Light microenvironment and single-cell gradients of carbon
fixation in tissues of symbiont-bearing corals
Daniel Wangpraseurt1,#, Mathieu Pernice1,#, Paul Guagliardo2, Matt R. Kilburn2, Peta
L. Clode,2,3, Lubos Polerecky4, Michael Kühl1,5*
1
Plant Functional Biology and Climate Change Cluster, University of Technology
Sydney, New South Wales 2007, Australia
2
Centre for Microscopy, Characterisation and Analysis, The University of Western
Australia, 35 Stirling Highway, Crawley, Western Australia 6009, Australia.
3
Oceans Institute, The University of Western Australia, 35 Stirling Highway,
Crawley, Western Australia 6009, Australia.
4
Universiteit Utrecht, Department of Earth Sciences, Utrecht, Netherlands
5
Marine Biological Section, Department of Biology, University of Copenhagen,
Strandpromenaden 5, DK-3000 Helsingør, Denmark
#
Shared first authorship
*
Corresponding author: mkuhl@bio.ku.dk
Supplementary Information
Supplementary Text S1: Materials and Methods
Isotopic labelling experiment
In order to quantify the assimilation of dissolved inorganic carbon in
symbiotic dinoflagellate cells, coral fragments were exposed to isotopically enriched
(NaH13CO3) artificial sea water (ASW) (and control non-enriched ASW, NaH12CO3).
Corals were incubated for 24h with the incubation starting and ending during the peak
of the photoperiod (see Figure S1 for details). Coral fragments were randomly
distributed among the treatment and control aquaria (10 L; closed water system;
continuously stirred using one power head pump for each tank and aerated with air
stones). The temperature of the tanks was maintained at 26˚C by placing experiment
and control tanks in flow-through ambient seawater. Isotopically enriched ASW was
made in accordance with Pernice et al. (2014) with the addition of NaH13CO3 (13C
isotopic abundance of 99%, Sigma) to a final concentration of 2 mM. Control nonenriched ASW contained NaH12CO3 in the same concentrations as enriched media
(Figure S3). A subset of coral fragments was removed from the treatment and control
tanks at T = 48 h. The successive steps used for sample fixation and preparation prior
to NanoSIMS analysis could have affected isotopic measurements by (i) diluting the
13
C signal and (ii) partially extracting sugars and other soluble molecules poor in
amino groups. Therefore, it is likely that the absolute
13
C concentrations reported in
our NanoSIMS analyses are underestimates, but the relative difference between
isotopic enrichment of Symbiodinium cells in different coral microenvironments
remains valid.
TEM and NanoSIMS sample preparation
Each coral fragment was chemically fixed for 24 h at 4˚C by immersion in a
solution containing 2.5% glutaraldehyde and 1% formaldehyde in PBS-sucrose buffer
(0.1 M phosphate, 0.65 M sucrose, 2.5 mM CaCl2), pH 7.5. Samples were then
washed 3 times in PBS-sucrose buffer (0.1 M phosphate, 0.65 M sucrose, 2.5 mM
CaCl2), pH 7.5 at 4˚C and processed for TEM and NanoSIMS analyses.
Coral fragments were embedded in 1.5% agarose prior to decalcification at
4°C using a solution of 90% formic acid diluted into the fixative solution described
above (1:3 mixture) with a solution change every 12 h until total dissolution of the
skeleton.
After
decalcification,
coral
samples
were
dissected
under
a
stereomicroscope into small pieces containing the oral and aboral parts of polyp and
coenosarc, respectively. The tissue parts were post-fixed 1 h at room temperature in
1% OsO4 in Sörensen phosphate buffer (0.1 M), and were then dehydrated in an
increasing series of ethanol concentrations (50%, 70%, 90% and 100%) and stored in
acetone until resin embedding. Samples were embedded in Spurrs resin, cut to 100120 nm-thick sections using an Ultracut E microtome (Leica Microsystems,
Australia), mounted onto finder grids for transmission electron microscopy
(ProSciTech, Australia), and counterstained with 2% aqueous uranyl acetate (10 min)
and Reynold’s lead citrate (10 min).
Regions of interest within the tissue sections were mapped and imaged at the
Centre for Microscopy and Microanalysis (University of Sydney, Sydney, Australia)
using a JEOL JEM1400 Transmission Electron Microscope (JEOL, Japan LTD)
operated at 80 kV accelerating voltage. These TEM Grids were then mounted in a
specific grid holder and gold-coated for subsequent NanoSIMS analyses.
