Matching Items (8)

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Single cell RT-qPCR based ocean environmental sensing device development

Description

This thesis research focuses on developing a single-cell gene expression analysis method for marine diatom Thalassiosira pseudonana and constructing a chip level tool to realize the single cell RT-qPCR analysis.

This thesis research focuses on developing a single-cell gene expression analysis method for marine diatom Thalassiosira pseudonana and constructing a chip level tool to realize the single cell RT-qPCR analysis. This chip will serve as a conceptual foundation for future deployable ocean monitoring systems. T. pseudonana, which is a common surface water microorganism, was detected in the deep ocean as confirmed by phylogenetic and microbial community functional studies. Six-fold copy number differences between 23S rRNA and 23S rDNA were observed by RT-qPCR, demonstrating the moderate functional activity of detected photosynthetic microbes in the deep ocean including T. pseudonana. Because of the ubiquity of T. pseudonana, it is a good candidate for an early warning system for ocean environmental perturbation monitoring. This early warning system will depend on identifying outlier gene expression at the single-cell level. An early warning system based on single-cell analysis is expected to detect environmental perturbations earlier than population level analysis which can only be observed after a whole community has reacted. Preliminary work using tube-based, two-step RT-qPCR revealed for the first time, gene expression heterogeneity of T. pseudonana under different nutrient conditions. Heterogeneity was revealed by different gene expression activity for individual cells under the same conditions. This single cell analysis showed a skewed, lognormal distribution and helped to find outlier cells. The results indicate that the geometric average becomes more important and representative of the whole population than the arithmetic average. This is in contrast with population level analysis which is limited to arithmetic averages only and highlights the value of single cell analysis. In order to develop a deployable sensor in the ocean, a chip level device was constructed. The chip contains surface-adhering droplets, defined by hydrophilic patterning, that serve as real-time PCR reaction chambers when they are immersed in oil. The chip had demonstrated sensitivities at the single cell level for both DNA and RNA. The successful rate of these chip-based reactions was around 85%. The sensitivity of the chip was equivalent to published microfluidic devices with complicated designs and protocols, but the production process of the chip was simple and the materials were all easily accessible in conventional environmental and/or biology laboratories. On-chip tests provided heterogeneity information about the whole population and were validated by comparing with conventional tube based methods and by p-values analysis. The power of chip-based single-cell analyses were mainly between 65-90% which were acceptable and can be further increased by higher throughput devices. With this chip and single-cell analysis approaches, a new paradigm for robust early warning systems of ocean environmental perturbation is possible.

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Agent

Created

Date Created
  • 2013

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Evaluating the Use of Surrogates of Marine Mammal Species Representation in Biodiversity Conservation Planning

Description

Biodiversity is required to guarantee proper ecosystem structure and function. However, increasing anthropogenic threats are causing biodiversity loss around the world at an unprecedented rate, in what has been deemed

Biodiversity is required to guarantee proper ecosystem structure and function. However, increasing anthropogenic threats are causing biodiversity loss around the world at an unprecedented rate, in what has been deemed the sixth mass extinction. To counteract this crisis, conservationists seek to improve the methods used in the design and implementation of protected areas, which help mitigate the impacts of human activities on species. Marine mammals are ecosystem engineers and important indicator species of ocean and human wellbeing. They are also disproportionally less known and more threatened than terrestrial mammals. Therefore, surrogates of biodiversity must be used to maximize their representation in conservation planning. Some of the most effective surrogates of biodiversity known have only been tested in terrestrial systems. Here I test complementarity, rarity, and environmental diversity as potential surrogates of marine mammal representation at the global scale, and compare their performance against species richness, which is the most popular surrogate used to date. I also present the first map of marine mammal complementarity, and assess its relationship with environmental variables to determine if environmental factors could also be used as surrogates. Lastly, I determine the global complementarity-based hotspots of marine mammal biodiversity, and compare their distributions against current marine protected area coverage and exposure to global indices of human threats, to elucidate the effectiveness of current conservation efforts. Results show that complementarity, rarity, and environmental diversity are all efficient surrogates, as they outcompete species richness in maximizing marine mammal species representation when solving the minimum-set coverage problem. Results also show that sea surface temperature, density, and bathymetry are the top environmental variables most associated with complementarity of marine mammals. Finally, gap analyses show that marine mammals are overall poorly protected, yet moderately exposed to hotspots of cumulative human impacts. The wide distribution of marine mammals justify global studies like the ones here presented, to determine the best strategy for their protection. Overall, my findings show that less popular surrogates of biodiversity are more effective for marine mammals and should be considered in their management, and that the expansion of protected areas in their most important habitats should be prioritized.

