Endosymbiotic green algae in European Hydra strains show quantitative difference on morphological and isoenzyme level

Abstract

The process of endosymbiosis is one of the promoters of speciation. The green hydra (Hydra viridissima Pallas, 1766) is a typical example of endosymbiosis. Its gastrodermal myoepithelial cells harbour the individuals of unicellular photoautotrophic algae. In this study we have developed healthy laboratory cultures of endosymbiotic green algae isolated from green hydra strains. The Hydra strains were collected from four different geographical localities, two of which were in Croatia (strains BV, T), one in Israel (strain M9) and one in Germany (strain HV). For the first time, endosymbiotic algae isolated from green hydra strains have been visualized and described by scanning and transmission electron microscopy. Isolated endosymbiotic algae were characterized by cytological morphometric parameters, enzyme activity and isoenzyme pattern analysis (catalase, peroxidase and esterase). In addition, cells of endosymbiotic algae were characterized by morphometric measurements of diameter, perimeter and area, and chloroplast area. Endosymbiotic algae HV (collected in Germany) and M9 (collected in Israel) were significantly different when compared with endosymbiotic algae T and BV collected in Croatia. The endosymbionts were more different with referent alga Parachlorella kessleri than with referent alga Chlorella vulgaris. The results obtained by isoenzyme pattern analysis also suggested that there was a difference between Croatian and European algal endosymbionts, i.e. the results indicate biological diversity among algal symbionts isolated from the green hydras from different geographical localities. Ultimately, five isoenzymes of catalase and five isoenzymes of peroxidase were resolved by PAGE electrophoresis. Catalase isoenzyme K1 appeared only in endosymbiotic alga M9 collected in Israel and catalase isoenzyme K3 only in endosymbiotic alga T collected in Croatia. Peroxidase isoenzyme P1 was observed only in endosymbiont HV while peroxidase isoenzyme P5 was observed in both endosymbionts HV and M9. Peroxidase isoenzymes P3 and P4 were visible in endosymbiotic algae BV and T collected in Croatia.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10

References

  1. Aebi H (1984) Catalase in vitro. Methods Enzymol 105:121–126

    CAS  Article  Google Scholar 

  2. Balen B, Krsnik-Rasol M, Simeon-Rudolf V (2003) Isoenzymes of peroxidase and esterase related to morphogenesis in Mammillaria gracilis Pfeiff. tissue culture. J Plant Physiol 160:1401–1406

    CAS  Article  Google Scholar 

  3. Beijerinck MW (1890) Culturversuche mit Zoochlorellen, Lichenene-gonidien und anderen niederen Algen I-III. Bot Ztg 48:726–740

    Google Scholar 

  4. Bradford MM (1976) A rapid and sensitive assay for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    CAS  Article  Google Scholar 

  5. Burlina A, Galzigna L (1972) A new and simple procedure for serum arylesterase. Clin Chim Acta 39:255–257

    CAS  Article  Google Scholar 

  6. Burlina A, Michielin E, Galzigna L (1999) Characteristics and behaviour of arylesterase in human serum and liver. Eur J Clin Investig 7:17–20

    Article  Google Scholar 

  7. Chance B, Maehly AC (1955) Assay of catalases and peroxidases. In: Colowick SP, Kaplan NO (eds) Methods in enzymology. Academic Press, New York, pp 764–775

    Google Scholar 

  8. Chelikani P, Fita I, Loewen PC (2004) Diversity of structures and properties among catalases. Cell Mol Life Sci 61:192–208

    CAS  Article  Google Scholar 

  9. Collins AG, Schuchert P, Marques AC, Jankowski T, Medina M, Schierwate B (2006) Medusozoan phylogeny and character evolution clarified by new large and small subunit rDNA data and an assessment of the utility of phylogenetic mixture models. Syst Biol 55:97–115

    Article  Google Scholar 

  10. Douglas AE (1994) Symbiotic interactions. Oxford University Press, New York

    Google Scholar 

  11. Dunahay TG, Jarvis EE, Zeiler KG, Roessler PG, Brown LM (1992) Genetic engineering of microalgae for fuel production. Appl Biochem Biotechnol 34(35):331–339

