the Expedition ARKTIS-XIX/4 of the research vessel POLARSTERN ...

A surface sediment sample was taken from 2-3 Multicorer tubes. The uppermost
centimeter of each tube was taken and immediately sieved under cold seawater after
sampling. 5 fractions were gained: >2mm, >500µm, >200µm, >112µm and >63µm.
Samples were put in 100 ml Kautex bottles, filled with the supernatant water of the
Multicorer tube and immediately frozen at –20°C until further processing.
Methodology
For further treatment, the sample was thawed and a sub sample was examined using
a stereomicroscope with a Nikon Coolpix 995 digital camera. Samples were sorted in
a Petri dish containing water using a brush. Specimens considered as „alive hat the
moment of sampling“ were picked out and subjected to further treatments.
Characters for the physiological state of the organisms are: intact state of the test/
mechanical damage, first chamber empty, other chambers filled, extended
pseudopodia (not observed), attachment to stones or other organisms, colour and
state of the first chamber (agglutinated specimens), also see figures 4.3-1 and -2 for
details. Specimens were determined using classical morphological methods and
Robert Wynn Jones´ „The Challenger Foraminifera”. Additionally specimens were
documented by the Nikon Coolpix 995 camera before further treatments.
DNA-Extraction
After sorting and documentation each single specimen was put into an 1.5 ml
Eppendorf-cup and 40 µl DOC-solution were added. For DNA-extraction, the cells
were crushed by mini-pistils and the samples were incubated for 1h at 60°C. After
incubation, samples were frozen at –20°C until further processing. As a whole, 376
specimens were extracted and treated by PCR.
PCR
PCR (Polymerase Chain Reaction) is a cyclic process to amplify a selected DNAfragment
between two primers (Primer: short DNA fragments used as start and end
point in conservative DNA regions). For phylogenetic analysis of relationships among
eukaryotes, the gene encoding the small subunit of the ribosomes (18S rDNA, SSU
rDNA) is commonly used. Primers used here were specifically designed for analysis
of the foraminiferal 18S rDNA. PCR was conducted using two different primer pairs,
Cibi fwd/Cibi rev for rotaliid foraminifera and F4/R1b for Astrorhizidae and
Textulariidae. PCR was performed using puRE Taq Ready-to-Go PCR Beads from
Amersham Biosciences and a Techne Progene thermocycler with the following
protocol:
Initial Denaturing: 94°C for 2 min
Primer Annealing: 55°C for 40 sec
Elongation: 72°C for 1 min 20 sec
Denaturing: 91°C for 40 sec
Final Elongation: 72°C for 5 min
Cooling: 4°C
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