Expression of autophagy 8 (Atg8) and its role in the midgut and other organs of the greater wax moth, Galleria mellonella, during metamorphic remodelling and under starvation.

A 345 base pair cDNA encoding autophagy 8 (Atg8) of Galleria mellonella (GmAtg8) was cloned and sequenced. The deduced protein was estimated to be 118 amino acids long. Structural comparison and phylogenetic analysis showed that GmAtg8 belong to the Atg8 family of ubiquitin-like proteins. It is predicted to contain four ß-sheets and four α-helices. It also contains a highly conserved glycine residue at the C-terminal, as well as highly conserved Phe77 and Ph79 at a recognition cleavage site of Atg4 and Tyr49, and Leu50 at a site for activation of the lipidated form of Atg8 by Atg7 and Atg3. The developmental expression profile demonstrated that GmAtg8 transcript and its protein products are expressed in such organs as the midgut, ovary, Malpighian tubules, fat body and silk gland. In the midgut and silk gland, GmAtg8 transcript and its protein products increased during metamorphosis and under starvation, but decreased after re-feeding. Expression of autophagy seems to precede apoptosis in the midgut transformation from larva to pupa and pupa to adult during metamorphosis. Some waves overlap with apoptotic waves, particularly at early stages, but others are unique in terms of site and timing.

Expression analyses of caspase-1 and related activities in the midgut of Galleria mellonella during metamorphosis.

The cDNA encoding caspase-1, a main protease involved in apoptosis, was cloned and sequenced from the midgut of the greater wax moth, Galleria mellonella. The open reading frame contains 879 nucleotides, encodes 293 amino acids, and was registered as Gmcaspase-1. The sequence comparison showed a high homology to lepidopteran caspase-1, human caspase-3, and ced-3 of Caenorhabditis elegans. Gmcaspase-1 is predicted to contain a short prodomain, large subunit, and small subunit domain. It also exhibits all characteristics of caspase, including three conserved cleavage sites after Asp-25, Asp-192, and Asp-181, three active site residues including a highly conserved QACQG pentapeptide active-site motif, and four substrate binding sites. The expression profiles during development showed that the transcript of Gmcaspase-1 and its protein products appeared in two or more waves in the midgut during metamorphosis. Immunohistochemistry, in situ hybridization, and TUNEL analyses revealed that apoptosis occurred first at the basal, then middle and then apical regions in the midgut epithelium and the yellow body is formed in the lumen. At least three waves of mitosis and differentiation follow the apoptosis waves from the basal and middle to apical parts to form the adult epithelium.