Requirement for sequential enzymatic activities for penetration of the integument of the corn earworm (Heliothis zea)

When larvae of the corn earworm, Heliothis zea, are boiled in 1% sodium dodecyl sulfate for 2 to 4 hr, larval contents are solubilized leaving a thin and transparent but intact skeletal structure that we are naming ghosts. The thinnest and most transparent ghosts are produced by boiling first-instar larvae. As larvae mature, later instar forms possess substantial quantities of melanized areas that remain largely undissolved in the detergent. Chemical analyses reveal that ghosts consist largely, and perhaps exclusively, of chitin and protein. Scanning electron microscopy shows that the chitin-protein complex is a continuous layer that does not possess holes sufficiently large for fungal hyphae to pass through without some type of accompanying enzymatic activity. Digestion of ghosts is possible using the combination of a proteolytic enzyme followed by chitinase. Because this sequence is necessary for dissolution of the structure, the protein material is most likely structural and wrapped about the chitin. Such an arrangement would allow protection of the structure from direct chitinase attack. The number of proteins involved is not known; however, because they survive the detergent-heat treatment this strongly suggests that the protein(s) is covalently bonded to chitin. Several different proteolytic enzymes allow attack of the underlying chitin by chitinase. This indicates that the accompanying protein(s) is exposed and therefore accessible to several types of proteolytic enzymes. Such a conclusion may not be warranted, however, if the accessibility and conformational state of the protein(s), and therefore digestibility, is affected by the relatively vigorous preparative procedure.