Article Figures & Data
- FIG 1
Nitrogen incorporation and storage within the coral-dinoflagellate endosymbiosis. (A) Coral nubbin (height, ~5 cm) of P. damicornis, showing individual polyps with extended tentacles (inset). (B) Optical image of a coral tissue section (methylene blue-Azur II staining) with abundant dinoflagellate cells (red arrow) in the oral gastroderm. Scale bar, 50 µm. SW, seawater; OE, oral epiderm; OG, oral gastroderm; Dino, dinoflagellate; Coel, coelenteron; AG, aboral gastroderm; Cal, calicodermis; Sk, skeleton. (C) NanoSIMS measurements of the dynamics of 15N incorporation by the dinoflagellates and the four coral epithelia during 90 min of incubation with 20 µM [15N]ammonium. Asterisks indicate significant incorporation of 15N compared to that by the unlabeled control coral. TEM micrograph (D) and corresponding NanoSIMS isotopic 15N/14N image (E) of a dinoflagellate after 90 min of exposure to [15N]ammonium. 15N hot spots are spatially correlated with crystalline deposits (white arrows). Scale bar, 2 µm. ab, accumulation body; nu, nucleus; pl, plastid; pyr, pyrenoid; st, starch. (F) Variations of 15N enrichment along the profile depicted in panel E passing through two 15N hot spots (black arrows). Electron diffraction pattern (G) and corresponding zero-loss (elastically scattered) TEM micrograph (H) and N map (I) of a cluster of dinoflagellate crystalline inclusions. Scale bars, 2 nm−1 (G) and 100 nm (H and I). GC-MS spectra of a uric acid standard (J) and of uric acid from the dinoflagellates of P. damicornis (K).
- FIG 2
Dynamics of N storage and remobilization in the dinoflagellates. Each panel represents a TEM image of a dinoflagellate (left) and its corresponding 15N/14N NanoSIMS image (right), obtained at 15 min (A), 30 min (B), 45 min (C), and 60 min (D) of coral incubation in 20 µM [15N]ammonium (pulse phase) and after 1 h (E), 5 h (F), 11 h (G), 23 h (H), 47 h (I), and 95 h (J) of subsequent coral incubation in unlabeled seawater (chase phase). White arrows indicate highly labeled crystalline uric acid inclusions in the dinoflagellates. Scale bar, 2 µm.
- FIG 3
Ammonium assimilation and N translocation from the dinoflagellates to the coral tissue. NanoSIMS measurements of 15N partitioning between dinoflagellates and coral tissue during the 95-h chase phase under standard light/dark (14 h/10 h) cycling, following a 1-h pulse-labeling in light with [15N]ammonium (20 µM) (A) and during a 95-h chase phase under constant darkness, following a 1 h pulse-labeling in darkness with [15N]ammonium (20 µM) (corals were preacclimatized to darkness for 24 h) (B). (C) Comparison of coral host 15N enrichment levels between the two experimental conditions.
- FIG 4
Nitrate assimilation by the dinoflagellates and N translocation to their coral host. (A) NanoSIMS measurements of 15N partitioning between dinoflagellates and coral tissue during an 84-h chase under standard light/dark (14 h/10 h) cycling, following a 12-h pulse (30 µM) of [15N]nitrate labeling in light. Significant labeling is indicated for the dinoflagellates (*) and the coral host tissue (+) compared to that of the unlabeled control corals. (B to F) TEM micrographs (left) and corresponding NanoSIMS isotopic 15N/14N images (right) of the dinoflagellate-containing oral gastroderm at 2, 6, and 12 h in the pulse (panels B, C, and D, respectively) and at 36 and 84 h in the chase (panels E and F, respectively). White arrows highlight nitrogenous compounds transferred from the dinoflagellates to their adjacent coral cells during the pulse. Scale bar, 5 µm. lb, coral lipid bodies; Dino, dinoflagellate.
