Global transcriptional profiling of the toxic dinoflagellate Alexandrium fundyense using Massively Parallel Signature Sequencing

BACKGROUND: Dinoflagellates are one of the most important classes of marine and freshwater algae, notable both for their functional diversity and ecological significance. They occur naturally as free-living cells, as endosymbionts of marine invertebrates and are well known for their involvement in "red tides". Dinoflagellates are also notable for their unusual genome content and structure, which suggests that the organization and regulation of dinoflagellate genes may be very different from that of most eukaryotes. To investigate the content and regulation of the dinoflagellate genome, we performed a global analysis of the transcriptome of the toxic dinoflagellate Alexandrium fundyense under nitrate- and phosphate-limited conditions using Massively Parallel Signature Sequencing (MPSS). RESULTS: Data from the two MPSS libraries showed that the number of unique signatures found in A. fundyense cells is similar to that of humans and Arabidopsis thaliana, two eukaryotes that have been extensively analyzed using this method. The general distribution, abundance and expression patterns of the A. fundyense signatures were also quite similar to other eukaryotes, and at least 10% of the A. fundyense signatures were differentially expressed between the two conditions. RACE amplification and sequencing of a subset of signatures showed that multiple signatures arose from sequence variants of a single gene. Single signatures also mapped to different sequence variants of the same gene. CONCLUSION: The MPSS data presented here provide a quantitative view of the transcriptome and its regulation in these unusual single-celled eukaryotes. The observed signature abundance and distribution in Alexandrium is similar to that of other eukaryotes that have been analyzed using MPSS. Results of signature mapping via RACE indicate that many signatures result from sequence variants of individual genes. These data add to the growing body of evidence for widespread gene duplication in dinoflagellates, which would contribute to the transcriptional complexity of these organisms. The MPSS data also demonstrate that a significant number of dinoflagellate mRNAs are transcriptionally regulated, indicating that dinoflagellates commonly employ transcriptional gene regulation along with the post-transcriptional regulation that has been well documented in these organisms

The Phytoplankton Taxon-Dependent Oil Response and Its Microbiome: Correlation but Not Causation

Phytoplankton strongly interact with their associated bacteria, both attached (PA), and free-living (FL), and bacterial community structures can be specific to phytoplankton species. Similarly, responses to environmental stressors can vary by taxon, as exemplified by observed shifts in phytoplankton community structure from diatoms to phytoflagellates after the Deepwater Horizon (DWH) oil spill. Here, we assess the extent to which associated bacteria influence the phytoplankton taxon-specific oil response by exposing xenic and axenic strains of three phytoplankton species to oil and/or dispersant. The dinoflagellates Amphidinium carterae and Peridinium sociale, and the diatom Skeletonema sp., all harbored significantly distinct bacterial communities that reflected their host oil response. Oil degrading bacteria were detected in both PA and FL communities of the oil resistant dinoflagellates, but their FL bacteria were more efficient in lipid hydrolysis, a proxy for oil degradation capability. Inversely, the growth rate and photosynthetic parameters of the diatom Skeletonema sp. was the most impacted by dispersed oil compared to the dinoflagellates, and oil-degrading bacteria were not significantly associated to its microbiome, even in the dispersed oil treatment. Moreover, the FL bacteria of Skeletonema did not show significant oil degradation. Yet, the lack of consistent significant differences in growth or photosynthetic parameters between the xenic and axenic cultures after oil exposure suggest that, physiologically, the associated bacteria do not modify the phytoplankton oil response. Instead, both oil resistance and phycosphere composition appear to be species-specific characteristics that are not causally linked. This study explores one aspect of what is undoubtedly a complex suite of interactions between phytoplankton and their associated bacteria; future analyses would benefit from studies of genes and metabolites that mediate algal-bacterial exchanges

Natural Sunlight Shapes Crude Oil-Degrading Bacterial Communities in Northern Gulf of Mexico Surface Waters

