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Biology

基于气相色谱-质谱的硬珊瑚样品靶向代谢组学研究

Published: October 13, 2023
doi:
10.3791/65628
, , , ,
1Climate Change Cluster (C3),University of Technology Sydney, 2Metabolomics Australia, Bio21 Molecular Science and Biotechnology Institute,The University of Melbourne, 3School of Biological Sciences,Victoria University of Wellington

Summary

在这里，我们介绍了从珊瑚全生物中提取和制备极性和半极性代谢物，以及分离的珊瑚宿主组织和共生菌科细胞组分，用于气相色谱-质谱分析。

Abstract

基于气相色谱-质谱（GC-MS）的方法已被证明在阐明刺胞动物-甲藻共生的代谢基础以及珊瑚如何对压力做出反应（即在温度诱导的漂白过程中）方面非常有效。珊瑚全息的稳态代谢物分析，包括刺胞动物宿主及其相关微生物（共生菌科和其他原生生物、细菌、古细菌、真菌和病毒），已成功应用于环境和压力条件下，以表征珊瑚的整体代谢状态。

然而，要回答围绕共生相互作用的问题，有必要独立分析珊瑚宿主及其藻类共生体的代谢物谱，这只能通过对组织进行物理分离和分离，然后进行独立的提取和分析来实现。虽然代谢组学的应用在珊瑚领域相对较新，但研究小组的持续努力已经开发出分析珊瑚中代谢物的可靠方法，包括珊瑚宿主组织和藻类共生体的分离。

本文介绍了用于GC-MS分析的全生物分离和代谢物提取的分步指南，包括需要考虑的关键优化步骤。我们证明了，一旦独立分析，两个部分（珊瑚和共生菌科）的组合代谢物谱如何与整体（holobiont）的图谱相似，但通过分离组织，我们还可以获得有关两个伴侣之间的代谢和相互作用的关键信息，这些信息无法单独从整体中获得。

Introduction

代谢物代表细胞过程的最终产物，代谢组学 – 研究给定生物体或生态系统产生的一系列代谢物 – 可以提供生物体功能的直接测量1.这对于探索生态系统、共生相互作用和恢复工具尤为重要，因为大多数管理战略的目标是保护（或恢复）特定的生态系统服务功能2。珊瑚礁是一种水生生态系统，它证明了代谢组学在阐明共生相互作用以及将珊瑚生理反应与群落水平和生态系统水平影响联系起来方面的潜在价值3.高通量气相色谱-质谱联用 （GC-MS） 的应用尤其受到重视，因为它能够以高选择性和灵敏度同时快速分析多种代谢物类别，在光谱库可用时提供快速化合物鉴定，并提供高水平的重现性和准确性，每个样品的成本相对较低。

珊瑚是由珊瑚动物、光合甲藻内共生体（科：共生虫科4）和复杂的微生物组 5,6 组成的全生物体。总体而言，全息生物的适应性主要通过小分子和元素的交换来维持，以支持每个成员的代谢功能7,8,9,10。代谢组学方法已被证明在阐明共生特异性的代谢基础方面特别有效 9,11、对热应激的漂白反应7、8、12、13、疾病反应 14、污染暴露反应15、光适应 16 和珊瑚中的化学信号传导 17，以及帮助生物标志物发现 18，19.此外，代谢组学可以为从基于DNA和RNA的技术推断的结论提供有价值的确认9,20。因此，使用代谢组学来评估珊瑚礁健康和开发珊瑚礁保护工具具有相当大的潜力3，例如通过检测压力的代谢生物标志物18,19 和研究营养补贴等积极管理策略的潜力21.

