Water for incubation and filtration was collected via Niskin bottles mounted on a rosette, equipped with a CTD for Western North Atlantic, Danish Coastal Seawater, and Abyssopelagic Waters off the Eastern Coast of Japan. Additionally, in the Western North Atlantic, water was collected via an in-situ syringe-based sampler and incubator developed by the Danish Center for Hadal Research.

Niskin-collected water was used to measure cell counts and investigate selfish polysaccharide uptake under near-surface (0.1 MPa), or bathy- and abyssopelagic hydrostatic pressures (20, 40, 42 and 52 MPa). Water at each station was dispensed into smaller glass Duran bottles that were cleaned and pre-rinsed three times with water from the Niskin prior to dispensing. To 350 mL of bulk seawater or 100 mL of autoclaved seawater, substrate was added at 3.5 μM monomer-equivalent concentrations, except for fucoidan, which was added at 5 μM concentrations (a higher concentration was necessary for sufficient fluorescence signal). From these bottles, nine 6 mL Exetainer vials were filled with seawater for each substrate, replicate, and time point, and four 6 mL Exetainer vials were filled with autoclaved seawater to serve as a killed control. Sets of vials were then pressurized to either 0.1, 20, or 40 MPa in separate pressure vessels for each substrate and time point and stored in the dark at 4ºC for 0, 2, or 5 days.

When collected via an in-situ syringe sampler (ISS), glass syringes fitted to the sampler were preloaded with individual polysaccharide substrates to 3.5 μM monomer-equivalent concentrations or preloaded with autoclaved seawater to serve as a negative control (blank). Once lowered to the desired depth of 2000, 4000, 4200, or 5200 m (depending on station depth), half of the syringes were triggered so that they drew in surrounding water that mixed with the preloaded substrate or autoclaved seawater. The ISS was held at depth for approximately 24 hours of incubation. While still at depth, shortly prior to retrieval, the second half of the syringes were triggered to draw in the surrounding water. These syringes served to measure the quantity of selfish uptake occurring briefly at depth, and then during the upcast.

After sample incubation, approximately 25 mL of incubated sample was filtered through a 25 mm 0.2 µM nucleopore filter for molecular analysis, placed in cryovials, and promptly frozen at -80C.

Another approximately 25 mL of incubated sample was incubated with fixative for two hours, then filtered through a 25 mm 0.2 µM nucleopore filter. After filtration, the filters were dried in a laboratory hood, then each filter was labeled along the outer edge, placed in a Petri dish, and stored at -80C for total cell counts, and quantification of 'selfish' uptake.

Polysaccharide used for incubation:

ara = arabinogalactan
chn = chondroitin sulfate
fuc = fucoidan
lam = laminarin
man = mannan
pul = pullulan
xyl = xylan
muc = mucin