Analysis of toxic and non-toxic Alexandrium (Dinophyceae) species using ribosomal RNA gene sequences

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 November 1992Sequences of small subunit (Ss) and large subunit (Ls) ribosomal RNA genes (rDNA) from the marine dinoflagellates Alexandrium tamarense, A. catenella, A. fundyense, A. affine, A. minutum, A. lusitanicum and A. andersoni were compared to assess the organisms' relationships. Cultures represent isolates from North America, Western Europe, Thailand, Japan, Australia and the ballast water of several cargo vessels, and include both toxic and non-toxic strains. An emphasis was placed on the A. tamarense/catenella/fundyense "species complex," a group of morphotypically-similar organisms found in many regions of the world. Two distinct SsrRNA genes, termed the "A gene" and the "B gene," were found in a toxic A. fundyense isolated from eastern North America. The B gene is considered to be a pseudogene. A restriction fragment length polymorphism (RFLP) assay developed to detect the A and B genes revealed five distinct groups of Alexandrium isolates. Three subdivide the A. tamarense/catenella/fundyense complex, but do not correlate with morphospecies designations. The two remaining groups are associated with cultures that clearly differ morphologically from the A. tamarense/ catenella/fundyense group: the fourth group consists of A. affine isolates, and the fifth group is represented by A. minutum, A.lusitanicum and A. andersoni. The B gene was only found in A. tamarense/catenella/ fundyense, but not in all members of this species complex. The B gene is not uniformly distributed among global populations of Alexandrium. All A. tamarense/catenella/fundyense isolates from North America harbor this gene, but it has also been found in some A. tamarense from scattered locations in Japan, as well as in A. tamarense from the ballast water of one cargo vessel which was on a defined run from Japan to Australia. The B gene may be endemic to North American populations of A. tamarense/catenella/fundyense. It is possible that in the recent past North American A. tamarense were introduced to Japanese waters, and cysts of these organisms have been transported from Japan to Australia. A subset of isolates examined using the the RFLP assay were also compared by cloning and sequencing a fragment of their LsrDNA. Eight major classes of LsrDNA sequences, termed "ribotypes," were identified. Five ribotypes subdivide members of the A. tamarense/catenella!fundyense complex; all isolates containing the B gene cluster as one ribotype. The three remaining ribotypes are typified by: 1) A. affine; 2) A. minutum and A. lusitanicum; and, 3) A. andersoni. LsrDNAs from A. minutum and A. lusitanicum are indistinguishable. A. minutum!lusitanicum/andersoni may represent another Alexandrium species complex, analogous to the A. tamarense/catenella/fundyense group. An organisms' ability to produce toxin appears to be correlated with its LsrDNA phylogenetic lineage. Ribotypes ascribed by the LsrDNA sequences are in complete agreement with, and offer a finer-scale resolution of, groups defined by SsrDNA restriction patterns. The SsrDNA RFLP groups and LsrDNA ribotypes are useful species- and population-specific markers. Alexandrium tamarense/catenella/fundyense exist as geneticallydistinct "strains" (populations), not three genetically-distinct species: representatives collected from the same geographic region appear the most similar, regardless of morphotype, whereas those from geographicallyseparated populations are more divergent even when the same morphospecies are compared. Contrary to this general pattern, A. tamarense/catenella from Japan were found to be exceptionally heterogeneous. Ballast water samples show that viable cysts (resting spores) of toxigenic A. tamarense/catenella are being discharged into Australian ports from multiple, genetically-distinct source populations. The rDNA sequences were also used to test theories accounting for the evolution and global dispersal of A. tamarense/catenella/fundyense. Results suggest a monophyletic radiation of these organisms from a common ancestor that included, or gave rise to, multiple morphotypes. Populations appear to have diverged as a result of vicariance (geographic isolation). The co-occurrence of genetically-distinct strains of these organisms is an indication of dispersal. An example of this is seen in Japan where an introduction of North American A. tamarense appears likely. Determining the timing of dispersal events is problematic if based strictly on rDNA sequence similarities, since these molecules undergo change on a scale of millions of years.This work was supported in part by a grant from the National Science Foundation to D. M. Anderson (contract number OCE89- 11226), the Woods Hole Oceanographic Institution Ocean Ventures Fund and the Woods Hole Oceanographic Institution Education Office

Diel transcriptional response of a California Current plankton microbiome to light, low iron, and enduring viral infection.

