Effects of protease inhibitors on the activities of midgut proteases of larval Grapholitha molesta(Lepidoptera: Tortricidae)

[Aim] Grapholita molesta(Busck) is a fruit pest that causes extremely serious damage. Intestinal protease plays an important role in its growth and development processes. To provide new ideas for using protease inhibitors to control pest insects, the effects of the optimum pH, different inhibitors and activators on the midgut protease activities of G. molesta larvae were studied in this study. [Methods] Enzyme fluids were extracted from the midgut of the 3rd instar larvae of G. molesta. Using enzyme specificity substrates, the optimum pH of the midgut proteases of G. molesta larvae was detected in three different buffer solutions (ddH sub(2) O as the CK), the effect of protease inhibitors and activators on the midgut protease activities were determined. At the same time, the effect of the different protease inhibitors (PMSF, TLCK, TPCL and STI) on the midgut protease activities was also determined whenG. molesta larvae were fed with them. [Results] The optimum pH levels of midgut total protease of G. molesta larvae in the three buffers Tris-HCl, KH sub(2) PO sub(4)/NaOH and glycine/NaOH were 10.5, 11.0 and 11.0, respectively, those of high-alkaline trypsin were 10.5, 11.0 and 11.0, respectively, those of low-alkaline trypsin were 8.5, 9.0 and 9.0, respectively, and those of chymotrypsin were 8.5, 9.0 and 9.5, respectively. Of the five protease inhibitors (DTT, PMSF, TLCK, TPCL and STI), except that TLCK activated the chymotrypsin activity, the other inhibitors inhibited the activities of all proteases; and the higher the inhibitor concentration was, the more significant the inhibitory effects were. The inhibition effects of the inhibitor DTT on the activities of total protease and low-alkaline trypsin were greater than those of the other inhibitors. Of the four protease activators (MgCl sub(2), CaCl sub(2), EDTA and EGTA), MgCl sub(2) inhibited the activities of total protease and chymotrypsin but activated the trypsin activity, CaCl sub(2) activated the activities of casein enzyme and low-alkaline trypsin but inhibited the activities of high-alkaline trypsin and chymotrypsin, EDTA inhibited the activities of the four proteases, and EGTA inhibited the activities of the other three proteases except high-alkaline trypsin. When G. molesta larvae were fed with different protease inhibitors (PMSF, TLCK, TPCL and STI), all inhibitors could inhibit the activities of the four proteases, with obviously different inhibitory activities at different sampling time poin