No description available.

With the global drive to find a renewable liquid fuel, attention has turned to macroalgae cultivation as a feasible approach for the production of a third generation biofuel. Additionally, the increasing demand for macroalgal extracts, and growing interest in their bioremediation role at aquaculture sites in an Integrated Multi-Trophic Aquaculture (IMTA) system, is driving the expansion of macroalgae cultivation across Europe. The suitability of kelp species to a large proportion of the European coastline, combined with high carbon, high value extract content and well-established cultivation techniques already demonstrated in Asia, makes them strong candidates for the expansion of macroalgae cultivation. Although large-scale kelp cultivation in Europe is still in its infancy, it is essential we understand the ecological impacts that such largescale sites may have on economically important coastal waters, before extensive production goes ahead. This thesis highlights the link between dissolved inorganic and organic matter exuded by candidate European kelp cultivation species Saccharina latissima and the microplankton assemblages. Laboratory incubation experiments using kelp exudates and microplankton were compared to fieldwork carried out at the world's largest kelp cultivation site in Rongcheng, China. Small-scale incubations have shown that resources in kelp exudates can be utilised by the bacterioplankton and mixotrophic Harmful Algal Species (HAS) of phytoplankton, and fieldwork at a working large-scale Asian kelp cultivation site, provides significant evidence impact of kelp cultivation on the bacterioplankton assemblage. Kelp exudates at large-scale cultivation sites will influence the microplankton assemblage, and this research is the first evidence of the impact that large-scale coastal aquaculture of kelp can have on bacterioplankton assemblages. A large knowledge gap in current fish, shellfish, invertebrate and IMTA aquaculture systems exists in understanding the fate of waste at cultivation sites in the microbial food web. Understanding the role of the microbial loop in aquaculture will be essential in managing the sustainability of aquaculture in an era of global expansion.

Viruses are the most abundant biological agents in the global marine environment. Through cellular lysis viruses influence many biogeochemical and ecological processes, including energy and nutrient cycling, host distribution and abundance, algal bloom control and genetic transfer. Nano- and picophytoplankton are ubiquitous in the world’s oceans and are responsible for a high proportion of the annual global carbon fixation. However, relatively few viruses have been isolated and described that infect these key primary producers and little is known of their diversity, dynamics or propagation strategies. The aims of this study were to detect, isolate and characterise novel marine viruses that infect these important members of the phytoplankton assemblage. Screening of seawater samples for viruses that infect a broad representation of nano and picophytoplankton species was undertaken here. To enable this, a large culture collection of 106 phytoplankton species was established and used to screen seawater samples for viruses on a weekly basis over a two year period. A total of 12 novel viruses infecting the prasinophyte species’ Ostreococcus tauri and Micromonas pusilla were isolated from seawater sampled in coastal waters of the Western English Channel. Viruses were purified by plaque purification or liquid serial dilution techniques. Characterisation of novel virus isolates included growth kinetics, visualisation using transmission electron microscopy, host range analysis and estimates of viral genome sizes using pulsed field gel electrophoresis. Phylogenetic analysis of these viruses was conducted based on the sequence of the conserved DNA polymerase gene. Genome sequencing of two of the viruses infecting O. tauri was completed and revealed many exciting features, including a suite of genes hitherto unreported, or with rare occurrence, in viruses. Evidence is presented for horizontal gene transfer between viruses isolated in this study and their hosts, as well as between other eukaryotic and bacterial sources. Functional characterisation of the viral genomes sequenced and described in this study will provide clearer insights into viral dynamics and evolutionary history.