Phytoplankton and associated microbial communities provide organic carbon to oceanic food webs and drive ecosystem dynamics. However, capturing those dynamics is challenging. Here, an in situ, semi-Lagrangian, robotic sampler profiled pelagic microbes at 4 h intervals over ~2.6 days in North Pacific high-nutrient, low-chlorophyll waters. We report on the community structure and transcriptional dynamics of microbes in an operationally large size class (>5 μm) predominantly populated by dinoflagellates, ciliates, haptophytes, pelagophytes, diatoms, cyanobacteria (chiefly Synechococcus), prasinophytes (chiefly Ostreococcus), fungi, archaea, and proteobacteria. Apart from fungi and archaea, all groups exhibited 24-h periodicity in some transcripts, but larger portions of the transcriptome oscillated in phototrophs. Periodic photosynthesis-related transcripts exhibited a temporal cascade across the morning hours, conserved across diverse phototrophic lineages. Pronounced silica:nitrate drawdown, a high flavodoxin to ferredoxin transcript ratio, and elevated expression of other Fe-stress markers indicated Fe-limitation. Fe-stress markers peaked during a photoperiodically adaptive time window that could modulate phytoplankton response to seasonal Fe-limitation. Remarkably, we observed viruses that infect the majority of abundant taxa, often with total transcriptional activity synchronized with putative hosts. Taken together, these data reveal a microbial plankton community that is shaped by recycled production and tightly controlled by Fe-limitation and viral activity

Microbial community transcriptional networks are conserved in three domains at ocean basin scales

Planktonic microbial communities in the ocean are typically dominated by several cosmopolitan clades of Bacteria, Archaea, and Eukarya characterized by their ribosomal RNA gene phylogenies and genomic features. Although the environments these communities inhabit range from coastal to open ocean waters, how the biological dynamics vary between such disparate habitats is not well known. To gain insight into the differential activities of microbial populations inhabiting different oceanic provinces we compared the daily metatranscriptome profiles of related microbial populations inhabiting surface waters of both a coastal California upwelling region (CC) as well as the oligotrophic North Pacific Subtropical Gyre (NPSG). Transcriptional networks revealed that the dominant photoautotrophic microbes in each environment (Ostreococcus in CC, Prochlorococcus in NPSG) were central determinants of overall community transcriptome dynamics. Furthermore, heterotrophic bacterial clades common to both ecosystems (SAR11, SAR116, SAR86, SAR406, and Roseobacter) displayed conserved, genome-wide inter- and intrataxon transcriptional patterns and diel cycles. Populations of SAR11 and SAR86 clades in particular exhibited tightly coordinated transcriptional patterns in both coastal and pelagic ecosystems, suggesting that specific biological interactions between these groups are widespread in nature. Our results identify common diurnally oscillating behaviors among diverse planktonic microbial species regardless of habitat, suggesting that highly conserved temporally phased biotic interactions are ubiquitous among planktonic microbial communities worldwide.Gordon and Betty Moore Foundation (3777)National Science Foundation (U.S.) (Grant EF0424599)Simons Foundation (Simons Collaboration on Ocean Processes and Ecology

Involvement of alcohol in injury cases in rural Sri Lanka:Prevalence and associated factors among in-patients in three primary care hospitals

