MIDGUT PROTEINASES IN 3 DIVERGENT SPECIES OF COLEOPTERA

Cysteine proteinases have been found in some families of Coleoptera an d, based on this, these enzymes were supposed to be characteristic of Coleoptera. To test this hypothesis, we studied midgut homogenates of three phylogenetically distant Coleoptera species: Tenebrio molitor (T enebrionidae) larvae, Pyrearinus termitilluminans (Elateridae) larvae, and Pheropsophus aequinoctialis (Carabidae) adults. T. molitor displa y two cysteine proteinases (pHo 6.8) resolved in Superose (FPLC) with Mr 31,000 and 51,000. These enzymes are inhibited by E-64 and pHMB, ar e activated by EDTA + cysteine and hydrolyze benzoyl-DL-arginine-beta- naphthylamide. T. molitor enzymes differ from a cysteine proteinase (M r 64,000 using Superose) present in the wheat meal ingested by the ins ect. The cysteine proteinases predominate in the anterior two thirds o f T. molitor midgut, probably because they are unstable in the higher luminal pH observed in the posterior third of the midgut. P. termitill uminans and P. aequinoctialis do not display cysteine proteinases, alt hough they have trypsins (Mr 15,000, 25,000 and 41,000 for P. termitil luminans; Mr 26,000, 33,000 and 52,000 for P. aequinoctialis) and chym otrypsins (Mr 38,000 and 25,000 for P. aequinoctialis and Mr 15,000 fo r P. termitilluminans). Our results, together with literature data, su ggest that cysteine proteinases occur in the Cucujiformia ancestor, wh ich corresponds to the ancestor of most Coleoptera which ingest seeds rich in serine proteinase inhibitors.