The Alexandrium tamarense species complex is a group of economically and ecologically important marine dinoflagellates. The complex is comprised of three morphospecies A. tamarense, A. fundyense and A. catenella grouped according to ribosomal DNA or ‘ribotype’. There are five ribotype type groups (I-V) each consisting entirely of toxic or non-toxic isolates. Toxic isolates are associated with harmful algal blooms (HAB’s) due to their ability to produce powerful neurotoxins, which are responsible for outbreaks of paralytic shellfish poisoning in areas of shellfish production. Sexual reproduction in A. tamarense has important implications for the initiation and termination of HAB’s associated with these species. Resistant, long-lived hypnozygotes are formed during blooms through gamete fusion and deposited in sediments. These hypnozygotes provide the source of inoculum of motile vegetative A. tamarense cells in temperate zones during subsequent spring/summer blooms. This study provides further insight into the mating interactions between toxic Group I and non-toxic Group III isolates primarily from UK coastal waters. Study of the effect of temperature on the mating interactions of A. tamarense Group I and Group III in culture showed that temperature had a significant effect on both groups. Co-cultures of compatible Group III isolates showed a significant decrease (p<0.05) in hypnozygote yield at 15°C, compared to 20°C. In contrast the mating compatibility of co-cultures of Group I isolates showed significant increase (p<0.05) at 15°C, compared to 20°C. Similar to other studies, compatible Group I and Group III isolates formed non-viable hybrid hypnozygotes in co-culture. Comparison of the average vigour of inbred Group I crosses and outbred Group I/III crosses suggest that Group I isolates are more likely to out-breed with a compatible Group III isolate. A finding that may have significance in areas where the two groups co-occur. Preliminary data suggesting the presence of both Group I and Group III ribotypes in some isolates has been generated from a nested single cell PCR/qPCR protocol using group specific primers. These data were compared to a dual probe whole cell fluorescent in situ hybridisation (whole cell FISH) assay of isolates. Whole cell FISH showed no dual expression of ribosomal RNA. This suggests that some A. tamarense Group I and Group III isolates may have rDNA pseudogenes corresponding to different ribotypes. If correct this could have implications for the overestimation of A. tamarense group diversity in natural populations when using rDNA sequences for identification.

Fucales are brown macroalgae that play a relevant structural and functional role in marine ecosystems but are experiencing a huge decline in many areas of the Mediterranean Sea. Despite the long tradition of phycological studies in the Gulf of Naples (Italy), a gap of knowledge on their dynamics and current status has been highlighted for the last 60 years in this area. This thesis aimed to provide an overview on long-term changes in the distribution ad abundance of Cystoseira spp. and Sargassum spp. along different sectors of the Gulf of Naples in the bathymetric range between 0 and 50 m depth. Research was focused into three main objectives: 1) to assess the historical decline of Fucales the Gulf of Naples and their putative causes; 2) to map their current distribution; 3) to investigate the role of consumers in their loss. Results highlighted a decline of Cystoseira and Sargassum spp. mainly in the most urbanized areas of the gulf. Seven of the eighteen species recorded in the first half of the 20th century were no longer recorded; for other species the local decline could end up with their extinction if no action will be pursued for their conservation. A detailed georeferenced map (1:2,500) of Fucales has been elaborated for the shallow species; SCUBA surveys were performed for deep algae. Some human actions, such as coastal artificialization, can affect species richness and distribution in shallow sublittoral stands. Another mechanic pressure that can act on deep species is the intense fishing activities performed with fishing gears. Probably the interaction between the physic damage and the increase of water turbidity, related to a high eutrophication, can affect the vitality of deep species and their successful spread. The occurrence of patches of Cystoseira, among very dense and deep stands of Posidonia oceanica meadows in the oligotrophic waters around the island of Capri, seems to confirm this hypothesis. Another interesting result was obtained by a feeding choice experiment. The sea urchin Paracentrotus lividus fed indiscriminately on different macrophytes according to the equal choice behaviour between macrophytes offered alone or together, regardless of differences in total phenolic compounds content and C:N ratio. In conclusion, results infer that the improvement and maintenance of the environmental quality of the Gulf of Naples is a major issue not only for the welfare of the coastal ecosystems, but also for its social and economic spin off.

