Isolated whole integuments from L. cuprina larvae rapidly incorporate radioactivity from both N-acetyl[I-'4C]glucosamine and [1-'4C]glucosamine into alkali-insoluble material, a reaction which does not require preincubation of the tissue with /i-ecdysone. The labelled product was degraded to N-acetylglucosamine during digestion with chitinase, establishing that it consists mainly of chitin. Incorporation was inhibited by polyoxin-D (Iso, 6 x 10-7 M) and diflubenzuron (Iso, 7 x 10-7M) but was not inhibited to any marked extent by isoprothiolane, Vetrazin or a-methyl-DOPA. The effectiveness of diflubenzuron as an inhibitor of chitin synthesis in this system (Iso, 7 x 10-7 M) correlates well with its potency as a larvicide (LDso, 2·1 x 10-6 M), providing additional support for the proposal that this compound kills larvae by interfering with chitin deposition in the cuticle. Polyoxin-D was much more effective as an inhibitor of chitin synthesis (Iso, 6 x 10-7 M) than as a larvicide (LDso, 2·0x 10-5 M). It was established that the final intermediate of chitin biosynthesis (UDP-N-acetylglucosamine) was formed in the isolated integuments in the presence of diflubenzuron and polyoxin-D. It seems likely therefore that both compounds interfere with the final polymerization step of the chitin biosynthesis pathway.
Chitin synthase activity has been demonstrated in crude homogenates of larval integuments from L. cuprina and in similar preparations from Musca domestica and Calliphora erythrocephala. This is the first report of an insect integumental chitin synthase. This activity brings about the incorporation of radioactivity from UDP-N-acetyl-[14C]glucosamine into an ethanol-and alkali-insoluble form. A major part of this labelled product has been characterized as chitin by its insolubility in alkali, resistance to degradation by proteases and its susceptibility to digestion by chitinase and RCI. Most of the radioactivity solubilized during digestion by chitinase co-migrates with N-acetylglucosamine, glucosamine and chitobiose during paper chromatography. Some radioactivity also becomes incorporated into non-chitin products in this system. There is substantial evidence that incorporation is not brought about by whole epidermal cells or by microbial contamination in the homogenates. The extent of incorporation obtained with the homogenates is limited by the presence of degradative enzymes which rapidly break down the substrate (UDP-N-acetylglucosamine). The incorporation was partially inhibited (50-70%) by both polyoxin-D (apparent Ki 0'04I1M) and diftubenzuron (apparent . This is the first report of a cell-free chitin-synthesizing system derived from insect tissue which is sensitive to inhibition by diftubenzuron.
Inhibitors of DOPA decarboxylase, the key enzyme in the formation of the sclerotizing agent (N-acetyl dopamine) of the blowfly cuticle, have been tested for larvicidal activity against L. cuprina. A significant level of DOPA decarboxylase activity has been shown to be present throughout larval life in this species. Four potent in vitro inhibitors of L. cuprina larval DOPA decarboxylase (carbidopa, benserazide, methyl tyrosine and methyl DOPA) have been shown to be effective larvicides when fed to first-or second-instar larvae. However, no correlation is seen between the apparent K t and LDso values for these compounds. Treated larvae are observed to die at the next moult but death can be averted by the addition of N-acetyl dopamine to the food. Thus the toxic effects of the DOPA decarboxylase inhibitors appear to result from an inhibition of the formation of the sclerotizing agent in the cuticles of treated larvae.
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