Dissertations / Theses on the topic 'Marine algal toxins'

Algal toxins can have a significant impact on human and ecological health as the toxins accumulate in the food chain and are consumed by both humans and marine organisms. This study focussed on the following marine algal toxins that were present at the study sites: okadaic acid (OA), domoic acid (DA), gymnodimine (GD), pectenotoxin-e (PTX-2) and PTX-2 seco acid (PTX-2SA). The study sites investigated for potential algal toxin exposure were selected from the waters around North Stradbroke Island, Queensland, Australia, where shellfish are harvested by the local population, and where dugongs are known to feed on seagrass. Samples were collected monthly for two consecutive years. The species of toxinproducing algae present at the sites studied were Pseudo-nizschia sp., Dinophysis caudata, D. acuminata and Prorocentrum lima. The occurrence of Dinophysis species was observed to be dependent on the season while Pseudo-nitzschia sp was present both in colder and warmer months. Data on the dose-response analysis were extracted from published literature. This data was categorised into whole organisms, human and animal cell lines, and compared to one another. For further toxicodynamic studies, human cell lines were dosed with known concentrations of the toxins: OA, DA and GD. These cytotoxicity and microarray analyses were performed to observe the effects of toxins on gene regulation. A more extensive analysis was performed using GD alone. Expression of numerous genes was affected, and real time polymerase chain reaction reactions were performed to confirm the regulation of those genes. Gymnodimine was demonstrated to affect genes within pathways relating to oxidative phosphorylation, apoptosis, MAPK and Wnt signalling pathways. The cytotoxicity and microarray data and the data accumulated from the published literature were combined to form a comprehensive database of both chronic and acute effects. The database was then referred to for the dose-response analysis for the risk assessment. The exposure data attained from field sampling in the current study was analysed against the doses for any shown effects. Total daily intake for humans and dugongs sourcing food from around the island were calculated and health risks were estimated by incorporating tolerable daily intake, guideline values and total daily intake. The risk characteristics of algal toxins on the health of humans (consuming shellfish) and dugongs (consuming seagrass) indicated that acute health risks were unlikely, unless an outbreak of toxic algae (algal bloom) were to occur. Since there were no occurrences of algal blooms during the study period, high levels of toxins were not detected in any of the collected shellfish, phytoplankton or seagrass samples. However, if such blooms were to occur around the island, the phytoplankton could potentially produce algal toxins at high enough concentrations to cause acute toxic effects in the consumers. The current study has also demonstrated that there is a potential for chronic, long term health effects from consuming shellfish and seagrass around the island. The presence of low-level concentrations of algal toxins in the food sources can lead to chronic effects. Toxins such as OA are known tumour promoters. For dugongs, which feed on seagrass on daily basis, the potential for chronic effects is high. It was demonstrated that GD also possesses toxicological characteristics that may enhance the possibility of tumour promotion because of its effect of down-regulating parts of the apoptosis pathway, which may prevent cell death and as a consequence, lead to uncontrolled cell growth.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
Griffith School of Environment
Faculty of Science, Environment, Engineering and Technology
Full Text

