chattonella marina (Endo et al. 1985) which is also associated with the NSP syndrome. Although damage to fish gills upon exposure to C. marina is thought to be due to the production of highly reactive oxygen radicals (Tanaka et al. 1992) this was ruled out as the cause of his. topathology in this study as oxygen radicals are short lived and appear to only occur in photosynthetically active cultures. The toxic principle(s) of A. minutum remained active in the culture-filtrate even after the cells were removed and stored frozen, in the dark for several weeks. The present work on novel ichthyotoxins in A. minutum culture medium adds to preliminary reports of such activity by both this species (Bagoien etal. 1996) as well as A. tamarense (Hansen 1989, Ogata and Kodama 1986), suggesting that this phenomenon may be more widespread.