Harmful phytoplankton occur naturally in British waters. However, little is known about the environmental drivers that lead to the formation and advection of harmful algae blooms (HABs). To minimise adverse effects of HABs it is necessary to improve our understanding of links between advection, environmental changes and HAB development. The aims of this PhD project were therefore to: 1) Provide a better understanding of the relationship between harmful phytoplankton, environmental drivers and key hydrodynamic features such as the shelf edge; 2) Determine the role of advection in bloom transport and development; 3) Utilise computational modelling to study environmental drivers and advection of HABs. To achieve these aims, field data was collected from two cruises and a glider mission. During the cruises, data on phytoplankton community was collected alongside physical data with a focus on key features such as the shelf edge and seasonal coastal fronts. This data provided an updated, detailed assessment of phytoplankton across the Hebridean and Malin Shelves. Field data showed that the European Slope Current (ESC) and Islay front can separate phytoplankton communities on the shelf. Lack of community differences along the ESC suggested stronger transport and exchange of phytoplankton within the ESC than adjacent shelf waters. Nutrients, nutrient ratios and light conditions were also found to play major structuring roles in determining phytoplankton assemblage. Data collected during field work can also be useful for regulatory assessment of shelf seas by providing baseline information about phytoplankton communities in the area. The glider mission provided an additional high resolution dataset of biological and physical water column properties across the Malin shelf. Vertical resolution showed that phytoplankton distribution was strongly linked to thermal stratification and temperature changes. Horizontal resolution was highly patchy, suggesting that scientific cruises could easily miss high density blooms with small spacial extent. The glider successfully monitored a high density HAB, suggesting that gliders could potentially be used for phytoplankton surveillance and detection of high biomass blooms. In addition to field data, a bio-physical individual based model (IBM) was used to simulate HAB progression. The IBM was coupled with a hydrodynamic ocean model to show the role of advection and importance of offshore seed populations in coastal HAB development. Model output was compared to coastal count data and satellite images whenever possible. Running the model under different conditions for phytoplankton growth and behaviour, suggest that it was crucial to include Abstract 5 biological processes to simulate HABs. The IBM could be initialised with data from satellite images, field data or discrete seed populations. Model simulations with different initial cell concentrations and locations could help to explain observed bloom pathways and suggest possible offshore origins for observed exceptional HABs. The results from field work and model simulations showed the role of the ESC in structuring phytoplankton community and transporting seed populations of HABs along the Scottish west coast. This suggests that future cruises and offshore monitoring should focus on the ESC and shelf break region. The bio-physical model could hindcast HAB pathways along the ESC, suggesting that modelling of HAB pathways of known seed populations could be integrated into an early warning system for aquaculture sites along the Scottish west coast in the future. Such an early warning system would allow the protection, relocation or early harvesting of affected aquaculture sites.

Marine dinoflagellates are an ecologically important phytoplanktonic group that accounts for two thirds of all known harmful algal bloom (HAB) species. This study explores the population ecology of Lingulodinium polyedrum (F. Stein) J.D. Dodge, a common bloom-forming dinoflagellate species in Southern California. Lingulodinium polyedrum is not considered a HAB species, but functions as one of the main model organisms for dinoflagellate biology. As such, knowledge about this species also contributes significantly to the understanding of dinoflagellate population dynamics at a more general level. In an attempt to understand some of the complex interactions that govern L. polyedrum population ecology, laboratory experiments of life cycle control and intraspecific phenotypic diversity were linked with an in situ study of the population dynamics and the intraspecific genetic diversity of this species in coastal waters of Southern California. The life cycle experiments showed that processes such as gametogenesis and ecdysis of L. polyedrum are influenced by photon flux density (PFD) and gave a first indication for an involvement of the photosynthetic apparatus in the induction of gametogenesis in dinoflagellates. The light acclimation experiments revealed, for the first time, intraspecific phenotypic diversity in L. polyedrum. The two studied strains differed distinctly in their light requirements and light acclimation ‘strategies’. For the study of intraspecific genetic diversity in L. polyedrum a novel method was developed that allowed the genotyping of individual cells. The application of this novel approach to natural populations showed that population genetic exchange of L. polyedrum in the Southern California Bight is tied to water circulation patterns and that both habitat structure and environmental change leave their signatures in the population genetic composition of L. polyedrum. This thesis represents one of the most comprehensive studies of dinoflagellate population ecology and builds the basis for the development of a holistic concept of the population ecology of L. polyedrum and other dinoflagellate species.