BACKGROUND: Injuries account for a major proportion of global morbidity and mortality related to alcohol use. Information on the prevalence of alcohol-related injury in rural Sri Lanka is limited. The aims of this study were to determine the burden of alcohol-related injury in a hospital-based sample in rural Sri Lanka and explore factors associated with an increased risk of alcohol-related injury. METHODS: Involvement of alcohol in injury amongst in-patients was assessed in three hospitals in the North Central Province of Sri Lanka over 6 months. Adult (≥ 18 years) patients were eligible. Patients were assessed for: injury characteristics, current alcohol use (in the past year) using the Alcohol Use Disorder Identification Test (AUDIT), and acute intoxication. Patients with a blood alcohol concentration (BAC) reading equivalent of 10 mg/dL (2.17 mmol/L) were considered as having an alcohol-related injury. Binary logistic regression was used to explore association between alcohol-related injury and demographic and injury characteristics. RESULTS: A total of 883 injured patients were eligible and consented to the study. No alcohol use was reported by 487 (55.2%) of patients (35.6% of men, 95.2% of women). Prevalence of alcohol-related injuries was 14.8% overall and 32.8% among current alcohol users. Almost all patients with an alcohol-related injury were male (122/123; 99.2%); 24 (18.8%) of these patients scored positive for possible alcohol dependence. Patients with an alcohol-related injury had significantly higher AUDIT scores (median = 15 vs 6, p < 0.001), were significantly more likely to be aged 26–40 (OR 2.29, 95% CI:1.11, 4.72) or 41–55 years (OR 2.76, 95% CI: 1.29, 5.90) (compared to 18–25 years), to have a transport-related injury (OR 5.14, 95% CI: 2.30, 11.49) (compared to animal/plant sting/bite), and have intentional injuries (OR 3.47, 95% CI: 1.01, 11.87). CONCLUSIONS: One in three injuries among people who drank alcohol in this sample were alcohol-related. In addition, problematic alcohol use was higher among those with alcohol-related injury. Further work is needed to explore whether this prevalence of alcohol-related injury is reflected in other rural settings in Sri Lanka. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12889-022-12958-8

Multispecies diel transcriptional oscillations in open ocean heterotrophic bacterial assemblages

Oscillating diurnal rhythms of gene transcription, metabolic activity, and behavior are found in all three domains of life. However, diel cycles in naturally occurring heterotrophic bacteria and archaea have rarely been observed. Here, we report time-resolved whole-genome transcriptome profiles of multiple, naturally occurring oceanic bacterial populations sampled in situ over 3 days. As anticipated, the cyanobacterial transcriptome exhibited pronounced diel periodicity. Unexpectedly, several different heterotrophic bacterioplankton groups also displayed diel cycling in many of their gene transcripts. Furthermore, diel oscillations in different heterotrophic bacterial groups suggested population-specific timing of peak transcript expression in a variety of metabolic gene suites. These staggered multispecies waves of diel gene transcription may influence both the tempo and the mode of matter and energy transformation in the sea.Gordon and Betty Moore Foundation (GBMF 492.01)Gordon and Betty Moore Foundation (GBMF 3777)National Science Foundation (U.S.) (Grant EF0424599)David & Lucile Packard Foundatio

Underwater Application of Quantitative PCR on an Ocean Mooring

The Environmental Sample Processor (ESP) is a device that allows for the underwater, autonomous application of DNA and protein probe array technologies as a means to remotely identify and quantify, in situ, marine microorganisms and substances they produce. Here, we added functionality to the ESP through the development and incorporation of a module capable of solid-phase nucleic acid extraction and quantitative PCR (qPCR). Samples collected by the instrument were homogenized in a chaotropic buffer compatible with direct detection of ribosomal RNA (rRNA) and nucleic acid purification. From a single sample, both an rRNA community profile and select gene abundances were ascertained. To illustrate this functionality, we focused on bacterioplankton commonly found along the central coast of California and that are known to vary in accordance with different oceanic conditions. DNA probe arrays targeting rRNA revealed the presence of 16S rRNA indicative of marine crenarchaea, SAR11 and marine cyanobacteria; in parallel, qPCR was used to detect 16S rRNA genes from the former two groups and the large subunit RuBisCo gene (rbcL) from Synecchococcus. The PCR-enabled ESP was deployed on a coastal mooring in Monterey Bay for 28 days during the spring-summer upwelling season. The distributions of the targeted bacterioplankon groups were as expected, with the exception of an increase in abundance of marine crenarchaea in anomalous nitrate-rich, low-salinity waters. The unexpected co-occurrence demonstrated the utility of the ESP in detecting novel events relative to previously described distributions of particular bacterioplankton groups. The ESP can easily be configured to detect and enumerate genes and gene products from a wide range of organisms. This study demonstrated for the first time that gene abundances could be assessed autonomously, underwater in near real-time and referenced against prevailing chemical, physical and bulk biological conditions

