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Flow Cytometry as a Method to Study Marine Unicellular Algae: Development, Problems, and Prospects

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Abstract

For more than the past three decades, flow cytometry has been successfully applied in various, including phytoplankton, studies that require rapid qualitative assessment of the physiological condition, chemical composition, and number of microalgal cells. The potential of this method for solving applied and fundamental problems in microalgae research, the parameters of main fluorescent dyes used for this method, the features of microalgae as study objects, as well as the challenges associated with the use of the method, are discussed in the present review.

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REFERENCES

  1. Zueva, E.E., Kurtova, A.V., Rusanova, E.B., et al., Diagnostika onkogematologicheskikh zabolevanii s pomoshch’yu protochnoi tsitometrii (Diagnostics of Oncological and Hematological Disorders Using Flow Cytometry), Emmanuel’, V.L., Ed., St. Petersburg: SpetsLit, 2017.

  2. Zurochka, A.V., Khaidukov, S.V., Kudryavtsev, I.V., and Chereshnev, V.A., Protochnaya tsitometriya v meditsine i biologii (Flow Cytometry in Medicine and Biology), 2nd ed., Yekaterinburg: Redaktsionno-Izdatel’sky Otdel, Ural. Otd., Ross. Akad. Nauk, 2014.

  3. Knyazev, N.A., Pechkovskaya, S.A., Skarlato, S.O., et al., The impact of temperature stress on DNA and RNA synthesis in potentially toxic dinoflagellates Prorocentrum minimum, J. Evol. Biochem. Physiol., 2018, vol. 54, no. 5, pp. 383–389.

    Article  CAS  Google Scholar 

  4. Markina, Zh.V. and Aizdaicher, N.A., Copper influence on different taxonomic groups marine microalgae cultures estimation with flow cytometry, Voda: Khim. Ekol., 2018, nos. 10–12, pp. 43–50.

  5. Solomonova, E.S., Dynamics of physiologically active cells of pico- and nanophytoplankton in the coastal waters of Black Sea, Vestn. S.-Peterb. Univ., Ser. 3: Biol., 2016, no. 1, pp. 62–72.

  6. Solomonova, E.S. and Akimov, A.I., The assessment of functional status of Chlorella vulgaris suboblonga by flow cytometry and variable fluorescence, Morsk. Ekol. Zh., 2012, vol. 11, no. 4, pp. 78–84.

    Google Scholar 

  7. Solomonova, E.S. and Akimov, A.I., Relation of live and dead components of suspension in some microalgae’ cultures in dependence on growth stage and different illumination, Morsk. Ekol. Zh., 2014, vol. 13, no. 1, pp. 73–81.

    Google Scholar 

  8. Alemán-Nava, G.S., Cuellar-Bermudez, S.P., Cuaresma, M., et al., How to use Nile Red, a selective fluorescent stain for microalgal neutral lipids, J. Microbiol. Methods, 2016, pp. 128, pp. 74–79.

    Article  PubMed  CAS  Google Scholar 

  9. Balduyck, L., Veryser, C., Goiris, K., et al., Optimization of a Nile Red method for rapid lipid determination in autotrophic, marine microalgae is species dependent, J. Microbiol. Methods, 2015, vol. 128, pp. 152–158.

    Article  CAS  Google Scholar 

  10. Børsheim, K.Y., Harboe, T., Jonsen, T., et al., Flow cytometric characterization and enumeration of Chrysochromulina polylepis during a bloom along the Norwegian coast, Mar. Ecol.: Prog. Ser., 1989, vol. 54, pp. 307–309.

    Article  Google Scholar 

  11. Carrier, G., Baroukh, C., Rouxel, C., et al., Draft genomes and phenotypic characterization of Tisochrysis lutea strains. Toward the production of domesticated strains with high added value, Algal Res., 2018, vol. 29, pp. 1–11.

    Article  Google Scholar 

  12. Chew, K.W., Yap, J.Y., Show, P.L., et al., Microalgae biorefinery: High value products perspectives, Bioresour. Technol., 2017, vol. 229, pp. 53–62.