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Agent

Created

Date Created
  • 2019

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Temporal and light-dependent variability of algal communities in land-fast Arctic sea ice

Description

Sea ice algae dominated by diatoms inhabit the brine channels of the Arctic sea ice and serve as the base of the Arctic marine food web in the spring. I

Sea ice algae dominated by diatoms inhabit the brine channels of the Arctic sea ice and serve as the base of the Arctic marine food web in the spring. I studied sea ice diatoms in the bottom 10 cm of first year land-fast sea ice off the coast of Barrow, AK, in spring of 2011, 2012, and 2013. I investigated the variability in the biomass and the community composition of these sea-ice diatoms between bloom phases, as a function of overlying snow depth and over time. The dominant genera were the pennate diatoms Nitzschia, Navicula, Thalassiothrix, and Fragilariopsis with only a minor contribution by centric diatoms. While diatom biomass as estimated by organic carbon changed significantly between early, peak, and declining bloom phases (average of 1.6 mg C L-1, 5.7 mg C L-1, and 1.0 mg C L-1, respectively), the relative ratio of the dominant diatom groups did not change. However, after export, when the diatoms melt out of the ice into the underlying water, diatom biomass dropped by ~73% and the diatom community shifted to one dominated by centric diatoms. I also found that diatom biomass was ~77% lower under high snow cover (>20 cm) compared to low snow cover (<8 cm); however, the ratio of the diatom categories relative to particulate organic carbon (POC) was again unchanged. The diatom biomass was significantly different between the three sampling years (average of 2.4 mg C L-1 in 2011, 1.1 mg C L-1 in 2012, and 5.4 mg C L-1 in 2013, respectively) as was the contribution of all of the dominant genera to POC. I hypothesize the latter to be due to differences in the history of ice sheet formation each year. The temporal variability of these algal communities will influence their availability for pelagic or benthic consumers. Furthermore, in an Arctic that is changing rapidly with earlier sea ice and snowmelt, this time series study will constitute an important baseline for further studies on how the changing Arctic influences the algal community immured in sea ice.

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Agent

Created

Date Created
  • 2014

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Bottom-up and top-down controls on the microzooplankton community in the Sargasso Sea

Description

Microzooplankton, mainly heterotrophic unicellular eukaryotes (protists), play an important role in the cycling of nutrients and carbon in the sunlit (euphotic) zone of the world’s oceans. Few studies have investigated

Microzooplankton, mainly heterotrophic unicellular eukaryotes (protists), play an important role in the cycling of nutrients and carbon in the sunlit (euphotic) zone of the world’s oceans. Few studies have investigated the microzooplankton communities in oligotrophic (low-nutrient) oceans, such as the Sargasso Sea. In this study, I investigate the seasonal and interannual dynamics of the heterotrophic protists, particularly the nanoflagellate, dinoflagellate, and ciliate communities, at the Bermuda Atlantic Time Series site and surrounding areas in the Sargasso Sea. In addition, I test the hypotheses that the community is controlled though bottom-up and top-down processes. To evaluate the bottom-up hypothesis, that the protists are controlled by prey availability, I test whether the protist abundance co-varies with the abundance of potential prey groups. Predation experiments with zooplankton were conducted and analyzed to test top-down control on the protists. I found distinguishable trends in biomass of the different protist groups between years and seasons. Nanoflagellates and dinoflagellates had higher biomass during the summer (28 ± 5 mgC/m2 and 44 ± 21 mgC/m2) than during the winter (17 ± 8 mgC/m2 and 30 ± 11 mgC/m2). Ciliates displayed the opposite trend with a higher average biomass in the winter (15 ± 9 mgC/m2) than in summer (5 ± 2 mgC/m2). In testing my bottom-up hypothesis, I found weak but significant positive grazer/prey relationships that indicate that nanoflagellates graze on picophytoplankton in winter and on the pico-cyanobacterium Prochlorococcus in summer. I found evidence that ciliates graze on Synechococcus in winter. I found weak but significant negative correlation between dinoflagellates and Prochlorococcus in summer. The predation experiments testing the top-down hypothesis did not show a clear top-down control, yet other studies in the region carried out during our investigation period support predation of the protists by the zooplankton. Overall, my results suggest a combination of bottom-up and top-down controls on these heterotrophic protists, however, further investigation is necessary to reveal the detailed trophic dynamics of these communities.

