Abstract: 【Aim】 The aim of this study is to investigate the effects of different protease inhibitors on the protease activities in the larval midguts of Janus piri and Dasineura pyri. 【Methods】 The midgut pH values and the activities of four proteases (total protease, high-alkaline trypsin, low-alkaline trypsin and chymotrypsin) in the midguts of the 2nd-5th instar larvae of J. piri and the 1st-4th instar larvae of D. pyri were determined by biochemical techniques. The effects of 10 inhibitors on the protease activities in the larval midguts of the two insect species were further determined at the optimum pH and larval instar. 【Results】 The mean pH values in the larval midguts of J. piri and D. pyri were 8.2 and 7.6, respectively, and the activities of the four midgut proteases were the highest in the 3rd instar larvae of the two insect species. The most effective inhibitor for total protease activity in the midgut of J. piri larvae was 5 mmol/L EDTA, followed by 1 mmol/L TPCK. For high-alkaline trypsin activity in the midgut of J. piri larvae, the most effective inhibitor was 1 mmol/L TPCK, while for low-alkaline trypsin activity, the best inhibitory effect was achieved with 10 mmol/L PMSF. The most effective inhibitor for chymotrypsin activity in the midgut of J. piri larvae was 5 mmol/L EGTA. In the midgut of D. pyri larvae, the most effective inhibitor for total protease activity was 1 mmol/L TPCK, followed by 50 μg/mL STI. For high-alkaline trypsin activity in the midgut of D. pyri larvae, the best inhibitory effect was achieved with 5 mmol/L EDTA, while for low-alkaline trypsin activity, 10 mmol/L EDTA showed the best inhibitory effect. The most effective inhibitor for chymotrypsin activity in the midgut of D. pyri larvae was 50 μg/mL STI. 【Conclusion】 TPCK, STI and EDTA had better inhibitory effects on the total protease activities in the larval midguts of J. piri and D. pyri, EDTA and EGTA had better inhibitory effects on their larval midgut high-alkaline trypsin activities, PMSF, EDTA and TPCK had better inhibitory effects on their larval midgut low-alkaline trypsin activities, and EGTA had a better inhibitory effect on their larval chymotrypsin activities. The results provide some theoretical reference value for the development of new biopesticides related to insect midgut proteases and inhibitors.

Key words: Janus piri, Dasineura pyri, midgut protease, intestinal pH, protease inhibitors, enzyme activity