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

Summary

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.

Abstract

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.

Introduction

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 ....

Protocol

NOTE: The experimental design, sample collection and storage have been described in detail elsewhere^2,30,31. 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.......

Discussion

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 .......

Materials

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	G1152	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 13C5–15N 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