Intergenic Regions and Repeating Gene Sequences in the Dinoflagellate \u3ci\u3eKarenia brevis\u3c/i\u3e

Karenia brevis is a species of dinoflagellates that is responsible for “red tides” (more formally known as harmful algal blooms) found in the Gulf of Mexico. The blooms have adverse effects on humans and marine animals. Karenia brevis produces toxins that, in humans, can cause upper and lower respiratory problems as well as nausea and vomiting. For marine mammals, birds, fish, and other marine organisms, exposure to brevetoxins can result in morbidity and even death. Even with the technology and resources we have today, scientist do not fully understand the mechanisms behind toxin production and bloom initiation. Because cellular processes may play a major role in Karenia brevis blooms, attempts have been made to understand K. brevis at the molecular level, but studies are still ongoing due to lack of understanding gene organization and expression. Thus, our goal was to help guide the reconstruction or sequencing of the genome. The purpose of my research was to find if there are multiple copies of certain gene sequences, and what variability exists between and within the genes. I attempted determine the copy number of a suspected multi-copy gene and if any sequence variability existed within the gene and the intergenic regions. Focus was placed on the gene for proliferating cell nuclear antigen (PCNA) whose known function is to aid in DNA replication and repair, because the PCNA is present in multiple copies and (Zhang et al. 2006) and the EST collection of K. brevis contained several PCNA single nucleotide polymorphisms (Lidie et al, 2005; McLean, unpublished data). Techniques involved were DNA extraction, PCR (polymerase chain reaction) primer design, PCR amplification of genic and intergenic regions of the chromosomes, and PCR purification and ligation. Research stopped at this point due to time constraints. Southern blotting (to quantify the number of copies) was to be performed and then the PCRs products sent to another lab to be sequenced. Simple bioinformatic gene sequence analysis would have determine if there were base changes that could result in changes in amino acids. Intergenic sequence analysis would have determined if each copy of a gene was capable of being expressed or regulated. Results of further research could advance understanding of the genetics of K. brevis and dinoflagellates in general. Additionally, results could shape further predictions on mutation rates and variability in the dinoflagellate genome

Molecular Mechanisms Regulating Chronological Aging and Cell Death in the Toxic Dinoflagellate, Karenia brevis

The toxic dinoflagellate, Karenia brevis, forms nearly annual blooms in the Gulf of Mexico that persist for many months in coastal waters, causing extensive marine animal mortalities and human health impacts. The molecular mechanisms that contribute to cell survival in high density, low growth blooms, and the mechanisms leading to often rapid bloom demise are not well understood. The studies presented in this dissertation investigate the existence and involvement of a programmed cell death-like (PCD-like) pathway in the demise of K. brevis cultures following oxidative stress and chronological aging. Firstly, to gain an understanding of the molecular processes that underlie chronological aging in this dinoflagellate, a microarray study was carried out and identified extensive transcriptomic remodeling during the transition into stationary phase indicative of a shift in the metabolic and signaling requirements for survival in a quiescent non-dividing phase. To better understand the connection between the transcriptomic context identified in the microarray study and the presence of a PCO-like pathway in K. brevis, hallmark morphological and biochemical changes (DNA fragmentation, caspase-like activity, and caspase 3-like protein expression) were used to define PCD-like morphological changes following chronological aging and oxidative stress. Targeted in silico bioinformatic mining was used to identify enzymes potentially responsible for the activities observed, as well as the substrates. Finally, K. brevis S-adenosylmethionine synthetase (KbAdoMetS), a putative caspase substrate predicted from the bioinformatics screen, was examined using MALDI-TOF MS to confirm the validity of the bioinformatics approach. Taken together, this work identified that K. brevis contains morphological changes indicative of a caspase-dependent PCD-like pathway and that KbAdoMetS is a caspase 3-like substrate. Finally, we sought to characterize the presence of metacaspases in Karenia brevis, and specifically evaluated the role of metacaspase 1 (KbMC1) during chronological aging and death in culture. Immunocytochemistry, subcellular fractionation, and western blotting results using a custom KbMC1 peptide antibody indicate that KbMC1 may be involved in PCD-like execution through its chloroplastic localization with proposed interactions with the photosynthetic machinery. This study provides the first comprehensive investigation of the molecular processes regulating chronological aging and execution of PCD-like death in a toxic dinoflagellate

The effects of the red tide producing dinoflagellate, Karenia brevis, and associated brevetoxins on viability and sublethal stress responses in scleractinian coral: a potential regional stressor to coral reefs

