Journal of Bioenergetics and Biomembranes

, Volume 50, Issue 2, pp 143–152 | Cite as

Alternative mitochondrial respiratory chains from two crustaceans: Artemia franciscana nauplii and the white shrimp, Litopenaeus vannamei

  • Chrystian Rodriguez-Armenta
  • Salvador Uribe-Carvajal
  • Monica Rosas-Lemus
  • Natalia Chiquete-Felix
  • Jose Angel Huerta-Ocampo
  • Adriana Muhlia-Almazan


Mitochondrial ATP is synthesized by coupling between the electron transport chain and complex V. In contrast, physiological uncoupling of these processes allows mitochondria to consume oxygen at high rates without ATP synthesis. Such uncoupling mechanisms prevent reactive oxygen species overproduction. One of these mechanisms are the alternative redox enzymes from the mitochondrial respiratory chain, which may help cells to maintain homeostasis under stress independently of ATP synthesis. To date, no reports have been published on alternative redox enzymes in crustaceans mitochondria. Specific inhibitors were used to identify alternative redox enzymes in mitochondria isolated from Artemia franciscana nauplii, and the white shrimp, Litopenaeus vannamei. We report the presence of two alternative redox enzymes in the respiratory chain of A. franciscana nauplii, whose isolated mitochondria used glycerol-3-phosphate as a substrate, suggesting the existence of a glycerol-3-phosphate dehydrogenase. In addition, cyanide and octyl-gallate were necessary to fully inhibit this species’ mitochondrial oxygen consumption, suggesting an alternative oxidase is present. The in-gel activity analysis confirmed that additional mitochondrial redox proteins exist in A. franciscana. A mitochondrial glycerol-3-phosphate dehydrogenase oxidase was identified by protein sequencing as part of a branched respiratory chain, and an alternative oxidase was also identified in this species by western blot. These results indicate different adaptive mechanisms from artemia to face environmental challenges related to the changing levels of oxygen concentration in seawater through their life cycles. No alternative redox enzymes were found in shrimp mitochondria, further efforts will determine the existence of an uncoupling mechanism such as uncoupling proteins.


Alternative enzymes Artemia Mitochondria Shrimp Branched respiratory chain 



We thank Consejo Nacional de Ciencia y Tecnologia (CONACyT, National Council for Research and Technology, Mexico) for the grant 241670 to AMA and the scholarship to CMRA. Thanks to Enrique De La Re-Vega and Alfredo Cabrera-Orefice for technical assistance.


