This dataset includes RNA-seq differential expression results of Pfam domains within mixotrophs from an iron incubation experiment using upwelled waters sampled in the California Current System (CCS) during the PUPCYCLE II cruise with Chief Scientist Adrian Marchetti. PUPCYCLE II (Phytoplankton response to the UPwelling CYCLE) took place onboard the R/V Sally Ride from May 29th to June 10th, 2023. Freshly upwelled seawater for the incubation experiment was collected within the northern CCS and placed into designated cubitainers. Three cubitainers were immediately harvested for the initial timepoint (T0). The remaining twenty-seven cubitainers were assigned treatments: nine were unamended (Ctrl), nine were amended with 5 nM FeCl2 (Fe), and nine were amended with 200 nM Desferrioxamine B, a strong iron chelator (DFB). Three cubitainers from each treatment were harvested for each of the three subsequent timepoints: 48 hours (T1), 168 hours (T2), and 264 hours (T3) after incubation. Approximately 4 liters of each sample was harvested for RNA-seq of the natural phytoplankton community, or metatranscriptomics. The dataset provides differential expression results of Pfam domains within mixotrophs (and which genera they map to), specifically comparing expression levels between +Fe and DFB at T1 and T2. This data includes domains that significantly differ in expression between treatments in mixotrophs, and results were used to understand phagotrophic versus phototrophic gene expression based on iron bioavailability.

Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • Data Processing Description
  • BCO-DMO Processing Description
• Related Publications
• Related Datasets
• Parameters
• Instruments
• Deployments
• Project Information
• Funding

This dataset is one of many generated from an iron incubation experiment conducted as part of the PUPCYCLE II cruise in May and June 2023. The different analyses are listed here, with links to other datasets from this study in the Related Datasets section.

Analyses include:

• Physiology (dataset 994607)
  • Dissolved inorganic nutrients
  • Size-fractionated chlorophyll a
  • Size-fractionated isotope uptake rates
  • Flow cytometry
  • FlowCAM analysis
  • FLP experiments for mixotroph grazing
• RNA taxonomy data (dataset 1003525)
• Differential expression: Pfam
  • Mixotroph Pfam (this dataset 1004659)
  • Diatom Pfam (dataset 1004674)
• Differential expression: KEGG gene (dataset 1004689)
• Differential expression: KEGG pathways (dataset1004704)
• Dissolved iron concentrations (dataset 1004007)

See Related Datasets section below for links to above mentioned datasets.

Data collection took place on the R/V Sally Ride from May 29th to June 10th, 2023. To simulate upwelling conditions under different iron treatments, an onboard incubation experiment was conducted. Seawater for the incubation experiment was collected within the northern CCS, off the southern coast of Oregon at 43°02'42.7"N, 124°33'07.2"W. The collected seawater was deemed as freshly upwelled water and taken from a depth of 55 meters – which corresponds to a depth slightly below the euphotic zone receiving less than 1% irradiance – using trace-metal clean techniques on May 30th, 2023, 13:30 GMT. The seawater was pumped and homogenized using trace metal clean techniques, then transferred into a total of thirty 20 L low-density polyethylene cubitainers. Three cubitainers were immediately harvested for the initial timepoint (T0). The remaining twenty-seven cubitainers were assigned treatments: nine were unamended (Ctrl), nine were amended with 5 nM FeCl₂ (+Fe), and nine were amended with 200 nM desferrioxamine B, a strong iron chelator that inhibits dissolved iron uptake (DFB). The cubitainers were placed in an on-deck incubator covered with two layers of neutral density screening to simulate 26% of incidence irradiance supplied with flow-through surface seawater to maintain ambient surface temperature. Three cubitainers from each treatment were harvested for each of the three subsequent timepoints: 48 hours (T1), 168 hours (T2), and 264 hours (T3) after incubation.

RNA Collection and Metatranscriptomics. Cubitainers were harvested for RNA by collecting and filtering approximately 2.5 to 4 L of each sample onto 0.8 μm Pall Supor filters (142 mm) using a peristaltic pump; samples were then immediately flash frozen in liquid nitrogen and stored at -80°C. RNA was extracted using the RNAqueous-4PCR kit, per instructions from the manufacturer with few modifications – filters were cut up due to their large size, 200 μL of glass beads were added, and 3 mL of lysis buffer was added. RNA samples were sent to GENEWIZ for library preparation and sequencing with PolyA tail selection. Sequencing was performed on an Illumina HiSeq 4000 with a 2x150 bp configuration. GENEWIZ provided raw paired-end read sequences for each sample. 

Raw reads were trimmed using Trim Galore v0.6.10 (Krueger, 2015) and quality control was assessed through FastQC (Martin, 2011). A de novo metatranscriptome assembly was conducted using rnaSPAdes v3.15.5 for individual assemblies (Bushmanova et al., 2019) and CD-HIT v4.8.1 for a grand assembly (Li & Godzik, 2006). TransDecoder v5.7.1 was used to determine the most likely open reading frame (ORF) for each contig (Haas, 2023). ORFs were annotated using the Marine Functional Eukaryotic Reference Taxa (MarFERReT) database v1.1.1 (e-value < 1e^-06) (Groussman et al., 2023) for NCBI taxonomies (Federhen, 2012), PR² taxonomies (Guillou et al., 2012) and Pfam 34.0 functions (Mistry et al., 2021). To remove any potential bacterial contamination, ORFs were also annotated against the PhyloDB v1076 database using EUKulele (e-value < 1e^-06) (Krinos et al., 2020). Any ORF with a higher bitscore for a bacteria or virus compared to its MarFERReT annotation was removed (resulting in < 3% of ORFs removed). To maximize functional annotation coverage, ORFs were annotated using eggNOG-mapper v2.1.12 (Cantalapiedra et al., 2021) to obtain annotations from the Kyoto Encyclopedia of Genes and Genomes (KEGG) (Kanehisa, 2000). Trimmed samples were then aligned to the grand assembly using Salmon v1.10.3 (Patro et al., 2017) and exported into a comprehensive counts table using tximport (Soneson et al., 2016). Reads not mapping to a protist group as defined by MarFERReT annotations were removed before downstream analysis. 

