Rozprawy doktorskie na temat „Dinoflagellates”

Dinoflagellates are an abundant and diverse group of aquatic eukaryotes, with members that have photosynthetic, heterotrophic, or mixotrophic life strategies, as well as a number of unique cytological features. My thesis focuses on two groups of closely related dinoflagellates: polykrikoids and warnowiids. Both include heterotrophic as well as plastid-bearing members, though the number of times photosynthesis has been lost (or gained) in each group is unclear, and the presence and provenance of plastids in some species (e.g., Nematodinium sp. and Polykrikos lebouriae) have been debated. Polykrikoids and warnowiids also contain some of the most complex subcellular structures described--such as nematocysts and, in warnowiids, eye-like ocelloids. Yet these groups are rare in nature and uncultivated, and as such, the origins of their complex organelles are unclear. For my thesis, I modified existing techniques for use on single-cell environmental isolates, and applied these techniques to wild polykrikoid and warnowiid cells. By exploiting the common splice leader sequence found on dinoflagellate transcripts, I was able to amplify a single-cell transcriptome from Polykrikos lebouriae—a dinoflagellate with aberrant plastids. Coupled with single-cell genomics using multiple displacement amplification (MDA), I demonstrated that Polykrikos lebouriae has retained peridinin-type plastids, while photosynthesis has been lost in multiple other polykrikoid species independently. Using MDA and single-cell transmission electron microscopy, I also determined that the eye-like ocelloid of Nematodinium sp. is made in part from a peridinin plastid, and also from mitochondria. Specifically, single-cell focused ion beam scanning electron microscopy (FIB-SEM) allowed me to demonstrate that a retina-like portion of the ocelloid is a small part of a much larger peridinin-plastid that ramifies throughout the Nematodinium cell. Lastly, I investigated the evolution of nematocysts in Polykrikos spp. and Nematodinium sp. using a combination of transcriptomics, TEM, SEM, and FIB-SEM, and inferred that “nematocysts” in these groups evolved independently from those in cnidarians. Thus, nematocyst-like extrusive organelles appear to have evolved multiple times in eukaryotes. The data presented in this thesis show how extreme subcellular complexity has evolved in dinoflagellates through both endosymbiotic and autogenous origins.
Science, Faculty of
Zoology, Department of
Graduate

Doutoramento em Biologia
Os dinoflagelados são um grupo muito diverso de protistas que possuem um conjunto de características pouco comuns. Os peridinióides são dinoflagelados com teca que é formada por seis séries latitudinais de placas, incluindo a série cingular e um anel incompleto de placas intercalares anteriores, embora as últimas estejam ausentes em algumas espécies de Peridiniopsis. São dinoflagelados com simetria bilateral em relação ao plano apical que contem o eixo dorso-ventral. Na série sulcal há apenas uma placa posterior que contacta com o limite ventral de duas grandes placas antapicais. Entre os peridinióides, a presença ou ausência de um poro apical e o número de placas no cíngulo são geralmente consideradas marcas filogenéticas importantes ao nível de género ou família. Actualmente, a definição de Peridinium Ehrenberg, o dinoflagelado mais comum de água doce, inclui organismos com combinações diferentes destas duas características. Trabalhos anteriores sobre a ultrastrutura e afinidade filogenética das espécies tipo de Peridinium, P. cinctum, e Peridiniopsis Lemmermann, P. borgei também sugerem a necessidade de reexaminar as relações taxonómicas dos peridinióides. Esta tese combina o estudo ultrastrutural de uma selecção de espécies com hipóteses filogenéticas baseadas nas sequências de LSU rDNA, para aumentar o nosso conhecimento das diferenças e afinidades dentro dos peridinióides. Tem como objectivo aumentar o nosso conhecimento das características individuais das células que possam levar a reconhecer sinapomorfias que possam ser usadas como marcadores dos peridinióides como um todo e dos seus subgrupos. As espécies escolhidas para exame pormenorizado foram: Peridinium palatinum Lauterborn, de um grupo com duas placas intercalares anteriores, seis placas cingulares e sem poro apical; Peridinium lomnickii Wo!oszy"ska, de um grupo com poro apical, três placas intercalares e seis cingulares; Peridiniopsis berolinensis (Lemmermann) Bourrelly, uma espécie heterotrófica com poro apical, sem placas intercalares e com seis placas cingulares; e Sphaerodinium cracoviense Wo!oszy"ska, um membro de um género de formas com teca com um tipo de tabulação marginalmente peridinióide, com um suposto poro apical e quatro placas intercalares anteriores. Peridinium palatinum difere de Peridinium e Peridiniopsis típicos, quer em características da teca, quer internas. As diferenças estimadas entre as sequências parciais de LSU rDNA de P. palatinum e a espécie próxima P. pseudolaeve, relativamente a P. cinctum são comparativamente grandes e, juntamente com a topologia da árvore filogenética, apoiam a separação de P. palatinum e formas próximas ao nível de género. Palatinus nov. gen. foi, então, descrito com as novas combinações Palatinus apiculatus nov. comb. (espécie tipo; sin. Peridinium palatinum), P. apiculatus var. laevis nov. comb. e P. pseudolaevis nov. comb.. As características distintivas de Palatinus incluem uma superfície das placas lisa ou um tanto granulosa, mas não areolada, um grande pirenóide central penetrado por canais citoplasmáticos e de onde radiam lobos plastidiais, e a presença de uma fiada microtubular homóloga à de um pedúnculo. As células de Palatinus saem da teca pela zona antapicalpos- cingular. Peridinium lomnickii apresenta tabulação semelhante às formas marinhas, produtoras de quistos calcários, do género Scrippsiella A.R. Loeblich. Para comparação, adicionámos novas observações ultrastruturais de S. trochoidea. Peridinium lomnickii tem uma combinação de características diferente de Peridinium, Peridiniopsis e Scrippsiella. As hipóteses filogenéticas baseadas em DNA colocam P. lomnickii no mesmo ramo que Pfiesteria Steidinger et Burkholder, Tyrannodinium e outras Pfiesteriaceae, com as quais partilha um "microtubular basket" e uma ligação peculiar entre duas placas do sulco. As características distintivas do novo género proposto Chimonodinium gen. ined. incluem, além da tabulação, a ausência de pirenóides, a presença de um "microtubular basket" com quatro ou cinco fiadas sobrepostas de microtúbulos associados a um pequeno pedúnculo, um sistema pusular com tubos pusulares bem definidos ligados aos canais flagelares, e a produção de quistos não calcários. Peridiniopsis berolinensis partilha várias características significativas com Pfiesteria e afins, como um "microtubular basket" com a capacidade de suportar um tubo de alimentação, quimiossensibilidade para encontrar presas apropriadas, o modo de natação junto às presas e a organização geral da célula. Hipóteses filogenéticas com base em LSU rDNA confirmam a afinidade entre P. berolinensis e Pfiesteria bem como a relação mais remota com a espécie tipo de Peridiniopsis, P. borgei. Estas razões justificam a proposta de Tyrannodinium gen. nov., uma nova Pfiesteriaceae que difere de outros membros do grupo por viver em água doce e nos pormenores da tabulação. Sphaerodinium cracoviense revelou a tabulação típica do género Sphaerodinium, que apresenta um número de placas intercalares superiores e pos-cingulares maior que o que é típico em peridinióides: 4 e 6, respectivamente. Observações em SEM mostraram uma estrutura apical diferente da dos peridinióides, e um sulco apical numa das placas fazendo lembrar a área apical de alguns woloszynskióides. Os pormenores do aparelho flagelar e do sistema pusular ligam o Sphaerodinium aos woloszynskióides em geral e ao género Baldinia em particular, mas não aos peridinióides. O volumoso estigma de S. cracoviense revelou ser extraplastidial e de um modelo único, composto por elementos que se encontram em woloszynskióides, mas nunca encontrados anteriormente juntos. A análise filogenética baseada nas sequências parciais de LSU rDNA também sugerem uma maior proximidade de S. cracoviense com os woloszynskióides do que com os peridinióides. Futuras análises pormenorizadas de dinoflagelados peridinióides, em especial entre os do numeroso grupo de espécies com poro apical, serão necessárias para clarificar as suas relações taxonómicas; e a produção de descrições melhoradas das características finas particulares das células serão um requisito para perceber a evolução dos caracteres dos peridinióides por forma a podermos identificar marcadores filogenéticos.
Dinoflagellates are a diverse and widespread group of protists that combine a number of unusual features. Peridinioids are thecate dinoflagellates with six latitudinal series of plates, including the cingular series and the incomplete ring of anterior intercalary plates, although the latter is absent in some species currently classified as Peridiniopsis. They tend to be bilaterally symmetrical in relation to the apical plane containing the dorsiventral axis. In the sulcal series there is only one posterior plate, which contacts with the ventral edge of two large subequal antapical plates. Among peridinioids, the presence or absence of an apical thecal pore and the number of plates in the cingulum are often considered important phylogenetic markers at genus or family level. As currently delimited, Peridinium Ehrenberg, the most widely represented dinoflagellate genus in freshwater, includes organisms with different combinations of these features. Previous studies on the fine-structure and phylogenetic affinites of the type species of Peridinium, P. cinctum, and of Peridiniopsis Lemmermann, P. borgei, likewise suggested the need for reexamination of the taxonomical relationships of peridinioids. This thesis combines the ultrastructural examination of selected species with phylogenetic hypothesis based on partial LSU rDNA sequences to extend our knowledge of variation and affinities within the peridinioid group. It aims to advance our understanding of individual cell features that may lead to the recognition of synapomorphies that may be used as markers for the peridinioid group as a whole and for its subgroups. The species targetted for detailed examination were: Peridinium palatinum Lauterborn, representative of a group with two anterior intercalary plates, six cingular plates and no apical pore complex; Peridinium lomnickii Wo!oszy"ska, of a group with apical pore complex, three anterior intercalary and six cingular plates; Peridiniopsis berolinensis (Lemmermann) Bourrelly, an heterotrophic species with apical pore complex, zero anterior intercalary and six cingular plates; and Sphaerodinium cracoviense Wo!oszy"ska, a member of a genus of thecate forms with a marginally peridinioid type of tabulation, with a putative apical pore complex and four anterior intercalary plates. Peridinium palatinum was found to differ from typical Peridinium and Peridiniopsis in both thecal and internal features. The relatively large estimated differences in the partial LSU rDNA sequences of P. palatinum and its close relative P. pseudolaeve compared to P. cinctum, together with the topology of the molecular tree, supported the separation of P. palatinum and related forms at the generic level. Palatinus nov. gen. was therefore described with the new combinations Palatinus apiculatus nov. comb. (type species; syn. Peridinium palatinum), P. apiculatus var. laevis nov. comb. and P. pseudolaevis nov. comb.. Distinctive characters for Palatinus include a smooth or slighty granulate, but not areolate, plate surface, a large central pyrenoid penetrated by cytoplasmic channels and radiating into chloroplast lobes, and the presence of a peduncle-homologous microtubular strand. Palatinus cells exit the theca through the antapical-postcingular area. Peridinium lomnickii has a similar tabulation to the mostly marine, calcareous cyst producers of the genus Scrippsiella A.R. Loeblich and fine-structural observations on S. trochoidea were added for comparison. Peridinium lomnickii showed a different combination of features from Peridinium, Peridiniopsis and Scrippsiella. Interestingly, the DNA-base phylogenetic hypothesis placed P. lomnickii in the same clade as Pfiesteria Steidinger et Burkholder, Tyrannodinium and other pfiesteriaceans, with which it shares a microtubular basket and a peculiar connection between two plates in the sulcus. Distinctive characters of the proposed new genus Chimonodinium gen. ined., include, in addition to the tabulation, the absence of pyrenoids, the presence of a microtubular basket with four or five overlapping rows of microtubules associated with a small peduncle, a pusular system with well-defined pusular tubes connected to the flagellar canals, and the production of non-calcareous cysts. Peridiniopsis berolinensis shares a number of important features with Pfiesteria and its allies, including a microtubular basket with the capacity of driving and supporting a feeding tube, the ability to follow chemical clues to find suitable prey, the swimming behaviour near the prey and the general organization of the cell. Partial LSU rDNA-based phylogenetic hypotheses strongly confirm the close affinity between P. berolinensis and Pfiesteria and the more remote relationship with the type species of Peridiniopsis, P. borgei. These reasons justify the proposal of Tyrannodinium gen. nov., a new pfiesteriacean that differs from other genera in the group in being a freshwater form and in details of the plate arrangement. Sphaerodinium cravoviense showed the tabulation typical of its genus, which extends beyond normal peridinioid tabulation numbers in the anterior intercalary and in the postcingular series, with 4 and 6 plates, respectively. SEM observations revealed that the apical structure differed from the typical arrangement seen in peridinioids and included a furrow with knob-like protuberances reminiscent of the apical area of the thinly thecate woloszynskioids, which usually possess larger numbers of amphiesmal vesicles. Details of the flagellar apparatus and associated pusular system link Sphaerodinium to the woloszynskioids in general and to Baldinia anauniensis in partidular, rather than to peridinioids. The prominent eyespot found in S. cracoviense was shown by TEM to be extraplastidial and of a unique type, made of two components, each known from some eyespot types found in woloszynskioids, but not previously found together. A closer relationship of S. cracoviense with woloszynskioids than with peridinioids was also suggested by a phylogenetic analysis based on LSU rDNA. Further analyses of peridinioids, particularly within the sizeable group of species with an apical pore complex, is needed before general taxonomic relationships become clear; and improved descriptions of fine-structural features of cells are required to unravel the evolution of particular characters, allowing phenotypic phylogenetic markers to be identified.

