Eur. J. Entomol. 108 (3): 481-485, 2011 | DOI: 10.14411/eje.2011.061

Fluorimetric determination of hydrogen peroxide production by the haemocytes of the wax moth Galleria mellonella (Lepidoptera: Pyralidae)

Ondřej VAŠÍČEK1, Ivana PAPEŽÍKOVÁ1, Pavel HYRŠL2
1 Department of Free Radical Pathophysiology, Institute of Biophysics, Academy of Sciences of the Czech Republic, Královopolská 135, 612 65 Brno, Czech Republic; e-mails: ondrej.vasicek@ibp.cz, papezikovai@email.cz
2 Department of Animal Physiology and Immunology, Institute of Experimental Biology, Faculty of Science, Masaryk University, Kotlářská 2, 611 37, Brno, Czech Republic; e-mail: hyrsl@mail.muni.cz

The aim of this study was to investigate whether haemocytes of Galleria mellonella (Lepidoptera: Pyralidae) larvae produce reactive oxygen species (ROS) like human blood phagocytes. The production of ROS was measured first using luminol-enhanced chemiluminescence of un-stimulated and stimulated (four activators with different modes of action) haemolymph or isolated haemocytes. However, spontaneous and activated production of ROS remained at the background level. In subsequent experiments an ultrasensitive fluorescence method using Amplex Red reagent to detect hydrogen peroxide (H2O2) was used. After optimization, Amplex Red was successfully used for determining H2O2 production by both un-stimulated and stimulated haemocytes. To determine the affect of pH and ions on the measurement, several diluent solutions were tested. This revealed that Ca2+ and Mg2+ ions are less important for the reaction in insect than mammalian cells. Among the activators tested, phorbol myristate acetate (PMA) and calcium ionophore (Ca-I) had the best stimulatory effect on insect samples, while opsonised zymosan particles (OZP) was the best activator for human phagocytes. In conclusion, the haemocytes of G. mellonella produce H2O2 as an important innate immunity factor, but under different conditions and in different amounts, which probably results in them being less effective in killing microbes than human phagocytes.

Keywords: Lepidoptera, Pyralidae, Galleria mellonella, chemiluminescence, fluorescence, hydrogen peroxide, reactive oxygen species, Amplex Red, haemocytes, phagocytes, leukocytes, phagocytosis