- FIG 5
Coral host utilization of N assimilated from [15N]ammonium under light/dark conditions. TEM micrograph (A) and corresponding NanoSIMS isotopic 15N/14N image (B) of the oral epiderm 11 h into the chase phase, showing 15N accumulation in mucus chambers of a mucocyte (white arrows). Scale bar, 5 µm. (C) High-magnification TEM view of the labeled structures (loculae). Scale bar, 2 µm. (D) 15N quantification of the profile indicated in panel B. TEM micrograph (E) and corresponding NanoSIMS isotopic 15N/14N image (F) of the oral gastroderm 45 min into the pulse (under light), showing 15N accumulation in a Golgi body (white arrows). Scale bar, 2 µm. (G) High-magnification TEM view of the labeled structure. Scale bar, 1 µm. (H) 15N quantification of the profile indicated in panel F. TEM micrograph (I) and corresponding NanoSIMS isotopic 15N/14N image (J) of the aboral epithelia 5 h into the chase phase, showing 15N accumulation in calicoblastic cells (white arrows). Scale bar, 5 µm. (K) High-magnification TEM view of the calicoblastic cell showing 15N incorporation in large vesicles (white arrows). Scale bar, 500 nm. (L) 15N quantification of the profile indicated in panel J. OE, oral epiderm; OG, oral gastroderm; AG, aboral gastroderm; Cal, calicodermis; Coel, coelenteron; Dino, dinoflagellate; is, intercellular space; m, mesoglea; mu, mucocyte; nu, nucleus; sj, septate junction; Sk, skeleton; SW, seawater.
Supplemental Material
Figure S1
Principles of NanoSIMS imaging and quantification of 15N distribution in coral tissue. Here, we use coral tissue containing dinoflagellates after 1 h of exposure to [15N]ammonium (20 µM) under light to illustrate NanoSIMS imaging procedures. (A) Ultrathin sections (~70 nm thick) of coral tissue previously observed with TEM are imaged with NanoSIMS. A cesium (Cs+) primary-ion beam focused to a spot size of about 100 to 150 nm raster the sample surface, producing 12C14N− (B) and 12C15N− (C) images of the same field of view. Note from the 12C14N− image that the dinoflagellates contain relatively more N than their surrounding coral cells. (D) The 15N/14N ratio distribution is obtained by taking the ratio of the two 12C15N− and 12C14N− images. Using the L’IMAGE software, regions of interest (ROIs) are defined directly on the 15N/14N isotopic ratio images by drawing the contours of the dinoflagellate cells (ROIs 1 and 2) and coral cells (ROIs 3 to 5). The δ15N values extracted from these ROIs are presented in Table S1. (E) Quantification of the 15N enrichment can also be obtained by extracting profiles from the NanoSIMS 15N/14N isotopic images. Scale bar, 5 µm. Cal, calicodermis; Dino, dinoflagellates; m, mesoglea; Sk, skeleton. Download Figure S1, JPG file, 0.4 MB.
Copyright © 2013 Kopp et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Table S1
NanoSIMS quantification of 15N enrichment using ROIs, defined as described in the legend of Fig. S1D. Table S1, PDF file, 0.2 MB.
Copyright © 2013 Kopp et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S2
NanoSIMS measurements of the dynamics of 15N incorporation in the dinoflagellate endosymbionts and the oral epithelia of the coral host over a 4-h incubation under light with 2 µM [15N]ammonium. Significant labeling relative to the unlabeled control coral is indicated for the dinoflagellates (*) and the coral host (+). Download Figure S2, JPG file, 0.2 MB.
Copyright © 2013 Kopp et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S3
EELS spectra and GC-MS data. (A) Both dinoflagellate crystalline inclusions and commercial uric acid standard display very similar spectra with a pronounced N ionization K edge at an ~400 eV energy loss, which is not apparent (arrow) for the spectrum obtained from amorphous cellular material. No Ca L2,3 ionization edge at ~345 eV was observed for the dinoflagellate crystals, ruling out calcium oxalate. Spectra are displaced on the intensity axis for easier comparison. (B) Total-ion chromatogram from a commercial uric acid standard showing a retention time (RT) of 20.8 min. (C) Corresponding mass spectrum from the peak of a uric acid standard at a RT of 20.8 min. The uric acid standard displays characteristic peaks at m/z 73, 147, 382, 441, and 456. (D) The mass spectrum of the dinoflagellate extract with a RT of 20.8 min is similar to that of the uric acid standard, with the same characteristic peaks. Download Figure S3, JPG file, 0.5 MB.
