*4.7. Testing of a Gulf of Maine Dinophysis norvegica Culture for DST Production*

Two new cultured clonal isolates of *D. norvegica* (DNBH-FB4 and DNBH-B3F) were established in culture in May 2018 from surface water collected from Blue Hill Falls, Maine, following the single-cell isolation methods described by [43]. At the time of water collection, the salinity was 30 psu and the water temperature was 12 ◦C. During isolation and maintenance of the culture, *D. norvegica* were fed *Mesodinium rubrum,* which had been previously raised on *Teleaulax amphioxeia* isolated from Japan [44] following the protocols of [45] as modified by [46]. The dinoflagellate, ciliate, and cryptophyte cultures were grown in modified f/6-Si medium [47] and a salinity of 30 at 15 ◦C in dim light (40 µmol photons·m-2·sec-1) under a 14 h:10 h light:dark photocycle.

To assess the toxigenicity of DNBH-FB4 and DNBH-B3F, cells were inoculated into fresh medium, salinity 30 psu, at 400 cells·mL−<sup>1</sup> , fed *M. rubrum* at a 1:10 ratio of prey to predator, and monitored

every 3 days for the complete consumption of *M. rubrum* by examining 1 mL subsamples in a Sedgewick-Rafter counting cell at 100x using an Olympus CX31 light microscope (Olympus America, Waltham, MA, USA). Three days after all ciliate prey were consumed, (i.e., during late exponential growth of the dinoflagellate) the culture was harvested for toxin analysis.

The harvested cultures were gently separated into cells (intracellular toxins) and medium (extracellular toxins) using a 10 µm sieve and the components were extracted and analyzed for toxins separately. Cells and medium were bath-sonicated at room temperature for 15 min (Branson 5800 Ultrasonic Cleaner, 5800) and loaded onto an Oasis HLB 60 mg cartridge (Waters Corporation, Millford, MA, USA) that was previously equilibrated with 3 mL of MeOH and 3 mL of GenPure water. The cartridge was then washed with 6 mL of GenPure water, blown dry, and eluted with 1 mL of 100% MeOH into a glass 1.5 mL high recovery LC vial and stored at −20 ◦C until analyzed. A portion of the sample underwent alkaline hydrolysis to enable the quantitation of total DSP toxins (free plus esterified) following [48]. Extracts, original and alkaline hydrolyzed, were analyzed using an Acquity liquid chromatography system coupled with a Xevo mass spectrometer with electrospray ionization (Waters, Milford, MA, USA) following the DSP and PTX2 analytical methods described by [49].

Dihydro-DTX1 was detected using selected reaction monitoring (SRM) in negative ion mode with the transitions *m*/*z* 819.5→255.1, 819.5→819.5, 819.5→151.1, and 819.5→113.1. Quantitation was performed using the former SRM transition; concentrations were calculated using an external DTX1 standard curve with MassLynx 4.1 software (Waters Corporation, Millford, MA, USA). Matrix spikes, final concentration of 12.5 ppb DTX1, were conducted to confirm separation from DTX1. Toxin data are presented as toxin concentration per mL of culture, and free vs. esterified, with the latter being calculated through the subtraction of free toxins from total toxins.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2072-6651/12/9/533/s1, Table S1: Sampling site coordinates for shellfish and phytoplankton samples collected during the 2016 *Dinophysis norvegica* bloom depicted in Figures 1 and 2. Figure S1: MS/MS spectrum of *m*/*z* 819.5 peak detected in the 26–27 min fraction from the bioactivity guided fractionation extract of mussel (*Mytilus edulis*) collected during 2016 *Dinophysis norvegica* bloom in the central coast of the Gulf of Maine, USA; Figure S2: Structure of precursor ions and proposed product ion structures for okadaic acid (OA), dinophysistoxin 1 (DTX1), and dinophysistoxin 2 (DTX2).

**Author Contributions:** Conceptualization, J.R.D., W.L.S., J.M., and J.L.S.; Investigation, J.R.D., W.L.S., M.D.C., J.M., A.E.H., J.L.S., D.M.K., P.M., C.D.R., C.A.B., and S.K.L.; Methodology, J.R.D., W.L.S., M.D.C., J.L.S., D.M.K., and P.M.; Resources, J.M., A.E.H., B.J.L., and D.W.M.; Supervision, K.K., S.D.A., J.B., and S.K.L.; Writing—original draft, J.R.D., W.L.S., M.D.C., A.E.H. and J.L.S.; Writing—review and editing, J.M., D.M.K., P.M., S.D.A., and S.K.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** Partial support for this research was received from the National Oceanic and Atmospheric Administration, National Centers for Coastal Ocean Science Competitive Research, Ecology and Oceanography of Harmful Algal Blooms Program under awards NA17NOS4780184 and NA19NOS4780182 to Juliette Smith (VIMS) and Jonathan Deeds (US FDA), and Prevention, Control, and Mitigation of Harmful Algal Blooms program award NA17NOS4780179 to Stephen Archer. This paper is ECOHAB publication number EC0956.

**Acknowledgments:** The authors would like to express their gratitude to Marta Sanderson and Han Gao (VIMS) for their assistance in culture maintenance and toxin analysis, Satoshi Nagai (National Research Institute of Fisheries Science, Japan) for his sharing of *Mesodinium rubrum* and *Teleaulax amphioxeia* cultures, Stephen Conrad (CFSAN) for assistance with figure preparation, and Claude Mallet (Waters Inc.) and Sabrina Giddings (Biotoxin Metrology, NRC Canada) for their technical assistance.

**Conflicts of Interest:** The authors declare no conflict of interest

## **References**


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