Dataset: Group II Syndiniales infected host and dinospore counts determined from CARD-FISH hybridization carried out on samples collected at the Martha's Vineyard Coastal Observatory (MVCO) monthly or bimonthly from September 2019 to October 2020

Final no updates expectedDOI: 10.26008/1912/bco-dmo.897734.1Version 1 (2023-06-21)Dataset Type:Other Field Results

Principal Investigator: Rebecca J. Gast (Woods Hole Oceanographic Institution)

BCO-DMO Data Manager: Shannon Rauch (Woods Hole Oceanographic Institution)


Project: Trojan Horses in the Marine Realm: Protist Parasite-host Dynamics in Coastal Waters (Coastal Parasites)


Abstract

This dataset includes Group II Syndiniales infected host and dinospore counts determined from CARD-FISH hybridization carried out on water samples collected at the Martha's Vineyard Coastal Observatory (MVCO) monthly or bimonthly from September 2019 to October 2020 from about 2 meters below the surface. Samples were collected using bucket casts or a CTD. The detailed hybridization method using the ALV01 (Group II) probe can be found at dx.doi.org/10.17504/protocols.io.bsxmnfk6.

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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.


Related Datasets

IsRelatedTo

Dataset: MVCO Syndiniales amplicon ASV counts
Gast, R. J. (2023) Relative abundances of different Syndiniales groups from surface water samples collected at the Martha's Vineyard Coastal Observatory (MVCO) monthly or bimonthly between 2013 and 2021. Biological and Chemical Oceanography Data Management Office (BCO-DMO). (Version 1) Version Date 2023-06-14 doi:10.26008/1912/bco-dmo.897547.1
IsRelatedTo

Dataset: https://doi.org/10.6073/pasta/ca34be7554ddc67c9fa0f8dea01f375b
Sosik, H. M., Crockford, E. T., & Peacock, E. (2022). Dissolved inorganic nutrients from the Martha's Vineyard Coastal Observatory (MVCO), including 4 macro-nutrients from water column bottle samples, ongoing since 2003 (NES-LTER since 2017) [Data set]. Environmental Data Initiative. https://doi.org/10.6073/PASTA/CA34BE7554DDC67C9FA0F8DEA01F375B

Related Publications

Methods

Sehein, T., Gast, R., Pachiadaki, M., Guillou, L., & Edgcomb, V. (2021). Group II Syndiniales ALV01 CARD-FISH v1. https://doi.org/10.17504/protocols.io.bsxmnfk6