Plankton

P. aeruginosa PAO1 was propagated on trypticase soy agar (TSA) for plate-based assays or in trypticase soy broth (TSB) for liquid culture. M9 growth media supplemented with 0.4% (w/v) glucose and 0.4% (wt/v) casamino acids was used for biofilm formation experiments. Culture media (TSB, TSA, M9 salts and casamino acids) were obtained from Difco/Becton Dickinson (Franklin Lakes, NJ, USA) and all other reagents (phosphate buffered saline, glucose, ethanol and crystal violet) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Corning 35–1172 flat-bottomed polystyrene 96-well plates were used for biofilm formation experiments and optical density measurements were performed in a Tecan M-200 (Durham, NC, USA) plate reader. Optical micrographs of biofilms were obtained using a Nikon Eclipse 80i microscope.
A microplate based assay, modified from Junker et al.[32] (link) was used to screen compounds for QSI. Briefly, P. aeruginosa PAO1 was grown in TSB for 18 h at 37°C with rotary shaking at 225 rpm. The culture was then centrifuged at 14,000 rpm and rinsed with phosphate buffered saline (PBS, pH 7.4) three times, then was re-suspended in M9 minimal growth media to approximately 1×10⁷ cfu/ml (determined by OD and plate count assay). Test compounds were dissolved in DMSO and were added to sterile distilled water to achieve concentrations ranging from 0.1–10 mM while keeping DMSO at a maximum of 1% (v/v). P. aeruginosa inocula (360 µl) were then pre-mixed with 40 µl of the test compound solutions to achieve final compound concentrations ranging from 0.01–1 mM. An aliquot (100 µl) of this cell/compound mixture was then added to three separate wells in a 96-well microplate for replicate testing. For control wells (no inhibitor), dilute DMSO was added to the inocula instead of test compounds, to a final concentration of 1% (v/v). Optical density (OD_600nm) measurements were performed immediately after inoculation and after 24 h incubation at 37°C (without shaking) to monitor planktonic cell growth. To determine the amount of biofilm formation, supernatant from the microplate wells was gently removed and the wells were washed twice with 150 µl of PBS using a multichannel pipette. The remaining biofilm was then stained using 100 µl of a 0.2% (w/v) crystal violet solution for 15 min at room temperature. The crystal violet was then removed from the wells, the wells were rinsed four times with PBS, and then 100 µl of 95% ethanol was added to extract the crystal violet solution from the biofilm. The OD_600nm of the extracted crystal violet was then measured, yielding a measure of biofilm formation (relative to the control). For optical imaging, crystal violet stained biofilms were washed with distilled water and no ethanol extraction was performed.
In addition to crystal violet based quantification of biofilm biomass, cell viability within biofilms exposed to inhibitor compounds was determined using the formazan dye-based MTT assay (Cell Proliferation Kit I, Roche Diagnostics, Mannheim, Germany). This assay has previously been described for determination of biofilm cell viability [43] (link)–[45] (link). Briefly, biofilms were grown in 96 well microplates for 24 h as described above, in the presence and absence of inhibitor compounds. After this initial inoculation period, planktonic cells were removed and the remaining biofilm was gently rinsed three times with 100 µl of PBS. After rinsing, 100 µl of PBS and 10 µl of the MTT labeling reagent were added and the suspension was incubated for 4 h at 37°C, followed by addition of 100 µl of solubilization solution. Plates were then incubated for 24 h at 37°C and absorbance measurements were taken using a Tecan M-200 plate reader at 560 nm (peak absorbance for the formazan dye breakdown product) and at 700 nm (reference wavelength for the intact dye).

The quantification of antibiofilm activity was based on the research by Waturangi et al. [4 (link)] with some modifications. The activities of antibiofilm were divided into inhibition and destruction activity. For inhibition activity, B. cereus and B. subtilis inoculated into Brain Heart Infusion Broth (BHIB) and incubated at 37 °C, 120 rpm 24 h, whereas S. putrefaciens inoculated into BHIB and incubated at 28 °C, 120 rpm 24 h. A total of 100 μL cultures (OD₆₀₀ = 0.132) and 100 μL crude extract were transferred into a 96-wells microplate and incubated at 37 °C 24 h for B. cereus and B. subtilis and 28 °C for S. putrefaciens. For destruction activity, a total of 100 μL cultures (OD₆₀₀ = 0.132) were transferred into a 96-wells microplate and incubated to form biofilms. After 24 h, 100 μL crude extract was added to each well then reincubated. Overnight culture of food spoilage bacteria without any treatment were used as a positive control, while sterile BHIB was used as a negative control.
After incubation, the planktonic cells and media were discarded and the adherent cells were rinsed twice with deionized water and allowed to air dry. A total of 200 μL of 0.4% crystal violet was used to stain the adherent cells for 30 min. Thereafter, the dye was discarded and the wells were rinsed five times with deionized water and allowed to air dry. Afterward, 200 μL of ethanol was added to solubilize the crystal violet. The optical density was determined at 595 nm with a microplate reader (TECAN M200 PRO). The percentage of inhibition or destruction activity calculated using the following equation:
$$\%\text{inhibition or destruction}=\frac{\mathrm{OD}\text{positive control}-\mathrm{OD}\text{sample}}{\mathrm{OD}\text{positive control}}\mathrm{x}100\%$$