Macroalgae are useful as model organisms because their simple physiology and modular growth allows their investments into growth, defence, and reproduction to be quantified. Ascophyllum nodosum is a dioecious perennial which responds clearly to environmental stressors, and Sargassum muticum is an invasive pseudo-annual which grows in discrete populations of known time-since-invasion. Two chapters investigate the occurrence of sexual dimorphism in A. nodosum under stress, and incorporate surveys, chemical analyses, and feeding trials to demonstrate that females invest more into sexual reproduction at sites where stressors impact juvenile mortality, and compensate by reducing investment into defence, leaving them more vulnerable to grazers. At sites where stressors impact mainly on adult mortality and performance with lower influence on juvenile mortality, limited sexual dimorphism is observed. Similar results have been demonstrated in terrestrial plants, but none have quantified responses to stress in such detail. The second two chapters test the Enemy Release Hypothesis, the Evolution of Increased Competitive Ability hypotheses, and the Biotic Resistance hypothesis using surveys, chemical analyses, and feeding trials with S. muticum and its associated grazers from populations established for different lengths of time, over 40 years. Grazers from older S. muticum were more likely to feed upon it in the laboratory, but surveys of chemical defences did not reflect any increased pressure over time in the field. Instead, S. muticum increased its defensive investment in the presence of a greater diversity of grazers, irrespective of the length of time those grazers had been exposed to it. Therefore although grazers can learn to consume S. muticum, they are still unlikely to do so in the field, such that many species must be present before at least some begin to impart top-down pressure upon it. Collectively these investigations demonstrate the high value of using macroalgae in wider ecology.

The optimization of fixation protocols was undertaken for Dunaliella salina, Nannochloropsis oculata and Pseudostaurosira trainorii to investigate two different aspects of microalgal biology. The first was to evaluate the effects of the infochemical 2, 4-decadienal as a potential lipid inducer in two promising lipid-producing species, Dunaliella salina and Nannochloropsis oculata, for biofuel production. D. salina fixed well using 1% glutaraldehyde in 0.5 M cacodylate buffer prepared in F/2 medium followed by secondary fixation with 1% osmium tetroxide. N. oculata fixed better with combined osmium-glutaraldehyde prepared in sea water and sucrose. A stereological measuring technique was used to compare lipid volume fractions in D. salina cells treated with 0, 2.5, and 50 μM and N. oculata treated with 0, 1, 10, and 50 μM with the lipid volume fraction of naturally senescent (stationary) cultures. There were significant increases in the volume fractions of lipid bodies in both D. salina (0.72%) and N. oculata (3.4%) decadienal-treated cells. However, the volume fractions of lipid bodies of the stationary phase cells were 7.1% for D. salina and 28% for N. oculata. Therefore, decadienal would not be a suitable lipid inducer for a cost-effective biofuel plant. Moreover, cells treated with the highest concentration of decadienal showed signs of programmed cell death. This would affect biomass accumulation in the biofuel plant, thus further reducing cost effectiveness. The second investigation used the cell structure and gametogenesis of Pseudostaurosira trainorii to evaluate the cytoplasmic support for the molecular phylogeny of diatoms proposed by Medlin and Kaczmarska (2004) which divides diatoms into three clades that differ in their ultrastructure, where Clade 1 contains centric diatoms, Clade 2a centric bipolar and multipolar diatoms and Clade 2b pennate diatoms. P. trainorii is classified as araphid diatom which has a thread-like structure reported by Sato et al. (2011) that could resemble the flagella in centric diatoms. P. trainorii fixed better with combined osmium-glutaraldehyde prepared in sea water and sucrose, and it was found that its Golgi apparatus and auxospore were resemble those of Clade 2b, while the pyrenoid structure might represent a variation of the structures described for other members of Clade 2b. In addition, this study proved that the thread-like structures in P. trainorii were not flagella.

