Name: Präparation von Metaboliten aus einem Korallenholobionten, separiertem Korallenwirtsgewebe und Symbiodiniaceae-Zellen für die Gaschromatographie-Massenspektrometrie-Analyse
Uploaded: 2023-10-13T13:30:00
Description: Watch this Scientific Journal Video about Preparation of Metabolites from a Coral Holobiont, Separated Coral Host Tissue, and Symbiodiniaceae Cells for Gas Chromatography-Mass Spectrometry Analysis at

preparation of metabolites from a coral holobiont, separated coral host tissue, and symbiodiniaceae cells for gas chromatography-mass spectrometry analysis

Metaboliten-Profilierung von Steinkorallenproben mittels GC-MS

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.

Autor im Rampenlicht: Trennung von Korallenwirtsgeweben und Algensymbionten und Analyse ihrer Metaboliten 

Gaschromatographie-Massenspektrometrie-basierte gezielte Metabolomik von Hartkorallenproben

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.

Watch this Scientific Journal Video about Preparation of Metabolites from a Coral Holobiont, Separated Coral Host Tissue, and Symbiodiniaceae Cells for Gas Chromatography-Mass Spectrometry Analysis at

Präparation von Metaboliten aus einem Korallenholobionten, separiertem Korallenwirtsgewebe und Symbiodiniaceae-Zellen für die Gaschromatographie-Massenspektrometrie-Analyse

Um intrazelluläre Metaboliten aus lyophilisiertem Holobiont zu extrahieren, fügen Sie dem lyophilisierten Holobiont 400 Mikroliter 100% kaltes Methanol mit internen Standards hinzu. Fügen Sie dann 10 Milligramm säuregewaschene Glasperlen hinzu und legen Sie das Röhrchen in eine vorgekühlte Perlenmühle. Bei 50 Hertz für drei Minuten einführen.Nach der Lyse 600 Mikroliter kaltes Methanol und Wirbel für eine Minute hinzufügen. Legen Sie das Röhrchen 30 Minuten lang bei vier Grad Celsius auf einen Drehspießschüttler. Um Metaboliten aus getrennten lyophilisierten Symbiodiniacae-Zellen zu extrahieren, fügen Sie 200 Mikroliter kaltes Methanol mit internen Standards zu den getrockneten Symbiodiniacae-Zellen hinzu.Dann fügen Sie säuregewaschene Glasperlen hinzu und legen Sie das Rohr in einen vorgekühlten Perlmühleneinsatz mit 50 Hertz. Nach drei Minuten 800 Mikroliter Methanol zugeben und 30 Sekunden lang vortexen. Für die Metabolitenextraktion aus abgetrenntem lyophilisiertem Wirtsgewebe wird ein Milliliter kaltes Methanol mit internen Standards in das getrocknete Wirtsmaterial gegeben und 20 Sekunden lang vorgewirbelt.Legen Sie dann das Röhrchen in einen schwimmenden Röhrchenhalter für die Beschallung bei vier Grad Celsius für 30 Minuten. Als nächstes zentrifugieren Sie für die Reinigung des Metabolitenextrakts alle Proben bei 3000 G für 30 Minuten bei vier Grad Celsius. Den Überstand vorsichtig in ein neues Zwei-Milliliter-Mikrozentrifugenröhrchen überführen, ohne das Pellet zu stören.Suspendieren Sie das Pellet erneut in einem Milliliter 50%igem kaltem Methanol und wirbeln Sie es eine Minute lang kräftig vor. Erneut zentrifugieren, dann den Überstand der polaren Extrakte sammeln und mit den halbpolaren Extrakten aus derselben Probe zusammenführen. Zentrifugieren Sie die gepoolten Extrakte bei 16, 100 G für 15 Minuten, um Ausfällungen zu entfernen.Für die Analyse werden 50 Mikroliter jedes Extrakts in einen Glaseinsatz aliquotiert und mit einem Vakuumkonzentrator 30 Minuten lang bei 30 Grad Celsius konzentriert. Die GCMS-Analyse ergab 107 annotierte Metaboliten über alle Behandlungen hinweg, darunter eine Reihe von Aminosäuren, organischen Säuren, Kohlenhydraten, Fettsäuren und sterilen Metaboliten. K bedeutet, dass drei verschiedene Cluster von Proben identifiziert wurden.Die holobionten Proben waren ein Zwischenprodukt zwischen den getrennten Wirts- und Symbiontenfraktionen. Obwohl K Clusterverteilungen, parallele Koordinaten und die Visualisierung von Heatmaps in der Metaboliten-relativen Häufigkeit darauf hindeuteten, dass das Holobiontenprofil näher mit dem Wirtsfraktionsprofil übereinstimmte, unterschied sich signifikant sowohl vom Wirts- als auch vom Symbiontenprofil. Die Wirts- und Symbiontenprofile unterschieden sich signifikant voneinander mit 100 einzelnen Metaboliten, wobei sich die Wirts- und Symbiontenfraktionen signifikant unterschieden.

Research

JoVE Journal

Biology

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