coral metabolism quenching and coral tissue removal from coral skeleton

Perfil de metabolitos de muestras de corales duros utilizando 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 destacado: Separación de los tejidos del huésped de coral y los simbiontes de algas y análisis de sus metabolitos 

Cromatografía de gases-Espectrometría de masas basada en metabolómica dirigida de muestras de coral duro

Watch this Scientific Journal Video about Coral Metabolism Quenching and Coral Tissue Removal from Coral Skeleton at JoVE.com

Coral Metabolism Quenching and Coral Tissue Removal from Coral Skeleton  

Enfriamiento del metabolismo de los corales y eliminación de tejido de coral del esqueleto de coral

Para comenzar, drene el exceso de agua de mar de la bolsa de recolección de muestras que contiene fragmentos de coral. Sumerja las muestras en nitrógeno líquido durante al menos 30 segundos. Para eliminar el tejido de coral del esqueleto, coloque una bolsa de recolección de muestras estéril sobre hielo asegurándose de que esté estable y abierta, pero no sumergida en el hielo.Agregue 10 mililitros de agua ultrapura fría a la bolsa. Con unas pinzas estériles, seleccione un fragmento de coral colocado en hielo y enjuáguelo con agua fría ultrapura hasta que no quede ningún residuo de agua de mar. Coloque el fragmento de coral enjuagado en la bolsa que contiene el agua ultrapura.A continuación, corte aproximadamente cinco milímetros del extremo de la punta de pipeta de un mililitro y péguelo con cinta aislante sobre el extremo de una pistola de aire comprimido. Apunte la pistola de aire sobre el fragmento de coral con la bolsa medio sellada y el flujo de aire en medio bajo para eliminar suavemente el tejido con un movimiento circular de agua sobre el fragmento. Después de tres minutos, o cuando se haya retirado todo el tejido del esqueleto, apague el aire y retire el aerógrafo.Selle la bolsa completamente y apriete todo el tejido de coral eliminado en la esquina inferior de la bolsa. Corta la esquina opuesta de la bolsa y vierte suavemente el contenido en un tubo de 15 mililitros sobre hielo.

coral-metabolism-quenching-coral-tissue-removal-from-coral

Research

JoVE Journal

Biology

Coral Metabolism Quenching and Coral Tissue Removal from Coral Skeleton

137 vistas. Para comenzar, drene el exceso de agua de mar de la bolsa de recolección de muestras que contiene fragmentos de coral. Sumerja las muestras en nitrógeno líquido durante al menos 30 segundos. Para eliminar el tejido de coral del esqueleto, coloque una bolsa de recolección de muestras estéril sobre hielo asegurándose de que esté estable y abierta, pero no sumergida en el hielo.Agregue 10 mililitros de agua ultrapura fría a la bolsa. Con unas pinzas estériles, seleccione un fragm...

Video: Coral Metabolism Quenching and Coral Tissue Removal from Coral Skeleton  

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 cnidarian-dinoflagellate symbiosis and how coral responds to stress (i.e., during temperature-induced bleaching). Steady-state metabolite profiling of the coral holobiont, which comprises the cnidarian host and its associated microbes (Symbiodiniaceae and other protists, bacteria, archaea, fungi, and viruses), has been successfully applied under ambient and stress conditions to characterize the holistic metabolic status of the coral.
However, to answer questions surrounding the symbiotic interactions, it is necessary to analyze the metabolite profiles of the coral host and its algal symbionts independently, which can only be achieved by physical separation and isolation of the tissues, followed by independent extraction and analysis. While the application of metabolomics is relatively new to the coral field, the sustained efforts of research groups have resulted in the development of robust methods for analyzing metabolites in corals, including the separation of the coral host tissue and algal symbionts.
This paper presents a step-by-step guide for holobiont separation and the extraction of metabolites for GC-MS analysis, including key optimization steps for consideration. We demonstrate how, once analyzed independently, the combined metabolite profile of the two fractions (coral and Symbiodiniaceae) is similar to the profile of the whole (holobiont), but by separating the tissues, we can also obtain key information about the metabolism of and interactions between the two partners that cannot be obtained from the whole alone.

Here, we present the extraction and preparation of polar and semi-polar metabolites from a coral holobiont, as well as separated coral host tissue and Symbiodiniaceae cell fractions, for gas chromatography-mass spectrometry analysis.

Gas chromatography-mass spectrometry (GC-MS)-based approaches have proven to be powerful for elucidating the metabolic basis of the ...

Investigación

Biología

Separation of Coral Host Tissues and Symbiodiniaceae Cells for Metabolite Extractions

Separación de tejidos hospederos coralinos y células simbiodiáceas para extracciones de metabolitos

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

Preparación de metabolitos a partir de un holobionte de coral, tejido huésped de coral separado y células de simbiodiáceas para el análisis de cromatografía de gases-espectrometría de masas