Coral cover is in decline on a global scale, with increased mortality events being attributed to a number of global and regional stressors. While the impacts of global stressors (e.g. sea surface temperature rise, ocean acidification) are well documented, there is growing interest in identifying and understanding the impacts of regional stressors. The reason for this change in focus is that regional stressors can often work in combination, sometimes synergistically, with global stressors and that stressors on a regional scale tend to be more easily mitigated by management practices. One regional stressor that impacts a myriad of marine organisms in the southeastern United States is the annual red tide blooms produced by the dinoflagellate, Karenia brevis. Their impacts, along with the lipid soluble polyether neurotoxins they produce, termed brevetoxins, are well studied in economically important organisms, such as bivalves. However, little is known of their impacts on organisms that possess ecological importance, such as species of scleractinian coral. To address this gap in knowledge, a multifaceted study is discussed herein, which evaluated the effects of ecologically relevant concentrations of K. brevis and associated brevetoxins on different coral life history stages and coral species. The second chapter addresses the impacts of red tide on larval behavior, settlement and survival of the coral species Porites astreoides, as well as impacts of photochemical efficiency and oxidative stress within different coral species (P. astreoides larvae, P. astreoides adults, Acropora cervicornis, Cladocora arbuscula, and Phyllangia americana). The third chapter confers the use of broad-scale proteomic analysis to identify the cellular response of the non-model coral species, P. astreoides, following exposure to red tide. Coral larvae actively avoided both medium and high bloom conditions of K. brevis and brevetoxins, while percent larval settlement and survival were impacted following exposure to high bloom concentrations of K. brevis. Photochemical efficiency of in hospite Symbiodinium was reduced following exposure to both K. brevis and brevetoxin in P. astreoides larvae, as well as exposure to K. brevis in P. astreoides adults, while being unimpacted in A. cervicornis. Compared to controls, high bloom conditions resulted in an increase in biomarkers of lipid peroxidation in C. arbuscula. This was also seen in P. astreoides larvae at 24 hours; however, this difference was indistinguishable following 48 hours. Surprisingly, no other biomarker of oxidative stress analyzed were impacted. Broad-scale proteomic analysis of P. astreoides following exposure to red tide conditions revealed variable changes in proteome expression depending on if the corals were exposed to K. brevis or brevetoxins. Exposure to brevetoxins resulted in differential expression of proteins related to DNA organization, chromatin formation and transcription expression; while exposure to K. brevis resulted in differential expression of proteins related to redox homeostasis, protein folding, energy metabolism, and production of reactive oxygen species. The results of this study demonstrate the potential for annual red tide blooms to act as a regional stressor on coral species. They highlight the ability of red tide conditions to negatively impact coral at multiple life history stages and that the extent of these effects may be species specific. They also provide further incite of coral’s response to red tide exposure at the cellular level

Transcriptomic characterisation and genomic glimps into the toxigenic dinoflagellate Azadinium spinosum, with emphasis on polykeitde synthase genes

BACKGROUND: Unicellular dinoflagellates are an important group of primary producers within the marine plankton community. Many of these species are capable of forming harmful algae blooms (HABs) and of producing potent phycotoxins, thereby causing deleterious impacts on their environment and posing a threat to human health. The recently discovered toxigenic dinoflagellate Azadinium spinosum is known to produce azaspiracid toxins. These toxins are most likely produced by polyketide synthases (PKS). Recently, PKS I-like transcripts have been identified in a number of dinoflagellate species. Despite the global distribution of A. spinosum, little is known about molecular features. In this study, we investigate the genomic and transcriptomic features of A. spinosum with a focus on polyketide synthesis and PKS evolution. RESULTS: We identify orphan and homologous genes by comparing the transcriptome data of A. spinosum with a diverse set of 18 other dinoflagellates, five further species out of the Rhizaria Alveolate Stramelopile (RAS)-group, and one representative from the Plantae. The number of orphan genes in the analysed dinoflagellate species averaged 27%. In contrast, within the A. spinosum transcriptome, we discovered 12,661 orphan transcripts (18%). The dinoflagellates toxins known as azaspiracids (AZAs) are structurally polyethers; we therefore analyse the transcriptome of A. spinosum with respect to polyketide synthases (PKSs), the primary biosynthetic enzymes in polyketide synthesis. We find all the genes thought to be potentially essential for polyketide toxin synthesis to be expressed in A. spinosum, whose PKS transcripts fall into the dinoflagellate sub-clade in PKS evolution. CONCLUSIONS: Overall, we demonstrate that the number of orphan genes in the A. spinosum genome is relatively small compared to other dinoflagellate species. In addition, all PKS domains needed to produce the azaspiracid carbon backbone are present in A. spinosum. Our study underscores the extraordinary evolution of such gene clusters and, in particular, supports the proposed structural and functional paradigm for PKS Type I genes in dinoflagellates

The Relevance of Marine Chemical Ecology to Plankton and Ecosystem Function: An Emerging Field

