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, Australia2 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, Netherlands5 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 non-

enriched 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

13C signal and (ii) partially extracting sugars and other soluble molecules poor in

amino groups. Therefore, it is likely that the absolute 13C 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 100-

120 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 13C. 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 12C12C- and 12C13C-, 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

13C/12C ratio images were obtained by dividing the 12C13C- image by the 12C12C 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 13C-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 12C14N-images, which allows

finding the contours of the dinoflagellate cells regardless of whether there was any

13C-label present. Significant levels of 13C-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). 13C/12C ratios derived from the 12C13C- and

12C12C secondary ions signals were converted by dividing the ratio by 2. 13C-

enrichments were expressed in the delta notation (δ13C in ‰) as follows:

Where, Cmes is the measured 13C/12C ratio and Cnat is the natural 13C/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, enrichmentsδ13C) 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 non-labelled 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= 6m.

Supplementary Figure S3Quantification 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).