Supplementary material and methods
(a) Insect material
H. sjostedti termites and logs were collected on Yakushima Island, Kagoshima
Prefecture, Japan. Termites were maintained with logs and soil obtained from the
collection site and stored at room temperature until use. Mature worker-caste termites
were used for all experiments.
(b) Preparation of 13C-cellulose
Acetobacter xylinum (NBRC 15237) were cultured in sterilized Hestrin–Schramm
medium [1] containing
13
C6-glucose. This culture medium was incubated statically at
30°C for 14 days. After cultivation, cellulose pellicles were washed with 10% Purelox,
4% NaOH, isopropanol, and distilled water to remove residual metabolites, proteins,
cell walls, and membranes. Purity of
13
C-cellulose pellicles was confirmed by
measuring 13C cross-polarization magic-angle spinning (CP/MAS) of dried samples and
comparing with our previous results [2]. The degree of
13
C-incorporation to the
cellulose was determined with isotope-ratio mass spectrometry (IR-MS) to be
approximately 93% as described below. Residual water on
by dabbing with paper towels, and the resulting
13
C-cellulose was removed
13
C-cellulose was applied to feeding
experiments.
(c) Isotope-ratio mass spectrometry (IR-MS)
Incorporation rate of 13C into the termite guts and degutted bodies without heads during
the experiments was confirmed by IR-MS (Supplementary figure S4). For each sample,
2 mg of dry powder (in case of nitrogen-starved sample such as wood was 20 mg) was
weighed into a tin cup and folded following the standard procedure to extrude any air.
The levels of carbon stable isotopes were measured using a vario MICRO cube
1
elemental analyzer (Elementar Analytical, Hanau, Germany) connected to an
IsoPrime100 gas IR-MS (IsoPrime Ltd, Cheadle, UK).
(d) 13C-cellulose feeding experiments
Twenty-five termites were maintained in a 100-ml plastic cup with approximately 10 g
of
13
C-cellulose for 24 h. Then, 20 termites were randomly selected and dissected to
examine foreguts, midguts, and hindguts (Supplementary figure S1). Hindguts were
further divided into anterior and posterior portions on dry ice (Supplementary figure S1).
Equal numbers (i.e. 20) of termites prior to feeding 13C-cellulose also were dissected as
controls. Two replicates of each gut segment were collected in 2-ml microtubes on dry
ice. All samples were kept at −80˚C until use.
(e) Sample preparation and measurements using 1D and 2D NMR
Termite samples stored at −80˚C were freeze-dried and homogenized using an auto-mill
(Tokken, Chiba, Japan). Subsequently, 4–10 mg of the freeze-dried tissue samples were
extracted with 400–1000 μl phosphate buffer solution (0.1 M K2HPO4/KH2PO4, 0.01%
NaN3,
pH
7.0),
containing
90%
D2O
and
1
mM
sodium
2,2-dimethyl-2-silapentane-5-sulfonate (DSS) at 65°C for 15 min. In addition to
metabolites, crude extracts of biological samples generally contain ions and
biopolymers that have the potential to cause unpredictable fluctuations in the chemical
shifts of metabolites. To ensure the reliability of the NMR detections, we prepared the
samples in appropriate dilutions using standards based on our previous method that had
fluctuations of biological samples within 0.03 ppm and 0.54 ppm for both 1H and 13C,
respectively [3]. After centrifugation, the extracted supernatants were transferred into
5-mm Φ NMR tubes for measurements. All 2D 1H-13C hetero-nuclear single quantum
coherence (HSQC) and J-resolved (2D-J) spectra were recorded on a Bruker DRU-700
NMR spectrometer (Bruker Biospin, Rheinstetten, Germany) equipped with a 1H
inverse cryogenically cooled probe with a z-axis gradient as previously described [4-6].
2
In brief, 128 complex f1 (13C) and 1024 complex f2 (1H) points were recorded at 8
scans per f1 increment for
13
C-HSQC measurements. Spectral widths were 7,042 Hz
and 9,389 Hz for f1 and f2, respectively. All NMR spectra were processed using
NMRPipe [7] and rNMR [8] and were assigned using the SpinAssign program on the
PRIMe Website [9]. Intensity values of all metabolite signals were initially normalized
using 1 mM DSS. Intensity values of each gut position were then divided by the gut
volume (µL) to represent relative intensities per µL of the metabolites (represented as
a.u., arbitrary units). The volume of each gut position was obtained by estimating
volumes of circular truncated cones by measuring gut diameters (widths) every 500 µm
of longitudinal length. The resulted gut volumes were 1.7 ± 0.2 µL (6.3%) for the
foregut, 2.0 ± 0.4 µL (7.5%) for the midgut, 16.9 ± 2.4 µL (59.6%) for the anterior
hindgut, and 6.9 ± 1.7 µL (26.6%) for the posterior hindgut, respectively (n=10).
(f) Data analysis
For clustering analyses, metabolites were grouped based on a hierarchical cluster of
temporal variation in the signals. Distances among metabolites were evaluated using
Euclidean distances after standardization of the data. Average linkage was used as the
clustering method. Significance among groups of interest was examined by a multiple
response permutation procedure (MRPP) [10] based on 9,999 permutations.
For ordination of metabolite groups in each gut regions, Kruskal’s non-metric
multidimensional scaling (NMDS) [10, 11] based on distances among metabolites was
performed with some modifications. In brief, dissimilarities in the temporal variations
of the signals were evaluated using Euclidean distance following standardization. The
number of dimensions used in the scaling was K = 2. Scaling was performed using
statistical software R.
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