Labelling kelps with 13C and 15N for isotope tracing or enrichment experiments
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Anton Kuech
,
Ursula Witte
and
Inka Bartsch
Abstract
Isotope tracing experiments can be used to trace organic material flow through the ecosystem by artificially adding labelled biomass into a system. The advantage of this process is the direct control of carbon and nitrogen addition to the system for measuring uptake rates by consumers, which can substantially reduce the uncertainties associated with food web models. This article details and discusses the steps involved in successfully culturing and isotopically enriching (13C and 15N) juvenile sporophytes of two common North Atlantic kelp species (Laminariales): Saccharina latissima and Laminaria digitata. A first successful isotopic enrichment study of S. latissima, as well as the first inclusion of 15N enrichment for L. digitata, are detailed. This protocol provides a comprehensive description of the stable isotope enrichment process in two kelp species, potentially serving as a foundation for its application in other macroalgal taxa.
Funding source: Natural Environment Research Council
Award Identifier / Grant number: NE/S007377/1
Acknowledgments
We gratefully acknowledge the technical support by Andreas Wagner (Alfred Wegener Institute) for the duration of the experiment.
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Research ethics: The Norwegian regulation on the access and benefit-sharing of genetic resources has not yet entered into force. We complied with our due diligence by sending an inquiry to the national authorities. In 2020, they replied that no permits are required and this statement was officially confirmed by the Norwegian National Focal Point in late 2024.
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Informed consent: Not applicable.
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Author contributions: IB and UW conceptualised the enrichment studies. AK, IB, and UW designed the final experiment and study. AK conducted the laboratory work, collected and analysed the data, and was supervised by UW and IB. IB provided laboratory facilities and technical and scientific supervision for the main experimental work. AK drafted the manuscript, while IB and UW critically revised it. All authors have accepted responsibility for the entire content of this manuscript and approved its submission.
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Use of Large Language Models, AI and Machine Learning Tools: None declared.
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Conflict of interest: The authors state no conflict of interest.
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Research funding: This work was supported by the Natural Environment Research Council [grant number NE/S007377/1].
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Data availability: All relevant data are within the paper and its Supporting Material files. Data used for growth rate calculations are stored in Table S2 and Table S3 for Saccharina latissima and Laminaria digitata, respectively. Labelling uptake results are provided in Table S4.
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Associated content: The protocol described in this peer-reviewed article is published on protocols.io https://doi.org/10.17504/protocols.io.8epv597rdg1b/v4. Associated labelling medium solution protocols have been uploaded as attachments to the main protocol and to protocols.io https://doi.org/10.17504/protocols.io.14egn77emv5d/v1 and https://doi.org/10.17504/protocols.io.rm7vzyyo5lx1/v1.
References
Ahn, O., Petrell, R.J., and Harrison, P.J. (1998). Ammonium and nitrate uptake by Laminaria saccharina and Nereocystis luetkeana originating from a salmon sea cage farm. J. Appl. Phycol. 10: 333–340.10.1023/A:1008092521651Search in Google Scholar
Andersson, J.H., Woulds, C., Schwartz, M., Cowie, G.L., Levin, L.A., Soetaert, K., and Middelburg, J.J. (2008). Short-term fate of phytodetritus in sediments across the Arabian sea oxygen minimum zone. Biogeosciences 5: 43–53.10.5194/bg-5-43-2008Search in Google Scholar