The interface between urban combinations and associated industry with fragile ecosystems delivering significant ecosystem services denotes one of the critical frontiers for ecological genomic investigation. The major issue when evaluating diffuse pollution generated at this interface revolves around the possible interactions between mixtures of contaminates that individually remain below trigger level but together may result in significant environmental impact. To determine whether mixture effects need to be considered, it is essential to define geochemical parameters by performing a survey for major classes of contaminates and to evaluate their penetrance into the food chain. The coastal marine environment of the Saudi Red Sea is subject to direct and indirect influences of major populations and industrial facilities found along the coast such as those discovered in proximity to Jeddah City in Saudi Arabia. Sampling of both sediment and sea water was performed at contrasting sites representative of near-shore with off-shore locations. Possible food-chain transference of any contaminates was evaluated by sampling fish (L.nebulosus) and plankton at the off-shore sites. Biomarkers are mostly useful in the evaluation of progressive diseases that apparent their symptoms long after exposure to the initiating factor. In such cases, traditional early warning symptoms of developing disease may be lacking. Thus, detection of earlier events can provide a valuable timely warning of risk. It is important to identify and address the growing environmental problems being faced by the community and address it before it takes the shape of an epidemic. To assess toxicity of single and paired metals to the nematode C. elegans, toxicity tests were designed to first determine the impact of single metal exposure Copper, Zinc and Aluminium and then nematodes were exposed to paired combinations. Exposures with paired metals showed a variety of interactions which ranged from antagonistic to synergistic effects.

Phytoplankton across the majority of the world’s oceans are thought to be limited by the availability of either nitrate or iron (Fe). However, the spatial resolution of experiments confirming this is low. Two thesis chapters present the results of bottle enrichment experiments at high spatial resolution across (i) the South Subtropical Convergence (SSTC) in the South Atlantic, and (ii) the Scotia Sea-Drake Passage sector of the Southern Ocean. These studies have added detail to the boundaries of limiting nutrients in these regions. Patterns of Fast Repetition Rate fluorometry (FRRf) derived parameters, physiological regulation of these parameters including influences of community structure, and the environmental controls driving them are analysed. Given its role as an essential micronutrient, there has been much effort in constraining potential sources of bioavailable Fe to the ocean, with one such source receiving recent interest: erupted ash from volcanoes. Bottle-scale ash-incubation experiments alongside conventional iron additions and laboratory ash-leaching experiments were conducted, the results of which suggest phytoplankton would respond strongly to ash deposition in the High Nitrate, Low Chlorophyll (HNLC) areas of the Southern Ocean. Particularly notable was the evidence these experiments provided for potential (co-)limitation of phytoplankton in these waters by the micronutrient manganese. The first three chapters of this thesis highlight a number of biogeochemical implications of trace metal stress, particularly that of Fe stress. Therefore, the ability to map the oceanographic extent of Fe-stressed regions using remote sensing would represent a particularly useful advance in marine biogeochemistry. Theoretically it could be possible to map Fe stress from space using satellite images of chlorophyll fluorescence, yet there are important uncertainties that need to be addressed before this can be carried out. In particular, a better understanding of the midday non-photochemical quenching driven reductions in chlorophyll fluorescence occurring at the time satellite images are captured is required. Analysis of over 200 non-photochemical quenching experiments collected over three research cruises, has allowed us to explore non-photochemical quenching and its relevance for using sunlight induced chlorophyll fluorescence to assess broad patterns of Fe stress. Our results have confirmed that satellite fluorescence quantum yields have the potential to reveal broad regions of Fe stress, however a dynamic non-photochemical quenching correction derived from our experiments and analysis was necessary to achieve this.

