Hostname: page-component-586b7cd67f-rdxmf Total loading time: 0 Render date: 2024-11-26T21:50:21.990Z Has data issue: false hasContentIssue false
  • English
  • Français

Coronatin-2 from the entomopathogenic fungus Conidiobolus coronatus kills Galleria mellonella larvae and incapacitates hemocytes

Published online by Cambridge University Press:  22 July 2016

M.I. Boguś ,
W. Wieloch  and
M. Ligęza-Żuber
M.I. Boguś*
Affiliation:
W. Stefański Institute of Parasitology, Polish Academy of Sciences, Twarda 51/55, 00-818 Warszawa, Poland BIOMIBO, Strzygłowska 15, 04-872 Warszawa, Poland
W. Wieloch
Affiliation:
W. Stefański Institute of Parasitology, Polish Academy of Sciences, Twarda 51/55, 00-818 Warszawa, Poland
M. Ligęza-Żuber
Affiliation:
W. Stefański Institute of Parasitology, Polish Academy of Sciences, Twarda 51/55, 00-818 Warszawa, Poland
*
*Author for correspondence Phone: (+48 22) 6978973 Fax: (+48 58) 6206227 E-mail: [email protected]
Get access Rights & Permissions [Opens in a new window]

Abstract

Coronatin-2, a 14.5 kDa protein, was isolated from culture filtrates of the entomopathogenic fungus Conidiobolus coronatus (Costantin) Batko (Entomophthoramycota: Entomophthorales). After LC–MS/MS (liquid chromatography tandem mass spectrometry) analysis of the tryptic peptide digest of coronatin-2 and a mass spectra database search no orthologs of this protein could be found in fungi. The highest homology was observed to the partial translation elongation factor 1a from Sphaerosporium equinum (protein sequence coverage, 21%), with only one peptide sequence, suggesting that coronatin-2 is a novel fungal protein that has not yet been described. In contrast to coronatin-1, an insecticidal 36 kDa protein, which shows both elastolytic and chitinolytic activity, coronatin-2 showed no enzymatic activity. Addition of coronatin-2 into cultures of hemocytes taken from larvae of Galleria mellonella Linnaeus (Lepidoptera: Pyralidae), resulted in progressive disintegration of nets formed by granulocytes and plasmatocytes due to rapid degranulation of granulocytes, extensive vacuolization of plasmatocytes accompanied by cytoplasm expulsion, and cell disintegration. Spherulocytes remained intact, while oenocytes rapidly disintegrated. Coronatin-2 produced 80% mortality when injected into G. mellonella at 5 µg larva−1. Further study is warranted to determine the relevance of the acute toxicity of coronatin-2 and its effects on hemocytes in vitro to virulence of C. coronatus against its hosts.

Keywords

insecticidal proteinmycotoxincytotoxicityHPLCLC–MS/MS
Type
Research Papers
Information
Bulletin of Entomological Research , Volume 107 , Issue 1 , February 2017 , pp. 66 - 76
Check for updates
Copyright
Copyright © Cambridge University Press 2016 