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Agent

Created

Date Created
  • 2016

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Aggregation of marine pico-cyanobacteria

Description

Marine pico-cyanobacteria of the genera Synechococcus and Prochlorococcus carry out nearly two thirds of the primary production in oligotrophic oceans. These cyanobacteria are also considered an important constituent of the

Marine pico-cyanobacteria of the genera Synechococcus and Prochlorococcus carry out nearly two thirds of the primary production in oligotrophic oceans. These cyanobacteria are also considered an important constituent of the biological carbon pump, the photosynthetic fixation of CO2 to dissolved and particulate organic carbon and subsequent export to the ocean’s interior. But single cells of these cyanobacteria are too small to sink, so their carbon export has to be mediated by aggregate formation and/or consumption by zooplankton that produce sinking fecal pellets. In this dissertation, I investigated for the first time the aggregation of these cyanobacteria by studying the marine Synechococcus sp. strain WH8102 as a model organism. I first found in culture experiments that Synechococcus cells aggregated and that such aggregation of cells was related to the production of transparent exopolymeric particles (TEP), known to provide the main matrix of aggregates of eukaryotic phytoplankton. I also found that despite the lowered growth rates, cells in the nitrogen or phosphorus limited cultures had a higher cell-normalized TEP production and formed a greater total volume of aggregates with higher settling velocities compared to cells in the nutrient replete cultures. I further studied the Synechococcus aggregation in roller tanks that allow the simulation of aggregates settling in the water column, and investigated the effects of the clays kaolinite and bentonite that are commonly found in the ocean. In the roller tanks, Synechococcus cells formed aggregates with diameters of up to 1.4 mm and sinking velocities of up to 440 m/d, comparable to those of larger eukaryotic phytoplankton such as diatoms. In addition, the clay minerals increased the number but reduced the size of aggregates, and their ballasting effects increased the sinking velocity and the carbon export potential of the aggregates. Lastly, I investigated the effects of heterotrophic bacteria on the Synechococcus aggregation, and found that heterotrophic bacteria generally resulted in the formation of fewer, but larger and faster sinking aggregates, and eventually led to an enhanced aggregation of cells and particles. My study contributes to the understanding of the role of marine pico-cyanobacteria in the ecology and biogeochemistry of oligotrophic oceans.

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Agent

Created

Date Created
  • 2016

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Growth and grazing mortality of pico- and nano-phytoplankton and their role in the carbon export in the Sargasso Sea

Description

The ocean sequesters more than 25% of the carbon released by anthropogenic action every year, and oligotrophic oceans, such as the Sargasso Sea, are responsible for about 50% of the

The ocean sequesters more than 25% of the carbon released by anthropogenic action every year, and oligotrophic oceans, such as the Sargasso Sea, are responsible for about 50% of the global carbon export. Pico- and nano-phytoplankton (cells < 5 µm), mostly unicellular eukaryotes (protists) and cyanobacteria, dominate the primary production in the Sargasso Sea; however, little is known about their contribution to the export of carbon into the deep ocean via sinking particles. The overall goal of this study is to examine the link between growth and grazing rates of pico- and nano-phytoplankton and the carbon export in the Sargasso Sea. I investigate three aspects: 1) how microzooplankton grazing and physical forcing affect taxon-specific primary productivity in this region, 2) how these microbial trophic dynamics impact their contribution to the export of particulate matter, and 3) how much pico-phytoplankton, specifically the pico-cyanobacteria Synechococcus and Prochlorococcus, contribute to the carbon export. I collected seawater samples within the sunlit (euphotic) zone, and sinking particles at 150 m depth using particle traps in the Sargasso Sea during the winter and summer seasons of 2011 and 2012. I conducted dilution experiments to determine the growth and grazing rates of the pico- and nano-phytoplankton community, and used 454 pyrosequencing and quantitative Polymerase Chain Reaction to measure the relative and absolute contribution of these primary producers to the plankton community within the euphotic zone and in the sinking particles. I found that micrograzing controls taxon-specific primary production, and that microbial trophic dynamics impact directly the taxonomical composition of the sinking particles. For the first time, I was able to quantify clade-specific carbon export of pico-cyanobacteria and found that, despite their small size, these tiny primary producers are capable of sinking from the surface to the deeper oceans. However, their contribution to the carbon flux is often less than one tenth of their biomass contribution in the euphotic zone. Our study provides a comprehensive approach to better understand the role of pico- and nano-phytoplankton in the carbon cycle of oligotrophic oceans, and a baseline to study changes in the carbon export in future warmer oceans.

