Dataset: Exudate Uptake Incubations - Microbial Abundances (Flow Cytometry)

Experiments were conducted to monitor responses of reef seawater microbial communities to concentrated exudates from P. astreoides and G. ventalina. After the organism incubations were conducted, excess filtrate (2 l) from 3 of the 6 colony/fragment incubations (selected randomly) were pooled into an acid-washed, 10 l carboy and mixed. Excess filtrate (~2 l) from the three control incubations were also pooled into a second, acid-washed, 10 l carboy and mixed.

For the P. astreoides experiment, surface seawater was collected from the offshore site and coarsely filtered through a GF/A filter (1.6 µm nominal pore size) using peristalsis to remove larger cells and minimize heterotrophic grazing, but retain bacteria and archaea. Approximately 2.4 l of this inoculum was added separately to each of the 10 l carboys (pooled coral and control filtrate) to create a 5:2 ratio of filtrate: inoculum. After this addition, each carboy was mixed and a suite of samples were collected for different analyses including cell counts, inorganic and organic macronutrient quantification, and 16S rRNA gene sequencing. This collection marked the first timepoint (0 hours [hrs]) for the exudate uptake experiment. For the G. ventalina experiment, reef seawater inoculum was collected from Tektite reef (Table S1).

For each experiment, coral metabolite or control filtrate seawater were transferred into separate 1 l acid-washed polycarbonate bottles (6 bottles per treatment). Within each treatment (coral and control), 3 of the 6 bottles were blackened to block light. The bottles were placed into a flow-through seawater table. PAR readings in the seawater table for the P. astreoides and G. ventalina experiments ranged from 250 – 1000 and 164 - 530 µmol quanta m -2s -1, respectively, with variation caused by shading and cloud cover. Over the course of 48 hours, samples were collected for cell counts (1 ml) at all timepoints (0, 12, 24, 36, and 48 hrs), macronutrient analyses (30 – 40 ml) at 0 and 48 hrs, and microbial community analyses (60 – 300 ml) at 0, 24, and 48 hrs.

Samples collected for cell counts were fixed to 1% (v/v) paraformaldehyde, refrigerated for 20 minutes in the dark, and frozen in a charged dry shipper.

Seawater samples (60 ml for P. astreoides) collected for microbial community analyses were obtained using 60 ml sterile, Luer-lock syringes (Becton Dickinson, Franklin Lakes, NJ, USA) and filtered using positive pressure onto 25 mm, 0.2 m pore-size filters. The amount of volume filtered was increased for the G. ventalina exudate experiment because of biomass concerns and ranged from 180 (0 and 24 hrs) to 300 ml (48 hrs). Previous work has demonstrated that sample volume does not significantly influence microbial community composition when beta diversity comparisons are made [6]. Alpha diversity analysis was avoided due to the differences in seawater volumes. Filters were then transferred into cryovials and placed in the charged dry shipper, followed by -80 °C storage until DNA extraction.