  1. Abatzopoulos THJ, Beardmore JA, Clegg, JS Sorgeloos, P (2002) Artemia: basic and applied biology. Springer science business media Dordrecht. ISBN 978–90–481-6073-0Google Scholar
  2. Abele D, Philipp E, Gonzalez PM, Puntarulo S (2007) Marine invertebrate mitochondria and oxidative stress. Front Biosci 12:933–946CrossRefGoogle Scholar
  3. Baradaran R, Berrisford JM, Minhas GS, Sazanov LA (2013) Crystal structure of the entire respiratory complex I. Nature 494:443–448. CrossRefGoogle Scholar
  4. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254. CrossRefGoogle Scholar
  5. Cabrera-Orefice A, Guerrero-Castillo S, Diaz-Ruiz R, Uribe-Carvajal S (2014) Oxidative phosphorylation in Debaryomices hansenii: physiological uncoupling at different growth phases. Biochimie 102:124–136. CrossRefGoogle Scholar
  6. Cardenas-Monroy CA, Pohlmann T, Piñon-Zarate G, Matus-Ortega G, Guerra G, Feldbrügge M, Pardo JP (2017) The mitochondrial alternative oxidase Aox1 is needed to cope with respiratory stress but dispensable for pathogenic development in Ustilago maydis. PLoS One 12:e0173389. CrossRefGoogle Scholar
  7. Clegg JS (1964) The control of emergence and metabolism by external osmotic pressure and the role of free glycerol in developing cyst of Artemia salina. Exp Biol 41:879–892Google Scholar
  8. Clegg JS (1997) Embryos of Artemia franciscana survive four years of continuous anoxia: the case for complete metabolic rate depression. J Exp Biol 200:467–475Google Scholar
  9. Considine MJ, Daley DO, Whelan J (2001) The expression of alternative oxidase and uncoupling protein during fruit ripening in mango. Plant Physiol 126:1619–1629. CrossRefGoogle Scholar
  10. Duarte M, Peters M, Schulte U, Videira A (2003) The internal alternative NADH dehydrogenase of Neurospora crassa mitochondria. Biochem J 371:1005–1011. CrossRefGoogle Scholar
  11. Elthon TE, Nickels RL, McIntosh L (1989) Monoclonal antibodies to the alternative oxidase of higer plant mitochondrial. Plant Physio l89:1311–1317. CrossRefGoogle Scholar
  12. Grecay PA, Stierhoff KL (2002) A device for simultaneously controlling multiple treatment levels of dissolved oxygen in laboratory experiments. J Exp Mar Biol Ecol 280:53–62. CrossRefGoogle Scholar
  13. Guerrero-Castillo S, Vazquez-Acevedo M, González-Halphen D, Uribe-Carvajal S (2009) In Yarrowia lipolytica mitochondria, the alternative NADH dehydrogenase interacts specifically with the cytochrome complexes of the classic respiratory pathway. Biochim Biophys Acta 1787:75–85. CrossRefGoogle Scholar
  14. Guerrero-Castillo S, Araiza-Olivera D, Cabrera-Orefice A, Espinasa-Jaramillo J, Uribe-Carvajal S (2011) Physiological uncoupling of mitochondrial oxidative phosphorylation. Studies in different yeast species. J Bioenerg Biomembr 43:323–331. CrossRefGoogle Scholar
  15. Guerrero-Castillo S, Cabrera-Orefice A, Vazquez-Acevedo M, Gonzalez-Halphen D, Uribe-Carvajal S (2012) During the station growth phase, Yarrowia lipolytica prevents the overproduction of reactive species by activating an uncoupled mitochondrial respiratory pathway. Biochim Biophys Acta 1817:353–362. CrossRefGoogle Scholar
  16. Hoefnagel MHN, Wiskich JT, Madgwick SA, Patterson Z, Oettmeier W, Rich P (1995) New inhibitors of the ubiquinol oxidase of higher plant mitochondria. Eur J Biochem 233:531–537. CrossRefGoogle Scholar
  17. Jimenez-Gutierrez LR, Uribe-Carvajal S, Sanchez-Paz A, Chimeo C, Muhlia-Almazan A (2014) The cytochrome c oxidase and its mitochondrial function in the whiteleg shrimp Litopenaeus vannamei during hypoxia. J Bioenerg Biomembr 46:189–196. CrossRefGoogle Scholar
  18. Joaquin-Ramos A, Huerta-Ocampo JA, Barrera-Pacheco A, De Leon-Rodriguez A, Baginsky S, Barba de la Rosa AP (2014) Comparative proteomic analysis of amaranth mesophyll and bundle sheath chloroplasts and their adaptation to salt stress. J Plant Physiol 171:1423–1435. CrossRefGoogle Scholar
  19. Kadenbach B (2003) Intrinsic and extrinsic uncoupling of oxidative phosphorylation. Biochim Biophys Acta 1604:77–94. CrossRefGoogle Scholar
