Scottish harmful phytoplankton monitoring programmes, and previous research studies, have shown that the saxitoxin producing (Group I) and, recently, the non saxitoxin producing (Group III) ribotypes of A. tamarense are present in Scottish waters, co-occurring in some locations. Shellfish toxcity events may have been in decline in recent years, possibly due to increasing presence of the benign Group III ribotypes. This thesis investigated the interactions between these two A. tamarense ribotypes. To study these morphologically identicle organisms a fluorescence in situ hybridization-flow cytometry (FISH-FC) based method of cell identification and enumeration was developed, allowing, for the first time, Group I and Group III A. tamarense strains to be studied in co-culture. In isolation, toxin production of the Group I strain was elevated under P limitation as well as at low temperature and short light (spring) conditions. In co-culture a competitive interaction between Group I and Group III ribotypes was clear, with growth rates, maximum cell densities and total toxicity per cell showing marked changes compared to monocultures. Both strains showed a preference for the intermediate temperatures of 15 and 18 °C. Yet despite this, strain differences did occur. The Group III strain out-competed the Group I strain, having a higher biomass and maximum specific growth rate across most of the temperatures studied. The Group III strain also appeared to be able take up and utilise P more effectively that the Group I strain. Mathematical modelling revealed that the observed growth patterns (i.e. dominance of the Group III strain) in mixed culture was not solely due to more efficient nutrient uptake but, additional interaction(s) such as allelopathy were also involved. Laboratory studies assessing a number of Scottish sediment samples identified the presence of Group I, Group III and hybrid cysts, highlighting the possibility that the more dominant ribotype may outbreed the other over time, if as the literature suggests, hybrid crosses fail to produce viable progeny.

Paralytic shellfish poisoning (PSP) and ciguatera fish poisoning (CFP) are the two major contributors to illnesses caused by dinoflagellate toxins. Paralytic shellfish poisoning toxins (PSTs) are produced by dinoflagellates in the genera Alexandrium, Gymnodinium, and Pyrodinium while ciguatera fish poisoning toxins, such as ciguatoxins (CTXs), are originated from benthic toxic dinoflagellates (Gambierdiscus, Prorocentrum, Ostreopsis, and Coolia species). These toxins are responsible for human intoxication syndromes to the nervous system and muscles. This study optimized the protein extraction for proteomics analysis of dinoflagellate. The protein and toxin profiles of Alexandrium tamarense CI01 at different toxin biosynthesis phases were compared; differentially expressed proteins in highly toxic algae were identified using matrix-assisted laser desorption/ionization time-of–flight (MALDI-TOF) spectrometry. Some potential proteins involved in the toxin biosynthesis of A. tamarense CI01 were also identified. The protein preparation method from dinoflagellates for proteomics is selective for proteins with different pIs and molecular weights. The Lysis method may cause variation of the target proteins, but Trizol and Tris do not. Trizol method is good at analyzing hydrophobic, high molecular weights or binding proteins on the membrane. These PST synthesis-related enzymes are common in dinoflagellates, thereby 2-DIGE and Trizol are the best staining methods for dinoflagellates toxin synthase proteomics research. Through a comparative study in A. tamarense CI01 under different nutrient conditions and growth phase, the potential toxin synthesis-related proteins were investigated. Based on proteomics results, methionine, ornithine, arginine metabolism-related proteins and photosynthesis-related proteins may be related to the PSTs biosynthesis. Results also identified a similar pathway of PST biosynthesis in both dinoflagellate and cyanobacteria. A comparative proteomics study was applied to identify proteins of biomarkers for CTX accumulation in hepatic tissue of grouper, Cephalopholis argus, and PSTs accumulation in shellfish, Saxidomus giganteus. In C. argus with elevated CTXs, ATP synthase subunit beta and cytochrome c, ubiquitin enzymes, ATP related enzymes, and telomerase reverse transcriptase were greatly reduced. The comparative proteomic analysis revealed that PSTs and CTXs induced influx/efflux of Na+ or Ca2+ disorders in fish and shellfish. ATP synthase can control the concentration and influx/efflux of Na+. Alterations of Na+/K+ adenosine triphosphatase, liagand gated ion channel and sarcoplasmic calcium-binding protein in toxic shellfish was believed to reduce damage that PSTs (sodium channel blockers) bring to shellfish by controlling the concentration and influx/efflux of Na+ or Ca2+. Two identified resistant mechanisms to Na+ channel toxins are amino acid mutation and toxin affinity-binding proteins. However, neither of them was confirmed in the present study, but the proteins controlling the concentration and influx/efflux of Na+ or Ca2+ in this study may be due to new mechanisms. In conclusion, our comparative proteomic analysis revealed that CTXs and PSTs induced influx/efflux of Na+ or Ca2+ changes in toxic samples with a concomitant interference with signal transduction, metabolomics processes, detoxification, and anti-apoptosis. The physiological roles of ion concentration controlling and ion signal-related proteins in toxic fish and shellfish species should be further studied for their potential roles in resistance mechanisms to CTXs and PSTs. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Thesis (M. Phil.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

published_or_final_version / abstract / Ecology and Biodiversity / Master / Master of Philosophy

Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2002. / Includes bibliographical references. Also available in electronic version. Access restricted to campus users.

Phytoplankton produce allelochemicals; excreted chemical substances that are affecting other microorganisms in their direct environment. In my thesis, I investigated strain specific variability in the expression of allelochemicals of the harmful flagellate Prymnesium parvum, that is euryhaline but mainly bloom forming in brackish water. I found a large variation among strains, but further showed that all strains of P. parvum were more allelopathic in brackish water compared to marine water. In a marine microbial community, allelochemicals can affect prey, competitors and grazers both, directly and indirectly. For instance, in a food web where grazing controls prey abundance, the negative direct effect of allelochemicals on grazers will positive affect their prey. During my thesis, I investigated how marine microbial communities respond to the addition of allelochemicals. I performed field experiments with microbial communities from seawater collected from different places over Europe, and tested how this communities respond to the addition of allelochemicals from the dinoflagellate Alexandrium tamarense. Before I incubated the microbial communities for several days with A. tamarense algal filtrate, I evaluated the allelopathic efficiency of the algal filtrates with an algal monoculture of Rhodomonas spp. This allowed me to compare the effect of A. tamarense filtrate between the different microbial communities. In general, bacteria reached higher abundances when allelochemicals were present. As allelochemicals also inhibited nanoflagellates and ciliates, we concluded, that allelochemicals indirectly benefit bacteria by reducing grazing pressure. In microbial food webs with many heterotrophic grazers, allelochemicals further benefitted other phytoplankton by inhibiting grazers. It was also shown that bioavailable DOM is released from a microbial community when allelochemicals are present. As most DOM was released from the seawater fraction > 60 μm, we concluded, that larger microorganisms are more affected by allelochemicals than smaller microorganisms. The results can be explained by the surface to volume ratio of microorganisms: Larger organisms provide more contact surface for allelochemicals, and therefore, are probably more vulnerable towards allelochemicals. In conclusion, the effect of allelochemicals on a microbial community depends among others on the structure of the microbial food web, the amount of available DOM, the particle density in the seawater and the composition of the phytoplankton community.