Dataset: Dissolved organic matter and nutrient data from water samples collected in Dec 2020 from six sponge species at two coral reef sites in the Florida Keys

Incurrent and excurrent water samples were taken from six sponge species from the Florida Keys (USA) using a modified version of the vacuSIP (Morganti et al. 2016). Briefly, acid cleaned and combusted bottles were negatively pressurized and sealed. PEEK tubing lines were connected to the pressurized bottles via a needle, and this pulled seawater from the opening of the sponge (“excurrent”) or nearby seawater (“incurrent”) at a slow and steady rate to fill the bottles. Nutrient data from the seawater samples were compiled and organized by sponge species and water type (incurrent or excurrent) in an Excel file. All seawater samples were filtered through 0.2 µm Teflon Omnipore filters. Filtrate was then saved for inorganic nutrients and the rest was acidified and saved for organic nutrient analysis. From this filtrate, inorganic nutrient, fluorescent Dissolved Organic Matter (fDOM), and targeted and untargeted metabolomics data were collected. Flow cytometry of phytoplankton and bacteria data was collected from pre-filtered seawater and preserved in 0.5% paraformaldehyde final concentration. Seawater for total organic carbon (TOC) and total nitrogen (TN) was also collected prior to filtration and was acidified to ~pH 2 using concentrated HCl. Metabolomics and TOC analysis were performed at Woods Hole Oceanographic Institution at the Mass Spectrometry Facility.

Dissolved Organic Matter Extraction:

Filtered seawater was processed using PPL solid phase extraction following the protocol by Dittmar et al. (2008). Extracts were dried to nearly completeness, leaving a small viscous drop in the vial. These extracts were then shipped to WHOI for metabolomics analysis.

Targeted Metabolite Analysis by UPLC-MS:

DOM extracts were reconstituted in 200 μl MilliQ water with 50 ng/ml isotopically-labeled injection standards d₂ biotin, d₆ succinic acid, d₄ cholic acid, and d₇ indole 3 acetic acid. We used ultra-performance liquid chromatography (Accela Open Autosampler and Accela 1250 Pump, Thermo Scientific) coupled to a heated electrospray ionization source (H-ESI) and a triple quadrupole mass spectrometer (TSQ Vantage, Thermo Scientific) operated under selected reaction monitoring (SRM) mode. We performed chromatographic separation with a Waters Acquity HSS T3 column (2.1 × 100 mm, 1.8 μm) equipped with a Vanguard pre-column and maintained at 40°C. We eluted the metabolites from the column with (A) 0.1% formic acid in water and (B) 0.1% formic acid in acetonitrile at a flow rate of 0.5 mL min^-1, according to the gradient: 0 min, 1% B; 1 min, 1%B; 3 min, 15%B; 6 min, 50%B; 9 min, 95%B; 10 min, 95%B; 10.2 min, 1%B; 12 min, 1%B (total run time = 12 min). Settings for source gases were 55 (sheath), 20 (auxiliary) and 0 (sweep), and these settings are presented in arbitrary units. The heated capillary temperature was 375 °C and the vaporizer temperature was 400 °C. For positive and negative modes, we performed separate autosampler injections of 5 μL each. 

Untargeted Metabolite Analysis by UPLC-MS:

DOM extracts were reconstituted in 200 μl MilliQ water with 50 ng/ml isotopically-labeled injection standards d₂ biotin, d₆ succinic acid, d₄ cholic acid, and d₇ indole 3 acetic acid. A pooled samples was made using 40 uL of each experimental sample. Untargeted metabolite analysis was performed using an ultra-high performance liquid chromatography system (Vanquish UHPLC, Thermo Scientific™) coupled with an Orbitrap Fusion Lumos Tribid mass spectrometer (Thermo Scientific™). A Waters Acquity HSS T3 column (2.1 × 100 mm, 1.8 μm), equipped with a Vanguard pre-column, was used for chromatographic separation and maintained at 40 °C. The column was eluted at 0.5 mL min-1 with a combination of solvents: A) 0.1% formic acid in water and B) 0.1% formic acid in acetonitrile. We eluted the metabolites from the column with (A) 0.1% formic acid in water and (B) 0.1% formic acid in acetonitrile at a flow rate of 0.5 mL min^-1, according to the gradient: 0 min, 1% B; 1 min, 1%B; 3 min, 15%B; 6 min, 50%B; 9 min, 95%B; 10 min, 95%B; 10.2 min, 1%B; 12 min, 1%B (total run time = 12 min). The column was washed and re-equilibrated with 1% B (2 min) between injections. Individual autosampler injections (5 μL each) were taken for negative and positive ion mode analyses. The electrospray voltage was set to 2600 V and 3600V for negative and positive modes, respectively. Settings for source gases were 55 (sheath), 20 (auxiliary) and 1 (sweep), and these settings are presented in arbitrary units. The heated capillary temperature was 350 °C and the vaporizer temperature was 400 °C. MS data were collected in the Orbitrap analyzer with a mass resolution of 120,000 FWHM at m/z 200. The automatic gain control (AGC) target was 4e5, the maximum injection time was 50 seconds, and the scan range was 100 – 1000 m/z. Data-dependent MS/MS data were also acquired in the Orbitrap analyzer using higher energy collisional dissociation (HCD) with a normalized collision energy of 35%. The AGC target value for fragmentation spectra was 5e4 and the intensity threshold was 2e4. Cycle time was set at 0.6 seconds. Precursor selection was performed within the quadrupole with a 1 m/z isolation window. All data were collected in profile mode. Pooled samples were run after every six samples to assess instrument performance and drift over the sample run and samples were run in a random order. 

Flow Cytometry:

Samples were shipped to the Center for Aquatic Cytometry at Bigelow Laboratory for Ocean Sciences where they were stored at -80 C until analysis. Picophytoplankton (less than 3 µm) and nanophytoplankton (3-20 µm) were analyzed using a slight modification of the method described in Lomas et al., 2010. Immediately after thawing at room temperature, 300-400 µl of sample was prescreened through 70 µm mesh and run at a flow rate of 1 µl sec^-1. Particles were excited with a 488 nm blue laser and data acquisition was triggered on red fluorescence. Signals were recorded from detectors with bandpass filters for forward scatter (FSC), right angle light scatter (SSC) and fluorescence emission in red (692/80 nm) indicative of chlorophyll a, and orange (593/52 nm) for phycoerythrin. Data files were analyzed from logarithmic dot plots based on fluorescence and characteristic light scattering properties (DuRand & Olson, 1996) using FlowJo 10.6 software (FlowJo, 2023) (Becton Dickinson & Company, San Jose, CA, USA). Total pico and nano phytoplankton populations were identified based upon cell size and red fluorescence. Phycoerythrin containing cell populations were determined by orange fluorescence. Based upon these gating criteria, the number of cells in each identified population was enumerated and converted to cell abundances using the processed sample volume and adjusted for dilution by preservative.

For total bacteria analysis, samples were thawed, diluted 1:10 with Tris EDTA (TE) Buffer pH 8.0 and stained using a 10x working stock of SYBR Green I Nucleic Acid Stain (Thermofisher Scientific, USA) at room temperature in the dark for 15 min using the protocol of Marie et al. (2005). At a flow rate of 0.5 µl sec^-1, 180 µl of the diluted sample was run. Particles were excited with a 488 nm blue laser and data acquisition was triggered on green fluorescence. Signals were recorded from detectors with bandpass filters for forward scatter (FSC), right angle light scatter (SSC) and fluorescence emission in green (525/35nm). Data files were analyzed from two logarithmic scatter plots based on fluorescence and characteristic light scattering properties. Total bacteria counts were identified based on size and presence of green fluorescence and counts were converted to cell abundances using the volume of sample processed including adjustments for preservation, dilution and staining.