分离宿主细胞和共生体细胞并独立分析其代谢物谱，而不是作为全生物一起分析，可以产生更多关于伴侣相互作用、独立生理和代谢状态以及潜在的适应分子机制的信息11,12,22,23,24.如果不分离珊瑚和共生鞭毛虫科，几乎不可能独立阐明珊瑚和/或共生菌科的贡献和代谢，除了复杂的基因组重建和代谢建模25，但这尚未应用于珊瑚-甲藻共生。此外，试图从全生物体的代谢物谱中提取有关宿主或藻类共生体个体代谢的信息可能会导致误解。

例如，直到最近，珊瑚和全生物组织的提取物中存在的 C18：3n-6、C18：4n-3 和 C16 多不饱和脂肪酸被认为是来自藻类共生体，因为珊瑚被认为不具有产生 omega-3 （ω3） 脂肪酸所必需的 ωx 去饱和酶;然而，最近的基因组证据表明，多个刺胞动物有能力从头产生ω3 PUFA，并进一步生物合成ω3长链PUFA26。将 GC-MS 与稳定同位素标记（例如，13C-碳酸氢盐、NaH13CO3）相结合，可用于在控制条件下和响应外部压力源的情况下通过珊瑚全生物代谢网络跟踪光合固定碳的命运27,28。然而，追踪 13C 命运的一个关键步骤是将珊瑚组织与藻类细胞分离——只有这样，珊瑚宿主部分中 13C 标记化合物的存在才能被明确地指定为共生菌科衍生的代谢物易位到珊瑚或易位标记化合物的下游产物。该技术通过挑战长期以来的假设，即甘油是光合作用从共生体转移到宿主的主要形式 29，以及阐明漂白过程中伴侣间营养通量如何变化27,28 和响应不相容的共生菌科物种11，证明了其力量。

虽然分离组织的决定主要由研究问题驱动，但这种方法的实用性、可靠性和潜在的代谢影响是需要考虑的重要因素。在这里，我们提供了详细的、演示的方法，用于从全生物中提取代谢物，以及单独的宿主和共生体部分。我们独立比较宿主和共生体的代谢物谱，以及这些谱与全息代谢物谱的比较。

Protocol

注：实验设计、样品收集和储存已在别处详细描述2,30,31。在收集和实验之前，必须获得收集野生珊瑚的许可批准。这里的样本是从巴达维亚珊瑚养殖场（华盛顿州杰拉尔顿）进口的Montipora mollis（绿色形态）群落中采集的，最初是从阿布罗洛斯群岛（西澳大利亚州;28°52’43.3“S 114°00’17.0”E）附近的一个珊瑚礁中采集的，?…

Representative Results

这项工作产生的所有数据均可在补充信息中找到。 宿主-共生体分离 图 1：珊瑚宿主组织和 Symbiodiniaceae 细胞分离的设置和验证。 （A） 用于从珊瑚骨架上去除珊瑚组织的气枪装置。用电工胶带将移?…

Discussion

宿主和共生体的分离可以通过简单的离心轻松快速地实现，这里的结果表明，分离馏分可以提供有价值的信息，表明特定的全生物成员的贡献，这有助于珊瑚健康的功能分析。在成年珊瑚中，脂质合成主要由常驻藻类共生体 40 进行，它提供脂质（例如，三酰基甘油和磷脂）41 和可以促进应激恢复的脂肪酸 11,42。</su…

Disclosures

The authors have nothing to disclose.