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第8563号
農博第1146号
新制||農||809(附属図書館)
学位論文||H12||N3442(農学部図書室)
UT51-2000-M27
京都大学大学院農学研究科応用生物科学専攻
(主査)教授 内田 有恆, 教授 中原 紘之, 教授 大山 莞爾
学位規則第4条第1項該当

University of Technology, Sydney. Faculty of Science.
Global climate change is leading to the rise of ocean temperatures and is triggering mass coral bleaching events on reefs around the world. This involves the expulsion of the symbiotic dinoflagellate algae, known as zooxanthellae, from the coral host. Coral bleaching is believed to occur as a result of damage to the photosynthetic apparatus of these symbionts, although the specific site of initial impact is yet to be conclusively resolved. This thesis examined a number of sites within the light reactions of photosynthesis and evaluated the efficiency of photoprotective heat dissipating pathways. Upon expulsion, the capacity for long-term survivorship of expelled zooxanthellae in the water column was also assessed. A reduction in photosystem II (PSII) photochemical efficiency during exposure to elevated temperature and high light (bleaching conditions) was found to be highly dependent upon the increase in abundance of QB non-reducing PSII centres (inactive PSII centres), indicating damage to the site of the secondary electron acceptor, QB, resulting in a limited capacity for its reduction. Therefore, this reduced the rate of the reoxidation of the primary electron acceptor, QA-. Fast induction curve (FIC) analysis of the rise from minimum fluorescence to maximum fluorescence revealed a lower amplitude in the J step along this curve, which was consistent with a reduction in the rate of QA reoxidation. This photoinhibition of PSII was found to occur once the effectiveness of excess energy dissipation through energy-dependent quenching and state-transition quenching was exceeded, suggesting that these mechanisms were incapable of preventing photodamage. Antenna size heterogeneity showed little change under bleaching conditions with a significant increase in PSIIbeta only apparent in one species of coral. The thermostability of the oxygen evolving complex (OEC) and thylakoid membrane were found to increase during exposure to bleaching conditions and exceeded bleaching thresholds of corals. This rapid rise in temperature-dependent thermostability also occurred over seasons, where variation in ocean temperatures was matched by gradual shifts in OEC and thylakoid membrane thermotolerance. Variation in thermostability between species was not found to be linked to zooxanthellae genotype, and instead was related to the bleaching susceptibility of the host. Despite this capacity for resilience to bleaching conditions, the PSII reaction centres did not exhibit such a mechanism for rapid acclimatisation. Corals can only be as tolerant to bleaching conditions as their most sensitive component allows. The formation of nonfunctional PSII centres is therefore suggested to be involved in the initial photochemical damage to zooxanthellae which leads to a bleaching response. Zooxanthellae were found to be expelled irrespective of OEC function and thylakoid membrane integrity, as these sites of the photosynthetic apparatus were still intact when cells were collected from the water column. Although zooxanthellae were photosynthetically competent and morphologically intact upon expulsion, their longevity in the water column was dependent on the time of expulsion following the onset of bleaching and the ambient water temperatures. The survivorship of these zooxanthellae was restricted to a maximum of 5 days in the water column which suggests that unless expelled zooxanthellae inhabit other environs of coral reefs which may be more favourable for survival, their capacity for persistence in the environment is extremely limited. Chlorophyll a fluorescence measurements are a common tool for investigating photosynthetic impacts to in hospite zooxanthellae of corals. Pathways causing dark-reduction of the plastoquinone pool are shown to be active in corals and affect measurements which require dark-adaptation. Pre-exposure to far-red light was found to be an effective procedure to oxidise the inter-system electron transport chain and ensure determination of the true maximum quantum yield of PSII and accurate FICs. It is concluded that the trigger for coral bleaching lies in the photosynthetic apparatus of zooxanthellae and evidence is presented in support of this impact site not being the OEC or thylakoid membrane.