Copyright © 2013 Kopp et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S4
Nitrogen storage by the dinoflagellates in response to a pulse of [15N]nitrate or [15N]aspartic acid labeling under light and ultrastructure of crystalline inclusions within dinoflagellates. TEM micrograph (A) and corresponding NanoSIMS isotopic 15N/14N image (B) of a dinoflagellate after 6 h of incubation with [15N]nitrate (30 µM). Scale bar, 2 µm. Highly enriched hot spots of 15N are spatially correlated with crystal-containing vesicles (white arrows). (C) The variations of 15N-labeling along the profile depicted in panel B show enrichments of up to 40,000‰ of these structures (black arrows). TEM micrograph (D) and corresponding NanoSIMS isotopic 15N/14N image (E) of a dinoflagellate after 6 h of exposure to [15N]aspartic acid (20 µM). Scale bar, 2 µm. Highly enriched hot spots of 15N are spatially correlated with crystal-containing vesicles (white arrows). (F) The variations of 15N labeling along the profile depicted in panel E indicate enrichments of up to 16,000‰ for these 15N hot spots. (G) Dynamics of 15N accumulation into the dinoflagellates and the four coral epithelia during a pulse of labeling with [15N]aspartic acid (20 µM). The algal endosymbionts and all cellular layers incorporated the 15N tracer at roughly similar rates. Significant 15N enrichment compared to that of the unlabeled control coral is indicated for the dinoflagellates and the coral epithelia (*). (H) TEM view of a dinoflagellate cell showing the spatial distribution of the crystals (white arrows) within the cytosol. Scale bar, 2 µm. (I) Higher-magnification view of the area defined in panel H. Scale bar, 500 nm. The tiny rod-shaped crystalline inclusions are contained in single-membrane vesicles, bearing one or more crystals, usually embedded in an amorphous matrix with a moderate-to-high electron density. Crystals were often lost during TEM sectioning, leaving holes (*). In thin sections, the crystals were relatively bright under the electron beam. ab, accumulation body; li, lipids; m, mitochondria; nu, nucleus; pl, plastid; pyr, pyrenoid; st, starch. Download Figure S4, JPG file, 0.8 MB.
Copyright © 2013 Kopp et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S5
Nitrogen assimilation and translocation in coral epithelia. (A to D) Compared 15N enrichments measured by NanoSIMS in coral cells of all epithelia between the two pulse-chase experiments performed in this study with [15N]ammonium, i.e., under a standard light/dark cycle (14 h/10 h) and constant darkness. A clear difference in 15N enrichment appears after about 6 h during the experiments and can be ascribed to N translocation from the dinoflagellates to the coral cells of all tissue layers. (E, F) 15N enrichment measured by NanoSIMS in the four coral epithelia during an 84-h chase phase under a standard light/dark cycle (14 h/10 h) following a 12-h pulse of [15N]nitrate (30 µM) labeling in light. Significant 15N enrichment is detected for each epithelium after about 12 h, except for the oral gastroderm (6 h), indicating N transfer from the endosymbiotic dinoflagellates. Significant 15N enrichment compared to that of the unlabeled control coral is indicated for the oral epiderm and the calicodermis (*) and for the oral and aboral gastroderms (+). Download Figure S5, JPG file, 0.7 MB.
Copyright © 2013 Kopp et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Table S2
GC-C-IRMS quantification of 15N enrichment in dinoflagellate uric acid molecules from corals exposed to various incubations with 15N-dissolved inorganic nitrogen. Table S2, PDF file, 0.2 MB.
Copyright © 2013 Kopp et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Table S3
Time course of 15N enrichment in the dinoflagellates during parallel pulse-chase experiments with [15N]ammonium, with chase under light/dark cycling versus constant darkness. Bulk analyses were carried out following conventional coral tissue dissociation and density separation of the dinoflagellate fraction from their animal host. Table S3, PDF file, 0.1 MB.
Copyright © 2013 Kopp et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Table S4
Best BLAST hits (E value < 1e−20 using the psi-BLAST algorithm) for proteins with high similarities to the domains of xanthine dehydrogenase and uricase present in 2 Symbiodinium species known to establish stable symbioses with coral hosts (Symbiodinium clade C3 and Symbiodinium sp. strain Mf1.05b). Table S4, PDF file, 0.1 MB.
Copyright © 2013 Kopp et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Text S1
Supplementary materials and methods. (a) Experimental design; (b) TEM ultrastructural observations; (c) NanoSIMS isotopic imaging; (d) NanoSIMS data processing and region of interest (ROI) definitions; (e) bulk 15N/14N isotopic measurements; (f) electron diffraction, EFTEM spectrum imaging, and EELS; (g) GC-MS and GC-C-IRMS analyses of uric acid. Download Text S1, PDF file, 0.2 MB.
Copyright © 2013 Kopp et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Supplementary Data
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