Acknowledgements

J.L.M. 得到了悉尼科技大学校长研究奖学金的支持。

Materials

100% LC-grade methanol Merck 439193 LC grade essential
2 mL microcentrifuge tubes, PP Eppendorf 30121880 Polypropylene provides high resistance to chemicals, mechanical stress and temperature extremes
2030 Shimadzu gas chromatograph Shimadzu GC-2030
710-1180 µm acid-washed glass beads Merck
G1152		 This size is optimal for breaking the Symbiodiniaceae cells
AOC-6000 Plus Multifunctional autosampler Shimadzu AOC6000
Bradford reagent Merck B6916 Any protein colourimetric reagent is acceptable
Compressed air gun Ozito 6270636 Similar design acceptable. Having a fitting to fit a 1 mL tip over is critical.
 DB-5 column with 0.25 mm internal diameter column and 1 µm film thickness Agilent 122-5013
DMF Merck RTC000098
D-Sorbitol-6-13C and/or 13C5–15N Valine Merck 605514/ 600148 Either or both internal standards can be added to the methanol.
Flat bottom 96-well plate Merck CLS3614
Glass scintillation vials Merck V7130 20 mL, with non-plastic seal
Immunoglogin G Merck 56834 if not availbe, Bovine Serum Albumin is acceptable
Primer v4
R v4.1.2
Shimadzu LabSolutions Insight software v3.6
Sodium Hydroxide Merck S5881 Pellets to make 1 M solution
tidyverse v1.3.1 R package
TissueLyser LT Qiagen 85600 Or similar
TQ8050NX triple quadrupole mass spectrometer Shimadzu GCMS-TQ8050 NX
UV-96 well plate Greiner M3812
Whirl-Pak sample bag Merck WPB01018WA Sample collection bag; Size: big enough to house a ~5 cm coral fragment, but not too big that the water is too spread
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References