2011/2012
SUMMARY Biotoxins produced by harmful algae during their proliferation can be accumulated by filter feeding organisms, such as bivalve shellfish, within their flesh. Furthermore, these toxins gradually are transferred to the higher trophic levels in the food chain, posing a threat to human health, after consumption of contaminated seafood. Filter-feeding invertebrates are organisms in which the toxin accumulation is a well-known phenomenon, especially during harmful algal blooms. Mussels, cockles, oysters, and scallops feed on toxic dinoflagellates, transferring them from the gills to digestive organs where the toxins accumulate. Different algal toxins can simultaneously contaminate edible shellfish, representing a world-wide sanitary and economic problem. Among them, Palytoxin (PLTX) is a highly toxic polyhydroxylated compound associated to human seafood intoxications in tropical and subtropical areas, but recently it has been detected also in microalgae and shellfish from temperate areas, as Mediterranean Sea. In the last years, also Yessotoxin (YTX) was frequently detected in mussels from Mediterranean Sea and a possible co-exposure to both PLTX and YTX can occur through contaminated seafood consumption. Therefore, the research was initially focused on the detection and quantification of PLTX and YTX in mussels collected in the Gulf of Trieste in order to verify the simultaneous shellfish contamination by these toxins and, subsequently, to study their toxic effects after simultaneous oral exposure. Analyses by liquid chromatography coupled mass spectrometry (LC-MS) did not reveal the presence of PLTX in mussels but identified the presence of YTX together with the diarrheic toxins okadaic acid (OA) and its acyl esters. Consequently, the final goal of this part of the research was the determination of these toxins in mussels from the Gulf of Trieste by LC-MS as well as that of OA and its esters by a functional assay, the protein phosphatase 2A (PP2A) inhibition assay (comparing the results obtained from two different protocols for the PP2A assay). These assays were used to analyse both cooked and uncooked mussel samples, to verify the influence of the heating procedure on the toxin content of mussels. Globally, no significant difference in toxins concentration between uncooked and cooked mussels was observed. However, comparing the data of single samples, a slight increase (not statistically significant) of toxin concentrations was detected in the cooked mussels with respect to the uncooked ones. The mussel analysis by LC-MS/MS detected also the presence of significant amounts of yessotoxin. Contrary to OA group toxins, yessotoxin was slightly less concentrated in the cooked mussels, probably because of its higher polarity that allows a dissolution in the water lost during the cooking procedure. In the other hand and as it was mentioned before, the palytoxin produced by Ostreopsis sps. microalgae have become a problem in more occasions for the attendance of the coastal environment, both for recreation and for business, representing a loss, as well as to public health, even for the tourism and the aquaculture industry. World market globalization, climatic changes and increasing overseas traffic are considered the main responsible for the appearance of these toxins. The expand of these toxins to temperate regions may be due in part to ballast water of ships and also to general changes in climate conditions, enough to induce bloom formation. Despite the extent of the contaminated area, few methods are currently available for palytoxins detection and quantitation in seafood. Moreover, among palytoxins, only palytoxin is commercially available, though expensive, and no certified standard material is currently sold. For monitoring purposes, a combination of screening methods followed by a chemical confirmatory analysis, such as LC-MS, is commonly used to detect palytoxins. Other methods for PLTX analysis include mouse bioassay, cytotoxicity assays, haemolysis assays, receptor binding assays, and immunoassays. Even if there were no food poisoning from palytoxin in the Mediterranean countries, the toxin was detected in shellfish, which gave positivity to the official test for the lipophilic toxins of algal origin (okadaic acid and derivatives, azaspiracids, yessotoxins and pectenotoxins). Some different experiments were applied in the evaluation of palytoxin toxicity in this study (Haemolytic assay, sandwich ELISA, LC-MS/MS and cytotoxicity studies were carried out). Haemolytic assay, carried out incubating mouse erythrocytes with palytoxin for 4 h (standard assay) or for 1 h in diluted PBS (abbreviated assay) is able to detect palytoxin at picomolar concentrations. Nevertheless, with the aim to detect the toxin in mussels, a significant matrix effect impairing the toxin quantification was observed already at the mussel extract concentration of 0.048 mg edible tissues equivalents/mL, which was more evident by the abbreviated assay. However, most of the experiments lack specificity or have other limitations. Thus, an indirect sandwich ELISA has been set up. The ELISA assay (indirect sandwich) was developed using the monoclonal antibody 73D3, and a rabbit polyclonal antibody produced at the University of Trieste. The assay detects the PLTX in a range of concentrations ranging from 1.25 to 40 ng/ml and is able to quantify with very similar sensitivity also biotinilated PLTX as well as 42-OH-PLTX, this latter isolated and characterized from the chemical point of view during the latter years from the group of prof. E. Fattorusso (University of Naples Federico II), in a sample of palytoxin kindly provided by Dr. M. Poly (Maryland, USA). The incapacity to detect okadaic acid (OA), domoic acid (DA), brevetoxin-3 (PbTx-3), saxitoxin (STX) and yessotoxin (YTX)(toxins that may be present along with PLTX in fish contaminated) indicates the specificity of the assay. The structure of Palytoxins is very complex. In addition to this structural complexity, there is still a lack of knowledge about the different congeners involved in this contamination and therefore there is still a very limited availability of standards and reference materials. These issues made difficult the advances in the development and optimization of analytical methods, particularly in the case of LC-MS/MS. Despite of this, a significant progress has been made over the last few years in the development of analytical techniques, particularly on LC-MS/MS approaches. In this part of the study, a LC-MS/MS method was optimized for the analysis of PLTXs in order to be able to detect, quantify and confirm the presence of this toxins in natural samples. During the PhD period, there was the possibility to get some natural contaminated samples to be evaluated by ELISA developed assay and then compare the results with the analysis by the developed LC-MS/MS method. The study was carried out with samples from 3 distinct sites characterized by having different coastal morphologies and continental hydrodynamic conditions: i) Madeira Islands’ archipelago in the NE Atlantic Ocean, Selvagens island in particular (Long, Lat) during the upwelling of August 2008; ii) Cascais, on the west coast of Portugal mainland, located at the northern side of Lisbon bay during the upwelling occurred during the favourable northerly wind periods (from April to September 2011); and iii) Algarve, Lagos, on the South Portuguese coast, also in 2011.
RIASSUNTO Biotossine prodotte da alghe nocive durante la loro proliferazione possono essere accumulati da organismi di alimentazione per filtrazione, come molluschi bivalvi, nell'ambito della loro carne. Inoltre, queste tossine vengono trasferite gradualmente ai livelli trofici superiori della catena alimentare, che rappresenta una minaccia per la salute umana, dopo il consumo di frutti di mare contaminati. Invertebrati con filtro-alimentazione sono organismi in cui l'accumulo di tossine, è un fenomeno ben noto, soprattutto durante fioriture algali nocive. Cozze, vongole, ostriche e capesante si nutrono di dinoflagellati tossici, trasferendoli dalle branchie agli organi digestivi, dove le tossine si accumulano. Diverse tossine algali possono contemporaneamente contaminare molluschi commestibili, che rappresenta un problema mondiale sanitario ed economico. Tra questi, Palitossina (PLTX) è un composto altamente tossico poliossidrilato associato ad intossicazioni ittici dell'uomo nelle zone tropicali e subtropicali, ma recentemente è stato rilevato anche in microalghe e molluschi dalle zone temperate, come il Mare Mediterraneo. Negli ultimi anni, anche yessotossina (YTX) è stato spesso rilevato nei mitili dal Mare Mediterraneo e una possibile co-esposizione sia PLTX e YTX può avvenire attraverso il consumo di frutti di mare contaminati. Pertanto, la ricerca è stata inizialmente concentrata sul rilevamento e la quantificazione di PLTX e YTX nei mitili raccolti nel Golfo di Trieste, al fine di verificare la contaminazione simultanea nei frutti di mare da queste tossine e, in seguito, per studiare i loro effetti tossici dopo esposizione orale simultanea. Analisi mediante cromatografia liquida accoppiata alla spettrometria di massa (LC-MS) non ha rivelato la presenza di PLTX in mitili ma è stata identificata la presenza di YTX insieme alle tossine diarroiche acido okadaico (AO) e suoi esteri. Di conseguenza, l'obiettivo finale di questa parte della ricerca è stata la determinazione di queste tossine nei mitili del Golfo di Trieste mediante LC-MS così come quella di AO e suoi esteri da un saggio funzionale, il Saggio d’inibizione Proteina Fosfatasi 2A (PP2A)(confrontando i risultati ottenuti da due diversi protocolli per il saggio PP2A). Questi test sono stati utilizzati per l'analisi di campioni di mitili sia crude e cotte, per verificare l'influenza della procedura di riscaldamento sul contenuto di tossina nei mitili. A livello globale, nessuna differenza significativa nella concentrazione di tossine tra cozze crude e cotte è stata osservata. Tuttavia, confrontando i dati dei singoli campioni, un lieve aumento (non statisticamente significativo) delle concentrazioni di tossine è stato rilevato nelle cozze cotte rispetto a quelli crude. L'analisi dei mitili per LC-MS/MS rilevò anche la presenza di quantità significativa di yessotossina. Contrariamente alle tossine gruppo OA, yessotossina era leggermente meno concentrata nelle cozze cotte, probabilmente a causa della sua polarità superiore che permette una dissoluzione in acqua persa durante la cottura. Altrimenti e come è stato detto prima, la palitossina prodotta da Ostreopsis sps. microalghe sono diventate un problema in più occasioni per la partecipazione dell'ambiente costiero, sia per la ricreazione e per le imprese, con una perdita, così come per la salute pubblica, anche per il turismo e l'industria dell'acquacoltura. Globalizzazione del mercato mondiale, i cambiamenti climatici e l'aumento del traffico all'estero sono considerati il principale responsabile della comparsa di queste tossine. L'espansione di queste tossine per le regioni temperate può essere dovuto in parte alla acque di zavorra delle navi e anche a cambiamenti delle condizioni climatiche generali, tanto da indurre la formazione di fioritura. Nonostante e dovuto alla estensione dell'area contaminata, alcuni metodi sono disponibili per il rilevamento e la quantificazione di palitossina in frutti di mare. Inoltre, tra palitossine, solo palitossina è disponibile in commercio, anche se costoso, e nessun materiale standard certificato è attualmente venduto. A scopo di monitoraggio, una combinazione di metodi di screening seguita da una analisi chimica di conferma, ad esempio LC-MS, è comunemente utilizzato per rilevare palitossine. Altri metodi di analisi includono PLTX biotest sui topi, saggi di citotossicità, saggi emolici, saggi di legame al recettore e saggi immunologici. Anche se non ci sono stati identificate intossicazione alimentare da palitossina nei paesi del Mediterraneo, la tossina è stata rilevata nei molluschi, che ha dato positività al test ufficiale per le tossine lipofile di origine algale (acido okadaico e derivati, azaspiracidi, yessotossine e pectenotossine). Alcuni esperimenti diversi sono stati applicati nella valutazione della tossicità della palitossina in questo studio (saggio emolitico, ELISA, LC-MS/MS e studi di citotossicità sono state effettuate). Saggio emolitico, effettuato incubando eritrociti di topo con palitossina per 4 h (saggio standard) o per 1 h in PBS diluito (saggio abbreviato) è in grado di rilevare la palitossina a concentrazioni picomolari. Tuttavia, con lo scopo di rilevare la tossina nelle cozze, un significativo effetto matrice ledere la quantificazione di tossina è stata osservata già alla concentrazione di estratto di cozze 0,048 mg equivalenti tessuti commestibili/mL, che è stato più evidente con il saggio abbreviato. Tuttavia, per la maggior parte degli esperimenti mancano specificità o hanno altre limitazioni. Così, un indiretto sandwich ELISA è stato istituito. Il saggio ELISA (sandwich indiretto) è stato sviluppato utilizzando gli 73D3 anticorpi monoclonali, e un anticorpo policlonale di coniglio prodotto nella Università di Trieste. Il saggio rileva la PLTX in un intervallo di concentrazioni variabili 1,25-40 ng / ml ed è in grado di quantificare con sensibilità molto simile anche PLTX biotinilata così come 42-OH-PLTX, quest'ultimo isolata e caratterizzata dal punto di vista chimico durante gli ultimi anni dal gruppo del prof. E. Fattorusso (Università degli Studi di Napoli Federico II), in un campione di palitossina gentilmente fornito dal Dr. M. Poli (Maryland, USA). L'incapacità di individuare acido okadaico (AO), acido domoico (AD), brevetossina-3 (PbTx-3), saxitossina (STX) e yessotossina (YTX) (tossine che possono essere presenti insieme a PLTX nel pesce contaminato) indica la specificità del dosaggio. La struttura della palitossina è molto complessa. In aggiunta a questa complessità strutturale, vi è ancora una mancanza di conoscenza sui diversi congeneri coinvolti in questa contaminazione e quindi c'è ancora molto limitata disponibilità di standard e materiali di riferimento. Questi problemi reso difficili gli progressi nello sviluppo e ottimizzazione di metodi analitici, in particolare nel caso di LC-MS/MS. Nonostante, un progresso significativo è stato compiuto negli ultimi anni allo sviluppo di tecniche analitiche, in particolare su approcci LC-MS/MS. In questa parte dello studio, un metodo LC-MS/MS stato ottimizzato per l'analisi di PLTXs per essere in grado di rilevare, quantificare e confermare la presenza di queste tossine in campioni naturali. Durante il periodo di dottorato di ricerca, c’è stata la possibilità di ottenere alcuni campioni naturali contaminati da valutare tramite il saggio ELISA sviluppato e poi confrontare i risultati con l'analisi con il metodo sviluppato di LC-MS/MS. Lo studio è stato effettuato con campioni da 3 posti diversi caratterizzati d’avere diverse morfologie e condizioni idrodinamiche costiere continentali: i) arcipelago Isole Madeira nel nord-orientale dell'Oceano, Selvagens isola in particolare durante il mese di agosto 2008; ii) Cascais, sulla costa occidentale del Portogallo continentale, che si trova sul lato settentrionale della baia di Lisbona durante i periodi favorevoli di vento dal nord (da aprile a settembre 2011), e iii) Algarve, Lagos, sulla costa sud-portoghesa, anche nel 2011.
XXV Ciclo
1983