Brown algae have been the focus of metal ecotoxicology research for over 60 years, mainly because of their high metal accumulation capacity and reputed resistance. Now that Ectocarpus siliculosus has been positioned as a model for the study of brown algae, and that the genome has been recently sequenced and annotated, new lines of research have been made possible on these ecologically and economically important organisms, including the field of ecotoxicology. Several strains of E. siliculosus have been collected and isolated from locations around the world, thus providing the opportunity to study inter-population differences in their responses to environmental stress. This investigation can be split into three main sections. In the first part Cu exposure experiments were carried out under laboratory conditions using three strains of E. siliculosus: Es524 from a Cu polluted location in Chile, REP10-11 from a metal polluted (including Cu) location in England and LIA4A from a pristine site in Scotland. Strains were exposed for 10 d to concentrations ranging between 0 and 2.4 μM Cu. We measured different parameters: relative growth rates; metal accumulation (extracellular and intracellular); phytochelatins and the expression of related enzymes; oxidative stress responses as manifested in lipid peroxidation and levels of H2O2, and levels of pigments; levels of antioxidants glutathione and ascorbate (in reduced and oxidised forms), and phenolic compounds; and the activity of the antioxidant enzymes superoxide dismutase, catalase, and ascorbate peroxidise. Strain Es524 was the most efficient in counteracting the effects of Cu stress as manifested by a combination of Cu exclusion production of metal chelators, upregulation of oxidative enzymes, and strong antioxidant metabolism. REP10-11 also showed effective Cu defences, especially related to glutathione-ascorbate interactions. LIA4A was the least tolerant strain, with metabolic defences significantly less effective against Cu exposure. In part two a novel transplantation experiment was developed to compare responses in the field with those obtained in the laboratory. The study was carried out at a metal polluted and a low-impacted site in central Chile using strain Es524 (as in the laboratory experiments) and Es147, isolated from a low metal-polluted site in Chile. From the biomass, we conducted similar measurements of Reactive Oxygen Metabolism (ROM) as for the laboratory experiments described in the first part. In agreement with the laboratory experiments, strain Es524 displayed a higher resistance to metal stress. Because they behaved similarly between strains, the best suggested biomarker candidates for future assessments are metal accumulation, glutathione and ascorbate in reduced and oxidised forms, phenolic compounds, and the activity of superoxide dismutase. The method is simple, widely applicable in temperate environments, cost-effective, and provides a reliable representation of metal bioavailability in the environment. In the final part of the study a novel technique for the co-extraction of RNA and DNA, using a high pH Tris-HCl buffer, from small amounts of biomass of different strains of E. siliculosus was successfully developed. The extraction of nucleic acids from brown algae is considered to be difficult and the product is of poor quality due to the high concentrations of interfering secondary metabolites such as phenolics and polysaccharides. The protocol devised here provided high yields of pure RNA and DNA that are suitable for molecular analyses. This investigation provides new insights on metal stress metabolism in brown algae, and demonstrates that metal resistance is dependent on inherited defences developed over a long history of exposure. Furthermore, the good agreement between the results obtained in the laboratory with those from the field study confirms that the responses expressed under controlled laboratory conditions are representative of stress metabolism of E. siliculosus under natural conditions.