  1. Bundy, J. G., Davey, M. P., Viant, M. R. Environmental metabolomics: A critical review and future perspectives. Metabolomics. 5 (1), 3-21 (2008).
  2. Matthews, J. L., Beale, D. J., Hillyer, K. E., Warden, A. C., Jones, O. A. H., et al. The metabolic significance of symbiont community composition in the coral-algal symbiosis. Applied Environmental Metabolomics. , 211-229 (2022).
  3. Lawson, C. A., van Oppen, M. J. H., Aranda Lastra, M., et al. Informing coral reef conservation through metabolomic approaches. Coral Reef Conservation and Restoration in the Omics Age. Coral Reefs of the World. , 179-202 (2022).
  4. LaJeunesse, T. C., et al. Systematic revision of Symbiodiniaceae highlights the antiquity and diversity of coral endosymbionts. Current Biology. 28 (16), 2570-2580 (2018).
  5. Rohwer, F., Seguritan, V., Azam, F., Knowlton, N. Diversity and distribution of coral-associated bacteria. Marine Ecology Progress Series. 243, 1-10 (2002).
  6. Maire, J., et al. Intracellular bacteria are common and taxonomically diverse in cultured and in hospite algal endosymbionts of coral reefs. The ISME Journal. 15 (7), 2028-2042 (2021).
  7. Hillyer, K. E., et al. Metabolite profiling of symbiont and host during thermal stress and bleaching in the coral Acropora aspera. Coral Reefs. 36, 105-118 (2016).
  8. Hillyer, K. E., Tumanov, S., Villas-Bôas, S., Davy, S. K. Metabolite profiling of symbiont and host during thermal stress and bleaching in a model cnidarian-dinoflagellate symbiosis. Journal of Experimental Biology. 219 (4), 516-527 (2016).
  9. Matthews, J. L., et al. Optimal nutrient exchange and immune responses operate in partner specificity in the cnidarian-dinoflagellate symbiosis. Proceedings of the National Academy of Sciences of the United States of America. 114 (50), 13194-13199 (2017).
  10. Rosset, S. L., et al. The molecular language of the cnidarian-dinoflagellate symbiosis. Trends in Microbiology. 29 (4), 320-333 (2020).
  11. Matthews, J. L., et al. Partner switching and metabolic flux in a model cnidarian-dinoflagellate symbiosis. Royal Society. 285 (1892), 20182336 (2018).
  12. González-Pech, R. A., et al. Physiological factors facilitating the persistence of Pocillopora aliciae and Plesiastrea versipora in temperate reefs of south-eastern Australia under ocean warming. Coral Reefs. 41, 1239-1253 (2022).
  13. Williams, A., et al. Metabolomic shifts associated with heat stress in coral holobionts. Science Advances. 7 (1), (2021).
  14. Deutsch, J. M., et al. Metabolomics of healthy and stony coral tissue loss disease affected Montastraea cavernosa corals. Frontiers in Marine Science. 8, 1421 (2021).
  15. Stien, D., et al. A unique approach to monitor stress in coral exposed to emerging pollutants. Scientific Reports. 10 (1), 9601 (2020).
  16. Lohr, K. E., et al. Resolving coral photoacclimation dynamics through coupled photophysiological and metabolomic profiling. Journal of Experimental Biology. 222 (8), (2019).
  17. Jorissen, H., et al. Coral larval settlement preferences linked to crustose coralline algae with distinct chemical and microbial signatures. Scientific Reports. 11 (1), 14610 (2021).
  18. Roach, T. N., Dilworth, J., Jones, A. D., Quinn, R. A., Drury, C. Metabolomic signatures of coral bleaching history. Nature Ecology & Evolution. 5 (4), 495-503 (2021).
  19. Parkinson, J. E., et al. Molecular tools for coral reef restoration: Beyond biomarker discovery. Conservation Letters. 13 (1), 12687 (2020).
  20. Jiang, J., et al. How Symbiodiniaceae meets the challenges of life during coral bleaching. Coral Reefs. 40, 1339-1353 (2021).
  21. Guerra, F. D., Attia, M. F., Whitehead, D. C., Alexis, F. Nanotechnology for environmental remediation: materials and applications. Molecules. 23 (7), 1760 (2018).
  22. Matthews, J. L., et al. Metabolite pools of the reef building coral Montipora capitata are unaffected by Symbiodiniaceae community composition. Coral Reefs. 39, 1727-1737 (2020).
  23. Papina, M., Meziane, T., van Woesik, R. Symbiotic zooxanthellae provide the host-coral Montipora digitata with polyunsaturated fatty acids. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology. 135 (3), 533-537 (2003).
  24. Kellogg, R., Patton, J. Lipid droplets, medium of energy exchange in the symbiotic anemone Condylactis gigantea: A model coral polyp. Marine Biology. 75, 137-149 (1983).
  25. Ankrah, N. Y., Chouaia, B., Douglas, A. E. The cost of metabolic interactions in symbioses between insects and bacteria with reduced genomes. mBio. 9 (5), e01433 (2018).
  26. Kabeya, N., et al. Genes for de novo biosynthesis of omega-3 polyunsaturated fatty acids are widespread in animals. Science Advances. 4 (5), (2018).
  27. Hillyer, K. E., Dias, D., Lutz, A., Roessner, U., Davy, S. K. 13C metabolomics reveals widespread change in carbon fate during coral bleaching. Metabolomics. 14 (1), 12 (2018).
