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Abstract

Dinoflagellate species such as Gymnodinium catenatum, Alexandrium
minutum and Alexandrium catenella produce potent neurotoxins, the causative
agents of Paralytic Shellfish Poisoning (PSP). Molecular genetic research on
these species is complicated by factors such as their symbiotic association
with bacteria, unusual chromosome structure, tough cellulose cell walls
(Alexandrium), and large amount of genomic and repetitive DNA. Little is
known about how, where and when PSP toxins (PSTs) are synthesised. The
basic precursors of the PST molecule(s) have been hypothesised, but no
genes coding for toxin production have been definitively identified. The
application of molecular methods to study armoured and unarmoured marine
dinoflagellates was assessed and techniques successfully refined, including
DNA and RNA isolation, flow cytometry, primer design, PCR, quantitative real
time PCR, molecular cloning and sequence analysis.

Methods for detecting intra- and extra-cellular bacteria were examined,
including fluorescence in situ hybridisation, light microscopy, agar plating and
PCR. Prolonged antibiotic treatment of G. catenatum, A. minutum and A.
catenella cultures reduced bacterial load but resulted in poor growth and cell
death of dinoflagellates. Close bacterial associations with dinoflagellates
may have an important and as yet poorly understood role in dinoflagellate
health and toxicity. A dinoflagellate (eukaryotic) origin of candidate PST
genes was confirmed by development of methods to isolate polyadenylated
RNA not contaminated with prokaryotic (bacterial) genes. This technique
was also crucial for gene expression studies.
Production of S-adenosylmethionine (SAM) is catalysed by the enzyme SAM
synthetase, which is encoded by Sam. This enzyme is involved in many
cellular metabolic processes, including the biosynthesis of PSTs. Sam was
characterised for the first time in toxic dinoflagellates, with multiple copies of
Sam present in individual strains. The most frequently identified copy of Sam
was highly conserved between dinoflagellates, but dissimilar to Sam
sequences from non-dinoflagellates. Two other candidate PST genes,
S-adenosylhomocysteine hydrolase (Sahh) and methionine aminopeptidase (Map), previously identified in the PSP dinoflagellate Alexandrium fundyense
were cloned in A. catenella.
Toxin dynamics and expression of Sam, Sahh and Map were examined
concurrently over the cell division cycle in A. catenella. The toxin profile was
constant over the cell cycle but cellular toxin content decreased during
division, suggesting that toxin was partitioned in dividing cells. Expression of
Map and Sahh appeared to follow a similar pattern to rate of endocellular
toxin production throughout the cell cycle. Positive toxin production occurred
in the absence of light, suggesting that light was not a direct trigger for toxin
production. The molecular techniques developed and sequence information
and knowledge of cellular toxin dynamics gained will facilitate further
characterisation of novel dinoflagellate genes.

Item Type:	Thesis - PhD
Authors/Creators:	Harlow, LD
Keywords:	Dinoflagellates, Paralytic shellfish poisoning
Copyright Holders:	The Author
Copyright Information:	Copyright 2005 the Author - The University is continuing to endeavour to trace the copyright owner(s) and in the meantime this item has been reproduced here in good faith. We would be pleased to hear from the copyright owner(s).
Additional Information:	Thesis (PhD)--University of Tasmania, 2006. Includes bibliographical references
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