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Gas Chromatography-Mass Spectrometry-Based Targeted Metabolomics of Hard Coral Samples

Published: October 13th, 2023



1Climate Change Cluster (C3), University of Technology Sydney, 2Metabolomics Australia, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, 3School of Biological Sciences, Victoria University of Wellington

Here, we present the extraction and preparation of polar and semi-polar metabolites from a coral holobiont, as well as separated coral host tissue and Symbiodiniaceae cell fractions, for gas chromatography-mass spectrometry analysis.

Gas chromatography-mass spectrometry (GC-MS)-based approaches have proven to be powerful for elucidating the metabolic basis of the cnidarian-dinoflagellate symbiosis and how coral responds to stress (i.e., during temperature-induced bleaching). Steady-state metabolite profiling of the coral holobiont, which comprises the cnidarian host and its associated microbes (Symbiodiniaceae and other protists, bacteria, archaea, fungi, and viruses), has been successfully applied under ambient and stress conditions to characterize the holistic metabolic status of the coral.

However, to answer questions surrounding the symbiotic interactions, it is necessary to analyze the metabolite profiles of the coral host and its algal symbionts independently, which can only be achieved by physical separation and isolation of the tissues, followed by independent extraction and analysis. While the application of metabolomics is relatively new to the coral field, the sustained efforts of research groups have resulted in the development of robust methods for analyzing metabolites in corals, including the separation of the coral host tissue and algal symbionts.

This paper presents a step-by-step guide for holobiont separation and the extraction of metabolites for GC-MS analysis, including key optimization steps for consideration. We demonstrate how, once analyzed independently, the combined metabolite profile of the two fractions (coral and Symbiodiniaceae) is similar to the profile of the whole (holobiont), but by separating the tissues, we can also obtain key information about the metabolism of and interactions between the two partners that cannot be obtained from the whole alone.

Metabolites represent the end products of cellular processes, and metabolomics - the study of the suite of metabolites produced by a given organism or ecosystem - can provide a direct measure of organismal functioning1. This is particularly critical for exploring ecosystems, symbiotic interactions, and restoration tools, as the goal of most management strategies is to preserve (or restore) specific ecosystem service functions2. Coral reefs are one aquatic ecosystem that demonstrates the potential value of metabolomics for elucidating symbiotic interactions and linking coral physiological responses to community-level and ....

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NOTE: The experimental design, sample collection and storage have been described in detail elsewhere2,30,31. Permit approval for the collection of wild corals must be obtained prior to collection and experimentation. The samples here were collected from colonies of Montipora mollis (green colour-morph) imported from Batavia Coral Farms (Geraldton, WA), originally collected from a reef off the Abrohlos Islands (Western A.......

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All the data produced during this work are available in the supplementary information.

Host-symbiont separation

Figure 1
Figure 1: Setup and validation of the separation of coral host tissues and Symbiodiniaceae cells. (A) The air gun setup for the removal of coral.......

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The separation of the host and symbiont is easily and rapidly achievable via simple centrifugation, and the results here show that separating the fractions can provide valuable information indicative of specific holobiont member contributions, which can contribute toward the functional analysis of coral health. In adult corals, lipid synthesis is primarily performed by the resident algal symbiont40, which supplies lipids (e.g., triacylglycerol and phospholipids)41 .......

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J.L.M. was supported by a UTS Chancellor's Research Fellowship.


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Name Company Catalog Number Comments
100% LC-grade methanol Merck 439193 LC grade essential
2 mL microcentrifuge tubes, PP Eppendorf 30121880 Polypropylene provides high resistance to chemicals, mechanical stress and temperature extremes
2030 Shimadzu gas chromatograph Shimadzu GC-2030
710-1180 µm acid-washed glass beads Merck
This size is optimal for breaking the Symbiodiniaceae cells
AOC-6000 Plus Multifunctional autosampler Shimadzu AOC6000
Bradford reagent Merck B6916 Any protein colourimetric reagent is acceptable
Compressed air gun Ozito 6270636 Similar design acceptable. Having a fitting to fit a 1 mL tip over is critical.
 DB-5 column with 0.25 mm internal diameter column and 1 µm film thickness Agilent 122-5013
DMF Merck RTC000098
D-Sorbitol-6-13C and/or 13C515N Valine Merck 605514/ 600148 Either or both internal standards can be added to the methanol.
Flat bottom 96-well plate Merck CLS3614
Glass scintillation vials Merck V7130 20 mL, with non-plastic seal
Immunoglogin G Merck 56834 if not availbe, Bovine Serum Albumin is acceptable
Primer v4
R v4.1.2
Shimadzu LabSolutions Insight software v3.6
Sodium Hydroxide Merck S5881 Pellets to make 1 M solution
tidyverse v1.3.1 R package
TissueLyser LT Qiagen 85600 Or similar
TQ8050NX triple quadrupole mass spectrometer Shimadzu GCMS-TQ8050 NX
UV-96 well plate Greiner M3812
Whirl-Pak sample bag Merck WPB01018WA Sample collection bag; Size: big enough to house a ~5 cm coral fragment, but not too big that the water is too spread

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