Algal and cyanobacterial1 blooms are increasingly becoming a problem to water utilities in the UK and around the world. Conditions such as increasing eutrophication and poor management of water bodies are consequently leading to increases in the number and severity of algal bloom events. These blooms present serious issues to both utilities and the environment; problems include deoxygentation of water bodies leading to fish kill and rapid algal growth leads to overgrowing of other plant species in the water, while blooms of toxic cyanobacteria can lead to the closure of reservoirs and a number of algae can lead to pump and filter blockages in treatment works. The problems created by algal and cyanobacterial blooms are becoming increasingly expensive to offset and while some technologies such as dissolved air flotation exist to control them at treatment level, there are few effective options to tackle blooms where control would be most effective, at the source. What methods there are for source control are often unreliable or almost as bad for the environment as the blooms themselves such as, in the use of chemical pesticides. Barley straw has been shown to have to potential to be an effective control of algal and cyanobacterial blooms at the reservoir level, being able to inhibit a variety of species at practical straw concentrations, but little is known about how it functions or how its ‘effect’ is developed. This study identified spcific areas which when investigated provided insight into these gaps in current knowledge. An analysis of the chemicals proposed as released by decaying barley straw has shown that they can control algal blooms at concentrations similar to what has been detected in the field. Investigations into the decay of barley straw has shown that barley straw decays in water in a way consistent with species that breakdown the lignin section of the straw potentially releasing the proposed chemicals. This finding was supported by the effect being consistent when field rotted straw, fresh straw rotted in the lab, and straw cultured in the lab with species specifically adapted to breaking down the lignin section of the straw were compared. Attempts at pre-treating the straw have shown that microbiological activity is important in developing the algistatic effect, which could lead to the possibility of keeping straw ‘ready for use’ thus removing the current need of several months wait before barley becomes effective. These findings have also provided a definitive route by which barley straw develops its effect, namely, microbiological decay of the lignin fraction of the straw leading to the production of phenol chemical release.

The applicability of HPLC and PCR-DGGE was tested on water samples from Loch Rannoch during a spatio-temporal assessment of the plankton communities. The deep oligotrophic lake, which stratified over summer, contained a low phytoplankton biomass, as revealed by microscopy and HPLC. Variations in the phytoplankton data were related to changes in light, temperature and nutrients. Spatial heterogeneity in the BCCs across and down the water column was mainly associated with stratification and dissolved organic carbon. An assessment of changes in plankton communities was made fortnightly in mesotrophic Loch Calder. Major peaks in cyanobacteria and diatoms occurred in summer 2009 and spring 2010 respectively, with lower phytoplankton biomasses noted over autumn and winter. Aspects of plankton eco-physiology were assessed by the quantification of pigment degradation products and carotenoids, which showed evidence of photo-protection, grazing and senescence. Bacterioplankton density was positively correlated to phytoplankton biomass and BCC appeared to shift in accordance with transitions in ecological seasons. A geospatial study of 20 Highland lakes revealed a diverse range of lake types. The pigment profiles corroborated their respective microscopy data, with lakes with pronounced differences in pH having distinct phytoplankton compositions. BEPM and BFC in Loch Calder and the Highland lakes showed some discrepancies which could be explained by factors such as sample processing and the sensitivities of the respective techniques. The presence of at least 60 dominant bacterioplankton taxa in the northern Highland lakes showed that diversity in Scottish lakes was comparable to that in some northern European lakes. This work is the first to demonstrate the applicability of HPLC and PCR-DGGE as viable tools for the assessment of biomass, composition and diversity of phytoplankton and bacterioplankton in Scottish freshwater lakes. Furthermore, the study highlights the potential of the pigment data in assessing the ecological status of the freshwater lakes.

