SUPPLEMENTARY ONLINE MATERIAL
Linking metabolite production to taxonomic identity in environmental samples by
(MA)LDI-FISH
Martin Kaltenpoth1,2,*, Kerstin Strupat3, and Aleš Svatoš4,*
1
Max Planck Institute for Chemical Ecology, Research Group Insect Symbiosis, Hans-Knöll-Str. 8, D-
07745 Jena, Germany
2
Present address: Johannes Gutenberg University, Department for Evolutionary Ecology, Johann-
Joachim-Becher-Weg 13, 55128 Mainz, Germany
3 Life
Science Mass Spectrometry, Thermo Fisher Scientific, Hanna-Kunath-Str. 11, 28199 Bremen,
Germany
4 Max
Planck Institute for Chemical Ecology, Research Group Mass Spectrometry, Hans-Knöll-Str. 8, D-
07745 Jena, Germany
*Corresponding authors:
Martin Kaltenpoth
Aleš Svatoš
Max Planck Institute for Chemical Ecology
Max Planck Institute for Chemical Ecology
Research Group Insect Symbiosis
Research Group Mass Spectrometry
Hans-Knoell-Str. 8
Hans-Knoell-Str. 8
07745 Jena, Germany
07745 Jena, Germany
Phone: +49-3641-571800
Phone: +49-3641-571700
Email: mkaltenpoth@ice.mpg.de
Email: svatos@ice.mpg.de
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Supplementary methods
AP-SMALDI-MSI of piericidin A1 and B1 produced by ‘Streptomyces philanthi’ colonies in vitro
In order to test whether PA1 and PB1 are produced by the same sub-populations of symbiont cells under
in vitro conditions, we performed AP-SMALDI-MSI (Roempp et al 2010, Roempp and Spengler 2013) on
cultures of ‘Streptomyces philanthi’. To this aim, we first pre-grew ‘Streptomyces philanthi biovar
triangulum’ tri23Af2 – a strain previously isolated from the antennae of a female European beewolf
(Philanthus triangulum) – from glycerol stocks in liquid Grace’s medium for about 10 days (Nechitaylo et
al 2014). Subsequently, symbiont biomass was inoculated onto an Express Plus® Membrane (0.22 µm
pore size, cat. no. GPWP01300, Merck Millipore, Darmstadt, Germany), which was placed on top of an
agar plate containing modified Grace’s medium (reconstituted by combining amino acids, salts, and
biotin, and adding 10% FBS). After two weeks, the membrane was carefully detached from the agar plate
and fixed with double-sided adhesive tape to a microscope slide for MS imaging.
An atmospheric pressure scanning microprobe AP-SMALDI10 (TransMIT GmbH, Giessen, Germany)
attached to a Q Exactive Plus (Thermo Fisher Scientific GmbH, Bremen, Germany) was used for mass
spectrometric imaging (MSI) experiments. A nitrogen laser (λ = 337 nm) at a repetition rate of 60 Hz was
used for desorption/ionization. The target voltage was set to 4.0 kV. The step size of the sample stage
was set to 10 μm. Samples mounted on microscope slides were used without further treatment for laser
desorption/
ionization
(LDI).
For
matrix-assisted
laser
desorption/ionization
(MALDI),
2,5-
dihydroxybenzoic acid was sublimed on the samples at 140°C and 1×10-3 Torr for five minutes. The Q
Exactive Plus instrument was operated in positive-ion mode at m/z 200–800 mass range. MSI
measurements were performed using the Orbitrap detector with a mass resolving power of 70,000 at
m/z = 400. Thermo Scientific Xcalibur software (Thermo) and Mirion (TransMIT) were used for data
collection and processing, respectively. The instrument was externally calibrated using a standard
calibration mixture, and masses were corrected on-line using lock-mass.
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High-resolution AP-SMALDI-MSI of piericidin A1 and B1 on beewolf cocoons
In order to assess the distribution of antibiotics on beewolf cocoons (P. triangulum) in more detail, highresolution AP-SMALDI-MSI experiments were performed on pieces of beewolf cocoons. The samples
were attached to microscope slides using double-sided adhesive tape and used for AP-SMALDI-MSI
without further treatment (LDI), or after application of 2,5-dihydroxybenzoic acid by sublimation (MALDI).