Nitrogen-replete cells of Amphidinium carterae took up ammonium in the light at a rate 5 - 6 times that of nitrate even though exponential growth rates were similar on these two N-sources. A. carterae exhibited a capability for enhanced initial ammonium uptake, particularly when deprived of nitrogen. Enhanced initial rates of ammonium uptake were also observed in a natural population of Gyrodinium aureolum. Initially ammonium accumulated within the cells of A. carterae but was assimilated into organic-N within a matter of hours; increases in total cellular-N, total free amino acids, glutamine and cellular protein were observed 4 h after an ammonium addition. In comparison, very little nitrate was accumulated. Ammonium (250 μM) inhibited reversibly the uptake of nitrate; the rapidity of the response suggests a direct effect on uptake. Prior nitrogen deprivation of the cells did not affect this inhibition. Rates of ammonium uptake were similar in the light and dark but nitrate uptake was completely inhibited by darkness in nitrogen replete cells of A. carterae and in a natural population of G. aureolum. Dark uptake of nitrate was stimulated by a period of nitrogen deprivation. Ammonium uptake in darkness by A. carterae was accompanied by the utilization of cellular polysaccharide, mainly glucose polysaccharide. Most of this carbon was unavailable for the assimilation of nitrate in the dark. It is suggested that a control mechanism is in operation, via a product of ammonium assimilation, on one or more of the enzymes concerned with polysaccharide breakdown, e.g. α-amylase or phosphorylase. Ammonium addition caused a marked enhancement of dark CO₂ fixation in several nitrogen-replete dinoflagellates. Nitrate addition produced little enhancement in comparison. The amount of enhancement was dependent on species, age of culture and period of diel cycle. Nitrogen deprivation caused a 2-3 fold increase in enhancement in all species tested. The measurement of dark ¹⁴CO₂ fixation shows promise as a technique for determining the nitrogen status of phytoplankton in both the laboratory and in the field. A natural population of Gyrodinium aureolum appeared to be slightly N-limited using this technique, an observation supported by other field data. The use of this technique as a tool to determine the nitrogen status of phytoplankton in culture and in the field is discussed.

Marine dinoflagellates are an ecologically important group of protists within the plankton, performing key process such as photosynthesis, heterotrophy and toxin production. Some dinoflagellates are also capable of producing bioluminescence and they are the most abundant protists that produce light in the surface waters of the oceans. This study employed molecular tools to investigate the identity of bioluminescent species, the genetic basis, diversity and functional regulation of bioluminescence, and the distribution of marine bioluminescent dinoflagellate populations. Using “universal” and taxon specific PCR primers designed to amplify the luciferase gene (lcf), the distribution of this gene within dinoflagellates was found to be taxon specific and the first lcf sequences obtained from four genetically distant genera revealed a previously unknown high diversity of this gene. The luciferin binding protein gene (lbp) was detected for the first time in three genera of ecologically important gonyaulacoid dinoflagellates, showing that this understudied gene is common in dinoflagellate bioluminescence systems. Phylogenetic analyses of both lcf and lbp provided new insight on species divergence within the toxic genus Alexandrium and pointed out important pitfalls in using protein coding genes for phylogenetic studies. The lcf PCR primers were employed in the first field study using a molecular approach to detect natural populations of bioluminescent dinoflagellates, showing that this approach outperforms optical bioluminescence measurements and that their distribution and composition is intimately tied to hydrographic patterns that create distinct environmental zones. The lcf PCR primers were also employed to study the regional variation in bioluminescence of Noctiluca scintillans, showing for the first time that the environment maintains or eliminates bioluminescence, by finding that a hydrographically isolated non bioluminescent variety in the west coast of the USA has “switched off” its bioluminescence system while diverging to possibly become a different species. Also, a novel lbp was discovered in N. scintillans which improved the current model for the evolution of bioluminescence genes in dinoflagellates. This thesis is the first study to employ molecular tools in a comprehensive and multifaceted investigation of dinoflagellate bioluminescence and has contributed significantly to building a fundamental understanding of this remarkable phenomenon.

Studies on the growth dynamics and physiology of the paralytic shellfish toxin producing dinoflagellates Alexandrium fundyense, A. minutum and Gymnodinium catenatum were conduced under a number of different nutrient regimes, light, temperature and salinity levels. G. catenatum was very slow to respond to N-refeeding, with relatively low internal amino acid concentrations, a low glutamine:glutamate ratio, and a stable toxin content, showing little variation with N- and P-limitation or with decreased salinity. In contrast Alexandrium sp. respond to N-refeeding with an increased internal amino acid pool, an increased glutamine: glutamate ratio, especially with ammonium as a N-source, and an increased toxin content. P-limitation in A. fundyense leads to a significant increase in the toxin content but only when cells are also N-limited. Sub-optimal temperature and light levels lead to decreased growth rates: low temperature results in larger cells and an increased toxin content in A. fundyense, whilst the effect of low light on A. minutum cells varies with the N-source available. The N-uptake kinetics of G. catenatum revealed that the maximum uptake rates (V_mas) and half-saturation constants (K_s) for transport were higher for ammonium when compared with nitrate. In A. funyense values of K_s were similar at 1 μM for both nitrate and ammonium whilst V_max for the latter was up to 5-times greater. A. fundyense was shown to be able to utilize organic-N in the form of dissolved free amino acids. Maximum uptake rates of 0.85 pmol cell^-1 h^-1 occurred during exponential growth and differed from the uptake capabilities of other phytoplankton in that uptake was not enhanced by N- or C-stress. A preliminary model is presented and is capable of simulating the relationship between N-refeeding and P-limitation and cellular toxin content in Alexandrium sp.

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

The ecology and taxonomy of dinoflagellates was investigated in 1982 and 1983 in sea-lochs and coastal waters of north-western Scotland, in order to provide a better understanding of conditions leading to blooms harmful to mariculture. Because such blooms are rare the ecology of all common thecate dinoflagellates was studied, particular attention being paid to the cyst stage and its role in bloom initiation. Creran, a fjordic sea-loch near Oban, was the main site of study; water column sampling was carried out regularly in 1982 and 1983 and the sediments were sampled for cysts in 1983. Distribution of motile dinoflagellates and their cysts on the west coast of Scotland was also investigated on cruises of the R.V. Calanus in the summers of 1982 and 1983. Identification of cells was carried out by light microscopy supplemented by studies using scanning electron microscopy. Links between cysts and motile stages were made by hatching experiments, and these and scanning electron microscopy have led to the discovery of some new cyst-theca relationships. In Loch Creran the first peak of dinoflagellate numbers occurred in May or June with warming of the water column and was dominated by Scrippsiella spp., accompanied in 1983 by Gonyaulax tamarensis. During July and August there was a mixed population, including Serippsiella spp., Heterocapsa triquetra, Protoperidinium spp. and Diplopsalis spp. Salinity stratification at the end of August preceded an autumn succession of G. polyedra, Prorocentrum micans and Ceratium species. Observations suggest that G. polyedra cysts sink rapidly and are retained within the loch, seeding blooms in subsequent years. Two other life strategies were hypothesized as the causes for different observed patterns of seasonal distribution. One involves non-cyst-forming species (for example Protoperidiniumbipes and Prorocentrum micans) present in the loch as motile cells in small numbers throughout the year, and occasionally reaching high concentrations. The other involves species (for example Protoperidinium depressum and Ceratium furca) whose motile cells are transported to the loch by tidal exchange or estuarine circulation from the sea outside. Seventy-two species of motile thecate dinoflagellates were found in the distributional study. Dinoflagellate abundance and variety were greatest in well- or partially-stratified water columns. Of potentially toxic species only Gonyaulax tamarensis was found to be widespread in 1983. Cysts varied in their distribution, with numbers higher in sea-lochs than in Firths and Sounds. Considerable sub-surface populations of cysts were noted in the sediments and viable cysts were encountered at 5 cm depth.