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Advisory Committee of Pesticides (1994) Evaluation on chlorfenvinphos. Available online at http://www.pesticides.gov.uk/Resources/CRD/ACP/107_chlorfenvinphos.pdf.Google Scholar
Arama, E., Agapite, J. & Steller, H. (2003) Caspase activity and a specific cytochrome C are required for sperm differentiation in Drosophila . Developmental Cell 4, 687–697.CrossRefGoogle Scholar
Bania, J., Samborski, J., Boguś, M.I. & Polanowski, A. (2006) Specificity of an extracellular proteinase from Conidiobolus coronatus and its inhibition by an inhibitor from insect hemolymph. Archives of Insect Biochemistry and Physiology 62, 186–196.Google Scholar
Boguś, M.I. & Scheller, K. (2002) Extraction of an insecticidal protein fraction from the pathogenic fungus Conidiobolus coronatus . Acta Parasitologica 47, 66–72.Google Scholar
Boguś, M.I., Kędra, E., Bania, J., Szczepanik, M., Czygier, M., Jabłoński, P., Pasztaleniec, A., Samborski, J., Mazgajska, J. & Polanowski, A. (2007) Different defense strategies of Dendrolimus pini, Galleria mellonella, and Calliphora vicina against fungal infection. Journal of Insect Physiology 53, 909–922.Google Scholar
Boguś, M.I., Czygier, M., Gołębiowski, M., Kędra, E., Kucińska, J., Mazgajska, J., Samborski, J., Wieloch, W. & Włóka, E. (2010) Effects of insect cuticular fatty acids on in vitro growth and pathogenicity of the entomopathogenic fungus Conidiobolus coronatus . Experimental Parasitology 125, 400–408.CrossRefGoogle ScholarPubMed
Boguś, M.I., Ligęza-Żuber, M., Wieloch, W. & Włóka, E. (2012) Mechanizm porażania owadów przez pasożytniczy grzyb Conidiobolus coronatus oraz reakcje obronne owadów pp. 225–248 in Skrzecz, I. & Sierpińska, A. (Eds) Kierunki Rozwoju Patologii Owadów w Polsce. Sękocin Stary, IBL. ISBN 978-83-62830-11-4.Google Scholar
Callaghan, A.A. (1969) Light and spore discharge in entomophthorales. Transactions of the British Mycological Society 53, 87–97.Google Scholar
Castrillo, L.A., Roberts, D.W. & Vandenberg, J.D. (2005) The fungal past, present, and future: germination, ramification, and reproduction. Journal of Invertebrate Pathology 89, 46–56.Google Scholar
Clarkson, J.M. & Charnley, A.K. (1996) New insight into the mechanisms of fungal pathogenesis in insects. Trends in Microbiology 4, 197–203.CrossRefGoogle ScholarPubMed
Czygier, M., Dzik, J.M., Wałajtys-Rode, E. & Boguś, M.I. (2000) Cuticle degrading enzymes from pathogenic fungus Conidiobolus coronatus . Acta Parasitologica 45, 247.Google Scholar
Domsch, K.H., Gams, W. & Anderson, T.-H. (1980) Compendium of Soil Fungi, Vol. 1 and 2. New York and London, Academic Press.Google Scholar
Dromph, K.M., Eilenberg, J. & Esbjerg, P. (2001) Natural occurrence of entomophthoralean fungi pathogenic to collembolans. Journal of Invertebrate Pathology 78, 226–231.Google Scholar
Dubovskiy, I.M., Whitten, M.M.A., Yaroslavtseva, O.N., Greig, C., Kryukov, V.Y., Grizanova, E.V., Mukherjee, K., Vilcinskas, A., Glupov, V.V. & Butt, T.M. (2013) Can insects develop resistance to insect pathogenic fungi? PLoS ONE 8, e60248.Google Scholar
Evans, H.C. (1989) Mycopathogens of insects of epigeal and aerial habitats pp. 205–238 in Wilding, N., Collins, N.M., Hammond, P.M. & Webber, J.F. (Eds) Insect-Fungus Interactions. London, Academic Press.Google Scholar
Fan, Y., Borovsky, D., Hawkings, C., Ortiz-Urquiza, A. & Keyhani, N.O. (2012) Exploiting host molecules to augment mycoinsecticide virulence. Nature Biotechnology 30, 35–37.Google Scholar
Fang, W., Azimzadeh, P. & St. Leger, R.J. (2012) Strain improvement of fungal insecticides for controlling insect pests and vector-borne diseases. Current Opinion in Microbiology 15, 232–238.Google Scholar
Faria, M. & Wraight, S.P. (2007) Mycoinsecticides and mycoacaricides: a comprehensive list with worldwide coverage and international classification of formulation types. Biological Control 43, 237–256.Google Scholar
Freimoser, F.M., Screen, S., Hu, G. & St. Leger, R. (2003) EST analysis of genes expressed by the zygomycete pathogen Conidiobolus coronatus during growth on insect cuticle. Microbiology 149, 1893–1900.Google Scholar
Fröbius, A., Kanost, M.R., Götz, P. & Vilcinskas, A. (2000) Isolation and characterization of novel inducible serine protease inhibitors from larval hemolymph of the greater wax moth Galleria mellonella . European Journal of Biochemistry 267, 2046–2053.Google Scholar