The nature and mechanisms of adaptive changes in the fatty acid composition of membrane glycerolipids in laboratory cultures of the marine cyanobacterium <I>Aphanizomenon </I>sp. induced by temperature transitions similar to those experienced by natural population of <I>Aphanizomenon</I> in the Baltic Sea were investigated. At 28° palmitic acid (16:0) predominated (35%) over α-linolenic acid (α-18:3; 23%), whilst in cells grown at 15°, α-18:3 (38%) predominated over 16:0 (25%). Changes in fatty acid composition of the major glycerolipids monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG0, sulfoquinovosyldiacylglycerol (SL) and phosphatidylglycerol (PG) were studied after temperature shifts from 28° to 15° and from 15° to 28°. After lowering the temperature, the level of 16:0 and 18:1 decreased in MGDG, DGDG and SL fractions, whilst the content of 16:3 and α-18:3 increased. In PG however the level of 16:1 and 18:1 increased whilst the level of α-18:3 decreased. After a temperature shift from 15° to 28° the level of 16:0 remained unchanged in MGDG and SL whilst α-18:3 decreased and 16:1 increased. Determination of the positional distribution of fatty acids present in the individual glycerolipid classes using <I>Rhizopus</I> lipase hydrolysis established the predominance of C-18 fatty acids at the <I>sn-</I>1 position and C-16 fatty acids at the <I>sn</I>-2 position; during the adaptation to lower temperature the most prominent change at the <I>sn-</I>1 position was an increase in 18:1, 18:3 and 16:3 levels in MGDG.

The green seaweed, Ulva, is highly valued in terms of animal feed, food and biofuel, as well in the delivery of crucial remediation services including wastewater treatment and CO2 removal. Accordingly, Ulva cultivation has gained significant research interest worldwide. Notwithstanding these research efforts, Ulva cultivation is still in its infancy and knowledge to underpin such developments remains limited. A common challenge in Ulva cultivation is the fluctuating productivity with time due to vegetative fragmentation and/or periodic reproduction. In this study, three methods were employed to address this challenge. Firstly, culture conditions were optimised to establish a balance between growth and reproduction. Secondly, a refined culture method was developed, which more than tripled growth of Ulva over an 18-day cultivation as compared to a standard method. Thirdly, a sterile strain was obtained by mutating a wild strain with ultraviolet radiation. This new strain grew five times faster over an 18-day cultivation and absorbed nitrate and phosphate 40.0% and 30.9% quicker compared to the wild strain respectively. The chemical composition of the sterile strain showed a lipid content of more than double that of the wild strain, while the protein content was 26.3% lower than the wild strain. Several tissue preservation techniques were developed to enable settlement and growth trials to be conducted on demand. The merits or otherwise of the preservation techniques were determined for gametes, germlings and thalli. In addition to cultivation-related techniques, the co-effects of climate change factors (global warming and ocean acidification) and eutrophication on Ulva cultivation were investigated. These three variables interacted in a complex pattern to differentially affect life history stages, as well as altering the chemical composition and functional properties of Ulva. These findings make tangible contributions to the ability to successfully and commercially cultivate Ulva in terms of culture conditions, tissue preservation and the development of mutant strains. Further, by placing Ulva culture in a climate change context, this work provides valuable insight into the limits to resilience of Ulva to a changing climate. This will inform the future development of the Ulva culture industry over the coming decades.

No description available.

Sexual reproduction is a fundamental phase in the life cycle of diatoms, linked to the production of genotypic diversity and the formation of large-sized initial cells that ensure population persistence. It occurs only within cells below a certain threshold size and, in heterothallic diatoms, only between strains of opposite mating types. We aim at identifying genes involved in mating type determination in the manne planktonic diatom Pseudo-nitzschia multistriata. This species is recorded in coastal waters worldwide and produces the neurotoxin domoic acid. A reference genome has been generated and transcriptomes have been produced for strains of opposite mating type (MT + and MT-). Differential expression analysis provided a list of candidate MT -biased genes validated with qPCR. Four MT-biased genes were identified, two in MT+ and two in MT-. The expression pattern of the candidate genes was followed in a 24 hours' time course experiment to verify whether they were regulated in dependence of light or cell cycle phases. Experimental evidences demonstrated their involvement during mating recognition in early stages of sexual reproduction while preliminary genetic analyses excluded that they could be the master gene responsible for mating type determination. The description of the four genes was improved through computational characterization to understand their role in the chemical communication occurring between opposite mating types. A further step towards the identification of the MT locus will include a Bulked Segregant Analysis applied to a library of 30 MT+ and 30 MT- Fl strains obtained through DNA deep sequencing. Elucidating the molecular and genetic basis of MT determination and sexual reproduction in diatoms will contribute to a better understanding of the regulation and evolution of their life cycles and reproductive strategies. Results from this study could also provide molecular markers to trace the distribution of MT + and MT - cells in environmental samples.