Supplementary Text S1
Supplementary information for Material and Methods
Tissue preparation for TEM and NanoSIMS analyses
Coral samples were fixed at 4˚C by immersion in a fixative solution
containing 1.25% of glutaraldehyde and 0.5% of paraformaldehyde in filtered
seawater (pH 7.4) This fixative mixture takes advantage of the rapid penetration of
paraformaldehyde, which initiate the structural stabilization of the tissue and allows
rapid and thorough cross-linking of proteins and amino groups by glutaraldehyde.
Coral fragments were then embedded in 1.5% agarose (Bythell et al 2002) prior to
decalcification using a solution of 90% formic acid : fixative (1:3) with a solution
change every 12h until total dissolution of the skeleton (Clode et al 2007). After
decalcification, coral samples were washed in fixative and dissected under light
microscope to obtain small piece of tissue containing few individual polyps. Tissue
samples 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 Araldite, cut into 250 nm sections using an Ultracut E microtome (Leica
Microsystems, Australia) and mounted onto finder grids for Transmission electron
Microscopy (ProsciTech, Australia).
Nitrogen content and uptake rate
Prior to nitrogen content analysis, pellets of dinoflagellate cells and samples of
coral host tissue were freeze-dried and weighted. Nitrogen content (%) was then
analyzed in both coral tissue and dinoflagellate in duplicate at the Analytical service
of School of Land, Crop and Food Sciences (the University of Queensland, Brisbane,
Australia) by using combustion (950°C) method with a LECO TruSpec analyzer
(LECO; St. Joseph, Mich.).
The Increase in
15
N/14N ratio (δ15N) for each sample was calculated in ‰ as
follows:
15N= ((Nmes/Nnat) – 1) x 103
where,
Nmes: 15N/14N measured in labeled samples by NanoSIMS
Nnat: natural abundance of 15N/14N measured in unlabeled samples by NanoSIMS.
The nitrogen uptake rate per mg of host tissue or dinoflagellate () was
expressed in ng N h-1 mg-1 and was calculated by normalizing the
15
N-incorporation
measured by using NanoSIMS to the average nitrogen content (% of dry mass) of the
animal tissue or the dinoflagellate symbionts and to the time of incubation according
to the equation of Dugdale and Wilkerson (1986) (Dugdale and Wilkerson 1986),
presented in Grover et al, (2002) (Grover et al 2002):
 = ((Nmes-Nnat)/((Nenr-Nnat) x Tinc)) x Ncontent x 103
where,
Nmes: 15N/14N measured in labeled samples by NanoSIMS
Nnat: natural abundance of 15N/14N measured in unlabeled samples by NanoSIMS
Nenr: 15N-enrichment of the incubation medium
Tinc: incubation time
Ncontent: average nitrogen content (%) measured by using combustion (950°C) method
with a LECO TruSpec analyzer.
Glutamine and Glutamate composition
Amino acids in the samples were separated by reverse-phase high
performance liquid chromatography (HPLC) on a Shimadzu SCL-10 HPLC
(Shimadzu, Tokyo, Japan) according to the method of Swanson and Hoegh-Guldberg
(Swanson and Hoegh-Guldberg 1998). Briefly, amino acids in samples were
prederivatised with ortho-phthaldialdehyde (OPA) and -mercaptoethanol in 0.5 M
potassium borate buffer (pH 10) prior to automated sample injection onto the column.
Amino acids were then detected with excitation at 338 nm and emission at 425 nm
(RF-10AXL fluorescence detector, Shimadzu, Japan). A Nova-Pak C-18 analytical
column (Waters, Country), protected by a GuardPak with Resolve C18 inserts, was
used to separate amino acids eluted at 1 ml.min-1 with a linear ± concave gradient of
potassium phosphate buffer (pH 6.65) and an organic mobile phase containing
isopropyl alcohol acetonitrile and methanol. Glutamine and Glutamate concentrations
in dinoflagellate were determined based on standard curves relating peak area to
known standard concentrations using a Pierce H standard (Waters, Australia).
Supplementary references
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