Global change poses new threats for life in the oceans forcing marine organisms to respond through molecular acclimatory and adaptive strategies. Although bivalve molluscs are particularly tolerant and resilient to environmental stress, they must now face the challenge of more frequent and severe Harmful Algal Blooms (HABs) episodes. These massive outbreaks of microalgae produce toxins that accumulate in the tissues of these filter-feeder organisms, causing changes in their gene expression profiles, which in turn modify their phenotype in order to maintain homeostasis. Such modifications in gene expression are modulated by epigenetic mechanisms elicited by specific environmental stimuli, laying the foundations for long-term adaptations. The present work aims to examine the links between environmental stress in bivalve molluscs (with especial emphasis on Harmful Algal Blooms) and specific epigenetic marks triggering responses through modifications in gene expression patterns. Overall, a better understanding of the molecular strategies underlying the conspicuous stress tolerance observed in bivalve molluscs will provide a framework for developing a new generation of biomonitoring strategies. In addition, this strategy will represent a valuable contribution to our knowledge in acclimatization, adaptation and survival. With that goal in mind, the present work has generated transcriptomic data using RNA-Seq and microarray technologies, facilitating the characterization and investigation of the epigenetic mechanisms used by the Mediterranean mussel Mytilus galloprovincialis during responses to HAB exposure. That information was made publicly available through a specialized online resource (the Chromevaloa Database, chromevaloa.com) assessing the response of chromatin-associated transcripts to Okadaic Acid. Specific epigenetic marks have been assessed under lab-controlled exposure experiments simulating the natural development of the HAB Florida Red Tide (FRT). Results demonstrate a role for the phosphorylation of histone H2A.X and DNA methylation in the response to FRT in the Eastern oyster Crassostrea virginica. Lastly, the study of co-expression networks based on RNA-Seq data series from the Pacific oyster Crassostrea gigas reveals dynamic transcriptomic patterns that vary with time, stressor and tissue. However, consistent functional profiles support the existence of a core response to general conditions of environmental stress. Such response involves metabolic and transport processes, response to oxidative stress and protein repair or disposal, as well as the activation of immune mechanisms supporting a tightly intertwined neuroendocrine-immune regulatory system in bivalves.

These (Ph.D.)--University of Massachuesetts Lowell, 2008.
Title from PDF title page. Available through UMI ProQuest Digital Dissertations. Includes bibliographical references (leaves 55-66). Also issued in print.

Marine micro- and macro-algae produce wound-activated compounds (WACs) as a means to reduce the grazing pressure. However, the primary ecological role of WACs may have been modified by detoxification processes and/or co-evolutionary relationships to play physiologic and behavioural effects on consumers. This study aimed at investigating the possible roles of WACs produced by micro- and macro-algae, i.e., their toxic, physiologic and behavioural effects. Choice experiments revealed the effects as infochemicals of selected microalgae W ACs on invertebrates living in the same community. In parallel, the toxicity of benthic micro- and macro-algae was investigated by means of a standard test on sea urchin embryos. Finally, the physiologic role of diatoms was investigated, testing the effect of Cocconeis scutellum parva on the sex reversal of Hippolyte inermis. The comprehension of these three effects allowed us to draw the main plant-animal relationships ruling various ecological systems and demonstrate that algal WACs play mainly the role of infochemicals for invertebrates living in their typical environment, while they function mainly as allelochemicals for invertebrates living in other environments. The specific toxicity of WACs was shown to be inversely correlated to the perception ability of selected invertebrates, depending on their life strategy: when a recognition of plant-produced volatile compounds is evolved by a given invertebrate, their original power as toxicants and grazing deterrents may be altered, so they become infochemicals or physiologic regulators. The influence of seawater acidification (as forecasted for the end of this century) on the ability of marine invertebrates to perceive WACs' was also investigated and we found influences of pH on invertebrates' odour perception ability. A simulation model was built based on these data and it might find useful application in conservation policies, while the important advancements reached in the identification of the apoptogenic power of diatom's WACs could prime interesting biotechnological applications

Master of Science
Food Science Institute
Curtis Kastner
Harmful Algal Blooms (HABs) including Cyanobacteria and other toxic marine biota are responsible for similar harmful effects on human health, food safety, ecosystem maintenance, economic losses and liability issues for aquaculture farms as well as the food industry. Detection, monitoring and mitigation are all key factors in decreasing the deleterious effects of these toxic algal blooms. Harmful algal blooms can manifest toxic effects on a number of facets of animal physiology, elicit noxious taste and odor events and cause mass fish as well as animal kills. Such blooms can adversely impact the perception of the efficacy and safety of the food industry, water utilities, the quality of aquaculture and land farming products, as well as cause ripple effects experienced by coastal communities. HABs can adversely impact coastal areas and other areas reliant on local aquatic ecosystems through the loss of revenues experienced by local restaurants, food manufacturers as well as seafood harvesting/processing plants; loss of tourism revenue, decreased property values and a fundamental shift in the lives of those that are reliant upon those industries for their quality of life. This paper discusses Cyanobacteria, macroalgae, HABs, Cyanobacteria toxins, mitigation of HAB populations and their products as well as the ramifications this burgeoning threat to aquatic/ landlocked communities including challenges these toxic algae pose to the field of food science and the economy.

The marine diatom genus Pseudo-nitzschia includes species that produce domoic acid, a neurotoxin responsible for illness and mortality in both humans and marine wildlife. Because of the expertise and time required for the microscopic discrimination of species, molecular methods that monitor environmental concentrations of Pseudo-nitzschia provide a rapid alternative for the early detection of blooms and prediction of toxin accumulation. We have developed a nucleic acid sequence-based amplification with internal control RNA (IC-NASBA) assay and a quantitative reverse transcription PCR (qRT-PCR) assay for the detection of the toxic species P. multiseries targeting the ribulose- 1,5-biphosphate carboxylase/oxygenase small subunit (rbcS) gene. Both methods use RNA amplification and fluorescence-based real-time detection. Due to a limited rbcS sequence database, primers were designed and used to sequence this gene from 14 strains of Pseudo-nitzschia (including four P. multiseries) and 19 other marine diatoms. The IC-NASBA and qRT-PCR assays had a limit of detection of one cultured cell of P. multiseries and were linear over four and five orders of magnitude, respectively (r2 ! 0.98). Neither of the assays detected closely related organisms outside the Pseudo-nitzschia genus, and the qRT-PCR assay was specific to P. multiseries. While cross-reactivity of primers with unknown species prevented reliable detection of P. multiseries in spiked environmental samples using IC-NASBA, the qRT-PCR assay had positive detection from 107 cells/L to 103 cells/L. Nearly a 1:1 relationship was observed between predicted and calculated cell concentrations using qRT-PCR. Based on a diel expression study, the rbcS transcript copy number per cell ranged from 2.16 x 104 to 5.35 x 104, with the highest expression during early to mid photoperiod. The rbcS qRT-PCR assay is useful for the detection and enumeration of low concentrations of P. multiseries in the environment.

Seven species of marine phytoplankters assigned to different taxonomic divisions were tested for toxic responses to two different molecular species of selenium known to be prevalent in seawater, selenite and selenate. Selenate proved to be more toxic than selenite, although severe toxicity was only observed at high concentrations (10⁻² and 10⁻³ M) of both selenate and selenite. At these concentrations, growth was completely or severely inhibited in most species tested. In some of the species that remained viable, both the percentage of motile cells and their swimming speed were drastically reduced. Scanning electron microscopy revealed that, under these circumstances, Dunaliella tertiolecta cells possessed much shorter flagella compared to the controls, while those that became non-motile lacked flagella altogether. Despite these striking alterations in both growth and morphology, cells of Amphidinium carterae, Dunaliella tertiolecta and Pavlova lutheri showed, after prolonged exposures, signs of adaptation to high selenium concentrations. Lower concentrations of selenium were generally non-toxic and frequently even stimulatory to growth. These observations suggest that for meaningful inferences on selenium toxicity both the concentration range and the length of the studies must be considered and the potential for adaptation to high selenium concentrations taken into consideration. The main ultrastructural and physiological changes in cells of Dunaliella tertiolecta, Pavlova lutheri and Amphidinium carterae treated with selenite or selenate involved the cell coat, mitochondria, chloroplasts as well as the respiratory and photosynthetic rates. Other changes were observed in the nucleus, lipids, vacuoles, nitrogen and carbon contents, but these showed greater variability among the microalgae studied. The major alterations suggested that energy transducing systems were severely affected by selenium toxicity. These led to significant decreases or even elimination of storage products which were indicative of severe shortage in energy and produced major reductions in growth. These occurred later upon exposure to the toxicant and coincided with the loss of cell coat material, suggesting that the shedding of cell surface material might play a major role in the detoxification and adaptation of the microalgae to toxic concentrations of selenium.
Science, Faculty of
Botany, Department of
Graduate