The inhibition of biofilm formation was assessed using methods that were described previously [20 (link)]. Briefly, 90 μL of a bacterial suspension (final OD₆₀₀ = 0.01), prepared by diluting an overnight culture grown in LB broth into the medium of interest, was added to the interior wells of a 96-well polystyrene microtitre plate containing 10 μL of peptide at 10× the final desired concentration, or 10 μL of vehicle control. A diagram depicting a typical microtitre plate layout for this experiment is shown in Figure S1. The plates were incubated overnight at 37 °C under static conditions to allow for bacterial growth and biofilm maturation. The following day, bacterial growth was quantified by recording OD₆₀₀ of each well using an Epoch Microplate Spectrophotometer (BioTek Instruments Inc., Winooski, VT, USA). The planktonic cells and the spent medium were discarded, and the adhered biomass was rinsed three times with distilled water. The biomass was stained with 0.1% CV solution for 20 min and then rinsed three times with distilled water to remove unbound dye. The bound CV dye was resuspended in 70% ethanol with gentle mixing and the A₅₉₅ was recorded in the same sample plate. The amount of biofilm inhibition was calculated relative to the amount of biofilm that was grown in the absence of peptide (defined as 100% biofilm) and the media sterility control (defined as 0% biofilm). Results from at least three separate biological replicates were averaged.

The abundance of krill was studied with a Simrad EK80 research echosounder with six frequencies. For this study, we focused on the 38 kHz frequency. It was scrutinized with the Large Scale Survey System (LSSS) software version 2.5.0^{68 (link)}. During the campaign, we used two transducers: one on the drop-keel (3 m from the hull when down) and one hull-mounted. The latter was used in ice-covered areas, with low impact on the detection of krill swarms^{15 (link)}. The density of krill was calculated as nautical area scattering coefficient (NASC)^{69 (link)}.
Krill swarms were sampled with a Macroplankton trawl at two locations within the bloom region (the positions of the two trawling stations are indicated in Fig. 4a). The trawl is a fine-meshed plankton trawl with a 36 m² mouth-opening and 3 × 3 mm diamond shaped mesh (7 mm stretched) from mouth to rear. Towing speed was normally 2.5–3 knots. The velocity of the trawl through water and depth of the trawl were monitored by a depth sensor (SCANMAR) attached to the headline. From each trawl, a subsample of approximately 150 individuals of Euphausia superba was taken, and the length of the individual krill was measured (±1 mm) from the anterior margin of the eye to tip of telson excluding the setae. Sex and maturity stages of E. superba were determined using the classification methods according to Makarov and Denis⁷⁰ .

The variation of ciliate vertical distribution was addressed by conducting two time-series sampling in the upper 500 m at two distinct sites, Station (St.) S1 in nSCS and St. P1 in tWP, during two different cruises (Fig. 7). St. S1 was visited from 29 to 31 March 2017 aboard R.V. “Nanfeng”, and St. P1 from 2 to 3 June 2019 aboard R.V. “Kexue”. During 48 h (St. S1) or 24 h (St. P1) sampling periods, seawater samples were collected by using a CTD (Sea-Bird Electronics, Bellevue, WA, USA)—rosette carrying 12 Niskin bottles of 12 L each (Supplementary Table S5). In the nSCS, the sampling depths were 3, 10, 25, 50, DCM (deep Chl a maximum layer), 100, 200 and 500 m; in the tWP, the sampling depths were surface (3), 30, 50, 75, DCM, 150, 200, 300 and 500 m. Casts were approximately launched every 6 h, the CTD determining vertical profiles of temperature, salinity and chlorophyll a in vivo fluorescence (Chl a). A total of 117 seawater samples were collected for planktonic ciliate community structure analysis. For each depth, 1 L seawater sample was fixed with acid Lugol’s (1% final concentration) and stored in darkness at 4 °C during the cruise.Figure 7

Survey stations in the northern South China Sea (nSCS) and tropical West Pacific (tWP).