Samples of fluorescent and non-fluorescent specimens of Montastraea cavernosa were

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1 Online Materials and Methods Sample collection Samples of fluorescent and non-fluorescent specimens of Montastraea cavernosa were collected at depths ranging from 15 to 30 meters in the waters around Lee Stocking Island (LSI), Bahamas ( Lat, Long). These samples were processed for analysis within six hours of collection. Fluorescence spectroscopy Fluorescence emission spectra were measured on corals in situ with a diver-operated spectrofluorometer while higher resolution fluorescence excitation and emission spectra were measured in the laboratory with a SPEX FluoroMax-2 spectrofluorometer fitted with a fiber optic probe. Flow Cytometry Samples of fluorescent and non-fluorescent corals approximately 1-2 cm 2 were lightly airbrushed (80 psi, ~10 cm distance to coral) with filtered (0.22 µm) seawater remove excess mucous and associated microbiota. Corals were then vigorously airbrushed (80 psi, < 1 cm distance to coral) to remove coral soft tissues with a small volume (1-2 ml) of filtered seawater. The homogenates were fixed at a final concentration of 0.5% with electron microscopy grade glutaraldehyde and frozen at 80 C. Samples were sent frozen to the Bigelow Laboratory for Ocean Sciences Flow Cytometry Facility. Each sample was analyzed using a Becton Dickinson FACScan flow cytometer equipped with a 15 mw, 488 nm, air-cooled Argon ion laser. Simultaneous measurements of forward light scatter (FSC, relative size), 90 degree light scatter

2 (SSC), chlorophyll fluorescence (>650 nm), and phycoerythrin fluorescence ( nm) were made on all samples. Light and Electron Microscopy Small samples (50 µl) of the homogenates described above were placed on microscope slides and photographed using a Zeiss Axiophot microscope at 100 x using FITC filters with an excitation filter of nm and barrier filter of nm. Intact pieces of coral samples of fluorescent and non-fluorescent Montastraea cavernosa were collected, airbrushed (80 psi, ~10 cm distance to coral), and fixed in 3% glutaraldehyde and filtered (0.22 µm) seawater. Postfixation (1% osmium tetroxide), dehydration, and de-calcification (5% EDTA) were carried out and the samples embedded in Epon-Araldite, and thin sections obtained. Thin sections were placed on formvar-backed grids, stained with uranyl acetate and lead citrate and viewed using an electron microscope (Hitachi H600 STEM) at 80 kv. Additional sections were placed on nickel grids, probed for phycoerythrin and nitrogenase primary antibodies and secondary antibodies (anti-rabbit IgG) conjugated to 20 nm gold particles, and post stained for 5 min in uranyl acetate. Ten random cells were chosen from immunogold labeled and control sections and the number of spheres per cell counted, expressed as spheres cell -1 ± SE, and analyzed using ANOVA. Western Blots Samples of fluorescent and non-fluorescent corals approximately 1-2 cm 2 were airbrushed (80 psi, < 1 cm distance to coral) in a small volume (1-2 ml) of 10 mm HEPES buffer (ph 7.5) with dithiothreitol (DTT) and phenylmethylsulfonyl fluoride (PMSF) to prevent protein oxidation and protease activity. Homogenates were centrifuged at 500 g for 10 min and the supernatants separated from the pellet of zooxanthellae. The supernatant was then sonicated at 2

3 4 C for 1 min (four 15 s bursts with 15 s between bursts) and centrifuged at 10,000 g for 10 min. The soluble proteins (25 µg) were then separated on SDS gradient (4-15%) polyacrylamide gels (SDS-PAGE). Duplicate gels were run, one gel was stained with Coomassie blue for protein and the other electrophoretically transferred to PVDF membranes (0.2 µm). The membrane was blocked with 5% instant milk and immunoblotting of the membrane was completed and the immunoblot developed using a secondary antibody at a titer of 1:2000 labeled with horseradish peroxidase. The transferred proteins were probed using polyclonal antibodies against β- phycoerythrin (1:1000), and the Fe protein of nitrogenase (1:5000) with negative controls (no primary antibody). Amplification and Sequencing of 16S rdna Samples of Montastraea cavernosa were collected from around Lee Stocking Island, Bahamas and stored frozen in 20% DMSO, 0.25 M EDTA, and saturated NaCl at ph 8.0. Genomic DNA was isolated from small pieces (0.5 x 0.5 mm) of coral using the Qiagen DNeasy Tissue Kit following the manufacturers protocol except for the elution volumes that were adjusted to 100 µl. DNA concentration was determined fluorometrically using a DyNA Quant 200 fluorometer (Hoefer, Inc.). Cyanobacterial-specific primers 22 (CYA 106F and CYA781R) or primers designed for Microcystis sp. (Ma-F1, 5 -CTA GGG GAG CGA AAG GGA TTA-3 and MA-R1, 5 -TAT GCT GAC CGG CGA TTA CTA -3 ) were used to amplify and sequence approximately 650 bp of cyanobacterial 16S rdna from the genomic extracts described above. Fluorescent In Situ Hybridization (FISH) Samples of Montastraea cavernosa were fixed as described above except that post fixation in osmium was eliminated and the samples were embedded in JB-4 (methacrylate) medium. 3

4 Thick sections (~1 µm) were placed on poly-l-lysine coated glass slides, dehydrated, treated with 10% SDS to permeabilize the tisues, and gently rinsed with sterile distilled water. A cyanobacterial-specific oligonucleotide probe CYA762 (3 -CTG CTT TCG ATC CCC TCG C- 5 ) was used as it cross reacts with several members of the Order Chroococcales 22. The probe was labeled at the 5 end with Oregon Green with an excitation at 488 nm. Probe preparation and hybridization was carried out on thick sections and mounted in non-fluorescent immersion oil and viewed by epifluorescence microscopy using FITC excitation/emission filters (490/515 nm). 4

5 Supplemantal Figure. Electron micrograph of epithelial tissues of M. cavernosa. Scale bar = 1.0 µm. C= cyanobacteria, N=nematocyst, HM=host membrane, SV=secretory vessicles.