Time pattern of appearance and disappearance of active molting chitinase in Manduca cuticle. The endogenous activity

Localization of insect molting chitinase in the old Truman’s terminology. An additional check to assure cuticle of pharate Munduca pupae has recently been that the pharate pupae analyzed were developing at reported from this laboratory [l] . It was shown that comparable rates was afforded by the fact that events highly active chitinase is held tenaciously in the cuticle late in the pupal molt, i.e. within the final 32 hr so that its activity remains by comparison low in preceding ecdysis, can be timed approximately by molting fluid and epidermal extracts. Cuticle chitinase observation of the extent of tanning in the new cuticle preparations may release over 200 pg N-acetylglucosamine which at that stage is visible through the old cuticle per milligram cuticle protein per hour at the pH 8.2, of enzymatically active Munduca molting fluid, and were shown to utilize endogenous cuticle chitin preferentially in presence of exogenous colloidal chitin prepared from Manduca cuticles. At the same time, however, endogenous chitinase activity in newly synthesized cuticle was found to be absent. It was of interest, therefore, to determine at what time endogenous chitinase activity arises in insect cuticle relative to the molt. Specific endogenous cuticle chitinase activity rises sharply about 30 hr before pupal ecdysis; it disappears as abruptly about 12 hr later. A similar but lower endogenous peak in old cuticle chitinase activity occurs just prior to the preceding larval molt. Larvae of Munducu sex&, the tobacco homworm, were reared on an artificial diet from eggs obtained through the courtesy of Dr R. A. Bell of the U.S.D.A. Metabolism and Radiation Research Laboratory, Fargo, North Dakota. -The larvae were observed carefully as the 4th larval molt approached, timed with 10 min of ecdysis, and harvested at various times following ecdysis. As previously reported [2] , larval ecdysis of Lepidopteru is associated with a circadian clock. However, animals from the same hatch do not all develop at the same rate, as already described by Truman [2]. The larvae used in the experiments here reported were chosen from the most rapidly developing group, i.e. from those belonging to Gate 1 in