Following the Deepwater Horizon (DWH) spill in 2010, an enormous amount of oil was observed in the deep and surface waters of the northern Gulf of Mexico. Surface waters are characterized by intense sunlight and high temperature during summer. While the oil-degrading bacterial communities in the deep-sea plume have been widely investigated, the effect of natural sunlight on those in oil polluted surface waters remains unexplored to date. In this study, we incubated surface water from the DWH site with amendments of crude oil, Corexit dispersant, or both for 36 d under natural sunlight in the northern Gulf of Mexico. The bacterial community was analyzed over time for total abundance, density of alkane and polycyclic aromatic hydrocarbon degraders, and community composition via pyrosequencing. Our results showed that, for treatments with oil and/or Corexit, sunlight significantly reduced bacterial diversity and evenness and was a key driver of shifts in bacterial community structure. In samples containing oil or dispersant, sunlight greatly reduced abundance of the Cyanobacterium Synechococcus but increased the relative abundances of Alteromonas, Marinobacter, Labrenzia, Sandarakinotalea, Bartonella, and Halomonas. Dark samples with oil were represented by members of Thalassobius, Winogradskyella, Alcanivorax, Formosa, Pseudomonas, Eubacterium, Erythrobacter, Natronocella, and Coxiella. Both oil and Corexit inhibited the Candidatus Pelagibacter with or without sunlight exposure. For the first time, we demonstrated the effects of light in structuring microbial communities in water with oil and/or Corexit. Overall, our findings improve understanding of oil pollution in surface water, and provide unequivocal evidence that sunlight is a key factor in determining bacterial community composition and dynamics in oil polluted marine waters

Insights into the loss factors of phytoplankton blooms : the role of cell mortality in the decline of two inshore Alexandrium blooms

Author Posting. © The Author(s), 2017. This is the author's version of the work. It is posted here under a nonexclusive, irrevocable, paid-up, worldwide license granted to WHOI. It is made available for personal use, not for redistribution. The definitive version was published in Limnology and Oceanography 62 (2017): 1742–1753, doi:10.1002/lno.10530.While considerable effort has been devoted to understanding the factors regulating the development of phytoplankton blooms, the mechanisms leading to bloom decline and termination have received less attention. Grazing and sedimentation have been invoked as the main routes for the loss of phytoplankton biomass, and more recently, viral lysis, parasitism and programmed cell death (PCD) have been recognized as additional removal factors. Despite the importance of bloom declines to phytoplankton dynamics, the incidence and significance of various loss factors in regulating phytoplankton populations have not been widely characterized in natural blooms. To understand mechanisms controlling bloom decline, we studied two independent, inshore blooms of Alexandrium fundyense, paying special attention to cell mortality as a loss pathway. We observed increases in the number of dead cells with PCD features after the peak of both blooms, demonstrating a role for cell mortality in their terminations. In both blooms, sexual cyst formation appears to have been the dominant process leading to bloom termination, as both blooms were dominated by small-sized gamete cells near their peaks. Cell death and parasitism became more significant as sources of cell loss several days after the onset of bloom decline. Our findings show two distinct phases of bloom decline, characterized by sexual fusion as the initial dominant cell removal processes followed by elimination of remaining cells by cell death and parasitism.This article is a result of research funded by the National Oceanic and Atmospheric Administration Center for Sponsored Coastal Ocean Research ECOHAB program under award no. NA09NOS4780166 to the University of Texas Marine Science Institute (D.L.E) and the Woods Hole Center for Oceans and Human Health by National Science Foundation (NSF) award no. OCE-1314642 and National Institute of Environmental Health Sciences (NIEHS) award no. 1-P01-ES021923-014 to D.M.A. and M.L B

Fiber-optic microarray for simultaneous detection of multiple harmful algal bloom species

Author Posting. © American Society for Microbiology, 2006. This article is posted here by permission of American Society for Microbiology for personal use, not for redistribution. The definitive version was published in Applied and Environmental Microbiology 72 (2006): 5742-5749, doi:10.1128/AEM.00332-06.Harmful algal blooms (HABs) are a serious threat to coastal resources, causing a variety of impacts on public health, regional economies, and ecosystems. Plankton analysis is a valuable component of many HAB monitoring and research programs, but the diversity of plankton poses a problem in discriminating toxic from nontoxic species using conventional detection methods. Here we describe a sensitive and specific sandwich hybridization assay that combines fiber-optic microarrays with oligonucleotide probes to detect and enumerate the HAB species Alexandrium fundyense, Alexandrium ostenfeldii, and Pseudo-nitzschia australis. Microarrays were prepared by loading oligonucleotide probe-coupled microspheres (diameter, 3 μm) onto the distal ends of chemically etched imaging fiber bundles. Hybridization of target rRNA from HAB cells to immobilized probes on the microspheres was visualized using Cy3-labeled secondary probes in a sandwich-type assay format. We applied these microarrays to the detection and enumeration of HAB cells in both cultured and field samples. Our study demonstrated a detection limit of approximately 5 cells for all three target organisms within 45 min, without a separate amplification step, in both sample types. We also developed a multiplexed microarray to detect the three HAB species simultaneously, which successfully detected the target organisms, alone and in combination, without cross-reactivity. Our study suggests that fiber-optic microarrays can be used for rapid and sensitive detection and potential enumeration of HAB species in the environment.This work was funded by the Sea Grant Technology Program (NA16RG2273)