The ecological success and also the susceptibility of corals to bleaching have been attributed to their obligate relationship with Symbiodinium and its functional diversity. Deeper understanding of this symbiosis is essential to enable the existence of reef ecosystems. This thesis aimed to determine how the environment and the host exert control over the proteome of Symbiodinium and how this is related to the maintenance of symbiosis. Firstly, it was explored how the proteome related to the photoacclimation strategies of Symbiodinium, and its regulation under different light environments. An initial assessment of the proteome regulation using shotgun proteomics over Symbiodinium type C1 cultured at two experimental photon fluxes displayed differences in proteins associated with light harvesting, electron transport, carbon fixation and protein modification. Subsequent two-dimensional gel electrophoresis (2-DE) analysis between three Symbiodinium types (A1, A13 and C1) cultured at the two previous photon fluxes revealed unique proteome differences across these types and between both experimental light treatments. Database searches of the isoelectric point (pI) and the molecular weight (MW) tagged these proteins also associated with the same processes matched by previous shotgun analysis over C1 type. These results are in agreement with previous hypothesis on different photoacclimation strategies displayed among different types of Symbiodinium. These strategies comprised differential proteomic modifications in the photosynthetic unit (PSU) of the experimental types of Symbiodinium to balance ATP production and achieve homeostasis at different photon flux environments. The host control effect and regulation of the proteome and physiology of Symbiodinium ex- hospite was studied using two host release factors (HRFs) a free aminoacid mix (FAA) and the host tissue (HT) homogenate from the coral Pocillopora damicornis. To comprehend the relation between the proteome and physiology various parameters related with the photosynthate production and translocation of Symbiodinium were measured including: glucose released, excitation pressure over photosystem two (PSII), total protein, total glucose and chlorophyll a concentration in the presence and absence of the HRFs. The measured parameters showed variability among types. In general, the parameters directly related to photochemistry: Chl a and excitation pressure over PSII were controlled by photon flux and inversely correlated with each other. Protein and glucose also correlated with each other and were controlled by host factors although HT and FAA had an antagonistic effect of stimulation and inhibition of these parameters respectively. The principal component analysis (PCA) evidenced the existence of a general mechanism of glucose release which correlated with protein expression. The HRFs control over the proteome of Symbiodinium explored by SDS-PAGE analysis displayed different bands with differential expression among types and treatments. Some of these proteins may be related with the stimulation and inhibition of the mechanism of translocation of glucose that were found. Identification of these proteins would probably clarify the physiology behind the translocation of glucose from the Symbiodinium cell to its coral host. The proteome response and physiology of Symbiodinium was assessed by the interaction of different light environments with different temperatures. Growth patterns and photochemical measurements confirmed unique acclimation processes to light and temperature conditions across the three Symbiodinium types. These acclimation strategies appeared regulated by the differential expression of proteins involved in photosynthesis, protein modification, energy metabolism and cell maintenance. This differential expression of the proteome related to these processes seemed associated with the activation of alternative electron transport pathways to balance ATP production, regulate energy metabolism and fuel the cell at the different experimental light and temperature conditions. The results obtained provide a broader view on how the differential regulation of the proteome mediates the physiological plasticity across types of Symbiodinium to photoacclimate to different light enviroments, to acclimate to high temperature and to respond to stressful conditions determining the functional diversity existing in this genus of coral symbionts.

Toxic/mutagenic potentials of Gambierdiscus toxicus (GT) and Prorocentrum lima (PL) methanol extracts (CME) were determined using brine shrimp (Artemia salina), mouse, chicken embryo and Salmonella microsomal assays. PL-CME and GT-CME were toxic to shrimp and mice. Isolation and separation were accomplished using ether/water, hexane/methanol partition and butanol extraction. Toxic fractions were purified using column and thin layer chromatography (TLC). GT-CME showed low levels of mutagenic potential. For GT isolated fractions and PL-CME, no mutagenic effects were observed. Both CMEs showed embryotoxicity, with no teratogenic effects. Ether/methanol and water/butanol fractions showed shrimp toxicity. These fractions were purified by treatment with warm/cold acetone. Acetone insoluble precipitates were obtained. Ether soluble acetone filtrate (ESAF) and butanol soluble acetone precipitate (BSAP) showed shrimp and mouse toxicity. GT-BSAP produced temperature depression in mice. Three toxic isolates were obtained from PL-ESAF, four from GT-ESAF and one from both BSAPs columns. TLC preparative plates showed at least 12 toxic isolates for PL-ESAF, 8 for GT-ESAF and 4 for GT-BSAP.

Dinoflagellates are a diverse group of flagellated eukaryotes, the majority of which are marine plankton, and are one of the most important groups of primary producers in the world’s oceans. The dinoflagellate fossil record is based on their zygotic resting cysts; this record indicates that the Bajocian of the Mid Jurassic (~170–168 Ma) represents a critical interval in dinoflagellate evolutionary history, marked by a rapid increase in the diversity of cysts from the family Gonyaulacaceae. From the Bajocian onwards, the Gonyaulacaceae have remained one of the most diverse and abundant groups of dinoflagellate cysts in the fossil record. Even so, Bajocian dinoflagellate cysts themselves have received relatively little study, leaving the patterns of this radiation unresolved. In this thesis, I examine the Bajocian diversification of gonyaulacacean dinoflagellate cysts in northwest Europe via quantitative palynological analysis, and relate this into a broader stratigraphic and palaeoenvironmental context. The dinoflagellate cyst record of the three key study areas demonstrates an increase in gonyaulacacean dinoflagellate cyst diversity through the Bajocian, irrespective of differing palaeoenvironmental settings. However, palynological and sedimentological data record systematic changes in lithostratigraphic composition and/or depositional environment which reflect changes in sea level. The integration of these data with biostratigraphic records indicates that the pattern of the radiation in Europe was strongly influenced by sea level, with the increase in gonyaulacacean diversity mirroring a major second-order transgression. On a finer scale, the main pulses of first appearances correlate with third-order transgressive episodes. A rise in sea level, coupled with changes in the tectonic configuration of ocean gateways, appears to have controlled the pattern of diversification in Europe. These palaeoceanographic changes may have enhanced water-mass transfer between Europe, the northwest Tethys Ocean, and the Hispanic Corridor, which promoted the floral interchange of dinoflagellates. Comparison to global data demonstrates that gonyaulacacean dinoflagellate cysts increased in diversity on a global scale through the Mid Jurassic. Whilst sea level rise and associated large-scale palaeoenvironmental shifts appear to have controlled the pattern of dinoflagellate cyst appearances in several regions (e.g. eastern Canada, Israel), there is no direct correlation between dinoflagellate cyst diversity and sea level rise on a global scale. Within dinoflagellate cyst assemblages from Europe, the spatial and temporal distribution of key taxa can be related to differing palaeoenvironmental settings of the basins studied; but certain patterns may reflect wider palaeoenvironmental drivers. Carbon isotope records generated for this thesis, and their correlation to other European sections, demonstrate that the Early Bajocian in Europe was marked by a positive shift in δ13C. Previous work has linked this carbon cycle perturbation to a phase of enhanced continental weathering and associated run-off. In several European basins, the Early Bajocian was marked by an acme of the genus Dissiliodinium; this genus may have bloomed in response to elevated nutrient levels. Intriguingly, a similar pattern is seen within dinoflagellate cyst assemblages from Australia, indicating this interval of palaeoenvironmental change may have had a global extent.