Axelsson, L., Mercado, J., and Figueroa, F. (2000). Utilization of HCO3 − at high pH by the brown macroalga Laminaria saccharina. Eur. J. Phycol. 35: 53–59.10.1017/S096702629900253XSearch in Google Scholar
Bailes, I.R. and Gröcke, D.R. (2020). Isotopically labelled macroalgae: a new method for determining sources of excess nitrogen pollution. Rapid Commun. Mass Spectrom. 34: e8951.10.1002/rcm.8951Search in Google Scholar PubMed
Bartsch, I. (2018). Derivation of clonal stock cultures and hybridization of kelps: a tool for strain preservation and breeding programs. Protoc. Macroalgae Res.: 61–78.10.1201/b21460-3Search in Google Scholar
Braeckman, U., Pasotti, F., Vázquez, S., Zacher, K., Hoffmann, R., Elvert, M., Marchant, H., Buckner, C., Quartino, M.L., Mác Cormack, W., et al.. (2019). Degradation of macroalgal detritus in shallow coastal Antarctic sediments. Limnol. Oceanogr 64: 1423–1441.10.1002/lno.11125Search in Google Scholar PubMed PubMed Central
Braga, C. and Yoneshigue-Valentin, Y. (1996). Nitrogen and phosphorus uptake by the Brazilian kelp Laminaria abyssalis (Phaeophyta) in culture. In: Fifteenth international seaweed symposium 1996, Valdivia, Chile. Kluwer, Dordrecht, pp. 445–450.10.1007/978-94-009-1659-3_64Search in Google Scholar
Fernández, P.A., Hurd, C.L., and Roleda, M.Y. (2014). Bicarbonate uptake via an anion exchange protein is the main mechanism of inorganic carbon acquisition by the giant kelp Macrocystis pyrifera (Laminariales, Phaeophyceae) under variable pH. J. Phycol. 50: 998–1008.10.1111/jpy.12247Search in Google Scholar PubMed
Glibert, P.M., Middelburg, J.J., McClelland, J.W., and Jake Vander Zanden, M. (2019). Stable isotope tracers: enriching our perspectives and questions on sources, fates, rates, and pathways of major elements in aquatic systems. Limnol. Oceanogr. 64: 950–981.10.1002/lno.11087Search in Google Scholar
Guiry, M.D. and Cunningham, E.M. (1984). Photoperiodic and temperature responses in the reproduction of north-eastern Atlantic Gigartina acicularis (Rhodophyta: Gigartinales). Phycologia 23: 357–367.10.2216/i0031-8884-23-3-357.1Search in Google Scholar
Haines, K.C. and Wheeler, P.A. (1978). Ammoniun and nitrate uptake by the marine macrophytes Hypnea musvuformis (Rhodophyta) and Macrocystis pyrifera (Phaeophyta) 1, 2. J. Phycol. 14: 319–324.10.1111/j.1529-8817.1978.tb00305.xSearch in Google Scholar
Hardison, A.K., Canuel, E.A., Anderson, I.C., and Veuger, B. (2010). Fate of macroalgae in benthic systems: carbon and nitrogen cycling within the microbial community. Mar. Ecol. Prog. Ser. 414: 41–55.10.3354/meps08720Search in Google Scholar
Hellebust, J.A. and Haug, A. (1972). Photosynthesis, translocation, and alginic acid synthesis in Laminaria digitata and Laminaria hyperborea. Can. J. Bot. 50: 169–176.10.1139/b72-022Search in Google Scholar
Herman, P.M.J., Middelburg, J.J., Widdows, J., Lucas, C.H., and Heip, C.H.R. (2000). Stable isotopes’ as trophic tracers: combining field sampling and manipulative labelling of food resources for macrobenthos. Mar. Ecol. Prog. Ser. 204: 79–92.10.3354/meps204079Search in Google Scholar
Hunter, W., Levin, L., Kitazato, H., and Witte, U. (2012). Macrobenthic assemblage structure and organismal stoichiometry control faunal processing of particulate organic carbon and nitrogen in oxygen minimum zone sediments. Biogeosciences 9: 993–1006.10.5194/bg-9-993-2012Search in Google Scholar
Kamp, A. and Witte, U. (2005). Processing of 13C-labelled phytoplankton in a fine-grained sandy-shelf sediment (North Sea): relative importance of different macrofauna species. Mar. Ecol. Prog. Ser. 297: 61–70.10.3354/meps297061Search in Google Scholar
Karlson, A.M.L., Pilditch, C.A., Probert, P.K., Leduc, D., and Savage, C. (2021). Large infaunal bivalves determine community uptake of macroalgal detritus and food web pathways. Ecosystems 24: 384–402.10.1007/s10021-020-00524-5Search in Google Scholar