Targeted sampling by autonomous underwater vehicles

© The Author(s), 2019. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Zhang, Y., Ryan, J. P., Kieft, B., Hobson, B. W., McEwen, R. S., Godin, M. A., Harvey, J. B., Barone, B., Bellingham, J. G., Birch, J. M., Scholin, C. A., & Chavez, F. P. Targeted sampling by autonomous underwater vehicles. Frontiers in Marine Science, 6 (2019): 415, doi:10.3389/fmars.2019.00415.In the vast ocean, many ecologically important phenomena are temporally episodic, localized in space, and move according to local currents. To effectively study these complex and evolving phenomena, methods that enable autonomous platforms to detect and respond to targeted phenomena are required. Such capabilities allow for directed sensing and water sample acquisition in the most relevant and informative locations, as compared against static grid surveys. To meet this need, we have designed algorithms for autonomous underwater vehicles that detect oceanic features in real time and direct vehicle and sampling behaviors as dictated by research objectives. These methods have successfully been applied in a series of field programs to study a range of phenomena such as harmful algal blooms, coastal upwelling fronts, and microbial processes in open-ocean eddies. In this review we highlight these applications and discuss future directions.This work was supported by the David and Lucile Packard Foundation. The 2015 experiment in Monterey Bay was partially supported by NOAA Ecology and Oceanography of Harmful Algal Blooms (ECOHAB) Grant NA11NOS4780030. The 2018 SCOPE Hawaiian Eddy Experiment was partially supported by the National Science Foundation (OCE-0962032 and OCE-1337601), Simons Foundation Grant #329108, the Gordon and Betty Moore Foundation (Grant #3777, #3794, and #2728), and the Schmidt Ocean Institute for R/V Falkor Cruise FK180310. Publication of this paper was funded by the Schmidt Ocean Institute

Diversity and toxicity of Pseudo-nitzschia species in Monterey Bay : perspectives from targeted and adaptive sampling

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 Harmful Algae 78 (2018): 129-141, doi:10.1016/j.hal.2018.08.006.Monterey Bay, California experiences near-annual blooms of Pseudo-nitzschia that can affect marine animal health and the economy, including impacts to tourism and commercial/recreational fisheries. One species in particular, P. australis, has been implicated in the most toxic of events, however other species within the genus can contribute to widespread variability in community structure and associated toxicity across years. Current monitoring methods are limited in their spatial coverage as well as their ability to capture the full suite of species present, thereby hindering understanding of HAB events and limiting predictive accuracy. An integrated deployment of multiple in situ platforms, some with autonomous adaptive sampling capabilities, occurred during two divergent bloom years in the bay, and uncovered detailed aspects of population and toxicity dynamics. A bloom in 2013 was characterized by spatial differences in Pseudo39 nitzschia populations, with the low-toxin producer P. fraudulenta dominating the inshore community and toxic P. australis dominating the offshore community. An exceptionally toxic bloom in 2015 developed as a diverse Pseudo-nitzschia community abruptly transitioned into a bloom of highly toxic P. australis within the time frame of a week. Increases in cell density and proliferation coincided with strong upwelling of nutrients. High toxicity was driven by silicate limitation of the dense bloom. This temporal shift in species composition mirrored the shift observed further north in the California Current System off Oregon and Washington. The broad scope of sampling and unique platform capabilities employed during these studies revealed important patterns in bloom formation and persistence for Pseudo-nitzschia. Results underscore the benefit of expanded biological observing capabilities and targeted sampling methods to capture more comprehensive spatial and temporal scales for studying and predicting future events.This work was supported by the National Oceanic and Atmospheric Administration (NA11NOS4780055, NA11NOS4780056, NA11NOS4780030) and a fellowship to H. Bowers from the Packard Foundation

Diel transcriptional response of a California Current plankton microbiome to light, low iron, and enduring viral infection

