Life cycle studies of the red tide dinoflagellate species complex Alexandrium tamarense

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the Massachusetts Institute of Technology and the Woods Hole Oceanographic Institution February 2011Blooms of toxic species within the algal dinoflagellate species complex Alexandrium tamarense may cause Paralytic Shellfish Poisoning, a significant and growing environmental threat worldwide. However, blooms of closely related nontoxic A. tamarense also occur, sometimes in close geographical proximity to toxic blooms. This thesis explores the interactions between toxic and nontoxic blooms by examining sexual crosses between each of five ribosomal clades within the A. tamarense complex (termed Groups I-V). Several lines of evidence argue that these clades represent separate species. Particular emphasis was given to interactions between toxic Group I and nontoxic Group III species because they are among the most closely related A. tamarense clades and because they share a natural range boundary in several parts of the world. Interspecies hybridization appeared widespread between different clades and between geographically dispersed isolates. However, subsequent germination studies of hypnozygotes produced from combinations of Group I and Group III clones failed to yield new vegetative cultures in multiple trials. The possibility that these hypnozygotes were actually inbred (i.e. the result of pairs of only Group I or only Group III gametes) was considered and rejected using a nested PCR assay that was developed to assess the parentage of individual cysts. The assay was also suitable for analyzing cysts collected from the field and was applied to individual cysts collected from Belfast Lough, an area where both Group I and Group III blooms were known to occur. Two Group I/Group III hybrids were detected in fourteen successful assays from the Belfast sample, showing that hybridization does occur in nature. These findings have several important implications. First, the failure of Group I/Group III hybrids to produce new vegetative cultures serves as a proof that the A. tamarense clades represent cryptic species because they are unable to produce genetic intermediates. Second, the presence of hybrid cysts in Belfast Lough indicates ongoing displacement of a nontoxic population by a toxic one (or vice versa) in that region. Third, the inviability of toxic/nontoxic hybrids suggests a remediation strategy whereby the recurrence of toxic A. tamarense blooms might be combated through the introduction of nontoxic cells.research support from NSF (grants OCE-0402707 and OCE- 9808173 awarded to Don Anderson), the Woods Hole Center for Oceans and Human Health (NSF Grant no. OCE-0430724 and NIEHS Grant no. P50ES012742-0), an EPA STAR graduate fellowship (FP-91688601), and the Coastal Ocean Institute at WHOI. I’ve also benefitted greatly from conference travel funds provided by the NOAA Center for Sponsored Coastal Ocean Research, the MIT Student Assistance Fund, and the Academic Programs Office at WHOI

Growing degree-day measurement of cyst germination rates in the toxic dinoflagellate Alexandrium catenella

© The Author(s), 2022. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Fischer, A., & Brosnahan, M. Growing degree-day measurement of cyst germination rates in the toxic dinoflagellate Alexandrium catenella. Applied and Environmental Microbiology, 88(12), (2022): e02518-21, https://doi.org/10.1128/aem.02518-21.Blooms of many dinoflagellates, including several harmful algal bloom (HAB) species, are seeded and revived through the germination of benthic resting cysts. Temperature is a key determinant of cysts’ germination rate, and temperature–germination rate relationships are therefore fundamental to understanding species’ germling cell production, cyst bed persistence, and resilience to climate warming. This study measured germination by cysts of the HAB dinoflagellate Alexandrium catenella using a growing degree-day (DD) approach that accounts for the time and intensity of warming above a critical temperature. Time courses of germination at different temperatures were fit to lognormal cumulative distribution functions for the estimation of the median days to germination. As temperature increased, germination times decreased hyperbolically. DD scaling collapsed variability in germination times between temperatures after cysts were oxygenated. A parallel experiment demonstrated stable temperature–rate relationships in cysts collected during different phases of seasonal temperature cycles in situ over three years. DD scaling of the results from prior A. catenella germination studies showed consistent differences between populations across a wide range of temperatures and suggests selective pressure for different germination rates. The DD model provides an elegant approach to quantify and compare the temperature dependency of germination among populations, between species, and in response to changing environmental conditions.We gratefully acknowledge support to A.D.F. and M.L.B. through the Woods Hole Center for Oceans and Human Health (National Science Foundation grants OCE‐0430724, OCE‐0911031, OCE‐1314642, and OCE-1840381 and National Institutes of Health grants NIEHS‐1P50‐ES02192301 and P01ES028938)

