Samples were selected from the NES-LTER transect and various time series sites in Narragansett Bay, Rhode Island (NB) during winter and summer periods from January 2018 through February 2023 to compare seasonal and regional patterns of Pseudo-nitzschia species composition and DA, as well as environmental drivers. NB and NES will henceforth be referred to as subregions of the larger Northeast U.S. Continental Shelf region, with NES specifically referring to the area spanned by the NES-LTER transect. Samples were collected on NES-LTER cruises (R/V Endeavor, R/V Atlantis) from 11 stations along a 150 km transect (n=77) each winter (January-February) and summer (July-August). Samples from three to four stations per cruise were used in this dataset spanning innershelf (L1), midshelf (L3, L4) and outershelf (L7, L8, L10) sections of the transect. The northernmost station, L1, is about 50 km from the mouth of NB. To collect plankton biomass for nucleic acid isolation, CTD rosette seawater from the surface and subsurface chlorophyll maximum (SCM) were passed via peristaltic pump over 25 mm 5 µm pore size filters (Sterlitech, Kent, WA, USA). Biomass filters were either flash frozen in liquid nitrogen (2018-2022) or placed in DNA/RNA shield (winter 2023; Zymo Research, Irvine, CA, USA) and stored in a -80°C freezer. The SCM depth varied as observed by in situ chlorophyll fluorescence, with a median depth of 28 m for summer and 19 m for winter samples. In cases where the SCM was not well defined due to water column mixing that typically took place in winter at nearshore stations, a sampling depth between 20 and 30 m was targeted.

In NB, surface seawater samples were collected from various sites in the East and West Passages including the Narragansett Bay Long-Term Plankton Time Series (NBPTS) site, Whale Rock (WR), Castle Hill Beach (CHB), East Passage (EP), and University of Rhode Island Graduate School of Oceanography (GSO) dock. Seawater was transported back to the laboratory and passed over 25 mm 5 µm pore size filters (Sterlitech, Kent, WA, USA) using a peristaltic pump before flash freezing in liquid nitrogen and storage at -80°C. To fill in several missing dates from this time series, six samples collected separately in the NBPTS (https://web.uri.edu/gso/research/plankton/) sampling program were used. These samples differed in collection methodology only by the filter pore size used (0.22 µm, Express Plus, Millipore Sigma) and vacuum as opposed to peristaltic filtration.

DNA was extracted from most NB and NES samples (n=219) using a modified version of the DNeasy Plant Kit (Qiagen, Germantown, MD, USA) that included a 1-minute bead beating step (0.1 mm and 0.5 mm Zirconia/Silica beads, BioSpec Products, Bartlesville, OK, USA) and two part elution into a total of 45 µL Buffer AE. Similarly, the six NBPTS samples were extracted using a modified version of the DNeasy Blood & Tissue kit (Qiagen, Germantown, MD, USA) with a 1-minute bead beating step and final elution into 50 µL Buffer AE. Some NES samples (n=18) were extracted using the Quick-DNA/RNA Miniprep Plus Kit (Zymo Research, Irvine, CA, USA) with a 1-minute bead beating step (0.4 mm Zirconium Beads, OPS Diagnostics, Lebanon, NJ, USA) and final elution into 50 µL nuclease-free water. DNA from each sample was amplified with a primer set that targets the eukaryotic internal transcribed spacer region 1 (ITS1) and effectively distinguishes Pseudo-nitzschia species (White et al., 1990; Sterling et al., 2022). Briefly, DNA was diluted to 1-4 ng/µL and 2 µL of template was added to 25 µL PCR reactions with Phusion Hot Start High-Fidelity Master Mix (Thermo Fisher Scientific Inc., Waltham, MA, USA) and HPLC-purified forward and reverse primers at 0.5 µM concentration with Illumina MiSeq adapters (Integrated DNA Technologies, Coralville, IA, USA). A stepwise thermocycle was used as described in Sterling et al. (2022).

