Dataset: Pseudo-nitzschia counts, nutrients and more
Deployment: EN644

For ease of comparison to previous enumeration data in the URI GSO LTPTS dataset, Pseudo-nitzschia spp. cells were enumerated using similar methods with the same counting volume. Between 15 and 50 mL of whole seawater was preserved with 1% acidic Lugol’s solution in glass or plastic containers (with a silica bead later added). Samples were stored at 4 °C for less than a year before being counted. One mL of sample was placed in a Sedgewick-Rafter counting chamber (Science First / Wildco, Yulee, FL, USA), Pseudo-nitzschia spp. cells were identified at the genus level and counted using 20x phase contrast light microscopy on a BX40 (Olympus America Inc., Melville, NY, USA).  Pseudo-nitzschia spp. cell counts from the LTPTS were conducted on live samples using the same enumeration protocol on an Eclipse E800 (Nikon Instruments Inc., Melville, NY, USA). Cell counts by same-day LTPTS on the Eclipse E800 were preferentially used over our counts performed in the BX40. Cell counts from the LTPTS are available at  https://web.uri.edu/gso/research/plankton/data/.

Samples for nutrient analysis were collected from 0.2 mm polyethersulfone filter (Sterlitech, Kent, WA, USA) filtrate, and frozen at -20 °C until analysis within approximately a year.

Extracted chlorophyll a measurements were taken by vacuum filtering surface seawater in triplicate onto GF/F filters (0.6 – 0.8 µm particle retention; Whatman n.k.a. Cytiva, Marlborough, MA, USA). The filters were placed in a glass tube with 90% acetone and extracted in the dark at -20 °C. After 24 hours, samples were equilibrated at room temperature for 20 minutes, vortexed, filter removed, and extract transferred to a sample reading tube for the fluorometer. After the first reading, samples were acidified with 3 drops of 10% hydrochloric acid. Fluorometers were calibrated with commercially purchased chlorophyll a standards in 90% acetone solution (P/N: 10-850, Turner Designs, Inc., San Jose, CA, USA). Measurements from the Trilogy were taken as relative fluorescent units (RFUs) and then pigments were determined using an external calibration calculation following the manufacturer’s protocol from standards previously measured. The 10AU fluorometer is maintained by URI GSO.

At each sampling station, approximately 2 L of the surface seawater collected was filtered across a 47 mm, 5.0 mm polyester membrane filter (Sterlitech, Kent, WA, USA) to collect phytoplankton biomass. These filters were flash frozen in liquid nitrogen and stored at -80 °C until extraction. Phytoplankton-containing filters were extracted in 0.1 M acetic acid for four hours, vigorously vortexing each hour. Following extraction, samples were filtered using a 0.2 μm syringe filter directly into a 1.5 mL LC-MS vial for LC-MS/MS analysis. The LC-MS/MS method utilized a Prominence UFLC system (Shimadzu, Kyoto, Japan) coupled to a SCIEX 4600 Qtrap mass spectrometer (Sciex, Framingham, MA, USA). A Kinetex C18 column (150 mm x 4.6 mm, 2.6 μm) (Phenomenex, Torrance, CA, USA) was used for chromatographic separation of samples. The flow rate was 0.4 mL min-1 and the mobile phase solvents were H2O (A) and MeOH (B) each modified with 0.05% formic acid. A gradient method was employed for analysis: Initial conditions of 95% A and 5% B were held for 5 min with the eluent sent to waste for the first 2 min. Next, the % of B was increased to 50% from 5 to 15 min, with a final change to initial conditions (95% A and 5% B) from 16 to 20 min. The peak of DA eluted at 11.00 min. LC-MS/MS with MRM was employed for sensitivity and selectivity in DA detection and quantification. Analysis was carried out in positive mode, and three transitions from the protonated DA molecule were used: m/z 312 → 266, m/z 312 → 248, and m/z 312 → 193. DA was quantified during each sample set analysis period using an external calibration curve generated using pure DA standards of increasing concentrations (Sigma-Aldrich, St. Louis, MO, USA). Phytoplankton-associated DA measurements are described as ng pDA L-1 of filtered seawater.

The software KorDSS was used to access data from the YSI ProDSS.

Problem report: In general, sites were sampled weekly or twice a week, with higher frequency in the spring, summer, and fall and less frequently in the winter. Some sites only have short-term sampling efforts like from Fort Wetherill. Some samples from this dataset were selected for sequencing of the Pseudo-nitzschia species assemblages from corresponding plankton biomass filters. However, some of the sequenced samples do not have corresponding environmental metadata including Library IDs AS 467 – 489 & AS 493 – 495. 

Also see: NCBI Bioproject PRJNA690940 which contains the high throughput sequences associated with the SequenceSample_ID column of this datasheet and the resulting amplicon sequence variants (ASVs) of the Pseudo-nitzschia taxa recovered (NCBI Accession Numbers MW447658 - MW447770). The NCBI Bioproject and Accession Numbers will be active in January 2025 or upon publication of the associated manuscript, whichever occurs first.