Life cycle studies of the red tide dinoflagellate species complex Alexandrium tamarense

Thesis (Ph. D.)--Joint Program in Oceanography/Applied Ocean Science and Engineering (Massachusetts Institute of Technology, Dept. of Biology; and the Woods Hole Oceanographic Institution), 2011.Cataloged from PDF version of thesis.Includes bibliographical references.Blooms of toxic species within the algal dinoflagellate species complex Alexandrium tamarense may cause Paralytic Shellfish Poisoning, a significant and growing environmental threat worldwide. However, blooms of closely related nontoxic A. tamarense also occur, sometimes in close geographical proximity to toxic blooms. This thesis explores the interactions between toxic and nontoxic blooms by examining sexual crosses between each of five ribosomal clades within the A. tamarense complex (termed Groups I-V). Several lines of evidence argue that these clades represent separate species. Particular emphasis was given to interactions between toxic Group I and nontoxic Group III species because they are among the most closely related A. tamarense clades and because they share a natural range boundary in several parts of the world. Interspecies hybridization appeared widespread between different clades and between geographically dispersed isolates. However, subsequent germination studies of hypnozygotes produced from combinations of Group I and Group III clones failed to yield new vegetative cultures in multiple trials. The possibility that these hypnozygotes were actually inbred (i.e. the result of pairs of only Group I or only Group III gametes) was considered and rejected using a nested PCR assay that was developed to assess the parentage of individual cysts. The assay was also suitable for analyzing cysts collected from the field and was applied to individual cysts collected from Belfast Lough, an area where both Group I and Group III blooms were known to occur. Two Group I/Group III hybrids were detected in fourteen successful assays from the Belfast sample, showing that hybridization does occur in nature. These findings have several important implications. First, the failure of Group I/Group III hybrids to produce new vegetative cultures serves as a proof that the A. tamarense clades represent cryptic species because they are unable to produce genetic intermediates. Second, the presence of hybrid cysts in Belfast Lough indicates ongoing displacement of a nontoxic population by a toxic one (or vice versa) in that region. Third, the inviability of toxic/nontoxic hybrids suggests a remediation strategy whereby the recurrence of toxic A. tamarense blooms might be combated through the introduction of nontoxic cells. The results from these experiments also highlighted several shortfalls in our understanding of the mechanisms governing sexual compatibility between clones and also our ability to replicate these organisms' sexual cycle in the laboratory. Two initiatives were begun with the ultimate goal of better characterizing sexual processes in natural populations. The first initiative was the application of an imaging flow cytometer to detect sexual events in natural blooms. An existing instrument, the Imaging FlowCytobot, was adapted to positively identify A. tamarense Group I cells in mixed species assemblages and measure cell DNA content. A collection of four samples were analyzed, three from the development and decline of a local Group I bloom and one from a Group I red tide that occurred near Portsmouth, NH and led to a major deposition of new cysts in the southern Gulf of Maine. Several unanticipated patterns were revealed including the discovery of a persistent layer of cells with 2c DNA content near the surface and disproportionately high rates of infection by an Amoebophrya sp. parasite in large A. tamarense planozygotes. The second initiative was the application of high throughput Illumina sequencing to define the transcriptomes of toxic Group I, nontoxic Group III, and toxic Group IV clones. Additional steps were taken to isolate RNA and prepare a cDNA library from a natural sample of Group I hypnozygotes. The applications for these data are expected to be extensive and include the discovery of sexual biomarkers and further characterization of the differences between toxic and nontoxic A. tamarense species. Preliminary results from the sequencing of these libraries and their initial assembly are described.by Michael L. Brosnahan.Ph.D

