Chitinolytic activities in Heligmosomoides polygyrus and their role in egg hatching

The occurrence of chitin in the eggshell of Heligmosomoides polygyrus has been determined by histochemical and biochemical techniques. Approximately 5% of the egg dry weight was chitin. Staining with Calcofluor white showed the chitin in the eggshell to be more accessible to the stain after hatching or rupturing of the eggshell. Chitinolytic activity has been detected using fluorescent substrates in extracts of adult males (at low levels), females and eggs. Enzyme activity in situ, within the developing larvae, was visualised with the same substrates. It was localized in discrete granules about 1 micron in diameter which occurred as groups in areas of about 5 microns in diameter, in the posterior third of the larvae. The chitinolytic activity in the eggs increased with the age of the egg and was released into the medium when the eggs hatched. The chitinase activities were very sensitive to inhibition by allosamidin, a specific chitinase inhibitor, with an IC50 for the crude egg extract of 2.2 nM. However, treatment of eggs with 250 microM allosamidin resulted in a slowing but not cessation of egg hatching.