Gillespie, J.P., Bailey, A.M., Cobb, B. & Vilcinskas, A. (2000) Fungi as elicitors of insect immune responses. Archives of Insect Biochemistry and Physiology 44, 49–68.Google Scholar
Glare, T., Caradus, J., Gelernter, W., Jackson, T., Keyhani, N., Köhl, J., Marrone, P., Morin, L. & Stewart, A. (2012) Have biopesticides come of age? Trends in Biotechnology 30, 250–258.Google Scholar
Gołębiowski, M., Maliński, E., Boguś, M.I., Kumirska, J. & Stepnowski, P. (2008) The cuticular fatty acids of Calliphora vicina, Dendrolimus pini and Galleria mellonella larvae and their role in resistance to fungal infection. Insect Biochemistry and Molecular Biology 38, 619–627.CrossRefGoogle ScholarPubMed
Gołębiowski, M., Boguś, M.I., Paszkiewicz, M. & Stepnowski, P. (2011) Cuticular lipids of insects as a potential biofungicides: methods of lipids composition analysis. Analytical and Bioanalytical Chemistry 399, 3177–3191.Google Scholar
Hajek, A.E. & St. Leger, R.J. (1994) Interactions between fungal pathogens and insect hosts. Annual Review of Entomology 39, 293–322.CrossRefGoogle Scholar
Harding, C.R., Schroeder, G.N., Collins, J.W. & Frankel, G. (2013) Use of Galleria mellonella as a model organism to study Legionella pneumophila infection. Journal of Visualized Experiments 81, e50964.Google Scholar
Hoffmann, J. (1995) Innate immunity of insects. Current Opinion in Immunology 7, 4–10.CrossRefGoogle ScholarPubMed
Keyhani, N.O. (2012) Using host molecules to increase fungal virulence for biological control of insects. Virulence 4, 415–417.Google Scholar
Khachatourians, G.G. (1996) Biochemistry and molecular biology of entomopathogenic fungi pp. 331–363 in Howard, D.H. & Miller, J.D. (Eds) The Mycota VI. Berlin, Heidelberg, Springer.Google Scholar
Kucera, M. (1981) The production of toxic protease by the entomopathogenous Metarhizium anisopliae in submerged culture. Journal of Invertebrate Pathology 38, 33–38.Google Scholar
Kucera, M. & Samsinakova, A. (1968) Toxins of the entomopathogenous fungus Beauveria bassiana . Journal of Invertebrate Pathology 12, 316–320.Google Scholar
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T-4. Nature 227, 680–685.Google Scholar
Morrissey, J.H. (1981) Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Analytical Biochemistry 117, 307–310.Google Scholar
O'Meara, R.W., Michalski, J.-P. & Kothary, R. (2011) Integrin signaling in oligodendrocytes and its importance in CNS myelination. Journal of Signal Transduction, Article ID 354091, 11 pages. doi: 10.1155/2011/354091 CrossRefGoogle ScholarPubMed
Ortiz-Urquiza, A. & Keyhani, N.O. (2013) Action on the surface: entomopathogenic fungi versus the insect cuticle. Insects 4, 357–374.CrossRefGoogle ScholarPubMed
Ottaviani, E. (2005) Insect immunorecognition. Invertebrate Survival Journal 2, 142–151.Google Scholar
Phadatare, S.U., Srinivasan, M.C. & Desphande, V.V. (1992) Evidence for controlled autoproteolysis of alkaline protease. A mechanism for physiological regulation of conidial discharge in Conidiobolus coronatus . European Journal of Biochemistry 205, 679–686.Google Scholar
Prasertphon, S. & Tanada, Y. (1969) Mycotoxins of entomophthoraceous fungi. Hilgardia 39, 581–600.Google Scholar
Purwar, J.P. & Sachan, G.C. (2006) Synergistic effect of entomogenous fungi on some insecticides against Bihar hairy caterpillar Spilarctia oblique (Lepidoptera: Arctiidae). Microbiological Research 161, 38–42.Google Scholar
Samson, R.A., Evans, H.C. & Latge, J.-P. (1988) Atlas of Entomopathogenic Fungi. Berlin Heidelberg, Springer-Verlag.CrossRefGoogle Scholar
Schleif, R.F. & Wensink, P.C. (1981) Practical Methods in Molecular Biology. New York, Heidelberg, Berlin, Springer-Verlag.Google Scholar
Schrank, A. & Vainstein, M.H. (2010) Metarhizium anisopliae enzymes and toxins. Toxicon 56, 1267–1274.CrossRefGoogle Scholar
Shah, P.A. & Pell, J.K. (2003) Entomopathogenic fungi as biological control agents. Applied Microbiology and Biotechnology 61, 413–423.Google Scholar
Shevchenko, A., Tomas, H., Havli, J., Olsen, J.V. & Mann, M. (2007) In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nature Protocols 1, 2856–2860.Google Scholar
Srivastava, C.N., Mohan, L., Sharma, P. & Maurya, P. (2011) A review on prospective of synergistic approach in insect pest management. Journal of the Entomological Research Society 35, 255–266.Google Scholar