Quantitative response of Alexandrium catenella cyst dormancy to cold exposure

Author Posting. © The Author(s), 2018. 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 Protist 169 (2018): 645-661, doi:10.1016/j.protis.2018.06.001.Many dinoflagellate cysts experience dormancy, a reversible state that prevents germination during unfavorable periods. Several of these species also cause harmful algal blooms (HABs), so a quantitative understanding of dormancy cycling is desired for better prediction and mitigation of bloom impacts. This study examines the effect of cold exposure on the duration of dormancy in Alexandrium catenella, a HAB dinoflagellate that causes paralytic shellfish poisoning (PSP). Mature, dormant cysts from Nauset Marsh (Cape Cod, MA USA) were stored at low but above freezing temperatures for up to six months. Dormancy status was then determined at regular intervals using a germination assay. Dormancy timing was variable among temperatures and was shorter in colder treatments, but the differences collapse when temperature and duration of storage are scaled by chilling-units (CU), a common horticultural predictor of plant and insect development in response to weather. Cysts within Nauset meet a well-defined chilling requirement by late January, after which they are poised to germinate with the onset of favorable conditions in spring. Cysts thus modulate their dormancy cycles in response to their temperature history, enhancing the potential for new blooms and improving this species’ adaptability to both unseasonable weather and new habitats/climate regimes.This work was supported by the National Science Foundation [OCE-0430724, OCE-0911031]; the National Institute of Environmental Health Sciences [1P50-ES01274201, 1P01ES021923]; the National Park Service Cooperative Agreement [H238015504]; and the Friends of Cape Cod National Seashore

Horizontal gene transfer is a significant driver of gene innovation in dinoflagellates

© The Author(s), 2013. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Genome Biology and Evolution 5 (2013): 2368-2381, doi:10.1093/gbe/evt179.The dinoflagellates are an evolutionarily and ecologically important group of microbial eukaryotes. Previous work suggests that horizontal gene transfer (HGT) is an important source of gene innovation in these organisms. However, dinoflagellate genomes are notoriously large and complex, making genomic investigation of this phenomenon impractical with currently available sequencing technology. Fortunately, de novo transcriptome sequencing and assembly provides an alternative approach for investigating HGT. We sequenced the transcriptome of the dinoflagellate Alexandrium tamarense Group IV to investigate how HGT has contributed to gene innovation in this group. Our comprehensive A. tamarense Group IV gene set was compared with those of 16 other eukaryotic genomes. Ancestral gene content reconstruction of ortholog groups shows that A. tamarense Group IV has the largest number of gene families gained (314–1,563 depending on inference method) relative to all other organisms in the analysis (0–782). Phylogenomic analysis indicates that genes horizontally acquired from bacteria are a significant proportion of this gene influx, as are genes transferred from other eukaryotes either through HGT or endosymbiosis. The dinoflagellates also display curious cases of gene loss associated with mitochondrial metabolism including the entire Complex I of oxidative phosphorylation. Some of these missing genes have been functionally replaced by bacterial and eukaryotic xenologs. The transcriptome of A. tamarense Group IV lends strong support to a growing body of evidence that dinoflagellate genomes are extraordinarily impacted by HGT.J.H.W. was supported by the NSF IGERT Program in Comparative Genomics at the University of Arizona (grant number DGE-0654435). This work was supported by grants from the National Science Foundation (grant numbers OCE-0723498, EF-0732440) and funding provided by the BIO5 Institute at the University of Arizona to J.D.H

Effects of Two Toxin-Producing Harmful Algae, Alexandrium catenella and Dinophysis acuminata (Dinophyceae), on Activity and Mortality of Larval Shellfish