Experience montrant une succession de peuplements algaux et faunistiques pendant les treize mois de colonisation. L'analyse des donnees n'etablit pas de correlation entre la presence de g. Toxicus, la densite et les fluctuations des parametres du milieu. Une correlation nette est mise en evidence entre l'accroissement de la population de g. Toxicus et le developpement de l'algue rouge melobesia farinosa, a la surface de laquelle il adhere et qui, de ce fait, joue le role d'algue-support

Bivalves play a marked role in coastal marine ecosystems, impacting primary productivity with a strong top-down control on phytoplankton communities. In addition, they are an important food source for higher trophic levels, including humans. However, bivalves can be impaired by several external factors, either biotic or abiotic, natural or anthropogenic. Exposure to these stressors, coupled with intensive farming, raised the need for adequate management of the wild populations and farmed production. To improve the difficult balance between ecological and socio-economic interests, it must be investigated how bivalves cope with external stressors. For this reason, the effects caused by the following stressors were studied in the present thesis: (i) harmful algal blooms (HABs) and (ii) climate change drivers in the seawater, such as warming (W) and acidification (A). HABs are increasing in frequency, intensity, persistence and geographic distribution, but are ambiguous regarding their ecological impacts and physiological effects on bivalves. A strong relationship is emerging between HABs and climate change drivers, what may affect both shellfisheries and wild populations. Hence, the objectives of this work are: 1) To determine whether native and invasive species cope with exposure to HABs differently, providing new insights on species-specificities and ecosystem functioning fragilities in the presence of invasive species and HABs; 2) To evaluate how commercially valuable bivalve species cope with simultaneous exposure to several climate change drives and HABs, providing new insights on how environmental changes affects toxicokinetics, physiological and genotoxic bivalves responses, under HABs; 3) To assess, marine biotoxins bioaccessibility, contributing for new information relevant for risk assessment. Different approaches were used to achieve these goals. Complementary field and laboratory works were performed to assess toxicokinetics patterns of marine biotoxins in native vs. invasive bivalve species. Bivalves were exposed to toxic blooming algae species under climate change scenarios in laboratory controlled conditions to assess toxicokinetics as well as physiological and genotoxic responses. Naturally contaminated bivalves were used to investigate their role as vectors of marine biotoxins to humans through in vitro digestion methodology. Relevant new data were obtained regarding the effects of several marine biotoxins in bivalves. Assessing accumulation of regulated and non-regulated biotoxins in native (Ruditapes decussatus) and invasive (R. philippinarum) clam species from Aveiro Lagoon revealed that higher toxin content, particularly regarding OA-group, the most abundant and frequent toxins in the Portuguese coast, is reached by native species. Accumulation of lower toxin levels by invasive clam may then favour farmers interest for their production. The kinetics and genotoxicity study in native and invasive clams reinforced the existence of species-specific behaviours. Exposure to the dinoflagellate Prorocentrum lima, an OA and dinophysistoxin-1 producer, increases the pressure over native clams, with higher toxin accumulation and genetic damage, as well as early and increased induction of DNA repair activity. Invasive clams, on the other hand, are better adapted to cope with these challenges. Investigation of the effects of combined exposure to multiple stressors revealed that W, A and HABs alter the accumulation/elimination dynamics of Paralytic Shellfish Poisoning (PSP) toxins in mussels (Mytilus galloprovincialis). Lower accumulation levels and slower elimination rates were observed. The predicted climate change scenarios and exposure to HABs may then lead to lower contamination levels but to longer harvesting closures. Simultaneous exposure to altered environmental conditions and HABs also had significant impacts in the antioxidant system and DNA integrity, resulting in an organ-specific modulation of the antioxidant response, increasing genetic damage and preventing/retarding DNA damage repair. However, the DNA damage observed seems to be non-oxidative. While the investigation of factors enhancing the elimination of toxins was not a main objective of this study, it was shown that acidification might promote PSP toxins elimination in mussels. Finally, the bioaccessibility studies revealed a significant reduction in the OA group content available after in vitro digestion, suggesting an overestimation of exposure to these biotoxins and an over-conservative approach in safety levels definition and risk assessment studies. This was the first study assessing bioaccessibility of OA-group toxins, pointing to in vitro digestion as a promising tool to obtain accurate data regarding toxin ingestion from bivalves to the consumer. Overall, the complexity of the relation between HABs, bivalves and the environmental factors to consider in marine biotoxins management is increasing, and only continuous and extensive monitoring of environmental, biological and anthropogenic conditions may allow for a healthier balance between environmental and socio-economic interests.
Os bivalves desempenham um papel fundamental nos ecossistemas marinhos costeiros, limitando a produtividade primária através de um forte mecanismo de controlo descendente (top-down) sobre as comunidades fitoplanctónicas e constituindo uma importante fonte de alimento para níveis tróficos superiores, incluindo o Homem. No entanto, os bivalves são afetados negativamente por diversos fatores externos, sejam estes bióticos ou abióticos, naturais ou antropogénicos. A exposição a estes fatores, juntamente com a exploração intensiva, leva a que seja necessária a implementação de uma gestão adequada, quer das populações selvagens, quer da produção em viveiros. Para melhorar esta gestão e alcançar o difícil equilíbro entre os interesses ecológicos e socioeconómicos é então necessário entender como os bivalves lidam com estes fatores. Na presente tese foram estudados os efeitos provocados por (i) blooms de algas tóxicas (HABs do inglês harmful algal blooms) e (ii) alterações das condições climáticas da água do mar, nomeadamente o aumento da temperatura (W) e acidificação (A). Os HABs são fenómenos que estão a aumentar em frequência, intensidade, persistência e abrangência geográfica, mas cujos impactos ecológicos e efeitos fisiológicos em bivalves são ainda ambíguos. Concomitantemente com a pressão causada pelo aumento da procura de bivalves como alimento para as populações humanas, fortes indícios apontam para uma relação entre a ocorrência de HABs e os parâmetros de alterações climáticas, o que pode afectar quer a indústria, quer as populações de bivalves selvagens. Assim, os objetivos deste trabalho são: 1) Determinar se as espécies nativas e invasoras lidam com a exposição a HABs de maneira diferente, dando particular atenção às respostas específicas de cada espécie e às fragilidades do ecossistema em relação à presença de espécies invasoras e ocorrência dos referidos blooms; 2) Avaliar como espécies de bivalves de elevado valor comercial lidam com a exposição simultânea a alterações climáticas e HABs, focando o efeito destas modificações ambientais na toxicocinética, respostas fisiológicas e genotoxicidade exibida pelos bivalves; 3) Avaliar a bioacessibilidade das biotoxinas marinhas, focando a obtenção de informação relevante para estudos de avaliação de risco. Foram realizados trabalhos complementares de campo e laboratório, de modo a avaliar os padrões de toxicocinética de biotoxinas marinhas em espécies nativas e invasoras. Em laboratório, foram expostos bivalves a algas tóxicas em condições alteradas, simulando a ocorrência de HABs sob cenários previstos de alterações climáticas, de modo a avaliar toxicocinética, respostas fisiológicas e genotoxicidade. Exemplares naturalmente contaminados foram utilizados para investigar o papel dos bivalves como vectores de biotoxinas marinhas para humanos, através da simulação da digestão in vitro. O estudo da acumulação de biotoxinas marinhas regulamentadas e não regulamentadas em espécies de amêijoa nativa (Ruditapes decussatus) e invasora (R. philippinarum) na Ria de Aveiro revelou que a espécie nativa acumula, recorrentemente, concentrações de toxinas mais elevadas, particularmente no que toca a toxinas grupo do ácido ocadaico (OA do inglês Okadaic acid), sendo estas as toxinas mais abundantes e frequentes na costa Portuguesa. A acumulação de menores concentrações por parte da amêijoa invasora pode promover o interesse dos produtores na sua exploração. A avaliação da cinética e da genotoxicidade em amêijoas nativas e invasoras reforçou a existência de comportamentos específicos para cada espécie. A exposição a Prorocentrum lima, dinoflagelado produtor de OA e dinofisistoxina 1, aumenta a pressão sobre a espécie nativa, uma vez que esta apresenta as concentrações de toxina mais elevadas, os maiores níveis de dano genético e ainda uma indução mais precoce e intensa dos mecanismos de reparação de dano no ADN. A espécie invasora, por outro lado, está melhor adaptada para lidar com estes desafios. O estudo dos efeitos de fatores múltiplos combinados revelou que a exposição a W, A e HABs altera a dinâmica de acumulação/eliminação de toxinas paralisantes (PSP) em mexilhões (Mytilus galloprovincialis). Os cenários previstos de alterações climáticas e a exposição a HABs sugerem níveis de contaminação mais baixos, mas períodos de interdição de apanha mais prolongados. A exposição simultânea a condições ambientais alteradas e HABs também teve um impacto significativo no sistema antioxidante e na integridade do ADN, resultando numa modulação da resposta antioxidante especifica para cada órgão, aumentando o dano genético e prevenindo/atrasando a sua reparação. O dano encontrado no ADN parece, no entanto, não ser oxidativo. Embora a investigação de fatores que favoreçam a rápida eliminação de toxinas não constasse dos objetivos deste estudo, foi demonstrado que a acidificação pode promover a eliminação de toxinas PSP em mexilhões. Por último, os estudos de bioacessibilidade revelaram uma redução na quantidade de toxinas do grupo-OA disponível após a digestão in vitro, sugerindo uma sobrestimação da exposição a estas biotoxinas e recomendando uma abordagem conservadora na definição dos níveis de segurança e na subsequente avaliação do risco. Este foi o primeiro estudo a determinar a bioacessibilidade das toxinas do grupo-OA e sugere a técnica de digestão in vitro como uma ferramenta promissora na obtenção de dados rigorosos no que diz respeito à ingestão de toxinas através de bivalves e a acessibilidade destes compostos no organismo humano. De um modo geral, a complexidade da relação entre HABs, bivalves e os fatores ambientais a serem considerados na gestão de biotoxinas marinhas está a aumentar, e apenas uma contínua e extensiva monitorização das condições ambientais, biológicas e antropogénicas pode permitir um melhor equilíbrio entre os interesses ambientais e socio-económicos.
Programa Doutoral em Ciência, Tecnologia e Gestão do Mar