DESeq2 was used to obtain normalized counts and conduct differential expression analyses (Love et al., 2014). Differential expression analysis for Pfam protein domains was performed for reads mapping to mixotrophs. Reads not mapping to mixotrophs were removed prior to normalization and statistical testing. Mixotrophs were identified based on constitutive mixotroph genera within the Mixoplankton Database (MDB) (Mitra et al., 2023). Reads were summed based on annotation ID prior to entering DESeq2 analysis; if a contig annotated to multiple IDs, its reads were evenly distributed between the mapped annotations. The differential expression analyses only focused on comparing +Fe vs DFB at T1 and T2. Normalized counts for each Pfam protein domain were also obtained to determine relative contributions of each mixotroph genera to the annotation ID. 

The metatranscriptomic pipeline code used for this project is publicly available at https://github.com/emilyspeciale/Speciale-Metatranscriptomics.

The following software was used:

Trim Galore v0.6.10
CD-HIT v4.8.1 
TransDecoder v5.7.1 
Marine Functional Eukaryotic Reference Taxa (MarFERReT) database v1.1.1 
PhyloDB v1076 database using EUKulele 
eggNOG-mapper v2.1.12 
Salmon v1.10.3 
tximport 
DESeq2

CURATION ACTIONS PERFORMED ON DATA
- Loaded source file "BCO_DMO_PUPCYCLEII_MixoPfamDE.csv" into the BCO-DMO system converting missing data identifier NA to empty cell
- Set data types for mixotroph columns as number, others to string
- Exported final file as "1004659_v1_mixotroph_pfam.csv"

CURATION ACTIONS PERFORMED ON METADATA
- Changed in-line citations to have format of (author, year) replacing number references.
- Added details to the parameter description for log2FoldChange -->Base-2 logarithm of the ratio of normalized read abundance under the iron-amended (+Fe) treatment to normalized read abundance under the desferrioxamine B (DFB) treatment, calculated for each Pfam domain
- Corrected minor typos and punctuation

ISSUES POTENTIALLY IMPACTING REUSE
N/A

Coverage: California Upwelling Zone

NSF Award Abstract:
Upwelling zones are hotspots of photosynthesis that are very dynamic in space and time. Microsocopic algae, known as phytoplankton, bloom when deep, nutrient-rich waters are upwelled into sunlit surface layers of the ocean, providing nourishment that supports productive food webs and draws down carbon dioxide (CO2) from the atmosphere to the deep ocean. Photosynthetic microbes in these regions must constantly adapt to changes in their chemical and physical environments. For example, subsurface populations respond to changes in light as they approach the surface. When upwelled waters move offshore, cells sink out of the illuminated zone, establishing seed populations that remain inactive until the next upwelling event. This process is called the upwelling conveyor belt cycle (UCBC). How phytoplankton respond to these changes in environmental conditions and how they may influence their nutrient requirements remains unknown. With future ocean changes predicted to alter seawater chemistry, including ocean acidification and decreased iron availability, some phytoplankton groups may be more vulnerable than others. Accompanying educational activities provide learning experiences to enhance understanding and awareness of marine microbes. The development of a research hub at UNC aims to provide infrastructure and support for scientists and students conducting research on environmental genomics. A laboratory component for an upper-level undergraduate course focused on marine phytoplankton is being developed. Educational outreach activities to broader communities include creation of a lesson plan on phytoplankton in upwelling zones and a virtual research cruise experience for middle-school students, as well as a hands-on lab activity for a local museum focused on marine phytoplankton and the important roles they play in shaping our planet.

The project examines how phytoplankton respond at the molecular and physiological level to the different UCBC stages, which seed populations (i.e., surface versus subsurface) contribute most to phytoplankton blooms during upwelling events of varying intensity, how phytoplankton elemental compositions are altered throughout UCBC stages, and how future predicted ocean conditions will affect the phytoplankton responses to UCBC conditions. This project contains both laboratory and fieldwork. In the laboratory, phytoplankton isolates recently obtained from upwelling regions are exposed to simulated UCBC conditions to examine changes in gene expression, growth and photosynthetic characteristics and elemental composition. Cultures are subjected to both current and future ocean conditions, including reduced iron availability and higher CO2. In the field, research cruises within upwelling regions study the dynamics of natural phytoplankton communities (both surface and subsurface) experiencing upwelling and relaxation and within simulated upwelling incubation experiments. Knowledge of how phytoplankton are affected by UCBC conditions at an integrated molecular, physiological and elemental level under both current and future scenarios is imperative for the proper conservation and management of these critically important ecosystems.