0.22 μm polycarbonate filter

Biomass freshly collected from the bioreactor on day 1,256, and sludge collected on the same day and location as the inoculum for the enrichment culture, was fixed with 2% paraformaldehyde as described elsewhere^{90 (link)}. Fixed samples were filtered onto 0.22 μm polycarbonate filters (Millipore) and stored at −20 °C until analysis.

Prokaryotic cells were counted using a flow cytometer (FacsCalibur, Becton Dickinson, Heidelberg, Germany) following the method of Gasol et al. (1999) (link). Calculations were performed using the software program “Cell Quest Pro,” plotting the emission fluorescence of SYBR Green I (488 nm) vs. the side scatter. Picocyanobacteria were similarly counted on the basis of their signature in a plot of orange (FL2) vs. red (FL3) fluorescence.
The incorporation of ³H-leucine (140 Ci mmol^-1) was measured to estimate heterotrophic bacterial productivity in 10-mL water samples. Triplicate samples were incubated at a final concentration of 100 nM for at least 1 h at the in situ temperature in the dark. Incorporation was stopped by fixing the cells with formaldehyde (5% v/v). A fourth sample, serving as a blank, was fixed for at least 10 min prior to the addition of the radioactively labeled substrate. The samples were filtered onto 0.22-μm polycarbonate filters (Millipore), which were then placed in 4 mL of scintillation cocktail. The incorporated substrate was counted in a scintillation counter (Packard). Bacterial carbon production was calculated from ³H-leucine incorporation according to Simon and Azam (1987) (link), using a leucine mol% value of 7.3 and a carbon conversion factor of 0.86.

Six sponge adults of Aplysina aerophoba were collected by SCUBA diving in June 2014 in Cala Montgó, Spain (42°06′ 52.6″N, 3°10′ 02.0″E) at 7.8 to 12.7 m depth. Individual samples were taken at locations between 3 to 15 meters apart from each other. They were collected using gloves, cutting approximately 5 cm³ of the sponge tissue and placing it in 50 mL sterile tubes. The in situ identification was done visually by Dr. Detmer Sipkema. Seawater was collected in the immediate vicinity of the sponges (at minimally 1 m of distance), in four locations, using 3 L sterile containers to compare putative sponge-associated bacterial proteins to bacterioplankton proteins. Immediately after collection, the sponge pieces were flash-frozen in liquid nitrogen and then stored at −80 °C. Seawater samples were filtered through a 0.22 μm polycarbonate filter (Millipore) and the filters were preserved at −80 °C.

Individual cell morphology was determined via scanning electron microscopy (SEM; Fig. 1). Bacterial samples for SEM were initially fixed in 3% glutaraldehyde in 0.1mol L^‐1 cacodylate for 72 h at 4°C. Samples were then pulled onto a 0.22 μm polycarbonate filter (Millipore, Billerica, MA), washed in a graded ethanol series (50%, 75%, and 100%) and placed in hexamethyldisilazane for 1 h at room temperature. Filters were then mounted, palladium‐coated (Hummer VI, Union City, CA), and visualized using a Phenom desktop SEM (FEI Instruments, Hillsboro, OR).

Shrimps and rearing water were collected from a commercial marine shrimp hatchery from 10 March to 28 April, 2014 in Hainan, China. Healthy shrimp and rearing water were taken from ponds where shrimp larvae had normal feeding behavior, black intestine and/or no apparent sign of disease by visual inspection. Samples covered all key developmental periods: zoea 1 (Z1), zoea 3 (Z3), mysis 1 (M1), mysis 3 (M3), postlarvae 1 (P1), postlarvae 3 (P3), and postlarvae 6 (P6) (a developmental time line was shown in Figure 1). Diseased shrimps and rearing water were obtained from ponds where shrimps presented poor growth, inactivity, lack of appetite, empty digestive tracts and/or low survival rate. Shrimp larvae were collected randomly from each pond. Details of the experimental design for sampling was shown in Supplementary Table S1. The surface of shrimp larvae was sprayed with 75% ethanol, and then washed with sterile seawater three times to remove adherent microorganisms. Rearing water was collected with 250 ml sterilized beaker from four different locations in each pond and then pooled. One liter of pooled rearing water was filtered through a 0.22 μm polycarbonate filter (Millipore). All the samples were stored at -80°C for 2 months until DNA extraction.