MARINE PHYTOPLANKTON TAXONOMY In 1978, I joined CSIRO Division of Fisheries & Oceanography in Cronulla, NSW, as a Postdoctoral Fellow with a freshly awarded Ph.D. from the University of Amsterdam, Netherlands, for a 1976 thesis on Photosynthetic Pigments, Biomass Estimates and Species Diversity of Freshwater Phytoplankton Populations. While the original purpose of my Australian visit was to work on phytoplankton pigments (work not included here), I quickly recognised the complete absence of modern phytoplankton taxonomic work in the Australian region, which had not been conducted since CSIRO scientist Ferguson Wood left Australia in the 1950s to take up a professorial position at the University of Miami. In the period 1978 —1983, I therefore developed skills in electron microscopy (University of Sydney) and phytoplankton taxonomy (training courses at University of Oslo and University of Washington (Friday Harbour)). This led to the first modern characterization of phytoplanIcton populations of tropical and temperate, offshore and inshore Australian waters. The phytoplankton ecology of Sydney coastal waters [publications no.2,7,32], the East Australian Current [1,9], the North West Shelf and Gulf of Carpentaria [6], and Coral Sea were documented during this period. My 1978-79 phytoplankton studies in NSW waters have subsequently served as an important baseline against which to assess to impact of the later Sydney sewage outfalls [54]. An important early discovery was the recognition of the importance of nanoplankton (2- 20 gm size) in Australian waters [2], and a number of definitive taxonomic works on a range of microalgal classes (diatoms [5,35], dinoflagellates [13,14,24] coccolithophorids [4], prymnesiophytes and prasinophytes [3]) were published. A very significant proportion of these species were new records for the Australian region. Important taxonomic revisions were instigated which have now become widely accepted, such as the transfer of the diatom Thalassiothrix frauenfeldii to Thalassionema [8], the separation of the dinoflagellate genera Dinophysis and Phalacroma [14]. A new freshwater dinoflagellate genus Thecadiniopsis was described, which is closely related to marine benthic dinoflagellates from which it is thought to have been derived [10, 69]. A new dinoflagellate species Gymnodinium microreticulatum was described which has close affinities to the toxic dinoflagellate Gymnodinium catenatum [48] and two new nanoplankton diatoms Navicula jeffreyi and Fallacia carpentariae were described which have important applications in abalone aquaculture [38]. HARMFUL ALGAL BLOOMS Upon transfer of CSIRO Marine Laboratories from Cronulla to Hobart in 1984, I devoted myself to the phytoplankton ecology of inshore Tasmanian waters. In my first plankton haul in the Derwent River I newly recognised vast numbers of the toxic dinoflagellate Gymnodinium catenatum which has the potential to kill human consumers of shellfish (causative organism of Paralytic Shellfish Poisoning, PSP). As leader of a small research team (4 staff) , the ecology of this species was elucidated and bloom triggers identified [37], cultures were established and the life cycle determined [17], its unknown toxin chemistry defined (collaboration with Toholcu University, Japan [11,33]) and unambiguous evidence was obtained from the identification of cysts in dated sediment depth cores that this nuisance organism had been -introduced— into Tasmanian-waters-after-19'73439], As a- result -of-my-work, Tasmania was the first state in Australia to start monitoring for dinoflagellate toxins in shellfish (collaboration with Tasmanian Departments of Sea Fisheries and Public Health) and this subsequently was extended ta Victoria, South Australia, Western Australia, and Queensland. My expertise in harmful algal blooms has also been called upon to address similar toxic dinoflagellate problems in Port Phillip Bay, Melbourne (first identification of the PSP dinoflagellate Alexandrium catenella [13], and Port River, Adelaide (first identification of the PSP dinoflagellate Alexandrium minutum, not previously known to be toxic [13, 16]) .I was also called upon to coordinate a training workshop on PSP problems caused by the dinoflagellate Pyrodinium bahamense in the tropical Indo-West Pacific (Brunei, Philippines [88]), and was the first worker to identify a bloom of the fish-killing raphidophyte Chattonella marina in Australian waters and circumstantially link it to a major tuna aquaculture mortality ($45M loss) event in South Australia [42,68] . An invitation to present a plenary lecture at the 1991 International Phycological Congress, North Carolina USA (supported by a Senior Fullbright Fellowship) on the apparent global increase in harmful algal blooms led to a state-of-the-art review in the Journal Phycologia [31]. This undoubtedly has become my most cited publication. The three key mechanisms raised (increased awareness of previously cryptic species, stimulation by eutrophication or unusual climate conditions; spreading by ballast water and aquaculture translocation) have become standard questions asked with any new unusual algal bloom event. I am well-recognised internationally for my innovative and perceptive work in the harmful algal bloom field, and have been elected on a number of national and international committees: *UNESCO group of international experts on Harmful Algal Blooms, which led to my editorship-in-chief of an authoritative 551 pp. Manual on Harmful Algal Blooms (first edition 1995 [90]; second edition to appear as a Monograph on Oceanographic Methodology in late 2002 [94]). * SCOR Working Group on the Physiological Ecology of Harmful Algal Blooms, which led to my associate editorship of an authoritative 662 pp. text on the physiological ecology of harmful algal blooms (first edition 1998 [91]). * To top this off, I was elected to host the 9th International Conference on Toxic Marine Phytoplankton in Hobart in February 2000, attended by 526 participants from 47 countries, which led to my editorship-in-chief of a major 518 pp.Conference Proceedings volume [submitted to the printer in May 2001, but published in late 2002 [92] and guest editorship of a special 2001 issue of the international journal Phycologia [93]. * Nationally, my production of an Aquaculturist's Guide to Harmful Australian Marine Microalgae [1 st edition 1991 [89]; 2 ' edition 2002] as well as production of a 1988 coffee-table book Plankton: A Microscopic World [87] have been most successful. ROLE OF SHIP'S BALLAST WATER IN GLOBAL SPREADING OF ALGAL BLOOMS My Tasmanian studies on Gymnodinium catenatum dinoflagellate cysts in dated sediment depth cores [39] triggered a major survey of toxic dinoflagellate cysts in ship ballast water [23,26]. My detection of three species of toxic dinoflagellate cysts in ships' ballast waters led in 1990 to the introduction in Australia of special ballast water quarantine regulations (collaboration with the Australian Quarantine and Inspection Service) which are now increasingly being adopted on an international basis. I played a key role in an AQIS Australian Government Scientific Working Group on Ballast Water (since 1989) and am a member of the advisory board of the CSIRO Centre for Research on Introduced Marine Pests (CRIMP) in obart (since 1995). Australia has become a leader in the field of management of marine bioinvasions, and the actions now pursued by the International Maritime Organisation (IMO) are largely based on the Australian model. To track down the overseas source populations of introduced Australian toxic dinoflagellate populations, I pioneered the application of molecular genetic markers, initially working with Chris Scholin at Woodshole Oceanographic Institution (in an award winning paper, [36]), and later with my Ph.D. students Chris Bolch (47) and Miguel de Salas (73). In order to assess what constitute baseline dinoflagellate species communities in Australian waters, I and my students instigated Australia-wide surveys of dinoflagellate cysts in sediments from major shipping ports, starting with Hobart [18] and eventually covering Devonport, Launceston, Port Lincoln, Albany, Bunbury, Fremantle, Port Hedland, Darwin, Karumba, Cape Flattery, Moorilyan, Lucinda, Hay Point, Townsville, Gladstone, Newcastle, Port Botany, Sydney Harbour and Eden. In a very successful collaboration with BHP shipping engineers Geoff Rigby and Alan Taylor, I pursued a number of ballast water treatment and management options using resistant dinoflagellate cysts as a model organism. These ranged from environmentally friendly biocides [30; too expensive], the application of mid-ocean exchange [34; which has become an IMO standard], to (what I advocate is the most effective) the use of ship engine waste heat to kill dinoflagellate cysts at temperatures as low as 38°C [41, 52]. These studies were published in three papers in the prestigious journal Marine Ecology Progress Series in 1997,1998, 2000 [39,45,52] and were widely reported also in New Scientist and Science. This work has been nominated by the Institute of Marine Engineers for the inaugural Stanley Gray award. RESEARCH IN PROGRESS My current research interests (June 2002) are addressing the widespread potentially ichthyotoxic dinoflagellate Pfiesteria in Australian waters, toxic PSP dinoflagellate blooms of Gymnodinium catenatum in New Zealand waters, the incidence of toxic benthic dinoflagellates (Prorocentrum, Cooha, Ostreopsis) in East Coast Tasmanian waters, the phylogeny and morphotaxonomy of fish-killing gymnodinioid dinoflagellates (including new species as well as genus descriptions ), and elucidation of the toxic principle of the ichthyotoxic raphidophyte Chattonella marina.