Harmful algal bloom (HAB) species Alexandrium catenella and Dinophysis acuminata are associated with paralytic shellfish poisoning (PSP) and diarrhetic shellfish poisoning (DSP) in humans, respectively. While PSP and DSP have been studied extensively, less is known about the effects of these HAB species or their associated toxins on shellfish. This study investigated A. catenella and D. acuminata toxicity in a larval oyster (Crassostrea virginica) bioassay. Larval activity and mortality were examined through 96-h laboratory exposures to live HAB cells (10–1000 cells/mL), cell lysates (1000 cells/mL equivalents), and purified toxins (10,000 cells/mL equivalents). Exposure to 1000 cells/mL live or lysed D. acuminata caused larval mortality (21.9 ± 7.0%, 10.2 ± 4.0%, respectively) while exposure to any tested cell concentration of live A. catenella, but not lysate, caused swimming arrest and/or mortality in \u3e50% of larvae. Exposure to high concentrations of saxitoxin (STX) or okadaic acid (OA), toxins traditionally associated with PSP and DSP, respectively, had no effect on larval activity or mortality. In contrast, pectenotoxin-2 (PTX2) caused rapid larval mortality (49.6 ± 5.8% by 48 h) and completely immobilized larval oysters. The results indicate that the toxic effects of A. catenella and D. acuminata on shellfish are not linked to the primary toxins associated with PSP and DSP in humans, and that PTX2 is acutely toxic to larval oysters

Effects of two toxin-producing harmful algae, Alexandrium catenella and Dinophysis acuminata (Dinophyceae), on activity and mortality of larval shellfish

© The Author(s), 2022. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Pease, S. K. D., Brosnahan, M. L., Sanderson, M. P., & Smith, J. L. Effects of two toxin-producing harmful algae, Alexandrium catenella and Dinophysis acuminata (Dinophyceae), on activity and mortality of larval shellfish. Toxins, 14(5), (2022): 335, https://doi.org/10.3390/toxins14050335.Harmful algal bloom (HAB) species Alexandrium catenella and Dinophysis acuminata are associated with paralytic shellfish poisoning (PSP) and diarrhetic shellfish poisoning (DSP) in humans, respectively. While PSP and DSP have been studied extensively, less is known about the effects of these HAB species or their associated toxins on shellfish. This study investigated A. catenella and D. acuminata toxicity in a larval oyster (Crassostrea virginica) bioassay. Larval activity and mortality were examined through 96-h laboratory exposures to live HAB cells (10–1000 cells/mL), cell lysates (1000 cells/mL equivalents), and purified toxins (10,000 cells/mL equivalents). Exposure to 1000 cells/mL live or lysed D. acuminata caused larval mortality (21.9 ± 7.0%, 10.2 ± 4.0%, respectively) while exposure to any tested cell concentration of live A. catenella, but not lysate, caused swimming arrest and/or mortality in >50% of larvae. Exposure to high concentrations of saxitoxin (STX) or okadaic acid (OA), toxins traditionally associated with PSP and DSP, respectively, had no effect on larval activity or mortality. In contrast, pectenotoxin-2 (PTX2) caused rapid larval mortality (49.6 ± 5.8% by 48 h) and completely immobilized larval oysters. The results indicate that the toxic effects of A. catenella and D. acuminata on shellfish are not linked to the primary toxins associated with PSP and DSP in humans, and that PTX2 is acutely toxic to larval oysters.This research was partially funded by the National Oceanic and Atmospheric Administration, National Centers for Coastal Ocean Science Competitive Research, Ecology and Oceanography of Harmful Algal Blooms Program under award #NA19NOS4780182 to J.L.S. (VIMS) and M.L.B (WHOI), and by a William & Mary, School of Marine Science, Student Research Grant to S.K.D.P. (VIMS). This paper is ECOHAB publication number 1022

A quantitative assessment of the role of the parasite Amoebophrya in the termination of Alexandrium fundyense blooms within a small coastal embayment