Small non-coding RNAs such as microRNAs (miRNAs), small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs) play a big role in regulating gene expression in cells. In my work, I focus primarily on miRNAs, which represses the expression of the mRNA targets post-transcriptionally. For Drosophila melanogaster, I predicted the tissue-specific expression of several miRNAs based on the expression levels of the predicted mRNA targets in those tissues. The computational predictions are then followed up by quantitative PCR validation of miRNA expression levels in dissected fly tissues. For Stylophora pistillata (a species of coral found in the Red Sea) and Symbiodinium sp. (a photosynthetic, symbiotic algae present in the coral cell), my collaborators and I strived to study the genome, transcriptome and proteome of both organisms. At present, there is another coral genome available — from Acropora digitifera — but the large evolutionary distance between both corals (about 240 million years apart) warrants in-depth study of our coral of interest. On the other hand, our Symbiodinium genome will be the first of its kind for any dinoflagellate. My role in the project was to investigate the small RNAome of both organisms via small RNAseq. As the presence of a thick cell wall in Symbiodinium sp. poses a unique challenge to RNA extraction, and compounded by the dearth of literature regarding RNA extraction from the dinoflagellate, we optimised a procedure that consistently produced high quality RNA for downstream sequencing. From our draft proteome, I showed that the RNA interference (RNAi) machinery is very likely to be present in both organisms. Based on our short RNAseq data, I predicted miRNAs in both organisms. Two of the predicted miRNAs in S. pistillata have been identified in other organisms, while all of the predicted miRNAs in Symbiodinium sp. were novel.

Phytoplankton populations are integral to the structure and function of aquatic ecosystems, and phytoplankton are an excellent study system for exploring ecological questions. Reservoirs often exhibit high horizontal (inflow to dam) and vertical (surface to sediments) environmental heterogeneity, which plays a large role in determining phytoplankton population dynamics. In this thesis, I explore how three bloom-forming phytoplankton taxa, the dinoflagellates Peridinium and Gymnodinium, and the cyanobacterium Planktothrix, respond to horizontal and vertical environmental gradients, respectively. First, I monitored recruitment, or the process of leaving the sediments and entering the pelagic life stage, of dinoflagellates across a horizontal reservoir ecosystem gradient. Surprisingly, coupling of dinoflagellate biology with reservoir physics and chemistry varied along this continuum; recruiting cells were sensitive to reservoir physics (e.g., flow rate, solar radiation) in the upstream riverine zone, while recruitment was related to reservoir chemistry (e.g., dissolved oxygen, nutrients) in the downstream lacustrine zone. This study indicates that upstream habitats should be monitored when studying reservoir phytoplankton dynamics. Next, I investigated the environmental drivers of the vertical distribution and biomass of a hypolimnetic cyanobacterial bloom over two consecutive summers. I collected high-resolution in situ phytoplankton data, and measured environmental variables throughout the water column. Across both years, the vertical distribution of this population was determined by light availability, while the cyanobacterial biomass was predicted by both light and nutrients. These two studies demonstrate that changing physics and chemistry across environmental gradients can regulate phytoplankton dynamics in reservoirs, and phytoplankton monitoring should include more spatially comprehensive sampling approaches.
Master of Science

Thesis (Ph.D.) - University of Glasgow, 2004.
Ph.D. thesis submitted to the Division of Environmental and Evolutionary Biology, Institute of Biomedical and Life Sciences, University of Glasgow, 2004. Includes bibliographical references. Print version also available.

This study documents the occurrence of dinoflagellate cysts from the Middle Eocene Tallahatta and Lisbon Formations in the Baldwin County, Alabama, Core. This is part of a larger project being conducted by the ARCO Depositional Sequence Analysis Group to gather paleontological, sedimentological, paleomagnetic, geophysical, and geochemical data from the Alabama Coastal Plain for the purpose of calibrating the Tertiary time scale with the sequence stratigraphy of the Gulf region. A total of 101 taxa were identified, including 55 genera, 91 species, and 10 subspecies. Statistical analyses were run on the data collected to identify paleoecologic patterns and to relate these patterns, if possible, to sedimentary sequences. Diversity curves generated from the data show that there is usually a rise in diversity at sequence boundaries and at introsequence surfaces. Q-mode cluster analysis and detrended correspondence analysis indicate that samples tend to cluster within sequences. R-mode cluster analysis was performed and live informal cyst associations are discussed. Comparison of this studies assemblage with ranges published by Goodman and Stover (1975, 1983), and Edwards (1982) confirm the Middle Eocene age assigned to these units.
Master of Science

My dissertation focuses on genome and functional evolution of photosynthetic eukaryotes and the design and implementation of computational methods and tools to enable genome-wide studies to investigate these taxa. The work described here is grouped into two major topics, 1) endosymbiosis and genome evolution, and 2) harmful algal blooms. I discuss my work related to endosymbiosis and genome evolution in chapters 2-4. Chapters 5-6 cover the work related to harmful algal blooms. In chapter 1, I introduce the state-of-art of what is known about the history of plastids and evolution of photosynthesis in eukaryotes, an overview of marine harmful algae, and the specific aims of my dissertation. In chapter 2, I describe the design and implementation of the phylogenetic sorting tool, PhyloSort and the assembly of a high-throughput phylogenomic pipeline. Together, PhyloSort and the pipeline has become a key tool for multiple subsequent studies. chapter 2 also presents a case study using these tools in which we provide an estimate of the number of cyanobacterial genes that have been transferred to the nuclear genome of Plantae through primary endosymbiotic gene transfer; I use the model unicellular green alga Chlamydomonas reinhardtii for this purpose. In chapter 3, I discuss another case of prokaryotic contribution to the nucleus of photosynthetic eukaryotes. Here, the intriguing relationship of Chlamydiae-like bacteria and plants and algae is examined in a large-scale analysis, in which we scanned all available genomes of the primary photosynthetic organisms for genes of potential Chlamydiae origin. Surprisingly, we identified more than fifty Chlamydiae-derived genes in plants and algae. Here, we propose a model for the role that a Chlamydiae-like symbiont might have played in the establishment of the primary plastid in the common ancestor of Plantae. In chapter 4, I describe a study in which we explored the complete protein models of two diatom organisms as representative for photosynthetic chromalveolates and looked for genes that might have been acquired through endosymbiotic (secondary) or horizontal transfers from red or green algae. In contradiction of the “chromalveolate hypothesis” which states that photosynthesis in chromalveolates originated via the engulfment of a red alga symbiont, our study shows an unexpected green algal contribution that is fourfold greater than that of the canonical red algal symbiont. Our data suggest that the chromalveolate history includes a previously unrecognized green algal endosymbiont that was captured and lost prior to the more recent establishment of the red alga plastid, which is widespread in extant photosynthetic chromalveolates. In chapter 5, I discuss the identification of the phylogenetic origin of the genes involved in the biosynthetic pathway of saxitoxin in cyanobacteria. Here, we used a pyrosequencing approach to sequence de novo genomes of two strains of Anabaena circinalis, one of which is saxitoxin-producing and the other is non-toxic. Using comparative and phylogenetic analyses, I show that, within the saxitoxin gene cluster, genes that encode the key and unique enzymes in the pathway are of foreign origin that originated via horizontal transfer from non-cyanobacterial sources. These genes introduced the ability to produce saxitoxin in the ancestor of the toxic cyanobacterial clade. In chapter 6, I describe a gene expression study in which we used massively parallel signature sequencing (MPSS) to investigate RNA abundance patterns in the toxic dinoflagellate Alexandrium tamarense. This work provides the first clear evidence for the utilization by dinoflagellates of transcriptional to regulation. Moreover, using MPSS, we provide an estimate of the number of the distinct genes in Alexandrium tamarense; i.e., remarkably 40,000 loci. Taken together, our data indicate that dinoflagellates possess a great metabolic flexibility that allows them to efficiently toggle between photoautotrophy and heterotrophy based on the environmental conditions.

