Appendix 2 Detailed description of sampling and analysis procedures for viral, bacterial and
zooplankton communities.
Prokaryotic and viral abundance
Duplicate 1.8-mL samples were fixed with glutaraldehyde (0.5% final concentration) in the dark for
15 min, flash-frozen in liquid nitrogen, and stored at -80°C until analysis. For analysis, samples were
stained with the nucleic acid dye SYBR Green I (Invitrogen) and estimates of the abundances of
prokaryotic cells (Bacteria and Archaea) and viruses were determined using a FACSCalibur (BD
Biosciences) flow cytometer as previously described (Brussaard, 2004; Brussaard et al., 2010). The
abundance of prokaryotes and viruses is given as the average of duplicate measurements from each
experimental replicate.
Bacterial community composition
We assessed bacterial community composition (BCC) using terminal restriction fragment length
polymorphism (T-RFLP) following the protocol of (Moeseneder et al. 1999). Samples (50-100 mL,
depending on filter clogging) were filtered through 0.22 µm pore-size filters, flash-frozen in liquid
nitrogen and stored at -80°C until analysis. The nucleic acids were extracted from the filters using a
PowerSoil DNA Isolation kit (Mo Bio) according to the manufacturer’s instructions and using the
alternative lysis protocol. The extracts had a final volume of 100 µl in solution S6 (contains no EDTA)
and subsamples were used directly in subsequent PCR amplifications.
The primer pair 27F (5’-AGA GTT TGA TCC TGG CTC AG-3’) and 1492R (5’-GGT TAC CTT GTT
ACG ACT T-3’) was used to amplify fragments of the bacterial 16S rRNA gene (Lane 1991). The
forward primer was fluorescently labeled on the 5’-end with 6-carboxyfluorescein (6-FAM; Thermo
Scientific). PCR chemicals were purchased from Invitrogen and cycling was performed in a Hybaid
PCR Express cycler (ESBE Scientific). Each 50 µL PCR reaction contained 5 µL of 10× Taq buffer (200
mM Tris-HCl [pH 8.4], 500 mM KCl), 1.5 µL of a 50 mM MgCl2 solution, 1 µL of 10 mM
deoxyribonucleotide triphosphate (dNTP) mix, 2.5 µL of each 10 µM primer solution, and 0.25 µL of 5
units µL-1 Platinum Taq DNA polymerase. Thermocycling started with an initial denaturation at 95°C
for 5 min, followed by 30 cycles with denaturation at 95°C for 1 min, annealing at 55°C for 1 min, and
elongation at 72°C for 1 min. The final elongation step was performed at 72°C for 30 min to prevent
the formation of artifacts (Janse et al., 2004). Subsequently, PCR fragments were cleaned and
concentrated using a QIAquick PCR purification kit (Qiagen) resulting in a final volume of 28 µL in
elution buffer. Agarose gel electrophoresis was used to size and standardize the concentration of PCR
products in the individual reactions in comparison with a molecular mass standard (Low DNA Mass
Ladder; Invitrogen) to prepare for restriction digestion and T-RFLP analysis.
Each 15 µL restriction digest contained 1.5 µL of 10×NEBuffer 4 (200 mM Tris-acetate, 500
mM potassium acetate, 100 mM magnesium acetate, 10 mM Dithiothreitol, pH 7.9), 0.15 µL of 100
µg mL-1 bovine serum albumin, 0.5 µL of 20 units µL-1 HhaI restriction enzyme, and 10 µL of
fluorescently labeled PCR products. Digests were performed in a PCR cycler at 37°C for 12 h, followed
by 65°C for 20 min to inactivate the restriction enzyme. Five µL of the digests were directly used in TRFLP analysis. Analyses were performed on an ABI 3730 DNA analyzer (Applied Biosystems) equipped
with 50 cm long capillaries running POP7 polymer (Applied Biosystems). The fluorescent signals from
the forward primer of the digests were collected and the peaks sized using Peak Scanner software
(version number 1.0; Applied Biosystems) against an internal size standard (Genescan 1200 LIZ;
Applied Biosystems). The peak patterns were analyzed for the number of peaks per sample and
translated into a binary data matrix (presence vs. absence).
