separation of coral host tissues and symbiodiniaceae cells for metabolite extractions

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 Separation of Coral Host Tissues and Symbiodiniaceae Cells for Metabolite Extractions at JoVE.com

Trennung von Korallenwirtsgewebe und Symbiodiniaceae-Zellen für Metabolitenextraktionen

Homogenisieren Sie die Korallenprobe mit einem mechanischen Sägezahnhomogenisator für eine Minute, bis die Probe vollständig homogenisiert ist und keine Klumpen mehr sichtbar sind. Für die Normalisierung wird ein Ein-Milliliter-Aliquot aus dem homogenisierten Gewebe für die Symbiodiniaceae-Zellzahl, die Analyse des Proteingehalts des Korallengewebes und die Chlorophyll-A-Schätzung entnommen. Bei der Trennung von Wirtsmaterial und Algenzellen zentrifugiert man das verbleibende Korallenhomogenat bei 2.500 G fünf Minuten lang bei vier Grad Celsius.Den Überstand, der das Wirtsmaterial enthält, in ein neues 15-Milliliter-Röhrchen überführen. Geben Sie zwei Milliliter kaltes, hochreines Wasser in das Algenpellet und wirbeln Sie sowohl das Wirtsmaterial als auch das Algenpellet zwei Minuten lang vor, um es zu resuspendieren. Zentrifugieren Sie alle Proben erneut und überführen Sie den Überstand, der das Wirtsmaterial enthält, in ein neues 15-Milliliter-Röhrchen.Nachdem Sie den Überstand aus dem Algenpellet entfernt haben, bewahren Sie das Pellet im ursprünglichen 15-Milliliter-Röhrchen auf. Brise entweder das Holobion-Homogenat oder den abgetrennten Wirt in Symbiodiniaceae-Fraktionen bei minus 80 Grad Celsius für mindestens zwei Stunden. Anschließend werden die Proben über Nacht mit einem 0,01-Millibar-Vakuum bei minus 85 Grad Celsius lyophilisiert.Nach dem Trocknen wird jede Probe auf einer Laborwaage in separate, weichmacherfreie Zwei-Milliliter-Mikrozentrifugenröhrchen gewogen. Die mikroskopische Visualisierung zeigte nach drei Waschschritten keine Symbiodiniaceae-Zellen in Wirtsgewebeproben. In ähnlicher Weise wurde minimales Wirtsgewebe in Symbiontenfraktionen gefunden.Das Holobiont-Homogenat deutete jedoch darauf hin, dass intrazelluläre Symbiodiniaceae nicht durch einfaches Airbrushen aus ihren Symbiosomen befreit worden waren.

separation-coral-host-tissues-symbiodiniaceae-cells-for-metabolite

Research

JoVE Journal

Biology

Separation of Coral Host Tissues and Symbiodiniaceae Cells for Metabolite Extractions

134 Views.  University of Technology Sydney. 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 supe...