The heterotrophic dinoflagellates Pfiesteria piscicida and Pseudopfiesteria shumwayae (here referred to as Pfiesteria) have been reported to secrete potent toxins responsible for inducing lesions in Atlantic menhaden, causing deaths of fishes in natural systems and laboratory assays and impacting human health. Many aspects of Pfiesteria biology and ecology have been questioned, including its complicated life-cycle, its role in fish kills, fish lesion events and human health effects as well as its ability to produce toxins. Although the involvement of Pfiesteria in major estuarine fish kills and lesion events has been disputed, there is a demonstrated ability of these organisms to cause fish pathology and mortality in laboratory bioassays. Little evidence exists however, to support the conclusion that these effects are due to a potent exotoxin. Many other dinoflagellates are morphologically and genetically similarity to Pfiesteria and have been referred to as "Pfiesteria -like", although the pathogenic ability of these other species has not been investigated. In this work we address various aspects of the behavior and biology of Pfiesteria and related dinoflagellates in order to determine their ability to impact the health of fishes and to determine if any adverse effects are caused by a toxin or if of some other pathogenic mechanism is involved. Aquarium-format bioassays commonly used to detect and determine the toxin-producing status of Pfiesteria are subject to impediments that can make determination of the cause of fish mortality difficult. We developed a sensitive, 96 hr larval fish bioassay for assessing Pfiesteria pathogenicity using 2-10 mL volumes and larval cyprinodontid fishes. This assay was a highly effective method to verify and evaluate ichthyocidal activity in these dinoflagellates. Results from the larval assays, together with histopathological analysis, electron microscopy and direct observations of the interactions of Pfiesteria and fish, demonstrated the ability of dinoflagellates of the Pfiesteriaceae (including several Pfiesteria-like species) to cause pathology and mortality by feeding on the epidermal tissues of live fish. For some species, this process of micropredatory feeding resulted in fish mortality in laboratory bioassays comparable to that reported for purportedly "toxic" strains, but without the involvement of a toxin. Thus, the assertion that Pfiesteria produces a potent, fish-killing toxin is disputed. In laboratory assays with larval fish, P. shumwayae was consistently and significantly more ichthyocidal than P. piscicida. This differential pathogenicity was observed to be unrelated to chemoattraction of the dinoflagellates to fish tissues. Instead, as determined with comparative morphometric analyses, differences in ichthyocidal activity between these two species were related to their capacity to grow and reproduce after feeding on fish or algal prey. The results of these studies suggest that the perception of toxicity in these dinoflagellates is erroneous and related to different life history strategies and associated differences in growth rate in response to prey type. There is no direct evidence that these heterotrophic dinoflagellates impact fish health in the natural environment.

Dinoflagellates are one of the most abundant and diverse groups of microalgae. Many dinoflagellates are covered with cellulose plates, whereas others lack these plates and are hence referred to as athecate or unarmoured. Unarmoured species have been historically poorly characterized because they deform when fixed with traditional methods. Most unarmoured genera are included within the order Gymnodiniales and are differentiated by morphological characters, but the recent combination of morphological observations with phylogenetic data concludes that molecular phylogenies do not support the "classical" morphological criteria used to distinguish the genera. It is also known that the order Gymnodiniales is not monophyletic. Consequently, the taxonomy of dinoflagellates has shifted to a combination of morphology and molecular information. The widespread use of molecular techniques has enabled detailed studies on the systematics of a lot of groups. Although many dinoflagellates are not easily cultivable and cannot be sequenced by standard techniques, the single-cell PCR technique has allowed obtaining sequences from a single cell by taking advantage of the large copy number of the ribosomal genes. Extensive identifications of dinoflagellates present in the NW Mediterranean Sea were carried out between the 1960s and 1980s. However, the diversity of unarmoured dinoflagellates was not suitably characterized because of the use of fixed samples and the lack of molecular tools. This thesis studies the diversity of dinoflagellates belonging to the Gymnodiniales order from the Catalan coast, as a representative site of the NW Mediterranean Sea. This thesis presents a revision of their taxonomy by combining morphological studies of live specimens with the respective phylogenetic information. Given that the phylogeny of most of the studied organisms had not been previously determined and the evidence that Gymnodiniales is not monophyletic, a secondary objective was to study the phylogenetic relationships of species. The D1-D2 region of the LSU rDNA sequence was selected to conduct the SC-PCR, but SSU rDNA sequences were obtained when necessary. The combination of morphological and molecular data has led to the identification of 58 unarmoured species belonging to the order Gymnodiniales. Ten morphospecies are detected for the first time in the Mediterranean Sea, and eight, for the first time along the Catalan coast (Chapter 1 and 2). Additionally, the application of single-cell PCR has resulted in the sequencing of 43 unarmoured species, 25 of them for the first time (Chapters 1 and 2). It has also allowed the detection and characterization of species previously undescribed, resulting in the erection of two new species: Gymnodinium litoralis (Chapter 3) and Polykrikos tanit (Chapter 4). Additionally, the toxic species Cochlodinium polykrikoides was detected for the first time along the Catalan coast. Most populations formed a newly differentiated ribotype, but others were included within a previously known one, demonstrating their coexistence in the Mediterranean Sea (Chapter 5). Finally, a Ceratoperidinium specimen was sequenced for the first time and a new phylogenetic clade was obtained, along with other unarmoured dinoflagellates, including Ceratoperidinium margalefii, Gyrodinium falcatum, which was transferred to the genus Ceratoperidinium, three Cochlodinium species, and two Gymnodinium-like organisms. This resulted in the emendation of the Ceratoperidiniaceae family and the genus Ceratoperidinum (Chapter 6). The correct identification of the species has allowed to conclude that there is a high diversity of unarmoured dinoflagellates in the Catalan coast, and to discuss the implications on the distribution and biogeography of the species at a Mediterranean level, as C. polykrikoides (Chapter 5), or globally, as for Gyrodinium spirale (Chapter 2)
Les dinoflagel•lades són un dels grups més abundants i diversos de microalgues. Moltes espècies estan cobertes amb plaques de cel•lulosa, mentre que d’altres que no en tenen són conegudes com a atecades o nues. Les espècies atecades han estat generalment mal caracteritzades ja que es deformen quan són fixades amb mètodes tradicionals. La majoria de gèneres atecats s'inclouen dins l’ordre Gymnodinials i es distingeixen per caràcters morfològics, però la combinació recent d'observacions morfològiques amb dades filogenètiques conclou que les filogènies moleculars no suporten els criteris morfològics clàssics utilitzats per distingir els gèneres. També es coneix que l’ordre Gymnodinials no és monofilètic. Per tant, la correcta taxonomia de dinoflagel•lades requereix una combinació d’informació morfològica i molecular. L'ús generalitzat de tècniques moleculars ha permès estudis detallats sobre la sistemàtica de molts grups. Tot i que moltes dinoflagel•lades no són fàcilment cultivables i no es poden seqüenciar mitjançant tècniques estàndards, la tècnica de single-cell PCR ha permès obtenir seqüències a partir d'una sola cèl•lula, aprofitant el gran nombre de còpies dels gens ribosòmics que les dinoflagel•lades contenen. Entre els anys 1960 i 1980 es van dur a terme identificacions extensives de les dinoflagel•lades existents al litoral català. Tanmateix, no es va caracteritzar adequadament la diversitat de dinoflagel•lades atecades degut a l’ús de mostres fixades i la manca d'eines moleculars. Aquesta memòria presenta l'estudi de la diversitat d'espècies de l’ordre Gymnodinials a la costa catalana com a representativa del NO del Mar Mediterrani, entre els anys 2010-2013. L'estudi presenta una revisió de la seva taxonomia pel que s'han combinant estudis morfològics d’exemplars vius amb la corresponent informació filogenètica. Atès que la filogènia d’alguns dels organismes estudiats no s'havia determinat amb anterioritat i l'evidència que l’ordre Gymnodinials no és monofilètic, el segon objectiu ha estat estudiar les relacions filogenètiques de les espècies. Es va seleccionar la regió D1-D2 del 28s rDNA per dur a terme els anàlisis amb SC-PCR, però també es van obtenir seqüències del 18s rDNA quan ha estat necessari. La combinació de les dades morfològiques i moleculars ha permès la identificació inequívoca de 58 espècies atecades pertanyents a l’ordre Gymnodinials. D’aquestes, es detecten deu morfoespècies per primera vegada a la mar Mediterrània, i vuit per primera vegada al litoral català (Capítols 1 i 2). A més, l'aplicació de la SC-PCR ha permès seqüenciar 43 espècies atecades, 25 de les quals per primera vegada (Capítols 1 i 2). També ha permès la detecció i caracterització d’espècies no descrites prèviament, que ha resultat en la descripció de les noves espècies Gymnodinium litoralis (Capítol 3) i Polykrikos tanit (Capítol 4). A més, es va detectar per primera vegada l'espècie tòxica Cochlodinium polykrikoides al litoral català. La majoria d’aquests organismes pertanyien a un nou ribotip, però d’altres quedaren inclosos en un ribotip ja conegut, demostrant la seva coexistència al Mar Mediterrani (Capítol 5). Finalment, es va seqüenciar per primera vegada un espècimen del gènere Ceratoperidinium i es va obtenir un nou grup filogenètic, juntament amb d’altres dinoflagel•lades atecades, incloent Ceratoperidinium margalefii, Gyrodinium falcatum, que va ser transferit al gènere Ceratoperidinium, tres espècies de Cochlodinium, i dos organismes semblants a Gymnodinium. Això va donar lloc a l'esmena de la família Ceratoperidiniaceae i del gènere Ceratoperidinum (Capítol 6). La correcta identificació de les espècies ha permès concloure que la costa catalana presenta una gran diversitat de dinoflagel•lades atecades, i discutir les implicacions en la distribució i biogeografia de les espècies a nivell de la Mediterrània, com el cas de C. polykrikoides (Capítol 5), o global, com el cas de Gyrodinium spirale (Capítol 2)

