Multi-omics determination of metabolome diversity in natural coral populations in the Pacific Ocean

Coral reefs, vital ecosystems facing climate change threats, are increasingly understood through the lens of the coral holobiont—the coral host, its symbiotic dinoflagellates (Symbiodiniaceae), and its microbiome. Rising seawater temperatures and acidification pose significant challenges, particularly heat stress leading to coral bleaching. Metabolomics, studying the complete set of metabolites, offers valuable insights into the physiological state of the coral holobiont and its response to environmental change. Previous metabolomic studies on corals have used various extraction and analytical techniques, lacking standardization. This study, part of the Tara Pacific expedition, aims to optimize a scalable metabolomic approach to analyze a large dataset of coral samples collected across a wide geographical range, investigating the metabolome variability among coral genera and across different geographic locations and environments, providing a baseline for future studies on the adaptive capacity of these vital organisms.

The literature review highlights the growing understanding of the coral holobiont and its response to environmental stress, primarily focusing on the role of metabolomics in characterizing this response. A significant gap identified was the lack of a standardized methodology for metabolomic analysis in corals. Existing studies employed diverse extraction methods (Folch, Gordon, Matyash) and analytical techniques (GC-MS, LC-MS, ¹H NMR), each with limitations in terms of coverage and annotation. This inconsistency hinders comparative studies and limits our comprehensive understanding of coral metabolome diversity and its ecological significance. The review also underscored the limited knowledge about the functional roles of different metabolites, particularly in natural populations, further emphasizing the need for the current study.

This study employed a standardized, scalable multi-omics approach to analyze the metabolome of three coral genera (*Pocillopora* spp., *Porites* spp., and *Millepora* cf. *platyphylla*) collected during the Tara Pacific expedition. A modified Matyash biphasic extraction method using MTBE/MeOH/H₂O (7:4:4) maximized extraction yields for both polar and non-polar metabolites. The lipidome (non-polar) was analyzed using LC-HRMS/MS, and the hydrophilic metabolome (polar) was analyzed using ¹H NMR. The LC-MS data was processed using W4M, including steps for peak detection, clustering, retention time correction, annotation, and quality control. Molecular networking analysis using GNPS was employed to annotate lipids. The NMR data was processed using NMRProcFlow, Pareto scaling, and statistical analysis in MetaboAnalyst. Metabolite annotation in the NMR data was done using reference compounds, including those synthesized in the laboratory. Statistical analyses (PCA, PPLS-DA) were used to compare metabolomic profiles among genera and across geographical locations.

The study identified significant differences in metabolomic profiles among the three coral genera but not within genotypes of the scleractinian corals. LC-MS analysis revealed distinct lipidome profiles among genera, with shared lipid families present at different concentrations and exhibiting structural variations in fatty acid chains. Specific betaine lipids (MGCC, DGCC, PAF, lyso-PAF) were identified and showed distinct distributions across genera. NMR analysis identified 24 metabolites (18 annotated, 6 unknown), predominantly amino acids and their betaine derivatives. The study revealed a high degree of genus-specificity in these polar metabolites. Geographical variability was also observed. Significant differences were detected in the lipidomes of the same coral genera across two islands in different marine provinces and also among sites. Geographic differences in the polar metabolome (NMR) were primarily observed in *Porites* spp. A novel family of sulfolipids was identified in *Millepora* cf. *platyphylla* from one location.

The findings highlight the significant role of genus-specific metabolic profiles in the coral holobiont, indicating distinct metabolic strategies employed by different genera, and the influence of environmental factors (primarily temperature) on the metabolome. While Symbiodiniaceae play a central role, they do not solely determine the observed differences. The observed variations suggest that both holobiont composition and environmental interactions drive metabolic diversity. The study underscores the importance of studying natural coral populations to understand adaptive responses to environmental changes. The differences observed in betaine concentration across genera suggest variations in osmoregulation mechanisms. Geographic differences in lipidomes highlight environmental adaptation, with the observed sulfolipid variations possibly indicating heat stress response.

This study provides a standardized metabolomic approach for coral research, revealing significant genus-specific and geographic metabolic variability in three Pacific coral genera. While Symbiodiniaceae contribute substantially, the combined effect of host, symbiont, and environment shapes the coral holobiont's metabolic profile. Future research should focus on expanding the analysis to the entire Tara Pacific dataset to better understand the relationship between metabolome, genotype, and environmental variables. Further characterization of novel metabolites is crucial for understanding their functional roles in coral adaptation and survival.

The study analyzed only a subset of samples from the Tara Pacific expedition. While the methodology was designed for scalability, the conclusions drawn are based on a limited geographic representation. Additionally, full structural confirmation of some compounds, particularly the novel sulfolipids, requires further investigation. The study focuses on the metabolome, neglecting other –omics data that could provide additional insights into the complex coral holobiont interaction.