Inorganic nutrients:

Inorganic nutrients included phosphate, nitrate+nitrite, nitrite, ammonia, and silicic acid. The phosphate method is a modification of the molybdenum blue procedure of

Bernhardt and Wilhelms (1967), in which phosphate is determined as reduced phosphomolybdic acid employing hydrazine as the reductant. The nitrate + nitrite analysis uses the basic method of Armstrong et al. (1967), with modifications to improve the precision and ease of operation.  Sulfanilamide and N-(1-Napthyl)ethylenediamine dihydrochloride react with nitrite to form a colored diazo compound. For the nitrate + nitrite analysis, nitrate is first reduced to nitrite using an OTCR and imidazole buffer as described by Patton (1983). Nitrite analysis is performed on a separate channel, omitting the cadmium reductor and the buffer. The method is based on that of Armstrong et al. (1967) as adapted by Atlas et al. (1971).  Addition of an acidic molybdate reagent forms silicomolybdic acid which is then reduced by stannous chloride. This indophenol blue method is modified from ALPKEM RFA methodology which references Methods for Chemical Analysis of Water and Wastes, March 1984, EPA-600/4-79-020, "Nitrogen Ammonia", Method 350.1 (Colorimetric, Automated Phenate) A detailed description of the continuous segmented flow procedures used can be found in Gordon et. al. (1994). 

Dissolved Combined Neutral Sugars (DCNS):

Dissolved combined neutral sugars (DCNS) were measured from frozen 20 µl aliquots of 0.22 µm filtered seawater from each sample at the Complex Carbohydrate Research Center, University of Georgia. Each sample was first desalted and hydrolyzed. For desalting, each sample was loaded onto a gravity column, containing forty milliliters of mixed ion exchange resins (AG 501-X8, 20-50 mesh, Bio-Rad), that was packed and prewashed with 200 ml (5x bed volume) of nano-pure water. Samples were then eluted with 120 ml (3x bed volume) of nano-pure water. The resulting flowthrough and wash solution were lyophilized. Following lyopholization, the recovered materials were hydrolyzed wth 2 ml of 2 N TFA at 100ºC until high-performance anion exchange chromatography (HPAEC) analysis. By employing a specific HPAEC program, as detailed below, the neutral monosaccharides can be separated allowing the measurement of carbohydrates in each sample. Monosaccharide standards, including fucose (Fuc), rhamnose (Rha), arabinose (Ara), glucose (Glc), galactose (Gal), xylose (Xyl), mannose (Man), and fructose (Frc), were hydrolyzed in the same manner and at the same time as the samples. Three concentrations of the standard mixture were prepared serially to establish a calibration equation. The quantity of each residue in the sample was calculated by linear interpolation of respective residue area units into the calibration equation. Monosaccharides from each sample were analyzed by HPAEC with pulsed amperometric detection (HPAEC-PAD) using a DIONEX ICS3000 system (Thermo Fisher Scientific) equipped with a gradient pump and an electrochemical detector. The carbohydrates were separated by a Dionex CarboPac PA20 (3x150mm) analytical column with an amino trap column and eluted with degassed 12 mM NaOH. Injections were made every 40 min. Under the HPAEC conditons, Xyl and Man cannot be separated6 and are presented as combined results. Samples were analyzed in triplicate and mean values were reported as ng ml-1 based on the volume analyzed.

TOC and TN:

Seawater for total organic carbon (TOC) and total nitrogen (TN) was also collected prior to filtration and was acidified to ~pH 2 using concentrated HCl. Metabolomics and TOC analysis were performed at Woods Hole Oceanographic Institution at the Mass Spectrometry Facility following methods of Longnecker et al. (2015).

Fluorescent DOM:

Following the methods from Nelson et al. (2015), samples were analyzed with a Horiba Aqualog scanning fluorometer with 150 watts Xe excitation lamp, Peltier-cooled CCD emission detector, and simultaneous absorbance spectrometer. Quartz cuvettes of 1-centimeter (cm) diameter, which were DIW-leached and rinsed, were used to measure fluorescence. Samples were brought to room temperature while the Xe bulb warmed up.

Organisms:

Organism identifiers (Life Science Identifier (LSID)) for organisms represented in this dataset:

• Xestospongia muta, urn:lsid:marinespecies.org:taxname:166894
• Ircinia strobilina, urn:lsid:marinespecies.org:taxname:165051
• Niphates digitalis, urn:lsid:marinespecies.org:taxname:166775
• Iotrochota birotulata, urn:lsid:marinespecies.org:taxname:169379
• Agelas tubulata (formerly conifera), urn:lsid:marinespecies.org:taxname:164845
• Verongula rigida, urn:lsid:marinespecies.org:taxname:169675