Kelly, L.J. and Martínez del Rio, C. (2010). The fate of carbon in growing fish: an experimental study of isotopic routing. Physiol. Biochem. Zool. 83: 473–480.10.1086/649628Search in Google Scholar PubMed
Krause-Jensen, D., Lavery, P., Serrano, O., Marba, N., Masque, P. and Duarte, C.M. (2018). Sequestration of macroalgal carbon: the elephant in the blue carbon room. Biol. Lett. 14.10.1098/rsbl.2018.0236Search in Google Scholar PubMed PubMed Central
Lüning, K., Schmitz, K., and Willenbrink, J. (1973). CO2 fixation and translocation in benthic marine algae. III. Rates and ecological significance of translocation in Laminaria hyperborea and L. saccharina. Marine Biol. 23: 275–281.10.1007/BF00389334Search in Google Scholar
Macreadie, P.I., Anton, A., Raven, J.A., Beaumont, N., Connolly, R.M., Friess, D.A., Kelleway, J.J., Kennedy, H., Kuwae, T., Lavery, P.S., et al.. (2019). The future of Blue Carbon science. Nat. Commun. 10: 3998.10.1038/s41467-019-11693-wSearch in Google Scholar PubMed PubMed Central
Mäkelä, A. (2017). Diversity and functioning of Arctic benthic ecosystems and their resilience to climate change driven alterations in food supply. University of Aberdeen, Aberdeen.Search in Google Scholar
Mäkelä, A., Witte, U., and Archambault, P. (2017). Ice algae versus phytoplankton: resource utilization by Arctic deep sea macroinfauna revealed through isotope labelling experiments. Mar. Ecol. Prog. Ser. 572: 1–18.10.3354/meps12157Search in Google Scholar
Mäkelä, A., Witte, U., and Archambault, P. (2018). Short-term processing of ice algal- and phytoplankton-derived carbon by Arctic benthic communities revealed through isotope labelling experiments. Mar. Ecol. Prog. Ser. 600: 21–39.10.3354/meps12663Search in Google Scholar
Martínez del Rio, C., Wolf, N., Carleton, S.A., and Gannes, L.Z. (2009). Isotopic ecology ten years after a call for more laboratory experiments. Biol. Rev. 84: 91–111.10.1111/j.1469-185X.2008.00064.xSearch in Google Scholar PubMed
McMeans, B., Rooney, N., Arts, M., and Fisk, A. (2013). Food web structure of a coastal Arctic marine ecosystem and implications for stability. Mar. Ecol. Prog. Ser. 482: 17–28.10.3354/meps10278Search in Google Scholar
Middelburg, J.J. (2014). Stable isotopes dissect aquatic food webs from the top to the bottom. Biogeosciences 11: 2357–2371.10.5194/bg-11-2357-2014Search in Google Scholar
Middelburg, J.J., Barranguet, C., Boschker, H.T.S., Herman, P.M.J., Moens, T., and Heip, C.H.R. (2000). The fate of intertidal microphytobenthos carbon: an in situ C-13-labeling study. Limnol. Oceanogr. 45: 1224–1234.10.4319/lo.2000.45.6.1224Search in Google Scholar
Moodley, L., Middelburg, J.J., Soetaert, K., Boschker, H.T.S., Herman, P.M.J., and Heip, C.H.R. (2005). Similar rapid response to phytodetritus deposition in shallow and deep-sea sediments. J. Mar. Res. 63: 457–469.10.1357/0022240053693662Search in Google Scholar
Murie, K.A. and Bourdeau, P.E. (2020). Fragmented kelp forest canopies retain their ability to alter local seawater chemistry. Sci. Rep. 10.10.1038/s41598-020-68841-2Search in Google Scholar PubMed PubMed Central
Oakes, J.M., Bautista, M.D., Maher, D., Jones, W.B., and Eyre, B.D. (2011). Carbon self‐utilization may assist Caulerpa taxifolia invasion. Limnol. Oceanogr. 56: 1824–1831.10.4319/lo.2011.56.5.1824Search in Google Scholar
Parker, B.C. (1965). Translocation in the giant kelp Macrocystis I. Rates, direction, quantity of C14-labeled products and fluorescein. J. Phycol. 1: 41–46.10.1111/j.1529-8817.1965.tb04554.xSearch in Google Scholar
Parnell, A.C., Inger, R., Bearhop, S., and Jackson, A.L. (2010). Source partitioning using stable isotopes: coping with too much variation. PloS One 5: e9672.10.1371/journal.pone.0009672Search in Google Scholar PubMed PubMed Central
Peterson, B.J. (1999). Stable isotopes as tracers of organic matter input and transfer in benthic food webs: a review. Acta Oecol. 20: 479–487.10.1016/S1146-609X(99)00120-4Search in Google Scholar