NanoSIMS analyses
The same regions of interest mapped with TEM within the tissue sections
were imaged with the NanoSIMS ion probe (NanoSIMS 50, CAMECA, Paris,
France) in order to quantify the distribution of newly fixed
13
C. Preliminary analysis
was done at the National Facility for High-Resolution In Situ Isotope and Element
Analysis (Utrecht University, Netherlands), while final analysis was performed at the
Australian Microscopy and Microanalysis Research Facility (AMMRF) at the Centre
for Microscopy, Characterisation, and Analysis (University of Western Australia,
Perth). Samples were bombarded with a 16 keV primary ion beam of (1-3 pA) Cs+
focused to a spot size of about 100-150 nm on the sample surface. Secondary
molecular ions 12C12C-, 12C13C-, 12C14N- and 31P- were simultaneously collected using
electron multipliers, at a mass resolution (ΔM/M, according to the manufacturer’s
definition) of about 9000, enough to resolve the 12C13C- peak from the 12C12C1H- peak
on mass 25. Carbon was measured on mass 24 and 25 due to the higher intensity of
the C2 signal compared to the single C ions. Charge compensation was not necessary.
Typical images of 35×35 μm with 256×256 pixels for
12 12
C C- and
12 13
C C-, were
obtained by rastering the primary beam across the sample with a dwell-time of 5
milliseconds; a total of 6 planes were acquired for each raster and used to create an
image stack. Images were dead-time corrected at the pixel level, and the image stack
was drift-corrected to account for movement of the ion beam between analyses. The
13
C/12C ratio images were obtained by dividing the
12
C13C- image by the
12 12
C C
image. The HSI images displayed in Figure 1 were produced using OpenMIMS image
processing software (www.nrims.harvard.edu/software.php) in ImageJ. The ratio
scale factor was 10000, and the minimum and maximum values were set to 220 (to
represent a natural abundance for the C2 ion, which corresponds to the natural 13C/12C
isotopic ratio of 0.0110– see below) to 1000 (which corresponds to a 13C/12C isotopic
ratio of 0.05, ~4.5 times above the natural 13C/12C isotopic ratio), respectively. Levels
of
13
C-enrichment were determined from the images for individual dinoflagellate by
extracting the number of counts recorded within Regions of Interest (ROIs) in
OpenMIMS. ROIs were drawn on the NanoSIMS
12 14
C N-images, which allows
finding the contours of the dinoflagellate cells regardless of whether there was any
13
C-label present. Significant levels of
13
C-enrichment were also measurable in the
surrounding host tissue indicating transfer of 13C-labelled compounds from symbionts
to the coral host. However, in this study, we focus our quantitative analyses on the
carbon fixation by individual Symbiodinium within their local microenvironment as
the temporal and spatial dynamics of carbon translocation have been described
previously in details (Kopp et al. 2015).
13
C/12C ratios derived from the
12 13 -
12 12
C C and
C C secondary ions signals were converted by dividing the ratio by 2.
13
C-
enrichments were expressed in the delta notation (δ13C in ‰) as follows:
13C  (
Where, Cmes is the measured
13
Cmes
 1)  103
Cnat
C/12C ratio and Cnat is the natural
13
C/12C ratio
measured in non-labelled coral samples as controls. Cnat was quantified on unlabelled
control samples each day during the NanoSIMS analyses and served as an internal
standard. The isotopic ratios, enrichmentsδ13C) and relative contribution to atomic
mass by 13C (at %) measured for each ROI are reported in Table S1. For each ROI the
total number of counts corresponding to the different mass measurements (Total 24
and Total 25) are indicated as given directly by the software OpenMIMS.
Supplementary Figures
Supplementary Figure S1
Design of the coral isotopic labelling experiment. Fragments of coral Favites sp.
were exposed to isotopically labelled (NaH13CO3) artificial sea water and control nonlabelled artificial sea water (NaH12CO3). Coral fragments were randomly distributed
among the treatment and control aquaria. Isotopic labelling lasted for 24h starting at
14:00 and finishing at 14:00 the next day. Arrows mark the sampling time (T=0 and
24h) under isotopic labelling treatment (top) and control treatment (bottom).
Supplementary Figure S2
Quantification of 13C-bicarbonate uptake by a single Symbiodinium cell within the
reef-building coral Favites sp. (a) TEM image of an individual Symbiodinium cell
within the tissue of coral Favites sp. (b) NanoSIMS image of the ultrastructural
distribution of 13C/12C in the same Symbiodinium cell showing the high variation in
the 13C signal, with hotspots corresponding to subcellular compartments such as the
central starch-filled pyrenoid (pyr). (b) Spatial fluctuations of 13C/12C ratios over the
transect indicated in (b). Scale bar= 6m.
Supplementary Figure S3
Quantification of 13C-bicarbonate uptake in control sample. Scale bar = 10 µm (same
as in Figure 1). The mean 13C enrichment (δ13C) for control samples was 3‰ (±13
SD, n=20).