    Article  CAS  PubMed  Google Scholar 

  13. Chioccioli, M., Hankamer, B., and Ross, I.L., Flow cytometry pulse width data enables rapid and sensitive estimation of biomass dry weight in the microalgae Chlamydomonas reinhardtii and Chlorella vulgaris, PLoS One, 2014, vol. 9, art. ID e97269. https://doi.org/10.1371/journal.pone.0097269

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Cid, A., Fidalago, P., Herrero, C., and Abalde, J., Toxic action of copper on the membrane system of a marine diatom measured by flow cytometry, Cytometry, Part A, 1996, vol. 25, pp. 32–36.

    Article  CAS  Google Scholar 

  15. Cid, A., Torres, E., Herrero, C., and Abalde, J., Disorders provoked by copper in the marine diatom Phaeodactylum tricornutum in short-time exposure assays, Cah. Biol. Mar., 1997, vol. 38, pp. 201–206.

    Google Scholar 

  16. Cooksey, K.E., Guckert, G.B., Williams, S.A., and Callis, P.R., Fluorometric determination of the neutral lipid content of microalgal cells using Nile Red, J. Microbiol. Methods, 1987, vol. 6, pp. 333–345.

    Article  CAS  Google Scholar 

  17. Cunningham, A. and Buonnacorsi, G.A., Narrow-angle forward light scattering from individual algal cells: implications for size and shape discrimination in flow cytometry, J. Plankton. Res., 1992, vol. 14, pp. 223–234.

    Article  Google Scholar 

  18. Cunningham, A. and Leftley, J.W., Application of flow cytometry to algal physiology and phytoplankton ecology, FEMS Microbiol. Rev., 1986, vol. 1, pp. 159–164.

    Article  Google Scholar 

  19. Dashkova, V., Malashenkov, D., Poulton, N., et al., Imaging flow cytometry for phytoplankton analysis, Methods, 2017, vol. 112, pp. 188–200.

    Article  CAS  PubMed  Google Scholar 

  20. Debelius, B., Forja, J.M., DelValls, T.A., and Lubián, L.M., Toxicity of copper in natural marine picoplankton populations, Ecotoxicology, 2009, vol. 18, pp. 1095–1103.

    Article  CAS  PubMed  Google Scholar 

  21. Doan, T.-T.Y. and Obbard, J.P., Improved Nile Red staining of Nannochloropsis sp., J. Appl. Phycol., 2011, vol. 23, pp. 895–901.

    Article  CAS  Google Scholar 

  22. Dorsey, J., Yentsch, C.M., Mayo, S., and McKenna, C., Rapid analytical technique for the assessment of cell metabolic activity in marine microalgae, Cytometry, Part A, 1989, vol. 10, pp. 622–628.

    Article  CAS  Google Scholar 

  23. Eleršek, T., The advantages of flow cytometry in comparison to fluorometric measurement in algal toxicity test, Acta Biol. Slov., 2012, vol. 55, no. 2, pp. 3–11.

  24. Esperanza, M., Houde, M., Seoane, M., et al., Does a short-term exposure to atrazine provoke cellular senescence in Chlamydomonas reinhardtii?, Aquat. Toxicol., 2017, vol. 189, pp. 184–193.

    Article  CAS  PubMed  Google Scholar 

  25. Figueroa, R.I., Garces, E., and Bravo, I., The use of flow cytometry for species identification and life-cycle studies in dinoflagellates, Deep Sea Res., Part II, 2010, vol. 57, pp. 301–307.

    Article  CAS  Google Scholar 

  26. Forget, N., Belzile, C., Rioux, P., and Nozais, C., Teaching the microbial growth curve concept using microalgal cultures and flow cytometry, J. Biol. Educ., 2010, vol. 44, pp. 185–189.

    Article  Google Scholar 

  27. Franklin, N.M., Stauber, J.L., and Lim, R.P., Development of flow cytometry-based algal bioassays for assessing toxicity of copper in natural waters, Environ. Toxicol. Chem., 2001, vol. 20, pp. 160–170.

    Article  CAS  PubMed  Google Scholar 

  28. Franqueira, D., Orosa, M., Torres, E., et al., Potential use of flow cytometry in toxicity studies with microalgae, Sci. Total Environ., 2000, vol. 247, pp. 119–126.

    Article  CAS  PubMed  Google Scholar 

  29. Gomes, A., Ferdandes, E., and Lima, J.L.F.C., Fluorescence probes used for detection of reactive oxygen species, J. Biochem. Biophys. Methods, 2005, vol. 65, pp. 45–80.

