Methodology from the IRNBRU cruise report

During the IRNBRU (MV1405) cruise, multiple incubation experiments were performed at different locations in relation to varying iron environments, including regions of high iron, upwelled waters as well as relatively low iron waters. Incubated phytoplankton community response was assessed using physiological and molecular methods in order to examine the effects of varying biogeochemical environments on metabolism and gene expression. Each experiment contained several treatments and time points consisting of the following: a 5 nmol L-1 FeCl3 addition, a 200 nmol L-1 desferroxamine B (DFB) addition, an initial 5 nmol L-1 FeCl3 addition followed by a 200 nmol L-1 DFB addition during the midpoint of the experiment, and an unamended control. For three of the experiments (sites 1, 2, and 4), seawater was collected from the near surface (2-5m) using a trace-metal clean sampling system which consisted of a towed GeoFish sampler attached to KevlarTM line, PFA Teflon tubing and a Teflon dual-diaphragm pump that pumped seawater directly into a positive pressure trace-metal clean bubble constructed in the main laboratory of the ship. The seawater was placed into a large 55 gallon acid-cleaned HDPE drum for homogenization before being distributed into10L flexible acid-cleaned polyethylene cubitainers and placed in on-deck plexiglass incubators with flow-through seawater to maintain near-ambient surface temperatures. For the experiment at site 3, seawater was obtained from the depth corresponding to the 10 deg C isotherm (91 m) in order to simulate an upwelling event. Cleaning protocols for all cubitainers included soaking the inside walls in 1.2 mol L-1 hydrochloric acid (reagent grade) for 3 d followed by three rinses with Milli-Q H20, soaking in 1.2 mol L-1 hydrochloric acid (trace metal grade) for 1 week followed by three rinses with Milli-Q H20, and soaking in 0.1 mol L-1 acetic acid (trace-metal grade). Prior to filling the cubitainers with seawater, the dilute acetic acid was removed and the cubitainers were rinsed thoroughly three times with ambient, low-iron seawater. Incubators were covered with neutral density screening to reduce irradiance to ca. 30% of the incident. For sample collection from initial conditions, triplicate cubitainers were immediately filtered. All incubations were initiated and terminated just prior to dawn. Following 96 hours of incubation, the seawater was removed from the incubators and stored in a dark, cool room until filtration. Subsamples for dissolved nutrients, size-fractionated chlorophyll a ,Fv/Fm, particulate nutrients, nutrient uptake, biogenic silica, domoic acid, dissolved iron, and RNA were collected from each cubitainer. Methods performed on the ship for each measurement are briefly explained below.

Dissolved nitrate + nitrite (NO3- + NO2-), phosphate (PO43-), and silicic acid (H4SiO4) concentrations were measured shipboard using a Lachat Quick Chem 8000 Flow Injection Analysis system. Particles were removed by filtering the sample through a GF/F filter using a syringe prior to analysis. Reference materials for nutrients in seawater (Lots BY and CA, KANSO Technos, Osaka, Japan) were run alongside samples for quality control.

For biomass determination, 400 mL of seawater was gravity-filtered through a 5 um polycarbonate filter (47 mm) followed by a GF/F filter (25 mm) under gentle vacuum pressure (<100 mm Hg) using a series filter cascade for size fractionation. Filters were rinsed with 0.45 um filtered seawater and immediately frozen at -80 deg C until analysis. Chlorophyll a extraction was performed on the ship using 90% acetone at -20 deg C for 24 hours and measured via in vitro fluorometry using a Turner Designs 10-AU fluorometer.

In order to assess changes in photophysiology among treatments, the maximum photochemical yield of Photosystem II (Fv:Fm) was measured shipboard using a Satlantic FIRe. Before each measurement, a subsample (5 mL) of each culture was placed in the dark for 20 minutes. The resulting Fv:Fm was derived from the induction profile using a saturating pulse (20,000 umol photons m2 s-1) for a duration of 100-200 us.

Particulate nitrate (PN), carbon (PC) and nitrate (NO3-) uptake was obtained by spiking 500 mL of seawater with 15N-NH4Cl at no more than 10% of ambient nitrate concentration and incubated for 8 hours in the flow-through plexiglass incubator. Following incubation, seawater filtration commenced immediately and was performed by gravity through a 5 um polycarbonate filter (47 mm), and with an in-line vacuum (<100 mm Hg) onto a precombusted (450 deg C for 5 hours) GF/F filter (25 mm). Cells on the 5 um polycarbonate filter were then rinsed onto an additional precombusted GF/F filter (25 mm) using an artificial saline solution. Filters were then stored at -20 deg C. In the laboratory, filters were heated at 50 deg C for 24 hours, wrapped in tin capsules, and pelletized in preparation for analysis of the atom % 15N, PN, and PC using an elemental analyzer paired with an isotope ratio mass spectrometer (EA-IRMS).

For inorganic carbon uptake, or measurements of primary productivity, 60mL samples from each cubitainer were distributed into a light and dark bottles cleaned with 10% HCl. For each bottle, 1.2 uCi of NaH14CO3 was added. A 1 mL subsample was taken and added to vials containing NaOH. The light and dark bottles were incubated on-deck for 6.5-8 hours and stacked polycarbonate filters (5 um and 1 um) with a mesh spacer between them. The blanks were filtered onto a GF/F filter after 5 minutes. Filters were vacuumed dried, placed in scintillation vials with 0.5 mL of 6M HCl, permitted to degas for 24h, and counted using a Beckman Coulter LS 6500 scintillation counter.

Biogenic silica was determined shipboard via filtration of 335 mL onto 1.2 um polycarbonate filters (45 mm). Concentrations were measuring using a NaOH digestion in teflon tubes3 and a colorimetric ammonium molybdate method.

Samples for iron (Fe) analysis were acidified at sea with the equivalent of 4 mL 6N quartz-distilled HCl per liter of seawater (to pH 1.7-1.8) and stored in LDPE bottles that had been cleaned as per the GEOTRACES cookbook (http://www.geotraces.org/science/‌intercalibration/222-sampling-and-sample-handling-protocols-for-geotraces-cruises). Samples were analyzed by preconcentrating the Fe on 2 cm columns of Nobias chelate PA1 resin, and analyzing the eluent on the Thermo-Element high-resolution XR ICP-MS at UC Santa Cruz. The resin columns are rinsed and conditioned with 0.05 M NH4Ac buffer at pH 6.0, and the samples are buffered to pH 6.0 with NH4Ac immediately before loading. The samples are eluted from the columns with ~1 mL of 1N quartz distilled HNO3. One to three days before preconcentration, samples were irradiated with UV light for 2 hours to ensure full recovery of other metals of interest.

Seawater for RNA analysis was filtered directly onto 0.8 um Pall Supor filters (142 mm) using a peristaltic pump. Filters were placed in cyrovials and immediately flash frozen in liquid nitrogen. Samples were later transferred to storage in -80 deg C freezers until RNA extractions were performed. Filters were briefly thawed on ice before being extracted individually using the ToTALLY RNA Total RNA Isolation Kit (Ambion). RNA-Seq library prep was conducted with the Illumina TruSeq Stranded mRNA Library Preparation Kit and HiSeq v4 reagents. Bioinformatic pipeline consisted of the following: trimming of reads for quality and removal of adapters, contig assemblies, read mapping for quantitative analysis, taxonomic and functional assignment, and differential gene expression determination.

Q_incubations.csv