© The Author(s), 2013. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in PLoS One 8 (2013): e81150, doi:10.1371/journal.pone.0081150.Parasitic dinoflagellates of the genus Amoebophrya infect free-living dinoflagellates, some of which can cause harmful algal blooms (HABs). High prevalence of Amoebophrya spp. has been linked to the decline of some HABs in marine systems. The objective of this study was to evaluate the impact of Amoebophrya spp. on the dynamics of dinoflagellate blooms in Salt Pond (MA, USA), particularly the harmful species Alexandrium fundyense. The abundance of Amoebophrya life stages was estimated 3–7 days per week through the full duration of an annual A. fundyense bloom using fluorescence in situ hybridization coupled with tyramide signal amplification (FISH- TSA). More than 20 potential hosts were recorded including Dinophysis spp., Protoperidinium spp. and Gonyaulax spp., but the only dinoflagellate cells infected by Amoebophrya spp. during the sampling period were A. fundyense. Maximum A. fundyense concentration co-occurred with an increase of infected hosts, followed by a massive release of Amoebophrya dinospores in the water column. On average, Amoebophrya spp. infected and killed ~30% of the A. fundyense population per day in the end phase of the bloom. The decline of the host A. fundyense population coincided with a dramatic life-cycle transition from vegetative division to sexual fusion. This transition occurred after maximum infected host concentrations and before peak infection percentages were observed, suggesting that most A. fundyense escaped parasite infection through sexual fusion. The results of this work highlight the importance of high frequency sampling of both parasite and host populations to accurately assess the impact of parasites on natural plankton assemblages.L. Velo-Sua´rez was supported by a Marie Curie International Outgoing Fellowship (IOF; grant agreement: MOHAB PIOF-GA-252260). This work was supported in part by NSF grants OCE-0430724 and OCE-0911031 and National Institute of Environmental Health Sciences grants 1P50-ES01274201 and 1P01ES021923-01 to D.M. Anderson and D.J. McGillicuddy through the Woods Hole Center for Oceans and Human Health, National Park Service Cooperative Agreement H238015504 to D.M. Anderson

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

Evaluation of sxtA and rDNA qPCR assays through monitoring of an inshore bloom of Alexandrium catenella Group 1

© The Author(s), 2019. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Murray, S. A., Ruvindy, R., Kohli, G. S., Anderson, D. M., & Brosnahan, M. L. Evaluation of sxtA and rDNA qPCR assays through monitoring of an inshore bloom of Alexandrium catenella Group 1. Scientific Reports, 9(1), (2019): 14532, doi:10.1038/s41598-019-51074-3.Alexandrium catenella (formerly A. tamarense Group 1, or A. fundyense) is the leading cause of Paralytic Shellfish Poisoning in North and South America, Europe, Africa, Australia and Asia. The quantification of A.catenella via sxtA, a gene involved in Paralytic Shellfish Toxin synthesis, may be a promising approach, but has not been evaluated in situ on blooms of A. catenella, in which cell abundances may vary from not detectable to in the order of 106 cells L−1. In this study, we compared sxtA assay performance to a qPCR assay targeted to a species-specific region of ribosomal DNA (rDNA) and an established fluorescent in situ hybridization (FISH) microscopy method. Passing-Bablok regression analyses revealed the sxtA assay to overestimate abundances when <5 cell equivalents A. catenella DNA were analysed, but otherwise was closer to microscopy estimates than the rDNA assay, which overestimated abundance across the full range of concentrations analysed, indicative of a copy number difference between the bloom population and a culture used for assay calibration a priori. In contrast, the sxtA assay performed more consistently, indicating less copy number variation. The sxtA assay was generally reliable, fast and effective in quantifying A. catenella and was predictive of PST contamination of shellfish.We thank the Australian Research Council for Funding (FT120100704). We thank Chowdhury Sarowar for the toxicity measurements, at the Sydney Institute of Marine Science. Support to MB and DA was provided by MIT Sea Grant (NA14OAR4170077) and the Woods Hole Center for Oceans and Human Health (National Science Foundation award OCE-1840381 and National Institute of Environmental Health Sciences award 1-P01-ES028938–01). We are grateful for assistance from David Kulis, Claire Mullen, and Isaac Rosenthal for assistance in the collection and processing of Salt Pond samples