To establish microcosms for the experiment, water samples were collected from the Apalachicola Bay (N 30° 4' 26.436", W 84° 10' 48.874") in northwest Florida, USA. About 20 liters of seawater (SW) were collected in sterile plastic bottles. The samples were transported on ice (4 ℃) to the Microbial Ecology Laboratory at Florida A&M University within two hours. To establish the microcosms, water samples were first filtered through 5 μm membrane filters (Whatman Laboratory, NJ) to remove debris and larger organisms. Then 600 ml was dispensed into each of three (A, B, and C) 1-liter flasks to create the various microcosms as shown in Figure 1. For treatment D, 600 ml of SW samples were filtered through 0.8 μm membrane filters to remove protist. One subsample labeled W was unfiltered. All microcosm flasks were shaken at 80 rpm at 25 ℃ for 120 h. Samples (5 ml) were collected every 3 hours, dispensed in a 96-well microtiter plate and the OD measured at 600 nm using an Absorbance Microplate Reader Q4 (BIO-TEK Instruments Inc., USA). Population changes in HBx and V. parahaemolyticus were monitored by culture- and qPCR-based methods as described below. The protocol described here is similar to that published by Williams et al. (2016). A key difference is those investigators also filtered the microcosm samples through a 0.1 µm filter to allow viruses to pass into the filtrate as their investigation included monitoring of virus numbers in the various microcosm treatments, and did not include protists.

Enumeration of HBx and V. parahaemolyticus by culture-dependent method

At 3-h intervals, 1 ml was collected from all microcosms (A, B, D, and W) and 10-fold dilutions of these prepared in sterile SW.  One tenth (0.1) ml taken from three of these dilutions (10-6, 10-7, 10-8) was inoculated on seawater yeast extract (SWYE) agar in duplicate to obtain countable numbers of CFU of V. parahaemolyticus. All plates were incubated at 28°C ± 0.5°C for 24 hours, and colony-forming units (CFU) were counted. To culture HBx requires a prey bacterium suspension. The suspension was prepared by flooding a SWYE agar plate with an overnight-grown culture of Vibrio parahaemolyticus with 5 ml of autoclaved SW. The fluid remained undisturbed on the surface of the plate for 5 minutes. The culture was then suspended in the fluid using s circular motion with a sterile L-shaped spreader. The resulting suspension was transferred into a 15 ml tube and used as the stock prey suspension. One ml of the prey stock suspension was inoculated into tubes along with samples (5, 1, 0.1 ml) from each of the microcosms. In cases where the sample volume was less than 5 ml, autoclaved SW was added to bring the final sample volume to 5 ml). The suspension was added to top agar, and the mixture mixed mechanically and overlaid onto the surface of bottom agar in agar plates. The plates were cultured at room temperature for 3 to 5 days and examined daily for plaques typical of HBx, Plaque –forming units (PFU).were counted. The daily counts of HBx and V. parahaemolyticus were plotted and changes over the course of the experiment were observed and compared with counts of protist during the same time period.  

DNA extraction from water samples for qPCR

For DNA extraction, a 1 ml sample was collected from each microcosm at each time interval, and 250 μl was used for DNA extraction. Genomic DNA was isolated using the PowerSoil® DNA Isolation Kit (Mo Bio Laboratories, Inc., Carlsbad, CA) according to the manufacturer’s instructions and the DNA was quantified with a NanoDrop ND-1000 UV spectrophotometer (Thermo Scientific, Wilmington, USA). The DNA samples were stored at -20 °C prior to use.