Benthic exometabolites and their ecological significance on threatened Caribbean coral reefs

Abstract

Benthic organisms are the architectural framework supporting coral reef ecosystems, but their community composition has recently shifted on many reefs. Little is known about the metabolites released from these benthic organisms and how compositional shifts may influence other reef life, including prolific microorganisms. To investigate the metabolite composition of benthic exudates and their ecological significance for reef microbial communities, we harvested exudates from six species of Caribbean benthic organisms including stony corals, octocorals, and an invasive encrusting alga, and subjected these exudates to untargeted and targeted metabolomics approaches using liquid chromatography-mass spectrometry. Incubations with reef seawater microorganisms were conducted to monitor changes in microbial abundances and community composition using 16 S rRNA gene sequencing in relation to exudate source and three specific metabolites. Exudates were enriched in amino acids, nucleosides, vitamins, and indole-based metabolites, showing that benthic organisms contribute labile organic matter to reefs. Furthermore, exudate compositions were species-specific, and riboflavin and pantothenic acid emerged as significant coral-produced metabolites, while caffeine emerged as a significant invasive algal-produced metabolite. Microbial abundances and individual microbial taxa responded differently to exudates from stony corals and octocorals, demonstrating that exudate mixtures released from different coral species select for specific bacteria. In contrast, microbial communities did not respond to individual additions of riboflavin, pantothenic acid, or caffeine. This work indicates that recent shifts in benthic organisms alter exudate composition and likely impact microbial communities on coral reefs.

Fig. 1. Heatmap of average log₁₀(fold changes) between organism and control incubations demonstrates that specific targeted metabolites are significantly enriched and depleted and vary by species.

Colors correspond to log₁₀(fold change) and asterisks reflect significantly enriched (black) or depleted (outlined) metabolites in organism compared to control incubations as determined using Welch’s t-tests.

Fig. 2. Non-metric multidimensional scaling analysis (NMDS) of Bray-Curtis dissimilarities calculated for untargeted metabolite features ionized in positive mode are significantly influenced by species and sample type (organism vs. control incubations).

A Global NMDS of control and organism incubations is displayed for all species and the results of the constrained adonis test are included above the plot. B–G NMDSs of control and organism incubations are displayed separately for each coral species. Symbol shape corresponds to species and color corresponds to sample type. Ellipse shapes indicate covariances among samples in each grouping.

Fig. 3. Abundances of Prochlorococcus, Synechococcus, and unpigmented cells respond differently to exudates from Porites astreoides and Gorgonia ventalina.

Line graphs of normalized cell counts of Prochlorococcus (A, B), Synechococcus (C, D), and unpigmented cells (E, F) show differing responses of microbial communities to Porites astreoides (A, C, E) and Gorgonia ventalina (B, D, F) exudates. Color corresponds to sample type, symbols represent average cell counts across replicates, and error bars reflect standard error across replicates (n = 3 per treatment type). Dashed lines represent controls and solid lines represent coral exudate addition treatments.

Fig. 4. Relative abundances of specific microbial taxa increase or decrease in the presence of Porites astreoides and Gorgonia ventalina exudates.

Log₁₀(fold change) plots of averaged microbial 16 S rRNA gene ASV relative abundances (normalized to the initial time point at 0 h) that significantly and consistently increased or decreased in the presence or absence of Porites astreoides (A) and Gorgonia ventalina (B) exudates determined using corncob analysis. Only ASVs with coefficients of variation less than –2 (depleted in coral exudate additions) and greater than 0 (enriched in coral exudate additions) were included to focus on ASVs displaying the most distinct changes. Symbol color reflects the time that samples were collected during the incubations and shape reflects light conditions.

Fig. 5. Relative abundances of specific microbial taxa exhibit similar or different responses to exudates from Porites astreoides and Gorgonia ventalina overtime.

Boxplots display relative abundances of ASVs that exhibit different (A–D) or similar (E, F) trends in response to coral exudates. Plots (A, C, and E) show trends in response to P. astreoides exudates and plots (B, D, and F) show trends in response to G. ventalina exudates. Symbol color indicates time and shape reflects light conditions.

Fig. 6. Concentrations of caffeine, pantothenic acid, and riboflavin exhibit different trends in the presence or absence of reef seawater microbial communities overtime.

Variations in A caffeine, B pantothenic acid, and C riboflavin concentrations overtime show that metabolites experience different trends in the presence/absence of reef seawater microbial communities. Color corresponds to sample type, symbols represent average cell counts across replicates, and error bars reflect standard error computed across replicates.