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Chitin is a major component of the cuticle of arthropods. However, the synthesis of chitin is poorly understood. Feeding larvae of the insect Lucilia cuprina on the fungal chitin synthase competitive inhibitor, nikkomycin Z resulted in strong concentration-dependent mortality of the larvae (LD50 = 280 nM). This result demonstrates that chitin is an essential component of this insect. The complete cDNA and deduced amino-acid sequences of the first arthropod chitin synthase-like protein, LcCS-1, from the larvae of the insect L. cuprina have been determined. The cDNA sequence is 5757 bp in length and codes for a large complex protein containing 1592 amino acids (Mr = 180 717). Analysis of the whole protein sequence reveals low, but significant, similarity to yeast chitin synthases with stronger areas of conservation centred on local regions implicated in the active sites of the yeast enzymes. Strikingly, LcCS-1 contains 15-18 potential transmembrane segments, indicating that the protein is an integral membrane protein. Two alternative topographical models of LcCS-1 are described, which involve its association with either the plasma membrane or the membrane of intracellular vesicles. LcCS-1 mRNA is produced in all life stages of the insect with expression in the larval stage limited to the integument and trachea. In a third instar larva the mRNA was localized to a single layer of epidermal cells immediately underlying the procuticle region of the integument. cDNA or genomic sequences that are highly related to fragments of LcCS-1 were demonstrated in three insect orders, one arachnid and Caenorhabditis elegans, thereby attesting to the importance of this enzyme in these chitin-producing organisms. Bioinformatics has been used to deduce the gene sequence and organization of the highly homologous Drosophila melanogaster orthologue of LcCS-1, DmCS-1.

Two chymotrypsin-like proteases were purified from the secretory and excretory material of first-instar larvae of Lucilia cuprina. The hydrolysis of N-succinyl-L-phenylalanine-nitroanilide was used to monitor the purification of these proteases which was achieved by affinity chromatography on soybean trypsin inhibitor-Sepharose followed by anion exchange and hydrophobic interaction chromatographies. The enzymatic specificity of the most abundant protease (Lucilia chymotrypsin b; LCTb) was further defined by determining the amino acid sequence of peptides released from insulin B chain after incubation with LCTb. Peptide amino acid sequences obtained from LCTb were used to design degenerate oligonucleotide primers which, in conjunction with the polymerase chain reaction, enabled cDNA coding for LCTb to be cloned and sequenced. The deduced amino acid sequence of LCTb showed many of the structural features of serine proteases as well as significant amino acid sequence homology with chymotrypsins from a diverse range of species. It is probable that LCTb plays an important role in establishing the myiasis-causing larvae of L. cuprina on host skin as well as providing nutrients for the rapidly growing larvae.

Various protease inhibitors active against both trypsin- and chymotrypsin-like serine proteases were used to characterize gut proteases from Lucilia cuprina by in vitro feeding assays. Significant larval growth retardation was observed on feeding first-instar larvae with trypsin inhibitors, particularly soybean trypsin inhibitor. Feeding of chymostatin, a specific chymotrypsin inhibitor, resulted in no significant growth retardation. This information suggests that trypsin-like serine proteases are probably the major gut digestive enzymes. A DNA fragment obtained by PCR which coded for part of a putative trypsin gene from L. cuprina was used to isolate a four-member multigene family of trypsins. The full nucleotide sequence of one of the genes and partial sequence from the other three genes were determined. Transcription of at least one of the genes has been confirmed. All four of the genes appear to have arisen by two separate gene duplication events.