A wide range of organisms host photosynthesizing symbionts. In these animals the metabolic exchange between host and symbionts has prevented in situ host anabolic turnover to be studied without the confounding effect of translocated photosynthates. Using the symbiotic coral
Gibbin et al. use [1–13C]-pyruvate and [2,3–13C]-pyruvate in different incubation conditions (light, light+DCMU, and dark) to discern and quantify coral host anabolism, with and without translocated metabolites from their photosynthesizing symbionts.
Aerobic cell metabolism consists of two complementary processes: catabolism, i.e., the ATP-producing oxidation of complex carbon molecules into CO2, and anabolism, i.e., the synthesis of building block molecules into compounds that have higher structural complexity. The tricarboxylic acid (TCA) cycle in mitochondria drives both catabolic and anabolic reactions
Recent advances in ion microprobe (NanoSIMS) imaging allows the in situ distribution of enriched stable isotopes in biological tissue to be imaged at a spatial resolution of ~100 nm. The resulting isotopic maps can be correlated directly with ultrastructural observations of the cell by electron microscopy (EM) of the same thin or semi-thin section
Corals are meta-organisms (so-called holobionts
In organisms hosting photosynthesizing symbionts, both the 13CO2 produced as a by-product of the formation of the Acetyl-CoA complex (C-position 1) and during the TCA cycle (C-positions 2 and 3) can be assimilated by the symbionts. Photosynthates that are labelled in 13C and translocated back to the host tissue then make it impossible to obtain a measure of pure host-cell anabolic turnover. This problem of photosynthate translocation can be avoided either by conducting experiments in the dark and/or by blocking photosynthesis, rendering it impossible for translocated photosynthates to contribute to 13C-enrichment in the host tissue. Here we have used symbiotic, reef-building corals as the model system to illustrate these strategies.
When photosynthesis was active, [1-13C]-pyruvate produced 13C-labelling patterns qualitatively similar to those previously observed in corals exposed to 13C-labelled bicarbonate in seawater, with preferential accumulation in the pyrenoid and starch granules of the dinoflagellates, and translocated 13C-labelled lipids in the coral host tissue Corals were incubated with differentially labelled pyruvate, in the presence or absence of the photosynthetic inhibitor DCMU, for 12 h. Scanning electron microscopy (top row) images and their correlative NanoSIMS image (bottom row) are shown for corals labelled with Shown is the relative assimilation of 13C (mean ± SE of
Incubation with [2,3-13C]-pyruvate resulted in strong, heterogeneously distributed 13C-labelling of the host tissues in all conditions (Figs.
The [2,3-13C]-pyruvate incubations permit several novel observations. Comparison between anabolic 13C-assimilation in the light versus the light + DCMU and night incubations, permits quantification of the symbiont boost to host anabolism via translocation. In our experiments, translocation increased 13C-incorporation by 13–22% in the epidermis (Fig.
To test for differences in 13C-labelled pyruvate assimilation between different regions of interest (ROIs) in the coral polyp, data were first analysed by two-way ANOVA with region of interest and colony as factors (Supplementary Table Individual polyps ( Four regions of interest were identified in the coral polyp: the aboral tissue layers
The tentacles also contain mucocytes and epithelio-muscular cells to facilitate their retraction
Another interesting observation was the presence of 13C-rich hotspots in all of the ROIs studied (Fig.
In this study, we show that pyruvate is an effective isotopic marker of anabolism in photosymbiotic holobionts. When combined with NanoSIMS isotopic imaging, isotopically labelled pyruvate provides quantitative information at the tissue- and single cell level and can be applied to disentangle complex host–symbiont metabolic interactions. Of particular current interest, the method can be used to throw light on how reef-building corals respond to temperature-induced bleaching: i.e., when corals expel or lose their symbiotic algae
Three
12 h isotopic pulses were conducted in 250 mL glass beakers, set atop a submersible magnetic stir-plate, which was placed in a flow-through aquarium. Day (light) and night incubations were conducted in ambient thermal conditions (26 ± 1 °C) in accordance with the diel light cycle in Eilat (day: 06:30–18:30). Light incubations were conducted under natural, but shaded light (mean: 144 ± 230 μmol photons m−2 s−1) conditions (Supplementary Fig.
During the isotopic-labelling experiments, water changes were performed and fresh isotopic labels were added every 3 h to ensure stable water chemistry. At the end of the labelling experiment, the apical tip of each coral fragment was removed and a 1 cm coral piece was clipped off and immersed in fixative (0.5% formaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer with 0.6 M sucrose, pH 7.4–7.6) for 24 h at room temperature
Samples were dissected into small tissue pieces containing a single polyp and post-fixed for 1 h at room temperature with 1% osmium tetroxide in 0.1 M phosphate buffer. The samples were then dehydrated in a graded series of ethanol (50%, 70%, 90%, and 100%), and embedded in Spurr resin blocks. Thin (200 nm) and semi-thin (500 nm) sections were cut using a 45° Diatome diamond knife and mounted on round glass slides (10 mm) for scanning transmission electron microscopy (GeminiSEM 500, Carl Zeiss Microscopy GmbH, Jena, DE), or NanoSIMS imaging.
All NanoSIMS images (40 × 40 μm, 256 × 256 pixels, 5 ms pixel−1 dwell time, five layers) were obtained using a 16 keV Cs+ primary ion beam, focused to a spot-size of about 120 nm. Secondary ions (12C2−, 13C12C−) were simultaneously counted in individual electron-multiplier detectors, with a mass resolving power of ~9000 (Cameca definition). Isotopic ratios were formed from drift-corrected ion images using the ratio of 13C12C− to 12C2− and expressed as parts-per-thousand (‰) deviation relative to an isotopically unlabelled coral tissue sample prepared and analysed in an identical manner.
Two separate experiments were performed: (1) A
1.
To test for differences in 13C-labelled pyruvate assimilation between [1-13C] and [2,3-13C]-pyruvate, separate Student’s
2
To test for patterns in pyruvate-derived 13C-assimilation between structures from different colonies, data were first analyzed by two-way ANOVA with structure (aboral tissues, tentacle, pharynx, mesentery filament) and colony (1–3) as fixed factors (Supplementary Table
Further information on research design is available in the
We would like to thank Drs. Louise Jensen and Stéphane Escrig (EPFL, Switzerland) for assisting with the correlative SEM and NanoSIMS imaging, respectively. We also thank Prof. Isabelle Domart-Coulon (Muséum National d'Histoire Naturelle, Paris) for insightful comments on a draft of this manuscript, and Dr. Thomas Krueger (University of Cambridge, UK) for discussions.
E.G., A.C., A.M. and M.F. designed the experiment. E.G. and G.B.-P. conducted the experiment. E.G. analyzed the samples and performed data analysis. E.G. and A.M. produced the first draft of the manuscript and all authors contributed to writing of the manuscript.
All data generated or analysed during this study are included in this published article (and its Supplementary Information files).
The authors declare no competing interests.
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