Development of microsatellite markers in the toxic dinoflagellate Alexandrium minutum (Dinophyceae)

Author Posting. © Blackwell, 2006. This is the author's version of the work. It is posted here by permission of Blackwell for personal use, not for redistribution. The definitive version was published in Molecular Ecology Notes 6 (2006): 756-758, doi:10.1111/j.1471-8286.2006.01331.x.Outbreaks of paralytic shellfish poisoning caused by the toxic dinoflagellate Alexandrium minutum (Dinophyceae) are a worldwide concern from both the economic and human health points of view. For population genetic studies of A. minutum distribution and dispersal, highly polymorphic genetic markers are of great value. We isolated 12 polymorphic microsatellites from this cosmopolitan, toxic dinoflagellate species. These loci provide one class of highly variable genetic markers, as the number of alleles ranged from 4 to 12, and the estimate of gene diversity was from 0.560 to 0.862 across the 12 microsatellites; these loci have the potential to reveal genetic structure and gene flow among A. minutum populations.Support for this research provided in part (to DMA) by U.S. National Science Foundation grants OCE-0136861 and OCE-0430724, and the National Institute of Environmental Health Sciences Grant 1 P50 ES012742-01

Comparison of Spatial and Temporal Genetic Differentiation in a Harmful Dinoflagellate Species Emphasizes Impact of Local Processes

Population genetic studies provide insights into intraspecific diversity and dispersal patterns of microorganisms such as protists, which help understanding invasions, harmful algal bloom development and occurrence of seafood poisoning. Spatial genetic differentiation has been reported in many microbial species indicating significant dispersal barriers among different habitats. Temporal differentiation has been less studied and its frequency, drivers, and magnitude are thus relatively poorly understood. The toxic dinoflagellate species Gambierdiscus caribaeus was sampled during 2 years in the Florida Keys, and repeatedly from 2006 to 2016 at St. Thomas, US Virgin Islands (USVI), including a 3-year period with monthly sampling, enabling a comparison of spatial and temporal genetic differentiation. Samples from the USVI site showed high temporal variability in local population structure, which correlated with changes in salinity and benthic habitat cover. In some cases, temporal variability exceeded spatial differentiation, despite apparent lack of connectivity and dispersal across the Greater Caribbean Region based on the spatial genetic data. Thus, local processes such as selection might have a stronger influence on population structure in microorganisms than geographic distance. The observed high temporal genetic diversity challenges the prediction of harmful algal blooms and toxin concentrations, but illustrates also the evolutionary potential of microalgae to respond to environmental change

Transcriptome profiling of a toxic dinoflagellate reveals a gene-rich protist and a potential impact on gene expression due to bacterial presence

© The Authors, 2010. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in PLoS One 5 (2010): e9688, doi:10.1371/journal.pone.0009688.Dinoflagellates are unicellular, often photosynthetic protists that play a major role in the dynamics of the Earth's oceans and climate. Sequencing of dinoflagellate nuclear DNA is thwarted by their massive genome sizes that are often several times that in humans. However, modern transcriptomic methods offer promising approaches to tackle this challenging system. Here, we used massively parallel signature sequencing (MPSS) to understand global transcriptional regulation patterns in Alexandrium tamarense cultures that were grown under four different conditions. We generated more than 40,000 unique short expression signatures gathered from the four conditions. Of these, about 11,000 signatures did not display detectable differential expression patterns. At a p-value < 1E-10, 1,124 signatures were differentially expressed in the three treatments, xenic, nitrogen-limited, and phosphorus-limited, compared to the nutrient-replete control, with the presence of bacteria explaining the largest set of these differentially expressed signatures. Among microbial eukaryotes, dinoflagellates contain the largest number of genes in their nuclear genomes. These genes occur in complex families, many of which have evolved via recent gene duplication events. Our expression data suggest that about 73% of the Alexandrium transcriptome shows no significant change in gene expression under the experimental conditions used here and may comprise a “core” component for this species. We report a fundamental shift in expression patterns in response to the presence of bacteria, highlighting the impact of biotic interaction on gene expression in dinoflagellates.This work was primarily funded by a collaborative grant from the National Institutes of Health (R01 ES 013679-01A2) awarded to DB, DMA, and M. Bento Soares. Funding support for DMA and DLE was also provided from the Woods Hole Center for Oceans and Human Health from the NSF/NIEHS Centers for Oceans and Human Health program, NIEHS (P50 ES 012742) and (NSF OCE-043072). Additional support came from the National Science Foundation (EF-0732440) in a grant awarded to F. Gerald Plumley, DB, JDH, and DMA. AM was supported by an Institutional NRSA (T 32 GM98629)

