Water samples were collected monthly or bimonthly (September 2019 to October 2020) at the Martha's Vineyard Coastal Observatory (MVCO) from about 2 meters below the surface using bucket casts or a CTD deployed from R/V Tioga. Water was processed within 2 hours of collection. One liter of whole seawater was collected onto 47-millimeter (mm) 0.2-micrometer (µm) Millpore Isopore filters and stored at -20 degrees Celsius (°C) for DNA extraction and amplicon sequencing. For in situ hybridization, variable amounts of whole water were collected by gentle filtration onto 47mm 3 µm Millipore Isopore filters for detection of infected hosts, and onto 47mm 0.2 µm Millipore Isopore filters (black) for the detection of free dinospores (see Supplemental File "MVCO_water_amt.csv"). Filtration was stopped when about 20 milliliters (ml) of water remained in the filter tower, and 20 ml of 8% formaldehyde (in filter sterile seawater) was added to fix the cells. After an hour, the solution was filtered, and the samples were rinsed three times with 20 ml of phosphate buffered saline (1x PBS). Filters were stored at -20°C until in situ hybridization.

The detailed hybridization method can be found at protocols.io at doi: 10.17504/protocols.io.bsxmnfk6. A one-eighth pie-shaped section was cut from each filter and placed on a glass slide. 20 µl of hybridization buffer (40% formamide) containing the horseradish peroxidase labeled probe (ALV01; 5’-GCC TGC CGT GAA CAC TCT-3’; Chambouvet el al. 2008) in a 9:1 ratio (final probe concentration 50 nanograms per microliter (ng µl-1)) was placed on the filter slice. Slides were put into 50ml centrifuge tubes containing a hybridization buffer saturated paper towel and incubated overnight at 37°C. Filter pieces were washed twice in 3 ml of wash solution at 46°C, then allowed to soak in 3 ml of TNT buffer at room temperature. Filters were placed on a new glass slide and the fluorescence signal was amplified using a tyramide signal amplification kit (Akoya Biosciences TSA Plus Fluorescein; NEL741001KT). After signal amplification, filters were washed twice in 3 ml of TNT buffer at 55°C to neutralize any residual fluorescein. Filter pieces were transferred to new glass slides and cellulose was stained using 20 µl of Calcofluor White for 7 minutes. Filter pieces were washed twice in 3 ml of water, then placed on new glass slides for counterstaining and mounting with a mixture of propidium iodide and Citiflour media (20 µl per slide). Hybridized filters were held overnight at 4°C, and then at -20°C for long-term storage.