Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • Data Processing Description
  • BCO-DMO Processing Description
  • Problem Description
• Parameters
• Instruments
• Project Information
• Funding

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).

Growth was monitored daily at the same time of day by in vivo chlorophyll fluorescence (relative fluorescence units, RFUs) on a Turner Designs Aquafluor handheld fluorometer (in vivo chlorophyll a channel). Briefly, each culture was mixed by swirling 3 times clockwise and then 3 times counterclockwise before aseptically removing a 2 mL subsample with a serological pipette. The subsample was transferred to a 10 mm × 10 mm methacrylate cuvette and placed in the Aquafluor for RFU measurement. The efficiency of photosystem II (PSII), Fv/Fm, was determined daily for each replicate using a Turner Designs AquaFlash handheld fluorometer (factory calibrated). Again, each culture was mixed (as described above) before aseptically removing a 3 mL subsample with a serological pipette. The subsample was transferred to a 10 mm × 10 mm quartz cuvette and placed in the AquaFlash for Fv/Fm measurement. If in vivo chlorophyll a fluorescence exceeded the maximum linear range, samples were diluted with artificial seawater L1-Si media containing the corresponding nitrogen concentration to bring the reading within range.

Data was processed in R version 4.4.0. To arrive at a final in vivo chlorophyll a RFU, readings were multiplied by dilution factors, if applied.

- Loaded data from "Chla_FvFm_GhuxleyiCultures_WHOI_Oct2023-Jan2024.xlsx" (sheet 1), treating empty strings and "nd" as missing values
- Converted the `date` column from format `%m-%d-%y` to ISO date format `%Y-%m-%d`, output as date type
- Renamed column `Fv/Fm` to `Fv_Fm`
- Set column types: `F0` and `Fm` as string; `Fv_Fm`, `chla_read`, `chla_total`, and `dilution_factor` as number; `date` as date with format `%Y-%m-%d`; `nitrogen` and `strain` as integer; `replicate` as string; `temperature_c` as integer
- Output written to "999184_v1_G_huxleyi_chlorophyll_and_photosynthetic_efficiencies.csv"

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.