    Article  Google Scholar 

  12. Falkowski PG, Raven JA (1997) Aquatic photosynthesis. Blackwell Scientific, Oxford

    Google Scholar 

  13. Felsenstein J (1975) The genetic basis of evolutionary change (1975). Evolution 29:587–590

    Google Scholar 

  14. Galliot B, Schmid V (2002) Cnidarians as a model system for understanding evolution and regeneration. Int J Dev Biol 46:39–48

    PubMed  Google Scholar 

  15. Gaspar T, Penel C, Hagege D, Greppin H (1991) Peroxidases in plant growth, differentiation and developmental processes. In: Lobarzewski J, Greppin H, Penel C, Th G (eds) Biochemical, molecular and physiological aspects of plant peroxidases. University M. Curie-Skłodowska. University of Geneva, Lublin, Geneva, pp 249–280

    Google Scholar 

  16. Graham LE, Graham JM, Wilcox LW (2009) Algae, 2nd edn. Pearson Education, San Francisco

    Google Scholar 

  17. Habetha M, Anton-Erksleben F, Neumann K, Bosch TCG (2003) The Hydra viridis/Chlorella symbiosis. Growth and sexual differentiation in polyps without symbionts. Zoology 106:1–8

    Article  Google Scholar 

  18. Hoshina R, Imamura N (2008) Multiple origins of the symbioses in Paramecium bursaria. Protist 159:53–63

    CAS  Article  Google Scholar 

  19. Huss VAR, Holweg C, Seidel B, Reich V, Rahat M, Kessler E (1993/1994) There is an ecological basis for host/symbiont specificity in Chlorella/Hydra symbioses. Endocytobiosis. Cell Res 10:35–46

    Google Scholar 

  20. Kawaida H, Ohba K, Koutake Y, Shimizu H, Tachida H, Kobayakawa Y (2013) Symbiosis between hydra and chlorella: molecular phylogenetic analysis and experimental study provide insight into its origin and evolution. Mol Phylogenet Evol 66:906–914

    Article  Google Scholar 

  21. Kessler E, Huss VAR (1992) Comparative physiology and biochemistry and taxonomic assignment of the Chlorella (Chlorophyceae) strains of the culture collection of the University of Texas at Austin. J Phycol 28:550–553

    Article  Google Scholar 

  22. Kovačević G, Franjević D, Jelenčić B, Kalafatić M (2010a) Isolation and cultivation of endosymbiotic algae from green hydra and phylogenetic analysis of 18S rDNA sequences. Folia Biol (Kraków) 58:135–143

    Article  Google Scholar 

  23. Kovačević G, Radić S, Jelenčić B, Kalafatić M, Posilović H, Pevalek-Kozlina B (2010b) Morphological features and isoenzyme characterization of endosymbiotic algae from green hydra. Plant Syst Evol 284:33–39

    Article  Google Scholar 

  24. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    CAS  Article  Google Scholar 

  25. Laloue H, Weber-Lotfi F, Lucau-Danila A, Guillemaut P (1997) Identification of ascorbate and guaiacol peroxidases in needle chloroplasts of spruce trees. Plant Physiol Bioch 35:341–346

    CAS  Google Scholar 

  26. Lebeda A, Luhová L, Sedlářová M, Jančová D (2001) The role of enzymes in plant-fungal pathogens interactions. Z Pflanzenk Pflanzen 108:89–111

    CAS  Google Scholar 

  27. Leliaert F, Verbruggen H, Zechman FW (2011) Into the deep: new discoveries at the base of the green plant phylogeny. BioEssays 33:683–692

    Article  Google Scholar 

  28. Lewis LA, Muller-Parker G (2004) Phylogenetic placement of "zoochlorellae" (Chlorophyta), algal symbiont of the temperate sea anemone Anthopleura elegantissima. Biol Bull 207:87–92

    CAS  Article  Google Scholar 

  29. Mittler R, Zilinskas B (1993) Detection of ascorbate peroxidase activity in native gels by inhibition of the ascorbate-dependent reduction of nitroblue tetrazolium. Anal Biochem 212:540–546