Toxic dinoflagellate blooms of Gymnodinium catenatum Graham (causative organism of paralytic shellfish poisoning) can have major impacts on aquaculture, human health and the aquatic environment. In south-east Tasmanian estuaries, these blooms are preceeded by rainfall events and the associated input of freshwater and humic substances (from soil leachates). This study examined the potential nutritive role of terrestrially-derived humic substances / micronutrients and the importance of vertical water column stratification in the development of G. catenatum blooms. Laboratory experiments showed that humic additives (standard aquatic humic acid purchased from the International Humic Substances Society, and dissolved organic matter isolated from the Huon Estuary by ultrafiltration) stimulated G. catenatum growth and biomass production. Under these conditions biomass was limited by the macro-nutrients nitrate and phosphate, but in seawater medium with no humic additives the micro-nutrient selenium was limiting. This suggests that humic substances change overall nutrient availability and / or uptake by G. catenatum, either by adding nutrients or by interaction with other nutrients. Bioassays indicated that 1 - 100 nM selenium (IV) stimulated G. catenatum growth and biomass production. However not all strains of this dinoflagellate species tested (including isolates from Tasmania, Japan and Spain) had an obligate selenium requirement. Another PSP dinoflagellate Alexandrium minutum showed a selenium requirement similar to G. catenatum, but the bloom forming diatom, Chaetoceros cf. tenuissimus showed no reduction in growth or biomass production under seleniumdeficient conditions. Inorganic selenium (selenite and selenate) concentrations in the Huon Estuary were commonly <0.01 nM (below detection) and thus are potentially limiting for G. catenatum. Since selenium enters estuarine systems primarily through river run-off, rainfall may be a crucial trigger for dinoflagellate blooms in these waters by increasing selenium levels. Water column stability and stratification, also often associated with rainfall, occurs in south-east Tasmanian coastal waters during summer, and this is also critical in the development of G. catenatum blooms. Laboratory experiments in stratified laboratory water columns (0.1 x 1m) examined the migration behaviour of G. catenatum and showed that nutrient-deficient cells migrate downwards to access nutrients from bottom layers at night, while nutrient-replete cells tend to remain at the surface. This migration pattern was similar when using humic and non-humic surface waters, indicating that humic substances do not enhance cellular nutrient concentrations to such a degree as to preclude the need for deep nutrient uptake. Diel vertical migration to facilitate dark nutrient assimilation provides an ecological advantage for G. catenatum over other nonmigratory species, particularly during late summer in Tasmanian estuaries when surface nutrients are depleted. This study indicates that selenium and humic substances contained in river run-off are stimulatory to G. catenatum growth and biomass production, providing essential micro-nutrients during bloom initiation. Furthermore, water column stability and vertical stratification caused by river run-off are of paramount importance in maintaining G. catenatum blooms. During this latter stage, vertical migration by nutrient-deficient cells forms an integral part of the successful ecological strategy utilised by this red tide dinoflagellate.

The Huon Estuary is a micro-tidal estuary in south-east Tasmania that is an important area for salmon aquaculture. In 2008 the salmon aquaculture industry in Australia is worth $260M (AUD) per year. Salmon aquaculture began in the Huon Estuary in the 1980's and production has since increased significantly. The Huon Estuary is nitrogen (N) limited and salmon farming is a significant input of N to this ecosystem. Both industry and government regulators are alert to the potential for eutrophication and increased harmful algal blooms if the assimilative capacity for N of the estuary is exceeded. As part of a larger project on the ecology of the Huon Estuary, this PhD research has two main objectives; firstly to determine whether phytoplankton in the Huon Estuary are using nitrogen that had, primarily, an oceanic source (e.g. nitrate) or was more locally supplied or regenerated (e.g. ammonium and urea) and secondly to examine the physiology of G. catenatum a toxic dinoflagellate that dominates the summer and autumn Huon Estuary phytoplankton biomass in many years. Uptake rates of NO\(_3\)\(^-\), NH\(_4\)\(^+\) and urea were measured on four occasions (28-29 May 2003, 23-24 Sept 2003, 18-19 Nov 2003, and 24-25 Feb 2004) in the Huon Estuary using a \(^{15}\)N tracer technique. Uptake rates were measured at Garden Island and Hideaway Bay in the lower estuary and at 5 and 20 m during the day and also at 5 and 20 m during the night. The mean uptake rates (mean across time of year, site, time of day and depth) for NH\(_4\)\(^+\) (0.13μg N μg chl α h\(^{-1}\) and urea (0.09 μg N μg chl α h\(^{-1}\)1) were 4.5 and 3.2 times higher than the uptake of NO\(_3\)\(^-\) ( 0.3 μtg N μg ch la h\(^{-1}\)). Overall NH\(_4\)\(^+\), NO\(_3\)\(^-\) and urea were responsible for 52, 37.5 and 10.5% of N uptake respectively. Gymnodinium catenatum is a toxic dinoflagellate that blooms periodically in the Huon Estuary and in years that it blooms it dominates the phytoplankton biomass and has caused closure of shellfish farms in the area. Laboratory experiments on effect of temperature and irradiance on growth rate, effect of different nitrogen species on growth rate and preferential uptake of different nitrogen species by G. catenatum were undertaken to better understand the physiology of this species and to test a hypothesis that G. catenatum vertically migrates to access NH\(_4\)\(^+\) at depth during summer. The effect of 12 different temperatures ranging from 11.9-25.2 °C and irradiances from 5-283 gmol photons m\(^{-2}\)s\(^{-1}\) on growth and biochemical composition of G. catenatum. The highest predicted growth rates (>0.2 d\(^{-1}\)) occurred during summer and autumn as might be expected based on observations of bloom dynamics of this species in the Huon Estuary which supports both a summer and autumn bloom in many years. G. catenatum was able to grow using NO\(_3\)\(^-\), NH\(_4\)\(^+\) or urea as its sole nitrogen source. There was no significant difference in growth on any of these nitrogen sources. Preferential uptake of NH\(_4\)\(^+\), NO\(_3\)\(^-\) or urea was examined by growing G. catenatum on a mixture of NO\(_3\)\(^-\), NH\(_4\)\(^+\) and urea. The results clearly showed that NH\(_4\)\(^+\) was taken up first, followed by NO\(_3\)\(^-\) and finally urea. Maximum mean uptake rates were 170, 98 and 30 pg cell\(^{-1}\) hour\(^{-1}\) respectively for NH\(_4\)\(^+\), NO\(_3\)\(^-\) and urea. In addition to the laboratory experiments the nitrogen uptake characteristics of a bloom of G. catenatum was examined at Pelican Island, Southport (30-31/03/2004) nearby the Huon Estuary. Mean urea uptake was greatest (0.045 μgN μg chl α h\(^{-1}\)) followed by NH\(_4\)\(^+\) (0.029 μgN μg chl α h\(^{-1}\)) and the lowest uptake was of NO\(_3\)\(^-\) (0.025 μgN μg chl α h\(^{-1}\)). For G. catenatum growing in the Huon Estuary it seems increasingly apparent that it functions as a nitrogen scavenger. When N concentrations are exhausted, it is able to migrate through the water column seeking whatever form of nitrogen is available.

