Expression if the insect metalloproteinase inhibitor IMPI in the fat body of Galleria mellonella exposed to infection with Beauveria bassiana

  • Lidiia Vertyporokh
  • Iwona Wojda


The inducible metalloproteinase inhibitor (IMPI) discovered in Galleria mellonella is currently the only specific inhibitor of metalloproteinases found in animals. Its role is to inhibit the activity of metalloproteinases secreted by pathogenic organisms as virulence factors to degrade immune-relevant polypeptides of the infected host. This is a good example of an evolutionary arms race between insect hosts and their natural pathogens. In this report, we analyze the expression of genes encoding an inducible metalloproteinase inhibitor (IMPI) in fat bodies of the greater wax moth larvae Galleria mellonella infected with an entomopathogenic fungus Beauveria bassiana. We use natural infection i.e. covering larval integument with fungal aerospores as well as injection of fungal blastospores directly into the larval hemocel. We compare the expression of IMPI with the expression of genes encoding proteins with fungicidal activity, gallerimycin and galiomycin, the expression of which reflects the stimulation of Galleria mellonella defense mechanisms. Also, the expression of the genes is analyzed in the light of survival of animals after spore injection.


Barelli L, Moonjely S, Behie SW, Bidochka MJ (2015) Fungi with multifunctional lifestyles: endophytic insect pathogenic fungi. Plant Mol Biol 90: 657–664. doi: 10.1007/s11103-015-0413-z

Bergin D, Murphy L, Keenan J, Clynes M, Kavanagh K (2006) Pre-exposure to yeast protects larvae of Galleria mellonella from subsequent lethal infection by Candida albicans and is mediated by the increased expression of antimicrobial peptides. Microb Infect 8: 2105–2112

Bolouri Moghaddam MR, Tonk M, Schreiber C, Salzig D, Czermak P, Vilcinskas A, Rahnamaeian M (2016) The potential of the Galleria mellonella innate immune system is maximized by the co-presentation of diverse antimicrobial peptides. Biol Chem 397: 939–945

Butt TM, Coates CJ, Dubovskiy IM, Ratcliffe NA (2016) Entomopathogenic fungi: new insights into host-pathogen interaction in Genetics and Molecular Biology of Entomopathogenic Fungi. First Edition. Lovett B, St. Leger R, eds, pp 307–364. Elsevier

Clermont A, Wedde M, Seitz V, Podsiadlowski L, Lenze D, Hummel M, Vilcinskas A (2004) Cloning and expression of an inhibitor of microbial metalloproteinases from insects contributing to innate immunity. Biochem J 382: 315–322

Cytryńska M, Wojda I, Jakubowicz T (2016) How insects combat infections in Lessons in Immunit. From single-cell organisms to mammals.

Ballarin L, Cammarata M eds. pp 117–124. Academic Print, Elsevier Dubovskiy IM, Whitten MM, Yaroslavtseva ON, Greig C, Kryukov VY, Grizanova EV, Mukherjee K, Vilcinskas A, Glupov VV, Butt TM (2013) Can insects develop resistance to insect pathogenic fungi? PLOS One 8: e 60248. doi:10.1371/annotation/3c61c1d6-7981- 4f3e-a690-1ce7a4d89285

Goel MK, Khanna P, Kishore J (2010) Understanding survival analysis: Kaplan-Meier estimate. Int J Ayurveda Res 1: 274–278. doi: 10.4103/0974-7788.76794

Griesch J, Wedde M, Vilcinskas A (2000) Recognition and regulation of metalloproteinase activity in the haemolymph of Galleria mellonella: a new pathway mediating induction of humoral immune responses. Insect Biochem Mol Biol 30: 461–472

Fan Y, Pei X, Guo S, Zhang Y, Luo Z, Liao X, Pei Y (2010) Increased virulence using engineered protease-chitin binding domain hybrid expressed in the entomopathogenic fungus Beauveria bassiana. Microb Pathog 49: 376–80. doi: 10.1016/j.micpath.2010.06.013

Fehlbaum P, Bulet P, Michaut L, Lagueux M, Broekaert WF, Hetru C, Hoffmann JA (1994) Insect immunity. Septic injury of Drosophila induces the synthesis of a potent antifungal peptide with sequence homology to plant antifungal peptides. J Biol Chem 269: 33159– 33163

Joop G, Vilcinskas A (2016) Coevolution of parasitic fungi and insect hosts. Zoology 119: 350–358

Krautz R, Arefin B, Theopold U (2014) Damage signals in the insect immune response. Front Plant Sci 5: e342. doi: 10.3389/ fpls.2014.00342

Lee YS, Yun EK, Jang WS, Kim I, Lee JH, Park SY, Ryu KS, Seo SJ, Kim CH, Lee IH (2004) Purification, cDNA cloning and expression of an insect defensin from the great wax moth, Galleria mellonella. Insect Mol Biol 13: 65–72

Lemaitre B, Reichhart JM, Hoffmann J.A. (1997) Drosophila host defense: Differential induction of antimicrobial peptide genes after infection by various classes of microorganisms. Proc Nat Acad Sci USA 94: 14614–14619.

