J.L.M. was supported by a UTS Chancellor&#39;s Research Fellowship.

Metabolite Profiling of Hard Coral Samples Using GC-MS

The authors have no conflict of interests to disclose.

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 ...

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 ecosystem-level impacts3. The application of high-throughput gas chromatography-mass spectrometry (GC-MS) is especially valued due to its capacity to rapidly analyze a broad range of metabolite classes simultaneously with high selectivity and sensitivity, provide rapid compound identification when spectral libraries are available, and provide a high level of reproducibility and accuracy, with a relatively low cost per sample.
Corals are holobionts consisting of the coral animal, photosynthetic dinoflagellate endosymbionts (family: Symbiodiniaceae4), and a complex microbiome5,6. Overall, the fitness of the holobiont is maintained primarily through the exchange of small molecules and elements to support the metabolic functioning of each member7,8,9,10. Metabolomic approaches have proven especially powerful for elucidating the metabolic basis of symbiosis specificity9,11, the bleaching response to thermal stress7,8,12,13, disease responses14, pollution exposure responses15, photoacclimation16, and chemical signalling17 in corals, as well as aiding in biomarker discovery18,19. Additionally, metabolomics can provide valuable confirmation of the conclusions inferred from DNA- and RNA-based techniques9,20. There is, therefore, considerable potential for the use of metabolomics for assessing reef health and developing tools for reef conservation3, such as through the detection of metabolic biomarkers of stress18,19 and for examining the potential of active management strategies such as nutritional subsidies21.
Separating the host and symbiont cells and analyzing their metabolite profiles independently, rather than together as the holobiont, can yield more information about the partner interactions, independent physiological and metabolic statuses, and potential molecular mechanisms for adaptation11,12,22,23,24. Without separating the coral and Symbiodiniaceae, it is almost impossible to elucidate the contribution and metabolism of coral and/or Symbiodiniaceae independently, except for with complex genome reconstruction and metabolic modeling25, but this has yet to be applied to the coral-dinoflagellate symbiosis. Furthermore, attempting to extract information about the individual metabolism of the host or algal symbiont from the metabolite profile of the holobiont can lead to misinterpretation.
For example, until recently, the presence of C18:3n-6, C18:4n-3, and C16 polyunsaturated fatty acids in extracts from coral and holobiont tissues was thought to be derived from the algal symbiont, as corals were assumed to not possess the &#969;x desaturases essential for the production of omega-3 (&#969;3) fatty acids; however, recent genomic evidence suggests that multiple cnidarians have the ability to produce &#969;3 PUFA de novo and further biosynthesize &#969;3 long-chain PUFA26. Combining GC-MS with stable isotopic labeling (e.g., 13C-bicarbonate, NaH13CO3) can be used to track the fate of photosynthetically fixed carbon through coral holobiont metabolic networks under both control conditions and in response to external stressors27,28. However, a critical step in the tracking of 13C fate is the separation of the coral tissue from the algal cells-only then can the presence of a 13C-labeled compound in the coral host fraction be unequivocally assigned as a Symbiodiniaceae-derived metabolite translocated to the coral or a downstream product of a translocated labeled compound. This technique has demonstrated its power by challenging the long-held assumption that glycerol is the primary form in which photosynthate is translocated from symbiont to host29, as well as elucidating how inter-partner nutritional flux changes during bleaching27,28 and in response to incompatible Symbiodiniaceae species11.
While the decision to separate tissues is primarily driven by the research question, the practicality, reliability, and potential metabolic impacts of this approach are important to consider. Here, we provide detailed, demonstrated methods for the extraction of metabolites from the holobiont, as well as the separate host and symbiont fractions. We compare the metabolite profiles of the host and symbiont independently and how these profiles compare to the holobiont metabolite profile.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100% LC-grade methanol</td>
			<td>Merck</td>
			<td>439193</td>
			<td>LC grade essential</td>
		</tr>
		<tr>
			<td>2 mL microcentrifuge tubes, PP</td>
			<td>Eppendorf</td>
			<td>30121880</td>
			<td>Polypropylene provides high resistance to chemicals, mechanical stress and temperature extremes</td>
		</tr>
		<tr>
			<td>2030 Shimadzu gas chromatograph</td>
			<td>Shimadzu</td>
			<td>GC-2030</td>
			<td></td>
		</tr>
		<tr>
			<td>710-1180 &#181;m acid-washed glass beads</td>
			<td>Merck</td>
			<td> 
			G1152</td>
			<td>This size is optimal for breaking the Symbiodiniaceae cells</td>
		</tr>
		<tr>
			<td>AOC-6000 Plus Multifunctional autosampler</td>
			<td>Shimadzu</td>
			<td>AOC6000</td>
			<td></td>
		</tr>
		<tr>
			<td>Bradford reagent</td>
			<td>Merck</td>
			<td>B6916</td>
			<td>Any protein colourimetric reagent is acceptable</td>
		</tr>
		<tr>
			<td>Compressed air gun</td>
			<td>Ozito</td>
			<td>6270636</td>
			<td>Similar design acceptable. Having a fitting to fit a 1 mL tip over is critical.</td>
		</tr>
		<tr>
			<td>&#160;DB-5 column with 0.25 mm internal diameter column and 1 &#181;m film thickness</td>
			<td>Agilent</td>
			<td>122-5013</td>
			<td></td>
		</tr>
		<tr>
			<td>DMF</td>
			<td>Merck</td>
			<td>RTC000098</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Sorbitol-6-13C and/or 13C5&#8211;15N Valine</td>
			<td>Merck</td>
			<td>605514/ 600148</td>
			<td>Either or both internal standards can be added to the methanol.</td>
		</tr>
		<tr>
			<td>Flat bottom 96-well plate</td>
			<td>Merck</td>
			<td>CLS3614</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass scintillation vials</td>
			<td>Merck</td>
			<td>V7130</td>
			<td>20 mL, with non-plastic seal</td>
		</tr>
		<tr>
			<td>Immunoglogin G</td>
			<td>Merck</td>
			<td>56834</td>
			<td>if not availbe, Bovine Serum Albumin is acceptable</td>
		</tr>
		<tr>
			<td>Primer</td>
			<td></td>
			<td>v4</td>
			<td></td>
		</tr>
		<tr>
			<td>R</td>
			<td></td>
			<td>v4.1.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Shimadzu LabSolutions Insight software</td>
			<td></td>
			<td>v3.6</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Merck</td>
			<td>S5881</td>
			<td>Pellets to make 1 M solution</td>
		</tr>
		<tr>
			<td>tidyverse</td>
			<td></td>
			<td>v1.3.1</td>
			<td>R package</td>
		</tr>
		<tr>
			<td>TissueLyser LT</td>
			<td>Qiagen</td>
			<td>85600</td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>TQ8050NX triple quadrupole mass spectrometer</td>
			<td>Shimadzu</td>
			<td>GCMS-TQ8050 NX</td>
			<td></td>
		</tr>
		<tr>
			<td>UV-96 well plate</td>
			<td>Greiner</td>
			<td>M3812</td>
			<td></td>
		</tr>
		<tr>
			<td>Whirl-Pak sample bag</td>
			<td>Merck</td>
			<td>WPB01018WA</td>
			<td>Sample collection bag; Size: big enough to house a ~5 cm coral fragment, but not too big that the water is too spread</td>
		</tr>
	</tbody>
</table>

