Evidence that glucose is the major transferred metabolite in dinoflagellate-cnidarian symbiosis

Abstract

Reef-building corals and many other cnidarians are symbiotic with dinoflagellates of the genus Symbiodinium. It has long been known that the endosymbiotic algae transfer much of their photosynthetically fixed carbon to the host and that this can provide much of the host's total energy. However, it has remained unclear which metabolite(s) are directly translocated from the algae into the host tissue. We reexamined this question in the small sea anemone Aiptasia using labeling of intact animals in the light with (13)C-bicarbonate, rapid homogenization and separation of animal and algal fractions, and analysis of metabolite labeling by gas chromatography-mass spectrometry. We found labeled glucose in the animal fraction within 2 min of exposure to (13)C-bicarbonate, whereas no significant labeling of other compounds was observed within the first 10 min. Although considerable previous evidence has suggested that glycerol might be a major translocated metabolite, we saw no significant labeling of glycerol within the first hour, and incubation of intact animals with (13)C-labeled glycerol did not result in a rapid production of (13)C-glucose. In contrast, when Symbiodinium cells freshly isolated from host tissue were exposed to light and (13)C-bicarbonate in the presence of host homogenate, labeled glycerol, but not glucose, was detected in the medium. We also observed early production of labeled glucose, but not glycerol, in three coral species. Taken together, the results suggest that glucose is the major translocated metabolite in dinoflagellate-cnidarian symbiosis and that the release of glycerol from isolated algae may be part of a stress response.

Fig. 1.

GC-MS chromatograms showing detection of multiple metabolites with a wide range of GC elution times in an anemone homogenate. CC7 anemones were incubated with ¹³C-bicarbonate in the light for 10 min, and a homogenate was prepared and analyzed as described in the Materials and methods. (A) For each GC elution time, the height shown is the sum of all of the MS intensities recorded for m/z 90–650. Inset: magnified image of the results for elution times of 29.5–30.5 min; a glucose standard eluted at 29.95±0.05 min on the column used (supplementary material Fig. S2C). (B) The same chromatogram after log transformation to deemphasize the background (intensities <6000 are not displayed), compress the peaks, and thus better show the multitude of peaks detected. Intensity_displayed=log₁₀(intensity recorded/1000–5) or simply 0 when intensity recorded was ≤6000.

Fig. 2.

GC-MS identification of photosynthetically produced glucose and of a glucose fragment useful in quantitating the incorporation of ¹³C into newly synthesized glucose. CC7 anemones were incubated with 10 mg ml^{−1 12}C-bicarbonate in the light for 10 min (A,D), with 10 mg ml^{−1 13}C-bicarbonate in the dark for 10 min (B,E) or with 10 mg ml^{−1 13}C-bicarbonate in the light for 10 min (C,F), and homogenates were prepared and analyzed as described in the Materials and methods. (A–C) Chromatograms produced by summing the MS peak intensities from 310 to 330 m/z for each elution time shown, showing a peak identified as glucose by comparing its elution time and MS spectrum (shown in part in D–F) to those observed in previous work and in our own glucose-standard runs (supplementary material Fig. S2). (D–F) Portions of the MS spectrum (m/z 310–330) of the material eluting from the GC at 29.95 min. Note that the m/z values are those for the derivatized glucose fragment; the rightward tail on each peak is due to naturally occurring ¹³C (~1.1%) and the isotopic distribution of the silicon in the silate groups used for derivatization (see supplementary material Fig. S2 legend for more details).

Fig. 3.

Retention of labeled glucose by isolated Symbiodinium cells. Algal cells freshly isolated from CC7 anemones (see Materials and methods) were incubated in artificial seawater (ASW) with 10 mg ml^{−1 13}C-bicarbonate for 60 min in the light (190 μE m⁻² s⁻¹) and then rinsed and resuspended in Milli-Q water. The samples were then treated with the homogenizer, filtered, and analyzed as performed with samples of host material (see Materials and methods). The peak-ratio method (see Materials and methods and Fig. 2) was then used to determine the distribution of newly synthesized, labeled glucose. Error bars represent 95% confidence intervals based on the three biological replicates.

