Name: Separasjon av korallvertsvev og symbiodiniaceae-celler for metabolittekstraksjoner
Uploaded: 2023-10-13T13:30:00
Description: Watch this Scientific Journal Video about Separation of Coral Host Tissues and Symbiodiniaceae Cells for Metabolite Extractions at JoVE.com

separation of coral host tissues and symbiodiniaceae cells for metabolite extractions

Metabolittprofilering av harde korallprøver ved bruk av 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.

Forfatter Spotlight: Separasjon av korallvertsvev og algesymbionter og analyse av deres metabolitter 

Gasskromatografi-massespektrometri-basert målrettet metabolomikk av harde korallprøver

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

Separasjon av korallvertsvev og symbiodiniaceae-celler for metabolittekstraksjoner

Homogeniser korallprøven med en mekanisk sagtannhomogenisator i ett minutt til prøven er fullstendig homogenisert og ingen klumper er synlige. For normalisering, samle en milliliter aliquot fra homogenisert vev for Symbiodiniaceae celletall, korallvev protein innhold analyse, og klorofyll-A estimering. Hvis du separerer vertsmaterialet og algecellene, sentrifugerer det gjenværende korallhomogenatet ved 2.500 G i fem minutter ved fire grader Celsius.Overfør supernatanten som inneholder vertsmaterialet til et nytt 15-milliliters rør. Tilsett to milliliter kaldt, ultrarent vann til algepelleten og virvelen både vertsmateriale og algepellet i to minutter for å resuspendere. Sentrifuger alle prøvene igjen og overfør supernatanten som inneholder vertsmaterialet til et nytt 15 milliliters rør.Etter å ha kastet supernatanten fra algepelleten, beholder du pelleten i det originale 15 milliliters røret. Bris enten holobiont homogenat eller separert vert i Symbiodiniaceae fraksjoner ved minus 80 grader Celsius i minst to timer. Deretter fryser du prøvene over natten med et vakuum på 0,01 millibar ved minus 85 grader Celsius.Etter tørking, vei hver prøve på en laboratoriebalanse i separate to-milliliter, myknerfrie mikrosentrifugerør. Mikroskopisk visualisering viste ingen Symbiodiniaceae-celler i vertsvevsprøver etter tre vasketrinn. Tilsvarende ble det funnet minimalt vertsvev i symbiontfraksjoner.Imidlertid indikerte holobionthomogenatet at intracellulær Symbiodiniaceae ikke hadde blitt frigjort fra deres symbiosomer gjennom enkel airbrushing.

Research

JoVE Journal

Biology

Separation of Coral Host Tissues and Symbiodiniaceae Cells for Metabolite Extractions

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 ...

Coral Metabolism Quenching and Coral Tissue Removal from Coral Skeleton

Korallmetabolisme-slukking og fjerning av korallvev fra korallskjelett

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