  20. Korshunov SS, Skulachev VP, Starkov AA (1997) High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria. FEBS Lett 416:15–18. CrossRefGoogle Scholar
  21. Marres CAM, de Vries S, Grivell LA (1991) Isolation and inactivation of the nuclear gene encoding the rotenone-insensitive internal NADH: ubiquinone oxidoreductase of mitochondria from Saccharomyces cerevisiae. Eur J Biochem 195:857–862. CrossRefGoogle Scholar
  22. McDonald AE, Vanlerberghe GC, Staples JF (2009) Alternative oxidase in animals: unique characteristics and taxonomic distribution. J Exp Biol 212:2627–2634. CrossRefGoogle Scholar
  23. Menze MA, Hutchinson K, Laborde SM, Hand SC (2005) Mitochondrial permeability transition in the crustacean Artemia franciscana: absence of a calcium-regulated pore in the face of profound calcium storage. Am J Physiol Regul Integr Comp Physiol 289: R68-R76.
  24. Nicholls DG, Ferguson SJ (2003) Bioenergetics 3. Academic Press, London ISBN 0125181213Google Scholar
  25. Patil YN (2012) Metabolic downregulation during diapause in embryos of Artemia franciscana. Louisiana State University, DissertationGoogle Scholar
  26. Perez-Rostro C, Racotta I, Ibarra AM (2004) Decreased genetic variation in metabolic variables of Litopenaeus vannamei shrimp after exposure to acute hypoxia. J Exp Mar Biol Ecol 302:189–200. CrossRefGoogle Scholar
  27. Puente E (2009) Physiological responses of juvenile white shrimp Litopenaeus vannamei, to oscillating conditions of dissolved oxygen and temperature. Dissertation La Paz BCS Interdisciplinary Marine Science Center-IPNGoogle Scholar
  28. Rasmusson AG, Soole KL, Elyhon TE (2004) Alternative NAD(P)H dehydrogenase of plant mitochondria. Annu Rev Plant Biol 55:23–39. CrossRefGoogle Scholar
  29. Towbin H, Staehelint T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Biochemistry 76:4350–4354. Google Scholar
  30. Tschischka K, Abele D, Pörtner HO (2000) Mitochondrial oxyconformity and cold adaptation in the polychaete Nereis pelagica and the bivalve Arctica islandica from the Baltic and white seas. J Exp Biol 203:3355–3368Google Scholar
  31. Umbach AL, Siedow JN (2000) The cyanide-resistant alternative oxidases from the fungi Pichia stipites and Neurospora crassa are monomeric and lack regulatory features of the plant enzyme. Arch Biochem Biophys 378:234–245. CrossRefGoogle Scholar
  32. Wittig I, Schägger H (2007) Electrophoretic methods to isolated protein complexes from mitochondria. Methods Cell Biol 80:723–741. CrossRefGoogle Scholar
  33. Wittig I, Karas M, Schägger H (2007) High resolution clear native electrophoresis for in-gel functional assays and fluorescence studies of membrane protein complexes. Mol Cell Proteomics 6:1215–1225. CrossRefGoogle Scholar
  34. Wittig I, Beckhaus T, Wumaier Z, Karas M, Schägger H (2010) Mass estimation of native proteins by blue native electrophoresis. Mol Cell Proteomics 9:2149–2161. CrossRefGoogle Scholar
  35. Zerbetto E, Vergani L, Dabbeni-Sala F (1997) Quantification of muscle mitochondrial oxidative phosphorylation enzymes via histochemical staining of blue native polyacrylamide gels. Electrophoresis 18:2059–2064. CrossRefGoogle Scholar
  36. Zimin AV, Delcher AL, Florea L, Kelley DR, Schatz MC, Puiu D, Hanrahan F, Pertea G, Van Tassell CP, Sonstegard TS, Marcais G, Roberts M, Subramanian P, Yorke JA, Salzberg SL (2009) A whole-genome assembly of the domestic cow, Bos Taurus. Genome Biol 10:R42. CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Chrystian Rodriguez-Armenta
    • 1
  • Salvador Uribe-Carvajal
    • 2
  • Monica Rosas-Lemus
    • 3
  • Natalia Chiquete-Felix
    • 2
  • Jose Angel Huerta-Ocampo
    • 4
  • Adriana Muhlia-Almazan
    • 1
  1. 1.Bioenergetics and Molecular Genetics LaboratoryCentro de Investigacion en Alimentacion y Desarrollo (CIAD)HermosilloMexico
  2. 2.Department of Molecular Genetics, Instituto de Fisiologia CelularUniversidad Nacional Autonoma de MexicoMexico CityMexico
  3. 3.Illinois Institute of TechnologyChicagoUSA
  4. 4.Protein Biochemistry LaboratoryCONACYT-Centro de Investigacion en Alimentacion y Desarrollo (CIAD)HermosilloMexico

Personalised recommendations