Trilogy laboratory fluorometer

Chlorophyll a was extracted with 90% acetone for 18–24 h at 4°C in the dark and measured by a Turner Designs Trilogy Laboratory Fluorometer (Turner Designs, San Jose, CA, United States) with CHL-A NA model following the work of Welschmeyer (1994) (link). Dissolved inorganic nutrients including nitrate, nitrite, ammonium, phosphate, and silicate were analyzed with a Technicon AA3 Auto-Analyzer (Bran + Luebbe, Hamburg, Germany) on the basis of the standard procedures (Hansen and Koroleff, 2007 ). Synechococcus cells were enumerated using a flow cytometer (Becton-Dickinson Accuri C6) equipped with a 488-nm laser. A total volume of 198 μl of samples was analyzed with a flow rate of 66 μl/min for 3 min, and 2-μm fluorescent beads (Polysciences, Warrington, PA, United States) were added as the internal standard (Olson et al., 1993 ).

Zooxanthellae cells were counted immediately after sample collection using a Neubauer hemocytometer, and normalized to the coral surface area. Surface area was obtained by using the single wax dipping method, according to Veal et al. (2010) (link). For the calibration curve, we used 15 spheres ranging from ∼1.0 to 18.2 cm². The wax dipping was conducted using paraffin wax (Lamers & Indemans BV, ’s-Hertogenbosch, The Netherlands) and heated to 65 °C using an MGW Lauda MT thermostat (Beun de Ronde BV, La Abcoude, The Netherlands). Coral skeletons and calibration objects were kept at room temperature and weighed before and after wax dipping using an EMB 600-2 digital scale (Kern, Balingen, Germany) (accuracy of 0.01 g).
For chlorophyll a measurements, we added 1.35 mL of 90% acetone to a 0.15 mL zooxanthellae suspension with known cell count and stored samples in the dark at −20 °C for 48 h (after Welschmeyer, 1994 (link)). Samples were centrifuged for 10 min at 18,000 rcf. Chlorophyll a concentrations were measured using 1.2 mL of supernatant with a Trilogy Laboratory Fluorometer (Turner Designs, San Jose, CA, USA), and further normalized per zooxanthella cell.

Pigments were yielded by extraction from 1 ml sediment with 8 ml acetone and disruption in a cell mill. The supernatant was collected after centrifugation and the extraction of the sediment was repeated twice. Two milliliter aliquots of the pooled extracts were subjected to fluorescence measurements [34 (link)] using a Trilogy Laboratory Fluorometer (Turner Designs; excitation = 428 nm; emission = 671 nm) and measured before and after acidification with 100 µl 20% HCl. The concentration of chlorophyll a (Chla), and phaeopigments (PhP) were determined according to $\begin{array}{ccc}\hfill \mathrm{Chla}\left[\mathrm{\mu }\mathrm{g}\right]\hfill & \hfill =\hfill & \hfill \left({\mathrm{RFU}}_{\mathrm{b}}-{\mathrm{RFU}}_{\mathrm{a}}\right)\times CF\times AF\times f\hfill \\ \hfill \mathrm{PhP}\left[\mathrm{\mu }g\right]\hfill & \hfill =\hfill & \hfill \left({\mathrm{RFU}}_a\times \mathrm{AR}-{\mathrm{RFU}}_{\mathrm{b}}\right)\times CF\times AF\hfill \end{array}$ with the relative fluorescence units before acidification (RFU_b) and after acidification (RFU_a) and the calibration factor (CF) and the dilution factor (f) determined from chlorophyll standards as described by Lorenzen [34 (link)].

Whole seawater for nutrients measurement were collected in triplicates in 50 mL conical tubes and kept in −20° C until flow-injection analysis at the Marine Sciences Institute Analytical Lab at University of California, Santa Barbara (http://www.msi.ucsb.edu/services/analytical-lab). Bacteria and viruses per ml seawater were counted on duplicate slides using SYBR green epifluorescence microscopy (Noble & Fuhrman, 1998 (link); Patel et al., 2007 (link)). For chlorophyll-a measurement, triplicates of 50–500 mL of whole seawater were filtered onto 25 mm GF/F filters and stored at −20 °C until analysis within the week. Filters were extracted with 4 mL of 100% acetone at −20 °C overnight in the dark, and processed on a calibrated Trilogy Laboratory Fluorometer (Turner Designs, San Jose, CA, USA) using the non-acidification method (Welschmeyer & Naughton, 1994 ). See Table S1 for all measured values.

Approximately 1–10 mL of water from each sample bottle was filtered onto 0.7 µm Whatman GF/Fs, and the filters stored at −20 °C until analysis. Filters were thawed and suspended in 7 mL of 100% acetone, sonicated for 5 s at 50% intensity (Fisher Scientific, Hampton, NH, USA, Model 120 Sonic Dismembrator), and extracted in the dark at −20 °C for 24 h [63 ]. Samples were run fluorometrically (Turner Designs Trilogy Laboratory Fluorometer) using the non-acidification method detailed by Welschmeyer [64 (link)].

Hydrochemistry and nutrients analyses were assayed by the Commonwealth Scientific and Industrial Research Organisation (CSIRO) Hydrochemistry team as described in Raes et al. (2018) (link). In this paper, all presented salinities are based on the PSS-78 reference (S_P) and are therefore unitless. The latitude, longitude, and absolute pressure values at the depths of sample collection (i.e., the sample “depths” in pressure units of dbar) provided with the data enable Absolute Salinity (S_A) to be calculated via the TEOS-10 equation² of state for seawater. Chlorophyll a profiles were generated using extracts from 0.525 liters of sample water from five sampling depths within the upper water column. Extraction was done using gentle vacuum filtration (pressure drop < 10 kPa) using 25-mm GF/F grade Whatman^® glass microfiber filters (Merck, Germany) and samples were measured on a Trilogy laboratory fluorometer (Turner Designs, United States). Chlorophyll a data is as presented in Raes et al. (2017) (link). Physical, biogeochemical, nutrient and metadata reported or discussed here can be accessed through the CLIVAR and Carbon Hydrographic Data Office (CCHDO) webpage³.

On the first day of incubation, triplicate 200–250 mL samples were taken immediately from the original carboy to measure initial chlorophyll a concentration (Chl a). On the last day (at 96 h of incubation), 200–250 mL samples were taken from each of all the 18 bottles. Chl a concentration was measured fluorometrically after filtration of the sample through 0.2-µm polycarbonate filters. The filters were stored in Eppendorf tubes and frozen at −20 °C until analysis. Chlorophyll a was then extracted in 6 mL of 90% HPLC-grade acetone and stored at 5 °C overnight. Chl a concentration was determined with a Trilogy fluorometer (Trilogy Laboratory Fluorometer, Turner Designs) that had been calibrated with pure Chl a.