Location
\nWater collection sites in the northern Gulf of Mexico, particularly at the mouth of Mobile Bay and Little Lagoon, AL.
\nWater Collection
\nBriefly, water was collected from the field using a 5-gallon bucket, pre-screened with a 200 \u00b5m nitex mesh, and gently poured into 10-20 L carboys and kept in the dark until returning to the laboratory for same-day processing.
\nTerminology
\ndDA \u2013 dissolved Domoic Acid
\npDAa \u2013 particulate Domoic Acid (algal fraction)
\npDAOP \u2013 particulate Domoic Acid (bound to organic polymers)
\ncDA \u2013 Domoic Acid in copepods
\nPOC \u2013 Particulate Organic Carbon
Field-simulation experiments
\nField water used for grazing experiments was collected during spring and summer (2017, 2018) from designated monitoring sites at Little Lagoon (Gulf Shores, Alabama, USA). The water was prefiltered with a 200 \u00b5m mesh, gently poured into carboys, and kept in the dark until the start of the laboratory experiment. Laboratory-reared adult Acartia tonsa, with no prior exposure to DA, were provided by the University of Southern Mississippi Gulf Coast Research Laboratory\u2019s Thad Cochran Marine Aquaculture Center and starved for 24 hours prior to experiment initiation.
\nInitial samples for Pseudo-nitzschia abundance and DA were collected. For cell abundance, 50 mL of seawater was preserved with 2 mL of Bouin\u2019s solution and stored at 4\u00b0C. A Sedgewick rafter slide was used to count cells in a 1 mL subsample. DA was measured in two forms, dDA and pDA. Seawater was filtered using a 25 mm glass fiber filter and 30 mL of filtrate was collected in a polypropylene conical tube and stored at -20\u00b0C for later analysis of dDA. pDA was sampled by filtering 100 mL of seawater under low vacuum through a 25 mm glass fiber filter and stored in a cryovial at -20\u00b0C.
\nThe experimental design consisted of four treatments, each containing triplicate 1-L polycarbonate bottles. Two treatments contained seawater filtered through a 0.2 \u00b5m polycap filter (as described above); these treatments tested whether copepods could assimilate dDA through the proposed organic polymer-bound pathway. The remaining treatments contained seawater with a natural phytoplankton community that was concentrated by a factor of three, using a 20 \u00b5m mesh. After the bottles were filled with the appropriate water, 30 copepods were added to the necessary treatments and the experiments started. After 24 hours the copepods were collected on a 200 \u00b5m screen, gently rinsed with filtered seawater, placed in fresh filtered artificial seawater, and allowed to evacuate their guts for ~1 hour. Afterwards, copepods were once again screened and rinsed three times, and then stored in a cryovial at -20\u00b0C until analysis.
\nLiquid chromatography-mass spectrometry method for domoic acid quantification
\nLC-MS sample preparation followed was modified from Wang et al. (2012) for the determination of dDA, pDA, pDAOP and cDA. The samples for DA determination were cleaned and concentrated using Bond Elut LRC - C18, 200 mg, solid-phase extraction (SPE) columns from Agilent Technologies. For dDA, 30 mL seawater samples were filtered using a 47 mm glass fiber filter; the filtrate was collected and acidified with formic acid to yield a 0.2% final solution. SPE columns were conditioned with one column volume of HPLC-grade methanol followed by one column volume of HPLC-grade water. Samples were then loaded on the SPE column and filtered at ~1 mL min-1 using a vacuum manifold, followed by 10 mL of 0.2% formic acid as a rinse for the sample tube and SPE column. The SPE column was then allowed to go dry and was eluted with 1.5 mL of 20 mM ammonium acetate in 50% methanol (pH 8) and collected in a glass tube. The tubes were centrifuged for 5 minutes at ~1300 x g, supernatant was transferred into an LC vial with a Pasteur pipette, and stored at 4\u00b0C until further analysis. For pDAa 100 mL of seawater were filtered through a 5 \u00b5m polycarbonate filter and stored in a 50 mL polypropylene tube at -20\u00b0C. Similarly, for pDAOP 150 mL of seawater was filtered through a pre-combusted 25 mm glass-fiber filter and stored at -20\u00b0C. Prior to concentration and clean-up for pDA, pDAOP, and cDA, the filters were submerged in 2 mL of 80% methanol and sonicated to ensure cells and copepods were lysed. Sonication pulses were done for a total of 45 seconds (5 seconds on/off) on a Sonics Materials Ultrasonic Processor (model - VCX 130) at 75% power. Subsequent clean-up using the SPE column is the same as for the dDA samples.
\nAn ultra-performance liquid chromatography (UPLC) \u2013 tandem mass spectrometry (MS) system was used for the quantification of DA.The LC-MS system consisted of Acquity UPLC system (Waters, Milford, MA) coupled to a 5500 QTRAP triple quadrupole / linear ion trap mass spectrometer equipped with a TurboIonSpray interface (Sciex, Foster City, CA, USA). The analytes were separated on a Luna C18 (2), 2.0 x 100 mm column (Phenomenex, Torrance, CA, USA) with column temperature held at 40\u00baC. The mobile phase was water (A) and 95% aqueous acetonitrile (B) with 0.1% formic acid additive and the flow rate was 0.4 ml/min. Gradient program was: 5% B for 3 min, linear gradient to 60% B at 10 min, 95% B at 10.1 min, hold at 95% B for 2 min. MS was operated in positive ion mode. Ion spray voltage was 5 kV and declustering potential was 80 V. Gas parameter settings were: nebulizer gas, 50 psi; turbo gas, 50 psi at 500\u00baC; curtain gas, 20 psi; and collision gas, medium setting. The collision energy applied was 25eV. The transitions used for selected reaction monitoring were m/z 312\u2192266, 193, 220. The transition m/z 312\u2192266 was used for quantitation.
\nFor organic polymer formation and sorption of DA results and methodology see https://www.bco-dmo.org/dataset/808280.
Domoic acid assimilation in copepods by consuming organic polymers and Pseudo-nitzschia. Results from experiments designed to investigate the contribution of organic polymers and Pseudo-nitzschia to domoic acid trophic transfer. Water samples were collected in the northern Gulf of Mexico in 2017 and 2018.
\nRelated datasets:
\nOrganic polymers and domoic acid https://www.bco-dmo.org/dataset/808280
\nDomoic acid assimilation in copepods https://www.bco-dmo.org/dataset/808402
BCO-DMO Data Manager Processing Notes:
\n* Data from sheet \"Field_DA-P.nitz\" from originally submitted Excel file \"DATASET_DA-org polymers-cop grazing_BCO-DMO_params_IAM.xlsx\" extracted to csv.
\n* Column names modified to meet BCO-DMO naming conventions designed for interoperability (only letter, numbers, and underscores, no spaces).
\n* Data loaded into the bco-dmo data system with -999 as a missing data identifier.
\n* Year in ISO timestamp changed from 2071 to 2017 in rows 17-31 of originally submitted Excel sheet.