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


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Lush, Graeme John 1999 , 'Novel exotoxic principle(s) produced by the toxic dinoflagellate Alexandrium minutum : effects on brine shrimp (Artemia salina) and larval fish (Rhombosolea taparina)', PhD thesis, University of Tasmania.

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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 GTX2and GTX3were dominant early in culture growth (36-
64 mole%, epimeric total on day 48) but as time progressed GTX 1and GTX4became
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 a/. 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

Item Type: Thesis - PhD
Authors/Creators:Lush, Graeme John
Keywords: Dinoflagellate blooms, Paralytic shellfish poisoning, Algal blooms, Toxic marine algae
Copyright Holders: The Author
Additional Information:

Thesis (Ph.D.)--University of Tasmania, 1999. Includes bibliographical references

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