  28. Hillyer, K. E., Dias, D. A., Lutz, A., Roessner, U., Davy, S. K. Mapping carbon fate during bleaching in a model cnidarian symbiosis: the application of 13C metabolomics. New Phytologist. 214 (4), 1551-1562 (2017).
  29. Burriesci, M. S., Raab, T. K., Pringle, J. R. Evidence that glucose is the major transferred metabolite in dinoflagellate-cnidarian symbiosis. Journal of Experimental Biology. 215 (19), 3467-3477 (2012).
  30. Thurber, R. V., et al. Unified methods in collecting, preserving, and archiving coral bleaching and restoration specimens to increase sample utility and interdisciplinary collaboration. PeerJ. 10, 14176 (2022).
  31. Grottoli, A. G., et al. Increasing comparability among coral bleaching experiments. Ecological Applications. 31 (4), 02262 (2020).
  32. Mushtaq, M. Y., Choi, Y. H., Verpoorte, R., Wilson, E. G. Extraction for metabolomics: access to the metabolome. Phytochemical Analysis. 25 (4), 291-306 (2014).
  33. Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry. 72 (1), 248-254 (1976).
  34. Peterson, G. L., et al. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Analytical Biochemistry. 83 (2), 346-356 (1977).
  35. Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry. 193 (1), 265-275 (1951).
  36. Zamer, W. E., Shick, J. M., Tapley, D. W. Protein measurement and energetic considerations: Comparisons of biochemical and stoichiometric methods using bovine serum albumin and protein isolated from sea anemones. Limnology and Oceanography. 34 (1), 256-263 (1989).
  37. Smart, K. F., Aggio, R. B., Van Houtte, J. R., Villas-Boas, S. G. Analytical platform for metabolome analysis of microbial cells using methyl chloroformate derivatization followed by gas chromatography-mass spectrometry. Nature Protocols. 5 (10), 1709-1729 (2010).
  38. Pang, Z., et al. Using MetaboAnalyst 5.0 for LC-HRMS spectra processing, multi-omics integration and covariate adjustment of global metabolomics data. Nature Protocols. 17 (8), 1735-1761 (2022).
  39. Tibshirani, R., Walther, G., Hastie, T. Estimating the number of clusters in a data set via the gap statistic. Journal of the Royal Statistical Society: Series B (Statistical Methodology). 63 (2), 411-423 (2001).
  40. Chen, W. -. N., et al. Diel rhythmicity of lipid-body formation in a coral-Symbiodinium endosymbiosis). Coral Reefs. 31 (2), 521-534 (2012).
  41. Imbs, A. Fatty acids and other lipids of corals: composition, distribution, and biosynthesis. Russian Journal of Marine Biology. 39 (3), 153-168 (2013).
  42. Rosset, S., et al. Lipidome analysis of Symbiodiniaceae reveals possible mechanisms of heat stress tolerance in reef coral symbionts. Coral Reefs. 38 (6), 1241-1253 (2019).
  43. Carreón-Palau, L., Parrish, C. C., Del Angel-Rodriguez, J. A., Perez-Espana, H. Seasonal shifts in fatty acids and sterols in sponges, corals, and bivalves, in a southern Gulf of Mexico coral reef under river influence. Coral Reefs. 40 (2), 571-593 (2021).
  44. Imbs, A. B., Dang, L. T. Seasonal dynamics of fatty acid biomarkers in the soft coral Sinularia flexibilis, a common species of Indo-Pacific coral reefs. Biochemical Systematics and Ecology. 96, 104246 (2021).
  45. Oku, H., Yamashiro, H., Onaga, K., Sakai, K., Iwasaki, H. Seasonal changes in the content and composition of lipids in the coral Goniastrea aspera. Coral Reefs. 22 (1), 83-85 (2003).
  46. Weis, V. M. Cell biology of coral symbiosis: foundational study can inform solutions to the coral reef crisis. Integrative and Comparative Biology. 59 (4), 845-855 (2019).
  47. Oakley, C., Davy, S., van Oppen, M., Lough, J. Cell biology of coral bleaching. Coral Bleaching. , 189-211 (2018).
  48. Lu, W., et al. Metabolite measurement: Pitfalls to avoid and practices to follow. Annual Review of Biochemistry. 86, 277-304 (2017).
  49. Lawson, C. A., et al. Heat stress decreases the diversity, abundance and functional potential of coral gas emissions. Global Change Biology. 27 (4), 879-891 (2021).
  50. Olander, A., et al. Comparative volatilomics of coral endosymbionts from one-and comprehensive two-dimensional gas chromatography approaches. Marine Biology. 168 (5), 76 (2021).
  51. Wuerz, M., et al. Symbiosis induces unique volatile profiles in the model cnidarian Aiptasia. Journal of Experimental Biology. 225 (19), (2022).

Tags

Keywords: Gas Chromatography-mass SpectrometryTargeted MetabolomicsHard Coral SamplesCoral HolobiontSymbiotic MicroorganismsSymbiodiniaceaeMetabolic InteractionsBiomarkersCoral Reef ConservationCoral RestorationVolatile Metabolite Emissions
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Matthews, J. L., Bartels, N., Elahee Doomun, S. N., Davy, S. K., De Souza, D. P. Gas Chromatography-Mass Spectrometry-Based Targeted Metabolomics of Hard Coral Samples. J. Vis. Exp. (200), e65628, doi:10.3791/65628 (2023).
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