The chain-forming toxic dinoflagellate Gymnodinium catenatum Graham is a known causative organism of paralytic shellfish poisoning (PSP). During the mid-1970's the geographic extent of G. catenatum plankton blooms increased dramatically, causing toxic episodes in Spain, Portugal, Mexico, Venezuela, Argentina, Uruguay, Japan, Korea and southern Australia (Tasmania). Studies of the distinctive microreticulate resting cysts of G. catenatum in Tasmania suggest it was introduced to southern Tasmania during the early 1970's, possibly via ballast water from either Japanese, Korean or Spanish populations. The cysts of this species have now been widely reported, even from areas where G. catenatum has rarely, or never, been detected in the plankton. The reported cyst diameter varies considerably (17-63 p,m diameter), displaying a bimodal size distribution at some localities, suggesting that cysts may belong to a species complex of two or more related species. This work examines the distribution, and morphological and genetic variation, of the Gymnodinium catenatum species complex to resolve three distinct microreticulate cystforming species. Resolution of the species complex allowed the elucidation of population genetic relationships between strains of the toxic "true G. catenatum" isolated from Japan, Spain and Portugal, and Australia, to examine the hypothesis that Tasmanian G. catenatum was introduced to Australia (Tasmania) from one of these two potential source populations. Mapping the distribution of G. catenatum by examining sediments for the microreticulate cysts is hampered by their low abundance in coastal sediments and the low proportion of intact and viable specimens. Cyst concentration methods (sodium polytungstate density centrifugation) and PCR-based genetic identification methods were developed to improve cyst survey detection limits. Sediment surveys of 105 sampling sites at 17 estuarine and coastal locations demonstrated that microreticulate cysts are widely distributed in Australian coastal sediments. Two distinct morphotypes were noted: a "small-form" cyst (17-28 p,m) widely distributed in temperate and tropical Australian estuaries and a "large-form" typical of G. catenatum ( 37-62 p.m) which was restricted to the coasts of south-eastern Tasmania, southern Victoria, Port Lincoln (South Australia) and the Hawkesbury Estuary (NSW). Germination experiments showed that the smaller cyst-type belonged to a species that was morphologically and genetically distinct from both G. catenatum and the European rnicroreticulate cyst species, G. nolleri Ellegaard et Moestrup. This new species is described herein as Gymnodinium microreticulatum sp. nov. Bolch et Hallegraeff. The possible origin and evolution of G. catenatum was investigated by examining the genetic relationships among 27 species of gymnodinoid, prorocentroid and peridinoid dinoflagellates using partial sequences of the large sub-unit ribosomal RNA gene. The phylogenies constructed confirmed earlier findings from small sub-unit (SSU) RNA studies. The relationships of the 21 free-living gymnodinoids correlated with their morphological and cytological features with the loop-apical-grooved species forming four clusters delineated by chloroplast structure and arrangement, and resting cyst morphology. The G. catenatum complex (G. catenaturn, G. nolleri and G. microreticulatum) formed a distinct monophyletic lineage arising from the base of this group. Within the complex, G. microreticulatum diverged earliest, followed by G. nolleri and G. catenatum. Comparison of nuclear volumes of the three species suggests that the group may have evolved by polyploidy from a G. microreticulatum-like ancestor. The abundant LSU-rDNA sequence variation among G. microreticulaturn isolates contrasted the absence of verifiable sequence variation among G. catenaturn from Australia, Hong Kong, Japan, Spain and Uruguay Analysis of allozymes and large-subunit ribosomal RNA sequences failed to reveal genetic polymorphism among G. catenatum strains from Australia, Japan, Spain and Portugal. However, reproductive compatibility analysis demonstrated extensive intrapopulation compatibility and an outbreeding, multiple-group mating system. Mating success analysis (by cyst production) and variation in post-meiotic progeny viability from inter-population crossing experiments indicated that Japanese and Spanish strains were more closely related to each other than to Australian strains. Mating studies were supported by genetic studies using RAPD-PCR. Genetic variation was partitioned primarily within populations (87%), consistent with a sexually outbreeding species, as confirmed by mating studies. The G. catenatum strains could be clearly separated into regional clusters: Australia, Japan and Spain/Portugal. The Spanish/Portuguese and Japanese clusters were most closely related with the Australian cluster more distant and almost equally related to the others. The similarity between Japanese and Spanish G. catenatum compared to Australian strains suggests recent dispersal between these two populations. The source population for Australian G. catenatum remains unclear, however, the data support a secondary relocation of Tasmanian G. catenatum to mainland Australia, possibly via a domestic shipping vector. Geographic and temporal clustering of Tasmanian strains by isolation location and bloom year indicates that genetic exchange between neighbouring estuaries is limited and that Tasmanian G. catenatum blooms are composed of localised, estuary-bound sub-populations. Re-examination of the dinoflagellate fossil record in light of recent molecular phylogenetic data suggests that the G. catenatum complex evolved near the start of the Cretaceous period [circa 150 million years ago (Mya)]. Using a G. catenatum complex "molecular clock" based on LSU-rDNA sequences, it is estimated that the common ancestor of the complex (probably a G. microreticulatum-like species) evolved around 130-140 Mya and that the G.nolleri and G. catenatum lineages diverged about 16-19 Mya. The known modern distributions of the three species suggest a European evolutionary origin of G. catenation followed by a geologically recent global dispersal. Considering the lack of rDNA variation among the five populations examined, this dispersal is conservatively estimated to have occurred well within the last 25 thousand years. Whether dispersal has been by natural processes or assisted by human means (or a combination of both) is not yet clear. This study demonstrates that PCR-fingerprinting methods, such as RAPD-PCR, can discriminate fine-scale genetic population structure and discriminate global population clusters. Comparative genetic studies of more G. catenation populations would allow a better assessment of global relationships and may identify population genetic dines that correlate with G. catenatum natural dispersal corridors, or discontinuities that imply trans-oceanic transfer and human introduction.

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.

A taxonomic survey of scale-bearing nanoflagellate algae from southern Tasmanian coastal waters was undertaken. Observations were made on 52 samples collected from 21 different sites (June 1994 - 1995) and resulting enrichment cultures. Scale morphology was examined using transmission electron microscopy. Over 70 species of scale-bearing nanoflagellates from four classes and 17 genera were illustrated, namely: Chrysophyceae - Apedinella, Chrysolepidomonas, Meringosphaera, Paraphysomonas (8 spp); Prymnesiophyceae - Chrysochromulina (32 spp), Corymbellus, Imantonia, Pavlova, Phaeocystis (2 spp), Prymnesium (2 spp); Prasinophyceae - Dolichomastix (2 spp), Mamiella, Mantomiella, Pyramimonas (7 spp); Dinophyceae - Heterocapsa; and, of uncertain taxonomic affinities, Petasaria and Thaumatomastix (3 spp). Seventeen of the nanoflagellate species found were new records for Australian waters, specifically: Chrysochromulina acantha, C. ahrengotii, C. aff. brachycylindra, C. aff. camella, C. mactra, C. aff. scutellum, Pavlova pin guis, Chrysolepidomonas cf. marina, Paraphysomonas antarctica, P. bandaiensis, P. foraminfera, P. cf. takahashii, Dolichomastix nummulifera, D. aff. tenuilepis, Mamiella gilva, Petasaria heterolepsis and Thaumatomastix cf. thomseni. All species, except one (Chrysochromulina novae-zelandiae), are also known from the northern hemisphere. Two known toxic species, Chysochromulina polylepsis and C. leadbeateri, responsible for massive fish kills in Scandinavia, were found in this survey, as well as the known fish-killing species, Prymnesium patellife rum, observed from two important Tasmanian oyster-growing area, and subsequently cultured. The biodiversity of scale-bearing nanoflagellates in Tasmanian waters was highlighted by the large number of previously unreported scale types seen, including over 17 Chrysochromulina-like scale types, five prasinophyte box scale types and five Thaumatomastix-like scale types, but lack of complete cells prevented new species decriptions. However, sufficient material was available to fully characterise two new Chrysochromulina species. Full species descriptions will be prepared in the primary literature in the near future. The biodiversity of scale-bearing nanoflagellates in Tasmanian waters was highlighted by the large number of previously unreported scale types seen, including over 17 Chrysochromulina-like scale types, five prasinophyte box scale types and five Thaumatomastix-like scale types, but lack of complete cells prevented new species decriptions. However, sufficient material was available to fully characterise two new Chrysochromulina species. Full species descriptions will be prepared in the primary literature in the near future. Over 20 unialgal strains, with representatives from each observed class, were isolated from enrichment cultures and maintained; these included Chrysochromulina (8 strains), Pavlova (4 strains), Prymnesium (6 strains), Phaeocystis globosa, Pyramimonas grossii, Heterocapsa rotundata and Chrysolepidomonas cf. marina. Toxicity testing of 26 scale-bearing nanoflagellate strains was undertaken, using larval brine shrimp (Artemia) bioassays. Prymnesium patelliferum, was found to be toxic, with 100% mortality of Artemia nauplii after 24 hours exposure to stationary phase cultures, and toxicity of this species was enhanced when grown in phosphate-deplete media. Heterocapsa rotundata caused low percentage mortality (12.5%) of Artemia nauplii under the same bioassay conditions. This is a novel record of toxicity for this widespread dinoflagellate, and agrees with recent confirmation of toxicity to bivalves by the related species, H. circularisquama. None of the other nanoflagellate strains tested were found to be toxic. A potential new live food species for aquaculture, Pavlova pin guis, was identified using a combination of ultrastructural and morphological features. This species is now used in commercial Tasmanian oyster hatcheries as a valuable algal diet.