Viral community composition
Viral community composition (VCC) was assessed using randomly-amplified polymorphic DNA
polymerase chain reaction (RAPD-PCR; Winget & Wommack 2008). The filtrate from the filtration
step used to collect bacteria (see above; 50-100 mL, depending on filter clogging) was also flashfrozen in liquid nitrogen and stored at -80C until analysis. Upon thawing, the 0.22 µm filtrates
were further concentrated to 200 µL using Amicon Ultra-15 centrifugal filtration devices (30 kDa
molecular weight cut-off; Millipore) in a swing-out rotor at 3000×g and 4°C. Nucleic acids were
extracted from 200 µL of this viral concentrate using a QIAmp MinElute Virus Spin Kit (Qiagen). The
extracts had a final volume of 30 µL in AVE buffer (Qiagen).
Two to five µl of viral DNA extracts from each sample were subjected to RAPD-PCR reactions
with primer OPA-13 (5’-CAG CAC CCA C-3’; Neilan 1995). PCR chemicals were purchased from
Invitrogen and cycling was performed in a HYBAID PCR Express cycler (ESBE Scientific). Each 50 µL
PCR reaction contained 5 µL of 10× Taq buffer (200 mM Tris-HCl [pH 8.4], 500 mM KCl), 1.5 µL of a 50
mM MgCl2 solution, 1 µL of 10 mM dNTP mix, 5 µL of a 10 µM solution of either primer CRA-22 or
OPA-13, and 0.25 µL of 5 units µL-1 Platinum Taq DNA polymerase. Initial denaturation was at 94°C
for 10 min followed by 30 cycles with denaturation at 94°C for 30 s, annealing at 35°C for 3 min, and
elongation at 72°C for 1 min. The final elongation step was performed at 72°C for 30 min (Janse et al.,
2004). Subsequently, 5 µL of each RAPD-PCR reaction were loaded onto 2 % agarose gels run in
1×TBE buffer (89 mM Tris-HCl [pH 8.3], 89 mM boric acid, 2 mM EDTA, pH 8.3) at 3 V cm-1 electrode
distance for 120 min. Gels were stained with SYBR Gold (1:10,000 dilution of stock solution;
Invitrogen) for 30 min and digital images were obtained on a gel documentation system equipped
with a charge-coupled device camera. The images were analyzed for the number of bands per sample
(presence vs. absence). The band patterns were translated into a binary data matrix for further
analysis (Winter and Weinbauer 2010).
Zooplankton abundance and community composition
Three samples from the top and bottom of each mesocosm were collected with a Schindler Patalas
sampler and pooled (48 L total volume; mesh size: 60 µm), and crustacean zooplankton was counted
and identified to the genus level using a stereo microscope.
References
Brussaard CPD (2004). Optimization of procedures for counting viruses by flow cytometry. Applied
and Environmental Microbiology 70: 1506-1513.
Brussaard CPD, Pajet JP, Winter C, Weinbauer MG (2010). Quantification of aquatic viruses by flow
cytometry. In: Wilhelm SW, Weinbauer MG, Suttle CA (eds). Manual of Aquatic Viral Ecology.
American Society of Limnology and Oceanography: Waco, TX. pp 102-109.
Janse I, Bok J, Zwart G (2004). A simple remedy against artifactual double bands in denaturing
gradient gel electrophoresis. Journal of Microbiological Methods 57: 279-281.
Lane DJ (1991). 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M. (eds). Nucleic acid
techniques in bacterial systematics. John Wiley & Sons, Inc.: New York, NY. pp: 115-176.
Moeseneder MM, Arrieta JM, Muyzer G, Winter C, Herndl GJ (1999). Optimization of terminalrestriction fragment length polymorphism analysis for complex marine bacterioplankton
communities and comparison with denaturing gradient gel electrophoresis. Applied and
Environmental Microbiology 65: 3518-3525.
Neilan BA (1995). Identification and phylogenetic analysis of toxigenic cyanobacteria by multiplex
randomly amplified polymorphic DNA PCR. Applied and Environmental Microbiology 61:
2286-2291.
Winget DM, Wommack K E (2008). Randomly amplified polymorphic DNA PCR as a tool for
assessment of marine viral richness. Applied and Environmental Microbiology 74: 2612-2618.
Winter C, Weinbauer MG (2010). Randomly Amplified Polymorphic DNA Reveals Tight Links between
Viruses and Microbes in the Bathypelagic Zone of the Northwestern Mediterranean Sea.
Applied and Environmental Microbiology 76: 6724-6732.