PROTEIN HYDROLYSIS IN GRASSHOPPER CUTICLES BY ENTOMOPATHOGENIC FUNGAL EXTRACELLULAR PROTEASES

Several regions of unsclerotized (teneral) cuticle from the migratory grasshopper, Melanoplus sanguinipes, were treated with extracellular protease-containing culture supernatants of the entomopathogenic fungi, Beauveria bassiana or Metarhizium anisopliae, or purified extracellular B. bassiana protease. Treatment of the various cuticles with the culture supernatants resulted in a 41.1% (forewing) to 83.0% (hindwing) loss in cuticle dry weight. Addition of phenylmethylsulfonyl fluoride (PMSF), a protease inhibitor, to the culture supernatants, resulted in a substantial retention of cuticle dry weight after the treatment. The urea-soluble proteins from untreated cuticles or cuticles treated with B. bassiana or M. anisopliae culture supernatants or purified B. bassiana protease were characterized by two-dimensional (2D) gel electrophoresis. An initial reduction in the number of acidic proteins was observed in the 2D gels from cuticles previously treated with fungal culture supernatants or purified B. bassiana protease. High-molecular-weight (>31 kDa) basic cuticular proteins were also susceptible to degradation by proteases. Addition of PMSF to the B. bassiana or M. anisopliae supernatants previous to incubation with the cuticle resulted in only minor qualitative changes in cuticular protein 2D patterns. The action of B. bassiana or M. anisopliae proteases toward acidic cuticular proteins, and to a lesser extent the high-molecular basic proteins, is discussed in light of what is known of the biochemistry of entomopathogenic fungal proteases. © 1994 Academic Press, Inc.