gas chromatography-mass spectrometry-based targeted metabolomics of hard coral samples

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.

Author Spotlight: Separation of Coral Host Tissues and Algal Symbionts and Analyzing Their Metabolites 

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

As with every organism, corals depend on superlative nutrition to grow and construct reefs for immunity and reproduction and to withstand changing environmental conditions. But we know very little about optimal nutrition for corals under normal or changing conditions, and how each member of the coral holobiont contributes to this nutritional status. Corals are holobionts comprised of the host and various symbiotic microorganisms.These include Symbiodiniaceae, protists, bacteria, viruses, and fungi. The fitness of the holobiont depends on the metabolic interactions between these members. For instance, Symbiodiniaceae provide photosynthate to support the host's metabolism and help it adapt to changing conditions.To understand this crucial partnership, we need to study each partner's chemical and metabolic composition separately. These findings demonstrated that more information could be obtained by separating host and symbiont fractions for metabolic analysis. However, the decision to analyze separate fractions versus the holobiont is ultimately governed by the research question.So in cases where analysis of the holobiont is preferable, we provided a methodology for holobiont metabolite extractions, and suggestions to obtain as much information as possible during analysis. The methods used in this study open up new avenues for research. These include revealing potential biomarkers, identifying key metabolites during specific life stages or stress conditions, understanding the advantages of associating with certain species, and exploring interactions within the holobiont.Future research will focus on applying the protocols established here to several research areas, including the detection of biomarkers with applications in coral reef conservation and restoration. This methodology can also be applied where the separation of fractions isn't possible for other physiological measures. For example, when analyzing volatile metabolite emissions from coral colonies.

All the data produced during this work are available in the supplementary information.
Host-symbiont separation
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65628/65628fig01.jpg" /> 
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...

Watch this Scientific Journal Video about Separation of Coral Host Tissues and Algal Symbionts and Analyzing Their Metabolites at JoVE.com

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 ...

