separation of coral host tissues and symbiodiniaceae cells for metabolite extractions

Metabolite Profiling of Hard Coral Samples Using 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.

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.

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

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.

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

Research

JoVE Journal

Biology

133 visualizações.  University of Technology Sydney. Homogeneizar a amostra de coral com um homogeneizador mecânico de dente de serra durante um minuto até que a amostra esteja totalmente homogeneizada e não sejam visíveis aglomerações. Para normalização, coletar uma alíquota de um mililitro do tecido homogeneizado para contagem de células de Symbiodiniaceae, análise do conteúdo de proteína do tecido coral e estimativa de clorofila-A. Se separar o material hospedeiro e as células de algas, centrifugar o homogeneizado de coral rest...