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Date Created
  • 2016

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Protist and cyanobacterial contributions to particle flux in oligotrophic ocean regions

Description

The oceans play an essential role in global biogeochemical cycles and in regulating climate. The biological carbon pump, the photosynthetic fixation of carbon dioxide by phytoplankton and subsequent sequestration of

The oceans play an essential role in global biogeochemical cycles and in regulating climate. The biological carbon pump, the photosynthetic fixation of carbon dioxide by phytoplankton and subsequent sequestration of organic carbon into deep water, combined with the physical carbon pump, make the oceans the only long-term net sink for anthropogenic carbon dioxide. A full understanding of the workings of the biological carbon pump requires a knowledge of the role of different taxonomic groups of phytoplankton (protists and cyanobacteria) to organic carbon export. However, this has been difficult due to the degraded nature of particles sinking into particle traps, the main tools employed by oceanographers to collect sinking particulate matter in the ocean. In this study DNA-based molecular methods, including denaturing gradient gel electrophoresis, cloning and sequencing, and taxon-specific quantitative PCR, allowed for the first time for the identification of which protists and cyanobacteria contributed to the material collected by the traps in relation to their presence in the euphotic zone. I conducted this study at two time-series stations in the subtropical North Atlantic Ocean, one north of the Canary Islands, and one located south of Bermuda. The Bermuda study allowed me to investigate seasonal and interannual changes in the contribution of the plankton community to particle flux. I could also show that small unarmored taxa, including representatives of prasinophytes and cyanobacteria, constituted a significant fraction of sequences recovered from sediment trap material. Prasinophyte sequences alone could account for up to 13% of the clone library sequences of trap material during bloom periods. These observations contradict a long-standing paradigm in biological oceanography that only large taxa with mineral shells are capable of sinking while smaller, unarmored cells are recycled in the euphotic zone through the microbial loop. Climate change and a subsequent warming of the surface ocean may lead to a shift in the protist community toward smaller cell size in the future, but in light of these findings these changes may not necessarily lead to a reduction in the strength of the biological carbon pump.

Contributors

Agent

Created

Date Created
  • 2011

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Seasonal and interannual variability of the Sargasso Sea plankton community

Description

Phytoplankton comprise the base of the marine food web, and, along with heterotrophic protists, they are key players in the biological pump that transports carbon from the surface to the

Phytoplankton comprise the base of the marine food web, and, along with heterotrophic protists, they are key players in the biological pump that transports carbon from the surface to the deep ocean. In the world's subtropical oligotrophic gyres, plankton communities exhibit strong seasonality. Winter storms vent deep water into the euphotic zone, triggering a surge in primary productivity in the form of a spring phytoplankton bloom. Although the hydrographic trends of this "boom and bust" cycle have been well studied for decades, community composition and its seasonal and annual variability remains an integral subject of research. It is hypothesized here that proportions of different phytoplankton and protistan taxa vary dramatically between seasons and years, and that picoplankton represent an important component of this community and contributor to carbon in the surface ocean. Monthly samples from the Bermuda Atlantic Time-series Study (BATS) site were analyzed by epifluorescence microscopy, which permits classification by morphology, size, and trophic type. Epifluorescence counts were supplemented with flow cytometric quantification of Synechococcus, Prochlorococcus, and autotrophic pico- and nanoeukaryotes. Results from this study indicate Synechococcus and Prochlorococcus, prymnesiophytes, and hetero- and mixotrophic nano- and dinoflagellates were the major players in the BATS region plankton community. Ciliates, cryptophytes, diatoms, unidentified phototrophs, and other taxa represented rarer groups. Both flow cytometry and epifluorescence microscopy revealed Synechococcus to be most prevalent during the spring bloom. Prymnesiophytes likewise displayed distinct seasonality, with the highest concentrations again being noted during the bloom. Heterotrophic nano- and dinoflagellates, however, were most common in fall and winter. Mixotrophic dinoflagellates, while less abundant than their heterotrophic counterparts, displayed similar seasonality. A key finding of this study was the interannual variability revealed between the two years. While most taxa were more abundant in the first year, prymnesiophytes experienced much greater abundance in the second year bloom. Analyses of integrated carbon revealed further stark contrasts between the two years, both in terms of total carbon and the contributions of different groups. Total integrated carbon varied widely in the first study year but displayed less fluctuation after June 2009, and values were noticeably reduced in the second year.

Contributors

Agent

Created

Date Created
  • 2010