    CAS  Article  Google Scholar 

  30. Miyazawa Y, Murayama T, Ooya N, Wang LF, Tung YC, Yamaguchi N (1988) Immunomodulation by a unicellular green algae (Chlorella pyrenoidosa) in tumor-bearing mice. J Ethnopharmacol 24:135–146

    CAS  Article  Google Scholar 

  31. Pardy RL (1983) Preparing aposymbiotic hydra. In: Lenhoff HM (ed) Hydra: research methods. Plenum Press, New York, pp 394–395

    Google Scholar 

  32. Pröschold T, Darienko T, Silva PC, Reisser W, Krienitz L (2011) The systematics of Zoochlorella revisited employing an integrative approach. Environ Microbiol 13:350–364

    Article  Google Scholar 

  33. Rahat M (1991) An ecological approach to hydra-cell colonization by algae-algae/hydra symbioses. Oikos 62:381–388

    Article  Google Scholar 

  34. Rahat M, Reich V (1986) Algal endosymbiosis in brown hydra: host/symbiont specificity. J Cell Sci 86:273–286

    CAS  PubMed  Google Scholar 

  35. Rajević N, Kovačević G, Kalafatić M, Gould SB, Martin WF, Franjević D (2015) Algal endosymbionts in European Hydra strains reflect multiple origins of the zoochlorella symbiosis. Mol Phylogenet Evol 93:55–62

    Article  Google Scholar 

  36. Reynolds ES (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol 17:208–212

    CAS  Article  Google Scholar 

  37. Samec P, Posvec Z, Stejskal J, Nasinec V, Griga M (1998) Cultivar identification and relationship in Pisum sativum L. based on RAPD and isozymes. Biol Plantarum 41:39–48

    CAS  Article  Google Scholar 

  38. Sharma P, Jha AB, Dubey RS, Pessarakli M (2012) Reactive oxygen species, oxidative damage, and antioxidative defense mechanism in plants under stressful conditions. J Botany 2012, Article ID e217037, 26 pages. https://doi.org/10.1155/2012/217037

    Article  Google Scholar 

  39. Tanaka K, Koga T, Konishi F, Nakamura M, Mitsuyama M, Himeno K, Nomoto K (1986) Augmentation of host defense by a unicellular green alga, Chlorella vulgaris, to Escherichia coli infection. Infect Immun 53:267–271

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Technau U, Steele RE (2011) Evolutionary crossroads in developmental biology: Cnidaria. Development 138:1447–1458

    CAS  Article  Google Scholar 

  41. Woodbury W, Spencer AK, Stahmann MA (1971) An improved procedure using ferrycyanide for detecting catalase isozymes. Anal Biochem 44:301–305

    CAS  Article  Google Scholar 

Download references

Acknowledgements

Very special thanks to Ms. Ivana Rajević for technical support with photographs and to Prof. Ivana Bočina for proofreading the manuscript. This work was supported by Adris project and Ministry of Science, Education and Sport of the Republic of Croatia project number 119-1193080-1214 “Molecular phylogeny, evolution and symbiosis of freshwater invertebrates”.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Nives Kević.

Electronic supplementary material

S1
figure11

(a-b) Laboratory cultivation of green hydra strains in Division of Zoology, Department of Biology, Faculty of Science, University of Zagreb (PNG 3371 kb)

S2
figure12

Endosymbiotic algae isolated from different green Hydra hosts in growing cultures. (a) Endosymbiotic alga isolated from green hydra strain T (b) Endosymbiotic alga isolated from green hydra strain BV (c) Endosymbiotic alga isolated from green hydra strain M9 (d) Endosymbiotic alga isolated from green hydra strain HV. Scale bar 1 cm (PNG 7866 kb)

High resolution image (TIF 7256 kb)

High resolution image (TIF 16718 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Kević, N., Brkanac, S.R., Vincek, N. et al. Endosymbiotic green algae in European Hydra strains show quantitative difference on morphological and isoenzyme level. Symbiosis 77, 161–175 (2019). https://doi.org/10.1007/s13199-018-0579-7

Download citation

Keywords

  • Endosymbiotic algae
  • Green hydra
  • Symbiosis
  • Electron microscopy
  • Isoenzyme analysis
  • Morphometry