Name: Coral Metabolism Quenching and Coral Tissue Removal from Coral Skeleton
Uploaded: 2023-10-13T13:30:00
Description: 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

GC-MS를 사용한 경산호 샘플의 대사산물 프로파일링

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.

저자 스포트라이트: 산호 숙주 조직과 조류 공생체의 분리 및 이들의 대사 산물 분석 

가스 크로마토그래피 - 경산호 시료의 질량분석법 기반 표적 대사체학

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 Coral Metabolism Quenching and Coral Tissue Removal from Coral Skeleton at JoVE.com

Coral Metabolism Quenching and Coral Tissue Removal from Coral Skeleton  

산호 신진대사 담금질 및 산호 골격에서 산호 조직 제거

시작하려면 산호 조각이 들어 있는 샘플 수집 백에서 과도한 바닷물을 배출합니다. 샘플을 액체 질소에 최소 30초 동안 담그십시오. 골격에서 산호 조직을 제거하려면 멸균 샘플 수집 백을 얼음 위에 올려 놓고 안정적이고 열려 있지만 얼음에 잠기지 않도록 합니다.가방에 차가운 초순수 10ml를 넣습니다. 멸균 핀셋을 사용하여 얼음 위에 놓인 산호 조각을 선택하고 바닷물 잔여물이 남지 않을 때까지 차가운 초순수로 헹굽니다. 헹군 산호 조각을 초순수가 담긴 가방에 넣습니다.다음으로, 1밀리리터 피펫 팁의 약 5밀리미터 끝을 자르고 전기 테이프를 사용하여 에어건 끝에 테이프로 붙입니다. 가방을 반쯤 밀봉하고 공기 흐름을 낮은 매체로 하여 산호 조각에 에어건을 조준하여 조각 위로 원을 그리며 물을 움직여 조직을 부드럽게 제거합니다. 3분 후 또는 골격에서 모든 조직이 제거되면 공기를 끄고 에어브러시를 제거합니다.가방을 완전히 밀봉하고 제거된 모든 산호 조직을 가방의 하단 모서리에 짜냅니다. 가방의 반대쪽 모서리를 잘라내고 얼음 위의 15밀리리터 튜브에 내용물을 부드럽게 붓습니다.

Research

JoVE Journal

Biology

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 ...

연구

생물학

Separation of Coral Host Tissues and Symbiodiniaceae Cells for Metabolite Extractions

대사산물 추출을 위한 산호 숙주 조직과 Symbiodiniaceae 세포의 분리

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