Marine chemical ecology comprises the study of the production and interaction of bioactive molecules affecting organism behavior and function. Here we focus on bioactive compounds and interactions associated with phytoplankton, particularly bloom-forming diatoms, prymnesiophytes and dinoflagellates. Planktonic bioactive metabolites are structurally and functionally diverse and some may have multiple simultaneous functions including roles in chemical defense (antipredator, allelopathic and antibacterial compounds), and/or cell-to-cell signaling (e.g., polyunsaturated aldehydes (PUAs) of diatoms). Among inducible chemical defenses in response to grazing, there is high species-specific variability in the effects on grazers, ranging from severe physical incapacitation and/or death to no apparent physiological response, depending on predator susceptibility and detoxification capability. Most bioactive compounds are present in very low concentrations, in both the producing organism and the surrounding aqueous medium. Furthermore, bioactivity may be subject to synergistic interactions with other natural and anthropogenic environmental toxicants. Most, if not all phycotoxins are classic secondary metabolites, but many other bioactive metabolites are simple molecules derived from primary metabolism (e.g., PUAs in diatoms, dimethylsulfoniopropionate (DMSP) in prymnesiophytes). Producing cells do not seem to suffer physiological impact due to their synthesis. Functional genome sequence data and gene expression analysis will provide insights into regulatory and metabolic pathways in producer organisms, as well as identification of mechanisms of action in target organisms. Understanding chemical ecological responses to environmental triggers and chemically-mediated species interactions will help define crucial chemical and molecular processes that help maintain biodiversity and ecosystem functionality

The Relevance of Marine Chemical Ecology to Plankton and Ecosystem Function: An Emerging Field

Marine chemical ecology comprises the study of the production and interaction of bioactive molecules affecting organism behavior and function. Here we focus on bioactive compounds and interactions associated with phytoplankton, particularly bloom-forming diatoms, prymnesiophytes and dinoflagellates. Planktonic bioactive metabolites are structurally and functionally diverse and some may have multiple simultaneous functions including roles in chemical defense (antipredator, allelopathic and antibacterial compounds), and/or cell-to-cell signaling (e.g., polyunsaturated aldehydes (PUAs) of diatoms). Among inducible chemical defenses in response to grazing, there is high species-specific variability in the effects on grazers, ranging from severe physical incapacitation and/or death to no apparent physiological response, depending on predator susceptibility and detoxification capability. Most bioactive compounds are present in very low concentrations, in both the producing organism and the surrounding aqueous medium. Furthermore, bioactivity may be subject to synergistic interactions with other natural and anthropogenic environmental toxicants. Most, if not all phycotoxins are classic secondary metabolites, but many other bioactive metabolites are simple molecules derived from primary metabolism (e.g., PUAs in diatoms, dimethylsulfoniopropionate (DMSP) in prymnesiophytes). Producing cells do not seem to suffer physiological impact due to their synthesis. Functional genome sequence data and gene expression analysis will provide insights into regulatory and metabolic pathways in producer organisms, as well as identification of mechanisms of action in target organisms. Understanding chemical ecological responses to environmental triggers and chemically-mediated species interactions will help define crucial chemical and molecular processes that help maintain biodiversity and ecosystem functionality

Molecular techniques for investigating toxic dinoflagellate species in the western English Channel, UK and in Bahrain coastal waters of the Arabian Gulf

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Transcriptomic and Epigenetic Responses to Environmental Stress in Marine Bivalves with a Focus on Harmful Algal Blooms

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

Spliced Leader RNAs, Mitochondrial Gene Frameshifts and Multi-Protein Phylogeny Expand Support for the Genus Perkinsus as a Unique Group of Alveolates

The genus Perkinsus occupies a precarious phylogenetic position. To gain a better understanding of the relationship between perkinsids, dinoflagellates and other alveolates, we analyzed the nuclear-encoded spliced-leader (SL) RNA and mitochondrial genes, intron prevalence, and multi-protein phylogenies. In contrast to the canonical 22-nt SL found in dinoflagellates (DinoSL), P. marinus has a shorter (21-nt) and a longer (22-nt) SL with slightly different sequences than DinoSL. The major SL RNA transcripts range in size between 80–83 nt in P. marinus, and ∼83 nt in P. chesapeaki, significantly larger than the typical ≤56-nt dinoflagellate SL RNA. In most of the phylogenetic trees based on 41 predicted protein sequences, P. marinus branched at the base of the dinoflagellate clade that included the ancient taxa Oxyrrhis and Amoebophrya, sister to the clade of apicomplexans, and in some cases clustered with apicomplexans as a sister to the dinoflagellate clade. Of 104 Perkinsus spp. genes examined 69.2% had introns, a higher intron prevalence than in dinoflagellates. Examination of Perkinsus spp. mitochondrial cytochrome B and cytochrome C oxidase subunit I genes and their cDNAs revealed no mRNA editing, but these transcripts can only be translated when frameshifts are introduced at every AGG and CCC codon as if AGGY codes for glycine and CCCCU for proline. These results, along with the presence of the numerous uncharacterized ‘marine alveolate group I' and Perkinsus-like lineages separating perkinsids from core dinoflagellates, expand support for the affiliation of the genus Perkinsus with an independent lineage (Perkinsozoa) positioned between the phyla of Apicomplexa and Dinoflagellata