MSI was carried out as described for the symbiont cultures (see above), but the step size of the sample
stage was set to either 20 µm (low resolution) or 5 µm (high resolution).
Localization of ‘S. philanthi’ and other bacteria on beewolf cocoons by FISH
Additional FISH experiments were performed to test for the presence of bacteria other than ‘S. philanthi’
on beewolf cocoons. To this aim, pieces of P. triangulum cocoons were subjected to FISH with the ‘S.
philanthi’-specific probe SPT177-Cy5 (Kaltenpoth et al 2005, Kaltenpoth et al 2006) and the general
eubacterial probe EUB338-Cy3 (Amann et al 1990) as described previously (Kaltenpoth et al 2010).
Fluorescence images of both fluorochromes were recorded on a Zeiss AxioImager Z.1 (Zeiss, Jena,
Germany), using both the mosaic and z-stack options for obtaining high-resolution images with increased
focusing depth. An overlay of images obtained for both probes allowed for assessing the presence of
non-symbiotic cells, which would be labeled by EUB338-Cy3, but not SPT177-Cy5.
Supplementary results
AP-SMALDI-MSI of ‘S. philanthi biovar triangulum’ tri23Af2 colonies in vitro revealed the presence of high
concentrations of PA1 and PB1 in the periphery of the colonies (Fig. 1 K-N). Both antibiotics showed
almost perfect co-localization, supporting the hypothesis that individual symbiont cells produce multiple
compounds simultaneously.
AP-SMALDI-MSI of beewolf cocoons at 5080 dpi resolution (step size 5 µm) revealed high concentrations
of PA1 and PB1 along some of the silken cocoon threads (Fig. S1 D-F). This distribution agrees with the
results of FISH experiments recording the highest symbiont cell densities along certain silk threads (Fig.
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S2), which we interpret to be the outermost cocoon threads that are the first to be spun by the beewolf
larva. While very apparent in high-resolution MSI, the pattern of high antibiotic concentrations along the
outer cocoon threads was less obvious at a lower resolution of 1270 dpi (step size 20 µm, Fig. S1 A-C).
Unfortunately, we were unable to combine high-resolution MSI with FISH, as the high laser intensities
significantly affected the quality of the samples and prevented subsequent molecular analyses.
FISH experiments with the ‘S. philanthi’-specific probe SPT177-Cy5 and the general eubacterial probe
EUB338-Cy3 revealed the perfect co-localization of both probes on pieces of several beewolf cocoons
(Fig. S2), indicating that the symbionts essentially occur as a monoculture on the cocoon surface. This
may be partially mediated by the production of the antibiotic cocktail, which exhibits antifungal as well as
antibacterial activity (Kroiss et al 2010).
References
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ribosomal RNA targeted oligonucleotide probes with flow-cytometry for analyzing mixed microbial
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Supplementary figure legends
Figure S1: AP-SMALDI-MSI of antibiotics produced by symbiotic ‘Streptomyces philanthi’ bacteria on a
beewolf cocoon (Philanthus triangulum), measured at a step size of 20 µm (A-C) and 5 µm (D-F),
respectively. Ion intensity maps of (A, D) Piericidin A1 (PA1, m/z 416 [M+H]+), (B, E) piericidin B1 (PB1,
m/z 430 [M+H]+), and (C, E) overlay of PA1 (green) and PB1 (blue). Scale bars represent 200 µm.
Figure S2: Fluorescence in situ hybridization (FISH) micrographs of three pieces from different beewolf
cocoons, using the general eubacterial probe EUB338-Cy3 (left panel), and the symbiont-specific probe
SPT177-Cy5 (middle panel). The overlay of both probes is given in the right panel. Note the perfect
congruence of the signals obtained from both probes, indicating that there are no (or very few) nonsymbiotic bacteria present on the beewolf cocoons. Scale bars for each row represent 100 µm.
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