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 photosynthates to support the host 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 symbion 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.

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 sea water 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. Homogenize the coral sample with a mechanical saw tooth 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 re-suspend. 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.

Freeze 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 drawing, weigh each sample on a laboratory balance into separate two milliliter plasticizer free micro centrifuge 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 hollow homogenate indicated that intracellular Symbiodiniaceae had not been released from their symbiosomes through simple airbrushing.

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 Symbiodiniaceae cells, add 200 microliters of cold methanol with internal standards to the dried Symbiodiniaceae 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 degrees Celsius for 30 minutes.

Next, for metabolite extract purification, centrifuge all samples at 3000 G 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, 100 G 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 sterols. 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.