SUMMARY Chitin is one of the most important biopolymers in nature. It is mainly produced by fungi, arthropods and nematodes. In insects, it functions as scaffold material, supporting the cuticles of the epidermis and trachea as well as the peritrophic matrices lining the gut epithelium. Insect growth and morphogenesis are strictly dependent on the capability to remodel chitin-containing structures. For this purpose, insects repeatedly produce chitin synthases and chitinolytic enzymes in different tissues. Coordination of chitin synthesis and its degradation requires strict control of the participating enzymes during development. In this review, we will summarize recent advances in understanding chitin synthesis and its degradation in insects.

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To start investigation of chitin synthesis and peritrophic membrane formation in the midgut of Manduca sexta, we have cloned a cDNA fragment encoding chitin synthase. Northern blots with a corresponding RNA probe revealed a single transcript of 4.7 kb, which was most prominent in poly(A) RNA isolated from the anterior and median midgut as well as from tracheal cells. In situ hybridization showed that the amount of chitin synthase transcripts in the cytoplasm of columnar cells decreased from the anterior to the posterior midgut. Moreover, in the anterior midgut they were localized in the apical region of columnar cells. Southern blots suggested more than one gene locus for chitin synthase in the Manducagenome. To analyze the distribution of chitin synthases on the protein level, we expressed a polymerase chain reaction (PCR) fragment of 119 amino acids in Escherichia coli and generated polyclonal antibodies to the purified recombinant protein. In immunoblots of crude extracts derived from the anterior midgut as well as from partially purified brush border membranes of columnar cells the affinity-purified anti-chitin synthase antiserum labeled a single protein with an apparent molecular mass of 150-200 kDa. Immunohistochemistry showed intense labeling in midgut brush border membranes. Immunofluorescence was restricted to the apical ends of microvilli. Apical membranes of salivary glands and tracheal cells were labeled as well, but not those of Malpighian tubules. This is the first time that chitin synthase expression has been visualized in insect tissues on the level of proteins.

The chitin portion of the peritrophic matrix in the midgut of the tobacco hornworm, Manduca sexta, is produced by chitin synthase 2 (CHS2), a transmembrane family II glycosyltransferase, located at the apical tips of brush border microvilli. To look for proteins that potentially interact with CHS2, we performed yeast twohybrid screening, identifying a novel chymotrypsin-like protease (CTLP1) that binds to the extracellular carboxyterminal domain of CHS2. The occurrence of this interaction in vivo is supported by co-localization and coimmunoprecipitation data. Based on our findings we propose that chitin synthesis is controlled by an intestinal proteolytic signalling cascade linking chitin synthase activity to the nutritional state of the larvae.

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