Dataset: Seawater nutrient/metabolite and flow cytometry metadata - Picoplankton incubation experiment 2020

Collection Media and inoculum:

Sponge exhalent water and reef sea water, for use as media in the incubation experiment, were collected on 9-December-2020 from Looe Key Reef Sanctuary Preservation Area (24.54605, -81.40610) in the Florida Keys National Marine Sanctuary (FKNMS). Sponge exhalent water was collected from each of 4 individuals of two sponge species, Niphates digitatalis and Xestospongia muta. Exhalent water collection was completed by scuba divers using a three-way valve with one valve attached to a ~8cm piece of PharMed L/S 25 tubing (Masterflex® L/S® Precision Pump Tubing, Radnor, PA, USA) that was placed into the excurrent osculum of the sponge to collect exhalent water, a second valve attached to a syringe to create suction and pull in exhalent water, and the final valve attached to ~8cm piece of Teflon tubing that further attaches to the stainless-steel fitting on a 1 L FlexFoil PLUS bag (SKC, Eighty Four, PA, USA) for final exhalent water storage. Exhalent water was pulled into the syringe at a rate of ~2ml min-1, below the estimated pumping rate of both sponge species. Two bags (~1L) were filled per sponge and all sponges were located within 4 meters of one another at ~8m depth. Upon return to the boat all bags were sealed and placed in an ice-filled cooler until further processing in the lab (~2 hrs). 

Reef surface water, for use as both reef metabolome media and picoplankton inoculum during the incubation experiment, was collected in 6 acid-washed 2 L Nalgene© HDPE bottles (Thermo Fisher Scientific, Waltham, MA, USA) from ~1 m below the surface just above the reef at Looe Key. Metabolome bottles were immediately placed in a cooler filled with ice until return to the lab (~1 hr). The reef picoplankton inoculum was kept in the dark at ambient seawater temperature.

Picoplankton incubation experimental setup:

The 48-hour experiment included two treatments, reef (i.e., control) and sponge metabolome, that were sampled at 0 hours, 24 hours, and 48 hours (T0, T24, T48, respectively). Surface seawater (6 L) for picoplankton inoculum was filtered through three 1.6 µm, 47 mm pre-combusted GF/A filter to remove larger eukaryotic plankton and particular matter. Filters were housed in a 47 mm acid-washed in-line PFA filter holder (Advantec, Cole-Parmer, Vernon Hills, IL, USA). Filtering was completed via peristalsis (MasterFlex L/S pump and pump heads, Cole-Parmer, Vernon Hills, IL, USA). Samples were filtered slowly (50 ml min-1) to avoid bursting of microbial cells on the filter membrane and filters were changed every 2 L. PharMed L/S 25 (Masterflex® L/S® Precision Pump Tubing, Radnor, PA, USA) acid-washed tubing was used for all filtration. The sponge and reef metabolome media (6 L each) were both passed through 0.22 µm, 47 mm polytetrafluoroethylene (PTFE) filters (Omnipore, EMD Millipore Corporation, Billerica, MA, USA) using the same process detailed above. Sponge metabolome media consisted of a 3:1 ratio of filtered sponge exhalent water (1:1 exhalent water from X. muta and N. digitalis) to 0.22 µm filtered sea water.

Following filtering, two 20 L food grade containers were used, one per treatment, to pool the filtered inoculum with the filtered media. Pooling was done to ensure consistent starting mixtures for each incubation bottle. Following initial (T0) sampling as detailed below, pooled treatments were evenly distributed into four 2 L Nalgene© bottles per treatment for an approximate ratio of 1:2 filtered picoplankton inoculum to filtered media. All incubation bottles were kept in a dark incubator at 26ºC for the duration of the 48-hr experiment and only removed for picoplankton community sampling at T24. Water chemistry and cell abundance was assessed for both the sponge and reef metabolome treatments at the start (T0) and end (T48) of the incubation experiment. Samples were also taken at T24, but they were only used to obtain cell abundance.