Received: October 29, 2010; Accepted: December 3, 2010; Published: July 1, 2011

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References

  1. BANFI B., CLARK R.A., STEGER K. & KRAUSE K.H. 2003: Two novel proteins activate superoxide generation by the NADPH oxidase NOX1. J. Biol. Chem. 278: 3510-3513 Go to original source...
  2. BERGIN D., REEVES E.P., RENWICK J., WIENTJES F.B. & KAVANAGH K. 2005: Superoxide production in Galleria mellonella hemocytes: identification of proteins homologous to the NADPH oxidase complex of human neutrophils. Infect. Immun. 73: 4161-4170 Go to original source...
  3. BOMAN H.G. & HULTMARK D. 1987: Cell-free immunity in insects. Annu. Rev. Microbiol. 41: 103-126 Go to original source...
  4. BROOKES P.S., YOON Y., ROBOTHAM J.L., ANDERS M.W. & SHEU S.S. 2004: Calcium, ATP, and ROS: a mitochondrial lovehate triangle. Am. J. Physiol. Cell Physiol . 287: C817-C833 Go to original source...
  5. DAIBER A., AUGUST M., BALDUS S., WENDT M., OELZE M., SYDOW K., KLESCHYOV A.L. & MUNZEL T. 2004: Measurement of NAD(P)H oxidase-derived superoxide with the luminol analogue L-012. Free Radic. Biol. Med. 36: 101-111 Go to original source...
  6. DUBOVSKII I.M., GRIZANOVA E.V., CHERTKOVA E.A., SLEPNEVA I.A., KOMAROV D.A., VORONTSOVA YA.L. & GLUPOV V.V. 2010: Generation of reactive oxygen species and activity of antioxidants in larva hemolymph of galleria mellonella (L.) (Lepidoptera: Pyralidae) at development of process of encapsulation. J. Evol. Biochem. Physiol . 46: 35-43 Go to original source...
  7. ELROD-ERICKSON M., MISHRA S. & SCHNEIDER D. 2000: Interactions between the cellular and humoral immune responses in Drosophila. Curr. Biol. 10: 781-784 Go to original source...
  8. FLORKIN M. & JEUNIAUX C. 1974: Hemolymph: Composition. In: Rockstein M. (ed.): The Physiology of Insecta. 2nd ed. Vol. V. Academic Press, New York, pp. 255-308
  9. GEISZT M., LEKSTROM K., WITTA J. & LETO T.L. 2003: Proteins homologous to p47phox and p67phox support superoxide production by NAD(P)H oxidase 1 in colon epithelial cells. J. Biol. Chem. 278: 20006-20012 Go to original source...
  10. GLUPOV V.V., KHVOSHEVSKAYA T.L., LOVINSKAYA Y.L., DUBOVSKI I.M., MARTEMYANOV V.V. & SOKOLOVA J.Y. 2001: Application of the nitroblue tetrazolium reduction method for studies on the production of reactive oxygen species in insect haemocytes. Cytobios 106: 165-178
  11. HAYDAK M.H. 1936: A food for rearing laboratory insect. J. Econ. Entomol. 29: 1026
  12. HOIDAL J.R., REPTNE J.E., BEALL G.D., RASP F.L. & WHITE J.G. 1978: The effect of phorbol myristate acetate on the metabolism and ultrastructure of human alveolar macrophages. Am. J. Pathol. 91: 469-482
  13. HYRSL P., CIz M., KUBALA L. & LOJEK A. 2004: Silkworm (Bombyx mori) hemocytes do not produce reactive oxygen metabolites as a part of defense mechanisms. Folia Microbiol. 49: 315-319 Go to original source...
  14. KAVANAGH K. & REEVES E.P. 2004: Exploiting the potential of insects for the in vivo pathogenicity testing of microbial pathogens. FEMS Microbiol. Rev. 28: 101-112 Go to original source...
  15. KAVANAGH K. & REEVES E.P. 2007: Insect and mammalian innate immune responses are much alike. Microbe 2: 596-599
  16. KONRAD M., SCHLINGMANN K.P. & GUDERMANN T. 2004: Insights into the molecular nature of magnesium homeostasis. Am. J. Physiol. Renal. Physiol . 286: F599-F605 Go to original source...
  17. KRISHNAN N., HYRSL P. & SIMEK V. 2006: Nitric oxide production by hemocytes of larva and pharate prepupa of Galleria mellonella in response to bacterial lipopolysaccharide: Cytoprotective or cytotoxic? Comp. Biochem. Physiol. (C) 142: 103-110
  18. KRISHNAN N., DAVIS A.J. & GIEBULTOWICZ J.M. 2008: Circadian regulation of response to oxidative stress in Drosophila melanogaster. Biochem. Biophys. Res. Commun. 374: 299-303 Go to original source...
  19. LAD P.M., OLSON C.V. & SMILEY P.A. 1985: Association of the N-formyl-Met-Leu-Phe receptor in human neutrophils with a GTP-binding protein sensitive to pertussis toxin. Proc. Natl. Acad. Sci. USA 82: 869-873 Go to original source...
  20. MOSS B. & ALLAM B. 2006: Fluorimetric measurement of oxidative burst in lobster hemocytes and inhibiting effect of pathogenic bacteria and hypoxia, J. Shellfish Res. 25: 1051-1058 Go to original source...
  21. MULLINS D.E. 1985: Chemistry and physiology of the hemolymph. In Kerkut G.A. & Gilbert L.I. (eds): Comprehensive Insect Physiology, Biochemistry and Pharmacology. Vol. 3. Pergamon Press, Elmsford, NY, pp. 355-400
  22. NAPPI A.J. & VASS E. 1998: Melanogenesis and the generation of cytotoxic molecules during insect cellular immune reactions. Pigment Cell Res. 6: 117-126 Go to original source...
  23. PAVELKOVA M. & KUBALA L. 2004: Luminol-, isoluminol- and lucigenin-enhanced chemiluminescence of rat blood phagocytes stimulated with different activators. Luminescence 19: 37-42 Go to original source...
  24. POHL P., ANTONENKO Y.N. & YAGUZHINSKY L.S. 1990: Kinetic properties of cation/H(+)-exchange: calcimycin (A23187)-mediated Ca2+/2H(+)-exchange on the bilayer lipid membrane. Biochim. Biophys. Acta 1027: 295-300 Go to original source...
  25. PRZYGODZKI T., SOKAL A. & BRYSZEWSKA M. 2005: Calcium ionophore A23187 action on cardiac myocytes is accompanied by enhanced production of reactive oxygen species. Biochim. Biophys. Acta 1740: 481-488 Go to original source...
  26. RATCLIFFE N.A. 1993: Cellular defence responses in insects: unresolved problems. In Bechage N.E., Thompson S.N. & Federace B.A. (eds): Parasites and Pathogens of Insects. Vol. 1. Academic Press, San Diego, CA, pp. 267-304
  27. SALZET M. 2001: Vertebrate innate immunity resembles a mosaic of invertebrate immune responses. Trends Immunol. 22: 285-288 Go to original source...
  28. SLEPNEVA I.A., GLUPOV V.V., SERGEEVA S.V. & KHRAMTSOV V.V. 1999: EPR detection of reactive oxygen species in hemolymph of Galleria mellonella and Dendrolimus superans sibiricus (Lepidoptera) larvae. Biochem. Biophys. Res. Commun. 264: 212-215 Go to original source...
  29. THEOPOLD U., LI D., FABBRI M., SCHERFER C. & SCHMIDT O. 2002: The coagulation of insect hemolymph. Cell. Mol. Life Sci. 59: 363-372 Go to original source...
  30. TOUYZ R.M. 2004: Magnesium in clinical medicine. Front Biosci. 9: 1278-1293 Go to original source...
  31. VILMOS P. & KURUCZ E. 1998: Insect immunity: Evolutionary roots of the mammalian innate immune system. Immunol. Lett. 62: 59-66 Go to original source...
  32. WANG Z., WILHELMSSON C., HYRSL P., LOOF TORSTEN G., DOBES P., KLUPP M., LOSEVA O., MORGELIN M., IKLE J., CRIPPS R.M., HERWALD H. & THEOPOLD U. 2010: Pathogen entrapment by transglutaminase - a conserved early innate immune mechanism. PLoS Pathogens 6(2): doi:10.1371/journal.ppat. 1000763 Go to original source...