A series of onboard incubations were performed in surface waters of the Southern California coastal system (CCS) to measure bulk community responses (chlorophyll a and POC/PON concentrations), bulk and UCYN-A/haptophyte symbiosis cell-specific N2 fixation and CO2 fixation rates, bulk and UCYN-A/haptophyte symbiosis cell-specific NO3-/NH4+ uptake rates, and UCYN-A/haptophyte symbiosis growth rates under DIN deplete and replete (NO3- or NH4+ amendments) conditions. The experiments were designed to investigate whether UCYN-A continues to fix N2 when NO3- and NH4+ are readily available and if the haptophyte host takes up NO3- and NH4+. Additionally, UCYN-A/haptophyte sublineage-specific responses to NO3-/NH4+ additions were investigated. 

Four experimental manipulations were conducted during 2017 and 2018; three experiments (NO3.1, NO3.2, NO3.3) were NO3- addition experiments and one was an NH4+ addition experiment (NH4.1). NO3.1-3 were conducted at three different stations aboard the R/V Gordon Sproul during two research cruises in 2017 that transited off the coast of Southern California and Baja California Sur, Mexico, while NH4.1 was conducted on the Scripps Institute of Oceanography pier. 

For NO3.1-3, surface water was pumped into 40 L carboys, housed in an on-deck laboratory container, using a pneumatic diaphragm pump PVDF and Teflon (Wilden Pump and Engineering, Grand Terrace, CA), to allow mixing of the seawater before being randomly dispensed into acid-cleaned 4 L polycarbonate bottles (Thermo Scientific™ Nalgene™, Waltham, MA). Grazers were removed using 150 µm nitex plankton netting (BioQuip, Rancho Dominguez, CA). The bottles were then incubated in triplicate with or without a 2 µmol L-1 addition of NO3- at T0. Incubation bottles were placed in a flow-through surface seawater incubator, amended with neutral density screening to attenuate incident light to 20% of the surface irradiance. Incubations lasted 48 h, with initial rate measurements between 0-24h and final rate measurements between 24-48 h. At each time point, bottles were sacrificed and subsampled for measuring chlorophyll a concentration, particulate nutrient concentrations, bulk CO2 and N2 fixation rates, inorganic N uptake rates, and UCYN-A/haptophyte symbiosis cell-specific N2 fixation, CO2 fixation and NO3- uptake rates (CARD-FISH nanoSIMS). Unlabeled initial samples were used to determine the atom% 15N- and 13C-normal of the unenriched bulk community and UCYN-A/haptophyte symbioses.  

For NH4.1, surface water was pumped into 40 L carboys from the waters surrounding the SIO Pier using a pneumatic diaphragm pump PVDF and Teflon (Wilden Pump and Engineering), then randomly dispensed into acid-cleaned 2 L polycarbonate bottles (Thermo Scientific™ Nalgene™). Grazers were removed using 150 µm nitex plankton netting (BioQuip, Rancho Dominguez, CA). The bottles were then incubated with or without a 2 µmol L-1 addition of NH4+ at T0. Incubation bottles were placed in a flow-through surface seawater incubator, amended with neutral screening to attenuate incident light to 20% of the surface irradiance. Incubations lasted 48 h, with N2 fixation initial rate measurements between 0-24h and final rate measurements between 24-48 h. For NH4+ uptake rates, initial rates were measured between 0-6 h, and final rates in NH4+-treatments were measured between 45-51 h. Incubation times (6 h) were chosen to ensure detection of isotope enrichments while minimizing isotope dilution. At each time point, bottles were sacrificed and subsampled for chlorophyll a concentration, particulate nutrient concentrations, bulk CO2 and N2 fixation rates, inorganic N uptake rates, and UCYN-A/haptophyte symbiosis cell-specific N2 fixation, CO2 fixation and NO3- uptake rates (CARD-FISH nanoSIMS). Unlabeled initial samples were used to determine the atom% 15N- and 13C-normal of the unenriched bulk community and UCYN-A/haptophyte symbioses.