DNA amplicons were sequenced at the Rhode Island-INBRE Molecular Informatics Core on the Illumina MiSeq platform (Illumina, Inc., San Diego, CA, USA). There, libraries were prepared by cleaning ITS1 PCR products with KAPA pure beads (KAPA Biosystems, Woburn, MA, USA) and attaching sequencing indices and adapters using PCR. This amplification was performed with the Illumina Nextera XT Index Kit (Illumina, San Diego, CA, USA) and Phusion High Fidelity Master Mix, followed by a second round of cleaning with KAPA pure beads and visualization with gel electrophoresis. The quality of select samples was assessed on a Bioanalyzer DNA1000 chip (Agilent Technologies, Santa Clara, CA, USA) and all samples were quantified on a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA). The final library was pooled, quantified with qPCR on a LightCycler480 (Roche, Pleasanton, CA, USA) using a KAPA Biosystems Illumina Kit (KAPA Biosystems, Woburn, MA, USA), and sequenced on the Illumina MiSeq using v3 chemistry and 2x250 paired-end reads. Samples were sequenced across five separate MiSeq runs using identical methods and negative controls.

Various biological, chemical, and physical data were collected during each sampling event. In NB, surface temperature and salinity were measured using multiparameter sondes (6920 V2 for samples collected at NBPTS and WR; ProDSS for samples collected at CHB and GSO dock; YSI, Yellow Springs, Ohio, USA). During NES sampling, temperature and salinity were measured using two SBE911 CTD sensors (Sea Bird Electronics, Bellevue, WA, USA) and the mean of the two measurements was used in the final analysis. Dissolved macronutrient samples were collected by freezing 0.2 µm seawater filtrate at -20°C. NB site nutrients were analyzed at the University of Rhode Island Marine Science Research Facility (URI MSRF, Narragansett, RI, USA) on a QuickChem 8500 (Lachat, Milwaukee, WI, USA) while NES samples were measured at the Woods Hole Oceanographic Institution’s Nutrient Analytical Facility (Woods Hole, MA, USA) on a four-channel segmented flow AA3 HR Autoanalyzer (SEAL Analytical, Mequon, WI, USA). Both instruments measured nitrite + nitrate, ammonium, silicate, and phosphate. Nitrite + nitrate and ammonium values were summed and used in the analysis as dissolved inorganic nitrogen (DIN). Any measurements below each instrument’s limit of detection for each nutrient type were replaced with zero.

Biomass for particle-associated DA analysis was collected, extracted, and analyzed via liquid chromatography with tandem mass spectrometry (LC-MS/MS) with multiple reaction monitoring. All samples were chromatographically separated in an identical fashion to Sterling et al. (2022), and the majority of samples (n=167) were analyzed using a 4500 QTRAP mass spectrometer (SCIEX, Framingham, MA, USA). A subset of samples (n=42) were measured using a 1290 Infinity II UHPLC system coupled to a 6470 Triple Quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). The peak of DA eluted at 10.41 min. Analysis was carried out in positive mode, and three transitions from the protonated DA molecule were used and optimized for quantification: m/z 312 → 266, m/z 312 → 161, and m/z 312 → 105 determined by MassHunter Optimizer (Agilent, Santa Clara, CA, USA) including the optimized fragmentor (112), collision energy (20, 28, 48), and cell accelerator voltage (4) settings. The m/z 312 → 266 transition was used for quantification following acquisition from both mass spectrometry instruments. Particle-associated DA was quantified to ng particulate DA L^-1 of filtered seawater using an external calibration curve created with pure DA standards of increasing concentrations included in each analysis (DA Certified Reference Material, National Research Council Canada, Halifax, Nova Scotia).

Pseudo-nitzschia spp. abundance was quantified using light microscopy cell counts of live (NBPTS site) and preserved (all other sites) samples. For preserved samples, acidic Lugol’s solution was added to whole seawater for a final concentration of 1% Lugol’s and stored at 4°C until enumeration. A Sedgewick-Rafter counting chamber (Science First/Wildco, Yulee, FL, USA) and a BX40 light microscope (Olympus, Tokyo, Japan) were used to identify and enumerate cells at the genus level, since many Pseudo-nitzschia species are morphologically cryptic under light microscopy (Bates et al., 2018).

Sampling Locations

Narragansett Bay sites:

Narragansett Bay Long Term Plankton Time Series (41.57 N -71.39 W)

Whale Rock (41.43 N -71.42 W)

East Passage (41.45 N -71.38 W)

Castle Hill Beach (41.46 N -71.36 W)

Graduate School of Oceanography dock (41.49 N -71.42 W)

NES stations:

L1 (41.20 N -70.88 W), L3 (40.86 N -70.88 W), L4 (40.70 N -70.88 W), L7 (40.23 N -70.88 W), L8 (40.14 N -70.77 W), L10 (39.93 N -70.88 W)

936856_v1_pseudo_nitzschia_environmental.csv