balthica, and (2) to determine the quantitative contribution of b

balthica, and (2) to determine the quantitative contribution of both species to the Baltic protistan community via fluorescently labelled specific probes. Moreover, both cultivated species are ideal model organisms for future studies on temporary anaerobic metabolism using derived mitochondria. Methods Sampling, isolation/cultivation and counting of choanoflagellates Strains of the newly described

Codosiga spp. were obtained from untreated plankton samples taken in the central Baltic Sea at the Gotland (IOW-station 271; 57° 19.2′ N; 20° 03′ E) and the Landsort Deep (IOW-station 284; 58° 35.0′ N; 18° 14.0′ E) in May 2005 during an expedition with the RV Alkor. Clonal cultures were obtained from a single cell shortly after sampling, which was isolated using a micromanipulator fitted with glass micropipette [54]. The cultures were deposited as part of the IOW culture collection, and were routinely kept in sterile 50-ml tissue culture flasks (Sarstedt, Nümbrecht, Germany) in F2 medium [55] (salinity 8–12 ‰) on a mixture AZD6738 order of bacteria grown on a

wheat grain. Altogether four choanoflagellate cultures could be established (Table 1). Samples for cell-counts of HNF were obtained on board the RV Poseidon in August 2008 (Gotland Deep) and the RV Maria S. Merian in September 2009 (Gotland and Landsort Deep). Water from different depths (GD 2008: 114–137 m, GD 2009: 90–140 m, LD 2009: 70–120 m) was collected in 10 l free-flow bottles attached to a conductivity, temperature and depth rosette (CTD) with a coupled oxygen sensor. In all cases, oxygen and hydrogen sulfide were measured immediately

on board according to standard methods [56]. In order to avoid potential Adenosine triphosphate oxygen contamination during emptying of the free-flow bottles, for experimental purposes only the bottom 5 l of water from 10 l free-flow bottles was employed. Molecular biological investigations DNA was extracted from cells harvested from 20–30 ml of dense cultures (8000 g, 20 min, 4°C) using a CTAB extraction as described previously [57]. The 18S rRNA gene was amplified by polymerase chain reaction (PCR) using eukaryotic specific primers 18SFor-n2 (5′- GAT CCT GCC AGT AGT CAT AYG C – 3′) and 18SRev-Ch (5′- TCC TTC TGC AGG TTC ACC TAC GG – 3′). The mixture containing 0.1 mM of each primer, 200 mM dNTPs, 10 mM Tris pH 8.3, 1.5 mM MgCl2, 50 mM KCl, and 1 unit of Taq DNA polymerase (Fermentas) was heated to 95°C for 2 min, and the 18S rRNA gene was amplified in 35 cycles of 95°C for 30 s, 52°C for 45 s, and 72°C for 2 min, followed by 10 min at 72°C. PCR 4SC-202 manufacturer products were purified with the Nucleospin II Kit (Machery Nagel). Sequencing was carried out by a company (Qiagen) with the primers used for PCR and four different internal sequencing primers (590F: 5′- CGG TAA TTC CAG CTC CAA TAG C – 3′, 600R: 5′- GCT ATT GGA GCT GGA ATT ACC G – 3′, 1280F: 5′- TGC ATG GCC GTT CTT AGT TGG TG – 3′, 1300R: 5′- CAC CAA CTA AGA ACG GCC ATG C – 3′).

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