Dynamic regulation of coral energy metabolism throughout the diel cycle

Abstract

Coral reefs are naturally exposed to daily and seasonal variations in environmental oxygen levels, which can be exacerbated in intensity and duration by anthropogenic activities. However, coral's diel oxygen dynamics and fermentative pathways remain poorly understood. Here, continuous oxygen microelectrode recordings in the coral diffusive boundary layer revealed hyperoxia during daytime and hypoxia at nighttime resulting from net photosynthesis and net respiration, respectively. The activities of the metabolic enzymes citrate synthase (CS), malate dehydrogenase, and strombine dehydrogenase remained constant throughout the day/night cycle, suggesting that energy metabolism was regulated through adjustments in metabolite fluxes and not through changes in enzyme abundance. Liquid chromatography-mass spectrometry analyses identified strombine as coral's main fermentative end product. Strombine levels peaked as oxygen became depleted at dusk, indicating increased fermentation rates at the onset of nightly hypoxia, and again at dawn as photosynthesis restored oxygen and photosynthate supply. When these peaks were excluded from the analyses, average strombine levels during the day were nearly double those at night, indicating sifnificant fermentation rates even during aerobic conditions. These results highlight the dynamic changes in oxygen levels in the coral diffusive boundary layer, and the importance of fermentative metabolism for coral biology.

Figure 1

O₂ concentration in the coral diffusive boundary layer (DBL) throughout a diel cycle. (A) Continuous [O₂] measurements in the DBL of coral branches from the 2014 experiment (n = 4). (B) Same as in A, but from the 2016 experiment (n = 7). (C) Average [O₂] in the coral DBL during day and night. The white circles and grey squares show the average [O₂] measurements for each coral branch during day and night, and the red lines represent mean ± SEM. Two-tailed paired t-test, n = 11 coral branches measured during 2014 and 2016. The dashed and dotted lines show normoxic [O₂] in the tank (235 μM) and nominal hypoxia (62.5 μM), respectively. (D) Picture of an O₂ microsensor in the DBL of an A. yongei branch taken through a dissecting scope (photo credit: L.B. Linsmayer).

Figure 2

Metabolic enzyme activity throughout a diel cycle. (A) Citrate synthase (CS); (B) Malate dehydrogenase (MDH); (C) Strombine dehydrogenase (SDH) activities (n = 6–8). The only significant difference between timepoints was for CS between 6.40 h and 10.40 h. One-way ANOVA on square root-transformed data (p = 0.0265) followed by Tukey’s post-test (which corrects for multiple comparisons) (p < 0.05).

Figure 3

LC–MS analysis of unfiltered and Celite-filtered strombine standard and coral extract. Liquid chromatography extracted ion chromatograms (EICs; left column) and MS/MS spectra (right column) of unfiltered (A–D) and Celite-filtered (E–H) pure strombine (A,B,E,F) and coral extract (C,D,G,H). Retention times are provided for the most intense peaks on the EICs. For both experiments, MS/MS was performed on the parent mass of strombine, m/z = 146, and the resulting MS/MS spectra of the extract (D,H) matches that of the standard (B,F), with daughter ions at m/z 102 and m/z 128 and the m/z 146 unfragmented parent ion (A. yongei photo credit: G.T. Kwan).

Figure 4

Strombine and alanopine abundances in coral tissues throughout a diel cycle. (A) Strombine abundance at the different time points (n = 6–7). (B) Pooled values from the day times 14.40 and 18.40 h were compared to pooled values from the night times 2.40 and 6.40 h (n = 12–13). The peaks at 10.40 and 6.40 h were omitted in this analysis. (C) Alanopine abundance at the different time points (n = 6–7). (D) Same as in (B), but for alanopine (n = 12–14). Data in (A) and (C) was squared-root transformed and analyzed by one-way ANOVA (p < 0.0001) followed by Tukey post-test (which corrects for multiple comparisons) (p < 0.05). Data in (B) was analyzed by Mann–Whitney test (p = 0.0002). Data in (D) was analyzed by unpaired t-test (p = 0.25).