Effects of Several Larvicidal Compounds on Chitin Biosynthesis by Isolated Larval Integuments of the Sheep Blowfly Lucilia cuprina

Abstract

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.

Integumental Chitin Synthase Activity in Cell-free Extracts of Larvae of the Australian Sheep Blowfly, Lucilia cuprina, and Two Other Species of Diptera

“…Isolated intact integuments from early third-instar larvae of L. cuprina incorporate [14C]NAG or [14C]glucosamine into chitin when incubated in insect tissue culture medium (Turnbull and Howells 1982) and, as demonstrated in this article, homogenates of these integuments form chitin from [14C]UDPAG. Thus some of the chitin synthase activity present in the intact integuments is preserved in active form in the homogenates.…”

“…Secondly, gassing the incubations with nitrogen or the addition of KCN did not reduce incorporation. Both treatments effectively eliminate chitin synthesis by intact epidermal cells (Turnbull and Howells 1982). Thirdly, incubations in which [14C]UDPAG was replaced with either e 4 C]NAG or [14C]glucosamine (at the same concentration and specific activity as [14C]UDPAG) gave only 11 and 5/,;;, respectively, of the normal level of incorporation.…”

“…This insecticide, which kills larvae of L. cuprina and inhibits chitin synthesis in intact larval integuments from this insect (Turnbull and Howells 1982), also inhibited incorporation by homogenates of integuments. As with polyoxin-D, inhibition was not complete; although there was variation in the level of inhibition between different homogenates, inhibition was always obtained.…”

“…Since labelled chitin is synthesized from [14C]NAG by intact epidermal cells from early third-instar L. cuprina larvae (Turnbull and Howells 1982), it was important to consider the possibility that the relatively low incorporations obtained with the homogenates might be due to residual intact cells (particularly since e 4 C]NAG was shown to be produced during incubation, as a breakdown product of the substrate e 4 C]UDPAG-see below). Consideration was also given to the problem of microbial contamination, because fungi synthesize chitin and bacteria may convert UDPAG to UDP-N-acetylmuramic acid.…”

“…The ethanolinsoluble material from large-scale (three times norma\) incubations were deproteinized by digestion first with trypsin (5 mg/ml, final concentration) for 24 hr and then with pepsin (5 mg/ml, final concentration) for 24 h as described by Turnbull and Howells (1982). The residual ethanol-insoluble material was resuspended in 1 ml concentrated HCI and incubated for 16 h at 80°C.…”