0.2 μm pore size membrane filters

Ten water samples were collected from the Delaware Bay in March, August/September, and November 2014, and six samples were collected from the Chesapeake Bay in April and August 2015, on board the RV Hugh R Sharp. Samples were collected to reflect different salinity gradients in each estuarine ecosystem (Fig. 7). The overall sampling strategy was to collect viral communities across a wide spatial and temporal scale in both estuaries. Additional information about environmental conditions can be found in Table 2 and in Table S1 in the supplemental material. Samples DB8.2A and DB8.2B are diel samples; samples CB8.2S, CB8.2M, and CB8.2D were taken at different depths (∼1, 13, and 22 m, respectively).
At each of the sampling sites, water samples were collected using a Niskin bottle on a Sealogger conductivity-temperature-depth rosette water sampler. For each sample, 10 liters of seawater was prefiltered through 0.2-μm-pore-size membrane filters (Millipore Corporation, Billerica, MA) to remove bacteria and larger organisms. Viral communities were concentrated from the 0.2-μm filtrates by following the FeCl₃ flocculation method described by John et al. (71 (link)). Viral dsDNA was extracted using the phenol-chloroform/isoamyl method (72 (link)).

Tiamulin, tilmicosin, valnemulin and lincomycin were obtained from Fluka Analytical (St Louis, Missouri, USA). Tylosin was obtained from Serva (Heidelberg, Germany) and tylvalosin from Eco Animal Health (London, UK). All antibiotic stock solutions were sterilised by filtration through 0.2 μm pore size membrane filters (Millipore).

Water samples were collected every 2 m from top to bottom using a Plexiglass^® water sampler (LB-800, Qingdao, China) at the center of the three lakes in July 2017. Due to the different depth of the lake, there are 3, 4, and 5 sampling points in PPH, MYH, and GH, respectively (Table 1). Water was sampled three times at each sampling point and each repetition took 1 L water. In total, nine samples were collected from PPH, 12 samples from MYH, and 15 samples from GH. Approximately 2.5 L of the water sample from each sampling site was filtered in the laboratory using a sterile 0.2 μm pore size membrane filters (Millipore, Jinteng, Tianjin, China) for DNA extraction. Filters with retaining biomass were sealed and stored at −80°C until analysis. The remaining 0.5 L water was used for analysis of physicochemical properties.