Brown algae are important primary producers and habitat formers in coastal environments and are believed to have evolved multicellularity independently of the other eukaryotes. The phaeoviruses that infect them form a stable lysogenic relationship with their host via genome integration, but have only been extensively studied in two genera: Ectocarpus and Feldmannia. In this study I aim to improve our understanding of the genetic diversity, host range and distribution of phaeoviruses. Sequencing and phylogenetic analysis of amplified fragments of three core phaeoviral genes (encoding major capsid protein (MCP), DNA polymerase and superfamily III helicase) of phaeovirus infected algae confirmed the suspected phaeoviral identity of viruses infecting E. fasciculatus, F. simplex, Pilayella littoralis, Myriotrichia clavaeformis and Hincksia hincksiae. Furthermore, this approach revealed multiple virus sequence variants within individual strains, and moreover that the variants formed two distinct subgroups. Subgroup A was highly conserved and observed in multiple algal genera, whereas subgroup B was much more diverse, but only found in Feldmannia species. Transcriptome sequencing of an actively infected F. irregularis strain revealed polymorphisms within key viral genes, suggesting that multiple variants were indeed active within this strain. High resolution melt curve (HRM) technology was used to develop a high throughput screening method for detecting phaeoviral MCP as a proxy for detection of phaeoviruses. This technique was also able to assign 88% of those detected to one of the subgroups, based on their differing melting temperature distributions. This was then applied to 1034 Ectocarpus isolates collected from around Europe and South America, and in accordance with previous studies of phaeoviral infection, 43-79% of strains contain virus sequence (depending on species). 17% of the isolates tested even contained sequence from both subgroups. 82 Laminariales strains, close relatives of the Ectocarpales, were also screened because they comprise commercially important kelp species but are not known to be infected by viruses. 10-17% of these tested positive for phaeoviral MCP, which when sequenced formed a separate group within the phaeoviruses. This finding could have a major impact on the kelp farming industry if the viruses are found to affect reproduction as happens in the Ectocarpales. The discovery of two subgroups is contrary to current beliefs that the phaeoviruses are a single monophyletic group, and that each species of alga has its own phaeovirus, casting doubt on the usefulness of the current convention of naming each phaeovirus after its host. It appears that the subgroup B viruses have begun to evolve away from the stable, K-selected subgroup A viruses towards a more r- type strategy with higher mutation and diversification. This study has identified potential mechanisms that may influence this shift, including mutations in a region of the DNA polymerase known to negatively affect DNA replication fidelity, combined with an active integrase and lack of a proofreading exonuclease, along with the observed infection of individuals with both phaeovirusal subgroups. The resulting mutations and recombinations could lead to the diversity observed here, and may provide a suitable model for the study of other emergent virus infections.

Small but mighty phytoplankton can be used as excellent model organisms to answer questions that are of importance to marine biologists and researchers in experimental evolution alike. For example, marine biologists are interested in finding out, how, in a changing ocean, the phytoplankton foundation of the ocean ecosystem is going to change - can we use short-term data to extrapolate to longer timescales? What are the physiological consequences of selection in stable and fluctuating high-pCO2 environments? From a more evolutionary perspective, is elevated pCO2 alone enough to drive evolution in marine algae? Can we select organisms to maintain plasticity in fluctuating environments, and how does selection in a fluctuating environment affect their ability to evolve? Can we detect a cost of plasticity? I have used theoretical and practical approaches from both disciplines to answer these questions, as they are ultimately similar questions that are important to address, and the lack of communication between disciplines has lead to conflicting predictions on the fate of populations in changing environments. Using evolutionary theory and applying it to an organism with a known function in the marine environment allows us to make ecologically relevant predictions while also enabling us to disentangle the underlying evolutionary mechanisms. While there have been some studies focusing on evolution of marine algae in climate change scenarios since I started my PhD, my study is the first to test the link between phenotypic plasticity and adaptation empirically, and it is also the first to use 16 rather than single or few genotypes of an algae, thereby creating the statistical power necessary to make any predictions. In a short-term CO2 enrichment study, and a selection experiment, those 16 physiologically and genetically distinct lineages of Ostreococcus, the smallest free living eukaryote, were selected for 400 generations in fluctuating and stable high pCO2 environments. I have shown that short-term plastic responses in phenotype can predict the magnitude of long-term evolutionary ones. Ostreococcus lineages in fluctuating environments evolve to be more plastic with no associated costs, and the adaptive response to selection in a high pCO2 environment is to grow more slowly in monoculture, but to be more successful competitors in mixed culture. High-pCO2 evolved lineages are genetically and physiologically different from their ancestors. Importantly, their quality as a food source for zooplankton will change, with possible repercussions for the ocean ecosystem at a whole. Furthermore, the lineages’ ability to perceive pCO2 levels in the surrounding medium is altered after evolution in fluctuating and high pCO2 environment, allowing them to broaden the window in which they can respond to changes in their environment without suffering metabolic stress.