    Article  CAS  PubMed  Google Scholar 

  30. Govender, T., Ramanna, L., Rawat, I., and Bux, F., BODIPY staining, an alternative to the Nile Red fluorescence method for the evaluation of intracellular lipids in microalgae, Bioresour. Technol., 2012, vol. 114, pp. 507–511.

    Article  CAS  PubMed  Google Scholar 

  31. Grégori, G., Denis, M., Lefèvre, D., and Beker, B., A flow cytometric approach to assess phytoplankton respiration, Methods Cell Sci., 2002, vol. 24, pp. 99–106.

  32. Günerken, E., D’Hondt, E., Eppink, M., et al., Flow cytometry to estimate the cell disruption yield and biomass release of Chlorella sp. during bead milling, Algal Res., 2017, vol. 25, pp. 25–31.

    Article  Google Scholar 

  33. Guzmán, H.M., de la Jara Valido, A., Duarte, L.C., and Presmanes, K.F., Estimate by means of flow cytometry of variation in composition of fatty acids from Tetraselmis suecica in response to culture conditions, Aquacult. Int., 2010, vol. 18, pp. 189–199.

    Article  CAS  Google Scholar 

  34. Hallenbeck, P.C., Grogger, M., Mraz, M., and Veverka, D., The use of Design of Experiments and Response Surface Methodology to optimize biomass and lipid production by the oleaginous marine green alga, Nannochloropsis gaditana in response to light intensity, inoculums size and CO2, Bioresour. Technol., 2015, vol. 184, pp. 161–168.

    Article  CAS  PubMed  Google Scholar 

  35. Hamed, I., The evolution and versatility of microalgal biotechnology: a review, Compr. Rev. Food Sci. Food Saf., 2016, vol. 15, pp. 1104–1123.

    Article  PubMed  Google Scholar 

  36. Hejazi, M.A., Kleinegris, D., and Wijffels, R.H., Mechanism of extraction of β-carotene from microalga Dunaliella salina in two-phase bioreactors, Biotechnol. Bioeng., 2004, vol. 88, pp. 593–600.

    Article  CAS  PubMed  Google Scholar 

  37. Hong, H.-H., Lee, H.-G., Jo, J., et al., The exceptionally large genome of the harmful red tide dinoflagellate Cochlodinium polykrikoides Margalef (Dinophyceae): determination by flow cytometry, Algae, 2016, vol. 31, pp. 373–378.

    Article  CAS  Google Scholar 

  38. Hyka, P., Lickova, S., Přibyl, P., et al., Flow cytometry for development of biotechnological processes with microalgae, Biotechnol. Adv., 2013, vol. 31, pp. 2–16.

    Article  CAS  PubMed  Google Scholar 

  39. Jamers, A.N., Lenjou, M., Deraedt, P., et al., Flow cytometric analysis of the cadmium-exposed green alga Chlamydomonas reinhardtii (Chlorophyceae), Eur. J. Phycol., 2009, vol. 44, pp. 541–550.

    Article  CAS  Google Scholar 

  40. de la Jara, A., Mendoza, H., Martel, A., et al., Flow cytometric determination of lipid content in a marine dinoflagellate, Crypthecodinium cohnii, J. Appl. Phycol., 2003, vol. 15, pp. 433–438.

    Article  CAS  Google Scholar 

  41. Jochem, F.J., Probing the physiological state of phytoplankton at the single-cell level, Sci. Mar., 2000, vol. 64, pp. 183–195.

    Article  Google Scholar 

  42. Kapuscinski, J., DAPI: a DNA-specific fluorescent probe, Biotech. Histochem., 1995, vol. 70, pp. 220–233.

    Article  CAS  PubMed  Google Scholar 

  43. Kleinegris, D.M., van Es, M.A., Janssen, M., et al., Carotenoid fluorescence in Dunaliella salina, J. Appl. Phycol., 2010, vol. 22, pp. 645–649.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Koch, F., Kang, Y., Villareal, T.A., et al., A novel immunofluorescence flow cytometry technique detects the expansion of brown tides caused by Aureoumbra lagunensis to the Caribbean Sea, Appl. Environ. Microbiol., 2014, vol. 80, pp. 4947–4957.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Levy, J.L., Stauber, J.L., and Jolley, D.F., Sensitivity of marine microalgae to copper: The effect of biotic factors on copper adsorption and toxicity, Sci. Total Environ., 2007, vol. 387, pp. 141–154.