Thecate gonyaulacoid dinoflagellates referable to the genus Protogonyaulax Taylor cause paralytic shellfish poisoning (PSP) in coastal regions throughout the world. Isolates of the Protogonyaulax tamarensis/ catenella species complex from diverse geographical regions, including ten contemporaneous isolates from the same location, were subjected to chemotaxonomic analysis of soluble isozymes by gel electrophoresis, an analysis of toxin components using high-pressure (performance) liquid chromatography and quantitative nuclear DNA determinations by epifluorescence microphotometry. The results were compared with conventional morphological criteria used to discriminate among species, to establish taxonomic linkages and to estimate phenotypic and genotypic variation within this group. These biochemical methods, along with measurements of acclimated growth rate, offered a means of distinguishing between isolates of this species complex, for which the thecal plate patterns were substantially the same. The isozyme patterns revealed a high degree of genetic polymorphism within and among morphotypes and geographical populations. Yet, within the tamarensoid morphotype, isolates from the same location were more similar than to those from elsewhere. This general trend was supported by evidence from toxin profiles and DNA analysis, although toxin heterogeneity was a more conservative measure of variation than isozyme diversity. Protogonyaulax isolates varied markedly in total toxin concentration and toxicity, even through the culture cycle of individual isolates, but the toxin ratios were distinctive and relatively constant. The catenelloid and tamarensoid forms, the dominant morphotypes within this species complex, were not well correlated with the biochemical characters investigated. Given the occasional presence of morphological intermediates, the morphological features presently used to identify P. catenella and P. tamarensis cannot always be used to reliably discriminate between these morphospecies, and appear to be inadequate as stable species descriptors. At least two smaller isolates from allopatric populations exhibited morphological and biochemical differences large enough to indicate possible species divergence. The high level of genetic diversity reflected in the biochemical heterogeneity within populations from a given geographical area suggests that sibling species may also have arisen within sympatrically distributed Protogonyaulax populations.
Science, Faculty of
Earth, Ocean and Atmospheric Sciences, Department of
Graduate

Thesis (Ph. D.) -- University of Maryland, College Park, 2006.
Thesis research directed by: Cell Biology & Molecular Genetics. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.

Despite the critical role algae serve as primary producers, increases or accumulation of certain algae may result in Harmful Algal Blooms (HABs). Algal toxins from these blooms contribute significantly to incidences of food borne illness, and evidence suggests HABs are expanding in frequency and distribution. Mitigation of these HABs without knowledge of the ecological purpose and biochemical regulation of their toxins is highly unlikely. The production, function, and potential of secondary metabolites produced by the dinoflagellates Karenia brevis and Pyrodinium bahamense, were investigated. Brevetoxins were demonstrated by two different methods to localize within the cytosol of Karenia brevis. Differential and density-dependent centrifugation followed by Enzyme Linked Immunosorbant Assays (ELISAs) indicated that brevetoxin was not contained by any cellular organelles. Light microscopy of brevetoxin immunolabeled preserved cells visually confirmed these results, showing stain to be distributed throughout the cytosol and notably absent from the nucleus. These results have implications for brevetoxin synthesis and function. The complex cyclization process of brevetoxin therefore likely occurs in the cytosol after export of a polyketide precursor from the chloroplast. Functionally, this cellular location suggests use of brevetoxin in cytosolic functions such as signaling and chelation. Culture experiments of Pyrodinium. bahamense var. bahamense were undertaken to determine the effects of nutrients and environmental conditions on growth requirements and toxin production. HPLC analysis was employed to separate and quantify the saxitoxins. As eutrophication is a concern where this species is most problematic, in the Indian River Lagoon area of Florida, utilization of urea and ammonium were explored and compared to nitrate. While all nitrogen conditions yielded similar growth curves in P. bahamense, the cultures using urea contained a substantially lesser amount of the potent STX congener. This difference implies the urease enzyme utilized by P. bahamense is inefficient and urea based fertilizers are unlikely to create blooms with greater toxicity. Cyst production in P. bahamense was found to depend on nutrient limitation. Cultures utilizing ammonium displayed a smaller proportion of cysts, presumably attributable to the bioavailablility of ammonium. The total toxin content of P. bahamense was found to vary inversely with growth rate, although mole percents of the saxitoxins were largely unchanged over a suite of environmental parameters including temperature, salinity, and pH. Possible reasons for the reported increase in HABs include global warming, dumping of ballast water, and nutrient influx. These studies outline controls on toxin synthesis and production and conditions needed for growth and will aid in predicting environmental and human health effects pending these global changes. Extracts of K. brevis and P. bahamense cultures were assayed against various pathogenic agents. Growth of K. pneumoniae was inhibited by extracts of both K. brevis and P. bahamense. An extract of K. brevis additionally inhibited MRSA, while a P. bahamense extract additionally inhibited both S. aureus and MRSA as well as the most common protozoan vector of malaria, P. falciparum. The activity of a dinoflagellate against an Apicomplexan (P. falciparum) found in this study is especially interesting as the phyla are closely phylogenetically related. Differences in activity of extracts against P. falciparum between a clonal culture on P. bahamense from the Indian River Lagoon and a 2011 bloom sampled from Tampa Bay were observed. Drugs are losing their effectiveness against these infectious agents, making pursuit of new drugs an important field. These results suggest that HAB dinoflagellates hold promise in drug discovery similar to other phytoplankton.

The small dinoflagellate Azadinium spinosum produces azaspiracid (AZA) toxins which can contaminate filter feeding shellfish to dangerous levels. Toxin-contaminated shellfish flesh, when consumed by humans, can cause acute intense illness and chronic health issues. Shellfish biotoxins are monitored in Scottish shellfish by Food Standards Scotland (FSS), and the concurrent monitoring of harmful phytoplankton in the water column acts as an important early warning system of future shellfish toxin contaminations. Since A. spinosum is very small (12-16 μm long) it is difficult to identify using a light microscope, therefore molecular techniques have been developed to detect species-specific environmental DNA from phytoplankton samples. In this thesis the application and verification of quantitative real time polymerase chain reaction (qPCR) is discussed in detail and documents its first use in Scottish waters to survey A. spinosum abundance and seasonality. The limit of detection of the method was found to be 2000 ±5600 cells L-1, however it is unclear whether this is adequate for regulatory monitoring because it is not yet understood how cell density in the water column relates to AZA shellfish toxicity. The qPCR probe and primer sequences were also found to be too specific to detect all strains of the A. spinosum species, as new strains have been isolated since their development. This is a significant hindrance to the application of the tool for monitoring which will need to be addressed in the future through the isolation of local A. spinosum strains. Over a year long sampling period, A. spinosum was detected only twice (maximum cell density of 2545 ±5600 cells L-1, August 2014) off the Shetland Islands. The seasonality of the species in Scottish waters could not be assessed with so little data, however other observed harmful species of importance to shellfish regulatory monitoring are discussed; of particular note an unusual bloom of Dinophysis acuta as its association with a temperature front at the mouth of Loch Fyne. This thesis critiques the use of this qPCR technique for A. spinosum detection at high-throughput. The issues which have been highlighted do not prevent its future use by FSS, but highlight specific areas of development which need addressed before national monitoring can occur.

Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2003.
Includes bibliographical references (leaves 118-130). Also available in electronic version. Access restricted to campus users.

Thesis (Ph. D.)--Joint Program in Biological Oceanography (Massachusetts Institute of Technology, Dept. of Biology; and the Woods Hole Oceanographic Institution), 2005.
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Includes bibliographical references.
Marine thecate heterotrophic dinoflagellates likely play an important role in the consumption of primary productivity and in the trophic structure of the plankton, yet we know little about these species. This thesis expanded our understanding of the autecology and evolutionary history of the Protoperidinium and diplopsalids. The distributions of Protoperidinium species off the southwestern coast of Ireland were influenced by physical oceanographic conditions coupled with the availability of preferred prey. The distributions of individual Protoperidinium species varied widely from the distribution of total Protoperidinium, indicating differences in ecologies among species. Certain species of Protoperidinium co-occurred with known preferred phytoplankton prey species. Concentrations of other Protoperidinium species were not related to those of any particular phytoplankton species, indicating that these Protoperidinium may rely on phytoplankton or other food sources beyond those already known, may not be species specific selective feeders, or may have become uncoupled from their preferred prey. The description of the sexual and asexual life history of Protoperidinium steidingerae provided the first account of the life history of any Protoperidinium species.
(cont.) Asexual division occurred by eleutheroschisis within a temporary, immotile cyst, yielding two daughter cells. Daughter cells were initially round and half to two-thirds the size of parent cells, then rapidly increased in size, forming horns before separating. Sexual reproduction was constitutive in clonal cultures, indicating that the species may be homothallic. Fusing gametes were isogamous, and resulted in a planozygote with two longitudinal flagella. Hypnozygotes had a mandatory dormancy period of ca. 70 days. Germination resulted in planomeiocytes with two longitudinal flagella. Nuclear cyclosis may occur in the planomeiocyte stage. A high level of morphological diversity among life history stages of P. steidingerae has led to mis-classification and taxonomic inaccuracy of Protoperidinium species identified from field samples. The large subunit ribosomal DNA (LSU rDNA) molecular phylogeny of the heterotrophic dinoflagellates revealed that the genus Protoperidinium appeared to be recently diverged within the dinoflagellates. In maximum parsimony and neighbor joining analysis, Protoperidinium formed a monophyletic group, evolving from diplopsalid dinoflagellates.
(cont.) In maximum likelihood and Bayesian analyses, however, Protoperidinium was polyphyletic, as the lenticular, diplopsalid heterotroph, Diplopsalis lenticula Bergh, was inserted within the Protoperidinium clade basal to Protoperidinium excentricum (Paulsen) Balech, and Preperidinium meunieri (Pavillard) Elbrichter fell within a separate clade as a sister to the Oceanica section and Protoperidinium steidingerae Balech. In all analyses, the Protoperidinium were divided into two major clades, with members in the Oceanica group and subgenus Testeria in one clade, and the Excentrica, Conica, Pellucida, Pyriforme, and Divergens sections in another clade. The LSU rDNA molecular phylogeny supported the historical morphologically determined sections, but not a simple morphology-based model of evolution based on thecal plate shape. LSU rDNA gene sequences are frequently used to infer the phylogeny of organisms. The many copies of the LSU rDNA found in the genome are thought to be kept homogenous by concerted evolution. In Protoperidinium species, however, there was high intragenomic diversity in the D1-D6 region of the LSU rDNA. For each species, the clone library was usually comprised of one highly represented copy and many unique sequences.
(cont.) Sequence differences were primarily characterized by single base pair substitutions, single base pair insertion/deletions (indels), and/or large indels. Phylogenetic analysis of all clones gave strong support for monophyly of the polymorphic copies of each species, and recovered the same species tree as an analysis using just one sequence per species. Analysis of LSU rDNA gene expression in three species by RT-PCR indicated that copies with fewer substitutions and fewer and smaller indels are expressed, and that 50% or more of the copies are pseudogenes. High intraspecific and intraindividual LSU rDNA sequence variability could lead to inaccurate species phylogenies and over-estimation of species diversity in environmental sequencing studies. This thesis has explored the ecology, life history, molecular phylogeny, and intraspecific DNA sequence variability of marine thecate heterotrohic dinoflagellates using a wide range of methodologies, including field sampling, culturing, microscopy, morphological analyses, histological staining, and molecular biology. The work here has broadened our understanding of the Protoperidinium and diplopsalids, providing new insights into the ecological and evolutionary relationships of these heterotrophs with other plankton species.
by Kristin Elizabeth Gribble.
Ph.D.

Interest in the effect of fish farming practices on the marine environment has arisen because there is concern that the wastes that fish farms produce may be contributing to eutrophication in coastal areas and the problem of harmful algal blooms. The focus of this thesis is an examination of phytoplankton distribution and abundance in relation to tuna fish farms in Boston Bay and near-shore Spencer Gulf. This is the first study in South Australia to define the short-term biomass fluctuations of chlorophyll and in vivo fluorescence, identify phytoplankton species distribution and abundance, including two potentially toxic dinoflagellates, and describe patch distribution relative to tuna fish farms in Boston Bay and the near-shore waters of Spencer Gulf. An ecological interpretation of phytoplankton distribution and abundance is determined and shows that community composition was different in lower Spencer Gulf compared to Boston Bay and upper Spencer Gulf sites. Pico- and nanophytoplankton were often the most abundant organisms. Diatoms and gymnoids were most common. Season and currents predominantly influenced the distribution of phytoplankton in Boston Bay and Spencer Gulf. Individual species may be influenced by inputs from the fish farms. Chlorophyll levels were different between the Spencer Gulf and Boston Bay sites and no differences were recorded, using mean levels of chlorophyll, between tuna cages and controls. Chlorophyll levels were higher east of Boston Island in autumn of 1999. Chlorophyll levels appeared to show a slight increase between years. This may have been an anomalous natural variation and future research may investigate this in the long term. In addition, Principal Components Analysis (PCA) was used to investigate differences between treatments and the functional grouping model supported an ecological interpretation of the factors from the PCA. A total of 131 taxa of phytoplankton were identified in this study. The 14 dominant taxa were used in the PCA and of these, 9 were diatoms. Phytoplankton abundance was not different between tuna cages and controls. However, when examining individual species, Karenia mikimotoi was more prevalent at tuna cages, close to shore, east and west of Boston Island than at other sites. PCA showed how different species bloomed together and were seasonal. Karenia brevis and K. mikimotoi featured predominantly in the PCA with K. brevis the dominant organism during summer and autumn along with Gyrodinium spp. and smaller gymnoids. K. brevis blooms were most likely influenced by water temperatures and fixation of nitrogen from a Trichodesmium erythaeum bloom. K. mikimotoi bloomed bimodally and may be influenced by ammonia excreted from fish from the tuna farms but , on the other hand, may be limited by the high salinities of South Australian waters. Currents in the region distribute both organisms. The final aspect of this study assessed finer temporal and spatial sampling using directional transects around tuna cages and controls using in-vivo fluorescence and size fractionated chlorophyll. The chlorophyll a sampling showed little spatial variability within a site in the 1000 m2 that the sampling area covers but far greater temporal variability (days). In contrast, fluorescence `mapping' expands the window of variability both spatially (within a site) and temporally (along transects and between days). This has given a spatial definition, which is unavailable from a single point sample, and thereby leaves room for much greater interpretation. Small patches are evident from the fluorescence mapping where this is impossible to detect from the single point samples. Therefore, the fluorescence `mapping' and patch definition show that the trend is widespread (spatially) and quite persistent (temporally) around the fish farm area. Size fractionated chlorophyll samples provided further insight into phytoplankton dynamics in this study where diatoms were favored over dinoflagellates and were responsible for the larger fraction of chlorophyll found at the tuna cage one (TC1) site. We suggest that seasonal fluctuations, high nutrient input from the farm activities and turbulence may be responsible for the different chlorophyll/fluorescent structures found at TC1. Future research may look at the long-term regional impact on phytoplankton size structure, biomass and communities from fish farm activities. As a good part of this journey involved counting phytoplankton using the Utërmohl technique, a short paper, published in the Journal of Plankton Research, on reducing the settling time of this method, is presented in Appendix.