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Research

JoVE Journal

Biology

Coral Metabolism Quenching and Coral Tissue Removal from Coral Skeleton

To begin, drain excess sea water from the sample collection bag containing coral fragments. Submerge the samples in liquid nitrogen for at least 30 seconds. To remove coral tissue from the skeleton, place a sterile sample collection bag on ice ensuring it is stable and open but not submerged in the ice.Add 10 milliliters of cold ultrapure water to the bag. Using sterile tweezers, select a coral fragment placed on ice and rinse it with cold ultrapure water until no seawater residue remains. Place the rinsed coral fragment in the bag containing the ultrapure water.Next, cut approximately five-millimeter end of the one milliliter pipette tip and tape it over the end of an air gun using electrical tape. Aim the air gun onto the coral fragment with the bag half sealed and the airflow on low medium to gently remove tissue using a circular water movement over the fragment. After three minutes, or when all tissue is removed from the skeleton, turn off the air and remove the airbrush.Seal the bag completely and squeeze all the removed coral tissue into the bottom corner of the bag. Cut off the opposite corner of the bag and gently pour the content into a 15-milliliter tube on ice.

Separation of Coral Host Tissues and Symbiodiniaceae Cells for Metabolite Extractions

Homogenize the coral sample with a mechanical sawtooth homogenizer for one minute until the sample is fully homogenized and no clumps are visible. For normalization, collect a one-milliliter aliquot from the homogenized tissue for Symbiodiniaceae cell counts, coral tissue protein content analysis, and chlorophyll-A estimation. If separating the host material and algal cells, centrifuge the remaining coral homogenate at 2, 500 G for five minutes at four degrees Celsius.Transfer the supernatant containing the host material to a new 15-milliliter tube. Add two milliliters of cold, ultrapure water to the algal pellet and vortex both host material and algal pellet for two minutes to resuspend. Centrifuge all samples again and transfer the supernatant containing the host material to a new 15-milliliter tube.After discarding the supernatant from the algal pellet, retain the pellet in the original 15-milliliter tube. Breeze either the holobiont homogenate or the separated host in Symbiodiniaceae fractions at minus 80 degrees Celsius for at least two hours. Then, lyophilize the samples overnight with a 0.01-millibar vacuum at minus 85 degrees Celsius.After drying, weigh each sample on a laboratory balance into separate two-milliliter, plasticizer-free microcentrifuge tubes. Microscopic visualization revealed no Symbiodiniaceae cells in host tissue samples after three wash steps. Similarly, minimal host tissue was found in symbiont fractions.However, the holobiont homogenate indicated that intracellular Symbiodiniaceae had not been released from their symbiosomes through simple airbrushing.

Preparation of Metabolites from a Coral Holobiont, Separated Coral Host Tissue, and Symbiodiniaceae Cells for Gas Chromatography-Mass Spectrometry Analysis

To extract intracellular metabolites from lyophilized holobiont add 400 microliters of 100%cold methanol with internal standards to the lyophilized holobiont. Then add 10 milligrams of acid washed glass beads and place the tube in a pre chilled bead mill. Insert at 50 hertz for three minutes.After lysis, add 600 microliters of cold methanol and vortex for one minute. Place the tube on a rotisserie shaker at four degrees celsius for 30 minutes. To extract metabolites from separated lyophilized symbiodiniacae cells, Add 200 microliters of cold methanol with internal standards to the dried symbiodiniacae cells.Then add acid washed glass beads and place the tube in a pre chilled bead mill insert at 50 hertz. After three minutes, add 800 microliters of methanol and vortex for 30 seconds. For metabolite extraction from separated lyophilized host tissue, add one milliliter of cold methanol containing internal standards to the dried host material and vortex for 20 seconds.Then place the tube in a floating tube holder for sonication at four degree Celsius for 30 minutes. Next for metabolite extract purification, centrifuge all samples at 3000G for 30 minutes at four degrees Celsius. Carefully transfer the supernatant into a new two milliliter micro centrifuge tube without disturbing the pellet.Re-suspend the pellet in one milliliter of 50%cold methanol and vortex vigorously for one minute. Centrifuge again, then collect and pool the polar extracts supernatant with the semi-polar extracts from the same sample. Centrifuge the pooled extracts at 16, 100G for 15 minutes to remove precipitates.For analysis, aliquot 50 microliters of each extract into a glass insert and concentrate using a vacuum concentrator for 30 minutes at 30 degrees celsius. GCMS analysis revealed 107 annotated metabolites across all the treatments, including a suite of amino acids, organic acids, carbohydrates, fatty acids, and sterile. K means clustering identified three distinct clusters of samples.The holobiont samples were intermediate between the separated host and symbiont fractions. Although K means cluster distributions, parallel coordinates, and heat map visualization in the metabolite relative abundance indicated that the holobiont profile more closely matched the host fraction profile significantly differed from both the host and symbiont profiles. The host and symbiont profiles were significantly distinct from each other with 100 individual metabolites, significantly different between the host and symbiont fractions.

1.3K Views.  University of Technology Sydney.  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.

Metabolite Profiling of Hard Coral Samples Using GC-MS

Summary