Methods Sampling The seawater-brine interfaces (haloclines) of the DHABs Tyro, Thetis, and Medee in the Mediterranean Sea were sampled on the cruise aboard the R/V Urania in 2009. Samples from the DHAB Urania were collected in 2009 on the R/V Oceanus. Sampling sites are depicted in Figure 1 and coordinates with environmental data for each DHAB halocline

and brine are provided in Table 3. The positions of the interfaces were determined using a SBE911plus CTD (Sea-Bird Electronics, Bellevue, WA, USA) equipped with an SBE43 oxygen sensor (Sea-Bird Electronics, USA). Samples were collected from the interface and brine of each basin using a rosette equipped with 12-L Niskin bottles. The Sapitinib supplier salinity gradient from the top to the bottom of individual Niskin bottles was confirmed on board the ship using a WTW portable sensor for conductivity, pH and selleck chemical temperature (WTW, Weinheim, Germany). Water samples were collected from Niskin bottles into 50-L Nalgene bottles flushed with argon gas and 6–10 L water were filtered immediately onto Durapore Buparlisib membranes (47 mm; 0.65 μm; Millipore, USA) under gentle vacuum (flow rate: ca. 50 ml/min) and under argon in the case of anoxic samples [2], followed by storage in RNAlater (Ambion, Applied Biosystems, USA). According to Ambion’s RNAlater manual,

the filters were stored at 4°C for 24 hours prior to freezing at −20°C until RNA extraction. RNA was used to ensure that samples were not contaminated by settling DNA from above

the investigated layers. Table 3 Coordinates, sampling depths and physico-chemical data of the brines (B) and halocline interfaces (IF) of the different DHABs under study   Coordinates (Long, Lat) Depth (m) Salinitya(PSU) Conductivitya(S/m) Oxygena(ml/l) Na+(mmol) Mg2+(mmol) SO4 2-(mmol) HS-(mmol) MIF 22.312124 E, 34.19468 N 2924 70 7.7 0.5 847 161 41 n.a. TIF 26.21962 E, 33.524236 N 3327 67 7.8 0.5 1111 15 11 0.07 ThIF 22.084368 E, 34.401134 N 3259 80 8.2 0.68 1368 174 76 0.11 UIF 21.283252 E, 35.13528 N 3468 63 7.8 1.22 876 79 42 0.66 MB 22.312124 E, Adenosine 34.19468 N 2950 320 16.7 0 4818 792 201 2.9 TB 26.21962 E, 33.524236 N 3448 321 16.7 0 5300b 71b 53b 2.1b ThB 22.084368 E, 34.401134 N 3380 348 16.7 0 4760b 604b 265b 2.1b UB 21.283252 E, 35.13528 N 3493 240 15.6 0 3505b 315b 107b 15 M Medee, T Tyro, Th Thetis, U Urania. Data are from the literature and from this study (measured as described in [5]). n.a. not available. afrom [54]; bfrom [5]. Environmental RNA Isolation, transcription and PCR amplification of ciliate SSU rRNAs The method for the extraction and reverse transcription of environmental RNA (envRNA) from protistan plankton collected on membranes has been described in detail previously [2].