Phytoplankton and associated microbial communities provide organic carbon to oceanic food webs and drive ecosystem dynamics. However, capturing those dynamics is challenging. Here, an in situ, semi-Lagrangian, robotic sampler profiled pelagic microbes at 4 h intervals over ~2.6 days in North Pacific high-nutrient, low-chlorophyll waters. We report on the community structure and transcriptional dynamics of microbes in an operationally large size class (>5 μm) predominantly populated by dinoflagellates, ciliates, haptophytes, pelagophytes, diatoms, cyanobacteria (chiefly Synechococcus), prasinophytes (chiefly Ostreococcus), fungi, archaea, and proteobacteria. Apart from fungi and archaea, all groups exhibited 24-h periodicity in some transcripts, but larger portions of the transcriptome oscillated in phototrophs. Periodic photosynthesis-related transcripts exhibited a temporal cascade across the morning hours, conserved across diverse phototrophic lineages. Pronounced silica:nitrate drawdown, a high flavodoxin to ferredoxin transcript ratio, and elevated expression of other Fe-stress markers indicated Fe-limitation. Fe-stress markers peaked during a photoperiodically adaptive time window that could modulate phytoplankton response to seasonal Fe-limitation. Remarkably, we observed viruses that infect the majority of abundant taxa, often with total transcriptional activity synchronized with putative hosts. Taken together, these data reveal a microbial plankton community that is shaped by recycled production and tightly controlled by Fe-limitation and viral activity

Dynamics of Alexandrium fundyense blooms and shellfish toxicity in the Nauset Marsh System of Cape Cod (Massachusetts, USA)

Author Posting. © The Author(s), 2011. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Harmful Algae 12 (2011): 26–38, doi:10.1016/j.hal.2011.08.009.Paralytic Shellfish Poisoning (PSP) toxins are annually recurrent along the Massachusetts coastline (USA), which includes many small embayments and salt ponds. Among these is the Nauset Marsh System (NMS), which has a long history of PSP toxicity. Little is known, however, about the bloom dynamics of the causative organism Alexandrium fundyense within that economically and socially important system. The overall goal of this work was to characterize the distribution and dynamics of A. fundyense blooms within the NMS and adjacent coastal waters by documenting the distribution and abundance of resting cysts and vegetative cells. Cysts were found predominantly in three drowned kettle holes or salt ponds at the distal ends of the NMS - Salt Pond, Mill Pond, and Town Cove. The central region of the NMS had a much lower concentration of cysts. Two types of A. fundyense blooms were observed. One originated entirely within the estuary, seeded by cysts in the three seedbeds. These blooms developed independently of each other and of the A. fundyense population observed in adjacent coastal waters outside the NMS. The temporal development of the blooms was different in the three salt ponds, with initiation differing by as much as 30 days. These differences do not appear to reflect the initial cyst abundances in these locations, and may simply result from higher cell retention and higher nutrient concentrations in Mill Pond, the first site to bloom. Germination of cysts accounted for a small percentage of the peak cell densities in the ponds, so population size was influenced more by the factors affecting growth than by cyst abundance. Subsurface cell aggregation (surface avoidance) limited advection of the vegetative A. fundyense cells out of the salt ponds through the shallow inlet channels. Thus, the upper reaches of the NMS are at the greatest risk for PSP since the highest cyst abundances and cell concentrations were found there. After these localized blooms in the salt ponds peaked and declined, a second, late season bloom occurred within the central portions of the NMS. The timing of this second bloom relative to those within the salt ponds and the coastal circulation patterns at that time strongly suggest that those cells originated from a regional A. fundyense bloom in the Gulf of Maine, delivered to the central marsh from coastal waters outside the NMS through Nauset Inlet. These results will guide policy decisions about water quality as well as shellfish monitoring and utilization within the NMS and highlight the potential for “surgical” closures of shellfish during PSP events, leaving some areas open for harvesting while others are closed.This work was supported by NOAA Grant NA06OAR4170021, NPS Grant H238015504 and by the Woods Hole Center for Oceans and Human Health through NSF Grants OCE-0911031 and OCE-0430724 and NIEHS Grant 1P50-ES01274201. B.G.C. was supported by a Xunta de Galicia Ángeles Alvariño fellowship and the Stanley W. Watson Chair for Excellence in Oceanography under a Postdoctoral program at the Woods Hole Oceanographic Institution