Investigation of larvae digestive β-glucosidase and proteases of the tomato pest Tuta absoluta for inhibiting the insect development

The tomato leaf miner Tuta absoluta is one of the most devastating pests for tomato crops. Digestive proteases and beta-glucosidase enzymes were investigated using general and specific substrates and inhibitors. Maximal beta-glucosidase and proteolytic activities occurred at temperature and pH optima of 30 and 40 degrees C, 5 and 10-11 unit of pH, respectively. Zymogram analysis showed the presence of distinguished beta-glucosidase exhibiting a specific activity of about 183 +/- 15 mu mol min(-1) mg(-1). In vitro inhibition experiments suggested that serine proteases were the primary gut proteases. Gel based protease inhibition assays demonstrated that the 28 and 73 kDa proteases might be trypsin-like and chymotrypsin-like enzymes, respectively. Overall gut trypsin-like and chymotrypsin-like activities were evaluated to be about 27.2 +/- 0.84 and 1.68 +/- 0.03 mu mol min(-1) mg(-1), respectively. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed that T. absoluta gut serine proteases are responsible for Bacillus thuringiensis Cry insecticidal proteins proteolysis. Additionally, bioassays showed that T. absoluta larvae development was more affected by the beta-glucosidases inhibitor (D-glucono-delta-lactone) than the serine proteases inhibitor (soybean trypsin inhibitor). These results are of basic interest since they present interesting data of beta-glucosidases and gut serine proteases of T. absoluta larvae.