Production of Cuticle-degrading Enzymes by the Entomopathogen Metarhizium Anisopliae during Infection of Cuticles from Calliphora Vomitoria and Manduca Sexta
SUMMARY: A biochemical and histochemical investigation with specific substrates and inhibitors was used to visualize protease, esterase and aminopeptidase activities produced in situ during penetration of Calliphora vomitoria and Manduca sexta cuticles by hyphae of the entomopathogenic fungus Metarhizium anisopliae. Two endoproteases, and aminopeptidase and esterase activities, were mainly localized in simple and complex appressoria and germinating conidia. The effect of inhibitors on two characterized proteases (Pr1 and Pr2) and aminopeptidase activity in appressorial plates was quantified by microdensitometric measurement of reaction products. Pr1 and Pr2 activities were differentially inhibited by various protease inhibitors. Pr1, Pr2, esterase, aminopeptidase and N-acetylglucosaminidase (exochitinase) activities were present during penetration as detected directly following desorption from fungal and cuticle components. The proteases produced in situ were fractionated, and were shown by immunological and enzymological criteria to be the same as those produced in culture media. The sequence of enzyme appearance in situ showed that production of proteolytic enzymes precedes exochitinase production. No production of endochitinase was found before or during hyphal penetration of the cuticle.
BellR. A.,
JoachimF. G.1976; Techniques for rearing laboratory colonies of tobacco hornworms and pink bollworms. Annals of the Entomological Society of America 69:365–375
CharnleyA. K.1984; Physiological aspects of destructive pathogenesis in insects by fungi: a speculative review. In Invertebrate-Microbial Interactions pp. 229–270 British Mycological Society Symposium 6AndersonJ. M.,
RaynerA. D. M.,
WaltonD. W. H.
Edited by Cambridge: Cambridge University Press;
FarguesJ.1984; Adhesion of the fungal spore to the insect cuticle in relation to pathogenicity. In Infection Processes of Fungi pp. 90–111RobertsD. W.,
AistJ. R.
Edited by New York: The Rockefeller Foundation;
GomoriG.1945; The microtechnical demonstration of sites of lipase activity. Proceedings of the Society for Experimental Biology and Medicine 58:362–364
RataultC.,
VeyA.1977; Production d’esterases et de N-acetyl-β-d-glucosaminidase dans le tegument du Coleoptere Oryctes rhinoceros par le champignon entomopathogene Metarhizium anisopliae. Entomo-phaga 22:289–294
SmithL. R.,
GilkersonE.1979; Quantitation of glycosaminoglycan hexosamine using 3-methyl-2-benzothiazolone hydrazone hydrochloride. Analytical Biochemistry 98:478–480
StaplesR. C.,
HochH. C.1984; A sensing mechanism in rust uredospore germlings responsive to host morphology starts the cell cycle. In Infection Processes of Fungi pp. 126–136RobertsD. W.,
AistJ. R.
Edited by USA: The Rockefeller Foundation;
St LegerR. J.,
CharnleyA. K.,
CooperR. M.1986a; Cuticle degrading enzymes of entomo-pathogenic fungi. Synthesis in culture on cuticle. Journal of Invertebrate Pathology 48:85–95
St LegerR. J.,
CooperR. M.,
CharnleyA. K.1986b; Cuticle degrading enzymes of entomo-pathogenic fungi: cuticle degradation in vitro by enzymes from entomopathogens. Journal of Invertebrate Pathology 47:167–177
St LegerR. J.,
CooperR. M.,
CharnleyA. K.1986C; Cuticle-degrading enzymes of entomo-pathogenic fungi : regulation of production of chitin-olytic enzymes. Journal of General Microbiology 132:1509–1517
St LegerR. J.,
CharnleyA. K.,
CooperR. M.1986d; Cuticle-degrading enzymes of entomo-pathogenic fungi : mechanisms of interaction between pathogen enzymes and insect cuticle. Journal of Invertebrate Pathology 47:295–302
St LegerR. J.,
CharnleyA. K.,
CooperR. M.1987; Characterization of cuticle-degrading proteases produced by the entomopathogen Metarhizium anisopliae. Archives of Biochemistry and Biophysics 253:221–232
Production of Cuticle-degrading Enzymes by the Entomopathogen Metarhizium Anisopliae during Infection of Cuticles from Calliphora Vomitoria and Manduca Sexta