Post, D.M. (2002). Using stable isotopes to estimate trophic position: models, methods, and assumptions. Ecology 83: 703–718.10.1890/0012-9658(2002)083[0703:USITET]2.0.CO;2Search in Google Scholar
Provasoli, L. (1968). Media and prospects for the cultivation of marine algae. Cult. Collect. Algae: 63–75.Search in Google Scholar
Queirós, A.M., Stephens, N., Widdicombe, S., Tait, K., McCoy, S.J., Ingels, J., Rühl, S., Airs, R., Beesley, A., Carnovale, G., et al.. (2019). Connected macroalgal‐sediment systems: blue carbon and food webs in the deep coastal ocean. Ecological Monographs 89: e01366.10.1002/ecm.1366Search in Google Scholar
Ramirez-Llodra, E., Pedersen, T., Dexter, K.F., Hauquier, F., Guilini, K., Mikkelsen, N., Borgersen, G., Van Gyseghem, M., Vanreusel, A., and Vilas, D. (2021). Community structure of deep fjord and shelf benthic fauna receiving different detrital kelp inputs in northern Norway. Deep-Sea Res. Part I: Oceanogr. Res. Pap. 168: 103433.10.1016/j.dsr.2020.103433Search in Google Scholar
Ravaglioli, C., Bulleri, F., Rühl, S., McCoy, S.J., Findlay, H.S., Widdicombe, S., and Queirós, A.M. (2019). Ocean acidification and hypoxia alter organic carbon fluxes in marine soft sediments. Global Change Biol. 25: 4165–4178.10.1111/gcb.14806Search in Google Scholar
Rees, A.P., Woodward, E.M.S., and Joint, I. (2006). Concentrations and uptake of nitrate and ammonium in the Atlantic Ocean between 60∘ N and 50∘ S. Deep Sea Res. Part II: Top. Stud. Oceanogr. 53: 1649–1665.10.1016/j.dsr2.2006.05.008Search in Google Scholar
Renaud, P.E., Lã¸Kken, T.S., Jã¸Rgensen, L.L., Berge, J.R., and Johnson, B.J. (2015). Macroalgal detritus and food-web subsidies along an Arctic fjord depth-gradient. Front. Mar. Sci. 2.10.3389/fmars.2015.00031Search in Google Scholar
Sun, M.-Y., Carroll, M.L., Ambrose, W.G., Clough, L.M., Zou, L., and Lopez, G.R. (2007). Rapid consumption of phytoplankton and ice algae by Arctic soft-sediment benthic communities: evidence using natural and 13C-labeled food materials. J. Mar. Res. 65: 561–588.10.1357/002224007782689094Search in Google Scholar
Thomas, F., Le Duff, N., Leroux, C., Dartevelle, L., and Riera, P. (2020). Isotopic labeling of cultured macroalgae and isolation of 13C-labeled cell wall polysaccharides for trophic investigations. Adv. Bot. Res. 95: 1–17.10.1016/bs.abr.2019.11.005Search in Google Scholar
Thomas, F., Le Duff, N., Wu, T.-D., Cébron, A., Uroz, S., Riera, P., Leroux, C., Tanguy, G., Legeay, E., and Guerquin-Kern, J.-L. (2021). Isotopic tracing reveals single-cell assimilation of a macroalgal polysaccharide by a few marine Flavobacteria and Gammaproteobacteria. ISME J.: 1–14.10.1038/s41396-021-00987-xSearch in Google Scholar PubMed PubMed Central
Tyler, A.C. and McGlathery, K.J. (2006). Uptake and release of nitrogen by the macroalgae Gracilaria vermiculophylla (Rhodophyta). J. Phycol. 42: 515–525.10.1111/j.1529-8817.2006.00224.xSearch in Google Scholar
Van Engeland, T., Bouma, T., Morris, E., Brun, F., Peralta, G., Lara, M., Hendriks, I., Van Rijswijk, P., Veuger, B., Soetaert, K., et al.. (2013). Dissolved organic matter uptake in a temperate seagrass ecosystem. Mar. Ecol. Prog. Ser. 478: 87–100.10.3354/meps10183Search in Google Scholar
Voigt, C.C., Rex, K., Michener, R.H., and Speakman, J.R. (2008). Nutrient routing in omnivorous animals tracked by stable carbon isotopes in tissue and exhaled breath. Oecologia 157: 31–40.10.1007/s00442-008-1057-3Search in Google Scholar PubMed
Wada, E., Mizutani, H., and Minagawa, M. (1991). The use of stable isotopes for food web analysis. Crit. Rev. Food Sci. Nutr. 30: 361–371.10.1080/10408399109527547Search in Google Scholar PubMed
Yorke, C.E., Page, H.M., and Miller, R.J. (2019). Sea urchins mediate the availability of kelp detritus to benthic consumers. Proc. R. Soc. B: Biol. Sci. 286: 20190846.10.1098/rspb.2019.0846Search in Google Scholar PubMed PubMed Central
Supplementary Material
This article contains supplementary material (https://doi.org/10.1515/bot-2025-0029).
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