Studies On Protease Activity In Midgut Of Five Common Orchard Pests

The planting scale and total output of fruit trees in China remains in a world-leading position.Orchard pests have become a major problem restricting the development of the fruit tree planting industry in China.In recent years,the 3R problem of pesticides has become increasingly prominent,and traditional chemical control cannot meet the needs of the development of the green fruit industry.A series of insect physiological activities such as insect growth,development and reproduction require energy supplied from external feeding.The insect midgut is the main location of food digestion.Proteolytic activity of the midgut is an important index for measuring the digestive ability of insects.Different insects feed on different host plants,and their protease activities are different.Protease inhibitors have certain inhibitory effects on the activity of enzymes.In this study,the intestinal pH values and the activities of total protease,high-alkaline trypsin,low-alkaline trypsin and chymotrypsin in the larvae of five common fruit tree pests,Adoxophyes orana,Carposina sasakii Matsumura,Dasineura pyri Bouche,Janus piri and Rhvnchites faldermanni Schoenherr,were determined and compared using biochemical techniques.At the same time,the inhibitory effects of various protease inhibitors were determined,and the corresponding protease inhibitors were selected to provide a theoretical basis for the development of new biological pesticides related to protease inhibitors and the cultivation of transgenic insect-resistant plants.The main results of this study are as follows:1.The midgut pH values of five orchard pests were obtained,and the results showed that the midgut pH of the 2^nd,3^rd and 4^th instar was 7.5 and the 5^th was 8.1 for the peach-fed Adoxophyes orana;the midgut pH of the 2^nd,3^rd and 4^th instar was 7.1 and the 5^th was 7.6for the plum-fed Adoxophyes orana;the midgut pH values of the Janus piri and the Dasineura pyri Bouche were 8.2 and 7.6,respectively;the midgut pH value of the Carposina sasakii Matsumura was 8.3;the midgut pH of the Rhvnchites faldermanni Schoenherr was 8.0 at the 2^nd,3^rd and 4^th instar larvae,and 8.9 at the 5^thh instar.The midgut pH values of the five insects ranged from 7 to 9.The results showed that the intestinal environment of the five pests selected in this study was alkaline.The Adoxophyes orana peach-hosted and plum-hosted and the Rhvnchites faldermanni Schoenherr had a sudden increase in the midgut pH values of the last instar.The results can provide a basis for rational use of gastrotoxic pesticides with different acidity and alkalinity.2.The activities of total protease,high-alkaline trypsin,low-alkaline trypsin and chymotrypsin in the midgut of five orchard pests at different instars were determined.It was concluded that the activity of four midgut proteases of these five insects was related to the larval instar.The highest enzymatic activity was found in the 3^rd instar of the peach-fed Adoxophyes orana,the 5^thh instar of the plum-fed Adoxophyes orana,the 3^rd instar of the Janus piri and Dasineura pyri Bouche,the 4^th instar of the Carposina sasakii Matsumura,and the 2^nd instar of the Rhvnchites faldermanni Schoenherr.This study provides a basis for pest control at specific larval instars.3.The activity of four midgut proteases of the peach-fed and plum-fed Adoxophyes orana was compared.It was found that the activity of four midgut proteases of the peach-fed Adoxophyes orana was higher than that of the plum-fed.Host species were identified as a factor affecting protease activity in the midgut of insect pests.4.The midgut protease activities of five orchard pest larvae were compared.It was found that among the five insect species,the highest protease activity of four proteases was for the Adoxophyes orana,in which the activity of peach-fed was slightly higher than that of the plum-fed,and the lowest activity was for the Janus piri.5.Protease inhibitors with obvious inhibitory effects on the midgut protease of five orchard pests were screened.It was concluded that the types and concentrations of inhibitors could affect their inhibitory effects on the four midgut proteases.The specific results are as follows:5mmol/L PMSF,2 mmol/L IAA and 1 mmol/L TLCK for Adoxophyes orana;5 mmol/L EGTA,10 mmol/L EDTA and 2 mmol/L TPCK for Janus piri;5mmol/L EDTA,50μg/mL STI and 10 mmol/L EDTA for Dasineura pyri Bouche;5mmol/L PMSF and 1 mmol/L TPCK for Carposina sasakii Matsumura;and 10 mmol/L DTT and 1 mmol/L TLCK for Rhvnchites faldermanni Schoenherr,respectively.