Evidence for embryonic haemoglobins from Sparus aurata under normal and hypoxic conditions

  • Manuela Mania
  • Giuseppe Bruschetta
  • Angela Avenoso
  • Angela D’Ascola
  • Michele Scuruchi
  • Adele Campo
  • Giuseppe Acri
  • Salvatore CampoEmail author


Teleost haemoglobins vary in polymorphisms and primary structure, although display similar functional properties. Key amino acids for Root effect (a reduction in oxygen-carrying capacity and loss of cooperativity with declining pH) are conserved throughout fish evolution. For the first time, we cloned and characterised Sparus aurata L. embryonic globin chains (eα1, eα2, eβ). We also studied haemoglobins (eHbI, eHbII) behaviour in normal and low-oxygen conditions. Several amino acids in fry globins are different in chemical type (e.g. polar → non-polar and vice versa), compared to adult globins. His55α1, crucial for Root effect, is substituted by Ala in fry, presumably enhancing oxygen capture, transport and reducing the dependence of Root effect from pH. Phylogenetic trees demonstrate that eα1 globin diversified more recently than eα2; moreover, eα1, eα2 and eβ globins evolved earlier than adult α and β globins. In low-oxygen conditions, fry haemoglobins display the same behaviour of the adult haemoglobins (probably, embryonic and adult-type I Hbs display a higher oxygen affinity than type II Hbs, operating through a rapid cycle of heme-Fe auto-oxidation/reduction). Therefore, based on our results and on the comparison with adult haemoglobins, we hypothesise that embryonic haemoglobins have evolved to better adapt fry to variable habitats. We studied Sparus aurata for its economical relevance in Mediterranean aquaculture. The information we provide can help understand Sparus aurata behaviour in the wild and in rearing conditions. Further studies with functional assays will deepen the knowledge on the molecular mechanisms of fry haemoglobin physiology.


Teleost Fish culture Haemoglobins Embryonic globin chains Phylogenesis 



Embryonic alpha 1


Embryonic alpha 2

Embryonic beta


Haemoglobin I


Haemoglobin II


Linnaeus (1758)


Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Baldwin JM (1980) The structure of human carbonmonoxy haemoglobin at 2.7 a resolution. J Mol Biol 136:103–128CrossRefGoogle Scholar
  2. Berenbrink M, Koldkjaer P, Kepp O, Cossins AR (2005) Evolution of oxygen secretion in fishes and the emergence of a complex physiological system. Science 307:1752–1757. CrossRefPubMedGoogle Scholar
  3. Bernard P, Gabant P, Bahassi EM, Couturier M (1994) Positive-selection vectors using the F plasmid ccdB killer gene. Gene 148:71–74CrossRefGoogle Scholar
  4. Boechi L, Marti MA, Vergara A, Sica F, Mazzarella L, Estrin DA, Merlino A (2011) Protonation of histidine 55 affects the oxygen access to heme in the alpha chain of the hemoglobin from the Antarctic fish Trematomus bernacchii. IUBMB Life 63:175–182. CrossRefPubMedGoogle Scholar
  5. Bonaventura C, Crumbliss AL, Weber RE (2004) New insights into the proton-dependent oxygen affinity of root effect haemoglobins. Acta Physiol Scand 182:245–258. CrossRefPubMedGoogle Scholar
  6. Brunori M (1975) Molecular adaptation to physiological requirements: the hemoglobin system of trout. Curr Top Cell Regul 9:1–39CrossRefGoogle Scholar
  7. Camardella L, Caruso C, D’Avino R, di Prisco G, Rutigliano B, Tamburrini M, Fermi G, Perutz MF (1992) Haemoglobin of the antarctic fish Pagothenia bernacchii. Amino acid sequence, oxygen equilibria and crystal structure of its carbonmonoxy derivative. J Mol Biol 224:449–460CrossRefGoogle Scholar
  8. Campo S, Nastasi G, D’Ascola A, Campo GM, Avenoso A, Traina P, Calatroni A, Burrascano E, Ferlazzo A, Lupidi G, Gabbianelli R, Falcioni G (2008) Hemoglobin system of Sparus aurata: changes in fishes farmed under extreme conditions. Sci Total Environ 403:148–153. CrossRefPubMedGoogle Scholar
  9. Campo S, Nastasi G, Fedeli D, D’Ascola A, Campo GM, Avenoso A, Ferlazzo A, Calatroni A, Falcioni G (2010) Molecular cloning and characterization of adult Sparus aurata hemoglobin genes. Omics 14:187–200. CrossRefPubMedGoogle Scholar
  10. Evans CJ, Hartenstein V, Banerjee U (2003) Thicker than blood: conserved mechanisms in Drosophila and vertebrate hematopoiesis. Dev Cell 5:673–690CrossRefGoogle Scholar
  11. Giardina B, Messana I, Scatena R, Castagnola M (1995) The multiple functions of hemoglobin. Crit Rev Biochem Mol Biol 30:165–196. CrossRefPubMedGoogle Scholar
  12. Ito N, Komiyama NH, Fermi G (1995) Structure of deoxyhaemoglobin of the antarctic fish Pagothenia bernacchii with an analysis of the structural basis of the root effect by comparison of the liganded and unliganded haemoglobin structures. J Mol Biol 250:648–658. CrossRefPubMedGoogle Scholar
  13. Kleszczyńska A, Vargas-Chacoff L, Gozdowska M, Kalamarz H, Martínez-Rodríguez G, Mancera JM, Kulczykowska E (2006) Arginine vasotocin, isotocin and melatonin responses following acclimation of gilthead sea bream (Sparus aurata) to different environmental salinities. Comp Biochem Physiol A Mol Integr Physiol 145:268–273. CrossRefPubMedGoogle Scholar
  14. Maruyama K, Sugano S (1994) Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides. Gene 138:171–174CrossRefGoogle Scholar
  15. Maruyama K, Yasumasu S, Iuchi I (2002) Characterization and expression of embryonic and adult globins of the teleost Oryzias latipes (medaka). J Biochem 132:581–589CrossRefGoogle Scholar
  16. Mazzarella L, Bonomi G, Lubrano MC, Merlino A, Riccio A, Vergara A, Vitagliano L, Verde C, di Prisco G (2006a) Minimal structural requirements for root effect: crystal structure of the cathodic hemoglobin isolated from the antarctic fish Trematomus newnesi. Proteins 62:316–321. CrossRefPubMedGoogle Scholar
  17. Mazzarella L, Vergara A, Vitagliano L, Merlino A, Bonomi G, Scala S, Verde C, di Prisco G (2006b) High resolution crystal structure of deoxy hemoglobin from Trematomus bernacchii at different pH values: the role of histidine residues in modulating the strength of the root effect. Proteins 65:490–498. CrossRefPubMedGoogle Scholar
  18. Mylvaganam SE, Bonaventura C, Bonaventura J, Getzoff ED (1996) Structural basis for the root effect in haemoglobin. Nat Struct Biol 3:275–283CrossRefGoogle Scholar
  19. Nagai K, Perutz MF, Poyart C (1985) Oxygen binding properties of human mutant hemoglobins synthesized in Escherichia coli. Proc Natl Acad Sci U S A 82:7252–7255CrossRefGoogle Scholar
  20. Perutz MF, Brunori M (1982) Stereochemistry of cooperative effects in fish an amphibian haemoglobins. Nature 299:421–426CrossRefGoogle Scholar
  21. Petruzzelli R, Barra D, Sensi L, Bossa F, Brunori M (1989) Amino acid sequence of alpha-chain of hemoglobin IV from trout (Salmo irideus). Biochim Biophys Acta 995:255–258CrossRefGoogle Scholar
  22. Powers DA, Lauerman T, Crawford D, DiMichele L (1991) Genetic mechanisms for adapting to a changing environment. Annu Rev Genet 25:629–659. CrossRefPubMedGoogle Scholar
  23. Randall DJ, Rummer JL, Wilson JM, Wang S, Brauner CJ (2014) A unique mode of tissue oxygenation and the adaptive radiation of teleost fishes. J Exp Biol 217:1205–1214. CrossRefPubMedGoogle Scholar
  24. Roesner A, Mitz SA, Hankeln T, Burmester T (2008) Globins and hypoxia adaptation in the goldfish, Carassius auratus. FEBS J 275:3633–3643. CrossRefPubMedGoogle Scholar
  25. Ronda L, Merlino A, Bettati S, Verde C, Balsamo A, Mazzarella L, Mozzarelli A, Vergara A (2013) Role of tertiary structures on the root effect in fish hemoglobins. Biochim Biophys Acta 1834:1885–1893. CrossRefPubMedGoogle Scholar
  26. Rummer JL, Brauner CJ (2015) Root effect haemoglobins in fish may greatly enhance general oxygen delivery relative to other vertebrates. PLoS One 10:e0139477. CrossRefPubMedPubMedCentralGoogle Scholar
  27. Rummer JL, McKenzie DJ, Innocenti A, Supuran CT, Brauner CJ (2013) Root effect hemoglobin may have evolved to enhance general tissue oxygen delivery. Science 340:1327–1329. CrossRefPubMedGoogle Scholar
  28. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Book 2nd. ed. Cold Spring Harbor Laboratory, New YorkGoogle Scholar
  29. Schaefer BC (1995) Revolutions in rapid amplification of cDNA ends: new strategies for polymerase chain reaction cloning of full-length cDNA ends. Anal Biochem 227:255–273. CrossRefPubMedGoogle Scholar
  30. Vergara A, Vitagliano L, Merlino A, Sica F, Marino K, Verde C, di Prisco G, Mazzarella L (2010) An order-disorder transition plays a role in switching off the root effect in fish hemoglobins. J Biol Chem 285:32568–32575. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Manuela Mania
    • 1
  • Giuseppe Bruschetta
    • 2
  • Angela Avenoso
    • 1
  • Angela D’Ascola
    • 1
  • Michele Scuruchi
    • 3
  • Adele Campo
    • 1
  • Giuseppe Acri
    • 1
  • Salvatore Campo
    • 1
    Email author
  1. 1.Department of Biochemical and Dental Sciences and Morphofunctional ImagesUniversity of MessinaMessinaItaly
  2. 2.Department of Veterinary SciencesUniversity of MessinaMessinaItaly
  3. 3.Department of Clinical and Experimental MedicineUniversity of MessinaMessinaItaly

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