During 2012 an extensive bloom of the toxic dinoflagellate Alexandrium catenella occurred on the east coast of Tasmania causing paralytic shellfish toxins (PST) to accumulate in bivalve shellfish at 12 times the regulatory maximum level (ML; 0.8 mg STX.2HCl equiv. kg\(^{−1}\)). Southern Rock Lobster, Jasus edwarsdii, accumulated PST in the hepatopancreas to 3.9 mg STX.2HCl equiv. kg\(^{−1}\), resulting in the first closure of an Australian lobster fishery due to marine biotoxins. Recurrent blooms since 2012 have had an on-going impact on both the commercial fishery in Tasmania, valued at AUD 93 M, and the significant recreational fishing sector. The present body of work aimed to address the following knowledge gaps exposed by this novel risk: (1) the toxicokinetics associated with PST uptake and depuration in J. edwardsii from A. catenella blooms; (2) the impact of PST on lobster health; (3) the supply chain risk of PST accumulation in J. edwarsdii; and (4) to determine and validate where appropriate cost-effective methods to appropriately monitor and manage PST accumulation in lobster the field. In elucidating these questions, information was sought that could inform management of public health and market access risks and determine whether PST accumulation could adversely affect lobster performance, health and catchability. To examine toxicokinetics of PST in J. edwardsii (Aim 1), an experimental study was undertaken in a biosecure aquaculture facility in South Australia. Adult male lobsters were fed highly toxic mussels (6 mg STX.2HCl equiv. kg\(^{−1}\)) sourced from the impacted area in Tasmania for four weeks, then allowed to depurate for a further 5 weeks. Control (fed non-toxic mussels) and exposed lobster were harvested at regular intervals, tissues dissected and analysed for PST. The lobsters rapidly accumulated PST in the hepatopancreas (exponential rate of 6% per day), exceeding the bivalve ML within one week, and reaching a maximum of 9.0 mg STX.2HCl equiv. kg\(^{−1}\). Once toxic feed was removed, the lobster depurated at a rate of 7% per day. Toxins were found in lobster antennal glands at concentrations two orders of magnitude lower than found in the hepatopancreas. This is the first report of PST in lobster antennal glands which, along with the gills, represent possible excretion routes for PST. However, PST were not detected at significant levels in the lobster haemolymph, which means there is no option for non-destructive sampling of lobster for PST. During the experiment, lobster health was assessed to determine the impact of PST accumulation in the hepatopancreas (Aim 2). A comprehensive range of behavioural (vitality score, righting ability and reflex impairment score), health (haemocyte count, bacteriology, gill necrosis and parasite load), nutritional (hepatopancreas index and haemolymph refractive index) and haemolymph biochemical (21 parameters including electrolytes, metabolites, and enzymes) parameters were examined. Accumulation of PST increased the apparent feed intake but did not result in mortality nor significant changes in the behavioural, health, or nutritional measures suggesting limited gross impact on lobster performance. Furthermore, most haemolymph biochemical parameters measured exhibited no significant difference between control and exposed animals. However, in the lobsters fed toxic mussels, the concentration of potassium in the haemolymph increased with PST, whilst the concentration of lactate and the sodium:potassium ratio decreased with PST. In addition, lobsters with elevated levels of PST in the hepatopancreas showed a hyperglycaemic response, indicative of stress. These findings suggest that PST accumulation results in some measurable indicators of stress for lobsters, but these changes appear to be within the adaptive range for J. edwardsii and do not result in a significant impairment of gross performance. To determine whether J. edwardsii could accumulate PST in the supply chain (Aim 3), a controlled experiment was conducted where adult male lobsters were exposed to highly toxic cultures of A. catenella at field relevant concentrations (2 × 10\(^5\) cells L\(^{−1}\)) over a period of 21 days. In contrast to the feeding experiment, no PST accumulated in lobster from exposure to toxic algal cells. The same lobster health parameters were assessed, with no change seen in any of the behavioural, health, nutritional or haemolymph biochemical parameters examined. To assess appropriate management strategies (Aim 4), regulatory monitoring results since September 2012 were combined with field studies to examine uptake and depuration of toxins in J. edwardsii on the east coast of Tasmania during A. catenella blooms. Results from 496 lobster hepatopancreas PST samples were analysed. A high degree of variability was seen across years, months, sites and between individuals. The highest risk sites are on the central east coast, with exceedances of the bivalve ML occurring between July and January. Mussel sentinel lines were installed in each lobster management zone on the east coast of Tasmania and monitored fortnightly during high-risk periods. These lines proved effective in indicating a risk of elevated PST in lobster hepatopancreas. Field uptake and depuration rates of PST in lobster were similar to those seen during the experimental studies (maximum of 2% and 3% per day respectively), but always less than rates measured simultaneously in mussels. Analysis of hepatopancreas PST levels during bloom development and decline occurred to determine the level of confidence in the regulatory sampling regime (5 individual lobster hepatopancreas samples are analysed from each site during an event). When PST in the hepatopancreas of all lobsters sampled is < 0.5 mg STX.2HCl equiv. kg\(^{−1}\) there is 97.5% confidence that any lobster from that site would be below the bivalve ML. In conducting this research, it became apparent that international regulations for maximum levels of PST in seafood and research into PST accumulation vary in the units used for PST concentration. Some standards/research reports use mg STX.2HCl equiv. kg\(^{−1}\), some mg STX equiv. kg\(^{−1}\) (effectively producing total PST results that are 24% lower), and some only state mg kg\(^{−1}\). Similarly, the toxic equivalency factors (TEF) used to convert analogue concentrations to total PST in toxicity equivalents are varied, and often not stated. A call for uniform reporting units was published in Toxicon correspondence to highlight this issue, recommending countries and researchers adopt the Codex Alimentarius Commission protocols of reporting in mg STX.2HCl equiv. kg\(^{−1}\), using FAO/WHO TEF. The combined field and experimental work detailed in this thesis has informed improvements to the biotoxin risk management program for J. edwardsii in Tasmania. Implications for biotoxin risk monitoring are: (1) lobsters continue to feed during bloom periods and high concentrations of PST can occur; (2) animal toxin monitoring should be frequent at the start of a bloom in case of a rapid accumulation of PST; (3) mussel sentinel lines are a cost-effective early warning system for toxin accumulation; (4) it is adequate to sample 5 individuals per site as long as a reduced trigger level of closure of harvest is employed; (5) depuration is relatively quick at up to 7% per day so that sampling to confirm re-opening should occur soon after bloom collapse (as indicated by mussel PST levels); (6) non-lethal sampling is not possible as haemolymph PST levels do not reflect levels in the hepatopancreas. Importantly, (7) lobsters exposed to toxic algae during wet storage in long supply chains (on vessel, in sea-cages or at processing facilities) do not take up PST. Furthermore, (8) survival, quality, and safety of this high-value product are not impacted by accumulation of PST or by exposure to toxic cells in the water.