Fig. 4.

Detection of labeled glucose, but not of other compounds, in host tissue at early times after the exposure of symbiotic anemones to light and ¹³C-bicarbonate. (A–C) CC7 anemones were incubated for various times in the light or dark with ¹³C-bicarbonate or ¹²C-bicarbonate, as indicated, and were then homogenized and analyzed as described in the Materials and methods. The peak-ratio method was used to determine the distributions of ¹³C-labeled glucose (A), succinate (B) and glycerol (C) in the total homogenate (circles), the filtrate (triangles), and the material retained on the filter (squares). (1) Dark pre-incubation followed by incubation in the light with ¹³C-bicarbonate; (2) light pre-incubation for ~2 h followed by incubation in the light with ¹³C-bicarbonate; (3) dark pre-incubation followed by incubation in the dark with ¹³C-bicarbonate; (4) dark pre-incubation followed by incubation in the light with ¹²C-bicarbonate. (D) As in panel A1 except using aposymbiotic anemones. Time axes are in log or linear scales, as indicated. Error bars (all panels) represent 95% confidence intervals based on the three biological replicates in each case; some error bars in A2, B1 and C1 are offset from the corresponding data points to allow easier discrimination between the symbols.

Fig. 5.

Rapid transfer of newly synthesized glucose into host tissue in other Symbiodinium–cnidarian symbioses. (A) Aiptasia strains CC7 (gray bars) and H1 (white bars) were pre-incubated in the dark, exposed to ¹³C-bicarbonate in the light for 5 min, and homogenized and analyzed for glucose and other potentially labeled compounds as described in the Materials and methods and Fig. 2. (B,C) Coral samples (see Materials and methods) were exposed to ¹³C-bicarbonate in the light for 5 min, homogenized and analyzed as in panel A. (B) Glucose peak ratios [323/(319+323)] in total homogenates of (1) Discosoma sp., (2) Cladiella sp. and (3) Acropora millepora. (C) The Discosoma sample was fractionated by filtration (as with anemone samples) and the peak-ratio method was used to determine the distribution of newly synthesized glucose. Error bars (all panels) represent 95% confidence intervals based on the three biological replicates in each case.

Fig. 6.

Blockage of glucose labeling by an inhibitor of photosynthesis but not by an inhibitor of gluconeogenesis. (A) CC7 anemones were treated with 50 μmol l⁻¹ DCMU (white bars) or not (gray bars) for 15 min before and during 5 min of exposure to ¹³C-bicarbonate in the light. The animals were then homogenized, fractionated and analyzed by GC-MS for the distribution of newly synthesized glucose as described in the Materials and methods and Fig. 2. (B) CC7 anemones were treated with 25 mmol l⁻¹ 1-thioglycerol (TG) (white bars) or not (gray bars) for 15 min before and during 15 min of exposure to ¹³C-bicarbonate in the light. The animals were then analyzed as in panel A. Error bars (both panels) represent 95% confidence intervals based on the three biological replicates in each case.

Fig. 7.

Release of glycerol but not glucose by freshly isolated dinoflagellates and its stimulation by host homogenate. Freshly isolated algae in ASW, host homogenate without algae, and freshly isolated algae mixed with host homogenate were prepared and incubated with ¹³C-bicarbonate in the light as described in the Materials and methods. Each sample was then centrifuged, and the algal pellets (where present) and supernatants were analyzed by GC-MS. (A,C) Total amounts of glycerol (A) and glucose (C) in pellets (gray bars) and supernatants (white bars) as determined by integration under the GC peaks (see Materials and methods). n.q., the amounts of glucose found in the supernatants were not large enough to be detected above background by the automated peak-integration software. (B) Glycerol labeling in samples from A as determined by the 221/(221+218) peak ratio. Error bars in A and C are 95% confidence intervals calculated from the standard errors of the means of the three biological replicates; error bars in B are 95% confidence intervals determined using an exact binomial test on the data from the three biological replicates (see Materials and methods).