St Leger, R.J. & Bidochka, M.J. (1996) Insect-fungal interactions pp. 443–479 in Söderhäll, K., Iwanaga, K.S. & Vasta, G.R. (Eds) New Directions in Invertebrate Immunology. Fairhaven, New York, SOS Publications.Google Scholar
St Leger, R.J., Bidochka, M.J., Joshi, L. & Roberts, D.W. (1996) Construction of an improved mycoinsecticide overexpressing a toxic protease. Proceedings of the National Academy of Sciences of the United States of America 93, 6349–6354.Google Scholar
Sugumaran, M. (1998) Unified mechanism for sclerotization of insect cuticle. Advances in Insect Physiology 27, 229–334.Google Scholar
Sutar, I.I., Srinivasan, M.C. & Vartak, H.G. (1991) A low weight alkaline proteinase from Conidiobolus coronatus . Biotechnology Letters 13, 119–124.Google Scholar
Tanksale, A.M., Vernekar, J.V., Ghatge, M.S. & Deshpande, V.V. (2000) Evidence for tryptophan in proximity to histidine and cysteine as essential to the active site of an alkaline protease. Biochemical and Biophysical Research Communications 270, 910–917.CrossRefGoogle Scholar
Vilcinskas, A. & Götz, P. (1999) Parasitic fungi and their interactions with the insect immune system. Advances in Parasitology 43, 267–313.Google Scholar
Vilcinskas, A., Matha, V. & Götz, P. (1997) Effects of the entomopathogenic fungus Metarhizium anisopliae and its secondary metabolites on morphology and cytoskeleton of plasmatocytes isolated from the greater wax moth, Galleria mellonella . Journal of Insect Physiology 43, 1149–1159.CrossRefGoogle Scholar
Vilcinskas, A., Jegorov, A., Landa, Z., Götz, P. & Matha, V. (1999) Effects of beauverolide L and cyclosporin A on humoral and cellular immune response of the greater wax moth, Galleria mellonella . Comparative Biochemistry and Physiology Part C: Pharmacology, Toxicology and Endocrinology 122, 83–92.Google ScholarPubMed
Wang, C. & St. Leger, R.J. (2006) A collagenous protective coat enables Metarhizium anisopliae to evade insect immune responses. Proceedings of the National Academy of Sciences of the United States of America 103, 6647–6652.Google Scholar
Wieloch, W. (2006) Toksyczne metabolity wytwarzane przez pasożytniczy grzyb Conidiobolus coronatus . PhD Thesis, Institute of Parasitology Polish Academy of Sciences, Warszawa.Google Scholar
Wieloch, W. & Boguś, M.I. (2005) Exploring pathogenicity potential of Conidiobolus coronatus against insect larvae in various infection conditions. Pesticides 4, 133–137.Google Scholar
Wieloch, W. & Boguś, M.I. (2007) Proteo-, chitino- and lypolitic enzymes production by entomopathogenic fungus Conidiobolus coronatus . Acta Biochimica Polonica 54, 79. Available online at http://www.science24.com/paper/10860.Google Scholar
Wieloch, W., Boguś, M.I., Ligęza, M., Koszela-Piotrowska, I. & Szewczyk, A. (2011) Coronatin-1 isolated from entomopathogenic fungus Conidiobolus coronatus kills Galleria mellonella hemocytes in vitro and forms potassium channels in planar lipid membrane. Toxicon 58, 369–379.Google Scholar
Włóka, E. & Boguś, M.I. (2010) Enzymatic activity of Conidiobolus coronatus under stress conditions. Acta Biochimica Polonica 57, 111. Available online at http://www.actabp.pl/pdf/Supl4_10/S6.pdf Google Scholar
Włóka, E., Bielski, P., Gajos, A., Nowak, A., Walkowiak, R., Gołębiowski, M. & Boguś, M.I. (2013) Susceptibility and resistance of various insect species to fungal infection may result from differential hydrolysis rates of their cuticle by fungal proteases affected by cuticular lipids. In MIKROBIOT 2013 – The 3rd Workshop on Microbiology in Health and Environmental Protection September 17–20, 2013.Google Scholar
Włóka, E., Gołębiowski, M. & Boguś, M.I. (2014) Fatty acids present in insect epicuticle affect activity of entomopathogenic fungus Conidiobolus coronatus enzymes engaged in cuticle degradation. In 1st Congress of the Polish Biochemistry, Cell Biology, Biophysics and Bioinformatics BIO 2014Warsaw, 9–12 September 2014. Acta Biochimica Polonica 61, Supplement 1/2014, 108. Available online at http://www.actabp.pl/pdf/Supl1_14/Session_5.pdf Google Scholar
Wojda, I., Kowalski, P. & Jakubowicz, T. (2009) Humoral immune response of Galleria mellonella larvae after infection by Beauveria bassiana under optima and heat-shock conditions. Journal of Insect Physiology 55, 525–531.Google Scholar