    Article  CAS  PubMed  Google Scholar 

  46. Long, M., Paul-Pont, I., Hégaret, H., et al., Interactions between polystyrene microplastics and marine phytoplankton lead to species-specific hetero-aggregation, Environ. Pollut., 2017, vol. 228, pp. 454–463.

    Article  CAS  PubMed  Google Scholar 

  47. MacIntyre, H.L. and Cullen, J.J., Classification of phytoplankton cells as live or dead using the vital stains fluorescein diacetate and 5-chloromethylfluorescein diacetate, J. Phycol., 2016, vol. 52, pp. 572–589.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Marie, D., Rigaut-Jalabert, F., and Vaulot, D., An improved protocol for flow cytometry analysis of phytoplankton cultures and natural samples, Cytometry, Part A, 2014, vol. 85, pp. 962–968.

    Article  CAS  Google Scholar 

  49. Marie, D., Simon, N., Guillou, L., et al., DNA/RNA analysis of phytoplankton by flow cytometry, Curr. Protoc. Cytom., 2000, vol. 11, pp. 11.12.1–11.12.14. https://doi.org/10.1002/0471142956.cy1112s11

  50. Mazálova, P., Šarhanova, P., Ondřej, V., and Poulíčková, A., Quantification of DNA content in freshwater microalgae using flow cytometry: a modified protocol for selected green microalgae, Fottea, 2011, vol. 11, pp. 317–328.

    Article  Google Scholar 

  51. Mendoza, H., de la Jara, A., Freijanes, K., et al., Characterization of Dunaliella salina strains by flow cytometry: a new approach to select carotenoid hyperproducing strains, Electron. J. Biotechnol., 2008, vol. 11, pp. 2–13.

    Article  CAS  Google Scholar 

  52. Oda, T., Nakamura, A., Shikayama, M., et al., Generation of reactive oxygen species by raphidophycean phytoplankton, Biosci., Biotechnol., Biochem., 1997, vol. 61, pp. 1658–1662.

    Article  CAS  Google Scholar 

  53. Peperzak, L. and Brussaard, C.P.D., Flow cytometric applicability of fluorescent vitality probes on phytoplankton, J. Phycol., 2011, vol. 47, pp. 692–702.

    Article  PubMed  Google Scholar 

  54. Pereira, H., Schulze, P.S.C., Schüler, L.M., et al., Fluorescence activated cell-sorting principles and applications in microalgal biotechnology, Algal Res., 2018, vol. 30, pp. 113–120.

    Article  Google Scholar 

  55. Pérez-Pérez, M.E., Lemaire, S.D., and Crespo, J.L., Reactive oxygen species and autophagy in plants and algae, Plant Physiol., 2012, vol. 160, pp. 156–164.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  56. Picot, J., Guerin, C.L., Kim, C.L.V., and Boulanger, C.M., Flow cytometry: retrospective, fundamentals and recent instrumentation, Cytotechnology, 2012, vol. 64, pp. 109–130.

    Article  PubMed  PubMed Central  Google Scholar 

  57. Satpati, G.G. and Pal, R., Rapid detection of neutral lipid in green microalgae by flow cytometry in combination with Nile red staining—an improved technique, Ann. Microbiol., 2015, vol. 65, pp. 937–949.

    Article  CAS  Google Scholar 

  58. Segovia, M. and Berges, J.A., Inhibition of caspase-like activities prevents the appearance of reactive oxygen species and dark-induced apoptosis in the unicellular chlorophyte Dunaliella tertiolecta, J. Phycol., 2009, vol. 45, pp. 1116–1126.

    Article  CAS  PubMed  Google Scholar 

  59. Seoane, M., Esperanza, M., Rioboo, C., et al., Flow cytometric assay to assess short-term effects of personal care products on marine microalga Tetraselmis suecica, Chemosphere, 2017, vol. 171, pp. 339–347.

    Article  CAS  PubMed  Google Scholar 

  60. Shapiro, H.M., Practical Flow Cytometry, 4th ed., Hoboken, N.J.: Willey, 2003.

    Book  Google Scholar 

  61. Skarlato, S., Filatova, N., Knyazev, N., et al., Salinity stress response of the invasive dinoflagellate Prorocentrum minimum, Estuarine, Coastal Shelf Sci., 2018, vol. 211, pp. 199–207.

