Species of the Diatom Genus Pseudonitzschia in Australian Waters

An Australia-wide taxonomic survey for species of the potentially toxic diatom genus Pseudonitzschia Peragallo (causative organisms of amnesic shellfish poisoning) was carried out using both light and transmission electron microscopy. Samples studied derived from the Gulf of Carpentaria, North West Shelf, Coral Sea, East Australian Current, Tasmanian, Victorian and Western Australian coastal waters. The dominant bloom-forming Pseudonitzschia species in Australian coastal waters were P. fraudulenta (New South Wales), P. pungens f. pungens and P. pseudodelicatissima (Tasmanian and Victorian waters). Other representatives of this diatom genus in Australian waters include P. americana, P. lineola, P. subpacifica and P. turgidula, but these were usually only present in low concentrations. While populations of P. pseudodelicatissima from the Bay of Fundy (Canada) can be weakly toxic, wild and cultured diatom populations of this species from Tasmanian and Victorian coastal waters were consistently nontoxic. The major toxic Pseudonitzschia species of concern in Canadian waters, P. pungens f. multiseries was detected as a minor component (5% of total biomass) of a dense P. pungens f. pungens bloom in a New South Wales estuary. Pseudonitzschia australis, a toxic species in Californian waters, was never detected in Australian waters. Surveys to identify bloom-forming Pseudonitzschia species in Australian waters, and monitoring of these species for toxicity, will continue. Introduction seriata Cleve (broad cells) and Nitzschia delicatissima ΛΛ v Cleve (narrow cells) (for example, see Dakin and ColThe diatom genus Pseudonitzschia (HPeragallo 1900) ^ ^ Wood ^ HaUe_ includes some 20 manne planktomc species with Hallegraeff 1981, for Australian waters). In pointed, fusiform valves which unite into chams by & carefol taxonomic study> Hasle (1972) demonstrated overlapping of their cell ends (so-called 'stepped' colon^ ̂ ̂ Pseudonitzschia seriata (Cleve) Peragallo ies). Until recently these species were included as a sec. jg ̂ ^^ ^ ̂ ̂ ^ ̂ rf ̂ ̂ ^ Remi_ tion within the large and complex diatom genus Nitzs^& ̂ ̂ & number rf other ̂ ^ faave been schia Hassall (see Hasle 1965), but they are now being consistently CQnfused with ̂ taxon recognised as a separate genus (Hasle 1993). Pseudonitzschia species are among the most difficult diatoms In the past five years, the recognition of toxic blooms to identify to species level. Their taxonomy is based on of Pseudonitzschia species has focused considerable atcolony type as seen in girdle view, valve outline, prestention on this diatom genus. First reported in November ence of a central nodule ('pseudonodulus' in older litera1987, Atlantic Canada was hit by a mysterious outbreak ture), and ratio between the number of fibulae ('keel of toxicity in cultured blue mussels that led to 3 human puncta' in older literature) and the number of transapical deaths and 105 cases of acute human poisoning (Bates costae ('interstriae')· Species of this group can often not et al 1989). Because of the neurological symptoms inbe accurately identified by light microscopy alone, and volved (permanent damage to short term memory), this are widely recorded in the older literature as Nitzschia new illness was called amnesic shellfish poisoning Botanica Marina / Vol. 37 / 1994 / Fasc. 5 Copyright © 1994 by Walter de Gruyter · Berlin '· New York 398 Hallegraeff: Pseitdonitzschia species in Australian waters (ASP). The causative toxin, the excitatory amino acid domoic acid, proved to have been produced by the diatom Pseudonitzschia pungens (Grunow) Hasle forma multiseries (Hasle) Hasle. This incident was followed in 1988 by a toxic bloom of P. pseudodelicatissima (Hasle) Hasle in the Bay of Fundy, Canada (Martin et al 1990). In September 1991, a massive diatom bloom of P. australis Frenguelli along the Californian coast was implicated in causing mortalities of brown pelicans and cormorants via foodchain transfer of domoic acid contained in their anchovy diet (Fritz et al. 1992, Garrison et al. 1992, Work et al. 1993). Some related Pseudonitzschia species, such as P. fraudnlenta (Cleve) Hasle, P. pungens forma pungens and P. seriata have proved to be non-toxic (Hargraves et al. 1993). Pseudonitzschia species can be a dominant component of the phytoplankton of Australasian waters, during the spring bloom in Sydney coastal waters (Hallegraeff 1981, Hallegraeff and Jeffrey 1993), and enrichment phenomena in East Australian Current eddy systems (Jeffrey and Hallegraeff 1980, 1987) and are a regular component of tropical communities in the Coral Sea (Hallegraeff and Jeffrey, unpublished) and equatorial Pacific (Hasle 1960). Thus, questions were raised both by the Australian shellfish industry and by the American shellfish export market as to whether similar diatom toxins could be present in Australian seafood products. In the present taxonomic survey, Australia-wide plankton samples collected from the Gulf of Carpentaria, North West Shelf, Coral Sea, East Australian Current, Tasmanian, Victorian and Western Australian coastal waters were reexamined in detail for the identity of Pseudonitzschia species. Material and Methods Collection of samples Seawater samples were taken throughout the water column using 8 L Niskin bottles or a 37 μ mesh free-fall plankton net. Collections were made from tropical, subtropical and temperate Australian waters as summarised in Table I. Enrichment cultures were initiated from selected seawater samples according to Jeffrey (1980 a) and grown at 17.5 °C and a light intensity of 80 μΕ m~ s" (12:12, L:D). In many cases Pseudonitzschia species could readily be established in culture using medium K (Keller et al 1987), but, for unknown reasons, over time most of these species were eventually lost from culture. Light microscopy and species counts The phytoplankton from 5 L of seawater was concentrated in a continuous centrifuge (15 000 g; flow rate l L 6 min — 1) to about 10 mL within 2—3 h of collection. The concentrated algae' were fixed with 3% glutaraldehyde buffered with 0.01 M phosphate (pH = 7.8) and stored at 5 °C for 3—5 weeks until quantitative species counts were completed. Some 30—50 random fields of the bottom area of a counting chamber (5 mm deep) were examined at 800 X magnification with an inverted microscope using phase-contrast illumination. Transmission electron microscopy Phytoplankton net samples, preserved with 2% formalin neutralized with hexamine, were rirised with distilled water to remove salt and preservatives, and then cleaned of organic material by overnight oxidation with potassium peroxodisulphate at 60 °C (Ma and Jeffrey 1978). Small drops were pipetted on to parlodion-coated copper grids (400 mesh/inch) and examined in a Philips 410 transmission microscope (TEM) at 100 kV. Selected cleaned diatom cells were also mounted in coumarone and inspected by phase-contrast light-microscopy. Diatom terminology follows Anonymous (1975) and Ross et al (1979) and supersedes terminology for Pseudonitzschia used by Hasle (1965).