Additionally, individual flagellate cells were isolated by means of a specially constructed micropipette , and cultured in 96-well plates or petri-dishes, with sterile autoclaved Baltic Sea water as medium and Pseudomonas putida MM-1 as food source. Dried whole mount preparations of these flagellates were later examined with a JEM-1011 transmission electron microscope (JEOL Ltd.; Tokyo, Japan) as previously described . For HNF cell counts in 2008 and
2009, 100 ml samples were fixed with a final concentration of 1% particle free formaldehyde in brown glass bottles, at 4°C, between 2 and 24 h. Subsamples were filtered onto black polycarbonate filters (0.8 μm pore-size; 25 mm diameter; Whatman GmbH, Dassel, Germany), which were stored at −20°C or −80°C. Filters were later stained with DAPI at a concentration of 0.01 mg ml−1, mounted, and observed under a Zeiss Axioskop 2 mot plus epifluorescence ERK inhibitor microscope (Carl Zeiss MicroImagimg GmbH, Gottingen, Germany). A minimum of 100 cells per filter were counted at 630X using filter set 02 Epacadostat (Carl Zeiss MicroImagimg GmbH). Aloricate choanoflagellates were clearly distinguishable and therefore counted as a separate
group. Acknowledgements We are indebted to Ronja Breitkopf and Bärbel Buuk for excellent technical support, as well as Dr. Konstantin Khalturin for transport of cultured strains to St. Petersburg. Sincere thanks are given to Dr. Cedric Berney for provision of a primer sequence. We would like to thank Olivia Diehr and Jürene Bruns-Bischoff for their sedulous support in providing a lot of references. We are grateful to Felix Weber for helpful discussions of the data and the manuscript. This work was funded by grant from the German Science Foundation (DFG) (JU 367/11–1) and the RAS Presidium program “Problems of life origin and biosphere development”. References Liothyronine Sodium 1. Adl SM, Simpson AGB, Farmer M, Andersen RA, Anderson OR, Barta JR, Bowser S, Brugerolle G, Fensome RA, Fredericq S, James T, Karpov S, MI-503 Kugrens P, Krug J, Lane CE, Lewis LA, Lodge J, Lynn DH, Mann DG, McCourt RM, Mendoza L, Moestrup Ø, Mozley-Standridge SE, Nerad
TA, Shearer CA, Smirnov AV, Spiegel FW, Taylor MJR: The new higher level classification of eukaryotes with emphasis on the taxonomy of protists. J Eukaryot Microbiol 2005, 52:399–451.PubMedCrossRef 2. King N, Carroll SB: A receptor tyrosine kinase from choanoflagellates: molecular insights into early animal evolution. Proc Natl Acad Sci USA 2001, 98:15032–15037.PubMedCrossRef 3. Steenkamp ET, Wright J, Baldauf SL: The protistan origins of animals and fungi. Mol Biol Evol 2006, 23:93–106.PubMedCrossRef 4. Ruiz-Trillo I, Lane CE, Archibald JM, Roger AJ: Insights into the evolutionary origin and genome architecture of the unicellular opisthokonts Capsaspora owczarzaki and Sphaeroforma arctica . J Eukaryot Microbiol 2006, 53:379–384.PubMedCrossRef 5.