    Article  CAS  Google Scholar 

  62. Stauber, J., Franklin, N., and Adams, M., Microalgal toxicity tests using flow cytometry, in Small-Scale Freshwater Toxicity Investigations, vol. 1: Toxicity Test Methods, Blaise, C. and Ferard, J.-F., Eds., Netherlands: Springer-Verlag, 2005, pp. 203–241.

  63. Stauffer, B.A., Schaffner, R.A., Wazniak, C., and Caron, D.A., Immunofluorescence flow cytometry technique for enumeration of the brown-tide alga, Aureococcus anophagefferens, Appl. Environ. Microbiol., 2008, vol. 74, pp. 6931–6940.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Tobin, E.D., Grünbaum, D., Patterson, J., and Cattolico, R.A., Behavior and physiological changes during benthic-pelagic transition in the harmful alga, Heterosigma akashiwo: Potential for rapid bloom formation, PLoS One, 2013, vol. 8, art. ID e76663. https://doi.org/10.1371/journal.pone.0076663

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Trask, B.J., van den Engh, G.J., and Elgershuizen, J.H.B.W., Analysis of phytoplankton by flow cytometry, Cytometry, Part A, 1982, vol. 2, pp. 258–264.

    Article  CAS  Google Scholar 

  66. van den Engh, G.J., Doggett, J.K., Thompson, A.W., et al., Dynamics of Prochlorococcus and Synechococcus at station ALOHA revealed through flow cytometry and high-resolution vertical sampling, Front. Mar. Sci., 2017, vol. 4, no. 359. https://doi.org/10.3389/fmars.2017.00359

  67. Vaulot, D., Olson, R.J., and Chrisholm, S.W., Light and dark control of the cell cycle in two marine phytoplankton species, Exp. Cell Res., 1986, vol. 167, pp. 38–52.

    Article  CAS  PubMed  Google Scholar 

  68. Veldhuis, M.J.W. and Kraay, G.W., Application of flow cytometry in marine phytoplankton research: current applications and future perspectives, Sci. Mar., 2000, vol. 64, pp. 121–134.

    Article  Google Scholar 

  69. Veldhuis, M., Kraay, G., and Timmermans, K., Cell death in phytoplankton: correlation between changes in membrane permeability, photosynthetic activity, pigmentation and growth, Eur. J. Phycol., 2001, vol. 36, pp. 167–177.

    Article  Google Scholar 

  70. Vigani, M., Parisi, C., Rodríguez-Cerezo, E., et al., Food and feed products from micro-algae: Market opportunities and challenges for the EU, Trends Food Sci. Technol., 2015, vol. 42, pp. 81–92.

    Article  CAS  Google Scholar 

  71. Yentsch, C.M., Mague, F.C., Horan, P.K., and Muirhead, K., Flow cytometric DNA determinations on individual cells of the dinoflagellate Gonyaulax tamarensis var. excavata, J. Exp. Mar. Biol. Ecol., 1983, vol. 67, pp. 175–183.

    Article  CAS  Google Scholar 

  72. Zetsche, E.-M. and Meysman, F.J.R., Dead or alive? Viability assessment of micro- and mesoplankton, J. Plankton Res., 2012, vol. 34, pp. 493–509.

    Article  CAS  Google Scholar 

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ACKNOWLEDGMENTS

The author is grateful to A.V. Boroda, a senior researcher of the Cell Technology Laboratory, Zhirmunsky National Scientific Center of Marine Biology, Far East Branch, Russian Academy of Sciences, for his assistance in the preparation of this article.

Funding

The work was supported with grant no. 18-4-050 in the framework of the program Priority research in the interest of integrated development of the Far Eastern Branch of RAS.

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  1. Zhirmunsky National Scientific Center of Marine Biology, Far East Branch, Russian Academy of Sciences, 690041, Vladivostok, Russia

    Zh. V. Markina

  2. Far Eastern Federal University, 690091, Vladivostok, Russia

    Zh. V. Markina

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Markina, Z.V. Flow Cytometry as a Method to Study Marine Unicellular Algae: Development, Problems, and Prospects. Russ J Mar Biol 45, 333–340 (2019). https://doi.org/10.1134/S1063074019050079

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