Ment D, Gindin G, Rot A, Soroker V, Glazer I, Barel S, Samish M (2010) Novel technique for quantifying adhesion of Metarhizium anisopliae conidia to the tick cuticle. Appl Environ Microbiol 76: 3521– 3528. doi: 10.1128/AEM.02596-09

Mowlds P, Barron A, Kavanagh K (2008) Physical stress primes the immune response of Galleria mellonella larvae to infection by Candida albicans. Microbes Infect 10: 628–634

Ortiz-Urquiza A, Riveiro-Miranda L, Santiago-Álvarez C, Quesada- Moraga E (2010) Insect-toxic secreted proteins and virulence of the entomopathogenic fungus Beauveria bassiana. J Invert Pathol 105: 270–178. doi: 10.1016/j.jip.2010.07.003

Ortiz-Urquiza A, Luo Z, Keyhani NO (2015) Improving mycoinsecticides for insect biological control. Appl Microbiol Biotechnol 99: 1057– 1068. doi: 10.1007/s00253-014-6270-x

Ortiz-Urquiza A, Keyhani NO (2016) Molecular genetics of Beauveria bassiana infection of insects. Adv Gen 94: 165–249. doi: 10.1016/ bs.adgen.2015.11.003

Schuhmann B, Seitz V, Vilcinskas A, Podsiadlowski L (2003) Cloning and expression of gallerimycin, an antifungal peptide expressed in immune response of greater wax moth larvae. Galleria mellonella. Arch Insect Biochem Physiol 53: 125–133

Shai Y (1999) Mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes by alpha-helical antimicrobial and cell non-selective membrane-lytic peptides. Biochim Biophys Acta 1462: 55–70

Travis J, Potempa J, Maeda H (1995) Are bacteria proteinases pathogenic factors? Trends in Microbiol 3: 405–407

Valero-Jiménez CA, Wiegers H, Zwaan BJ, Koenraadt CJ, van Kan JA (2016) Genes involved in virulence of the entomopathogenic fungus Beauveria bassiana. J Invert Pathol 133: 41–49. doi: 10.1016/j. jip.2015.11.011

Vertyporokh L, Taszłow P, Samorek-Pieróg M, Wojda I (2015) Shortterm heat shock affects the course of immune response in Galleria mellonella naturally infected with the entomopathogenic fungus Beauveria bassiana. J Invert Pathol 130: 42–51. doi: 10.1016/j.jip.2015.07.001

Wedde M, Weise C, Kopacek P, Franke P, Vilcinskas A (1998) Purification and characterization of an inducible metalloproteinase inhibitor from the hemolymph of the grater wax moth larvae. Galleria mellonella. Eur J Biochem 255: 535–543

Wedde M, Weise C, Rolf N, Altincicek B, Vilcinskas A (2007) The insect metallopreoteinase inhibitor gene of the lepidopteran Galleria mellonella encodes two distinct inhibitors. Biol Chem 388: 119–127.

Wojda I, Jakubowicz T (2007) Humoral immune response upon mild heat-shock conditions in Galleria mellonella larvae. J Insect Physiol 53: 1134–1144

Wojda I, Kowalski P, Jakubowicz T (2009) Humoral immune response of Galleria mellonella larvae after infection by Beauveria bassiana under optimal and heat-shock conditions. J Insect Physiol 55: 525–531

Wojda I, Taszłow P (2013) Heat shock affects host-pathogen interaction in Galleria mellonella infected with Bacillus thuringiensis. J Insect Physiol 59: 894–905. doi: 10.1016/j.jinsphys.2013.06.011

Wojda I (2016) Immunity of the greater wax moth Galleria mellonella. Insect Sci. doi: 10.1111/1744-7917.12325

Xiao G, Ying SH, Zheng P, Wang ZL, Zhang S, Xie XQ, Shang Y, St Leger RJ, Zhao GP, Wang C, Feng MG (2012) Genomic perspectives on the evolution of fungal entomopathogenicity in Beauveria bassiana. Sci Rep 2: e483. doi: 10.1038/srep00483