Centers for Oceans and Human Health : a unified approach to the challenge of harmful algal blooms

© 2008 Author et al. This is an open access article distributed under the terms of the Creative Commons Attribution License The definitive version was published in Environmental Health 7 (2008): S2, doi:10.1186/1476-069X-7-S2-S2.Harmful algal blooms (HABs) are one focus of the national research initiatives on Oceans and Human Health (OHH) at NIEHS, NOAA and NSF. All of the OHH Centers, from the east coast to Hawaii, include one or more research projects devoted to studying HAB problems and their relationship to human health. The research shares common goals for understanding, monitoring and predicting HAB events to protect and improve human health: understanding the basic biology of the organisms; identifying how chemistry, hydrography and genetic diversity influence blooms; developing analytical methods and sensors for cells and toxins; understanding health effects of toxin exposure; and developing conceptual, empirical and numerical models of bloom dynamics. In the past several years, there has been significant progress toward all of the common goals. Several studies have elucidated the effects of environmental conditions and genetic heterogeneity on bloom dynamics. New methods have been developed or implemented for the detection of HAB cells and toxins, including genetic assays for Pseudo-nitzschia and Microcystis, and a biosensor for domoic acid. There have been advances in predictive models of blooms, most notably for the toxic dinoflagellates Alexandrium and Karenia. Other work is focused on the future, studying the ways in which climate change may affect HAB incidence, and assessing the threat from emerging HABs and toxins, such as the cyanobacterial neurotoxin β-N-methylamino-L-alanine. Along the way, many challenges have been encountered that are common to the OHH Centers and also echo those of the wider HAB community. Long-term field data and basic biological information are needed to develop accurate models. Sensor development is hindered by the lack of simple and rapid assays for algal cells and especially toxins. It is also critical to adequately understand the human health effects of HAB toxins. Currently, we understand best the effects of acute toxicity, but almost nothing is known about the effects of chronic, subacute toxin exposure. The OHH initiatives have brought scientists together to work collectively on HAB issues, within and across regions. The successes that have been achieved highlight the value of collaboration and cooperation across disciplines, if we are to continue to advance our understanding of HABs and their relationship to human health.This work was funded through grants from the NSF/NIEHS Centers for Oceans and Human Health, NIEHS P50 ES012742 and NSF OCE-043072 (DLE and DMA), NSF OCE04-32479 and NIEHS P50 ES012740 (PB and RRB), NSF OCE-0432368 and NIEHS P50 ES12736 (LEB), NIEHS P50 ES012762 and NSF OCE-0434087 (RCS, KAL, MSP, MLW, and KAH). Additional support was provided by the ECOHAB Grant program NSF Grant OCE-9808173 and NOAA Grant NA96OP0099 (DMA), NOAA OHHI NA04OAR4600206 (RRB) and Washington State Sea Grant NA16RG1044 (RCS). KAL and VLT were supported in part by the West Coast Center for Oceans and Human Health (WCCOHH) as part of the NOAA Oceans and Human Health Initiative

Diversity and dynamics of a widespread bloom of the toxic dinoflagellate Alexandrium fundyense

© The Author(s), 2011. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in PLoS One 6 (2011): e22965, doi:10.1371/journal.pone.0022965.Historically, cosmopolitan phytoplankton species were presumed to represent largely unstructured populations. However, the recent development of molecular tools to examine genetic diversity have revealed differences in phytoplankton taxa across geographic scales and provided insight into the physiology and ecology of blooms. Here we describe the genetic analysis of an extensive bloom of the toxic dinoflagellate Alexandrium fundyense that occurred in the Gulf of Maine in 2005. This bloom was notable for its intensity and duration, covering hundreds of kilometers and persisting for almost two months. Genotypic analyses based on microsatellite marker data indicate that the open waters of the northeastern U.S. harbor a single regional population of A. fundyense comprising two genetically distinct sub-populations. These subpopulations were characteristic of early- and late-bloom samples and were derived from the northern and southern areas of the bloom, respectively. The temporal changes observed during this study provide clear evidence of succession during a continuous bloom and show that selection can act on the timescale of weeks to significantly alter the representation of genotypes within a population. The effects of selection on population composition and turnover would be magnified if sexual reproduction were likewise influenced by environmental conditions. We hypothesize that the combined effects of differential growth and reproduction rates serves to reduce gene flow between the sub-populations, reinforcing population structure while maintaining the diversity of the overall regional population.This work was supported by the National Institute of Environmental Health Sciences (1-P50-ES012742 to DMA and DLE), by the National Science Foundation through the Woods Hole Center for Oceans and Human Health (OCE-0430724), and by the ECOHAB program (NOAA Grant NA06NOS4780245)