| Contributors | Affiliation | Role |
|---|---|---|
| Alexander, Harriet | Woods Hole Oceanographic Institution (WHOI) | Co-Principal Investigator |
| Dyhrman, Sonya T. | Lamont-Doherty Earth Observatory (LDEO) | Co-Principal Investigator |
| Haley, Sheean | Lamont-Doherty Earth Observatory (LDEO) | Scientist |
G. huxleyi strains RCC874 and RCC914 were obtained from the Roscoff Culture Collection (RCC). The cultures were transferred from the maintenance stock into artificial seawater (40 g Instant Ocean® Sea Salt per L Milli-Q® water) amended with nutrients, trace metals, and vitamins according to the L1 recipe (https://ncma.bigelow.org/algae-media-recipes) without silica (herein reported as L1-Si). The temperatures were selected to simulate no heat stress (18°C), moderate heat stress (22°C), and high heat stress conditions (25°C and 28°C for RCC874 and RCC914, respectively). The cultures were acclimated to the temperature conditions under a 14:10 light/dark cycle for approximately 10 days prior to the start of the experiment.
Following acclimation, cultures were transferred in triplicate into 2.8 L Fernbach flasks at an initial concentration of 1.0 ✕ 10⁵ cells mL⁻¹. The cultures were divided into two conditions: nitrogen-replete (N replete; L1-Si with standard 8.82 ✕ 10⁻⁴ M NaNO₃) and nitrogen-limited (N limited; L1-Si with 8.82 ✕ 10⁻⁵ M NaNO₃). When N-limited cell counts significantly deviated from the N-replete condition, between 7–9 days after inoculation, the cultures were sampled for particulate nutrients. Samples for particulate nutrient composition were filtered onto muffled 0.7-µm Whatman GF/F filters using gentle vacuum pressure, sampling 25–100 mL of culture depending on cell density. Samples were processed for particulate carbon (PC), particulate nitrogen (PN), and particulate phosphorus (PP) at the University of Maryland Center for Environmental Science Chesapeake Biological Laboratory Nutrient Analytical Services Laboratory.
FCS data were processed using flowCore version 2.18 in R version 4.4.0. Maximum exponential phase-specific growth rate in cells per day was calculated in R version 4.4.0 using the growthrates package version 0.8.4, using the function all_splines() (spar = 0.5) (Petzoldt, 2022).
- Loaded data from "ParticulateNutrients_GhuxleyiCultures_WHOI_Oct2023-Jan2024.xlsx" (sheet 1) with headers on row 1, treating "" and "nd" as missing values
- Converted Sample_date column from format "%m-%d-%y" to ISO date format "%Y-%m-%d", outputting as date type
- Renamed column Unit to Result_Unit
- Set types for multiple columns: MDL_RL, Method, Method_of_preservation, Parameter, Result_Unit, rep as string; Result as number; Sample_date as date ("%Y-%m-%d"); n_conc as integer; strain and temp as integer
- Output saved to "999174_v1_G_huxleyi_particulate_nutrients.csv"
| Dataset-specific Instrument Name | BD Accuri C6 Plus equipped with a C-Sampler (BD, USA) |
| Generic Instrument Name | BD Biosciences Accuri C6 Plus flow cytometer |
| Dataset-specific Description | Cell abundances and optical properties were measured daily using flow cytometry with a BD Accuri C6 Plus equipped with a C-Sampler (BD, USA). |
| Generic Instrument Description | A flow cytometer designed to detect fluorochromes (including FITC, PE, APC), polymer dyes, and fluorescent proteins (including GFP, YFP, mCherry). The instrument is equipped with a blue and red laser, two light scatter detectors, and four fluorescence detectors with user-interchangeable filters. A non-pressurised peristaltic pump system drives the fluidics. The system monitors the sample volume pulled per run and can calculate absolute counts or sample concentration per microliter (uL). In the standard configuration (3-blue/1-red), three detectors read fluorescence emissions from fluorochromes excited by the blue laser, while a fourth detector reads emissions from fluorochromes excited by the red laser. The optional Selectable Laser Module allows the system to operate in 2-blue/2-red and 4-blue configurations. The minimum detectable particle size and sample volume are 0.5 micrometers (um) and 0.5 uL respectively. The maximum number of events is 1 million with an acquisition rate of up to 10,000 events/second. |
| Dataset-specific Instrument Name | CE-440 Elemental Analyzer (Exeter Analytical, Inc., USA) |
| Generic Instrument Name | Elemental Analyzer |
| Dataset-specific Description | PC and PN concentration were determined using a CE-440 Elemental Analyzer (Exeter Analytical, Inc., USA) (Reference Method: EPA 440.0). |
| Generic Instrument Description | Instruments that quantify carbon, nitrogen and sometimes other elements by combusting the sample at very high temperature and assaying the resulting gaseous oxides. Usually used for samples including organic material. |
NSF Award Abstract:
Emiliania huxleyi is a numerically and ecologically important phytoplankton species in the ocean known for its cosmopolitan distribution and ability to form large blooms in coastal and open ocean regions. Studies of E. huxleyi variants in culture have found differences in growth, function and activity potential among them. The E. huxleyi variants also differ in some of the genes they carry. It has been hypothesized that this genomic variability may underlie the global success of this phytoplankton species by allowing adaptation of variants to diverse environments. Yet, the direct connection between genomic content and ecological success remains unclear. This project investigates how the conserved and variable portions of E. huxleyi genome may be connected to its success and its dynamics under varied environmental conditions. This work is critical to our understanding of how this important phytoplankton species may shift and respond to future changes. This project also supports the development of a series of hands-on activities designed to teach middle school students advance computational data analysis in ocean science. These activities are in collaboration with the Girls Who Code Club at the Our Sisters School, a tuition-free, non-sectarian, independent school for girls from low-income families, located in New Bedford, MA.
Understanding how phytoplankton diversity and phenotype are driven by changes in the environment is crucial for better predicting carbon cycle dynamics in the future ocean. While much work has investigated competition among phytoplankton species, intraspecific diversity and dynamics remain largely unknown for many eukaryotic phytoplankton. The overarching goal of this project is to define the role of the pan genome (set of variable genes) in E. huxleyi ecology and biogeography through a series of genomic analyses, computational field surveys, and laboratory-based experiments. This project is sequencing the genomes of several E. huxleyi isolates from across the global ocean and combining them with existing genome sequences to constrain the core and variable portions of the pan genome. Using this new pan genome reference database and leveraging global scale metagenomics and metatranscriptomic surveys, this project is estimating ecotype diversity of E. huxleyi across ocean regions to identify patterns of environmental selection. This project additionally focuses on identifying the physiological and transcriptional responses of a selection of sequenced strains and their responses to shifts in their nutrient environments in controlled laboratory studies. As E. huxleyi plays such a significant role in marine ecosystems and the global carbon cycle it is important that its pan genome and its impact on the biogeography and ecology of E. huxleyi is taken into consideration. It is likely that these dynamics are acutely important to predicting how this genus, and perhaps others, respond to changing environmental conditions in the future ocean.
This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.
| Funding Source | Award |
|---|---|
| NSF Division of Ocean Sciences (NSF OCE) | |
| NSF Division of Ocean Sciences (NSF OCE) |